The Antihypertensive Effect from Aqueous Extract of Oxalis corniculata by In Vitro Antihypertensive Activity Assay

 

H. M. Moyeenudin*, S. Vijayalakshmi

School of Hotel and Catering Management, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai-600117, Tamil Nadu, India

*Corresponding Author E-mail: moyeenudin@velsuniv.org

 

ABSTRACT:

The fundamental element for hyper tension is stress and food habits which are mediated for causing hyper tension by the consumption of food loaded with high sodium, fats and also with a stressful life style, the poor intake of vegetables and fruits are the another factor that leads to hyper tension. Establishing a good food habits with physical exercises will be an alternate treatment to control and heal this condition. There are many herbs has their ability on healing and reducing hypertension symptoms. In this circumstances, the findings from In Vitro antihypertensive activity assay conducted on Oxalis corniculate indicates that the consumption of this leaves will lead to antihypertensive that was proved by the ability of Oxalis corniculate in inhibiting Angiotensin Converting Enzyme. Thus, the aim of the study is focused to prepare aqueous extract of Oxalis corniculata, to screen its phytochemical contents of these leaves, to analyze the inorganic elements present in Oxalis corniculate and to find out the property of anti-hypertensive by In Vitro Antihypertensive Activity Assay. The findings of this study shows that the aqueous extract was found to have the ACE inhibitory activity. Hence it is concluded that Oxalis corniculata could be included in the diet on a regular basis to prevent and manage hyper tension.

 

KEYWORDS: Hypertension, Puliyarai, Oxalis corniculata, medicinal herb.

 

 


1.      INTRODUCTION:

The major prevalent cardiovascular risk through hypertension in developing countries[1] food is considered as one of the major source. It is noticed that the foods which are loaded with sodium, fat, and calories acts as a primary source of hypertension[2]. In recent days people prefer to have foods which are easily prepared and served fast, and the main reasons for most of the health implications are food habits and the type of food consumed will leads to unhealthy life style. This study was done in order to find the food which is suitable to reduce hypertension. Herbs Puliyarai has the tendency to control the hypertension and bring it to normal[3], Puliyarai herb can be mixed or consumed with any food and it is suitable for all age groups.

 

 

The common reasons for most of the coronary diseases is hypertension which also affects the other organs of the body and leads to stroke, kidney disease, and poor vision[4][5]. Although stress, depression and work pressure can induce the hypertension, foods habits are the major treat in present scenario with growth of junk foods in market and also with the fast availability. There are various carried to cure this disease and there is no any permanent cure as this is related with daily food habits, avoiding sodium in food can control temporarily because it is also induced by depression, stress and work pressure. Daily exercise and low calorie food with less fat is also an alternative method. Addition of Herb Puliyarai (Oxalis corniculata) with the daily food may result in curing this disease, since it has the tendency to control the hypertension and bring it to normal.

 

1.1 Oxalis corniculata (Puliyarai):

Puliyarai (Oxalis corniculata) has the origin from South Europe and Hawaii. This herb is also found in tropical regions of Canada and North America as well as in India, Pakistan, Afghanistan, China, Indonesia and Taiwan. This perennial herb belongs to oxalidacea family consist of thin and slender primary roots. Puliyarai is an herb grows in gardens and in favorable temperatures for creepers as this weed has creeping tendency often found in dense forest, unused lands, near roadside fences. The branch of this herb always lie on the ground because the branches are thin, it aid in forming a new plant and finally become a cluster of creeper. Puliyarai herb has the tendency to grow up to height of 30 centimeter by creeping on wood, The length of this herbs branches may grow up to 20 inches cm in length the branches looks fibrous. The leaf of this herb looks like a heart shape and there is three leafs combined in each branch petiole. The size of these Leaves will be around 5-11 mm long 7- 18 mm broad. There are yellow color flowers found in this herb puliyarai mostly in between the combined leaflets[6].

 

Botanical Description and Distribution

Botanical name

Oxalis corniculata

 

Genus

Oxalis

Family

Oxalidacea

Common Name

Creeping wood sorrel

Species

Kingdom

Vernacular Names

O. corniculata

Plantae

Tamil: Puliyarai  English: Indian Sorrel Hindi: Amrul Sanskrit: Amlalonika; Amlika

Habit

Herb

Traditional Uses

Medicinal Plant

Figure 1: Puliayarai Keerai

 

1.2 Aim of the Study:

The major objective and aim of this research is to determine the presence of antihypertensive property in puliyarai (Oxalis corniculata) by preparing an aqueous extract of puliyarai (Oxalis corniculata), in order to screen its phytochemical contents of these leaves, to analyze the inorganic elements present in puliyarai (Oxalis corniculata) by In Vitro Antihypertensive Activity Assay.

 

1.3 Collection of plant:

The Puliyarai herbis collected in March 2018 from Thiruvayaru, Thanjavur district, Tamil Nadu, India. The Puliyarai keeraileaves are washed to remove the impurities and other elements with the help distilled water several times. The leaves of puliyarai herb is placed on a plain tissue paper and allowed to dry for around 15 days in room temperature for about in order to prepare a fine powder of this herb puliyarai it is grinded nicely. The powder obtained by grinding is used for the research.

 

 

1.4 Preparation of plant extract:

The Puliyarai keerai leaves powder around 3 gram is sent in to various conical glass containers measured up to 300ml. The glass conical containers are filled with 50ml of water. The glass conical container need to be shake vigorously for around 40 minutes using free hand then later it has to be filtered after one day or 24 hours, the extracts are filtered using Whatman filter paper No.1 and the filtrate extracted is used for further study.

 

1.5 Phytochemical screening:

Chemical tests were carried out on the extract using standard procedures to identify the constituents as described by Sofowara (1993), Trease and Evans (1989) and Harborne (1973 and 1984)[7] [8] [9].

 

1.6   Assessment for Saponin:

About 2 ml of sample is boiled in 20 ml of distilled water in a water bath and filtered. 10ml of the filtrate is mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth. The frothing is mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion.

 

1.7   Assessment for Flavonoids:

5 ml of dilute ammonia solution were added to a portion of the aqueous filtrate of each plant extract followed by addition of concentrated H2S04. A yellow color obtained from the puliyarai herb that shows the occurrence of flavonoids. The yellow color formed was vanished after some time during stable.

 

1.8   Assessment for Steriods:

The 4 ml of acetic anhydride is mixed with the puliyarai extract 2 ml of each sample along with 4 ml H2SO4. The colour turned into purple to blue and green in some samples which indicates the existence of steroids.

 

1.9Assessment for Terpenoids (Salkowski test):

The addition of 4 ml puliyarai extract is added with 2 ml of chloroform along with 3ml of concentrated sulfuric acid is cautiously mixed to obtain a line of layer during this analysis. There is a reddish brown colouration of the line is obtained in results which indicates the existence of terpenoids in puliyarai Extract.

 

1.10Assessment for alkaloids:

Mayer’s test: From the corner of test tube to 2 ml of puliyarai extract few or two drops of Mayer’s reagent is mixed. There is a white or creamy hasty mixture is obtained which indicates the assessment for alkaloids is positive.

 

1.11Assessment for carbohydrates:

Benedict’s test is conducted with 1 ml of the filtrate obtained from puliyarai along with 1 ml of Benedict’s reagent. This combination was heated on boiling water bath for 3 min. A characteristic hasty red colored is obtained that indicates the presence of sugar.

 

1.12 Assessment for Proteins:

The 2ml of Puliyarai extract was taken and mixed with 2 ml of 40% Sodium hydroxide is mixed slowly from the corner of test tubes as few drops. The appearance of copper sulphate in to violet or pink colour shows that there is an existence of protein.

 

1.13Assessment for Polyphenols:

The 8 ml of ethanol is mixed with the 2 ml of each puliyarai extracts and the final solution is transferred in test tubes and heated with a water bath for around 20 minutes. Finally few drops of freshly prepared ferric cyanide solution were added to the puliyarai extract solution. In order to obtain a blue green colour, that is showing the presence of polyphenols.

 

1.1.1 Qualitative analysis of inorganic elements:

The drug material was obtained and a test conducted with 6:2 ratio of Nitric acid and Hydrochloric acid for 40 to 55 minutes. Gradually the blend was filtered and the final solution extracted through the filtration is used carry out further analysis[10].

 

1.1.2 Calcium:

The diluted form NH40H one drop is used for this test and it is added with the saturated form of 10ml C2H8N2O4 solution along with the filtrate obtained for this study. There is a white hasty form of calcium oxalate is obtained that is soluble with HC1but insoluble inCH3COOH.

 

1.1.3 Magnesium:

The hasty CaC2O4was removed through the filtration of the extracted solution then it is allowed to warm then with the assistance of burner it has been heated finally allowed to room temperature by cooling it, this extractedNa3PO4 is added with dilutedNH3. There is a crystalline white precipitate is obtained finally.

 

1.1.4 Sodium:

A small amount ofC8H12MgO10U is added as a testing agent with the 4 ml of the extracted final solution and it is shaken well, later allowed to rest for 20 minutes. There is a pale yellow crystalline precipitate of sodium C8H12MgO10U is obtained.

 

1.1.5 Potassium:

The three drops of Na3Co (NO2)6 solution is mixed with 4ml of the Extracted solution. There is a final hasty yellow of K3 [Co (NO2)6] is obtained.

 

1.1.6 Iron:

A two drops of K4Fe (CN) 6·3H2O is mixed with 6ml of the extracted final solution. There is a dark blue colour tone is obtained in the end shows there is an iron content in the present extract.

 

 

1.1.7 Sulphate:

To identify the presence of sulphate, a 6ml of puliyarai extract solution is taken and added with, Pb(C2H3O2)2. There is a white colour precipitate is obtained, which is soluble in NaOH.

 

1.1.8 Phosphate:

The 6ml of puliyarai extract was prepared added with two drops of HNO3 and few drops of (NH4)6Mo7O24 solution were added and it is gradually heated for minimum 5to 8 minutes and rest it to get cool. There is a yellow crystalline precipitate of ammonium MoO4-2 is obtained.

 

1.1.9 Chloride:

The 6ml of Pb (C2H3O2)2 solution is mixed and blended with the 8 ml of puliyarai filtrate. There is a white precipitate formed after the blend, which is observed as soluble.

 

1.1.10 Carbonate:

The 6 ml of puliyarai extract is used as a testing solution and it is added with dilute acid and then there is Carbon dioxide is liberated from the puliyarai extract finally.

 

1.1.11 Nitrates:

The ferrous sulfate is added with 6ml of Puliyarai Extract. There is no brown colour is obtained in the end, although when H2SO4 acid was added (slowly from the side of the test tube), a brown colored ring was produced at the junction of two liquids.

 

2. In Vitro Antihypertensive Activity Assay:

The Antihypertensive activity through In Vitro method was signified with the capability of Puliyarai keerai in order identify the ability of Angiotensin Converting Enzyme (ACE). ACE inhibition action is determined using UV spectrophotometer established through the amount of formation of C9H9NO3 from hippuryl-L-histidine -L-2-Amino-4-methylpentanoic acid (HHL) mobilized by Angiotensin Converting Enzyme[11] The little amount of ACE (50 mU/mL) is produced with 80 micro liter of puliyarai extract (Different concentration 30, 60, 80 mg/ml) at 33°C temperature of for 15 minutes, later 200 µL of the extracted solution is added with (8.3 Mm of HHL in 50 mM Na2[B4O5(OH)4]·8H2O a shield holding 0.5 M sodium chloride, pH 8.3) is mixed with the blend for about 40 minutes at 33°C. This gives a response on stoppage of mixing 300 µL of 1.0 M hydrogen chloride. The addition of 1 mLC4H8O2 is mixed with the blend and centralized for 15 minutes. The partial amount of 0.3 mL from the top layer is composed and sent into a test tube and vaporized using vacuity form. The C9H9NO3 that is obtained is mixed in 2 mL of distilled water and the absorbance was determined with the spectrophotometrically at 348 nm.C9H15NO3S (Different concentration 20, 40, 60/ml) is used as normal with a concentration of 4.5 ng/ mL. The rate of ACE inhibition is measured by the below mentioned formula.

 

              Control Absorption–Sample Absorption

% Inhibition=--------------------------------------×100

                            Control Absorption

 

 

3. RESULTS AND DISCUSSION:

The Puliyarai herb has nutritional properties and consumed with daily diet, which has protein, carbohydrate, fats and oils minerals, vitamins and water required for the growth and progress of man and animals. There are phytochemical available in this puliyarai extract otherwise known as plant chemicals. The term “Phyto” is obtained from Greekword for plant. Phytochemicals are categorized as fundamental or ancillary elements, in regard with plant growth. The fundamental growth and development of plants that contains the collective saccharides, amino acids, proteins, which has the aromatic heterocyclic organic compound of biopolymers with chlorophyll’s pigments. The ancillary growth of a plant acts as a main part in the continuous existence of a plant with atmosphere and the surroundings. The ability of cross breed with ordinary protection structure compared to raiders and infections, etc., are considered as an ancillary cases in regard with the growth[7].

 

The ancillary intermediate end of metabolism is produced which is an imperative characteristic for the food plants like (palatability, pigment, flavor, etc.). In addition the consumption of herb also loaded with many ancillary plant metabolites such as active water soluble compounds, alkaloids, tannins, saponins, steroids, anthocyanins, terpenoids, rotenoids etc. acts as a drug inviable applications, dye, flavour, fragrance, insecticide, etc. Such fine chemicals are extracted and purified from puliyarai solution[12].

 

The presence of catechins of polyphenolic compounds with bioflavonoids such as quercetin, is recognized by possessions of free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action. The polyphenolic combinations of 15 carbon composites that is usually disseminated all over the plant realm. There are few phytoestrogens comprehensively assist in controlling pesticides. This compounds has a major part in resisting disease and few polyphenolic compounds like quercetin and plant pigment are useful in supporting health of a human by anti-inflammatory, antihistaminic and antiviral agents which aided by various properties of the herb puliyarai[13].The polyphenolic compounds shows inhibitory properties in contrast to numerous viruses. There are several studies has recognized the efficacy of polyphenolic compounds, namely glycyrrhizin and chrysin[14]. polyphenolic compounds have been represented to as environment’s natural reaction transformers, since the natural capacity to change the body’s response towards the sensitivities and viral infections which exhibits its anti-allergic, anti-inflammatory and anti-microbial properties[14].

 

The nitrogenous organic compounds like alkaloids has a traditional wide range of antibacterial properties and it is also acts as a pain reliever. These compounds are vital in medicine and establish the maximum valued medicine. The compounds such as solasodium is specified as an initial chemical that is used in manufacturing the steroidal drugs. The unadulterated remote polyphenolic compounds has weird results acts as an initial curative agents due to its pain relief properties, assist in treating bladder spasm and also preventing the growth of bacteria and kills. This shows the biological activity when managed in animals[13].

 

The qualitative analysis of aqueous extract of Puliyarai keerai leaves showed that tannin, flavonoids, steroids, terpenoids, carbohydrate, polyphenol and alkaloids present in both extract (Table 1).

 

 

Table 1 Qualitative phytochemical screening of Puliyarai keerai leaf extract

S. No.

Name of the Test

Aqueous extract

1.

Tannin

++

4.

Flavonoids

+

5.

Steroids

+

6.

Terpenoids

++

8.

Alkaloids

++

9.

Carbohydrate

+

10.

Protein

+

12.

Polyphenol

++

(+) moderately present (--) Indicates absent (++) Indicates Present

 

 

There are some minerals present in body tissues as an inorganic substances with fluids and their existence is essential for the upkeep of definite physicochemical processes in a human survival. The minerals are constituted by chemicals and aid in growth and survival by several ways. Even if there no production of energy, it carryout a great responsibility in playing multiple roles in human survival[15]. Each system of survival process need these inorganic compounds to run a regular life routine[16]. The prominence of these compounds in human nutrition has been unanimously accepted[17]. The insufficiencies in nutrition is due to food borne diseases of multiple reasons[18]. In the present study to investigate the inorganic elements in Puliyarai keerai leaf and represent in table 2

 

 

 

 

Table 2 Qualitative elemental analysis of Puliyarai keerai leaf extract

Inorganic Elements

Puliyarai keerai leaf

Calcium

+

Magnesium

++

Sodium

+

Potassium

++

Iron

++

Sulphate

+

Phosphate

+

Chloride

+

Nitrate

++

(++ indicates high concentration, + indicates presences and - Indicates absence)

 

3.1Angiotensin Converting Enzyme:

The primary threat factor of deaths throughout the world is high blood pressure. It is observed that there are more than 16 million deaths a year due to cardiovascular diseases, in comparison with that a 9.4 million are lead though hypertension according to the World Health Organization[1]. The communal threat is concerned with unhealthy diet, too much of sodium in food and along with alcohol intake, There are other factors like continuous worries, anxieties and obesity lead to hypertension. The pervasiveness of high blood pressure is observed from the majority due to ageing among the growing population, this high blood pressure has the character of “silent killer” since there is no any specific symptoms in its initial stage only seen during the chronic stages. As it is mostly comes without any signs and symptoms the high blood pressure gives substantial impairment to the human metabolism by harming the organs of body. In order to diminish the high blood pressure threat and which is linked with other health issues a specific life style must be followed with a good dietary practices and with regular aerobic exercises. Nevertheless, the medication is proved to be necessary for those whose way of life alterations seems unsuccessful and insufficient[19].

 

The high blood pressure medication has commonly starts through reducing sodium intake in dietary practices with an enormous collection of recent medicines. The drug which increase the production of waste fluids and exerts through urine are diuretic also by blocking adrenaline through beta blockers, that is also added with calcium channel blockers are used in treating high blood pressure. Even though the combination of these drugs are established as exceptionally effective in treating high blood pressure, but their efficiency are repeatedly spoiled with their linked harmful side effects on regular consumption. The Renin-Angiotensin-Aldosterone System (RAAS) assists in regulating arterial blood pressure which is the major and most substantial process of coronary system as it is targeted towards the chasing the objects cause high blood pressure in order to regulates the blood pressure through Renin-Angiotensin-Aldosterone System also by means of angiotensin release with the body that acts as electrolyte gratified via aldosterone release, the idea of preventing ACE has become a standard and operative healing method in treating high blood pressure and diseases in relation with heart[20].

 

The primary direct ACE blocking through captopril, it is measured as a development in managing hypertension and also regarded as an early example of a structure-based drug design. The supplementary artificial ACE inhibitors are also used widely for clinical purpose for the medication of high blood pressure, which is comprised with enalapril, alcacepril, and lisinopril, in association of early antihypertensive medicine, the artificial ACE inhibitors are also accompanied with some side effects like angioedema and dry cough. The current trend in anti-hypertensive drug research is finding potential ACE inhibitors from natural products that mimic synthetic ACE inhibitors and provide health benefits without adverse effects[21]. There have been several bioactive compound sextracted from plants which were found to possess in vitro ACE inhibitory activity such as favonoids, hydrolasable tannins, phenylpropanes, xanthones, fatty acids, terpenoids, alkaloids, proanthocyanidins, oligosaccharides, and peptide amino acids [22].

 

In the present study to investigate the Angiotensin Converting Enzyme (ACE) inhibiting (Antihypertensive) Activity of Puliyarai keerai extract and represent in table 3. The results shows dose dependent inhibition of ACE. The lowest dose shows 21.30% and highest dose shows 85.6% while standard shows 23.44 and 94.67 for lowest and highest doses. The Angiotensin Converting Enzyme (ACE) inhibiting (Antihypertensive) Activity of Puliyarai keerai extract nearest to the standard.

 

Table 3: Angiotensin Converting Enzyme (ACE) inhibiting (Antihypertensive) Activity of Puliyarai keerai extract

Samples

Concentrations (mg/ml)

10

20

30

40

50

Extract (% of inhibition)

21.30

43.50

59.62

75.44

85.64

Standard as Captopril acid (% of inhibition)

23.44

48.50

64.76

79.20

94.67

 

Fig 2: Angiotensin Converting Enzyme (ACE) inhibiting (Antihypertensive) Activity of Puliyarai keerai extract

4. CONCLUSION:

The preliminary evaluation of the Puliyarai keerai aqueous extract for antihypertensive characteristics of Oxalis corniculata through In Vitro antihypertensive activity assay indicated that the plant is a potential source of bioactive compounds and elements that can inhibit the activity of ACE. The aqueous extract was found to have the ACE inhibitory activity. The anti-hypertensive activities of Puliyarai keerai extract observed in this study may have resulted from one of the mechanisms in inhibiting ACE activity. This study establish the antihypertensive effect of Puliyarai keerai and the foods could be prepared by the addition of this herb and that can be followed in regular diet in order to treat the hypertension with the day to day food.

 

5. REFERENCES:

1.     WHO World Health Organization. 2016. [Internet source] cardiovascular diseases (CVDs). Retrieved.2016 from WHO website http://www.who.int/mediacentre/factsheets/fs317/en/.

2.     Bazzano, Lydia. A, et al. “Dietary approaches to prevent hypertension” Current hypertension reports volume15, Issue 6 (2013): Pages 694-702.

3.     K. J. Achola, et al, Pharmacological Activity of Oxalis corniculata, International Journal of Pharmacognosy Volume 33, 1995 - Issue 3, Sep 2008, Pages 247-249.

4.     Jan A. Staessen et al.“Blood Pressure Measurement Anno 2016” American Journal of Hypertension, Volume 30, Issue 5, 1 May 2017, Pages 453–463,https://doi.org/10.1093/ajh/hpw148

5.     Wu, Chen-Yi et al. “High Blood Pressure and All-Cause and Cardiovascular Disease Mortalities in Community-Dwelling Older Adults” Medicine volume. 94, issue 47 (2015): e2160.

6.     https://en.wikipedia.org/wiki/Oxalis_corniculata [Internet Source]

7.     Sofowara A, Medicinal plants and Traditional medicine in Africa. Spectrum Books Ltd, Ibadan, Nigeria. (1993). page. 289.

8.     Trease GE, Evans WC. Pharmacognosy. 11th Edn. Brailliar Tiridel Can. Macmillan publishers1989.

9.     Harborne JB Phytochemical methods, London. Chapman and Hall, Ltd. (1973). pp. 49-188.

10.   Khandelwal, K.R Practical Pharmacognosy (16th ed.,) Macmillan publishers Nirali, Prakashan, Pune.(2006).Pages 98-106.

11.   Chaudhary SK., et al. A Potential Angiotensin Converting Enzyme (ACE) Inhibitor Useful in Hypertension. Indian J Nat Prod Res. 2014; 5: 83–87.

12.   Das K, et al, Techniques for evaluation of medicinal plant products as antimicrobial agent: Current methods and future trends. Journal of Medicinal Plants Research 2010; 4(2): Pages 104-111.

13.   Okwu D.E. Phytochemical and vitamin content of indigenous spices of south eastern Nigeria. J. Sustain Adric. Environ. Volume 6 Issue 1: (2004). Pages 30-37.

14.   Duraipandiyan V,M. et. al. Antimicrobial activity of some ethnomedicinal plants. Asian Journal of Microbiology. Volume 5: (2006). Pages 334-337.

15.   Eruvbetine D Canine Nutrition and Health. A paper presented atthe seminar organized by Kensington Pharmaceuticals Nig. Ltd. Lagos on August 21, 2003.

16.   Ozcan M . Mineral Contents of some Plants used as condiments in Turkey. Journal of Food Chemistry Volume 84: Pages: 437-440.

17.   Darby WJ et al. Trace elements in human health and disease, (Academic Press, New York, San Francisco, London) (1976). Pages 1: 17.

18.   Gordon RF Poultry Diseases. The English Language Book Society and Bailliere Tindall, London.(1977).

19.   Bazzano LA, et al. Hypertension. In Reference Module in Biomedical Sciences. International Encyclopedia of Public Health, United States: Elsevier. 2008.pages.123-33.

20.   Bavishi C, et al. Renin Angiotensin Aldosterone System Inhibitors in Hypertension: Is There Evidence for Benefit Independent of Blood Pressure Reduction? From Progress in Cardiovascular Diseases 2016. http://www.onlinepcd.com/article/S0033-0620(16)30111-6/references.

21.   Antonios TF, MacGregor GA. Angiotensin converting enzyme inhibitors in hypertension: Potential problems. Journal of Hypertension.1995; Volume 13(Supple 3):S11-16.

22.   Braga FC, et al. Angiotensin-converting Enzyme Inhibition by Brazilian Plants. Fitoterapia. 2007; Volume 78 Issue 5 Pages: 353-8.

 

 

 

 

 

Received on 20.02.2019          Modified on 02.03.2019

Accepted on 06.04.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(6): 2981-2986.

DOI: 10.5958/0974-360X.2019.00504.3