Extraction, characterization and intestinal anti-inflammatory and anti-oxidative activities of polysaccharide from stems and leaves of Chuanminshen violaceum M. L. Sheh & R. H. Shan.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanminshen violaceum M. L. Sheh & R. H. Shan (CV) is used as a medicine with roots, which have the effects of benefiting the lungs, harmonizing the stomach, resolving phlegm and detoxifying. Polysaccharide is one of its main active components and has various pharmacological activities, but the structural characterization and pharmacological activities of polysaccharide from the stems and leaves parts of CV are still unclear. AIM OF THE STUDY: The aim of this study was to investigate the optimal extraction conditions for ultrasound-assisted extraction of polysaccharide from CV stems and leaves, and to carry out preliminary structural analyses, anti-inflammatory and antioxidant effects of the obtained polysaccharide and to elucidate the underlying mechanisms. MATERIALS AND METHODS: The ultrasonic-assisted extraction of CV stems and leaves polysaccharides was carried out, and the response surface methodology (RSM) was used to optimize the extraction process to obtain CV polysaccharides (CVP) under the optimal conditions. Subsequently, we isolated and purified CVP to obtain the homogeneous polysaccharide CVP-AP-I, and evaluated the composition, molecular weight, and structural features of CVP-AP-I using a variety of technical methods. Finally, we tested the pharmacological activity of CVP-AP-Ⅰ in an LPS-induced model of oxidative stress and inflammation in intestinal porcine epithelial cells (IPEC-J2) and explored its possible mechanism of action. RESULTS: The crude polysaccharide was obtained under optimal extraction conditions and subsequently isolated and purified to obtain CVP-AP-Ⅰ (35.34 kDa), and the structural characterization indicated that CVP-AP-Ⅰ was mainly composed of galactose, galactose, rhamnose and glucose, which was a typical pectic polysaccharide. In addition, CVP-AP-Ⅰ attenuates LPS-induced inflammation and oxidative stress by inhibiting the expression of pro-inflammatory factor genes and proteins and up-regulating the expression of antioxidant enzyme-related genes and proteins in IPEC-J2, by a mechanism related to the activation of the Nrf2/Keap1 signaling pathway. CONCLUSION: The results of this study suggest that the polysaccharide isolated from CV stems and leaves was a pectic polysaccharide with similar pharmacological activities as CV roots, exhibiting strong anti-inflammatory and antioxidant activities, suggesting that CV stems and leaves could possess the same traditional efficacy as CV roots, which is expected to be used in the treatment of intestinal diseases.

Ultrasonic-assisted extraction of polysaccharide from Paeoniae Radix alba: Extraction optimization, structural characterization and antioxidant mechanism in vitro.

Paeoniae Radix alba is used in Traditional Chinese Medicine for the treatment of gastrointestinal disorders, immunomodulatory, cancer, and other diseases. In the current study, the yield of Paeoniae Radix alba polysaccharide (PRP) was significantly increased with optimal ultrasound-assisted extraction compared to hot water extraction. Further, an acidic polysaccharide (PRP-AP) was isolated from PRP after chromatographic separation and was characterized as a typical pectic polysaccharide with side chains of arabinogalactans types I and II. Moreover, it showed antioxidant effects on LPS-induced damage on IPEC-J2 cells determined by qRT-PCR and ELISA, including decreasing the pro-inflammatory factors' expressions and increasing the antioxidant enzymes activities, which was shown to be related to the Nrf2/Keap1 pathway modulated by PRP-AP. The metabolites change (such as itaconate, cholesterol sulfate, etc.) detected by untargeted metabolomic analysis in cells was also shown to be modulated by PRP-AP, and these metabolites were further utilized and protected cells damaged by LPS. These results revealed the cellular active mechanism of the macromolecular PRP-AP on protecting cells, and supported the hypothesis that PRP-AP has strong benefits as an alternative dietary supplement for the prevention of intestinal oxidative stress by modulating cellular metabolism.

Ultrasound-assisted extraction and characterization of Penthorum chinense polysaccharide with anti-inflammatory effects.

Penthorum chinense has been used in both food and medication for many years, and polysaccharide of which was considered as one of the bioactive compounds. However, the extraction process of polysaccharide from P. chinense (PCP) was not well optimized. Ultrasound-assisted extractionhas been widely employed in the extraction of natural products for its compliance with the concept of green and economic chemistry. To better investigate the structure and biology activity of PCP, response surface methodology was employed to optimize the ultrasound-assisted extraction conditions of PCP. The optimum extraction for the ultrasound-assisted extraction of PCP were obtained as ratio of solvent to material 40 mL/g, ultrasonic power 380 W, and extraction time of 50 min. The yield of PCP reached 8.71% under these optimized conditions. PCP was further purified by using anion exchange chromatography and gel filtration, an acidic fraction PCP-AP-1 was hereby obtained. The results of structural elucidation indicated that PCP-AP-1 was a typical pectic polysaccharide with a molecular weight of 66360 Da, mainly composed of galacturonic acid (68.5 mol%), followed by arabinose (9.8 mol%), rhamnose (9.4 mol%), glucose (7.7 mol%), with homogalacturonan region and rhamnogalacturonan I regions. In vitro study showed that PCP-AP-1 could improve the inflammation induced by lipopolysaccharide in intestinal epithelial cells, which was probably performed through the inhibition of multiple signaling pathways including the inhibition of TLR4, NOD1/2 and NF-κB pathway, as well as the reduction of NLRP3 inflammasome. This study defined the type of polysaccharide present in P. chinense and revealed a potential of application this plant in the prevention of intestinal inflammatory diseases.

Extraction, characterization and anti-oxidant activity of polysaccharide from red Panax ginseng and Ophiopogon japonicus waste.

Red ginseng and Ophiopogon japonicus are both traditional Chinese medicines. They have also been used as food in China for thousands of years. These two herbs were frequently used in many traditional Chinese patent medicines. However, the carbohydrate compositions of these two herbs were not normally used during the production of said medicine, such as Shenmai injection, resulting in a large amount of waste composed of carbohydrates. In this study, the extraction conditions were optimized by response surface methodology. The Shenmai injection waste polysaccharide was extracted by using distilled water that was boiled under the optimized conditions. The Shenmai injection waste polysaccharide (SMP) was thereby obtained. SMP was further purified by anion exchange chromatography and gel filtration. With this method, a neutral polysaccharide fraction (SMP-NP) and an acidic polysaccharide fraction (SMP-AP) were obtained. The results of structure elucidation indicated that SMP-NP was a type of levan, and SMP-AP was a typical acidic polysaccharide. SMP-NP exhibited potential stimulation activity on the proliferation of five different Lactobacilli strains. Therefore, SMP-AP could promote the antioxidant defense of IPEC-J2 cells. These findings suggest that Shenmai injection waste could be used as a resource for prebiotics and antioxidants.

Polysaccharide from aerial part of Chuanminshen violaceum alleviates oxidative stress and inflammatory response in aging mice through modulating intestinal microbiota.

Aging is a biological process of progressive deterioration of physiological functions, which poses a serious threat to individual health and a heavy burden on public health systems. As population aging continues, research into anti-aging drugs that prolong life and improve health is of particular importance. In this study, the polysaccharide from stems and leaves of Chuanminshen violaceum was obtained with water extraction and alcohol precipitation, and then separated and purified with DEAE anion exchange chromatography and gel filtration to obtain CVP-AP-I. We gavaged natural aging mice with CVP-AP-I and performed serum biochemical analysis, histological staining, quantitative real-time PCR (qRT-PCR) and ELISA kit assays to analyze inflammation and oxidative stress-related gene and protein expression in tissues, and 16SrRNA to analyze intestinal flora. We found that CVP-AP-I significantly improved oxidative stress and inflammatory responses of the intestine and liver, restored the intestinal immune barrier, and balanced the dysbiosis of intestinal flora. In addition, we revealed the potential mechanism behind CVP-AP-I to improve intestinal and liver function by regulating intestinal flora balance and repairing the intestinal immune barrier to regulate the intestinal-liver axis. Our results indicated that C. violaceum polysaccharides possessed favorable antioxidant, anti-inflammatory and potentially anti-aging effects in vivo.

Angelica sinensis aboveground part polysaccharide and its metabolite 5-MT ameliorate colitis via modulating gut microbiota and TLR4/MyD88/NF-κB pathway.

The roots of Angelica sinensis have been used in Traditional Chinese Medicine for thousands of years. However, tons of aerial parts of this herb (aboveground part) are commonly discarded during the process of root preparations. A polysaccharide (ASP-Ag-AP) in the aboveground parts of A. sinensis was isolated and preliminarily characterized as typical plant pectin. ASP-Ag-AP exhibited noticeable protective effects against dextran sodium sulfate (DSS)-induced colitis, including reduction of colonic inflammation, modulation of barrier function, and alteration of gut microbiota and serum metabolite profile. Anti-inflammatory effects of ASP-Ag-AP were observed by inhibiting TLR4/MyD88/NF-κB signaling pathway in vitro and in vivo. Additionally, the level of serum metabolite 5-methyl-dl-tryptophan (5-MT) was reduced by DSS and restored by ASP-Ag-AP, which also negatively correlated with Bacteroides, Alistipes, Staphylococcus and pro-inflammatory factors. The protection from inflammatory stress on intestinal porcine enterocytes cells (IPEC-J2) of 5-MT was observed through the inhibition of TLR4/MyD88/NF-κB pathway. Besides, 5-MT also exhibited robust anti-inflammatory effect in colitis mice with improving colitis symptoms, barrier function and gut microbiota, which was the same as presented by ASP-Ag-AP. Therefore, ASP-Ag-AP could be a promising agent for colitis prevention and 5-MT could be the signal metabolite of ASP-Ag-AP on defending against intestinal inflammatory stress.

Salvia miltiorrhiza polysaccharide and its related metabolite 5-methoxyindole-3-carboxaldehyde ameliorate experimental colitis by regulating Nrf2/Keap1 signaling pathway.

The roots of Salvia miltiorrhiza have been used in Traditional Chinese Medicine for thousands of years. However, tons of aerial parts of this plant are usually discarded in the production of roots preparation. To make better use of these plant resources, the polysaccharide isolated from the aerial part of S. miltiorrhiza was investigated for its potential protection against intestinal diseases. A pectic polysaccharide (SMAP-1) was isolated and characterized being composed of homogalacturonan as the main chain and rhamnogalacturonan type I as ramified region, with side chains including arabinans and possible arabinogalactan type I and II. SMAP-1 exhibited robust protective effects against dextran sodium sulfate (DSS)-induced colitis and restored colitis symptoms, colonic inflammation, and barrier functions. Anti-oxidative effects were also observed by up-regulating Nrf2/Keap1 signaling pathway. Additionally, the level of serum 5-methoxyindole-3-carboxaldehyde (5-MC) was restored by SMAP-1 identified in metabolomic analysis, being correlated with the aforementioned effects. Protection against oxidative stress on intestinal porcine enterocyte cells (IPEC-J2) by 5-MC was observed through the activation of Nrf2/Keap1 system, as also shown by SMAP-1. In conclusion, SMAP-1 could be a promising candidate for colitis prevention, and 5-MC could be the signal metabolite of SMAP-1 in protecting against oxidative stress in the intestine.

[Recent research progress of selenium polysaccharides from medicinal plants].

Selenium polysaccharides (Se polysaccharides) are a kind of organic selenium compounds which obtain the activities from Se and polysaccharides. Comparing to Se or polysaccharides, Se polysaccharides exhibit improved biological activities and are more prone to be absorbed by human bodies, therefore, they have been widely used in medical applications, such as immunomodulation, anti-tumor, anti-oxidation, anti-aging. Due to their unique pharmacological activities, Se polysaccharides from medicinal plants have gradually become a research hotspot. However, only a few of Se polysaccharides have been separated and purified in recent years. The structure of polysaccharides is also very complex, therefore, determination of the chemical structure and mechanism of bioactivity of Se polysaccharide in vivo remain to be further studied. This article systematically introduced the main source and biological activities of Se polysaccharides from medicinal plants. The purpose of this review is to provide a basis for the further research of Se polysaccharides.

[Diversity of agronomic traits of Paris polyphylla var. chinensis and their correlation with rhizome yield and active compositions].

The supply deficiency of crude medicinal plant of Paris polyphylla var. chinensis has become a bottleneck for related medicinal industry. An important approach to increase herbal production is to breed high-yield cultivated variety, which characterized ideal plant morphology. In the present study, we collected 99 wild germplasm resources of P. polyphylla and then measured their 12 main agronomic traits and contents of polyphyllin Ⅶ,Ⅵ,Ⅱ,Ⅰ. Followed analyses were used to characterize those traits and explore the potential connection with herbal yield or quality. The results showed that: ①There was ample morphological diversity in wild P. polyphylla, whose variation of agronomic traits reduced according to followed order: content of polyphyllin, weight of dry rhizome, petiole length, stem length, petal length, pedicel length, sepal length, leaf width, leaf length, sepal width, leaf number, stamen number, petal number. ② Most of those traits were significantly correlated to each other and generally represented the characterization of photosynthetic organs or reproductive organ. ③The total content of polyphyllin Ⅶ,Ⅵ,Ⅱ,Ⅰvaried between 0.02% and 0.87% and averagedat 0.13%, which showed no significant correlation with any agronomic trait. ④Plant breeders should play more attention on those germplasm resources with large leaves, large sepals and high stem.

Metabolomics approach reveals annual metabolic variation in roots of Cyathula officinalis Kuan based on gas chromatography-mass spectrum.

BACKGROUND: Herbal quality is strongly influenced by harvest time. It is therefore one of crucial factors that should be well respected by herbal producers when optimizing cultivation techniques, so that to obtain herbal products of high quality. In this work, we paid attention on one of common used Chinese herbals, Cyathula officinalis Kuan. According to previous studies, its quality may be related with growth years because of the variation of several main bioactive components in different growth years. However, information about the whole chemical composition is still scarce, which may jointly determine the herbal quality. METHODS: Cyathula officinalis samples were collected in 1-4 growth years after sowing. To obtain a global insight on chemical profile of herbs, we applied a metabolomics approach based on gas chromatography-mass spectrum. Analysis of variance, principal component analysis, partial least squares discriminant analysis and hierarchical cluster analysis were combined to explore the significant difference in different growth years. RESULTS: 166 metabolites were identified by using gas chromatography-mass spectrum method. 63 metabolites showed significant change in different growth years in terms of analysis of variance. Those metabolites then were grouped into 4 classes by hierarchical cluster analysis, characterizing the samples of different growth ages. Samples harvested in the earliest years (1-2) were obviously differ with the latest years (3-4) as reported by principal component analysis. Further, partial least squares discriminant analysis revealed the detail difference in each growth year. Gluconic acid, xylitol, glutaric acid, pipecolinic acid, ribonic acid, mannose, oxalic acid, digalacturonic acid, lactic acid, 2-deoxyerythritol, acetol, 3-hydroxybutyric acid, citramalic acid, N-carbamylglutamate, and cellobiose are the main 15 discrimination metabolites between different growth years. CONCLUSION: Harvest time should be well considered when producing C. officinalis. In order to boost the consistency of herbal quality, C. officinalis is recommended to harvest in 4th growth year. The method of GC-MS combined with multivariate analysis was a powerful tool to evaluate the herbal quality.

[Cloning, subcellular lacalization and expression analysis of chloroplast-targeted Obg gene in Cyathula officinalis].

According to ObgC gene sequences from Cyathula officinalis genomic data, the specific primers were designed, and a full-length CoObgC cDNA (2 226 bp) was obtained by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methord. Sequence alignment showed that CoObgC gene contained a 1 818 bp open reading frame (ORF) encoding 605 amino acids. Sequence analysis predicted that molecular weight of CoObgC protein was about 66.39 kDa, the academic isoelectric point was 5.35, and the protein was stable protein. Then multiple sequence alignment was applied to construct phylogenetic tree. The real-time fluorescence quantification PCR (RT-qPCR) demonstrated that a high expression level in leaf, followed by root and flower, the low transcription was in stem. The recombinant vector pCABIA2300-CoObgC was constructed and introduced into tobacco epidermal cells by agrobacterium-mediated transformation, green fluorescence was tested and targeted to chloroplast under a laser scanning confocal microscope. These findings will be helpful to lay a foundation for studying the structure and function of CoObgC gene, and elucidating C. officinalis molecular biology experiment.

[Physiological and biochemical change of Paris seed in after-ripening during variable temperature stratification].

In order to explore the dormancy physiological and biochemical mechanism of Paris seeds, the seed embryo growth courses, and the dynamic change of 5 enzymes, include SOD, POD, CAT, MDH, G-6-PDH were measured during variable temperature stratification. The results indicated that Paris seeds embryo grew quickly after 40 d in warm-stratification (18 ± 1) °C, at the meantime the metabolic activity was significantly strengthened. These facts showed that Paris seeds turned into physiological after-ripening process. After 60-80 d, the morphological embryo after-ripping process basically completed, and the following cold-stratification (4 ± 1) °C furthered Paris seed to finish physiological after-ripening. After 40 d, the activity of MDH decreased while G-6-PDH increased significantly. This showed that the main respiratory pathway of seed changed from TCA to PPP, which benifited breaking seed dormancy. In the whole period of stratification process, the activity variation of SOD and CAT was insignificantly and the activity of POD was enhanced significantly after shifting the seed in cold stratification process. This showed that SOD, CAT had no direct effects on breaking Paris seed dormancy but keeping the seed vigor, while the POD might involve in the process of Paris seed dormancy breaking.

[Effects of low-light stress on photosynthetic characteristics of Paris polyphylla var. chinensis in artificial domestication cultivation].

OBJECTIVE: To decide on the optimum artificial domestication cultivation light environment for Paris polyphylla var. chinensis through investigating the effect of light intensity on leaf's gas exchange parameters, photosynthetic parameters, light saturation point and compensation point of Paris polyphylla var. chinensis. METHODS: Different low-light stress gradients' effect on the growth of Paris polyphylla var. chinensis was compared with no low-light stress treatment through calculating leaf's gas exchange parameters, photosynthetic parameters, light saturation point and compensation point, and then all these parameters were statistically analyzed. RESULTS: Light intensity had significant influence on the photosynthetic characteristics of Paris polyphylla var. chinensis. With the strengthening of the low-light stress, chlorophyll content, gas exchange parameters, photosynthetic parameters P., AQY and light saturation point all gradually increased at first, and then decreased. However, both photosynthetic parameters Rd and light compensation point firstly decreased and then rose again. These results showed that too strong or too weak light intensity affected the optimization of photosynthetic parameters of Paris polyphylla var. chinensis. The optimal illuminance for each parameter was not completely same, but they could all reach a relative ideal state when the shading ranges between 40% and 60%. However, photosynthetic parameters deteriorated rapidly when the shading surpass 80%. CONCLUSION: For artificially cultivating Paris polyphylla var. chinensis in Baoxing,Sichuan or the similar ecological region, shading 40%-60% is the optimal light environment, which can enhance the photosynthesis of Paris polyphylla var. chinensis and promote the accumulation of photosynthetic products.