An updated and comprehensive review on the ethnomedicinal uses, phytochemistry, pharmacological activity and toxicological profile of Tinospora crispa (L.) Hook. f. & Thomson

Tinospora crispa (L.) Hook. f. & Thomson (Menispermaceae) is a plant indigenous to Africa and South-East Asia. It is widely used in ethnomedicine to alleviate various diseases including hypertension, diabetes, rheumatism, jaundice, inflammation, fever, fractures, scabies, and urinary disorders. A total of 167 phytoconstituents, belonging to 12 different chemical categories, including alkaloids, flavonoids, terpenoids, and phenolic compounds have thus far been isolated from various parts of T. crispa. Numerous in vitro and in vivo investigations have already established the antidiabetic, anticancer, antiparasitic, antimicrobial, immunomodulatory, hepatoprotective, analgesic, antipyretic, antihyperuricemic, and pesticidal activity of this plant, as well as its effects on the cardiac and the central nervous system. Most pharmacological investigations to date have been carried out on plant extracts and fractions. The exact identity of the phytoconstituents responsible for the observed biological effects and their mode of action at the molecular level are yet to be ascertained. Toxicological studies have demonstrated that T. crispa is relatively safe, although dose-dependent hepatotoxicity is a concern at high doses. This review presents a comprehensive update and analysis on studies related to the ethnomedicinal uses, phytochemistry, pharmacological activity and toxicological profile of T. crispa. It provides some critical insights into the current scientific knowledge on this plant and its future potential in pharmaceutical research.

This study aims to provide a detailed account of the taxonomy, phytochemistry, pharmacology, and toxicology relevant to T. crispa, so that it may serve as a valuable resource providing future direction for researchers. Electronic versions of tertiary literature sources (e.g. Google Scholar, PubMed, ScienceDirect, Scopus, Wiley Online Library, SpringerLink, Semantic Scholar, Web of Science and MEDLINE) were used to retrieve data on the ethnopharmacology, phytochemistry, pharmacology, and toxicology of T. crispa published within 1930-2021.

Vernacular names
The following vernacular names for T. crispa have been reported (Quisumbing 1951;Forman 1981;Noor et al. 1989;Longuefosse and Nossin 1996;Ahmad and Ismail 2003;Grenand et al. 2004;Dweck and Cavin 2006;Hout et al. 2006;Li et al. 2006;Roosita et al. 2008;Islam et al. 2011;Rahmatullah et al. 2011;Koay and Koay 2013;Haque et al. 2017;Dapar 2020;Dapar et al. 2020): • Bangladesh: Guloncho-ban, Golonchi, Khorosh, Guntai • India: Dier, Faridbuti, Dagadi, Chipuru-tige, Kattle-ti, Giloya • Malaysia: Brotowali, Akar Patawali, Patawali, Akar Seruntum, Seruntum, Sapai, Daun akar walli Taxonomy Tinospora crispa is one of the 34 species that belong to the genus Tinospora. All species of this genus are found in tropical and subtropical regions of Asia, Africa and Australia. Most species produce bioactive constituents (especially diterpenoids and alkaloids) and are used widely in ethnomedicine (Chi et al. 2016). Tinospora crispa is also known as Chasmanthera crispa Baill., Cocculus crispus DC., Cocculus verrucosus Wall., Menispermum crispum L., Menispermum rimosum Blanco, Menispermum tuberculatum Lam., Menispermum verrucosum Roxb., Menispermum verrucosum Roxb. ex Fleming, Tinospora crispa Diels, Tinospora gibbericaulis Hand.-Mazz., Tinospora mastersii Diels, Tinospora rumphii Boerl., Tinospora thorelii Gagnep. and Tinospora tuberculata Beumée ex K. Heyne. (The Plant List 2013;Global Biodiversity Information Facility 2021;World Flora Online 2021). This species has a generally fleshy, with older stems being fleshier than younger ones. Younger stems present a thin membranous and glabrous epidermis is characteristic of younger stems, while tubercles are observed on older ones. The stem contains a bitter, milky sap. Tinospora crispa has long, filamentous, aerial roots. The leaves are cordiform in shape and are usually 6-12 cm long and 7-12 cm wide. They are marginally fleshy with chartaceous leaf-blades. The dried leaves are quite delicate. Domatia are not usually observed, but a flat pocket appears intermittently in the axis of the basal nerves on the ventral surface. The leaf petioles are 5-15 cm long and glabrous. The flowers are fascicled and greenish-yellow or yellow. The male inflorescences are taller and thinner compared to the female counterparts, 5-10 cm versus 2-6 cm respectively. Both male and female flowers share morphological similarities in terms of sepals and petals with six green sepals in two verticils. The inner three sepals are obovate while the rest are ovate. Both male and female flowers have 3-6 yellow petals. The fruits are vermillion or scarlet, with a pale white endocarp. They are ellipsoidal, 7-8 mm long, and feature a distinctive dorsal ridge with a small ventral aperture and a deeply seed-cavity intrusive condyle. The seeds are curved, bean-shaped, and white. The root is succulent (Forman 1981;Patel et al. 2013;Haque et al. 2017). Tinospora crispa and its various parts are illustrated for identification in Fig. 1.

Ethnomedicinal uses
Tinospora crispa is used in ethnomedicine predominantly in South-East Asia. Some of its uses are common across multiple ethnicities (e.g. diabetes) while others are reserved to certain regions only. In Bangladesh, various preparations are used for fever, body pain, rheumatism, skin diseases, paralysis, abdominal pain, intestinal disorders and leprosy (Rahmatullah et al. 2009(Rahmatullah et al. , 2011Islam et al. 2011;Kadir et al. 2014). In Malaysia, infusions of the stems and of the whole plant are used as a postpartum remedy and to treat type-2 diabetes mellitus,  (Forman 1981;Noor et al. 1989;Ahmad and Ismail 2003;Mohamad et al. 2011;Dapar 2020). In the Philippines, the stems and leaves are employed for fever, indigestion, flatulence, intestinal disorders, diarrhea, vomiting, ulcer, body ache, rheumatism, toothache, ocular soreness, scabies, lacerations and boils (Quisumbing 1951;Dapar 2020;Dapar et al. 2020). In Thailand, the leaves, stems, roots and seeds are prepared into powders, infusions and decoctions to treat wounds, itches, cholera, diabetes, fever, rheumatism, intestinal parasites, snake-bites, syphilitic sores, sore eyes, and alcohol or drug-induced poisoning (Dweck and Cavin 2006). People in the Yao community in China use hot infusions of the stems as bath water to treat fractures, contusions, furuncles, carbuncles and viper-bites (Li et al. 2006). In China again, the plant is used for fever, septicemia, scabies and ulcers (Koay and Koay 2013). In the South Kerala region of India, locals use the plant as an antidiabetic (Thomas et al. 2016). The use of T. crispa as an antimalarial agent is widespread in Malaysia, the Philippines, Indonesia, Vietnam, Southern Laos and the Republic of Guinea (Forman 1981; Ahmad and Ismail 2003;Bertani et al. 2005;Elkington et al. 2014;Ramadani et al. 2018;Dapar 2020;Dapar et al. 2020). Indonesians also employ the plant for hyperglycemia, inflammation, fever and rheumatism. The last two uses are also reported in Cambodia (Hout et al. 2006;Adnan et al. 2016;Ramadani et al. 2018). Apart from the aforementioned uses, T. crispa stems are also employed to treat jaundice and fever in Vietnam (Forman 1981). The Kadayan Malay community in the Sengkurong mukim region of Brunei use the stems for hypertension and abdominal ache (Dapar 2020). In Guyana, a bitter beverage produced from T. crispa macerated stems, combined with Quassia amara bark, is taken for albuminuria and diabetes (Grenand et al. 2004; Thomas et al. 2016). In Martinique, the leaves and stems are used in decoctions and tinctures to treat diabetes (Longuefosse and Nossin 1996). The ethnomedicinal uses of T. crispa are listed in Table 1.

Phytoconstituents
Extensive phytochemical investigations on the aerial parts of T. crispa, both as a whole and as individual parts (stems, leaves, and vines), led to the identification of 167 phytoconstituents belonging to diverse chemical classes. Clerodane-type furanoditerpenoids are the most abundant phytoconstituents in this species. A considerable number of alkaloids, flavonoids, and steroidal compounds have also been reported. Other classes of secondary metabolites, present to a lesser extent, include triterpenes, phenolic compounds, nucleosides, aromatic constituents, volatile terpenoids and long-chain fatty acid derivatives. All compounds reported from T. crispa to date are listed in Table 2 and their structures are illustrated in Figs. 2, 3, 4, 5, 6, 7 and 8.

Clerodane-type furanoditerpenoids
Furanoditerpenoids are a class of compounds that features at least one furan ring as part of their core skeleton. The outstanding significance of this class lies in its pharmacological potential, which is primarily be attributed to the biologically-interactive furan ring. The clerodane-type furanoditerpenoids are based on a decahydronaphthalene skeleton with a furan ring attached to it via a two-carbon bridge. Based on the number of lactone rings present, these compounds have been further categorized into three major subgroups viz. A, B and C, featuring zero, one and two lactone rings, respectively (Bao et al. 2017). A total of 38 clerodane-type furanoditerpenoids have been identified in T. crispa (1-38) (Fig. 2). Among them, only two (1, 2) were of type A (Hossen et al. 2016;Noman et al. 2018), while 28 compounds (3-30) featured one lactone ring in their structures and belonged to type B Lokman et al. 2013;Abood et al. 2014;Langrand et al. 2014;Hamid et al. 2015;Mantaj et al. 2015;Adnan et al. 2016;Gao et al. 2016;Xu et al. 2017;Rahman et al. 2020). Five of the furanoditerpenoids (31-35) were of type C with two lactone rings (Ahmed et al. 2006;Choudhary et al. 2010b;Lam et al. 2012;Praman et al. 2012). Compounds from both type B and C exhibited further structural diversification in terms of the position of the lactone ring(s), extent of hydroxylation and presence of monosaccharides at different positions. A total of 21 furanoditerpenoids (4-6, 8, 13-15, 17-27, 30, 32, 35) were characterized as glycosides. While most of the glycosidic constituents contained a single b-D-glucose moiety in their structure, two of them (6, 21) featured two saccharide moieties (Gao et al. 2016), and one of them (21) included an a-D-xylose moiety (Choudhary Stems (Choudhary et al. 2010a) 46 N-acetylnornuciferine Stems (Pachaly et al. 1992;Bakhari et al. 2005Bakhari et al. , 2013Lin 2009) 47 N-demethyl-N- Enzyme inhibitory assay (Hamid et al. 2015) 63 N-cis-feruloyltyramine Stems (Langrand et al. 2014)

Pharmacological activity
Tinospora crispa has been extensively studied in vitro, in vivo and in silico to scientifically validate its use in ethnomedicine. Most studies have focussed on the antidiabetic and cardiac activity, including the mechanisms of action at the molecular level, of T. crispa extracts and phytoconstituents. Significant evidence to support the anticancer, antiparasitic, antimicrobial, antioxidant and immunomodulatory potential of this plant has also been obtained. Preliminary evidence of its hepatoprotective, analgesic, antipyretic, anticholinesterase, central nervous system, antihyperuricemic and pesticidal activity has been reported. Such effects, however, remain comparatively unexplored and require further exhaustive investigations. A concise summary of the pharmacological activities of the plant is presented in Table 3.

Antidiabetic activity
The aqueous extract of T. crispa has been evaluated for its activity on diabetic male Wistar albino rats, on rat and human islets of Langerhans, and on HIT-T15 cells. A week after administration of the extract (4 mg/ mL), lowered blood glucose levels (10.4 ± 1.0 mmol/ L) were observed compared to the control group (17.4 ± 1.7 mmol/L). Additionally, insulinotropic activity was also evident with comparatively greater insulin levels in the test group than in the control (12.8 ± 1.1 lU/mL and 8.0 ± 0.7 lU/mL, respectively). In the rat islets, the extract (0.01-1 mg/mL) led to a dose-dependent enhancement of basal insulin secretion up to a maximum of fivefold. The extract also potentiated (1.5-fold) the glucose-mediated induction of basal insulin secretion. Similar results were obtained in the human islet system as the extract (1 mg/mL) induced insulin release similar to that of a high dose of glucose (20 mmol/L). The extract also further potentiated glucose-mediated insulin release.
In HIT-T15 cells, the extract (0.01-4.00 mg/mL) boosted the basal insulin release sevenfold, along with a 1.5-fold enhancement of glucose-induced insulin secretion. This was the first evidence of the plant acting as an oral hypoglycemic and insulinotropic agent (Noor et al. 1989). The in vivo antidiabetic effect was further confirmed by multiple subsequent studies in other animal models (Arcueno et al. 2015;Hassani et al. 2016;Arundina et al. 2017;Firdausa et al. 2020). Antidiabetic mechanisms other than an insulinotropic effect were evaluated in another study using the aqueous extract. It was found that the extract (1 mg/ mL) played no significant role in intestinal or adipocyte glucose uptake. In HIT-T15 cells, the insulinotropic activity was inhibited by adrenaline (5 mM), somatostatin (1 mg/mL), verapamil (50 mM) and nifedipine (50 mM). Cyclic AMP concentration (cAMP) and 86 Rb efflux were further measured and it was hypothesized that the insulinotropic effect of T. crispa was the result of calcium ion transport across the membrane of pancreatic b cells, and possibly closure of ATP-mediated potassium channels (Noor and Ashcroft 1998a). This was confirmed by a later study which revealed that the extract increased HIT-T15 cell sensitivity to extracellular calcium ions and resulted in increased intracellular accumulation of these ions caused by increased uptake and suppressed efflux. The physiological nature of the underlying mechanism suggested the presence of individual compounds in T. crispa which may serve as potential insulin secretagogues (Noor and Ashcroft 1998b). It was found in a later study that the administration of T. crispa powder in capsule form (1 g thrice daily) could not induce hypoglycemia in type-2 diabetic patients non-responsive to oral hypoglycemic drugs. It was postulated that these results reaffirm the insulinotropic nature of the antidiabetic activity of T. crispa (Sangsuwan et al. 2004).
This ability to reverse the insulin resistance was also demonstrated in a study using Wistar rats fed a high fat diet. The aqueous extract of T. crispa at a dose of 1 g/mL resulted in a significant decrease in glucose (8.50 ± 0.30 mmol/L compared to 13.75 ± 0.25 m mol/L in the untreated group). Serum glucose, cholesterol and triglycerides levels decreased with the treatment, along with a fall in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, creatine and urea (Abu et al. 2015). A subsequent investigation established the capacity to abolish insulin resistance in insulin resistant IR-HEP-G2 cells using rosiglitazone maleate as a standard. It was observed that T. crispa methanol extract and the standard (both at doses of 100 lg/mL) led to a 2.5-and 1.5-fold increase in 2-DG uptake, respectively. It was found that the insulin receptor was upregulated, ultimately recruiting the PI3K/Akt pathways. Subsequent increase of GLUT4 expression was also observed resulting in a boosted 2-DG uptake. Additionally, T. crispa methanol extract triggered apoptosis in the IR-HEPG2 cells stimulated with insulin (Abu et al. 2017).
Another study revealed that an ethanol extract of T. crispa stems displayed a-glucosidase inhibitory activity, with a 78.34% inhibition at a concentration of 450 ppm compared to 81.01% when using the standard acarbose. The IC 50 values for the extract and acarbose were 237 and 116 ppm, respectively (Tambunan et al. 2013). In a recent study, the ethanol and aqueous extracts of the stem have also been observed to inhibit the enzyme a-amylase in vitro with an IC 50 of 10.348 ± 0.313 and 11.660 ± 0.310 mg/mL, respectively (Hartini et al. 2022). Interestingly, endophytic fungi isolated from T. crispa have been found to exhibit a amylase and a glucosidase inhibitory activity (Lestari et al. 2015;Pramitasari et al. 2017). The aqueous extract of the plant at a dose of 500 mg/kg has been reported to increase superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels in streptozotocin-treated diabetic Sprague Dawley rats, thereby boosting antioxidant activity (Firdausa et al. 2018). The ethanol extract of T. crispa has showed an ability to increase lymphocytes, fibroblasts and enhanced healing activity in diabetic male Wistar rats with oral mucosal ulcers Roestamadji et al. 2017).
As there have been numerous studies on the antidiabetic potential of T. crispa extracts, the same can also be said for its phytoconstituents. Particularly, a number of clerodane type furanoditerpenoids and their glycosides have been reported to have significant hypoglycemic activity. Borapetosides A (32) and C (14) at a dose of 5 mg/kg significantly decreased blood glucose levels in normal and type-1 diabetic mice compared to the standard metformin (200 mg/kg). Borapetoside C (14) at a dose of 3 mg/kg also displayed activity against type-2 diabetes, evident from its insulin secretagogue activity. This was comparable to that of glibenclamide (5 mg/kg) and was exerted through an increased peripheral tissue glucose uptake and suppressed hepatic gluconeogenesis ). Borapetoside C (14) (0.1 mg/ kg) is also capable of increasing glycogen synthesis in skeletal muscles when given in combination with insulin in normal, type-1 and type-2 diabetic mice. It increased the serine phosphorylation of Akt, phosphorylation of the insulin receptor, and GLUT2 levels by 3.0, 1.4 and 1.3-fold when administered with insulin ). This demonstrated the versatility of this compound in terms of antidiabetic activity. Another compound with established insulin secretagogue activity is borapetol B (16), which was assessed on normoglycemic Wistar and spontaneously type-2 diabetic Goto-Kakizaki (GK) rats at a dose of 0.1 mg/kg. In the Oral Glucose Tolerance Test (OGTT), a significant decrease in glucose levels was observed in both animal models. This compound also enhanced insulin secretion in isolated pancreatic islets (Lokman et al. 2013). In a later study, borapetoside C (14) (IC 50 value of 0.527 ± 0.008 mg/mL) and 4-hydroxybenzaldehyde (130) (IC 50 value of 0.557 ± 0.004 mg/mL) were found to be the most potent a-glucosidase inhibitors. The alkaloids liriodenine (49), lysicamine (50) and N-formylanonaine (39) also strongly inhibited this enzyme, with IC 50 values ranging from 0.5 to 0.8 mg/mL. Borapetoside C (14) (IC 50 value of 0.775 ± 0.005 mg/mL) displayed the most prominent activity against a-amylase alongside notable activity observed for N-trans-feruloyltyramine (62), dihydrodiscretamine (53) and magnoflorine (51) (IC 50 value of 0.8 to 0.9 mg/mL). It was suggested that the ring hybridization of these alkaloids allowed them to interact with the aforementioned enzymes, but that the presence of different functional groups weakened their activity (Hamid et al. 2015). Another clerodane furanoditerpenoid, borapetoside E (4) (40 and 80 mg/kg), caused stark improvements in hyperglycemia, insulin resistance, hyperlipidemia, hepatic steatosis and oxygen consumption in high fat diet-fed mice compared to the standard metformin (400 mg/kg). This compound also reduced the expression of sterol regulatory element binding proteins (SREBPs), which are important transcription factors in lipid synthesis and have emerged as novel targets for the treatment of type-2 diabetes (Xu et al. 2017). Tinosporol A (8) induced dose-dependent hypoglycemic activity in type-1 diabetic ICR (Institute of Cancer Research) mice and type-2 diabetic db/db mice, although it was found that the type-1 model was more sensitive to this compound than the type-2 one (Gao et al. 2016). In a study investigating the aglucosidase inhibitory activity of acylated glucosylflavones (tested at a concentration of 10 lg/mL), isovitexin-2''-(E)-p-coumarate (89) displayed maximum inhibition (IC 50 value of 4.3 ± 1.4 lM) compared to the standard acarbose (IC 50 value of 0.033 ± 0.006 lM) (Chang et al. 2015). Some clinical studies have been conducted to evaluate the effect of T. crispa on healthy volunteers, on patients with diabetes and patients with high risks of developing diabetes. For example, a clinical study conducted in Thailand, showed that pre-prandial administration of T. crispa (250 mg capsule twice daily for two months) in patients with metabolic syndrome resulted in a steady decrease in fasting blood sugar and triglyceride levels (Sriyapai et al. 2009). Another study reported a remarkable reduction in plasma glucose levels following oral administration of T. crispa powder (6 g) to healthy subjects (Rattanajarasroj et al. 2004). In both studies, however, T crispa caused a noticeable increase in ALT and AST serum levels, implying possible hepatotoxicity (Sriyapai et al. 2009;Rattanajarasroj et al. 2004). Other clinical studies also indicated the increased risk of hepatotoxicity associated with T. crispa and/or concluded that there was no evidence to support to use of this plant for the treatment of diabetes (Sangsuwan et al. 2004;Klangjareonchai and Roongpisuthipong 2012). In depth details and discussions on the clinical studies involving T. crispa can be found under the 'Clinical Trials' section.
In summary, the ethnomedicinal use of T. crispa in the treatment of diabetes has been underpinned by many scientific studies. The antihyperglycemic activity of this plant occurs mainly as a result of enhanced insulin secretion and inhibition of aglucosidase and a-amylase. The pathways involved in the antidiabetic mode of action of T. crispa extracts and its phytoconstituents are similar (Fig. 9). Selected clerodane-type furanoditerpenoids present in T. crispa have been reported to possess insulin secretagogue properties. Further structure activity relationships (SAR) studies on this class of phytochemicals should be undertaken to determine the pharmacophore(s) responsible for the modulation of intracellular calcium ion levels. Other phytochemicals such as flavonoids, for example, have strong inhibitory activity against a-glucosidase and aamylase and several SAR studies have been investigated these effects (Tadera et al. 2006;Proença et al. 2017Proença et al. , 2019Zhu et al. 2020). Further research work on the antidiabetic potential of the various flavonoids present in T. crispa should be conducted.

Cardiac activity and cardiovascular effects
Multiple extracts and fractions, at doses of 0.25-1 mg/ mL, were evaluated for their cardioactive potential in isolated atria and aorta of male Sprague Dawley rats. Extraction was performed with petroleum ether, chloroform, methanol and water; and four fractions derived from the chloroform extract obtained following flash chromatography using chloroform/n-hexane and chloroform/methanol combinations. The fractions derived from the chloroform extract were found to be the most active, inhibiting the isoprenaline-induced positive chronotropic response in the left atrium by 80% at a dose of 1 mg/mL. From the dose-response curve obtained, it was concluded that all the extracts and fractions mentioned above functioned as noncompetitive b-adrenoceptor antagonists. In the right atrium however, the extracts at high doses effectuated a complete inhibition of the isoprenaline-induced positive chronotropic response by suppressing the sinoatrial node. This could be rectified by high doses of isoprenaline. In the aorta, the fractions derived from the chloroform extract showed 85-99% inhibition of the noradrenaline-induced positive inotropic response, and the inhibition was commensurate with the increasing polarity of the fractions. The dose-response curve obtained suggested that these fractions acted as noncompetitive a adrenoceptor antagonists (Bakhari and Isa 2010). The n-butanol fraction of the aqueous extract of T. crispa (1-100 mg/kg) was also tested in normal and reserpine-induced female Wistar rats. Whilst this fraction produced significant hypotensive and positive chronotropic activity in normal rats, dual effects were obtained following reserpine induction with a transient decrease followed by an increase in hypotensive activity. Similar dual effects were obtained for the positive chronotropic action. The mechanism of action was unravelled using posttreatment with propranolol (0.6 mg/kg), phentolamine (2 mg/kg), atenolol (2 mg/kg), the b2 antagonist ICI-118,551 (0.01 mg/kg), atropine (0.6 mg/kg) and hexamethonium chloride (10 mg/kg), either individually or in various combinations. This revealed that the action of the active constituents was mediated via b2adrenergic receptors producing hypotension, as well as b1and b2-adrenergic receptors effectuating a positive chronotropic response. Additionally, some constituents caused hypertension and increased heart rate via modulation of a-adrenergic receptors. The authors further concluded that compounds acting via non-adrenergic and non-cholinergic pathways were also present to cause a reduction in mean arterial pressure and heart rate (Praman et al. 2011).
Other compounds from T. crispa have been investigated for their cardio-active potential. This includes cycloeucalenol (118) (5.6 9 10 -5 M) and cycloeucalenone (119) (5.6 9 10 -5 M). Both molecules had slightly positive inotropic activity in the isolated right atria of male Wistar rats. Conversely, these compounds initially demonstrated minimal negative inotropic activity, followed by significant negative inotropic activity in the left atria, thereby exhibiting mild cardiotonic activity compared to noradrenaline (1 9 10 -8 M) (Kongkathip et al. 2002). A synthetic racemic mixture of N-formylnornuciferine (43) produced a negative inotropic and chronotropic response in isolated rat heart (Imphanban et al. 2009). The identified mechanisms through which the T. crispa modulates cardio-activity are presented in Fig. 10. However, it should be noted that the cardiac potential of this plant cannot be attributed to a particular class of compounds with much confidence, other than the purinergic action of its nucleosides. Moreover, when administered to diabetic rats, T. crispa powder produced a significant increase in hemoglobin concentration and red blood cells (RBC) alongside a notable decrease in White Blood Cells (WBC) compared to control (Suchantabud et al. 2008).

Anticancer activity
The cytotoxic potential of various extracts and fractions of T. crispa has been reported by multiple investigators using the brine shrimp lethality assay method. A petroleum ether fraction of the methanol extract was reported to have strong cytotoxic activity with IC 50 of 173 ppm (Mackeen et al. 2000). Another study revealed that the methanol extract of the stem along with its chloroform and petroleum ether fractions at doses of 0.781-400 lg/mL showed comparable cytotoxicity (LC 50 of 12.0, 11.5, and 12.6 lg/mL, respectively). Vincristine sulfate was used as a standard with an LC 50 of 0.323 lg/mL (Haque et al. 2011). Stronger cytotoxicity (LC 50 values of 6.43, 4.58, and 0.80 lg/mL, respectively) was later reported in another study on the same stem extract and fractions tested within the same concentration range. This study also evaluated the aqueous extract which showed a LC 50 of 7.46 lg/mL (Islam et al. 2013). The ethanol extract of the leaves had a LC 50 of 62.75 lg/mL, which is notably weaker compared to the previously mentioned extracts (Tarukbua et al. 2018). The methanol extract of the stems was found to suppress the proliferation of HL-60, HEP-G2 and Hep3B cancer cells in a dose-and time-dependent manner (Ahmad et al. 2016a). The aqueous extract showed moderate antiproliferative activity against MCF-7, Caov-3, HeLa and HEP-G2 cells (IC 50 of 107, 100, 165 and 165 lg/mL, respectively) (Zulkhairi Jr et al. 2008). The aqueous, methanol and chloroform extracts of T. crispa stem revealed antiproliferative and cytotoxic activity against MCF-7, MDA-MB-231, 3T3 and HeLa cells. The extracts produced dosedependent cytotoxicity, with the methanol extract being the most potent (Ibahim et al. 2011). The ethanol extract (12.5, 25, 50, and 100 lg/mL) showed inhibition of head and neck squamous cell carcinoma (HNSCC) metastasis on HN22 and HSC3 cells. In a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, this extract, at the maximum concentration used, significantly decreased cell viability in the two cell lines to 50% and 60%, respectively compared to the negative control dimethyl sulfoxide (DMSO). Administration of this extract at concentrations of 12.5, 25, and 50 lg/mL also downregulated MMP-13 gene expression in both cell lines. A stronger reduction in secreted MMP-13 levels was observed in HN22 compared to that of HSC3 cells. In the latter cell line, the ethanol extract at 25 and 50 lg/ mL increased the expression of the tissue inhibitors of metalloproteinase-2 (TIMP-2). Moreover, pre-treatment with this extract (50 lg/mL) in a scratch wound healing assay using HN-22 cells caused cell migratory activity to drop to 65% compared to the control DMSO (Phienwej et al. 2015).
The chloroform extract of the stems was evaluated for its anti-angiogenic activity in the Chick embryo Chorioallantoic Membrane (CAM) induced by basic Fibroblast Growth Factor (bFGF) assay. Dose-dependent anti-angiogenic activity of 31.87 ± 9.01, 43.12 ± 8.01, 53.44 ± 2.70 and 62.81 ± 4.74% was obtained for concentrations of 15, 60, 240, and 960 lg/mL, respectively (Triastuti 2010). In contrast, no cytotoxic activity was reported for the methanol and aqueous extracts of the stems in a water-soluble tetrazolium (WST) or MTT assay employing HL-60, HEP-G2 and MCF-7 cancer cells (IC 50 [ 500 lg/mL) (Tungpradit et al. 2010). This apparent difference of activity on different cell lines may depend upon the nature of phytoconstituents present in the extracts. This, in turn, may be linked to differences in geographical areas of plant collection as has been reported previously when samples collected from different regions of the East Jawa province in Indonesia showed significant difference in cytotoxicity. The ethanol extract yielded LC 50 values ranging from 30.64 ± 2.18 (strong activity) to 254.15 ± 30.77 lg /mL (weak activity) in an MTT assay carried out on MCF-7 breast cancer cells (Mutiah et al. 2019). Tinocrisposide (14) (3.125-100 lg/mL) isolated from the dichloromethane fraction of the methanol stem extract was tested using an MTT assay on H1299 and MCF-7 cells. IC 50 values of 70.9 and [ 100 lg/ mL were obtained in these cell lines, respectively. It was suggested that this compound, whilst not a viable cytotoxic agent, could still prove useful as a chemopreventive agent (Adnan et al. 2016). The cis-clerodane furanoditerpenoid crispene E (10) isolated from the n-hexane fraction of the methanol stem extract   (Mantaj et al. 2015). A subsequent study isolated two related compounds, crispene F (2) and crispene G (11), which yielded IC 50 values of 42 and 17 lM, respectively, in the FP assay and 119% to 130% inhibition compared to STA-21, respectively. Both compounds had IC 50 values of 10 and 7.8 lM on MDA-MB-231 cells using the MTT assay. Weak activity on A4 (STAT-3 independent) colon cancer cells indicated that the compounds possibly induced STAT-3-specific inhibition. Comparatively, crispene E (10) was identified as the most potent among the three derivatives (Noman et al. 2018).
The in vitro anticancer activity of T. crispa has been demonstrated against several cancer cell lines. Its effects on gene expression and the underlying mechanisms are illustrated in Fig. 10. There have been no studies reported on the anticancer activity of the plant in vivo, which warrants further investigations. Interestingly, pure compounds such as clerodane-type furanoditerpenoids have displayed promising activity, particularly on STAT-3 inhibition. Quantitative SAR (QSAR) studies are now required into the 38 compounds of this class that have been isolated from the plant. This may help to focus on specific chemical moieties that can interact with the binding sites of interest in the STAT-3 protein. Although T. crispa has been reported as a traditional medicine against parasites, particularly Plasmodium (Vigneron et al. 2005;Malik 2015), investigations carried out to date have provided conflicting accounts on its antimalarial activity. The methanol stem extract (dose of 0.1-2.5 mg/mL) was evaluated for in vitro antiplasmodial activity against Plasmodium falciparum (FCR-3 strain). The highest dose of this extract showed 100% inhibition after 24 h of incubation. In vivo activity was further studied in adult female mice infected with Plasmodium berghei (chloroquine sensitive ANKA strain). At a dose of 5 mg/kg, the extract led to 0-32.7% parasitemia from days 1 to 5 post-infection, which was lower than the negative control. However, antiplasmodial activity was not considered to be significant (Rahman et al. 1999). Similarly, inconsequential results were obtained in another study testing the same extract against the same strain (Niljan et al. 2014). Tinospora crispa aqueous extract (1 mg/mL) yielded approximately 40% inhibition of P. falciparum and 80% inhibition of Babesia gibsoni in infected erythrocytes. In case of P. falciparum, the extract was considered to be inactive (Murnigsih et al. 2005). Similar inactivity against P. falciparum was also observed for the ethanol, ethyl acetate and n-hexane fractions of T. crispa stems (Ramadani et al. 2018). The methanol extract (0.5-3.0 mg/mL) showed IC 50 values between 0.27-0.29 mg/mL against P. falciparum 3D7 strain. Artemisin was used as a standard and showed an IC 50 of 10 -8 mg/mL. The 2 mg/mL dose was found to significantly lower the parasitic load, with the percentage parasitemia and parasite DNA concentration reduced by 47.12% and 56.83%, respectively. At doses above 2.0 mg/mL, these effects did not correlate with the dose administered. It was postulated that antioxidant activity was responsible for the observed effects (Ihwan et al. 2014). In a different study using the same model, the ethanol extract was found to be more potent (IC 50 of 0.344 ± 0.210 lg/mL). In the in vivo study using male Swiss mice infected with P. berghei NK65, the extract (doses of 50-400 mg/kg) had an ED 50 of 271.89 ± 4.32, and consequently the plant was deemed to possess moderate activity (Abdillah et al. 2015). In another in vitro assay, the methanol extract displayed an EC 50 value of 7.5 lg/ mL, indicating strong antimalarial activity (Tran et al. 2003). The ethanol extract when administered at doses of 20, 40 and 80 mg/kg to ICR mice infected with P. yoelii 17XL demonstrated dose-dependent activity, with 53.68% parasitemia on day 18 at the highest dose (Rungruang and Boonmars 2009). In another assay using ICR mice infected with P. berghei (ANKA strain), 13-hydroperoxyoctadeca-9,11-dienoic acid (159) was identified as a probable antimalarial compound (Lee et al. 2020). The aqueous extract of the plant also exerted hepatoprotection in ICR mice infected with P. berghei. The liver damage, indicated by increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, was inhibited by this extract at a dose of 500 mg/kg (Somsak et al. 2015). In the same model, the aqueous extract at doses of 500, 1000 and 2000 mg/kg displayed renoprotective and antihemolytic effects. At higher doses, the blood urea nitrogen (BUN) and creatinine levels decreased significantly compared to the negative control. For the highest dose, the hematocrit percentage increased significantly compared to the untreated group (Nutham et al. 2015).
Three combinations of artesunate (32 mg/kg) were prepared using three doses of the aqueous extract (2.5, 3 and 3.5 mg/kg) and administrated to C57BL/6 J mice infected with P. berghei. This caused a substantial inhibition of Nuclear Factor Kappa B (NFjB) and Intracellular Adhesion Molecule-1 (ICAM1) compared to the artesunate or extract only groups (Izzati et al. 2016). The aqueous extract of T. cripsa stems was also assessed against Brugia malayi, amongst other parasites, to evaluate its antifilarial potential. Following an incubation period of 24 h, the extract produced relative mobility values of 25, 7 and 0 at doses of 1, 5 and 10 mg/mL, respectively (Zaridah et al. 2001). Another study reported that an ointment prepared from an oil extract of the stem displayed significant activity against Pediculus humanus capitis compared to a shampoo used as a positive control and containing 1% permethrin (Torre et al. 2017). The ethanol extract of the stem (1.56-200 lg/mL) also proved to be active against Toxoplasma gondii (RH strain) compared to standards of veratrine and clindamycin used at the same concentrations. This extract did not display any cytotoxicity in an MTT assay against Vero cells (IC 50 value 179 lg/mL) compared to clindamycin (IC 50 of 116.5 lg/mL) and veratrine (IC 50 of 60.4 lg/mL). The antitoxoplasmic activity of the extract was established with an IC 50 of 6.31 lg/mL compared to that of clindamycin (8.33 lg/mL) and veratrine (14.25 lg/mL). The good selectivity index calculated for this extract (28.4) suggests it may represent a promising source of new antitoxoplasmic agents (Sharif et al. 2019).
Overall, T. crispa has demonstrated in vitro and in vivo activity against various parasites, but there have been contradictory reports regarding the potency of its extracts against Plasmodium species. Further pharmacological investigation and bio-assay guided isolation of active compounds are required in the future.

Antimicrobial activity
An in vitro disk diffusion assay was carried out to evaluate the antimicrobial activity of the aqueous, ethanol and chloroform extracts of T. crispa (25, 50, 75, and 100%) against various Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae, Corynebacterium diphtheriae, Bacillus cereus, Listeria monocytogenes) and Gram-negative (Escherichia coli, Salmonella typhi, Shigella flexneri, Klebsiella pneumoniae, Proteus vulgaris) bacteria using flemequine as a standard. All extracts dose-dependently inhibited S. pneumoniae, C. diphtheriae and S. flexneri compared to the standard. At concentrations above 50%, the aqueous and chloroform extracts inhibited S. aureus and E. coli. All extracts were ineffective against B. cereus and S. typhi (Zakaria et al. 2006). Additional testing of the aqueous extract on S. aureus and E. coli using an agar diffusion assay, led to a modest inhibitory effect with Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 227.27 mg/mL each (Zakaria et al. 2011). Another study showed that the aqueous, ethanol, methanol and chloroform extracts of the plant were active against S. pneumoniae, E. coli and Candida albicans compared to the standards tetracycline and fluconazole (Asif Iqbal et al. 2012). The ethanol extract at a dose of 1 mg/disk was also active against Methicillin Resistant S. aureus (MRSA) compared to the standard vancomycin in a disk diffusion assay (Al-alusi et al. 2010). Furthermore, the ethanol extract, when administered as ointment (9% v/v) with zeolite, showed bactericidal activity against S. aureus and Pseudomonas aeruginosa compared to a preparation containing gentamicin (Susanti et al. 2016). Another disk diffusion assay study confirmed the efficacy of the ethanol extract against E. coli (zone of inhibition of 20-22 and 22-30 mm at concentrations of 8% and 32%, respectively) compared to the standard amoxicillin (19 mm) (Muslimin et al. 2018). The aforementioned extract also showed strong antifungal activity against Trichophyton rubrum at concentrations C 40% (Erza et al. 2020). The n-hexane extract of the stem significantly inhibited the growth of S. aureus, Shigella boydii, S. dysenteriae, Vibrio mimicus, C. albicans and Aspergillus niger (Rahman et al. 2020). Two oxaporphine alkaloids isolated from the plant, namely lysicamine (50) and liriodenine (49), displayed activity on S. aureus and Enterococcus faecalis in a disk diffusion assay (Hamid et al. 2021). The plant ethanol extract, when employed as a 30% ointment, also revealed activity against Propionibacterium acnes (zone of inhibition of 9.13 mm), indicating its potential as an anti-acne treatment (Yusriani et al. 2018). One study tested the chloroform and petroleum ether fractions of the methanol extract of T. crispa using a disk diffusion assay against five Gram-positive bacteria (Bacillus subtilis, B. megaterium, B. cereus, S. aureus, Sarcina lutea), seven Gram-negative bacteria (E. coli, S. dysenteriae, S. typhi, S. paratyphi, S. boydii, V. mimicus, V. parahemolyticus) and three fungi (C. albicans, A. niger and Sacharomyces cerevisiae). The activity of the extract and fractions (400 lg/disc) was compared to that of the standard doxycycline (30 lg/ disc). Zones of inhibition, albeit negligible, were only observed for the chloroform fraction (Haque et al. 2011). The weak activity of the chloroform fraction was confirmed by another study testing the same fractions against the aforementioned microorganisms and P. aeruginosa, and using kanamycin (30 lg/disc) as a standard. This study reported no activity for the petroleum ether fraction (Islam et al. 2014). The antibacterial activity of the protein extract of T. crispa was evaluated against B. cereus, S. aureus, K. pneumoniae and Salmonella typhimurium. Only B. cereus was found to be sensitive to the extract (zone of inhibition of 9.7 ± 0.5 mm) (Zin et al. 2016).
The antiviral activity of T. crispa was evaluated for the ethanol and aqueous extracts (3-100 lg/mL) against HIV-1 integrase. Weak activity was obtained (IC 50 [ 100 lg/mL) (Bunluepuech and Tewtrakul 2009). Another study reported the use of a molecular docking approach to investigate the interactions of a variety of T. crispa constituents (putatively detected by GC-MS) with the SARS-CoV2 main protease. Imidazolidin-4-one and 2-imino-1-(4-methoxy-6dimethylamino-1,3,5-triazin-2-yl) (64) were found to bind with the active site of this enzyme in a similar manner to the standard nelfinavir (Rakib et al. 2020c).
Overall, T. crispa extracts have demonstrated in vitro activity against selected microorganisms, which should be further investigated particularly employing in vivo models of infection. Also noteworthy are bioassay-guided studies to identify the phytoconstituents responsible for such activity. Hamid et al. (2021) have reported that aporphine alkaloids had good activity against Gram-positive bacteria. A total of 13 alkaloids of this type have been isolated from T. crispa to date, warranting further testing and SAR studies. The molecular mechanisms underlying the antimicrobial activity of T. crispa extracts/constituents should also be elucidated. Considering the current global antimicrobial drug resistance issue, unravelling the specific microbial pathway(s) targeted and the chemical pharmacophores are particularly important as this may pave the way for future antibiotic design and development.

Immunomodulatory activity
The ability of T. crispa to modulate the innate and adaptive immune response has been demonstrated in several studies. The plant contains both anti-inflammatory and pro-inflammatory constituents. In the carrageenan-induced rat paw oedema model, the methanol extract of the stem at a dose of 10 mg/kg produced a 38% suppression of the oedema. The nbutanol fraction of the same extract was more effective than the diethyl ether and the aqueous fractions. When administered subcutaneously a dose of 3 mg/kg, the nbutanol fraction showed activity comparable to 250 mg/kg sulpyrine and 10 mg/kg diphenhydramine (Higashino et al. 1992). The anti-inflammatory     (Vikneswaran and Chan 2005) activity of the plant was also assessed using an antigen-induced rat basophilic leukemia (RBL)-2H3 cell line where release of b hexoaminidase was measured. The ethanol extract and aqueous extract of the stem (concentration range of 0-100 lg/mL) revealed dose-dependent inhibition up to 44% and 65%, respectively. However, their IC 50 values were higher ([ 100 lg/mL and 83 lg/mL, respectively) compared to the standard ketotifen fumerate (20.2 lg/ mL), suggesting weak activity. Interestingly, the ethanol extract of T. crispa combined with the ethanol extract of Piper nigrum (1:1, v/v) produced an IC 50 of 26.7 lg/mL (Kraithep et al. 2008). The methanol extract was evaluated for its ability to inhibit reactive oxygen species (ROS) in whole blood, polymorphonuclear (PMN) leukocytes and macrophages during phagocytosis using a luminol/lucigenin-based chemiluminescence assay. The extract produced significant suppression of ROS in the metabolic phase of phagocytosis (IC 50 of 0.6 ± 4.2 lg/mL compared to 3.0 ± 1.3 for the standard acetylsalicylic acid). It performed poorly in the other assays that were used in the study, including the PMN chemotaxis assay, compared to the standard ibuprofen (Jantan et al. 2011). Another study involving both the methanol and aqueous extracts of T. crispa stem was carried out on hydrogen peroxide-induced human umbilical vein endothelial (HUVEC) cells using a Tumor Necrosis Factor-a (TNF-a)-induced model of inflammation. The extracts inhibited Intracellular Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1) in a dose-dependent manner at concentrations ranging from 100-600 lg/mL. A significant and dose-dependent increase in Nitric Oxide (NO) production was observed in the presence of both extracts (Kamarazaman et al. 2012). In the carrageenan-induced paw oedema model, the aqueous extract of T. crispa (50, 100 and 150 mg/kg) showed inhibition comparable to ibuprofen (0.5%). In an in vitro membrane stabilization assay using hypotonic solution-induced lysis of human RBCs, the extract at a concentration of 2.5% was not active. At concentrations of 5 and 7.5%, however, it showed membrane stabilization comparable to ibuprofen (0.5%). The extract also dose-dependently inhibited the denaturation of protein in an albumin solution (Hipol et al. 2012). The ethanol extract (50, 100 and 200 mg/kg) was also tested on male Balb/C mice primed with sheep RBCs, using levimasole as a positive control. The results indicated that this extract increased peritoneal macrophage engulfment of E. coli, NO production, and lysozyme and myeloperoxidase serum levels. The extract at a dose of 200 mg/kg was equivalent to 2.5 mg/kg of levimasole. Upregulation of Immunoglobulin G (IgG) and Immunoglobulin M (IgM) also occurred, with the extract at the dose 100 mg/kg proving more potent than the standard. Dose-dependent delayed hypersensitivity was also observed in a footpad edema assay (Ahmad et al. 2016b).
A recent in silico study postulated that tyramine (67) may act as a COX-2 inhibitor and exert antiinflammatory activity (Widodo et al. 2021). While it is confounding that T. crispa phytoconstituents are able to both activate and suppress the immune system, it also opens up possibilities into designing new classes of immunomodulators. It is noticeable that the compounds of interest are not confined to a particular chemical class. This may also explain the marked diversity in the biochemical responses produced.

Antioxidant activity
The antioxidant activity of various extracts and fractions of the plant has been studied extensively. In this regard, the methanol extract was found to be more potent compared to the aqueous and chloroform extracts. In a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, the methanol extract had an IC 50 of 12 lg/mL which was comparable to the standard ascorbic acid. The resultant inhibition also approached 100%. Its total phenolic and flavonoid contents were found to be 255.33 ± 10.79 mg Gallic Acid Equivalent (GAE)/g sample and 9.53 ± 0.50 mg Quercetin Equivalent (QE)/g sample, respectively (Ibahim et al. 2011). Another study used a DPPH free radical scavenging assay on the ethanol extract, aqueous fraction and ethyl acetate fraction. The ethyl acetate fraction displayed the strongest activity (53.77% inhibition at 200 lg/mL) (Irianti et al. 2011). Several in vitro and in vivo studies were performed on the aqueous extract. The latter at a concentration of 10% produced DPPH inhibition, Thiobarbituric Acid (TBA) inhibition and displayed a Ferric Reducing Antioxidant Power (FRAP) value of 86.51 ± 0.07%, 39.2 ± 5.14% and 0.89 ± 0.07 mmol/L, respectively compared to the standards ascorbic acid (96.36 ± 0.90%, 73.2 ± 5.14% and 1.05 ± 0.00 mmol/L, respectively) and butylated hydroxytoluene (96.51 ± 0.95%, 75.8 ± 6.08% and 1.03 ± 0.03 mmol/L, respectively). An in vivo study was carried out on hypercholesterolemic rabbits using the aqueous extract at doses of 150, 300 and 450 mg/ kg. The extract reduced Total Cholesterol (TC), Triglyceride (TG) and Low-density Lipoprotein (LDL) while boosting High-density Lipoprotein (HDL) and restored malondialdehyde (MDA) levels to normal. Aortic atherosclerotic lesions were dosedependently lessened up to 100%. This suggests that the antioxidant potential of T. crispa is linked to its inhibition of atherosclerosis and plasma lipid peroxidation . The aqueous extract of T. crispa stem showed anti-atherosclerotic and antihypercholesterolemic activity in adult male New Zealand albino rabbits. The animals were first conditioned with a 0.5% high cholesterol diet, which caused an increase of C-Reactive Protein (CRP) levels. A dose-dependent reduction of CRP levels was observed following administration of the extract. At 200 mg/kg, the extract did not change the CRP levels. At 450 mg/ kg, it returned the CRP levels to normal levels while at 600 mg/kg it reduced the CRP levels to levels lower than normal. The extract also dose-dependently reduced atherosclerotic plaque coverage and foam cell formation to a considerable degree (Shah et al. 2021). Further investigations were carried out on the radical-scavenging activity of the methanol extract and its petroleum ether, chloroform, carbon tetrachloride and aqueous fractions, using a DPPH assay. The carbon tetrachloride fraction showed the strongest activity with an IC 50 value of 30 lg/mL compared to the standard ascorbic acid (15 lg/mL) and BHT (25 lg/mL) (Haque et al. 2011). In another study using a DPPH assay, the ethanol extract, its water fraction and selected subfractions, showed IC 50 values of 49.92 lg/mL, 38.25 lg/mL, 36.12 lg/mL, and 16.18 lg/mL, respectively. It was postulated that acid hydrolysis of the subfractions improved their antioxidant potential . Several other studies measuring the total phenolic content, total flavonoid content, DPPH free radical scavenging activity and Ferric Reducing Antioxidant Power of T. crispa all confirned the antioxidant potential of the plant (Zulkefli et al. 2013;Abood et al. 2014;Nguyen et al. 2020;Mahalle and Gupta 2021). In a metal chelating assay, the petroleum ether, chloroform, methanol and water extracts of the stem were first mixed and dried together. The mixed extract (1 mg/ mL) produced 81.97% inhibition of Ferrozine-Fe 2? complex formation compared to ethylenediaminetetraacetic acid (EDTA) at the same concentration (98.51% inhibition) (Zulkefli et al. 2013). In an MTT cell viability assay, pre-treatment with the aqueous extract (50-1000 lg/mL) and the methanol extract (600 lg/mL) of T. crispa boosted viability to 69% and up to 76%, respectively. When assessed for antioxidant activity in hydrogen peroxide-induced HUVEC cells, antioxidant enzymes including Catalase (CAT), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx) were increased by the aqueous extract in a dose-dependent manner. The methanol extract on the other hand showed maximum CAT and SOD activity at 400 lg/mL and potentiated GPx activity dosedependently. MDA levels were inhibited up to 58% and 60% for the aqueous and methanol extracts, respectively (Kamarazaman et al. 2012). A study using hyperlipidemic rabbits further confirmed the effect of the aqueous extract (administered at doses of 200, 450 and 600 mg/kg) on the cholesterol profile and the amelioration of atherosclerotic plaques compared to the standard simvastatin. Whilst SOD and GPx activity were also potentiated, the Total Antioxidant Status (TAS) did not improve substantially in the presence of T. crispa aqueous extract (Zamree et al. 2015). Three isolated constituents, N-trans-feruloyltyramine/moupinamide (62), N-cis-feruloyltyramine (63) and secoisolariciresinol (135) displayed stronger antioxidant activity than the standard BHT in a DPPH free radical scavenging assay (Cavin et al. 1998). Other compounds such as protoberberine alkaloids isolated from the plant, namely columbamine (54), dihydrodiscretamine (53) and 4,13-dihydroxy-2,8,9trimethoxydibenzo[a,g]quinolizinium (55) showed IC 50 [ 500-800 lg/mL in a DPPH free radical scavenging assay (Hamid et al. 2021).
Whilst the antioxidant potential of T. crispa has been established in multiple in vitro studies, further in vivo studies are warranted, particularly focussing on how T. crispa extracts/constituents may interfere with antioxidant enzymes (Fig. 11). The numerous flavonoids present in the plant may contribute to the modulation of these enzymes, but this has yet to be assessed. Alkaloids, of the protoberberine class and others present in the plant should also be evaluated for their antioxidant potential so as to gain valuable insights into structure-activity relationships.

Hepatoprotective activity
The hepatoprotective potential of T. crispa has been demonstrated in many studies (Lee et al. 2017;Rakib et al. 2020a). The methanol extract of T. crispa was found to increase the activity of phase-1 metabolic enzymes in male Sprague Dawley rat hepatocytes. The extract produced a substantial increase in aminopyrine N-demethylase activity at a dose of 0.001-1.0 mg/mL. At lower (but not higher) doses, this effect was mediated by the cAMP pathway (Tin et al. 2005). In an in vitro study, the same extract (0.5 mg/mL) produced 61.3% inhibition of the CYP3A4 enzyme compared to the standard troleandomycin (62.1%) in a timedependent manner (Usia et al. 2006) (Fig. 11). The activity of this extract on CYP3A4 and CYP2D6 yielded IC 50 values of 428 and 488 lg/mL, respectively (Subehan et al. 2006). The ethanol extract also acted against tert-butyl hydroperoxide-induced hepatotoxicity in HEP-G2 cells (EC 50 of 144.3 lg/mL). The underlying mechanism was established to be via the induction of Nrf2-mediated expression of HO-1 (Lee et al. 2017). Another study demonstrated that carbon tetrachloride-induced Swiss albino mice pretreated with the methanol extract (doses of 100-400 mg/kg body weight) resulted in noteworthy hepatoprotection. Levels of ALT, AST, Alkaline Phosphatase (AP), Malondialdehyde (MDA) and total bilirubin were reduced comparably to the standard silymarin (Rakib et al. 2020a). The enzyme modulatory and hepatoprotective activity of T. crispa warrants further investigations. In particular, bio-assay guided isolation studies should be performed to assess the activity of phytochemicals.

Analgesic activity
Although used traditionally for pain management, the analgesic activity of the plant is not well studied. An extract of T. crispa stems was reported to demonstrate central analgesic activity in a tail flick response to radiant heat (Almeida et al. 2001). The ethanol extract Fig. 11 Schematic diagram of the modulation of miscellaneous enzymes by Tinospora crispa. AE Aqueous Extract, EE Ethanol Extract, ME Methanol Extract, CAT Catalase, SOD Superoxide dismutase, GPx Glutathione Peroxidase, COX-2 Cyclooxygenase-2, XO Xanthine Oxidase (300 mg/kg) showed dose-dependent peripheral analgesia with 92% inhibition in the acetic acid-induced writhing test in mice, compared to the standard acetyl salicylic acid (81% inhibition at 100 mg/kg) (Sulaiman et al. 2008). In the same assay, the methanol extract, its petroleum ether and chloroform fractions (400 mg/kg) yielded 48.06, 51.94 and 43.41%, respectively, compared to 65.12% inhibition for the diclofenac sodium standard (100 mg/kg). The activity of the petroleum ether fraction was considered statistically significant (p \ 0.05) compared to the standard (Islam et al. 2014). The methanol extract and the chloroform fraction (at doses 200 and 400 mg/kg) also displayed significant antinociceptive activity in the acetic acidinduced writhing and formalin-induced paw-licking tests, compared to the standard diclofenac (Rakib et al. 2020b). Having said that, the analgesic potential of the plant still requires further exploration. Future work should focus on investigations that aim to identify the phytoconstituents responsible for such activity. Studies on the molecular mode of action of the analgesic constituents must also be undertaken.

Antipyretic activity
The n-butanol fraction of T. crispa stems (3 mg/kg) suppressed LPS-induced fever in rats when administered intravenously. The activity was equivalent to that of 100 mg/kg sulpyrine and 1 mg/kg morphine hydrochloride administered intraperitoneally (Higashino et al. 1992). In DPT (Diphtheria-Pertussis-Tetanus) vaccine-induced male Wistar rats, a 40% ethanol extract of the plant produced significant antipyretic effect at 90-and 120-min post-treatment (Wulandari and Bestari 2016). Significant antipyretic activity was also observed for a methanol extract and its petroleum ether and n-hexane subfractions administered at a dose of 400 mg/kg to Swiss albino mice with Brewer's Yeast-induced fever. The activity was found to be dose-dependent (Rakib et al. 2020a). These studies provide some evidence to support the ethnomedicinal use of T. crispa for the treatment of pyrexia. The specific molecular mode of action of such effects, however, remains to be elucidated.

Anticholinesterase activity
It is interesting to note that quaternary alkaloids are prevalent in T. crispa, indicating its probable acetylcholinesterase (AChE) inhibitory potential. One study assessed the potential of such alkaloids using a modified Ellman's colorimetric method with physostigmine as the standard. Among the seven alkaloids studied, the least polar one-columbamine (54)-displayed significant inhibitory activity with an IC 50 of 48.1 ± 1.3 lM compared to physostigmine (31.4 ± 0.5 lM). Dihydrodiscretamine (53) and Nformylanonaine (39) only showed moderate activity (Fig. 11). A preliminary SAR study was also performed on these alkaloids (Yusoff et al. 2014). QSAR studies employing the crystallized protein structure of AChE should be performed to gather information on the probable interactions of this target with bioactive ligands.

Central nervous system (CNS) activity
The activity of T. crispa on the CNS has not been studied extensively. A decoction of the plant was evaluated in a motor activity test, curiosity test, hanging test and rotary road test at various concentrations (6.5, 13 and 26%). It was found that the lowest concentration produced CNS-stimulant effects similar to the positive control caffeine (Merwanta et al. 2019). The methanol extract, its chloroform and n-hexane fractions at doses of 200 and 400 mg/kg were evaluated in the open field, hole board and elevated plus maze tests. A significant decrease in locomotion was observed in the open field test comparable to the standard diazepam (1 mg/kg). In the hole board test, the chloroform fraction at the highest dose yielded significant results, which indicated a reduced fearfulness. Additionally, the methanol extract (at the highest dose) and the chloroform extract (at the lowest dose) displayed anxiolytic activity in the elevated plus maze test comparable to the standard diazepam (1 mg/kg) (Rakib et al. 2020b). Additional investigations on the CNS activity of T. crispa are warranted, particularly focusing on the identification of the phytochemical(s) responsible for such activity.

Antihyperuricemic activity
The n-hexane insoluble fraction of the ethanol extract of T. crispa stem was evaluated in male BALB/C mice for its potential xanthine oxidase (XO) inhibitory activity. The extract reduced the levels of uric acid ranging from 49 to 78% at doses of 50-200 mg/kg. Peak activity was observed at the 100 mg/kg dose compared to the standard allopurinol (10 mg/kg)   (Fig. 11). These results contradict a previous study carried out using the root of the plant, which showed an IC 50 of 370.35 lg/mL compared to the standard allopurinol (0.022 lg/mL) (Vikneswaran and Chan 2005). This may suggest that the presence of phytoconstituents with prospective XO inhibitory activity is localized in certain parts of the plant. However, it is premature to drawing any conclusion on this aspect without supplementary evidence. Further identification of the phytoconstituents involved in the modulation of this enzyme are warranted.
Another study revealed that the aqueous extract (3.125, 6.25, 12.5, and 25 mg/L) of the stem produced LC 50 values of 16.95 and 30.12 mg/L, respectively (Jiraungkoorskul 2019). Additionally, the chloroform, n-hexane, methanol, and aqueous extracts of the stem displayed time-and concentration-dependent molluscicidal activity on Pomacea canaliculata. The nhexane, followed by the aqueous extract, were the least cytotoxic of all extracts tested. The chloroform and methanol extracts were more prominently molluscicidal than other extracts, with the methanol extract outperforming the rest (Aziz et al. 2021).
These studies suggest the usefulness of T. crispa as a biopesticide. Bio-assay guided isolation and analysis of active compounds should be carried out in the future in order to discover new natural chemical entities that could replace the harmful commercial pesticides currently used.

Clinical trials
The clinical trials conducted thus far with T. crispa have focused entirely on the assessment of its antidiabetic properties (Table 4). One placebo-controlled, double-blind, randomized trial was conducted on 20 type-2 diabetic patients who were non-responsive to oral antidiabetic drugs and did not receive insulin. Following administration of T. crispa (1 g dry powder thrice a day for 6 months), no significant differences were observed between the T. crispa-treated group and the control group in terms of fasting blood sugar, insulin, and glycosylated hemoglobin levels. Unexpectedly, the T. crispa-treated group displayed higher cholesterol and glycosylated hemoglobin concentrations. Interestingly, an average of 2 kg of body weight loss was observed among the treated patients (Sangsuwan et al. 2004). One placebo-controlled, doubleblind, randomized, crossover study conducted on 36 patients with metabolic syndrome revealed that treatment with T. crispa (250 mg capsules daily for two months) significantly lowered fasting blood sugar and triglyceride levels, but induced hepatotoxicity with ALT and AST levels noticeably increased in about 16.7% of the patients (Sriyapai et al. 2009). Another trial, conducted in Thailand, showed that T. crispa administered as a single dose (6 g) to non-diabetic healthy volunteers neither induced acute changes in glucose metabolism nor significantly improve glucose tolerance in 9 subjects. A similar single 6 g dose administered to 6 different healthy volunteers led to a significant decrease in blood glucose levels, but no changes in insulin levels. To check the biochemical and hematological effects of the plant, 12 subjects were treated with T. crispa 1 g thrice daily for 8 weeks while 13 others received 1.05 g doses in a similar fashion. Serum glucose and other hematological parameters were unchanged, except for AST and ALT levels which were noticeably increased, indicating hepatotoxicity (Rattanajarasroj et al. 2004). A more recent study to observe the effects of T. crispa ingestion employed 10 healthy and 10 diabetic subjects. The subjects received 75 g of glucose with or without 250 mg of T. crispa supplements after overnight fasting, and serum samples were collected every 30-60 min for 3 h. No significant changes in glucose or insulin levels were observed between the control and test groups (Klangjareonchai and Roongpisuthipong 2012).
The clinical trials carried out so far are preliminary with small sample size and non-systematic. To evaluate the plant as a safe and effective antidiabetic agent for human use, a thorough, serious and more operationally randomized controlled trial have to be performed.

Safety and toxicological profile
Many studies have indicated that T. crispa extracts are relatively safe for oral ingestion. However, some The extract caused significant increases in the serum levels of ALT, AST, AP, bilirubin, and G-glutamyl transferase, and histological features of hepatocytic degeneration were also observed (Kadir et al. 2011). Similar elevation of AST and ALT were also reported in two Thai clinical studies involving T. crispa (Sriyapai et al. 2009;Rattanajarasroj et al. 2004). Two cases of toxic hepatitis following the use of T. crispa have been reported to date. The first one was a 49-year-old male who had been using a T. crispa-containing herbal medication (Langrand et al. 2014). The second was a 57-year-old man who ingested the aqueous extract of the plant (Cachet et al. 2018). Clerodane-type furanoditerpenoids and borapetosides have been suggested as the constituents responsible for the observed in vivo toxicity of T. crispa. However, one study using a LPS-induced ND-4 mice model reported that borapetosides B (17), C (14), and F (25) did not produce hepatotoxicity, when administered both individually and in combination at a dose of 500 mg/kg for 21 days (Parveen et al. 2020). Tinospora crispa ethanol extract, and the n-hexane and chloroform fractions from its methanol extract, have been found to be quite safe in murine models. No harmful effects were observed neither following the administration of the ethanol extract (50-200 mg/kg) to male Balb/C mice, nor following the administration of fractions from the methanol extract (various doses with the maximal dose of 2000 mg/kg) on Swiss albino mice (Ahmad et al. 2016b;Rakib et al. 2020a). A dermal irritation test employing adult albino rabbits showed that a T. crispa-based ointment was nonirritant when administered topically (Torre et al. 2017).
Potential drug-drug interactions have been suggested between T. crispa and other co-administered drugs through its capacity of modulating the Pregnane X-receptor (PXR). In an in vitro luciferase reporter gene assay, the methanol extract of the plant and its chloroform and n-hexane fractions significantly activated PXR. Its ethyl acetate and the butanol fractions showed negligible activity. Several T. crispa constituents shared this activity, and the in vitro results were further reflected in silico (Parveen et al. 2022).
Thus, any drug being metabolized or activated through PXR might experience altered pharmacokinetics when co-administered with T. crispa. Given the potential of T. crispa as a source for novel therapeutic lead compounds, further comprehensive studies should be conducted to establish its absolute safety.

Conclusion and future prospects
Multiple in vitro and in vivo studies on T. crispa have demonstrated its remarkable medicinal potential, particularly in the treatment of diabetes and hypertension, providing support to justify some of its ethnobotanical uses. Several clerodane-type furanoditerpenoids in T. crispa have been reported to possess significant antidiabetic activity, which is worthy of further exploration for the discovery of novel antidiabetic drugs. In addition, the adrenergic activity of its alkaloids may provide new avenues for the treatment of high blood pressure. While the cytotoxic prospects of this plant have been studied at length, the specific molecular mechanisms involved in this effect have yet to be elucidated. Clerodane-type furanoditerpenoids have revealed noticeable results as chemopreventive agents, which is also worthy of further investigations. The widespread use of T. crispa as an antimalarial agent has been supported by many studies, but with conflicting reports on its efficacy. It is interesting to note, however, that various extracts provide protection against malaria, which may help to offset the detrimental effects of this disease. These observations suggest the need for further bioassay-guided isolation in order to identify the antiplasmodial phytoconstituent(s) of T. crispa. Likewise, the immunomodulatory activity of various T. crispa extracts and phytoconstituents demands scrutiny. One of the most notable aspects in this regard is the ability of T. crispa phytoconstituents to both activate and suppress the immune system. Alkaloids from T. crispa have been found to be particularly potent and deserve closer inspection. Among the other pharmacological effects displayed by the plant, the inhibition of acetylcholinesterase by quaternary alkaloids looks promising and should be investigated further. Although the hepatotoxicity of T. crispa has been attributed to the presence of selected clerodane-type furanoditerpenoids, there are other compounds within that class that are not hepatotoxic. Therefore, in-depth investigations into this class of phytochemicals are essential in order to evaluate their relative safety and toxicity. The characteristic phytoconstituents of T. crispa may overall play an important part in the discovery of new drug leads. In that respect, additional in vivo pharmacological and toxicological studies on T. crispa are warranted to provide assurance of adequate efficacy and safety.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data availability Not applicable.
Code availability Not applicable.

Declarations
Conflict of interest The authors have no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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