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Annexure – II


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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

Annexure – II



PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION


1.

NAME OF THE CANDIDATE AND ADDRESS

Mr. sandeep s. biradar


department OF pharmacology,

K.L.E. SOCIETY’S COLLEGE OF PHARMACY,

J.N.M.C CAMPUS, NEHRU NAGAR,

BELGAUM – 590010.



2.

NAME OF THE INSTITUTION


K.L.E.S’s COLLEGE OF PHARMACY, BELGAUM – 590010.

3.

COURSE OF STUDY & SUBJECT


MASTER OF PHARMACY In pharmacology

4.

DATE OF ADMISSION TO COURSE


JUNE 2008

5.

TITLE OF THE TOPIC:

SCREENING OF CYPERUS ESCULENTUS LINN. AND CYPERUS ROTUNDUS LINN. ESSENTIAL OILS ON VARIOUS PHARMACOLOGICAL ACTIVITIES.”




6.

BRIEF RESUME OF THE INTENDED WORK:




6.1 NEED FOR THE STUDY:


Essential oils are used extensively in aromatherapy and various traditional medicinal systems. Due to the numerous health benefits of essential oils. They are being explored by the scientific community for treating a variety of disease including cancer, HIV, bronchitis, heart strokes, etc. They are more than 90 essential oils each having its own benefits. Every essential oil blends well with many other essential oils enabling herbalist preparations. Most of these oils are strong in nature and can cause side effects, if they are not taken in appropriate manner and quantities.1

Cyperus esculentus Linn. and Cyperus rotundus Linn. Belonging to family Cyperaceae, found in Punjab, Nilgiri Hills and throughout India and it Contains volatile oil, sesquiterpenes, phenolic, flavonoids, hydrocarbons epoxide and ketones etc. The tubers are traditionally used as astringent, bitter, acrid, cooling, carminative, digestive, anthelmintic, galctogogue, aphrodisiac, antimicrobial, stimulant, febrifuge, and also used in diuretic, expectorant, dyspepsia, flatulence, colic, helminthiasis, diarrhea, dysentery, agalctia, bilious, hyperdipsia, burning sensation, opthalmopathy, leprosy, hepatopathy, cephalalgia, amentia, neurasthenia, constipation, epilepsy, anorexia, wounds, and malarial fever.2

Burn can be defined as tissues damage caused by a variety of agents such as heat, chemicals, electricity, sunlight or nuclear radiation. The most common are burn scalds, building fires, and flammable liquids and gases. Every year, about two million people receive medical treatment for burn injury.3

A burn is an injury caused by transfer of energy into tissue with a resulting disruption in its functional integrity.4 Various chemical mediators play role are peptides and low molecular weight substance that regulate the cellular function and microenvironment of tissue. 5

Antimicrobial drugs design to inhibit/kill the infecting organism and to have no/minimal effect on the recipient. This therapy is called chemotherapy.6 Chemotherapeutic agents, used orally or systemically for the treatment of microbial infections of humans or animal 7.Many effort have been done to discover new antimicrobial compounds from various kinds of sources such as soil. Microorganisms, animals and systematic screening of them may result in the discovery of novel effective compounds8. Antimicrobial activity will provide useful data for burn wound healing activity.

The antimicrobial properties of the plant volatile oils and their constituents from a wide variety of plants have been assessed and reviewed. Investigations into the antimicrobial activities, mode of action and potential uses of plant volatile oils have regained momentum.7

Anthelmintics are that drugs that either kill (vermicide) or expel (vermifuge) infesting helminthes. Helminthiasis is prevalent globally (1/3rd of world’s population harbours them), but is more common in developing countries with poorer personal and environmental hygiene.6 Helminthiasis is a major problem where a large proportion of mankind harbours helminths of one species or another. In some cases, as threadworms in children, in results in discomfort and do not cause substantial ill health while in others such as schistosomiasis and hookworm disease, may cause very serious morbidity. In many countries, particularly those in tropical and subtropical regions, almost all the indigenous population is infected with helminths and the problem of importance 9

However no scientific evidence is available on essential oils obtained from these plants on antimicrobial, burn wound healing and anthelmintic activity.

Therefore the present study will be designed to screen Cyperus esculentus Linn. and Cyperus rotundus Linn essential oils on antimicrobial, burn wound healing and anthelmintic activity.






6.2 REVIEW OF LITERATURE:

Plant description:

Cyperus esculentus Linn. and Cyperus rotundus Linn found in Punjab, Nilgiri Hills and throughout India as a weed in waste lands from sea level to 1,800m, belonging to family Cyperaceae.

Cyperus esculentus Linn. is erect perennial glabrous grass-like sedge. It is upto 50cm in height. Tubers are ovoid to cylindrical edible upto 2cm in length. Stem base are pale brown, triquetrous, smooth, finely striated, leaf sheaths are not fibrous, and leaf blades are shorter than stem. Linear and gradually tapering in the upper part to a fine acuminate apex, 3.5m, in width.

The tubers of cyperus esculentus Linn. are traditionally used as astringent, bitter, acrid, cooling, carminative, digestive, anthelmintic, galctogogue, aphrodisiac, stimulant, febrifuge and also used in dyspepsia, flatulence, colic, helminthiasis, diarrhea, dysentery, agalctia , constipation, bilious, hyperdipsia and antimicrobial property.

Composition

Volatile oil: On Stem distillation the dried tubers yield from 0.5-1.0 percent of volatile oil. α -pinene (70.5-75.5), α -thujene, α –cadinol, cineole, cyperol, α-cyperone.

Flavonoids: These are idenfied as Cyperene, caryophyllene, Υ- cadinene, karacolene.

Terpenoids: Monoterpens and Sequiterpenes like α-cadinene, adebic acid,

Bicyclic sequiterpene ketone, Tricyclic sequiterpene alcohol, Tricyclics sequiterpene phenol, etc.

Cyperus rotundus Linn is glabrous, stolons elongated, slender to 20cm long. Tubers are ovoid tunicate black fragrant tuber are 0.8-2.5cm in diameter, stems are subsolitary, 10-75cm long, triquetrous at the top, sometimes tuberous at the base. Leaves are shorter or longer than the stem, narrowly linear, 4-8mm broad, finely acuminate, flat,1-nerved. Spikelet’s are variable in length, 1.6-3.8cm by 2.5mm, linear, subacute red brown 10-50 flowered, compressed; rhachilla with hyaline wings.

The tubers of Cyperus rotundus Linn. are traditionally used as diuretic, nervine tonic, anti-inflammatory, astringent, bitter, acrid, cooling, carminative, digestive, anthelmintic, galctogogue, aphrodisiac, stimulant, febrifuge, antimicrobial property and also used in flatulence, wounds, ulcer, helminthiasis, constipation, colic, dyspepsia, diarrhea, dysentery, agalctia, bilious, epilepsy, anorexia and hyperdipsia.

Composition

Volatile oil: On Stem distillation the dried tubers yield from 0.5-1.0 percent of volatile oil. α -pinene (70.5-75.5) α-cadinol, cyperol, cyperene, α-cyperone, isolongifolen-5-one, rotundene and cyperorotundene.

Flavonoids: These are idenfied as caryophyllene, γ- cadinene, karacolene.

Terpenoids: Monoterpens and Sequiterpenes like α-cadinene, adebic acid,

Bicyclic sequiterpene ketone, Tricyclic sequiterpene alcohol, Tricyclics sequiterpene phenol, etc.1,2

The skin has been described as the largest organ in the body. Its functions are numerous and injury to its various layers increases the vulnerability of the organisms to additional biological and physical hazards resulting in wound. The pathophysiological reaction to a burn injury is complex and varies with cause. In thermal burn injury changes in the burn wound are mainly are mainly caused the direct effect of heat but superimposed on these are changes associated with an acute inflammatory process.10

Free radical as well as histamine and prostaglandin released from the burn wound cause lipid peroxidation in the skin. In consequence they cause burn edema, intravascular heamolysis, hypovolemic shock and adult respiratory distress syndromes. Lipid peroxidation occurs after burn and is continue during the whole post burn period. This cause damage of lipid part of biological membrane, which leads to loss its function and apoptosis. One of the major peroxidation is malondialdehyde, which is an index of free radical activity. Burn wound healing and repair can occur accordingly whenever the whole body substrate is adequate for survival and functioning of the specialized cell of organ parenchyma. This study can be clearly understood by performing the inflicting burn wound model by using rats.11

Anthelmentic drugs are used to eradicate or reduce the number of helminthic parasites in the intestinal tract or tissues of the body. These parasites have many biochemical and physiologic processes in common with their human hosts, yet there are subtle differences that are beginning to yield to pharmacologic investigation. 12 Due to discovery and development of anthelmintics, particularly from veterinary applications physicians now have effective, and in some cases, broad spectrum of agents that will cure or control most human infections caused by either flukes or intestinal helminths. But cysticercosis, echinococcosis, filariasis, and trichinosis are examples of systemic infections caused by tissue dwelling helminths that at best respond only partially to currently available drugs. Because Metazoan parasites are generally long-lived and have relatively complex life cycle, acquired resistance to anthelmintics in human beings has yet to become a major factor limiting clinical efficacy. It acts probably by blocking glucose uptake in the parasite and depletion of its glycogen store. Intracellular microtubules in the worm are gradually lost 13. This study can be clearly understood by performing Motility study and glucose uptake studies by using worms.

Antimicrobial agents (AMA) have specific effect on limited number of microbes. Antimicrobial agents binds with several sites at 30 S and 50 S subunits as well as to their interface-freeze initiation, interfere with polysome formation and cause misreading on mRNA code. Some of the AMA binds to 30S ribosome and inhibit aminoacyl t-RNA attachment to the ‘A’ site, and some of them binds to 50S subunit interferes with peptide bond. Formation and transfer of peptide chain for ‘P’ site and some of them binds to 50S ribosome and hinder translocation of the elongated peptide chain back from ‘A’ site to ‘p’ site and the ribosome does not move along the mRNA to expose the next codon, peptide synthesis may be prematurely terminated. The sensitivity testing is done by serial dilution method to determine the minimum inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration by using the bacteria and fungi.6

For determination of LD50, the OECD guidelines will be adopted (number 423 or 425).






6.3 OBJECTIVES OF THE STUDY:

Objective of the proposed study are



  1. Preliminary phytochemical investigation of oils.

  2. Determination of LD50.

  3. To screen activity of Cyperus esculentus Linn. and Cyperus rotundus Linn Essential oils on antimicrobial activity.

  4. To screen activity of Cyperus esculentus Linn. and Cyperus rotundus Linn Essential oils on burn wound healing activity.

  5. To screen activity of Cyperus esculentus Linn. and Cyperus rotundus Linn Essential oils on anthelmintics activity.

7.

MATERIALS AND METHODS

7.1 SOURCE OF DATA:

The source of data’s is from the laboratory experiments, which involves extraction of essential oils and their pharmacological activities on experimental animals.



7.2 METHOD OF COLLECTION OF DATA: (including sampling procedure if any)

1. Animal Selection:

Rats: Swiss Wister rats of either sex.

Mice: female albino mice

Microorganism:

Gram positive : Staphylococcus aureus, Staphylococcus albus.

Gram negative :Pseudomonas aeruginosa, E.coli.

Fungi: Candida albicans, Aspergillus.

Earthworm: Pheretima posthuma, Ascardia galli.



2. Chemicals: Molich reagent, Felhing’s reagent, Benedict’s reagent, Barfoed’s reagent, Millon’s reagent, Ninhydrine reagent, Salkowshi reagent, Libberman- Burchard reagent, Baljet reagent, Cobalt Nitrate, Iodine solution, Tannic acid reagent, Lead Acetate, Chloroform, Sodium Nitroprusside, Antimony Trichloride, Piperazine Citrate, Nutrient agar, gum acacia, NaOH, H2SO4, CMC, Mebendazole, Glucose Estimation kit,and essential oils of Cyperus esculentus Linn. and Cyperus rotundus Linn.
3.1 Phytochemical investigations.

3.2 Determination of LD50:

The guidelines described by OECD will be adopted for the determination of LD50 on female albino mice. And 1/10th of LD50 will be administered orally in 2% Tween 80.


3.3 Model for burn wound.

  1. Inflicting burn wound

Healthy Wistar albino rats weighing between 150 and 180g will be used. The dorsum of each rat will be shaved. Burn wounds will be inflicted on overnight-starved animals under anesthesia. A 2 × 2-cm metal cylinder will be placed on the shaven back of the animals. Melted wax at 80°C is poured into the metal cylinder and the wax was allowed to solidify. Eight minutes after this, until the wax will be completely solidified, the metal cylinder containing wax adhering to the skin will be gently removed to inflict a distinctly demarked burn wound.

Assessment of wound healing

After the drug administration, the wound inflicted areas of animals every day from day 1. Animals to be observed for wound healing by measuring the wound contraction (tracing the raw wound area first on a transparent polythene paper and then retraced on graph paper) up to the 12th day post wounding. The wound contraction to be calculated as percentage of original wound size for each animal of a group.



Histopathological studies:

Few animals will be scarified by deep anesthesia. The wounds will be excised, leaving a 5mm margin of normal skin around the edges of wound and placed in 10% formalin for histopathological examination. After the tissue will be processed, mid-wound vertical sections of each specimen were cut and stained with haematoxylin and eosin. The specimens will be assessed under light microscope for the progression of new epithelium, inflammation, vascular responses and the formation of collagen in the wound.14




  1. Model for antimicrobial activity:

Serial dilution method:

In this technique, various agar plates will be prepared by pouring melted nutrient agar media, each of them will be inoculated with a particular microorganisms like gram positive, gram negative bacteria and fungi.

After agar solidifies, cups will be made in nutrient agar. The antimicrobial substances are placed in the cups. The antimicrobial substance diffuses through agar around the cups. The plates will be inoculated at a temperature at 37°C for 24 hours for bacteria and 48 hours for fungi. The diameter of this zone to be measured for the degree of activity of antimicrobial substance.15
3) Model for antihelmintic activity:

Motility study

Samples of the anthelmintic study will be prepared by dissolving the essential oil in 25ml of 1% gum acacia solution prepared in normal saline to obtain a stock solution. From this stock solution the different working dilution will be prepared.

Five groups of approximate equal size Indian earthworm consisting of six earthworms in each group will be used for one of the following: vehicle (1% gum acacia normal saline), Piperazine Citrate (15mg/ml) and different concentration of the essential oils. Observation will be made for the time taken to paralyze and/or death of individual worms. Paralysis will be said to occur when the worm do not revive even in normal saline. Death will be concluded when the worms lost their motility, followed with fading away of their body colour.16

Ascardia galli worm will be incubated in different concentration of the essential oils Cyperus esculentus Linn. and Cyperus rotundus Linn and Mebendazole (100µg and 300 µg) and drug free tyrode medium (control) for 20 hours. The glucose estimation will be done by using glucose oxidase (GOD)/peroxidase (POD) methods.17




    1. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INVENTION TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO, PLEASE, MENTION BRIEFLY.

Above study requires screening of Cyperus esculentus Linn. and Cyperus rotundus Linn. essential oils on various pharmacological activities like antimicrobial, anthelmintics, burn wound healing activity in rats.


    1. HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION INCASE OF 7.3?

The study has to be cleared from the ethical committee of the institution.

.





REFERENCES




  1. Kubmarawa d, Ogunwande.A, Okorie D.A, Olawore N.O, Kasali A.A, Chemical constituents of volatile oil of Cyperus esculentus Linn. and Cyperus rotundus Linn from Nigeria, CENTRE NATIONAL DE LA RECHERCHÉ SCIENTIRIQUE 2005; 20(6):640-41.

  2. Sala AV, Indian Medicinal Plants, A Compendium of 500 Species, vol.III. Madras: Orient Longman, 1997, 293-296.

  3. Srivastava P, Durga Prasad S, Burn wound healing property of Cocos nucifera: An appraisal. Indian Journal of Pharmacology 2008; 40(4):144-146.

  4. Lee RC, Astumain RD. The Physiochemical Basis for Thermal and Non-Thermal ‘Burn’ injuries. Burns 1996; 22 506-19.

  5. Arturson G. Pathophysiology of the burn wound and pharmacological treatments, Burns 1996; 22:255-74.

  6. Tripathi KD, Essential of Medical Pharmacology, 6th ed, New Delhi: JAYPEE BROTHER’S MEDICAL PUBLISHERS (P) LTD 2008:667.

  7. Dorman HJD, Deans SSG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology 2000; 88:308-16.

  8. Parekh J, Chanda S, In-vitro Antimicrobial Activities of Extracts of Launaea procumbens Roxb. (Labiateae), vitis vinifera L. (Vitaceae) and Cyperus Rotundus L. (Cyperaceae). African Journal of Biomedical Research 2006;9:89-93

  9. Rang HP, Dale MM, Ritter JM, Moore PK, Pharmacology.5th Ed Churchill Livingstone; Elsevier sites publication 2003:687-92.

  10. Schilling IA. Wound healing. Physiological Reviews 1968:48(2):374-423.

  11. Andrezes M, Eva S, Krzysztof L, Jerzy B. Influence of early excision of burn wound on the lipid peroxidation in related rat tissue. Poland bullvet palway 2002: 46:255-65.

  12. Katzung BG, “Basic and Clinical pharmacology”. 8th ed Lange medical books, Mc Graw Hill. Medical Publishing Division 2002:903-20.

  13. Brunton LL, Lazo JS, Parker KL. Goodman & Gillman, The Pharmacological Basis of Therapeutics. 11th ed, New York: Mc-Graw Hill Medical Publishing Division 2006; 1073-93

  14. Malipeddi VR, Dua k, Udai Sara UVS, Malipeddi H, Agrawal A. Comparative Evaluation of Transdermal Formulations of Norfloxacin With Silver Sulfadiazine Cream, USP, for Burn Wound Healing Property. Journal of Burns Wounds 2006; 5:e4.

  15. Pelczar MJ, Chan ESC, Krieg NR, Microbiology,4th ed New Delhi: TATA Mc Graw Hill Company Limited 1993: 115-32.

  16. Mali RG, Mahajan S, Patil KS. ANTHELMINTIC ACTIVITY OF ROOT BARK OF CAPPARIS SPINOSA. Indian J. Nat. Prod. 2004; 21(4): 50.

  17. Nathan O. Kalpan. Enzymatic determination of free sugars. In: Colowik SP, Kalpan NO, eds. Methods in enzymology. New York: Academic Press Inc, 1975; 3:34-36.







9.

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 present study will be carried out under my supervision and guidance.


11.

NAME & DESIGNATION OF (in block letters)

11.1 Guide



Shri. V. P. Rasal. M.Pharm.

associate Professor

DEPARTMENT OF Pharmacology,

KLES'S COLLEGE OF PHARMACY,

BELGAUM-590010.





11.2 Signature






11.3 Co-guide (if any)

-----




11.4 Signature

-----




11.5 Head of the

Department

Prof. A.D. Taranalli. M.Pharm.
vice principal and head,

DEPARTMENT OF pharmacology,

KLES'S COLLEGE OF PHARMACY,

BELGAUM-590010.





11.6 Signature




12.

    1. Remarks of the Chairman & Principal


    1. Signature

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

Dr. F. V. Manvi. M.Pharm., Ph.D.

Principal,



K.L.E.S’s College of Pharmacy, Belgaum-590010.







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