Anti-oxidative effect of Salvia miltiorrhiza Bunge fermented with Shiraia bambusicola Henn. against H₂O₂-induced injury in PC12 cells

Abstract

Purpose

The objective of this investigation was to produce fermented solid-state products of Salvia miltiorrhiza Bunge (SMB) utilizing Shiraia bambusicola Henn. (FSSMB). Additionally, it was intended to further explore toxicity-reducing potential and antioxidant effects of FSSMB ethanol extract against H₂O₂-induced oxidative stress in rat pheochromocytoma cell line 12 (PC12).

Methods

PC12 cells were pre-treated with SMB or FSSMB ethanol extract for 24 h, followed by exposure to 1 mmol/L H₂O₂ for 3 h to induce oxidative stress. Subsequently, the cell viability, reactive oxygen species (ROS) level, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, p-p38/p38 and p-JNK/JNK proteins were assessed. Additionally, the content of antioxidant, including tanshinones (tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone) and salvianolic acids (danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B) were quantified by HPLC in SMB and FSSMB.

Results

Here, we found that FSSMB exhibited lower cytotoxicity compared to SMB in PC12 cells, and FSSMB instead of SMB significantly increased cell viability in H₂O₂-induced PC12 cells. Notably, FSSMB exhibited superior antioxidant properties in H₂O₂-induced PC12 cells, as evidenced by reduced levels of ROS, MDA, and enhanced SOD activity compared to SMB. Mechanistically, FSSMB reversed H₂O₂-induced increase of phosphorylation of p38 and JNK protein in mitogen-activated protein kinase (MAPK) signaling pathway, thereby protecting PC12 cells from oxidative stress-induced injury. Furthermore, we found a significant increase of tanshinone IIA and cryptotanshinone content, accompanied by decreased levels of tanshinone I, dihydrotanshinone and salvianolic acids in FSSMB compared to SMB.

Conclusions

Our study explores a new biological transformation approach through solid-state fermentation (SSF) with Shiraia bambusicola Henn. on SMB. This study demonstrated that the SSF may reduce the cytotoxicity and enhance the antioxidant capacity of SMB in PC12 cells, thus paving the way for safer and more efficacious applications in the future. FSSMB may be an effective substitute for traditional Chinese medicine SMB for resistance to oxidative stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-04956-1.

Introduction

Salvia miltiorrhiza Bunge (SMB), belonging to the family Lamiaceae and commonly known as danshen, is a perennial herb characterized by erect stems, pinnately compound leaves, and purple or blue flowers. Its primary medicinal part is the reddish-brown and branched roots, which are native to North-Central, South-Central and Southeast China as well as Vietnam, and has been introduced into Korea [1]. SMB possesses a mild aroma and a slightly bitter flavor, and its dried roots and rhizomes are widely utilized in traditional Chinese medicines (TCM) [2]. So far, numerous chemical compounds have been extracted from SMB, including polysaccharides, alkaloids, phenolics and diterpene [3]. Its principal bioactive constituents are lipophilic diterpene quinones, also named tanshinones (dihydrotanshinone, cryptotanshinone, tanshinone I, and tanshinone IIA et al.) [4] and hydrophilic polyphenolics, also named salvianolic acids (danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B et al.) [5]. SMB exhibit significant pharmacological activities, notably antioxidant effects, due to the presence of tanshinones and salvianolic acids [6–9]. Specifically, tanshinone IIA has been shown to enhance antioxidant enzyme activity and reduce oxidative stress markers like reactive oxygen species (ROS) and malondialdehyde (MDA) [10]. Nevertheless, recent studies focusing on the safe dosage of SMB have revealed that high-dose SMB injection can lead to toxicity. In particular, administration of high doses (5.76 g/kg/day, the maximum clinical dosage) of SMB injection triggered increased peripheral vascular toxicities in mice, and long-term tests on beagle dogs indicated that dosages exceeding 320 mg/kg were toxic [11, 12]. Furthermore, recent clinical investigations have also further detected minor adverse drug events associated with SMB injection, including stomach discomfort, itching, and local pain [13]. Therefore, developing novel approaches to reduce SMB toxicity without compromising efficacy are necessary.

Solid-state fermentation (SSF) technology plays a crucial role in processing agricultural products and their by-products, involving microbial fermentation on solid substrates to provide optimal growth conditions for microbe [14]. Initially applied in brewing, SSF gradually extended to TCM and food to yield medicinal or food fermentation products, which is important for the overall development of agriculture [15]. SSF has been shown to effectively reduce substrate toxicity through the degradation of toxic components. A successful SSF system employed Ganoderma lucidum (Curtis) P. Karst. to ferment Tripterygium wilfordii Hook.f. Levels of toxic compounds, including dibutyl-phthalate and diisobutyl-phthalate, were significantly reduced after SSF [16]. However, the potential of SSF to reduce SMB toxicity remains unexplored. Some studies have demonstrated that SSF significantly enhances the content of bioactive compounds in SMB, leading to improved antioxidant capacity and other biological activities. Fermentation of SMB roots with Aspergillus oryzae significantly enhanced their antioxidant and antibacterial activities by increasing total phenolic and flavonoid contents, primarily through the conversion of bioactive components into more polar compounds via acylation, dealkylation, and esterification processes [17]. Similarly, fermentation of SMB with Geomyces luteus markedly improved antioxidant activity and total phenolic content, particularly by elevating the concentration of salvianolic acids, as confirmed by spectroscopic and HPLC analyses [18]. Therefore, this study examined SSF-mediated enhancement of antioxidant capacity in SMB and its phytochemical basis. SSF has been widely applied to enhance bioactive compounds and pharmacological properties in various TCM and functional foods beyond SMB, as evidenced by numerous studies. The combined application of SSF with Bacillus subtilis and pulsed light significantly enhanced the flavonoid and phenolic content, the antioxidant levels and sensory evaluation scores in Ginkgo biloba dark tea [19]. The independent application of SSF technology also has many advantages. SSF can alter the microstructure of oats, releasing the bioactive compounds, thereby enhancing the nutritional value and the bioactive properties of oats, including antioxidant, antidiabetic, anti-cancer, and other properties [20]. Similarly, through SSF with autochthonous microorganisms, chickpeas gained a higher proportion of small peptides and functional bioactive peptides, witnessed an increase in both total phenolic and flavonoid content, and ultimately enhanced the nutritional quality of chickpeas [21]. One study demonstrated that SSF with Ganoderma lucidum (Curtis) P. Karst. increasing the content of triterpenoids and flavonoids, and activities of antioxidant and hypoglycemic in Tartary buckwheat by increasing the activity of glycosidases and cellulases to liberate the bioactive compounds [22]. The alteration of bioactive compounds and biological activities in substrates by SSF is closely related to the types of microbial strains. Shiraia bambusicola Henn., belonging to the family Shiraiaceae, is a parasitic fungus growing on bamboo species, manifested by its dark stromatic fruiting bodies and the production of bioactive secondary metabolites hypocrellins, predominantly found in the subtropical regions of Asia, especially on tender bamboo branches [23]. SSF using Shiraia bambusicola Henn. is commonly employed to enhance hypocrellins production [23, 24]. However, the antioxidant activity and toxicity of Shiraia bambusicola Henn.-derived SSF products remain poorly characterized.

Oxidative stress is implicated in a diverse range of various neuropsychiatric disorders, including Alzheimer's disease, Parkinson's syndrome, and depression [25, 26]. Elevated levels of ROS during oxidative stress could induce cellular damage to lipids, proteins, and DNA, disrupting neural growth and exacerbating pathophysiological processes [27]. Decreased H₂O₂-induced oxidative stress and ROS level enhances the viability of the rat pheochromocytoma cell line 12 (PC12) [28]. It has been shown that antioxidants are abundant in a diet, has a positive effect on maintaining the balance of ROS concentration and hold potential as therapeutics for neuropsychiatric disorders [27, 29]. It is widely acknowledged that extracts of Chinese herbal medicines serve as abundant sources of natural antioxidants and neuroprotective compounds [30]. Therefore, it is crucial to investigate the changes in the content of antioxidant substances before and after SSF of SMB.

In this study, we aim to clarify the effects of SSF on the cytotoxicity and antioxidant capacity of SMB in PC12 cells. Ethanol extractions were obtained from fermented solid-state products of SMB utilizing Shiraia bambusicola Henn. (FSSMB). The effective concentration and neuroprotective effects of the FSSMB in PC12 cells were evaluated and the content of key antioxidant substances (tanshinones and salvianolic acids) in SMB was detected to explore the possibility of FSSMB serving as an effective substitute for SMB, thus opening up possibilities for more secure and effective applications in the future.

Materials and methods

Materials

SMB is supplied by Tianjiang Pharmaceutical Co., Ltd. Shiraia bambusicola Henn. strain was kindly provided from senior experimentalist Guangming Huo of NanJing XiaoZhuang University. Fetal bovine serum (FBS) was purchased from Biochannel (Nanjing, China). DMEM was purchased from Gibco (United States). Dimethyl sulfoxide (DMSO) was purchased from Solarbio (Beijing, China). Tanshinone IIA, dihydrotanshinone, tanshinone I, cryptotanshinone, danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B standards were of chromatographic grade and purchased from Shanghai yuanye Bio-Technology (Shanghai, China). ROS, MDA, SOD detection kits, BCA Protein Assay Kit and BeyoECL Plus were purchased from Beyotime Biotechnology (Shanghai, China) models: S0033S, S0131S, S0109, P0010S, P0018S. Antibodies against p-p38 and p38 were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against p-JNK, JNK, GAPDH and Goat Anti-Rabbit IgG (H + L) HRP were purchased from Proteintech Technology (Wuhan, China). The PC12 cells were kindly provided by professor Jianmei Li of Nanjing Normal University.

Organism, media, and solid-state fermentation conditions

The Shiraia bambusicola Henn. strain was cultivated at 28 °C for 3 days on a potato dextrose agar plate. After picking up with inoculation rings, the mycelium was transferred to a 500 mL conical flask (Shubo Group, Chengdu, China) containing 200 mL potato dextrose broth media. The seed culture was cultivated at 26 °C for 3 days at 150 rpm in a rotary incubator (Zhichu, Shanghai, China). Twenty milliliter seed suspensions are inoculated at matrixes in disposable fermentation bags, each containing 50 g SMB with 50 mL water. After inoculation, fermentation was performed in an incubator (Huitai instrument, Shanghai, China) at 26 °C for 20 days.

Preparation of SMB, FSSMB ethanol extract

SMB and FSSMB were dried at 40 °C in an oven and subsequently smash into powder. An aliquot of 1 g the powder was mixed with 15 mL of 75% ethanol (1:15, v/v) and subjected to sonication at 60 °C with an ultrasonic power of 288 W for 30 min (SB-5200DTD, Xinzhi instrument, Ningbo, China). The mixture was then filtered, and the filtrate was concentrated using rotary evaporation (RE-3000, Yarong instrument, Shanghai, China) at 40 °C. The resultant extract was dissolved in DMSO.

PC12 cell culture

The PC12 cells were cultured in DMEM (10% FBS, 1% penicillin/ streptomycin) under 37℃, 5% CO₂. The cells were seeded in 96-well plates at a density of 3 × 10⁵ cells/mL (100 μL/well); 24-well plates at a density of 2 × 10⁵ cells/mL (500 μL/well) or 6-well plates at a density of 4 × 10⁵ cells/mL (2 mL/well) and grown at 37 °C with 5% CO₂ humidified atmosphere [31]. Experimental treatments were performed when cell density reached 50% confluency.

Cell viability assay

SMB and FSSMB ethanol extract were dissolved in DMSO and the final concentration of DMSO was taken up to less than 0.1% for all treatments. The same volume of DMSO (0.1% v/v) was added to the control groups. PC12 cells were cultured with or without H₂O₂ in the presence of SMB or FSSMB ethanol extract to explore the neuroprotective and antioxidative effect, respectively. At the time of cell harvest, 10 μL of Cell Counting Kit-8 (CCK-8) solution was added to each well in 96-well plate and incubated under 37 °C, 0.5 h. OD value was measured at 450 nm by a microplate reader [32].

ROS measurement

PC12 cells in the logarithmic growth phase were seeded in 96-well plates. After 24 h of adherent growth, modeling and drug administration were carried out in accordance with the experimental groups. As the manufacturer’s instructions (S0033S, Beyotime, China), the ROS fluorescent probe DCFH-DA was used to stain the cells. The fluorescence intensity was detected under a fluorescence microscope (Olympus Corp., Tokyo, Japan) with the excitation wavelength of 488 nm and the emission wavelength of the fluorescence microscope set to 525 nm [31].

MDA and SOD assay

PC12 cells in the logarithmic growth phase were seeded in either 6-well plates or 12-well plates, and after 24 h of adherent growth, modeling and drug administration were carried out according to experimental groups. The cells were washed twice with PBS, then cell lysate was added to lyse the cells and centrifuge it at 12,000 g for 10 min. The supernatant was collected as the sample to be tested. The intracellular MDA content was detected following the manufacturer's instructions (S0131M, Beyotime, China). Meanwhile, the intracellular SOD content was determined according to the manufacturer's instructions (S0109, Beyotime, China).

Western blot

PC12 cells in the logarithmic growth phase were seeded in 6-well plates, and after 24 h of adherent growth, modeling and drug administration were performed according to experimental groups. The total protein of the cells is lysed by cell lysis buffer (P0013, Beyotime, China) and the protein concentration is determined using the BCA Protein Assay Kit (PO010S, Beyotime, China). Then electrophoresis in 10% SDS-PAGE (E303, Vazyme, China), transfer at 120 V, and incubate with 5% BSA (SW3015, Solarbio, China) for 1 h at room temperature for sealing. Incubate the Polyvinylidene Fluoride (PVDF) membrane (ISEQ00010, MerckMillipore, USA) with the primary antibody overnight at 4 °C, wash 3 times with TBST (T1082, Solarbio, China), and incubate with the secondary antibody for 1 h at room temperature. Wash 3 times with TBST, detect the protein bands with the ECL kit (180–501, Tanon, China), and assess the protein intensity and quantify the proteins with GAPDH as an internal reference with ImageJ software [32]. Finally, an antibody stripping buffer (P0025N, Beyotime, China) was used to remove bound antibodies allows us to reuse the membrane for other protein detection.

Determination of tanshinones and salvianolic acids by HPLC

Tanshinones

One gram of SMB and FSSMB powder were placed in 15 mL of methanol solvent (1:15, v/v) and sonicated at 60 °C, 288 W for 30 min. The extract was evaporated under vacuum at 40 °C and the residue is re-dissolved in 1 mL of methanol. The solution filtered through 0.22 μm disposable oil filter analyzed by HPLC on an Agilent 1260 device fitted with a Waters reversed-phase C18 symmetry column (XDB-C18, 5 μm, 4.6 × 250 mm, Agilent). Acetonitrile and 0.02% phosphoric acid worked as the mobile phase, flowing at a flow rate of 1 mL/min with the detection wavelength set at 270 nm [33]. Four components, dihydrotanshinone, cryptotanshinone, tanshinone I, and tanshinone IIA, were detected and quantified by comparison with authentic standards. The sum of dihydrotanshinon, cryptotanshinon, tanshinone I, and tanshinone IIA was calculated as the total tanshinone content.

Salvianolic acids

One gram of SMB and FSSMB powder were placed in 20 mL of methanol solvent (1:20, v/v) and sonicated at 80 °C 288 W for 30 min. The extract was evaporated under vacuum at 40 °C and the residue is re-dissolved in 1 mL of methanol. The solution filtered through 0.22 μm disposable oil filter analyzed by HPLC on an Agilent 1260 device fitted with a Waters reversed-phase C18 symmetry column. Acetonitrile and 0.1% phosphoric acid worked (22:78, v/v) as the mobile phase, flowing at a flow rate of 1 mL/min with the detection wavelength set at 289 nm [34]. Four components, danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B were detected and quantified by comparison with authentic standards. The sum of danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B was calculated as the total salvianolic content.

Statistics

Data were shown as mean ± SEM. F-test is used for homogeneity of variance between two groups of samples. Two-tailed Student’s t test was used for comparisons between two groups, and data were analyzed statistically using one-way ANOVA for comparisons of three groups with single factor variance. HPLC chromatograms were draw with Origin Pro 2018. Statistical analyses were performed with GraphPad Prism Software (Graph Pad Software, San Diego, USA). Value of P < 0.05 was considered statistically significant.

Results

Neuroprotective effect of FSSMB against H₂O₂-induced injury in PC12 cells

To assess the effect of H₂O₂ on cell viability, PC12 cells were treated with concentration gradient of H₂O₂ (200, 400, 600, 800, 1000 and 1200 µM). The cell viability was reduced accompanied by the increase in H₂O₂ (Fig. 1a). It's worth noting that the cell viability declined to approximately 50% compared with control group when the H₂O₂ concentration reached 1000 µM (Fig. 1a). Thus, the concentration 1000 µM was selected for subsequent experiments to induce the H₂O₂-induced cell injury model. Evaluation of the neurotoxic effects of SMB extracts on PC12 cells revealed a significant decrease in cell viability at SMB concentrations exceeding 160 µg/mL (Fig. 1b). Shiraia bambusicola Henn. does not exhibit a significant effect on cell viability at the concentration range of 20–320 μg/mL (Fig. 1c). Notably, even at a higher concentration of 320 µg/mL, the viability of PC12 cells remained above 100%, indicating the favorable safety profile of FSSMB extract (Fig. 1d). As shown in Fig. 1e and f, compared to the H₂O₂ group, the cell viability has no amelioration after the SMB or Shiraia bambusicola Henn. treatment at the concentrations ranging from 20 to 320 µg/mL. Conversely, compared to the H₂O₂ group, the cell viability increased markedly to 88% and 79% after FSSMB treatment at the concentration of 160 and 320 µg/mL, respectively (Fig. 1g). These findings indicate that FSSMB extract exhibits lower toxicity than SMB and could alleviate H₂O₂-induced cytotoxicity.

Fig. 1.

The effect of FSSMB treatment on cell viability of PC12 cells. a The impact of H₂O₂ treatments on PC12 cells. The cell activity of PC12 cells by SMB (b), Shiraia bambusicola Henn. (c) and FSSMB (d) treatment. Effect of SMB (e), Shiraia bambusicola Henn. (f) and FSSMB (g) on cell viability damaged by H₂O₂ (1000 μM). CTL, control group; H₂O₂, H₂O₂ treated group; SMB, Salvia miltiorrhiza Bunge (ethanol extract); FSSMB, fermented solid-state products of SMB utilizing Shiraia bambusicola Henn. (ethanol extract). Values are represented as mean ± SEM. *Indicated significant difference (*P < 0.05, **P < 0.01, ***P < 0.001) between control and H₂O₂ groups. # represented a significant difference (^#P < 0.05, ^##P < 0.01, ^###P < 0.001) between H₂O₂ and SMB-H₂O₂ groups or FFSMB-H₂O₂ groups

The antioxidant effect of FSSMB on PC12 cells

It is widely acknowledged that oxidative stress represents a crucial factor in neurological disorders [35]. MDA is commonly utilized as an indicator of oxidative stress levels [36], whereas SOD functions to scavenge excess ROS and counteract oxidative stress within the human body [37]. Thus, those markers associated with oxidative stress were used to investigate the antioxidative potential of FSSMB. Relative to the H₂O₂ group, ROS level was decreased prominently in both the SMB and FSSMB groups (Fig. 2a-b). Notably, the ROS level in FSSMB group was lower than that in SMB group (Fig. 2a-b). Furthermore, the elevated MDA level caused by H₂O₂ decreased significantly after SMB and FSSMB treatment (Fig. 2c). Additionally, the reduced SOD activity induced by H₂O₂ was significantly improved after FSSMB treatment, while the SOD activity did not show a significant increase after SMB treatment (Fig. 2d). On the whole, FSSMB mitigated the occurrence of oxidative stress damage.

Fig. 2.

The antioxidative effect of FSSMB treatment on PC12 cells. a Representative images of ROS captured (Scale bars: 50 μm). b Relative fluorescence intensity was determined as a percentage of control using Image J software. The level of (c) MDA and (d) SOD in different treated groups. CTL, control group; H₂O₂, H₂O₂ treated group; SMB, Salvia miltiorrhiza Bunge (ethanol extract); FSSMB, fermented solid-state products of SMB utilizing Shiraia bambusicola Henn. (ethanol extract). *Indicated significant difference (*P < 0.05, **P < 0.01) between control and H₂O₂ groups. # represented a significant difference (^#P < 0.05, ^##P < 0.01) between H₂O₂ and SMB-H₂O₂ groups or FFSMB-H₂O₂ groups. $ represented a significant difference (^$P < 0.05, ^$$P < 0.01) between SMB-H₂O₂ groups and FFSMB-H₂O₂ groups

FSSMB protects PC12 cells from oxidative damage by regulating the MAPK signaling pathway

Accumulating evidence indicates the involvement of the mitogen-activated protein kinase (MAPK) signaling pathway in oxidative stress-induced damage induced by H₂O₂ [38]. Thus, the PC12 cells were collected and the total proteins related with MAPK signaling were extracted. The levels of total p38 and JNK protein remained unchanged in PC12 cells across all groups, whereas the ratios of p-p38/p38 and p-JNK/JNK in H₂O₂ group were 8.0- and 5.6-fold higher than those in the control group, respectively (Fig. 3). Compared with the H₂O₂ group, SMB and FSSMB could strikingly downregulate the p38/p-p38 ratio to 5.8- and 3.7-fold of the control group, respectively (Fig. 3a-c). Consistently, compared with the H₂O₂ group, SMB and FSSMB could exceedingly downregulate the p-JNK/JNK ratio to 3.5- and 1.6-fold that of the control group, respectively (Fig. 3d-f). Collectively, FSSMB significantly diminished the ratios of p-p38/p38 and p-JNK/JNK to a greater extent than SMB. All of these results suggested that FSSMB inhibits the activation of p38/JNK MAPK signaling pathway, thereby protecting PC12 cells from oxidative stress-induced injury.

Fig. 3.

Effects of FSSMB treatment on the activation of p38/JNK signaling in H₂O₂-treated PC12 cells. Representative images of p-p38, p38 (a), p-JNK, and JNK (d) protein expressions in PC12 cells cultured with or without 1 mmol/L H₂O₂ in the presence of SMB (160 μg/mL) or FSSMB (160 μg/mL), respectively. The densities of protein bands were quantitated, and the ratios of p-p38/p38 (b-c) and p-JNK/JNK (e–f) were calculated, as shown in the bottom panels. Relative protein levels of p-p38, p38, p-JNK, and JNK were normalized to GAPDH, respectively. CTL, control group; H₂O₂, H₂O₂ treated group; SMB, Salvia miltiorrhiza Bunge (ethanol extract); FSSMB, fermented solid-state products of SMB utilizing Shiraia bambusicola Henn. (ethanol extract). *P < 0.05, **P < 0.01, ***P < 0.001

Effect of Shiraia bambusicola Henn. fermentation on tanshinones content of FSSMB

In this article, Shiraia bambusicola Henn. served as the original strain, and mycelium suspension was obtained subsequent to the activation of the strain (Fig. S1a). The mycelium suspension was introduced into the fermentation medium of SMB, and the FSSMB were harvested after 20 days (Fig. S1b). Liposoluble tanshinones and water-soluble phenolic compounds represent the two main categories of bioactive constituents in SMB [33]. Tanshinones, a class of phenanthraquinone compounds in SMB, constitute an essential parameter for assessing SMB quality [39]. To investigate the impact of SSF of Shiraia bambusicola Henn. on SMB, the levels of dihydrotanshinone, cryptotanshinone, tanshinone I, and tanshinone IIA in both SMB and FSSMB were determined by HPLC (Fig. 4a-e). The levels of dihydrotanshinone and tanshinone I in FSSMB were marginally lower than those in SMB, whereas cryptotanshinone and tanshinone IIA levels were significantly higher in FSSMB compared to SMB (Fig. 4d). Total tanshinone content in FSSMB exhibited no discernible alteration compared to SMB (Fig. 4e). Generally, the tanshinone content exhibited modest changes following SSF.

Fig. 4.

HPLC analysis of tanshinones production in SMB and FSSMB. a Representative HPLC chromatograms of standards and extracts from SMB (b) and FSSMB (c). d Contents of tanshinones in SMB and FSSMB. e Total tanshinone content in SMB and FSSMB. SMB, Salvia miltiorrhiza Bunge; FSSMB, fermented solid-state products of SMB utilizing Shiraia bambusicola Henn. Values are represented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. DT, dihydrotanshinone; CT, cryptotanshinone; T1, tanshinone I; TIIA, tanshinone IIA

Effect of Shiraia bambusicola Henn. fermentation on salvianolic acids content of FSSMB

In this study, salvianolic acids (danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B) were examined in SMB and FSSMB. In general, the content of phenolic acids in FSSMB were observed to be lower compared to those in SMB (Fig. 5a-d). Specifically, danshensu, rosmarinic acid, lithospermic acid, and salvianolic acid B levels in FSSMB were determined to be 1.66, 13.02, 10.22, and 11.57 times lower than those in SMB, respectively (Fig. 5d). Additionally, in comparison to SMB, the total phenolic acid content exhibited a notable reduction of 1.92 times in FSSMB (Fig. 5e). Collectively, these findings suggest a significant decrease in phenolic acid levels following SSF.

Fig. 5.

HPLC analysis of phenolic acids content in SMB and FSSMB. Representative HPLC chromatograms of standards (a) and extracts from SMB (b) and FSSMB (c). d Total phenolic acids content in SMB and FSSMB. e Contents of phenolic acids in SMB and FSSMB. SMB, Salvia miltiorrhiza Bunge; FSSMB, fermented solid-state products of SMB utilizing Shiraia bambusicola Henn. Values are represented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Da, danshensu; Ro, rosmarinic acid; Li, lithospermic acid; SB, salvianolic acid B

Discussion

Most previous studies on SMB focused on traditional extraction methods or simple chemical modifications, while our study explores a new biological transformation approach through SSF. It was found that the product of SSF, FSSMB, has lower neurotoxicity and better antioxidant activity. Meanwhile, in terms of the mechanism, it was explored that FSSMB protects PC12 cells from oxidative damage by regulating the MAPK signaling pathway. Moreover, the contents of the antioxidant substances, tanshinone IIA and cryptotanshinone, in FSSMB are increased. It provides a research basis for the safer and more effective clinical application of SMB, and offers more evidence for the future research on enhancing the efficacy and reducing the toxicity of TCM through SSF.

Traditionally, SMB has been used to nourish blood and tranquilize the mind [6]. Emerging pharmacological evidence reveals that tanshinones, the primary bioactive constituents of SMB, exhibit neuroprotective effects against neurological disorders including depression, cerebral ischemia–reperfusion injury, and cognitive dysfunction [40]. Despite its therapeutic potential, clinical applications of SMB are partially limited by inherent side effects [13]. To address this issue, our study proposes SSF as a novel strategy to mitigate adverse reactions while preserving neuroactive components. As fundamental functional units of the nervous system, neurons are particularly vulnerable to oxidative stress, a key pathological mechanism underlying neurodegenerative diseases such as Alzheimer's disease and spinal cord injury [41, 42]. The PC12 cells, possessing typical neuronal characteristics, serves as a validated in vitro model for neuropharmacological research [43]. H₂O₂-induced oxidative damage in PC12 cells has been widely adopted to simulate neuronal injury mechanisms and evaluate neuroprotective agents [44]. Previous studies employing this model have demonstrated the efficacy of SMB-derived compounds, including protocatechuic aldehyde and ribisin A, in counteracting oxidative stress-mediated cellular damage [45, 46]. Based on this evidence, we established an H₂O₂-induced PC12 cell injury model to systematically investigate the neurotoxicity and efficacy of SSF on SMB, and evaluate the therapeutic enhancement achieved through SSF processing.

A study found that after chickpea flour was fermented by SSF, the contents of phenolic substances (such as γ-aminobutyric acid, proto-catechuic acid, and p-hydroxybenzoic acid) increased significantly, which was significantly and positively correlated with the enhancement of in vitro antioxidant activity [22]. However, this study found that compared with the chickpea flour, the content of salvianolic acid, a phenolic substance in the fermented chickpea flour, decreased significantly, but it showed better antioxidant activity. A number of studies have found that tanshinone IIA and cryptotanshinone have excellent anti-inflammatory and antioxidant activities [14, 47–50]. One study found that increasing the contents of tanshinone IIA and cryptotanshinone through SSF could improve the in vitro antioxidant capacity [17]. In this study, we found that compared with SMB, the contents of tanshinone IIA and cryptotanshinone in FSSMB increased, and the antioxidant capacity on PC12 cells was enhanced. Therefore, we speculate that this elevation in tanshinone IIA and cryptotanshinone may be the primary reason for the enhanced ability to resist oxidative stress observed in PC12 cells induced by H₂O₂.

It is delineated that tanshinone IIA enhanced the antioxidant enzymes activity of T-SOD and GSH-PX, while reducing the level of ROS and MDA to ameliorate oxidative stress [14]. Moreover, it is reported that indicate that tanshinone IIA prevents streptozotocin-induced Alzheimer's disease in mice by mitigating neuronal damage, attenuating oxidative stress, and inhibiting the activation of the p38 MAPK signaling pathway [47, 48]. In this study, following SSF, there was a notable increase in tanshinone IIA, while the total tanshinone content remained unchanged. This elevation in tanshinone IIA may be the primary reason for the enhanced ability to resist oxidative stress observed in PC12 cells induced by H₂O₂.

In this study, we exposed PC12 cells to 1 mmol/L H₂O₂ to examine the neuroprotective and antioxidative effects of FSSMB, along with the potential underlying mechanisms. Previous reports have shown that induced oxidative stress can activate the MAPK pathway and promote neuronal cell damage [51, 52]. Moreover, research has demonstrated that lithium pretreatment mitigates high glucose-induced neurotoxicity in PC12 cells by reducing JNK and p38 phosphorylation [53]. In line with these findings, we investigated the levels of phosphorylated JNK and p38 proteins and observed that FSSMB inhibited the activation of the p38 and JNK MAPK signaling pathways, thereby protecting PC12 cells from oxidative stress injury induced by H₂O₂. H₂O₂-mediated oxidative stress is known to trigger JNK activation through a tightly regulated molecular cascade involving redox-sensitive signaling nodes. Initially, extracellular H₂O₂ enters cells predominantly via aquaporin (AQP) channels, including AQP1, AQP3, AQP8 and AQP5 [54]. These AQPs localize to lipid rafts, forming signaling complexes that amplify oxidative stress responses. Once intracellular, H₂O₂ oxidizes specific molecular targets, initiating stress-responsive signaling. A critical mediator of H₂O₂-induced JNK activation is apoptosis signal-regulating kinase 1 (ASK1), a redox-sensitive MAP3K. H₂O₂ disrupts the interaction between ASK1 and reduced thioredoxin (Trx), triggering Trx dissociation and subsequent ASK1 oligomerization via its C-terminal coiled-coil domain [55]. This oligomeric ASK1 signalosome recruits adaptor proteins TRAF2 and TRAF6, thereby enabling phosphorylation and subsequent activation of downstream kinases MKK4 and MKK7 that directly phosphorylate JNK [56]. In parallel, p38 activation is mediated through phosphorylation by MKK3 and MKK6 [57]. Based on these studies, we propose that FSSMB potentially attenuates H₂O₂-induced JNK and p38 activation through modulation of key upstream regulators, including AQP channel-mediated H₂O₂ influx and/or ASK1 activation.

The limitation of this paper lies in the lack of exploration of the molecular mechanism by which Shiraia bambusicola Henn. regulates the synthesis of tanshinones and salvianolic acids in SMB. The biosynthesis of tanshinone occurs via the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways [58, 59]. Transcription factor SmERF73 activates DXR1, CPS1 and KSL1 gene expression to positively regulate tanshinones synthesis [60]. SmMYB4 negatively regulates cryptotanshinone, and tanshinone IIA synthesis by activating the expression of the GGPPS3 gene [61]. Our findings reveal that following SSF, the content of cryptotanshinone and tanshinone IIA significantly increased, whereas the content of dihydrotanshinone and tanshinone I notably decreased. Mechanistically, we speculate that Shiraia bambusicola Henn. may regulate the expression of key enzymes in the MVA and MEP pathways by influencing the transcription factors such as ERF and MYB in SMB, thereby affecting the synthesis of tanshinones and salvianolic acids. Additionally, our study observed a marked decrease in phenolic acid contents after fermentation, suggesting that Shiraia bambusicola Henn. primarily utilizes phenolic acids, possibly for its own growth and metabolic requirements. However, the precise regulatory mechanism remains unclear and warrants further investigation. Another limitation of this paper is that in our study, we found that the FSSMB extract exhibits lower toxicity than the SMB. It is also possible that the content of harmful substances decreased in SMB after SSF. Research has found that following SSF with medicinal fungus Ganoderma lucidum (Curtis) P. Karst., the levels of toxic chemicals such as dibutyl-phthalate and diisobutyl-phthalate in Tripterygium wilfordii Hook.f. were reduced [16].

Inherent differences in cellular sensitivity and metabolic activity, cytotoxic responses to compounds vary significantly across cell lines in vitro [62]. For in vivo applications, dose re-evaluation is essential, as factors such as bioavailability, metabolism, and systemic toxicity must be considered [63]. Therefore, we plan to explore these aspects in future studies using a broader range of cell lines and experimental animal models to establish a reliable dose and provide further evidence for the preclinical evaluation of FSSMB.

Conclusion

In conclusion, our study demonstrates the potential of SSF technology to address the limitations associated with SMB by enhancing its therapeutic properties and reducing adverse reactions. FSSMB exhibited lower cytotoxicity and superior antioxidant properties compared to SMB in PC12 cells, as evidenced by decreased levels of ROS and MDA, along with enhanced SOD activity. Mechanistically, FSSMB reversed H₂O₂-induced phosphorylation of p38 and JNK proteins. Additionally, FSSMB showed significant increases in tanshinone IIA and cryptotanshinone content, while reducing levels of tanshinone I, dihydrotanshinone, and salvianolic acids. These findings highlight the importance of enhancing the therapeutic efficacy and safety profile of FSSMB. In the future, we aim to utilize SSF to conduct research on enhancing the efficacy and reducing the toxicity of more TCM and their residues, and deeply analyze their material basis, providing a theoretical basis and technical support for expanding the application scope of TCM and the reuse of their residues (Fig. 6).

Fig. 6.

Proposed anti-oxidative effect of FSSMB against H₂O₂-induced injury in PC12 cells

Abbreviations

SMB

Salvia miltiorrhiza Bunge

FSSMB

Fermented solid-state products of SMB utilizing Shiraia bambusicola Henn.

PC12

Pheochromocytoma cell line 12

ROS

Reactive oxygen species

MDA

Malondialdehyde

SOD

Superoxide dismutase

SSF

Solid-state fermentation

p-p38

Phosphorylated protein-38

p-JNK

Phospho-c-Jun N-terminal kinases

MAPK

Mitogen-activated protein kinase

TCM

Traditional Chinese medicines

DMSO

Dimethyl sulfoxide

FBS

Fetal bovine serum

CCK-8

Cell Counting Kit-8

PVDF

Polyvinylidene Fluoride

DT

Dihydrotanshinone

CT

Cryptotanshinone

T1

Tanshinone I

TIIA

Tanshinone IIA

Da

Danshensu

Ro

Rosmarinic acid

Li

Lithospermic acid

SB

Salvianolic acid B

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

AQP

Aquaporin

ASK1

Apoptosis signal-regulating kinase 1

Trx

Reduced thioredoxin

MVA

Mevalonate

MEP

Methylerythritol phosphate

Authors’ contributions

WRL and LSJ designed the experiments. WQN, ZCH and SZR performed the experiments. WQN, HYF and HLY analyzed the data and prepared the manuscript. QYF, WRL and LSJ validated the data, edited the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (24KJA180006) and Jiangsu Provincial Key Construction Laboratory (No. SuJiaoKe [2024] 3) of Nanjing Xiaozhuang University.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.