*2.9. Statistical Analysis*

All measurements were carried out in triplicate. The 50% inhibitory concentration (IC50) was calculated by nonlinear regression analysis and expressed as mean ± standard deviation (SD). Means were compared for statistically significant differences by one-way analysis of variance (ANOVA) using GraphPad Prism 7 (La Jolla, San Diego, CA, USA) with a 95% confidence interval.

#### **3. Results and Discussion**

The results of the various phytochemical tests carried out on *A. bilimbi* crude methanolic leaves extract are shown in Table 1. The results indicate the presence of alkaloids, flavonoids, terpenoids, and saponins (which have positive inference), while the extract showed negative results for the presence of free and combined anthraquinones.


**Table 1.** Phytochemical screening for chemical class identification of *Averrhoa bilimbi* crude methanolic leaves extract.

+++: Strong intensity reaction, ++: Medium intensity reaction, +: Weak intensity reaction, −: Undetected.

#### *3.1. DPPH Radical Scavenging Activity*

The DPPH radical exhibits absorption maxima at *λ* = 517 nm in its stable state. This absorption decreases when the stable radical undergoes reduction to a hydrazine derivative in the presence of antioxidant agent; consequently, the purple colour of the DPPH reagen<sup>t</sup> is changed to pale yellow [26]. The reduction is achieved either through the donation of a hydrogen atom to DPPH, or the removal of oxygen. The results of the DPPH radical scavenging activity which demonstrated the antiradical effect for crude methanolic extract and its subsequent fractions, as well as reference standard (ascorbic acid), are shown in Table 2.

**Table 2.** Free radical scavenging (DPPH) activity of *Averrhoa bilimbi* crude methanolic leaves extract and its fractions.


Values are mean of triplicate experiments ± standard deviation. The results were analysed using one way Analysis of variance (ANOVA). The superscript (\*) indicates significant activity (*p* < 0.05). IC50 represents the concentration required to scavenge 50% of available radicals. The IC50 values were calculated by linear regression of plots.

The results of the DPPH assay showed that the n-butanol fraction displays the highest activity, followed by the crude methanolic leaves extract and the chloroform fraction, while the hexane fraction did not show any significant radical scavenging activity. The radical scavenging activity displayed by the n-butanol fraction was comparable to that of the ascorbic acid, which was used as reference standard. The substantial antioxidant activity of the n-butanol fraction could be due to the presence of phenolic constituents which are frequently soluble in polar solvents and are known to display significant radical scavenging activity [8,27]. The IC50 value for the chloroform (13.44 ± 1.00) fraction also signifies a significant radical scavenging effect, which suggests the presence of some active constituents.

#### *3.2. Xanthine Oxidase Inhibitory Activity*

Xanthine oxidase as the name implies is an enzyme that is responsible for the conversion of xanthine and hypoxanthine to uric acid through oxidation reaction. Increased activity of xanthine oxidase leads to over production of uric acid and resultant hyperuricemia [28]. Increased levels of uric acid in the blood are implicated in the development of gouty arthritis, as well as increased reactive oxygen species, which are involved in various pathological processes [29]. Table 3 shows the result for the xanthine oxidase inhibitory activity of *A. bilimbi* crude methanolic leaves extract and its resultant fractions. The n-butanol fraction displayed significant (*p* < 0.05) activity with 50% effective concentration (IC50) of 64.84 ± 3.93 μg/mL, while the hexane and chloroform fractions displayed very weak inhibitory activities. The xanthine oxidase inhibitory activity observed with this fraction suggests that it contains substantial amount of phytoconstituents with xanthine oxidase inhibitory potential.

**Table 3.** IC50 for xanthine oxidase inhibitory activity of *Averrhoa bilimbi* crude methanolic leaves extract and its fractions.


Values are mean of triplicate experimental results with standard deviation. The results were analysed using one way ANOVA. The symbol asterisk (\*) indicates significant activity (*p* < 0.05).

Xanthine oxidase is present in large quantities in the liver, where it plays vital role in purine nucleotide metabolism. The activity of xanthine oxidase has been linked to the generation of reactive oxygen species such as superoxide (O<sup>2</sup>−), which is known to play vital roles in the development and progression of important pathological conditions like diabetes mellitus and related complications [30]. This result suggests that inhibition of xanthine oxidase could be part of the mechanism of *A. bilimbi* activity against oxidative tissue damage.

#### *3.3. LC-QTOF-MS Analysis of n-Butanol Fraction of Averrhoa bilimbi Crude Methanolic Leaves Extract*

The results of the LC-QTOF-MS analysis of active n-butanol fraction of *A. bilimbi* crude methanolic leaves extract are shown in Figure 1 and Table 4. Several compounds which were detected in the active n-butanol fraction have been identified from the LC-MS compound library by matching their accurate mass. There were also some unknown compounds that could not be identified because they were not present in the compound database. The list of identified compounds include two phenolic constituents, namely, 5,7,4-trihydroxy-6-(1-ethyl-4-hydroxyphenyl) flavone-8-C-glucoside (cucumerin A) (Figure 2) and afzelechin 3-*O*-alpha-L-rhamnopyranoside (Figure 3) which could partly be responsible for the antiradical and xanthine oxidase inhibitory activity of the n-butanol fraction of the *A. bilimbi* crude methanolic leaves extract.

**Figure 1.** LC-MS spectrum of n-butanol fraction of *Averrhoa bilimbi* crude methanolic leaves extract (For compounds and retention time, please refer to Table 4).


**Figure 2.** Tentative structure of 5,7,4-trihydroxy-6-(1-ethyl-4-hydroxyphenyl)flavone-8-C-glucoside (cucumerin A).

**Figure 3.** Tentative structure of afzelechin 3-*O*-alpha-L-rhamnopyranoside.

The two compounds, viz. cucumerin A and afzelechin 3-*O*-alpha-L-rhamnopyranoside, are flavonoid glycosides. The antioxidant activities of flavonoid glycosides have been well studied. Based on our literature search, we discovered that there is no available literature focusing specifically on the radical scavenging effect or xanthine oxidase inhibitory activity of afzelechin 3-*O*-alpha-L-rhamnopyranoside and cucumerin A. However, previous research findings have shown that related flavonoid glycosides displayed significant antioxidant potential through DPPH radical scavenging effect and xanthine oxidase inhibitory activity [31,32]. Thus, it can be rightly construed that the antioxidant activity of the n-butanol fraction of *A. bilimbi* crude methanolic leaves extract could partly be due to the presence of phytoconstituents which were identified through the LC-QTOF-MS analysis.

#### *3.4. Identification of Compounds Structures through Fragmentation Analysis (LC-QTOF-MS/MS)*

A Q-TOF LC-MS system was used to analyze samples with some modifications [33]. LC-QTOF-MS/MS analysis was carried out to study the fragmentation pattern of 5,7,4-trihydroxy-6-(1-ethyl-4-hydroxyphenyl)flavone-8-glucoside (cucumerin A) (Figures 4 and 5) and afzelechin 3-*O*-alpha-L-rhamnopyranoside (Figures 6 and 7), in order to further characterize the structure of these compounds. The MS/MS spectrum and fragmentation pattern of cucumerin A are shown in Figures 4 and 5, respectively. The spectrum shows the presence of fragment ions that are the characteristic of

C-glycosides. The ion peaks at *m*/*z* 403 and *m*/*z* of 421 representing [M + H-150]+ and [M + H-132]+ are typical of flavonoids with C-glycosides [34]. The peak at *m*/*z* 421 (base peak) represents the most stable ion fragment. In addition, a neutral loss of water molecules was observed, which produced fragment ions at [M + H-18]+, [M + H-36]+ and [M + H-54]+. A peak at *m*/*z* 341 [M + H-132]+, which indicates the loss of the B ring of the flavone skeleton and 1-ethyl-4-hydroxyphenyl ring attached to C-6 of the compound, further confirmed its structure. Based on this fragmentation pattern, we resolve the structure of this compound as 5,7,4-trihydroxy-6-(1-ethyl-4-hydroxyphenyl) flavone-8-glucoside (cucumerin A). Figures 6 and 7 show the MS/MS spectrum and fragmentation pattern of afzelechin 3- *O*-alpha-L-rhamnopyranoside, respectively. The peak at *m*/*z* 275 represents the aglycone afzelechin ion, which was produced from the loss of the glycone moiety (O-rhamnosyl). This form of fragmentation pattern is characteristic of flavone-O-glycosides [35]. The aglycone ion underwent further fragmentation to yield an ion peak at *m*/*z* 107, which is characteristic of the flavan. The spectrum also revealed the loss of water molecules with peaks at *m*/*z* 403 and 367 representing [M + H-18]+ and [M + H-54]+. Based on this information, we tentatively identified this compound as afzelechin 3- *O*-alpha-L-rhamnopyranoside.

**Figure 4.** MS/MS spectrum of cucumerin A.

**Figure 5.** Fragmentation pattern of cucumerin A.

**Figure 6.** MS/MS spectrum of afzelechin 3-*O*-alpha-L-rhamnopyranoside.

**Figure 7.** Fragmentation pattern of afzelechin 3-*O*-alpha-L-rhamnopyranoside.
