Garcinia is widely distributed in tropical region and has been traditionally used in medicinal folklore as with little understanding of the actual mechanism of its therapeutic action. It is one of the most important medicinal plants that have been used traditionally for its medicinal value. Earlier studies have demonstrated that Garcinia possesses anti-bacterial, anti-cancer, antioxidant activities as well as emerging anti-inflammatory potential [31–33]. The pharmacological effects of Garcinia may be primarily due to presence of compounds such as hydroxycitric acid (HCA), garcinol, isogarcinol etc. [22–25]. From our previous studies, it has been established that GHE consists of several important phytocompounds and one of the potent active phytoconstituents is HCA. In our previous studies using the LPS-challenged rat model, it was established that GHE could potentially inhibit the inos and cox-2 expressions and decrease subsequent production of NO and PGE2 . Therefore, this study was undertaken to establish the inhibitory action of GHE and one of its important phytocompounds garcinol against iNOS and COX-2 targets in vitro. The present study investigated the inhibitory action of GHE and garcinol against inos and cox-2 mRNA expressions. In addition, quantitative analysis was done to study the effects of GHE and garcinol on the production of NO and PGE2 in lipopolysaccharide (LPS)-stimulated murine macrophage RAW264.7 cells. Also, molecular docking analysis was performed to study the interactions of garcinol and HCA with iNOS and COX-2 proteins. In this study, the test for Mycoplasma contamination was performed prior to the experiments and was found to be negative. Our results demonstrated the effects of GHE and garcinol on RAW264.7 cell viability. The present study reported significant (p < 0.05) inhibition of RAW264.7 cell viability post treatment with GHE and garcinol for 12hr. The IC50 of GHE and garcinol for RAW264.7 cells as calculated from the regression equation was found to be 460µg/ml and 24µM respectively (Figs. 4 and 5). Based on the IC50 two different doses for GHE (230µg/ml and 115µg/ml) and garcinol (6µM and 12µM) were selected for treatment of the cells. Similarly, the LPS dose was determined by nitric oxide assay. The cells on treatment with different concentrations of LPS after 24h showed significant increase in the production of nitric oxide. However, the maximum amount of nitric oxide produced 24h after LPS treatment was found at the concentration of 1µg/ml LPS by 6.99fold (p < 0.001) as compared to control. Therefore, the LPS dose selected for the present study was 1µg/ml (Fig. 6). The results of the nitric oxide assay and PGE2 estimation revealed that there was a significant increase in the NO level after 24h of LPS exposure as compared to control. On the other hand its levels were found to significantly decrease in GHE and garcinol pre-treated cells as compared to the LPS treated cells. The results of the nitric oxide assay revealed that there was a significant increase in the total NO level by 4.30 fold (p < 0.001) after 24h of LPS exposure as compared to control. On the other hand its levels was found to significantly decreased by -2.43fold (p < 0.05) in 230µg/ml GHE; -1.92fold (p < 0.05) and − 3.19 fold (p < 0.01) respectively in the 6µM and 12µM garcinol pre-treated cells as compared to the LPS treated cells (Fig. 7). It has been well established that NO is a pro-inflammatory mediator . The results of the PGE2 assay revealed that there was a significant increase in the PGE2 level by 13.33 fold (p < 0.001) 24h post LPS exposure as compared to control. On the other hand, its levels significantly decreased by -3.17 fold (p < 0.01) and − 5.28 fold (p < 0.01) respectively in the 115µg/ml and 230µg/ml GHE; -5.97 fold (p < 0.01) and − 7.31 fold (p < 0.01) respectively in the 6µM and 12µM garcinol pre-treated cells as compared to the LPS treated cells (Fig. 8).
Similarly, the results of the qPCR analysis revealed a significant increase in the transcript levels of inos and cox-2 after 24h of LPS treatment. Their transcript levels increased post LPS stimulation and found to decrease significantly in the GHE and garcinol pre-treated cells. The real-time qPCR studies revealed a significant increase in the transcript levels of inos, and cox-2 after 24h of LPS treatment. Their transcript levels increased by 46.68 fold (p < 0.001), and 12.98 fold (p < 0.01) post LPS-stimulation respectively as compared to control. The mRNA expressions of inos was however observed to be decreased significantly in the GHE (230µg/ml) pre-treated cells by -6.30 fold (p < 0.01) and cox-2 mRNA expression was found to be decreased significantly in both the concentrations of GHE (115µg/ml and 230µg/ml) by -1.90 fold (p < 0.05), and − 2.69 fold (p < 0.05) respectively as compared to the LPS treated cells (Fig. 8). Similarly, inos and cox-2 transcripts levels were found to be decreased in garcinol pre-treated cells respectively by -1.54 (p < 0.05), and − 3.47(p < 0.05) for 6µM; -2.89 (p < 0.01), and − 3.02(p < 0.05) for 12µM concentrations of garcinol as compared to the LPS treated cells (Fig. 9). The reduction of NO and PGE2 production in GHE and garcinol treated cells can thus be attributed to the decreased expression of inos and cox-2 respectively. iNOS is the enzyme principally responsible for NO in inflammation. iNOS is not usually expressed in resting cells but is however induced by some cytokines as well as microbial agents [35, 36]. Studies have reported stimuli like LPS and certain cytokines cause the expression of iNOS along with the production of pro-inflammatory mediators, such as prostaglandin and prostacyclin, through COX pathway [37, 38]. NO is known to elevate the synthesis of prostaglandin by activating the constitutive and inducible cyclooxygenases in many cells [38, 39]. Evidence suggests that in macrophages, the activity of iNOS and COX-2 induces the release of several pro-inflammatory mediators including NO and certain cytokines (Tumor necrosis factor-α and interleukins) [40, 41]. Hence, the inhibition of iNOS and COX-2 is an important step toward prevention of inflammation. The study by Liao et al. has shown that garcinol could suppress iNOS as well as COX-2 expression in LPS-stimulated macrophages and also inhibit the NFκB activation  which is in line with our study. Macrophages when stimulated by bacterial endotoxin (LPS) lead to induction of inflammatory response. Such responses involve the release of several pro-inflammatory mediators like NO and PGE2 whose production is induced by the expression of iNOS and COX-2 respectively [42, 43]. Inducible nitric oxide synthase catalyzes the production of a large amount of NO during the inflammatory condition. Therefore, iNOS inhibitors are essential for healing nitric oxide-mediated inflammatory responses . Moreover, herbal inhibitors such as GHE might play an important role as safe modulators of NO in the pathogenesis of inflammation. Similarly, COX-2 catalyzes the production of proinflammatory PGE2 and is known to be highly expressed during inflammation [45, 46]. From the present observations, it is found that Garcinia can act as a potential inhibitor of LPS-induced NO and PGE2 production. This inhibition might be due to the blocking of major downstream signaling involved in the production of these inflammatory mediators. However, the actual mechanism of inhibition is still unclear. It has been reported that Garcinia mangostana extracts induce anti-inflammatory action by decreasing the LPS-induced cytokine and PGE2 levels in immortalized human gingival fibroblasts cells . Similarly, Cho and Cho studied the antiinflammatory activities of ethanol extracts of Garcinia subelliptica in macrophages. They established that noncytotoxic concentrations of the extracts could decrease the NO and PGE2 generation by altering the iNOS and COX-2 expression respectively in LPS-induced RAW 264.7 cells. This observation is in line with our study. Further, they established that the decreased secretion of inflammatory mediators by Garcinia subelliptica was associated with a decrease in the activation of cJun Nterminal kinase (JNK) . Evidence suggests that LPS significantly induces the secretion of proinflammatory mediators in macrophages by triggering the Mitogen Activated Protein Kinase (MAPK) signaling. Therefore, blocking the downstream signaling including suppressing of p38, ERK, and JNK phosphorylation suggests a vital target for a therapeutic approach against inflammation [48–50].
Molecular docking studies were performed to elucidate the interaction between the targets (iNOS and COX-2) and garcinol as well as HCA as a potent inhibitor. The molecular properties of ligands (garcinol and HCA) such as LogP, number of hydrogen bond donors, number of hydrogen bond acceptors, molecular weight was calculated using Molinspiration tool (Table 2). HCA showed zero violations against the Lipinski’s rule of five. However, garcinol with molecular weight greater than 500 i.e., 602.81g/mol and logP value of 8.26 shows two violations against the rule of five. Therefore, it suggests that the bioavailability of HCA is more since it follows the Lipinski’s rule of five and garcinol is therefore considered to be poorly absorbed. Certain bioactive compounds having anti-inflammatory effects isolated from various medicinal plants have been studied through molecular docking against iNOS and COX-2 . In the present study, molecular docking analysis of garcinol and HCA against iNOS and COX-2 proteins showed good binding affinities. Molecular docking studies were performed to elucidate the interaction between the targets (iNOS and COX-2) and chief constituents (garcinol and HCA) of Garcinia as potent anti-inflammatory agent. The docking analysis clearly indicates significant binding affinities of garcinol and HCA with the protein targets. The present study revealed that garcinol showed hydrogen bonding interactions with Cys200, Ile201, Leu464 and hydrophobic interactions with Arg199, Gln263, Trp372, Pro350, Tyr373, Ala351, Met374, Val352, Trp463, Glu377, Val465, Met355, Pro466, Tyr491 residues of iNOS with a binding energy (ΔG) of -9.46kcal/mol (Fig. 10); hydrogen bonding interactions with Arg120 and hydrophobic interactions with Trp387, Ala516, Arg513, Tyr385, Leu384, Gly526, Ile517, Phe518, His90, Leu352, Val523, Ser353, Leu359, Val116, Leu531, Ser530, Val349, Tyr348, Ala527, Met522, Phe381 residues of COX-2 with a binding energy (ΔG) of -4.2kcal/mol (Fig. 11). Similarly, our study also revealed that HCA showed hydrogen bonding interactions with Thr121, Lys123, Thr126 and hydrophobic interactions with Thr109, Ile119, Pro122 residues of iNOS with binding energy (ΔG) of -3.11kcal/mol (Fig. 12); hydrogen bonding interaction with Lys83, Tyr115, Arg120, Glu524 and hydrophobic interactions with Pro84, Pro86, Ser 119, Tyr122 residues of COX-2 with binding energy (ΔG) of -3.15kcal/mol (Fig. 13). The H-bond formation together with the hydrophobic interactions indicates that garcinol as well as HCA other than the anti-inflammatory drugs could prove to be potent inhibitor of iNOS and COX-2. Studies have shown that anti-inflammatory drugs (NSAIDs) such as dexamethasone and indomethacin inhibit iNOS and COX-2 [5, 52]. NSAIDs like sodium diclofenac and ibuprofen have shown interaction with iNOS with binding energy (ΔG) of about − 6.7kcal/mol and − 7.50kcal/mol respectively [53, 54]. Also, it has been reported that diclofenac binds to Ser530 and Tyr385 residues of COX-2 active site . Our study has shown lower binding energy (ΔG= -9.46kcal/mol) between garcinol and iNOS rendering it a potent inhibitor. Several studies have reported many natural inhibitory ligands for iNOS and COX-2 by molecular docking analysis [51, 54, 56–59]. It is well established that non-steroidal anti-inflammatory drugs (NSAIDS) operate by suppressing the release of prostaglandins by inhibiting COX-2. Synthetic drugs such as Ibuprofen and Naproxen have been reported to prevent the release of prostaglandins. Reports suggest that molecular docking of inhibitory drugs such as Ibuprofen as well as Naproxen against COX-2 showed involvement of Arg120 and Tyr355 amino acid residues . Studies have also revealed Xanthone derivatives inhibit the COX enzyme that shows contact with Arg120, Ser 530, Met522, Tyr 355, Tyr385, Ser353 of the enzyme . Similarly, our findings demonstrate that HCA interacts with COX-2 forming H-bonds with Arg120. Studies have reported that molecular docking of certain flavonoids including quercetin against iNOS involved the interactions with the active site residues Ile119, Thr109, Ser118, Trp461, Met480 that suggested causing inhibition of iNOS . This is in agreement with the present study which showed favourable interaction of HCA with iNOS effectively involving Ile119 as well as Thr109 amino acid residues. Such potential molecular affinity of HCA provides a vast possibility for safe drug designing. Curcuminoids have been used as potential agents to block iNOS and COX-2 [53, 62]. Studies have shown that curcumin binds with iNOS with ΔG of-6.8 kcal/mol . Other phytocompounds like quercetin has been found to efficiently interact with iNOS active residues compared to tetrahydrobiopterin, an iNOS inhibitor . Similarly, curcumin analogues are known to interact with Ser530 residue of COX by H-bond . Also, Xanthone derivatives were observed to be potent inhibitors of COX. Studies have shown interaction of such derivatives with Arg120, Ser530, Met 522, Tyr355, Tyr 385, Ser353 residues of COX .This report is in agreement with the present study. These findings showed the inhibition of the target proteins by ligand binding, which is in line with our study. The present study therefore provides information suggesting the possible anti-inflammatory role of Garcinia and its important compound garcinol.