lcariin (ICA) Promotes Self-Renewal of Neural Stem Cells: An Involvement of Extracellular Regulated Kinase Signaling Pathway

HUANG J-h, CAI W-j, ZHANG X-m, and SHEN Z-y. Chinese Journal of Integrative Medicine. February 2014, Volume 20, Issue 2, pp 107-115

To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs). Methods: NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST)

and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin- 13 ill(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed.

Results: ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased opticalabsorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs. 

Conclusion:  ICA can promote the self­renewalof NSCs at least partially through ERK/MAPK signaling pathway.

Study Summary: Single cells derived from dissected neural tissue, when plated under non-adherent,permissive conditions, form floating balls of cells termed neurospheres. These highly heterogeneous structures contain neural stem cells as well as more restricted progenitors and even differentiated progeny. The presence of stem cells can be confirmed by dissociating these neurospheres into single cells and replating them. They can not or only re-form very small neurospheres. However, the neural stem cells will demonstrate strong self-renewal, and generate neurospheres in continuous passages. To test the self-renewal potential of cells within the neurosphere cultures, dissociated single cells need to be plated at a density at which a single cell gives rise to a single neurosphere. This is termed clonal analysis. When single cells were plated under medium to high cell densities,cells or small neurospheres can adhere to each other and combine to form larger neurospheres.

In this situation, the clonalanalysis is impossible,and the true proliferative capacity of stem cells can not properly be determined. But in drug screening assay, it is not necessary. Many reports employed a low plating cell density of 5 000 cells/m or 1 000 cells/m to measure the self-renewal of neural stem cells.(26 31> In our study, we plated the single cells at a density of 5 000 cells/ml. About 5% plated single cells re-formed neurospheres in consistency with previous studies.(25 ICA significantly increased the neurosphere formation. This increase in neurosphere formation by ICA may be derived from elevated cell proliferation of NSCs. To test this hypothesis, we investigated the proliferation after addition of ICA using EdU incorporation assay. As a result, ICA enhanced EdU incorporation significantly. In addition, the optical absorbance value was also significantly elevated by ICA treatment. Results above together suggested that ICA can promote the self­renewal of NSCs possibly through enhancement of proliferation of NSCs.

When NSCs enter into the process of differentiation, they generally and gradually exited from cell cycle. Studies have shown that the length of the neural progenitor cell cycle is directly coupled to cell fate choices, and that shortening the cell cycle could inhibit differentiative divisions. Then we observed the effects of ICA on the differentiation of NSCs. We found that ICA had no significant effects on the percentage of 13 - ill tubulin positive cells and GFAP positive cells in immunofluorescence assay. In Western blotting analysis, we also saw ICA had no effects on the 13 -ill tubulin and GFAP protein expression. So we thought ICA has no significant role in differentiation of NSCs. Next, using the microarray technique, we screened the differentially expressed genes induced by ICA . And we further analyzed the signaling pathways enriched by these genes. MPAK signaling pathway was of interest in which 10 differential lyregulated genes were enriched. The MAPK pathway exists in all eukaryotes, and controls such fundamental cellular processes as proliferation, differentiation, survival and apoptosis. ERK and p38 are two major subfamilies of MAPK signaling pathway.

ERKs are characteristically activated by various growth factors. Members of the p38 MAPK (p38} and c-Jun N-terminal kinase (JNK) are strongly activated in response to stress stimuli.c33 In our microarray study, the differentially regulated genes by ICA were enriched in both ERK/MAPK and p38/MAPK subfamily. In order to confirm which subfamily mediated the effects of ICA on self-renewal of NSCs, we observed whether ERK or p38 signaling specific inhibitor can block the effects of ICA. Consequently, ERK/MAPK signaling inhibitor abolished the effects of ICA on cell proliferation and self-renewal of NSCs. Meanwhile p38 inhibitor did not show this ability. Many other studies also indicated that ERK was an important mediator of effects of ICA.

Our study provides a further evidence for that ERK is involved in pharmacological effects of ICA. Recently, we also found ICA can stimulate silent neuralstem cells in hippocampus in aging rats and improve the memory and learning ability. Therefore, ICA maybe has the potential to be used in the treatment of degenerative diseases of central nervous system.

Cnidii extract contains Osthole 10%, Coumarines 1%

Osthole is a libido stimulator: researchers investigated the cavemosal relaxant effect of osthole, which has been long used in China as an herbal medicine to improve both male and female sexual dysfunction. The data suggested that osthole possessesa relaxant effect on rabbit corpus cavernosal tissues which is attributatble to the release of NO from sinusoidal endothelium and to the potentiation of the cGMP and/or cAMP signal mediating relaxation of cavernosal smooth muscle by inhibiting phosphodiesterase."" (Corpus cavernosum = one of the columns of erectile tissue forming the dorsum of the penis (one of the two columns of erectile tissue forming the clitoris)). Osthole Osthola, osthol, Osthol,osthenol, Ost, Cnidiadin, (C15H16O3), Coumarin, 7-methoxy-8-(3-methyl-2-butenyl); 2H-1-Benzopyran-2-one, 7-methoxy-8-(3-methyl-2-butenyl); 7-Methoxy-8-(3-methyl-2-butenyl)-coumarin; 7-Methoxy-8-isopentenylcoumarin; osthol; osthole; Ostol; Ostole."

Effects of osthol on androgen level and nitric oxide synthase activity in castrate rats

Yuan J, Xie J, Li A, Zhou F. Zhong Yao Cai. 2004 Jul;27(7):504-6.

OBJECTIVE: To investigate the effects of osthol on androgen level and nitric oxide synthase (NOS) activity in castrate rats. METHODS: Immature castrate male rats were treated with osthol daily for 20 d. The levels of testosterone(T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) in serum and the activity of NOS, the nitric monoxide (NO) level in penis tissue were determined. RESULTS: T(P< 0.05), LH(P< 0.05) and FSH(P< 0.01) were significantly increased at the high dose of osthol treatment. The high and low dose of osthol treatment significantly enhanced the activity of NOS (P< 0.01). CONCLUSION: Osthol could increase androgen, gonadotropin and nitric oxide synthase (NOS) activity. It suggested that osthol had androgen-like effect and gonadotropin-like effect.

Osthole improves aspects of spatial performance in ovariectomized rats.

Hsieh MT, Hsieh CL, Wang WH, Chen CS, Lin CJ, Wu CR. Am J Chin Med. 2004;32(1):11-20.

The present study was designed to investigate the ameliorating effects of Cnidiuim monnieri L. Cusson (CM) and osthole, a constituent of CM, on the spatial performance deficit in scopolamine (SCOP)-treated or ovariectomized (OVA) rats. CM improved the deficit of spatial performance, and reversed the lower plasma estradiol levels caused by SCOP in female rats. In addition, osthole (3 and 10 mg/kg, s.c.) improved the performance deficit in OVA rats. It (10 and 30 micrograms/brain, icv) also improved the performance deficit caused by SCOP in intact female rats, and at 30 micrograms/brain improved the deficit in OVA rats. However, osthole did not alter the latency swum to reach the visible target in SCOP-treated and OVA rats. Accordingly, we suggested that osthole is an active constituent of CM, and possesses ameliorating effects on the spatial performance deficits in SCOP-treated female rats or OVA rats. The action mechanism of the effects of osthole on performance deficits was related to the estrogen-like properties and activating the central cholinergic neuronal system.

Effects of osthole on postmenopausal osteoporosis using ovariectomized rats; comparison to the effects of estradiol.

Li XX, Hara I, Matsumiya T. Biol Pharm Bull. 2002 Jun;25(6):738-42.

The purpose of this study was to examine effects of osthole on postmenopausal osteoporosis using ovariectomized (OVX) rats. All of the rats were divided into sham and OVX groups. At 2 weeks post-operation, the sham-operated rats received solvent vehicle (97% corn oil and 3% ethanol, 1.0 ml/kg, subcutaneously); the OVX rats were divided into three groups which were treated with solvent vehicle (same the sham rats, 1.0 ml/kg, subcutaneously), 17beta-estradiol (30 microg/kg, subcutaneously) or osthole (9.0 mg/kg, orally) 5 d/week for 4 weeks, respectively. In OVX rats, the increases of body weight, spleen and thymus weight were significantly decreased and the atrophy of uterus was preserved by 17beta-estradiol treatment, but not by osthole. Treatment with either 17beta-estradiol or osthole significantly protected cancellous bone loss owing to estrogen deficiency and significantly increased the maximal load in the femoral neck of OVX rats. In addition, the increases of serum osteocalcin (OC) and urinary deoxypyridinoline (DPD) levels caused by ovariectomy were all significantly suppressed by 17beta-estradiol. However, only urinary DPD was significantly reduced by osthole and no change was found in serum OC. Our results demonstrate that osthole may be just as effective as 17beta-estradiol in suppressing bone loss due to ovariectomy but osthole perhaps does not work through the estrogen pathway.

Effects of coumarins from cnidium monnieri on the function of pituitary-adrenocortical axis in kidney yang deficiency rats

Qin LP, Zhang JQ, Shi HP. Zhongguo Zhong Xi Yi Jie He Za Zhi. 1997 Apr;17(4):227-9.

OBJECTIVE: To observe the effect of Cnidium monnieri in Kidney Yang Deficiency rats. METHODS: Rats were injected intraperitoneally with hydrocortisone acetate to replicate the animal model of Kidney Yang Deficiency. Osthol, total-coumarins (TCR) from the fruit of Cnidium monnieri and positive control drug (PCD) were administered orally to model rats. The plasma levels of corticosterone (B), adrenocorticotropic hormone (ACTH), renin (PRA), angiotensin-II (AT-II) and aldosterone (ALD) were determined by radioimmunoassay. RESULTS: Compared with those in normal rats, the levels of B and ACTH in the plasma of model rats lowered significantly (P< 0.01), the levels of B and ACTH of treatment groups were higher than those of model group, while the levels of PRA, AT-II and ALD had no significant difference (P > 0.05). CONCLUSION: The osthol, TCR have the effects in protecting and strengthening the function of pituitary-adrenocortex axis.

Lu Rong 

Lu Rong can boost strength and endurance, increase oxygen-carrying capacity of the blood, stimulate production and circulation of blood, increase blood count, and lower the level of free radicals. As an aphrodisiac, Lu Rong can treat sexual dysfunction, boost estrogen and testosterone levels. It can promote youthfulness, increase mental clarity, improve immune system functioning, and promote rapid recovery from illness. It also has anticancer and anti-inflammatory effects.

Effects of pilose antler and antler glue on osteoporosis of ovariectomized rats

Meng HY, Qu XB, Li N, Yuan S, Lin Z. Zhong Yao Cai. 2009 Feb;32(2):179-82.

OBJECTIVE: To observe the effects of pilose antler and antler glue on osteoporosis of ovariectomized rats. METHODS: 60 6-month-old female rats were divided randomly into sham-operated group, model group, the positive group, pilose antler high-dose group, pilose antler low-dose group and antler glue group. We extracted bilateral ovaries to establish osteoporosis model and treated at two weeks postoperationally for consecutively 13 weeks, then observed the effects of pilose antler and antler glue on rat bone mineral density, bone mineral content, serum biochemical indicators, bone tissue morphology and other indicators. RESULTS: After 13 weeks, the high-dose group and antler glue group could significantly improve the BMD and bone mineral content of the ovariectomized rats, reduce BGP and ALP content, and remarkably increase the width of trabecula bone and bone trabecula area percentage, increase osteoblasts significantly, and decrease osteoclast cells significantly. Pilose antler low-dose also had the phenomenon above, but the result was not so good as high-dose group. CONCLUSION: Pilose antler and antler glue can antagonize osteoporosis of the ovariectomized rats.

Testosterone, but not IGF-1, LH, prolactin or cortisol, may serve as antler-stimulating hormone in red deer stags (Cervus elaphus).

Bartos L, Schams D, Bubenik GA. Bone. 2009 Apr;44(4):691-8. Epub 2008 Dec 16.

The role of androgens and insulin-like growth factor 1 (IGF-1) in antler growth has been disputed. We predicted that the secretory of IGF-1 may be associated with an acceleration of body growth rather than with antler growth. Furthermore we anticipated a relationship between the increase of testosterone and the progress of antler growth. If IGF-1 is involved in the stimulation of antler growth, this should be more obvious in young than in mature stags. Eight two-year-old red deer stags (Cervus elaphus), and twelve adult red deer stags were blood sampled and the length of their velvet antlers was measured in one-week intervals during the period of antler growth. Concentrations of testosterone, cortisol, IGF-1, luteinizing hormone (LH), and prolactin were determined in plasma by enzyme immunoassay or radioimmunoassay. Antler growth per day was primarily dependent on changes in testosterone concentration per day in both groups of stags. As expected, only in two-year-old stags we detected a possible role of IGF-1 in the antler growth regulation, but that was not in agreement with previously published studies. Nevertheless, this effect was still utilized in interaction with testosterone. In addition to total antler length, only concentrations of testosterone and LH were significantly higher in adult males in comparison to two-year-old males. Our present results lead us to conclude that it is not IGF-1 but testosterone which is responsible for the intensity of antler growth in subadult and adult red deer stags.

Root Rehmannia glutinosa (Shu Di Huang)

Study on phytoestrogenic-like effects of four kinds of Chinese medicine including Radix Rehmanniae Preparata, Radix Paeoniae Alba, Radix Angelicae Sinensis, Rhizoma Chuanxiong

Hao Q, Wang J, Niu J, Zhao P, Cui Y, Sun L, Xue X. Zhongguo Zhong Yao Za Zhi. 2009 Mar;34(5):620-4.

OBJECTIVE: To explore the phytoestrogenic-like effects of four kinds of Chinese medicine including Radix rehmanniae preparata, Radix Paeoniae Alba, Radix Angelicae Sinensis, Rhizoma Chuanxiong. METHOD: Sixty immature female SD rats weighting (70 +/-5) g were randomly divided into six groups: normal control group, positive control group and 4 Chinese medicine groups. The rats in different groups were treated for 4 days. On the fifth day, animals were sacrificed and uteri were separated solely and weighed. The blood was collected, and serum was separated. The effect of the pharmacological serum on proliferation assay with human breast cancer cell line (MCF7) by MTT method. Cell-cycle analyses were carried out with propidium iodide staining by flow cytometer. The expressions of subtypes-estrogen receptor-alpha (ERalpha) were detected by flow cytometry. RESULT: Radix Rehmanniae Preparata, Radix Paeoniae Alba and Radix Angelicae Sinensis could increase the immature rat's uterus wet weight and the ratio of uterus to body weight (P< 0.05). The pharmacological serum of the four kinds of Chinese medicine stimulated proliferation of MCF7 cell respectively compared with normal control group (P< 0.01). The cell cycle was impulsed from G1 to S, DNA synthesizing was inhanced, and PI was also increased. The pharmacological serum of Radix Angelicae Sinensis and Rhizoma Chuanxiong could increase the expressions of (ERalpha) (P< 0.05). CONCLUSION: Radix Rehmanniae Preparata, Radix Paeoniae Alba and Radix Angelicae sinensis have phytoestrogenic effects. But the data werent consistent with in vitro and in vivo assay of Rhizoma Chuanxiong.

Effects of pilose antler and antler glue on osteoporosis of ovariectomized rats

Meng HY, Qu XB, Li N, Yuan S, Lin Z. Zhong Yao Cai. 2009 Feb;32(2):179-82.

OBJECTIVE: To observe the effects of pilose antler and antler glue on osteoporosis of ovariectomized rats. METHODS: 60 6-month-old female rats were divided randomly into sham-operated group, model group, the positive group, pilose antler high-dose group, pilose antler low-dose group and antler glue group. We extracted bilateral ovaries to establish osteoporosis model and treated at two weeks postoperationally for consecutively 13 weeks, then observed the effects of pilose antler and antler glue on rat bone mineral density, bone mineral content, serum biochemical indicators, bone tissue morphology and other indicators. RESULTS: After 13 weeks, the high-dose group and antler glue group could significantly improve the BMD and bone mineral content of the ovariectomized rats, reduce BGP and ALP content, and remarkably increase the width of trabecula bone and bone trabecula area percentage, increase osteoblasts significantly, and decrease osteoclast cells significantly. Pilose antler low-dose also had the phenomenon above, but the result was not so good as high-dose group. CONCLUSION: Pilose antler and antler glue can antagonize osteoporosis of the ovariectomized rats.

Testosterone, but not IGF-1, LH, prolactin or cortisol, may serve as antler-stimulating hormone in red deer stags (Cervus elaphus).

Bartos L, Schams D, Bubenik GA. Bone. 2009 Apr;44(4):691-8. Epub 2008 Dec 16.

The role of androgens and insulin-like growth factor 1 (IGF-1) in antler growth has been disputed. We predicted that the secretory of IGF-1 may be associated with an acceleration of body growth rather than with antler growth. Furthermore we anticipated a relationship between the increase of testosterone and the progress of antler growth. If IGF-1 is involved in the stimulation of antler growth, this should be more obvious in young than in mature stags. Eight two-year-old red deer stags (Cervus elaphus), and twelve adult red deer stags were blood sampled and the length of their velvet antlers was measured in one-week intervals during the period of antler growth. Concentrations of testosterone, cortisol, IGF-1, luteinizing hormone (LH), and prolactin were determined in plasma by enzyme immunoassay or radioimmunoassay. Antler growth per day was primarily dependent on changes in testosterone concentration per day in both groups of stags. As expected, only in two-year-old stags we detected a possible role of IGF-1 in the antler growth regulation, but that was not in agreement with previously published studies. Nevertheless, this effect was still utilized in interaction with testosterone. In addition to total antler length, only concentrations of testosterone and LH were significantly higher in adult males in comparison to two-year-old males. Our present results lead us to conclude that it is not IGF-1 but testosterone which is responsible for the intensity of antler growth in subadult and adult red deer stags.

Xian Ling Pi

Xian Ling Pi has hormone-like effects. It can decrease blood pressure and increase blood flow in coronary artery. 100% decoction of this herb can decrease lipoprotein, cholesterol and blood sugar. It also can improve and enhance physical stamina and immunity.

Differential Effects of Different Types of Tonifying Kidney on mRNA Expression of Estrogen Receptor of Amygdala and Cortical Parietal Lobe in Male Rats

CAI Jing, DU Jian, CAO Zhi-yun. Fu Jian Zhong Yi Xue Yuan Xue Bao. 2006; 16(1): 34-36.

Objective:To study the regulatory effects of two different herbs of tonifying kidney (Epimedium and Fructus Ligustri Lucidi) on mRNA expression of estrogen receptor a and b of amygdala and cortical parietal lobe in male rats from molecule level. Methods: The male SD rats of group A and B were perfused with Epimedium and Fructus Ligustri Lucidi for 14 days respectively, group C as the control, then the mRNA expression of ERa and ERb were detected by RT-PCR. Result: The expression of ERa had no obvious variation between group A and B, but ERb in group A was higher than that of group B and C (P<0.01), the expression ERb of amygdala in group B was higher than the control group (P<0.05). Conclusion. Tonifying kidney perhaps improve the cognitive function through regulating ERb gene. The result disagrees with the traditional theory of Chinese medicine and we will open out and make sure this novel theory.

Experimental studies on anti-Partial androgen deficiency effects of icariin

Zhang Zhenbao, Yang Qingtao. Zhong Guo Nan Ke Xue Za Zhi. 2006; 20(3): 47-50.

Objective To explore the protective effects oflcariin on partial androgen deficiency rat. Methods Reproduction system of 15 months male rat was first damaged by CYP. After ig. Icariin and ip. Propyltestosterone, the content of serum T, LH, FSH, BGP and StrACP were determined. The change of histology of the testis and the penis were observed by microscope with the method of H-E staining and TUNEL method respectively. Results (1) As to the contents of serum T, T group 04, 96±11.42)ng/ml and ICA group (10.94±2.04)ng/ml were statistically higher than C group 0.01±0.41)ng/mland M group (0.79±0.45)ng/m1. Furthermore, Tgroup as higher than ICA group, and C group as higherthan M group (P<0, 05); (2) Serum BGP, T group (4.50±1, 70)ng/ml and ICA group (3.44±0.51) ng/ml were statistically higher than C group (1. 82±0.50)ng/ml and M group (1.22±0.50) ng/ml (P<0.05); (3)Serum Str ACP, T group (25.73±7.57)U/L and ICA group (19.99±8.63)U/L were statistically lower than C group(51.32±14.16)U/L and M group(64.89±12.57)U/L, Furthermore, C group was lower than M group (P<0, 05); (4)As to the cell apoptosis rate in rat penis, T group (22.65±5.17)% and ICA group(34.26±3.45)%were statistically lower than M group (48.28±6.74)%, (P<0.05). Conclusion lcariin possesses theanti-partial androgen deficiency effects, and is beneficial to PADAM.

Comparative studies on protective effects on reproduction of Icariin and the substance extracted from the semen cuscutae in rats with partial androgen deficiency

ZHANG Zhen-Bao, YANG Qing-Tao, YANG Jing-Qiu, Zhen Junhong, Zhou Derong. Zhong Guo Lao Nian Xue Za Zhi. 2006; 26(10): 1389-1391.

Objective To evaluate the protective effects on reproduction of Icariin(ICA) and semen cuscutae(SSC) on partial androgen deficiency(PAD) rats. Methods The male rat model for impaired reproduction system and partial androgen deficiency were made by cyclophosphamide to determine the content of serum testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) after ig. ICA and SCC. The change of histology of the testis and the penis were observed by microscope with the method of H-E staining and TUNEL method respectively. Results (1)The contents of serum testosterone in ICA group were statistically higher than that in control group and model group, and were statistically lower than that in testosterone group (P<0.05). However, contents of serum testosterone in SSC group were higher than that in model group, and lower than that in testosterone group. But there had no statistically difference between SSC group and control group. (2)There had no statistically difference among every group as to the contents of LH and FSH. (1)The cell apoptosis rate in rat penis in testosterone group and ICA group were statistically lower than that in model group (P<0.05). But there had no statisticaldifference between SSC group and model group. Conclusions ICA and SSC both have the protective effects on reproduction on partial androgen deficiency rats, but ICA is much more beneficial to PAD in the aging male (PADAM).

Study on the estrogen-like effects of Epimedium extractive

YU Yan, YAN Hong, HU Sen ke, ZHANG Jing-hua. Xi An Jiao Tong Da Xue Xue Bao: Yi Xue Ban. 2009; 30(3): 373-376.

Objective To investigate the estrogen-like effects of Epimedium extractive. Methods By using the uterus growth test in mice and test of MCF-7 proliferation and adding estrogen receptor antagonist Icariin, we evaluated the estrogenic effects of Epimedium and Icariin and the possible mechanism. Ftesults Epimedium and lcariin increased significantly the uterus coefficient in mice from 0.097 ± 0.008 to 0.156 ± 0.018 and 0.152 ± 0.016, respectively; the weight increased significantly from (18.9±1.3)g to (19.8±1.3)g and (20.03± 1.3)g (P<0.05). Icariin incrcased significantly the serum level of E2 from (132.22 ± 11.45)ng/L to (147. 73 ± 12.43)ng/L and decreased FSH level from (50. 15 ± 3.36) mg/L to (44.47 ± 2.46) mg/L (P<0.05), but there was no significant difference in Epimedium group. Thcrc was obviously enhanced proliferation in MCF-7 in 24, 48 and 72 h (P<0.05), from 0.238±0.043, 0.387±0.051 and 0.563±0.083 to 0.362±0.045, 0.521±0.046 and 0.648±0.043 as well as 0. 324±0.043, 0.465±0.034 and 0.622±0.079, respectively. However, the cell proliferation was inhibited significantly by Icariin added in 72 h, the proliferation ratio decreased to 99. 461% and 94. 434%, respectively. Conclusion Epimedium and Icariin have estrogen-like effects and all of their effects arc realizcd through the estrogen receptor.

Dang Gui 

Dang Gui has bi-directional functions on the uterus: it can inhibit the uterine muscle with its volatile oil, and excite the uterus muscle with its water-soluble and alcohol-soluble nonvolatile substance. Dang Gui can dilate coronary arteries in isolated guinea pigs, and has been shown to increase blood flow and lower the blood pressure in clinical trials. It can decrease blood fat of experimental hyperlipoidemia and has a protective effect on experimental atheroma. Dang Gui and its ingredient ferulic acid can inhibit palate aggregation and antagonize formation of thrombosis. It also has anti-anemia, sedative, analgesic, anti-inflammatory and antibacterial effects. Besides, Dang Gui can promote the transformation of lymphocytes.

Estrogenic activity of standardized extract of Angelica sinensis.

Circosta C, Pasquale RD, Palumbo DR, Samperi S, Occhiuto F. Phytother Res. 2006 Aug;20(8):665-9.

Since ancient times, extracts of plants have been used for women's health to prevent menopausal symptoms. The symptoms of menopause have been attributed to a reduction in the amount of estrogen produced by the ovaries. In this study the estrogenic activity of a commercial standardized extract of the roots of Angelica sinensis, used to relieve climacteric symptoms was evaluated using in vivo tests such as the degree of cornification of vaginal epithelium, uterotrophic assays and serum LH concentration in ovariectomized rats. Furthermore, the effects on the estrous cycle in rat were investigated. The results obtained have shown that the administration of a standardized ethanol extract in ovariectomized rats exhibited a stimulation of the uterine histoarchitecture, a significant cornification in the vaginal epithelium and a reduction of serum LH concentration showing the estrogenic nature of the extract. Furthermore, the administration of the extract in intact female rats provoked a significant modification of the vaginal smear in 67% of treated rats. The estrous cycle thus modified was characterized by a prolonged estrus stage with a temporary reduction of the regular cyclicity.

The immediate effect of natural plant extract, Angelica sinensis and Matricaria chamomilla (Climex) for the treatment of hot flushes during menopause. A preliminary report.

Kupfersztain C, Rotem C, Fagot R, Kaplan B. Clin Exp Obstet Gynecol. 2003;30(4):203-6.

OBJECTIVE: To assess the efficiency of a medicinal herb extract preparation (Climex) for the treatment of menopausal symptoms. METHOD: In this placebo-controlled experiment on 55 postmenopausal women who complained of hot flushes and refused hormonal therapy. The women were randomly divided into two groups, one to receive Climex (5 chewable tablets daily between meals) and the other group to receive a placebo; both groups would take the tablets for 12 weeks. The women were asked to complete a daily structured (Kupperman) questionnaire assessing the frequency and intensity of menopausal symptoms, starting one week prior to treatment to the completion of the study. All women underwent hormone profile measurements and transvaginal ultrasonography evaluation before and after treatment. RESULTS: There was a significant difference between the study group and the control group in the decrease in number and intensity of hot flushes from baseline to completion of treatment (90-96% vs 15-25%, p< 0.001). In the study group, a response was already noted during the first month of treatment (68% +/-2% reduction of hot flushes during the day and 74% +/-4% during the night). There was also a marked alleviation of sleep disturbances and fatigue. CONCLUSIONS: Treatment with Climex seems to be effective for menopausal symptoms without apparent major adverse effects. This hormone-free preparation may be used as an important modality for menopausal women with contraindications for hormone replacement therapy.

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

Liu J, Burdette JE, Xu H, Gu C, van Breemen RB, Bhat KP, Booth N, Constantinou AI, Pezzuto JM, Fong HH, Farnsworth NR, Bolton JL. J Agric Food Chem. 2001 May;49(5):2472-9.

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

Fructus Cnidii

Fructus Cnidii is traditionally regarded as medicine of warming the kidney to activate YANG. Modern research proved that it has certain effects on animals with deficient Yang, which may be gained by acting on metabolism of cyclic nucleoside acid and prostaglandin. Coumarin/Osthole may be effective ingredient of Fructus Cnidii for warming the kidney to activate YANG. There is one specific prostaglandin that indeed plays a role in the male sexual tract, prostaglandin E1.

In the words of medical researcher A.Lea:" prostaglandin E1 is a vasodilating agent which acts by relaxing the smooth muscles of the corpus cavemosum and by increasing the diameter of cavemous arteries; this leads to erection."

Ren Shen 

Ren Shen can regulate nervous function and restore dysfunctional nervous progress caused by tension and then improve memory and to focus. It has anti-fatigue effects which helps improve working and functional ability. Ren Shen can dilate coronary arteries, cerebral blood vessels, ocular blood vessels and peripheral blood vessels of the whole organism, while contracting isolated auricular blood vessels of rabbits and isolated blood vessels of rats. Extract of Ren Shen can increase RBC, Hb and WBC of rabbits. Ginsenosides can increase content of ACTH and corticosteroid in plasma by exciting hypothalamic or pituitary function, it has gonadotropic effects. Ginsenosides can also promote metabolism of lipids and biologic synthesis of cholesterol and blood lipoprotein of rats. But both Ren Shen and ginsenosides can decrease hypercholesterolemia in animals. Besides, Ren Shen can reinforce phagocytic function of WBC and reticuloendothelial system. It also has diuretic, anti-shock and anti-tumor actions.

Study on the Changes of Rat Testis Androgen Receptor mRNA Expression and Plasma Testosterone after Cold Stress

WANG Hong-jun, FENG Li, CHU Zheng, YU Ning, YANG Qing, ZHANG zhi-qiang, WANG Li-qiang, WU Yi-min. Shi Zhen Guo Yi Guo Yao. 2008; 19(4): 929-930.

Objective To study the effect of ginseng polysugar on the mRNA expression of testis tissue androgen receptor and plasma testosterone change after cold stress. Methods Rats were divided into control group, cold stress experiment group and cold stress ginseng polysugar group, testis tissue androgen receptor mRNA expression was determined by rat androgen receptor cDNA probe dot blotting. Testosterone was determined by radioimmunoassay kit(RIA kit). Results The content of testosterone(nmol/ L) was 10.93 ± 1.44 in control group, 6.06 ± 1.78 in cold stress experiment group, 8. 01 ± 1.38 in cold stress ginseng polysugar group. There was a significant synthesis difference between cold stress group and control group(P<0.05). Dot-blot: the mRNA expression of androgen recepter in cold stress group was weaker than that in control group, cold stress ginseng polysugar group was more than that in cold stress group. Conclusion Testosterone has identical change with androgen receptor mRNA expression, ginseng polysugar has up regulation action for androgen receptor mRNA expression, and promote testosterone.

The Effect of Ginseng Polysugar on Secretion Function of Luteal and Granulose Cell of Rat in vitro

SUN Yan, FENG Li, WANG Hong-jun, NIAN Chun-zhi, CHU Zheng, WANG Li-qiang, ZHANG zhi-qiang, WU Yi-min. Shi Zhen Guo Yi Guo Yao. 2007; 18(8): 1925-1926.

Objective To study using of ginseng polysugar on secretion function of female rat ovary. The experiment had observed progesterone and cAMP by HCG inducing luteal and granulose cell as well as living cell rate of oocyte in vitro. Methods Rat luteal and granulose cell and oocyte was cultivated, cell counting was worked by. microscope, progesterone and cAMP content was determined by radioimmunoassay kit(RIA kit). Cell exist rate of oocyte was determined by MTF way, Results Progesterone: the luteal cell test groups compare with control group, 6573.46 ± 185.14 and(4509.55 ±126.82) pmol, The granulose cell test groups compare with control group, 197.16±42.87 and (320.42±12, 46)pmol. cAMP: the luteal cell test groups compare with control group, 31.20±17.13and(65.26±15.93) fmol, the granulose cell test groups compare with control group, 121.15 ±19.96 and (296.42 ± 27.28)fmol, Conclusion This treatise had demonstrated that ginseng polysugar can inhibit HCG inducing progesterone secretion and enhancing cAMP content of luteal and granulose cell of rat, ginseng polysugar has lower oocytes cell grow up inhibitory rate. It is evident that ginseng polysugar possesses characteristic of dose dependent.

Rhiz. Curculigo orchiodes (Xian Mao)

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

Vijayanarayana K, Rodrigues RS, Chandrashekhar KS, Subrahmanyam EV. J Ethnopharmacol. 2007 Nov 1;114(2):241-5. Epub 2007 Aug 12.

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.

Bark Eucommia ulmoides (Du Zhong)

Effect of Eucommia Bark on Weight of Femur, Mineral Content, Anti-bend Strength of Tibiae and Serum ALP in Ovariectomized Rats

CAI Jian-ping, ZHANG Xian, XIA Shu-lin, XIE Huan-song. Shi Zhen Guo Yi Guo Yao. 2009; 20(8): 1967-1969.

Objective To observe the effect of Eucommia Bark on weight of femur, mineral content, anti-bend strength of tibiae and serum ALP in ovariectomized (ovx) rats. Methods 80 femal SD rats were divided into 4 groups, sham, negatire, positive, and experimental groups. The rats in each group were ovarieetomized except sham operation group. 10 days after operation, intragastric administration was used to rats in positive and experimental groups with Alfaealeidol (ALF) and Eueommia Bark respectively once a day. After 3 months, femurs were separated and weight and mineral content were determined, and anti-bend strength of tibiae and serum alkaline phosphatase (ALP) were measured in each group. Results The weight of femur in experimental group was deereased obviously (P<0.05), and positive and experimental groups had no significant change (P>0.05) as compared with sham group. In experimental group, mineral content, activity of serum ALP and anti-bend strength of tibiae were increased compared to negative group (P<0.05), meanwhile activity of ALP in positive group was lower than negative (P<0.05) and sham groups (P<0.01). Conclusion Eucommia Bark can inhibit bone absorption and elevate the bone strength, and has protetive and therapeutic effects on osteoporosis of ovariectomized rats.

Novel phytoandrogens and lipidic augmenters from Eucommia ulmoides.

Ong VY, Tan BK. BMC Complement Altern Med. 2007 Jan 29;7:3.

BACKGROUND: Plants containing compounds such as the isoflavonoids, with female hormone-like effects that bind to human estrogen receptors, are known. But none has been previously shown to have corresponding male hormone-like effects that interact with the human androgen receptor. Here, we report that the tree bark (cortex) of the Gutta-Percha tree Eucommia ulmoides possesses bimodal phytoandrogenic and hormone potentiating effects by lipidic components. METHODS: The extracts of E. ulmoides were tested using in-vitro reporter gene bioassays and in-vivo animal studies. Key compounds responsible for the steroidogenic effects were isolated and identified using solid phase extraction (SPE), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), gas chromatography-mass spectroscopy (GC-MS), electron spray ionisation-mass spectroscopy (ESI-MS) and nuclear magnetic resonance (NMR). RESULTS: The following bioactivities of E. ulmoides were found: (1) a phenomenal tripartite synergism exists between the sex steroid receptors (androgen and estrogen receptors), their cognate steroidal ligands and lipidic augmenters isolated from E. ulmoides, (2) phytoandrogenic activity of E. ulmoides was mediated by plant triterpenoids binding cognately to the androgen receptor (AR) ligand binding domain. CONCLUSION: In addition to well-known phytoestrogens, the existence of phytoandrogens is reported in this study. Furthermore, a form of tripartite synergism between sex steroid receptors, sex hormones and plant-derived lipids is described for the first time. This could have contrasting clinical applications for hypogonadal-and hyperlipidaemic-related disorders.

Fruit Lycium chinense (Gou Qi Zi)

Effect of lycium barbarum polysaccharides on rats’ sexual organ indexes and serum sexual hormone levels

WANG Chun-xiang, JIANG Guo-rong, LIANG Guo-qiang, WU Hui-jiag. Jiang Su Da Xue Xue Bao: Yi Xue Ban. 2007; 17(2): 118-120.

Objective: To investigate the effect of Lycium barbarum Polysaccharides (LBP) on rats of different months and different sex. Methods: Twenty three SD female rats (15 months), 20 female rats (1.5 months) and 19 male rats (5.5 months) were divided into four, two and four groups according to their different doses of LBP. And the drug was poured into the rats’ stomach. Serum sexual hormone levels and sexual organ indexes were measured after two weeks. Results: Taken LBP two weeks later, the weight of uteruses and ovaries of female rats(15 months) were elevateci; the weight of uteruses and ovaries of female rats(1.5 months) were increased remarkably; the weight of muscle levafing anus of male rats(5.5 months) were increased remarkably. While the serum sex hormones of all the groups shown a lower level at normal ranges. Conclusion: LBP could elevate sexual organ index and keep a lower serum sex hormones level at the normal ranges, and further implied that LBP might exert the effects by affecting the sex hormone receptor.

Morinda officinalis (Ba Ji Tian)

Inhibitory effects of morinda officinalis extract on bone loss in ovariectomized rats.

Li N, Qin LP, Han T, Wu YB, Zhang QY, Zhang H. Molecules. 2009 Jun 8;14(6):2049-61.

The present study was undertaken to investigate the protective effects of ethanol extract from the root of Morinda Officinalis (RMO) on ovariectomy-induced bone loss. Administration of RMO extract increased trabecular bone mineral content and bone mineral density of tibia, improved the levels of phosphorus (P), calcium (Ca) and OPG, decreased the levels of DPD/Cr, TRAP, ACTH and corticosterone, but did not reverse the levels of ALP, TNF-alpha and IL-6 in serum of ovariectomized rats. These findings demonstrated that RMO extract reduced bone loss in ovariectomized rats, probably via the inhibition of bone resorption, but was not involved with bone formation. Anthraquinones and polysaccharides from Morinda officinals could be responsible for their antiosteoporotic activity, and the action mechanism of these constituents needs to be further studied. Therefore, RMO has the potential to develop a clinically useful antiosteoporotic agent.

Fruit Cornus officinalis (Shan Zhu Yu)

Effects of the extract of Fructus Corni on secretion of corticotrophin-releasing hormone(CRH) in the hypothalamus of animals with kidney-yang deficiency

JIANG Shujun, SUN Jianrong, Wang Tao, Li Baoyu. Bin Zhou Yi Xue Yuan Xue Bao. 2008; 31(3): 179-181.

Objective To explore effects of extra of Fructus Corni (FCE) on tonifying kidney-yang. Methods The animal model of kidney-yang deficiency with the hydrocortisone was made by exogenous glucocorticoid. Effects of the extract of Fructus Corni on the kidney-yang deficiency rats was observed. Results The FCE can decrease the levels of calmodulin(CaM) mRNA and increase the secretion of CRH in the hypothalamus. Conclusion The extract of Fructus Corni have significant protecting efficacy in the animal’s model with kidney-yang deficiency.

Bark Cinnamomum cassia (Rou Gui)

Stimulatory effects of extract prepared from the bark of Cinnamomum cassia blume on the function of osteoblastic MC3T3-E1 cells.

Lee KH, Choi EM. Phytother Res. 2006 Nov;20(11):952-60.

The ethanol extract from the bark of Cinnamomum cassia Blume (CCE) was tested for estrogenic activity. CCE (4-60 microg/mL) significantly induced the growth of MCF-7 cells, an ER-positive human breast cancer cell line, over that of untreated control cells (P< 0.05). In the ER competitive binding assay, CCE showed higher affinity with ERbeta compared with ERalpha. To investigate the bioactivities of CCE, which act on bone metabolism, the effects of CCE on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were studied. CCE (4-60 microg/mL) dose-dependently increased the survival of MC3T3-E1 cells. In addition, CCE (10 and 50 microg/mL) increased alkaline phosphatase (ALP) activity, collagen synthesis and osteocalcin secretion in MC3T3-E1 cells. Treatment with CCE (10 and 50 microg/mL) prevented apoptosis induced by TNF-alpha (10(-10) m) in osteoblastic cells. In the presence of TNF-alpha, culture with CCE (10-100 microg/mL) for 48 h inhibited the production of IL-6 and nitric oxide in osteoblastic MC3T3-E1 cells. These results suggest that Cinnamomum cassia has a direct stimulatory effect on bone formation in vitro and may contribute to the prevention of osteoporosis and inflammatory bone diseases.