Simultaneous quantitation of 15 bioactive components in Yupingfeng granules by LC-MS/MS.

Yupingfeng granules (YPFG) is commonly used in the treatment of immunological diseases, inflammations, and pulmonary diseases. Several studies have found that chromones, flavones, and saponins were the major bioactive compounds of YPFG. However, few studies have reported accurate quantification methods of these compounds. This study aimed to establish a simple and rapid method by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine 15 bioactive compounds in YPFG. The experimental parameters including extraction methods, extraction solvents, extraction time, solid-liquid ratio, and LC-MS/MS condition were optimized. The linearity, precision, repeatability, stability, and recovery of the established method were evaluated. The contents of 15 bioactive compounds in seven batches of YPFG samples were analyzed by the established method and the results were compared with the values determined by HPLC. The optimal extraction condition was to extract 0.1 g of YPFG by ultrasound with 50 mL 50% ethanol for 30 min. A Waters ACQUITY UPLCBEH C18 column using the 0.1% formic acid water solution and acetonitrile as mobile phase with a gradient elution was applied to the chromatographic separation. The linearity, precision, repeatability, stability, and recovery of the method were within acceptable ranges. Compared with HPLC analysis methods in Chinese Pharmacopoeia and literature, the established method was faster, simpler, more accurate, and more reliable. The method of simultaneous determination of 15 components in YPFG by LC-MS might provide a basis for the study of the bioactive compounds and the improvement of the quality standard of YPFG.

A novel flavonol-polysaccharide from Tamarix chinensis alleviates influenza A virus-induced acute lung injury. Evidences for its mechanism of action.

BACKGROUND: Tamarix chinensis Lour. is a Chinese medicine used for treating inflammation-related diseases and its crude polysaccharides (MBAP90) exhibited significant anticomplement activities in vitro. PURPOSE: To obtain anticomplement homogenous polysaccharides from MBAP90 and explore its therapeutic effects and potential mechanism on influenza A virus (IAV)-induced acute lung injury (ALI). METHODS: Anticomplement activity-guided fractionation of the water-soluble crude polysaccharides from the leaves and twigs of T. chinensis were performed by diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns to yield a homogeneous polysaccharide MBAP-5, which was further characterized using ultra-high-performance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MS) and nuclear magnetic resonance (NMR) analysis. In vitro, the anticomplement activity of MBAP-5 through classical pathway was measured using a hemolytic test. The therapeutic effects of MBAP-5 on ALI were evaluated in H1N1-infected mice. H&E staining, enzyme linked immunosorbent assay (ELISA), immunohistochemistry, and western blot were used to systematically access lung histomorphology, inflammatory cytokines, degree of complement component 3c, 5aR, and 5b-9 (C3c, C5aR, and C5b-9) deposition, and inflammasome signaling pathway protein expressions in lung tissues. RESULTS: MBAP-5 was a novel flavonol-polysaccharide with the molecular weight (Mw) of 153.6 kDa. Its structure was characterized to process a backbone of →4)-α-D-GlcpA-(1→, →6)-α-D-Glcp-(1→, →3,4)-α-D-Glcp-(1→, →3,4,6)-α-D-Glcp-(1→, and →4,6)-ß-D-Glcp-(1→, as well as branches of α-L-Araf-(1→ and ß-D-Galp-(1→. Particularly, O-3 of →3,4,6)-α-D-Glcp-(1→ was substituted by quercetin. In vitro assay showed that MBAP-5 had a potent anticomplement activity with a CH50 value of 102 ± 4 µg/ml. Oral administration of MBAP-5 (50 and 100 mg/kg) effectively attenuated the H1N1-induced pulmonary injury in vivo by reducing pulmonary edema, virus replication, and inflammatory responses. Mechanistically, MBAP-5 inhibited the striking deposition and contents of complement activation products (C3c, C5aR, and C5b-9) in the lung. Toll-like receptor 4 (TLR4) /transcription factor nuclear factor κB (NF-κB) signaling pathway was constrained by MBAP-5 treatment. In addition, MBAP-5 could suppress activation of the inflammasome pathways, including Nod-like receptor pyrin domain 3 (NLRP3), cysteinyl aspartate specific proteinase-1/12 (caspase-1/12), apoptosis­associated speck­like protein (ASC), gasdermin D (GSDMD), interleukin (IL)-1ß, and IL-18 expressions. CONCLUSIONS: A novel flavonol-polysaccharide MBAP-5 isolated from T. chinensis demonstrated a therapeutic effect against ALI induced by IAV attack. The mechanism might be associated with inhibition of complement system and inflammasome pathways activation.

Flavonoid substituted polysaccharides from Tamarix chinensis Lour. alleviate H1N1-induced acute lung injury via inhibiting complement system.

ETHNOPHARMACOLOGICAL RELEVANCE: Viral pneumonia is a highly pathogenic respiratory infectious disease associated with excessive activation of the complement system. Our previous studies found that the anticomplement polysaccharides from some medicinal plants could significantly alleviate H1N1-induced acute lung injury (H1N1-ALI). The leaves and twigs of Tamarix chinensis Lour. are traditionally used as a Chinese medicine Xiheliu for treating inflammatory disorders. Interestingly, its crude polysaccharides (MBAP90) showed potent anticomplement activity in vitro. AIM OF THE STUDY: To evaluate the therapeutic effects and possible mechanism of MBAP90 on viral pneumonia and further isolate and characterize the key active substance of MBAP90. MATERIALS AND METHODS: The protective effects of MBAP90 were evaluated by survival tests and pharmacodynamic experiments on H1N1-ALI mice. Histopathological changes, viral load, inflammatory markers, and complement deposition in lungs were analyzed by H&E staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC), respectively. An anticomplement homogenous polysaccharide (MBAP-3) was obtained from MBAP90 by bio-guided separation, and its structure was further characterized by methylation analysis and NMR spectroscopy. RESULTS: Oral administration of MBAP90 at a dose of 400 mg/kg significantly increased the survival rate of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with MBAP90 (200 and 400 mg/kg) could decrease the lung index, lung pathological injury, the levels of excessive proinflammatory cytokines (IL-6, TNF-α, MCP-1, IL-18, and IL-1ß), and complement levels (C3c and C5b-9). In addition, MBAP-3 was characterized as a novel homogenous polysaccharide with potent in vitro anticomplement activity (CH50: 0.126 ± 0.002 mg/mL), containing 10.51% uronic acids and 9.67% flavonoids, which were similar to the composition of MBAP90. The backbone of MBAP-3 consisted of →4)-α-D-Glcp-(1→, →3,4,6)-α-D-Glcp-(1→, and →3,4)-α-D-Glcp-(1→, with branches comprising α-L-Araf-(1→, α-D-GlcpA-(1→, →4,6)-α-D-Manp-(1→ and →4)-ß-D-Galp-(1 â†’ . Particularly, O-6 of →4)-ß-D-Galp-(1→ was conjugated with a flavonoid, myricetin. CONCLUSIONS: MBAP90 could ameliorate H1N1-ALI by inhibiting inflammation and over-activation of the complement system. These polysaccharides (MBAP90 and MBAP-3) with relative high contents of uronic acid and flavonoid substituent might be vital components of T. chinensis for treating viral pneumonia.

Structural Characterization and Anticomplement Activity of an Acidic Heteropolysaccharide from Lysimachia christinae Hance.

A novel acidic heteropolysaccharide (LCP-90-1) was isolated and purified from a traditional "heat-clearing" Chinese medicine, Lysimachia christinae Hance. LCP-90-1 (Mw, 20.65 kDa) was composed of Man, Rha, GlcA, Glc, Gal, and Ara, with relative molar ratios of 1.00: 3.00: 11.62: 1.31: 1.64: 5.24. The backbone consisted of 1,4-α-D-GlcpA, 1,4-α-D-Glcp, 1,4-ß-L-Rhap, and 1,3,5-α-L-Araf, with three branches of ß-D-Galp-(1 → 4)-ß-L-Rhap-(1→, α-L-Araf-(1→ and α-D-Manp-(1→ attached to the C-5 position of 1,3,5-α-L-Araf. LCP-90-1 exhibited potent anticomplement activity (CH50: 135.01 ± 0.68 µg/mL) in vitro, which was significantly enhanced with increased glucuronic acid (GlcA) content in its degradation production (LCP-90-1-A, CH50: 28.26 ± 0.39 µg/mL). However, both LCP-90-1 and LCP90-1-A were inactivated after reduction or complete acid hydrolysis. These observations indicated the important role of GlcA in LCP-90-1 and associated derivatives with respect to anticomplement activity. Similarly, compared with LCP-90-1, the antioxidant activity of LCP-90-1-A was also enhanced. Thus, polysaccharides with a high content of GlcA might be important and effective substances of L. christinae.

Trichosanates A-G and cucurbitacins W-Y, anticomplement monoterpenoids and cucurbitane-type triterpenoids from the pericarps of Trichosanthes kirilowii.

The pericarps of Trichosanthes kirilowii are often used to treat cough in traditional Chinese medicine, and its ethanol extract exhibited effective therapeutic effects on acute lung injury (ALI) in vivo caused by H1N1. An anticomplement activity-guided fractionation on the extract resulted in the isolation of ten new terpenoids, including seven monoterpenoids, trichosanates A-G (1-7), and three cucurbitane-type triterpenoids, cucurbitacins W-Y (8-10), as well as eleven known terpenoids (11-21). The new terpenoids' structures were determined by spectroscopic analysis, X-ray crystallographic analysis (1), electronic circular dichroism (ECD) analysis and calculations (2-10). Twelve monoterpenoids (1-7 and 11-15) and five cucurbitane-type triterpenoids (8-10, 18, and 20) exhibited anticomplement activity in vitro. For the monoterpenoids, the long aliphatic chain substituents might enhance their anticomplement activity. Additionally, two representative anticomplement terpenoids, 8 and 11, obviously attenuated H1N1-induced ALI in vivo by inhibiting complement overactivation and reducing inflammatory responses.

Therapeutic effects on H1N1-induced pneumonia in mice and intestinal bacteria biotransformation of four main flavonoids from Houttuynia cordata Thunb.

Flavonoids widely exist in a large number of Chinese herbal medicines with antiviral and anti-inflammatory properties. Houttuynia cordata Thunb. is a traditional Chinese herbal medicine for heat-clearing and detoxification. In our previous research, total flavonoids from H. cordata (HCTF) effectively alleviated H1N1-induced acute lung injury (ALI) in mice. In this study, 8 flavonoids were recognized from HCTF (containing 63.06 % ± 0.26 % of total flavonoids, as quercitrin equivalents) by UPLC-LTQ-MS/MS. Four main flavonoid glycosides in HCTF (rutin, hyperoside, isoquercitrin and quercitrin) and their common aglycone quercetin (100 mg/kg) all showed therapeutic effects on H1N1-induced ALI in mice. The two flavonoids (hyperoside and quercitrin) with higher contents and quercetin showed stronger therapeutic effects on H1N1-induced ALI in mice. Hyperoside, quercitrin and quercetin significantly reduced the levels of pro-inflammatory factors, chemokines, or neuraminidase activity compared with the same dose of HCTF (p < 0.05). The results of mice intestinal bacteria biotransformation in vitro showed that quercetin was the main metabolite. The conversion rates of hyperoside and quercitrin were significantly higher by the intestinal bacteria under the pathological state (0.81 ± 0.02 and 0.91 ± 0.01, respectively) than normal state (0.18 ± 0.01 and 0.18 ± 0.12, respectively, p < 0.001). Our findings showed that hyperoside and quercitrin were the main efficacious components of HCTF for treating H1N1-induced ALI in mice and could be metabolized to quercetin by intestinal bacteria in pathological state to exert their effects.

Structural Characterization and Anti-inflammatory Activities of Anticomplementary Polysaccharides from Rhododendron principis.

Rhododendron principis leaves have been used as "Dama", a Traditional Tibetan Medicine for treating inflammatory diseases. R. principis crude polysaccharides with anticomplementary activity demonstrated promising anti-inflammatory effects on acute lung injury induced by lipopolysaccharide. R. principis crude polysaccharides significantly decreased the levels of TNF-α and interleukin-6 in both serum and blood and bronchoalveolar lavage fluid in lipopolysaccharide-induced acute lung injury mice by intragastric administration (100 mg/kg). A heteropolysaccharide, ZNDHP, was obtained from R. principis crude polysaccharides with successive anticomplementary activity-guided separation. ZNDHP was characterized as a branched neutral polysaccharide with a backbone composed of → 2)-ß-Glcp-(1→, → 2,6)-α-Glcp-(1→, → 6,3)-ß-Galp-(1→, → 2,6)-α-Galp-(1→, → 6,2)-ß-Glcp-(1→, → 4)-α-Glcp-(1→, → 5)-ß-Araf-(1→, → 3,5)-α-Araf-(1→, and → 4,6)-ß-Manp-(1→, and the backbone structure was further confirmed by partial acid hydrolysis. In addition to anticomplementary and antioxidant activities, ZNDHP exhibited potent anti-inflammatory activity by significantly inhibiting the secretion of nitric oxide, TNF-α, interleukin-6, and interleukin-1ß of lipopolysaccharide-treated RAW 264.7 cells. However, all of these activities decreased greatly after partially hydrolyzing, indicating the importance of the multibranched structure for its bioactivity. Therefore, ZNDHP might be an important component of R. principis for treating inflammation.

In vitro nephrotoxicity and quantitative UPLC-MS analysis of three aristololactams in Houttuynia cordata.

Three analogues of aristololactam Ⅰ (AL Ⅰ), AL AⅡ, AL FⅠ and AL BⅡ, had been isolated from Houttuynia cordata, a commonly used medicinal and edible plant with heat-removing and toxin-removing functions. Considering the significant nephrotoxicity of AL Ⅰ, this study evaluated the toxicity of these three aristololactams (ALs) on human proximal tubular epithelial cells (HK-2) by MTT assay, ROS assay, ELISA tests and cytologic morphology observation. Furthermore, the distribution of the three ALs in H. cordata were investigated by UPLC-MSn recognition and quantitation in SIM mode, so as to estimate primarily the safety of the plant. The results showed that all the three ALs in H. cordata had comparative cytotoxicity as AL I with the IC50 values from 3.88 µM to 20.63 µM, caused high levels of cellular levels of reactive oxygen species (ROS) in HK-2 cells, exhibited the potential to cause renal fibrosis by remarkably increasing the levels of transforming growth factor-ß (TGF-ß1) and fibronectin (FN), and induced fibrous changes in morphology of HK-2 cells. The contents of the three ALs varied significantly in 30 batches of H. cordata from different regions and parts. Overall, the aerial part contained much more ALs (3.20 - 108.19 µg/g) than the underground part (0.95 - 11.66 µg/g), and flowers had the highest contents. Besides, no ALs were detected in the water extract of any part of H. cordata. This work revealed that the aristololactams in H. cordata had similar in vitro nephrotoxicity as AL Ⅰ and were mainly distributed in the aerial part of the plant.

The synergistic effect and mechanisms of flavonoids and polysaccharides from Houttuynia cordata on H1N1-induced pneumonia in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata Thunb. (HC, Saururaceae family) is a classical Traditional Chinese Medicine used to treat pneumonia clinically. The total flavonoids (HCF) and polysaccharides (HCP) are key medicinal components of H. cordata involved in its beneficial effect on viral pneumonia. AIM OF THE STUDY: The purpose of the study is to investigate the synergistic or complementary effects of combination of HCF and HCP on viral pneumonia as well as the mechanisms underlying. MATERIALS AND METHODS: HCF or HCP were administrated separately or combined in different proportions on influenza virus H1N1 - infected mice. The survival and lung weight of mice were recorded. The synergistic effect on HCF and HCP combination was calculated by Chou-Talalay method. H&E staining was performed to detect lung histomorphology. Western blot, immunohistochemistry and enzyme linked immunosorbent assay were done to analyze the representative protein expression in lung and intestine tissues. AB - PAS staining on intestine tissue sections was performed to evaluate the histopathology of intestines. Bacterial genomic DNA was extracted and sequenced for gut microbiota analysis. RESULTS: In H1N1 lethally infected mice, the combined administration of HCF and HCP significantly increased the survival rate and prolonged the life span of mice, compared with mono-drug therapy. The viral pneumonia was remarkably improved by HCF and HCP combination reflected by lower lung index, more intact lung morphology, and less inflammatory cells and mediators. Furthermore, the combination of HCF and HCP regulated intestinal microbiota, significantly reduced the proportion of pathogenic Proteobacteria and the secretion of proinflammatory cytokine in gut. The combined HCF and HCP showed synergistic effect on reducing lung and intestine injury. The complementary interaction was also found in HCF and HCP combined therapy, as HCF provided the significant antiviral activity and HCP markedly improved intestinal physical barrier and increased the protein expression involving removal of edema. CONCLUSIONS: Our findings indicated that combination of HCF and HCP from H. cordata synergistically alleviated H1N1-induced viral pneumonia in mice via multimodal regulation of both pulmonary and intestinal homeostasis, which might imply novel therapeutic strategy for treating viral pneumonia.

Exploring the anticomplement components from Fagopyrum dibotrys for the treatment of H1N1-induced acute lung injury by UPLC-Triple-TOF-MS/MS.

In the present study, a procyanidins-enriched fraction (PCE) from the rhizome of Fagopyrum dibotrys was obtained by anticomplement activity-guided fractionation. PCE could alleviate H1N1-induced ALI in mice by reducing weight loss, decreasing lung index, and regulating cytokine levels in lung tissue. PCE contained 76.5 ± 1.1% procyanidins with a mean degree of polymerization (mDP) of 5.24 ± 0.16. Meanwhile, thirty-three chemical constituents, including 27 procyanidins and 6 other compounds, were recognized by UPLC-Triple-TOF-MS/MS. Among them, twenty recognized procyanidins were composed of (epi)catechin with B-type link, while the rest consisted of (epi)catechin gallate. Furthermore, six compounds were obtained by preparative HPLC on a C18 column (250 × 10.0 mm, 5 µm), and their structures were confirmed by mass spectrum (MS), nuclear magnetic resonance (NMR), and specific rotation. The structure-activity relationship analysis indicated that DP and galloylation were closely related to the anticomplement activity of procyanidins. The obtained results revealed that anticomplement procyanidins were one kind of the potentially effective materials of F. dibotrys against H1N1 influenza virus infection, and the in vivo efficacy of these compounds was worthy of further investigation.

Houttuynia cordata polysaccharides alleviate ulcerative colitis by restoring intestinal homeostasis.

Houttuynia cordata is traditionally used as phytoantibiotics for treating lung disease in China. Houttuynia cordata polysaccharides (HCPs) have been reported to alleviate influenza virus-induced intestinal and lung immune injury by regulating the gut-lung axis. The present study aims to investigate the effects and mechanisms of HCPs on ulcerative colitis (UC). Male C57BL/6 mice were induced by dextran sodium sulfate (DSS) to establish the UC animal model. Our results showed that HCPs significantly reduced the weight loss and the shortening of colon length in colitis mice, and relieved the pathological damage of colon mucosa and inhibited the expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6, etc. It was suggested that HCPs could significantly improve DSS-induced colitis in mice. HCPs directly protected intestinal epithelial cells, ameliorated epithelial barrier dysfunction and cell apoptosis, which was also proved in H2O2 stimulated cell apoptosis model. HCPs inhibited inflammation in the colon, which was related to suppressing the infiltration of macrophages, inhibiting the expression of pro-inflammatory cytokines and proteins (TLR4, NF-κB), and restoring the dysfunction of Th17 and Treg cells. HCPs also restored the alteration of intestinal flora induced by DSS, increased the abundance ofFirmicutes and Bacteroides, and reduced the abundance of Proteobacteria. This study confirmed the protective effect of Houttuynia cordata polysaccharide extracted from traditional Chinese medicine on ulcerative colitis, of which the mechanism was closely related to the maintenance of intestinal homeostasis (intestinal barrier, immune cells, and intestinal bacteria).

Therapeutic Potential of 2-Methylquinazolin-4(3H)-one as an Antiviral Agent against Influenza A Virus-Induced Acute Lung Injury in Mice.

Qingdai-Mabo (QM), a traditional Chinese herbal formula composed of medicinal herb and fungus, has been used for treatment of cough and viral pneumonia. However, the underlying mechanism and bioactive components against anti-influenza A virus remain unclear. In the present study, ethyl acetate (EA) extract of QM decoctions was tested for its biological activity against acute lung injury (ALI) and its main components were identified using UPLC-MS/MS. In total, 18 bioactive components were identified, including 2-Methylquinaozlin-4(3H)-one (C1), which showed significant antiviral activity in vitro with an IC50 of 23.8 µg/mL. Furthermore, we validated the efficacy of C1 in ameliorating ALI lesions and inflammation in influenza A virus-infected mice. The results showed that C1 significantly reduced the lung index, downregulated neuraminidase (NA) and nucleoprotein (NP), and decreased the expression of pro-inflammatory molecules IFN-α, TNF-α, MCP-1, IL-6, and IL-8; however, they enhanced levels of IL-10 and IFN-γ in lung homogenate from mice infected by influenza A virus. In addition, C1 inhibited the recruitment of macrophages. These in vitro and in vivo studies suggested that the significant anti-influenza A virus activity contributed to its curative effect on lesions and inflammation of viral pneumonia in mice. Given its potential antiviral activity against influenza A virus, C1 is determined to be a main active component in the EA extract of QM. Taken together, the antiviral activity of C1 suggests its potential as an effective treatment against viral pneumonia via the inhibition of virus replication, but the mechanism C1 on antiviral research needs to be explored further.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

Integrated network pharmacology and intestinal flora analysis to determine the protective effect of Xuanbai-Chengqi decoction on lung and gut injuries in influenza virus-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanbai-Chengqi decoction (XBCQ) is a traditional Chinese medicine (TCM) compound used in the treatment of pulmonary infection in China. Despite the popular usage of XBCQ, its underlying protective roles and the associated molecular mechanisms with the gut-lung axis in influenza remain unclear. AIM OF THE STUDY: We aimed to explore the protective effects and the underlying mechanism of XBCQ efficacy on lung and intestine injuries induced by influenza A virus as well as to identify the main active components through integrated network pharmacology, intestinal flora analysis and pathway validation. MATERIALS AND METHODS: The potential active components and therapeutic targets of XBCQ in the treatment of influenza were hypothesized through a series of network pharmacological strategies, including components screening, targets prediction and bioinformatics analysis. Inflammatory cytokines and pathway proteins were assayed to validate the results of network pharmacology. Then the mechanism of XBCQ alleviating lung and intestine injuries was further explored via intestinal flora analysis. The important role of Rhubarb in the formula was verified by removing Rhubarb. RESULTS: XBCQ could significantly improve the survival rate in IAV-infected mice. The network pharmacology results demonstrated that JUN, mitogen-activated protein kinase (MAPK), and tumor necrosis factor (TNF) are the key targets of XBCQ that can be useful in influenza treatment as it contains the core components luteolin, emodin, and aloe-emodin, which are related to the pathways of TNF, T-cell receptor (TCR), and NF-κB. Verification experiments demonstrated that XBCQ could significantly alleviate the immune injury of the lungs and the gut of the mice, which is attributable to the inhibition of the release of inflammatory cytokines (such as TNF-α, IL-6, and IL-1ß), the downregulation of the protein expression levels of Toll-like receptors-7 (TLR7), MyD88, and p-NF-κB65, and the reduction in the relative abundance of Enterobacteriaceae and Proteus, while an increase in that of Firmicutes and Lachnospiraceae. The overall protective role of XBCQ contributing to the treatment of the lungs and the gut was impaired when Rhubarb was removed from XBCQ. CONCLUSIONS: Our results suggest that the efficacy of XBCQ is related to the inhibition of the immune injury and remodeling of the intestinal flora, wherein Rhubarb plays an important role, which cumulatively provide the evidence applicable for the treatment of viral pneumonia induced by a different respiratory virus with XBCQ.

Effects of Different Doses of Clopidogrel plus Early Rehabilitation Therapy on Motor Function and Inflammatory Factors in Patients with Ischemic Stroke.

This prospective randomized controlled study was intended to assess the effects of different doses of clopidogrel plus early rehabilitation therapy on motor function and inflammatory factors in patients with ischemic stroke. Between August 2018 and October 2020, 90 cases of ischemic stroke treated in the Second People's Hospital of Yibin were randomized at a ratio of 1 : 1 to receive either oral 50 mg/d clopidogrel plus early rehabilitation therapy (low-dose group) or oral 75 mg/d clopidogrel plus early rehabilitation therapy (high-dose group), with 45 cases in each group. The outcome measures including the Barthel Index (BI), National Institutes of Health Stroke Scale (NIHSS), Fugl-Meyer simplified scale, hypersensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and occurrence of adverse events were collected. After treatment, the high-dose group had higher BI results than the low-dose group. All eligible patients showed significantly declined NIHSS scores, and the high-dose group had markedly lower results (P < 0.05). After treatment, the Fugl-Meyer scores of both upper and lower extremities of the high-dose group were significantly higher than those in the low-dose group. The high-dose group achieved a greater decrease in inflammatory factor levels after treatment versus the low-dose group. The two groups showed a similar incidence of adverse events. High-dose clopidogrel plus early rehabilitation outperforms the low-dose treatment for patients with ischemic stroke by effectively mitigating the inflammatory response in the body, promoting the restoration of neurological function, improving the level of motor function, and enhancing the patient's quality of life, with manageable safety.

New Phenolic Glycosides and Lignans from the Roots of Lilium dauricum.

Three new phenolic glycosides, carvacrol-2-O-ß-D-apiofuranosyl-(1 → 6)-ß-D-glucopyranoside (1: ), 1-methyl-3-isopropylphenol-4-O-ß-D-apiofuranosyl-(1 → 6)-ß-D-glucopyranoside (2: ), p-methoxythymol-5-O-ß-D-apiofuranosyl-(1 → 6)-ß-D-glucopyranoside (3: ), and a pair of new 8-O-4' neolignan enantiomers (5A: /5B: ), together with 26 known compounds (4, 6:  - 30: ) were isolated from the roots of Lilium dauricum. The structures of the new compounds were elucidated based on extensive spectroscopic and chemical methods, and the absolute configurations of 5A: and 5B: were established by electronic circular dichroism analysis. Nine compounds (1, 3, 4, 8, 9, 17, 25, 29,: and 30: ) exhibited potent α-glucosidase inhibitory activity with IC50 values ranging from 73.4 µM to 988.2 µM. Besides, compound 19: displayed strong anticomplementary activity (CH50: 71.6 µM).

Glycoproteins From Rabdosia japonica var. glaucocalyx Regulate Macrophage Polarization and Alleviate Lipopolysaccharide-Induced Acute Lung Injury in Mice via TLR4/NF-κB Pathway.

Background and Aims: Rabdosia japonica var. glaucocalyx is a traditional Chinese medicine (TCM) for various inflammatory diseases. This present work aimed to investigate the protective effects of R. japonica var. glaucocalyx glycoproteins on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the potential mechanism. Methods: Glycoproteins (XPS) were isolated from R. japonica var. glaucocalyx, and homogeneous glycoprotein (XPS5-1) was purified from XPS. ANA-1 cells were used to observe the effect of glycoproteins on the secretion of inflammatory mediators by enzyme-linked immunosorbent assay (ELISA). Flow cytometry assay, immunofluorescence assay, and Western blot analysis were performed to detect macrophage polarization in vitro. The ALI model was induced by LPS via intratracheal instillation, and XPS (20, 40, and 80 mg/kg) was administered intragastrically 2 h later. The mechanisms of XPS against ALI were investigated by Western blot, ELISA, and immunohistochemistry. Results: In vitro, XPS and XPS5-1 downregulated LPS-induced proinflammatory mediators production including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and nitric oxide (NO) and upregulated LPS-induced IL-10 secretion. The LPS-stimulated macrophage polarization was also modulated from M1 to M2. In vivo, XPS maintained pulmonary histology with significantly reducing protein concentration and numbers of mononuclear cells in bronchoalveolar lavage fluid (BALF). The level of IL-10 in BALF was upregulated by XPS treatment. The level of cytokines including TNF-α, IL-1ß, and IL-6 was downregulated. XPS also decreased infiltration of macrophages and polymorphonuclear leukocytes (PMNs) in lung. XPS suppressed the expression of key proteins in the TLR4/NF-κB signal pathway. Conclusion: XPS was demonstrated to be a potential agent for treating ALI. Our findings might provide evidence supporting the traditional application of R. japonica var. glaucocalyx in inflammation-linked diseases.

Beneficial effect of Indigo Naturalis on acute lung injury induced by influenza A virus.

BACKGROUND: Infections induced by influenza viruses, as well as coronavirus disease 19 (COVID-19) pandemic induced by severe acute respiratory coronavirus 2 (SARS-CoV-2) led to acute lung injury (ALI) and multi organ failure, during which traditional Chinese medicine (TCM) played an important role in treatment of the pandemic. The study aimed to investigate the effect of Indigo Naturalis on ALI induced by influenza A virus (IAV) in mice. METHOD: The anti-influenza and anti-inflammatory properties of aqueous extract of Indigo Naturalis (INAE) were evaluated in vitro. BALB/c mice inoculated intranasally with IAV (H1N1) were treated intragastrically with INAE (40, 80 and 160 mg/kg/day) 2 h later for 4 or 7 days. Animal lifespan and mortality were recorded. Expression of high mobility group box-1 protein (HMGB-1) and toll-like receptor 4 (TLR4) were evaluated through immunohistological staining. Inflammatory cytokines were also monitored by ELISA. RESULT: INAE inhibited virus replication on Madin-Darby canine kidney (MDCK) cells and decreased nitric oxide (NO) production from lipopolysaccharide (LPS)-stimulated peritoneal macrophages in vitro. The results showed that oral administration of 160 mg/kg of INAE significantly improved the lifespan (P < 0.01) and survival rate of IAV infected mice, improved lung injury and lowered viral replication in lung tissue (P < 0.01). Treatment with INAE (40, 80 and 160 mg/kg) significantly increased liver weight and liver index (P < 0.05), as well as weight and organ index of thymus and spleen at 160 mg/kg (P < 0.05). Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were reduced by INAE administration (P < 0.05). The expression of HMGB-1 and TLR4 in lung tissue were also suppressed. The increased production of myeloperoxidase (MPO) and methylene dioxyamphetamine (MDA) in lung tissue were inhibited by INAE treatment (P < 0.05). Treatment with INAE reduced the high levels of interferon α (IFN-α), interferon ß (IFN-ß), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted factor (RANTES), interferon induced protein-10 (IP-10), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) (P < 0.05), with increased production of interferon γ (IFN-γ) and interleukin-10 (IL-10) (P < 0.05). CONCLUSION: The results showed that INAE alleviated IAV induced ALI in mice. The mechanisms of INAE were associated with its anti-influenza, anti-inflammatory and anti-oxidation properties. Indigo Naturalis might have clinical potential to treat ALI induced by IAV.

Molecular Identification Based on Chloroplast Sequences and Anti-complementary Activity Comparison of Juniperus Samples from the Qinghai-Tibet Plateau.

Juniperus (Cupressaceae, Pinales) plants are widely distributed in the Qinghai-Tibet Plateau of China. The leaves and twigs of at least 8 Juniperus species (J. pingii, J. pingii var. wilsonii, J. squamata, J. recurva var. coxii, J. saltuaria, J. indica, J. tibetica and J. convallium var. microsperma) have been used as the Tibetan medicine Xuba. At present, it is difficult to distinguish among the original species of Xuba based only on their similar morphological characteristics. However, in our previous studies, 4 Xuba samples from different Juniperus species exhibited significant differences in both anticomplementary activity in vitro and anti-inflammatory effects on acute lung injury in vivo. To identify the effective original species of Xuba reliably, in this study, we developed a sequencing-based DNA molecular technology to distinguish 14 populations of 8 Juniperus species collected from Tibet region, using trnS-G, trnD - T, and petN-psbM genomic regions to build phylogenetic trees. In addition, their anticomplementary activities were evaluated. The results showed that combined sequence of these 3 genomic regions could identify 8 Juniperus species clearly and clustered individuals of one species but from different locations, whichever phylogenetic tree was constructed. Moreover, the anticomplementary activities of the 8 species were clustered into 2 groups. Among them, J. saltuaria and J. recurva var. coxii, which formed an independent branch apart from the other 6 species in phylogenetic trees, were the most potent (CH50: 0.029 - 0.032 mg/mL). Consequently, DNA identification of Juniperus using the combined sequence could provide beneficial guidance for further efficacy evaluation and quality control of Xuba.

Anti-HIV tigliane diterpenoids from Reutealis trisperma.

Bioassay-guided fractionation of the n-butanol extract from the branches and leaves of Reutealis trisperma resulted in the isolation of six undescribed (crotignoids L ~ Q) together with two known (12-deoxyphorbol-13-hexadecanoate and 12-deoxyphorbol-13-myristate) tigliane diterpenoids. Their structures, especially the absolute configurations, were determined from extensive spectroscopic studies, including 2D NMR spectra, CD data analysis and electronic circular dichroism (ECD) calculations. All isolates were tested for anti-HIV activity against HL4-3 virus in MT4 cells. Except for crotignoid Q, the remaining seven tigliane diterpenoids exhibited potent anti-HIV activity with IC50 values ranging from 0.0023 to 4.03 µM.