Effect of Vitex pinnata L. leaf extract on estrogenic activity and lipid profile in ovariectomized rats
Wilawan Promprom1, Wannachai Chatan1, Phukphon Munglue2
1 Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham, Thailand
2 Department of Biology, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Ratchathani, Thailand
|Date of Submission||11-Oct-2019|
|Date of Decision||27-Dec-2019|
|Date of Acceptance||11-Aug-2020|
|Date of Web Publication||30-Nov-2020|
Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham, 44150
Source of Support: None, Conflict of Interest: None
| 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 profile 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 five 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 14 days. 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 significant (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 benefits for postmenopausal women.
Keywords: 17 β-estradiol, estrogenic activity, lipid profile, ovariectomized rats, Vitex pinnata
|How to cite this article:|
Promprom W, Chatan W, Munglue P. Effect of Vitex pinnata L. leaf extract on estrogenic activity and lipid profile in ovariectomized rats. Phcog Mag 2020;16, Suppl S2:492-7
|How to cite this URL:|
Promprom W, Chatan W, Munglue P. Effect of Vitex pinnata L. leaf extract on estrogenic activity and lipid profile in ovariectomized rats. Phcog Mag [serial online] 2020 [cited 2021 Dec 6];16, Suppl S2:492-7. Available from: http://www.phcog.com/text.asp?2020/16/5/492/301886
- 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 significant (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).
| Introduction|| |
The 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 Thailand. Chantaranothai recognized 18 species in Thailand. Several species of the genus, for example, Vitex negundo, Vitex doniana, Vitex polygama, Vitex trifolia, Vitex rotundifolia, Vitex altissima, Vitex peduncularis and Vitex agnus-castus have long histories of use as phytoestrogens,,,, in the alternative treatment of postmenopausal symptoms in many countries.Vitex pinnata is known to contain carbohydrates, phenolic compounds, alkaloids, flavonoids, saponins, tannins, steroids, amino acids, and proteins. The leaves of V. pinnata (syn Vitex pubescens Vahl.) have been applied on cuts and wounds and have been eaten to treat hypertension and fever. The root is consumed for backache, body pain, and fatigue. A previous phytochemical study reported the isolation of the ecdysteroids, pinnatasterone, 20-hydroxyecdysone, and turkesterone. Another study reported a new iridoid glucoside, pinnatoside, and three known flavonoids, namely viscioside, apigennin, and luteolin from the bark of V. pinnata. Moreover, Kamal et al. have reported phytochemicals of V. pinnata, principally stigmasterol, β-sitosterol and flavonoids (5-hydroxy-3, 7,4–trimethoxyflavone, 5-hydroxy-7,4-dimethoxy-flavone and 5-hydroxy-3, 3, 4, 7-tetramethoxyflavone). The three compounds (β-Sitosterol, flavonoids and phenolic compounds) are known to have the estrogenic activity., However, there is no data on the estrogenic properties and lipid profile of V. pinnata. Thus, we evaluated the estrogenic activity and lipid profile of V. pinnata leaf extract in ovariectomized (OVX) rats, an animal model of menopause.
Leaves of V. pinnata were sampled and collected from the northeast of Thailand (February to March). Identification of a voucher specimen (no. W. Chatan 1748) was performed by Assistant Prof. Dr. Wannachai Chatan, Department of Biology, Faculty of Science, Mahasarakhan University, Thailand, and kept in the Natural Medicinal Mushroom Museum or MSUT, Mahasarakham University, Maha Sarakham Province, Thailand. The leaves were washed, air-dried, powdered and ethanolic extraction. The extract was filtered by using filter 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). The identification of the extract composition was based on comparisons with mass spectra and retention indices of authentic reference compounds where possible.
The thirty female Wistar rats (200–230 g) were chosen in the present study. The rats were maintained by using the guidelines of the Committee on the Care and Use of Laboratory Animal Resources, National Research Council. The experimental protocol was approved by the Institutional Animal Care and Use Committee, Khon Kaen University, (approval no. 76/2017). The 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. The parameters assessed were histology of vaginal and uterine wet weight. The OVX rats were separated into five groups (n = 6). All the rats in each group received the treatment for 14 days. Group 1 (OVX [OVX]; control group) received 1 ml of 1% (v/v) Tween 80, Group 2 (standard group) received 17 β-estradiol at a dose 1 mg/kg. Group 3, Group 4, and Group 5 (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
After 14 days of treatment, the rats were sacrificed under CO2 anesthesia. The uterus and vagina were removed and weighed. Relative organ weight (%ROW) of the vaginal and uterine weights were calculated as in Eq. 1.
% ROW = ([OR/BW]) 100 (1)
Where OR = the absolute organ weight of the rat and BW = the body weight of the rat.
The six excised uteri and vagina from each group were fixed 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.
Lipid profile 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 was carried out using one-way (ANOVA) followed by Duncan's multiple comparison tests. The results are presented as mean ± standard error mean from six rats in each group. P < 0.05 (P <0.05) were considered significant.
| Results|| |
Gas chromatography-mass spectrometry chromatogram of ethanol leaf extract from Vitex pinnata
The 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. The details of the identified phytoconstituents and their therapeutic activities are presented in [Table 1] and [Table 2].
|Table 1: Identified phytoconstitutents from the ethanol leaf extract of V. pinnata|
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|Table 2: Therapeutic activity of the phytocompounds in Vitex pinnata leaf extract|
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The body weight and relative organ weight
The means of the initial body weights of OVX rats were not different among the groups after 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 final body weight. When compared to OVX, the standard drug 17 β-estradiol (1 mg/kg B. W.) produced statistically significant (P <0.05) decreases in body weight [Table 3].
V. pinnata extracts at 1000 mg/kg slightly increased uterine and vaginal weight and endometrial thickness [Table 3]. The effect was not dose dependent. The standard drug, 17 β-estradiol produced statistically significant (P <0.05), 1.40 ± 0.4 increase in uterine and vagina weight [Table 3].
[Figure 1] shows images of the representative of the transverse section of uteri taken from one animal per treatment group. The OVX rats present a typical atrophic condition of the uterine endometrium [Figure 1]a. The histology showed the typical atrophic features with thin endometrial layer of the uterus. This layer contained poor vascularity and atrophied uterine glands, which were covered by low cuboidal epithelial cells. Oral administration of 17 β-estradiol affected the structure and size of all uteri as illustrated by an expansion in endometrial thickness, an enlargement uterine gland and more numerous vascularity. The bulky epithelial layer was well enlarged, as showed by the columnar cell type in [Figure 1]b. While, V. pinnata extract at 100 and 500 mg/kg B. W. did not perform animportant change to the endometrial proliferation [Figure 1]c and d], at 1000 mg/kg B. W. It made a slight increase in endometrial proliferation, but pathology signs were not detected [Figure 1]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)|
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[Figure 2] shows images the representative of the transverse section of vagina taken from one rat per treatment group. The atrophic vaginal epithelium was detected in OVX rats. Normally this layer consisted of one or two shriveled cuboidal or flattened squamous cells with a diminutive mucous cells [Figure 2]a. The result determined that the groups treated with the 17 β-estradiol displayed a normal squamous multilayered epithelium [Figure 2]b, while the layer number was similar to the number in the OVX ratsand cornification was not found in the 100 and 500 mg/kg B. W. treatments [Figure 2]c and d]. For the group treated with 1000 mg/kg B. W. extract, the epithelium thickness was slightly expanded [Figure 2]e.
|Figure 2: 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)|
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The mean of triglycerides and serum total cholesterol were significantly (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].
|Table 4: Mean lipid levels (mg/dl) of ovariectomized rats supplemented with various doses of Vitex pinnata|
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| 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. 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 et al. 1998 found that phytochemicals such as flavonoids, steroids (phytosterols) and phenolic compounds are estrogenic substances. 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 for screening, identification and quantification of many bioactive compounds in plant extracts. 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. The estrogenic activity shown by the extract of V. pinnata can be attributed to the presence of unsaturated fatty acid. Thus this type of GC-MS analysis is the first step toward understanding the nature of the active principles in theethanolic extract of V. pinnata.
This study showed that feeding V. pinnata extracts at doses of 500 and 1000 mg/kg B. W resulted in significant decreases in total cholesterol and triglyceride and this might have been a consequence of feeding phytoestrogen. Hwang et al. 2001 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. The results indicate that the leaf extract fromV. 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 effects of phytoestrogen is not clear. Our results of gas chromatography-mass spectrometry verified that the phytoestrogen of V. pinnata L. Which is a technique used for screening/indentification/quantification of many chemical compounds in plant extracts.
| Conclusion|| |
This 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. These 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 profiles in menopause women.
Thanks 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, Thailand for financial support.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]