Journal of Interdisciplinary Dentistry

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 10  |  Issue : 1  |  Page : 24--28

In vitro assessment of cytotoxicity and anti-inflammatory properties of shilajit nutraceutical: A preliminary study


S Sarah Victoria Ezhilarasi, Rajkumar Kothandaraman, Ravikumar Nesamani, Saravanakarthikeyan Balasubramanian, Sekar Mahalaxmi 
 Department of Conservative Dentistry and Endodontics, SRM Dental College, Chennai, Tamil Nadu, India

Correspondence Address:
Dr. Saravanakarthikeyan Balasubramanian
Department of Conservative Dentistry and Endodontics, SRM Dental College, Bharathi Salai, Ramapuram, Chennai - 600 089, Tamil Nadu
India

Abstract

Background: Shilajit is a nutraceutical exudate found mainly in the Indian Himalayas, which is formed for centuries by the gradual decomposition of certain plants due to microbial action. It has been effectively used as a potent and safe dietary supplement, restoring the energetic balance and potentially able to prevent several diseases. Hence, the aim of the present preliminary in vitro study is to assess the cytotoxicity of shilajit extract and to comparatively evaluate the anti-inflammatory effect of shilajit and diclofenac sodium, a commonly used drug. Materials and Methodology: Shilajit was commercially procured in pure powder form which was dissolved in 10 ml of methanol and boiled for 80 min, followed by centrifugation (2500 rpm) for 10 min. The supernatant fluid thus obtained was used as an experimental solution. 25-mg diclofenac sodium was used as a control drug. The cytotoxicity of shilajit extract was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas the anti-inflammatory property of the test groups was comparatively evaluated using protein denaturation assay. Results: As the dilution of the extracts increased, the amount of cell viability was also increased, thereby showing that the diluted shilajit extract concentrations proved to be least cytotoxic. Different percentages of shilajit exhibited concentration-dependent inhibition of protein denaturation. Although shilajit extract exerted marginally better anti-inflammatory effect than diclofenac sodium, the effect being dose dependent; the protein inhibition values were not statistically significant (P > 0.05). Conclusion: Within the limitations of this in vitro preliminary study, it can be concluded that: (i) shilajit extract was found to be nontoxic when tested on L929 mouse fibroblast cell lines and (ii) the anti-inflammatory effect of shilajit was comparable to that of diclofenac sodium. Hence, this nutraceutical can be a viable alternative to conventional anti-inflammatory drugs in the field of medicine and dentistry.



How to cite this article:
Ezhilarasi S S, Kothandaraman R, Nesamani R, Balasubramanian S, Mahalaxmi S. In vitro assessment of cytotoxicity and anti-inflammatory properties of shilajit nutraceutical: A preliminary study.J Interdiscip Dentistry 2020;10:24-28


How to cite this URL:
Ezhilarasi S S, Kothandaraman R, Nesamani R, Balasubramanian S, Mahalaxmi S. In vitro assessment of cytotoxicity and anti-inflammatory properties of shilajit nutraceutical: A preliminary study. J Interdiscip Dentistry [serial online] 2020 [cited 2020 Oct 22 ];10:24-28
Available from: https://www.jidonline.com/text.asp?2020/10/1/24/283533


Full Text



 Clinical Relevance to Interdisciplinary Dentistry



The management of pre..and post.operative pain is a main prerequisite during and following periodontal, endodontic, and surgical procedures. Nonsteroidal anti.inflammatory drugs (NSAIDS) are commonly prescribed in dental practice for the management of pain and swelling. However, these drugs do exhibit side effects, the most frequent range from mild (e.g. nausea, vomiting, and dizziness) to severe problems (gastric bleeding and renal toxicity). In addition, their use is contraindicated during pregnancy. The currently studied shilajit extract derived from Indian mountain rocks (Ayurvedic rasayan) is a safe natural derivative which is reported to have significant anti.inflammatory properties, and hence, it can be a viable alternative to conventional NSAIDS in clinical practice

 Introduction



Inflammatory response is a crucial aspect of the tissue's reaction to injury resulting from any noxious stimuli and plays an inevitable role in the host defense.[1] The terminology “inflammation” is derived from a Latin word, “inflammare” meaning “to burn” (de oliveira). The mechanism of inflammation represents a chain of organized, dynamic responses including both cellular and vascular events with specific humoral secretions. The cells at the site of inflammation release specialized substances such as vasoactive amines and peptides, eicosanoids, pro-inflammatory cytokines, and acute-phase proteins which mediate the inflammatory process by preventing further tissue damage and ultimately resulting in healing and restoration of the tissue function. Basically, inflammation involves two processes with some overlapping, namely early inflammatory response and later, followed by healing. Nevertheless, inflammation regularly progresses to acute or chronic stages.[1],[2] Acute inflammation is due to an early response by the body and is of short duration, whereas chronic inflammation is of longer duration and occurs usually after a delay which is characterized by chronic inflammatory cells.

The initial inflammatory cell infiltrate consists primarily of neutrophils, macrophages, and lymphocytes.[3] As a response to injury, inflammation is initiated, in part, due to the release of reactive oxygen species (ROS) such as superoxide (O2−), hydroxyl (OH−), hypochlorous acid (HOCl), and peroxyl (ROO) radicals from activated neutrophils and macrophages. They are partially reduced metabolites of oxygen that possess strong oxidizing capabilities.[4] These ROS stimulate the release of a wide range of nonspecific mediators such as histamine, bradykinin, serotonin, interleukin, and arachidonic acid metabolites that recruit additional inflammatory cells. Hence, it can be inferred that ROS are important mediators that initiate and sustain inflammatory processes.[5]

Under normal conditions, the ROS and their by-products are neutralized by the body's natural antioxidant system. The antioxidant defense system is a highly complex biochemical organization that consists of numerous enzymes and a large number of scavenger molecules. Each of these enzymes and antioxidant molecules participates in highly specific reactions and, as such, is not interchangeable.[6] Oxidative stress is created when there is an imbalance between ROS production and antioxidant capacity, such as that occurs in inflammation.

The most commonly used nonsteroidal, anti-inflammatory agents in the field of dental medicine include diclofenac sodium, ibuprofen; although effective, they possess several side effects, from mild subjective symptoms such as nausea and dyspepsia to more serious gastroduodenal ulcerations and cardiovascular and renal problems that are dose dependent.[7]

Recently, there has been a growing interest in the phenolic compounds present in natural products such as fruits and vegetables. These are rich in antioxidants that, when used, can neutralize and scavenge the ROS to attenuate inflammation. Shilajit, used by the ayurvedic medicine since centuries, also known in North India as salajit, shilajatu, mimie, or mummiyo, is a blackish-brown exudate from high mountain rocks, found especially in the Himalayan mountains between India and Nepal. Latex-bearing plants, namely Euphorbia royleana and Trifolium repens, which occur in the vicinity of these rocks are thought to be the most likely source of shilajit. Other sources also include plant organisms such as, Barbula, Fissidens, Minium and Thuidium, Asterella, Pellia, Plagiochasma, and Stephenrencella-Anthoceros.[4],[5],[8],[9]

Purified shilajit is prepared from these exudates by a proprietary extraction process. Its main composition includes humic substances such as fulvic acids, humins, and humic acids. Fulvic acid, the biologically active compound, is well known for its memory enhancing property and is a potential anti-aggregation factor of tau protein in vitro. In addition, it possesses therapeutic properties such as antioxidant, anti-inflammatory, antiallergic, immunomodulatory, antidiabetic, anxiolytic, in addition, as a cognitive and memory enhancer with an ability to interact positively with other drugs.[9],[7] Ayurveda literature reports that shilajit has an influence on endocrine, autonomic, and brain functional changes, mediated by cytokines, released by activated immunological cells.[10] This opens up possibilities for a similar mechanism of action of shilajit in inflammatory processes.

Hence, this preliminaryin vitro study was undertaken to evaluate the cytotoxicity of shilajit extract by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and to comparatively evaluate the anti-inflammatory effect of shilajit with that of a commonly used anti-inflammatory drug, i.e. diclofenac sodium using protein denaturation method. The null hypothesis proposed was that shilajit extract does not exhibit any anti-inflammatory action when compared to that of diclofenac sodium.

 Materials and Methodology



This preliminaryin vitro study was conducted following consent from our institutional ethical clearance board, SRM Dental College, Ramapuram, Chennai, India (Ref No: SRMU/MandHS/SRMDC/2020/S/001).

Materials

Shilajit was obtained in pure powder form from Kishore Pharma Ltd., Punjab, India. 25 mg of shilajit powder was dissolved in 10 ml of methanol and boiled for 80 min. It was then centrifuged at 2500 rpm for 10 min. The supernatant fluid thus obtained was used in the present study. Diclomax 25 MG tablet (Torrent Pharmaceuticals Ltd., Ahmedabad, India) containing 25 mg diclofenac sodium was used as a control drug.

Grouping

Group I (Control): Diclofenac sodium 25 mgGroup II (Experimental): Shilajit extract.

Cytotoxicity assessment of the experimental shilajit extract using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Preliminary cytotoxicity tests were performed in order to assess the biocompatibility of the test groups. Cell viability was assessed on mouse fibroblast cell lines using a MTT-based colorimetric assay.[11],[12] L929 mouse fibroblast cell lines were obtained from National Centre for Cell Sciences, Pune, India, and the cells were maintained in minimal essential media (Sigma Aldrich, India) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 50 μg/ml CO2 at 37°C.

The test sample (shilajit extract) was serially diluted to 8 concentrations. 8 × 10[5] L929 mouse fibroblast cells, diluted with Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal calf serum, were pipetted into 96-well microliter plates and were incubated overnight at 37°C and 5% CO2. The cells were exposed to these dilutions for 3 h under the same conditions of incubation. The sample solutions were then removed from the wells by washing with phosphate-buffered saline (PBS). One milliliter/well (5 mg/ml) of 0.5% MTT solution dissolved in PBS was added to the wells. After further incubation of 4 h, 100 μl of dimethyl sulfoxide was added for the dissolution of formazan crystals. The absorbance of each well was then read at 570 nm using a microplate reader. The absorbance at 570 nm was measured with an ultraviolet (UV) spectrophotometer using wells without sample containing cells as blanks. The effect of the samples on the proliferation of L929 cells was expressed as the % cell viability, using the following formula:

Percentage (%) cell viability = A570 of treated cells/A570 of control cells × 100%.

Anti-inflammatory property evaluation using protein denaturation bioassay

The reaction mixture (5 ml) consisted of 0.2 ml of 1% aqueous solution of bovine egg albumin, 2.8 ml of PBS (pH: 6.4), and 2 ml of 1%, 2%, 3%, and 4% of shilajit. The pH of the reaction mixture was adjusted using small amount of HCl. Similar volumes of double-distilled water served as control. The mixtures were incubated at 37°C ± 2°C in a Bio-Oxygen Demand (BOD) incubator for 15 min and then heated at 70°C for 5 min. After cooling, the turbidity of the mixture was determined by measuring the absorbance at 660 nm using UV spectrophotometer. Diclofenac sodium tablet at the final concentration of (1%, 2%, 3%, and 4%) was used as reference drug and treated similarly for determination of absorbance. The experiment was performed in triplicates. The percentage inhibition of protein denaturation was calculated using the following formula:

Percentage (%) inhibition = 100 × (Vt/Vc – 1). where Vt = absorbance of test sample and Vc = absorbance of control.

 Results



[Table 1] depicts the cytotoxic effects of the shilajit extract on normal L929 mouse fibroblast cell lines. In this study, a series of extracts of different concentrations of shilajit were made to observe a possible dose-dependent relationship. According to the results, as the dilution of the extracts increased, the amount of cell viability was also increased, thereby showing that the diluted extract concentrations proved to be less cytotoxic. The anti-inflammatory property of the test agents was assessed by protein denaturation assay, and the results are depicted in [Table 2], wherein the average values of the different concentrations of shilajit and diclofenac sodium were mentioned. In the present study, different percentages of shilajit (Group II) exhibited concentration-dependent inhibition of protein denaturation, similar to that of diclofenac sodium (Group I). As the concentration of shilajit increased, there was a concomitant increase in its percentage (%) inhibition. The comparative overall % inhibition values of shilajit (221.29 ± 61.089) and diclofenac sodium (250.24 ± 56.52) were statistically analyzed. However, the results were not statistically significant (P > 0.05) between the test groups (P = 0.382).{Table 1}{Table 2}

 Discussion



The natural antioxidant defense system is a highly complex biochemical organization that consists of numerous enzymes and a large number of scavenger molecules.[7] Production of ROS is central to the progression of many inflammatory diseases.[13] The ROS acts as both a signaling molecule and a mediator of inflammation. The increase in the prevalence of multiple drug resistance has resulted in search for new anti-inflammatory from alternative sources.[14] In recent years, the search for phytochemicals possessing anti-inflammatory properties has been on the rise due to their potential use in the therapy of various chronic and infectious diseases in the field of medicine.[14] The role of anti-inflammatory and analgesic drugs is also crucially significant following periodontal, endodontic, and oral surgical procedures.

Shilajit is composed mainly of humic substances, including fulvic acid that accounts for around 60%–80% of the total components. Other molecules present in shilajit preparations are ellagic acid, some fatty acids, resins, latex, gums, albumins, triterpenes, sterols, aromatic carboxylic acids, 3,4-benzocoumarins, amino acids, polyphenols, and phenolic lipids. Of these, fulvic acid, the biologically active compound, is known for its antioxidant, anti-inflammatory, and memory-enhancing properties. Literature evidences were also reported pertaining to the fact that the ayurvedic rasayan, shilajit, is more effective than several currently available clinically efficacious immunomodulators.[15]

Based on the results of thisin vitro study, although shilajit extract exerted marginally better anti-inflammatory effect than diclofenac sodium, the effect being dose dependent; the values were not statistically significant, and thus, the proposed null hypothesis was rejected. Diclofenac sodium tablet is commonly available in various strengths such as 25, 50, and 75 mg. Diclofenac tablet (25 mg) is commonly recommended for pediatric patients greater than the age of 1. Hence, the present preliminary study involved 25 mg strength which is the safest and lowest dose preferred for patients of all age groups. Therefore, it can be inferred that shilajit is as equally effective as with even the lowest strength of diclofenac drug.

Since further studies are underway pertaining to the use of shilajit in endodontics, we have evaluated its cytotoxicity on mouse fibroblast cell lines, the results of which revealed that shilajit is biocompatible. Shilajit was also found to have potent anti-inflammatory activity in all three models of acute, subacute, and chronic inflammation. Hence, this phytotherapeutic extract which is a millenary product of nature can be potentially used as an anti-inflammatory drug alternative to traditional drugs in the field of dental medicine following endodontic and surgical procedures.

Although this study is a novel attempt to explore the cytotoxic and anti-inflammatory effects of shilajit extract for its effective use in dentistry, it is not without its limitations. This is only a preliminary study, and hence, further studies pertaining to its characterization, dose concentration, and evaluation of cytotoxic and anti-inflammatory effects on human periodontal fibroblast cell lines have to be elucidated before clinical evaluation.

Traditional medicine is an integral part of health-care systems in many developing countries.[16] Although shilajit is considered a panacea in oriental medicine as described in ancient texts, it is imperative that research should be conducted to validate these claims and uses. The vast majority of published papers on shilajit are of natural alternate medicine, and it is necessary that shilajit breaks the cultural paradigm and enters into the rest of the world by the hand of rigorous research at the molecular and cellular levels, which could elucidate the interactions of the active ingredients of the different shilajit preparations with biomolecules.[17]

 Conclusion



Within the limitations of thisin vitro preliminary study, it can be concluded that: (i) shilajit extract was found to be nontoxic when tested on L929 mouse fibroblast cell lines and (ii) the anti-inflammatory effect of shilajit was comparable to that of diclofenac sodium. Hence, this nutraceutical can be a viable alternative to conventional anti-inflammatory drugs in the field of medicine and dentistry.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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