Novel triterpenoids from Ganoderma resinaceum attenuate UV-induced photoaging via modulating Nrf2 and MAPK signaling pathways

1 Introduction

Ganoderma species, especial Ganoderma lucidum, G. sinense, and G. resinaceum have gained significant attentions for its bioactive compounds, including polysaccharides, triterpenoids, and phenolic acids [1–3]. These compounds exhibit diverse bioactivities, such as immunomodulation [4], anti-inflammatory effects [5], and ROS scavenging [6], making Ganoderma a candidate for functional food and medicine development. Besides, Ganoderma species has been used in cosmetics industry due to its safety and health benefits. Evidence from several studies demonstrates that Ganoderma extracts (GE) and polysaccharides (GP) confer antioxidant protection via the activation of antioxidant enzymes and the reduction of UVB-induced reactive oxygen species (ROS) [7]. Additionally, GP acts through the IL-6/STAT3 pathway to diminish melanogenesis in melanocytes, counteracting paracrine influences exerted by keratinocytes and fibroblasts [8]. Ganoderol A protects against UVA damage and exhibits anti-inflammatory activity [9]. The potential for Ganoderma triterpenoids (GTs), major active compounds in Ganoderma species, to confer anti-skin-aging benefits and the underlying mechanisms remain to be established.

The skin provides a visible model of the systemic aging process, reflecting contributions from both innate biological programs (intrinsic aging) and environmental exposures (extrinsic aging) [10–12]. A central mechanism of extrinsic aging, particularly photoaging, involves oxidative stress. Ultraviolet (UV) radiation triggers a damaging cycle by inducing overproduction of reactive oxygen species (ROS) in skin cells [11, 13]. This state of oxidative stress not only directly harms cellular components but also activates specific signaling pathways. It promotes the overexpression of matrix metalloproteinases (MMPs)—enzymes responsible for the degradation of the extracellular matrix (e.g., collagen, elastin)—largely through the activation of the MAPK pathways (ERK, JNK, p38) [14–16]. The downstream outcome of this signaling is increased activity of the transcription factor AP-1, leading to collagen breakdown and inflammation. Given this mechanism, targeting oxidative stress with antioxidants is a rational and crucial strategy for mitigating skin photoaging [17, 18].

A promising target for the prevention and treatment of oxidative stress-related chronic diseases is the Keap1-Nrf2 signaling pathway [19–21]. This pathway maintains cellular homeostasis by upregulating key protective proteins [22]. The transcription factor Nrf2 activates antioxidant response elements (ARE) in promoters, driving expression of detoxification and antioxidant genes. Keap1 directly regulates Nrf2 by sensing oxidative/chemical stress [22, 23]. As a result, enhancing Nrf2 activity is considered a potential strategy for preventing and treating various chronic diseases [20, 24, 25], including diabetes, cancer, and neurodegenerative disorders, which are linked to oxidative stress.

To investigate the anti-photoaging properties of GTs, we conducted a systematic study on the GTs from the fruiting bodies of Ganoderma resinaceum, a medicinal and edible mushroom [26, 27]. A total of 43 lanostane-type triterpenoids (1‒43) were isolated (Fig. 1), exhibiting various degrees of degradation, among which sixteen compounds (1‒11, 15, 31, 35, 37, and 42) were novel. We assessed their anti-photoaging effects and mechanisms of action using a UV-induced HaCaT cell model. Our findings suggest that ganoresinic acid B (42) may serve as a promising candidate for anti-photoaging treatment, as it demonstrated significant antioxidative capabilities by activating Nrf2 nuclear translocation, and protection against collagen degradation through the suppression of the MAPK signaling pathway.

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2 Results and discussion

2.1 Structural elucidation

The phytochemical investigation of the fruiting bodies of G. resinaceum was performed, leading to the isolation of 43 triterpenoids including 16 new compounds (1‒11, 15, 31, 35, 37, and 42).

Ganoresinol A (1) exhibited a molecular ion cluster at m/z 516.2364 [M + Cl]^- in negative HRESIMS, consistent with the molecular formula C₂₉H₃₈O₆ (calcd. for C₂₉H₃₈O₆Cl, 516.2362; Δ = 0.2 ppm). This formula implies 11 degrees of unsaturation. Key ¹H NMR features included five methyl singlets (δ_H 0.76–1.62), a triplet methyl (δ_H 0.89, J = 2.3 Hz), a dd oxymethine proton (δ_H 3.20, J = 11.9, 5.3 Hz), and a singlet olefinic/aromatic methine (δ_H 6.06). Analysis of the ¹³C-DEPT NMR data for 1 confirmed 29 distinct carbons: six methyl groups, seven methylene groups, four methines (including an oxymethine and an olefinic/aromatic methine), and twelve quaternary carbons—comprising four carbonyls, three sp² quaternary, one oxygenated, and four aliphatic quaternary carbons. Collectively, the NMR data support a 3-hydroxy-7,11,15-trioxo-lanosta-8-en skeleton, mirroring the structure of 21 [28]. The assignment was confirmed by HMBC correlations, including H₃-19 to C-9 (δ_C 153.6) and H₃-30 to C-15 (δ_C 208.8), while COSY spin systems (H₂-1/H₂-2/H-3) established proton-proton linkages (Fig. 2).

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The core framework explained eight degrees of unsaturation, with the ketone and double bond contributing two more, necessitating an additional ring in 1. This ring was identified as cyclopentenone through HMBC correlations (Fig. 2): from H-17 (δ_H 3.51, t, J = 8.9 Hz) to C-20 (δ_C 179.3), C-21 (δ_C 47.3), and C-22 (δ_C 129.3), and from H₂-21 (δ_H 2.58, m; 2.80, m) and H-22 (δ_H 6.06, s) to C-17 (δ_C 45.0), C-20 (δ_C 179.3), C-23 (δ_C 211.5), and C-24 (δ_C 79.2). Additional support for the ethyl linkage at C-24 came from HMBC interactions: H₃-26 (δ_H 0.89, t, J = 2.5 Hz) with C-24 and H₂-25 with C-21/C-22. These observations finalized the planar structure assignment.

Stereochemical features were resolved through complementary techniques: the β-orientation of 3-OH followed from the H-3/H-5 ROESY correlation (Fig. 3), while X-ray diffraction (P1211, Flack parameter 0.02; CCDC: 2,259,021, Fig. 4) defined C-24 as R-configuration. Ganoresinol A (1) thus emerges as 3β,24R-dihydroxy-27-nor-21,24-cyclolanosta-8,20(22)-dien-7,11,15,23-tetraone.

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Ganoresic acid A (2) exhibited an [M + Cl]^- peak at m/z 481.2364 (calcd. 481.2362) by HRESIMS, corresponding to C₂₆H₃₆O₆. Key ¹H NMR signals (Table 1) included five methyl singlets (δ_H 0.85, 0.85, 1.01, 1.29, 1.55), one methyl doublet (1.05, d, J = 6.3 Hz), and an oxymethine (3.20, dd, J = 11.6, 4.8 Hz). ¹³C/DEPT data confirmed 26 carbons: 6 × CH₃, 6 × CH₂, 4 × CH (including δ_C 78.1 oxymethine), and 10 × C (3 × ketone, 1 × carboxyl, 2 × sp², 4 × aliphatic quaternary). These features characterize 2 as a tetranorlanostane triterpenoid.

HMBC data confirmed the tetracyclic 3-hydroxy-7,11,15-trioxo scaffold in 2 (Fig. 2). Key observations included correlations from multiple protons to the carbonyl and oxygen-bearing carbons. The degraded side chain (C-24–C-27) was established by HMBC (H₃-21 → C-17/C-20/C-22) and the COSY correlations H₂-16/H-17/H-20/H₂-22. The ROESY correlation (H-3/H₃-28/H-5) defined the 3β-OH orientation. The structure of 2 is therefore 3β-hydroxy-24,25,26,27-tetranorlanosta-8-en-7,11,15-trioxo-23-oic acid.

HRESIMS analysis of ganoresinol B (6) showed a deprotonated molecular ion [M–H]^- at m/z 417.2646 (calculated 417.2649), corresponding to the molecular formula C₂₅H₃₈O₅. Key 1D NMR data (Table 2) of 6 displayed 6 × CH₃, 6 × CH₂ (one oxygenated), 5 × CH (two oxygenated), and 8 × C (two ketones and two sp² C). This pentanorlanostane triterpenoid framework resembles ganosineniol A [29], differing solely through carbonyl substitution at C-15 (replacing the oxygenated methine). The further HMBC correlations (Fig. 2) from H-17 and H₃-30 to C-15 supported this modification. Additionally, ROESY cross-peaks (H-3/H-5/H-7/H₃-30) (Fig. 3) confirmed the β-orientation of both the 3-OH and 7-OH groups. Compound 6 is therefore assigned as 3β,7β,22-trihydroxy-23,24,25,26,27-pentanorlanosta-8,9-en-11,15-dione.

Compound 9 shared a core structure with ganolucidoid B (32) [5] but lacked its C-12 acetyl group. This conclusion was underpinned by HMBC data (Fig. 2): the correlation from H-12 to C-9, among others, confirmed the substitution at C-12, while the H₃-18 to C-12 interaction was also consistent. The β-orientations of the 3-OH and 12-OH groups were concurrently verified by ROESY correlations (H-3/H-5 and H-12/H₃-30) (Fig. 3). Accordingly, the structure of 9 was defined as 3β,12β-dihydroxy-22,23,24,25,26,27-hexanorlanosta-8(9)-en-7,11,15,20-tetraone, for which we propose the name ganoresinone A.

HRESIMS analysis of 15 showed an [M + Na]⁺ ion at m/z 421.1988 (calcd. for C₂₄H₃₀O₅Na: 421.1985), confirming the molecular formula C₂₄H₃₀O₅, identical to compound 17 [30]. Comparative ¹H/¹³C NMR analysis revealed distinct chemical shifts at C-7 (δ_C 69.0 for 17; δ_C 67.8 for 15), suggesting differential stereochemistry. The ROESY spectrum of 15 showed that correlations of H-3/H-5, and of H-7/H-15/H₃-18 illustrated that 3-OH was β-oriented, whereas both 7-OH and 15-OH were α. Complementary X-ray crystallography (P1211, Flack parameter = 0.02, CCDC: 2,259,022, Fig. 4) unequivocally confirmed both planar structure and stereochemical assignments. Thus, 15 is characterized as 3β,7α,15α-trihydroxy-22,23,24,25,26,27-hexanorlanosta-8(9)-en-11,20-dione and assigned the trivial name ganoresinone D.

RESIMS data for ganoresinic acid A (31) displayed a sodium adduct peak [M + Na]⁺ at m/z 533.2517 (calculated 533.2510 for C₃₀H₃₈O₇Na), confirming its molecular formula. The ¹H and ¹³C NMR data of 31 were consistent with a C₃₀ lanostane-type triterpenoid closely related to ganoderic acid E [31] but featuring a Δ¹⁶⁽¹⁷⁾ double bond instead. Key HMBC correlations from H-16 and H₃-18 established the position of this double bond. The R configuration at C-25 was deduced from the identical chemical shifts of C-24–C-27 to those in resinacein T [26]. These data collectively determine the structure of 31 to be 3,7,11,15,23-pentaoxo-lanosta-8(9),16(17)-dien-26-oic acid, designated ganoresinic acid A.

Compound 37 was identified as methyl 3,7,11,15,23-pentaoxo-lanosta-8(9),20(22)-dien-26-oate, a methyl ester derivative of methyl ganoderate E (33) [5]. This assignment was based on NMR data that indicated the sole difference was an added methoxy group, which was shown by HMBC to be ester-linked to C-26. The E-configuration of the Δ^20,22 double bond was confirmed by ROESY data, leading to the name methyl ganoderenate F.

HRESIMS analysis established the molecular formula of ganoresinic acid B (42) as C₃₀H₄₂O₇ ([M + Na]⁺ at m/z 539.2972, calcd. 539.2979). Its 1D NMR data (Table 3) closely matched those of 41 and 36 [32], sharing the 7,8-epoxy-9(11)-en-12-one core but differing by a C-20/C-22 double bond. The side-chain structure was defined by key HMBC and COSY correlations (Fig. 2). ROESY data (Fig. 3) established the β-orientations of the 3-OH and 7,8-epoxy groups, while the R configuration at C-25 was confirmed by comparison with resinacein T [26]. The structure was thus determined to be 3β-hydroxy-7β,8β-epoxy-12,15,23-trioxo-lanosta-9(11)-en-27-oic acid.

Details of the structural characterization for the other new compounds can be found in the supplementary file. According to the number of carbons, these isolates can be divided into five subtypes, including C29 (Type Ⅰ: compound 1), C26 (Type Ⅱ: compounds 2‒5), C25 (Type Ⅲ: compounds 6‒8), C24 (Type Ⅳ: compounds 9‒20), and C30 (Type Ⅵ: compounds 21‒43). Compound 1 was the first example of the C29 lanostane-type nortriterpenoid with a rare 21,24-cyclo-five-membered carbon fraction. Ganoderma triterpenoids are belonging to lanostane-type triterpenoids, which were biosynthesized by isoprenoid pathways, starting from acetyl-coenzyme A [33]. Initially, the C30 skeleton was constructed, and subsequently, various nortriterpenoids can be generated through oxidative cleavage of the side chain (Scheme S1) [33].

2.2 Pharmacology

2.2.1 Selection of irradiation dose in HaCaT cells

Prior to assessing anti-photoaging compounds from G. resinaceum in HaCaT cells, we determined the appropriate UVA (365 nm) and UVB (312 nm) irradiation doses for model development. Viability assessment (Fig. 5) indicated that doses below 350/3594.0 mJ/cm² maintained viability > 80%. Therefore, a dose of 250/2567.5 mJ/cm² was utilized to create the photoaging model.

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2.2.2 Ganoderma triterpenoids prevent UV-induced oxidative stress in HaCaT cells

Given the limited content of certain compounds, the anti-photoaging potential of compounds 2–14, 16–25, 27–31, 34–37, and 41–43 was investigated. First, the cytotoxicity of these compounds on HaCaT cells with or without UV irradiation was tested, and none exhibited toxicity at concentrations below 25 μM (Fig. S117). As a result, a concentration of 25 μM was chosen for the follow-up experiments.

In skin cells, exposure to UV radiation triggers the generation of reactive oxygen species (ROS), resulting in DNA damage and the formation of senescent cells due to oxidative stress [10]. Malondialdehyde (MDA), a key byproduct of lipid peroxidation, is primarily formed through the degradation of polyunsaturated fatty acids in cell membranes when attacked by ROS and free radicals. The measurement of MDA levels is widely utilized as an indicator of oxidative stress intensity. As a critical component of the primary antioxidant defense mechanism, superoxide dismutase (SOD) was important in preserving the cellular redox balance [10]. Consequently, in our research, we assessed the impact of isolates on ROS levels, MDA production, and SOD activity. Our results (Fig. 6) showed the elevation of the intracellular ROS content in UV-irradiated HaCaT cells, whereas the significant decrease after pretreatment with compounds 2‒4, 13, 17, 35, 36, and 42 at the concentration of 25 μM. Meanwhile, these active compounds also significantly suppressed the production of MDA. The activity of SOD was dramatically reduced by UV irradiation, but compounds 2‒4, 13, 17, 35, and 42 significantly reversed this tendency.

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As we know, UV irradiation causes a reduction in collagen levels within the skin. Hydroxyproline (HYP), the most abundant amino acid in collagen, serves as an indicator of total collagen content [34]. Therefore, we also assessed the effects of various compounds on HYP levels. Our results showed that the HYP content within HaCaT cells declined following UV exposure. However, the HYP levels increased in the groups treated with compounds 4, 13, 35, 36, and 42 (Fig. 6). Based on the comprehensive analysis of the above indicators, Compound 42 is considered the most significantly active compound. Therefore, further activity studies were conducted on compound 42.

2.2.3 Protective effect of compound 42 against UV-induced oxidative stress in HaCaT cells

The cytotoxic effects of compound 42 on both normal and UV-induced HaCaT cells were examined. The results revealed that compound 42 exhibited no cytotoxic effect on HaCaT cells with or without UV irradiation, even at a high concentration of 100 μM. Compound 42 dose-dependently reduced ROS and MDA levels. At 40 μM, its inhibition matched the positive control resveratrol (10 μM) (Fig. 6).

2.2.4 Compound 42 protects against UV-induced photoaging by inhibiting MAPK signaling

Under UV exposure, reactive oxygen species (ROS) accumulate, prompting the activation of key MAPK signaling proteins—ERK, p38, and JNK. This cascade further stimulates AP-1, leading to the upregulation of matrix metalloproteinases (MMPs). The increased MMP activity, in turn, accelerates the degradation of collagen and other extracellular matrix (ECM) proteins—a key process in skin aging [16]. As shown in Fig. 7, UV-treated cells exhibited markedly higher levels of phosphorylated ERK, JNK, and p38 compared to untreated controls. Notably, compound 42 at 40 μM demonstrated the strongest inhibitory effect, substantially reducing the phosphorylation of all three MAPK proteins. Furthermore, compound 42 suppressed the mRNA expression of MMP-1 and MMP-3 in a concentration-dependent manner. By attenuating MAPK activation, compound 42 led to reduced MMP expression and consequently inhibited ECM degradation, suggesting a potential mechanism for its mitigation of UV-induced photoaging.

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2.2.5 Compound 42 protects against UV-induced oxidative stress via activating NRF2

Nrf2 functions as a central regulator within the KEAP1/NRF2 signaling pathway. Under basal conditions, KEAP1 constitutively suppresses NRF2 activity by facilitating its ubiquitination and subsequent proteasomal degradation. Electrophilic stimuli, however, interfere with KEAP1’s inhibitory control over NRF2, enabling its stabilization and translocation to the nucleus. In the nucleus, NRF2 functions as a transcription factor to activate cytoprotective antioxidant genes such as superoxide dismutase (SOD) [23]. Thus, NRF2 has been considered as an important pharmacological target. To investigate the effect of compound 42 on NRF2 activation, we assessed its subcellular localization in HaCaT cells by Western blotting. Treatment with 40 μM compound 42 increased NRF2 levels in the nucleus while decreasing them in the cytoplasm compared to the model group (Fig. 7), indicating its translocation. Together with the observed suppression of MDA and activation of SOD, these results suggest that compound 42 mitigates oxidative stress by promoting NRF2 nuclear translocation, thereby activating antioxidant defense systems to neutralize ROS.

3 Conclusion

In summary, this study reports the isolation of forty-three lanostane-type triterpenoids with varying degrees of degradation, which included C30, C29, C26, C25, and C24 triterpenoids from the fruiting bodies of G. resinaceum. Notably, sixteen of these triterpenoids were identified as new compounds, significantly expanding the structural diversity of Ganoderma triterpenoids. Additionally, in line with the known anti-aging effects of Ganoderma species, we assessed the anti-photoaging activities of compounds 2‒14, 16‒25, 27‒31, 34‒37, and 41‒43. Among them, compound 42 significantly alleviated UV-induced oxidative stress in HaCaT cells by reducing ROS and MDA levels while enhancing SOD activity and hydroxyproline content. Mechanistic studies revealed that these protective effects are mediated through the promotion of Nrf2 nuclear translocation and the concurrent suppression of the MAPK signaling pathway, as evidenced by reduced phosphorylation of ERK, JNK, and p38. Consequently, the downstream mRNA expression of collagen-degrading enzymes MMP-1 and MMP-3 was downregulated. Collectively, our findings indicate that compound 42 exerts anti-photoaging effects by activating the Nrf2-antioxidant axis and inhibiting the MAPK-MMPs pathway. This work not only expands the structural diversity of Ganoderma triterpenoids (GTs) but also provides scientific evidence for the anti-aging potential of Ganoderma species.

4 Materials and methods

4.1 General information

Refer to the Supplementary materials for details regarding the instruments and reagents employed in isolation and structural characterization.

4.2 Fungi material

The fruiting bodies of Ganoderma resinaceum (10 kg) were collected from Kunming’s Luoshiwan Traditional Chinese Medicine Market (June 2016). Authentication was conducted by Prof. Yang Zhuliang (Kunming Institute of Botany, CAS), with a voucher specimen (KGR-201606) archived for reference.

4.3 Extraction and isolation

10 kg of powdered, air-dried G. resinaceum fruiting bodies were exhaustively extracted via reflux with methanol (3 × 50 L). The resultant extract was aqueous-suspended and subjected to sequential solvent extraction with petroleum ether followed by ethyl acetate. The ethyl acetate-soluble material (300 g) was purified by D101 macroporous resin chromatography employing a stepped methanol/water gradient (0% → 20% → 50% → 70% → 90% MeOH). The 50% MeOH fraction (64.2 g) was further separated by silica gel column chromatography using dichloromethane/methanol step gradients (50:1 to 5:1, v/v) to afford three subfractions (Fr.1–Fr.3). Details are shown in Supplementary materials.

4.4 X-ray crystallography data

X-ray data of compounds 1 and 15 are shown in Supplementary materials.

4.5 Cell culture and treatment

HaCaT keratinocytes (Kunming Zoo Institute, Chinese Academy of Sciences) were maintained in DMEM (Servicebio) containing 10% FBS (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 ℃ under 5% CO₂. For UV exposure, cells at ~ 80% confluency were pretreated with compounds for 24 h, followed by irradiation using a solar simulator (UVA: 250 mJ/cm²; UVB: 25.67 J/cm²). After the radiation, the cells were subsequently replaced with a fresh DMEM and incubated for 24 h before the cell viability analysis.

4.6 Cytotoxic assay

We evaluated cytotoxicity using the MTT assay (Beyotime Institute of Biotechnology, C0009M) with modifications based on earlier descriptions [35]. Detailed experimental protocol is attached in the Supplementary materials.

4.7 Quantitative reverse transcription-PCR (qRT-PCR)

HaCaT cells (1 × 10⁶ cells/mL) were plated in 6-well plates and pretreated with compound 42 (10, 20, or 40 μM) for 24 h. Cells were then UV-irradiated and incubated for an additional 24 h. Total RNA isolation was performed with Biosharp RNA Extraction Reagent (BLI665A), followed by cDNA synthesis, and then quantitative PCR was carried out. Gene-specific primers of MMP-1, MMP-3, and β-actin (sequences in Table S3) were amplificated and detected. β-actin was normalized for gene expression.

4.8 Western blots

Protein samples for Western blotting were prepared following established protocols from prior work [36].

4.9 Determination of ROS

ROS generation was assessed with a standardized assay (Catalog E004-1-1, NanJing JianCheng Bioengineering Institute, China). The procedure adhered to the manufacturer’s protocol but included minor adaptations from prior methodologies [37]. Details are shown in the Supplementary materials.

4.9.1 MDA, T-SOD, hydroxyproline generation assay

Following treatment, cell supernatants were collected and transferred to fresh microcentrifuge tubes for analysis. Total Superoxide Dismutase (T-SOD) activity, Malondialdehyde (MDA) levels, and hydroxyproline content were quantified using a commercial assay kit and quantified strictly referring to the manufacturer's instructions and supplemented by established methodology [15, 37, 38].

4.9.2 Statistical analysis

GraphPad Prism (v.10.1.2) was used for statistical analyses. Data are shown as mean ± SD (n = 3). Significance was tested with one-way ANOVA and Tukey’s post hoc test (P < 0.05).

Notes

Acknowledgements

This study was financially supported by the Basic Research Program of Yunnan Province (202301AT070332), the Yunnan Revitalization Talent Support Program “Young Talent” Project (XDYC‑QNRC‑2022‑0480).

Author contributions

Yi Luo and Xiaocui Liu: Writing—original draft, Methodology, Formal analysis, Data curation, Conceptualization. Yujie Li: Methodology, Data curation. Minghua Qiu: Writing—review. Xingrong Peng: Project administration, Funding acquisition, Writing—review and editing.

Data availability

All data is confidential.

Declarations

Competing interest

There is no competing interests.