A Review of Cytotoxic Plants of the Indian Subcontinent and a Broad-Spectrum Analysis of Their Bioactive Compounds.

Cancer or uncontrolled cell proliferation is a major health issue worldwide and is the second leading cause of deaths globally. The high mortality rate and toxicity associated with cancer chemotherapy or radiation therapy have encouraged the investigation of complementary and alternative treatment methods, such as plant-based drugs. Moreover, over 60% of the anti-cancer drugs are molecules derived from plants or their synthetic derivatives. Therefore, in the present review, an attempt has been made to summarize the cytotoxic plants available in the Indian subcontinent along with a description of their bio-active components. The review covers 99 plants of 57 families as well as over 110 isolated bioactive cytotoxic compounds, amongst which at least 20 are new compounds. Among the reported phytoconstituents, artemisinin, lupeol, curcumin, and quercetin are under clinical trials, while brazilin, catechin, ursolic acid, β-sitosterol, and myricetin are under pharmacokinetic development. However, for the remaining compounds, there is little or no information available. Therefore, further investigations are warranted on these subcontinent medicinal plants as an important source of novel cytotoxic agents.


Introduction
Cancer is a severe metabolic disorder and the leading cause of death worldwide [1]. It involves unrestrained proliferation of normal cells, caused by genetic alterations and instabilities, resulting in the generation of malignant cells and initiation of metastasis or tissue invasion. The genetic alterations include mutation of tumor suppressor genes (NF1, NF2, p53 etc.), DNA repair genes (tool box for DNA and p21, p22, p27, p51, p53), oncogenes (MYC, RAS, Bcl-2, RAF), and genes involved in cell growth and metabolism [2]. These mutations are caused by internal factors, such as alteration in hormonal balance and immune system as well as by external factors, such as radiation and pesticide exposure, tobacco smoking, and ingestion of carcinogenic chemicals or metals [1]. The incidence and geographic distribution of cancer are also related to parameters such as gender, age, race, genetic predisposition, and exposure to environmental carcinogens including azo dyes, aflatoxins, petrol, and mutagenic agents [3].
Since time immemorial, drugs of natural origin have formed the basis of traditional medicine in different cultures and in present times, medicinal plants occupy the most important position as

Acanthaceae
Barleria grandiflora, the only plant belonging to the Acanthaceae family with reported cytotoxic potential, demonstrated in vitro and in vivo anti-tumor activity against Dalton's Lymphoma Ascites (DLA), A-549, and Vero cell lines. Efficacy as an anticancer agent was proved through the prolongation of animal lifespan. An upsurge in ascitic fluid, the major nutritional source of tumor, was witnessed in DLA bearing mice. Treatment with B. grandiflora leaves extract in these mice led to the reduction in ascetic fluid as well as cessation of tumor growth, thereby increasing animal lifespan [17,18]. While the most significant adverse effects associated with cancer chemotherapy include anemia and myelosuppression, mice treated with B. grandiflora exhibited significant improvement in the red blood cell count and hemoglobin content, and a reduction in white blood cell count, thus demonstrating its antitumor potential [17,19].

Actinidiaceae
Saurauja roxburghii, belonging to the Actinidiaceae family, was assessed for cytotoxic activity. Ursolic acid and corosolic acid (Figure 1), obtained from the plant, were tested for cytotoxicity against A431 human epidermoid carcinoma and C6 rat glioma cell lines. Corosolic acid was effective against both cancer cells, while ursolic acid was cytotoxic against C6 glioma cells [20,21].

Amaryllidaceae
Narcissus tazetta was evaluated for cytotoxic potential against MCF-7 (human breast adenocarcinoma) and Hep-2 (human epithelial type 2) cells, and the flower and aerial part extracts led to reduction of cell viability by 40% and 20%, respectively [22].
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Amaryllidaceae
Narcissus tazetta was evaluated for cytotoxic potential against MCF-7 (human breast adenocarcinoma) and Hep-2 (human epithelial type 2) cells, and the flower and aerial part extracts led to reduction of cell viability by 40% and 20%, respectively [22].
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Aristolochiaceae
In the Aristolochiaceae family, Aristolochia ringens and Aristolochia longa root extracts exhibited cytotoxic effect against multiple cancer cell lines. A. ringens was reported to be effective against HeLa, A-431, A-549, PC-3 (prostate cancer), HCT-116, and THP-1 (human acute monocytic leukemia) cells, with IC50 values ranging between 3-24 µ g/mL, while A. longa was found to be active against MCF-7, HT-29, H5-6, and N2A cells, with 22-83% inhibition of tumor cell growth compared to control. Moreover, while testing against S 180 ascites and S 180 solid tumor model, A. ringens demonstrated significant tumor growth inhibition in a dose-dependent manner. Furthermore, A. ringens also demonstrated a rise in mean survival time (MST) in in vivo models of L1210 lymphoid leukemia [33,34].

Asphodelaceae
Whole plant of Aloe vera of Asphodelaceae was examined against HepG2 cell lines and showed time and dose-dependent inhibition of cancer cells (IC50 value: 10.5 µ g/mL). Cytotoxicity was demonstrated by apoptosis of HepG2 cells through increased expression p53 and decreased expression of Bcl-2 genes [35].

Aristolochiaceae
In the Aristolochiaceae family, Aristolochia ringens and Aristolochia longa root extracts exhibited cytotoxic effect against multiple cancer cell lines. A. ringens was reported to be effective against HeLa, A-431, A-549, PC-3 (prostate cancer), HCT-116, and THP-1 (human acute monocytic leukemia) cells, with IC 50 values ranging between 3-24 µg/mL, while A. longa was found to be active against MCF-7, HT-29, H5-6, and N2A cells, with 22-83% inhibition of tumor cell growth compared to control. Moreover, while testing against S 180 ascites and S 180 solid tumor model, A. ringens demonstrated significant tumor growth inhibition in a dose-dependent manner. Furthermore, A. ringens also demonstrated a rise in mean survival time (MST) in in vivo models of L1210 lymphoid leukemia [33,34].

Asphodelaceae
Whole plant of Aloe vera of Asphodelaceae was examined against HepG2 cell lines and showed time and dose-dependent inhibition of cancer cells (IC 50 value: 10.5 µg/mL). Cytotoxicity was demonstrated by apoptosis of HepG2 cells through increased expression p53 and decreased expression of Bcl-2 genes [35].

Asteraceae
Artemisinin (Figure 4), the designated phytoconstituent of Artemisia annua, was reported to be active against two human osteosarcoma cell lines, 148B and MG63 (IC 50 values were 167 µM and 178 µM, respectively) [37]. Dihydroartemisinin (Figure 4), a derivative of the compound was evaluated against canine OSA (Osteosarcoma) cell lines, D-17, OSCA2, OSCA16, and OSCA50 and the respective IC 50 values were 8.7, 43.6, 16.8 and 14.8 µM [38]. Apart from this, other bioactive compounds of A. annua were also tested by evaluating the cytotoxicity of its hydro-alcoholic, dichloromethane, and methanol extracts against D-17, HeLa, and TC221 cell lines, and a dose-dependent anticancer activity was observed [39,40]. For Bidens pilosa, different fractions of whole plant were evaluated, using the MTT and Comet assays, against HeLa and KB cell lines. The chloroform fraction was found to be most effective against KB cells, while the water fraction was most active against HeLa cells, with CC 50 values (concentration of the sample tolerated by 50% of the cultures exposed) of 88.6 and 372.4 µg/mL, respectively [41]. Aerial parts of both Centaurea antiochia and Centaurea nerimaniae were evaluated against Vero and HeLa cell lines and showed moderate cytotoxicity against Centaurea antiochia and significant cytotoxicity against Centaurea nerimaniae [23,42].
active against two human osteosarcoma cell lines, 148B and MG63 (IC50 values were 167 μM and 178 μM, respectively) [37]. Dihydroartemisinin (Figure 4), a derivative of the compound was evaluated against canine OSA (Osteosarcoma) cell lines, D-17, OSCA2, OSCA16, and OSCA50 and the respective IC50 values were 8.7, 43.6, 16.8 and 14.8 μM [38]. Apart from this, other bioactive compounds of A. annua were also tested by evaluating the cytotoxicity of its hydro-alcoholic, dichloromethane, and methanol extracts against D-17, HeLa, and TC221 cell lines, and a dosedependent anticancer activity was observed [39,40]. For Bidens pilosa, different fractions of whole plant were evaluated, using the MTT and Comet assays, against HeLa and KB cell lines. The chloroform fraction was found to be most effective against KB cells, while the water fraction was most active against HeLa cells, with CC50 values (concentration of the sample tolerated by 50% of the cultures exposed) of 88.6 and 372.4 µ g/mL, respectively [41]. Aerial parts of both Centaurea antiochia and Centaurea nerimaniae were evaluated against Vero and HeLa cell lines and showed moderate cytotoxicity against Centaurea antiochia and significant cytotoxicity against Centaurea nerimaniae [23,42].

Berberidaceae
Berberis aristata stem extract was found to be cytotoxic against MCF-7 human breast cancer cell (IC 50 of 220 µg/mL) [47]. The roots of the plant was found to be cytotoxic against DWD (oral), Hop62 (lungs), and A2780 (ovary) cancer cell lines, with the lowest IC 50 value of 71 µg/mL observed against DWD [48].

Bignoniaceae
Tecoma stans was tested against A549 (human lung adenocarcinoma) cell line and cell viability was reduced in a dose-dependent manner, indicating a moderate cytotoxic effect [49]. Another study showed that the plant extract was cytotoxic against HepG2 cells where a reduction in viability of cancer cells was observed [50]. The major anti-proliferative phytoconstituents of the plant included rutin, luteolin, diosmetin, and skytanthine [51].

Boraginaceae
Cordia dichotoma, belonging to the Boraginaceae family, was tested against prostate carcinoma cell line (PC3). In addition, some bioactive flavonoids were isolated. The cytotoxic activity of the plant extract (IC 50 value of 74.5 µg/mL) was exerted through apoptosis, nuclear condensation, and ROS (reactive oxygen species) production [52].

Berberidaceae
Berberis aristata stem extract was found to be cytotoxic against MCF-7 human breast cancer cell (IC50 of 220 μg/mL) [47]. The roots of the plant was found to be cytotoxic against DWD (oral), Hop62 (lungs), and A2780 (ovary) cancer cell lines, with the lowest IC50 value of 71 μg/mL observed against DWD [48].

Bignoniaceae
Tecoma stans was tested against A549 (human lung adenocarcinoma) cell line and cell viability was reduced in a dose-dependent manner, indicating a moderate cytotoxic effect [49]. Another study showed that the plant extract was cytotoxic against HepG2 cells where a reduction in viability of cancer cells was observed [50]. The major anti-proliferative phytoconstituents of the plant included rutin, luteolin, diosmetin, and skytanthine [51].

Boraginaceae
Cordia dichotoma, belonging to the Boraginaceae family, was tested against prostate carcinoma cell line (PC3). In addition, some bioactive flavonoids were isolated. The cytotoxic activity of the plant extract (IC50 value of 74.5 μg/mL) was exerted through apoptosis, nuclear condensation, and ROS (reactive oxygen species) production [52].

Caesalpiniaceae
Heartwood and leaf extracts of Caesalpinia sappan were examined for cytotoxic potential against MCF-7 and A-549 cell lines. Brazilin A (Figure 6) was also isolated and evaluated against MCF-7. Significant cytotoxic property was observed for both crude extracts and bioactive compound. Further molecular docking proved the effectiveness of Brazilin A in the reduction of Bcl-2 apoptotic inhibitor [54]. Saraca asoca (Caesalpiniaceae family) was reported cytotoxic against AGS cell lines (IC 50 value: 20 µg/mL). Further evaluation of the bioactive compounds was recommended [24].
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Caesalpiniaceae
Heartwood and leaf extracts of Caesalpinia sappan were examined for cytotoxic potential against MCF-7 and A-549 cell lines. Brazilin A (Figure 6) was also isolated and evaluated against MCF-7. Significant cytotoxic property was observed for both crude extracts and bioactive compound. Further molecular docking proved the effectiveness of Brazilin A in the reduction of Bcl-2 apoptotic inhibitor [54]. Saraca asoca (Caesalpiniaceae family) was reported cytotoxic against AGS cell lines (IC50 value: 20 μg/mL). Further evaluation of the bioactive compounds was recommended [24].

Dilleniaceae
The stem and bark extracts of Dillenia pentagyna (Dillenaceae family) were found to have cytotoxic activity against DL, MCF-7, and HeLa cell lines (IC50 values of 25.8, 41.6 and 76.8 µ g/mL, respectively). Reduction in glutathione (GSH) level was also observed in D. pentagyna treated animal. GSH is related with onset of tumor cell proliferation through regulation of PKC (protein kinase C). Thus, D. pentagyna was reported to be beneficial in cancer treatment through depletion of GSH [46,56]. In addition, Dillenia indica leaves have also been reported to be cytotoxic against MCF-7 and MDA-MB-231 cell lines (IC50 of 340 and 540 μg/mL, respectively) [24].

Dilleniaceae
The stem and bark extracts of Dillenia pentagyna (Dillenaceae family) were found to have cytotoxic activity against DL, MCF-7, and HeLa cell lines (IC 50 values of 25.8, 41.6 and 76.8 µg/mL, respectively). Reduction in glutathione (GSH) level was also observed in D. pentagyna treated animal. GSH is related with onset of tumor cell proliferation through regulation of PKC (protein kinase C). Thus, D. pentagyna was reported to be beneficial in cancer treatment through depletion of GSH [46,56]. In addition, Dillenia indica leaves have also been reported to be cytotoxic against MCF-7 and MDA-MB-231 cell lines (IC 50 of 340 and 540 µg/mL, respectively) [24].

Euphorbiaceae
Croton caudatus was evaluated against DL, MCF-7, and HeLa cell lines. Potent cytotoxic effect against DL cell line was observed (IC 50 value: 29.7 µg/mL) [46]. The cytotoxic effect of Euphorbia tirucalli was evaluated against pancreatic cancer cell line (MiaPaCa-2) and a dose-dependent cytotoxic effect was observed with a reduction in quantity of viable cells (7% for 200 µg/mL) [58].

Fabaceae
Adenanthera pavonina of family Fabaceae was evaluated in vivo for its cytotoxic potential in DLA induced ascetic mice model. Significant reduction in volume of tumor and number of viable tumor cells were observed in mice treated with the plant extract [18]. For Ononis sicula and Ononis hirta,

Hypericaceae
Hypericum kotschyanum aerial part was reported to have moderate cytotoxic effects against HeLa and Vero cell lines (IC 50 values were 507 and 367 µg/mL, respectively) [23].

Lamiaceae
Lavandula angustifolia, belonging to the Lamiaceae family, was found to have negligible or no activity against MCF-7 and Hep-2 cell lines [22]. On the other hand, Plectranthus stocksii stem and leaf extract was significantly cytotoxic against RAW264.7, Caco-2, and MCF-7 cell lines. The least IC 50 value for RAW264.7 was 9 mg/mL (stem ethyl acetate fraction), for Caco-2 it was 36.1 mg/mL (leaves ethyl acetate fraction), and for MCF-7, it was 48.9 mg/mL (leaf ethyl acetate fraction) [61]. For Salvia hypargeia, cytotoxic activity was examined against Vero and HeLa cell lines and the IC 50 values were over 1000 µg/mL against both cell lines [23]. In contrast, Salvia officinalis was found to be significantly cytotoxic against MCF-7, B16F10, and HeLa cells (IC 50 = 14-36 µg/mL). Two bioactive compound i.e., α-humulene and trans-caryophyllene ( Figure 7) were also isolated and tested against MCF-7 cell line and the IC 50 values were found to be 81 and 114 µg/mL, respectively [62]. the aerial parts of Teucrium sandrasicum were examined for cytotoxic effects against HeLa and Vero cell lines and the reported IC 50 values were 513 and 593 µg/mL, respectively [23].

Malvaceae
Three species of Hibiscus (Malvaceae family) i.e., Hibiscus micranthus, Hibiscus calyphyllus, and Hibiscus deflersii were evaluated for their cytotoxic effect against HepG2 and MCF-7 cell lines, using the MTT assay. The petroleum ether fraction of H. deflersii extract was found to be most potent amongst the three species against HepG2 and MCF-7 cell lines (IC50 values were 14.4 and 11.1 μg/mL, respectively). Three cytotoxic biomarkers i.e., ursolic acid, β-sitosterol, and lupeol ( Figure 8) were quantified through HPLC analysis and highest concentration of these biomarkers were obtained from the petroleum ether fraction of H. deflersii extract.
Again, the petroleum ether fraction of H. calyphyllus extract (IC50 values were 14.5 and 25.1 μg/mL against HepG2 and MCF-7, respectively) and the chloroform fraction of H. micranthus extract (IC50 values were 27.6 and 24.1 μg/mL against HepG2 and MCF-7, respectively) had the most potent cytotoxic effect [67]. Both extracts were then analyzed with HPLC for the quantification of the aforementioned biomarkers. Among the three biomarkers, the apoptotic effect of ursolic acid was reported to be mediated by cytochrome c-dependent caspase-3 activation, inhibition of DNA replication through topoisomerase I cleavage, and increase in the expression of p21 WAF1 cell-cycle regulator [68]. β-Sitosterol induced apoptosis via caspase-3, caspase-9 activation and poly (ADP-ribose)-polymerase cleavage. In addition, reduction in anti-apoptotic Bcl-2 protein expression and increase in pro-apoptotic Bax protein expression were reported [69]. The cytotoxic effects of lupeol were attributed to the inhibition of topoisomerase II, DNA polymerase, angiogenesis, and induction of apoptosis through caspases activation, poly (ADP-ribose)-polymerase cleavage, and decreased Bcl-2 expression [70]. Nepeta italica, belonging to Lamiaceae family, was found to have moderate cytotoxicity against HeLa and Vero cell lines (IC 50 values of 980 and >1000 µg/mL, respectively) [23]. In a DMBA (7,12-dimethylbenz[a]anthracene)-induced hamster buccal pouch carcinogenesis model, Ocimum sanctum demonstrated inhibition of tumor development and early events of carcinogenesis [63]. In S 180 induced mice model, an increase in survival rate was observed without any impact on tumor volume. These effects were attributed to its indirect or direct impact on the immune system through modulation or regulation of humoral immunity and stimulation of cell-mediated immunity, thereby resulting in inhibition of neoplasm [64][65][66]. Origanum sipyleum was found to have minimal cytotoxic effect against both HeLa and Vero cell lines, with IC 50 values over 1000 µg/mL. [23]. Teucrium polium (Laminaceae family) was examined against MCF-7, Hep-2, T47D, CACO-2, HRT18, A375.S2, and WM1361A cell lines. The extract was found to be most effective against MCF-7, Hep-2, and A375.S2 cell lines (percent of remaining viable cells were 78, 58, and 61%, respectively) [22,43].

Malvaceae
Three species of Hibiscus (Malvaceae family) i.e., Hibiscus micranthus, Hibiscus calyphyllus, and Hibiscus deflersii were evaluated for their cytotoxic effect against HepG2 and MCF-7 cell lines, using the MTT assay. The petroleum ether fraction of H. deflersii extract was found to be most potent amongst the three species against HepG2 and MCF-7 cell lines (IC 50 values were 14.4 and 11.1 µg/mL, respectively). Three cytotoxic biomarkers i.e., ursolic acid, β-sitosterol, and lupeol ( Figure 8) were quantified through HPLC analysis and highest concentration of these biomarkers were obtained from the petroleum ether fraction of H. deflersii extract.
Again, the petroleum ether fraction of H. calyphyllus extract (IC 50 values were 14.5 and 25.1 µg/mL against HepG2 and MCF-7, respectively) and the chloroform fraction of H. micranthus extract (IC 50 values were 27.6 and 24.1 µg/mL against HepG2 and MCF-7, respectively) had the most potent cytotoxic effect [67]. Both extracts were then analyzed with HPLC for the quantification of the aforementioned biomarkers. Among the three biomarkers, the apoptotic effect of ursolic acid was reported to be mediated by cytochrome c-dependent caspase-3 activation, inhibition of DNA replication through topoisomerase I cleavage, and increase in the expression of p21 WAF1 cell-cycle regulator [68]. β-Sitosterol induced apoptosis via caspase-3, caspase-9 activation and poly (ADP-ribose)-polymerase cleavage. In addition, reduction in anti-apoptotic Bcl-2 protein expression and increase in pro-apoptotic Bax protein expression were reported [69]. The cytotoxic effects of lupeol were attributed to the inhibition of topoisomerase II, DNA polymerase, angiogenesis, and induction of apoptosis through caspases activation, poly (ADP-ribose)-polymerase cleavage, and decreased Bcl-2 expression [70]. Molecules 2020, 25, x FOR PEER REVIEW

Menispermaceae
The aerial parts of Cocculus hirsutus were evaluated for cytotoxic effects against MCF-7 cell line using in vitro MTT assay. The methanol extract of the plant demonstrated cytotoxic potential (IC50 value: 39.1 μg/mL). Multiple bioactive anticancer compounds were isolated from this plant, including coclaurine, haiderine, and lirioresinol, which exerted anti-tumor effects by interacting with cell-cycle regulatory proteins, such as Aurora kinase, c-Kit, FGF, Nuclear Factor-Kappa B (NF-kB), Bcl-xL, and VEGF [78,79].

Menispermaceae
The aerial parts of Cocculus hirsutus were evaluated for cytotoxic effects against MCF-7 cell line using in vitro MTT assay. The methanol extract of the plant demonstrated cytotoxic potential (IC 50 value: 39.1 µg/mL). Multiple bioactive anticancer compounds were isolated from this plant, including coclaurine, haiderine, and lirioresinol, which exerted anti-tumor effects by interacting with cell-cycle regulatory proteins, such as Aurora kinase, c-Kit, FGF, Nuclear Factor-Kappa B (NF-kB), Bcl-xL, and VEGF [78,79].

Myristicaceae
Myristica fragrans was found to have cytotoxic activity against KB cell lines (IC50 value: 75 μg/mL). Cytotoxicity was induced by apoptosis of cancer cells through interaction with Bcl-2 protein [10]. Other studies demonstrated 2 new phenolic and 38 essential oils from M. fragrans which were tested against K-562, HCT-116, and MCF-7 cells, and the IC50 values were found to be within the range 2.11-78.15 μg/mL [90,91].

Myristicaceae
Myristica fragrans was found to have cytotoxic activity against KB cell lines (IC 50 value: 75 µg/mL). Cytotoxicity was induced by apoptosis of cancer cells through interaction with Bcl-2 protein [10]. Other studies demonstrated 2 new phenolic and 38 essential oils from M. fragrans which were tested against K-562, HCT-116, and MCF-7 cells, and the IC 50 values were found to be within the range 2.11-78.15 µg/mL [90,91].

Nyctaginaceae
The aerial parts of Mirabilis jalapa were tested for cytotoxic activity against MCF-7 and Hep-2 cell lines and the percentage of remaining viable cells after treatment were 60 and 78, respectively [22]. Studies have reported the presence of bioactive rotenoids and Mirabilis antiviral protein (MAP) in M. jalapa, which were found to be cytotoxic against HeLa, Raji, A549, HCT 116, and Vero cell lines. The IC 50 values of MAP against HCT116, MCF-7, and A549 cell lines were 150, 175, and 200 µg/mL, respectively [95,96].
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Nyctaginaceae
The aerial parts of Mirabilis jalapa were tested for cytotoxic activity against MCF-7 and Hep-2 cell lines and the percentage of remaining viable cells after treatment were 60 and 78, respectively [22]. Studies have reported the presence of bioactive rotenoids and Mirabilis antiviral protein (MAP) in M. jalapa, which were found to be cytotoxic against HeLa, Raji, A549, HCT 116, and Vero cell lines. The IC50 values of MAP against HCT116, MCF-7, and A549 cell lines were 150, 175, and 200 µ g/mL, respectively [95,96].

Phyllanthacae
Phyllanthus emblica, belonging to the Phyllanthacae family, was found to be cytotoxic against HT-29 colon cancer cell lines (IC50 value: ~35 µ g/mL) [100]. Amongst the bioactive compounds, a new apigenin glucoside and 14 sterols have been isolated (including two new compounds) and screened for cytotoxic activity against HL-60 and SMMC-7721. Among these compounds, trihydroxysitosterol ( Figure 11

Pinaceae
The cytotoxic activity of Cedrus deodara, belonging to the Pinaceae family, was tested against multiple cancer cell lines. It was found to be cytotoxic against Mia-Pa-Ca-2, PC3, and A-2780 (ovary) cancer cells (IC 50 of 74, 77, and 63 µg/mL, respectively) [48]. In addition, the plant extract was found to have cytotoxic potential against Molt-4 cancer cells (IC 50 value: 15 µg/mL). Cytotoxicity was induced by apoptosis of cancer cells through interaction with caspase 3, 8, and 9 proteins [106].

Polygonaceae
Calligonum comosum, belonging to the Polygonaceae family, was found to be cytotoxic against HepG2 cancer cell lines (IC 50 value: 9.60 µg/mL). The major cytotoxic phytoconstituents of the plant were catechin and its derivatives as well as kaempferol and its derivatives, including mequilianin. Cytotoxicity was induced by apoptosis through increased expression of p53 and reduced expression of Bcl-2 gene [35,111].

Rubiaceae
The bark and wood of Hymenodictyon excelsum, belonging to the Rubiaceae family, were screened for cytotoxicity against Vero, NIH3T3, AGS HT-29, MCF-7, and MDA-MB-231 cell lines, and significant cytotoxic effects of the bark were observed against all cell types (IC 50 values were 230, 70, 90, 160, 80, and 440 µg/mL, respectively) [24]. The cytotoxicity was induced by DNA fragmentation and apoptosis of cancer cells [117]. The leaves of Oldenlandia corymbosa were reported to be cytotoxic against K562 cells (IC 50 of 114.4 µg/mL) and the toxicity was induced by apoptosis [118].

Salicaceae
The flowers of Populus alba were reported to be moderately cytotoxic against MCF-7 and Hep-2 cells. The percentage of residual cell viability was 100% for both cell lines at 100 µg/mL dose [22]. Another study revealed the cytotoxicity of the essential oils from the plant against A549, H1299 (human non-small cell lung cancer), and MCF-7 cancer cells (IC 50 values were 12.05, 10.53, and 28.16 µg/mL, respectively) [119].

Saururaceae
Saururus chinensis roots were found to be cytotoxic against MCF-7 breast cancer cell lines in the MTT assay. The lowest IC 50 value calculated was 91.2 µg/mL (water fraction). The major bioactive constituents included aristolactram, dihydroguaiatric acid, and sauchinone [121]. Saucerneol D, manassantin A and B, and saucerneol F were some of the lignans isolated from S. chinensis, which had cytotoxic effects against HT-29 and HepG2 cells (IC 50 : 10-16 µg/mL). The cytotoxicity was due to the inhibition of DNA topoisomerase I and II [122].

Scrophulariaceae
The flowers and aerial parts of Verbascum sinaiticum were reported to have moderate cytotoxicity against MCF-7 and Hep-2 cancer cells (cell viability of 60 and 80%, respectively [22]. Another study reported cytotoxic effects of two flavonolignans, novel sinaiticin and hydrocarpin, obtained from V. sinaticum. They were tested against P-388 cells and found cytotoxic with ED 50 of 1.2 and 7.7 µg/mL for hydrocarpin and sinaiticin, respectively [123].

Sterculiaceae
Helicteres isora whole plant was screened for cytotoxicity against HeLa-B75, HL-60, HEP-3B, and PN-15 cells. Moderate effectiveness was observed against all cell types. The major cytotoxic components of this plant were cucurbitacin B and isocucurbitacin B [127,128].

Thymelaeaceae
Aquilaria malaccensis, belonging to the Thymelaeaceae family, was found to be cytotoxic against DLA and EAC cancer cells (IC 50 values were 72 and 79 µg/mL, respectively). However, the cytotoxicity against normal cells was negligible [129]. Another study evaluated the cytotoxicity of the oil fraction of the plant extract against HCT116 colon cancer cells and reported an IC 50 value 4 µg/mL [130]. The major phytoconstituents of the plant were benzaldehyde, pinene, octanol, germacrene, and hexadecanal [131].

Verbenaceae
Clerodendrum viscosum leaf extract was screened against VERO, NIH3T3, AGS, HT-29, MCF-7, and MDA-MB-231 cancer cell lines and was found to have cytotoxic effects against MCF-7 and HT-29 cells (IC 50 of 50 and 880 µg/mL, respectively) [24]. The roots of C. infortunatum, belonging to the same family, exhibited in vivo cytotoxicity in DLA-induced ascetic mice model and the induction of apoptosis was observed through interaction with Bax, Bcl-2, caspases 8, and 10 proteins [132].

Zingiberaceae
Rhizomes of Curcuma longa, belonging to the Zingiberaceae family, were found to have 97% cytotoxicity against Hep-2 cell line, at a dose of 1000 µg/mL [136]. The major phytoconstituent of this plant is curcumin (Figure 12), which has been reported to have anticancer activity in previous studies [137,138]. Amongst other phytoconstituent in C. longa, β-sesquiphellandrene ( Figure 13) has also been reported to possess anticancer potential [139]. The cytotoxic effect of curcumin was through the induction of apoptosis [140].
Rhizomes of Curcuma longa, belonging to the Zingiberaceae family, were found to have 97% cytotoxicity against Hep-2 cell line, at a dose of 1000 μg/mL [136]. The major phytoconstituent of this plant is curcumin (Figure 12), which has been reported to have anticancer activity in previous studies [137,138]. Amongst other phytoconstituent in C. longa, β-sesquiphellandrene ( Figure 13) has also been reported to possess anticancer potential [139]. The cytotoxic effect of curcumin was through the induction of apoptosis [140]. The major constituents of Zingiber officinale, i.e. gingerol ( Figure 13) and its derivatives were screened against A549, SK-OV-3, SK-MEL-2, and HCT15 and were found to be cytotoxic (IC50 < 50 μM). Many essential oils of Z. officinale have also been reported to have cytotoxic effects [141,142]. The combination effect of C. longa and Z. officinale were observed to have synergistic cytotoxic effect against PC-3M cancer cells [143].

Reported Cytotoxic Constituents: Therapeutic Perspective and Future Directions
The aforementioned cytotoxic phytoconstituents, isolated from different plants of Indian subcontinent, were further examined for their significance in cancer chemotherapy. The DTP database of the National Cancer Institute (NCI), USA, was utilized to assess the therapeutic effects of these bioactive constituents [144].
Ursolic acid, a pentacyclic triterpenoid, was found to have significant anticancer effect against MCF-7 and MDA-MB-231 breast cancer cell lines. The reported mechanisms of cytotoxicity included reduction in cyclin D1, STAT3 (signal transducer and activator of transcription), CDK4 (cyclin dependent kinase), Bcl-2, AKT, and MMP-2 (matrix metallopeptidases) proteins as well as activation of Bax, caspase 3, caspase 8, caspase 9, PARP (poly(ADP-ribose)polymerase), p53, and p21 proteins ( Figure 13, 14) [145]. DTP database revealed several in vivo animal model studies against L1210, P388, and B16 cancer cells. Although the compound showed low toxicity in in vivo studies, the major impediment in the therapeutic development of ursolic acid was its poor bioavailability and short plasma-half life. Currently, attempts are being made to improve its pharmacokinetic parameters by utilizing nano-particle-based drug delivery techniques [146].
Artemisinin, a well-known anti-malarial drug and its derivative dihydroartemisinin, were found to have anticancer activity in numerous in vitro and in vivo studies. The cytotoxic effects were attributed to DNA damage by base excision or homologous recombination, cell death by apoptosis, autophagy, and necrosis; inhibition of angiogenesis via AMPK (AMP activated protein kinase) pathway; reduction in CyclinD1, CyclinE, and Bcl-2; and activation of Bax, Caspase 3, Caspase 8, The major constituents of Zingiber officinale, i.e., gingerol ( Figure 13) and its derivatives were screened against A549, SK-OV-3, SK-MEL-2, and HCT15 and were found to be cytotoxic (IC 50 < 50 µM). Many essential oils of Z. officinale have also been reported to have cytotoxic effects [141,142]. The combination effect of C. longa and Z. officinale were observed to have synergistic cytotoxic effect against PC-3M cancer cells [143].

Reported Cytotoxic Constituents: Therapeutic Perspective and Future Directions
The aforementioned cytotoxic phytoconstituents, isolated from different plants of Indian subcontinent, were further examined for their significance in cancer chemotherapy. The DTP database of the National Cancer Institute (NCI), USA, was utilized to assess the therapeutic effects of these bioactive constituents [144].
Ursolic acid, a pentacyclic triterpenoid, was found to have significant anticancer effect against MCF-7 and MDA-MB-231 breast cancer cell lines. The reported mechanisms of cytotoxicity included reduction in cyclin D1, STAT3 (signal transducer and activator of transcription), CDK4 (cyclin dependent kinase), Bcl-2, AKT, and MMP-2 (matrix metallopeptidases) proteins as well as activation of Bax, caspase 3, caspase 8, caspase 9, PARP (poly(ADP-ribose)polymerase), p53, and p21 proteins (Figures 13 and 14) [145]. DTP database revealed several in vivo animal model studies against L1210, P388, and B16 cancer cells. Although the compound showed low toxicity in in vivo studies, the major impediment in the therapeutic development of ursolic acid was its poor bioavailability and short plasma-half life. Currently, attempts are being made to improve its pharmacokinetic parameters by utilizing nano-particle-based drug delivery techniques [146].
Caspase 9, PARP, p53, and p21 (Figure 13, 14). Currently, clinical trials are being conducted to establish artemisinin as a potential anticancer agent [147]. Brazilin was reported to have cytotoxic effects against TCA8113, MG-63, and T24 cancer cells. The reported mechanisms of cytotoxicity included interaction with c-Fos, inhibition of Bcl-2, p62, and p-mTOR as well as enhancement of Bax, caspase-3, LC3B, and p-AMPK (Figure 13, 14) [148,149]. Future studies should focus on the pharmacokinetic evaluation of this compound. β-Sitosterol was found to be have anti-cancer effects against breast, prostate, lung, colon, stomach, and ovarian cancers as well as leukemia. The established mechanisms of cytotoxicity were through interaction with cell signaling pathway, apoptosis, invasion, angiogenesis, metastasis, and proliferation. Although the compound was reported to be nontoxic, it was found to be less potent. The use of cell-specific or liposome-based drug delivery is proposed for the successful application of this compound as an anticancer agent [150].  Artemisinin, a well-known anti-malarial drug and its derivative dihydroartemisinin, were found to have anticancer activity in numerous in vitro and in vivo studies. The cytotoxic effects were attributed to DNA damage by base excision or homologous recombination, cell death by apoptosis, autophagy, and necrosis; inhibition of angiogenesis via AMPK (AMP activated protein kinase) pathway; reduction in CyclinD1, CyclinE, and Bcl-2; and activation of Bax, Caspase 3, Caspase 8, Caspase 9, PARP, p53, and p21 (Figures 13 and 14). Currently, clinical trials are being conducted to establish artemisinin as a potential anticancer agent [147].
Brazilin was reported to have cytotoxic effects against TCA8113, MG-63, and T24 cancer cells. The reported mechanisms of cytotoxicity included interaction with c-Fos, inhibition of Bcl-2, p62, and p-mTOR as well as enhancement of Bax, caspase-3, LC3B, and p-AMPK (Figures 13 and 14) [148,149]. Future studies should focus on the pharmacokinetic evaluation of this compound.
β-Sitosterol was found to be have anti-cancer effects against breast, prostate, lung, colon, stomach, and ovarian cancers as well as leukemia. The established mechanisms of cytotoxicity were through interaction with cell signaling pathway, apoptosis, invasion, angiogenesis, metastasis, and proliferation. Although the compound was reported to be nontoxic, it was found to be less potent. The use of cell-specific or liposome-based drug delivery is proposed for the successful application of this compound as an anticancer agent [150].
Lupeol was reported to be cytotoxic against HeLa, CWR22Rt1, A549, 451Lu, SMMC7721, and WM35 cancer cells by downregulation of cyclin D1, CDK2, and Bcl-2 and upregulation of Bax, caspase-3, and p38 (Figures 13 and 14). The compound is currently under investigation in various clinical trials, and if successful, it might be used as a novel adjuvant therapeutic agent for treating multiple cancers in human [151].
Curcumin is a prominent natural phytoconstituent used in the treatment of several types of cancers, including prostate, pancreatic, colorectal, breast, and lung cancers as well as multiple myeloma and leukemia. Reported molecular targets for curcumin include Bcl-2, Bax, p53, caspases, p38, NF-kB, PARP, and cyclin proteins (Figures 13 and 14). The limited bioavailability of curcumin, owing to poor absorption and rapid metabolism, has been improved by synthesizing structural analogues and development of liposomal, nanoparticle, and phospholipid complex-based drug delivery systems. Several clinical trials have been conducted to evaluate the efficacy of curcumin. Further studies are required to enhance the bioavailability of this compound [152].
A large number of studies have reported the cytotoxicity of catechins, such as epicatechin, epigallecatechin-3-gallate etc. Possible mechanisms of cytotoxicity include interaction with Bcl-2, STAT, FYN, p53, CDKs, and vascular endothelial growth factor (VEGF). Several in vivo studies have also demonstrated the effectiveness of catechins in animal models. Additional pharmacokinetic and pharmacodynamic studies in humans are required [153].
Myricetin is a natural flavonoid that has extensively been reported for its antitumor and cytotoxic potential against gastric, esophageal, ovarian, colon, and cervical cancers and multiple leukemia, melanoma, and sarcoma. The compound was tested against many cell lines, including HGC-27, MCF-7, HCT-15, OVCAR-3, HepG2, A549, HeLa, and PC3. Myricetin was found to interact with multiple proteins, such as Bcl-2, Bax, Caspases, Bcl-xl, p53, p21, NF-kB, and cyclin, IL (Figures 13 and 14) [154]. Despite being a potential cytotoxic agent, poor aqueous solubility and poor absorption of this compound has led to its reduced bioavailability. Formulation into nano-suspensions or micro-emulsions was proposed to enhance its absorption. More studies are required in this regard [155].
Quercetin, another naturally occurring polyphenolic flavonoid, has already been reported to be effective against breast, colon, pancreatic, lung, liver, prostate, bladder, gastric, bone, blood, brain, cervical, skin, eye, ovarian, thyroid, and kidney cancers. The cytotoxicity of the compound has been attributed to its interaction with p51, p21, caspases, TNF, IL, Bcl-2, Bax, p53, myeloid cell leukemia (MCL), and cyclin proteins (Figures 13 and 14) [156]. Despite of its low toxicity, multiple clinical trials have been conducted with quercetin. However, extensive studies are required before the development of an established anticancer quercetin formulation [157].
Among the other reported phytoconstituents, caryophyllene, campesterol, rutin, gallic acid, and caffeic acid have been assessed in vivo by DTP of NCI, USA in mice models of P388, L1210, Friend virus leukemia, and Lewis lung carcinoma. However, data regarding the remaining bioactive cytotoxic phytoconstituents reported in this review article, is very limited. Hence, in future, further in vivo as well as mechanistic studies are warranted to assess the activity of these cytotoxic compounds.
Moreover, out of almost 50,000 plant species found in the Indian subcontinent, only a handful of them have been properly examined for their cytotoxic potentials [158]. The diverse environmental conditions such as dry forests, mangrove forests, deserts, aquatic reservoirs, and hill tracts have made the Indian subcontinent a natural treasure. However, the studies conducted so far have just explored the tip of the iceberg. Therefore, the Indian subcontinent still remains a vast unexplored region with great number of exclusive medicinal plants. Further studies should investigate these endemic plants with respect to their cytotoxic potential, mechanism of action, and isolation of active constituents.  Acknowledgments: The authors wish to acknowledge the support of Osaka University, Osaka, Japan and Jashore University of Science and Technology, Jashore, Bangladesh for providing software access and other technological assistances.

Conflicts of Interest:
The authors declare no conflict of interest.