The present study investigated the possible protective effect of Helicteres isora (Sterculiaceae) bark extracts on certain biochemical markers in streptozotocin (STZ)-induced diabetes in rats. STZ treatment (60 mg/kg/i.p) caused a hyperglycemic state that led to various physiological and biochemical alterations. Blood levels of glucose, urea, uric acid and creatinine, plasma levels of albumin and albumin/globulin ratio and the activities of diagnostic marker enzymes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and ?-glutamyl transpeptidase (? -GT) in plasma, liver and kidney were markedly altered in STZ diabetic rats. Oral administration of H. isora (100, 200 mg/kg/p.o) for 21 days restored all these biochemical parameters to near normal levels. Thus, the present results have shown that H. isora bark extract has the antihyperglycemic effect and consequently may alleviate liver and renal damage associated with STZ-induced diabetes in rats.
aManonmaniam Sundaranar University, Sri Paramakalyani
Centre for Environmental Science, Alwarkurichi, Tamilnadu, India.
bCentre for Biotechnology, Muthayammal College of Arts and Sciences,
Kakkaveri, Rasipuram, Namakkal, Tamilnadu, India.
The present study investigated the possible protective effect of
isora (Sterculiaceae) bark extracts on certain biochemical markers in
streptozotocin (STZ)-induced diabetes in rats. STZ treatment (60 mg/kg/i.p)
caused a hyperglycemic state that led to various physiological and biochemical
alterations. Blood levels of glucose, urea, uric acid and creatinine, plasma
levels of albumin and albumin/globulin ratio and the activities of diagnostic
marker enzymes including aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transpeptidase
(γ -GT) in plasma, liver and kidney were markedly altered in STZ diabetic rats.
Oral administration of H. isora (100, 200 mg/kg/p.o) for 21 days restored all
these biochemical parameters to near normal levels. Thus, the present results
have shown that H. isora bark extract has the antihyperglycemic effect and
consequently may alleviate liver and renal damage associated with STZ-induced
diabetes in rats.
Keywords: Helicteres isora; Sterculiaceae; Streptozotocin; Diabetes mellitus;
Oxidative stress is currently suggested as one of the mechanism underlying
diabetes mellitus, which affects carbohydrate, lipid and protein metabolism.
Several alterations in diabetic individuals are oxidative in nature or may
depend on increased oxidative stress (1). Glycation (2) and hyperglycemic
pseudohypoxia (3) can generate a redox imbalance inside the cells, especially in
the liver (4). In a recent paper, we reported the elevated levels of plasma
thiobarbituric acid reactive substance (TBARS) and hydroperoxide (an index of
tissue injury) in streptozotocin (STZ) diabetic rats. The higher levels of these
substances suggest an increased rate of tissue injury in STZ diabetic rats.
Diabetes mellitus (DM) is also grossly reflected by profound changes in protein
metabolism and by a negative nitrogen (N) balance and loss of nitrogen from most
organs (5). Increased urea nitrogen production in diabetes may be accounted for
by enhanced catabolism of both liver and plasma proteins (6, 7). Indian
traditional medicine has used different herbs for the treatment of a broad
spectrum of ailments such as inflammation, DM and the management of various
hepatic and renal disorders. Ayurveda, the ancient system of Indian medicine,
has identified hepatic and renal diseases quite early and recommended a number
of herbal drugs, which are a good source of natural antioxidants believed to
exert their effects by reducing the formation of the final active metabolite of
the drug-induced systems or by scavenging the reactive molecular species to
prevent their reaching a target site (8-10). In addition, Bopanna et al. (11)
and Eskander et al. (12) demonstrated that the administration of several herb
extracts could restore the changes in the activities of serum enzymes like
alkaline phosphatase (ALP), acid phosphatase and transaminases: aspartate
aminotransferase (AST) and alanine aminotransferase (ALT).
The Helicteres isora Linn. (Sterculiaceae) is a shrub or small tree available in
forests throughout Central and Western India. In traditional Medicine the root
juice is claimed to be useful in diabetes, empyema and a favorite cure for
snakebite (13, 14). The root and bark are expectorant, demulcent, constipating
and are useful in colic, scabies, gastropathy, diarrhoea and dysentery (15). The
fruits are astringent, refrigerant, stomachic, vermifuge, vulnerary and useful
in griping of bowels, flatulence of children (16) and have antispasmodic effect
(17). The bark and leaves are used as tan and the wood which is twice as hard as
teak, is extensively used for boat-building, carts, carriages, firewood, bow of
violin, planking, tool-handles, beams and fence posts (18). The root of H.isora
was undertaken to verify the claim and evaluate the anti-diabetic property (19).
In the current literature, there is no much data concerning the effect of the
H.isora on the biochemical parameters and the activities of enzymes which are
abnormally altered due to DM. Therefore, the present study aims to examine the
influence of oral administration of the bark extract of H. isora on the levels
of some biochemical parameters and the activities of some enzymes in serum,
liver, and kidney in STZ-induced diabetic rats.
Male Wistar albino rats (160-200g) were procured from the animal house,
Bharathidasan University, Tiruchirapalli under standard environmental conditions
(12-h light/dark cycles at 25-28?C, 60-80% relative humidity), and fed with a
standard diet (Hindustan Lever, India) and water ad libitum. All the studies
were conducted in accordance with the National Institute of Health ?Guide for
the Care and Use of Laboratory Animals? (20).
Preparation of plant extract
The dried bark of H.isora was ground into fine powder with auto-mix blender.
Then, the fine powder was suspended in equal amount of water and stirred
intermittently and left overnight. The macerated pulp was then filtered through
a coarse sieve and the filtrate was dried at reduced temperature. This dry mass
(yield 85 g/kg of powdered bark) was served as the aqueous extract of H. isora
Induction of diabetes
Rats were made diabetic by single administration of STZ (60 mg/kg/i.p) obtained
from Sigma Chemical Co. (St. Louis, MO, USA) dissolved in 0.1-M citrate buffer,
pH 4.5. After 48 h, blood sample were collected and glucose levels were
determined to confirm the development of diabetes. Only those animals which
showed hyperglycemia (blood glucose levels>240 mg/dl) were used in the
Diabetes was induced in animals 2 weeks before starting the treatment. After the
induction of diabetes, rats were divided into 4 groups of 6 animals each (group
II-V). Group I received the vehicle alone and was served as the control. Group
II received STZ (60 mg/kg/i.p) dissolved in 0.1-M citrate buffer. Group III &
Group IV received the bark extract of H. isora (100 mg, 200 mg/kg/p.o) once
daily for 21 days. Group V received tolbutamide (250 mg/kg/p.o) once daily for
Animals described as fasting were deprived of food for 12 h but allowed free
access to drinking water. After 21 days of treatment, the animals were killed by
cervical dislocation. Blood was collected into heparinized tubes, and the plasma
and serum were separated by centrifugation. The liver and kidney were quickly
removed, washed in ice cold, isotonic saline and blotted individually on
ash-free filter paper and weighed were measured. The tissues were then
homogenized in 0.1 M Tris-HCl buffer, pH 7.4. The homogenate was used for the
estimation of proteins, enzymes, and other parameters.
Estimation of biochemical parameters
Plasma glucose level was measured by the method of Sasaki and Sonae (21) and
serum concentration of urea, uric acid and creatinine were determined by
Autoanalyser using reagent kit obtained from Boehringer (Mannheim, Germany). The
protein content in plasma, liver and kidney was measured by the method of Lowry
et al. (22). The albumin and globulin content in plasma were determined by the
method described by Reinhold (23). The enzymes (AST, ALT and ALP) were assayed
by the method of King and Armstrong (24) and γ-glutamyl transpeptidase (γ -GT)
was assayed by the method of Rosalki and Rau (25).
Values were presented as Means?SD. Data were analyzed using analysis of variance
(ANOVA) and group means were compared with Duncan?s multiple range test (DMRT)
using SPSS (Statistical Package for Social Science).
Table 1 shows the liver and kidney weights in control and STZ diabetic rats.
isora restored the liver weight to near normal. The kidney weight was increased
in diabetic rats and H. isora normalized the kidney weight in STZ diabetes.
Table 2 shows the blood levels of urea, uric acid, and creatinine in plasma of
normal and STZ diabetic rats. These biochemical variables were significantly
elevated in STZ diabetic rats (p<0.05) when compared to control animals. Oral
administration of H. isora bark extract (100, 200 mg/kg/p.o) for 21 days
significantly lowered urea, uric acid and creatinine levels in STZ diabetic
The levels of protein, plasma albumin and albumin/globulin ratio in normal and
STZ diabetic rats are shown in Table 2. The level of protein in plasma was found
to be reducing in diabetic animals (p<0.05) when compared to control animals.
The lowered level of protein, after bark extract treatment, increased to near
normal. The levels of albumin and albumin/globulin ratio in plasma were
decreased in diabetic animals. These lowered levels of plasma albumin and
albumin/globulin ratio level were reverted back significantly in H. isora
treated diabetic rats.
Tables 3, 4 and 5 show the activities of AST, ALT, ALP and γ -GT in plasma,
liver and kidney of the control and STZ diabetic rats, respectively. The
activities of these enzymes were found to be significantly increased (p<0.05) in
plasma and liver of diabetic rats. In the kidney of diabetic animals, the
activities of ALP and γ-GT were increased while the activities of AST and ALT
were not altered. Oral administration of H. isora (100, 200 mg/kg/p.o) for 21
days resulted in the near normalization of the activities of AST, ALT, ALP and γ
-GT in plasma, liver and kidney of diabetic rats.
The present investigation indicates the hypoglycemic and protective effects of
H. isora in the liver and kidney of STZ diabetic rats. We have observed a
significant weight gain in H. isora treated diabetic rats when compared with
untreated animals. This observation shows the anabolic effect of H. isora on
body weight of the diabetic animals. A decrease in the liver weight observed in
diabetic animals might be due to an increased breakdown of glycogen and/or
pronounced gluconeogenesis. After 21 days of treatment with H. isora in diabetic
animals, an increase in the liver weight was observed. This result agrees well
with the result of Jefferson et al. (26) who has reported that insulin therapy
can increase the accumulation of glycogen in diabetic liver. Seyer-Hansen (27)
and Esterby and Gundersen (28) reported 15% rise in whole kidney weight within
72 h of induction of STZ in experimental diabetic rats. We have also observed an
increased whole kidney weight in diabetic animals when compared with normal
control animals. This is due to the glomerular cell proliferation accompanying
glomerular enlargement in the early phase of STZ-induced diabetes in rats. In
our present study, oral administration of H. isora significantly decreases the
kidney weight to near normal value. This might be due to the protective effect
of H. isora on glomerular cells in STZ-induced diabetic rats.
The diabetic hyperglycemia induces the elevation of plasma levels of urea, uric
acid and creatinine, which are considered as the significant markers of renal
dysfunction (5). The results in Table 2 show significant (p<0.05) increase in
the level of plasma urea and creatinine in the diabetic rats when compared with
respective control rats, while after the treatment of STZ diabetic rats with the
bark extract of H. isora (100, 200 mg/kg), the levels of urea, uric acid and
creatinine were significantly (p<0.05) decreased. These results are in agreement
with the previous studies on the mesocarp extract of B. aegyptiaca (29, 30) and
root extract of Panax ginseng (31).
As illustrated in Table 2, a marked (p<0.05) reduction in plasma total protein
(TP) and albumin (A) levels was observed in diabetic rats and this is consistent
with the results obtained by Bakris (32) and Tuvemo et al. (33). The decrease in
TP and A may be due to microproteinuria and albuminuria, which are important
clinical markers of diabetic nephropathy (34), and/or may be due to increased
protein catabolism (35). The results of the present study demonstrated that the
treatment of diabetic rats with the bark extract of H. isora caused a noticeable
elevation in the plasma TP and A levels as compared with their normal levels.
Such improvement of serum protein and albumin was previously observed after the
oral administration of B. aegyptiaca to experimentally diabetic rats (29). It
has been established that insulin stimulates the incorporation of amino acids
into proteins (5). The enzymes directly associated with the conversion of amino
acids to ketoacids are aspartate transferase and alanine transferase, and these
enzymes are increased in diabetic condition. Begum and Shanmugasundaram (36)
also reported an increase in the activities of aspartate transaminase and
alanine transaminase in the liver of diabetic animals. The rise in the activity
of alanine transaminase is due to hepatocellular damage and is usually
accompanied with aspartate transaminase (37). Treatment with H. isora (100 and
200 mg/kg) or tolbutamide (250 mg/kg) normalized these enzymes activities.
Similarly, increased activities of aspartate and alanine transaminase in
diabetic liver were also reported by Jorda et al. (7).
Elevated activity of ALP was observed in STZ diabetic rats. Prince et al. (38)
have also reported increased ALP activity in experimentally diabetic rats. The
increased activity of this enzyme in plasma may be a result of diabetes-induced
damage to the tissues. H. isora (100, 200 mg/kg) treatment restored the activity
of these enzymes to near normal by reducing their induction in diabetes. γ -GT
catalyzes the transfer of the γ -glutamyl group from γ -glutamyl peptides to
another peptide or L-amino acids or to water. The assay of γ-GT is a helpful
adjunct in detecting hepatic damage (39). A highly significant elevation in the
activity of γ-GT was observed in plasma, liver and kidney of STZ induced
diabetic rats. This is in accordance with earlier investigations (40), wherein
there has been shown a dramatic increase in γ-GT expression in the liver of
diabetic rats. In addition, hepatocellular damage or cholestasis may also
contribute to the elevation of this activity. Increased activity of γ-GT in
STZ-induced diabetic rats was lowered to near normal by H. isora (100, 200
mg/kg/p.o) treatment that indicates the possible prevention of necrosis by H.
From the roots of H. isora, cucurbitacin B and isocucurbitacin B have been
isolated and reported (41). Neolignans, helisterculins A, B and helisorin have
been separated from the fruits, while, betulic acid, daucosterol, sitosterols,
isorin (42) have been isolated from the roots and barks of H. isora. It is
interesting to note that many plant sitosterols have been reported to exhibit
hypoglycemic effects (43-46). However, the full potential of sitosterol as the
hypoglycaemic agent can only be realized after further comprehensive
pharmacological and toxicological investigations.
In conclusion, the present study demonstrated that the bark extract of
could influence the protein metabolism and marker enzymes in STZ induced
diabetic rats. Further, bark extract of H. isora ameliorated the impaired renal
function and inhibited the liver damage associated with STZ diabetes in rats.
(1) Baynes JW. Role of oxidative stress in development of
complication in diabetes. Diabetes (1991) 40: 405-412
Vlassara H, Bucala R and Striker L. Pathogenic effects of advanced
glycosylation: biochemical, biological and clinical implications for diabetes
and aging. Lab. Invest. (1994) 70: 138-151
Williamson JR and Lilo G. Capillary basement membrane in diabetes. Diabetes
(1993) 32: 96-100
Gallou G, Ruelland A, Legras B, Maugendre D, Allanic H and Cloarec L. Plasma MDA
in type 1 and type 2 diabetes. Clin. Chim. Acta (1993) 214: 227-234
Almdal TP and Vilstrup H. Effect of streptozotocin-induced diabetes and diet on
nitrogen loss from organs and the capacity of urea synthesis in rats.
Diabetologia (1987) 30: 952-956
Jorda A, Cabo J and Grisolia S. Changes in the levels of urea cycle enzymes and
their metabolites in diabetes. Enzyme (1981) 26: 240-244
Jorda A, Gomez M, Cabo J and Grisolia S. Effect of streptozotocin diabetes on
some urea cycle enzymes. Biochem. Biophys. Res. Commun. (1982) 106: 37-43
Shanmugasundaram ERB, Rajeswari G and Baskaran K. Use of Gymnema sylvestre leaf
extract in the control of blood glucose in insulin-dependent diabetes mellitus.
J. Ethnopharmacol. (1990) 30: 281-94
Srivastava Y, Venkatakrishnan Bhatt H and Verma Y. Antidiabetic and adaptogenic
properties of Momordica charantia extract: an experimental and clinical
evaluation. Phytother. Res. (1993) 7: 285-289
Sharma SR, Dwivedi SK and Swarup D. Hypoglycaemic, antihyperglycaemic and
hypolipidaemic activities of Casealpina bonducella seeds in rats. J.
Ethnopharmacol. (1997) 58: 39-44
Bopanna KN, Kannan J, Godgil S, Balaraman R and Rathod SP. Antidiabetic and
antihyperlipidemic effects of neem seed kernel powder on alloxan diabetic
rabbits. Ind. J. Pharmacol. (1997) 29: 162-172
Eskander EF, Jun HW, Ibrahim KA and Abdelal WE. Hypoglycemic effect of an herbal
formulation in alloxan induced diabetic rats. Egypt J. Pharm. Sci. (1995) 36:
Kirtikar KR and Basu BD. Indian Medicinal Plants. Vol.1, International Book
Distributors, Dehradun (1995) 371-372
Singh SB, Singh AK and Thakur RS. Chemical Constituents of the leaves of
Helicteres isora. Ind. J. Pharm. Sci. (1984) 46: 148-149
Prajapathi ND, Purohit SS, Sharmi AK and Kumar T. A Handbook of Medicinal
Plants: A Complete Source Book. Shyam Printing Press, Jodhpur (2003) 265
Chopra RN, Nayar SL and Chopra IC. Glossary of Indian Medicinal Plants. 1st ed.
CSIR, New Delhi (1956) 131.
Pohocha N and Grampurohit ND. Antispasmodic activity of the fruits of Helicteres
isora Linn. Phytother. Res. (2001) 15: 49-52
Nadkarni KM and Nadkarni AK. Indian Materia Medica. Vol.1, Popular Prakashan,
Bombay (1976) 615-616
Venkatesh S, Dayananda Reddy G, Madhava Reddy B and Lakshman M. Antidiabetic
effect of Helicteres isora root. J. Ethnopharmacol. (2003) 90: 225-228
National Institutes of Health. Guide for the Care and Use of Laboratory Animals.
DRR/NIH, Bethesda (1985) 585
Sasaki TM and Sonae A. Effect of acetic acid concentration on the color,
reaction in the o-toluidine boric acid method for blood glucose estimation.
Rinsho Kagaku (1972) 1: 346-353
Lowry OH, Rosenbrough NJ, Farr AL and Randall RL. Protein measurement with the
Folin-phenol reagent. J. Biol. Chem. (1951) 193: 264-275
Reinhold J. Determination of serum total protein, albumin and globulin fractions
by the biuret method. In: Varley H, Gowen Lock AH and Bell M. (eds.) Practical
Clinical Biochemistry. 5th ed. Vol I, William Heinemann Medical Book Limited,
London (1980) 545-547
King KJ and Armstrong AL. Calcium, phosphorus and phosphatase. In: Varley H.
(ed.) Practical Clinical Biochemistry. 4th ed. CBS Publishers, New Delhi (1988)
Rosalki SB and Rau D. Serum γ-glutamyl transpeptidase activity in alcoholism.
Clin. Chim. Acta (1972) 39: 41- 47
Jefferson LS, Liao WST, Peavy DE, Miller JB, Appel MC and Jaylor JM. Diabetes
induced interactions in liver protein synthesis: Changes in the relative
abundance of mRNAs for albumin and other plasma proteins. J. Biol. Chem. (1983)
Seyer-Hansen K. Renal hypertrophy in streptozotocin diabetic rats. Clin. Sci.
Mal. Med. (1976) 51: 551-555
Esterby R and Gundersen HJG. Glomerular size and structure in diabetes mellitus
I. Early abnormalities. Diabetologia (1975) 11: 225-229
Mansour HA, Newairy AA. Amelioration of impaired renal function associated with
diabetes by Balanites aegyptiaca fruits in streptozotocin-induced diabetic rats.
J. Med. Res. Inst. (2000) 21: 115-125
Saeed A, Ibrahim N, Bashandy S and El-Gengaihi S. Saponin of Balanites
aegyptiaca Del fruits and biological evaluation. Bull. Fac. Pharm. (2000) 33:
Badr El-Din NK. Effect of Panax ginseng extract on the nephrotoxicity of
streptozotocin-induced experimental diabetes. Egypt J. Biochem. (1997) 15: 29-52
Bakris GL. Diabetic nephropathy. What you need to know to preserve kidney
function. Postgrad. Med. (1997) 93: 89
Tuvemo T, Ewald U, Kobboh M and Proos LA. Serum magnesium and protein
concentrations during the first five years of insulin-dependent diabetes in
children. Acta Paediatr. Suppl. (1997) 418: 7-10
Almdal JP and Vilstrup H. Strict insulin therapy normalizes organ nitrogen
contents and the capacity of urea nitrogen synthesis in experimental diabetes in
rats. Diabetologia (1988) 31: 114-118
Mauer SM, Steffes MW and Brown DM. The kidney in diabetes. Am. J. Med. (1981)
Begum N and Shanmugasundaram KR. Transaminases in experimental diabetes. Arogya
J. Health Sci. (1978) 4: 116-122
Mohan Rao GM, Morghom LO, Kabar MN, Benmohamud BM and Ashibani K. Serum glutamic
oxaloacetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels
in diabetes mellitus. Indian J. Med. Sci. (1989) 5: 118-122
Prince PSM, Menon VP and Pari L. Effect of Syzigium cumini extract on hepatic
hexokinase and glucose 6- phosphatase in experimental diabetes. Phytother. Res.
(1997) 11: 529-531
Prakasam A, Sethupathy S and Pugalendi KV. Influence of Casearia esculenta root
extract on protein metabolism and marker enzymes in streptozotocin-induced
diabetic rats. Pol. J. Pharmacol. (2004) 56: 587-593
McLennan SV, Heffernan S, Wright L, Rae C, Flsher E, Yue DK and Tortle JR.
Change in hepatic glutathione metabolism in diabetes. Diabetes (1991) 40:
Bean MF, Antoun M, Abramson D, Chang CJ, Mc Laughlin JL and Cassady JM.
Cucurbitacin B and isocucurbitacin B cytotoxic components of Helicteres isora.
J. Nat. Prod. (1985) 48: 500-503
Qu WH, Li JG and Wang MS. Chemical studies on the Helicteres isora. Zhongguo
Yaoke Daxue Xuebao (1991) 22: 203-206
Lotlikar MM and Rajaramma R. Pharmacology of hypoglycaemic principle isolated
from the fruits of Momordica charantia Linn. Ind. J. Pharm. (1966) 28: 129-133
Raman A and Lau C. Antidiabetic properties of phytochemistry of Momordica
charantia L. (Cucurbitaceae). Phytomedicine (1996) 2: 349-362
Perez GRM, Zavala SMA, Parez GS and Parez GC. Antidiabetic effect of compounds
isolated from plants. Phytomedicine (1998) 5: 55-75
Kumar G, Murugesan AG and Rajasekara Pandian M. Effect of Helicteres isora bark
extract on hepatic enzymes in experimental diabetes. Pharmazie (2006) 61: