Distilled technical grade methanol, hexane, ethyl acetate, and ethanol were used to produce the crude plant extracts and the preliminary fractions by vacuum liquid chromatography (VLC). Analytical reagent (AR) grade hexane (JT Baker), ethyl acetate (JT Baker), ethanol (Scharlau, JT Baker), methanol (JT Baker), acetonitrile (RCI Labscan, JT Baker), DMSO (RCI Labscan), and ultrapure water (18.2 MΩ) (OmniaTap, stakpure GmbH) were used for the further fractionation of the VLC fractions via various separation methods, and as needed for the bioassays.

C. intermedia stems (accession number UPB-0090) were collected from Halsema Highway, Atok, Benguet. The stems were washed and air-dried for 5–7 days, and were then homogenized in a stainless-steel pulverizer.

D. dao bark (accession number PUH 13423) were collected from the University of the Philippines Diliman Campus, Quezon City. The bark was washed, dried at 40°C in an oven for at least 3 days, and ground in a stainless-steel pulverizer.

The extraction and solvent partitioning procedures used were optimized in the laboratory and adapted from previous protocols (Punzalan and Villaseñor, 2019a). Approximately 1.9 kg of dried C. intermedia stems and approximately 1.4 kg of dried D. dao bark were soaked in distilled methanol, separately, for at least 3 days. The methanol soakings for both plants were then separately filtered and concentrated using a rotary evaporator (BÜCHI Rotavapor R-100). The water bath was set to 40°C and the chiller (BÜCHI Recirculating Chiller F-105) was at 2.5°C. The extraction process was repeated at least 7 times for each plant.

Solvent partitioning was performed on the methanolic extracts of C. intermedia stem and D. dao bark. Approximately 25–26 g of methanolic extract was dissolved in 500 mL ultrapure water and then partitioned with 500 mL distilled hexane. The hexane extract was separated and concentrated using a rotary evaporator, and the addition of distilled hexane to the aqueous mixture was repeated at least 6 times. To extract the slightly polar components, 500 mL distilled ethyl acetate was shaken with the remaining aqueous portion from the initial aqueous mixture. Similarly, extraction of the ethyl acetate extract was performed at least 6 times, and the extract was subsequently concentrated. The remaining aqueous mixture was then filtered to remove insoluble particles and stored at −20°C. A total of 53.30 g and 76.91 g of methanolic extract were partitioned for C. intermedia stem and D. dao bark, respectively.

The fractionation procedures used were optimized in the laboratory and adapted from previous protocols (Abaya and Villaseñor, 2019; Punzalan and Villaseñor, 2019b). The ethyl acetate extracts of C. intermedia stem and D. dao bark were subjected to VLC. The samples were prepared by mixing the extracts with silica gel 60G for thin layer chromatography (Merck) in a 1:1 sample to silica weight ratio. Silica gel 60G was also used as packing material for the stationary phase, using a 1:30 sample to silica weight ratio. Dry silica was loaded and packed with the assistance of a vacuum pump onto a glass column with a radius of 4.25 cm and a medium porosity glass frit. Prior to loading the sample, the stationary phase was equilibrated using distilled hexane. Stepwise gradient elution was employed for both samples using a solvent system of distilled hexane and ethyl acetate, starting with 100% hexane, then changing the concentration of each solvent in 10% increments, until 100% ethyl acetate was reached. Fractions were collected by volume for each solvent system and concentrated using the rotary evaporator.

For C. intermedia stem, 5.492 g of ethyl acetate extract was used for VLC with a packed column height of 6.5 cm. The total volume used per solvent system is 500 mL. To recover highly polar compounds, stepwise gradient elution using a solvent system of distilled ethyl acetate and distilled ethanol was performed, starting from 100% distilled ethyl acetate, until 100% distilled ethanol, in 25% increments. The collected fractions were concentrated.

D. dao bark was subjected to VLC twice (5.038 g and 4.681 g), both times with a column height of 5.5 cm and a mobile phase of 250 mL per solvent system. Compounds with higher polarity were recovered by using 50% distilled ethyl acetate:50% distilled ethanol mixture, followed by 100% distilled ethanol. The collected fractions from both runs were pooled and concentrated.

Size-exclusion chromatography (SEC) was performed to fractionate the 13th VLC fraction (which eluted from the 50% ethyl acetate:50% ethanol solvent system) of the C. intermedia stem ethyl acetate extract. The packing material used for the stationary phase was Sephadex LH-20 (Sigma-Aldrich, LH20100), made to swell overnight in AR grade methanol. The stationary phase was loaded onto a glass column with a radius of 1 cm and a medium porosity glass frit. The resulting packed stationary phase had a height of 58.5 cm. The sample was prepared by dissolving 105 mg of the 13th VLC fraction in 1.5 mL AR grade methanol, which was then centrifuged prior to loading. AR grade methanol was used as the mobile phase, and the fractions were collected by band and volume. Based on the obtained UHPLC-MS profiles of the SEC fractions, fractions 24 to 31 were pooled together and concentrated using a rotary evaporator. Fractionation of the 13th VLC fraction using SEC was repeated three times. The total weight of sample used for SEC was 315 mg.

The pooled C. intermedia stem SEC fraction was then fractionated further through solid phase extraction (SPE). The fraction was dissolved in 100% ultrapure water. The SPE cartridge (Bond Elut C18 500 mg/3 mL; Agilent) was activated using AR grade acetonitrile, then equilibrated with ultrapure water. The sample was loaded onto the cartridge, then washed with ultrapure water. Stepwise gradient elution was performed, starting with 100% ultrapure water until 100% AR grade acetonitrile, in 10% increments. All the fractions were collected per solvent system and concentrated using a refrigerated centrifugal vacuum concentrator (Labconco). The first fraction is henceforth referred to as CINS.

Further fractionation of the 12th VLC fraction (eluted from 100% ethyl acetate) of D. dao bark ethyl acetate extract was done through gravity column chromatography (GCC). For the stationary phase, silica gel 60 (0.063–0.200 mm) for column chromatography (Merck) was used as packing material. A 1:100 sample to silica weight ratio was followed. The stationary phase was loaded onto a glass column with a radius of 1 cm, and a medium porosity glass frit. The solvent used for column equilibration and as the initial solvent was 30% hexane:70% ethyl acetate. The fraction (142.2 mg) was dissolved with 30% hexane:70% ethyl acetate and loaded onto the column through wet loading. Stepwise gradient elution and collection by band and volume were employed, which resulted in a total of 15 GCC fractions. Based on the profiles of the 15 fractions obtained using UHPLC-MS, similar fractions were pooled together. Pooled GCC fraction 4 (henceforth referred to as DDAB) was then subjected to UHPLC-MS/MS analysis.

All the extracts and fractions generated were subjected to α-glucosidase and 15-lipoxygenase-1 inhibition assays.

The α-glucosidase inhibitory activities of the extracts and fractions were determined via a spectrophotometric enzyme assay based on the Tuklas Lunas Protocols for Drug Discovery and Development in the Philippines (Naing et al., 2019). The assay was based on the procedure of Shobana et al. (2009) with modifications. Modifications of the assay were established from optimization experiments such as determination of the optimal enzyme concentration, K_m and V_max determination, determination of the IC₅₀ of the positive control, and solvent tolerance of the enzyme.

The reaction mixture contained phosphate buffer (50 mM NaH₂PO₄ (Loba Chemie Pvt. Ltd.)/Na₂HPO₄ (Loba Chemie Pvt. Ltd.) with 100 mM NaCl (Scharlau) buffer, pH = 6.8), α-glucosidase from Saccharomyces cerevisiae recombinant (EC 3.2.1.20; Sigma-Aldrich, G0660), sample/inhibitor, and p-nitrophenyl-α-D-glucopyranoside (p-NPG) (Merck). The enzyme inhibitory activity was tested by plating 10 µL of the sample (300 ppm), 216 µL of the phosphate buffer, and 24 µL of the α-glucosidase solution (120 mU/mL) into a 96-well quartz microplate (Hellma Analytics). After incubation at 37°C for 10 min, 50 µL of p-NPG (11.34 mM) was added to start the reaction. Using a microplate spectrophotometer (Thermo Scientific Multiskan GO) and the software SkanIt RE v7.0, the absorbance was monitored at 405 nm every 30 s for 30 min. The positive control used was acarbose (Sigma-Aldrich, A8980-1G, SLCF5122), and the negative control used was 5% DMSO in phosphate buffer.

The % inhibition was calculated using the following equation: %   I n h i b i t i o n   p e r   r e p l i c a t e = A b s o r b a n c e u n i n h i b i t e d   −   A b s o r b a n c e i n h i b i t e d   A b s o r b a n c e u n i n h i b i t e d × 100

Dose-response curves for CINS, DDAB and acarbose against the α-glucosidase assay were constructed, Supplementary Figures S13–S15 and the IC₅₀ values were calculated. For the effective concentrations of CINS, DDAB and acarbose used in the determination of the IC₅₀ values, refer to Supplementary Table S1.

A spectrophotometric assay for the determination of lipoxygenase inhibitory activity was employed following the Tuklas Lunas Protocols for Drug Discovery and Development in the Philippines (Allanigue and Hernandez, 2019). The assay was based on the procedures of Auerbach et al. (1992) and Axelrod et al. (1981) with modifications. Modifications of the assay were established from optimization experiments such as determination of the optimal enzyme concentration, K_m and V_max determination, determination of the IC₅₀ of the positive control, and solvent tolerance of the enzyme.

The reaction mixture contained phosphate buffer (0.1 M KH₂PO₄ (Loba Chemie Pvt. Ltd.)/K₂HPO₄ (Loba Chemie Pvt. Ltd.), pH = 7.4), lipoxidase (15-lipoxygenase-1) from Glycine max (soybean) Type I-B (EC 1.13.11.12; Sigma-Aldrich L7395), sample/inhibitor, and linoleic acid (Sigma-Aldrich, L1376). The enzyme inhibitory activity was tested by plating 10 µL of the sample (1,000 ppm), 260 µL of the phosphate buffer, and 15 µL of the enzyme solution (1896 U/mL) into a 96-well quartz microplate (Hellma Analytics). After incubation at 25°C for 5 min, linoleic acid (1.6085 mM) was added to start the reaction. Using a microplate spectrophotometer (Thermo Scientific Multiskan GO) and the software SkanIt RE v7.0, the absorbance was monitored at 234 nm every 10 s for 5 min. The positive control used was nordihydroguaiaretic acid (NDGA) (Sigma-Aldrich, 74,540-1G, BCBX4406), and the negative control used was 10% DMSO in phosphate buffer.

The % inhibition was calculated using the following equation: %   I n h i b i t i o n   p e r   r e p l i c a t e = A b s o r b a n c e u n i n h i b i t e d   −   A b s o r b a n c e i n h i b i t e d   A b s o r b a n c e u n i n h i b i t e d × 100

Dose-response curves for CINS, DDAB and NDGA against the 15-lipoxygenase-1 assay were constructed, Supplementary Figures S16–S18 and the IC₅₀ values were calculated. For the effective concentrations of CINS, DDAB and NDGA used in the determination of the IC₅₀ values, refer to Supplementary Table S2.

For both the α-glucosidase and the 15-lipoxygenase-1 assays, extracts and fractions that exhibit ≥50% inhibition were considered to be active against the enzyme.

The assays were performed with three trials, with 4 replicates per trial. The enzyme inhibitory activities are presented as average ± standard deviation. IBM SPSS Statistics 26 was used to conduct the statistical analysis of the assay data. One-sample Kolmogorov-Smirnov test was done to check the normality of the distribution of slopes for each replicate, followed by Levene’s test to check the equality of the variances from the different test groups. Brown-Forsythe and Welch tests were done afterward, since the variances were unequal (p < 0.05). The data was then analyzed using Tamhane T2 - One-way ANOVA with unequal variances to check if the samples are considered active. Grubbs’ test (Graphpad outlier calculator) was used to determine any outliers in the replicates. GraphPad Prism 9 was used to calculate the IC₅₀ values. The results of the statistical analyses of the assay data can be found in Supplementary Table S3–8.

The UHPLC-MS/MS analysis and dereplication procedures were optimized in the laboratory and adapted from previous protocols (Junio et al., 2019).

Fractions were dissolved in LC-MS grade methanol (Merck) at a concentration of 500 μg/mL. The sample solutions were centrifuged, and the supernatants were transferred to LC-MS-certified vials (Waters). UHPLC-MS experiments were conducted on a Waters H-Class series system with a Xevo G2-XS quadrupole time of flight (QToF) mass spectrometer. A 0.3 mL/min flow rate was used with an Acquity UPLC HSS T3 1.8 µm (2.1 × 100 mm) column (Waters). The mobile phase was composed of varying ratios of LC-MS grade acetonitrile (Merck) and water (Merck), both with 0.1% formic acid. The composition of the acetonitrile used was as follows: 10% at 0.00–2.00 min, increasing from 10% to 100% at 2.00–8.00 min, 100% at 8.00–10.00 min, decreasing from 100% to 10% at 10.00–11.00 min, and 10% at 11.00–12.00 min. The injection volumes used for the samples were 1 µL for the positive mode and 3 µL for the negative mode.

Base peak intensity (BPI) chromatograms were obtained for 12 min per sample and spectra were acquired in ESI positive and negative modes within the range of 50–1,200 Da. ESI source parameters for the positive mode were set as follows: capillary voltage = 2.80 kV, sampling cone voltage = 40 V, source offset = 80 V, source temperature = 120°C, desolvation temperature = 450°C, cone gas = 50 L/h, and desolvation gas = 800 L/h. For the negative mode, ESI source parameters were set as follows: capillary voltage = 1.50 kV, sampling cone voltage = 30 V, source offset = 80 V, source temperature = 120°C, desolvation temperature = 500°C, cone gas = 50 L/h, and desolvation gas = 1000 L/h. Mass spectrometry data were collected using two modes: MS and data dependent acquisition (DDA). For the DDA mode, MS/MS acquisition was done on ions that exceeded an intensity threshold of 1.0 × 10⁵ and 1.0 × 10⁴ for the positive and negative modes, respectively. A maximum of 10 ions were selected for MS/MS acquisition from a 0.5-s MS scan. Three collision energy ramps (15–30 eV, 30–45 eV, and 45–60 eV) were set to acquire extensive data on the small molecules.

MassLynx v4.1 was used to view and obtain the BPI chromatograms of the fractions. Peak alignment and conversion of the RAW data file to mgf file were executed using MSDIAL, v4.9.221218 (Tsugawa et al., 2015). The data were then uploaded to the Global Natural Products Social Molecular Networking (GNPS) platform (Wang et al., 2016) using WinSCP v6.1.1.

The feature-based molecular networking (FBMN) module of the GNPS platform (Nothias et al., 2020) was used to match the data acquired using DDA mode with existing spectral databases to obtain the putative identities of the compounds found in the active fractions. The criteria for matching were also adapted from a previous study (Molino et al., 2021) and optimized in the laboratory. The parameters for the FBMN were set as follows: precursor ion mass tolerance = 0.02 Da and fragment ion mass tolerance = 0.50 Da, a maximum of 5.00 for the ppm error, a minimum cosine score of 0.70, and a minimum of 6 for the matched fragment ions. The mirror matches of the experimental spectra with the reference spectra were also considered as criteria for the determination of the putative hits.

The preliminary results of the bioassays done on C. intermedia stem and D. dao bark extracts and fractions can be found in Supplementary Figures S7-12. The methanolic extract of C. intermedia stem (12.20% yield) both exhibited α-glucosidase (AGLUC) (99.91% ± 0.04%) and 15-lipoxygenase-1 (LOX) (91.44% ± 0.11%) inhibitory activities. Solvent partitioning was performed on the methanolic extract to separate the nonpolar, slightly polar, and the polar extracts. C. intermedia stem ethyl acetate extract was fractionated using VLC because it exhibited higher inhibition against both AGLUC (99.85% ± 0.03%) and LOX (92.90% ± 0.28%). Moreover, the ethyl acetate extract also had a higher yield (20.63%) compared to the hexane extract (5.29%). A total of 15 fractions were produced from the VLC. Out of the 15 fractions, 5 fractions were active against AGLUC only, and 1 fraction was active against LOX only. Six fractions were found to be active against both AGLUC and LOX and the 13th VLC fraction was selected since it had the highest yield (26.56%).

SEC was used to further fractionate the 13th VLC fraction. From this, 31 fractions were generated through isocratic elution. Thirteen out of the 31 fractions exhibited AGLUC inhibitory activity only. Eight fractions (fractions 24–31) exhibited inhibitory activity against both AGLUC (96.57% ± 0.65% to 99.73% ± 0.11%) and LOX (48.26% ± 2.47% to 91.96% ± 0.53%). Upon UHPLC-MS profiling, it was observed that all eight fractions have almost the same chromatogram. Since all eight fractions contained all the major peaks, they were pooled together into 1 fraction. The pooled fraction was then fractionated using a C18 SPE cartridge, which generated 12 fractions. The first SPE fraction (CINS) was chosen for dereplication and IC₅₀ screening since it had the highest yield among the 12 fractions.

The AGLUC and LOX inhibitory activities of D. dao bark methanolic extract (12.82% yield) were 99.24% ± 0.22% and 99.26% ± 1.16%, respectively. Similar to the C. intermedia stem ethyl acetate extract, the D. dao bark ethyl acetate extract was pursued for fractionation since it exhibited high inhibitory activities against AGLUC (97.02% ± 1.59%) and LOX (97.73% ± 1.12%). Moreover, the D. dao bark ethyl acetate extract also had a higher yield (18.14%) than the hexane extract (8.87%).

The ethyl acetate fraction was fractionated through VLC. Out of the 25 VLC fractions of D. dao bark ethyl acetate, 12 fractions were found to be active against AGLUC and LOX. Two fractions exhibited inhibition against AGLUC only. The 12th VLC fraction was prioritized due to its high AGLUC (99.90% ± 0.03%) and LOX (108.22% ± 1.03%) inhibitory activities.

The 12th VLC fraction was further fractionated through GCC. Out of the 15 GCC fractions, 11 fractions showed inhibition against both AGLUC and LOX. One fraction was active against AGLUC only, and 1 fraction was active against LOX only. Based on the BPI chromatograms of the fractions obtained through UHPLC-MS, GCC fractions 6–11 (pooled GCC fraction 4) were pooled together. The pooled GCC fraction 4 (DDAB) had the highest yield among all the pooled fractions. DDAB was also selected for dereplication and IC₅₀ screening.

Table 1 summarizes the IC₅₀ results from the enzyme assays that were done on the prioritized active fractions. The IC₅₀ values reported here for each sample/control are the average of the three trials per sample/control.

It can be observed that both CINS and DDAB exhibited markedly lower IC₅₀ values as compared to acarbose in the AGLUC inhibition assay. On the other hand, both CINS and DDAB exhibited greater IC₅₀ values compared to NDGA in the LOX inhibition assay. This implies that the compounds present in these fractions are more potent inhibitors of AGLUC compared to acarbose but are less potent inhibitors of LOX compared to NDGA.

Multiple compounds were putatively identified through the dereplication of CINS and DDAB. These compounds are summarized in Table 2; Figure 1 for CINS, and Table 3; Figure 2, 3 for DDAB.

Based on the dereplication results, a literature search was conducted on the putative hits for both CINS and DDAB to see whether there have been previous studies on their antidiabetic and/or anti-inflammatory activities, which might explain the fractions’ bioactivities that were observed in the enzyme inhibition assays.

Ellagic acid (compound 1) is a phenolic compound that was previously identified in the hexane fraction from the methanolic extract of C. intermedia leaves and roots (Chang et al., 1996), and can be found in numerous plants such as the leaves of Tectaria subtriphylla (Hook. and Arn.) Copel. (Feng-Lin and Jhy-Yih, 1993), the leaves and bark of Acer negundo L. (Saleh et al., 1969), and the bark of Mallotus japonicus (L.f.) Müll.Arg. (Yoshida et al., 1982). It also has anti-diabetic and anti-inflammatory properties (Ríos et al., 2018). More specifically, it was found to exhibit inhibitory activity against AGLUC with an IC₅₀ value of 2.18 μg/mL (You et al., 2012). Its anti-inflammatory mechanisms include the inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-6 (IL-6) production (BenSaad et al., 2017). Compound 1 was putatively identified from both CINS and DDAB fractions.

Corilagin (compound 2) is a hydrolysable tannin that was previously isolated from the shoot of Geranium thunbergii Siebold & Zucc. (Okuda et al., 1975), the roots of Phyllanthus emblica L. (Zhang et al., 2000) and the leaves of Phyllanthus niruri L. (Colombo et al., 2009). In addition, it was also isolated from the leaves of Terminalia macroptera Guill. and Perr. and exhibited AGLUC and LOX inhibitory activities with IC₅₀ values of 2.58 ± 0.08 µM and 41 ± 4 μM, respectively (Pham et al., 2014).

For (1S,2S,6R,7R, 9R)-6-methyl-10,12-dioxatricyclo [7.2.1.0 < 2,7>]dodec-4-en-8-one (compound 3), no previous studies have been conducted which quantified its antidiabetic properties using the AGLUC inhibition assay, and its anti-inflammatory activity using the LOX inhibition assay. Furthermore, this compound has not been previously identified from a plant source.

Isoscopoletin (compound 4) is a hydroxycoumarin that has been isolated from various plants such as the aerial parts of Tagetes lucida Cav. (Céspedes et al., 2006), Euphorbia hirta L. (Wu et al., 2012) and from the stem wood of Zanthoxylum integrifoliolum (Merr.) Merr. (Chen et al., 2007). Furthermore, it also has antidiabetic activities that is exemplified by a variety of mechanisms such as the inhibition of advanced glycation end products formation (IC₅₀ = 18.78 ± 0.50 µM) (Jung et al., 2012). Moreover, it also exhibited inhibitory activities towards α-amylase, maltase, and sucrase with % inhibition values of 0.2%, 32.2%, and 20.9%, respectively, at a concentration of 400 µM (Kato et al., 2008). Its anti-inflammatory properties can be attributed to its ability to inhibit LOX (IC₅₀ = 15.1 µM) (Deng et al., 2007).

2-O-galloylhyperin (compound 5) is a flavonoid glycoside that was isolated from the aerial parts of Rubus amabilis Focke (Chen et al., 2001), Pyrola incarnata (DC.) Fisch. ex Freyn (Yazaki et al., 1989), Euphorbia lunulata Bunge (Nishimura et al., 2005) and the leaves of Eucalyptus globulus Labill. (Park et al., 2023).

Oleamide (compound 6) is a fatty acid amide that is found in numerous plants such as the stem of Desmos cochinchinensis Lour. Sun et al. (1995), the leaves of Polygonum maritimum L. (Rodrigues et al., 2017) and the leaves of Lithocarpus polystachyus (Wall. ex A. DC.) Rehder (Fang et al., 2022). It was reported with an IC₅₀ value of 30.69 ± 1.47 μg/mL against AGLUC (Fang et al., 2022). Moreover, it demonstrated anti-inflammatory effects through various mechanisms such as the inhibition of LPS-induced nuclear factor kappa B (NF-κB) activation (Oh et al., 2010) and the suppression of p38, ERK, and PI 3-kinase/Akt and ROS accumulation (Moon et al., 2018). Compound 6 was putatively identified from both CINS and DDAB fractions.

1,2,3,6-tetra-O-galloyl-beta-D-glucose (compound 7) is a tannin that was putatively identified in Corchorus olitorius L. leaf extract through UHPLC-MS (Alara et al., 2023). It was also isolated from the stem bark of Bersama abyssinica subsp. abyssinica and Bersama abyssinica subsp. paullinioides (Nyamboki et al., 2021) and the leaves and branches of Phyllanthus emblica L. Zhang et al. (2002).

7,2′,3′-trimethoxyflavanone (compound 8) is a flavanone. Flavanones are responsible for the bitter taste of citrus peels and fruits, and they exhibit antioxidant, anti-inflammatory, blood lipid-lowering, and cholesterol-lowering properties (Panche et al., 2016).

Abscisic acid (compound 9) is an isoprenoid phytohormone that is responsible for several regulatory plant functions such as growth. It can be found in numerous plants such as the leaves of Cinnamomum subavenium Miq. Kuo et al. (2008) and the epiphyte, Cuscuta pentagona Engelm (Kimura et al., 1982). Moreover, this compound can be a promising treatment for diabetes as it regulates glucose uptake in vitro and can stimulate insulin production in beta pancreatic cells (Xu et al., 2018). In addition, it was found that abscisic acid intake reduces neuroinflammation (Jeon et al., 2020) and colon inflammation (Guri et al., 2011).

Pseudoanisatin (compound 10) is a lactone that was isolated from various Illicium species. It was isolated from the leaves of Illicium parviflorum Michaux ex Ventenat (Schmidt, 1999) and the pericarps of Illicium oligandrum Merr. and Chun (Zhu et al., 2009) and Illicium dunnianum Tutcher (Jianmei and Chunshu, 1996).

1,3,6-Tri-O-galloyl-beta-D-glucose (compound 11) is a glycoside which was isolated from the leaves of Terminalia bellirica (Gaertn.) Roxb. (Kim et al., 2018; Manome et al., 2022).

Similarly, 1,2,4,6-tetra-O-galloyl-beta-D-glucose (compound 12) is another glycoside that was isolated from the leaves of Phyllanthus emblica L. (Zhang et al., 2000), the fresh pericarps of Juglans sigillata Dode (Si et al., 2011) and the leaves of Melastoma malabathricum L. (Yoshida et al., 1982).

Pyrocatechuic acid (compound 13) is a phenolic acid which was isolated from the ethyl acetate extract of Mangifera casturi bark (Pardede and Koketsu, 2016). Pyrocatechuic acid was also isolated from the fruit extract of Flacourtia inermis Roxb (George et al., 2011).

Pyrogallol (compound 14) belongs to the class of phenols and has been previously isolated from the stem and bark of Barringtonia asiatica (L.) Kurz (Umaru, 2020). It was also isolated from the rhizomes of Bergenia ciliata (Haw.) Sternb (Zafar et al., 2019). In a previous study, the interaction of pyrogallol with AGLUC was evaluated through computational simulations and it was found that pyrogallol inhibits AGLUC with an IC₅₀ value of 0.72 ± 0.051 mM (Zheng et al., 2018). Pyrogallol was also found to have an IC₅₀ value of 8.7 mM against LOX (Yasumoto, Yamamoto, and Mitsuda, 1970).

There have been no previous studies conducted that quantified antidiabetic properties using the AGLUC inhibition assay, and anti-inflammatory activity using the LOX inhibition assay for compounds 3, 5, 7, and 10–13.