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ANTIDIABETIC EFFICACY OF LEAVES AND CALLUS OF PEDALIUM
MUREX L. ON ALLOXAN INDUCED DIABETIC ALBINO RATS
1
INTRODUCTION
Diabetes is a serious metabolic disorder with micro and macro vascular
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
also recommended the evaluation of the effectiveness of plants in condition where we
lack safe modern drugs (Upathaya et al.,1991 ). The pharmaceutical drugs are either too
expensive or have undesirable side effects. Treatment with sulphonylureas and
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
to sugar(Akhtar and Hussain,1992).
Diabetes is defined as a state in which homeostasis of carbohydrate and lipid
metabolism is improperly regulated by insulin. This results primarily in elevated fasting
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
agent substantially capable of preventing this type of disease(Rabinovitch et al., 1998 ;
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
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 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
complications such as retinopathy , microangiopathy , and nephropathy (Rotshteyn 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
300 million by 2025 (Porter and Barret, 2005).
Diabetes Mellitus is a heterogenic metabolic disorder characterized by chronic
hyperglycemia and disturbance of carbohydrate, fat and protein metabolism.The term is
derived from Greek words "diabetes" means to pass through," Mellitus" means honey or
related to sugar(Akhtar and Hussain, 1992).
Diabetes is characterized by hyperglycemia resulting in various short term metabolic
changes in lipid and protein and long term irreversible vascular changes .These include
diabetes specific complication of the micro vascular system (retinopathy,
nephropathy,and neuropathy)and complications of macrovascular system (atherosclerosis
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)
Diabetes mellitus is a depilating and often life-threatening disorder with
increasing disorder with increasing incidence throughout the world (WHO-
1985).Diabetes is a chronic disease without a cure, however, with proper management
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
carbohydrates or from the breakdown of complex carbohydrates such as starch. After
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.
1. Cells may become starved for energy
2. Overtime, high blood glucose levels may harm kidney, heart, eyes or
nerves
A complex disease like diabetes mellitus, where little is talked about in
aspects of prevention and curation, but rather management, there is an increased focus on
plants in the search for appropriate hypoglycemic/antihyperglycemic agents. Firstly,
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
thought to act on a variety of targets by various modes and mechanisms (Tiwari et
al.,2002)-given the multi-factorial pathogenicity of the disorders.
Diabetes is becoming something of a pandemic and despite the recent
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
carbohydrate hydrolysing enzymes (pseudosaccharides), release of insulin from
pancreatic b-cells (sulphonyl urea), glucose utilization (biguanides), insulin sensitizers,
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
manipulating carbohydrate metabolism by various mechanisms, preventing and restoring
integrity and function of b-cells, insulin-releasing activity, improving glucose uptake and
utilization and the antioxidant properties present in medicinal plants offer exciting
opportunity to develop them into novel therapeutics. The multifactorial pathogenicity of
diabetes demands multi-modal therapeutic approach. Thus, future therapeutic strategies
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
traditional medicinal practice, which prescribes polyherbal formulations. The theories of
6
polyherbal formulation have the synergistic, potentiative, agonistic/antagonistic
pharmacological agents within themselves due to incorporation of plant medicines with
diverse pharmacological actions. These pharmacological principles work together in a
dynamic way to produce maximum therapeutic efficacy with minimum side effects.
Traditional medicinal preparations therefore, should not be considered just as a collection
of therapeutic recipes. They are formulated and prepared keeping in mind the conditions
of sickness and the healing properties of individual ingredients. It is important therefore,
that herbal medicines and preparations should be taken with the consideration of their
holistic therapeutic approach. The multiple activities of plant-based medicinal
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
in terms of multiple therapeutic approaches of herbal medicines, well-organized, rigorous
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
growing, it is also being simultaneously counterbalanced by inadequate regulation (Ernst,
2000).
Ayurveda is a traditional system of medicine using a wide range of modalities
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 ).
Pedalium murex L. (pedaliaceae) commenly known as "yanai nerungi" /
"peru nerungi" is an erect ' much branched , foetid smelling succulent annual herb . The
leaves contain pedalitin , diosmetin , dianatin,pedalin , dianatin-7-glucoronide and
diosmetin-7-glucuronide(Subramanian and Nair, 1972).Decoction of the root is used an
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
aqueous and antipyretic activities (Muralidharan and Balamurugan, 2008)
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
flavonoids and other constituents with diuretic, antispasmodic and aphrodisiac
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
and Dalziel 1964).
Medicinal plants are of great interest to the researchers in the
field of biotechnology as most of the drug industries depend in part, on plants for the
production o pharmaceutical compounds (Chand et al., 1997). The endeavor is to adopt
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
materials produced through tissue culture are deficient in secondary chemicals of
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
ethanomedicine around the world ,(Thomson, 1978;Stockwell, 1988). The multidrug
resistant strain of many microorganisms has revealed exploration of alternative
antimicrobial agent. Medicinal plants have become the focus of intense study in terms of
validation of their traditional uses through the determination of their actual
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
need to search new infection fighting strategies to control microbial infections
(Sieradzki et al., 1999)
In our present study is standard protocol for Pedalium murex in
tissue culture technique and anti microbial ,anti diabetic activity of invitro and invivo
plant extract of Pedalium murex
10
.
11
Review
Diabetes mellitus is a metabolic disorder featured by hyperglycemia and
alterations in carbohydrate, fat and protein metabolism associated with absolute or
relative deficiency of insulin secretion and /or insulin action, (Kameswara Rao et
al.,2003) It is one of the oldest diseases affecting millions of people all over the world.
(Andallu et al., 2002). Different types of oral hypoglycaemic agents such as biguanides
and sulphonylurea are available along with insulin for the treatment of diabetes mellitus
(Holman et al.,1991), but the side effects associated are unavoidable with their uses
(Kameshwara Rao, et al., 1997). Herbal drugs are widely prescribed today because of the
biologically active compounds are having minimal adverse effects and low costs.
Valiathan,(1998). In recent years, numerous traditional medicinal plants were tested for
their antidiabetic potential in the experimental animals, ( Srivastava et al.,1993). World
Health Organization (WHO), also permites the use of plant drugs for different disease,
including diabetes mellitus, (Gupta et al., 2005).
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).
The elevated levels of blood glucose in diabetes produce oxygen-free
radicals that cause membrane damage due to peroxidation of membrane lipids and protein
glycation (Baynes 1991). Glucose auto-oxidize in the presence of transition metal ions
generates oxygen-free radicals, which make the membrane vulnerable to oxidative
damage (Hunt et al. 1990). The oxidative stress and resultant tissue damage are important
component in the pathogenesis of diabetic complications (Baynes 1991). The free
radicals react with biomembrane causing oxidative destruction of polyunsaturated fatty
acids forming cytotoxic aldehydes by a process known as Lipid Peroxidation (LPO)
(Wolff 1993). The extent of LPO was measured in terms of ThioBarbituric Acid Reactive
Substances (TBARS) and lipid hydroperoxides (HPX), which are the end products of
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).
Diabetes mellitus is associated with augmented oxidative stress
(Menon et al., 2004) which leads major chronic complications namely retinopathy,
neuropathy, nephropathy, atherosclerotic coronary artery disease, and peripheral
atherosclerotic vascular disease (Kaczmar ,1998). Hyperglycemia increases the
production of reactive oxygen species (ROS) inside the aortic endothelial cells. ROS-
induced activation of protein kinase-C isoforms, increased formation of glucose-derived
advanced glycation end products, increased glucose flux through aldose reductase
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).
Allium cepa significantly reduced blood glucose level of alloxan induced
diabetic rats (Sheela etal., 1995). Alloxan induced diabetic rats fed a diet containing
Allium sativum (12.5%) for 15 days were able to reduce blood glucose as compare to
control group (Jelodar et al., 2005). Herbal extract of garlic produced hypoglycemia,
probably by interfering with food intake of both normal and STZ-diabetic rats
(Musabayane et al., 2006).
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).
Andrographis paniculata extract effectively produced hypoglycemic and
anti-hyperglycemic activity in normal rats (Borhanuddin et al., 1994),and different doses
of Andrographis paniculata extract effectively reduced the fasting serum glucose level
of STZ-diabetic rats (Zhang et al., 2000a). Significant reduction in blood glucose level
(52.90%) observed when hyperglycemic rats treated with aqueous extract of
Andrographis paniculata, (Husen et al. 2004).
Annona squamosa aqueous leaf extract produces hypoglycemic activity
in streptozotocin-nicotinamide induced diabetic rats (Shirwaikar et al., 2004), and the
fruit pulp extract has been observed to improve the glucose tolerance of alloxan diabetic
rats, (Gupta et al., 2005). Oral administration of aqueous leaf-extract to diabetic rats for
16
30 days significantly reduced the levels of blood glucose and increased the activity of
plasma insulin and antioxidant enzymes (Kaleem et al., 2006).
Aqueous extract Azadirachta indica to produce antihyperglycemic and
hypoglycemic activity in diabetic dogs (Satyanarayan et al., 1978), fresh leaves decoction
induced antihyperglycemic activity (Chattopadhyay et al., 1987a) and increased the
peripheral glucose utilization in normal rats (Chattopadhyay et al., 1987b). Leaf extract
of Azadirachta indica has been reported to produce the hypoglycemic activity in normal
rats (Chattopadhyay et al., 1999) and the crude ethanol extract of Azadirachta indica
potentially lowered the blood sugar level of alloxan diabetic rats , (Kar et al., 2003), and
also it produce anti-hyperglycemic activity in streptozotocin diabetic rats without altered
serum cortisol level (Gholap et al., 2004).
Ethanolic extract of Cinnamomum tamala leaves induced potential
hypoglycemic effect in 18 hours fasted albino rats (Tripathi et al., 1990),and it produced
hypoglycemic activity in alloxan induced diabetic rats when administered orally for two
weeks at a dose of 250mg/kg (Kar et al., 2003). Ethanol extract of Coccinia indica
produced hypoglycemic activity in fasted, glucose fed and diabetic albino rats ,
(Mukherjee et al., 1988), and hypoglycemic and hypercholesterolemic effect of aqueous
Ficus bengalensis bark extract was observed in alloxan induced mild and severe diabetic
rabbits, (Gupta et al., 2002).
Alcoholic leaf extract of Gymnema sylvestre lowered maximum blood sugar in
fasted, glucose fed and diabetic rats along with insulin released from pancreatic ß-cells
(Chatopadhyay et al., 1993), and Gymnemic acid isolated from leaves Gymnema
sylvestre to produced potent hypoglycemic effect in STZ-diabetic mice (Sugihara et al.,
2000). Gymnema sylvestre leaf extract shown anti-hyperglycemic activity (Gholap et
al., 2003) and hypoglycemic (Gholap et al., 2004) effects of in corticosteroid-induced
diabetes mellitus,
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).
Janapati et al.,(2008) reported that alcoholic extract of the Talinum
cuneifolium leaves have significantly lower the blood sugar level of hyperglycemic
rats.Aqua solution of Artemisia herba-alba, possessed antidiabetic activity in alloxan
induced diabetic rats,(Didem Tastenkin et al.,2006). Tanko et al .,(2008) reported that
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).
The diabetogenic effects of alloxan are attributed to a specific cytotoxic action
mediated by hydroxyl radical generation on pancreatic β-cells. This damages a large
number of β-cells resulting in a decrease in endogenous insulin release. Alloxan
administered rats therefore become hyperglycaemic in a short period of time, followed by
hepatic glucose over production. (Milgro and Martinez, 2000)
The increase in oxygen free radicals in diabetes could be due to increase in
blood glucose levels, which upon autooxidation generate free radicals. Streptozotocin has
been shown to produce oxygen free radicals (Ivorra et al., 1989). Lipid peroxide
mediated tissue damages have been observed in the development of type I and type II
diabetes mellitus (Feillet-Coudray et al., 1999). Previous studies have reported that there
was an increased LPO in liver and kidney of diabetic rats (Pari and Latha 2002;
Venkateswaran and Pari 2002). Peroxidation of membrane lipids associated with increased membrane
rigidity, and reduced cells survival has been implicated in diabetes mellitus (Selvam and Anuradha, 1988).
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
peroxidation and reduced antioxidant reserve (Panneerselvam and Govindasamy, 2004) .
Phyllanthus fraternus possessed antidiabetic and antioxidant activity (Munish
Garg et al.,2008). Annona squamosa leaf extract prevents diabetic complications from
lipid peroxidation and antioxidant systems in experimental diabetic rats. Anthocephalus
indicus, root as a potent sugar, lipid lowering and antioxidant (Vishnu Kumar et al.,
2009). vitamin E prevents stress-induced elevation of lipid peroxidation.
(Olanlokun ,2008). Decreased lipid peroxides and tissue lipids clearly showed the
antihyperlipidemic and antiperoxidative effect of Diasulin apart from its antidiabetic
effect (Ramalingam Saravanan and Leelavinothan Pari 2005). Diospyros peregrine
possessed considerable antioxidant activity in alloxan induced diabetic rats. (Dewanjee et
al., 2007). Scoparia dulcis, possessed an antidiabetic effect in addition to antioxidant
activity, (Pari and Latha,2005). Nymphaea stellata flower extracts exhibited
antihyperglycaemic and antihyperlipidaemic effects of on alloxan-induced diabetic rats.(
Rajagopal and Sasikala ,2008),an Tephrosia purpurea has potent antihyperglycemic and
antilipidperoxidative effects in streptozotocin induced diabetic rats. (Pavana et al., 2007).
20
Methanolic extracts of Cleome droserifolia exhibited a significant antidiabetic and
antiperoxidative activity in alloxan diabetic rats, (Naggari et al., 2005).
A significant increase in lipid peroxidation in diabetic rats suggests that
increased generation of free radicals by hyperglycemia related to glucose auto-oxidation
(Woeff and Dean, 1987). The antioxidant activity of olive oil has been reported that its
elevates the activities of hepatic antioxidant enzymes such as catalase, superoxide
dismutase and glutathione peroxidase, (Ruiz-Gutierrez et al.,1999; (Aguilera et al.,
2003)). Olive oil is highly enriched in oleic acid (unsaturated fatty acid) which results in
a decrease of LDL, cholesterol and triglycerides levels of hypercholesterolemic patients,
(Sirtori et al., 1992). Polyunsaturated fatty acids have a hypocholesterolemic activity
(Reaven et al., 1993).
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)
Experimental diabetes, induced by streptozotocin provoked hyperglycemia
accompanied by symptoms like loss of weight, polydipsia and polyphagia (Szkudelski
and Szkudeska ,2002 ) . Sundaram et al. (1996) have reported that the concentration of
lipid peroxides increases in the kidney of diabetic rats. An increased level of TBARS is
an index of lipid peroxidation Casearia esculenta extract decreased level of TBARS
diabetic kidney and liver (Prakasam et al., 2005)
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).
Liver and kidney exhibits numerous morphological and functional
alterations during diabetes (Sochar et al.,1985). Trigonella foenum-graecum L. helps to
recover the pathological effects of diabetes on liver and kidney of streptozotocin induced
diabetic rats(Naveen et al.,2007). Ayesha Noor et al .,(2008) reported that changes in the
histology of kidney and stomach sections after feeding with A. vera extract to diabetic
rats.
During diabetes the excess of glucose present in blood react with
heamoglobin to form glycosylated haemoglobin. (Alyassin and Ibrahim,1981;Sheela and
Augusti,1992).Glycosylated haemoglobinhas been found to be increased over a long
period time in the diabetes mellitus.(Bunn et al.,1978). There is an evidence that
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.
Plant-based antimicrobials and antibacterials represent a vast untapped source
for medicines and hence have enormous therapeutic potential (Phillipson, 1994). They
are effective in the treatment of infections while mitigating many of the side effects
associated with synthetic antimicrobial and antibacterial (Mathews et al., 1999; Bagghi,
2000). Lauric, palmitic, linolenic, linoleic, oleic, stearic and myristic acids are known to
have potential antibacterial and antifungal agents. (McGaw et al.,2002; Seidel et
al.,2004).
Aqueous extract of roots of the Hemidesmus indicus exhibited
bacteriostatic activity in mice infected with Mycobacterium leprae, Pmethoxy salicylic
aldehyde present in the extract was considered to be responsible for the activity (Gupta,
1981). Essential oil of H .indicus exhibited marked antibacterial activity against both
gram positive and gram negative bacteria even at concentration of 0.2%. The oil however
failed to show appreciable antifungal activity against fungi tested (Prasad et al., 1983).
Chloroform and ethanol (95%) extracts of H .indicus showed antifungal activity against
A. niger (Hiremath et al.,1997). The methanolic extract of root was proved to possess
anti-diarrhoeal activity in in-vivo and in-vitro studies (Das et al., 2003).
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
Medicinal plants are gifts of nature to cure limitless number of diseases
among human beings (Bushra and Ganga, 2003). The abundance of plants on the
earth’s surfaces has led to an increasing interest in the investigation of different
extracts obtained from traditional medicinal plants as potential sources of new
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
fraction submitted to biological or pharmacological screening is even smaller
(Mahesh and Sathish,2008).Thus,any phytochemical investigation of a given
plant will reveal only a very narrow spectrum of its constituents . Historically
pharmacological screening of compounds of natural or synthetic origin has been
the source of innumerable therapeutic agents. Random screening as tool in
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
continue to occupy an important niche in modern medicine. On a global basis, at
least 130 drugs, all single chemical entities extracted from higher plants, or
modified further synthetically for economic reasons .Medicinal plants represent a
rich source of antimicrobial agents.
Plants are used medicinally in different countries and are
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
effective compounds (Janovska et al., 2003).
Infectious diseases are the leading cause of death world-wide.
Antibiotic resistance has become a global concern (Westh et al., 2004). The
clinical efficacy of many existing antibiotics is being threatened by the emergence
of multidrug-resistant pathogens (Bandow et al., 2003). Many infectious diseases
have been known to be treated with herbal remedies throughout the history of
mankind. Natural products, either as pure compounds or as standardized plant
extracts, provide unlimited opportunities for new drug leads because of the
unmatched availability of chemical diversity. There is a continuous and urgent
need to discover new antimicrobial compounds with diverse chemical structures
and novel mechanisms of action for new and re-emerging infectious diseases
(Rojas et al ., 2003). Therefore, researchers are increasingly turning their attention
to folk medicine, looking for new leads to develop better drugs against microbial
infections (Benkeblia , 2004).
29
The increasing failure of chemotherapeutics and antibiotic
resistance exhibited by pathogenic microbial infectious agents has led to the
screening of several medicinal plants for their potential antimicrobial activity
(Colombo and Bosisio ,1996; Iwu et al.,1996). India is a varietal emporium of
medicinal plants and is one of the richest countries in the world in regard to
genetic resources of medicinal plants. It exhibits a wide range in topography and
climate, which has a bearing on its vegetation and floristic composition.
Moreover, the agro-climatic conditions are conducive for
introducing and domesticating new exotic plant varieties (Martins et al., 2001). In
recent years, secondary plant metabolites (phytochemicals), previously with
unknown pharmacological activities, have been extensively investigated as a
source of medicinal agents (Krishnaraju et al., 2005).
Thus, it is anticipated that phytochemicals with adequate antibacterial
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
treatment and prevention of various ailments (Tanaka et al., 2002).
In many developing countries, a large proportion of the
population relies heavily on traditional practitioners, who are dependent on
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
has also prompted researcher to screen scientifically various claims regarding
properties and uses of medicinal plant materials.
Presently, both, common consumers and healthcare professionals
seek updated, authoritative information towards safety and efficacy of any
recommended medicinal plant as drug prior to its use. (Manish et
al.,2007).Medicinal plants are of great interest to the researchers in the field of
biotechnology as most of the drug industries depend in part , on plants for the
production of pharmaceutical compounds .Tissue culture techniques are being
used globally for the exist conservation of plants . (Amutha et al., 2008).
Medicinal plants are of great interest to the researchers in the field
of biotechnology as most of the drug industries depend in part, on plants for the
production o pharmaceutical compounds (Chand et al., 1997).
Many higher plants are major sources of natural products used as
pharmaceuticals, agrochemicals, flavours and fragrances ingredients, food
additives, and pesticides (Balandrin and Klocke, 1988). It has been mentioned that
natural habitats for medicinal plants are disappearing fast and together with
environmental and geopolitical instabilities; it is increasingly difficult to acquire
plant derived compounds.
This has prompted industries, as well as scientists to consider the
possibilities of investigation into cell cultures as an alternative supply for the
production of plant pharmaceuticals (Mulabagal and Tsay, 2004).
31
In the search for alternative to production of desirable medicinal
compounds form plants, biotechnological approaches, especially, plant tissue
cultures, are found to have potential as a supplement to traditional agriculture in
the industrial production of bioactive plant metabolites (Dicosmo and Misawa,
1995; Ramanchandra Rao and Ravishankar, 2002).
Pedalium murex is a valuable plant source of medically useful
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
compounds and good antibacterial properties .particularly the gram positive
organisms ( Nagaraj et al.,2008).The present investigation is callus induction of
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:
The glassware used for culture work comprised of 6”x1” Riviera
and Borosil test tubes, 100 ml, 250 ml, 500 ml, and 1000 ml corning and Borosil
flasks, pipettes, and measuring cylinders (100 ml, 500 ml). Before use, glassware
were thoroughly brushed with alkaline detergent teepol and then washed in
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).
Constituents Molecular formula Concentration (mg/l)
Macronutrients Ammonium nitrate Potassium nitrate Calcium chloride Magnesium sulphate Potassium dihydrogen ortho Phosphate
NH4NO3 KNO3
CaCl2.2H2O MgSO4.7H2O KH2PO4
1650 1900 440 370 170
Micronutrients Ferrous Sulphate Disodium Ethylene Diamine Tetra Acetic Acid Boric acid Potasium iodide Manganese sulphate Zinc sulphate Sodium molybdate Cobalt chloride Copper sulphate
FeSO4. 7H2O Na2EDTA
H3BO3
KI MnSO4. 4H2O ZnSO4.7H2O Na2MoO4. 2H2O CoCl2. 6H2O CuSO4.5H2O
27.6 37.4
6.2
0.83 22.3 8.6
0.25 0.025 0.025
Vitamins Nicotinic acid Pyridoxine HCl Thiamine HCl Myo-inositol
100 0.5 0.5 0.1
Amino acids : Glycine 2.0
35
Sucrose 30,000
Agar 80,000
Table 2. Preparation of stock solutions of MS medium
Stock ingredients Quantity (mg)
Volume of stock/L of medium (ml)
Stock A (20X)
NH4NO3 KNO3
CaCl2. 2H2O MgSO4.7H2O
For 500 ml.
16500 19000 4400 3700
50
Stock B (100X)
MnSO4. 4H2O ZnSO4. 7H2O H3BO3 KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O
For 100 ml.
223 86 62 8,3 2.5 0.25 0.25
10
Stock C(100 X)
Nicotinic Acid Pyridoxine HCl Thiamine HCl Glycine
For 200 ml.
10 10 2 40
10
Stock D (100X) For 100 ml 10
36
KH2PO4 1700
Stock E (200X)
FeSO4. 7H2O Na2EDTA
For 100 ml.
556 746
5
Table 3. Preparation of plant growth regulator stock solution
Plant growth regulator (PGR)
Common abbreviation
Quantity (mg) for 10 ml.stock
Solvents Concentration of stocks
Auxins Indole -3- acetic acid α-Naphthalene acetic acid 2,4–Dichlorophenoxy acetic Acid Indole -3- butyric acid
IAA NAA 2,4 – D
IBA
10
Ethanol /1N NaOH 1 N
NaOH 1m1 = 1 mg/l
Cytokinins 6-Benzyl amino purine kinetin
BAP KIN
101 N HCl 1 ml = 1 mg/l
Gibberellic acid GA3 10 1 N NaOH 1 ml = 1 mg/l
The medium was supplemented with growth harmones
such as α-Naphthalene Acetic Acid (NAA), Indole Acetic Acid (IAA) , 2,4-
37
Dichlorophenoxy Acetic Acid (2,4-D), 6-Benzyl Amino Purine (BAP) and
Kinetin (KIN) either alone or in combinations at various concentrations.
The reagents used were of Analytical Reagent Grade. Each salt
was dissolved separately one after one to avoid precipitation. All the constituents
except agar were mixed and then the pH of the solution was adjusted to 5.5 - 5.8.
Later, agar was added and the medium was heated to boil so as to homogenize
agar. Following are some of the supplements which were used either singly or in
combination for the induction of callus, differentiation and multiple shoot
formation.
After the preparation of the medium, water was poured out of the
autoclaved glassware. Definite aliquots of the medium were then added depending
upon the capacity of the culture vessel. Generally 25 ml, 50 ml, 100 ml of the
medium was distributed into the test tubes, 100 ml and 250 ml flasks respectively.
After plugging the glassware with cotton plugs, media ware steam-sterilized at 15
lb/in (121oC) for 15 - 20 minutes. After Autoclaving, tubes were placed in stands
to prepare the slants. These were then left to cool and solidify.
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 Ao). The floor of the chamber was thoroughly
scrubbed with cotton dipped in alcohol.
The surface of all the vessels and other accessories such as
instruments (spatula, forceps, scalpels, blade etc.), gas burner, lighter, tube
containing absolute alcohol etc were also cleaned with alcohol. The fresh material
to be inoculated was kept in a Petri dish covered with a piece of black paper in
order to protect it from the harmful effects of U. V. rays. Alcohol was then
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.
Hands and arms which were to be used inside the inoculation
chamber were scrubbed with alcohol before inoculation. The rims of the test tubes
and the sides of the plugs were flame sterilized. Instruments (like forceps, scalpels,
spatula etc.) were all sterilized by dipping in the alcohol and flaming a number of
times. Care was taken to cool the instruments before putting into operation. The
explants taken from field borne plants were treated with 0.01 - 0.1% mercuric
chloride solution for 5-10 minutes respectively depending upon the explants.
Shoot apices of Nasturtium were treated with 0.1% mercuric chloride for 4 - 5
minutes.
The explants like stems and leaves were treated with 0.1% Hgcl 2 for 5 –
6 minutes. The explants were then thoroughly washed (4 - 5 washings) with
sterilized distilled water to remove the traces of Hgcl2. Fresh cuts were given to
the stem explants after sterilization to remove undesirable or dead portions. The
explants were then planted on variously augmented MS medium. Seeds were
surface sterilized with 0.1% Hgcl2 for 7 - 8 minutes. Constant shaking was done
during this period to get thorough sterilization. Rinsing with sterile distilled water
4 - 5 times was necessary for the removal of sterilant from the seeds. These were
then planted on Basal MS medium for germination. Leaves were excised from 4
weeks old seedlings and transferred separately to different experimental media.
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
Column:Elite-1 (100% Dimethyl poly siloxane),30m χ 0.25mm ID ×1µm df
Equipment:GC Clarus 500 Perkin Elmer
Carrier gas : Helium 1ml/min
Detector :Mass detector-Turpo mass gold –Perkin Elmer, Software-Turpo mass
5.1
Sample injected:2 µl
Split: 10:1
Oven Temperature programme:
110○C-2min hold
Up to 200 ○C at the rate of 10○C/min
Up to 280 ○C at the rate of 5 ○C/min-9 min hold
Total GC time: 36 min
Injector temp: 250 ○C
MS Programme
Library used : NIST Ver.2.0-year 2005
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
tested against following organisms: Escherichia coli ,Pseudomonas
aeruginosa ,Salmonella typhi Enterococcus facalis ,Klebsiella
Pneumoniae ,Basillus species Staphylococcus aures , Streptococcus epidermidis.
These culture were collected 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
product of Himedia Laboratories Mumbai (India) were used in this study.
Preparation of inoculum
41
Each organism was recovered by sub-culturing on fresh media. A loopful
inoculum of each bacterium was suspended in 5 ml of nutrient broth and incubated
overnight at 37○C .These overnight cultures were used as inoculums.
Sub-culturing of microorganisim
The pure cultures of microorganism were maintained on nutrient agar slants by
frequent sub-culturing. These cultures were stored at 4○C.
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
Initially callus was initiated form leaf explants Of Pedalium murex on
basal MS medium supplemented with 2,4-D and IAA at different concentrations
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 .
Based on the results the maximum response and well growth of
callus were produced by the combination of MS medium supplemented with IAA
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
and concentration of growth regulators .This combination producing callus was
taken for the further pharmacological studies .
Percentage of the main types of compound which were identified
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 :20.24),and three minor compounds are 2,3 dihydro benzofuran (RT:7.16),
2,propenoic acid ,3,phenyl (RT :10.10) ,and octadeconoic acid (RT:20.56). GC
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
minor compounds are 2-Furancarboxaldehyde,5-methyl (RT: 7.09) , octadecanoic
acid (RT:20.58)
Alcohol extract of the leaf and leaf derived callus of Pedalium murex
showed a well profound activity against Escherichia coli ,Pseudomonas
aeruginosa ,Salmonella typhi Enterococcus facalis ,Klebsiella
Pneumoniae ,Basillus species Staphylococcus aures , Streptococcus
epidermidis.Results 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 inhibition activity in Escherichia coli , Pseudomonas
aeruginosa ,salmonella typhi ,and staphylococcus aureus among the ten bacterial
culture .
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
leaf extract of Pedalium murex through GCMS analysis. The result of
chromatogram of callus/cell samples showed most of compounds present in leaf
extract chromatogram .Interestingly, additional compounds were found in ethanol
extract .The accumulation of active principles in cultured cells at higher level than
those in native plants through optimization of cultural conditions has been
observed in Panax ginseng (Ushiyama, 1991).Rosmarinic acid by Colleus blumei
(Ulbrich et al.,1983) . Shikonin by Lithospermum erythrorhizon (Takahashi and
Fujita 1991),diosgenin by Dioscorea (Rokem et al .,1984),ubiquinone-10 by
Nicotiana tabacum (Matsumoto et al.,1980) were accumulated in much higher
levels in cultured cells than intact plants .
Sometimes cultured plant cells often produce reduced quantities and
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
seen in leaf ethanol extract sample.
Plants are important source of potentially useful structures for
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.
45
Infection associated with Proteus sp. (Madigan et al.,
2000). Both leaves and leaves derived callus ethanol extracts of the Pedalium
murex can be used in the treatment of boils, sores and wounds, since
Staphylococcus aureus and P. aeruginosa have been implicated as causative
agents of these diseases (Braude, 1982). Since the demand in pharmaceutical
industries for plant based raw materials is ever increasing .
The present study is a stepping stone for in vitro production Of
required active principles of Pedalium murex .So far, there is no known report of
active principles of Pedalium murex by callus .
Organic solvent extracts exhibited a higher degree of antimicrobial
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
for S. aureus and C. albicans .
Antibacterial effects of these plants on Staphylococcus aureus, E. coli,
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
46
Pedalium murex is a valuable plant source of medically useful
compounds that has been used in several trational ailment preparation .Leaf and
leaf derived callus extract in organic solvent showed good source for the bioactive
compounds and good antibacterial properties .However, a detailed study is
required to find out the specific bioactive compounds responsible for the
antimicrobial properties through various advanced techniques.
47
INTRODUCTION
Diabetes mellitus is a group of metabolic disorders characterized by
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
a heterogeneous group of syndromes characterized by an elevation of blood glucose
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
48
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;
UKPDS, 1997). Orthodox treatment of diabetes mellitus includes a modification of life
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
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,
requirement of constant refrigeration and in the event of excess dosage-fatal
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
antidiabetic activity .further, most of marketed medicine are distillations, combinations,
reproductions or variations of substances that are found in nature. Our forefathers
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).
49
Insulin therapy affords glycemic control in IDDM yet its short comings include
ineffectiveness on oral administration, fatal hypoglycemia in the event of exess dosage
(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
is a disease of disordered metabolism of carbohydrate, protein and fat which is caused by
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,
oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol,
phospholipids and triglycerides. Thus it is expected to have changes in liver and kidney
during diabetes (Seifter and England, 1982).
MATERIALS AND METHODS
50
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.
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
Induction of Diabetes mellitus in rats
The rats were injected alloxan monohydrate dissolved in sterile
normal saline at a dose of 150mg/kg body weight , intraperitoneally .Since alloxan
is capable producing fatl hypoglycemia as a result of massive insulin release rats
were treated with 20% glucose solution bottles in their cages to prevent
hypoglycemia(Stanely Mainzen Prince,1998).After a fornight ,rats with moderate
51
diabetes having glycosuria (indicated by Benedicts test for urine) and hyper
glycemia with blood glucose range of 250±30mg/dl were used for the treatment.
Collection and identification of plant
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.
Preparation of the Pedalium murex extract (PME)
The leaves were collected from Sivapuram, Pudukkottai District. The leaves and
leaves derived callus were collected and dried in shade for 15 days and made to
coarse powder. The power was passed through sieve No.40 to achieve uniform
particle size and then used for extraction process. A weighed quantity of the
powder was subjected to continuous hot extraction in soxhlet apparatus with 99%
ethanol.The extract was evaporated under reduced pressure using rotovac
evaporator until all solvent was removed to give a molten extract with a yield of
36% w/w.The ethanolic extract of of Pedalium murex was used for the study
Acute toxicity study:
Albino rats of either sex weighing 230-250 g selected by random sampling
technique were used in the study. Acute oral toxicity was performed as per OECD-
423 guidelines (Ecobichon,1997).The animals were fasted overnight ,provided
only water ,after which the drug PME was administered to the respective groups
orally at the dose level of 5 mg/kg body weight by gastric intubations and the
groups observed for 14 days .If mortality was observed in 2 or 3 animals ,then the
52
dose administered was assigned as a toxic dose. If mortality was observed in one
animal,then the same dose was repeated again to confirm the toxic dose .If
mortality was not observed ,the procedure was repeated for further higher doses
such as 50,300,and 2000 mg/kg body weight .The animals were observed for toxic
symptoms such as behavioral changes, locomotion ,convulsions and mortality for
72 h.
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
53
body weight daily for 21 days, once a day.
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 .After 3 weeks of treatment the rats were sacrificed by cervical
dislocation.Blood was collected and processed for the estimation of blood
glucose and glycosylated haemoglobin in separate tubes.Blood collected in
another set of tubes without 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 ice cold saline , patted dry and weighed.
Estimation of Blood glucose
Blood glucose was estimated by O-toluidine method (Sasaki et al., 1972).
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.
Glucose concentration was expressed as mg/dL of blood
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.
The values were expressed as g/dL.
Estimation of liver Glycogen
Hepatic glycogen content was estimated by the method of Morales
et al.,(1973).
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
55
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
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
Determination of the activity of Hexokinase
Hexokinase (ATP: D-hexose-6-phosphotransferase) was determined by the
method of Brandstrup et al., (1957).
Procedure
The reaction mixture contains 1.0 ml of glucose solution, 0.5 ml of ATP,
0.1 ml of magnesium chloride, 0.4 ml of dipotassium hydrogen phosphate, 0.4
ml of potassium chloride, 0.1 ml sodium fluoride and 2.5 ml of Tris-HCl buffer
(pH 8.0), was pre incubated at 37ºC for 5 minutes. The reaction was initiated by
the addition of 2.0 ml of tissue homogenate. 1.0 ml aliquot of the reaction mixture
was taken immediately (zero time) to tube containing 1.0 ml of 10% TCA. A
second aliquot was removed after 30 minutes of incubation at 37ºC. The
precipitated protein was removed by centrifugation and the residual glucose in the
supernatant was estimated by the O-toluidine method of Hyvarinen and Nikkila
(1962) as described previously. A reagent blank was run with each test. The
difference between the two values gave the amount of glucose phosphorylated.
56
The enzyme activity was expressed as nM of glucose-6-phosphate formed
per min per mg of protein.
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.
The enzyme activity was expressed as µM of phosphate liberated /min/mg protein
Determination of activity of Fructose-1, 6-diphosphatase
Fructose-1, 6-diphosphatase was determined by the method of Fiske and
subbarow(1925)
Procedure
The incubation mixture contains 1.5 ml buffer, 0.1 ml substrate, 0.25 ml MgCl2,
0.1 ml KCl, 0.25 ml EDTA, and 0.1 ml tissue homogenate.The reaction mixture
57
was incubated at 37ºC for 15 minutes. The reaction was terminated by the addition
of 1.0 ml of 10% TCA. The suspension was centrifuged and the supernatant was
made upto 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.
The enzyme activity was expressed as µM of phosphate liberated /min/mg protein
Estimation of Total Cholesterol
Cholesterol was estimated by the method of Parekh and Jung (1970).
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
58
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
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
Phospholipids was expressed as mg/g in wet tissue
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.
60
The level of protein was expressed as g per dL in serum and mg per g wet
tissue of liver and kidney.
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
Aspartate amionotransferase (2-oxyglutrate amino transferase) (AST)
The activity of aspartate aminotransferase was estimated by the method of
King (1965a).
Procedure
About 1.0 ml of substrate was incubated at 37oC for 10 minutes. Then 0.2
ml of enzyme solution was added and the mixture was incubated at 37oC for one
61
hour. To the control tubes, enzyme solution was added after the reaction and it was
arrested by the addition of 1.0 ml of DNPH reagent. The tubes were kept at room
temperature for 30 minutes. Then 5.0 ml of 0.4N sodium hydroxide was added. A
set of standard pyruvate solution was also treated in a similar manner. The colour
developed was read at 540 nm using spectrophotometer.
The enzymes activity was expressed as IU per L in serum and in tissue as
µM of pyruvate liberated per min per mg of protein.
Alanine aminotransferase (L-Alanine: 2-oxyglumate amino
transferase) (ALT)
The activity of alanine aminotransferase was assayed by the method of
King (1965a).
Procedure
About 1.0 ml of substrate was incubated at 37oC for 10 minutes. Then 0.2
ml of enzyme solution was added. The tubes were incubated at 37oC 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.
The enzymes activity was expressed as IU per L in serum and in tissues as
µM of pyruvate liberated per min per mg of protein.
Estimation of Urea
62
Urea in serum was determined by Diacetyl Monoxime method
(Natelson, 1957).
Procedure
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 serum urea levels were expressed as mg/dl
Estimation of Uric acid
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.
The values expressed as mg/dL
Estimation of creatinine
Serum creatinine was estimated by the method of Slot(1965).
Procedure
63
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
picric acid .The tubes were kept in a boiling water bath for 15minutes.The colour
developed was read at 470nm in a spectrophotometer.
Serum creatinine level was expressed as mg/dL
Determination of activity of Alkaline Phophatase (ALP)
(Orthophosphoric-monoester phosphohydrolase) (EC. 3.1.3.1)
The activity of alkaline phosphatase was assayed by the method of Moog
(1946) as modified by King (1965c).
Procedure
The assay mixture containing 1.5 ml of buffer, 1.0 ml of substrate and 0.1
ml of magnesium chloride were pre-incubated at 37oC for 10 minutes. Then 0.1 ml
of enzyme was added and incubated at 37oC for 15 minutes. The reaction was
arrested by 1.0 ml of 10% TCA. Control without enzyme was also incubated and
the enzyme was added after the addition of TCA solution. Then 1.0 ml of sodium
carbonate and 0.5 ml of Folin’s phenol reagent were added. After 10 minutes the
blue colour developed was read at 640nm using a spectrophotometer.
The enzyme activity was expressed as IU per Lit in serum
Determination of activity of acid phospatase (ACP)
The activity of acid phospatase was determined by the method of gutman
and gutman (1938,1940)
Procedure
64
To 6.0 ml of buffered substrate 0.3 ml of serum was added and incubated for
1 hr .at 37 0C . After 1 hr the tube was taken out , at the same time maintained a
separate tube containing 6 ml buffered substrate, 0.3 ml of serum was added to
which after incubation 2.7 ml of folin ciocataue reagent was added ,mixed and
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
tissue homogenate (Tris-HCl buffer, pH 7.5) was treated with 2 ml of (1 : 1 : 1 ratio)
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
expressed as mmol/100 g tissue.
HPX were expressed as mmol/100 g tissue. 0.1 ml of tissue homogenate
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
read colorimetrically at 560 nm.
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
65
deviations, and the data were analyzed using analysis of variance (ANOVA). The group
means were compared with Duncan's Multiple Range Test (DMRT).
RESULTS
In all groups prior to Alloxan administration, the basal levels of blood glucose of the rats
were not significantly different. However, 48 h after streptozotocin administration, blood
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
mg/kg/day of Pedalium murex leaves and leaves derived callus.
Table shows plasma insulin glycosylated haemoglobin in normal
and experimental group. While the level of plasma inulin was decreased and
glycosylated heamoglobin was significant elevation during diabetes when compared to
66
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.
Effects on the administration of Pedalium murex leaves ,leaves derived
callus and glibenclamide on hepatic hexokinase and glucose-6-phosphatase, fructose-1,
6-bisphosphatase of liver are presented in Table 3. table – shows the activity of these
enzymes in kidney of diabetic rats The activity of hepatic hexokinase is significantly
decreased while glucose-6-phosphatase and fructose- 1, 6-bisphosphatase are
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
normal values as that of standard drug glibenglamide treatment.
Effects on the administration of Pedalium murex leaves ,leaves derived callus
and glibenclamide on protein,albumin and haemoglobin of serum are presented in Table
3.the activity of protein,albumin and haemoglobin are significantly decreased in allaxon
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
drug glibenglamide treatment.
Triglycerides are a group of lipids absorbed from the diet and produced
endogenously from carbohydrates .hyperlipidemia ,a common feature of diabetes ,is
evidenced by the increased serum cholesterol, triglycerides and phospholipids in diabetic
rats compared to normal control rats .table shows the result for the level of serum
67
cholesterol, triglycerides and phospholipids .the activity of serum cholesterol,
triglycerides and phospholipids significantly increased alloxan diabetic rats when
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.
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
the diabetic rats.
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
extract maintained the level to near normal status.
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
tends to bring the values to near normal.
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
(Bhattaram et al., 2002). Furthermore, an increasing reliance on the use of medicinal
68
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
effect of Pedalium murex leaves and leaves derived callus on ethanol extract in alloxan
induced diabetic rats.
Alloxan , a β cytotoxin induces chemical diabetes in a wide variety of animal
species by damaging the insulin secreting cells of the pancreas . This damages a large
number of β-cells resulting in decrease in endogenous insulin release .Alloxan
administered rats therefore become hyperglycemic in short period of time ,followed by a
hepatic glucose over production .(Matinez and Milagro, 2000). Numerous studies
demonstrated that a variety of plant extracts effectively lowered the glucose level in
alloxan-induced diabetic animal (Vijayvargia et al.,2000 ; Satyanarayana et al .,2005).
Hyperglycemia causes oxidative damage by the generation of reactive oxygen species
(Mohamed et al., 1999) and results in the development of diabetic complications
(Donnini et al.,1996; Baynes et al.,1999 ).
Oral administration of Pedalium murex leaves and callus (200mg/kg body
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
69
protect from maximum body weight loss seems to be due to its ability to reduce
hyperglycemia.
Further, the antihyperglycemic activity of Pedalium murex was associated with an
increase in plasma insulin level suggesting an insulinogenic activity of the Pedalium
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,
2002; chattopadhyay, 1999).
The observed increase the levels of glycosylated haemoglobin in diabetic
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
glycosylated haemoglobin with a concomitant increase in the level of haemoglobin in the
diabetic rats standard drug glibenglamide also showed the same results.
The liver is regarded as one of the central metabolic organs in the
body, regulating and maintaining homeostasis. Diabetes results in a decrease in glucose
utilization and an increase in glucose production in insulin-dependent tissues such as
liver.( Seifter et al.,1982) . Decreased glycolysis, impeded glycogenesis and increased
70
gluconeogenesis are some of the changes of glucose metabolism in the diabetic liver.
(Baquer ,1998) Hexokinase is an insulin-dependent and insulin-sensitive enzyme and are
almost completely inhibited or inactivated in diabetic rat liver in the absence of insulin.
(Gupta ,1997) Decreased enzymatic activity of hexokinase and phosphofructokinase has
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
decrease in hepatic as well as renal hexokinase activity in alloxandiabetic rats.
Administration of P.murex leaves and callus to alloxan treated rats resulted in an
increased activity of hexokinase in liver and kidney. This increased activity of
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
in the concentration of glucose in alloxan-treated rats given P.murex may be as a result of
increased glycolysis (increased liver hexokinase activity).
Two gluconeogenic enzymes, glucose-6-phosphatase and fructose-1,6-
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
changes in the allosteric effectors of the enzymes namely fructose-2,6-bisphosphate,
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
71
of ATP is more favorable for fructose-1,6-bisphosphatase activation. (Baquer ,1998) .
The reduction in the activities of these gluconeogenic enzymes can result in decreased
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-
phosphatase and fructose-1, 6-bisphosphatase in alloxan diabetic rats.
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 aegyptiaca (B.
aegyptiaca) to experimentally diabetic rats.( Mansour et al .,2000) It has been established
that insulin stimulates the incorporation of amino acids into proteins.(Almdal et al.,1988).
Lipid profile ,which I altered in serum of diabetic patients
( orchard,1990 ;Betteridge ,1994) , appears to be a significant factor in the development
of premature atherosclerosis and includes an increase in triglycerides and total cholesterol
levels .In this study the extract significantly reduces the triglycerides ,phospholipids and
total cholesterol. This reduction could be beneficial in preventing diabetic complication
as well as improving lipid metabolism in diabetes (cho et al., 2002).
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
significant reduction in the levels of plasma total cholesterol, triglycerides ,and
phospholipids .the same results were obtained with the fruit ethanol extract of Pedalium
murex ,which showed hypolipidemic effect in diabetic rats.(Balasubramanian et
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 increased levels of serum total cholesterol, triglycerides , and phospholipids .
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
impaired in diabetic rats.
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
levels during diabetes, the measurement of enzymatic activities of phosphatases such as
acid phosphatase (ACP) and alkaline phosphatase (ALP) is of clinical and toxicological
importance as changes in their activities are indicative of tissue damage by toxicants.
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
extensive damage to liver in the experimental animals by alloxan. Treatment with
P.murex callus ethanol extract in alloxan-induced diabetic rats produces a more
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.
A marked increase in the concentration of TBARS and
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
glibenglamide tends to bring the increased concentration of lipid peroxidation products to
near normal level.
75
INTRODUCTION
Diabetes mellitus, a disease of metabolic disorders is associated with a number of
chronic complications like nephropathy, neuropathy, retinopathy and cardiovascular
diseases (Mahdi et al., 2003). Implication of oxidative stress in the pathogenesis of
diabetes is suggested not only by oxygen free-radical generation but also due to non
enzymatic protein glycosylation and alteration in antioxidant enzymes (Mullarkey et al.,
1990; Gillery et al., 2006). Several herbal drugs in different formulations have been
experimented in search of an effective treatment. However, hyperglycemia-induced
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
therapy needs to be improved either older antioxidants such as vitamin E, L. A. (lipoic
acid), and NAC (N-acetyl-L-cysteine) needs to be reformulated, or newer antioxidants
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
,2002), anthocyanins and other phenolic compounds (Sanchez-Moreno et al., 1998).
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
event in the pathogenesis of secondary diabetic complications (Ceriello, 2000). Free
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
production or reduced activity of antioxidant defenses or both. Implication of oxidative
stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free
radical generation but also due to non-enzymatic protein glycosylation, auto-oxidation of
glucose, impaired glutathione metabolism, alteration in antioxidant enzymes and
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
catalase (CAT), whose activities contribute to eliminate superoxide, hydrogen peroxide
and hydroxyl radicals (Soto et al .,2003).
Many of the complications of diabetes mellitus, including retinopathy 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
(Cunningham et al.,1998). Plants often contain substantial amounts of antioxidants,
flavonoids and tannins and the present study suggests that antioxidant action may be an
important property of plant medicines associated with the hypoglycemic effect on
diabetes mellitus (Larson, 1988).
Materials methods
Determination of the activity of Superoxide Dismutase (SOD)
The activity of superoxide dismutase was determined by the method of
Kakkar et al., (1984)
Procedure
Preparation of enzyme source
To 0.5 ml of tissue homogenate, 0.5 ml of distilled water was added followed
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
activity of the supernatant was determined.
ii. Assay of the activity of SOD
The assay mixture contains 1.2 ml of sodium phosphate buffer, 0.1 ml of
phenazine methosulphate, 0.3 ml of nitroblue tetrazolium and 0.5 ml of 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
intensity of the chromogen in butanol layer was read at 560nm in a
spectrophotometer. A system devoid of enzyme source was maintained as control.
The activity of SOD was expressed as the amount of enzyme required to give
a 50% inhibition of the reduction of nitroblue tetrazolium per minute per mg of
protein.
Determination of the activity of Catalase (CAT)
The activity of catalase was determined by the method of Sinha (1972).
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
withdrawn and blown into 2.0 ml of dichromate/acetic acid reagent at 1 minute
intervals. Then the mixture was heated for 10 minutes in a boiling water bath. After
cooling, the absorbance was measured at 570nm in spectrophotometer.
The activity of catalase was expressed as M of H2O2 utilized
per min per mg of protein.
Determination of the activity of Glutathione Peroxidase (GPx)
The activity of glutathione peroxidase was assayed by the method of Rotruck
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
tubes were centrifuged at 2000 rpm. To 0.5 ml of supernatant, 3.0 ml of disodium
hydrogen phosphate and 0.5 ml DTNB were added and the colour developed was
read at 420 nm immediately.
80
The activity of GPx was expressed as M of glutathione oxidized per min per
mg of protein.
Determination of the activity of Glutathione-S-transferase (GST)
The activity of Glutathione-S-transferase was determined by the method of
Habig et al., (1974).
Procedure
To 1.0 ml of phosphate buffer, 0.1 ml of CDNB, 1.7 ml of water and 0.1 ml
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
internal of 1 minute for 3 minutes at 340nm in a UV-vis spectrophotometer. A
complete assay mixture without enzyme was used a control.
Activity of glutathione S-transferase was expressed as nM of CDNB-GSH
conjugate formed per min per mg of protein.
Estimatima
tion of non-enzymatic antioxidants and glutathione
recycling enzymes
Estimation of reduced glutathione
Reduced glutathione was estimated by method of Moron et al., (1979).
Procedure
81
About 0.5 ml of sample (plasma/homogenate) was precipitated with 1.0
ml of 10% TCA and the precipitate was removed by centrifugation. To 0.5 ml
of the supernatant 1.0 ml of DTNB was added and the total volume was made up
to 3.0 ml with phosphate buffer. The absorbance was read at 412nm.
The level of glutathione was expressed as mg per dL in plasma and mg per
g tissue in liver and kidney.
Estimation of vitamin E (α-Tocopherol)
The level of vitamin E was estimated by the method of Baker et al., (1980).
Procedure
To 0.5 ml of sample (plasma/homogenate), 1.5 ml of ethanol was added,
mixed and centrifuged. The supernatant was dried at 80oC. 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.
The level of -tocopherol was expressed as mg per dL in plasma and mg
per g tissue in liver and kidney.
Estimation of ascorbic acid (Vitamin C)
82
The level of ascorbic acid was estimated by the method of Omaye et al.,
(1979).
Procedure
To 0.5 ml of sample (plasma/tissue), 0.5 ml of water and 1.0 ml of 5%TCA
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
520nm using a spectrophotometer.
The level of ascorbic acid was expressed as mg per dL in plasma and mg
per g tissue in liver and kidney.
RESULT
The concentration of tissues SOD, CAT, GSH, GST, and GPx were significantly
decreased in diabetic rats when compared to the control group. Administration of
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
liver and kidney of Alloxan-diabetic rats when compared to controls. Administration of
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
adaptive response to condition of increased peroxidative stress (Matkovics et al. 1982).
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
related to scavenging activity. Increased lipid peroxidation in diabetes can be
due to enhance oxidative stress in the cells as a result of depletion of
antioxidant scavenger system. Reduced glutathione is a major
endogenous antioxidant which counteracts free radical mediated
damage. Depletion of liver and kidney reduced glutathione levels
represents enhanced oxidative stress (Anuradha and Selvam ,1993).
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 status on extract treatment.
The decrease could have been due to increased utilization of ascorbic
acid as an antioxidant defense against increased reactive oxygen species or to a decrease
in the GSH level, since GSH is required for the recycling of ascorbic acid Hunt, (1996).
α-Tocopherol, a lipid soluble, chainbreaking antioxidant was significantly decreased in
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
radical related metabolism in diabetes. Impaired generation of naturally-occurring
antioxidants (GSH, ascorbic acid, and α-tocopherol) results in increased oxidative injury
by failure of protective mechanisms. There is increased flux of glucose through the
polyol pathway, which is hyperactive in hyperglycemia (Moncada and Higgs ,1993).
Vitamin E is one of the most important free radical scavenging chain-breaking
antioxidant within biomembrane (Parks and Traber, 2002). Reduced glutathione, a major
86
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
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
Diabetes mellitus is the most common disease associated with
carbohydrate metabolism, affecting about 200 million people worldwide. Extracts of
88
various plant materials capable of decreasing blood sugar have been tested in
experimental animal models and their effects confirmed. Many unknown and lesser
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
including hematological, gastro-intestinal reactions, hypoglycemic coma, and
disturbances in liver and kidney metabolisms. In addition, these preparations are not ideal
for use during pregnancy [Altan and Kilic .,1997).
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
many features seen in human subjects with uncontrolled diabetes mellitus(Chattopadhyay
et al ., 1997). liver and kidney in some cases (Ghosh, 2001). Patients depend on insulin
for management of IDDM. Without insulin, they develop degenerative complications
such as microangiopathy, nephropathy and retinopathy.
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).
Oxidative stress has been considered as a common pathogenetic factor in
diabetic nephropathy and other complications (Baynes ,1991; Larkins, and
Dunlop,1992,). Diabetic nephropathy is characterized by glomerular hypertrophy,
thickening of glomerular and tubular basement membranes, increased amounts of
extracellular matrix (ECM) in the mesangium, and increased glomerular permeability
(Zatz et al.,1986 ; Steffes et al.,1992). Excessive excretion of glycogen through the
glomeruli is reabsorbed into the cytoplasm of tubules. These phenomena are especially
prominent in the straight portion of proximal tubules. In histological preparations, these
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
morphological characteristic of a diabetic kidney (Watanabe and Hotta, 1997).
Lipid peroxidation is a free radical mediated process leading to oxidative
deterioration of polyunsaturated lipids. Under normal physiological conditions, low
90
concentrations of lipid peroxide are found in plasma and tissues. The possible source of
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
diabetes.(Sundaram et al.,1996) have reported that the concentration of lipid peroxides
increases in the kidney of diabetic rats.
Liver plays an important role in the maintenance of blood glucose level by
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
in carbohydrate metabolism and regulation of blood sugar.
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, oxidation and metabolic conversion of free
fatty acids, synthesis of cholesterol, phospholipids and triglycerides. Thus it is expected
to have changes in liver and kidney during diabetes (Seifter and England ,1982).. Liver
during diabetes, showed a relatively severe impairment in antioxidant capacity than
kidney. The kidney exhibits a characteristic pattern of changes during diabetes (Sharma
91
et al., 2003). The present study demonstrates the efficacy of P.murex leaves and leaves
derived callus (ethanol extract) in reducing diabetes-induced functional and histological
alterations in the kidneys.
MATERIALS AND METHODS
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.
Induction of Diabetes mellitus in rats
The rats were injected alloxan monohydrate dissolved in sterile
normal saline at a dose of 150mg/kg body weight , intraperitoneally .Since alloxan
is capable producing fatl hypoglycemia as a result of massive insulin release rats
were treated with 20% glucose solution bottles in their cages to prevent
hypoglycemia(Stanely Mainzen Prince,1998).After a fornight ,rats with moderate
diabetes having glycosuria (indicated by Benedicts test for urine) and hyper
glycemia with blood glucose range of 250±30mg/dl were used for the treatment.
Experimental designs
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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.
Histopathological studies
One portion of liver and kidney tissues were removed after sacrificed and rapidly
placed in 10% phosphate buffered-formalin for histological examination. Tissues were
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dehydrated in alcohol, embedded in paraffin wax, sectioned in 5m and stained with
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
removal from the animal.
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.
The tissues were dehydrated by ascending grades of isopropyl alcohol by immersing in
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
morented and cut with rotary microtome at 3 micron thickness.
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
The sections were deparaffinised by immersing in xylene for 10 min in horizontal
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
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hematoxylin, the sections were washed in tap water and dipped in acid alcohol to remove
excess stain (8.3% HCl in 70% alcohol).
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 were allowed to dry.
Complete dehydration of stained sections was ensured by placing 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
observed under high power dry objective (Dunn 1974).
RESULT
Kidney sections of diabetic animals showed thickening on the walls of nephrons
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
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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
liver, when compared with group I (figure 1 e and f).
Kidney sections of diabetic animals showed thickening on the walls of nephrons
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,
when compared with group I (Figure 2 e and f).
DISSCUSION
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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
to be associated with functional and/or morphological changes in the kidney (Badole et
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
measurements of urinary total protein are insufficiently sensitive (Harycy, 2002).
Microalbuminuria and proteinuria typically reflect the presence of moderate and
advanced lesions, respectively, in kidney disease (Roy, 2004; Van den Born et al., 1995).
However, the development of diabetic nephropathy is characterised by a progressive
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
kidneys section of STZ-diabetic control rats showed marked multifocal clarifications,
vacuolations and abundance of mucopolysaccharide in diabetic rats’ kidneys. Moreover,
it has been reported that streptozotocin does not possess any significant nephrotoxic
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potential (Floretto et al., 1998). All structural changes in kidneys resulting from STZ
administration in rats can thus be attributed to altered metabolism in diabetes (Rasch,
1980). Normoglycaemia with A. occidentale treatment could ameliorate the glomerular
and tubular lesions that characterise diabetic nephropathy. The improvement of renal
morphology and function associated with STZ-induced diabetes and P.murex treatment
in the present investigation could be attributed to its antidiabetic action resulting in
alleviation of altered metabolic status in animals. An infusion of extract prepared from
the leaves ,stem, and fruits of P.murex in cold water is a demulcent and a diuretic found
useful in the disorder of urinary ststems.such as gonorrhea , dysuria and incontinence of
urine etc.(Chopra et al.,1999; Shukla and khanuja,2004).
The action by which the extract lowered the blood glucose is not well
known; it may increase glycogen level in liver by an increase in glycogenesis and/or a
decrease in glycogenolysis. Since P.murex did not significantly reduce glycaemia in non-
diabetic animals, it is possible that its mechanism of action is similar to that of
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
by the stimulation of regeneration process and revitalisation of remaining β cells
(Diatewa et al., 2004).
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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.
Accelerated chemical modification of proteins by glycosidation and accumulation of
AGE (Advanced Glycation End-products) are implicated in diabetic nephropathy and
Hennebele et al. (2004) suggested that these molecules can be inhibited with
polyphenols. Standard antidiabetic drugs such as insulin and sulfonylureas cause
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.
In the liver of diabetic rats (group-III) shrunken nuclei, granular cytoplasm
(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-
II diabetic rat models.( Can et al.,2004 ).observed an increase in degeneration in central
veins to portal veins, excess vacuolization, granular appearance in the cytoplasm,
dilations in the sinusoids and moderate hyperemia.(Ayesha et al.,2008).P.murex leaves
and leaves derived callus appears to be an attractive material for further studies leading to
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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.
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