Effecacy of Biejia (Carapax Trionycis) and Ezhu (Rhizoma Curcumae Phaeocaulis) couplet medicine on epithelial-mesenchymal transition, invasion and migration of MDA-MB-231 triple negative breast cancer cells via PI3K/Akt/mTOR signaling pathway.

OBJECTIVE: To investigate the efficacy of Biejia (Carapax Trionycis) and Ezhu (Rhizoma Curcumae Phaeocaulis) couplet medicine on epithelial-mesenchymal transition (EMT), invasion and migration of MDA-MB-231 triple negative breast cancer (TNBC) cells based on PI3K/Akt/mTOR signaling pathway. METHODS: MDA-MB-231 cells were treated with different medicated serum as Biejia-, Ezhu-, Biejia-Ezhu (BJ-, EZ-, BJ-EZ-) groups, intervened with no drug rat serum and paclitaxel with final concentration of 33 nM (IC50) as negative and positive control (NC and PC) groups. CCK-8 assay, scratch test, and Transwell assay were used to examine cell proliferation, invasion, and migration. The expression of E-cadherin, N-cadherin, Vimentin, MMP-2, MMP-9, PI3K, Akt, p-Akt, mTOR, and p-mTOR was determined by Western blot, and the mRNA expression of PI3K, Akt and mTOR was determined by real-time polymerase chain reaction. RESULTS: BJ-EZ group inhibited proliferation after 24, 48, and 72 h compared with the NC group (P < 0.05, < 0.01 or < 0.001) and reduced the invasion and migration of MDA-MB-231 cells (P < 0.01 or < 0.001). In addition, BJ-EZ group upregulated the expression of E-cadherin, downregulated the expression of N-cadherin, Vimentin, MMP-2, and MMP-9 (P < 0.05, P < 0.01 or P < 0.001), and inhibited the mRNA and protein expression of PI3K, Akt (p-Akt), mTOR (p-mTOR) (P < 0.05, < 0.01 or < 0.001). CONCLUSION: Biejia (Carapax Trionycis) and Ezhu (Rhizoma Curcumae Phaeocaulis) couplet medicine can inhibit the proliferation, invasion, migration and EMT of MDA-MB-231 cells through PI3K/Akt/mTOR signaling pathway, and the effect is better than that of Biejia (Carapax Trionycis) or Ezhu (Rhizoma Curcumae Phaeocaulis) alone.

The immunomodulatory effects of Carapax Trionycis ultrafine powder on cyclophosphamide-induced immunosuppression in Balb/c mice.

BACKGROUND: There are abundant resources of Carapax Trionycis from soft-shelled turtle processing wastes each year in China. Our preliminary work showed that Carapax Trionycis ultrafine powder (CTUP) obtained using ball-milling with a particle size of 2.24 µm (D0.025) contained more active ingredients. The CTUP D0.025 has a good bioaccessibility, but there has been no report about the immunomodulatory function of CTUP. Therefore, using a cyclophosphamide-induced immunosuppression mice model, we investigated the immunomodulatory effects of CTUP D0.025. RESULTS: The results indicated that CTUP D0.025 administration significantly improved the immune organ (bone marrow, thymus and spleen) indices, ameliorated spleen tissue morphology and increased the capacity of splenocyte proliferation and the activity of macrophage phagocytosis. CTUP D0.025 also significantly promoted the secretion of cytokines (IL-2, IL-4, IL-10, IFN-γ and TNF-α), improved the related mRNA expression levels of IL2, IFN-γ, T-bet and GATA3 in immunosuppressed mice and increased the production of serum hemolysin and the levels of IgG, IgM as well as complement C3 . Moreover, CTUP D0.025 administration enhanced the antioxidant capacity of mice, exhibited a moderating effect on the damage of bone and skeletal muscle and improved the recovery of bone mineral density and calcium metabolism. CONCLUSIONS: These findings demonstrated that CTUP D0.025 had an effective immune-enhancing function in immunosuppressive Balb/c mice and also exhibited anti-osteoporosis properties. © 2020 Society of Chemical Industry.

Bioavailability and prebiotic potential of Carapax Trionycis, a waste from soft-shelled turtle processing.

BACKGROUND: Carapax Trionycis is the shell of the soft-shelled turtle. It is rich in minerals, amino acid, peptides, and other nutrients. Current processing and consumption of soft-shelled turtle leads to the waste of huge quantities of Carapax Trionycis in the form of spent materials. In this study, the bioavailability, prebiotic activity, and physicochemical properties of Carapax Trionycis using different processing methods were investigated. The vinegar-quenched Carapax Trionycis (V-CT), fine powders (D0.18, D0.10), and superfine powders (D0.05, D0.025) of Carapax Trionycis were prepared by the vinegar-quenching method, common grinding, and the superfine grinding method. RESULTS: The average particle sizes of D0.18, D0.10, D0.05, and D0.025 were 147.82, 77.35, 36.65, and 2.24 µm, respectively. Superfine grinding changed the surface morphology of Carapax Trionycis and promoted the release of active ingredients. D0.025 had the highest polypeptide (8.15%), polysaccharide (1.21%), total free amino acid (232.36 mg 100 g-1 ) and water-soluble extract content (10.74%), and showed the highest calcium release rate (55.64%) after in vitro digestion. The apparent permeability (PAPP ) of the resulting Carapax Trionycis samples in the dialysis tubing model and the everted intestinal sac model increased significantly with the decrease in the Carapax Trionycis particle sizes. Furthermore, the five Carapax Trionycis samples significantly stimulated the growth of the tested probiotics and increased lactic acids production after 48 h fermentation compared to the control. The Carapax Trionycis powder prepared by superfine grinding displayed better prebiotic activity than other samples as it significantly induced a greater proliferation of probiotic bacteria and higher production of lactic acid, as well as greater release of free calcium. CONCLUSIONS: The results showed that Carapax Trionycis superfine powder D0.025 had the highest active ingredient content, calcium bioavailability, and prebiotic activity. Our approach of developing Carapax Trionycis superfine powder as natural calcium supplement or potential prebiotic would therefore broaden the scope of soft-shelled turtle processing waste utilization in an eco-friendly, cost-effective, and sustainable approach in the future. © 2020 Society of Chemical Industry.

Isolation and Characterization of Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from the Enzymatic Hydrolysate of Carapax Trionycis (the Shell of the Turtle Pelodiscus sinensis).

Carapax Trionycis (the shell of the turtle Pelodiscus sinensis) was hydrolyzed by six different commercial proteases. The hydrolysate prepared from papain showed stronger inhibitory activity against angiotensin I-converting enzyme (ACE) than other extracts. Two noncompetitive ACE inhibitory peptides were purified successively by ultrafiltration, gel filtration chromatography, ion exchange column chromatography, and high-performance liquid chromatography (HPLC). The amino acid sequences of them were identified as KRER and LHMFK, with IC50 values of 324.1 and 75.6 µM, respectively, confirming that Carapax Trionycis is a potential source of active peptides possessing ACE inhibitory activities. Besides, both enzyme kinetics and isothermal titration calorimetry (ITC) assay showed that LHMFK could form more stable complex with ACE than KRER, which is in accordance with the better inhibitory activity of LHMFK.

Carapax Trionycis extracts inhibit fibrogenesis of activated hepatic stellate cells via TGF-ß1/Smad and NFκB signaling.

Carapax Trionycis is used as a traditional Chinese medicine with a long history of clinical application in China, and it represents an essential medication used for liver fibrosis treatment. Previous studies demonstrated that Carapax Trionycis extracts protect liver against fibrosis in CCL4-induced animal models. This study investigated the anti-fibrotic molecular mechanisms exerted by Carapax Trionycis extracts with molecular weight less than 6 KD (CT6) in rat hepatic stellate cell line HSC-T6 activated by TGF-ß1. HSC-T6 cells induced by TGF-ß1 were used to evaluate CT6 anti-fibrotic effect in vitro. CCK8 was used to evaluate cell viability and CT6 effect on HSC-T6 proliferation. ELISA was performed to detect the presence of inflammatory cytokines. Western blot and q-PCR were performed to explore the molecular mechanisms. Our data demonstrated that CT6 did not clearly affect cell viability but suppressed TGF-ß1-induced HSC-T6 proliferation. Collagen I and α-smooth muscle actin (α-SMA) protein levels were decreased by CT6 in TGF-ß1-induced HSC-T6, followed by the inhibition of TIMP1, TIMP2 and TGF-ß1/Smad pathway. Furthermore, CT6 decreased Jun D and p-p65 protein levels, down-regulated Tgf-ß1, Tnf-α, Il-1ß, Il-6 mRNA and TNF-α, IL-1ß and IL-6 expression in TGF-ß1-treated HSC-T6. These results suggested that CT6 inhibited HSC-T6 activation induced by TGF-ß1, indicating the potential therapeutic effect of these extracts against liver fibrosis.

Study on the Improvement of Processing Method of Carapax Trionycis / 中国药房

OBJECTIVE:To explore the optimal steaming time of Carapax trionycis during cleansing period,and to optimize and improve production technology of Carapax trionycis recorded by current Chinese Pharmacopoeia. METHODS:The mechanical processing replaced the artificial processing method in Chinese Pharmacopoeia. The content of protein and the appearance of Cara-pax trionycis were investigated after steaming for 30,60,90,120,180,240 min during cleansing period. The extract,decoction, ash content,appearance and property of Carapax trionycis decoction piece processed with vinegar were also investigated after cleansed Carapax trionycis decoction piece was processed by sand scalding and vinegar quenching method. RESULTS:The differ-ent steaming time obtained different quality of cleansed Carapax trionycis decoction piece and Carapax trionycis decoction piece pro-cessed with vinegar. Compared with decoction piece steamed for other duration,when the steaming time was 90 min,the content of protein in cleansed Carapax trionycis decoction piece was higher(31.16%),and its appearance was up to the requirement. Cara-pax trionycis decoction piece processed with vinegar had higher contents of extract and decoction(9.13%,11.39%)and lower con-tent of ash(66.29%),and its appearance was up to the requirement. CONCLUSIONS:Different steaming time have certain effect on the quality of cleansed Carapax trionycis and Carapax trionycis processed with vinegar,the optimal steaming time of Carapax tri-onycis is about 90 min during cleansing. The mechanical processing method maybe replace the artificial processing on Carapax tri-onycis for improving its production efficiency.

Effects of Oligo-peptide I-C-F-6 in Carapax Trionycis on Rats with Liver Fibrosis Induced by CCl4 / 中国中医药信息杂志

Objective To study effects of oligo-peptide I-C-F-6 in carapax trionycis on rats with liver fibrosis induced by CCl4;To discuss its anti-liver fibrosis effects and possible mechanisms. Methods Forty-eight SD male rats were randomly divided into normal control group, model group, bifendate group, and oligo-peptide I-C-F-6 group, 12 in each group.CCl4 was injected intraperitoneally to build rat liver fibrosis model.Oligo-peptide I-C-F-6 group and bifendate group were given subcutaneous injection of oligo-peptide I-C-F-6 (0.12μg/g) or bifendate (0.12μg/g). At the same time, normal control group and model group were giventhe same volume of saline for seven weeks. The levels ofALT, AST,MDA, SOD, IL-4, IL-10 and TNF-α were tested.The histomorphology changes were observed under optical microscopeby HE, and the expressions of transforming growth TGF-β1 were determined by immunohistochemistry.Results Compared with model group, serum levels of ALT and AST were reduced evidently in oligo-peptide I-C-F-6 group. Hepatic content of MDA, IL-4 and TNF-α decreased, while SOD activity and IL-10 were found significantly increased. Liver fibrosis was ameliorated significantly. Hepatic expressions of TGF-β1 were weakly positive.Conclusion Oligo-peptide I-C-F-6 can ameliorate hepatocyte damage of model rats, thus it has anti-oxidative and anti-liver fibrosis effects on liver fibrosis in rats.

Authentication of TCM Carapax Trionycis by allele-specific diagnostic polymeras chain reaction / 中草药

Object\ To develop a convenient and practical method for the identification of Carapax Trionycis Methods\ Based on the sequence variations of 12S rRNA gene between Pelodiscus sinensis and other softshell turtles, a pair of allele specific primers was designed to distinguish P. sinensis from other species of Trionychidae. DNA were extracted and anplified and Carapax Trionycis could be identified accurately by polymerase chain reaction (PCR) using the primers Results\ Ten samples of turtle shell from different sources were indentified by the allele specific PCR with the primers The result indicated that three samples were substitutes of Carapax Trionycis, consilient with the result from DNA sequence analysis The mitochondrial 12S rRNA gene fragment of P. maculatus and a faked imitation had also been sequenced Conclusion\ The primers could be used as key components in Carapax Trionycis identification kit