1H NMR-Based Metabolomics of Clinacanthus nutans Leaves Extracts in Correlation with Their Anti-neuroinflammation Towards LPS-Induced BV2 Cells

Abstract

The metabolomics approach successfully explained the possible neuroprotective effect of Clinacanthus nutans (Burm. f.) Lindau (CN) leaf extracts. Forty-four metabolites were putatively identified via proton Nuclear Magnetic Resonance ( 1 H NMR and J-resolved NMR) metabolic profiling of CN leaf extracts in three types of solvents, namely water, 50% ethanol, and ethanol. Metabolite fingerprinting has efficaciously differentiated aqueous between the other two extracts. The variable importance of projection (VIP) showed that 30 metabolites were responsible for the discrimination of the extracts by component 1 in the Partial Least Square (PLS) score plot. The lipopolysaccharides (LPS)-induced murine microglial of the BV2 cell line successfully exhibited aqueous CN as the closest extract related to the nitrite oxide (NO) inhibitory activity via PLS biplot, with an IC50 value of 336.2 ± 4.7 µg/mL through Griess assay. The cytotoxicity assay also indicated that all CN extracts were non-toxic. Schaftoside, acetate, propionate, alanine, and clinacoside C were identified as the most potential biomarkers in the anti-inflammatory assay. Hence, the aqueous CN extract could be further investigated, particularly relating to the anti-neuroinflammation study.

The anti-neuroinflammatory effects of Clinacanthus nutans leaf extract on metabolism elucidated through 1H NMR in correlation with cytokines microarray

“…Putative identification revealed forty-four metabolites including triterpenes, sulfur-containing glycosides, C -glycosyl flavones, and a variety of other compounds including sugars, amino acids, and phenolics [ 15 – 17 , 21 ]. The present study of CNE showed a similar 1 H NMR spectroscopic pattern [ 15 ], with a high intensity of the shaftoside resonance [ 16 , 17 ]. In our recent report on CNE phytoconstituents with NO inhibitory activities in the BV2 cell assay revealed 30 key chemical markers, namely schaftoside, acetate, propionate, alanine, clinacoside C, monoacyl-monogalactosylglycerol, fructose, clinacoslides A and B, ascorbic acid, a mixture of cerebrosides, choline, stigmasterol- β -glucoside, citric acid, valine, catechin, orientin, chlorogenic acid, leucine, butyrate, cycloclinacosides A1 and A2, sucrose, vitexin, β -sitosterol, β -glucose, vanillic acid, gendarucin A, betulin, and isoleucine [ 16 ].…”

“…Khoo et al . also successfully demonstrated through HPLC-DAD-ESI-MS/MS quantification that four metabolites, namely shaftoside (0.65±0.03 mg/g), isovitexin (0.128±0.007 mg/g), orientin (0.005±0.00 mg/g), and isoorientin (0.004±0.000 mg/g), were present in the CNE, wherein all these four constituents were also identified in the current study [ 15 , 16 ].…”

“…The pooled CN extracts were concentrated by removal of the solvent using a rotary evaporator at 40°C. The extraction method is the same as the published report on CNE by this laboratory [ 16 , 17 ], which was slightly modified from the procedure of Khoo and colleagues [ 15 , 21 ]. The lyophilized CN crude extract (CNE) (extraction yield; water: 30% w/w of the actual percentage of dry leaf weight) was kept frozen until further use.…”

“…f.) Lindau (Acanthaceae) (CN) are a well-established therapeutic alternative for inflammation [ 14 , 15 ]. Hence, the potential of CN as an anti-inflammatory agent in brain-induced inflammation was explored in this laboratory [ 16 , 17 ]. A bioactivity study of CN crude aqueous extract (CNE) on nitric oxide inhibition in in vitro LPS-induced BV2 cells (rat microglia) revealed the extract had potential as an anti-neuroinflammatory source [ 16 ].…”

“…Hence, the potential of CN as an anti-inflammatory agent in brain-induced inflammation was explored in this laboratory [ 16 , 17 ]. A bioactivity study of CN crude aqueous extract (CNE) on nitric oxide inhibition in in vitro LPS-induced BV2 cells (rat microglia) revealed the extract had potential as an anti-neuroinflammatory source [ 16 ]. Nevertheless, the use of various matrices, such as cells, tissues, and biofluids offer much richer information source for metabolic profiling in direct diagnosis, therapeutic strategies, and system biology studies [ 18 ].…”