Uncovering the pharmacological mechanisms of Zizhu ointment against diabetic ulcer by integrating network analysis and experimental evaluation in vivo and in vitro.

Diabetic ulcer (DU) has been recognized as one of the most prevalent and serious complications of diabetes. However, the clinical efficacy of standard treatments for DU remains poor. Traditional Chinese medicine (TCM) shows a positive therapeutic effect on DU. Specifically, Zizhu ointment (ZZO) has been widely used to treat DU in long-term clinical practice, but the exact mechanism by which it promotes DU wound healing remains unknown. In this study, network analysis and high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) were conducted to identify the active compounds of ZZO. We detected isovalerylshikonin (ISO), mandenol, daidzein, kaempferol, and formononetin in both network analysis and UPLC-HRMS. Moreover, ZZO could ameliorate DU by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and inflammation signaling pathways, according to the results of KEGG analysis. We established a DU mouse model with a high-fat diet and streptozotocin injection in vivo to evaluate the network analysis result. The experimental results showed that ZZO could inhibit inflammation, remodel fibrous tissue, and promote angiogenesis in the DU area, facilitating wound healing in DU mice. Moreover, the PI3K/AKT signaling pathway was indeed activated by ZZO treatment, promoting macrophage M2 polarization. In addition, we used molecular docking technology to evaluate the binding sites between ZZO and the PI3K/AKT pathway. The results showed that ISO has a good binding interaction with AKT. Moreover, ISO promoted M2 polarization in macrophages in a dose-dependent manner in vitro. Our study found that ZZO could promote DU wound healing by inhibiting inflammation, which was achieved by macrophage M2 polarization through activating the PI3K/AKT pathway. Further studies have demonstrated that ISO plays major role in the above process. These findings provide a theoretical basis for further preclinical evaluation and lay a foundation for nano-gel compound treatment with ZZO.

Optimization of ultrasonication-assisted extraction conditions using RSM-I-Optimal experimental design to recover vitamin D2 and K1 from selected green leafy vegetable samples

This study employed the response surface methodology to optimize the extraction conditions for recovering vitamins D2 and K1 from green leafy vegetables using ultrasonication-assisted extraction. The vitamin content was determined using an Accucore C18 column and a UPLC-Q-ToF/MS method. An RSM-I-Optimal design was used for designing the experiment to find the best combination of solvent level (mL), sonication time (min), sonication frequency (kHz), and temperature (°C). The experimental data from a 25-sample set were fitted to a second-order polynomial equation using multiple regression analysis. The extraction models had R2 values of 0.895 and 0.896, respectively, and the probability values (p < 0.0001) indicated that the regression model was highly significant. The optimal extraction conditions were: solvent level of 65 mL, sonication time of 45 min, sonication frequency of 70 kHz, and temperature of 45 °C. Under these conditions, the predicted recovery (%) values for vitamins D2 and K1 were 90.7% and 90.4%, respectively. This study has the potential to use the reported extraction method for routine quantification of vitamins D2 and K1 in the laboratory using UPLC-Q-ToF/MS.

Anti-inflammatory and antiviral activities of flavone C-glycosides of Lophatherum gracile for COVID-19.

Lophatherum gracile (L. gracile) has long been used as a functional food and herbal medicine. Previous studies have demonstrated that extracts of L. gracile attenuate inflammatory response and inhibit SARS-CoV-2 replication; however, the underlying active constituents have yet to be identified. This study investigated the bioactive components of L. gracile. Flavone C-glycosides of L. gracile were found to dominate both anti-inflammatory and antiviral effects. A simple chromatography-based method was developed to obtain flavone C-glycoside-enriched extract (FlavoLG) from L. gracile. FlavoLG and its major flavone C-glycoside isoorientin were shown to restrict respiratory bursts and the formation of neutrophil extracellular traps in activated human neutrophils. FlavoLG and isoorientin were also shown to inhibit SARS-CoV-2 pseudovirus infection by interfering with the binding of the SARS-CoV-2 spike on ACE2. These results provide scientific evidence indicating the efficacy of L. gracile as a potential supplement for treating neutrophil-associated COVID-19.

Metabolite profile of COVID-19 revealed by UPLC-MS/MS-based widely targeted metabolomics.

The metabolic characteristics of COVID-19 disease are still largely unknown. Here, 44 patients with COVID-19 (31 mild COVID-19 patients and 13 severe COVID-19 patients), 42 healthy controls (HC), and 42 patients with community-acquired pneumonia (CAP), were involved in the study to assess their serum metabolomic profiles. We used widely targeted metabolomics based on an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The differentially expressed metabolites in the plasma of mild and severe COVID-19 patients, CAP patients, and HC subjects were screened, and the main metabolic pathways involved were analyzed. Multiple mature machine learning algorithms confirmed that the metabolites performed excellently in discriminating COVID-19 groups from CAP and HC subjects, with an area under the curve (AUC) of 1. The specific dysregulation of AMP, dGMP, sn-glycero-3-phosphocholine, and carnitine was observed in the severe COVID-19 group. Moreover, random forest analysis suggested that these metabolites could discriminate between severe COVID-19 patients and mild COVID-19 patients, with an AUC of 0.921. This study may broaden our understanding of pathophysiological mechanisms of COVID-19 and may offer an experimental basis for developing novel treatment strategies against it.

Inhibitors of HIV-1 and Cathepsin L Proteases Identified from the Insect Gall of Hypericum kouytchense.

Hypericum kouytchense Lévl is a semi-evergreen plant of the Hypericaceae family. Its roots and seeds have been used in a number of traditional remedies for antipyretic, detoxification, anti-inflammatory, antimicrobial and antiviral functions. However, to date, no bioactivity compounds have been characterized from the insect gall of H. kouytchens. In this study, we evaluated the antiviral activities of different extracts from the insect gall of H. kouytchen against cathepsin L, HIV-1 and renin proteases and identified the active ingredients using UPLC-HRMS. Four different polar extracts (HW, H30, H60 and H85) of the H. kouytchense insect gall exhibited antiviral activities with IC50 values of 10.0, 4.0, 3.2 and 17.0 µg/mL against HIV-1 protease; 210.0, 34.0, 24.0 and 30.0 µg/mL against cathepsin L protease; and 180.0, 65.0, 44.0 and 39.0 µg/mL against human renin, respectively. Ten compounds were identified and quantified in the H. kouytchense insect gall extracts. Epicatechin, eriodictyol and naringenin chalcone were major ingredients in the extracts with contents ranging from 3.9 to 479.2 µg/mg. For HIV-1 protease, seven compounds showed more than 65% inhibition at a concentration of 1000.0 µg/mL, especially for hypericin and naringenin chalcone with IC50 values of 1.8 and 33.0 µg/mL, respectively. However, only hypericin was active against cathepsin L protease with an IC50 value of 17100.0 µg/mL, and its contents were from 0.99 to 11.65 µg/mg. Furthermore, we attempted to pinpoint the interactions between the active compounds and the proteases using molecular docking analysis. Our current results imply that the extracts and active ingredients could be further formulated and/or developed for potential prevention and treatment of HIV or SARS-CoV-2 infections.

Characterization of Planktochlorella nurekis Extracts and Virucidal Activity against a Coronavirus Model, the Murine Coronavirus 3.

Certain members of the Coronaviridae family have emerged as zoonotic agents and have recently caused severe respiratory diseases in humans and animals, such as SARS, MERS, and, more recently, COVID-19. Antivirals (drugs and antiseptics) capable of controlling viruses at the site of infection are scarce. Microalgae from the Chlorellaceae family are sources of bioactive compounds with antioxidant, antiviral, and antitumor activity. In the present study, we aimed to evaluate various extracts from Planktochlorella nurekis in vitro against murine coronavirus-3 (MHV-3), which is an essential human coronavirus surrogate for laboratory assays. Methanol, hexane, and dichloromethane extracts of P. nurekis were tested in cells infected with MHV-3, and characterized by UV-vis spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy, ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), and the application of chemometrics through principal component analysis (PCA). All the extracts were highly efficient against MHV-3 (more than a 6 Log unit reduction), regardless of the solvent used or the concentration of the extract, but the dichloromethane extract was the most effective. Chemical characterization by spectrophotometry and NMR, with the aid of statistical analysis, showed that polyphenols, carbohydrates, and isoprene derivatives, such as terpenes and carotenoids have a more significant impact on the virucidal potential. Compounds identified by UPLC-MS were mainly lipids and only found in the dichloromethane extract. These results open new biotechnological possibilities to explore the biomass of P. nurekis; it is a natural extract and shows low cytotoxicity and an excellent antiviral effect, with low production costs, highlighting a promising potential for development and implementation of therapies against coronaviruses, such as SARS-CoV-2.

Systematic identification of chemical components in Fufang Shuanghua oral liquid and screening of potential active components against SARS-CoV-2 protease.

Coronavirus disease (COVID-19) caused by SARS-COV-2 infection has been widely prevalent in many countries and has become a common challenge facing mankind. Traditional Chinese medicine (TCM) has played a prominent role in this pandemic, and especially TCM with the function of "heat-clearing and detoxifying" has shown an excellent role in anti-virus. Fufang Shuanghua oral liquid (FFSH) has been used to treat the corresponding symptoms of influenza such as fever, nasal congestion, runny nose, sore throat, and upper respiratory tract infections in clinic, which are typical symptoms of COVID-19. The content of chlorogenic acid, andrographolide and dehydrated andrographolide as the quality control components of FFSH is not less than 1.0 mg/mL, 60 µg/mL and 60 µg/mL respectively. In this study, UPLC-Q-TOF-MS/MS was employed to describe the chemical profile of FFSH. Virtual screening and fluorescence resonance energy transfer (FRET) were used to screen the effective components of FFSH acting on SARS-CoV-2 main protease (Mpro). As a result, 214 compounds in FFSH were identified or preliminarily characterized by UPLC-Q-TOF-MS/MS, and 61 active ingredients with potential inhibitory effects on Mpro were selected through receptor-based and ligand-based virtual screening. In particular, quercetin, forsythoside A, and linoleic acid showed a good inhibitory effect on Mpro in FRET evaluation with IC50 values of 26.15 µM, 22.26 µM and 47.09 µM respectively, and had a strong binding affinity with the receptor Mpro (6LU7) in molecular docking. CYS145 and HIS41 were the main amino acid residues affected by small molecules in the protein binding domain. In brief, we characterized, for the first time, 214 chemical components in FFSH, and three of them, including quercetin, forsythoside A and linoleic acid, were screened out to exert beneficial anti-COVID-19 effects through CYS145 and HIS41 sites, which may provide a new research strategy for TCM to develop new therapeutic drugs against COVID-19.

Unraveling the mystery of efficacy in Chinese medicine formula: New approaches and technologies for research on pharmacodynamic substances.

Traditional Chinese medicine (TCM) is the key to unlock treasures of Chinese civilization. TCM and its compound play a beneficial role in medical activities to cure diseases, especially in major public health events such as novel coronavirus epidemics across the globe. The chemical composition in Chinese medicine formula is complex and diverse, but their effective substances resemble "mystery boxes". Revealing their active ingredients and their mechanisms of action has become focal point and difficulty of research for herbalists. Although the existing research methods are numerous and constantly updated iteratively, there is remain a lack of prospective reviews. Hence, this paper provides a comprehensive account of existing new approaches and technologies based on previous studies with an in vitro to in vivo perspective. In addition, the bottlenecks of studies on Chinese medicine formula effective substances are also revealed. Especially, we look ahead to new perspectives, technologies and applications for its future development. This work reviews based on new perspectives to open horizons for the future research. Consequently, herbal compounding pharmaceutical substances study should carry on the essence of TCM while pursuing innovations in the field.

Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, a Potential Oral Drug Candidate for COVID-19 Treatment.

Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

Dual Inhibition of HIV-1 and Cathepsin L Proteases by Sarcandra glabra.

The COVID-19 pandemic continues to impose a huge threat on human health due to rapid viral mutations. Thus, it is imperative to develop more potent antivirals with both prophylactic and treatment functions. In this study, we screened for potential antiviral compounds from Sarcandra glabra (SG) against Cathepsin L and HIV-1 proteases. A FRET assay was applied to investigate the inhibitory effects and UPLC-HRMS was employed to identify and quantify the bioactive components. Furthermore, molecular docking was carried out to get a glimpse of the binding of active compounds to the proteases. Our results showed that the SG extracts (SGW, SG30, SG60, and SG85) inhibited HIV-1 protease with an IC50 of 0.003~0.07 mg/mL and Cathepsin L protease with an IC50 of 0.11~0.26 mg/mL. Fourteen compounds were identified along with eight quantified from the SG extracts. Chlorogenic acid, which presented in high content in the extracts (12.7~15.76 µg/mg), possessed the most potent inhibitory activity against HIV-1 protease (IC50 = 0.026 mg/mL) and Cathepsin L protease (inhibition: 40.8% at 0.01 mg/mL). Thus, SG extracts and the active ingredients could potentially be used to prevent/treat viral infections, including SARS-CoV-2, due to their dual-inhibition functions against viral proteases.

Rapid and ecofriendly UPLC quantification of Remdesivir, Favipiravir and Dexamethasone for accurate therapeutic drug monitoring in Covid-19 Patient's plasma.

Innovative therapeutic protocols to the rapidly spreading coronavirus disease (COVID19) epidemic is highly required all across the world. As demonstrated by clinical studies, Favipiravir (FVP) and Remdesivir (REM) are new antiviral medicines that are effective against COVID-19. REM is the first FDA approved antiviral medicine against COVID-19. In addition to antivirals, corticosteroids such as dexamethasone (DEX), and anticoagulants such as apixaban (PX) are used in multidrug combinations protocols. This work develops and validates simple and selective screening of the four medicines of COVID -19 therapeutic protocol. FVP, REM, DEX, and PX as internal standard in human plasma using UPLC method by C18 column and methanol, acetonitrile, and water acidified by orthophosphate (pH = 4) in a ratio of (15: 35: 50, by volume) as an eluate flowing at 0.3 mL/min. The eluent was detected at 240 nm. The method was linear over (0.1-10 µg/mL) for each of FVP, REM, and DEX. The validation of the UPLC method was assessed in accordance with FDA guidelines. The method can detect as low as down to 0.1 µg/mL for all. The recoveries of the drugs in spiked human plasma ranged from 97.67 to 102.98 percent. Method accuracy and precision were assessed and the drugs showed good stability. The method was proven to be green to the environment after greenness checking by greenness profile and Eco-Scale tool.

Antimicrobial and anticancer activities of Hainan dry noni fruit alcoholic extracts and their novel compounds identification using UPLC-Q-Exactive Obitrap-MS/MS.

Morinda citrifolia Linn (noni) is an important plant in the Pacific Asian region. The fruit has been used as a food source and has shown therapeutical benefits for health. Recently, it has become a source for bioactive compounds. In this study, we investigated the antimicrobial and anticancer activities of alcoholic extracts of Hainan dry noni fruit with machinery assistance and identified their novel compounds by UPLC-Q-Exactive Obitrap-MS/MS. By IE extractor aided method, the extraction of both NFE (Noni Fruit Ethanol) and NFM (Noni Fruit Methanol) solvent crude sample extracts were obtained with recovery yields of 98.48% and 71.65%, respectively. The antimicrobial effect of the crude extracts was subjected to disc diffusion test screening against two microbial strains bacterium SA (Staphylococcus aureus) and, fungal CA (Candida albicans). The MIC values of SA and CA were 35.34 and 47.80 mg/mL for NFE, 117.40 and 108.01 mg/mL for NFM, respectively. Further on, cell viability assay showed that IC50 values of extract NFE and NFM on human UMUC-3 bladder carcinogenic cells were 865.1 and 789.1 µg/mL with less effect to human SVHUC-1 normal cell line for 72hr incubation. Using UPLC-Q-exactive Orbitrap-MS/MS, ten compounds were identified in the noni extracts and confirmed from the HMDB and FooDB. Five known bioactive compounds had been used for treatments in anti-cancer, anti-obesity, and Covid-19 patients. The remaining five compounds were found novel in noni fruit. They were Cyanidin 3-(2 G-xylosylrutinoside), Inulobiose, Clausarinol, Pectachol, and 4,7-Megastigmadien-9-ol. The potential bioactivities of these novel compounds will be studied in the near future. These findings form a basis on screening natural medicinal plant extracts for beneficial use as a food and health source.

Comparative pharmacokinetics of 24 major bioactive components in normal and ARDS rats after oral administration of Xuanfei Baidu granules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu prescription, consisting of 13 Chinese medicines, was formulated by academicians Boli Zhang and Professor Qingquan Liu based on their experience in first-line clinical treatment of COVID-19. Xuanfei Baidu granules (XFBD granules) are a proprietary Chinese medicine preparation developed based on Xuanfei Baidu prescription. It is recommended for the treatment of patients with the common wet toxin and lung stagnation syndrome of COVID-19. However, the pharmacokinetic characteristics of its major bioactive components in rats under different physiological and pathological conditions are unclear. MATERIALS AND METHODS: A rapid and sensitive analytical method, ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS), was developed and applied to 24 major bioactive components in normal and ARDS rats after oral administration of XFBD granules. We studied the metabolic process of XFBD granules in vivo to compare the differences in pharmacokinetic parameters between normal and model metabolic processes. RESULTS: This method was successfully applied to the pharmacokinetic investigation of 24 major components of XFBD granules following oral administration in normal and ARDS rats. Eight components, including ephedrine and amygdalin, were more highly absorbed and had shorter Tmax values than the model group; the absorption of six components, such as rhein, decreased in ARDS rats, and there was no significant difference in the absorption of ten components, such as verbenalin and naringin, between the normal and ARDS rats. The results showed that the peak times of other analytes were very short, and 80% of these target constituents were eliminated in both normal and ARDS rats within 6 h except for liquiritigenin and 18ß-glycyrrhetinic acid. CONCLUSIONS: In this study, a rapid and sensitive UPLC-MS/MS analytical method was developed and applied to 24 major bioactive components in normal and ARDS rats after the oral administration of XFBD granules. This will serve to form the basis for further studies on the pharmacokinetic-pharmacodynamic correlation of XFBD granules.

Mycotoxins in Maize Silage from China in 2019.

Animal feed (including forage and silage) can be contaminated with mycotoxins. Here, 200 maize silage samples from around China were collected in 2019 and analyzed for regulated mycotoxins, masked mycotoxins (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside), and emerging mycotoxins (beauvericin, enniatins, moniliformin, and alternariol). Deoxynivalenol and zearalenone were detected in 99.5% and 79.5% of the samples, respectively. Other regulated mycotoxins were detected in fewer samples. The highest deoxynivalenol and zearalenone concentrations were 3600 and 830 µg/kg, respectively. The most commonly detected masked mycotoxin was 15-acetyldeoxynivalenol, which was detected in 68.5% of the samples and had median and maximum concentrations of 61.3 and 410 µg/kg, respectively. The emerging mycotoxins beauvericin, alternariol, enniatin A, enniatin B1, and moniliformin were detected in 99.5%, 85%, 80.5%, 72.5%, and 44.5%, respectively, of the samples but at low concentrations (medians <25 µg/kg). The samples tended to contain multiple mycotoxins, e.g., the correlation coefficients for the relationships between the concentrations of beauvericin and deoxynivalenol, deoxynivalenol and zearalenone, and zearalenone and beauvericin were 1.0, 0.995, and 0.995, respectively. The results indicated that there needs to be more awareness of the presence of one or more masked and emerging mycotoxins in maize silage in China.

Bioavailability Enhancement of Cepharanthine via Pulmonary Administration in Rats and Its Therapeutic Potential for Pulmonary Fibrosis Associated with COVID-19 Infection.

Cepharanthine (CEP) has excellent anti-SARS-CoV-2 properties, indicating its favorable potential for COVID-19 treatment. However, its application is challenged by its poor dissolubility and oral bioavailability. The present study aimed to improve the bioavailability of CEP by optimizing its solubility and through a pulmonary delivery method, which improved its bioavailability by five times when compared to that through the oral delivery method (68.07% vs. 13.15%). An ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method for quantification of CEP in rat plasma was developed and validated to support the bioavailability and pharmacokinetic studies. In addition, pulmonary fibrosis was recognized as a sequela of COVID-19 infection, warranting further evaluation of the therapeutic potential of CEP on a rat lung fibrosis model. The antifibrotic effect was assessed by analysis of lung index and histopathological examination, detection of transforming growth factor (TGF)-ß1, interleukin-6 (IL-6), α-smooth muscle actin (α-SMA), and hydroxyproline level in serum or lung tissues. Our data demonstrated that CEP could significantly alleviate bleomycin (BLM)-induced collagen accumulation and inflammation, thereby exerting protective effects against pulmonary fibrosis. Our results provide evidence supporting the hypothesis that pulmonary delivery CEP may be a promising therapy for pulmonary fibrosis associated with COVID-19 infection.

Polyphenolic HRMS Characterization, Contents and Antioxidant Activity of Curcuma longa Rhizomes from Costa Rica.

Curcuma longa constitutes an important source of secondary metabolites that have been associated with multiple health benefits. For instance, curcumin, demethoxycurcumin and bisdemethoxycurcumin, have been found to perform important biological activities, such as anti-inflammatory, antioxidant, anticancer, antimicrobial, antihypertensive and anticoagulant. These promising results prompted this research to evaluate the polyphenols of C. longa rhizomes in Costa Rica. The present work reports a comprehensive study on the polyphenolic profile and the contents of the three main curcuminoids as well as the antioxidant activity of extracts from C. longa rhizomes (n = 12) produced in Costa Rica. Through UPLC-QTOF-ESI MS, a total of 33 polyphenols were identified, grouped in eight types of structures. In addition, our findings on the main curcuminoids using UPLC-DAD show all rhizomes complying with total curcuminoids (TC) content established by the United States Pharmacopeia (USP). At an individual level, samples NW-3 and NE-1 show the higher contents (118.7 and 125.0 mg/g dry material), representing more than twice the average values of the lowest samples. These samples also exhibit the highest Folin-Ciocalteu (FC) reducing capacity results as well as the best DPPH (IC50 15.21 and 16.07 µg extract/mL) and NO (IC50 between 52.5 and 54.3 µg extract/mL) antioxidant values. Further, Pearson correlation analysis findings indicated positive correlation (p < 0.05) between TC, CUR with FC results (r = 0.833 and r = 0.867 respectively) and negative correlation (p < 0.05) between CUR, TC and FC with DPPH results (r = -0.898, r = -0.911, and r = -0.890, respectively) and between NO results and DPPH (r = -0.805, p < 0.05). Finally, results for Principal Component Analysis (PCA) showed composition variability associated with their region of origin with products from the Northeastern (NE) region exhibiting higher average values for FC, TC and antioxidant activities. Further, PCA confirmed that two samples, namely NE-1 and NW-3, stand out by presenting the highest PC1 due to their particularly high TC, CUR and antioxidant activities. Consequently, our findings agree with previous results indicating the importance of C. longa extracts to elaborate products with potential benefits for health, while delivering extracts with higher levels of curcuminoids than previous reports and exhibiting high antioxidant activity.

Use of hyphenated analytical techniques to identify the bioactive constituents of Gunnera perpensa L., a South African medicinal plant, which potently inhibit SARS-CoV-2 spike glycoprotein-host ACE2 binding.

SARS-CoV-2, the causative agent of COVID-19, continues to cause global morbidity and mortality despite the increasing availability of vaccines. Alongside vaccines, antivirals are urgently needed to combat SARS-CoV-2 infection and spread, particularly in resource-limited regions which lack access to existing therapeutics. Small molecules isolated from medicinal plants may be able to block cellular entry by SARS-CoV-2 by antagonising the interaction of the viral spike glycoprotein receptor-binding domain (RBD) with the host angiotensin-converting enzyme II (ACE2) receptor. As the medicinal plant Gunnera perpensa L. is being used by some South African traditional healers for SARS-CoV-2/COVID-19 management, we hypothesised that it may contain chemical constituents that inhibit the RBD-ACE2 interaction. Using a previously described AlphaScreen-based protein interaction assay, we show here that the DCM:MeOH extract of G. perpensa readily disrupts RBD (USA-WA1/2020)-ACE2 interactions with a half-maximal inhibition concentration (IC50) of < 0.001 µg/mL, compared to an IC50 of 0.025 µg/mL for the control neutralising antibody REGN10987. Employing hyphenated analytical techniques like UPLC-IMS-HRMS (method developed and validated as per the International Conference on Harmonization guidelines), we identified two ellagitannins, punicalin (2.12% w/w) and punicalagin (1.51% w/w), as plant constituents in the DCM:MeOH extract of G. perpensa which antagonised RBD-ACE2 binding with respective IC50s of 9 and 29 nM. This good potency makes both compounds promising leads for development of future entry-based SARS-CoV-2 antivirals. The results also highlight the advantages of combining reverse pharmacology (based on medicinal plant use) with hyphenated analytical techniques to expedite identification of urgently needed antivirals.

Chemical profiling and unraveling of anti-COVID-19 biomarkers of red sage (Lantana camara L.) cultivars using UPLC-MS/MS coupled to chemometric analysis, in vitro study and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Red sage (Lantana camara L.) (Verbenaceae) is a widely spread plant that was traditionally used in Brazil, India, Kenya, Thailand, Mexico, Nigeria, Australia and Southeast Asia for treating several ailments including rheumatism and leprosy. Despite its historical role in relieving respiratory diseases, limited studies progressed to the plant's probable inhibition to respiratory viruses especially after the striking spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. AIM OF THE STUDY: This study aimed to investigate the inhibitory activity of different L. camara cultivars to SARS-CoV-2, that was not previously inspected, and clarify their mechanisms of action in the metabolomics viewpoint, and to determine the biomarkers that are related to such activity using UPLC-MS/MS coupled to in vitro-studies and chemometric analysis. MATERIALS AND METHODS: Chemical profiling of different cultivars was accomplished via UPLC-MS/MS. Principle component analysis (PCA) and orthogonal projection to latent structures (OPLS) models were built using SIMCA® (multivariate data analysis software). Cytotoxicity and COVID-19 inhibitory activity testing were done followed by TaqMan Real-time RT-PCR (Reverse transcription polymerase chain reaction) assay that aimed to study extracts' effects on RNA-dependent RNA polymerase (RdRp) and E-genes expression levels. Detected biomarkers from OPLS analysis were docked into potential targets pockets to investigate their possible interaction patterns using Schrodinger® suite. RESULTS: UPLC-MS/MS analysis of different cultivars yielded 47 metabolites, most of them are triterpenoids and flavonoids. PCA plots revealed that inter-cultivar factor has no pronounced effect on the chemical profiles of extracts except for L. camara, cultivar Drap d'or flowers and leaves extracts as well as for L. camara cv Chelsea gem leaves extract. Among the tested extracts, flowers and leaves extracts of L. camara cv Chelsea gem, flowers extracts of L. camara cv Spreading sunset and L. camara cv Drap d'or showed the highest selectivity indices scoring 12.3, 10.1, 8.6 and 7.8, respectively, indicating their relative high safety and efficacy. Leaves and flowers extracts of L. camara cv Chelsea gem, flowers extracts of L. camara cv Spreading sunset and L. camara cv Drap d'or were the most promising inhibitors to viral plaques exhibiting IC50 values of 3.18, 3.67, 4.18 and 5.01 µg/mL, respectively. This was incremented by OPLS analysis that related their promising COVID-19 inhibitory activities to the presence of twelve biomarkers. Inhibiting the expression of RdRp gene is the major mechanism behind the antiviral activity of most extracts at almost all concentration levels. Molecular docking of the active biomarkers against RdRp revealed that isoverbascoside, luteolin-7,4'-O-diglucoside, camarolic acid and lantoic acid exhibited higher docking scores of -11.378, -10.64, -6.72 and -6.07 kcal/mol, respectively, when compared to remdesivir (-5.75 kcal/mol), thus these four compounds can serve as promising anti-COVID-19 candidates. CONCLUSION: Flowers and leaves extracts of four L. camara cultivars were recognized as rich sources of phytoconstituents possessing anti-COVID-19 activity. Combination of UPLC-MS/MS and chemometrics is a promising approach to detect chemical composition differences among the cultivars and correlate them to COVID-19 inhibitory activities allowing to pinpoint possible biomarkers. Further in-vitro and in-vivo studies are required to verify their activity.

Exploring the Anti-Pulmonary Fibrosis Mechanism of Jingyin Granule by Network Pharmacology Strategy.

Pulmonary fibrosis (PF) is a clinically common disease caused by many factors, which will lead to lung function decline and even respiratory failure. Jingyin granule has been confirmed to have anti-inflammatory and antiviral effects by former studies, and has been recommended for combating H1N1 influenza A virus (H1N1) infection and Coronavirus disease 2019 (COVID-19) in China. At present, studies have shown that patients with severe COVID-19 infection developed lung fibrotic lesions. Although Jingyin granule can improve symptoms in COVID-19 patients, no study has yet reported whether it can attenuate the process of PF. Here, we explored the underlying mechanism of Jingyin granule against PF by network pharmacology combined with in vitro experimental validation. In the present study, the active ingredients as well as the corresponding action targets of Jingyin granule were firstly collected by TCMSP and literature data, and the disease target genes of PF were retrieved by disease database. Then, the common targets were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and then a PPI network and an ingredient-target network were constructed. Next, UPLC-MS was used to isolate and identify selected representative components in Jingyin granule. Finally, LPS was used to induce the A549 cell fibrosis model to verify the anti-PF effect of Jingyin granule in vitro. Our results indicated that STAT3, JUN, RELA, MAPK3, TNF, MAPK1, IL-6, and AKT1 were core targets of action and bound with good affinity to selected components, and Jingyin granule may alleviate PF progression by Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3), the mammalian nuclear factor-κB (NF-κB), the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), tumor necrosis factor (TNF), and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathways. Overall, these results provide future therapeutic strategies into the mechanism study of Jingyin granule on PF.

Development and Validation of a Sensitive UPLC-Q-TOF-MS/MS for the Measurement of Nine Components in Rat Plasma and Tissues and its Application to Pharmacokinetics and Tissue Distribution Studies with Xuanfei Baidu Granules.

BACKGROUND: Xuanfei Baidu granules (XFBD granules) are based on the prescription of Xuanfei Baidu, which showed promise as a first-line treatment against Corona Virus Disease 2019 (COVID-19) in Wuhan, Hubei. On March 2, 2021, XFBD granules were marketed as a novel drug for epidemic diseases. However, there is little information about the pharmacokinetics and tissue distribution of the main constituents in XFBD granules. METHODS: A sensitive analytical method was developed for detecting the marker components of XFBD granules by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOFMS/ MS), and for studying its pharmacokinetics and tissue distribution by UPLC-QDa. RESULTS: Following an oral administration of a single granule in experimental rats at a dose of 14 g/kg for pharmacokinetic and tissue distribution studies, 42 compounds and nine analytes were identified in XFBD granules. Nine compounds were detected in the lungs and the liver of the rats. Six compounds were detected in the kidneys. Five compounds were detected in the spleen and three were detected in the heart. As it went undetected in the brain, XFBD granules are considered unable to cross the blood-brain barrier. CONCLUSION: A sensitive UPLC-Q-TOF-MS/MS method was established and validated for the quantification of nine components in rat plasma and tissue samples. This method was successfully applied to study the pharmacokinetics and tissue distribution profiles of XFBD granules after their oral administration.