Evaluation of Effect on Combination of Five Medicinal Plants Extracts on Denaturation of Protein - A Remedy of Arthritis Disease

Introduction

Arthritis is one amongst the oldest diseases. It is a systemic disease, affecting mainly joints. Internationally, the prevalence of arthritis is believed to range from 0.4 to 1.3% and incidences increases with age, women being affected thrice over men [1]. In adult Indian population a prevalence of arthritis rate of 0.75% was observed. Arthritis is one amongst the foremost common system disorder which will affect many tissues and organs but principally attack synovial joints. Arthritis can affect at any age but more common within the age range of 25-50 years [2]. Arthritis could be autoimmune of disease and a significant reason behind disability throughout the World. It is a chronic multisystem disease characterized by hyperactivity of certain immune reactions, persistent synovitis with diffuse proliferation, and in most of the cases deposition of autoantibodies to immunoglobulins called autoantibody [3]. In severe cases, the synovial inflammation results in articular cartilage damage, bone erosion, and subsequent changes in joint integrity usually peripheral joints are involved.

Some literature Ammon, et al. reported that in autoimmune disorder the denaturation of protein is one amongst the causes. Production of auto-antigens in certain rheumatic diseases is additionally due to in vivo denaturation of proteins [4]. Mechanism of denaturation probably involves alteration in hydrogen, hydrophobic electrostatic and disulphide bonding [5]. Various anti-inflammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation. In our present study, methanolic extract of combination of plant extracts inhibited heat induced protein denaturation and will be one amongst the explanations of possessing anti-arthritic activity [6]. Formation of blood clots is one amongst the vital reasons of blood circulation problem. Thrombi or emboli can shack a vas and block the flow of blood in this location depriving tissues of normal blood flow and oxygen. This will lead to damage, destruction (infarction), or perhaps death of the tissues in this area. A blood is made from fibrinogen by thrombin and is lysed by plasmin which is activated from plasminogen by tissue proteinase (TPA). Fribrinolytic drugs are wont to dissolve thrombi in acutely occluded coronary arteries there by restoring blood supply to ischemic myocardium to limit necrosis and to boost prognosis [7].

Streptokinase is an antigenic clot buster used for the treatment of acute infarction. It reduces mortality as effectively because the non-antigenic altreplase in most infarct patients while having the benefits of being much more cost-effective. Tissue-type protease (TPA) is usually preferred as being effective and safer than either urokinase or streptokinase type activators. All available thrombolytic agents still have significant shortcomings, including the requirement for giant doses to be maximally effective, limited fibrin specificity and a major associated bleeding tendency. Due to the shortcomings of the available thrombolytic drugs, attempts are underway to develop improved recombinant variants of those drugs [8, 9, 10, 11, 12].

The plant represents an infinite reservoir of biologically active compounds with various chemical structures and protective/disease preventive properties (phytochemicals). Nearly 50% of medication employed in medicine is of plant origin and only a little fraction of plants with medicinal activity has been assayed. Therefore much current research has been dedicated to the phytochemical investigation of upper plants which have ethno-botanical information related to them. The phytochemicals isolated are then screened for various forms of biological activity like thrombolytic potentials [13]. Herbal preparations are used potential source of medication since times of yore to keep up health and regain healthy state of mind. Herbs showing thrombolytic activity are studied and a few significant observations.

The use of herbal medicines in India represents a protracted history of human interactions with the environment. Plants used for traditional medicine contain a good range of drugs that may be substances treat chronic diseases. The traditionally many plant reported to use as treatment of autoimmune disorder in Indian literature Deodhar, et al. [14, 15, 16]. But scientifically very less works in those plants. It is urgent must base upon traditionally knowledge prepare the formulation and their see the active ingredients. Plant derived drugs function a prototype to develop simpler and fewer toxic medicines. Among them Tinospora cordifolia, Pongamia pinnata, Curcuma longa, Emblica officinalis, Piper nigrum incorporates a big selection of bioactive principles in addition because it has been proven medicinally important plants for arthritis disease curing.

The modern medicine uses non-steroidal anti- inflammatory drugs, diseases modifying anti-rheumatic drugs, TNF- α antagonists and immunosuppressant drugs for the treatment of rheumatoid arthritis. These drugs have potent activity and long term administration is required for the treatments of chronic diseases. Furthermore, they produce some side effects include gastric ulcer, renal damage etc., and toxicity. Therefore there is a need for a new alternative natural therapy. Nowadays herbal plant extracts formulation and their isolated compounds were used both internally as well as externally for the treating inflammatory conditions like arthritis. The goal of the treatment is to cut back joint inflammation and pain, maximize joint function, and forestall joint destruction and deformity. The present study for screening of different formulations for drug development is to cure the rheumatoid arthritis using the protein denaturation method.

Total Flavonoid Assay

Total flavonoid contents were measured with the aluminium chloride colorimetric assay. Methanolic extracts or standard solution of gallic acid (100ug mlˉ¹) was added to 10 ml volumetric flask containing 4ml of water. To the above mixture, 0.3ml of 5% NaNO2 was added. After 5 minutes, 0.3ml of 10% AlCl3 was added. After 6 min, 2ml of 1 mol L–1 NaOH was added and the total volume was made up to 10ml with water. The solution was mixed well and the absorbance was measured against a prepared reagent blank at 510 nm. Total flavonoid content of plants extracts was measured using standard readings [19].

Analytical Thin Layer Chromatography

Analytical Thin Layer Chromatography was performed to find out suitable solvent system for the development of chromatogram. The following solvent mixtures were tried on precoated TLC plates (Merck, silica gel 60 F254 plate, 0.25mm). Plate were developed using glass beaker which was pre-saturated with mobile phase for 20min. After that sample were applied from 2cm above of the plate using micro-capillaries. The samples were run up to height of 10cm. Methanol: Acetic acid: Water mobile phase used with composition (6.9:0.1:3). After development plate were removed and dried and visualize under UV light.

HPTLC Analysis

For chemical profile analysis, plant leaves extract formulation 10mg/10ml was taken. The sample used for the HPTLC analysis (Camag system equipped with a sample applicator Linomat-5, twin development chamber, TLC scanner-3 and integration software, documentation systemReprostar-3 with G5 digital camera) (Camag, Switzerland). HPTLC aluminium sheet pre-coated with silica gel 60 (1.05547 E Merck) was used as the adsorbent. Methanol: Acetic acid: Water (6.9:0.1:3) was used as the mobile phase. The chromatographic development chamber was saturated with mobile phase for 10 min prior to placement of the plates. The plates were run up to 8 cm height and derivatized (10% H2SO4in methanol). The derivatized plates were heated at 100°C for 4 min, bands were observed and scanned at 366 nm and photographs were taken for record.

Gas Chromatography Mass Spectroscopy (Gc-Ms)

The GC-MS analysis was done by electron impact ionization (EI) method on Auto system XL gas chromatography (Perkin Elmer Instrument, Germany) coupled to a Turbo Mass Spectrophotometer (Perkin Elmer

Instrument, Germany) at Sophisticated and Instrumentation Centre for Applied Research and Training (SICART), Vallabh Vidyanagar, Gujarat. The column was fused silica capillary column, 30 x 0.25 mm ID; coated with D-I, 0.25 μm film thickness. The temperature of column was programmed at 70 to 250° C at the rate of 10ºC /min increase, injection port temperature at 250°C. Helium was used as carrier gas at constant pressure of 100 kpa and flow rate of 20ml/min. Samples which dissolved in methanol was run fully at range of 60-550 amu and the results were compared by using NIST 107 Spectral library search programme.

Result and Discussion

Arthritis is one of the oldest diseases. It is a systemic inflammatory disease, affecting mainly joints. Internationally, the prevalence of arthritis is believed to range from 0.4 to 1.3%.In adult Indian population a prevalence of arthritis rate of 0.75% was observed.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by systemic features and joint involvement which affects 1% of the world’s adults. It is one of the most common musculoskeletal system disorder that may affect many tissues and organs but principally attack synovial joints. In 1st stage swelling of the synovial lining, causing pain, stiffness, redness and swelling around the joints. In the 2nd stage the rapid division of cells which leads to the synovium to thicken. In last stage the inflamed cells releases the enzymes that may digest bone and cartilage, often causing the joint to lose its shape and alignment, more pain and loss of movement.

Medicinal plants have been used as natural medicines.

This practice has been living since prehistoric times. There are alternative ways during which plants are found useful in medicines like crude extract of plants has been used directly due to the presence of natural chemical constituents and natural compounds for the synthesis of medication like, colchicine, nicotine, quinine etc. for therapeutic purpose by folk people.

However, it’s been felt that a review with almost fuller information on the disease with focus to the event of satisfactory plant based drugs including combination therapy for successful treatment of all kinds of arthritis is required. The present study for screening of different formulations for drug development is to cure the rheumatoid arthritis using the protein denaturation method.

Extractive Yield (%) of Medicinal Herbs

Before evaluating the knowledge regarding the yield of extract from each herb is important. Lower extract yielding plants are not commonly preferred by the pharmaceutical industry though they are rich in their potency [18]. So, the work was carried out with yield calculation. We have selected methanol solvent for extracting the plant constituents. The organic solvent methanol eluted most of the phytoconstituents from the plant.

The yield of the solvent extracts is mentioned in the Table 3. The methanol extract showed maximum extractive yield of Tinospora cordifolia, Curcuma longa Pongamia pinnata, Emblica officinalis, Piper nigrum, that are 11.88 %, 49.2 %, 72.76%, 48%, 24.88% respectively Table 3. Environmental factors and the location of plants also play a significant role.

Protein Denaturation by Using Bovine Albumin of Different Formulations

The effect of methanolic extract of combination of plants was evaluated against denaturation of bovine serum albumin. The results are summarized in Figure 1. The present findings exhibited a concentration dependent inhibition of protein denaturation by combination of plant extracts throughout the range of 100 to 400μg/ml. It was effective in inhibiting heat induced albumin denaturation. In BSA denaturation method at concentration of 100,200,300,400µg/ml methanolic extract of F2 formulation (10mg/ml) showed 70.83%, 75%, 87.5%, and 95.83% respectively and reference drug Diclofenac sodium. The present study finding that show the F2 formulation possessed maximum anti-arthritic effect against denaturation of protein in vitro.

F2 formulation (10mg/ml) present the phytochemical constitute of flavonoids, alkaloids and terpenoids. The F2 formulation it may be useful for curing of anti-rheumatism Figure 1.

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Some literature reported that in rheumatoid arthritisthe denaturation of protein is one of the causes. Production of auto-antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins [19]. Mechanism of denaturation probably involves alteration in hydrogen, hydrophobic electrostatic and disulphide bonding. Various anti-inflammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation [20]. In our present study, methanolic extract of combination of plant extracts inhibited heat induced protein denaturation and may be one of the reasons of possessing anti-arthritic activity. Choudary, et al. [12] Review Tinospora cordifolia contain high fiber (15.9%), sufficient protein (4.5-11.2%), sufficient carbohydrate (61.66%), and low fat (3.1%) [21]. It has high potassium (0.845%), high chromium (0.006%), sufficient iron (0.28%), and sufficient calcium (0.131%) and important in various regulatory functions. Guduchi means to rejuvenate dead cells. It is widely used in veterinary folk medicine and has also claimed to be beneficial according to Ayurveda for the cure of skin diseases, diabetes and various infections for its anti- inflammatory, anti- arthritic [22]. It enhances Vitamin C and so function as an effective antioxidant [23]. In our present study, methanolic extract of combination of plant extracts inhibited heat induced protein denaturation and may be one of the plant materials of possessing anti- arthritic activity.

Arora, et al. [2] investigated the anti-inflammatory activity in different fractions of the petroleum ether extract of the rhizomes of turmeric (two constituents) in animals [24]. They found that the extracts reduced the granuloma growth and no toxic effects were observed. Chandra and Gupta (1972) demonstrated the anti-inflammatory and anti- arthritic actions of volatile oil of C. longa [25]. Pharmacological

actions of curcumin as an anti-inflammatory agent have been examined by Srimal [13, 14, 26]. Huang, et al_. [15] examined the inhibitory effects of curcumin on the proliferation of blood mononuclear cells and vascular smooth muscle cells [27]. The investigators suggested that curcumin could be use clinically in transplant atherosclerosis. Administration of 1200 mg C. _longa a day caused improvement of morning stiffness, walking duration, and relief of joint pain and swelling compared to phenylbutazone (30 mg) [28, 29]. In our present study, methanolic extract of combination of plant extracts inhibited heat induced protein denaturation and may be one of the plant materials of possessing anti- arthritic activity. There are data in the literature showing the administration of Curcuma longa powder in different patients with respiratory diseases and it was observed that these treated patients have relief in symptoms like cough and sputum or physical signs. Other authors reported the treatment of 18 patients with rheumatoid arthritis and found a real improvement on them, treated with 120 mg/day of the drug, and administered orally in patients [30] our result revealed that other author comparative better performance of methanolic extract of F2 formulation.

Mature seeds of karanja have recently gained a great commercial relevance owing to their high oil content, which is explored as an alternate source of fuel and energy [31]. The seeds are reported to contain on average about 28– 34% oil with high percentage of polyunsaturated fattyacids [32]. All parts of the plant have been used as a crude drug for the treatment of tumours, piles, skin diseases, itches, painful rheumatic joints wounds, ulcers, diarrhea etc., [33]. It has also been recognized to possess applications in agriculture and environmental management, with insecticidal activity. More recently, the effectiveness of P. pinnata as a source of biomedicines has been reported [34]. The leaves are hot, digestive, laxative, anthelmintic and cure piles, wounds and other inflammations. A hot infusion of leaves is used as a medicated bath for relieving rheumatic pains [35].

According to Bharat and review, the fruit is rich in, phyllaemblic compounds, gallic acid, tannins, flavonoids, pectin and vitamin C and also contains various polyphenolic compounds [36]. A wide range of phytochemical components including terpenoids, alkaloids, flavonoids, and tannins have been shown to posses’ useful biological activities. Many pharmacological studies have demonstrated the ability of the fruit shows antioxidant, anti-carcinogenic, anti-tumour, antigenotoxic, anti-inflammatory activities, supporting its traditional uses, which have been already discussed in detail by Shah NC [7, 37]. Stating about the nutritional and health care benefits of Amla fruit juice, its other products in which Chyavnaprash is the main poly herbal preparation, which constitutes 70% of the Amla formulations is immune enhancer and a health tonic for both children and elders [38]. Amla is a rare fruit which contains all tastes except salty. With sourness as the foremost taste, it is at the same time sweet, astringent, bitter and pungent. It is light, dry and cold in effect and the richest source of vitamin C. which has already been stated earlier, however, the laboratory tests show that every 100 gm of fresh amla provides nearly 700 mg of this vitamin which is 20 times higher than what is found in an orange. Our result revealed that other author comparative better performance of methanolic extract of F2 formulation.

The phytochemical investigations of Piper nigrum revealed that it contains variety of phytochemicals. Piperine was the first pharmacologically active compound isolated from different members of Piperaceae family. Many investigators isolated different types of compounds viz Phenolics, flavonoids, alkaloids, steroids, terpenes etc and many other compounds. The different pharmacological activities were reported due to the presence of these phytochemicals. Piperine reported to have four isomers viz; Piperine, Isopiperine, Chavicine and Isochavicine. Among all isolated compounds isolated from Piper nigrum Piperine, piperamide and piperamine were found to possess diverse pharmacological activities [39]. The pain and arthritic symptoms in rats were significantly reduced by piperine. It was concluded that piperine showed anti-inflammatory, analgesics and anti-arthritic activities in arthritis model of rats [40]. In fact, herbal plants are being used widely as medicine around decades for treatment of arthritic disease [41]. Herbal medicine constitutes important resources for the treatment of various ailments. Our result revealed that other author comparative better performance of methanolic extract of F2 formulation.

Conclusion

Methanolic extract of different formulation for their anti-arthritic activity against denaturation of bovine serum albumin. Result and attributed the 15 formulation out of F2 formulation possessed highly denaturation of protein. The denaturation of protein F2 formulation presence of active principle such as alkaloids, flavonoids, saponins and terpenoids which are responsible for this activity. F2 formulation (10mg/10ml) based on HPTLC analysis major 2 bioactive compounds present. GC-MS analysis of methanol extracts identified major bioactive with peak area 36.92 % is maximum in F2 formulation (10mg/10ml). In fact, herbal plants formulation of five plants for the F2 formulation are being used widely as medicine around decades for treatment of arthritic disease. Herbal medicine constitutes important resources for the treatment of various ailments. This formulation is further requirement for study of the cyto– toxicity and compound identification in F2 formulation.

Acknowledgements

Authors are thankful and acknowledge the Sophisticated Instrument Centre for Applied Research and Testing (SICART), DST, India for HPTLC and GC-MS analyses. Authors are also thankful to Charutar Vidya Mandal (CVM), VallabhVidyanagar, Gujarat, India and Director of Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences (ARIBAS), New Vallabh Vidyanagar -388121, Gujarat, India, for providing necessary support research and laboratory facility.