*4.2. Tc-Based Extracts*

The various parts of *Tc* were subjected to different types of extracts for isolating the bioactive phytocomponents, including the alcoholic extract and aqueous extract. For alcoholic extracts, the parts were dried, ground in an electrical grinder and dissolved in either ethanol or methanol. Soxhlet apparatus was used for the extracts. For aqueous extracts, either distilled water or double-distilled water was used. Phytocomponents isolation was also accomplished using solvents like petroleum ether, chloroform, ethyl acetate, and acetone with alcohol [8,10]. Some authors preferred using both aqueous as well as alcoholic extracts [7,10]. Hexane extracts utilized solvents including hexane, benzene, chloroform and ethyl acetate [24] Singh B et al. used phenol to extract the dried leaves and stems of *Tc* [18]. Overall, most of the included studies preferred alcoholic extracts.

### *4.3. Dose E*ff*ect of Phytocomponents*

According to the US National Cancer Institute, an IC50 value (drug concentration required for 50% inhibition in vitro) of less than 100 μg/mL from a medicinal plant is sufficient to be considered as an anticancer agen<sup>t</sup> [29]. Components of such plants are isolated and characterized to delineate their bioactive molecules. The methanolic extracts of *Tc* have shown to exhibit an IC50 value of less than 100 μg/mL [29]. Sharma N used 1.5 kg of dried, crushed *Tc* soaked in 4.5 liters and found an IC50 value of less than 100 μg/mL [6]. Priya M S et al. used varying dosage (200, 400 and 600 μg/mL) of *Tc*, revealing dose-dependent inhibition [7]. Bala M et al. used 2 kg of dried stem in 80% ethanol extract. They extracted three phytocomponents and elicited an IC50 value of less than 100 μg/ml [9]. Maliakkal et al. used 2 kg of the dried crushed stem through alcohol extract and observed a dose which depended on cytotoxicity, with an ideal IC50 value of less than 100 μg/mL. They also showed that combining different phytocomponents resulted in a profound anti-carcinogenic effect [11]. Ansari et al. used 10 kg of 50% methanolic extract of *Tc*. The extract showed the anti-carcinogenic effect, while its rutin concentration was found to be higher than quercetin [15]. Ali H et al. found the phytocomponent palmatine showed anticancer effect against environmentally induced carcinogenesis. Jagetia et al. observed that combining the various alkaloid with berberine increased the antineoplastic effect. Thus, in addition to the dose-dependent effect, the overall anti-carcinogenic effect also depended on the type, number and dosage of the used phytocomponents [7,14,15,22].

#### *4.4. Phytocomponents and Its Mechanism of Action against Cancer Cells*

The active phytocomponents of *Tc* include alkaloids, glycosides, steroids, aliphatic compounds, essential oils, a mixture of fatty acid, calcium, phosphorous, protein and polysaccharides [4]. The various phytocomponents identified from the studies analyzed in the systematic review included berberine, new clerodane furanodiptherineglycosidae, elligic acid, kaempferol, N-formylannonain, magnoflorine, jatrorrhizine palmatine, 11-hydroxymustakone, cordifolioside A, tinocordiside, yangambin, anthraquinones, terpenoids, saponins and phenol, pyrrole-based small molecules, quercetin and rutin, arabinogalectian, palmatine, clerodane-derived diterpenoids and hexane fractions. These

phytocomponents induced anticancer effect via mitochondrial-mediated apoptosis, cytotoxic activity, mutagenic activity, reduction in tumor size, triggering reactive oxygen species, decreased gene expression of the cell cycle, effectively inhibiting cancer proliferation [5–23]. The mechanism of action of *Tc* depends on the phytocomponents used. The new clerodane furanodiptherineglycosidae exhibits anticancer activity through induction of mitochondrial-mediated apoptosis by triggering reactive oxygen species and autophagy [6]. Phenolic compounds have genoprotective and antioxidant effects on cancer cells [8]. *Tc* ethanolic extracts induced apoptosis via. increased sub G0 phase without altering cell cycle [11]. Arabinogalactans present in aqueous extracts of *Tc* shown to produce immunological activity and cytotoxic activity. Phenols have shown antimutagenic and anti-malignant effects. Flavanoids have a chemopreventive role in cancer. Pyrrole-based molecules induced apoptosis and cytotoxic effects [13]. Palmatine showed enhanced antioxidant activity by the increase in the level of antioxidant enzymes and also showed inhibition of lipid peroxidation showing role in detoxification pathway [20]. Berberine has shown to inhibit tumor cell growth by a reduction in the secretion of growth factors [5]. Hexane fractions have induced apoptosis via caspase 3-activated DNase [24]. Leyo et al. reported the polysaccharides in *Tc* to show antineoplastic effect by reducing the protein levels [25]. Epoxy-clerodane-diterpene blocks the carcinogen metabolic activation and enhances carcinogen detoxification. Singh N et al. reported *Tc* extracts have shown anticancer effect by direct tumoricidal actions [28]. Thus, *Tc* extracts have shown anti-carcinogenic properties through several mechanisms including induction of DNA damage, apoptosis, inhibiting topoisomerase II, clonogenicity, antioxidant activity, glutathione S transferase activity and increasing lipid peroxidase activity.

### *4.5. Anti-Carcinogenic E*ff*ect of Tc Phytocomponents*

In vitro studies: In the included studies, the *Tc*-extracted phytocompounds were used in combination with conventional chemotherapeutics including: fluorouracil, cisplatin, paclitaxel, suramin, doxorubicin, mitomycin, adriamycin, and methotrexate. The *Tc* extract berberine (an alkaloid) has shown to inhibit cell cycle, differentiation, and epithelial–mesenchymal transition on HEP2 human laryngeal cancer cell lines [5]. New clerodane-furano-diterpene-glycoside obtained as an aqueous-alcoholic extract through bioassay-guided fractionation exhibited significant cytotoxic effect and induced apoptosis in human lung carcinoma (A549), prostate (PC-3), SF-269(CNS), melanoma (MDA-MB-435), colon cancer (HCT-116) and breast cancer (MCF-7) cell lines. It was observed the induction of apoptosis was Reactive oxygen species-mediated through mitochondria by activation of the caspase pathway [6]. Singh B et al. identified phenolic compounds from a fungal extract of endophytic fungus *Cladosporium velox* TN-9S isolated from the stem of *Tc*. Total phenol content was 730 μg gallic equivalents/mL as determined by Folin Ciocalteu reagent. The IC50 value was less than 100 μg/mL. These phenolic compounds have shown to exhibit a mild genoprotective potential against DNA damage on Chinese hamster ovary cell lines after the treatment with non-ionic surfactant nonylphenol. It was also noted that the endophyte's capability to synthesize phytocomponent was similar to the host plant. Their non-mutagenic and non-cytotoxic nature was suggested to enhance the antioxidant and genoprotective potential [8]. Bala et al. identified the phytocompounds from *Tc* extracts such as N-formylannonain, magnoflorine, jatrorrhizine palmatine, 11-hydroxymustakone, cordifolioside A, tinocordiside and yangambin through spectroscopic analysis. These phytocompounds were shown to exhibit anti-cancer properties on several human cancer cell lines including KB (human oral squamous carcinoma), CHOK-1 (hamster ovary), HT-29 (human colon cancer) and SiHa (human cervical cancer). Bala et al. compared the anticancer activity for different fractions of the *Tc* extract. It was noted that combining the phytocompounds increased the anti-carcinogenic properties through a synergistic effect [9]. The ethanol phytofraction obtained from plant samples of *Tc* by Mishra R et al. were cytotoxic to IMR- 32 human neuroblastoma cancer cell lines. Analysis of the cellular and nuclear morphology through immunostaining revealed Tc induced apoptosis, increased expression of senescence markers. Anti-metastatic activity in the form of a reduced cell migration capacity was also observed. Protein assays result expressed the arrest of cells in the Go/G1 phase. Mishra R et al. extracted

phytocomponents from Tc plant including anthraquinones, terpenoids, saponins and phenol. This component e ffectively inhibited the growth of prostate, ovary and breast cancer cell lines. *Tc* extracts were also shown to exhibit antiproliferative, apoptotic-inducing, anti-migratory and antimetastatic potential on glioma cells. [12]. Butanoic fractions (pyrrole-based small molecules) were shown to induce apoptosis on breast cancer cells. Rashmi KC et al. determined apoptotic induction by evaluation by various apoptotic markers, ROS generation, caspase activity, and cell cycle analyzing. They found phytocomponents of *Tc* extract having anticancer activity and also observed inhibition of tumor proliferation. Despite promising results, the major limitation of most of the abovementioned studies is that several key aspects, including the complete mechanism of action, signaling, and pharmacological actions were nor clearly delineated [13]. Quercetin and rutin belong to phenolic phytocomponents extracted from Tc showed antiproliferative activity and was confirmed on human breast cancer MDA-MB-231 cells through induction of apoptosis, expression of altered genes and checking for the levels of intracellular ROS. The pharmacokinetics profiles, pharmacodynamic profiles and preclinical evaluation are some examples of the ongoing research by the authors [15]. Table 1 provides a summary of the overall e ffects elicited by *Tc* against the various cell lines.

In vivo studies: Animals that are used for the study in this review include male or female Swiss albino mice injected with Ehrlich ascites cells; male BALB/c mice with benzopyrene-induced pulmonary tumor; DABA-induced mammary carcinogenesis female Sprague Dawley rats; freshwater air-breathing fish *C. punctatus*; Swiss albino with DABA-induced carcinoma, C57BL/6 mice injected by B16F-10 melanoma cell lines; and male Wistar albino-strain rats with hepatocellular carcinoma. Arabinogalactan, a polysaccharide, was shown to inhibit cancer in male BALB/c mice. The arabinogalactan and the stem extract of *Tc* were shown to have a higher anticancer e ffect than only *Tc* [18]. Mishra A et al. conducted a scientific evaluation of phenolic components such as ellagic acid and kaempferol obtained from *Tc* extracts. These components were shown to have a genoprotective e ffect on fresh-air-breathing fish as elicited by the observations made on the morphology of the nucleus. Injecting the extract of fungus of *Tc* plant and nonylphenol caused a drastic reduction in the nuclear abnormalities. [19] Hall et al. extracted alkaloid phytocomponent palmative from *Tc* and studied anticancer property against DMBA induced skin cancer. Palmative caused a gradual decrease in the bodyweight of tumor size. Palmatine phytocomponent was shown to enhance the antioxidant enzyme levels and also inhibit carcinogenesis when administrated orally. [20] Another alkaloid phytocomponent, berberine, showed tumor remission on Swiss albino mice. The study was conducted by Jagetia G C et al. The anticancer e ffect was dose dependent. The exact mechanism was unknown, and authors concluded that the combinational e ffect of the alkaloids caused a higher anticancer e ffect [21]. Phytocomponents including triterpenoids, alkaloids, pyrrole-based small molecules, hexane fraction and clerodane-derived diterpenoids were also shown to exhibit significant anti-carcinogenic e ffect in di fferent cancer induced in animals by showing reduction in solid tumor growth [22–24,26–28]. Leyon P V and Kuttan G extracted polysaccharide from *Tc* to observe the metastatic e ffect on C55BL/6 mice and the highly metastatic melanoma cell line B16F-10. Significant inhibition was noted, and although the exact mechanism of action was not known, an antimitogenic e ffect through natural killer cell-mediated immune modulation was suggested as a possible pathway [25]. Table 2 provides a summary of the overall e ffects elicited by *Tc* against the various in vivo animal models.
