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S492 © 2020 Pharmacognosy Magazine | Published by Wolters Kluwer - Medknow
ABSTRACT
Background: The genus Vitex contains about 300 species distributed
around the world. These genuses are used in the treatment of premenstrual
syndrome. However, their estrogenic activity is not well understood.
Objectives: To compare the estrogenic activity and lipid prole of ethanol
extracts of leaves of Vitex pinnata L. with 17 β‑estradiol in bilaterally
ovariectomized (OVX) rats. Methods: Ethanol extracts were analyzed
by gas chromatography‑mass spectrometry (GC‑MS). Bilaterally OVX
rats were divided into ve groups,(n= 6) receiving different treatments,
consisting of a vehicle(1% Tween), ethanol extract of V. pinnata at three
different doses(100, 500, 1000 mg/kg) and standard drug, 17 β‑estradiol at
a dose of 1 mg/kg. All groups were administered orally, daily for 14days.
Results: GC‑MS data revealed that the major chemical constituents
of the extract were 3, 7, 11, 15‑Tetramethylhexadecen‑2‑en‑1‑ol,
Gamma‑Stigmasterol, 9,12,15‑octadecatrienoic acid and n‑hexadecanoic
acid. V. pinnata extracts at 1000 mg/kg slightly increased uterine and
vaginal weight and endometrial thickness. Doses of extract at 500 and
1000 mg/kg induced a signicant(P<0.05) decrease of triglycerides and
total cholesterol in serum of OVX rats. Conclusion: V. pinnata leaf extract
exhibits estrogenic activity and reduces levels of serum triglycerides and
cholesterol. The understanding of such activity of V. pinnata leaf extract has
benets for postmenopausal women.
Key words:17 β‑estradiol, estrogenic activity, lipid prole, ovariectomized
rats, Vitex pinnata
SUMMARY
• GC‑MS data revealed that the major chemical constituents of the extract
were 3, 7, 11, 15‑Tetramethylhexadecen‑2‑en‑1‑ol, Gamma‑Stigmasterol, 9,
12, 15‑octadecatrienoic acid and n‑hexadecanoic acid
• Vitex pinnata extracts at 1000 mg/kg slightly increased uterine and vaginal
weight and endometrial thickness
• Vitex pinnata extracts at 500 and 1000 mg/kg induced a signicant(P<0.05)
decrease of total cholesterol and triglycerides in serum of ovariectomized
rats.
Abbreviations used: GC‑MS: Gas chromatography‑mass spectrometry;
OVX: Ovariectomized; V. pinnata: Vitex pinnata;
(H and E):(Hematoxylin and eosin).
Correspondence:
Dr. Wilawan Promprom,
Department of Biology, Faculty of Science,
Mahasarakham University, Kantharawichai
District,
Maha Sarakham, 44150 Thailand.
E‑mail:wipromprom@gmail.com
DOI: 10.4103/pm.pm_443_19
ORIGINAL ARTICLE
INTRODUCTION
e genus Vitex L. is in the plant family Lamiaceae which comprises
about 300 species in the tropics. It can be found in Malaysia, Indonesia,
Philippines, Cambodia, and ailand.[1] Chantaranothai recognized
18 species in ailand.[2] Several species of the genus, for example,
Vite x negundo, Vitex doniana, Vitex polygama, Vitex trifolia, Vitex
rotundifolia, Vitex altissima, Vitex peduncularis and Vitex agnus‑castus
have long histories of use as phytoestrogens[3‑7] in the alternative
treatment of postmenopausal symptoms in many countries.[8] Vitex
pinnata is known to contain carbohydrates, phenolic compounds,
alkaloids, avonoids, saponins, tannins, steroids, amino acids, and
proteins.[9] e leaves of V. pinnata (syn Vite x pubescens Vahl.) have
been applied on cuts and wounds[10] and have been eaten to treat
hypertension and fever. e root is consumed for backache, body
pain, and fatigue.[1] A previous phytochemical study reported the
isolation of the ecdysteroids, pinnatasterone, 20‑hydroxyecdysone,
and turkesterone.[11] Another study reported a new iridoid glucoside,
pinnatoside, and three known avonoids, namely viscioside,
apigennin, and luteolin from the bark of V. pinnata.[12] Moreover,
Kamal etal.[13] have reported phytochemicals of V. pinnata,
principally stigmasterol, β‑sitosterol and avonoids (5‑hydroxy‑3,
7,4–trimethoxyavone, 5‑hydroxy‑7,4‑dimethoxy‑avone and
Pharmacogn. Mag.
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Cite this article as: Promprom W, Chatan W, Munglue P. Eect of Vitex pinnata
L. leaf extract on estrogenic activity and lipid prole in ovariectomized rats. Phcog
Mag 2020;16:S492-7.
Eect of Vitex pinnata L. Leaf Extract on Estrogenic Activity and
Lipid Prole in Ovariectomized Rats
Wilawan Promprom, Wannachai Chatan, Phukphon Munglue1
Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham, 1Department of Biology, Faculty of Science, Ubon
Ratchathani Rajabhat University, Ubon Ratchathani, Thailand
Submitted: 11-Oct-2019 Revised: 27-Dec-2019 Accepted: 11-Aug-2020 Published: 30-Nov-2020
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WILAWAN PROMPROM, etal.: Estrogenic Activity of Vitex pinnata L.
Pharmacognosy Magazine, Volume 16, Issue 5, July-September 2020 (Supplement 3) S493
5‑hydroxy‑3,3,4,7‑tetramethoxyavone). e three compounds
(β‑Sitosterol, avonoids and phenolic compounds) are known to
have the estrogenic activity.[14,15] However, there is no data on the
estrogenic properties and lipid prole of V. pinnata. us, we evaluated
the estrogenic activity and lipid prole of V. pinnata leaf extract in
ovariectomized(OVX) rats, an animal model of menopause.
Experimental
Plant materials
Leaves of V. pinnata were sampled and collected from the northeast of
ailand(February to March). Identication of a voucher specimen(no.
W. Chatan 1748) was performed by Assistant Prof. Dr. Wannachai
Chatan, Department of Biology, Faculty of Science, Mahasarakhan
University, ailand, and kept in the Natural Medicinal Mushroom
Museum or MSUT, Mahasarakham University, Maha Sarakham Province,
ailand. e leaves were washed, air‑dried, powdered and ethanolic
extraction. e extract was ltered by using lter paper. Evaporation was
done in a rotary evaporator. It was dried with a lyophilizer and then kept
at−20°C until use.
Characterization of the extract
Composition of leaf extract of V. pinnata was analyzed for its chemical
constituents by GC‑MS(GC‑MS)(GC‑MS 7890A Agilent Technology).
e identication of the extract composition was based on comparisons
with mass spectra and retention indices of authentic reference
compounds where possible.
Used animals
e thirty female Wistar rats(200–230 g) were chosen in the present
study. e rats were maintained by using the guidelines of the Committee
on the Care and Use of Laboratory Animal Resources, National Research
Council.[16] e experimental protocol was approved by the Institutional
Animal Care and Use Committee, Khon Kaen University,(approval no.
76/2017). e rats were kept in polypropylene cages(under the standard
conditions 12 h light and 12 h dark cycles; 25°C± 2°C) and had free
access to water ad libitum and a commercial pellet diet.
Design of experiments
Estrogenic activity was evaluated in bilaterally OVX rats.[17] e
parameters assessed were histology of vaginal and uterine wet weight.
e OVX rats were separated into ve groups(n = 6). All the rats in
each group received the treatment for 14days. Group1 (OVX[OVX];
control group) received 1 ml of 1%(v/v) Tween 80, Group2(standard
group) received 17 β‑estradiol at a dose 1 mg/kg. Group3, Group4, and
Group5(test group) received plant extract(1 ml) at doses of 100, 500,
and 1000 mg/kg B. W., respectively.
Body weight and relative organ weight
Aer 14days of treatment, the rats were sacriced under CO2 anesthesia.
e uterus and vagina were removed and weighed. Relative organ
weight(%ROW) of the vaginal and uterine weights were calculated as
in Eq. 1.[18]
% ROW =([OR/BW]) 100 (1)
Where OR=the absolute organ weight of the rat and BW= the body
weight of the rat.
Histological examination
e six excised uteri and vagina from each group were xed in formalin
and processed for histological preparations. Slides stained with
hematoxylin and eosin were examined under microscope for the changes
in cellular organization of the uterus and vagina.[19]
Lipid prole assessment
Serum levels of total cholesterol, triglyceride, high‑density lipoprotein
(HDL)‑cholesterol, and low‑density lipoprotein (LDL)‑cholesterol
were measured by using the automated enzymatic method of
SYNCHRONLX20Pro, at Khon Kaen University Community Outreach
Center.
Statistical analysis
Statistical analysis was carried out using one‑way(ANOVA) followed
by Duncan’s multiple comparison tests. e results are presented
as mean±standard error mean from six rats in each group. P< 0.05
(P<0.05) were considered signicant.
RESULTS
Gas chromatography-mass spectrometry
chromatogram of ethanol leaf extract from Vitex
pinnata
e GC‑MS analysis has shown the presence of 20
compounds: benzoic acid, methyl 4‑ethoxybenzoate,
4‑((1E)‑3‑hydroxy‑1‑propenyl)‑2‑methoxyphenol, 2 (4H)‑benzofuranone,
5,6,7,7a‑tetrahydro‑6‑hydroxy‑4,4,7a‑trimethyl‑, (6S‑cis), hexadecanoic
acid, methyl ester, n‑hexadecanoic acid, hexadecanoic acid, ethyl ester,
9,12,15‑Octadecatrienoic acid, 3,7,11,15‑Tetramethylhexadecen‑2‑en‑1‑ol,
9,12,15‑Octadecatrienoic acid, cis‑Vaccenic acid, Stearic acid,
2‑Palmitoylglycerol, all‑trans‑Squalene, 1‑Hexacosanol, alpha‑tocopherol,
Ergost‑5‑en‑3beta‑ol, Beta‑Stigmasterol, gamma‑sitosterol, and
maragenin I. e details of the identied phytoconstituents and their
therapeutic activities are presented in Table s1 and 2.
The body weight and relative organ weight
e means of the initial body weights of OVX rats were not dierent
among the groups aer the administration of V. pinnata for fourteen
days. However, at the end of the experiment, the results showed that the
bilateral OVX enhanced the increase of the nal body weight. When
compared to OVX, the standard drug 17 β‑estradiol(1 mg/kg B. W.)
produced statistically signicant(P < 0.05) decreases in body weight
[Table3].
V. pinnata extracts at 1000 mg/kg slightly increased uterine and vaginal
weight and endometrial thickness[Table 3]. e eect was not dose
dependent. e standard drug, 17 β‑estradiol produced statistically
signicant (P < 0.05), 1.40 ± 0.4 increase in uterine and vagina
weight[Table3].
Histology
Figure1 shows images of the representative of the transverse section of
uteri taken from one animal per treatment group. e OVX rats present
a typical atrophic condition of the uterine endometrium[Figure1a]. e
histology showed the typical atrophic features with thin endometrial
layer of the uterus. is layer contained poor vascularity and atrophied
uterine glands, which were covered by low cuboidal epithelial cells.
Oral administration of 17 β‑estradiol aected the structure and size
of all uteri as illustrated by an expansion in endometrial thickness,
an enlargement uterine gland and more numerous vascularity. e
bulky epithelial layer was well enlarged, as showed by the columnar
cell type in Figure1b. While, V. pinnata extract at 100 and 500 mg/
kg B. W. did not perform animportant change to the endometrial
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WILAWAN PROMPROM, etal.: Estrogenic Activity of Vitex pinnata L.
S494 Pharmacognosy Magazine, Volume 16, Issue 5, July-September 2020 (Supplement 3)
proliferation[Figure 1c and d], at 1000 mg/kg B. W. It made a slight
increase in endometrial proliferation, but pathology signs were not
detected[Figure1e].
Figure2 shows images the representative of the transverse section of
vagina taken from one rat per treatment group. e atrophic vaginal
epithelium was detected in OVX rats. Normally this layer consisted
of one or two shriveled cuboidal or attened squamous cells with a
diminutive mucous cells Figure 2a. e result determined that the
groups treated with the 17 β‑estradiol displayed a normal squamous
multilayered epithelium[Figure2b], while the layer number was similar
to the number in the OVX ratsand cornication was not found in the
100 and 500 mg/kg B. W. treatments[Figure2c and d]. For the group
treated with 1000 mg/kg B. W. extract, the epithelium thickness was
slightly expanded[Figure2e].
Lipid prole
e mean of triglycerides and serum total cholesterol were
signicantly (P < 0.05) decreased in the17 β‑estradiol and the V.
pinnata(500 and 1000 mg/kg B. W.) groups when compared to the OVX
control group[Table 4].
Table2: Therapeutic activity of the phytocompounds in Vitex pinnata leaf extract
RT Name of the compound Therapeutic activity**
4.20
10.77
10.95
13.30
13.70
14.16
15.46
15.80
15.98
16.34
29.65
31.95
Sodium benzoate
4‑((1E)‑3‑Hydroxy‑1‑propenyl)‑2‑me thoxyphenol
2(4H)‑Benzofuranone,
5,6,7,7a‑tetrahydro‑6‑hydroxy‑4,4,7a‑trimethyl‑,(6S‑cis)
Hexadecanoic acid, methyl ester
n‑Hexadecanoic acid
Hexadecanoic acid, ethyl ester
9,12,15‑Octadecatrienoic acid, methyl ester(Z, Z, Z)‑
3,7,11,15‑Tetramethylhexadecen‑2‑en‑1‑ol
cis‑Vaccenic acid
Stearic acid
Alpha‑tocopherol
Beta‑Stigmasterol
Antifungal, preservative
Anticancer, antitumor
Anti‑HIV‑integrase, increase tyrosine hyroxylase activity
Antibacterial, antifungal,[20] increase aromatic amino acid decarboxylase activity
Antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant
Antiandrogenic, avor, hemolytic 5‑alpha reductase inhibitor
Antioxidant, hypocholesterolemic, nematicide, pesticide
Antiinammatory, hyopcholesterolemic, cancer preventive, hepatoprotective, nematicide,
insectifuge, antihistaminic
Antimicrobial
Anhibit production of uric acid
Inhibit oroduction of uric acid
Antiageing, analgesic, antidiabetic, anttiinammatory, antioxidant, antidermatitic,
antileukemic, antitumor, anticancer, hepatoprotective, hypocholesterolemic,
antiulcerogenic, vasodilator, antispasmodic, antibronchitic, anticoronary
Antiamyloid‑beta, hypocholesterolemic
**Source: Dr. Duke’s Phytochemical and Ethnobotanical Databases.[20] RT: Retention time
Table1: Identied phytoconstitutents from the ethanol leaf extract of V. pinnata
Retention time Name of the compounds Molecular formula Molecular weight Area
4.20 Sodium benzoate C7H5NaO2144.10 2.31
7.50 Benzoic acid, 4‑hydroxy‑, methyl ester C8H8O3152.15 0.53
10.77 4‑((1E)‑3‑Hydroxy‑1‑propenyl)‑2‑me thoxyphenol C10H12O3180.20 1.71
10.95 2(4H)‑Benzofuranone, 5,6,7,7a‑
tetrahydro‑6‑hydroxy‑4,4,7a‑trimethyl‑,(6S‑cis) C11H16O196.243 0.38
13.30 Hexadecanoic acid, methyl ester C17H34O 270.451 0.33
13.70
14.16
15.46
15.80
15.93
15.98
16.34
16.85
20.89
25.63
29.45
29.65
31.29
31.95
33.23
34.78
35.913
n‑Hexadecanoic acid
Hexadecanoic acid, ethyl ester
9,12,15‑Octadecatrienoic acid methyl ester(Z, Z, Z)‑
3,7,11,15‑Tetramethylhexadecen‑2‑en‑1‑ol
9,12,15‑Octadecatrienoic acid
cis‑Vaccenic acid
Stearic acid
Stearic acid
2‑Palmitoylglycerol
All‑trans‑Squalene
1‑Hexacosanol
Alpha‑Tocopherol
Ergost‑5‑en‑3beta‑ol
Beta‑Stigmasterol
Gamma‑Sitosterol
Maragenin I
Alpha‑Tocopherol
C16H32O
C18H36O
C18H30O
C20H40O
C18H30O
C18H34O
C18H36O
C18H36O
C19H38O
C30H50
C26H54O
C29H50O2
C28H48O
C29H48O
C29H50O
C29H46O2
C29H50O2
256.424
284.477
278.430
296.531
278.430
282.468
284.477
284.477
330.503
410.718
382.706
430.717
400.69
412.702
414.706
426.685
430.717
3.31
1.73
0.26
16.39
3.87
1.18
2.14
1.46
0.37
0.71
0.46
0.44
0.99
2.80
7.58
0.74
0.46
RT: Retention time
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WILAWAN PROMPROM, etal.: Estrogenic Activity of Vitex pinnata L.
Pharmacognosy Magazine, Volume 16, Issue 5, July-September 2020 (Supplement 3) S495
DISCUSSION
Ovariectomy can cause estrogen deficiency, the reproductive
cycle stops, and an increase in body weight and the changes in the
plasma lipid levels(a risk factor for cardiovascular disease). When
female rats do not have an estrogen, the vagina and uterus undergo
atrophy of the endometrium. However, giving estrogenic substances
helps to prevent atrophic changes of the organ and also stimulates
mitosis in the epithelia of the endometrium in OVX females.[21] In
the present study, treatment with 17 β‑estradiol showed evidence of
uterotrophic activity as indicated by uterine and vagina weight and
histological changes of the uterus and vagina in OVX rats. Low and
medium doses(100 and 500 mg/kg of extract had not effect on the
uterus and vagina. Interestingly, the result showed that the ethanolic
plant extract at high dose(1000 mg/kg) produced slightly increased
the weight of the uterus and vagina and epithelium in OVX rats.
GC‑MS analysis has shown the presence of V. pinnata leaf extract
found few phytosterols such as β‑stigmasterol and gamma‑sitosterol.
Kuiper etal. 1998 found that phytochemicals such as flavonoids,
steroids (phytosterols) and phenolic compounds are estrogenic
substances.[15] Thus, the effects of the plant extract can be attributed
to its weak estrogenic activity.
Phytochemical analysis of the leaves of V. pinnata revealed the
presence of constituents that are known to exhibit medicinal as well
as physiological activities. GC‑MS has been the best technique used
Table3: Body and relative organ weight
Treatment
group
Final body
weight(g)
Uterus and
vagina weight(g)
Uterus and vagina
weight/body weight(%)
OVX control 267.00±1.21b0.22±0.1b0.08±0.0b
17β‑estradiol 213.33±50.7a1.40±0.4a0.67±0.2a
V. pinnata 100 269.83±2.62b0.22±0.2b0.08±0.0b
V. pinnata 500 276.33±10.76b0.25±0.0b0.09±0.0b
V. pinnata 1000 269.00±5.27b0.32±0.7b0.12±0.3b
a,bMean within a column with dierent letters are dierent(P<0.05). V. pinnata: Vitex pinnata; OVX: Ovariectomized
Figure2: Photomicrograph of haematoxylin and eosin stained transverse
section of vagina of Vitex pinnata; (a) OVX, (b) 17β-estradiol, (c) Vitex
pinnata (100 mg/kg), (d) Vitex pinnata (500 mg/kg.), (e) Vitex pinnata
(1000 mg/kg b.w.)(H and E, ×40)
d
c
b
a
e
Figure 1: Photomicrographs of haematoxylin and eosin stained
transverse section of uterus of Vitex pinnata;(a) OVX,(b) 17β-estradiol,
(c) Vitex pinnata (100 mg/kg), (d) Vitex pinnata (500 mg/kg), (e) Vitex
pinnata (1000 mg/kg)(H and E, ×40)
d
c
b
a
e
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WILAWAN PROMPROM, etal.: Estrogenic Activity of Vitex pinnata L.
S496 Pharmacognosy Magazine, Volume 16, Issue 5, July-September 2020 (Supplement 3)
Table4: Mean lipid levels(mg/dl) of ovariectomized rats supplemented with various doses of Vitex pinnata
Treatment Lipid prole(mg/dl)
Total cholesterol Triglyceride HDL‑cholesterol LDL‑cholesterol
OVX control 52.50±1.91c93.17±17.90a14.17±0.65a17.67±3.81b
17β‑estradiol 31.33±3.15a57.83±7.96b18.00±1.86a1.77±2.36a
V. pinnata 100 48.33±5.58c57.33±10.38b19.00±2.96a17.87±2.86b
V. pinnata 500 40.50±4.16b52.67±4.88b17.50±1.54a10.57±5.88b
V. pinnata 1000 41.00±1.78b51.33±6.49b19.50±0.76a13.07±3.71b
a,b,cMean within a column with dierent letters are dierent(P<0.05). V. pinnata: Vitex pinnata; OVX: Ovariectomized; LDL: Low‑density lipoprotein; HDL: High‑density
lipoprotein
for screening, identication and quantication of many bioactive
compounds in plant extracts.[22] GC‑MS data revealed that the
ethanolic extract of V. pinnata contains n‑Hexadecanoic acid, 9, 12,
15‑Octadecatrienoic acid and the unsaturated fatty acid stearic acid,
which is known to have estrogenic activity. e estrogenic activity
shown by the extract of V. pinnata can be attributed to the presence
of unsaturated fatty acid.[23] us this type of GC‑MS analysis is the
rst step toward understanding the nature of the active principles in
theethanolic extract of V. pinnata.
is study showed that feeding V. pinnata extracts at doses of 500 and
1000 mg/kg B. Wresulted in signicant decreases in total cholesterol
and triglyceride and this might have been a consequence of feeding
phytoestrogen. Hwang etal. 2001[23] showed that phytoestrogens
are potent low density lipoprotein antioxidants. Other reports have
demonstrated that V. agnus‑castus phytoestrogen also reduced
LDL‑cholesterol and triglyceride and produced HDL‑cholesterol in
OVX rats.[24] e results indicate that the leaf extract from V. pinnata
possess hypolipidemic activityis more likely associated with the
presence of unsaturated fatty acid which is known to have estrogenic
activity. However, the mechanism of the lipid‑lowering eects of
phytoestrogen is not clear. Our results of gas chromatography‑mass
spectrometry veried that the phytoestrogen of V. pinnata L. Which is
a technique used for screening/indentication/quantication of many
chemical compounds in plant extracts.[25]
CONCLUSION
is study showed that a higher dose V. pinnata L.
extract(1000 mg/kg) possesses the estrogenic activity and resulted in
decreases of total cholesterol and triglycerides in OVX rats. GC‑MS
analysis data revealed that the ethanolic extract of V. pinnata contains
phytosterols and unsaturated fatty acids. ese phytosterols and
unsaturated fatty acids are known to possess estrogenic activity.
Furthermore, the results lend some support for the traditional use of
this plant in the management of gynecologic disorders and lipid proles
in menopause women.
Acknowledgements
anks for linguistic advice from Dr Adrian Roderick Plant, Division
of Research Facilitation and Dissemination, Mahasarakham University.
Financial support and sponsorship
We gratefully acknowledge Mahasarakham University 2017, ailand
for nancial support.
Conicts of interest
ere are no conicts of interest.
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