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Micrococca Mercurialis Benth– Pharmacognostic Analysis and Antimicrobial Activity of an Important Folk Medicinal Plant Sutapa Choudhury Chowdhury Habibur Rahaman Sudhendu Mandal* Department of Botany,School of Life Science, Visva Bharati, Sanitiniketan-731235, India. Email: smandalbot@ gmail.com Abstract The drug evaluation and bioassay of traditional herbs of various herbal systems is now getting more momentum throughout the world. It is the high time now to evaluate scientifically the information stored in different herbal medicine systems of the world in terms of their pharmacognostic, phytochemical and pharmacological characterization.The evaluation of quality and purity of crude drugs by means of various parameters is the most important aspects of pharmacognosy. The present study deals with different pharmacognostic parameters of Micrococca mercurialis, an ethnomedicinally important plant of the family Euphorbiaceae. The leaves are traditionally used by the tribal people to cure old sores, rheumatic pain, constipation, etc. The parameters like micromorphological, organoleptic evaluation, physical and chemical evaluation, antimicrobial activity studies, etc. of powder drugs have been considered for the pharmacognostical evaluation of the plant. The plant bears amphistomatic leaves and stomata are paracytic type with occurrence of few anisocytic and anomocytic types. Palisade ratio is 8 and stomatal index is 13 and 17 on the upper and lower surfaces respectively. Dried leaf powder is dark olive in colour and gives characteristic colouration under UV light. Methanolic extract of the leaf indicates the presence of alkaloids, flavonoids, reducing sugars, gums, tannins and saponins, etc. Ash value and moisture content of the leaves are 28.48% and 72.59% respectively. This plant seems to be very potent against different human pathogenic bacteria as the leaf extracts in different solvents (methanol, water, ethyl acetate extract) gave characteristic inhibition zone (Bacillus subtilis ATCC 11778– 16mm. in methanolic extract; Escherichia coli NCTC 10418– 11mm. in water extract; Enterococcus faecalis ATCC 19433– 40mm. in ethyl acetate extract, etc.). This study will throw some scientific knowledge to herbalists and pharmacologists for proper evaluation and validation of folk drug. Background Medicinal plant research has now got a momentum among the scientists of the world. The scientific evaluation of ethnomedicinally important plants is now being done thoroughly covering various aspects of their study like efficacy of the crude drugs, chemistry of active principles, different pharmacognostic parameters, etc. Chemical analyses and biological assay of medicinal plants are the important factors for identification of novel bioactive phytochemicals and drug discovery. Use of micromorphology and anatomy is now a recognised tool in the field of plant systematics. Importance of epidermal characters in general and those of trichomes in particular are widely recognised in taxonomic consideration of angiosperms [1, 2, 3, 4]; Ontogeny and structure of stomata are now also considered as an important taxonomic character for many of the angiospermic taxa [5, 6, 7, 8, 9, 10]. The members of different genera and families of angiosperm have been studied anatomically by various workers with special emphasis on leaf epidermal micromorphology [11, 12, 13, 14, 15]. Only to some extent, the ontogeny, structure of stomata and phytochemical studies of different members of Euphorbiaceae have been studied by different workers [16]. But the detailed foliar epidermal characters including trichomes, stomata, chemical analysis, physical evaluation, antimicrobial activity, etc. of many members of the family Euphobiaceae have not yet been studied. Chemical analysis and biological assays are very important aspects in pharmacognostic evaluation of medicinal plants [17].
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Materials And Methods Plant material
Micrococca mercurialis Benth. (Family: Euphorbiaceae) Common name: Badam, Desei badam. Botanical Characteristics: Erect, 2.5-5.0 x 1.25-2.5 cm, ovate to ovate- lanceolate, rounded or acute at base, glabrous. Flowers few, distant, in slender racemes longer than the leaves; female flowers usually solitary, with several males. Sepals glabrous. Capsules sub-globose, of three hairy cocci. Seeds globose deeply ovulate, palebrown. Flowering and fruiting time: August to October. Distribution: In India and Sri Lanka; also in Burma, Arabia and Tropical Africa.. Habit and habitat: Annual, erect herb. Terrestrial, wild, common, grows mainly along the shady wall sides; in waste places and along railway tracks. Useful part: Leaf. Medicinal use: Purgative. Chemical constituents: Information not available. Methods For the study of foliar epidermis, leaf samples were cleared by Bokhari’s method (1970) [5]. The cleared leaf samples were then mounted on the slide with a drop of 10% glycerine and 1% aqueous safranin solution and observed under compound light microscope. The drawings of the leaf epidermal micromorphological characters were made with the camera lucida and measurements were taken with standardized ocular micrometer in each cases. Finally, the leaf powders were extracted (soxhlet extraction) with 90% methanol and these extracts were used for different chemical colour reaction tests for identification of different phytochemical groups. Screening of antimicrobial activity was carried out by agar diffusion method. Nutrient agar medium was prepared by suspending nutrient agar (Merk) 20g/L in distilled water. The pH value of the media was adjusted to 7.0, autoclaved, and allowed to cool up to 45OC. 50 µl of extract was poured in 6mm wells punched in test culture seeded assay plates. 24 h old bacteria cultures in nutrient broth were used to seed the assay plates containing NAM. Assay plates seeded with bacterial cultures were incubated at 37° C, for 24 hours. After incubation antimicrobial activity was determined by measuring the zone of inhibition. The selected bacteria eight common human pathogenic bacterial strains are Bacillus subtilis ATCC 11778, Enterococcus faecalis ATCC 19433 , Escherichia coli NCTC 10418, Klebsiella aerogenes W70, Micrococcus luteus NCTC2665, Pseudomonas aeruginosa NCTC10662, Salmonella typhi ATCC 19430 and Streptococcus faecalis MTCC 439. Results Macromorphology i) Leaf: Herbaceous, 2.5-5.0 x 1.25-2.5 cm, ovate to ovate-lanceolate, rounded or acute at base, glabrous. ii) Stem: Erect, branched, glabrous, hairy. Micromorphology General description and measurement of the epidermal cells, stomata, trichomes and crystals of the investigated plant are represented in Tables 1, 2, 3 and 4. 1. Epidermis: Epidermal cell walls are irregular in shape and the out lines are wavy on both the surfaces. Epidermal cell size is 26.64 µm x 53.28 µm on the upper surface and 30.64 µm x 96.57 µm on the lower surface. Frequency is 903.08 /mm2 on the upper surface and 814.97 /mm2 on the lower surface. Palisade ratio is 08.23 (Table-1; Fig. I- A, B, 123
Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.4, No.27, 2014
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C, D). 2. Stomatal Complex: Leaves are amphistomatic i.e. stomata are present on both the surfaces. On the upper surface paracytic stomata are predominant with few anomocytic types. On the lower surface mainly paracytic stomata are mixed with few anisocytic and anomocytic types. Size of the stomata is 24.97 µm x 16.65 µm on the upper surface and 28.30 µm x 19.98 µm on the lower surface. Frequency of the stomata is 132.16 /mm2 and 210.61 /mm2 on the upper and lower surfaces respectively. Stomatal index is 17.39 (Table-2; Fig. I- A, B, C, D). 3. Trichomes: Nonglandular, unicellular, straight trichomes with pointed apex are present on both the epidermal layers. Size is 266.40 µm x 10.82 µm and frequency is 11.01 /mm2. Trichome index is 01.71 (Table-3; Fig. I- E, F, G, H, I). 4. Crystals: Small spheroidal Ca-oxalate crystals are found on both the epidermal layers (Table-4). Organoleptic Features of the Crude Drug Colour: Dark olive; Odour: No specific odour; Taste: No specific taste; Texture: Herbaceous, glabrous (in fresh form). Microchemical Evaluation of the Powdered Drug Through the phytochemical tests of the methanolic extract of leaf, the detected phytochemical groups are alkaloids, flavonoids, reducing sugars, gums, tannins and saponins, etc. (Table 5). Physical Evaluation [I] PHYSICAL CONSTANT i) Ash Value: a) Total ash - 28.48% b) Water soluble ash- 09.23 c) Acid insoluble ash- 11.56% ii) Moisture Content- 72.59 % (in fresh form). [II] FLUORESCENCE ANALYSIS Here in this study it is observed that drug powder treated with different chemical reagents gives characteristic colourations when seen under UV light and it is compared with the colourations observed under ordinary light. In some cases there are marked differences in colour (Table 6). Antimicrobial Activity Methanolic and ethyl acetate soluble foliar extracts of Micrococca mercurialis exhibited extreme potency against the selected bacteria. All the selected eight bacteria were successfully inhibited by the methanolic and ethyl acetate soluble foliar extracts. Water soluble extract was inhibitory against Escherichia coli only. Methanolic extract exhibited maximum inhibitory activity against Bacillus subtilis ATCC 11778 (inhibition zone, 16 mm). Water extract showed 11 mm of inhibition zone against Escherichia coli NCTC 10418. Ethyl acetate extract exhibited maximum inhibitory effect to Enterococcus faecalis ATCC 19433 (inhibition zone, 40 mm). Tables Table1: EPIDERMAL CELL CHARACTERS OF THE INVESTIGATED PLANT * Plant Leaf Cell Cell Cell Cell Cell Palisade Surface Shape Length Width Frequency Wall Ratio (µm) (µm) 2 Outline /mm Upper Irregular 26.64 53.28 903.08 Wavy 8 Micrococca mercurialis Lower Irregular 30.64 96.57 814.97 Wavy
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Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.4, No.27, 2014
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Table2: STOMATAL FEATURES OF THE INVESTIGATED PLANT* Leaf Stomatal Type Stomatal Stomatal Width Stomatal Surface Length (µm) Index (µm) (%) Upper Mainly paracytic; 24.97 16.65 few anomocytic
Plant
Micrococca mercurialis Lower
Plant
Micrococca mercurialis
Mainly paracytic; few anomocytic and anisocytic
28.30
19.98
17
210.61
Table 3: TRICHOME FEATURES OF THE INVESTIGATED PLANT * Leaf Types Trichome Trichome Trichome Surface Length Width Frequency (µm) (µm) /mm2 Nonglandular, 266.40 10.82 11.01 Upper unicellular, straight Lower Nonglandular, 266.40 10.82 11.01 unicellular, straight
Plant Micrococca mercurialis
Stomatal Frequency /mm2 132.16
Trichome Index %
1
Table- 4: CRYSTAL FEATURES OF THE INVESTIGATED PLANT * Surface Types Dissolved in Upper Small spheroidal HCl Lower
Small spheroidal
HCl
* Data presented in the tables are averages of 20 observations Table 5: MICROCHEMICAL TESTS OF LEAF AND STEM EXTRACTS OF THE INVESTIGATED PLANT Tests/ Reagents Tests For Nature of Changes Degree of Changes Dragendroff’s reagent Alkaloids Orange brown ppt + Wagner’s reagent Alkaloids _ _ Shinoda’s tests Flavonoids Magenta colour + 10% NaOH Flavonoids Magenta colour + Steroids and Salkowski test _ _ triterpenoids Benedict’s reagent Reducing sugars Brick red ppt +++ Fehling’s reagent Reducing sugars Brick red ppt +++ Molish’s test Gums Red-violet ring + 10% aqueous potassium Tannins Yellowish-brown ppt _ dichromate solution 10% aqueous lead acetate Tannins Yellow ppt ++ solution 5% aqueous ferric chloride Tannins Greenish-black colour +++ solution 1% lead acetate Saponins White ppt +++ Borntrager’s test Anthraquinones _ _ + = Present; - = Absent
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Table 6: UV FLUORESCENCE NATURE OF THE INVESTIGATED PLANT Materials and treatment
In fluorescence light
In ordinary light
Powder as such Treated with dilute nitric acid Treated with sodium hydroxide in water Treated with hydrochloric acid
Black green Orange Light yellowish pink
Dark olive Orange Light reddish brown
Brilliant yellow green
Yellowish green
Treated with dilute sulphuric acid Treated with antimony trichloride
Very pale blue Greyish purplish blue
Blackish red Yellowish brown
Table 7: ZONE OF INHIBITION (mm) FOR Micrococca mercurialis LEAF EXTRACTS AGAINST SOME HUMAN PATHOGENIC BACTERIA *
METHANOLIC EXTRACT
INHIBITION ZONE (mm) WATER ETHYL ACETATE EXTRACT EXTRACT
SELECTED BACTEIA
Bacillus subtilis ATCC 11778 Enterococcus faecalis ATCC 19433 Escherichia coli NCTC 10418 Klebsiella aerogenes W70 Micrococcus luteus NCTC2665 Pseudomonas aeruginosa NCTC10662 Salmonella typhi ATCC 19430 Streptococcus faecalis MTCC 439 * = Values are mean of three replicates - = No inhibition zone ± = Standard error
72 Hrs
72 Hrs
72 Hrs
16±0.05 12±0.03 14±0.02 11±0.02 10±0.023 14±0.03
9±0.01 -
33±0.04 40±0.02 17±0.03 15±0.03 35±0.03 36±0.04
15±0.02
-
34±0.02
13±0.032
-
30±0.03
DISCUSSION Pharmacognosy implies a particular knowledge of methods of identification and evaluation of crude drugs obtained from plants which include macromorphology, phytochemical and pharmacological studies. The major problem of the commercial supply of crude drugs is identification of genuine drug. Crude drugs may easily be adulterated or substituted by or confused for other ones because neither it has any trade name printed on it nor it carries any identifying structure for the easy identification of it by the plant taxonomists, rather drug samples supplied are shrunken, rolled, twisted, deformed and discoloured. So, pharmacognostic evaluation of crude drugs with macromorphology, micromorphology, microchemical colour reaction tests, organoleptic tests, ash value UV fluorescence study, etc will help in identifying genuine drugs and thus in checking adulteration, because the tests are very specific for a particular drug. Leaf micromorphology of this investigated plant show the general features of Micrococca mercurialis which conform to the characters reported in earlier works [17, 18]. Importance of epidermal characters in general is widely recognized in taxonomic considerations of angiosperm [19, 20, 21] and in many cases they have successfully used in identification of taxa at genus as well as species levels. Leaves are amphistomatic i.e. stomata are present on both the surfaces. On the upper surface paracytic stomata are predominant with few anomocytic types. Studies in stomata can have a great taxonomic as well as pharmacognostic value in proper identification of different plant taxa including medicinal plants [22, 23]. Size of the stomata is 24.97 µm x 16.65 µm on the upper surface and 28.30 µm x 19.98 µm on the lower surface. Trichome features are also very 126
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important in proper identification of the plants and considered as one of the valuable taxonomic marker now. Here nonglandular type of trichomes is found. Small spheroidal crystals are present. In case of crude drug evaluation, ash value plays a very important role which includes % of total ash, water soluble ash and acid insoluble ash [24]. Ash value gives a maker character for identification of crude drugs obtained from the investigated taxa. Here ash value of Micrococca mercurialis is 28.48% which is very distinct and will be successfully used in evaluation of drug quality of the plant. Similarly fluorescence characters of the crude drugs are considered very important marker in making distinction among the drugs. Here some fluorescence features have been identified which are very much distinctive in identifying the respective drug obtained from this plant. Resistance to antimicrobial agents is emerging in a wide variety of microorganisms and multiple drug resistant organisms pose serious threat to the treatment of infectious diseases [25]. Keeping this problem in mind various workers are actively involved in bioprospection of phytochemicals as potent antimicrobials from the ethnomedicinal plants with the help of ethnobotanical leads [26, 27, 28, 29, 30, 31]. Kirby-Bauer (1966) elaborately showed the methods of detecting antimicrobial activity of certain specimens[32]. Recently floral and foliar extracts are screened by different workers against several pathogenic and nonpathogenic microorganisms [33]. In this context, antimicrobial activity of the selected plant has been done. The leaf extracts in different solvents (methanol, water, ethyl acetate extract) gave characteristic inhibition zone. [V] CONCLUSION The results obtained from this investigation are very authentic and can be used as an effective tool in proper identification of the crude drugs produced from this plant. It will also be very useful in quality control of the genuine drugs plant and for detection of adulterants. The information generated in this study will enrich the data base of Indian Pharmacopoeia by incorporating the pharmacognostic information of these selected ethnomedicinal plants. The information regarding the ethnomedicinal uses of these respective plants will also be included in the Indian national ethnomedicinal inventory. Such findings will have a great value not only to the botanists, ethnobilogists, chemists but also to the rural poor people of our country fot their economic development if these plants are cultivated commercially. Finally it will highlight some promising areas for further investigations to the researchers in the fields of ethnobotany, phytochemistry, pharmacology, pharmaceutical science and molecular biology. FINANCIAL DISCLOSURE We are very thankful to the University Grant Commission, Govt. of India for financial assistance. ACKNOWLEDGEMENT We are very grateful to the Head of the Botany Department, Visva-Bharati and Subhashree Biotech, Kolkata for providing the necessary laboratory facilities. REFERENCES [1] Banerjee, A.; Rahaman, C. H.; Kar, R.K. and Mandal, S. [2002] Micromorphology of foliar epidermis of some tropical tree legumes. Phytomorphology 52(2 & 3): 223-230. [4] Mukherjee, K.K. , Roy, M. Saha, P.K. , Ganguly, S.N. [2000] Surface morphology of tea (Camellia sinensis L.) leaves. Phytomorphology 50: 125-131. [3] Ogundipe, O.T. and Olatunji, O.A. [1991] The leaf anatomy of the species of Cochlospermum Kunth (Cochlospermaceae) in West Africa. Feddes Repertorium 102: 183-187. [4] Parveen, N.S.; Murthy, K.S.R. and Pullaiah, T. [2000] Leaf epidermal characters in Crotalaria sp. (Papilionoideae) from Eastern Ghats. Phytomorphology 50: 205-212. [5] Ahmed, K.J. [1979] Stomatal features of Acanthaceae. In Structure, Function and Ecology of Stomata (ed. D.N. Sen). Bishen Singh Mahendra Pal Singh, Deheradun, India. pp. 43-60. [6] Carpenter, S.B. and Smith, N.D. [1975] Stomatal distribution and size in Southern Appalachian hardwoods. Can. J. Bot. 53: 1153 – 1156. [7] Inamdar, J.A. [1970] Epidermal Structure and Ontogeny of Caryophyllaceous stomata in some Acanthaceae. Bot. Gaz. 131: 261-268. [8] Kothari, M.J. and Shah, G.L. [1975] Epidermal structure and ontogeny of stomata in the Papilionaceae. Bot. Gaz. 136: 372-379. [9] Paliwal, G.S. [1966] Structure and ontogeny of stomata in some Acanthaceae. Phytomorphology 16: 533539. [10] Rajagopal, T. [1979] Distributional patterns and taxonomic importance of foliar stomata. Indian J. Bot. 2: 63-69. [11] Carlquist, S.[1961] Comparative Plant Anatomy, Holt Rinechart and Winston, New York, USA. [12] Hagarup, O. [1953] The Morphology and Systematics of the leaves of Ericales. Phytomorphology 3: 459 – 127
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464. [13] Hossian, A.B.M.E. and Khan, S.A. [1994] Use of epidermal character for identification of Blumea (Compositae) species from Bangladesh. Bang. J. Bot. 23: 147 – 154. [14] Krishnamurthy, K.H. and Kannabiran, B.[ 1970] Histomorphology of foliar epidermis and pharmacognosy in Asclepiadaceae. J. Indian Bot. Soc. 49: 105-113. [15] Metcalfe, C.R. and L. Chalk, [1950] Anatomy of Dicotyledons. Vol 2, Clarendon Press, Oxford, UK. [16] Sharma, K. Surendra, Sheela, M. A. [2011] Pharmacognostic evaluation of leaves of certain Phyllanthus species used as a botanical source of Bhumyamalaki in Ayurveda. International Quarterly Journal of Research in Ayurveda. 32(2): 250–253. [17] Trease, G.E. and Evans, W.C., [1983] Pharmacognosy, 12th edn. English Language Book Society / Bailliere Tindall. [18] Chakravarty, H.L. [1937] Physiological anatomy of leaves of Cucurbitaceae. Philipp. J. Sci. 63 (4): 409431. [19] Metcalfe, C.R. and L.Chalk, [1979] Anatomy of Dicotyledons. Vol 1, 2nd edn, Clarendon Press, Oxford, UK. [20] Rao, R.S. and Ramayya, N. [1987] Trichome types and their taxonomic importance in the Tiliaceae. Indian J. Bot. 10: 65-73. [21] Stace, C.A. [1965a] Cuticular studies as an aid to Plant Taxonomy. Bull. Br. Mus. Nat. Hist. Botany 4: 378. [22] Stace, C.A. [1965b] The taxonomic importance of the leaf surface. In Current Concepts of Plant Taxonomy (eds. V.H. Heywood and D.F. Moore). Academic Press, London. pp. 67-94. [23] Bokhari, M.H. [1970] Morphology and Taxonomic significance of foliar sclereids in Limonium . Notes Royal Bot. Gard. Edinburgh. 30: 43-53. [24] Pant, D.D. and Mehra, B. [1963] Development of Caryophyllaceous stomata in Asteracantha longifolia Nees. Ann. Bot. 27: 647-652. [25] Indian Pharmacopoeia (IP). [1996] 4 th edn.Vol. 1, The Controller of Publications, New Delhi. pp. A 100-A124. [35] Tomir, E. and Tomasz, A. [1986] β- lactum specific resistant mutant of Staphylococcus aureus. Antimicrob. Agents. Chemother. 30: 577-583. [26] Cox, P.A., Balick, M.J. [1994] The ethnobotanical approach to drug discovery. Sci. Am 270: 82-87. [27] Favel, A., Steinmetz, M.D., Regli, P., Olivier, E.V., Elias, R. and Balansard, G. [1994] In vitro antifungal activity of triterpenoid saponins. Planta Med. 60: 50-53. [28] Hiremeth, S.P., Swamy, H.K.S., Shishailappa, B. and Meena, S. [1996] Antibacterial and antifungal activities of Striga densiflora and Striga orobanchioides. Indian J. Pharm. Sci. 174-176. [29] Hostettmannn, K. and Marston, A. [1995] Saponins (Chemistry and Pharmacology of Natural Products). University Press, Cambridge. [30] Majorie, M.C. [1999] Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12(4): 564-582. [31] Sukul, S. and Chaudhari, S. [2001] Antimicrobial natural products from leaves of Lantana camara with activity comparable to some therapeutically used antibiotics. Ind. J. Pharm. Sci. 63: 20-23. [32] Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Truck, M. [1966] Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45: 493-496. [33] Akhtar, N., Ali, M. and Das, A. [2006] Antimicrobial activity of Inula cuspidata leaves. Indian J.Nat. Prod. 22 (3): 33-34.
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