Pharmacological study on Samia cynthia ricini in old female mice

Journal of Ethnopharmacology 105 (2006) 210–214

Pharmacological study on Samia cynthia ricini in old female mice Mingzhe Gan a , Deguang Wan b , Xiaona Qin a , Ping Gao a,b,∗ a

Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, Sichuan University, Chengdu 610064, China b College of Pharmacy, Chengdu Chinese Medical Science University, Chengdu, China Received 17 April 2005; received in revised form 19 October 2005; accepted 24 October 2005 Available online 1 December 2005

Abstract The effect of the extraction from female moths Samia cynthia ricini (family Saturniidae) on menopausal syndrome was studied in order to search for an effective traditional Chinese medicine to menopausal syndrome and offer a theoretical support for the further research on Samia cynthia ricini. Aged, nonreproductive female mice was used as the model and randomly divided into three groups: control, ethanol extraction group and diethylstilbestrol (DES) group according to the medicine applied. After 4 weeks of treatment, keratinization rates of vagina epithelia, indexes of organs, serum concentrations of estradiol (E2) and progesterone (P), the bone mineral contents (BMC) and the morphological changes of ovary and uterus were measured. And the fingerprint of the extracts was obtained by gas chromatography–mass spectrometry (GC–MS). The results showed that the moth extraction could obviously accelerate the keratinization of aged mice’s vagina epithelia, increase the weight of the ovary and ameliorate its degenerative process. It could also, to some extent, increase the serum concentrations of estradiol, progesterone concentrations as well as the bone mineral contents. Furthermore, there is no obvious side effect detected on immune system in our research. The extraction from female moths Samia cynthia ricini could ameliorate the menopause symptom of aged female mice and thus be a potential remedy for it. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Samia cynthia ricini; Female moths; Menopausal syndrome; Mice

1. Introduction The menopausal syndrome, a disease characterized by the vasomotor-vegetative and psychological disorders, mainly results from the decline of estrogen level during menopause (Fischl, 2001). It often occurs in middle-aged women and the symptom includes vasomotor-vegetative disorders, psychological disorders, etc. The estrogen replacement therapy (ERT), the supplement of estrogen for those women, is confirmed as an effective remedy for menopausal syndrome. Among the different kinds of estrogen in ERT, natural estrogen is regard as the best one because it has a short half life, easy to decompose in tissue and innocuous to liver (Tapiero et al., 2002) although long-term use of a single estrogen will cause endometrial hyperplasia and increase women’s risks of ovarian cancer (Lacey et al., 2002). However, adding progestogen in ERT could efficiently reduce these risks, particularly in those with intact uterus (Rice, 2002). It is reported some analogs of estradiol (E2) and progesterone (P) as well as some other kinds of hormone, such ∗

Corresponding author. Tel.: +86 28 85251989. E-mail address: [email protected] (P. Gao).

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.10.024

as ecdysone and juvenile hormone can be extracted from some variety of insects (Keshan and Ray, 2001). The natural combination of some major insect hormones with analogs of estradiol and progesterone might be a potential natural ERT drug of medical value. Samia cynthia ricini (family Saturniidae) has been recorded as Chinese materia medica in Zhong Hua Ben Cao (Administer Office of Herbalist Doctor and Medicine on China, 1999). The extraction from female moths Samia cynthia ricini, as shown in our previous studies, with advanced estrogen-like activity and the ability to revive the reproductive system during the menopause without side effect on immune organs, might be developed as a new drug. Therefore, we conducted a further pharmacological study of the Samia cynthia ricini in aged mice in order to search for an ideal drug for ERT and offer a theoretical support for the further research. 2. Materials and methods 2.1. Sample and reagents Female moths of Samia cynthia ricini (the yellow and white species) were provided by the main station of the exten-

M. Gan et al. / Journal of Ethnopharmacology 105 (2006) 210–214

sion of sericultural techniques, Guangxi. The ethanol extraction of female moths (EEFM) was prepared in our laboratory according to Yao’s method (Yao, 1992). Diethylstilbestrol (DES) containing active component 1 mg per pill was purchased from Guangdong Huanan Pharmacy, which was diluted to the required concentration with saline before the treatment.

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Department, the Hospital of Chengdu Chinese Medical Science University). 2.7. Bone mineral content (BMC) evaluation Bone mineral content of mice’s left femurs, which is defined as percentage of ratio of bone mineral weight to bone dry weight, was measured according to Liu’s method (Liu et al., 2003).

2.2. Subject animals 2.8. Statistics Female mice of Kunming strain, aged more than 12 months and weighed 30–35 g, were purchased from the Experimental Animal Center of Chengdu Chinese Medical Science University, all of which were housed in a controlled environment (temperature 20 ± 2 ◦ C, natural illumination) with free access to food and water. After vagina smear performed in 7 days, all mice were confirmed to have irregular sexual cycle and no reproductive ability. 2.3. Group treatment After 7 days adaptive feeding, the mice were randomly divided into three groups: control group (n = 8), saline 0.5 ml/day; DES group (n = 8), DES 0.085 mg/(kg day); EEFM group (n = 8), EEFM 0.4 g/(kg day). Each mouse was gavaged 0.5 ml per 10 g body weight once a day. All mice were sacrificed after 4 weeks. 2.4. Vagina smear Vagina smear was performed as reported (Vogel, 2001) on each mouse before it was killed. The number of positive response is counted if the percentage of vaginal keratinized epithelial cell exceeds 50%. 2.5. Visceral coefficient measurement and section observation All mice were weighted 24 h after last gavage, and then killed by decapitation. Ovaries, uteruses, spleens, adrenals and thymuses were removed, respectively, then weighted immediately on the electronic scales and the visceral coefficient was calculated. The visceral coefficient = organ weight (mg)/body weight (g) × 100. Ovaries and uteruses were cut into pieces, fixed in 10% formalin and processed for paraffin sections. Tissue sections were stained with hematoxylin and eosin. Cross-sections were examined through an optic microscope. 2.6. Serum estradiol and progesterone levels measurements Blood samples were obtained from each mouse before sacrifice to determine the serum levels of estradiol and progesterone. Blood collected in heparinized tubes was centrifuged at 6000 rpm for 10 min and then the supernatant serum was stored at −20 ◦ C until assay. The serum levels of estradiol and progesterone were determined using radioimmunoassay (Radiation

All data were analyzed using SPSS 10.0 and expressed as the mean ± standard deviation. The intergroup differences were demonstrated for significance using the χ2 -test in vagina smear and t-test in others. 2.9. The fingerprint of EEFM established by gas chromatography–mass spectrometry (GC–MS) A Hewlett-Packard 6890 gas chromatograph, coupled with a Hewlett-Packard 5973N mass spectrometer was used for the establishment of fingerprint of EEFM. The separation was performed on HP-5MS column, 0.25 mm i.d. × 30 m, 0.25 ␮m coating thickness. The temperature of the column programmed from 150 to 258 ◦ C at 6 ◦ C min−1 , 258 to 278 ◦ C at 10 ◦ C min−1 and 278 to 300 ◦ C at 40 ◦ C min−1 . The injector temperature and the detector temperature were 300 ◦ C. Helium was used as carrier gas with a constant flow rate of 0.8 ml min−1 . The mass spectrometer was operated at 70 eV, scan range 20–450 amu. All separated compounds were identified from the recorded mass spectra by comparison with the mass spectra from the NIST and Wiley libraries. 3. Results 3.1. Vaginal epithelial keratinization None of the control group showed the positive results in contrast to all in DES group. The percentage of positive results in EEFM group is 62.5%. Compared with control group, there is a significant difference confirmed by χ2 -test, which shows EEFM can obviously accelerate the vaginal epithelial cell maturation and keratinization (Table 1). 3.2. Indexes of organs The mean index of ovary in EEFM group is significantly higher than it in control group. And the mean indexes of uterus Table 1 The effects of EEFM on the percentage of vaginal keratinized epithelial cell of old female mice Group

Positive number

Negative number

Control DES EEFM

0 8 5

8 0 3

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M. Gan et al. / Journal of Ethnopharmacology 105 (2006) 210–214

Table 2 The effects of EEFM on ovary, uterus, spleen, adrenal and thymus of old female mice Group

Control DES EEFM *

¯ ± S) The visceral coefficient (mg/100 g) (X Ovary

Uterus

Spleen

Adrenal

Thymus

22.05 ± 6.68 18.93 ± 8.96 42.93 ± 17.71*

204.4 ± 136.3 587.4 ± 169.8* 327.7 ± 98.5

456.8 ± 188.4 505.1 ± 178.8 520.3 ± 135.9

27.07 ± 8.74 27.26 ± 5.18 33.76 ± 7.41

149.4 ± 87.9 110.4 ± 53.0 230.5 ± 72.2

Compared with control, P < 0.05.

Fig. 1. The ovary of control (H.E. stain ×160).

and thymus are about 60 and 55% higher than those in control group, but with no significance in statistics. The mean index of thymus in DES group is only 70% of that in control. All the indexes of spleen and adrenal are nearly the same (Table 2). 3.3. Histological findings Histology of ovary shows more follicles in different stages in EEFM group (Fig. 1) than those in control (Fig. 2). The uterus of the control group shrank and endometrial epithelial cells were

Fig. 2. The ovary of EEFM (H.E. stain ×160).

Fig. 3. The uterus of control (H.E. stain ×160).

arranged irregularly (Fig. 3). In contrast, the uterine cavity in EEFM group was much greater and endometrium was thicker but with no hyperplasia (Fig. 4). The uteruses in the DES group were permeated with large amount of transparent fluid and showed serious hyperplasia. In view of the obvious pathological changes, we cancelled the observation of slices. 3.4. Concentrations of estradiol and progesterone in serum Both estradiol and progesterone levels in serum taken from EEFM group were higher than those from control. The con-

Fig. 4. The uterus of EEFM (H.E. stain ×160).

M. Gan et al. / Journal of Ethnopharmacology 105 (2006) 210–214 Table 3 The effects of EEFM on the concentrations of estradiol and progesterone in serum of old female mice Group

¯ ± S) E2 (pg/ml, X

¯ ± S) P (ng/ml, X

Control DES EEFM

9.57 ± 1.91 8.52 ± 4.03 12.68 ± 3.75

5.04 ± 2.85 7.14 ± 3.31 15.62 ± 8.23*

*

Compared with control, P < 0.05.

Table 4 The compounds identified in EEFM by GC–MS Retention time (min)

Area (%)

Compound

16.67 19.10 19.24 19.68 19.85 20.14

0.30 14.85 0.85 1.21 1.14 6.05

Cholesta-3,5-diene Cholest-5-en-3-ol Vitamin E Ergost-5-en-3-ol Stigmasta-5,22-dien-3-ol Stigmasta-5-en-3-ol

tent of progesterone exhibited the most significant increase (Table 3). 3.5. Bone mineral content The mean BMC values of control and DES group are, respectively, 61.8 and 65.2% lower than that, 71.5%, in EEFM group. 3.6. Fingerprint of EEFM The fingerprint of EEFM was obtained by GC–MS (Fig. 5). From the chromatogram of the mixture, 33 component peaks were detected. Six compounds, whose retention times were

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between 16 and 21 min, were identified as steroids and Vitamin E (Table 4). 4. Discussion and conclusions When female enters menopause, the final stage of female reproductive age, the ovary and uterus begins to shrink and degenerate. Thus, estradiol and progesterone production sharply declines, which disrupts the feedback control mechanisms of the hypothalamic–pituitary–ovarian axis. The increase in folliclestimulating and luteinizing hormone level, together with the decrease in estrogen level, lead to other menopausal disorders, such as heart disease, osteoporosis, etc. (Tsavachidou and Liebman, 2002). Using single estrogen would significantly increase the risk of cancer. However, the combined use of estrogen and progestogen could alleviate menopausal syndrome with no obvious adverse side effect (Freedman, 2002). Our pervious research has demonstrated obvious estrogenlike effect of the ethanol extraction of female moths of Samia cynthia ricini without no adverse side effect on immune system in mouse. Compared with those in ovariectomized mature female mouse, the physiological conditions in aged female mouse are much more similar to menopause conditions and are more sensitive to drugs (Ye et al., 2003). So we choose the aged female mouse as the object animal for the research on medical effect of menopausal syndrome. The accelerating effect of estrogen on vaginal epithelial keratinization is especially evident in rodents. Due to its high speciality, vaginal epithelial keratinization is regarded as the primary criterion to evaluate the estrogen-like activity. The level of estrogen could be evaluated by the degree of keratinization (Korach and Mclachlan, 1995). In our current research, we found all mice in control group were still in dioestrus because of insufficient level of estrogen production. In contrast, the high degree of keratinization in EEFM group indicated there

Fig. 5. The fingerprint of EEFM.

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is strong estrogen-like activity of EEFM. EEFM obviously promotes the index of ovary. Through histological inspection we found the development of follicles is much more advanced. From these results we presume the EEFM could alleviate the ovary function degeneration. The index of uterus in EEFM group was enhanced a little but not significantly. This indicated that the estrogen-like activity was still limited on aged female mice. In DES group, glands in uterus increased and endometrium became thicker with no hyperplasia. It suggested that EEFM could alleviate the atrophy of uterus without causing hyperplasia of endometrium. Results of RIA showed there was obvious increase in progesterone compared with control group, which could reduce the risk of endometrial cancer associated with estradiol and may also weaken the effect of estrogen on uterus to some extent. The serum levels of estradiol in EEFM group increased with no significance in statistics. According to our present and previous research, we presumed that estradiol may not be the only estrogen-like component in EEFM and there may be some other way to demonstrate the estrogenlike effect of EEFM than detecting serum estradiol level. The reduced index of thymus in DES group compared with the control group fits well with our previous findings and other reports (Luster et al., 1984). EEFM had no adverse side effect on the weight of immune organs from which no pathological changes were observed. The fingerprint of EEFM was established and analyzed by GC–MS. According to the fingerprint, we found there was considerable proportion of steroids in EEFM. The main compounds of steroids were cholesterin, stigmasterol and their derivatives. However, there was still a large proportion of unknown components in EEFM which needs further investigation. Our experiment revealed EEFM had considerable medical value in that it could alleviate some pathologic menopausal changes in old female mouse. However, the effective components in EEFM and its working mechanism still need further investigation. The established fingerprint of EEFM may provide some reference for the advanced research.

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