6. Discussion
In this study, chemical isolation from the triterpenoid-rich fractions of the extracts of the vines and leaves of M. charantia yielded predominantly four cucurbitane-type triterpenoids: momordicines I, II, and IV, and TCD. Momordicine I exerted deleterious effects on the growth of normal cell lines, momordicine II had a milder adverse effect on cells, and momordicine IV and TCD were not harmful to normal cells at 0.1–100 μM. Therefore, in view of safety, momordicine IV and TCD are more appropriate for potential development in biomedical applications than are momordicine I or II. However, these findings suggest that the extract of vines and leaves from M. charantia contains potentially toxic chemicals and thus should be used with caution.
The anti-inflammatory potential of TCD was more notable than those of momordicines II or IV; thus, we further characterized it. To reiterate, TCD was indicated to have anti-inflammatory activity both in vivo and in vitro because it inhibited LPS-induced phagocytosis and the expression of iNOS, NO, TNF-α, and IL-6 in a macrophage cell model. Furthermore, it ameliorated ear edema in an animal model. The underlying mechanism likely hinges on the suppression of the IKK/NF-κB pathway. However, LPS enhanced the expression of Nrf2, HO-1, and GCLM, which may provide protection against excessive inflammatory responses
[17], whereas TCD also increased the expression of these proteins whether when used alone or together with LPS. HO-1 reduces cellular oxidative damage and mitigates inflammatory responses
[18][19][20]. Thus, TCD likely protected cells from oxidative damage by activating the Nrf2/HO-1 pathway, which also helped reduce inflammation. However, TCD activated MAPKs and did not suppress MAPK activation by LPS. MAPKs increase the expression of some proinflammatory cytokines, which may explain why TCD only partially inhibited LPS-induced production of inflammatory cytokines, as shown in
Figure 5E,F.
MAPKs are suggested to activate the Nrf2/HO-1 pathway
[14][15][16], and ERK1/2 and p38 have been reported to activate Nrf2
[18][21][22], whereas the role of JNK is less clear. TCD and LPS both activated p38, ERK1/2, and JNK. Our data suggest that p38 and ERK1/2 are both involved in LPS- or TCD-induced activation of Nrf2, whereas JNK is not. TCD- or LPS-promoted Nrf2 expression was both partially yet significantly inhibited by either p38 inhibitor or ERK inhibitor, though the suppressive effects of these inhibitors declined on the Nrf2 downstream factors HO-1 and GCLM. Theoretically, if p38 and ERK1/2 are both involved in activating Nrf2, when one of these kinases is inhibited, Nrf2 can still be activated by the other one. Therefore, the observed partial decrease of Nrf2 level by either p38 or ERK1/2 inhibitor is reasonable. In TCD and LPS-cotreated cells (
Figure 8F–H), LPS and TCD may synergistically activate p38 or ERK1/2 when one of these kinases is inhibited, resulting in a higher extent of Nrf2 activation. Consequently, significant Nrf2 suppression by either inhibitor could not be observed in
Figure 8F.
On the basis of our findings, we propose the anti-inflammatory mechanism of TCD (Figure 10). TCD can reduce LPS-induced inflammation through inhibiting IKK activation by LPS. Moreover, TCD and LPS both activate ERK1/2 and p38. These kinases activate Nrf2 and increase the expression of the antioxidant enzymes HO-1 and GCLM. Inflammation can exacerbate oxidative stress; conversely, oxidative stress can aggravate inflammation. Antioxidant enzymes can protect cells from oxidative damage and thus prevent excessive amounts of inflammation. In other words, TCD reduces the extent of inflammation and protects cells from oxidative damage.
Figure 10. The proposed anti-inflammatory mechanism of TCD. TCD inhibits the LPS-induced activation of IKK. Moreover, TCD, together with LPS, activates ERK1/2 and p38, resulting in elevated expression of Nrf2 and the antioxidant enzymes HO-1 and GCLM to protect cells from oxidative damage. P-IκB, phosphorylated IκB.
Momordicines II and IV are the glucosides of momordicine I. In cytotoxicity assays, the GI50 of momordicine I was lower than that of momordicine II in IEC-18 cells. Moreover, momordicine I was lethal to FL83B cells and RAW 264.7 cells in concentrations higher than 10 μM, whereas 20–100 μM momordicine II did not inhibit the growth of these cells. Momordicine IV exhibited no harmful effect on any of the three tested cell lines. Thus, the order of cytotoxicity is momordicine I > momordicine II > momordicine IV. This suggests that glycosylation reduces the cytotoxicity of momordicine I, and that glycosylation on the –OH group of C-7 (momordicine IV) reduces toxicity to a greater extent than does glycosylation on the –OH group of C-23 (momordicine II).
TCD and momordicine I are isomers with similar structures, but their cytotoxic effects differ substantially. TCD exerts considerably less deleterious effects on cells than does momordicine I. These two isomers differ structurally between C-23 and C-25. The –OH group on C-25 of TCD is shifted to C-23 in momordicine I; the double bond between C-23 and C-24 in TCD is moved to between C-24 and C-25. This structural rearrangement notably affects the cytotoxicity. Future studies should investigate the cellular targets that interact with momordicine I and cause the cytotoxic effect.
Although cytotoxic, momordicine I exhibited a potential anti-inflammatory effect in that it reduced the LPS-induced iNOS expression at its sublethal concentration (10 μM). Thus, momordicine I and TCD both likely contribute to the anti-inflammatory activity of the extract of vines and leaves from M. charantia. However, momordicine I may also contribute to the adverse effects of the extract.
High lipophilicity may facilitate the permeation of a compound through biomembranes, resulting in higher bioactivity or cytotoxicity of the compound
[23][24][25]. The lipophilicity of the compounds reported here is TCD > momordicine I > momordicine II = momordicine IV according to their logP values. This lipophilicity order may explain why TCD and momordicine I are more bioactive than momordicine II and momordicine IV, but it cannot explain why momordicine I is the most cytotoxic among them. Moreover, momordicine I exhibited a potential anti-inflammatory effect at its sublethal concentration, suggesting that momordicine I may be as effective as or even better than TCD in anti-inflammation. Therefore, in addition to lipophilicity, there are likely other factors that influence the bioactivity or cytotoxicity of these compounds. For example, MAPKs regulate mitochondrial metabolism
[26], and some compounds were reported to target mitochondria for their cytotoxic or anti-inflammatory effects
[24][27][28]. Thus, whether the triterpenoids reported in this study target mitochondria deserves to be addressed. Moreover, some lipophilic compounds including nobiletin, kaempferol, luteolin, quercetin, and tangeretin were suggested to enter cells through an energy independent carrier-mediated system
[29]. It cannot be excluded that such a system also exists for the compounds reported here.
TCD was also found in the fruits of
M. charantia L.
[30]. This compound has been reported to reduce blood glucose levels in diabetic animal models
[30] and serve as an insulin sensitizer and insulin substitute
[31]. Our findings further demonstrate that it is an anti-inflammatory agent. Moreover, TCD was also isolated from the leaves of wild varieties of
M. charantia previously
[32] and was demonstrated to reduce periodontal pathogen- or
Cutibacterium acnes-induced inflammatory responses in human monocytic THP-1 cells in vitro and ameliorate inflammation in the corresponding pathogen-stimulated mouse models in vivo
[32][33]. Therefore, studies from different groups all support that TCD is an anti-inflammatory agent. In sum, TCD, which is distributed in the fruits, vines, and leaves of
M. charantia L., has hypoglycemic effects and anti-inflammatory functions. It can be regarded as a potential new agent for developing therapeutic or health-care products of anti-inflammation or glycemic control, or as an index ingredient for the bioactivity of bitter melon extracts.
Our results indicate that although momordicine I is cytotoxic, TCD is relatively safe and exerts notable anti-inflammatory effects. Therefore, a protocol that can enrich the extraction of TCD from the vines and leaves of M. charantia but remove momordicine I would likely enhance the anti-inflammatory effect and improve the safety of the extract.