Evaluation of estrogenic activity of alcoholic extract of rhizomes of Curculigo orchioides

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Journal of Ethnopharmacology 114 (2007) 241–245

Evaluation of estrogenic activity of alcoholic extract of rhizomes of Curculigo orchioides K. Vijayanarayana a,∗ , Rashmi S. Rodrigues b , K.S. Chandrashekhar a , E.V.S. Subrahmanyam a a

N.G.S.M Institution of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore 574160, Karnataka, India b Lotus Labs Private Limited, Bangalore 560052, Karnataka, India Received 3 April 2007; received in revised form 3 July 2007; accepted 3 August 2007 Available online 12 August 2007

Abstract The rhizomes of Curculigo orchioides Gaertn. (Amaryllidacea) is an important Ayurvedic as well as Unani drug. It is present in several drug formulations used in the treatment of menorrhagia and other gynecological problems. In this study, we conducted a comparative study of estrogenic activity of alcoholic extract of Curculigo orchioides with diethylstilbestrol in bilaterally ovariectomized young albino rats. Bilaterally ovariectomized albino rats were divided into five groups (n = 9) receiving different treatments, consisting of vehicle (0.6% w/v sodium carboxy methyl cellulose), ethanolic extract of rhizomes of Curculigo orchioides at three different doses (viz., 300, 600, 1200 mg/kg body weight) and standard drug diethylstilbestrol (DES) at a dose of 2 mg/kg body weight. All these were administered orally daily for 7 days. Estrogenic activity was assessed by taking percentage vaginal cornification, uterine wet weight, uterine glycogen content and uterine histology as parameters of assessment. Alcoholic extract of Curculigo orchioides showed a significant increase in percentage vaginal cornification, uterine wet weight (P < 0.001), uterine glycogen content (P < 0.001) and a proliferative changes in uterine endometrium compared to the control. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Curculigo orchioides; Estrogenic activity; Ovariectomized rats; Vaginal cornification

1. Introduction Curculigo orchioides Gaertn. (Amaryllidacea) is a small perennial herb, with an elongated tuberous rootstock, found wild in sandy regions of hotter parts of India and Srilanka. The rhizome is an important Ayurvedic as well as Unani drug. It is present in several drug formulations for a wide variety of ailments, especially as a general tonic, as an aphrodisiac and in the treatment of bone fracture (Nadkarni, 1976). Its use in the treatment of menorrhagia and other gynecological problems is mentioned in ‘Dravyangunavignyana’ (Gogate, 1982). The rhizomes were known to contain resin, tannin, mucilage, fat and ash containing oxalate of calcium. Various flavonoid, phenolic and triterpenoid glycosides were isolated from the rhizomes of Curculigo orchioides. ␤-Sitosterol and crystalline

∗

Corresponding author. Tel.: +91 944 8255124; fax: +91 824 2203992. E-mail address: [email protected] (K. Vijayanarayana).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.08.009

needles of sapogenin have also been detected (Krishna Kumar and Vaidya, 1992; Mashelkar et al., 2000). Formulations containing Curculigo orchioides are being promoted for use in conditions like irregular menses, menopause, breast cancer and infertility (Sharma et al., 1991; Prema, 2002). Thus the evaluation of the estrogenic activity of Curculigo orchioides was carried out to know whether its beneficial effect in various gynecological problems and breast cancer is due to its estrogenic activity. 2. Materials and methods 2.1. Material Rhizomes of Curculigo orchioides were collected from the field areas of Manjeshwar in the month of December. The plant was identified and confirmed by Dr. Noeline J. Pinto, Head of the Department of Botany, St. Agnes College, Mangalore. A voucher specimen (V. no. NGSM 3561) has been deposited in the pharmacognosy department of N.G.S.M. Institute of

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Table 1 Effect of alcoholic extract of Curculigo orchioides on uterine wet weight and uterine glycogen content in bilaterally ovariectomized albino rats Group

Treatment (route)

Dose (mg/kg)

Uterine wet weight (mg)

1 2 3 4 5

Control 0.6% w/v Sod. CMC (p.o.) Standard DES (p.o.) Curculigo orchioides extract (p.o.)

– 2 300 600 1200

98.7 221.3 162.55 184.95 262.2

± ± ± ± ±

3.1 8.3b 4.03b 6.34b 7.2b

Uterine glycogen content (␮g/mg of uterine tissue) 0.4387 1.0270 0.7284 0.9254 1.2169

± ± ± ± ±

0.024 0.046b 0.036a 0.050b 0.118b

Values are mean ± S.E.M. of nine animals in each group. Data were analysed by one-way ANOVA followed by Dunnett’s t-test. a P < 0.01 compared to control group. b P < 0.001 compared to control group.

Pharmaceutical Sciences, Mangalore. They were dried under shade, powdered and subjected to Soxhlet extraction with 80% ethanol. The extract was concentrated to get a brownish sticky mass. The yield was 8.4%.

estimation by anthrone method (Dayton et al., 1980). This study was conducted in accordance with the latest CPCSEA guidelines and the experimental protocol was approved by Institutional Animals Ethics Committee.

2.2. Animals and experimental set-up

2.3. Statistical analysis

Estrogenic activity of the alcoholic extract was assessed in bilaterally ovariectomized immature female Sprague–Dawley rats of 20–30 days old (weighing 50–60 g) using a standardized method with few modifications, taking percentage vaginal cornification, uterine wet weight, uterine glycogen content and uterine histology as parameters of assessment (Jonathan et al., 1995). The ovariectomized rats were divided into 5 groups each consisting of nine animals. Estrogenic activity of phytoestrogens ranges from 1/500 to 1/1000 to the activity of diethylstilbestrol (DES) (Cassidy, 1999). Based on this assumption a dose range between 300 and 1200 mg/kg of Curculigo orchioides extract was used:

One-way analysis of variance (ANOVA) followed by Dunnett’s t-test was used to analyze the difference in uterine wet weight, uterine glycogen content between different groups of treatments. Data of vaginal cornification were analyzed by twoway ANOVA followed by Bonferroni test.

• Group 1 (control): received 0.6% (w/v) Sod. CMC suspension at a dose of 10 ml/kg. • Group 2 (standard): received aqueous suspension of diethylstilbestrol (NEMESTROLTM ) in 0.6% (w/v) Sod. CMC at a dose of 2 mg/kg. • Group 3 (test): received aqueous suspension of alcoholic extract of Curculigo orchioides in 0.6% (w/v) Sod. CMC at a dose of 300 mg/kg. • Group 4 (test): received aqueous suspension of alcoholic extract of Curculigo orchioides in 0.6% (w/v) Sod. CMC at a dose of 600 mg/kg. • Group 5 (test): received aqueous suspension of alcoholic extract of Curculigo orchioides in 0.6%(w/v) Sod. CMC at a dose of 1200 mg/kg. All these were administered orally daily for 7 days. Vaginal cornification was examined daily. After 24 h of last treatment, hysterectomy was performed in all rats under pentobarbitone anaesthesia. Harvested uteri were cleaned carefully from adhering connective tissue and weighed. The three excised uteri from each group were fixed in Bouin’s fluid and processed for histological preparations. Haematoxylin and eosin stained slides were examined under microscope for changes in cellular organization. The remaining uteri were used for glycogen

3. Results Assessment of estrogenic activity of alcoholic extract of Curculigo orchioides was done by taking percentage vaginal cornification, uterine wet weight, uterine glycogen content and uterine histology as parameters. The alcoholic extract of Curculigo orchioides showed a dose dependent, statistically significant (P < 0.001) increase in uterine wet weight and uterine glycogen content compared to control (Table 1). The standard drug DES produced statistically significant (P < 0.001), 2.24-fold increase in uterine wet weight. The alcoholic extract of Curculigo orchioides-induced proliferative changes in the uterine endometrium as evidenced by

Fig. 1. Photomicrograph of haematoxylin and eosin stained transverse section of uterus of Curculigo orchioides extract (1200 mg/kg, p.o.) treated rat, showing proliferative stage (i.e., stimulated endometrium with loose stroma).

0 99.8 ± 2.81a 94.8 ± 2.52a 97.1 ± 1.62a 99.8 ± 0.16a 0 99.2 ± 2.95a 91.1 ± 2.21a 94 ± 3.21a 89.5 ± 2.9a 0 98.88 ± 1.61a 77.6 ± 5.56a 80 ± 4.65a 77 ± 4.87a Control, 0.6% w/v Sod. CMC (p.o.) Standard, DES (p.o.) Curculigo orchioides extract (p.o.)

Values are mean ± S.E.M. of nine animals in each group. Data were analysed by two-way ANOVA followed by Bonferroni test. a P < 0.001 compared to control.

0 98.67 ± 2.47a 64 ± 4.5a 70 ± 3.59a 72.5 ± 3.74a 0 87.82 ± 4.38a 53.1 ± 3.47a 61.67 ± 4.22a 61.8 ± 2.27a 0 74.33 ± 1.87a 42.1 ± 3.69a 54 ± 4.33a 51 ± 2.25a 0 0 0 0 0 0 0 0 0 0

Day 3 Day 2 Day 1

Vaginal cornification (%) Dose (mg/kg)

1 2 3 4 5

Fig. 4. Photomicrograph of methylene blue and eosin stained vaginal smear (in diestrous) of control rat, showing only leukocytes (arrow mark).

Treatment (route)

increased height of luminal epithelium, with loose stroma and increased number of glands (Fig. 1), compared to control. The control animals presented a typical infantile condition (Fig. 2). DES also induced similar proliferative changes (Fig. 3). The vaginal smear of ovariectomized control did not show any vaginal cornification (Fig. 4). The percentage of vaginal corni-

Group

Fig. 3. Photomicrograph of haematoxylin and eosin stained transverse section of uterus of diethylstilbestrol (2 mg/kg, p.o.) treated rat, showing proliferative stage (i.e., stimulated endometrium with loose stroma and glands).

Table 2 Effect of alcoholic extract of Curculigo orchioides on vaginal cornification in bilaterally ovariectomized albino rats

Day 4

Day 5

Fig. 2. Photomicrograph of haematoxylin and eosin stained transverse section of uterus of control rat, showing disintegrated endometrium (arrow mark).

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– 2 300 600 1200

Day 6

Day 7

Day 8

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Fig. 5. Photomicrograph of methylene blue and eosin stained vaginal smear (in estrous) of Curculigo orchioides extract (1200 mg/kg, p.o.) treated rat, showing only cornified epithelial cells (arrow mark).

fication (Table 2) obtained at 1200 mg/kg of alcoholic extract of Curculigo orchioides (Fig. 5) was seen to be equivalent to that of DES at a dose of 2 mg/kg (Fig. 6). 4. Discussion Uterus and the female reproductive tract undergo innumerable physiologic and biochemical changes under the influence of ovarian hormones such as estrogen (Prakash and Mathur, 1979). If female rats are ovariectomized, the resultant lack of estrogen causes atrophy of the uterus and the reproductive tract; administration of estrogenic substances to ovariectomized rats leads to uterotrophic effects, vaginal cornification, increase in uterine glycogen content and proliferative changes in uterine endometrium (Williamson and Okpako, 1996). Estrogenic compounds are known to cause the keratinization and cornification of the vaginal epithelium, causing the superficial cells to be shed into the lumen to form large squamous cells (Burn, 1952).

Fig. 6. Photomicrograph of methylene blue and eosin stained vaginal smear (in estrous) of diethylstilbestrol (2 mg/kg, p.o.) treated rat, showing only cornified epithelial cells (arrow mark).

Thus the dose dependent increase in percentage vaginal cornification shown by the alcoholic extract of Curculigo orchioides can be attributed to its estrogenic activity. Estrogenic potency and efficacy have traditionally been expressed in terms of uterotrophic effects in immature or ovariectomized female rats (Ruentiz, 2003). The increase in uterine wet weight was successive and gradual with increase in the dose of the extract of Curculigo orchioides The histological examination of uterus of extract treated rats showed estrogenic influence, as evidenced by increased height of luminal epithelium with loose stroma and increased number of glands. Increase in uterine glycogen content in ovariectomized rats under the influence of alcoholic extract of Curculigo orchioides may be due to their estrogenic activity since estrogens have been reported to increase the hexose transport into the rat uterus and thereby increase the synthesis of glycogen in uterus (Tripathi, 1983). Literature review conducted on Curculigo orchioides indicated the presence of flavonoids, phytosterols and phenolic compounds (Krishna Kumar and Vaidya, 1992; Mashelkar et al., 2000). Flavonoids and phenolic compounds are known to possess estrogenic activity (Murad and Jeffrey, 1991; Kuiper et al., 1998). Thus the estrogenic activity shown by the extract of Curculigo orchioides can be attributed to the presence of flavonoids and phenolic compounds. Further, isolation of active constituents and ER selectivity studies of such isolated compounds are in progress. Acknowledgements Authors are grateful to the principal of Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, for his support throughout the study. This work was carried out with the financial support of Nitte Education Trust. References Burn, J.H., 1952. Biological Standardization. Oxford University Press, London, pp. 240–256. Cassidy, A., 1999. Dietary phytoestrogens–potential anti-cancer agents? British Nutrition Foundation Bulletin 24, 22–30. Dayton, S.S., Novice, S.B., Muntwryler, E., 1980. The estimation of glycogen with anthrone reagent. Archives of Biochemistry 25, 191– 195. Gogate, V.M., 1982. Dravyangunavignyana. Continental Prakashan, Pune, 55–56. Jonathan, S., Dehadrai, S., Prakash, A.O., 1995. Estrogenic activity in the ethanolic extract of Bupleurum marginatum. Indian Journal of Pharmacology 27, 258–261. Krishna Kumar, P.P., Vaidya, D.S.A., 1992. Selected Medicinal Plants of India. Chemexcil, Bombay, pp. 119–120. Kuiper, G.G., Lemmen, J.G., Carlsson, B.O., 1998. Interaction of estrogenic chemicals and physoestrogens with estrogen receptor ␤. Endocrinology 139, 4252–4263. Mashelkar, R.A., Hajra, P.K., Kumar, V., Babu, C.R., Bennet, S.S.R., 2000. The Wealth of India 1 (Suppl). National Institute of Science Communication, CSIR, New Delhi, pp. 258–259. Murad, F., Jeffrey, A.K., 1991. Estrogens and progestins. In: Alfred, G.G., Theodore, W.R., Alan, S.N., Palmer, T. (Eds.), Goodman and Gilman’s The

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