Kaempferol exerts antioxidant effects in age-related diminished ovarian reserve by regulating the HSP90/NRF2 pathway.

Kaempferol is the active ingredient of Er-Xian decoction (EXD), a traditional Chinese medicine formula used clinically to treat ovarian dysfunction, but the mechanism of kaempferol relieving age-related diminished ovarian reserve (AR-DOR) is still unclear. In this study, 36 volunteers and 78 DOR patients (37 patients with EXD treatment) were enrolled in the clinical research. Meanwhile, 32-week-old female mice were used to establish the AR-DOR model, and these model mice were intragastrically administered with 100 mg/kg kaempferol in the presence or absence of 200 mg/kg geranylgeranylacetone (GGA) or 1 mg/kg geldanamycin (GDA). The effects of kaempferol on serum hormone levels and oxidative stress-related indexes were detected by enzyme-linked immunosorbent assay. Antral follicle count (AFC) was determined by hematoxylin-eosin staining. The protein levels of HSP90 and nuclear factor erythroid 2-related factor 2 (NRF2) were assayed by Western blot. This study displayed that the serum anti-Mullerian hormone (AMH) level in DOR patients with EXD treatment was higher than that in DOR patients without EXD treatment. Kaempferol treatment reversed the low levels of AMH, estradiol (E2), AFC, superoxide dismutase (SOD), and catalase (CAT), as well as the high levels of follicle-stimulating hormone (FSH), reactive oxygen species (ROS), and malonaldehyde (MDA). The results showed that HSP90 was predicted to have high affinity with kaempferol, and its expression was inhibited by kaempferol, while the expression of NRF2, the target of HSP90, was up-regulated by kaempferol. However, the above effects of kaempferol were reversed by GGA. On the contrary, GDA enhanced the therapeutic effects of kaempferol on AR-DOR mice. Moreover, the treatment of kaempferol resulted in a reduction in the phosphorylation level of heat shock factor 1 (HSF1), the transcription factor associated with HSP90, and an increase in the phosphorylation level of Src, a client protein of HSP90. In summary, kaempferol exerts an antioxidant effect on AR-DOR by inhibiting HSP90 expression to up-regulate NRF2 expression. This study provides a theoretical basis for the clinical application of kaempferol in AR-DOR.

CangFu Daotan decoction improves polycystic ovarian syndrome by downregulating FOXK1.

Objective: Polycystic ovarian syndrome (PCOS) is a prevalent gynecologic disorder, often associated with abnormal follicular development. Cangfu Daotan decoction (CFD) is a traditional Chinese medicine formula that is effective in alleviating PCOS clinically, but the specific mechanism remains unclear. Forkhead box K1 (FOXK1) is associated with cellular function. This study aimed to explore the effects of CFD and FOXK1 on PCOS.Methods: High-fat diet and letrozole were combined to establish PCOS rat models. Next, primary GCs were extracted from those PCOS rats. Then, GC cells were transfected with si-FOXK1 or oe-FOXK1. CFD-contain serum was prepared, and experiments were conducted to investigate the regulation of FOXK1 by CFD.Results: FOXK1 was highly expressed in GCs of PCOS rats. Further investigation revealed that FOXK1 overexpression resulted in inhibition of proliferation and DNA synthesis, along with promotion of apoptosis and autophagy in GCs. Additionally, it was found that FOXK1 promoted the expressions of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway-related proteins. Interestingly, treatment with CFD reversed all the effects of FOXK1 overexpression in GCs. Conclusion: This study demonstrated that CFD exerted a protective role in PCOS by inhibiting FOXK1, which provided a research basis for the application of CFD in PCOS, and suggested that FOXK1 is a novel therapeutic target in PCOS treatment.

Investigation of the mechanisms and experimental verification of Cuscuta-Salvia in the treatment of polycystic ovary syndrome (PCOS) via network pharmacology.

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with reproduction. The Cuscuta-Salvia formula has been widely used to treat for PCOS in clinic. However, its chemical and pharmacological properties remain unclear. We identified the active components and related targets of Cuscuta-Salvia using UHPLC-ESI-Q-TOF-MS and TCMSP database. Disease targets were obtained from the DisGeNET and GeneCards databases. Subsequently, common targets between Cuscuta-Salvia and PCOS were identified using a Venn diagram. PPI network was established. Core genes were selected using a Cytoscape software plugin. GO and KEGG enrichment analyses were performed for common targets using the "pathview" package in R. Several core targets were verified using molecular and Immunological methods. By combining UHPLC-ESI-Q-TOF-MS with a network pharmacology study, 14 active components and a total of 80 common targets were obtained. Ten core genes were regulated by Cuscuta-Salvia in PCOS, including IL6, AKT1, VEGFA, TP53, TNF, MAPK1, JUN, EGF, CASP3, and EGFR. GO results showed that cellular response to drugs, response to oxygen levels, response lipopolysaccharides, and response to molecule of bacterial origin in BP category; membrane, transcription regulator complex, nuclear chromatin, postsynaptic membrane, and vesicle lumen in CC category; DNA-binding transcription factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, DNA-binding transcription activator activity, RNA polymerase II-specific, DNA-binding transcription activator activity, and cytokine receptor binding in MF terms. The KEGG enrichment pathway was mainly involved in the PI3K - Akt, MAPK, TNF, IL-17 signalling pathways, and in cellular senescence. Furthermore, the results of the experimental study showed that Cuscuta-Salvia ameliorated the pathological changes in the ovaries, liver and adipose tissue. And it improved the expressions of the genes or proteins. Our results demonstrate that Cuscuta-Salvia may provide a novel pharmacological basis in an experimental model of PCOS by regulating gene expression. This study provides a basis for future research and clinical applications.

In vivo and in vitro protective effects of the Wuzi Yanzong pill against experimental spermatogenesis disorder by promoting germ cell proliferation and suppressing apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuzi Yanzong pill (WZYZP) is a classical traditional Chinese medicine (TCM) formula originated from the Tang dynasty. WZYZP has a long history of use for reinforcing kidney and alleviating male infertility in China. AIM OF THE STUDY: The effect of WZYZP on male infertility and the mechanism underlying this effect was not clarified clearly. Therefore, this study aimed to investigate the protective effect of WZYZP in experimental spermatogenesis disorder via in vivo and in vitro studies, to promote the use of this formula for the treatment of spermatogenesis disorder. MATERIAL AND METHODS: Male SD rats were exposed to tripterygium glycosides to induce experimental spermatogenesis disorder, and WZYZP was subsequently administrated at different dosages for treatment. Sperm counts, sperm motility, and serum hormone levels were detected. HE staining and TUNEL staining were performed to evaluate the pathological lesions and apoptosis of testes, respectively. Next, germ cells were isolated from spermatogenesis disorder-model rats and treated with WZYZP- containing serum at different concentrations. CCK-8 assay and flow cytometry assay were performed to detect cell proliferation and apoptosis. Immunofluorescence assay, qRT-PCR and Western blotting analyses were performed to detect the expression of Beclin 1, LC3 and TGF-ß-PI3k/AKT-mTOR pathway - related factors, including TGF-ß, PI3K, AKT, mTOR, 4 EBP-1 and p70S6K. RESULTS: In vivo experiments showed that WZYZP protected against spermatogenesis disorder in model rats by improving sperm count and motility, as well as restoring serum hormone levels. HE and TUNEL staining demonstrated that the pathological injuries and cell apoptosis in testes of the model rats were alleviated by WZYZP treatment. Moreover, in vitro experiments of germ cells isolated from spermatogenesis disorder-model rats showed that WZYZP treatment increased the cell proliferation, inhibited cell apoptosis and autophagy. qRT-PCR and Western blotting assay results showed that this protective effect was associated with the regulation of the TGF-ß/PI3K/AKT/mTOR signaling pathway. The expression levels of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, 4 EBP-1 and p70S6K were increased, while TGF-ß was inhibited in the WZYZP treated groups. CONCLUSION: The results showed that WZYZP could protect against experimental spermatogenesis disorder by increasing the germ cell proliferation and inhibiting their apoptosis. Our support the clinical use of this formula for the management of spermatogenesis disorder.

Protective effects of berberine in a rat model of polycystic ovary syndrome mediated via the PI3K/AKT pathway.

BACKGROUND: Ber, a Chinese herbal monomer has been reported to exhibit an array of pharmacological activities related to the lowering of blood glucose and the treatment of polycystic ovarian syndrome (PCOS). In the present study, we aimed to elucidate the effect of berberine (Ber) on a rat model of PCOS mediated via the PI3K/AKT signaling pathway. METHODS: A PCOS animal model was induced with the administration of letrozole, and animals were then randomized into untreated or Ber and metformin hydrochloride treated groups. After administration, fasting blood glucose, HOMA-IR, fasting insulin (FINS) values, and the serum hormone levels were measured in PCOS rats. The ovarian tissues were stained with hematoxylin and eosin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) for pathological and apoptosis examination. Moreover, the effect of Ber on the proliferation and apoptosis of granulosa cells was detected by CCK-8 assays and flow cytometry. The influence of Ber on granulosa cells was confirmed by blockade of the PI3K/AKT pathway. In addition, the modulatory effect of the blockade of the PI3K/AKT pathway on the expression of related proteins was demonstrated via western blotting. RESULTS: We found that Ber was able to restore the serum hormone levels and improve IR in a PCOS rat model. The morphological lesions and apoptosis of the ovary were also restored by the Ber treatment. Blockade of the PI3K/AKT pathway attenuated the influences of Ber on the proliferation and apoptosis of granulosa cells. CONCLUSION: The beneficial effects of Ber on PCOS included alterations of the serum hormone levels, recovery of morphological lesions in the ovary, improvement of insulin resistance, and cell viability and inhibition of apoptosis, which were all mediated through the PI3K/AKT pathway.

Cangfudaotan Decoction Alleviates Insulin Resistance and Improves Follicular Development in Rats with Polycystic Ovary Syndrome via IGF-1-PI3K/Akt-Bax/Bcl-2 Pathway.

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder prevalent in females of reproductive age; insulin resistance (IR) is the major pathogenic driver. Pharmacology is a basic option for PCOS therapy; traditional Chinese medicine (TCM), as a significant part of complementary and alternative medicine, has a long history in the clinical management of PCOS. Cangfudaotan decoction (CFD) has been used clinically for gynaecological diseases especially PCOS. In this study, first, chemical components in CFD were clarified using UPLC-Q/TOF-MS analysis. Then, an animal model of PCOS was established, granular cells were also isolated from the rats with PCOS, and CFD was administrated at different dosages in PCOS rats and granular cells, to investigate the therapeutic effect and mechanisms of CFD for PCOS treatment. The result showed that CFD treatment is effective in PCOS rats and granulosa cells. CFD was able to improve IR, restore the serum hormone levels, inhibit the inflammatory cytokines in PCOS rat, and alleviate ovary morphological injury and apoptosis in PCOS rats. In granulosa cells of PCOS, the result showed that the cell viability was improved, and cell apoptosis was inhibited after CFD administration. Further experiments suggested that CDF improves IR, follicular development, cell apoptosis, and inflammatory microenvironment, and this was associated to the regulation of IGF-1-PI3K/Akt-Bax/Bcl-2 pathway-mediated gene expression. Given that CFD sufficiently suppresses insulin resistance and improves follicular development in this study, exploring these mechanisms might help to optimize the therapeutic treatment of CFD in PCOS patients.

Wuzi Yanzong Pill-Based on Network Pharmacology and In Vivo Evidence-Protects Against Spermatogenesis Disorder via the Regulation of the Apoptosis Pathway.

The crisis of male infertility is an issue of human reproductive health worldwide. The Wuzi Yanzong pill (WZYZP) is a traditional Chinese medicine prescription that shows efficacy in kidney reinforcement and essence benefit to ameliorate male reproductive dysfunctions. However, the pharmacological mechanisms of the WZYZP on male infertility have not been investigated and clarified clearly. This study was designed to investigate the effects of the WZYZP on spermatogenesis disorder and explore its underlying pharmacological mechanisms. First, based on a network pharmacology study, 39 bioactive compounds and 40 targets of the WZYZP associated with spermatogenesis disorder were obtained, forming a tight compound-target network. Molecular docking tests showed tight docking of these compounds with predicted targeted proteins. The protein-protein interaction (PPI) network identified TP53, TNF, AKT1, Bcl-XL, Bcl-2, and IκBA as hub targets. The Kyoto Encyclopedia of Genes and Genomes pathway network and pathway-target-compound network revealed that the apoptosis pathway was enriched by multiple signaling pathways and multiple targets, including the hub targets. Subsequently, the chemical characterization of WZYZP was analyzed using liquid chromatography to quadrupole/time-of-flight mass spectrometry, and 40 compounds in positive ion mode and 41 compounds in negative ion mode in the WZYZP were identified. Furthermore, based on the prediction of a network pharmacology study, a rat model of spermatogenesis disorder was established to evaluate the curative role and underlying mechanisms of the WZYZP. The results showed that WZYZP treatment improved rat sperm quality and attenuated serum hormone levels, reversed histopathological damage of the testis, reduced cell apoptosis in testis tissues, and ameliorated the expression of the predicted hub targets (TP53, TNF-α, AKT1, NFκB, and IκBA) and the apoptosis related proteins (Bcl-XL, Bcl-2, Bax, Caspase 3, and Caspase 9). These results indicated that the WZYZP has a protective effect on spermatogenesis disorder, suggesting that it could be an alternative choice for male infertility therapy.

[Combined therapy of Compound Xuanju Capsules and traditional Chinese medicinal formula for infertility in male smokers].

OBJECTIVE: To investigate the clinical effects of the combined therapy of Compound Xuanju Capsules (CXJC) and traditional Chinese medicinal formula on infertility in male smokers. METHODS: A total of 176 male infertility patients were divided into a smoking and a non-smoking group and the former further divided into mild, moderate and heavy smokers according to the daily consumption of cigarettes and the length of smoking history. The patients were treated with CXJC combined with traditional Chinese medicinal formula for 3 four-week courses and the therapeutic results were evaluated by comparing the indicators of the traditional Chinese medicine (TCM) syndrome, routine semen parameters, sperm morphology, and sperm DNA fragmentation index (DFI) among different groups before and after treatment. RESULTS: The baseline TCM syndrome scores were remarkably higher in the heavy smokers than in the non-smoking group (P < 0.05) but showed no statistically significant differences between the mild and moderate smokers (P > 0.05). The baseline percentage of sperm head defects and DFI were also markedly higher in the heavy and moderate smokers than in the non-smoking group (P < 0.05). Compared with the baseline, significant improvement was achieved after treatment in the TCM syndrome, routine semen parameters, sperm morphology and sperm DFI, especially in the heavy smokers in the percentages of grade a+b sperm (ï¼»17.12 ± 2.54ï¼½ vs ï¼»30.15 ± 3.10ï¼½%, P < 0.05), morphologically normal sperm (ï¼»15.54 ± 1.98ï¼½ vs ï¼»26.82 ± 3.52ï¼½%, P < 0.05), and head-defective sperm (ï¼»27.02 ± 2.14ï¼½ vs ï¼»22.07 ± 1.52ï¼½%, P < 0.05). CONCLUSIONS: Sperm quality is significantly decreased while the risk of infertility remarkably increased in moderate and heavy smokers. The combined therapy of CXJC and traditional Chinese medicinal formula can effectively improve semen quality, sperm morphology and sperm DFI in male smokers with infertility, though more evidence is to be collected from further studies.

Effect of Acupuncture and Clomiphene in Chinese Women With Polycystic Ovary Syndrome: A Randomized Clinical Trial.

Importance: Acupuncture is used to induce ovulation in some women with polycystic ovary syndrome, without supporting clinical evidence. Objective: To assess whether active acupuncture, either alone or combined with clomiphene, increases the likelihood of live births among women with polycystic ovary syndrome. Design, Setting, and Participants: A double-blind (clomiphene vs placebo), single-blind (active vs control acupuncture) factorial trial was conducted at 21 sites (27 hospitals) in mainland China between July 6, 2012, and November 18, 2014, with 10 months of pregnancy follow-up until October 7, 2015. Chinese women with polycystic ovary syndrome were randomized in a 1:1:1:1 ratio to 4 groups. Interventions: Active or control acupuncture administered twice a week for 30 minutes per treatment and clomiphene or placebo administered for 5 days per cycle, for up to 4 cycles. The active acupuncture group received deep needle insertion with combined manual and low-frequency electrical stimulation; the control acupuncture group received superficial needle insertion, no manual stimulation, and mock electricity. Main Outcomes and Measures: The primary outcome was live birth. Secondary outcomes included adverse events. Results: Among the 1000 randomized women (mean [SD] age, 27.9 [3.3] years; mean [SD] body mass index, 24.2 [4.3]), 250 were randomized to each group; a total of 926 women (92.6%) completed the trial. Live births occurred in 69 of 235 women (29.4%) in the active acupuncture plus clomiphene group, 66 of 236 (28.0%) in the control acupuncture plus clomiphene group, 31 of 223 (13.9%) in the active acupuncture plus placebo group, and 39 of 232 (16.8%) in the control acupuncture plus placebo group. There was no significant interaction between active acupuncture and clomiphene (P = .39), so main effects were evaluated. The live birth rate was significantly higher in the women treated with clomiphene than with placebo (135 of 471 [28.7%] vs 70 of 455 [15.4%], respectively; difference, 13.3%; 95% CI, 8.0% to 18.5%) and not significantly different between women treated with active vs control acupuncture (100 of 458 [21.8%] vs 105 of 468 [22.4%], respectively; difference, -0.6%; 95% CI, -5.9% to 4.7%). Diarrhea and bruising were more common in patients receiving active acupuncture than control acupuncture (diarrhea: 25 of 500 [5.0%] vs 8 of 500 [1.6%], respectively; difference, 3.4%; 95% CI, 1.2% to 5.6%; bruising: 37 of 500 [7.4%] vs 9 of 500 [1.8%], respectively; difference, 5.6%; 95% CI, 3.0% to 8.2%). Conclusions and Relevance: Among Chinese women with polycystic ovary syndrome, the use of acupuncture with or without clomiphene, compared with control acupuncture and placebo, did not increase live births. This finding does not support acupuncture as an infertility treatment in such women. Trial Registration: clinicaltrials.gov Identifier: NCT01573858.

[Effect of modified cangfu daotan decoction in improving endometrial receptivity in infertility patients with polycystic ovarian syndrome].

OBJECTIVE: To study the effect and potential mechanism of Modified Cangfu Daotan Decoction (MCDD) on endometrial receptivity in infertility patients with polycystic ovarian syndrome (PCOS). METHODS: Totally 298 women having normal ovulation who underwent artificial insemination were recruited as the control group, and they received no drug therapy. Another 355 infertility patients with PCOS who received ovarian stimulation therapy were recruited as the treatment group. Then they were further assigned to the treatment group I (195 cases) and the treatment group II (160 cases) according to random digit table. Patients in the treatment group I received clomiphene (CC) + human menopause gonadotropin (HMG) +human chorionic gonadotropin (HCG), while those in the treatment group II received CC + HMG + HCG and additionally took modified MCDD. The therapeutic course for all was three menstrual cycles. The pregnancy ratio, the endometrial thickness, and spiral artery pulsatility index (PI), resistance index (RI), and homeostasis model assessment-insulin resistance (HOMA-IR) were measured. Furthermore, the uncoupling protein 2 (UCP2) level was tested by Western blot. RESULTS: Compared with the control group, the endometrial thickness decreased and PI and RI increased in the treatment group I (all P < 0.05). Compared with the treatment group I , the endometrial thickness increased and PI and RI decreased in the treatment group II (all P < 0.05). Compared with before treatment, HOMA-IR levels were significantly decreased in the treatment group II after treatment (P < 0.05). Compared with the control group before treatment, the HOMA-IR level increased in the treatment group I and the treatment group II before treatment (P < 0.05). Compared with the control group after treatment, the HOMA-IR level increased in the treatment group I (P < 0.05). But there was no statistical difference in the post-treatment HOMA-IR level between the control group and the treatment group II (P < 0.05). Compared with the control group, the post-treatment UCP2 level was increased in the treatment group II (P < 0.05). After one year follow-up, the pregnancy rate was 16.1% (48/298) in the control group, 23.1% (37/160) in the treatment group I, and 33.8% (66/195) in the treatment group II. Compared with the control group, the pregnancy rate was significantly increased in the treatment group II (P < 0.05). CONCLUSION: MCDD was found to be capable of increasing the pregnancy rate of infertility patients with PCOS, which might be associated with improving endometrial blood flow and insulin resistance, increasing the UCP2 expression, and finally improving the endometrial receptivity.

[Extract from Morindae officinalis against oxidative injury of function to human sperm membrane].

OBJECTIVE: To study the intervention of Morindae officinalis extract in human sperm membrane, and to study the treatment of male infertility and asthenoospermia by M. officinalis. METHOD: To select sperm with normal physiological function using the Percoll gradient centrifugation for the normal sperm model. Then separating the sperm suspension into the normal, model, and control group (Vitamin C group), and the large, medium and small dose of M. officinalis. The ROS was made from hypoxanthine-xanzine xanzine (HX-XO), and ROS, different concentrations (0.125, 0.25, 0.5 mg x mL(-1) of the extract were hatched with sperm in the oxygen environment, the sperm membrane Lipid peroxide injury were analyzed, and the function of sperm membrane were analyzed by sperm Hypoosmoticswelling (HOS) and compared with the controlled group. RESULT: In the same conditions, all the small, medium and large extracts of M. officinalis (0.125, 0.25, 0.5 g x mL(-1)) improved SOD vitality of sperm suspension, reduced the content of MDA, intervened in the injury of sperm membrane by ROS to some extent and protected some function of sperm membrane. The 0.125 mg x mL(-1) extract had no obvious difference (P > 0.05) with Vitamin C in it, but the (0.25, 0.5 mg x mL(-1)) concentration of the extract is significantly better than control Vitamin-C (P < 0.01, P < 0.001). Furthermore, there was a dependence on the dosage, the large dose (0.5 mg x mL(-1)) of M. officinalis especially protected the function of sperm membrane. CONCLUSION: The extract from M. officinalis can significantly intervene in lipin peroxidation in sperm membrane by guarding against oxidation, and protect the structure and function of sperm membrane, that is one of the mechanisms for treating male's infertility and asthenoospermia with M. officinalis.

[Clincal observation of qingre yulin decoction in treatment of male infertility caused by accessory gland infection].

OBJECTIVE: To study the effect of Qingre Yulin Decoction (QYD) on male infertility caused by accessory gland infection (AGI) with randomized controlled trial (RCT). METHODS: Sixty infertility outpatients were equally divided into two groups randomly, the QYD group treated with modified QYD and the control group with antibiotic plus vitamin E, both for 3 months with another 6 months' follow-up. Pregnant rates, routine test of sperm and expressed prostatic secretion (EPS) were determined. RESULTS: The healed rate was 26.7% (8 cases), the markedly effective rate was 43.3% (13 cases), the effective rate was 16.7% (5 cases), and the total effective rate was 86.7% in the QYD group, while in the control group it was 6.7% (2), 30.0% (9), 40.0% (12) and 76.7% respectively, showing higher healed rate and total effective rate in the former than those in the latter. Sperm quality of infertility patients with AGI decreased obviously, manifesting short ened average liquefaction time, reduced concentration, survival rate and vitality of sperm. These abnormal changes were improved after treatment in both groups, and the efficacy was better in the QYD group than that in the control group. CONCLUSION: Infertility patients with AGI were manifested as oligospermatism and asthenospermia, which may not be the definite outcome of AGI. QYD is able to improve sperm quality, especially sperm vitality in infertility patients with AGI and therefore increase pregnant rate of their wives.

[Protection of extract from Cuscuta japonica on human sperm acrosome and ultrastructure].

OBJECTIVE: To study the oxidation damage of active oxygen (ROS) to human sperm acrosome and ultrastructure, and study the function mechanism about Cuscuta japonica treating male's infertility and asthenoospermia. METHOD: By using the Percoll gradient centrifugation, the sperm with normal physiological function were selected for the normal sperm model, and the sperm suspension were divided into the normal group, the model group, the positive control group (Vitamin C group), and the lugh, the median and the low dose gvoups of C. japonica. The ROS made from hypoxanthine-xanzine xanzine(HX-XO) and different content (0.125, 0.25, 0.5 g x mL(-1)) of extract were incubated with sperm in the oxygen environment. The acrosomic integrity rate were calculated and the sperm acrosome and ultrastructure were observed. RESULT: The content (0.125, 0.5 g x mL(-1)) of extract had no obvious difference as compared with Vitamin C (0.25 mg x mL(-1)) in protecting the acrosome and ultrastructure, but the content (0.25 mg x mL(-1)) of extract was significantly better than Vit C (P < 0.001). CONCLUSION: The suitable content of extract from C. japonica can significantly protect the sperm membrane, the acosomic structure and the mitochondrion function from the damage caused by ROS.