Chondroprotective and anti-inflammatory effects of ChondroT, a new complex herbal medication

Background Ganghwaljetongyeum (GHJTY) is a complex herbal decoction comprising 18 plants; it is used to treat arthritis. In order to develop a new anti-arthritic herbal medication, we selected 5 out of 18 GHJTY plants by using bioinformatics analysis. The new medication, called ChondroT, comprised water extracts of Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex. This study was designed to investigate its chondroprotective and anti-inflammatory effects to develop an anti-arthritic herb medicine. Methods ChondroT was validated using a convenient and accurate high-performance liquid chromatography–photodiode array (HPLC–PDA) detection method for simultaneous determination of its seven reference components. The concentrations of the seven marker constituents were in the range of 0.81–5.46 mg/g. The chondroprotective effects were evaluated based on SW1353 chondrocytes and matrix metalloproteinase 1 (MMP1) expression. In addition, the anti-inflammatory effects of ChondroT were studied by Western blotting of pro-inflammatory enzymes and by enzyme-linked immunosorbent assay (ELISA) of inflammatory mediators in lipopolysaccharides (LPS)-induced RAW264.7 cells. Results ChondroT enhanced the growth of SW1353 chondrocytes and also significantly inhibited IL-1β-induced MMP-1 expression. However, ChondroT did not show any effects on the growth of HeLa and RAW264.7 cells. The expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was induced by LPS in RAW264.7 cells, which was significantly decreased by pre-treatment with ChondroT. In addition, ChondroT reduced the activation of NF-kB and production of inflammatory mediators, such as IL-1β, IL-6, PGE2, and nitric oxide (NO) in LPS-induced RAW264.7 cells. Conclusions These results show that ChondroT exerted a chondroprotective effect and demonstrated multi-target mechanisms related to inflammation and arthritis. In addition, the suppressive effect was greater than that exhibited by GHJTY, suggesting that ChondroT, a new complex herbal medication, has therapeutic potential for the treatment of arthritis.


Background
Arthritis is the most common inflammatory disease and a major public concern in elderly individuals. The symptoms include joint pain, tenderness, and joint inflammation. Although rheumatoid arthritis and osteoarthritis differ fundamentally in several respects, they result in cartilage degradation, which in turn leads to cartilage bone damage [1]. Matrix metalloproteases (MMPs) play a critical role in the breakdown of cartilage [2,3]. In particular, MMP-1 is known to decompose type II collagen, which is a major component of chondrocytes [4]. Cartilage degradation in arthritis is recognized to be induced by inflammatory cytokines, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α) [5][6][7]. Recently, prostaglandin E 2 (PGE 2 ) or nitric oxide (NO) has been shown to play key roles in the induction of MMP expression in chondrocytes [8,9]. In addition to chondrocytes, macrophages contribute to inflammation and matrix degradation in osteoarthritis tissues, and inflammatory mediators such as IL-1β, TNF-α, IL-6, PGE 2 , and NO represent potential targets for osteoarthritis disease modification [9].
Proinflammatory enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) that cause pain and inflammation, provide a measure to assess the effect of drugs for the treatment of arthritis [10]. Nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors are pharmacological treatments used for arthritis. Some oriental medicines have been used to treat arthritis [11][12][13]. We reported that Ganghwaljetongyeum (GHJTY), a traditional Korean herbal medicine used to treat severe joint pain, limitation of motion, fever, and swelling, inhibited inflammatory processes associated with arthritis [14]. Because GHJTY is a complex herbal decoction composed of 18 plants, we selected 5 effective herbs, i.e., Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex, using bioinformatics analysis to develop a new anti-arthritic herbal medication [15]. In the present study, the water extracts of these 5 herbs named ChondroT was evaluated as an anti-arthritic herb drug. To develop a multi-functional herbal medicine for arthritis, we tested the effects of ChondroT on various arthritis-related pathomechanisms. The effects of ChondroT were evaluated on SW1353 chondrocyte protection and IL-1β-induced MMP1 expression. In addition, the inhibitory effects of ChondroT were studied on the expression of inflammatory enzymes COX-2 and iNOS and on the production of inflammatory mediators such as IL-1β, TNF-α, IL-6, PGE 2 , and NO in RAW264.7 macrophage cells.

Plant materials
The five herbal medicines forming ChondroT -Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortexlisted in Table 1 were purchased from Omniherb (Yeongcheon, Korea). The origin of the five herbal medicines was confirmed taxonomically by Professor Jong-Kil Jeong, Dept. of Herbology, College of Oriental Medicine, Dongshin University, Republic of Korea. Voucher specimens (KYR2014-020) have been deposited at college of Pharmacy, Chonnam National University.

Preparation of ChondroT
We combined 5 herbs containing Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex in a ratio listed in Table 1. ChondroT herb material composed of above 5 herbs was extracted once using 10-fold water solvent at 100°C for 3 h and then filtered (180 mesh). The water extract solution of Chon-droT was concentrated using a continuous vacuum evaporator (around 55~60°C, 670 mmHg) followed by lyophilization using a vacuum drier (720 mmHg) for 8 h. The water extract from GHJTY herbs was prepared as previously described [14]. Stock solutions of ChondroT and GHJTY were prepared in a concentration of 50 mg/mL using phosphate buffered saline (PBS) and filter-steriled.    (5), decursin (6), and decursinol angelate (7) Reagents and high-performance liquid chromatography (HPLC) analysis Seven reference compounds used for quality control of ChondroT are shown in Table 2. The chemical structures of the seven marker compounds are shown in

Western blot analysis of MMP1
Human SW1353 chondrosarcoma cells were cultured in a 6-well plate (SPL life sciences Co., Pocheon, Korea) at 5 × 10 5 /well for 24 h. The cells were pretreated with

Griess assay
The NO in the culture supernatant was measured using Griess Reagent (1 % sulfanil-amide in 2. ChondroT did not show any cytotoxic effect on HeLa and RAW264.7 cells. * P < 0.05 and *** P < 0.001 compared to the untreated group Reagent for 30 min, and the absorbance was read at 570 nm using an ELISA microplate reader (ELx808).

Statistical analysis
All studies were repeated at least three times. Statistical differences were evaluated using one way ANOVA. P value <0.05 was considered significant.

The quality assessment of seven marker components in ChondroT
The HPLC-PDA method was developed for simultaneous determination of the quality assessment of seven marker components in ChondroT, which was composed of five medicinal herbs, Ostericum koreanum Maximowicz (Osterici Radix), Lonicera japonica Thunberg (Lonicerae Folium), Angelica gigas Nakai (Angelicae Gigantis Radix), Clematis mandshurica Ruprecht (Clematidis Radix), and Phellodendron amurense Ruprecht (Phellodendri Cortex). In this study, the seven compounds, oxypeucedanin hydrate from Osterici Radix, chlorogenic acid from Lonicerae Folium, nodakenin, decursin, and decursinol angelate from Angelicae Gigantis Radix, isoferulic acid from Clematidis Radix, and berberine Cl from Phellodendri Cortex were selected as marker compounds for quality control of ChondroT. The calibration curves of the seven marker components showed good linearity with a correlation coefficient (r 2 ) ≥ 0.9996 in the different concentration ranges, and the other parameters were shown in Table 3. Using optimized chromatography conditions, the seven marker compounds were separated within 30 min. The typical HPLC chromatogram of ChondroT is shown in Fig. 1b and 1c Table 4.

Effects of ChondroT on the proliferation of SW1353 cells
The most predominant pathological change during osteoarthritis is cartilage degradation. To evaluate the effect on cartilage protection, SW1353 cells were treated with ChondroT or GHJTY for 2 days, and the cell proliferation was evaluated by MTS. ChondroT significantly increased the proliferation of SW1353 cells at concentrations of 0.1 1.0 mg/mL (Fig. 2a). In contrast, ChondroT and GHJTY did not show any cytotoxic effect on other origin cells, RAW264.7 and HeLa cells, at concentrations of 1, 0.3, and 0.1 mg/mL ( Fig. 2b and 2c). This result suggests that ChondroT has specific proliferation activity on humanborn fibroblast SW1353 cells.
Effects of ChondroT on MMP1 expression in IL-1β-or PMA-induced SW1353 cells MMPs play a critical role in cartilage destruction in arthritic joints, and MMP-1 is known to decompose a major component of chondrocytes. The effect of Chon-droT was evaluated on MMP1 expression in SW1353 cells induced by IL-1β or PMA using Western blotting. MMP-1 expression was significantly increased by IL-1β or PMA in SW1353 cells, which was decreased by pretreatment with ChondroT (Fig. 3). ChondroT showed inhibitory effect on MMP1 expression in SW1353 cells greater than GHJTY (Fig. 3).  (Fig. 4). The inhibitory effect of Chon-droT on COX-2 and iNOS expression was greater than that exhibited by GHJTY (Fig. 4).

Effects of
Effects of ChondroT on the production of inflammatory mediators in LPS-activated RAW264.7 cells RAW264.7 cells were pretreated with ChondroT or GHJTY for 2 h prior to addition of LPS (500 ng/mL). IL-1β, TNF-α, IL-6, and PGE 2 cytokines were assayed using ELISA kits. ChondroT significantly decreased the production of IL-1β, IL-6, and PGE 2 in RAW264.7 cells activated with LPS ( Fig. 5a, b, and c). NO production in LPS-activated RAW264.7 cells was also decreased by the pretreatment of ChondroT (Fig. 5d). The inhibitory effect of ChondroT on inflammatory mediator production was greater than that exhibited by GHJTY.

Effects of ChondroT on PMA-induced NF-kB activation in 293T cells
NF-kB is a transcription factor related to inflammation and arthritis. The effect of ChondroT was tested on NF-kB activation in 293T cells. PMA increased NF-kB transcription, which was decreased by ChondroT pretreatment (Fig. 6). The inhibitory effect of ChondroT on PMA-induced NF-kB activation was greater than that exhibited by GHJTY.

Anti-oxidant activity of ChondroT
To study the anti-oxidant activity of the ChondroT, the DPPH scavenging potential was determined. Vitamin C and BHA were used as the positive controls. DPPH free radicals were decreased by approximately 95 % at 0.3 mg/mL ChondroT and the inhibitory effect was greater than that exhibited by GHJTY (Fig. 7).

Discussion
In this study, we evaluated the multifunctional effects of ChondroT, a new complex herbal medication, for arthritis pharmacologic treatment. ChondroT is a water extract of 5 herbs, Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex. We previously reported that Gangwhaljetongyeum a b c d Fig. 5 Effects of ChondroT on the production of pro-inflammatory cytokines in LPS-activated RAW264.7 cells. RAW264.7 cells were pretreated with ChondroT for 2 h and then incubated with LPS for 24 h. Inflammatory mediators were measured in the cell supernatants by ELISA. ChondroT decreased the LPS-induced production of IL-1β (a), IL-6 (b), and PGE 2 (c). NO in the supernatant was detected by Griess reagent (d). LPS-induced NO production in RAW264.7 cells was decreased by ChondroT treatment. + P < 0.01 and +++ P < 0.001 compared with the untreated group. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the LPS-treated group (GHJTY), a traditional Korean herbal medicine comprised with 18 herbs, attenuates synoviocyte proliferation and reduces the production of proinflammatory mediators in macrophages [14]. GHJTY as an anti-arthritic drug may be limited because it is composed of 18 plants. To reduce the numbers of plants and to increase the potency as multifunctional anti-arthritic drugs, we conducted bioinformatics analysis [15]. In addition, oriental clinical doctors also suggested 4 complex herb medication candidates according to their clinical experiences. We compared the inhibitory effects of GHJTY and 4 complex herb medications on COX-2 and iNOS expression. Among them, an herb material ChondroT showed the most potent activity in chondrocyte protective effects and anti-inflammatory effects. ChondroT enhanced the proliferation chondrocytes (Fig. 2a) and also significantly inhibited IL-1β-or PMAinduced MMP-1 expression in the chondrocytes (Fig. 3). In addition, ChondroT decreased the expression of inflammatory enzymes COX-2 and iNOS (Fig. 4) and reduced the production of inflammatory mediators, such as IL-1β, IL-6, PGE 2 , and NO, thereby playing important roles in arthritis (Fig. 5). ChondroT also decreased the PMAinduced activation of NF-kB, a transcription factor related to inflammation and arthritis (Fig. 6). These results show that ChondroT exerted a chondroprotective effect and demonstrated a multi-target mechanism on inflammation and arthritis.
Oxidative stress was reported to induce apoptic cell death of chondrocytes and excessive production of various inflammatory cytokines in osteoarthritis, which further promote the expression of MMPs [16,17]. Polysaccharide from Angelica sinensis was reported to protect chondrocytes from H 2 O 2 -induced apoptosis through its antioxidant effects in vitro [18]. The anti-oxidant effects of ChondroT (Fig. 7) can help patients suffering arthritis. These results suggest that ChondroT composed of 5 herbs has therapeutic potential for the treatment of arthritis.
Arthritis has become a significant clinical problem worldwide with an increase in the aging populations. NSAIDs and selective COX-2 inhibitors are used for pharmacologic treatment of arthritis. Recent study showed that celecoxib, a COX-2 inhibitor decreased NO production in chondrocytes from osteoarthritic rat joints and reduced inflammation by blocking NF-kB activation in a murine model [19][20][21][22]. However, some patients treated with these drugs complained of side effects such as gastrointestinal, cardiovascular, and other complications. Our results show that the suppressive effect of ChondroT on COX-2 and iNOS expression was similar that exhibited by celecoxib, a COX-2 inhibitor used for the treatment of arthritis (Fig. 4). In addition, ChondroT did not show any cytotoxicity to various origin cells (Fig. 2a, b, and c). Traditional herb medications have been concerned for the treatment of arthritis. Recently, SKI306X (Joins), an oriental herbal mixture were developed for osteoarthritis patients [1,[23][24][25].
ChondroT is a water extract of 5 herbs, Osterici Radix, Lonicerae Folium, Angelicae Gigantis Radix, Clematidis Radix, and Phellodendri Cortex. Effect of Phellodendri Cortex was reported in protecting human osteoarthritic and cartilage and chondrocytes [26]. Anti-inflammatory effects of ChondroT can be attributed to the action of all 5 herbs [27][28][29][30][31]. We evaluated the remedial value of ChondroT compared with GHJTY. The suppressive effect of ChondroT was greater than that exhibited by GHJTY, and it showed multifunctional therapeutic effects on inflammation and arthritis. For standard validation of ChondroT, a  Fig. 7 Anti-oxidant activity of ChondroT. ChondroT and other drugs dissolved in methanol were mixed with DPPH (0.15 mM in methanol) in 96-well plate for 30 min and then the absorbance was measured at 470 nm using an ELISA microplate reader. BHA and vitamin C were used as positive anti-oxidant drugs. DPPH free radicals were decreased by approximately 95 % at 0.3 mg/mL ChondroT, and the inhibitory effect was greater than that exhibited by GHJTY. *** P < 0.001 compared with the control group convenient and accurate HPLC-PDA detection method was used for simultaneous determination of seven reference components. In conclusion, ChondroT treatment increased chondrocyte proliferation, in part through a reduction in oxidative damage. In addition, ChondroT attenuated the severity of cartilage degradation factor. With its anti-inflammatory and antioxidative properties, ChondroT may constitute a promising therapeutic option for the management of arthritis.

Conclusion
ChondroT exerted a chondroprotective effect and demonstrated a multi-target mechanism involving effects on inflammation and arthritis. In addition, the suppressive effect was greater than that exhibited by GHJTY, suggesting that ChondroT, a new complex herbal medication, has therapeutic potential for the treatment of arthritis.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.