Estrogenic Activities of Pueraria candollei Grah. Tuberous Roots

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1 Universities Research Journal 2011, Vol. 4, No. 1 Estrogenic Activities of Pueraria candollei Grah. Tuberous Roots Wai Wai Thein Abstract Comparative study of estrogenic activity of 70% ethanolic extract and dried powder of Pueraria candollei Grah was conducted with premarin cream in albino mice. Mice were divided into twelve groups (n=5) receiving different treatments, consisting of 0.1 ml/kg distilled water, dried powder and 70% ethanolic extract of Tuberous roots of Pueraria candollei Grah at five different doses (viz., 50, 100, 200, 400 and 500 mg/kg body weight) and standard drug premarin cream at a dose of 50 mg/kg body weight. The dried powder and 70% ethanolic extract were administered orally and 50 mg/kg BW (Premarin cream) was applied intravaginally for five consecutive days. Estrogenic activity was assessed by counting the percentages of vaginal epithelial maturation. The significant differences at p<0.01 level were found in 200 mg/kg and 400 mg/kg of 70% ethanolic extract compared to the control. Key words: estrogenic activity, Pueraria candollei Grah Introduction The plant Pueraria candollei Grah. belongs to the family Fabaceae. The English name of Pueraria candollei Grah.is Burmese Kudzu. In Thailand, the plant is known as Kua ta lan and Ma-U-Nwe in Myanmar. They are widely distributed in Myanmar as wild plants, especially in Yangon, Bago Region and Mon State. The habit of Pueraria candollei Grah. is twining herbs or shrubs with tuberous roots. The leaves of Pueraria candollei Grah. are alternate and pinnately tri-foliolate. Leaflets are subcoriaceous, entire, rarely faintly lobed and roundish with an acute point. Lateral leaflets are oblique and smaller than terminal leaflet. The tuberous roots are up to 60 cm long and up to 38 cm in diameter. Tuberous roots are in different sizes depending on the soil condition or the environment and the time of cropping. The tuberous roots are sweet, watery and cool in taste. In Thailand, the tubers are used as a tonic and aphrodisiac, to treat mammary gland expansion and for their oestrogenic effect (Praptiwi, Lecturer, Department of Botany, Maubin University

2 140 Universities Research Journal 2011, Vol. 4, No ). The tuber extracts of Pueraria candollei Grah. contain phytoestrogens (hormone from plant) comprising isoflavones (daidzein and genistein). Estrogens are responsible for the normal development of the female genital tract, the breast and the female secondary sex characters. The estrogen affects the first visible sign of puberty in female mammals and maintain throughout the reproductive life span, those structures necessary for pregnancy. The estrogen production does decline to some extent after menopause but progesterone production declines much more because the adrenals are the only producers of progesterone after menopause (Shorr, 1940). Under the influence of estrogen hormones all layers of the squamous epithelium of vagina become thickened and proliferated, most marked in the intermediate and superficial (Wachtel and Plester, 1954). The aim of this study was to extend what is currently known about estrogenic effects and following the specific research line, to investigate their possible effects in postmenopausal women. Materials and Methods The tuberous roots of phytoestrogen-rich Pueraria candollei Grah. were collected from Hlawgar, Mingaladon Township and Thardukan,Shwe- Pyi-Thar Township. The dried powder and 70% ethanolic extract obtained from tuberous roots for estrogenic activity was evaluated by counting the percentages of vaginal epithelial maturation in albino mice (Krantz, 1959). The level of cellular maturation index was attained by a differential count of the four major cell-types shed from the stratified squamous epithelium, viz: parabasal, intermediate, superficial and anucleate squamous (Novak and Woodruff, 1962). The necessary reagents for Papanicolaou s staining are 70%, 80% and 95 % ethyl alcohol, 1 % acid alcohol, Harris hematoxylin, Orange G6 (OG 6), Eosin-Azure 50 (EA 50), Xylene, Distilled water and DPX mountant (Papanicolaou, 1954). The sixty female albino mice weighing g were separated into twelve groups. Group I served as control and administered 0.1 ml/kg distilled water only. The dried powder were prepared and administered orally to Group II to VI and 70% ethanolic extracts to Group VII to XI for five consecutive days at dosages of 50, 100, 200, 400 and 500 mg/kg BW respectively. The mice were individually marked and administered orally

3 Universities Research Journal 2011, Vol. 4, No with intragastric dosing canula syringes in various concentrations of the test drug. For standard group (Group XII), 50 mg/kg BW (Premarin cream) was applied intravaginally for five consecutive days (Yochin and Karthy, 1964). Vaginal smears were taken daily from twelve groups for 5 consecutive days. Non-absorbent cotton wrapped around the end of the slender wooden stick (swab) and dipped into normal saline and then the swab was inserted into the vagina and turned around inside the vagina (Wied, 1955). The material obtained on the swab was spread thinly on the slide and then immediately fixed in the fixative (95% ethyl alcohol) for overnight. After adequate fixation the slides were taken out and thoroughly dried in the air. Then the dried film was stained according to the Papanicolaou s staining procedure. After that the slides were examined under the light microscope, interpretation was performed by Cytopathologist (Wachtel and Plester, 1954). This experiment was carried out at National Health Laboratory. The significant differences at p<0.05, p<0.01 level and mean ± standard deviations were calculated by pairedsamples T test and descriptive statistics (SPSS 15.0 for Windows Evaluation). The results are shown in Tables (1 to 5) and Figures (1 to 11). Results The outstanding characters of Pueraria candollei Grah. were shrub plant with various sizes of tuberous roots as shown in Figures (1 and 2). Fig. 1 Habit Fig. 2 Tuberous roots Table 1 shows the estrogenic activity of control mice. In this table, the significant differences at p < 0.05 level were observed in mean values of superficial. In control group (treatment with 0.1 ml/kg distilled water), estrogens cause superficial maturation (M.I. to the right). When

4 142 Universities Research Journal 2011, Vol. 4, No. 1 estrogenic effect is extreme, many of the percentage of superficial become anucleate, with the resultant shift to the right (68/19/11/2) to (0/10/0/90). In this case, the maturation index showed a mean of 58.6 (SD = 42.26, n =1) and mean of (SD = 10.89, n =5). The results are shown in Tables (1 and 5) and Figures (5 and 6). Table 1. Estrogenic activity of control mice Control Maturation Index (in %) group (n =5) 10± ± ± ±13.28 Distilled water 18.8± ±1.41 2±1.41* 78.2± ± ± ± ± ± ± ±1.92* 84.8± ± ± ±5.26* 58.6±42.26 The values are expressed as mean ± SD * significantly different from control group (p < 0.05) ** significantly different from control group (p < 0.01) Table 2 shows the estrogenic activity of standard drug (Premarin cream). In this table, the significant differences at p < 0.05 level were observed in mean values of superficial and anucleate and p<0.01 level in mean values of anucleate compared to the control. In standard group (treatment with 50 mg/kg of Premarin cream), estrogens cause superficial maturation (M.I. to the right) while a large dose produce a complete shift to the right (M.I. 0/0/0/100). In this case, the maturation index showed a mean of 92.0 (SD = 14.19, n =1) and mean of 92.0 (SD= 3.44, n =5). The results are shown in Tables (2 and 5) and Figures (5 and 7).

5 Universities Research Journal 2011, Vol. 4, No Table 2. Estrogenic activity of standard drug (Premarin cream) Standard Group (n = 5) Maturation Index ( in % ) 8± ± ±1.09* 90.8±14.99 Premarin Cream 50 mg/kg BW 10± ± ±2.19* 87.2± ± ±2.19 1±1.00* 93.4±5.59** 6.6± ±3.13 0±0.00* 92± ± ± ±2.68* 96.6±6.50* The values are expressed as mean ± SD * significantly different from control group (p < 0.05) ** significantly different from control group (p<0.01) Table 3 shows the estrogenic activity of dried powder at various dosages. In group II to VI (treatment with dried powder at dosages of 50, 100, 200, 400 and 500 mg/kg BW respectively), estrogens cause superficial maturation (M.I. to the right) while a large dose produce a complete shift to the right (M.I. 0/0/0/100). The significant differences at p < 0.05 level were observed in mean values of superficial of five groups and anucleate of group III and V and p< 0.01 level in mean values of anucleate of group IV compared to the control. In these cases, the maturation index showed a mean of 99.8 (SD= 0.44, n=1) and mean of (SD= 13.82, n=5) at dosage of 200 mg/kg BW and mean of 97.4 (SD= 4.21, n=1) and mean of 89.6 (SD= 4.79, n=5) at dosage of 400 mg/kg BW. The results are shown in Tables (3 and 5) and Figures (5, 10and 11). Table 3. Estrogenic activity of dried powder at various dosages Dosages of Dried powder (n = 5) Maturation Index ( in % ) 50 mg/kg BW 12.24± ± ± ±8.43

6 144 Universities Research Journal 2011, Vol. 4, No. 1 Dosages of Dried powder (n = 5) Maturation Index ( in % ) 100 mg/kg BW 12.8± ± ± ± mg/kg BW 8.68± ±2.1 1± ± mg/kg BW 4.96± ± ±2 89.6± mg/kg BW 9.88± ± ± ±2.36 The values are expressed as mean ± SD Maturation index (%) Dosages of dried powder (mg/kg BW) Fig. 3. Estrogenic activity of dried powder at various dosages Table 4 shows the estrogenic activity of ethanolic extract at various dosages. In this table, the significant differences at p < 0.05 level were observed in mean values of intermediate (0 to 1.8) at dosage of 200 mg/kg BW and parabasal (1 to 10.2) at dosage of 400 mg/kg BW and p< 0.01 level in mean values of parabasal (0.8 to 3.6) and anucleate (92.6 to 97) at dosage of 200 mg/kg BW and anucleate (87.6 to 97) at dosage of 400 mg/kg BW compared to the control. In group VII to XI (treatment with ethanolic extract at dosages of 50, 100, 200, 400 and 500 mg/kg BW respectively), estrogens cause superficial maturation (M.I. to the right) while a large dose produce a complete shift to the right (M.I. 0/0/0/100). The significant differences at p < 0.05 were observed in mean values of superficial and anucleate and p<0.01 level in mean values of anucleate compared to the control. In this case, the

7 Universities Research Journal 2011, Vol. 4, No maturation index showed a mean of 94.8 (SD= 6.53, n=1) and mean of (SD= 1.78, n=5) at dosage of 200 mg/kg BW. The results are shown in Tables (4 and 5) and Figures (5, 8 and 9). Table 4. Estrogenic activity of ethanolic extract at various dosages Dosages of ethanolic extract (n = 5) Maturation Index ( in % ) 50 mg/kg BW 5.7± ± ± ± mg/kg BW 200 mg/kg BW 400 mg/kg BW 6.4± ±2.93 6± ± ±1.35** 0.36±0.8* 2.24± ±1.78** 4.92±3.86* 0.72±1.02 3± ±3.76** 500 mg/kg 7.88± ± ± ±6.72 BW The values are expressed as mean ± SD * significantly different from control group (p < 0.05) ** significantly different from control group (p<0.01) Maturation index (%) Dosages of ethanolic extract (mg/kg BW) Fig. 4 Estrogenic activity of ethanolic extract at various dosages

8 146 Universities Research Journal 2011, Vol. 4, No. 1 Table 5 shows the comparison of dried powder and ethanolic extract with control and standard drug (Premarin cream). In this table, ethanolic extract at a dosage of 200 mg/kg BW showed a significant increase in maturation index (mean = 95.28, SD= 1.78, n=5) compared to the control.the estrogenic activity at dosages of 200 mg/kg and 400 mg/kg of dried powder was nearly similar to estrogenic activity of standard drug, 50 mg/kg of premarin cream. Similarly, the estrogenic activity at dosage of 400 mg/kg of ethanolic extract was also nearly similar to estrogenic activity of standard drug. Among all of them, the estrogenic activity at dosage of 200 mg/kg BW was slightly higher than those of standard drug. The results are shown in Table (5) and Figure (5). maturation index (%) Control (DW) Premarin cream 50 mg/kg BW Dried Powder Dried Powder 200 mg/kg BW 400 mg/kg BW Ethanolic extract 200 mg/kg BW Ethanolic extract 400 mg/kg BW Fig. 5 Comparison of dried powder and ethanolic extract with control and standard drug (Premarin cream) Table 5. Comparison of dried powder and ethanolic extract with control and standard drug (Premarin cream) Groups (n-5) Maturation Index (in %) Control (DW) 15.4 ± ± ± ± Premarin cream 50 mg/kg BW 6.08 ± ± ± ± 3.44 Dried Powder 200 mg/kg BW 8.68 ± ± ± ± 13.82

9 Universities Research Journal 2011, Vol. 4, No Groups (n-5) Dried Powder 400 mg/kg BW Ethanolic extract 200 mg/kg BW Ethanolic extract 400 mg/kg BW The values are expressed as mean ± SD Maturation Index (in %) 4.96 ± ± ± ± ± ± ± ± ± ± ± ± 3.76 Fig. 6 Vaginal cytological picture seen in control (X400) Fig. 7 Vaginal cytological picture seen after treating with 50 mg/kg BW Premarin cream (X400) Fig. 8 Vaginal cytological picture seen after treating with 200 mg/kg ethanolic extract (X400) Fig. 9 Vaginal cytological picture seen after treating with 400 mg/kg BW ethanolic extract (X400)

10 148 Universities Research Journal 2011, Vol. 4, No. 1 Fig. 10 Vaginal cytological picture seen after treating with 200 mg/kg BW dried powder (X400) Fig. 11 Vaginal cytological picture seen after treating with 400 mg/kg BW dried powder (X400) Discussion and Conclusion Maturation Index (M.I) gives a more complete cytological picture and is useful in more clinical situations. The significant differences at p < 0.01 level was found in mean values of anucleate treated with 200 and 400 mg/kg BW of ethanolic extract when compared with control group. In the comparison of estrogenic activity of dried powder and 70% ethanolic extract with standard Premarin cream 50 mg/kg BW, the M.I of ethanolic extract was higher than of dried powder according to the statistical analysis. When compared with standard, there was a slight increase in M.I of ethanolic extract. The estrogenic activity at dosages of 200 and 400 mg/kg BW of dried powder and 400 mg/kg BW of ethanolic extract was nearly similar to estrogenic activity of standard drug, 50 mg/kg of premarin cream. Among all of them, the estrogenic activity at dosage of 200 mg/kg BW of ethanolic extract was slightly higher than those of standard drug, 50 mg/kg of premarin cream. The highest estrogenic activity was observed at dosages of 200 and 400 mg/kg BW of dried powder and ethanolic extract. The dried powder is suitable for oral administration as traditional medicine. Ethanolic extract is recommended to use as external application. In addition, the highest estrogenic activity was constantly found at dosage

11 Universities Research Journal 2011, Vol. 4, No of 200 mg/kg BW of both dried powder and ethanolic extract. Therefore, 200 mg/kg dosage of both dried powder and ethanolic extract from tuberous roots of Pueraria candollei Grah. can be safely applied in therapeutic purposes especially for menopausal women. Thus, tuberous roots of Pueraria candollei Grah. are as qualified as standard drug, premarin cream. They can be scientifically and systematically used in hormone replacement therapy especially for postmenopausal women. It can also be used locally as a rejuvenator and antiaging agent such as breast-firming cream, skin-nourishing cream, night cream and so on. Acknowledgements First and foremost I would like to express my sincere gratitude to Rector Dr. Htay Linn Maung, Maubin University and Professor Dr. Yee Yee Myint, Head of Botany Department, Maubin University for their permission to do the research paper. I would like to express special thanks to Dr. Daw Aye Kyi, Professor and Head (Retired), Department of Botany, Yangon University and Daw Naw Wah Wah Paw, Professor and Head (Retired), Department of Botany, Dagon University for their instructions and advice. My warm thanks are also extended to Dr. Than Aung Soe, Veterinarian, Administrative Officer of National Health Laboratory, and Professor Dr. Thein Thein Aye (Lt. Col., Retired), Consultant Pathologist, for their supervision and research facilities. References Krantz, K. E. (1959). The gross and microscopic anatomy of the vagina. Ann. N. Y. Acad. Sci., The Vagina, 83: Novak, A. B. and B. S. Woodruff (1962). Gynecologic and obstetric pathology with clinical and endocrine relations. (5 th Ed.). W. Saunder Company. Philadelphia-London. Papanicolaou, G. N. and E. Shorr (1936). The action of ovarian follicular hormone in the menopause as indicated by vaginal smears. Amer. Obst. Gynec., 31:806. Papanicolaou, G. N. (1954). Atlas of exfoliative cytology. Harrard Univ. Press. Cambridge, Mass. Also supplements. Praptiwi (1999). Medicinal and poisonous plants. Plant resources of South-East Asia. No. 12 (1). Prosea Foundation, Bogor, Indonesia. Pp Shorr, E. (1940). The menopause. Bull. N.Y. Acad. Med., 16:453.

12 150 Universities Research Journal 2011, Vol. 4, No. 1 Wachtel, E. and J. A. Plester (1954). Hormonal assessment by vaginal cytology. J.Obstet. Gynaec. Brit. Emp., 61: 155. Wied, G. L. (1955). Importance of the site from which vaginal cytologic smears taken. Amer. J. Clin. Path., 25: 742. Yochin, M. X. and I. L. Mc. Karthy (1964). Experimental endocrinology. Academic Press. NY & London.