Identification of Flavonoids from Scutellaria barbata D. Don as Inhibitors of HIV-1 and Cathepsin L Proteases and Their Structure-Activity Relationships.

Scutellaria barbata D. Don (SB, Chinese: Ban Zhi Lian), a well-known medicinal plant used in traditional Chinese medicine, is rich in flavonoids. It possesses antitumor, anti-inflammatory, and antiviral activities. In this study, we evaluated the inhibitory activities of SB extracts and its active components against HIV-1 protease (HIV-1 PR) and SARS-CoV2 viral cathepsin L protease (Cat L PR). UPLC/HRMS was used to identify and quantify the major active flavonoids in different SB extracts, and fluorescence resonance energy transfer (FRET) assays were used to determine HIV-1 PR and Cat L PR inhibitions and identify structure-activity relationships. Molecular docking was also performed, to explore the diversification in bonding patterns of the active flavonoids upon binding to the two PRs. Three SB extracts (SBW, SB30, and SB60) and nine flavonoids inhibited HIV-1 PR with an IC50 range from 0.006 to 0.83 mg/mL. Six of the flavonoids showed 10~37.6% inhibition of Cat L PR at a concentration of 0.1 mg/mL. The results showed that the introduction of the 4'-hydroxyl and 6-hydroxyl/methoxy groups was essential in the 5,6,7-trihydroxyl and 5,7,4'-trihydroxyl flavones, respectively, to enhance their dual anti-PR activities. Hence, the 5,6,7,4'-tetrahydroxyl flavone scutellarein (HIV-1 PR, IC50 = 0.068 mg/mL; Cat L PR, IC50 = 0.43 mg/mL) may serve as a lead compound to develop more effective dual protease inhibitors. The 5,7,3',4'-tetrahydroxyl flavone luteolin also showed a potent and selective inhibition of HIV-1 PR (IC50 = 0.039 mg/mL).

Metabolic Insight Into the Neuroprotective Effect of Tao-He-Cheng-Qi (THCQ) Decoction on ICH Rats Using Untargeted Metabolomics.

Tao-He-Cheng-Qi decoction (THCQ) is an effective traditional Chinese medicine used to treat intracerebral hemorrhage (ICH). This study was performed to investigate the possible neuroprotective effect of THCQ decoction on secondary brain damage in rats with intracerebral hemorrhage and to elucidate the potential mechanism based on a metabolomics approach. Sprague-Dawley (SD) rats were randomly divided into five groups: the sham group, collagenase-induced ICH model group, THCQ low-dose (THCQ-L)-treated group, THCQ moderate-dose (THCQ-M)-treated group and THCQ high-dose (THCQ-H)-treated group. Following 3 days of treatment, behavioral changes and histopathological lesions in the brain were estimated. Untargeted metabolomics analysis with multivariate statistics was performed by using ultrahigh-performance liquid chromatography-mass spectrometry (UPLC-Q-Exactive Orbitrap MS). THCQ treatment at two dosages (5.64 and 11.27 g/kg·d) remarkably improved behavior (p < 0.05), brain water content (BMC) and hemorheology (p < 0.05) and improved brain nerve tissue pathology and inflammatory infiltration in ICH rats. Moreover, a metabolomic analysis demonstrated that the serum metabolic profiles of ICH patients were significantly different between the sham group and the ICH-induced model group. Twenty-seven biomarkers were identified that potentially predict the clinical benefits of THCQ decoction. Of these, 4 biomarkers were found to be THCQ-H group-specific, while others were shared between two clusters. These metabolites are mainly involved in amino acid metabolism and glutamate-mediated cell excitotoxicity, lipid metabolism-mediated oxidative stress, and mitochondrial dysfunction caused by energy metabolism disorders. In addition, a correlation analysis showed that the behavioral scores, brain water content and hemorheology were correlated with levels of serum metabolites derived from amino acid and lipid metabolism. In conclusion, the results indicate that THCQ decoction significantly attenuates ICH-induced secondary brain injury, which could be mediated by improving metabolic disorders in cerebral hemorrhage rats.

Asperochratides A-J, Ten new polyketides from the deep-sea-derived Aspergillus ochraceus.

Ten new C9 polyketides (asperochratides A-J, 1-10) and 14 known miscellaneous compounds (11-24) were isolated from the deep-sea-derived fungus Aspergillus ochraceus. Structures of the new compounds were elucidated by extensive spectroscopic analyses, modified Mosher's method, Mo2(OAc)4 induced circular dichroism (ICD) experiments, and ECD calculations. Structurally, compounds 1-11 and 16-18 share the same polyketide origin of the skeleton and belong to aspyrone co-metabolites. All isolates were tested for cytotoxic, anti-food allergic, anti-H1N1 virus, anti-microbe, and anti-inflammatory activities in vitro. Results showed that compounds 5-8 and 13-17 exerted significant cytotoxic effects on BV-2 cell line, and compound 16 showed the potential of anti-inflammatory activities.

An ethanol extract of the rhizome of Atractylodes chinensis exerts anti-gastritis activities and inhibits Akt/NF-κB signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Atractylodes chinensis (DC.) kodiz (Compositae) has traditionally been used to treat inflammatory disorders such as arthritis and stomach ache, but scanted report has been issued on its anti-inflammatory mechanisms. AIM OF THE STUDY: Here, we investigated the anti-gastritis activities and explored the mechanism of action of an ethanolic extract of the herb (Ac-EE). MATERIALS AND METHODS: Ac-EE was prepared with 95% ethanol. To determine its in vivo effects, we employed an HCl/EtOH-induced gastritis rat model. We used a lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage model for in vitro assays. Griess and MTT assays were used to measure nitric oxide (NO) production and cell viability, respectively. We used real-time PCR to determine mRNA levels. To measure prostaglandin E2 (PGE2) production we used a PGE2 EIA kit. To estimate protein levels and enzyme activities, we employed immunoblotting. Luciferase assays were used to examine nuclear transcription factor (NF)-κB activities. RESULTS: Intragastric administration of Ac-EE (30 mg/kg) ameliorated HCl/EtOH-induced stomach tissue damages in SD rats. Ac-EE inhibited the levels of NO and PGE2, down regulated mRNA and protein levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. Ac-EE suppressed the nuclear level of NF-κB (p50), and inhibited NF-κB luciferase activity. The Phosphorylation of Akt and IκBα was also inhibited by Ac-EE both in vivo and in vitro. CONCLUSION: Ac-EE treatment exerts an anti-gastritis effect in rats. Inhibition of the Akt/IκBα/NF-κB signaling pathway is associated with this effect, providing a pharmacological basis for the clinical application of the rhizome of A. chinensis in the treatment of inflammatory diseases.

In vitro assays suggest Shenqi Fuzheng Injection has the potential to alter melanoma immune microenvironment.

ETHNOPHARMACOLOGICAL RELEVANCE: A modern agent Shenqi Fuzheng Injection (SFI), prepared from Codonopsis Radix and Astragali Radix, has been commonly used as a supplementary therapy for cancers including melanoma. This agent was derived from a formula documented in the "National Collection of Chinese Medicine Prescriptions". The formula has long been used as a remedy for Qi deficiency that is closely associated with cancer-related fatigue and poor quality of life. However, the antimelanoma mechanisms of SFI remain unclear. Here we tested if SFI exerted antimelanoma effects by reprograming the tumour immune microenvironment using in vitro assays. MATERIALS AND METHODS: The cytotoxic activities of Jurkat T cells when co-cultured with A375 cells were determined in the presence or absence of SFI. The migratory activities of Jurkat T cells were examined in the transwell assay system. The mRNA expression and production of cytokines (IL-10, TGF ß and VEGF) in A375 cells in the presence or absence of SFI were determined by real-time PCR and ELISA, respectively. RESULTS: When A375 cells were co-cultured with Jurkat T cells in the presence of SFI (220µg/mL), a potent cytotoxicity effect against A375 cells was observed. Supernatants from A375 cells that were treated with SFI (110 and 220µg/mL) significantly increased the migratory capacity of Jurkat T cells in transwell assays. SFI also markedly reduced the mRNA expression levels and the release of immunosuppressive cytokines IL-10, TGF-ß and VEGF in A375 cells in a concentration-dependent manner. CONCLUSIONS: SFI enhanced the cytotoxic and migratory activities of Jurkat T cells towards A375 melanoma cells. The effects were associated with SFI's suppression on immunosuppressive cytokines for their release from and gene expressions in A375 melanoma cells. These in vitro findings suggested that SFI might reprogramme the immunosuppressive melanoma microenvironment in vivo to enhance the cytotoxicity of tumour-infiltrating immune cells. This study provides a pharmacological basis for the adjunctive use of SFI in melanoma treatment.

[Rapid Identification of Chemical Components in Polygonum multiflorum Formula Granules by UPLC/Q-TOF MS].

OBJECTIVE: To establish a simple and reliable method for rapid separation and identification of chemical components in Polygonum multiflorum Formula Granules. METHODS: An ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method( UPLC/Q-TOF MS) was used. The separation was performed on an Agilent Eclipse Plus C18 RRHD(100 mm x 2.1 mm, 1.8 µm) column with a mobile phase of water and acetonitrile in a gradient elution mode. The flow rate was 0.4 mL/min and the column temperature was maintained at 25 degrees C. TOF MS was applied for qualitative analysis under positive ion mode. RESULTS: Five compounds were identified by the time of flight mass spectrometry and literature data. CONCLUSION: This method is accurate, rapid and sensitive, it can provide reference for the quality control of Polygonum multiflorum Formula Granules.

Chemical constituents of Abies delavayi.

Systematic phytochemical investigations on Abies delavayi afforded 110 compounds, including 49 terpenoids, 13 lignans, 20 flavonoids, three coumarins, and 25 other chemical constituents. By detailed analysis of one- and two-dimensional NMR spectroscopic and high-resolution mass spectrometric data, 10 previously unreported compounds were identified: they comprised three sesquiterpenoids, two diterpenoids, one triterpenoid, one monoterpenoid, one flavonoid, and two phenols. These 10 compounds and some previously known ones were subjected to two cytotoxic bioassays against three human tumor cell lines and NO production inhibition on RAW264.7 macrophages, respectively. (25R)-24,25-Dihydroabieslactone had the strongest cytotoxic activity against Colo-205 cells with an IC50 value of 19.0±3.7µg/mL. (+)-T-cadinol, 8,11,13-abietatrien-15-ol-18-yl acetate, 18-acetoxy-13-epi-manool, imperatorin, bergapten, and 5,7-O-dimethyl poriol exhibited weak inhibitory activity against LPS-induced NO production in RAW264.7 macrophages with IC50 values of approximately 50µg/mL.

UHPLC UHD Q-TOF MS/MS analysis of the impact of sulfur fumigation on the chemical profile of Codonopsis Radix (Dangshen).

Over recent decades sulfur fumigation has been becoming abused in processing some freshly harvested Chinese medicinal herbs, although it is questioned whether sulfur fumigation can result in changes in efficacy and safety of the herbs. One of the herbs commonly processed by sulfur fumigation is Codonopsis Radix (Dangshen). A report showed that lobetyolin content in sulfur-fumigated Dangshen was lower than in air-dried Dangshen. Whereas there is no investigation designed to compare the chemical profiles of the sulfur-fumigated Dangshen and the air-dried Dangshen. In the present study, a rapid and versatile ultra-high-performance liquid chromatography coupled with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for comprehensive analysis of the chemical profiles of sulfur-fumigated and air-dried Dangshen samples. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) demonstrated that there were significant chemical differences between sulfur-fumigated and air-dried Dangshen samples. Among the changed components, 57 compounds were identified, in which 15 sulfur-containing compounds were detected only in sulfur-fumigated samples. The established methods were successfully applied to discriminate sulfur-fumigated Dangshen among commercial samples. Whether the chemical changes caused by sulfur fumigation affect the clinical efficacy and safety of Dangshen needs to be further investigated.

Tracing antibacterial compounds from Acalypha australis Linn. by spectrum-effect relationships and semi-preparative HPLC.

Several Acalypha australis Linn. species are used in traditional medicine in Southeast Asia. In this work, the ultra-performance liquid chromatography/mass spectrometry fingerprints and the antibacterial activities of A. australis Linn. were investigated. An in-depth discussion on the reliability of identifying and obtaining potentially active compounds by spectrum-effect relationship and semi-preparative high performance liquid chromatography was conducted. The result shows that gallic acid and a compound with molecular weight of 634.1 in the fingerprints were the main antibacterial compounds. Compared to the crude extract of A. australis Linn., both compounds increase the antibacterial efficacy 10 to 20 times. Compounds with molecular weights of 154.0, 292.0, and 485.1 in the fingerprints were the auxiliary antibacterial compounds. Through the entire isolation procedure, we obtained these antibacterial compounds with purities of 92.53, 87.98, 90.73, 89.36, and 88.14%, respectively. This work provides a general model of the combination of ultra-performance liquid chromatography/mass spectrometry fingerprinting and antibacterial test to study the spectrum-effect relationships of A. australis Linn. This model can be used to discover further the active compounds of this herb.

Mono- and sesquiterpenoids, flavonoids, lignans, and other miscellaneous compounds of Abies georgei.

A systematic phytochemical investigation of the aerial parts of Abies georgei yielded nine new and 72 known compounds, including four monoterpenes, four sesquiterpenes, 25 flavonones, 14 lignans, and 34 other chemical constituents. The new compounds included two monoterpenes (1 and 2), two sesquiterpenes (3 and 4), three flavonones (5, 6, and 7), and two other components (8 and 9). Their chemical structures were established on the basis of various spectroscopic data. All the isolates were tested for antitumor and anti-inflammatory activities. The new compound 9,4'-dihydroxy-5,7-dimethoxy-8-methylchalcone (7) demonstrated a moderate antiproliferative effect on QGY-7703 tumor cells (IC (50)  = 17.6 µg/mL). The known compound isoferulaldehyde (67) exhibited the strongest inhibitory activity against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages (IC (50) = 19.0 µg/mL). Abies georgei may be a significant source of beneficial pharmaceutical compounds.

Abiesatrines A-J: anti-inflammatory and antitumor triterpenoids from Abies georgei Orr.

A novel spiro-lanostane (abiesatrine A, 1) was isolated from the aerial parts of Abies georgei together with 9 new (abiesatrines B-J, 2-10) and 10 known triterpenes (11-20). The new structures were established by the extensive analysis of their spectroscopic data. The configuration of 1, featuring a unique spirolactone formed by C-13 and C-23 via oxygen-bridge, was confirmed by X-ray crystallography, and its biopathway was tentatively proposed. Among these isolates, compound 16 showed the strongest inhibitory activity against LPS-induced NO production in RAW264.7 macrophages (IC(50) = 8.9 microg mL(-1)). While compounds 1 and 20 exhibited potent anti-proliferative effects on QGY-7703 cells with IC(50) values of 9.3 and 7.6 microg mL(-1), respectively. Preliminary structure-activity relationship (SAR) investigations defined structural feature of the 24Z-olefinic bond key to the lanostane and cycloartane pharmacophore.

Chemical constituents of Dracocephalum forrestii.

A systematic phytochemical examination of the whole plant Dracocephalum forrestii led to the isolation of 4 new and 65 known chemical constituents. By detailed 1D and 2D NMR spectroscopic analyses, the new compounds were identified as 4-hydroxy-3-methoxyphenylethanol 8- O-[(6- O-syringoyl)- beta- D-glucopyranoside] (1), 3,4,5-trimethoxyphenylethanol beta- D-glucopyranoside ( 2), 4- O-[ beta- D-glucopyranosyl-(1 --> 3)- alpha- L-rhamnopyranosyl]phenylethylcinnamamide (3), and 9''- O- N-butyl lithospermate (4). The new isolates were evaluated for inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages. Compound 2 revealed a moderate effect without any cytotoxicity under the assayed concentrations.

Two new spirobiflavonoids from Abies chensiensis with moderate NO production inhibitory activity.

Phytochemical investigation of the aerial parts of Abies chensiensis afforded two new (compounds 2 and 3) and 27 known compounds, including the related compound larixinol ( 1). The structures of spirobiflavonoids 1- 3 were established using 1D and 2D NMR spectroscopic techniques. In addition, the structure of larixinol ( 1) was confirmed by X-ray crystallographic analysis. Compounds 1- 3 were evaluated for inhibitory activities against LPS-induced NO production in macrophages. Larixinol ( 1) showed moderate effects, with an IC(50) value of 60.0 microg/mL. In addition, it did not show any cytotoxicity on RAW 264.7 macrophages at 100 microg/mL.

Terpenoid constituents of Abies chensiensis with potential anti-inflammatory activity.

Six new triterpenes (neoabieslactones A-F, 1-6) and 17 known compounds were isolated from the aerial parts of Abies chensiensis. The structures of the new triterpenes were proposed by 1D and 2D NMR spectroscopy. Compound 1 was confirmed structurally by X-ray crystallography. In a bioassay against LPS-induced NO production in RAW264.7 macrophages, three compounds, neoabieslactone E (5), (12R,13R)-8,12-epoxy-14-labden-13-ol (7), and manool (8), exhibited IC(50) values of 9.1, 1.9, and 9.6 microg/mL, respectively.

Anti-inflammatory and anti-tumour effects of Abies georgei extracts.

Chloroform (AGC), ethyl acetate (AGE) and n-butanol (AGB) extracts of Abies georgei were investigated for anti-tumour and anti-inflammatory activities in-vitro and in-vivo. AGC exhibited potent antiproliferative effects against A549, LOVO, QGY-7703 and 6T-CEM tumour cells, with EC50 values of 77.5, 7.8, 11.1 and 32.8 microg mL(-1), respectively. It also inhibited the growth of S180 sarcoma implanted into mice; tumour growth inhibition ratios were 46.7, 53.1 and 31.0% of controls at doses of 100, 200 and 400 mgkg(-1), respectively. AGE showed significant anti-inflammatory activities in the carrageenin-induced acute pedal oedema model in rats and dimethylbenzene-induced ear oedema in mice at doses of 140 mgkg(-1) and 200 mgkg(-1) p.o., respectively. Primary mechanism studies in-vitro showed that AGE inhibited platelet aggregation induced in rabbits by arachidonic acid (AA), with an IC50 of 14.4 microg mL(-1). Its effect on AA metabolism was also studied in mouse peritoneal macrophages stimulated by A23187. Formation of prostaglandin E(2), leukotriene B(4) and 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) was significantly inhibited in a concentration-dependent manner. In addition, AGE inhibited lipopolysaccharide-induced nitric oxide production in RAW246.7 macrophages and nuclear factor kappaB activation induced in 293 cells by tumour necrosis factor alpha.