In-Vitro Evaluation Of Anti-Inflammatory Property Of Sesamum Indicum Seeds And Withania Somnifera Roots

Abstract

Methanol, Chloroform, and water extract of Withania Somnifera and Sesamum Indiucum (Family-Solanaceae and Pedaliaceae) was assessed for its anti-inflammatory activity by in-vitro methods. In-vitro anti-inflammatory activity was evaluated using albumin denaturation assay, proteinase inhibitory activity, membrane stabilization, and anti-lipoxygenase activity at different concentrations. Aspirin and Diclofenac sodium were used as standard drugs. The results showed that Withania somnifera and sesamum seeds of Methanol, Chloroform Water Extract (EAME) at a concentration range of 100-500µg/ml significantly protects the heat induced protein denaturation. At the concentration of 400 and 500 ?g/ml, showed significant inhibition of 55.22%and 62.71%of proteinase inhibitory action, but at the concentration of 100 and 200 µg/ml did not show significant activity. Heat induced hemolysis of erythrocyte was significantly inhibited at the concentration of 400 and 500ug/ml. Hypotonicity induced hemolysis and lipoxygenase activity were significantly inhibited at the concentration range of 200-500ug/ml and 400, 500µg/ml respectively. The results obtained in the present study indicate that methanol extracts of Withania somnifera and sesamum indicum can be a potential source of anti- inflammatory agents.

Keywords

Introduction

Since ancient times, researchers have been exploring nature in search of new drugs. Useful products can be derived from any part of the plant like bark, leaves, flowers, seeds, etc. plant products have been part of phytomedicines since times immemorial. Due to this many medicinal plants with curative properties have been utilized. For primary health care, around 80% of world population releases on traditional medicines, involving plant extracts. In traditional systems on Unani, Ayurveda, Homeopathy, and Siddha, almost 90% of prescriptions were based on drugs obtained from plants. Drugs from the plant sources are easily available, are less expensive, safe, and efficient and rarely have side effects. Knowledge of chemical constituents of secondary metabolites in plants is desirable because such information will be of value for the synthesis of complex chemical substances. A complete and detailed study of secondary metabolites of medicinal plants found in India needs to be done as they are responsible for the medicinal activity of plants.Plants are a source of large number of drugs comprising to different groups such as antispasmodics, emetics, anticancer, anti microbial, etc. many the plants are clamed to possess antibiotic properties in the traditional system and are also used extensively by the tribal people worldwide. Plants have been known to relive various diseases in Ayurveda. Therefore the researchers today are emphasizing on evaluation and characterization of various plants and plant constitutions against a number of diseases based on the traditional claims of the plant given in ayurved. Extraction of the bioactive plant constitutions as always been a challenging task for the researchers.

Anti-Inflammatory: Anti-inflammatory, make up about half of analgesics. These drugs remedy pain by reducing inflammation as opposed to opioids, which affect the central nervous system to block pain signaling to the brain. Inflammation is a normal, protective response to tissue injury caused by physical trauma, noxious chemicals, or microbiologic agents. Inflammation is the body effort to inactive or destroy invading organisms, remove irritants, and set the stage for tissue repair. When healing is complete, the inflammatory process usually subsides. However, inflammation is sometimes inappropriately triggered by an innocuous agent, such a pollen, or by an autoimmune response as in asthma or rheumatoid arthritis. Inflammation is triggered by the release of chemicals mediators from injured tissues and migrating cells include amines, such as histamine and 5-hydroxytriptamine, lipids, such as the prostaglandins, small peptides such as bradykinin, and larger peptides such as Interleukin-I . There are various medicines for controlling and suppressing inflammatory crisis; steroids, nonsteroid anti-inflammatory drugs, and immunosuppressant are the practical examples of these medications which are associated with adverse effects while in practice our goal is to apply minimum effective dose by the highest efficacy with the least adverse effects. Thus, we need to apply natural anti-inflammatory factors within medication therapy to achieve increased pharmacological response and the lowest degree of unwanted side effects. Herbal medicines are promoting subjects in medicine and, of course, we have to increase our knowledge about them. Complementary, alternative, and traditional medicines are the pivotal source of herbal medication guidance, but surely modern medicine must prove these guidelines through scientific methods before using them in practice. In this review, we have endeavored to assess the plants and the most clinical evidence of their anti-inflammatory effects.           

Withania somnifera: Ashwagandha (Withania somnifera), also known as Indian ginseng, and as Indian Winter Cherry is an important ancient plant, the roots of which have been employed in Indian traditional systems of medicine, Ayurveda, and Unani. It grows in dry parts in subtropical regions. Rajasthan, Punjab, Haryana, Uttar Pradesh, Gujarat, Maharashtra, and Madhya Pradesh are the major Ashwagandha producing states of the country. The estimated production of Ashwagandha roots in India is more than 1500 tones and the annual requirement is about 7000 tones necessitating the increase in its cultivation and higher production.^[1]

Morphology: A dense, hairy erect grayish to mentose herb or under shrub. The roots are stout, long tuberous, fleshy, whitish brown, and aromatic. The leaves are simple, alternate or sub-opposite, round-oval shaped. The flowers are greenish-yellow and found in few flowered clusters in axils. The fruit is a round orange-red berry, enclosed in green enlarged calyx. The fruit resembles that of red cherries. The seeds are many, yellow kidney shaped and discoid.

Taxonomical Classification:

• Kingdom  : Plantae (Plants)
• Sub kingdom: Tracheobronchial
• Division   : Magnoliophyte
• Class        : Magnoliopsida (Dicotyledone)
• Sub class  : Asteridae
• Order       : Solanales
• Family     : Solanaceae
• Genus       : Withania
• Species    : Withania Somnifera

Chemical Constituents: The methanol, hexane and diethyl ether extracts from both leaves and roots of Ashwagandha were found. Alkaloid percentage in roots ranges from 0.13 to 0.31%. The roots of Withania somnifera are alterative, aphrodisiac, deobstruent, diuretic, narcotic, sedative, and restorative in nature. The pharmacological activity of the root is attributed to the alkaloids and steroidal lactones. Indian ginseng’s pharmacological activity has been attributed to two main withanolide, withaferin A and withanolide D. ginseng’s pharmacological activity has been attributed to two main withanolide, withaferin A and withanolide D. Ashwagandha as medicinal herb, ashwagandha is one of the best rejuvenating agents in Ayurveda. Its roots, seeds and leaves are used in Ayurvedic and Unani medicines. Ashwagandha root drug finds an important place in treatment of rheumatic pain, inflammation of joints, nervous disorders, and epilepsy. Dried roots are used as tonic for hiccup, cold, cough, female disorders, as a sedative, in care of senile debility, ulcers, etc. Leaves are applied for carbuncles, inflammation, and swellings. Leaf juice is useful in conjunctivitis. Ashwagandha has anti- inflammatory, anti-tumor, anti-stress, antioxidant, mind-boosting, immune-enhancing, and rejuvenating properties. Ashwagandha root has also been noted to have sex-enhancing properties.

Medicinal Properties:

• It has anti-stress, adaptogenic, aphrodisiac, sedative, diuretic, antispasmodic, germicidal, anti-inflammatory action.
• It is a nervine tonic.
• It enhances immunity and endurance.
• It is a natural nutrient for insomnia.
• It is good hypnotic in Alcoholism.
• It is bitter in taste and hot in potency, so it alleviates vata and kapha.^[1]

Sesamum Indicum Seed: Sesame (Sesamum indicum) is one of about 15 species of herbaceous plants of the genus ‘Sesamum’ native to Africa and Asia and is the most widely cultivated species for its nutritious seeds and oil. Historians believe that the original homeland of the sesame seed is the Indian subcontinent. The seed has been called the “Queen of oilseed crops” because of the high yield of oil and the quality of seed, oil, and meal. Sesame oil is a non-drying oil, highly stable, rarely turning rancid in hot climates. Sesame is an herbaceous annual and tropical plant which reaches a height of 1-2 meters. The leaves have varied shapes; they are either oval or lanceolate and fluffy on both sides. The flowers are either violet or white and at their end, one can find pods of 3cm containing many seeds. These are small and have a length of 3.5 mm. Their color varies from yellowish white to red, brown, or black.^[4]

Taxonomical Classification:

• Kingdom  : Plantae
• Division   : Angiosperms
• Order       : Lamiales
• Family     : Pedaliaceae
• Genus       : Sesamum
• Species    : S. indicum.

Chemical constituents: Lignans and lignan glycosides present in sesame appear to be the important functional components. The main sesame lignans are sesamin and sesamolin which are found in sesame oil. Carbohydrates in sesame seeds are composed of 3.2% glucose, 2.6% fructose and 0.2% sucrose and the remaining quantities seem to be dietary fibers. Sesame is rich in sulfur containing amino acids and limited in lysine and contains significant amounts of oxalic (2.5%) and phytic acids (5%). The plant needs full sun and well-drained soil. Sesame has been cultivated for its edible seed for over 5,000 years and is still widely grown in tropical and warm temperate zones.The seed contains about 21.5% protein, 60.8?t, 8.9?rbohydrate, 3.4% ash. It is a rich source of unsaturated fatty acids, calcium, vitamins A, B and E. The calcium is absorbed well by the body, making sesame an excellent dietary source. Sesame seed has lecithin which is effective for dermatitis and dry skin. A poultice of sesame seeds can be applied externally with beneficial results over ulcers, burns and scalps. Crushed leaves of sesame are beneficial in treatment of dandruff. It is said to prevent premature greying of hair and promote their growth. Research suggests that sesame oil may have potential as a cancer fighter. It contains large amounts of linoleate in triglyceride form which selectively inhibited malignant melanoma growth.

Sesame seeds are valuable in treating respiratory disorders like preventing airway spasm in asthma, pneumonia, acute and chronic bronchitis. Sesame seeds are a good source of magnesium which supports respiratory health. Black sesame seeds, as rich source of iron, are valuable in treating anemia. Sesame oil is known to reduce cholesterol due to high polyunsaturated fat content in oil. Also called healing oil due to its ability to unblock arteries. Sesame seeds are a very good source of copper which is known for its use in reducing some of the pain and swelling of rheumatoid arthritis. Sesame seeds are a good source of calcium which helps prevent bone loss that can occur because of menopause. Another reason for older men to make zinc rich foods such as sesame seeds as a regular part of their healthy way of eating will increase bone mineral density Although osteoporosis is often found in postmenopausal women, it is also a potential problem for older men. Sesame seeds are useful in treating dysentery and diarrhea. Its centuries old reputation as a laxative perish to this day. It is also used to treat blurred vision, dizziness, and headache. The oil fraction shows a remarkable stability to oxidation. This could be attributed to endogenous antioxidants (lignans) together with tocopherols. The stability is more pronounced in case of unsaponifiable matter extracted from roasted sesame seeds due to synergistic role.^[4]

In-vitro Analysis: "In-vitro," is a Latin word that means "within the glass." Therefore, the studies which are done outside the living organism, inside glass (test tubes or Petri dishes) are known as in vitro studies. It is the experiment or observations done on the tissue outside of the living organism in a controlled environment, usually using Petri dishes and test tubes. Most experiments in cellular biology are done through in vitro studies and are not conducted in the organism's natural environment or inside a living organism. This results in the limited success of the experiments in simulating the actual conditions inside an organism and makes its outcome less precise.

The term In-vitro is used in cell biology to explain the techniques which are performed in a controlled environment outside a living cell or organism. In in vitro experiments, researchers optimize the conditions very similar to cellular conditions to study the actual activities. However, in vitro experiments have less success due to the inability to provide the precise cellular conditions of the cells or the organisms under laboratory conditions. In in vitro processes, conditions are artificial, and they are reconstructions of in vivo environments. Artificial conditions are formed by mixing the necessary components and reagents under controlled conditions inside a glassware in the laboratory.^[9]

Example: Examples of in-vitro studies include: the isolation, growth and identification of cells derived  from multicellular  organisms (in cell or tissue   culture);   subcellular   components  (e.g. mitochondria or ribosomes); cellular (e.g. wheat germ or reticulocyte extracts); purified molecules (such as proteins, DNA, or RNA); and the commercial production of antibiotics and other pharmaceutical products. Viruses, which only replicate in living cells, are studied in the laboratory in cell or tissue culture.

Applications of in-vitro studies in pharmacology

• In-vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells.
• In-vitro (literally 'in glass') testing methods are employed primarily: to identify potentially hazardous chemicals to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals, and food additives.
• In-vitro toxicity testing methods can be more useful and cost-effective than toxicology studies in living animals (which are termed in vivo or "in life" methods).
• Cell viability and Cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals. This in vitro test evaluates the potential of the materials or their extracts to cause damage to cells in culture. (Useful in evaluating the toxicity or irritancy potential of materials and chemicals.)
• Cell cultures are suitable test systems for the determination of cytotoxic reactions such as changes in cell cycle, inhibition of cell division, and cell death. (Caused by eluates or extracts of products either natural or processed.)

Advantages of in-vitro testing:

• In vitro methods reduce the use of mice at the antibody-production stage (but can use mice as a source of feeder cells when antibody generation is under way).
• In vitro methods are usually the methods of choice for large-scale production by the pharmaceutical industry because of the ease of culture for production, compared with use of animals, and because of economic considerations.
• In vitro methods avoid the need to submit animal protocols to IACUCS.
• In vitro methods avoid or decrease the need for laboratory personnel experienced in animal handling.
• In vitro methods using semipermeable-membrane-based systems produce mAb in concentrations often as high as those found in ascitic fluid and are free of mouse ascitic fluid contaminants.

Disadvantages of in-vitro testing:

• Some hybridomas do not grow well in culture or are lost in culture.
• In vitro methods generally require the use of FBS, which limits some antibody uses. The use of in vitro methods for maybe production generally requires the use of FBS, which is a concern from the animal-welfare perspective.
• The loss of proper glycosylation of the antibody (in contrast with in vivo production) might make the antibody product unsuitable for in vivo experiments because of increased immunogenicity, reduced binding affinity, changes in biologic functions, or accelerated clearance in vivo.^[9]

Experimental work:

Materials and methods:

Plant material:

1. Withania Somnifera:

Withania somnifera roots were purchased form a reputed vendor of herbal material (Dr. Bhoite’s Charak Ayurved) in Satara.

Preparation of plant extract: The purchased Withania somnifera roots were cut into small pieces. These roots were air dried thoroughly under shade (at room temp.) for 2 to 3 weeks to avoid direct loss of phytoconstituents from sunlight. The shade dried materials were powered using the pulverized and sieved up to 80 meshes. It was then homogenized to fine powder and stored in airtight bottle for future use.

Hot extraction method:

A total of 10 gm of powdered sample was taken and mixed with 50 ml distilled water in round bottom flask and gentle refluxed for one and half hour separately. The residue was removed by filtration through Whatmann no.1 filter paper and the aqueous extract was concentrated used on rotatory evaporator for just as long as was required to remove the solvent and re-dissolved the residue in small of water.^[2]

2. Sesamum Indicum:

Sesamum Indicum (black sesame seed) was purchased from local market of Satara Preparation of seed extract: 10 gm of a seeds of each of the selected varieties were finely powdered and socked for 24 hours minimum in 50 ml of methanol, chloroform, and distilled water each.

Next day all the extracts were filtered using vacuum pump through Whatmann no.1 filter paper. Methanol and chloroform residues were taken to dryness and then residues were dissolved in 2N HCl.^[3]

Anti-inflammatory property of test drugs is screened by:

1. Inhibition of protein denaturation (using egg albumin)
2. Membrane stabilization test (using Human red blood cells)
3. Inhibition of 5- Lipoxygenase
4. T-cell proliferations
5. Cox – enzyme inhibition
6. DPPH radical scavenging assay
7. Hydroxy radical assay.

The Inhibition of Protein Denaturation:

Concentration : Chosen for study 100,200,300,400,500µg/ml

Standard          : Diclofenac sodium

Chemical: Phosphate buffer saline pH7.5, egg albumin serum.

Instrument required: Incubator, UV spectrophotometer (660nm)

The following three solutions were prepared for the test.

Test solution: 5ml test solution consist of 0.2 ml egg albumin and 2.8 ml of phosphate buffer saline 2ml in various concentrations of extracts (100,200,300,400,500µg/ml).

Test control solution: 5ml test solution consists of 0.2 ml egg albumin and 2.8 ml of phosphate buffer saline and 2ml distilled water.

Standard solution: 5ml standard solution consists of 0.2 ml egg albumin and 2.8 ml of phosphate buffer saline and Aspirin 100µg/ml.

Procedure:

The pH of the above solution adjusted 6.4 using small amount of 1NHCL.the sample were incubated at 37°C for 20min.and heated 70°C for 5min. After cooling their absorbance was measured 660nm using pure blank Aspirin (standard drug) was reference drug and treated as such for the determination of absorbance. The percentage inhibition of protein denaturation was calculated as,^[10]

Membrane Stabilization Test:

Concentration : Chosen for study 100,200,300,400,500µg/ml

Standard          : Diclofenac sodium

Chemical        : Phosphate buffer saline pH 7.5, Hyposaline, Isosaline.

Instrument required    : Incubator, UV spectrophotometer (560nm), Centrifuge.

The following three solutions were prepared for the test.

Preparation of red blood cells (RBC) suspension

Fresh whole human blood (10ML) was collected and transferred to the Heparin zed centrifuged tubes. The tubes were centrifuge 2500 rpm for 10 min. and were washed three times with equal volume of normal saline. The volume of the blood was measured and reconstituted as 10% v/v suspension with normal saline.

Heat induced hemolysis.

The reaction mixture (2ml) consisted of 1ml of test drug solution and 1ml of 10% RBC suspension, instead of drug only saline was added to the control test tube. Diclofenac was taken as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in a water bath at 56°C for 30min.At the end of the incubation, the tubes were cooled under running tap water. The reaction mixture was centrifuge at 2500rpm for 10 min. and the absorbance of the supernatants was taken at 560nm. Percent membrane stabilization activity was calculated by the formula mentioned above.^[7]

RESULTS AND DISCUSSION:

Inhibition of albumin denaturation:

Protein denaturation is a process in which proteins lose their tertiary and secondary structure by application of external stress or compound such as strong acid or base a concentrated inorganic salt and organic solvent or heat. Most biological proteins lose their biological functions when denatured. Denaturation of the proteins is a well-documented cause of inflammation. As part of the investigation on the mechanisms of the anti-inflammation activity ability of plant extract to inhibit protein denaturation was studied.it was effective in inhibiting it induced albumin denaturation. Maximum inhibition of 55.55% was absorbed at 500µg/ml. aspirin is a standard inflammation drug showed the maximum inhibition 62.71% at the concentration of 500µg/ml compared with control.

Membrane Stabilization: From the result of present study, the root extract of Withania somnifera and seeds of sesamum indicum was subjected to In-vitro Anti-inflammatory activity in various concentration i.e.,100,200.300.400,500µg/ml and the percentage stabilization of a different extracts by HRBC membrane stabilization method shown in following table. The extract of anti-inflammatory activity at all the doses tested compared to control. The percentage membrane stabilization shows increases with the increases in concentration of the extract.

DISCUSSION: Different concentration of its extract has been taken for assessing the in-vitro anti- inflammatory activity. Roots of Withania somnifera and seeds od sesamum indicum exhibited membrane stabilization effect by inhibiting hypotonicity induced lysis of erythrocyte membrane. The erythrocyte membrane is analogues to lysosomal membrane and its stabilization implies that extract may stabilize lysosomal membrane. Stabilization of lysosomal membrane is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil such as bactericidal enzyme and proteases which cause further tissue inflammation and damage upon extracellular release. Denaturation of a protein is one of the causes of anti-inflammatory that is documented production of auto antigen in certain anti-inflammatory diseases may be due to denaturation of protein. Agents that can prevent protein denaturation therefore would be worthwhile for anti- inflammatory drug development. From the present study it can be stated that the extract of withania somnifera and sesamum indicum is capable of controlling the production of autoantigen and they’re by inhibit denaturation of protein and its effect was compared with the standard drug.

CONCLUSION: The in-vitro study on roots of Withania somnifera and seeds of Sesamum indicum show the presence of significant anti- inflammatory activity due to the presence of active principles. Methanol extract of Withania somnifera is showed better anti-inflammatory activity than the methanol and water extracts. This study gives on idea that the compound of the plant Withania somnifera and seeds of Enicostemma littorale cane be used as lead compound for designing a potent anti-inflammatory drug which can be used for treatment of various diseases such as cancer, neurological disorder.

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9. H. Gerhard Vogel. Drug Discovery and Evaluation. March 2002.
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