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. 2026 Sep 24. Online ahead of print. doi: 10.1039/d6ra05046h

Discovery of bioactive compounds from Thai medicinal plants as potential SARS-CoV-2 main protease inhibitors

Kiattawee Choowongkomon a,b, Thi-Minh Dinh Tran c, Thuc-Huy Duong d, Thi-Hoai-Thu Nguyen e, Thi-Kim-Dung Le f,g, Boi-Phong Vuong d, Xuan-Dai Nguyen d, Jirapast Sichaem h, Ngoc-Hong Nguyen i,✉, Huy Truong Nguyen g,✉
PMCID: PMC13602124  PMID: 42787705

Abstract

Thai medicinal plants represent valuable sources of structurally diverse secondary metabolites with potential antiviral relevance. In this study, a phytochemical investigation of three Thai medicinal plants, Phyllanthus emblica, Averrhoa bilimbi, and Helicteres isora, was conducted to identify natural products with inhibitory activity against SARS-CoV-2 main protease (Mpro). The crude MeOH extracts of the three plants showed moderate Mpro inhibition at 100 µg mL−1, with inhibition values of 61.7 ± 1.5%, 56.9 ± 3.8%, and 53.3 ± 1.7% for P. emblica, A. bilimbi, and H. isora, respectively. Bioactivity-guided fractionation and repeated chromatographic separation led to the isolation of 24 compounds, including nine constituents from P. emblica (P1–P9), six compounds from A. bilimbi (A1–A6), and nine compounds from H. isora (H1–H9). These metabolites comprised phytosterols, lupane-type triterpenoids and triterpenoid esters, lignans, phenolic esters, flavonoids, and related oxygenated derivatives. Among them, A1 was identified as a new phenolic glycoside and named demethoxybilimoside A, while stigmastane-3β,6α-diol 3-O-tetradecanoate (P1) was isolated and characterized as a pure compound. All isolated compounds were evaluated for SARS-CoV-2 Mpro inhibition. High apparent inhibition was observed for (stigmastane-3β,6α-diol 3-O-tetradecanoate) P1, lupeol (P4), kaempferol (A5), and quercetin (A6), with inhibition values of 99.3 ± 2.0%, 88.5 ± 0.9%, 95.8 ± 1.0%, and 99.5 ± 1.1%, respectively. Compound P1 showed an apparent IC50 value of 86.5 ± 3.8 µM and was subsequently selected for molecular docking analysis. These findings expand the phytochemical knowledge of Thai P. emblica, A. bilimbi, and H. isora and identify several compounds for further biochemical evaluation.

Thai medicinal plants represent valuable sources of structurally diverse secondary metabolites with potential antiviral relevance.graphic file with name d6ra05046h-ga.webp

1. Introduction

Although the acute phase of the COVID-19 pandemic has declined, SARS-CoV-2 continues to circulate globally, and COVID-19 remains a relevant public health issue because of viral evolution, recurrent infections, and the continuing need for effective antiviral agents (World Health Organization, 2025). The SARS-CoV-2 main protease (Mpro), also known as 3C-like protease (3CLpro) or non-structural protein 5 (nsp5), plays an essential role in viral polyprotein processing and replication, making it one of the most validated molecular targets for anti-COVID-19 drug discovery.1,2 The clinical relevance of Mpro inhibition is supported by the development of small-molecule Mpro inhibitors, including ensitrelvir, an orally active antiviral agent approved for SARS-CoV-2 infection.3 However, the emergence of viral variants, potential resistance-associated mutations, and limitations of current antiviral regimens continue to justify the search for structurally diverse Mpro inhibitors with new chemical scaffolds.4,5 Natural products are attractive in this context because they provide chemically diverse and stereochemically complex structures that may serve as starting points for antiviral lead discovery. Recent structure-guided studies have yielded orally bioavailable Mpro inhibitors with strong binding affinity and reduced susceptibility to resistance-associated mutations.5 These advances reinforce the therapeutic relevance of Mpro inhibition while also highlighting the importance of identifying additional structurally diverse inhibitor scaffolds. Natural products are therefore particularly attractive as a reservoir of chemically diverse molecules for protease-targeting lead discovery and future anti-COVID-19 drug development.4,6

Averrhoa bilimbi, commonly known as bilimbi, is a perennial woody plant cultivated in many tropical countries. It has been used in traditional medicine for the management of diabetes, hypertension, infections, inflammation, and other disorders.7 Previous studies have reported that A. bilimbi contains diverse classes of secondary metabolites, including flavonoids, phenolics, triterpenoids, steroids, alkaloids, and other oxygenated compounds, which may contribute to its antioxidant, antimicrobial, anti-inflammatory, antihyperglycemic, hepatoprotective, and cytotoxic properties.7,8 Recently, a bioassay-guided investigation of Thai medicinal plants reported the isolation of five compounds from the branches of A. bilimbi collected in Uttaradit Province, Thailand, including the new compound bilimoside A, and demonstrated nitric oxide inhibitory activity of its extracts and isolated constituents.9

Although several investigations have focused on the fruits and leaves, the chemical constituents of the branches remain less explored. Phyllanthus emblica, commonly known as Indian gooseberry or amla, is another tropical medicinal and edible plant that has attracted considerable attention because of its rich phytochemical composition and broad pharmacological potential. The plant is traditionally used for the treatment of fever, cough, asthma, diarrhea, sore throat, inflammation, urinary disorders, constipation, cardiovascular disorders, and other ailments.10,11 Phytochemical studies have revealed numerous phenolic compounds, steroids, bisabolane sesquiterpenoid glycosides, norsesquiterpenoid glycosides, flavonoids, chalconoids, triterpenoids, and phytosterols from different parts of P. emblica, including the roots, stems, leaves, bark, fruits, and seeds.12–17 These metabolites have been associated with antioxidant, hepatoprotective, anti-inflammatory, antidiabetic, cytoprotective, and antiviral-related activities. The genus Helicteres is distributed mainly in tropical Asia, Australia, and the Americas, and several species have been recorded in Thailand.18H. isora is traditionally used for gastrointestinal disorders, diabetes, inflammation, infections, and liver-related conditions. Previous studies on H. isora and related Helicteres species have reported phenylethanoids, phenolics, flavonoids, steroids, triterpenoids, alkaloids, and other metabolites.19,20 Pharmacological investigations have also demonstrated antispasmodic, antioxidant, hypoglycemic, antimicrobial, cytotoxic, hepatoprotective, and nephroprotective activities.21,22

Recent studies continue to highlight Thailand as an important source of medicinal-plant diversity and traditional ethnopharmacological knowledge. Ethnobotanical investigations in northeastern Thailand have documented a high diversity of edible and medicinal plants used by local communities, emphasizing the value of Thai plant resources for food, health care, and traditional medicine.23 In parallel, Thai herbal medicine has attracted renewed attention in antiviral and COVID-19-related research. Selected Thai medicinal plants have been reported to contain extracts and phytoconstituents with potential anti-SARS-CoV-2 activity, supporting the relevance of Thai plant resources for COVID-19-oriented natural-product screening.24,25 More directly related to the present study, recent investigations have reported bioactive constituents from Thai-collected A. bilimbi and H. isora, including nitric oxide inhibitory compounds from A. bilimbi branches and α-glucosidase inhibitory constituents from H. isora fruits.9,26 The previous study focused on A. bilimbi branches and nitric oxide inhibition,9 whereas the present work investigates the leaves for SARS-CoV-2 Mpro inhibition. Both studies used plant material from the same authenticated collection; however, the branches and leaves were processed independently, and no isolation or biological data from the previous study were reused. Although P. emblica has been widely investigated for its phytochemistry and pharmacological properties, studies focusing specifically on Thai-collected specimens remain comparatively limited.27,28 Therefore, focusing on Thai-collected A. bilimbi, P. emblica, and H. isora is scientifically justified because Thailand provides both rich ethnomedicinal knowledge and distinct tropical growing conditions that may influence secondary-metabolite profiles. Based on this rationale, the present study was designed as a COVID-19-oriented phytochemical investigation of Thai-collected medicinal plants. The study aims to identify secondary metabolites from A. bilimbi, P. emblica, and H. isora and to evaluate their potential relevance to SARS-CoV-2 Mpro inhibitor discovery. By combining chromatographic isolation, spectroscopic structure elucidation, and COVID-19-targeted biological evaluation, this work seeks to expand the phytochemical knowledge of Thai medicinal plants and provide new chemical evidence supporting the future development of natural-product-based anti-COVID-19 lead compounds (Fig. 1).

Fig. 1. Chemical structures of compounds P1–P9, A1–A6, and H1–H9.

Fig. 1

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2. Results and discussion

2.1. Biological activity of extracts

The crude MeOH extracts of three medicinal plants, Phyllanthus emblica, Averrhoa bilimbi, and Helicteres isora, were first screened for their inhibitory activity against SARS-CoV-2 main protease (Mpro) at a concentration of 100 µg mL−1. Among them, the crude MeOH extract of P. emblica showed the strongest inhibition, with an inhibition rate of 61.7 ± 1.5%, followed by those of A. bilimbi (56.9 ± 3.8%) and H. isora (53.3 ± 1.7%) (Fig. 2). These results indicated that all three crude MeOH extracts exhibited moderate Mpro inhibitory activity and were therefore selected for further solvent partitioning. Each crude MeOH extract was subsequently fractionated into three solvent-partitioned extracts, including n-hexane, n-hexane : EtOAc, and EtOAc extracts. The inhibitory activity of these partitioned extracts was then compared with that of the corresponding crude MeOH extract within each plant species to identify the most active fraction for chemical investigation. For P. emblica, the crude MeOH extract remained the most active sample (61.7 ± 1.5%), followed by the n-hexane : EtOAc extract (51.5 ± 2.2%) and the n-hexane extract (40.9 ± 0.7%), whereas the EtOAc extract showed weaker activity (23.4 ± 3.7%). For A. bilimbi, solvent partitioning resulted in a clear enhancement of activity in the EtOAc extract, which showed the strongest Mpro inhibition (71.2 ± 4.4%) and was more active than the corresponding crude MeOH extract (56.9 ± 3.8%). In contrast, the n-hexane : EtOAc extract showed only weak activity (14.5 ± 1.8%), while the n-hexane extract was inactive. For H. isora, the crude MeOH extract showed the highest activity, with 53.3 ± 1.7% inhibition, while the EtOAc extract exhibited comparable but slightly lower activity (48.8 ± 2.8%). The n-hexane : EtOAc extract was weakly active (5.0 ± 2.0%), and the n-hexane extract was inactive. Accordingly, the n-hexane : EtOAc extract of P. emblica, the EtOAc extract of A. bilimbi, and the EtOAc extract of H. isora were selected as the most suitable fractions for subsequent chemical analysis.

Fig. 2. SARS-CoV-2 main protease (Mpro) inhibitory activity of extracts from Phyllanthus emblica, Averrhoa bilimbi, and Helicteres isora at a concentration of 100 µg mL−1. Ritonavir was used as the positive control. Data are presented as mean ± SD.

Fig. 2

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2.2. Phytochemical investigation and structural elucidation

Multiple chromatographic methods were applied to n-hexane : EtOAc and EtOAc extracts of Phyllanthus emblica, Averrhoa bilimbi, and Helicteres isora, resulting in the isolation of 24 compounds (P1–P9, A1–A6, and H1–H9). These metabolites represent diverse carbon skeletons, including phytosterols (P1–P9), lupane-type triterpenoids and triterpenoid esters (P4, P5, and H3–H5), a chromene-5,8-dione derivative (P6), a diterpenoid-derived phenolic compound (P7), phenolic glycosides (A1 and A2), flavonoids (A3–A6, H8, and H9), lignans (H1, and H2), and simple monocyclic phenolic esters (P8, P9, H6, and H7). The isolated compounds were identified as stigmastane-3β,6α-diol 3-O-tetradecanoate (P1), previously reported as a component of an inseparable steryl ester mixture and isolated here as a pure compound,29 β-sitosterol palmitate (P2),30 β-sitosterol (P3),16 lupeol (P4),31 lupenone (P5),32, (2R,4aR,8aR)-3,4,4a,8a-tetrahydro-4a-hydroxy-2,6,7,8a-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-chromene-5,8-dione (P6),33 phyllane C (P7),34 methyl 4-hydroxybenzoate (P8),17 and methyl vanillate (P9)35 from Phyllanthus emblica; demethoxybilimoside A (a new compound), bilimoside A (A2),9 ayanin (A3),36 3-O-methylkaempferol (A4),37 kaempferol (A5),38 and quercetin (A6)39 from Averrhoa bilimbi; and lignans, triterpenoid esters, gallic acid derivatives, and flavone derivatives, including pinoresinol (H1),40 syringaresinol (H2),41 3β-acetoxy-27-benzoyloxylup-20(29)-en-28-oic acid methyl ester (H3),42 3β-hydroxy-27-trans-caffeoyloxylup-20(29)-en-28-oic acid methyl ester (H4),43 3β-methoxy-27-trans-caffeoyloxylup-20(29)-en-28-oic acid methyl ester (H5),43 gallic acid (H6),39 methyl gallate (H7),44 genkwanin (H8),45 and 8-sulfonylgenkwanin (H9),46 from Helicteres isora. Although stigmastane-3β,6α-diol 3-O-tetradecanoate (P1) was previously described by Luo et al.29 (2006) as one component of an inseparable mixture of three homologous steryl esters, the present study reports its isolation and characterization as a pure compound.

Compound A1 was obtained as a colorless oil. The molecular formula of compound A1 was assigned as C22H26O11 by HRESIMS from the deprotonation peak at m/z 465.1415 [M–H]− (calcd for [C22H26O11-H]−, 465.1397). The 1H NMR spectrum in combination with HMBC correlations of compound A1 indicated the presence of a 1,3,5-trisubstituted benzene ring (so-called A-ring), characterized by three mutually meta-coupled aromatic protons at δH 6.39 (1H, t, 2.0, H-2′), 6.43 (1H, t, 2.0, H-4′), 6.46 (1H, t, 2.0, H-6′), and another 1,2,4-trisubstituted benzene ring (so-called B-ring), characterized by the aromatic proton signals coupled each other at δH 7.54 (1H, d, 2.0, H-2‴), 6.92 (1H, d, 8.5, H-5‴), 7.64 (1H, dd, 8.5, 2.0, H-6‴). In addition, the 1H NMR spectrum showed the presence of a methylene group at δH 2.62 (2H, m, H-1a/H-1b), an oxymethylene group at δH 3.70 (2H, m, H-2a/H-2b), and a methoxy group at δH 3.87 (3H, s). Furthermore, 1H NMR signals attributable to an anomeric proton at δH 4.98 (1H, d, 7.5, H-1″), four oxymethine groups at δH 3.54 (1H, m, H-2″), 3.48 (1H, m, H-3″), 3.63 (1H, m, H-4″), and 3.90 (1H, m, H-5″), and an oxymethylene group at δH 4.34 (1H, dd, 11.5, 6.5, H-6″a) and 4.71 (1H, dd, 11.5, 2.0, H-6″b) established the presence of a glucose moiety. The 13C NMR spectrum in combination with HMBC correlations of compound A1 showed the presence of an ester carbonyl signal at δC 165.7; six aromatic methine carbons at δC 108.1 (C-2′), 109.8 (C-4′), 101.5 (C-6′), 112.4 (C-2‴), 114.8 (C-5‴), and 124.0 (C-6‴), six quaternary aromatic carbons at δC 158.8 (C-1′), 141.8 (C-3′), 158.2 (C-5′), 121.6 (C-1‴), 147.1 (C-3‴), and 151.0 (C-4‴), one methylene carbon at δC 39.5 (C-1), one oxymethylene carbon at δC 62.8 (C-2), and one aromatic methoxy carbon at δC 55.5. In addition, the 13C NMR spectrum also displayed signals assignable to a glucose unit, including an anomeric carbon at δC 100.8 (C-1″), four oxymethine carbons at δC 74.2 (C-2″), 76.6 (C-3″), 70.7 (C-4″), and 73.8 (C-5″), as well as one oxymethylene carbon at δC 64.0 (C-6″). The HMBC spectrum of compound A1 showed correlations from the methylene protons H2-1 (δH 2.62) to the oxymethylene carbon C-2 (δC 62.8) and three aromatic carbons C-2′ (δC 109.8), C-3′ (δC 141.8), and C-4′ (δC 108.1) and from the oxymethylene protons H2-2 (δH 3.70) to C-1 (δC 39.5) and C-3′ (δC 141.8). These correlations established that the position of 2-hydroxyethan-1-yl group at C-3′ in the A-ring. The HMBC spectrum further showed a correlation from the characteristic anomeric proton H-1″ (δH 4.98) to C-1′ (δC 158.8) of ring A. This correlation established that the sugar moiety formed an ether linkage with the A-ring at C-1′. In addition, on A-ring, the position of the exchangeable hydroxy proton (δH 8.26) was located at C-5′ by the HMBC correlations of this proton to three aromatic carbons at C-4′ (δC 108.1), C-5′ (δC 158.2) and C-6′ (δC 101.5) were observed. In a B-ring, the HMBC correlations of the methoxygroup (δH 3.87) and C-3‴ (δC 147.1), of the hydroxy proton (δH 8.42) to three carbons C-3‴ (δC 147.1), C-4‴ (δC 151.0) and C-5‴ (δC 114.8), and of the two aromatic protons H-2‴ (δH 7.54) and H-6‴ (δH 7.64) with an ester carbonyl carbon 1‴-COO (δC 165.7) established the chemical structure of the B-ring. The HMBC spectrum of compound A1 also showed correlations from the two oxymethylene protons of the glucose unit, H-6″a [δH 4.34 (1H, dd, 11.5, 6.5)] and H-6″b [δH 4.71 (1H, dd, 12.0, 2.0)] to the ester carbonyl carbon 1‴-COO, establishing the linkage between the B-ring and the glucose moiety through an ester bond between 1‴-COO and C-6″. The 1H–1H COSY spectroscopic data enabled the assignment of the chemical shifts of the hydroxy groups on the β-hexopyranose moiety. Specifically, the correlation between the hydroxy proton signal at δH 4.53 (1H, d, 4.5) and H-2″ [δH 3.54 (1H, m)] the COSY cross-peak between the hydroxy proton signal at δH 4.62 (1H, d, 4.5) and H-3″ [δH 3.48 (1H, m)] as well as the correlation between the hydroxy proton signal at δH 4.44 (1H, d, 4.5) and H-4″ [δH 3.63 (1H, m)], clarified the positions of the three exchangeable proton signals on the sugar unit. The stereochemistry of A1 was determined by NOESY correlations and analysis of the J coupling constants. First, the coupling constant of H-1″ (J = 7.5 Hz) indicated that this proton was in an axial orientation. The NOESY correlations between H-2″ and H-4″, as well as between H-2″ and H-6″, showed that H-2″, H-4″, and H-6″ were located on the same face in space. Similarly, the NOESY correlations between H-1″ and H-5″, and between H-3″ and H-5″, indicated that these three protons were on the same face and all occupied axial positions. These spectroscopic data established that the sugar moiety was β-glucopyranose. Based on its close spectroscopic similarity to bilimoside A (A2)9 and biogenetic considerations supported by previously reported d-glucose-containing glycosides from A. bilimbi,9 the sugar unit of A1 was tentatively assigned as β-d-glucopyranose. The high similarity between A1 and bilimoside A (A2)9 was observed with the only difference being the disappearance of the methoxy group in the B-ring (Fig. 3). Accordingly, A1 was identified as a new compound and was named demethoxybilimoside A.

Fig. 3. Chemical structures of A1 and bilimoside A and key HMBC, NOESY and COSY correlations of compound A1.

Fig. 3

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2.3. Biological activity of isolated compounds

The isolated compounds from Phyllanthus emblica (P1–P9), Averrhoa bilimbi (A1–A6), and Helicteres isora (H1–H9) were evaluated for their inhibitory activity against SARS-CoV-2 Mpro using a FRET-based assay at 100 µM. The results revealed clear differences in activity depending on both plant origin and structural class. Among the P. emblica constituents, P1 (stigmastane-3β,6α-diol 3-O-tetradecanoate), P2 (β-sitosterol palmitate), and P4 (lupeol) exhibited strong inhibition, with inhibition values of 99.3 ± 2.0%, 76.1 ± 1.3%, and 88.5 ± 0.9%, respectively, whereas P3 (β-sitosterol) and P7 (phyllane C) showed weaker activity, with 41.6 ± 0.9% and 21.5 ± 0.6% inhibition, respectively. The remaining compounds, including P5 (lupenone), P6 [(2R,4aR,8aR)-chromene-5,8-dione derivative], P8 (methyl 4-hydroxybenzoate), and P9 (methyl vanillate), were inactive or nearly inactive under the tested conditions. Among the P. emblica constituents, P1 and P4 showed the highest apparent inhibition in the initial FRET screening. P1 and P2 produced marked inhibition in the initial FRET screening. Since long-chain sterol derivatives have rarely been investigated against SARS-CoV-2 Mpro, and P1 was obtained here as a pure compound of particular phytochemical interest, we selected P1 for further IC50 evaluation. At the same time, the high lipophilicity of these compounds raises the possibility of nonspecific effects. Because detergent-based controls and independent aggregation assays were not included, aggregation-related interference cannot be ruled out, and the observed activities should therefore be regarded as preliminary. Among the lupane-type triterpenoids, P4 showed higher apparent inhibition than P5 under the present screening conditions; however, additional analogues and concentration-response data would be required to establish a structure–activity relationship (Fig. 4).

Fig. 4. SARS-CoV-2 main protease (Mpro) inhibitory activity of isolated compounds from Phyllanthus emblica (P1–P9), Averrhoa bilimbi (A1–A6), and Helicteres isora (H1–H9) at 100 µM. Ritonavir was used as the positive control. Data are presented as mean ± SD.

Fig. 4

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For the compounds isolated from Averrhoa bilimbi, flavonoids represented the dominant active class. A5 (kaempferol) and A6 (quercetin) showed the highest Mpro inhibitory activity in the FRET-based screening assay, with inhibition values of 95.8 ± 1.0% and 99.5 ± 1.1%, respectively. Under the present single-concentration screening conditions, A5 and A6 showed higher apparent inhibition than other phenolic compounds A1–A4. Molecular docking further indicated plausible interactions of both compounds within the Mpro active site, with docking scores of −7.4 and −7.1 kcal mol−1 and Prime MM-GBSA values of −43.8 and −45.1 kcal mol−1 for A5 and A6, respectively (Fig. 5). Both compounds formed predicted hydrogen-bond interactions involving Glu166, Asn142, and Thr26. These findings are generally consistent with previous computational and experimental studies of kaempferol and quercetin against SARS-CoV-2 Mpro,50–52 in which interactions with residues within the substrate-binding pocket, including Asn142 and Glu166, have also been reported. Nevertheless, the present docking results should be considered preliminary structural support rather than evidence of a specific binding mechanism or a quantitative explanation of the apparent FRET-based inhibitory activity, particularly given the potential interference of flavonoids in fluorescence-based assays. For the H. isora constituents, the isolated compounds showed variable apparent inhibition in the single-concentration FRET screening. H4 showed the highest apparent inhibition within this series (67.0 ± 0.4%), followed by H1 and H8, whereas H3 and H6 showed intermediate responses and H2, H5, H7, and H9 showed lower apparent inhibition. These results suggest that the triterpenoid ester framework contributes to moderate Mpro inhibition, but the activity is highly dependent on the ester substituent and oxygenation pattern.

Fig. 5. Two-dimensional binding interactions of the native ligand and compounds P1, P4, A5, and A6 with SARS-CoV-2 Mpro (PDB ID: 9HAK), showing docking scores, MM-GBSA binding free energies, and key protein–ligand interactions.

Fig. 5

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Several of the identified constituents have previously been investigated in the context of SARS-CoV-2 Mpro. Lupeol (P4) has been evaluated mainly by molecular docking against SARS-CoV-2 Mpro, with a reported docking score of −7.17 kcal mol−1 (ref. 47, 48) and experimental cell-based antiviral evaluation.49 Quercetin (A6) has likewise been reported as an Mpro-interacting metabolite in previous in silico studies, with a docking score of −7.5 kcal mol−1.50 In addition, kaempferol (A5) and quercetin (A6) have been evaluated experimentally against SARS-CoV-2 Mpro, with reported IC50 values of 34.46 µM (ref. 51) and 9.44 µM,52 respectively. Because P4, A5, and A6 have already been investigated in previous SARS-CoV-2 studies, additional IC50 determination was not pursued for these known compounds in the present study. Instead, P1 was prioritized for further characterization because its long-chain steryl-ester scaffold has been much less explored in relation to Mpro inhibition and because P1 was isolated here as a pure compound of particular phytochemical interest. Nevertheless, the literature IC50 values cited above were obtained under different experimental conditions and are therefore not directly compared quantitatively with the present single-concentration FRET screening data. The high apparent inhibition observed for kaempferol (A5) and quercetin (A6) should also be interpreted cautiously. Flavonoids may interfere with FRET-based assays through fluorescence quenching and/or nonspecific aggregation. Because dedicated fluorescence-interference controls and orthogonal cleavage assays were not performed in the present study, the apparent inhibition of A5 and A6 cannot be conclusively attributed to specific Mpro active-site inhibition. Accordingly, A5 and A6 are regarded as preliminary screening hits rather than confirmed Mpro inhibitors, and further orthogonal validation is required to establish their specific inhibitory activity.

Compound P1 was further evaluated by concentration-response analysis and showed an apparent IC50 value of 86.5 ± 3.8 µM. This compound was further investigated by molecular docking analysis. Ritonavir, used as a reference compound, exhibited an IC50 value of 33.2 ± 3.5 µM under the present assay conditions, providing a benchmark for comparison with P1.

The docking protocol was validated using the SARS-CoV-2 Mpro structure (PDB ID: 9HAK). Redocking of the native ligand gave an RMSD value of 0.8 Å, which is below the generally accepted threshold of 2.0 Å, confirming the reliability of the docking procedure. The native ligand showed a docking score of −9.3 kcal mol−1 and an MM-GBSA binding free energy of −108.9 kcal mol−1. The binding mode of the native ligand was stabilized by hydrogen-bond interactions between the amide carbonyl oxygen and Gly143, the carbonyl oxygen and Cys145, and the secondary amine group and His41. In addition, the bromine substituent on the aromatic ring formed a halogen-bond interaction with the side-chain amide of Gln192, further contributing to the stabilization of the ligand within the Mpro binding pocket. Compound P1 was then docked into the same Mpro binding pocket. P1 showed a docking score of −5.0 kcal mol−1 and an MM-GBSA binding free energy of −51.8 kcal mol−1. Although these values were weaker than those of the native ligand, P1 retained a hydrogen-bond interaction with Glu166. The lower docking score of P1 may be associated with its bulky hydrophobic steryl ester framework, which limits optimal fitting within the catalytic pocket despite contributing favorable hydrophobic contacts. Therefore, the docking result suggests that P1 can interact with SARS-CoV-2 Mpro, but its binding affinity is weaker than that of the native co-crystallized ligand (Fig. 5). Yet, prime MM-GBSA calculations were performed using a single energy-minimized docked pose rather than an ensemble of conformations sampled from an equilibrated molecular dynamics trajectory. Consequently, protein and ligand flexibility, solvent reorganization, and the time-dependent stability of the predicted interactions were not fully represented. This limitation is particularly relevant to P1 because of its bulky, highly hydrophobic, and conformationally flexible steryl ester structure, for which a single-structure calculation may overestimate the magnitude of the predicted binding energy. Accordingly, the reported MM-GBSA values should be regarded only as preliminary post-docking rescoring estimates for qualitative comparison and not as absolute binding free energies or quantitative predictors of Mpro inhibitory activity. Future studies incorporating molecular dynamics simulations followed by ensemble-based MM-GBSA calculations will be required to evaluate the stability of the predicted complexes and provide a more rigorous assessment of their relative binding tendencies.

3. Experimental

3.1. General experimental procedures

NMR spectra were recorded on a Bruker Avance III spectrometer at frequencies of 500 MHz for 1H NMR and 125 MHz for 13C NMR, using CDCl3 (δH 7.26, δC 77.16 ppm) and acetone-d6 (δH 2.05, δC 206.26 & 29.84 ppm) as the internal standard. High-resolution electrospray ionization mass spectra (HRESIMS) were acquired on a MicrOTOF-Q mass spectrometer coupled to an Agilent 1100 LC-MSD Trap system. Thin-layer chromatography (TLC) analyses were conducted on precoated silica gel 60 F254 plates, with visualization achieved by spraying with 10% H2SO4 followed by heating. Column chromatography was performed on silica gel 60 (0.040–0.063 mm, Himedia) using gravity flow.

3.2. Plant material

The leaves of Averrhoa bilimbi L. were collected from Uttaradit Province, Thailand, in October 2023, while the stem bark of Phyllanthus emblica L. and the roots of Helicteres isora L. were collected from Lampang Province, Thailand, in January and August 2024, respectively. These species were authenticated by Asst. Prof. Dr Kanit Wangwasit, Department of Biology, Faculty of Science, Mahasarakham University, Thailand. Voucher specimens [K. Wangwasit 240807-1 (A. bilimbi), K. Wangwasit 260623-1 (P. emblica), and K. Wangwasit 240811-1 (H. isora) have been deposited at the herbarium of Mahasarakham University, for future reference.

3.3. Extraction and isolation

The stem bark of Phyllanthus emblica L. (1.5 kg) was macerated with MeOH (5 × 10 L) at room temperature to afford a crude MeOH extract (260.0 g) after solvent removal under reduced pressure. The crude extract was subjected to liquid–liquid partitioning successively with n-hexane, n-hexane–EtOAc (1 : 1, v/v), and EtOAc to yield three extracts, namely the n-hexane extract (H, 2.5 g), n-hexane–EtOAc extract (HEA, 30.1 g), and EtOAc extract (EA, 78.8 g). The HEA extract (30.1 g) was dissolved in MeOH (3 × 500 mL) to give a MeOH-soluble portion (27.5 g) and an insoluble solid. The MeOH-soluble portion was subjected to silica gel column chromatography (CC), eluted with n-hexane–EtOAc (10 : 1 to 1 : 1, v/v), to afford six fractions, A (8.2 g), B (1.1 g), C (2.7 g), D (10.6 g), E (3.2 g), and F (1.2 g). Fraction A (8.2 g) was further separated by silica gel CC using n-hexane–DCM (2 : 1, v/v) to yield subfractions A1–A10. Subfraction A8 (350 mg) was purified by CC using n-hexane–DCM (2 : 1, v/v) to afford compounds P2 (4.5 mg) and P3 (35.0 mg). Subfraction A10 (660 mg) was further separated by CC using n-hexane–DCM–EtOAc–acetone (300 : 1 : 1, v/v/v) to give four subfractions, A10.1 (80 mg), A10.2 (100 mg), A10.3 (120 mg), and A10.4 (255 mg). Further purification of A10.3 by reversed-phase CC using H2O–MeOH (1.5 : 1, v/v) yielded compounds P4 (22.0 mg) and P5 (6.0 mg). Fraction B (1.1 g) was subjected to silica gel CC using n-hexane–EtOAc (3 : 1, v/v) to give six subfractions, B1–B6. Subfraction B4 (130 mg) was further purified by CC using n-hexane–DCM (2 : 1, v/v) to afford compounds P1 (3.4 mg) and P8 (20.0 mg). Fraction C (2.7 g) was separated by silica gel CC using n-hexane–DCM (1 : 1, v/v) to yield six subfractions, C1–C6. Subfraction C3 (100 mg) was further purified by CC using n-hexane–DCM (1 : 2, v/v) to afford compounds P6 (2.2 mg), P7 (4.0 mg), and P9 (15.0 mg). The isolation procedure was summarized in Fig. 6.

Fig. 6. Isolation procedure of P1–P9.

Fig. 6

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The dried powdered leaves of Averrhoa bilimbi L. (2.2 kg) were macerated with MeOH (5 × 10 L) at room temperature. The combined extract was concentrated under reduced pressure to remove the solvent, yielding a crude MeOH extract (188.0 g). This extract was then subjected to liquid–liquid partitioning successively with n-hexane, n-hexane–EtOAc (1 : 1, v/v), and EtOAc to afford three organic fractions, namely the n-hexane extract (H, 3.0 g), n-hexane–EtOAc extract (HEA, 19.0 g), and EtOAc extract (EA, 34.0 g). The EA fraction (34.0 g) was chromatographed over Sephadex LH-20 using MeOH as the eluent to afford five subfractions, EA1 (3.0 g), EA2 (13.5 g), EA3 (2.6 g), EA4 (4.5 g), and EA5 (9.7 g). Fraction EA1 (3.0 g) was further subjected to Sephadex LH-20 column chromatography using MeOH as the eluent to yield three subfractions, EA1.1 (1.2 g), EA1.2 (0.6 g), and EA1.3 (0.95 g). Fraction EA1.3 was subjected to silica gel CC using a gradient system of n-hexane–EtOAc (5 : 1 to 0 : 1, v/v) to provide four fractions, T1–T4. Fraction T1 (18 mg) was purified by RP-C18 CC using MeOH–H2O (5 : 1, v/v) to obtain compounds A3 (2.0 mg) and A4 (2.5 mg). Fraction T4 (55 mg) was purified by RP-C18 CC using MeOH–H2O (2 : 1, v/v) to obtain compounds A5 (15.0 mg) and A6 (30.0 mg). Fraction EA3 (2.6 g) was further subjected to Sephadex LH-20 column chromatography using MeOH as the eluent to yield four subfractions, EA3.1 (0.22 g), EA3.2 (0.73 g), EA3.3 (0.45 g), and EA3.4 (1.31 g). Fraction EA3.3 (0.45 g) was then separated by normal-phase silica gel CC using a gradient system of n-hexane–EtOAc–MeOH–H2O (3 : 9 : 1 : 0.01 to 0 : 1 : 1 : 0.01, v/v/v/v) to give six fractions, S1–S6. Fraction S6 (25 mg) was further purified by normal-phase silica gel CC using EtOAc–MeOH (15 : 1 to 0 : 1, v/v) as the eluent to afford compounds A1 (1.5 mg) and A2 (2.1 mg). The isolation procedure was summarized in Fig. 7.

Fig. 7. Isolation procedure of A1–A6.

Fig. 7

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The pulverized roots of Helicteres isora L. (1.9 kg) were extracted by maceration with MeOH (5 × 10 L). After filtration, the combined extract was concentrated under reduced pressure using a rotary evaporator to yield a crude MeOH extract (74.0 g). This extract was subsequently subjected to liquid–liquid partitioning successively with n-hexane, n-hexane–EtOAc (1 : 1, v/v), and EtOAc to yield three extracts, namely the n-hexane extract (H, 5.2 g), n-hexane–EtOAc extract (HEA, 11.8 g), and EtOAc extract (EA, 30.1 g). The EA extract (30.1 g) was subjected to silica gel column chromatography (CC) using a step gradient of n-hexane–EtOAc (10 : 1 to 0 : 1, v/v) as the mobile phase to afford ten fractions, T1–T10 (1.5, 2.1, 0.9, 3.5, 1.5, 2.8, 0.4, 4.1, 4.9, and 6.4 g, respectively). Fraction T3 (0.9 g) was further separated by normal-phase silica gel CC using a multicomponent solvent system of n-hexane–CH2Cl2–EtOAc–acetone, with a gradient from 12 : 1 : 1 : 0.5 to 0 : 1 : 1 : 0.5 (v/v/v/v), to yield five subfractions, T3.1–T3.5. Subfraction T3.3 (31 mg) was purified by reversed-phase CC using MeOH–H2O (10 : 1, v/v) to afford compounds H3 (3.0 mg) and H7 (15.0 mg). Subfractions T3.4 (12 mg) and T3.5 (29 mg) were separately purified by normal-phase silica gel CC using CH2Cl2–EtOAc–acetone–H2O–MeOH (110 : 9 : 1 : 0.01 : 0.9, v/v/v/v/v) to afford compounds H4 (2.0 mg) and H5 (8.0 mg), respectively. Fraction T4 (3.5 g) was subjected to normal-phase silica gel CC using CH2Cl2–EtOAc–acetone–MeOH–H2O (120 : 110 : 1 : 1 : 0.2, v/v/v/v/v) as the eluent to afford five subfractions, T4.1–T4.5. Subfraction T4.2 (156 mg) was further separated by normal-phase silica gel CC using CH2Cl2–EtOAc–acetone–MeOH–H2O (120 : 110 : 100 : 10 : 0.2, v/v/v/v/v) to yield six subfractions, S1–S6. Further purification of subfraction S2 (21 mg) by normal-phase silica gel CC using CH2Cl2–EtOAc–acetone–MeOH–H2O (110 : 110 : 100 : 1 : 0.2, v/v/v/v/v) afforded compounds H1 (3.5 mg) and H2 (2.4 mg). Subfraction S3 (30 mg) was purified by reversed-phase silica gel CC using MeOH–H2O (1.5 : 1, v/v) to afford compounds H6 (9.0 mg), H8 (3.0 mg), and H9 (1.5 mg). The isolation procedure was summarized in Fig. 8.

Fig. 8. Isolation procedure of H1–H9.

Fig. 8

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Demethoxybilimoside A (A1). Colorless oil. [α]20D + 220 (c 0.1, MeOH); 1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz): See Table 1. HRESIMS m/z: [M–H]− 465.1415 (calcd for [C22H26O11-H]−, 465.1397, mass error +3.9 ppm). IR (neat) νmax 3644, 1705, 1520, 1368, 1228, 1043 cm−1.

Table 1. 1H- and 13C-NMR spectroscopic data of A1 in acetone-d6 (δ in ppm and J values in Hz)a.

No. δ H δ C No. δ H δ C
1 2.62 (2H, m) 39.5 1‴ 121.6
2 3.70 (2H, m) 62.8 2‴ 7.54 (1H, d, 2.0) 112.4
1′ 158.8 3‴ 147.1
2′ 6.39 (1H, t, 2.0) 109.8 4‴ 151.0
3′ 141.8 5‴ 6.92 (1H, d, 8.5) 114.8
4′ 6.43 (1H, t, 2.0) 108.1 6‴ 7.64 (1H, dd, 8.5, 2.0) 124.0
5′ 158.2 –COO− 165.7
6′ 6.46 (1H, t, 2.0) 101.5 –OCH3 3.87 (3H, s) 55.5
1″ 4.98 (1H, d, 7.5) 100.8 2″-OH 4.53 (1H, d, 4.5)
2″ 3.54 (1H, m) 74.2 3″-OH 4.62 (1H, d, 4.5)
3″ 3.48 (1H, m) 76.6 4″-OH 4.44 (1H, d, 4.5)
4″ 3.63 (1H, m) 70.7
5′′ 3.90 (1H, m) 73.8
6″ 4.71 (1H, dd, 11.5, 2.0) 64.0
4.34 (1H, dd, 11.5, 6.5)
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a

Measured at 500 MHz (1H NMR) and 125 MHz (13C NMR).

3.4. Bioactivity assay

The SARS-CoV-2 Mpro inhibitory activity of the crude extracts, fractions, and purified compounds was determined using a fluorescence resonance energy transfer (FRET)-based enzymatic assay. All samples were initially evaluated at final concentrations of 100 µg mL−1 for extracts and fractions and 100 µM for purified compounds. The enzyme and fluorogenic substrate, Dabcyl-KTSAVLQSGFRKM{Glu(Edans)}, were freshly prepared in the corresponding assay buffers prior to use. Buffer X contained 20 mM Tris–HCl, 100 mM NaCl, 2 mM DTT, and 0.4 mM EDTA at pH 7.5, whereas buffer Y had the same composition but without DTT and was used for sample preparation. The assay was performed in black 384-well plates with a final reaction volume of 25 µL per well. Each reaction contained 10 µL of buffer X, 5 µL of SARS-CoV-2 Mpro solution (10 µM), and 5 µL of the test sample. The mixture was pre-incubated at room temperature for 10 min, after which the reaction was initiated by adding 5 µL of the fluorogenic FRET substrate (200 µM). Under these conditions, the final concentrations of Mpro and substrate were 2 µM and 40 µM, respectively. Fluorescence signals were recorded every 60 s for 30 min using a Tecan Infinite 200 Pro multimode microplate reader at excitation and emission wavelengths of 340 and 430 nm, respectively. The inhibitory activity was calculated from the initial reaction slopes according to the following equation: Mpro inhibition (%) = [1 − (Ss − Sb)/(Sc − Sb)] × 100, where Ss is the slope obtained in the presence of the test sample, Sc is the slope of the control reaction, and Sb is the slope of the enzymatic blank. Each concentration was tested in three technical replicate wells on the same assay plate. For P1, the IC50 value was determined using a two-fold serial dilution over the concentration range of 17–2200 µM. Dose–response curves were generated using GraphPad Prism 8.0.1 and fitted by nonlinear regression with a variable-slope model. Ritonavir was used as a reference inhibitor.53,54 The substrate Km and assay Z′-factor were not experimentally determined in the present study, which represents a limitation of the assay protocol. Assay performance was monitored using ritonavir as the reference inhibitor under the same experimental conditions.

4. Conclusion

In conclusion, phytochemical investigation of Thai-collected Phyllanthus emblica, Averrhoa bilimbi, and Helicteres isora afforded structurally diverse natural products, several of which showed preliminary Mpro inhibition under the present FRET screening conditions. Chromatographic separation of the active extracts afforded 24 secondary metabolites, including phytosterols, lupane-type triterpenoids, triterpenoid esters, lignans, phenolic esters, flavonoids, and related oxygenated compounds. Among them, demethoxybilimoside A (A1) was identified as a new phenolic glycoside, while stigmastane-3β,6α-diol 3-O-tetradecanoate (P1) was isolated as a pure compound. Biological evaluation identified several compounds showing apparent Mpro inhibition. Among them, P1 was of particular interest because long-chain sterol derivatives have rarely been examined for this activity. It showed an apparent IC50 value of 86.5 ± 3.8 µM and was further examined by molecular docking. Further orthogonal biochemical validation is required to establish whether the observed apparent inhibition represents specific SARS-CoV-2 Mpro inhibition.

Author contributions

Conceptualization, K. C., H. T. N. and N. -H. N.; methodology, H. T. N., T. -M. D. T., T. -H. D. and J. S.; software, K. C., T. -K. -D. L. and N. -H. N.; validation, K. C., H. T. N., N. -H. N. and J. S.; formal analysis, H. T. N., T. -H. D., T. -M. D. T., B. -P. V. and X. -D. N.; investigation, T. -M. D. T., T. -H. D., T. -H. -T. N., T. -T.-T. N., B. -P. V. and X. -D. N.; resources, J. S., K. C., H. T. N. and N. -H. N.; data curation, H. T. N., T. -H. D., T. -M. D. T., B. -P. V., X. -D. N. and T. -K. -D. L.; writing – original draft preparation, H. T. N., T. -H. D. and T. -M. D. T.; writing – review and editing, K. C., N. -H. N., H. T. N., T. -H. D. and J. S.; visualization, H. T. N., T. -H. D., B. -P. V. and X. -D. N.; supervision, K. C., H. T. N., N. -H. N. and J. S.; project administration, K. C., H. T. N. and N. -H. N.; funding acquisition, K. C., H. T. N. and N. -H. N. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

The authors gratefully acknowledge the Faculty of Pharmacy, Ton Duc Thang University, and Thammasat University, for supporting laboratory facilities and research conditions. The authors also thank Asst. Prof. Dr Kanit Wangwasit, Department of Biology, Faculty of Science, Mahasarakham University, Thailand, for the authentication of the plant materials. This research was funded by the National Foundation for Science & Technology Development (NAFOSTED), Vietnam, grant number 108.05-2023.35. This work was also supported by Kasetsart University Research and Development Institute, KURDI (FF(KU)54.69).

Data availability

The data that support the findings of this study are available in the supplementary information (SI). Supplementary information: NMR spectra, IR and HRESIMS spectrum of the new compound A1, IC50 curve of compound P1 against SARS-CoV-2 Mpro, and NMR data of P1. See DOI: https://doi.org/10.1039/d6ra05046h.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available in the supplementary information (SI). Supplementary information: NMR spectra, IR and HRESIMS spectrum of the new compound A1, IC50 curve of compound P1 against SARS-CoV-2 Mpro, and NMR data of P1. See DOI: https://doi.org/10.1039/d6ra05046h.

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