Phenylethanoid Glycosides Of Cistanche On Menopausal Syndrome Model in Mice
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Shuo Tian, Mingsan Miao, Ming Bai, Zhenzhen Wei
Abstract
Cistanche is the traditional and precious Chinese herbal, with two thousand years of use history in China. It has the effect on tonifying the kidney, strong supplements to the liver and kidney, and replenishing essence and blood, known as the "desert ginseng”. Here, we explored the mechanism of Phenylethanoid Glycosides of Cistanche (PGC) to the model mice of menopausal syndrome, as well as the therapeutic effect and characteristics of PGC to the menopausal syndrome. In this study, KM mice were reproduced by the complete resection of the ovaries on both sides of the back to establish the model mice of the menopausal syndrome (MPS), and received distilled water or drugs, respectively. Model mice received distilled water. Mice received 200 mg/(kg day) high doses of Phenylethanoid Glycosides of Cistanche (HPGC), and 100 mg/(kg day) medium doses of Phenylethanoid Glycosides of Cistanche (MPGC), and 50 mg/(kg day) low doses of Phenylethanoid Glycosides of Cistanche (LPGC). After 21 days, it could determine the number of independent activities and the number of standing, the latent period of first entering the darkroom, and the electric number. It also calculated the viscera index of the uterus, thymus, spleen, measured the levels of estradiol (E2), testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum. Furthermore, it observed the pathological changes of the uterus, thymus, spleen and pituitary of mice. The results showed that behavioral indicators: Compared with the model group (MG), HPGC, MPGC, LPGC could increase the independent activities (P < 0.01); HPGC, MPGC could increase the number of standing, the latent period of first entering the darkroom, and reduce the electric number (P < 0.01); LPGC could increase the number of standing (P < 0.05); Viscera index: Compared with MG, HPGC, MPGC could increase the viscera index of the uterus, thymus, spleen (P < 0.01); LPGC could increase the viscera index of the uterus (P < 0.05); Serum index: Compared with MG, all groups could decrease the levels of LH in the serum (P < 0.01); HPGC, MPGC could improve the levels of E2, T and decrease the levels of LH, FSH in the serum (P < 0.01); LPGC could improve the levels of E2 and decrease the levels of FSH in the serum (P < 0.05). Meanwhile, it had the trend to improve the levels of T in the serum. Pathological changes: Compared with MG, HPGC could significantly improve the pathological changes of the uterus, thymus, spleen and pituitary of mice; other groups also have a certain effect. The results indicated that PGC could improve the sex hormone disorder of MPS, and restore the function of the uterus, thymus and spleen, with better therapeutic effect on MPS.
Introduction
Cistanche, the dried fleshy stems with scaly leaves of Cistanche deserticola Y. C. Ma and C. tubulosa (Schrenk) Wight (Chen et al., 2013), is a famous drug and widely used for thousands of years. It is originated from the "Shen Nong’s Herbal Classic” (Liu et al., 2013), salty in flavor and warm in nature. It has the effect of tonifying the kidney, strong supplement to the liver and kidney, replenishing essence and blood, moistening intestines to relieve constipation, and other efficacy. It is mainly used to treat the deficiency of kidney-yang (leading to impotence), prospermia, cold sperm, and sterility due to cold in the uterus, constipation, enuresis and frequency of micturition, and another disease. Modern research shows that it can enhance human immunity, memory and learning ability with anti-aging, anti-inflammatory, anti-fatigue, and other effects.
MPS is a syndrome of autonomic nervous system dysfunction disorder caused by estrogen secretion disorder and accompanied by neuropsychiatric symptoms. It has different degrees of hot flashes, irritability, dizziness, tinnitus, palpitations, insomnia, etc., with visible osteoporosis, memory loss, cognitive impairment, cardiovascular and cerebrovascular diseases in the late period. The main reason is the gradual decline and disappearance of ovarian function (Ia, 2016). Currently, the main use of hormone replacement therapy (HRT) for the treatment of MPS, direct supplement estrogen, long-term HRT can cause vaginal bleeding, breast tenderness, endometrial cancer, breast cancer and other adverse effects. In addition, the effect is not yet with satisfaction—the dose of estrogen on the immune system still has inhibited effect (Ma et al., 2016; Nawaz et al., 2017). Especially the recent discovery shows that it can increase the risk of cardiovascular and cerebrovascular disease and other serious side effects significantly limiting the clinical application. In the urgent market demand, more and more attention from the traditional Chinese (TCM) medicine to search for the treatment of MPS drugs (Halim and Phang, 2017; Mustafa et al., 2017). TCM treats the MPS with a long history (Zhang and Miao, 2011), by regulating the hypothalamic-pituitary-ovarian axis (HPOA), the ovarian function recovery, and the ovarian aging delay. It has been found that the majority of drugs of kidney yang have a better therapeutic effect on the MPS (Wei and Miao, 2013). Cistanche is the highest frequency of drugs replenishing kidney yang for the past dynasties (Tu et al., 2011). The main effective components of Cistanche are phenylethanoid glycosides, with an androgen effect. It is the embodiment of modern medicine kidney yang (Zhao and Pan, 2013), with the highest active ingredients in the Cistanche (Yan et al., 2012). It is mainly through two ways to function (Wumaierjiang and Yao, 2016). Firstly, it has the function to strengthen the hypothalamus-pituitary-adrenal function, and promote the release of neurotransmitters and hormones in the body; secondly, it has the function of anti-fatigue, improving body function. The effect of the replenishing kidney of Cistanche is different from the general kidney drugs such as Epimedium brevicornum Maxim, Morinda Officinalis How. Cistanche does not hurt the yin, but replenishes the kidney. It does not appear with firmness, dry mouth and other symptoms for the long-term use
2. Material and methods
2.1. Material and reagents
Cistanche (Batch No.20130501) was purchased from Anhui, Dechang Pharmaceutical Pieces Co., Ltd. The samples were identified by Professor Chen Suiqing (the Henan University of Chinese Medicine, identification of Chinese drug discipline) as the dried fleshy stems with scaly leaves of Cistanche deserticolaY. C. Ma, as well as the dried fleshy stems with scaly leaves of C. tubulosa (Schrenk) Wight, respectively. GC (Batch No.120303) was supplied by Shanxi star Pharmaceutical Co., Ltd.; Echinacoside reference substance (Batch No.111670-200503) was supplied by the National Institute for the Control of Pharmaceutical and Biological Products; AB-8 macroporous absorption resin (Batch No.20130618) was supplied by the Tianjin Guangfu of Institute of Superfifine Chemical Industry; Sodium carboxymethyl cellulose (Batch No.20120418, Tianjin Hengxing Chemical Reagent Co., Ltd.), Benzylpenicillin Sodium for Injection (Batch No.c1206807, North China Pharmaceutical Co., Ltd., the specification: 4 million units), 0.9% Sodium Chloride Injection (Batch No.1301265303, Chen Xin Pharmaceutical Co., Ltd; specifications: 250 ml), Chloral hydrate (Batch No.20120827, Tianjin Institute of Fine Chemical Industry), E2 ELISA assay kit (Batch No.20131001A, R&D Systems China), T ELISA assay kit (Batch No.20131001A, R&D Systems China), LH ELISA assay kit (Batch No. 20131001A, R&D Systems China), FSH ELISA assay kit (Batch No. 20131001A, R&D Systems China).
2.2. Sample preparation
2.2.1. Preparation methods
The procedure for sample preparation was as follows: By the literature methods (Gu et al., 2011), we were under the guidance of Feng Suxiang (Pharmaceutical analysis course discipline, Henan University of Chinese Medicine). The Cistanche was crushed to the meal, by the methods of reflux extraction to extract 2 times with the amount of ethyl alcohol (the content of the ethyl alcohol was 70%). The time of reflux extraction was 1.5 h for once and then combined the alcohol extraction liquid 2 times. The extraction liquid was decompressed and enriched without the alcohol flavor, and the distilled water was used to disperse (the concentration is 0.5 g/ml, as the sample solution. The sample solution was installed into the AB-8 resin with the flow (the ratio of the sample solution to resin was 1:10), after standing for 5 h until the sample solution was fully adsorbed. And then, first with 10 times of the column volume of distilled water to wash the AB-8 resin with the sample solution, it was abandoned to the water; Secondly, with 10 times of the column volume of the ethyl alcohol (the content of the ethyl alcohol is 10%), the impurities were removed; Thirdly, with 7 times of the column volume of the ethyl alcohol (the content of the ethyl alcohol was 60%) to elution, we collected the eluate and dried the eluate, that is the powder of Phenylethanoid Glycosides of Cistanche.
2.2.2. Selection of determining wavelength
Select 0.5 mL control product solution, and add 5% sodium nitrite solution 1 mL to the control product. Then shake and quiet place for 6 min. After that, add 10% aluminum nitrate solution to the above mixture. Then, shake and quiet place for 6 min. Add 10% sodium hydroxide 10 mL to the above mixture, the volume of the mixture was fixed to 25 mL with the water. Shake and quiet place for 18 min, as control product solution. Choose 0.5 mL Phenylethanoid Glycosides of Cistanche solution, as the above method configuration, as the test solution. The blank sample was the blank solution except for the control product solution and the sample solution, like the above method configuration. In the UV spectrophotometer with the wavelength range of 200–800 nm, the full wavelength was used to scan the above 3 solutions. The control product solution and the sample solution had the maximum absorption peak at 507 nm, so the wavelength of 507 nm was determined as the absorption wavelength. After the Phenylethanoid Glycosides of Cistanche solution (1) and the Echinacoside reference substance (2) (superposition contrast), the color appeared (See the next Figure)
2.2.3. Method of the content detection
Precise weigh 1 mL waiting for the determination of solution, add 1 mL 5% sodium nitrite solution to the control product, shake and quiet place for 6 min. And then add 10% aluminum nitrate solution to the above mixture. Shake and quiet place for 6 min. Add 10 ml 10% sodium hydroxide to the above mixture, and the volume of the mixture was fixed to 25 mL with the water. Shake and quiet place for 18 min; the blank solution was prepared by the same method as the blank sample without sample solution. With the UV spectrophotometry to determine at the spectrophotometry of 507 nm.
2.2.4. Methodology examination
2.2.4.1. Preparation of the control solution and the sample solution.
Preparation of the control solution: Precise weighs 3.06 mg echinacoside with drying to constant weight as the reference substance, and put it into the measuring flask of 25 mL. Then use 70% ethanol solution diluted to the scale, and shake the mixture, as the control solution. The content of the control solution was 0.1224 mg/ml. Preparation of the sample solution: Precise weigh 5 mg Phenylethanoid Glycosides of Cistanche and put it into the measuring flask of 10 mL. Then use 70% ethanol solution diluted to the scale, and shake the mixture, as the sample solution.
2.2.4.2. Preparation of standard curve.
Accurately draw the control solution of 0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL, according to the ‘‘Selection of determining wavelength”, and measured the absorbance. The regression equation was obtained by using the concentration to the absorbance: Y = 20.296X 0.0015 (r = 0.9994), and the linear range was 4.90–29.4 lg/ml.
2.2.4.3. Precision test.
Parallel determined the absorbance of the sample solution 6 times according to the item of ‘‘Method of the content detection”. After calculating the relative standard deviation of the absorbance, the absorbance was 0.299, 0.299, 0.298, 0.297, 0.297, and 0.297, and the average absorbance was 0.298, with RSD = 0.330% (n = 6).
2.2.4.4. Stability test.
Determine the absorbance of the sample solution after the solution was fully colored for 0, 15, 30, 50, and 60 min according to the item of ‘‘Method of the content detection”. After calculating the relative standard deviation of the absorbance, the absorbance was 0.300, 0.296, 0.295, and 0.295, and the average absorbance was 0.299, with RSD = 1.362% (n = 5).
2.2.4.5. Repetitive testing.
Prepare 6 sample solutions according to the item of Preparation of the sample solution”, and calculate the absorbance and the content according to the item of ‘‘Method of the content detection”. The contents were 65.2%, 65.6%, 66.7%, 67.2%, and 66.5%, respectively, and the average content was 66.2%, with RSD = 1.142% (n = 6).
3. Results
3.1. The effect of the independent activities and the number of standing in mice
Figs. 1 and 2 show the data of the independent activities and the number of standing. It demonstrates that MG mice are remarkably reduced compared with the BC (P < 0.01), reflecting the curiosity to the fresh environment is reduced. The mice in GC, HPGC, MPGC, and LPGC can remarkably improve the independent activities compared with the MG (P < 0.01); those in GC, HPGC and MPGC can improve the independent activities compared with the MG (P < 0.01); those in LPGC can improve the number of standing a little compared with the MG (P < 0.05).
3.2. Effect of the latent period of first entering the darkroom and the electric number
As shown in Figs. 3 and 4, the data of the latent period of first entering the darkroom and the electric number demonstrated that MG mice are remarkably reduced compared with the BC (P < 0.01),
reflecting the decline of memory in the MPS mice. The mice in GC, HPGC, and MPGC can remarkably increase the latent period of first entering the darkroom and reduce the electric number compared with the MG (P < 0.01), as well as improve the memory.
3.3. Effect of the viscera index
As shown in Fig. 5, the data of the viscera index of the uterus, thymus and spleen demonstrate that MG mice is remarkably reduced compared with the BC (P < 0.01), reflecting the uterus and the immune organ of MPS mice are atrophied. The mice in GC, HPGC, and MPGC can remarkably improve the viscera index of the uterus, thymus, and spleen compared with the MG (P < 0.01). The mice in LPGC is a bit improved the viscera index of the uterus compared with the MG (P < 0.05).
3.4. Effect of the content of E2, T, LH, and FSH
As shown in Fig. 6, the data of the level of E2, and T demonstrate that MG mice are remarkably reduced, and the data of the level of LH, and FSH demonstrate that MG mice is remarkably increased compared with the BC (P < 0.01), it reflected the sex hormone levels in serum is disordered in the MPS mice. All groups of mice are significantly decreased with the level of LH in serum compared with the BC (P < 0.01). The mice in GC, HPGC, and MPGC can remarkably improve E2, T and decrease the level of LH, FSH in serum compared with the MG (P < 0.01). The mice in LPGC can a bit improve E2 and decrease the level of FSH in serum compared with the MG (P < 0.05).
3.5. Observation of the pathological morphological changes of the uterus
As shown in Figs. 7 and 8, with the micrometer scale, the morphological changes of the uterus demonstrate that MG mice have significant pathological changes in the uterus compared with the BC (P < 0.01). The mice in GC, HPGC can remarkably improve the pathological morphological changes compared with the MG (P < 0.01). The mice in MPGC can improve the pathological morphological changes compared with the MG (P < 0.05). The changes of the pathological morphological are reported as the semi-quantitative criteria to measure the uterus, and the pathological organization morphology can be divided into four levels for each group and calculate the number of mice.
3.6. Measurement of the thickness of the thymic cortex
As shown in Figs. 9 and 10, with the micrometer scale, the date of the thickness of the thymic cortex demonstrate that MG mice are significantly reduced compared with the BC (P < 0.01), indicating that the volume of the thymus is decreased after establishing the MPS model mice. The mice in HPGC can remarkably increase the thickness of the thymic cortex compared with the MG (P < 0.01). The mice in MPGC can increase the thickness of the thymic cortex compared with the MG (P < 0.05).
3.7. Measurement the thickness of splenic nodule
As shown in Figs. 11 and 12, with the micrometer scale, the date of the thickness of splenic nodule demonstrates that MG mice are significantly reduced compared with the BC (P < 0.01), indicating that the volume of the spleen is decreased after establishing the MPS model mice. The mice in HPGC can remarkably increase the thickness of the splenic nodule compared with the MG (P < 0.01). The mice in MPGC can increase the thickness of the splenic nodule compared with the MG (P < 0.05).
3.8. Calculating the number of the basophilic cell and the anterior pituitary cell
As shown in Figs. 13 and 14, the date of the number of the basophilic cell and the anterior pituitary cell demonstrate that MG mice are significantly reduced compared with the BC (P < 0.01), indicating that the number of the cell source of secretory gonadotropin is decreased after establishing the MPS model mice. All received drugs groups can remarkably increase the number of the basophilic cell compared with the MG (P < 0.01). The mice in GC, HPGC, and MPGC can remarkably increase the number of the anterior pituitary cell compared with the MG (P < 0.01).
4. Discussion
Records of the menopausal syndrome in ancient books are mainly scattered in the description of the "lily disease”, "zangzao” syndrome, "melancholia”, "abnormal menstruation” and others (Chen et al., 2015; Ma and Chen, 2015). For the woman over forty years, the Yin Qi was cut a half; Tiangui has dried up; the kidney Qi was gradually declined; the Yin Jing was deficient; the balance of Yin and Yang were lost. Its main performance was as the menstrual disorders or menopause appear such as tidal fever and sweatiness, emotional irritability, restless, palpitation and insomnia, back pain, edema of the face and limbs, dizziness, tinnitus, and skin feeling like ant crawled disease. The strong decline of the kidney Qi is the intrinsic reason of the menstruation come or cut off. Kidney deficiency is the root reason of the menopausal syndrome (Tan et al., 2013), and the kidney is the origin of human life, called the "congenital foundation”. The rise and fall of kidney essence dominate the growth and reproductive function of mature and decline (Wang and Huang, 2011). Studies have shown that the decline of ovarian function is the essence of kidney deficiency syndrome, and the imbalance of Yin and Yang of the kidney is closely related to the disorder of endocrine secretion and autonomic nervous system dysfunction. The change of E2, FSH, and LH is an expression of kidney deficiency and kidney Yin and Yang imbalance (Shi et al., 2007). Tonifying kidney yang drugs are used to regulate the hormone level in the hypothalamus-pituitary ovary axis or play a direct role in regulating ovarian function. Modern Chinese medicine believes that the etiology and pathogenesis of menopausal syndrome is kidney deficiency (Razali and Said, 2017). The study used the complete resection of the ovaries on both sides of the back to reproduce the MPS mice model. 90% estrogen of females was secreted by the ovary, and the ovary was removed. It artificially caused the level of estrogen that was decreased by a sudden and then to simulate the MPS. This model was the classic model, with a high success rate, stability and reliability and so on. Through independent activities and the number of standing, the latent period of first entering the darkroom and the electric number, we observed the improvement of the learning and memory function and the curiosity of the fresh environment by the medicine. The ovarian function was declined with the sex hormone secretion decreased. Furthermore, the gonadotropin was increased with MPS patients. The levels of E2, T in serum were lower than that person without the MPS symptoms, while the levels of LH, FSH were significantly higher than that person without the MPS symptoms. Therefore, through the measurement of E2, T, FSH and LH levels in serum, it could be used the visual indicators for identification of the MPS, reflect the effect of drugs on the MPS disease. The study showed that the ovarian function decline was the connotation of kidney deficiency, and the change of E2, T, FSH and LH was a kind of expression of kidney deficiency (Li et al., 2014; Cheng and Tian, 2012). Cistanche was through the effect of replenishing the kidney to enhance immunity (Zhang et al., 2009; Shareef et al., 2017). We used the kidney-replenishing medicine to treat MPS, which was innovative. The results also showed that PGC could improve the disorder hormone level of MPS mice, and increase the levels of E2, T, LH, and FSH in serum. Meanwhile, it could improve the pathological changes of the uterus, thymus, spleen and pituitary of mice, conforming to the literature that kidney deficiency is fundamental in MPS.
5. Conclusion
Our study results indicated that the PGC had a good therapeutic effect for MPS—it could increase the independent activities and the number of standing, the latent period of first entering the darkroom, the viscera of thymus, spleen, and uterus, E2, T as well as reduce the electric number, LH, and FSH. The phenylethanoid glycosides were selected in Cistanche for the high efficiency and convenience of the MPS research to improve the treatment of the perimenopausal syndrome. Its quality standards are easy to control, conducive to the innovative research and industrialization development of drugs. It provides a new way of thinking for the treatment of MPS.
