[Spatial Distribution Patterns of Soil Organic Carbon in Karst Forests of the Lijiang River Basin and Its Driving Factors].

The aim of this study was to explore the carbon storage potential of karst forest soils in the Lijiang River Basin, reveal the spatial pattern of soil organic carbon (SOC), investigate the contributions and pathways of each driving factor to the spatial distribution of soil organic carbon, and provide a scientific basis for assessing the carbon cycle function of karst forests in the region. We employed structural equation modeling (SEM) and correlation analysis to investigate the spatial distribution characteristics of forest soil organic carbon in different basin sections (upper, middle, and lower reaches) and soil layers at different depths of the Lijiang River. Additionally, the direct and indirect ratios of each factor were quantified. The results showed that the overall soil layer of karst forest soils in the Lijiang River Basin was shallow, and soil organic carbon was phenoconcentric. The distribution of soil organic carbon content in different watershed sections was upstream > downstream > midstream, and the distribution of readily oxidizable carbon (ROC) and dissolved organic carbon (DOC) was consistent, whereas the distribution of microbial biomass carbon (MBC) was upstream > midstream > downstream. The contribution of various biotic and abiotic factors to the spatial distribution of soil organic carbon in karst forests in the watershed was different, and their contributions were ranked in descending order as:soil physicochemical factors > soil organic carbon active fraction > sample elevation > sample species diversity, with the total effects of 1.148, 0.574, 0.284, and -0.013, respectively. Among them, the sample site elevation had only an indirect effect on soil organic carbon, and the soil organic carbon active fraction had only a direct effect on soil organic carbon. Among the driving factors, total soil nitrogen, soil oxidizable organic carbon, sample site species richness, and soil soluble organic carbon could be used as important predictors of soil organic carbon content in karst forests in the Lijiang River Basin. Therefore, it is necessary to establish an effective eco-environmental protection mechanism covering the whole Lijiang River Basin, to reduce and control the impact of anthropogenic disturbances (especially in the middle urban section of the Lijiang River Basin), and to enhance and protect the species diversity of karst forests in the basin in order to improve soil physicochemical properties, improve and enhance the content of the soil organic carbon active fraction, and enhance the soil organic carbon stocks of karst forests in the Lijiang River Basin through other effective ways, as well as to promote the enhancement of the regional forest carbon sink function.

Screening of active fractions of Jiegu ointment for promoting fracture healing in New Zealand rabbits / 中国药房

OBJECTIVE To screen the active site of Jiegu ointment in promoting fracture healing in New Zealand rabbits. METHODS The ethanol extract of Jiegu ointment， as well as the ethyl acetate and n-butanol parts， were prepared and mixed with honey to form a plaster with appropriate viscosity. The radial fracture model of left forelimb in New Zealand rabbit was established and divided into model control group， ethanol extract group， ethyl acetate fraction group and n-butanol fraction group， with 6 rabbits in each group. Except for model control group， rabbits of all other groups were treated with corresponding polar part of Jiegu ointment for external application， for 4 weeks. The radial fracture healing of rabbits was studied by X-ray examination. Enzyme-linked immunosorbent assay was used to detect the serum levels of interleukin-6 （IL-6）， tumor necrosis factor α （TNF-α）， osteocalcin （OC）， vascular endothelial growth factor A （VEGFA）， basic fibroblast growth factor 2 （bFGF2） and alkaline phosphatase （ALP）. HE staining was adopted to observe the changes of pathological morphology of rabbit fracture site， and immunohistochemical method was used to detect the protein expression of bFGF2 in fracture site of rabbits. RESULTS The healing speed of the fracture site in the n-butanol fraction group was the fastest， followed by ethanol extract group， and the ethyl acetate fraction group was the slowest； the serum levels of TNF-α and IL-6 in n-butanol fraction group decreased the fastest， while the levels of ALP， bFGF2， OC and VEGFA increased the fastest [significant increase compared with ethanol extract group （P＜0.01）]； the chondrocytes at the fracture fraction completely disappeared， forming a large number of bone marrow cavities， and the bone trabeculae in the bone marrow cavity were officially formed. The expression level of bFGF2 was also higher than ethanol extract group. CONCLUSIONS The effect of n-butanol fraction on promoting fracture healing is more significant than ethyl acetate fraction and ethanol extract， and n-butanol fraction is the active fraction of Jiegu ointment to promote fracture healing.

Study on material basis of cathepsin K targeted inhibitor in Erzhi Wan / 药学实践杂志

Objective To investigate the active ingredients and components that inhibiting cathepsin K activity in Erzhi Wan, a classic kidney-tonifying formula. Methods Then-butanol, dichloromethane, ethyl acetate and petroleum ether parts and 30 active components in Erzhi Wan were screened by established high throughput fluorescence methods of inhibit the binding activity of CTSK with Z-FR-MCA substrate, the formation of CTSK and chondroitin sulfate A (CSA) complex activity, and the activity of substrate type I collagen degradation by CTSK. Molecular docking and insoluble collagen substrate binding assays were applied to verify the potential CTSK inhibitors. Results The n-butanol and petroleum ether parts of Erzhi Wan inhibited the formation of CTSK and CSA* complex by more than 90%, the petroleum ether part inhibited the binding of CTSK to substrate Z-FR-MCA by more than 90%, the collagen degradation inhibition rate of CTSK in n-butanol part was more than 95% and that in petroleum ether part was 58.6%. Among the 30 active components, 11 showed that the inhibition rate of CTSK and CSA* complex formation was more than 50%, and 5 components with the inhibition rate of Z-FR-MCA binding activity more than 50%. Finally, there were four components including eclalbasaponin Ⅸ, (-)-epicatechin gallate, nuezhenoside and wedelolactone. The inhibition rate of collagen degradation was more than 50%. Eclipta saponin IX inhibited the binding rate between collagen fibers and CTSK, up to 60%, but all of them failed to dock with CTSK active site. Conclusion There are active components that inhibiting cathepsin K in Erzhi Wan, which mainly exists in the n-butanol ingredients, but the active components is not an active-site inhibitor. It might inhibit the binding of CTSK with oligosaccharides by binding to other sites of CTSK, and then reduce the collagen degradation activity of CTSK.

Anti-asthmatic fraction screening and mechanisms prediction of Schisandrae Sphenantherae Fructus based on a combined approach.

Objective: Schisandrae Sphenantherae Fructus (SSF) is a traditional Chinese medicine used to treat coughs and pulmonary inflammatory diseases. However, the pharmacodynamic material basis and mechanisms for SSF in asthma treatment remain unclear. This study aims to screen the anti-asthmatic fraction and verify the pharmacodynamic material basis, predict the potential mechanism, and verify the interaction ability between compounds and core targets. Methods: First, three fractions from SSF were compared in terms of composition, comparison, and anti-asthmatic effects. Then, the ultra-performance liquid chromatography-quadrupole/time-of-flight-mass spectrometry/mass spectrometry (UPLC-Q/TOF-MS/MS) strategy was used to identify the compounds from the active fraction, and the anti-asthmatic efficacy of the active fraction was further studied by the ovalbumin (OVA)-induced asthma murine model. Finally, network pharmacology and molecular methods were used to study the relationships between active compounds, core targets, and key pathways of PEF in asthma treatments. Results: The petroleum ether fraction (PEF) of SSF showed better effects and could significantly diminish lung inflammation and mitigate the level of serum immunoglobulin E (IgE), interleukin (IL)-4, IL-5, IL-6, IL-13, and IL-17 in mice. A total of 26 compounds from the PEF were identified, among which the main compounds are lignans and triterpenes. Moreover, 21 active compounds, 129 overlap-ping targets, and 10 pathways were screened by network pharmacology tools. The top five core targets may play a great role in asthma treatment. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the PEF can treat asthma by acting on multiple asthma pathological processes, including the IL-17 signaling pathway, T helper (Th) 17 cell differentiation, and the calcium signaling pathway. Molecular docking was performed to evaluate the interactions of the protein-ligand binding, and most docked complexes had a good binding ability. Conclusion: The present results might contribute to exploring the active compounds with anti-asthmatic activity.

Efficacy and Mechanism of Active Fractions in Fruit of Amomum villosum Lour. for Gastric Cancer.

Amomi Fructus is the dried ripe fruit of Amomum villosum Lour. (A. villosum). It is a well-known traditional Chinese medicine widely used to treat gastrointestinal diseases, while the efficacy or mechanism of main components in Amomi Fructus on cancer treatment remains unknown. In this study, volatile oil of A. villosum (VOAV), total flavonoids of A. villosum (FNAV) and the other residue of A. villosum (RFAV) were distilled, extracted and separated as different active fractions of A. villosum. The cell toxicity test results indicated that VOAV and FNAV can effectively inhibit the cell growth of MFC cells. Flow cytometry test results confirmed that MFC cells were caused apoptosis after being treated with VOAV, FNAV or RFAV. VOAV, FNAV or RFAV induced MFC cells apoptosis through reactive oxygen species (ROS)-mediated mitochondrial pathway, evident by the increase of endogenous ROS and mitochondrial membrane potential collapse. In addition, FNAV exhibited robust inhibitory effects on MFC tumor growth, and could improve the health status of mice compared to that of mice in 5-FU treated group. To sum up, all the above results suggest that FNAV may be a good candidate for the development of new drugs for the treatment of gastric cancer.

Artocarpus sericicarpus stem bark contains antimalarial substances against Plasmodium falciparum.

OBJECTIVES: The finding of alternative medicine for malarial treatment still has become a substantial demand. The plant is one of the potential sources of drugs, among other natural sources. Artocarpus species showed great potential as the antimalarial source. This study aims to obtain active antimalarial fractions from Artocarpus sericicarpus stem bark. METHODS: Stem bark of A. sericicarpus was extracted by ultrasonic-assisted extraction method using n-hexane, dichloromethane, and methanol as solvents. Fractionation of dichloromethane extract was conducted by open column chromatography using octadecyl silica as a stationary phase and gradient acetonitrile-water as a mobile phase. The antimalarial activity was determined by lactate dehydrogenase (LDH) assay against Plasmodium falciparum 3D7 strain. RESULTS: A. sericicarpus n-hexane, dichloromethane, and methanol extracts were showed antimalarial activity with an IC50 value of >4, 2.11, and >4 µg/mL, respectively. Fractionation of dichloromethane extract was obtained 13 fractions. Seven of the 13 fractions tested showed antimalarial activity. Fraction-6 performed the highest inhibition with an IC50 value of 1.53 ± 0.04 µg/mL. Phytochemistry screening revealed that Fraction-6 contains flavonoid, polyphenol, and terpenoid compounds that can take a role in its antimalarial activity. CONCLUSIONS: A. sericicarpus contains antimalarial substances mainly in Fraction-6, which strongly inhibited the growth of P. falciparum. The flavonoid, polyphenol, and terpenoid compounds were identified in Fraction-6, which need to be further isolated to obtain and elucidate the active antimalarial compounds.

In vitro and in vivo anthelmintic study of Sesbania sesban var. bicolor, a traditionally used medicinal plant of Santhal tribe in Assam, India.

The leaf decoction of Sesbania sesban var. bicolor is used traditionally by Santhal tribe of Assam, India, for the treatment of intestinal helminthic infections. This study was conducted to evaluate the in vitro and in vivo anthelmintic efficacy of methanolic extract of S. sesban var. bicolor leaves using Hymenolepis diminuta-rat (cestode) and Syphacia obvelata-mice (nematode) as test parasites and models. Praziquantel (PZQ) and albendazole (ABZ) were used as reference drugs. At the highest concentration of 30 mg/ml of the plant extract, H. diminuta and S. obvelata showed mortality at 0.81 ± 0.01 h and 15.17 ± 0.05 h, respectively. The in vivo results substantiated the in vitro findings, and the extract showed a better cestocidal efficacy in a dose-dependent manner, whereby treatment of rats with 400 mg/kg of the plant extract caused 65.10% reduction in eggs per gram (EPG) of faeces and 56% reduction in worm counts. S. obvelata-infected mice treated at the same dose showed 34.32% and 47.08% reduction in EPG and worm counts at necropsy, respectively. The methanolic extract was subjected to bioassay-guided fractionation using different solvents and the ethyl acetate fraction proved to be the most active. This active fraction was subjected to column chromatography using varying concentrations of hexane:ethyl acetate. Maximum efficacy was observed in 7:3 hexane:ethyl acetate, where H. diminuta and S. obvelata showed mortality at 3.56 ± 0.12 h and 9.21 ± 0.02 h, respectively. This indicates that the isolated fraction contained the active component responsible for its anthelmintic activity, which substantiates the medicinal usage in traditional practice.

[Inhibition effect of active fraction from clove on PI3K/Akt/mTOR signaling pathway to induce apoptosis of human colon cancer HCT116 cells].

To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 µg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P<0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/ß-actin after treatment of AFC(100 µg·mL~(-1)) were significantly different from those in the control group(P<0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 µg·mL~(-1)) were significantly lower than those in the control group(P<0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P<0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P<0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.

Phytochemical Profile and Anti-Inflammatory Activity of the Fraction from Artemisia lavandulaefolia.

Artemisia lavandulaefolia, a traditional herbal medicine, has been utilized as anti-inflammatory and analgesia agent in clinic. Bioassay-guided fractionation resulted in a fraction (ALDF) with anti-inflammatory effect obtained from A. lavandulaefolia. Its main constituents were analyzed and identified by UPLC-ESI-Q-TOF-MS technology. ALDF showed the strong inhibitory activity on the nitrogen oxide (NO) production in LPS-induced RAW 264.7 macrophages with an IC50 value of 1.64±0.41 µg/mL. Further results displayed that ALDF also significantly suppressed the secretion of key pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2 ) and interleukin-1ß (IL-1ß), and the increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression induced by LPS stimulation. Mechanism study indicated that ALDF was able to block NF-κB signaling pathway through inhibiting IκB and p65 phosphorylation, as well as NF-κB p65 nuclear translocation. Furthermore, in vivo results in mice revealed that treatments with ALDF evoked significant inhibition on ear edema induced by xylene and on the writhing responses induced by acetic acid. These results suggest that ALDF holds great potential in the prevention and treatment of inflammatory disorders.

Molecular level changes during suppression of Rhizoctonia solani growth by humic substances and relationships with chemical structure.

Elucidation of the inhibitory effects of humic substances (HSs) on phytopathogenic fungi and the underlying molecular mechanisms are highly important for improved biocontrol. In this study, we investigated the growth suppression, morphological characteristics, transcriptomic sequence, and radical signals of Rhizoctonia solani following HS addition (50 mg/L). Through mycelial cultured experiment, mycelia growth of R. solani had been suppressed with HS addition, and the inhibition rate was 24.88 ± 0.11% compared to the control. Field emission-scanning electron microscopy showed increased and superimposed branching mycelial growth, with a shriveled appearance. RNA samples of R. solani cultured with or without HSs were both extracted to examine the sequence on molecular level by Illumina HiSeq sequencing platform. RNA sequencing analysis revealed 175 differentially expressed genes (DEGs; 111 upregulated and 64 downregulated) between the HSs treatment and control. The upregulated unigenes were annotated and significantly enriched to three molecular processes: vitamin B6 metabolism, ABC transporters, and glutathione metabolism, while the downregulated unigenes were annotated to carbohydrate metabolism, but not significantly enriched. Real time-quantitative polymerase chain reaction analysis showed that the unigenes related to hexokinase, glucose-6-phosphate isomerase, glutathione synthase, and glutathione reductase were significantly decreased (by 60.03%, 70.70%, 60.33%, and 57.59%, respectively), while those related to glutathione S-transferase were significantly increased (2.66-fold). The electron paramagnetic resonance spectra showed that HSs induced increased the intensity of radical signals of R. solani in a cultured system increased by 59.56% compared to CK (without HSs addition). Network analysis based on DEGs expression and the chemical structure of HSs revealed that the carbonyl moiety in HSs formed the most links with nodes of the DEGs (sum of the links of positive and negative effects = 70), implicating this structure as the active fraction responsible for the inhibitory effect. This study provides molecular and chemical evidence of the biofungicidal activity of HSs with the potential for practical application.

Screening of Hepatoprotective Activity Fractions from Ixeris chinensis and Study on Its Chemical Compositions / 中国药房

OBJECTIVE： To screen the hepatoprotective active fractions from Ixeris chinensis and study its chemical constituents. METHODS：The petroleum ether，ethyl acetate，n-butanol and residual water fractions from 70％ ethanol extract of I.chinensis were extracted by systematic solvent method. Human hepatocytes HL-7702 were induced by acetaminophen to induce liver injury model. MTT method was used to detect the protective effect of the above fractions（40 μg/mL，by the dosage of crude drug）on injured cells，and the active fractions were screened. The active fractions were separated and purified by silica gel column and Sephadex column chromatography. The structure of the compounds were identified by physical and chemical properties and spectral data （hydrogen spectrum，carbon spectrum）. RESULTS：After treated with different fractions of I. chinensis，the cell survival rate of each administration group was increased significantly，compared with model group（P＜0.01），and the n-butanol and water fractions had the strongest activity （the cell survival rates were 49.3％ and 52.2％ ，respectively）. Six compoundswere isolated from n-butanol fraction and identified as sonchifolignan A（Ⅰ），apigenin-7-O-β-D-glucopyranoside methyl ester（Ⅱ），luteolin-7-O-β-D-glucuronopyranoside methyl ester （Ⅲ），luteolin-7-O-β-D-glucopyranoside （Ⅳ），apigenin-7-O-β-Dglucopyranoside（Ⅴ）and luteolin（Ⅵ）. CONCLUSIONS：The n-butanol fraction is regarded as an effective position for protecting liver，and flavonoids are the main active omponents.KEYWORDS Ixeris chinensis；Hepatoprotective activi

Inhibition effect of active fraction from clove on PI3K/Akt/mTOR signaling pathway to induce apoptosis of human colon cancer HCT116 cells / 中国中药杂志

To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 μg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P<0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/β-actin after treatment of AFC(100 μg·mL~(-1)) were significantly different from those in the control group(P<0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 μg·mL~(-1)) were significantly lower than those in the control group(P<0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P<0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P<0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.

Lectins from the Edible Mushroom Agaricus bisporus and Their Therapeutic Potentials.

The mushroom Agaricus bisporus secretes biologically active compounds and proteins with benefits for human health. Most reported proteins from A. bisporus are tyrosinases and lectins. Lectins are of therapeutic or pharmaceutical interest. To date, only limited information is available on A. bisporus lectins and lectin-like proteins. No therapeutic products derived from A. bisporus lectin (ABL) are available on the market despite its extensive exploration. Recently, A. bisporus mannose-binding protein (Abmb) was discovered. Its discovery enriches the information and increases the interest in proteins with therapeutic potential from this mushroom. Furthermore, the A. bisporus genome reveals the possible occurrence of other lectins in this mushroom that may also have therapeutic potential. Most of these putative lectins belong to the same lectin groups as ABL and Abmb. Their relationship is discussed. Particular attention is addressed to ABL and Abmb, which have been explored for their potential in medicinal or pharmaceutical applications. ABL and Abmb have anti-proliferative activities toward cancer cells and a stimulatory effect on the immune system. Possible scenarios for their use in therapy and modification are also presented.

Active fractions of mannoproteins derived from yeast cell wall stimulate innate and acquired immunity of adult and elderly dogs.

Nutritional intervention in older dogs aims to increase lifespan and improve life quality as well as delay the development of diseases related to ageing. It is believed that active fractions of mannoproteins (AFMs) obtained through extraction and fractionation of yeast cell walls (Saccharomyces cerevisiae) may beneficially modulate the immune system. However, studies that have evaluated this component and the effects of ageing on the immune system of dogs are scarce. This study aimed to evaluate the immunological effects of AFMs in adult and elderly dogs. Three extruded iso-nutrient experimental diets were formulated: without addition of AFM (T0); with AFM at 400 mg/kg (T400); and with AFM at 800 mg/kg (T800). Thirty-six beagle dogs were used, and six experimental treatments, resulting in combinations of age (adult and elderly) and diet (T0, T400, and T800), were evaluated. On days zero, 14, and 28, blood samples were obtained for leucocyte phenotyping and phagocytosis assays. On days zero and 28, a lymphoproliferation test, quantification of reactive oxygen (H2O2) and nitrogen (NO) intermediate production, evaluation of faecal immunoglobulin A (IgA) content, and a delayed cutaneous hypersensitivity test (DCHT) were performed. Statistical analyses were performed with SAS software. Repeated measure variance analyses were performed, and means were compared by the Tukey test. Values of P ≤ 0.05 were considered significant, and values of P ≤ 0.10 were considered tendencies. Dogs fed T400 tended to have higher neutrophilic phagocytic activity than dogs fed T800 (P = 0.073). Regarding reactive oxygen intermediates, bacterial lipopolysaccharide (LPS)-stimulated neutrophils from animals that were fed T400 had a tendency to produce more H2O2 than those from animals fed the control diet (P = 0.093). Elderly dogs, when compared to adult dogs, had lower absolute T and B lymphocyte counts, lower auxiliary T lymphocyte counts, and higher cytotoxic T lymphocyte counts (P < 0.05). A significant effect of diet, age, and time with saline inoculation was noted for the DCHT. There was no effect of diet or age on faecal IgA content in dogs. This study suggests beneficial effects of mannoproteins on the specific and nonspecific immune responses in adult and elderly dogs.

Active fraction combination from Liuwei Dihuang decoction (LW-AFC) ameliorates corticosterone-induced long-term potentiation (LTP) impairment in mice in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Liuwei Dihuang decoction (LW), a classic formula in Traditional Chinese medicine (TCM), has been used for nearly one thousand years for various diseases with characteristic features of kidney yin deficiency. LW consists of 6 herbs including Dihuang (prepared root of Rehmannia glutinosa (Gaertn.) DC.), Shanyao (rhizome of Dioscorea polystachya Turcz.), Shanzhuyu (fruit of Cornus officinalis Siebold & Zucc.), Mudanpi (root bark of Paeonia × suffruticosa Andrews), Zexie (rhizome of Alisma plantago-aquatica L.) and Fuling (scleorotia of Wolfiporia extensa (Peck) Ginns). LW-active fraction combination (LW-AFC) is extracted from LW, it is effective for the treatment of kidney yin deficiency in many animal models. Recent researches indicate that the "kidney deficiency" is related to a disturbance in the neuroendocrine immunomodulation (NIM) network, and glucocorticoids play an important role in kidney deficiency. AIM OF THE STUDY: This study evaluated the effects of LW-AFC and the active fractions (polysaccharide, LWB-B; glycoside, LWD-b; oligosaccharide, CA-30) on corticosterone (Cort)-induced long-term potentiation (LTP) impairment in vivo. MATERIALS AND METHODS: In this study, LTP was used to evaluate the synaptic plasticity. LW-AFC was orally administered for seven days. The active fractions were given by either chronic administration (i.g., i.p., 7 days) or single administration (i.c.v., i.g., i.p.). Cort was injected subcutaneously 1 h before the high-frequency stimulation (HFS) to induce LTP impairment. Moreover, in order to research on the possible effective pathways, an antibiotic cocktail and an immunosuppressant were also used. RESULTS: Chronic administration (i.g.) of LW-AFC and its three active fractions could ameliorate Cort-induced LTP impairment. Single administration (i.c.v., i.g., i.p.) of any of the active fractions had no effect on Cort-induced LTP impairment, while chronic administration (i.g., i.p.) of LWB-B or LWD-b showed positive effects against Cort. Interestingly, CA-30 only showed protective effects via i.g. administration, and there was little effect when CA-30 was administered i.p. In addition, when the intestinal microbiota was disrupted by application of the antibiotic cocktail, CA-30 showed little protective effects against Cort. The effects of LW-AFC were also abolished when the immune function was inhibited. In the hippocampal tissue, Cort treatment increased corticosterone and glutamate, and LW-AFC could inhibit the Cort-induced elevation of corticosterone and glutamate; there was little change in D-serine in Cort-treated animals, but LW-AFC could increase the D-serine levels. CONCLUSION: LW-AFC and its three active fractions could ameliorate Cort-induced LTP impairment. Their protective effects are unlikely by a direct way, and immune modulation might be the common pathway. CA-30 could protect LTP from impairment via modulating the intestinal microbiota. Decreasing corticosterone and glutamate and increasing D-serine in the Cort-treated animals' hippocampal tissue might be one of the mechanisms for the neural protective effects of LW-AFC. Further study is needed to understand the underlying mechanisms.

Evaluation and characteristics of immunological adjuvant activity of purified fraction of Albizia julibrissin saponins.

PURPOSE: The purified fraction of Albizia julibrissin saponins (AJSAF) was evaluated and characterized for the adjuvant activity on porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. METHODS: The effects of AJSAF on serum PRRSV N protein-specific antibody titers, splenocyte proliferation, natural killer (NK) cell activity, mRNA expression of cytokines and transcription factors, secretion of cytokines, T cells response in splenocytes, as well as delayed type hypersensitivity (DTH) in the mice immunized PRRSV vaccine were determined by ELISA, MTT assay, flow cytometry and quantitative real-time PCR (qRT-PCR). RESULTS: AJSAF not only significantly enhanced the serum PRRSV N protein-specific IgG, IgG1, IgG2a and IgG2b antibody titers in the mice immunized with PRRSV CH-1R modified live vaccine (CH-1R MLV), inactivated vaccine (CH-1R IAV), and highly pathogenic JXA1-R modified live vaccine (JXA1-R MLV), but promoted the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and PRRSV N protein-stimulated splenocyte proliferation, the activities of NK cells and delayed type hypersensitivity (DTH) in the mice immunized CH-1R MLV. AJSAF also remarkably induced the production of both Th1 (IFN-γ) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 cytokines (IFN-γ and IL-2) and transcription factors (T-bet and STAT4) as well as Th2 cytokines (IL-4 and IL-10) and transcription factors (GATA-3 and STAT6) in splenocytes from the CH-1R MLV-immunized mice. Furthermore, AJSAF markedly increased the frequencies of PRRSV N protein-specific Th1 (INF-γ+ and IL-2+) and Th2 (IL-4+ and IL-10+) CD4 T cells as well as Tc1 (INF-γ+ and IL-2+) and Tc2 (IL-4+ and IL-10+) CD8 T cells in splenocytes from the CH-1R MLV-immunized mice. CONCLUSIONS: Our results demonstrated that AJSAF had a potential to enhance and improve immune responses and elicit both Th1/Th2 and Tc1/Tc2 response to PRRSV vaccine, and that AJSAF would be a promising adjuvant candidate for PRRSV vaccine.

Effect and Mechanism of Active Fraction of Polyrhachis vicina in Treatment of Systemic Lupus Erythematosus(SLE) / 中国实验方剂学杂志

Objective: To observe the effect of an active fraction of Polyrhachis vicina (AFPV) on systemic lupus erythematosus (SLE) and its possible mechanism based on animal and cell models. Method: Totally 60 SD rats were randomly divided into normal control group, model group, prednisone acetate group (5 mg·kg-1), and high, medium and low-dose AFPV groups (400, 200, 100 mg·kg-1). SLE model was replicated with bovine serum albumin-Freund's complete (incomplete) adjuvant. Arthus reaction was observed to study the effect of AFPV on the diameter of back skin redness in rats with SLE. The expressions of anti-double-stranded DNA (dsDNA) antibody, complements 3 (C3), complement 4 (C4), immunoglobulin M (IgM), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-31 (IL-31) and interleukin-33 (IL-33) in serum were detected by enzyme-linked immunosorbent assay. CD4+T cells were isolated from the spleens of MRL/lpr and C57BL/6J mice at the age of 16 to 18 weeks by immunomagnetic beads method. The expressions of miR-200a and miR-155 and the levels of zinc-finger-enhancer binding protein 1(ZEB1) and suppressor of cytokine signaling1(SOCS1) in CD4+T cells were observed to explore the effect of AFPV on SLE and its possible mechanism. Result: Compared with the normal group, the diameter of back skin swelling in the model group was significantly increased (PPPPPPP+T cells of MRL/lpr lupus mice. Compared with the model group, the expression of microRNA-200a increased significantly, the expression of microRNA-155 decreased significantly (PPConclusion: AFPV has therapeutic effect on rats with SLE, its mechanism may be related to the regulation of miR-200a/ZEB1 and miR-155/SOCS1.

Quantitative Determination of Four Active Constituents in Anti-inflammatory and Analgesic Active Fraction of Gaultheria leucocarpa var. yunnanensis / 中国实验方剂学杂志

Objective:To establish a high performance liquid chromatography (HPLC) method for determination of four active constituents, namely MSTG-A,MSTG-B,gaultherin and chlorogenic acid in the anti-inflammatory and analgesic active fraction (ARF) of the ethnic medicine Gaultheria leucocarpa var. yunnanensis, in order to provide a methodological basis for the in-depth study and quality control of G. leucocarpa var. yunnanensis,and lay a foundation for later preparation and clinical application. Method:The determination was performed on COSMOSIL 5C18-PAQ (4.6 mm×250 mm,5 μm) column with methanol-0.2% glacial acetic acid (gradient elution) as the mobile phase at a flow rate of 1 mL·min-1. The column temperature was 25℃. The detection wavelength was set at 294 nm. Result:The linear range of MSTG-B,MSTG-A,gaultherin and chlorogenic acid were 0.014 06-0.450 00,0.007 81-0.250 00,0.003 13-0.100 00,0.000 94-0.030 00 g·L-1 (r ≥ 0.999 7),respectively,with a good precision,repeatability and stability. And the average recoveries were 100.81%,98.99%,96.12% and 102.56%,respectively. RSDs were 1.4%,0.7%,0.7%,2.4%,respectively. The contents of MSTG-B,MSTG-A,gaultherin and chlorogenic acid in ARF fraction of G. leucocarpa var. yunnanensis were 23.608,41.973,8.282,2.673 mg·g-1,respectively. Conclusion:The established method was simple and accurate, with a high repeatability. It can be used for determination of four active constituents in ARF fraction of G. leucocarpa var. yunnanensis,so as to provide a reference for the in-depth research,quality control and comprehensive evaluation of G. leucocarpa var. yunnanensis and lay a solid foundation for preparation and clinical application.

Identification of Chemical Components in Active Fraction of Xiaozhong Zhitong Lotion by UPLC-Q-TOF-MS / 中国药房

OBJECTIVE： To analyze chemical components in active fraction of Xiaozhong zhitong lotion， to clarify the material basis of its efficacy， and to provide reference for the second development of ointment preparation. METHODS： UPLC-Q-TOF-MS was adopted to analyze the chemical components of active fraction （40％ ethanol elution site separated by D101 macroporous resin） of Xiaozhong zhitong lotion. The determination was performed on Hypersil GOLD aQ C18 column with mobile phase consisted of 0.1％ formic acid acetonitrile （A）-0.1％ formic acid water （B） （gradient elution） at the flow rate of 0.4       mL/min. The sample size was 4 μL， and the column temperature was 30 ℃. The condition of mass spectrometry was ESI detection in positive and negative scanning ion mode （ESI+/ESI－）. The scanning range was 100-2 000 Da. The collision energy was 45/－45 eV， and the energy of the extended collider was 10/15 eV. The accurate molecular weight， retention time and multi-stage fragment ion information of the compounds were collected after obtaining the chromatogram， and the chemical components were identified by comparing with the mass spectrum information of reference materials and references. RESULTS： A total of 48 compounds were identified， and 9 and 39 compounds were identified under ESI+/ESI－ ion mode， mainly including 10 phenolic acids， 8  phenylpropanoids， 9 anthraquinones， 3 flavones， 7 alkaloids， 5 tannins and 6 other categories. CONCLUSIONS： UPLC-Q-TOF- MS method is rapid， efficient and accurate for identify chemical components from active fraction of Xiaozhong zhitong lotion. Main chemical components of the active fraction are phenolic acids， phenylpropanoids， anthraquinones， alkaloids and tannins.

Antitussive effect and mechanism study of effective fractions from Sauropus rostratus / 中草药

Objective: To study the antitussive effect of Sauropus spatulifolius, screen the active part of its antitussive effect, and study its antitussive mechanism. Methods The acute toxicity of different extraction sites of S. spatulifolius were studied by modified Karber’s method; The model was made with ammonia liquor to induce cough. The spray time that caused half of the mice to cough was calculated by sequential method with aim to screen the active sites. Capsaicin was used to induce cough, and the mechanism of action of extracts from various parts of S. spatulifolius on opioid receptor and ATP-sensitive potassium channel (KATP) of mice was explored. Results The LD50 of 75% ethanol, ethyl acetate, n-butanol, and 95% ethanol extracts was 7.30, 17.00, 69.68, and 75.88 g/kg, respectively; The maximum tolerance dose (MTD) of petroleum ether extracts was 117.71 g/kg; Extracts from 75% ethanol and ethyl acetate had antitussive effects, and its antitussive effect was related to opioid receptor and KATP pathway. Conclusion The fractions from 75% ethanol and ethyl acetate are the active parts of S. spatulifolius for relieving cough, and its antitussive mechanism is related to the KATP pathway and opioid receptors in the excited central system.