Effects of different postharvest drying processes on flavonoid content and enzymatic activity of Styphnolobium japonicum (L.) Schott flowers for industrial and medicinal use.

Traditionally, fresh S. japonicum flowers (SJF) and S. japonicum flowers buds (SJFB) are dried prior to further processing and use. Here, we investigated the ways in which drying techniques, including sun drying (SD), steam drying (STD), microwave drying (MD), hot air drying (HAD, 40 °C, 60 °C, 80 °C, 100 °C), and freeze drying (FD), alter the flavonoid composition of freshly-harvested SJF and SJFB. The flavonoid content of dried samples was determined by Ultra High Performance Liquid Chromatography-Diode Array Detector (UPLC-DAD). Overall, different drying techniques had significantly different effects on the RU content, ranging from 10.63 % (HAD-80 °C) to 34.13 % (HAD-100 °C) in SJF and from 18.91 % (HAD-100 °C) to 29.16 % (HAD-40 °C) and 30.53 % (SD) in SJFB. To clarify the mechanism by which drying affects the RU content of S. japonicum flowers, we studied the activity of a rutin-hydrolyzing enzyme (RHE) isolated from SJF and SJFB using multiple separation and assay methods. According to the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) results, the apparent molecular weight of the purified RHE was approximately 38 kDa. According to UPLC-DAD, RHE catalyzes the production of quercetin (QU) from rutin (RU), but not from other flavonoid glycosides. Drying fresh SJF and SJFB at low and high temperatures can inhibit RHE activity and prevent RU hydrolysis. Therefore, subjecting freshly-harvest SJF to HAD-100 °C, and freshly-harvest SJFB to SD or HAD-40 °C, can greatly increase the RU content. In particular, HAD is viable for large-scale application due to its simplicity and industrial feasibility.

Effect of ultrasonic degradation on the physicochemical characteristics, GLP-1 secretion, and antioxidant capacity of Polygonatum cyrtonema polysaccharide.

This study aimed to evaluate the influence of ultrasonic degradation on the physicochemical and biological characteristics of Polygonatum cyrtonema polysaccharide (PCP, 8.59 kDa). PCP was subjected to ultrasonic treatment for 8, 16, and 24 h and yielded the degraded fractions PCP-8, PCP-16, and PCP-24 (5.06, 4.13, and 3.69 kDa), respectively. Compared with the intact PCP, PCP-8, PCP-16 and PCP-24 had a reduced particle size (decrements of 28.03 %, 46.15 % and 62.54 %, respectively). Although ultrasonic degradation did not alter the primary structure of PCP, its triple helical and superficial structures were disrupted, with degraded fractions demonstrating reduced thermal stability and apparent viscosities compared with those of the intact PCP. Furthermore, the functional properties of the degraded fractions were different. PCP-16 most favourably affected GLP-1 secretion, while PCP-8 and PCP-24 exhibited the strongest antioxidant and enzyme inhibitory activities, respectively. Hence, controlled ultrasound irradiation is an appealing approach for partially degrading PCP and enhancing its bioactivity as a functional agent.

Two new sesquiterpene glycosides from the stems of Dendrobium henanense and their anti-inflammatory activity.

Two new sesquiterpene glycosides, 8α,12,15ß-trihydroxycopacamphan-15-O-ß-D-glucopyranoside (1) and dendrobiumane C-11-O-ß-D-glucopyranoside (2), along with three known terpenoids (3-5) were isolated from the aerial stems of Dendrobium henanense. Their structures were elucidated based on NMR-spectroscopic and HR-MS analyses. All compounds could reduce the levels of NO, TNF-α and IL-1ß in LPS-induced RAW264.7 cells with IC50 values ranging from 10.37 to 34.55 µΜ.

Three new isocoumarin analogues from an endolichenic fungus Aspergillus flavus CPCC 400810.

Five isocoumarin derivatives including three new compounds, aspermarolides A-C (1-3), and two known analogues, 8-methoxyldiaporthin (4) and diaporthin (5) were obtained from the culture extract of Aspergillus flavus CPCC 400810. The structures of these compounds were elucidated by spectroscopic methods. The double bond geometry of 1 and 2 were assigned by the coupling constants. The absolute configuration of 3 was determined by electronic circular dichroism experiment. All compounds showed no cytotoxic activities against the two human cancer cells HepG2 and Hela.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside.

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 µmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

Physicochemical, morpho-structural, and biological characterization of polysaccharides from three Polygonatum spp.

Polygonatum species, including P. cyrtonema, P. kingianum, and P. sibiricum, are edible plants with medicinal purposes, which have long been consumed as food due to their high nutritional value. In this study, polysaccharides from P. cyrtonema (PCP), P. kingianum (PKP) and P. sibiricum (PSP) were obtained, and their physicochemical properties and in vitro biological activities were investigated. Our results demonstrated that PCP, PKP, and PSP consist of major fructose and minor glucose, galacturonic acid, and galactose in different molar ratios with the molecular weights of 8.5 × 103 Da, 8.7 × 103 Da, and 1.0 × 104 Da, respectively. The three polysaccharides had triple-helical structures with ß-d-Fruf, α-d-Glcp, α-d-Galp sugar residues, and an O-acetyl group, and displayed peak-shaped structures in different sizes. They also exhibited thermal, shear-thinning behavior and viscoelastic properties, and PCP presented the highest viscoelasticity. Moreover, they exerted strong free radical-scavenging abilities, and significant reducing capacity. PCP was the strongest, followed by PSP and then PKP. They significantly promoted the polarization of the M1 macrophage, with the effect of PCP ranking first. All three had similar effects on GLP-1 secretion. It is, therefore, necessary to identify the various roles of these three Polygonatum polysaccharides as functional agents based on their bioactivities and physicochemical properties.

Establishment of an oligoasthenospermia mouse model based on TAp73 gene suppression.

Background: Oligoasthenospermia is one of the main causes of male infertility. Researchers usually use chemical drugs to directly damage germ cells to prepare oligoasthenospermia models, which disregards the adhesion and migration between spermatogenic cells and Sertoli cells. TAp73 is a critical regulator of the adhesin of germ cell; thus, we sought to explore a novel oligoasthenospermia model based on TAp73 gene suppression. Methods: Mice in the Pifithrin-α group were injected intraperitoneally with 2.5 mg/kg Pifithrin-α (TAp73 inhibitor) daily for 30 consecutive days. Reproductive hormone levels and epididymal sperm quality, as well as the network morphology of Sertoli cells were tested. Results: Sperm density, motility, and the relative protein and mRNA expression of TAp73 and Nectin 2 were obviously decreased in the Pifithrin-α group compared with the normal control group. No significant distinction was observed in the relative mRNA and protein expression of ZO-1. Furthermore, the tight junctions (TJs) and apical ectoplasmic specialization (ES) were destroyed in the Pifithrin-α group. Conclusion: The above results indicate that we successfully established a new oligoasthenospermia mouse model. This study provides a foundation for further exploration of the roles of TAp73 genes during spermatogenesis and provides new research objects for further oligospermia research and future drug discovery.

Two new acetoisovanillone glycosides from the water-soluble fraction of Paeonia ostii.

The cortex root of Paeonia ostii, is used as a traditional Chinese medicine for treating female diseases. Phytochemical investigation of the water-soluble fraction of the plant led to the isolation of two new acetoisovanillone glycosides: acetoisovanillone-3-O-ß-D-glucopyranoside (1) and 2-hydroxy-acetoisovanillone-3-O-ß-D-glucopyranoside (2). Their structures were elucidated by extensive spectroscopic methods.

NMR-based metabolomics approach to study the effects of Wu-Zi-Yan-Zong-Wan on triptolide-induced oligospermia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Wu-Zi-Yan-Zong-Wan (WZYZW) is a commonly used Chinese medicinal recipe for oligozoospermia. Oligozoospermia is a common disease that harms human fertility, there is no effective therapeutic medicine at present. However, the underlying pharmacological mechanism remains unclear. METHODS: Oligozoospermia rats model induced by Tripterygium glycosides (TG) was established to inspect the efficiency of WZYZW in the treatment of oligozoospermia by traditional pharmacodynamics combined with NMR-based metabolomics. Multivariate statistics were used to extracted the underlying biomarkers and metabolic pathways of WZYZW in the treatment of oligozoospermia. RESULTS: The results showed that TG disturbed many metabolites and metabolic pathways such as oxidative stress (choline, O-phosphocholine, betaine and ascorbate), energy metabolism in mitochondria (glucose, lactate, succinate, fumarate, 3-hydroxybutyrate and alanine), mitochondrial apoptosis markers (Bax and Bcl-2) and amino acids metabolisms (arginine, branched-chain amino acids, taurine and myo-inositol). CONCLUSIONS: WZYZW could significantly reverse the disturbed metabolites to their normal status by their abilities of anti-oxidation, anti-apoptosis, balancing the osmotic pressure regulatory molecules and regulating the amino acids metabolism. This study provides pharmacological basis and guidance for the clinical usage of WZYZW.

[Chemical constituents from ethyl acetate extraction of Prunus mume].

Eight compounds were isolated from the ethyl acetate extraction of Prunus mume by column chromatography. On the basis of physicochemical properties and spectrum analysis, these compounds were identified as isoquercitrin-6″-O-benzoate(1), pinoresinol(2), naringin(3), ethyl-ß-D-glucopyranoside(4), astragalin(5), quercetin(6), hypericin(7), and rutin(8). Among them, compound 1 was a new natural product, and compounds 2-5 were isolated from this plant for the first time. In vitro study, compounds 1, 3, 5-8 could significantly increase the cell survival ratio.

Anti-inflammatory bibenzyls from the stems of Dendrobium huoshanense via bioassay guided isolation.

The stems of Dendrobium huoshanense have long been used to prevent various diseases, including inflammatory diseases. This study was aimed to explain the anti-inflammatory effect of D. huoshanense stems in LPS-induced RAW 264.7 macrophages and to discover potential anti-inflammatory compounds. Results exhibited that D. huoshanense stems ethanol extract could significantly inhibit LPS-induced production of NO, TNF-α and IL-1ß. Based on bioassay guided strategy, four bibenzyls (1-4) were isolated from D. huoshanense stems for the first time. Anti-inflammatory assay showed 1-4 could remarkably inhibit the production of NO in LPS-induced macrophages. Moreover, quantitative RT-PCR analysis displayed that the mRNA levels of iNOs, TNF-α and IL-1ß could also be significantly reduced by 1-4. These results suggested that D. huoshanense stems ethanol extract and bibenzyls 1-4 might be well developed as therapeutic agent to prevent inflammatory diseases.

One new benzophenone and one new 1,3-diphenylpropane from the fibrous roots of Anemarrhena asphodeloides Bge. and their cytotoxicity.

One new benzophenone (1) and one new 1,3-diphenylpropane (2) were obtained from the fibrous roots of Anemarrhena asphodeloides Bge. Their structures were determined by comprehensive 1D, 2D NMR and HRESIMS data. By comparing the calculated ECD curves and OR with the experimental data the absolute configurations were determined. The antitumor activity of all isolates was evaluated against two human hepatoma carcinoma cells (HepG2 and Hep3B) in vitro. The results demonstrated that compound 1 and 2 showed potent cytotoxicity against HepG2 and Hep3B cells.

Structural features and anti-gastric cancer activity of polysaccharides from stem, root, leaf and flower of cultivated Dendrobium huoshanense.

The structure features and anti-gastric cancer activities in vitro of stem, root, leaf and flower polysaccharides from cultivated Dendrobium huoshanense were investigated systematically. Stem polysaccharide (cDHPS) was composed of →4)-ß-D-Glcp-(1→, →4)-ß-D-Manp-(1→, →4)-3-O-acetyl-ß-D-Manp-(1→ with the molecular weight of 2.59 × 105 Da; root polysaccharide (cDHPR) was composed of →3,5)-α-L-Araf-(1→, →4)-ß-D-Glcp-(1→, →4)-ß-D-Manp-(1→, →4,6)-ß-D-Manp-(1→, →6)-α-D-Galp-(1→ and terminal ß-L-Araf with the molecular weight of 1.41 × 104 Da; leaf polysaccharide (cDHPL) was composed of →4)-ß-D-Glcp-(1→, →4)-ß-D-Manp-(1→, →4)-3-O-acetyl-ß-D-Manp-(1→, →3,6)-ß-D-Manp-(1→ and terminal α-D-Galp with the molecular weight of 2.09 × 105 Da; and flower polysaccharide (cDHPF) was composed of →4)-ß-D-Glcp-(1→, →4)-ß-D-Manp-(1→, →3,6)-ß-D-Manp-(1→ and terminal α-D-Galp with the molecular weight of 4.78 × 105 Da. Among these four polysaccharides, cDHPS showed the best anti-gastric cancer activity evidenced by the inhibited growth and c-myc expression as well as the enhanced apoptosis and p53 expression of murine forestomach carcinoma (MFC) cells, suggesting their difference in anti-gastric cancer activity should be contributed to their difference in structure features.

[Improvement researches on quality standard of Anemarrhenae Rhizoma and its raw processed products].

This study is to improve the quality standard and supply the scientific basis for Anemarrhenae Rhizoma and its raw processed products. Steroidal saponin including timosaponin BⅡ, timosaponin AⅢ and flavonoids including neomangiferin and mangiferin were selected as the indicative components. Silica gel G thin layer chromatography(TLC) and polyamide TLC were used to detect the two types of compounds, respectively. The contents of timosaponin BⅡ and timosaponin AⅢ were determined by HPLC-ELSD and the content of neomangiferin, mangiferin and isomangiferin were determined by HPLC-UV. Moisture, total ash and acid insoluble ash were determined according to Chinese Pharmacopoeia(2015 edition). And 80% ethanol was selected as the solvent and the content determination of total extract were determined. The fingerprints of Anemarrhenae Rhizoma and its raw processed products were established by HPLC-UV and HPLC-ELSD. The results showed that the methods of TLC and HPLC have been successfully stablished. There are 2 and 3 peaks which have been identified by HPLC-ELSD and HPLC-UV, respectively. The HPLC fingerprint methods are specific and can be used to identify and quality control for Anemarrhenae Rhizoma and its raw processed products in the mass. Comparing to Chinese Pharmacopoeia(2015 edition), the TLC identification and content determination were revised and the total extract determination and HPLC fingerprints were added in the present study. Our results can be used as the scientific basis of quqlity control for Anemarrhenae Rhizoma and its raw processed products.

Benzophenones from Anemarrhena asphodeloides Bge. Exhibit Anticancer Activity in HepG2 Cells via the NF-κB Signaling Pathway.

A chemical investigation of the fibrous roots of Anemarrhena asphodeloides Bge. led to the isolation of four benzophenones, including one new compound (1) and three known ones (2-4). Comprehensive 1D, 2D NMR and HRESIMS data established the structures of the isolated compounds. The absolute configurations were determined by comparison of the calculated optical rotation (OR) with experimental data. All the isolates were evaluated for their cytotoxicities on hepatocellular carcinoma cell lines (HepG2 and Hep3B). Compound 1 showed strong cytotoxicity against HepG2 and Hep3B cells, with IC50 values at 153.1 and 180.6 nM. Through MTT assay, flow cytometry and Western blot analysis, compound 1 demonstrated the ability to stimulate apoptosis via the NF-κB signaling pathway in HepG2 cells. These benzophenones are potential lead compounds for the development of better treatments for hepatocellular carcinoma.

[A new benzophenone isolated from fibrous roots of Anemarrhena asphodeloides].

Five compounds were isolated from the fibrous roots of Anemarrhena asphodeloides by silica gel, Sephadex LH-20 and semi-HPLC column chromatography. On the basis of physic-chemical properties and spectroscopic data analysis, these compounds were identified as methyl 2-[2,4-dihydroxy-3-(4-hydroxybenzoyl)-6-methoxyphenyl]acetate(1), 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoate(2), perlolyrine(3),syringaresinol-4'-O-ß-D-glucoside(4) and 4',6-dihydroxy-4-methoxybenzophenone-2-O-(2″),3-C-(1″)-1″-desoxy-α-L-fructofuranoside(5). Among them, 1 was a new benzophenone. Compounds 2-5 were isolated from this plant for the first time. Compound 1 was tested for neuroprotective effects against H_2O_2-induced damage in SH-SY5 Y cells.

Dendrobium huoshanense polysaccharide regulates hepatic glucose homeostasis and pancreatic ß-cell function in type 2 diabetic mice.

In the present study, the hypoglycemic mechanism of a homogeneous Dendrobium huoshanense polysaccharide (GXG) was investigated using type 2 diabetic (T2D) mouse model. With a 5-week oral administration of GXG, the levels of fasting blood glucose, glycosylated serum protein and serum insulin in T2D mice were decreased, and the glucose tolerance and the insulin sensitivity were improved. The histological analysis, the periodic acid-schiff staining and the immunofluorescence staining of insulin, glucagon and apoptosis showed that the hypoglycemic effect of GXG was related to the improvement of pancreatic ß-cell quantity and function and the regulation of hepatic glucose metabolism. Western blot analysis indicated that the up-regulated IRS1-PI3K-Akt phosphorylation followed by the down-regulated FoxO1/GSK 3ß phosphorylation contributed to the enhanced glycogen synthesis and the decreased gluconeogenesis by GXG, suggesting that the response of insulin-mediated IRS1-PI3K-Akt-FoxO1/GSK 3ß signaling to GXG might be the required mechanism for GXG-ameliorated development of type 2 diabetes.

Mimengosides J and K: two new neuroprotective triterpenoids from the fruits of Buddleja lindleyana.

Two new 11-methoxyl substituted triterpenoids, named as mimengosides J (1) and K (2), along with seven known compounds, were isolated from the fruits of Buddleja lindleyana. Their structures were elucidated on the basis of spectroscopic analysis. In addition, the new ones were evaluated for protective effects against damage of SH-SY5Y cells induced by 1-methyl-4-phenylpyridinium ion (MPP+) and the results indicated that those may be one of the candidate compositions of Buddleja lindleyana for the treatment of neurodegenerative disease.

The effect of ethyl acetate extract from persimmon leaves on Alzheimer's disease and its underlying mechanism.

BACKGROUND: Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders characterized by neuronal loss in the brain and cognitive impairment. AD is now considered to be the third major cause of death in developed countries, after cardiovascular disease and cancer. Persimmon leaves are used as a popular folk medicine to treat hypertension, angina and internal haemorrhage in Cyangbhina, and it has been reported that ethyl acetate extract of persimmon leaves (EAPL) displays a potential therapeutic effect on neurodegenerative diseases. HYPOTHESIS/PURPOSE: This study was designed to investigate the effects of EAPL on AD, to clarify the possible mechanism by which EAPL exerts its beneficial effects and prevents AD, and to determine the major constituents involved. STUDY DESIGN: AD model was established by bilateral injection of Aß1-42 into the hippocampus of rats. The cognitive performance was determined by the Morris water maze and step-down tests. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), apoptosis, total and phosphorylated c-Jun NH2-terminal kinase (JNK/p-JNK), caspase-3, Bax and Bcl-2 were determined. In addition, a sensitive and reliable LC-QTOF-MS method was applied to identify the major compounds present in EAPL. RESULTS: EAPL at doses of 200mg/kg, 400mg/kg could markedly reduce the latency, significantly increase the time in the first quadrant and number of the target crossing times in Morris water maze test, markedly increase the latency and reduce the number of errors in the step-down test, significantly inhibit the reductions in SOD and GSH-Px activities, and increase the level of MDA. In addition, EAPL treatment attenuated neuronal apoptosis in the hippocampus, reduced the expression of p-JNK, caspase-3, and the relative ratio of Bax/Bcl-2. Meanwhile, 32 constituents were identified by LC-QTOF-MS/MS assays. CONCLUSION: The results indicate that EAPL has a potent protective effect on cognitive deficits induced by Aß in rats and this effect appears to be associated with the regulation of the antioxidative defense system and the mechanism of mitochondrial-mediated apoptosis. Furthermore, analysis of the LC-MS data suggests that flavonoids and triterpenoids may be responsible for the potential biological effects of EAPL.