Exploring Steroidal Saponin Composition and Morphometric Characteristics of Rhizomes from Trillium govanianum Wall. ex D. Don: Inference for Medicinal Properties and Genetic Stock Improvement.

Trillium govanianum, a medicinal herb, exhibiting diverse morphometric traits and phytochemicals across developmental stages of plants. The changes in the chemical profile and steroidal saponin levels in the rhizome of T. govanianum across different developmental stages were previously unknown. This study categorizes rhizomes into three types based on scar presence: juvenile (5-10 scars, Type I), young (11-19 scars, Type II), and mature (21-29 scars, Type III). Rhizomes show varying sizes (length 1.2-4.7 cm, girth 0.3-1.6 cm), weight (0.18-5.0 g), and extractive yields (9.7-16.1 % w w-1), with notable differences in saponin content (5.95-21.9 mg g-1). Ultra-high performance liquid chromatography-MS/MS (UHPLC-QTOF-MS/MS)-based chemical profiling identifies 31 phytochemicals, mainly including diverse saponins. Ultra-high performance liquid chromatography coupled with evaporative light scattering detection (UHPLC-ELSD)-based quantitative analysis of seven key saponins reveals stage-specific accumulation patterns, with protodioscin (P) and dioscin (DS) predominant in mature rhizomes. Statistical analysis confirms significant variation (p=0.001) in saponin levels across developmental stages with chemical constituent protodioscin (P=4.03±0.03-15.76±0.14 mg g-1, PAve=9.79±3.03 mg g-1) and dioscin (DS=1.23±0.06-3.93±0.07 mg g-1, DSAve=2.59±0.70 mg g-1), with acceptable power (p=0.738; |δ|>0.5) statistics for effective sample size (n=27 samples used in the study) of T. govanianum. Principal Component Analysis (PCA) and Euclidean clustering further highlighted chemotype distinctions.

NADES extraction, UHPLC-ELSD-based quantification, and network pharmacology-guided target identification of fourteen specialised metabolites from Trillium govanianum Wall. ex D.Don.

INTRODUCTION: Trillium govanianum Wall. ex D.Don is a folk medicinal herb rich in structurally diverse steroidal saponins. The annual demand for this herb in India is about 200-500 metric tons, highlighting the need for a thorough quality assessment. OBJECTIVE: The objective of this study is to develop an easy and reliable ultrahigh-performance liquid chromatography-evaporative light scattering detector (UHPLC-ELSD)-based quality assessment method with 14 specialised metabolites of T. govanianum and identify the potential targets of this herb using network pharmacology. MATERIAL AND METHODS: A UHPLC-ELSD method was developed and validated with 14 markers of T. govanianum. The developed method and natural deep eutectic solvent (NADES)-assisted extraction were utilised for the recovery enhancement study of targeted specialised metabolites from rhizome samples (collected from five geographically distinct areas). In addition, the network pharmacology approach was performed for these 14 markers to predict the plausible biological targets of T. govanianum. RESULT: The developed method showed good linearity (r2: 0.940-0.998), limit of detection (LOD) (2.4-9.0 µg), limit of quantification (LOQ) (7.92-29.7 µg), precision (intra-day relative standard deviations [RSDs] 0.77%-1.96% and inter-day RSDs 2.19-4.97%), and accuracy (83.24%-118.90%). NADES sample TG-1* showed the highest recovery (yield: 167.66 ± 4.39 mg/g of dry weight) of total saponin content (TSC) as compared to its hydroethanolic extract (yield: 103.95 ± 5.36 mg/g of dry weight). Sample TG-1* was the most favourable (yield: 167.66 ± 4.39 mg/g) in terms of TSC as compared to other analysed samples (32.68 ± 1.04-88.22 ± 6.79 mg/g). Govanoside D (yield: 3.43-28.06 mg/g), 22ß-hydroxyprotodioscin (yield: 3.22-114.79 mg/g), and dioscin (yield: 1.07-20.82 mg/g) were quantified as the major metabolites. Furthermore, network pharmacology analysis of targeted 14 markers indicated that these molecules could be possible therapeutic agents for managing neuralgia, diabetes mellitus, and hyperalgesia. CONCLUSION: The current study represents the first report for the simultaneous quantification and a network pharmacology-based analysis of 14 chemical marker compounds isolated from T. govanianum.

Chemometric guided isolation of new triterpenoid saponins as acetylcholinesterase inhibitors from seeds of Achyranthes bidentata Blume.

Achyranthes bidentata Blume (Amaranthaceae) is an annual or perennial herb widely used as ethnomedicine in Traditional Chinese Medicine for treating fever, cold, ulcers, mensural pain, dementia, and osteoporosis. In the current study, UPLC-IM-Q-TOF-MS/MS-based chemometric approach was adopted for the tentative identification of fifty-six compounds in the extract and fractions of A.bidentata seeds. Further, the chemometric-guided isolation led to the isolation of two previously undescribed oleanane-type triterpenoid saponins, named achyranosides A-B (27 and 30), along with three known compounds (31, 44, and 23) from water fraction of A. bidentata seeds. The structures of new compounds were elucidated based on the detailed analysis of NMR, HR-ESI-MS, FT-IR spectral data, and GC-FID techniques. The isolated compounds in vitro acetylcholinesterase inhibitory activity revealed the promising activity of chikusetsusaponin IVa (23) (IC50 = 63.7 µM) with mixed type of AChE inhibition in enzyme kinetic studies. Additionally, in silico binding free energy of isolated compounds disclosed the greater stability of enzyme-ligand complex owing to underlying multiple H-bond interactions. Overall, the study demonstrates the effectiveness of a chemometric-guided approach for the phytochemical exploration and isolation of new oleanane-type triterpenoid saponins from A. bidentata seeds.

Cyperus rotundus L.: Invasive weed plant with insecticidal potential against Aphis craccivora Koch and Planococcus lilacinus (Cockerell).

Cyperus rotundus L. is a widely distributed invasive weed plant with vast traditional medicinal uses. Herein, the methanolic root extract of C. rotundus and its fractions (n-hexane, chloroform, n-butanol, and aqueous) were evaluated for insecticidal activity against nymphs of Aphis craccivora Koch and crawlers of Planococcus lilacinus (Cockerell) to find promising lead (s). In contact topical assay, among extract/fractions, n-hexane fraction exhibited more toxicity against A. craccivora (LD50 = 1.12 µg/insect) and P. lilacinus (LD50 = 0.94 µg/insect). The chemical analysis of n-hexane fraction revealed a volatile composition similar to that of the essential oil (EO) of C. rotundus roots. Hence, EO was extracted using water and deep eutectic solvents (DESs) as cosolvent, which revealed enhancement in EO yield (from 0.28 to 0.46% w/w) on implementing DESs. A total of 35 diverse volatile metabolites were identified in all EO samples, accounting for 85.0 to 91.8% of chemical composition, having cyperotundone, cyperene mustakone, isolongifolen-5-one, boronia butenal as major constituents. The EO obtained with DES-7 [choline chloride: ethylene glycol (1:4)] and DES-6 [choline chloride: lactic acid (1:3)] were found effective against A. craccivora (LD50 = 0.62-0.87 µg/insect) and P. lilacinus (LD50= 0.59-0.67 µg/insect) after 96 h. NMR analysis of EO revealed cyperotundone as a major compound, which was isolated along with cyperene and cyperene epoxide. All the molecules were found effective against P. lilacinus, whereas against A. craccivora cyperotundone, cyperene and cyperene epoxide showed promising toxicity (LD50 = 0.74-0.86 µg/insect). Extract/fractions, EO, and isolated molecules showed a significant reproductive inhibition rate of A. craccivora at higher concentrations. All the tested concentrations of cyperotundone showed significant inhibition of acetylcholinesterase (AChE) and glutathione-S-transferase (GST) in A. craccivora and P. lilacinus. Based upon the present study, C. rotundus can be recommended to control targeted insects in the greenhouse/field conditions after performing bio-efficacy and phytotoxicity studies.

Shatavarin-IV saponin adjuvant elicits IgG and IgG2b responses against Staphylococcus aureus bacterin in a murine model.

Asparagus adscendens Roxb. also known as "safed musli" or "shatavari" is a medicinal plant commonly found in South Asian countries. Shatavari is effective for the treatment of gastric ulcers, renal stones, bronchitis, diabetes, diabetic neuropathy, irritable bowel syndrome, alcohol withdrawal and has reported immunostimulatory effects. In this study, the adjuvant potential of Shatavarin-IV saponin against Staphylococcus aureus bacterin in mice was investigated. Shatavarin-IV was evaluated for its toxicity and immunomodulatory potential against S. aureus bacterin in mice. Cellular and humoral immune responses were assessed. Shatavarin-IV was isolated from the fruit extract of Asparagus adscendens. The confirmation of the isolated molecule as Shatavarin-IV was done via TLC-based comparison with the standard molecule. Further, the structure was confirmed by using extensive spectroscopic analyses and comparing the observed data with literature reports. It was found safe up to the dose of 0.1 mg in the mice model. Shatavarin-IV adjuvant elicited IgG and IgG2b responses at the dose of 40 µg against S. aureus bacterin. However, the cell-mediated immune response was lesser as compared with the commercial Quil-A saponin . We demonstrated that Shatavarin-IV saponin adjuvant produced an optimum humoral immune response against S. aureus bacterin. These results highlight the potential of Shatavarin-IV as an adjuvant in a combination adjuvant in vaccine formulations for induction of potent immune response.

Antiplasmodial activity of the bulbs of Fritillaria cirrhosa D.Don (Syn: Fritillaria roylei Hook.): UPLC-IM-Q-TOF-MS/MS-based biochemometric approach for the identification of marker compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillaria cirrhosa D.Don (Syn: Fritillaria roylei Hook.) (Hindi name: Kshirakakoli) is a critically endangered Himalayan medicinal plant, well documented in Ayurveda for its therapeutic uses against various disorders such as jvara (fever), kasa (respiratory tract disease) etc. Its bulbs are also used as Szechuan-Pei-Mu for their antipyretic properties in the traditional Chinese medicine. However, despite its ethnomedicinal usage, the therapeutic use of F. cirrhosa bulbs for jvara (fever) related conditions such as malaria has remained unexplored. Hence in the context of increasing global concerns about drug-resistant malaria, it is important to investigate the antiplasmodial activity of F. cirrhosa bulbs for novel antimalarial agents. AIM OF THE STUDY: To investigate the antiplasmodial effects of the extracts/fractions of F. cirrhosa bulbs by the biochemometric approach and to rationalize its ethnopharmacological usage for jvara (fever) related conditions such as malaria. MATERIAL AND METHODS: This study involves the UHPLC-MS-based plant material selection, preparation, quantification, and assessment of F. cirrhosa bulb extracts against CQ-sensitive Pf 3D7 & CQ-resistant Pf INDO strains. Further, UPLC-IM-Q-TOF-MS-based biochemometric approach has been applied for the identification of marker compounds responsible for the observed antiplasmodial effects. The identified marker compounds were also assessed for their in silico ADMET properties and binding efficacy with the drug transporter Pf CRT. RESULTS: Different F. cirrhosa bulb extracts/fractions showed promising antiplasmodial activity with IC50 values 2.71-19.77 µg/mL for CQ-resistant Pf INDO strain and 1.76-21.52 µg/mL for CQ-sensitive Pf 3D7 strain. UPLC-IM-Q-TOF-MS/MS-based biochemometric analysis revealed four marker compounds i.e., peimine (m/z 432.3448), peimisine (m/z 428.3504), puqiedinone (m/z 414.3379), and puqiedine (m/z 416.3509) responsible for the observed antiplasmodial activity. The identified marker compounds showed excellent binding efficacy with Pf CRT and suitable drug-like properties in silico. CONCLUSIONS: The study demonstrated promising antiplasmodial activity of the chloroform and alkaloid enriched fractions of F. cirrhosa bulbs and further identified the four marker compounds responsible for the promising antiplasmodial activity. These marker compounds i.e., peimine, peimisine, puqiedinone and puqiedine were identified by the biochemometric analysis as the putative antiplasmodial constituents of the F. cirrhosa bulbs. Further, in silico studies indicated the good binding affinity of the marker compounds with Pf CRT along with suitable ADMET properties. Overall, the study elucidates the antiplasmodial activity of F. cirrhosa bulbs from the western Himalayan region and provides nascent scientific evidence for their ethnopharmacological usage in jvara (fever) related conditions such as malaria.

Bisbenzylisoquinolines from Cissampelos pareira L. as antimalarial agents: Molecular docking, pharmacokinetics analysis, and molecular dynamic simulation studies.

Malaria is a major global health issue due to the emergence of resistance to most of the available antimalarial drugs. There is an urgent need to discover new antimalarials to tackle the resistance issue. The present study aims to explore the antimalarial potential of chemical constituents reported from Cissampelos pareira L., a medicinal plant traditionally known for treating malaria. Phytochemically, benzylisoquinolines and bisbenzylisoquinolines are the major classes of alkaloids reported from this plant. In silico molecular docking revealed prominent interactions of bisbenzylisoquinolines such as hayatinine and curine with Pfdihydrofolate reductase (-6.983 Kcal/mol and -6.237 Kcal/mol), PfcGMP-dependent protein kinase (-6.652 Kcal/mol and -7.158 Kcal/mol), and Pfprolyl-tRNA synthetase (-7.569 Kcal/mol and -7.122 Kcal/mol). The binding affinity of hayatinine and curine with identified antimalarial targets was further evaluated using MD-simulation analysis. Among the identified antimalarial targets, the RMSD, RMSF, the radius of gyration, and PCA indicated the formation of stable complexes of hayatinine and curine with Pfprolyl-tRNA synthetase. The outcomes of in silico investigation putatively suggested that bisbenzylisoquinolines may act on the translation of the Plasmodium parasite to exhibit antimalarial potency.

Comparative phytochemical analysis of Ferula assa-foetida with Ferula jaeschkeana and commercial oleo-gum resins using GC-MS and UHPLC-PDA-QTOF-IMS.

Ferula assa-foetida is an important species of the genus Ferula, best known for its oleo-gum resin, mainly used as a flavoring agent. Ferula jaeschkeana is another Himalayan medicinal plant of this genus, known for its contraceptive effect but not used in food applications. This study aimed to do a detailed phytochemical analysis of F. assa-foetida growing under controlled conditions in India using GC-MS/headspace and UHPLC-PDA-QTOF-IMS. Further, a comparative analysis of F. assa-foetida was performed with F. jaeschkeana (collected from its natural habitat) and commercial samples of F. assa-foetida oleo-gum resin (collected from the local market). UHPLC-QTOF-IMS profiling of F. assa-foetida led to the identification of foetisulfide C, assafoetidnol A, gumosin, flabellilobin (A/B), and foetisulfide A. In total, 141 metabolites were identified, including vitamins, nucleosides, sulfur compounds, flavonoids, sugars derivatives, and others, using METLIN database. Serine, arginine, asparagine, isoleucine, and phenylalanine were major amino acids quantified among the samples for the nutritional aspect. Characteristic sulfurous compounds (n-propyl-sec-butyl disulfide, trans-propenyl-sec-butyl disulfide, cis-propenyl-sec-butyl disulfide, and bis[1-(methylthio)propyl] disulfide) were identified in all samples except F. jaeschkeana. PCA and cluster analysis showed a significant difference in the volatile constituents of rhizomes of both species. Metabolomics studies also revealed the association of sesquiterpenoid and triterpenoid biosynthesis, phenylpropanoid, flavon, and flavanol biosynthesis. The current study demonstrates, "why only F. assa-foetida is used in culinary applications instead of F. jaeschkeana"?

Insecticidal potential of extracts, fractions, and molecules of Aconitum heterophyllum Wall ex. Royle against aphid Aphis craccivora Koch (Hemiptera: Aphididae).

BACKGROUND: Aphis craccivora is the major sap-sucking pest of leguminous crops and vector of plant viruses that cause damage to plants and reduce yield. Indiscriminate and nonjudicious use of synthetic insecticides led to resistance development and harmful to environment. Therefore, it is important to discover plant-based lead(s) which can replace synthetic insecticides. In the current study the residual toxicity of extracts, fractions, and isolated compounds of Aconitum heterophyllum were evaluated against A. craccivora to identify lead(s) for further development of botanical formulation. RESULTS: In residual contact assay, ethanolic (LC50  = 2837.17 mg L-1 ) and aqueous methanolic extracts (LC50  = 2971.59 mg L-1 ) were effective against A. craccivora. Among fractions, the n-butanol fraction of the aqueous methanolic extract (LC50  = 986.96 mg L-1 ) was found to be most effective, followed by the ethyl acetate fraction of the ethanolic extract (LC50  = 1037.52 mg L-1 ) and the n-hexane fraction of both extracts (LC50  = 1113.85 to 1233.11 mg L-1 ). Among pure molecules, aconitic acid was found to be the most effective (68% mortality; LC50  = 1313.19 mg L-1 ) and was on a par with azadirachtin 0.15% EC (66% mortality; LC50  = 1921.10 mg L-1 ). Furthermore, from the effect of ethanoic extract on detoxification enzyme inhibition in A. craccivora we concluded that the target site of action of this extract in A. craccivora might be glutathione S-transferase. CONCLUSIONS: The parent extract/fractions of A. heterophylum showed promising activity against A. craccivora. Among phytoconstituents of the active extract and fractions, aconitic acid was found to be on a par with azadirachtin 0.15% EC. © 2022 Society of Chemical Industry.

Iridoid glycosides from Picrorhiza genus endemic to the Himalayan region: phytochemistry, biosynthesis, pharmacological potential and biotechnological intercessions to boost production.

Iridoid glycosides are monoterpenoids synthesized in several plant species known to exhibit a diverse range of pharmacological activities. They are used as important bioactive ingredients in many commercially available drug formulations and as lead compounds in pharmaceutical research. The genus Picrorhiza comprises two medicinally important herbs endemic to the Himalayan region viz. Picrorhiza kurrooa Royle and Picrorhiza scrophulariiflora Hong. The medicinal properties of these two species are mainly due to iridoid glycosides present in their root, rhizome, and leaves. Unregulated harvesting from the wild, habitat specificity, narrow distribution range, small population size and lack of organized cultivation led to the enrolling of these species in the endangered category by the International Union for Conservation of Nature and Natural Resources (IUCN). Therefore, there is a need for immediate biotechnological and molecular interventions. Such intercessions will open up new vistas for large-scale propagation, development of genomic/transcriptomic resources for understanding the biosynthetic pathway, the possibility of genetic/metabolic manipulations, and possible commercialization of iridoid glycosides. The current review article elucidates the phytochemistry and pharmacological importance of iridoid glycosides from the genus Picrorhiza. In addition, the role of biotechnological approaches and opportunities offered by next-generation sequencing technologies in overcoming challenges associated with the genetic engineering of these species are also discussed.

A novel extraction method enhanced the osteogenic and anti-osteoporosis effect of tea extract without any hepatotoxicity in ovariectomized rats.

Tea (Camellia sinensis) has several reported health benefits, including that on bone health attributed to catechins of which the most abundant is epigallocatechin-3-gallate (EGCG). However, several preclinical and clinical studies raise safety concerns about EGCG in tea extract causing acute liver failure. Tea also contains kaempferol, albeit scanty, and it has hepatoprotective and osteogenic effects. Here, we utilized a novel extraction procedure of acid hydrolysis to enhance the osteogenic effect of tea extract while reducing its hepatotoxicity. The resultant extract (USKECSE) has a ~40-fold increase in kaempferol and a 2.5-fold reduction in EGCG content compared with the hydroethanolic extract (USCSE). In a female Sprague Dawley (SD) rat femur osteotomy model, USKECSE (100 mg/kg) but not USCSE promoted bone regeneration. In a rat postmenopausal osteoporosis model induced by bilateral ovariectomy (OVX), USKECSE through an osteogenic mechanism maintained bone mass, strength, and microarchitecture to the levels of ovary-intact rats with no hepatotoxic effect. After a single oral dose (100 mg/kg) of USKECSE to adult rats, kaempferol was detectable for 48 hours, suggesting its significant absorption and distribution in plasma. Peak kaempferol concentration in plasma (Cmax) was 483 ng/ml (2 µM), and at this concentration, kaempferol induces osteoblast differentiation. USKECSE had no genotoxicity, and its safety index assessed by preclinical toxicity studies, including safety pharmacology, was >20-fold. Taken together, we report a novel extraction process that enhanced the osteogenicity and concomitantly reduced hepatotoxicity of tea extract with significant kaempferol bioavailability and a favorable systemic safety profile. Based on these data, we propose assessing the USKECSE effect for postmenopausal osteoporosis treatment.

Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by connectivity map-based analysis and in vitro studies.

BACKGROUND: Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds such as Oseltamivir, extracted using shikimic acid from star anise (Illicium verum) and Acyclovir from Carissa edulis are FDA (Food and Drug Administration) approved antiviral drugs. In this study, we dissect the anti-coronavirus infection activity of Cissampelos pareira L (Cipa) extract using an integrative approach. METHODS: We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map ( https://clue.io/ ). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. Molecular docking analyses of constituents of with key targets of SARS-CoV2 protein viz. spike protein, RNA­dependent RNA­polymerase (RdRp) and 3C­like proteinase. was also performed. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. RESULTS: Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g. pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa. Amongst all the tested compounds, Magnoflorine and Salutaridine exhibited the most potent and consistent strong in silico binding profiles with SARS-CoV2 therapeutic targets.

Insecticidal Activity of Extracts, Fractions, and Pure Molecules of Cissampelos pareira Linnaeus against Aphid, Aphis craccivora Koch.

Aphis craccivora Koch is a polyphagous and major pest of leguminous crops causing significant damage by reducing the yield. Repeated application of synthetic insecticides for the control of aphids has led to development of resistance. Therefore, the present study aimed to screen the insecticidal activity of root/stem extracts/fractions, and pure molecules from Cissampelos pareira Linnaeus against A. craccivora for identification of lead(s). Among root extract/fractions, the n-hexane fraction was found most effective (LC50 = 1828.19 mg/L) against A. craccivora, followed by parent extract (LC50 = 2211.54 mg/L). Among stem extract/fractions, the n-hexane fraction (LC50 = 1246.92 mg/L) was more effective than the water and n-butanol fractions. Based on GC and GC-MS analysis, among different compounds identified in the n-hexane fraction of root and stem, ethyl palmitate (known to possess insecticidal activity) was present in the highest concentration (24.94 to 52.95%) in both the fractions. Among pure molecules, pareirarineformate was found most effective (LC50 = 1491.93 mg/L) against A. craccivora, followed by cissamine (LC50 = 1556.31 mg/L). Parent extract and fractions of C. pareira possess promising activity against aphid. Further, field bio-efficacy studies are necessary to validate the current findings for the development of botanical formulation.

Antiplasmodial diterpenoid alkaloid from Aconitum heterophyllum Wall. ex Royle: Isolation, characterization, and UHPLC-DAD based quantification.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum heterophyllum Wall. ex Royle is a traditionally important medicinal plant having numerous therapeutic actions as documented in Ayurveda. This plant is traditionally known for combating worm infestation, fever, respiratory tract disease, vomiting, diarrhoea, diabetes, skin disorders, anaemia, and joint disorders. Further, it has been used alone and in combination with other plants to prepare various anti-malarial formulations. However, there is no report on the assessment of its anti-plasmodial activity, and the metabolite(s) responsible for this activity. AIM OF THE STUDY: The main aim of this study was to conduct phytochemical investigation of A. heterophyllum roots for the preparation of extract, fractions, and isolation of pure molecules to identify active fractions/molecules responsible for the anti-plasmodial activity, and development of UHPLC-DAD based analytical method which can be used for the quantification of marker compounds in the extracts and fractions. MATERIALS AND METHODS: Hydroalcoholic extract (1:1 v/v) and fractions (n-hexane, chloroform, ethyl acetate, n-butanol, and water) were prepared from the dried powdered roots of A. heterophyllum. Fractions were further subjected to silica gel column chromatography to isolate pure specialized secondary metabolites from this plant. All extracts, fractions, and pure molecules were evaluated against the chloroquine resistant Pf INDO and chloroquine sensitive Pf3D7 strains in culture for calculating their IC50 values. UHPLC-DAD based analytical method was also developed for the first time for the quantification of marker compounds and quality assessment of this commercially important Himalayan medicinal plant. RESULTS: Phytochemical investigation of A. heterophyllum root led to the isolation of six specialized metabolites viz. 2-O-cinnamoyl hetisine (1), atisinium (2), 4-oxabicyclo [3.2.2] nona-1(7),5,8-triene (3), atisinium cinnamate (4), aconitic acid (5), and atisinium formate (6). Compound 1 is a new hetisine type diterpenoid alkaloid, compounds 4 and 6 are new counter ionic forms observed with atisinium ion, and compound 3 is being reported for the first time from this genus. Chloroform fraction was found to be the most active with IC50 (µg/mL) 1.01 (Pf INDO) and 1.32 (Pf3D7). The molecule 2-O-cinnamoyl hetisine (1), a new diterpenoid alkaloid isolated from chloroform fraction, showed promising antiplasmodial activities with IC50 (µM) 1.92 (Pf INDO) and 10.8 (Pf 3D7). The activity of chloroform fraction was further validated by the developed UHPLC-DAD based method as the quantity of 2-O-cinnamoyl hetisine (1) was higher in the chloroform fraction (≅200 mg/g) than in all other fractions (<7 mg/g). Atisinium (2) and 2-O-cinnamoyl hetisine (1) were found to be the main marker compounds of this plant based on quantity and antiplasmodial activity, respectively. CONCLUSION: This study provides the scientific rationale for the traditional use of this plant in treating malaria. Further, this study revealed that the anti-malarial potential of this plant might be due to the presence of diterpenoid alkaloids.

Transcriptome analysis and connectivity mapping of Cissampelos pareira L. provides molecular links of ESR1 modulation to viral inhibition.

Bioactive fractions obtained from medicinal plants which have been used for the treatment of multiple diseases could exert their effects by targeting common pathways. Prior knowledge of their usage could allow us to identify novel molecular links. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira L. (Cipa), used for the treatment of female hormone disorders and fever. Transcriptomic studies on MCF7 cell lines treated with Cipa extract carried out using Affymetrix arrays revealed a downregulation of signatures of estrogen response potentially modulated through estrogen receptor α (ERα). Molecular docking analysis identified 38 Cipa constituents that potentially bind (ΔG < - 7.5) with ERα at the same site as estrogen. The expression signatures in the connectivity map ( https://clue.io/; ) revealed high positive scores with translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is a reported ERα coactivator. Further, gene knockdown experiments revealed that Cipa exhibits antiviral activity in dengue infected MCF7 cells potentially modulated through estrogen receptor 1. This approach reveals a novel pathway involving the ESR1-RPL7 axis which could be a potential target in dengue viral infection.

Steroidal saponins from Trillium govanianum as α-amylase, α-glucosidase, and dipeptidyl peptidase IV inhibitory agents.

OBJECTIVE: To provide the scientific basis for the utility of rhizome of Trillium govanianum as nutraceutical supplements in managing physiological glycemic levels. METHODS: The in vitro enzyme inhibitory activity of the extract, fractions, and the isolated steroidal saponins from the rhizome part of T. govanianum was carried out against α-amylase, α-glucosidase, and dipeptidyl peptidase IV. The molecular interactions, binding score, and pharmacokinetic parameters (absorption, distribution metabolism, and excretion) of steroidal saponins were analyzed by the Schrodinger molecular docking software. KEY FINDINGS: Current study explained that the extract, fractions, and isolated steroidal saponins from T. govanianum possess good α-amylase and α-glucosidase inhibitory activity while moderate dipeptidyl peptidase IV inhibitory activity. Moreover, in vitro results revealed that borassoside E (IC50 7.15 ± 1.78 µM), protodioscin (IC50 6.72 ± 0.04 µM), and diosgenin (IC50 12.75 ± 2.70 µM) are most effective in inhibiting the activity of α-amylase, α-glucosidase, and dipeptidyl peptidase IV, respectively. Current in silico and in vitro studies established an association between the steroidal saponins from T. govanianum and their molecular interactions with α-amylase, α-glucosidase, and dipeptidyl peptidase IV. CONCLUSION: The results of this investigation suggest that fractions and steroidal saponins from T. govanianum exhibit good antidiabetic activity which could be used as nutraceutical supplements for the management of systemic glucose level.

Cissampelos pareira L.: A review of its traditional uses, phytochemistry, and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Cissampelos pareira, a well-known medicinal climber-plant of the Menispermaceae family, has been extensively used in the traditional medicinal system since the ancient time for the treatment of numerous diseases such as ulcer, wound, rheumatism, fever, asthma, cholera, diarrhoea, inflammation, snakebite, malaria, rabies, and also recommended for blood purification. AIM OF THE REVIEW: The main purpose of this review is to provide updated information on ethnopharmacology, phytochemistry, chromatographic and spectroscopic analysis, pharmacology, and toxicology of C. pareira along with the possible future research. This information will help to provide a foundation for plant-based drug discovery in the near future. MATERIAL AND METHODS: The online databases such as Scifinder, Web of Science, PubMed, and Google Scholar were used to collect electronically available literature data on C. pareira. Ayurveda text is searched for the traditional uses of this plant in India. The published books are also searched for the information on this plant. Our search was based on traditional uses, botany, phytochemistry, and pharmacological potential by using "Cissampelos pareira" as the keyword. RESULTS: To date, approximately 54 phytomolecules have been isolated and characterized from C. pareira including mainly isoquinoline alkaloids along with few flavonoids, flavonoid glycosides, and fatty acids. The crude extracts of C. pareira have shown various pharmacological activities such as antipyretic, anti-inflammatory, antiarthritic, antiulcer, antidiabetic, anticancer, antifertility, antimicrobial, antioxidant, antivenom, antimalarial, and immunomodulatory, etc. The chemical fingerprinting of C. pareira carried out using HPTLC, HPLC, UPLC, LC-MS, and GC-MS, revealed the presence of alkaloids (isoquinoline alkaloids), fatty acids, and flavonoid glycosides. Moreover, the toxicological assessment of C. pareira has been moderately investigated, which requires further comprehensive studies. CONCLUSION: Comprehensive literature survey reveals that till date, remarkable growth has been made on phytochemistry and pharmacology of C. pareira reflecting the great medicinal potential of this plant. Although some of the traditional uses have been well clarified and documented by modern pharmacological analysis, the correlation between its pharmacological activities and particular phytoconstituents still needs to be validated. Furthermore, there is partial data available on most of the pharmacological studies, along with incomplete toxicological screening. Future research needs to pay more attention to pharmacological studies of C. pareira via pre-clinical and clinical trials. Additionally, scientific validation of traditional knowledge of C. pareira is vital for ensuring safety, efficacy, and mechanism of action before clinical uses.

Insecticidal activity of the extract, fractions, and pure steroidal saponins of Trillium govanianum Wall. ex D. Don for the control of diamondback moth (Plutella xylostella L.) and aphid (Aphis craccivora Koch).

BACKGROUND: Plutella xylostella L. is the major pest of crucifers globally, causing significant yield loss. Aphis craccivora Koch is the main sucking pest of legumes that transmit viral diseases, leading to economic yield reduction. To minimize loss due to pests, farmers/growers use synthetic insecticides frequently for their control, which led to insecticide resistance, detrimental to natural enemies of pest, environment, etc. Therefore, in this study, the insecticidal activity of plant extract, fractions, and pure steroidal saponins from Trillium govanianum was evaluated for their bio-efficacy against targeted pests. RESULTS: Parent extract was found more effective (LC50 = 1541.2 mg L-1 ) against larvae of P. xylostella after 96 h than n-butanol, n-hexane, and ethyl acetate fractions (LC50 = 3030, 3578 and 3878.1 mg L-1 , respectively). For A. craccivora, ethyl acetate fraction (LC50 = 2186.3 mg L-1 ) was most effective after 96 h than n-hexane fraction (LC50 = 2234.6 mg L-1 ), n-butanol fraction (LC50 = 2696.3 mg L-1 ) and parent extract (LC50 = 3709.1 mg L-1 ). Among pure molecules, govanoside B was found more effective (76% mortality, LC50 = 3279.5 mg L-1 ) followed by borassoside E (74%, LC50 = 3467.1 mg L-1 ) against A. craccivora after 96 h. CONCLUSION: Parent extract/fractions of T. govanianum showed promising efficacy against larvae of P. xylostella and A. craccivora. Further, field study is required for its bio-efficacy against targeted pests for validation and formulation development.

Chemical profiling and quantification of potential active constituents responsible for the antiplasmodial activity of Cissampelos pareira.

ETHNOPHARMACOLOGICAL RELEVANCE: Cissampelos pareira is used traditionally in India as a remedy for the treatment of various diseases including malaria but the active ingredients responsible for antiplasmodial activity have not yet been investigated. AIM OF THE STUDY: The identification and quantification of compounds responsible for antiplasmodial activity in different parts (leaf, stem and root) of C. pareira is the target of current study. MATERIAL AND METHODS: The hydro ethanolic parent extracts of different parts of C. pareira and fractions prepared from these extracts were evaluated against Pf3D7 (chloroquine sensitive) and PfINDO (chloroquine resistance) strains in culture to quantify the IC50 for extracts and fractions. Promising fractions of root part of plant were subjected to silica gel column chromatography to obtain pure compounds and their structures were elucidated by detailed spectroscopic analysis. Pure compounds were also tested against Pf3D7 and PfINDO strains. A rapid and simple UPLC-DAD method was developed for the identification and quantification of pharmaceutically important metabolites of C. pareira. RESULTS: Among different extracts, the hydro ethanolic extract of root part of C. pareira was found most active with IC50 values (µg/ml) of 1.42 and 1.15 against Pf 3D7 and Pf INDO, respectively. Tested against Pf 3D7 the most potent fractions were root ethyl acetate fraction (IC50 4.0 µg/ml), stem water fraction (IC50 4.4 µg/ml), and root water fraction (IC50 8.5 µg/ml). Further, phytochemical investigation of active fractions of root part led to the isolation and characterization of a new isoquinoline alkaloid, namely pareirarine (8), along with five known compounds magnoflorine (5), magnocurarine (10), salutaridine (11), cissamine (13) and hayatinine (15). Hayatinine (15), a bisbenzylisoquinoline alkaloid, isolated from root ethyl acetate fraction was most promising compound with IC50 of 0.41 µM (Pf INDO) and 0.509 µM (Pf 3D7). Magnocurarine (10) and cissamine (13) were also found active with IC50 values of 12.51 and 47.34 µM against Pf INDO and 12.54 and 8.76 µM against Pf 3D7, respectively. A total of thirty compounds were detected in studied extracts and fractions, structures were assigned to 15 of these and five of these biologically important compounds were quantified. Isolation of saluteridine (11) from C. pareira and the evaluation of antiplasmodial activity of pure compound from C. pariera is disclosed for the first time. CONCLUSION: This study concludes that the antimalarial potential of C. pareira may be attributed to isoquinoline type alkaloids present in this plant and also provides the scientific evidence for the traditional use of this plant in treatment of malaria.

Antioxidant, Antiproliferative and Apoptosis-Inducing Efficacy of Fractions from Cassia fistula L. Leaves.

: Cassia fistula L. is a highly admirable traditional medicinal plant used for the treatment of various diseases and disorders. The present study was performed to divulge the antioxidant, antiproliferative, and apoptosis-inducing efficacy of fractions from C. fistula leaves. The hexane (CaLH fraction), chloroform (CaLC fraction), ethyl acetate (CaLE fraction), n-butanol (CaLB fraction), and aqueous (CaLA fraction) were sequentially fractionated from 80% methanolic (CaLM extract) of C. fistula leaves. The CaLE fraction was fractionated using column chromatography to yield a pure compound, which was characterized as Epiafzelechin (CFL1) based on 1H, 13C, and DEPT135 NMR. Among these fractions, CaLE and isolated CFL1 fractions exhibited an effective antioxidant potential in Ferric ion reducing power, (2,2'-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid)) cation radical scavenging, and nitric oxide radical scavenging assays. Epiafzelechin was investigated for its antiproliferative effects against MG-63 (osteosarcoma), IMR-32 (neuroblastoma), and PC-3 (prostate adenocarcinoma), and was found to inhibit cell proliferation with a GI50 value of 8.73, 9.15, and 11.8 µM respectively. MG-63 cells underwent apoptotic cell death on treatment with Epiafzelechin as the cells showed the formation of apoptotic bodies, enhanced reactive oxygen species (ROS) generation, mitochondrial membrane depolarization along with an increase in early apoptotic cell population analyzed using Annexin V-FITC/PI double staining assay. Cells showed cell cycle arrest at the G0/G1 phase accompanied by a downregulation in the expression levels of p-Akt (Protein kinase B), p-GSK-3ß (Glycogen synthase kinase-3 beta), and Bcl-xl (B-cell lymphoma-extra large) proteins. RT-PCR (Real time-polymerase chain reaction) analysis revealed downregulation in the gene expression level of ß-catenin and CDK2 (cyclin-dependent kinases-2) while it upregulated the expression level of caspase-8 and p53 genes in MG-63 cells.