Evaluation of diuretic,anti pyretic and anti acne activities of unriped pulp extracts of cucumis sativus L




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EVALUATION OF DIURETIC,ANTI PYRETIC AND ANTI ACNE ACTIVITIES OF UNRIPED PULP EXTRACTS OF CUCUMIS SATIVUS L



SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO


RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BENGALURU

BY
Mr. ASHIK NAVAS .T

M. PHARM- PART I

DEPARTMENT OF PHARMACOLOGY

(2012-2013)

UNDER THE GUIDANCE OF

Dr.D. GNANASEKARAN M. Pharm., Ph.D.,

PROFESSOR,AND HOD

DEPARTMENT OF PHARMACOLOGY

BHARATHI COLLEGE OF PHARMACY, BHARATHINAGARA (K.M.DODDI)-571422











RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE – II




PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION



1.


NAME OF THE CANDIDATE

AND ADDRESS


Mr . ASHIK NAVAS.T

PERMANENT ADDRESS:

S/O JAMAL MUHAMMED .T ,

THEKKEN KUTTUR POST,TIRUR Tq,

MALAPPURAM Distri , KERALA




2


NAME OF THE INSTITUTION

BHARATHI COLLEGE OF PHARMACY,

BHARATHINAGARA, (K.M.DODDI),MADDUR TQ, MANDYA DIST-571422



3.


COURSE OF STUDY AND SUBJECT


MASTER OF PHARMACY (PHARMACOLOGY)


4.



DATE OF ADMISSION

12/DEC/2012




5.


TITLE OF THE DISSERTATION WORK:

EVALUATION OF DIURETIC, ANTI PYRETIC AND ANTI ACNE ACTIVITIES OF UNRIPED PULP EXTRACTS OF CUCUMIS SATIVUS L .





6


6.1 BRIEF RESUME OF THE INTENDED WORK;


Nature always stands as a golden mark and it has provided the natural products from plant, animal and minerals to cure all aliments of mankind (Verma and Singh, 2008). India is a home of traditional medicine systems which possesses large extent of higher plants approximately 400,000 species in the world as compared to animal’s species that are about 5-10 millions (El Saed and Al Barak, 2011). About 80% of world population utilize plant as their primary source of medicinal agents (Shafaei et al., 2011). Medicinal plants are moving from fringe to mainstream use with a greater number of people seeking remedies and health approaches free from side effects caused by synthetic chemicals (Dubey et al., 2004). The research into plants with alleged folkloric use as pain relievers, anti-inflammatory agents, should therefore, be viewed as a fruitful and logical research strategy in the search for new analgesic and anti-inflammatory drugs (Jothimanivannan et al., 2010).The universal role of plants in the treatment of diseases is established by their employment in all important systems of medicine. There are many plants which has been unexplored in the field of medicine or Science (Reddy et al., 2010). One of such plant is Cucumis sativus l . The fruit of which is mainly consumed as a vegetable. It is belonging to the family Cucurbitaceae. It is commonly called as wild cucumberThe cucumber (Cucumis sativus) is a widely cultivated plant in the gourd family Cucurbitaceae. It is a creeping vine which bears cylindrical edible fruit when ripe. There are three main varieties of cucumber: "slicing", "pickling", and "burpless". Within these varieties, several different cultivars have emerged. The cucumber is originally from the Indian subcontinent but is now grown on most continents. Many different varieties are traded on the global market . The fruit is usually consumed as a vegetable due to its good nutritional value

Cucumbers grow on vines




Scientific classification

Kingdom:

Plantae

(unranked):

Angiosperms

(unranked):

Eudicots

(unranked):

Rosids

Order:

Cucurbitales

Family:

Cucurbitaceae

Genus:

Cucumis

Species:

sativus

Binomial name Cucumis sativus L



Cucumber, with peel, raw

Nutritional value per 100 g (3.5 oz)

Energy

65 kJ (16 kcal)

Carbohydrates

3.63 g

Sugars

1.67

Dietary fiber

0.5 g

Fat

0.11 g

Protein

0.65 g

Water

95.23

Thiamine (vit. B1)

0.027 mg (2%)

Riboflavin (vit. B2)

0.033 mg (3%)

Niacin (vit. B3)

0.098 mg (1%)

Pantothenic acid (B5)

0.259 mg (5%)

Vitamin B6

0.04 mg (3%)

Folate (vit. B9)

7 μg (2%)

Vitamin C

2.8 mg (3%)

Vitamin K

16.4 μg (16%)

Calcium

16 mg (2%)

Iron

0.28 mg (2%)

Magnesium

13 mg (4%)

Manganese

0.079 mg (4%)

Phosphorus

24 mg (3%)

Potassium

147 mg (3%)

Sodium

2 mg (0%)

Zinc

0.2 mg (2%)

Fluoride

1.3 µg


DIURETIC ACTIVITY

Diuretics are the drugs which increses the flow of urine. The diuretics which are therapeutically important, produce a loss of both water and solutes. Many drugs know to cause diuretic action, act by either of the following ways.

1) They increase the renal blood flow and glomerular filteration rate. Although, such action leads to diuretic effect exerted by such drugs. All three xanthines (theobromine, caffeine and theophyllin) have diuretic properties, which may validate the traditional use of the plant a s a diuretic.

2) The other drugs increase the solute excretion in the glomerular filtrate and tubular fluid. This group includes drugs acting in two different ways. Some are called osmotic diuretics which are not electrolytes. They are freely filtered at the glomerulus and they are very little reabsorbed through the renal tubule. The other drugs inhibit sodium reabsorption from the glomerular filtrate. They increase the amount of solute excreted and thus, of water too.

3) Some plants produce diuretic effects comparable to the high ceiling diuretic furosemide.

Drug-induced diuresis is beneficial in many life threatening disease conditions such as congestive heart failure, nephritic syndrome, cirrhosis, renal failure, hypertension, and pregnancy toxaemial14. Most diuretic drugs have the adverse effect on quality of life including impotence, fatigue, and weakness15. Although most of the diuretics proved to be very effective in promoting sodium excretion, all cause potassium loss and this prompted the search for potassium sparing diuretics14,15. Hence a search for new diuretic agents that retain therapeutic efficacy and yet devoid of potassium loss is justified.



ANTI PYRETIC ACTIVITY

Today it has become essential to screen plant drugs for its efficacy Fever, also known as pyrexia or controlled hyperthermia [16], is a common medical sign characterized by an elevation of temperature above the normal range of 36.5–37.5°C (98– 100°F) due to an increase in the regulatory set-point [17]. This increase in set-point triggers increased muscle tone and shivering. Fever is one of the body's immune responses that attempts to neutralize a bacterial or viral infection [18], which releases pyrogens, causing a release of prostaglandin E2 (PGE2) which in turn causes heat-creating effects (vasoconstriction, shivering and increased metabolic activity) to match a new temperature level [18]. Fever is a symptom of many diseases worldwide, the most common cause in Africa being malaria, lobar pneumonia, typhoid, urinary tract infection, brucellosis, typhus or septicaemia and infective endocarditis [18].



ANTI ACNE ACTIVITY

Indroduction to acne vulgaris

Acne vulgaris, a chronic inflammatory disorder in adolescents consists of the pilosebaceous follicles, characterized by comedones papules, cysts,nodules and oftem scars, chiefly on face, neck etc the microorganisms involved include propionibacterium and staphylococcus epidermidis. The comedo, commonly known as ‘ black-head’, is the basic lesion in acne. It is produced by hyperkeratosis of the lining of the follicles, which retains keratin, sebum and some microorganisms. Thus acne is a disorder resulting from the action of hormones on the oil glands (sebaceous glands) of skin which leads to plugged pores and outbreak of lesions commonly called pimple or zits Propionibacterium acnes and propionibacterium granulosum are representative anerobes in the sin and they are the predominant organisms growing in the sebaceous region. The recognized case acne includes as heredity hormonal activity (puberty, menstruation and pregnancy), stress certain medication and skin irritation. Skin is the largest organ of the body it is made up of two layers outer epidermis and inner dermis. The epidermis of the skin is formed by the stratified epithelium which is made up of five layers. They are stratum corneum, stratum incidum, stratum granulosam, stratum spinosum and stratum germinativum. The layer of dermis are superficial papillary layer and deeper recticulum. The skin contains two types of glands sebaceousglands and the sweat glands. Skin provides a protective layer for organs and tissues from pathogens,heat and light. It regulates body temperature, it stores water, fat and vitamin D has touch receptors that scnse pain or pleasure. Skin is a excretory and absorption organ, it gives better appearance.



Incidence Of Acne

Acne is a tremendously common condition affecting millions of adolescents and adults. Up to 80% of teenage girls and 90% of teenage boys are afflicted with acne. Adults acne, although less common than adolescent acne, is a significant problem for 3% to 6% of adult men and 5% to 12% of adult women . Although the presence of acne does not physically impair patients, it can have a remarkable psychological effect. Thirty to fifty precent of adolescents experience psychiatric disturbance as the result of acne. Studies have shown that acne causes similar level of social, psychological, and emotional impairment as asthma and epilepsy. Studies have also shown that unemployment is grater among adults with acne than among adults without acne.



Classification of acne

Acne fulminas:it is a rare, acute from of acene, found usally in young adult males. The etiology of this condition is known although there is speculation that immune mechanisms are involved.

Acne necrotica: it is rare dermatosis of adults, consisting of crops of erythematous,follicle-based papules that become superficially necrotic, umbilicated and crusted,with subsequent healing which produces a depressed varioliform scar thin condition has been regarded as nothing more than neurotic excoriation superimposed on a bacterial folliculitis, an etiology that has been proposed for acne necrotica itself.

Chlorance: it is an acne form eruption caused by systemic poisoning by halogenated aromatic compounds. Cutaneous lesions may persist for long periods after the last exposure to the offending chemical.



Etiology and pathogenesis

Follicular keratinisation, seborrhea, and colonization of the pilosebaceous unit with propionibacterium acne are central to the development of lesions. Genetic and hormonal changes related to menstrual periods,pregnancy,birth control pills, psychological stress,certain drugs (such as steroids,testosterone, estrogen and phenytoin), high level of humidity and sweating, grasy and oily cosmetics and hair products also play a role, possibly by optimizing the follicular environment suitable for the growth of p.acne or by influencing the inflammatory response and thus the nature of the lesions.

Acene valgaris is of multifactorial origin with both intrinsic and extrinsic factor contributing to final outcome. There are four major etiological factors, i.e,,abnormal bacterial function, increased sebum production, hypercornification of pilosebaceous duct and inflammation. Propionibacterium acnes are the bacterial species which performs abnormal bacterial function by producing biolagically active substances which activate the complement system and stimulate the release of hydrolyses from neutrophils. These in turn damage the follicular wall, leading to the libaration of the contents of the follicle in to the dermis, stimulating the chemotaxis, and thus resulting the production of inflamed lesions.(21)

Development of acne

Acene is a disease that affects the skin’s oil glands. The small holes in the skin (pores) connect to oil gands under the skin. These glands make an oily substance Called sebum. The pores connect to the glands by a canal called a follicle. Inside the follicle, oil carries dead skin cells to the surface of the skin. A thin hair also grows through the follicle and out to the skin. When the follicle of a skin gland clogs up, a pimple grows.

Most pimples are found on the face, neck, back,chest,and shoulders. Acene is not a serious health threat but, it can cause scars. Sometimes,the hair,sebum, and skin cells clumb together in to a plug. The bacteria in the plug cause swelling.then when the plug starts to break down, a pimple grows.(20)

Develop ment of inflammatory lesions:

Comedogenes is Inflammatory lesions develop from comedons arisind due an abnormal pattern of kertnisation in the sebaceous follicle.comedogenesis was once thought not to be part of imflammatory process,but detailed immunihistological studies that have followed the development of inflammatory acene lesions have shown this to be the first step toward formation of an inflammatory lesions. The micro comodon is known believed to be the earliest type subclinical acene lesion.

Microcomodones develop into full comedons, which may be either open (“blackhead”) or closed (“whitehead”),and inflammatory lesions. Several different types of inflammatory lesions may arise that can be distinguish clinically.papules and pustules may be upto 5 mm in size with a raised area of erythema. Nodules and cysts are larger lesions that are associated with more several acne. Nodules may have an inflammatory area of 5mm or more and persist for several weaks. Cysts may be as large as a few centimeters in size: it is these lesions that often lead to scaring .the resolving stage of acene of an lesion is the macule,which is seen clinically as area of erythema Three stage of formation of an aceneic inflammatory lesions.(1) the pilosebaceous unit is normally producing sebum and differentiating keratinocytes,in the presence of the cutaneous flora. (2)hyperkeratinization and excess sebum provoke the clogging of the pore, (3) anaerobic bacteria proliferate,recruit inflammatory cells which release inflammatory signals (IL-1b, TNF a ,etc.)

Pathological changes

The three major components of acene vulgaris are comedones, inflammatory lesions and scars. A comedo is an impaction of horny cells in the lumen of a sebaceous follicle. Preceding this is the microcomedo, a clilically invisible lesion in which there is only minimal distension of infrainfunibular canal of sebaceous follicle,accompanied by increased retention of horny cells and a prominenet underlying granular layer.



Inflammatory lesion have traditionally been attributed to the accumulation of neutrophil with in microcomedones or comedones with subsequent rupture of the follicle and the formation of a putule in the dermis . it now appear that there is an earlier stage which involves the transmigration of lymphocyte into the wall of follicle associated with increasing spongiosis of follicular epithelium this change has been likened to an allergic contact sensitivity reaction. This is followed after 24-72 hours by the accumulation of nutrophils within the follicle,leading to its distension and subsequent repture. They may be localized loss of the granular layer in the region of eventual repture, suggesting a defect in keratinization in this regian. A perifollicular pustule develops following the repture of the comedo. Lymphocytes, plasma cell and foreign body gaint cells subsequently appear.



6.2 REVIEW OF LITERATURE:

Anti bacterial activity

Ankita Sood et al was conducted the antimicrobial activity of CS against 4 human microbial pathogens. Antimicrobial assay was performed by Agar well diffusion method. Specific concentration of seed extract was showed highest zone of inhibition against S. aureus. These pathogens were highly sensitive to the methanol extract also except E. coli (enteropathogen) and P. aeruginosa. Finally they concluded that CS seeds possess potential broad spectrum antimicrobial activity. [3-4]

Antifungal activity

Ankita Sood et al was conducted the antifungal activity of CS against two potent fungus .Finally they concluded that CS seeds possesses potential antifungal activity. [3-4]

Jony Mallik et al was performed an study on the antifungal activity of the ethanolic extracts of Cucumis sativus . The antifungal potentials of the ethanol extract of cucumis sativus Linn. (30μg/disc) were assessed against six fungus. The results (diameter of zone of inhibition) were compared with the activity of the standard drug, Griseofulvin (30μg/disc). At 80μg/disc, the ethanol extracts of Cucumis sativus Linn. [5]

Cytotoxic activity

Jony Mallik et al were performed an study on the cytotoxic activity of the ethanolic extracts of Cucumis sativus. In brine shrimp lethality bioassay, the Ethanol extract showed lethality against the brine shrimp nauplii. It showed different mortality rate at different concentrations. From the plot of percent mortality versus log concentration on the graph paper, LC50 (μg/ml) and LC90 (μg/ml) of the ethanol extract of Cucumis sativus Linn. Were deduced respectively. [5]

Antacid & Carminative activity

Swapnil Sharma et al was investigate with the aqueous extract fruit pulp of C. sativa significantly neutralized acid and showed resistance against change in pH and also illustrate good carminative potential. The extract of C. sativa, has shown to possess significant carminative and antacid property. [6]

Activity against ulcerative colitis

Patil et al was descrives after an authentic investigation with the aqueous extract of Cucumis sativus Linn. fruit in ulcerative colitis in laboratory animals. In this investigation, the aqueous extract of C. sativus L. selected for screening against experimentally induced bowel disease. The extract of C. sativa, has shown to possess significant property against ulcerative colitis. [7]

Hepatoprotective activity

H. Heidari et al was studied the effect of Cucumis sativus against cumene hydroperoxideinduced-oxidative stress. Results showed that aqueous extract of Cucumis sativus acts as ahepatoprotective and antioxidant agent against CHP-induced hepatotoxicity suggesting that antioxidants and radical scavenging components of Cucumis sativus fruit extract can easily cross the cell membrane and cope with the intracellular ROS formation. [8]

Hypoglycemic and Hypolipidemic activity

R. Sharmin et al was studied Hypoglycemic and Hypolipidemic Effects of Cucumber in Alloxan Induced Diabetic Rats. It was concluded that the ethanol extracts of Cucurbitaceae family fruits, cucumber, white pumpkin and ridge gourd has significant antihyperglycemic effects in AIDRs. They also have the capacity to reduce the elevated lipid profiles in AIDRs. Ridge gourd has also significant effects to restore the depressed hepatic glycogen levels in AIDRs. Therefore, we believe that these fruits extracts can be useful, at least as an adjunct, in the therapy of diabetes, a condition in which hyPerglycemia and hyperlipidemia coexist quite often. However, further study is necessary for the screening of chemical compounds and the structure elucidation of the respective antidiabetic leads as well as their exact mechanism. [9]

. Wound healing activity

Patil et al were studied on pharmacological evaluation of wound healing potential of Cucumis sativus. He stated that aqueous extracts of Cucumis sativus have proper efficacy onwound healing. Herbal paste preparation showed significant (P<0.05) improvement onmaturation, wound contraction and epithelialization. [10]
All the above mentioned activities are done in fruit,seed and peel extracts only. No research work was done with unriped pulp extract of CUCUMIS SATIVUS L.
6.3 DRUG PROFILE:
CUCUMIS SATIVUS L



Cucumis sativus is used as a vegetable. It is a creeping vine that roots in the ground and climbs up trellises or other supporting frames, wrapping around ribbing with thin, spiraling tendrils. The plant has large leaves that form a canopy over the fruit. It is an annual climber growing to 2 m.The fruit is roughly cylindrical, elongated, with tapered ends, and may be as large as 60 cm long and measuring 10 cm in diameter. C. sativus are eaten fresh and pickled. In temperate zones, it is in flower from July to September, and the seeds ripen from August to October. The flowers are monoecious (individual flowers are either male or female, but both sexes can be found on the same plant) and are pollinated by insects. The plant undergoes self-fertilization.

Plant Part Used

Fruits, leaves, seeds.



Chemical Constituents

Seeds: 1,3-Diamino-propane, 22-dihydrobrassicasterol, 2,4-methylene-cholesterol 24-beta-ethyl-25(27)-dehydrolathosterol, 24-methyl-25(27)-dehydrocycloartanol, 24-methyl-cholest-7-en-3-beta-ol 25(27)-dehydro-chondrillasterol, 25(27)-dehydro-fungisterol 24-epsilon-ethyl-25(27)-dehydrolophenol 24-methyl-lathosterol, 24-methylene-24-dihydro-lanosterol, 24-methylene-24-dihydro-parkeol, 24-methylene-cycloartenol , 25(27)-dehydro-poriferasterol, 7-dehydro-avenasterol, alpha-amyrin, avenasterol, beta-pyrazol-1-yl-alanine, butyric-acid, campesterol, cucurbitin, cycloartenol, cycloeucalenol, euphol, gramisterol, isomultiflorineol, lupeol, lysolecithin, multiflorineol, obtusifoliol, phosphatidic-acid, phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidyl-glycerol, phosphatidyl-inositol, spermidine, stigmast-7,22,25-trien-3-beta-ol, stigmast-7,25-dien-3-beta-ol, stellasterol, taraxerol, tirucallol. (1)

Leaves: 22-Dihydro-spinasterol, alpha-spinasterol, isoorientin, meloside-a, stigmast-7-en-3-beta-o.l (1)

Fruit: Alanine,  alpha-linolenic-acid, alpha-tocopherol, aluminum arginine, arsenic, ash, aspartic-acid barium, beta-amyrin, beta-carotene, beta-sitosterol boron, cadmium, caffeic-acid, calcium, carbohydrates, chlorogenic-acid, chromium, citrulline, cobalt, copper, cucurbitacin-a, cucurbitacin-b, cucurbitacin-c, cucurbitacin-e, cystine, ferulic-acid, fiber, fluorine, folacin, gamma-glutamyl-beta-pyrazol-1-yl-alanine, glutamic-acid glycine, hexanal, hexen-(2)-al-(1), histidine, iron, isoleucine, lead, leucine, lithium, lysine, magnesium, manganese, mercury, methionine, mevalonic-acid, myristic-acid, niacin, nickel, nitrogen, non-trans-2-en-al, nonadien-2,6-al-1, nonadien-2,6-ol-2, nonen-2-al-1, pantothenic-acid, pentadec-cis-8-en-1-al, phosphorus, phytosterols, potassium, proline, propanal, protein, riboflavin, rubidium, selenium, serine, silicon, silver, sodium, squalene, strontium, sugar, sulfur, thiamin threonine, titanium, tryptophan, tyrosine, valine, vitamin b6, zinc, zirconium. [1]

Seeds of C. sativus were found to contain gibberellins A1, A3, A4, and A7 with A1 being the predominant specie. [2]








6.4 OBJECTIVES OF THE STUDY:
To carry out the acuteoral toxicity study of Cucumis sativus L [ unriped pulp extract].

To carry out efficacy study of the unriped pulp extracts of Cucumis sativus L for Diuretic, Antipyretic and Anti acne activities.







7

7. MATERIALS AND METHODS:

7.1 SOURCE OF THE DATA:

The drugs are procured from the retailers under specified terms and conditions. The whole work is planned to generate data from laboratory studies. The intended research work will be performed as described in the standard bibliography, may be obtained from standard journals and text books available within the college, National Institutes or through online data in the world wide web:



  • Bharathi College of Pharmacy Library

  • Websites:

      • www.sciencedirect.com

      • www.ncbi.nlm.nih.gov/pubmed

      • http://www.drugabuse.gov/nidahome.html

      • www.accessdata.fda.gov/scripts/cder/drugsatfda

      • www.springerlink.com/

      • http://europa.eu.int/comm/food/fs/sc/scf/index_en.html

Above sites will be used to obtain related information regarding this research protocol.
7.2 METHODS OF COLLECTING DATA:

The entire study is divided into the following phases:



  1. PHYTOCHEMICAL ANALYSIS:

Phytochemical analysis of the drug was done following the standard methods of Trease and Evans and Kokate, C.K., Purohit A. P., Gokhale S.B [11-12].

  1. ANIMALS:

All animal experiment will be performed in accordance with Institutional Animal Ethical Committee guidelines .

Healthy Wistar rats of either sex weighing between 180-250 gm were used in this study. Animals were maintained at 25±20°C and kept in well ventilated animal house under natural photoperiodic condition in polypropylene cages with paddy husk as bedding with free access to food and water ad libitum.



  1. ACUTE TOXICITY STUDIES

Acute oral toxicity study was conducted according to the OECD test guideline 423- Acute toxic class method.

  1. PHARMACOLOGICAL STUDIES;

Pharmacological screening done by In-vitro,In-vivo and intact animal models.

PHARMACOLOGICAL SCREENING

DIURETIC POTENTIAL STUDY;IN-VIVO;

Experimental Protocol: The rats were randomly divided into 5 groups of 5 animal each as Group I- vehicle control given 2 % tragacanth suspension in distilled water 1 ml/kg (p.o); Group II- standard drug frusemide (20 mg/kg, p.o); Group III- normal; Group IV- test(low dose). Group V-Test(high dose) Animal were fasted for 18 hr prior to the experimentation but with free access to water only. All the rats received priming dose of normal saline 25 ml/kg orally. Immediately after administration vehicle, standard drug and different doses of extracts according to body weight all the rats were placed in metabolic cages (group wise) specially designed to separate urine and faeces at room temperature of 25±5°C(19). Urine was collected in a graduated cylinder and its volume was measured for next 5 hr. During this period no food and water was made available to animals. Collection time for first drop of urine and total volume of urine collected from both control and treated groups were measured. Cumulative urine excretion was calculated in relation to body weight and expressed as ml/100 gm body weight. Electrolyte (Na+ and K+) concentration and pH of collected urine was estimated at the end of the experimental period and expressed as Meq/100 gm body weight.
Measurement of Urine output and Analysis of Electrolytes: Na+ and K+ concentrations were measured using a digital flame photometer (Elico, India). The instrument was calibrated with standard solutions containing different concentration of Na+ and K+. Chloride ion concentration was estimated by titration with silver nitrate solution (N/50) using 3 drops of potassium chromate solution as indicator 25. A pH meter (Jyoti Scientific, India) was used to measure the pH of freshly collected urine sample.
ANTI PYRETIC ACTIVITY

Antipyretic activity was determined by a modified method previously described14. Briefly, basal rectal temperatures of the rabbits were taken using a probe thermometer (model Panlab-0331). Thereafter, pyrexia was induced by injecting 105 CFU of E. coli/kg into the marginal ear vein of each rabbit. The rectal temperature of the rabbits was again taken 1 h after induction of pyrexia and animals having temperature between 38.7 and 39.7 °C(representing significant temperature increase from basal values of 37.41 ± 0.58°C) were regarded as hyperthermic and were randomly assigned to five groups of five rabbits each. Doses of test of extract dissolved in distilled water were administered orally to the rabbits ingroups A and B respectively, using an orogastric tube. A dose of 100 mg/kg ofacetylsalicylic acid (ASA) constituted in 10 % ethanol was administered orally to animals in group C which served as positive control. The rabbits in group D received 2 ml/kg of the vehicle for ASA (10 % ethanol) orally and served as negative control. The post-drug treatment rectal temperature of each rabbit was recorded at time points 30, 60, and 90 min. The differences in temperature before and after the administration of the extract or ASA were recorded as dT.



ANTI ACNE ACTIVITY:
IN VITRO

Fresh cultures of the isolates of bacteria were suspended in nutrient broth and reinforced clostridium medium, S. epidermis cultures were incubated for 24 at 370C. P. acnes culture was incubated in an anaerobic chamber at 37°C consisting of 15% CO2, 15% H2 and 70% N2 for 24 hrs. Antibacterial activity of extracts was tested using agar disc diffusion method4. Fresh culture suspension (100 μl) of test bacteria was evenly spread on nutrient agar and reinforced clostridial agar plates. The concentration of cultures was 5 × 105 CFU/ml. For screening, 6 mm diam. filter paper disc, impregnated with 15 μl of extract solution equivalent to 0.5 mg of extract was placed on the surface of inoculated media agar plates. Incubation was done at 37°C or 30°C for 24 .Clear zones of inhibition were measured in mm, including the diameter of disc. Zone measuring 15 mm or more was considered as effective against test organisms. Clindamycin (15 μg/disc) was used as positive control and the respective solvents, which were used for extraction, served as negative control.



Minimum inhibitory concentrations using micro dilution:

Minimum inhibitory concentration (MIC) of active methanolic extracts (AME) was studied by using broth micro dilution method with slight modify cations5. Extracts were dissolved in(DMSO) dimethyl sulfoxide (15% of total volume) and two folddilutions were done using pre-sterilized culture broth to give final concentrations ranging from 3-0.21 mg/ml. 100 μl of each dilution was distributed in 96 well plates. Sterility control (sterilized nutrient broth) and growth control (culture broth with DMSO) were also set up. Each test and growth control well was inoculated with 2 μl of a bacterial suspension (5x105 CFU/ml). All experiments were performed in triplicate and the micro dilution plates were incubated under optimum conditions. Bacterial growth was detected after the addition of 15 μl of 85% alcoholic solution of INT (0.3 mg/ml) into each well followed by incubation for 30 minutes. Colour change from yellow to purple indicated the presence of microbial growth.



  1. DESIGN OF THE STUDY;

DETAIL RESEARCH PLAN

The proposed research work will emphasize on the following:


Identification and collection of plant, Authentication of the part, Extraction of the plant material: The authenticated parts to be exhaustively extracted in a Soxhlet apparatus using Ethyl Alcohol,Chloroform and Ethyl acetate as solvents.


  1. STATISTICAL ANALYSIS:

Values will be expressed as mean ± SEM. Statistical difference in mean will be analyzed using one way ANOVA followed by Dunnet’s multiple comparison tests. P< 0.05 will be considered statistically significant.


7.3


7.3 Does the study require any investigation or interventions to be conducted on patients or

the humans or animals? If so please describe briefly.

Yes. The study requires investigations on animals. The effects of the drugs will be studied on various parameters using Rats ,Rabbits as experimental animals.

7.4


7.4 Has ethical clearance been obtained from your institute?

Yes. Institutional animal ethical committee approval letter will be obtained from Bharathi College of Pharmacy and submitted.







8 . REFERENCES:


    1. Duke, JA. 2007. Dr. Duke's Phytochemical and Ethnobotanical Databases. http://www.ars-grin.gov/cgi-bin/duke/farmacy2.pl.

    2. Hemphill, DD, Baker, LR, Sell, HM. Isolation and Identification of the Gibberellins of Cucumis sativus and Cucumis melo. Planta (Berl.),103: 241-248,1972.

    3. Ankita S., Kaur P., and Gupta R. Phytochemical screening and antimicrobial assay of various seeds extracts of Cucurbitaceae Family. International Journal of Applied Biology & Pharmaceutical Technology. Volume 3, Issue 3 (2012): 401-409

    4. Ankita S., Kaur P., and Gupta R. Phytochemical screening and antimicrobial assay of various seeds extracts of Cucurbitaceae Family. International Journal of Applied Biology & Pharmaceutical Technology. Volume 3, Issue 3 (2012): 401-409

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09



SIGNATURE OF CANDIDATE



10.

REMARK OF THE GUIDE:


The above information and literature has been extensively investigated, verified and was found to be correct. The experimental plan is justifiable and feasible. The present study will be carried out under my supervision and guidance.


11.


11.1 NAME AND DESIGNATION OF GUIDE


Dr.D.GNANASEKARAN, M.Pharm., Ph.D.,

PROFESSOR AND HOD,

DEPT.OF PHARMACOLOGY,

BHARATHI COLLEGE OF PHARMACY, BHARATHINAGARA-571422.




11.2 SIGNATURE


11.3 CO-GUIDE

NA

11.4 SIGNATURE

NA


11.5 HEAD OF THE DEPARTMENT


Dr.D.GNANASEKARAN, M.Pharm.,Ph.D.,

PROFESSOR AND HOD,

DEPT.OF PHARMACOLOGY,

BHARATHI COLLEGE OF PHARMACY, BHARATHINAGARA-571422.




11.6 SIGNATURE


12.



12.1 PRINCIPAL

Dr. T. TAMIZ MANI, M.Pharm., Ph.D.,

PRINCIPAL,

BHARATHI COLLEGE OF PHARMACY, BHARATHINAGARA-571422


12.2 SIGNATURE





12.3 REMARK OF THE CHAIRMAN AND PRINCIPAL

The above mentioned information is correct and I recommend the same for approval.





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