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Background: The composition and bioactivity of natural plant extract strongly depend on the extraction technique employed. Clinacanthus nutans Lindau (C. nutans) is a well-known medicinal plant in South-East Asia that has been traditionally used for the treatment of various diseases. Several conventional methods have been using for the extraction of bioactive compounds from C. nutans. However, extraction of fatty acids using supercritical carbon dioxide was not reported yet from this medicinal herbs. Objective: The main objective of the study is to examine the potential of supercritical carbon dioxide (scCO2) extraction of fatty acids from leaves and stems of C. nutans. Method: Fatty acid compositions were determined from leaves and stems of C. nutans oil extracted by scCO2 (temperature 45-65 °C, pressure 25-35 MPa) and compared to the results of Soxhlet extraction. Results: Supercritical CO2 extraction shows the highest oil recovery in both leaves (3.7%) and stems (1.6%) at pressure 35 MPa, temperature 65°C and 2 ml/min flow rate, which was closer to the yield of Soxhlet. The scCO2 yields presented a higher percentage of polyunsaturated fatty acids (PUFA), especially linoleic acid (C18:2n-6). Palmitic acid ranging from 42%- 47% in leaves and stems of C. nutans was found dominant saturated fatty acids (SFA) in both scCO2 and Soxhlet method. Conclusion: The current results indicate that leaves and stems of C. nutans could be a potential source of fatty acids, especially biologically active compounds.

The development of new and effective pain medications that are natural is expected to have the benefits of treatment. The purpose of this study was to determine the antinociceptive effectiveness of a combination of 75% ethanol extract from dandang gendis leaves and 96% ethanol fraction of bakung leaf. In this study was used chemical method induction (Sigmund's method) in 25 male mice which were divided into 5 treatment groups, namely treatment group I as negative control was given CMC 0.5%, treatment group II as positive control was given acetosal 100 mg / kg BW, treatment groups III, IV and V were given combinations extract. Each group was given treatment orally, thirty minutes then induced with 0.6% acetic acid intraperitonially, after which it was observed and counted for the amount of stretching for sixty minutes. Data were analyzed by one-way ANOVA test. The results of this study showed that all treatment groups had a significant difference (p

Silver nanoparticles (AgNPs) previously synthesised using leaf (AgNP-L) and stem (AgNP-S) extracts of Clinacanthus nutans (C. nutans) were tested to evaluate antimicrobial, antioxidant, and cytotoxicity activities. The AgNPs showed good inhibition against bacteria, but not fungi. The inhibition results showed the highest activity against Staphylococcus aureus (S. aureus) with 11.35 mm (AgNP-L) and 11.52 mm (AgNP-S), while the lowest inhibition was against Escherichia coli (E. coli) with 9.22 mm (AgNP-L) and 9.25 mm (AgNP-S) in the disc diffusion method. The same trend of results was noted in the well diffusion method. The IC50 of AgNP-L and AgNP-S in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays was 417.05 μg/mL and 434.60 μg/mL, as well as 304.31 μg/mL and 326.83 μg/mL, respectively. Ferric reducing power (FRAP) assay showed that AgNP-L [872.389 μmol/L Fe(II)] and AgNP-S [612.770 μmol/L Fe(II)] exhibited significantly ( p p

Context Atherosclerosis predisposes individuals to adverse cardiovascular events. Clinacanthus nutans L. (Acanthaceae) is a traditional remedy used for diabetes and inflammatory conditions. Objectives To investigate the anti-atherosclerotic activity of a C. nutans leaf methanol extract (CNME) in a type 2 diabetic (T2D) rat model induced by a high-fat diet (HFD) and low-dose streptozotocin. Materials and methods Sixty male Sprague-Dawley rats were divided into five groups: non-diabetic fed a standard diet (C), C + CNME (500 mg/kg, orally), diabetic fed an HFD (DM), DM + CNME (500 mg/kg), and DM + Metformin (DM + Met; 300 mg/kg). Treatment with oral CNME and metformin was administered for 4 weeks. Fasting blood glucose (FBG), serum lipid profile, atherogenic index (AI), aortic tissue superoxide dismutase levels (SOD), malondialdehyde (MDA), and tumour necrosis factor-alpha (TNF-α) were measured. The rats’ aortas were stained for histological analysis and intima-media thickness (IMT), a marker of subclinical atherosclerosis. Results The CNME-treated diabetic rats had reduced serum total cholesterol (43.74%; p = 0.0031), triglycerides (80.91%; p = 0.0003), low-density lipoprotein cholesterol (56.64%; p = 0.0008), AI (51.32%; p

Clinacanthus nutans was found to possess anti-venom, anti-inflammatory, analgesic, anti- diabetic, anti-rheumatism, antiviral and antioxidant properties. Silver nanoparticles are nanoparticles between 1nm to 100nm in size and play significant role in medicinal fields. Silver nanoparticles exhibit unique properties, such as excellent conductivity, chemical stability, catalytic and antimicrobial activity. Streptococcus mutans is usually discovered in human oral cavity and the main aetiological of tooth decay. There is no study on antibacterial effect of silver nanoparticles Clinacanthus nutans (AgNP-CN) against Streptococcus mutans reported to date. Therefore, objective of this study is to investigate antibacterial properties of silver nanoparticles Clinacanthus nutans against Streptococcus mutans. Streptococcus mutans was subcultured in brain heart infusion (BHI) broth and agar. AgNP-CN with different concentrations was tested against Streptococcus mutans via disc diffusion assay, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Amoxicillin was used as positive control while DMSO and blank silver nanoparticles were used as negative control. Disc diffusion assay shows AgNP-CN inhibit Streptococcus mutans growth. AgNP-CN shows the greatest inhibition properties (15.3±1.53 mm) in comparison to Clinacanthus nutans leaves extract (6.0±0.01 mm) and blank silver nanoparticles (6.0±0.01 mm). MIC value for AgNP-CN is 2.5±0.01 mg/mL while amoxicillin is 0.007±0.01 mg/mL. Whereas MBC values for AgNP-CN is 2.5 mg/mL. Results are concentration dependent, with higher concentration shows better inhibition property. It can be concluded AgNP-CN possesses bactericidal properties against Streptococcus mutans.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1β, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1β transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1β mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1β, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1β siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1β transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

Objective: The present study aims to evaluate the antioxidant, antimicrobial and cytotoxic properties of the secondary metabolites and proteins extracted from Clinacanthus nutans. Methods: Methanol (ME) and acetone (AC) and also protein (PE) extracts were obtained from Clinacanthus nutans. Antioxidant activity of sample extracts was examined by using 1,1-Diphenyl-2-Picrydyl Hydrazyl (DPPH) assay and antimicrobial activity was examined by using the agar diffusion method against Bacillus subtilis, Bacillus cereus, Streptococcus pyrogenes, Escherichia coli, and Pseudomonas aeroginosa bacterial strain. The cytotoxic potential was determined by brine shrimp lethality assay. Results: Methanol extract (ME) demonstrated to have the highest antioxidant activity with IC50 value of 98.84 μg/ml, followed by acetone extract (AE) with IC50 value of 134.83µg/ml, and protein extract (PE) with IC50 value of 353.49 µg/ml. Among all of the sample extracts, only methanol crude extracts (ME) displayed moderate inhibition against Gram-positive B. cereus (7.33±1.15 mm) and S. pyrogenes (8.67±0.57 mm) at concentration of 100 mg/ml, while both acetone (AE) and protein (PE) extracts had no activity against all tested microorganisms. All extracts from methanol (ME), acetone (AE) and protein (PE) exhibited significant cytotoxic activity against brine shrimps at LC50 7.2 μg/ml, 1.42 μg/ml and 70.6 μg/ml respectively. Conclusion: These data proved that methanol extract from the leaves of Clinacanthus nutans is the most potent among all samples tested and has the potential to be developed as an antioxidant, antimicrobial and cytotoxic agents. Whilst, acetone and protein crude extracts have potent antioxidant and cytotoxicity activities.

PurposeThe purpose of this paper is to evaluate the antioxidant activity and toxicity of Clinacanthus nutans leaves from three conventional extraction methods, i.e. maceration, Soxhlet and magnetic stirring.Design/methodology/approachTotal flavonoid content (TFC) and phenolic content (TPC) were determined using colorimetric method of aluminum chloride and Folin-Ciocalteu (FC) assay, respectively. Antioxidant property of C. nutans was evaluated using 2,2'-diphenyl-1-pierylhydrazyl (DPPH) free radical scavenging assay. Cytotoxic activity of C. nutans against brine shrimp was evaluated based on LC50 (lethality concentration) after 24 h exposure to the plant extract.FindingsThe highest TPC of C. nutans was observed with Soxhlet extraction method (98.87 ± 10.43 mg of gallic acid equivalents (GAE/g) followed by maceration (68.77 ± 2.45 mg of GAE/g) and magnetic stirring (46.75 ± 2.45 mg of GAE/g). Interestingly, remarkable highest TFC was observed with magnetic stirring (568.90 ± 4.85 mg of rutin equivalent (RE)/g) followed by maceration (249.60 ± 2.79 mg of RE/g) and Soxhlet (174.8 ± 1.74 mg of RE/g). On the other hands, the extract obtained using maceration method showed the highest antioxidant activity (IC50: 14.18 mg/mL compared to ascorbic acid 144.36 µg/mL). Cytotoxicity of C. nutans from all extraction methods showed similar LC50 values with maceration (3.81 mg/mL), Soxhlet (2.61 mg/mL) and magnetic stirring (4.56 mg/mL), respectively.Originality/valueBoth phenolic and flavonoids are responsible for the antioxidant activity, of C. nutans extracts. Based on Meyer's toxicity index, all extracts were nontoxic (LC50>1 mg/mL).

Clinacanthus nutans is a great medicinal herb with high polyphenols content and had been used in its infusion form to treat illness. In this paper, microwave-assisted extraction of C. nutans tea infusion was investigated by using central composite design combined with response surface methodology. The influence factors of microwave power (80 - 800 W); brewing time duration (0 – 240 s) and extraction time duration (60 s – 300 s) on total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (DPPH) and acetylcholinesterase inhibition activity (AChE) were investigated. The optimum extraction conditions were determined as follows: microwave power X1, 560 W; brewing duration, X2, 60 s and extraction time, X3, 240 s. Under this optimal extraction conditions, the responses were as followed: Total phenolic content (251.58±10.98 mg GAE/L); Total flavonoid content (102.21±5.16 mg RE/L); DPPH (62.22±0.03%) and acetylcholinesterase inhibition activity (AChE) of 24.77±0.12%. Results demonstrated that MAE could be used to extract bioactive constituents having antioxidant and anti-acetylcholinesterase properties that may benefit in Alzheimer’s disease.

BACKGROUND: Osteoporotic-osteoarthritis is an incapacitating musculoskeletal illness of the aged. OBJECTIVES: The anti-inflammatory and anti-catabolic actions of Diclofenac were compared with apigenin-C-glycosides rich Clinacanthus nutans (CN) leaf extract in osteoporotic-osteoarthritis rats. METHODS: Female Sprague Dawley rats were randomized into five groups (n = 6). Four groups were bilateral ovariectomised for osteoporosis development, and osteoarthritis were induced by intra-articular injection of monosodium iodoacetate (MIA) into the right knee joints. The Sham group was sham-operated, received saline injection and deionized drinking water. The treatment groups were orally given 200 or 400 mg extract/kg body weight or 5 mg diclofenac /kg body weight daily for 28 days. Articular cartilage and bone changes were monitored by gross and histological structures, micro-CT analysis, serum protein biomarkers, and mRNA expressions for inflammation and catabolic protease genes. RESULTS: HPLC analysis confirmed that apigenin-C-glycosides (shaftoside, vitexin, and isovitexin) were the major compounds in the extract. The extract significantly and dose-dependently reduced cartilage erosion, bone loss, cartilage catabolic changes, serum osteoporotic-osteoarthritis biomarkers (procollagen-type-II-N-terminal-propeptide PIINP; procollagen-type-I-N-terminal-propeptide PINP; osteocalcin), inflammation (IL-1β) and mRNA expressions for nuclear-factor-kappa-beta NF-κβ, interleukin-1-beta IL-1β, cyclooxygenase-2; and matrix-metalloproteinase-13 MMP13 activities, in osteoporotic-osteoarthritis rats comparable to Diclofenac. CONCLUSION: This study demonstrates that apigenin-C-glycosides at 400 mg CN extract/kg (about 0.2 mg apigenin-equivalent/kg) is comparable to diclofenac in suppressing inflammation and catabolic proteases for osteoporotic-osteoarthritis prevention. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40199-020-00343-y) contains supplementary material, which is available to authorized users.

Limitations in existing anticoagulants have prompted a search for the natural origin of anticoagulants. Since there is an increasing need to source for pharmacological and medicinal materials from a plant source, thus an exploratory effort towards identifying and characterizing new anticoagulants from plants is worthwhile. Clinacanthus nutans (C. nutans) commonly known as Sabah snake grass belongs to Acanthaceae family was chosen since it has therapeutic and medicinal benefits. This plant possessed anti-oxidant, anti-microbial, anti-inflammatory, and anti-diabetic activities. This study was carried out to identify the anticoagulant activities of C. nutans leaves in aqueous and methanol extracts at different concentrations based on prothrombin time (PT) and activated partial thromboplastin time (aPTT). The dried leaves of C. nutans were ground into a fine powder and extracted using aqueous and methanol. Anticoagulant assays of PT and aPTT were done on three different concentrations of C. nutans aqueous and methanol extracts (10 mg/mL, 20 mg/mL, and 30 mg/mL) and control group respectively. PT and aPTT of plasma samples were prolonged in both types of extracts (p

Isolation of anti-proliferative compounds from plants is always hindered by the complexities of the plant’s nature and tedious processes. Clinacanthus nutans (Burm. f.) Lindau is a medicinal plant with reported anti-proliferative activities. Our study aimed to isolate potential anti-proliferative compounds present in C. nutans plant. To start with, for our study, we came up with a strategy by first profiling the volatile compounds present in the leaf, stem and root of C. nutans using GC-MS. Comparing the plant’s volatile profiles greatly narrowed down our target of study. We decided to start with the isolation and characterization of a pentacyclic terpenoid, i.e., lupeol from the roots of C. nutans, as this compound was found to present abundantly in the roots compared to the leaf or stem. We developed a simple maceration and re-crystallization method, without the necessity to go through the fractionation or column chromatography for the isolation of lupeol. Characterizations of the isolated compound identified the compound as lupeol. The anti-proliferative activity of the isolated lupeol was further investigated against the MCF-7 cell line, which showed comparable anti-proliferative activity with the authentic lupeol and camptothecin. Our strategy to profile every part of the plant first, followed by selection of the most suitable plant part and targeted compound proved useful for further isolation and characterization bioactive compound from C. nutans.

The lack of specific treatment for chikungunya fever makes the need for anti-chikungunya virus agents more crucial. This study was conducted to evaluate 132 extracts obtained by sequential solvent extraction from 21 medicinal plants for cytopathic effect inhibitory activity using virus-infected Vero cells in two different sample introduction modes. Among the extracts, 42 extracts (31.8%) from 12 plants in the concurrent mode and three extracts (2.3%) from a plant in the non-concurrent mode displayed strong cytopathic effect inhibitory activity (cell viability ≥70%). Viral load quantification analysis unveiled that the extracts of Clinacanthus nutans (chloroform, ethyl acetate, and ethanol), Hydrocotyle sibthorpioides (ethanol), and Ocimum americanum (ethanol and methanol) hindered the release of viral progeny from the infected cells while the extracts of Ficus deltoidea (ethanol), Gynura bicolor (water), H. sibthorpioides (water), and O. americanum (chloroform and ethyl acetate) blocked the entry of virus into the cells. The extracts of Diodella sarmentosa (ethyl acetate), Diplazium esculentum (chloroform, ethyl acetate, and ethanol), and G. bicolor (ethanol) possessed virucidal effect and caused 5.41-log to 6.63-log reductions of viral load compared to the virus control. The results indicate that these medicinal plants are potential sources of anti-chikungunya virus agents that have varied modes of action.

The use of metabolomics as a comprehensive tool in the analysis of metabolic profiles in disease progression and therapeutic intervention is rapidly advancing. Yet, a single analytical platform could not be applied to cover the entire spectrum of a biological sample’s metabolome. In the present paper, multi-platform metabolomics approaches were explored to determine the diverse rat sera metabolites extracted from intracerebroventricular lipopolysaccharides (LPS)-induced neuroinflammed rats treated with oral therapeutic interventions of positive drug (dextromethorphan, 5 mg/kg BW); with Clinacanthus nutans (CN) aqueous extract (CNE, 500 mg/kg BW); and with phosphate buffer saline (PBS) as the control group for 14 days. Analyzed by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) techniques, this study depicted the potential of metabolites associated with neuroinflammation and verified by MetDisease. The key observations in the perturbed metabolic pathways that showed ameliorative effects were linked to the class of amino acid and peptide metabolism involving valine, leucine, and isoleucine biosynthesis; phenylalanine, tyrosine, and tryptophan biosynthesis; and phenylalanine metabolism. Lipid metabolism of arachidonic acid metabolism, glycerophospholipid metabolism, terpenoid backbone biosynthesis, and glycosphingolipid metabolism were also affected. Current findings suggested that the putative biomarkers, especially lysophosphatidic acid (LPA) and 5-diphosphomevalonic acid from glycerophospholipid and squalene/terpenoid and cholesterol biosynthesis, respectively, showed the ameliorative effects of the drug and CN treatments by controlling cell differentiation and proliferation. Our study proved that the complex and dynamic sera profiling affected during the CN treatment was greatly influenced by the analytical platform selection as integration between the two data yielded a more holistic summary of the metabolite pattern changes. Hence, an evidence-based herb, such as CN, can be used for novel diagnostic tools in the quest for ethnopharmacological studies.

Cervical cancer is a leading cause of death in women, worldwide, and second in Malaysia. Nowadays, modern cancer treatment comes with negative and adverse side effects to the patients. Traditional medicinal plants such as Clinacanthus nutans has been locally recognized for its medicinal properties and was claimed for cancer treatment. According to our pilot study, a fraction of C. nutans has shown a potent inhibition on human cervical cancer cells, SiHa, in vitro. Hence, this study aimed to investigate the underlying anticancer mechanism of a semi-purified fraction (SF1) of C. nutans towards cervical cancer cells. Bioassay guided fractionation and further purification was carried out. All collected fractions of C. nutans were examined for cytotoxicity activity towards HeLa, SiHa and NIH cells by MTT Assay. SF1 exhibited better cytotoxic activity with best growth inhibition against SiHa cells (IC50 value = 9.60 μg/mL). Cytoselective effects were shown as no cytotoxicity observed in the treated normal NIH cells. SF1 induced early apoptosis event in SiHa cells as resulted by FITC-Annexin V/propidium iodide staining by flowcytometric analysis. The protein expression by flowcytometry using specific antibody conjugated fluorescent dye showed up-regulation of Bax and down-regulation Bcl-2 as well as increment of cytochrome C levels upon treatment with SF1. In conclusion, the findings suggested that SF1 demonstrated antiproliferative effects and induced apoptosis in SiHa cells through up-regulation of Bax and down-regulation of Bcl-2 via mitochondrial-dependent pathway.

The present study used in vitro and in silico techniques, as well as the metabolomics approach to characterise α-glucosidase inhibitors from different fractions of Clinacanthus nutans. C. nutans is a medicinal plant belonging to the Acanthaceae family, and is traditionally used to treat diabetes in Malaysia. n-Hexane, n-hexane: ethyl acetate (1:1, v/v), ethyl acetate, ethyl acetate: methanol (1:1, v/v), and methanol fractions were obtained via partitioning of the 80% methanolic crude extract. The in vitro α-glucosidase inhibitory activity was analyzed using all the fractions collected, followed by profiling of the metabolites using liquid chromatography combined with mass spectrometry. The partial least square (PLS) statistical model was developed using the SIMCA P+14.0 software and the following four inhibitors were obtained: (1) 4,6,8-Megastigmatrien-3-one; (2) N-Isobutyl-2-nonen-6,8-diynamide; (3) 1′,2′-bis(acetyloxy)-3′,4′-didehydro-2′-hydro-β, ψ-carotene; and (4) 22-acetate-3-hydroxy-21-(6-methyl-2,4-octadienoate)-olean-12-en-28-oic acid. The in silico study performed via molecular docking with the crystal structure of yeast isomaltase (PDB code: 3A4A) involved a hydrogen bond and some hydrophobic interactions between the inhibitors and protein. The residues that interacted include ASN259, HID295, LYS156, ARG335, and GLY209 with a hydrogen bond, while TRP15, TYR158, VAL232, HIE280, ALA292, PRO312, LEU313, VAL313, PHE314, ARG315, TYR316, VAL319, and TRP343 with other forms of bonding., Graphical abstract fx1, Highlights • The antidiabetic potential of various extracts of C. nutans leaf has been evaluated. • Multivariate data analysis identified 4 α-glucosidase inhibiting compounds. • In silico study predicted the protein-inhibitors interaction.

Clinacanthus nutans (Burm. f.) Lindau (C. nutans) has been reported to lower blood glucose level; however, evidence on its efficacy in lowering diabetic complications is limited. The antidiabetic properties of C. nutans aqueous leaf extract on serum metabolic indices, sorbitol production, and aldose reductase enzyme activities in the kidneys, lens, and sciatic nerve of type II diabetic (T2D) rats were evaluated. All rats except normal control rats were fed with a high‐fat diet for 8 weeks to induce obesity and subsequently injected with 35 mg/kg streptozotocin to induce type II diabetes. Aqueous leaf extract of C. nutans (100 and 200 mg kg−1 day−1) and quercetin (10 mg kg−1 day−1) were fed orally for 4 weeks. Diabetic rats administered with C. nutans at 100, 200 mg kg−1 day−1 and quercetin had significantly (p

Clinacanthus nutans, an herbal shrub belonging to the Acanthaceae family, is traditionally used as a functional food to treat various ailments in Malaysia and Indonesia. Although the polar fraction of this plant shows non-toxic effect, the toxicity of the non-polar extract is not reported so far. The present study aimed to assess the toxic effect and determine the lethal concentration of this non-polar fraction using zebrafish embryos. The n-hexane fraction was partitioned from the crude extract of C. nutans obtained using 80% methanolic solution. After spawning of the adult male and female zebrafish, the eggs were collected, transferred into a 96-well plate and incubated with the n-hexane fraction at concentrations of 15.63 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml and 500 μg/ml in 2% DMSO. The survival and sublethal endpoint were assessed, the mortality and hatchability rates were calculated based on microscopic observation, while the heartbeat rate was measured using DanioScope software. The median lethal concentration (LC50) of the C. nutans n-hexane fraction, which was determined using probit analysis, was calculated to be 75.49 μg/mL, which is harmful. Moreover, gas chromatography-mass spectrometry (GC–MS) analysis revealed the presence of palmitic acid, phytol, hexadecanoic acid, 1-monopalmitin, stigmast-5-ene, pentadecanoic acid, heptadecanoic acid, 1-linolenoylglycerol and stigmasterol in the n-hexane fraction. Keywords: Clinacanthus nutans, Danio rerio, DanioScope, Median lethal concentration, Teratogenicity, Probit analysis

Diabetes mellitus is a common metabolic disease marked by hyperglycemia. Diabetes also can be caused by oxidative stress. The administration of antioxidants, whether it’s from food or supplement can inhibit the formation of free radicals. Clinacanthus nutans is reported to possess various medicinal properties including blood glucose lowering effect, alpha-glucosidase inhibition activity, antioxidant activities, anti-cancer properties and anti-inflammatory effects. Based on research Clinacanthus nutans leaf extracts fractions has several shortcomings, amongst them are its viscosity that gave difficulties in formulation and its astringent and bitter taste that made it hard to use orally. To overcome these shortcomings, microencapsulation can be used. The purpose of this research is to find the blood glucose level lowering effect of the aqueous fraction of microcapsules and to find the effective dose on fructose and fat-induced Wistar strain rat. The induction using fructose and high-fat feed for 55 days was applied. Rats were divided into 8 groups: normal control (aquadest), negative control (Arabic gum and maltodextrin), positive control (metformin), dose I 15,89 mg/ kgBW (microcapsule of aqueous fraction of Clinacanthus nutans extract), dose II 31,78 mg/ kgBW, dose III 47,67 mg/ kgBW, dose IV 63.56 mg /kgBW, and dose V 79.45 mg/ kgBW. The result showed that the microcapsules of the aqueous fraction of Clinacanthus nutans (Burm.f) extract could lower Wistar strain rat blood glucose level in the dose of 15.89 mg/kg of the rat’s body weight.

Surface sterilization is a vital step in preparation of healthy and viable explants in tissue culture. Most surface contaminants can be eliminated by surface sterilization with a suitable sterilizing agent. The study aimed to present an effective disinfection method for Clinacanthus nutans shoot regeneration using nodal segments. A total of four different sterilization approaches were conducted by treating nodal explants with various concentrations of sterilizing agent. Sterilizing agents used were Rhizophora apiculata Pyroligneous acid (PA), sodium hypochlorite (Clorox) thiophanate-methyl (fungicide), and Mercuric chloride (HgCl2). Nodal explant then was cultured on plant growth regulator-free Murashige and Skoog (MS) basal medium. This study sterilizing agents revealed that PA showed strong bactericidal activity. However, it led to a high number of fungal contaminations. The pyroligneous acid did not exhibit a strong potential as a disinfectant for C. nutans nodal explant. Overall, HgCl2 exhibits the best reduction in fungal contamination and gives a significant result with thiophanate-methyl fungicide. Surface sterilization with mercuric chloride (0.2%) for 1 hour was the optimum concentration and duration, which resulted in the highest percentage of nodal explant survival and viability. All viable nodal segments developed into shoots. It had been concluded that the best surface sterilization agent was HgCl2.

Clinacanthus nutans Lindau has been traditionally used for healing of bone fragility, but the mechanism of actions has not been clarified yet. In this study, the bone regeneration activity of lupeol derived from C. nutans was assessed using an in vitro model of osteoblast cells MC3T3-E1. The finding revealed that the compound was not significantly toxic to osteoblast cells at concentration of ≤40 μg/mL. Lupeol demonstrated the osteogenic activity through enhancement of alkaline phosphatase (ALP) of osteoblast cells up to 31.2%, 21%, and 12% at concentrations of 5, 10, and 20 µg/mL, respectively ( p p p

Background Clinacanthus nutans (C. nutans) Lind. locally known as Belalai Gajah or Sabah snake grass is a medicinal plant belonging to Acanthaceae family. In Asia, this plant is traditionally used for treating skin rashes, insects and snake bites, diabetes mellitus, fever and for diuretic effect. C. nutans has been reported to possess biological activities including anti-oxidant, anti-inflammation, anti-cancer, anti-diabetic and anti-viral activities. Methods Proton Nuclear Magnetic Resonance (1H NMR) and Liquid Chromatography Mass Spectroscopy (LCMS) coupled with multivariate data analysis were employed to characterize the metabolic variations of intracellular metabolites and the compositional changes of the corresponding culture media in rat renal proximal tubular cells (NRK-52E). Results NMR and LCMS analysis highlighted choline, creatine, phosphocholine, valine, acetic acid, phenylalanine, leucine, glutamic acid, threonine, uridine and proline as the main metabolites which differentiated the cisplatin-induced group of NRK-52E from control cells extract. The corresponding media exhibited lactic acid, glutamine, glutamic acid and glucose-1-phosphate as the varied metabolites. The altered pathways perturbed by cisplatin nephrotoxic on NRK-52E cells included changes in amino acid metabolism, lipid metabolism and glycolysis. Conclusion The C. nutans aqueous extract (1000 μg/mL) exhibited the most potential nephroprotective effect against cisplatin toxicity on NRK-52E cell lines at 89% of viability. The protective effect could be seen through the changes of the metabolites such as choline, alanine and valine in the C. nutans pre-treated samples with those of the cisplatin-induced group.

Clinacanthus nutans, commonly known as Sabah snake grass, is one of the more important medicinal plants in Malaysia&rsquo, s herbal industry. C. nutans has gained the attention of medical practitioners due to its wide range of bioactive compounds responsible for various biological activities, such as anti-cancer, anti-venom and anti-viral activities. Due to its high pharmacological properties, the species has been overexploited to meet the demands of the pharmaceutical industry. The present study was conducted to establish a suitable in vitro culture procedure for the mass propagation of C. nutans. Murashige and Skoog (MS) basal medium, supplemented with different types of cytokinins, auxins, basal medium strength and sucrose concentrations, were tested. Based on the results, a full-strength MS basal medium supplemented with 12 &micro, M 6-benzylaminopurine (BAP) and 30 g/L sucrose was recorded as the best outcome for all the parameters measured including the regeneration percentage, number of shoots, length of shoots, number of leaves and fresh weight of leaves. In the analysis of the phenolics content and antioxidant activities, tissue-cultured leaf extracts assayed at 100 &deg, C exhibited the highest phenolic content and antioxidant activities. The propagation of C. nutans via a plant tissue culture technique was recorded to be able to produce high phenolic contents as well as exhibit high antioxidant activities.

Sabah snake grass or Clinacanthus nutans has drawn public interest having significant economic benefits attributable to the presence of phytochemicals and several interesting bioactive constituents that may differ according to harvesting age and harvesting frequency. The current study was aimed to evaluate the effect of harvesting age and harvesting frequency towards herbal yield, antioxidant activities, phytochemicals synthesis, and bioactive compounds of C. nutans. A factorial randomized completely block design with five replications was used to illustrate the relationship between herbal yield, DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ferric reducing antioxidant power (FRAP) assays, total phenolic and flavonoid content affected by harvesting age (week 8, 12, and 16 after transplanting), and harvesting frequency (harvest 1, 2, and 3). The bioactive compounds by HPLC were also determined to describe the interaction effect between both harvesting age and harvesting frequency. The yield, antioxidant activities, and phytochemical contents were gradually increased as the plant grew, with the highest recorded during week 16. However, the synthesis and activities of phytochemicals were reduced in subsequent harvests despite the increment of the herbal yield. All bioactive compounds were found to be influenced insignificantly and significantly by harvesting age and harvesting frequency, respectively, specifically to shaftoside, iso-orientin, and orientin. Among all constituents, shaftoside was the main compound at various harvesting ages and harvesting frequencies. These results indicated that harvesting at week 16 with 1st harvest frequency might enhance the yield while sustaining the high synthesis of polyphenols and antioxidant activities of C. nutans.

Medicinal plants are commonly used in complementary and alternative medicine to treat several chronic diseases. Gynura procumbens, Hibiscus sabdariffa and Clinacanthus nutans are believed to contain high level of antioxidants including phenolic acids that may contribute to antioxidant activities. Thus, the objectives of this study were to determine total phenolic content (TPC) and antioxidant scavenging capacity of these plants using three different extracts as well as to determine the relationship between TPC and antioxidant activity. The quantification of phenolic compound was conducted by analyzing the compounds at three different fractions (free, insoluble bound and soluble bound). Three replicates of the medicinal plants were extracted with 80% methanol (free fraction) and undergone hydrolysis with HCl (soluble bound) and NaOH (insoluble bound). The TPC was determined using Folin-Ciocalteau reagent while antioxidant radical scavenging capacities were analyzed using DPPH assay. Results showed that G. procumbens and C. nutans of the insoluble bound extracts had the highest amount of TPC compared to the free phenolic and soluble bound (p

Clinacanthus nutans is a well-known herb that has been used as an alternative and therapeutic medicine, however more selective C. nutans extracts are needed. In this study, leaves were extracted with 80% methanol and further fractionated with n-hexane, dichloromethane, chloroform, n-butanol, and aqueous residue. Subsequently, the total phenolic content (TPC), total flavonoid content (TFC), antioxidant scavenging activity, and antiproliferative effects on breast cancer (Michigan Cancer Foundation-7 [MCF7]) and normal breast (Michigan Cancer Foundation-10A [MCF 10A]) cells of the extracts were measured. Additionally, molecular docking simulation of the major compounds from C. nutans extracts was conducted. The aqueous residue had the highest TPC and TFC, whereas the crude extract had the highest scavenging activity. Among the extracts, dichloromethane extract (CN-Dcm) was selected as it had the highest selectivity index (SI) (1.48). Then, the chosen extract (CN-Dcm) was proceed for further analysis. The compounds from CN-Dcm were identified using gas chromatography&ndash, mass spectrometry (GC&ndash, MS). The major compounds from CN-Dcm were further investigated through molecular docking studies. Palmitic acid and linolenyl alcohol were the compounds found in the CN-Dcm extract that exhibited the highest binding affinities with p53-binding protein Mdm-2. These results highlight the potential of C. nutans as a source of anticancer activities.

Background: Clinacanthus nutans, also known as ‘Sabah snake grass’ or ‘Belalai gajah’, is a herb well known locally for its medicinal values. The primary chemical constituents of the leaves are schaftoside, vitexin, isovitexin, orientin and isoorientin, and antiviral activity is shown by two glycoglycerolipids. Despite the importance of C. nutans, complete information with respect to commercial production and postharvest handling of the herb in the local herbal industry is still lacking. Thus, the objective of this study was to determine the optimum postharvest handling processes that could retain the phytochemicals quality of C. nutans. Materials and Methods: The flavonoid compounds of C. nutans were analysed by using ultra fast liquid chromatography (UFLC). Total phenolic content and antioxidant activity were determined using a spectrophotometer. Results: The total phenolic compounds and antioxidant activity in C. nutans were found to be higher in the young vegetative stage than in the mature vegetative stage. Flavonoid compounds (schaftoside, isovitexin, vitexin and orientin) were also found to be highest in the young vegetative plant compared to the mature vegetative plant. All of the assayed phytochemicals and flavonoid compounds levels were found to be highest in oven dried samples compared to the sun, air and solar dried samples. Conslusion: This study suggests that oven-drying young vegetative C. nutans plant material is the optimum method to retain postharvest quality.

INTRODUCTION Clinacanthus nutans, a small shrub that is native to Southeast Asia, is commonly used in traditional herbal medicine and as a food source. Its anti-inflammation properties is influenced by the metabolites composition, which can be determined by different binary extraction solvent ratio and extraction methods used during plant post-harvesting stage. OBJECTIVE Evaluate the relationship between the chemical composition of C. nutans and its anti-inflammatory properties using nuclear magnetic resonance (NMR) metabolomics approach. METHODOLOGY The anti-inflammatory effect of C. nutans air-dried leaves extracted using five different binary extraction solvent ratio and two extraction methods was determined based on their nitric oxide (NO) inhibition effect in lipopolysaccharide-interferon-gamma (LPS-IFN-γ) activated RAW 264.7 macrophages. The relationship between extract bioactivity and metabolite profiles and quantifications were established using 1 H-NMR metabolomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The possible metabolite biosynthesis pathway was constructed to further strengthen the findings. RESULTS Water and sonication prepared air-dried leaves possessed the highest NO inhibition activity (IC50 = 190.43 ± 12.26 μg/mL, P

The present study sought to identify the key biomarkers and pathways involved in the induction of allergic sensitization to ovalbumin and to elucidate the potential anti-anaphylaxis property of Clinacanthus nutans (Burm. f.) Lindau water leaf extract, a Southeast Asia herb in an in vivo ovalbumin-induced active systemic anaphylaxis model evaluated by 1H-NMR metabolomics. The results revealed that carbohydrate metabolism (glucose, myo-inositol, galactarate) and lipid metabolism (glycerol, choline, sn-glycero-3-phosphocholine) are the key requisites for the induction of anaphylaxis reaction. Sensitized rats treated with 2000 mg/kg bw C. nutans extract before ovalbumin challenge showed a positive correlation with the normal group and was negatively related to the induced group. Further 1H-NMR analysis in complement with Kyoto Encyclopedia of Genes and Genomes (KEGG) reveals the protective effect of C. nutans extract against ovalbumin-induced anaphylaxis through the down-regulation of lipid metabolism (choline, sn-glycero-3-phosphocholine), carbohydrate and signal transduction system (glucose, myo-inositol, galactarate) and up-regulation of citrate cycle intermediates (citrate, 2-oxoglutarate, succinate), propanoate metabolism (1,2-propanediol), amino acid metabolism (betaine, N,N-dimethylglycine, methylguanidine, valine) and nucleotide metabolism (malonate, allantoin). In summary, this study reports for the first time, C. nutans water extract is a potential anti-anaphylactic agent and 1H-NMR metabolomics is a great alternative analytical tool to explicate the mechanism of action of anaphylaxis.

One new furofuran lignan 2-methoxy-9β-hydroxydiasesamin (1), and four analogues (2–5), together with eight alkaloids (6–13), were isolated from the ethanol extract of the aerial part of Clinacanthus nutans. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. All the compounds were isolated from the genus of Clinacanthus for the first time. In addition, lignans isolated from C. nutans was reported for the first time. Compound 1 exhibited modest activity against three human tumor cell lines Hela, MCF-7, and A549, with IC50 values of 68.55, 60.00, and 59.17 μM, respectively.

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L−1 2,4-D and 0.75 mg L−1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L−1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L−1 2,4-D and 0.50 mg L−1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.

Clinacanthus nutans (C. nutans) leaf extracts have been widely used by cancer patients in Malaysia and local practice claims a cure to cancer. There were several studies done to determine the cytotoxicity potency of C. nutans extracts on various types of cells. However, there is still lacking on the knowledge regarding the combination effect of C. nutans with anticancer drugs. Thus, the study was carried out to determine the cytotoxicity potency of C. nutans extracts and paclitaxel (PTX) alone and, in combination on MDA-MB-231 cells. The cells were treated with 100% ethanol extract of C. nutans (CNE) and water extract of C. nutans (CNA), PTX and combination of both extracts and PTX for 72 hours and the cytotoxic activity was determined using SRB assay. Result showed that CNE had little cytotoxic activity, whereas CNA showed no cytotoxic activity on MDA-MB-231 cells. For combination treatment of C. nutans extracts and PTX, only CNE showed significant enhanced PTX-induced cytotoxicity (p < 0.05), meanwhile CNA inhibited PTX-induced cytotoxicity significantly (p < 0.05). As a conclusion, CNE was able to increase PTX potency to inhibit the viability of MDA-MB-231 cells. DOI : http://dx.doi.org./10.17576/JSKM-2018-14

The application of solar drying (SD) and heat pump-assisted solar drying (HPSD) on the retention of flavonoid components, total color changes, and water activity of Clinacanthus nutans Lindau leaves were investigated. Analysis of data shows significantly higher extractable yield and flavonoid (orientin and vitexin) percentage during the drying with HPSD. The same drying technique also revealed optimum color values and low water activity. Thin-layer models fitted to the experimental data show that Hii and Law model is suitable for SD, while logarithmic model is able to give a good fit to HPSD.

Clinacanthus nutans (Burm. f.) Lindau is a traditional medicinal plant belonging to the Acanthaceae family. Its therapeutic potentials have been increasingly documented particularly the antiviral activity against Herpes Simplex Virus (HSV), anti-cancer, anti-oxidant, anti-inflammatory and immunomodulatory activities. However, majority of these studies used crude or fractionated extracts and not much is known about individual compounds from these extracts and their biological activities. In the present study, we have isolated four compounds (CN1, CN2, CN3 and CN4) from the hexane fractions of C. nutans leaves. Using NMR spectroscopic analysis, these compounds were identified to be shaftoside (CN1), stigmasterol (CN2), β-sitosterol (CN3) and a triterpenoid lupeol (CN4). To determine the immunosuppressive potential of these compounds, their effects on mitogens induced T and B lymphocyte proliferation and the secretion of helper T cell cytokines were examined. Among the four compounds, stigmasterol (CN2) and β-sitosterol (CN3) were shown to readily inhibit T cell proliferation mediated by Concanavalin A (ConA). However, only β-sitosterol (CN3) and not stigmasterol (CN2) blocks the secretion of T helper 2 (Th2) cytokines (IL-4 and IL-10). Both compounds have no effect on the secretion of Th1 cytokines (IL-2 and IFN-γ), suggesting that β-sitosterol treatment selectively suppresses Th2 activity and promotes a Th1 bias. CN3 was also found to significantly reduce the proliferation of both T helper cells (CD4+CD25+) and cytotoxic T cells (CD8+CD25+) following T cell activation induced by ConA. These results suggested that phytosterols isolated from C. nutans possess immunomodulatory effects with potential development as immunotherapeutics.

Subcritical carbon dioxide Soxhlet extraction of biologically active compounds from Clincanthus nutans was investigated by full factorial design to identify and optimize the factors (particle size and co-solvent) affecting extract yield, antioxidant activity, total phenolic content, total flavonoid content, and α-glucosidase inhibitory activity. An average of 3.103% yield, 98.90% antioxidant activity, 49.40 mg/g (GAE) TPC, 43.76 mg/g (RE), and 88.58% AGI activity can be achieved using the optimum levels of independent variables. The GC-Q-TOF MS identification of optimized extract shown that different classes of phytoconstituents were successfully separated by CO2-Soxhlet to produce potential antioxidant and α-glucosidase inhibitory activity.

Context:Clinacanthus nutans Lindau (Acanthaceae) is a medicinal plant that has been reported to have anti-inflammatory, antiviral, antimicrobial and antivenom activities. In Malaysia, it has been widely claimed to be effective in various cancer treatments but scientific evidence is lacking. Objective: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts. Materials and methods: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 μg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS. Results: The proliferation of MCF-7 cells was inhibited with the IC50 values of 35 and 30 μg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols. Discussion and conclusions: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this.

Clinacanthus nutans (CN) (Acanthaceae) is well-known for its anti-inflammatory properties among Asian communities; however, there are currently no data specifically focused on the anti-inflammatory effects of CN on the brain tissue. Neuroinflammation is a common consequence of toxin intrusion to any part of the central nervous system (CNS). As an innate immune response, the CNS may react through both protective and/or toxic actions due to the activation of neuron cells producing pro- and/or anti-inflammatory cytokines in the brain. The unresolved activation of the inflammatory cytokines' response is associated with the pathogenesis of neurological disorders. The present study aimed to decipher the metabolic mechanism on the effects of 14 days oral treatment with CN aqueous extract in induced-lipopolysaccharides (LPS) rats through 1H NMR spectroscopic biomarker profiling of the brain tissue and the related cytokines. Based on the principal component analysis (PCA) of the nuclear magnetic resonance (NMR) spectral data, twenty-one metabolites in the brain tissue were profiled as biomarkers for the LPS (10 μL)-induced neuroinflammation following intracerebroventricular injection. Among the twenty-one biomarkers in the neuroinflammed rats, CN treatment of 1000 and 500 mg/kg BW successfully altered lactate, pyruvate, phosphorylcholine, glutamine, and α-ketoglutarate when compared to the negative control. Likewise, statistical isolinear multiple component analysis (SIMCA) showed that treatments by CN and the positive control drug, dextromethorphan (DXM, 5 mg/kg BW), have anti-neuroinflammatory potential. A moderate correlation, in the orthogonal partial least squares (OPLS) regression model, was found between the spectral metabolite profile and the cytokine levels. The current study revealed the existence of high levels of pro-inflammatory cytokines, namely IL-1α, IL-1β, and TNF-α in LPS-induced rats. Both CN dose treatments lowered IL-1β significantly better than DXM Interestingly, DXM and CN treatments both exhibited the upregulation of the anti-inflammatory cytokines IL-2 and 4. However, DXM has an advantage over CN in that the former also increased the expression of IL-10 of anti-inflammatory cytokines. In this study, a metabolomics approach was successfully applied to discover the mechanistic role of CN in controlling the neuroinflammatory conditions through the modulation of complex metabolite interactions in the rat brain.

The present work showed the preparation of gold (Au NPs) and silver nanoparticles (Ag NPs) using aqueous leaf extract of Clinacanthus nutans Lindau (C. Lindau). The prepared NPs were studied using various microscopic and spectroscopic techniques, which confirmed the formation of crystalline Ag NPs, Au NPs that are stabilized with C. Lindau extract polyphenols. The prepared Au NPs and Ag NPs are studied to assess their comparative analgesic and muscle relaxant activities conducted on BALB/c mice. The muscle relaxant studies displayed that Ag NPs were comparatively higher efficient than Au NPs and methanolic C. Lindau extract in traction examination. Additionally, the analgesic studies exhibited that Ag NPs, Au NPs showed maximum percentage reduction in acetic acid induced writhing at the concentrations of 50, 100 and 150 mg/kg body weight. Further, these results conclude that as prepared Au NPs and Ag NPs prepared from C. Lindau leaf extract demonstrated very good analgesic and muscle relaxant activities for the use of pain management in nursing care.

Background Methanol extract (MECN) of Clinacanthus nutans Lindau leaves (family Acanthaceae) demonstrated peripherally and centrally mediated antinociceptive activity via the modulation of opioid/NO-mediated, but cGMP-independent pathway. In the present study, MECN was sequentially partitioned to obtain petroleum ether extract of C. nutans (PECN), which was subjected to antinociceptive study with aims of establishing its antinociceptive potential and determining the role of opioid receptors and l–arginine/nitric oxide/cyclic-guanosine monophosphate (l–arg/NO/cGMP) pathway in the observed antinociceptive activity. Methods The antinociceptive potential of orally administered PECN (100, 250, 500 mg/kg) was studied using the abdominal constriction-, hot plate- and formalin-induced paw licking-test in mice (n = 6). The effect of PECN on locomotor activity was also evaluated using the rota rod assay. The role of opioid receptors was determined by pre-challenging 500 mg/kg PECN (p.o.) with antagonist of opioid receptor subtypes, namely β–funaltrexamine (β–FNA; 10 mg/kg; a μ-opioid antagonist), naltrindole (NALT; 1 mg/kg; a δ-opioid antagonist) or nor–binaltorphimine (nor–BNI; 1 mg/kg; a κ-opioid antagonist) followed by subjection to the abdominal constriction test. In addition, the role of l–arg/NO/cGMP pathway was determined by prechallenging 500 mg/kg PECN (p.o.) with l–arg (20 mg/kg; a NO precursor), 1H-[1, 2, 4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 2 mg/kg; a specific soluble guanylyl cyclase inhibitor), or the combinations thereof (l–arg + ODQ) for 5 mins before subjection to the abdominal constriction test. PECN was also subjected to phytoconstituents analyses. Results PECN significantly (p 0.05) affect the locomotor activity of treated mice. The antinociceptive activity of PECN was significantly (p 0.05) affected by ODQ. HPLC analysis revealed the presence of at least cinnamic acid in PECN. Conclusion PECN exerted antinocicpetive activity at peripheral and central levels possibly via the activation of non-selective opioid receptors and modulation of the NO-mediated/cGMP-independent pathway partly via the synergistic action of phenolic compounds.

Clinacanthus nutans has attracted Malaysian public interest due to its high medicinal value in the prevention of cancer. Currently, the specific compound or compounds giving rise to the anticancer potential of C. nutans has not been investigated thoroughly. The extraction was carried out by MeOH at room temperature using the powdered bark of C. nutans, while chromatography was carried out on a silica gel RP-18 column using the crude methanolic extract. Six fractions collected from column chromatography were evaluated by MTT assay against two breast cancer cell lines: MDA-MB-231 and MCF-7. Amongst the fractions, A12 and A17 were shown to exhibit the highest activity. Two sulphur-containing compounds, viz., entadamide C (1) and clinamide D (2), were isolated from these fractions. Molecular docking simulation studies revealed that entadamide C and clinamide D could bind favourably to the caspase-3 binding site with the binding energy of −4.28 kcal/mol and −4.84 kcal/mol, respectively. This study provides empirical evidence for the presence of sulphur-containing compounds in the leaves of C. nutans that displayed anticancer effects which explains its ethnomedicinal application against breast cancer. The docking simulation study showed that both compounds could serve as important templates for future drug design and development.

Sabah Snake Grass (Clinacanthus nutans) is an interesting traditional medicine because of pharmacology characteristics that contain rich in phenolic content. The aim of the study was to determine the optimum flavonoid compound on the antioxidant activities. The optimum extraction parameters for optimum recovery of phenolic content from Clinachantus nutans leaves were determined using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed to investigate the effects of ultrasonic frequency (X1: 25 - 40 kHz), temperature (X2: 40 - 80 °C) and solid-to-solvent ratio (X3: 10 - 30 ml/g) on the recovery of flavonoid vitexin (Y1) and orientin (Y2). The optimal parameter achieved based on combination of the extraction parameters are as follows: X1 = 25 kHz, X2 = 40 °C and X3 = 15 g/ml. These optimum conditions yielded vitexin (2.25 mg/g) and orientin (3.75 mg/g). Our results revealed that distilled water may be a good choice for extracting the phenolic compound of Clinacanthus nutans due to costing and environmental issue. Vitexin and orientin have potential of antiproliferate activity on the cancer cell. The concentration of extracted compound (orientin and vitexin) from the optimum parameters had produced high in phenolic content. In conclusion, the application of response surface method successfully provide the high extraction parameters with high total phenolic content and antioxidant activity.

Methanolic extract of Clinacanthus nutans Lindau leaves (MECN) has been reported to exert antinociceptive activity. The present study aimed to elucidate the possible antinociceptive mechanisms of a lipid-soluble fraction of MECN, which was obtained after sequential extraction in petroleum ether. The petroleum ether fraction of C. nutans (PECN), administered orally to mice, was (i) subjected to capsaicin-, glutamate-, phorbol 12-myristate 13-acetate-, bradykinin-induced nociception model; (ii) prechallenged (intraperitoneal (i.p.)) with 0.15 mg/kg yohimbine, 1 mg/kg pindolol, 3 mg/kg caffeine, 0.2 mg/kg haloperidol, or 10 mg/kg atropine, which were the respective antagonist of α2-adrenergic, β-adrenergic, adenosinergic, dopaminergic, or muscarinic receptors; and (iii) prechallenged (i.p.) with 10 mg/kg glibenclamide, 0.04 mg/kg apamin, 0.02 mg/kg charybdotoxin, or 4 mg/kg tetraethylammonium chloride, which were the respective inhibitor of ATP sensitive-, small conductance Ca2+-activated-, large conductance Ca2+-activated-, or nonselective voltage-activated-K+ channel. Results obtained demonstrated that PECN (100, 250, and 500 mg/kg) significantly (P+ channel blockers. However, only pretreatment with apamin and charybdotoxin caused full inhibition of PECN-induced antinociception. The rest of the K+ channel blockers and all antagonists caused only partial inhibition of PECN antinociception, respectively. Analyses on PECN’s phytoconstituents revealed the presence of antinociceptive-bearing bioactive compounds of volatile (i.e., derivatives of γ–tocopherol, α–tocopherol, and lupeol) and nonvolatile (i.e., cinnamic acid) nature. In conclusion, PECN exerts a non-opioid-mediated antinociceptive activity involving mainly activation of adenosinergic and cholinergic receptors or small- and large-conductance Ca2+-activated-K+ channels.

Clinacanthus nutans (Burm.f.) Lindau is a valuable medicinal plant that has gained interest as a side treatment for cancer in Southeast Asia. Phenolic and flavonoid compounds identified in this plant have been linked to anti-cancer properties. However, quantification of such metabolites in plants varies depending on cultivation methods and conditions resulting in inconsistent yield. This study aims to establish cell suspension culture of C. nutans, evaluate the accumulation of phenolics, flavonoids and to assess the antioxidant activity of extracts from in vitro cultures. Callus was induced from leaf explants of C. nutans and proliferated on Murashige & Skoog (MS) medium supplemented with different combinations of plant growth regulators (2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine and kinetin). Assessment of growth kinetics for the suspension culture was performed. The total phenolic, flavonoid contents and antioxidant activity of the extracts were evaluated followed by high-performance liquid chromatography (HPLC) to detect selected flavonoids. MS medium supplemented with 0.25 mg/L 2, 4-D and 0.25 mg/L BAP was optimal for callus induction, proliferation and suspension cultures. The highest total phenolic content was obtained from suspension cells (55.35 mg GAE/g DW) whereas leaf showed the highest flavonoid content (25.13 mg QE/g DW). Leaf extract demonstrated the strongest antioxidant activity with the lowest IC50 value (117.42 μg/mL). HPLC analysis revealed the presence of catechin, luteolin, quercetin and kaempferol in the suspension cells and the leaf. The present study indicated that cell suspension cultures are able to accumulate higher phenolic compounds and possess all four selected flavonoids similar to the outdoor grown plant.

Breast cancer is one of the most common cancers with a relatively high mortality rate. Despite the advancement of its medical treatments, many patients are still seeking complementary alternative medicines, namely Clinacanthus nutans which is found mainly in South-East Asian countries. We aim to find the antioxidant properties and cytotoxic activity of the plant extract toward breast cancer cell lines Michigan Cancer Foundation-7 (MCF7) and T47D individually and in combination with doxorubicin. Extractions of C. nutans with ethanol, n-hexane, and ethyl acetate were done using rotatory vacuum evaporators with the reflux method. Screening of biochemical properties was conducted. Antioxidant activity was measured toward α, α-diphenyl-β-picrylhydrazyl (DPPH) with IC50 scores were shown to be highest in ethyl acetate extract. Cytotoxic effects of all three extracts were shown to be low in both MCF7 and T47D cells. However, combinations of 125 μg/ml n-hexane extract of C. nutans, and 0.1 μg/ml doxorubicin in T47D cancer cells showed further proliferation reduction compared to the single administration. The results suggested possible synergisms of the treatment combination.

Clinacanthus nutans has been used in traditional herbal medicine for cancer prevention, but the specific bioactive compounds responsible for the observed activities have not been explored. Different polar solvents such as methanol, chloroform, ethyl acetate, and hexane were used for the extraction. The extracts, fractions, and isolated compounds were subjected to DPPH and ferric reducing antioxidant potential (FRAP) assays. Methanol extracts show significant free-radical scavenging activity of 69.09% in DPPH and 56.49% FRAP. Purification of MeOH extracts afforded the fraction FB28 and two new sulfur-containing compounds, named clinamide D and E (1, 2). Compound (1) proved to be more active with an IC50 value for DPPH radical scavenging of 118.27 ± 0.01 µg/mL and reduction of Fe3+–TPTZ complex of 386.24 ± 0.02, higher than that of the standard ascorbic acid. Sulfur-containing compounds isolated from C. nutans is a potential natural antioxidant.

Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

Objective To evaluate the monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) from Clinacanthus nutans ( C. nutans ) for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by plaque reduction assay. Methods MGDG and DGDG were extracted with chloroform from C. nutans leaves. MGDG and DGDG were separated from chloroform crude extract using column chromatography, characterized by thin layer chromatography and quantified by high performance liquid chromatography. The anti HSV-1 and 2 activity against pre-treatment and post-treatment of the compounds was evaluated using plaque reduction assay. The cytotoxicity of the extract and the compounds on Vero cells were performed by MTT assay. Results MGDG and DGDG obtained by column chromatography showed identical profiles as standard MGDG and standard DGDG using thin layer chromatography and high performance liquid chromatography. MGDG and DGDG from C. nutans showed 100% inhibition of HSV-1 replication at the post step of infection at noncytotoxic concentration with IC 50 values of 36.00 and 40.00 μg/mL, and HSV-2 at 41.00 and 43.20 μg/mL, respectively. Moreover, MGDG and DGDG from C. nutans were demonstrated to have anti-herpes simplex activity at the same level as standard synthetic compounds. In contrast, pre-treatment of Vero cells with MGDG and DGDG before HSV-1 and HSV-2 infection did not show inhibitory effect against these viruses. MGDG and DGDG exhibited antiviral activity against HSV-1 with selectivity index of 26.00 and 23.00 and HSV-2 of 23.30 and 21.30. Conclusions MGDG and DGDG from C. nutans , a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.