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Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

EFFECT OF COMMIPHORA MUKUL GUM RESIN ON POLYOL PATHWAY AND


INTESTINE DISACCHARIDASES ENZYMES OF INSULIN DEFICIENT AND FRUCTOSE
FED INSULIN RESISTANT RATS
Ramesh B1#, Sainath S.B2#, Karuna R3, Sreenivasa Reddy S4, Manjunatha B4, Sudhakara G4, Shashi
Bhushan Rao B4 , Raveendranath D5, Saralakumari D4*,
1

Sri Venkateswara University, Tirupati - 517502, Andhra Pradesh, India


Vikrama Simhapuri University, S.P.S.R.Nellore - 524003, Andhra Pradesh, India
3
University of Nebraska medical Centre,Omaha, NE, USA
4
Sri Krishnadevaraya University, Anantapur - 515003, Andhra Pradesh, India
5
JNTU Hyderabad, Hyderabad-500085, Telangana State, India
2

Corresponding author
Prof.D.Saralakumari
Professor, Department of Biochemistry,Sri Krishnadevaraya University,
Anantapur-515 003, IndiaVenkateswara puram,A.P, Indian
+91-08554-255879+91-08554-255805,skumari1@yahoo.co.in
Please cite this article in press as Ramesh B et al. Effect of Commiphora mukul gum resin on polyol pathway and intestine
disaccharidases enzymes of insulin deficient and fructose fed insulin resistant rats. Indo American Journal of Pharm
Research.2014:4(12).
Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Keywords
Commiphora Mukul,
Intestine Disaccharidases,
Polyol Pathway,
High Fructose Diet,
Phytochemicals.

ABSTRACT
Diabetes causes increased oxidative stress, which is thought to play an important role in the
pathogenesis of various diabetic complications. Commiphora mukul (CM) gum resin
ethanoilc extract has several biological properties including hypolipidemic, amtidabetic and
antioxidant activity. This study was undertaken to evaluate the effects of CM gum resin
ethanolic extract on polyol path way enzymes and intestinde disaccharidases in
streptozotocin (STZ) induced diabetic and high fructose diet (HFD) induced insulin resistant
rats. The male Wistar albino rats were randomly divided into six groups of eight animals
each: two of these groups (groups C and C+CM) were fed with standard pellet diet and the
other two groups (groups F and F+CM) were fed with high fructose diet (HFD) (66 %), other
two groups (D group and D+CM) were induced by intraperitoneal injection of streptozotocin
(STZ, 55 mg/kg body wt) to male Wistar rats. C. mukul gum resin ethanolic extract in water
(200 mg/kg body weight/day) was administered orally to group C+CM, D+CM and group
F+CM. At the end of 60-day experimental period biochemical parameters related to Polyol
pathway enzymes like liver, pancreas and heart tissues of aldose reductase (AR) and sorbital
dehydrogenase (SDH) and intestine disccharidases enzymes like maltase, sucrose and lactase
enzymes were performed. The STZ induced diabetic and fructose fed insulin resistant rats
showed increased levels of enzymatic activities aldose reductase (AR) and sorbital
dehydrogenase (SDH) and intestine disccharidases enzymes like maltase, sucrose and lactase
enzymes. Administration of C.mukul (200 mg/kg bw) to STZ induced and fructose fed insulin
resistant rats for 60 days significantly reversed the above parameters towards normally. In the
present study the enhanced or elevated activities of polyol pathway enzymes and intestinal
disaccharidases of insulin deficient diabetic and insulin resistant rats were prevented by the C.
mukul treatment. It may also contribute to intestinal disaccharidase inhibitory activity; another
beneficial property of C. mukul in decreasing the polyol pathway towards preventing the
oxidative stress induced diabetic complications, and hence this plant can be used as an
adjuvant for the prevention and management of insulin resistance and insulin deficient to it.

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ARTICLE INFO
Article history
Received 10/12/2014
Available online
31/12/2014

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ISSN NO: 2231-6876

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INTRODUCTION
One of the consequences of hyperglycemia in human DM is increased metabolism of glucose by sorbitol (polyol) pathway.
Aldose reductase catalyses the reduction of glucose to sorbitol. Sorbitol doesnt readily diffuse across the cell membrane and tends to
accumulate in the cell. Under hyperglycemic condition, high glucose flux through the sorbitol pathway accounts for one-third of
glucose metabolism. This has important implications in terms of redox changes of NADP + and NAD+ couples and metabolism of
glucose by alternative pathways [1]. Conversion of glucose to sorbitol by aldose reductase requires NADPH and forms NADP + and
thereby competes with other NADPH requiring reactions. Conversion of sorbitol to fructose by sorbitol dehydrogenase is coupled to
reduction of NAD+ to NADH which inhibits glycolysis at the glyceraldehyde dehydrogenase step for NAD +. Increased flux of glucose
via polyol pathway has also consequences for the overall antioxidant status leading to depletion of glutathione (GSH) as a result of
competition between aldose reductase and glutathione reductase for NADPH.
In addition, glycated proteins may serve as a source of oxygen radicals. Amadori adducts of protein glycation may undergo
oxidation in the presence of oxygen and transition metals leading to the formation of O2 , the release of erythronic acid and the
products of oxidative cleavage of the glycated protein-carboxymethylysine, carboxymethylhydroxylysine, and pentosidine. These
compounds termed glycoxidation products were used as the biomarkers of glucose-dependent protein damage [2]. Besides affecting
the functions of these molecules, oxidative stress also triggers a series of cellular responses including the activation of protein kinase
C (PKC), the transcription nuclear factor B (NF- B), and JNK stress associated kinases [3]. In appropriate activation of these
important regulatory molecules can have deleterious effects on cellular functions and is thought to contribute to the pathogenesis of
various diabetic vascular complications. Through subsequent ROS-induced DNA damage and poly-ADP ribose polymerase activation,
ADP-ribose polymers attach to and inhibit the cytosolic glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. This
inhibition in turn mediates the activation of four proposed mechanisms of hyperglycemia-associated tissue damage- the polyol
pathway, the hexosamine pathway, protein kinase C activation, and formation of advanced glycation end products [4]. Although there
is controversy about the antioxidant status in diabetes, several studies report decreased plasma or tissue concentration of superoxide
dismutase, CAT, GSH and ascorbic acid in both diabetic animals and patients [5]. Thus enhanced oxidant production with decreased
antioxidant potential of diabetes intensifies the oxidative stress.
Traditional (India) uses of C. mukul (CM) are for its anti-inflammatory, antispasmodic, carminative, emmenagogue,
hypoglycemic, alternative, antiseptic, and astringent, a thyroid stimulant, anthelminitic and antihyperlipidemia properties. It is an
important herb used in the treatment of several degenerative disorders in modern medicine too and established as hypolipidemic drugs
[6]. Ayurvedic medicines containing gum guggul often contain guggul in their names, such as in Shunthi-guggul, and Yogaraja
guggul. All the formations used for traditional Ayurvedic treatments for obesity contained gum guggul among its herbal ingredients.
In addition, it was found that guggulsterone treatment increased lipolytic enzyme activity as well as receptor-mediated
catabolism of LDL [7]. Two stereo isomers, E- and Z-guggulsterone (cis- and trans- 4, 17(20)-pregnadiene-3, 16-dione, respectively),
are the most important constituents studied in detail for their therapeutic potential from C. mukul. Various studies have been
conducted to understand and illustrate the mechanism of action and potential of guggulsterone as a therapeutic agent using synthetic E
and Z isomers [8]. The mechanisms implicated for lipid-lowering effect of guggulipid are stimulation of hepatic lipases and receptor
mediated catabolism of low-density lipoproteins, and suppression of hepatic cholesterol biosynthesis [9]. Guggulsterones inhibited
cholesterol synthesis in the liver via antagonism of the forsenoid X receptor and the bile acid receptor [10]. A number of clinical trials
have been conducted to evaluate the hypolipidemic effect of guggulipid. Most of these studies were carried out in India and one in the
United States. Consistent with the preclinical data, most of these studies demonstrated hypolipidemic activity of guggul or gugulipid
with an average of 1030 % and 1020 % decrease in total cholesterol and triglyceride, respectively. With proven hypolipidemic
efficacy in rats, guggulsterone was used as a positive control to assess the hypolipidemic activity of other chemical compounds [11].
It also increased fibrinolytic activity and decreased platelet adhesiveness and scavenges free radicals in patients with
atherosclerotic ischemic diseases, thus reducing cellular damage from ischemia [12]. Clinical trials have also indicated C. mukul as an
effective medicine for angina pectoris, atherosclerosis and myocardial infarction [13], and known to cause thyrogenic effect [14].
Guggulsterone reversed the myocardial damage and the induced metabolic changes [15]. In more recent studies, the cardioprotective
activity of guggulsterone was compared with that of a hypolipidemic drug, gemfibrozil.
In a recent study, C. mukul and guggulsterone were effective antioxidants against LDL oxidation [16]. Hepatic microsomal
lipid peroxidation was also significantly reduced by guggulipid. In addition, guggulipid significantly reversed the cardiac damage and
biochemical changes induced by isoproterenol [17]. In one study, guggulsterone isomers (Z- and E-forms) exhibited potent inhibitory
activity against the production of nitric oxide induced by bacterial lipopolysaccharides (LPSs) in macrophages with IC50 values of 1.1
and 3.3 M, respectively [18]. This finding indicated that guggulsterone may be of therapeutic benefit in diseases associated with
oxidative stress, such as myocardial ischemia and neurodegenerative diseases. Our previous work demonstrated that C. mukul gum
resin ethanolic extract has antidiabetic activity in streptozotocin (STZ) - induced insulin-dependent diabetes mellitus and fructoseinduced insulin resistance in animals [19, 20].

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Isomers of guggulsterone.
Gugulipid significantly increased the level of catecholmine and the activity of dopamine -hydroxylase in normal rabbits and
decreased those in cholesterol-fed rabbits. The plethora of pharmacological effects of guggulu particularly anti-dyslipidemic effect is
suggestive of its potential as cognitive enhancer.
Chemical Constituents
The chemical composition of C. mukul is very complex and has not been well defined. It contains sugars (sucrose, fructose),
amino acids, camphorene, cembrene, allycembrol, resin, oil and several steroids or Sterones. Many compounds have been isolated
from guggul. Besides gum and resin, it contains perfumed essential oil. Detailed chemical studies have revealed that guggulu is a good
source of several useful steroids. These compounds are diterpene hydrocarbon, diterpenealcohol, Z-guggulsterone, E-guggulsterone,
Guggulsterol besides sesaminetic. Till date, several chemical components such as diterpenes, sterols, steroids, esters and higher
alcohols have been identified in C. mukul [16]. The active ingredients responsible for the use of the plant in maintenance of healthy
cholesterol levels is guggulsterones 4, 17(20)-pregnadiene-3, 16-dione], specifically guggulsterone E and guggulsterone Z [16].
MATERIALS AND METHODS
Plant material
Ethanolic extract of Commiphora mukul gum resin (CM; brown, dry powder with Lot No. L5111031) was obtained from the
manufacturers and exporters of herbal extracts, Ms Plantex Pvt. Ltd., Vijayawada, Andhra Pradesh, India. Procedure followed by the
firm for the preparation of extract is as follows: the plant was identified by Dr. K. Narasimha Reddy, Taxonomist, Lailaimpex R & D
Center, Vijayawada. The collected plant sample (resin) was washed thoroughly with tap water, dried at room temperature away from
sun light, cut into small pieces, and then powdered. Ethanolic extract was prepared by cold maceration of gum resin powder in ethanol
for 7 days. The extract was filtered, concentrated under reduced pressure and finally dried in vacuum desiccators. Herb-to-product
ratio was 8:1. A voucher specimen has been deposited in the Department of Biochemistry, Sri Krishnadevaraya University,
Anantapur, under number DSK-CM-09. The extract was stored at 04C and dissolved in water just before use.
Chemicals
Streptozotocin, DL-glyceraldehyde was obtained from Sigma-Aldrich (St.Louis, MO, USA). All other chemicals and
solvents were analytical grade and were obtained from Sisco Research Laboratory (P) Ltd. (Mumbai, india)
Phytochemical Screening
Chemical test were carried out on the aqueous & hydroalcholic extract and on the powdered specimen using standard
procedures to identify the constituent as described by Sofowara (1993) [21].
Carbohydrates
Molischs Test
In a test tube containing 2 ml of extract, 2 drops of freshly prepared 10 percent alcoholic solution of - naphthol was added.
Then it was shacked and 2 ml of conc. sulphuric acid was added from sides of the test tube. So the violet ring was formed at the
junction of two liquids, indicated the presence of carbohydrates.

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Test for Saponin


About 2g of the powdered sample was boiled in 20 ml of distilled water in a water bath and filtered. 10ml of the filtrate was
mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil
and shaken vigorously, then observed for the formation of emulsion.

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Glycosides
Borntrager Test
3 ml of extract was treated with dilute Sulphuric acid then boil and filtered. Cold filtrate was treated with chloroform (equal
volume) and shacked for some time. The organic layer is separated and treated withdilute ammonia. Pinkish colour of ammonical
layer indicated anthraquinone glycoside.

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Test for Flavonoids


Three methods were used to determine the presence of flavonoids in the plant sample (Sofowara, 1993) [21]. 5 ml of dilute
ammonia solution were added to a portion of aqueous filtrate of plant extract followed by addition of concentrated H2SO4. A yellow
colouration observed in extract indicates the presence of flavonoids. The yellow colour disappeared on standing. Few drops of 1%
aluminium solution were added to a portion of filtrate. A yellow colouration was observed indicating the presence of flavonoids. A
portion of the powdered plant sample was heated with 10ml of ethyl acetate over a steam bath for 3min.
The mixture was filtered and 4 ml of the filtrate was shaken with 1ml of dilute ammonia solution. A yellow colouration was observed
indicating a positive test for flavonoids. A piece of mg ribbon was added to 1ml ethanolic extract of Shirishadi Group and then 1ml of
conc.HCL was added. Pink or red colour indicates the presence of flavonoids.
Test for Steriods
Two ml of acetic anhydride was added to 1 ml (0.05g) of ethanolic extract of Shirishadi & Bharangadi compound separately
and 2ml of H2SO4 was then added to it. The colour changed from violet to blue or green in sample indicating the presence of steriods.
Test for Alkaloids
A drop of ethanolic extract of each compound was spotted on pre-coated TLC plate and sprayed by modified Dragendroffs reagent.
Orange colour of the spot indicates the presence of alkaloids.
Animals
Male Wistar rats weighing (160-190 g) were procured from Sri Venkateswara Enter prises (Bangalore, India), acclimatized
for 7 days in our animal house (Regd. no. 470/01/a/ CPCSEA) before dietary manipulation. Animals were housed two per cage in an
air-conditioned room (2220C) with 12 h light/dark cycle and had free access to standard pellet diet and water. All the procedures
were per formed in accordance with the Institutional Animal Ethics Committee.
Experimental animal models
STZ induced diabetic animal model
Diabetes was induced in rats by a single intraperitoneal injection of freshly prepared STZ with a dosage of 55 mg/ kg body
weight, in 0.05 M citrate buffer pH 4.5 in a volume of 0.1 ml. STZ was first weighed individually in Eppendorf tubes for each animal
according to the weight and then solubilized, just prior to injection in buffer. Seventy-two hours after STZ administration, plasma
glucose level of each rat was determined for confirmation of diabetes. They were allowed for a window period of 10 days before
commencement of treatment. Rats with fasting plasma glucose greater than 300 mg/100 ml were considered diabetic and included in
the present study.
Insulin resistance animal model
Insulin resistance was induced by a fructose-enriched diet22. This diet contained 66 % fructose, 18 % protein, 8 % fat, 4 %
cellulose, 3 % mineral and 1 % vitamin mix which was procured from National Centre for Animal Science, National Institute of
Nutrition (Hyderabad, India).

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Sample collection and preparation for biochemical estimations and assays


Sacrifice of animals and organ collection
After the experimental period of 60 days, animals from all six groups were sacrificed by cervical dislocation and liver,
pancreas, kidneys, heart and small intestine were removed immediately and washed thoroughly with ice-cold 0.9 % NaCl (saline).
Each organ of every animal was suspended in 0.15 M KCl in polypropylene containers, sealed with parafilm, labelled carefully and
frozen at -80o C until assays were carried out.

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Experimental design
In the present study a total of 48 male Wistar albino rats were acclimatized to our animal house before induction of type-1
diabetes/IR. One third of the experimental animals were made diabetic by STZ injection and maintained on standard pellet diet and
used for further studies in the present investigation. Insulin resistance was induced in another one third of the experimental animals by
feeding fructose-enriched diet throughout the experimental period. The remaining one third of the animals served as controls and was
maintained on standard pellet diet. Each set of animals (type-1 DM, IR and control) was further subdivided into two groups thus
comprising total of six groups-control group (C-group), control rats treated with C. mukul (C + CM-group), STZ diabetic group (Dgroup), STZ diabetic rats treated with C. mukul (D + CM-group), fructose fed rats (F-group) and fructose fed rats treated with C.
mukul (F + CM-group). Groups- C + CM, D + CM and F + CM rats were administered C. mukul gum resin ethanol extract (2 ml of
5% Tween-80) (200 mg/kg body weight in ~2 ml of water/day) through gastric intubation for 60 days[19].
The dose of C. mukul ethanol extract in the current study is based on preliminary experiments on dose-dependent
antihyperglycemic effect in STZ induced diabetic rats. A dose less than 200 mg/kg body weight was not found to effective in rats.
Body weight and plasma glucose, insulin and lipids in plasma were monitored at 15 day intervals till the end of study. After 60 days of
treatment, all the rats were sacrificed following 12 h of fasting.

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Intestinal Disaccharidases
Collection of Intestinal mucosa and preparation of enzyme source for disaccharidases
Intestinal mucosa was collected by following the procedure outlined by Ravinder (1989) [23]. A long segment 20 - 25 cm of
small intestine between jejunum and caecum leaving about 5 cm on either side was excised. The intestine was flushed with ice-cold
saline and cut open longitudinally. The mucosa scraped with a microscopic slide was homogenized in ice cold saline, filtered and used
as enzyme source for maltase, sucrase and lactase assays.
Maltase, Sucrase and Lactase
The intestinal disaccharidases were assayed by following the method outlined by Dahlqvist, (1968) [24] with slight
modification.
After incubation of the enzyme with the appropriate substrate the reaction was arrested by the addition of Tris buffer and the
liberated glucose was assayed by GOD-POD enzymatic method using Span Diagnostic kit [25]. The reaction was started by mixing
0.1 ml of enzyme source with 0.1 ml of appropriate substrate (0.056 M maltose/sucrose/lactose in 0.1M sodium maleate buffer, pH
6.0). After incubation for 60 min at 37 C, 2.0 ml of 0.5M Tris buffer, pH 7.0 was added to arrest the reaction. To all the tubes 1.0 ml
of glucose oxidase reagent was added, mixed well and incubated at 37 C for 10 min and the colour intensity was read at 505 nm
against reagent blank. A series of standard glucose (20-100 g) were treated in a similar manner. The activity was expressed in terms
of nmol of disaccharide hydrolyzed/min/mg protein.
Sorbitol dehydrogenase (E.C 1.1.1.14)
Sorbitol dehydrogenase activity was measured by the method of Asada and Galambos, (1963) [26]. SDH catalyses the
reduction of fructose to sorbitol in presence of NADH as reducing agent. The activity was measured by monitoring the decrease in the
absorbance at 340 nm using fructose as substrate.
The assay system contained 1.6 ml of 107 mM triethanolamine buffer (pH 7.4), 0.1 ml of 0.4 mM NADH. 0.1 ml of enzyme
source and incubated at 25 C for 30 min. The reaction was initiated by adding 0.3 ml of 4 M fructose. The decrease in absorbance per
min at 340 nm was monitored for 5 min. The activity was calculated using extinction co-efficient of NADH as 6.22 cm-1 mmol-1.
Aldose reductase (E.C 1.1.1.21)
Aldose reductase catalyses the NADPH linked reduction of aldose. The activity was measured by monitoring the decrease in
the absorbance at 340 nm using glyceraldehyde as a substrate according to the method of Hayman and Kinoshita (1965) [27].
The assay system contained 0.4 ml of 50 Mm DL-glyceraldehydes, 0.4 ml of 5 mM mercaptoethanol, 0.1 ml of enzyme
source and incubated at 37 C for 5 min. The reaction was initiated by adding 0.1 ml of 0.1 mM NADPH. The decrease in absorbance
was monitored at 1 min interval for 5 min at 340 nm. The activity was calculated using extinction co-efficient of NADPH as 6.22 cm-1
mmol-1.
Statistical Analysis
The results were expressed as meansSEM. Data were analyzed for significant difference using Duncans Multiple Range
(DMR) test (P <0.05) [28].
RESULTS
The phytochemical screening of C.mukul gum resin ethanolic extract revealed the presence of alkaloids, carbohydrates,
glycosides, sapononins, steroids, tannins and resins indicating the presence of pharmacologically important phytochemicals (Table 1).

The activities of AR and SDH in liver, pancreas and heart tissues of six experimental groups are given in Table.2. Aldose
reductase enzyme activity was the highest in liver, followed by pancreas and heart. A significant increase in the activity of AR was
observed in liver (39.1 %), pancreas (13.8 %) and heart (20.4 %) of D-rats compared to C-rats whereas a slight but not significant
increase in the AR activity was observed in F-group compared to C-group. A significant increase in SDH activities of liver, pancreas
and heart tissues was observed both in D and F-groups compared to C-group. Per cent increase in the activity of SDH in liver,

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Phytochemicals Presence/Absence
Alkaloids
+
Carbohydrates
+
Flavonoids
+
Glycosides
+
Phenols
Saponins
+
Steroids
+
Tannins
Triterpenes
+
Where + denotes presence and -denotes absence C. mukul gum resin ethanol extract

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Table 1. Phytochemicals profile of C. mukul.

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pancreas and heart are 39.5, 19.0 and 13.0 % respectively in D-group and 12.5, 9.5 and 13.0 % respectively in F-group compared to Cgroup. Thus the increase in SDH activity is more prominent in D-group than F-group.
Table 2. Effect of C. mukul treatment on activities of aldose reductase and sorbitol dehydrogenase enzyme activities in STZ
diabetic and fructose fed IR rats.
Parameter
Aldose reductase
mol of NADPH
oxidized/min/mg protein
Sorbitol dehydrogenase
mol of NADH
oxidized/min/mg protein
Values are mean S.E.M., (n=8
(Duncans multiple range test).

Tissue
C
C + CM
D
D + CM
F
F + CM
Liver
0.920.023a 0.890.037a 1.280.032b 0.900.038a 0.980.043c
0.930.030a
a
a
b
a
b
Pancreas 0.720.035 0.710.023 0.820.022 0.730.016 0.780.0255 0.720.014a
Heart
0.490.013a 0.480.016a 0.590.019b 0.510.03a
0.540.05c
0.500.03a
a
a
b
a
c
Liver
4.800.076 4.920.04
6.720.13
4.90.12
5.630.07
5.100.069a
a
a
b
a
c
Pancreas 2.140.29
2.090.04
2.530.07
2.180.04
2.300.04
2.100.03a
a
a
b
a
c
Heart
2.300.029 2.200.03
2.660.03
2.210.03
2.600.03
2.540.02a
animals). Values with different superscripts within the row are significantly different at P<0.05

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Change in intestine disaccharidases enzymes levels in stz induced diabetic and fructose fed insulin resistant rats after 60 days
of treatment with C.mukul extract. Data are the Mean (Value n=8) with S.E. Data are significant at P<0.05 compared to
diabetic and insulin resistant control.
DISCUSSION
Flavonoids, sterols, triterpenoids, alkaloids, saponins and phenolics are reported as bioactive antidiabetic principles [29].
Flavonoids can regenerate the damaged -cells in the alloxan induced diabetic rats [30]. Polyphenolics, apart from their much-cited
antioxidant and -amylase and sucrase inhibitory activities, have been shown to be the principal substances for antihyperglycemic
activity [31]. Saponins, polyphenolic compounds and gallic tannins possess potent inhibition of intestinal glucose transport by
inhibiting sodium glucose co-transporter-1 (S-GLUT-1) of intestines. Numerous epidemiological studies suggest that herbs/diets rich
in phytochemicals and antioxidants execute a protective role in health and disease [32] .
Polyol pathway also known as sorbitol pathway basically involves the conversion of glucose to fructose via sorbitol. Aldose
reductase (AR) reduces glucose to sorbitol with the aid of co-factor NADPH and the second enzyme sorbitol dehydrogenase (SDH)
with its co-factor NAD+, converts sorbitol to fructose.

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The data presented in Fig.1 reveals the activities of intestinal disaccharidases (maltase, sucrase and lactase) of the six
experimental groups. Both STZ diabetic group (D-group) and insulin resistant rats (F-group) showed significantly enhanced activities
of maltase (149.7 and 71.8 %), sucrase (14.5 and 8.8 %) and lactase (20.0 and 16.6 %) respectively when compared to C-group. Thus
STZ diabetic rats showed significantly higher maltase and lactase activities than F-group rats whereas no significant variation was
observed in intestinal sucrase activity between D and F-groups. C. mukul administration for 60 days caused no significant alteration in
the activities of intestinal disaccharidases in C + CM-group from C-group. However it caused significant decreases in the activities of
intestinal disaccharidases in D + CM-group and F + CM-group compared to D and F-groups respectively. Thus C. mukul treatment
resulted in the restoration of enhanced intestinal disaccharidases observed in SZT diabetic condition and fructose fed conditions to the
normal values.

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Under normal conditions the bulk of glucose is metabolized through the glycolytic pathway and the pentose shunt. Under
normal glycemic conditions, approximately 3 % of the glucose metabolized is routed via the polyol pathway. However, under
hyperglycemic conditions this pathway accounts for more than 30 % of the glucose utilized [33]. The polyol pathway enzyme AR
catalizes the reduction of aldose sugars and other saturated and unsaturated aldehydes [34]. This enzyme constitutes the first and the
rate limiting step of the polyol pathway. This pathway has been suggested to play an important role in the development of vascular
and neurological complications in diabetes [35]. Inhibition of aldose reductase ameliorated vascular and other complications in
diabetes [36].
In the pathogenesis of diabetic complications, the polyol pathway is important and it is the one of the major sources of
diabetes induced oxidative stress. There are three potential mechanisms for the polyol pathway to contribute to oxidative stress. (1).
AR activity depletes its co-factor NADPH, which is also required for GR to regenerate GSH. Under hyperglycemic condition, glucose
is channelled into the polyol pathway causing a substantial depletion of NADPH and consequently a significant decrease in the GSH
level. Thus, during hyperglycemia, AR activity diminishes the cellular antioxidant capacity [37]. (2). Oxidation of sorbitol to fructose
by SDH causes oxidative stress because its co-factor NAD+ is converted to NADH in the process, and NADH is the substrate for
NADH oxidase to generate ROS [38]. Oxidation of sorbitol by NAD + increases the cytosolic NADH: NAD+ ratio tends to inhibit
glyceraldehydes phosphate dehydrogenase activities. Which can lead to increased levels of triose phosphates, methylglyoxal, and
diacylglycerol? This chain of events is also associated with consumption of NAD+ by activated poly (ADP-ribose) polymerase. This
in turn is associated with intracellular AGE formation and activation of PKC, the hexosamine pathway and NF-B [39] . (3). the
polyol pathway converts glucose to fructose. Because fructose and its metabolites fructose-3-phosphate and 3-deoxyglucose are more
potent nonenzymatic glycation agents than glucose, the flux of glucose through the polyol pathway would increase AGE formation.
AGES, as well as binding of AGE to their receptors, are known to cause oxidative stress [40].
It is well established that glucose flux through AR is increased in diabetes since aldose reductase has a high Km for glucose
[41]. It would mean that increase flux via polyol pathway proportionately. The degree of hyperglycemia is significantly greater in
STZ diabetic rats than in fructose fed rats. The greater glucose availability time (duration) is in part a key reason for the increased flux
through aldose reductase in STZ diabetic rats compared to fructose fed rats. Polyol pathway dependent alterations in the cytosolic
redox state could potentially account for increased free radical generation, AGE accumulation, protein kinase C activation and
hexosamine formation by causing decreased NADPH and GSH. Reduction of NADH/NAD + by the oxidation of glucose-derived
sorbitol to fructose would favour lactate production [42] and divert glycolytic intermediates to the synthesis of phospholipid
precursors such as -glycerophosphate, phosphatidic acid, diacylglycerol, while at the same time interfering with -oxidation of longchain fatty acids giving a metabolic profile similar to that of ischemia [43] .
C. mukul treatment resulted in a significant decrease in tissue AR activity in D + CM-group compared to D-group, thus
resulting in normalizing the tissue AR activity of D + CM-group whereas F + CM and C + CM-groups showed no significant change
in tissue AR activities from C-group. C. mukul treatment resulted in a significant decrease in tissue SDH activity in D + CM and F +
CM-groups compared to D and F-groups respectively. Thus C. mukul treatment restored the SDH activity of pancreas and heart
tissues to the normal values in D + CM and F + CM-groups. However, the decreased activity of hepatic SDH by C. mukul treatment in
D + CM and F + CM-groups did not reach the normal levels. Control rats treated with C. mukul (C + CM-group) showed no
significant change in tissue SDH activity when compared to C-group.
Thus restoration of enhanced polyol pathway enzymes to normal levels by C. mukul supplementation in diabetic and fructose
fed animals (D + CM and F + CM) avoided the deleterious alterations due to enhanced operation of polyol pathway towards oxidative
stress.
Excessive activation of the polyol pathway increases intracellular and extracellular sorbitol concentrations. The sorbitol can
not cross cell membranes, and when it accumulates, it produces osmotic stresses on cells by drawing water. Osmotic stress, from the
accumulation of sorbitol, is more an important factor for the development of diabetic cataract [44]. Earlier studies demonstrated that
increased cellular polyol pathway enzymes and increased ischemic injury with duration of diabetes. Further inhibition of AR and SDH
resulted in reduced ischemic injury and improved functional recovery [45].
Beneficial effects of AR inhibitors (ARIs) that block conversion of glucose to sorbitol in animal and human diabetes strongly
implicate the sorbitol pathway as a significant factor in the pathogenesis of the long-term complications of diabetes mellitus,
particularly diabetic polyneuropathy [46]. Hotta and Kakuta (1981) [47] demonstrated that AR activity of the polyol pathway is not
affected directly by insulin. It is likely that the specific activities of several enzymes regulated by insulin are altered and that glucose
utilization is diverted to metabolic pathways other than the polyol pathway.
Reports are available on polyol pathway inhibiting activity of many plant extracts and phytochemicals. Aqueous extracts of
Ocimum sanctum, Withania somnifera, Curcuma longa, Azadirachta indica inhibit AR activity [48]. Flavonoid treatment inhibited the
polyol pathway in experimental diabetes [49]. Turmeric and curcumin supplementation also reduced the oxidative stress encountered
by the diabetic rats. This was demonstrated by the lower levels of TBARS which may have been due to the decreased influx of
glucose into the polyol pathway [50]. Results from our study revealed another beneficial property of C. mukul in decreasing the polyol
pathway towards preventing the oxidative stress induced diabetic complications.
The intestinal digestive and absorptive processes of carbohydrates are mediated by the disaccharidases and hexose
transporters that are localized in the brush-border membranes of intestinal absorptive cells [51].
Diabetes mellitus is a state of nutrient starvation that frequently results in severe metabolic imbalances and pathological
changes in many tissues. In the small intestine this severe disease causes significant changes in the morphology and function of the
mucosa [52]. Diabetes stimulates the functional activity of the intestinal brush border membrane with enhancement of both hydrolytic
enzyme activity [53] and membrane transport system [54].

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So the present study was extended to understand the efficacy of C. mukul administration in regulating the intestinal
disaccharidases activity both under insulin deficient and insulin resistance conditions. The increased activities of intestinal
disaccharidases (maltase, sucrase and lactase) observed in present study are in agreement with the earlier reports in STZ diabetic
induced insulin deficient rats [55] and fructose feed induced insulin resistant rats [56].
Hormone secretion by the gut and the pattern of response after feeding may be abnormal in diabetes and might be regulatory
for disaccaridases. The intestinal effects are prevented or markedly diminished by insulin therapy [57] Caspary et al., 1972 suggesting
that they are in fact the result of insulin deficiency or insulin resistance. In addition, change in the pattern of food consumption i.e.,
polyphagia of diabetes might also stimulate the intestinal disaccharidases in STZ diabetic rats.
Previous studies demonstrated the adaptable nature of intestinal disaccharidase activity in rats [58]. Specific dietary sugars
can alter enzyme activity in the small intestine of man in a specific fashion. Sucrose and fructose are able to regulate sucrase and
maltase activity [59]. Yasutake et al. (1995) [60] reported that dietary sucrose enhanced both sucrase-isomaltase mRNA and
sodium/D-glucose transporter-1 (SGLT-1) mRNA levels in the rat jejunum within 12 h. Similarly, Miyamoto et al. (1993)
demonstrated that the mRNA levels of jejunal hexose transporters (SGLT-1, GLUT-5, and GLUT-2) are elevated by feeding fructose
diet to rats for 5 days.
The -glucosidase inhibitors are a new class of antihyperglycemic drugs that have a unique effect on the glycemic profile.
Their major action is to lower postprandial plasma glucose levels by inhibiting the breakdown of complex carbohydrates within the
intestine resulting in delayed absorption of glucose with the small bowel and a consequent reduction is postprandial glucose levels.
The -glucosidase inhibitors not only delay carbohydrate absorption but they also alter the gastrointestinal hormonal axis. These drugs
decrease postprandial secretion of gastric inhibitory polypeptide (potentiates insulin secretion under hyperglycemic condition) and
increases postprandial levels of glucagon-like peptide which may play a role in the regulation of insulin secretion [61]. Therefore, glucosidase inhibitors such as acarbose, voglibose and miglitol are widely used either alone or in combination with insulin
secretagogues in patient with Type-2 diabetes [62]. Acarbose is the first commercially available -glucosidase inhibitor and inhibits
both amylase and membrane bound -glucosidase with approximately equal affinity. It has a potent inhibitory effect on sucrase but
weak effect on the maltase and no effect on -glucosidase such as lactase. Thus lactose is digested normally[63]. Acarbose does not
interfere with the sodium dependent glucose transporter thereby not affecting absorption of glucose. Friedman et al. (1991) [64]
reported that the decreased levels of GLUT-4 of fatty Zucker rats were normalized by the treatment with a disaccharidase inhibitor,
acarbose. Since GLUT 4 level is the rate-limiting step of insulin stimulated glucose disposal, normalization of GLUT-4 level might
considerably improve in vivo insulin resistance of fatty Zucker rats.
Voglibose, an N-substituted derivative of valiolamine isolated from the fermentation broth of Streptomyces hygroscopicus, is
a potent and structurally novel inhibitor of the intestinal disaccharidases. It had a potent inhibitory effect on maltase but with a short
inhibitory duration [65]. Shinozaki et al. (1996) [66] found an improvement in in vivo sensitivity to insulin in non-diabetic
hyperinsulinemic patients by administration of a disaccharidase inhibitor A0-128 (voglibose). It is hardly thought that a
disaccharidase inhibitor by itself corrects the abnormality of post-receptor pathway of both the peripheral tissue and liver in insulin
resistant diabetic rats. Tominaga et al. (1997) [67] also reported that insulin resistance of Wistar fatty rats was improved by a
disaccharidase inhibitor. Even though acarbose and voglibose are included in the drug list of diabetes management of either Type-1 or
Type-2, they show some common side effects. The most common adverse effects of these drugs are gastrointestinal disturbance
induced by fermentation of unabsorbed carbohydrate in the bowel, and increments of gastrointestinal motility and hepatotoxicity [68].
Unfortunately, the disturbance of digestive system also exists in diabetics [69]. Luo et al. (2001) [70] reported combined effect of
these drugs with gymnemic acid (GA), a mixture of triterpene glucuronides, found in the leaves of the Indian plant Gymenema
sylvestre which augmented their intestinal disaccharidases inhibitory effects with diminished adverse effects [71].
In this category, a majority of the recent studies report the potential of antidiabetic medicinal plants on inhibition of
carbohydrate hydrolyzing enzymes, -amylase and -glucosidase and manipulation of intestinal glucose transporters. A wealth of
literature has emerged now showing the potential effect of phytochemicals in inhibiting -amylase [72] and
-glucosidase.
Watanabe et al. (1997) [73] also further, the potential effect of phytochemicals in inhibiting -glucosidase activity resulting in
lowering of in vivo postprandial hyperglycemia has also been reported [73].

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Disclosure of interest
The authors declare that they have no conflicts of interest con-cerning this article.

5905

CONCLUSIONS
In the present study the enhanced or elevated activities of intestinal disaccharidases of insulin deficient diabetic and insulin
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