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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
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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
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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
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Figure 1. Metabolic disturbances leading to diabetic coma in type I DM
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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.
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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
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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
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Figure 2. Late complications of DM (Kumar et al., 2005)
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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.
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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)
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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)
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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).
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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.
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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 - -
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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 - -
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sanctum ;Tulsi
Phyllanthusniruri
Jangli amla
Hypoglycemicand antidiabetic activity
Bioflavanoids,Vitamin C,
emblicanin A, B
-
Punica grantumAnar
Antidiabetic-
-
S wertia chiraytiaChirata
Hypoglycemic actionAntihyperglycemic action
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
Hypoglycemic actionAntihyperglycemic action
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 - -
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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
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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)
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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.
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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
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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.
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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
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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
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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
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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
Division - Magnoliophyta
Class - Liliopsida
Order - Myrtates
Family - Myrtaceae
Genus -Eugenia
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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
Division - Magnoliophyta
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
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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
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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.
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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)
Determination of percentage yield
The percentage yield of each extract was calculated by using following formula: -
Weight of Extract
Percentage yield = -------------------------------------- x 100
Weight of powder drug Taken8
Percentage yield = -------------------------------------- x 100100
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Table 3 Percentage Yield of hydro alcholic Extracts (%w/w)
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)
Determination of percentage yield
The percentage yield of each extract was calculated by using following formula: -
Weight of Extract
Percentage yield = -------------------------------------- x 100
Weight of powder drug Taken
3
Percentage yield = -------------------------------------- x 100
60
Table 3 Percentage Yield of hydro alcholic Extracts (%w/w)
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
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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
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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.
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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.
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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)
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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.
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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.
Following procedure was followed
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.
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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.
Following procedure was followed
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.
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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
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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
tabulated in figure 26 and table 22 respectively.
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.
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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
tabulated in figure 27 and table 22 respectively.
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
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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
tabulated in figure 28 and table 22 respectively.
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
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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
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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
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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
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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.).
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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 +
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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
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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
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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
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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
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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
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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
ppr r eeppaar r aattiioonn. 200
157.00 33..3399** 69.33 ..007766** 5.90
G-V Drug Treated
PPoollyyhheer r bbaall
ppr r eeppaar r aattiioonn. 400
124.00 11..3399** 53.67 1.20* 6.39
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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
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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
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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
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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
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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
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