Phytochemical Identification and Anti-Oxidative Stress Effects Study of Cimicifugae Rhizoma Extract and Its Major Component Isoferulic Acid

Abstract

Background and Objectives: Cimicifugae Rhizoma, also known as ‘Sheng ma’ in Madeiran, is a widely used Chinese herbal medicine that has several pharmacological qualities, one of which is its antioxidant activity. Isoferulic acid, a prominent phenolic compound found in Cimicifugae Rhizoma, has potent antioxidant properties. This study was aimed to comprehensively analyze the components in Cimicifugae Rhizoma and rat plasma to evaluate the in vitro antioxidant and anti-inflammatory properties of Cimicifugae Rhizoma extract and Isoferulic acid as potential candidates for developing herbal formulations targeting podocyte injury in diabetic nephropathy for further clinical utilization. Materials and Methods: UPLC/Q-TOF-MS and HPLC were utilized as analytical tools to identify components of Cimicifugae Rhizoma extract or rat plasma after administrating it. MPC5 cells were cultured with H₂O₂ and high glucose and subjected to oxidative stress injury. The CXCL12/CXCR4 system plays a crucial role at certain stages of multiple kidney diseases’ injury. Apoptosis-related and target CXCL12/CXCR4/mTOR/Caspase-3 and Cask protein levels were assessed, and the levels of inflammatory-related factors, motility, morphology, ROS level, and apoptosis in podocytes were tested. Results: A total of 82 and 39 components were identified in the Cimicifugae Rhizoma extract and plasma, and Isoferulic acid content was determined as 6.52 mg/g in the Cimicifugae Rhizoma extract. The Cimicifugae Rhizoma extract (1 μg/mL) and Isoferulic acid (10, 25, 50 μM) considerably decreased high glucose and oxidative-stress-mediated toxicity, impaired mobility and adhesion and apoptotic changes in MPC5 cells, and reversed inflammation response. Moreover, the Cimicifugae Rhizoma extract and Isoferulic acid down-regulated Cask, mTOR, and Caspase-3, while significantly blocking the overactivation of CXCL12/CXCR4 in podocytes stimulated by oxidative stress and high glucose. Conclusions: These results indicate that the renal protective mechanism of the Cimicifugae Rhizoma extract and Isoferulic acid on simulating H₂O₂-induced podocyte injury involves mainly the of CXCL12/CXCR4 pathways and the inactivation of oxidative-stress-mediated apoptotic pathways after comprehensive qualitative and quantitative research by UPLC/Q-TOF-MS and HPLC. These findings provide an important efficacy and ingredient basis for further study on the clinical utilities of Cimicifugae Rhizoma and Isoferulic acid on podocyte and kidney impairment.

1. Introduction

Cimicifugae Rhizoma (Figure 1a) is the dry rhizome of Cimicifuga heracleifolia Kom., Cimicifuga dahurica (Turcz.) Maxim., or Cimicifuga foetida L. of Ranunculaceae. Cimicifugae Rhizoma. Cimicifugae Rhizoma shows great antioxidant capacity and contains triterpenoid saponins, phenolic acids, chromogenic ketones, alkaloids and so on, with caffeic acid, ferulic acid, isoferulic acid, and ascophyllin as the main components [1]. And the antioxidant effect may come from the phenolic acids that Cimicifugae Rhizoma contains. The amount of phenolic acid in Cimicifugae Rhizoma is strongly linked to its antioxidant effect [2]. A previous study identified the most important beneficial components are caffeic acid, ferulic acid, and Isoferulic acid, which work by metal-chelating and radical-scavenging to reduce oxidative damage [3]. Isoferulic acid (Figure 1b), which is the representative phenolic acid component of Cimicifugae Rhizoma and the specified quality control target component of Cimicifugae Rhizoma, according to the 2020 Edition of the Chinese Pharmacopoeia, can exert antioxidant, anti-inflammatory, and anti-apoptosis activities and a potential role in regulating blood glucose [4,5,6]. Isoferulic acid has been found to have a mixed-inhibition effect on intestinal α-glucosidase by kinetic study, which also showed that Isoferulic acid would block intestinal maltase [7]. Isoferulic acid may help stop the glycation and oxidation of bovine serum albumin, which is linked to diabetes complications [8]. This is shown by lowering the levels of fructosamine, carboxymethyllysine, and amyloid cross-β structure, as well as the protein carbonyl content and stopping thiol group modification in bovine serum albumin [9]. Methylglyoxa causes failure, ROS production, and death in INS-1 cells. Isoferulic acid pretreatment decreases these effects by increasing GLO1 activity and the mitochondrial survival pathway [10]. Administering streptozotocin and Isoferulic acid (5.0 mg/kg) three times in one day intravenously to diabetic rats results in reduced levels of glucose in the blood plasma and alterations in the quantities of GLUT4 and PEPCK proteins. These findings indicate that Isoferulic acid may have potential benefits in the treatment of diabetes [11]. In the subsequent investigation of Isoferulic acid, it was determined that the decline in plasma glucose levels in streptozotocin -diabetic rats might perhaps be attributed to the activation of opioid mu-receptors, leading to an augmented release of beta-endorphins and alterations in glucose utilization or hepatic glucose production [12]. Due to its potential anti-inflammatory and anti-glycemic properties, Isoferulic acid might potentially be used for the treatment of diabetic nephropathy. Diabetic nephropathy is the primary factor leading to end-stage renal disease globally and is a prevalent vascular complication of both type 1 and type 2 diabetes mellitus. Elevated levels of TNF-α and IL6 are strongly associated with oxidative stress and inflammation, resulting in the development of diabetic nephropathy [13]. To enhance diabetic nephropathy, it is possible to inhibit the cell signaling pathways responsible for the production of ROS [14]. Elevated levels of ROS activate cellular pathways that trigger apoptosis and necrosis [15]. Exogenous H₂O₂ triggers a signaling cascade by mimicking the endogenous ROS signaling pathway, leading to ROS accumulation, which inhibits cell viability and triggers mitochondria-dependent apoptosis. Meanwhile, high glucose induction could lead MPC5 podocytes to the situation of diabetic nephropathy.

Cimicifugae Rhizoma has also been found to have good anti-inflammatory and antioxidant activity in previous studies. Phenolic acid compounds such as Cimidahuside E and Cimidahuside D can inhibit the production of PGE2 in LPS-stimulated RAW 264.7 macrophages, down-regulate the expression of COX-2, and then exert anti-inflammatory activity [16]. Caffeic-acid-containing cinnamic acid derivatives (such as Isoferulic acid) with potential anti-inflammatory activity were screened by UPLC/Q-TOF-MS-integrated NF-κB luciferase reporter gene detection system. Caffeic acid significantly inhibited the activation of NF-κB in TNF-α-induced injury of BEAS-2B cells in a dose-dependent manner [17]. The ethanol extract of Cimicifugae Rhizoma can effectively alleviate OVA ovalbumin-induced inflammatory and oxidative stress injury by up-regulating Nrf2/HO-1/NQO1 signaling and down-regulating NF-κB phosphorylation [18]. The previous studies focused on the quality markers and components of Cimicifugae Rhizoma from original plants [19,20]. But our study aimed to comprehensively identify the components absorbed into the plasma after administration of Cimicifugae Rhizoma and screen out the component with the best antioxidant effect on podocytes impaired by oxidative stress, so as to provide the potential evidence of the renal protection of Cimicifugae Rhizoma.

Isoferulic acid (6.52 mg/g) was screened for the strongest antioxidant activity from 82 and 39 components identified by the UPLC/Q-TOF-MS of the Cimicifugae Rhizoma extract and plasma after administration of Cimicifugae Rhizoma extract combined with MTT assay. The objective of this research was to determine the protective effects of Cimicifugae Rhizoma and Isoferulic acid on oxidative-stress-damaged podocytes. This was carried out by examining the influence of Cimicifugae Rhizoma extract and Isoferulic acid on MPC5 cells that were subjected to high levels of glucose and H₂O₂. Researchers discovered that it operates by specifically targeting the CXCL12/CXCR4 pathway and its association with Caspase-3 and mTOR, hence alleviating cell death and oxidative stress. Consequently, the Cimicifugae Rhizoma extract and Isoferulic acid should be useful for devising a prospective component for the treatment.

2. Materials and Methods

2.1. Reagents and Chemicals

The conditionally immortalized mouse podocyte cell line (MCP5) was purchased from the BeNa Culture Collection (BNCC342021, Beijing, China). Cell line identification was performed by the same company. Isoferulic acid was obtained from China National Institute for Food and Drug Control (Lot: 111698-201904, purity = 99.3%, Beijing, China). Ferulic acid (Lot: B20007, purity ≥ 98%), Methyl ferulate (Lot: B30655, purity ≥ 98%), Caffeic acid (Lot: B20660, purity ≥ 98%), Paeonol (Lot: B20266, purity ≥ 98%), Ethyl caffeate (Lot: B20661, purity ≥ 98%), Methyl caffeate (Lot: B25588, purity ≥ 98%), Sinapic acid (Lot: B30104, purity ≥ 98%), 4-Hydroxyphenylacetic acid (Lot: B30104, purity ≥ 98%), Cimifugin (Lot: B21156, purity ≥ 98%), Cimigenol (Lot: B23157, purity ≥ 98%), and Acetylcimigenol 3-O-alpha-L-arabinopyranside (Lot: B21504, purity ≥ 98%) were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cimicifugae Rhizoma-prepared herbal medicine was purchased from Beijing TaiRenKang Pharmaceutical Co. (Beijing, China) (Lot: 22081501): Roswell Park Memorial Institute 1640 (RPMI 1640). The 0.05% trypsin and penicillin/streptomycin solutions were Gibco products. Fetal bovine serum (FBS), Hydrogen peroxide (H₂O₂), and DMSO were products of Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and methyl thiazolyl tetrazolium (MTT) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Anti-beta Actin antibody (ab8227), Anti-SDF1 antibody (ab155090), Anti-CXCR4 antibody (ab181020), Anti-mTOR antibody (ab134903), Anti-Caspase-3 antibody (ab184787), and Anti-CASK antibody (ab3383) were obtained from Abcam (Cambridge, UK). Mouse IL-6 ELISA kit (SEA079Mu), CXCR4 ELISA kit (SEA940Mu), CXCL12 ELISA kit (SEA122Mu), and TNF-α ELISA kit (SCA133Mu) were products from Cloud-Clone Corp. (Wuhan, China), while IL-1β ELISA kit (E-EL-R0012c) was from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Annexin V/FITC apoptosis assay kit, DCFH-DA assay kitand Actin-Tracker Red-594 were purchased from Beyotime Biotechnology (Shanghai, China).

2.2. Extract Samples Preparation and Components Identification

2.2.1. HPLC Parameters and Cimicifugae Rhizoma Extract Preparation

Cimicifugae Rhizoma extracts were prepared into 5 mg/mL methanol solution, filtered by a 0.22 μm filter member, and analyzed by HPLC (LC-20AT HPLC with SPD-20A UV-VIS detector, Shimadzu, Kyoto, Japan). A C18 (4.6 × 250 mm, 5 μm) column (Agilent Eclipse XDB-C18) was used to perform compounds’ separations. The mobile phase consisted of water containing acetonitrile (A) and 0.1%phosphoric acid (B). The gradient program was as follows: 0~5 min, 5% A, 5~20 min, 5% → 20% A, 20~60 min, 20% → 40% A. Injection volume was 10 μL, and the flow rate was 0.8 mL/min and under 320 nm.

The crude powder of Cimicifugae Rhizoma was taken and soaked in 50 times the amount of 70% ethanol for about 12 h. It was extracted twice by ultrasonication (37 °C, 200 W, 40 kHz) for 60 min and dried to the extract. Isoferulic acid was prepared with methanol and stored in cold storage at 4 °C for later use.

2.2.2. UPLC/Q-TOF-MS Parameters and Sample Preparations

The Cimicifugae Rhizoma extract sample was prepared by dissolving 50 mg in 1 mL of menthol via ultrasonication at 200 W and 40 kHz for 30 min and centrifugation at 12,000 r/min for 10 min at 4 °C. The solution was diluted tenfold, and the supernatant was centrifuged at a low temperature and high speed (12,000 r/min, 10 min). The resultant supernatant was the final sample to do the analysis. The standards of Isoferulic acid, ferulic acid, methyl ferulate, caffeic acid, paeonol, ethyl caffeate, methyl caffeate, sinapic acid, and 4-hydroxyphenylacetic acid were weighed precisely and prepared into 1 mg/mL single standard solution with methanol. Fifty μL of each single standard solution was added into a 50 mL measuring flask and, then, made up with methanol solution to form a 1 μg/mL mixed standard solution. The solution was diluted into 10 ng/mL, and the supernatant was centrifuged at a low temperature and high speed (12,000 r/min, 10 min).

In this study, 3 SD rats (SiPeiFu (Beijing) Biotechnology Co., Ltd. (Beijing, China)) were administered the Cimicifugae Rhizoma extract at a dose of 0.253 g/kg for the calculations of body areas of humans and rats (based on the maximum dosage of 10 g for human use as stipulated in the 2020 edition Chinese Pharmacopoeia). Rats were fasted for 24 h before drug administration. Blank blood samples were obtained before drug administration. Plasma was taken from the orbital plexus at 0.5 h, 1 h, 2 h, 4 h, and 6 h after administration and before administration as blank comparison. The blood samples were centrifuged at 4000 r/min for 10 min, and the plasma samples (supernatants) were kept at −80 °C prior to analysis. For plasma processing methods, refer to the previous study [21].

The experiments were approved by the Laboratory Animal Ethics Subcommittee, Academic Committee, Beijing University of Chinese Medicine (BUCM) under the Animal Ethics Licence No. BUCM- 202350804- 2115.

SYNAPT G2-Si Mass Spectrometer (Waters Corp., Milford, MA, USA) and I-Class Ultra High-Performance Liquid Chromatography (Waters Corp., Milford, MA, USA) were used. Column: ACQUITY UPLC^TM T3 C₁₈ column (100 × 2.1 mm, 1.7 μm); mobile phase: 0.1% formic acid in water (A) − 0.1% formic acid in acetonitrile solution (B); column temperature: 35 °C; sample holding temperature in the instrument: 10 °C; flow rate: 0.25 mL.min⁻¹; injection volume: 3 μL. In both the positive and negative mode, elution was set as the gradient program (0–3 min, 0% B; 3–70 min, 0–100% B; 70–76 min, 100% B; 76–80 min, 100–0% B).

The MS parameters in the positive mode were as follows: mass scan range, 50 Da to 1500 Da; capillary voltage, 3000 V; lock spray capillary, 3000 V; desolvation temperature, 500 °C; gas flow rate, 500 L/Hr; cone gas flow rate, 50 L/Hr. Most MS parameters in the negative mode were same as those in the positive mode except for gas flow rate (800 L/Hr), capillary voltage (2000 V).

The identification was performed using the UNIFI Scientific Information System with the commercial Traditional Chinese Medicine database, online databases (e.g., Massbank), relative data from reports or articles, and results obtained from mixed standard solutions (

Table A3. The identification results of mixed standards.

Label	Component	Formula	Mass (Da)	m/z (Precursor Ion)	Mass Error (ppm)	Observed RT (min)	Adducts	MS/MS Fragment (Product Ion)
1	Caffeic acid	C9H8O4	180.04226	179.0351	0.7	12.56	−H	135.0447
2	Ferulic acid	C10H10O4	194.0579	193.051	1.8	19.17	−H	133.0294
3	Paeonol	C9H10O3	166.063	165.0561	2.3	25.41	−H	133.0295
4	4-Hydroxyphenylacetic acid	C8H8O3	152.0473	151.0401	0.3	11.46	−H	107.0500
5	Sinapic acid	C11H12O5	224.0685	223.0617	2.3	16.53	−H	149.0242
6	Methyl caffeate	C10H10O4	194.0579	193.0507	0.4	16.22	−H	121.0291/93.0340
7	Ethyl caffeate	C11H12O4	208.0736	207.0668	2.4	22.84	−H	178.0826/135.0448/133.0294
8	Isoferulic acid	C10H10O4	194.0579	193.0505	−0.5	16.89	−H	178.0286/135.0431/134.0380
9	Methyl ferulate	C11H12O4	208.0736	207.0667	2.0	23.51	−H	133.0293

) in this study.

2.3. Cell Cultures and Treatment

MPC5 cells were resuspended and inoculated in RPMI 1640 containing 10 U/mL interferon-gamma and 10% FBS and cultured at 33 °C with 5% CO₂ for proliferation and passaging. The cells were transferred to RPMI 1640 without IFN-γ and allowed to differentiate for 7–12 days at 37 °C with 5% CO₂.

Fully differentiated podocytes were divided into the low glucose group (LG), high glucose with H₂O₂-Induced group (HG + H₂O₂), and drug group. The cells in LG were treated with 5.6 mM glucose medium as the control group. HG + H₂O₂ cells were treated with 30 mM glucose medium and 150 μM H₂O_2. The drug group cells were treated with 10, 25, 50 μM Isoferulic acid or 1 μg/mL Cimicifugae Rhizoma extract under the same condition of HG + H₂O₂ or LG, respectively. The whole treatment time was 24 h.

2.4. MTT Assay on Cell Viability

The MTT assay was applied to assess the influence of Isoferulic acid and Cimicifugae Rhizoma extract on HG + H₂O₂ MPC5 cells. Harvested cells (6 × 10³ cells/well) were seeded in 96-well plates, followed by incubation for 18 h. Thereafter, the cells were subjected to 24 h incubation after treatment as described in Section 2.3. Then, the cells were exposed to 100 μL of MTT (0.5 mg/mL) for 4 h, followed by replacement of the medium with 150 μL DMSO to solubilize the resultant formazan crystals. The absorbance of each well was read at 490 nm in a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.5. Transwell Test for Podocyte Migration

MPC5 cells were seeded in 6-well plates at 5 × 10⁵ cells/well and treated as described in Section 2.3 after 18 h incubation. Treated cells were washed by PBS and collected after digestion by trypsin. Then, 2 × 10⁴ cells/well podocytes with 200 μL RPMI 1640 from different groups were seeded onto Transwell culture inserts (pore size 5 μm, Costar Corporation, Richmond, VA, USA), while the inserts were flowed into 750 μL RPMI 1640 with 10% FBS. After 24 h incubation, the inserts were emptied of medium and washed by PBS twice. Migrated podocytes were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet solution. The number of migrated cells was counted randomly using a phase-contrast microscope with a ×20 objective (BH2-RFCA; Olympus, Tokyo, Japan).

2.6. Podocyte Adhesion Ability Test

Firstly, 2 × 10⁴ cells/well treated podocytes with 100 μL RPMI 1640 were seeded into pre-coated 96-well plates and incubated for 2 h. Cell staining solution (10 μL) was added and incubated for another 2 h. Absorbance was measured at 450 nm. The adhesion ratio was calculated by dividing OD of HG or drug group podocytes minus OD of blank group by OD of LG podocytes minus OD of blank group.

2.7. Determination and of ROS Levels

MPC5 cells were seeded in 6-well plates at 5 × 10⁵ cells/well and treated as described in Section 2.3after 18 h incubation. Treated cells were washed by PBS and collected after being digested by trypsin. Intracellular ROS levels were assessed with the DCFH-DA method. Briefly, the cells were seeded and incubated as mentioned earlier. Finally, incubation was carried out in darkness for 10–20 min with DCFH-DA at 37 °C. This was followed by rinsing and re-suspension with PBS or RPMI 1640. And they were analyzed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) which was used to quantitate the relative FITC-A Mean values.

2.8. 4′,6-Diamidino-2-phenylindole (DAPI) Staining and Flow Cytometry Apoptosis

MPC5 cells were seeded in 24-well plates at 5 × 10⁴ cells/well and treated as described in Section 2.3 after 18 h incubation. Thereafter, the medium was removed, and the cells were fixed with 4% paraformaldehyde solution at room temperature for 10 min. Subsequently, the cells were washed twice with PBS, and 0.3 mL of DAPI reagent was added, followed by reacting with the stain in the dark for 3 min. Finally, the changes in the shape of the nuclei were observed under a fluorescence microscope (in the center fields while at ×100 magnification).

MPC5 cells were seeded in 6-well plates at 5 × 10⁵ cells/well and treated as described in Section 2.3 after 18 h incubation. Treated cells were washed by PBS and collected after digested by trypsin.

Annexin FITC-Annexin V/PI assay distinguished between the apoptotic and viable cells. After treatment as described in Section 2.3, the cells were incubated with the fluorescent probe, followed by using a flow cytometer to determine apoptotic rates.

2.9. Enzyme Linked Immunosorbent Assay (ELISA) Assay

The ELISA kits were used to detect the levels of inflammatory cytokines, including TNF-α, IL-6, and IL-1β. CXCR4 and CXCL12 levels of podocytes were preliminarily determined by ELISA kits at the same time.

2.10. Observation of Actin-Tracker Red-594 Phalloidin Staining Podocyte Cytoskeleton

The fully developed and specialized MPC5 cells were placed in 12-well plates with a concentration of 5 × 10⁴ cells per well. Each group received the respective intervention medium for 24 h, with a total volume of 600 μL supplied to each well. The intervention medium was aspirated and rinsed three times with 300 μL of PBS for 3 min each time. Following the washing process, a solution of 300 μL of 4% paraformaldehyde was applied and allowed to undergo fixation at room temperature for 20 min. Rinse three times with 300 microliters of PBS for 3 min each time. Following the washing step, a solution containing 0.5% TritonX100 diluted with PBS was applied. A volume of 0.5 mL was used. The membrane was then pierced at room temperature for 30 min. It is important to note that in this case, membrane penetration was not necessary since the target protein was already a membrane protein. The Actin-Tracker Red-59 dilution (1:50) was introduced and allowed to incubate at a temperature of 37 °C for a duration of 50 min. Subsequently, it was rinsed twice with 300 μL of PBS for 3 min each time. After applying 300 μL of DAPI staining solution for 1 min, the sample was rinsed twice with 300 μL of PBS for 3 min each time. A single droplet of a solution that inhibits fluorescence was applied and introduced again.

2.11. Western Blot Analysis

Following the incubation of cells with different treatments, the cells were collected and PBS-rinsed. The protein concentration was quantified by the Bradford Protein Quantification Kit. Protein samples were mixed with 5× loading buffer and were heated for 5 min for protein denaturation, followed by an ice bath for 5 min. The samples were loaded onto 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes at 350 mA for 2 h. The membranes were incubated overnight with the following primary antibodies: Cask, CXCL-12, CXCR4, mTOR, Caspase-3 (1:1000). Then, the immunoblots were incubated with corresponding secondary antibodies and developed using Western Lightning^TM Chemiluminescence Reagent (PerkinElmer, Waltham, MA, USA) and analyzed with Labworks^TM 4.0 digital quantification software (UVP, Upland, CA, USA).

2.12. Statistical Analysis

Results are presented as mean ± S.D. Multiple-group comparison was performed with one-way ANOVA. All statistical analyses were carried out with GraphPad Prism 8.0 device. A statistically significant difference was assumed at p < 0.05.

3. Results

3.1. Identification of the Components in Cimicifugae Rhizoma Extract by UPLC-MS/MS and Quantification of Isoferulic Acid by HPLC

General ion flow diagrams (Figure 2a–d) were collected in UPLC-MS/MS positive and negative ion modes, and the UNIFI scientific information system was used for data analysis (Cimicifugae Rhizoma (Cimicifuga heracleifolia Kom., Cimicifuga dahurica (Turcz.) or Cimicifuga foetida L.)). Rapid identification was assisted by UNIFI platform or Masslynx 4.1. The secondary fragment ion peaks were compared with the data in the references and database (https://massbank.us/ (accessed on 27 February 2024)) or the test results of the mixed standard solution.

As shown in

Table 1. The identification results of Cimicifugae Rhizoma extract in the plasma.

Label	Component	Formula	Mass (Da)	m/z (Precursor Ion)	Mass Error (ppm)	Observed RT (min)	Adducts	MS/MS Fragment (Product Ion)	Reference
1	12β-O-Acetyl-Liposide B	C37H58O11	678.3979	677.3907	0.0	36.76	−H	617.3701	[19]
2	12β-O-Acetylcimifugoside A	C37H56O11	676.3823	677.3870	−3.7	27.88	+H	541.3164/195.1206/181.1043	[19]
3	12β-Hydroxystigmasterolxyloside	C35H56O10	636.3874	635.3801	0.0	31.04	−H	577.3384	[19]
4	15α-HydroxycimifugosideH2	C35H54O11	650.3666	649.3609	2.4	25.09	−H	531.2990	[22]
5	24-O-Acetyl-7,8-didehydroclerosterolxyloside	C37H58O11	678.3979	677.3901	−0.8	33.58	−H		[20]
6	24-O-Acetyl-Cresolxyloside	C37H60O11	680.4136	679.4059	−0.5	37.16	−H		[20]
7	7,8-Didehydroclerosterolxyloside	C35H54O9	618.3768	619.3830	−1.6	31.06	+H	469.3325/451.3226/433.3109	[20]
8	Acetylcimigenol 3-O-alpha-L-arabinopyranside	C37H58O10	662.4030	663.4090	−1.9	37.16	+H	623.3551	[22]
9	25-Anhydrocimigenol-xyloside	C35H54O8	602.3819	603.3875	−2.8	33.68	+H, +Na	583.3613	[22]
10	12β-Hydroxycitrinol	C30H48O6	504.3451	503.3370	−1.6	36.73	−H	503.3370	[19]
11	Arcotoxin	C37H56O11	676.3823	677.3888	−1.1	26.69	+H	131.0894
12	Cimigenol xyloside	C35H56O9	620.3924	643.3838	3.4	28.02	+Na	435.3249
13	Cimiside A	C35H56O10	636.3874	635.3808	1.2	28.16	−H	445.8311	[19]
14	Cimiside C	C43H70O16	842.4664	841.4598	0.8	31.41	−H	637.3959/447.3112
15	Cimiside H1	C35H52O9	616.3611	615.3545	1.1	36.33	−H	251.2021	[19]
16	Cimiside H2	C35H54O10	634.3717	633.3642	−0.3	27.75	−H	501.2875/369.2438	[19]
17	Cimicifugine	C35H51NO8	613.3615	614.3693	0.9	22.45	+H	446.3062/184.0776	[23]
18	Cimaroside V	C47H76O19	944.4981	943.4898	−1.0	68.58	−H	303.2335	[22]
19	Cimaroside I	C36H58O11	666.3979	665.3910	0.5	33.69	−H		[20]
20	Cimiacerin A	C35H54O9	618.3768	619.3831	−1.5	35.79	+H, +Na	451.3225	[22]
21	Cimidahuside E	C35H52O8	600.3662	601.3728	−1.2	35.77	+H	451.3195	[19]
22	Cimidahuside F	C35H52O9	616.3611	617.3687	0.5	27.77	+H	451.3328	[22]
23	Cimidahuside G	C35H56O9	620.3924	621.3972	−4.0	31.51	+H	453.3379
24	Cimidahuside H	C35H54O9	618.3768	641.3644	−2.5	35.29	+Na	451.3223/433.3122	[22]
25	Cimicidanol	C30H44O5	484.3189	485.327	1.6	27.76	+H	449.3901	[22]
26	Isodahurinol/Cimigenol	C30H48O5	488.3502	489.3563	−2.4	31.51	+H	453.3379	[22]
27	Ferulic acid	C10H10O4	194.0579	193.0495	−5.9	19.52	−H		standards data
28	Cimidahurinine	C14H20O8	316.1158	315.1069	−5.3	8.11	−H	153.9128/149.2179
29	Ethyl caffeate	C11H12O4	208.0736	207.0664	0.8	22.80	−H		standards data
30	Cimicifugic acid E	C21H20O10	432.1057	431.0983	−0.1	21.93	−H	165.0557	[22]
31	Isocimicifugamide	C25H31NO10	505.1948	504.1861	−2.8	21.81	−H	343.9951	[22]
32	Cimicifugaside B	C33H40O18	724.3962	723.3952	−3.6	33.62	−H	723.3952	[16]
33	Shomaside C	C27H30O15	594.3390	617.3677	−3.0	27.73	+Na, +H	193.9128/177.0620	[19]
34	Methyl-ferulic acid	C11H12O4	208.0679	207.0673	−6.8	22.93	−H	194.1196/193.0924
35	Caffeicacid-β-D-glucopyranosylester	C15H18O9	342.0951	341.0893	4.5	10.54	−H
36	Cimicifugamide	C25H31NO10	505.1948	504.1860	−3.1	17.76	−H		[22]
37	Cimidahurinine	C14H20O8	316.1158	315.1077	−2.7	9.09	−H	153.0564
38	Isoferulic acid	C10H10O4	194.0579	193.0505	−0.5	16.89	−H		standards data
39	Cimifugin	C16H18O6	306.2462	305.2379	2.1	53.65	−H	235.9243	[19]

Note: Reference, i.e., relevant research literature and standard data referenced on behalf of the constituents, so as to screen and analyze the constituents of Cimicifugae Rhizoma extract in the plasma.

and Table S1, 82 and 39 components were identified in the medication and plasma, respectively. Most components in the Cimicifugae Rhizoma extract are filed under triterpene and triterpenoid saponins (46) and phenolic acid (30), respectively. In the plasma, most components are still filed under triterpene and triterpenoid saponins (26) and phenolic acid (12), respectively.

3.1.1. Identification of Phenolic Acids in Cimicifugae Rhizoma Extract

The fragment of phenolic acids was characterized by neutral losses of 15.02 Da (–CH₃), 45.01 Da (–COOH), and 29.06 Da (–C₂H₅). Based on the same fragment ions m/z 193 as ferulic acid, components 33 and 34 were characterized as Shomaside C and Methyl-ferulic acid. A fragment response at m/z 177 was expected to occur when a methoxy group was present on the benzene ring together, which could be found in Shomaside C. The same fragment ions m/z 178 as caffeic acid could also be observed in Ethyl caffeate (Figure 2e). A fragment response at m/z 135 was captured in caffeic acid and its derivatives and Isoferulic acid. To distinguish Ferulic acid and Isoferulic acid, the fragment ions m/z 133 could be seen in Ferulic acid and its derivatives (Table A3).

3.1.2. Identification of Triterpene in Cimicifugae Rhizoma Extract

Triterpene and triterpenoid saponins were very common in the Cimicifugae Rhizoma extract. A lot of triterpene and triterpenoid saponins were liable to form [M + H]⁺ ion and [M + Na]⁺ ion in positive mode. The main cleavage pathways of triterpenoid saponins were prone to lose 18.01 Da (H₂O), 17.01 Da (OH⁻), and 132.05 Da (arabinose). Components 21–24 were the specific triterpenoid saponin constituents of Cimicifuga dahurica (Turcz.) Maxim. The following were examples of the conventional fragmentation pathways for triterpenoid saponins. For example, m/z 451 ([M + H − H₂O − Ara]⁺ and [M + H − 2H₂O − Ara]⁺) was detected in Cimidahuside E and Cimidahuside F (Figure 2f).

3.1.3. Identification of Isoferulic Acid Content in Cimicifugae Rhizoma Extract by HPLC

This HPLC methodological result was sufficient to meet the standard which was shown in Appendix A. In the HPLC analysis, both standard Isoferulic acid and the Cimicifugae Rhizoma extract exhibited the same retention time (<0.1 min). This matching retention time indicates the same chemical component. The Isoferulic acid concentration in the Cimicifugae Rhizoma extract was calculated to be 6.52 mg/g (n = 3, calculated by standard curve from 1.56 μg/mL to 50.00 μg/mL). This quantification was confirmed through the HPLC analyses, as depicted in Figure 2g,h.

3.2. Cimicifugae Rhizoma Extract and Isoferulic Acid Inhibit H₂O₂ + HG Cytotoxicity in MPC5 Cells

To optimize the dose of H₂O₂ in the HG environment, the effects of 50~200 μM H₂O₂-inducion were tested. And the result (Figure 3a) showed MPC5 cells’ viability reduced significantly and stably at the 150 μM H₂O₂ level. Under the condition of H₂O₂ (150 μM) + HG (30 mM), the Cimicifugae Rhizoma extract was able to significantly increase cell survival at a dose of 1 μg/mL (Figure 3b). We performed MTT screening of three representative phenolic acid constituents and triterpenoid constituents of the Cimicifugae Rhizoma extract (Figure 3c–h) and found that two phenolic acid constituents, ferulic acid and Isoferulic acid, exhibited antioxidant properties. And Isoferulic acid had the strongest antioxidant property at 10–40 μM, which might be the main antioxidant component of the Cimicifugae Rhizoma extract.

The repeated experiment showed that 10, 25, and 50 μM of Isoferulic acid recovered the podocytes viability, which could protect most podocytes from H₂O₂ + HG injury (Figure 3i). On the other hand, the solution DMSO itself, 1 μg/mL Cimicifugae Rhizoma extract, or the different levels of Isoferulic acid could not result in the abnormal proliferation of MPC5 cells in the LG environment (Figure 3j). The above two results meant that Isoferulic acid was moderating H₂O₂ + HG injury and protecting the podocytes.

3.3. Isoferulic Acid Shows Potential in Improving MPC5 Cells’ Impaired Mobility and Adhesion

The Cimicifugae Rhizoma extract could increase the migration of podocytes, but there was no statistical difference from the H₂O₂ + HG group (Figure 4a,b). The Cimicifugae Rhizoma extract could restore damaged podocyte adhesion (Figure 4c). Isoferulic acid treatment improved MPC5 and regained the migration (Figure 4d,e) and cell adhesion (Figure 4f) of conditionally immortalized podocytes cultured in high-glucose and H₂O₂ media in comparison with normal-glucose media.

3.4. Isoferulic Acid Inhibits H₂O₂ + HG-Mediated Cell Apoptosis and Oxidative Stress

3.4.1. Isoferulic Acid Inhibits H₂O₂ + HG -Mediated Cell Apoptosis

The changes in the nucleus of MPC5, as well as apoptosis, were primarily assessed through the DAPI staining. Based on the results (Figure 5a), the nucleus of cells in the LG and Cimicifugae Rhizoma extract (1 μg/mL) and Isoferulic acid (10–50 μM) group were uniformly maintained. Stronger fluorescence intensity and blue blot with chromosome fragmentation and chromatin condensation could be observed in the HG + H₂O₂ group. The Cimicifugae Rhizoma extract (1 μg/mL) and Isoferulic acid (10–50 μM) reduced the above nuclear morphologies and apoptotic changes.

The results in Figure 5b,c showed that the Cimicifugae Rhizoma extract (1 μg/mL) had a favorable inhibitory effect on podocyte apoptosis induced by high-glucose H₂O₂. The number of viable cells was significantly increased (79.13 ± 2.47% to 89.03 ± 0.96%), the amount of early apoptotic cells was significantly reduced (13.20 ± 2.51% to 7.36 ± 0.21%), and the number of late apoptotic versus early necrotic cells was also significantly reduced (from 7.20 ± 0.36% to 3.13 ± 0.60%), returning to the same level as the LG control (p > 0.05).

As shown in Figure 5d,e, the number of viable cells in the HG + H₂O₂-injured group was significantly reduced (94.53 ± 1.00% versus 70.99 ± 2.74%), while the amount of early apoptotic cells and late apoptotic and early necrotic cells was significantly increased (4.38 ± 0.56% versus 22.13 ± 3.08%; 0.87 ± 0.39% versus 6.50 ± 1.76%) compared to the LG group. Isoferulic acid (50, 25, 10 μM) exhibited anti-apoptotic capability by reducing the rate of apoptosis: the number of viable cells rebounded significantly (90.68 ± 1.57%, 89.23 ± 3.90%, 87.13 ± 5.93%), and the number of early apoptotic cells decreased significantly (6.70 ± 0.96%, 7.53 ± 2.31%, 8.97 ± 3.49%). In the meantime, the number of late apoptotic and early necrotic cells decreased (2.45 ± 0.80%, 2.93 ± 1.45%, 3.70 ± 2.39% respectively). The induction of HG + H₂O₂ could produce cytotoxicity to MPC5 cells that further leads to cell apoptosis. The results showed that Isoferulic acid, especially at 50 μM, was able to inhibit the apoptosis of podocytes.

3.4.2. Isoferulic Acid Inhibits H₂O₂ + HG -Mediated Oxidative Stress and Inflammatory Response in MPC5 Cells

Oxidative stress which can affect triggering of the apoptosis signal is indicated by enhanced ROS levels. In order to further explore whether Isoferulic acid protects MPC5 cells through antioxidant activity, after the podocytes were exposed to HG + H₂O₂ in the absence or presence of Isoferulic acid (10–50 µM), the relative ROS levels were quantified by flow cytometry. Results in Figure 6 indicate that intracellular ROS which was induced by HG + H₂O₂ were suppressed by Isoferulic acid. These results suggest that the anti-apoptotic protective effect of Isoferulic acid on MPC5 cells may be involved in oxidative-stress-mediated apoptosis.

3.5. Effects of Cimicifugae Rhizoma Extract and Isoferulic Acid on the Morphology of the Damaged Podocyte Cytoskeleton

The results depicted in Figure 7 demonstrated that in the LG normal control group, the podocyte exhibited prominent pedicle protrusions with clear edges and neatly arranged actin fiber bundles. However, after subjecting the cells to H₂O₂-induced injury in a high sugar environment for 24 h, the HG + H₂O₂ injury group displayed the absence of pedicle protrusions, inward rolling of the podocyte cell edges, reduced cell volume, and fragmented or even vanished actin fiber bundles. Consequently, this led to a significant decrease in fluorescence intensity. Following the intervention of the Cimicifugae Rhizoma extract and Isoferulic acid, the podocyte protrusions were successfully repaired; the size of the podocyte cells remained within the normal range, and the edge of the cells was clearly defined. The actin fiber bundles were less fragmented; the fluorescence intensity significantly increased, and both the podocyte protrusions and actin fiber bundles exhibited a favorable morphology.

3.6. Cimicifugae Rhizoma Extract and Isoferulic Acid Inhibit H₂O₂ + HG -Mediated Inflammatory Response and CXCL12/CXCR4 Signaling Pathway in MPC5 Cells

TNF-α, IL-6, and IL-1β belong to the pro-inflammatory cytokine family. Figure 8a–c demonstrated that TNF-α, IL-6, and IL-1β exhibited excessive production in the cell medium treated with high glucose and hydrogen peroxide (HG + H₂O₂). The ELISA findings demonstrated that the administration of several dosages of Isoferulic acid (10, 25, 50 μM) in combination with the Cimicifugae Rhizoma extract effectively reduced the excessive release of inflammatory factors induced by high-glucose H₂O₂. Every quantity of Isoferulic acid effectively decreased the aberrant production of IL-6 (p < 0.05). One μg/mL Cimicifugae Rhizoma extract and 50 μM Isoferulic acid reduced the abnormal release of TNFα (p < 0.05). Additionally, the Cimicifugae Rhizoma extract and Isoferulic acid reduced the abnormal release of IL-1β (p < 0.05) and relieved the inflammatory phenomena. Out of all the options, the intervention impact on inflammatory variables was most significant at a concentration of 50 μM Isoferulic acid.

The ELISA findings shown in Figure 8d,e reveal an ample increase in the expression of CXCL12 and CXCR4 in podocytes stimulated by HG + H₂O₂. The administration of an Cimicifugae Rhizoma extract and Isoferulic acid effectively controlled the levels of CXCL12/CXCR4 in podocytes, indicating that the protective actions of podocytes are linked to the targets CXCL12 and CXCR4. Fifty μM Isoferulic acid could regulate the CXCL12/CXCR4 expression level back to LG group.

3.7. Cimicifugae Rhizoma Extract and Isoferulic Acid Regulates Protein Levels in MPC5 Cells That Were Influenced by HG + H₂O₂

As we can see in the Figure 9, CXCL12 and CXCR4 were abnormally up-regulated in podocytes induced by HG + H₂O₂ induction, and the intervention of Isoferulic acid significantly regulated the levels of these target proteins in podocytes, which matched the results of the previous ELISA experiments. Consistent with the results of ELISA, Cimicifugae Rhizoma extract also inhibited the aberrant expression of the CXCL12/CXCR4 pathway in injured podocytes, but 50 μM Isoferulic acid had a stronger inhibitory effect on the CXCR4 target.

The increased activity of Caspase-3 appeared to be detected after 24 h HG + H₂O₂ incubation, suggestive of the cells’ entry point into the apoptotic-signaling pathway after exposure to HG + H₂O₂. And 50 μM Isoferulic acid treatment could down-regulate the apoptosis-related protein Caspase-3 in the HG + H₂O₂ environment.

In the present study, the podocyte injury caused by high-glucose H₂O₂ resulted in a rise of mTOR in the cells of the model group, and the down-regulation of mTOR protein levels by Isoferulic acid may indicate that mTOR occupies a major role in the apoptotic pathway in the present model of injury. Also, the Cimicifugae Rhizoma extract inhibited apoptosis and podocyte motility and affected the overexpression of the targets mTOR, Caspase-3, and Cask. The inhibitory effects of mTOR and Cask were consistent with that of 50 μM Isoferulic acid, and the inhibitory effect of 50 μM Isoferulic acid was better in regulating the apoptosis-related target Caspase-3 that surged in response to the damage by oxidative stress.

4. Discussion

This study identified 82 and 39 components in a Cimicifugae Rhizoma extract and plasma after Cimicifugae Rhizoma extract administration by UPLC/Q-TOF and screening for the effective antioxidant component Isoferulic acid (6.52 mg/kg). We found the cleavage pattern of triterpene glycoside components and phenolic acid components in the Cimicifugae Rhizoma extract, which led to a rapid and comprehensive analysis of the active components of the Cimicifugae Rhizoma extract. The in vitro part explored the renoprotective role of Cimicifugae Rhizoma extract and Isoferulic acid in mice podocyte cells. Under the condition of H₂O₂ (150 μM) + HG (30 mM), the cytotoxicity, cell apoptosis, oxidative stress, and inflammatory response of MPC5 cells aggravated immediately. Through MTT assay, flow cytometry apoptosis assay, and ELISA test, the Cimicifugae Rhizoma extract and Isoferulic acid were found to alleviate cytotoxicity, cell apoptosis, ROS level, and pro-inflammatory cytokines and maintain the healthy state of podocyte morphology. Also, the unique features of podocyte cells needed to be explored. The migration and adhesion abilities of MPC5 cells were degraded in the HG + H₂O₂-treated cell group. Isoferulic acid could reverse the decline in the migration and adhesion abilities of MPC5 cells.

However, the mechanisms and pathways behind the therapeutic efficacies of Cimicifugae Rhizoma extract and Isoferulic acid still need to be further studied. Not many studies have tried to investigate this area. The CXCL12/CXCR4 system is vital in the regulation of stem and progenitor cell trafficking in some processes of kidney injuries. CXCL12/CXCR4 signaling leads to several different intracellular signaling pathways and can culminate in multiple downstream effects like PI3K/Akt/mTOR and β-catenin [24]. Renal CXCR4 expression was observed to be increased in diabetic rats, while CXCL12/CXCR4 antagonistAMD3100 unmasked albuminuria and accelerated tubular epithelial cell death while reducing podocyte VEGF secretion. CXCR4 blockade prevented high-glucose/CXCL12-augmented phosphorylation of the pro-survival kinase. MMP9 was also up-regulated in diabetic nephropathy biopsies, and up-regulated MMP9 could prevent CXCL12/CXCR4 signaling in tubular epithelial cells [25]. On the other hand, DPP4 inhibition contributed to protection of the diabetes-injured kidney through CXCL12-dependent antioxidative and antifibrotic effects and the amelioration of adverse renal hemodynamics [26].

In the present study, we found that Cimicifugae Rhizoma extract and Isoferulic acid reversed the HG + H₂O₂-induced increase in CXCL12 and CXCR4 levels in MPC5 cells, while indicating a significant activation of CXCL12/CXCR4 system might relate to the diabetic nephropathy injury in mice podocyte cells. Consequentially, Cimicifugae Rhizoma extract and Isoferulic acid down-regulated mTOR, Caspase-3, and Cask. As the potential downstream target, mTOR differentially affects both podocyte apoptosis and autophagy in diabetic nephropathy. Caspase 3 has been shown to recognize and cleave a conserved DXXD common tetrapeptide motif, and the putative recognition site for Caspase 3 is found in the amino acid sequence of RelA/p65 [27]. Partial p65 cleavage-activated fragmentation by caspase 3 interferes with anti-apoptotic transcription conferred by RPS3/NF-κB, leading to programmed cell death [28]. High glucose induces podocyte apoptosis in the apoptotic pathway, and the expression of AMPK in apoptotic cells is inhibited and activates mTOR, whereas the inhibition of mTOR has no effect on the phosphorylation of AMPK, suggesting that mTOR is downstream of these signaling molecules [29]. Also, mTOR promotes the oxidative-stress-induced apoptosis of thylakoid cells in diabetic nephropathy [30]. The roles of mTOR in autophagy include rapamycin’s ability to effectively inhibit the mTOR/P70S6K/4EBP1 signaling pathway and activate pedicellular autophagy, thereby reducing pedicellular apoptosis [31]. Complex interactions between nephrin and adhesion junction proteins mediated by the scaffolding proteins Cask, CD2AP, and ZO-1 may be critical for maintaining the structural integrity and signaling properties of glomerular podocyte [32]. This is the first study linking Cask overexpression in podocytes to oxidative stress and high glucose damage.

The anti-oxidative stress properties of Isoferulic acid and Cimicifugae Rhizoma extract were examined. It was found that Isoferulic acid at the doses of 10, 25, and 50 μM and the Cimicifugae Rhizoma extract at the dose of 1 μg/mL could alleviate the oxidative stress damage induced by H₂O₂ in the high-glucose environment and could reduce the apoptosis and the level of intracellular ROS, increase the survival rate of the podocytes, maintain the morphology of the podocyte cytoskeleton, alleviate the burst of inflammation factors, and improve the motor ability of the damaged podocytes. And in the connection of apoptosis as well as inflammatory factor inhibition, 50 μM Isoferulic acid exhibited greater efficiency. In the modulation of CXCL12/CXCR4 signaling pathway and other associated proteins, the effectiveness of Isoferulic acid revealed a dose-dependent impact. Cimicifugae Rhizoma extract demonstrated a similar impact on the CXCL12/CXCR4 signaling pathway, although the modulatory effect on CXCR4 and Caspase-3 target proteins was marginally lower than that of 50 μM Isoferulic acid.

The research indicates that Isoferulic acid has a substantial antioxidant impact on H₂O₂-induced oxidative stress damage in the simulated diabetic nephropathy environment, which may preserve the podocytes and minimize the inflammatory response. The phenolic acid-richness of Cimicifugae Rhizoma also demonstrates effective mitigation of oxidative stress damage.

5. Conclusions

Overall, this study identified 82 components in Cimicifugae Rhizoma extract and 39 components in rat plasma. Cimicifugae Rhizoma extract (1 μg/mL) and Isoferulic acid (10, 25, 50 μM) significantly lowered high glucose and H₂O₂-mediated toxicity, reduced movement and binding and apoptotic changes in MPC5 podocytes, and reversed oxidative-stress-induced inflammation response. Cimicifugae Rhizoma extract and Isoferulic acid down-regulated Cask, mTOR, and Caspase-3. Moreover, Cimicifugae Rhizoma extract and Isoferulic acid treatment significantly stopped overactivation of CXCL12/CXCR4 in podocytes triggered by oxidative stress and high glucose. These results suggest that the protective mechanism of Cimicifugae Rhizoma extract and Isoferulic acid on MPC5 cells modelling diabetic nephropathy symptoms includes mainly the of CXCL12/CXCR4 pathways and the inhibition of oxidative-stress-mediated apoptotic pathways. Overall, our work found novel mechanisms by which Cimicifugae Rhizoma extract and its bioactive component detected in identification, Isoferulic acid, reduce oxidative stress reactions brought on in podocytes, suggesting their therapeutic potential in diabetic nephropathy. An in vivo experiment on streptozotocin-induced mice to confirm the renal and podocyte protection of Cimicifugae Rhizoma and Isoferulic acid and the pharmacokinetics study of phenolic acid components in Cimicifugae Rhizoma will be conducted in the future.