Effect of ivermectin, amitraz and fipronil on midgut epithelium and digestive enzyme profile in Rhipicephalus microplus ticks (Acari: Ixodidae).

Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and fipronil, are known to act on the central nervous system, resulting in the mortality of ticks. The present study is focused on the effect of these acaricides on the midgut and gut enzymes of Rhipicephalus microplus. The ultra-thin sections of midgut of ivermectin-treated ticks showed irregular basal membrane and ruptured digestive vesicles. Amitraz treatment resulted in a notable decrease in digestive cells with pleats in the basal membrane, while fipronil-exposed ticks exhibited reduced digestive cells, loss of cellular integrity, and disintegration of the basal membrane and muscle layer. The gut tissue homogenate of ivermectin and fipronil treated ticks showed a significant reduction of cathepsin D level, 76.54 ± 3.20 µg/mL and 92.67 ± 3.72 µg/mL, respectively, as compared to the control group (150.0 ± 3.80 µg/mL). The leucine aminopeptidase level (4.27 ± 0.08 units/mL) was significantly decreased in the ivermectin treated ticks compared to other treatment groups. The acid phosphatase activity (29.16 ± 0.67 µmole/min/L) was reduced in the ivermectin treated group whereas, increased activity was observed in the fipronil and amitraz treated groups. All the treatment groups revealed increased alkaline phosphatase levels (17.47-26.72 µmole/min/L). The present finding suggests that in addition to the established mechanism of action of the tested acaricides on the nervous system, the alterations in the cellular profile of digestive cells and enzymes possibly affect the blood digestion process and thus the synthesis of vital proteins which are essential for vitellogenesis, and egg production in ticks.

6-Shogaol Exhibits Anti-viral and Anti-inflammatory Activity in COVID-19-Associated Inflammation by Regulating NLRP3 Inflammasomes.

Recent global health concern motivated the exploration of natural medicinal plant resources as an alternative target for treating COVID-19 infection and associated inflammation. In the current study, a phytochemical, 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one; 6-SHO] was investigated as a potential anti-inflammatory and anti-COVID-19 agent. In virus release assay, 6-SHO efficiently (94.5%) inhibited SARS-CoV2 replication. When tested in the inflammasome activation model, 6-SHO displayed mechanistic action by regulating the expression of the inflammasome pathway molecules. In comparison to the existing drugs, remdesivir and hydroxy-chloroquine, 6-SHO was not only found to be as effective as the standard anti-viral drugs but also much superior and safe in terms of predicted physicochemical properties and clinical toxicity. Comparative molecular dynamics simulation demonstrated a stable interaction of 6-SHO with NLRP3 (the key inflammasome regulator) in the explicit water environment. Overall, this study provides important cues for further development of 6-SHO as potential anti-inflammatory and anti-viral therapeutic agents.

Synergistic property of piperonyl butoxide, diethyl maleate, triphenyl phosphate and verapamil hydrochloride with deltamethrin and ivermectin against Rhipicephalus microplus ticks.

The present study was taken up to evaluate the synergistic properties of piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP) and verapamil (VER) with deltamethrin (DLM) and ivermectin (IVM) against DLM and IVM resistant tick populations collected from Madhya Pradesh and Punjab states of India. The collected field tick populations were resistant to DLM (Resistance Factor [RF] in the range of 21.71-32.98) and IVM (RF in the range of 1.89-4.98). A strong synergism between DLM and, IVM with PBO and IVM with VER was noticed. The synergistic efficacy of PBO and VER with IVM in reducing the lethal concentration 50 (LC50) value (1.69-5.72 times for PBO and 3.00-10.62 times for VER) of IVM in resistant ticks suggest that a combination of these synergists with IVM can significantly enhance the effectiveness of IVM against IVM-resistant Rhipicephlaus microplus populations gradually establishing in the different parts of the country. The synergistic efficiency of PBO with DLM in reducing the LC50 value was 2.65 and 18.01 times, respectively, against DLM- resistant two R. microplus populations (KTN and LDH). The study revealed the gradual establishment of DLM and IVM resistant populations in the surveyed states suggesting the need to adopt required resistance management strategies. The use of synergists with DLM and IVM has emerged as an effective approach for controlling the acaricide-resistant ticks.

Evaluation of acaricidal resistance status of Rhipicephalus microplus ticks from the hilly state (Uttarakhand) of India and evaluation of efficacy of a natural formulation for the management of resistant ticks.

The resistance status against deltamethrin, cypermethrin, coumaphos and ivermectin was assessed of Rhipicephalus microplus from five districts of Uttarakhand, through adult immersion test and larval packet test. The field isolates were highly resistant to deltamethrin (median resistance ratio [RR50] = 9.10-29.13-fold) followed by cypermethrin (2.23-3.55). Surprisingly the isolates were susceptible to coumaphos (0.34-3.17). Emerging resistance against ivermectin (1.55-3.27) was also observed in all the isolates. Elevated levels of esterases (enzyme ratio = 2.93-5.84-fold), glutathione S-transferases (5.10-10.06) and monooxygenases (1.68-4.02) in resistant fields isolates were highly correlated (47.4-86.0%) with the resistant factor (RR50) determined by bioassay. All the isolates except Uttarkashi possess mutation at the 190th position in domain II of the sodium channel gene. As a mitigation strategy an Ageratum conyzoides-based characterized natural formulation was tested against all the isolates and was found effective at the concentration of 10.1-11.5%. The possibility of using the natural formulation for the management of multi-acaricide resistant ticks is discussed.

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.

Protective effect of standardised fruit extract of Garcinia cowa Roxb. ex Choisy against ethanol induced gastric mucosal lesions in Wistar rats.

BACKGROUND AND AIM: The fruit of Garcinia is a rich and valuable source of bioactive compounds and is traditionally used for treating wounds and ulcers. The present study was carried out to investigate the protective effect of chromatographically standardized fruit extract of Garcinia cowa (GCE) on ethanol-induced gastric lesions in rats and its possible mechanisms. METHODS: The effect of GCE (200 and 400 mg/kg body weight) was evaluated by determining various gastric ulcer parameters like gastric wall mucus, non-protein sulfhydryls (NP-SH) content, microvascular permeability, endogenous antioxidant enzyme, and gastric histopathological study. RESULTS AND CONCLUSIONS: Oral administration of GCE at doses of 200 and 400 mg/kg exhibited significant (p < .01) dose-dependent inhibition of ulcer index by 18.94-44.02%, respectively. Pre-treatment of rats with GCE (400 mg/kg) significantly restored the depleted gastric wall mucus level by 34.09% and NP-SH content by 33.35% induced by ethanol administration. In addition, GCE (400 mg/kg) showed a significant decrease in microvascular permeability of Evans Blue by 47.43%, rationalizing its protective effect. Furthermore, a significant increase in oxidative enzyme levels with reduction in malondialdehyde level and elevation of superoxide dismutase (SOD) activity was observed in the GCE treated group as compared to the ulcer control group. The histopathological assessment also confirmed the protective nature of GCE. HPTLC analysis showed the presence of 0.27%, 0.11% w/w gallic acid, and amentoflavone, respectively in GCE. The content of α-mangostin and xanthochymol in the G. cowa extract sample quantified by HPLC-PDA method was 0.72 and 8.46%, respectively. The results obtained indicate that the protective effect of GCE against gastric ulcers in rats through multiple actions confirmed by the reduction of oxidative stress and restoration of adhered gastric mucus, NP-SH content, and histological architecture.KEY MESSAGESEthanol is the most typical ulcerogenic agent and has been shown to extend the risk of ulcer in humans.Natural products are promising alternative medication for the development of new drugs to regulate gastrointestinal diseases.Garcinia cowa protects the gastric mucosa through multiple actions that include restoration of adhered gastric mucus and inhibition of lipid peroxidation.

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.

Comparative Profiling of Four Lignans (Phyllanthin, Hypophyllanthin, Nirtetralin, and Niranthin) in Nine Phyllanthus Species from India Using a Validated Reversed Phase HPLC-PDA Detection Method.

BACKGROUND: Phyllanthus species exhibit a wide range of in vitro and in vivo pharmacological activities; however, little is known about the compounds present in the extracts that are responsible for such actions. OBJECTIVE: Development and validation of a simple reversed phase HPLC-PDA method for profiling of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of Phyllanthus species was carried out. METHODS: Separation was achieved using an XBridge column® (150 × 4.6 mm, 5.0 µm id) in an isocratic elution mode with mobile phase comprising of a mixture of acetonitrile and water with TFA (0.05%, v/v, pH = 2.15) at ambient temperature with a flow rate of 1 mL/min. RESULTS: Phyllanthin, hypophyllanthin, nirtetralin, and niranthin were eluted at mean retention times of 10.47, 11.10, 13.67, and 14.53 min, respectively. LOD and LOQ for all four analytes were 0.75 and 3.00 µg/mL, respectively. RSDr values for intraday and interday precision for phyllanthin, hypophyllanthin, nirtetralin, and niranthin were 0.38-1.32 and 0.45-1.77%; 0.22-3.69 and 0.24-3.04%, 0.73-2.37 and 0.09-0.31%, and 1.56-2.77 and 0.12-0.68%, respectively. CONCLUSIONS: The developed and validated HPLC-PDA method was applied for identification and quantification of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of different plant parts of selected Phyllanthus species. The outcome of the present investigation could be useful for selection of best species to promote its commercial cultivation and suitable extraction solvent for preparation of lignan-enriched fractions. This HPLC-PDA method could be useful for quality control of herbal formulations containing plants from Phyllanthus species.

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.

Simultaneous Identification and Quantification of Three Xanthones and Two Polyisoprenylated Benzophenones in Eight Indian Garcinia Species Using a Validated UHPLC-PDA Method.

Background: Xanthones and polyisoprenylated benzophenones (PIBs) are two important classes of plant secondary metabolites with a wide range of bioactivities. Garcinia species synthesize numerous xanthones and PIBs. As per the literature, no data claiming simultaneous identification and quantification of three xanthones, α-mangostin, ß-mangostin, γ-mangostin, and two PIBs, xanthochymol, isoxanthochymol, were found. Methods: A validated ultra-HPLC (UHPLC)-photodiode array (PDA) method for the simultaneous identification and quantification of five compounds in different extracts of eight Indian Garcinia species was developed. The compounds were separated on a Waters ACQUITY™ UPLC H-Class column using a mobile phase consisting of solvents 0.1% formic acid in water (A) and methanol (B) in gradient elution mode. The total run time was 9 min. Results: From fruit rinds of eight Indian Garcinia species, namely Garcinia cambogia, G. cowa, G. indica, G. loniceroides, G. mangostana, G. morella, G. pedunculata, and G. xanthochymus, extracts were prepared using solvents of varying polarity. These extracts were analyzed for five biologically important compounds, namely α-mangostin, ß-mangostin, γ-mangostin, xanthochymol, and isoxanthochymol. The results revealed that there is a wide variation in concentration of these compounds in extracts of Garcinia species. Conclusions: The developed and validated UHPLC-PDA method could be used for simultaneous identification and quantification of these five compounds for bioprospection of other Garcinia species.

Ovicidal and larvicidal effects of extracts from leaves of Andrographis paniculata (Burm. f.) Wall.ex Nees against field isolates of human hookworm (Ancylostoma duodenale).

ETHNOPHARMACOLOGICAL RELEVANCE: The whole plant of Andrographis paniculata (Burm. f.) Wall.ex Nees is used traditionally in different forms by the local people of Asian countries owing to its myriad medicinal properties. Its use as an anthelmintic has been mentioned in literature but has not been well elucidated. AIM OF THE STUDY: To determine anthelmintic effects of extracts from leaves of A.paniculata against human hookworm species based on a standard assay system and to establish the effects of major active compounds responsible for the effects. MATERIALS AND METHODS: Ovicidal and larvicidal activities of extracts of leaves of A.paniculata in different solvents ethanol (Et), methanol (Met), ethyl acetate (EA) and petroleum ether (PE) was studied against field isolates of Ancylostoma duodenale collected and cultivated from hookworm infected human stool samples by egg hatch and larval motility assays. Major active compounds namely andrographolide (AP1), neoandrographolide (AP2) and andrograpanin (AP3) were estimated quantitatively in all the extracts by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Anthelmintic effects (ED50, LC50) and presence of the marker compounds in each extract was statistically analyzed by principal component analysis (PCA). Further, biological activities of pure compounds of AP1, AP2, AP3 were assessed to validate the results of the study. RESULTS: Extracts in ethanol and methanol showed highest activity in inhibition of egg hatching with lowest ED50 values (0.017 and 0.02 mg/mL respectively) while ethyl acetate extract had the highest activity against larval motility (0.001 mg/mL) followed by ethanol (0.019 mg/mL). On HPLC analysis, andrographolide content (%), the major diterpene compound, in Met and Et was 0.85 and 1.43 respectively. On PCA, andrographolide component in the extracts was associated with significant inhibitory effects both on egg hatching and larval motility. Pure compound AP1 also showed significant ovicidal and larvicidal activities at concentrations 0.125 µg/mL and 0.019 mg/mL respectively. CONCLUSION: Andrographolide is one of the main phytochemical responsible for significant ovicidal and larvicidal activity against field isolates of A.duodenale from human infections and can be developed as a potential therapeutic choice.

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC-Photodiode Array Detection Method.

Background: Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective: The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods: The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results: The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84-3.13%, and total withanolide content showed wide variability. Conclusions: The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera-enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights: A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera. Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera, crop improvement, bio-prospection studies and quality control.

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of Rhipicephalus microplus tick collected from a northern state of India.

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9-58.6 and to one of the OP compounds, diazinon having RF of 3.5-13.7 but susceptible to coumaphos (RF < 1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372 bp) and acetylcholinesterase 2 (1692 bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.

Extraction optimization of gallic acid, (+)-catechin, procyanidin-B2, (-)-epicatechin, (-)-epigallocatechin gallate, and (-)-epicatechin gallate: their simultaneous identification and quantification in Saraca asoca.

The objective of the present investigation was to optimize extraction conditions for maximum recovery of bioactive phenolics from different parts of Saraca asoca. Extraction recovery was optimized using a mixture of methanol and water in different proportions. For identification and quantification of six analytes, a rapid reversed phase ultra-performance liquid chromatography (UPLC) photo diode array detection method was developed. UPLC separation was achieved in a gradient elution mode on a C18 column with acetonitrile and aqueous phosphoric acid (0.1%, pH = 2.5). Extraction solvent for maximum recovery of analytes varied depending on the nature of matrices. The developed UPLC method was validated in accordance with International Council for Harmonisation (ICH) guidelines. Wide linearity range, sensitivity, accuracy, short retention time, and simple mobile phase composition implied that the method could be suitable for routine analysis of all six analytes with high precision and accuracy. The uniqueness of this study is the determination of the distribution of these compounds in the various parts of S. asoca.

Comparison on extraction yield of sennoside A and sennoside B from senna (Cassia angustifolia) using conventional and non conventional extraction techniques and their quantification using a validated HPLC-PDA detection method.

Senna is an important medicinal plant and is used in many Ayurvedic formulations. Dianthraquinone glucosides are the main bioactive phytochemicals present in leaves and pods of senna. The extraction efficiency in terms of yield and composition of the extract of senna prepared using both conventional (cold percolation at room temperature and refluxing) and non conventional (ultrasound and microwave assisted solvent extraction as well as supercritical fluid extraction) techniques were compared in the present study. Also a rapid reverse phase HPLC-PDA detection method was developed and validated for the simultaneous determination of sennoside A and sennoside B in the different extracts of senna leaves. Ultrasound and microwave assisted solvent extraction techniques were more effective in terms of yield and composition of the extracts compared to cold percolation at room temperature and refluxing methods of extraction.

Accumulation of three important bioactive compounds in different plant parts of Withania somnifera and its determination by the LC-ESI-MS-MS (MRM) method.

A comprehensive experiment was conducted to study the accumulation pattern and determination of three important bioactive compounds namely withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanolide-A (WD) and its determination by the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method in root, stem, fruits and leaves of Withania somnifera. A rapid and sensitive LC-ESI-MS-MS method was developed and validated for the determination of these three important bioactive compounds, having same molecular weight. The multiple reaction monitoring method was established by two transitions for each analyte and intense transition used for quantification. Separation of the three analytes was achieved within a run time of 5 min on an RP-18 column using a mobile phase consisting of acetonitrile and 0.1% acetic acid in water in an isocratic condition. The developed method was validated as per the ICH guidelines. The developed method was found to be suitable for identification and quantification of WA, WO and WD in different plant parts such as roots, stems, fruits and leaves of W. somnifera. The accumulation of WA was highest in leaves samples (8.84 ± 0.37 mg/g) and it was 2.23, 5.85 and 27.26 times higher than its concentration in fruits, stems and roots, respectively. WO and WD contents were highest (0.44 ± 0.016 and 0.72 ± 0.016 mg/g, respectively) in root.

A validated high-performance liquid chromatography method for determination of tannin-related marker constituents gallic acid, corilagin, chebulagic acid, ellagic acid and chebulinic Acid in four Terminalia species from India.

A validated rapid HPLC-PDA method was developed for identification and quantification of five tannin-related constituents gallic acid (GA), corilagin (CL), chebulagic acid (CB), ellagic acid (EA) and chebulinic acid (CN) in the extracts prepared from the bark and fruits of four Terminalia species available in India. The separation of the five analytes was achieved on an RP-18 column (4.6 × 250 mm, 5 µm) at 25°C using a solvent mixture comprising of acetonitrile and (0.05%) trifluoroacetic acid-water in a gradient elution mode. Limit of detection was 1.0, 0.5, 1.0, 0.5 and 1.0 µg/mL for GA, CL, CB, EA and CN, respectively. Similarly, limit of quantification was 2.5, 1.0, 2.5, 1.0 and 2.5 µg/mL for GA, CL, CB, EA and CN, respectively. Good linearity (r(2) > 0.992) was observed for all the five compounds in wide concentration range. Using the developed HPLC method, the five analytes were identified and quantified in bark and fruit extracts of Terminalia chebula, Terminalia bellirica, Terminalia arjuna and Terminalia catappa. This is the first report of identification and quantification of the five tannin-related marker constituents in the bark and fruit extracts of T. chebula, T. bellirica, T. arjuna and T. catappa.

Novel chemical constituents with anti-inflammatory activity from the leaves of Sesbania aculeata.

From the hexane and ethyl acetate extracts of the leaves of Sesbania aculeata, three novel chemical compounds were isolated and fully characterized as compound 1, (ceramide type); compound 2, (cerebroside type) and compound 3 as a triterpene acid 3-O-α-L-rhamnopyranoside along with nine known compounds (Tricontanol, Lauric acid, Palmitic acid, Heptadecanoyl-1-tridecanoic acid, ß-sitosterol, stigmasterol, poriferasterol glucoside, ononitol and pinitol). The anti-inflammatory potential of all three compounds were evaluated using in vitro target based anti-inflammatory activity in LPS-stimulated macrophages. TNF-α is one of the mediators of various chronic inflammatory disorders and treatment of hexane leaf extract (HL), Ethyl acetate leaf extract (EAL) and compounds 1, 2 and 3 at a dose of 10 µg/mL showed significant (P<0.001) inhibition of TNF-α, a pro-inflammatory cytokine. IL-6 was significantly (P<0.05) inhibited by compound 1 and HL at a dose of 10 µg/mL as compared with vehicle treatment. In-vitro cell cytotoxicity study using MTT assay revealed that these compounds were non toxic to the normal cells.

Extraction of three bioactive diterpenoids from Andrographis paniculata: effect of the extraction techniques on extract composition and quantification of three andrographolides using high-performance liquid chromatography.

Andrographis paniculata (Burm.f.) wall.ex Nees (Acanthaceae) or Kalmegh is an important medicinal plant finding uses in many Ayurvedic formulations. Diterpenoid compounds andrographolides (APs) are the main bioactive phytochemicals present in leaves and herbage of A. paniculata. The efficiency of supercritical fluid extraction (SFE) using carbon dioxide was compared with the solid-liquid extraction techniques such as solvent extraction, ultrasound-assisted solvent extraction and microwave-assisted solvent extraction with methanol, water and methanol-water as solvents. Also a rapid and validated reverse-phase high-performance liquid chromatography-diode array detection method was developed for the simultaneous determination of the three biologically active compounds, AP, neoandrographolide and andrograpanin, in the extracts of A. paniculata. Under the best SFE conditions tested for diterpenoids, which involved extraction at 60°C and 100 bar, the extractive efficiencies were 132 and 22 µg/g for AP and neoandrographolide, respectively. The modifier percentage significantly affected the extraction efficiency.

A validated HPLC-PDA method for identification and quantification of two bioactive alkaloids, ephedrine and cryptolepine, in different Sida species.

A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species.