Journal of Ethnopharmacology 76 (2001) 269277

Effect of an antidiabetic extract of Catharanthus roseus on enzymic

activities in streptozotocin induced diabetic rats
Som Nath Singh *, Praveen Vats, Shoba Suri, Radhey Shyam, M.M.L. Kumria,
S. Ranganathan, K. Sridharan
Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi 110054, India
Received 9 October 2000; received in revised form 1 April 2001; accepted 24 April 2001
Abstract
Hypoglycemic activity was detected in dichloromethane:methanol extract (1:1) of leaves and twigs of Catharanthus roseus
(family Apocynaceae), a traditionally used medicinal plant, using streptozotocin (STZ) induced diabetic rat model. Extract at dose
500 mg/kg given orally for 7 and 15 days showed 48.6 and 57.6% hypoglycemic activity, respectively. Prior treatment at the same
dose for 30 days provided complete protection against STZ challenge (75 mg/kg/i.p. 1). Enzymic activities of glycogen synthase,
glucose 6-phosphate-dehydrogenase, succinate dehydrogenase and malate dehydrogenase were decreased in liver of diabetic
animals in comparison to normal and were signicantly improved after treatment with extract at dose 500 mg/kg p.o. for 7 days.
Results indicate increased metabolization of glucose in treated rats. Increased levels of lipid peroxidation measured as
2-thiobarbituric acid reactive substances (TBARS) indicative of oxidative stress in diabetic rats were also normalized by treatment
with the extract. 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Antidiabetic activity; Catharanthus roseus; Streptozotocin; Glucose metabolism; Glutathione; Lipid peroxidation
www.elsevier.com/locate/jethpharm
1. Introduction
Diabetes mellitus is a metabolic disease as old as
mankind and its incidence is considered to be high
(45%) all over the world (Pickup and Williams, 1997).
In spite of the introduction of hypoglycemic agents,
diabetes and related complications continue to be a
major medical problem. Since time immemorial, pa-
tients with non-insulin requiring diabetes have been
treated orally in folk medicine with a variety of plant
extracts. In India a number of plants are mentioned in
ancient literature (Ayurveda) for the cure of diabetic
conditions known as madhumeha and some of them
have been experimentally evaluated and the active prin-
ciples isolated (Chopra et al., 1956; Rajashekharan and
Tuli, 1976; Chattopadhyay et al., 1993; Pugazhenthi
and Murthy, 1996; Chattopadhyay, 1999; Joy and Kut-
tan, 1999)
Cajtharanthus roseus belonging to family Apocy-
naceae is known with various names in India and all
over the world. Hot water decoction of the leaves
and/or the whole plant is used for treatment of diabetes
in several countries i.e. Brazil, Cook Islands, Dominica,
England, Jamaica, Mozambique, Pakistan, Taiwan,
Thailand and West Indies (Don, 1999). In India seven
owers/leaves are used at a time whereas in The Cook
Islands 18 leaves boiled in a kettle of water and in The
West Indies roots of plants infused in whiskey are used
traditionally. Preliminary reports indicate blood glucose
lowering activity in alcoholic extract of leaves (Ghosh
and Gupta, 1980; Chattopadhyay et al., 1991, 1992). In
the present study we have evaluated antidiabetic activ-
ity of a dichloromethanemethanol (DCMM) extract
of leaves and twigs (L&T) and its effect on enzymes of
carbohydrate metabolism to nd out possible mecha-
nism of hypoglycemic action. In addition to this the
effect of extract was evaluated on glutathione levels,
related enzymes and lipid peroxidation as oxidative
stress is known to occur in diabetes. Effect of extract on
other enzymes of pharmacological importance i.e. as-
* Corresponding author. Fax: +91-11-3932869.
E-mail address: shoba72@yahoo.com (S.N. Singh).
0378-8741/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0378- 8741( 01) 00254- 9
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 270
partate aminotransferase (AST), alanine aminotrans-
ferase (ALT), acid and alkaline phosphatases were
also evaluated.
2. Materials and methods
2.1. Plant material
Flowering twigs of C. roseus were collected after
authentication by Dr. J. Yadava, Defence Agricultural
Research Laboratory, Pithoragarh. Voucher specimen
of plant sample is available in herbarium le of the
institute (HERB/DIP 99/Vinca 13). Collected plant
material was washed thoroughly with water and dried
in the shade. Dried leaves, twigs and owers were
made into powder in a grinder and were extracted
with 10 volumes of dichloromethane:methanol (1:1)
by continuous stirring for 48 h. After 48 h the solvent
was ltered and remaining parts were re-extracted
with the same volume of solvent. Extracts were
pooled and evaporated to dryness at 60 C. The yield
of this extract was 17% on dry weight basis. Extracts
thus prepared were stored at 4 C. For preparation of
aqueous crude extract, known quantities of wet leaves
and twigs were homogenized with distilled water in a
mixer grinder and centrifuged at 1000g for 15 min,
supernatants were freeze dried and used as crude ex-
tract (yield 2024% of wet wt).
2.2. Experimental animals and induction of diabetes
Male SpragueDawley rats weighing 250300 g
were used in the present study. Animals were main-
tained at 2292 C with 12 h light and dark cycle, fed
on standard pellet diet supplied by Lipton India Ltd.
Animals had free access to diet and water. After ini-
tial determination of 12 h fasting blood glucose levels
(blood drawn from retro orbital plexus) animals were
given single i.p. injection of streptozotocin at dose 75
mg/kg (freshly dissolved in physiological saline) and
blood glucose was monitored after 24 h and thereafter
at weekly intervals after 12 h fasting in case of all the
experiments reported here.
2.3. Determination of antidiabetic acti6ity and change
in body mass
Animals showing blood glucose levels \200 mg/dl
48 h after STZ treatment were selected for study.
Animals were treated orally for different duration at
different doses of test extracts suspended in distilled
water. Fasting blood glucose levels were monitored
weekly along with untreated controls. Any reduction
in blood sugar level in comparison to that of un-
treated controls was taken as antidiabetic activity.
Five animals at a time were used and experiments
were repeated twice in identical conditions with same
number of control animals making a total of 810
animals for each study parameter. Changes in body
weight of untreated controls and experimental animals
were recorded at the same time, i.e. in fasting state.
2.4. Prophylactic acti6ity against STZ challenge
For determination of prophylactic activity, normal
animals were treated with test extracts (aqueous ex-
tract and DCMM) at a dose of 500 mg/kg (p.o.) for
30 days. After monitoring blood glucose levels on day
30, animals were challenged to streptozotocin at a
dose of 75 mg/kg1 (i.p.). Blood glucose levels after
48 h were monitored for induction of hyperglycemia
along with untreated control animals (fed with dis-
tilled water) challenged with STZ.
2.5. E6aluation of effect on biochemical 6ariables
For in vivo study of effect of DCMM extract on
biochemical variables, diabetic animals were treated
(n=8-10) with the extract at a dose of 500 mg/kg for
7 consecutive days which showed around 50% hypo-
glycemic activity. Diabetic animals with similar ele-
vated blood glucose levels and normal animals were
kept as diabetic and normal controls respectively (n=
810). All the three groups were sacriced by cervical
dislocation on day 8 post treatment after fasting
overnight. Blood was drawn from the heart. The liver
was removed, washed with chilled saline, small
weighed portion of the liver were processed for deter-
mination of glycogen and glutathione immediately af-
ter their removal. Ten percent homogenate (w/v) of
liver was prepared in 150 mM KCl using Potter
Elvehjem homogenizer at 4 C. Two milliliter aliquots
of crude liver homogenates were used for assay of
lipid peroxidation and rest of the homogenates were
centrifuged at 3000g for 15 min at 4 C and super-
natants were divided into aliquots and frozen at
20 C until assayed for different enzymes. Blood
plasma was recovered by centrifugation at 1000 g for
10 min at 4 C.
Effect of extract on activities of different enzyme of
glucose metabolism were evaluated in 6itro using liver
homogenate/plasma of normal animals as enzyme
source. Extract was dissolved in methanol (10 mg/ml).
An aliquot of 1030 ml as required was added to the
assay mixture to give a nal concentration of 100
mg/ml. Samples were incubated for 10 min at 37 C
before assay. All assays were performed in triplicate
with suitable amount of enzyme and assay mixtures
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 271
containing solvent (methanol) in place of extract served
as control.
Blood glucose was estimated by method of Nelson as
described by Ashwell (1957). Effect on oral glucose
tolerance was evaluated by feeding 10 g/kg glucose after
3.5 h of treatment with 500 mg/kg DCMM extract
(Chattopadhyay et al., 1991). Plasma fructosamine was
determined by the method of Johnson et al. (1982) with
slight modication. In brief, reduction of nitroblue
tetrazolium (NBT) was monitored at 530 nm using an
assay mixture containing 50 ml plasma, 1.75 ml Tris
buffer pH 10.6 (pH adjusted with 0.1 N NaOH) and 0.2
ml of NBT (1 mg/ml). Liver glycogen was measured by
method of Montgomery (1957). The specic activities
of enzymes viz. succinate dehydrogenase (EC 1.3.99.1)
(Slater and Bonner, 1952), glucose-6-phosphate dehy-
drogenase (EC 1.1.1.49), malate dehydrogenase (EC
1.1.1.37) (Shonk and Boxer, 1964), glucokinase (EC
2.7.1.2) (Parry and Walker, 1966), glycogen synthase
(EC 2.4.1.11) (Leloir and Goldenberg, 1962), lactate
dehydrogenase (EC 1.1.1.27), aspartate aminotrans-
ferase (EC 2.6.1.1), alanine aminotransferase (EC
2.6.1.2) (King, 1965), acid phosphatase (EC 3.1.3.2),
alkaline phosphatase (EC 3.1.3.1) (Lowry et al., 1954),
g-glutamyl transpeptidase (EC 2.3.2.2) (Orlowsky and
Meister, 1963), glutathione S-transferase (EC 2.5.1.18)
(Habig and Jakoby, 1981) were estimated with standard
colorimetric and photometric techniques. Glutathione
in blood and liver was estimated colorimetrically by
using DTNB (Ellman, 1959) and lipid peroxidation in
crude homogenates as 2-thiobarbituric acid reactive
substances (TBARS) by method of Utley et al., (1967).
Protein content of enzyme samples were determined
colorimetrically (Lowry et al., 1951).
2.6. Statistical analysis
Values are expressed as mean9SEM. Unpaired Stu-
dent t-test was used for statistical comparison. In case
of in vivo studies comparison were made between nor-
mal and diabetic, diabetic versus diabetic treated ani-
mals. Changes were considered signicant if the
P-value was less than 0.05.
3. Results
3.1. Antidiabetic and prophylactic acti6ity
Diabetic rats treated with crude aqueous extract at
oral dose of 1 g/kg for 21 days showed 20.2% reduction
in blood glucose in comparison to untreated diabetic
rats. Blood glucose levels of control diabetic and
treated animals were 315915.7, 215.3925.0 mg/dl
respectively (PB0.05).
DCMM extract showed 57.6 and 48.6% antidiabetic
activity (PB0.001) at dose 500 mg/kg given orally for
15 and 7 days respectively (Table 1). Changes in body
weight and nitroblue tetrazolium (NBT) reduction as-
say a measure of fructosamine in plasma are depicted in
Fig. 1. Normal animals treated with extract at dose 500
mg/kg (p.o.) and given oral glucose 10 g/kg showed
delay in peak blood glucose levels by 30 min in com-
parison to untreated rats (Fig. 2).
Prior to treatment for 30 days with aqueous and
DCMM extract at dose of 500 mg/kg (p.o.) before STZ
challenge provided 27.7 and 100% protection, respec-
tively, in comparison with control animals which re-
ceived saline daily (Table 2).
3.2. Effect on biochemical 6ariables
3.2.1. Effect on glucose metabolism
In vivo effect of treatment with DCMM extract at
dose 500 mg/kg for 5 days on glycogen levels and
glucose metabolizing enzymes are given in Tables 3 and
4.
In vitro effect of DCMM extract at conc. 100 mg/ml
in assay mixture on enzymic activities are given in
Table 5. There was signicant stimulation of glucoki-
nase activity whereas other enzymic activities remain
almost unaffected.
3.2.2. Effect on glutathione and other biochemical
6ariables
No signicant change in glutathione (GSH) levels of
diabetic, diabetic DCMM treated animals were ob-
served in comparison to normal animals. Lipid peroxi-
dation was 22.7% more in diabetic animals in
comparison with normal animals but was reduced in
animals treated with extract at 500 mg/kg (p.o.) 7
days. Enzymic activities of g-glutamyl transpeptidase,
glutathione S-transferase (GST) and enzymes of toxico-
logical importance, i.e. AST, ALT, acid and alkaline
phosphatase activities are given in Table 6.
4. Discussion
For the study of antidiabetic agents, STZ induced
hyperglycemia in rodents is considered to be a good
Table 1
Effect of DCMM extract of C. roseus (leaves and twigs) on blood
glucose levels (mg/dl) of STZ induced diabetic rats
Blood glucose Dose (mg/kg p.o.days)
Treated diabetic Control diabetic
291.36914.6 5007 149.87925.99
a
50015 237.98919.17 100.80916.9
a
Values are expressed as mean9SEM.
a
PB0.001 in comparison with control diabetics.
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 272
Fig. 1. Effect of DCMM extract on change in body weights and plasma NBT reduction. Values are mean (n=10), * PB0.05, in comparison with
initial and normal in case of body weight and NBT reduction respectively. PB0.05 in comparison with diabetic.
preliminary screening model (Ivorra et al., 1989) and is
widely used. STZ, N-[methylnitrocarbamoyl]-D-glu-
cosamine is a potent methylating agent for DNA and
acts as nitric oxide donor in pancreatic cells. b cells are
particularly sensitive to damage by nitric oxide and free
radicals because of their low levels of free radical
scavenging enzymes (Lukic et al., 1998; Spinas, 1999).
Leaves and owers of C. roseus are used traditionally
by diabetic patients in India and are taken as water
decoction. Due to this reason crude aqueous extract was
given orally at dose of 1 g/kg (generally used oral dose
for primary testing of crude products at our laboratory)
for a period of 21 days and 20.2% glucose lowering
activity was observed. Although the activity is appar-
ently low, it has signicance for further studies as STZ
treated animals represent chronic model of IDDM and
marked destruction of pancreatic structures was ob-
served (slide not shown). Signicant hypoglycemic activ-
ity was detected in DCMM extract of leaves and twigs
(Table 1) along with 100% prophylactic action against
STZ challenge during primary screening and need spe-
cial attention (Table 2). DCMM extract was better
tolerated in primary toxicity study and oral LD
50
was
found more than 5000 mg/kg body weight in rats. Other
studies on hydroalcoholic extracts of leaves and twigs
also indicate fairly higher margin of safety (Chattopad-
hyay et al., 1992; Chattopadhyay, 1999)
Nitroblue tetrazolium (NBT) reduction assay as a
marker of fructosamine levels (proteinketoamine
product) also showed decrease in animals treated with
DCMM extract (Fig. 1). This assay is a measure of long
term glycemic control (Baker et al., 1985).
The extract was evaluated in vitro as well as in vivo
on several biochemical variables. Glycogen levels in liver
which were low in diabetic animals, increased several
folds in DCMM treated diabetic animals (Table 3).
Although glycogen synthetase activity decreased in dia-
betic animals signicantly, the treatment however could
not normalize activity. Glycogen content of normal
animals in fasting stage was only slightly higher than
diabetic animals and this may be due to degradation of
glycogen to maintain normal blood glucose levels,
whereas glycogen levels in diabetics were found to be
very low despite high blood glucose levels possibly due
to lower levels of glycogen synthase activity. Accumula-
tion of glycogen in liver of treated animals is somewhat
similar to that reported during insulin therapy. When
insulin therapy is instituted, hepatic glycogen accumula-
tion begins rapidly and glycogen content rises to 300%
of normal levels within 24 h and this inordinate accumu-
lation of glycogen may account for upto 60% of dry liver
weight in diabetic animals (Osborn et al., 1953; Spiro et
al., 1958; Steiner and King, 1964; Anderson, 1974).
Activity of glucose-6-phosphate dehydrogenase, the
rst regulatory enzyme of pentose phosphate pathway
was found to be decreased in diabetic animals and
increased in DCMM extract treated animals, the activity
was higher in comparison to untreated diabetic animals
indicating improvement in glucose utilization by this
pathway (Table 4).
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 273
Fig. 2. Effect of DCMM extract on blood glucose levels of normal rats after feeding glucose (10 g/kg). Values are mean (n=8), PB0.05 in
comparison with control.
Table 2
Prophylactic activity of C. roseus extracts against STZ induced diabetes in rats (n=10)
No. of animals % Animals showing Blood glucose (mg/dl) % Protection in comparison to
diabetic/total untreated control hyper-glycemia
48 h Post STZ Initial
challenge
83 74.1494.05 350.0910.44 Untreated control 10/12
85.0093.11 6/10 60 27.7 Aqueous extract 336.5697.94
91.7691.77
a
70.5094.81 0/12 0 100 Dichloromethane:
methanol extract
Animals were treated with extract at dose of 500 mg/kg, p.o. for 30 consecutive days prior to STZ challenge. Values expressed as mean9SEM
(n=10).
a
PB0.001 in comparison with untreated control group.
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 274
Table 3
Effect of DCMM extract of C. roseus on liver glycogen levels and
glycogen synthase activity of STZ induced diabetic rats
Liver glycogen Glycogen synthase Group
(mg/g wet tissue) (mmol UDP
formed/min/mg
protein)
Normal 4.8490.74 3.0190.19
Diabetic 0.7790.18
a
4.3790.93
22.1996.69
b
1.2390.19
a, NS
Diabetic treated (500
mg/kg P.O. x 7
days)
Values expressed as mean9SEM (n=8).
a
PB0.001 in comparison with normal.
b
PB0.05 and NS-not signicant when compared with diabetic.
unit B and isozymes 2,3 and 5 in STZ induced diabetes
in Chinese hamsters is reported. Increase in lactate
dehydrogenase activity is also reported in hypoglycemic
rats (Chang et al., 1977; Lemieux et al., 1984). Malate
dehydrogenase plays an important role in the citric acid
cycle by providing oxaloacetate for the formation of
citrate with acetyl-CoA for generating malate which
can feed the cytosolic gluconeogenic pathway (Murray
et al., 1998). In the present study, decrease in malate
dehydrogenase in liver and plasma of diabetic animals
was observed. Levels increased signicantly in liver of
treated animals whereas in plasma it reached almost
normal level. Succinate dehydrogenase activity which
was decreased to almost half in diabetic animals was
found to be increased in treated animals and levels were
even more than normal (Table 5). Increase in succinate
and malate dehydrogenase activities in treated animals
indicates better utilization of energy yielding intermedi-
ates by TCA cycle. These enzymes are reported to be
inhibited in tissues of diabetic animals in several studies
(Chen and Ianuzzo, 1981; Lemieux et al., 1984; Ianuzzo
and Armstrong, 1976). Our results indicate that treat-
Activity of glucokinase, the rst regulatory enzyme
of glycolytic pathway was also increased by extracts in
vitro (Table 5). Decreased activity of glucokinase is
reported in diabetes (Storey and Bailey, 1978; Chang et
al., 1977). We found no change in lactate dehydroge-
nase activity in liver of diabetic animals in the present
study (Table 5). Increase in lactate dehydrogenase sub-
Table 4
Effect of DCMM extract of C. roseus on enzymes of glucose metabolism
Enzyme Specic activity
Diabetic Normal Diabetic treated
Glucose-6-phosphate dehydrogenase
14.3291.09** Liver 15.4690.76*
a,NS
19.3391.31
(nmol NADP
+
reduced/min/mg protein)
Lactate dehydrogenase
Liver 62.8095.93 65.4992.84 63.1692.58
(nmol pyruvate formed/min/mg protein)
Succinate dehyrogenase
4.1690.40 Liver 2.9290.39* 5.8490.68
b
(nmol Pot. Ferricyanide reduced/min/mg protein)
Malate dehydrogenase
Liver 1.5690.13 1.1690.07* 3.8590.21
***,

(mmol NADH oxidized/min/mg protein)

Plasma
(mmol NADH oxidized/min/ml) 0.8890.13 0.5290.10* 0.8090.18
Values expressed as mean9SEM (n=10).
a
* PB0.05, ** PB0.01, *** PB0.001 in comparison with normal.
b
PB0.01, PB0.001, NS not signicant in comparison with diabetic.
Table 5
In vitro effect of DCMM extract of C. roseus on enzymatic activities of liver
Addition Glycogen synthase
1b
Glucokinase
2
Lactate dehydrogenase
3
Succinate dehydrogenase
4
Malate dehydrogenase
5
3.0290.03 1.7790.04 4.1690.49 4.6290.37 65.7492.47 Control MeOH
5.0190.29 4.0290.03
a
62.2492.77 Extract (100 mg) 4.7390.74 1.4990.17
Values expressed as mean9SEM (n=10)
a
PB0.001 in comparison with MeOH control.
b
(1) mmol UDP formed/min/mg protein, (2) nmol NADP converted to NADPH/min/mg protein, (3) nmol pyruvate formed/min/mg protein,
(4) nmol potassium ferricyanide reduced/min/mg protein, (5) mmol NADH oxidized/min/mg protein.
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 275
Table 6
Effect of DCMM extract of C. roseus on glutathione, MDA levels and activities of g-GT, GST, AST, ALT, acid and alkaline phosphatase
Variable Normal Diabetic Diabetic treated (500 mg/kg (p.o.)7 days)
Glutathione (GSH)
105.699.5 121.5924.2 Blood 108.8919.7
(mg/dl)
2822.49186.0 Liver 3262.59292.0 3381.59544.0
(mg/g wet tissue)
Lipid peroxidation
13.7090.40 Liver 16.8490.20** 33.8891.40
(mmol MDA/g wet tissue)
g-Glutamyl transpeptidase
4.6190.27 Liver 2.7690.68*
a
5.7590.95

(nmol p-nitroaniline released/min/mg protein)

22.3492.05** 14.1991.35 17.5893.00 Plasma
(nmol p-nitroaniline released/min/ml)
Glutathione S-transferase
0.4590.06 0.5990.05 0.4490.07 Liver
(mmol thioester formed/min/mg protein)
Aspartate aminotransferase
73.4798.37 88.8897.79 78.1497.03 Liver
(nmol pyruvate /min/mg protein)
163.73924.25** 79.1897.03 107.09927.21 Plasma
(nmol pyruvate formed/min/ml)
Alanine aminotransferase
79.2698.25 84.5592.22 80.3398.94 Liver
(nmol pyruvate /min/mg protein)
Alkaline phosphatase
1.0690.10*** 0.5490.06 1.1790.15*** Liver
(nmol p-nitrophenol released/min/mg protein)
Acid phosphatase
9.4690.83 9.2690.16 9.9890.67 Liver
(nmol p-nitrophenol released/min/mg protein)
Values expressed as mean9SEM (n=10)
a
* PB0.05, ** PB0.01, *** PB0.001 in comparison with normal, PB0.05 in comparison with diabetic.
ment with DCMM extract of C. roseus increases utiliza-
tion of circulating glucose by liver.
Oxidative stress appears to be a key element of the
production of secondary complications in diabetes
(Wohaieb and Godin, 1987; Godin et al., 1988; Wolff et
al., 1991; Thomson and McNeil, 1993; Thornalley et
al., 1996). Glutathione, a tripeptide present in millimo-
lar concentrations in all the cells is an important an-
tioxidant (Meister and Anderson, 1983; DeLeve and
Kaplowitz, 1991; Lu, 1999). Decreased glutathione lev-
els in diabetes have been considered to be an indicator
of increased oxidative stress (Wolff, 1987; McLennan et
al., 1991). In the present study not much change was
observed in GSH levels either in blood or liver of
diabetic animals, however in treated animals GSH lev-
els were marginally high in both blood as well as liver.
Lipid peroxidation was found to be increased in liver of
diabetic animals which became normal in DCMM
treated animals. This indicates that the extract may be
helpful in the prevention of damage caused by oxygen
free radicals. We have not studied oxidized glutathione
levels and this is a limitation of this study.
There was not much change in glutathione S-trans-
ferase activity in diabetic and diabetic treated animals.
g-Glutamyl transpeptidase activity was decreased sig-
nicantly in liver of diabetic animals and with DCMM
extract treatment activity increased (Table 6). g-Glu-
tamyl transpeptidase has a key role in amino acid
transport across membranes and catalyzes the initial
step in breakdown of glutathione, i.e. transfer of g-glu-
tamyl moiety of glutathione to a variety of amino acids
and peptides (Meister, 1983). Increase in g-glutamyl
transpeptidase activity in plasma is an indicator of
impairment in liver function. In the present study there
was an increase in g-glutamyl transpeptidase activity in
plasma of diabetic animals. In C. roseus treated animals
activity of this enzyme shows a decrease in plasma and
was close to normal activity.
Measurement of enzymic activities of aminotrans-
ferases (AST and ALT) and phosphatases (acid and
alkaline) is of clinical and toxocological importance as
changes in their activities are indicative of tissue dam-
age by toxicants or in disease conditions. AST and
ALT activities in liver of diabetic animals remain un-
changed in liver though AST activity was little less than
normal. Plasma levels of AST were increased around
S.N. Singh et al. / Journal of Ethnopharmacology 76 (2001) 269277 276
twice that of normal in diabetic animals and diabetic
animals treated with extract show improvement. Recov-
ery of plasma AST levels of diabetic rats towards
normal shows that the DCMM extract has no adverse
effect on liver functions. Liver alkaline phosphatase
activity was found to be signicantly increased in dia-
betic animals. Treatment with extract further caused
increase in activity (Table 6). Increase in alkaline phos-
phatase activity in testes and prostate at 300 mg/kg for
24 days by ethanolic extract of C. roseus leaves is
reported in normal animals (Chauhan et al., 1979).
Acid phosphatase activity of liver of diabetic rats was
also found to be increased. At low dose 75 mg/kg for 24
days ethanolic extract is reported to inhibit acid phos-
phatase activity and at higher doses i.e. 300 mg/kg
stimulation is reported (Chauhan et al., 1979).
Detection of hypoglycemic activity in DCMM ex-
tract along with protective effect against STZ challenge
and preventive action on lipid peroxidation provides
scientic rationale of use of C. roseus as antidiabetic
plant. Antidiabetic activity seems to be a result of
increase in glucose utilization. Further chromato-
graphic fractionation of the extract may be useful in
improving activity and reduction of dose. In prelimi-
nary toxicity study it is safe, however chronic toxicity
evaluation will be required for human use. Prophylactic
activity observed in present study is of much impor-
tance and there is a need for further studies on this
aspect.
Acknowledgements
The investigators express their sincere thanks to Dr.
P.K.Banerjee, ScF and Dr. W. Selvamurthy, Director
DIPAS, for their constant interest and encouragement
throughout the present study.
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