Deciphering the Underlying Mechanisms of Formula Le-Cao-Shi Against Liver Injuries by Integrating Network Pharmacology, Metabonomics, and Experimental Validation.

Le-Cao-Shi (LCS) has long been used as a folk traditional Chinese medicine formula against liver injuries, whereas its pharmacological mechanisms remain elusive. Our study aims to investigate the underlying mechanism of LCS in treating liver injuries via integrated network pharmacology, metabonomics, and experimental validation. By network pharmacology, 57 compounds were screened as candidate compounds based on ADME parameters from the LCS compound bank (213 compounds collected from the literature of three single herbs). According to online compound-target databases, the aforementioned candidate compounds were predicted to target 87 potential targets related to liver injuries. More than 15 pathways connected with these potential targets were considered vital pathways in collectively modulating liver injuries, which were found to be relevant to cancer, xenobiotic metabolism by cytochrome P450 enzymes, bile secretion, inflammation, and antioxidation. Metabonomics analysis by using the supernatant of the rat liver homogenate with UPLC-Q-TOF/MS demonstrated that 18 potential biomarkers could be regulated by LCS, which was closely related to linoleic acid metabolism, glutathione metabolism, cysteine and methionine metabolism, and glycerophospholipid metabolism pathways. Linoleic acid metabolism and glutathione metabolism pathways were two key common pathways in both network pharmacology and metabonomics analysis. In ELISA experiments with the CCl4-induced rat liver injury model, LCS was found to significantly reduce the levels of inflammatory parameters, decrease liver malondialdehyde (MDA) levels, and enhance the activities of hepatic antioxidant enzymes, which validated that LCS could inhibit liver injuries through anti-inflammatory property and by suppressing lipid peroxidation and improving the antioxidant defense system. Our work could provide new insights into the underlying pharmacological mechanisms of LCS against liver injuries, which is beneficial for its further investigation and modernization.

Annular oxygenation and rearrangement products of cryptotanshinone by biotransformation with marine-derived fungi Cochliobolus lunatus and Aspergillus terreus.

Structural modification of natural products by biotransformation with fungi is an attractive tool to obtain novel bioactive derivatives. In the present study, cryptotanshinone (1), a quinoid abietane diterpene from traditional Chinese medicine Salvia miltiorrhiza (Danshen), was transformed by two marine-derived fungi. By using Cochliobolus lunatus TA26-46, one new oxygenated and rearranged product (2), containing a 5,6-dihydropyrano[4,3-b]chromene moiety, together with one known metabolite (10), were obtained from the converted broth of cryptotanshinone (1) with the isolated yields of 1.0% and 2.1%, respectively. While, under the action of Aspergillus terreus RA2905, seven new transformation products (3-9) as well as 10 with the fragments of 2-methylpropan-1-ol and oxygenated p-benzoquinone were produced and obtained with the isolated yields of 0.1%-1.3%. The structures of the new compounds were elucidated by comprehensive spectroscopic analysis including High Resolution Electrospray Ionization Mass Spectroscopy (HRESIMS), Nuclear Magnetic Resonance (NMR) and Electronic Circular Dichroism (ECD). The metabolic pathways of cryptotanshinone by these two fungi were presumed to be the opening and rearrangement of furan ring, and/or oxygenation of cyclohexane ring. Cryptotanshinone (1) and its metabolites displayed anti-inflammatory activities against NO production in LPS-stimulated BV-2 cells and antibacterial activities towards methicillin-resistant Staphylococcus aureus. These findings revealed the potential of marine fungi to transform the structures of natural products by biotransformation.

The oxygenated products of cryptotanshinone by biotransformation with Cunninghamella elegans exerting anti-neuroinflammatory effects by inhibiting TLR 4-mediated MAPK signaling pathway.

Cryptotanshinone (1), a major bioactive constituent in the traditional Chinese medicinal herb Dan-Shen Salvia miltiorrhiza Bunge, has been reported to possess remarkable pharmacological activities. To improve its bioactivities and physicochemical properties, in the present study, cryptotanshinone (1) was biotransformed with the fungus Cunninghamella elegans AS3.2028. Three oxygenated products (2-4) at C-3 of cryptotanshinone (1) were obtained, among them 2 was a new compound. Their structures were elucidated by comprehensive spectroscopic analysis including HRESIMS, NMR and ECD data. All of the biotransformation products (2-4) were found to inhibit significantly lipopolysaccharide-induced nitric oxide production in BV2 microglia cells with the IC50 values of 0.16-1.16 µM, approximately 2-20 folds stronger than the substrate (1). These biotransformation products also displayed remarkably improved inhibitory effects on the production of inflammatory cytokines (IL-1ß, IL-6, TNF-α, COX-2 and iNOS) in BV-2 cells via targeting TLR4 compared to substrate (1). The underlying mechanism of 2 was elucidated by comparative transcriptome analysis, which suggested that it reduced neuroinflammatory mainly through mitogen-activated protein kinase (MAPK) signaling pathway. Western blotting results revealed that 2 downregulated LPS-induced phosphorylation of JNK, ERK, and p38 in MAPK signaling pathway. These findings provide a basal material for the discovery of candidates in treating Alzheimer's disease.

Hepatoprotective effects of total phenylethanoid glycosides from Acanthus ilicifolius L. against carbon tetrachloride-induced hepatotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Acanthus ilicifolius L. has been used as a folk medicine in the treatment of acute and chronic hepatitis in China for a long time. Phenylethanoid glycosides are one of main components in A. ilicifolius L. AIM OF THE STUDY: The aim of present study was to assess the hepatoprotective activities of total phenylethanoid glycosides from A. ilicifolius L. (APhGs) against carbon tetrachloride (CCl4)-induced liver injury in vivo and in vitro. MATERIALS AND METHOD: The APhGs was separated by resin column chromatography. The purity of total phenylethanoid glycosides was determined by UV-Vis spectrophotometry using acteoside as a standard. The hepatoprotective activities of APhGs against CCl4-induced liver injury were performed on experimental mice and L-02 hepatocytes. Moreover, the antioxidant activities of APhGs were tested in vitro. RESULTS: The results showed that pre-administration of APhGs to mice decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum, and improved superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) level in serum and liver tissue induced by CCl4. Specifically, the SOD activities of APhGs-H and APhGs-M treatment groups were stronger than that of silymarin treatment group. The protective activities of APhGs were confirmed by histopathological results. Moreover, immunohistochemical analysis showed that APhGs could remarkably down-regulate the protein expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). In vitro experiment, APhGs was observed to increase L-02 hepatocyte viability against CCl4-induced hepatotoxicity. In addition, antioxidation assays revealed that APhGs showed 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and ferric reducing ability. CONCLUSION: Overall, total phenylethanoid glycosides from A. ilicifolius L. displayed promising hepatoprotective effects. These results offer a support for the medicine uses of A. ilicifolius L.

Simultaneous Quantification of Four Phenylethanoid Glycosides in Rat Plasma by UPLC-MS/MS and Its Application to a Pharmacokinetic Study of Acanthus Ilicifolius Herb.

Acanthus ilicifolius herb (AIH), the dry plant of Acanthus ilicifolius L., has long been used as a folk medicine for treating acute and chronic hepatitis. Phenylethanoid glycosides (PhGs) are one family of the main components in AIH with hepatoprotective, antioxidant, and anti-inflammatory activities. In this study, the pharmacokinetics of AIH was investigated preliminarily by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). A simultaneously quantitative determination method for four PhGs (acteoside, isoacteoside, martynoside, and crenatoside) in rat plasma was first established by UPLC-MS/MS. These four PhGs were separated with an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) by gradient elution (mobile phase: MeCN and 0.1% formic acid in water, 0.4 mL/min). The mass spectrometry detection was performed using negative electrospray ionization (ESI-) in multiple reaction monitoring (MRM) mode. By the established method, the preliminary pharmacokinetics of AIH was elucidated using the kinetic parameters of the four PhGs in rat plasma after intragastric administration of AIH ethanol extract. All four PhGs showed double peaks on concentration-time curves, approximately at 0.5 h and 6 h, respectively. Their elimination half-lives (t1/2) were different, ranging from 3.42 h to 8.99 h, although they shared similar molecular structures. This work may provide a basis for the elucidation of the pharmacokinetic characteristics of bioactive components from AIH.

Hepatoprotective effects of a traditional Chinese medicine formula against carbon tetrachloride-induced hepatotoxicity in vivo and in vitro.

Le-Cao-Shi (LCS), a formula of Traditional Chinese Medicine (TCM), has been used as a folk medicine for protection and treatment of liver injury. However, scientific evidences on its hepatoprotective effects have not been investigated. In this study, hepatoprotective activities of LCS water extracts (LCS-W) and ethanol extracts (LCS-E) against carbon tetrachloride (CCl4)-induced liver damage were investigated in vivo and in vitro. In vivo experiments, pretreatment of LCS-W and LCS-E to rats significantly declined the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and markedly increased the activity of superoxide dismutase (SOD) and ameliorated the level of malondialdehyde (MDA) induced by CCl4 treatment. Especially, LCS-WM group significantly prevented the elevation of lipid peroxidation level induced by CCl4, with the MDA level closed to that of normal group. Histopathological examinations further confirmed that LCS-W and LCS-E could protect the liver cells from CCl4-induced damage. In addition, immunohistochemically analysis revealed that LCS-W could significantly down-regulated the hepatic protein expression of necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Correspondingly, LCS-W and LCS-E were observed to promote cell viability and decline the levels of ALT, AST, and lactate dehydrogenase (LDH) in vitro. It could be concluded that LCS can exert a protective effect against CCl4-induced hepatotoxicity, which might be a potential therapeutic prescription for preventing or treating liver injury. Notably, LCS-W displayed better hepatoprotective activity against CCl4-induced injury than that of LCS-E, suggesting that LCS extracted by water decoction has good development prospects. Our results contribute towards the validation of the traditional use of LCS in the treatment of liver disorders.

Effects of traditional Chinese medicine formula Le-Cao-Shi on hepatitis B: In vivo and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Formula Le-Cao-Shi (LCS) is a traditional Chinese medicine (TCM), which has long been used as a folk remedy against hepatitis B in China. The present study was conducted to evaluate the anti-hepatitis B effects of aqueous extract of LCS in vivo and in vitro. MATERIALS AND METHOD: we investigated the anti-HBV effects of LCS in vivo and in vitro with duck hepatitis B model and HepG2.2.15 cell line model, respectively. The serologic and cellular biomarkers and the histopathological changes were examined. RESULTS: By a duck hepatitis B model, the extract of LCS was found to restrain the expressions of duck hepatitis B surface antigen (DHBsAg), hepatitis B e antigen (DHBeAg), and HBV-DNA (DHBV-DNA). Moreover, LCS could decrease the levels of aspartate and alanine aminotransferases (AST and ALT) and ameliorate duck liver histological lesions. Correspondingly, in a HepG2.2.15 cellular model, LCS could also significantly inhibit the secretions of HBsAg and HBeAg. CONCLUSION: LCS exerted potent anti-hepatitis effects against the infection of HBV. The above results demonstrated the first-hand experimental evidences for the anti-hepatitis B efficiency of LCS. Our study provides a basis for further exploration and development of this promising compound prescription to treat hepatitis B disease.

Xylapeptide A, an Antibacterial Cyclopentapeptide with an Uncommon L-Pipecolinic Acid Moiety from the Associated Fungus Xylaria sp. (GDG-102).

Two new cyclopentapeptides, xylapeptide A (1) with an uncommon L-pipecolinic acid moiety, and xylapeptide B (2) having a common L-proline residue were identified from an associated fungus Xylaria sp. isolated from the Chinese medicinal plant Sophora tonkinensis. Their planar structures were elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra. The absolute configurations were determined by Marfey's method and single-crystal X-ray diffraction (Cu Kα) analysis. Xylapeptide A (1) is the first example of cyclopentapeptide with L-Pip of terrestrial origin and showed strong antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 µg/mL.

Chinese Marine Materia Medica Resources: Status and Potential.

Chinese marine materia medica (CMMM) is a vital part of traditional Chinese medicine (TCM). Compared with terrestrial TCM, CMMM, derived from specific marine habitats, possesses peculiar chemical components with unique structures reflecting as potent pharmacological activities, distinct drug properties and functions. Nowadays, CMMM appears to be especially effective in treating such difficult diseases as cancers, diabetes, cardio-cerebrovascular diseases, immunodeficiency diseases and senile dementia, and therefore has become an important medicinal resource for the research and development of new drugs. In recent years, such development has attracted wide attention in the field of medicine. In this study, the CMMM resources in China were systematically investigated and evaluated. It was found that the historic experiences of Chinese people using CMMM have continuously accumulated over a period of more than 3600 years, and that the achievements of the research on modern CMMM are especially outstanding. By June 2015, 725 kinds of CMMMs from Chinese coastal sea areas have been identified and recorded, covering 1552 organisms and minerals. More than 3100 traditional prescriptions containing CMMMs have been imparted and inherited. However, the number of CMMMs is less than the 8188 terrestrial TCMs, from more than 12,100 medicinal terrestrial plants, animals and minerals. In the future, the research and development of CMMM should focus on the channel entries (TCM drug properties), compatibility, effective ingredients, acting mechanisms, drug metabolism and quality standard. This study reveals the high potential of CMMM development.

Chemical constituents of the Gorgonian Dichotella fragilis (Ridleg) from the South China Sea.

Two new steroidal glycosides, fragilioside A (1) and fragilioside B (2), along with five known compounds (3-7) were isolated from the gorgonian Dichotella fragilis (Ridleg) collected from the South China Sea. The structures of the new compounds (1 and 2) were elucidated by comprehensive analysis of spectral data, especially 2D NMR. The brine shrimp lethality and antifouling activity of the isolated compounds were also evaluated.

Two new derivatives of griseofulvin from the mangrove endophytic fungus Nigrospora sp. (strain No. 1403) from Kandelia candel (L.) Druce.

Two new compounds, methyl 3-chloro-6-hydroxy-2-(4-hydroxy-2-methoxy-6-methylphenoxy)-4-methoxybenzoate (1) and (2 S,5' R,E)-7-hydroxy-4,6-dimethoxy-2-(1-methoxy-3-oxo-5-methylhex-1-enyl)-benzofuran-3(2H)-one (2), together with four known compounds, griseofulvin (3), dechlorogriseofulvin (4), bostrycin (5), and deoxybostrycin (6), were isolated from the marine endophytic fungus NIGROSPORA sp. (No. 1403) collected from the South China Sea. The structures were elucidated by spectroscopic methods, 1D, 2D NMR, and HREIMS. Compounds 5 and 6 showed moderate antitumor and moderate antimicrobial activity.

Chemical constituents of the soft coral Sarcophyton infundibuliforme from the South China Sea.

One new cembrane diterpenoid, named sarcolactone A (1), along with five known compounds (2-6) were isolated from the soft coral Sarcophyton infundibuliforme collected from the South China Sea. The structure of sarcolactone A (1) was elucidated by comprehensive analysis of spectral data, especially 2D-NMR spectra (1H-1H COSY, HMBC and NOESY). All the compounds were evaluated for their brine shrimp lethality and antifouling activities.