Proteins from Blackberry Seeds: Extraction, Osborne Isolate, Characteristics, Functional Properties, and Bioactivities.

Blackberry fruit contains high levels of nutrients and phenolic compounds. Blackberry pomace accounts for 20~30% of its whole fruit during processing and is generally treated as fertilizer. Blackberry pomace has many seeds that contain carbohydrates, polyphenols, flavonoids, pectin, protein, and other bioactive nutrients. However, its functional properties and seed protein compositions have not been reported. We used a single-factor experiment, response surface, and Osborne isolate method to extract protein isolate, albumin, globulin, glutelin, and prolamin from blackberry seeds for the first time and evaluated their characteristics and functional properties. Glutelin and protein isolate showed good water-holding capacity, emulsification, and foaming capacity, while albumin and globulin showed good oil-holding capacity and thermal stability. They were found to have good antioxidant activities that might be good DPPH free radical scavengers, especially prolamin, which has the lowest IC50 value (15.76 µg/mL). Moreover, globulin had the lowest IC50 value of 5.03 µg/mL against Hela cells, 31.82 µg/mL against HepG2 cells, and 77.81 µg/mL against MCF-7 cells and a high selectivity index (SI), which suggested globulin had better anti-cervical, antihepatoma, and anti-breast activity but relatively low cytotoxicity. These seed proteins may have great prospects for the development and application of food and drugs in the future.

Blueberry and Blackberry Anthocyanins Ameliorate Metabolic Syndrome by Modulating Gut Microbiota and Short-Chain Fatty Acids Metabolism in High-Fat Diet-Fed C57BL/6J Mice.

In this study, blueberry (Vaccinium ssp.) anthocyanins (VA) and blackberry (Rubus L.) anthocyanins (RA) were used to investigate the effects on metabolic syndrome (MetS) and the potential mechanisms. Importantly, all of the data presented in this study were obtained from experiments conducted on mice. As a result, VA and RA reduced body weight gain and fat accumulation while improving liver damage, inflammation, glucose, and lipid metabolism induced by a high-fat diet. Moreover, VA and RA regulated the gut microbiota composition, decreasing the pro-obesity and proinflammation bacteria taxa, such as the phylum Actinobacterium and the genera Allobaculum and Bifidobacterium, and increasing those negatively associated with obesity and inflammation, such as the phylum Bacteroidetes and the genera Prevotella and Oscillospira. Additionally, the supplementation with VA and RA reversed the elevated levels of valeric, caproic, and isovaleric acids observed in the high-fat diet (HFD) group, bringing them closer to the levels observed in the Chow group. This reversal indicated that alterations in the composition and abundance of gut microbiota may contribute to the restoration of short-chain fatty acids (SCFAs) levels. Additionally, PICRUSt2 exhibited that cyanamino acid metabolism and betalain biosynthesis might be the major metabolic pathways in the HVA group compared with the HFD group, while in the HRA group, it was the phosphotransferase system. These findings suggest that VA and RA can ameliorate MetS by modulating the gut microbiota and production of SCFAs.

Analysis of the Current Situation and Influencing Factors of Coping Styles in Ischemic Stroke Patients.

Objective: To explore the current situation and influencing factors of coping styles in ischemic stroke patients. Methods: 250 ischemic stroke patients admitted to our hospital from September 2019 to September 2021 were selected as the study subjects to obtain the general information of patients, and the corresponding indexes of patients were evaluated by the method of document investigation to analyze the current situation and influencing factors of coping styles in ischemic stroke patients. Results: Under stressful conditions, patients with no dependence on life-support level, without anxiety and depression, enjoying a high quality of life, and with high self-efficacy were more likely to adopt the positive coping styles (P < .05). Logistic regression analysis showed that infarction area, life-support level, and self-efficacy were independent risk factors for coping style in patients with ischemic stroke (all P < .05). Conclusion: Ischemic stroke patients tend to adopt negative coping styles. Infarction area, life-support level, and self-efficacy of ischemic stroke patients are found to be the main factors affecting their coping styles.

Polyphenols from Prunus mume: extraction, purification, and anticancer activity.

Prunus mume is an ancient medicinal herb and food that are commonly used in Asian countries with high nutritional ingredients and biological activities. Polyphenols are important functional components in Prunus mume. To obtain a more efficient extraction process of Prunus mume polyphenols, a single-factor test and response surface method were used. After extraction and purification, the final polyphenol content of Prunus mume (L1) was up to 90%. Biological experiments showed that L1 had high anticancer activity against HeLa (125.28 µg mL-1), HepG2 (117.24 µg mL-1), MCF-7 (170.19 µg mL-1), and A549 (121.78 µg mL-1) in vitro by MTT assay. The combination of DDP and DOX significantly enhanced the anticancer activity of the four cell lines, especially L1-DOX had the smallest IC50 value of 0.04 µg mL-1 against HepG2 cells, indicating the combination of drugs had synergistic effects. It is further demonstrated that L1 could inhibit cell proliferation by inducing apoptosis with ROS detection and confocal fluorescence images. The relative tumor proliferation rate (T/C) was 40.6%, and the tumor inhibition rate was 57.9%, indicating L1 to have no significant toxicity but high anti-HepG2 activity in vivo. Although the study is very limited, it is anticipated to provide a reference for further exploration of the functionality of the plant.

The Extraction and High Antiproliferative Effect of Anthocyanin from Gardenblue Blueberry.

Blueberries are rich in flavonoids, anthocyanins, phenolic acids, and other bioactive substances. Anthocyanins are important functional components in blueberries. We collected 65 varieties of blueberries to investigate their nutritional and functional values. Among them, Gardenblue had the highest anthocyanin content, with 2.59 mg/g in fresh fruit. After ultrasound-assisted solvent extraction and macroporous resin absorption, the content was increased to 459.81 mg/g in the dried powder. Biological experiments showed that Gardenblue anthocyanins (L1) had antiproliferative effect on cervical cancer cells (Hela, 51.98 µg/mL), liver cancer cells (HepG2, 23.57 µg/mL), breast cancer cells (MCF-7, 113.39 µg/mL), and lung cancer cells (A549, 76.10 µg/mL), and no apparent toxic effects were indicated by methyl thiazolyl tetrazolium (MTT) assay, especially against HepG2 cells both in vitro and in vivo. After combining it with DDP (cisplatin) and DOX (doxorubicin), the antiproliferative effects were enhanced, especially when combined with DOX against HepG2 cells; the IC50 value was 0.02 µg/mL. This was further evidence that L1 could inhibit cell proliferation by inducing apoptosis. The detailed mechanism might be L1 interacting with DNA in an intercalation mode that changes or destroys DNA, causing apoptosis and inhibiting cell proliferation. The findings of this study suggest that L1 extract can be used as a functional agent against hepatoma carcinoma cells.

[Effect of Antibiotic-Degrading Bacteria on Maturity and Bacterial Community Succession During Pig Manure Composting].

The inoculation of antibiotic-degrading bacteria into manure could promote the removal of antibiotics during composting. However, knowledge on the impact of inoculating these antibiotic-degrading bacteria on the composting process and indigenous microbial community succession is still limited. This study assessed the antibiotic removal efficiency in pig manure after inoculating a microbial inoculum with antibiotic-degrading bacteria as the key component. The effect of inoculating this microbial inoculum on the physicochemical dynamics and the succession of the manure bacterial community during composting was also analyzed. The results showed that the antibiotic degradation in pig manure reached 81.95% after inoculating the microbial inoculum. When compared with that in the control, the total concentration of antibiotic residues in manure with the microbial agent inoculated was decreased by 42.18%. During composting, inoculating the microbial inoculum accelerated the temperature rise of compost, favored water loss, and alleviated the release of NH3 and H2S. Moreover, the total nutrient content (nitrogen, phosphorus, and potassium) in the final compost and the germination index of radish seeds increased by 6.80% and 68.33%, respectively, after inoculating this microbial inoculum. Furthermore, inoculating the microbial inoculum increased the content of stable organic carbon in the final compost and decreased the content of recalcitrant substances such as cellulose and hemicellulose. The analysis of the manure bacterial community showed that inoculating the microbial inoculum increased the relative abundances of Actinomycetes and Firmicutes in the compost. In particular, the thermophilic bacteria that was positively related to the compost temperature was increased significantly (P<0.01) after inoculating the microbial inoculum, whereas the relative abundance of pathogenic bacteria was correspondingly decreased. Network analysis of the bacterial coexistence pattern showed that inoculating this microbial inoculum also changed the interaction pattern of indigenous manure bacterial communities, which greatly reduced the complexity and connectivity of the bacterial interaction and improved the ecological relationship between beneficial bacteria and other bacterial communities. The effect of this microbial inoculum on the interaction with manure bacterial community laid a foundation for the establishment of a new and healthier composting bacterial community. This study provides a scientific basis for the application and development of multifunctional antibiotic-degrading microbial agents in manure treatments.

The complete chloroplast genome of Gynura japonica and its phylogenetic implications.

Gynura japonica (Asteraceae) is a folk herbal medicine with multi-pharmacological functions involving analgesic, hemostatic and antiangiogenic activities. The study was conducted to assemble the complete chloroplast (cp) genome of G. japonica through a genome-skimming approach. The assembled cp genome was 151,023 bp in size, with 62.8% AT content, consisting of a large single copy (LSC) of 83,185 bp, two copies of inverted repeat (IRa and IRb) of 24,847 bp, and a small single copy (SSC) of 18,144 bp. The cp genome of G. japonica contained 133 genes, including eight ribosomal RNA genes (rRNAs), 37 transfer RNA genes (tRNAs), 86 protein-coding genes (PCGs), and two pseudogenes (ψycf1 and ψrps19). Our phylogenomic analysis based on whole plastid genomes strongly supports G. japonica is a sister to the clade including Crassocephalum crepidioides and Jacobaea vulgaris.

Effects of Acupuncture in Ischemic Stroke Rehabilitation: A Randomized Controlled Trial.

Background: Acupuncture is a well-known treatment option for ischemic stroke recovery, but evidence of its effectiveness remains limited. This is a randomized controlled trial to evaluate the effectiveness of acupuncture treatment for ischemic stroke rehabilitation. Methods: Rehabilitation training was provided to the control group. In acupuncture arm 1, these acupoints were derived from the ancient books, including GV20 (baihui), GV26 (shuigou), PC9 (zhongchong), ST6 (jiache), ST4 (dicang), LI15 (jianyu), LI11 (quchi), LI4 (hegu), GB30 (huantiao), GB31 (fengshi), GB34 (yanglingquan), and GB39 (xuanzhong). In acupuncture arm 2, the acupoints used were GV20 (baihui), PC6 (neiguan), LI11 (quchi), LI10 (shousanli), SJ5 (waiguan), LI4 (hegu), GB30 (huantiao), ST36 (zusanli), GB34 (yanglingquan), SP6 (sanyinjiao), ST41 (jiexi), and LR3 (taichong), which were extracted from Acupuncture and Moxibustion Science. After acupuncture, the needles were left in for 30 min and manually manipulated every 10 min. The three groups received treatment once a day, 5 times a week for 2 weeks. The primary outcome was the National Institutes of Health Stroke Scale (NIHSS), and the secondary outcomes were the Barthel Index (BI) and the Modified Ashworth Scale (MAS). Outcomes were measured in patients both before and after treatment. Results: A total of 497 patients with ischemic stroke were randomized into either arm 1 (159 cases), arm 2 (173 cases), or the control group (165 cases). After 2 weeks of treatment, the NIHSS scores for arm 1 were lower than those of the control group (P = 0.017); the BI scores were higher in arm two than that in the control group at T2 (P = 0.016) and follow-up (P = 0.020). Additionally, there was no significant difference between arm one and the control group for either the BI scores or the MAS scores (P > 0.05) and no significant difference between arm two and the control group for the MAS scores or the NIHSS scores (P > 0.05). Conclusion: The clinical efficacy of arm 1 and arm 2 (acupuncture groups) was superior to that of the control group, but there was no difference between the effects of the two acupuncture groups. Clinical Trial Registration: http://www.chictr.org.cn/index.aspx, identifier: ChiCTR-IOR-16008627.

Phenolic profiles, antioxidant activity and inhibition of digestive enzymes of water caltrop pericarps.

BACKGROUND: Water caltrop (Trapa natans L.) is widely cultivated as a popular vegetable or fruit in Asian countries. In China, water caltrop pericarp is also used as a functional food to treat metabolic syndrome. However, the profiling of bioactive substances and their pharmacological activities in different water caltrop varieties remains to be investigated. In the present study, three varieties of water caltrop pericarps collected from 13 origins in China were analyzed for their phenolic substances. To investigate the pharmacological activities, samples were tested for their free radical scavenging capacity and inhibitory potency against α-glucosidase and pancreatic lipase. RESULTS: In total, 46 phenolic compounds were identified in the ethanol extract of water caltrop pericarp using a liquid chromatography-quadrupole time of flight-tandem mass spectrometry method, most of which were hydrolyzable tannins. Two cultivated varieties samples exhibited a relatively higher phenolic content and stronger antioxidant and inhibitory activities against α-glucosidase and pancreatic lipase compared to those from the wild variety. Correlation analysis between phenolic contents and biological activities suggested that phenolic compounds exhibited potential free radical scavenging capacity, α-glucosidase and pancreatic lipase inhibitory activities. CONCLUSION: It is concluded that the phenolic compounds of water caltrop pericarp are promising sources of natural antioxidants, α-glucosidase and pancreatic lipase inhibitors. © 2021 Society of Chemical Industry.

Total Sesquiterpene Glycosides from Loquat Leaves Ameliorate HFD-Induced Insulin Resistance by Modulating IRS-1/GLUT4, TRPV1, and SIRT6/Nrf2 Signaling Pathways.

Loquat (Eriobotrya japonica Lindl.), a subtropical fruit tree native to Asia, is not only known to be nutritive but also beneficial for the treatment of diabetes in the south of China. To expand its development, this study was undertaken concerning the potential therapeutic role of total sesquiterpene glycosides (TSGs) from loquat leaves in insulin resistance (IR), the major causative factor of type 2 diabetes mellitus (T2DM). Male C57BL/6 mice were fed on high-fat diet (HFD) to induce IR and then were given TSG by oral administration at 25 and 100 mg/kg/day, respectively. TSG notably improved metabolic parameters including body weight, serum glucose, and insulin levels and prevented hepatic injury. Moreover, inflammatory response and oxidative stress were found to be remarkably alleviated in IR mice with TSG supplement. Further research in liver of IR mice demonstrated that TSG repaired the signalings of insulin receptor substrate-1 (IRS-1)/glucose transporter member 4 (GLUT4) and AMP-activated protein kinase (AMPK), which improved glucose and lipid metabolism and prevented lipid accumulation in liver. It was also observed that TSG suppressed the expression of transient receptor potential vanilloid 1 (TRPV1), whereas the signaling pathway of sirtuin-6 (SIRT6)/nuclear factor erythroid 2-related factor 2 (Nrf2) was significantly promoted. Based on the results, the current study demonstrated that TSG from loquat leaves potentially ameliorated IR in vivo by enhancing IRS-1/GLUT4 signaling and AMPK activation and modulating TRPV1 and SIRT6/Nrf2 signaling pathways.

FK506 ameliorates osteoporosis caused by osteoblast apoptosis via suppressing the activated CaN/NFAT pathway during oxidative stress.

OBJECTIVE: Osteoporosis is affecting the health of postmenopausal women in the world. In case of that, we explored whether FK-506 could ameliorate osteoporosis by inhibiting the activated CaN/NFAT pathway during oxidative stress. METHODS: First, the castrated rat model is constructed through the bilateral ovariectomy. Hologic Discovery (S/N 80347) dual-energy X-ray absorptiometry assessed bone mineral density (BMD) implemented at left femur of rats. Next, hematoxylin-eosin (H&E) staining observed and calculated the changes of bone trabecular, mean trabecular plate separation (Tb.Sp), mean trabecular plate thickness (Tb.Th), and bone volume fraction (BV/TV). Then, CCK-8 assay, TUNEL assay, ALP kit and alizarin red staining detected the viability, apoptosis, alkaline phosphatase (ALP) activity, and capacity of mineralization respectively. At last, commercially available kits detected the levels of ROS and SOD in transfected MC3T3-E1 cells and bone tissues, and Western blot analysis detected proteins related to apoptosis and CaN/NFAT pathway. RESULTS: FK-506 increased the BMD and changes of bone trabecular in female castrated rats. FK-506 inhibited the oxidative stress and apoptosis by suppressing the activated CaN/NFAT pathway. Low dose of FK-506 improved the viability, ALP activity, and mineralization capacity. What's more, it suppressed the apoptosis of H2O2-induced MC3T3-E1 cells, which was deteriorated by the high dose of FK-506. Briefly, low dose of FK-506 inhibited the oxidative stress by suppressing the activated CaN/NFAT pathway, while high dose of that further inhibited the oxidative stress by suppressing the CaN/NFAT pathway. CONCLUSION: FK-506 ameliorates osteoporosis resulted from osteoblastic apoptosis which caused by suppressing the activated CaN/NFAT pathway during oxidative stress.

Traditional uses, phytochemistry, pharmacology and toxicology of the genus Gynura (Compositae): A comprehensive review.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura cass., belonging to the tribe Senecoineae of the family Compositae, contains more than 40 accepted species as annual or perennial herbs, mainly distributed in Asia, Africa and Australia. Among them, 11 species are distributed in China. Many of the Gynura species have been used as traditional herbal medicines for the treatment of diabetes mellitus, rheumatism, eruptive fever, gastric ulcer, bleeding, abscesses, bruises, burning pains, rashes and herpes zoster infection in tropical Asia countries such as China, Thailand, Indonesia, Malaysia, and Vietnam. Some of the species have been used as vegetables, tea beverage or ornamental plants by the local people. AIM OF THE STUDY: A more comprehensive and in-depth review about the geographical distribution, traditional uses, chemical constituents and pharmacological activities as well as safe and toxicity of Gynura species has been summarized, hoping to provide a scientific basis for rational development and utilization as well as to foster further research of these important medicinal plant resources in the future. MATERIALS AND METHODS: A review of the literature was performed based on the existing peer-reviewed researches by consulting scientific databases including Web of Science, PubMed, Elsevier, Google Scholar, SciFinder and China National Knowledge Infrastructure. RESULTS: Many of the Gynura species have been phytochemically studied, which led to the isolation of more than 338 compounds including phenolics, flavonoids, alkaloids, terpenoids, steroids, cerebrosides, aliphatics and other compounds. Pharmacological studies in vitro and in vivo have also confirmed the various bioactive potentials of extracts or pure compounds from many Gynura plants, based on their claimed ethnomedicinal and anecdotal uses, including antioxidant, anti-inflammation, anticancer, antidiabetic, antihypertension, antibacterial and other activities. However, pyrrolizidine alkaloids (PAs) pose a threat to the medication safety and edible security of Gynura plants because of toxicity issues, requiring the need to pay great attention to this phenomenon. CONCLUSION: The traditional uses, phytochemistry and pharmacology of Gynura species described in this review demonstrated that these plants contain a great number of active constituents and display a diversity of pharmacological activities. However, the mechanism of action, structure-activity relationship, potential synergistic effects and pharmacokinetics of these components need to be further elucidated. Moreover, further detailed research is urgently needed to explain the mechanisms of toxicity induced by PAs. In this respect, effective detoxification strategies need to be worked out, so as to support the safe and reasonable utilization of Gynura plant resources in the future.

In Colorectal Cancer Cells With Mutant KRAS, SLC25A22-Mediated Glutaminolysis Reduces DNA Demethylation to Increase WNT Signaling, Stemness, and Drug Resistance.

BACKGROUND & AIMS: Mutant KRAS promotes glutaminolysis, a process that uses steps from the tricarboxylic cycle to convert glutamine to α-ketoglutarate and other molecules via glutaminase and SLC25A22. This results in inhibition of demethylases and epigenetic alterations in cells that increase proliferation and stem cell features. We investigated whether mutant KRAS-mediated glutaminolysis affects the epigenomes and activities of colorectal cancer (CRC) cells. METHODS: We created ApcminKrasG12D mice with intestine-specific knockout of SLC25A22 (ApcminKrasG12DSLC25A22fl/fl mice). Intestine tissues were collected and analyzed by histology, immunohistochemistry, and DNA methylation assays; organoids were derived and studied for stem cell features, along with organoids derived from 2 human colorectal tumor specimens. Colon epithelial cells (1CT) and CRC cells (DLD1, DKS8, HKE3, and HCT116) that expressed mutant KRAS, with or without knockdown of SLC25A22 or other proteins, were deprived of glutamine or glucose and assayed for proliferation, colony formation, glucose or glutamine consumption, and apoptosis; gene expression patterns were analyzed by RNA sequencing, proteins by immunoblots, and metabolites by liquid chromatography-mass spectrometry, with [U-13C5]-glutamine as a tracer. Cells and organoids with knocked down, knocked out, or overexpressed proteins were analyzed for DNA methylation at CpG sites using arrays. We performed immunohistochemical analyses of colorectal tumor samples from 130 patients in Hong Kong (57 with KRAS mutations) and Kaplan-Meier analyses of survival. We analyzed gene expression levels of colorectal tumor samples in The Cancer Genome Atlas. RESULTS: CRC cells that express activated KRAS required glutamine for survival, and rapidly incorporated it into the tricarboxylic cycle (glutaminolysis); this process required SLC25A22. Cells incubated with succinate and non-essential amino acids could proliferate under glutamine-free conditions. Mutant KRAS cells maintained a low ratio of α-ketoglutarate to succinate, resulting in reduced 5-hydroxymethylcytosine-a marker of DNA demethylation, and hypermethylation at CpG sites. Many of the hypermethylated genes were in the WNT signaling pathway and at the protocadherin gene cluster on chromosome 5q31. CRC cells without mutant KRAS, or with mutant KRAS and knockout of SLC25A22, expressed protocadherin genes (PCDHAC2, PCDHB7, PCDHB15, PCDHGA1, and PCDHGA6)-DNA was not methylated at these loci. Expression of the protocadherin genes reduced WNT signaling to ß-catenin and expression of the stem cell marker LGR5. ApcminKrasG12DSLC25A22fl/fl mice developed fewer colon tumors than ApcminKrasG12D mice (P < .01). Organoids from ApcminKrasG12DSLC25A22fl/fl mice had reduced expression of LGR5 and other markers of stemness compared with organoids derived from ApcminKrasG12D mice. Knockdown of SLC25A22 in human colorectal tumor organoids reduced clonogenicity. Knockdown of lysine demethylases, or succinate supplementation, restored expression of LGR5 to SLC25A22-knockout CRC cells. Knockout of SLC25A22 in CRC cells that express mutant KRAS increased their sensitivity to 5-fluorouacil. Level of SLC25A22 correlated with levels of LGR5, nuclear ß-catenin, and a stem cell-associated gene expression pattern in human colorectal tumors with mutations in KRAS and reduced survival times of patients. CONCLUSIONS: In CRC cells that express activated KRAS, SLC25A22 promotes accumulation of succinate, resulting in increased DNA methylation, activation of WNT signaling to ß-catenin, increased expression of LGR5, proliferation, stem cell features, and resistance to 5-fluorouacil. Strategies to disrupt this pathway might be developed for treatment of CRC.

Flavonoids isolated from loquat (Eriobotrya japonica) leaves inhibit oxidative stress and inflammation induced by cigarette smoke in COPD mice: the role of TRPV1 signaling pathways.

Chronic obstructive pulmonary disease (COPD) is a chronic, progressive lung disease with few successful treatments, and is strongly associated with cigarette smoking (CS). Since the novel coronavirus has spread worldwide seriously, there is growing concern that patients who have chronic respiratory conditions like COPD can easily be infected and are more prone to having severe illness and even mortality because of lung dysfunction. Loquat leaves have long been used as an important material for both pharmaceutical and functional applications in the treatment of lung disease in Asia, especially in China and Japan. Total flavonoids (TF), the main active components derived from loquat leaves, showed remarkable anti-inflammatory and antioxidant activities. However, their protective activity against CS-induced COPD airway inflammation and oxidative stress and its underlying mechanism still remain not well-understood. The present study uses a CS-induced mouse model to estimate the morphological changes in lung tissue. The results demonstrated that TF suppressed the histological changes in the lungs of CS-challenged mice, as evidenced by reduced generation of pro-inflammatory cytokines including interleukin 6 (IL-6), IL-1ß, tumor necrosis factor α (TNF-α), nitric oxide (NO), and inducible nitric oxide synthase (iNOS) and diminished the protein expression of transient receptor potential vanilloid 1 (TRPV1). Moreover, TF also inhibited phosphorylation of IKK, IκB and NFκB and increased p-Akt. Interestingly, TF could inhibit CS-induced oxidative stress in the lungs of COPD mice. TF treatment significantly inhibited the level of malondialdehyde (MDA) and increased the activity of superoxide dismutase (SOD). In addition, TF markedly downregulated TRPV1 and cytochrome P450 2E1 (CYP2E1) and upregulated the expression of SOD-2, while the p-JNK level was observed to be inhibited in COPD mice. Taken together, our findings showed that the protective effect and putative mechanism of the action of TF resulted in the inhibition of inflammation and oxidative stress through the regulation of TRPV1 and the related signal pathway in lung tissues. It suggested that TF derived from loquat leaves could be considered to be an alternative or a new functional material and used for the treatment of CS-induced COPD.

Trapa natans pericarp extract ameliorates hyperglycemia and hyperlipidemia in type 2 diabetic mice

Abstract The pericarp of Trapa natans L., an annual aquatic floating herb belonging to Lythraceae family, is used as a folk medicine in China. In this study, extracts of Trapa natans pericarp were tested both in vitro and in vivo through a high-fat diet with a single medium dosage streptozotocin injection induced type 2 diabetic mice. Different solvent extracts of Trapa natans pericarp showed α-amylase and α-glucosidase inhibitory activity. After four weeks administration, the ethyl acetate extract of Trapa natans pericarp (50 and 100 mg/kg b.w.) reduced fasting blood glucose level, ameliorated oral glucose tolerance and insulin resistance, improved serum lipids alterations in type 2 diabetic mice as well. Additionally, ethyl acetate extract significantly elevated the insulin receptor substrate 1 and Akt serine/threonine kinase phosphorylation compared to diabetic group. HPLC-MS and HPLC-DAD analysis showed that the ethyl acetate extract was rich in hydrolysable tannins. Results support the notion that Trapa natans pericarp extract has a potential hypoglycemic activity.

Hepatoprotective Effect of Seed Coat ofEuryale ferox Extract in Non-alcoholic Fatty Liver Disease Induced by High-fat Diet in Mice by Increasing IRs-1 and Inhibiting CYP2E1.

Non-alcoholic fatty liver disease (NAFLD), a common chronic liver disease characterized by hepatic steatosis, affects 30-40% of the population in the world. The seed of Euryale ferox salisb. possesses several pharmacological actions, including metabolic syndrome. However, the seed coat of E. ferox was usually discarded as waste, which contains comparatively abundant polyphenols, and its biological activity has been rarely investigated. In this work, we evaluate the hepatoprotective effect of E. ferox seed coat extract (EFSCE), in NAFLD mice induced by high-fat diet (HFD). The HPLC-MS analysis indicated that the main components of EFSCE were polyphenols. And then, mice were treated with HFD for 4 weeks to induce NAFLD. The result showed that the body weight, weight of adipose tissue, the ratio of liver to body weight in NAFLD mice increased compared with control group. In addition, blood lipids parameters including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL) also increased in NAFLD mouse model. It was showed that, after treated with EFSCE (15 and 30 mg/kg/day) for 4 weeks, the body weight, lipids deposition in the liver and blood lipids in HFD-induced NAFLD mice markedly reduced. Compared with NAFLD mice, EFSCE administration could also prevent malondialdehyde (MDA) overproduction and strengthen Superoxide Dismutase (SOD) activity to counteract oxidative stress. Moreover, EFSCE was also found effective in reducing alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in HFD-induced NAFLD model, which indicated liver injury in NAFLD. Therefore, EFSCE (rich in polyphenols) is indicated as bioactive nature product for HFD-induced NAFLD treatment, by eliminating lipid accumulation and oxidative stress via regulation of IRs-1 and CYP2E1.

Antitussive and expectorant properties of growing and fallen leaves of loquat (Eriobotrya japonica)

ABSTRACT Folium Eriobotryae, the dried leaves of loquat (Eriobotrya japonica, (Thunb.) Lindl., Rosaceae), is a traditional Chinese medicine used to treat cough with phlegm in China. Fallen and growing loquat leaves were tested for their effect on coughing and expectoration in mice. HPLC-ELSD and HPLC-MS analyses of aqueous and ethanol extracts of fallen or growing leaves were used to identify the chemical components responsible for this effect. Both the aqueous and ethanol extracts of growing and fallen leaves of loquat contained antitussive and expectorant activities. Moreover, an aqueous extract of growing loquat leaves with a higher flavonoid content displayed a stronger expectorant activity while the ethanol extract of fallen loquat leaves that contained a higher content of triterpenoid acids induced a stronger antitussive activity.

A novel sesquiterpene glycoside from Loquat leaf alleviates oleic acid-induced steatosis and oxidative stress in HepG2 cells.

Loquat (Eriobotrya japonica) leaf has displayed beneficial effect on metabolic syndrome. In our previously study, total sesquiterpene glycosides (TSG) isolated from Loquat leaf exhibited therapeutic effect on Non-alcoholic fatty liver disease (NAFLD) in vivo, but the accurate active compound remains unknown. Sesquiterpene glycoside 1 (SG1) is a novel compound, which is exclusively isolated from Loquat leaf, but its biological activity has been rarely reported. The present study was designed to evaluate the pharmacological effect of SG1, the main component of TSG, in oleic acid (OA)-induced HepG2 cell model of NAFLD with its related mechanisms of action. In this study, both SG1 and TSG were found to significantly reduce the lipid deposition in the cell model. They could also decrease total cholesterol (TC), triglyceride (TG) and intracellular free fatty acid (FFA) contents. Compared with OA-treated cells, the superoxide dismutase (SOD) level increased, and the malondialdehyde (MDA) and 4-hydroxynonenal levels respectively decreased after the administration of SG1 or TSG. The high dose of SG1 (140 µg/mL) displayed a similar therapeutic effect as TSG at 200 µg/mL. Both SG1 and TSG were found to suppress the expression of cytochrome P450 2E1 (CYP2E1) and the phosphorylation of c-jun terminal kinase (JNK) and its downstream target c-Jun in OA-treated cell. These results demonstrate again that TSG are probably the main responsible chemical profiles of Loquat leaf for the treatment of NAFLD, for which it can effectively improve OA-induced steatosis and reduce oxidative stress, probably by downregulating of CYP2E1 expression and JNK/c-Jun phosphorylation, while SG1 may be the principle compound.

Identification and analysis of CYP450 genes from transcriptome of Lonicera japonica and expression analysis of chlorogenic acid biosynthesis related CYP450s.

BACKGROUND: Lonicera japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of L. japonica are mainly due to its rich natural active ingredients, most of which are secondary metabolites. CYP450s are a large, complex, and widespread superfamily of proteins that participate in many endogenous and exogenous metabolic reactions, especially secondary metabolism. Here, we identified CYP450s in L. japonica transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis. METHODS: The recent availability of L. japonica transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in L. japonica transcriptome. Then, phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation analyses were conducted to characterize the identified CYP450s. qRT-PCR was used to explore expression patterns of five CGA biosynthesis related CYP450s. RESULTS: In this study, 151 putative CYP450s with complete cytochrome P450 domain, which belonged to 10 clans, 45 families and 76 subfamilies, were identified in L. japonica transcriptome. Phylogenetic analysis classified these CYP450s into two major branches, A-type (47%) and non-A type (53%). Both types of CYP450s had conserved motifs in L. japonica. The differences of typical motif sequences between A-type and non-A type CYP450s in L. japonica were similar with other plants. GO classification indicated that non-A type CYP450s participated in more molecular functions and biological processes than A-type. KEGG pathway annotation totally assigned 47 CYP450s to 25 KEGG pathways. From these data, we cloned two LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of L. japonica. qRT-PCR results indicated that two LjC3Hs exhibited oppositing expression patterns during the flower development and LjC3H2 exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three LjC4Hs were quite different and the expression pattern of LjC4H3 was quite similar with that of LjC3H1. DISCUSSION: Our results provide a comprehensive identification and characterization of CYP450s in L. japonica. Five CGA biosynthesis related CYP450s were cloned and their expression patterns were explored. The different expression patterns of two LjC3Hs and three LjC4Hs may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjC3H1 and LjC4H3 strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica.

Flavonoids Isolated From the Flowers of Limonium bicolor and their In vitro Antitumor Evaluation.

BACKGROUND: Limonium bicolor, a halophytic species, can grow in saline or saline-alkali soil, is well known as a traditional Chinese medicine. Recently it attracted much attention for its treatment for cancer. OBJECTIVE: The present study was performed to evaluate this species from the phytochemical standpoint and the possible relationship between the antitumor activity and its natural products. MATERIALS AND METHODS: The chemical constituents from the flowers of L. bicolor were investigated through bioassay-guided fractionation and isolation. All the individual compounds were characterized by spectroscopic analysis and their potential antitumor activity was tested against three different human tumor cell lines by MTT assays. RESULTS: The EtOAc extract was proven as the most potent fraction and further fractionation led to the isolation of 15 natural flavonoids, which were characterized as luteolin (1), acacetin (2), quercetin (3), isorhamnetin (4), kaempferol (5), eriodictyol (6), kaempferol-3-O-α-L-rhamnoside (7), kaempferol-3-O-ß-D-glucoside (8), quercetin-3-O-α-L-rhamnoside (9), quercetin-3-O-ß-D-glucoside (10), quercetin-3-O-ß-D-galactoside (11), myricetin-3-O-α-L-rhamnoside (12), kaempferol-3-O-(6″-O-galloyl)-ß-D-glucoside (13), hesperidin (14) and rutin (15). The biotesting results demonstrated that both compounds 1 and 3 showed good cytotoxicity against human colon cancer cells (LOVO). Compound 5 exhibited relative greater growth inhibition against both human breast cancer cells (MCF-7) and osteosarcoma cell lines (U2-OS) at the concentration of 100 µg/mL. CONCLUSION: On the basis of these findings, the flavonoids were deduced to be potentially responsible for the antitumor activity of L. bicolor. The preliminary structure-activity relationship analysis suggests that the 3-O-glycosylation moiety in natural flavonoids was not essential for the antiproliferative activity on LOVO and U2-OS cells. SUMMARY: The phytochemical investigation of Limonium bicolor led to the isolation of 15 flavonoids.The biotesting of the isolates against three different human tumor cell lines was evaluated.The structure-antitumor activity relationship between the isolated flavonoids was discussed. Abbreviation used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EtOAc: Ethyl acetate; LOVO: human colon cancer; MCF-7: human breast, cancer; U2-OS: human osteosarcoma; 5-FU: 5-Fluorouracil; DMSO: dimethyl sulfoxide, NMR: nuclear magnetic resonance; HR-ESI-MS: high resolution electrospray ionization mass chromatography, HPLC: high performance liquid chromatography, EtOH: ethanol; n-BuOH: n-butanol; CC: column chromatography, TLC: thin layer chromatography; PBS: phosphate-buffered saline.