Clinacanthus nutans: a review on ethnomedicinal uses, chemical constituents and pharmacological properties

Abstract Context: Medicinal plants have attracted global attention for their hidden therapeutic potential. Clinacanthus nutans (Burm.f) Lindau (Acanthaceae) (CN) is endemic in Southeast Asia. CN contains phytochemicals common to medicinal plants, such as flavonoids. Traditionally, CN has been used for a broad range of human ailments including snake bites and cancer. Objectives: This article compiles the ethnomedicinal uses of CN and its phytochemistry, and thus provides a phytochemical library of CN. It also discusses the known pharmacological and biological effects of CN to enable better investigation of CN. Methods: This literature review was limited to articles and websites published in the English language. MEDLINE and Google Scholar databases were searched from December 2014 to September 2016 using the following keywords: "Clinacanthus nutans" and "Belalai gajah". The results were reviewed to identify relevant articles. Information from relevant selected studies was systematically analyzed from contemporary ethnopharmacological sources, evaluated against scientific literature, and extracted into tables. Results: The literature search yielded 124 articles which were then further scrutinized revealing the promising biological activities of CN, including antimicrobial, antiproliferative, antitumorigenic and anti-inflammatory effects. Few articles discussed the mechanisms for these pharmacological activities. Furthermore, CN was beneficial in small-scale clinical trials for genital Herpes and aphthous stomatitis. Conclusion: Despite the rich ethnomedicinal knowledge behind the traditional uses of CN, the current scientific evidence to support these claims remains scant. More research is still needed to validate these medicinal claims, beginning by increasing the understanding of the biological actions of this plant.


Introduction
Natural product drug discovery is a vibrant research area traversing nearly all scientific fields. Natural products, such as medicinal plants, serve as a rich potential source of new therapeutic compounds (Ramesh et al. 2014). In many countries, traditional medicinal plants have been used to treat a plethora of ailments, and this precious ethnomedicinal knowledge has been passed on over many generations. For as long as they have inhabited the earth, humans have used plants as a medicinal source. Plant-based drug development has grown more sophisticated, with modern chemists using compounds isolated from plants as structural leads to generate novel compounds with additional benefits, such as lower toxicity or higher efficacy in drug-resistant diseases.
Most medicinal plants have been studied for a range of biological activities including anticarcinogenic, anti-inflammatory and antimicrobial activities. These activities were further evaluated to identify potential therapeutic benefits for various human ailments such as cancers, autoimmune diseases and chronic infections. Continuing the quest for plant-based natural products is critical because plants contain many potentially novel therapeutic compounds. Clinacanthus nutans (Burm.f) Lindau (Acanthaceae) (CN) has been selected for the focus of this review as the plant has garnered much attention from social media about its potential therapeutic benefits. CN is one of many medicinal plants traditionally used to treat various diseases and injuries such as skin rashes, burns, fever and snakebite (Aslam et al. 2015). Some cancer patients have claimed that CN leaf consumption has helped in treating their cancer and improving their health, although there is a lack of clinical studies to support these claims (Shim et al. 2013).
To undertake a systematic and extensive review, literature was searched from various computerized databases (MEDLINE and Google Scholar) up to September 2016 as available on PubMED. Moreover, this literature review was limited to websites and articles published in the English language. The following keywords were used: "Clinacanthus nutans", "Sabah Snake Grass" and "Belalai gajah". The results were reviewed to identify relevant articles. Contemporary sources of knowledge were also used to compare the ethnopharmacological information against the scientific literature available. Data from the selected studies was extracted systematically into tables for analysis. A total of 124 articles were found using the search method described above revealing promising biological activities of CN, including antimicrobial, antiproliferative, antitumorigenic and anti-inflammatory effects.

Ethnomedicinal uses of CN
CN is commonly used in traditional medicine in the Southeast Asian region, particularly in Thailand and Malaysia. Many traditional therapeutic uses for CN have been reported (Table 2), but clinical and scientific data support only a few of these. In general, only the leaves of CN are used in traditional medicine. The use of CN to treat Herpes virus infections is supported by the results of various scientific studies and clinical trials (Sangkitporn et al. 1993(Sangkitporn et al. , 1995Yoosook et al. 1999;Lipipun et al. 2011;Kongkaew (Sakdarat et al. 2009;Sittiso et al. 2010; Kongkaew and Chaiyakunapruk 2011;Roeslan et al. 2012;Kunsorn et al. 2013;Rathnasamy et al. 2013;Arullappan et al. 2014) Skin rashes NAD NAD NAD (Sakdarat et al. 2009;Sittiso et al. 2010;Kunsorn et al. 2013;Lau et al. 2014) Varicella zoster, herpes simplex and herpes genitalis lesions Fresh leaves Ethanol extract Topical use as a cream (Direkbusarakom et al. 1998;Lipipun et al. 2003;Sakdarat et al. 2009;Sittiso et al. 2010;Roeslan et al. 2012;Kunsorn et al. 2013;Rathnasamy et al. 2013;Chelyn et al. 2014) Pruritic rash NAD Ethanol extract Topical use as a cream (Chotchoungchatchai et al. 2012) Aphthous ulcers NAD Ethanol extract in glycerin solution Topical use as a cream (Chotchoungchatchai et al. 2012) Burns NAD Oil extract Topical use as a cream (Chotchoungchatchai et al. 2012) Inflammation Whole plant NAD NAD (Rathnasamy et al. 2013;Arullappan et al. 2014;Ghasemzadeh et al. 2014) Dysentery Fresh leaves Decoction of leaves boiled in water Oral ingestion; Handful of fresh leaves is boiled in 5 glasses of water until water level reduces to 3 glasses. Dosage is 1 glass. (Roosita et al. 2008;Roeslan et al. 2012;Arullappan et al. 2014;Globinmed 2015) Diabetes

Fresh leaves Decoction of leaves boiled in water
Oral ingestion; 7-12 fresh leaves boiled in 2 glasses of water, until water level reduces to 1 glass. Dosage is 1 glass, twice daily (Roosita et al. 2008;Roeslan et al. 2012;Ching et al. 2013;Arullappan et al. 2014;Globinmed 2015) Dysuria  Kunsorn et al. 2013). CN leaves were initially consumed for general health in various countries in Southeast Asia (Shim et al. 2013;Siew et al. 2014). However, CN is gaining popularity in Malaysia and Singapore because of claims of its anticancer properties (Table 2). This has led to the availability of a wide variety of commercial products, including teas, drinks and powders ( Figure 2). Dampawan et al. (1977) isolated lupeol and b-sitosterol crystals using ether and light petroleum solvent system, after having extracted the stem of CN in a Soxhlet apparatus with light petroleum. X-ray diffraction of these crystals revealed discrete molecules without short intermolecular contacts. Teshima et al. (1998) isolated sulfur-containing glucosides, clinacoside A and clinacoside B, from the colorless butanol soluble fraction of the methanol extract prepared from the CN stem and leaves. These phytoconstituents were identified using chemical and spectroscopic methods.
Using GC-MS, Yong et al. (2013) identified 14 chemical constituents (n-pentadecanol; eicosane; 1-nonadecene; heptadecane; dibutylphthalate; n-tetracosanol-1; heneicosane; behenic alcohol; 1-heptacosanol; 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester; nonadecyl heptafluorobutyrate; eicosayl trifluoroacetate; 1,2-benzenedicarboxylic acid, dinonyl ester; and phthalic acid dodecyl nonylester) from chloroform, methanol and aqueous leaf extracts of CN. The 1,2-benzenedicarboxylic acid mono (2-ethylhexyl) ester was the major chemical constituent, with a relative peak area of approximately 28.6% while those of the others were each less than 2%. Chelyn et al. (2014) identified C-glycosidic flavones, including isovitexin, vitexin, isoorientin, orientin and shaftoside, in an ethanol extract of CN leaves. These flavones were optimized and validated with an HPLC method for quantification and quality control of herbal materials. Tu et al. (2014) isolated phytochemicals containing sulfur, clinamides A, B and C and 2-cis-entadamide A, from ethanol extracts of the aerial parts of CN. Clinamide A, a pale yellow oil, was assigned the molecular formula, C 6 H 11 NO 4 S by 1 H-NMR spectroscopy and exhibited a methylsulfonyl signal and two methylene signals, including a trans-disubstituted double bond. Clinamide B was assigned a molecular formula of C 8 H 13 NO 4 S by HRESIMS (high-resolution electrospray ionization mass spectrometry), and its 13 C-NMR spectrum was similar to that of entadamide C, but with an additional acetyl group. The partial NMR spectrum of clinamide C, a pale yellow oil, also indicated a close structural similarity to entadamide A and HRESIMS determined its molecular formula C 12 H 22 N 2 O 4 S 2 . HRESIMS assigned the molecular formula C 6 H 11 NO 2 S to both 2-cis-entadamide A and entadamide A, indicating that they are geometric isomers. Yang et al. (2013) isolated saponins, phenolics, flavonoids, diterpenes and phytosterols from methanol extracts of CN leaves with a 15% w/w yield and with approximately 1.77 mg gallic acid equivalents per g of total phenolic content.
Use of CN leaves for various pharmacological purposes has increased exponentially because of information on the internet. Plant harvesting and preparation of its parts prior to extraction are of paramount importance, influencing the quantity and quality of the phytoconstituents extracted. Raya et al. (2015) assessed effects of storage duration on the phytochemical content of CN stems and leaves at different stages of harvesting. Phenolic content was 26% and 90% higher in younger leaves and stems, respectively, compared with their mature counterparts. Moreover, parts of mature plants had lower contents of phytochemicals, chlorophyll, and ascorbic acid compared with those from young plants. Also, prolonged storage reduced levels of these CN constituents. After storage for 4 d, the contents of total phenolics and chlorophyll were reduced to 50% and 25%, respectively, of the amounts in freshly prepared CN parts. Such evidence demands that fresh plant parts be used to avoid phytochemical loss and, thereby, optimize efficacy. Huang et al. (2015) extracted dried aerial parts of CN using ethanol, purified the crude extract and identified compounds by HPLC with tandem mass spectrometry (LC/MS/MS). 1 H-NMR analysis provided additional confirmation of compound identity. Flavonoids such as shaftoside, apigenin 6,8-C-a-L-pyranarabinoside, orientin, isoorientin, vitexin and isovitexin were observed in this study.
Several parameters such as solvent characteristics, prior plant preparation and thermal degradation influence the phytochemicals extracted from the plant. The extraction technique is a key factor determining the amounts and the natures of the phytoconstituents obtained.  compared extraction efficiencies with microwave-assisted extraction (MAE), pressurized microwave-assisted extraction (P-MAE), supercritical carbon dioxide extraction (SFE) and the Soxhlet method. They reported on yields, extraction times and recovery of phytoconstituents, specifically, phenols, flavonoids, phytosterols and b-sitosterol. While MAE resulted in the highest yields of polyphenol and flavonoids, SFE was the best method for extracting phytosterols and b-sitosterol. P-MAE resulted in slightly improved yields of polyphenol and flavonoids. Overall, the study concluded that MAE was the most efficient extraction technique for CN, giving high extraction efficiency and better selectivity, compared with the other techniques, for compounds of nutraceutical interest, including those with anti-inflammatory, antioxidant and antimicrobial activities . The study further explored the influence of ethanol concentration and applied microwave energy and solvent-to-feed ratio on CN extraction using a microwave-assisted technique. Microwave pretreatment improved extraction rates by a factor of 2-5 fold, with water: ethanol (1:1) solvent (Mustapa, Martin, Gallego et al. 2015). Table 3 shows the chemical structures discussed in the phytochemistry section.
Metabolomics generate metabolic fingerprints of an organism by identifying and quantifying its metabolites, which enhances the understanding of chemical variability among various organisms. Khoo et al. (2015) used nuclear magnetic resonance (NMR) to analyze the metabolite profile of CN leaves and stems, which were stratified based on two techniques: firstly, drying including air, oven, and freeze; secondly, extraction including soaking and sonication methods. Compared to leaves, stems contained a higher amount of terpenoids and phenolic compounds, correspondingly the activity levels of total phenolic content, a-glucosidase inhibition and antioxidant were higher as well, confirming to the well-established compound-activity correlation. Drying and extraction methods affect the yield of various phytoconstituents in extracts and thus its pharmacological activity. Partial least-squares analysis (PLS) biplot model analysis of the NMR revealed the superiority of oven and air drying methods over freeze drying and soaking methods for their yield of terpenoids, phenolic compounds, and glucosides, which were further confirmed by their corresponding better biological activities. Huang et al. (2016) successfully attempted the purification and analysis of a novel polysaccharide-peptide complex from the leaves of CN, which showed significant promising results for gastric cancer cells SGC-7901 inhibition. The monosaccharide analysis, FTIR, 1H NMR and methylation analysis revealed the presence of CN polysaccharide which includes L-rhamnose and a backbone 1-6 linked Galp residues, while atomic force microscopy (AFM) displayed the entangled and branched structure of the compound. Identification of compounds will lead to the determination of the structure-activity relationship to develop it into a lead molecule. Multi-targeted therapy by plant extract as a single herb regimen either as the adjuvant or main treatment therapy calls for the systematic investigation of purified compounds of their beneficial therapeutic activities and mechanism to optimize them toward clinical studies. Table 3. Chemical structures of compounds isolated from Clinacanthus nutans.

Antioxidant activity
Antioxidants are substances that neutralize potentially damaging oxidizing agents or free radicals, which are thought to cause chronic health problems in diseases such as cancer, cardiac disease, and aging-related disorders. Pannangpetch et al. (2007) reported that CN extracts significantly reduced oxidative free-radical production by phorbol 12-myristate 13-acetate (PMA)-stimulated rat macrophages. Furthermore, the extract showed a substantial inhibitory effect (98%) on haemolysis in a 2,2 0 -azobis(2-amidinopropane) dihydrochloride (AAPH)-induced cell lysis model. AAPH causes lysis of red blood cells through oxidation of lipids and proteins in the blood cell membranes. Subsequent studies demonstrated in vitro antioxidant effects of CN based on various criteria, as summarized in Table 4. Collectively, data from these studies showed clearly that CN extracts could have antioxidant properties.
Antiproliferative and cytotoxic activity       MTT assay    Apigenin6-C-b-D-glucopyranosyl-8-C-a-L-arabiopyranoside 6,8-Apigenin-C-a-L-pyranarabinoside (Huang et al. 2015) Cytokine production Ethanol extraction IL-4 production increased at 2.5 mg/ml and 5 mg/ml, but the extract had no effect on IL-2 levels NR (Chompuki et al. 1996) Extraction  (Chompuki et al. 1996) Lymphocyte proliferation response assay Ethanol extraction Lymphocyte proliferation was significantly increased at extract concentrations below 5 lg/ml and significantly decreased at those above 2.5 mg/ml NR (Chompuki et al. 1996) Macrophage activation Extraction in distilled water, precipitated with ethanol and fractionated using Superdex 200 Macrophage activation was measured via production of nitric oxide Incubation of RAW264.7 cells in CN extract for 48 hours showed a dose-dependent increase in the production of nitric oxide Polysaccharide-peptide complex (Huang et al. 2016) Myeloperoxidase production Extraction in methanol and dissolution in acetone Ã MPO production was significantly reduced by extract in a concentration dependent manner, with an IC 50 of 219.5 lg/ml NR (Wanikiat et al. 2008) Neutrophil chemokinesis assay Extraction in methanol and dissolution in acetone Ã Neutrophil chemokinesis was significantly suppressed by extract in a concentration-dependent manner NR (Wanikiat et al. 2008) Neutrophil chemotaxis assay Extraction in methanol and dissolution in acetone Ã Neutrophil chemotaxis was significantly suppressed by extract in a concentration-dependent manner NR (Wanikiat et al. 2008) NK cell activity assay Ethanol extraction Significant reduction in NK activity at 1 mg/ml of crude extract and no detectable activity at 5 mg/ml of crude extract NR (Chompuki et al. 1996) Superoxide anion generation Extraction in methanol and dissolution in acetone Ã Superoxide anion generation was significantly reduced after incubation with extract for 10 min, in  Apigenin6-C-b-D-glucopyranosyl-8-C-a-L-arabiopyranoside 6,8-Apigenin-C-a-L-pyranarabinoside (Huang et al. 2015) Acetylcholinesterase activity In vivo assay in mice (Ellman method) Maceration, then crude methanol extraction Acetylcholinesterase activity in Balb/C male mice liver, kidney and heart was significantly higher in the extract treated than in the control group Acetylcholinesterase activity in the brain showed no significant differences between groups NR (Lau et al. 2014) Toxicity studies In vivo subacute oral toxicity study in rats Ethanol extract No signs of toxicity seen in mice after feeding the extract at the highest dose of 1.3g/kg of body weight Platelet counts of rats fed with CN extract were significantly higher However, creatinine levels were lower for CN-treated rats No histopathological changes were detected NR (Chavalittumrong et al. 1995) Maceration with methanol No significant changes were seen in serum biochemical parameters, relative organ weight, body weight gain, food intake and water consumption with extract treatment  extracts on these cell lines are summarized in Table 4. All these studies used the MTT assay as a standard measure of cell proliferation. This is a colorimetric assay measuring cellular metabolic activity. Few studies have tested the antiproliferative effects of CN on primary cell types, although one study tested CN extracts on primary human gingival fibroblasts and detected no antiproliferative activity (Roeslan et al. 2012).

Antitumorigenic activity
While antiproliferative compounds exert their anticancer effects through the inhibition of cell proliferation (Zulkipli et al. 2015), compounds with antitumorigenic activities may have a myriad of effects, which may prevent the development, maturation or spread of cancerous cells. Only one study has shown the potential antitumorigenic activity of CN. A CN ethanol extract, compared with a control treatment, significantly reduced tumour growth in a mouse HepA hepatoma model (Huang et al. 2015).
The antitumorigenic effect in this model was significantly greater than that of fluorouracil, an established chemotherapeutic drug.
Western blotting analysis showed that high levels of the proapoptotic mediator, Bax and apoptotic executioner protein, caspase 3 in tumours extracted from CN-treated tumour-bearing HepA mice. This suggests that CN could induce apoptosis in cancer cells as a mechanism to halt cell proliferation during tumour growth. However, more conclusive data obtained in multiple types of tumour models will be needed to prove that CN has antitumorigenic activity.

Antimicrobial activity
With the rise of antibiotic-resistant strains of bacteria in the clinical environment, scientists have turned to natural compounds in medicinal plants to identify potential new antibacterial compounds. The antibacterial effects of CN extracts have been tested on microbial strains (Yang et al. 2013;Arullappan et al. 2014).
Several studies have reported that CN extracts inhibited bacterial growth and survival, while other studies have reported no antibacterial activities of CN extracts against similar species of bacteria (Table 4). Overall, these mixed findings suggest that the antimicrobial effects of CN extracts may be selective for only certain microorganisms. However, the exact mode of action of CN on bacteria killing is yet to be defined.

Antiviral activity
One of the most common ethnomedicinal use for CN is for treating Herpes infections (Sangkitporn et al. 1993(Sangkitporn et al. , 1995. It is not surprising that there has been much interest in identifying a potential anti-Herpes agent from CN. The early work by Jayavasu et al. (2013) showed that CN leaf extracts inhibited plaque formation by HSV-2 in a baby hamster kidney cell line, suggesting that these extracts may contain antiviral components. The majority of work on the antiviral effects of CN has focused on infections with HSV, the causative agent for genital Herpes in cell lines and HSV-infected animals (Table 4). CN extract-based topical formulations were shown to be effective against the development and progression of skin lesions in a mouse model of cutaneous HSV-1 infection (Lipipun et al. 2011). Recently, Pongmuangmul et al. (2016 showed that purified monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) from CN leaves resulted in 100% inhibition of HSV viral plaque formation. Glycoglycerolipids, such as MGDG, have previously been shown to exert anti-HSV effects (Janwitayanuchit et al. 2003). Although the mechanism of anti-HSV activity of these glycoglycerolipids is still not clear, the known antiviral effects of monoglycerides against enveloped viruses such as HSV (Thormar et al. 1994) makes these glycoglycerolipids potential drug candidates against HSV. Interestingly, work by several groups showed that compounds from CN leaf extracts were able to inhibit dengue virus activity (Sittiso et al. 2010;Tu et al. 2014). CN extracts were also shown to be effective against a fish virus, the yellow-head virus, in CHSE-214-infected cells (Direkbusarakom et al. 1998). This suggests that CN-derived products could be effective not only against viruses other than HSV but also against viruses from various animal hosts.

Anti-inflammatory activity and immune-modulatory effects
Extracts from CN leaves have been used to reduce symptoms of inflammation in insect bites, Herpes infection and allergic responses in traditional medicine. A few reports have also described the effects of CN extracts on the immune system. CN extracts at low doses increased peripheral blood mononuclear cells (PBMC) proliferation, suggesting potential mitogenic properties . Interestingly, levels of interleukin-4 IL-4, an anti-inflammatory cytokine, were elevated only at higher CN doses, suggesting that inflammatory effects could be dampened with such doses. However, in a hepatocarcinoma (HepA) tumour model in mice, IL-4 induction by CD4 þ T-helper 1 lymphocytes (Th1 cells) was not affected by treatment with a CN ethanol extract, compared with vehicle (Huang et al. 2015). Th1 cells primarily secrete IL-2 and interferon (IFN)-c, which can suppress tumour growth by promoting CD8 þ cytotoxic T lymphocyte (CTL) function (Dunn et al. 2006). Indeed, treatment with a CN ethanol extract-induced IL-2 and IFN-c release and promoted CD8 þ CTL infiltration into the tumour tissue in the HepA tumour-bearing mice (Huang et al. 2015). This suggests that CN could modulate the adaptive immune system by skewing the immune system toward a Th1biased response, which would favour tumour suppression. In contrast, CN was also shown to act on the innate immune system. In two rat models of inflammation, ethyl phenylpropionateinduced ear oedema, and carrageenan-induced hind paw oedema, CN extracts significantly reduced oedema in the ears and paws, respectively (Wanikiat et al. 2008). The same study also reported attenuation of N-formyl-methionyl-leucyl-phenylalanine (fMLP)mediated migration (chemotaxis and chemokinesis) and function (myeloperoxidase production and elastase release) in neutrophils treated with CN extract. These studies suggest that bioactive compounds found in CN leaves may have multiple effects on the immune system and any resulting inflammation, depending on the model system used. Additional studies are summarized in Table 4, and key studies have been highlighted here.

Toxicity studies
The purpose of toxicity studies as per Barle et al. (2012) is, 'to determine the effect of an action on a biological system which can be used later to extrapolate the doses and effects on humans'. This data is essential to identify the optimal therapeutic dose and the highest dose up to which the extract can be given, above which lethality would be expected. The toxicity studies can be acute, sub-acute and chronic, where the former is the most commonly used type to evaluate the dose to be used for testing the dose required for preliminary testing.
Sub-chronic toxicity study for 90 days of ethanol CN extract exhibited similar food consumption in control and test yet body weight was significantly lower in male rats with 1 g/kg bw. Although toxicity was not observed at the given dose, platelet and creatinine levels were altered in different ways, with the former being higher than control (Chavalittumrong et al. 1995).
Sub-acute toxicity study for 14 days of methanol CN extract with a maximum 0.9 g/kg dose did not display any toxic effect in rats. Compounds administered by an oral route of administration are, in general, metabolized by the liver and eliminated by the kidney, thus are investigated during oral toxicological experiments. While AST and ALT are markers for hepatocyte integrity, blood urea and creatinine are biochemical indicators for renal function. Consecutive administration of the intervention substance in rats is equivalent to less than 7 days human consumption (World Health Organization 2004). 'Acceptable daily intake is the level that is harmless to humans based on the non-observable adverse effect level value obtained from animal study', as mentioned by Food and Agricultural Materials Inspection Center was calculated to be 9 mg/kg in humans (P'ng et al. 2013;FAMIC 2014).
The mice were orally administered 1000 mg/kg of methanol CN crude extract for 14 days and were observed to have normal behaviour related to central nervous system despite having significantly elevated levels of acetylcholinesterase enzyme in liver, heart and kidney but not in the brain (Lau et al. 2014). It would be beneficial to elucidate the compounds responsible and the mechanism of the elevated enzyme levels in various organs.
Farsi investigated the correlation between dose and exposure period and included male and female rats. The longer treatment duration, induced decreased ALP at 2000 mg/kg oral dose of aqueous CN, yet within the physiological range and is clinically insignificant. There was an increase in body weight on day 42. However, the contrary was true on day 77 in rats administered with 500 mg/kg. Nevertheless, significant changes were not observed in the group administered with 2000 mg/kg. Mutagenicity, the tendency of a test compound to induce DNA changes, was measured by the bacteria reverse mutation test and mutagenicity was not observed with CN (Farsi et al. 2016).
Methanol, ethanol and aqueous extracts of CN extracts were evaluated for toxicity in rats or mice to enrich the understanding of CN safety profile (summarized in Table 4). The animal toxicity experiments provide only preliminary information and must be followed by a battery of tests in animals. Subsequently, chronic and toxicokinetic assessments must be carried out to confirm the safety profile of CN.

Clinical trials
In addition to in vitro work, small-scale clinical trials performed using a topical formulation of a CN extract showed significant resolution of clinical symptoms of genital Herpes (Sangkitporn et al. 1993) and Herpes zoster (Sangkitporn et al. 1995;Charuwichitratana et al. 1996), as compared with patients treated with a placebo. A meta analysis of patients with hepatitis genitalis and Herpes zoster infections indicated that treatment with creams containing CN extracts resulted in faster healing of infection-induced lesions (Kongkaew & Chaiyakunapruk 2011).
The review mentions about its three limitations, namely: inability to perform a test of publication bias, the influence of small study effect and overestimate of clinical trials effect due to non-blinding. Due to these limitations, the results are to be interpreted with caution and summons for well-designed, robust, systematic, randomized controlled trials, which is either double or triple blinded. However, use of acyclovir with CN with its potential synergistic effect beckons for use as an adjuvant therapy in H. genitalis treatment. Patients with minor recurrent aphthous stomatitis, which is benign mouth ulcer, were recruited for the randomized, double-blind controlled trial to assess the efficacy of CN (Buajeeb & Kraivaphan 1994). Three arms were recording the duration of ulcers and pain, with CN, triamcinolone acetonide, and placebo, wherein the interventions were given in Orabase V R , an oral pain reliever. CN was better than placebo in shortening the ulcer duration than placebo, albeit triamcinolone acetonide was the best. Despite this evidence, the precise molecular mechanism of how CN extracts kill HSV is not known and warrants further elucidation in this area.

Future directions
CN has been demonstrated to have pharmacological effects on both host cells and microbes. The potential use of CN as an anti-HSV agent is promising. Future studies should investigate the antiviral activity of CN against other types of viruses, especially those endemic to regions where CN is abundant. Studies on the molecular mechanism of how CN extracts kill HSV are also needed.
Research to date has shown the antibacterial activity of CN only in vitro, in minimum inhibitory concentration assays, and the mechanism of this antibacterial activity is still unknown. Also, effects of CN in vivo in animal infection models have not yet been demonstrated. The lack of observed toxicities when various oral doses of CN leaf methanol extracts were given to Sprague-Dawley rats (P'ng et al. 2013;Farsi et al. 2016), and Balb/c mice (Lau et al. 2014) suggested that testing CN in more in vivo studies is feasible.
The anti-inflammatory effects of CN suggest that it has the potential to modulate the immune system. However, there is little current evidence addressing this and studies measuring cytokine production in immune cells have conflicting results. Therefore, more investigations would be required to establish the mechanisms by which CN dampens inflammatory activity in immune cells.
The mechanism for anti-proliferation by CN also remains to be determined. A link between antioxidant activity and anticancer effects has been suggested, particularly for phenolic compounds, such as flavonoids, from medicinal plants (Kumar & Pandey 2013;Roleira et al. 2015). Therefore, it is possible that phenolic compounds from CN could possess such biological activities. Although in vivo studies showed that CN has potential as an anticancer agent, more evidence from additional in vivo tumor models will be required conclusively to prove the biological relevance of the anticancer properties of CN.
Most of the studies investigating the antiproliferative effects of CN extracts on cells have focused on using the MTT colorimetric assay. Alternative lines of investigation for future studies could include investigating whether the antiproliferative effects of CN involve a cell division blockade or initiation of a cell death pathway (Zulkipli et al. 2015). Interestingly, in a recent study, high levels of the pro-apoptotic mediator, Bax, and apoptotic executioner protein, caspase 3, were detected by western blotting in tumor tissue from HepA tumor-bearing mice treated with a CN extract (Huang et al. 2015). Such data suggest that CN might induce apoptosis in cancer cells as a mechanism to halt tumor growth. The reported potent antiproliferative and antioxidant activities suggest that CN could be a good source of anticancer therapies.
There is a discrepancy in the procurement and processing of CN leaves used for various purposes. It is interesting to note that most CN users prefer fresh leaves while most researchers have experimented with dried plant parts. Thus, more experimentation by researchers on the effects of storage conditions on extract efficacy is needed. Many websites including Singapore Sabah Snake Grass (2011) have described a regimen of treatment for cancer and diabetes with fresh CN leaves (Roosita et al. 2008;Singapore Sabah Snake Grass 2011;Ching et al. 2013;Globinmed 2015). Standardization of the chemical constituents present in the leaves, their interaction with chemical constituents present in other plants and the efficacy of CN-derived preparations for the different stages of cancer have yet to be confirmed. Moreover, researchers also need to consider the selection of plant parts as well as the method of extraction and suitable solvents, to isolate the chemical constituents present in CN fresh leaves. Further research on the harvesting of CN parts and their proper storage will be needed to minimize loss of essential phytochemicals. These isolated compounds should be analyzed to compare their efficacy with the CN preparations used traditionally. Although it has been claimed that ingestion of CN prevents cancer, its prophylactic action has not been demonstrated. These measures would be beneficial to the community of CN users. If a potent activity is identified, the common process of drug discovery may then be applied such as what has been done for Viscum album mistletoe plant . Any potential drug arising from CN extracts would need to be investigated to determine the best formulation, dosage and delivery route. Once the mechanisms of the antitumor and other pharmacological properties of CN are better understood, it will be possible to identify molecular targets for upstream drug discovery research.