1. Introduction
The use of plant-based products in both traditional and modern societies as herbal remedies or crude drugs, or as purified compounds have a long history [
1]. Phytoresearches provide significant classes of drugs that are used in the treatment of various illnesses in both humans and animals including cancers, malaria, HIV/AIDS and diabetes. In the recent past, medicinal plants, such as
C. nutans, are gaining increasing attention in both traditional and modern societies [
1]. Most of these plants are widely distributed and consumed globally, particularly in developing countries, where larger proportion depend on plants and their products for their primary health care challenges [
2] and chronic diseases. This is perhaps because of poverty, the increasing cost of modern medicines and little awareness of the plants’ side effects [
3].
Clinacanthus nutans is a known herbal plant that belongs to the family Acanthaceae, which consists of 250 genera and about 2500 species, which are mostly tropical herbs or shrubs, and epiphytes in some cases [
2]. The genus Clinacanthus consists of two species,
C. nutans (Burm. F.) Lindau and
C. siamensis Bremek, both of which are found throughout Southeast Asia. The two species have different pharmacological characteristics, molecular aspect and anti-herpes simplex virus (HSV) types 1 and 2 activities [
4].
C. nutans is locally known as “Sabah snake grass” or “rumput belalai gajah” (in Malaysia), “Phayo yo” (in Thailand) and “Dandang Gendis” (in Indonesia) [
5]. The plant has been initially used to treat poisonous snake bites; however, it was later identified to possess potential antiviral properties [
6]. It is traditionally used in Thailand for the treatment of various illnesses including cancers, skin rashes, snake and insects bites, diabetes mellitus, diarrhoea, as an anti-viral agent against HSV and varicella-zoster virus (VZV) [
2,
7]. The mechanism of action of this plant is attributed to its anti-cell lysis property rather than an antineuromuscular transmission blocker [
6]. The wide range of pharmacological activities associated with
C. nutans could be attributed to the bioactive compounds reported in different parts of the plant, including saponins, glycosides, steroids and flavonoids [
8,
9]. Recently, Hao et al. [
10] reported that preparations of silver and gold nanoparticles using aqueous leaf extract of
C. nutans Lindau demonstrated higher analgesic and muscle relaxant activities at concentrations of 50, 100 and 150 mg/kg body weight in BALB/c mice, compared to the methanolic extract of the plant at concentrations of 100, 200 and 400 mg/kg [
10].
Toxicity evaluation of
C. nutans therefore becomes very necessary in order to ascertain its safety levels that should be used for the treatment of various illnesses. The current literature mostly reported the toxicity effects of the aqueous leaf extracts of the plants [
11,
12], while a few others that employed the methanolic/ethanolic leaf extracts reported that the plant is not associated with toxicity effects mostly based on the evaluation of body weight changes, haematological and serum biochemical parameters [
9,
13,
14], while the effects of the extracts on the histology of liver and kidneys, which were the primary organs involved in the detoxification and excretion of potential toxic substances from the body, were not reported.
Murugesu at al. [
15] investigated the in vitro toxic effect and the lethal concentration of non-polar fraction of
C. nutans at concentrations of 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL on zebrafish embryos at 72 hpf. The results showed that the median lethal concentration (LC
50) was found to be 75.49, which was harmful [
15] according to the organization for economic co-operation and development (OECD) guideline. The concentration of 500 µg/mL was toxic to the embryos showing 100% mortality after 24 h. Other malformations reported by Murugesu et al. [
15] due to hexane extract of
C. nutans in zebrafish embryos included spinal curvature, oedema, malformed yolk sac, as well as reduced hatchability and growth retardation [
15]. These in vitro toxic lesions make it necessary to study further the in vivo toxicity effects of
C. nutans extract in laboratory animals.
Currently, there is paucity of information on the bioactive compounds, nutritional composition and toxicity effects of
C. nutans cultivated in Pahang-Malaysia. Some of the previous studies [
3,
9] reported the groups of phytochemical compounds (e.g., saponins, glycosides, steroids, flavonoids, etc.) in
C. nutans. However, the specific phytocompounds in this plant cultivated in Pahang-Malaysia have not been adequately investigated. Besides, there is no uniformity in the previous reports that investigated the phytochemical compounds of the plant [
16,
17,
18], possibly due to the differences in agro-climatic conditions, genetic factors, differences in cultivation techniques, drying and extraction methods employed [
19,
20]. Förster et al. [
21] reported that the secondary metabolites of plants are highly susceptible to various environmental conditions, including temperature regulation, water quality and cycling, carbon and nutrient cycle, etc. This makes it necessary to verify the phytochemical compounds of medicinal plants in each field trial in the various cultivation areas [
21].
Consequently, this study investigated the bioactive compounds of C. nutans ethanolic leaf extract cultivated and collected from Pahang-Malaysia using a liquid chromatography-electrospray ion mass spectrometer (LCESI-MS/MS). Moreover, the detailed effects of single as well as repeated oral administration of CELE on the blood parameters as well as histology of liver and kidney of female institute of cancer research (ICR) mice were investigated in this study. This study would provide reliable data on the bioactive compounds of C. nutans cultivated in Pahang-Malaysia, at the same time provide details possible toxic effects of CELE on the histology of liver and kidneys of female ICR mice, which could be useful as a guide in selecting appropriate doses for future treatment studies using the plant extract.
3. Discussion
Analysis of bioactive compounds from a plant (also known as metabolomics) is considered a powerful discipline in plant sciences that is applicable in many aspects of plant biology, including aspects of growth and development, responses to external stimuli, genetics as well as nutritional requirements [
22,
23]. Phytochemical compounds are biologically important chemicals that occur naturally in plants. They may be responsible for colour and other organoleptic properties of plants. Phenolics, alkaloids, saponins, glycosides, terpenes, tannins, anthraquinones and steroids are some of the groups of phytochemical compounds identified in most medicinal plants [
24,
25]. These phytochemicals have been reported to be responsible for biological activities, including antioxidant, tissue protective, analgesic, antiulcer, antihypertensive, radioprotective and immunomodulatory effects, associated with most of the medicinal plants including
C. nutans in both humans and animal studies [
26]. Previous phytochemical investigation of
C. nutans extracts revealed the presence of various bioactive compounds including C-glycosyl flavones [
27], phytosterols, triterpenoid [
28], stigmasterol, glycoglycerolipids [
29], lupeol, b-sitosterol, belutin, sulphur-containing glycosides and some compounds related to chlorophyll a and chlorophyll b. The extraction method has a significant role on the phytochemical yield of
C. nutans extract [
30]. Alam et al. [
6] reported that vitexin, iso-vitexin, schaftoside, isomollupentin 7-
O-bglucopyranoside, orientin and iso-orientin were isolated from the
n-butanol and water-soluble fractions of methanolic extract of this plant in Thailand.
The results of the LC/MS/MS analyses of the
C. nutans ethanolic leaf extract in this study demonstrated the presence of myricetin, orientin, iso-orientin, vitexin, iso-vitexin, isookanin, apigenin and ferulic acid as some of the important bioactive compounds of the plant. However, three of these compounds—myricetin, isookanin and ferulic acid—were additional compounds identified in the
C. nutans ethanolic leaf extract cultivated in Pahang-Malaysia, which were not among the compounds identified earlier by Huang et al. [
17] and Chelyn et al. [
18], as well as other recent literatures as reported by Khoo et al. [
31].
The identification of orientin, isoorientin, vitexin and isovitexin in this study corroborates the earlier report by Huang et al. [
17] where a total of seven compounds including shaftoside (apigenin-6-
C-β-
d-glucopyranosyl-8-
C-α-
l-arabinopyranoside), apigenin 6,8-
C-α-
l-pyranarabinoside, orientin, isoorientin, isovitexin and vitexin were identified from 30% ethanol extract of
C. nutans. Similarly, Chelyn et al. [
18] reported that shaftoside, iso-orientin, orientin, iso-vitexin and vitexin were the major flavonoids found in the leaves of
C. nutans cultivated in Perak, Johor, and Negri Sembilan, Malaysia [
18].
Vitexin and isovitexin are bioactive compounds found in many traditional Chinese medicine and in various medicinal plants [
32]. Vitexin, which is otherwise known as apigenin-8-
C-glucoside, has nowadays gained increasing attention because of its diverse pharmacological activities, including antioxidant, anticancer, anti-inflammatory, neuroprotective, etc. Isovitexin, on the other hand, is an isomer of vitexin also known as apigenin-6-
C-glucoside, and it is also associated with wide range of biological activities [
32].
Orientin is a water-soluble flavonoid
C-glycoside that is chemically known as 2-(3,4-dihydroxyphenyl)-5,7-dihydrox-y-8[(2
S,3
R,5
S,6
R)-3,4,5-trihydroxy-6-(hydroxymethyl)]chromane-4-one. The compound has been isolated from different medicinal plants including
Ocimum sanctum,
Phyllostachys species (bamboo leaves),
Passiflora species (passion flowers),
Trollius species (Golden Queen) and
Jatropha gossypifolia (Bellyache Bush) [
33]. Orientin has been reported to be associated with various pharmacological activities including antibacterial, antiviral, anti-inflammatory, antioxidant and antiageing, among others [
33]. Moreover, isoorientin is a flavone that is a 6-
C-glucoside of luteolin. It has been reported to have antidiabetic, anti-inflammatory, proapoptotic and antioxidant activities.
The three additional compounds identified in this study were also reported to have various pharmacological activities. Myricetin is one of the common plant derived flavonoids that is well known for its nutraceutical activities [
34]. It is one of the naturally occurring phenolic compounds in fruits, berries, vegetables, teas, medicinal plants and wines produced from various plants [
34,
35]. Myricetin was first isolated in India from the bark of
Myrica nagi Thunb. (Myricaceae) as light yellow-coloured crystals [
36]. The compound is recognised mainly for its iron-chelating, anticancer, antioxidant, anti-inflammatory and antidiabetic activities among others [
34,
37].
Ferulic acid belongs to the phenolic acids group that are commonly found in plant tissues [
38]. It is mostly found in whole grains, grapes, parsley, spinach, oats, rhubarb and barley [
39]. Similarly, ferulic acid was reported to have several physiological functions ranging from antioxidant, anti-inflammatory, antimicrobial, antidiabetic and anticancer activities [
39]. It is also widely used in skin care formulations as a photoprotective agent and as an anti-skin photoageing process. However, ferulic acid is reported to have a high tendency for rapid oxidation [
39,
40,
41,
42], which could make it prone to toxicity and oxidative stress.
The presence of these compounds could be responsible for most of the pharmacological activities associated with
C. nutans, including antitumor, anti-snake and insect bites, anti-VZV lesions and anti-hepatitis activities reported in the literature [
17,
43,
44]. Although these compounds are reported to have potential pharmacological activities, it should be noted that the compounds could also have some adverse effects on normal cells, especially when consumed in large quantities.
The assessment of the toxic effect of plant is very necessary in evaluating its safety for both human and animal use. Evaluation of acute toxicity study of a plant or substance may perhaps provide significant information for identification of the targeted organs of the test substances following acute exposure [
45]. The acute toxicity study of CELE in this study revealed that the extract at 2000 mg/kg did not cause any mortality or any signs of acute toxicity in the treated mice. The appetite and activities of the mice in both the two experimental groups were not affected by the administration of the extract throughout the period of observation. This report corroborates with the earlier reports by P’ng et al. [
14], who reported that administration of
C. nutans methanolic leaf extract at 900 and 1800 mg/kg is not associated with either mortality or any signs of acute toxicity in the treated mice. Similarly, Sajjaratul et al. [
7] and Khoo et al. [
12], respectively, reported that administration of 2000 mg/kg ethanolic extract and 5000 mg/kg aqueous extract of
C. nutans did not cause any toxicity nor mortality in Sprague Dawley rats throughout the 14-day period of observation.
The significant fluctuations in the body weight gain of the mice treated with 2000 mg/kg CELE once in this study could suggests that the extract could have affected the appetite of the mice at the beginning, but shortly after, the animals have adjusted, and the extract resulted in significant weight gain at the end of the experimental period. This is in agreement with P’ng et al. [
14] and Khoo et al. [
12], who also recorded a significant increase in the body weight of mice and rat, respectively, administered with methanolic and aqueous
C. nutans extracts. Furthermore, the changes in the body weight gain pattern were similar to that reported by Khoo et al. [
12], who observed significant alteration in carbohydrate metabolism, energy metabolism and amino acid metabolism in Sprague Dawley rats treated with 5000 mg/kg
C. nutans aqueous extract 2 h post administration. Nonetheless, the metabolic expression collected 24 h, 5, 10 and 15 days post-administration showed that the rats have overcome the effects of the extracts and did not show accumulation of any toxicity biomarkers [
12].
The oral administration of a high dose of the extract (CELE) at 2000 mg/kg once did not affect the haematological parameters of the ICR mice significantly in this study. This may suggest further that the extract at 2000 mg/kg may not have significant effects on the metabolism [
46,
47] of the treated mice. Correspondingly, Khoo et al. [
12] reported that administration of
C. nutans aqueous extract even at high dose of 5000 mg/kg did not affect the blood profile of Sprague Dawley rats following 14 days of observation.
Conversely, administration of the extract at 2000 mg/kg resulted in a significant increase in the levels of ALT, AST and CK, which may suggest that the extract has some adverse effects on the liver [
48,
49] of the treated mice. Correspondingly, histopathological evaluation of liver and kidneys suggested that the extract at this high dose of 2000 mg/kg might have induced mild histopathological lesions in the liver and kidneys of the treated mice. The histopathological lesions observed in this study, including cytoplasmic vacuolation and eosinophilic cytoplasm, although not significant, may suggest that the plant extract at 2000 mg/kg exhibited some degree of degenerative and necrotic effects [
50,
51] on the liver and kidney of the treated mice. The significant differences in the liver injury markers observed in this study contradicts the earlier reports by Khoo et al. [
12], where administration of 5000 mg/kg aqueous extract of
C. nutans did not cause any significant changes in the levels of AST, ALT, ALP and total bilirubin between the treated and untreated groups of rats; this may perhaps be due to the differences in the extraction solvents, as water has less ability to extracts phytochemicals compared to ethanol [
30,
52,
53].
The subacute toxicity study of this research revealed significant alterations in certain parameters of the treated mice. The significant decrease in weight gain observed in the groups of mice treated with 500 mg/kg and 1000 mg/kg daily for 28 days could suggest that the extract affected the mice’s feed intake [
54] or has resulted in the reduction in the deposition of fats [
46,
55]. Reduction in the body weight gain has been reported to be associated with toxicity following exposure to potential toxic chemical or substances in animals [
56,
57]. These findings are in agreement with the report of Chavalittumrong et al. [
58], where a significant decrease body weight of male rats treated with 1.0 g/kg of
C. nutans ethanolic extract daily for 90 days compared to the control group was reported. However, it is contrary to the report of Zakaria et al. [
9], who reported that repeated oral administration of methanolic extract of
C. nutans daily for 28 days did not affect the body weight of the mice even at the highest dose of 2500 mg/kg. This perhaps may be due to shorter duration of exposure [
56,
57] compared to the present study.
The repeated oral administration of the extract at 125 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg daily for 28 days in this study did not affect the haematological parameters of the ICR mice significantly, suggesting that the metabolic processes of the mice were not significantly affected [
59,
60] by the administration of the extract.
The mean corpuscular volume (MCV) is used to classify anaemia as microcytic (below the normal range), normocytic (within the normal range) or macrocytic (above the normal range) [
61,
62]. Therefore, the significant alterations in the values of MCV observed in this study could suggest that the extract has macrocytic effect on the RBC of the treated mice. Similarly, the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC) are also used as indices for diagnosis of anaemia. Decreased level of MCHC may indicate hypochromasia in early iron deficiency anaemia [
63]. Therefore, the significant increase in the level of MCH observed in this study could suggests that the extract might have certain compounds capable of promoting haemoglobin production and this is in agreement with the report of Archibong et al. [
61].
The results of plasma biochemical parameters showed that daily oral administration of CELE for 28 days at 500 and 1000 mg/kg doses has effect on some plasma biochemical parameters. The significant increase in creatinine level detected in the group of mice treated with 1000 mg/kg daily for 28 days could suggest that the extract especially at higher doses of 500 and 1000 mg/kg may possess some adverse effects on the kidneys of the ICR mice [
48,
64,
65]. This is because creatinine, which is the product of creatine metabolism, is solely excreted by the kidneys; therefore, any injury to the kidneys may result in hypercreatinaemia [
66]. These findings were similar to the report of Zakaria et al. [
9], where there was significant increase in the level of creatinine in both male and female mice treated with both 500 and 1000 mg/kg
C. nutans repeatedly for 28 days. However, Chavalittumrong et al. [
58] reported a significant decrease in creatinine levels in the male rats treated with 1000 mg/kg
C. nutans ethanolic extract daily for 90 days compared to the control. The significant increase in the levels of ALT observed in this study at 500 and 1000 mg/kg, could suggest that the extract at this high doses might have affected the normal function of liver in the treated mice [
48,
49,
67]. This was further supported by the results of the histopathological evaluation of liver and kidney in this study.
The histopathological evaluation of liver and kidney of the experimental mice in this study revealed that daily oral administration of CELE for 28 days at 1000 mg/kg dose resulted in various hepatic and renal lesions. The significant hepatic degeneration and necrosis observed histopathologically in the group of mice administered with daily oral doses of CELE at 1000 mg/kg for 28 days were suggestive of toxic or adverse effects [
7,
50,
51,
68,
69,
70] of the extract on the animals, as earlier observed in the results of plasma biochemical parameters for hepatic injury markers (ALT and AST). Similarly, the renal tubular degeneration and necrosis observed in this research further supported the results of plasma biochemical parameters, providing further indication that administration of the extract at 1000 mg/kg is not safe [
50,
51] for the female ICR mice. Severe hepatic damage has been reported to be associated with oxidative stress and depletion of ATP leading to necrosis of the hepatocytes [
71,
72].
P’ng et al. [
13] reported that Food and Agricultural Materials Inspection Centre (FAMIC) uses the acceptable daily intake (ADI) to determine the non-toxic level of a test substance to humans based on the non-observable adverse effect level (NOAEL) value obtained from animal trials [
13]. Based on the results of this study, the NOAEL of ethanolic leaf extract of
C. nutans was 250 mg/kg in mice, whereas the low observable adverse effect level was 500 mg/kg in mice. Therefore, the ADI of
C. nutans ethanolic leaf extract was determined to be 2.5 mg/kg, according to the method described by P’ng et al. [
13]