Enclosure-i brief resume of the intended work: 1 general discussion[1][2]




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ENCLOSURE-I
BRIEF RESUME OF THE INTENDED WORK:

6.1 GENERAL DISCUSSION[1][2] :

The liver performs many vital functions, including metabolic and detoxification activities. Beyond the treatment of liver disorders, everyday care of the liver lays a cornerstone for total body health. More than 500 vital functions have been identified with the liver. The liver is important because a person’s nutritional level is not only determined by what he or she eats, but by what the liver processes. Unfortunately it is extremely difficult to detect early warning symptoms specific to liver metabolic imbalances. People can suffer for a long time from a liver ailment without knowing of it. The incredible complexity of liver chemistry and its fundamental role in human physiology is so daunting to researchers thought that simple plant remedies might have something to offer is astonishing and incredible!

After extensive search of the modern and traditional literature on this subject we are proud to present a range of safe herbal alternatives from India that can serve as an excellent hepatoprotectives if consumed as per directions. Therefore, many folk remedies from plant origin are tested for its potential antioxidant and hepatoprotective liver damage in experimental animal model. Paracetamol induced hepatotoxicity model is widely used for the study of hepatoprotective effects of drugs and plant extracts.

The botanical name of Myrtle is Myrtus communis and it belongs to family Myrtaceae. Ripe berries contain an essential volatile oil (oil of myrtle) resin, tannin [2], citric acid, malic acid, sugar etc. Plant is stimulant and astringent. The plant myrtle leaves contains fragnant volatile oil, which is distilled from the leaves is antiseptic and rubefacient, it is generally employed for the perfumery. It is used in infections of respiratory organs and bladder and oil is a local application in rheumatic infections. A fixed oil is obtained from the berries; it strengthens and promotes the hair growth. Powder of leaves is useful application in eczema, wounds and ulcers.

The fruit myrtle berry is carminative and given in diarrhoea and dysentery in the form of infusion. It is also used in haemorrhages, internal ulcerations, deep sinuses, leucorrhoea and prolapsus of the uterus. A powder made by taking two drachms of the berries, one drachm of gum acacia and two drachms of kharamubasmi and reducing them to a fine powder is also useful for chronic dysentery, diarrhoea, and scorpion-sting.

6.2 NEED FOR THE STUDY[3] :

Medicinal practioners have prescribed Ayurveda and drug from herbal origin as a system of medicine in India over centuries. Many of the modern drug mainly based on synthetic chemical compounds, however have been found to have harmful side effects on the human system. This has triggered off extensive research and development in the field of herbal medicine. In fact, there is a growing demand for herbal medicines in most of the developed and developing countries of the world today. Although there is several medicinal plants mentioned in Ayurveda and folklore medicine are used as house hold remedies, the chemical investigation or biochemical studies or pharmacological studies were not fully established. Such a study may yield useful results in our quest to discover antihepatotoxic property.

The predominant type of liver disease varies according to country and may be influenced by local factors. The causative factors of liver disorders include virus infection, exposure to or consumption of certain chemicals. The substance that injures the liver cells in some people and results serious harm to the liver caused by drugs and by the combination of drugs and other substances is an important health problem. Physician and patients are in need of effective therapeutic agents with a low incident of side effects. There are few effective plants that cure liver disease. So a considerable interest have developed in the examination of those numerous worldwide traditional plant remedies which are useful for such treatment and that in recent years investigation carried out to provide experimental evidence which conforms that many of these plants do indeed have anti hepatotoxic properties.

So plants traditionally used in the allieviation of liver dysfunctions might therefore provide a useful source of new hepatoprotective compounds for development as pharmaceutical entities or as simple adjuncts to existing therapies, With this perspective the present study is undertaken to evaluate the antihepatotoxic effect.

From the traditional knowledge it is very clear that the plant Myrtus communis have the hepatoprotective activity [4][5]. Hence, the purpose of this study is to evaluate for Myrtus communis for its hepatoprotective activity against paracetamol induced hepatotoxicity in rats.

6.3 REVIEW OF LITERATURE :

Botanical name: Myrtus communis

Family : Myrtaceae

Common name: Dutch-myrtle


Habitat:

The plant grows all over India in tropical areas. It is also cultivated in gardens.


Chemical constituents:

Isolation and structure elucidation of two acylphoroglucinols A&B.Limonene, linanol, α-pinenine, cineol, p-cymol, camphene, β-pinene, treces of car-3-ene found in leaf essential oil[6]. The leaves also contain tannins and polyphenolics[7][8].



Ethnomedical Uses:[2][4]

Ripe berries is used in infections of respiratory organs, gallbladder. Myrtus communis is used as a stimulant and astringent. Fragrant volatile oil is distilled from the leaves is antiseptic and rubefacient, it is generally employed for perfumery. A fixed oil obtained from berries strengths and promotes the hair growth. Powder of leaves is useful in eczema and wounds and also ulcers. The volatile oil of the Myrtus communis is used as for foetid ulcers .Infusion or decoction is useful as a mouth wash. Syrup made by ounces of the Myrtus communis brusied seeds in twelve ounces of distilled water for 3 hours and then adding sugar and boiling for half an hour over a gentle heat is useful for in dysentery.



Reported biological activities:

Anti inflammatory effect[9], Anti mutagenic activity[10], Antioxidant activity[11], Antibacterial activity[12], Hypoglycemic activity[13], Apoptosis in cancer cells[14] and Antiulcer activity.[15]



6.4 OBJECTIVES OF THE STUDY:

  • Collection and Authentication of plant materials.

  • Preparation of plant extract.

  • Evaluation of Hepatoprotective activity using various parameters.

  1. Biochemical Parameters.

  1. Aspartate amino transferase (AST).

  2. Alanine amino transferase (ALT).

  3. Alkaline Phosphatase (ALP).

  4. Gamma Glutamate transpeptidase (GGT).

  5. Total bilirubin.

  6. Total protein.

  1. Histopathological studies.

  2. Anti-oxidant studies.



ENCLOSURE-II

MATERIALS AND METHODS:

7.1. Source of Data:

  1. Library, Bharathi College of Pharmacy.

  2. E-library, Bharathi College of Pharmacy.

  3. Library, RGUHS, Bangalore.


7.2. Experimental animals:

Adult albino rats weighing approximately 150-200 g will be used. The animals will be

fed with standard feed and will be given water ad libitum.
7.3. Plant material:

Dried leaves of Myrtus communis were powdered and sequentially extracted in a soxhlet with petroleum ether (60–80°C), chloroform and alcohol. The alcoholic extract will be dried under vaccum and suspended in carboxy methyl cellulose before use.


7.4. Selection of Dose of myrtus communis alcoholic extract:

From literature survey the alcoholic extract of Myrtus communis was found to be effective in 2000 mg/kg of body weight [27]. Hence 200, 400 mg/kg/bw doses of extract will be administered.


7.5 METHOD:

Rats were divided into five groups, each group consisting of six animals.



Group I: Control received the vehicle viz. normal saline (2 ml/kg).

Group II: Received paracetamol 500 mg/kg body wt by p.o, at every 72 h for 10 days [16].

Group III: Received silymarin 50 mg/kg p.o. for 10 days and simultaneously administered

paracetamol 500 mg/kg body wt by p.o 1 h after the respective assigned treatments for every 72 h.



Group IV: Received alcohol extract of Myrtus communis 200 mg/kg p.o. for 10 days and simultaneously administered paracetamol 500 mg/kg body wt by p.o., every 72 h.

Group V: Received alcohol extract of Myrtus communis 400 mg/kg p.o. for 10 days and simultaneously administered paracetamol 500 mg/kg body wt by p.o.,every 72 h.
At the end of experimental period, all the animals were sacrificed by cervical decapitation. Blood samples were collected, allowed to clot. Serum was separated by centrifuging at 2500 rpm for 15 min and analyzed for various biochemical parameters.
Assessment of liver function:

Biochemical parameters i.e., aspartate amino transferase(AST)[17], alanine amino transferase(ALT)[17], alkaline phosphatase (ALP)[18], γ-glutamate transpeptidase (GGTP)[19], total bilirubin [20] and total protein [21] were analyzed according to the reported methods. The liver was removed, weighed and morphological changes were observed. A 10% of liver homogenate was used for antioxidant studies such as lipid peroxidation (LPO) [22], superoxide dismutase (SOD) [23], and catalase [24] and glutathione peroxidase (GPx) [25]. A portion of liver was fixed in 10% formalin for histopathological studies.


Histopathological studies:

Liver slices fixed for 12 hrs in Bovin’s solution were processed for paraffin embedding following standard micro techniques [26]. 5μm sections of liver grained with alum haematoxylin and eosin were observed microscopically for histopathological changes.


7.6 Experimental design:


S. No

Treatment

Dose (mg/kg)

Route of administration

No. of.

Animals

Parameters

1

Control (Distilled water)

2ml

Oral


6

1.Aspartate amino transferase

2.Alanine amino transferase

3.Alkaline Phosphatase

4.Gamma Glutamate transpeptidase

5. Total bilirubin.

6.Total protein

7. Histopathological studies.


2

Paracetamol

500

Oral


6

3

Standard

50

Oral

6

4

M.communis

200

Oral

6

5



M.communis

400

Oral

6



7.6. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO

BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS

-YES-

Hepatoprotective activity test: Adult albino rats weighing approximately 150-200 g will be



used to check hepatoprotective study of Myrtus communis.
7.7. HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION

IN CASE OF 7.6?


The study is cleared from ethical committee of institution.

(Reg. no. 1135/a/07/CPCSEA Dated 13/02/2008)—Copy enclosed



ENCLOSURE-III

REFERENCES:

  1. www.agriinfotech.com.

  2. Nadkarni KM. Indian Materia Medica.1st ed. Bombay: popular Prakashan Pvt.Ltd; 1976.vol 1.p.838.1683-5.

  3. http://antihepatoprotective activity.blogspot.com/2008/01.

  4. Trease and Evanas. Pharmacognosy. 15th ed. Elsevier publishers. p. 477.

  5. Dr.K.Madhava chetty. Flowering plants of chittor district, Andhra Pradesh.India.2nd ed. Tirupathi.p.128.

  6. T.K. Chatterjee. Herbal options 1st ed. Calcutta. Eastern traders. p. 180-1.

  7. Tuberso CI, Barra A, Angioni A, Sarritzu E, Pirisi FM. Chemical composition of volatile oil in Sardian myrtle(Myrtus communis) alcoholic extracts and essential oils. J Agric Food Chem.2006; 54:1420-6.

  8. El-Sissi HI, Ansary MA. Tannis and polyphenolics of the leaves of Myrtus communis. Planta Med. 1967; 15:41-51.

  9. Rossi A, Di Paola R, Mazzon E, Genovese T, Caminiti R. Myrtucommulone from Myrtus communis exhibits potent anti-inflammatory effectiveness in vivo. J Pharmacol Exp Ther. 2009; 329:76-86.

  10. Hayder N, Kilani S. Antimutagenic activity of aqueous extracts and essential oil isolated from Myrtus communis.Pharmzie. 2003; 58:523.

  11. Mimica-dukic N, Bugarin D, Grbovic S, Mitic –Culafic D. Essential oil of Myrtus communis L. as a potent antioxident and antimutagenic agents. Molecules. 2010; 15:2759-70.

  12. Alem G, Mekonennen Y, Tirunesh M, Mulu A. In vitro anti bacterial activity of crude preparation of myrtle (Myrtus communis) on common human pathogens. Ethiop Med J.2008; 46:63-9.

  13. Sepici A, Gürbüz I, Cevik C, Yesilada. Hypoglycemic effects of myrtle oil in normal and alloxan-diabetic rabbits. E J Ethnopharmacol. 2004; 93:311-8.

  14. Tretiakova I, Blaesius D, Maxia L, Myrtucommulone from Myrtus communis induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9. Apoptosis. 2008; 13:119-31.



  1. Sumbul S, Ahmad MA, Asif M, Saud I, Akhtar M.H Evaluation of Myrtus communis Linn.berries (common myrtle) in experimental ulcer models in rats. ExpToxicol. 2010; 2:2-4.

16. Pandey G.P, Srinivasthava D.N. Effect of livol and some of its ingredients on paracetamol induced hepatotoxicity in mice. Indian J pharmacology 1990; 2291:12.

17. Reitman S, Frankel S. A colorimetric method for determination of serum glutamate

oxalo acetate and glutamic pyruvate transaminase. Amer J Clin Path 1957, 28: 56-58.

18. Kind PR, King EJ, Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrin. J Clin Path 7: 322- 326.

19. Szaszi G, A kinetic photometric method for serum gamma–glutamyl transpeptidase. Clin Chem 1969; 15: 124 -126.

20. Mallay HT, Evelyn KA, Estimation of serum bilirubin level with the photoelectric colorimeter. J.Biochem 1973; 119: 481 – 484.

21. Lowry OH, Rosenbrough NT, Farr AL Protein measurement with Folin –Phenol

reagent. J. Bichem 1951; 173: 265-275.

22. Devasagayam, TPA, and Tarachand U, Decreased lipid peroxidation in the rat kidney during gestation. Biochem. Biophys. Res.Commun, 1987; 56, 836-842.

23. Marklund S. and Marklund G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem.1974; 47, 469-474

24. Sinha A.K, Colorimetric assay of catalase. Anal. Biochem.1972; 47, 389-394.

25. Rotruck JT, Pope AL, Ganther, HL, and Swanson AB, Selenium: biochemical role as a component of glutathione peroxidase. Science, 1973, 179, 588-590.

26. Galigher AE and Kozloff EN; Essential Practical Micro technique, 2nd ed, Lea and Febiger, Philadelphia1971; p.77.

27. Elfellah MS, Akhter MH, Khan MT. Anti-hyperglycaemic effect of an extract of Myrtus communis in streptozotocin-induced diabetes in mice. J Ethnopharmacol. 1984; 11:275-81.







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