Estrogen-Like Activity of Volatile Components From Vitex Rotundifolia L.
By Hu, Yuan Hou, Ting-Ting; Xin, Hai-Liang; Zhang, Qiao-Yan; Et al
Background & objectives: The fruits of Vitex rotundifolia L. are used as a traditional medicine in China. Dried fruits of V. rotundifolia (rich in essential oils) have been known to show strong estrogenic activity. Therefore, we investigated volatile components in the essential oil isolated from the fruits of V. rotundifolia and its estrogen-like biological activity in human breast adenocarcinoma cells. Methods: Essential oil was extracted by SFE (supercritical fluid extraction) and its chemical composition was analysed by gas chromatography-mass spectrometry (GC-MS). The estrogenic activity of essential oil was evaluated in the MCF-7 cells by proliferation assay.
Results: The chemical composition of 40 compounds was determined and this represented 84.1 per cent of the total oil which was characterized by a high content of unsaturated fatty acids (54.84% of total oil). The oil contained mainly linoleic acid (47.46%), palmitic acid (5.18%), untriacontane (2.28%), bicetyl (2.61%) and stearic acid (2.2%). The essential oil strongly stimulated the proliferation of MCF-7 cells, and this effect was significantly inhibited by the specific estrogen receptor antagonist ICI 182,780.
Interpretation & conclusion: Our results showed estrogen-like biological activity of the essential oil of the fruits from V. rotundifolia, and in future has a potential medical application.
Key words Essential oil – phytoestrogens – SFE – Vitex rotundifolia
Vitex, a genus of the Verbenaceae family, contains about 250 species many of which have been used as traditional medicinal plants. One of these, Vitex rotundifolia L., grows to a height of 2- 3 m, and is distributed in the Mediterranean region, Central Asia, and along the seacoast from south to north of China. The fruits of V. rotundifolia has been used as a folk medicine for the treatment of headache, cold, migraine, eye pain, asthma, chronic bronchitis, and gastrointestinal infections such as bacterial dysentery and diarrhoea due to gastroenteritis1-3. V. rotundifolia is the same genus as me commonly planted ornamental, chaste-tree (Vitex agnus- castus). This is probably one of the most important herbs used to treat female hormonal disorders such as pre menstrual syndrome (PMS) and is considered to be a hormonal modulator4,5. Essential oil of V. agnus- castus has been shown to affect female hormones6-7, Liu et al8 reported that linoleic acid present in the fruits of V. agnus- castus can bind to estrogen receptors and induce certain estrogen inducible ER mRNA up-regulation8.
We undertook the present study to examine the chemical composition of essential oil isolated from the fruits of V. rotundifolia and to evaluate if this essential oil has estrogenic activity in human breast adenocarcinoma (MCF-7) cells in culture.
Material & Methods
Plant material: V. rotundifolia L. was collected at a lakefront in Xinjian, Jiangxi province, in China and authenticated by Prof. Hanchen Zheng, Second Military Medical University, Shanghai, People Republic of China. The voucher specimens of these plants were deposited at the Herbarium of Department of Pharmacognosy, Second Military Medical University.
Chemicals: ICI 182, 780 [7 a-{9(4,4,5,5,5-pentafluoropentyl) sulphfinyl} nonyl] -estra- 1,3,5(10) -triene-3,17beta-diol], a steroidal antiestrogen was purchased from Tocris, Cookson, United Kingdom. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsuphoxide (DMSO), 17-beta estradiol, phenol red-free Dulbecco’s modified Eagle’ medium (phenol red-free DMEM), charcoal-dextrantreated foetal bovine serum (CD-FBS) were purchased from Sigma-Aldrich (St. Louis. MO, USA). DMEM medium and foetal bovine serum were obtained from Gibco BRL Life Technologies, Scotland.
Preparation and analysis of essential oil: Extraction of essential oil from the fruits of V. rotundifolia was done using the Speed supercritical fluid extraction (SFE) instrument (HA-121-50- 01, China)9-10. The extraction was performed by placing 20kg of the plant fruits in a 54L volume extraction vessel. The samples were extracted for 90 min at an extraction pressure of 20 MPa, 40[degrees]C at static extraction step and were further separated at a pressure of 8 MPa, 50[degrees]C and 6 MPa 35 [degrees]C for the two dynamic extraction steps.
Gas chromatography-mass spectrometry (GC-MS) : GC/MS analyses were performed using the Finnigan Vayager equipped with a VF-5ms fused-silica capillary column (30 x 0.25 mm i.d., film thickness 0.25 [mu]m, VARIAN,USA). The column temperature was programmed at 70[degrees]C (2 min), increasing to 300[degrees]C at rate of 20[degrees]C/min, injector temperature was 250[degrees]C; carrier gas (helium) was set at a flow rate of 1.0 ml/min; ionization energy 70 eV, and scan mode EI. 1[mu]l of sample of essential oil extract was injected with split ratio of 1:20 (v/v) and the compounds were identified by matching their mass spectra and retention times with standard obtained from NIST (National Institute of Standards and Technologies USA) whenever possible.
Assay of estrogen-like biological activity of essential oil extracted from V. rotundifolia : Cell culture-Estrogen receptor- positive human breast adenocarcinoma MCF-7 cells provided by the Institute of Bio chemistry and Cell Biology were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing antibiotics (100 IU/ml penicillin and 100 ug/ml streptomycin), supplemented with 10 per cent foetal bovine serum. Cells were grown at 37[degrees]C in a humidified atmosphere of 95 percent air:5 percent CO2. Medium was renewed 3-4 times per week.
Charcoal-dextran stripped human serum preparation: In order to minimize the estrogenic activity of serum, steroid hormones were extracted from pooled human serum by treatment with charcoal and dextran (purchased from Gibco BRL). The charcoal- dextran stripped human serum was filtered and stored at -20[degrees]C until used.
Proliferation assay of MCF-7 cells- Confluent MCF-7 cells were washed twice with D-Hanks solution before the addition of 0.25 per cent trypsin-EDTA. The flask was left for 2-3 min at room temperature (approx. 20[degrees]C), after which the cells were detached, resuspended in full medium, counted and seeded in a 25ml bottle at a density of 1 x 10^sup 4^ cells/well in normal growth medium. The cells were completely attached to the well bottom after 48 h after which the cells were washed with D-Hanks, and the estrogen-free medium (phenol red free DMEM with 5 per cent charcoal- dextran stripped human serum) was then added. Following another 24h of pre-treatment, the cells were exposed to increasing concentrations of estradiol (1 nM) and the different dosages of essential oil extract (5mg/l to 500 mg/l). The concentration of essential oil was set at 50 mg/1 to assay the effect of co- treatment with the pure estrogen receptor antagonist-0.1 [mu]M ICI 182,780 on cell proliferation with the MTT method” after 7 days, with the medium being changed every 3 days. In the assessment method, the cells were incubated with 100 [mu]l of 5 mg/ml MTT solution for 4 h and following this, the medium was discarded, and 600[mu]l DMSO was added. The absorbance at 540 nm was determined with an ELx800 universal microplate reader (Bio-TEK, USA) and the cell numbers were correlated with absorbance values by the MTT method. The results are expressed as proliferation compared with that induced by treatment with 1 nM estradiol.
Result & Discussion
Chemical composition of the essential oil : V. rotundifolia fruits extraction produced dark yellow and viscous essential oil, the physico-chemical characteristics were as follows: oil yield (w/ w%) was 4.4, refractive index (RF) at 20[degrees]C was 1.7065 and density was 1.810 g/ml; chemical composition is shown in the Table. Although the essential oil of V. rotundifolia fruits extracted using steam distillation has been previously investigated 11, the composition of V. rotundifolia fruits essential oil from China using SFE process has not been studied previously.
The extraction of essential oil components using solvent at high pressure, or supercritical fluids, has received much attention in the past several years, especially in food, pharmaceutical and cosmetic industries, because it presents an alternative to conventional processes such as organic solvent extraction and steam distillation12,13. Our experiment showed that the SFE process can extract many high polarity compounds. By comparing the composition of the product with hydrodistilled oil11,14, higher levels of the camphene and pinene were found in the hydrodistilled oil. By using hydrodistillation method mainly delta3-carene(C^sub 10^H^sub 16^), beta-phellandrene (C^sub 10^H^sub 16^), camphene, 1,8-caieole(C^sub 10^H^sub 18^O), and isoterpinolene(C^sub 10^H^sub 16^) were isolated, however, these compounds were not found in the SFE products. In contrast, fatty acids such as linoleic acid, palmitic acid, stearic acid and some sterols were found in the SFE products (Table), which in were present in negligible amounts in the distilled oils.
Stimulation of MCF-7 cell proliferation: The proliferative effect of fruit extract relative to that of estradiol (1 nM, 100%) was expressed as relative proliferative effect (RPE) (Fig. 1). The essential oil was able to significantly stimulate MCF-7 cell proliferation at concentrations of 50mg/l (P<0.05) and 500mg/l (/ MLOl) (Fig. 1). The maximal proliferative effect of the oil was achieved at 50mg/l and was almost equivalent to the effect displayed by 1 nM estradiol (RPE was 96.7+-15.3%).The proliferative effects of 1 nM estradiol, 50mg/l concentration of essential oil were blocked by addition of 100 nM ICI 182,780 (Fig. 2). The RPE of estradiol and essential oil after treatment with ICI 182,78 decreased to 17.0+- 7.0 per cent. Phytoestrogens are polyphenols non-steroidal plant derived compounds with estrogen-like biological activity, which have been associated with a variety of changes in the reproductive system and certain hormone-dependent diseases, such as prostate cancer, colon cancer, breast cancer and PMS15,16. Essential fatty acids, such as palmitic acid, oleic acid, linoleic acid, stearic acid were also found in V. agnuscastus17. Among them, linoleic acid was reported to display anti-PMS activity8. These fatty acids are similar to those found in the essential oil of the fruits of y rotundifolia extracted by SFE. Our results showed that the essential oil of V. rotundifolia could significantly stimulate the growth of MCF-7 cells and the proliferation stimulatory effect could be reversed by co-administration of a pure anti-estrogen ICI 182,780.
In conclusion, our study showed that the essential oil of V. rotundifolia possess potential estrogen-like activity as shown in MCF-7 cells, and thus has a potential to be used as a medicine or added to food as a health care products which have a role to play in balancing the changes in the reproductive system and for treating certain hormone-dependent diseases.
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Yuan Hu, Ting-Ting Hou, Hai-Liang Xin, Qiao-Yan Zhang, Han-Chen Zheng, Khalid Rahman* & Lu-Ping Qin
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China & *School of Biomolecular Science, Faculty of Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, England, UK
Received October 12. 2006
Reprint requests: Dr Lu-Ping Qin, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University
325, Guohe Road, Shanghai 200433. China
e-mail: ipqin@smmu.edu.in, lupingqin@hotmail.com
Copyright Indian Council of Medical Research Jul 2007
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