Component Analysis Based on UHPLC‐Q‐Exactive‐Orbitrap/MS and Immune Regulatory Activity Assessment by an LPS‐Stimulated Macrophages Model of Sacha Inchi Shell (Plukenetia volubilis Linneo) Extract

ABSTRACT

Sacha Inchi shell (SIS) is the fruit shell of Sacha Inchi (SI), a perennial woody vine belonging to the Euphorbiaceae family and native to the Amazon tropical rainforest region. Due to its various pharmacological effects such as antioxidant, anti‐inflammatory, and immune modulation, it has also attracted the attention of numerous scholars. We characterized and identified the Sacha Inchi shell water extract (SISWE) using UHPLC‐Q‐Exactive‐Orbitrap/MS technology. A total of 44 compounds were identified, which 30 components were detected in SISWE for the first time. We investigated the anti‐inflammatory and immunomodulatory effects of SISWE via using the normal RAW264.7 macrophage model and LPS‐induced inflammatory macrophage model. The research results indicated that SISWE enhanced the immune response by promoting the phagocytic ability, production of NO, TNF‐α, and IL‐6 in normal RAW264.7 cells, suppressed the excessive release of NO, IL‐6, and TNF‐α in LPS stimulated RAW264.7 cells, decreased the expression level of iNOS protein, and markedly inhibited the phosphorylation of p65, ERK, and JNK, demonstrating good immune regulatory effects. In this study, we demonstrated the bidirectional immunomodulatory function of SISWE. These findings highlight the therapeutic potential of SISWE for both immunodeficiency‐related diseases and immune hyperactivation‐related disorders.

The extract of SISE was characterized by LC‐MS, and 44 constituents were identified. Immune function evaluation confirmed that SISWE has a bidirectional immune regulation effect. Further mechanistic studies revealed that SISWE alleviates inflammatory response via suppressing NF‐KB and MAPK signaling pathways.

1. Introduction

Immunity is an extremely important self‐defense function of the human body, to maintain normal functions and resist various immune‐related diseases. An imbalance in immune regulation lead to a series of inflammatory and immune diseases, such as rheumatoid arthritis [1, 2, 3]. In recent years, the incidence rates of various immune diseases has gradually increased. According to statistics, immune diseases affect about 5%–8% of the world's population, causing great pain to patients [4].

Immune dysregulation can disrupt immune homeostasis, resulting in either excessive or insufficient immune responses that contribute to a wide range of immune‐related diseases. Immune overactivity is caused by the over protective response of the immune system when the body is invaded by pathogens, allergens, and so forth, for example, the overactivation of macrophages can lead to immune overactivity [5]. On the contrary, the decreased phagocytic ability of immune cells such as macrophages can lead to a decline in the body's immune function, resulting in immune deficiency syndromes and various immune deficiency diseases. Therefore, health problems caused by immune dysfunction have also received widespread attention from clinical doctors [6, 7].

Macrophages are immune cells specialized in the innate immune system, with a long survival time and phagocytic activity. They, together with neutrophils, is the first responders to infection and a key cell in the innate immune system of the human body. Macrophages participate in the recognition, phagocytosis, and degradation of cell debris and pathogens [8]. On the one hand, the sentinel function of macrophages can recruit and guide innate and adaptive immune cells, while they act as effectors to engulf pathogens, upregulate antimicrobial peptides, or trigger cell death to limit intracellular pathogens. This function relies on macrophages secreting inflammatory cytokines and presenting antigens to adaptive immune cells to initiate and coordinate local or systemic immune activation [9]. The ability of macrophages to engulf particles and secrete inflammatory cytokines reflects, to some extent, the immune function of the body [10]. A weak ability of macrophages to engulf and secrete inflammatory cytokines leads to weakened immune function. On the other hand, inducers such as Interferon‐γ (IFN‐γ) or LPS in the body can overactivate macrophages, causing them to release a large amount of inflammatory cytokines, which lead to immune dysregulation and trigger inflammatory immune diseases such as rheumatoid arthritis (RA). This study aims to explore the anti‐inflammatory and immune‐enhancing effects of SISWE based on two different states: activated macrophages and normal macrophages.

The occurrence of inflammation is a complex pathophysiological response. Normally, inflammation is beneficial to the body because it is an automatic defense and self‐protection behavior [11]. However, excessive or chronic inflammation can cause damage to various tissues in the human body, thereby participating in the occurrence and development of various diseases, including rheumatoid arthritis and cancer [12, 13]. In the inflammatory response, activated macrophages release a large amount of inflammatory factors such as IL‐6, TNF‐α. And promote the release of NO from cells, further exacerbating inflammation and leading to a cascade reaction of inflammation. The activation of Toll‐like receptors 4 (TLR‐4) receptors on the surface of macrophages initiates downstream inflammatory signaling pathways, including nuclear factor‐kappa B (NF‐κB), mitogen‐activated protein kinase (MAPK), and leads to increased protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) [14]. The MAPK and NF‐κB signaling pathways play a significant role in regulating inflammation [15, 16, 17]. Currently, NF‐κB and MAPK inhibitors play a significant role in the treatment of inflammation‐related diseases, including rheumatoid arthritis, in clinical practice [18]. Continuous activation of the NF‐κB and MAPK signaling pathways often leads to autoimmune diseases, chronic inflammation, and even cancer [19]. Therefore, inhibiting the activation of the NF‐κB signaling pathway, reducing the release of pro‐inflammatory factors such as NO, TNF‐α, and IL‐6, and effectively suppressing the inflammatory response constitute one of the strategies for intervening in the treatment of various diseases. Therefore, studying the bidirectional immune regulatory effects of drugs shows broad prospects in preventing and improving immune suppression and inflammatory diseases.

Sacha Inchi (Plukenetia volubilis Linneo), also known as South American oil vine, Inca fruit, or star oil vine, is a perennial woody vine in the Euphorbiaceae family, native to the Amazon plain [20]. Some studies have reported that SI is rich in various substances such as alpha linolenic acid, vitamin E, plant sterols, tocopherols, phenolic acids, and so forth [21, 22]. It has multiple effects such as antioxidant, anti‐inflammatory, lipid‐lowering, blood pressure‐lowering, immune enhancement, and memory improvement [23]. At present, there are relatively few reports on the composition of SISWE. Studies have shown that the main components of SISWE are sugars and proteins. It can reduce hypertension, improve oxidative stress damage, lower levels of inflammatory cytokines, and restore the intestinal barrier in lipopolysaccharide‐induced intestinal injury in mice [24]. The immune regulatory function of SISWE has attracted the attention of many scholars, but there are few reports on it, and its immune regulatory substance composition and mechanism are still unclear. Therefore, this study aims to further elucidate the chemical composition and immune regulatory activity of SISWE. In the early stages of research and development, researchers mainly focused on the kernels of SI, but neglected the development and utilization of SISWE, which are usually discarded as waste or used as fertilizers for plant cultivation. Based on the research idea of “turning waste into treasure,” the research team has visited the SI planting area in Yunnan province multiple times and found that SISWE are used by some local residents to brew tea for long‐term consumption (Figure 1). Their immunomodulatory and anti‐inflammatory effects have been widely spread among the people, indicating that SISWE have good medicinal development value and prospects, and are worthy of further research.

FIGURE 1.

SI planted in Simao district, Yunnan province (A), Sacha Inchi shell (B), Sacha Inchi shell powder (C), Sacha Inchi shell water extract (D).

2. Materials and Methods

2.1. Chemicals and Reagents

Chromatographic pure methanol, acetonitrile (Merck, Germany), liquid grade formic acid (Thermo Fisher Scientific.). All other reagents are analytical grade, quinic acid (batch number: A06N11L130218) was purchased from Shanghai Yuanye Biotechnology, purity is ≥98% (COA‑verified). Luteolin (batch number: M‐007‐190422), purity is ≥98% (COA‑verified), Kaempferol (batch number: S‐014‐171216), purity is ≥98% (COA‑verified) were purchased from Chengdu Ruifensi Biotechnology. Apigenin (batch number: 22042001) was purchased from Chengdu Pufide Biotechnology Co., Ltd, purity is ≥98% (COA‑verified). Chlorogenic acid (batch number: L‐007‐171216), purity is ≥98% (COA‑verified), isoquercitrin (batch number: Y‐076‐181025), purity is ≥98% (COA‑verified), cynaroside (batch number: M‐025‐181025), purity is ≥98% (COA‑verified) was purchased from Chengdu Ruifensi Biotechnology Co. Ltd. Gallic acid (batch number: PCS0637) was purchased from Chengdu Zhibiao Pure Biotechnology Co., Ltd, purity is ≥98% (COA‑verified). Caffeic acid (batch number: C108306), purity is ≥98% (COA‑verified), quercetin (batch number: Q111274), purity is ≥95% (COA‑verified), naringenin (batch number: N164488), purity is ≥97% (COA‑verified) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Rutin (batch number: A05GB144263), purity is ≥98% (COA‑verified) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Hyperoside (batch number: 111521‐201507), purity is ≥98% (COA‑verified) was purchased from China National Institute for Food and Drug Control. SISWE were purchased from Pu'er Lianzhong Biological Resources Development Co., Ltd. Dulbecco's modified Eagle medium (DMEM) culture medium (batch number: C1195500BT), FBS fetal bovine serum (batch number: 10091‐148), LPS lipopolysaccharide (batch number: L2880‐100MG), and DEX dexamethasone (batch number: D1756‐1G) were purchased from Gibco Corporation in the United States, dimethyl sulfoxide (DMSO) (batch number: 1084ML500) and phosphate buffered saline (PBS) (batch number: BL601A) were purchased from Guangzhou Saiguo Biotechnology Co., Ltd, MTT (batch number: A600799‐0005) was purchased from Shanghai Shenggong Bioengineering Co., Ltd., penicillin streptomycin (batch number: SV30010) from Hyclone Corporation in the United States, the mouse TNF‐α ELISA kit (batch number: 88‐7324‐88) and mouse IL‐6 ELISA kit (batch number: 88‐7064‐88) were purchased from Invitrogen in the United States, the NO detection kit (batch number: S0021M) and BCA protein quantification kit (batch number: P0010S) were purchased from Shanghai Biyuntian Biotechnology Research Institute. Loading buffer 5× (batch number: BL502B) purchased from Biosharp Life Science anti‐mouse IgG (batch number: 7076S), anti‐rabbit IgG (batch number: 7074P2), mouse anti‐β‐actin polyclonal antibody (batch number: 3700S), INOS (batch number: 2982S), COX‐2 (batch number: 1282S) rabbit antibody was purchased from Cell Signaling Technology in the United States.

2.2. Extraction of SISWE

The dried SIS powder was ground into a fine powder, and a 500 g aliquot was subjected to sequential reflux extraction with 15 volumes (v/w) of distilled water in a 20 L round‐bottom flask. The extraction was performed three times, each for 2 h under heating and refluxing conditions. After each cycle, the supernatant was collected and filtered. The combined filtrates were concentrated under reduced pressure using a rotary evaporator, followed by freeze‐drying to obtain the final extract. The yield of the water‐extracted SIS (SISWE) was 56 g, corresponding to an extraction rate of 11.2% (w/w).

2.3. Chromatographic Conditions

Waters ACQUITY UPLC BEH C₁₈ chromatographic column (100 mm × 2.1 mm, 1.7 µm), using a mobile phase of 0.1% formic acid aqueous solution (A)—acetonitrile (B) for gradient elution (0–3 min, 95% A, 3–8 min, 95%∼90% A, 8–12 min, 90%∼75% A, 12–16 min, 75%∼70% A, 16–21 min, 70%∼45% A, 21–25 min, 45%∼5% A, 25–26 min, 5%∼95% A, 26–30 min, 95% A). The flow rate is 0.3 mL min⁻¹, the injection volume is 2 µL, and the column temperature is 40°C.

2.4. Mass Spectrometry Conditions

The electric spray ion source is used, and the scanning mode of positive and negative ions is adopted. The resolution is 70 000, the sheath gas flow rate is set at 30 L/min, the auxiliary gas flow rate is 10 L/min, the capillary voltage is 3.50 kV, the temperature of the ion transfer tube is 320°C, the auxiliary gas temperature is 350°C, the mass spectrum scanning range is m/z 100∼1500, and the data acquisition method is Full MS‐ddMS₂.

2.5. Sample Preparation

The lyophilized SISWE powder (20 mg) was dissolved in 1 mL of ultrapure water. The resulting solution was centrifuged at 12 000 r/min for 20 min at 4°C (centrifuge radius: 3.385 cm). The supernatant was then collected and filtered through a 0.22 µm organic‐phase microporous membrane prior to UHPLC‐MS analysis.

2.6. Preparation of Mixed Standard Solution

Each of the 16 reference standards was accurately weighed and individually dissolved in methanol to prepare separate stock solutions at appropriate concentrations. From these stock solutions, aliquots were combined and diluted with methanol to obtain a series of mixed standard solutions. In the final mixed solutions, the mass concentration of each individual reference compound was adjusted to approximately 20 µg/mL.

2.7. Analysis Strategy

The purpose of this study was to use UHPLC‐Q‐Exactive Orbitrap MS to identify the components in SISWE. The prepared SISWE sample was injected into the UHPLC‐Q‐Exactive Orbitrap mass spectrometer to obtain high‐resolution MS data. In addition, UHPLC‐Q‐Exactive Orbitrap MS was combined with PRM scanning to collect the MS₂ data of the components. Finally, potential chemicals are identified by comparing them with standard samples, analyzing secondary ion fragment losses, and referencing the literature.

2.8. Cell and Culture

Mouse peritoneal macrophages RAW264.7 (RRID: CVCL_0493) were purchased from Wuhan Ponosi Life Technology Co., Ltd. and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PS). After the cell density reached 80%, cells in good condition were selected for the experiment [25].

2.9. MTT Assay for Detecting Cell Viability

RAW264.7 cells in good condition were taken and inoculated into 96‐well culture plates at a density of 5000 cells/well, with 100 µL per well. After cell adhesion, the cells were pre‑treated with different concentrations of SISWE (0–1600 µg/mL) for 2 h, and then incubated with LPS at a final concentration of 100 ng/mL for cultivation. After 24 h, the cell supernatant was discarded, and add 100 µL of DMEM culture medium and 10 µL of MTT were added to each well. After 2 h of reaction, measure the absorbance (OD value) was measured using a microplate reader at 490 nm [26].

2.10. Griess Method for Detecting NO Levels

To validate the effectiveness of the inflammatory cell model and evaluate the intervention effect of positive drugs, this study established dexamethasone (DEX) as a positive control drug. The cell grouping is as follows: normal group, model group, positive drug dexamethasone group (0.5 µM), SISWE (25, 100, and 400 µg/mL), with three wells in each group and three parallel experiments. Inoculate the cell suspension into a 48 well plate at a density of 1 × 10⁵ cells/well, with 300 µL per well, and culture overnight. Add the prepared drug to a 48 well plate at a rate of 300 µL/well, and add the normal and model groups to the complete culture medium. After 2 h, 3 µL of 10 µg/mL LPS (final concentration of 100 ng/mL) was added to the wells of cells other than the normal group, and cultured in an incubator for 18 h. For NO quantification, the Griess reagent assay was employed. A standard curve was established using serial dilutions of the 0.1 M nitrite standard provided in the NO detection kit, yielding final concentrations ranging from 0 µM to 100 µM. Both cell culture supernatants (samples) and the serially diluted standards were dispensed into a 96‐well plate at 50 µL per well. Subsequently, 50 µL of Griess Reagent I was added to each well, and the plate was incubated at room temperature in the dark for 10 min. Thereafter, 50 µL of Griess Reagent II was added, followed by a second incubation under the same conditions for another 10 min. The absorbance of each well was measured at 540 nm using a microplate reader. The NO release level in each group was then calculated by interpolating the sample absorbance values against the nitrite standard curve [25].

2.11. ELISA Detection of IL‐6, TNF‐α Secretion Level

RAW264.7 cells were cultured as described in Section 2.10. The cell supernatant was collected, and the absorbance was measured at 450 nm using an ELISA reader according to the instructions of the ELISA kit.

2.12. Neutral Red Experiment to Detect the Phagocytic Ability of SISWE

RAW264.7 cells in the logarithmic growth phase were selected from a cell culture bottle, counted, and resuspended in complete culture medium to prepare a cell suspension at a density of 2 × 10⁵ cells/well. Then, 100 µL of cell suspension was added to each well of a 96 well plate for cell plating. The plate was then incubated at 37°C in a 5% CO₂ incubator for 24 h. Carefully aspirate the cell culture supernatant and discard. RAW264.7 macrophages were seeded into 96‐well plates and incubated at 37°C in a 5% CO₂ atmosphere for 24 h. The culture supernatant was then aspirated and discarded. The cells were treated separately with various concentrations of SISWE (6.25, 25, 50, 100, 200, 400, and 800 µg/mL) or with LPS (1 µg/mL) as a positive control for phagocytosis. Following another 24 h of incubation, the drug‐containing supernatants were removed, and 200 µL of 0.1% neutral red solution was added to each well to assess cell viability. After 2 h of incubation, the neutral red solution was aspirated, and the cells were washed with PBS for further analysis [27]. After cultivation for 24 h, the cell culture supernatant containing different drugs was discarded from each well. Then 200 µL of 0.1% neutral red solution was added to each well, and the cells were observed under a microscope to check for any cell loss. After being incubated in the incubator for 2 h, the cells were removed and washed three times with warm 1 × PBS. Subsequently, 200 µL of cell lysate was added to each well and the plate was placed back in the incubator for 12 h. The absorbance was measured at 540 nm using a microplate reader [28]. The relative phagocytic rate was calculated according to the following formula:

$$\begin{array}{ccc}\hfill \text{Relative phagocytic rate}(\%)& =& \frac{A_{\text{experimental}}-A_{\text{blank}}}{A_{\text{control}}-A_{\text{blank}}}\times 100\%\hfill \end{array}$$

2.13. Western Blot Detection of COX‐2, iNOS, P‐P65, P65, ERK, P‐ERK, JNK, P‐JNK, p‐p38, p38 Protein Expression Levels in Cells

To validate the effectiveness of the inflammatory cell model and evaluate the intervention effect of positive drugs, this study established dexamethasone (DEX) as a positive control drug. The cell grouping is as follows: normal group, model group, positive drug dexamethasone group (0.5 µM), SISWE (25, 100, 400 µg/mL), with three wells in each group and three parallel experiments. Cells were inoculated into 6‑well plates at a density of 1 × 10⁶ cells/well, culture for 24 h, and the cell supernatant was discarded. After pretreatment with SISWE and the positive drug for 2 h, LPS at a final concentration of 100 ng/mL was added for stimulation for 24 h or 15 min. Then, RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF) was added to the plates to extract total protein. The protein concentration was measured using a bicinchoninic acid (BCA) assay kit. Protein samples of 20 µg were subjected to electrophoresis on 8%–10% polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies overnight. The next day, the membranes were incubated with secondary antibodies at room temperature for 1 h. Finally, protein bands were visualized using an electrochemiluminescence (ECL) detection solution with a chemiluminescence imaging system, and the relative expression levels of the target proteins were calculated.

2.14. Statistical Analysis

GraphPadPrism was used for statistical processing. The obtained data is expressed as mean ± standard error of mean (SEM), and one‐way ANOVA and t‐test are performed between groups. p < 0.05 Indicates that the difference is statistically significant.

3. Results

3.1. Identification of the Composition of SISWE

Through comparison with reference standards, databases, and literature, a total of 44 chemical components were identified from SISWE, including 22 phenolic acid compounds, 17 flavonoid compounds, 1 organic acid, 2 coumarins, and 2 lipid compounds. Among them, 13 were accurately identified, and the presence of 30 components was first reported in SISWE. The total ion chromatogram is shown in Figure 2, and the retention times, high‐resolution mass spectrometry information, and identification results for each chemical component are shown in Table 1.

FIGURE 2.

The components of SISWE were verified by UHPLC‐Q‐Exactive Orbitrap MS. (A) Negative ionization mode‐analysis of SISWE. (B) Positive ionization mode analysis of the SISWE.

TABLE 1.

Identification of 44 compounds from SISWE.

No.	TR (min)	Identification	Category	Formula	Ion mode	Theo. mass (m/z)	Obser. mass (m/z)	Error (ppm)	MS/MS fragments (m/z)	Refs.
1 [Link] , [Link]	0.84	Quinic acid	Phenolic acids	C7H12O6	[M‐H]−	191.05611	191.05515	−5.032	85.02804, 87.00735, 57.03316, 93.03299
2 b	0.97	Citric acid	Organic acid	C6H8O7	[M‐H]−	191.01972	191.01877	−5.004	111.00732, 87.00736	[29]
3 b	1.09	1′‐O‐galloylsucrose	Phenolic acids	C13H16O10	[M‐H]−	331.06706	331.06683	−0.724	169.01317, 125.02312, 107.01255, 69.03300	[30]
4	1.48	Vanillic acid	Phenolic acids	C8H8O4	[M‐H]−	167.03498	167.03391	−6.418	123.00724	[31]
5 a	1.64	Gallic acid	Phenolic acids	C7H6O5	[M‐H]−	169.01424	169.01315	−6.488	124.01543, 97.02814, 79.01749, 69.03313
6 b	1.91	4‐O‐Methylgallic acid	Phenolic acids	C8H8O5	[M‐H]−	183.02989	183.02898	−5.008	124.01523, 69.03294	[32]
7	2.78	Vanillin acid glucoside	Phenolic acids	C14H18O9	[M‐H]−	329.0878	329.08792	0.349	108.02027, 123.04391, 152.01033, 167.03380	[33]
8	2.96	Protocatechuic acid	Phenolic acids	C7H6O4	[M‐H]−	153.01933	153.0183	−6.744	109.02832, 91.01754	[34]
9 b	3.18	4‐Hydroxybenzoic acid glucoside	Phenolic acids	C13H16O8	[M‐H]−	299.07724	299.07718	−0.203	137.02336, 93.03315	[32]
10 b	3.55	1‐Caffeoylquinic acid	Phenolic acids	C16H18O9	[M‐H]−	353.0878	353.08765	−0.44	191.05507, 135.04419, 179.03380	[35]
11 b	3.65	Dihydroxybenzoic acid‐O‐glucoside	Phenolic acids	C13H16O9	[M‐H]−	315.07215	315.07211	−0.144	137.02318, 93.03314	[36]
12 b	3.85	4‐Hydroxybenzoic acid glucoside isomer	Phenolic acids	C13H16O8	[M‐H]−	299.07724	299.07697	−0.905	137.02324, 93.03316	[32]
13 b	4.74	Caffeic acid‐4‐O‐glucoside	Phenolic acids	C15H18O9	[M‐H]−	341.08780	341.08743	−1.1	161.02336, 179.03375, 59.01241, 135.04388	[32]
14	4.67	4‐Hydroxybenzoic acid	Phenolic acids	C7H6O3	[M‐H]−	137.02441	137.02319	−8.975	93.03314, 94.03661, 65.03822	[36]
15	5.99	Chlorogenic acid‌	Phenolic acids	C16H18O9	[M‐H]−	353.0878	353.08759	−0.61	191.05519, 173.04449, 135.04370, 161.02356, 93.03335	[35]
16 b	6.11	Brevifolincarboxylic acid	Phenolic acids	C13H8O8	[M‐H]−	291.01464	291.01431	−1.135	247.02431, 203.03354, 219.02917, 191.03406, 173.02336, 145.02834	[37, 38]
17 a	6.41	Caffeic acid	Phenolic acids	C9H8O4	[M‐H]−	179.03498	179.03397	−5.635	135.04398, 117.03302, 107.04881, 89.03821
18 b	7.37	Taxfolin‐7‐o‐rhamnoside	Flavonoids	C21H22O11	[M‐H]−	449.10893	449.10889	−0.099	151.00240, 121.02800, 107.04893, 259.06079	[30]
19 b	8.6	Taxfolin‐7‐o‐rhamnoside isomer	Flavonoids	C21H22O11	[M‐H]−	449.10893	449.10858	−0.79	125.02309, 259.00079, 151.00240, 121.02800, 107.04893	[30]
20 b	8.67	Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate	Lipid	C12H16O5	[M‐H]−	239.09249	239.09249	−2.245	59.01242, 109.06404, 95.04861, 133.06529	HMDB
21	9.20	p‐Coumaric acid	Phenolic acids	C9H8O3	[M‐H]−	163.04006	163.03889	−7.222	119.04885, 93.03308	[39]
22 b	9.93	Neochlorogenic acid	Phenolic acids	C16H18O9	[M‐H]−	353.0878	353.08517	−7.464	191.03488, 135.08025, 161.02139, 179.03162
23 b	11.57	Dihydroquercetin	Flavonoids	C15H12O7	[M‐H]−	303.05102	303.05081	−0.713	151.03868, 125.02310, 121.02814, 107.01255
24 b	11.84	Daphnetin	Coumarin	C9H6O4	[M‐H]−	179.03388	179.03368	−1.146	179.03371, 91.01820, 103.05408, 77.03909, 65.03924, 51.02366	PubChem
25 b	12.12	Naringin‐O‐glucoside	Flavonoids	C21H22O10	[M‐H]−	433.11402	433.11371	−0.716	271.06091, 151.00249, 119.04886	[32]
26 b	12.13	Apiin	Flavonoids	C26H28O14	[M‐H]−	563.14062	563.14069	0.109	353.06818,	[39]
27 b	12.32	Rutin	Flavonoids	C27H30O16	[M‐H]−	609.1461	609.14594	−0.276	285.03934, 151.00253, 178.99767, 300.99911, 255.03011, 227.03300
28 b	12.63	Naringenin 7‐O‐gentiobioside	Flavonoids	C27H32O15	[M‐H]−	595.16684	595.16693	0.146	151.00262, 271.06073	[32]
29 b	12.73	Ellagic acid	Phenolic acids	C14H6O8	[M‐H]−	300.99899	300.99854	−1.496	283.99622, 257.00842, 229.01358, 185.02298
30 b	12.79	Hyperoside	Flavonoids	C21H20O12	[M‐H]−	463.08819	463.08716	−2.244	300.02753, 151.00246, 178.99695, 271.02432,
31 b	13.11	Isoquercitrin	Flavonoids	C21H20O12	[M‐H]−	463.08819	463.0881	−0.214	300.02753, 301.03424, 271.02432, 151.00246, 178.99695
32 b	13.74	Kaempferol‐3‐O‐glucoside	Flavonoids	C21H20O11	[M‐H]−	447.09328	447.09286	−0.950	135.04410, 153.05464, 285.17050, 271.06082, 151.00246, 119.04890	[39]
33 b	13.93	Aromadendrin	Flavonoids	C15H12O6	[M‐H]−	287.05611	287.05579	−1.119	153.01785, 135.04373, 125.02310, 107.01250, 151.00243, 177.05467, 173.05988	[39]
34 b	14.09	Luteolin‐7‐O‐glucoside	Flavonoids	C21H20O11	[M‐H]−	447.09328	447.09305	−0.525	284.03235, 285.03864, 151.00255, 271.06152, 255.02946
35 b	14.92	6‐Methoxytaxifolin	Flavonoids	C16H14O7	[M‐H]−	317.06667	317.06644	−0.744	300.99454, 301.99695, 151.00220, 178.99731, 107.01230	[30]
36 b	15.21	4‐p‐Coumaroylquinic acid	Phenolic acids	C16H18O8	[M‐H]−	337.09289	337.09293	0.117	191.03412, 163.03896	[40]
37 a	16.28	Quercetin	Flavonoids	C15H10O7	[M‐H]−	301.03537	301.03531	−0.219	151.00258, 107.01249, 121.02820
38 b	16.76	3,3'‐Di‐O‐methyltannic acid.	Phenolic acids	C16H10O8	[M‐H]−	329.03029	329.02985	−1.339	312.99881, 298.98291, 270.98813, 285.00491, 271.99261, 242.99239	[41]
39 a	17.19	Naringenin	Flavonoids	C15H12O5	[M‐H]−	271.06119	271.06091	−1.058	119.04887, 107.01246, 151.00253, 93.03311
40	17.65	Coumarin	Coumarin	C9H6O2	[M + H]−	147.04405	147.04375	−2.081	147.09062, 119.04877, 103.05398, 91.05452, 77.03870, 65.03917, 51.02369	[35]
41 a	17.78	Apigenin	Flavonoids	C15H10O5	[M‐H]−	269.04554	269.0455	−0.173	117.03322, 151.00227, 107.01273, 121.02824
42 a	17.89	Kaempferol	Flavonoids	C15H10O6	[M‐H]−	285.04046	285.04007	−1.373	135.00754, 211.03992, 229.04938, 185.05995, 151.00246, 187.03897
43 b	18.93	3,3',4‐Tri‐O‐methylellagic acid	Phenolic acids	C17H12O8	[M‐H]−	343.04594	343.04547	−1.372	328.02112, 298.97952, 312.99893, 297.97513, 285.00357, 269.98031	[41]
44	19.27	3‐Hydroxydecanoic acid	Fatty acyls	C10H20O3	[M‐H]−	187.13396	187.13307	−4.797	59.01242	PubChem

^aComparison with standard samples.

^bFirst identification in SISWE.

3.1.1. Identification of Phenolic Acids

A total of 22 phenolic acid compounds were identified in SISWE, among which compounds 1, 5, 17, and 29 were identified by standard comparison, with retention times of 0.84, 1.64, 6.41, and 12.73 min, respectively. By comparison of the retention times and mass spectrometry data with those of the corresponding standards, they were identified as quinic acid, gallic acid (Figures S1 and S2), caffeic acid (Figures S3 and S4), and tannic acid, respectively.

The retention time of compound 16 is 6.11 min, and its fitted molecular formula was C₁₃H₈O₈, with the quasi‑molecular ion peak observed at m/z 291.01431 [M‐H]⁻. Upon loss of a carboxyl group (─COOH), the fragment ion at m/z 247.02431 [M‐H–COOH]⁻ is obtained. Successive losses of two CO groups then yielded product ions at m/z 219.02917 [M‐H–COOH‐CO]⁻ and m/z 191.03406 [M‐H–COOH‐2CO]⁻, additionally, a fragment ion corresponding to the loss of 2× COO was observed at m/z 203.03354 [M‐H–2COO]⁻. This may be due to the instability of carboxyl and carbonyl groups, and combined with literature reports, it is speculated to be brevifolincarboxylic acid, the fragmentation pattern is shown in Figure 3 [42].

FIGURE 3.

Possible cleavage pathways of brevifolincarbolic acid.

The molecular formula of C₁₇H₁₂O₈ was inferred based on the parent ion at m/z 343.04547 [M‐H]⁻ for compound 43, and the MS/MS fragment ion were analyzed. The following fragment ions were detected: m/z 328.02112 [M‐H–CH₃]⁻, m/z 312.99893 [M‐H–2CH₃]⁻, m/z 297.97513 [M‐H–3CH₃]⁻, 269.98031 [M‐H–3CH₃‐CO]⁻. Successive neutral losses of CH₃ groups were observed, which may be attributed to the instability of the methoxy groups on the benzene ring [43]. Based on these data, the fragment ions at m/z 343.04594 [M‐H]⁻, 329.03029 [M‐H]⁻, and 300.99899 [M‐H]⁻ are attributed to 3,3',4‐tri‐O‐methylellagic acid, respectively, the fragmentation pattern is shown in Figure 4.

FIGURE 4.

Possible cleavage pathways of 3,3'‐di‐O‐methyltannic acid.

3.1.2. Identification of Flavonoids

In SISWE, a total of 17 flavonoids were identified, including compounds 18, 19, 23, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 39, 41, and 42. Among them, compounds 23, 27, 30, 31, 34, 37, 39, 41, and 42 were identified by standard comparison, with retention times of 11.57, 12.32, 13.11, 13.11, 14.09, 16.28, 17.19, 17.78, and 17.89 min, respectively. By comparing their retention time and mass spectrometry data with standard samples, they were identified as dihydroquercetin, rutin, hyperoside, isoquercitrin, luteolin‐7‐O‐glucoside, quercetin, naringenin, apigenin, and kaempferol, respectively. Flavonoids mainly undergo reverse Diels–Alder reactions, loss of functional groups such as CO, CO₂, and H₂O, as well as rearrangement reactions.

The retention time of compound 35 is 14.92 min, and its fitted molecular formula was C₁₆H₁₄O₇, with the quasi‑molecular ion peak observed at m/z 317.06644 [M‐H]⁻. Successive loss of a CH₃ group and a H atom yielded ions at m/z 301.99695 [M‐H–CH₃]⁻ and m/z 300.99454 [M‐H–CH₃‐H]⁻. Additional MS/MS fragment ions were observed at m/z 151.00220, 107.01230, which are characteristic ions of quercetin, based on these data, compound 35 was tentatively identified as 6‑methoxytaxifolin, and its fragmentation pattern is shown in Figure 5.

FIGURE 5.

Possible cleavage pathways of 6‐methoxytaxifolin.

3.1.3. Identification of Organic Acid

One organic acid compound, compound 2, was identified in SISWE. Organic acid compounds tend to lose neutral fragments such as CO₂ and H₂O in negative ion mode. The retention time of compound 2 had a retention time of 0.97 min, and its molecular formula was determined as C₆H₈O₇, with the quasi molecular ion peak is m/z 191.01877 [M‐H]⁻. Upon successive loss of two H₂O molecules and one CO₂ molecule, the fragment ion at m/z 111.00732 [M‐H–2H₂O‐CO₂]⁻ was obtained. Based on literature reports, it is speculated to be citric acid.

3.1.4. Identification of Coumarins

Two coumarin compounds were identified in SISWE, namely compounds 24 and 40. Compounds 24 and 40 were discovered at 11.84 and 17.65 min, respectively. For compound 40, fragment ions were observed at m/z 103.05398 [M‐H–CO₂]⁻, m/z 77.03870 [M‐H–CO₂‐C₂H₂]⁻, and m/z 51.02369 [M‐H–CO₂‐2C₂H₂]⁻ indicating successive loss of one CO₂ molecule followed by two C₂H₂ molecules. In addition, fragmentation of the parent ion yielded ions at 119.04877 [M‐H‐CO]⁻, m/z 91.05452 [M‐H‐2CO]⁻, with further loss of one C₂H₂ group giving m/z 65.03917 [M‐H‐2CO‐C₂H₂]⁻, which is speculated to be coumarin. its proposed cleavage pathway is shown in Figure 6. Similarly, compound 24 exhibited fragment ions at m/z 179.03371 [M‐H]⁻, m/z 91.01820 [M‐H‐2CO]⁻, m/z 103.05408 [M‐H–CO₂]⁻, m/z 77.03909 [M‐H‐CO₂‐C₂H₂]⁻, m/z 65.03924 [M‐H–2CO‐C₂H₂]⁻, m/z 51.02366 [M‐H–CO₂‐2C₂H₂]⁻, it is speculated to be daphnetin.

FIGURE 6.

Possible cleavage pathways of coumarin.

3.2. The Effect of SISWE on the Proliferation of RAW264.7 Cells

Compared with the control group, when the concentration of SISWE was in the range of 25–800 µg/mL, the cell viability significantly increased. When the concentration increased to 1600 µg/mL, the cell survival rate significantly decreased, indicating that high concentrations of SISWE exhibited certain cytotoxicity (Figure 7A). After LPS stimulation, there was no significant difference between the SISWE groups at different concentrations and the LPS group, indicating that SISWE had no significant effect on LPS‐induced RAW264.7 cell viability (Figure 7B). Therefore, concentrations of 25, 100, and 400 µg/mL of SISWE were selected as the treatment concentrations for subsequent experiments.

FIGURE 7.

The effect of SISWE on the survival rate and NO release of normal macrophages and LPS‐induced RAW264.7 macrophages. (A) Effect of SISWE on RAW264.7 cell viability. RAW264.7 cells treated with SISWE were subjected to MTT assay at a dose of 6.25–1600 µg/mL for 24 h. (B) Effect of SISWE on LPS‐induced RAW264.7 cell viability. LPS‐induced RAW264.7 cells treated with SISWE were subjected to MTT assay at a dose of 6.25–1600 µg/mL for 24 h. (C) RAW264.7 cells treated with SISWE were stimulated at doses of 25, 100, and 400 µg/mL for 18 h, and the NO content was detected. (D) LPS‐induced RAW264.7 cells treated with SISWE were stimulated at doses of 25, 100, and 400 µg/mL for 18 h, and the NO content was detected. Data are presented as the means ± SEM (n = 3). Compared with the control group, #p < 0.05, ##p < 0.01, and ###p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, and ***p < 0.001.

3.3. The Effect of SISWE on NO Secretion Levels in Normal RAW264.7 Cells and LPS‐Induced RAW264.7 Cells

Different concentrations of SISWE (25, 100, 400 µg/mL) dose dependently inhibited the release of NO in LPS‐induced RAW264.7 cell inflammation model after 24 h (Figure 7C). After stimulation with LPS (100 ng/mL), the NO concentration in the model group was significantly increased. Compared with the model group, both the SISWE and positive dexamethasone groups significantly inhibited the concentration level of NO in the LPS‐induced RAW264.7 cell model. These results indicates that SISWE exerts a significant inhibitory effect on the concentration of NO from LPS‐induced inflammatory cells. Our study also found that SISWE promoted the release of NO in normal RAW264.7 cells at different concentrations of 400 µg/mL (Figure 7D). These results indicate that SISWE can promote the concentration of NO in normal RAW264.7 cells, suggesting that SISWE may enhance immune function. These research results indicate that SISWE exhibits bidirectional immune regulatory effects related to context‐dependent under both basal and LPS stimulation conditions.

3.4. Effects of SISWE on IL‐6 and TNF‐α Secretion Levels in Normal RAW264.7 Cells and LPS‐Induced RAW264.7 Cells

The research results showed that compared with the blank control group, treatment with SISWE for 24 h significantly increased the levels of IL‐6 and TNF‐α in non‐activated RAW264.7 cells at a concentration of 400 µg/mL (Figure 8A,B). After establishing the LPS (100 ng/mL)‐induced RAW264.7 inflammation model, the levels of IL‐6 and TNF‐α in the model group were significantly increased. Compared with the model group, different concentrations of SISWE (final concentrations: 25, 100, and 400 µg/mL) significantly reduced the levels of IL‐6 and TNF‐α in activated RAW264.7 cells after 24 h of treatment. At a concentration of 400 µg/mL, the levels of IL‑6 and TNF‑α were significantly reduced. Both the SISWE groups and the positive drug group exhibited significant inhibitory effects. These results indicate that SISWE inhibits the secretion of IL‐6 and TNF‐α in activated RAW264.7 cells induced by LPS (Figure 8C,D).

FIGURE 8.

Effects of SISWE on the secretion of inflammatory factors in the supernatant of cell culture. (A) RAW264.7 cells treated with SISWE were stimulated at doses of 25, 100, and 400 µg/mL for 18 h, and TNF‐α levels were detected. (B) RAW264.7 cells treated with SISWE were stimulated at doses of 25, 100, and 400 µg/mL for 18 h, and IL‐6 levels were detected. (C) LPS‐induced RAW264.7 cells treated with SISWE were stimulated at doses of 25, 100, and 400 µg/mL for 18 h, and TNF‐α levels were detected. (D) LPS‐induced RAW264.7 cells treated with SISWE were stimulated at doses of 25, 100, and 400 µg/mL for 18 h, and IL‐6 levels were detected. Data are presented as the means ± SEM (n = 3). Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. The Effect of SISWE on the Phagocytic Ability of RAW264.7 Cells

When the concentration of SISWE ranged between 6.25 µg/mL and 100 µg/mL, the relative phagocytic rate of RAW264.7 cells toward neutral red increased gradually in a concentration‑dependent manner. At a concentration of 50 µg/mL, SISWE significantly enhance the phagocytic activity of RAW264.7 cells toward neutral red, and the relative phagocytic rate was not significantly different from that of the positive control LPS group (100 ng/mL). The relative phagocytosis rate of neutral red by SISWE at 100 µg/mL was significantly higher than that of the LPS group. These results indicate that SISWE can significantly enhance the phagocytic ability of RAW264.7 cells and exhibits high immune modulatory activity (Figure 9).

FIGURE 9.

RAW264.7 cells were treated with various concentrations of SISWE (6.25–800 µg/mL) in the presence or absence of LPS (1 µg/mL). LPS (1 µg/mL) was used as the positive control for phagocytic activation. Data are presented as means ± SEM (n = 3). *p < 0.05, **p < 0.01 versus control group; #p < 0.05 versus LPS group.

3.6. Effect of SISWE on iNOS and COX‐2 Protein Expression in LPS‐Induced RAW264.7 Cells

Compared with the blank control group, treatment of RAW264.7 cells with LPS for 24 h increased the expression levels of COX‑2 and iNOS proteins. Compared with the LPS group, different concentrations of SISWE (25, 100, 400 µg/mL) in the presence of LPS  significantly reduced iNOS protein expression, but did not significantly affect COX‐2 protein expression (Figure 10).

FIGURE 10.

Effects of SISWE on the level of iNOS and COX‐2 proteins in LPS‐induced RAW264.7 cells. Western blot was used to detect the expression levels of p‐p65 and p65 proteins in RAW264.7 cells induced by LPS stimulation at 25, 50, and 100 µg/mL SISWE for 18 h. Data are presented as the means ± SEM (n = 3). Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

3.7. The Effect of SISWE on NF‐κB Signaling Pathway

To investigate the effect of SISWE on LPS‑induced NF‑κB activation, all experimental groups (except the blank control) were stimulated with LPS (100 g/mL). Compared with the blank control group cells, treatment of RAW264.7 cells with LPS for 24 h increased the protein expression level of phosphorylated p‑p65. Compared with the LPS group, different concentrations of SISWE (100, 400 µg/mL) significantly reduced the expression of p‐p65 protein in a concentration‐dependent manner. These findings indicate that the SISWE may exert anti‐inflammatory effects by affecting the phosphorylation of p65 in the NF‐κB signaling pathway (Figure 11).

FIGURE 11.

Effects of SISWE on the level of p‐p65 and p65 proteins in LPS‐induced RAW264.7 cells. Western blot was used to detect the expression levels of p‐p65 and p65 proteins in RAW264.7 cells induced by LPS stimulation at 25, 50, and 100 µg/mL SISWE for 0.5 h. Data are presented as the means ± SEM (n = 3). Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compare with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

3.8. The Effect of SISWE on MAPK Signaling Pathway

To investigate the effect of SISWE on LPS‑induced NF‑κB activation, all experimental groups (except the blank control) were stimulated with LPS (100 g/mL). As shown in Figure 12, compared with the blank control group cells, treatment of RAW264.7 cells with LPS for 25 min significantly increased the phosphorylation levels of ERK, JNK, and p38 in the MAPK signaling pathway. Compared with the LPS group, different concentrations of SISWE (25, 100, and 400 µg/mL) significantly reduced the phosphorylated protein expression levels of ERK and JNK in a concentration‐dependent manner, but had no significant effect on the phosphorylation of p38. These findings indicate that SISWE may exert its anti‑inflammatory effects by modulating the phosphorylation of ERK and JNK in the MAPK signaling pathway.

FIGURE 12.

Effects of SISWE on the level of ERK, p‐ERK, JNK, p‐JNK, p‐p38, p38 proteins in LPS‐induced RAW264.7 cells. Western blot was used to detect the expression levels of ERK, p‐ERK, JNK, p‐JNK, p‐p38, p38 proteins in RAW264.7 cells induced by LPS stimulation at 25, 50, and 100 µg/mL SISWE for 0.5 h. Data are presented as the means ± SEM (n = 3). Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

A total of 44 compounds were identified using UHPLC‑Q‑Exactive Orbitrap MS. It has been shown that studies have purified phenolic compounds from the shell of SI using macroporous resin vanillic acid glucoside, vanillic acid, p‐hydroxybenzoic acid and p‐coumaric acid. A study has identified kaempferol, gallic acid, 4‐hydroxybenzoic acid, caffeic acid, p‐coumaric acid, quercetin, and naringenin in SIS including quinic acid, citric acid, 1′‐O‐galloylsucrose, 4‐O‐methylgallic acid, 4‐hydroxybenzoic acid β‐glucoside, 1‐caffeoylquinic acid, dihydroxybenzoic acid‐O‐glucoside, 4‐hydroxybenzoic acid β‐glucoside, caffeic acid‐O‐glucoside, brevifolincarboxylic acid, taxfolin‐7‐o‐rhamnoside, taxfolin‐7‐o‐rhamnoside isomer, isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate, neochlorogenic acid, dihydroquercetin, daphnetin, naringenin‐glucoside, apiin, rutin, naringenin 7‐O‐gentiobioside, ellagic acid, hyperoside, isoquercitrin, kaempferol‐3‐O‐glucoside, aromadendrin, luteolin‐7‐O‐glucoside, 6‐methoxytaxifolin, 4,5‐di‐p‐cis‐coumaroylquinic acid, 3,3′‐di‐O‐methylellagic acid, trimethylellagic acid and first identified from SISWE. Notably, most of these compounds are polyphenols and flavonoids, which are known to possess good immune‑regulatory properties. On this basis, we propose that SISWE also exhibits good immune‑regulatory activity.

Inflammation is an immune response that can be triggered by various factors. The inflammatory response is an important self‑protective mechanism of the body, in which macrophage activation represents the initiating process of the inflammatory response [19]. After activation, macrophages exhibit enhanced functional activities. To a certain extent, this enhancement is beneficial to the body. However, if this enhancement exceeds the physiological limit, it may lead to functional impairment and tissue damage. Previous studies have reported that during a short period, the body releases large amounts of NO, which can exert cytotoxic effects against tumor cells. Nevertheless, prolonged accumulation of NO can cause persistent oxidative damage and cellular dysfunction, which may contribute to pathological tissue injury when the inflammatory response exceeds the physiological limit. At a certain concentration range, TNF‑α is beneficial to the body, as it helps regulate the body's immune response. When the body is infected, TNF‐α exerts anti‐infection effects and promote tissue repair. In the early stages of cancer, TNF‐α can directly induce apoptosis of tumor cells, However, despite these important functions, once its level exceeds the physiological threshold, it can also cause damage to the body, such as the pathological damage associated with post‑traumatic inflammatory responses [44]. The LPS‐induced RAW264.7 cell model is a commonly used in vitro inflammatory cell model, and the bioactivity of many traditional Chinese medicine extracts has been confirmed using this model [45]. In the present experiment, we used LPS to stimulate RAW264.7 macrophages to establish an inflammatory model, in order to further investigate the possible anti‐inflammatory mechanism of action of SISWE [46]. When RAW264.7 cells are stimulated by external factors, they promote the production of pro‐inflammatory mediators such as NO, TNF‐α, IL‐6, and so forth. In this study, we first evaluated the cytotoxicity of SISWE on RAW 264.7 cells using MTT colorimetric assay, with the aim of establishing a concentration range without cytotoxic effects for subsequent immune regulatory function evaluation. The results showed that when the concentration of SISWE was below 400 µg/mL, the cell viability remained above 90%. Therefore, 25, 100, and 400 µg/mL were selected as safe concentrations for subsequent NO and cytokine detection. NO is a dual regulator of inflammation, which can promote synovial blood flow, regulate synovial function, and participate in the pathological process of RA, is production is regulated by iNOS expression. COX‐2 is a key enzyme in prostaglandin biosynthesis of prostaglandins, catalyzing the formation of prostaglandins from arachidonic acid, which generates a series of inflammatory mediators and participates in various physiological and pathological processes through inflammatory cascade reactions [47]. TNF‐α is the earliest inflammatory mediator released during the inflammatory response. When the body is exposed to LPS, TNF‑α is rapidly and massively produced, and it can enhance the chemotaxis of neutrophils and synergize with IL‑6, thereby enhancing macrophage activity and amplifying the inflammatory response. IL‐6 can work synergistically with other inflammatory mediators, further amplifying the inflammatory response and inducing tissue damage. It can also serve as a biomarker for assessing the severity of inflammation [48]. One study has investigated the anti‐inflammatory activity of physalis and neophysalins from the calyx of Physalis alkekengi by establishing an LPS‐induced RAW264.7 cell model, which may inhibit the expression levels of NO and IL‐6, iNOS, and COX‐2 [49]. Therefore, suppressing iNOS expression and subsequently reducing the release of pro‑inflammatory mediators is regarded as one of the key strategies for the treatment of inflammation. The results of the present study indicate that the SISWE significantly prevented LPS‐induced concentration of NO, IL‐6, and TNF‐α in RAW264.7 cells at ranging from 25 µg/mL to 400 µg/mL, while also significantly downregulating the expression level of iNOS protein. In parallel, in normal RAW264.7 macrophages, SISWE promoted macrophage proliferation, enhanced phagocytic activity, and increased the production of NO, IL‑6, and TNF‑α, thereby enhancing immune function.

Many traditional Chinese medicines have also been proven to possess bidirectional immune‐regulatory properties, such as Huangqi, which exerts effects including immune enhancement and anti‑inflammatory/immune modulation [50]. The present research aims to provide a theoretical basis for developing functional foods with bidirectional immunomodulatory activity derived from SISWE. SISWE was found to upregulate the NO production ability of normal RAW264.7 cells to a certain extent, and also downregulate the excessive production of NO, TNF‐α, IL‐6 in LPS‑induced RAW264.7 cells to a certain extent. Therefore, it can be concluded that SISWE exerts a bidirectional regulatory effect on the production of NO, TNF‐α, IL‐6 by RAW264.7 cells. When the production of these cytokines is markedly enhanced, as in the case of LPS stimulation, which already triggers an inflammatory response in RAW264.7 cells. SISWE can only enhance the production of NO, TNF‑α, and IL‑6 to a limited extent, suggesting that it may enhance the normal immunity without inducing inflammatory reactions. On the one hand, SISWE predominantly enhances the proliferation and phagocytic capacity of macrophages, thereby strengthening innate immune function by promoting NO, TNF‑α, and IL‑6 production. On the other hand, in hyperactivated models induced by LPS, SISWE simultaneously reduces excessive immune responses by inhibiting NO, TNF‑α, and IL‑6 production. Therefore, it can be considered that SISWE exerts immunoenhancing effects in normal organisms, whereas it exerts potent immunosuppressive effects in organisms with hyperactive immunity.

The results of this study indicate that SISWE exhibits dual characteristics in regulating the function of macrophages. On the one hand, at rest, SISWE exhibits a typical toxic excitatory effect on phagocytic function, which increases phagocytic activity in a concentration‐dependent manner within the range of 6.25–100 µg/mL. After exceeding 100 µg/mL, the enhancing effect weakens and shows an inhibitory trend. At the same time, inflammatory factors such as TNF‐α, IL‐6, and NO also show a certain degree of concentration (25–400 µg/mL)‐dependent increase under resting conditions, indicating that SISWE has a broad positive regulatory effect on immune function in a steady‐state environment. On the other hand, in the LPS‐induced inflammatory microenvironment, the direction of action of SISWE is reversed, significantly inhibiting the excessive production of the above‐mentioned inflammatory factors, reflecting the environment dependent bidirectional immune regulatory ability, that is, enhancing immune surveillance in steady state and inhibiting pathological response during inflammation [51]. The above two effects may stem from the background dependent regulation of key signaling pathways such as MAPK/NF‐κB by SISWE, which provides mild stimulation to enhance basal function in resting cells and inhibits signal hyperactivity in overactivated inflammatory cells. In summary, SISWE has dual characteristics of bidirectional immune regulation, suggesting that it may serve as an “intelligent regulator” that dynamically adjusts its direction of action based on the body's immune status, and has potential application value in both immunocompromised and hyperactive states.

Nuclear transcription factor kappa B is a family of transcription factors with pleiotropic regulatory effects, and plays important roles in the transcription and expression of multiple genes as well as in the regulation of inflammatory responses [52]. Upon LPS stimulation of cells, NF‐κB is released and rapidly translocated to the nucleus, where it activates transcription of its target genes and modulates the release of pro‑inflammatory factors [53]. Previous studies have shown that hyperactivation of NF‐κB is an important regulatory hub for degenerative diseases including intervertebral disc degeneration, osteoporosis, and osteoarthritis. Our findings demonstrate that the expression level of phosphorylated p65 (p‑p65) was significantly upregulated in LPS‑induced RAW264.7 cells, whereas the expression of p‑p65 was significantly downregulated in cells treated with SISWE (100–400 µg/mL), suggesting that SISWE can inhibit the activation of the NF‑κB signaling pathway in RAW264.7 cells (Figure 11). Meanwhile, studies have shown that the compounds identified in SISWE are mostly polyphenolic and flavonoid compounds, which usually have good anti‐inflammatory and antioxidant effects. Research reported that rutin inhibits BHBA‐triggered hepatocyte inflammation and lipid accumulation by modulating the NF‐κB signaling pathway, implying that rutin may be a promising target for ameliorating damage in when subclinical ketosis (SCK) cows [54]. Kaempferol is a natural flavonoid with anti‐inflammatory properties. Research has shown that kaempferol significantly inhibits inflammation, CD36 expression, mitochondrial membrane potential elevation, ROS production, and MAPK/NF‐κB expression [55]. Natural flavonoid quercetin exhibits various biological activities and has good anti‐inflammatory activity. Quercetin restored excessive activation of inflammation in high‐fat diet mice by eliminating phosphorylation of NF‐κB p65. By regulating hepatic lipid metabolism, oxidative stress, and inflammatory response, it exerts liver protective effects in metabolic associated fatty liver disease mice [56]. Ellagic acid is a natural polyphenolic organic compound with various biological activities such as antioxidant, anticancer, whitening, and anti‐inflammatory. Research has shown that ellagic acid significantly inhibits the expression of TLR4/NF‐κB signaling pathway and suppresses inflammatory response [57]. Neochlorogenic acid is a natural polyphenolic compound with excellent anti‐inflammatory properties. Research has shown that neochlorogenic acid enhances Nrf2 activation, upregulates antioxidant genes, and inhibits ROS accumulation. It inhibits NF‐κB signaling and reduces inflammatory proteins [58].

MAPK plays a crucial role in organisms, as it not only promotes growth, development, division, differentiation, and apoptosis, but also maintains normal cellular functions. Upon exposure to external inflammatory stimuli, it is rapidly activated and exerts a key regulatory role in the formation and progression of inflammation [59]. The experimental results showed that although the water extract of SISWE did not affect the phosphorylation of p38, it significantly inhibited the expression of ERK and JNK phosphorylation proteins, indicating that SISWE (25–400 µg/mL) can inhibit the activation of ERK and JNK pathways in RAW264.7 cells (Figure 12). Studies have shown that rutin exerts anti‐inflammatory and analgesic effects by reducing the levels of inflammatory cytokines such as TNF‐α and interleukin‐1β inhibiting COX‐2 and iNOS expression, as well as regulating MAPK, NF‐κB, and Nrf‐2/HO‐1 signaling [60]. Preliminary studies have shown that kaempferol can regulate multiple signaling pathways involved in inflammation, making it a candidate drug for treating cholecystitis. Kaempferol inhibits LPS‐induced inflammatory response in HGBECs, possibly by suppressing MAPK/NF‐κB signaling [61]. Studies have shown that quercetin is a natural flavonoid with multiple bioactive properties that can improve temporomandibular joint synovitis by targeting the p38 MAPK pathway. Quercetin exhibits stability in binding to key targets such as MAPK14 and leads to downregulation of phosphorylated p38 MAPK and JNK [62]. Studies have shown that daphnetin provides beneficial therapeutic effects for septic lung injury by upregulating CD38 and inhibiting the MAPK/NF‐κB/NLRP3 pathway [63]. We further speculate that the immune regulatory effect of SISWE may stem from the synergistic action of multiple active ingredients, rather than a single compound.

5. Conclusion

A total of 44 compounds were identified using UHPLC‐Q Exactive Orbitrap MS, including 22 phenolic acid compounds, 17 flavonoid compounds, 1 organic acid, 2 coumarins, and 2 lipid compounds. Among them, 30 compounds were identified for the first time in SISWE. In vitro studies have shown that SISWE can effectively stimulate cell proliferation and enhance phagocytic ability in the RAW264.7 cell model from 6.25 µg/mL to 100 µg/mL. SISWE can increase the production of NO in normal cells and promote the secretion of cytokines (IL‐6 and TNF‐α) from 25 µg/mL to 400 µg/mL. In the macrophage inflammation model, SISWE dose dependently reduced the excessive secretion of IL‐6 and TNF‐α at 25–400 µg/mL, and alleviated LPS‐induced inflammation of RAW264.7 macrophages by downregulating iNOS protein expression and inhibiting NO release at 25‐400 µg/mL. This study also showed that SISWE has a significant inhibitory effect on the phosphorylation expression of P65 at 100–400 µg/mL, and a significant inhibitory effect on the phosphorylation expression of ERK and JNK at 25–100 µg/mL, indicating that SISWE may exert anti‐inflammatory effects by blocking the NF‐κB and MAPK inflammatory signaling pathways (Figure 13). Overall, our study demonstrates the bidirectional immune regulatory function of SISWE, highlighting its potential in treating immunosuppressive and inflammatory diseases. The aim of this study is to provide theoretical basis for SISWE as a source for developing bidirectional immune regulatory functional foods.

FIGURE 13.

Proposed molecular mechanism by which SISWE inhibits LPS‐stimulated macrophages. LPS, which is a common macrophage activator, can activate macrophages through the NF‐κB and MAPK signaling pathway. Activation of the NF‐κB signaling pathway leads to increase the levels of proinflammatory mediators (iNOS) and cytokines (TNF‐α and IL‐6). Activation of the MAPK signaling pathway leads to the activation of JNK, p38, and ERK, which increase the levels of proinflammatory mediators (iNOS) and cytokines (TNF‐α and IL‐6). SISWE can inhibit the activation of JNK, p38, and ERK, thereby decreasing the levels of proinflammatory mediators (iNOS) and cytokines (TNF‐α and IL‐6). In addition, SISWE‐mediated inhibition of the expression of proinflammatory mediators (iNOS) and cytokines (TNF‐α and IL‐6) can ultimately suppress the release of NO.

Author Contributions

Min Wang: conceptualization, data curation, investigation, writing – original draft, methodology, software, formal analysis. Yin‐hong Xiang: writing – original draft, conceptualization. Hui Li: investigation, software. Qin‐yi Li: writing – original draft, conceptualization. Fan Ou‐Yang: software, data curation, investigation. Mei Liu: formal analysis, investigation. Yao He: formal analysis, investigation. Jing Wen: data curation, methodology, investigation. Mei‐qin Huang: data curation, methodology, investigation. Hui‐feng Sun: writing – review and editing, supervision. Zhi‐Gang Wang: writing – review and editing, supervision. Ning Zhang: writing – review and editing, supervision. Jian‐xin Liu: funding acquisition, writing – review and editing, writing – original draft, supervision.

Funding

This research was financially supported by the National Natural Science Foundation of China (Project No. 82274178), the Hunan Provincial Natural Science Foundation of China (Project No. 2026JJ80539), the 2020 Hunan Province Science and Technology Innovation Key Projects (Project No. 2020SK1020), the Wuxi Young Talent Program of Huaihua City, the Hunan University of Medicine High‐Level Talent Introduction Startup Funds and the Hunan Province College Students Innovation and Entrepreneurship Training Program (Project No. S202412214035), and the Furong Young Scholar Program of Hunan Province.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71796‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.