Diverse phloroglucinols with hAChE inhibitory and anti-VRE effects from Rhodomyrtus tomentosa fruits

1 Introduction

Phloroglucinols derived from species within the Myrtaceae family have garnered significant attention from scientific community due to their complex molecular architectures and notable biological activities [1]. Rhodomyrtus tomentosa, a medicinal and edible plant of this family, is extensively cultivated across Thailand, Vietnam, and southeastern China [2]. The R. tomentosa fruits are popularly used as ingredients of pies and jams in these regions [3]. Furthermore, local populations traditionally infuse the fruits into white spirits to prepare herbal liquors, which are valued for their enhanced flavor and health-promoting and disease-preventive properties. Historically, the fruits, have also been utilized for the management of diarrhea and dysentery [3]. Previous studies have demonstrated that crude extracts and phenolic constituents from R. tomentosa fruits exhibit antioxidant [4–6], anti-inflammatory [7, 8], hepatoprotective effects against nonalcoholic fatty liver disease [9], and general health-promoting benefits [10]. Despite these findings, only one phloroglucinol homodimer with anti-inflammatory activity, namely watsonianone A [11], has been identified from the fruits of title plant, leaving the broader phloroglucinol composition largely unexplored. In contrast, phloroglucinol compounds isolated from R. tomentosa leaves in tropical regions of China have been characterized for both structures and their bioactivities [12–16]. Therefore, further elucidation of novel bioactive phloroglucinol components from its fruits represents a valuable research direction. To obtain additional active phloroglucinol constituents, the fruits were subjected to ultraviolet-guided employing a DAD detector. Consequently, eight newly reported polymethylated phloroglucinols (1–8) and six known analogues (9–14) were obtained (Fig. 1). Selected compounds were examined for their hAChE inhibitory and anti-VRE activities. For the most potent hAChE inhibitor, molecular docking analysis was conducted to elucidate the possible interaction mechanism. This study presents the isolation, comprehensive structural characterization, and preliminary pharmacological assessment of the isolated metabolites.

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2 Results and discussion

The phloroglucinol containing moiety of R. tomentosa fruits was subjected to sequential chromatographic separation on silica gel, reversed-phase C18, Sephadex LH-20, and semi-preparative HPLC, leading to the isolation of one new polymethylated phloroglucinol meroterpenoid (PPM, 1), one new polymethylated phloroglucinol homodimer (PPHMD, 2), and six polymethylated phloroglucinol heterodimers (PPHTDs, 3–8). In addition, six previously reported congeners were structurally identified as myrciarone B (9) [17, 18], rhodotomentodimer D (10) [13], isomyrtucommulone B (11) [17, 18], rhodomyrtone (12) [19], myrtucommulone B (13) [20], and callistenone A (14) [21], based on detailed comparison of their NMR and mass spectrometry (MS) profiles with reference data previously documented in the literature.

Phloroglucinol 1 was assigned the molecular formula of C₂₅H₃₈O₃, as determined by an high-resolution electrospray ionization mass spectrometry (HRESIMS) with an [M + H]⁺ ion at m/z 387.2904 (calcd for C₂₅H₃₉O₃, 387.2894), indicating seven degrees of unsaturation. The ¹H NMR spectrum (Table 1) displayed signals for nine methyl groups at δ_H 0.85 (d, J = 6.8 Hz, H₃-10'), 0.86 (d, J = 6.8 Hz, H₃-9'), 0.89 (d, J = 6.6 Hz, H₃-11), 0.95 (d, J = 6.6 Hz, H₃-10), 1.33 (s, H₃-13), 1.34 (s, H₃-12), 1.38 (s, H₃-15), 1.40 (s, H₃-14), and 1.42 (s, H₃-7′), along with two strongly shielded methylene protons at δ_H 0.51 (2H, brd, J = 5.4 Hz, H₂-5'), characteristic of a cyclopropane moiety. Analysis of the ¹³C NMR data of phloroglucinol 1 (Table 1), supported by DEPT and HSQC experiments, disclosed the presence of 25 distinct carbon signals, including two keto carbonyl groups at δ_C 198.3 (C-2) and 213.6 (C-4), two olefinic quaternary carbons at δ_C 109.2 (C-1) and 168.8 (C-6), and additional four quaternary carbons, one of which was oxygenated at δ_C 87.3 (C-1'). The remaining signals were attributed to five methines, three methylenes, and nine methyl groups. Three of the seven degrees of unsaturation were attributed to the presence of two ketone functionalities and a single olefinic bond, while the remaining unsaturation implied the existence of a tetracyclic core structure. The planar architecture of phloroglucinol 1 was determined through comprehensive analyses of ¹H–¹H COSY and HMBC spectra (Fig. 2). The ¹H–¹H COSY experiment established an isopentyl unit of δ_H 2.64 (H-7)/1.29 (H₂-8a) and 1.23 (H₂-8b)/1.67 (H-9)/0.95 (H₃-10) and 0.89 (H₃-11). Key HMBC interactions, from δ_H 2.64 (H-7) to δ_C 109.2 (C-1), 168.8 (C-6), and 198.3 (C-2); from δ_H 1.34 (H₃-12) and 1.33 (H₃-13) to δ_C 55.5 (C-3), 98.3 (C-2) and 213.6 (C-4); and from δ_H 1.40 (H₃-14) and 1.38 (H₃-15) to δ_C 47.7 (C-5), 168.8 (C-6), and 213.6 (C-4), strongly supported the presence of a β-triketone core. Likewise, apart from three structural fragments of δ_H 1.85 (H-2')/1.34 and 1.74 (H₂-3'), 0.51 (2H, H₂-5')/1.36 (H-6'), and 1.29 (H-8')/0.85 (H₃-9') and 0.86 (H₃-10') as indicated by the ¹H–¹H COSY spectrum, HMBC correlations (Fig. 2), specifically from δ_H 1.74 (H-3'a) to δ_C 14.2 (C-5') and 32.4 (C-4'), from δ_H 1.42 (H₃-7') to δ_C 34.9 (C-6'), 37.6 (C-2'), and 87.3 (C-1'), and from δ_H 0.86 (H₃-9') and 0.85 (H₃-10') to δ_C 32.4 (C-4') confirmed the presence of a thujene unit. Additional HMBC cross-peaks from δ_H 2.64 (H-7) to δ_C 34.0 (C-3') and 87.3 (C-1') indicated that the β-triketone moiety and the thujene fragment was connected through a C-6‒O‒C-1' ether linkage and a C-7‒C-2' bond. ROSEY correlations between δ_H 1.85 (H-2') and 0.51 (H-5'), 1.42 (H₃-7'), and 2.64 (H-7) supported an α orientation of all these protons. The assignment of absolute configuration for phloroglucinol 1 was achieved by comparing its measured ECD spectrum with that simulated by TDDFT B3LYP/6–31++ G(2d, p) using Gaussian 16. The experimental curve (Fig. 4) exhibited negative Cotton effects at 205 and 271 nm and a positive signal at 311 nm, closely matching the calculated spectrum, thereby confirming the (7R,1'R,2'S,4'R,6'R) configuration. Based on combined spectroscopic and computational evidence, phloroglucinol 1, was structurally identified as a novel β-triketone‒thujene meroterpenoid and designated rhodotomentodione F.

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Phloroglucinol 2 was determined to possess the molecular formula C₂₁H₂₆O₆, as indicated by HRESIMS, which displayed a [M + H]⁺ ion at m/z 375.1802 (cacld for C₂₁H₂₇O₆, 375.1802). The ¹H NMR spectrum of phloroglucinol 2 (Table 2) featured a downfield singlet at δ_H 8.00 (1H, s, H-1”) and eight singlet methyl groups at δ_H 1.08 (H₃-9), 1.38 (H₃-8), 1.40 × 2 (6H, H₃-7'/H₃-8'), 1.41 (H₃-7), 1.50 (H₃-10), and 1.53 × 2 (6H, H₃-9'/H₃-10'). The ¹³C NMR data revealed four ketone carbonyl signals at δ_C 194.9 (C-2'), 197.1 (C-2), 210.5 (C-4'), and 210.7 (C-4), two double bonds at δ_C 108.3 (C-1'), 122.1 (C-1), 127.7 (C-1”), and 172.9 (C-6'), a hemiketal carbon at δ_C 100.8 (C-6), four quaternary carbons at δ_C 48.0 (C-5'), 53.9 (C-5), 54.8 (C-3), and 56.1 (C-3'), and eight methyl groups, indicating a tricyclic scaffold. Key HMBC correlations (Fig. 2) from δ_H 1.41 (H₃-7) and 1.38 (H₃-8) to δ_C 54.8 (C-3), 197.1 (C-2), and 210.7 (C-4), from δ_H 1.50 (H₃-10) and 1.08 (H₃-9) to δ_C 53.9 (C-5), 100.8 (C-6), and 210.7 (C-4), and from δ_H 8.00 (H-1”) to δ_C 100.8 (C-6), 122.1 (C-1), and 197.1 (C-2) supported a β-triketone motif incorporating a hemiketal at C-6 and a C-1 and C-1” double bond. Similarly, HMBC cross-peaks from δ_H 1.40 (6H, H₃-7' and H₃-8') to δ_C 56.1 (C-3'), 194.9 (C-2'), and 210.5 (C-4'), from δ_H 1.53 (6H, H₃-9'/ H₃-10') to δ_C 48.0 (C-5'), 172.9 (C-6'), and 210.5 (C-4'), and from δ_H 8.00 (H-1”) to δ_C 108.3 (C-1'), 172.9 (C-6'), and 194.9 (C-2') indicated a second β-triketone fragment. Additional HMBC data indicated linkage of the two fragments via a C-6‒O‒C-6' ether bond and a C-1‒C-1”‒C-1' connection. Phloroglucinol 2, identified as a racemic mixture, was resolved into enantiomers (+)-2 and (−)-2 using a Daicel CHIRALPAK IC column. ECD spectral analysis (Fig. 4) established the absolute configurations for (+)-2 and (−)-2 to be (6R) and (6S), respectively. Thus, phloroglucinol 2 was structurally characterized as a PPHMD and designated rhodotomentodimer H.

Phloroglucinol 3 was determined to possess the molecular formula C₂₅H₃₂O₆, as established by its HRESIMS, which exhibited a [M + H]⁺ ion at m/z 429.2279 (calcd for C₂₅H₃₃O₆, 429.2272). The ¹H NMR spectrum of phloroglucinol 3 (Table 3) indicated four singlet methyl groups at δ_H 1.39 (H₃-12), 1.42 (H₃-14), 1.43 (H₃-13), and 1.61 (H₃-15), three doublet methyl groups at δ_H 0.74 (J = 6.8 Hz, H₃-9), 1.24 (J = 7.3 Hz, H₃-10'), and 1.25 (J = 6.7 Hz, H₃-9'), a triplet methyl group at δ_H 0.90 (J = 7.3 Hz, H₃-11). An aromatic proton at δ_H 6.26 (s, H-5'), and a strongly deshielded hydroxyl proton at δ_H 13.30 (s, OH-6') were also observed. The ¹³C NMR spectrum (Table 3) displayed 25 carbon resonances, including eight methyl groups at δ_C 12.3 (CH₃-11), 14.9 (CH₃-9), 17.7 (CH₃-9'), 20.8 (CH₃-10'), 24.3 (CH₃-12), 24.5 (CH₃-13), 24.9 (CH₃-14) and 25.1 (CH₃-15), one methylene group at δ_C 26.1 (CH₂-10), three methine groups at δ_C 30.9 (CH-7), 39.7 (CH-8'), and 42.1 (CH-8), two aliphatic quaternary carbons at δ_C 47.2 (C-5) and 56.2 (C-3), a pair of olefinic carbons at δ_C 112.7 (C-1) and 167.5 (C-6), six aromatic carbons at δ_C 100.6 (CH-5′), 103.0 (C-3′), 104.0 (C-1′), 153.8 (C-2′), 159.3 (C-4′), and 164.6 (C-6′), and three ketone carbonyl groups at δ_C 197.9 (C-2), 208.9 (C-7′), and 211.7 (C-4). Comparison of the abovementioned spectral data with those of myrtucommulone B (phloroglucinal 13) [20] revealed close similarity, with the exception that a 2-methylbutyl unit replaced an isobutyl at C-1 in myrtucommulone B. This structural feature was supported by a 4.35 (H-7)/1.56 (H-8)/1.50 (H₂-10a) and 1.03 (H₂-10b) and 0.74 (H₃-9)/0.90 (H₃-11) spin system identified through ¹H–¹H COSY analysis, along with key HMBC cross-peaks (Fig. 2) from δ_H 4.35 (H-7) to δ_C 103.0 (C-2'), 112.7 (C-1), 153.8 (C-2'), 159.3 (C-4'), 167.5 (C-6), and 197.9 (C-2). The H-7 was stochastically assigned as a β configuration, and the relative configuration for the side chain at C-7 was determined by a theoretical NMR calculation method (Fig. 3) and DP4+ probability analysis. Finally, the absolute configuration of compound 3 was established as (7S,8S) based on the ECD calculations (Fig. 4). Thus, phloroglucinol 3 was identified as a PPHTD and designated rhodotomentodimer I.

Phloroglucinols 4 and 5 were both assigned shared the molecular formula C₂₅H₃₂O₆, supported by HRESIMS [M + H]⁺ peaks at m/z 429.2275 and 429.2277 (both calcd for C₂₅H₃₃O₆, 429.2272), respectively. Further examination of 1D NMR data (Table 3) with that of callistenone B (14) [20] revealed a substitution C-1', where the isovaleryl group in 14 was replaced by a 2-methylbutanoyl moiety in both compounds. This structural change was validated by HMBC correlation between Me-9' and C-7' and ¹H–¹H COSY cross-peaks linking H-8'/H₂-10' and H₃-9'/H₃-11' (Fig. 2). The relative configurations for H-7 in compounds 4 and 5 were assigned as α and β configurations, respectively, and the relative configurations for the C-7′ side chains were determined according to further NMR calculations and DP4+ probability analysis (Fig. 3). In light of ECD calculations (Fig. 4), the absolute configuration of phloroglucinol 4 was established as (7R,8′S), and comparison of experimental ECD spectra of 5 and 4 led to the establishment of absolute configuration for 5 to be (7S,8′S). Based on spectral and computational evidence, the two structures were characterized as two PPHTDs and designated rhodotomentodimer J and rhodotomentodimer K, respectively.

Phloroglucinols 6 and 7 shared the molecular formula C₂₆H₃₄O₆, as indicated by their HRESIMS [M + H]⁺ signals at m/z 443.2435 and 443.2438 (both calcd for C₂₆H₃₅O₆, 443.2428), respectively. Their 1D NMR spectra (Table 4) were highly similar to those of phloroglucinol 3, suggesting structural similarity to PPHTD congeners. A distinguishing feature was the presence of a 2-methylbutyl moiety at C-1', evidenced by disclosed by HMBC correlations of H₃-9' with C-7' and of OH-6' with C-1' as well as a COSY defined spin system of H₃-9'/H-8'/H₂-10'/H₃-11' (Fig. 2). The relative configurations for H-7 in compounds 6 and 7 were randomly assigned as α and β configurations, respectively, and further NMR calculations and DP4+ probability analysis allowed the establishment of the relative configurations for the C-7′ side chains (Fig. 3). Owing to ECD calculations (Fig. 4), the absolute configurations of phloroglucinols 6 and 7 were established to be (7R,8S,8′S) and (7S,8R,8′S), respectively. Based on these spectroscopic and computational results, phloroglucinols 6 and 7 were identified as two two PPHTDs and designated rhodotomentodimer L and rhodotomentodimer M, respectively.

Phloroglucinol 8 was assigned the same molecular formula C₂₆H₃₄O₆, identical to that of 7, as indicated by its HRESIMS peak at m/z 443.2431 [M + H]⁺ (calcd for C₂₆H₃₅O₆, 443.2428). Analysis of the ¹³C NMR spectrum (Table 4), supported by HSQC and DEPT data, revealed 26 carbon resonances, including three keto carbonyls, an aromatic ring, four quaternary carbons (two olefinic ones), three methine groups, two methene groups, and eight methyl groups. Comparison of phloroglucinol 8 and rhodotomentodimer D (10) [13] indicated the replacement of the isopentyl side chain at C-1 by a 2-methylbutyl group. This substitution was corroborated by a ¹H–¹H COSY spin system (Fig. 2) of δ_H 4.32 (H-7)/1.60 (H-8)/1.45 (H₂-10a) and 1.01 (H₂-10b) and 0.71 (H₃-9)/0.89 (H₃-11) and HMBC correlations of δ_H 4.32 (H-7) with δ_C 104.4 (C-3'), 112.6 (C-1), 156.6 (C-4'), 162.3 (C-2'), 167.6 (C-6), and 197.6 (C-2). The relative configuration of H-7 in compound 8 was stochastically assigned to be β orientated, the β configuration for the C-8 and C-8' ethyl groups were determined by theoretical NMR calculations and DP4+ probability analysis (Fig. 3). Subsequent ECD calculations (Fig. 4) permitted the assignment of the absolute configuration for phloroglucinol 8 to be (7S,8R,8′S). Hence, phloroglucinol 8 was identified as a PPHTD and named rhodotomentodimer N.

Given that rhodotomentodimer D (10) [16], a structurally related analogue, exhibited inhibitory activity against human acetylcholinesterase (hAChE), further screening was conducted to assess this activity among related compounds (Table 5). All PPHTDs (3‒14) demonstrated significant hAChE inhibition, with IC₅₀ values between 1.04 and 3.35 μM. In contrast, phloroglucinols 1 and 2 were inactive, with IC₅₀ values exceeding 40.0 μM.

To gain insights into the binding modes of excellent hAChE inhibitor 13, molecular docking was carried out using the crystal structure of recombinant hAChE complex with (‒)-galantamine (PDB code: 4EY6, 2.40 Å resolution) [22]. The docking results indicated that phloroglucinol 13 fits well within the active site pocket of hAChE (Fig. 5). Apart from a π‒σ stacking interaction with Tyr337 (3.61 Å), five hydrogen bonds were observed between phloroglucinol 13 and the residues Gly120 (2.46 Å), Tyr133 (1.67 Å), Glu202 (2.13 Å), Ser203 (2.24 Å), and His447 (2.39 Å).

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In light of well-documented antibacterial activity of rhodomyrtone (12) [23–26], other phloroglucinol compounds were also assessed for their inhibitory effects on vancomycin-resistant Enterococci (VRE). Phloroglucinols 11 and 12 exhibited strong activity (MIC = 1 μg/mL), outperforming ampicillin (AMP, MIC = 2 μg/mL). Similarly, phloroglucinols 8‒10 showed comparable potency (MIC = 2 μg/mL). In contrast, phloroglucinols 1‒7, 13, and 14 were inactive (MIC > 32 μg/mL). These findings suggested that the different connection patterns of PPHTDs can significantly affect their antibacterial properties, thereby offering valuable insights for the design and synthesis of novel antibacterial agents.

3 Conclusions

A total of 14 diverse phloroglucinol derivatives (1‒14), including eight newly identified isolates (1‒8), were obtained from R. tomentosa fruits. The absolute configurations of the side chains in phloroglucinols 3‒8 were unambiguously determined through a combination of ECD computations, NMR calculations, and DP4+ probability assessments. In addition, the previously reported phloroglucinol dimers (9–14) were obtained from R. tomentosa fruits for the first time, and several of them represent characteristic and major bioactive constituents of both R. tomentosa and Myrtus communis. Notably, many of the isolated compounds demonstrated potent hAChE inhibitory activities, highlighting their potential as lead candidates for the treatment of neurological disorders, including Alzheimer's disease [27]. Furthermore, several isolates exhibited significant anti-VRE effects, underscoring the therapeutic promise of R. tomentosa fruits for combating drug-resistant bacterial infections [28, 29]. Given the limited prior knowledge of the phloroglucinol constituents of R. tomentosa fruits, the present findings significantly expand current understanding and provide a scientific basis for the future development of nutraceutical and medicinal food products.

4 Experimental procedures

4.1 General experimental procedures

The experimental procedures followed those previously reported in our recent publication [30].

4.2 Plant material

The mature fruits of R. tomentosa were collected in Wanning, Hainan province, China, in September 2023. The plant material was authenticated by Xin-Quan Yang (Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences). A voucher specimen (KIB-Q-202301) has been deposited at the State Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences.

4.3 Extraction and isolation

The air-dried and powdered fruits of R. tomentosa (19.5 kg) were extracted thrice with ethanol at room temperature (48 h). The ethanol extracts were concentrated under reduced pressure, to afford a crude residue (1.56 kg), which was partitioned with petroleum ether (PE) and ethyl acetate (EtOAc), yielding PE (331 g) and EtOAc-soluble (282 g) fractions. To eliminate lipoid components, the PE extract was further partitioned with MeOH‒H₂O (8:2, v/v), yielding a phloroglucinol enriched moiety (58.0 g). This enriched extract was subjected to silica gel column chromatography with a PE–EtOAc (100:1 → 1:1, v/v) gradient, yielding seven main fractions (Fr. A‒Fr. G). Fr. A (28.6 g) was further separated on silica gel (PE‒EtOAc, 100:0 → 0:100, v/v), producing three subfractions (Fr. A₁–Fr. A₃). Fr. A₁ (6.7 g) was purified by an RP-C18 column (MeOH–H₂O, 70:30 → 100:0, v/v), producing six subfractions (Fr. A_1.1–Fr. A_1.6). Compound 12 (4 mg) was obtained from Fr. A_1.1 via recrystallization d (Acetone–MeOH, 10:1, v/v), while 10 (2.8 mg, t_R = 36 min) was isolated from Fr. A_1.2 (0.05 g) by semi-preparative HPLC (MeOH–H₂O, 83:17, v/v). Fr. B (13 g) was separated by silica gel chromatography (PE–EtOAc, 15:1 → 0:16, v/v), resulting in seven subfractions (Fr. B₁–Fr. B₇). Fr. B₁ (0.3 g) was further purified via semi-preparative HPLC (MeCN‒H₂O, 52:48, v/v) to afford compounds 9 (3.1 mg, t_R = 33 min) and 13 (10.5 mg, t_R = 49 min). Fr. C (12 g) underwent RP-C₁₈ chromatography (MeOH–H₂O, 70:30 → 100:0, v/v), yielding five fractions (Fr. C₁‒Fr. C₅). Fr. C₂ (0.18 g) was further purified by a semi-preparative HPLC (MeCN–H₂O, 80:20, v/v), affording compounds 3 (1.4 mg, t_R = 18 min) and 5 (3.0 mg, t_R = 15 min). Fr. D (16 g) was fractionated by an RP-C₁₈ column (MeOH–H₂O, 75:25 → 100:0, v/v), resulting in six subfractions (Fr. D₁‒Fr. D₆). Fr. D₂ (0.92 g) afforded compounds 1 (2.0 mg, t_R = 35 min) and 8 (2.2 mg, t_R = 40 min) by semi-preparative HPLC (MeCN–H₂O, 73:27, v/v). Likewise, compound 2 (3.2 mg, t_R = 42 min) was isolated from Fr. D₄ (0.65 g) by a semi-prep. HPLC (MeCN–H₂O, 65:35, v/v). Fr. E (2 g) was separated on Sephadex LH-20 (CHCl₃–MeOH, 1:1, v/v), giving three fractions (Fr. E₁‒Fr. E₃). Fr. E₁ (0.39 g) was subjected to semi-preparative HPLC (MeCN–H₂O, 60:40, v/v), yielding compounds 4 (3 mg, t_R = 32 min), 11 (7.2 mg, t_R = 40 min), 6 (2.6 mg, t_R = 44 min), 7 (3 mg, t_R = 42 min), and 14 (7.0 mg, t_R = 37 min). Chiral separation of compound 2 was achieved using a Daicel CHIRALPAK IC column (n-hexane–isopropanol, 95:5 → 85:15, v/v), furnishing enantiomers (+)-2 (1.0 mg, t_R = 12 min) and (−)-2 (1.0 mg, t_R = 13 min).

Rhodotomentodione F (1): Colorless gum; [α] + 28.1 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 268 (3.68) nm; ECD (MeOH) λ_max (Δε) 205(–5.36), 241 (+ 1.25), 271 (–4.41), 311 (+ 9.76) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 1; (+)-HRESIMS m/z 387.2904 [M + H]⁺ (calcd for C₂₅H₃₈O₃, 387.2894).

Rhodotomentodimer H (2): Colorless gum; [α] + 49.0 (c 0.10, MeOH) for (+)-2; [α] –49.0 (c 0.10, MeOH) for (–)-2; UV (MeOH) λ_max (log ε) 217 (2.68), 272 (2.52) nm; ECD (MeOH) λ_max (Δε) 239 (‒4.49), 271 (+4.56), 301 (−5.83), 333 (+ 1.61) nm for (+)-2; ECD (MeOH) λ_max (Δε) 239 (+4.49), 271 (−4.56), 301 (+ 5.83), 333 (−1.61) nm for (–)-2; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 2; (+)-HRESIMS m/z 375.1802 [M + H]⁺ (calcd for C₂₁H₂₇O₆, 375.1802).

Rhodotomentodimer I (3): Colorless gum; [α] − 85.4 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (4.08), 216 (4.06), 295 (3.87) nm; ECD (MeOH) λ_max (Δε) 209 (+0.34), 222 (‒1.01), 238 (+1.26), 280 (+3.70), 314 (‒4.94) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 3; (+)-HRESIMS m/z 429.2279 [M + H]⁺ (calcd for C₂₅H₃₃O₆, 429.2272).

Rhodotomentodimer J (4): Colorless gum; [α] + 149.4 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (4.27), 217 (4.26), 295 (4.10) nm; ECD (MeOH) λ_max (Δε) 209 (‒1.19), 221 (+3.65), 237 (−3.38), 280 (−10.08), 313 (+8.51) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 3; (+)-HRESIMS m/z 429.2275 [M + H]⁺ (calcd for C₂₅H₃₃O₆, 429.2272).

Rhodotomentodimer K (5): Colorless gum; [α] –186.6 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (4.26), 216 (4.27), 294 (4.16) nm; ECD (MeOH) λ_max (Δε) 208 (+ 1.49), 221 (‒4.50), 238 (‒3.40), 283 (+ 8.83), 316 (‒10.50) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 3; (+)-HRESIMS m/z 429.2277 [M + H]⁺ (calcd for C₂₅H₃₃O₆, 429.2272).

Rhodotomentodimer L (6): Colorless gum; [α] + 83.7 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (4.10), 217 (4.10), 295 (3.98) nm; ECD (MeOH) λ_max (Δε) 209 (‒0.79), 221 (+ 2.42), 237 (‒2.24), 278 (‒4.85), 313 (+ 5.65) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 4; (+)-HRESIMS m/z 443.2435 [M + H]⁺ (calcd for C₂₆H₃₅O₆, 443.2428).

Rhodotomentodimer M (7): Colorless gum; [α] ‒85.4 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 205 (4.25), 218 (4.27), 297 (4.15) nm; ECD (MeOH) λ_max (Δε) 209 (+ 1.22), 222 (–2.88), 238 (+ 2.69), 283 (+ 7.78), 316 (–9.79) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 4; (+)-HRESIMS m/z 443.2438 [M + H]⁺ (calcd for C₂₆H₃₅O₆, 443.2428).

Rhodotomentodimer N (8): Colorless gum; [α] ‒60.2 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 202 (4.02), 225 (3.99), 263 (3.73), 301 (3.88) nm; ECD (MeOH) λ_max (Δε) 207 (+ 0.20), 222 (‒2.94), 262 (+ 11.13), 297 (‒4.81) nm; ¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR data, see Table 4; (+)-HRESIMS m/z 443.2431 [M + H]⁺ (calcd for C₂₆H₃₅O₆, 443.2428).

4.4 ECD and NMR calculations

ECD and NMR computations were carried out based on previously reported methods using the Gaussian 16 software package [14]. Three-dimensional molecular structures were constructed based on ROESY spectra (for compound 1) or randomly generated (for compounds 2–8), followed by conformational analysis using CONFLEX with the MMFF94S force field. ECD and NMR chemical shift calculations were conducted at the B3LYP/6–311++ G(2d, p) and mPW1PW91/6–31+ G(d, p) levels, respectively. The DP4+ probability analyses were accomplished utilizing a published computational template [31].

4.5 Human AChE inhibitory assay

hAChE inhibition assays were performed according to a reported protocol, with galantamine as the positive control [32]. All assays were performed in triplicate. Compounds with IC₅₀ values exceeding 40 μM were considered inactive.

4.6 Anti-VRE assay

As described in earlier studies, the antibacterial activities against vancomycin-resistant Enterococci (VRE) were achieved by a standard broth microdilution method in 96-well plates [33]. Ampicillin (AMP) was a positive control, and all tests were performed three times.

4.7 Molecular docking

Molecular docking simulations were performed utilizing the AutoDock software suit [34], following established protocols. The docking complexes of phloroglucinol 13 with hAChE (PDB ID: 4EY6) were generated, and the resulting binding poses were visualized utilizing the PyMOL Molecular Graphics System.

Notes

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (32270425 and 31970377), the Yunnan Fundamental Research Projects (202501AS070093, 202201AT070181, and 202401AT070191), the Top Young Talent of Ten Thousand Talents Program of Yunnan Province (YNWR-QNBJ-2020-253), the Reserve Talents of Young and Middle-Aged Academic and Technical Leaders of Yunnan Province (202105AC160023), and CAS Light of West China Program. We are grateful to Analysis and Testing Centre (Kunming Institute of Botany, Chinese Academy of Sciences) for NMR, HRESIMS, CD, and bioactive measurement.

Author contributions

Ling-Yun Chen carried out the experiments and prepared the original draft; Mu-Yuan Yu participated in the main experiments and revised the manuscript; E-E Luo and Wen-Ying Zong performed the ECD calculations; Shu-Mei Lei and Yu Pan performed the NMR calculations; Ai-Chun Lu and Cheng-Qin Liang revised the manuscript; Xu-Jie Qin conceived and supervised the study, revised the manuscript, and secured project funding. All authors reviewed and approved the final version of the manuscript.

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.