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Abstract

Objective: Propolis of Tetragonula iridipennis, which is one of the most popular stingless bees found in Vietnam, has been used to promote health and prevent disease. This study aims to identify the chemical constituents of T iridipennis propolis and their cytotoxic activity. Methods: The propolis sample was extracted with ethanol. The crude extract was isolated by combined chromatographic methods. The structures of isolated compounds were determined by electrospray ionization mass spectrometry and nuclear magnetic resonance spectra. The cytotoxicity was evaluated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide method. The protein interactions of active compounds were explored by molecular docking study. Results: Sixteen known compounds, including 11 triterpenes, 3 xanthones, a sterol, and a flavonoid were isolated and identified. The propolis ethanol extract displayed good cytotoxicity against A-549 and KB cancer cell lines. 24S,25-Dihydroxytirucall-7-en-3-one was the most active compound with IC₅₀ values ranging from 18.75 ± 0.21 to 19.62 ± 0.36 µg/mL. Conclusions: Sixteen known constituents were identified from T. iridipennis propolis, of which 8 compounds were discovered for the first time. The propolis of T iridipennis has multiple sources of plant resin, among that Ailanthus triphysa (Simaroubaceae) was identified as a new plant source. A molecular docking study revealed that cytotoxic effects of 24S,25-dihydroxytirucall-7-en-3-one, α-mangostin, 6-methoxy–bispyrano xanthone, and 3-isomangostin related to the inhibition of all 3 targets epidermal growth factor receptor, human epidermal growth factor receptor 2, and mammalian target of rapamycin (mTOR), while other isolated triterpenes exert their cytotoxicity by the inhibition of mTOR protein activity.

Keywords

triterpene xanthone cytotoxicity molecular docking

Introduction

Stingless bees belong to the Apidae family, distributed mainly in tropical and subtropical regions with more than 600 different species. The size of stingless bees is smaller than that of honey bees, ranging from 2 to 5 mm and they do not have a functional sting.^1,2 Currently, stingless bee keeping (Menipoliculture) is practiced in many places worldwide to produce a considerable amount of honey and propolis. The honey and propolis of stingless bees possess medicinal properties because they collect nectar and pollen selected from medicinally valuable plants. Propolis, a special resin that stingless bees produce by mixing their secretions with resin collected from leaves, trees, and buds, has been used to protect their hives.³ Propolis possesses many biological activities such as antibacterial, antioxidant, antiviral, anti-inflammatory, and anticancer properties. Previous studies revealed that stingless bee propolis contained several compound classes such as triterpenes, coumarins, xanthones, flavonoids, and phenolic compounds.^4–13 The chemical composition and biological activity of propolis vary depending on its botanical sources, geographical location, and climate.⁴

Tetragonula iridipennis (Smith, 1854) is one of the most popular stingless bees found in India, Nepal, Indonesia and many provinces of Vietnam.^14–17 In Vietnam, T. iridipennis propolis is used to promote health and prevent disease. The ethanol (EtOH) extract of T. iridipennis propolis showed antioxidant, antimicrobial, and anticancer activities.^15–19 We have analyzed the chemical composition of several T. iridipennis propolis samples by gas chromatography/mass spectrometry to reveal the presence of sugars, sugar alcohols, fatty acids, aromatic acids, phenolic lipids, and triterpenes.¹⁹ Further chemical and biological studies are needed to explore the chemical constituents and plant sources for propolis and to identify bioactive compounds.

In our preliminary results, 4 xanthones including cochinchinone A, cratoxylumxanthone B, cochinchinone K, and γ-mangostin were isolated from the propolis of T. iridipennis collected in Van Canh district, Binh Dinh province.²⁰ Herein, we wish to describe the chemical constituents and their cytotoxicity from T. iridipennis propolis. Sixteen constituents were isolated and identified including 11 triterpenes malabaricol (1), (14S,17R,20S)-14,17-epoxy-24-malabaricen-3β,20-diol (2), ocotillone II (3), mangiferonic acid (4), 24S,25-dihydroxytirucall-7-en-3-one (5), mangiferolic acid (6), 2α,3α,24-trihydroxyurs-12-en-28-oic acid (7), asiatic acid (8), occotilol (9), jatrogossol A (10), and hispidol B (11), 3 xanthones α-mangostin (12), 6-methoxy–bispyrano xanthone (13), and 3-isomangostin (14), a sterol 3β,5α,6α-trihydroxyergosta-7,22-diene (15) and a flavonoid 7-O-methyl kaempferol (16). The cytotoxicity of the EtOH extract and several isolated compounds were tested. The binding modes of active compounds with different cancer targets such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and mammalian target of rapamycin (mTOR) proteins were performed by molecular docking studies.

Materials and Methods

General Experimental Procedures

Spectrometers Bruker AVANCE NEO 600 MHz and Bruker AVANCE III HD 500 MHz (Bruker, Billerica, MA, USA) were used to record nuclear magnetic resonance spectra. An Agilent 6530 Accurate-Mass Q-TOF liquid chromatography/mass spectrometry (LC/MS) system and Agilent 1260 series single quadrupole LC/MS system (Agilent, CA, USA) were used to obtain high-resolution mass spectrometry and electrospray ionisation mass spectrometry spectra. Silica gel 230-400 mesh (Merck), Sephadex LH-20 (Sigma), and reversed-phase silica gel RP-18 (YMC*GEL, ODS-A, 30-50 μm) were used for column chromatography (CC). Analytical thin-layer chromatography was carried out on aluminum plates of silica gel 60 F₂₅₄ (Merck).

Propolis Material

The propolis of Tetragonula iridipennis was collected from a stingless bee hive in Van Canh district, Binh Dinh province, Vietnam, in March 2022. The stingless bee species was confirmed as Tetragonula iridipennis (Smith, 1854) by Dr Nguyen Thi Phuong Lien, Institute of Ecology and Biological Resources, VAST.

Extraction and Isolation

The propolis of Tetragonula iridipennis (255 g) was macerated with EtOH (4 × 2.5 L for 24 h) at room temperature. The EtOH solvents were filtered and evaporated under a vacuum. The crude extract (152 g) was suspended in distilled water (1 L) and extracted with EtOAc to give EtOAc residue (128 g). The EtOAc residue was subjected to a silica gel CC eluting with a solvent gradient of hexane/EtOAc (100/1-0/100) to yield 15 fractions (F1-F15). Fraction F6 (3.96 g) was isolated by silica gel CC (hexane/EtOAc v/v, 19:1) to afford 7 fractions F3A-E3G. Fraction E3A (53.7 mg) was purified by silica gel CC (hexane/EtOAc v/v, 9:1) to give 1 (4.1 mg). Fraction F7 (3.0 g) was fractionated by silica gel CC (hexane/acetone v/v, 19:1) to yield 8 fractions F7A-F7H. Fraction F7C (90 mg) was separated by silica gel CC (hexane/EtOAc v/v, 9:1) to yield 2 (4.1 mg). Fraction F7H (621 mg) was purified by silica gel CC (hexane/EtOAc v/v, 9:1) to give 3 (26 mg). Fraction F9 (5.0 g) was applied on silica gel CC (hexane/EtOAc v/v, 19:1) to give 10 fractions F9A-F9 K. Compound 4 (6.1 mg) was obtained from fraction F9D. Fraction F9H (1.87 g) was purified by Sephadex® LH-20 CC, and eluted with MeOH to give 12 (6.7 mg). Fraction F10 (5.1 g) was fractionated by silica gel CC (hexane/EtOAc v/v, 19:1) to afford 12 fractions F10A-F10M. Fraction F10I (1.92 g) was purified by Sephadex® LH-20 CC, and eluted with MeOH to yield 3 fractions F10I1-F10I3. Fraction F10I1 was subjected to reversed-phase silica gel CC (MeOH/H₂O v/v, 3:1) to give 11 fractions F10I1A-F10I1K. Fraction F10I1E (160 mg) was separated by Sephadex® LH-20 CC, and eluted with MeOH to yield 5 (7.8 mg) and 6 (3 mg). Fraction F13 (2.45 g) was fractionated by silica gel CC (hexane/EtOAc v/v, 9:1) to yield 6 fractions F13A-F13F. Fraction F15C (200 mg) was separated by Sephadex® LH-20 CC, and eluted with MeOH to yield 16 (7.1 mg). Fraction F13D (850 mg) was purified by Sephadex® LH-20 CC, and eluted with MeOH to yield 3 fractions F13D1-F13D3. Fraction F13D2 (104 mg) was subjected to reversed-phase silica gel CC (acetone/H₂O v/v, 2:1) to yield 14. Fraction F13E (210 mg) was purified by silica gel CC (CH₂Cl₂/MeOH v/v, 15:1) to give 3 fractions F13E1-F13E3. Fraction F13E3 (24 mg) was subjected to reversed-phase silica gel CC (acetone/H₂O v/v, 2:1) to give 7 (3.5 mg). Fraction F14 (2.5 g) was applied to a silica gel CC (CH₂Cl₂/EtOAc v/v, 9:1) to afford 9 fractions F14A-F14I. Fraction F14H (36 mg) was purified by Sephadex® LH-20 CC, eluted with MeOH to yield 8 (6.6 mg). Fraction F14E (204 mg) was purified by reversed-phase silica gel CC (acetone/H₂O v/v, 2:1) to yield 4 fractions F14E1-F14E4. Fraction F14E2 (45 mg) was separated by silica gel CC (CH₂Cl₂/EtOAc v/v, 2:1) to give 9 (2.9 mg). Fraction F14E3 (35 mg) was purified by Sephadex® LH-20 CC, and eluted with MeOH to yield 10 (6.7 mg). Fraction F14E4 (56 mg) was purified by silica gel CC (CH₂Cl₂/MeOH v/v, 30:1) to give 15 (5.0 mg). Fraction F14G (800 mg) was purified by silica gel CC (hexane/EtOAc v/v, 4:6) to yield 5 fractions F14G1-F14G5. Fraction F14G2 (40 mg) was purified by Sephadex® LH-20 CC, eluted with MeOH give 13 (3.3 mg). Fraction F14G5 (67 mg) was purified by Sephadex® LH-20 CC, eluted with MeOH give 11 (3.3 mg).

Cytotoxicity Assay

The cytotoxic evaluations were performed in triplicate using the MTT method, which was documented in our previous study.¹⁰ Three cancer cells (KB (epidermal carcinoma), HepG-2 (human hepatoma), and A-549 (human lung adenocarcinoma) obtained from ATCC) were grown in Dulbeccos’ DMEM medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, at 37 °C in a humidified atmosphere (95% air and 5% CO₂). Human cancer cell lines (3 × 10⁵ cells/mL) were treated with solutions of isolated compounds in DMSO. After 48 h of incubation, 10 µL of MTT solution in sodium phosphate buffer (5 mg/mL) was then added and the cells were incubated at 37 °C for 4 h. Cell growth was estimated by colorimetric measurement of formazan at 570 nm using a microplate reader. The results were expressed as a percentage of growth inhibition and IC₅₀ was calculated for each compound using Rawdata software. Ellipticine was used as a positive control.

Molecular Docking

Protein structures (EGFR, HER2, and mTOR) were obtained from the RCSB Protein Data Bank with IDs 4HJO, 3PP0, and 4DRI, respectively. After removing co-ligands and water molecules, hydrogen atoms were added, followed by assigning partial charges using Autodock Tool 1.5.6. Then, the resulting structures were saved as pdbqt files. Molecular docking was performed using Autodock Vina,²¹ following our established protocol.²² For EGFR and HER2, a 20 × 20 × 20 Å³ grid box was used, while for mTOR, we utilized a 22.5 × 20 × 20 Å³ grid box. Grid box centers were defined as follows: EGFR (x = 24.135, y = 9.847, and z = 0.741), HER2 (x = 15.809, y = 16.895, and z = 27.075), and mTOR (x = 35.461, y = 48.585, and z = 35.327). Positive controls included erlotinib for EGFR, 03Q for HER2, and everolimus for mTOR. Co-ligand redocking was performed to validate the method, resulting in RMSD values of 1.39, 0.46, and 1.47 Å for the 3 co-ligands, demonstrating the method's accuracy and reliability.

Statistical Analysis

In the cytotoxic assays, the IC₅₀ is presented as the mean ± standard deviation (SD) by using Microsoft Excel (Microsoft Corporation, 2013). The statistical significance of all treatment effects was evaluated by Student's t-test with a probability limit for the significance of P < .05.

Results and Discussion

Compound Identification and Plant Sources

The phytochemical separation led to the isolation of 16 compounds (Figure 1) including 11 triterpenes: malabaricol (1),²³ (14S,17R,20S)-14,17-epoxy-24-malabaricen-3β,20-diol (2),²³ ocotillone II (3),⁵ mangiferonic acid (4),⁹ 24S,25-dihydroxytirucall-7-en-3-one (5),²⁴ mangiferolic acid (6),⁹ 2α,3α,24-trihydroxyurs-12-en-28-oic acid (7),²⁵ asiatic acid (8),²⁶ occotilol (9),²⁷ jatrogossol A (10),²⁸ and hispidol B (11),²⁹ 3 xanthones: α-mangostin (12), 6-methoxy–bispyrano xanthone (13),³⁰ and 3-isomangostin (14),¹⁰ a sterol 3β,5α,6α-trihydroxyergosta-7,22-diene (15),³¹ and a flavonoid 7-O-methyl kaempferol (16).³² The chemical structures of isolated constituents were identified by spectral data and comparison with the previously reported literature.

Figure 1.

Chemical structures of isolated compounds from Tetragonula iridipennis propolis.

This is the first investigation of isolating of individual constituents from T. iridipennis propolis. Triterpenes 3, 4, and 6, xanthones 12, 14, and flavonoids 16 were previously found in propolis samples of Vietnam and the Philippines.^6,7,9,10,32 Triterpenes 1, 2, 5, 7, 8, 10, and 11 and a sterol 15, were discovered from stingless bee propolis for the first time.

The chemical results indicate that the propolis of T. iridipennis has multiple sources of plant resin. The presence of prenyl/geranyl xanthones (compounds 12-14 and 4 previously reported ones) and cycloartane-type triterpenes (compounds 4 and 6) suggested Cratoxylum cochinchinense (Hypericaceae) and Mangifera indica (Anacardiaceae) were plant sources for propolis. These 2 plants were commonly found as resin sources for stingless bee propolis in Vietnam and in other countries.^{6,7,9,10,19,33}

The finding of jatrogossol A (10) indicates Jatropha sp. (Euphorbiaceae)²⁸ could be one of the plant sources. Independently with our study, Layek et al have reported Jatropha curcas and J. gossypiifolia, that widely distributed in Vietnam, were resin sources for T. iridipennis in West Bengal, India by the direct observation method.³⁴

The malabaricane-type triterpenes 1 and 2 were isolated from Ailanthus triphysa (synonym: Ailanthus malabarica) and A. excelsa.^28,35 Furthermore, triterpenes 3, 5, 9, and 11 were also found in Ailanthus exelsa and Ailanthus altissima plants.^35–37 In the book “An Illustrated Vietnamese Flora,” Ho reported 4 Ailanthus species in Vietnam, among which Ailanthus triphysa was distributed in Binh Dinh and neighboring provinces.³⁸ Therefore, our results suggested that A. triphysa (Simaroubaceae) might also be one of the plant sources. On the other hand, the sources for compounds 7-8 and 15-16 are still being determined since these compounds occurred in many plant extracts. In summary, the propolis of T. iridipennis collected in this study has at least 4 different plant resin sources, among that Ailanthus triphysa was identified as a new one.

Cytotoxicity

The EtOH extract and several isolated compounds were evaluated for their cytotoxic activity in HepG-2, KB, and A-549 cancer cell lines. The cytotoxicity of several known compounds, which were previously isolated from different propolis samples,^10,22 were also presented in Table 1. As a result, the EtOH extract showed cytotoxicity against A-549, KB, and HEPG-2 cancer cell lines with IC₅₀ values of 24.84 ± 0.95, 31.17 ± 1.14, and 60.44 ± 1.63 µg/mL, respectively. Among the tested compounds, triterpene 5 displayed good cytotoxicity against all 3 cancer cell lines with IC₅₀ values ranging from 18.75 ± 0.21 to 19.62 ± 0.36 µg/mL. Other triterpenes 1, 2, 7, 8, 10, and xanthone 13 exhibited moderate activity with IC₅₀ values ranging from 42.08 ± 2.70 to 79.00 ± 1.50 µg/mL. However, several xanthones like α-mangostin (12), 3-isomangostin (14), cochinchinone A (17), cratoxylumxanthone B (18), and γ-mangostin (20) were reported in our publications^10,22 to have good cytotoxicity, that make contribution to the activity of the propolis EtOH extract (Table 1).

Table 1.

Cytotoxicity of the Ethanol (EtOH) Extract and Isolated Compounds.

Compounds	IC₅₀ (µg/mL)
Compounds	KB	Hep-G2	A-549
1	81.11 ± 2.33	70.89 ± 1.72	75.29 ± 0.49
2	79.0 ± 1.5	77.95 ± 0.75	76.38 ± 0.78
3	> 128	> 128	> 128
4	> 128	> 128	> 128
5	18.75 ± 0.21	19.35 ± 0.19	19.62 ± 0.36
6	> 128	> 128	> 128
7	76.73 ± 1.54	77.41 ± 0.77	76.36 ± 1.57
8	68.83 ± 0.52	54.47 ± 1.61	76.93 ± 0.77
9	> 128	> 128	> 128
10	77.18 ± 0.34	64.0 ± 1.20	73.74 ± 1.3
11	> 128	> 128	> 128
12	9.28 ± 0.58^a	12.90 ± 0.68^a	7.55 ± 0.25^a
13	52.15 ± 0.78	42.08 ± 2.70	71.61 ± 1.34
14	10.45 ± 0.74^b	4.23 ± 0.21^b	–
15	> 64	> 64	> 64
16	104.0 ± 2.61	86.35 ± 2.39	99.28 ± 1.82
Cochinchinone A (17)	29.27 ± 2.07^a	17.36 ± 1.03^a	20.33 ± 1.34^a
Cratoxylumxanthone B (18)	7.61 ± 0.43^b	2.1 ± 0.15^b	–
Cochinchinone K (19)	>64^b	>64^b	–
γ-Mangostin (20)	10.62 ± 0.65^b	7.19 ± 0.63^b	–
EtOH extract	31.17 ± 1.14	60.44 ± 1.63	24.84 ± 0.95
Ellipticine	0.41 ± 0.02	0.42 ± 0.02	0.42 ± 0.03

Values are adopted from Phuong et al.²²

Values are adopted from Oanh et al.¹⁰

– : not tested.

Molecular Docking Studies

In our previous study of Homotrigona apicalis propolis, 2 xanthones, namely α-mangostin (12) and cochinchinone A (17), exhibited promising potential in the inhibition of the growth of cancer cell lines such as KB, A-549, and HepG2. This observed effect might be linked to the capability to inhibit EGFR and HER2 of these xanthones.²² Furthermore, a study conducted by Zulkipli et al³⁹ reported that asiatic acid (8) could possess promising cytotoxic properties by inhibiting mTOR. Therefore, in this study, molecular docking between active compounds against EGFR, HER2, and mTOR was performed to explore the cytotoxic mechanism of these substances.⁴⁰

The results of the docking analysis revealed that all xanthone compounds showed strong interactions with both EGFR and HER2 proteins with binding energy values ranging between −9.3 and −10.7 kcal/mol. Among the potentially active compounds, xanthones 13 and 14 exhibited the highest binding affinities to both EGFR and HER2 proteins, with binding energy values of −10.7 and −10.1 kcal/mol for 13 and −10.5 and −10.6 kcal/mol for 14, respectively (Table 2). Among the triterpenes, compound 5 displayed the lowest binding energies, with values of −8.7 kcal/mol for EGFR, and −8.6 kcal/mol for HER2. The binding modes between xanthone 13 and EGFR/HER2, as well as triterpenes 5 and EGFR/HER2, are illustrated in Figure 2.

Figure 2.

Binding mode of (a) xanthone 13 and (b) triterpene 5 with (1) EGFR and (2) HER2.

Table 2.

Binding Energy of Isolated Compounds and 3 Protein Targets.

No.	Compounds	Binding energy (kcal/mol)
No.	Compounds	EGFR	HER2	mTOR
1	1	−8.6	−5.9	−11.5
2	2	−8.5	−6.8	−10.6
3	5	−8.7	−8.6	−10.6
4	7	−5.8	−2.5	−11.2
5	8	−6.3	2.6	−10.6
6	10	−8.7	−7.1	−10.6
7	12	−9.3^a	−9.7^a	−9.7
8	13	−10.7	−10.1	−11.8
9	14	−10.5	−10.6	−10.2
10	17	−9.7^a	−9.9^a	−9.9
11	18	−9.3	−9.3	−9.7
12	20	−9.5	−10.4	−9.7
13	Control	−7.2	−11.3	−10.3

Abbreviations: EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; mTOR, mammalian target of rapamycin.

Values are adopted from Phuong et al.²²

As could be seen from Figure 2, the interactions between xanthone 13 and triterpene 5 with EGFR prominently involved key residues, including Leu694, Val702, Cys773, Met769, Thr766, and Leu820. On the other hand, regarding HER2, the interactions occurred with residues such as Leu726, Leu852, Cys805, Leu807, Arg849, Thr862, and Val734. Among these residues, Met769 and Thr766 were notably involved in establishing hydrogen bonds between compounds 5, 13 and EGFR. In the case of HER2, Thr862 played a significant role in these interactions. Other residues contributed to the formation of hydrophobic interactions, including Van der Waals forces, π-π, and π-alkyl interactions between these 2 targets and isolated substances. These findings also aligned with our previous investigation²² into the binding modes of xanthones, cochinchinone A, and α-mangostin, with EGFR, as well as with HER2. In other words, this alignment suggested that the cytotoxic mechanism attributed to xanthone 13 and triterpene 5, could potentially be linked to their ability to inhibit both EGFR and HER2.

Regarding the affinity of the isolated triterpenes toward EGFR and HER2, the docking results (Table 2) suggested that, except for compound 5, EGFR and HER2 may not be the primary targets corresponding to their cytotoxic activities with notably low binding energies observed between these compounds and 2 proteins. On the other hand, our findings indicated that all isolated triterpenes exhibited strong binding with mTOR, with binding energy values ranging from −10.6 to −11.5 kcal/mol, compared to everolimus at −10.3 kcal/mol. Among the isolated xanthones, compound 13 displayed the highest activity with an affinity of −11.8 kcal/mol. For the remaining xanthones, while their binding energy values for mTOR were not lower than that of the positive control (everolimus), the low binding energy values ranging between −9.9 and −10.2 kcal/mol also suggested a strong interaction with mTOR which aligned with previous studies on the mTOR inhibitory activity of xanthones, including research by Liu et al,⁴¹ Nguyen et al,⁴² and Tang et al.⁴³

Further analysis of the interactions between malabaricol (1), 24S,25-dihydroxytirucall-7-en-3-one (5), xanthone 13, and mTOR (Figure 3) indicated that these compounds could potentially inhibit mTOR activity through a series of interactions involving key residues such as Gln85, Trp90, Tyr57, Phe130, Phe77, Phe2039, Trp2101, Tyr2105, and Phe2108. The molecular docking results also indicated that the primary interactions between the isolated compounds and mTOR included Van der Waals forces, hydrogen bonding, π-σ, π-π, and π-alkyl interactions (Figure 3). Specifically, Gln85 played a crucial role in establishing hydrogen bonds between mTOR and triterpenoids 1 and 5. In contrast, mTOR forms hydrogen bonds with xanthone 13 through residues Ser2035 and Tyr113. The other residues, including Phe77, Phe130, Tyr57, Trp90, Phe2039, Tyr2105, Phe2108, and Trp2101, are primarily involved in generating hydrophobic interactions with the isolated compounds. Remarkably, these results were consistent with the study conducted by Zulkipli et al,³⁹ which revealed the importance of residues such as Asp68, Phe77, Trp2101, Tyr2105, Phe2039, and Phe2108 in the context of mTOR inhibitory action. These findings suggested that mTOR might be a pivotal target contributing to the cytotoxic effects of isolated triterpenes, while the potential cytotoxic effects of isolated xanthones might come from the simultaneously inhibitory activities of these compounds on all 3 targets: EGFR, HER2, and mTOR.

Figure 3.

Binding mode of mammalian target of rapamycin (mTOR) with (a) triterpene 1, (b) 5, and (c) xanthone 12.

Regarding the cytotoxic effects of the isolated compounds, an interesting result concerning 2 triterpenoid compounds, 9 and 10 was observed. Although these 2 substances share a dammarane-type structure, their biological activities diverged significantly. Specifically, compound 10 exhibited notable inhibitory effects on 3 different cell lines, KB, Hep-G2, and A-549, with IC₅₀ values of 77.18 ± 0.34, 64.0 ± 1.20, and 73.74 ± 1.3 μg/mL, respectively. On the contrary, triterpenoid 9 displayed no activity on these same cell lines. The structural differences between these 2 compounds lay in the addition of a 2-OH group (in 10) as well as the replacement of an ether group (in 9) with a 24-OH group (in 10). To explain this result, molecular docking between these 2 substances and mTOR was performed (Figure 4). The findings indicated that while both compounds could effectively bind to the target, the interaction between mTOR and triterpenoid 9 primarily involved π-alkyl interactions, whereas compound 10 demonstrated the ability to form a hydrogen bond between Asp2102 and Arg73 residues and the OH group at C-25, as well as a hydrogen bond between Trp2101 residue and the OH group at C-24. These results implied that hydrogen bonding between the isolated compounds and mTOR may play a pivotal role in their mTOR inhibitory effects. Furthermore, the docking results also suggested that the OH group at C-24 might be particularly influential in the mTOR inhibitory effect, while the OH group at C-2 appeared to have a less pronounced role in this regard.

Figure 4.

Binding mode of mammalian target of rapamycin (mTOR) with (a) triterpene 9 and (b) 10.

In this study, we isolated 16 compounds and assessed their cytotoxic effects on 3 cell lines: KB, A-549, and HEP-G2. Molecular docking was also performed to gain preliminary insights into how these isolated compounds might interact with the EGFR, HER2, and mTOR targets. While our findings obtained certain valuable results, this research also got some limitations. One such shortcoming concerned the choice of protein targets. In fact, cancer pathogenesis is a highly complex process with numerous potential targets. Therefore, relying solely on previous studies on the cytotoxic effects of similar substances to select targets may inadvertently overlook other important connections between the isolated compounds and these diseases. On the other hand, it is important to note that molecular docking only provides a theoretical estimation of the binding affinity. Therefore, in some instances, the docking results may not accurately reflect how these compounds behave in a biological system. To ensure the reliability of our molecular docking results, it is essential to complement them with other computational methods, such as molecular dynamics simulations, and experimental validation.

Conclusion

Phytochemical study of Tetragonula iridipennis propolis leads to the isolation and identification of 16 known compounds including 11 triterpenes (1-11), 3 xanthones (12-14), a sterol (15), and a flavonoid (16). Seven triterpenes and a sterol compound 15 were isolated for the first time from stingless bee propolis. The propolis of T. iridipennis collected in this study has at least 4 different plant resin sources, including Cratoxylum cochinchinenese, Mangifera indica, Jatropha sp., and Ailanthus triphysa. A. triphysa (Simaroubaceae) was identified as a new plant source for stingless bee propolis. The EtOH extract of T. iridipennis showed good cytotoxicity against A-549 and KB cancer cell lines with IC₅₀ values of 24.84 ± 0.95, and 31.17 ± 1.14, respectively. Triterpene 5 was the most active compound with IC₅₀ values ranging from 18.75 ± 0.21 to 19.62 ± 0.36 µg/mL. Docking experiments showed that xanthone derivatives and triterpene 5 may exert their activity by simultaneously inhibition of all 3 targets EGFR, HER2, and mTOR while other triterpenes exert their activity by the inhibition of mTOR protein.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X231219088 - Supplemental material for Chemical Constituents, Cytotoxicity, and Molecular Docking Studies of Tetragonula iridipennis Propolis

Supplemental material, sj-doc-1-npx-10.1177_1934578X231219088 for Chemical Constituents, Cytotoxicity, and Molecular Docking Studies of Tetragonula iridipennis Propolis by Diep Thi Lan Phuong, Nguyen Thanh Cong, Nguyen Van Phuong, Nguyen Thi Hue, Nguyen Quoc Vuong, Nguyen Thi Phuong Lien, Duc Viet Ho, Nguyen Le Tuan, Nguyen Thị Nghia, Vo Thi Thanh Tuyen, Nguyen Thu Hang and Le Thanh in Natural Product Communications

Footnotes

Acknowledgments

The authors thank Mr Dang Vu Luong for performing NMR experiments and Dr Nguyen Thi Thu Ha, Institute of Chemistry, VAST for the cytotoxic evaluation.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Vietnamese Ministry of Education and Training (grant number B2022-DQN-08.

Ethical Approval

Ethical approval is not applicable to this article.

ORCID iDs

Nguyen Thi Hue

Duc Viet Ho

Le Thanh

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Chemical Constituents,Cytotoxicity,and Molecular Docking Studies of Tetragonula iridipennis Propolis

Abstract

Keywords

Introduction

Materials and Methods

General Experimental Procedures

Propolis Material

Extraction and Isolation

Cytotoxicity Assay

Molecular Docking

Statistical Analysis

Results and Discussion

Compound Identification and Plant Sources

Cytotoxicity

Molecular Docking Studies

Conclusion

Supplemental Material

sj-doc-1-npx-10.1177_1934578X231219088 - Supplemental material for Chemical Constituents, Cytotoxicity, and Molecular Docking Studies of Tetragonula iridipennis Propolis

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

Ethical Approval

ORCID iDs

Statement of Human and Animal Rights

Statement of Informed Consent

Supplemental Material

References

Supplementary Material