Apium graveolens. L




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Effect of apium graveolens.L in Diabetic, Cataract and as Anti-oxidants in alloxan induced diabetic rats.
M. Pharmacy Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore – 560 041
By

Mr. KAMALESH.D.R B.pharam

Under the Guidance of


Mr.T.PRAKASH

Asst. Professor

Department of pharmacology







P.G. Department of Pharmacology,

Acharya & B.M.Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road, Bangalore – 560 090
2007-2008

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. KAMALESH.D.R

S/O RADHA KRISHNA SETTY.D.A

Venkateshwara Nilaya.

5th Main, Bhuvaneshwari Nagar,

T-Dasarahalli, Bangalore -57.

Phone No: 080-28376800










2

NAME OF THE INSTITUTION


Acharya & B.M. Reddy College Of Pharmacy.

Soldevanahalli, Hesaraghatta, Road, Chikkabanavara (Post),

Bangalore -560090.

Phone No: 080-65650815










3

COURSE OF STUDY AND SUBJECT

M. PHARM.

PHARMACOLOGY.








4

DATE OF ADMISSION

July-2007.










5

TITLE OF THE TOPIC

Effect of apium graveolens.L in Diabetic, Cataract and as Anti-oxidants in alloxan induced diabetic rats.









6.

BRIEF RESUME OF INTENDED WORK


6.1 Need of the work
6.2 Review of Literature
6.3 Aim and Objectives of the study


Enclosure I


Enclosure II
Enclosure III


7.

MATERIALS & METHODS


7.1 Source of data
7.2 Methods of collection of data

( Including sampling ,procedure

if any)
7.3 Does the study require any investigation on animals?

If yes give details


7.4 Has ethical clearance been obtained from your institution in case of 7.3



Enclosure IV
Enclosure V

Enclosure VI


Yes (Copy Enclosed)




8.

LIST OF REFERENCES


Enclosure VII




9.

SIGNATURE OF THE CANDIDATE






10.

REMARKS OF THE GUIDE







11.

NAME & DESIGNATION OF


11.1 Guide

11.2 Signature of Guide

11.3 Head of the Department

11.4 Signature of HOD





Mr. T.PRAKASH

Asst. Professor

P.G. Department of Pharmacology,

Acharya & B.M.Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post),

Hesaraghatta Main Road , Bangalore – 560 090


Prof. Kalyani Divakar M.Pharm. (Ph.D)

P.G. Department of Pharmacology,

Acharya & B.M.Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post),

Hesaraghatta Main Road , Bangalore – 560 090




12.

REMARKS OF THE PRINCIPAL

12.1 Signature



PRINCIPAL

Dr. GOLI DIVAKAR M.Pharm. PhD

Acharya & B.M.Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara (Post),

Hesaraghatta Main Road , Bangalore – 560 090



Enclosure - I
6. BRIEF RESUME OF INTENDED WORK
6.1 NEED OF THE WORK:

Human beings are affected with several diseases from time immemorial by various means. The diseases and their proper treatment occupy the center stage of medicinal therapy. The revolutionized growth in research and development has led to the discovery of potential medicinal moieties certainly associated with some adverse effects. As a matter of fact different traditional systems of medicine offer unique advantage over modern medicine. One such time tested holistic system of medicine is Ayurveda, which provides alternative treatment for human ailment with assured safety and least or literally no side effects. For the last two decades the herbal medicines derived from medicinal herbs have been receiving worldwide attention for the exploration of their properties. At present the challenge is how to carry out research and evaluate the safety and efficacy of herbal medicines. The details provided for the identification of medicinal plants and drugs in the highly popular Ayurvedic treatises like Sushrutasamhita, Charakasamhita, and Astanga hridaya are not easily decipherable which necessitates to carry out a thorough scientific investigation. On one such drug Apium graveolens.L is selected to investigate the screening for its anti-diabetic, anti-cataract and anti-oxidant pharmacological activity.

There are probably at least 100 million people in the world suffering from diabetes mellitus and the incidence is increasing rapidly. Diabetes occurs when the blood glucose level increases and a state of hyperglycemia is produced, as a result of a deficiency of available and effective insulin. Patient with juvenile onset diabetes mellitus have impaired insulin secretion or excessive hepatic glucose production, which develops as a result of synergistic effects of genetic, environmental and immunological factors. Patients have normal β-cell mass at birth but infection or environmental factor may led to autoimmune destruction of β-cell destruction of about 80% leads to feature of diabetes mellitus genetic defects may not manifest unless environmental or other factors like obesity are superimposed.

A key goal of diabetes treatment is to prevent complications because, over time, diabetes can damage the heart, blood vessels, eyes, kidneys, and nerves although the person may not know damage is taking place. It's important to diagnose and treat diabetes early. How diabetes causes long-term problems is unclear. However, changes in the small blood vessels and nerves are common. These changes may be the first step toward many problems that diabetes causes. Diabetes will develop complications, but complications are most likely to occur in someone who has had diabetes for many years.

Diabetes can affect the eyes in several ways. Frequently, the effects are temporary and can be corrected with better diabetes control. However, long-term diabetes can cause changes in the eyes that threaten vision. Stable blood glucose levels and yearly eye examinations can help reduce the risk of serious eye damage. The reason may be that changing levels of glucose in blood also can affect the balance of fluid in the lens of the eye, which act as a flexible camera lens to focus images. If the lens absorbs more water than normal and swells, it’s focusing power changes. Diabetes affect the function of nerves that control eyesight, causing blurred vision11.

Cataract and glaucoma are eye diseases that occur more frequently in people with diabetes. Cataract is a clouding of the normally clear lens of the eye. Glaucoma is a condition in which pressure within the eye can damage the optic nerve that transmits visual images to the brain. Early diagnosis and treatment of cataract and glaucoma can reduce the severity of these disorders14.

An Anti-oxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Anti-oxidants terminate these chain reactions by removing radical intermediates and inhibit other oxidation reactions by being oxidized themselves. Although oxidation reactions are crucial for life, they can also be damaging hence, plants and animals maintain complex systems of multiple types of anti-oxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of anti-oxidants, or inhibition of the anti-oxidant enzymes causes oxidative stress and may damage or kill cells which also lead to apoptosis in conditions like diabetes12.

Apium graveolens is a plant consists of a important chemical constituents like glycosides, flavonides, coumarine, vitamins like A, B, C and some minerals. Which are effective in treating diabetics, cataract due to its antioxidant property so the plant has been selected for the following work. According to the review of literature the following work on the plant apium graveolens is not carried out.



Enclosure – II

6.2 REVIEW OF LITERATURE:

Apium graveolens - L.(Wild Celery)


Kingdom : plantae

Division : Magnoliophyta

Class : magnoliopsida

Order : Apiales

Family : Apiaceae

Genus : Apium L.


Names in different language

Sanskrith : Ajmoda

English : Wild celery, Smallage
Description of plant:

Apium graveolens.L is used around the world as a vegetable, either for the crisp stems or fleshy taproot.

Wild Celery is widely cultivated, biennial plant which also grows wild in salty soils of North and South America, Europe, and Africa. The fleshy, bulbous root sends up, in the second year, an angular, furrowed, branched stem from 1 to 3 ft high. Celery leaves are opposite, dark green, shiny, and pinnate, the leaflets wedge-shaped, incised, coarsely toothed. The white to gray-white flowers bloom from July to November13.


Geographical distribution of the plant:

Apium graveolens L. occur in the following: Arizona, California, Connecticut, Florida, Idaho, Illinois, Louisiana, Massachusetts, Missouri, Mississippi, Himalaya.



Chemical constituents:


From the water-soluble portion of the methanol extract of celery seed (fruit of Apium graveolens L.) five sesquiterpenoid glycosides (celerioside A-E) and three phthalide glycosides (celephtalide A-C) were isolated together with six aromatic compound glycosides, two norcarotenoid glucosides and a lignin glucoside. Coumarins (seselin,osthenol, opigravin, celerin, umbeliferon) furono coumarin Flavonoids (apigenin, apiin) phenolic compound fatty acid, calcium ions, Vit A, B, C, magnesium,phosphorus.The leaf mainly constitute of arabinose, car-3-ene, caffeic acid, bata elemene,mucilage. The roots main consists are of molybdenum, p-coumaric acid, rubidium, pentosane, sesalin, shikimic acid, zinc14,13.

Traditional therapeutic uses:

In temperate countries, celery is also grown for its seeds, which yield a valuable volatile oil used in the perfume and pharmaceutical industries. Celery seeds can be used as flavoring or spice either as whole seeds or ground and mixed with salt, as celery salt. Celery salt can also be made from an extract of the roots. Wild celery has a long history of medicinal and food use. It is an aromatic bitter tonic herb that reduces blood pressure, relieves indigestion, stimulates the uterus and is anti-inflammatory. The ripe seeds, herb and root are apatite, carminative, diuretic, emmenagogue, galactogogue, nervine, stimulant and tonic. Wild celery is said to be useful in cases of hysteria, promoting restfulness and sleep and diffusing through the system a mild sustaining influence. The herb should not be prescribed for pregnant women. An essential oil obtained from the plant has a calming effect on the central nervous system. Some of its constituents have antispasmodic, sedative and anticonvulsant actions. It has been shown a value in treating high blood pressure. It is used in treating rheumatism and kidney complaints 16.




Therapeutic uses documented by research:

  1. Inhibitory effect of celery seeds extract on chemically induced hepatocarcinogenesis: modulation of cell proliferation, metabolism and altered hepatic foci development1.

  2. Hepatoprotective activity of two plants belonging to the Apiaceae and the Euphorbiaceae family2

  3. Antioxidant, cyclooxygenase and topoisomerase inhibitory compounds from Apium graveolens Linn. Seeds3.

  4. Apium graveolens modulates sodium valproate-induced reproductive toxicity in rats4.

  5. Anti-nociceptive and anti-inflammatory effects of some Jordanian medicinal plant extracts5.

  6. Anti-oxidant, cyclooxygenase and topoisomerase inhibitory compounds from Apium graveolens Linn. Seeds6.

  7. Hepatoprotective activity of Apium graveolens and Hygrophila auriculata against paracetamol and thioacetamide intoxication in rats7.


Enclosure – III

6.3 AIM AND OBJECTIVES OF THE STUDY:

AIM: Study of Anti-diabetic and Anti-cataract activity of

apium graveolens.L with relation to Anti-oxidant activity in alloxan induced diabetic rat.


  1. Extraction of plant.

  2. Phytochemical analysis.

  3. Toxicity analysis and calculation of LD-50.

  4. Evaluating the efficiency of apium graveolens.L extract for

    • Anti-diabetic

    • Anti-cataract

    • Anti-oxidant activity.


Enclosure – IV


7. Materials and Methods
7.1 SOURCE OF DATA:

        The source of data will be obtained from experiments which involves-

  1. Laboratory based studies.

  2. Literature survey, CD ROM, Chemical abstracts.

  3. National & International Journal.

  4. Text books.

  5. Internet.


Enclosure – V

7.2 METHOD OF COLLECTION OF DATA (including sampling procedure if any)
Field and laboratory studies

Field work:

The selected plant of Apium graveolens.L will be collected from the forests of Bangalore rural district and it will be authenticated.


Laboratory work:
Animals

Wistar albino Rats of Either sex (200-250 g) will be selected for Anti-diabetic,

Anti-cataract and Anti-oxidant activity. The animals will be housed in standard environmental condition & provided with food & water ad libitum.

 

Plant

              The plant of apium graveolens.L will be collected, shade dried, coarsely powdered & stored in an airtight container. The plant includes seeds, leaf and roots. 

Solvents


  1. Aqueous solvent.

  2. Methanolic solvent8.


Phytochemical tests

Phytochemical tests for detection of organic constituents will be done as per Kokate 15.


Toxicity study

Minimal lethal dose of the drug will be carried out following Ghosh10.


Pharmacological studies

Animal experimental models will be devised to assess the Anti-diabetic, Anti-cataract and Anti-oxidant activity.


Data analysis

Statistical analysis of the data will be evaluated by analysis of variance (ANOVA) followed by Dunnets test.


Methodology





  1. Extraction:

The plant of apium graveolens.L will be collected, shade dried, coarsely powdered & extracted with aqueous & alcoholic solvents to get sufficient crude extract. Extract is subjected to evaporation to obtain a concentrate. Then aqueous & alcoholic extracts are subjected for pharmacological evaluation of Anti-diabetic, Anti-cataract and anti-oxidant activity7.


  1. Toxicity study:

Determination of LD50 carried as per CPCSEA guidelines.12 Mice will be used. For the toxicological study and dose fixation.

(3) PHARMACOLOGICAL STUDY DESIGN.

I . EVALUATION OF ANTI-DIABETIC ACTIVITY.

ALLOXAN INDUCED DIABETES:

Rats of Wistar albino Rats strain weighing 150-200 g are injected subcutaneously with 100-175 mg/kg Alloxan monohydrate. This single dose of alloxan produced persistent hyperglycemia after 24 h. Rats showing fasting blood glucose levels are selected for the study 9.



    1. Rats are divided into nine groups consisting of six animals.

Groups 1: Normal control (Saline).

2: Diabetic control.

3: Alloxan glibenclamide at a dose of 10 mg/kg.

Methanolic extract group 4: Alloxan + Apium graveolens (low dose).

5: Alloxan + Apium graveolens (medium dose).

6: Alloxan + Apium graveolens (high dose).

Aqueous extract group 7: Alloxan + Apium graveolens (low dose).

8: Alloxan + Apium graveolens (medium dose).

9: Alloxan + Apium graveolens (high dose).


ESTIMATION OF BIOCHEMICAL PARAMETERS:

The blood samples are withdrawn from rats by tail vein puncturing using hypodermic needle at ‘0’ and the end of 15th days are analyzed .Urine sample are also collected and analyzed for parameters of urine.


a) LIPID PROFILE

  • Total cholesterol

  • High density lipid ( HDL )

  • Low density lipid ( LDL )

  • Very low density lipid ( VLDL )

  • Triglycerides.

b) LIVER PROFILE



  • Serum albumin

  • Total-protein

c) RENAL PROFILE



        • Calcium

        • Blood urea

        • Serum creatinine

d) KIDNEY LEAK PROFILE IN URINE



  • Urine micro albumin

  • Urine creatinine


II . EVALUATION OF ANTI-CATARACT ACTIVITY.

Alloxan monohydrate is used to induce cataract in a batch of Wistar albino Rats by injecting intraperitoneal a single dose (20mg/kg body weight/24 h) of 2% alloxan monohydrate solution in saline, the animal is tested for following parameters after particular group treatment.



Rats are divided into thirteen groups consisting of six animals14.

Groups 1: Control group animals given normal saline.

First week 2: Diabetic control

3: Alloxon + Apium graveolens extract (low dose)

4: Alloxon + Apium graveolens extract(medium dose)

5: Alloxon + Apium graveolens extract (high dose)

Third week 6: Diabetic control

7: Alloxon + Apium graveolens extract (low dose)

8: Alloxon + Apium graveolens extract(medium dose )

9: Alloxon + Apium graveolens extract (high dose)

Sixth week 10: Diabetic control

11: Alloxon + Apium graveolens extract (low dose)

12: Alloxon + Apium graveolens extract(medium dose)

13: Alloxon + Apium graveolens extract (high dose)


ESTIMATION OF BIOCHEMICAL PARAMETERS 17:

  • Ophthalmic examination after homatropinization.

  • Con of DNA, in blood, aqueous humor, lens.

  • Con of RNA, in blood, aqueous humor, lens.

  • Total soluble protein content in blood, aqueous humor, lens.

  • Na, K, Ca, water content.

  • Gluthathione reductase (GSH).

  • Gluthation peroxidase (SOD).

  • Catalase.

III . EVALUATION OF ANTI-OXIDANT ACTIVITY18.
ESTIMATION OF BIOCHEMICAL PARAMETERS :
In Vitro

  • Total polyphenolic compound.

  • Determination of total flavanoids.

  • Super-oxide anion scavenger activity.

  • Hydroxyl radical scavenging activity.

  • Nitrous oxide scavenging.

  • Hydrogen peroxide scavenger activity.

  • Fe2+ chelating activity.

In Vivo12

  • Glutathione peroxidase (SOD).

  • Glutathione reductase.

  • Catalase.

  • Tiobarbituric acid reactive substance (TBRAS).

  • Ascorbic acid

The rats are weighed at the beginning and at the end of the experiment. Test drug at various concentration depending on the design of experiment is administered. Animals are sacrificed liver and kidney are removed, weighed, and part used thereof for ascorbic acid determination. The rest of the tissue are frozen not more than72 h to analysis the remaining in vivo experiment.


  • The data obtained from the above study will be subjected to statistical analysis using ‘t’ test.




  • Total duration for the completion of whole project may be 11 months

    1. Duration of experimentation Six months.

    2. Literature survey two & half months.

    3. Thesis writing two & half months.


ENCLOSURE – VI
7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.
The above study requires investigation on Wistar albino Rats for Anti-diabetic, Anti-cataract and Anti-oxidant activity .


7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The study is cleared from Ethical Committee of the institution. (Certificate enclosed).

ENCLOSURE – VII

LIST OF REFERENCES: -


  1. Sarwat Sultana, Salahuddin Ahmed, Tamanna Jahangir and Sonia Sharma.

Inhibitory effect of celery seeds extract on chemically induced hepatocarcinogenesis: modulation of cell proliferation, metabolism and altered

Hepatic foci development.


Cancer Letters April 2005;Volume 221, Issue 1, 18: P 11-20

  1. Bahar Ahmed, Tanveer Alam, Manoj Varshney and Shah Alam Khan.

Hepatoprotective activity of two plants belonging to the Apiaceae and the Euphorbiaceae family.
Journal of Ethnopharmacology March 2002;Volume 79, Issue 3:P 313-316

  1. R.A. Momin and M.G. Nair. Antioxidant, cyclooxygenase and topoisomerase inhibitory compounds from Apium graveolens Linn. Seeds.
    Phytomedicine 2002;Issue 4,Volume:P 312-318

  2. Apium graveolens modulates sodium valproate-induced reproductive toxicity

in rats.
J Exp Zool Part A Ecol Genet Physiol 2007 Apr 1;307(4):p 199-206.

  1. A. H. Atta and A. Alkofahi. Anti-nociceptive and anti-inflammatory effects of some Jordanian medicinal plant extracts
    Journal of Ethnopharmacology  March 1998; Volume 60, Issue 2: P 117-124

  2. roopm and roos. Antioxidant, cyclooxygenase and topoisomerase inhibitory compounds from Apium graveolens Linn. seeds.
    Phytomedicine 2002 May; 9(4):P 312-8.

  3. Hepatoprotective activity of Apium graveolens and Hygrophila auriculata

against paracetamol and thioacetamide intoxication in rats.
J Ethnopharmacol 1995 Dec 15; 49(3): P 119-26.

  1. Anonymous. Indian Pharmacopoeia. New Delhi: Controller of Publications ;1996.

  2. H.Gehard Vogel(Ed), Drug discovery and evaluation, Springer, anti diabetics;1996, p-947-1051.

  3. Ghosh MN. Fundamental methods in Pharmacology. 3rd ed. Calcutta: Hilton and company; 2005,P 190-197.

  4. Goodman and Gilman. The pharmacological Basic of Therapeutics. 10th ED New Delhi; Mc GrawHill Medical Publishing Division, New York; 2001.P1686-1692.

  5. Pulak. K.Mukherjee. Quality control of Herbal drugs An approach to evaluation of botanicals, Pharmacological Screening of herbal drugs, Delhi; 2003,P 562-571.

  6. Free patents [on line], Herbal Composition having anti allergic properties and a process for the preparation there of [cited 2007 sep21]; Available from; URL:http:www.freepatentonline.com/6730332.htm.

  7. S.K.Guptha Drug screening methods, Jaypee,I edition, ant cataract activity;2002.P 334-339.

  8. Kokate CK. Practical Pharmacognosy. New Delhi: Vallabh Prakashan; 1999,P 149-156.

  9. Namo _amitabha [on line],herbal store [cited 2007 sep20]; Available from; URL:http:www. Namo _amitabha/herbstore/order_apium graveolen_ajmoda-wildcelery.htm.

  10. U.M.Rawal and D.N.Gandhi, studies on cataractocenesis in rat with alloxon induced diabetes, Indian journal of pharmacology (1986);18:P 73-77.

  11. S.Velavan, K.Nagulendran, R.Mahesh, V.Hazeena begam. Invitro antioxidant activity of asparagus racamous root. Pharmacogonosy magazine mar 2007;vol 3, issue 9:P 26-33.







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