Protective effect of Salvia miltiorrhiza in rheumatoid arthritis patients: A randomized, single-blind, placebo- controlled trial

Purpose: To study the protective effect of Salvia miltiorrhiza (SM) against rheumatoid arthritis (RA) in RA patients. Methods: Sixty RA patients were divided into two groups: SM (n = 30) and placebo (n =30) groups given SM at a dose of 250 mg/kg (3 capsules/day), and placebo (3 capsule/day), respectively, for 12 weeks. Patient responses based on American College of Rheumatology (ACR), health assessment questionnaire (HAQ) score, and global assessment of disease (GAD) were recorded. Moreover, Disease Activity Score (DAS) 28, pain score (visual analogue score, VAS), rheumatoid factor (Rh factor), and inflammatory cytokines (markers) were determined. Results: After 12 weeks of intervention with SM, ACR20 (30 %)/ACR50 patient response (13.3%, i.e., score for swelling and tenderness of joints), was significantly improved. There were considerable reductions in GAD, HAQ, DAS 28, pain score (VAS), and levels of erythrocyte sedimentation rate (ESR), acute phase reaction protein (CRP), Rh factor (IgM) and inflammatory cytokines (IL-1β, IL-6 and TNF-α), when compared to placebo (p < 0.01). Treatment with SM produced milder adverse effects than treatment with placebo (p < 0.01). Conclusion: Overall, SM produces better anti-RA effect than placebo by significantly altering ACR patient response, reducing GAD, HAQ, DAS 28 scores, Rh factor, ESR, CRP and inflammatory cytokines in RA patients. However, a large-scale clinical trial is needed before SM can be recommended for combating RA and its related symptoms.


INTRODUCTION
Rheumatoid arthritis (RA) is a chronic and destructive auto-immune disease characterized by increased inflammation of the joints and adverse effects on movement. The major symptoms of RA are swollen joints, arthralgia (joint pain), and stiff joints due to damage to cartilage and bone of joints, especially at the knees, ankles, feet, and elbow [1]. The disease affects about 0.5-2 % of the global population, and is associated with high morbidity (deformity and disability), especially amongst elderly women [2]. The quality of life of RA patients is considerably hampered due to disability and deformity. Hence, most RA patients need extensive care and assistance even in the performance of basic day-to-day activities, leading to serious economic burden (absence from work) [3].
The pathophysiology of RA is still not fully understood.
However, oxidative stress (excessive free radical generation), altered immune response and inflammatory events are considered as pivotal events in the pathogenesis of RA [1]. Currently, disease-modifying antirheumatic drugs (DMARDs), non-steroidal antiinflammatory drugs (NSAIDs), and steroids are recommended for the treatment or management of RA and its related symptoms. These treatments are effective only at the early stages of the disease, but they are expensive in the long run, and they also result in serious adverse reactions, apart from being ineffective in some RA patients [4]. Therefore, many researchers are shifting their focus to Traditional or Complementary or Alternative medicine which have lower adverse effects, and are more affordable and more effective in most RA patients [5].
Salvia miltiorrhiza (SM) Bunge is a well-known Traditional Chinese Medicinal (TCM) plant (Danshen/Reg sage) which belongs to the family Lamiaceae. The dried roots are recommended for treatment of cold, fever, nausea, joint pain and sprain, and for enhancement of blood circulation [6]. The dried roots of SM rhizome are rich in diterpenoids (tanshinone), phenolic compounds (salvianolic acid A/B), and flavonoids [7]. Salvia miltiorrhiza (SM) has a wide spectrum of therapeutic and beneficial properties such as anti-inflammatory, immunosuppressive, antioxidant, anti-tumor, pro-apoptotic, antidiabetic, anti-rheumatoid arthritis, as well as cardio-, hepato-and reno-protective effects. These properties are due to the presence of various bioactive phytocomponents such as tanshinones (I, IIA/IIB), salvianolic acid A/B, and lithospermic acid [8]. Previous studies on cell lines and animal models have revealed that SM and its bioactive components (tanshinone and salvianolic acid) possess potent antiinflammatory, immune-suppressive, antiadhesion and anti-rheumatoid arthritis properties [9][10][11]. Nevertheless, to date, no clinical trials have been conducted with SM in RA patients. Hence, in the present investigation, a singleblinded and randomized placebo-control clinical trial was conducted to study the protective effect of SM in RA patients.

Recruitment of subjects and ethical approval
This single-blind, randomized placebo-controlled clinical trial was conducted by initially recruiting 78 RA patients (outpatients) aged between 22 and 72, based on American College of Rheumatology (ACR) criteria and Disease Activity Score (DAS 28) > 5.1, with the help of flyers/posters and newspaper advertisements. All patients underwent basic physical examination, vitals and biochemical parameters to cross-check their health status and wellness. Moreover, information was obtained especially about their medication before enrolling the participants for this trial. The inclusion criteria covered only RA subjects (determined based on ACR criteria) with at least 5 swollen joint/pain score, and who were on DMARDs. Heavy smokers or alcohol drinkers, drug addicts, cancer patients, and patients who had hepatic, cardio-pulmonary, gastro-intestinal or renal disorders/diseases, were excluded. Moreover, subjects allergic to plant products and other auto-immune disorders were not included. Based on these exclusion and inclusion criteria, only 60 RA subjects were finally enrolled in the trial.
This clinical trial was conducted at Beijing Ditan Hospital Capital Medical University from March to December 2018, and was approved by the ethical clearance board members of Beijing Ditan Hospital of Capital Medical University, China (approval no. = TMBD-1546/12-2017). All the procedures used in this trial were conducted in accordance with the guidelines of the Declaration of Helsinki [4]. All the 60 RA subjects enrolled in this study were asked to sign a consent form before explaining the details of this trial. The flow chart of the clinical trial is shown in Figure 1.

Experimental protocol/intervention
The 60 RA patients were randomly divided into two groups: SM (n = 30) and placebo (n = 30) groups, using computerized random digital numbers. Each participant in SM group was given 3 capsules every day at a dose of 250 mg/kg for 12 weeks. The SM/Danshen extract and placebo capsules were provided by Wuxi Gorunjie Natural-Pharma Co. Ltd, Wuxi, China. The dose of SM was chosen based on previous studies conducted by van Poppel et al [12]. There were no differences between the SM and placebo capsules in terms of color and flavor. The placebo was filled with starch, and it had 1% SM which gave it a flavor similar to that of SM.

Sample collection and measurement of various parameters
Basic medical checkup covering all the vitals were done to confirm the health status. Then, blood samples were collected by a phlebotomist, at baseline and at the 12 th week (end of the study). The blood samples were kept at -80 °C prior to analysis. Serum samples were obtained after centrifugation of clotted whole blood, and subjected to various biochemical analyses. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using ultracentrifugation technique, based on the Salunkhe method [13]. The PBMCs were subjected to rheumatoid (Rh) factor-like IgM analysis using immune-nephelometry with BNII analyzer kits (Siemens Medical Solutions Diagnostics; Munich; Germany) based on the manufacturer's protocol. Erythrocyte sedimentation rate (ESR) was calculated using Westergren method. Serum high sensitivity C reactive protein (hs-CRP) was determined using commercial ELISA CRP kit (MYBioSource; CA, USA). Various inflammatory markers, viz, interleukin 1 beta/6 (IL-1β/6) and tumor necrosis factor-alpha (TNF-α), were quantified with commercial (Quantikine) ELISA kit (R&D System, MN, USA) method based on manufacturer's instruction.
Other major factors such as those associated with global assessment of disease (GAD, on a scale of 0 -100), including core disability Health Assessment Questionnaire (HAQ) score/index (score range of 0 -3, with 20 different questions on dressing, walking, grooming, eating and rising) were calculated [14,15]. Moreover, ACR criteria i.e. ACR 20/50 patient response (positive improvement) which refers to reduction of 20 or 50 % in the number of swollen and tender joints; patient pain (VAS), hs-CRP, as well as GAD and HAQ were determined with DAS 28-ESR (including 28 joints/tender-radiographic examination, ESR, and VAS pain scale of 0 -100) by a trained rheumatologist and physician, at baseline and at the 12 th week [16,17]. Finally, the incidence of various adverse or side effects such as headache, anemia, dizziness, nausea/vomiting, dyspepsia, anorexia, diarrhea, insomnia, and skin rashes/itching were recorded after 12 weeks of intervention.

Statistical analysis
Changes in the various parameters after the intervention (at the12 th week of the study) between the placebo and SM groups were analyzed using a one-way ANOVA and Student's t-test, while intra-group comparisons between baseline and 12 th week in each group (SM or placebo) were conducted using paired t-test. All statistical analyses were carried out with SPSS software (ver 21 from IBM Corp, CA, USA). A probability value of < 0.05 was considered statistically significant.

Baseline demographic characteristics
Only 56 out of the 60 RA patients completed the full 12 weeks of intervention. The other patients (3 from placebo group, and 1 from SM group) withdrew during the trial due to dissatisfaction with treatment. Table 1 shows the baseline demographic characteristics of the RA patients. The values of various baseline demographic characteristics including age, gender, age at disease onset, duration of RA and body mass index (BMI) were similar in both groups.  Table 2 shows the ACR20/50 patient response and the related score/index in RA patients after SM and placebo treatments. The ARC 20 and ARC 50 responses of RA patients (30 and 13.3%, respectively) were markedly improved (p < 0.01) after 12 weeks of treatment with SM, when compared with the placebo group. Moreover, levels of the various ARC-related scores/indices such as GAD and HAQ, as well as acute phase reaction protein hs-CRP, were significantly decreased (p < 0.01) as a result of supplementation with SM, relative to placebo.

Level of VAS, Rh factor, DAS 28 and inflammatory markers
As shown in Table 3, VAS, Rh factor (IgM) and DAS 28 were significantly reduced in RA patients after SM administration for 12 weeks, relative to placebo. Likewise, the levels of VAS, Rh factor (IgM) and DAS 28 were gradually decreased (p < 0.01) in the SM group at the 12 th week, when compared with baseline values. Table 4 shows the levels of inflammatory markers (proinflammatory cytokines) in RA patients after SM and placebo treatments. The concentrations of all pro-inflammatory cytokines i.e. IL-1β, Il-6 and TNF-α were markedly lowered in RA patients in the SM group, when compared with the patients that received placebo (p < 0.01). Table 5 shows the incidence of various adverse effects in RA patients after SM and placebo treatments. During and after SM and placebo interventions, some minor adverse effects were observed. In the SM group, the adverse effects comprised headache, anemia, dizziness, nausea/vomiting, dyspepsia and skin rashes (although minimal). The adverse events seen in the placebo group were anemia, nausea/vomiting, dizziness, anorexia, headache and skin rashes/itching. Overall, anemia was the major adverse event noted in both groups (13.3 % in SM group, and 16.6 % in placebo group). The other adverse events were minimal, implying the safety of SM. 1.58 ± 0.1b # 3.10±0.30a Data are expressed as mean ± SD. For ACR, values are expressed as % of patient response. Values within the same experimental group (SM or placebo) bearing different co-script letters are significantly different (p < 0.05). *P < 0.05; # p < 0.01, SM vs placebo at the 12 th week: SM = Salvia miltiorrhiza; ACR = American College of Rheumatology; GAD = global assessment of disease; HAQ = Health assessment questionnaires; hs-CRP = high sensitivity C reactive protein 3.89± 0.5b # 6.15± 0.5a Data are expressed as mean ± SD. Values within the same experimental group (SM or placebo) bearing different co-script letters differ significantly (p < 0.05). *P < 0.05; # p < 0.01, SM vs placebo at the 12 th week: SM = Salvia miltiorrhiza; VAS = visual analogue score; IgG = immunoglobulin G; ESR = erythrocyte sedimentation rate; DAS 28 = disease activity score .55a Data are expressed as mean ± SD. Values within the same experimental group (SM or placebo) bearing different subscript letters differ significantly (p < 0.05). *P < 0.05; # p < 0.01, SM vs placebo at the 12 th week: SM = Salvia miltiorrhiza; IL-1β = interleukin 1 beta; IL-6 = interleukin 6; TNF-α = tumor necrosis factor-alpha; pg = picogram

DISCUSSION
This is the first single-blind, placebo-controlled, randomized clinical trial conducted with SM alone (extract capsule) to assess the protective effect of SM through determination of ACR response and GAD, HAQ and DAS 28 scores, as well as levels of Rh factor, ESR, CRP and inflammatory cytokines in RA patients. The outcome of the trial showed that intake of SM capsule for 12 weeks markedly improved ACR 20/50 response of RA patients, with concomitant reductions in the levels of GAD, HAQ, DAS 28 scores, and decreases in the concentrations of Rh factor, ESR, CRP and inflammatory cytokines (markers). Patients in the placebo and SM groups showed similar baseline demographic characteristics. This helped to determine the impact of SM by comparing with a placebo group. The modified ACR classification criteria were developed in 2010 to plan a new set of criteria for diagnosing and classifying RA patients based on the various specifications which aid in early diagnosis and therapeutic intervention [18]. The ACR 20/50 patient response (positive improvement) represents a reduction of 20 or 50 % in the number of swollen (tender joints) in RA patients. Hence, ACR criteria and its response (ACR 20/50/70) are considered the primary outcomes of any anti-RA drug. In this study, RA subjects who ingested SM capsule for 12 weeks had significantly enhanced ACR20/50 patient response, relative to placebo-treated RA patients. Previously, Jie and his colleagues also demonstrated that treatment of RA patients with injection of salvia and complementary medicine considerably improved ACR 20/50 patient response [19]. During this clinical trial, there was no positive response in ACR70. Hence, it was not included. Furthermore, the ACR-related scores/indices such as GAD and HAQ were significantly lowered upon treatment with SM, when compared with the placebo group.
The levels of acute reaction protein hs-CRP and rheumatoid factors (Rh factors) are highly associated with joint damage especially during RA, Thus, hs-CRP and Rh factor (IgM) are clinically considered as markers for RA [20]. Significant decrease in the levels of hs-CRP, Rh and IgM were seen in the SM group (after 12 weeks, relative to baseline. Likewise, when compared to placebo on the 12 th week, hs-CRP and IgM levels were markedly decreased. A study on rabbits conducted by Zhang and his coworkers also reported that treatment with salvianolic acid and tanshinone (active components of SM) significantly lowered hs-CRP level [21]. In addition, pain scores (VAS and DAS-28 scores) were decreased by administration of SM capsule.
During RA, the pro-inflammatory cytokines IL-1β/6 and TNF-α are exponentially synthesized (produced) by macrophages and lymphocytes, and they subsequently infiltrate the synovium to trigger inflammatory events involved in the pathophysiology of RA [22]. Similarly, in the present study, the baseline levels of the inflammatory markers IL-1β/6 and TNF-α in the placebo and SM groups were high, but on intervention with SM the levels of the inflammatory markers were markedly decreased. In the case of placebo, no difference was seen between values at baseline and values at the 12 th week. These results are in agreement with those reported by Xia and his co-workers, who also observed that treatment with salvianolic B (major phytocomponent of SM) significantly lowered the production of various pro-inflammatory cytokines i.e. IL-1β, IL-6, and TNFα in a collagen-induced rheumatoid arthritis rat model [11].
The safety of SM was evaluated by checking various common adverse effects encountered during an anti-RA intervention. As mentioned earlier, in both groups, few minor adverse events were observed, especially headache, anemia, dizziness, nausea/vomiting, and skin rashes. However, the SM group showed more minimal levels of adverse effects than the placebo group. This shows that SM which has been widely used in TCM for many years, is safe. Moreover, these adverse events (anemia, headache and skin rashes) are common in RA patients. However, these adverse effects were noted, but they were lower in the SM group than in placebo group.

Limitations of the study
The major limitations of this study were the involvement of a small number of RA patients and the short duration of intervention (12 weeks). These might affect the interpretation of the results. In addition, the usage of SM (TCM) might make it harder to pinpoint the exact mechanism involved, since SM has many bioactive phytocomponents.

CONCLUSION
Based on the outcome of this single-blinded, placebo-controlled randomized clinical trial, SM may be recommended for combating RA and its related symptoms via significant changes in ACR response and DAS 28 scores in RA patients, along with conventional anti-RA drugs. Nevertheless, in the future, there is need for a large-scale study of a longer duration which might give a more precise conclusion about the mechanism underlying the anti-RA effect of SM.