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.

Two new alkaloids from the roots of Cocculus hirsutus (L.) W. Theob.

Two undescribed alkaloids, 15-carboxydihydroerysotrine (1) and (14 R)-4-methoxy-13,14-dihydrooxypalmatine (2), along with six known compounds, 1,6-didehydro-3,15,16-trimethoxy-9-methylerythrinanium (3), 8-oxytetrahydropalmatine (4), 20-hydroxyecdysone (5), makisterone A (6) turkesterone (7) and magnoflorine (8) were isolated from the root part of Cocculus hirsutus (L.) W. Theob. Their structures were established based on detailed analysis of NMR, UV-Vis, HRESIMS, and single-crystal XRD spectroscopic experiments. Compounds 3, 4 and 7 were reported for the first time from the genus Cocculus. All the compounds were analysed in silico to investigate their human acetylcholinesterase inhibition potential. This analysis revealed that compounds 1 and 8 interacted well with the selected protein, which suggested their further exploration as acetylcholinesterase inhibitors via in vitro and in vivo investigation.

Govanosides C-F, unprecedented steroidal saponins with rare sugars from rhizomes of Trillium govanianum and their antagonistic effects on acetylcholinesterase.

Four previously undescribed steroidal saponins named govanosides C-F (1-4) and nine known compounds (5-13) were isolated from the rhizomes of Trillium govanianum Wall. ex D.Don. Govanosides C-E contained a rare sugar moiety i.e., 6-deoxy allose, while govanoside F has acetylated rhamnose moiety in its glycan part. Also, this is the first report on the isolation of feruloyl sucrose derivatives (11-12) and (E)-4-hydroxy-dodec-2-enedioic acid (13) from the Trillium genus. The structure of isolated compounds was deduced using 1D and 2D NMR, HRESIMS, LC-MS/MS, GC-MS, and saccharide linkage analysis. Steroidal scaffold isolates (1-10) were evaluated for their antagonistic effects on acetylcholinesterase inhibitory activity. Govanoside C (1) significantly inhibited acetylcholinesterase (IC50: 2.38 µM). Molecular docking experiments have also been performed to depict the molecule's interaction and binding free energy with acetylcholinesterase.

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.

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"?

Targeting Akt/NF-κB/p53 Pathway and Apoptosis Inducing Potential of 1,2-Benzenedicarboxylic Acid, Bis (2-Methyl Propyl) Ester Isolated from Onosma bracteata Wall. against Human Osteosarcoma (MG-63) Cells.

Onosma bracteata Wall. is an important medicinal and immunity-enhancing herbs. This plant is commonly used in the preparation of traditional Ayurvedic drugs to treat numerous diseases. Inspired by the medicinal properties of this plant, the present study aimed to investigate the antiproliferative potential and the primary molecular mechanisms of the apoptotic induction against human osteosarcoma (MG-63) cells. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in superoxide radical scavenging assay and lipid peroxidation assay with an EC50 value of 95.12 and 80.67 µg/mL, respectively. Silica gel column chromatography of ethyl acetate (Obea) fraction of O. bracteata yielded a pure compound, which was characterized by NMR, FTIR, and HR-MS analysis and was identified as 1,2-benzene dicarboxylic acid, bis (2-methyl propyl) ester (BDCe fraction). BDCe fraction was evaluated for the antiproliferative potential against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung carcinoma A549 cell lines by MTT assay and exhibited GI50 values of 37.53 µM, 56.05 µM, and 47.12 µM, respectively. In MG-63 cells, the BDCe fraction increased the level of ROS and simultaneously decreased the mitochondria membrane potential (MMP) potential by arresting cells at the G0/G1 phase, suggesting the initiation of apoptosis. Western blotting analysis revealed the upregulation of p53, caspase3, and caspase9 while the expressions of p-NF-κB, p-Akt and Bcl-xl were decreased. RT-qPCR studies also showed upregulation in the expression of p53 and caspase3 and downregulation in the expression of CDK2, Bcl-2 and Cyclin E genes. Molecular docking analysis displayed the interaction between BDCe fraction with p53 (-151.13 kcal/mol) and CDK1 (-133.96 kcal/mol). The results of the present work suggest that the BDCe fraction has chemopreventive properties against osteosarcoma (MG-63) cells through the induction of cell cycle arrest and apoptosis via Akt/NF-κB/p53 pathways. This study contributes to the understanding of the utilization of BDCe fraction in osteosarcoma treatment.

Insecticidal and Enzyme Inhibition Activities of Leaf/Bark Extracts, Fractions, Seed Oil and Isolated Compounds from Triadica sebifera (L.) Small against Aphis craccivora Koch.

Aphid, Aphis craccivora Koch (Hemiptera: Aphididae), is a major sap-sucking insect pest of leguminous crops and also transmits plant viruses, leading to economic yield loss. Indiscriminate and repeated use of insecticides for control of aphid leads to the development of resistance, and is harmful to the environment, non-target organisms, etc. Plant-based extracts/seed oils (SO) are the best alternatives to insecticides. Insecticidal activities of Triadica sebifera have not been reported against A. craccivora and other insect pests to date. In the current study, the main objective was to study the insecticidal activities of leaf/bark extracts/fractions, seed oil, isolated compounds, and their combinations against A. craccivora. Results showed that, among the extracts, ethanolic bark extract 80% (LC50 = 5115.98 mg/L) was more effective against A. craccivora. Among fractions, the n-hexane fraction of leaves (LC50 = 425.73 mg/L) and the ethyl acetate fraction of bark (LC50 = 813.45 mg/L) were promising. Among compounds, gallic acid was the most effective (LC50 = 1303.68 mg/L) compared to shikimic acid and quercetin. SO (LC50 = 850.94 mg/L) was superior compared to extracts/fractions/compounds. All the combinations showed toxicity and synergistic activity. Leaf/bark extracts and SO significantly inhibited the AChE and GST activity in A. craccivora. Based on field bio-efficacy, the leaf extract/SO or their combinations can be recommended for the control of aphids.

Govanoside B, a new steroidal saponin from rhizomes of Trillium govanianum.

Trillium govanianum, commonly known as Nag Chhatri and Teen Patra, is a popular herbal supplement traditionally used for curing different inflammatory and sexual disorders, infection and wound healing. Steroidal saponins are considered as active components of this species. The present study demonstrated the isolation of nine steroidal saponins, including one new compound named as govanoside B (9) and eight known, pregna-chacotrioside (1), pennogenin-triglycoside (2), borassoside E (3), pennogenin-tetraglycoside (4), protodioscin (5), clintonioside B (6), pennogenin-diglycoside (7) and borassoside D (8). This is the first report on the isolation of 1, 2, 4, 5, 6, 7 and 8 from rhizomes of T. govanianum. The extract, fractions and isolated compounds were further evaluated for their DPPH and ABTS radical scavenging activity.

Tinospora cordifolia activates PPARγ pathway and mitigates glomerular and tubular cell injury in diabetic kidney disease.

BACKGROUND: Diabetic Kidney Disease (DKD) is a common complication of diabetes and a leading cause of end-stage renal disease progression. Therefore, therapeutic strategies are desirable to mitigate the progression of disease into more severe consequences. Hypothesis/Purpose:Tinospora cordifolia is a traditionally known antidiabetic plant; however, its effect against DKD remains unexplored. Therefore, in the present study, we assessed the efficacy and mechanism of action of Tinospora cordifolia extract (TC) against DKD. METHODS: The molecular interaction of the various phytoconstituents of TC with PPARγ were analyzed in silico. The effect of TC was studied on the viability, cell cycle, and gene expressions (PPARγ, TGFß, and αSMA) in high glucose treated NRK-52E and SV40 MES13 cells. Further, streptozotocin-induced diabetic rats were treated with TC for eight weeks, and the effects on different biochemical, histological and molecular parameters were studied. RESULTS: In silico analysis revealed the integration of various phytoconstituents of TC with PPARγ. It further increased PPARγ and decreased TGFß and αSMA expressions in NRK-52E and SV40 MES13 cells. In diabetic rats, TC improved the fasting blood glucose, serum urea, and creatinine levels. It also lowered the urine microalbumin and advanced glycation end products (AGEs) levels. Histopathological studies revealed the preventive effect of TC on degenerative changes, mesangial proliferation and glomerular hypertrophy. Further, it reduced the inflammation and fibrotic changes in the kidney tissue estimated by various markers. The kidney tissue and gene expression analysis revealed the augmented levels of PPARγ after TC treatment. CONCLUSION: In conclusion, TC exerted the protective effect against DKD by inhibiting inflammation and fibrogenesis through the activation of PPARγ dependent pathways.

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.

Qualitative and quantitative determination of steroidal saponins in Trillium govanianum by UHPLC-QTOF-MS/MS and UHPLC-ELSD.

INTRODUCTION: Trillium govanianum (Nag Chhatri and Teen Patra) is traditionally used for curing joint pains, wounds, and sexual disorders. Steroidal saponins are the main active components of this species. However, only a small amount of information is available about steroidal saponins of this plant. OBJECTIVE: To develop an ultra-high-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and ultra-high-performance liquid chromatography-evaporative light scattering detector (UHPLC-ELSD) methods for the qualitative and quantitative determination of steroidal saponins in T. govanianum. METHOD: The dried rhizomes of T. govanianum (100 mg) were extracted with ethanol-water (80:20, 10 mL) by ultrasonic treatment for 30 min at 40°C. The prepared sample was analysed by UHPLC-QTOF-MS/MS and UHPLC-ELSD for the qualitative and quantitative determination of steroidal saponins. RESULT: A total of 24 saponins were identified using UHPLC-QTOF-MS/MS; seven of them were characterised by comparing with standards. Furthermore, five saponins [govanoside B (2), protodioscin (6), pennogenin tetraglycosides (11), borassoside E (21) and borassoside D (24)] were quantified using UHPLC-ELSD method in different extracts and fractions of T. govanianum. The method showed good linearity (R2 ≥ 0.993), limit of detection (0.92-4.09 µg/mL), limit of quantification (3.1-13.5 µg/mL), precision [intra-day relative standard deviations (RSDs) < 4.3% and inter-day RSDs < 5.5%], and accuracy (84.0-110.3%). This is the first report on the quantification of 2, 6, 11, 21 and 24 in T. govanianum. CONCLUSION: The present study provides an efficient analytical method for the identification and quantification of steroidal saponins and will be helpful for the quality evaluation of T. govanianum.