Identification of Phytochemicals from Arabian Peninsula Medicinal Plants as Strong Binders to SARS-CoV-2 Proteases (3CLPro and PLPro) by Molecular Docking and Dynamic Simulation Studies.

We provide promising computational (in silico) data on phytochemicals (compounds 1-10) from Arabian Peninsula medicinal plants as strong binders, targeting 3-chymotrypsin-like protease (3CLPro) and papain-like proteases (PLPro) of SARS-CoV-2. Compounds 1-10 followed the Lipinski rules of five (RO5) and ADMET analysis, exhibiting drug-like characters. Non-covalent (reversible) docking of compounds 1-10 demonstrated their binding with the catalytic dyad (CYS145 and HIS41) of 3CLPro and catalytic triad (CYS111, HIS272, and ASP286) of PLPro. Moreover, the implementation of the covalent (irreversible) docking protocol revealed that only compounds 7, 8, and 9 possess covalent warheads, which allowed the formation of the covalent bond with the catalytic dyad (CYS145) in 3CLPro and the catalytic triad (CYS111) in PLPro. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) analysis from molecular dynamic (MD) simulations revealed that complexation between ligands (compounds 7, 8, and 9) and 3CLPro and PLPro was stable, and there was less deviation of ligands. Overall, the in silico data on the inherent properties of the above phytochemicals unravel the fact that they can act as reversible inhibitors for 3CLPro and PLPro. Moreover, compounds 7, 8, and 9 also showed their novel properties to inhibit dual targets by irreversible inhibition, indicating their effectiveness for possibly developing future drugs against SARS-CoV-2. Nonetheless, to confirm the theoretical findings here, the effectiveness of the above compounds as inhibitors of 3CLPro and PLPro warrants future investigations using suitable in vitro and in vivo tests.

Covalent Inhibitors from Saudi Medicinal Plants Target RNA-Dependent RNA Polymerase (RdRp) of SARS-CoV-2.

COVID-19, a disease caused by SARS-CoV-2, has caused a huge loss of human life, and the number of deaths is still continuing. Despite the lack of repurposed drugs and vaccines, the search for potential small molecules to inhibit SARS-CoV-2 is in demand. Hence, we relied on the drug-like characters of ten phytochemicals (compounds 1-10) that were previously isolated and purified by our research team from Saudi medicinal plants. We computationally evaluated the inhibition of RNA-dependent RNA polymerase (RdRp) by compounds 1-10. Non-covalent (reversible) docking of compounds 1-10 with RdRp led to the formation of a hydrogen bond with template primer nucleotides (A and U) and key amino acid residues (ASP623, LYS545, ARG555, ASN691, SER682, and ARG553) in its active pocket. Covalent (irreversible) docking revealed that compounds 7, 8, and 9 exhibited their irreversible nature of binding with CYS813, a crucial amino acid in the palm domain of RdRP. Molecular dynamic (MD) simulation analysis by RMSD, RMSF, and Rg parameters affirmed that RdRP complexes with compounds 7, 8, and 9 were stable and showed less deviation. Our data provide novel information on compounds 7, 8, and 9 that demonstrated their non-nucleoside and irreversible interaction capabilities to inhibit RdRp and shed new scaffolds as antivirals against SARS-CoV-2.

Compounds Related to Saudin and Three New Series of Diterpenoids from Clutia lanceolata.

Clutia lanceolata is a medicinal plant native to Ethiopia and sub-Saharan Africa and to the Arabian Peninsula. It is used traditionally in Saudi Arabia for the treatment of diabetes. Previous phytochemical analysis of this species has been limited to the identification of methylthiocoumarins. Further work has led to isolation of 19 new diterpenoids in three structural classes. Their structures were established by HRMS and by a range of NMR techniques (1H, 13C, COSY, NOESY, HSQC, HMBC), with confirmation for some examples by X-ray crystallography. NOESY and 1H-1H NMR coupling constants gave the relative stereochemical configurations and conformational information, with absolute configurations being established through X-ray crystallography. One example closely related to the known hypoglycemic compound saudin (found in C. richardiana and also in C. lanceolata) and one with a different core tetracycle were found to enhance strongly the glucose-triggered release of insulin from murine pancreatic islets. Biosynthetic proposals for the three groups of new diterpenoids by alternative cyclization of a common precursor are put forward. Lanceolide P (16) is proposed as a lead compound for further development for the treatment of diabetes.

The anti-oxidative, anti-cell proliferative and anti-microbial efficacies of cold-adapted Crepis flexuosa: HPTLC and GC/MS analyses.

The genus Crepis constitutes cold-adapted plant spp., of these some are traditionally used in folk medicine against inflammation or fungal infections without scientific validations. Here, we report the biological activities of Crepis flexuosa total ethanol-extract (CF-EtOH) and its hexane (CF-Hex), ethyl acetate (CF-EtOA), butanol (CF-ButOH), and aqueous (CF-Aqua) fractions. Our in vitro DPPH and ABTS radical-scavenging assays showed CF-EtOH, CF-ButOH and CF-Aqua with maximal, CF-EtOA with moderate, and CF-Hex with mild anti-oxidant activities. When tested on human cancer cell lines, high cytotoxicity was demonstrated by CF-EtOH (IC50: 42.45 µg/ml) and CF-Aqua (IC50: 46.37 µg/ml) on HepG2, followed by CF-Hex (IC50: 63.24 µg/ml) and CF-ButOH (IC50: 65.32 µg/ml) on MCF7 cells. The human primary cell line (HUVEC) had comparatively lower cytotoxicity for the tested samples. Moreover, when assessed for anti-microbial efficacy, CF-ButOH and CF-Aqua exhibited the strongest activity (MIC: 156.25 µg/ml) against S. aureus, E. faecalis and C. albicans. Further, while the developed RP-HPTLC identified the bioactive flavonoid luteolin-7-O-glucoside (17.58 mg/g), GS/MS analysis revealed sixteen compounds in C. flexuosa extract. In conclusion, we for the first time show the promising anti-oxidative, anti-cell proliferative and anti-microbial efficacies of C. flexuosa. This warrants further phytochemical and bio-efficacy studies towards isolations and identifications of active principles.

A neoclerodane orthoester and other new neoclerodane diterpenoids from Teucrium yemense chemistry and effect on secretion of insulin.

Teucrium yemense, a medicinal plant commonly grown in Saudi Arabia and Yemen, is traditionally used to treat infections, kidney diseases, rheumatism, and diabetes. Extraction of the dried aerial parts of the plant with methanol, followed by further extraction with butanol and chromatography, gave twenty novel neoclerodanes. Their structures, relative configurations and some conformations were determined by MS and 1-D and 2-D NMR techniques. Most were fairly conventional but one contained an unusual stable orthoester, one had its (C-16)-(C-13)-(C-14)-(C-15) (tetrahydro)furan unit present as a succinic anhydride and one had a rearranged carbon skeleton resulting from ring-contraction to give a central octahydroindene bicyclic core, rather than the usual decalin. Mechanisms are proposed for the biosynthetic formation of the orthoester and for the ring-contraction. Four novel neoclerodanes increased the glucose-triggered release of insulin from isolated murine pancreatic islets by more than the standard drug tolbutamide, showing that they are potential leads for the development of new anti-diabetic drugs.

Identification of potential TNF-α inhibitors: from in silico to in vitro studies.

Tumor Necrosis Factor Alpha (TNF-α) is a pleiotropic pro-inflammatory cytokine. It act as central biological regulator in critical immune functions, but its dysregulation has been linked with a number of diseases. Inhibition of TNF-α has considerable therapeutic potential for diseases such as cancer, diabetes, and especially autoimmune diseases. Despite the fact that many small molecule inhibitors have been identified against TNF-α, no orally active drug has been reported yet which demand an urgent need of a small molecule drug against TNF-α. This study focuses on the development of ligand-based selective pharmacophore model to perform virtual screening of plant origin natural product database for the identification of potential inhibitors against TNF-α. The resultant hits, identified as actives were evaluated by molecular docking studies to get insight into their potential binding interaction with the target protein. Based on pharmacophore matching, interacting residues, docking score, more affinity towards TNF-α with diverse scaffolds five compounds were selected for in vitro activity study. Experimental validation led to the identification of three chemically diverse potential compounds with the IC50 32.5 ± 4.5 µM, 6.5 ± 0.8 µM and 27.4 ± 1.7 µM, respectively.

Stimulation of insulin secretion by 5-methylcoumarins and its sulfur analogues isolated from Clutia lanceolata Forssk.

Clutia lanceolata Forssk. (C. lanceolata) is a medicinal plant native to sub-Saharan Africa and the Arabian Peninsula. Phytochemical investigation of the aerial parts of C. lanceolata yielded twenty-one coumarins including methylthio and methylsulfinyl-coumarins. Thirteen of these compounds are reported here for the first time, named as cluteolin A to M. The remaining eight compounds are known but have not been associated previously with C. lanceolata. The structures of the undescribed compounds were elucidated from their 2D NMR and MS spectra. Single crystal X-ray analyses confirmed the structures of eleven compounds. As, in Saudi Arabian tradition, C. lanceolata has been reported to have anti-diabetic and anti-fungal properties, the coumarins were examined for their biological activity. Seven compounds strongly enhanced the glucose-triggered release of insulin by murine pancreatic islets, with two compounds showing more than two-fold enhancement of insulin secretion, compared with the standard drug glimepiride.

New flavonoids from the Saudi Arabian plant Retama raetam which stimulates secretion of insulin and inhibits α-glucosidase.

Retama raetam is a bush which is a member of the family Fabaceae. It is used traditionally in North Africa and Saudi Arabia for the treatment of diabetes. Several flavonoids and alkaloids are already known from this plant. Chromatographic fractionation and purification led to the isolation of three new derivatives of prenylated flavones, retamasin C-E, and four new derivatives of prenylated isoflavones, retamasin F-I, in addition to two isoflavones which have not been previously reported in this plant. Particularly interesting structures included isoflavones containing 3,5-dihydro-2H-2,5-methanobenzo[e][1,4]dioxepine and 3a,8b-dihydro-7-hydroxyfuro[3,2-b]benzo[2,1-d]furan units, both of which are new amongst natural product flavonoids. Five new examples (two flavones and three isoflavones) strongly enhanced the glucose-triggered release of insulin by murine pancreatic islets and one isoflavone was a potent inhibitor of α-glucosidase. This study may rationalise the traditional medicinal use of R. raetam and provide new leads for drug design in the treatment of diabetes.

High-performance thin-layer chromatography based concurrent estimation of biomarkers ent-phyllanthidine and rutin in the dried aerial parts of Flueggea virosa.

The biomarkers are needed to be defined for standardization purposes so that safe and effective herbal formulations can be catered to the society. There is an urgent need for statistical support of herbal drugs because most of the herbal products are still used in the non-standardized form. This study is based on the development of a simple and sensitive RP-HPTLC method for concurrent estimation of two biomarkers ent-phyllanthidine and rutin in the methanol extract of aerial parts of Flueggea virosa. The developed method was found to be simple, economic and sensitive. Separation and quantification were performed with acetonitrile: water (4:6 V/V) used as the mobile phase on glass-backed RP-HPTLC plate. Detection of absorption maxima and quantification was done at 310 nm of UV region. The developed chromatographic system was found to give a sharp band for ent-phyllanthidine and rutin at Rf 0.73 ± 0.01 and 0.68 ± 0.01, respectively. The linearity ranges for ent-phyllanthidine, and rutin were found to be 200-1600 ngband-1 and 100-1400 ngband-1, respectively, with correlation coefficients (r2 values) of 0.998 and 0.997, respectively. The percentage of ent-phyllanthidine and rutin was found to be 9.121 ± 0.02% and 1.018 ± 0.04% (w/w), respectively. The resolution of bands and separation of constituents in FVME exhibited the perfect optimization of the developed method. The validation statistics supports the proposed method for standardizing crude drugs as well as formulations of a natural product containing ent-phyllanthidine and rutin.

Neoclerodane Diterpenoids from Reehal Fatima, Teucrium yemense.

Teucrium yemense (Defl), locally known as Reehal Fatima, is a medicinal plant commonly grown in Saudi Arabia and Yemen. Phytochemical investigation of the aerial parts of T. yemense yielded six new neoclerodane diterpenoids, namely fatimanol A-E (1, 2, 3, 5, and 6) and fatimanone (4), and the known teulepicephin (7). As both the Teucrium genus and the related Lamiaceae family have previously been widely reported to possess anthelmintic and antimicrobial activities, the structural and biological characterization of the seven diterpenoids was pursued. The structures of the new compounds were elucidated from their 2D NMR and MS profiles and by comparison to related compounds. The structure of fatimanol D (5) was confirmed by X-ray crystallographic analysis. The new structures contribute to the breadth of knowledge of secondary metabolites in this genus.

Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita.

CONTEXT: Extensive research on Rhus (Anacardiaceae) shows their antioxidant potential, which warrants further evaluation of its other species. OBJECTIVE: To perform a comparative antioxidant assay on extracts of R. retinorrhoea and R. tripartita, including sakuranetin quantification by a validated HPTLC method. MATERIALS AND METHODS: In vitro antioxidant assay was performed on chloroform and ethanol extracts of R. retinorrhoea Steud. ex Oliv. (RRCE and RREE) and R. tripartita (Ucria) Grande (RTCE and RTEE) by DPPH radical scavenging (at 31.25, 62.5, 125, 250 and 500 µg/mL concentrations) and ß-carotene-linoleic acid bleaching methods at 500 µg/mL concentration. Densitometric HPTLC method was developed and validated using toluene: ethyl acetate: methanol (8:2:0.2; v/v/v) as mobile phase, executed on glass-backed silica gel F254 plate and scanned at 292 nm. RESULTS: Antioxidant activity of Rhus extracts tested by the two methods (DPPH/BCB) was found in order of RTEE > RREE > RTCE > RRCE with IC50 118.67/256.26, 315.75/82.35, 827.92/380.0 and 443.69/292.75, respectively. Scanning of the HPTLC plate provided an intense peak of sakuranetin at Rf = 0.59. The estimated sakuranetin content in the dry weight of the extracts was highest in RREE (27.95 µg/mg) followed by RRCE (25.22 µg/mg), RTEE (0.487 µg/mg) and RTCE (0.0 µg/mg). Presence of sakuranetin in RREE, RRCE and RTEE supported the highest antioxidant property of the two Rhus species. Nonetheless, low sakuratenin in R. tripartita indicated the presence of other bioactive constituents responsible for synergistic antioxidant activity. CONCLUSION: The developed HPTLC method therefore guarantees its application in quality control of commercialized herbal drugs and formulations containing sakuranetin.

STUDIES ON WOUND HEALING ACTIVITY OF SOME EUPHORBIA SPECIES ON EXPERIMENTAL RATS.

BACKGROUND: Plants of Euphorbiaceae are used in folkloric medicines in variety of ailments and well known for chemical diversity of their isoprenoid constituents. This study was carried out to explore the preliminary wound healing potential of four Euphorbia species (E. consorbina 1, E. consorbina 2, E. inarticulata, E. balsamifera and E. schimperi). MATERIALS AND METHODS: Excision wound surface of the animals were topically treated with ethyl acetate and methanol extracts of plants at a dose of 400 mg/kg body weight for twenty days. Povidone-iodine ointment was used as a reference drug. Wound contraction measurement and period of epithelialization were used to assess the effect of plants extracts on wound repairing. RESULTS: The groups treated with methanol extracts of E. balsamifera and E. schimperi showed profound effects, high rate of wound contraction (100%) and decrease in epithelization period 19.00±0.40 and 18.50±0.64 respectively, followed by methanol extracts of E. consorbina 2, ethyl acetate extract of E. inarticulata and ethyl acetate extracts of E. consorbina 2 which showed significant (P <0.001) wound contraction and decrease in epithelization period. Conversely ethyl acetate extract of E. consorbina 1, E. balsamifera and E. schimperi and methanol extract of E. Consorbina 1 and E. Inarticulata treated groups was not showing significant wound healing. Methanol extracts of E. balsamifera and E. schimperi were also tested for their safety margin and found safe up to dose of 2000mg/kg body weight. CONCLUSION: Topical application of methanol extracts of E. balsamifera and E. schimperi have potential wound healing activity which is identical with standard drug Povidone-iodine.

Operculina turpethum attenuates N-nitrosodimethylamine induced toxic liver injury and clastogenicity in rats.

The root extract of Operculina turpethum (OTE) has been used as an anti-inflammatory, purgative, and hepato-protective agent. N-Nitrosodimethylamine (NDMA) is a potent hepatotoxin that induces fibrosis of the liver. In the present study, we examined the therapeutic effects of OTE root extract against NDMA-induced hepatotoxicity and clastogenicity in rats. Hepatic fibrosis was induced in adult male albino rats through serial intraperitoneal administrations of NDMA at a concentration of 10mg/kg body weight on three consecutive days of each week over a period of three weeks. A group of rats received OTE orally in doses of 75, 150 and 200mg/kg body weight at 5h after the administration of NDMA. The controls and treated animals were sacrificed on days-7, 14 and 21 after the start of the administration of NDMA. The progression of hepatic fibrosis as well as the amelioration effect of OTE was evaluated through histopathologically as well as by immunohistochemical staining for the activation of hepatic stellate cells. Alterations in serum and liver biochemical parameters and LDH isoenzymes were also studied. Serial administration of NDMA resulted in well formed fibrosis in the liver and induction of micronuclei in the bone marrow cells. Staining of alpha-SMA demonstrated activated stellate cells from day-7 onwards which was dramatically increased on day-21. An elevation of micronuclei count, liver function enzymes, serum hydroxyproline levels and LDH isoenzymes 4 and 5 were also observed. All these changes were remarkably reduced in OTE administered animals and fibrogenesis was completely absent. Our results suggest that OTE has hepatoprotective and anti-clastogenic effects against NDMA-induced hepatic fibrosis. Therefore OTE may be used as a hepatoprotective agent against various liver diseases including toxic liver injury.