Serine-glycine-betaine, a novel dipeptide from an endophyte Macrophomina phaseolina: isolation, bioactivity and biosynthesis.

AIMS: Endophytes are a rich source for structurally complex chemical scaffolds with interesting biological activities. Endophytes associated with Brugmansia aurea L. (family: Solanaceae), a medicinal plant, have not yet explored for the bioactive metabolites. METHOD AND RESULTS: Hence, Macrophomina phaseolina, a fungal endophyte, was isolated from the roots of the plant. Its methanolic extract was found active against human cancer cell lines with IC50 <20 µg ml-1 . Later, a di-peptide compound, serine-glycine-betaine, was isolated and characterized. Serine-glycine-betaine consists of a unit of an N-trimethyl glycine attached to serine. It exhibited potent activity against MIA PaCa-2 and HCT-116 cell lines with IC50 8·9 and 15·16 µmol l-1 , respectively. Furthermore, it induced apoptosis in MIA PaCa-2 cells confirmed by microscopy. The apoptotic cell death in MIA PaCa-2 cells was evidenced biochemically with the generation of intracellular reactive oxygen species level and leading to loss of mitochondrial membrane potential due to activation of the intrinsic pathway. This study describes the plausible biosynthesis of serine-glycine-betaine based on genomics (genome sequencing, annotation and genes alignment). CONCLUSIONS: A novel di-peptide, serine-glycine-betaine isolated from M. phaseolina induced apoptosis in MIA-Pa-Ca-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that dipeptides like serine-glycine-betaine and tyrosine-betaine might be specific to fungal genera, hence being used for diagnostic purposes.

Synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus ticks from north-western Himalayas, India.

In the present study, adult immersion test (AIT) was used for evaluation of resistance against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India. Resistance factors (RFs) were calculated between 0.94 to 50.71 for deltamethrin and 0.32 to 13.18 for cypermethrin. Resistance to deltamethrin was detected at level I in two, level II in four, level III and level IV in one isolate each while one isolate was susceptible. Against cypermethrin, resistance at levels I and II was detected in three isolates each while three isolates were found susceptible. The low altitude sub-tropical zone revealed higher density of resistant ticks where intensive animal husbandry practices were followed and the synthetic pyrethroid usage was common. Data generated on pyrethroid resistance status of ticks in north-western Himalayan region will provide new insights in acaricidal resistance particularly from remote areas of this region and will help in formulating suitable control measures.

In vitro acaricidal activity of Piper nigrum and Piper longum fruit extracts and their active components against Rhipicephalus (Boophilus) microplus ticks.

In vitro acaricidal activity of Piper nigrum and P. longum fruit extracts and their active components (piperine for P. nigrum and piperine and piperlonguminine for P. longum) was evaluated against adults engorged females of Rhipicephalus (Boophilus) microplus using adult immersion test. Three concentrations of each extract with four replications were used in the bioassay. Extracts significantly affected mortality rates of ticks in dose-dependent manner ranged 12.5-95.8% for P. nigrum and 29.2-87.5% for P. longum, with an additional effect on the reproductive physiology of ticks by inhibiting oviposition (28.1-96.9% by P. nigrum and 36.1-89.3% by P. longum). However, the acaricidal and oviposition limiting properties were decreased significantly when the active component(s) of each extract was tested separately. However, the combination of piperine and piperlonguminine (obtained from P. longum extract) caused 79.2% mortality of ticks which is equivalent to the corresponding concentration (~ 5%) of the extract. It can be concluded that the fruit extracts of P. nigrum and P. longum had both acaricidal and oviposition limiting actions against the adults of R. (B.) microplus which could make it a valuable component of developing sustainable strategy for integrated tick management.

A strain of Streptomyces sp. isolated from rhizospheric soil of Crataegus oxycantha producing nalidixic acid, a synthetic antibiotic.

AIM: Exploration of microbes isolated from rhizospheric soil of Crataegus oxycantha for bioactive natural products. METHODS AND RESULTS: A strain of Streptomyces sp. (C-7) was isolated from rhizospheric soil of C. oxycantha. The 16S rRNA gene sequence of strain C-7 displayed 99% sequence similarity with different Streptomyces species. The highest score was displayed for Streptomyces sp. strain Chy2-8 followed by Streptomyces violarus strain NBRC13104 and Streptomyces arenae strain ISP5293. The position of C-7 in the phylogenetic tree suggested uniqueness of the strain. Nalidixic acid (1), a quinolone antibiotic, was isolated from Streptomyces sp. strain (C-7) for the first time and characterized by NMR and chemically analysed. Compound 1 exhibited antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. The production of compound 1 was also validated by repeating fermentation of strain C-7 and compound isolation in a separate natural product laboratory with no prior information. Furthermore, Compound 1 showed a cytotoxic effect against human prostate cancer cell line PC3 with an IC50 11 µg ml-1 . CONCLUSION: To the best of our knowledge, this is the first report showing production of nalidixic acid naturally by a strain of Streptomyces sp. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated a strain of Streptomyces sp. producing nalidixic acid, which was otherwise only obtained through chemical synthesis.

Fungal endophytes associated with Viola odorata Linn. as bioresource for pancreatic lipase inhibitors.

BACKGROUND: As per the recent statistical reports of World Health Organisation (WHO), 13% of total global population is obese. Orlistat remains to be the only drug approved for the long term treatment of obesity. Recent findings highlighted severe adverse effects of orlistat that included hepatotoxicity, gall stones, kidney stones and acute pancreatitis. Therefore, search for new drug is required. The investigations based on endophytic natural products would prove pivotal in the global fight against this health issue. METHODS: Obesity is associated with lipid metabolism involving pancreatic lipase enzyme. The inhibition of pancreatic lipase is demonstrated by using the extracts of endophytes isolated from Viola odorata Linn. In addition, endophytes were identified using ITS based rDNA sequencing. RESULTS: Present study involves the isolation and identification of 27 endophytes from V. odorata. All the endophytes were evaluated for lipase inhibitory activities. The extracts of seven endophytes exhibited lipase inhibitory activity with IC50 < 10 µg/mL. The extract of VOLF4 (Aspergillus sp.) displayed promising lipase inhibitory activity (IC50 3.8 µg/mL). CONCLUSION: The present study demonstrates that V. odorata harbors endophytic community with potent lipase inhibitory activity. VOLF4 is the potential endophyte. The extract of VOLF4 can be used to develop the potential drug to treat obesity.

Detection of amitraz and malathion resistance in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Jammu region of India.

Detection of resistance levels against amitraz and malathion in Rhipicephalus (Boophilus) microplus collected from four districts of Jammu region (India) was carried out using the adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentration of drugs were utilised for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index and percentage inhibition of oviposition) the resistance level was categorised as I, II, III and IV. Out of these four districts, resistance to amitraz was detected at level I in Udhampur (RF = 2.81), Jammu (RF = 2.53) and Samba isolates (RF = 2.24) whereas Rajouri isolate was found susceptible (RF = 1.0). Resistance to malathion was detected at level I in Udhampur (RF = 4.01) and Jammu isolates (RF = 1.76) whereas Rajouri (RF = 0.472) and Samba (RF = 0.199) isolates were found susceptible. The data generated on amitraz and malathion resistance status will help in formulating a tick control strategy in the region.

Deltamethrin resistance in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Jammu and Kashmir, India.

Detection of resistance levels against deltamethrin in Rhipicephalus (Boophilus) microplus collected from six districts of Jammu and Kashmir (India) was carried out using the adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentration of drug were utilised for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on mortality, egg mass weight, reproductive index and percentage inhibition of oviposition, the resistance level was categorised as I, II, III and IV. Out of these six districts, resistance to deltamethrin at level I was detected in one district (RF = 1.9), at level II in two districts (RF = 7.08-10.07) and at level IV in three districts (RF = 96.08-288.72). The data generated on deltamethrin resistance status will help in formulating tick control strategy in the region.

In vitro acaricidal activity of ethanolic and aqueous floral extracts of Calendula officinalis against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus.

Detection of resistance levels against deltamethrin and cypermethrin in Rhipicephalus (Boophilus) microplus collected from Jammu (India) was carried out using larval packet test (LPT). The results showed the presence of resistance level II and I against deltamethrin and cypermethrin, respectively. Adult immersion test (AIT) and LPT were used to evaluate the in vitro efficacy of ethanolic and aqueous floral extracts of Calendula officinalis against synthetic pyrethroid resistant adults and larvae of R. (B.) microplus. Four concentrations (1.25, 2.5, 5 and 10 %) of each extract with four replications for each concentration were used in both the bioassays. A concentration dependent mortality was observed and it was more marked with ethanolic extract. In AIT, the LC50 values for ethanolic and aqueous extracts were calculated as 9.9 and 12.9 %, respectively. The egg weight of the live ticks treated with different concentrations of the ethanolic and aqueous extracts was significantly lower than that of control ticks; consequently, the reproductive index and the percent inhibition of oviposition values of the treated ticks were reduced. The complete inhibition of hatching was recorded at 10 % of ethanolic extract. The 10 % extracts caused 100 % mortality of larvae after 24 h. In LPT, the LC50 values for ethanolic and aqueous extracts were determined to be 2.6 and 3.2 %, respectively. It can be concluded that the ethanolic extract of C. officinalis had better acaricidal properties against adults and larvae of R. (B.) microplus than the aqueous extract.

Acaricidal activity of ethanolic extract of Artemisia absinthium against Hyalomma anatolicum ticks.

The aim of the study was to evaluate the in vitro efficacy of different concentrations of ethanolic extract obtained from the aerial parts of Artemisia absinthium in comparison to amitraz on adults, eggs and larvae of Hyalomma anatolicum using the adult immersion test (AIT), egg hatchability test and larval packet test (LPT), respectively. Four concentrations of the extract (2.5, 5, 10 and 20%) with three replications for each concentration were used in all the bioassays. In AIT, the mortality rates at 2.5, 5 and 10% were significantly different (p < 0.05) in comparison to the control group; however, at 20%, it was similar to the positive control group. Maximum mortality of 86.7% was recorded at 20%. The LC50 and LC95 values were calculated as 6.51 and 55.43%, respectively. The oviposition was reduced significantly by 36.8 and 59.1% at concentrations of 10 and 20%, respectively. Egg hatchability was reduced significantly at all concentrations (2.5-20%) in comparison to the control. In LPT, the extract caused 100% mortality of larvae at all the concentrations after 24 h. The results show that ethanolic extract obtained from the aerial parts of A. absinthium has acaricidal properties and could be useful in controlling H. anatolicum.

In vitro evaluation of ethanolic extracts of Ageratum conyzoides and Artemisia absinthium against cattle tick, Rhipicephalus microplus.

In vitro efficacy of ethanolic extracts obtained from the aerial parts of Ageratum conyzoides and Artemisia absinthium was assessed on Rhipicephalus microplus using adult immersion test (AIT). Five concentrations of the extract (1.25%, 2.5%, 5%, 10%, and 20%) with three replications for each concentration were used in the bioassay. In AIT, the maximum mortality was recorded as 40% and 66.7% at 20% concentration for A. conyzoides and A. absinthium, respectively. Acaricidal activity was found to be higher in the extract of A. absinthium with LC50 and LC95 values of 11.2% and 61.7%, respectively. Egg mass weight of the live ticks treated with different concentrations of the extracts was significantly (P<0.05) lower than that of control ticks; consequently, the reproductive index and oviposition values of the treated ticks were reduced significantly (P<0.05). The A. conyzoides inhibited 90% hatching of eggs at the 20% concentration, whereas A. absinthium showed 100% inhibition at 5%, 10%, and 20% concentrations. The results show that A. absinthium has better acaricidal properties than A. conyzoides and could be useful in controlling R. microplus.

In vitro acaricidal activity of Atropa belladonna and its components, scopolamine and atropine, against Rhipicephalus (Boophilus) microplus.

In vitro efficacy of methanolic extract of Atropa belladonna and its components scopolamine and atropine was assessed against Rhipicephalus (Boophilus) microplus. Five concentrations of the extract (1.25%, 2.5%, 5%, 10%, and 20%) were used whereas scopolamine and atropine were each tested at 0.1%. In adult immersion test, the extract was lethal to ticks at 20% concentration. The LC50 and LC95 values were determined as 6.875% and 17.306%, respectively. The extract caused a significant reduction (P < 0.05) in egg mass production at 10% concentration. In larval packet test, the extract was lethal to larvae in the concentrations of 10% and 20% after 24 h, with LC50 and LC95 values of 1.321% and 4.935%, respectively. Scopolamine and atropine showed 93.3% and 60.0% mortality of adult ticks, respectively, but they caused complete (100%) blocking of hatching as well as 100% larval mortality. Scopolamine and atropine were observed to be more potent than the crude extract at an equivalent concentration in both the bioassays.

Acaricidal activity of extract of Artemisia absinthium against Rhipicephalus sanguineus of dogs.

The objective of the study was to evaluate the in vitro efficacy of different concentrations of chloroform extract obtained from the aerial parts of Artemisia absinthium in comparison to amitraz on adults, eggs and larvae of the dog tick Rhipicephalus sanguineus using the adult immersion test (AIT), egg hatchability test (EHT) and larval packet test (LPT), respectively. Five concentrations of the extract (1.25, 2.5, 5, 10 and 20 %) with three replications for each concentration were used in all the bioassays. A control group was established (water + dimethylsulphoxide) together with a positive control group (amitraz), with three repetitions each. In AIT, the mortality rates were 0.0, 13.3, 16.7, 33.3 and 93.3 % in concentrations of 1.25, 2.5, 5, 10 and 20 %, respectively, and the variation was significant (p=0.0151). The LC50 (CI) and LC95 (CI) values were calculated as 8.793 % (8.217-9.408) and 34.59 % (29.71-40.26), respectively. The egg production was reduced by 6.6, 6.6, 18.3, 42.5 and 85.1 % in the concentrations of 1.25, 2.5, 5, 10 and 20 %, respectively, and it was statistically significant (p=0.0274). In EHT, hatching was completely inhibited at 5, 10 and 20 % displaying 100 % ovicidal action while at the concentrations of 1.25 and 2.5 %, the inhibition rates were 20 and 60 %, respectively. In LPT, the extract caused 100 % mortality of larvae in the concentrations of 5, 10 and 20 % after 24 h while at the concentrations of 1.25 and 2.5 %, the mortality rates were 54.3 and 96.7 %, respectively. The LC50 (CI) and LC95 (CI) values were determined to be 1.11 % (1.099-1.121) and 2.37 % (2.328-2.423), respectively. The results show that the extract of A. absinthium has acaricidal properties and could be useful in controlling R. sanguineus which is an efficient vector of pathogens both in dogs and humans.

Histological and biochemical changes in testis of zinc deficient BALB/c strain of mice.

Zinc, protein, cholesterol, phospholipids, alkaline phosphatase (AlPase), acid phosphatase (AcPase), adenosine-5-triphosphatase(ATPase) and histology were studied in testis of zinc-deficient mice. Zinc and protein decreased in the 3-week experiment whereas they increased in the 6-week experiment. Zinc is involved in several functions of the cell and is regulated by hormones. Inhibition of spermatogenesis indicates for decreased zinc levels in 3-week whereas the increase in 6-week experiment indicates for accumulation of zinc in oedomatous fluid and uncontrolled diffusion of zinc across the blood testis barrier. Glycogen decreased in the 3-week as well as 6-week experiments due to blockage of androgen and spermatogenesis. Cholesterol and phospholipids increased in the 3-week experiment and decreased in 6-week experiment as both the parameters are related to steroidogenesis. Zinc deficiency leads to aspermatogenic condition and comparatively less injury to non-germinal cells. This could have blocked the transport of material across the testis barrier and therefore might have increased AlPase levels. Increased AcPase, probably represents lysosomal enzymes, as the cell debris of disorganised epithelium are to be digested and removed. ATPase increased in 3-week experiment and can be correlated to increased demands of energy of testicular cells to overcome the insults of zinc deficiency whereas the decrease in 6-week experiment could be as a result of inhibition of spermatogenesis.