Professional Documents
Culture Documents
Ravi Kumar PHD Theisis
Ravi Kumar PHD Theisis
Ravi Kumar PHD Theisis
1
INTRODUCTION
complications that results in significant morbidity and mortality. The increasing number
of ageing population, consumption of calories rich diet, obesity and sedentary life style
have led to increase the number of diabetes worldwide. The current treatment, although
provide a good glycemic control but do a little in preventing complications (Vats et al.,
2004). Besides, these drugs are associated with side effects (Rang et al., 1991). There is
an increased demand to use natural products with antidiabetic activity due to the side
effects associated with the use of insulin and oral hypoglycemic agents (Holman et al
1991& Kameswara Rao et al 1997).The World Health Organization (WHO) (1980) has
lack safe modern drugs (Upathaya et al.,1991 ). The pharmaceutical drugs are either too
biguanides are also associated with side effects. (Rang et al., 1991). The term is derived
from Greek words "diabetes" means to pass through," Mellitus" means honey or related
2
and postprandial blood glucose levels. If this imbalanced homeostasis does not return to
normalcy and continues for a protracted period of time, it leads to hyperglycemia that in
due course turns into a syndrome called diabetes mellitus. There are two main categories
of this disease. Type 1 diabetes mellitus also called insulin-dependent diabetes mellitus
(IDDM) and Type 2, the noninsulin dependent diabetes mellitus (NIDDM). IDDM
represents a heterogenous and polygenic disorder, with a number of non-HLA loci (about
20) contributing to the disease susceptibility Lernmark and ott (1998). Though this form
of diabetes accounts for 5 to 10% of all cases, the incidence is rapidly increasing in
specific regions. It is estimated that incidence of Type 1 diabetes will be about 40%
higher in the year 2010 than in 1997 (Onkamo et al.,1999) and yet there is no identified
Schatz et al., 2000). NIDDM is far more common and results from a combination of
defects in insulin secretion and action. This type of disease accounts for 90 to 95% of all
secretion, reduced insulin-mediated glucose uptake and utilization (De Fronzo, 1997;
Polonsky ,1996 )
Diabetes mellitus is a metabolic disorder in which the body does not produce or properly
use insulin . It causes disturbances in carbohydrate , protein , and lipid metabolism and
Zito, 2004) In practical terms diabetes mellitus is condition in which cells are starting in
the set of glucose. During diabetes a profound alteration in the concentration and
3
composition of lipid occurs. The global figure of people with Diabetes was estimated to
affect 177 million people worldwide in 2000 and this figure is projected to increase to
derived from Greek words "diabetes" means to pass through," Mellitus" means honey or
changes in lipid and protein and long term irreversible vascular changes .These include
leading to heart diseases, stroke and peripheral vascular disease) which are present in the
non-diabetic population but have a 2-5 fold increase in diabetic subjects (zimmet and
Alberti., 1997)
and treatment diabetics can live normal, healthy lives .Normally, the body gets a major
source of energy from glucose ,a single sugar that comes from foods high in simple
4
sugar and starch are digested in stomach, they enter the bloodstream in the form of
glucose .The glucose in the blood stream becomes a potential source of energy for the
entire of body. In diabetes, there is too much glucose in the blood .When glucose builds
in the blood, instead of going into the cells, it can cause two problems.
2. Overtime, high blood glucose levels may harm kidney, heart, eyes or
nerves
aspects of prevention and curation, but rather management, there is an increased focus on
because of leads provided by traditional medicine to natural products that may be better
treatments than currently used conventional drugs (Rates , 2001). Secondly the plants by
secondary metabolic means contain a variety of herbal and non-herbal ingredients that are
surge in new drugs to treat and prevent the condition, its prevalence continues to soar.
Perhaps the most worrying aspect of all is that the rise is even reflected in children ( Yost
et al.,2001; Ludwig and Ebbeling 2001). Although several drugs targeted for
5
PPARg agonists (glitazones) are in clinical practice, the growing diabetes market
observes a number of changes. The glitazones are meant to target the problem
of insulin resistance and enhance insulin action at the cellular level; however, some of
these drugs are linked to liver toxicity (troglitazone), including a number of deaths from
hepatic failure( Krische and West, 2000; Stern, 1999 )and raising the symptoms and risk
factors of heart disease leading to heart failure (rosiglitazone) (Gale, and Lancet., 2001).
Therefore, as the long term of risk and effect on the complications of diabetes related
with these drugs are not yet clear, UK Drug and Therapeutic Bulletin warrants that
patients taking glitazones be monitored for signs of heart failure (Scrip, 2001 ) On the
other hand, traditional medicinal plants with various active principles and properties as
discussed in this article have been used since ancient times by physicians and laymen to
treat a great variety of human diseases such as diabetes, coronary heart disease and
cancer . ( Middleton et al., 2000; Havsteen 1984) . The beneficial multiple activities like
integrity and function of b-cells, insulin-releasing activity, improving glucose uptake and
utilization and the antioxidant properties present in medicinal plants offer exciting
require the combination of various types of multiple agents. (Gale and Lancet, 2001)
laments that ‘. . the rise of modern medicine has largely been based on new drugs, and
most of us can expect to hobble to our graves on the crutch of polypharmacy’. However,
medicatrix naturae – the power of self-preservation or adjustment has been the motto of
6
polyherbal formulation have the synergistic, potentiative, agonistic/antagonistic
dynamic way to produce maximum therapeutic efficacy with minimum side effects.
of therapeutic recipes. They are formulated and prepared keeping in mind the conditions
that herbal medicines and preparations should be taken with the consideration of their
preparations meant for diabetes offer enormous scope for combating the threat of the
diabetic epidemic. To achieve a blockbuster status, clear evidence of the advantage over
the existing therapy is the most important requirement of the day. The ability of modern
medicine and health care systems to adequately manage symptoms of chronic and
terminal disease is a central theme. The systematic reviews and Meta analysis of clinical
trials are the foundation of their success. Unfortunately, despite the apparent supremacy
clinical trail evidences are not adequately available in order to advocate their scientific
merit and supremacy over the existing drugs. Though the markets for herbal medicines
are booming (Brevoort and Herbalgram, 1998) and evidence for effectiveness is
2000).
to create health and well being. The primary aim of Ayurveda health care is to restore the
7
physicalmental and emotional balance in patients, thereby improving health, preventing
disease and also treating any current illness. The number of patients seeking alternate and
herbal therapy is growing exponentially. Herbal medicines are now in great demand in
the developing world for primary healthcare not because they are inexpensive but also for
better cultural acceptability, better compatibility with the human body and minimal side
effects. Herbal medicine is still the mainstay of about 75–80% of the world population,
mainly in the developing countries for primary healthcare . However among the
estimated 250,000-400,000 plant species, only 6% have been studied for biological
activity, and about 15% have been investigated phytochemically (Balandrin et al., 1985;
Cragg et al .,1997 ).
"peru nerungi" is an erect ' much branched , foetid smelling succulent annual herb . The
antibilious agent , while the juice of the fruit is used as an emmenagogue and to promote
lochial discharge (Satyavathi et al., 1987). Leves are applied for healing ulcers. The fruits
and leaves of pedalium murex yield a number of phenolic acids.(Das et al ., 1996). The
decoction of pedalium murex and glycoside obtained from it showed mild diuretic
activity (Harvey, 1996). Diuretic activity was also reported in ethanolic extract(50%)
.The extracted was devoid of anthelmintic, and anti cancer activities (Dhar et al., 1974)
8
Pedalium murex, commenly called Gukhru in India belonging to the
family Pedaliaceae is distributed in the coastal areas of south India (Nadkarani, 1982). An
infusion or extract prepared from the leaves, stem and fruits in cold water is demulcent
and a diuretic found useful in the disorders of urinary systems such as gonorrhea, dysuria
and incontinence of urine etc (Chopra et al., 1999; Shukla and Khanuja, 2004). An
The medicinal and culinary uses of members of the family are well‐documented
in literatures. Bhakuni et., al. (1992) reported that fruits of Pedalium murex Linn, possess
properties. Also, Kothari & Moorthy (1994) stated that Pedalium murex is used for the
treatment of urinogenital system diseases in India while Shah et al., (1997) reported that
P. murex contains male contraception properties hence it can be used for fertility
regulation. Ecologically, the plant is a saline soil indicator in coastal regions (Hutchinson
field of biotechnology as most of the drug industries depend in part, on plants for the
the method to multiply the medicinal herbs and monitor their secondary metabolites.
Conservation of endangered medicinal plants has also been achieved through cell cultures
with significance (Rao et al., 1996). Reports of in vitro plant regeneration from tissues of
medicinal plants are available (Gupta et al., 1997; Verma and Kant, 1996; Hoque et al.,
2000; Nichol et al., 1991 ; Palai et al., 2000). Plant tissue culture is a boon and can help
9
produce large quantities of the herbal material. However, it is speculated that plant
therapeutic importance
The use of medicinal plants as a source for relief from illness can be traced back
over five millennia to written documents of the early civilization in India ,china, and the
near east ,but it is doubtless an art as old as mankind.Plants are still widely used in
antimicrobial agent. Medicinal plants have become the focus of intense study in terms of
pharmacological effects. Synthetic drugs are not only expensive and inadequate for the
treatment of diseases but also often with adulterations and side effects. Therefore, there is
tissue culture technique and anti microbial ,anti diabetic activity of invitro and invivo
10
.
11
Review
There are two main categories of this disease. Type 1 diabetes mellitus is
called insulin-dependent diabetes mellitus (IDDM) and Type 2 is the non insulin
dependent diabetes mellitus (NIDDM). IDDM represents a heterogenous and polygenic
12
disorder, with a number of non-HLA loci (about 20) contributing to the disease
susceptibility (Lernmark, 1998). Though this form of diabetes accounts for 5 to 10% of
all cases, the incidence is rapidly increasing in specific regions. It is estimated that
incidence of Type 1 diabetes will be about 40% higher in the year 2010 than in 1997
(Onkamo et al.,1999), and yet there is no identified agent substantially capable of
preventing this type of disease (Rabinovitch, and Skyler,1998;Schatz et al., 2000
;Atkinson et al.,2001). NIDDM is far more common and results from a combination of
defects in insulin secretion and action. This type of disease accounts for 90 to 95% of all
diabetic patients. Treatment of Type 2 diabetes is complicated by several factors inherent
to the disease process, typically, insulin resistance, hyperinsulinemia, impaired insulin
secretion, reduced insulin-mediated glucos uptake and utilization ( De Fronzo , 1997;.
Polonsky et al.,1996; Groop et al., 1989).
Despite the great strides that have been made in understanding and management
in this disease, serious problems like diabetic retinopathy (Ferris et al., 1999), diabetic
nephropathy (Ritz et al., 1999) and lower extremity amputation (Reiber et al., 1995)
continue to confront patients and physicians. The graph of diabetes-related mortality is
rising unabated (Olefsky, 2000). The level of serum lipids is usually raised in diabetes
and such an elevation represents a risk factor for cardiovascular disease
(shamaony et al., 1994). The chronic hyperglycemia of diabetes is associated with long
term damage, dysfunction and failure of various organs (Lyra et al., 2006).
13
LPO. Several studies on human and animal models, using TBARS assay have shown
increased LPO in membranes and lipoproteins in the diabetic state, (Griesmacher et al.
1995; Krishnakumar et al., 1999).
HPX formed by LPO have direct toxic effects on endothelial cells and also
degrade to form hydroxyl radicals (OH-) (Testafamariam 1993). The action of
streptozotocin and alloxan produces reactive free radicals, which have been shown to be
cytotoxic to the B-cells of the pancreas (Ivorra et al., 1989; Lenzen, and Panten, 1988).
As the diabetogenic action of streptozotocin is preventable by SuperOxide Dismutase
(SOD), Catalase (CAT) and other OH - scavengers such as ethanol and dimethyl urea,
there is evidence to suggest that the action of streptozotocin and alloxan involve a
superoxide anion and OH (Asplund et al. 1984). Thus, alloxan-induced diabetes could
elicit changes in the antioxidant defense systems in response to increased oxidative stress.
The deleterious effects of superoxide radicals (O.− 2) and OH- in oxidative
stress can be counteracted by antioxidant enzymes such as SOD, CAT and glutathione
peroxidase (GPx). In addition to these enzymes, glutathione-S-transferase (GST)
provides glutathione (GSH) and help to neutralize toxic electrophiles. There is an
evidence to show the role of free radicals in diabetes and studies indicate that tissue
injury in diabetes may be due to free radicals (Wohaieb and Godin 1987; Kakkar et al.
1995). Diabetes is becoming pandemic and despite the recent surge in new drugs to treat
and prevent the condition, its prevalence continues to soar ( Tiwari and Madhusudana
Rao, 2002).
14
pathways, and activation of cytokines are some of the known biochemical mechanisms of
hyperglycemia-induced tissue and cell injury (Koya et al., 1998; Brownlee, 1995).
The mammalian cells are operational with both enzymatic and nonenzymatic
antioxidant defenses which minimize ROS mediated cellular damage (Haliwell and
Gutteridge,1994). The majority of plasma antioxidants are depleted in diabetes patients
(Valabhji et al.,2001). Thus antioxidant therapy in diabetes may be helpful in relieving
symptoms and complications observed in diabetes patients. As plants often contain a
substantial amount of antioxidants, so herbal hypoglycemic coupled with antioxidant
property may serve as a wonderful antidiabetic agent (Larson, 1998).
Different medicinal systems are using the active plant constituents, which
discovered as natural hypoglycemic medicine, came from the virtue of traditional
knowledge. Herbal drugs are considered free from side effects than synthetic one and
they are less toxic, relatively cheap and popular (Moming, 1987). In India, medicinal
plants have been used as natural medicine since the days of Vedic glory. Many of these
medicinal plants and herbs are part of our diet as spices, vegetables and fruits.
Historically, in ‘Atharva-Veda’ (about 200 B.C.) description of medicinal plants was
made under a separate chapter ‘Ayurveda’. Sushruta (about 400 B.C.) compiled
classification of 700 herbal drugs under 37 classes in ‘Sushruta Samhita’ (A compendium
of ancient Indian surgery). Charak (about 600 B.C.) made the scientific classification of
herbal drugs based on remedial properties in his renowned treatise ‘Charaka Samhita’ (A
compendium of general medicine). In which, it described 50 classes of herbal remedies
comprising 500 crude drugs (Mukherjee, 1983; Saxena et al., 2006). The medicinal
values of plants have been tested by trial and error method for a long time by different
workers. Indian medicinal plants having blood sugar lowering potentials (Mukherjee et
al., 1981; Grover et al., 2002; Saxena et al., 2004; Mukherjee et al., 2006).
Aegle marmelos is widely used in Indian Ayurvedic medicine for the treatment
of diabetes mellitus (Kamalakkanan et al., 2003). Hypoglycemic effect of Aegle
marmelos root bark decoction (Karunanayake et al., 1984), leaf extract of Aegle
15
marmelos produced anti hyperglycemic activity in alloxan diabetic rats (Ponnachan et
al., 1993),and produced hypoglycemic effect and increased plasma insulin level of STZ-
diabetic rats, (Sharma et al., 1996).
Aloe vera leaf pulp extract showed hypoglycemic activity on type 1 and type 2
diabetic rats, the effect being enhanced in type 2 diabetes as compared with
glibenclamide (Okyar et al., 2001). Oral administration of ethanolic extract to STZ-
diabetic rats for 21 days resulted in a prominent reduction of fasting blood glucose along
with improved plasma insulin level of diabetic rats (Rajsekaran et al., 2005). Oral
administration of Aloe vera gel extract to STZ-diabetic rats resulted in a significant
reduction of fasting blood glucose and improved the plasma insulin level
(Rajsekaran et al., 2006).
16
days significantly reduced the levels of blood glucose and increased the activity of
plasma insulin and antioxidant enzymes (Kaleem et al., 2006).
17
Oleanolic acid and momordin from Momordica charantia plant, produced
antihyperglycemic effect by inhibiting glucose transport in intestine of rat (Matsuda et
al., 1988). Fruit aqueous extract (200mg/kg, orally for 6 weeks), and exercise potentially
lowered blood sugar of type 2 diabetic and hyperinsulinemic (insulin resistance) rats
(Miura et al., 2004). Oral administration of alcoholic extract of leaves of Ocimum
sanctum lowered blood sugar level in normal; glucose fed hyperglycemic and STZ-
diabetic rats, (Chattopadhyay, 1993). Aqueous extract of Pterocarpus marsupium (1g/kg,
orally) bark has been observed to produce antidiabetic activity in alloxan diabetic rats
(Vats et al., 2004). Alcoholic extract of Swertia chirayita exhibited hypoglycemic effect
in alloxan induced diabetic rats (Kar et al., 2003).
Aqueous and ethanolic extracts of Syzygium cumini fruit-pulp has been
produces antihyperglycemic effect in alloxan induced diabetic rats, (Sharma et al., 2006).
The methanol extract of Mallotus roxburghianus leaves have the antidiabetic properties on
streptozocin induced diabetic rats (Lalhlenmawia et al., 2007). Eucalyptus globulus Labill.
(Tasmanian Bleu Gum) when given to streptozotocin-diabetic mice reduced the level of
hyperglycaemia.(Swatson-Flatt et al.,1990). Tournefortia hirsutissima Linn. decreased
the hyperglycaemic level in rabbits,( Alarcon-Aguilar et al.,1998).
Similarly the plant Guazuma ulmifolia Wall. also significantly decreased the
hyperglycaemic peak in rabbits.( Roman-Ramos et al., 1991) The root mucilages of
Glossostemon bruguieri Desf. (Moghat) had remarkable hypoglycaemic activity
decreasing the blood glucose levels in diabetic rats by 54.5% within 15 days.
(Ibrahim et al.,1997). The aqueous extract of Camellia sinensis L. (black tea)
significantly reduced the blood glucose levels of streptozotocin-induced diabetic rats
(Gomes et al., 1995).
18
methanolic extract of Adansonnia digitata stem bark have a anti-diabetic activity in
streptozotocin-induced rats.
Ethyl acetate and ethanol extracts of the Momordica dioica fruits have
been shown significant anti diabetic activity (Reddy et al., 2006). Oral administration of
the extract of Asteracantha longifolia Nees. can significantly improve glucose tolerance
in healthy human subjects and diabetic patients,(Fernando et al.,1991). Methanol and
water extract of the Achyranthes aspera L produced a significant dose-related
hypoglycaemic effect in normoglycaemic and alloxan-induced diabetic rabbits. (Akhtar
and Iqbal,1991). Antidiabetic activity of Mangifera indica leaves in rat (Aderibigebe and
Lawal ,1999) . Nymphaea stellata flower extracts exhibited antihyperglycaemic and
antihyperlipidaemic effects of on alloxan-induced diabetic rats, (Rajagopal and Sasikala,
2008).
GSH acts as an antioxidant and its decrease was reported in diabetes mellitus
(Baynes and Thorpe 1999). The decrease in GSH levels represents increased utilization
due to oxidative stress (Anuradha and Selvam 1993). The depletion of GSH content may
also lower the GST activity as GSH is required as a substrate for GST activity
19
(Rathore et al., 2000). Depression in GPx activity was also observed in liver and kidney
during diabetes. GPx has been shown to be an important adaptive response to condition
of increased peroxidative stress (Matkovics et al., 1982).
SOD and CAT are the two major scavenging enzymes that remove toxic free
radicals in vivo. Previous studies have reported that the activity of SOD is low in diabetes
mellitus (Vucic et al., 1997). Reduced activities of SOD and CAT in liver and kidney
have been observed during diabetes and this may result in a number of deleterious effects
due to the accumulation of O.− 2 and H2O2 (Searle and Wilson, 1980). Scoparia dulcis
possessed an antidiabetic effect in addition to antioxidant activity, which may be
attributed to its protective action on LPO and to the enhancing effect on cellular
antioxidant defense contributing to the protection against oxidative damage in
streptozotocin diabetes, (Pari and Latha, 2005). Diabetes represents a state of increased lipid
20
of Cleome droserifolia exhibited a significant antidiabetic and antiperoxidative activity in
alloxan diabetic rats, (Naggari et al., 2005).
The level of serum lipids is usually raised in diabetes and such an elevation
represents a risk factor for cardiovascular disease (shamaony et al.,1994). Lowering of
serum lipid levels through dietary or drug therapy seems to be associated with a decrease
in risk of vascular disease (Rhoads et al., 1976). Anthocephalus indicus root possessed
hypoglycemic and hyperlipidemic activity in alloxan induced diabetic rats. (Vishnu
Kumar et al.,2009). Hypoglycemic and hypolipidemic action of alcohol extract of
Tinospora cordifolia roots in chemical induced diabetes in rats were studied.
(Stanley et al., 2003)
21
In the liver, the enzyme is an important regulator of glucose storage and
disposal (Robert and Christopher, 1999). Insulin decreases gluconeogenesis by
decreasing the activities of key enzymes such as glucose-6-phosphatase, fructose 1,6,
bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase ( Murray
et al.,2000). Glucose 6-phosphatase, one of the key enzymes in the homeostatic
regulation of blood glucose level, catalyzes the terminal step in both gluconeogenesis and
glycogenolysis (Beaudet et al 1991; Hers et al.,1989) and fructose 1,6-bisphosphatase,
catalyzes one of the irreversible step in gluconeogeneis, and serves as a site for the
regulation of process (Jejwani and Horecker,1976). Ethanolic extract (200mg/kg) of
Momordica charantia was produced hypoglycemic activity in normal and streptozotocin
diabetic rats; this was occurred possibly due to inhibiting glucose-6-phosphatase and
fructose-1,6-biphosphatase in liver, and stimulating hepatic glucose-6- phosphate
dehydrogenase activities (Shibib et al., 1993). Alcoholic leaf extract produced
hypoglycemic effect in normal fed and 48 hours fasted rats, response mediated by
suppression of gluconeogenic enzyme glucose-6-phosphatase (Hossain et al., 1992).
Ethanol (60%) leaf extract (200mg/kg, orally) lowered the blood sugar level of diabetic
rats due to suppressed glucose synthesis, through depression of glucose-6-phosphatase,
fructose-1-6-biphosphatase and enhanced glucose oxidation by shunt pathway through
activation of glucose-6-phosphate dehydrogenase (Shibib et al., 1993).
22
glycation may it self induce the generation of oxygen derived free radicals in diabetic
condition. (Gupta et al., 1997).
Many active compounds have been isolated from the plant and herb species
of India. These active principles are dietary fibres, alkaloids, flavonoids, saponins, amino
acids, steroids, peptides and others. These have produced potent hypoglycemic, anti
hyperglycemic and glucose suppressive activities (Saxena et al., 2006). The above effects
achieved by either insulin release from pancreatic ß-cells, inhibited glucose absorption in
gut, stimulated glycogenesis in liver or increased glucose utilization by the body (Grover
et al., 2002; Saxena et al., 2004). These compounds also exhibited their antioxidant,
hypolipidemic, anticataract activities, restored enzymatic functions, repair and
regeneration of pancreatic islets and the alleviation of liver and renal damage (Mukherjee
et al., 2006). Some active constituents have been obtained from plants possess insulin
like activity and could be provide alternate for insulin therapy.
23
Terminalia chebula exhibited antibacterial activity against a number of bacterial
species (Ahmad et al.,1998). One group of researchers found that it is effective in
inhibiting the urease activity of Helicobactor pyroli (H. pyroli), an ubiquitous bacterium
implicated in the development of gastritis, ulcers and stomach cancers, (Malckzadeh et
al.,2001). Antibacterial activity of Terminalia chebula against both Gram positive and
Gram negative human pathogenic bacteria has also been reported (Phadke and Kulkarni ,
1989). Gallic acid and its ethyl ester isolated from ethanolic extract of Terminalia
chebula showed antimicrobial activity against methicillin-resistant Staphylococcus
aureus (Sato et al.,1997). Diffusate of Terminalia chebula showed an inhibitory effect
against strain XC-100 of the bacterium Xanthomonas Campestris pv. Citri indicating its
usefulness for the management of citrus canker disease (Afzalakhtar et al.,1997). It has
also growth inhibitory action against Salmonella typhi (Rani, and Khullar ,2004) and
intestinal bacteria (Kim et al.,2005).Nayeemulla et al .,(2006) reported that Rauvolfia
tetrophylla and Physalis minima leaf and callus extracts inhibited bacterial and fungal
growth. Y. Rajeshwar et al., (2005) reported the antimicrobial activity of the methanolic
extract of Mucuna pruriens.
In vitro propagation of medicinal plants could help in raising disease free health
clones in a large scale for extraction of pure drug. To date only one report available on
regeneration for this important medicinal plant (Thulaseedharan and Vaidyanathan 1990)
reported callus induction and plant regeneration in Vicoa indica. callus and cell
suspension culture of several plant species and extraction of medicinally important
compounds (Mulabagal et al., 2004). In vitro callus culture of Aegle marmelos has as
much potential in diabetes management ( Sevugan Arumugam et al.,2008). Allium cepa
callus cultures showed greater hypoglycemic potential over natural onion bulb (Kelkar et
al., 2001).
The medicinal and culinary uses of members of the Pedalaceae family are
well-documented in literatures. Kothari and Moorthy (1994),stated that Pedalium murex
is used for the treatment of urinogenital system diseases in India .While Shah et al.,
24
(1997) reported that P. murex contains male contraception properties hence it can be used
for fertility regulation. Ecologically, the plant is a saline soil indicator in coastal regions
(Hutchinson & Dalziel 1963).Pedalium murex leaves extract possessed antimicrobial
activity.(Nagaraj et al.,2008). Sahayaraj et al., (2008). Reported anti hyperlipidemic
activity of Pedalium murex fruits.Aqueous extract from Pedalium murex has been
evaluated for its analgesic and antipyretic activities (Muralidharan and
Balamurugan,2008).An infusion or extract prepared from leaves ,stem, and fruits in cold
water is demulcent and diuretic.
Present study we are investigated anti microbial activity and antidiabetic
activity of pedalium murex leaves and leaves derived callus on Alloxan induced rats.
25
26
27
INTRODUCTION
among human beings (Bushra and Ganga, 2003). The abundance of plants on the
antimicrobial agents. (Bonjar and Farrokhi, 2004). The potential as a source for
new drugs is still largely unexplored. Among the estimated 250,000-500,000 plant
species ,only a small percentage has been investigated phytochemically and the
plant will reveal only a very narrow spectrum of its constituents . Historically
discovering new biologically active molecules has been most productive in the
28
area of antibiotic. Even now, contrary to common belief, drugs from higher plants
least 130 drugs, all single chemical entities extracted from higher plants, or
source of many potent and powerful drugs. One of such resources is folk
medicine and systematic screening of them may result in the discovery of novel
Antibiotic resistance has become a global concern (Westh et al., 2004). The
have been known to be treated with herbal remedies throughout the history of
extracts, provide unlimited opportunities for new drug leads because of the
and novel mechanisms of action for new and re-emerging infectious diseases
to folk medicine, looking for new leads to develop better drugs against microbial
29
The increasing failure of chemotherapeutics and antibiotic
medicinal plants and is one of the richest countries in the world in regard to
introducing and domesticating new exotic plant varieties (Martins et al., 2001). In
efficacy will be used for the treatment of bacterial infections (Balandrin et al.,
1985). Since time immemorial, man has used various parts of plants in the
medicinal plants to meet the primary health care needs. Although, modern
medicines are available, herbal medicines have often retained popularity for
historical and cultural reasons. Since the usage of these herbal medicines has
increased, the issues regarding their safety, quality, and efficacy in industrialized
30
and developing countries are cropped up (Anonymous, 1999). Growing interest
biotechnology as most of the drug industries depend in part , on plants for the
used globally for the exist conservation of plants . (Amutha et al., 2008).
of biotechnology as most of the drug industries depend in part, on plants for the
additives, and pesticides (Balandrin and Klocke, 1988). It has been mentioned that
natural habitats for medicinal plants are disappearing fast and together with
31
In the search for alternative to production of desirable medicinal
compounds that has been used in several trational ailments preparations. Leaf part
of the plant extract in organic solvents showed good source for the bioactive
the Pedalium murex leaves used MS media ,Phytochemical analysis and anti
microbial activiy of leaf callus and field grown leaves of the Pedalium murex.
32
MATERIALS AND METHODS
MATERIALS
CALLUS INDUCTION
Plant material
. Field grown plants were used as source of explants. Leaf, of four weeks
old seedlings were selected as explants for callus induction. The explants were
washed in running tap water for 30 minutes. Then they were washed in an agitated
solution of liquid detergent 2 %( v/v) (Teepol) for two minutes and rinsed in
distilled water three times. Surface sterilization was performed by immersion of
the explants in 70% (v/v) aqueous ethanol for 40 seconds followed by 0.1% (w/v)
mercuric chloride for five minutes. Finally, the materials were thoroughly rinsed
with sterile distilled water five times to remove the traces of mercuric chloride. All
the explants were cut into pieces approximately 10–15 mm long for inoculation.
Glassware:
33
running water. These were then treated with hot Chromic acid (mixture of
K2Cr2O7 + H2SO4 + H2O) followed by thorough washing with tap water. The
glasswares were then inverted in a clean tray and left to dry in the oven. Plugs for
the tubes and flasks were made out of absorbent surgical cotton wrapped in
muslin. 5 - 10 ml water was then poured into every culture vessel which was
tightly plugged. The glasswares were then steam sterilized in an autoclave at a
pressure of 15 lb/in at 121o C for 15 - 20 minutes.
Culture Medium:
The media formulation described as Murashige and Skoog
(1962) referred as MS medium was selected as the optimal culture medium. Stock
solutions of generally 4 times major elements, 1000 times minor elements, 100
times organic constituents were prepared. These stock solutions were stored in a
freeze chest at - 4oC and were mixed in desired proportions only before use. None
of the stock solutions were stored for more than 15 days.
34
Table 1. Composition of MS medium (Murashige and Skoog’s, 1962).
Macronutrients NH 4NO
Ammonium nitrate 3 KNO3 1650
Potassium nitrate CaCl2.2H2O 1900
Calcium chloride MgSO4.7H2O 440
Magnesium sulphate KH2PO4 370
Potassium dihydrogen ortho 170
Phosphate
Micronutrients
Ferrous Sulphate FeSO4. 7H2O 27.6
Disodium Ethylene Diamine Na2EDTA 37.4
Tetra Acetic Acid
Boric acid H3BO3
Potasium iodide KI 6.2
Manganese sulphate MnSO4. 4H2O
Zinc sulphate ZnSO4.7H2O 0.83
Sodium molybdate Na2MoO4. 2H2O 22.3
Cobalt chloride CoCl2. 6H2O 8.6
Copper sulphate CuSO4.5H2O
0.25
0.025
0.025
Vitamins
Nicotinic acid 100
Pyridoxine HCl 0.5
Thiamine HCl 0.5
Myo-inositol 0.1
35
Sucrose 30,000
Agar 80,000
Nicotinic Acid 10
10
Pyridoxine HCl 10
Thiamine HCl 2
Glycine 40
36
KH2PO4 1700
Cytokinins
6-Benzyl amino purine BAP 10
kinetin KIN 1 N HCl 1 ml = 1 mg/l
37
Dichlorophenoxy Acetic Acid (2,4-D), 6-Benzyl Amino Purine (BAP) and
Kinetin (KIN) either alone or in combinations at various concentrations.
Inoculations:
All the experimental manipulations were carried out under strictly
aseptic conditions in laminar air flow bench fitted with a bactericidal U. V. tube
(15 W, peak emission 2637 A o). The floor of the chamber was thoroughly
scrubbed with cotton dipped in alcohol.
38
sprayed in the chamber with the help of an atomizer. The chamber was then
sterilized with U.V. rays continuously on for one hour. The explant like leaves
and were taken from the plants growing under the in vivo conditions. The leaves,
were placed in different bottles and covered with net and washed for 30 minutes
under running tap water to remove all the adhering dust particles and microbes
from the surface. The explants were then washed with liquid detergent (teepol) for
another 15 minutes and then washed properly to remove the detergent. The
explants were then treated with Bavistin (fungicide) for another 20-30 minutes to
remove the fungus and then washed properly to remove the fungicide.
39
Cultural conditions:
All the cultures were maintained in an air conditioned culture room
at a temperature of 25 ± 4oC. The source of illumination consisted of 2.5 feet wide
fluorescent tubes (40 watt) and incandescent bulb (25 watt). The intensity of
illumination was 3500 lux at the level of cultures and a 12 hour light regime was
followed by 12 hour darkness.
GC-MS Programme
MS Programme
40
Inlet line temperature: 200 ○C
Source temperature: 200○C
Electron energy: 70 eV
Mass scan: (m/z) 45-450
MS Time: 36 min
Antimicrobial activity
Microorganisms
The activity of the Pedalium murex leaves and leaves derived callus extract was
from the Depatment of microbiology ,JJ college of Arts and Science ,Pudukkottai ,
Tamilnadu,S.India.
Media:
Nutrient broth, Nutrient agar, Malt extract broth and Sabouraud dextrose agar, all
Preparation of inoculum
41
Each organism was recovered by sub-culturing on fresh media. A loopful
Sub-culturing of microorganisim
Antibacterial assay
Agar well diffusion
The assay was conducted as described by Perez et al., (1990).
Procedure
Microorganisms from growth on nutrient agar incubated at 37 ○C for 18 h were
suspended in saline solution o.85% NaCl and adjusted to a turbididy of 0.5 Mac
farland standards(108 ctu/ml).The suspension was used to inoculate 90mm
diameter Petri plates with a sterile non toxic cotton swab on a wooden applicator.
Six millimeter diameter wells were punched in the agar and filled with 50µl of
2000µg/ml extract. Plates were incubated in air at 37 ○C for 24 hours. Antibacterial
activities were evaluated by measuring inhibition zone diameters. The experiments
conducted thrice.
RESULTS
42
(1,1.5,2,2.5,and 3.0 mg/l). In order to study the effects of plant growth regulators
(PGRs) on callus culture, different PGRs were tried at different concentration and
various combinations. Based on the results the growth of callus was obtained in
MS medium individually amended PGRs such as IAA and 2,4 D the callus
response was low .The combination of auxins and cytokinins in MS medium were
tested for initiate callus from the leaves . The combination of IAA and BAP in MS
medium produced white friable callus and MS medium supplemented with IAA
and kinetin produced brown callus.2,4 D was tested for the same .
and BAP growth regulators .The high percentage of callus growth was found in
auxin such as 2,4-D (2.5 mg/l )with BAP (0.5 mg/l) among the other combination
by GC-MS in ethanol extract from Pedalium murex leaves and leaves derived
callus .GC chromatogram as shown on Figure 1 yielded two major peaks and three
minor peaks identified from the field grown leaf extract .Two major compounds
are n-Hexadecanoic acid (retention time (RT):17.46 and a- linolenic acid (RT :
chromatogram as shown on Figure 2 yielded two major peaks and two minor
43
peaks identified from the leaf derived callus ethanol extract. Two major
compounds are n-Hexadecanoic acid (RT:17.47), and oleic acid (RT:20.29), two
acid (RT:20.58)
Alcohol extract of the leaf and leaf derived callus of Pedalium murex
obtained in the present study relieved that the tested Pedalium murex extract
posses potential anti microbial activity against pathogenic bacteria . The field
grown leaves and leaves derived callus extract of Pedalium murex high effective
DISCUSSION
Based on the results of the previous experiments, the high biomass yielding
concentration of 2,4-D (2.5 mg/l) with BAP (0.5 mg/l) were selected for the
synergistic effects of auxins wih cytokinin on callus culture .The culture grew very
fast within 15 days in all combinations tested . in order to determine the active
principles present, callus/cell was extracted by using ethanol and compared with
44
chromatogram of callus/cell samples showed most of compounds present in leaf
extract .The accumulation of active principles in cultured cells at higher level than
different profiles of secondary metabolites when compared with the intact plant
(Whitaker et al.,1986).This report coincides with our where there were additional
peaks in GCMS chromatogram of callus ethanol extract sample which was not
the development of new chemotherapeutic agents. The first step towards this goal
is the in vitro antibacterial activity assay (Tona et al., 1998). Many reports are
available on the anti microbial activity plants (Bylka et al., 2004.). Some of these
observations have helped in identifying the active principle responsible for such
activities and in the developing drugs for the therapeutic use in human beings.
2000). Both leaves and leaves derived callus ethanol extracts of the Pedalium
45
murex can be used in the treatment of boils, sores and wounds, since
required active principles of Pedalium murex .So far, there is no known report of
activity. Several workers have reported that many plants possess antimicrobial
properties including the parts which include; flower, bark, stem, leaf, e.t.c. It has
been shown that when solvents like ethanol, hexane and methanol are used to
extract plants, most of them are able to exhibit inhibitory effect on both gram
positive and gram negative bacteria (Bushra and Ganga, 2003). Similar work by
Omonkhelin et al., showed that ethanolic extract of- - Kigelia africana has
minimum inhibitory concentration of 6.25 + 1.07 mg/ml and 7.92 + 1.52 mg/ml
and Pseudomonas aeruginosa showed that the plants can be used in the treatment
of gastrointestinal infection and diarrhea in man and skin diseases (Rogger et al.,
1990) and they can also be used in the treatment of urinary tract
compounds that has been used in several trational ailment preparation .Leaf and
46
leaf derived callus extract in organic solvent showed good source for the bioactive
required to find out the specific bioactive compounds responsible for the
47
INTRODUCTION
hyperglycemia and defective metabolism of glucose and lipids. Diabetes was estimated to
affect 177 million people world wide in 2000 and this figure is projected to increase to
300 million by 2025 (Porter and Barret, 2005). Diabetes is not a single disease rather it is
caused by relative or absolute deficiency of insulin. Diabetes can be divided into two
main groups based on their requirements of insulin: insulin dependent diabetes mellitus
(Type 1), and non-insulin dependent diabetes mellitus (Type 2). However, other types of
diabetes have also been identified. Maturity Onset Diabetes of the Young (MODY) is
now classified as Type 3 and gestational diabetes classified as Type 4. NIDDM type 2
diabetes account for about 90 percent of diabetic cases (WHO, 2002). Insulin resistance
and β-Cell dysfunction are the metabolic abnormalities in the type 2 diabetes (Sa’ad et
al.,1991). Glycemic control is one of the targets for managing diabetes mellitus. Studies
have confirmed that for the type 2 diabetes, effective control of blood glucose
substantially decrease the risk of developing diabetic complications (Ohkubo et al., 1995;
style, such as diet and exercise and the use of insulin and/or oral hypoglycaemic drugs.
These pharmacologic agents target increased insulin secretion, decreased hepatic glucose
48
production and increased sensitivity to insulin (Kelly and Mandarino, 2000).
Management of this disease with insulin and/or oral hypoglycaemic agents have certain
drawbacks (University Group Diabetes Program, 1974; Knatterud et al., 1978). For
insulin such drawbacks include ineffectiveness on oral administration, short shelf life,
hypoglycaemia. The use of oral hypoglycaemic drugs like sulfonylureas and biguanides
is also associated with side effects such as propensity to gain weight (Rang and Dale,
1991).
Throughout the world many traditional plants have been found successful
recommended some of the substances, which are abundantly found in nature. Long before
their value was demonstrated and understood by scientific methods. How ever , few have
received scientific or medical scrutiny and the World Health Organization (WHO) has
recommended the trational plant treatment for diabetes warrant further evaluation (WHO
, 1980) .More ever today it is justified to use a plant or its active principles for treatment
(Singh et al.,2006).
Insulin therapy affords glycemic control in IDDM yet its short comings include
(Senthilkumar et al.,2006) .Diabetes is one of the oldest known diseases of the man
whose devastating effect is increasing by the day and severity almost at epidemic level. It
49
the complete or relative insufficiency of insulin secretion and /or insulin action (Balkau
et al., 2000).
Experimental diabetes in animals has provided considerable insight into the physiologic
and biochemical derangement of the diabetic state. Many of this derangement were in the
form of significant changes in lipid metabolism and structure (Sochar et al., 1985). These
structural changes are clearly oxidative in nature and are associated with development of
vascular disease (Baynes et al., 1999) .In diabetic rats, increased lipid peroxidation was
also associated with hyperlipidemia (Morel and Chisolm ,1989). During diabetes, a
profound alteration in the concentration and composition of lipids occurs. Liver and
kidney are important for glucose and lipid homeostasis, they participates in the uptake,
phospholipids and triglycerides. Thus it is expected to have changes in liver and kidney
Animals
Healthy male adult albino rats (Wistar strain) of 6-7 weeks old, weighing
150 ± 20 g was procured from “Sri Venkateswara Enterprises”, Bangalore, India.
They were housed in clean sterile polypropylene cages with proper aeration and
lighting (12 ± 1 hr day / night rhythm) throughout the experimental period. During
the course of the experiments, the temperature was maintained between 27ºC ±
2ºC. The animals were fed with commercially available pelleted rat feed (Sri Sai
Durga feeds Bangalore, India.Under the trade name “Sri Sai Durga feed and
50
food”) and water ad libitum. The usage and handling of experimental rats was
followed as per the rules and regulations given by the Institutional Ethics
Committee for the purpose.
Chemicals
Alloxan was purchased from sigma chemicals company, St .Louis Mo.,
USA Thiobarbituric acid (TBA), pyrogallol, hydrogen peroxide, 1-chloro 2,4-
dinitro benzene(CDNB), dithio dinitro benzoic acid (DTNB), glutathione,trichloro
acetic acid, cholesterol,thio urea,palmitic acid and O-toluidine were purchased
from E.Merck,India.All other chemicals and reagents used in this study are
analytical grade purchased from Fine chemicals (P).Ltd., Mumbai
51
Fresh whole plants of Pedalium murex were collected from Sivapuram
Pudukkottai District, during the months of September-December 2006 and
identified by Dr.P.Jayaraman (Director), Plant Anatomy Reasearch centre,
Chennai.
52
Experimental designs
Experimental design
The rats were divided into seven groups, each group consists of six animals.
Group I :Served as control and received normal feed and water ad libitum .
Group II : served as a normal rats received 200 mg/kg /bw Pedalium murex
leaves extract and water ad libitum.
Group III : served as normal rats received 200 mg/kg /bw Pedalium murex
leaves derived callus extract and water ad libitum.
Group IV :Served as diabetic control and received feed and water ad libitum
Group V : Diabetic rats and were treated orally with ethanol extract of
Pedalium murex leaves at the dose of 200 mg/kg body weight daily
for 21 days, once a day.
Group VI : Diabetic rats and were treated orally with ethanol extract of
Pedalium murex leaves derived callus at the dose of 200 mg/kg
body weight daily for 21 days, once a day.
Group VII :Diabetic rats given glibenglamide orally at the dose of 0.6 mg/kg
body weight daily for 21 days, once a day.
53
The animals were carefully monitored everyday and weighted every
week and blood glucose of all rats were determined .animals described as fasted
were deprived of food for at least 12h but allowed free access to drinking water
was collected and processed for the estimation of blood glucose and glycosylated
anticoagulant was allowed to stand for 30minutes and centrifuged at 3000rpm for
15minutes to separate the serum. Liver and kidney were dissected out, washed in
Procedure
0.1ml of freshly drawn blood was immediately mixed with 1.9ml of 10%
TCA to precipitate the proteins and then centrifuged.To 1ml of the supernatant
,added 4.0ml of O-toluidine reagent and kept in a boiling water bath for 15
minutes .The green colour developed was read colorimatrically at 620 nm .A set of
standard glucose(20-100g) were treated simultaneously using reagent blank.
54
Estimation of Glycosylated Haemoglobin (HBA1c)
The estimation of glycosylated haemoglobin was done by the method of
Sudhakar Nayak and Pattabiraman (1971) with modification according to Bannon
(1982).
Procedure
5ml of blood was collected with EDTA and plasma was separated .To
0.5ml of packed cell,5 ml of citrate buffer was added .mixed and incubated at
37○ C for 15 minutes .After centrifugation the supernatant was discarded. To the
aliquot ,4.0ml of (1M) oxalate in (2M) HCl solution was added,mixed and heated
at 100○C for 4 hours,cooled and precipitated with 2.0 ml of 40% TCA.The mixture
was then centrifuged .To the aliquot,0.05 ml of 80% phenol and 3.0ml of
conc.H2SO4 were added .A set of standards (10-50mg) were also treated in the
similar manner. The colour developed was read at 480 nm after 30 minutes.
Procedure
A weighted amound of the tissue was subjected to alkali digestion in a
boiling water bath for 20 minutes after addition of 5ml of 30% potassium
hydroxide.The tubes were cooled and 3ml of absolute ethanol and a drop of
ammonium acetate were added .The tubes were then placed in a freezer overnight
to precipitate the glycogen .The precipitated glycogen was collected after
centrifugation at 3000 g for 10 minutes . The precipitate was washed thrice with
55
alcohol and dissolved in 3ml of water .Aliquotes were taken and made up to 1ml
with water .4ml of anthrone reagent was added to the tubes kept in an ice
bath,mixed and heated in a boiling water bath for 20 minutes.The green colour
developed was read at 640 nm .Working standard glucose and a blank were treated
similarly.
The values were expressed as mg/g tissue
56
Determination of activity of Glucose-6-Phosphatase
The enzyme activity was determined by the method of Fiske and subbarow(1925)
Procedure
Incubation mixture contains 0.7ml of citrate buffer ,0.3ml of substrate and
0.3ml tissue homogenate.The reaction mixture was incubated at 37 ○ C for 1
hour .Addition of 1ml of 10% TCA to the reaction tubes terminates the reaction of
the enzyme .The suspension was centrifuged and phosphorus content of the
supernatant was estimated by the method of Fiske and Subbarow.The supernatant
was madeup to known volume.To this 1ml of ammonium molybdate was added
followed by 0.4ml ANSA.The blue colour developed after 20 minutes was read at
640nm colorimetrically.
57
The enzyme activity was expressed as µM of phosphate liberated /min/mg protein
Procedure
About 0.1 ml of aliquot was taken and it was evaporated to dryness. The
dried extract and standard were made up to 3.0 ml with ferric chloride- uranyl
acetate reagent. Then 2.0 ml of sulphuric acid ferrous sulphate reagent was added
to all the tubes and the contents were mixed well. After 20 minutes the colour was
read at 540nm in a spectrophotometer.
Total cholesterol level was expressed as mg per dL for serum and mg per g
of wet tissue for liver and kidney.
Estimation of Triglycerides
Triglycerides were estimated by the method of Rice (1970) based on the
method of Van Handel (1961).
Procedure
About 0.1 ml of the lipid extract was mixed with 1.0 ml of chloroform-
methanol mixture and 50 mg of activated silicic acid was added, shaken
vigorously, allowed to stand for 30 minutes and centrifuged. To 0.5 ml of
supernatant, as well as standard and blank, 0.5 ml of alcoholic potassium
hydroxide was added and the mixture was saponified in a 60-70ºC water bath for
20 minutes. To this 0.5 ml of 0.2 N sulphuric acid was added and kept in a boiling
58
water bath for 10 minutes. After cooling the tubes, 0.1 ml of sodium meta
periodate was added and allowed to stand for 10 minutes.
The excess periodate was reduced by the addition of 0.1 ml of sodium meta
arsenite. Then 5.0 ml of chromotrophic acid was added, mixed thoroughly, and
kept in a boiling water bath for 30 minutes. After cooling 0.5 ml of thiourea
solution was added and the colour developed was read at 570nm using
spectrophotometer.
The triglyceride level was expressed as mg per dL for serum and mg per g
of wet tissue.
Estimation of phospholipids
Phospholipids in tissue was estimated by the method of Rouser et al.,
(1970)
Procedure
0.1ml of sample was diluted to 2.0ml with 10%TCA.The precipitated proteins
were sedimented by centrifugation .The supernatant was discarded.1.0ml of 70%
perchloric acid was added to the residue and digested on a sand bath till the
solution become colourless .After cooling ,the solution was made up to 5.0ml with
distilled water.Standard phosphate solution and blank containing water were
mixed with 0.8ml of perchloric acid and the final volume was made up to 5.0ml
distilled water .0.5ml of ammonium molybdate and ascorbic acid were added and
the mixture was kept in a boiling water bath for 6 minutes.The blue colour
developed was read at 710nm .
59
Estimation of HDL cholesterol
HDL cholesterol was estimated by the method of Allin (1974)
Procedure
1. Precipitation
0.5ml of serum was mixed with 1.0ml of precipitant and it was allowed to stand
for 10 minutes at 4000 rpm. After centrifugation ,the clear supernatant containing
HDL cholesterol was separated .
2. Estimation
0.1ml of the supernatant was mixed with 2.0ml of diluted reagent (1:1)
and incubated for 5 minutes at 37○C .The absorbance of sample against reagent
blank was read at 505nm. HDL cholesterol levels were expressed as mg/g in wet
tissue
Estimation of Protein
The Protein content of serum and the tissue homogenates of liver and
kidney were estimated by the method of Lowry et al., (1951).
Procedure
To 0.1 ml of suitably diluted serum/homogenate, 0.9 ml of water and 4.5 ml
of alkaline copper reagent were added and kept at room temperature for 10
minutes. Then 0.5 ml of Folin’s phenol reagent was added and the blue colour
developed was read after 20 minutes at 640nm using a spectrophotometer.
The level of protein was expressed as g per dL in serum and mg per g wet
tissue of liver and kidney.
60
Estimation of serum Albumin
Albumin in serum was estimated by Biuret method(Reinhold,1953).
Procedure
0.5 ml of sample was layered on to 9.5 ml of sodium sulphite in a centrifuge
tube and it was inverted to mix.2ml of mixture was taken immediately and marked
as total protein .The rest of the solution was allowed to stand for 10-15minutes for
precipitation of globulin and filtered using Whatman filter paper.The filterate
contains the albumin and 2ml of filtrate was taken and marked as total albumin
.The contents of all the tubes were made up to 2.5ml of distilled water .About
2.5ml of distilled water served as blank.Then 5ml of Biuret reagent was added to
all the tubes .Mixed the contents and kept for 10 minutes.A series of standard were
prepared and treated as the test .The purple or violet colour developed was read
colorimatrically at 540nm
The serum albumin levels were expressed as g/dL
61
set of standard pyruvate solution was also treated in a similar manner. The colour
developed was read at 540 nm using spectrophotometer.
Procedure
About 1.0 ml of substrate was incubated at 37 oC for 10 minutes. Then 0.2
ml of enzyme solution was added. The tubes were incubated at 37 oC for 30
minutes. To the control tubes enzymes were added after arresting the reaction with
1.0 ml of DNPH reagent. The tubes were kept at room temperature for 20 minutes.
Then 5.0 ml of 0.4 N sodium hydroxide was added and the colour developed was
read at 540nm using spectrophotometer.
Estimation of Urea
62
0.1ml of serum and 3.3ml of water was taken ; 0.3ml of 10% sodium
tungstate and 0.3ml of 2/3N sulphuric acid was added and centrifuged .To 1ml of
supernatant fluid added 1ml of water ,0.4ml diacetyl monoxime and 1.6ml of the
sulphuric acid –phosphoric acid mixture ,cooled ,and read against water blank at
480nm .
The level of uric acid in serum was estimated by the method of Caraway(1963).
Procedure
4.5ml of tungstic acid was added 0.5ml of samples were added and centrifuged .
3ml of the supernatant was added to 0.6 ml of sodium carbonate and followed with
0.6ml of diluted phosphotungstate .Mixed and placed in water bath at 25 oC for
30min.Then read within 15 minutes at 700nm.
Estimation of creatinine
Procedure
1.0ml of serum was mixed with 7.0ml of distilled water .1.0ml of sodium
tungstate and 1.0 ml of sulphuric acid were added and centrifuged. From this
4.0ml of supernatant was mixed with 1.0ml of sodium hydroxide and 1.0ml of
63
picric acid .The tubes were kept in a boiling water bath for 15minutes.The colour
developed was read at 470nm in a spectrophotometer.
64
centrifuged. About 4 ml supernatant from each tube (test,control , and standard ) in
a boiling water bath for 15 min ,cooled and the color developed was read at 680
nm
The activity of enzyme is expressed in IU/L
Estimation of LPO
LPO in tissues were estimated colorimetrically by TBARS and HPX by the method
of Nehius and Samuelson (1968) and Jiang et al. (1992), respectively. In brief, 0.1 ml of
TBA-TCA-HCl reagent (TBA 37%, TCA 15% and 0.25 N HCl) and placed in water bath
for 15 min, cooled and centrifuged at room temperature for 10 min at 1,000 rpm. The
absorbance of clear supernatant was measured against reference blank at 535 nm and
was treated with 0.9 ml of fox reagent (88 mg butylated hydroxytoluene, 7.6 mg xylenol
orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml
250 mmol/l sulphuric acid) and incubated at 37◦C for 30 min. The colour developed was
Statistical analysis
Statistical analysis was performed using the SPSS software package (Statistical Package
for the Social Sciences, United States). Data are presented as means with their standard
deviations, and the data were analyzed using analysis of variance (ANOVA). The group
RESULTS
65
In all groups prior to Alloxan administration, the basal levels of blood glucose of the rats
glucose levels were significantly higher in rats selected for the study. In contrast, non-
diabetic controls remained persistently euglycaemic throughout the course of the study.
Table shows the change in body weight gain to control and experimental groups of
rats .There was a signifigant decrease in the body weight of diabetic rats compared with
control rats .Upon the treatment of Pedalium murex leaves and leaves derived callus and
glibenglamide .the body weight gain was improved but the effect was more pronounced
in Pedalium murex leaves callus treated rats then leaves and glibenglamide
Table 1 shows the effect of treatment with extracts on blood glucose levels.
In all the Pedalium murex leaves, callus ethanol extract -treated groups a significant
antihyperglycaemic effect was evident from first week onwards the decrease in blood
sugar being maximum on completion of the third week in the group receiving 200
and experimental group. While the level of plasma inulin was decreased and
control group .Oral administration of Pedalium murex leaves and leaves derived callus
brought back to near normal values as that of standard drug glibenglamide treatment.
66
Effects on the administration of Pedalium murex leaves ,leaves derived
6-bisphosphatase of liver are presented in Table 3. table – shows the activity of these
significantly elevated in allaxon treated diabetic rats as compared to normal rats. Oral
administration of Pedalium murex leaves and leaves derived callus brought back to near
treated diabetic rats as compared to normal rats. Oral administration of Pedalium murex
leaves and leaves derived callus brought back to near normal values as that of standard
Triglycerides are a group of lipids absorbed from the diet and produced
rats compared to normal control rats .table shows the result for the level of serum
compare with control rats .administration of Pedalium murex leave and leaves derived
callus and glibenglamide to diabetic rats tends to bring the values to near normal.
67
Table shows the level of urea ,uric acid ,and creatinine in normal control and
diabetic control rats. The level of urea ,uric acid and creatinine were significantly
reduced in the Pedalium murex leaves and callus extract treated rats as compared with
Table shows the level of AST ,ALT,ACP and ALP control and diabetic treated
rats .the diabetic rats showed a significant increase in AST ,ALT,ACP AND ALP as
compared to normal control rats. by supplementing Pedalium murex leaves and callus
Table shows the concentration of TBARS and hydro peroxides in tissues of normal
control and experimental animals. There was a significant elevation in tissues TBARS
and HPx during diabetes when compared to the corresponding control group.
Administration of Pedalium murex leaves and leaves derived callus and glibenglamide
DISCUSSION
The use of traditional medicine and medicinal plants in most developing countries, as a
normative basis for the maintenance of good health, has been widely observed
plants in the society has been traced to the extraction and development of several drugs
and chemotherapeutics from these plants as well as from traditionally used rural herbal
remedy (Bhattaram et al., 2002). This study was under taken to asses the antidiabetic
68
effect of Pedalium murex leaves and leaves derived callus on ethanol extract in alloxan
species by damaging the insulin secreting cells of the pancreas . This damages a large
hepatic glucose over production .(Matinez and Milagro, 2000). Numerous studies
demonstrated that a variety of plant extracts effectively lowered the glucose level in
wt./day) resulted in a significant reduction in the blood glucose and improvement in body
weight. The decrease in body weight in diabetic rats clearly shows a loss or degradation
of structural proteins due to diabetes. The structural proteins are known to contribute for
the body weight (Rajkumar and Govindarajulu, 1991). Protein synthesis is decreased in
all tissues due to absolute or relative deficiency of insulin (an anabolic hormone) in
alloxan-induced diabetic rats. The ability of the Pedalium murex leaves and callus to
protect from maximum body weight loss seems to be due to its ability to reduce
hyperglycemia.
69
Further, the antihyperglycemic activity of Pedalium murex was associated with an
murex leaves and callus extract. The observed increase in the level of plasma insulin
indicates that P.murex leaves and callus extract stimulates insulin secretion from
regenerated β-cells. In this context, a number of other plants have also been reported to
extent hypoglycemic activity through insulin release stimulatory effect .(Pari and latha,
control group of rats is due to the presence of excessive amount of blood glucose. During
diabetes the excess of glucose present in blood react with haemoglobin to form
glycosylated haemoglobin .(Alyassin and Ibrahim , 1981; sheela and Augusti , 1992).
glycosylated haemoglobin has been found to be increased over a long period of time in
the diabetes mellitus (Bunn et al .,1978). There is evidence that glycation may itself
induce the generation of oxygen derived free radicals in diabetic condition. (Gupta et
al.,1997). Treatment with P.murex leaves and callus extract showed a decrease in the
diabetic rats standard drug glibenglamide also showed the same results.
gluconeogenesis are some of the changes of glucose metabolism in the diabetic liver.
70
almost completely inhibited or inactivated in diabetic rat liver in the absence of insulin.
also been reported in diabetic animals, resulting in depletion of liver and muscle
glycogen. (Laakso et al.,1995 ; Murray et al.,2000). In our study, we also have observed
hexokinase can cause the increased utilization of glucose for energy production. P.murex
leaves and callus has been observed to decrease the level of blood glucose. The decrease
bisphosphatase have been measured in the liver and kidney of diabetic animals and those
treated with P.murex. Both enzymes showed an increase in activity during diabetes in the
liver. Administration of P.murex leaves and callus was found to be more effective in
reversing both the enzymes to normal levels in the liver of alloxan-diabetic rats. The
increased hepatic as well as renal fructose1,6- bisphosphatase activity may be due to the
ATP, AMP and citrate. In a diabetic state, there is more lipolysis than lipogenesis,
especially in liver, which will result in the formation of more AMP and lower utilization
of citrate for lipogenesis leading to high energy state in the cell, i.e. higher concentration
The reduction in the activities of these gluconeogenic enzymes can result in decreased
71
concentration of blood glucose. Administration of P.murex leaves and callus had
increased the activity of hexokinase and decreased the activities of both glucose-6-
Reduction in plasma total protein and albumin level was observed in diabetic rats
and this is consistent with the results obtained by Bakris,1997 ; Tuvemo et al.,1997.The
decrease in protein and albumin may be due to microproteinuria and albuminuria, which
are important clinical markers of diabetic nephropathy, ( Mauer et al. ,1981) and/or may
be due to increased protein catabolism.( Almdal et al.,1988) The results of the present
study demonstrated that the treatment of diabetic rats with the aqueous extract of
P.murex leaves and callus caused a noticeable elevation in the plasma total protein and
albumin levels as compared with their normal levels. Such improvement of serum protein
and albumin was previously observed after the oral administration of Balanites
been established that insulin stimulates the incorporation of amino acids into proteins.
(Almdal et al.,1988).
atherosclerosis and includes an increase in triglycerides and total cholesterol levels .In
this study the extract significantly reduces the triglycerides ,phospholipids and total
72
The levels of serum lipids is usually elevated in diabetes mellitus ,and such an
elevation represents the risk factor for coronary heart disease ( Davidson ,1981).
Lowering of serum lipids concentration through dietary with or drug therapy seems to
be associated with a decrease in the risk of vascular disease (Rhoads et al., 1976).Several
investigation demonstrated that near normalization of the blood glucose level resulted in
phospholipids .the same results were obtained with the fruit ethanol extract of Pedalium
al.,2008). The result of this study show that continuous administration of P.murex
leaves and leaves derived callus extract at the dose of 200mg /kg/day significantly lower
The plasma levels of urea, uric acid and creatinine levels were measured, as DM
also causes renal damage due to abnormal glucose regulation, including elevated glucose
and glycosylated protein tissue levels, haemodynamic changes within the kidney tissue,
and increased oxidative stress.( Aurell and Bjorck ,1992) The Alloxan-induced diabetic
rats exhibited significantly higher plasma urea, uric acid and creatinine levels compared
to the DM group. However, the P.murex leaves and callus supplement lowered these
plasma values to a control range. A significant elevation in serum creatinine and urea
levels indicate an impaired renal function of diabetic animals.( Shinde and Goyal ,2003))
Thus, it would appear that the P.murex leaves ,callus supplement lowered the plasma
urea, uric acid and creatinine levels by enhancing the renal function that is generally
73
The serum AST and ALT levels increase as a result of metabolic changes
in the liver, such as administration of toxin, cirrhosis of the liver, hepatitis and liver
cancer including diabetes.( Chalasani et al .,2004) Similarly in the present study, it was
observed that the levels of serum AST and ALT in alloxan induced diabetic rats were
elevated. It may be due to leaking out of enzymes from the tissues and migrating into the
circulation by the adverse effect of alloxan.( Stanely et al .,1999).AST and ALT were
used as markers to assess the extent of liver damage in streptozotocin induced diabetic
rats.( Hye-Jin Hwang et al .,2005). In this study, the administration of P.murex callus
ethanol extract to alloxan-induced diabetic rats reduces AST and ALT levels efficiently
than P.murex leaves extract treated rats. In addition to the assessment of AST and ALT
acid phosphatase (ACP) and alkaline phosphatase (ALP) is of clinical and toxicological
Singh et al.,2001). In our study, serum ACP and ALP increased considerably in alloxan
induced diabetic rats. Elevated level of these enzymes in diabetes may be due to
significant decline in these levels than the leaves extract treated rats. From the present
observation, it was evident that P.murex leaves and callus ethanol extract protects the
adverse effects of lipid peroxide mediated tissue damage in alloxan induced diabetic rats.
hydroperoxides are observed in liver and kidney of diabetic rats (Latha and Pari, 2003;
74
Sathish and Pari, 2004). In this study shows that P.murex leaves and callus and
75
INTRODUCTION
diabetes is suggested not only by oxygen free-radical generation but also due to non
1990; Gillery et al., 2006). Several herbal drugs in different formulations have been
76
oxidative stress ultimately leads to tissue damage has advanced considerably in recent
years. Effective therapeutic strategies to prevent or delay the development of this damage
remain limited and the American Diabetes Association recommended that antioxidant
need to be identified (Evans et al. , 2003). Plants constitute an important source of active
natural products, which differ widely in terms of structure and biological properties. They
have a remarkable role in the traditional medicine in different countries. The protective
effects of plant products are due to the presence of several components, which have
distinct mechanisms of action; some of them are enzymes and proteins and others are low
molecular weight compounds such as vitamins, carotenoids, flavonoids (Zhang and Wang
In recent years, much attention has been focused on the role of oxidative
stress, and it has been reported that oxidative stress may constitute the key and common
radicals are continuously produced in the body as a result of normal metabolic processes
and interaction with environmental stimuli. Oxidative stress results from an imbalance
between radical-generating and radical-scavenging systems that has increased free radical
stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free
77
formation of lipid peroxides (Mullarkey et al .,1990; Lennan et al., 1991). In addition to
reduced glutathione (GSH), there are other defense mechanisms against free radicals,
such as the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and
atherosclerotic vascular disease, the leading cause of mortality in diabetes mellitus, have
been linked to oxidative stress, and antioxidants have been considered as treatments
flavonoids and tannins and the present study suggests that antioxidant action may be an
Materials methods
Procedure
by the addition of 2.5 ml of ethanol and 1.5 ml of chloroform (all reagents were
78
chilled). The mixture was shaken for 1 minute at 4oC , centrifuged and the enzyme
preparation. The reaction was started by the addition of 0.2 ml of NADH and
incubated at 30oC for 90 seconds. Then the reaction was arrested by the addition of
1.0 ml of glacial acetic acid. The contents were mixed and shaken with 4.0 ml of n-
butanol. The mixture was allowed to stand for 10 minutes, centrifuged and the colour
The activity of SOD was expressed as the amount of enzyme required to give
protein.
Procedure
79
The assay mixture contains 4.0 ml of hydrogen peroxide, 5.0 ml of phosphate
buffer and 1.0 ml of homogenate. 1.0 ml portion of the reaction mixture was
intervals. Then the mixture was heated for 10 minutes in a boiling water bath. After
et al., (1973).
Procedure
The reaction mixture consisted of EDTA, sodium azide and H2O2, (each 0.2
ml) 0.4 ml of phosphate buffer and 0.1 ml homogenate was incubated at 37C for 10
minutes. The reaction was arrested by the addition of 0.5 ml of 10%TCA and the
hydrogen phosphate and 0.5 ml DTNB were added and the colour developed was
80
The activity of GPx was expressed as M of glutathione oxidized per min per
mg of protein.
Procedure
of enzyme source were added. After 5 minutes of incubation at 37C, 0.1 ml of GSH
was added and the change in optical density was measured immediately with an
Estimatima
recycling enzymes
Procedure
81
About 0.5 ml of sample (plasma/homogenate) was precipitated with 1.0
of the supernatant 1.0 ml of DTNB was added and the total volume was made up
The level of vitamin E was estimated by the method of Baker et al., (1980).
Procedure
mixed and centrifuged. The supernatant was dried at 80 oC. To the tubes after
dried, 0.2 ml of 2,2'-dipyridyl and 0.2 ml of ferric chloride solutions were added.
Mixed well and 4.0 ml of butanol was added. The red colour developed was read
at 520nm.
82
The level of ascorbic acid was estimated by the method of Omaye et al.,
(1979).
Procedure
were added, mixed thoroughly and centrifuged for 20 minutes. To 1.0 ml of the
supernatant, 0.2 ml of DTC reagent was added and incubated at 37C for 3 hrs. Then
1.5 ml of sulphuric acid was added, mixed well and the solutions were allowed to
stand at room temperature for another 30 minutes. The colour developed was read at
RESULT
The concentration of tissues SOD, CAT, GSH, GST, and GPx were significantly
PEDALIU MUREX extract and insulin to diabetic rats tend to bring the activities of these
enzymes to near normal level . The extent of increase was higher in groups treated with
ethanol extract of Pedalium murex leaves and callus than glibenclamide treated groups.
Treatment with P.murex leaves and callus to normal animals did not show any significant
alterations.
83
Table – shows significant reduction in non-enzymatic antioxidants in
P.murex leaves and callus extract (200 mg/kg body weight) and glibenclamide for a
period of 3 weeks decreased the glucose levels significantly and improved the tissue
antioxidant status significantly (p < .05). p.murex callus extract at a dose of 200 mg/kg
body weight was more effective then the other dose of leaves extracted 200 mg/kg body
weight.
DISCUSSION
Under in vivo conditions, GSH acts as an antioxidant and its decrease was
reported in diabetes mellitus (Baynes and Thorpe 1999). We have observed a significant
decrease in GSH levels in liver and kidney during diabetes. The decrease in GSH levels
represents increased utilization due to oxidative stress (Anuradha and Selvam 1993). The
depletion of GSH content may also lower the GST activity as GSH is required as a
substrate for GST activity (Rathore et al. 2000). Depression in GPx activity was also
84
observed in liver and kidney during diabetes. GPx has been shown to be an important
The increased GSH content in the liver and kidney of the rats treated with P.murex
leaves, callus and glibenclamide may be one factor responsible for inhibition of LPO.
SOD and CAT are the two major scavenging enzymes that remove toxic free radicals in
vivo. Previous studies have reported that the activity of SOD is low in diabetes mellitus
(Vucic et al. 1997). Reduced activities of SOD and CAT in liver and kidney have been
observed during diabetes and this may result in a number of deleterious effects due to the
accumulation of O.− 2 and H2O2 (Searle and Wilson 1980). Administration of Pedalium
murex leaves and callus increased the activity of enzymes and may help to control free
radical, which scavenge the free radicals generated during diabetes. Any compound,
natural or synthetic, with antioxidant properties, might contribute towards the partial or
total alleviation of this damage. Therefore, removing O.− 2 and OH∗ is probably one of
the most effective defenses against diseases (Lin et al., 1995). The result of the SOD
and CAT activity suggest that P.murex leaves and callus contains a free radical
scavenging activity, which could exert a beneficial action against pathological alterations
caused by the presence of O.− 2 , H2O2 and OH∗. This action could involve mechanisms
radical mediated damage. Depletion of liver and kidney reduced glutathione levels
85
Superoxide dismutase is an antioxidant enzyme which reduces superoxide radicals to water
and molecular oxygen (McCord et al., 1976) whilst catalase reduces hydrogen peroxide
(Gutteridge, 1995). Diminished activity of these antioxidant enzymes result elevation of ROS
and ROS mediated cell destruction. Reduced activities of superoxide dismutase and catalase
in liver and kidney were observed in diabetic rats and these were reverted to near normal
The decrease could have been due to increased utilization of ascorbic acid as
GSH level, since GSH is required for the recycling of ascorbic acid Hunt, (1996). α-
liver and kidney of STZ-diabetic rats. P.murex leaves,callus and glibenclamide treatment
tends to bring the α tocopherol levels to near normal value. Higuchi (1982) observed a
decreased hepatic α-tocopherol in rats with STZ-induced diabetes. These results suggest
that the demand for the antioxidant vitamin E is increased due to the activation of free
antioxidants (GSH, ascorbic acid, and α-tocopherol) results in increased oxidative injury
antioxidant within biomembrane (Parks and Traber, 2002). Reduced glutathione, a major
endogenous antioxidant, plays a crucial role in the antioxidant defense (Anuradha and
Selvam ,1993). Vitamin C, a major extra cellular non-enzymatic antioxidant, has crucial
86
role in scavenging several reactive oxygen species. The observed increase in antioxidant
status P.murex leaves and callus extact to treated diabetic rats suggests its potent
antioxidative effects. Furthermore the plant drug was found to be as effective as that of
the reference drug glibenclamide. Further studies are therefore needed to isolate and
characterize the bioactive antidiabetic principles from P.murex leaves and leaves derived
callus.
87
INTRODUCTION
various plant materials capable of decreasing blood sugar have been tested in
experimental animal models and their effects confirmed. Many unknown and lesser
88
known plants are used in folk and tribal medicinal practices in India. The medicinal
values of these plants are not much known to the scientific world.
Today, more than 200 traditional medicinal plants have been used for the
treatment of diabetes mellitus and widely practiced in South India. Plant drugs are
frequently considered to be less toxic and more free from side-effects than synthetic ones
[Momin , 1985]. Synthetic oral hypoglycemic agents can produce a series of side-effects
disturbances in liver and kidney metabolisms. In addition, these preparations are not ideal
Liver disease is one of the leading causes of death in persons with type 2
diabetes. The standardized mortality rate for death from liver disease is greater than that
of cardiovascular disease. The spectrum of liver disease in type 2 diabetes ranges from
nonalcoholic fatty liver disease to cirrhosis and hepatocellular carcinoma (Keith et al.,
2004). Experimental type 1 diabetes induced with streptozotcin or alloxan in rats display
et al ., 1997). liver and kidney in some cases (Ghosh, 2001). Patients depend on insulin
89
Diabetic nephropathy is the most important cause of death in type 1 diabetic patients,
of whom, 30 – 40% eventually develop end stage renal failure (Giorgino et al., 2004).
Liver disease is one of the leading cause of death in persons with type 2 diabetes. The
standardized mortality rate for death from liver disease is greater than that of
cardiovascular disease. The spectrum of liver disease in type 2 diabetes ranges from
nonalcoholic fatty liver disease to cirrhosis and hepatocellular carcinoma (Keith et al.,
2004).
glomeruli is reabsorbed into the cytoplasm of tubules. These phenomena are especially
glycogen inclusions are washed out and result in a clearing effect within the tubular
epithelial cells. This phenotype is referred to as Almanni- Ebstein cells and is a clear
concentrations of lipid peroxide are found in plasma and tissues. The possible source of
90
oxidative stress in diabetes includes shifts in redox balance resulting from altered
carbohydrate and lipid metabolism, increased generation of reactive oxygen species, and
decreased level of antioxidant defenses such as GSH and ascorbic acid (Baynes, 1991).
Increased levels of TBARS suggest increasing oxygen free radicals. Lipid peroxide-
mediated tissue damage has been observed in the development of Type II and Type I
regulating its metabolism, hexokinase ,which brings about the first phosphorilation.stepof
glues metabolism is reduced significantly in the diabetic rats (Nehal and Baquer,1989).In
the liver the enzymes is an important regulator of glucose storage and disposal . Attention
has long centered on the liver in diabetes mellitus because of the importance of this organ
composition of lipids occurs. Liver and kidney are important for glucose and lipid
homeostasis, they participates in the uptake, oxidation and metabolic conversion of free
to have changes in liver and kidney during diabetes (Seifter and England ,1982).. Liver
kidney. The kidney exhibits a characteristic pattern of changes during diabetes (Sharma
et al., 2003). The present study demonstrates the efficacy of P.murex leaves and leaves
91
derived callus (ethanol extract) in reducing diabetes-induced functional and histological
Animals
Healthy male adult albino rats (Wistar strain) of 6-7 weeks old, weighing
150 ± 20 g was procured from “Sri Venkateswara Enterprises”, Bangalore, India.
They were housed in clean sterile polypropylene cages with proper aeration and
lighting (12 ± 1 hr day / night rhythm) throughout the experimental period. During
the course of the experiments, the temperature was maintained between 27ºC ±
2ºC. The animals were fed with commercially available pelleted rat feed (Sri Sai
Durga feeds Bangalore, India.Under the trade name “Sri Sai Durga feed and
food”) and water ad libitum. The usage and handling of experimental rats was
followed as per the rules and regulations given by the Institutional Ethics
Committee for the purpose.
92
The rats were divided into seven groups, each group consists of six animals.
Group I :Served as control and received normal feed and water ad libitum .
Group II : served as a normal rats received 200 mg/kg /bw Pedalium murex
leaves extract and water ad libitum.
Group III : served as normal rats received 200 mg/kg /bw Pedalium murex
leaves derived callus extract and water ad libitum.
Group IV :Served as diabetic control and received feed and water ad libitum
Group V : Diabetic rats and were treated orally with ethanol extract of
Pedalium murex leaves at the dose of 200 mg/kg body weight daily
for 21 days, once a day.
Group VI : Diabetic rats and were treated orally with ethanol extract of
Pedalium murex leaves derived callus at the dose of 200 mg/kg
body weight daily for 21 days, once a day.
Group VII :Diabetic rats given glibenglamide orally at the dose of 0.6 mg/kg
body weight daily for 21 days, once a day.
Histopathological studies
One portion of liver and kidney tissues were removed after sacrificed and rapidly
dehydrated in alcohol, embedded in paraffin wax, sectioned in 5m and stained with
93
haematoxylin and eosin for light microscopy. The sections were examined at 40x. in liver
,10x in kidney
Histopathology
The liver, kidney and pancreas were preserved in 20% formalin immediately after
Tissue processing
Liver, kidney and pancreatic tissues were placed in 10% formalin (diluted to 10%
with normal saline) for 1 hr to rectify shrinkage due to high concentration of formalin.
80% isopropanol overnight and 100% isopropyl alcohol for 1 hour. The dehydrated
tissues were cleared in two changes of xylene, 1 hour each. The wax impregnated tissues
were embedded in paraffin blocks using the same grade wax. The paraffin blocks were
The sections were floated on a tissue floatation bath at 40 °C and taken on glass
slides and smeared with equal parts of egg albumin and glycerol. The sections were then
melted in an incubator at 60°C and after 5 min the sections were allowed to cool.
Tissue staining
staining jar. The deparaffinised sections were washed in 100% isopropyl alcohol and
stained in Ehrlich’s hematoxylin for 8 min in horizontal staining jar. After staining in
94
hematoxylin, the sections were washed in tap water and dipped in acid alcohol to remove
The sections were then placed in running tap water for 10 min for
blueing (slow alkalization). The sections were counter stained in 1% aqueous eosin (1 gm
in 100 ml tapwater) for 1 min and the excess stain was washed in tap water and the
sections in the incubator at 60°C for 5 min. When the sections were cooled, they were
mounted in DPX mount having the optical index of glass (the sections were wetted in
xylene and inverted on to the mount and placed on the cover slip). The architecture was
observed low power objective under microscope. The cell injury and over aspects were
RESULT
filling their lumen and glomerulopathy. The thickening of the walls was reversed by the
P.murex leaves and leaves derived callus (200 mg/kg) treatment, but the glomerulus
remain expanded Liver tissue of diabetic rat showed distortion in the arrangement of cells
around the central vein, enlargement and thickening of the walls of veins, capillaries, and
development of fibrosis in the degenerated cells. P.murex leaves and leaves derived
callus (200 mg/kg) treatment almost restored the cellular arrangement of hepatocytes
95
around the central vein and reduced fibrosis. It also helped to bring the blood vessels to
normal condition.
In the normal liver tissue section shows sinusoidal cards of hepatocytes with
central vein and portal tracts. The portal tracts show portal triad with portal vein, hepatic
artery and bile duct, where as the diabetic rat liver tissue section shows distortion in the
arrangement of cells around the central vein, periportal fatty infiltration with focal
necrosis of hepatocytes (Figure 1 a and b). The leaf extract of Pedalium murex (100 and
200 mg / kg body weight) treated brought back the cellular arrangement around the
central vein and reduced necrosis. Also it helped to bring the blood vessels to normal
condition (Figure 1 c and d). The group V and VI did not show any significant change of
filling their lumen and glomerulopathy. Kidney sections of diabetic rat showed tubular
damage, proteinuria and haemorrhage. Haemorrhage is seen with in the Bowman’s space
due to glomerular damage (Figure 2 a and b). In Pedalium murex leaf extract and callus
extract (200 and 200 mg / kg body weight) treated diabetic kidney, the damaged capillary
loops with increase in the thickness of the wall, glomeruli and tubules without proteinuria
and haemorrhage (Figure 2 c and d) Group V and VI did not alter the structure of kidney,
DISSCUSION
96
The main function of the kidneys is to excrete the waste products of metabolism
and to regulate the body concentration of water and salt. The morphological changes in
alloxan diabetic rats in the present investigation is associated with significant increased of
total protein excreted, albuminuria, glycosuria, and urinary urea levels, indicating
impaired renal function of diabetic rats. Alloxan-induced diabetes in rats had been shown
al., 2006).In our study, treatment of alloxan-diabetic rats with P.murex leaves and leaves
derived callus and glibenglamide induced a fall in the level of all these metabolic
parameters. However, the improvement in urinary protein, albumin, glucose and urea
excretion with P.murex extract were not sufficient to reach the levels observed in the
non-diabetic rats; moreover P.murex did not alter any biochemical kidney function
variables in non diabetic rats. Similar results were obtained with diabetic rabbits treated
with Eugenia jambolana (Kedar and chakrabarti, 1983). and non-diabetic rats treated
with Bauhinia forficata (Pepato et al, 2002). Albumin measurements are required, as
advanced lesions, respectively, in kidney disease (Roy, 2004; Van den Born et al., 1995).
increase in urinary protein particularly albumin and a late decline in glomerular filtration
rate, leading eventually to end-stage renal failure (Salah et al., 2004). Histologically, the
it has been reported that streptozotocin does not possess any significant nephrotoxic
97
potential (Floretto et al., 1998). All structural changes in kidneys resulting from STZ
and tubular lesions that characterise diabetic nephropathy. The improvement of renal
morphology and function associated with STZ-induced diabetes and P.murex treatment
the leaves ,stem, and fruits of P.murex in cold water is a demulcent and a diuretic found
The action by which the extract lowered the blood glucose is not well
decrease in glycogenolysis. Since P.murex did not significantly reduce glycaemia in non-
glibenclamide and insulin. Similar results have been observed with the treatment of STZ-
induced diabetic rats with Cassia kleinii leaf extract and glibenclamide (Babu et al.,
2003). In another hand, the chemical substances therapeutic properties could be mediated
98
The phytochemical analysis had revealed the presence of alkaloids,
polyphenols and saponins in the plant extract. Based on the increasing number of reports
on blood glucose reduction associated with some saponins (Diatewa, 2004) and alkaloids
(Bolkent et al., 2000) isolated from other medicinal plants, it is likely that the active
principle (s) could be present in one or the two families of chemical substances.
Hennebele et al. (2004) suggested that these molecules can be inhibited with
hypoglycaemia when taken in excessive doses and hypoglycaemia is the most worrisome
effect of these drugs P.murex did not cause any hypoglycaemia, therefore, it could be an
effective treatment for early renal disease and possibly other diabetic complications.
(Figure 2 b), dilatation in the sinusoids and inflammation were noticed (Figure 2 c).
These changes were reduced in A. vera-fed rats of group-IV (Figure ). This may be due to
beneficial and protective effect of A. vera extract on liver tissues of diabetic rats. Our
histological findings are in agreement with the degenerative structural changes reported
in liver tissues as result of insulin depletion in neonatal STZ (100 mg/kg) - induced type-
and leaves derived callus appears to be an attractive material for further studies leading to
99
possible drug development for diabetes. Development of phytomedicines is relatively
inexpensive and less time consuming; it is more suited to our economic conditions than
allopathic drug development which is more expensive and spread over several years.
100