Effect of Stewartia koreana Boiling Extract (SKBE) on Osteoarthritis and Purification of Spinasterol From SKBE

Abstract

Osteoarthritis is to the abnormality of the inflammatory response of joint tissue caused by various causes such as aging, and muscle loss. In this study, the activity in joint inflammation was verified using SKBE, a plant extract, and the expression levels of arthritis-inducing proteins including MMP-1, MMP-3, MMP-13, and collagen type II in vitro were compared and analyzed. Furthermore, we synthesized α-spinasterol, an active ingredient of SKBE, by the previously reported synthesis method and these findings could provide a new starting point for the development of treatments for osteoarthritis.

INTRODUCTION

Osteoarthritis is the most common joint disease in the world that causes the loss of joint cartilage due to aging and a variety of causes such as obesity, injury, and muscle weakness. It is also known that inflammation in the loss of the joint area causes deterioration of the function of the joint cartilage.^1,2 The progression of osteoarthritis initiates inflammatory responses in the joint area, and the pro-inflammatory molecules of these regions caused pain and swelling in the joint.^3,4 Many studies have reported an increase in the expression of pro-inflammatory molecules, including interleukin (IL)-1β, IL-6, interferon-gamma (IFN-γ), and especially, various extracellular matrix-degrading enzymes, such as MMP-1, MMP-3, and MMP-13 in the synovial fluids of osteoarthritis patients.^5,6 Therefore, regulation of abnormal inflammatory responses and inhibition of extracellular matrix degradation proteins expression could be an ideal treatment strategy for osteoarthritis.

A variety of natural products, herbs, and plant extracts have long been used in many countries as medicine, food, and beverage materials.^7,8 Stewartia koreana is a native tree in Korea and Stewartia koreana leaf MeOH extract (SKE) exhibits various biological activities, such as anti-inflammatory activity, inhibition of osteoclast differentiation, and angiogenic activity.^9-11 In a previous study, we reported that the α-spinasterol from SKE is an active ingredient in anti-inflammatory activity.¹² Alpha spinasterol is known as phytosterol with blood-brain barrier (BBB) permeability and antioxidant activity. In addition, it has been reported that the ligand of transient receptor potential vanilloid 1 (TRPV1) has anticonvulsant and antidepressant effects.^13,14 In our previous report, we isolated α-spinasterol from SKE, and we confirmed that α-spinastrol inhibits MMP-1 expression in UVB-treated human fibroblast and has TNF-α-induced chemokine expression such as CCL17 and CCL22 inhibition activity.^15,16

Although some methods for synthesizing α-spinasterol are known, there are problems such as low yield, low reproducibility, and no ready availability of starting materials.^17-19 In this study, we synthesized the α-spinasterol according to the published method¹⁹ and analyzed the anti-arthritis effect of Stewartia koreana leaf boiling extract (SKBE) using the osteo-defected rat model.

EXPERIMENTAL

Materials and Cell Culture

The leaves of Stewartia koreana were collected from Gokseong-gun, Jeollanam-do, Korea, and identified by Lifetree Co., Korea. Samples were dried at room temperature in the shade.

The dried Stewartia koreana leaves (14 kg) were extracted using 100 ℃ water (10 times), and filtered with filter paper. The resultant water extract was evaporated under reduced pressure at 45 ℃ using a rotary evaporator (Heidolph, Germany) and freeze-dried (SKBE).

Human chondrocytes were cultured in DMEM (Welgen, Seoul, Korea) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen) in a humidified incubator at 37 ℃ with 5% CO₂.

Isolation and Analysis of Spinasterol from SKE by HPLC

Spinasterol and samples were dissolved in methanol at a concentration of 1 mg/mL, and the solutions were filtered through a 0.45 μm PTFE syringe filter. The HPLC system consisted of a 1200 HPLC system with a 1200 binary pump (Agilent Technologies, Palo Alto, CA). The chromatographic separation of the compounds was achieved using an XBridge^® Shield RP18 (4.6 mm × 150 mm, 3.5 μm, Waters, USA), and keeping the column oven temperature at 40 ℃. The mobile phase consisted of 0.1% Phosphoric acid (solvent A) and acetonitrile (solvent B). The mobile phase flow rate was 1 mL/min with isocratic elution: 95% of solvent B. The injection volume was 5 μL, and the UV detection wavelength was set at 205 nm.

MTT Assay

Assay Human chondrocytes were stabilized for 24 h at a density of 5 × 10³ cells/well in a 96-well culture plate. After the chondrocytes were stabilized, all growth media were discarded and rinsed more than three times using a medium that did not contain FBS. And then, SKBE was treated on chondrocytes at the indicated concentrations in DMEM without FBS for 48 h and maintained under the same culture conditions as previously described. After incubation, cells were treated with 100 μg/mL of 3-[4,5-dimetnythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) for 15 mins. The formazan precipitate was dissolved in 200 μL of DMSO and the absorbance at 560 nm was determined by UV-spectrophotometer. Analyses were repeated three times and the results are expressed as the means of three independent experiments.

Profiling of Pro-inflammatory Cytokine Expression

The chondrocytes (1 × 10⁶ cells/well) were seeded and stabilized in culture plates (60 mm) and treated with or without TNF-α (10 ng/mL). The cells were maintained with indicated concentrations of SKBE for 24 h. After the incubation, the cell culture medium was collected, and the protein concentrations were determined. The quantification of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, and IFN-γ were confirmed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol.

Matrix Metalloproteinases and Collagen Expression

Cell maintenance and SKE treatment were carried out under the same method and conditions as in the cell culture and ELISA experiment. Total RNA was isolated using the TRIzol reagent kit (Invitrogen, Carlsbad, CA) in chondrocytes according to the manufacturer’s protocol. A standard experimental process of securing to obtain cDNA from total RNA (2 μg) was done using M-MuLV reverse transcriptase (Fermentas Life Science, Burlington, Canada). The obtained cDNA was used to amplify target mRNAs with MMP-1specific primers using the AccuPower PCR-pre-mix kit (Bioneer, Daejeon, Korea).

Cell culture and samples were treated in the same condition as the RT-PCR method, and then the culture media of each well were harvested to recover MMP proteins secreted to the outside of the cell. After washing the plate from which media was recovered with 1 mL PBS, cell proteins were separated using 400 μL of RIPA buffer containing protease inhibitor. Western blot was performed by a general method using 20 to 80 μg of prepared protein samples, and then the degree of expression was confirmed using specific antibodies of each protein. In the case of collagen type II, a new intracellular protein, β-actin protein was used as internal control, and the intensity of the protein band was measured by Image Jsoftware after three or more repetitive experiments in the same method, and in the case of MMPs, the expression level of each protein for positive control was compared as a fold value. In addition, the measured intensity value was quantified and statistically processed to analyze the degree of protein expression and calculate the p-value.

Animal Experiment

The animal experiments were approved by Institutional Animal Care and Use Committee at Kyung Hee university (KHUASP(SE)-15-027). As a result of biological analysis of SKBE using a human chondrocyte, a non-toxicity in the cell viability was found at the highest treatment concentration of 1000 μg/mL, and for using the animal test, the samples of the concentration to be 5 and 50 times the highest concentration of in vitro were dissolved in 5 mg/kg DMSO. 12 SD rats were divided into 4 groups (control, vehicle, 5 mg/kg, 50 mg/kg) and adapted for 1 to 2 weeks. After adaptation, nine rats excluding three control groups were drilled with a 2 mm diameter drill in one knee joint to build an osteochondral defect (OD) model with damaged joint cartilage. From the next day, after weight measurement, oral administration of vehicle or SKE of 5 mg/kg and 50 mg/kg was performed for 5 weeks every 2 days. After 5 weeks, bone tissue, including the damaged area of the bone joint, was separated at the expense of the animal model. The separated bone tissue was fixed using formalin for 2 weeks. De-calcification was performed using 14% EDTA (pH 7.5) to remove calcium from fixed bone tissue and make it smooth. Calcium removal proceeded for 4 to 8 weeks, grinding the solution every 3 days until the bone tissue became completely soft. The calcium-removed bone tissue was paraffin-embedded to form a block after undergoing a tissue process. The prepared paraffin block was sectioned around the damaged area of the bone joint to prepare a slide for staining. Cartilage damage and recovery were compared by dyeing the prepared tissue slide with safranin/fast green staining method (orange to red: cartilage, mucin, mast cell granules/blue green-background: cytoplasm/black: nuclei). A stained tissue slide was randomly selected for each group and the thickness of the stained cartilage was measured by at least 8 points. The value was quantified, and the degree of cartilage damage and recovery compared to the control group was analyzed and expressed as a relative value, and the p-value was calculated by statistical processing.

Statistical Analysis

The data are presented as the means ± SD. Statistical comparisons between groups were performed using one-way ANOVA followed by the Student’s t-test.

RESULTS AND DISCUSSION

First, the effect of SKBE on cell viability in human chondrocytes was evaluated. Treatment with SKBE up to 1,000 μg/mL showed no significant effect on cell viability for 48 h (Fig. 1A). Next, we verified the regulatory effect of SKBE on TNF-α induced inflammatory cytokine expression such as IL-1β, IL-6, and IFN-γ in human chondrocytes using ELISA method. Pro-inflammatory cytokine expression levels of IL-6, IL-1β, and IFN-γ in TNF-α-induced human chondrocyte increased about 90 times, 3 times, and 2 times, respectively, compared to the mediumonly negative control, and the SKBE-treated group showed a decrease in the expression of pro-inflammatory cytokines to normal levels in a dose-dependent manner (Fig. 1 B-D).

Figure 1. Effects of SKBE on pro-inflammatory cytokines production and cell viability in TNF-α induced human chondrocytes. The data represent the mean ± SD of triplicate experiments. *p < 0.01; **p < 0.001.

Next, the effect of SKBE on mRNA and protein expression levels such as MMP-1, MMP-3, MMP-13, and collagen type II a 1 (Col2a1) molecules was evaluated in TNF-α induced human chondrocyte. It was confirmed that the mRNA expression inhibitory activity for MMPs including MMP-1, MMP-3, and MMP-13 showed a similar tendency to the pro-inflammatory cytokine expression inhibition activity. These results confirmed that the increase in the mRNA expression level of MMPs increased by TNF-α, and decreased depending on the dose-dependent concentration manner by SKBE treatment (Fig. 2A-C). In the case of Col2a1, the mRNA expression level has decreased in TNF-α induced chondrocytes and has increased depending on the concentration by SKBE treatment (Fig. 2D). In Fig. 3, the expression level of MMPs (MMP-1, MMP-3, and MMP13) and Col2a1 proteins in TNF-α induced chondrocytes were confirmed by performing western blotting. As a result, it was confirmed that it showed a very similar pattern to the mRNA expression result.

Figure 2. Effects of SKBE on mRNA expression of MMPs and Col2a1 in TNF-α induced human chondrocytes. The data represent the mean ± SD of triplicate experiments. ^#p < 0.05; *p < 0.01; **p < 0.001.

Figure 3. Effects of SKBE on protein expression of MMPs and Col2a1 in TNF-α induced human chondrocytes. The data represent the mean ± SD of triplicate experiments. ^#p < 0.05; *p < 0.01; **p < 0.001.

Next, we evaluated the effect on knee joint damage for SKBE using the OD models made as described in the materials and methods section. The OD rat model experiment was conducted in accordance with the procedure shown in Fig. 4A, and the shape of the knee was confirmed by the photo after making a hole in the knee joint (Fig. 4B). After the decalcification process was completed, the tissue was dyed to observe the degree of cartilage regeneration. The results showed that the degree of cartilage regeneration in the knee joint was increased in the SKBE-treated group compared to the vehicle group (Fig. 4C). In addition, quantitative evaluation of cartilage regeneration showed that cartilage regeneration was significantly higher than that of the vehicle group, and in the case of the high-concentration SKBE treatment group (50 mg/kg), cartilage regeneration was more significant than a sham group.

Figure 4. Effects of SKBE on cartilage regeneration in rat-OD animal model. The data represent the mean ± SD of triplicate experiments. ^#p < 0.05; **p < 0.01.

According to the previously reported synthesis method¹⁹, α-spinasterol was prepared from commercially available stigmasterol by a modified synthesis procedure including acetylation, allylic oxidation using CrO₃ condition,²⁰ hydrogenation, hydrazone formation, elimination, and decarboxylation. As a result of analysis by the HPLC system, the spinasterol standard was found to have a spinasterol peak at a retention time of 9.7 min, and the synthesized spinasterol sample also had a peak at the same time (Fig. 5).

Figure 5. Extraction and preparation of spinaterol. Comparison activity of isolated and synthetic α-spinasterol on MMP-1 mRNA expression.

Since ancient times, natural products have been used to treat various diseases in many Asian countries, including South Korea. The plants are known to generate and utilize various secondary metabolic products to be used for their growth and protection. We use these secondary metabolic products to develop medicines, and in particular, we use new structures as key backbone structures for new drug development. The stewartia koreana tree is a plant native to Korea with a very beautiful woody tree and has been mainly used for ornamental purposes. However, recent studies have shown that the stewartia koreana leaf MeOH extract (SKE) contains various secondary metabolites and also has functions such as anti-inflammatory, bone loss protection in LPS-induced bone loss animal models, and skin protection.¹⁰

According to the previous study, SKE is known to concentrate-dependently inhibit the production of LPS-induced nitrogen oxide (NO) in murine macrophage RAW264.7 cells. It was also found that SKE alleviates the symptoms of arthritis in the CIA-induced arthritis animal model.⁹ As shown in Fig. 1, it was confirmed that the high concentration of SKBE did not affect the survival of human chondrocytes, and this result was similar to the results confirmed in various cells through many previous studies. Therefore, SKBE can be considered safe for in vitro tests up to at least 500 μg/mL. In our results (Fig. 2 and Fig. 3), SKBE can be seen that inhibits the expression of MMPs including MMP-1, MMP-3, and MMP-13 induced by TNF-α, and it is known that these protein transcriptions were controlled by NF-κB in a key transcription factor. That is, it may be inferred that the expression of MMPs is suppressed by the MAPK/IKK/IκB signal transduction inhibition mechanism. We have confirmed through our previous studies that SKE inhibits the phosphorylation of MAPK/IKK/IκB induced by TNF-α in human keratinocytes.^9,10,12 Therefore, it may be that the expression of MMPs will be suppressed through the same mechanism in human chondrocytes. Osteoarthritis refers to the inflammatory condition of cartilage tissue caused by abnormal conditions around cartilage tissue due to the aging of cartilage, and cartilage loss is known to be the most important cause of osteoarthritis. In this study, an artificial knee joint inflammation model was created using the rat-OD model and the activity of SKBE was compared. As shown in Fig. 4, articular cartilage tissue regeneration could be confirmed. But, these processes are known that cartilage regeneration proceeds easily in rat studies, but in humans, cartilage regeneration does not occur well. Also, although the rat-OD model cannot be a perfect model to verify the therapeutic efficacy of human osteoarthritis, it is an experimental method that can explain the bone-strengthening of SKBE activity and has already proven the bone-strengthening of SKE efficacy in the LPS-induced rat-bone loss model through our previous study.¹⁰ Therefore, SKBE may consider that animal models have definite bone-strengthening activity, and further studies in humans are needed.

In our previous study, spinasterol-Glc was confirmed to be an active ingredient by purification/structural analysis of secondary metabolites from SKE.¹² Alpha-spinasterol is a phytosterol found in various plants and is known to exist in the form of glycosides in spinach leaves, cucumber fruits, seeds of pumpkin and watermelon, argan seed oil, and cactus pear seed oil.¹³ Recent studies have shown that α-spinasterol is a molecule with various physiological activities such as anti-inflammatory and anti-diabetes, and in particular, it is reported that it acts as a strong antagonist for TRPV-1 receptors through BBB.^13,14 As described above, although α-spinasterol is a compound of high utilization value, research on the synthesis of α-spinastrol derivatives has not been actively conducted. In this study, α-spinasterol was synthesized based on a previously known synthesis method,^17-20 and the MMP-1 mRNA expression activity of α-spinasterol isolated from SKE was compared (Fig. 5). Therefore, we believe that our study is meaningful to secure more derivative synthesis using α-spinasterol in the future and to derive a drug candidate that can present a new backbone material for the development of osteoarthritis treatments.

CONCLUSION

In conclusion, this study confirmed that the plant extract, SKBE, promotes the regeneration of cartilage tissue in rat-OD animal models and it has the inhibitory activity of MMPs expression including MMP-1, MMP-3, and MMP-13 in vitro. It was also confirmed to increase the production of collagen type II which induces the normalization of joint tissue. Furthermore, α-spinasterol, known as the active ingredient of SKE, was synthesized and its activity was compared with α-spinasterol isolated from SKE. These findings are considered important in deriving new drugcandidate materials for inhibiting joint inflammation.