Antimicrobial, Antioxidant, and α-Glucosidase-Inhibitory Activities of Prenylated p-Hydroxybenzoic Acid Derivatives from Oberonia ensiformis

Abstract

Seven previously undescribed prenylated p-hydroxybenzoic acid derivatives, oberoniaensiformisins A–G, were isolated from an EtOH extract of the whole plant Oberonia ensiformis. Their structures were determined through spectroscopic analyses (IR, NMR) and HRESIMS analysis. The isolated compounds were tested for their antimicrobial, antioxidant, and α-glucosidase-inhibitory activity. Among them, compounds 6 and 12 exhibited potential antioxidant activity, while compounds 5, 6, 12, 13, and 15 showed varying degrees of α-glucosidase-inhibitory activity, with IC₅₀ values ranging from 34.03 to 106.10 μg/mL.

1. Introduction

The Orchidaceae family represents the second-largest angiosperm family following Asteraceae, comprising approximately 28,000 species classified into 736 genera [1]. As a tribute to the 50th anniversary of the founding of New China in the 20th century, the monumental compendium of Chinese materia medica “Chinese Materia Medica “ documented as many as 155 medicinal orchid species belonging to 56 genera [2]. Beyond the orchid-based medicinal materials recorded in the Pharmacopoeia of the People’s Republic of China, an additional 47 species from 18 genera of Orchidaceae have been officially documented as source plants for traditional Chinese medicines and ethnic medicines in various regional medicinal material quality standards. Notably, many of these species are commonly used in local traditional and ethnic medicine practices, with Oberonia ensiformis being an example [3]. The whole herb of O. myosurus Lindl. has the effect of clearing heat and removing toxins, dispelling wind, and activating blood circulation. It is used by local ethnic minorities as a folk medicine mainly for the treatment of pneumonia, bronchitis, hepatitis, cystitis, and urinary tract infections and externally for the treatment of bruises, swelling and pain, and rheumatic bone pain [4]. In our previous study, we conducted a systematic investigation into the chemical constituents of O. myosurus Lindl [4]. To further explore and utilize the resources of plants within this genus, we carried out a detailed chemical study on the congeneric plant, O. ensiformis. As a result, 18 prenylated p-hydroxybenzoic acid derivatives, including 7 new compounds (1–7), were isolated and identified (Figure 1). All isolated compounds were evaluated for potential antimicrobial activity, antioxidant activity, and α-glucosidase-inhibitory activity, and the detailed results are presented in this paper.

2. Results

2.1. Structural Elucidation of the Isolated Compound

Compound 1 was isolated as a light-yellow oil. Its molecular formula, C₁₃H₁₆O₅, was determined using positive-ion HRESI-MS, with an [M + Na]⁺ peak at m/z 275.0901 (calcd. for C₁₃H₁₆O₅Na; 275.0895). The ¹H NMR spectrum (

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR data for compounds 1 and 2.

Position	1		2
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	126.7		119.1
2	115.2	7.49, d (2.1)	150.0
3	148.9	7.43, d (2.1)	142.8
4	149.2		150.0
5	134.1		123.7
6	122.9		121.5	7.69, s
7	166.6		165.3
8
9
1′	23.7	2.93, t (7.8)	137.1	7.73, d (16.4)
2′	47.6	2.79, t (7.8)	128.5	6.77, d (16.4)
3′	207.5		198.6
4′	30.0	2.17, s	27.7	2.39, s
COOCH3	52.1	3.88, s	52.5	3.93, s
OCH3-2			62.7	3.95, s
OCH3-4	61.3	3.84, s	61.5	4.00, s

Measured in CDCl₃ (δ_H 7.26 ppm, δ_C 77.0 ppm).

) displayed two metacoupled aromatic doublets at δ_H 7.49 (d, J = 2.1 Hz, 1H) and 7.43 (d, J = 2.1 Hz, 1H), a methyl singlet at δ_H 2.17 (s, 3H), and two methoxy groups at δ_H 3.84 and 3.88 (each 3H, s). The ¹³C spectrum showed a total of 13 carbon resonances, including an aromatic ring [δ_C 126.7 (C-1), 115.2 (C-2), 148.9 (C-3), 149.2 (C-4), 134.1 (C-5), and 122.9 (C-6)], a carbonyl carbon δ_C 166.6, a methyl singlet δ_C 30.0, an ester methoxy group δ_C 52.3, and an aromatic methoxy group (δ_C 61.3). The NMR data of compound 1 were compared with those of methyl (E)-3-hydroxy-4-methoxy-5-(3-oxo-1,3-butadienyl) benzoate, a known compound from O. myosurus [4]. The key difference lies in the observation that both H-1′ and H-2′ in compound 1 exhibited methylene (CH₂) signals rather than olefinic proton signals. Finally, compound 1 was identified as methyl 3-hydroxy-4-methoxy-5-(3-oxobutyl) benzoate and named as oberoniaensiformisin A.

Compound 2 was isolated as a light-yellow oil. Its molecular formula was established as C₁₄H₁₆O₆ based on positive-ion HRESIMS analysis, which revealed a [M + H]⁺ peak at m/z 281.1021 (calcd. for C₁₄H₁₇O₆, 281.1025), corresponding to seven degrees of unsaturation. Comparative analysis of the NMR data between compounds 2 and 1 revealed the presence of an additional olefinic methylene proton pair and an aromatic methoxy signal in compound 2, indicating that 2 is a structural analog of 1. The ¹H NMR spectrum of 2 exhibited a pair of vinyl protons at δ_H 7.73 (d, J = 16.4 Hz, 1H) and 6.77 (d, J = 16.4 Hz, 1H), a methyl singlet at δ_H 2.39 (s, 3H), and three methoxy singlets at δ_H 3.93, 3.95, and 4.00 (s, each 3H). The ¹³C NMR spectrum revealed signals corresponding to a benzene ring, two aromatic methoxy groups (δ_C 62.7 and 61.5), and an isoprene chain featuring a double bond (δ_C 128.5 and 137.1). These NMR data, combined with key HMBC correlations—such as those from the methoxy group at δ_H 3.95 (s, 3H) to C-2, from δ_H 4.00 (s, 3H) to C-4, from H-2′ to C-1′, C-3′, C-4′, C-4, and C-5, and from H-6 to C-1, C-3, and C-7—collectively indicated that compound 2 is a 2,3,4,5-trisubstituted benzoic acid methyl ester derivative. The structure features a hydroxy group and two methoxy groups positioned at C-3, C-2, and C-4, respectively. The side-chain structure was confirmed by the HMBC (Figure 2) correlations from H-4′ (δ_H 2.39) to C-1′ (δ_C 137.1), C-2′ (δ_C 128.5), and C-3′ (δ_C 198.6); from H-2′ (δ_H 6.77) to C-1′ (δ_C 137.1), C-3′ (δ_C 198.6), C-4′ (δ_C 27.7), C-4 (δ_C 150.0), and C-5 (δ_C 123.7); and from H-1′ (δ_H 7.73) to C-4 (δ_C 150.0), C-6 (δ_C 121.5), C-2′ (δ_C 128.5), and C-3′ (δ_C 198.6). The diagnostic coupling constant (J = 16.4 Hz) between H-1′ and H-2′ indicated that the double bond between C-1′ and C-2′ adopts an E-configuration. Therefore, the structure of compound 2 was determined to be methyl (E)-3-hydroxy-2,4-dimethoxy-5-(3-oxobut-1-en-1-yl) benzoate, and it was assigned the trivial name oberoniaensiformisin B.

Compound 3 was obtained as a colorless oil. Its molecular formula, C₁₃H₁₄O₅, was established by HR-ESI-MS analysis, which showed an [M + H]⁺ peak at m/z 251.0892 (calcd. for C₁₃H₁₅O₅, 251.0919). The ¹H NMR spectrum of 3 revealed signals characteristic of a 1,2,4,5-tetrasubstituted aromatic benzene ring, including two broad singlets at δ_H 7.30 (br s, H-7, 1H) and 7.01 (br s, H-4, 1H). Additionally, a methoxy group was observed at δ_H 3.95 (s, -OCH₃, 3H). The spectrum also displayed signals for a terminal olefinic group at δ_H 4.94 and 5.10 (br s, each 1H) and a methyl group at δ_H 1.75 (s, 3H), indicating the presence of an isopropylene moiety (

Table 2. ¹H (600 MHz) and ¹³C (150 MHz) NMR data for compounds 3 and 4.

Position	3 a		4 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
2	92.7	4.85, d (1.0)	167.0
3	76.7	5.12, br s	101.8	6.77, d (1.0)
4	114.3	7.01, s	124.4	8.28, d (1.8)
5	136.6		126.2
6	157.0		126.7	7.98, dd (8.6, 1.8)
7	109.5	7.30, s	111.9	7.54, d (8.6)
8	152.3		158.8
9	113.3		130.1
1′	141.4		69.7
2′	113.0	4.94, br s	28.9	1.65, s
		5.10, br s
3′	17.6	1.75, s	28.9	1.65, s
COOCH3	52.6	3.95, s	52.6	3.94, s
-C=O	170.3		168.8

^a Measured in CDCl₃ (δ_H 7.26 ppm, δ_C 77.0 ppm). ^b Measured in CD₃OD (δ_H 3.31 ppm, δ_C 49.0 ppm).

). The ¹³C NMR spectrum exhibited 13 carbon signals, including 8 characteristic benzofuran signals attributed to six aromatic carbons at δ_C 114.3 (C-4), 136.6 (C-5), 157.0 (C-6), 109.5 (C-7), 152.3 (C-8), and 113.3 (C-9) in the low-field region; one oxygenated carbon at δ_C 92.7 (C-2); one methine at δ_C 76.7 (C-3); and a carbonyl carbon (δ_C 170.3). Analysis of the NMR data of 3 revealed that compound 3 shares significant structural similarities with compound 4, and compound 3 lacks a hydroxyl group at the C-6 position compared to compound methyl (2R,3S)-2,3-dihydro-3-hydroxy-2-(1-methylethenyl)-5-benzofurancarboxylate [5]. The presence of a hydroxyl group at the C-3 position was confirmed by HMBC correlations, from H-3 to C-2 and C-9, as well as from H-2 to C-3, C-8, C-9, and C-12. These correlations further indicated that the isopropene moiety was attached at the C-2 position. Based on the combined analysis of these data, the planar structure of the compound 3 was determined to be methyl 3,6-dihydroxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-5-carboxylate. The relative configuration of compound 3 was assigned as 2S*3R*, supported by remarkable consistency in NMR chemical shifts with graphostrin D [5,6] and a computational analysis revealing a characteristic H-2-C-2-C-3-H-3 dihedral angle of 110° (Supplementary Materials). In addition, through DP4+ analysis of the carbon NMR data, the relative configuration of compound 3 was also determined to be 2S*3R* (Supplementary Materials). Further comparison between the experimental and calculated ECD spectra confirmed its absolute configuration as 2S3R (Figure 3). Thus, the structure of compound 3 was determined as shown in Figure 1 and was assigned the trivial name oberoniaensiformisin C.

Compound 4 was obtained as a colorless oil. The ¹H NMR spectrum displayed signals corresponding to two methyl groups [δ_H 1.65 (s, H-2′, H-3′, 6H)], a methoxy group [δ_H 3.94 (s, -OCH₃, 3H)], and an ABX-type benzene ring system [δ_H 8.28 (d, J = 1.8 Hz, H-4, 1H), 7.98 (dd, J = 8.6, 1.8 Hz, H-6, 1H), 7.54 (d, J = 8.6 Hz, H-7, 1H)]. Additionally, an alkene hydrogen signal was observed at δ_H 6.77 (s, H-3, 1H). The ¹³C NMR and HSQC spectra revealed 14 carbon signals, including a carbonyl carbon [δ_C 168.8 (C=O)], six aromatic carbons [δ_C 158.8(C-8), 130.1(C-5), 126.7(C-6), 126.2(C-9), 124.4(C-4), 111.9(C-7)], two alkene carbons [δ_C 167.0(C-2), 101.8(C-3)], a quaternary carbon signal [δ_C69.7 (C-1′)], a methoxy carbon signal [δ_C 52.6 (-OCH₃)], and two methyl carbon signals [δ_C 28.9 (C-2′, 3′)]. The key HMBC correlations from H-3 to C-2/C-5/C-8/C-9, along with those from H-2′ to C-2/C-1′/C-3′, unequivocally confirmed the attachment of the methyl group at the C-1′ position. This compound differs from compound 10 exclusively in possessing a methyl acetate substituent at C-5 [7]. Hence, compound 4 was assigned as methyl 2-(2-hydroxypropan-2-yl) benzofuran-5-carboxylatev and named as oberoniaensiformisin D.

Compound 5 was isolated as an apparent single component after repeated HPLC purification. Its molecular formula (C₂₂H₃₀O₇) was established by HRESIMS and NMR data. Additionally, the ¹H and ¹³C NMR spectra indicated that compound 5 exists as a mixture of two prenylated p-hydroxybenzoic acid derivatives in a 1:1 ratio, suggesting the presence of two stereoisomers in solution. Structural analysis was subsequently conducted on one of the configurations, designated as 5a. The ¹H NMR spectrum (

Table 3. ¹H (600 MHz) and ¹³C (150 MHz) NMR data for compound 5a–5b.

Position	5a		5b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	120.6		120.6
2	129.4	7.58, s	129.4	7.58, s
3	128.4		128.3
4	158.0		158.1
5	125.9		126.0
6	130.9	7.60, s	131.0	7.59, s
7	165.1		165.1
1′	28.2	3.26, d (6.9)	28.2	3.26, d (6.9)
2′	122.4	5.24, t (7.4)	122.4	5.24, t (7.4)
3′	131.9		131.9
4′	25.5	1.70, s	25.5	1.70, s
5′	17.7	1.69, s	17.7	1.68, s
1″	37.7	2.80, d (6.6)	37.7	2.80, d (6.6)
2″	74.9	4.23, d (18.8)	74.7	4.23, d (18.8)
3″	147.1		147.2
4″	110.4	4.75, d (3.7)	110.3	4.75, d (3.7)
		4.90, d (6.2)		4.90, d (6.2)
5″	18.0	1.73, s	18.1	1.73, s
1‴	175.8		175.8
2‴	75.3		75.3
3‴	74.2	5.09, d (6.4)	74.2	5.09, d (6.4)
4‴	13.6	1.22, d (6.3)	13.6	1.22, d (6.3)
5‴	22.3	1.27, s	22.3	1.27, s

Measured in DMSO-d₆ (δ_H 2.49 ppm, δ_C 39.5 ppm).

) displayed characteristic signals corresponding to one set of 1,3,4,5-tetrasubstituted benzene rings at δ_H 7.58 (s, H-2) and 7.58 (s, H-6). Additionally, signals corresponding to five methyl groups were observed at δ_H 1.73 (s, H-5″, 3H), 1.70 (s, H-4′, 3H), 1.69 (s, H-5′, 3H), 1.27 (s, H-4‴, 3H), and 1.22 (s, H-5‴, 3H). Three methylene groups were also identified: δ_H 3.26 (d, J = 6.9 Hz, H-1′, 2H), 2.80 (m, H-1″, 2H), and 4.90, 4.75 (d, J = 6.2 Hz, H-4″, 2H). A carbonyl resonance was detected at δ_C 165.1, along with a prenyl group characterized by the following signals: δ_H 3.26 (d, J = 6.9 Hz, 2H), 5.25 (t, J = 7.4 Hz, 1H), 1.70 (s, 3H), 1.68 (s, 3H), and δ_C 28.2, 122.3, 131.0, 25.5, and 17.6. The remaining signals at δ_H 2.81 (d, H-1″, 2H), 4.23 (d, J = 18.8 Hz, H-2″, 1H), 4.75, 4.90 (d, J = 6.2,3.7 Hz H-4″, each 1H), and 1.73 (3H, s, H-5″) and δ_C 37.7, 74.9, 147.2, 110.4, and 18.1 were assigned as a 2-hydroxy-3-methylbut-3-en-1-yl unit, which was confirmed by the HMBC correlations (Figure 2) from H-1″ to C-2″ and C-3″ and from H-5″ to C-2″, C-3″, and C-4″. The esterification group was established as a 2,3-dihydroxy-2-methylbutyryloxy unit based on HMBC correlations from H-4‴ (δ_H 1.22, d, J = 6.3 Hz) to C-3‴ (δ_C 74.2) and C-2‴ (δ_C 75.3) and from H-5‴ (δ_H 1.26) to C-1‴ (δ_C 175.8), C-2‴, and C-3‴. The chemical shift in H-3‴ (δ_H 5.09) indicated that the hydroxy group at C-3‴ was esterified, which was further confirmed by the HMBC correlation from H-3‴ to C-7 (δ_C 165.1).

Attempts to separate this mixture were consistently unsuccessful. Based on the HPLC chromatogram, the mixture exists in an approximate 1:1 ratio of the R-epimer and its stereoisomer [8,9,10].

Compound 6 was obtained as a colorless oil. Its molecular formula, C₂₄H₃₀O₈, was established by positive-ion HRESIMS, which displayed a [M + H]⁺ peak at m/z 447.2022 (calcd. for C₂₄H₃₁O₈, 447.2019), corresponding to ten degrees of unsaturation. The ¹H and ¹³C NMR spectra exhibited 24 carbon signals, including those characteristic of a tetrasubstituted benzene ring: δ_C 120.3, 129.1, 128.2, 158.1, 126.1, and 130.7, along with corresponding proton signals at δ_H 7.49 (s, 1H) and 7.54 (s, 1H). The esterification group was established as a (4-hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-butenyl)benzoic acid unit according to the HMBC correlations (Figure 2) from H-2 (δ_H 7.49, s) to C-1″ (δ_C 28.0) and C-6 (δ_C 130.7); from H-1′ (δ_H 3.25, d) to C-4 (δ_C 158.1), C-2′ (δ_C 122.1), C-4′ (δ_C 25.4), and C-3′ (δ_C 132.2); and from H-1″ (δ_H 2.75, d) to C-2″(δ_C 74.6), C-3″ (δ_C 147.1), and C-5 (δ_C 126.1) [11]. The remaining signals [δ_H 2.21, br dd, (J = 18.3, 4.4 Hz), 2.62, br. dd, (J = 18.3, 4.4 Hz), δ_C 27.4; δ_H 3.79, (s), δ_C 67.5; δ_H 5.15, dt, (J = 5.9); δ_C 70.2; δ_H 4.27 (br s), δ_C 65.5, δ_H 6.68 (br s); δ_C 138.8] could be recognized as a shikimic acid ester [12]. The esterification process involving the C-5‴ hydroxy group and the C-7 carboxyl of (4-hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-butenyl)benzoic acid was substantiated through the chemical shift observed at H-5‴ (δ_H 5.15) and the HMBC correlation from H-5‴ to C-7. Consequently, the structure of compound 6 was elucidated and designated as oberoniaensiformisin F.

Similar to compound 5, compound 6 was also identified as a mixture. Repeated attempts to separate this mixture proved unsuccessful. HPLC chromatographic analysis demonstrated an approximate 3:1 ratio between the target compound and its co-existing mixture components.

Compound 7 had a molecular formula of C₂₅H₃₂O₇, as determined by its HRESIMS, ¹H-NMR, and ¹³C-NMR data. A comprehensive analysis of the 1D and 2D NMR data (

Table 4. ¹H (600 MHz) and ¹³C (150 MHz) NMR data for compounds 6 and 7.

Position	6 a		7 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	120.3		122.1
2	129.1	7.49, s	129.9	7.57, s
3	128.2		129.5
4	158.1		158.7
5	126.1		129.5
6	130.7	7.54, s	129.9	7.57, s
7	165.3		167.7
1′	28.0	3.25, d (7.6)	29.2	3.30, d (1.6)
2′	122.1	5.23, t (7.6)	122.8	5.30, m
3′	132.2		134.3
4′	25.4	1.68, s	25.9	1.76, s
5′	15.6	1.64, s	17.8	1.70, s
1″	37.4	2.75, d (4.3) 2.80, d (8.3)	29.2	3.30, d (1.6)
2″	74.6	4.19, dd (8.3)	122.8	5.30, m
3″	147.1		134.3
4″	18.1	4.88, s	25.9	1.77, s
		4.73, s
5″	110.2	1.71, s	17.8	1.70, s
1‴	128.0		129.3
2‴	138.8	6.68, br s	139.5	6.86, br s
3‴	65.5	4.27, br s	67.4	4.41, br s
4‴	67.5	3.79, s	69.2	3.98, dd (4.4)
5‴	70.2	5.15, dt (5.9)	71.5	5.34, dt (4.3)
6‴	27.4	2.21, br dd (18.3)	28.3	2.41, dd (19.5)
		2.62, dd (18.3)		2.85, dd (18.5, 2.4)
7‴	167.7		168.3
COOCH3			52.4	3.76, s

^a Measured in DMSO-d₆ (δ_H 2.49 ppm, δ_C 39.5 ppm). ^b Measured in CD₃OD (δ_H 3.31 ppm, δ_C 49.0 ppm).

) revealed the presence of a nervogenic acid moiety. Furthermore, the NMR data of compound 7 indicated that its structure closely resembles that of oberoniamyosurusin K, with the key difference being the replacement of the shikimic acid ester in oberoniamyosurusin K with a methyl shikimate moiety [4]. Hence, the structure of compound 7 was established and named as oberoniaensiformisin G.

The known compounds (8–18) were identified by comparing their experimental NMR spectral data with the corresponding data reported in the literature. These compounds were characterized as methyl 4-hydroxy-3-(2-methylbut-3-en-2-yl)benzoate (8) [13], (E)-3-hydroxy-4-methoxy-5-(3-oxo-1,3-butadienyl)benzoic acid methyl ester (9) [4], liparacid A (10) [14], oberoniamyosurusin E (11) [4], nervogenic acid (12) [15], oberoniamyosurusin L (13) [4], oberoniamyosurusin K (14) [4], oberoniamyosurusins I (15) [4], 1iparacids C (16) [14], oberoniamyosurusin F (17) [3], and liparacid B (18) [14].

2.2. In Vitro Antioxidant, Antibacterial, and α-Glucosidase Activity of Compounds 1–18

The antibacterial and antioxidant activities of all compounds (compounds 5 and 6 were performed using their epimeric mixtures) were evaluated using the 96-well plate microbroth dilution method and DPPH radical scavenging assay, respectively. However, none of the compounds exhibited bactericidal activity against the three tested bacterial strains: Escherichia coli ATCC 25922, Staphylococcus aureus subsp. Aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853.

In the antioxidant assay, sodium L-ascorbate (positive control) demonstrated potent activity with an IC₅₀ of 2.37 ± 0.11 μM. Among the tested compounds, only 6 and 12 exhibited weak DPPH radical scavenging activity (>50%), with IC₅₀ values of 173.76 ± 23.92 μM and 185.36 ± 1.96 μM, respectively (

Table 5. Clearance rate of compounds on DPPH (mean ± SD).

No.	Compound	IC50 (μg/mL) *
positive control	L-ascorbate	2.37 ± 0.11
6	oberoniaensiformisin F	173.76 ± 23.92
12	nervogenic acid	185.36 ± 1.96

* IC₅₀: Clearance rate of DPPH 50% activity (concentration in µg/mL required for a 50% reduction in antioxidant activity).

, Figure 4).

All isolated compounds were screened for α-glucosidase-inhibitory activity using the PNPG method, with acarbose as the positive control (IC₅₀ = 0.66 ± 0.36 μg/mL). Among them, compounds 5, 6, 12, 13, and 15 showed moderate inhibitory effects, with IC₅₀ values ranging from 34.03 to 106.10 μg/mL (

Table 6. Inhibitory effects of compounds on α-glucosidase (mean ± SD).

No.	Compound	IC50 (μg/mL) *
positive control	acarbose	0.66 ± 0.36
5	oberoniaensiformisin F	106.10 ± 2.12
6	oberoniaensiformisin F	58.07 ± 6.22
12	nervogenic acid	34.03 ± 0.16
13	oberoniamyosurusin L	86.53 ± 1.08
15	oberoniamyosurusin I	38.80 ± 4.43

* IC₅₀: inhibition of α-glucosidase 50% activity (concentration in µg/mL required for a 50% reduction in α-glucosidase-inhibitory activity).

, Figure 5).

3. Discussion

The experiment was conducted to evaluate the antibacterial activity of three bacterial strains using the microbroth two-fold dilution method. The findings revealed that none of the tested compounds exhibited significant antibacterial activity. In the DPPH antioxidant assay, compounds 6 and 12 demonstrated a scavenging effect on DPPH radicals. In the α-glucosidase inhibition assay, compounds 5, 6, 12, 13, and 15 displayed varying degrees of inhibitory activity against α-glucosidase. Specifically, compounds 12 and 15 exhibited strong inhibitory effects, compounds 6 and 13 showed moderate activity, and compound 5 displayed weak activity. Preliminary structure–activity relationship (SAR) analysis suggests that the presence of isopentenyl chains at the C-3 and C-5 positions of the aromatic rings is a critical factor for α-glucosidase inhibition in isopentenylated benzoic acid derivatives. This conclusion is supported by the observation that compounds with isopentenyl chains at these positions tend to exhibit lower IC₅₀ values, indicating higher inhibitory potency. Notably, although other derivatives (e.g., compounds 1–4, 7–9) also possess isopentenyl chains at either the 3 or 5 position, they were inactive. This suggests that carbonyl O-methylation at position 7 may negatively impact their activity [16,17].

Additionally, compounds 10, 11, 16, 17, and 18 also showed no significant activity against the enzyme. We hypothesize that this could be attributed to the specific positioning of the isopentenyl chain substitution and the presence of substituent groups on the chain, which may adversely affect the inhibitory activity. Further studies are warranted to explore additional in vitro activities of these isolated compounds and to validate the proposed SAR hypotheses.

4. Materials and Methods

4.1. General Experimental Procedures

NMR spectra were carried out on Bruker Avance III 600 spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany) with deuterated solvent signals used as internal standards, and chemical shifts (δ) are expressed in ppm. Electrospray ionization mass spectrometry (ESIMS) and high-resolution (HR)-ESIMS analyses were performed using an Agilent G6230 time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The infrared (IR) spectra were recorded using a Nicolet FTIR-iS20 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Circular dichroism spectra were obtained using a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). Optical rotation was measured using a high-sensitivity polarimeter, MCP-150 (Anton Paar, Graz, Austria). Column chromatography (CC) was performed using silica gel (100–200 mesh and 300–400 mesh, Shanghai Haohong Scientific Co., Ltd., Shanghai, China), octadecylsilyl silica gel (12 nm, S-50 μm, YMC, Komatsu, Japan) and Sephadex LH-20 (25–100 µm, Cytiva, Uppsala, Sweden), and MCI-gel (75–150 µm, Mitsubishi Chemical Corporation, Tokyo, Japan). Thin-layer chromatography (TLC) was performed using silica gel GF254-precoated plates (Shanghai Haohong Scientific Co., Ltd., Shanghai, China), and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH.

4.2. Plant Material

The whole O. myosurus plants were collected in October 2021 from the Xishuangbanna Dai and Yi Autonomous Prefecture, Yunnan Province, People’s Republic of China. The plant was identified by Dr. Fucai Ren of Anhui Medical University. A voucher specimen (AHMU-R02) was deposited at the School of Pharmacy of Anhui Medical University, China.

4.3. Extraction and Isolation

The O. myosurus whole herbs (5.0 kg) were air-dried, extracted three times with 95% EtOH (20 L × 3) at room temperature, and then filtered. The extracts were combined and concentrated to afford an organic extract (ca. 620 g), which was mixed with silica gel, loaded onto a silica gel column, and eluted with CH₂Cl₂-CH₃OH (1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1) to obtain 15 fractions (Fr. 1–12) (Figure 6.) Fraction 3 (16.7 g) was separated on a Sephadex LH-20 (CH₂Cl₂/MeOH 1:1) column, and Fraction C3-2 was further fractionated using silica gel CC and eluted with CHCl₃–methanol (MeOH) (80:1–10:1, v/v) to obtain six subfractions (3–1–3–6). Subfraction 3–4 was isolated using Sephadex LH-20 CC (CH₂Cl₂/MeOH; 1:1) to obtain compound 8 (9.6 mg).

Fraction 5 (86 g) was further fractionated using silica gel CC and eluted with CHCl3–methanol (MeOH) (100:1–10:1, v/v) to obtain five subfractions (5–1–5–5). Subfraction 5–5 was isolated using Sephadex LH-20 CC (Sigma-Aldrich, St. Louis, MO, USA, CH₂Cl₂/MeOH; 1:1), repeated silica gel CC (CH₂Cl₂/MeOH; 50:1→10:1), and preparative TLC (petroleum ether/ethyl acetate; 10:1), resulting in the isolation of compound 18 (6.5 mg).

Fraction 6 (28 g) was subjected to column chromatography (MCI, MeOH/H₂O 10% to 100%) and further fractionated using Sephadex LH-20 (CH₂Cl₂/MeOH 1:1), yielding six fractions (Fr.1-Fr.6). Fraction C3 (1.2 g) were separated by silica gel CC (CH₂Cl₂/MeOH from 50:1 to 10:1) and purified by MPLC (ODS, MeOH/H2O from 10% to 100%) to yield compounds 3 (4.5 mg) and 4 (3.8 mg).

Fraction seven (64 g) was further fractionated using silica gel CC and eluted with CHCl₃–methanol (MeOH) (100:1–10:1, v/v) to obtain six subfractions (7–1–7–6). Subfraction 7–5 was isolated using Sephadex LH-20 CC (CH₂Cl₂/MeOH; 1:1), repeated silica gel CC (CH₂Cl₂/MeOH; 60:1→10:1), and preparative TLC (petroleum ether/ethyl acetate; 1:1), resulting in the isolation of compounds 1 (6.9 mg), 2 (5.1 mg), 9 (4.6 mg), and 10 (8 mg).

Fraction 8 (64 g) was subjected to silica gel CC (CH₂Cl₂/MeOH from 50:1 to 10:1) and Sephadex LH-20 (MeOH) to produce two fractions (C8-1 and C8-5). Fraction C8-1 was purified by HPLC (MeOH/H2O 65:35, 2.5 mL/min), and compounds 15 (62.7 mg, tR 14.6 min), 17 (9.1 mg tR 33.5 min), and 16 (8.2 mg tR 40.7 min) were obtained.

Fraction 9 (80 g) was further fractionated using silica gel CC and eluted with CHCl₃–methanol (MeOH) (60:1–10:1, v/v) to obtain ten subfractions (9–1–9–10). Subfraction 9–3 was isolated using Sephadex LH-20 CC (CH2Cl2/MeOH; 1:1), repeated silica gel CC (CH₂Cl₂/MeOH; 100:1→10:1), MCI resin (MeOH–H₂O, 100:0 → 1:1), and preparative TLC (petroleum ether/ethyl acetate; 1:1), resulting in the isolation of compounds 5 (28.2 mg), 11 (21.4 mg), 12 (39.3 mg), 13 (28.7 mg), and 14 (36.8 mg).

Fraction 11 (79 g) was separated sequentially using silica gel (CHCl₃–MeOH, 100:0 → 10:1), MCI resin (MeOH–H₂O, 100:0 → 1:1), and Sephadex LH-20 (MeOH) and then preparative HPLC to afford compounds 6 (18.6 mg) and 7 (3.2 mg).

4.4. Spectroscopic Data

Oberoniaensiformisin A (1): light-yellow oil; (+)-HRESIMS m/z 275.0901 [M + Na]⁺ (calcd. for C₁₃H₁₆O₅Na, 275.0895); IR νmax:3396, 2922, 2851, 1747, 1552, 1435, 1384, 1224, 902, 772, 581, 473 cm⁻¹, for ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃); data shown in Table 1.

Oberoniaensiformisin B (2); light-yellow oil; (+)-HRESIMS m/z 281.1021 [M + H]⁺ (calcd. for C₁₄H₁₇O₆, 281.1025); IR νmax: 3267, 2922, 2850, 1726, 1666, 1643, 1600, 1470, 1427, 1362, 1336, 1302, 1246,1208, 1095, 1035, 999, 975, 928, 796, 771, 675, 555, 470 cm⁻¹, for ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃); data are shown in Table 1.

Oberoniaensiformisin C (3); colorless oil; [α${]}_D^{25}$ −4.0 (c 0.1, MeOH); (+)-HRESIMS m/z 251.0892 [M + H]⁺ (calcd. for C₁₃H₁₅O₅, 251.0919); IR νmax: 3399, 2923, 2852, 1601, 1553, 1442, 1360, 1240, 1067, 902, 791, 644, 480 cm⁻¹. for ¹H NMR (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃); data are shown in Table 2.

Oberoniaensiformisin D (4); colorless oil; (+)-HRESIMS m/z 235.0979 [M + H]⁺ (calcd. for C₁₃H₁₅O₄, 235.0970); IR νmax: 3433, 2926, 1638, 1384, 1104, 469 cm⁻¹, for ¹H NMR (600 MHz, MeOH) and ₁₃C NMR (150 MHz, MeOH); data are shown in Table 2.

Oberoniaensiformisin E (5); yellow amorphous powder; [α${]}_D^{25}$ −4.0 (c 0.1, MeOH); (+)-HRESIMS m/z 429.1902 [M + Na]⁺ (calcd. for C₂₂H₃₀O₇Na, 429.1889); IR νmax: 3428, 2926, 1712, 1602, 1555, 1453, 1355, 1304, 1202, 1102, 1061, 771 cm⁻¹, for ¹H NMR (600 MHz, DMSO) and ¹³C NMR (150 MHz, DMSO); data are shown in Table 3.

Oberoniaensiformisin F (6); yellow amorphous powder; [α${]}_D^{25}$ −14.0 (c 0.1, MeOH); (+)-HRESIMS m/z 447.2022 [M + H]⁺ (calcd. for C₂₄H₃₁O₈, 447.2019); IR νmax: 3855, 3752, 3737, 3553, 3415, 2921, 2850, 1638, 1618, 1030, 623, 481 cm⁻¹, for ¹H NMR (600 MHz, DMSO-d₆) and ¹³C NMR (150 MHz, DMSO-d₆); data are shown in Table 4.

Oberoniaensiformisin G (7); light-yellow oil; [α${]}_D^{25}$ −78.7 (c 0.1, MeOH); (+)-HRESIMS m/z 445.2249 [M + H]⁺ (calcd. for C₂₅H₃₃O₇, 445.2226); IR νmax: 3394, 2922, 2850, 1716, 1552, 1436, 1354, 1258, 1107, 1100, 902, 770, 646, 473 cm⁻¹, for ¹H NMR (600 MHz, MeOH) and ¹³C NMR (150 MHz, MeOH); data are shown in Table 4.

4.5. Quantum Chemical Calculations

Conformational analysis was initially performed using the GMMX module in GaussView with the MMFF94 force field, considering all conformers within a 3.5 kcal/mol energy window. The resulting low-energy conformers were further optimized at the B3LYP/6-311+G(2d,p) level of theory using Gaussian 16. To be compared with experimental data, electronic circular dichroism (ECD) spectra were calculated at the TDDFT/B3LYP/6-311+G(2d,p) level in MeOH, while NMR chemical shifts were calculated at the mPW1PW91/6-311+G(d,p) level in CHCl₃. Finally, the theoretical values were Boltzmann-averaged based on the relative populations of the optimized conformers and compared with experimental observations. The DP4+ probability analysis was performed on the calculated ¹³C NMR chemical shifts to assess the confidence level of each proposed stereochemical configuration [18,19].

4.6. Antibacterial Assay

In accordance with the background of folk medicine application of O. myosurus, all isolated compounds were evaluated for their potential antibacterial activity against the three bacterial strains Escherichia coli ATCC25922, Staphylococcus aureus subsp. aureus ATCC29213, and Pseudomonas aeruginosa ATCC27853. Ciprofloxacin was used as a positive control. Targeted microbes were cultivated in LB medium (yeast extract 5 g/L, peptone 10 g/L, NaCl 10 g/L, pH = 7.4) overnight at 37 °C and diluted bacterial suspension (5 × 10⁵ CFU/mL) for the test. The minimum inhibitory concentrations (MICs) of samples and positive control were determined in sterile 96-well plates by the modified broth dilution test [20,21]. All wells were filled with 196 μL of bacterial suspension containing 5 × 10⁵ CFU/mL. Test samples (4 μL) with concentrations of serial dilutions were added to each well. The negative control was a medium containing 1% DMSO, and the positive control used was ciprofloxacin. The final concentrations of test compounds were 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg/mL in medium. After incubation, the minimum inhibitory concentration (MIC) was defined as the lowest test concentration that completely inhibited the growth of the test organisms.

4.7. Antioxidant Assay

The activity screening of 18 compounds isolated from O. ensiformis was carried out using 96-well plates and an enzyme labeler with DPPH methanol solution as substrate (0.15 mmol/L) and sodium ascorbate as positive control [22]. The gradient mass concentration of the samples to be tested was 200, 100, 50, 25, 12.5 μg/mL and then divided into a sample group (100 μL DPPH solution + 100 μL sample, A), a sample control group (100 μL methanol + 100 μL sample, A₀), and a negative control (100 μL DPPH solution + 100 μL methanol, A₁). The samples to be tested were fully reacted under light-avoiding conditions, and the OD values were determined at 517 nm after 30 min [23]. The experiments were performed three times in parallel, and the DPPH radical scavenging rate was calculated. The scavenging rate of DPPH radical was calculated according to the following equation [24].

E = [1 − (A − A_a)/A_b] × 100%

A:100 μL of test compounds or L-Ascorbic Acid Sodium Salt and 100 μL of DPPH.

A_a: 100 μL of test compounds or L-Ascorbic Acid Sodium Salt and 100 μL of MeOH.

A_b: 100 μL of MeOH and 100 μL of DPPH.

4.8. α-Glucosidase Inhibition Assay

The inhibitory activity of the obtained compounds on α-glucosidase was evaluated using the method mentioned in previous studies with minor modifications [25,26,27]. The α-glucosidase inhibitor acarbose was used as a positive control, and the total experimental system was 200 µL. The following sentences describe the assay process in brief: The compounds were first dissolved in DMSO, then diluted with 0.1 M phosphate buffer (PBS, pH 6.8, DMSO concentration lower than 1%); 50 μL compounds or acarbose (dissolved in 0.1 M PB, pH 6.8) with different concentrations were mixed with 20 μL of 0.2 U/mL α-glucosidase (dissolved in 0.1 M PB, pH 6.8), and then incubated at 37 °C for 10 min. Subsequently, 50 μL of 5.0 mM p-Nitrophenyl α-D-glucopyranoside (pNPG, dissolved in 0.1 M PB, pH 6.8) was added to the mixture. After incubation at 37 °C for 30 min, the reaction was stopped by adding 80 μL of 0.2 M sodium carbonate solution (dissolved in ultrapure water) to the reaction system. The absorbance value was measured at 405 nm using a microplate reader. The inhibitory activity of compounds or acarbose on α-glucosidase was calculated by the formula

Inhibition rate (%) = 1 − (A_a − A_b)/A_c − A_d × 100

A_a: Absorbance of the reaction system containing 20 μL of test compounds or acarbose at different concentrations, 50 μL of α-glucosidase, and 50 μL of pNPG.

A_b: Absorbance of the reaction system containing 70 μL of test compounds or acarbose at different concentrations and 50 μL of α-glucosidase.

A_c: Absorbance of the reaction system containing 20 μL PBS, 50 μL α-glucosidase, and 50 μL pNPG.

A_d: Absorbance of the reaction system containing 70 μL of PBS and 50 μL of α-glucosidase.

5. Conclusions

In conclusion, our investigation led to the identification of 18 tandem prenylated p-hydroxybenzoic acid derivatives from O. ensiformis, including 7 previously undescribed compounds (1–7). The intricate stereochemical configurations of these isolates were successfully determined through comprehensive spectroscopic analyses, complemented by comparative studies between experimental and calculated ECD spectra. In addition, all the compounds were investigated for their biological activities, and in the antioxidant assay, compounds 6 and 12 showed scavenging of DPPH radicals above 50%, with IC₅₀ values of 173.76 ± 23.92 and 185.36 ± 1.96, respectively. The in vitro hypoglycemic activity of the obtained compounds was evaluated using an α-glucosidase inhibition assay. The results demonstrate that some of the compounds exhibit varying degrees of inhibitory activity against α-glucosidase, with IC₅₀ values ranging from 34.03 to 106.10 μg/mL. The current study significantly expands our understanding of the in vitro biological activities associated with isoprenylated p-hydroxybenzoic acid derivatives. While these findings establish a solid theoretical foundation for further research on O. ensiformis, additional investigations are warranted to explore potential alternative biological activities of the isolated compounds and to validate their pharmacological relevance.