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Year : 2018  |  Volume : 50  |  Issue : 1  |  Page : 22--29

Reduced synovial inflammation and inhibition of matrix metalloproteinases explicates anti-osteoarthritis activity of polyherbal formulations

Dhanashri R Ingale1, Priya G Kulkarni1, Soumya J Koppikar1, Abhay M Harsulkar1, Alpana S Moghe2, Suresh D Jagtap3,  
1 Department of Cell and Molecular Biology, Interactive Research School for Health Affairs, Bharati Vidyapeeth Deemed to be University Medical College Campus, Pune, Maharashtra, India
2 Department of Cell and Molecular Biology, Rajiv Gandhi Institute of IT and BT, Bharati Vidyapeeth Deemed to be University Medical College Campus, Pune, Maharashtra, India
3 Department of Herbal Biotechnology, Interactive Research School for Health Affairs, Bharati Vidyapeeth Deemed to be University Medical College Campus, Pune, Maharashtra, India

Correspondence Address:


OBJECTIVES: Current osteoarthritis (OA) research experiences an incline toward Ayurveda to attain a complete cure without notable adverse effects. Ayurveda uses natural products, which are known to perform the multi-faceted role, a much demanding approach for OA management. However, lack of scientific evidence is a major drawback hindering their wider use. The present work investigated the anti-arthritic potential of Ashwagandharishta, Balarishta, Dashmoolarishta, and Triphala-extract to establish molecular-evidence for their clinical use. MATERIALS AND METHODS: Rabbit synoviocytes were induced using interleukin-1 beta (IL-1 β) and lipopolysaccharide (LPS) separately and were further treated with study formulations to test anti-inflammatory and anti-oxidant potential, using nitric oxide (NO) and malondialdehyde (MDA) assays. Collagenase inhibition activity was estimated with N-(3-[2-Furyl] acryloyl)-Leu-Gly-Pro-Ala (FALGPA)-substrate and gelatinase spot assays. Data were analyzed with GraphPad Prism using one-way ANOVA followed by Bonferroni's multiple comparison. RESULTS: The study formulations were effective against synovitis, oxidative-stress, and inhibiting collagenase. They caused NO reduction in selected concentrations. DA showed the maximum NO decline of 0.02 ± 0 and 0.97 ± 0.62 μM/ml with IL-1 β and LPS induction at 5 and 20 μg/ml concentrations, respectively. Estimated by FALGPA assay, increasing collagenase inhibition was observed as the function of concentration. All formulations showed a significant MDA decline, in dose-dependent manner. CONCLUSION: We assessed the anti-OA efficacy of conventionally prescribed Ayurvedic drugs using relevant biochemical assays. The studied formulations revealed potential to restrain synovitis, cartilage degeneration and to reduce oxidative stress, and the signature OA features. With established molecular authenticity, Ayurvedic drugs can offer a safer and affordable therapeutic option for OA.

How to cite this article:
Ingale DR, Kulkarni PG, Koppikar SJ, Harsulkar AM, Moghe AS, Jagtap SD. Reduced synovial inflammation and inhibition of matrix metalloproteinases explicates anti-osteoarthritis activity of polyherbal formulations.Indian J Pharmacol 2018;50:22-29

How to cite this URL:
Ingale DR, Kulkarni PG, Koppikar SJ, Harsulkar AM, Moghe AS, Jagtap SD. Reduced synovial inflammation and inhibition of matrix metalloproteinases explicates anti-osteoarthritis activity of polyherbal formulations. Indian J Pharmacol [serial online] 2018 [cited 2023 Feb 5 ];50:22-29
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Osteoarthritis (OA), the most disabling arthritic affection, is characterized by progressive loss of hyaline cartilage, osteophyte formation, and subchondral sclerosis. In India, the prevalence of OA is reported in the range of 17%–60.6%.[1] Although the disease pathology is still indistinct, very recent research has established its strong linkage with inflammation. Furthermore, catabolic and proinflammatory mediators such as cytokines, nitric-oxide (NO), prostaglandin E2, and neuropeptides alter the balance of cartilage matrix remodeling. This process leads to an excess production of matrix metalloproteinases (MMPs), which are responsible for cartilage breakdown. Further, cartilage alteration, amplifies synovitis, creating a vicious circle of inflammation and degradation.[2]

In a nutshell, OA is a multifactorial and polygenic disease. The comprehensive analysis of molecular pathways involved in OA within different joint tissues is needed to be well perceived to develop new approaches for prevention and treatment for the disease. Current medical treatments for OA exclusively focus on pain reduction and control of inflammation. These approaches, however, have a little effect on the natural course of the disease. Frequently, the ultimate management for OA is a joint replacement; however, associated with the inherent risks and cost that come with surgery. Modern drugs, being associated with single-molecular approach have unacceptable side effects, and have limited scope in treating polygenic disorders such as OA; hence, multi-targeted approach is required.[3]

Ayurveda has been a popular choice to treat chronic noncommunicable diseases. This ancient medical science invariantly uses natural remedies, derived from medicinal plants, and minerals to treat chronic disorders. Plants are rich in polyphenolic compounds, which perform a multi-faceted role to cure chronic diseases such as OA. However, lack of scientific evidences has been listed as the most common limitation of Ayurveda. This scenario certainly demands a thorough the evaluation of Ayurveda formulations for their action at the molecular level to establish them for OA management. Therefore, current research interest experiences a shift from modern drugs to traditional wisdom, as a ray of hope, for effective pharmacological intervention without many adverse effects.

The present study is planned to investigate anti-OA potential of four selected Ayurveda formulations – Ashwagandharishta (AA), Balarishta (BA), Dashmoolarishta (DA), and Triphala extract (TE). Ayurvedic literature suggests that these are the commonly prescribed formulations for OA treatment; however, their mechanistic action has never been evaluated in the frame of the disease. To be precise, although the clinical efficacy of these compounds is proven, molecular evidences are yet to be established. To bridge this gap, we conducted a series of experiments to evaluate anti-inflammatory and antioxidant potential for these formulations using a range of relevant biochemical assays. Further, collagenase inhibition activity was determined using synthetic chromogenic substrate, N-(3-[2-Furyl] acryloyl)-Leu-Gly-Pro-Ala (FALGPA) and dot-blot assays of collagenase inhibition activity. The objective was to provide biochemical basis for the use of Ayurveda formulations, which could offer an effective therapeutic option.

 Materials and Methods

Ayurvedic formulations

Commercially available Ayurveda formulations AA, BA, DA, and TE were procured from Green Pharmacy (Pune, Maharashtra, India).

Cell line

Rabbit synoviocytes (HIG-82) cell line was procured from the National Centre for Cell Science, Pune, India.


Tissue culture medium, fetal bovine serum (FBS), FALGPA (N-[3-(2-Furyl) acryloyl]-Leu-Gly-Pro-Ala), the chromogenic substrate of collagenase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylthiazolium bromide (MTT) dye, sodium dodecyl sulfate (SDS), dimethylformamide (DMF), interleukin-1 beta (IL-1 β), and lipopolysaccharide (LPS) and were ordered from Sigma-Aldrich, USA. Butylated hydroxyltoluene (BHT) and thiobarbituric acid (TBA) were purchased from Himedia Corporation, Mumbai, India, Sulfanilic acid (Qualigens, Navi Mumbai, Maharashtra, India), N-[1-napthyl] ethylenediamine dihydrochloride (NEDD), Tri-chloroacetic acid (TCA) Merck, and Collagenase type II were purchased from Life Technologies Gibco, USA. X-ray films (Fuji films, Japan) were obtained from local market, Pune. Tissue culture plastic ware was bought from Axygen Scientific, USA.

Preparation of TE extract

Triphala powder was stirred in triple distilled water in a ratio of 1:10 (w/v) and kept overnight at 37°C at 100 rpm. The extract was centrifuged at 5000 rpm for 10 min and filtered through 0.22 μm filter (Millipore) and stored in aliquots at −80°C. This work was carried out with the same batch of extract for biochemical assays and in vitro experiments.

Yield of extraction of the study formulations for its further experimental usage

The yield extraction percentage was calculated for all the four study formulations. The extraction yield was a measure of the solvent efficiency to extract dissolvable solids from the original material. In this study, glass petri dishes were weighed before the addition of 1 ml aliquots of commercially available AA, BA, DA and the TE and were dehydrated at 50°C for 30 min, and the mass of solids for each sample was carefully determined. The ratio of the mass of dissolved solids to the amount of dry powder, used in the equivalent volume of extract, was then calculated, providing a measure of the quantity of dissolved solids, extracted per mass of AA, BA, DA, and TE.

Cell viability assay

Cell viability of the four study-formulations was determined using MTT assay with some modifications as described in Maioli et al.[4] At first, HIG-82 cells were seeded in 96-well plates at density 1 × 105 cells/ml; cells were grown on Nutrient Mixture Ham's F12 medium containing 2 mM L-glutamine, 10% FBS, and 100U/ml of penicillin-streptomycin. The cells were incubated in a humidified 5% CO2 incubator at 37°C. An untreated group was used as a negative control. AA, BA, and DA were added at concentrations 50, 100, and 500 ng/ml and 1, 5, 10, and 20 μg/ml in triplicates. For TE, concentrations used were 10, 20, 40, 80, 360, and 640 μg/ml in triplicates for 24, 48, and 72 h. MTT solution (5 mg/ml) was added after 24, 48, and 72 h in each well, cells were cultured for 4 h at 37°C in 5% CO2 incubator. The formazan crystals formed were dissolved by addition of 90 μl of SDS-DMF (20% SDS in 50% DMF). After 15 min, the amount of colored formazan derivative was determined by measuring optical density (OD) using ELISA microplate reader (Bio-rad, Hercules, CA, USA) at 570 nm (OD 570–630 nm). The percent viability was calculated as follows:

% Viability = (OD of treated cells/OD of control cells) × 100

Gelatinase spot assay of collagenase type II enzyme

Collagenase type II (0.125% w/v in 50 mMTris–HCl buffer, pH 6.8) was assayed after preincubation with varying concentrations of AA, BA, DA, and TE with collagenase type II as a control (0.125%) for 10 min. Further, a 3 μl spot of each reaction mixture was pipetted onto a prewashed, X-ray film in duplicate. After incubation for 10 min, films were washed to visualize enzyme activity, as indicated by clear spots of hydrolyzed gelatin. This assay was standardized in our laboratory,[5] and was performed with some modification for the present study.

Nitric-oxide assay

NO levels were measured using Griess-reduction method,[6] with the following modifications. HIG-82 cells were seeded in 96-well plate (1 × 105 cells/ml) and were grown overnight at 37°C in 5% CO2. After 24 h, cells were induced using human recombinant IL-1 β (100 ng/ml) or LPS (10 μg/ml) for 72 h. Further, induced HIG-82 cells were treated with the selected concentrations of the herbal formulations (AA, BA, DA, and TE). The conditioned media were collected after treatment of 24 h and appropriately diluted, further added with 1% Sulfanilic acid and 0.1% NEDD prepared in 5% phosphoric acid in 1:1 ratio. After Incubation of 10 min at room temperature, absorbance was measured at 540 nm in spectrophotometer (Perkin Elmer, Massachusetts, USA). Final calculations were based on a standard curve made with linear concentrations of sodium nitrate.

Collagenase inhibitory activity N-(3-[2-Furyl] acryloyl)-Leu-Gly-Pro-Ala assay

Collagenase activity was measured using FALGPA, a specific chromogenic substrate as described.[7] For all the study-formulations, 5.5 mg of collagenase enzyme (1.4 FALGPA units) were dissolved in 50 mM tricine buffer (with 0.4 M NaCl and 0.01 M CaCl2, pH 7.5). FALGPA 2 mM was prepared in above tricine buffer, 50 μl of extract; tricine buffer and collagenase enzyme solution were added in the vial. This vial was incubated for 15 min at room temperature, and then 100 μl of FALGPA solution (2 mM) was added, OD was immediately measured at 340 nm using spectrophotometer (Perkin Elmer, Massachusetts, USA). Collagenase inhibitory activity was calculated by as following formula:

Collagenase inhibitory activity (%) = (OD Control–OD Sample/OD Control) × 100

Lipid peroxidation assay

Lipid peroxidation (LPO) activity of the cell membrane was calculated regarding generation of TBA reactive substances (TBARS) as per the protocol;[8] however, with some modifications. To achieve this, the cells were seeded at a density of 1 × 105 cells/ml in 96-well plates and were grown overnight. Cellular inflammation was induced using IL-1 β (100 ng/ml) for 72 h, which was followed by treatments of the study formulations for 24 h. For AA, BA, and DA concentrations used were, 500 ng/ml, 5 and 20 μg/ml, whereas for TE, 20, 80, and 160 μg/ml doses were used. The cells were harvested and resuspended in 120 μl of 1X PBS. They were homogenized on ice for 10 min using a micro-pestle and then centrifuged at 10,000 rpm for 10 min. This was followed by addition of 100 mM BHT (1.5 μl), 15% TCA (50 μl), 0.25 mM BHT (50 μl), 0.375% TBA (50 μl), and 8.5% SDS (2 μl). The samples were vortex-mixed for 5 min and incubated at 80°C for 120 min, reaction was stopped by cooling on ice for 10 min. The samples were centrifuged at 10,000 rpm for 10 min and supernatant from each tube was transferred to a 96-well plate. The OD was measured at 540 nm using ELISA plate reader (Bio-rad, Hercules, CA, USA).

Statistical analysis

All the experiments were performed three times in triplicates. Data were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 5 Program (San Diego, CA, USA). One-way ANOVA test was applied followed by Bonferroni's multiple comparison test to determine the significance.


Cell viability assay

Rabbit synoviocytes were treated with four Ayurvedic formulations namely AA, BA, DA, and TE. Among the four AA, BA, and DA showed cell viability up to 20 μg/ml, whereas in case of TE, it was up to concentration of 160 μg/ml for 24, 48, and 72 h [Figure 1]. We used this data to determine concentrations for spot test, NO, malondialdehyde (MDA), and FALGPA assays.{Figure 1}

Gelatinase spot assay of collagenase type II enzyme

All the study formulations showed complete enzyme inhibition of collagenase activity at specific concentrations. AA, BA, DA, and TE showed complete enzyme inhibition at 75, 50, 39, and 20 mg/ml, respectively [Figure 2]. TE showed a total collagenase inhibition at the lowest concentration of 20 mg/ml, while AA showed inhibition at the highest concentration (75 mg/ml).{Figure 2}

Nitric-oxide assay

Significant NO reduction was observed after treating the cells with the study formulations, wherin inflammation was induced by treatment of IL-1 β and LPS. For AA, BA, and DA, used concentrations were 500 ng/ml, 5 and 20 μg/ml; however, for TE 20, 80, and 160 μg/ml concentrations were used. These concentrations were finalized with the reference to the cell viability study, as mentioned above. For AA treated cells, induced by IL-1 β, the concentration 500 ng/ml, 5 and 20 μg/ml caused a drop in NO levels as 5.53 ± 0.41, 14.65 ± 2.07, and 6.12 ± 1.24 μM/ml, respectively. For the inflammation generated by LPS, it was observed as 5.53 ± 0.41, 4.50 ± 0.20, and 6.28 ± 1.8 μM/ml, respectively. In case of BA, IL-1 β induced cells showed a significant decrease in NO release 2.88 ± 2.49, 16.71 ± 0, and 5.09 ± 1.45 μM/ml for the respective concentrations of 500 ng/ml, 5 and 20 μg/ml. Cells, wherein inflammation was generated by LPS, reduced NO levels were calculated as 4.50 ± 4.36, 3.18 ± 3.32, and 3.76 ± 2.07 μM/ml for the respective concentrations 500 ng/ml, 5 and 20 μg/ml of BA. Further for DA, in IL-1 β induced cells, NO estimated was 3.1 ± 0.4, 0.02 ± 0, and 0.97 ± 0.20 μM/ml for the respective concentration of 500 ng/ml, 5 and 20 μg/ml of DA. On the other hand, in LPS treated cells, decreased NO was observed as 10.08 ± 0.20, 7.44 ± 1.03, and 0.97 ± 0.62 μM/ml for the selected concentrations, respectively. In case of TE, decreased NO calculated, for both IL-1 β and LPS induced cells, at the concentrations of 20, 80, and 160 μg/ml, was 5.53 ± 2.07, 1.85 ± 1.03, and 1.41 ± 0.8 μM/ml; whereas 4.94 ± 0.83, 0.97 ± 0.20, and 1.4 ± .0.8 μM/ml, respectively.

To summarize, for AA maximum decrease in NO levels was at the concentration 500 ng and with the inflammation induction by IL-1 β and for LPS induction, it was for the concentration of 5 μg/ml. For BA, similar to AA, the maximum decrease was for the concentration of 500 ng/ml and for IL-1 β and LPS induction and in case of LPS; it was for the concentration of 5 μg/ml. DA treatment caused the maximum NO decrease at the dose of 5 μg/ml when the cells were induced by IL-1 β; whereas for LPS induction, the maximum decrease was for 20 μg/ml. Interestingly, TE showed a maximum reduction in NO levels at concentration of 160 μg/ml on IL-1 β treated cell lines and on LPS induction, it was for the dose of 80 μg/ml [Figure 3] and [Figure 4].{Figure 3}{Figure 4}

FALGPA assay

Results of assay were expressed in percentage (%) of collagenase inhibition. IC50 values were calculated for all the four extracts. Percentage inhibition increased with the increasing concentration of formulations. AA showed the highest IC50 (95.54 mg/ml), while TE revealed the lowest (4.24 mg/ml). For BA and DA, IC50 were 93.02 and 27.76 mg/ml, respectively.

Malondialdehyde assay

In the present study, MDA levels found significantly reduced after the treatment of AA, BA, DA, and TE as compared to the control (untreated IL-1 β induced cells). The decrease was in a dose-dependent manner for the concentrations of 500 ng/ml and 20 μg/ml; for AA, the decline was calculated as 2.4–6.7-fold (P < 0.001), BA exhibited 2.3–6.7-fold (P < 0.001) drop, while DA revealed the maximum decrease of 4-fold to 12.2 (P < 0.001)-fold in MDA. TE showed a drop in MDA levels in the range from 2-fold to 8-fold (P < 0.001) for the studied concentrations [Figure 5].{Figure 5}


According to the Ayurveda philosophy, OA (known as “sandhigatvata”) is a cluster of diseases where vata dosha (the principal of force and motion in the body) prevails. Particularly, in knee OA, (jaanusandhigatvata), vatadosha occupies knee joints; where the causes, most often attributed are, improper diet, unfavorable lifestyle, trauma, aging processes, and constitutional predispositions. Further, the aggravated vaat is responsible for dryness (rukshata), lightness (laghutva), porosity (saushirya), coarseness (kharatva), and ultimately a loss of function of knee joint. A reduction and regulation of the aggravated vayudosha thus, stands as a central principle of Ayurveda treatment approach for knee OA.[9]

The selected herbal compounds in the present study (AA, BA, DA, and TE), are well-known for their vata alleviation potential, and thus has remained as conventional therapy for OA in routine clinical practice. In other words, clinical evidences for these formulations are well established; however, molecular authentication for the same is deficient. The present communication is attributed to assess the molecular mechanism of these formulations to establish their anti-OA activity, and thus to support their clinical usage. To achieve this, we selected biomarkers which address the key pathologies involved in OA, such as synovitis, cartilage degeneration by the collagenolytic enzyme and oxidative stress.

NO plays a chondroprotective role when present in a limited amount; however, excess contributes to OA pathogenesis [10] by mediating inflammation as well as cartilage destruction.[11],[12] Excessive NO production further inhibits matrix synthesis and promotes its degradation.[13] Thus, intercepting NO production is thought to be a promising target in OA treatment,[14],[15] which will ultimately result in reducing inflammation and slowing down the destructive process in diseased cartilage. In the present set of experiments, NO release was measured after inducing inflammation in HIG-82 cell, using IL-1 β and LPS and treating them with the selected formulations of extracts. All the Ayurvedic compounds were observed to cause NO reduction in inflamed cells, albeit with different concentrations. AA showed a maximum decline in NO levels (5.53 ± 0.41 and 4.50 ± 0.20 μM/ml), for IL-1 β and LPS treated cells respectively, for the concentration of 500 ng and 5 μg/ml. In case of BA, DA and TE, the lowest NO was recorded for the concentration of 500 ng/ml and 5 μg/ml, 5 and 20 μg/ml, and 160 and 80 μg/ml, when the cells were induced with IL-1 β and LPS, respectively [Figure 3] and [Figure 4].

FALGPA is a synthetic collagenase-specific chromogenic substrate,[16] and previously has been used to determine the collagenase-inhibitory activity of different herbs.[7],[17] Our previous study showed that elevated level of collagenase type II enzyme contributes to the cartilage degradation.[18] We, therefore, carried out gelatinase (gelatin is a degenerated collagen) spot test assay to detect collagenase inhibitory potential of the study formulations. Gelatinase spot assays are validated, reproducible, reliable, and sensitive method for measuring collagenase type II inhibition.[5] All the studied compounds showed a significant inhibition of collagenase type II further, measured by FALGPA.

In the present work, MDA was one of the selected oxidative stress biomarker, as in OA, it not only influences the degree of cell damage but also reflects a severity of LPO.[19] Higher levels of LPO are responsible for altered cell structure and function.[20] This LPOs production can be induced by reactive oxygen species (ROS).[21] In OA, ROS are released during inflammation of the synovium, which further inhibits collagen as well as proteoglycan synthesis, activate MMPs production that finally results in apoptosis.[22] Here, we have measured MDA from inflamed untreated cells as well as after treating them with the selected Ayurveda formulations. A significant decline was observed in MDA levels after the treatment with studied compounds, in a dose-dependent manner.

To recapitulate, all the selected Ayurveda formulations were found to be effective in controlling synovitis, to reduce oxidative stress and to arrest cartilage-loss; three pivotal pathologies associated with OA. However, evaluation of these formulations using critical imaging diagnostic tools and clinical parameters, addressing the above-mentioned pathologies will ascertain their efficacy in real means, as potential candidates for OA management.


We believe that the traditional knowledge of Ayurveda with its holistic approach, supported by experimental base will serve as the powerful and innovative foundation for newer, safer as well as affordable medicines. Thus, the plant species mentioned in the ancient text of Ayurveda if explored with the modern scientific approaches, will certainly be of assistance for the better-leads in healthcare. The present study, therefore, was trusted as an initial step toward this goal; where we have tried to set up molecular evidences for the commonly practiced Ayurvedic formulations in OA treatment. The development and validation of traditional medicinal systems with the perspectives of safety, efficacy, and quality will help not only to preserve this traditional heritage but also to rationalize the use of natural products in the health care.

Till date, a single molecular approach to treat, polygenic disease like OA, has rented to provide only a palliative care instead of alleviate. Hence, in recent time, there is a growing interest in exploring biological activities of different Ayurvedic herbs. Being natural and polyphenol in nature, these herbs are thought to have a potential to provide a better alternative for OA; however, molecular evidences for the same are scarce. In the present communication, we have assessed the anti-OA efficacy of the most commonly prescribed Ayurvedic drugs using relevant biochemical assays. To our findings, the formulations were efficient to restrain synovitis, reduce oxidative stress and can protect cartilage the signature features of OA pathogenesis. To establish molecular authenticity, Ayurvedic herbs can offer safer, yet an affordable therapeutic option for OA management.


The authors are thankful to Director, Interactive Research School of Health Affairs, BharatiVidyapeeth Deemed University, Pune for his constant encouragement and support.

Financial support and sponsorship

This study was financially supported by Interactive Research School for Health Affairs, Bharati Vidyapeeth Deemed University, Pune, Maharashtra, India.

Conflicts of interest

There are no conflicts of interest.


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