Chemical fingerprint analysis and quality assessment of Tibetan medicine Triphala from different origins by high-performance liquid chromatography.

INTRODUCTION: Although the Tibetan medicine Triphala (THL) is widely used in many countries, insufficient progress has been made in quality control. OBJECTIVES: The present study aimed to propose a methodology for quality control of THL based on HPLC fingerprinting combined with an orthogonal array design. METHODS: Seven identified peaks were used as indicators to examine the effects of temperature, extraction time, and solid-liquid ratio on the dissolution of active ingredients in THL. Fingerprint analysis was performed on 20 batches of THL from four geographical areas (China, Laos, Thailand, and Vietnam). For further chemometric assessment, analysis techniques including similarity analysis, hierarchical clustering analysis, principal component analysis, and orthogonal partial least squares discrimination analysis (OPLS-DA) were used to classify the 20 batches of samples. RESULTS: Fingerprints were established and 19 common peaks were identified. The similarity of 20 batches of THL was more than 0.9 and the batches were divided into two clusters. Four differential components of THL were identified based on OPLS-DA, including chebulinic acid, chebulagic acid, and corilagin. The optimal extraction conditions were an extraction time of 30 min, a temperature of 90°C, and a solid-liquid ratio of 30 mL/g. CONCLUSION: HPLC fingerprinting combined with an orthogonal array design could be used for comprehensive evaluation and quality assessment of THL, providing a theoretical basis for further development and utilization of THL.

Targeted modulation of gut microbiota by traditional Chinese medicine and natural products for liver disease therapy.

The gut microbiota not only constitutes intestinal microenvironment homeostasis and human health but also exerts indispensable roles in the occurrence and progression of multiple liver diseases, including alcohol-related liver disease, nonalcoholic fatty liver disease, autoimmune liver disease and liver cancer. Given the therapeutic status of these diseases, their prevention and early therapy are crucial, and the detailed mechanism of gut microbiota in liver disease urgently needs to be explored. Meanwhile, multiple studies have shown that various traditional Chinese medicines, such as Si Miao Formula, Jiangzhi Granules, Liushen Capsules, Chaihu-Shugan Power, Cassiae Semen and Gynostemma, as well as some natural products, including Costunolide, Coprinus comatus polysaccharide, Antarctic krill oil, Oridonin and Berberine, can repair liver injury, improve fatty liver, regulate liver immunity, and even inhibit liver cancer through multiple targets, links, and pathways. Intriguingly, the aforementioned effects demonstrated by these traditional Chinese medicines and natural products have been shown to be closely related to the gut microbiota, directly driving the strategy of traditional Chinese medicines and natural products to regulate the gut microbiota as one of the breakthroughs in the treatment of liver diseases. Based on this, this review comprehensively summarizes and discusses the characteristics, functions and potential mechanisms of these medicines targeting gut microbiota during liver disease treatment. Research on the potential effects on gut microbiota and the regulatory mechanisms of traditional Chinese medicine and natural products provides novel insights and significant references for developing liver disease treatment strategies. In parallel, such explorations will enhance the comprehension of traditional Chinese medicine and natural products modulating gut microbiota during disease treatment, thus facilitating their clinical investigation and application.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

[Adenosine Al Receptor Mediated Neuroprotection of Shenmai Injection on Rat Cerebral Ischemia/Reperfusion Injury: an Experimental Study].

OBJECTIVE: To observe whether adenosine Al receptor (Al R) mediated neuroprotection of Shenmai Injection (SI) on rat cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral I/R model was established by middle cerebral artery occlusion (MCAO). Totally 60 successfully modeled rats was divided into 5 groups according to randomized block principle, i.e., the model group, the SI group, the SI + AlR antagonist (1,3-dipropyl-8-cyclopentylxanthine, DPCPX) group, the AlR antagonist control group, and the dimethyl sulfoxide (DMSO) control group, 12 in each group. Besides, a sham-operation group was set up (n =12). SI at 15 mL/kg was peritoneally injected to mice in the SI group immediately after cerebral I/R. Equal volume of normal saline was injected to mice in the model group and the sham-operation group. DPCPX at 1 mg/mL was peritoneally injected to mice in the Al R antagonist control group 30 min before peritoneal injecting SI. DPCPX at 1 mg/kg and DMSO at 1 mL/kg were peritoneally injected to mice in the AlR antagonist control group and the DMSO control group 30 min immediately before cerebral I/R. Rats' neurobehavioral scores were assessed after 24 h reperfusion. The volume of cerebral infarction and Bcl-2 protein expression of cerebral infarction penumbra were also detected. Results Compared with the sham-operation group, neurobehavioral scores, the volume of cerebral infarction, and Bcl-2 protein expression increased (all P <0. 05). Compared with the model group, neurobehavioral scores and the volume of cerebral infarction obviously decreased, but Bcl-2 protein expression increased in the SI group (all P <0. 05). Compared with the SI group, neurobehavioral scores increased, the volume of cerebral infarction was obviously enlarged, and Bcl-2 protein expression was obviously reduced in the A1R antagonist control group (all P <0. 05). CONCLUSIONS: SI's neurobehavioral scores could be partially reversed in the Al R antagonist control group, the volume of cerebral infarction and Bcl-2 protein expression improved. AlR might possibly meditate neuroprotection of SI on MACO mire

[Local adenosine A1 receptors of baihui acupoint mediate cerebral ischemia tolerance induced by Electroacupuncture].

OBJECTIVE: To investigate whether local A1R of Baihui acupoint mediate cerebral ischemia tolerance induced by electro-acupuncture (EA). METHODS: Sixty SD rats were randomly divided into five groups, i.e., the sham-operation (S) group, the model group (M), the electroacupuncture (E) group, the CCPA group and the DMSO group. The focal cerebral ischemia/reperfusion model was established by middle cerebral artery occlusion (MCAO) in rats. Rats in the E group were received EA pretreatment baihui acupoint at 2 h before established MCAO. The rats in DMSO group and the CCPA group were injected with DMSO (20 µl) and CCPA (0.1 mmol/L) 20 µl into Baihui, respectively, at 2 h before established MCAO. After 24 h reperfusion, the rats' behavior, cerebral infarct volume, the cerebral Bcl-2 protein expression were assessed. RESULTS: Compared with M group, the rats' behavior was improved, the cerebral infarct volume was decreased and the Bcl-2 protein expression was up-regulated (P < 0.05) in the E group. Compared with M and DMSO group, the rats' behavior was improved, the cerebral infarct volume was decreased and the Bcl-2 protein expression was up-regulated (P < 0.05) in the CCPA group. There were no statistical differences between CCPA and E group. CONCLUSIONS: EA induced cerebral ischemia tolerance. Local A1R of Baihui acupoint possible mediate cerebral ischemia tolerance induced by Electroacupuncture.

Anticancer potential of Euphorbia helioscopia L extracts against human cancer cells.

Euphorbia helioscopia L is considered a traditional Chinese herb which is widely distributed in China. The active anticancer fractions and anticancer mechanism of the herb are unclear. In this study, we evaluated the growth inhibitory effects of Euphorbia helioscopia L extracts on five different human cancer cell lines for screening the main active fraction with antitumor effect. In this regard, the ethyl acetate extract (EAE) was found to markedly inhibit the proliferation of SMMC-7721 cells in a time- and dose-dependent manner. EAE treatment arrested cell cycle in G-1 phase and EAE used at the concentration range of 100-200 µg/mL induced a marked increase of subdiploid peak. After EAE treatment at the concentrations of 150 and 200 µg/mL, the percentage of apoptotic cells was increased. At the EAE concentration of 200 µg/mL, the typical morphology of early apoptotic change was observed in SMMC-7721 cells. Since tumorigenesis is often defined by an uncontrolled proliferation and transplantability, we also determined the anti-invasive effects of EAE. The EAE treatment displayed a dose-dependent inhibitory effect on tumor cell invasion and MMP-9 expression. Also, the major active fraction was assayed using high-performance liquid chromatography (HPLC). The data showed that the flavonoids could be the main constituents of EAE. Based on the evidence from these data, we inferred that the EAE of Euphorbia helioscopia L could have chemopreventive potential against the human cancer.

Quantitative analysis of quercetin in Euphorbia helioscopia L by RP-HPLC.

Euphorbia helioscopia L is widespread in China and has a large number of flavonoids. Quercetin glycosides, having useful biological activities, are abundant in this plant, and no validated analytical method has so far been developed for their determination. We, therefore, standardized a reversed-phase high-performance liquid chromatography (RP-HPLC) assay for quercetin detection. For this, the plant was locally procured and identification was confirmed based on its morpho-histological characteristics. Ethyl acetate extracts of leaves, stems, and roots were analyzed by RP-HPLC using Agilent 1120 HPLC TC-C(18) column (250 × 4.6 mm; 5 µm) with UV-detector system. The mobile phase of methanol-0.2% phosphoric acid (65:35) solution was used with the flow rate of 1.0 ml min(-1) at 30°C, and the detection was performed at 360 nm wavelength. Our data show that the linear range of quercetin was 0.025-0.150 mg.ml(-1) (r = 0.9995; n = 6) with the recovery rate of 97.50-103.30% (average 100.40%; RSD = 2.28%). The target component was baseline separated during only the period of 9 min. The repeatability of RP-HPLC analysis was demonstrated with an RSD of 1.77% (n = 6), and the highest quercetin content (average 1.42 mg g(-1)dry-weight) was present in leaves. It was, therefore, concluded that RP-HPLC is a simple, rapid, accurate, and sensitive method for the detection of quercetin from Euphorbia helioscopia L.

Antioxidant enzymes from the crab Scylla paramamosain: gene cloning and gene/protein expression profiles against LPS challenge.

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551bp with an open reading frame of 1551bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections.

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide.

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.