Recent Advancement in Anticancer Activity of Clinacanthus nutans (Burm. f.) Lindau

Clinacanthus nutans is a traditional medicinal herb that is applied for the therapy of snake bites, skin infection, herpes infection, burns, scalds, dysentery, and diabetes. Clinacanthus nutans is also used to treat several cancers, including breast, cervical, colon, gastric, head and neck, liver, lung, pancreatic, and skin cancers, as well as lymphoma and leukemia; however, the underlying mechanisms of its anticancer activity remained undetermined. We searched PubMed and Google with key words “Clinacanthus nutans and cancer” and collected recent papers of Clinacanthus nutans with anticancer activity. We focused on the preparation, effects, and action mechanisms of Clinacanthus nutans extracts on various types of cancers. We hope that this mini review can help update our knowledge about active components, effects, and molecular mechanisms of extracts from this promising herb Clinacanthus nutans for ongoing studies and speed up its clinical application in the future.


Background
Clinacanthus nutans (Burm. f.) Lindau (C. nutans for abbreviation thereafter) is a perennial herb that belongs to the Acanthaceae family, which consists of many medicinal species of plants with high value [1]. For detailed description of morphology and characteristics of this plant, please refer to previous reviews [1][2][3]. C. nutans is a vegetable and traditional herb in Southern Asian countries such as ailand, Indonesia, and Malaysia [4]. is plant is usually used for the treatment of snake bites, skin infection, burns, scalds, hurts, dysentery, diabetes, virus infection, and even cancers [5][6][7].
A flurry of research indicated that C. nutans possesses various biological activities including antimicrobial, antiviral, antioxidant, antidiabetic, anti-inflammatory, immunomodulatory, antihyperlipidemic, and anticancer activities [2]. C. nutans now becomes a multifunctional plant that draws attention of scientists in various research areas. A bunch of reviews on the detailed phytochemistry and pharmacology of C. nutans have been provided [1,4,8,9]. Here we intend to focus on recent progress of the anticancer activity of C. nutans. We searched PubMed for most related literature by the key words "Clinacanthus nutans and cancer" from the year 2015 to the year 2020, and 27 articles were selected, including 5 reviews.
ose articles that are not related to anticancer activity were sacrificed due to the scope of this mini review. Some dated studies were also included in order to have a more complete story of C. nutans on various cancers. We also performed Google search and a thesis and a conference paper were also retrieved, giving that they have contribution on the studies of anticancer activity of C. nutans.
Cancer patients that were subjected to oral administration of C. nutans extract, blended with fruit juice to reduce the bitterness, have been shown to be recovered from a variety of cancers [10,11]. Toxicity studies have shown that C. nutans extracts were more or less nontoxic [9]. ese studies highlight the potential of C. nutans for cancer therapy. To keep track of the progress of anticancer activity of C. nutans and point out a new direction for further research and future clinical application, we aimed to review up-to-date publications about the effects of C. nutans on different types of cancer, including breast, cervical, colorectal, gastric, liver, lung, head and neck, skin, and pancreatic cancers, as well as lymphoma and leukemia, primarily based on cell and animal studies. A novel way of extract package such as nanoencapsulation has also been tried in a cell-based study [12]. Preparation of extract and the investigation of their anticancer activities and possible underlying mechanisms of C. nutans extracts are listed in Table 1. Two interesting studies about combination therapy of C. nutans extracts with current anticancer drugs are listed in Table 2.

Breast Cancer.
According to the literature, the different parts of whole plants of C. nutans, including leaves, stem, roots, and even bark, have been used for combating diseases [13]. erefore, Teoh and colleague isolated methanol and ethyl acetate extracts from C. nutans roots and tested antiproliferative as well as apoptosis-inducing activities of these extracts on MCF-7 human breast cancer cells. Both extracts inhibited proliferation of MCF-7 cells with the IC 50 of 35 and 30 μg/mL for methanol and ethyl acetate extracts, respectively. Induction of apoptosis was evidenced by chromatin condensation and downregulation of BCL2. However, the expression level of BAX was not changed. Further evidences showed that ethyl acetate extract was able to decrease the mitochondrial membrane potential by JC-1 staining. GC-MS identification revealed that these root extracts are rich in terpenoids and phytosterols, so their anticancer activities need to be further analyzed [14]. In addition to root extract, methanol extract from leaves of C. nutans has been isolated and the antioxidant and antitumor activities in 4T1 tumor-bearing mice of breast cancer using low-dose concentration (200 mg/kg) and highdose concentration (1000 mg/kg) of extracts were determined. Both dosages of extract significantly decreased the blood nitric oxide (NO) and malondialdehyde (MDA) levels. High-dose extract effectively reduced the number of mitotic cells, tumor weight, and tumor volume of 4T1 breast cancer model mice. However, extract dosage as high as 1000 mg/kg did not elicit adverse effect and inflammatory response.
ese data indicate that methanol extract from leaves of C. nutans would be potential for the therapy of breast cancer due to its antitumor activities and nontoxicity at given dosages [15].
Besides, crude methanol extract of bark powder from C. nutans was isolated and used for further fractionation by chromatography, and six fractions were obtained. ese fractions were then tested for their cytotoxicity toward MDA-MB-231 and MCF-7 breast cancer cell lines by dimethylthiazol diphenyltetrazolium bromide (MTT) assay. Two fractions, A12 and A17, which harbor sulfur-containing compounds, including entadamide C and clinamide D, were found to have strong cytotoxic activities. e molecular target of these two compounds was accessed to be caspase-3 by molecular docking simulation, which will pave the way for the design and development of more effective drugs based on structures of entadamide C and clinamide D against breast cancers and other cancers [16].
Recently, Ismail and his colleagues obtained 80% methanol extract from C. nutans leaves and subsequently the crude extract was fractionated by n-hexane, dichloromethane, chloroform, n-butanol, and water. en a series of analyses including total phenolic content (TPC), total flavonoid content (TFC), and antioxidant and antiproliferation activities were performed on MCF-7 breast cancer cells and normal MCF 10A cells. Crude extract (CN-Crd) itself had the strongest antioxidant scavenging activity, while water fraction (CN-Aqu) possessed the highest amount of TPC and TFC. Hexane fraction (CN-Hex) displayed the highest antiproliferative effects on MCF-7 cells, with the IC 50 of 50.34 ± 0.11 μg/mL, followed by the dichloromethane fraction (CN-Dcm) (IC 50 : 65.95 ± 0.14 μg/mL). As dichloromethane fraction (CN-Dcm) had the highest selectivity index (SI � 1.48), it was chosen for further analyses. GC-MS identification indicated that linolenyl alcohol (29.10% at 12.023 min) and palmitic acid (23.84% at 11.133 min) are the most abundant among 14 compounds. Molecular docking studies showed that p53-binding protein Mdm-2 is the cellular target of these two major components. eir data suggested that methanolic extract from C. nutans leaves possesses potential antiproliferative activities, as reported by other laboratories [17,18]. Besides, Quah et al. reported the antiproliferative activity of methanolic extract from C. nutans leaves on MDA-MB-231 cells with IC 50 of 18.67 μg/mL. However, the same extract was much less toxic to normal 3T3 cells. Further analyses showed that the antiproliferative effects were possibly attributed to the inhibition in activities of CYP3A4 and CYP2E1, two liver enzymes for detoxifying drugs [19].

Cervical Cancer.
To combat cervical cancer, petroleum ether, ethyl acetate, and methanol crude extracts of C. nutans leaves and stems were produced and subsequently isolated by bioassay-guided fractionation. ese fractions were then tested for antioxidant and antitumor activities against HeLa cervical cancer cells. Among these extracts, petroleum ether extract displayed the strongest cytotoxic activity against HeLa cells with IC 50 of 18.0 μg/mL. Petroleum ether extract also possessed the highest radical scavenging activity. ese data suggest that active compounds in C. nutans extracts, especially petroleum ether extract, are health-promoting reagents with antioxidant activity and potential cytotoxic activity to cancer cells [20]. Another laboratory reported the further fractionation of C. nutans methanol extracts by hexane (methanol-hexane), dichloromethane (methanoldichloromethane; DCM), and water (methanol-water) and evaluated the effects of these extracts on various biological 2 Evidence-Based Complementary and Alternative Medicine    Evidence-Based Complementary and Alternative Medicine    Evidence-Based Complementary and Alternative Medicine   Evidence-Based Complementary and Alternative Medicine activities, including cell proliferation, apoptosis, and cell cycle progression to HeLa cells. ey found out that all extracts displayed antiproliferative activities to HeLa cells, and DCM extract had the highest activity with the IC 50 to be 70 μg/mL at 48 hours. DCM extract also induced apoptosis and cell cycle arrest at S phase. GC-MS analysis revealed that at least 28 compounds exist in DCM extract and most of them are fatty acids. e anticancer effects of these compounds or combination of compounds are pending [17].
Yusmazura et al. isolated aqueous and methanol extracts from C. nutans leaves and tested their cytotoxicity to HeLa cells. ey found out that aqueous extract showed great cytotoxic effects (IC 50 � 13 ± 0.82 μg/mL) and induced apoptosis of HeLa cells, while both extracts were not toxic to normal kidney Vero cells [21]. Moreover, Ghasemzadeh and his colleagues compared the methanol extracts of leaves and buds of different plant ages, including onemonth, six-months, and one-year-old C. nutans. e sixmonth-old buds contain highest total flavonoid (TF) (6.32 mg/g dry weight) and total phenolic (TP) compounds (18.21 mg/g dry weight). Highest contents of caffeic acid (0.307 mg/g dry weight) and gallic acid (5.96 mg/g dry weight) phenolic acids were detected in extracts of one-year and six-month-old buds, respectively. e activity of chalcone synthase (CHS, EC 2.3.1.74), a key enzyme for flavonoid production, was the highest in six-month-old buds (9.5 nkat/mg protein). e 1, 1-diphenyl-2-picrylhydrazyl (DPPH) activity was the highest in the extract of one-year-old buds with 50% of free radical scavenging (IC50) values of 64.6 μg/mL. However, the ferric reducing antioxidant power (FRAP) activity was higher in sixmonth-old buds (488 μM of Fe(II)/g). MTT assay showed that the extract of six-month-old bud significantly decreased cell viability of HeLa cells with IC50 of 56.8 μg/mL. ese analyses indicate that six-month-old buds of C. nutans harbor substantial amount of secondary metabolites, providing valuable antioxidant and anticancer compounds [22].

Colorectal Cancer.
Leaf extracts from five different reagents, including ethanolic, methanolic, 50% ethanolic, 50% methanolic, and water extracts, of C. nutans were produced and tested on HCT-116 colorectal carcinoma cells, and CCD-18Co normal colon fibroblasts. Extract concentrations at 200 and 100 μg/mL showed no significant cytotoxicity on tested cell lines. Further fractionation of methanolic extract (CN-M) by silica gel flash column chromatography showed that fractions 3, 4, 14, and 16 displayed significant cytotoxicity to HCT-116 cells at the concentration of 200 μg/mL. Among them, fraction 14 showed highest growth inhibition activity (84 ± 1.1% at 100 μg/mL) [23,24]. Besides, ethanol extract and thereafter the hexane, ethyl acetate, and aqueous fractions of C. nutans leaves were obtained by another laboratory and their biological activities were tested on HCT-116 cells. C. nutans ethyl acetate fraction (CNEAF) displayed the strongest cytotoxicity (IC 50 � 48.81 ± 1.44 µg/mL) to HCT-116 cells. CNEAF induced cell apoptosis, dissipated mitochondrial membrane potential, and elevated the reactive oxygen species (ROS) level, accompanied by the increase in expression of Bax and the decrease in expression of Bcl-2 and Bcl-X2, resulting in the activation of caspase−3, −9, −8, and −10. Upregulation in death receptor 5 was also detected. ese data suggest that both intrinsic and extrinsic apoptosis pathways are involved in CNEAFelicited effects. It is known that dysregulation of autophagy contributes to cancer; therefore, targeting autophagy would be a promising way for cancer treatment [25]. CNEAF also induced autophagy by the increase in LC-3 and decrease in p62 expression. CNEAF-induced elevation in the reactive oxygen species (ROS) level can be alleviated by N-acetylcysteine. In addition, N-acetylcysteine treatment also decreased both CNEAF-induced apoptosis and autophagy. ese evidences showed that both apoptosis and autophagy elicited by CNEAF are ROS-dependent in HCT-116 cells [26].

Gastric Cancer.
A novel polysaccharide-peptide complex CNP-1-2 with a molecular weight of 9.17 × 10 4 Da was isolated by a series of complex purification methods from C. nutans leaves. CNP-1-2 displayed the strongest growthinhibition effect on SGC-7901 human gastric cancer cells and was able to stimulate the activation of macrophages among all prepared polysaccharide fractions. CNP-1-2 consists of about 87.25% carbohydrate and 9.37% protein.
e structural moieties of CNP-1-2 were determined by several approaches including monosaccharide analysis, methylation analysis, FT-IR, 1 H NMR spectroscopy analysis, and atomic force micrograph (AFM) analysis [27]. Novel polysaccharide-peptide complex such as CNP-1-2 would open a new avenue to combat cancers, including gastric cancer [27].

Liver Cancer.
A previous study has identified several phytochemicals and fractions, including β-sitosterol-3-O-β glucopyranoside, β-sitosterol-3-O-β glucoside, and subfraction F-III from the ethyl acetate extract of C. nutans. Among these isolates, the subfraction F-III exhibited strong cytotoxicity against HepG2 human hepatoma cells, with IC 50 of 36.80 µg/mL, but with no effect of this subfraction on antioxidant assay [28]. However, no further action mechanism was provided in this study. ereafter, 30% ethanol extract (CN30) from aerial parts of C. nutans was produced and the active components in CN30 were furtherly fractionated and purified by using a bioassay that analyzes upregulation of the immune response of hepatoma cells-injected mice. High-performance liquid chromatography (HPLC) and mass spectrometry (LC/MS/MS) analyses showed that the main components of CN30 were identified to be (1) apigenin 6-C-β-D-glucopyranosyl-8-Cα-L-arabinopyranoside (known as shaftoside), (2) apigenin 6,8-C-α-L-pyranarabinoside, (3) orientin, (4) isoorientin, (5) vitexin, (6) isovitexin, and (7) gallic acid. CN30 at doses of 3 and 10 mg/kg induced 8.2% and 58.6% of inhibition in tumor size and tumor weight of the HepA xenograft model mouse. CN30 at dose of 10 mg/kg displayed stronger inhibition (58.6%) than that (37.1%) of the anticancer drug fluorouracil (20 mg/kg) in tumor weight. Further analyses showed that CN30 treatment induced significant apoptosis, decreased protein levels of proliferation markers PCNA and p-AKT, increased protein levels of apoptosis markers BAX and cleaved caspase-3, increased the number of IFN-c + T cells, decreased the number of IL-4 + T cells, and increased the levels of IFN-c and interleukin-2 in serum of hepatoma-bearing mice. ese data indicated that 30% ethanol extract (CN30) of C. nutans displays antitumor activity by augmenting the immune response and inducing apoptosis in vivo [29].
It has been reported that phytochemicals, including the total phenolic content (TPC) and total flavonoid content (TFC), were extracted from C. nutans by using organic solvents that include hexane, methanol, chloroform, and ethyl acetate and the cytotoxicity against HepG2 cells was tested. As compared to extracts of other solvents, methanol extract displayed strongest cytotoxic activity against HepG2 cells, with 74.17 ± 0.50% of inhibition at 100 μg/mL after 24 hours of treatment. Further analyses showed that chloroform extract harbors highest total phenolic content (TPC), which is 119.29 ± 0.07 mg of gallic acid equivalent (GAE). Methanol extract contains highest total flavonoid content (TFC), which is 937.67 mg of butylated hydroxytoluene (BHT). ese compounds could function as chemosensitizers [30] and provide sources of chemotherapeutic ingredients for cancer therapy in the future [31]. Quah also reported the antiproliferative activity of methanolic extract from C. nutans leaves on HepG2 cells with IC 50 of 13.33 μg/ mL. However, the methanolic extract was much less toxic to normal 3T3 cells. e antiproliferative effect was possibly through inhibition in the enzymatic activities of two detoxifying enzymes CYP3A4 and CYP2E1 [19]. Another study reported the preparation of C. nutans extracts with five different solvents (hexane, chloroform, ethyl acetate, methanol, and water) and examined the anticancer activities on HepG2 cells. ey found out that hexane and chloroform  ese studies suggested the involvement of both intrinsic and extrinsic apoptosis pathways underling the effects of hexane extract of C. nutans [32].
2.6. Lung Cancer. Fazil et al. evaluated the maximum yield and time of exhaustive extraction of flavonoids from C. nutans using Peleg's model and thereafter examined the antiproliferative activity of water extract on twodimensional culture of A549 lung cancer cells. ey obtained the predicted maximum extract density to be 29.20 ± 4.54 hours. However, the exhaustive time of extraction to have maximum flavonoids content was determined to be 18 hours and the best antiproliferative effects (IC 50 ) on A549 cells was observed at 138.82 ± 0.60 μg/mL. Such kinetics extraction modeling with modification could allow us to obtain the best timing with best yield for flavonoid water extraction from C. nutans or other herbal plants [33]. Another laboratory isolated hexane, chloroform, ethyl acetate, methanol, and water extracts from C. nutans and showed that hexane extract had highest cytotoxic activity to A549 cells, with the IC 50 of 74 μg/mL. Further evidences showed that hexane extract induced cell apoptosis, increased the percentage of cells at sub-G1 phase, and increased the levels of reactive oxygen species. Hexane extract also upregulated caspases 8, 9, and 3/7 activities at concentrations of more than 100 μg/mL [32].

Lymphoma and Leukemia.
Studies have shown that C. nutans chloroform extract displayed higher antiproliferative activities on K-562 human erythroleukemia cells (91.28 ± 0.03%) and Raji human Burkitt's lymphoma cells (88.97 ± 1.07%) at 100 μg/mL, as compared to the rest of cell lines [10]. Moreover, Arullappan et al. found out that C. nutans petroleum ether extract displayed the strongest cytotoxic activity against K-562 cells with IC 50 of 20.0 μg/mL at 72h, as compared to other extracts [20]. However, the underlying mechanisms are not yet investigated. In addition to these cells, the activity of C. nutans extract to SUP-T1 human lymphoma cells and MOLT-4 leukemia cells was also reported by another laboratory. ey obtained several fractions from C. nutans by extraction of leaf powder with different organic solvents and examined the effects of these extracts on various biological activities in SUP-T1 and MOLT-4 cells. e results showed that methanol-hexaneacetone extract (MHA) had the highest antiproliferative activities to SUP-T1 and MOLT-4 cells. Further studies showed that MHA increased cell apoptosis, reactive oxygen species, and calcium flux; arrested cell cycle at G2/M phase; and decreased the mitochondrial membrane potential and ER stress as evidenced by the increase in expression of CHOP and IRE-1α proteins, suggesting that MHA of C. nutans could possess anticancer activity in lymphoma and leukemia [34].

Head and Neck
Cancer. Ng isolated C. nutans extracts of whole plants by hexane, chloroform, ethyl acetate, methanol, and water. ey showed that hexane extract displayed strongest cytotoxicity to CNE-1 human nasopharyngeal carcinoma cells with IC 50 of 116.7 μg/mL. Hexane extract elicited cell apoptosis, increased the levels of reactive oxygen species and the percentage of cells at sub-G1 stage, and activated caspases 8, 9, and 3/7 at concentrations of ≥100 μg/ mL. Besides, ethyl acetate extract inhibited cell proliferation of CNE-1 cells at the concentration of 300 μg/mL [32]. e extracts of Clinacanthus nutans are rich in flavonoids and polyphenols, which suffer from low solubility, poor permeability, and low bioavailability [35]. Nanoencapsulation is a good way of packaging drugs to overcome these drawbacks and increase drug efficacy [36,37]. To this purpose, Yakop and colleagues reported the preparation of silver nanoparticle Clinacanthus nutans (AgNps-CN) of leaves aqueous extract and tested the anticancer activity including cytotoxicity, apoptosis, cell cycle progression, and expression of key proteins involving apoptosis in HSC-4 human oral squamous cell carcinoma cells. AgNps-CN inhibited the cell viability of HSC-4 cells with IC 50 of 1.61 μg/ mL, while AgNps-CN is not harmful to normal 3T3-L1 cells at all tested concentrations. AgNps-CN induced cell apoptosis and cell cycle arrest at G0/G1 stage. Western blotting analyses showed that the ratio of Bax/Bcl-2 was increased by AgNps-CN. Due to its toxicity to HSC-4 cells and nontoxicity to normal cells, silver nanoparticles of C. nutans extracts could have the potential to treat cancers [12].

Skin Cancer.
In order to know whether the extracts of plants from different geographical environment were different in their anticancer activities against skin cancer, Fong and his colleagues reported the production of crude methanol leaf extracts of C. nutans from different locations in Malaysia, ailand, and Vietnam and examined the effects of extracts on cytotoxicity, apoptosis, and cell morphology of D24 melanoma cells. ey found out that the extract from higher elevations with lower temperature was more toxic to D24 cells. Highest cytotoxic activities were found in extract from Chiang Dao and Chiang Mai, ailand, with the half maximal effective concentration (E50) of 0.95 mg/mL for 24 hours and E50 of 0.77 mg/mL for 72 hours. Chiang Dao extract also induced apoptosis in biochemical and morphological analyses of D24 cells [38]. ese data suggest that C. nutans extracts and extracts of plants in higher elevations and lower temperature could be more promising for the treatment of skin cancer.

Anticancer Activity of C. nutans (CN) Extract
Combined with Current Anticancer Drug

Combined Effects of CN Extract with Cisplatin.
Combination therapy with current anticancer drugs would be another way for C. nutans extracts to treat cancers. To this purpose, methanolic (MCN) and aqueous (WCN) extracts of C. nutans were obtained and combined treatment of extracts with anticancer drug cisplatin on MCF-7 breast cancer cells was tested. ey found out that MCN displayed stronger cytotoxicity to MCF-7 cells than WCN. Most combined treatment of MCN and WCN extracts with cisplatin displayed strong antagonism. However, combined treatment of cisplatin with high concentration of MCN is additive (combination index � 1) against cancer cells [39].

Combined Effects of CN Extract with Gemcitabine.
In addition to the cotreatment of C. nutans extracts with cisplatin described above, cotreatment with gemcitabine was also reported. Hii and his colleagues reported the isolation of four extracts, which include polar leaf extracts (LP), nonpolar leaf extracts (LN), polar stem extract (SP), and nonpolar stem extracts (SN) from C. nutans, and analyzed the antiproliferative activities of drug combination of C. nutans extracts with gemcitabine, on different cancer cell lines, including human breast, colorectal, lung, endometrial, nasopharyngeal, and pancreatic cancer cells by MTT assay. Among all cancer cell lines, AsPC1, BxPC3, and SW1990 pancreatic ductal adenocarcinoma (PDAC) cell lines were most sensitive to nonpolar stem extracts (SN). Addition of SN extract can reduce the dosage of gemcitabine with the same effect and can potentiate the killing activity of gemcitabine to PDAC by apoptosis through upregulation of Bax, as well as downregulation of bcl-2, cIAP-2, and XIAP levels in SW1990 and BxPC3 cells independent of the TRAIL-4 expression [40].

Concluding Remarks
C. nutans extracts display strong anticancer activities, including inhibition of cell proliferation, retardation of cell cycle progression, and induction of apoptosis and autophagy, in addition to its antiviral, antibacterial, and antioxidant effects. is review provides up-to-date information about anticancer activities and action mechanism of various C. nutans extracts, which were obtained by different solvents and different isolation methods, from different parts of the plants, different plant age, and plants of different geographical environment. Moreover, nanoencapsulation of C. nutans extract and combined treatment of C. nutans extracts with current anticancer drugs are evaluated in order to increase the drug efficacy and decrease possible side effects. ese studies would shed light on the discovery of bioactive components of C. nutans and development of nature product-based drug using novel technologies [41], which could be applied for the therapy of various cancers in the future.

Conflicts of Interest
e authors report no conflicts of interest.