2.2.1. Anti-Inflammatory Activity

The anti-inflammatory activity of compounds **2**–**6** was analyzed. Compound **1** was assessed in our previous publication, and the low levels of compound **7** did not allow for further in-vitro assessments [18]. Interestingly, all the purified compounds exhibited anti-inflammatory properties by promoting the down-regulation of the pro-inflammatory gene markers *IL-6*, *TNF-α*, *COX-2,* and *iNOS* (Figure 3). All the compounds purified from the Indian bitter melon significantly decreased the expression of *IL-6* compared to LPS treated cells. The lowest expression of *IL-6* was observed in the cells treated with karaviloside VI, karaviloside VIII, momordicoside L, and momordicoside A. The downregulation of *iNOS* was also observed in all the compounds except momordicoside A. Similarly, momordicoside A, along with momordicoside L, significantly decreased *TNF-α* mRNA expression. Momordicoside L and karaviloside VI are also considerably reduced the expression of *COX-2* mRNA expression.

**Figure 3.** Effect of purified compounds on mRNA expression of *IL-6*, *TNF-α*, *iNOS*, and *COX-2* in LPS-induced murine macrophage RAW 264.7 cells. The cells were pretreated with 50 μM karaviloside VI (**2**), karaviloside VIII (**3**), momordicoside L (**4**), momordicoside A (**5**), and charantoside XV (**6**) followed by LPS (1 μg/mL) stimulation. Data are expressed as mean ± SD (*n* = 9) and analyzed by one-way ANOVA with a Tukey post hoc test. Different letters within the same plot indicate the significant differences at *p* < 0.05.

Pro-inflammatory cytokines mediate the inflammatory response in humans. LPS is a potent pro-inflammatory agent causing an increase in the expression of pro-inflammatory genes such as *IL-6*, *TNF-α*, *COX-2*, and *iNOS*. High levels of *IL-6* and *TNF-α* have been reported in patients with type-2 diabetes and insulin resistance [15]. Additionally, *COX- 2* has been reported to be induced under hyperglycemic conditions [16]. Any agent that promotes the decrease in the expression of these genes has the potential to work as a viable anti-inflammatory agent. Our results indicate that the compounds isolated from bitter melon may have potential antidiabetic activities by modulating the inflammatory process. In past studies, various bitter melon extracts and compounds have been reported to have anti-inflammatory activities in different cell models [21,31–33]. Bitter melon ethyl acetate extracts have been reported to decrease the expression of *iNOS*, *COX-2*, *IL-6,* and *TNF-α* in RAW 264.6 macrophages, but purified compounds derived from those extracts were not evaluated [34]. As such, the anti-inflammatory activity presented in this study suggests that bitter melon compounds are potential agents against inflammation and possible diseases that arise from chronic inflammation, such as diabetes.

#### 2.2.2. α-Amylase and α-Glucosidase

Several strategies have been explored in the management of diabetes mellitus for either reducing glucose production by the liver or enhancing insulin sensitivity or secretion [35]. Among these approaches, the inhibition of the *α*-amylase and *α*-glucosidase enzymes are directed to manage the post-prandial hyperglycemia by delaying starch hydrolysis by cleaving 1,4-glucosidic linkages [36,37]. In this sense, the α-amylase inhibitory effect of compounds **2**–**6** was also evaluated in this paper. The inhibitory effect ranges from 68.0 to 76.6%, but no significant statistical difference was observed among the compounds (Figure 4A). Additionally, the α-glucosidase inhibitory effect ranges from 23.7 to 56.5%. Karaviloside VIII was the most active compound, followed by karaviloside VI (40.3%), while the less active compounds were momordicoside L (23.7%), momordicoside A (33.5%) and charantoside XV (23.9%). Assayed compounds showed a lower α-glucosidase inhibitory effect than acarbose (Figure 4B).

**Figure 4.** α-Amylase (**A**) and α-glucosidase (**B**) inhibitory effects of purified compounds: karaviloside VI (**2**), karaviloside VIII (**3**), momordicoside L (**4**), momordicoside A (**5**) and charantoside XV (**6**). Compounds were assayed at 0.67 mM and data are expressed as mean ± SD (*n* = 3), and analyzed by one-way ANOVA with a Tukey post hoc test. Different letters within the same plot indicate there were significant differences at *p* < 0.05.
