Triterpenoid Compounds from Cyclocarya paliurus: A Review of Their Phytochemistry, Quality Control, Pharmacology, and Structure-Activity Relationship.

Cyclocarya paliurus (Batalin) Iljinskaja (C. paliurus) is a single species of Cyclocarya paliurus in Juglandaceae. It is a unique rare medicinal plant resource in China that is mainly distributed in the south of China. The leaves of C. paliurus, as a new food ingredient, are processed into tea products in daily life. Triterpenoids are the main active ingredient in C. paliurus. So far, 164 triterpenoid compounds have been isolated and identified from C. paliurus, which are included 3,4-seco-dammaranes, dammaranes, oleanane, ursane, lupinanes, taraxeranes, and norceanothanes. Modern pharmacological studies manifested that these ingredients have a wide range of pharmacological activities both in vitro and in vivo, such as reducing blood sugar, lowering blood lipids, and anti-tumor, anti-inflammatory, anti-oxidant, and other activities. In addition, current results indicate that the pharmacological mechanisms of triterpenoids were closely related to their chemical structure, molecular signaling pathways, and the expression of related proteins. In order to further study C. paliurus based on the current research situation, this review summarizes the prospect and systematic summary of the triterpenes of C. paliurus from the aspects of structural characteristics, quality control, biological activity, and the structure-activity relationship, which provide a reference for further research and application of the triterpenoids from C. paliurus in the field of functional food and medicine.

[Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy].

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1ß(IL-1ß) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1ß and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

[Study on protective effect of water extract from Sabia parviflora on liver injury in mice induced by acetaminophen].

The aim of this study was to observe the protective effect of water extract from Sabia parviflora on mice with acute liver injury induced by acetaminophen, and investigate its possible mechanism. Fifty-eight Kunming mice were divided into 6 groups, 8 in the normal group, 10 in the model group, 10 in the biphenyl diester group, and 10 each in the low, medium and high dose groups. After adaptive feeding for one week, the mice in normal group were intragastrically administered with an equal volume of 0.5% sodium carboxymethylcellulose sodium(CMC-Na), and the mice in other groups were intragastrically administered with corresponding drugs at 20 mL·kg~(-1) once a day. Then acetaminophen(200 mg·kg~(-1)) was administered after the above drug administration except the normal group. The behavior and signs of the experimental animals were observed every day and the samples were taken for experiments on the next day of the final administration. The liver mass and mass index were calculated. The blood was collected from the abdominal aorta and centrifuged to obtain the serum for detecting aspartate aminotransferase(AST) activity and alanine aminotransferase(ALT) activity. The liver tissue homogenate was used to detect superoxide dismutase(SOD) activity, glutathione(glutathione, r-glutamyl cysteingl+glycine, GSH) activity and malondialdehyde(MDA) content. Liver tissue was analyzed for histological analysis. The results showed that S. parviflora could alleviate the lipid peroxidation damage in the liver caused by acetaminophen, reduce the ALT and AST activities in serum, increase the levels of SOD and GSH in liver tissue, decrease the content of MDA in liver tissue, and inhibit the apoptosis. S. parviflora could also improve the live histopathological profile, protect liver cells and restore liver function. Among them, the high dose had the most significant effect and showed dose-effect relationship. This study indicated that S. parviflora had a significant protective effect on acetaminophen-induced liver injury in mice, and its mechanism may be related to its anti-oxidation effect and inhi-bitory effect on apoptosis.

[Therapeutic effect of total triterpenoids of Chaenomeles speciosa combined with omeprazole on gastric ulcer induced by indomethacin in rats].

The aim of this paper was to observe the combination therapy with total triterpenoids of Chaenomeles speciosa and omeprazole on indomethacin-induced gastric ulcer in rats, and explore its possible mechanism. Rats were randomly divided into normal group, model group, omeprazole monotherapy(3.6 mg·kg~(-1)) group, total triterpenoids of C. speciosa monotherapy(100 mg·kg~(-1)) group, total triterpenoids of C. speciosa and omeprazole combination therapy(100 mg·kg~(-1)+3.6 mg·kg~(-1)) group. Except for the normal group, the other groups were given indomethacin(20 mg·kg~(-1)) by oral once a day for 7 consecutive days. Then the treated groups were given corresponding drugs by gavage, once a day for 14 consecutive days. The next day after the last administration, half of the rats in each group were measured the gastric mucosal blood flow, gastric juice volume and serum TNF-α, IL-1ß, IL-6, IL-4 and IL-10. After the remaining rats in each group were underwent pyloric ligation 4 hours after the last administration, the gastric endocrine volume, pH value and total acidity of gastric secretion were measured, then histological analysis was performed, MPO activity, cAMP content and histomorphological analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of gastric tissue TNF-α,IL-1ß, IL-6, IL-4, IL-10, VEGFA, A_(2A)R; the protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue were detected by Western blot. The results indicated that total triterpenoids of C. speciosa and omeprazole combination therapy might significantly increase gastric mucosal blood flow, gastric mucus volume, reduce gastric endocrine volume, secretion acidity and mucosal damage, decrease the levels of TNF-α,IL-1ß and IL-6, increase the levels of IL-4 and IL-10 in blood and gastric tissue, inhibit the activity of MPO, increase the content of cAMP in gastric tissue, up-regulate the mRNA expressions of VEGFA, A_(2A)R and protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue, elevate p-PKA/PKA, p-CREB/CREB and p-EFGR/EFGR. Moreover, the combination therapy with total triterpenoids of C. speciosa and omeprazole was more obvious than those of two monotherapies. These aforementioned findings suggested that the combination therapy with total triterpenoids of C. speciosa and omeprazole on indomethacin-induced gastric ulcer have significant therapeutic effect on indomethacin induced gastric ulcer in rats, its mechanism might be related to regulating A_(2A)R/AKT/CREB, A_(2A)R/VEGFA, EGF/EGFR and MUC6/TFF2 signaling pathways, inhibiting pro-inflammatory factors, increasing gastric mucosal blood flow, up-regulating mucosal cell proliferation factors and promoting mucosal protective factors.

Methyl-Hispolon from Phellinus lonicerinus (Agaricomycetes) Affects Estrogen Signals in MCF-7 Breast Cancer Cells and Premature Aging in Rats.

We studied Phellinus lonicerinus to determine the cytotoxic effect and the dual estrogenic activities of methyl-hispolon and their relation to estrogen signals in vivo and in vitro. The Glide scores of methyl-hispolon-estrogen receptor α (ERα) and methyl-hispolon-ERß docked complexes were -7.29 kcal/mol and -6.68 kcal/mol in docking simulations. Methyl-hispolon had a significant antiproliferative effect for estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen, and it exhibited dual estrogen activities. Methyl-hispolon increased the serum E2 in rats with premature ovarian failure and fulfilled the estrogenic function in the uterus and ovary. Methyl-hispolon significantly inhibited the expression of Ras, API, ERα, C-myc, and cyclinDl, as well as their gene transcription in RL95-2 cells. The phosphorylation of ERK1/2 was inhibited by methyl-hispolon. Thus, methyl-hispolon has potential use in treating estrogen deficiency-related diseases, with good antitumor effects and estrogenic activity.

Gastroprotective effect of araloside A on ethanol- and aspirin-induced gastric ulcer in mice: involvement of H+/K+-ATPase and mitochondrial-mediated signaling pathway.

The aim of this study was to elucidate the gastroprotective activity and possible mechanism of involvement of araloside A (ARA) against ethanol- and aspirin-induced gastric ulcer in mice. The experimental mice were randomly divided into control, model, omeprazole (20 mg/kg, orally) and ARA (10, 20 and 40 mg/kg, orally). Gastric ulcer in mice was induced by intragastric administration of 80% ethanol (10 mL/kg) containing 15 mg/mL aspirin 4 h after drug administration on day 7. The results indicated that ARA could significantly raise gastric juice volume and acidity; ameliorate gastric mucosal blood flow, gastric binding mucus volume, ulcer index and ulcer inhibition rate; suppress H+/K+-ATPase activity, which was confirmed by computer-aided docking simulations; inhibit the release of mitochondrial cytochrome c into the cytoplasm; inhibit caspase-9 and caspase-3 activities and down-regulate mRNA expression levels; down-regulate the mRNA and protein expressions of apoptosis protease-activating factor-1 and protein expression of cleaved poly(ADP ribose) polymerase-1; and up-regulate Bcl-2 mRNA and protein expressions and down-regulate Bax mRNA and protein expressions, thus elevating the Bcl-2/Bax ratio in a dose-dependent manner. Histopathological observations further provided supportive evidence for the aforementioned results. The results demonstrated that ARA exerted beneficial gastroprotective effects on alcohol- and aspirin-induced gastric ulcer in mice, which was related to suppressing H+/K+-ATPase activity as well as pro-apoptotic protein expression, and promoting anti-apoptotic protein expression, thus alleviating gastric mucosal injury and cell death.

[Effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by DSS in mice].

To observe the effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by dextran sulfate sodium (DSS) in mice, C57BL/6 mice were randomly divided into normal group, model group, total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) groups and sulfasalazine (250 mg·kg⁻¹) group. The ulcerative colitis (UC) model was induced by orally administering 2.5% DSS to the experimental mice, and the corresponding drugs were given to each group 3 days before the administration with 2.5% DSS. The normal group and the model group were given the equal volume of 0.5% carboxymethyl cellulose sodium solution by gavage continuously for 10 days, q.d. The general conditions of the mice were observed on a daily basis, and the disease activity index (DAI) score was recorded. On the 10th day after the treatment, mice were put to death, the contents of TNF-α, IL-1ß, IL-6, IFN-γ, IL-4 and IL-10 in the blood were detected, colon length was measured, colon mucosa damage index (CMDI) score was calculated, and MPO activity detection and histomorphology analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of E-cadherin, occluding,MUC2 and TFF3; the protein expressions of SIRT1, IKKß, p-IKKß, IκBα, p-IκBα and cytosol and nucleus PPARγ, NF-κBp65 in intestinal tissue were detected by western blot. The results indicated that total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) could significantly improve the general conditions of UC mice, reduce the DAI, CMDI and histopathological scores, increase the colon length, reduce the colonic mucosa ulcers, erosion and inflammatory infiltration, restore the normal intestinal mucosal barrier function, reduce the contents of TNF-α, IL-1ß, IL-6, IFN-γ, increase the contents of IL-4 and IL-10 in the blood, inhibit MPO activity in colon tissue, up-regulate the mRNA expressions of E-cadherin, occludin, MUC2 and TFF3 in colon tissue, down-regulate the protein expressions of cytosol PPARγ, tissue p-IKKß, p-IκBα and nucleus NF-κBp65 in the colon tissue, decrease the p-IKKß/IKKß and p-IκBα/IκBα ratios, up-regulate the protein expressions of nucleus PPARγ, tissue SIRT1 and cytosol NF-κBp65 (P<0.05 or P<0.01, respectively), with a dose-effect relationship between the total triterpenoids of C. speciosa treated groups. These findings suggested that total triterpenoids of C. speciosa had a significantly therapeutic effect on UC mice induced by DSS, its mechanism might be related to the regulation of PPARγ/SIRT1/NF-κBp65 signaling pathway, the inhibition of pro-inflammatory factor formation and the up-regulation of protein expression of protective factors.

Effect of Hispolon from Phellinus lonicerinus (Agaricomycetes) on Estrogen Receptors, Aromatase, and Cyclooxygenase II in MCF-7 Breast Cancer Cells.

Phytoestrogen has previously been proposed as an alternative to hormone replacement therapy. Hispolon has been found to have phytoestrogenic properties. However, the possible effects of hispolon on estrogen receptors and other related molecules remain to be determined. This study was performed mainly to confirm the estrogenic function of hispolon as it relates to estrogen receptors, aromatase, and cyclooxygenase 2 (COX-2). Hispolon was shown to increase the serum 17ß-estradiol in vivo. Immunohistochemical staining methods showed that hispolon exhibited a biphasic effect on ERα/ß and aromatase expression in MCF-7 cells. Hispolon could also significantly inhibit aromatase activity, assessed using the enzyme-linked immunosorbent assay. Western blotting showed that COX-2 and aromatase could be inhibited by hispolon. These results further prove the phytoestrogenic features of hispolon and explore some pharmacological mechanisms that suggest that hispolon could be useful in the treatment of breast cancers or other gynecologic diseases.

Two new steroidal glycosides with unique structural feature of 14α-hydroxy-5ß-steroids from Reineckia carnea.

Two new steroidal glycosides (1-2) were isolated from the roots of Reineckia carnea, together with three known compounds (3-5). Their structures were determined on the basis of chemical methods and spectral data. Compounds 1-2 were the unique steroidal glycosides possessing structural feature of 14α-hydroxy-5ß-steroids, and compounds 4-5 were isolated from R. carnea for the first time. The isolated compounds (1-5) were tested in vitro for their cytotoxic activities against the A549, HepG 2 and Caski cancer cell lines. Among them, the compounds 2 and 3 showed cytotoxicity against Caski cancer cell line with IC50 values of 34.4 and 3.7µM, respectively.

Anti-proliferative effect of RCE-4 from Reineckia carnea on human cervical cancer HeLa cells by inhibiting the PI3K/Akt/mTOR signaling pathway and NF-κB activation.

Cervical cancer is the second leading cause of cancer deaths in women worldwide. In recent years, the studies find that inflammation is a critical component of tumor progression, and the ideal therapeutic methods should be aimed at the inflammation reaction triggers. (1ß,3ß,5ß,25S)-spirostan-1,3-diol1-[α-L-rhamnopyranosyl-(1 → 2)-ß-D-xylopyranoside] (RCE-4) was the main active composition of Reineckia carnea (Andr.) Kunth. It significantly induced apoptosis in cervical cancer Caski cells through the mitochondrial pathway in our previous studies; however, its underlying mechanism remains poorly understood. This study aimed to further evaluate the effect of RCE-4 on human cervical cancer HeLa cells. Based on this observation, we investigated the anti-cervical cancer effect of RCE-4 by modulating phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway, nuclear factor-kappa B (NF-κB) activation, and inflammation-related key factors in HeLa cells. The results indicated that the HeLa cell was the most sensitive with an IC50 of 7.01 µM; RCE-4 significantly promoted the release of cellular lactate dehydrogenase (LDH); increased DNA fragmentation and apoptosis; reduced PI3K, Akt, mTOR, and NF-κBp65 phosphorylation levels; increased the Bax and cleaved poly (ADP-ribose) polymerase (PARP) protein levels; suppressed Bcl-2 protein expression; elevated the Bax/Bcl-2 expression ratio; and decreased the interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) mRNA expressions in HeLa cells in a concentration-dependent manner. These findings suggest that RCE-4 exerted beneficially anti-cervical cancer effect on HeLa cells, mainly inhibiting PI3K/Akt/mTOR signaling pathway phosphorylation and NF-κB activation, promoting HeLa cell apoptosis. Graphical abstract Anti-tumor effect of RCE-4 on HeLa cells.

Protective Effect of Eburicoic Acid of the Chicken of the Woods Mushroom, Laetiporus sulphureus (Higher Basidiomycetes), Against Gastric Ulcers in Mice.

In this study, we investigated the anti-inflammatory and tumor-inhibiting effects of eburicoic acid, the main bioactive component in the Laetiporus sulphureus, on gastric ulcers. A total of 48 Kunming mice were randomly divided into six groups: control, model, OL (omeprazole, 20 mg/kg/day, orally), EA-L (eburicoic acid, 10 mg/kg/day, orally), EA-M (eburicoic acid, 20 mg/kg/day, orally), and EA-H (eburicoic acid, 40 mg/kg/day, orally). Gastric ulcers were induced in mice by administering 80% ethanol containing 15 mg/mL aspirin (10.0 mL/kg, i.g.) 4 hours after drug administration on day 5. The ulcer index and H+/K+-ATPase activity were evaluated in vivo. Computer-aided molecular docking simulated the interaction between eburicoic acid and H+/K+-ATPase. The results showed that the oral administration of eburicoic acid protected the gastric mucosa from gastric lesions morphologically and especially attenuated H+/K+-ATPase activity. The results of this study indicate that the gastric protective effect of eburicoic acid might inhibit gastric acid.

Rapid Identification and Verification of Indirubin-Containing Medicinal Plants.

Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ) phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC) was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture), but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.

Trametenolic acid B reverses multidrug resistance in breast cancer cells through regulating the expression level of P-glycoprotein.

Trametenolic acid B (TAB) is the main active composition of Trametes lactinea (Berk.) Pat which possesses antitumor activities. There was no report its antitumor effect through regulating P-glycoprotein (P-gp) so far, due toP-gp over expression is one of the most important mechanisms contributing to the multiple drug resistance phenotype. The present aim was to investigate the effects of TAB on P-gp in multidrug-resistant cells;Paclitaxel-resistant cell line MDA-MB-231/Taxol was established by stepwise exposure for 10 months.MDA-MB-231 cells and MDA-MB-231/Taxol cells were treated with TAB, and their growth was evaluated using MTT assays. Paclitaxel accumulation in the cells was analyzed by high performance liquid chromatogram(HPLC). The activity of P-gp was detected by intracellular accumulation of rhodamine 123 (Rho123), and the protein expression of P-gp was evaluated using western blot. Results indicated that the IC50 of MDA-MB-231/Taxol to paclitaxel (Taxol) was 33 times higher than that of nature MDA-MB-231. TAB increased the intracellular concentration of Taxol and inhibited the activity of P-gp and suppressed the expression of P-gp in MDA-MB-231/Taxol cells. Our present results showed that TAB could reverse Taxol resistance in MDA-MB-231/Taxol cells,mainly inhibiting the activity of P-gp and down-regulating the expression level of P-gp, and then enhancing the accumulation of chemotherapy agents.

Protective effect of saponins extract from Panax japonicus on myocardial infarction: involvement of NF-κB, Sirt1 and mitogen-activated protein kinase signalling pathways and inhibition of inflammation.

OBJECTIVE: Inflammation is widely acknowledged to increase morbidity and mortality in myocardial infarction (MI), and the ideal therapeutic methods should be aimed at the inflammation reaction triggers. The aim was to evaluate the protective effect of saponins extracted from Panax japonicus (SPJ) on MI, and based on these results investigate the possible involvement mechanism of the nuclear factor-kappa B (NF-κB), sirtuin1 (SIRT1) and mitogen-activated protein kinases (MAPKs) signalling pathways. METHODS: Sprague-Dawley rats were randomly divided into sham, MI, MI + SPJ 50 and SPJ 100 mg/kg groups. After administration for 3 days, MI rats were created by ligaturing coronary artery, and then underwent the same administration for 7 days as before. Cardiac function and the expressions of pro-apoptosis protein Bax, anti-apoptosis protein Bcl-2, NF-κB, SIRT1, MAPKs signal pathway-related proteins and inflammatory factor, such as tumour necrosis factor alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1), were assessed. KEY FINDINGS: SPJ might significantly improve cardiac function, decrease the serum MCP-1 and TNF-α levels, ameliorate the increased Bax protein expression and decrease Bcl-2 protein expression, and suppress the protein expressions of NF-κBp65 subunit, extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK, but have no effect on c-Jun NH2-terminal kinase, and increase the expression of SIRT1. Histopathological observations provided supportive evidence for aforementioned results, and with the dose of SPJ increasing, the aforesaid improvement became more and more strong. CONCLUSION: The studies demonstrated that SPJ exerted beneficially cardioprotective effects on MI rats, mainly inhibiting NF-κB, ERK1/2 and p38 MAPK activation, but increased the expression of SIRT1, alleviating MI injury and cardiac cell death.

Protective effects of total flavonoids from Epimedium on the male mouse reproductive system against cyclophosphamide-induced oxidative injury by up-regulating the expressions of SOD3 and GPX1.

Total flavonoids of Epimedium (TFE) is the main active composition of Epimedium that has been used to treat male reproductive problems. The present aim was to investigate the protective effects of TFE on male mice reproductive system against cyclophosphamide (CP)-induced oxidative injury. The animals were treated with CP to make testicular injury model and the protective effects of TFE were observed. In the CP-treated group, testicular and epididymal weights, sperm count and motility significantly decreased relative to the control group (P < 0.05 and P < 0.01, respectively). Compared with the CP-treated group, TFE (200 and 400 mg/kg) treated mice increased testicular weights by 21.6% and 28.4% (P < 0.05), sperm counts by 81.7% and 148.3% (P < 0.01) and sperm motility by 47.2% and 61.3% (P < 0.01). Meanwhile, the CP-treated group showed enhancement of lipid peroxidation leading to testicular reproductive toxicity. TFE restored these oxidative damages by up-regulating the expression of antioxidant enzymes, especially SOD3 and GPX1. TUNEL assay and histopathological observations provided supportive evidence for above results, and when the dose of TFE increased, the aforesaid improvement became more and more strong. These results demonstrated that TFE exerted beneficially protective effects on the structural and functional damage of male mice reproductive system and reduced apoptosis in spermatogenic cells by inhibiting CP-induced oxidative stress.

A Study on the Mechanism of Cinobufagin in the Treatment of Paw Cancer Pain by Modulating Local ß -Endorphin Expression In Vivo.

Background. Cinobufagin has been widely used in the treatment of carcinoma and plays an important role in the relief of cancer pain. But the involved mechanism remains unknown. Aim. To investigate the changes in thermal and mechanical hyperalgesia in paw cancer pain in mice and the action mechanism of cinobufagin using a paw cancer pain model. Methods. 60 female mice were randomly divided into 5 groups: control group, model group, cinobufagin group, cinobufagin +NAL-M group, and morphine group; except ones in control group, mice were inoculated with H22 hepatoma cells in the right hind paw. From the 9th day after inoculation, mice were administrated drug once daily lasting for 8 days. The pain behavior was determined on the 2nd, 4th, 6th, and 8th days before and after administration. On the last day, they were sacrificed. The levels of ß -END, CRF, and IL-1 ß were analyzed by ELISA; immunohistochemistry was performed to detect the expressions of ß -END, POMC, and µ -OR in the tumor and adjacent tissue. Results. The thresholds of thermal pain and mechanical pain were significantly increased by cinobufagin. Moreover, the expressions of ß -END, CRF, POMC, and µ -OR were significantly upregulated by cinobufagin. The analgesic effect of cinobufagin was blocked by the peripheral opioid receptor antagonist NAL-M. Conclusions. Cinobufagin significantly relieved cancer pain in mice and raised their pain threshold, mainly upregulating the expression levels of ß -END and µ -OR in the hind paw tumor and adjacent tissue.

The H+/K+-ATPase inhibitory activities of Trametenolic acid B from Trametes lactinea (Berk.) Pat, and its effects on gastric cancer cells.

Trametenolic acid B (TAB), the bioactive component in the Trametes lactinea (Berk.) Pat, was reported to possess cytotoxic activities and thrombin inhibiting effects. This study was performed to investigate the effects of TAB on H(+)/K(+)-ATPase and gastric cancer. The H(+)/K(+)-ATPase inhibitory activity was determined by gastric parietal cells. Compared to the normal control group, TAB (10, 20, 40 and 80 µg/mL) inhibited the H(+)/K(+)-ATPase activity by 15.97, 16.96, 24.86 and 16.25%, respectively. In the study, 36 Kunming mice were randomly divided into six groups: control, model, TAB-L (TAB, 5 mg/kg/day, i.g.), TAB-M (TAB, 20 mg/kg/day, i.g.), TAB-H (TAB, 40 mg/kg/day, i.g.) and omeprazole (OL, 10 mg/kg/day, i.g.). All mice except the control group were administrated with anhydrous alcohol (5.0 mL/kg, i.g.) for induced gastric-ulcer 1h after the 5th day. At the same time, the control mice were given the same volume of physiological saline. After 4h, TAB was evaluated for H(+)/K(+)-ATPase inhibitory activities of ulcerative gaster, gastric ulcer index and ulcer inhibition. In vitro, the anti-proliferation effect of TAB to gastric cancer cell (HGC-27) in acid environment was detected by MTT, and the apoptosis morphological changes were also observed by Hoechst 33258 dye assay. The results indicated that TAB inhibited moderately H(+)/K(+)-ATPase activity in vitro. Compared to the model group, TAB showed anti-ulcer effects in gastric tissue with the dosages of 20 and 5 mg/kg in vivo. Apart from that, TAB could selectively inhibit gastric cancer cell viability and reduce cell apoptosis against HGC-27 cells at low doses in acid environment.

Growth inhibition and apoptosis-inducing effect on human cancer cells by RCE-4, a spirostanol saponin derivative from natural medicines.

Reineckia carnea has been used to treat several diseases in folk remedies. RCE-4 has been isolated from several plants of the family Liliaceae, but its biological activity has not yet been reported. In the present study, we found that RCE-4 exhibited potent cytotoxicity to the tested human cancer cell lines, and the CaSki cell line was the most sensitive with an IC50 of 3.37 µM. Thus, we presented the apoptosis-inducing effect of RCE-4 on CaSki cervical cancer cells and investigated the relevant mechanisms. Based on observations using transmission electron microscopy, RCE-4-treated cells manifested nuclear shrinkage, condensation and fragmentation. Annexin V/PI dual staining flow cytometry assay further confirmed that RCE-4 caused a dose-dependent early apoptotic effect. Prior to these events, RCE-4 triggered a rapid decrease of the mitochondrial membrane potential and caused the release of cytochrome c from the mitochondria into the cytoplasm. RCE-4 increased the expression of Bax and decreased the expression of Bcl-2, thus augmenting the Bax/Bcl-2 ratio. These findings suggest that RCE-4 induces mitochondrial-mediated apoptosis in CaSki cells and has the potential to be developed as an anticancer agent.

[Effects of total saponin from Panax japonicus on acute myocardial ischemia injury induced by ligating coronary artery left anterior descending branch in rats].

OBJECTIVE: To investigate the cardioprotective effects of total saponin from Panax japonicus pretreating on acute myocardial ischemia injury induced by ligating coronary artery left anterior descending branch (LAD) in rats. METHODS: Rats were divided into sham group, model group and total saponin group. The treatment group was given total saponin of Panax japonicus. After pretreating for 7 days, the rats' acute myocardial ischemia model was induced by LAD. The electrocardiogram, hemodynamics, infarct size and histomorphology were assessed 12 h after LAD, the serum levels of phosphokinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were also measured. RESULTS: Compared with the model group, total saponin from Panax japonicus significantly improved heart function, heart histomorphology, opposed ECG T wave decreased and decreased infarct size; Remarkably decreased the contents of LDH, CK and MDA, increased the activity of SOD, CAT. CONCLUSION: Total saponin from Panax japonicus has cardioprotective effects on acute myocardial ischemic injury and the mechanism may be related to antioxidation.

Hepatoprotective effects of phloridzin on hepatic fibrosis induced by carbon tetrachloride against oxidative stress-triggered damage and fibrosis in rats.

The present study was to study the hepatoprotective effects of phloridzin (PHL) on hepatic fibrosis induced by carbon tetrachloride (CCl4) in rats, on the basis of this investigation, the possible mechanism of PHL was elucidated. Male Sprague Dawley (SD) rats were randomly divided into six groups: control, model, PHL-L, PHL-M, PHL-H and colchine. All rats except control group were intraperitoneally injected with CCl4, and control rats were injected with olive oil, twice a week for eight weeks. At the same time, the rats were orally given homologue drugs once a day, respectively. Hepatoprotective effects of PHL were evaluated by liver weight indexes, biochemical values, total antioxidant capacity and total-superoxide dismutase, histopathological observations, hepatic fibrosis, and the hepatic fibrosis relative gene and protein expressions. PHL significantly improved hepatic function; remarkably decreased serum hyaluronic acid (HA), transforming growth factor-ß1 (TGF-ß1), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and liver tissues hydroxyproline, malondialdehyde (MDA) levels, increased glutathione peroxidase (GSH-Px), total-antioxygen capacity (T-AOC) and total-superoxide dismutase (T-SOD) contents of liver tissues; Real-time polymerase chain reaction (PCR) and immunohisto-chemical results showed PHL might markedly reverse the up-regulated mRNA and protein expressions of the α-smooth muscle actin (SMA), TGF-ß1 and tissue inhibitor of metalloproteinase-1 (TIMP1), up-regulate the matrix metalloproteinase-1 (MMP1) mRNA and protein expressions. Histopathological observations provided supportive evidence for biochemical analyses and the hepatic fibrosis relative gene and protein expressions, and with the dose of PHL increasing, the aforesaid improvement became more and more strong. The studies demonstrated that PHL exerted beneficially hepatoprotective effects on hepatic fibrosis induced by CCl4, mainly enhancing antioxidant capacity of liver organizations, reduce the level of lipid peroxidation induced by CCl4, and protect hepatocyte membranes from damage, and alleviate hepatic fibrosis.