2.1 Extraction and isolation of C. canum stem bark
The extraction of the C. canum stem bark has yielded oily yellowish-brown n-hexane (22.0 g), light brown dichloromethane (15.1 g), dark brown chloroform (24.8 g), reddish-brown ethyl acetate (10.9 g), and reddish-black methanol (21.3 g) crude extracts. The percentage yield of the crude extracts was tabulated in Table 1. The compounds from the hexane and chloroform extracts have been previously reported (Lizazman et al. 2023).
Meanwhile, the isolation of ethyl acetate extract has identified l-hydroxy-7-methoxyxanthone (1) (Charoensup et al. 2022), caloxanthone C (2) (Lizazman et al. 2023), trapezifolixanthone (3) (Lizazman et al. 2022), ananixanthone (4) (Karunakaran et al. 2020), euxanthone (5) (Lim et al. 2019), gentisin (6) (Zheng et al. 2014), 2-hydroxyxanthone (7) (Amanatie et al. 2017), canumolactone (8), α-mangostin (9) (Morelli et al. 2015; Abate et al. 2022) as shown in Figure 1. The structural elucidation of the compounds was achieved using 1D, 2D NMR, and comparison of the spectral data with the previous literature. The spectral data for compounds 1-9 and the flow chart of the isolation works were reported in the supplementary data (3.4.1-3.4.9) and Figure S1 respectively. The 1D and 2D NMR spectra of canumolactone (8) were also reported (Figure S4-S8).
Table 1. Extraction yield of C. canum extracts
Crude extracts
|
Dry yield (g)
|
Percentage yield (%)
|
n-hexane
|
22.0
|
2.20
|
dichloromethane
|
15.1
|
1.51
|
chloroform
|
24.8
|
2.48
|
ethyl acetate
|
10.9
|
1.09
|
methanol
|
21.3
|
2.13
|
2.2 Antioxidant assay
The antioxidant activity of the extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay (Trinh et al. 2014). The radical scavenging activity (RSA), which indicates the ability of the extracts to neutralize free radicals, varied notably across the extracts. The RSA percentages were ranged from 2.65 to 97.14 % as tabulated in Table 2. The ethyl acetate extract exhibited the highest RSA (97.14 ± 0.24 %), followed closely by the methanolic extract (86.89 ± 0.12 %). The lowest activity was demonstrated by the n-hexane extract with 2.65 ± 1.19 %. Meanwhile, comparable radical scavenging activity was exhibited by chloroform extract (19.46 ± 1.26 %) and dichloromethane extract (15.83 ± 2.15 %). These findings suggest that certain extracts possess superior antioxidant capabilities, potentially due to the presence of specific bioactive compounds. The results were tabulated in Table 2.
2.3 Total phenolic content
The total phenolic content of the extracts was determined using the Folin-Ciocalteu (FC) method (Naves et al. 2019). The total phenolic content (TPC) of the crude extracts was quantified using the established standard calibration curve of gallic acid depicted in Figure S2 (y = 0.0079x – 0.0773, R2 = 0.9697). The result obtained revealed significant differences among the extracts, and expressed as milligrams of gallic acid equivalent per gram of dried weight extract (mg GAE/ g extract). The TPC was ranged from 83.07 to 277.33 mg GAE/g extract as tabulated in Table 2. The ethyl acetate extract contained the highest concentration of phenolic compounds (277.33 ± 9.65 mg GAE/g extract), followed by the methanolic extract (223.48 ± 12.88 mg GAE/g extract). The chloroform, dichloromethane, and n-hexane extracts displayed progressively lower concentrations, with values of 96.06 ± 2.64 mg GAE/g extract, 92.35 ± 3.43 mg GAE/g, and 83.07 ± 1.84 mg GAE/g extract, respectively. Phenolic compounds are well-known for their antioxidant properties, and the higher TPC values observed in certain extracts align with their enhanced RSA. The results were tabulated in Table 2.
2.4 Total flavonoid content
The total flavonoid content of the extracts was determined using the aluminium chloride calorimetric method (Sakai et al. 2022). The total flavonoid content (TFC) of the crude extracts was quantified using the established standard calibration curve of quercetin depicted in Figure S3 (y = 0.0321x – 0.0703, R2 = 0.9548). The result highlighted considerable variability across the extracts, and expressed as milligrams of quercetin equivalent per gram of dried weight extract (mg QE/ g extract). The TFC ranging from 11.55 to 139.56 mg QE/ g extract as tabulated in Table 2. The highest TFC was found in the chloroform extract (139.56 ± 3.19 mg QE/g extract), followed by the dichloromethane extract (117.96 ± 3.35 mg QE/g extract). Subsequently, the n-hexane extract exhibited a falovonoid content of 11.55 ± 0.32 mg QE/g extract, while the ethyl acetate and methanolic extracts showed values of 87.66 ± 0.36 mg QE/g extract and 43.51 ± 2.22 mg QE/g extract, respectively. Flavonoids, like phenolic compounds, possess notable antioxidant properties, contributing to the overall RSA of the extracts. The results were tabulated in Table 2.
Table 2. Radical scavenging activity, total phenolic content, and total flavonoid content of the C. canum crude extracts
Crude
extracts
|
Radical scavenging activity (%)
|
Total phenolic content (mg GAE/ g extract)
|
Total flavonoid content (mg QE/ g extract)
|
n-hexane
|
2.65 ± 1.19
|
83.07 ± 1.84
|
11.55 ± 0.32
|
dichloromethane
|
15.83 ± 2.15
|
92.35 ± 3.43
|
117.96 ± 3.35
|
chloroform
|
19.46 ± 1.26
|
96.06 ± 2.64
|
139.56 ± 3.19
|
ethyl acetate
|
97.14 ± 0.24
|
277.33 ± 9.65
|
87.66 ± 0.36
|
methanol
|
86.89 ± 0.12
|
223.48 ± 12.88
|
43.51 ± 2.22
|
An intriguing correlation between RSA and phytochemical content of the C. canum crude extracts was observe as in Figure 2. Notably, there appears to be a consistent trend between the levels of certain phytochemicals and the antioxidant potential of the extracts. When examining the RSA (%), we found that extracts with higher TPC (mg GAE/g extract) tended to exhibit greater RSA. This suggests that the presence of phenolic compounds may contribute significantly to the antioxidant potential of the extracts. On the other hand, the relationship between RSA (%) and TFC (mg QE/g extract) appeared less pronounced, indicating that flavonoid content may not play as substantial a role in determining the antioxidant activity of the extracts compared to phenolic compounds.
Based on the Pearson correlation coefficient (r) and the associated significance levels (p-values), the relationship between RSA (%) and TPC exhibits contrasting patterns (Table 3). The strong positive correlation coefficient of 0.9875 (p<0.05) suggests a highly significant linear relationship between RSA (%) and TPC. These findings support the notion that phenolic compounds contribute significantly to the antioxidant activity exhibited by C. canum extracts, with higher levels of phenolic content corresponding to enhanced RSA. However, the correlation coefficient of 0.1338 (p>0.05) between RSA (%) and TFC suggests a weak and statistically insignificant linear relationship. This indicates a negligible linear relationship between the antioxidant potential and the concentration of flavonoids in the extracts. While flavonoids are recognized for their antioxidant properties, the results suggest that, in the context of C. canum extracts, phenolic compounds may play a more significant role in determining the antioxidant activity compared to flavonoids.
Table 3. Pearson correlation coefficients between radical scavenging activity (%) and phytochemical content
Phytochemical content
|
Pearson correlation coefficient (r)
|
Significant level (p-value)
|
TPC
|
0.9875
|
0.002
|
TFC
|
0.1338
|
0.830
|
2.5 Discussion
Xanthones are commonly found in Calophyllum plants, known for their diverse biological activities. Previous studies on C. canum have identified several xanthones from different parts of plant. For instance, (Carpenter et al. 1969) isolated osajaxanthone, 2- (3,3-dimethylallyl) -1,3,7- trihydroxyxanthone, and 2- (3,3-dimethylallyl) -1,3,5,6-tetrahydroxyxanthone from the heartwood, while (Taher et al. 2020; Lizazman et al. 2023) reported biscaloxanthone, trapezifolixanthone A, 5-methoxytrapezifolixanthone, 5-methoxyananixanthone, 1,5-dihydroxy -3- methoxy-4 -isoprenylxanthone, and 6-deoxyisojacareubin from the stem bark. The current study focused on isolating compounds from the ethyl acetate extract of C. canum, resulting in the identification of 1-hydroxy-7-methoxyxanthone (1), caloxanthone C (2), trapezifolixanthone (3), ananixanthone (4), euxanthone (5), gentisin (6), 2-hydroxyxanthone (7), canumolactone (8), and α-mangostin (9).
In terms of radical scavenging activity (RSA), the n-hexane extract exhibited the lowest activity (2.65 ± 1.19 %) consistent with previous findings indicating a higher half response concentration (RC50 85 ± 4.35 µg/mL) compared to the methanol extract (Alkhamaiseh et al. 2012). (Susanti et al. 2011) also reported weak antioxidant (IC50 0.26 mg/mL) for the n-hexane extract of C. canum stem bark. Similarly, the dichloromethane portrayed weak RSA with 15.83 ± 2.15 % which is comparable with the previous study indicating RC50 91.6 ± 6.65 µg/mL. In contrast, the methanolic extract displayed high RSA (86.89 ± 0.12 %), supported by its low IC50 11.89 ± 0.74 µg/mL (Ramli et al. 2019) and RC50 < 31.25 µg/mL (Alkhamaiseh et al. 2012).
Additionally, the n-hexane, dichloromethane, and chloroform extracts exhibited similarly low total phenolic content (TPC), with the n-hexane extract displaying the lowest TPC at 83.07 ± 1.84 mg gallic acid equivalents per gram (GAE/g) of extract. This finding is consistent with a previous report (Alkhamaiseh et al. 2012), which indicated a TPC of 1.292 ± 0.001 µg gallic acid per 10 mg of extract. Notably, the TPC of the extracts increased with the polarity of the solvents used for extraction. The dichloromethane extract (92.35 ± 3.43 mg GAE/g extract) exhibited a higher TPC than the n-hexane extract, as observed in previous research (1.992 ± 0.002 µg gallic acid/10 mg extract) (Alkhamaiseh et al. 2012). However, the chloroform extract demonstrated a slightly higher TPC value than that of the dichloromethane extract. The ethyl acetate extract displayed the highest TPC, followed by the methanolic extract (223.48 ± 12.88 mg GAE/g extract). In comparison with previous research (Alkhamaiseh et al. 2012), our results align with the observation that methanol yielded the highest TPC, with 3.517 ± 0.017 µg gallic acid/10 mg extract. However, another study reported a lower TPC for the methanolic extract at 77.11 ± 2.78 mg GAE/g of extract (Ramli et al., 2019).
Moreover, the n-hexane extract exhibited the lowest amount of total flavonoid compounds, measuring 11.55 ± 0.32 mg quercetin equivalents per gram (QE/g) of extract. This finding aligns with a previous report by (Alkhamaiseh et al. 2012), which indicated a total flavonoid content (TFC) of 5.040 ± 0.184 µg quercetin per 10 mg of extract. In contrast, the dichloromethane extract displayed the highest TFC value at 117.96 ± 3.35 mg QE/g extract. However, (Alkhamaiseh et al. 2012) reported a significantly lower TFC for the dichloromethane extract at 11.201 ± 2.446 µg quercetin per 10 mg of extract, indicating a substantial difference in the observed TFC. Furthermore, our study revealed that the methanolic extract showcased a TFC of 43.51 ± 2.22 mg QE/g extract. This contrasts with the findings of the previous report, which documented a TFC of 14.643 ± 1.222 µg quercetin per 10 mg of extract (Alkhamaiseh et al. 2012). These inconsistencies in TFC values between our study and previous reports emphasize the importance of methodological differences, plant variability, and extraction techniques, which can significantly influence the quantification of bioactive compounds in natural extracts. Importantly, the antioxidant activity, TPC and TFC of the chloroform and ethyl acetate extracts is reported here for the first time.
According to the Pearson’s correlation analysis, the statistical data indicate a positive relationship between high total phenolic content (TPC) and strong antioxidant activity within the extracts. This suggests that phenolic compounds play a significant role in the antioxidant activity measured by the DPPH method. Antioxidant compounds possess varying polarities, leading to differences in their concentrations depending on the solvent used for extraction or isolation. Solvents can generally be categorized as polar (such as ethanol, methanol, and aqueous solutions) or nonpolar (including acetone, ethyl acetate, and hexane), based on their ability to form ions in solution (Nwozo et al. 2023).