Phenolic metabolites and flavonoids show potent antioxidant activity by scavenging ROS and free radicals through enzyme inhibition, metal chelation, and hydrogen donation. These metabolites regulate the metabolism, inflammation, and immune responses and are also utilized in the treatment of diabetes, cardiovascular disorders, and viral infections (Yao et al., 2024).

Various metabolites belonging to different groups, including phenolics (vanillic acid and its derivatives, n-eicosanyl cinnamate), were isolated from the methanolic extract of the stem bark using column chromatography followed by thin-layer chromatography. The study used triple chemical fingerprinting methods to characterize the isolated metabolites (Ali et al., 2017). Alkaloid extract, when analyzed by GC–MS, yielded several phenolic metabolites, including veratric acid, benzoic acid, and phthalic acid, from different plant parts (Bhardwaj, 2018). In another study, the root was successively extracted with petroleum ether and acetone. The former extract yielded acidic and neutral metabolites. The latter showed the presence of tectol. Joshi et al. (1986) further identified tectol and octacosanyl ferulates in the heartwood using the above methodology. On extracting the ether-soluble fraction of the stem bark with sodium carbonate, further acidification showed the presence of veratric acid using TLC and other spectral techniques (Singh et al., 1972). Different extraction techniques were employed to isolate flavonoids from the hydroethanolic extracts of T. undulata flowers and leaves using HPLC–ESI–MS/MS. In all the techniques, leaves had more content of individual flavonoids except 5,6-dimethyoxy-3′,4′-dioxymethylene-7-O-(6″-β-D-glucopyranosyl-β-D-glucopyranosyl) flavanone, which had less content in leaves when extracted through Soxhlet, marination, sonication, and reflux. The highest content of rutin was found to be in flowers (28.1%) and leaves (28.2%). The lowest content was that of luteolin glucoside in leaves processed through marination (Laghari et al., 2013). Among the various extraction techniques, microwave-assisted extraction (MAE) yielded the highest flavonoid content in the shortest time. The MAE technique significantly reduces energy consumption, solvent usage, and processing time, making it a sustainable and environmentally friendly methodology. It should be preferably coupled with conventional extraction to enhance the extraction yield and purity (Alvi et al., 2022). Cirsimaritin and cirsilineol were reported in the petroleum ether extract of leaves separated through column chromatography (Azam and Ghanim, 2000). The ethyl acetate fraction of the stem bark was found to contain ferulic acid (4.95%), quercetin (0.72%), and rutin (0.18%), all of which have been shown to have anti-obesity activity (Alvala et al., 2013). Quercetin was also identified in the stem bark, having antiproliferative activity (Ravi et al., 2011).

The perhydrocyclopentanophenanthrene nucleus forms the basic skeleton of steroid molecules. Numerous types of steroids exist due to variations in this fundamental skeleton, and the attachment of different functional groups results in structural diversity and biological activities among steroids (Atanasov et al., 2021).

The petroleum ether extract of heartwood (3 kg) was dissolved in ethyl acetate and extracted with sodium carbonate (Bhardwaj, 2018). The sodium carbonate-insoluble fraction yielded stigmasterol (1 g) and β-sitosterol (1.5 g). Sitosterol was commonly observed in the bark (Singh et al., 1972) and root (Joshi et al., 1977) following an almost similar extraction procedure. Steroidal arabinosyl diester was characterized from the methanolic extract of the stem bark using column chromatography with petroleum ether and chloroform in equal proportion, followed by TLC for further purification (Ali et al., 2017). Stigmasterol is utilized in the synthesis of semi-synthetic and synthetic pharmaceutical compounds. It demonstrates a broad spectrum of pharmacological effects. Similarly, β-sitosterol, a common dietary phytosterol, is found to inhibit tumor metastasis by enhancing gut immunity and also contributes to blood sugar regulation, immunomodulation, reproductive protection, and fever reduction (Rani et al., 2025).

Ethyl hexadecanoate was commonly identified in the stem, root, and bark, with the highest content found in the stem bark. The GC–MS data revealed the maximum diversity in terms of the presence of fatty acids and their esters in various plant parts that were tested (Bhardwaj, 2018). Out of the five identified fatty aldehydes, the maximum content was found to be of cis-9-hexadecenal in the stem, and the minimum content was of octadecanal in the leaves. Two fatty alcohols, viz., 1-undecanol and trans-2-dodecen-1-ol, trifluoroacetate, were reported in the roots with peak areas of 2.38% and 3.71%, respectively. Triacontanol was reported by Joshi et al. (1977) in the roots of the plant. There are some limitations to the analysis of plant metabolites using GC–MS as it can only separate volatile metabolites, which are typically of low molecular weight. Non-volatile and polar metabolites should preferably be derivatized before analysis. Moreover, for proper chemical fingerprinting, it is always suggested to identify the metabolites using multiple spectral and chromatographic techniques (Heinrich et al., 2022).

Alkaloids are nitrogen-containing bioactive substances that are promising candidates for drug development due to their significant biological and structural activity. These metabolites possess diverse therapeutic potential and are used in the treatment of cancer, inflammation, malaria, hypertension, diabetes, etc. (Rajput et al., 2022).

The flower (20 g) was processed to obtain an alkaloid fraction (0.5 g), which was then analyzed using GC–MS. During extraction, n-hexane was used instead of chloroform, which facilitated the removal of fatty metabolites and other interfering metabolites. Derivatization of the alkaloid fraction is a standard process used to achieve better results with GC–MS as it enhances the volatility, detection, and separation efficiency by chemically altering the functional groups on the original molecule (Wang et al., 2025). However, in this study, derivatization was not included. Almost 50% of the alkaloids were present in the fraction, and they were structurally diverse, comprising aromatic, cyclic, and bicyclic compounds, which is quite rare (Laghari et al., 2014). Out of 11 alkaloids identified, the largest peak area was observed for 4-formyl-1,3-dihydro-1,3-dimethyl-2H-imidazole-2-thione (16.63%), while the smallest area was for 1-piperidineethanol (1.44%). In leaves and stems, 1-docosene (Bhardwaj, 2018) and n-hexadecanyl caffeate, respectively, have also been reported (Ali et al., 2017). However, some alkaloids also have side effects/toxicity on human health (Rajput et al., 2022); thus, the use of plant-based medicines rich in alkaloids needs to undergo meticulous safety assessments.

The defatted stem bark powder was extracted with methanol, leading to the isolation of betulinic acid, which was further purified (98%) using column chromatography and preparative TLC (Jain et al., 2012). Betulinic acid is a lupane-type pentacyclic triterpene having several bioactivities, including antidiabetic, anticancer, diuretic, antiviral, and immunomodulatory activities (Oliveira-Costa et al., 2022). Bhardwaj (2018) reported the presence of terpenoids in the alkaloid-rich fraction of plant parts. 3,7,11,15-Tetramethyl-2-hexadecen-1-ol and 2,6,10-trimethyl,14-ethylene-14-pentadecane were found to be present in all the plant parts. This study, compared to the work of Laghari et al. (2014), clearly demonstrates the importance of selecting and processing samples appropriately to obtain a fraction rich in the metabolite of interest. Moreover, the inclusion/modification of steps that remove most of the interfering molecules becomes crucial. Both studies aimed to identify alkaloids; however, Bhardwaj et al. (2014) found the presence of very few alkaloids in the alkaloid-rich fraction.

The petroleum ether extract of the heartwood (3 kg) showed the presence of seven quinones. Radermachol (70 mg; rare pigment) and 2-isopropenylnaphtho [2,3-b]furan-4,9-quinone (30 mg) were reported for the first time in the genus Tecomella (Singh et al., 2008). Other naphthoquinone derivatives were also reported. Naphthoquinone derivatives (Tecomella naphthoquinone A and Tecomella naphthoquinone B) were reported for the first time in the stem bark of the plant by Ali et al. (2017). Dehydrotectol was reported in the bark (Singh et al., 1972) and root (Joshi et al., 1977; Joshi et al., 1986) of the plant. Lapachol is another quinone commonly found in the root, bark, and heartwood (Table 3). This metabolite has been reported to be toxic to monkeys (Willard and Murray, 2020).

Undulatin, an iridoid glucoside, was identified in the stem bark of the plant. The defatted powdered sample was extracted with ethanol, followed by ethyl acetate, to yield undulatin (50 mg). The metabolite was characterized using IR, UV, and ¹H NMR spectroscopy (Verma et al., 1986). Another iridoid glucoside, 6-0-veratryl catalposide, was isolated from the ether-insoluble acetone extract of the heartwood (Joshi et al., 1975) and the root of the plant (Joshi et al., 1977). Undecanyl stearate was identified from the methanolic extract of the stem bark with a 0.034% yield. Using GC–MS, Bhardwaj (2018) identified diacetylene (stem), cyclopropane derivatives (stem and bark), alkene (stem and root), ketone (stem), primary alcohol (root), phthalate esters (root, bark, and leaves), alkene (bark), anhydride (bark), fluoroalkyl (leaves), and esters (leaves).

T. undulata is reported to have hepatoprotective activity against isoniazid-induced liver damage. The stem bark extract significantly lowered the elevated levels of AST (aspartate aminotransferase), ALT (alanine transaminase), ALP (alkaline phosphatase), GGT (gamma-glutamyl transferase), and bilirubin (Zehri et al., 2020). These enzymes are released due to the membrane damage of liver cells. Thus, the plant has a membrane-stabilizing effect. In a damaged state, the liver cannot properly release bilirubin through the bile. This may lead to its leakage into the blood. High levels of bilirubin may lead to jaundice (Bajaj et al., 2022). However, there is no clarity regarding the determination of the dose and the authentication of the plant samples. Additionally, the inclusion of histopathological studies and other markers of liver damage could have made the investigation more comprehensive. The methanolic extract of the plant reduced the levels of liver enzymes, including cholesterol, in the experimental model compared to those in the CCl₄ group. However, the amount of protein and albumin increased. It is reported that damage to the ER leads to a loss of P540, resulting in lower protein synthesis. Moreover, CCl₄ inhibits the synthesis of bile from cholesterol, resulting in its accumulation. Histological sections revealed that the cellular architecture improved following the administration of the plant extract (Rana et al., 2008).

In related studies, the ameliorative effect of T. undulata stem bark on acetaminophen-induced toxicity in rats was observed. Acetaminophen forms N-acetyl P benzoquinone (NAPQI), which is toxic. Overproduction of NAPQI generates free radicals, which damage mitochondrial DNA and increase membrane permeability, thereby adversely affecting hepatocytes. The bark extract improved liver histology, which was characterized by a reduced presence of inflammatory cells (Fatima et al., 2022), and lowered hepatic enzyme levels (Fatima et al., 2023). The leaves of the plant showed protective effects against alcohol-induced hepatotoxicity. The occurrence of liver marker enzymes decreased in the serum, and the liver GSH (glutathione), GPx (glutathione peroxidase), and SOD (superoxide dismutase) levels increased. Moreover, lipid peroxidation was also reduced. Fatima et al. did not justify the need to present the related data (histopathological and liver marker enzymes) in separate articles, which is questionable. Similar effects of the leaf extract were observed in rats with paracetamol-induced liver damage (Singh and Gupta, 2011). The extract may possess antioxidants that minimize lipid peroxidation of the membrane, and thus, the presence of marker enzymes is reduced in the serum. Alcohol decreases the production of antioxidant enzymes due to the adverse effects of free radicals or due to the production of acetaldehyde as a result of alcohol oxidation (Das et al., 2005). Jain et al. (2012) evaluated the hepatoprotective activity of the plant’s stem bark on HepG2 cells and rats and concluded that this may be due to the presence of betulinic acid (triterpenoid). TSB-2 fraction showed the highest degree of cytotoxicity, which may be due to the presence of lapachol (Almeid, 2009). TSB-7 fraction (containing betulinic acid) showed lower cytotoxicity and was assessed in an animal model. Betulinic acid is reported to be cytotoxic against several animal cell lines (Eichenmüller et al., 2009); however, HepG2 cells were less adversely affected, which may be due to the expression of survivin and Bcl2 (survival factors). The positive effect of the plant extract on liver marker enzymes is indicative of reduced liver damage and a membrane-stabilizing effect.

Non-alcoholic fatty liver disease (NAFLD) is a primary concern globally, which is caused by poor eating habits, a sedentary lifestyle, and obesity. It mainly leads to non-alcoholic steatohepatitis (NASH). Mice were fed with a Western diet sugar water (WDSW), leading to nonalcoholic steatohepatitis. In a preclinical study, the stem bark showed a positive effect on liver marker enzymes, total cholesterol levels, triglyceride levels, and insulin resistance (Srinivas et al., 2023). The mouse model mimics the effects of diet and the incidence and progression of disease in humans. The dose of the experiment was determined according to guidelines established by the US Food and Drug Administration. A pharmacopoeia evaluation of the plant material was conducted to assess its pharmaceutical quality. Overall, important factors such as the dose, mode of administration, timing of the intervention, extent of exposure, and endpoint assessments were meticulously selected to evaluate the effect of Tecomella in treating NASH. Oxidative stress, inflammation, and ER stress were found to be reduced, which was nearly equivalent to that of saroglitazar. Downregulation of ER stress markers [C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (Grp78), and unfolded protein response (UPR)] was observed. The reduction in inflammation was primarily due to a decrease in pro-inflammatory markers, specifically tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). The expression of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK1/2), which are markers of inflammation and steatosis, was observed (Urano et al., 2000). Thus, a reduction in oxidative and ER stress could have lowered the levels of cholesterol and lipids in the liver.

T. undulata exhibits notable pain-relieving properties. In a study, the whole plant was processed to obtain an extract using absolute methanol. Significant analgesic activity was observed, as assessed by the hot-water tail immersion test in mice. However, the results were not dose-dependent. The extract was not able to exert significant anti-inflammatory activity on paw edema induced by carrageenan (Ahamad et al., 1994). Carrageenan, as an irritant substance, is used to cause edema. It induces the secretion of cytokinins under the influence of bradykinin (Vats et al., 2024). The variation in results may be due to the presence and concentration of bioactive metabolite/s and pharmacokinetic variations. Since the methanolic extract was administered to animal models, it becomes imperative to include a negative control, which was not mentioned in the study.

Arsalan et al. (2023) highlighted the anti-inflammatory potential of T. undulata leaves. The study revealed that the plant extract was effective in both the initial and later phases of edema (Akinloye et al., 2020; Zahra et al., 2020). This may be due to the presence of phenolic metabolites in the ethanolic extract, which might have worked synergistically and antagonistically with anti- and pro-inflammatory markers. The study was carried out on formalin- and carrageenan-induced paw edema in rats. However, the study is too preliminary, and further molecular and biochemical studies are needed to establish the efficacy of the plant. However, the paw edema test is a very preliminary study and does not conclusively establish the bioactivity of the plant extract.

Ravi et al. (2011) studied the anti-proliferative activity of the plant bark against cancer cell lines with a promising IC₅₀ value (30 µg/mL) in K562 cells. The cell line exhibited characteristic features of apoptotic cells, including membrane blebs, cell shrinkage, and DNA damage. Moreover, phosphatidylserine (PS) residues were bound to Annexin V, which enhanced the uptake of 7-aminoactinomycin (7-AA). In normal cells, PS is present on the inner surface of the membrane; however, during early apoptosis, it becomes exposed on the cell surface. Annexin V is a phospholipid-binding protein with high affinity to PS (Robinson et al., 2020). On the other hand, uptake of 7-AA signifies a late apoptotic event (Wang T. et al., 2022). The chloroform extract showed the presence of a metabolite with an identical retention time to quercetin. Isolation, characterization, quantification, and evaluation of the antitumor potential of these metabolites may result in the identification of the lead target molecule.

Riaz et al. (2022) reported a bioassay-guided study of T. undulata, which showed significant cytotoxic, antimutagenic, and anticancer potential. The hexane extract had the greatest effect on the locomotion of Caenorhabditis elegans, while the methanolic extract had the least. Salmonella typhimurium strains TA98 and TA100 were modified for frame-shift and base-pair substitutions, respectively. The growth of these strains is inhibited in the absence of histidine in the culture medium. Plant extracts are tested in the presence of a mutagen to evaluate their antimutagenic potential, which is calculated according to the number of revertant colonies. The methanolic extract of Tecomella showed the highest antimutagenic potential, which may be due to the presence of flavonoids and other phenolic metabolites in the extract. Thus, the plant showed potential to prevent or inhibit the carcinogenic effect of mutagens (De Silva and Alcorn, 2019).

An MTT assay was performed to assess the cellular toxicity. The methanolic extract showed good activity against HepG2 tumor cell lines (68.17%). A significant difference in the inhibition of HepG2 cell lines was observed when comparing the data of the chloroform (Ravi et al., 2011) and dichloromethane extracts (Riaz et al., 2022) of the plant. These two solvents differ slightly in their polarity index, and the difference in their activity may be due to the time of collection and geographical location, which affect the concentrations of plant metabolites (Heinrich et al., 2022). The resazurin assay (simple, rapid, and sensitive) was performed on various cancer cell lines. The methanolic extract was found to be significantly effective on liver HepG2 and lung A549 cell lines, with CC₅₀ values of 117.37 and 142.01 µg/mL, respectively. Resazurin, also known as Alamar blue, is an indicator dye used to measure cell viability. The extract was found to possess a decent selectivity index when its cytotoxicity on cancer cells was compared with that on normal cells. Such an extract or metabolite is considered suitable for further anticancer studies. The metabolites present in the extract also showed potential against cancer-related proteins, as revealed through docking studies.

The plant was reported to have a mild blood glucose-lowering effect in an acute study. The effect was quite significant in a chronic study wherein the ethanolic leaf extract was administered for 30 days. Moreover, glucose tolerance improved in streptozotocin-treated rats (Kumar et al., 2012). This may be due to the increased secretion of insulin from pancreatic cells or the peripheral utilization of glucose. In the long term, diabetes glycosylation of proteins, including hemoglobin, is observed. A high level of glycosylated hemoglobin (HbA1c) is a marker for poor glycemic control and is associated with diabetes-related disorders (Lau and Aw, 2020). The plant extract helped bring the enhanced levels of HbA1c to nearly normal levels. Insulin activates glycogen synthase to form more glycogen (Norton et al., 2022). Hepatic glycogen was found to increase after the treatment with the plant extract. This may be attributed to the reactivation of glycogen synthase in test animals. Lipid peroxidation was observed to be reduced, as evidenced by the lower levels of malondialdehyde in diabetic rats treated with T. undulata. Additionally, GSH levels increased in streptozotocin-induced diabetic rats. The data projects the antioxidant potential of the plant. It is known that oxidative stress occurs in diabetes. Glycosylation of proteins can lead to the generation of reactive oxygen species (ROS) (Gupta et al., 1997). In individuals with diabetes, glucose is channeled toward a pathway that requires NADPH. GSH reductase forms reduced glutathione, involving NADPH. Thus, diabetes leads to GSH depletion and enhances oxidative stress (Sha et al., 2021). However, the study authors did not highlight dose determination and toxicity analyses, which are essential in animal studies using plant extracts.

In a study conducted by Das et al. (2015), the hydroethanolic extract of the heartwood of T. undulata lowered blood glucose levels and serum triglycerides, total cholesterol, and low-density lipoproteins and increased high-density lipoproteins. Increased free fatty acids in diabetes are associated with decreased glucose tolerance and impaired β-cell function (Wismayer et al., 2023). Bioactives present in the plant extract may have partially reversed the damage caused to the pancreas by streptozotocin (Papuc et al., 2021), as evidenced by the histopathological study of the pancreas. A similar effect of the hydroethanolic extract was reported in streptozotocin-induced diabetic rats using the leaves (Lal et al., 2017a) and roots (Lal et al., 2017b) of the plant. Tecomella extract induced the rearrangement of peripheral tissue and the normalcy of islets. The biochemical studies also supported the plant’s antidiabetic efficacy. However, the mechanism of action and the identification of lead antidiabetic metabolites still need to be explored.

Free radicals are byproducts of cellular metabolism. However, if their production exceeds the cell’s neutralization capacity, it leads to oxidative stress (Masenga et al., 2023). Thus, external supplementation with antioxidants is recommended, and the use of plant-based products is a matter of personal choice (Vats, 2016; Baroni et al., 2021). Bhardwaj et al. (2014) analyzed the methanolic extract of different plant parts of T. undulata for its antioxidant activity. The total phenolic content was found to be the highest in the stem (12.70 GAE/g DW). Meanwhile, the maximum flavonoid content was observed in leaves (71.87 mg QE/g DW). The best ferric reducing antioxidant power was observed in leaves (96.66 mM/L/g). The best antioxidant potential with respect to the ABTS assay was shown by the leaves. The results of the antioxidant assays are in accordance with the occurrence of the highest total flavonoid content in leaves. These in vitro antioxidant assays only define the chemical profile of a preparation and require further evaluation through pharmacological experiments to validate its antioxidant efficacy.

The methanolic extract of leaves showed better activity than the aqueous extract against Staphylococcus epidermidis and Bacillus subtilis (Parekh et al., 2005). This clearly reveals the importance of solvent selection in extracting active metabolites against a specific microbe (Nortjie et al., 2022). In another study, Sharma A. et al. (2013) reported that, among the different solvents tested, the methanolic extract exhibited the best antimicrobial potential. The least MIC (0.01 mg/mL) was observed against Klebsiella pneumoniae, and the highest MIC (4 mg/mL) was observed against B. subtilis. The antimicrobial potential of the methanolic extract of the stem bark was found to be better than that of the chloroform and aqueous extracts, as reported by Pandya et al. (2019). It is essential to note that the former study did not demonstrate any positive effect against Salmonella typhi; however, the latter study was found to be effective against the test organism of typhoid. The difference in the results may be due to the solubility of different bioactive metabolites in both solvents. Additionally, different plant parts may vary in terms of the type and concentration of metabolites. Moreover, the better diffusion of the methanolic extract in the microbial medium may be another reason for the differential activity (Parekh et al., 2005). Suganya et al. (2011) reported the antibacterial potential of PCL/PVP (polycaprolactone/polyvinylpyrrolidone) mats loaded with T. undulata extract. The medicated fibers remained stable even after high electrical voltage. The study highlighted the use of mats as both a drug carrier and a wound dressing material loaded with antimicrobials.

Khan et al. (2013) observed the maximum acaricidal activity of the methanolic extract in goats and camels, followed by humans. However, the results were better in the in vitro assay, wherein the plant extract (30%) showed 80% mortality. The miticidal activity may be due to the presence of lapachol, which interferes with cellular respiration and the generation of free radicals (Rahman et al., 2022). However, the actual molecular mechanism involved in the acaricidal activity needs to be further explored. Antiplasmodial activity of 17 plants was studied by Sachdeva et al. (2023). It was observed that T. undulata was the most effective with IC₅₀ values of 15 and 15.3 µg/mL against Plasmodium falciparum 3D7 and P. falciparum INDO, respectively. Some metabolites, such as quercetin, rutin, ursolic acid, lapachol, and betulinic acid (Table 4), may be responsible for the antimalarial activity of the plant extract.

Obesity is prevalent in most parts of the world. Obesity is associated with the incidence of other disorders, viz., diabetes, cardiac disorders, and cancer (Chen et al., 2020). It has been reported that during weight loss, there is mainly a reduction in the volume of adipocytes rather than a reduction in their number (Spalding et al., 2008). The expansion of adipose tissue through hyperplasia is quite challenging to reverse as adipocytes are resistant to apoptosis. Thus, anti-obesity drugs essentially should target hyperplasia. Alvala et al. (2013) sequentially extracted the bark of T. undulata and further fractionated the ethyl acetate fraction. Fraction 1 (F1) significantly inhibited the division and accumulation of triglycerides in adipocytes (3T3-L1 mouse fibroblast cells). This was achieved through the activation of Sirtuin 1 (SIRT1) mRNA and proteins. In addition, downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) was observed. A reduction in the mRNA expression of E2F1, leptin, FAS, and LPL was also observed. On the other hand, levels of adiponectin increased. SIRT1 is known to regulate transcription factors, which affect the expression of PPARγ, C/EBPα, and leptin, among others, which in turn regulate fat metabolism. PPARγ and C/EBPα stimulate adipocyte-specific genes and regulate adipocyte differentiation. LPL expression is a marker of lipid accumulation in adipocytes. FAS enzyme catalyzes the synthesis of long-chain fatty acids, and it is upregulated during adipogenesis (Ortega et al., 2010). E2Fs regulate adipogenesis through changes in the expression of the nuclear receptor PPARγ (Chen et al., 2020). Leptin is a hormone that plays a significant role in energy balance (Tucker et al., 2024). Adiponectin is a hormone derived from fat that has been shown to affect obesity negatively (Maeda et al., 2020). Thus, agents that regulate SIRT1 activity can be essential candidates for treating obesity and its related disorders. An in vivo study supported the data obtained in cell lines. A significant decrease in cholesterol, triglycerides, LDL, LDL/cholesterol, and VLDL was noticed. Additionally, an increase in HDL and HDL/cholesterol was observed in mice treated with the plant extract compared to obese mice fed a high-fat diet. The F1 was found to be rich in ferulic acid (4.95%), and the metabolite has been reported to possess anti-obesity properties (Wang O. et al., 2022). However, the study did not specify how the dose administered to mice was determined.

A stomach ulcer was induced in rats using ethanol, and the ulcer index was found to be 7, with an ulcerated area of 1.10 cm²–0.3 cm² (Arsalan et al., 2023). T. undulata extract significantly reduced ulcer index (2) and ulcerated area (0.3 cm²–0.1 cm²). Ethanol-induced gastric ulcers cause a decline in bicarbonate secretion and a reduction in the mucus present in the gastric wall (Ibrahim et al., 2022). Ethanol induces the production of free radicals, such as the superoxide anion and hydroxyl radical, and enhances lipid peroxidation, leading to the impairment of the stomach mucosa (Ibrahim et al., 2022). Thus, the plant extract may have reduced oxidative stress, leading to decreased capillary injury, vascular permeability, and the production of inflammatory markers (Akmal et al., 2023). Furthermore, constipation was induced in rats using loperamide (Arsalan et al., 2023), which reduces peristaltic movement in the intestine, including water secretion (Parkar et al., 2024). The plant extract increased the weight and number of fecal matter, suggesting improved colon movement (Katsirma et al., 2021). This may be due to the presence of glycosides, which possess laxative activity, and their presence was also reported in the study.

The plant was projected as an antifertility agent by Soni and Mali (2016). Administration of T. undulata extract resulted in a 70% reduction in the weight of male reproductive organs, accompanied by a decrease in fertility rate. The sialic acid content, which facilitates the seamless movement of sperm, was found to be reduced. This may have affected the acrosomal membrane and the fertilization ability of sperm (Aslan Çetin et al., 2022). Reduced levels of luteinizing hormone and testosterone were observed. LH induces the production of testosterone. Testosterone plays a crucial role in spermatogenesis (Oduwole et al., 2021). The analysis of the phytochemicals present in the extract and the determination of the dose were not mentioned by the authors.

The ameliorative effect of T. undulata on CdCl₂ (cadmium chloride)-induced splenomegaly was investigated by Nagpal et al. (2019). In rats treated with the plant leaf extract, the size of the spleen was comparable to that of the control group. The complete bold count suggested a positive effect of the plant’s extract. The hemoglobin, red blood cell, platelet, and packed cell volume data were found to be almost equal to those of the control group and better than those of the CdCl₂-treated group. The histological studies revealed that the spleen of rats administered with CdCl₂ showed necrosis, hyperplasia, swollen and dead cells in the pulp, and sinus congestion. These features were improved in the group treated with T. undulata. In the above-studied parameters, a lower dose of the plant’s extract did not prove to be effective; however, a higher dose (>600 mg/kg/day) showed promising results.

Botanical extracts, in general, are complex, and seasonal/geographical differences significantly affect the concentrations of individual metabolites, thereby affecting their efficacy. These changes are also due to the use of agrochemicals during cultivation and other variables affecting plant growth (Heinrich et al., 2022). However, none of the above-mentioned studies have provided these details.

Explants collected in August and September showed the best response and the highest number of shoot inductions. The maximum number of shoots was observed in MS + BAP (5 mg/mL) + IAA (0.05 mg/mL); however, the shoots were very short in length. This shows that higher concentrations of BAP inhibited the adequate differentiation of shoots (Rathore et al., 1991). It was observed that the response was better at 31 °C, possibly due to the physiological nature of the plant, which propagates optimally in a semi-arid environment. Kinetin-induced leafy shoots were not found to be better for further micropropagation steps. Furthermore, the sub-culturing of the shoots was performed on a medium with lower concentrations of auxins to inhibit callusing (Vats and Kamal, 2013; 2014; Vats, 2018). Aghdaei et al. (2012) investigated the impact of silver nanoparticles on plant culture. Nanoparticles, when used alone, showed a mild response in terms of the induction of the number of shoots (<2). In combination with hormones (BAP + IAA), the response was even poorer. When nanoparticles were used in combination with thidiazuron, the response was found to be slightly better with two shoots per explant. Thus, silver nanoparticles had no significant effect on shoot induction. This may be due to seasonal variations in terms of explant collection, region of collection, and the hormones used (Cheng et al., 2024). Aslam et al. (2006) observed a positive effect of IAA and BAP on shoot induction from cotyledonary nodes. Better shoot induction was observed when IAA and BAP were used in combination, with a superior percentage of explant response. A higher concentration of cytokinin than auxin generally promotes shoot growth (Wu et al., 2022). Chhajer and Kalia (2017) used Schenk and Hilderbrandt medium supplemented with BAP, KN, and ascorbic acid for multiple shootings. Antioxidants have been used in the medium to avoid the production of phenolic metabolites in the culture and leaching in the medium (Vats, 2018).

Rooting from shoots was induced using a liquid medium supplemented with IBA for 2 days, and then the plants were transferred to half-strength MS medium (Rathore et al., 1991). The survival rate after acclimatization was evaluated to be 46%, which is comparatively lower. Aslam et al. (2006) and Aslam et al. (2009) reported a stepwise process for efficient rooting, which involved transferring in vitro shoots to a liquid medium containing different concentrations of NAA, IAA, and IBA, either alone or in combination, for 36 h, followed by half-strength MS medium without hormones. Many workers suggest that the two-step method reduces the number of days needed for root initiation and yields a better average number of shoots (Rathore et al., 1991; Bhansali, 1993). In vitro-generated shoots were cultured on Schenk and Hildebrandt (SH) medium supplemented with IBA and ascorbic acid for rooting. According to Tyagi and Tomar (2013), the most suitable medium for in vitro rooting was ½ B5 medium, possibly due to its lower content of ammonium nitrate and potassium nitrate than that of the MS medium. The addition of ascorbic acid improved the rooting because it acts as an antioxidant, which minimizes phenolic production involved in retarding the growth of cultures.

Indirect micropropagation was carried out using seedlings and nodal explants from the tree. Callus obtained from explants of the tree was hard, compact, and green; however, it was fragile and light brown when seedlings were used as explants (Nandwani et al., 1996). NAA, in combination with kinetin, had no effect on shoot induction in callus generated from both explants; however, IAA and BAP proved to be effective in this regard. Better shooting was observed in the seedling-derived callus. The two-step method was followed for root induction. Instead of supplementing hormones in agar containing MS medium, pretreatment was carried out in a hormone-containing liquid medium, and thereafter, the cells were transferred to ½ MS medium. Auxins (NAA, IBA, and IAA) were used either alone or in combination, and the best response was observed when all three hormones were used in combination at a concentration of 5 ppm. As mentioned earlier, Rathore et al. (1991) also used a two-step method; however, the response was poor. Variation in the result may be due to the difference in genotype (Holmes et al., 2021).