Preparative separation of structural isomeric pentacyclic triterpene oleanolic acid and ursolic acid from natural products by pH-zone-refining countercurrent chromatography

In this work, pH-zone-refining countercurrent chromatography was investigated in the preparative separation of two bioactive components, oleanolic acid and ursolic acid, from three different natural products, Aralia chinensis, apple peels and Eriobotrya japonica Thunb. Oleanolic acid and ursolic acid are structurally isomeric pentacyclic triterpene acids that are widely distributed in many natural products. However, it was difficult to separate these components with high purity by conventional methods. A biphasic solvent system composed of n-hexane–dichloromethane–methanol–water (7 : 3 : 2 : 8, v/v) was selected, in which an optimized concentration of 10 mmol L⁻¹ trifluoroacetic acid was added in the upper phase as the retainer and 10 mmol L⁻¹ ammonia (with 25–28% NH₃) was added in the aqueous phase as the eluter. Consequently, 38.56 mg of oleanolic acid with 99.01% purity was separated from 100 mg of the crude extract of Aralia chinensis, while 65.6 mg of a mixture of ursolic acid (90.98%) and oleanolic acid (6.51%) and 46.6 mg of a mixture of ursolic acid (74.35%) and oleanolic acid (23.61%) were separated from 100 mg of the crude extract of apple peels and 100 mg of the crude extract of Eriobotrya japonica Thunb., respectively, by pH-zone-refining countercurrent chromatography using the above selected biphasic solvent system. The results showed that pH-zone-refining countercurrent chromatography is an efficient method for the preparative separation of pentacyclic triterpene acids from natural products.

pH-zone-refining countercurrent chromatography was investigated in preparative separation of oleanolic acid and ursolic acid from three different natural products, Aralia chinensis, apple peels and Eriobotrya japonica Thunb.     

Synthesis,cytotoxicity and anti-inflammatory activity of rhamnose-containing ursolic and betulinic acid saponins

Betulinic acid and ursolic acid are ubiquitous, naturally-occurring triterpenoids exhibiting various pharmacological activities including cytotoxic and anti-inflammatory activities. However, these triterpenoids display unfavorable pharmacokinetic properties as well as low aqueous solubility. It has been shown that the presence of α-l-rhamnose moieties positively modulates the anticancer activity of secondary metabolites. Herein we report the synthesis and in vitro evaluation of cytotoxic and anti-inflammatory activities of a series of rhamnose-containing ursolic and betulinic acid saponins. Relying on Schmidt''s normal and inverse procedures, monorhamnosides, (1→4)-linked dirhamnosides as well as branched trirhamnosides and tetrarhamnosides were synthesized in high yields with full control of stereoselectivity. A betulinic acid saponin bearing a 3-O-α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl residue was found to be a potent cytotoxic agent against human colorectal adenocarcinoma cells without damaging the healthy cells (selectivity ratio > 20) whereas rhamnose-containing ursolic acid saponins potently inhibited NO overproduction induced by LPS-stimulated macrophages. Our results reveal that rhamnose-containing ursolic and betulinic acid saponins represent promising therapeutic agents.

Rhamnose-containing saponins featuring betulinic and ursolic acid as aglycones were synthesized using both Schmidt''s normal and inverse procedures. Some of these synthetic saponins exhibited selective cytotoxic and/or anti-inflammatory activities.     

Sub–acute oral toxicity study of methanol leaves extract of Catharanthus roseus in rats

ObjectiveTo examine the sub-acute (14 d) oral toxic effects of methanol leaves extract of Catharanthus roseus (C. roseus) (Family: Apocynaceae) on liver and kidney functions in Sprague Dawley (SD) rats.MethodsTwenty four female SD rats were used throughout the experiment. The first group was orally treated with distilled water and served as control, whereas the remaining three groups were orally treated with single dose daily of 0.1 g/kg, 0.5 g/kg, 1 g/kg of C. roseus extract, respectively for 14 d. Cage-side observations were done daily. Any animal died during the experiment was dissected for gross organ examination. Body weight changed, food consumption and water intake were recorded weekly. Blood was collected via cardiac puncture on day-15 and used for determination of serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine and urea. The relative organ weights were also measured. All results were expressed as mean ± S.E.M and analysed using Dunnett's test. The level of significance was set at P<0.05 when compared to the control group.ResultsRepeated oral administration of 0.5 g/kg and 1 g/kg of methanol leaves extract of C. roseus caused mortality and diarrhoea in rats after few days of treatment. There were no significant changes observed in serum biochemical markers, body weight changed, water and food intake and relative organ weight in rats treated with a single dose daily of 0.1 g/kg of C. roseus extract treatment for 14 d when compared to control group.ConclusiondsFourteen days repeated oral administration of 0.1 g/kg of methanol leaves extract of C. roseus was safe in female SD rats without causing any significant damages to liver and kidney.     

Biocatalysis of ursolic acid by the fungus Gliocladium roseum CGMCC 3.3657 and resulting anti-HCV activity

Biocatalysis of ursolic acid (UA 1) by Gliocladium roseum CGMCC 3.3657 was investigated. Baeyer–Villiger oxidation was found to occur during the reaction. Four metabolites were isolated from the cultures and their structures were identified as 21-oxo,A-homo-3a-oxa-urs-12-en-3-one-28-oic acid (2), 21-oxo-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid (3), 21β-hydroxyl-A-homo-3a-oxa-urs-12-en-3-one-28-oic acid (4) and 21β-hydroxyl-3,4-seco-ursan-4(23),12-dien-3,28-dioic acid (5), based on their NMR and MS spectral data. All of the four metabolites were new and their anti-HCV activity was tested. Their biotransformation pathway was also proposed.

Biocatalysis of ursolic acid (UA 1) by Gliocladium roseum CGMCC 3.3657 was investigated.     

Penicacids H–J,three new mycophenolic acid derivatives from the marine-derived fungus Rhizopus oryzae

Chemical investigation of secondary metabolites in crude methanol extract of a solid rice medium of a marine-derived fungus, Rhizopus oryzae, has enriched the metabolic profile of this genus by affording three mycophenolic acid derivatives recognized as new fungal metabolites trivially named as penicacids H–J (1–3), along with two known naphtho-γ-pyrone dimers, asperpyrone A (4) and dianhydroaurasperone C (5). Structure elucidation of isolated compounds was unambiguously determined based on extensive 1D and 2D NMR spectroscopic analyses together with comparing coupling constant and optical rotation values with those reported for related congeners in literature. All isolated compounds were assessed for their antibacterial activity against four different bacterial microorganisms and they revealed moderate to weak activities with minimum inhibitory concentration (MIC) values ranging from 62.5 to 250 μg mL⁻¹.

Penicacids H–J (1–3), three new natural MPA derivatives, were purified from a marine-derived fungus, Rhizopus oryzae, together with two known naphtho-γ-pyrone dimers, asperpyrone A (4) and dianhydroaurasperone C (5).     

Determination of valganciclovir and ganciclovir in human plasma by liquid chromatography tandem mass spectrometric detection

ObjectivesThe aim of this work was to develop a simple, sensitive and selective LC/MS/MS method for the assay of valganciclovir and ganciclovir in human plasma.Design and methodsSample preparation involved solid phase extraction on mix mode cation exchanger. Separation was performed on Chromolith RP18e column using water, trifluoroacetic acid (1 M, pH 4.4) and methanol (29.9:0.1:70, v/v) as mobile phase. Both analytes were detected by electro spray ionization mass spectrometry in positive ion multiple reaction monitoring mode.ResultsCCs with good linearties having r ≥ 0.9990 and ≥ 0.9992 were obtained in the range of 5–800 ng/mL and 70–11,200 ng/mL for valganciclovir and ganciclovir, respectively. The extraction recoveries were around 85% for both the analytes.ConclusionThe method provided a simple and selective procedure that can be easily used for the evaluation of the pharmacokinetic profile of valganciclovir and ganciclovir in human plasma.     

Synergistic effect of ursolic acid and piperine in CCl4 induced hepatotoxicity

BackgroundUrsolic acid (UA) is a potent plant-based hepatoprotective agent having poor bioavailability, which hampers its therapeutic efficacy. The present study tries to overcome this limitation by combining it with piperine (PIP), a proven bioenhancer and hepatoprotective agent.MethodsThe type of interaction (synergism, addition, or antagonism) resulting between UA and PIP was analyzed and quantified by isobologram and combination index analysis. The hepatoprotective activity of UA and PIP was evaluated by measuring the level of hepatic marker enzymes. Pharmacokinetic analysis was carried out to ascertain the improvement of bioavailability.ResultsThe combinations significantly decrease the enzyme levels, which indicate better hepatoprotective activity compared to single drugs. The relative oral bioavailability of UA was increased about tenfold (from AUC_0–t =12.78 ± 2.59 µg/h/ml to 125.15 ± 1.84 µg/h/ml) along with the improvement of plasma concentration and elimination half-life.ConclusionsThe findings indicated that the combination of PIP and UA is an effective strategy in enhancing the bioavailability and hepatoprotective potential of UA.

KEY MESSAGES

• Ursolic acid in a combination with piperine provides a synergistic hepatoprotective effect in carbon tetrachloride induced liver damage in rats.
• Piperine improves the pharmacokinetic properties of ursolic acid when given in combination.
• Piperine improves the relative oral bioavailability of ursolic acid by tenfold when combined together.

     

Drimia calcarata Bulb Extracts Deactivate the PI3K Signalling Pathway in Cervical HPV-18 Positive HeLa Cells

BackgroundThe efficacy of Drimia calcarata against the human cervical cancer remains unexplored and less understood.ObjectiveThe present study focused on investigating the cytotoxic effect of D. calcarata bulb extracts on cervical cancer cells.MethodsThe growth inhibitory effects of D. calcarata extracts were determined using the MTT, Ki67 and PI3K Activation assays. Apoptosis induction was assessed using fluorescence microscopy, the Muse^? Cell Analyser and gene expression analysis by RT-PCR. The cytotoxicity of the fractions was evaluated using MTT assay and the apoptosis induction using Muse^? Cell Analyser.ResultsBoth methanol extract (ME) and water extract (WE) showed safety against noncancerous KMST-6 and HEK-293 cells. The extracts exhibited anticancer activity against HeLa, with no significant cytotoxic effect against the Ca-Ski cells. The WE increased the Ki67 positive Ca-Ski population, while both ME and WE arrested HeLa cells at G2/M phase, and Ca-Ski cells in G0/G1 phase. AO/EB staining, Annexin V and Caspase 3/7 Activation revealed that the extracts significantly induced apoptosis in HeLa cells. In HeLa cells, the ME downregulated TP53 variants, while WE upregulated both TP53 variants in HeLa cells. Both extracts decreased the STAT5A and STAT5B mRNA expression in HeLa cells; however, these extracts upregulated cancer-promoting STAT3 in Ca-Ski cells. Additionally, these extracts inactivated the PI3K signalling pathway in HeLa cells but not in Ca-Ski cells. The resistance of the Ca-Ski cells to the D. calcarata extracts may be due to the upregulation of STAT3 and activated PI3K signalling pathway. The cytotoxicity and apoptosis induction in HeLa cells by D. calcarata extracts may be attributed due to downregulation of STAT5A survival mechanisms. Water fractions 1, 2, 3 and 6 and methanol fractions 1 and 2 reduced cell viability of HeLa cells. Water fractions 2, 3 and 6 and methanol fractions 1 and 3 induced apoptosis, which was preceded by secondary necrosis. However, water fraction 1 and methanol fraction 2 led to most cells undergoing necrotic cell death. There are several compounds that can be credited with the anticancer activities of the ME and WE extracts since several fractions exhibited cytotoxicity against the HeLa cells.ConclusionThese findings suggest that the D. calcarata extracts have anticancer activities, and thus, could be useful for therapeutic purposes against human HPV-18 positive gynaecologic cancers.     

Evaluation of the efficacy of Withania somnifera (Ashwagandha) root extract in patients with obsessive-compulsive disorder: A randomized double-blind placebo-controlled trial

BackgroundObsessive-compulsive disorder (OCD) is a chronic psychiatric disorder that is causally linked to dysregulation of the serotonergic system. The aim of this study is to investigate the efficacy of Withania somnifera (W. somnifera) root extract as an adjunct therapy to standard OCD treatment.MethodsThirty patients with a confirmed diagnosis of OCD according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria participated in this randomized double-blind placebo-controlled trial and were randomly assigned to the treatment group (W. somnifera extract, 120 mg/day; n = 15) or the placebo group (n = 15). All patients were under treatment with Selective Serotonin Re-uptake Inhibitors (SSRIs), and were instructed to take 4 capsules of the extract or placebo per day, preferably after meals, for a period of six weeks. The Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) was used in order to assess the severity of OCD symptoms at baseline and at the end of the trial. Statistical analyses were performed using SPSS software and Y-BOCS values were presented as median and range (Min-Max).ResultsComparison of the change in Y-BOCS score during the course of the trial revealed a significantly greater effect of W. somnifera (26 (14–40) [pre-treatment] versus 14 (4–40) [post-treatment]; change: −8 (−23 to 0)) versus placebo (18 (11–33) [pre-treatment] versus 16 (10–31) [post-treatment]; change: −2 (−4 to 0)) (P < 0.001). The extract was safe and no adverse event was reported during the trial.ConclusionW. somnifera extract may be beneficial as a safe and effective adjunct to SSRIs in the treatment of OCD.     

Utilization of mesoporous phosphotungstic acid in nanocellulose membranes for direct methanol fuel cells

Nanocellulose (NC) composite membranes containing novel ternary materials including NC, imidazole (Im), and mesoporous phosphotungstic acid (m-PTA) were successfully fabricated by a phase inversion method. The single-particle size of NC was 88.79 nm with a spherical form. A m-PTA filler with a mesopore size of 4.89 nm was also successfully synthesized by a self-assembly method. Moreover, the fabricated membrane NC/Im/m-PTA-5 exhibited the best performances towards its proton conductivity and methanol permeability at 31.88 mS cm⁻¹ and 1.74 × 10⁻⁶ cm² s⁻¹, respectively. The membrane selectivity was 1.83 × 10⁴ S cm⁻³.

A NC/Im/m-PTA membrane was fabricated for direct methanol fuel cell applications.     

Simultaneous determination of Levetiracetam and its acid metabolite (ucb L057) in serum/plasma by liquid chromatography tandem mass spectrometry

ObjectiveLevetiracetam and its acid metabolite have almost identical MRMs. They therefore need to be separated chromatographically prior to quantitation.Research design and methodsThe sample is deproteinized with acetonitrile containing Ritonavir as internal standard, centrifuged and the supernatant diluted with water (1:2 v/v). Sixty microliters of the supernatant is injected into the LC–MS/MS and Levetiracetam (LEV) and LEV metabolite separated chromatographically at room temperature employing a Supelco C₁₈ column and a 0.1% formic acid methanol gradient at pH of 2.5.ResultsThe retention times for LEV metabolite, LEV and Ritonavir were 4.50, 5.38 and 9.18 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 0–50 μg/mL for LEV and 0.0–5.0 µg/mL for LEV metabolite. Intra- and inter-run imprecision (n = 10) gave CVs of 2.3–4.7%, 3.4–8.9% for LEV and 2.9–3.9%, 3.3–7.4% for LEV metabolite. Recoveries of both LEV and LEV metabolite were close to 100%. Results for LEV were compared with those obtained by a commercial reference laboratory (r = 0.974).ConclusionThe procedure is reliable, quick, and inexpensive. LEV and LEV metabolite co-elute using C-18 columns at pHs > 3.0 and previously published methods employing these conditions could therefore be subject to metabolite interference. In this method LEV and LEV metabolite are separated at pH 2.5. The total run time including the washing step is 10 min/sample, making this method suitable when moderate throughput is needed such as in clinical or commercial reference laboratories.     

Metabolomics and Type 2 Diabetes Risk: An Updated Systematic Review and Meta-analysis of Prospective Cohort Studies

BACKGROUNDDue to the rapidly increasing availability of metabolomics data in prospective studies, an update of the meta evidence on metabolomics and type 2 diabetes risk is warranted.PURPOSETo conduct an updated systematic review and meta-analysis of plasma, serum, and urine metabolite markers and incident type 2 diabetes.DATA SOURCESWe searched PubMed and Embase until 6 March 2021.STUDY SELECTIONWe selected prospective observational studies where investigators used high-throughput techniques to investigate the relationship between plasma, serum, or urine metabolites and incident type 2 diabetes.DATA EXTRACTIONBaseline metabolites per-SD risk estimates and 95% CIs for incident type 2 diabetes were extracted from all eligible studies.DATA SYNTHESISA total of 61 reports with 71,196 participants and 11,771 type 2 diabetes cases/events were included in the updated review. Meta-analysis was performed for 412 metabolites, of which 123 were statistically significantly associated (false discovery rate–corrected P < 0.05) with type 2 diabetes risk. Higher plasma and serum levels of certain amino acids (branched-chain, aromatic, alanine, glutamate, lysine, and methionine), carbohydrates and energy-related metabolites (mannose, trehalose, and pyruvate), acylcarnitines (C4-DC, C4-OH, C5, C5-OH, and C8:1), the majority of glycerolipids (di- and triacylglycerols), (lyso)phosphatidylethanolamines, and ceramides included in meta-analysis were associated with higher risk of type 2 diabetes (hazard ratio 1.07–2.58). Higher levels of glycine, glutamine, betaine, indolepropionate, and (lyso)phosphatidylcholines were associated with lower type 2 diabetes risk (hazard ratio 0.69–0.90).LIMITATIONSSubstantial heterogeneity (I² > 50%, τ² > 0.1) was observed for some of the metabolites.CONCLUSIONSSeveral plasma and serum metabolites, including amino acids, lipids, and carbohydrates, are associated with type 2 diabetes risk.     

Pharmacokinetics of depside salts from Salvia miltiorrhiza in healthy Chinese volunteers: A randomized, open-label, single-dose study

Background: Depside salts from Salvia miltiorrhiza, with active components of lithospermic acid B (LSB), rosmarinic acid (RA), and lithospermic acid (LA), are a multicomponent drug marketed in China for the treatment of coronary heart disease.Objectives: The aims of this study were to determine the concentrations of LSB, RA, and LA in human plasma and urine, and to compare the pharmacokinetic properties of depside salts from S miltiorrhiza in healthy Chinese volunteers.Methods: A randomized, open-label, single-dose study was conducted in healthy Chinese volunteers. Participants were randomly assigned to receive a single intravenous infusion of 100 or 200 mg of depside salts from S miltiorrhiza. Blood was collected through a venous cannula prior to study drug administration (0 min) and at 10, 20, 30, 60, 65, 70, 80, and 90 minutes and 2, 3, 4, 6, 8, 12, and 24 hours after study drug administration. Urine samples were taken before study drug administration (0) and at 0 to 12 and 12 to 24 hours after study drug administration. LSB, RA, and LA concentrations in serum and urine were analyzed by an LC-MS/MS method. Tolerability was determined by clinical assessment; vital signs (ie, blood pressure, heart rate, breathing rate, body temperature) monitoring at baseline and at the end of the study, clinical laboratory tests (ie, hematology, blood biochemistry, hepatic function, renal function, urinalysis), 12-lead ECG measurements, and physical examinations at baseline and after completion of the study.Results: Twelve Chinese volunteers (6 males, 6 females; mean [SD] age, 25.2 [3.8] years; mean height, 165.7 [8.9] cm; mean body mass index, 21.6 [2.5] kg/m²) were enrolled in the study. Peak plasma concentrations of LSB, RA and LA were observed at 0.3 to 1 hour following the 1-hour intravenous infusion, with respective mean (SD) Cmax of 4925 (1861), 174 (61), and 361 (101) ng/mL for the 100-mg dose and 10,285 (2259), 308 (77), and 674 (85) ng/mL for the 200-mg dose. The AUC_last values for LSB, RA, and LA were 4537 (1265), 129 (28), and 1229 (330) ng/mL/h, respectively, for the 100-mg dose and 10,426 (2589), 260 (53), and 2792 (729) ng/mL/h for the 200-mg dose. No significant difference in pharmacokinetic parameters was observed between male and female subjects. Three metabolites were found in the plasma with low concentrations. The urinary excretion recoveries of LSB, RA, and LA were 0.58% (0.42%), 25.21% (20.61%), and 10.02% (7.72%) for the 100-mg dose and 0.38% (0.18%), 20.11% (10.50%), and 6.34% (3.20%) for the 200-mg dose. No adverse events were reported by the subjects or found by the investigators in the analysis of vital signs, 12-lead ECG measurements, physical examinations, or clinical laboratory tests.Conclusions: Following single intravenous infusion of 100 or 200 mg of depside salts from S miltiorrhiza to healthy Chinese subjects, no statistical differences in pharmacokinetic parameters were observed between males and females. The 2 doses of depside salts from S miltiorrhiza were clinically well tolerated during the study.     

Phytochemical standardization of Aloe vera extract by HPTLC techniques

ObjectiveTo examine the phytochemical parameters of Aloe vera (A. vera) L. which can be used as a tool for its standardization.MethodsThe phytochemical analysis, solubility test, heavy metal analysis, antimicrobial study and quantitative analysis of gallic acid and berberine by HPTLC method were included in present study.ResultsPhytochemical analysis revealed the presence of alkaloid, carbohydrate, tannin, steroid, triterpenoid and glycoside. Total flavonoid and phenol content was found to be 1.9% and 13.11%. Concentartion of lead, arsenic, mercury and cadmium was found to be under the limit. Total bacterial count, yeast and moulds contents were found to be under the limit whereas Escherichia coli (E. coli) and salmonella was found to be absent in the extract. Quantitative analysis through HPTLC revealed the presence of 2.74% and 0.543% w/w of berberine and gallic acid.ConclusionsThe results indicate that the plant extract are rich in berberine and gallic acid implying their importance to human health. This investigation could be used as source of standard parameters which can play an important role in its standardization.     

An improved HPLC-MS/MS method for simultaneous quantification of propranolol and its two phase I metabolites in plasma of infants with hemangioma and its application to a comparative study of plasma concentrations

Propranolol is now a preferred treatment for infantile hemangioma. However, there are no published papers on the metabolism and concentrations of propranolol in the plasma of infants with hemangioma. In the present study, a sensitive, simple and reliable method was developed and validated for the simultaneous quantification of propranolol and its metabolites 4-hydroxypropranolol (M1) and N-desisopropylpropranolol (M2) in infants'' plasma for the first time by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A volume of 100 μL plasma was prepared by one-step protein precipitation with acetonitrile (300 μL), followed by its separation on an Hypersil GOLD C₁₈ column maintained at 40 °C with gradient mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.3 mL min⁻¹. The quantification was performed via multiple reaction monitoring (MRM) by a triple quadrupole mass spectrometer under positive electrospray ionization (ESI) mode. Bisoprolol was chosen as the internal standard. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect and stability. The matrix-matched calibration curves for propranolol ranging from 1 to 500 ng mL⁻¹, for M1 ranging from 0.2 to 100 ng mL⁻¹ and for M2 ranging from 0.2 to 100 ng mL⁻¹ were all linear, with correlation coefficients calculated using weighted (1/x²) least square linear regression analysis. The lower limits of quantification (LLOQs) were 1 ng mL⁻¹, 0.2 ng mL⁻¹ and 0.2 ng mL⁻¹ for propranolol, M1 and M2, respectively. The intra-day and inter-day precisions were less than 7.1% and relative errors were all less than 9.8%. This validated method was successfully applied to quantify the concentrations of propranolol and its metabolites 4-hydroxypropranolol (M1) and N-desisopropylpropranolol (M2) in the plasma of infants with hemangioma after oral administration of different doses of propranolol for the first time.

Comparative study of propranolol, 4-hydroxypropranolol and N-desisopropylpropranolol in the plasma of infants with hemangioma after oral administration of different doses of propranolol.     

Anti-SARS-CoV-2 activities of tanshinone IIA,carnosic acid,rosmarinic acid,salvianolic acid,baicalein, and glycyrrhetinic acid between computational and in vitro insights

Six compounds namely, tanshinone IIA (1), carnosic acid (2), rosmarinic acid (3), salvianolic acid B (4), baicalein (5), and glycyrrhetinic acid (6) were screened for their anti-SARS-CoV-2 activities against both the spike (S) and main protease (Mpro) receptors using molecular docking studies. Molecular docking recommended the superior affinities of both salvianolic acid B (4) and glycyrrhetinic acid (6) as the common results from the previously published computational articles. On the other hand, their actual anti-SARS-CoV-2 activities were tested in vitro using plaque reduction assay to calculate their IC₅₀ values after measuring their CC₅₀ values using MTT assay on Vero E6 cells. Surprisingly, tanshinone IIA (1) was the most promising member with IC₅₀ equals 4.08 ng μl⁻¹. Also, both carnosic acid (2) and rosmarinic acid (3) showed promising IC₅₀ values of 15.37 and 25.47 ng μl⁻¹, respectively. However, salvianolic acid (4) showed a weak anti-SARS-CoV-2 activity with an IC₅₀ value equals 58.29 ng μl⁻¹. Furthermore, molecular dynamics simulations for 100 ns were performed for the most active compound from the computational point of view (salvianolic acid 4), besides, the most active one biologically (tanshinone IIA 1) on both the S and Mpro complexes of them (four different molecular dynamics processes) to confirm the docking results and give more insights regarding the stability of both compounds inside the SARS-CoV-2 mentioned receptors, respectively. Also, to understand the mechanism of action for the tested compounds towards SARS-CoV-2 inhibition it was necessary to examine the mode of action for the most two promising compounds, tanshinone IIA (1) and carnosic acid (2). Both compounds (1 and 2) showed very promising virucidal activity with a most prominent inhibitory effect on viral adsorption rather than its replication. This recommended the predicted activity of the two compounds against the S protein of SARS-CoV-2 rather than its Mpro protein. Our results could be very promising to rearrange the previously mentioned compounds based on their actual inhibitory activities towards SARS-CoV-2 and to search for the reasons behind the great differences between their in silico and in vitro results against SARS-CoV-2. Finally, we recommend further advanced preclinical and clinical studies especially for tanshinone IIA (1) to be rapidly applied in COVID-19 management either alone or in combination with carnosic acid (2), rosmarinic acid (3), and/or salvianolic acid (4).

Tanshinone IIA shows the most promising anti-SARS-CoV-2 biological activity: molecular docking, molecular dynamics, in vitro, and SAR studies.     

The effect of green coffee extract supplementation on anthropometric measures in adults: A comprehensive systematic review and dose-response meta-analysis of randomized clinical trials

Background and aimTwo meta-analyses summarized data on the effects of green coffee extract (GCE) supplementation on anthropometric measures. However, the accuracy of those meta-analyses is uncertain due to several methodological limitations. Therefore, we aimed to conduct a comprehensive systematic review and dose-response meta-analysis to summarize all available evidence on the effects of GCE supplementation on anthropometric measures by considering the main limitations in the previous meta-analyses.MethodsWe searched available online databases for relevant publications up to January 2020, using relevant keywords. All randomized clinical trials (RCTs) investigating the effects of GCE supplementation, compared with a control group, on anthropometric measures [including body weight, body mass index (BMI), body fat percentage, waist circumference (WC) and waist-to-hip ratio (WHR)] were included.ResultsAfter identifying 1871 studies from our initial search, 15 RCTs with a total sample size of 897 participants were included in the systematic review and meta-analysis. We found a significant reducing effect of GCE supplementation on body weight (weighted mean difference (WMD): −1.23, 95 % CI: −1.64, −0.82 kg,P < 0.001), BMI (WMD: −0.48, 95 % CI: −0.78, −0.18 kg/m², P = 0.001), and WC (WMD: −1.00, 95 % CI: −1.70, −0.29 cm, P = 0.006). No significant effect of GCE supplementation on body fat percentage and WHR was seen. In the dose-response analyses, there was no significant association between chlorogenic acid (CGA) dosage, as the main polyphenol in green coffee, and changes in anthropometric measures.ConclusionWe found that GCE supplementation had a beneficial effect on body weight, BMI and WC. It provides a cost-effective and safe alternative for the treatment of obesity. Additional well-designed studies are required to further confirm our findings.     

Pharmacokinetic profile and metabolite identification of bornyl caffeate and caffeic acid in rats by high performance liquid chromatography coupled with mass spectrometry

Bornyl caffeate was initially discovered as a bioactive compound in medicinal plants. Despite the promising pharmacological activities including anti-tumor and antibacterial activities, the pharmacokinetics of the compound remain open. This work developed a high performance liquid chromatography-tandem mass spectrometric method for the determination of bornyl caffeate and caffeic acid (major metabolite and a main unit of bornyl caffeate) in vivo. Successful application of the method included identification of its metabolites and investigation on the drug pharmacokinetics. A total of 30 compounds were identified as the metabolites of bornyl caffeate in rats. We attributed these metabolites to phase I metabolic routes of reduction, oxidation, hydrolysis and phase II metabolic reactions of glucuronidation, sulfation, O-methylation and glycine. Glucuronidation, sulfation, O-methylation and reduction were the main metabolic pathways of bornyl caffeate. The method presented a linear range of 1–4000 ng mL⁻¹. The pharmacokinetic profile of bornyl caffeate was found to be a three compartment open model, while caffeic acid fitted to a two compartment open model when it was administered alone or served as the main metabolite of bornyl caffeate. The time to peak concentration (T_max) and the maximum plasma concentration (C_max) of bornyl caffeate were 0.53 h and 409.33 ng mL⁻¹. Compared with original caffeic acid, the compound displayed an increased half-life of elimination (T_1/2β), area under the concentration time curve from 0 to t (AUC_0–t) and area under the concentration time curve from 0 to ∞ (AUC_0–∞), a decreased half-life of absorption (T_1/2α) and an identical C_max. Taking together, we concluded that bornyl caffeate is able to rapidly initiate therapeutic effect and last for a relatively long time in rats; metabolic pathways of O-methylation and reduction is key to interpret the mechanism and toxicity of bornyl caffeate.

We revealed the metabolic profile of bornyl caffeate by HPLC-Q-TOF/MS, and then simultaneously examined the pharmacokinetics of bornyl caffeate and CA after administration of a single dose of bornyl caffeate by HPLC ion trap MS.