Application of enzyme-assisted extraction of baicalin from Scutellaria baicalensis Georgi.

Endophytes may depend on degrading the plant cell wall with cellulases for their survival. Therefore, cellulase produced by endophytes may be useful in releasing the active ingredient of medicinal plants. Scutellaria baicalensis Georgi is a traditional Chinese medicinal plant widely used in China and baicalin is one of its main active ingredients. In this study, fresh S. baicalensis Georgi was used to isolate endophytes, Congo red staining was used to screen cellulase-producing strains, and HPLC was used to determine the content of baicalin in S. baicalensis Georgi. As a result, a highly active strain of endophyte capable of the extraction of high levels of baicalin was obtained. The strain was named HG-5 and identified as Bacillus sp. Scanning electron microscopy analysis confirmed that the enzyme better promotes the dissolution of plant active ingredients. After optimizing the enzyme production and extraction processes, we found that when compared with the traditional extraction method, the baicalin yield was increased 79.31% after extraction with the HG-5 enzyme. The current study provides a novel approach and method for the use of endophyte cellulase to improve the extraction of compounds from medicinal plants.

Diversity and Bioactivity of Endophytes From Angelica sinensis in China.

Plant seeds are not merely reproductive organs, they are also carriers of microorganism, particularly, inherent and non-invasive characteristic endophytes in host plant. Therefore, in this study, the endophytic diversity of Angelica seeds was studied and compared with endophytes isolated from healthy leaves, stems, roots, and seeds of A. sinensis using 20 different media. The metabolites of endophytic strains were evaluated with six different methods for their antioxidant activity and the paper disc diffusion method for antimicrobial activities. As a result, 226 endophytes were isolated. Compared with the biodiversity and abundance of uncultured fungi from Angelica seed, the result showed that the most frequent endophytic fungi were Alternaria sp. as seen in artificial media; moreover, compared with artificial media, the pathogenic fungi, including Fusarium sp. and Pseudallescheria sp., were not found from the Angelica seed, the results suggested it may not be inherent endophytes in plants. In addition, bacteria from seven phyla were identified by high-throughput sequencing, while five phyla of endophytic bacteria were not isolated on artificial media including Proteobacteria, Actinobacteria, Bacteroidetes, Microgenomates, and Saccharibacteria. Furthermore, the sample JH-4 mycelium displayed the best antioxidant activity, and the active constituent may be a flavonoid as determined by total phenol and flavonoid content. Moreover, YH-12-1 mycelium had strong inhibitory activity against the five tested strains and the minimum inhibitory concentration (MIC) against Pseudomonas aeruginosa and Streptococcus pneumoniae was found to be 25 µg/mL. Our results confirm that plant endophytes are rich in biodiversity and contain important resource of many uncultured microorganisms.

A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds.

Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.

Reynoutria Japonica from Traditional Chinese Medicine: A Source of Competitive Adenosine Deaminase Inhibitors for Anticancer.

BACKGROUND: Adenosine deaminase (ADA) is an important enzyme in purine metabolism and is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Traditional Chinese Herbal Medicine (TCHM) is widely used alone or in combination with chemotherapy to treat cancer, due to its ability to deliver a broad variety of bioactive secondary metabolites as promising sources of novel organic natural agents. OBJECTIVE: In the present study, 29 varieties of medicinal plants were screened for the presence of ADA inhibitors. RESULTS: Extracts from Reynoutria japonica, Glycyrrhiza uralensis, Lithospermum erythrorhizon, Magnolia officinalis, Gardenia jasminoides, Stephania tetrandra, Commiphora myrrha, Raphanus sativus and Corydalis yanhusuo demonstrated strong ADA inhibition with rates greater than 50%. However, Reynoutria japonica possessed the highest ADA inhibitory activity at 95.26% and so was used in our study for isolating the ADA inhibitor to be further studied. Eight compounds were obtained and their structures were identified. The compound H1 had strong ADA inhibitory activity and was deduced to be emodin by 1H and 13C-NMR spectroscopic analysis with an IC50 of 0.629 mM. The molecular docking data showed that emodin could bind tightly to the active site of ADA. Our results demonstrated that emodin displayed a new biological activity which is ADA inhibitory activity with high cytotoxic activity against K562 leukemia cells. The bioactivity of cordycepin was significantly increased when used in combination with emodin. CONCLUSION: Emodin may represent a good candidate anti-cancer therapy and adenosine protective agent.

Retinoic acid receptor α mediates all-trans-retinoic acid-induced Klf4 gene expression by regulating Klf4 promoter activity in vascular smooth muscle cells.

The transcription factor Krüppel-like factor 4 (KLF4) plays a critical role in vascular smooth muscle cell (VSMC) differentiation induced by all-trans-retinoic acid (ATRA). Although it has been demonstrated that ATRA stimulation augments both KLF4 protein and mRNA levels in VSMCs, the molecular mechanisms by which ATRA regulates Klf4 transcription are unknown. In this study, we examined the roles of ATRA-selective nuclear retinoic acid receptors (RARs) in the transcriptional regulation of Klf4. The introduction of small interfering RNA and an RAR antagonist demonstrated that RARα, but not RARß or RARγ, mediated ATRA-induced Klf4 expression. A luciferase assay for the Klf4 promoter showed that three GC boxes in the proximal Klf4 promoter were indispensible for ATRA-induced Klf4 transcription and that RARα enhanced Klf4 promoter activity in a GC box-dependent manner. Furthermore, chromatin immunoprecipitation and oligonucleotide pulldown assays demonstrated that the transcription factors KLF4, Sp1, and YB1 directly bound to the GC boxes of the proximal Klf4 promoter. Upon RARα agonist stimulation, RARα was recruited to the Klf4 promoter through its interaction with KLF4, Sp1, and YB1 to form a transcriptional activation complex on the three GC boxes of the Klf4 promoter. These results suggest that RARα serves as an essential co-activator for ATRA signaling and that the recruitment of RARα to the KLF4-Sp1-YB1 complex, which leads to Klf4 expression in VSMCs, is independent of a retinoic acid response element.

Flavonoids from Inula britannica reduces oxidative stress through inhibiting expression and phosphorylation of p47(phox) in VSMCs.

CONTEXT: Inula britanica Linn. (Compositae) is a traditional Chinese medicinal herb that has been used to treat bronchitis and inflammation. The total flavonoid extracts (TFEs) isolated from its flowers can inhibit neointimal formation induced by balloon injury in vivo. OBJECTIVE: To investigate the mechanism by which TFE suppresses oxidative stress generation and the subsequent inflammation response in vitro. MATERIALS AND METHODS: The cultured vascular smooth muscle cells (VSMCs) form rats were exposed to oxidative stress following pretreatment with or without TFE at different concentration. Then, fluorescence staining was used to detect superoxide anion (O2(˙-)) production, and the lever of maleic dialdehyde (MDA) and superoxide dismutase (SOD) was measured at the same time. Furthermore, tumor necrosis factor-α (TNF-α) was measured by enzyme linked immunosorbent assay (ELISA), reverse transcription-PCR and western blot were performed to detect the expression activity of p47(phox) gene, and immunoprecipitation was used to test the level of p47(phox) phosphorylation. RESULTS: TFE inhibited the production of O2(˙-) induced by H2O2 in VSMCs, with decrease in secretion of TNF-α; elevated the activity of SOD in the medium, similar to the effect of quercetin; reduced the level of MDA in culture medium of VSMCs. The pretreatment with TFE resulted in decrease the level of p47(phox) mRNA and protein, and even p47(phox) phosphorylation in VSMCs, compared with H2O2 control. DISCUSSION AND CONCLUSION: These findings demonstrate that TFE is capable of attenuating the oxidative stress generation and the subsequent inflammation response via preventing the overexpression and activation of p47(phox) and the increased TNF-α secretion in VSMCs in vitro.

[Survey on the influencing factors of malnutrition in rural children under 7 years of age in Gansu Province].

OBJECTIVE: To study the status and influencing factors of malnutrition in rural children under 7 years of age in Gansu Province. METHODS: By stratified random cluster sampling, 9 390 rural children under 7 years of age from 4 counties of Gansu Province were enrolled. A cross-sectional investigation on children's nutritional status was performed. The major influencing factors of malnutrition were evaluated by logistic regression analysis. RESULTS: The detection rate of under-weight was 3.60% (338 cases). The growth retardation occurred in 992 cases (10.56%). Athrepsy was found in 196 cases (2.49%) out of 7 868 children under 5 years of age. Gender, age, birth weight, life style, parents' educational level and family income were influencing factors of malnutrition. CONCLUSIONS: More attention should be paid to the issue of malnutrition of rural children under 7 years of age in Gansu Province. The prevalence of malnutrition may be reduced through increasing family income and nutrition education, and improving parents' education level and children's life style.