Site-selective synthesis and pharmacological elucidation of novel semi-synthetic analogues of koenimbine as a potential anti-inflammatory agent.

Koenimbine (1), a carbazole alkaloid isolated from Murraya koenigii, belongs to the Rutaceae family. Various pharmacological effects such as anti-diabetic, melanogenesis inhibition, anti-diarrheal, anti-cancer, and anti-inflammatory properties of koenimbine have already been reported. In the current study, we investigated the anti-inflammatory role of koenimbine (1) and its novel semi-synthetic derivative 8-methoxy-3,3,5-trimethylpyrano[3,2-a] carbazole-11(3H)-yl) (3-(trifluoromethyl) phenyl) methanone (1G) in both in vitro and in vivo biological systems. Our results demonstrated that the anti-inflammatory activity of 1G significantly lowered the production of NO, pro-inflammatory cytokines (IL-6, TNF-α & IL-1ß), LTB4 following LPS stimulation in RAW 264.7 macrophages. Furthermore, 1G significantly attenuated the expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner and also decreased the production of reactive oxygen species (ROS) in LPS-activated RAW 264.7 cells. In addition, the oral administration of 1G reduced the inflammatory response in carrageenan-induced paw edema in BALB/C mice. Moreover, it effectively reduced NO, IL-6, IL-1ß & TNF-α levels, liver markers (AST, ALT), and kidney markers (BUN, CRE, and Urea). Also, 1G reverted the infiltration of inflammatory cells and tissue damage in lungs, liver and kidney enhanced the survival rate in LPS-challenged mice. 1G blocks NF-κB p65 from translocating into the nucleus and activating inflammatory gene transcription. These results illustrated that 1G suppresses the inflammatory effects both in-vitro and in-vivo studies via downregulating the nuclear factor kappa-B (NF-κB) signaling pathway. In conclusion, our results demonstrate that semi-synthetic derivative 1G can effectively attenuate the inflammatory response via NF-κB and MAPK signaling pathways; suggesting 1G is a potential novel anti-inflammatory drug candidate in treating inflammatory disorders.

Anti-inflammatory and anti-arthritic potential of methotrexate in combination with BA-25, an amino analogue of ß-boswellic acid in the treatment of rheumatoid arthritis.

ß- boswellic acid, a pentacyclic triterpene derived from Boswellia serrata is extensively known for its anti-inflammatory potential. BA-25 (3-α-o-acetoxy-4ß-amino-11-oxo-24-norurs-12-ene) is an amino analogue of ß-boswellic acid that has shown anti-inflammatory potential in LPS-induced macrophages and animal models. The present study aims at investigation of the combination of BA-25 with the conventional gold standard DMARD methotrexate (MTX) for its anti-inflammatory and anti-arthritic potential using in vitro and in vivo experimental models. The anti-inflammatory potential of MTX versus the combination (BA-25 + MTX) was investigated for inhibition of NO, ROS and pro-inflammatory cytokines including TNF-α and IL-6 using ELISA in LPS-stimulated RAW-264.7 cells. The results demonstrated significant reduction in NO, ROS, TNF- α and IL-6 production with the combination treatment in comparison to MTX alone. The cytokine inhibition potential of the combination was further validated in-vivo using balb/c wherein the combination restored LPS-induced increase in pro-inflammatory cytokines. The toxicological aspect of the in vivo doses of the combination was also investigated in mice after dosing for 28 days wherein the results suggested no significant change in the hematological parameters and serum biochemical parameters in the combination versus the vehicle group. The effect of BA-25 was also investigated on MTX-induced increase in liver function tests and the expression of Bax and blc2. The results demonstrated decrease in the production of liver enzymes with BA-25 administration along with downregulating the expression of apoptotic protein Bax while increasing the expression of anti-apoptotic protein Bcl2. Furthermore, pharmacokinetic studies of BA-25 were conducted in Balb/c mice wherein the compound showed rapid absorption, high volume of distribution and a t1/2 of 13.08. Finally the anti-arthritic effect of the combination of MTX + BA-25 vs MTX alone was investigated using CIA model in DBA/1 mice wherein the treatment with the combination resulted in significant reduction in paw inflammation, IL-6 and IL-1ß levels. Furthermore, the western blot analysis demonstrated considerable decrease in the expression of p-NF-κB p65 and p-IκB in the ankle-joint tissue of the CIA mice treated with the combination therapy. The results insinuated increased anti-inflammatory and anti-arthritic potential of the combination of MTX with BA-25 as evident from in to vitro and in-vivo studies.

Arteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling.

Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway.

Novel alantolactone derivative AL-04 exhibits potential anti-inflammatory activity via modulation of iNOS, COX-2 and NF-κB.

Natural compounds and their synthesized analogues continue to be valuable sources in the discovery and development of novel anti-inflammatory agents. AL-04 is a thiol analogue derived from a natural sesquiterpene alantolactone, that demonstrated potential anti-inflammatory activity in vitro in comparison to its parent compound. However, the anti-inflammatory mechanism of action of AL-04 has not been elucidated. In this context, we investigated the signaling pathway that primarily mediate the anti-inflammatory activity of AL-04 and its effect on principal inflammatory mediators including iNOS, COX-2 and ROS. Furthermore, the anti-inflammatory activity was investigated in vivo in carrageenan induced paw oedema model in addition to the exploration of anti-nociceptive activity and acute toxicity. The results suggested that treatment with AL-04 significantly decreased the LPS-induced upregulation of pro-inflammatory cytokines and mediators in addition to the downregulated transcription of TNF-α and IL-6 in RAW 264.7 cell line. Furthermore, mRNA and the protein expression of COX-2 and iNOS were also significantly attenuated with AL-04 at a concentration of 10 µM. Western blot studies further suggested that AL-04 downregulated LPS-stimulated NF-κB p65 expression. In addition to this the anti-inflammatory activity of AL-04 was demonstrated in carrageenan induced paw oedema model with significant inhibition of oedema in a dose-dependent manner. The anti-inflammatory activity of AL-04 was further demonstrated in balb/c mice by inhibition of leukocyte migration and vascular permeability. Besides, AL-04 also inhibited thermally and chemically induced pain in tail-flick and acetic acid induced writing assays respectively in balb/c mice suggesting the analgesic potential of the compound. Acute toxicity studies further suggested the appreciable safety of AL-04 at high dose of 2000 mg/kg with no indications of toxicity or changes in biochemical and haematological parameters. Overall, the study insinuates the anti-inflammatory potential of AL-04 and paves way for further exploration of the compound as a safer therapeutic anti-inflammatory agent.