Structure-Based Virtual Screening to Discover Potential Lead Molecules for the SARS-CoV-2 Main Protease.

The COVID-19 disease is caused by a new strain of the coronavirus family (SARS-CoV-2), and it has affected at present millions of people all over the world. The indispensable role of the main protease (Mpro) in viral replication and gene expression makes this enzyme an attractive drug target. Therefore, inhibition of SARS-CoV-2 Mpro as a proposition to halt virus ingression is being pursued by scientists globally. Here we carried out a study with two objectives: the first being to perform comparative protein sequence and 3D structural analysis to understand the effect of 12 point mutations on the active site. Among these, two mutations, viz., Ser46 and Phe134, were found to cause a significant change at the active sites of SARS-CoV-2. The Ser46 mutation present at the entrance of the S5 subpocket of SARS-CoV-2 increases the contribution of other two hydrophilic residues, while the Phe134 mutation, present in the catalytic cysteine loop, can cause an increase in catalytic efficiency of Mpro by facilitating fast proton transfer from the Cys145 to His41 residue. It was observed that active site remained conserved among Mpro of both SARS-CoVs, except at the entrance of the S5 subpocket, suggesting sustenance of substrate specificity. The second objective was to screen the inhibitory effects of three different data sets (natural products, coronaviruses main protease inhibitors, and FDA-approved drugs) using a structure-based virtual screening approach. A total of 73 hits had a combo score >2.0. Eight different structural scaffold classes were identified, such as one/two tetrahydropyran ring(s), dipeptide/tripeptide/oligopeptide, large (approximately 20 atoms) cyclic peptide, and miscellaneous. The screened hits showed key interactions with subpockets of the active site. Further, molecular dynamics studies of selected screened compounds confirmed their perfect fitting into the subpockets of the active site. This study suggests promising structures that can fit into the SARS-CoV-2 Mpro active site and also offers direction for further lead optimization and rational drug design.

Synthesis of N-substituted indole derivatives as potential antimicrobial and antileishmanial agents.

Leishmaniasis and microbial infections are two of the major contributors to global mortality and morbidity rates. Hence, development of novel, effective and safer antileishmanial and antimicrobial agents having reduced side effects are major priority for researchers. Two series of N-substituted indole derivatives i.e. N-substituted indole based chalcones (12a-g) and N-substituted indole based hydrazide-hydrazones (18a-g, 19a-f, 21 a-g) were synthesized. The synthesized compounds were characterized by 1H NMR, 13C NMR, Mass and FT-IR spectral data. Further these derivatives were evaluated for their antimicrobial potential against Escherichia coli, Bacillus subtilis, Pseudomonas putida and Candida viswanathii, and antileishmanial potential against promastigotes of Leishmania donovani. Compounds 18b, 18d and 19d exhibited significant activity with an IC50 of 0.19 ± 0.03 µM, 0.14 ± 0.02 µM and 0.16 ± 0.06 µM against B. subtilis which was comparable to chloramphenicol (IC50 of 0.25 ± 0.03 µM). Compounds 12b and 12c exhibited an IC50 of 24.2 ± 3.5 µM and 21.5 ± 2.1 µM in the antileishmanial assay. Binding interactions of indole based hydrazide-hydrazones were studied with nitric oxide synthase in silico in order to understand the structural features responsible for activity.

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220.

MsrA, an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8-fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.

Influence of Drug-Polymer Interactions on Dissolution of Thermodynamically Highly Unstable Cocrystal.

Solubility advantage of thermodynamically highly unstable cocrystals, which undergo solution-mediated phase transformation (SMPT) in less than 1 min, does not translate to enhanced dissolution. The present study was aimed to understand the impact of polymeric additives on dissolution of thermodynamically highly unstable cocrystal with specific emphasis on influence of drug-polymer interactions. Exemestane-maleic acid was selected as a model cocrystal with SMPT time of <30 s and eutectic constant ( Keu) of 75475. Hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and polyvinylpyrrolidone (PVP) were selected as polymers for a dissolution study based on measurement of induction time using precipitation study. In the presence of 0.2% w/v of HPC, the cocrystal showed significantly higher drug release (∼3-fold) as compared with the cocrystal in the absence of predissolved polymers. Differential dissolution profiles of the cocrystal were observed with each polymer and the order of increasing dissolution rate was found to be HPC ≈ HPMCAS > PVP. The molecular basis of the differential dissolution performance was investigated using infrared spectroscopy, solution-state nuclear magnetic resonance spectroscopy, and nuclear Overhauser effect spectroscopy (NOESY). The polymers with stronger interactions with drug in the cocrystal (HPMCAS and HPC) displayed higher dissolution rate as compared with that of no intermolecular interaction (PVP). The study also highlighted that, despite no influence of the polymers on the cocrystal SMPT, dissolution enhancement was achieved. This was attributed to small-sized drug crystals (1-3 µm) generated from the supersaturation-mediated crystallization and improved solvation due to drug-polymer interactions. These findings have implications on development of drug products using thermodynamically unstable cocrystals.

Effect of crude extracts and purified compounds of Alpinia galanga on nutritional physiology of a polyphagous lepidopteran pest, Spodoptera litura (Fabricius).

Secondary plant metabolites play an important role in providing protection to plants against herbivore insect pests. Keeping in view the increasing importance of biopesticides, the crude extracts from different plants are being investigated for insecticidal activities. Alpinia galanga, a medicinal plant belonging to family Zingiberaceae exhibits a wide range of biological activities. In the present study, crude extracts of A. galanga and its purified compounds i.e. 1'-acetoxychavicol acetate and galangin were evaluated for their effect on various nutritional parameters of Spodoptera litura (Fab.). All the extracts exhibited a significant influence on relative growth and consumption rates as well as efficiency of conversion of ingested and digested food. Ethyl acetate extract was found to be the most effective causing significant reduction in values of RGR, RCR, ECI and ECD of S. litura larvae in comparison to control larvae. The highest concentration of the ethyl acetate extract (2500 ppm) resulted in 44.95%, 10.99%, 38.08% and 37.04% decrease respectively in RGR, RCR, ECI and ECD in comparison to control. The purified compounds also showed inhibitory effects on various nutritional parameters. 1'-Acetoxychavicol acetate was found to be more effective in comparison to galangin.

Synthesis of C-2 and C-3 substituted quinolines and their evaluation as anti-HIV-1 agents.

A plenty of natural products and synthetic derivatives containing quinoline moiety have been reported to possess various pharmacological activities. Quinolines such as 2-styrylquinolines and 8-hydroxyquinolines are extensively studied for their anti-HIV-1 activity and found to act mainly through HIV-1 integrase enzyme inhibition. In continuation of our efforts to search for newer anti-HIV-1 molecules, thirty-one quinoline derivatives with different linkers to ancillary phenyl ring were synthesized and evaluated for in vitro anti-HIV-1 activity using TZM-bl assays. Compound 31 showed higher activity in TZM-bl cell line against HIV-1VB59 and HIV-1UG070 cell associated virus (IC50 3.35 ±â€¯0.87 and 2.57 ±â€¯0.71 µM) as compared to other derivatives. Compound 31 was further tested against cell free virus HIV-1VB59 and HIV-1UG070 (IC50 1.27 ±â€¯0.31 and 2.88 ±â€¯1.79 µM, TI 42.20 and 18.61, respectively). This lead molecule also showed good activity in viral entry inhibition assay and cell fusion assay defining its mode of action. The activity of compound 31 was confirmed by testing against HIV-1VB51 in activated peripheral blood mononuclear cells (PBMCs). Binding interactions of 31 were compared with known entry inhibitors.

From Leaf Metabolome to In Vivo Testing: Identifying Antifeedant Compounds for Ecological Studies of Marsupial Diets.

Identifying specific plant secondary metabolites that influence feeding behavior can be challenging, but a solid understanding of animal preferences can guide efforts. Common brushtail possums (Trichosurus vulpecula) predominantly eat Eucalyptus species belonging to the subgenus Symphyomyrtus, and avoid eating those belonging to the Monocalyptus subgenus (also called subgenus Eucalyptus). Using an unbiased (1)H NMR metabolomics approach, a previous study identified unsubstituted B ring flavanones in most species of monocalypts examined, whereas these compounds were absent from symphyomyrtles. We hypothesised that unsubstituted B ring flavanones act as feeding deterrents for common brushtail possums. In the current study, we tested this hypothesis by comparing how much possums ate of a basal diet, with diets containing one of four structurally related compounds; pinocembrin, flavanone (unsubstituted B ring flavanones), chrysin (the flavone analogue of pinocembrin), and naringenin (a flavanone with B ring substitution). We found that pinocembrin and flavanone deterred feeding relative to the basal diet, but that chrysin and naringenin did not at equivalent concentrations. Thus, unsubstituted B-ring flavanones may explain why brushtail possums avoid eating monocalypt species. Furthermore, small differences in the structure of secondary compounds can have a large impact on antifeedant properties. These results demonstrate that metabolomics can be a valuable tool for ecologists seeking to understand herbivore feeding preferences.

Qualitative and quantitative analysis of Potentilla fulgens roots by NMR, matrix-assisted laser desorption/ionisation with time-of-flight MS, electrospray ionisation MS/MS and HPLC/UV.

INTRODUCTION: Potentilla fulgens is a commonly used folk medicine by natives of northeast India, Nepal and Bhutan and is rich in polyphenolic and triterpene constituents. OBJECTIVE: To identify chemomarkers in the roots of P. fulgens by an interplay of (13)C-NMR, matrix-assisted laser desorption/ionisation with time-of-flight (MALDI/TOF) MS, electrospray ionisation (ESI) MS/MS and HPLC/UV. MATERIAL AND METHODS: The (13)C-NMR spectrum of crude methanolic extract was recorded in deuterated dimethyl sulphoxide. For MALDI/TOF/MS analysis, 2,5-dihydroxybenzoic acid was used as the matrix. For determination of chemical constituents, two independent simple isocratic HPLC/UV methods for monomeric/oligomeric flavanols and triterpene acids were developed and validated. RESULTS: The (13)C-NMR spectrum of the methanolic extract indicated the presence of B-type oligomeric polyphenolics containing mainly epicatechin/catechin (epicat/cat) and epiafzelechin/afzelechin (epiafz/afz) as the monomeric units. Several isobaric monomeric and oligomeric flavanols and triterpenoids were tentatively identified by MALDI/TOF/MS and ESI/MS/MS. Fourteen compounds (four monomeric and five dimeric flavanols and five triterpene acids) were isolated using repeated column chromatography and semi-preparative HPLC, and were quantitated using HPLC/UV. CONCLUSION: It is evident from these analyses that roots of P. fulgens contain flavans, including oligomeric flavanols, as major constituents followed by triterpene acids. The methods described can be applied to other Potentilla species to identify their constituents.

Antileishmanial compounds from Moringa oleifera Lam.

The antileishmanial activity of extracts and phytoconstituents of Moringa oleifera Lam. was investigated in vitro against promastigotes of Leishmania donavani. The 70% ethanolic extract of roots and the methanolic extract of leaves showed moderate inhibitory activity with IC50 values of 83.0 microg/ml and 47.5 microg/ml, respectively. Antileishmanial activity of the methanolic extract of leaves increased upon fractionation, as its ethyl acetate fraction was found to be more active with an IC50 value of 27.5 microg/ml. The most active antileishmanial compound niazinin, a thiocarbamate glycoside isolated from this fraction, showed an IC50 value of 5.25 microM. Results presented in this study indicate that extracts from M. oleifera may be developed as an adjuvant therapy for the treatment of leishmaniasis.

Tetrahydroisoquinolines - an updated patent review for cancer treatment (2016 - present).

INTRODUCTION: Cancer is a prominent cause of death globally, triggered by both non-genetic and genetic alterations in genes influenced by various environmental factors. The tetrahydroisoquinoline (THIQ), specifically 1,2,3,4-tetrahydroisoquinoline serves as fundamental element in various alkaloids, prevalent in proximity to quinoline and indole alkaloids. AREA COVERED: In this review, the therapeutic applications of THIQ derivatives as an anticancer agent from 2016 to 2024 have been examined. The patents were gathered through comprehensive searches of the Espacenet, Google patent, WIPO, and Sci Finder databases. The therapeutic areas encompassed in the patents include numerous targets of cancer. EXPERT OPINION: THIQ analogues play a crucial role in medicinal chemistry, with many being integral to pharmacological processes and clinical trials. Numerous THIQ compounds have been synthesized for therapeutic purposes, notably in cancer treatment. They show great promise for developing anticancer drugs, demonstrating strong affinity and efficacy against various cancer targets. The creation of multi-target ligands is a compelling avenue for THIQ-based anticancer drug discovery.

An adamantyl-caffeoyl-anilide exhibits broad-spectrum antibacterial activity by inhibiting FtsZ assembly and Z-ring formation.

Several harmful bacteria have evolved resistance to conventional antibiotics due to their extensive usage. FtsZ, a principal bacterial cell division protein, is considered as an important drug target to combat resistance. We identified a caffeoyl anilide derivative, (E)-N-(4-(3-(3,4-dihydroxyphenyl)acryloyl)phenyl)-1-adamantylamide (compound 11) as a new antimicrobial agent targeting FtsZ. Compound 11 caused cell elongation in Mycobacterium smegmatis, Bacillus subtilis, and Escherichia coli cells, indicating that it inhibits cell partitioning. Compound 11 inhibited the assembly of Mycobacterium smegmatis FtsZ (MsFtsZ), forming short and thin filaments in vitro. Interestingly, the compound increased the rate of GTP hydrolysis of MsFtsZ. Compound 11 also impeded the assembly of Mycobacterium tuberculosis FtsZ. Fluorescence and absorption spectroscopic analysis suggested that compound 11 binds to MsFtsZ and produces conformational changes in FtsZ. The docking analysis indicated that the compound binds at the interdomain cleft of MsFtsZ. Further, it caused delocalization of the Z-ring in Mycobacterium smegmatis and Bacillus subtilis without affecting DNA segregation. Notably, compound 11 did not inhibit tubulin polymerization, the eukaryotic homolog of FtsZ, suggesting its specificity on bacteria. The evidence indicated that compound 11 exerts its antibacterial effect by impeding FtsZ assembly and has the potential to be developed as a broad-spectrum antimicrobial agent.

Wound-healing effect of topical nanoemulsion-loaded cream and gel formulations of Hippophae rhamnoides L. (sea buckthorn) fruit oil and their acute dermal toxicity study on female SD rats.

OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of topical nanoemulsion (NE)-loaded cream and gel formulations of Hippophae rhamnoides L. (sea buckthorn [SBT]) fruit oil for wound healing. MATERIALS AND METHODS: The NE-loaded cream and gel formulations of H. rhamnoides L. (SBT) fruit oil (IPHRFH) were prepared and evaluated for their wound-healing activity on female Sprague-Dawley (SD) rats. They were further divided into groups (seven) and the wound-healing activity was determined by measuring the area of the wound on the wounding day and on the 0th, 4th, 8th, and 10th days. The acute dermal toxicity of the formulations was assessed by observing the erythema, edema, and body weight (BW) of the rats. RESULTS: The topical NE cream and gel formulations of H. rhamnoides L. (SBT) fruit oil showed significant wound-healing activity in female SD rats. The cream formulation of IPHRFH showed 78.96%, the gel showed 72.59% wound contraction on the 8th day, whereas the positive control soframycin (1% w/w framycetin) had 62.29% wound contraction on the 8th day. The formulations also showed a good acute dermal toxicity profile with no changes significantly affecting BW and dermal alterations. CONCLUSIONS: The results of this study indicate that topical NE-loaded cream and gel formulation of H. rhamnoides L. (SBT) fruit oil are safe and effective for wound healing. The formulations showed no signs of acute dermal toxicity in female SD rats.

Determining and reporting purity of organic molecules: why qNMR.

Although NMR has been routinely used to determine/estimate relative number of protons for structure elucidation, it has been rarely used to determine and report the purity of organic compounds. Through this paper, we want to emphasize on routine use of quantitative NMR (qNMR) for this purpose. The results of qNMR can be routinely considered as documentation of purity much like other established methods (HPLC, elemental analysis and differential scanning calorimetry). qNMR is a fast, easy, accurate and non-destructive alternate to speed up the whole analytical process and serves the purpose of both identification and purity determination of compounds using single technique.

Simultaneous quantitation of nine carbazole alkaloids from Murraya koenigii (L.) Spreng by 1H qNMR spectroscopy.

Murraya koenigii leaves are widely used as a spice and also have several biological activities. The major active constituents are carbazole alkaloids. Quantitation by HPLC or HPTLC requires pure marker compounds, whereas nuclear magnetic resonance spectroscopy can be used as a quantitative technique without the requirement of a pure marker compound. An alkaloid-rich fraction was prepared from the leaves and a validated qNMR method was developed for the quantitation of nine carbazole alkaloids, namely mahanimbine, girinimbine, koenimbine, koenine, kurrayam, mukonicine, isomahanimbine, euchristine B and bismahanine. One of the major compounds, koenimbine, was isolated and quantified by HPTLC to compare the results. The results obtained by qNMR were compared with the reported yields of these compounds.

Cisplatin conjugation with an exopolysaccharide extracted from Lactobacillus gasseri potentiates its efficacy and attenuates its toxicity.

The development of newer cisplatin analogs is constantly being investigated owing to its low solubility, poor pharmacokinetics, and dose-related toxicity. In order to address the limitations of current cisplatin therapy, the present study was undertaken. Cisplatin conjugation with an exopolysaccharide extracted from Lactobacillus gasseri (LG-EPS) showed remarkably enhanced and selective anticancer activity by targeting tumor cells overexpressing glucose transporter 1 (GLUT1). The EPS-cisplatin complex exhibited a 600-fold increase in aqueous solubility with a better pharmacokinetic profile (longer half-life) in comparison to cisplatin. Cell viability assay and western blotting demonstrated a strong correlation between the cytotoxicity profile and GLUT1 expressions in different cell lines. The concentration of DNA-bound platinum was also found to be significantly higher in EPS-cisplatin-treated cells. Quercetin, a competitive inhibitor of GLUTs, was shown to prevent this selective uptake of EPS-cisplatin complex. Surprisingly, EPS-cisplatin complex showed an exceptionally safer profile (4 times the maximum tolerated dose of cisplatin) in the acute toxicity study and was also more efficacious against the xenograft mice model. The study suggests that this green glycoconjugation can be an effective and safer strategy to broaden the therapeutic potential of anti-cancer drugs in general and cisplatin in particular.

Antioxidant and antiproliferative activities of anthocyanin/ellagitannin-enriched extracts from Syzygium cumini L. (Jamun, the Indian Blackberry).

Colored fruits, particularly berries, are highly chemoprotective because of their antioxidant, antiproliferative, and antiinflammatory activities. We report the cancer chemoprotective potential of Syzygium cumini L., commonly known as jamun or Indian blackberry. Anthocyanins and other polyphenolics were extracted with acidic ethanol and enriched by amberlite XAD7/HP20 (1:1). The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins, and 1.15% total polyphenolics. Jamun seed contained no detectable anthocyanins but had higher amounts of ellagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) than the pulp powder. Upon acid hydrolysis, the pulp extract yielded 5 anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%), delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Extracts of both jamun pulp (1,445 ± 64 µmol of trolox equivalent (TE)/g) and seeds (3,379 ± 151 µM of TE/g) showed high oxygen radical absorbance capacity. Their high antioxidant potential was also reflected by 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)- and 2,2-diphenyl-1-picrylhydrazyl-scavenging and ferrous ion-chelating activities. We also analyzed antiproliferative activity of jamun extracts against human lung cancer A549 cells. The hydrolyzed pulp and seed extracts showed significant antiproliferative activity. However, unhydrolyzed extracts showed much less activity. These data showed that in addition to 5 anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.

Heat stable antimicrobial activity of Burkholderia gladioli OR1 against clinical drug resistant isolates.

BACKGROUND & OBJECTIVES: Drug resistant microbes are a serious challenge to human health. During the search for novel antibiotics/inhibitors from the agricultural soil, a bacterial colony was found to inhibit the growth of clinical isolates including Staphylococcus (resistant to amikacin, ciprofloxacin, clindamycin, clinafloxacin, erythromycin, gentamicin and methicillin) and Candida (resistant to fluconazole and itraconazole). The culture was identified as Burkholderia gladioli and produced at least five different antimicrobial compounds which were highly stable at high temperature (121 o C) and in the broad pH range (3.0-11.0). We report here the antimicrobial activity of B. gladioli against drug resistant bacterial pathogens. METHODS: The bacterial culture was identified using morphological, biochemical and 16S rRNA gene sequencing techniques. The antimicrobial activity of the identified organism against a range of microbial pathogens was checked by Kirby-Bauer's disc diffusion method. The antimicrobial compounds in the cell free supernatant were chloroform-extracted and separated by thin layer chromatography (TLC). RESULTS: B. gladioli OR1 exhibited broad spectrum antimicrobial activity against drug resistant clinical isolates belonging to various genera of bacteria (Staphylococcus, Enterobacter, Enterococcus, Acinetobacter and Citrobacter) and a fungus (Candida). Based on TLC profile and bioautography studies, the chloroform extract of B. gladioli OR1 consisted of at least three anti-staphylococcal and two anti-Candida metabolites. The antimicrobial activity was heat stable (121 o C/20 min) as well as pH stable (3.0-11.0). INTERPRETATION & CONCLUSIONS: The bacterial soil isolate, B. gladioli OR1 possessed the ability to kill various drug resistant bacteria and a fungus. This organism produced many antimicrobial metabolites which might have the potential to be used as antibiotics in future.

Isolation and quantitation of ecologically important phloroglucinols and other compounds from Eucalyptus jensenii.

INTRODUCTION: Eucalyptus jensenii has not been explored for its phytoconstituents except for its essential oil although a formylated phloroglucinol compound jensenone has been the focus of several ecological studies. OBJECTIVE: i) To optimise the extraction conditions for preparative scale isolation of jensenone and other secondary metabolites from leaves. (ii) To develop an RP-HPLC method for simultaneous determination of phloroglucinols and other compounds in Eucalyptus leaves. METHODOLOGY: Jensenone and other compounds were isolated from acetone extract using VLC over TLC grade silica. HPLC quantitation of isolated compounds was undertaken on a C18-column using acetonitrile-water (2% formic acid) gradient elution. RESULTS: Extraction conditions for isolation of jensenone were optimised and more than 99% pure jensenone was obtained in a yield of 2.1% from the dried leaves. Ten phloroglucinol compounds, including a new nuclear methylated phloroglucinol named 4-O-demethyl miniatone, and two triterpenoids were also isolated. A RP-HPLC method was developed for simultaneous quantitation of phloroglucinols and other compounds in different extracts of E. jensenii leaves. A total of 19 compounds reported from different species of Eucalyptus was separated by this method. CONCLUSION: The method described for isolation of jensenone is a simple, rapid and low-cost procedure amenable to the preparative scale. A new monomeric phloroglucinol compound was isolated and characterised from the acetone extract of E. jensenii leaves. The HPLC method developed can be applied successfully to different eucalypt matrices for precise and accurate determination of 19 secondary metabolites belonging to different chemical classes.

Quantitative NMR: an applicable method for quantitative analysis of medicinal plant extracts and herbal products.

INTRODUCTION: Quantitative analysis and standardisation of plant extracts or herbal products is a tedious process requiring time-consuming sample preparation and analytical method development for the resolution of analyte peaks from the complex natural extract. Quantitative analysis by HPLC requires a pure authentic standard of the compound being quantified. We report here a quantitative NMR (qNMR) method for quantitative analysis of three medicinal plant extracts and their herbal products without the need of authentic standards. Quantitation can be done by using any commercially available pure sample as an internal reference standard. OBJECTIVE: To develop a reliable method for standardisation and quantitative analysis of extracts from medicinal plants Eugenia jambolana, Withania somnifera and Aegle marmelos and their herbal products using qNMR. METHODOLOGY: The (1) H-NMR spectra of known amounts of crude plant extracts with internal standards were recorded in deuterated solvents and quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds and the internal reference standard. Anthocyanins [delphinidin-3,5-diglucoside (1), petunidin-3,5-diglucoside (2) and malvidin-3,5-diglucoside (3)] for E. jambolana fruit extract and imperatorin (4) for A. marmelos fruit extract were selected as marker constituents for quantitation and 1,3,5-trimethoxybenzene (TMB) was used as an internal reference standard. Total withanolide content was determined for W. somnifera using 2,4-diformyl phloroglucinol as an internal reference standard. RESULTS: The (1) H-NMR gave a linear response for the marker constituents, anthocyanins, withaferin A and imperatorin. Using the described method, the amount of anthocyanins in Amberlite(R) XAD7HP and Sephadex enriched extracts of E. jambolana was 3.77% and 9.57% (delphinidin-3,5-diglucoside), 4.72% and 12.0% (petunidin-3,5-diglucoside), 6.55% and 15.70% (malvidin-3,5-diglucoside), respectively. The imperatorin content was 0.424% in A. marmelos fruit and 0.090 % and 0.114% in sharbat and candies. Total withanolides content was 0.191% in the chloroform extract and 0.234% in the capsule extract. These values are in accordance with HPLC results. CONCLUSION: This qNMR technique could be used for NMR fingerprinting and quantitation for the purpose of quality control and standardisation of many plant-based herbal products and medicines and has certain advantages over HPLC.

Terpenoidal constituents of Eucalyptus loxophleba ssp. lissophloia.

CONTEXT: Eucalyptus has been a source of a number of biologically active compounds. The anti-leishmanial activity of terpenoids from Eucalyptus loxophleba (Benth.) ssp. lissophloia (Myrtaceae) has not yet been investigated. OBJECTIVE: Isolation of the terpenoidal constituents for evaluation of in vitro anti-leishmanial activity against the Leishmania donovani (Dd8 strain) promastigotes. MATERIALS AND METHODS: The chloroform-methanol (8:2) extract of dried leaves of Eucalyptus loxophleba was used to isolate terpenoidal constituents employing solvent partitioning, column chromatography and preparative high performance liquid chromatography and characterized from spectral data. The anti-leishmanial activity of the isolated compounds was tested in vitro using an Alamar blue assay against a culture of L. donovani (Dd8 strain) promastigotes. RESULTS: Two new naturally occurring triterpenes, named loxanic acid and 3-acetyl loxanic acid together with four known ursane triterpenoids and one bis-monoterpene glycoside, cuniloside B isolated from the leaves showed anti-leishmanial activity (IC(50) 133 to 235 µM) against the promastigotes of the tested strain. CONCLUSION: The terpenes isolated from the leaves of E. loxophleba showed moderate anti-leishmanial activity.