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Objectives Hydrolysable tannin fraction (HTF) derived from Terminalia chebula fruit pericarps was assessed for its anti-inflammatory potential in LPS-induced RAW 264.7 cells. Its molecular mechanism was also established and compared with individual tannins – chebulagic acid (CH) and corilagin (CO). Methods The effect of HTF on LPS-stimulated RAW 264.7 cells was studied by estimating the release of NO, ROS, cytokines and changes in nuclear morphology by DAPI staining. Furthermore, the effect of HTF, CO and CH was compared with the expression of p65, p38 and pERK proteins by immunoblotting and the mRNA transcript level of COX-2, iNOS and TNF-α by quantitative PCR. The in-silico interactions of various hydrolysable tannins present in HTF with molecular targets of inflammation were studied using Maestro software. Key findings HTF at the dose levels of 25, 50 and 100 µg/ml was able to decrease the release of NO, ROS and cytokines from LPS-induced RAW 264.7 cells without disturbing the cell nuclear morphology. Investigation of molecular mechanism revealed that inhibition of NF-κB and MAPK signalling pathways was responsible for its anti-inflammatory action. The effect of HTF was higher than the individual tannins CH and CO. Conclusion HTF can be developed as an effective anti-inflammatory agent.

The immunomodulating effect of Phenolic acid bound arabinoxylans (PCA-AXs) extracted from Little (PCA-AX-L) and Kodo (PCA-AX-K) millet seeds in RAW 264.7 cells were investigated. The PCA-AXs were extracted from millets and their chemical characterization were carried out by GC-MS, HPLC, and FT-IR. The immunomodulatory effect of PCA-AXs in RAW 264.7 cells were investigated by estimating ROS, NO, and cytokines TNF-α, IL-1β, IL-6, and evaluation of molecular mechanism by q-PCR & western blotting techniques. The xylose: arabinose ratio of PCA-AX-L and PCA-AX-K were 1.48:1.0 and 2.26:1.0, respectively. The phenolic acids content was higher in PCA-AX-K than PCA-AX-L determined by HPLC. FT-IR analysis confirms the presence of α-glucosidic linkage with the degree of substitution of xylan backbone by arabinose residues. The evaluation of immunomodulating effect of PCA-AXs revealed that the PCA-AX-L-treated cells showed higher release of NO, ROS and cytokines than PCA-AX-K-treated cells. The mRNA expressions of TNF-α, iNOS and COX-2 were upregulated by PCA-AX-L and downregulated by PCA-AX-K in dose-dependent manner. Furthermore, in western blotting, the ERK and NF-κB were found to be activated by PCA-AX-L and inhibited by PCA-AX-K. Our findings suggest that the high branched arabinoxylans of PCA-AX-L could modulate the immune response in RAW 264.7 cells through activation of ERK and NF-κB signaling pathways and acts as an immunostimulant. The higher phenolic content in PCA-AX-K could modulate the immune response by downregulation of ERK and NF-κB signaling pathways and thus, it could act as an immunomodulator. PRACTICAL APPLICATIONS: Millets are the richest source of arabinoxylans in which they are known to be bound with phenolic acids (PCA-AX). Arabinoxylans derived from rice and wheat is known immunomodulators. This study was focused to evaluate the immunomodulatory property of PCA-AX derived from two different millets little and kodo. The study results clearly indicated the immune stimulatory action of PCA-AX-L and immunomodulatory action of PCA-AX-K. The explored mechanism indicated that the PCA-AXs modulate NF-κB & ERK pathways for their immunomodulatory action.

The chemical characterization and evaluation of immunostimulating effect of phenolic acid bound arabinoxylan (PA-AXs) isolated from barnyard (PA-AX-B) and foxtail (PA-AX-F) millets were performed. The sugar composition analysis and bound phenolic acids' (caffeic acid, p-coumaric acid, and ferulic acid) content of PA-AXs were examined by gas chromatography and mass spectroscopy (GC-MS) and high performance liquid chromatography (HPLC), respectively. The immunostimulatory activity of PA-AXs was evaluated by studying the effect of PA-AXs on the release of nitric oxide (NO), ROS, and cytokine (TNF-α, IL-1β, and IL-6) in RAW 264.7 murine macrophage cells. The GC-MS results revealed the xylose: arabinose ratio of PA-AX-F and PA-AX-B as 1.96:1.0 and 1.64:1.0, respectively. In HPLC analysis, PA-AX-B showed higher phenolic acid content than PA-AX-F. In RAW 264.7 cells, immunostimulatory activity was established by its increased release of NO, ROS, and cytokine (TNF-α, IL-1β, and IL-6) in a dose-dependent manner. Both PA-AX-B and PA-AX-F exhibited significant immunostimulation in in vitro studies. PRACTICAL APPLICATIONS: Millets are known for the higher content of phenolic acid bound arabinoxylans (PA-AX). The composition of PA-AX varies with different types of millets. In general, rice bran and wheat arabinoxylans are well reported to have significant immunostimulatory and antitumor properties. The bound ferulic acid with arabinoxylan isolated from finger millet bran also possesses immunostimulatory property. As the millets grains, foxtail and barnyard are also rich in PA-AXs, the present study was focused to evaluate the immunostimulatory property of PA-AX derived from two different millets. The study results indicated the immune stimulatory action of millet PA-AX's and thus the purified PA-AX can be explored further to identify the mechanism of action with respect to its immune stimulation property.

Terminalia chebula Retz. (Fam: Combretaceae) (TC) is widely used in traditional system for the treatment of fever, asthma, urinary diseases, and rheumatism. The aim of the present study is to evaluate the efficacy of hydrolysable tannin-rich fraction (HTF) isolated from TC fruits in collagen-induced arthritic BALB/c mice. Type II collagen-induced arthritis was attained in BALB/c mice. HTF treatment at three gradual doses (100, 200, and 400 mg/kg) was started from 24th day on daily administration up to 4 weeks. Body weight, food intake, and increase in hind paw were monitored at weekly basis. At the end of the study, serum and joint tissues were estimated for proinflammatory cytokines and biochemical parameters and the hind limbs were removed for radiological and histopathological evaluations. Oral administration of HTF could significantly protect CIA in BALB/c mice by reducing paw volume, arthritic score, spleen index, serum and joint cytokine level, and biochemical markers. The anti-inflammatory efficacy of HTF was further demonstrated by morphological observation, histopathological , and radiological evaluations. HTF is a good therapeutic agent for the treatment of joint inflammatory condition, as its action is mediated through cytokine inhibition.