Seedpod extracts of Wrightia tinctoria shows significant anti-inflammatory effects in HepG2 and RAW-264.7cell lines

Abstract W. tinctoria, an Indian herb Indrajao, has significant therapeutic potential. While studies have highlighted the anti-inflammatory potential of the leaves and bark of this plant, similar efficacy of the seed-pods remains unexplored. We demonstrate significant anti-inflammatory effects of the hexane fraction (Fr-B) of ethyl acetate extract of the seedpods in reducing lipopolysaccharide and palmitate mediated inflammation in RAW264.7 macrophages and HepG2 cells. GC-MS and NMR profiling of Fr-B revealed the existence of hexadecanoic acid, ethyl hexadecanoate, 9,12-octadecanoic acid, 9,12,15-octadecatrienoic acid, 9,12,15-octadecatrienoic acid ethyl ester, ethyl linoleate and octadecanoic acid ethyl esters. Graphical Abstract


Introduction
Research over several years positioned chronic inflammatory response as an underlying cause for a vast continuum of detrimental disorders ranging from diabetes, cardiovascular ailments, neurodegenerative disorders, and aggressive cancers (Glass et al. 2010;Koene et al. 2016) The obnoxious side effects of long-term application of anti-inflammatory drugs necessitated the requirement for herbal drugs with minimal toxicity. Since ancient times, plants served as a trustable source of therapeutic agents and several plant parts in the form of extract exhibited potential in preventing inflammation (Rodr ıguez-Yoldi 2021). Empirical evidence of such plants with significant therapeutic potential includes Wrightia tinctoria of the Apocynaceae family, which showed beneficial effects in treating diseases like jaundice, dysentery, haemorrhage, and psoriasis (Kumar and Sinha 2004;Anusharaj et al. 2013). While significant antiinflammatory effects of the leaves and bark of this plant is already known, similar effects of its seedpods is yet to be reported. We studied the anti-inflammatory effects of seedpod extracts of this plant in RAW 264.7 and HepG2 cell lines, mediated by palmitate (PA) and lipopolysaccharide (LPS).

Fractions of W. tinctoria seedpod exhibit anti-inflammatory properties
The anti-inflammatory effects of ethyl acetate extract (Ex-A) obtained from the seedpod and its fractions, hexane (Fr-B) and ethyl acetate (Fr-C) were evaluated in RAW264.7 (mouse macrophage cell line) and HepG2 (hepatoma cell line). The cytotoxic effects were evaluated by cell viability percentage using MTT assay. Data shows that each of the three extract/fractions possess no such significant cytotoxic effect at a concentration range of 10-300 mg/ml in both the cells ( Figure S1A and B). Further we checked the effect of extract/fractions on inhibition of cellular nitrates, and nitrites (RNS) produced by LPS (E. Coli LPS, Sigma) stimulation (1 mg/ml) for 22 hr by using Griess reagent on RAW264.7 macrophage cells. A range of different concentrations of sodium nitrite was used to generate a standard curve and a reduction in the level of RNS was correlated with reduction in cellular nitrite level. Our data reveals that pretreatment with hexane (Fr-B) fraction significantly reduces RNS generation with an IC 50 of 139.83 mg/ml as compared to ethyl acetate extract (Ex-A; IC 50 : 422.93 mg/ml) and other fraction (Fr-C; IC 50 : 428.71 mg/ml) ( Figure S2A). Subsequently, we estimated total cellular ROS level using DCFH-DA after pre-treating cells with the extract/fraction followed by LPS treatment for 16 hr. Fr-B significantly reduced the level of total cellular ROS at 200 mg/ml concentration in RAW264.7 macrophage cells ( Figures 1A and S2B). However, Ex-A and Fr-C were not so effective in reducing ROS, thereby hinting at the overall efficacy of Fr-B. Since Fr-B showed a potent effect in reversing elevated ROS and RNS level upon induction by LPS, it prompted us to check the expression of proinflammatory cytokines IL-6, IL1b, and TNFa (Table S1). Since the IC 50 dose of Fr-B emerged as 139.83 mg/ml in case of Griess Assay (inhibition of RNS) which was also supported by DCFH-DA assay (inhibition of ROS), we selected the dose of 200 mg/ml for further confirmatory anti-inflammatory studies. The expression of intracellular protein levels of IL-6 and IL1b was checked by flow cytometry treating HepG2 cells with PA (0.75 mM). The results indicate that in presence of PA, Fr-B (200 mg/ml) decreases IL6, IL1b expression ( Figure S3A). Subsequently, mRNA expression was checked by real-time qPCR in RAW264.7 macrophage cells which revealed a marked decrease in the expression of pro-inflammatory cytokines viz, IL1b, TNFa ( Figure 1B) and IL6 ( Figure S3B) when treated with Fr-B (200 mg/ml) upon LPS induction for 16 hr.
Additionally, a decrease in secreted IL-1b protein concentration was noticed on pretreating RAW264.7 cells with Fr-B (200 mg/ml) and stimulating with LPS for 6 hr ( Figure  S3C), measured by sandwich ELISA method. Studies earlier highlighted that LPS and saturated fatty acids like PA works through TLR4 mediated inflammatory pathway where NFjb acts as a major transcription factor (Korbecki and Bajdak-Rusinek 2019). After activation by proteasomal degradation, NFjb p65 translocate into the nucleus and regulates transcription of different set of genes, thereby triggering secretion of proinflammatory cytokines like TNFa, IL-6, and IL1b (Liu et al. 2017). Consistent to this, we checked the effect on overall expression of NFjb, treating both RAW264.7 and HepG2 cells with hexane (Fr-B) fraction (200 mg/ml) stimulating with LPS and PA respectively. Confocal imaging of cells showed a marked reduction in overall expression of NFjb in HepG2 cells and a prominent inhibition of expression as well as nuclear translocation in RAW264.7 cells, suggesting the plausible anti-inflammatory role of inhibition of NFjb in pro-inflammatory signalling cascade ( Figure S3D).

Experimental
Supplementary file.

Conclusions
The present study highlights that hexane fraction (Fr-B) obtained from ethyl acetate extract (Ex-A) exhibits potential in inhibition of inflammatory response induced by LPS and PA in HepG2 and RAW264.7 cells. Since poly-unsaturated fatty acids (PUFA) are known to render protection against inflammation (Park et al. 2014;Pauls et al. 2018) we presume that the presence of the identified compounds might be responsible in delivering anti-inflammatory effects. It would be interesting to identify the anti-inflammatory effects of Fr-B in various animal models to substantiate the traditional uses and isolation of individual compound from the Fr-B and treating them separately or in combination in in-vitro and in-vivo models would further clarify its efficacy, dosing and therapeutic potential.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Instrumentation support from CIF division of the institute is appreciated. This work was supported by grants from in-house projects (CSIR-IICB).