4.1 Chemicals and reagents

Arachidonate 5-lipoxygenase (5-LOX), horseradish peroxidase (HRP), and arachidonic acid (AA) were purchased from Sigma-Aldrich (St. Louis, MO, USA), Xue Man Biotechnology Development Co., Ltd. (Shanghai, China) and TCI Development Co., Ltd. (Shanghai, China), respectively. 3′3′5′5-tetramethylbenzidine (TMB) and nordihydroguaiaretic acid (NDGA) were purchased from Adamas-beta Reagent Co., Ltd (Shanghai, China) and Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), respectively. The cyclooxygenase 2 (COX-2) inhibitor screening kit (S0168) and BCA Protein Assay Kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal bovine serum (FBS) 1% penicillin–streptomycin solution were and 1% GlutaMax was purchased from Procell Life Science and Technology Co., Ltd. (Wuhan, China). LPS (Escherichia coli 055: B5), dexamethasone, and dimethyl sulfoxide (DMSO) were purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Biosharp (Anhui, China). Enzyme-linked immunosorbent assay (ELISA) kits (interleukin (IL)-6, IL-10, tumour necrosis factor (TNF)-α) were purchased from Multi Sciences Biotech Co., Ltd. (Hangzhou, China). Griess Reagent Kit for Nitrite Determination was purchased from Thermo Fisher Scientific, Inc. (NY, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and toll-like receptor (TLR4) antibodies were purchased from GenScript Biotech Corp. (Nanjing, China) and Proteintech Group, Inc. (Wuhan, China), respectively. Inducible nitric oxide synthase (iNOS) and MyD88 antibodies were purchased from Affinity Biosciences LTD. (Jiangsu, China). P65 and P-p65 antibodies were purchased from Abcam (Shanghai, China). carrageenan was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

4.2 Plant materials

Fleagrass was collected in the Menglun Town, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China (21° 90′ N, 101° 27′ E) and identified by the prof. Pei Shengji of Kunming Institute of Botany (KIB), Chinese Academy of sciences (CAS) in October 2021. The plant specimens were stored at Herbarium of KIB-CAS (specimen number: 1341141).

4.3 Preparation of the extract

The fresh fleagrass plants were divided into two parts: the inflorescence and the stem and leaves. The two parts of the plant material were cut separately, heated in distilled water for 3 h at 100 ℃ according to traditional usage methods. The filtered aqueous solution was concentrated to obtain the aqueous extracts of the inflorescence (ST-F) and those of the stem and leaf (ST-SL).

Dried inflorescence and stem-leaf powders were soaked in 95% methanol with ultrasonic wave for 30 min, respectively. The filtrate was concentrated and then added to the mixture of distilled water and petroleum ether (1:1). After full extraction is completed, the aqueous part of the extraction is obtained, and an equal amount of ethyl acetate solution is added to this aqueous part for further extraction. After extraction, the ethyl acetate layer was collected, which was evaporated and concentrated to form paste, and then the extract of the ethyl acetate parts of inflorescence (YSYZ-F) and stem-leaf (YSYZ-SL) was obtained respectively. The above residue of 95% methanol was soaked again in 65% methanol with ultrasonic wave for 30 min, respectively. Then the collected filtrate was concentrated into inflorescence 65% methanol extract (JC-F) and stems-leaves 65% methanol extract (JC-SL).

4.4 5-LOX analysis

All test samples (ST-F, ST-SL, YSYZ-F, YSYZ-SL, JC-F, and JC-SL) were dissolved in 100% DMSO and diluted with Tris–HCl to 250 μg/mL. The final concentration of DMSO should not exceed 5%. Initially, the extract solution, HRP, and 5-LOX (each 10 μL) were incubated with 160 μL of Tris–HCl buffer (0.1 M PH = 7.0) at 37 ℃ for 10 min. Then, TMB and AA (10 μL each) were added to the mixture and incubated at 37 ℃ for 15 min. Finally, the reaction was terminated by adding 10 μL of H2SO4. Their optical density (OD) values were determined at 450 nm using an enzymatic assay system. The rate of the inhibition of 5-LOX activity was calculated as follows:

$$}\left( \% \right) = \left( }_}}} }_}} } \right)/\left( }_}}} }_}}} } \right) \times 00$$

4.5 COX-2 analysis

The test samples were prepared in the same manner as in the 5-LOX experiment and diluted to 50 μg/mL with a buffer solution. The inhibition of COX-2 activity in the test samples was determined using the COX-2 Inhibitor Screening kit [44]. Briefly, the blank control, 100% enzyme activity control, and sample group were set up, and the specific experimental steps were performed according to the manufacturer’s instructions. The rate of the inhibition of COX-2 activity was calculated as follows:

$$}\left( \% \right) = \left( }_00\% }}} }_}}} } \right)/\left( }_00\% }}} }_}} } \right) \times 00$$

4.6 Cell culture of RAW 264.7

The mouse monocyte–macrophage cell line, RAW264.7, was purchased from Procell Life Science and Technology Co., Ltd. (Wuhan, China). The cells were cultured in DMEM supplemented with 10% FBS and 1% double antibody (100 U/mL penicillin and 100 μg/mL streptomycin) in a 5% CO2 incubator at a constant temperature of 37 °C. Stock solutions of the extracts were prepared at a concentration of 50 mg/mL in 100% DMSO. Working concentrations of the extracts were then diluted using the culture medium. The final DMSO concentration was 0.2%.

4.7 Cell viability assays

The cytotoxic effects of the test samples (ST-F, ST-SL, YSYZ-F, YSYZ-SL, JC-F, and JC-SL) on RAW264.7 macrophage cells were investigated by performing the CCK-8 assay. The cells were randomly inoculated in 96-well plates (5 × 104 cells/well) for 24 h. Afterwards, the cells were co-incubated with the test samples, DEX, and LPS (1 μg/mL) for 24 h. Further, the CCK-8 solution (10 µL) was added to each well and incubated for another 1.5 h according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated according to the following formula:

$$}\left( \% \right) = \left( }_}}} }_}}} } \right)/\left( }_}}} }_}}} } \right) \times 00\% .$$

4.8 Nitric oxide (NO) analysis

The concentration of NO in the culture supernatant was measured using the Griess reagent system kit according to the manufacturer’s instructions. Briefly, the cells were randomly inoculated into 24-well plates (2 × 105 cells/well) for 24 h. After 2 h of treatment with the test samples (ST-F, ST-SL, YSYZ-F, YSYZ-SL, JC-F, and JC-SL) and DEX, LPS (1 μg/mL) was added to the RAW264.7 cells and cultured for 22 h. Supernatants were collected and measured according to the manufacturer’s instructions using a commercial kit.

4.9 Measurement of the levels of IL-6, TNF-α, and IL-10 by ELISA

The cells were randomly inoculated into a 6-well plate at a cell density of 5 × 105/well for 24 h, followed by incubation with different concentrations of YSYZ-F (4 and 12 μg/mL) and LPS (1 μg/mL) for 24 h. Supernatants were collected and used to measure the levels of IL-6, TNF-α, and IL-10 according to the manufacturer’s instructions.

4.10 Western blotting

Cell treatment was the same as that used in the ELISA. Cell proteins were prepared in cold phosphate-buffered saline (PBS) and radioimmunoprecipitation assay buffer containing phenylmethanesulfonyl fluoride. The protein samples were separated using sodium dodecyl sulphate–polyacrylamide gel (12%) and transferred to a polyvinylidene difluoride membrane. Further, the TBST membrane containing 5% skim milk power for 2 h and incubated with the corresponding primary antibodies (iNOS, P65, P-p65, TLR4, MyD88, and GAPDH) at 4 ℃ for 24 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h. The proteins were detected and colour development was observed using the ECL system.

4.11 Immunofluorescence assay

Coverslips were placed on 6-well plates containing the test samples and cell treatment was the same as that used in the ELISA. The cells on the slides were soaked in PBS, fixed with 4% paraformaldehyde, soaked with 0.5% Triton X-100 for 20 min and then closed with normal goat serum for 30 min at room temperature (25 ℃). Subsequently, the coverslips were cleaned with PBS and incubated with the corresponding primary antibodies (nuclear factor-kappa B (NF-κB) p65)) for 24 h at 4 °C, followed by incubation with a cy3-labeled secondary antibody for 1 h. 4′,6-diamidino-2-phenylindole staining was performed and images were analyzed under a fluorescence microscope.

4.12 Animals and treatment

Male Kunming mice (18–22 g) were obtained from the Experimental Animal Center of Kunming Medical University, Yunnan Province, China. Mice were exposed to specific pathogen free environment for 12 h dark/light cycle at a constant temperature and humidity. Mice are allowed free access to standard laboratory food and water. The animal experiments were approved by the Research Ethics Committee of the Kunming Institute of Botany (kib202303018), Chinese Academy of Sciences. The mice were acclimatised to their new environment for 1 week before the experiment.

4.13 Carrageenan-induced mouse paw oedema model

An experimental method reported in previous studies was used with slight modifications [45, 46]. Briefly, the mice were randomly divided into the following five groups (n = 5): model group, YSYZ-F groups, and positive control (DEX). YSYZ-F dissolved in acetone was used for the treatment. 20 µL each of different concentrations (4 mg/mL, 8 mg/mL and 12mg/mL) YSYZ-F and dexamethasone (0.1 mg/paw) were applied to the right hind paw of mice, and the model groups were treated with the same amount of acetone. After 1 h, each mouse was injected with carrageenan (1% in saline) (50 μL) on the right hind paw. Basal thickness (T0) was measured before injection, whereas pathological thickness (Tt) was measured 1, 2, 3, 4, 5, and 6 h after injection. The degree of swelling was calculated using the following formula:

$$}\left( \% \right) = \left[ }_}} }_ } \right)/}_ } \right] \times 00$$

4.14 Statistical analyses

All data are expressed as the mean ± SEM of at least three independent experiments. One-way analysis of variance with Tukey’s test was used for multiple comparisons. Paw oedema data in Fig. 6 was analyzed with Two-way analysis of variance with Tukey’s test. All analyses were performed using GraphPad Prism 9.0 (Graphpad Software, Inc., USA). A p-value less than 0.05 was considered statistically significant.