Vijay L. Maheshwari

Antidiarrheal activity, chemical and toxicity profile of Berberis aristata

Context: This study evaluated the in vitro and in vivo antidiarrheal activity, oral acute toxicological profile, and developed a chemical fingerprint of Berberis aristata Linn. (Berberidaceae). Materials and methods: The ethanol (by maceration) and aqueous (by Soxhlet) extracts of Berberis aristata bark were used for the study. The study involved the antimicrobial (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by micro dilution) and antidiarrheal (castor oil induced diarrhea, charcoal motility) tests. The active principle, berberine was characterized by different spectroscopic and chromatographic techniques. Results: The MIC and MBC (of both extracts) against all strains of Shigella were recorded between 125 to 500 µg/mL and 300 to 600 µg/mL, respectively. The MIC and MBC values of berberine are almost comparable to standard ciprofloxacin. UV and IR spectroscopy along with HPTLC and HPLC studies showed presence of berberine in the extracts. The onset of castor oil induced diarrhea was delayed and number of diarrheal episodes was reduced by both the extracts in a dose dependent manner. Similarly, the length of intestine traveled by the feed was also significantly reduced in the charcoal motility test by both the extracts. LD50 of >5000 mg/kg body weight was observed for both extracts in the acute oral toxicity studies with Swiss albino mice. Conclusion: The results validate in vivo and in vitro antidiarrheal activity of Berberis aristata extracts and provide its chemical fingerprint.

Withania somnifera (Ashwagandha): Regeneration through Meristem Culture

High frequency of callus induction was achieved from shoot tip explants (80%) of Withania somnifera (Hindi name-Ashwagandha) as compared to leaf explants (70%), on MS medium supplemented with IAA (56μM) and Kinetin (56μM). When hormone-free MS medium was fortified with vitamins [thiamine. HCI (3μM), nicotinic acid (40.7μM) and pyridoxine. HCI (24.3μM)], it was found suitable for complete plant regeneration through meristem culture.

Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose

Post-harvest insect infestation of stored grains makes them unfit for human consumption and leads to severe economic loss. Here, we report functional and structural characterization of two coleopteran α-amylases viz. Callosobruchus chinensis α-amylase (CcAmy) and Tribolium castaneum α-amylase (TcAmy) along with their interactions with proteinaceous and non-proteinaceous α-amylase inhibitors. Secondary structural alignment of CcAmy and TcAmy with other coleopteran α-amylases revealed conserved motifs, active sites, di-sulfide bonds and two point mutations at spatially conserved substrate or inhibitor-binding sites. Homology modeling and molecular docking showed structural differences between these two enzymes. Both the enzymes had similar optimum pH values but differed in their optimum temperature. Overall, pattern of enzyme stabilities were similar under various temperature and pH conditions. Further, CcAmy and TcAmy differed in their substrate affinity and catalytic efficiency towards starch and amylopectin. HPLC analysis detected common amylolytic products like maltose and malto-triose while glucose and malto-tetrose were unique in CcAmy and TcAmy catalyzed reactions respectively. At very low concentrations, wheat α-amylase inhibitor was found to be superior over the acarbose as far as complete inhibition of amylolytic activities of CcAmy and TcAmy was concerned. Mechanism underlying differential amylolytic reaction inhibition by acarbose was discussed.

Plant metabolites: an alternative and sustainable approach towards post harvest pest management in pulses

Grain legumes are an important source of proteins in vegetarian diet besides their role in biological nitrogen fixation. They are prone to heavy pest infestation both on and off the field. Pest associated losses are an important contributing factor towards declining per capita availability of grain legumes. Synthetic chemical pesticides have played an important role in crop preservation, however their incessant use has posed several environmental and human health concerns. Methyl bromide and phosphine are commonly used for the post harvest preservation of grain legumes. However, the former has to be phased out by 2015 as per the Montreal protocol whereas the latter is showing development of resistance to it by the insects. In the light of this, alternative, safer and sustainable strategies are needed for crop protection. Plants can serve as a rich source of bioactive chemicals for this purpose. Both primary as well as secondary metabolites can be evaluated against the target pests. The paper reviews the status of research in the area of use of plant metabolites in post harvest pest management of grain legumes.

Effect of Diphenyl Carbazide on the Metribuzin Induced Inhibition of Photosystem-II Photochemistry

Biochemical parameters with respect to inhibition of PS II photochemistry by metribuzin like Ki, Hill coefficient and quantum requirement have been determined. The effect of metribuzin in isolated chloroplasts was found to be independent of cation induced structural and functional changes in thylakoid membranes. DPC — an electron donor on the oxidizing side of PS ll, was found to relieve the metribuzin induced inhibition of PS II photochemistry. The results suggest that DPC may alter the binding of metribuzin presumably because of overlapping binding domain in the QB pocket as reported earlier for some other herbicides also.

In vivo antidiabetic and antioxidant activities of Coccinia grandis leaf extract against streptozotocin induced diabetes in experimental rats

Abstract Objective To evaluate antidiabetic and antioxidative properties of Coccinia grandis leaf extract in experimental diabetic rats and to study the co-relation of the two in vivo. Methods Ethanolic extract, showing the highest activity in in vitro experiments, was prepared in saline and given orally to streptozotocin induced albino Wistar diabetic rats for 21 days. Biochemical parameters, histopathological analyses, liver and muscles glycogen and in vivo antioxidant activity in normal, diabetic control, standard (metformin) and treated animals were determined and compared. Results Treatment of experimental rats with diabetes induced by streptozotocin by ethanolic leaf extract (500 mg/kg) caused significant (P Conclusions The results suggest that Coccinia grandis ethanolic leaf extract has strong antidiabetic activity and can be meaningfully utilized in the management of diabetes.

A glycoprotein α-amylase inhibitor from Withania somnifera differentially inhibits various α-amylases and affects the growth and development of Tribolium castaneum

BACKGROUND Identification and characterisation of plant defensive molecules enrich our resources to design crop protection strategies. In particular, plant-derived proteinaceous inhibitor(s) of insect digestive enzymes appear to be a safe, sustainable and attractive option. RESULTS A glycoprotein having non-competitive α-amylase inhibitory activity with a molecular weight of 8.3 kDa was isolated and purified from seeds of Withania somnifera α-amylase inhibitor (WSAI). Its mass spectrometry analysis revealed 59% sequence coverage with Wrightide II-type α-amylase inhibitor from Wrightia religiosa. A dose-dependent inhibition of α-amylases from Aspergillus oryzae, Bacillus subtilis, Helicoverpa armigera and Tribolium castaneum was recorded. Interestingly, WSAI did not inhibit human salivary α-amylase significantly. When adults of T. castaneum were fed with WSAI (1.6 mg g-1 ), decrease in consumption, growth and efficiency of conversion of ingested food was evident, along with over fourfold increases in feeding deterrence index. A decline in larval residual α-amylase activity after feeding of WSAI resulted in a reduction in longevity of T. castaneum. CONCLUSION The study reflects the significance of WSAI in affecting the overall growth and development of T. castaneum. Pre- and post-harvest pest resistive capability makes WSAI a potential candidate for insect pest management. Further, the effectiveness of this inhibitor could be explored either in formulations or through a transgenic approach.

Chapter 5 – Bioactive Secondary Metabolites From Endophytic Fungi: A Review of Biotechnological Production and Their Potential Applications

New viral diseases, multidrug-resistant microorganisms, and infections by uncommon pathogens are emerging as new threats to human, plant, and animal health. Therefore, there is continuous need of discovery and development of new, safer, and effective drugs to combat these diseases. In the recent past, our drug discovery strategies heavily relied on microorganism where nearly 80% of the worlds antibiotics have their origins. These microorganisms are mainly isolated from the soil and most of the worlds soils have been screened out for the search of new microbes. Therefore, there is an urgent need to explore new microbial habitats for the microorganisms that produce novel bioactive compounds. “Endophytes” are the microorganisms which are present inside the living tissues of the plant by establishing a variety of relationships such as symbiotic, mutualistic, or parasitic with their host plants. In their relationship with the host plant, endophytes contribute to the plant by producing a plethora of substances that provide protection and survival benefits to the host plant. Endophytic fungi have been recognized as a novel source of bioactive secondary metabolites with diverse biological activities such as novel antibiotics, antimycotics, immunosuppressant, anticancer compounds, and so on to name a few. Their bioactive potential was first recognized when worlds first multibillion dollar anticancer drug, paclitaxel (Taxol), was obtained from Taxomyces andreanae, an endophytic fungus isolated from the yew plant, Taxus brevifolia. However, it has been estimated that less than 1% of endophytic microorganisms are currently known, suggesting that a vast majority of them are yet to be discovered. Thus, endophytic microbes represent a potential source for the discovery of new and useful compounds, or new platforms for the organic synthesis of such compounds for the benefit of mankind. Moreover, plants located in the area of biodiversity hotspots of India, such as Eastern Himalaya, Western Ghats, and Andaman and Nicobar Island are believed to be host to novel endophytes with diverse chemical potential. The objective of the present article is to review the current state of information on the endophytes and their hosts, advances in isolation techniques, mode of cultivation, culture conditions, and biological activities of metabolites derived from endophytic microorganisms.

In Vitro Culture and Regeneration of Solanum khasianum and Extraction of Solasodine

Leaf explants of Solanum khasianum regenerated on MS medium containing 2, 4-D (3 mg/l) and kinetin (1 mg/l). Shoots could be induced from these calli on medium containing BAP (3 mg/l) alone. Rhizogenesis of these shoots occurred when transferred to medium containing 2 mg/l NAA. The yield of solasodine — a pharmaceutically important compound, from 4-month-old callus tissue was remarkable at 2 per cent of dry weight.

Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3′UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC50. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.