BACKGROUND: 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.
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.
RESULTS: 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), 22β-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.
CONCLUSIONS: 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.

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.

Two novel steroidal saponins, trilliumosides K (1) and L (2), were isolated from the rhizomes of Trillium govanianum led by bioactivity-guided phytochemical investigation along with seven known compounds: govanoside D (3), protodioscin (4), borassoside E (5), 20-hydroxyecdysone (6), 5,20-hydroxyecdysone (7), govanic acid (8), and diosgenin (9). The structure of novel compounds 1-2 was established using analysis of spectroscopic data including 1D and 2D nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-ESI-MS) data. All isolated compounds were evaluated for in vitro cytotoxic activity against a panel of human cancer cell lines. Compound 1 showed significant cytotoxic activity against the A-549 (Lung) and SW-620 (Colon) cancer cell lines with IC50 values of 1.83 and 1.85 µM, respectively whereas the IC50 value of Compound 2 against the A-549 cell line was found to be 1.79 µM. Among the previously known compounds 3, 5, and 9, the cytotoxic IC50 values were found to be in the range of 5-10 µM. Comprehensive anti-cancer investigation revealed that Compound 2 inhibited in vitro migration and colony-forming capability in the A-549 cell line. Additionally, the mechanistic analysis of Compound 2 on the A-549 cell line indicated distinctive alterations in nuclear morphology, increased reactive oxygen species (ROS) production, and decreased levels of mitochondrial membrane potential (MMP). By upregulating the pro-apoptotic protein BAX and downregulating the anti-apoptotic protein BCL-2, the aforementioned actions eventually cause apoptosis, a crucial hallmark in cancer research, which activates Caspase-3. To the best of our knowledge, this study reports the first mechanistic anti-cancer evaluation of the compounds isolated from the rhizomes of T. govanianum with remarkable cytotoxic activity in the desired micromolar range.

Trillium govanianum is a high-value medicinal herb, having multifunctional traditional and culinary uses. The present investigation was carried out to evaluate the phytochemical, biological and toxicological parameters of the T. govanianum Wall. ex D. Don (Family: Trilliaceae) roots collected from Azad Kashmir, Pakistan. Phytochemical profiling was achieved by determining total bioactive contents (total phenolic and flavonoid contents) and UHPLC-MS analysis. For biological evaluation, antioxidant activities (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, and metal chelation assays) and enzyme inhibition activities (against AChE, BChE, glucosidase, amylase, and tyrosinase) were performed. Moreover, cytotoxicity was assessed against three human carcinoma cell lines (MDA-MB-231, CaSki, and DU-145). The tested extract was found to contain higher total phenolics (7.56 mg GAE/g dry extract) as compared to flavonoid contents (0.45 mg RE/g dry extract). Likewise, for the antioxidant activity, higher CUPRAC activity was noted with 39.84 mg TE/g dry extract values. In the case of enzyme assays, higher activity was pointed out against the cholinesterase, glucosidase and tyrosinase enzymes. The plant extract displayed significant cytotoxicity against the cell lines examined. Moreover, the in-silico studies highlighted the interaction between the important phytochemicals and tested enzymes. To conclude, the assessed biological activity and the existence of bioactive phytochemicals in the studied plant extract may pave the way for the development of novel pharmaceuticals.

Understanding the genetic diversity and population structure of pharmaceutically important endangered plant species is crucial for their conservation and sustainable use. Despite the continuous population decline in Trillium govanianum Wall. ex D. Don, a highly prized medicinal plant endemic to the Himalaya, information regarding its conservation genetics has been lacking. Here, we employed a conservation genetics approach to investigate how drastically declining populations in natural habitats impact population genetic diversity and structure of this endangered species across the Kashmir Himalaya. We used Start codon targeted (SCoT) and Simple sequence repeat (SSR) markers to assess the intra- and inter-population genetic variation in seven sites across the study region. Based on these markers, we found a very low genetic diversity in T. govanianum populations. Very low levels of observed heterozygosity (Ho = 0.000) and that expected (He = 0.064) in the populations indicate high heterozygote deficiency and high levels of inbreeding depression (FIS = 1.000). A high genetic differentiation was observed among the populations for both SCoT (Gst = 0.719) and SSR (Fst = 0.707) markers. Both the markers showed low gene flow, SCoT (Nm = 0.195) and SSR (Nm = 0.119), depicting high among-population variation than within-population variation. Analysis of molecular variance also indicated a higher genetic variation between the populations than within populations. We also observed a significant positive correlation between genetic divergence and geographical distance, indicating that genetic differentiation in T. govanianum follows a pattern of isolation by distance. Bayesian structure and cluster analysis grouped the populations according to their geographical proximity. Further, redundancy analysis (RDA) revealed the presence of one polymorphic locus for each marker with high discriminatory power. Overall, our findings reveal a very low genetic diversity, high levels of inbreeding, and high genetic differentiation among the populations; likely resulting from habitat fragmentation, population isolation, bottleneck effect, low gene flow, and predominantly asexual reproduction currently operative in the species. Finally, based on the insights gained, we discuss the potential implications of our findings in guiding species recovery and habitat rehabilitation of T. govanianum in the Himalaya with conservation lessons for elsewhere in the world.

BACKGROUND: The current study aimed to develop an economic plant-based therapeutic agent to improve the treatment strategies for diseases at the nano-scale because Cancer and Diabetes mellitus are major concerns in developing countries. Therefore, in vitro and in vivo antidiabetic and anti-cancerous activities of Trillium govanianum conjugated silver nanoparticles were assessed.
METHODS: In the current study synthesis of silver nanoparticles using Trillium govanianum and characterization were done using a scanning electron microscope, UV-visible spectrophotometer, and FTIR analysis. The in vitro and in vivo anti-diabetic and anti-cancerous potential (200 mg/kg and 400 mg/kg) were carried out.
RESULTS: It was discovered that Balb/c mice did not show any major alterations during observation of acute oral toxicity when administered orally both TGaqu (1000 mg/kg) and TGAgNPs (1000 mg/kg), and results revealed that 1000 mg/kg is not lethal dose as did not find any abnormalities in epidermal and dermal layers when exposed to TGAgNPs. In vitro studies showed that TGAgNPs could not only inhibit alpha-glucosidase and protein kinases but were also potent against the brine shrimp. Though, a significant reduction in blood glucose levels and significant anti-cancerous effects was recorded when alloxan-treated and CCl4-induced mice were treated with TGAgNPs and TGaqu.
CONCLUSIONS: Both in vivo and in vitro studies revealed that TGaqu and TGAgNPs are not toxic at 200 mg/kg, 400 mg/kg, and 1000 mg/kg doses and possess strong anti-diabetic and anti-cancerous effects due to the presence of phyto-constituents. Further, suggesting that green synthesized silver nanoparticles could be used in pharmaceutical industries to develop potent therapeutic agents.

In an era of global environmental change, conservation of threatened biodiversity and ecosystem restoration are formidable ecological challenges. The forest understory strata and the belowground soil environment including rhizospheric microbial communities, which are crucial for ecosystem functioning and overall forest biodiversity maintenance, have remained understudied. Here, we investigate the soil microbiome of Trillium govanianum - an endangered Himalayan Forest herb, to unravel the underground diversity, drivers, and potential indicators of the microbial community. We collected rhizospheric and bulk soil samples for microbiome and physicochemical analysis at three sites along an elevation gradient (2500-3300 m) in Kashmir Himalaya. Amplicon sequencing of 16 S rRNA and ITS was used to identify the bacterial and fungal soil microorganisms. We found significant differences in the structure and diversity of microbial community (bacterial and fungal) between the rhizosphere and bulk soil along the altitudinal gradient, and noticeable shifts in the nutrient level in dominant microbial phyla associated with T. govanianum. A significant difference between soil physicochemical parameters and increasing altitude suggests that microbial community structure is determined by altitude and soil type. Similarly, the microbial communities showed a significant (P < 0.05) correlation with soil physicochemical variables along the altitudinal gradient. The moisture content in bacterial and total organic carbon in fungal communities showed the most substantial impact on the physiochemical drivers. We also identify potential bacterial and fungal plant growth promoter indicator species in the soil microbiome of T. govanianum. Overall, our findings provide novel research insights that can be pivotal in designing integrated species recovery programs and long-term restoration plans for T. govanianum, with learnings for biodiversity conservation elsewhere.

Trillium govanianum rhizomes are traditionally consumed as a raw powder and decoction for the treatment of health complications. Hence, the present study aimed to investigate whether aqueous and alcoholic extracts of T. govanianum rhizomes under hot and cold extraction conditions have similar or dissimilar chemical, nutrient, and antioxidant profiles. The total phenolics, flavonoids, carbohydrates, proteins, fats, and energy values were estimated in all the conditionally prepared samples. The total phenolics (21.23±1.4 mg GAE/g extract), flavonoids (70.57±3.24 mg RE/g extract) were found higher in hot ethanolic extract (TGHEt), while cold water extract (TGGC) showed higher nutrients including amino acids (10.545±0.219 mg/g) and nucleosides (1.803±0.018 mg/g). The nutrient energy value (2.60 and 2.49 Kcal/g extract) was higher in cold and hot ethanolic extracts. Further, TGHEt scavenged the DPPH. (IC50 ; 870±22 μg/mL) and ABTS.+ (IC50 ; 80±1.49 μg/mL) effectively and proved its highest antioxidant activity compared to other samples. In LC/MS/MS-based metabolite profiling, twenty-six metabolites (fatty acids, steroidal saponins, triterpene saponins, ecdysteroid hormones) were confirmed with mass fragmentation and literature, while one hundred nine metabolites were identified using the METLIN database. The principal component analysis showed clustering of hot condition extracts while cold extracts were differentially located in quadrants. The heatmaps exhibited the associations and differences between metabolite composition, solvents, and extraction conditions. The identified metabolites speculatively predicted the biosynthesis pathway of T. govanianum. Findings also illustrated that T. govanianum is a source of bioactive nutritional components and saponins. The current metabolite profiling of T. govanianum will help in its agricultural and biotechnological interventions for higher quality produce.

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.
CONCLUSIONS: 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.

BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.