SRL were sun-dried. After removing the damaged leaves, the dried intact leaves were pulverized in a food mill and filtered through a 150-μm mesh sieve to obtain the powder. The powder was boiled in pure distilled water, and the extract solution was centrifuged for 20 min at 2190 × g to remove the pellet. The supernatant was filtered through a 0.20-μm filter membrane and dried using a freeze dryer. We purchased commercial agents prepared from stems of SR, such as mangiferin (Sigma-Aldrich Co., USA), triptotriterpenic acid B (AApin Chemicals Abingdon, UK), and (−)-epicatechin (Sigma-Aldrich Co., USA). These agents were dissolved in ethanol. All samples were stored at −20°C until use.

Collagen antibody-induced arthritic (CAIA) mice were generated using DBA/1 J mice as reported previously [⁸]. Synovial tissues were obtained from the knee joints of these mice. The inflammatory synovial tissues were minced and stirred with type IV collagenase (Sigma-Aldrich Co., USA) in serum-free DMEM/F12 medium at 37°C for 3 h in an incubator shaker. The synovial tissue lysate was then filtered through a 40-μm nylon mesh, washed extensively, and seeded at 1 cell/well in 96-well microplates. The cells were cultured in DMEM/F12 supplemented with 10% FBS and benzylpenicillin potassium (100 units/mL) at 37°C/5% CO₂. The inflammatory synovial tissue has been reported that the gene of Synoviolin is over-expressed [¹²]. Therefore, the cell named MTS-C H7 of high-expressed genes of Synoviolin was established. The study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee of Josai University, Saitama, Japan.

MTS-C H7 cells were plated in 96-well microplates at a density of 5 × 10³/well. After 3 h, the SRL extract was added and cultured for 24 h. After incubattion, 10 μL of the cell proliferation reagent WST-1 (Roche Diagnostics, USA) was added and incubated for 30 min. Cell proliferation was measured at 450 nm by using a spectrophotometer.

MTS-C H7 cells were plated on 96-well microplates at a density of 5 × 10³/well. After 3 h, mouse IL-1β or other samples were added and cultured for 24 h. The cell proliferation assay was performed as describe above.

The HPLC system consisted of an LC-Organizer, L-6200 Intelligent pump, L-4200H UV–VIS Detector, and ELITE LaChrom Column Oven L-2350 (HITACHI, Japan). Separation was carried out on a Wakosil-II 5C18AR column (250 mm × 4.6 mm, 5 μm). HPLC conditions were performed according to the reference [¹³]. All samples were evaporated and dried, dissolved in ethanol. All samples were stored at −20°C until use.

The SRL extract was separated by hexane, diethyl ether, ethyl acetate, and n-butanol. The up layers and residual water fraction were collected. These samples were evaporated and dried, dissolved in ethanol or ultrapure water. All samples were stored at −20°C until use.

MTS-C H7 cells were cultured at approximately 2 × 10⁵/dish. After 24 h, mouse IL-1β and other samples were added and cultured for 24 h. RNA was extracted using the TRIzol reagent according to the manufacturer’s instructions. The term of genes expression has been reported previously [⁶].

SDS-PAGE was used to determine the molecular weight and purity of the protein isolated from SRL. The samples were run on a MULTIGEL Mini 15/25 (COSMO BIO, Tokyo, Japan) gradient gel with the SDS-PAGE buffer (0.1% SDS, 0.05 M Tris, 0.05 M tricine). After electrophoresis, the gel was fixed with ultrapure water for 15 min and stained with GelCode Blue Safe Protein Stain (Thermo Fisher Scientific K.K., Waltham, USA) for 1 h.

SRL extract was mixed with CPaseY incubated at 25°C from 0 to 60 min. MTS-C H7 cells were plated in 96-well microplates at a density of 5 × 10³/well. After 3 h, SRL + CPaseY and IL-1β were added and cultured for 24 h. The cell proliferation assay using samples were performed as describe above.

The results are expressed as the mean and standard deviation (SD) of three independent experiments. Statistical analysis was carried out with Stat-Mate III Version 3.18 (ATMS Co., Ltd., Japan). Data distributions were compared using the Analysis of variance.

As shown in Figure 1, cell viability was not changed by 25 and 100 μg dry matter/mL; however, it was decreased by 500 μg dry matter/mL and showed to decreased until 2000 μg dry matter/mL. We calculated the IC₅₀ according to the literature [¹⁴] and obtained the value of ~850 μg/mL.

As shown in Figure 2, MTS-C H7 cell proliferation in the presence of the SRL was significantly decreased compared with only IL-1β. Thus, the SRL clearly inhibits IL-1β-induced cell proliferation.

As shown in Figure 3, M, T, or E did not affect IL-1β-activated cell proliferation, which indicates that the 3 tested reagents are not among the active ingredients of SR.

The HPLC analysis data for 2 different extracts are shown in Figure 4A. The (−)-epicatechin (EC), epicatechin gallate, epigallocatechin, and epigallocatechin gallate (EGCG) catechins were detected in a green tea extract used as a catechin control. The EC and EGCG were detected in the SRL extract. These results show that the components of the hot-water extract from Camellia sinensis (Japanese green tea) leaves are different from those in the SRL extract.

As shown in Figure 4B, Cell proliferation in the presence of IL-1β did not change when F1, F2, or F3 were added to the culture. These results show that none of the HPLC polyphenolic fractions of the SRL extract suppressed the IL-1β-induced cell proliferation.

As shown in Figure 5A, cell proliferation in the presence of the residual water sample was decreased compared with IL-1β alone. These results show that the water-soluble component in the SRL extract attenuated the IL-1β-induced cell proliferation.

As shown in Figure 5B, SRL extract and residual water samples were significant decrease in the mRNA levels for MMP-3 and MMP-13. These results show the possibility that the active ingredients in the residual water fraction decrease the activation of gene expression by IL-1β.

As shown in Figure 6A, using only the SRL extract, a band of ~3 kDa was observed. In addition, in the sample treated with HCl, no band could be observed at any position. As shown in Figure 6B, at MTS-C H7 cell proliferation in the presence of CPaseY-treated SRL extract was not decreased compared with only IL-1β. Moreover,HCl-treated SRL extract showed a similar results (data not shown).