Herbo-mineral formulation ‘Ashwashila’ attenuates rheumatoid arthritis symptoms in collagen-antibody-induced arthritis (CAIA) mice model

Balkrishna, Acharya; Sakat, Sachin Shridhar; Joshi, Kheemraj; Paudel, Sandeep; Joshi, Deepika; Joshi, Kamal; Ranjan, Ravikant; Gupta, Abhishek; Bhattacharya, Kunal; Varshney, Anurag

doi:10.1038/s41598-019-44485-9

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Published: 29 May 2019

Herbo-mineral formulation ‘Ashwashila’ attenuates rheumatoid arthritis symptoms in collagen-antibody-induced arthritis (CAIA) mice model

Acharya Balkrishna^1,2,
Sachin Shridhar Sakat¹,
Kheemraj Joshi¹,
Sandeep Paudel¹,
Deepika Joshi¹,
Kamal Joshi¹,
Ravikant Ranjan¹,
Abhishek Gupta¹,
Kunal Bhattacharya^1,3 &
…
Anurag Varshney^1,2

Scientific Reports volume 9, Article number: 8025 (2019) Cite this article

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Rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder that affects joints of hands and feet and introduces injury in secondary organs such as cardiac tissue. In the present study, we induced RA in male Balb/c mice (CAIA) using collagen-antibody cocktail (C-Ab) and lipopolysaccharide intraperitoneal injections. Induction of RA in the animals was detected through the loss of body weight, food, and water consumption, pedal edema, increased arthritis score of the paw and ankle, increase in radiological and histological lesion score of ankle and knee joints and enhanced pain perception in the C-Ab induced RA animals. Ashwashila is a herbo-mineral medicine from Indian Ayurvedic system. Human equivalent doses of Ashwashila (ASHW) and standard of care, Methotrexate were given to the CAIA animals for two weeks. ASHW treatment significantly reversed the effect of C-Ab with reduced pedal edema, arthritis score, radiological and histological lesion scores in ankle-joint, knee-joint and articular cartilage, reduced pain perception. These effects were comparable with the Methotrexate treatment. In human monocytic (THP-1) cells, ASHW was found to be biocompatible at in-vitro test doses. The anti-arthritis mechanism of action for ASHW was established through the suppression of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α; and upstream regulator, NF-κB. Taken together, we show the pre-clinical efficacy of ASHW in reducing RA associated symptoms by controlling inflammation and suggest it as a potential therapeutic candidate for rheumatoid arthritis.

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Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects between 0.5–1% population of the world with women representing the majority of the diseased population¹. Several factors such as genetic, epigenetic, gender, environment and lifestyles play a crucial role as precursors for this disease. RA is characterized by local and systemic inflammation mostly occurring in the joints through the development of auto-antibodies to immunoglobulin G (IgG) such as rheumatoid factor (RF) and citrullinated proteins¹. Under chronic and untreated conditions, RA can lead to severe and irreversible damage of the joints through inflammation, edema, cartilage and bone damages.

The clinical symptoms of the RA disease are the soft synovial swelling along with morning stiffness and tenderness of metacarpophalangeal and proximal interphalangeal joints of the hands and feet, along with wrist, ankle, elbow, shoulder, knee and hip joints². Synovial region of the joint is the primary location for detectable RA. Pathogenic changes induced in the synovial area are mainly, increase and activation of synoviocytes A (macrophages) and B (fibroblast-like). Increased synoviocyte activity leads to the release of pro-inflammatory cytokines, chemokines and collagen-degrading proteases such as matrix metalloproteinases; and small molecule cell-signaling mediators such as prostaglandins and leukotrienes^3,4,5. Other changes associated with the development of RA are infiltration of immune cells such as CD4+ memory T cells, B cells, plasmablasts, and plasma cells into the synovial sub-lining, producing RF and citrullinated proteins¹. The cardinal signs of RA are damage to the articular cartilage and bone, along with visible pannus formation in the joints. Treatment of RA includes non-steroidal anti-inflammatory drugs (NSAIDs), disease modifying anti-rheumatic drugs (DMARDs), tumor necrosis factor alpha (TNF-α) inhibitors, IL-6 inhibitors, T-cell activation inhibitors, B-cell depletors, Kinase (JAK) inhibitors, immune-suppressants, and steroids. Other than these medications, appropriate changes in lifestyle such as regular exercise are also advisable.

Collagen type II is the major component of the joint’s cartilage matrix protein. The RA in animals models are induced by systemic administration of a cocktail of monoclonal antibodies (C-Ab) that target the various regions of collagen type II. Animals are further stimulated by lipopolysaccharide (LPS) for induction of joint inflammation⁶. Pathogenic features of collagen type II antibody induced RA in animal models include elevated arthritic scores, pedal edema, synovitis with infiltration of polymorphonuclear and mononuclear cells, pannus formation, collagen degradation, and bone erosion⁷.

‘Ashwashila’ (ASHW) is a herbo-mineral formulation containing aqueous extract of ‘Ashwagandha’ (Withania somnifera, family: Solanaceae) commonly known as ‘Indian Winter Cherry’ or ‘Indian Ginseng’; and dry powder of ‘Shilajit’ found as a blackish-brown exudate present on the rocks of the Himalayas between Arunachal Pradesh and Kashmir, in India⁸. Under severe RA conditions in animals, ‘Ashwagandha’ herbal extracts have been found to reduce inflammatory responses^9,10. ‘Ashwagandha’ herbal extracts have shown that its withanolides components modulate proliferation of breast cancer tissue through induction of FOXO3a protein and pro-apoptotic protein BIM, leading to induction of apoptosis in breast cancer cells¹¹. Similarly, ‘Withaferin-A’, a component of the ‘Ashwagandha’ has been reported to bind with the cysteine residues of the IKK-β kinase. This deactivation of the IKK-β kinase exerts anti-inflammatory response by blocking of downstream NFκβ activation¹². The second component of ASHW, ‘Shilajit’ is formed from gradual decomposition of plant matter contains both humic and non-humic constituents⁸. ‘Shilajit’ has been used extensively in ancient herbal formulations as a rejuvenator and anti-aging agent. Fulvic acid present in ‘Shilajit’ has been found to have immunomodulatory and psychoactive behavior¹³. Treatment of ‘Shilajit’ prevents self-aggregation of tau fibrils, that is responsible for the development of Alzheimer’s disease^14,15. ‘Shilajit’ also contains elemental Selenium that a has a significant anti-inflammatory function, as an inhibitor for COX-2 and TNF-α activity^16,17. Dietary supplement of Selenium also decreases mechanically induced osteoarthritis; and increases levels of anti-oxidative enzymes in the knee joints¹⁸.

Combined treatment with herbal extracts of both ‘Ashwagandha’ and ‘Shilajit’ has been found to work as a nootropic or psychoactive drug, reducing addiction to alcohol consumption in the Swiss albino mice¹⁹. Both ‘Ashwagandha’ and ‘Shilajit’ are present in the ASHW herbal formulation in equal quantity. However, no study has been reported to date to determine the efficacy of ASHW on RA and inflammation.

In the present study, the efficacy of ASHW herbo-mineral formulation in reducing the inflammatory response to RA in the joints of Balb/c mice has been tested. RA was induced in the Balb/c mice using a collagen-antibody cocktail (C-Ab) and lipopolysaccharide (LPS). The collagen antibody-induced arthritis (CAIA) animals were treated with ASHW and Methotrexate (MTX), as the reference standard of care for two weeks. These animals were studied for their feeding and water intake habits, body weight changes along with modifications in the symptoms for arthritic edema, pain perception, radiological and histopathological analysis of the ankle and knee joints. For determining the mechanism of action, we treated the LPS stimulated human monocytic (THP-1) cells with ASHW and studied the release of interleukin one beta (IL-1β), IL-6 and Tumor Necrosis Factor-alpha (TNF-α); and expression of upstream regulatory protein, NFκB. Our results showed that the ASHW exhibited promising efficacy in reducing the RA symptoms in the diseased animals through the modulation of cell-signaling components associated with inflammation.

Results

In the present study, disease control (DC) animal model, the collagen antibody-induced arthritis (CAIA) Balb/c mice, showed severe induction of rheumatoid arthritis (RA) and associated distress through rapid reduction in their body weight, feed, and water intake habits (Fig. 1A, Suppl. Fig. 1A–C). Treatment of the CAIA animals with 353 mg/kg dose of ASHW (mice equivalent human dose of 2000 mg/day) every day for two weeks did not affect the induced loss of body weight along with the reduced food and water consumption quantities (Suppl. Fig. 1A–C). Treatment of the CAIA animals with 0.38 mg/kg dose of standard of care drug, MTX every alternate day for two weeks showed significant recovery of body weight, as compared to the DC animals (Suppl. Fig. 1A–C), without any observable changes in the reduced food and water consumption habits.

The onset of RA in the DC animals following C-Ab and LPS treatments was also visible through a constant increase in arthritis score compared to the normal control (NC) animals (Fig. 1B). Treatment of the CAIA animals with the 353 mg/kg dose of ASHW or with 0.38 mg/kg dose of MTX induced a significant reduction in the arthritis score till the end of the study period (Fig. 1B). Statistically significant reduction of arthritis score in ASHW-treated diseased animals was found on days 14 (p-value ≤ 0.01) and 16 (p-value ≤ 0.05); and for reference drug MTX on days 8 (p-value ≤ 0.05), 10 (p-value ≤ 0.01), 12 (p-value ≤ 0.05), 14 (p-value ≤ 0.01) and 16 (p-value ≤ 0.05), when compared to the DC animals. The anti-arthritic activities shown by ASHW and reference drug MTX were found to be statistically indistinguishable on day 14 (Fig. 1C).

According to the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) on RA classification, swelling of at least one joint is a requisite criterion²⁰. In our study, edema examination of the paw and ankle joints showed increased swelling of the ankle, feet, and digits in the CAIA animals as compared to the NC animals (Fig. 2A,B). Treatment of the CAIA animals with herbal formulation ASHW and reference drug MTX prompted a reduction in the swelling in the ankle, feet, and digits (Fig. 2C,D). Visual observation of edema was further complemented by measuring the paw and ankle thickness in the animals (Fig. 2E,G). The DC animals showed significant (p-value ≤ 0.05) increase in the paw and ankle thickness (Fig. 2E,G). Treatment of the CAIA animals with ASHW (paw edema - Day 8: 37.0 ± 11.2%; Day 12: 30.5 ± 11.6%; Day 16: 34.9 ± 15.3%) and MTX showed a considerable decrease in paw and ankle edema measured at different days (Fig. 2E,G). Those observed reduction in paw edema following drug treatments were statistically not significant. However, reduction in the ankle edema on day five was statistically significant for both the ASHW (p-value ≤ 0.05) and MTX (p-value ≤ 0.01) treatments (Fig. 2G). ASHW showed equipotent efficacy in controlling pedal and ankle joint edema in comparison to the reference care drug MTX throughout the study period, except on day 5, wherein MTX displayed superior effects (p-value ≤ 0.05) in controlling ankle joint edema (Fig. 2F,H).

During the onset of RA, there is a significant induction of neuroinflammation and hyperalgesia, due to the release of pro-inflammatory cytokines and chemokines²¹. Clinical evidence for enhanced hyperalgesia has been demonstrated in RA patients²². Pain perception analysis of the CAIA animals was performed using Randall-Selitto test (Mechanical Hyperalgesia) and hot plate test (Thermal Hyperalgesia), on different days of treatments (Figs 1A and 2I,J). Treatment of the CAIA animals with ASHW showed noticeable reduction (p-value ≤ 0.01) in their pain sensitization towards Randall-Selitto test at days 10 and 14 (Fig. 2I). Similarly, treatment of the CAIA animals with the reference drug MTX decreased their pain sensitivity (p-value ≤ 0.01) at days 10 and 14 (Fig. 2I). Hot plate test further confirmed the reduction of pain sensitivity following ASHW or MTX treatments in the CAIA animals (Fig. 2J). Statistically significant decrease in the pain sensitivity of the CAIA animals tested by hot plate test was observed in ASHW treatment on day 11 (p-value ≤ 0.05) and for MTX treatment on days 11 (p-value ≤ 0.01) and 15 (p-value ≤ 0.01) (Fig. 2J).

The severity of the C-Ab induced RA disease in the animals was studied using X-ray techniques, and radiological scoring was enumerated, considering different parameters²³. CAIA animals showed induction of clinical symptoms such as periosteal reaction/hypertrophy (PR), bone erosion (B), soft tissue swelling (SS), narrowed joint space (JS) and osteoporosis (OP) in the knee- and ankle-joints (Fig. 3A–D). These were found to be ameliorated with the ASHW, and MTX treatments. The CAIA animals showed high radiological scores in the ankle region (p-value ≤ 0.05) (Fig. 3E). Treatment of the CAIA animals with ASHW (p-value ≤ 0.05) or MTX (p-value ≤ 0.01) showed a significant reduction in the radiological score in the ankle-joint (Fig. 3E). Standard of care drug, MTX showed higher efficacy compared to ASHW in reducing radiological scores in the ankle-joint of the CAIA animals (Fig. 3E insert).

Similarly, knee-joint also showed significant lesions development in the CAIA animals, with increased radiological scores, as compared to the NC animals. Treatment of the CAIA animals with ASHW showed a minor decrease in the radiological scores. However, MTX treatment significantly reduced (p-value ≤ 0.01) radiological scores, as compared to the DC animals (Fig. 3F). Similar to the ankle-joint, MTX treatment in the CAIA animals exhibited higher efficacy as compared to the ASHW in controlling the development of CAIA driven radiological changes in the knee-joints (Fig. 3F insert).

Histopathological analysis of the CAIA mice pedal was performed following fourteen days of treatment of ASHW and MTX (Figs 4 and 5). Ankle-joints analysis of the DC animals indicated the development of RA disease-associated lesions such as the moderately enlarged synovial membrane, hyperplastic synovium, increased synovial vascularity, the presence of inflammation and, bone and cartilage erosion (Fig. 4). Severe ankle-joint damages in the DC animals were observed through lesion score analysis for synovial-lining cell layer-hyperplasia, -vascularity, infiltration of inflammatory cells, pannus formation, cartilage, and bone erosion (Fig. 4E and Suppl. Fig. 2A–G). Significant reduction in the ankle-joint lesion scores (p-value ≤ 0.01) was observed in the CAIA animals following treatment with ASHW and MTX. Total and individual scoring showed that both the ASHW and MTX exhibited comparable lesion-reducing efficacy in the synovial membrane inflammation, pannus formation, cartilage, and bone erosions of the CAIA animals (Fig. 4F, Suppl. Fig. 2A–G).

Similarly, histopathological analysis of the CAIA animals knee-joints showed a significant increase (p-value ≤ 0.01) in the lesion scores (Fig. 5E). Treatment of the CAIA animals with ASHW and MTX significantly reduced the RA-associated lesions (p-value ≤ 0.01). Individual lesion score analysis in the CAIA animals following ASHW or MTX treatments indicated an equal reduction in synovial membrane inflammation and vascularity along with pannus formation, cartilage and bone erosion (Suppl. Fig. 3A–G). Comparative analysis of the drug efficacy of the MTX and ASHW indicated statistically comparable disease inhibition potentials (Fig. 5F).

Stimulation of RA in the CAIA animals induced severe damage and lesion formation in the articular cartilage of the ankle- and knee-joints, as well (Figs 6 and 7). Safranin ‘o’ staining of the proteoglycans present in the cartilage region, showed severe induction of damage to the articular cartilage (p-value ≤ 0.01) in the ankle joints of the DC animals (Fig. 6A,B,E). Treatment of the CAIA animals with ASHW and MTX showed a prominent decrease (p-value ≤ 0.05) in their mean lesion score (Fig. 6C–E). Both ASHW and MTX showed similar efficacy for reducing cartilage damage and lesion formations in the treated CAIA animals. Similar trends were observed in knee joints cartilage analysis. Treatments of the CAIA animal with ASHW and MTX significantly reduced (ASHW: p-value ≤ 0.05; MTX: p-value ≤ 0.01) the disease driven cartilage damages and the formation of lesions over two weeks (Fig. 7A–E). Finally, both the ASHW and MTX showed similar efficacies in modulating disease induced cartilage lesions in the knee-joint (Fig. 7F).

Basic liver functions were studied in the serum of the CAIA mice by analyzing enzymatic biomarkers: Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) (Figs 1 and 8). Balb/c mice treated with C-Ab antibody cocktail and LPS showed a significantly high stimulated release of both ALT and AST (p-value ≤ 0.01) (Fig. 8A,B). Treatment of the CAIA animal with ASHW significantly reduced the ALT back to basal levels (Fig. 8A), while no statistically significant change was observed for the AST biomarker for liver function (Fig. 8B). MTX treatment of the CAIA animals exhibited a substantial decrease in the release of both the ALT and AST biomarkers in the blood serum (Fig. 8A,B). It is noteworthy that ASHW treatment did not induce any additional elevation of serum ALT and AST levels, in comparison to DC animals; suggesting ASHW did not induce any gross level changes in the liver functions of Balb/c mice.

Cell viability analysis of the ASHW in the THP-1 cells showed no loss of cell viability up to 12.5 mg/mL (Fig. 9A). Based on the obtained results, ASHW concentration to induce a 20% loss in cell viability (IC₂₀) was calculated at 18.83 mg/mL and IC₅₀ at 42.29 mg/mL (Fig. 9A). Statistically significant mild toxicity was detected at the ASHW concentration of 25 mg/mL (p-value ≤ 0.01) (Fig. 9A). It is worthwhile to note that MTX is highly cytotoxic, with reported IC₅₀ in the range of 30 μM (equivalent to 13.6 μg/mL), across several cell lines²⁴. For elucidating the mechanism of action of the ASHW under in-vitro conditions, we studied the modulation of IL-1β, IL-6 and TNF-α cytokines in LPS stimulated human monocytic (THP-1) cells (Fig. 9B–D). All the pro-inflammatory cytokines were found to be upregulated in the THP-1 cells on stimulation with 500 ng/mL LPS. Treatment of the LPS stimulated cells with the ASHW between the concentration of 0.3–10 mg/mL significantly reduced the released levels of IL-1β, IL-6, and TNF-α cytokines in a dose-dependent manner (Fig. 9B). The highest reduction of IL-6 and TNF-α cytokines release in the LPS stimulated THP-1 cells were detected at the ASHW dose of 10 mg/mL (p-value ≤ 0.001) (Fig. 9C,D).

Modulation of the pro-inflammatory upstream gene regulatory protein, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), were studied using luminescent reporter gene assay for NFκB in THP-1 cells. LPS stimulation of the THP-1 cells induced a 3-fold increase in the expression of the NFκB protein (Fig. 9E). Treatment of the LPS stimulated THP-1 cells with ASHW significantly reduce the upregulated production of NFκB protein in a dose-dependent manner. Highest inhibition of NFκB expression by ASHW was found at the concentration of 10 mg/mL (Fig. 9E). Taken together, these in-vitro results complement well with the in-vivo study findings; and supplement the indication that ASHW is indeed a strong anti-inflammatory herbo-mineral formulation.

Discussion

Tradition Indian Medicines (TIM) have been widely accepted in the public domain as an excellent alternative or additive therapeutics²⁵. Disease-modifying anti-rheumatic drugs and non-steroidal anti-inflammatory medicines have been used as the principal therapy for controlling the clinical symptoms associated with RA²⁶. Compared to synthetic medicines; herbal formulations are considered to be rather holistic and safe²⁷. However, there are limited scientific studies performed on the pre-clinical efficacy of these TIMs in curing chronic and acute diseases. RA is a systemic inflammatory disease that induces inflammation, hyperplasia, auto-antibody production, cartilage and bone destruction, causing pain and immobility in the patients. Ashwashila (ASHW) has been broadly prescribed for the treatment of inflammation, neuropathy, strengthening the physiological and immune system by the traditional Ayurvedic practitioners.

In the present study, we determined the anti-arthritic efficacy of ASHW using collage antibody (C-Ab) induced arthritis (CAIA) Balb/c mice models. The mice dosage of ASHW selected in the study was 353 mg/kg/day (human equivalent dose of 2000 mg/day) given for two weeks; and the standard of care drug, MTX dosage was 0.38 mg/kg given every alternate day for two weeks. Our results showed that ASHW did not modulate the loss of weight, feeding, and water intake habit of the diseased animals, as compared to the MTX. However, both ASHW and MTX showed similar efficacy in reducing the arthritis score, paw and ankle edema, inflammatory lesions in the ankle and knee joints, and pain sensitivity in the CAIA animals. The mode of action for the MTX is well studied, such as, by reducing T-cell activity at the site of inflammation, blocking IL-1β surface receptors of target cells and reducing bone and cartilage damages through erosions^28,29. However, no information is available regarding the mode of action of ASHW herbal formulation in reducing RA symptoms.

ASHW herbal formulation is composed of an equal quantity of Ashwagandha aqueous extract and Shilajit. Ashwagandha or Withania somnifera has been extensively studied for its chemical composition, and its biologically active components identified are alkaloids, steroidal lactones, saponins containing additional acyl group and withanolides³⁰. Shilajit is composed of three oxygenated biphenyls and three oxygenated 3-4-benzcoumarins, several phenolics and amino acids and triterpenes³¹. Rasool and Varalakshmi (2007) studied the efficacy of root powder from Withania somnifera in modulating the inflammation, oxidative stress and cartilage erosion in adjuvant-induced arthritis in Wistar rat models³². The authors showed that the Withania somnifera root powder at the daily dose of 1000 mg/kg/day significantly reduced inflammation in the form of lipid peroxidation; and anti-oxidant levels returned to normal levels, as compared to the disease control animals³³. Other studies have shown the efficacy of Withania somnifera extract at the concentrations between 100–800 mg/kg to reduce RA symptoms such as oxidative stress, ankle swelling, lipid peroxidation, glutathione levels, arthritic and radiological score and arthritic pain in the collagen-induced arthritis rat models^9,33. Shilajit at the concentration of 50 mg/kg/day dose given for 14 days reduced pedal edema by 77% in the carrageenan-stimulated Wistar rats showing inflammation³¹.

In our study, reduction in arthritis score, along with paw, ankle and knee-joint inflammation, and edema in the CAIA animals was observed following a dose of 353 mg/kg/day oral dose of ASHW. Interestingly, since ASHW contains 1:1 ratio by weight of Withania somnifera and Shilajit, reduction in RA symptoms such as paw and ankle edema, arthritic and radiological scores were achieved at the individual daily doses of 176.5 mg/kg/day as compared to the other studies, suggesting a synergistic mechanism of action for both of the constituents. Furthermore, ASHW was observed to reduce liver toxicity and damages in the CAIA mice through a reduction in the stimulated levels of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), in blood serum. This in line with several other studies that have reported the role of Withania somnifera extracts in improving liver functions and in ameliorating chemicals induced hepatotoxicity^34,35,36.

Using the in-vitro cell-based studies, we were further able to elucidate that ASHW was not only biocompatible in the human monocytic (THP-1 cells) but also capable of reducing inflammatory reactions in the LPS-stimulated cells. The inhibitory concentration at 50% loss of cell viability (IC₅₀) for ASHW was extrapolated at 42.29 mg/mL. Compared to the RA standard of care drug MTX (IC₅₀: 13.6 μg/mL)²⁴, the IC₅₀ value for ASHW was found to be many order of magnitudes higher; indicating a better safety margin for ASHW.

Studies have demonstrated that pro-inflammatory cytokines play a major role in the severity of RA disease and their reduction also reduces the RA pathogenicity^37,38. Withanolides present in the Withania somnifera extracts are responsible for their anti-inflammatory properties, and in the Shilajit, their phenolic components are accountable for their anti-oxidant and anti-arthritic activities^39,40. Treatment of human subjects having RA with 5 grams twice daily dose of Withania somnifera powder followed by a mineral formulation (sidha makardhwaj) treatment significantly reduced the clinical RA factor; and reduced scores of tender joint counts, swollen joint counts, physician assessment score and pain⁴¹. Therefore, modulation of the pro-inflammatory cytokines along with attenuation of RA pathogenicity in the CAIA animals by ASHW indicate a direct relationship between the two events. This research paper is the first report on the combined behavior of Withania somnifera and Shilajit, in the form of ASHW, in modulating the RA symptoms in diseased animals as there are no scientific reports available on the combined effect of Withania somnifera and Shilajit on reducing CAIA systemic and joint inflammation, edema, articular cartilage erosion, and bone erosion.

Earlier studies have reported a direct correlation between the induction of RA pathogenicity, release of pro-inflammatory cytokines and the stimulated expression and release of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the CAIA animals⁴². Earlier Singh et al. (2007), have shown Withania somnifera plant crude ethanol extract to reduce LPS stimulated expression of NFκB and associated inflammatory responses⁴³. NF-κB protein plays an important role as a mediator for the release of pro-inflammatory cytokines in both innate and adaptive immune cells. In our study, reduction in expression of NFκB protein along with the downstream associated pro-inflammatory cytokines presents the mode of action of ASHW in attenuating RA pathogenicity. Root extracts from Withania somnifera have been observed to have chondroprotective effect in cases of other forms of arthritis such as osteoarthritis through the reduction of the nitric oxide in the joints and inhibition of gelatinase activity of the collagenase type 2^44,45. However, in our study the ASHW treatment did not induce relief from CAIA distress as observed through weight and feeding habit loss of the animals. It is, therefore, possible that the ASHW may require a longer duration of treatment to make significant gross level changes. Observed ASHW driven reversals of bone and cartilage erosion, which is comparable to the MTX treatment, substantiate this hypothesis. The release of various pro-inflammatory soluble mediators and oxidative stress during RA and other systemic inflammations have been implicated in neuroinflammation and cognitive impairments. Both, Withania somnifera and Shilajit have been individually observed to possess neuroprotective effect through their anti-oxidant and anti-inflammatory behaviors^8,46.

Primary afferent neurons perform three major nociceptor functions that are: detection of noxious elements or damaging stimuli (transduction); passage of the resulting sensory input from peripheral terminals to the spinal cord (conduction); and synaptic transfer of this input to neurons within specific laminae of the dorsal horn (transmission)⁴⁷. During severe inflammation such as RA, the threshold for nociceptor neurons to fire action potentials is reduced, leading to pain sensitivity or “hyperalgesia”⁴⁸. This further reduces tissue immune response to damaging stimuli and noxious elements. In the present study, using the Randall-Selitto test and hot plate test we could display that the CAIA animals showed higher pain sensitivity and movement. An oral dose of ASHW helped in the reduction of mechanical and thermal hyperalgesia similar to those observed using the reference drug MTX. This indicated that the ASHW has neuroprotective potentials, as expected.

Taken together, we can conclude that ASHW is capable of reducing RA associated inflammation, oxidative stress and symptoms through modulating the amount of pro-inflammatory mediators. While synthetic drugs may produce rapid relief from RA associated edema and pain, their long term usage and effects on health are always a concern. Herbal formulations, on the other hand, may have milder effects in modulating disease-associated symptoms, but due to their nature derived origin and long-term historical usage, no known side-effects are expected. Therefore, using the CAIA Balb/c mice model, we accentuate that Ashwashila can be a potential candidate for treating rheumatoid arthritis, as a standalone or companion therapy.

Materials and Methods

Chemicals and reagents

For the study, Ashwashila (Batch no: AH18/038, manufacturing date: April 2018) was sourced from the Divya Yoga Pharmacy, Haridwar, India, 5-Clone Cocktail antibodies (Cat No-53040) and LPS (Escherichia coli strain 0111: B4; Cat No-9028) were purchased from Chondrex, Inc. WA. Methotrexate (Cat No-M9929) was procured from Sigma Aldrich, St. Louis, MO. Haematoxylin, Potassium Aluminium Sulphate Dodecahydrate, Mercury (II) Oxide red were purchased from Merck India Pvt Ltd, Mumbai, India. Safranin and Fast green were procured from Loba Chemie Pvt. Ltd, Mumbai, India. Eosin Yellow and Ferric Chloride were purchased from Hi-Media Laboratories, Mumbai, India. For tissue culture work, RPMI-1640 cell culture media, Fetal bovine serum, antibiotics, and other reagents were purchased from Life Technologies, Delhi, India.

Experimental animals

Male Balb/c mice (20–30 g) were procured from Charles River Laboratory licensed supplier, Hylasco Biotechnology Pvt. Ltd, Hyderabad, India. All the animals were housed in polypropylene cages in a controlled room temperature 22 ± 1 °C and relative humidity of 60–70% with 12:12 h light and dark cycle in a registered animal house (Registration Number: 1964/PO/RC/S/17/CPCSEA). The animals were supplied with standard pellet diet (Purina Lab Diet, St. Louis, MO, USA) and sterile filtered water ad libitum. The study protocol was approved by the Institutional Animal Ethical Committee of Patanjali Research Institute vide IAEC approval number- PRIAS/LAF/IAEC-009; and all the experiments were performed following relevant guidelines and regulations.

Induction of arthritis in animals

Arthritis was induced in the Balb/c mice by intraperitoneal (I.P.) injection of a cocktail of 5 monoclonal antibodies to type II collagen (1.5 mg in PBS/mouse; day-0) followed by LPS I.P. injection of 50 μg of lipopolysaccharide (LPS from Escherichia coli strain 011B4; in a sterile normal saline) on day-3 (Fig. 1A). The normal control (NC) animals received an equal volume of PBS along with vehicle Na-CMC. On day-4, disease induced animals were selected and randomized into different groups for treatments:

Group I: NC (PBS+ 0.25% Na-CMC p.o.; every day for two weeks)

Group II: Disease Control (C-Ab + 0.25% Na-CMC p.o.; every day for two weeks)

Group III: C-Ab+ MTX (0.38 mg/kg p.o.; every alternate day for two weeks)

Group IV: C-Ab+ ASHW (353 mg/kg p.o.; every day for two weeks)

The vehicle or MTX or ASHW treatment was initiated from day-4 and continued for the next two weeks.

Assessment of severity of arthritis

The severity of arthritis in each mouse paw was scored every alternate day in a blinded manner according to the marginally modified method of Khachigian (2006) and Moore et al. (2014) on a 0–4 scale as follows: 0 = normal; 1 = mild redness, slight swelling of ankle or wrist; 2 = moderate swelling of ankle or wrist; 3 = severe swelling, including some digits, ankle and foot; 4 = maximally inflamed. The total maximum score for each mouse was 16^6,49. The anti-arthritic activity (%) was calculated for each animal using the following formula: ((Mean arthritis score of disease control animals − arthritis score of each test animal)/Mean arthritis score of disease control animals) × 100.

Assessment of body weight, feed and water intake habits

Body weight was measured every alternate day. The change in body weight of each animal was calculated before and after the onset of arthritis and after subsequent treatment. Animal feed and water consumption were recorded daily until the end of the experiment.

Assessment of inflammatory parameters: Measurement of paw and ankle joint thickness

Mouse paw thickness was measured using digital vernier caliper (Mitutoyo, Japan) on days-0, 2, 4, 8, 12, 16 for paw thickness, and days 0, 2, 5, 9, 13, 17 for ankle thickness. The paw and ankle joint edema were calculated by subtracting 0 h (basal) thickness from the respective thickness post-treatment thickness on each day of the study. The anti-inflammatory activity (%) was calculated for each animal using the following formula: ((Mean edema of disease control animals (mm) − edema of each test animal (mm))/Mean edema of disease control animals (mm)) × 100.

Assessment of pain behaviours

Randall Selitto Pressure Test

The Randall–Selitto pressure test was performed to measure static hyperalgesia in animals according to the modified methods of Randall and Selitto (1957) and Anthony et al. (2007)^50,51. The pain response as paw withdrawal threshold (PWT) to the mechanical stimulus was determined with a Randall and Selitto device on day-0, 2, 6, 10 and 14, after 1 hour of drug treatment. The paw withdrawal threshold was defined as the force applied to the dorsal surface of the hind paw that causes mouse to vocalize or withdraw the paw. A limit of 25 g was set to avoid tissue damage. The average of 3 readings with a gap of 5 min from each mouse was recorded in a blinded manner, by a researcher unknown to the treatment conditions.

Hot Plate Test

Thermal hyperalgesia was assessed using a hot plate test as described by the methods of Chagasa et al. (2017) and Eddy and Leimbach (1953) with minor modifications^52,53. All the animals were placed into the perspex cylinder of the hot plate (Ugo Saile, Italy) maintained at 55.0 ± 0.5 °C; and time to discomfort reaction (licking paws or jumping) was recorded as response latency. The hot plate test was performed on a day- 0, 2, 7, 11 and 15, after 1 hour of drug treatment. A maximal cut-off point of 20 sec was considered to avoid any possible accidental paw damage. The average of 3 readings with a gap of 5 min from each mouse was recorded in a blinded manner, by a researcher unknown to the treatment conditions.

Radiological analysis

X-ray analysis was used to assess the morphology of hind limb swelling. After two weeks of drug treatment, all the animals were humanely sacrificed; left hind limbs were isolated and processed for X-rays images using X-ray device (Siemens Heliophos-D Germany) with a 40 KV exposition 0.01 sec (at Department of Radiology, Patanjali Ayurveda Hospital, Haridwar, India). The radiological analysis was done in a blinded manner by the Veterinary Radiologist. Scoring of the abnormalities such as swelling of the soft tissue around the joints, periosteal hypertrophy, narrowing of the joint space, periarticular osteoporosis, bone erosions, and any other lesions were done on the severity level: 0 = Normal; 1 = Slight; 2 = Moderate and 3 = Severe. Knee and ankle joints were analysed separately⁵⁴. The % activity was calculated using the following formula: ((Mean knee or ankle joint radiological score of disease control animals − Knee or ankle joint radiological Score of each test animal)/Mean knee or ankle joint radiological score of disease control animals) × 100.

Histopathological examination

The mouse right limb was harvested immediately after humanely sacrificed, fixed in 10% buffered-neutral formalin, decalcified in 10% formic acid for four days, embedded in paraffin, sliced solid sections of 3–5 µm thickness and stained with hematoxylin-eosin for general evaluation and Safranine O dye for specific assessment of cartilage damage. Blinded examination of histological slides was performed by a veterinary pathologist to minimize bias. The severity of microscopic arthritic changes (enlargement in synovial lining cell layer, synovial hyperplasia, synovial vascularity, infiltration of inflammatory cells, pannus formation, cartilage erosion, and bone erosion) were evaluated in hematoxylin and eosin (H & E) stained slides using the following grades: 0 = No abnormality detected; 1 = minimal (<1%); 2 = mild (1–25%); 3 = moderate (26–50%); 4 = marked (51–75%); 5 = severe (76–100%). Distributions of the lesions were recorded as focal, multifocal and diffuse. Similarly, the severity of the cartilage degradation was scored as 0 = no apparent change; 1 = superficial fibrillation of articular cartilage; 2 = defects limited to uncalcified cartilage; 3 = defects extends into calcified cartilage; 4 = exposure of subchondral bone at the articular surface. The scoring of knee and ankle joints were recorded separately. Images of the histological slides (H & E and Safranine O) were captured using Olympus Magnus microscope camera, and processed by Olympus MagVision image analysis software.

Blood biochemistry

For analysis of the Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), the Balb/c mice blood serum was isolated and stored at −80 °C till further use. Commercially available kits for ALT (AL 8304) and AST (AS 8306) were purchased from Randox, USA and processed on RX Monaco technology platform (Randox, USA), as per manufacturer’s instructions.

Cell culture for in-vitro experiments

Human monocytic (THP-1) cells were obtained from the ATCC authorized cell repository, NCCS, Pune, India. THP-1 cells were cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (FBS), in presence of 100 units/mL concentrations of penicillin/streptomycin, 1 mM sodium pyruvate and 4 mM L-glutamine. Cells were grown at 37 °C in a 5% CO₂/air environment.

Cell viability analysis

One gram of the powdered ASHW was suspended in incomplete culture media (RPMI-1640) at 37 °C for two hours. The insoluble part was cleared by high-speed centrifuge at 14000 RPM. The cleared fraction was filtered with 0.2 μm filter and stored at 4 °C till further use. THP-1 cells were plated in a 96 well plate at the concentration of 10,000 cells per well in a 96 well plate. The cells were pre-incubated over night and exposed to the ASHW at the concentrations of 0.0, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25 mg/mL for a period of 24 h. At the end of the time period, the exposure medium was removed and the cells were washed with 100 μL PBS. 100 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well; and the plates were incubated for 3 hours at 37 °C. At the end of the exposure period, the dye was removed and each well was washed with 100 μL PBS. 100 μL of DMSO was added and the plates were placed on a shaker for 10 minutes. Absorbance of each well was then read using the Envision multiplate reader (PerkinElmer, USA) at λ _ABS = 595 nm; and cell viability percentage was calculated.

Cytokines level measurements

Human monocyte THP-1 cells (5 × 10⁵cells/well) were seeded in 24 well culture plates. To study cytokine modulations, media with different concentrations of ASHW was added to the wells at the concentrations of 0.1, 0.33, 1, 3.3 and 10 mg/mL. After treating cells for an hour, LPS was added at final concentration 500 ng/ml except in control wells. Consumed media or cell supernatants were collected after 24 h to measure different cytokines levels such as TNF-α, IL-1β, and IL-6 using standard ELISA kits (BD Biosciences). ELISA assay was performed according to the manufacturer’s protocol, and plates were read at 450 nm using Envision microplate reader (Perkin Elmer, USA).

Luciferase reporter NFκB gene assay

THP-1 cells were transiently transfected with luciferase reporter vector with NFκB promoter sequence upstream of the luciferase gene. Transfection was performed following the manufacturer’s instruction in 96 well plates using Lipofectamine 3000 (Invitrogen, USA). Two days after transfection, the experiment was performed as described earlier⁵⁵, with some modifications. Used media was replaced with media containing the test compound and control sample. After 1 hour LPS was added at a concentration of 500 ng/ml, where required and incubated further for 12 hours. D-Luciferin salt (Perkin Elmer, USA) at a final concentration of 150 μg/ml was added to the cells and incubated at 37 °C, protected from light. Relative percentage changes in light emission intensity were measured from each well, using Envision microplate reader (Perkin Elmer, USA), LPS induction alone was measured as 100% activity of the NFκB reporter gene⁵⁵.

Statistical analysis

The data are expressed as the mean ± standard error of the mean (SEM) for each experiment. Statistical analysis was done using GraphPad Prism version 7.0 software. A one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison t-test was used to calculate the statistical difference. Student unpaired t-test was used to calculate the statistical difference in comparison to MTX. Values of p < 0.05 were considered statistically significant.

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Acknowledgements

The authors are indebted to Param Shradhey Swami Ramdev ji for his financial and institutional supports to accomplish this research work. We acknowledge the contributions of Dr. Shobhit Chandra and Mr. Ranaji from the Department of Radiology, Patanjali Ayurveda Hospital, Haridwar, India for their support in X-ray imaging. We would like to thank Prof. Partha Roy, Indian Institute of Technology, Roorkee, India for his kind gift of reporter gene construct. We would also like to appreciate Mr. Bhanu Pratap, Mr. Pushpendra Singh, Mr. Vipin Kumar and Mr. Sonit Kumar for their excellent animal handling and maintenance; Ms. Monika Gupta for her support in performing in-vitro studies. We extend our gratitude to Ms. Babita Chandel, Mr. Brij Kishore, Mr. Pradeep Nain, Mr. Gagan Kumar and Mr. Lalit Mohan for their swift administrative supports. This presented work has been conducted using research funds from Patanjali Research Foundation Trust, Haridwar, India.

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Drug Discovery and Development Division, Patanjali Research Institute, NH-58, Haridwar, 249 405, Uttarakhand, India
Acharya Balkrishna, Sachin Shridhar Sakat, Kheemraj Joshi, Sandeep Paudel, Deepika Joshi, Kamal Joshi, Ravikant Ranjan, Abhishek Gupta, Kunal Bhattacharya & Anurag Varshney
University of Patanjali, Patanjali Yog Peeth, Roorkee-Haridwar Road, Haridwar, 249 405, Uttarakhand, India
Acharya Balkrishna & Anurag Varshney
Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health, 665 Huntington Avenue, Boston, 02115, MA, United States of America
Kunal Bhattacharya

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Acharya Balkrishna
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Contributions

A.B. provided broad direction for the study, prepared the formulations, generated resources and gave final approval for the manuscript; S.S.S. conducted the in-vivo study, analysed the data and helped in manuscript writing and reviewing; Kheemraj Joshi, D.J. and S.P. assisted in animal handling and in performing in-vivo studies; R.R. performed the in-vitro experiments; Kamal Joshi prepared the histopathological slides; A.G. supervised the studies and reviewed the manuscript; K.B. performed data curing and wrote the manuscript and its revisions; A.V. conceptualized and supervised overall studies, generated resources, reviewed and finally approved the manuscript.

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Correspondence to Anurag Varshney.

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Balkrishna, A., Sakat, S.S., Joshi, K. et al. Herbo-mineral formulation ‘Ashwashila’ attenuates rheumatoid arthritis symptoms in collagen-antibody-induced arthritis (CAIA) mice model. Sci Rep 9, 8025 (2019). https://doi.org/10.1038/s41598-019-44485-9

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Received: 23 October 2018
Accepted: 17 May 2019
Published: 29 May 2019
DOI: https://doi.org/10.1038/s41598-019-44485-9

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Comments

Commenting on this article is now closed.

Batuknath Choudhary 1 September 2019, 10:54

I was surprised to see this article "Herbo-mineral formulation
‘Ashwashila’ attenuates rheumatoid arthritis symptoms in collagen-antibody-induced arthritis (CAIA) mice model"
The first author of this article "Acharya Balkrishna" does not hold a degree in Science. He claims to hold a degree in Sanskrit Language. According to news even his High school degree and Sanskrit degree were forged to obtain Indian Passport "The CBI said that his passport was issued based on forged high school and graduation certificates https://www.huffingtonpost....
Now there are two main questions: How the first Author Acharya Balakrishna is published high qualities article in such a reputed journal as he does hold any degree even in general science.
How the corresponding author made Acharya Balakrishna first author . I am sure Mr Acharya Balakrishna cannot even understand the topic of this article.
Saurabh Singh Replied to Batuknath Choudhary 14 September 2019, 10:00

You don't need a degree to prove your knowledge. Albert Einstein was considered a slacker and was kicked out of school. Thomas Edison, who invented light bulb was a dropout. Wright Brothers never graduated High School. Maharshi Charak, one of the principal contributors to Ayurveda did not have any degree but wrote on Ayurveda and Medicines. There are such other examples of several noted people who never needed a paper or document to prove the knowledge and mastery they had.
You don't need a degree to attain knowledge. A degree is needed to just stay informed.
Batuknath Choudhary Replied to Saurabh Singh 19 September 2019, 22:22

Ha ha ha one person writing all comments by different names , Mr balakishan and his friend Ramdev claims cure of AIDS, Cancer , Homo sexuality. Does he even know how to read and write english
Rishi Verma Replied to Batuknath Choudhary 22 September 2019, 13:22

You should shame on you leg pulling mentality. You have done nothing for society, even self disgraced, not even capable to tell your identity. You are capable only for making such comment.
Vedpriya Arya Replied to Batuknath Choudhary 22 September 2019, 13:32

Seriously, shame on you. Acharya Balakrishna is a world fame personality of knowledge, wisdom and Science. Needs no introduction. I think you have no knowledge of scientific studies that's why you are raising questions on the journal too.
Grow up from your cockroach's mentality.
Manu Kumar Replied to Batuknath Choudhary 22 September 2019, 15:14

He, you or anybody doesn’t need to know English to express himself/herself.
Nishant Gupta Replied to Batuknath Choudhary 22 September 2019, 15:30

See, Mr. Batuknath, I am going to answer your each doubtful remark one by one.
I think you got confused about English and Knowledge ! According to you, English is necessary to prove intellectuals, but your favorite language still on 3th position in the world (1st is Chinese and 4th is Hindi ). Literally English is spoken and written by 335 million officially out of 7 billion population, means 6 billion 6 hundred sixty five millions peoples who never used English as official are illiterate ? How miserable your criteria to judge peoples ! Have you ever visited Patanjali Research Institute ? you should, if not. About treating AIDS, Cancer etc, yeas Acharaya Balkrishna ji and Swami ji successfully normalised thousands patients, with record; may be shocking for you but true. Now comes to above article, which is probably not digestible to you; initially, they did not wish to publish anything about their scientific work in Ayurveda and yoga, but peoples like you enforced them by stupid questioning so they decided and committed to explain everything to world in scientific manner. Now comes to their Alma Mater, Acharaya ji had been laureated by Doctorate degrees and many national and international awards; their life may be a good research topic for you; please come and join Patanjali to understand their work correctly and closely.
akanksha kathuria Replied to Saurabh Singh 23 September 2019, 05:24

Mr. Batuknath chaudhary, I am totally diagree with your argument. How can you blame such a noble soul like this?
For your kind information let me discuss some important facts with you.
1- In India earlier when we dont have such so kind of degrees and colleges, people used to take knowledge from GURUKULS by our noble RISHIS and MUNIS who wrote many ancient vedas like Ramayan, Bhagwat Geeta etc and this knowledge was known as VEDIC Science.
2- F
ew example of great souls without any so called research degrees- Aryabhatta was a master Astronomer and Mathematician, Acharya Charaka has been crowned as the Father of Medicine, Indian surgeon Sushruta is credited as being the founding father of surgery, Maharishi Patanjali gave the World the art of Yoga and showed how Yoga can improve not only one’s physical health but also mental health of a person, Acharya Kanad, an Indian philosopher and a great thinker far ahead of his time, developed what we today call the atomic theory, Chankayaji, Paniniji, Nagarjuna ji and tje list goes very long.
3 - They all are the exmaples of great contributors of knowledge and science without out holding any degree.
4 - And in todays modern world Swami Ramdev and Acharya Bal Krishan ji are the two noble souls who work selflessly and having ample of knowledge in the field of yoga and jadi buti science which we called Ayurveda and with these they treated millions of people and save their lives.
5- If we talk about the reserch paper, then it is one of the best research paper which throughly explain about the Chronic Diseases Rheumatoid arthritis.
6 - This paper is totally based on primary research which undergone experimentation process, which results and conclusion cant be challenge by any one globally.
And if still you are having any doubts, I will share lots of sources of our ancient vedic science who proves knowledge and intelligence only requires intellectual mind not any kind of degrees.
Jeevan Singh Replied to Batuknath Choudhary 15 September 2019, 06:21

If you have skill to do something new according to your interest then it is not compulsory to have degree reagarding to your work. Degree is not a confirmation of your skill and talent to gain knowledge about that particular area in which you have interest. If you have capability of doing your work then you automatically attain mastery and you do not need any kind of degree from university. Albert Einstein, Wright Brothers, Maharshi Charak all these did not have any degree in their area of interest but now they all are inspiration for us..
Sumit Kumar Replied to Batuknath Choudhary 15 September 2019, 07:15

I really feel pity for you because of the way you think. Whatever you just wrote above makes you the most illiterate person on this planet. If you think that a degree is directly proportional to knowledge then sorry to say this but that is very idiotic. The world has seen countless no. of people who havn't even gone to school or completed their college but still their names resonates in this world because of their work. Some of the great examples are Bill Gates, Mark Zuckerberg, Steve Jobs, James Cameron, Tiger Woods, Lady Gaga and the list goes on and on. In India too we had Late Mr.Dhirubhai Ambani Ji, who was the richest man in India or maybe even Asia. So how did all of them excelled in life even though they didn't had any college degree?? It was because of their knowledge, their passion to achieve, their skills, their thinking. Zuckerberg who didn't even had a degree in programming but still created the most complex coding through which Facebook was created and for that he was considered a prodigy in the programming world. Degree doesn't define your knowledge. If degree was so important then their would have been no job interviews in the world. Everyone would have been hired by just showing their degree. Am i right??
Sadhvi Devaditi Replied to Batuknath Choudhary 22 September 2019, 14:25

Well no wonder type of comment and information you have put here shows that you must be surprised with the limitation of your mind. You will get surprise even more with the article's link I have shared below. Because to the same person you have put allegations on, has been actually rewarded by the UNSDG an UN organization for his efforts made in healthcare at international level.
https://www.business-standa...
It's very easy for people like you to judge because of the limitation of knowledge you have blocked yourself with. And people like him are working very hard with broad mind which you can't even imagine of. May God bless people like you.
Mamta Replied to Batuknath Choudhary 22 September 2019, 14:33

You do not need a college degree to justify your knowledge in a particular field. If you have talent, skill, knowledge and passion to do something so degree is not matter. Pujya Acharya is one of them. He believes in her Skill, Knowledge and Karma that way things happen. And people like you only waste your time to do this types of rubbishing thing. One advice to you HONORABLE BATUKNATH CHOUDHARY JI do something for your nation and your Society. Do not waste your time to do such types of cheap things. Pujya Acharya ji is well known world famous personality and involve in the Nation building activities and the World at large through Karma Yoga (Self less action). And one more advice to you further do not try to use this types of words and statement against him.
Rashi Replied to Batuknath Choudhary 22 September 2019, 15:20

My question to you is who are you to ask these questions. What did you have done and what knowledge do you have about all this. Asking question to a these genius people who are already famous for their knowledge and work.
Vedpriya Arya Replied to Batuknath Choudhary 22 September 2019, 15:23

It seems that you are so much deprived of general knowledge. Sanskrit is the most versatile and influential language on this earth. Even NASA admitted it.....
https://www.google.com/amp/...
Acharya Balkrishna ji is making his day night efforts to preserve the ancient wisdom of Ayurveda which was unexplored or hidden till date due to some fools like you. He is preparing scholars who works in healthcare sector. He is the only person saving lives of crores of people by his effective scientific home remedies.
For your kind information, he is the guiding force of 300 plus scientists who are working on several projects with the world-class laboratory facilities.
Several highly scientific institutes of national and international level are taking his advice on healthcare. Even UNSDG recognised his potential on healthcare and awarded him.
But the illetrates like you are unaware of these facts and making rubbish. Rather than to support someone who work for the betterment of the society, you foolishly making comments
And yes wait for another two-three years, he is going to make india 'A Vishv guru'.
Manu Kumar Replied to Batuknath Choudhary 22 September 2019, 15:36

Hi Batuknath,
With all due respect to your right to accept or ignore this article and provide your opinions, I would like to explicitly put across that your comments are nothing but a personal attack on author based on your biased notion with moulded facts and half baked understanding of the law, literature, philosophy and academia.
The facts you have quoted are also one side story put forward in initial allegations made against the author which have been proven wrong in the court of law.
Amit Sharma Replied to Batuknath Choudhary 22 September 2019, 16:23

Hello Mr. Batuknath Choudahry,
I guess your Mom Dad gave lot of thoughts before naming you "Batuknath". Although from your name itself I can perceive a lot about your mentality level. But, then also I would like to bring your kind attention on the following points to answer your stupid and irrelevant two questions.
1.To write a research paper you don’t need a degree just adequate knowledge on the subject and new findings. That’s the reason this article is being published in such a reputed journal.
2.Acharya Balkrishna ji and Baba RamDev ji are the main reason behind the popularity of Yoga and Ayurveda at national and even at international levels. Need not to mention that lot people worldwide are now getting benefited out of it.
3.Also, to your statement of “I am sure Mr. Acharya Balakrishan cannot even understand the topic of this article”. I would like to suggest you to kindly go through his talks on Aastha channel and YouTube as well regarding Ayurveda and various jadibuttis. Also, this research paper itself is an appropriate response to your question sir.
Stop judging people and do something productive for the nation. By the way how many degrees do you hold and what are you doing? (Apart from this nonsense over internet)
May God Bless You with some wisdom (OM)
Tarun Patel Replied to Batuknath Choudhary 22 September 2019, 18:29

First you think how much knowledge you have to talk about acharya balkrishna ji . You need knowledge do such a great work .
VP SHARMA 22 September 2019, 15:11

Can Batuknath can explore & tell if the man who invented Steam Engine ever held any sort of degree. Were Write Brothers who flew a flying machine degree holders Aviators. Laymen constructed Taj Mahal were Architectures.
Practice makes a man perfect
Hardik Yadav 22 September 2019, 15:40

This should reach more and more people. RA is a serious problem with God knows how many people suffering from it. I hope this paper makes it more bearable.
praveenchander swabhimani 22 September 2019, 15:57

Acharya Balkrishna ji & team of patanjali research institute is serving to mankind by presenting various research paper, to fight against diseases.
Pradeep sharma 22 September 2019, 16:01

We can't judge a person's expertise with a piece of paper we were given while graduating. Knowledge can be gained by anyone and from anywhere.
Maybe he don't have a piece of paper to prove his education but people would surely have the medical documents to prove how he has helped no. of people through Ayurveda.
nilesh ranjan 22 September 2019, 16:17

According to you, people who know English can only cure people. Let me tell you that to cure diseases, you need to know about medicines, not any specific language. Swami Ramdev and Acharya Balakrishna recalled the culture of our country and showed the importance of Yoga and Ayurveda around the world. All this was due to their knowledge and experience.
Beena Choudhary 22 September 2019, 16:17

Mr Batuknath, you can't be serious. I totally disagree.
I am sorry but you really have a disgusting criteria of judging people. I feel so ashamed of you and also on your thinking. I don't think there is any connections of English with knowledge.
You have given no right to put such allegations on anyone.
My suggestion to you would be to grow on your mentality.
Rishi 22 September 2019, 16:28

There is a big differenece between well degreeate person and a well educated person ...you can not define a person with their degree ..for your kind info one of the millionaire the facebook owner get his degree after years of getting the postion .
The degree define the person but the education and knowledge of practice and work defines the person character. There is a proverbs that to whiten hair in the sun
Razia Parveen 22 September 2019, 16:41

Mr. Batuknath ji, English is not a degree or knowledge it is a language. And I think you don't know about Patanjali Yogpeeth. My special invitation for you please come and see thousands of critical diseases patient like Cancer, HIV, etc has been treated under Pujya Acharya Ji's treatment with good results (documented proved) by Ayurvedic and Sanskrit knowledge. I asked you one thing what are you doing for mankind with your degree.
Anantika Sharma 22 September 2019, 17:07

How could you be so injudicious to a person who is known globally for his contribution in the field of Ayurveda. Just because a person doesn't hold a degree to prove their knowledge you have no right to condemn their work.
Rather than surfing the internet to know about his education, try to look for all the work he has done. That would help you a lot more!
Pratyush Dubey 22 September 2019, 17:07

We can't rely only on degree sometimes we go to a doctor with lots of degree but there is no experience of work so we can't trust him totally i personally experienced when I went to a dr and he gave me a medicine which is reacted and because of this I m going through lots of troubles so there is a lot of things which gain by experience not only from degrees
Saurabh Singh 22 September 2019, 17:45

A degree cannot describe your knowledge and talent. Knowing how to do a job in actual practice is more important than understanding the proper steps in theory. Acharya Balakrishna has years of work experience with knowledge of Ayurveda medicines and cured millions of people. You need to check the work that he has done for the public before checking his degree to talk about him anywhere like this
Shivani sharma 22 September 2019, 17:51

Om Mr. Batuknath
Critics 90%, Talker 8.5% and doer only 1.5%. Please look at the mirror and ask yourself who you are??????
Sadhvi Devaditi 22 September 2019, 18:01

This research has fascinated me for the output is so relaxing. The output of such research are a big leap for the humankind and I m sure for more such researches will come up from the author which will prove to be great milestones in modern era of medicine in the healing of living beings.
VP SHARMA Replied to Sadhvi Devaditi 23 September 2019, 11:25

Kudos to you
I pray good senses may pervail on Bhatkunath.
He is seemed to be a dropout from Mckauly's school.
सुनील स्वाभिमानी 23 September 2019, 01:41

Mr. Batuknath
There is big difference between a well
degreeat person and a well educated person. इसीको पढ़े लिखे मुर्ख कहते हैं.
सुनील स्वाभिमानी 23 September 2019, 02:26

Mr. बटुक नाथ, shame on you.
आचार्य बालकृष्ण जी के नेतृत्व में 500 साइंटिस्ट कार्य कर रहे हैं.उनके डिग्री पर क्वेश्चन उठा रहे हैं. जिसने आयुर्वेद का गौरव बढ़ाया..100 से अधिक रिसर्च इंटरनेशनल जर्नल में प्रकाशित हुए .आइंस्टीन के पास क्या डिग्री था ???
Dr. Pallavi Thakur 23 September 2019, 02:48

Now a days, English has become a symbol of judging someone's knowledge...I have seen several cases, where the person knows perfect grammatically correct english, but is unaware about the normal facts of life, humanity and hospitality...In fact, Mr. Batuknath, you are a perfect example of what i have stated above...You can murmur your english, but you dont have the power to heal someone, to help someone and to boost up the hopes of millions out there...Our Acharya ji and Swami ji have all these powers, the ultimate power to invoke zeal and zenith in everyone directly or indirectly associated with them...Now, do you need a degree for further justification...Degree, and English language doesn't matter in the field of Science...All that matters is the 'true Science' which actually goes out in the society for the benefit of mankind...The two sages are, in true senses, the real epitope of wisdom...So rather than defaming them, you should try to focus on what you are doing on your part...Get well soon...
VP SHARMA Replied to Dr. Pallavi Thakur 23 September 2019, 11:17

I would suggest don't pay any heed to such persons who are always critical of others. They are zealous of the prominence is Swami Ramdevji & Acharaya Balkrishan hi, who committed to welfare of the mankind on the Universe.
Bhatkunath Nath is deemed to have strayed
Mayur Chauhan 23 September 2019, 03:40

It's good research by Patanjali... Ayurveda is best way to treat disease without any side effect....
Jyoti 23 September 2019, 03:46

You should ashamed of yourself. Acharya ji always think about nation and you belong to this nation too... So please think before you write.
Pankaj Kumar Tripathi 23 September 2019, 03:57

Mr. Batuk
I think you need some blessings according to your name (Batuk means boy). Ap unke barein me aisi bat bol rahe ho, jinke Naam Ka danka puri dunia me bajta hai, aur jinhe har field me maharat hasil hai. Mai bus itna kahna chahunga ki Bahgwan apko sadbuddhhi de..
Shantanu 23 September 2019, 03:59

Batuknath ji,
The paper has its detailed methods, references and results listed. Do some purusharth (hard work), rather than commenting on merits of the author. Merit of any good experiment is that, the same results can be reproduced using the same set of methods. Use scientific rigour and try to repeat the experiment at your end, and if you get different results, then you can make a counter claim. Without that any argument is just play of words, jealously or frustration.
Hemant Sharma 23 September 2019, 04:05

Those who understand English as a status language today may have forgotten that the mother of all is our Sanskrit language. Mr Batuknath, who understands this, cannot be like you. Many scientists are working together in many disciplines under the guidance of Acharya Shri and Swamiji. Which is based on the test of science evidence and is useful for the public.
Priyanka Chauhan 23 September 2019, 04:07

Mr. Batuknath ji
Patanjali has proved its mettle in the field of medicine, and Acharyaji has brought remedy for many life-threatening ailments at our doorsteps. So if you can't appreciate his selfless efforts, the least you can do is stop spreading hatred against the noble soul. And incase you are affected by any of those diseases mentioned, Visit Patanjali.
Dr. Rashmi Mittal 23 September 2019, 04:23

Around the globe in over more than 200 countries, millions of people are dying every year due to chronic diseases. Inspite of vast advancement in medical study, mortality rate is continuously increasing. Ayurveda, one amongst the most ancient system has well described the treatment measure for almost all the diseases. Pram Pujya Acharya Balakrishna Ji has tremendous knowledge of ‘jadi-buti vigyan’ and has in depth understanding of ‘vedas’ and ‘puranas’. Therapeutic measure proposed by him is not only based upon the historic facts but these facts have been scientifically proven in his laboratories. Huge number of patients are getting cured from his medicine regularly in ‘Patanjali Ayurved Hospital’ and the research work on which Mr. Batuknath has commented upon is just a demo of it. Mr. Batuknath if you ever get a chance to personally meet Acharya Balakrishna ji then try to realize his vision of healing process not only for the Indians but for the entire world. After seeing his efforts for establishing a disease free world, somewhere your conscience will raise a question over you that how you have judged such a great personality with a language. You can never define someone’s ability by restricting them with words which could have been framed in any language and moreover in all the languages it will reflect the same meaning. Somewhere to some extent this kind of comments reflects the inability of Mr. Batuknath to accept the successful efforts made by Acharya Balakrishna Ji for the well-being of humanity. Your comments will not stop the pace by which Acharya Ji is working day and night and nor it will bother him in any manner but the people those who believe in devoting their life towards the society will not tolerate this attitude towards Param Pujya Acharya ji, so next time before commenting on either Acharya Ji or any other such personality please ask your conscience whether it is true or not, please never write such post just to get fame.
VP SHARMA Replied to Dr. Rashmi Mittal 23 September 2019, 11:07

You have rightly pointed out immense strength of Ayurveda medicines, which have been in use for thousands of years.
People like Bhatkunath will not understand the essence of deep thoughtful formulae of Ayurveda.
Cat always wanders around the kitchen, which in return keep the the keeper vigilant against any default.
Let Patanjali alert of such type of mentalities.
akshay kumar 23 September 2019, 04:27

आचार्य बालकृष्ण जी आयुर्वेद शिरोमणि है, डिग्री किसी के ज्ञान से बड़ी नहीं हो सकती।
स्वामी रामदेव और आचार्य बालकृष्ण की देश सेवा अतुलनीय है। आपके या किसी और के कुछ के देने से कुछ फर्क पड़ेगा नहीं।
Nidhi Sharma 23 September 2019, 04:27

English is just a medium of communication. You can’t judge one’s knowledge or intelligence by his English speaking skills.I have one question for you...can you read and write Sanskrit which is the most scientific language of the world......surely you don't....even you don't know our ancient knowledge.when you don't know any thing how you comment on others knowledge or wisdom. 'Puff not against the wind' is appropriate for you..
Shantanu 23 September 2019, 04:29

Ayurveda principles are well established through thousands of years of study, and documented well in, Charak Sahinta and other texts. Now Acharya BalKrishna and Patanjali Research Institute are, proving the same principles again in the modern western scientific framework also.
Commendable work for Ayurveda and Humanity !
Sahil Arora 23 September 2019, 04:32

Everyone can have their own opinion and interest but you have no right to judge someone based on their language. If you don’t have believe on these research you have option to ignore this but asking such irrelevant questions to these highly respected people is not the right way.
Priyanka Rai 23 September 2019, 04:36

Acharya ji and swami ji are noble souls who are working selflessly day night for the betterment of this nation. They represented India globally in many areas and this would not be possible without having proper knowledge.. So you better think before criticising someone.
Vinod kumar pandey 23 September 2019, 04:37

Mr. Batuk , your are the pure example of UK slave who judge indian by foreign language and and you don't have capacity to challenge the person who work 24*7 to cure india, people like you Mr. Batuk who work for foreign medical companies which are struggling to sale their products due the great work of swami ramdev ji, and achary Balkrishna ji.
Our ayurveda still has the tough time because of the your mental issue. You are mentally disable person who judge the person's knowledge with their language subject, I think you must go and meet their student Kidd's in acharykulam they will teach the language.
Shantanu 23 September 2019, 04:39

Out of the three dozen comments in this thread, none of the comments really talk about the scientific merits of the paper, about its methods and results. Comments are largely about merit of the author, usage and understanding of English.
Does any one has any comment on the merit of paper ?
geetanjali adhikari 23 September 2019, 04:40

Mr. Batukatnath
shame on u.. english is just a language not a measurment of intelligence..
Aakash Arya 23 September 2019, 04:43

The thing that really matters is an experience. You can take the example of Bill Gates who started Microsoft when he was 19 and has since become one of the world’s wealthiest people. If only getting a higher education is important then he would never become that successful. You cannot criticise such knowledgeable people like Acharya Ji who is already well-known for his expertise in Ayurveda.
Rama Shankar 23 September 2019, 04:48

In fact Mr. Batuk Nath is absolutely confused. He need not to interfere not only in the matters of Patanjali, but others too. Generating thoughts is a priority than the language. At certain occasions some journals need German, French and Chinese languages for the abstracts and none of the authors know the concern language. He must keep in mind to control over such commenting. If he gets any scientific mistake only he can make comments.
animesh mishra 23 September 2019, 04:51

Do you even know about how to deal with such kind of dangerous diseases like cancer, Aids and all then stop blaming others if you do not have proper knowledge about those topics. If you think that one person writing all comments by different names, this shows your way of thinking and its your problem so dear Batuknath firstly figure out your problems.
Arun Kr. Ojha 23 September 2019, 05:24

To
Batuk nath
English man in india
Dear Batuknath, we indian beside the degree and language barrier we work to serve the world with Indian heritage which is highly acceptable by other countries, just take the example of YOGA and healing with Ayurveda products are highly demanding things in USA and UK.
If you judge the person by their degree then do you know the degree of swami vivekanand ji, who's speech is inspiration for whole world.
Degree is a just a piece of paper which can not assure the knowledge of any person, we Indian form ancient time we always believe on person's skills not on the piece of paper and Swami Ji and Achary ji has done the remarkable work not only for India also for across the globe. Your narrow mind can not understand the noble work done by Patanjali.
Get well soon.
Your well wisher
भारत स्वाभिमान न्यास फ़रीदाबाद 23 September 2019, 05:25

Mr. Batukatnath
Does Shri Sushruta and all respected Ayurved Acharyas need any degree for their dignified Work toward humanity
भारत स्वाभिमान न्यास फ़रीदाबाद 23 September 2019, 05:28

Mr. Batukatnath ,
People like you due to their negativity. They can fall so much it is not a surprise.
Please practice some Yoga change your narrow and negative mind to Broad and Positive.
Shobha Chaudhary 23 September 2019, 05:28

How you can blame someone without knowing about anything. Does you know about English and you have a degree in any particuar subject. Is knowing english or having degree is the criteria according to you about someones knowldege. I think you need to study more about such things. Mr Batuknath first you gain some more knowledge and then ask some relevant questions instead of such irrelevant ones.
akanksha kathuria 23 September 2019, 05:30

Mr. Batuknath chaudhary, I am totally diagree with your argument. How can you blame such a noble soul like this?
For your kind information let me discuss some important facts with you.
1- In India earlier when we dont have such so kind of degrees and colleges, people used to take knowledge from GURUKULS by our noble RISHIS and MUNIS who wrote many ancient vedas like Ramayan, Bhagwat Geeta etc and this knowledge was known as VEDIC Science.
2- Few example of great souls without any so called research degrees- Aryabhatta was a master Astronomer and Mathematician, Acharya Charaka has been crowned as the Father of Medicine, Indian surgeon Sushruta is credited as being the founding father of surgery, Maharishi Patanjali gave the World the art of Yoga and showed how Yoga can improve not only one’s physical health but also mental health of a person, Acharya Kanad, an Indian philosopher and a great thinker far ahead of his time, developed what we today call the atomic theory, Chankayaji, Paniniji, Nagarjuna ji and tje list goes very long.
3 - They all are the exmaples of great contributors of knowledge and science without out holding any degree.
4 - And in todays modern world Swami Ramdev and Acharya Bal Krishan ji are the two noble souls who work selflessly and having ample of knowledge in the fieldi buti science which we called Ayurveda and with these they treated millions of people and save their lives.
5- If we talk about the reserch paper, then it is one of the best research paper which throughly explain about the Chronic Diseases Rheumatoid arthritis.
6 - This paper is totally based on primary research which undergone experimentation process, which results and conclusion cant be challenge by any one globally.
And if still you are having any doubts, I will share lots of sources of our ancient vedic science who proves knowledge and intelligence only requires intellectual mind not any kind of degrees.
Ankit Sharma 23 September 2019, 06:24

Sanskrit is India's gift to the world and people from different countries come to India to learn this language. There are even some words in English that were originally Sanskrit words. So people like you need to understand the importance of Sanskrit which is known as “Mother of all languages”.
So try to broaden your mind a bit and make some sense.
Making this ill statements on such a great personality makes you look more illiterate.
ajay gusain 23 September 2019, 06:38

Firstly you said about degree and now you are talking about knowledge of English. So tell me what is your main problem degree or english. I think you are confuse and you only knows is how to blame someone. Problem is not degree and English but problem is your mentality.
VP SHARMA 23 September 2019, 07:08

Batuknath is seemed to be a confused person. He doesn't know what to talk & what not.
I would advise him to join Yoga classes somewhere.
Rajender Joshi 23 September 2019, 09:17

Mr. Batuknath, shame on you and your remark
Acharya Balkrishna person you have put allegations, has been rewarded by the UNSDG an UN organization for his efforts made in healthcare at international level.
Devika Sharma 23 September 2019, 09:18

A blind man always tend others to be blind. You feel that persons who are well versed with English language are most intelligent and other languages of the world have no place in academic arena. Let you be apprised of that the Sanskrit and Hindi are the richest languages of the world.
Ship in the sea is sailed by a Commander, who is assisted by dozen subordinates on board. But in the harbor and elsewhere its sailing is acknowledged by the name of the Commander only.
Rajender Joshi 23 September 2019, 09:33

Mr. Batuknath, shame on you and your remark
Acharya Balkrishna person you have put allegations, has been rewarded by the UNSDG an UN organization for his efforts made in healthcare at international level.
For more details Click the link below
http://www.divyayoga.com/tr...
https://www.aninews.in/news...
https://www.business-standa...
Anubha 23 September 2019, 09:38

Every people have their own language and it's not necessary to be english it's our knowledge.
VP SHARMA 23 September 2019, 10:26

It is easy to make comments on others' success.
It doesn't behove good to make adverse comments on the hard working team committed to do good for the ailing persons in particular & masses in general
Up till now, no efforts have been made either by any of the governmental agencies or entrepreneures to do a bit on research on medicinal properties found in flora and fauna & when someone is doing,why Batuknath Bhai burns of hate & zealousy.
Which was binding force in bringing yoga to every house hold
Who was the personality, who have been decorated with INSDC in the field of Health & hygiene.
Do you know '0' was invented by Non-profit degree holders from India .
Who counted the stars in the sky & who had measured the distances of various planets from the earth
These were all our Rishis, who even never attended the schools.
Ram H 23 September 2019, 10:49

This is exactly the propaganda of the Left liberal gang. Whatever are the positive achievements of the people if our country, these achiements as well as the people are criticized by them.
These left liberals are hell bent on proving how ineffective and wrong we indians are. Be it Swami Ramdev, Acharya Balkrishna or Prime Minister Narendra Modi, the leftists leave no stone unturned to defame and demean them. Also it's not just a coincidence that Batuknath is using a foreign funded news portal to support his claim.
With overwhelming criticism batuk nath is facing due to his post, we can rest assured that our people have enlightened and are giving fitting reply to such left liberals.
Jitendra Kumar Pandey 24 September 2019, 03:22

Mr. Batuknath Choudhary
Your comment doesn't tell the quality and ability of the person to whom you are addressing rather it is a reflection of your own ability, quality, thinking level, knowledge level, and mentality. I searched your name on Google Scholar and did not find even a single document. I really want to know your scientific profile and I request you please share your research articles, I am waiting.
Deepikainsa Insa 25 September 2019, 08:19

The information shared here z really useful... Ppl must go towards ayurveda for life longevity n healthy lifestyle. Thanks fr promoting ayurveda for what India has been known since ages