Modulation of NF-κB and MAPK signalling pathways by hydrolysable tannin fraction from Terminalia chebula fruits contributes to its anti-inflammatory action in RAW 264.7 cells.

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.

Identification of galangin as the bioactive compound from Alpinia calcarata (Haw.) Roscoe rhizomes to inhibit IRAK-1/ MAPK/ NF-κB p65 and JAK-1 signaling in LPS stimulated RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia calcarata (Haw.) Roscoe rhizomes are used to treat diabetes, rheumatism, gastrointestinal problems, inflammatory diseases, cough and respiratory problems in traditional practices. The primary objective of the study is to identify and isolate anti-inflammatory bioactive compounds from A.calcarata rhizomes and to assess its molecular mechanism. MATERIALS AND METHODS: The bioassay-guided fractionation of methanolic extract of A. calcarata rhizomes yielded chloroform fraction as the effective fraction and galangin as the bioactive compound identified by NMR studies. The anti-inflammatory action of galangin was evaluated by determining NO and cytokine production in LPS stimulated RAW264.7 cells. Further, its mechanism was studied on the expression levels of mRNA and protein targets by qPCR and Western blot analysis. RESULTS: Based on the MTT assay, the concentration of 3.1-25 µM of galangin was selected for further studies. Galangin reduced the levels of NO and proinflammatory cytokines (TNF-α, IL-1ß and IL-6) production in LPS induced RAW 264.7 cells in a dose-dependent manner. In addition, the qPCR analysis revealed a reduction in the mRNA expression levels of COX-2, IRAK 1 and JAK 1 in galangin treated LPS stimulated RAW 264.7 cells in a dose-dependent manner. Western blot analysis implicated that galangin has markedly reduced the protein expression levels of cell signaling regulators (JAK-1, IRAK-1, MyD88, MAPK (p38 and ERK) and NF-κB p65). CONCLUSION: From the results, it is evident that the inhibition of these cell signaling regulators has contributed to the anti-inflammatory effects of galangin. To our knowledge, we are the first to report IRAK-1 and JAK-1 as therapeutic targets of galangin for its anti-inflammatory effect.

Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Chemical characterization and immunostimulatory activity of phenolic acid bound arabinoxylans derived from foxtail and barnyard millets.

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-1ß, 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-1ß, 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.

Hydrolysable tannin-rich fraction from Terminalia chebula Retz. fruits ameliorates collagen-induced arthritis in BALB/c mice.

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.