Evaluation of iso-quinoline alkaloids by RP-HPLC-PDA in different Berberis species collected from Western Himalayan Region.

The genus Berberis has high significance in Indian and other traditional systems of medicine due to the presence of iso-quinoline alkaloids. This study was conducted to record the metabolic variation in eight Berberis spp. collected from the Western Himalayan region. The RP-HPLC-PDA chromatogram separates the markers jatrorrhizine, palmatine and berberine at specific retention time, 11.45, 16.41 and 18.15 min respectively. The method was validated on linearity, precision and recovery indices as per International Conference on Harmonisation (ICH) guidelines. The maximum content of berberine, palmatine and jatrorrhizine was found in Berberis asiatica (1.74% ± 0.032, 1.63% ± 0.028 and 0.264% ± 0.012, respectively) on % dry weight basis. This study will help in phyto-marker-based identification and differentiation of various Berberis species, which can be used as an alternative to the official drug Daruharidra i.e. Berberis aristata.

Anti-rheumatic potential vis-à-vis aconitine and hypaconitine content analysis in different Aconitum spp. from Sikkim Himalayas (India).

Aconitum spp. are important medicinal plants mentioned in Ayurveda as Ativisa or Vatsanabha. The present study aims to evaluate anti-rheumatic potential in seven Aconitum species and correlation with aconitine and hypaconitine content. Anti-rheumatic potential was analyzed through in vitro xanthine oxidase inhibition, anti-inflammatory and ROS scavenging assays; and quantification of aconitine and hypaconitine with RP-HPLC method validated as per ICH guidelines. The findings reveal that A. palmatum possessed the most promising response (IC50 =12.68±0.15 µg/ml) followed by A. ferox (IC50 =12.912±1.87 µg/ml) for xanthin oxidase inhibition. We observed a wide variation in aconitine and hypaconitine content ranging from 0.018 %-1.37 % and 0.0051 %-0.077 % respectively on dry weight basis. Aconitine and hypaconitine showed moderate positive correlation (r=0.68 and 0.59 respectively) with anti-rheumatic potential. The study identifies potential alternative species of Aconitum that can help in sustainable availability of quality raw material.

Nutritional characterization of an underutilized legume Entada rheedii Spreng. seeds and validation of its folklore uses.

Entada rheedii seeds are a rich source of protein (23.99% ± 0.07), starch (42.04% ± 0.05) and potassium (1670.15 ppm ± 116.732). HPTLC-UV analysis (seeds) reveals galactose in considerable amount, that is, 2.60% ± 0.008. Additionally, the species is low in fat and anti-nutrient metabolites like tannin. Interestingly, the proximates in Entada seeds was found comparable with the commonly consumed legumes like cowpea, green gram, and so on. The species exhibits promising anti-radical, anti-inflammatory and anti-diabetic activity. Data advocates the folklore use of E. rheedii seeds and can be a promising alternative source of dietary nutrition, fortified with medicinal value. Standardization and validation of scientific knowledge will bring such underutilized, low-cost legumes into daily dietary intake and are a promising source for Protein-Energy-Malnutrition.

In silico guided in vitro study of traditionally used medicinal plants reveal the alleviation of post-menopausal symptoms through ERß binding and MAO-A inhibition.

The slumping level of estrogen and serotonin in menopausal women is directly associated with the occurrence of menopausal symptoms where, estrogen receptor-ß (ERß) and monoamine oxidase-A (MAO-A) are directly involved. The present investigation aimed for validation of promising plants traditionally used to alleviate menopausal symptoms with ERß mediated MAO-A inhibition potential through in silico disease-target network construction using Cytoscape plugins followed by molecular docking of phytomolecules through AutoDock vina. ADMET parameters of identified bioactive phytomolecules were analysed through swissADME and ProTox II. The efficacy of promising plant leads was further established through in vitro ERß competitive binding, MAO-A inhibition, enzyme kinetics and free radical quenching assays. In silico analysis suggested glabrene (ΔG = -9.7 Kcal/mol) as most promising against ERß in comparison to 17ß-estradiol (ΔG = -11.4 Kcal/mol) whereas liquiritigenin (ΔG = -9.4 Kcal/mol) showed potential binding with MAO-A in comparison to standard harmine (ΔG = -8.8 Kcal/mol). In vitro analysis of promising plants segregated Glycyrrhiza glabra (IC50 = 0.052 ± 0.007 µg/ml) as most promising, followed by Hypericum perforatum (IC50 = 0.084 ± 0.01 µg/ml), Trifolium pratense (IC50 = 0.514 ± 0.01 µg/ml) and Rumex nepalensis (IC50 = 2.568 ± 0.11 µg/ml). The enzyme kinetics of promising plant leads showed reversible and competitive nature of inhibition against MAO-A. The potency of plant extracts in quenching free radicals was at par with ascorbic acid. The identified four potent medicinal plants with ERß selective, MAO-A inhibitory and free radical quenching abilities could be used against menopausal symptoms however, finding needs to be validated further for menopausal symptoms in in vivo conditions for drug development.Communicated by Ramaswamy H. Sarma.

Characterization of deltamethrin, cypermethrin, coumaphos and ivermectin resistance in populations of Rhipicephalus microplus in India and efficacy of an antitick natural formulation prepared from Ageratum conyzoides.

Rhipicephalus microplus is posing a serious threat to productive animal husbandry. Excessive use of synthetic chemicals in tick management has led to the development of resistant tick populations. Characterization of resistance to deltamethrin, cypermethrin, coumaphos and ivermectin in ticks is necessary to develop a suitable and sustainable control strategy. Based on adult immersion test and larval packet test, the resistance ratios (RR50) for adults and larvae of R. microplus populations from two Indian states ranged from 3.8 to 19.4 and 1.35-25.0 against deltamethrin, 0.061-26.3 and 0.22-19.2 against cypermethrin, and 0.2-9.5 and 0.01-3.1 against coumaphos, respectively, were recorded. Moreover, the RR50 for adults ranged from 0.212 to 3.87 against ivermectin. The RR50 for different acaricides was significantly (p<0.01) correlated with esterases, Glutathione S-transferase and monooxygenase activity. A point mutation at the 190th position of the domain II S4-5 linker region of the sodium channel gene in synthetic pyrethroids (SP) resistant populations was also detected. An antitick natural formulation prepared from the plant Azeratum conyzoides and containing two major compounds, Precocene-I (7­methoxy-2, 2-dimethyl 2H-chromene) and Precocene II (6, 7-dimethoxy-2, 2-dimethyl- 3-chromene), was developed and tested against the resistant ticks. The LC50 values of the natural formulation against the resistant populations were in the range of 4.31-5.33% irrespective of their RR50 values. Multi-acaricide resistant populations of R. microplus are established in India and the A. conyzoides based natural formulation can be used for its management.

Influence of Soil Variation on Diosgenin Content Profile in Costus speciosus from Indo-Gangetic Plains.

Costus speciosus is a rich source of commercially important compound Diosgenin, distributed in different regions of India. The present investigation was aimed to quantify diosgenin through High Performance Thin Layer Chromatography in 34 germplasms of Costus speciosus and also to identify the superior sources and to correlate the macronutrients of rhizospheric soil. The starch content varied in microscopic examination and correlated inversely (r=-0.266) with diosgenin content. Findings revealed that the extraction process with acid hydrolysis yielded higher diosgenin content (0.15-1.88 %) as compared to non-hydrolysis (0.009-0.368 %) procedure. Germplasms from Uttar Pradesh (NBCS-4), Jharkhand (NBCS-39) and Bihar (NBCS-2) were identified as elite chemotypes based on hierarchical clustering analysis. The phosphorous content of respective rhizospheric soil correlated positively (r=0.742) with diosgenin content. Findings of present study are useful to identify the new agrotechniques. The elite germplasms can also be used as quality planting material for large scale cultivation in order to assure a sustained supply to the herbal drug industry.

Chemodiversity and molecular variability in the natural populations (India) of Gloriosa superba (L.) and correlation with eco- geographical factors for the identification of elite chemotype(s).

Gloriosa superba L. has economic significance due to colchicine, a bioactive compound used for gout. In present study metabolic and molecular variability in natural population of species was analyzed and correlated with edaphic and climatic factors. Thirty populations (wild) of G. superba were mapped from 10 different eco-regions of India at an elevation range of 10-1526 m, having no morphotypic variations. The two known biologically active alkaloids colchicine (ranged from 0.015-0.516%) and gloriosine (0.19-0.44%) were significantly varied (p < 0.05) among populations, leading to the identification of four elite chemotypes. Molecular variability from ISSR data divides the population in different sub clusters at intra-specific level, presenting the high similarity percentage with bootstrap value of 66-100%. Principal component analysis (PCA) revealed that elite chemotypes are related to temperature, precipitation and aridity gradient. The rhizospheric soil selenium was significantly correlated with colchicine content in G. superba.

Development of an efficient antitick natural formulation for the control of acaricide-resistant ticks on livestock.

Animal production has a key role in global economic development and food security. Ticks, specifically Rhipicephalus microplus cause substantial economic and health impacts on more than eighty percent of the world cattle population. Though synthetic acaricides play a major role in tick management, their injudicious usage has caused environmental pollution and also promote the establishment of multi-acaricide resistant tick populations which is a matter of great concern. To provide an effective tool for controlling these resistant ticks, the present work was aimed to develop safe and inexpensive antitick natural formulations. Our bioprospection studies of Ageratum conyzoides plant established it as a species potentially having strong acaricidal activity due to the presence of potent acaricidal phyto-chemicals. To develop a suitable antitick natural formulation, 41 samples/fractions/formulations were prepared from the dry powder of the whole aerial part of the A. conyzoides plant using different techniques and delivery matrices. The strongest antitick effect was recorded for formulation ACF6, which demonstrated 87 ± 6% mean mortality with 57 % inhibition of oviposition in treated female ticks. Ticks treated with the ACF6 formulation showed a significant (p < 0.001) reduction in cuticular protein (1.238 ± 0.01 mg/mL) as compared to control ticks (2.928 ± 0.01 mg/mL) but no significant difference in chitin content of treated ticks and control ticks was observed. The formulation was found safe in a rat model as no significant differences in biochemical and haematological parameters among treated and control rats were noted. Histopathological studies indicated no sign of hepatocellular necrosis and no significant changes in the weights of liver and spleen was recorded. The overall in vivo efficacy of the formulation was 85 % for experimentally infested cattle with direct mortality of more than 80 % within 96 h post-application. The lethal effect of the formulation was in the form of drying and dead ticks 1-2 d after application. The developed formulation has the potential to be adopted as an alternative tick control measure in an ecofriendly manner.

In-silico efficacy of potential phytomolecules from Ayurvedic herbs as an adjuvant therapy in management of COVID-19.

The recent COVID-19 outbreak caused by SARS-CoV-2 virus has sparked a new spectrum of investigations, research and studies in multifarious directions. Efforts are being made around the world for discovery of effective vaccines/drugs against COVID-19. In this context, Ayurveda, an alternative traditional system of medicine in India may work as an adjuvant therapy in compromised patients. We selected 40 herbal leads on the basis of their traditional applications. The phytomolecules from these leads were further screened through in-silico molecular docking against two main targets of SARS-CoV-2 i.e. the spike protein (S; structural protein) and the main protease (MPRO; non-structural protein). Out of the selected 40, 12 phytomolecules were able to block or stabilize the major functional sites of the main protease and spike protein. Among these, Ginsenoside, Glycyrrhizic acid, Hespiridin and Tribulosin exhibited high binding energy with both main protease and spike protein. Etoposide showed good binding energy only with Spike protein and Teniposide had high binding energy only with main protease. The above phytocompounds showed promising binding efficiency with target proteins indicating their possible applications against SARS-CoV-2. However, these findings need to be validated through in vitro and in vivo experiments with above mentioned potential molecules as candidate drugs for the management of COVID-19. In addition, there is an opportunity for the development of formulations through different permutations and combinations of these phytomolecules to harness their synergistic potential.

Development and Validation of a Reversed Phase High Performance Liquid Chromatography-Photodiode Array Detection Method for Simultaneous Identification and Quantification of Coumarin, Precocene-I, ß-Caryophyllene Oxide, α-Humulene, and ß-Caryophyllene in Ageratum Conyzoides Extracts and Essential Oils from Plants.

BACKGROUND: Ageratum conyzoides is an aromatic plant. It is considered as an invasive and cosmopolite weed, widely spread in tropical and subtropical regions. Phytochemicals such as benzopyrenes, flavonoids, and terpenoids are reported from A. conyzoides. OBJECTIVE: Development and validation of a reversed-phase HPLC-photodiode array (PDA) detection method for simultaneous identification and quantification of coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene in extracts of A. conyzoides and essential oils was carried out. METHODS: Separation of analytes was achieved on a RP-18 (250 mm × 4.6 mm, 5 µm) column using a solvent system comprising of a mixture of acetonitrile and water with 0.05% trifluoroacetic acid in gradient elution mode at ambient temperature with flow rate of 1 mL/min. RESULTS: The retention time of coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene was 4.38, 12.86, 20.10, 33.34, and 35.11 min, respectively. Limits of detection for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene were 2.5, 2.5, 2.5, 0.025, and 2.5 µg/mL, respectively. Similarly, LOQ were 10, 10, 10, 0.10, and 10 µg/mL for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß- caryophyllene, respectively. Repeatabilities (RSD, %) values for intraday and interday precision for coumarin, precocene-I, ß-caryophyllene oxide, α-humulene, and ß-caryophyllene was 0.765-2.086 and 0.886-2.128; 0.879-1.672 and 0.979-1.825; 0.696-2.418 and 0.768-2.592; 1.728-2.362 and 1.965-2.378; 1.615-2.897 and 1.658-2.906, respectively. CONCLUSIONS: The separation of five analytes was achieved within 50 min. The developed and validated HPLC-PDA method was successfully applied for identification and quantification of above five analytes in A. conyzoides extracts and essential oils. The method could be used for meeting the characterization criteria of phytoformulations.

Characterization of acaricide resistance in Rhipicephalus microplus populations infesting cattle in northeastern India and assessment of local plant extracts for tick management.

In the present study, the tick isolates were collected from Assam state, of northeastern region (NER) and characterized using in vitro bioassay, biochemical and molecular assays. Comparing LC50 value of susceptible IVRI-I and larvae of field isolates, revealed that RF against deltamethrin was highest for Morigaon (MGN = 21.8) and lowest for Sonitpur (SNP = 3.3) isolate. The RF against cypermethrin was highest for Nagaon (NGO = 5.0) and lowest for Barpeta (BPT = 1.2) isolate. Against coumaphos, the highest RF of 4.5 was calculated for BPT (4.5) and lowest for NGO (1.3) isolate. While using adults based assay, highest RF of 24.68 against deltamethrin and lowest RF of 4.96 was determined for MGN and SNP isolate, respectively. In contrast to the results obtained using larvae, against cypermethrin, highest RF was recorded for Kamrup Metropolitan (KMP) while it was NGO isolate using larvae. In case of coumaphos, both larvae and adults of BPT isolate were also highly resistant and lowest RF was detected in SNP (2.30) isolate. All the isolates were susceptible to ivermectin. A significant correlation (p < 0.01) between deltamethrin resistance and higher expression of glutathioneS-transferase was observed while no correlation with esterase and monooxygenase enzymes activity was noted. For the development of possible ecofriendly control measure, different accessions of Argemone mexicana and Datura metel plant species were collected, extracted and screened against adult ticks. Two accessions, NEA-03 and NED-06 collected from Amlighat and Diphu (East Karbi Anglong) were more than 90 % effective. Further dose response study of these accessions determined the LC50 values of 4.86 and 3.96 %, respectively.The resistance status of the collected tick isolates was compared with the data generated from other regions having higher livestock population and possibility of exploitation of identified plant species for the development of natural antitick product is discussed.

High-performance Thin-layer Chromatographic-densitometric Quantification and Recovery of Bioactive Compounds for Identification of Elite Chemotypes of Gloriosa superba L. Collected from Sikkim Himalayas (India).

BACKGROUND: Gloriosa superba L. (Colchicaceae) is used as adjuvant therapy in gout for its potential antimitotic activity due to high colchicine(s) alkaloids. OBJECTIVE: This study aimed to develop an easy, cheap, precise, and accurate high-performance thin-layer chromatographic (HPTLC) validated method for simultaneous quantification of bioactive alkaloids (colchicine and gloriosine) in G. superba L. and to identify its elite chemotype(s) from Sikkim Himalayas (India). METHODS: The HPTLC chromatographic method was developed using mobile phase of chloroform: acetone: diethyl amine (5:4:1) at λmax of 350 nm. RESULTS: Five germplasms were collected from targeted region, and on morpho-anatomical inspection, no significant variation was observed among them. Quantification data reveal that content of colchicine (Rf: 0.72) and gloriosine (Rf: 0.61) varies from 0.035%-0.150% to 0.006%-0.032% (dry wt. basis). Linearity of method was obtained in the concentration range of 100-400 ng/spot of marker(s), exhibiting regression coefficient of 0.9987 (colchicine) and 0.9983 (gloriosine) with optimum recovery of 97.79 ± 3.86 and 100.023% ± 0.01%, respectively. Limit of detection and limit of quantification were analyzed, respectively, as 6.245, 18.926 and 8.024, 24.316 (ng). Two germplasms, namely NBG-27 and NBG-26, were found to be elite chemotype of both the markers. CONCLUSION: The developed method is validated in terms of accuracy, recovery, and precision studies as per the ICH guidelines (2005) and can be adopted for the simultaneous quantification of colchicine and gloriosine in phytopharmaceuticals. In addition, this study is relevant to explore the chemotypic variability in metabolite content for commercial and medicinal purposes. SUMMARY: An easy, cheap, precise, and accurate high performance thin layer chromatographic (HPTLC) validated method for simultaneous quantification of bioactive alkaloids (colchicine and gloriosine) in G. superba L.Five germplasms were collected from targeted region, and on morpho anatomical inspection, no significant variation was observed among themQuantification data reveal that content of colchicine (Rf: 0.72) and gloriosine (Rf: 0.61) varies from 0.035%-0.150% to 0.006%-0.032% (dry wt. basis)Two germplasms, namely NBG 27 and NBG 26, were found to be elite chemotype of both the markers.

Chemo-profiling and bioassay of phytoextracts from Ageratum conyzoides for acaricidal properties against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infesting cattle and buffaloes in India.

In India, Rhipicephalus (Boophilus) microplus populations have developed a certain level of resistance to most of the acaricides marketed against tick species. To manage the problem, the present study was undertaken to evaluate the acaricidal potential of Ageratum conyzoides plants against acaricides-resistant ticks infesting cattle and buffaloes. The regression analysis of dose-response data of ethanolic extract of A. conyzoides revealed LC90 value of 8.91% against reference susceptible IVRI-1 line of R.(B.) microplus. The ethanolic extract was found efficacious against 76.7-90% acaricides-resistant field ticks and adversely affected oviposition showing 7.04-31.3% reduction in egg laying capacity. The extract was also showed an in vitro efficacy of 52.5 and 76.7% against reference resistant IVRI-4 and 5 lines. The GC/MS/MS profiling of hexane extract, two bioactive sub-fractions and essential oils revealed the presence of 6,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran (precocene II) as a major phyto-compound. The bioactive sub-fractions showed 96.2-97.5% efficacy against larvae of IVRI-1 and 77.1-94.9% against multi-acaricide resistant larvae of IVRI-5 line of R.(B.) microplus. The results of this study provided significant support for the development of a phyto-formulation based on A. conyzoides species.

Identification of potential plant extracts for anti-tick activity against acaricide resistant cattle ticks, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

To develop an eco-friendly tick control method, seven plant extracts were prepared using 50 and 95% ethanol and evaluated for acaricidal activity against cattle tick, Rhipicephalus (Boophilus) microplus. The adult immersion test was adopted for testing different extracts. Based on 72 h screening criterion, 95% ethanolic extracts of Datura metel fruits and Argemone mexicana whole plant were found effective showing more than 50% mortality of treated ticks. The 95% ethanolic extracts of D. metel fruits and A. mexicana whole plant exhibited acaricidal and reproductive inhibitory effects on treated ticks. The LC90 values of D. metel and A. mexicana extracts were determined as 7.13 and 11.3%, respectively. However, although both the extracts were found efficacious against deltamethrin-resistant IVRI-4 and multi-acaricide resistant IVRI-5 lines of R. (B.) microplus, they caused less mortality than treated ticks of the reference IVRI-I line. Phytochemical studies indicated the presence of alkaloids and glucosides in D. metel fruits and alkaloids, terpenoids, flavonoids and phenolics in A. mexicana whole plant extracts. The results indicated that these botanicals may play an important role in reducing the use of chemicals for tick control and possibly to manage resistant tick population in environment friendly manner.