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1. INTRODUCTION

iabetes mellitus (DM) is the name given to a group of disorders characterized by

chronic hyperglycemia, polyurea, polydipsia, polyphagia, emaciation and

weakness due to disturbance in carbohydrate, fat and protein metabolism associated with

absolute or relative deficiency in insulin secretion and / or insulin action. DM is a

condition in which the sugar level is above the normal sugar level 80-120 mg/dl of the

whole blood (Deb and Dutta, 2006).

DM is the commonest disorder that affects more than 100 million people worldwide (6

per cent of the population) and in the next 10 years it may affect about five times morethan it does now (WHO/Acadia, 1992; ADA, 1997) (Grover et al., 2002). In India, the

prevalence rate of diabetes is estimated to be 1 to 5 per cent. The worldwide figure of

people with diabetes is set to raise from 150 million in the year 2000 to 300 million

in 2025 It is predicted that by 2030, India, China and the United States will have the

largest number of people with diabetes. Hyperglycemia and metabolic dysregulation may

be associated with secondary damage in multiple organ systems, especially the kidneys,

eyes, nerves, and blood vessel (Kumar et al., 2005).

The prevalence of DM increases with age in both sexes and is consistently higher in men

than in women of 20-49 year of age.

1.1 AN ETIOLOGIC CLASSIFICATION OF DIABETES MELLITUS (Harsh

Mohan., 2006)

Although all forms of DM share hyperglycemia a common feature, the pathogenic

processes involved in the development of hyperglycemia vary widely. The previous

classification schemes of DM were based on the age of onset or on the mode of therapy;

in contrast, the recently revised classification reflects our greater understanding of the

pathogenesis of each variant.

D



I. Type 1 diabetes (-cell destruction, usually leading to absolute insulin deficiency)

A. Immune-mediated

B. Idiopathic

II. Type 2 diabetes (may range from predominantly insulin resistance with relative

insulin deficiency to a predominantly insulin secretory defect with insulin resistance)

III. Other specific types of diabetes

A. Genetic defects of b-cell function characterized by mutations in:

1. Hepatocyte nuclear transcription factor (HNF) - 4 (MODY 1)

2. Glucokinase (MODY 2)

3. HNF-1 (MODY 3)

4. Insulin promoter factor (IPF) 1 (MODY 4)

5. HNF-1 (MODY 5)

6. Mitochondrial DNA

7. Proinsulin or insulin conversion

B. Genetic defects in insulin action

1. Type A insulin resistance

2. Leprechaunism

3. Rabson-Mendenhall syndrome

4. Lipoatrophic diabetes

C. Diseases of the exocrine pancreas

Pancreatitis, pancreatectomy, neoplasia, cystic fibrosis, hemochromatosis,

fibrocalculous pancreatopathy

D. Endocrinopathies

Acromegaly, Cushing's syndrome, glucagonoma, pheochromocytoma,

hyperthyroidism, somatostatinoma, aldosteronoma

E. Drug- or chemical-induced

Pentamidine, nicotinic acid, glucocorticoids, thyroid hormone, diazoxide, b-

adrenergic agonists, thiazides, phenytoin, -interferon, protease inhibitors,

clozapine, -blockers

F. Infections

Congenital rubella, cytomegalovirus, coxsackie



G. Uncommon forms of immune-mediated diabetes "stiff-man" syndrome, anti-

insulin receptor antibodies

H. Other genetic syndromes sometimes associated with diabetes

Down's syndrome, Klinefelter's syndrome, Turner's syndrome, Wolfram's

syndrome, Friedreich's ataxia, Huntington's chorea, Laurence-Moon-Biedl

syndrome, myotonic dystrophy, porphyria, Prader-Willi syndrome

IV. Gestational diabetes mellitus (GDM)

NOTE: MODY, maturity onset of diabetes of the young.

1.2 CLINICAL FEATURES (Kumar et al., 2005)

DM is not a single disease, but numerous disease and symptoms are associated with

hyperglycemia. The predominant clinical features of the two categories of DM are

outlined below.

1.2.1 TYPE I DIABETES (IDDM)

It usually manifest at early age, generally below the age of 40. The plasma insulin levels

are low and patients respond to exogenous insulin therapy. The onset is marked by

polyurea, polyphagia, and, with extreme derangement, ketoacidosis, all resulting from

metabolic dearrangements. A catabolic state is reached because of insulin deficiency

which results in glucose, fat and protein metabolism. Glucose assimilation in the muscle

and liver is greatly reduced and also the stores of glycogen are depleted by increased

glycogenolysis. This causes glycosuria. The glycosuria induces osmosis and thus results

in polyurea, causing profound loss of electrolytes and water. Such a renal water loss and

hyperosmolarity causes depletion of intracellular water provokes the osmoreceptors of

the thirst centers of brain and causes intense thirst i.e. polydipsia. Due to lack of insulin

catabolism of protein and fats occurs resulting in removal of gluconeogenic amino acid

from the liver, this result in negative energy balance which in turn leads to increasing

appetite i.e. polyphagia. Despite of increased appetite catabolic effect prevails resulting

in weight loss and fatigue but the patients are not obese. Thus type I DM is a classic triad

of polyurea, polydipsia and polyphagia, weight loss and fatigue as shown in figure 1



Figure 1. Metabolic disturbances leading to diabetic coma in type I DM



Diabetic ketoacidosis (DKA) is also a serious complication in type I DM. insulin

deficiency stimulates the release of epinephrine that stimulates the release of glucagons.

This disturbed insulin: glucagon ratio stimulates lipoprotein lipase, with the resultant

breakdown of adipose stores, and an increase in the levels of free fatty acids (FFA). FFA

reaches the liver get esterified to fatty acyl CoA. Oxidation of this fatty acyl CoA within

the mitochondria produces ketone bodies (acetoacetic acid and -hydroxybutyric acid.

Rate of formation of these ketone bodies exceeds their utilization in peripheral tissues

causing ketonemia and ketonuria as shown in above figure 1

1.2.2 TYPE II DIABETES (NIDDM)

It is basically generally manifested in the middle life or beyond, usually above the age of

40. Its onset is slow and insidious. Generally, the patient is a symptomatic when the

diagnosis is made on the basis of glycosuria or hyperglycemia. During physical

examination, the patients are frequently obese and may have polyurea, polydipsia,

unexplained weakness and normal to higher loss of weight. There is relative insulin

deficiency. Metabolic complications such as ketoacidosis are infrequent. Obesity

common (present in >75 per cent). Symptoms often mild, absent or unrecognized, insulin

resistance common and insulin treatment often required to maintain long term glycaemic

control.

In table 1 the pertinent clinical, genetic and histopathological features that distinguish

type I and type II diabetes are described.



Table 1. Comparison of Type I DM and Type II DM (Kumar et al, 2005)

TYPE I DM TYPE II DM

CLINICAL Onset <20years

Normal weight

Decreased insulin

anti-islet cell antibodies

Ketoacidosis common

Onset>30 years

Obese

Increased blood insulin

No anti-islet cell antibodies

Ketoacidosis rare; nonketotic

hyperosmolar coma

GENETICS 30-70 per cent concordance

in twins

Linkage to MHC Class II

HLA genes

50-90 per cent concordance in

twins

No HLA linkage

Linkage to candidate diabetogenic

genes (PPAR)

PATHOGENESIS Autoimmune destruction of

-cells mediated by T-cells,

absolute insulin deficiency

Insulin resistance in skeletal

muscle, adipose tissue and liver. -

cell dysfunction and relative insulin

deficiency

ISLET CELLS Insulitis early

Marked atropy and fibrosis

-cell deletion

No insulitis

Focal atropy and amyloiddeposition

Mild -cell depeletion

Methods to Induce Experimental Diabetes(Vogel HG )

Pancreatectomy in dogs

Alloxan induced diabetes

Streptozotocin induced diabetes

Other diabetogenic compounds

Hormone induced diabetes,Virus induced diabetes



1.3 COMPLICATIONS OF DM

Patients with long-standing diabetes may develop complications affecting the eyes,

kidneys or nerves (micro vascular complications) or major arteries. The major arteries are

affected in people with diabetes, causing a substantial increase in both in coronary artery

disease and strokes as well as peripheral vascular disease. The greatest risk of large

vessel disease occurs in those diabetic patients who develop proteinuria or micro

albuminuria, which are associated with widespread vascular damage. The complications

of DM are depicted in below mentioned figure 2



Figure 2. Late complications of DM (Kumar et al., 2005)



1.4 PATHOGENESIS OF DM

1.4.1 PATHOGENESIS OF TYPE I DM

This form of diabetes is because of severe lack of insulin caused by an immunologically

mediated destruction of -cells. Such an immunological destruction is caused primarily by T

lymphocytes reacting against poorly defined -cell antigens. Type I DM is basically caused

by the destruction of the -cells, genetic susceptibility and various environment factors.

Several mechanisms contribute to the destruction of the -cells. The tissue macrophages

recognize the -cell antigens and cause the cell damage. These tissue macrophages include

CD4+ T cells of the TH1 subset and CD8+ cytotoxic T lymphocytes which directly kill the -

cell and secrete cytokines that activate macrophages. Destructed islets detected early shows

cellular necrosis and lymphocytic infiltration called as insulitis. They were found to contain

CD4+ and CD8+ cells. Some studies have shown that a -cell enzyme, glutamic acid

decarboxylase (GAD) and insulin itself act as autoantibodies. The locally produced cytokines

such as IFN-, TNF and IL-1 produced by the tissue macrophages due immune reaction also

damage the -cells. Studies show that these cytokinins induce -cell apoptosis in culture

medium. Hence many of such immune mechanisms act together to cause progressive

destruction of the -cell.

Type I DM also has genetic causes. A number of complex patterns are involved in disease,

yet the most important is the class II MHC (HLA) locus which contributes for about half the

genetic susceptibility.

Various environmental causes are involved in triggering autoimmunity in type I diabetes and

other autoimmune diseases. Viral infections have along prevalence with seasonal trends e.g.

association of coxsakieviruses of group B and pancreatic disease along with diabetes. Other

implicated viral infections include mumps, measles, cytomegalovirus, rubella and infectious

mononucleosis. Such infections do not cause the disease directly by damaging the -cell

rather such infections causes issue damage and inflammation, leading to the release of -cell

antigens and recruitment and activation of lymphocytes and other inflammatory leucocytes

in the tissue. The other factor is that viruses produce proteins that mimic self antigens and

the immune response to the viral protein cross-reacts with the self tissue.



1.4.2 PATHOGENESIS OF TYPE II DM ( Kasper, 2001)

Type II DM is a heterogeneous disorder characterized by three pathophysiological

abnormalities: impaired insulin secretion, peripheral insulin resistance, and excessive hepatic

glucose production. However this type is not associated with the genes and there is no

evidence of autoimmune basis for type II DM. The two metabolic defects that characterize

type II DM are insulin resistance and -cell dysfunction. Insulin resistance is defined as

resistance of insulin on glucose uptake, metabolism, or storage.

The following figure depicts the insulin action on various tissues

Figure 3. Metabolic effects of insulin on various tissues(Kumar et al, 2005)



Insulin resistance leads to decreased uptake in muscle and adipose tissues and is manifestedby an inability of the hormone to suppress hepatic gluconeogenesis. Insulin resistance also

demonstrate various abnormalities in insulin signaling pathways, including down regulation

of the insulin receptor; decreased insulin receptor phosphorylation and decreased tyrosine

kinase activity; reduced levels of active intermediates in the insulin signaling pathways; and

impairments of translocation, docking, and fusion of GLUT-4 containing vesicles with the

plasma membrane as shown in figure 4

Figure 4 Insulin signal transduction pathway (Rang and Dale, 2007)



1.6 DRUG TREATMENT OF DIABETES (Rang et al., 2007)

A brief overview of drugs commonly used in clinic to treat or control DM is the following:

y I nsulin: There are many kinds of preparations

Fig 2 Shows Insulin synthesis and secretion

Insulin synthesis and secretion. Intracellular transport of glucose is mediated by

GLUT-2, an insulin-independent glucose transporter in cells. Glucose

undergoes oxidative metabolism in the cell to yield ATP. ATP inhibits an

inward rectifying potassium channel receptor on the cell surface; the receptor

itself is a dimeric complex of the sulfonylurea receptor and a K+ -channel

protein. Inhibition of this receptor leads to membrane depolarization, influx of

Ca2+ ions, and release of stored insulin from cells.

(Maitra, 2005).



y S ulfonylureas (SU ): Tolbutamide (D860, Orinase), Glibenclamide (Glyburide,

HB419,Micronase, Daonil), Gliclazide (Diamicron), Glibenese (Minidiab), Glurenorm(Gliquidone), Glutril (Glibornuride) and Glimepiride, and so on

y Biguanide (BG): Phenformin (Phenethyldiguanidi Hydrochloridum, Diabenide, DBI),

Dimethylbiguanide (FluamineMetformin, Diaformin, Diabex, Mellitin, Obin, Melbine,

Metformin, Hydrochloride, Glucophage, DMBG)

y -Glucosidase inhibitors (-GDI ): Glucobay (Acarbose), Voglibose, Miglitol,

Emiglitate, Glyset, Precose

y Aldose reductase inhibitor (ARI ): Tolrestat, Alredase, Epslstat, Kinedak, Imirestat,

Opolrestat.

y T hiazolidinediones (TZ D): Troglitazone, Rosigitazone, Pioglitazone, Englitazone

y C arbamoylmethyl benzoic acid (CM BA): Repaglinide

y I nsulin-like growth factor (I GF ): IGF-1

y Others: Dichloroacetic acid

Hence all newly diagnosed patients with type II DM should have an initial trial of dietary

and exercise modifications. However, many patients require pharmacological treatment

without an optional trial of nutrition and physical activity. Monotherapy with a sulfonylurea

or metformin can be used as first line pharmacotherapy. However, if FPG is >250 mg/dl or

random blood glucose is >400 mg/dl, insulin should be used as initial therapy. If elevations

in post-prandial glucose are problematic, meglitinites and -glucosidase inhibitors may be

beneficial. So when selecting an oral antidiabetic agent, the effect on glucose, lipids, adverse

effects and route of elimination should be considered. Pharmacotherapy therapy should be

tailored to the goals and needs of each individual patient.



1.7 HERBAL TREATMENT OF DIABETES MELLITUS

Table 2. Plants with potential antidiabetic activity

Botanical name

& local name

Reported Action Chemical

constituents

identified as

antidiabetic

Mechanism of action

Acacia arabica

Babul

Anti-diabetic action - Insulin release

Aegle marmelose Bael

Controlled the bloodglucose, urea, body weight

, liver glycogen , serumcholesterol

Alkaloids Insulin release,decreased malate

dehydrogenase

Allium cepa Pyaj

Hypoglycemic action(ethyl acetate fraction)

Antihyperglycemic action(petroleum ether)

S-methyl cysteine Normalized theactivities of liver

hexokinase, glucose 6-phosphatase.

Allium sativum

Garlic

Hypoglycemic action ,

hepatic glycogen action,fasting blood glucose

decreases, triglyceridesdecreases,

S containing amino

acid

Stimulates the synthesis

or release of insulin

Andrographispaniculata Nees

Andrographolide -

Artemisia pallensDavana

Antihyperglycemic action - Increased glucoseutilization or inhibited

glucose reabsorption

Areca catechu

Supari

Hypoglycemic effect Nitrosamines

Alkaloid

-

Azadirachtaindica

Neem

Hypoglycemic action andantihyperglycemic action Plant blocks the actionof epinephrine onglucose metabolism

Beta vulgaris

Chukkander

Inhibition of

non-enzymaticglycosylation

of skin proteins

Beta vulgarosides II,

III and IV

Increases the glucose

tolerance.inn OGTT.

Brassica juncea Hypoglycemic action - -



Seeds

C aesalpiniabonducella

Kantkarej

Hypoglycemic action andantihyperglycemic action

- -

C arica papaya

Linn.Papaya

Hypoglycemic action Glycosides, papain

mineral salts andpolysaccharides.

-

C itrulluscolocynthis

Badi indrayan

Hypoglycemic Glycosides,alkaloids, sapaonins

SignificantInsulin release.

Eucalyptusglobules

Safeda

No hypoglycemiaDecreased polydepsia

Prevented body loss

- Increased peripheralglucose utilization.

insulin secretion

F icus

bengalenesisBanyan tree

Hypoglycemic action Glucoside,

pelaronidine ,leucopelarogonodone

Increases the insulin

level and decreasing theinsulinase activity

Gymnemasylvestre

Gudmar

Antihperglycemic actionNormalized blood glucose

Decreased HbA1c, lipid

GymnemosidesGymnemic acid

Decrease ingluconeogenic enzymes

-cell regeneration,

Hibiscus rosa

Gudhal

Hypoglycemic action Increases the insulin

release by stimulationof pancreatic beta cells

or an increase of theglycogen deposition in

liver.

I pomeo batatasSakkargand

Reduces hyperinsulinemiain Zucker fatty rats

- Reduction in insulinresistance

Lantana camaraCaturang

Hypoglycemic actionBut hepatotoxic in nature.

- -

M angifera indica Aam or Amb

Antidiabetic action if givenconcurrently with glucose

Reduction in intestinalabsorption of glucose

M emecylon

umbellatum Anjani

Significant reduction in the

serum glucose levels

- -

M omordicacharantia

Karela

Hypoglycemic actionAntihyperglycemic action

Anticataract

Polypeptide,oleanolic acid

3-O-glucoronideMomordin Ic

Inhibited the glucose-6-phosphatase and

fructose-1,6-biphosphate

M orus albaShetut

Hypoglycemic action Alkaloids Glycosidase inhibitoryactivity.glucose

uptake.

M urray loeingii

Kurry patta

Hypoglycemic action - Insulin like action

Occimum Significant reduction - -



sanctum ;Tulsi

Phyllanthusniruri

Jangli amla

Hypoglycemicand antidiabetic activity

Bioflavanoids,Vitamin C,

emblicanin A, B

-

Punica grantumAnar

Antidiabetic-

-

S wertia chiraytiaChirata


Swerchirin Stimulated the insulinrelease

S yzigium cumini

Jamun

Hypoglycemic action

Reduced the glycosuriaRestored the hepatic

glycogen, hepaticglucokinase, hexokinase

- Increases the activity of

cathepsin BInhibited the isulinase

activity

T rigonellafoenum

Methi


Antiglycosoric effectNormaised the altered

creatinine kinase.

Fibres, saponins,poteins

4-hydroxyisoleucine

Stimulated the insulinsecretion in absence of

pancreatic pancrratic-pancreatic cells

T inospora

cordifoliaGuduci

Hypoglycemic action

Decresed the brain lipidlevel, alkaline and lactate

dehydrogenase

- -

Vinca rosea

Sadabahar

Antihyperglycemic - -



2.REVIEW OF LITERATURE

efore going to any further studies, it is necessary to refer some related past works

done. So, this chapter deals with the findings of previous works, carried out by the

researchers. Thus under this heading an attempt has been made to compile the material

reviewed related to diabetes Momordica charantia, Eugenia jambolana, Ocimum sanctum,

Allium cepa

2.1 Review related to diabetes

1. Sabu et al., 2002 evaluated the antidiabetic activity and its relationship with their

antioxidant property of methanolic extract (75%) of T erminalia chebula, T erminalia

belerica, Emblica officinalis and their combination ³Triphala´ (equal prorportion of

above three plants).

2 .Nagappa et al., 2003 evaluated the antidiabetic activity of petroleum ether, methanol,

and aqueous extracts of T erminalla catappa Linn fruit, on fasting blood sugar levels and

serum biochemical analysis in alloxan- induced (150 mg/kg) diabetic rats. All the three

extracts produced a significant antidiabetic activity at dose levels 1/5th

of their lethal doses

(LD50). Histological studies showed comparable regeneration by methanolic and aqueous

extracts.

2. Roy et al., 2005 studied the protective effect of dry latex (DL) of C alotropis procera

against alloxan-induced diabetes in rats.

3. Musabayane et al., 2005 studied the effects of S yzygium cordatum (Hochst.)

[Myrtaceae] leaf extract on plasma glucose and hepatic glycogen in streptozotocin-

induced diabetic rats.

B



4. Vats et al., 2004 studied the effect of administration of Ocimum sanctum (OS) on the

alteration in hepatic glycogen in streptozotocin induced diabetes in rats and effect on

carbohydrate metabolism.

5. Yazdanparast et al., 2007 examined possible protective effect of Achillea santolina L.

(Compositae) against pancreatic damage in streptozotocin (STZ)-treated diabetic rats.

6. Kalousová et al., 2002 determined advanced glycation end-products (AGEs) and

dvanced oxidation protein products (AOPP) in the sera of 52 patients with diabetes

mellitus (DM)- 18 with DM Type 1 and 34 with DM Type 2 a nd examined their

relationship to the compensation of the disease.

2.2 Review of Ethanomedical Use:

A.Momordica charantia:

The leaves are used as anthelmintic, antipyretic, emetic, purgative, they also used

inconstipation, intermittent fever, helminthiasis, burning sensation of the sole. The fruits are

stimulant, purgative, antidiabetic, carminative, digestive, stomachic, anthelmintic, anti-

inflammatory, febrifuge.they are useful in skin dieases, leprosy, ulcers, wounds

,burningsensation, constipation, anorexia, flatulence ,colic hepatomegaly, splenomegaly,

asthma. Seeds are useful in the treatment of ulcers, obstruction of the liver and spleen .[(Arya

Vaidya Sala 2004 vol 4. p 48).

B. Eugenia jambolana:-

The barkiscarminative, diuretic, digestive, anthelmentic, febrifuge, stomachic,

Antibacterial .It is useful in diabetes, fever, gastropathy, dermatopathy .the leaves are

antibacterialand are used for strengthening the teeth and gums.the fruits and seeds are used in

diabetes, diarrhea, pharyngitis, splenopathy, urethorrhea and ringworm (Arya Vaidya Sala.

2004.vol 5. p 225)



C. Ocimum sanctum:-

Demulcent, expectorant, and antiperiodic .Root is febrifuge; seeds are mucilaginous and

expectorant. Leaves are anti-catarrhal, expectorant, fragrant and aromatic.Persons affected

with bad skin diseases, such as itches,ringworm, leprosy, bad blood,etc .should drink the

juice of basil leaves and also apply the same by itself. Dried plant in decoction is a domestic

remedy for catarrh, bronchitis and diarrhea. Leaf-juice poured into the ear is first-rate

remedy for earache (Dr.K.M.Nadkarni 1982 P 865 )

D. Allium cepa:-

The bulbs are antiperiodic, antibacterial ,aphrodisiac, emmenagogue, emollient , expectorant,

carmimnative, stomachic, and diuretic.they are useful in haemorrhoids, dysentery,

flatulence, dyspepsia, colic, jaundice, splenopathy,hepatopathy, asthma, bronchitis,

opthalmia ,vomiting ,malarial fever, lumbago, epilepsy, tumors, wounds, paralysis,

arthralgia and skin diseases(Arya Vaidya Sala2004.vol 1. p 88)

2.3. Review of Biological Activities Reported of Momordica charantia:

1. .Day.c et al .(1990)[21]

Hypoglycemic effect of Momordica charantia.

2. Reyes. B.A.S et al . (2006) antidiabetic potential of Momordica charantia and

Andrographis paniculata.

3. Alessadra.B et al (2008) Antimicrobial activity of Momordica charantia..

4. Alessadra.B . Chemical composition and Antimicrobial activity of Momordica charantia.

Seed essential oil.Fitoterapia 2008; 79;123-125.

5. Batran et al., 2006 performed some toxicological studies of M omordica charantia L. on

albino rats in normal and alloxan diabetic rats.

6. Batran S A E S, El-Gengaihi S E, Shabrawy O A E, Some toxicological studies of

M omordica charantia L. on albino rats in normal and alloxan diabetic rats, Journal of

Ethnopharmacology, 108, 2006, 236±242.



7. Singh .J et al (2004) Momordica charantia fruit juice stimulates glucose and amin acid

uptakes in L6 myotubes

8. Hui-Ling Huang et al (2008) Bitter melon inhibits adipocyte hypertrophy.

9. Ajaya Kumar .S et al (2005) Momordica charantia modulates activities of intestinal and

renal disaccharidases.

10. Anila .L , Vijayalakshmi.N.R(2000) Beneficial effects of Flavonoids from Momordic

charantia.

11. Patil.S.b et al (1998) Antispermatogenic and androgenic activities of Momordica

charantia .

12. Patil.S.b .Antispermatogenic and androgenic activities of Momordica charantia in albino

rats. Journal of Ethanopharmacology 1998;61 ;9-16.

13. Bhavna S et al. (2008)[22]

hypoglycemic and hypolipidemic effect.

14. .Rajasekaran M et al .(1988)[23] antifertility effect in male rats.

2.4 Review of Biological Activities Reported of Eugenia jambolana;

1. Saha.B.P et al (1998) Anti-diarrhoeal activity of Eugenia jambolana.

2. Subramanian et al(2004) Hypoglycemic activity of Eugenia jambolana.

3. Partha.R et al(2008) Effect on Carbohydrate and Lipid metabolism of Eugenia jambolana.

4. Suman.B.S et al (2006)Antihyperglycemic Effect of the fruit pulp of Eugenia jambolana



2.5. Review of Biological Activities Reported of Ocimum sanctum;

1. Juntachote.T, Berghofer.E.(2005) Antioxidative properties of Holy basil and

Galangal.

2. Madhu Kumar et al.(2005) Protection against mercury-induced renal damage in swiss

albino mice by ocimum sanctum.

3. Reddy .S.S et al (2008) Prevention of Insulin resistance by Ocimum sanctum

4. Samson . J et al Biogenic amine changes in brian regions by Ocimum sanctum.

5.

Surender.S et al (1996) Anti-inflammatory activity of Ocimum sanctum.

6. Grover.J.K et al 9-(2004) Alteration in glycogen content and carbohydrate

metabolism in rats by Ocimum sanctum

7. Mohamed Anees.A (2008) Larvicidal activity of Ocimum sanctum.

8. Chattopadhytay RR. (1993)[24]

hypoglycemic effect of ocimum sanctum.

9. Grover j.k .(1990)[25] hepato & cardioprotective action of tulsi.

10. Surender S et al.(1996)[26]anti-inflammtory potential of tulsi.

11. Chattopadhyay RR .(1990)[27]

anti-ulcer activity of tulsi.

12. . Asha M.k et al .(2001)[28]

anthelmintic activity of essential oils.

2.6. Review of Biological Activities Reported of Allium cepa;

1. Helen A.et al .(2000)[29]

anti-oxidant activity of onion.

2. Danish S et al.(2004)[30]

antileishmanial activity of onion.

3. Richa S et al (2008)[31] neuroprotective effect of allium cepa.

4. pyun M.S .(2006)[32]

anti-fungal effect of allium plants.



5. Gabriella.C.et al (2005)Antispasmodic saponins from Allium cepa.

6. Freddy . A.R et al (2006)Antibacterial and Antioxidant of Allium cepa.

7. Dhan .P et al (2007) Free Radical Scavenging Activities of phenols from Onion.

8. Dhan .P Anti-oxidant and Free Radical Scavenging Activities of phenols from

Onion. Food Chemistry 2007;102;1389-1393.

9. Richa shri. Kundan.S.B(2008) Neuroprotective of Allium cepa.

4. RESEARCH ENVISAGED

ral synthetic agents currently being used as antidiabetic agents has severe undesirable

side effects and has failed to correct the fundamental lesion and diabetic complication.

This fact has provoked the WHO expert committee on DM to investigate on antidiabetic

agents from medicinal plants.

Based on literature survey Momordica (fruits),Eugenia jambolana(seeds), Tulsi (leaves),

Allium cepa(juice) have been used traditionally to alleviate increased blood glucose level.

But no such evidence is available for these plants to treat oxidative stress, tissue damage and

complications in DM.

Hence the current research is aimed to

1. Explore the antidiabetic effect of these plants and their combination in alloxan induced

diabetes mellitus in rats.

2. Explore the effect of these plants and their combinations in oxidative stress and tissue

damage in diabetes mellitus and their complications.

3. To establish a suitable mechanism of action of prevention of diabetes mellitus by these

plants.

O



5. Plan of work:

A. Selection of plants

B. Identification, collection of plant material

C. Phytochemical investigation

a. Extraction of the crude drug in a suitable solvent

b. Qualitative chemical examination

c. Quantitative chemical examination

D. Pharmacological evaluation

I. Acute toxicity study and determination of lethal dose

II. Antioxidant activity by-

a. DPPH free radical-scavenging activity

b. Nitric oxide radical-scavenging activity

c. N.B.T.superoxide-scavenging activity

d. Redused glutathione(GSH) activity

e. Lipid peroxidation

III. Antidiabetic activity

IV. Statistical analysis

V. Compilation of work



2. PLANT MATERIALS

Poly Herbal Preparation(Momordica charantia, Eugenia jambolana, Ocimum sanctum,

Allium cepa) :-

These plants were reported to have anti-diabetic activity with

different mechanism . so we have aimed to prepare a polyherbal preparation which could be

active against diabetes through different mechanism.

Momordica charantia (Edwin J E. Sheeja E J. 2006.p.179)

Family: Cucurbitacea

Hindi = karela

Telugu = kasara

English = bitter gourd.

Taxonomical Classification:-

Kingdom -Plantae

Division - Magnoliophyta

Class - Liliopsida

Order - Violales

Family - Cucurbitaceae

Genus -M omardica

Part Used---Dried fruits.

Habitat--- Bitter melon is a vegetable indigenous to subtropical and tropical regions of



South America and Asia This vegetable is also cultivated in the southern part of Kyushu,

Japan, due to its subtropical climate.

Constituents(kokate C.k. 2004.p219)

charantin (stearoidal saponin), momordicin, carbohydrates, mineral matter, alkaloids,

glucosides, saponins and mucilage.

Medicinal Action and Uses(. Grover . J. K. Yadav S. P. 2004)

Antibacterial ativity, Antiviral activity, Anti-HIV activity, Antiherpes activity,

Antipoliovirus activity, Anticancer activity, Abortifacient, antifertility, Anthelmintic study,

Antmalarial activity (Amorim et al), Immunomodulatory activity, Analgesic,

antinflammatory activity, Hypotensive and anti prothrombin activity, Hypocholesterolemic,

anti-oxidant potential and Antirheumatiod.

B. Eugenia jambolana

Taxonomical Classification(;Edwin J E. Sheeja E J. 2006.p235)

Kingdome - Plantae


Class - Liliopsida

Order - Myrtates

Family - Myrtaceae

Genus -Eugenia



Part Used---Dried seeds

Habitat² Throughout India, in forests upto 1800m usually along river blanks and moist

localities,also cultivated as shade trees along roadsides.

Constituents---It contains approximately 70% eugenol,carvacrol, and eugenol-methyl-ether.

Properties And Medicinal Uses(. Arya Vaidya Sala 2004.vol.5 .p225)

---The fruits and seeds are sweets, acrid, sour,tonic, diarrhea,

pharyngitis,splenopathy,urethrorrhea,and ringworm.

C.Ocimum sanctum

Botanical: Ocimum sanctum

Family: Labiatae

Taxonomical Classification(Edwin J E. Sheeja E J. 2006.p193)

Kingdome - Plantae


Class - Liliopsida

Order - Lamiaceae

Family - Lamiaceae

Genus -Ocimum

Part Used---Dried leaves

Habitat---It is a herbaceous, much branched annual plant found throughout india.

The plant is commonly cultivated in garden and also grown near temple.

Constituents(kokate C.k .2004.p.348)

-It contains approximately 70% eugenol,carvacrol, and eugenol-methyl-ether



Medicinal Action and Uses---Used as stimulant, aromatic, anticatarrhal,spasmolytic and

diaphoretic.

D.Allium cepa

Botanical: Allium cepa

Family: Liliaceae

Taxonomical Classification(Edwin J E. Sheeja E J 2006.p.17.).:-

Kingdome -Plantae

Division -Magnoliophyta

Class -Liliopsida

Order -Liliales

Family -Liliaceae

Genus -Allium

Part Used---onion bulbs.

Habitat--- It is richin flavonoids such as quercetin and sulfur compounds,such as allyl

propyl disulphide that have perceived benefitsto human health .

Constituents(Arya Vaidya Sala 2004.vol.1.p.88)

It contains approximately 70% eugenol,carvacrol, and eugenol-methyl-ether.

Medicinal Action and Uses² These compounds possess antidiabetic, antibiotic,

hypocholesterolaemic,fibrinolytic, and various other biological effects..

Onion was also a popular folk remedy. In addition,onion and garlic are rich in sulfur

containing compoundsmainly in the form of cysteine derivatives, viz.

In addition to volatile substances in alliums,there are nonvolatile sulfur-containing peptides

and proteinswhich have been shown to have potential health



benefits.

2.1 Collection and Identification:

Healthy dried fruits of Momordica charantia,dried seeds of Eugenia jambolana,Allium

cepa and dried leaves of Ocimum sanctum were collected from Mandsaur.The

authentification was done by Dr. R. N. Kanpure (Asstt. Prof. / Scientist, Dept. of Fruit

Science, K. N. K. College of Horticulture, Mandsaur). A voucher specimen of,

Momordica charantia.: BRNCP/ C/ 006/ 2008/ Momordica charantia/ hari

Eugenia jambolanaLinn.: BRNCP/ E/ 002/ 2008/ Eugenia jambolana Linn/hari Ocimum sanctum.: BRNCP/ E/ 002/ 2008/ Ocimum sanctum.: Linn/hari Allium cepa.: BRNCP/ E/ 002/ 2008/ Allium cepa.Linn/hari respectively has been

deposited in the Department of Pharmacognosy, BRNCP, Mandsaur.



3. PHYTOCHEMICAL INVESTIGATIONS

3.1 EXTRACTION OF THE CRUDE DRUGS IN A SUITABLE SOLVENT

5.2 EXTRACTION OF Momordica charantia;

The dried fruits of Momordica charantia were coarsely powdered, weighed and filled in

Soxhlet apparatus for extraction. The solvent used was hydroalcholic i.e. 70% ethanol and

30% water. % yield was calculated for each extract after drying. (Kokate. 2000)

Determination of percentage yield

The percentage yield of each extract was calculated by using following formula: -

Weight of ExtractPercentage yield = -------------------------------------- x 100

Weight of powder drug Taken

10Percentage yield = -------------------------------------- x 100

80

Table 3 Percentage Yield of hydro alcholic Extracts (%w/w)

5.EXTRACTION OF dried seeds of Eugenia jambolana

The dried seeds of Eugenia jambolana were coarsely powdered, weighed and filled in

Soxhlet apparatus for extraction. The solvent used was hydroalcholic i.e. 70% ethanol and

30% water. % yield was calculated for each extract after drying. (Kokate. 2000)



Weight of Extract

Percentage yield = -------------------------------------- x 100

Weight of powder drug Taken8





5.2 EXTRACTION of Ocimum sanctum leaves;

The dried Ocimum sanctum leaves were coarsely powdered, weighed and filled in Soxhlet

apparatus for extraction. The solvent used was hydroalcholic i.e. 70% ethanol and 30%

water. % yield was calculated for each extract after drying. (Kokate. 2000)



Weight of Extract


Weight of powder drug Taken

3


60


S.No. Extract % Yield Characteristic

1. Momordica

charantia

12.5 Semi-solid, brown with yellowish shade in

colour, characteristics odour

2. Eugenia

jambolana

8 Semi-solid, dark brown with yellowish shade

in colour, characteristics odour

3 Ocimum

sanctum

5 Semi-solid, dark green in colour ,characteristics odour



3.2 QUALITATIVE CHEMICAL EVALUATION (Khandelwal, 2006; Kokate, 2006)

3.2.1 Alkaloids

i. Dragendorff¶s test

To 2 mg of the ethanolic extract 5 ml of distilled water was added, 2M Hydrochloric acid

was added until an acid reaction occurs. To above solution 1 ml of Dragendorff¶s reagent

was added. Formation of orange or orange red precipitate indicated the presence of alkaloids.

ii. Hager¶s test

To 2 mg of the ethanolic extract taken in a test tube, a few drops of Hager¶s reagent were

added. Formation of yellow precipitate confirms the presence of alkaloids.

iii. Wagner¶s test

2 mg of ethanolic extract was acidified with 1.5 per cent v/v of hydrochloric acid and a few

drops of Wagner¶s reagent was added. Formation of yellow or brown precipitate indicates

the presence of alkaloids.

iv.M ayer¶s test

To few drops of the Mayer¶s reagent, 2 mg of ethanolic extract was added. Formation of

white or pale yellow precipitate indicates the presence of alkaloids.

3.2.2 Carbohydrates

i. Anthrone test

To 2 ml of anthrone reagent solution, 0.5 ml of aqueous extract was added. Formation of

green or blue colour indicated the presence of carbohydrates.

ii. Benedict¶s test

To 0.5 ml of aqueous extract, 5 ml of Benedict¶s solution was added and boiled for 5 min.

Formation of brick red coloured precipitate indicated the presence of carbohydrates.

iii. F ehling¶s test

To 2 ml of aqueous extract, 1 ml mixture of equal parts of Fehling¶s solution A and B were

added and boiled for few minutes. Formation of red or brick red coloured precipitate

indicated the presence of reducing sugar.

iv.M olisch¶s test

In a test tube containing 2 ml of aqueous extract, 2 drops of freshly prepared 20 per cent

alcoholic solution of - naphthol was added. 2 ml of conc. sulphuric acid was added so as to



form a layer below the mixture. Red-violet ring appeared, indicating the presence of

carbohydrates which disappeared on the addition of excess of alkali.

3.2.3 Flavonoids

i. S hinoda¶s test

In a test tube containing 0.5 ml of the ethanolic extract 10 drops of dilute hydrochloric acid

followed by a small piece of magnesium was added. Formation of pink, reddish or brown

colour indicated the presence of flavonoids.

3.2.4 Triterpenoids

i. Liebermann - Burchard¶s test

2 mg of dry extract was dissolved in acetic anhydride, heated to boiling, cooled and then 1

ml of concentrated sulphuric acid was added along the sides of the test tube. Formation of a

violet coloured ring indicated the presence of triterpenoids.

3.2.5 Proteins

i. Biuret¶s test

To 1 ml of hot aqueous extract, 5 to 8 drops of 10 per cent w/v sodium hydroxide solution,

followed by 1 or 2 drops 3 per cent w/v copper sulphate solution were added. Formation of a

violet red colour indicated the presence of proteins.

ii.M illon¶s test

1 ml of aqueous extract was dissolved in 1ml of distilled water and 5 to 6 drops of Millon¶s

reagent were added. Formation of white precipitate which turns red on heating indicated the

presence of proteins.

3.2.7 Saponins

In a test tube containing about 5 ml of an ethanolic extract, a drop of sodium bicarbonate

solution was added. The test tube was shaken vigorously and left for 3 min. Formation of

honeycomb like froth indicated the presence of saponins.



3.2.8 Steroids

i. Liebermann-Burchard¶s test

2 mg of dry extract was dissolved in acetic anhydride, heated to boiling, cooled and then 1

ml of concentrated sulphuric acid was added along the sides of the test tube. Formation of

green colour indicated the presence of steroids.

ii. S alkowski reaction

2 mg of dry extract was shaken with chloroform, to the chloroform layer sulphuric acid was

added slowly by the sides of test tube. Red colour indicated the presence of steroids.

3.2.9 Tannins

To 1-2 ml of the ethanolic extract, few drops of 5 per cent w/v FeCl3 solution were added. A

green colour indicated the presence of gallotannins, while brown colour indicated the

presence of pseudotannins.

3.2.10 Starch

0.01g of Iodine and 0.075 g of potassium iodide were dissolved in 5 ml of distilled water and

2 to 3 ml of an ethanolic extract was added. Formation of blue colour indicated the presence

of starch.



5.4 PHARMACOLOGICAL STUDIES

Procurement and selection of animals

Wistar albino rats of either sex weighing between 130 ± 180 gm of either sex were obtained

from B.R.N.C.P. Mandsaur animal house. These animals were used for the acute toxicity and

antidiabetic activity. The animals were stabilized for 1 week; they were maintained in

standard condition at room temp; 60 ± 5% relative humidity and 12 h light dark cycle. They

had been given standard pellet diet and water ad-libitum throughout the course of the study.

The animals were handled gently to avoid giving them too much stress, which could result in

an increased adrenal out put.

ACUTE TOXICITY STUDIES

The acute toxicity study was carried out in adult female albino rats by ³fix dose´ method of

OECD (Organization for Economic Co-operation and Development) Guideline No.420.

Fixed dose method as in Annex 2d: Test procedure with a starting dose of 2000 mg/Kg body

weight was adopted. The animals were fasted overnight and next day polyherbal preparation

(suspended in 0.5 % w/v sodium CMC) were administered orally at dose level 2000 mg/kg.

Then the animals were observed continuously for three hour for general behavioral,

neurological, autonomic profiles and then every 30 min for next three hour and finally for

mortality after 24 hour till 14 days. The observations were tabulated according to µIrwin¶s

Table¶

Selection of doses

For the assessment of antidiabetic activity, two dose level were chosen in such a way that,

one dose was approximately one tenth of the maximum dose during acute toxicity studies,

and a high dose, which was twice that of one tenth dose (200mg/kg, 400mg/kg)



4.2 EX VIVO STUDY

4.2.1 Determination of Glutathione (GSH)

Liver was made free from remaining tissues and kept in cold saline. It was then homogenized

in 2 ml of normal saline.

4.2.1.1 Solutions prepared

i. DTNB reagent: 4 mg of DTNB in 10 ml of 1 per cent solution of tri-sodium citrate.

ii. Trichloro acetic acid: 10 per cent solution in distilled water.

iii. Disodium hydrogen phosphate: 0.3 M solution was prepared in distilled water.

4.2.1.2 Preparation of stock solution

i. 30 mg of glutathione was accurately weighed and dissolved in distilled water and volume

made upto 100 ml with distilled water.

ii. 10 ml of above solution was futher diluted to 100 ml with distilled water to make a stock

solution of 30g/ ml.

iii. Aliquots of (0.3 to 6.9 ml) were taken and 2 ml of 0.3 M disodium hydrogen phosphate

was added to all aliquots.

iv. To all aliquots 0.5 ml of DTNB was added and volume was made up to 10 ml with

distilled water.

v. Absorbance was measured at max

= 410 nm as depicted in figure 18.

The standard curve of glutathione was tabulated in table 17 and calibration curve is depicted

in figure 19.

4.2.1.3 GSH determination in liver homogenates.

i. Liver tissue was homogenized in 1 ml of distilled water.

ii. Homogenate was centrifuged at 6000 g for 1 h.

iii. 0.5 ml of above supernatant was mixed with 1 ml of 0.6 m disodium hydrogen

phosphate.

iv. 0.5 ml of DTNB reagent was added.

v. Absorbance of this solution was measured against blank which was prepared similarly

but instead of liver homogenate distilled water was added.



Level of GSH in various treatment groups is tabulated in table 18 depicted in figure 20

4.2.2 Determination of Lipid Peroxidation (Yazdanparast et al., 2007)

Lipid peroxidation products such as MDA (Malondialdehyde) are generated under high

levels of un-scavenged free radicals. These products may be important in the pathogenesis of

vascular complications in diabetes mellitus. Increased MDA level is associated with tissue

damage. In DM the level of lipid peroxidation in the tissue is higher than non-diabetic rats.

Following procedure was followed

i. Liver was separated and kept in ice cold saline.

ii. It was homogenized in 10 volumes of 50 mm sodium phosphate buffer (pH 7.4)

iii. The homogenate was centrifuged at 10000 g for 15 min.

iv. 0.5 ml of supernatant of the tissue was mixed with 2.5 ml of 10 per cent TCA.

v. It was centrifuge to remove the proteins.

vi. To clear supernatant (2 ml) 1ml of 0.67 per cent w/v thiobarbituric acid was added.

vii. The absorbance of the mixture was taken at max = 532 nm.

viii. Sample reading (n mole/ml of the supernatant) was correlated with regression equation

y=0.0055x+0.0006 taken from the Zeptometrix assay kit.

The effect of various extracts on lipid peroxidation is tabulated in table 19 and depicted in

figure 21.

4.2.3 Determination of Advanced Oxidation protein Products (AOPP) (Yazdanparast

et al., 2007; Kalousová et al., 2002)

Structural changes in proteins are considered to be among the molecular mechanism leading

to progression and development of diabetes and its complication. An overload of ROS is

known to modify proteins and to generate AOPP products that are presently considered as

markers of oxidative injury to proteins. AOPP are also considered as novel markers to

estimate the degree of oxidant-mediated protein damages.


i. Liver was separated and kept in cold saline and trimmed of adipose tissue.

ii. Finely minced and homogenized in 50mM phosphate buffer, pH 7.4. It was homogenised

in 10 volumes.



iii. The homogenate was then centrifuged at 10,000 x g for 15 min.

iv. 0.4 ml of pancreatic supernatant was treated with 0.8 ml phosphate buffer saline (PBS)

solution.

v. After 2 min, 0.1 ml 1.16M potassium iodide (KI) was added to the tube followed by 0.2

ml of acetic acid.

vi. The absorbance of the reaction mixture was immediately recorded at 340 nm against the

blank solution containing 1.2 ml PBS, 0.1 ml of KI, and 0.2 ml of acetic acid.

vii. The concentration of AOPP for each sample was calculated by using the extinction

coefficient of 26l mMí1

cmí1

and the results were expressed as nmol/mg protein.

The effect of various extracts on AOPP is tabulated in table 20 and depicted in figure 22.

4.2.4 HEPATIC GLYCOGEN MEASUREMENT (Musabayane et al., 2005)

Measurement of glycogen is based on of digestion of the tissue in hot concentrated KOH and

then precipitation of the glycogen with ethanol. The precipitated glycogen was then weighed

by the method of subtraction.


i. 1 g of the animal liver tissue was digested with KOH (30 per cent) by heat the tube in

water bath for about 20 to 30 minutes.

ii. The homogenate was then centrifuged and the supernatant was decanted in another test

tube.

iii. From the supernatant the glycogen was precipated by adding absolute ethanol drop wise

till complete precipitation and kept in cold for 15 min.

iv. The precipitated glycogen was then weighed.

The glycogen content of liver of different groups is tabulated in table 21 and depicted in

figure 23.



4.2.5 SERUM BIOCHEMICAL PARAMETERS

4.2.5.1 DETERMINATION OF SERUM UREA (Beale, 1961)

4.2.5.1.1 PRINCIPLE

Urea reacts with ortho-pthalaldehyde and Napthylethylene diamine to form an orange

coloured complex. The rate of formation of this complex is directly proportional to urea

concentration and it is measured by a fixed time mode at 505 nm.

4.2.5.1.2 REACTION

Urea + OPA NH4 + + H2O

NH4 +

+ NED Orange Coloured Complex

4.2.5.1.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum urea level by Beacon,

U rea determination kit .

The serum urea concentration in normal, diabetic and diabetic treated rats is depicted and

tabulated in figure 24 and table 22 respectively.

4.2.5.2 DETERMINATION OF SERUM CREATININE (Owen, 1954)

4.2.5.2.1 PRINCIPLE

Creatinine is a waste product from muscle by way of high energy storage compound. Serum

creatinine levels are more specific and sensitive indicator of renal function rathet than urea.

Creatinine reacts with picric acid in alkaline medium to form an orange coloured complex

and the rate of change of its absorbance is measured at 505 nm.

4.2.5.2.2 REACTION Creatinine + Picric Acid

4.2.5.2.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum urea level by Beacon,

C reatine determination kit .

NaOH



The serum creatinine concentration in normal, diabetic and diabetic treated rats is depicted

and tabulated in figure 25 and table 22 respectively

4.2.5.3 DETERMINATION OF SERUM ASPARTATE TRANSAMINASE/

GLUTAMATE OXALO ACETATE ( AST/ SGOT) (Henry, 1959)

4.2.5.3.1 PRINCIPLE

Glutamate oxalo acetate transaminase is localized in normal muscle cells in mitochondria

and cytoplasm. When cells are damaged or cellular nutrition disturbed, the permeability of

the cell increases and transaminases are released into the blood stream. An increased serum

transaminases level indicates cellular death. Its determination is particularly significant in the

diagnosis of myocardial infarction. The kit works on kinetic estimation method in which the

reduction of substract NADH to NAD+

which is measured when the reaction is in progress at

340 nm.

4.2.5.3.2 REACTION

L-Aspartate + -Ketoglutarate Oxaloacetate + L- Glutamate

Oxaloacetate + NADH + H+

L-Malate + NAD+

4.2.5.3.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum AST level by Liquizyme ,S GOT determination kit .

The serum concentration in normal, diabetic and diabetic treated rats is depicted and


4.2.5.4 DETERMINATION OF SERUM ALANINE TRANSAMINASES/

GLUTAMATE PYRUVATE TRANSAMINASES (ALT/ SGPT) (Henry, 1959)

4.2.5.4.1 PRINCIPLE

Alanine transaminase is present in high concentration in liver, kidneys, heart and skeletal

muscle tissue. It is also present in lungs, spleen, pancreas, brain and erythrocytes at a lower

concentration. Primary liver disease (cirrhosis, obstructive jaundice, carcinoma, viral or toxic

hepatitis) as well as liver damage secondary to other causes result in elevated GPT levels.



4.2.5.4.2 REACTION

1. In this reaction, L-alanine and alpha-ketoglutarate react in the presence of GPT

L-Alanine Pyruvate

+ +

-Ketoglutarate

in the sample to yield pyruvate and L-glutamate.

2. Pyruvate is reduced by lactate dehydrogenase to yield lactate wit the oxidation of

NADH to NAD. The reaction is monitored by measurement of the decrease in absorbance to

NADH at 340 nm.

Pyruvate Lactate

+ +

NADH

The rate of reduction in absorbance is proportional to GPT activity in sample.

4.2.5.4.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum ALT level by Liquizyme ,

S GP T determination kit .

The serum concentration in normal, diabetic and diabetic treated rats is depicted and


4.2.5.5 DETERMINATION OF SERUM PROTEINS (Gornall, 1948)

4.2.5.5.1 PRINCIPLE

Serum Protein was estimated by 1948. An alkaline medium, total protein reacts with the

copper of biuret reagent causing an increase in absorbance, at 546 nm is due to formation of

the violet coloued complex and it is directly proportional to the concentration of protein

present in the sample.

4.2.5.5.2 REACTION

Total protein

GPT

LDH

Alk. Medium



4.2.5.5.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum protein level by

Liquizyme ,Protein determination kit .

The serum protein concentration in normal, diabetic and diabetic treated rats is depicted and


4.2.5.6 DETERMINATION OF SERUM CHOLESTEROL (Zurkowski, 1962)

4.2.5.6.1 PRINCIPLE

Cholesterol determination is used for the diagnosis and monitoring of lipid metabolism

disorders

4.2.5.6.2 REACTIONThe measurement of cholesterol is based on the following enzymatic reaction.

Cholesterol esters + H2O Cholesterol + fatty acids

Cholesterol + O2 Cholest-4-en-3-one + H2O2

H2O2 + hydroxybenzoate + 4-Aminoantipyridine Red complex2O

The intensity of the red complex is proportional to the total cholesterol present in the sample.

4.2.5.6.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum cholesterol level by

Beacon, C holesterol determination kit .

The serum triglycerides concentration in normal, diabetic and diabetic treated rats is depicted

and tabulated in figure 29 and table 22 respectively

4.2.5.6 DETERMINATION OF SERUM TRIGLYCERIDES (Bucolo, 1975)

4.2.5.6.1 PRINCIPAL

Increase in serum triglycerides levels are seen in cases of liver destruction due to hepatitis,

extra hepatic biliary obstructions as well as cirrhosis. An increase synthesis of VLDL

CHE

CHOD

POD



resulting from diabetes mellitus also plays roll in increase of serum triglycerides level.

Triglycerides incubated with lipoprotein lipase are hydrolysed to free fatty acids and

glycerol. Glycerol kinase catalyses the conversion of glycerol and ATP to glycerol-3-

phosphate and ADP. The glycerol-3-phosphate gets oxidized to dihydroxy acetone phosphate

by glycerol phosphate oxidase. Hydrogen peroxideformed in this reaction with the help of

peroxidase reacts with 4- aminoantipyrine to give a purple coloured complex which is read at

546 nm.

4.2.5.6.2 REACTION

Triglycerides

Glycerol + ATP

Glycerol-3-P + O2 DHAP + H2O2

H2O2 + 4-aminoantipyrine PurpleQuinonimine.

4.2.5.6.3 PROCEDURE

Unhaemolysed serum sample was used for determination of serum triglycerides level by

Liquizyme , T riglycerides determination kit .

The serum triglycerides concentration in normal, diabetic and diabetic treated rats is

depicted and tabulated in table 30 and table 22 respectively

4.3 IN VITRO STUDY

DPPH (2, 2-diphenyl-1-picrylhydrazyl) RADICAL SCAVENGING ACTIVITY

The molecule of 1,1-diphenyl-2-picrylhydrazyl (EE-diphenyl-F-picrylhydrazyl; DPPH) (1)

is characterised as a stable free radical by virtue of the delocalisation of the spare electron

over the molecule as a whole, so that the molecules do not dimerise, as would be the case

with most other free radicals. The delocalisation also gives rise to the deep violet colour, as

shown in figure 31 characterised by an absorption band in ethanol solution centred at about

LP Li ase

l cer l kina e

Gly-3-P Oxidase

POD



515 nm. When a solution of DPPH is mixed with that of a substance that can donate a

hydrogen atom, then this gives rise to the reduced form (2) with the loss of this violet colour

(although there would be expected to be a residual pale yellow colour from the picryl group

still present).

1: Diphenylpicrylhydrazyl (free radical) 2: Diphenylpicrylhydrazine

Following method was followed was followed

4.3.1 Preparation of DPPH stock solution

3.94 mg of DPPH was dissolved in q.s 100 ml of methanol to prepare 0.1 mM solution. The

solution was wrapped in aluminium foil and kept in dark.

4.3.2 Preparation of test solution

1. Firstly 100 mg/ml of stock solution was prepared and from it various concentrations of

extracts ranging from 2-60 mg/ml was prepared.

2. Then 2.5 ml each of DPPH and test solutions were mixed and then their absorbance was

measured at fixed max= 517 nm after incubation for 30 min in dark.

3. The concentration corresponding to 50 per cent reduction in the absorbance of DPPH

was considered as IC50.

B) N.B.T Superoxide Scavenging Activity

This was determined by the NBT (Nitro blue tetrazolium) reduction method. The assay was

based on the capacity of the sample to inhibit blue formazan formation by scavenging the

Yellow Violet



superoxide radical generated in riboflavin-light-NBT system. The reaction mixture contains

EDTA, riboflavin, nitro blue tetrazolium (NBT), various concentrations of extract and

phosphate buffer (pH 7.6) in a final volume of 3 ml. The tubes were uniformly illuminated

with an incandescent lamp for 15 min and absorbance was measured at 560 nm before and

after illumination. The percentage inhibition of superoxide generation was measured by

comparing the absorbance values of control and those of the test compound.

Preparation of the test sample

100 mg of aqueous extract was dissolved in100ml of ethanol separately to make stock

solution of 1000 Qg/ml, which is further diluted with ethanol to get 10, 20, 30, 40, 50,

100Qg/ml.

Preparation of reagents

Phosphate buffer: 200 ml of phosphate buffer of pH 7.6 was prepared according to

IP.

Riboflavin solution: 5 mg riboflavin was dissolved in 25 ml phosphate buffer.

EDTA solution: 402 mg EDTA was dissolved in 10 ml phosphate buffer.

NBT solution: 5 mg NBT was dissolved in 5 ml phosphate buffer.

Protocol for Estimation of Super Oxide scavenging Activity

100 Ql riboflavin solution 200 Ql EDTA solution, 200 Ql ethanol and 100 Ql NBT solution

were mixed in a test tube and the reaction mixture was diluted up to 3 ml with phosphate

buffer. The absorbance of solution was measured at 590 nm using phosphate buffer as blank

after illumination for 15 minutes. This was taken as control reading.

y Screening of test sample of different concentration of aqueous extracts: 100 Ql

test sample, 100 Ql riboflavin, 200 Ql EDTA, 200 Ql ethanol and 100 Ql NBT

solution were mixed in a test tube and the reaction mixture was diluted upto to



3 ml with phosphate buffer. The absorbance of solution was measured after

illumination for 15 minutes at 590 nm.

y Percentage reduction was calculated and this activity was expressed as an

effective concentration at 50% (IC50) i.e. the concentration of the test sample

required to give 50% decrease in the absorbance compared to that of control

reading. IC50 was calculated from the graph showing 50% inhibition (See Table:

7)

Control absorbance ± Test absorbance

% Reduction = ---------------------------------------------------------- x 100

Control absorbance

5.7 ANTIDIABETIC ACTIVITY

Diabetogenic agent: - alloxan monohydrate

Rationalized dose: - 125 mg/kg

Reference drug: - Glibenclamide

Induction of diabetesAnimals were fasted for 24 hours then a single intra peritoneal injection of freshly prepared

alloxan (125 mg/kg dissolved in 0.9% saline) was injected. After that the animals were left

aside for 4 hrs and then 10% glucose solution was placed in the cages for 24 hrs. The

diabetes was confirmed by estimation of blood glucose level (BGL) at 3 rd day. Rats having

BGL more than 250 mg/dl were used for study

Grouping of animals

Group I: Kept as Normal control i.e. neither treated with extract or standard.

Group II: Kept as Negative control i.e. treated with alloxan (125 mg/kg).i.p.

Group III: Treated with standard oral hypoglycemic drug i.e. Glibenclamide

(0.5 mg/kg) after 3rd

day of treatment with alloxan (125 mg/kg i.p.).

Group IV: Treated orally with 200 mg/kg of polyherbal preparation after 3rd

day of

treatment with alloxan (125 mg/kg i.p.).



Group V: Treated orally with 400 mg/kg of polyherbal preparation after 3rd

day of

treatment with alloxan (125 mg/kg i.p.).

Determination of Antidiabetic activity

Test samples were given orally using oral gastric gavages to the animals once before food

was given. The blood glucose concentrations of the animals were measured at the beginning

of the study and the measurements were repeated on 3rd, 7th and 10th day after the initial of

the experiment. The inference was made by comparing Blood Glucose Level, Body Weight,

Serum Creatinine, Blood Urea, Serum Triglycerides and Serum Total Cholesterol with

treated and negative control (alloxan treated). Observations mentioned in Table 9, 10, 11

( Vogel, 2002)

6. RESULTS

The following are the results of my work

Qualitative test shows presence of various biochemical as tabulated in table 5.

Table 5 Result of Qualitative Test

S.No. EXPERIMENT polyherbal

preparation

1. Test for carbohydrates +

2. Test for gum and mucilage +

3. Test for Proteins -

4. Test for Alkaloids +

5. Test for Glycosides +

6. Test for Steroids +



7. Test for Tannins +

8. Test for Saponins +

9. Test for Flavanoids +

10. Test for Anthraquinones +

11. Test for Furanoids -

12. Test for Coumarin -

13. Test for Terpenoids +

+ sign indicates presence where as ± indicates absence of constituents

Acute Toxicity studies:

Acute Toxicity studies on female rat¶s shows no mortality at a dose of 2000 mg/kg, during a

time period of 14 days (Table 6). The Behavioral, Neurological, Autonomic responses were

studied for a time period of 6 hrs of toxicity study. During the study no noticeable responses

were seen in the rats. This helps to predict that it does not contain any type of toxicity and is

safe .

Table 6 For Mortality in Acute Toxicity Study

S.No. Treatment

(hydroalcholicextract )

70:30

Dose

(mg/kg)

Number of

animals

Mortality

Toxicity

Profile

After 24

Hrs.

After7Days

After 14Days

1.

Leaves Extract.

orally

2000

5

0

0

0

Safe

2

Flower Extract.orally

2000

5

0

0

0

Safe



Invitro Antioxidant studies of polyhrbal preparation;

Table 8 DPPH Free Radical Scavenging Activity

Standard absorbance: - 0.973 nm of blank i.e. DPPH and 0.019 nm of standard i.e. ascorbic

acid (98.05% reduction). NN== 66

CCoonncc.. polyherbal preparation

AAbbssoorrbbaannccee %% R R eedduuccttiioonn

1100 ..663399 3333..1155

2200 ..554411 4433..4411

3300 ..446622 5511..6677

4400 ..332299 6655..5588

5500 ..223300 7755..9944

110000 ..112288 8866..6611

CCoonnttrrooll aabbssoorrbbaannccee==..995566

Table 9 N.B.T Superoxide Scavenging Activities

Standard absorbance 0.836 nm of blank and 0.035 nm of ascorbic acid (% reduction is 95.9)N=3

CCoonncc.. PPoollyyhheerrbbaall pprreeppaarraattiioonn

AAbbssoorrbbaannccee %% R R eedduuccttiioonn

1100 ..776622 1122..8811

2200 ..665577 2244..8822

3300 ..554499 3377..1188

4400 ..443366 5500..1111

5500 ..336677 5588..0000

110000 ..222266 7744..1144

Control absorbance=.874



Table 11 Antidiabetic activity of extracts on BGL (mg/dl)

GROUP REGIMEN 0 DAY 3 DAY 7 DAY

BGL BGL BGL

G-I

Normal control

Normal saline (0.9%) 8888..6666 11..2288 8888..8800 00..9944 8866..88 22..22

G-II

Negative control

Alloxan (125 mg/kg) 8877..3333 11..4400 335544..0000 1188..8844**** 440066..8833 1144..

G-IIIPositive control

Alloxan ( 125mg/kg) + Glibenclamide

(0.5mg/kg)

8877..5500 22..7711 333399..6677 1166..4488**** 113344..6677 33..11

G-IV

Drug Treated

Alloxan ( 125

mg/kg) + PPoollyyhheer r bbaall

ppr r eeppaar r aattiioonn (200 mg/kg)

8877..6666 22..2266 332277..6677 1166..1111**** 330000 1111..9933

G-V

Drug Treated

Alloxan ( 125

mg/kg) + PPoollyyhheer r bbaall

ppr r eeppaar r aattiioonn (400 mg/kg)

9900..5500 11..338899 332255..3333 1122..4488**** 227777..66 1111..11



N=5 ** p < 0.01, *** p < 0.001 vs Negative control Value expressed in means SEM

Table 12 Antidiabetic activity of extracts on basis of body wt. as parameter

GROUP Treated 0 DAY 3 DAY 7 DAY

G-I Normal control 116644..4433

55..2200116655..1177 44..7700 116677..5500 55..3344

G-II Negative control (alloxan) 116600 55..3333 112266..3333 55..1144 111166..6677 22..6622

G-III Positive control

(gilbenclamide)

115566..1177

33..335511113311..3333

44..8822****114433..5500

44..0066****

G-IV Drug Treated PPoollyyhheer r bbaall

ppr r eeppaar r aattiioonn 200

115533 55..77 112288..6677

55..1111****113388..3333

55..2277****

G-V Drug Treated PPoollyyhheer r bbaall

ppr r eeppaar r aattiioonn. 400

115599 77..4422 113333..11 77..99**** 115511..8800 66..9922

N=6 * p < 0.01 ** p < 0.001 vs Negative control Value expressed in ms SEM



Table 13 Antidiabetic activity of extracts on basis of biochemical parameter

GROUP TREATED UREA CREATINI

NE

TRIGLYCERID

ES

G-I Normal control 42.83 11..4400 .7 ..0077 86.83 22..0088

G-II Negative control 66.33 22..6600 1.75 ..1166 159.30 11..9900

G-III Positive control 25.67 11..5500** .65 ..0077** 73.17 22..2244**

G-IV Drug TreatedPPoollyyhheer r bbaall ppr r eeppaar r aattiioonn.

200

39.83 11..8811** 1.15 ..1122** 98.50 22..2299**

G-V Drug Treated

PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn.

400

31.67 11..1144** .71 ..0066** 87.67 11..7722**

N=6 * p < 0.01, vs Negative control Value expressed in means SEM



GROUP TREATED AST ALT TOTA

PROT

G-I Normal control 138.30 22..4455 54.83 2.12 5.80

G-II Negative control 341.80 66..4466 114.20 22..9977 3.71

G-III Positive control 115.30 22..8822** 48.67 1.99* 7.26

G-IV Drug Treated

PPoollyyhheer r bbaall


157.00 33..3399** 69.33 ..007766** 5.90

G-V Drug Treated

PPoollyyhheer r bbaall


124.00 11..3399** 53.67 1.20* 6.39



Table 17. Calibration curve for reduced glutathione (GSH)

S.No. Volume of Aliquot

(ml)

Concentration

(g/ml)

Absorbance

( max = 410 nm)

1 0 0 0

2 0.3 0.9 0.036

3 0.9 2.7 0.091

4 1.5 4.5 0.171

5 2.1 6.3 0.252

6 2.7 8.1 0.334

7 3.3 9.9 0.433

8 3.9 11.7 0.508

9 4.5 13.5 0.601

10 5.1 15.3 0.649

11 5.7 17.1 0.696

12 6.3 19.9 0.715

13 6.9 20.7 0.728

Table 18. GSH levels in various treatment groups

S.No. Groups Sample absorbance GSH concentration

(g/ml)

1 G-I 0.692 17.86

2 G-II 0.238 5.89

3 G-III 0.386 9.98

4 G-IV 0.314 7.86

5 G-V 0.365 8.78

**p<0.01 highly significant with respect to diabetic control,

**p< 0.001 highly significant with respect to diabetic control



Table 19. Effect of various extracts administration on lipid peroxidation

S.No. Groups Sample absorbance MDA concentration (n mole/ml)

1 G-I .006 1.28

2 G-II .128 23.15

3 G-III .038 6.18

4 G-IV .041 7.36

5 G-V .056 9.68

***p<0.001 highly significant with respect to diabetic control

Table 20. Effect of various extracts on Advanced Oxidation Protein Products.

Values are in mean

SEM; n=6

###p<0.001 significant with respect to non-diabetic control, *p<0.05 significant with respect to diabetic

control, ***p<0.001 significant with respect to diabetic control

Treatment

(400 mg/kg)

AOPP (nmol/mg protein)

G-I .48 ..0022

G-II .86 ..0033

G-III .67 ..0033

G-IV .73 ..1122

G-V .65 ..0033



Table 21. Effect of various extracts on the terminal hepatic glycogen content

Treatment (400 mg/kg) Liver glycogen mg/g tissue

G-I 13.86 ..1122

G-II 3.68 11..2200

G-III 16.74 ..6600

G-IV 11.63 22..66

G-V 14.86 66..3300

Values are in mean¡

SEM; n=6# p<0.05, ### p<0.001 values significant with respect to non-diabetic control, *p<0.05, ***p<0.001 values

significant with respect to diabetic control (One-way ANOVA followed by a Bonferroni test)

Antidiabetic activity

Poly herbal preparation at a dose of 400 mg/kg showed a decrease in glucose level

highly i.e. brought the BGL at near about normal on 11th day of diabetes.

The decreasing order of the BGL on 10th day of initiation of study is

PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn. 400 > PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn. 200

Effect on Body weight of animals during study period

Body weight of rats showed a decline initially but after 10 days of treatment, the

treated group of Poly herbal preparation showed increase in body weight.

The increase in body weight of animal treated with the leaf extract was more than

flower extract .

Effect on Blood profile of animals after study period

On estimation of blood biochemical parameters a varying effect has been obtained

Blood Urea estimation showed a great decrease in the urea level .PPoollyyhheer r bbaall

ppr r eeppaar r aattiioonn. 400 > PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn. 200

The serum creatinine level was maintained at a normal range i.e. 0.8-1.2 mg/100ml.

Poly herbal preparation 400 mg/kg has the same triglyceride level as of standard 7th

day. The activity in the order of PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn 400 > PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn.

200



On estimation of serum cholesterol results showed a decrease in the cholesterol level

but when compared with normal rats it was not in significant range i.e. it is far more

high near about 95-100. But in comparison with diabetic rats the extracts showed

LIST OF CHEMICALS

S .NO NAME OF THE

CHEMICAL

NAME OF THE MANUFACTURE GRADE

1 ALLOXAN Spectrochem Pvt. Ltd, Bombay Analytical

grade

2 GLIBENCLAMIDE Torrent Pharmaceutical Pvt.. Ltd.,

Mehsana

Analytical

grade3 NORMAL SALINE Inven Pharmaceutical pvt. Ltd. Analytical

grade

4 DTNB Merck Pvt. Ld Analytical

grade

5 GLUTATHIONE Merck Pvt. Ld Analytical

grade

6 THIOBARBITURIC ACID Loba Chemie Pvt. Ltd. Analytical

grade

7 DIETHYL ETHER Loba Chemie Pvt. Ltd. Analytical

grade

8 ACETONE Loba Chemie Pvt. Ltd Analytical

grade

9 SODIUM HYDROXIDE Loba Chemie Pvt. Ltd Analytical

grade

10 HEXANE Loba Chemie Pvt. Ltd Analytical

grade

11 PETROLEUM ETHER Loba Chemie Pvt. Ltd Analytical

grade



activity in the order as PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn. 400 > PPoollyyhheer r bbaall ppr r eeppaar r aattiioonn. 200

1. LIST OF EQUIPMENTS

12 ETHANOL Loba Chemie Pvt. Ltd Analytical

grade

S .NO. NAME OF EQUIPMENT U S ED NAME OF MANUFACTURE

1 Centrifugation machine Remi

2 Electronic balance Shimadzu

3 pH meter PE DPL Kota

4 Hot plate Lab House

5 Oven B House

6 Heating mantle Lab Hosp

7 UV chamber Perfit India

8 Vacuum Oven Lab House

9 Glucose Strips Accu- check

10 Autoclave Lab House

11 Soxhlet Apparatus ASGI

12 Heater Bajaj

13 Microtone American Optical Company, New York

14 Light Microscope BioXL- Research Microscope

15 Microtone Knife Sharpner Spencer

13 UV spectrophotometer Thermospectronic

final mahendra[1]

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