Vijayasal Book Eng

Bijaysal
A Monograph of Pterocarpus marsupium in Nepal
Government of Nepal
Ministry of Forests and Environment
Department of Plant Resources
National Herbarium and Plant Laboratories
Godawari, Lalitpur, Nepal
2021
xi
iv
Bijaysal
“A Monograph of Pterocarpus marsupium in Nepal”
Editors:
Lajmina Joshi
Sangeeta Rajbhandary
Buddi Sagar Paudel
Sanjeev Kumar Rai
Subhash Khatri
Government of Nepal
Department of Plant Resources
National Herbarium and Plant Laboratories
Godawari, Lalitpur, Nepal
2021
i
Bijaysal: A monograph of Pterocarpus marsupium in Nepal
ISBN: 978-9937-9476-0-2
Editors: Lajmina Joshi

Sangeeta Rajbhandary
Buddi Sagar Paudel
Sanjeev Kumar Rai
Subhash Khatri
Technical Coordinator: Pratikshya Chalise
Citation: Second name of author, Abbreviation of first name. Name of

Chapter. 2021. In: Joshi, L., Rajbhandary, S., Paudel, B.S., Rai,
S.K., and Khatri, S. (Eds.) Bijaysal: A Monograph of Pterocarpus
marsupium in Nepal. National Herbarium and Plant Laboratories,
Godawari, Lalitpur, Nepal. Pp.
(For example: Chalise, P. Taxonomy. 2021. In: Joshi, L.,
Rajbhandary, S., Paudel, B.S., Rai, S.K., and Khatri, S. (Eds.)
Bijaysal- A Monograph of Pterocarpus marsupium in Nepal.
National Herbarium and Plant Laboratories, Godawari, Lalitpur,
Nepal. Pp. 6-11.)
Cover design: Sangeeta Rajbhandary
Cover page: Mature tree (Ganga Datta Bhatt),

Winged pod (Amrit KC),
Kino gum releasing from bark (Yagya Raj Paneru),
Seeds (Ram Krishna Bhandari),
TLS of wood (Pratikshya Chalise),
Seedling (Ram Krishna Bhandari),
Anti-microbial test (Pramesh Bahadur Lakhey),
Wooden Tumblers (Dipesh Pyakurel).
No. of Copies: 500
© All rights reserved, 2021
National Herbarium and Plant Laboratories, Godawari, Lalitpur, Nepal
(www.kath.gov.np)
Printed at: Bonefied Printing Press, Nayabazar, Kathmandu, Nepal
ii
FOREWORD
Pterocarpus marsupium Roxb, locally known as “Bijaysal” is a medium to a large-sized
deciduous tree belonging to the family Fabaceae. The species is native to India, Nepal,
Bangladesh, Sri Lanka, and Taiwan. In Nepal the species is confined to an altitude of 100
to 640 m. The plant has a limited global distribution and due to its multipurpose uses,
especially being highly exploited for the extraction of Kino gum, the trees are disappearing
rapidly. The aqueous extract of its heartwood is considered as a miracle cure against diabetes
and several other uses are mentioned in traditional systems of medicine.
Being one of the prioritized species Government of Nepal has banned its cutting, collection,
transportation and export for commercial purpose. Recently, the Department of Forests has
prepared “Bijaysal Conservation Action Plan 2018” for the conservation of Bijaysal and is
the first conservation action plan for plant species in Nepal. Therefore, assessing the detailed
information on its taxonomy, reproductive biology, ecology, distribution, conservation status
of the species and developing an accurate species level identification through its anatomical
study is of utmost importance. Owing to the existing threats as well as poor seed viability,
slow growth and poor natural regeneration of this species; hunting for effective measures
to enhance the seed germination is also essential.
I am delighted to see the publication “Bijaysal: A monograph of Pterocarpus marsupium
in Nepal” and congratulate all the contributors of the manual, for their significant
contributions. The book includes detail information on taxonomy, reproductive biology,
anatomy, distribution, ecology, population status, germination, ethnobotany, phytochemistry,
pharmacology, anti-microbial activity, threats, conservation and trade of Bijaysal. I would
also like to express my sincere thanks to the editors- Lajmina Joshi, Former Scientific
Officer, Department of Plant Resources; Prof. Sangeeta Rajbhandary, Central Department
of Botany, Tribhuvan University; Buddi Sagar Paudel, Spokesperson, Ministry of Forests
and Environment; Sanjeev Kumar Rai, Director General, Department of Plant Resources;
and Subhash Khatri, Chief, National Herbarium and Plant Laboratories for their support
and effective coordination and hard work to bring this manual. I am also thankful to Shamik
Mishra for the Nepali translation of the relevant chapters of the book. I would like to thank
all the staffs of Department of Plant Resources, especially the staffs of National Herbarium
and Plant Laboratories, Godawari for their direct as well as indirect contribution to bring
out this book. I strongly believe that the information generated in the book will be helpful
to the researchers, local stakeholders, community forests, private nurseries as well as the
general public who have a keen interest towards the target species and will ultimately help
in proper identification, management, sustainable utilization and conservation of existing
populations of Bijaysal.
July, 2021
Dr. Pem Narayan Kandel

Secretary
Singhadurbar, Kathmandu
iii
iv
PREFACE
Pterocarpus marsupium commonly known as “Bijaysal” in Nepal is a deciduous tree

in the family Fabaceae. The aqueous extract of its wood was used against diabetes
since very long time and the species has been highly exploited for the heartwood
as well as for the Kino gum. Poor natural regeneration, poor seed viability and
germination, grazing, over exploitation of the tree have resulted massive decrease in
natural populations worldwide. The IUCN Red List of Threatened Species has enlisted
the tree in ‘Near Threatened’ category. Government of Nepal has declared Bijaysal
as a protected species and has banned its collection, fetching and transportation for
commercial purpose. A wide diversity of chapters that cover information on almost
every aspect of Bijaysal have been incorporated in the monograph.
We believe the book will be helpful to the scientific community, local stakeholders
as well as general public and will help in its proper identification, management,
sustainable utilization and conservation.
It is a team work, but still we would like to specially thank Pratikshya Chalise,
Research Officer, National Herbarium and Plant Laboratories, Godawari, for her
contribution in different chapters and all necessary requirements to bring out this book.
We would specially like to thank the contributing authors Mohan Dev Joshi, Secretary,
Ministry of Industry, Tourism, Forests and Environment, Karnali Province, Surkhet;
Pashupati Nath Koirala, Ministry of Forests and Environment, Singhadurbar,
Kathmandu; Sajita Dhakal, National Herbarium and Plant Laboratories, Godawari;
Parasmani Yadav, Devi Prasad Bhandari from Natural Products Research Laboratory,
Thapathali; Pramesh Bahadur Lakhe and Sachita Joshi from Department of Plant
Resources, Thapathali; Ram Krishna Bhandari from Plant Research Centre,
Nepalgunj, Banke; Prof. Suresh Kumar Ghimire and Neelam Pandey from Central
Department of Botany, Tribhuvan University; Dipesh Pyakurel from Resources
Himalaya Foundation, Sanepa and Yagya Raj Paneru, Capital College and Research
Centre, Koteshwor.
Several data were collected from the field by the authors to complete the chapters, so
we take this opportunity to thank Yagya Raj Paneru; Ghanashyam Chalise, Charpala
Community Forests User Group, Rupandehi; Parbati Chalise; Prabhat Chalise;
Pratima Chalise; Bashu Dev Bhatt; Parwati Bhatt for helping during the sample
collection from different places of Rupandehi, Kailali and Kanchanpur districts
and during ethnobotanical survey at Kailali and Kanchanpur districts. The local at
Malakheti, Khamaura, Kailali district and Bhimdattanagar, Kanchanpur district are
especially acknowledged for their warm hospitality and knowledge. Dambar Karki,
District Plant Resource Office, Kailali; Maheshwori Bhatt, Assistant Forest Officer,
v
Division Forest Office, Kanchanpur; Bodh Raj Subedi, Divisional Forest Officer,
Division Forest Office, Rupandehi; and Maha Laxmi Sharma, Assistant Forest Officer,
Division Forest Office, Rupandehi for their direct as well as indirect help during the
field visits and sample collection. We are greatly indebted to the entire management
committee of Charpala CFUG and Shankarnagar CFUG, Rupandehi and Gwalabari
CFUG, Bani, Kanchanpur for their direct as well as indirect help during the visits
to the respective community forests.
At the same we would also like to acknowledge all the staff of National Herbarium
and Plant Laboratories for their help in various ways for the book. We would like to
thank all the staffs of Department of Plant Resources for their valuable contribution
to bring out this book.
Photo credits are provided to these persons: Pratikshya Chalise, Yagya Raj Paneru,
Ghanashyam Chalise, Ganga Datta Bhatt, Amrit KC, Ram Krishna Bhandari, Neelam
Pandey, Dipesh Pyakurel and Pashupati Nath Koirala.
We are highly obliged to Shamik Mishra for the Nepali translation of the relevant
chapters of the book. We acknowledge Mahesh Maharjan for the current layout and
printing.
- Editors
vi
ACRONYMS AND ABBREVIATIONS
ANOVA Analysis of Variance

°C degree Celsius
CAMP Conservation Assessment and Management Prioritization
cm centimetres
CF(s) Community Forest(s)
CFUG Community Forests Users Group
DoF Department of Forests
DPR Department of Plant Resources
et al. ‘et alia’, ‘and others’
FAA Formalin- Acetic Acid- Alcohol
gm gram
GoN Government of Nepal
GoI Government of India
hrs hours
IKI Iodine- Potassium Iodide
IUCN International Union for Conservation of Nature and Natural
Resources
KATH National Herbarium and Plant Laboratories, Lalitpur, Nepal
mg/kg miligram per kilogram
mm milimetre
mg.ml-1 miligram per mililitre
µg microgram
µg/ml microgram per ml
µm micrometer
nm nanometre
RLS Radial Longitudinal Section
SF Stomatal Frequency
SI Stomatal Index
Subsp. Sub-species
TTC Tetrazolium Tetrachloride
TS Transverse Section
TLS Tangential Longitudinal Section
TUCH Tribhuvan University Central Herbarium
VS Vertical Section
vii
viii
CONTENTS
FOREWORD.......................................................................................................... iii
PREFACE............................................................................................................... iv
ACRONYMS AND ABBREVIATIONS................................................................ vi
CHAPTER-1 INTRODUCTION...........................................................................1
CHAPTER-2 TAXONOMY..................................................................................6
CHAPTER-3 REPRODUCTIVE BIOLOGY......................................................12
CHAPTER-4 ANATOMY...................................................................................18
CHAPTER-5 DISTRIBUTION...........................................................................25
CHAPTER-6 ECOLOGY AND POPULATION STATUS: A CASE STUDY

IN GWALABARI COMMUNITY FOREST, KANCHANPUR,
NEPAL..........................................................................................30
CHAPTER-7 PRETREATMENT METHODS FOR SEED GERMINATION...36
CHAPTER-8 ECONOMIC BOTANY AND ETHNOBOTANY........................45
CHAPTER-9 PHYTOCHEMISTRY, ANTIOXIDANT, ANTIDIABETIC

ACTIVITIES AND TOXICITY....................................................51
CHAPTER-10 ANTIMICROBIAL ACTIVITY...................................................60
CHAPTER-11 THREATS, CONSERVATION AND TRADE..............................69
RefereNCES .......................................................................................................74
appendICES . .....................................................................................................83
ix
x
CHAPTER - 1
INTRODUCTION
Pratikshya Chalise and Subhash Khatri
Fabaceae or Leguminosae with about 751 genera and about 19,000 known species,
is the third largest family of flowering plants after Orchidaceae and Asteraceae and
is one of the economically important family of flowering plants (Xu and Deng,
2017; Doyle, 2001). It is a very diverse family that includes a wide variety of growth
forms, including trees, shrubs, herbaceous plants and some vines or lianas; ranging
from tiny alpine ephemerals to large trees in the tropical rainforest canopies (Doyle,
2001). Economically, Leguminosae is considered as the second most important family
after Poaceae. Majority of the members of this family are important due to their
food value, fiber extraction, production of dyes and have also been used as natural
fertilizers since ages. This is because the members of this family are characterized
by the presence of root nodules that contain nitrogen-fixing bacteria (Xu and Deng,
2017) except in the genus Styphnolobium.
Genus Pterocarpus
Pterocarpus is a deciduous tree belonging to the family Fabaceae. The members of this
genus are characterized by the presence of compound leaves with alternate leaflets;
yellow flowers arranged in racemes; flat, orbicular, indehiscent pods surrounded
by circular wing, with one to two dolabriform seeds (Troup, 1921). The barks are
greyish brown in colour which peels off in irregular scales and bright red gum resin is
exuded when slashed (Troup, 1921; Gamble, 1922; Badakhane et al., 2010; DoF,
2018).
They are renowned for the “Padauk wood” which is obtained from several species
of the genus Pterocarpus (Pullaiah, 2019(b)). But, Troup (1921) considers only
three species namely; P. macrocarpus (Myanmar Padauk or, the True Padauk), P.
dalbergioides (Andaman Padauk) and P. indicus (Malay Padauk or, Indian Padauk)
as the actual “Padauk”. All padauks are of African or Asian origin and are valued
for their toughness, stability, durability and decorativeness, most having a reddish
wood (Azamthulla et al., 2015).
Most Pterocarpus woods contain either water or alcohol-soluble substances and can
be used as dyes; which is used to colour food, alcoholic beverages, wood polish,
metal varnishes, textiles, wool, silk, leather, jute, anti-sun tanning agents, hair dyes,
medicine coating and as dye in sensitized solar cells (Pulliah and Reddy, 2019).
1
List of species
The genus is pan-tropical in distribution, but the number of species has been
mentioned differently by various researchers. Allen and Allen (1981) reported 60-
70 species within the genus Pterocarpus. Later, Hooker (1879) as well as Brandis
(1907) reported the occurrence of 15 species. However, Gamble (1922), Troup (1921),
Pearson and Brown (1932) mentioned the occurrence of 35 species in the tropics
and subtropics world-wide. Similarly, The Plant List has included 235 scientific
plant names of species rank and 33 infraspecific taxa for the genus Pterocarpus.
Out of which, 72 are the accepted names, 155 are synonyms, two are misapplied
names and 39 names are unresolved names (http://www.theplantlist.org/tpl1.1/
search?q=pterocarpus, retrieved on 02-03-2021). However, The Plants of the World
online has mentioned the occurrence of 41 species within the genus Pterocarpus
(http://www.plantsoftheworldonline.org/taxon/urn:lsid:ipni.org:names:331884-2,
retrieved on 02-03-2021). However, Roskov et al. (2020) have reported 35 species
and seven subspecies existing worldwide except in Australia; with greatest diversity in
Africa (https://www.catalogueoflife.org/data/taxon/74N7, retrieved on 02-03-2021).
1. Pterocarpus acapulcensis Rose 19. Pterocarpus mutondo De Wild.
2. Pterocarpus albopubescens Hauman 20. Pterocarpus officinalis Jacq.
3. Pterocarpus amazonum (Benth.) Amshoff 21. Pterocarpus orbiculatus DC.
4. Pterocarpus angolensis DC. 22. Pterocarpus osun Craib
5. Pterocarpus antunesii (Taub.) Harms 23. Pterocarpus rohrii Vahl
6. Pterocarpus brenanii Barbosa and Torre 24. Pterocarpus rotundifolius (Sond.) Druce
7. Pterocarpus claessensii De Wild. 25. Pterocarpus santalinoides DC.
8. Pterocarpus dalbergioides DC. 26. Pterocarpus santalinus L.f.
9. Pterocarpus echinatus Pers. 27. Pterocarpus soyauxii Taub.
10. Pterocarpus erinaceus Poir. 28. Pterocarpus ternatus Rizzini
11. Pterocarpus gilletii De Wild. 29. Pterocarpus tessmannii Harms
12. Pterocarpus hockii De Wild. 30. Pterocarpus tinctorius Welw.
13. Pterocarpus homblei De Wild. 31. Pterocarpus velutinus De Wild.
14. Pterocarpus indicus Willd. 32. Pterocarpus villosus (Benth.) Benth.
15. Pterocarpus lucens Guill. and Perr. 33. Pterocarpus violaceus Vogel
16. Pterocarpus macrocarpus Kurz 34. Pterocarpus zehntneri Harms
17. Pterocarpus marsupium Roxb. 35. Pterocarpus zenkeri Harm.
18. Pterocarpus mildbraedii Harms
Most of these species are found in Africa, notably in Nigeria, Cameroon, Sierra
Leone and Equatorial Guinea (Pulliah, 2019(b)). However, only one species is found
in Nepal (https://www.efloras.org, retrieved on 02-03-2021).
2
Pterocarpus marsupium Roxb.
Pterocarpus marsupium Roxb., popularly known as “Bijaysal” is a deciduous tree
that grows up to 33 meters high (Barstow, 2017). It was first reported from Circar
Mountains in “Plants of the Coast of Coromandel, Volume 2” by William Roxburgh
from East India Company during 1798 which was officially published in 1799. It
was locally called as “Yangshaw of the Telingas” (Roxburgh, 1799).
Pterocarpus marsupium is a repository of numerous bioactive compounds and
possesses medicinal properties. The wooden tumbler (Figure 1d) made up of its
heartwood is used for drinking water as traditional remedy because of its medicinal
property (Badakhane et al., 2010; Joshi et al., 2012). Since long time, it has been used
in the treatment of krmiroga (worm infection), kustha (leprosy), prameha (diabetes),
pandu (anaemia), medodosa (obesity) as well as several other cases (GoI, 2013). It
is popularly known for a red gum resin (Kino) that exudes from the cuts and injuries
(Figure 1a) in the bark (Badakhane et al., 2010), which gets denser and appears blood
red when it comes in contact with air (Figure 1c). Bark is often slashed to collect the
Kino gum, which is an important remedy against diabetes (Khanal and Bhattarai,
2020). This species has medicinal, fodder as well as timber values. It has superlative
characteristics in its wood and has high medicinal value so it is considered as one of
the most prioritized woods for millennia (Badakhane et al., 2010).
The plant is an important tropical tree with a restricted population existing within
an altitudinal range of 100 to 640 m, predominantly in Western tarai compared to
Central and Eastern Nepal. IUCN Red list of threatened species has enlisted it as
a ‘Near Threatened’ tree because of its declining population globally (Barstow,
2017). Considering its limited distribution and declining population in Nepal, GoN
has also prioritized this species as an important floral component. Thus, to increase
the population of Bijaysal through both in-situ and ex-situ conservation (Figure 1b)
and to sustain its healthy population in natural habitats, the “Bijaysal Conservation
Action Plan 2018-2022” has also been prepared (DoF, 2018).
Bijaysal should have been a matter of concern to ecologists, forest scientists and
environmentalists, but comparatively very few works have been carried out regarding
this species in Nepal. Although GoN has prioritized this species for its conservation,
only few initiatives have been taken by Community Forest User Groups and other
stakeholders, therefore the conservation approaches need to be intensified. Restricted
distribution patterns, low germination and slow growth, long juvenile period and
tough access to mature flowering trees might have been the reasons for its limited
study (DoF, 2018; Pulliah, 2019(b)). To conserve the existing populations of Bijaysal,
we need to control illegal cutting and fetching, unsustainable harvesting, habitat
destruction, and trade inside as well as outside the country. Together, appropriate
3
measures should have to be adopted to promote natural regeneration, propagation
as well as plantation of Bijaysal in the lowlands of Nepal.
Figure 1: Pterocarpus marsupium a. Trunk showing deep cut for collection of Kino gum;
b. Conservation initiatives taken at Gwalabari CFUG for raising public awareness; c. Kino gum
exudating from the bark; and d. Wooden tumblers made from the heartwood of Bijaysal. (Photos: a.
and c. Yagya Raj Paneru; b. and d. Pratikshya Chalise).
4
Figure 2: Digitized herbarium specimens of Pterocarpus marsupium Roxb., housed at KATH. a.
Flowering state; and b. Fruiting state.
5
CHAPTER - 2
TAXONOMY
Pratikshya Chalise
Scientific and Vernacular names

Scientific name: Pterocarpus marsupium Roxb., Pl. Coromandel 2(1): 9, t. 116
(1799).
Nepali: Bijayashal, Bijaysal, Vijaysar, Bijayasar, Banduk pushp.
English: Gum Kino, Indian Kino Tree, Malabar Kino Tree, East Indian
Kino
Ayurveda: Biyo, Asana, Vijaysar, Pitasara, Asanam, Bijasal, Bijayasar
Sanskrit: Bijasra, Pitasara, Bijaka, Asanaka
Etymology
The genus Pterocarpus is derived from two Greek words “pteron” meaning “winged”
and “karpos” meaning “fruit” referring to the winged pod present in the members
of this genus (Hutchinson, 1964; Pulliah, 2019(b); Silva et al., 2019). Similarly,
“marsupium” refers to the “pocket in female plants for storing reproductive structures
i.e, seeds”. Thus, Pterocarpus marsupium is a tree with winged fruit that enclose the
seeds. Some literatures also refer the specific epithet to describe the distribution of
this tree in specific pocket areas (Duthie, 1915).
Taxonomy and Systematic position

Kingdom - Plantae
Phylum - Tracheophyta
Class - Magnoliopsida
Order - Fabales
Family - Fabaceae
Genus - Pterocarpus Jacq.
Species - P. marsupium Roxb.
The generic name “Pterocarpus” was given by Linnaeus in 1754. It was later
described by several researchers like Hooker (1879), Brandis (1907), Troup (1921)
and Brummitt (1992). However, the taxon “Pterocarpus marsupium” was first
described by William Roxburgh in 1799 from the Circar mountains, Coromandel
in “Plants of the Coast of Coromandel: selected from drawings and description
presented to the court of the East India Company” volume II (Roxburgh, 1799).
Polhill (1981) categorized two groups within Leguminosae, based on the nature of
fruits; winged fruits versus unwinged fruits, and placed the genus Pterocarpus in the
6
tribe Dalbergieae. Similarly, Wu and Raven (2010) in Flora of China volume 10 and
Pulliah (2019(b)) also placed the genus Pterocarpus within the tribe Dalbergieae of
the family Fabaceae.
Lavin et al. (2001) placed Pterocarpus under subfamily Faboideae and assigned to the
clade Pterocarpus, sub-tribe Pterocarpeae, within the tribe Dalbergieae. They placed
legumes with samaroids pods under Pterocarpeae, pods with very small marginal
wings under Lonchocarpeae and legumes with drupaceous fruits under Geoffroyeae.
Previously, Polhill (1971, 1981, and 1994) also used the evidences from morphology,
seed chemistry and wood anatomy and grouped the 19 woody genera from Bentham’s
Pterocarpeae and Geoffroyeae into new Dalbergieae.
The informal monophyletic clade Pterocarpus contains 22 genera and ca. 200 species
including genus Pterocarpus and the members of this clade are mostly centred in
the Neotropics, whereas Pterocarpus and Stylosanthes are pantropical, Inocarpus is
Asian, and Chapmannia is transatlantic in terms of distribution (Lavin et al., 2001).
Holotype of Pterocarpus marsupium Roxb.

A holotype of a species is the only specimen (or sometimes illustration) designated
by the author as the “nomenclatural type”. The type locality of Pterocarpus
marsupium Roxb. is Circar mountains of the Coromandel as per the protologue
(Roxburgh, 1799). The protologue of Pterocarpus marsupium Roxb. is based on an
illustration (Figure 1).
7
Figure 1: Holotype of Pterocarpus marsupium Roxb. (Source: Roxburgh, 1799).
8
Synonyms
Lingoum marsupium (Roxb.) Kuntze
Pterocarpos bilobus G. Don
Pterocarpos marsupium f. acuminata (Prain) St.-Lag.
Pterocarpos marsupium f. acuta Prain
Pterocarpos marsupium f. biloba (Roxb. Ex G. Don) Prain
Pterocarpos marsupium subsp. marsupium Roxb.
Pterocarpos marsupium var. acuminata Prain
Pterocarpos marsupius (Roxb.) St.-Lag.
Taxonomic description
Medium to large sized deciduous tree, bark grey to dark brown coloured, often
characterized by the presence of shallow cracks. Branches spreading, glabrous to
pubescent. Leaves alternate, imparipinnate, leaflets 5-7, alternate, rarely subopposite,
sparingly clothed beneath with persistent hairs; stipules small, caducous; petioles
rounded and smooth. Inflorescence a much branched terminal panicle with yellow
flowers; pedicels rusty puberulous; pedicels 0.2 cm, with two small ovate caducous,
bracteoles at the apex. Calyx often incurved, obconical, 0.5-0.7 cm in length,
5-toothed; teeth short, connate. Corolla papilionaceous, exserted beyond the calyx
tube, enclosing the stamens and carpel inside; petals long clawed, margin crisped;
standard ovate to orbicular, waved, reflexed and veined; wings slightly twisted and
reflexed; keels waved, fused at the tip. Stamens 10, united at the base and soon
dividing into two bundles with 5 stamens in each resulting (5+5) arrangement towards
apex; staminal sheath split open dorsally; anthers versatile, bulbous and bilobed.
Ovary elliptical, pedicellate, hairy, generally bi-celled, 2–6- ovuled; style incurved,
filiform, glabrous, ascending; stigma terminal. Legume indehiscent, orbicular,
compressed, broadly hardened, winged around margin, borne on a long pedicel,
usually 1-seeded, sometimes 2-seeded; style persistent, incurved. Seeds oblong
or sub-reniform, dolabriform, hilum small. Flowering occurs during September to
November and Fruiting from December to March (Figure 2 and Figure 3).
Infraspecific taxa
Pterocarpus marsupium subsp. acuminatus (Prain) Thoth.
Pterocarpus marsupium subsp. marsupium
9
Figure 2: Pterocarpus marsupium; a. Tree; b. Trunk with termite nest during summer; c. Slashed
bark releasing Kino gum; d. Seedling in wild; e. Flowering branch; f. Fruiting branch; g. and h.
Variation in leaf shapes; i. Branch with winged fruits; and j. Seeds. (Photos: a., c., g., and h. Ganga
Datta Bhatt; b. Ghanashyam Chalise; d. Pratikshya Chalise; e. and f. Neelam Pandey; i. Amrit KC;
and j. Ram Krishna Bhandari).
l.
i.
10
Figure 3: Illustration Pterocarpus marsupium Roxb.: a. Habit; b. Calyx tube (opened); c. Standard
petal; d. Wings petals; e. Keels; f. Staminal tube opened; g. Carpel; h. Pod; and i. Seed. (KATH070868,
Kurmi, P.P. 10084 and KATH070855, Bhatt, G.D. and Kurmi, P.P. 1006. Illustration: drawn by
Pratikshya Chalise).
11
CHAPTER - 3
REPRODUCTIVE BIOLOGY
Pratikshya Chalise
Reproductive biology broadly incorporates floral biology, phenology, pollination

mechanism, fruit and seed ontogeny of plants. These aspects must be clearly
understood to carry out a quality research on breeding programme and efficiently
manage seed orchards or maximize seed production (Pulliah, 2019(a)). Together,
the inflorescence structure also determines the spatiotemporal arrangement of the
flowers during anthesis and is therefore crucial for reproductive success in plants
(Prenner, 2013).
In Pterocarpus marsupium, the flowers are insect pollinated especially by honey
bees. Studies have shown that the species with yellow flowers are mostly pollinated
by different species of bees, as bees prefer yellow flowers (Endress, 1995). However,
successful pollination not only depends on the pollinator but it is also determined
by the flower-opening time and duration, anther dehiscence, stigma receptivity and
germination of pollen tubes.
Very little studies have been carried out on the sexuality as well as pollination
mechanism of this plant which indicates that P. marsupium exhibits mixed breeding
system with both self- pollination and cross-pollination (Pal and Mondal, 2018), but
both self- and cross-pollinations depend on pollen mediation by the flower visitors
(Pulliah, 2019(a)). However, Rao et al. (2001) also indicated that there is large-scale
abortion of flower buds, flowers and fruits. Being a multipurpose tree species, Bijaysal
should have been a matter of concern to researchers, ecologists, forest scientists,
environmentalists and policy makers but the reproductive biology of the plant is
poorly understood. Restricted distribution patterns, low germination rate, prolonged
dormancy period, slow growth, long juvenile period and tough access to mature
flowering trees may be reasons for its limited study (DoF, 2018; Pulliah, 2019(a)).
Materials and Methods

Present investigation was carried out based on the observations in Bijaysal trees
growing in different parts of the country from October 2019 to February 2021. The
records of herbarium specimens housed at KATH as well as relevant secondary
sources were also referred to draw the inferences. Similarly, to study the floral
structure as well as for palynological examination; flower samples were collected in
30% alcohol from Kailali district, Western Nepal during November 2020. Similarly, to
study the morphological structure of fruits and seeds, pods of Bijaysal were collected
from Nepalgunj during January, 2021.
12
Floral structures as well as palynological studies were carried out in National
Herbarium and Plant Laboratories (KATH), Godawari. Flower samples were
rehydrated in distilled water mixed with a drop of detergent (Rajbhandary, 2015)
and observed under stereomicroscope and individual floral parts were photographed
using Nikon Coolpix 2800 camera. For the study of pollen grains, the anthers were
taken by a dissecting needle, placed in glass slides, teased anthers were stained with
1% safraine solution, covered with cover slips and observed under the microscope
and measured using calibrated ocular. Pollen shape was determined based on the
values of P/E ratio as documented by Erdtman (1952).
Pollen viability analysis was carried out by following Iodine-Potassium Iodide
(IKI) and Tetrazolium test (TTC test). The total number of stained pollen grains per
microscopic field was counted for twenty observations and pollen viability (%) was
calculated using the following formula (Hauser and Morrison, 1964);
Pollen Viability (%) = No. of stained pollen grains X 100

Total no. of pollen grains per microscopic field
However, information on the pollination biology of Bijaysal was also gathered from
secondary sources.
Phenology
Bijaysal exhibits leaves fall by the end of February and undergoes a brief deciduous
phase between April to May and new foliage starts to appear from May to June.
Similar observations were made by Pal and Mondal (2018) from West Bengal, India.
Bijaysal is a mass blooming legume tree and flowering starts during August and lasts
up to November whereas fruiting during December to March.
Inflorescence structure and flower opening

The flowers in Bijaysal are minute ca. 1.6 cm, bright yellow coloured and are borne
in branched, terminal panicles. Pal and Mondal (2018) reported that these minute
flowers open at 05.30 h and continued upto 06:30 h during which they emit mild
fragrance as well as nectar.
Flower morphology and structure

The flowers are bright yellow, 16 mm long, typically papilionaceous, zygomorphic,
bisexual and pedicellate, and with mild fragrance. Sepals- five, green, joined at the
base to form a narrow tube, ca. 0.6 cm x 0.3 cm, slightly bent forming curvature,
persistent (Figure 1a,b). Petals-5, vexillary aestivation, yellow, standard petal
posterior, ca. 13 mm in length, long clawed, ovate with crisped margins, veins
13
Figure 1: Floral anatomy of Pterocarpus marsupium Roxb. (different floral parts observed under
stereomicroscope); a. Bud; b. Calyx tube dissected; c. Dissected flower; d. Standard petal; e. Wing
petals; f. Keels; g. Unequal stamens extending out of calyx tube; h. Stamens with versatile anther; i.
Stamens with 5+ 5 arrangement opened at the base; and j. Pedicellate carpel with capitate stigma, slender
style and ovary. All the structures photographed under stereomicroscope. (Photos: Pratikshya Chalise).
prominent, brownish; lateral two winged petals, ca. 7 mm in length, clawed, ovate
with crisped margins, veins prominent, brownish; the two keel petals, ca. 4.5 cm in
length, fused anteriorly, free at the base, margin curved, veined (Figure 1c,d,e,f).
Both androecium and gynoecium are concealed within the keels. Stamens unequal
in length, diadelphous, with ten anthers arranged in two bundles of five anthers each
14
(Figure 1g,h,i). Such 5+5 arrangement of stamens was also reported by Rao and
Raju (2002). Each stamen coherent with the adjacent stamen on both sides, except
for the extreme ones. Filaments are fused upto more than half of the length from
the base, forming a more or less tubular structure; anthers are dithecous, bulbous,
light yellowish to creamy coloured, versatile. Carpel ca. 0.9 cm, pedicelled, oblong,
slightly bent; ovary hairy, unilocular, superior ovary with two ovules; style ascending,
glabrous, stigma capitate (Figure 1j). Sinjushin (2019) also reported pentamerous
and pentacyclic flower with a monomerous gynoecium (K5 C5 A5+5 G1).
During bud stage, the standard petal encloses the wing and keel petals which in
turn enclose both the sex organs (Figure 1a). As the bud matures, the standard petal
gradually bulges out and protrudes outwards from the calyx tube. Both the style
and the staminal tube follow the keel’s curvature upwards, and thus appear slightly
bent; both anthers and stigma are positioned around the keel apex. Similar structure
was reported by Pinheiro et al. (2018) and Pulliah (2019(a)) in various species of
Pterocarpus.
Nectar is accumulated around the base of the gynoecium and is confined within the
chamber formed by the fusion of the filaments. Thus, it can be reached only by small
openings on the base of the upper side of the staminal tube, whose access is blocked
by the claw of the standard petal (Pinheiro et al., 2018). These specialized structures
make the flowers of Bijaysal mechanically isolated such that they require specific
pollinators for pollinating these complex flowers. Pal and Mondal (2018) reported
that flower visitors in Bijaysal include the members of Hymenoptera, Lepidoptera and
Thysanoptera where bees were found to be the most dominant and the most effective
one. These flower visitors are rewarded with both nectar and pollens. According to
Pal and Mondal (2018), during the time of flower opening nectar is absent but the
secretion of nectar increases gradually with time.
Anthesis and Palynological observation

In Bijaysal, the standard petal encloses the wings and keels which in turn enclose
both the reproductive structures, thus anther dehiscence can take place only after
the unfolding of these petals. Anthers are tetrasporangiate and dehiscence of anthers
takes place by the formation of longitudinal slits (Figure 2a). According to Pulliah
(2019(a)), dehiscence of anther takes place asynchronously; two anthers an hour
after anthesis, three anthers again after one hour and the remaining five again after
1.5 hours.
Pollen grains are medium sized, 19.64 - 22.58 µm, spheroidal, psilate, tricolporate with
rounded poles. Grain arrangement was found to be monad (Figure 2c,d,e). Panicker
(2004) considered monads are as the simplest arrangement in the evolutionary line
and polyads as the most advanced type of grain arrangement. Interestingly in-vivo
15
germination of pollen grains inside the anther was also observed, and the pollen
tubes were of variable length in different pollen grains. The TTC test for pollen
viability, viable pollens stained red while the non-viable did not take stain. The
stained pollen grains were counted to calculate the pollen viability which was found
to be 80% (Figure 2b). However, in IKI test, pollen did not take the stain. The result
is in accordance with Pal and Mondal (2018), which reported 82% pollen viability
using TTC test. Studies have shown that each anther produces approx. 3,518±12.79
pollen grains and a single matured flower produces approx. 35,180±127.95 pollen
grains (Pal and Mondal, 2018).
Figure 2: Palynological observation in Pterocarpus marsupium under Olympus E 329153

microscope; a. Longitudinal dehiscence of anther showing pollen grains; b. Pollen viability test
(v- viable pollen; nv- non viable pollen); c. Single, tricolporate pollen grain (equatorial view);
d. Tricolporate pollen (polar view with three colpi); and e. Tricolporate pollen showing rounded
poles (polar view with three poles). Magnification: a. 100X, b., c., d., e. 400X. (Photos: Pratikshya
Chalise).
16
Pollination biology
In Pterocarpus, the stigma becomes receptive by about the time the first two anthers
dehisce and remains so until late evening of the same day (Rao and Raju, 2002). Since,
the flowers are zygomorphic and have semi-closed petals, they are mechanically
strong with good landing platform (Pulliah, 2019(a)).
Bees land on the corolla wing-keel complex and direct their heads towards basal
portion of the staminal tube where they extend the proboscis through small openings
to reach the nectar (Pulliah, 2019(a)). This causes the flower keel to move down,
exposing anthers and stigma; which then touch the ventral side of the bee’s abdomen
such that pollen grains get attached on their underside and are transferred to other con-
specific stigmas during their subsequent visits. After the bees depart, the petals would
return to their original position. But sometimes the flowers may remain unvisited for
several days or even weeks because of the absence of pollinator activity, especially
during dry and hot conditions.
Morphology and Ontogeny of Fruits and Seeds
Fruits orbicular, ca. 6.2 cm long and 4.5 cm wide; winged, wing more or less rounded;
pedicellate; with persistent calyx and style even at the maturity of fruit (Figure 3b).
Style more distinct in young stage (Figure 3a). Seeds usually one, sometimes two,
mean size 5x3 mm, brownish, slightly kidney shaped, dolabriform and glabrous
(Figure 3c).
Figure 3: Morphology of fruit and Seed in Pterocarpus marsupium; a. Young fruit; b. Matured fruit
and; c. Seeds. (Photos: a. Pratikshya Chalise; b. Amrit KC; and c. Ram Krishna Bhandari).
Pterocarpus is able to fruit through both self- and cross-pollination so the fruit
production is high (Pal and Mondal, 2018). But, the offspring especially those
resulting from self-pollination may either become inviable or, become sterile and
these poor offspring are gradually and selectively eliminated from the population.
Thus, despite of very high flower production, the natural fruit set is very low
(Pulliah, 2019(a)).
17
c.
CHAPTER - 4
ANATOMY
Pratikshya Chalise and Lajmina Joshi
Several studies have been done on phytochemistry, pharmacognosy of Pterocarpus

marsupium but only few scientists (Rao and Juneja, 1971; Gamble, 1972; Pearson
and Brown, 1932) have done the anatomical studies of the wood. Metcalfe and
Chalk (1957, 1989) has also mentioned the anatomy of wood, stem, bark, leaf and
petiole in the members of several angiosperm families in his book “Anatomy of
Dicotyledons”. Chauhan and Rao (2003) has also mentioned about the anatomical
features of the legumes including genus Pterocarpus in their book “Wood Anatomy
of Legumes of India: Their Identification and Uses” but detailed study on Bijaysal
has not been done until now.
In present scenario, vigorous increase in global trade has resulted in over-exploitation
of forest resources imposing serious threats over the biodiversity. Therefore,
developing accurate species level identification through their anatomical studies is a
crucial and significant technical prerequisite. In Nepal, anatomical studies have not
been done so far regarding this species. The present study has thus been undertaken
to highlight the anatomy of different parts of P. marsupium such as wood, leaf, bark,
petiole, rachis and petiolule in order to give detail information about their anatomical
characteristics and to help in identification of the Kino tree.

For anatomical studies the samples of wood and leaves were collected from Rupandehi
in November, 2019 and Kailali and Kanchanpur districts in April, 2020. For the
wood sample, a small cubic block (2-3 cm in side length) of wood was taken from
the outermost part of the tree (GoN, 2012). The blocks, leaves, bark and petiole were
fixed and preserved in FAA (Formalin- Acetic acid- Alcohol). Before sectioning,
wood blocks were boiled in order to soften so that section can be taken easily. After
softening, thin sections (20-40 µm) of transverse, longitudinal and radial faces
were cut using a sliding microtome. The sections were stained in Safranin and Fast
green; dehydrated in alcohol series. Permanent slides were prepared by mounting
in DPX mountant. Maceration of wood chips was done using Jeffrey’s solution
and the temporary slides were prepared to observe the detailed structure of vessel,
phloem, fibres, etc. Similarly, transverse sections of petiole, rachis, petiolule and
leaves were also taken and permanent slides were prepared. Stomatal study was done
by peeling the epidermal layer, which was done by boiling small pieces of leaves
in Jefferey’s solution at 60ºC in hot air oven for softening. Epidermal peels were
washed thoroughly, stained in aqueous Safranin. Temporary slides were prepared
18
mounting in diluted glycerine and then sealed by nail polish. The permanent as well
as temporary slides were then studied under compound microscopes at different
magnification and photographs were taken.
Stomatal Index (SI) and Stomatal Frequency (SF) were calculated using the formulas
(Rajbhandary, 2015).
Stomatal Index (SI)= S X 100/ (E+S)
where, S= average no. of stomata in microscopic field
E= average no. of epidermal cells in microscopic field
Stomatal Frequency (SF)= S/A per mm2
where, S= average no. of stomata in microscopic field
A= area of microscopic field
Anatomy of Stem
The transverse section of stem is ribbed due to the presence of fissured bark. The
epidermis consists of a single row of rectangular cells that is covered with cuticle. In
young stem, some of the epidermal cells give rise to multicellular hairs. Epidermis
is followed by collenchymatous hypodermis which consists of three to four layers of
cells. Below hypodermis, multilayered cortex is present that consists of thin walled,
parenchymatous cells. Endodermis is single layered and is made up of elongated,
barrel shaped cells. The endodermal region is followed by pericycle that consists of
sclerenchymatous cells. The stele consists of conjoint, collateral and open vascular
bundles arranged in ring. The bundles are relatively different in size and number. There
are six large bundles located opposite ridges. A large pith is present in the centre of
stem which consists of polygonal parenchymatous cells that tend to decrease in size
towards the periphery. However, in matured stem, due to the presence of periderm,
the epidermal cells as well as hypodermal region seem to be rudimentary.
Wood anatomy
Wood very hard, close grained, somewhat heavy and durable. Heartwood yellowish
brown to brown, that gradually turns brown with age. Heartwood fluorescent,
staining yellow when damp and gives brownish colour in aqueous solution. The
colour of this aqueous solution becomes faint after frequent use. Sap wood pale
yellowish to yellowish-white, narrow, distinct from the heartwood. Growth rings
not very distinct. Pores usually minute, visible only under the lens but sometimes
large and clearly visible to the eyes.
19
Wood is diffuse porous type. Vessels few, 7-10 vessels per mm2, mostly solitary,
sometimes in short radial multiples of 2-4 (Figure 1a,b). Solitary vessels oval in
outline; 90-251 (165) µm and 74-177 (165) µm in radial and tangential diameter
respectively (Figure 1a,b). Vessel elements 62-79 (70) µm long, end wall oblique.
Perforation plates simple. Inter- vascular pits alternate, bordered, 4.8-5.2 (5) µm
in diameter (Figure 1i), but two distinct diameter classes of vessels are absent.
According to the Inside Wood Database (http://insidewood.lib.ncsu.edu, retrieved
on 02-03-2021), the vessels in P. marsupium range between 100-200 µm in size and
the number ranges between 5-20 vessels per mm2. However, Chalk (1989) considers
the size as well as number of vessels susceptible to environmental influence. Anoop
et al. (2019) also reported that vessel diameter as well as vessel element length both
decrease whereas the number of vessels increases with increase in aridity. Heartwood
vessels sometimes filled with gums and other deposits (Figure 1b), but tyloses are
absent. Chauhan and Rao (2003) reported the occurrence of brown or orange- brown
gummy deposits in vessels.
Fibre tracheids constitute the ground mass of the wood, square to polygonal in outline;
7-19 (12.8) µm in diameter, thick walled, 5-8 (6) µm; 520-780 (750) µm long, non-
septate (Figure 1i). Pits simple, small, round with slit like apertures. Fibres are often
attached with the ray parenchyma. Fibres greatly influence the strength as well as
shrinkage of the woods (Anoop et al., 2019).
Wood parenchyma paratracheal, vasicentric; aliform confluent; arranged in narrow
tangential bands, square, round or polygonal in outline; 12-36 (24) µm and 9-35 (18)
µm in radial and tangential diameter respectively; thin walled (Figure 1b); 1-2 celled,
septa nodular (Figure 1e); few storied (Figure 1c). Vessel parenchyma pit similar to
vessel pit, oval, bordered, alternate, 5-10 (8) µm in horizontal diameter (Figure 1h).
Crystals present (Figure 1e). Metcalfe and Chalk (1989) also reported the presence
of solitary crystal in majority of the members of family Fabaceae. Chalk (1989)
reported the aliform confluent parenchyma as the most advanced type of parenchyma.
Rays homogenous, 1-3 cells wide, made up of parenchymatous tissue; mostly
uniseriate, few multiseriate; very fine and distinct (Figure 1c,d,e), composed of
procumbent cells only. Uniseriate rays 4-5 celled, 85- 137 (104) µm in height (Figure
1d); multiseriate rays 2-3 celled, 14-31 (23) µm in width and 109-177 (134) µm in
height (Figure 1e). Gamble (1972) described these rays as fine, white, wavy, often
interrupted concentric lines visible in the cross-section of wood. Procumbent cells
oval, vertically elongated in tangential section, 60-100 (82) µm and 17-25 (20.5)
µm in radial and tangential diameter respectively (Figure 1d,e,g). Ray vessel pit
3.24- 5.91 µm (4.41µm) in diameter (Figure 1h). Crystals absent.
20
Figure 1: Wood anatomy of Pterocarpus marsupium a, b. Cross-section of wood (TS) showing
solitary as well as short radial multiple of vessels, paratracheal, aliform confluent arrangement of
parenchyma, vessels with deposits; c. d. e. h. TLS of wood showing vessels, fibres and few stories
parenchyma in (c); uniseriate rays in (d); multiseriate rays and two celled parenchyma in (e); f. g. RLS
of wood showing homogenous rays and crystals in parenchyma in (f); and procumbent cells with
pitted vertical and horizontal wall in (g); and i. Fibres in macerated wood samples. Magnification-
a. (4X+ 0.5X), b., c., f. (10X+ 0.5X), d., e., g., h., and i. (40X+ 0.5X) (Photos: Pratikshya Chalise).
e.
21
Anatomy of Petiole and Petiolule
The transverse section of petiole shows the following features. The epidermal region
is uniseriate, glabrous, with rectangular shaped cells and is covered with thick cuticle.
Collenchymatous hypodermis is located under the epidermis. The cortex consists
of orbicular parenchymatous cells. Cortical region consists of few darkly stained
secretory cells (Figure 2a). Endodermis is single layered, slightly wavy in outline
and is made up of slightly elongated parenchymatous cells. It is followed by patches
of sclerenchymatous pericycle (Figure 2b). Heneidak et al. (2008) also suggested
the presence of pericycle fibres in family Fabaceae including genus Pterocarpus.
Between sclerenchymatous patches some cells are enlarged. Vascular strands conjoint,
collateral and open type; with radially elongated xylem elements towards the inner
side and phloem towards the outer side. Cambium is present between xylem and
phloem. The centre of the petiole is occupied by parenchymatous pith. Tanniniferous
canals present in the pith just below the xylem elements (Figure 2b). Darkly stained
deposits present in cortex and phloem.
The transverse section of rachis shows the following features. Epidermis is single
layered, glabrous, with thick deposition of cuticle. Hypodermis is absent. Cortex is
highly reduced compared to petiole (Figure 2c). Cortex 2-3 layered, parenchymatous.
Endodermis is single layered; wavy in outline; made up of slightly elongated
parenchymatous cells. Pericycle is in sclerenchymatous patches. Vascular strands
conjoint, collateral and open type with radially elongated xylem elements towards
the inner side and phloem towards the outer side. Cambium is present between
xylem and phloem. The center of the petiole is occupied by parenchymatous pith.
Tanniniferous canals present in pith just below xylem elements (Figure 2c). Darkly
stained deposits present in cortex and phloem.
Anatomically petiolule is similar to the petiole. Unlike petiole, the transverse section
of petiolule shows wavy outline and is covered with dense trichomes.
Anatomy of Leaves
Leaves in P. marsupium are dorsiventrally flattened, hypostomatic due to the presence
of stomata on the lower epidermis, with prominent midrib (Figure 2d,e). Upper
epidermis (adaxial surface) is dark green in colour compared to lower epidermis
(abaxial surface). The upper epidermis is single layered, with square shaped cells,
thick walled, covered with thick cuticle (Figure 2e); anticlinal walls straight (Figure
2f). Lower epidermal cells oval, angular in outline; thick walled; undulate or slightly
sinuous anticlinal walls (Figure 2e,g,h). Stomata and trichomes present in lower
epidermis only.
22
Figure 2: Anatomy of petiole, rachis and leaf; a. b. TS of Petiole showing cortical region with
tanniniferous cells in (a); Stellar region with phloem and xylem, Tanniniferous canal in pith in (b); c.
TS of rachis showing sclerenchymatous pericycle, vascular strand, tanniferous canal in pith; d. VS of
leaf through mid-rib, showing single large C-shaped vascular bundle and a small vascular bundle, both
surrounded by sclerenchymatous bundle sheath; e. VS of leaf through leaf lamina, showing palisade
and spongy parenchyma; f. Upper epidermis with smooth anticlinal walls; g. Lower epidermis with
stomata and trichomes; h. Paracytic stomata with two subsidiary cells parallel to the guard cells,
epidermal cells with undulate anticlinal walls; and i. Unicellular, glandular trichome in lower epidermis.
Magnification: a, b (100X), c, d, e, f, g (10x+0.5X), h, i (40X+0.5X) (Photos: Pratikshya Chalise).
23
Stomata paracytic (Rubiaceous type). Each stoma is surrounded by two subsidiary
cells that lie parallel to the guard cells (Figure 2h). Guard cells typically kidney-
shaped and ostiole are located on the same level relative to the epidermal cells.
Stomatal length ranges from 20-27 (22.4) µm. Stomatal frequency 223.2 per mm2
and Stomatal index 40.21. Idu et al. (2006) also reported hypostomatic stomata
as the characteristic feature of family Fabaceae. They reported the occurrence of
anomocytic stomata with agenous ontogenic pathway as primitive character and the
occurrence of paracytic stomata with eumesogenous ontogenic pathway as advanced
characteristics within Fabaceae.
Trichomes unicellular, glandular; 96-188 (129) µm long, 18 per mm2; basal glands
28-38 (32) µm in diameter (Figure 2i). Trichomes are more clustered and shorter
in the region of veins, whereas longer and distantly present in the other part of leaf
blades compared to veins.
The mesophyll lies just beneath the epidermis; dorsiventral, heterogeneous;
differentiated into palisade and spongy parenchyma (Figure 2e). Palisade parenchyma
2-3 layered; made up of elongated cells; vertically arranged; parallel to each other.
Palisade zone does not exceed the upper epidermis of midrib. Spongy parenchyma
lies below palisade parenchyma; made of up round to oval cells; loosely arranged,
which extend upto the lower epidermis.
Midrib is externally bound by single layered epidermis. Mesophyll tissue is absent in
the midrib. Epidermis is followed by collenchymatous hypodermis. Cortex consists
of orbicular parenchymatous cells. Vascular system comprises of one large ‘C’-
shaped vascular bundle and one smaller vascular bundle lying opposite to the large
bundle (Figure 2d). Vascular bundles are conjoint, collateral and closed type with
varied shape and size from center to margin. Each vascular bundle consists of closely
arranged, radial files of narrow angular xylem elements towards the inner side and
phloem towards the outer side (Figure 2d). The cortical vascular bundles are smaller
in size; conjoint, collateral and closed type. At the center lies the parenchymatous
pith. Darkly stained deposits found in phloem and pith.
24
CHAPTER - 5
DISTRIBUTION
Yagya Raj Paneru, Pratikshya Chalise and Mohan Dev Joshi
Pterocarpus marsupium Roxb. (Bijaysal), is an important tree species occurring

in the lowlands of Nepal. Its distribution is restricted in patches and the natural
stands of the tree are disappearing rapidly (Anis et al., 2005). Bijaysal is confined
within an altitudinal range of 100 to 1200 m, and has a limited population in both
global as well as national level (Barstow, 2017; DoF, 2018; Sukhadiya et al., 2019).
Global distribution of Bijaysal is also limited as it extends across the Indian Peninsula
and northwards up to the foot of the central Himalayas (Troup, 1921). In Nepal, this
species is naturally distributed at the foothills of Siwalik in Kanchanpur, Kailali,
Bardia, Banke, Kapilvastu, Rupandehi and Nawalparasi (now Nawalpur and Parasi)
districts (Jha, 1999; DoF, 2018) and a few trees have also been traced in Palpa and
Arghakhachi districts as well (DoF, 2018). Records of herbarium specimens housed
at KATH also shows that natural populations of Bijaysal occur in the lowlands of
Western and Central Nepal. Similarly, in Eastern Nepal the species exists in planted
condition (Rajbhandari et al., 2020).
The IUCN Red List of Threatened species has listed Bijaysal in ‘Near Threatened’
category (Barstow, 2017). Similarly, the GoN has also prioritized this species for
its conservation and for sustaining its population in long run, the first species level
conservation action plan for plant species was developed in 2018 as "Bijaysal
Conservation Action Plan 2018-2022" (DoF, 2018). However, these attempts might
have often failed to be supported or have simply been super-shaded due to lack of
detailed information on their biology, distributions, level of trade, population status
and associated range reductions. Therefore, this chapter describes distribution and
the factors governing patchy distribution of Bijaysal in Nepal.

Present study was carried out to predict the suitable habitat of Bijaysal in Nepal
using Ecological niche modeling. A total of 30 presence points were obtained from
the herbarium records as well as from field observation. Similarly, 19 bioclimatic
variables were considered, out of which few significant variables were selected
considering correlation among the variables and biological significance for the
species. Modelling was done within the distribution range of the P. marsupium across
the tarai region of Nepal (Figure 1).
25
Figure 1: Map of Nepal showing the occurrence points of Pterocarpus marsupium in Nepal.
Potential distribution in Nepal

The field-based observation, herbarium data as well as personal communication
showed the distribution of Bijaysal in Central and Western Nepal from Parasi to
Kanchanpur districts. The study indicates the foothills of Siwalik and Tarai of Central
and Western Nepal as the potential area for distribution of P. marsupium (Figure 2).
High habitat suitability was found in Western Nepal as compared to Central Nepal.
However, in Eastern Nepal, habitat was found to be unsuitable for Bijaysal. The
four districts viz; Banke, Bardiya, Kailali and Kanchanpur of Western Nepal occupy
most of the probable potential area and also these districts consist of highly suitable
areas. Dang district shows very low potential area. Similarly, three districts, namely
Parsa, Bara and Rautahat also showed some potential area, but there is no record
of the presence of this plant in natural habitat in these areas. Presence points from
Banke and Bardiya were not found as most of the potential area lies within the two
protected areas; Banke and Bardiya National Parks.
26
Figure 2: Current potential habitat of Pterocarpus marsupium in Nepal based on MaxEnt model.
Factors governing Current distribution

Environmental factors play an important role in predicting the distribution of P.
marsupium. The jackknife test revealed that the variable with highest gain when
used in isolation was Soil type (Figure 3). Soil type also decreased the gain when it
was omitted from the analysis, thus it has importance in determining the potential
Figure 3: Average regularized training gain for each variable for 50 replicates (Jacknife test result).
27
habitat. Calcaric phaeozems soil with slope of 13°-18° were favorable for Bijaysal.
Precipitation of wettest quarter (Bio 16) and warmest quarter peaks around 2326.43
mm and 131.37 mm respectively. Most probable area for suitability were within
38.86° maximum temperature of warmest month and 28.14° mean temperature of
warmest quarter. This result from Jacknife test also justifies high suitability of the
studied species in Western Nepal since the temperature in Western Nepal falls within
this range.
Figure 4: Variation in precipitation trends in different ecological regions of Nepal.
28
This distribution trend might have been governed by the variation in precipitation
trend, length of growing seasons and temperature during summer in different
ecological regions of Nepal. There is an increasing precipitation trend in Eastern
Nepal (Figure 4) whereas Central and Western Nepal have decreasing precipitation
trends (Pokharel et al., 2019). Since Bijaysal is a deciduous tree species that requires
normal precipitation (Troup, 1921), excessive rainfall can damage the seedlings
resulting in water logged soil. Together, this could also be due to the variation in
length of growing season in the three ecological zones. Western Nepal has shorter
length of growing season as compared to Central and Eastern Nepal (Figure 3).
The present study revealed the potential distributional range of P. marsupium in
foothills and Tarai of Western and Central Nepal. The study shows that most of
the suitable habitat lies in Siwalik and Tarai region of Western Nepal compared to
Central and Eastern Nepal. Restricted distribution of Bijaysal is found with high
suitability in Western Nepal, moderate suitability in Central Nepal and no suitability
in Eastern Nepal. Results will help in fulfilling the gaps regarding the existing
population distribution in natural habitats of this species. These findings will be
helpful to identify the potential habitats in the areas previously not explored, which
might help in the conservation of Bijaysal in the wild and also in its reintroduction
into suitable habitats.
29
CHAPTER - 6
ECOLOGY AND POPULATION STATUS: A CASE STUDY IN
GWALABARI COMMUNITY FOREST, KANCHANPUR, NEPAL
Neelam Pandey and Suresh Kumar Ghimire
Pterocarpus marsupium Roxb. is a deciduous tropical tree species in the family

Fabaceae. It is native to Bangladesh, India, Nepal, Sri Lanka, and Taiwan (officially
the Republic of China) (Barstow, 2017). It is found mainly in lowland tropical areas,
at elevations from 100 to 500 m, but exceptionally reaches up to 1200 m (Troup,
1921; Sukhadiya et al., 2019; Ghosh et al., 2021). The tree is slow growing, attaining
a height up to 33 m, with spreading branches (Figure 1; Barstow, 2017). The tree can
blossom and bear fruit (pod) when reaching to 5–6 years of age (Xu et al., 2016).
The plant produces winged pods, each containing single seed (Figure 2). The tree has
very limited natural regeneration due to low seed viability and poor seed germination
mainly because of hard seed coat and lack of adequate exposure and sunlight in the
dense tropical deciduous forest (Kalimuthu and Lakshmanan, 1995).
P. marsupium is a multipurpose tree, highly valued for medicine and as an excellent
timber (Gairola et al., 2010; Sukhadiya et al., 2019). The older trees, when wounded,
exude a blood red gum resin known as ‘Kino’, which mainly contains kinotannic
acid and is used as an astringent (Troup, 1921; Gairola et al., 2010). It has suffered
from overexploitation for timber (handicrafts), medicine (kino gum and bark), and
fodder (DoF, 2018; Ahmad and Anis, 2019; Khanal and Bhattarai, 2020) resulting
in sharp decline of natural populations globally (Anis et al., 2005; Barstow, 2017).
It has been listed in the Red Data book as ‘Near Threatened’ by IUCN based on the
threats present to the species (Barstow, 2017). However, it has been argued that P.
marsupium almost meets the criteria for ‘Vulnerable’ category (Barstow, 2017).
In Nepal, P. marsupium is restricted towards the western Tarai and Siwalik region
where it occurs in small fragmented populations (DoF, 2018). Government of Nepal,
under the Forest regulation 1995, banned P. marsupium for felling, transportation
and export (MoFSC, 2002). P. marsupium has also been considered as ‘Critically
Endangered’ for Nepal under CAMP (Conservation Assessment and Management
Prioritization) threat category (Tandon et al., 2001).
The restricted range of distribution, along with its slow growth, poor regeneration,
and over-exploitation pose serious threat to the exiting wild populations (Tiwari et
al., 2004). This species needs immediate conservation effort, which should be based
on a detailed knowledge about its current status. Therefore, the chapter deals with
the ecology and population status of P. marsupium as a case study in Gwalabari
Community Forest, Kanchanpur District, Nepal.
30
b.
Figure 1: Pterocarpus marsupium Roxb. a. Adult reproductive; b. Adult vegetative; c. Young
sapling; d. Trunk with splashed bark releasing Kino gum; e. Epiphytic orchid on P. marsupium; and
f. Termite nest on P. marsupium (Photos: Neelam Pandey).
d.
Figure 2: Winged pod of Pterocarpus marsupium (Photos: Neelam Pandey).

The Gwalabari Community Forest (28°52' to 28°53'N latitudes and 80°25' to
80°26'E longitudes) is located in the Krishnapur municipality of Kanchanpur district,
Sudurpaschim province, Nepal (Figure 3). The total area of the community forest
is 253.44 ha. The elevation ranges from 170 to 215 m above sea level. The nearest
village to the community forest comprises approximately 500 households of the
31
native Tharu tribe with most households being dependent on forest for fuelwood,
fodder, vegetables and other non-timber forest products (NTFPs).
The entire area of the community forest was surveyed in November, 2019 to
enumerate individuals of P. marsupium for evaluating its population status. All
individuals of the target species with >10 cm DBH (diameter at breast height)
were categorized as reproductive adults (Xu et al., 2016). Individuals with DBH
≥3–10cm were categorized as vegetative adults (Ankalaih et al., 2017), and those
with <3cm DBH were categorized as recruits representing saplings and seedlings
(Tiwari et al., 2010; Kala and Dubey, 2012). Local people (n= 50) were interviewed
to understand the use pattern of the target species and its associated impact. Habitat
preference of P. marsupium was assessed by recording forest types and enumerating
vascular plant species associated with the forest types and the target species. In
addition, soil samples (at the depth of 15 cm) under the canopy of each adult tree of
P. marsupium were collected, air dried and analyzed for pH using an electronic pH
meter. Assessment of disturbance was made based on the presence of human and
animal trampling, browsed plants, broken aerial parts and logged and lopped trees.
Moreover, P. marsupium trees were observed for any kind of ecological association
with other life forms.
Figure 3: Map of the study area showing Gwalabari Community Forest, Bani, Kanchanpur.
32
Population density, Regeneration status, and Level of disturbance
A total of 177 P. marsupium individuals were recorded in the entire study area,
representing adult reproductive (17 number of individuals), adult vegetative (49),
sapling (102) and seedling (9) stages, which almost exhibited a unimodal frequency
distribution (Figure 4), agreeing to the findings of previous studies (Pyakurel and
Oli, 2014; Khanal and Bhattarai, 2020). While assessing the status of P. marsupium,
Pyakurel and Oli (2014) revealed that the number of seedlings was low in most of
the surveyed community forests of Kanchanpur district, Nepal. Furthermore, high
proportions of poles (68%) and saplings (28%), but with few adults (3%) and no
seedlings of P. marsupium were recorded by Khanal and Bhattarai (2020) in Hariyali
Community Forest, Kapilvastu, Nepal. These results suggest that the recruitment
of P. marsupium in western Tarai is quite low. This may be due to reduced density
of reproductive individuals and low seed germination and seedling recruitment
potentialities (Ankalaiah et al., 2017; Sukhadiya et al., 2019).
P. marsupium produces winged pods, which are 2.5–5 cm across, each containing
single seed. Seeds are very light, the average dry weight of five matured seeds was
calculated as 0.32 ± 0.02 gm (n =17). Most of the seeds can be carried long distances
by the wind. However, we did not observe sufficient recruitment, and this might be
related mostly to the lower density of reproductive adults. Only 17 reproductive trees
were recorded in this study for the entire 253.44 ha. forest area, comparable with
similar findings from other parts of Nepal (e.g., Khanal and Bhattarai, 2020), can be
regarded as quite low as compared to the studies from some of the tropical forests
in India (e.g., Sundarapandian and Swamy, 2013; Nag and Gupta Joshi, 2020). The
reduced density of matured reproductive trees may affect the seed output, ultimately
affecting the future recruitment (Ankalaiah et al., 2017).
The adult trees were subjected to logging for timber, lopping for fodder and bark
splashing and wounding practices for kino gum extraction (Figure 1). Browsed,
broken or defoliated aerial parts of many seedlings were observed, indicating
trampling being prevalent. Most of the adult reproductive trees (n = 12) were found
to be lopped for fodder or logged for heartwood. Similarly, approximately 50% of
adult vegetative individuals were heavily lopped for fodder. Almost 60% of the
respondents (n= 50) of Tharu community residing adjacent to the community forest
were found to be involved in the kino gum extraction. The gum collected mainly
during summer season was used as a traditional anti-diabetic medicine and also as a
cooling agent. Furthermore, it was also reported to be used for joint pain and fever.
33
Figure 4: Population structure of P. marsupium in Gwalabari Community Forest, Kanchanpur,
Nepal. Data shown are proportions of seedling (SD), sapling (SAP), adult vegetative (ADV) and
adult reproductive (ADR) stages.
Habitat preferences and other Biotic interactions

P. marsupium preferred weakly acidic soil (pH 6.51 ± 0.83) and the soil type was
sandy alluvial and clayey. These results are consistent with the findings of Pyakurel
and Oli (2014). Shorea- Terminalia, Shorea- Mallotus, and Shorea- Acacia forests
are the main vegetation types found in Gwalabari Community Forest. Altogether,
106 species of vascular plants belonging to 90 genera and 37 families were found
to be associated with P. marsupium. Fabaceae with 19 species was the dominant
family associated with P. marsupium, followed by Lamiaceae (9 species), Malvaceae
(8), Poaceae (8), Asteraceae (7) and others. Shorea robusta was the most frequently
observed tree species, followed by Schleichera oleosa and Terminalia alata.
Similarly, Phyllodium pulchellum, Desmodium gangeticum, Woodfordia fruticosa,
Mazus pumilus and Knoxia sumatrensis were among the dominant shrubs and herbs
associated with P. marsupium.
In the present study, P. marsupium was found being the host for epiphytic orchids
like Vanda tessellata and Pelatantheria insectifera (Figure 1). Moreover, during
flowering period, there was the presence of termite nest (Figure 1) on each flowering
individual of P. marsupium indicating some kind of mutualism probably related to
pollination (Ahmad et al., 2018). Recent study of Pal and Mondal (2018) has shown
that bees (Apis sp.) and ants (Camponotus sp.) of order Hymenoptera, butterflies
(species belonging to the genera Borbo, Catopsilia, Delias, Euploea and Pachliopta)
of Lepidoptera and thrips belonging to the order Thysanoptera are the insects to visit
flowers of P. marsupium. The association that we observed with termite is a subject of
34
further study. However, this observation added important insights to the postulation
that P. marsupium is one of the important components of tropical forest with high
ecological significance, which provide food to bees, ants, birds and mammals
(Pyakurel and Oli, 2014; Pal and Mondal, 2018). The tree has also been regarded as
a “vulture's vantage point and the climax partner of Shorea robusta” (DoF, 2018).
Conclusions and Recommendations

P. marsupium in Gwalabari community forest comprises a very small population with
lower proportions of seedlings and reproductive individuals and highest proportion of
saplings. The loss of reproductive stage indicates intense current harvesting of adults.
Therefore, to enhance fitness and ensure long-term viability, the existing population
of P. marsupium should be strictly protected from harvesting, overgrazing and other
human interferences. Particularly, protection and monitoring of seedlings and adult
reproductive trees in the natural habitat is the immediate need for its conservation.
Such monitoring should also take into consideration all the ecological factors affecting
population fitness. Establishment of a system for sustainable Kino gum extraction
is another important aspect to be focused. In addition, complementary approaches,
such as augmentation and reintroduction programs are needed to increase the size
of the existing populations and enhance its gene pool; and create new populations in
the ecologically suitable site within its historical range where the species no longer
occurs. Our study also provides some insights about the habitat preferences and
other biotic interactions highlighting the significance of the species to the ecosystem.
35
Chapter - 7
PRETREATMENT METHODS FOR SEED GERMINATION
Ram Krishna Bhandari and Pratikshya Chalise
Pterocarpus marsupium is an epigeal germinating plant and its seeds prefer moist
environment and needs loose weed free soil as well as light shade during germination
(Troup, 1921; DoF, 2018). It grows best in deep, well drained, less fertile soils and
can tolerate dry spells (Barstow, 2017). The suitable habitat is tropical region where
plants often grow taller than the Sal (Shorea robusta) that enable wide and distant
dispersal of seeds, which is further aided by its light, winged seed (DoF, 2018). But
the propagation of Pterocarpus species through seeds is not much successful because
of poor seed viability (Vikaspedia, 2021). However, in natural conditions, when the
pods are lying uncovered on the ground surface, the radicles dry up when exposed
to the intense sunlight, and are susceptible to insect damage. Similarly, the seedlings
are invariably killed by frost during winter (Troup, 1921). Owing to the hardness
of the pods, the seeds possess mechanical dormancy so that germination of seeds is
a matter of difficulty and thus, the percentage of success is also comparatively low
(Barmukh and Nikam, 2008). Besides this, poor pod set and delayed pod maturity
is also another reason (Dayanand and Lohidas, 1988).
Studies have also shown that the regeneration rate of leguminous trees in natural
habitats (Acharya et al., 2012) as well as through tissue culture is quite low
(Lakshmi et al., 1992; Dewan et al., 1992; Das and Chatterjee, 1993; Kalimuthu and
Lakshmanan, 1995). In tissue culture, rooting is very scarse due to which in vitro
propagation is seen to be less effective. Regarding natural regeneration, there are
a number of factors such as prolonged juvenile period, long time duration to reach
seed-bearing age, hardness of pod, low germination potential, poor seed viability
due to which the seed germination percentage is lower than 30% (Venkataramaiah
et al., 1980; Kalimuthu and Lakshaman, 1995). Poor natural regeneration is not only
due to low germination and prolonged dormancy but also because of fungal growth
inside the seed coat and seasonal fruit-bearing habit of the plant itself (Kumarasinghe
et al., 2003). However, sprouting from roots can take place after the existing plants
are damaged by fire (Anuradha et al., 2019).
After a series of struggle, some seeds hardly germinate and sprout into seedlings,
but these seedlings are again limited by a number of underlying factors such as
high temperature, water stress, predation, pod size, light intensity and seasonal
variations. The physical nature of pods as well as seeds may also result into poor
natural regeneration of the seedlings (Anuradha et al., 2019). Seedlings obtained
via tissue culture technique also die out quickly and their survival rate is hardly
10% in open field due to intolerance of high temperature (Vikaspedia, 2021). The
36
cumulative effect of all these factors in turn drives this species towards an unknown
bottle-neck resulting poor natural occurrences and a declining population of Bijaysal
in Nepal as well as worldwide. This chapter deals with several pretreatment methods
for seed germination in Bijaysal so as to propose a cost effective method for its seed
germination, propagation and commercial cultivation.

The present study was carried out in Dhakeri Botanical Garden, Nepalgunj. Pods
from a healthy, matured plant were collected. Three consecutive series of experiments
were carried out using different soil/ substrate types and seed pretreatment condition.
First experiment was carried out with seven different soil types and two conditions
of seeds pretreatment. Seeds were soaked in lukewarm water for 24 hours as well
as in normal tap water (room temperature) for 24 hours before sowing. Altogether,
14 experimental set up were carried out, each using 25 seeds (Table 1). The seeds
were sown in each soil type placed in polytubes of size 4X7 on 16th April, 2021 and
Khar (Eulaliopsis binata) was used for mulching the experimental set up. Each of
the experimental set up was irrigated on daily basis and germination of the seeds in
each set up was recorded.
Table 1. First experimental set up for seed germination in P. marsupium.
S.No. Soil/Substrate type Seeds pretreatment
1. Pure soil Treated in normal tap water for 24 hrs.
2. Soil+ Compost manure (1:1) Treated in normal tap water for 24 hrs.
3. Pure sand Treated in normal tap water for 24 hrs.
4. Sand+ Compost manure (1:1) Treated in normal tap water for 24 hrs.
5. Soil+ Sand+ Compost manure (1:1:1) Treated in normal tap water for 24 hrs.
6. Soil+ Urea+ Potash Treated in normal tap water for 24 hrs.
7. Sand+ Urea+ Potash Treated in normal tap water for 24 hrs.
8. Pure soil Treated in lukewarm water for 24 hrs.
9. Soil+ Compost manure (1:1) Treated in lukewarm water for 24 hrs.
10. Pure sand Treated in lukewarm water for 24 hrs.
11. Sand+ Compost manure (1:1) Treated in lukewarm water for 24 hrs.
12. Soil+ Sand+ Compost manure (1:1:1) Treated in lukewarm water for 24 hrs.
13. Soil+ Urea+ Potash Treated in lukewarm water for 24 hrs.
14. Sand+ Urea+ Potash Treated in lukewarm water for 24 hrs.
37
Second experiment was carried out with four different soil types and two conditions
of seed pre-treatment. In first pretreatment condition seeds were soaked in normal tap
water for 18 hours and in second pretreatment condition seedcase were incised but not
treated in water. In first pretreatment condition, the seeds that floated in the surface
of water and those which shrunk underneath were treated separately. Altogether, 12
experimental set up were carried out, each using 50 seeds (Table 2). The seeds were
sown in each soil type prepared in nursery beds on 2nd May, 2021 and germination
of the seeds in each set up was recorded.
Table 2. Second experimental set up for seed germination in P. marsupium.
Treated in normal tap water for 18 hrs, sunken seeds
1. Pure soil
only considered
2. Soil+ Compost manure (1:1)
only considered
Soil+ Sand+ Compost manure Treated in normal tap water for 18 hrs, sunken seeds
3.
(1:1:1) only considered
4. Soil+ Urea+ Potash
only considered
Treated in normal tap water for 18 hrs, floated seeds
5. Pure soil
only considered
only considered
Soil+ Sand+ Compost manure Treated in normal tap water for 18 hrs, floated
7.
(1:1:1) seeds only considered
8. Soil+ Urea+ Potash
only considered
9. Pure soil Seedcase incised but not treated in water
10. Soil+ Compost manure (1:1) Seedcase incised but not treated in water
Soil+ Sand+ Compost manure
11. Seedcase incised but not treated in water
(1:1:1)
12. Soil+ Urea+ Potash Seedcase incised but not treated in water
Third experiment was carried out with five different soil types and two conditions of
seed pre-treatment. Here, instead of seeds the whole pods were soaked in normal tap
water for 24 hours and the seeds were extracted from the soaked pods and floatation
test was carried out. In first pretreatment condition, the seeds that floated in the
surface of water were considered and in second pre-treatment condition, the seeds
that shrunk underneath were considered. Altogether, eight experimental set up were
carried out, each using 50 seeds (Table 3). The seeds were sown in each soil type
placed in polytubes of size 4X7 on 21st May, 2021 and germination of the seeds in
each set up was recorded.
38
Table 3. Third experimental set up for seed germination in P. marsupium.
Pods treated in normal tap water water for 24 hrs,
1. Pure soil
sunken seeds only considered
Soil+ Sand+ Compost manure Pods treated in normal tap water water for 24 hrs,
3.
(1:1:1) sunken seeds only considered.
4. Sand+ Compost manure (1:1:1)
Pods treated in normal tap water for 24 hrs, floated
5. Pure soil
seeds only considered
Soil+ Sand+ Compost manure Pods treated in normal tap water for 24 hrs, floated
7.
(1:1:1) seeds only considered
8. Sand+ Compost manure (1:1:1)
*All the pods were soaked in normal tap water for 24 hours and the seeds were extracted from the
soaked pods.
Seed germination experiment

Seed germination took place between 5 to 25 days of sowing. Although extraction
of seeds was a very tedious task, germination was faster while using the seeds rather
than using the pods. Treatment of seeds with cold water (at room temperature) was
an efficient method for the seed germination. Here, only the sunken seeds germinated
and germination took place between 5 to 25 days. However, in case of the seeds
that floated in water during pretreatment, germination was absent. Similarly, in the
seeds pretreated with luke warm water, germination was absent. Mulching with Khar
during the first set up, increased the infestation of termites which destroyed the seeds
as well as germinating seedlings. Similarly, excessive irrigation into the polytubes
also destroyed the seedlings. Germination percentage was maximum (30%) when
combination of soil, sand and compost (1:1:1) was used as the substrate/soil type
and the seeds were treated with cold water for 24 hours and only sunken seeds were
considered for sowing. The result of the germination study on the three consecutive
experiments is given in the Table 4, 5 and 6.
39
Table 4. Result of seed germination from the first experimental set up.
S. Seeds No. of Germinated
Soil/substrate type Remarks
No. pretreatment seeds seeds
Germinated on 23rd
April, 2021. (Leaf tip
1 Pure soil Normal tap water 25 1
turned black and died
on 2nd day)
2 Soil+ Compost manure Normal tap water 25 0 -
3 Pure sand Normal tap water 25 0 -
Sand+ Compost
4 Normal tap water 25 0 -
manure
Germinated on 23rd
Soil+ Sand+ Compost April, 2021. Later
5 Normal tap water 25 1
manure destroyed by heavy
rain and hail
6 Soil+ Urea+ Potash Normal tap water 25 0 -
7 Sand+ Urea+ Potash Normal tap water 25 0 -
8 Pure soil Luke warm water 25 0
9 Soil+ Compost manure Luke warm water 25 0
10 Pure sand Luke warm water 25 0
Seeds could not
Sand+ Compost germinate when
11 Luke warm water 25 0
manure treated with Luke
Soil+ Sand+ Compost warm water.
12 Luke warm water 25 0
manure
13 Soil+ Urea+ Potash Luke warm water 25 0
14 Sand+ Urea+ Potash Luke warm water 25 0
Table 5. Result of seed germination from the second experimental set up.
Soil/
S. Seeds No. of Germinated Germination
Substrate Remarks
No. pretreatment seeds seeds (%)
type
5 seeds
germinated
Normal tap water / between 5 to 12
1 Pure soil 50 4 8
sunken seeds days after sowing.
But 1 seedling
died by 20th day.
Seeds germinated
Soil+ between 5 to 17
Normal tap water /
2 Compost 50 7 14 days after sowing.
sunken seeds
manure All the seedlings
survived.
40
Soil/
S. Seeds No. of Germinated Germination
Substrate Remarks
No. pretreatment seeds seeds (%)
type
Soil+ Sand+ Seeds germinated
Normal tap water /
3 Compost 50 2 4 between 12 to 17
sunken seeds
manure days after sowing.
3 seeds
germinated within
Soil+ Urea+ Normal tap water /
4 50 1 2 12 days. But two
Potash sunken seeds
seedlings died on
20th day.
Normal tap water /
5 Pure soil 50 0 0
floated seeds
Soil+
Normal tap water /
6 Compost 50 0 0
floated seeds
manure Floated seeds did
Soil+ Sand+ not germinate.
Normal tap water /
7 Compost 50 0 0
floated seeds
manure
Soil+ Urea+ Normal tap water /
8 50 0 0
Potash floated seeds
Seeds germinated
Seedcase incised/
9 Pure soil 50 1 2 12 days after
no water treatment
sowing.
Soil+ Seedcase incised/
10 Compost no cold water 50 0 0 -
manure treatment
Soil+ Sand+ Seeds germinated
Seedcase incised/
11 Compost 50 1 2 17 days after
no water treatment
manure sowing.
3 seeds
germinated 12
Soil+ Urea+ Seedcase incised/ days after sowing.
12 50 1 2
Potash no water treatment But 2 seedlings
died within the 1st
week.
* All the seedlings were carefully transplanted from the nursery beds to polytubes on 4th June, 2021,
i.e, after 28 days.
41
Table 6. Result of seed germination from the third experimental set up.
Soil/ Substrate Seeds/ Fruits No. of Germinated Germination
S.No. Remarks
type pretreatment seeds seeds (%)
Seeds
germinated
Normal tap water/
1 Pure soil 50 5 10 between 5 to
sunken seeds
15 days after
sowing.
Seeds
germinated
Soil+ Compost Normal tap water/
2 50 4 8 between 10 to
manure sunken seeds
20 days after
sowing.
Seeds
Soil+ Sand+ germinated
Normal tap water/
3 Compost 50 15 30 between 5 to
sunken seeds
manure 25 days after
sowing.
Seeds
Sand+ germinated
Normal tap water/
4 Compost 50 6 12 between 5 to
sunken seeds
manure 15 days after
sowing.
Normal tap water/
5 Pure soil 50 0 0
floated seeds
Soil+ Compost Normal tap water/
6 50 0 0
manure floated seeds
Floated
Soil+ Sand+ seeds did not
Normal tap water/
7 Compost 50 0 0 germinate.
floated seeds
manure
Sand+
Normal tap water/
8 Compost 50 0 0
floated seeds
manure
* All the seeds that germinated survived during the third experiment.
Gamble (1972) reported that seeds of Bijaysal do not always germinate very well.
Germination is better when the seeds are taken out from the pod before sowing,
but extracting the true seeds from the pod was a tedious and labor intensive task.
Barmukh and Nikam (2008) also reported that seeds are prone to injury or, physical
damage while separating from the pods. Here, seed extraction was easier when the
pods were soaked in water for 24 hours before sowing. This also enhanced the seed
germination percentage.
42
Seeds of Pterocarpus marsupium germinated between 5 to 25 days. Similar time of
germination was reported by Van Dalean (1991) where seed germination took place
between one to six weeks. Seedlings initially have simple, unifoliate leaves which
later developed into compound, imparipinnate leaves (Figure 1).
Factors affecting Seed germination

Seed germination depend on several factors such as time of fruit harvest (Chauhan
et al., 2018), extraction of seeds, seed pretreatment, light, moisture (water supply/
irrigation), insect infestation. During first experiment, germination was affected by the
time of pod harvesting as well as seed sowing. Here, the pods were harvested during
first week and sown during second week of April, exhibiting very little germination.
This might be because seed germination increases with seed maturity (Ahmad et
al., 2015). Together, mulching was done which reduced the availability of light and
increased the possible infestation of termites. Excess irrigation also suppressed the
germination.
Figure 1: Study of seed germination in Pterocarpus marsupium; a. Seeds after extraction from pods;
b. Seeds; c. Floatation test of seeds; d. Germinating seed; e. Seedling with two cotyledonary leaves;
f. Seedling with two cotyledonary leaves and one true leaf; g. Seedling with two cotyledonary leaves
and a pair of true leaves; h. Seedling with two cotyledonary leaves and three true leaves; i. Seedling
with two cotyledonary leaves and four true leaves; j. Seedling with degenerating cotyledonary leaves
and five true leaves; and k. Seedlings of Bijaysal in 4X7 polytubes. (Photos: Ram Krishna Bhandari).
43
Seed germination was higher when the seeds were pretreated with normal tap water
for 24 hours. Vikaspedia (2021) also reported that germination is enhanced when the
seeds are treated with water before sowing. The seeds that shrunk underneath had
higher germination percentage compared to the seeds that float in water, in which
germination was completely absent. This might be because, seeds with high specific
gravity, larger size, good quality and viability shrunk underneath whereas the seeds
with low specific gravity, small size or, immature, poor quality, poor viability or,
nonviable seeds float on water. However, the seeds that were treated in lukewarm
water for 24 hours, did not germinate. The seed coat is not very hard but the hardness
is due to their pods; which after separating make the seeds susceptible to damage
by heat as well.
The plant does not require excess water if the seeds are treated with water before
sowing. Mulching is also not necessary as mulching increased insect infestation.
However, protecting the germinating seedlings from excess heat as well as
environmental extremes such as hail, thunderstorm, etc. are required for better growth.
Germination was comparatively higher when whole pods were pretreated with normal
tap water for 24 hours and only the sunken seeds were sown in substrate containing
equal proportion of soil, sand and compost manure. Troup (1921) also reported that
propagation of the plant can also be done by soaking the pods for few days before
sowing them into the ground. In this case, the margins of the pods should be cut so
that water enters inside the pods and are ultimately used up by the seeds. This could
be a fast, cost effective pretreatment method for the germination of the seeds; which
could provide new insights for the propagation as well as commercial cultivation
of Bijaysal in context of Nepal. However, there are several other techniques for
germination and propagation of tree species such as treatment with acids in different
concentration, physical scarification, heat treatment, etc. which were not considered
in this study, so further studies can be carried out for testing their validity.
44
CHAPTER - 8
ECONOMIC BOTANY AND ETHNOBOTANY
Pratikshya Chalise and Sajita Dhakal
Pterocarpus marsupium is a versatile plant with a broad spectrum of uses (Troup,

1921) that comprise of its medicinal, fodder, and timber values (Acharya et al., 2002).
It has been continuously mentioned in various traditional systems of medicine like
Ayurvedic, Unani and Homeopathic systems of medicine (Badkhane et al., 2010).
Almost every parts of the plant are used as primitive home remedies against various
human diseases (Manne et al., 2020; Prasad et al., 2007). Pant and Yadav (2013)
also reported Bijaysal to have high medicinal value and listed it among the 20 most
exploited plant species in Kanchanpur district, Western Nepal. Wood is very hard
and durable so it is considered as a multipurpose tree throughout India (Troup, 1921;
Troup, 1986). Wooden tumblers made from the wood of Bijaysal tree are still used
to control diabetes (Grover et al., 2002; Reddy et al., 2008; Badkhane et al., 2010;
Khare, 2010; Joshi et al., 2012; Khanal and Bhattarai, 2020; Vikaspedia, 2021) and
is referred to as ‘The miracle cure for diabetes’ (Katiyar et al., 2016). The water left
in these tumblers overnight (Joshi et al., 2012), when consumed daily twice for 30
days has shown beneficial effects in individuals suffering from diabetes (Katiyar
et al., 2016).
Stem bark is boiled and consumed as a tonic after child birth (Prasad et al., 2007).
Heartwood and kino gum are useful in the skin as well as blood-related diseases as
they have astringent, anti-diarrhoeal and anti-haemorrhagic properties (Badkhane
et al., 2010; Vikaspedia, 2021). Similarly, the leaves are used externally to treat
boils, sores and other skin diseases and the flowers are used as febrifuge (Seema
et al., 2010; Vikaspedia, 2021). The seed paste and decoction of heartwood is used
to cure Diabetic anaemia (Badkhane et al., 2010).
The bark of stem is often slashed for the Kino gum due to its medicinal properties.
Khanal and Bhattarai (2020) reported that lower parts of the tree trunk are usually
exposed and gums are collected. The synthesis of information in this chapter aims
to stimulate further research on economic and ethnobotanical uses of P. marsupium.

Present investigation was carried out based on the survey carried out in Malakheti
and Khamaura villages, Kailali district and Bhimdattanagar of Kanchanpur district,
Far West, Nepal during November, 2020 (Figure 1).
45
Figure 1: Map showing the study sites in Kailali and Kanchanpur districts of Sudurpaschim province.
46
Information on economic as well as ethno-botanical uses of Bijaysal was collected
from the study area using questionnaire method. In both Kailali as well as Kanchanpur
districts, 45 respondents with age ranged above 35 years were interviewed. In both
study sites, 20 respondents were male and 25 were female. The informants were also
asked to specify the parts used and the mode of using it. The major inhabitants of
the study area are Brahmin, Chhetri and Tharus.
Use Profile of Pterocarpus marsupium

Majority of the respondents interviewed mentioned Bijaysal to be an important plant
species since ages. The economic as well as ethnobotanical uses of Bijaysal reported
during this study are presented in table 1.
Table 1. Economic and ethnobotanical uses of Pterocarpus marsupium in Kailali and Kanchanpur
districts, Far West, Nepal.
S. Use Uses
Parts used Mode of use
No. category (against/as)
Medicinal Diabetes Water is filled in wooden tumblers and left
overnight and the next morning this water is
Body ache taken in empty stomach.
Joint pain Alternatively, wooden powder/ pieces is
1. Heartwood soaked in water overnight and the next
morning the water is filtered and consumed
Cough and cold in empty stomach.
Wood is used in making ploughs, toys,
Others Furniture furniture’s, etc.
Leaf paste is applied on the skin to cure
Skin diseases
skin problem
Medicinal
Leaf paste is applied on the skin in case of
2. Leaves Insect bites
insect bites.
Branches are lopped and leaves are given
Fodder Fodder for cattles
as fodder to domestic cattles.
Paste of bark is applied on the skin to cure
Skin infection
skin infection, scabies, etc.
Bark powder is soaked in water and
Diabetes
Medicinal consumed.
3. Bark Paste of bark is also applied in case of
Toothache
toothache.
Malaria and fever Paste of bark is consumed directly.
Others Dyes Fresh bark is used to extract the dyes.
47
S. Use Uses
Parts used Mode of use
No. category (against/as)
Bark of tree are slashed and gum is
Diabetes collected. Kino gum is stored in small
bottle and taken whenever necessary.
Body ache Kino gum is consumed directly. quantity
4. Kino gum Medicinal
Diarrhoea and
Kino gum is consumed directly.
Dysentery
Paste of bark is also applied in case of
Toothache
toothache.
Flowers are consumed directly to control
5. Flowers Medicinal Fever
fever.
Branches and twigs are dried properly and
Firewood Firewood
Twigs and used as firewood.
6.
branches Twigs of Bijaysal are used to brush teeth
Medicinal Toothache
due to their medicinal properties
Wood/ wood
7. - - Used as an alternative to tea leaves.
powder
Economic Aspects
Locally the heartwood is used for making wooden tumbler that is in high demand
due to its medicinal value. Several other utensils, musical instruments, cups, glasses,
gift and jewelry boxes, etc. are prepared and sold in the local market. The wood
powder is used as an alternative to tea and thus packets of wooden powder is also
sold in the market. The bark is also used for toothache as well as for dyeing purpose
(Sukhadaya et al., 2019). Similarly, Troup (1921) reported that the wood is used for
making buildings, agricultural equipments, carts, wheel-works, boats, etc. Khare
(2007) and Sukhadaya et al. (2019) reported that the wood of Bijaysal is well known
due to its excellent timber value and ranks next to teak and rosewood throughout
the peninsular India.
Ethnobotanical aspects
Locally, the residents of Western Tarai especially of Kailali and Kanchanpur districts
have been using heartwood as well as Kino gum for body ache, joint pain and
gastro-intestinal disorders. It is listed among the most exploited plant species in Far
Western Tarai (Pant and Yadav, 2013). Traditional medical practitioners like Baidhya/
Baidawa, local healers; Guruwa, etc. are engaged in folklore medicines similar to the
studies from other parts of the country (Khanal and Bhattarai, 2020). Several parts
of the plant such as heartwood, bark, leaves, flowers, gum resin, etc. are used since
many generations because of their inherent medicinal properties. The indigenous
48
Figure 2: Pterocarpus marsupium: field observation. a. Adult trees in the cultivable land; b. Local
people collecting fodder for cattle; c. Collecting information from the local stakeholders in Kanchanpur
district; d. Local in Kanchanpur district making wooden tumblers from heartwood Bijaysal; e. Making
wooden tumblers; and f. Wooden tumblers and other wooden utensils made from Bijaysal (Photos:
a., c., and f. Yagya Raj Paneru; b., d., and e. Pratikshya Chalise).
49
Tharus and Brahmins collect the Kino gum and store them in small plastic bottles
and consume it whenever and wherever necessary. Several traditional ploughs and
bullock carts are made from the wood of Bijaysal. But, some local healers were
reluctant to share their knowledge about the medicinal plants and their properties.
This is because they fear sharing their knowledge with other will make their gurus
angry, and they will lose their knowledge as well as the ability to heal forever.
Similipalkol tribes in Odisha, India make a paste of the barks of Pterocarpus
marsupium, Mangifera indica, Shorea robusta and Spondias pinnata to treat dysentery
and other diarrheal illnesses (Sharma and Gautam, 2017). Similarly, the Kannada
people of India believe that a poultice prepared from the bark and leaves of Bijaysal
possesses astringent properties so it is useful in treating skin infections (Sharma and
Gautam, 2017).
However, due to changing perception of the local people from generation to
generation; due to the ever increasing influence of global commercialization, socio-
economic transformation and modern medicinal practices; indigenous knowledge
on uses of plant resources is constantly diminishing (Gadgil et al., 1993; Kunwar
and Adhikari, 2005).
Conclusion and Recommendations

P. marsupium is an important tree species with high medicinal value; renowned as an
important remedy against diabetes. However, despite traditional claims, insufficient
scientific validation for the treatment of diabetes, dysentery, cough and cold, joint
pain, skin diseases, fever and toothache; require further examinations. It is under threat
of extinction due to increasing human pressure as well as habitat degradation due to
several anthropogenic activities such as grazing, lopping, expansion of agriculture
land, forest fire, etc. and improper collection of Kino gum locally. The present study
endeavors to document the traditional ethnobotanical information and the overall
usage of this multipurpose tree. Further, the information generated from this study
will give a premise to phytochemical and pharmaceutical studies.
50
CHAPTER - 9
PHYTOCHEMISTRY, ANTIOXIDANT, ANTIDIABETIC
ACTIVITIES AND TOXICITY
Parasmani Yadav and Devi Prasad Bhandari
Pterocarpus marsupium is an important therapeutic and medicinal plant (Rahman

et al., 2018). It is one of the most valuable multipurpose tropical tree and has great
importance of producing timbers. The bark and resin decoction is an astringent for
severe diarrhoea, dysentery, for the treatment of tumours of the glands, urethral
discharges, on ringworm of the scalp, chronic ulcers and abortifacient (Basu et al.,
1975). The heart wood is astringent, bitter acrid, anti-inflammatory, anthelmintic,
anodyne (Kritikar, 1987). It is also good for elephantiasis, leucoderma, diarrhoea,
rectalgia, cough and greyness of hair (Mankani et al., 2005). Besides these, the bark
of P. marsupium is found to be the most effective in preventing cataract development
and reducing hyperglycemia (Vats et al., 2004). It is safe and effective in wounds,
fever, stomach ache, diabetes, jaundice and ulcers (Jung et al., 2006).

The leaf and bark samples of Bijaysal were collected from the premises of Dhakeri
Botanical Garden, Banke and was identified at National Herbarium and Plant
Laboratories, Godawari, Lalitpur. Herbarium specimens were prepared and deposited
at National Herbarium and Plant Laboratories (KATH). Samples were cleaned and
shade dried before the extraction process.
Preparation of Plant extracts

Crude extracts of bark and leaves were prepared by Soxhlet extraction method. 10 gm
of powdered plant material was uniformly packed into a thimble and extracted with
100 ml of Methanol. The process of extraction continued for 18-24 hours. Chlorophyll
was removed wherever necessary by treating the methanolic extract with Hexane in
separating funnel. The extracts were concentrated by keeping beakers in water bath
at 55ºC till all the solvent got evaporated. Dried extract was kept in refrigerator at
4ºC for their future use in phytochemical analysis.
Qualitative Phytochemical Analysis

Qualitative phytochemical analysis was carried by Standard analytical procedures
as described by Harbone (1973), Trease and Evans (1989), Sofowora (1993) and
Aluko et al. (2012).
51
1. Test for reducing sugars (Fehling’s test)
Equal volume of Fehling A and Fehling B reagents were mixed together and 2
ml of it was added to crude extract and gently boiled. A brick red precipitate
appeared at the bottom of the test tube indicated the presence of reducing sugars.
2. Test for glycoside
4 ml of extract solution was dried till 2 ml and 1-2 ml of Ammonium hydroxide
was added. The resulting mixture was shaked. Appearance of Cherish red colour
indicates the presence of glycosides.
3. Salkowski’s test
Crude extract was mixed with 2 ml of Chloroform. Then 2 ml of concentrated
H2SO4 was added carefully and shaked gently. A reddish brown colour indicated
the presence of steroidal ring, i.e., glycone portion of the glycoside.
4. Keller-Kilani test
Crude extract was mixed with 2 ml of Glacial Acetic acid containing 1-2 drops of
2% FeCl3 solution. The mixture was then poured into another test tube containing
2ml of concentrated H2SO4. A brown ring at the interface indicated the presence
of cardiac glycosides.
5. Test for polyphenols and tannins
Crude extract was mixed with 2 ml of 2% FeCl3 solution. A blue-green or blue-
black coloration indicated the presence of polyphenols and tannins.
6. Test for flavonoids
Crude extract was mixed with few fragments of Magnesium ribbon and
concentrated HCl was added dropwise. Pink or magenta red colour appeared
after few minutes which indicated the presence of flavonoids.
7. Test for saponins
Crude extract was mixed with 5 ml of distilled water in a test tube and it was
shaked vigorously for 30 seconds. The formation of stable foam (1 cm height)
even after 30 minutes was taken as an indication for the presence of saponins.
8. Test for steroids
Crude extract was mixed with 2 ml of Chloroform and concentrated H2SO4 was
added sidewise. A red colour produced in the lower Chloroform layer indicated
the presence of steroids. Another test was performed by mixing crude extract
with 2 ml of Chloroform. Then, 2 ml of each, concentrated H2SO4 and Acetic
acid were poured into the mixture. The development of a greenish coloration
indicated the presence of steroids.
52
9. Test for terpenoids
Crude extract was dissolved in 2 ml of Chloroform and evaporated to dryness.
Then, 2 ml of concentrated H2SO4 was added; a reddish brown coloration at the
interface indicated the presence of terpenoids.
10. Test for alkaloids
Crude extract was mixed with 2 ml of 1% HCl and heated gently. Mayer's and
Wagner's reagents were then added to the mixture. Turbidity of the resulting
precipitate was taken as evidence for the presence of alkaloids.
11. Test for coumarins
Extract solution is concentrated to yield a residue. The residue is dissolved in
hot water. After cooling, the solution is divided in two test tubes. Then, to one
test tube 10% (w/v) Ammonium hydroxide is added whereas the other test tube
is used as control. Fluorescence colour indicates the presence of coumarins.
Observation
The change of colour was observed when the test reagent was added to the prepared
sample for the phytochemical test. The result was recorded as present (+) or absent
(-) depending on the outcome of the test.
Determination of Total Phenolic Content

Phenolic content was measured by using the Folin–Ciocalteu reagent in each extract
(Singleton et al., 1999). The results were derived from a calibration curve of different
concentration of Gallic acid and expressed in Gallic acid equivalents (GAE) per
gram dry extract weight.
Preparation of Reagent
1. Preparation of Sodium carbonate solution
1 Molar Sodium carbonate solution was prepared by dissolving 2.65g of Sodium
carbonate in 25 ml distilled water, 1 ml of FCR reagent was mixed with 10 ml
of water to prepare 1:10 FC reagent.
2. Preparation of Standard Gallic acid and sample solution
5 mg Gallic acid was dissolved in 5 ml of Ethanol to prepare the stock solution
of 1000 μg/ml concentration. The stock solution was further diluted into final
concentration of 10, 20, 30, 40, 50, 60, 70, 80 μg/ml. Different concentration of
the Gallic acid was used as positive control in total phenolic content test.
53
Similarly, plant extract of 5000 μg/ml concentration were prepared from stock
solution of crude extract in 50% DMSO solution.
Procedure
Total phenolic content of both plant extract was determined by using Folin-Ciocalteu
reagent. Gallic acid of different concentration 10, 20, 30, 40, 50, 60, 70, 80 μg/ml
was loaded triplicate used for the standard control. Plant sample of 5000 μg/ml was
loaded 20 μl triplicate. 100 μl of the FC reagent was added in each well containing
Gallic acid and plant sample. Initial reading of plate was taken at 765 nm using a
microplate reader. After initial reading 80 μl of Na2CO3 was added separately to each
well, and incubated for 15 minutes. After incubating the plate, the final absorbance
was taken 765 nm in (Epoch2, BioTek, Instruments, Inc, USA) microplate reader.
Standard curve of the Gallic acid was plotted as standard curve.
Determination of Total Flavonoid Content (TFC)

Total flavonoid contents in selected plant extracts were determined by using
Aluminium chloride in a colorimetric method (Atanassova et al., 2011). The results
were derived from the calibration curve of different concentration of quercetin and
expressed in quercetin equivalents (QE) per gram dry extract weight.
1. Preparation of Aluminum trichloride
10% Aluminum trichloride was prepared by dissolving 1 g of AlCl3 into 10 ml
distilled water and 1 M potassium acetate in 10 ml distilled water was prepared.
2. Preparation of standard Quercetin and sample solution
1.54 mg quercetin was dissolved in 10 ml water to prepare 154 μg/ml concentration
of stock solution. By diluting the stock solution of the Quercetin different
concentration of the quercetin 10, 20, 40, 60, 80, 100 μg/ml was prepared.
Similarly, plant sample was prepared by diluting stock solution in 50% DMSO.
Procedure
130 μl of different concentration (10, 20, 40, 60, 80, 100 μl) of the Quercetin was
loaded triplicate in 96 well plate. Similarly, 20 μl of the plant sample (5000 μg/ml)
was loaded triplicate; and 110 μl of distilled water was added in each well containing
plant sample. 60 μl of Ethanol was added to each well containing plant extract and
Quercetin. Initial reading was taken at wavelength 415 nm in microplate reader
(Epoch 2, BioTek, Instruments, Inc. USA). After initial reading of the plate, 5-5 μl
54
of AlCl3 was added in each plate. After addition of the reagent, plate was incubated
in dark for 30 minute and final reading of plate was taken at same the wave length.
Determination of Antioxidant activity

The antioxidant activity was determined as DPPH Radical Scavenging Activity (Hu
et al., 2011). All data were compared with the IC50 value of standard Quercetin.
1. Preparation of DPPH solution (0.1mM)
0.1 mM DPPH solution was prepared by dissolving 1.95 mg DPPH in 50 ml
Methanol in Dark volumetric flask.
2. Preparation of quercetin solution and plant sample
1 mg of Quercetin was dissolved in 10 mL Methanol to prepare stock solution of
100 μg/ml. Quercetin solutions of 20, 10, 5, 2.5, 1.25, 0.625 μg/ml concentration
were prepared by diluting the stock solution. Similarly, plant extracts of different
concentration (500, 250, 125, 62.5, 31.25 μg/ml) was prepared in 50% DMSO,
from stock solution of 5000 μg/ml.
Procedure
Radical scavenging activity of the plant sample was evaluated by modified
colorimetric method, from 96 well plate. For standard curve, Quercetin 100 μl of
different concentrations (20, 10, 5, 2.5, 1.25, 0.625 μg/ml) was loaded triplicate as
positive control, and 50% DMSO was loaded triplicate as negative control. Plant
samples of different concentration were loaded, 100 μl to each well and initial
absorbance was measured. After initial reading, 100 μl of DPPH solution was added
in each well and was incubated for 30 minutes in dark. After incubation, final reading
was taken and the percentage inhibition of the sample was calculated and compared
with standard curve of Quercetin.
% Inhibition = Control (A) - Sample (A) x 100

Control (A)
Where, A is the absorbance of the sample and control.
55
Anti-diabetic test (α-Glucosidase inhibition assay)
The α-glucosidase inhibitory activity was assessed by the standard method (Dong
et. al., 2012) with slight modifications.
1. Preparation of Buffer solution
7.393 gm of Potassium dihydrogen orthophosphate and 7.956 gm of Dipotassium
hydrogen orthophosphate were weighted and dissolved in 1 litre of distilled
water. Here, pH of the buffer was maintained 6.8 by adding mono-potassium or
di-potassium phosphate.
2. Preparation of Acarbose and plant extract
100 μg/ml concentration of Acarbose was prepared by mixing 1 mg Acarbose
in 10 ml phosphate buffer solution. Different concentrations of the Acarbose
solution (20, 10, 5, 2.5, 1.25 μg/ml) was prepared by diluting the stock solution
of Acarbose. 1000 μg/ml of plant extract was prepared in buffer solution and
was diluted to different concentrations (1000, 500, 250, 125, 62.5, 31.5 μg/ml)
using buffer solution.
Procedure
The α-glucosidase inhibition of the plant extract was analyzed by using substrate
PNPG. 20 μl plant extract and Acarbose of different concentrations (1000, 500,
250, 250,125, 62.5, 31.25 μg/ml) were loaded triplicate in microplate. 10 μl of the
α-glucosidase enzyme was loaded in each plate. Similarly, 130 μl was incubated for
15 minutes at 37°C. After initial incubation, initial reading of the plate was taken at
wavelength 405 nm. After incubating the solution, 20 μl of 5 mM PNPG was added
to each well on microplate. Then, the reaction mixture was incubated for 15 minutes
at 37°C. After final incubation, 20 μl of Na2CO3 was loaded to each well. Here, yellow
colour of the para-Nitrobenzene was determined at 410 nm by using a microplate
reader (Epoch2, BioTek, Instruments, Inc., USA). The percentage of α-glucosidase
activity was calculated by the following formula.
% Inhibition = Control (A) - Sample (A) x 100

Control (A)
Where, A is the absorbance of the sample and control.
56
Acute Oral Toxicity Test
The guidelines for Testing of Chemicals, Acute Oral Toxicity, Acute Toxic Class
Method 423 of the Organization for Economic Cooperation and Development
(OECD) was used. A total of 18 female swissabino mice (6 mice/group), were
randomly selected and marked for individual identification. All the animals were
subjected to 4 hours of fasting prior to the treatment. The test groups included a control
group (10 ml/kg distilled water) and two other treatment groups viz; Group I and
Group II for dose 300 mg/kg and 2000 mg/kg body weight of extracts respectively.
The animals were observed for 1 hour after treatment, and then intermittently for
4 hours, and thereafter the mice were further observed for up to 14 days following
treatment. Clinical signs such as weakness or aggressiveness, food refusal, loss of
weight, diarrhoea, discharge from eyes and ears, noisy breathing and the number
of deaths in each treated groups were monitored carefully. Body weight, food and
water intake were measured on 1st, 7th and 14th day.
Results: Phytochemistry, Antioxidant, Antidiabetic activities and Toxicity

Preliminary qualitative phytochemical analysis
The result of preliminary qualitative phytochemical analysis is given in table 1.
Table 1. Result of preliminary qualitative phytochemical analysis.
S.No. Parameters Phytochemical Test Leaf Bark
Volatile oils Spot test/Residue test - -

Alkaloids Mayer's Regent test, + ++
Shinoda test +++
Flavonoid +++
Alkaline Reagent test
Steroids Steroid test + +++
Terpenoids Terpenoids test + ++
Glycosides Salkowski’s test +++ +++
Phenols FeCl3 test +++ +++
Saponins Froth/ Foam test - -
Protein Ninhydrin test - -
Carbohydrate Molish test ++ -
*Note: Result + means presence in traces amount, ++ means presence in moderate amount, +++
means presence in adequate amount and – means absence.
57
Determination of Total Phenolic Content/Total Flavnoid Content, Antioxidant,
Antidiabetic and Acute Oral Toxicity
The TPC, TFC, Antioxidant, Antidiabetic activity and Oral toxicity values of the leaf
and bark extract is presented in table 2.
Table 2. Result of phytochemical tests for TPC, TFC, antioxidant, antidiabetic activity and oral toxicity.
Antioxidant Antidiabetic Oral toxicity

S. Plant TPC TFC
IC50 % inhibition LD50
No. (part used) (mgGAE/g) (mgQE/g)
(µg/mL) (µg/mL) (mg/kg)
1. P. marsupium (leaf) 1114.76 5.73 15.21222 98.73 >2000
2. P. marsupium (bark) 596.7 4.61 43.71872 98.32 >2000
Leaf extract showed comparatively higher total phenolic and total flavonoid contents
than that of bark. The IC50 value of Quercetin for antioxidant assay was found to
be 3.33. However, the antioxidant potential was higher in bark extract compared
to the leaves extract. Antidiabetic property was nearly equal in both leaf and bark
extracts. Similarly, toxicity values (the median lethal dose (LD50) of active principle
of extract) revealed that the doses of the LD50 were >2000 mg/kg, for both the bark
and leaf extract indicating non-toxicity at all.
Figure 1: a. Total Phenolic Content of Standard Gallic Acid; b. Total Flavonoid content (TFC) of
Standard Gallic Acid; c. Antioxidant Activity of Standard Quercetin against DPPH; and d. Antioxidant
Activity of Bark and Leaf of P. marsupium.
58
The overall laboratory tests suggested that P. marsupium is rich in phytochemicals and
possesses good antioxidant and antidiabetic properties. It possesses high antioxidant
activity that might be proposed for impeding toxic oxidation in nutraceuticals or
drugs for the treatment of coronary diseases. It has strong inhibitory activity against
α-glucosidase and can be used to formulate antidiabetic drugs. Non-toxicity of the
extracts indicates its applicability as a drug. Further investigations regarding the
isolation and identification of responsible antioxidants and antidiabetic components
and their mechanism of action is necessary. This will enable us to better understand
their ability to control diseases and could have a significant impact on the quality
of life.
59
CHAPTER - 10
ANTI-MICROBIAL ACTIVITY
Pramesh Bahadur Lakhey and Sachita Joshi
Various parts of the Pterocarpus marsupium tree have been used as traditional
ayurvedic medicine. The medicinal utilities have been described, especially for leaf,
fruit and bark. In vitro testing of antimicrobial activity of extracts from different parts
of this plant has been carried out by different authors. Bhat et al. (2014) reported
antimicrobial activity of heartwood extract. Similarly, Pant et al. (2017) evaluated
antimicrobial activity of stem wood. Manne et al. (2020) tested chitosan synthesized
nanoparticles from the heartwood extract, for its antimicrobial property. Antimicrobial
studies on its extracts have also been done by other authors including Deepa et al.
(2014), Gayathri and Kannabiran (2021) and Ramya et al. (2008).

Sample Source
Approximately, one-kilogram fresh weight of leaf-bearing twigs and bark samples
of P. marsupium were collected from Dhakeri Botanical Garden, Banke. After the
collection of samples, herbarium specimens were prepared and housed at National
Herbarium and Plant Laboratories, Godawari, Lalitpur.
Preparation of extract
The samples were washed in tap water to remove adhering soil particles. The leaves
were manually plucked from twigs. The twig and bark samples were cut into small
pieces using pruning shears. The leaves, pieces of twigs and bark were then spread
into a thin layer and shade dried. The dried samples were separately ground into
coarse powder in a mixer-grinder.
Extraction was done from the powdered samples by modified maceration process
(steady-state extraction) as described by Singh (2008) in three phases using Hexane,
Ethyl acetate and Methanol as menstruum for each phase. For the first extraction
phase, 15 gm of each sample was placed in a 250 ml conical flask and 150 ml
Hexane (ten times the weight of the sample) was added to it. The conical flasks
were agitated at 150 rpm in a rotary flask shaker for the extraction duration of a
week in a continuous cycle of 60 seconds agitation followed by 30 seconds rest. The
extraction was done in four replicates. After seven days, the mixture so formed was
allowed to stand for 24 hours and the supernatant was strained off into a separate
vessel. The solid residue or marc was transferred into three layers of muslin cloth
and was pressed to release as much occluded solution as possible. The strained and
60
filtering through Whatman No.1 filter paper under vacuum using a Buchner funnel. The
miscella, so obtained, was concentrated in a rotary vacuum evaporator. The concentrated
occluded
miscella liquids
was further were
dried onmixed and wasring
a concentric allowed
watertobath
standtofor 24 hours.
yield The mixture was
a dry extract.
further clarified by filtering through Whatman No.1 filter paper under vacuum using
a Buchner
Marc from funnel.
the first Theofmiscella,
phase so obtained,
extraction was shadewas concentrated
dried in a to
for 48 hours rotary vacuum
remove residual
evaporator. The concentrated miscella was further dried on a concentric ring water
solventbath
andtowas
yieldused forextract.
a dry second phase of extraction using Ethyl acetate as menstruum. The
same process was repeated in the third extraction phase with the marc from second phase
Marc from the first phase of extraction was shade dried for 48 hours to remove
using methanol as menstruum.
residual solvent Thefor
and was used extracts yielded
the second phasein of
each of the using
extraction three Ethyl
extraction phases
acetate
as menstruum.
were separately The same
weighed, processinwas
collected repeated in the
a McCartney third labelled
bottles, extractionand
phase with in a
stored
the marc from second phase using Methanol as menstruum. The resulting extracts
refrigerator at 2-8ºC
from the until further
three extraction use. were separately weighted, collected in a McCartney
phases
bottles, labelled and stored in a refrigerator at 2-8ºC until further use.
The yield of different extracts was calculated by using the given formula:
The yield of different extracts was calculated by using the given formula:

   

Screening of extracts
Screening for Antimicrobial
of extracts Activity
for Antimicrobial Activity
Test organisms
Test organisms
The extracts
The extracts were were
testedtested for antimicrobial
for antimicrobial activity
activity against
against eleven
eleven bacterial
bacterial strains/
strains/isolates
isolates belonging to 10 bacterial species and 2 fungal strains belonging to two fungal
belonging to 10
species bacterial
which have species and in
been listed 2 table
fungal1.strains belonging to two fungal species which
have been listed in Table 1
Table 1. List of bacterial and fungal strains used as test organisms.
S.No. Name of species
Table 1List ofbacterial and fungal strains usedReference No. Strains
as test organisms Source
1 Bacillus subtilis ATCC-6051 Gram positive
S.No. 2 ofEscherichia
Name species coli ATCC-8739
Reference No. Gram negative Microbiologics,
Strains Source
1 Bacillus
3 subtilis
Enterococcus faecalis ATCC-6051
ATCC-29212 Gram positive
Gram positive 200 Cooper
2 Escherichia coli pneumoniae ATCC-8739 Avenue North,
4 Klebsiella ATCC-700603 Gram negative
Gram negative Microbiologics,
St, Cloud
3 Enterococcus faecalis ATCC-29212 Gram positive 200 Cooper
4 5 Proteus
Klebsiella vulgaris
pneumoniae ATCC-6380 Gram
ATCC-700603 Gram negative Avenue
negative MN56303
North, St,
5 6
Proteus Pseudomonas
vulgaris aeruginosa ATCC-9027 Gram
ATCC-6380 Gram negative Cloud MN56303
negative
6 Pseudomonas
7 aeruginosa
Salmonella enterica enterica TyphiATCC-9027
Clinical Isolate Gram negative
Gram negative TU Teaching
7 Salmonella
8 enterica enterica
Shigella dysenteriae Typhi Clinical Gram
Clinical Isolate Gram negative
negative TU Teaching
Hospital,
Isolate Hospital,
Maharajgunj,
8 Shigella dysenteriae Clinical Gram negative Maharajgunj,
Kathmandu
9 Staphylococcus aureus ATCC 6538P Gram positive Microbiologics,
200 Cooper
Avenue North,
St, Cloud
MN56303
61
S.No. Name of species Reference No. Strains Source
10 Methicillin resistant S. aureus Clinical Isolate Gram positive Sukraraj
(MRSA) Tropical and
Infectious
Disease
Hospital, Teku,
Kathmandu
11 Staphylococcus epidermidis ATCC 1228 Gram positive Microbiologics,
12 Candida albicans ATCC.2091 Fungi 200 Cooper
Avenue North,
13 Saccharomyces cerevisae ATCC.18824 Fungi
St, Cloud
MN56303
Preliminary screening for antimicrobial activity

The extracts were screened for antimicrobial activities following modified Agar well
diffusion method, described by Nathan et al. (1978) and Holder and Boyce (1994).
Clinical and Laboratory Standards Institute (2012) and National Committee for
Clinical Laboratory Standards (2004) were also referred to.
Test solution of each extract of 100 mg.ml-1 concentrations was prepared in dimethyl
sulphoxide (DMSO). Inoculation suspension of each of the test organism with
turbidity equivalent to 0.5 McFarland Nephelometric Standard was prepared in
sterilized normal saline using 18 to 24 hours culture by direct colony suspension
method. A sterile cotton swab was used to evenly distribute bacterial or fungal
culture drawn from the respective inoculums over the appropriate medium, viz.
Muller-Hinton Agar (MHA) for bacteria and Muller-Hinton Agar with Glucose and
Methylene Blue (MHA.GMB) for fungi (Clinical and Laboratory Standards Institute,
2012; National Committee for Clinical Laboratory Standards, 2004). The inoculated
plates were left for maximum 15 minutes to allow absorption of excess surface
moisture. Using sterilized cork borer, agar wells of 6 mm diameter were made in
the inoculated plates, 4 to 5 wells per plate. Micropipettes were used to place 50 μl
of the test solution into each well. DMSO (50 μl) was also added to one of the wells
of each plate as negative control. Antibiotic sensitivity discs were used as positive
control for bacterial species; Ciprofloxacin 5 μg for Bacillus subtilis, Enterococcus
faecalis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas
aeruginosa, Salmonella enterica enterica Typhi and Shigella dysenteriae; Cefoxitin
30 μg and Azithromycin 30 μg for Staphylococcus aureus, Methicillin resistant S.
aureus (MRSA) and S. epidermidis. The plates were placed in obverse position until
the test solution was absorbed into agar and the wells appeared dry. The plates were
then incubated in inverted position at 35±2°C for 18 to 24 hours for all test organisms
except Saccharomyces cerevisiae which was incubated at 25±2°C for 24 to 48 hours.
62
After the incubation period, the diameters of zones of inhibition (ZOIs), interpreted
as the clear areas around the agar wells, were measured using a digital caliper. The
test for antimicrobial activity was done in five replicates for each of the test species.
Determination of minimum microbicidal concentration

Broth macro-dilution method described by National Committee for Clinical
Laboratory Standards (1999) was followed to determine minimum microbicidal
concentration (MMC) of the test extracts. Twelve vials, each containing 1 ml of
Muller Hinton Broth for bacteria and Sabauraud Dextrose Broth of fungi were
prepared and labelled from 0 to 11. In no. 0 vial, the media was removed and only
1 ml test solution of 100 mg.ml-1 concentrations was placed. In no. 1 vial, 1 ml of
the test solution was added to 1 ml of the medium and mixed evenly by vortexing
to obtain a solution of concentration 50 mg.ml-1; 1 ml of the 50 mg.ml-1 solution in
no. 1 vial was transferred to no. 2 vial and was mixed evenly to obtain a solution
of concentration 25 mg.ml-1. This process was repeated up to vial no. 10 to obtain
solutions of concentrations 12.5 mg.ml-1, 6.25 mg.ml-1, 3.125 mg.ml-1, 1.5625 mg.
ml-1, 0.78125 mg.ml-1, 0.390625 mg.ml-1, 0.1953125 mg.ml-1 and 0.09765625422 mg.
ml-1. Vial no. 11 was left with medium only. Each vial was inoculated with 20 μl
of the cell suspension of the organisms with turbidity equivalent to 0.5 McFarland
Nephelometric Standard and were incubated at 35±2°C for 18 to 24 hours for all
test species except Saccharomyces cerevisiae which was incubated at 25±2°C for
24 to 48 hours. The MIC of the samples were visually determined as the lowest
concentration that did not show any growth as indicated by turbidity in comparison to
the uninoculated medium. For the determination of MMC, the vials were sub-cultured
on nutrient agar (NA) for bacterial species and Sabauraud dextrose agar (SDA) plates
for fungal species to determine the viability of the inoculated organisms at different
concentrations of the extracts and were incubated at suitable temperature and for
suitable durations. MMC was determined as the concentration value between the
minimum concentration with no growth and maximum concentration showing growth.
Statistical analysis
The charts were generated using Microsoft Excel 2013. The extract yield percentages
were compared by ANOVA followed by Tukey HSD test, and ZOI values by paired
sample t-test using SPSS 23.0.
Results: Antimicrobial activity

The yield of extract from bark, leaves and twig samples of Pterocarpus marsupium in
Hexane, Ethyl acetate and Methanol differed significantly (df = 8,27, F = 19758.396,
p = 0.000, Figure 1). The maximum extract yield was observed in methanol extract
63
of bark (10.33±0.050%) followed by methanol extract of twigs (9.91±0.057%)
while the minimum yield was observed in Hexane and Ethyl acetate extracts of bark
(0.34±0.052% and 0.036±0.070% respectively). The high yields of methanol extracts
from the leaf, bark and twig samples of P. marsupium indicate that these parts are
rich in polar compounds. In comparison to bark and twigs, leaves contained more
non-polar and mid-polar components as evident from the yield of Hexane and Ethyl
acetate extracts of these samples.
During preliminary antimicrobial activity screening by agar well diffusion method,
only methanol extracts of bark and twigs showed antimicrobial activity against
Staphylococcus aureus, methicillin resistant S. aureus (MRSA) and S. epidermidis;
while methanol extract of leaves showed activity against S. aureus and S. epidermidis
(Plates 1a, 1b and 1c). However, all the tested plant extracts did not show any
antimicrobial activity against Bacillus subtilis, Enterococcus faecalis, Escherichia
coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella
enterica entrica Typhi, Shigella dysenteriae, Candida albicans and Saccharomyces
cerevisiae (Zone of Inhibition (ZOI) = 0 mm). In contrast to the findings of this study,
Patil and Gaikwad (2011) have reported antimicrobial activities of bark extract of
P. marsupium against Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoneae, Salmonella typhi, Proteus
mirabilis and Micrococcus sp. They concluded Staphylococcus aureus as being
most sensitive to the extract. Ramya et al. (2008) found hexane, ethyl acetate and
methanol extracts of bark and leaves of this plant species as effective against Bacillus
subtilis, Bacillus cereus, Staphylococcus aureus, Streptococcus pyrogens, Escherichia
coli, Salmonella typii, Serratia marcescens and Pseudomonas aeruginosa with
inhibitory activity being dose dependent. They found ethyl acetate and methanol
extracts to be more active towards the organisms tested than hexane extract.
Gayathri and Kannabiran (2021) reported antimicrobial activity of aqueous extract
of roots of this plant against Staphylococcus aureus, Pseudomonas aeruginosa and
Klebsiella pneumoniae. Bhat et al. (2014) found that the heartwood aqueous extract
of P. marsupium was effective against Enterococcus sp., Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Deepa et al.
(2014) reported antimicrobial activity of the ethanol extract of bark of this species
against Bacillus polymyxa, Vibrio cholera and Candida albicans.
64
Figure 1: Percentage yield of extracts from bark, leaves and twig samples. Different alphabets above
the error bars indicate significant difference at 5% level of significance as determined by Tukey's HSD
test. The numbers above the error bars are mean yield percentages. The error bars indicate ± 2SEM.
Cefoxitin 30 μg sensitivity disc has been recommended for determining antibiotic
resistance (methicillin/oxacillin resistance) in strains/isolates of S. aureus (Clinical
and Laboratory Standards Institute, 2012). During this study, the sensitivity of MRSA
to cefoxitin 30 μg disc, as indicated by ZOI, was found to be significantly less in
comparison to that of sensitive S. aureus strain ATCC 6538P (df = 2, t = 38.602,
p = 0.001, Figure 2, Plates 1a and 1b). On the other hand, there was no significant
difference between the antimicrobial activities shown by methanol extract of bark
against S. aureus ATCC 6538P and MRSA (df = 4, t = 1.075, p = 0.343, Figure
3, Plates 1a and 1b). Similar result was also observed in case of methanol extract
of twigs (df = 4, t = 0.480, p = 0.657, Figure 4, Plates 1a and 1b). However, the
methanol extract of leaves which showed antimicrobial activity against S. aureus
ATCC 6538P (ZOI=8.88±0.83 mm) did not show activity against MRSA (ZOI = 0).
65
Figure 2: ZOIs of cefoxitin 30 μg disc against S. aureus strains. Different alphabets to the right of
the error bar indicates significant difference between means as determined by paired-sample t test
at 1% level of significance. The number to the right of error bar is the mean ZOI. The error bars
indicates ± 2SEM.
Figure 3: ZOIs of methanol extract of bark against S. aureus strains. The same alphabet to the right
of the error bar indicates no significant difference between mean as determined by paired-sample t
test at 1% level of significance. The number to the right of error bar is the mean ZOI. The error bars
indicates ± 2SEM.
Figure 4: ZOIs of methanol extract of twigs against S. aureus strains. The same alphabet to the right
of the error bar indicates no significant difference between mean as determined by paired-sample t
test at 1% level of significance. The number to the right of error bar is the mean value. The number
to the right of error bar is the mean ZOI. The error bars indicates ± 2SEM.
66
Among the extracts tested for minimal microbicidal concentration (MMC), the least
MMC of 3.1256.25 mg.ml-1 was measured for methanol extract of bark against
S. aureus ATCC 6539P. The same extract showed higher MMC value between
6.2512.5 mg.ml-1 for MRSA. The highest MMC value was observed in case of
methanol extract of leaves against S. aureus ATCC 6539P (Table 1, Plates 3 and 4).
Gayathri and Kannabiran (2021) found ZOI values of aqueous extract of roots
of this tree species between 0.04 to 0.09 mg.ml-1 against Staphylococcus aureus,
Pseudomonas aeruginosa and Klebsiella pneumoniae. The ethanol extract of bark
has been shown to have minimum inhibitory concentration of 1.25 mg.ml-1 against
Bacillus polymyxa and Vibrio cholerae while inhibition of Candida albicans was
found at 25 mg.ml-1 through broth dilution method (Deepa et al., 2014; Gayathri and
Kannabiran, 2021; Katiyar et al., 2016).
Table 2. Minimum microbicidal concentrations (MMCs) of the extracts showing antimicrobial
activities in preliminary screening.
S. Minimum microbicidal
Extract Bacterial strain
No. Concentrations (mg.ml-1)
1 Methanol extract of Bark S. aureus ATCC 6539P 3.125-6.25
MRSA 6.25-12.5
S. epidermidis 6.25-12.5
2 Methanol extract of Leaves S. aureus ATCC 6539P 25-50
MRSA Not done due since ZOI = 0
S. epidermidis Not done due since ZOI = 0
3 Methanol extract of twigs S. aureus ATCC 6539P 6.25-12.5
MRSA 6.25-12.5
S. epidermidis 3.125-6.25
This study validates the traditional use of “Bijaysal” for medicinal purposes for skin
related ailments. It strongly indicates the suitability of methanol extracts of bark and
twigs for the treatment of diseases caused by Staphyloccus aureus and its antibiotic
resistant strain MRSA. The active ingredients in these extracts should be identified
and isolated so that the efficiency of this natural herbal medicine can be increased.
67
Plate: 1a. Preliminary screening of antimicrobial activity of methanolic extracts of Pterocarpus
marsupium bark, leaves and twigs samples against Staphylococcus aureus by agar well diffusion
method. The wells on each plate from top right hand side in clockwise direction contains methanolic
extract of bark (5M), methanolic extract of leaves (6M), methanolic extract of twigs (7M) and positive
control (azithromycin 30 μg disc in the upper two plates, cefoxitin 30 μg in lower 3 plates). The
central well contains DMSO as negative control; b. Preliminary screening of antimicrobial activity
of methanolic extracts of Pterocarpus marsupium bark, leaves and twigs samples against methicillin
resistant Staphylococcus aureus (MRSA) by agar well diffusion method; c. Preliminary screening of
antimicrobial activity of methanolic extracts of Pterocarpus marsupium bark, leaves and twigs samples
against Staphylococcus epidermidis by agar well diffusion method; d. Determination of minimum
microbicidal concentration (MMC) of methanolic extract of Pterocarpus marsupium bark against
Staphylococcus aureus by subculturing of two fold inoculated two-fold serial dilution vials in each
of the section of petridish labelled 0 to 11; e. Determination of minimum microbicidal concentration
(MMC) of methanolic extract of Pterocarpus marsupium bark against methicillin resistant
Staphylococcus aureus (MRSA); and f. Determination of minimum microbicidal concentration
(MMC) of methanolic extract of Pterocarpus marsupium bark against Staphylococcus epidermidis.
68
CHAPTER - 11
THREATS, CONSERVATION AND TRADE
Dipesh Pyakurel and Pashupati Nath Koirala
Ecological resources from forests such as timber, non-timber and medicinal plants
play a vital role in the livelihoods of rural communities around the globe. It has been
estimated that environmental income accounts for 28% of the total household income
in the developing world (Angelsen et al., 2014). The higher dependency may exert
direct pressure on the sustainability of species with high demand and price making
these species vulnerable. Examples of exploitation of high valued species are seen
from Africa (Gaoue and Ticktin, 2008), Nepal (Pyakurel et al., 2019), India (Chauhan
et al., 2018), and South America (Cruz-Garcia et al., 2015).
Pterocarpus marsupium Roxb. (Bijaysal) is a slow growing tree species with limited
geographical distribution from tropical South Asia to Taiwan (Barstow, 2017). In
Nepal, it is naturally distributed in foothills of Chure (Siwalik) in Kanchanpur, Kailali,
Bardiya, Banke, Kapilbastu, Rupandehi, Palpa and Nawalparasi districts within 100
to 500 m. The population, however, is very sparsely distributed (DoF, 2018).
This chapter identifies the threats, analyzes the conservation status, and discusses
about the trade of P. marsupium in Nepal. Both electronic and online database e.g.,
Google Scholar, PubMed, Research gate, Scopus, Science Direct were conducted by
typing key words like Pterocarpus marsupium, trade, threats, conservation. Online
searches were conducted for unpublished reports. Likewise, other sources like books,
reports, thesis, and newspaper articles were also referred to collect the information.
Entrepreneurs and traders from Sudurpaschim province were individually contacted
to generate trade related information regarding per unit price of different items and
annual turnovers.
Existing threats and mitigation measures

Low germination and slow growth, high grazing pressure, fodder collection,
and harvesting of wood (to manufacture glass, cups, bowls, lota1, amkhora2 and
agricultural equipment) and Kino gum for economical and medicinal use are the
major threats to P. marsupium, due to which the global population is declining
(Wilkins, 1991).
Bijaysal is sparsely distributed even in its area of natural occurrence. Only 395 mature
trees were recorded from 12 Community Forests (CF) of Kanchanpur district in
1 Lota: A traditional Nepalese utensil made by brass and used to carry small quantity of water.
2 Amkhora: A traditional Nepalese utensil made by brass and with a long spout/pourer; used to drink water.
69
2014. The average number of mature trees was 9- 10 per hectare (Pyakurel and Oli,
2014). Likewise, 500 mature trees were recorded from Saraswati CF, Buddhabhumi
Municipality-10 of Kapilbastu district (DoF, 2018), showing the sparse distribution
pattern of P. marsupium in Nepal. Aggravated to that, it is a slow growing species
taking at least 15 years to mature (Vikaspedia, 2021).
The tree has multiple uses ranging from medicinal, economic and ecological (Pyakurel
and Oli, 2014; Barstow, 2017; DoF, 2018). People rely on wild stands of Bijaysal
for medicinal and economical uses. The Kino gums are extracted from the trunk
of tree by making scars. The ecological implications of such practices are not well
documented. The trees are cut and fell — despite protected by the Government of
Nepal under Forest Regulation 1995, for the preparation of bijaysal cups, glasses,
bowls, and other items which are praised in Nepalese and Indian communities for
its health benefit and as a decorative item. Only dead and fallen trees are allowed
to collect by law but high demand of items made from its wood causes the looping
of standing trees, thereby maximizing the risk of disappearance from the wild. This
scenario shows that ban on products with huge commercial value may not be effective.
Alternatively, governments e.g., Division Forest Offices of western Tarai should start
raising seedlings and saplings of P. marsupium in government nurseries and promote
private nurseries to do so. Successful raising and plantation has been observed in
Rupandehi as a research plot established by Forest Research and Training Centre
(the then Department of Forest Research and Survey). Sparse plantation has been
initiated by the government in Bardiya (by Plant Research Centre, Kailali) and other
districts too. Private nurseries can be promoted to raise seedlings of P. marsupium
and later cultivation in private lands can be initiated.
Livestock, along with deer and other wild animals feed on seedlings and saplings of
P. marsupium as leaves are highly palatable. Communities also cut small branches for
fodder, thereby rendering its natural regeneration. However, sensing its ecological and
economical importance, some community forests have prohibited the open grazing
by promoting stall feeding of cattle, penalty on open grazing in CFs (Pyakurel and
Oli, 2014). Such successful practice adopted by Betkot CF, Baijanath CF, Mahakali
CF and Ramnagar CF in Kanchanpur districts should be replicated in other CFs and
natural forests where P. marsupium is naturally distributed.
Conservation status and Initiatives

Natural stands of Pterocarpus marsupium are said to be 'fast disappearing' in the
natural habitat (Anis et al., 2005). The major threat to its natural population is low
germination percentage and cutting and felling for various purposes. The natural
regeneration takes place by means of seed, but the germination percentage is only
30% and even low (Kalimuthu and Lakshaman, 1995). Due to overexploitation of
70
the tree for its various useful application coupled with low germination and very
limited distribution, P. marsupium is banned for felling, transportation, and export
by Government of Nepal under the Forest Regulation of 1995 (amended in 2001). It
falls in ‘Near Threatened’ category of International Union for Conservation of Nature
(IUCN) Red List because of the threats present to the species, decreasing population
and declining areas of occurrence (Barstow, 2017).
There are, however, few conservation initiatives taken by the government. The
Bijaysal Conservation and Action Plan, Nepal 2018-2022 was prepared by Department
of Forests for its long-term conservation, increasing the population in the wild, and
to strengthen the multi-stakeholder participation. Few other initiatives taken for
conservation of Pterocarpus marsupium are: (i) in-situ and ex-situ conservation
were initiated in few western Tarai districts (e.g., in Kanchanpur, Kapilbastu);
(ii) a breeding orchard was established by Department of Forest Research and
Survey in Jogikuti, Butwal; (iii) seedlings raised in nursery in Dhangadi (operated by
Department of Plant Resources and Division Forest Office) and Kanchanpur (Division
Forest Office) including other CF forest nurseries; and (iv) resource assessment of
P. marsupium was conducted by District Forest Office in twelve community forests
of Kanchanpur district (DoF, 2018) and due to conservation initiative adopted by
CFs, the number of P. marsupium is increasing (personal communication). Raising
seedling and plantation have been initiated in other districts too.
A few private nurseries are also raising seedlings of P. marsupium (e.g., in Bardiya
district) but the objective taken by Bijaysal Conservation and Action Plan, Nepal
2018-2022 in increasing the wild population by 15%, is not fulfilled— showing the
need of more coordinated work among the government, communities and private
sectors.
Figure 1: Bijaysal breeding seed orchard at Krishnapur, Kanchanpur district. (Photos: a. Pashupati
Nath Koirala; and b. Pratikshya Chalise).
71
Trade
Owing to its multiple medicinal use specially to treat gastro-intestinal disorder and
diabetes, Pterocarpus marsupium has been used in traditional, ayurvedic for many
centuries (Abirami et al., 2012; RPRC, 2014). As a result, items made from P.
marsupium have high demand, resulting into higher market price. Given below is the
range of products made from wood of P. marsupium in 2020, along with their price.
Table 3. Products and prices of items made from Bijaysal in 2020.
S. No. Product Name Selling price (NPR)
1 Amkhora 700 – 800
2 Lota 800
3 Bowl 700
4 Cup 400 – 500
5 Glass 600 – 700
6 Chips/ Piece 50 – 100
Out of those products, cups have high market demand (Figure 2), followed by
glass. Amkhora, Lota and Bowls are made in less quantity but per unit price is high.
Moreover, small chips/pieces of wood also have market value. In Nepal, there are
only a handful number of persons who are engaged in manufacturing these items.
A direct consultation with two entrepreneurs disclosed that only 4- 6 persons are
skilful to manufacture items made from P. marsupium, but now because of the lower
availability of the woods from the community forests, only two registered enterprises
are in operation: one is operating since 2000 and second one from 2015, both are
located in Kanchanpur district. Their individual annual turnover ranged between NPR
4- 5 hundred thousand. Apart from these two, a very handful number of unregistered
enterprises operate even in a smaller scale. Their cumulative annual trade does exceed
NPR 0.5 million. Thus, it can be conservatively estimated that the annual trade of
items made from P. marsupium in and from Nepal is around NPR 1- 1.5 million.
Figure 2. Cups made from Bijaysal wood. These cups have high demand in Nepal and India. (Photos:
a. Dipesh Pyakurel; and b. Pashupati Nath Koirala).
72
Due to the rising demand of items made from Pterocarpus marsupium, cultivation
initiative in private and community forests, as mentioned earlier will ensure the
supply of the raw material in a long run. Once the supply is ensured, new micro-
entrepreneurs will start manufacturing and existing entrepreneurs can upscale their
production, thereby contributing to household and local economy.
Owing to the multiple health benefits of Pterocarpus marsupium, the demand will
rise and to fulfil the growing demand, entrepreneurs will collect P. marsupium
trees, one way or other. Thus, a cultivation initiative is felt necessary which serves
multiple purposes: (i) ensuring the sustainability of trees in nature, (ii) increasing
the population of P. marsupium in Nepal and (iii) ensuring the sustainable supply of
wood to entrepreneurs in a longer run. Private nurseries should be promoted, along
with the nurseries managed by the government. The successful practice of prohibiting
open grazing in community forests should be replicated in other CFs and national
forests to promote natural regeneration of Pterocarpus marsupium.
73
REFERENCES
Abirami, B., Gayathri, P., and Uma, D. 2012. In vitro antioxidant potential of
Pterocarpus marsupium bark. Int. J. Chem. Pharm. Sci. 3: 17-24.
Acharya, K.P., Regmi, R., and Acharya, B. 2002. Growth performance of Bijaya Sal
(Pterocarpus marsupium) in the Terai of Nepal. Banko Janakari. 12(1): 69-71.
Ahmad, A., and Anis, M. 2019. Meta-topolin improves in vitro morphogenesis,
rhizogenesis and biochemical analysis in Pterocarpus marsupium Roxb.: a
potential drug-yielding tree. Journal of Plant Growth Regulation. 38: 1007-1016.
Ahmad, R.Z., Shaheen, M.N.K., Afzal, J., Siddique, S.U., Qamar, I.A., and Ahmed,
J. 2015. Improvement of seed germination in some important multipurpose
leguminous trees of Islamabad Area: An experimental study. Basic Research
Journal of Agricultural Science and Review. 4(7): 217-224.
Ahmad, S.K., Dawah, H.A., and Khan, M.A. 2018. Ecology of termites. In: Khan
M., Ahmad W. (eds.) Termites and Sustainable Management. Sustainability in
Plant and Crop Protection. Springer.
Allen, O.N., and Allen, E.K. 1981. The leguminosae: A Source Book of Characteristics,
Uses and Nodulation. Macmuan Publishers Ltd, London and Basingstoke, UK.
Aluko, B.T., Oloyede, O.I. and Afolayan, A.J. 2012. Phytochemical and nutrient
compositions of the leaves of Ocimum canum Sim. African Journal of
Biotechnology. 11(63): 12697-12701.
Angelsen, A., Jagger, P., Babigumira, R., Belcher, B., Hogarth, N.J., Bauch, S.
and Wunder, S. 2014. Environmental income and rural livelihoods: a global-
comparative analysis. World Development. 64: 12-28.
Anis, M., Hussain, M.K., and Shahzad, A. 2005. In vitro plantlet regeneration of
Pterocarpus marsupium Roxb., an endangered leguminous tree. Current Science.
88: 861-863.
Ankalaiah, C., Mastan, T., and Reddy, M.S. 2017. A study on the density, population
structure and regeneration of Red Sanders Pterocarpus santalinus (Fabales:
Fabaceae) in a protected natural habitat- Sri Lankamalleswara Wildlife Sanctuary,
Andhra Pradesh, India. Journal of Threatened Taxa. 9: 10669-10674.
Anoop, E.V., Rao, R.V., and Mukundan, G. 2019. Wood anatomy and wood property
variation in Red Sanders. In: Pullaiah T., Balasubramanya, S., Anuradha, M. (Eds.)
Red Sanders: Silviculture and Conservation. Springer. Pp. 131-152.
Anuradha, M., Indu, B.K., and Balasubramanya, S. 2019. Propagation of Red Sanders:
An Overview. In: Pullaiah T., Balasubramanya, S., Anuradha, M. (Eds.) Red
Sanders: Silviculture and Conservation. Springer. Pp. 85-100.
Atanassova, M., Georgieva, S., and Ivancheva, K. 2011. Total phenolic and total
flavonoid contents, antioxidant capacity and biological contaminants in medicinal
herbs. Journal of the University of Chemical Technology and Metallurgy. 46(1):
81-88.
74
Azamthulla, M., Balasubramanian, R., and Kavimani S. 2015. A review on
Pterocarpus santalinus Linn. World Journal of Pharmaceutical Research. 4(2):
282-292.
Badkhane, Y., Yadav, A.S., Sharma, A.K., Raghuwanshi, D.K., Uikey, S.K., Mir,
F.A., Lone, S.A., and Murab, T. 2010. Pterocarpus marsupium Roxb.- Biological
activities and medicinal properties. International Journal of Advances in
Pharmaceutical Sciences. 1: 350-357.
Barmukh, R.B., and Nikam, T.D. 2008. Promotion of seed germination in Pterocarpus
marsupium Roxb. Indian J. Plant Physiol. 13(2): 143-150.
Barstow, M. 2017. Pterocarpus marsupium. The IUCN Red List of Threatened
Species 2017:e.T34620A67802995(http://dx.doi.org/10.2305/IUCN.UK.20173.
RLTS.T34620A67802995.en) (retrieved on various dates).
Basu, K. 1975. Indian Medicinal Plant. Jayed Press, Delhi: Dehradun. Second
edition. Pp. 828.
Bhat, V., Ballal, M., and Baliga, S. 2014. Evaluation of phytochemical and
antimicrobial properties of heart wood of Pterocarpus marsupium Roxb.
(Fabaceae). World Journal of Pharmaceutical Research. 3(6): 1454-1458.
Brandis, D. 1907. Indian Trees. Archibald Constable and Co., Ltd. London.
Burmmitt, R.K. 1992. Vascular Plant Families and Genera. Printed and Bound in
Great Britain by Whitstable Litho Ltd., Whitstable, Kent.
Chalk, L. 1989. Vessels. In: Metcalfe, C.R., and Chalk, L. Anatomy of Dicotyledons
Wood Structure and Conclusion of the General Introduction. Second edition. Vol.
2. Oxford Science Publications, Great Britain.
Chauhan, H.K., Bisht, A.K., Bhatt, I.D., Bhatt, A., Gallacher, D., and Santo,
A. 2018. Population change of Trillium govanianum (Melanthiaceae) amid
altered indigenous harvesting practices in the Indian Himalayas. Journal of
Ethnopharmacology. 213: 302-310.
Chauhan, L., and Rao, R.V. 2003. Wood Anatomy of Legumes of India: Their
Identification, Properties and Uses. Bishen Singh Mahendra Pal Singh. New
Delhi, India.
Clinical and Laboratory Standards Institute. 2012. Performance standards for
Antimicrobial Disk Susceptibility Tests: Approved standard. Eleventh edition
(Vol. 32, Issue 1).
Cruz-Garcia, G., Lagunez-Rivera, L., Chavez-Angeles, M.G., and Solano-Gomez,
R. 2015. The wild orchid trade in a Mexican local market: diversity and
economics. Economic Botany. 69(4): 291-305.
Das, T., and Chatterjee, A. 1993. In vitro studies of Pterocarpus marsupium – An
endangered tree. Indian J. Plant Physiol. 36: 269-272.
Dayanand, T., and Lohidas, T. 1988. Studies on development and maturity of Red
sanders (Pterocarpus santalinus Linn. f.). Indian Journal of Forestry. 11: 207-208.
Deepa, R., Manjunatha, H., Krishna, V., and Kumara Swamy, B.E. 2014. Evaluation
75
of antimicrobial activity and antioxidant activity by electrochemical method of
ethanolic extract of Pterocarpus marsupium Roxb. bark. Journal of Biotechnology
and Biomaterials. 4(1): 1-4.
Dewan A., Nanda K., and Gupta S.C. 1992. In vitro micropropagation of Acacia
nilotica subsp. indica Brenan via cotyledonary nodes. Plant Cell Rep. 12: 18-21.
DoF, 2018. Bijaysal Conservation Action Plan for Nepal (2018-2022). Department
of Forest, Ministry of Forests and Environment, Kathmandu, Nepal.
Dong, H.Q., Li, M., Zhu, F., Liu, F.L., and Huang, J.B. 2012. Inhibitory potential
of trilobatin from Lithocarpus polystachyus Rehd. against α-glucosidase and
α-amylase linked to type 2 diabetes. Food Chemistrym. 130: 261-266.
Doyle, J.J. 2001. Leguminosae. Academic Press. (doi: 10.1006/rwgn.2001.1642).
Duthie, J.F. 1915. Flora of the Upper Gangetic Plain and of the adjacent Siwalik
and Sub-Himalayan Tracts. 3: 1-2.
Endress, P.K. 1995. Diversity and Evolutionary Biology of Tropical Flowers.
Cambridge University Press, Cambridge.
Erdtman, G. 1952. Pollen Morphology and Plant Taxonomy: Angiosperms. (An
introduction to Palynology I). Almqvist and Wiksell. Stockholm.
Gadgil, W., Berkes, F., and Folke, C. 1993. Indigenous Knowledge for Biodiversity
Conservation. Ambio. 22(2/3): 151-156.
Gairola, S., Gupta, V., Singh, B., Maithani, M., and Bansal, P. 2010. Phytochemistry
and pharmacological activities of Pterocarpus marsupium: a review. International
Research Journal of Pharmacy. 1: 100-104.
Gamble, J.S. 1922. A Manual of Indian Timbers. Second Ed. Sampson Low, Marston
and India Ltd. London.
Gamble, J.S. 1972. A Manual of Indian Timbers: An Account of the Growth,
Distribution, and Uses of the Trees and Shrubs of India and Ceylon, with
Descriptions of Their Wood-structure. Bishen Singh Mahendra Pal Singh,
Dehradun. Pp. 261-262.
Gaoue, O.G., and Ticktin, T. 2008. Impacts of bark and foliage harvest on Khaya
senegalensis (Meliaceae) reproductive performance in Benin. Journal of Applied
Ecology. 45(1): 34-40.
Gayathri, M., and Kannabiran, K. 2021. Antimicrobial activity of Hemidesmus
indicus, Ficus bengalensis and Pterocarpus marsupium Roxb. Indian J. Pharm.
Sci. 71(5): 578-581.
Ghosh, B.G., Garai, S., Rahaman, S.M., Khatun, M., Mohammad, N., Mishra, Y.,
Ranjan, A., and Tiwari, S. 2021. Assessing potential habitat distribution range
of the endangered tree species Pterocarpus marsupium Roxb. under the climate
change scenario in India. Trees, Forests and People. 6: 100-124.
GoI, 2013. The Ayurvedic Pharmacopoeia of India. Part 1, Vol 1. Government of
India, Ministry of Health and Family Welfare, Department of Ayush.
GoN, 2012. Wood Identification Manual of Important Timbers of Nepal. Vol. 1.
76
National Herbarium and Plant Laboratories, Godawari, Lalitpur, Nepal.
Grover, J.K., Yadav, S., and Vats, V. 2002. Medicinal plants of India with anti-diabetic
potential. Journal of Ethnopharmacology. 81: 81-100.
Harborne, J.B. 1973. Phytochemical Methods. Chapman and Hall Ltd., London.
Hauser, E.J.P., and Morrison, J.H. 1964. The cytochemical reduction of nitroblue
tetrazolium as an index of pollen viability. Am. J. Bot. 51: 748-752.
Heneidak, S., Samai, A., and Shaheen, M. 2008. Characteristics of the proximal to
distal regions of the petioles to identify 15 tree species of papilinoideae- Fabaceae.
Bangladesh Journal of Plant Taxonomy. 14(2):101-115.
Holder, I.A., and Boyce, S.T. 1994. Agar well diffusion assay testing of bacterial
susceptibility to various antimicrobials in concentrations non-toxic for human
cells in culture. Burns. 20(5): 426-429.
Hooker, J.D. 1879. Flora of British India Vol. 1, 2 and 3. L. Reeve and Company,
London.
Hu, Q., Xu, J., Chen, S., and Yang, F. 2004. Antioxidant activity of extracts of black
sesame seed (Sesamum indicum L.) by supercritical carbon dioxide extraction.
Journal of Agricultural and Food Chemistry. 52(4): 943-947.
Hutchinson, J. 1964. The Genera of Flowering Plants (Angiospermae) Dicotyledons.
Vol. 1 and 2. Oxford, at the Clarendon press.
Idu, M., Olorunfemi, D.I., and Omonhinmin, A.C. 2000. Systematics value of stomata
in some Nigerian hardwood species of Fabaceae. Plant Biosystem. 134(1): 53-60.
Inside Wood. 2004-onwards. (https://insidewood.lib.ncsu.edu/description?4)
(retrieved on 02-03-2021).
Jha, P.K. 1999. Inventory report of in-situ Gene Conservation Area of Pterocarpus
marsupium located at Banshakti Chhetra Forest, Dhankauli VDC of Kapilbastu
district. HMG/DANIDA/NARMSAP, Regional seed centre, Hetauda.
Joshi, K.R., Devkota, H.P., and Yahara, S. 2012. Chemical analysis of heartwood
of Bijaysal (Pterocarpus marsupium Roxb.). Nepal Journal of Science and
Technology. 13(2): 219-224.
Jung, M., Park, M., Lee, H.C., Kang, Y.H., Kang, E.S., Kang, S.K., and Sang, K.K.
2006. Antidiabetic agents from medicinal plants. Curr. Med. Chem. 13: 1203-1218.
Kala, C.P., and Dubey, Y. 2012. Anthropogenic disturbances and status of forest
and wildlife in the dry deciduous forests of Chhattisgarh state in India. Journal
of Forestry Research. 23: 45-52.
Kalimuthu K., and Lakshaman K.K. 1995. Effect of different treatments on the pod
germination of Pterocarpus species. Indian J. Forestry. 18: 104-106.
Katiyar, D., Singh, V., and Ali, M. 2016. Phytochemical and pharmacological profile
of Pterocarpus marsupium: A review. The Pharma Innovation Journal. 5(4): 31-39.
Khanal, S., and Bhattarai, S. 2020. Status and ethnobotanical uses of Pterocarpus
marsupium Roxb. in Hariyali Community Forest, Kapilvastu. International
Journal of Ecology and Environmental Sciences. 46(2): 167-171.
77
Khare, C.P. 2007. Indian Medicinal Plants, An Illustrated Dictionary. Springer-Verlag
Berlin/Heidelberg, Science+Business Media, LLC.
Kirtikar, B. 1987. Indian Medicinal Plants. Second Edition Vol 1. New Delhi, Materia
Medica. Pp. 826-827.
Kumarasinghe, H.K.M.S., Subasinghe, S., Aruna Kumara, K.K.I.U. 2003. Seed
propagation of Rath Handun (Pterocarpus santalinus L.) as affected by method of
soaking and scarification. In: Proceedings of 9th Annual Forestry and Environment
Symposium. University of Sri Jaye-wardenepura, Sri Lanka. Pp. 57.
Kunwar, R.M., and Adhikari, N. 2005. Ethnomedicine of Dolpa district, Nepal: The
plants, their vernacular names and uses. Lyonia. 8(1): 43-49.
Lakshmi, S., Sreenatha, K.S., and Sujata, S.G. 1992. Plantlet production from shoot
tip cultures of red sandalwood (Pterocarpus santalinus). Curr. Sci. 62: 532-535.
Lavin, M., Pennington, R.T., Klitgaard, B.B., Sprent, J.I., Lima, H.C. and Gasson,
P.E. 2001. The dalbergioid legumes (Fabaceae): Delimition of a pantropical
monophyletic clade. American Journal of Botany. 88(3): 503-533.
Mankani, K.L., Krishna, V., Manjunatha, B.K., Vidya, S.M., Manohara, Y.N., Aneesh,
Raheman, A., and Avinash, K.R. 2005. Evaluation of hepato-protective activity of
stem bark of Pterocarpus marsupium Roxb. Indian J. Phamacol. 37(3): 165-168.
Manne, A.A., Viswanath, V.K., Kumar, A. G., Mangamuri, U., and Podha, S.
2020. Pterocarpus marsupium Roxb. heartwood extract synthesized chitosan
nanoparticles and its biomedical applications. Journal of Genetic Engineering
and Biotechnology. 18: 19.
Metcalfe, C.R., and Chalk, L. 1957. Anatomy of the Dicotyledons. Vol. 2, Clarendon
Press, Oxford.
Metcalfe, C.R., and Chalk, L. 1989. Anatomy of Dicotyledons. Wood structure and
Conclusion of the General Introduction. Second Edition. Vol. 2. Oxford Science
Publications, Great Britain.
MoFSC. 2002. Nepal Biodiversity Strategy. Ministry of Forests and Soil Conservation
(MoFSC), Government of Nepal. Kathmandu, Nepal.
Nag, A., and Gupta Joshi, H. 2020. Population structure and regeneration status of
selected tree species in eight tropical dry deciduous forests of West Bengal, India.
International Journal of Botany Studies. 5(6): 621-627.
Nathan, P., Law, E.J., Murphy, D.F., and MacMillan, B.G. 1978. A laboratory method
for selection of topical antimicrobial agents to treat infected burn wounds. Burns.
4(3): 177-187.
National Committee for Clinical Laboratory Standards. 1999. Methods for
Determining Bactericidal Activity of Antimicrobial Agents, Approved Guideline
(Vol. 19, Issue 18). National Committee for Clinical Laboratory Standards.
National Committee for Clinical Laboratory Standards. 2004. Method for Antifungal
Disk Diffusion Susceptibility Testing of Yeasts. In Nccls Ma44-a. (Vol. 24, Issue
15).
78
Pal, S., and Mondal, S. 2018. Floral biology, breeding system and pollination of
Pterocarpus marsupium Roxb. The International Journal of Plant Reproductive
Biology. 10(2): 172-177.
Panicker, K.T.C. 2004. Studies in the pollen morphology of mimosaeae monads,
tetrad and octad taxa. Journal of Palynology. 40: 9-21.
Pant, D.R., Pant, N.D., and Saru, D.B. 2017. Phytochemical screening and study
of antioxidant, antimicrobial, antidiabetic, anti-inflammatory and analgesic
activities of extracts from stem wood of Pterocarpus marsupium Roxb. J.
Intercult. Ethnopharmacol. 12(6/2): 170-176. (https://doi.org/10.5455/jice.
20170403094055).
Pant, P., and Yadav, R.K. 2013. Plant resource and utilization: A case study in
Kanchanpur district, Far-western Tarai, Nepal. Ecoprint. 20: 89-95.
Patil, U.H., and Gaikwad, D.K. 2011. Phytochemical screening and microbicidal
activity of stem bark of Pterocarpus marsupium. International Journal of Pharma
Sciences and Research. 2(1): 36-40.
Pearson, R.S. and Brown, H.P. 1932. Commercial Timbers of India: Their Distribution,
Supplies, Anatomical Structure, Physical and Mechanical Properties and Uses.
Vol. I. Government of India, Central Publication Branch, Calcutta. Pp. 391- 395.
Pinheiro, M., de Brito, V.L.G., and Sazima, M. 2018. Pollination biology of
melittophilous legume tree species in the Atlantic forest in Southeast Brazil. Acta.
Bot. Bras. 32(3): 410-425.
Pokharel, B., Wang, S.Y.S., Meyer, J., Marahatta, S., Nepal, B., Chikamoto, Y.,
and Gillies, R. 2019. The east-west division of changing precipitation in Nepal.
International Journal of Climatology. 40(7). (doi: 10.1002/joc.6401)
Polhill, R.M. 1971. Some observations on generic limits in Dalbergiea
elonchocarpineae Benth. (Leguminosae). Kew Bulletin. 25: 259-273.
Polhill, R.M. 1981. Dalbergieae. In: Advances in legume systematics, part 1. Royal
Botanic Gardens, Kew, Richmond, Surrey, UK. Pp. 233-242.
Polhill, R.M. 1994. Classification of the Leguminosae. In: Phytochemical dictionary
of the Leguminosae, Vol. 1. Chapman and Hall, London, UK. Pp. 35-48.
Prasad, M.N.V., Padmalatha, K., Jayaram, K., Raju, N.L., and Silva, J.A.T.
2007. Medicinal plants from Deccan ecoregion, India: Traditional knowledge,
ethnopharmacology, cultivation, utilization, conservation and biotechnology-
opportunities and impediments. Medicinal and Aromatic Plant Science and
Biotechnology. 1(2): 155-208.
Prenner, G. 2013. Part of a special issue on inflorescences papilionoid inflorescences
revisited (Leguminosae-Papilionoideae). Annals of Botany. 112: 1567-1576.
Pulliah, T. 2019(a). Reproductive Biology. In: Pullaiah, T., Balasubramanya, S.,
Anuradha, M. (Eds.) Red Sanders: Silviculture and Conservation. Springer. Pp.
77-84.
Pulliah, T. 2019(b). Taxonomy and Distribution. In: Pullaiah, T., Balasubramanya,
79
S., Anuradha, M. (Eds.) Red Sanders: Silviculture and Conservation. Springer.
Pp. 7-16.
Pulliah, T., and Reddy, V.D. 2019. Colouring Principle (Dye). In: Pullaiah, T.,
Balasubramanya, S., Anuradha, M. (Eds.) Red Sanders: Silviculture and
Conservation. Springer. Pp. 49-56.
Pyakurel, D., and Oli, B.R. 2014. Resource Assessment of Bijaysal (Pterocarpus
marsupium) in Selected Community Forests of Kanchanpur District. District
Forest Office, Kanchanpur, Nepal.
Pyakurel, D., Smith-Hall, C., Bhattarai-Sharma, I. and Ghimire, S.K. 2019. Trade
and conservation of Nepalese medicinal plants, fungi, and lichen. Economic
Botany. 73(4): 505-521.
Rahman, M.S., Mujahid, M.D., Siddiqui, M.A., Rahman, M.A., Arif, M., Eram, S.,
Khan, A., and Azeemuddin, A. 2018. Ethnobotanical uses, phytochemistry and
pharmacological activities of Pterocarpus marsupium. Pharmacognosy Journal.
10(6): 1-8.
Rajbhandari, K.R., Tamang R., Chhetri, R., Khatri, S., Joshi, M.D., and Rai, S.K.
2020. Flowering Plants of Province 1 (East Nepal). Ministry of Industry, Tourism,
Forest and Environment Province No. 1, Biratnagar, Nepal.
Rajbhandary, S. 2015. Trends in Taxonomy: Main Tools in Taxonomic revision.
In: Siwakoti M. and Rajbhandary, S. (Eds.) Taxonomic Tools and Flora Writing.
Department of Plant Resources, MoFSC and Central Department of Botany, TU,
Kathmandu, Nepal. Pp. 143-163.
Ramya, S., Kalayansundaram, M., Kalaivani, T., and Jayakumararaj, R. (2008).
Phytochemical screening and antibacterial activity of leaf extracts of Pterocarpus
marsupium Roxb. (Fabaceae ). Ethnobotanical Leaflets. 12: 1029-1034.
Rao, K.B.S., and Juneja, R.K. 1971. A Handbook for Field Identification of Fifty
Important Timbers of India. Delhi, India.
Rao, S.P., Atluri, J.B., and Reddi, C.S. 2001. Intermittent mass blooming, midnight
anthesis and rockbee pollination in Pterocarpus santalinus (Fabaceae). Nord. J.
Bot. 21: 271-276.
Rao, S.P., Raju, A.J.S. 2002. Pollination ecology of the Red sanders Pterocarpus
santalinus (Fabaceae), an endemic and endangered tree species. Curr. Sci. 83:
1144-1148.
Reddy, K.N., Pattanaik, C., Reddy, C.S., Murthy, E.N., and Raju, V.S. 2008. Plants
used in traditional handicrafts in north eastern Andhra Pradesh. Indian Journal
of Indian Knowledge. 7(1): 162-165.
Roskov, Y., Ower, G., Orrell, T., Nicolson, D., Bailly, N., Kirk, P.M., Bourgoin,
T., DeWalt, R.E., Decock, W., Nieukerken, E. van, Zarucchi, J., and Penev, L.
Species 2000 and ITIS Catalogue of Life, 2020 Annual Checklist. (https://www.
catalogueoflife.org/ data/taxon/74N7, retrived on various dates).
Roxburgh, W. 1799. Plants of the Coast of Coromandel: selected from drawings and
80
descriptions Presented to the Hon. Court of the East India Company. Vol. 2. W.
Bulmer and Company, Shakespeare Printing Office, London.
RPRC (Regional Plant Resource Centre). 2014. Bijaysal (Pterocarpus marsupium
Roxb.)- Indian Kino Tree. (http://www.rprcbbsr.in/View/Downloads/news/
Pterocarpus%20 marsupium 17.12.14.pdf) (retrieved on 8th April 2021).
Seema, G., Vikas, G., Baljinder, S., Mukesh, M., and Parveen, B. 2010. Phytochemistry
and pharmacological activities of Ptercarpus marsupium- a review. International
Research Journal of Pharmacy. 1(1): 100-104.
Sharma, V.D., and Gautam, D. 2017. Engaging Community in Medicinal and Aromatic
Plant Conservation: An Experience from Patana Forest of Kapilvastu District.
SAGUN and United Nations Development Programme, Kathmandu, Nepal.
Silva, J.A.T., Kher, M.M., Soner, D., and Nataraj, M. 2019. Red sandalwood
(Pterocarpus santalinus L. f.): biology, importance, propagation and
micropropagation. J. For. Res. 30(3): 745–754.
Singh, J. 2008. Maceration, percolation and infusion techniques for the extraction
of medicinal and aromatic plants. In: Handa, S.S,. Singh Khanuja, S.P., Longo,
G. and Rakesh, D.D. (Eds.) Extraction Technologies for Medicinal and Aromatic
Plants. United Nations Industrial Development Organization and the International
Centre for Science and High Technology. Pp. 67-82.
Singleton, V. L., Orthofer, R., and Lamuela-Raventós, R.M. 1999. Analysis of total
phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu
reagent. Methods in Enzymology. Pp. 152-178.
Sinjushin, A.A. 2019. Notes on floral symmetry in the Pterocarpus clade
(Leguminosae: Papilionoideae: Dalbergieae). Wulfenia. 26: 1-30.
Sofowara, A. 1993. Medicinal Plants and Traditional Medicine in Africa. Spectrum
Books Ltd., Ibadan, Nigeria.
Sukhadiya, M., Dholariya, C., Behera, L.K., Mehta, A.A., Huse, S.A., and Gunaga,
R.P. 2019. Indian kino tree (Pterocarpus marsupium Roxb.): Biography of
excellent timber tree species. Mfp News. 29: 1-8.
Sundarapandian, S., and Swamy, S.P. 2013. Short-term population dynamics of
tree species in tropical forests at Kodayar in the Western Ghats of Tamil Nadu,
India. Proceedings of the International Academy of Ecology and Environmental
Sciences. 3(3): 191-207.
Tandon, V., Bhattarai, N.K., and Karki, M. 2001. Conservation Assessment and
Management Prioritization (CAMP) Report. International Development Research
Centre (IDRC), Canada and Ministry of Forest and Soil Conservation, Kathmandu,
Nepal.
Tiwari, G.P.K., Tadele, K., Aramde, F., and Tiwari, S.C. 2010. Community structure
and regeneration potential of Shorea robusta forest in subtropical submontane
zone of Garhwal Himalaya, India. Nature and Science. 8: 70-74.
Tiwari, S., Shah, P., and Singh, K. 2004. In vitro propagation of Pterocarpus
81
marsupium Roxb.: An endangered medicinal tree. Indian Journal of Biotechnology.
3: 422-425.
Trease, G.E., and Evans W.C. 1989. Pharmacognsy. 11th ed, Brailliar Tiridel Can.
Macmilliam publishers. Pp. 35-38.
Troup, R.S. 1921. The Silviculture of Indian Trees. Vol. 1, Clarendon Press, Oxford
University, UK. Pp. 336.
Troup, R.S. 1986. Indian Woods and their Uses. Soni Reprints Agency, Delhi, India.
Pp. 4- 222.
Van Daalen, J.C. 1991. Germination of Pterocarpus angolensis seed. Suid-Afrikaanse
Bosboutydskrif. 158: 33-36.
Vats, V., Yadav, S., Biswas, N., and Grover, J. 2004. Anti-cataract activity of
Pterocarpus marsupium bark and Trigonella foenum- graecum seeds extract in
alloxan diabetic rats. Journal of Ethnopharmacology. 93(2-3): 289-294.
Venkataramaiah, V., Prasad, S.V., Rajeswara, R.G., and Swamy, P.M. 1980. Level
of phenolic acids in Pterocarpus santalinus L. Indian J. Exp. Biol. 18: 887-889.
Vikaspedia, 2021. (https://vikaspedia.in/agriculture/crop-production/package-of-
practices/ medicinal-and-aromatic-plants/pterocarpus-marsupium) (retrieved on
various dates).
Wilkins, C.P. 1991. Conservation of tree crops. In: In vitro methods for conservation
of plant genetic resources. Dodd, J. H. (ed.), London: Chapman and Hall. Pp.
151-237.
Wu, Z., and Raven, P. H. 2010. Flora of China, Fabaceae. Volume 10. Missouri
Botanical Garden Press, St. Louis, USA.
Xu, C., Zeng, J., Cui, T., Chen, Q., and Ma, Y. 2016. Introduction, growth performance
and ecological adaptability of Hongmu tree species (Pterocarpus spp.) in
China. Journal of Tropical Forest Science. 28: 260-267.
Xu, Z., and Deng, M. 2017. Fabaceae or Leguminosae. In: Identification and Control
of Common Weeds: Vol. 2. Springer, Dordrecht. (https://doi.org/10.1007/978-94-
024-1157-7_43).
http://www.plantsoftheworldonline.org/taxon/urn:lsid:ipni.org:names:331884-2
(retrieved on various dates).
http://www.theplantlist.org/tpl1.1/search?q=pterocarpus (retrieved on various dates).
http://www.worldfloraonline.org/taxon/wfo-0000185089 (retrieved on various dates).
https://www.efloras.org(retrieved on various dates).
82
APPENDICES
Appendix 1. List of Herbarium specimens examined.
Western Nepal:
Baghphanta, Kanchanpur district, Western Nepal, 140m; 1981-10-02 AD; Kattel,

L.P., 747; (KATH016545).
Dudhiya Khanta, Kanchanpur district, Western Nepal, 160m; 1980-11-23 AD; Kattel,
L.P. and Malla, K.J., 50; (KATH015263).
Gaugi, Mahendranagar, Kanchanpur district, Western Nepal, 300m; 1999-10-22 AD;

Bhatta, G.D. and Kurmi, P.P., 1006; (KATH070856).
Kanchanpur district, Western Nepal; 2036- Poush; Bhattacharya., (KATH070860).
Daizi, Kanchanpur district, Western Nepal, 160m; 2049-7-27 BS; Joshi, C.M. and
Rijal, H.L., 470/49; (KATH070876).
Malakheti, Kailali district, Western Nepal; 2077-7-15 BS; Chalise, P., Paneru, Y.R.
and Bhatta, P., K0020.
Dewaria Botanical Garden, Kailali district, Western Nepal; 2077-7-13 BS; Chalise,
P. and Paneru, Y.R., K0014- Planted tree.
Dhakeri Botanical Garden, Banke district, Western Nepal; 2021-01-05 AD; Bhatt,
G.D. and Acharya, Y. 77906- Planted tree; (KATH086318).
Dhakeri Botanical Garden, Banke district, Western Nepal; 2021-01-05 AD; Bhatt,
G.D. and Acharya, Y. 77907- Planted tree; (KATH086322).
Samaiji Community Forest, Krishnapur-4, Kanchanpur district, Western Nepal, 207m;

2020-12-26; Bhatt, G.D. and Karkee, D., 77819; (KATH086302).



83
Central Nepal:
Bulakiya, Kapilbastu district, Central Nepal, 200m; 1996-3-1 AD; Kurmi, P.P.,
10084; (KATH070868).
Butwal, Rupandehi district, Central Nepal, 200m; 2062-9-3 BS; Adhikari, M.K.,
Joshi, L., Manandhar, V. and Kurmi, P.P., 17; (KATH070861).
Champapur, Kapilbastu district, Central Nepal, 200m; 1992-11-3 AD; Kurmi, P.P.,
KB451; (KATH070862).
Near Banaganga Rangepost, Kapilbastu district, Central Nepal, 180m; 1998-4-2 AD;
Kurmi, P.P. and Bhatta, G.D., 1003; (KATH070871).
Charpala CF, Tamnagar, Rupandehi district, Central Nepal, 2076-7-8 BS; Chalise,
P., Paneru, Y.R. and Chalise, G.S., 76K032.
Eastern Nepal:
Bhadrapur, Jhapa district, Eastern Nepal, 100m; 2003-1-26 AD; Thapa, N., Bhatta,
G.D. and Khatri, S., 2070; (KATH070878)- Planted tree.
84
Appendix 2. Some photographs collected during field observation, data collection,
locality tracing, and sample collection of Bijaysal.
85
Appendix 3. Authors/ Contributors and their affiliation.
S. No. Name Affiliation
1. Devi Prasad Bhandari Senior Scientific Officer, Natural Products Research

Laboratory, Thapathali, Kathmandu
2. Dr. Dipesh Pyakurel Researcher, Resources Himalaya Foundation,
Sanepa, Kathmandu
3. Lajmina Joshi Senior Plant Taxonomist and Anatomist
4. Mohan Dev Joshi Secretory,

a Ministry of Industry, Tourism, Forests
and Environment, Karnali Province, Surkhet
5. Neelam Pandey MSc. Graduate, Central Department of Botany,
Tribhuvan University, Kirtipur, Kathmandu
6. Parasmani Yadav Assistant Scientific Officer, Natural Products
Research Laboratory, Thapathali, Kathmandu
7. Pashupati Nath Koirala Ministry of Forests and Environment, Singadurbar,
Kathmandu
8. Pramesh Bahadur Lakhe Assistant Scientific Officer, Department of Plant
Resources, Thapathali, Kathmandu
9. Pratikshya Chalise Research Officer, National Herbarium and Plant
Laboratories, Godawari, Lalitpur
10. Ram Krishna Bhandari Garden Officer, Dhakeri Botanical Garden,
Nepalgunj, Banke
11. Sachita Joshi Senior Scientific Officer, Department of Plant
Resources, Thapathali, Kathmandu
12. Subhash Khatri Senior Research Officer, National Herbarium and
Plant Laboratories, Godawari, Lalitpur
13. Prof. Dr. Suresh Kumar Ghimire Central Department of Botany, Tribhuvan
University, Kirtipur, Kathmandu
14. Yagya Raj Paneru Lecturer/ Capital College and Research Centre,
Koteshwor, Kathmandu
(MSc. Graduate, Central Department of Botany,
Tribhuvan University, Kirtipur, Kathmandu)
15. Sajita Dhakal Research Officer, National Herbarium and Plant
Laboratories, Godawari, Lalitpur
86
iv
xii

Vijayasal Book Eng

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Vijayasal Book Eng

Uploaded by

Copyright:

Available Formats

Bijaysal

A Monograph of Pterocarpus marsupium in Nepal

Editors: Lajmina Joshi

Technical Coordinator: Pratikshya Chalise

Citation: Second name of author, Abbreviation of first name. Name of

Cover design: Sangeeta Rajbhandary

Cover page: Mature tree (Ganga Datta Bhatt),

Printed at: Bonefied Printing Press, Nayabazar, Kathmandu, Nepal

Dr. Pem Narayan Kandel

Pterocarpus marsupium commonly known as “Bijaysal” in Nepal is a deciduous tree

ANOVA Analysis of Variance

ACRONYMS AND ABBREVIATIONS................................................................ vi

CHAPTER-3 REPRODUCTIVE BIOLOGY......................................................12

CHAPTER-6 ECOLOGY AND POPULATION STATUS: A CASE STUDY

CHAPTER-7 PRETREATMENT METHODS FOR SEED GERMINATION...36

CHAPTER-8 ECONOMIC BOTANY AND ETHNOBOTANY........................45

CHAPTER-9 PHYTOCHEMISTRY, ANTIOXIDANT, ANTIDIABETIC

CHAPTER-10 ANTIMICROBIAL ACTIVITY...................................................60

CHAPTER-11 THREATS, CONSERVATION AND TRADE..............................69

Scientific and Vernacular names

Taxonomy and Systematic position

Holotype of Pterocarpus marsupium Roxb.

Reproductive biology broadly incorporates floral biology, phenology, pollination

Materials and Methods

Pollen Viability (%) = No. of stained pollen grains X 100

Inflorescence structure and flower opening

Flower morphology and structure

Anthesis and Palynological observation

Figure 2: Palynological observation in Pterocarpus marsupium under Olympus E 329153

Several studies have been done on phytochemistry, pharmacognosy of Pterocarpus

Materials and Methods

Pterocarpus marsupium Roxb. (Bijaysal), is an important tree species occurring

Materials and Methods

Potential distribution in Nepal

Factors governing Current distribution

Figure 4: Variation in precipitation trends in different ecological regions of Nepal.

Pterocarpus marsupium Roxb. is a deciduous tropical tree species in the family

Figure 2: Winged pod of Pterocarpus marsupium (Photos: Neelam Pandey).

Materials and Methods

Habitat preferences and other Biotic interactions

Conclusions and Recommendations

Materials and Methods

Seed germination experiment

Factors affecting Seed germination

Pterocarpus marsupium is a versatile plant with a broad spectrum of uses (Troup,

Materials and Methods

Use Profile of Pterocarpus marsupium

Conclusion and Recommendations

Pterocarpus marsupium is an important therapeutic and medicinal plant (Rahman

Materials and Methods

Preparation of Plant extracts

Qualitative Phytochemical Analysis

Determination of Total Phenolic Content

Determination of Total Flavonoid Content (TFC)

Determination of Antioxidant activity

% Inhibition = Control (A) - Sample (A) x 100

% Inhibition = Control (A) - Sample (A) x 100

Where, A is the absorbance of the sample and control.

Results: Phytochemistry, Antioxidant, Antidiabetic activities and Toxicity

S.No. Parameters Phytochemical Test Leaf Bark

Volatile oils Spot test/Residue test - -

Antioxidant Antidiabetic Oral toxicity

2. P. marsupium (bark) 596.7 4.61 43.71872 98.32 >2000

Materials and Methods