Mahendra Padurang Darokar

Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils

The presence of certain metabolites has been observed to interfere with DNA isolation procedures and downstream reactions such as DNA restriction, amplification and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yields with a single protocol, and thus, even closely related species may require different isolation protocols. Here we describe the essential steps of a rapid DNA isolation protocol that can be used for diverse medicinal and aromatic plants, which produce essential oils and secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides, terpenes and quinones. The procedure is applicable to dry as well as fresh plant tissues. This protocol, in our experiments, permitted isolation of DNA from tissues of diverse plant species and produced fairly good yields. The isolated DNA proved amenable to PCR amplification and restriction digestion.

Phenotypic and RAPD diversity among Cymbopogon winterianus Jowitt accessions in relation to Cymbopogon nardus Rendle

The species Cymbopogon winterianus Jowitt is believed to have originated from the well-known species Cymbopogon nardus, type Maha Pengiri, referred to as Ceylonese (Sri Lankan) commercial citronella. Cymbopogon winterianus Jowitt was named after Winter, who raised its population as a separate species in the 19th century. C. winterianus was introduced into Indonesia and became commercially known as the Javanese citronella. The Javanese type C. winterianus material was introduced into India for the commercial cultivation of this crop during 1959. Varieties of this species have been developed later by the use of breeding procedures from the same introduced material. A comparative analysis of yields of herb, oil percentage and oil constituents for eight prevalent C. winterianus cultivars comparing them between themselves as well as against an accession of C. nardus has been carried out. All these accessions were analyzed at the molecular level for the similarity and genetic distances through RAPD profiling, using 20 random primers. More than 50% divergence was observed for all the C. winterianus accessions in relation to C. nardus accession CN2. The clustering based on the similarity matrices showed a major cluster of six accessions, consisting of two sub-clusters. The accession C. nardus CN2 got carved out along with two C. winterianus accessions, CW2 and CW6. On the other hand, the accessions CW2 and CW6 demonstrated distinct identities compared to CN2 at the DNA level.

In vitro and in vivo synergistic interaction of substituted chalcone derivatives with norfloxacin against methicillin resistant Staphylococcus aureus

Thirty chalcone derivatives were synthesized via a base catalyzed Claisen Schmidt condensation and evaluated for their anti-methicillin-resistant Staphylococcus aureus (MRSA) activity alone and in combination with norfloxacin. Among these, 5 derivatives namely trans-3-(1H-indol-3-yl)-1-(4′-benzyloxyphenyl)-2-propen-1-one (2), 1-(4″-biphenyl)-3-(3′4′-dihydroxyphenyl)-2-propen-1-one (11), 1-(4″-hydroxy-3″-methylphenyl)3-(4′-hydroxyphenyl)-2-propen-1-one (14), 3-(4′-chlorophenyl)-1-(4″-hydroxyphenyl)2-propen-1-one (17), and LTG-oxime (27) showed significant antibacterial activity with MIC 12.5–50 µg mL−1 respectively. In combination studies, derivatives 2 and 14 significantly reduced the MIC of norfloxacin by up to 16 fold (FICI < 0.5), while derivatives 11, 17 and 27 reduced it by up to eight fold (FICI ≤ 0.5). Flow cytometry analysis results clearly indicated that derivatives 2 and 14 significantly promote the accumulation and inhibition of the Et-Br efflux, which was further validated through spectrofluorimeter using clinical isolate MRSA-ST2071. In systemically infected Swiss albino mice model, both the compounds significantly (P < 0.001, P < 0.01) lowered the systemic bacterial load in blood, liver, kidney, lung and spleen tissues. This study supports the promising use of chalcones in the development of economical antibacterial combinations.

Chemical composition and bioactivity of Boswellia serrata Roxb. essential oil in relation to geographical variation

Abstract Oleo–gum–resin of Boswellia serrata Roxb. (Burseraceae) was collected from Shivpuri forest situated in northwestern district of Madhya Pradesh, India. The commercial samples were purchased from Mandsaur and Neemuch districts (the major herbal markets of Madhya Pradesh) for comparative study of their essential oils. The average essential oil contents in the commercial Neemuch samples (11.1%) were much better as compared to the wild collection from Shivpuri (6.1%) as well as commercial collection from Mandsaur (6.9%). The commercial samples contained higher percentage of monoterpene hydrocarbons (81.9–88.1%) including α-thujene (61.4–69.8%) as the major compound. The wild habitat contained higher percentage of oxygenated monoterpenoids/benzenoids (15.7%) and sesquiterpenes (19.2%) including α-terpineol (7.8%), terpinyl isobutyrate (5.1%), and eudesmol (11.5%). Further, the antimicrobial activities of these collections showed remarkable variation among the essential oils. On the other hand, wild habitat contained improved percentage of semi-polar compounds (34.9%) which might be responsible for enhanced antibacterial activity. Similarly, the winter collection from Neemuch (BS-N2) showed significant antioxidant activity, whereas the wild habitat did not show any antioxidant activity. The higher percentage of monoterpene hydrocarbons in commercial samples, especially δ-3-carene, α-pinene, and α-thujene, might be responsible for enhanced antifungal and antioxidant activities. The Pearson correlation values justify the bioactivity relationship of the major compounds with different microbial strains. It also justified the interaction among the major individual compounds.

Drug Resistance Reversal Potential of Isoliquiritigenin and Liquiritigenin Isolated from Glycyrrhiza glabra Against Methicillin‐Resistant Staphylococcus aureus (MRSA)

Isoliquiritigenin (ISL) and liquiritigenin (LTG) are structurally related flavonoids found in a variety of plants. Discovery of novel antimicrobial combinations for combating methicillin‐resistant Staphylococcus aureus (MRSA) infections is of vital importance in the post‐antibiotic era. The present study was taken to explore the in vitro and in vivo combination effect of LTG and ISL with β‐lactam antibiotics (penicillin, ampicillin and oxacillin) against mec A‐containing strains of MRSA. Minimum inhibitory concentration (MIC) of both LTG and ISL exhibited significant anti‐MRSA activity (50–100 µg/mL) against clinical isolates of MRSA. The result of in vitro combination study showed that ISL significantly reduced MIC of β‐lactam antibiotics up to 16‐folds [∑ fractional inhibitory concentration (FIC) 0.312–0.5], while LTG reduced up to 8‐folds (∑FIC 0.372–0.5). Time kill kinetics at graded MIC combinations (ISL/LTG + β‐lactam) indicated 3.27–9.79‐fold and 2.59–3.48‐fold reduction in the growth of clinical isolates of S. aureus respectively. In S. aureus‐infected Swiss albino mice model, combination of ISL with oxacillin significantly (p < 0.05, p < 0.01, p < 0.001) lowered the systemic microbial burden in blood, liver, kidney, lung and spleen tissues in comparison with ISL, oxacillin alone as well as untreated control. Considering its synergistic antibacterial effect, we suggest both ISL and LTG as promising compounds for the development of novel antistaphylococcal combinations. Copyright

In vitro antimalarial activity and molecular modeling studies of novel artemisinin derivatives

Cerebral malaria is a serious and sometimes fatal disease caused by a Plasmodium falciparum parasite that infects a female anopheles mosquito which feeds on humans. The parasites responsible for mosquito-borne infectious diseases are increasingly resistant to current drug approaches, and almost half of the world is at risk of contracting an illness. A series of twenty five new ether and ester derivatives of dihydroartemisinin (DHA) have been prepared based on in silico studies and in vitro antimalarial activity and later assessed against the chloroquine sensitive NF-54 strain of Plasmodium falciparum. In general the incorporation of nitro functionality in ester derivatives enhances the activity relative to artemisinin. Most of the ether derivatives were found to be as active as DHA, while 11-OH ether derivatives were not as active as DHA. The most potent analogue in the series was compound 21 which was several fold more active than artemisinin against P. falciparum used in the study. Molecular docking and ADMET studies were performed to explore the possible mode of interaction of active compounds in to the binding site pocket of malaria parasite target enzyme plasmepsin-II and evaluated compliance with oral bioavailability and pharmacokinetics parameters. The ester derivatives 19 and 20 were found to be twice active than DHA, having nitro functionality showing IC50 10.58 nM and 8.54 nM respectively.

A cytotoxic agent from Strychnos nux-vomica and biological evaluation of its modified analogues

Although a number of chemicals have been isolated from Strychnos nux-vomica, only a few have been evaluated for their biological significance. As a part of our drug discovery programme for cytotoxic agents from Indian medicinal plants, a novel cytotoxic agent, loganin 1 was isolated from the fruit pulp of S. nux-vomica. The loganin 1 showed significant anticancer activity against the human liver (WRL-68), colon (COLO-320 and CaCo2), ovarian (PA-1) and breast (MCF-7) cancer cell lines. Loganin 1 was further chemically transformed into eleven semi-synthetic derivatives 2–12 of which 2′,3′,4′,7-tetra-O-acetyl-6′-O-(3′′′, 4′′′, 5′′′)-trimethoxybenzoyl loganin 11 and 2′,3′,4′,7-tetra-O-acetyl-6′-O-lauroyl loganin 6 derivatives showed eight, 13 and three times while 2′,3′,4′,7-tetra-O-acetyl-6′-O-lauroyl loganin 6 showed equal, 13 and two times more activity against human suspension colon (CaCo2), adherent colon (COLO-320) and liver (WRL-68) cancer cell lines, respectively, than the known anticancer agent, vinblastine. The other analogues (except 4, 8 and 9) and loganin also showed marginal to moderate anticancer activity against the five tested human cancer cell lines.

Glabridin-chalcone hybrid molecules: drug resistance reversal agent against clinical isolates of methicillin-resistant Staphylococcus aureus

A novel series of glabridin-chalcone hybrid molecules (GCHMs) were synthesized and evaluated for their antibacterial and resistance reversal activity against clinical isolates of a methicillin-resistant strain of Staphylococcus aureus (MRSA) alone and in combination with norfloxacin. Glabridin showed significant anti-staphylococcal activity against various MRSA clinical isolates with MICs of 12.5 μg ml−1. However, all its synthesized derivatives displayed moderate to weak activity (MICs ranging from 12.5 to >100 μg ml−1). Among all the synthesized hybrid molecules, compounds 6f, 6h, 8f and 8h along with glabridin were chosen for combination study with norfloxacin. Among all tested compounds, 8h exhibited marked synergism and up to 16-fold reduction in MICs with norfloxacin (FICI range from 0.312 to 0.375). In a systemically infected Swiss albino mice model, compound 8h significantly (p < 0.01, p < 0.05) lowered the systemic bacterial load in blood, liver, kidney, lung and spleen tissues. The present study reports the potential use of GCHMs in the development of economical anti-infective combinations for the treatment of infection caused by clinical MRSA isolates.