Anais da Academia Brasileira de Ciências (2017) 89(2): 927-938
(Annals of the Brazilian Academy of Sciences)
Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201720160411
www.scielo.br/aabc
Psychotria viridis: Chemical constituents from leaves and biological properties
Débora b.S. SoareS1, LuCienir P. Duarte1, anDré D. CavaLCanti2, FernanDo C. SiLva3,
ariaDne D. braga4, MiriaM t.P. LoPeS4, JaCqueLine a. takahaShi1 and SiDney a. vieira-FiLho5
1
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas
Gerais, Avenida Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
2
Setor Técnico-Científico do Departamento de Polícia Federal de Minas Gerais, Rua
Nascimento Gurgel, 30, 30441-170 Belo Horizonte, MG, Brazil
3
Universidade do Estado de Minas Gerais, Unidade de Divinópolis, Avenida Paraná, 3001, 35501-170 Divinópolis, MG, Brazil
4
Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil
5
Departamento de Farmácia, Escola de Farmácia, Universidade Federal de Ouro Preto,
Campus Universitário, 35400-000 Ouro Preto, Minas Gerais, Brazil
Manuscript received on June 27, 2016; accepted for publication on October 14, 2016
abStraCt
The phytochemical study of hexane, chloroform and methanol extracts from leaves of Psychotria viridis
resulted in the identification of: the pentacyclic triterpenes, ursolic and oleanolic acid; the steroids,
24-methylene-cycloartanol, stigmasterol and β-sitosterol; the glycosylated steroids 3-O-β-D-glucosyl-βsitosterol and 3-O-β-D-glucosyl-stigmasterol; a polyunsaturated triterpene, squalene; the esters of glycerol,
1-palmitoylglycerol and triacylglycerol; a mixture of long chain hydrocarbons; the aldehyde nonacosanal; the
long chain fat acids hentriacontanoic, hexadecanoic and heptadenoic acid; the ester methyl heptadecanoate;
the 4-methyl-epi-quinate and two indole alkaloids, N,N-dimethyltryptamine (DMT) and N-methyltryptamine.
The chemical structures were determined by means of spectroscopic (IR, 1H and 13C NMR, HSQC, HMBC
and NOESY) and spectrometric (CG-MS and LCMS-ESI-ITTOF) methods. The study of biologic properties
of P. viridis consisted in the evaluation of the acetylcholinesterase inhibition and cytotoxic activities. The
hexane, chloroform, ethyl acetate and methanol extracts, the substances 24-methylene-cycloartanol, DMT
and a mixture of 3-O-β-D-glucosyl-β-sitosterol and 3-O-β-D-glucosyl-stigmasterol showed cholinesterase
inhibiting activity. This activity induced by chloroform and ethyl acetate extracts was higher than 90%. The
methanol and ethyl acetate extracts inhibit the growth and/or induce the death of the tumor cells strains
B16F10 and 4T1, without damaging the integrity of the normal cells BHK and CHO. DMT also demonstrated
a marked activity against tumor cell strains B16F10 and 4T1.
key words: Psychotria viridis, Rubiaceae, N,N-dimethyltryptamine, B16F10, 4T1 tumor cells.
introDuCtion
Brazil has a large plant genetic diversity, with more
than 55,000 species cataloged (Nodari and Guerra
Correspondence to: Fernando César Silva
E-mail: fernando.cesar@uemg.br
1999, Veiga 2008). In the Amazon region, the use of
medicinal plants has its bases in indigenous practice
(Labate 2011). Many of these plants are also used in
religious rituals. This practice originated from the
great cultural influence of the ancient peoples of the
An Acad Bras Cienc (2017) 89 (2)
928
DÉBORA B.S. SOARES et al.
Amazon, which was mixed with African traditions
and European culture brought by the colonizers
(Almeida 2011). Ritualistic plants become sacred
when their use in ritual gives them a sacred value,
to be legitimized through rites of a given belief
model, philosophy or kind of thinking (Camargo
2006). These plants eliminate fatigue and insomnia,
relieve the feeling of hunger, stimulate or cancel
sexuality, induce depression or euphoria, provoke
visions and fantastic predictions. In general, they
are ingested orally in nature or prepared or enters
the body by means of the oral route by inhalation
of its fumes, or smelled; or topically applied to
healthy or scarified skin. As a result, the use of
such plants or its derivatives has been popularized
worldwide, both for religious purposes, or not
(Camargo 2007). The effect of these substances is
confused with religious mysticism of the Indians
and the wisdom of healers, shamans and “pajé”,
which usually play a dual role of religious guide and
medicine doctor in your tribe. Thus, it is difficult to
distinguish between the pharmacological actions
of these preparations and their placebo effects on
phenomenological healings that occur in rituals
(Callaway et al. 1994, Callaway and Grob 1998).
Most ritual plants used in healing ceremonies in
shamanism, umbanda and other rituals, acts in the
CNS in order to facilitate communication with spirit
guides (Corrêa et al. 1998, Camargo 2006, 2007).
Several studies have been conducted to discover
new compounds that reach pharmacological targets
in the CNS. The harmin represents an example of
β-carboline alkaloid isolated from Banisteriopsis
caapi (Malpighiaceae), which is a component
of ayahuasca (yagé, hoasca, daime or caapi), a
ritualistic beverage used in Brazil, Bolivia, Equador
and Peru (Callaway and Grob 1998). This alkaloid
acts on CNS inhibiting the monoamino-oxidase
(MAO) (Breakefield et al. 1976, Pires et al. 2010).
Inhibitors of MAO induce an enhancement of the
amine concentration in brain, mainly dopamine,
noradrenalin and serotonin, generating a state of
An Acad Bras Cienc (2017) 89 (2)
excitement, euphoria, increased psychomotor
activity (antidepressant), and others (Farzin and
Mansouri 2006). It is also observed hallucinogenic
effect attributed to structural similarity with
endogenous amines, such as tryptamine and
serotonin (Fortunato 2009). Other examples of
ritualistic plants that alter the SNC physiology
include Lophophora williamsii (Lem. ex SalmDyck J.M. Coult.) (Peiote) used in México and
Central America, the Mimosa hostiles Benth
(Jurema), in Northeast of Brazil, the Tabernanthe
iboga Baill (Iboga) in Africa and Peganum
harmala L. (Pégano) in the Middle East (Teixeira
et al. 2008).
Among the species of plants with potential
medicinal property are several members of the family
Rubiaceae, due to the large number of substances
with potential biological activity, including
compounds belonging to different chemical
classes, such as alkaloids and anthraquinones
(Chan-Blanco et al. 2006, Delprete and Jardim
2012). This family is constituted by the subfamilies
Ixoroideae, Rubioideae, Cinchonoideae Raf.
And Antirheoideae Raf., covering aproximatley
650 genera and 13,000 species (Lima 2011). In
South America, there are 30 % of all species of
the Rubiaceae family, which exceeds in number
of species, all regions of the Earth, being then in
South Asia and Africa. In Brazil, Rubiaceae family
is represented by about 2,000 species distributed
among 110 genera (Delprete and Jardim 2012). In
the most important Brazilian ecosystems such as the
Amazon and Cerrado regions and Atlantic Forest,
species of this family are the form of trees, shrubs,
subshrubs or perennial or annual herbs, and lianas
and epiphytes. The species of Rubiaceae have great
diversity of secondary metabolites such as indole
alkaloids, tannins, triterpenes, saponins, steroids
and flavonoids (Lima 2011). This justifies its use
in traditional medicine and in the manufacture of
herbal medicinal products. An example is the cat’s
claw “unha de gato”, Uncaria tomentosa (Willd.
P. viridis: CONSTITuENTS AND BIOLOGICAL PROPERTIES
ex Schult.) DC, whose extract obtained from its
roots is indicated for the treatment of rheumatism
and arthritis (Moraes 2013). The subfamily
Rubioideae is considered as the second source of
highest concentration of alkaloids (Delprete and
Jardim 2012) belonging to different classes, such as
isoquinoline (44 identified constituents) quinoline
(70 compounds) and indole (391 constituents)
(Cordell et al. 2001).
The species of subfamily Rubioideae are
divided into 15 tribes, the largest being the
Psychotrieae, comprised of approximately 50
genera widely distributed throughout the tropical
zone (Libot et al. 1987). The Psychotria genus was
first described by Linneaus in 1759, and actually is
considered as one of the largest plant genera with
flowers. Nowadays, in this genus are included about
1000 species (Steyermark 1974, Nepokroeff et al.
1999). According to morphological characteristics
and geographical distribution, it is divided into
three subgeneras: Psychotria (pantropical),
Heteropsychotria (including neotropical species
of Psychotria) and Tetramerae which includes
species of Africa and Madagascar (Faria 2009).
Different species of the genus Psychotria are
used in traditional medicine for the treatment of
disorders of the female reproductive tract and as an
aid to relieve symptoms that occur pre- and postpartum, and also to treat diseases of the bronchi and
gastrointestinal disorders (Lima 2011). Topically,
in the form of poultices or baths species of this
genus are used to treat skin disorders, “tumors”,
ulcer, ocular disorders, to relieve fever, headaches
and earache (Faria 2009). Bioactive materials are
obtained from various species of Psychotria such
as extracts of P. microlabastra (Khan et al. 2001)
and P. capensis that showed antibiotic activity, of
P. serpens that presented antiviral properties, of P.
hawaiiensis (Locher et al. 1995) and P. insularum,
to which antiviral, antifungal and anti-inflammatory
properties were attributed (Dunstan et al. 1997).
929
Psychotria viridis (Rubiaceae) is a species used
in the preparation of ayahuasca drink, in association
with Banisteriopsis caapi Morton (Callaway and
Grob 1998). The Shamans and Healers of the
Amazonia first used the drink. They practiced
traditional medicine based on plant extracts (Luna
1986). The use of N,N-dimethyltryptamine (DMT),
one constituent of this drink, due to its psychotropic
effect is banned in Brazil (ANVISA, Portaria,
344/98). However, in August 2006, was enacted
in Brazil the law n0 11.343, which allows its use
in “religious rituals” as long as authorized by the
State. This law is in accordance with the Vienna
Convention, promoted by the United Nations in
1971 with the aim of establishing the rules on the
use of “psychotropic substances” (Groisman 2013).
The release of scientific research and ritualistic
use of ayahuasca is regulated in Brazil, through
Resolution no 1/2010 of the National Council for
Drug Policy (Garrido and Sabino 2009).
P. viridis is commonly known as “chacrona”
or “chacruna” and is morphologically similar to
other species of the genus (Blackledge and Taylor
2003) and was firstly described in 1779 by Ruiz
and Pavón (Aranha et al. 1991). It is represented by
shrubs and grows naturally in flat and humid forest
areas from the northern region of Central America
to Central South America. It is most commonly
found in the Amazon region of Peru and northern
Bolivia (Blackledge and Taylor 2003).
Herein it is described a phytochemical study
of P. viridis aiming the isolation of bioactive
secondary metabolites, followed by the structural
identification by means of spectroscopic methods,
and evaluate the biological properties of extracts
and constituents present in its leaves.
MateriaLS anD MethoDS
PLANT MATERIAL
Leaves of P. viridis were collected in the morning,
at the extensive culture of the nucleus of “União do
An Acad Bras Cienc (2017) 89 (2)
930
DÉBORA B.S. SOARES et al.
Vegetal” located at Lagoa da Prata municipality,
Minas Gerais. P. viridis cultivated in this region
is originally from the State of Rondonia, Brazil,
which is located at the center of Amazon region.
The collected plant material was dried in a forced
ventilation oven at 40 °C for two days.
apparatus coupled to mass spectrometer Shimadzu
IT-TOF, hybrid with analyzers type ion trap and
TOF in sequence (LCMS-ESI-IT-TOF) was also
applied in some analysis of constituents from
leaves of P. viridis.
EXTRACTION AND ISOLATION OF COMPOuNDS
GENERAL PROCEDuRES
Silica gel 60 (Merck) [7g:15mL water] 0.25 mm
thickness was used for thin layer chromatography
(TLC) and 0.50 mm for preparative assays (PTLC),
after dried at 100 ºC/ 30 minutes. The plates were
revealed by means of UV light irradiation on
CHROMATO-uVE, ultra-Violet Products, Inc.,
and after treatment with chromogenic reactants.
Chromatographic columns (CC) were performed
on silica gel 60 (70-230 Mesh), with ratio sample/
stationary phase of 1:40 w/w. The elution solvents
were analytical grade pure or in mixtures of
enhanced polarity. Medium Pressure Column
Chromatography (MPLC) was performed on
Isolera-One, BiotageTM, equipped with uV detector
(UV1 254 nm, UV2 280 nm) and SNAPTM 100g
column, with 50 mL/min mobile phase. The melting
point of isolated compounds was determined using
a Microquímica MQAPF-302 apparatus. Fourier
transform infrared (FTIR) spectra were obtained for
solid samples dissolved in KBr [1 % (w/w)], using
Spectrum One Perkin Elmer spectrometer. The
1D/2D NMR spectra were performed on Bruker
AVANCE DPX-200 or DRX-400 spectrometers.
Deuterated solvents are indicated for each analyze.
Chemical shift assignments were recorded in ppm
(δ) using tetramethylsilane (TMS) as internal
reference (δH = δC = 0). The coupling constants (J)
were registered in Hertz (Hz). Gas chromatography
coupled with mass spectrometry (CG-MS) was
carried out on AGILENT 7890A GC FOR 5975
series MSD, equipped with HP 5MS (30 m ×0,25
mm × 0,25 μm) column. Helium (1.4 mL/min)
was used as carrier gas. A Shimadzu LC-20AD
An Acad Bras Cienc (2017) 89 (2)
The air-dried leaf (362.4 g) of P. viridis was
sequentially extracted with hexane, chloroform,
ethyl acetate, and methanol through maceration
at room temperature. Each extractor solvent was
recovered using IKA® Rotary Evaporator RV 10,
and the residual solvent was evaporated to dryness
under vacuum, producing the EHF, ECF, EAcF and
EMeF extracts.
The hexane extract (EHF, 4.3 g) was separated
in two aliquots: A (2.2 g) and B (2.1 g), due to
the maximum capacity of sample used in the
chromatographic column (CC). The samples A
and B were chromatographed separately using the
equipment Isolera-One and eluted with hexane,
chloroform and methanol pure or in mixtures of
increasing polarity. The fractions collected from A
and B were subjected to TLC and grouped in five
groups in accordance with the profile similarity.
All groups were subjected to silica gel CC and
eluted with a hexane, ethyl acetate and methanol
pure or in mixtures of increasing polarity. The
group 5 was also submitted to PTLC. The groups
1-5 affording the compounds (1; a mixture of long
chain hydrocarbons) (26.8 mg; eluted with hexane),
(2; 24-methylene-cycloartenol) (16.2 mg; eluted
hexane/ethyl acetate 97:3), (3; squalene) (4.2 mg;
eluted hexane/ethyl acetate 93:7), (4 and 5; a mixture
of β-sitosterol and stigmasterol, respectively) (20.6
mg; eluted with hexane/ethyl acetate 88:12), and
(6; triacylglycerol) (16.7 mg; eluted with hexane/
ethyl acetate 86:14), respectively.
The chloroform extract (ECF, 4.1 g) was
subjected to silica gel CC using as eluent
chloroform, ethyl acetate and methanol pure or in
P. viridis: CONSTITuENTS AND BIOLOGICAL PROPERTIES
mixtures of increasing polarity. The fractions were
grouped by means of TLC, furnishing six groups.
The group 1 was submitted to successive silica
gel CC affording the compound 7 (nonacosanal)
(10.6 mg; eluted with hexane/chloroform 75:25).
The group 2 was dissolved in acetone, furnishing
compound 8 (nonacosanol) (25.4 mg) as a
precipitated. The group 3 was dissolved in acetone,
furnishing compound 9 (hentriacontanoic acid)
(9.0 mg) as a precipitated, and the filtrated after
solvent evaporation was submitted to silica gel CC,
affording compound 10 (hexadecanoic acid) (3.3
mg; eluted with hexane/ethyl acetate 90:10). The
group 4 was submitted to silica gel CC, affording
compound 11 (heptadenoic acid) (7.8 mg; eluted
with hexane/ethyl acetate 80:20). The group 5 was
subjected to successive silica gel CC, affording a
mixture (4.5 mg; eluted with hexane/ethyl acetate
70:30) of compounds 12 and 13, respectively
identified as ursolic acid and oleanolic acid. The
group 6 was submitted to silica gel, affording
compound 14 (1-palmitoylglycerol) (5.5 mg; eluted
with hexane/ethyl acetate 50:50).
By means of TLC the ethyl acetate extract
(EAcF) was characterized as complex mixture, and
due to the small quantity obtained (<50 mg), it was
not subjected to phytochemical processes.
The methanol extract (EMeF, 43.6 g) was
separated in two aliquots, A (23.5 g) and B (20.1
g). Aliquot A was subjected to acid-base extraction
according to methodology suggested by Matos
(1988), and Wagner and Bladt (1996), providing
four fractions. The fraction 1 was subjected to silica
gel CC, affording a solid mixture (29.1 mg; eluted
with chloroform/methanol 70:30, and purified by
acetone) constituted by compounds 15 and 16,
respectively identified as 3-O-β- D-glucosyl-βsitosterol and 3-O-β-D-glucosyl-stigmasterol. The
fractions 2 and 3 were separately subjected to silica
gel CC, and purified by means of PTLC, affording
the compound 17 (N,N-dimethyltryptamine)
(52.9 mg; eluted with chloroform/methanol 97:3,
931
under atmosphere of ammonium hydroxide). The
fraction 4 was submitted to successive silica gel
CC, affording a solid mixture (15.7 mg; eluted
with chloroform/methanol 97:3 with atmosphere
of ammonium hydroxide) constituted by
compounds 17 and 18, respectively identified as
N,N-dimethyltryptamine and N-methyltryptamine.
The aliquote B was submitted to silica gel
CC, eluted with a chloroform, methanol and
ammonium hydroxide pure or in mixtures of
increasing polarity. The fractions were grouped
by means of TLC, furnishing two groups. Group
1 was dissolved in acetone, furnishing compound
19, (methyl 4-epi-quinate) (11.2 mg) as a solid
material. The group 2 was submitted to silica gel
CC followed by PTLC, affording the compounds
20, (methyl heptadecanoate) (41.4 mg; eluted
with chloroform), 17, (N,N-dimethyltryptamine)
(19.1 mg; eluted with chloroform/methanol 97:3
under atmosphere of ammonium hydroxide), and
18, (N-methyltryptamine) (8.5 mg; eluted with
chloroform/methanol 97:3 under atmosphere of
ammonium hydroxide).
BIOLOGICAL ASSAYS
Acetylcholinesterase inhibition assay
The acetylcholinesterase (AChE) inhibition
property of substances isolated from P. viridis
was performed in 96 wells microtiter plate
using Ellman’s spectrophotometric method
modified by Rhee et al. (2001). Thus, buffer A (50
mM Tris–HCl, pH 8, containing 0.1 M NaCl and
0.02 M MgCl2·6H2O), B (50 mM Tris–HCl, pH 8,
containing 0.1% bovine serum albumin), and C (50
mM Tris–HCl, pH 8) were prepared to the in vitro
AChE inhibitory activity assays. The volumes of
25 µL of acetylcholine iodide (15 mM in water),
125 µL of Ellman's reagent [5,5-dithiobis-(2nitrobenzoic acid)] (3 mM in buffer A), 50 µL of
buffer B, and 25 µL of sample (10 mg/mL in MeOH
An Acad Bras Cienc (2017) 89 (2)
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DÉBORA B.S. SOARES et al.
diluted by 10 times with buffer C, providing a final
concentration of 1.0 mg/mL) were added into each
well of a 96-well microtiter plate. Instead of adding
the sample solution, a volume of 25 µL of buffer C
was used to prepare the blank sample. The positive
control was prepared under same conditions. Each
assay was carried out in triplicate. The hydrolyze
of substrate generate compounds containing
sulfhydryl groups that reacts with the Ellman’s
reagent whose concentration can be colorimetric
evaluated at 405 nm (Trevisan and Macedo 2003).
Thus, the absorbance was measured every 60 s by
eight times using an Elisa Thermoplate microplate
reader. After addition of 25 µL of AChE solution
(0.226 U/mL in buffer B), the absorbance was
again read every 60 s, for ten times. The increase in
absorbance was corrected by reaction rate variation
before and after addition of the enzyme. The
inhibition percentage was calculated by comparing
the rates of the sample with the blank.
MTT Cell viability assay
Lines of normal and tumor cells were used to
evaluate the effects of P. viridis extracts upon cell
proliferation. The normal cells lines chosen BHK21 (Baby Hamster Kidney Fibroblast Cells) and
CHO (Chinese hamster ovary cell) were gifts from
the Pan-American FMD Center, Rio de Janeiro,
Brazil. The tumor cells lines used were B16F10
(murine melanoma) was a gift from the “Instituto
Ludwig de Pesquisa sobre o Câncer” (Sao Paulo,
Brazil) and 4T1 (murine tumor mammary gland)
(American Type Culture Collection, Manasssas,
VA). Originally, B16F10 were isolated clones
from spontaneous malignant melanoma (Fidler and
Kripke 1977), while 4T1 is a thioguanine resistant
variant that was obtained from 410.4 without
mutagenic exposure (Aslakson and Miller 1992).
Both of tumor cells types are able to form a tumor
mass and metastazise to lung tissue, once injected
in mice.
An Acad Bras Cienc (2017) 89 (2)
Firstly, cells were cultured in RPMI
1640 medium (GIBCO, Carlsbad, CA, USA)
supplemented with 10% of heat-inactivated fetal
bovine serum (FBS) (GIBCO), 1% penicillin/
streptomycin (Sigma-Aldrich, St. Louis,
MO) at 37 oC in a humidified atmosphere of
5% CO 2. Once confluent, the monolayer was
harvested by incubation with trypsin/EDTA;
ethylenediaminetetraacetic acid (0.025% and 0.02
w/v, respectively). The released cell suspension
(>90% of viability) were adjusted to the appropriate
cell density and then seeded in 96-polystyrene-well
plates. In the next day, the cells were exposed for
72 hours to increase concentrations of P. viridis
extracts or DMT (20-400 μg/mL). Control cells
were exposed to RPMI 1640 supplemented with 10
% of FBS added to 0.2% (v/v) DMSO (extracts and
DMT vehicle).
Cell viability was measured by means of
the MTT (3-(4,5)-dimethylthiahiazo(-z-y1)3,5-diphenytetrazoliumromide) method. In this
assay, the tetrazolium compound is reduced in the
intracellular medium, to formazan by mitochondrial
dehydrogenase. The conversion of blue tetrazolium
into purple formazan by metabolically active cells
indicates the extent of cell viability. Measure cells
viability was done by quantification of formazan
dye using a colorimetric method (Heo et al. 1990).
MTT assay and colorimetric method were used to
evaluate the effects of P. viridis extracts on cell
proliferation. So, after extracts or DMT exposure
period, 10 μL of 5 mg/mL MTT (Sigma Chemical
Co, St. Louis, MO, USA) was added onto each
well and incubated at 37 °C for 4 h. The cells were
then lysed with dimethyl sulfoxide (DMSO) and
the absorbance determined at 570 nm using an
ELISA reader. The results were plotted based on
percentage of cell death vs. concentration (μg/mL)
of the sample, using the software GraphPad Prism
5.0. A non-linear regression model (sigmoidal
dose-response option) was used to determine the
inhibitory concentration IC50 (i.e., 50% reduction
P. viridis: CONSTITuENTS AND BIOLOGICAL PROPERTIES
933
in cell viability) for methanol and ethyl acetate
extracts upon normal and tumor cells.
reSuLtS anD DiSCuSSion
From leaves of P. viridis were identified nineteen
constituents: a mixture of long chain hydrocarbons
(1), 24-methylene-cycloartenol (2) (De Pascual
et al. 1987), squalene (3) (Valladao et al. 2010),
β-sitosterol (4), stigmasterol (5) (Goulart et al. 1993),
triacylglycerol (6) (Gunstone 1991), nonacosanal
(7), nonacosanol (8) (Řezanka and Sigler 2007),
hentriacontanoic acid (9), hexadecanoic acid
(10), heptadenoic acid (11) (Gunstone 1991),
ursolic acid (12), oleanolic acid (13) (Valadares
2009), 1-palmitoylglycerol or monopalmitin
(14) (Gunstone 1991), 3-O-β- D -glucosyl-βsitosterol (15) (Silva et al. In press), 3-O-β-Dglucosyl-stigmasterol (16) (El-Askary 2005), N,Ndimethyltryptamine (17), N-methyltryptamine (18)
[40], 4-methyl-4-epi-quinate (19) (Gunstone 1991)
and methyl tetradecanoate (20) (Gunstone 1991).
The chemical structures of some constituents
isolated from leaves of P. viridis are presented in
Figure 1.
The compound 1 was identified as a mixture
of long chain hydrocarbons. The IR spectrum of
1 showed intense absorption bands in the region
of 2958 to 2850 cm-1, 1474 to 1464 cm-1 and a
double band at 730 cm-1. The observed profile of
this spectrum suggested the aliphatic nature of 1.
The 1H NMR spectra of 1 showed hydrogen signal
at δH 0.88 (t; J = 6.0 Hz) characteristic of methyl
groups of hydrocarbons. The IR spectrum of 2
showed intense absorption bands in the region of
3403 (OH), 1640 (C=C) and 1047 cm-1 (CO). The
1
H NMR spectra of 2 showed hydrogen signal at
δH 0.35 (d; J = 4.17 Hz) and δH 0.57 (d; J = 4.11
Hz) indicating a cyclopropane ring. The 13C NMR
data compared with the literature confirmed the
compound 2 as 24-methylene-cycloartenol (De
Pascual et al. 1987). The 1H and 13C NMR spectra
Figure 1 - Chemical structures of some constituents isolated
from leaves of P. viridis.
of compound 3 showed signals at δH 5.08-5.15 and
δC 124.31-135.12 that are characteristics of olefin
compounds. The 13C NMR data compared with the
literature confirmed the compound 3 as squalene
(Valladao et al. 2010). In the 1H NMR spectrum
of compounds 4, 5, 15 and 16 were observed the
signal at δH 5.2-5.3 attributed to olefin hydrogen,
and a multiplet at δH 3.4-3.5 correspondents to
hydroxylated carbon. The profile of these spectra
was similar to those observed for the steroidal
skeletons. In the spectra of compound 15 and 16
were observed signals at δ H 3.60-4.58 that are
characteristics of glucose hydrogen. The chemical
shift assignments of signals observed in the 13C
NMR spectra, mainly the signals around δC 121.72
and δC 140.78, and δC 129.30 and δC 138.31 (15 and
16, respectively) were attributed to olefin carbons.
The presence of signals at δC 102.01-102.4 in 13C
An Acad Bras Cienc (2017) 89 (2)
934
DÉBORA B.S. SOARES et al.
NMR spectra confirmed compounds 15 and 16 as
glucosylated steroids. The 13C NMR data compared
with the literature confirmed compound 4 as
β-sitosterol, 5 as stigmasterol (Goulart et al. 1993),
15 as 3-O-β-D-glucosyl-β-sitosterol (Silva et al.
2016), and 16 as 3-O-β-D-glucosyl-stigmasterol
(El Askary 2005).
The IR spectrum of 6 showed intense
absorption bands in the region of 2928-2856 (CH),
1742 and 1720 (C=O), and 1170 cm-1 (CO). The
absorption bands at 1742, 1720, and 1170 cm -1
indicate an ester group. The signals at δC 173.21
and δC 171.97 observed in 13C NMR spectra were
attributed to carbonyl groups. The 13C NMR data
compared with the literature confirmed compound
6 as triacylglycerol (Gunstone 1991). In the IR
spectra of 7 and 8 were observed intense absorption
bands in the region of 3346 (OH for compound 8),
2918-2915 (CH), 1720 (C=O for 7), and 720-730
cm-1 (long chain). The absorption bands in the
regions of 3346 cm-1 indicated 8 as an alcohol,
and at 1720 cm-1 indicated 7 as an aldehyde. The
1
H NMR and 13C spectra of compound 7 showed
signals at δH 9.7 (t; J = 1.6 Hz) and at δC 202.85
which are characteristics of aldehyde. The spectral
data compared with the literature confirmed
compound 7 as being nonacosanal (Řezanka
and Sigler 2007). The 1H and 13C NMR spectra
of compound 8 showed signals at δH 3.66 (t; J =
6.5 Hz) and δC 63.11 that are characteristics of
alcohol. The spectral data confirmed the compound
8 as nonacosanol. For compounds 9, 10 and 11
were observed signals at δH 0.88 (t; J = 6.3 Hz)
correspondent to methyl groups, and at δH 1.601.73 and δH 2.35 (t; J = 7.4 Hz) that are typical of
carbon β and α from carboxylic acid, respectively.
The presence of signals at δC 14.12-14.13 (methyl
group) and at δC 176.50-177.90 (carboxyl group)
contributed to identification of these compounds
as carboxylic acids (Gunstone 1991). The spectral
data confirmed compound 9, 10 and 11 as
hentriacontanoic, hexadecanoic and heptadenoic
An Acad Bras Cienc (2017) 89 (2)
acids respectively. In the 1H NMR spectra of
compound 12 and 13 were observed signals at δH
0.70-2.05 which are characteristics of triterpenes,
and the signals at δH 5.25-5.28 (t; J = 3.8 Hz), that
are attributed to hydrogen atoms of double bound
commonly found in ursane and oleanane skeleton.
The signals at δC16.93 and δC 21.17 confirmed the
structure of ursane triterpene for 12, and at δC 33.07
and δC 23.63 the structure of oleanane triterpene for
13. The 13C NMR data compared with the literature
confirmed compounds 12 as ursolic acid, and 13 as
oleanolic acid (Valadares 2009). In the IR spectrum
of 14 were observed intense absorption bands at
3472 (OH), 2928-2852 (CH), 1742 (C=O), and
at 722 cm-1 (long chain). In the 1H NMR spectra
of compound 14 the signals at δH 4,13-4,17 (dd;
J = 11,6 and 6,1 Hz) and 4,19-4,23 (dd; J = 11,6
and 4,6 Hz) were attributed to hydrogen atoms of
hydroxylated carbon (C1). In the 13C NMR spectra
of constituent 14, were attributed the signals at
δC 174.37 to carbonyl from ester, and at δC 65.19
and 63.36 to hydroxylated carbon atom. The 13C
NMR data compared with the literature confirmed
compound 14 as 1-palmitoylglycerol (Goulart et
al. 1993). The absorption band at 3500-3400 cm-1
observed in the IR spectra of 17 and 18 indicate
the presence of NH from indole ring and these
compounds as being alkaloids. The 1H NMR
and 13C spectra of compound 17 showed signals
at δH 6.92-7.09, δH 7.29 (d; J = 8.0 Hz) and 7.49
(d; J = 8.0 Hz), and δC 112.25 and 138.15 which
are characteristic of indole ring. The molecular
formula of 17 was established as C12H16N2 on the
basis of the molecular ion at m/z 189.1359 [M+H]
(calcd 189.1386) obtained in the LC/MS-TOF. The
spectral data compared with the literature confirmed
compound 17 as being the N,N-dimethyltryptamine
(Gaujac et al. 2013). By means of LCMS-ESIIT-TOF data, the compound 18 was identified as
N-methyltryptamine. Based on the molecular ion
at m/z 175.1214 [M+H], the molecular formula of
18 was determined as C11H14N2 (calcd 175.1229).
P. viridis: CONSTITuENTS AND BIOLOGICAL PROPERTIES
The IR spectrum of 19 showed absorption bands at
3446, 3408 and 3366, cm-1 (OH), 2924, 2958 cm1
, 1436 and 1370 cm-1 (CH), 1144 cm-1 and 11181078 cm-1 (C-O). The absorption band at 1736 cm-1
indicates an ester group. In the 1H NMR spectrum
of 19 were observed the signals at δH 1.66-1.71 (dd,
1H, H2eq, J = 13.7 and 5.1 Hz) and at δH 1.81-1,85
(dd, 1H, H6eq, J = 13.4 and 2.9 Hz) and a multiplet
at δH 2.28-2.37, attributed to hydrogen H6ax and
H2ax, a signal at δH 3.72-3.75 (dd, 1H, J = 5,7
and 3.0 Hz), associated to H4, a simplet at δH 3.77
correspondent to hydrogen atoms of methoxyl. The
multiplet at δH 4.01–4.05 (1H) was correlated to
H3 and at δH 4.12–4.16 (1H) and attributed to H5.
Based on the 13C NMR spectra including DEPT135 data were classified 3 CH, 2 CH2, 1 CH3 and
2 non-hydrogenated carbon atoms, one being
C=O (δH 176.12). The 1H and 13C NMR chemical
shift assignments of 19 were in accordance to the
data previously published for 4-metil-epi-quinate
(Armesto et al. 2006). Through the LCMSESI-IT-TOF the molecular formula of 19 was
confirmed based on the ion of m/z 229.0687 Da
(calcld. 229.0688). The IR spectrum of 20 showed
intense absorption bands at 2926-2854 (CH), 1744
(C=O), and 722 cm-1 (carbon long chain). In the
1
H NMR spectra of compound 20 were observed
signals at δH 3.67 characteristic of hydrogen atoms
of methoxyl groups, at δH 2.30 (t; J = 7.5 Hz)
attributed to hydrogen atoms linked to α carbon
atom, and a multiplet at δH 1.54-1.65 attributed to
hydrogen atoms linked to β carbon atom. In the
13
C NMR spectra of 20 were observed signals at δC
174.43 attributed to carbonyl from ester, δC 51.46
attributed to carbon atom from methoxyl group,
and δC 14.13 characteristic of methyl group. The 13C
NMR data compared with the literature confirmed
compound 20 as methyl tetradecanoate (Gunstone
1991). The compounds 1 to 20 isolated from leaves
of P. viridis were respectively identified by means
of melting point, spectral data from IR, NMR and
MS and comparison with the published data.
935
tabLe i
Percent inhibition of acetylcholinesterase (aChe) induced
by substances isolated from leaves of P. viridis.
Sample
Inhibitiona of AChE
(% ± sd)
Coefficient
of variation
Hexane extract
80.34 ± 1.33
0.017
Chloroform extract
91.76 ± 2.25
0.025
Ethyl acetate extract
91.21 ± 3.95
0.043
Methanol extract
85.28 ± 2.99
0.035
Cycloartenol
49.53 ± 3.37
0.068
DMT
19.84 ± 4.83
0.244
β-sitosterol and
stigmasterol
67.15 ± 4.85
0.072
Galantamine
(Standard)
90.31 ± 0.45
0.005
Eserine (Standard)
70.14 ± 0.85
0.012
a = Median of triplicate assays.
Sd = Standard deviation.
DMT = Dimethyltryptamine.
The hexane (EHF), chloroform (ECF), ethyl
acetate (EAcF), and methanol (EMeF) extracts and
some substances isolated from leaves of P. viridis
were subjected to the following biological assays:
acetylcholinesterase inhibition activity and on the
viability of normal and tumor cells line.
The hexane, chloroform, ethyl acetate and
methanol extracts and the constituents cycloartenol
(2), DMT (17) and a mixture of β-sitosterol (4)
and stigmasterol (5) isolated from the leaves of
P. viridis present significant acetylcholinesterase
inhibition properties, wherein the chloroform and
ethyl acetate extracts induced inhibition effect
above 90% (Table I).
In accordance with Trevisan and Macedo
(2003) extracts whose enzyme inhibition is greater
than or equal to 50% are considered as promising
candidates for future drugs. Thus, the results
open perspectives for the isolation of the most
active compounds from P. viridis, which may be
considered as templates to obtain drug candidate
compounds to be tested against the Alzheimer
disease.
An Acad Bras Cienc (2017) 89 (2)
936
DÉBORA B.S. SOARES et al.
Figure 2 - Representative curves of surviving B16F10 (A) and
4T1 cells (B) versus log of concentration (µg/mL) of EAcF,
EMeFand DMT, isolated from leaves of P. viridis. The IC50
value was evaluated by means of non-linear regression (n = 5).
By means of the IC50 of the extracts upon
B16F10 and 4T1 tumor cells was possible to
conclude that at lower concentrations EMeF induce
an incidence of cytotoxic effect higher than EAcF,
for both cell strains (Figure 2). EMeF was also
considered more cytotoxic than DMT that is the
principal constituent in the methanol extract from
leaves of P. viridis. It is possible to infer that the
cytotoxicity on tumor cells promoted by EMeF
involves the action of other chemical constituents
than the DMT.
Both ethyl acetate and methanol extracts
showed the property to reduce tumor cell viability
An Acad Bras Cienc (2017) 89 (2)
Figure 3 - Representative curves of surviving BHK-21 (A) and
CHO cells (B) versus log of concentration (µg/mL) of EAcF
and EMeF extracts from leaves of P. viridis. The IC50 value
was not determined in the concentration range used (n = 5).
(B16F10 and 4T1), although do not interfere in
viability of normal cells (BHK-21 and CHO).
The non-cytotoxicity on normal cell strains used
as reference shows a selectivity action of extracts
from leaves of P. viridis. This selective cytotoxic
effect is an important property to be achieved in
antitumor new drugs. The present study constitutes
perspectives for the investigations about how these
compounds act in viability reduction of B16F10
and 4T1 tumor cells.
aCknoWLeDgMentS
The authors thank the Fundação de Amparo à
Pesquisa do Estado de Minas Gerais (FAPEMIG)
for financial support and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)
P. viridis: CONSTITuENTS AND BIOLOGICAL PROPERTIES
for studentship (DBSS). The authors are also
thankful to Núcleo de Lagoa Dourada of união do
Vegetal for providing P. Viridis sample.
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