Journal of Ethnopharmacology 142 (2012) 65–71

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antidiabetic and antioxidant potential of Emblica officinalis Gaertn. leaves

extract in streptozotocin-induced type-2 diabetes mellitus (T2DM) rats
Parminder Nain a,n, Vipin Saini a, Sunil Sharma b, Jaspreet Nain a
a
M.M. College of Pharmacy, M.M. University, Mullana-Ambala, Haryana 133207, India
b
Department of Pharmaceutical Science, G.J. University, Hisar, Haryana 125001, India

a r t i c l e i n f o abstract

Article history: Ethnopharmacological relevance: In traditional Indian medicine, all parts of Emblica officinalis Gaertn
Received 30 December 2011 plant including the fruit, seed, leaves, root, bark and flowers are used in various herbal preparations for
Received in revised form the treatment of diabetes mellitus, chronic diarrhea, anti-inflammatory and antipyretic.
5 March 2012
Aim of the study: To evaluate the hypoglycemic and antioxidants effects of the hydro-methanolic
Accepted 8 April 2012
(20:80) extract of leaves of Emblica officinalis Gaertn. (HMELEO) in streptozotocin induced diabetic rats.
Available online 17 April 2012
Material and methods: The hypoglycemic effect was measured by blood glucose and plasma insulin
Keywords: level. The oxidative stress was measured in liver and kidney by level of antioxidant markers i.e. lipid
Antidiabetic peroxidation (LPO), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase
Emblica officinalis Gaertn
(GPx) and catalase (CAT), and the biochemical parameters, i.e. blood serum levels of creatinine, urea,
Antioxidant
serum glutamic pyruvic transaminases (SGPT), serum glutamic oxaloacetic transaminases (SGOT),
Streptozotocin
alkaline phosphatase (ALP), total cholesterol and triglyceride levels were the salient features observed
in diabetic control and treated rats.
Results: Oral administration of the HMELEO at a concentration of 100, 200, 300 and 400 mg/kg b.w.
daily for 45 days showed a significant (P o0.05) decrease in fasting blood glucose and increase insulin
level as compared with the diabetic rats. Also it significantly (P o 0.05) reduced all biochemical
parameters (serum creatinine, serum urea, SGOT, SGPT and lipid profile). The treatment also resulted in
a significant (P o 0.05) increase in reduced glutathione, glutathione peroxidase, superoxide dismutase,
catalase, and decrease LPO level in the liver and kidney of diabetic rats.
Conclusion: The results clearly suggest that the hydro methanolic extract of leaves of Emblica officials
Gaertn. treated group may effectively normalize the impaired antioxidant status in streptozotocin
induced diabetes at dose dependent manner than the glibenclamide-treated groups. The extract
exerted rapid protective effects against lipid peroxidation by scavenging of free radicals and reducing
the risk of diabetic complications.
& 2012 Elsevier Ireland Ltd All rights reserved.

1. Introduction It is a complex metabolic disorder of the endocrine system with

dynamic expression of pathological disequilibria, resulting in
Diabetes is a deadly disease that affected an estimated 285 various micro and macro vascular complications. It is character-
million people worldwide in 2010 and the number is increasing in ized by high blood glucose levels (hyperglycemia) due to the
rural and poor populations throughout the world and is projected inability of the body’s cells to utilize glucose properly (West,
to become one of the world’s main disablers and killers within the 2000). The increased blood glucose levels in diabetes produce
next 25 years (Shaw et al., 2010). The developed countries such as superoxide anions, which generate hydroxyl radicals via Haber–
India, China, and the U.S. are presently the countries with the Weiss reaction, resulting in peroxidation of membrane lipids and
leading number of diabetics. Furthermore, seven percent of the protein glycation. This leads to oxidative damage of cell mem-
residents of the United States are diabetic. Though it is a non- branes. These radicals further damage other important biomole-
communicable disease, but is considered to be one of the five cules including carbohydrates, proteins and deoxyribonucleic acid
leading causes of death world-wide (Chakraborty and Das, 2010). (DNA) (Baynes, 1991).
In diabetes, oxidative stress has been found mainly due to an
increased production of oxygen free radicals and a sharp reduc-
n
Corresponding author. Tel.: þ91 8059930159. tion of antioxidant defenses (Oberley, 1988). The endogenous
E-mail addresses: parminder.nain26@gmail.com (P. Nain),
vipinsaini31@rediffmail.com (V. Saini), sunilsgju@gmail.com (S. Sharma),
antioxidant enzymes (e.g. SOD, CAT, GSH and GPx) are respon-
preetisidana@gmail.com (J. Nain). sible for the detoxification of deleterious oxygen radicals

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.04.014
66 P. Nain et al. / Journal of Ethnopharmacology 142 (2012) 65–71

(Jacob, 1995). Antioxidants thus play an important role to protect and light cycle. Animals were fed pellet diet and water ad libitum.
the human body against damage caused by reactive oxygen The experimental protocol has been approved by the institutional
species (Baynes, 1991). Hence, compounds with both hypoglyce- animal ethics committee (Protocol no. MMCP/IEC/10/11).
mic and antioxidative properties would be useful antidiabetic
agents (Baynes, 1995). 2.3. Plant Materials
Experimental diabetes mellitus has been induced in laboratory
animals by several methods. The generally effective method is to take Leaves of Emblica officinalis Gaertn. were collected in the
the pancreas out of the body. The second method for creating month of June from national park, Yamunanagar and authenti-
diabetes in animals is injecting drugs such as alloxan or Streptozo- cated from NISCAIR, (Ref. no. NISCAIR/RHMD/CONSULT/2009-
tocin (STZ). Oxidative stress is increased in experimental models of 2010/1336/138), New Delhi. Leaves were air dried, and pulverized
streptozotocin induced diabetes mellitus. The cytotoxic action of (coarse power).
streptozotocin selectively destroys b-cells of pancreas by generating
excess ROS and carbonium ion (CH3þ ) leading to DNA breaks by 2.4. Preparation of the Hydro-methanolic extract
alkylating DNA bases causing oxidative damage (Szkudelski, 2001).
Now a days, scientists and researchers are very much trying on The coarse powder (250 g) was extracted with 500 ml of
research of natural plant products all over the World and a large hydro-methanol (20:80) using a soxhlet extractor for 7 h at a
number of substantiation have shown the immense potential of temperature (64 1C) not exceeding the boiling point of the
medicinal plants used traditionally (Habib et al., 2005). Ethnobo- solvents. The extract was filtrated using whatman filter paper
tanical information indicates that more than 800 plants are used (no. 1) and then concentrated at 40 1C using a rotary evaporator.
as traditional remedies for the treatment of diabetes. The evalua- The residue (21 g) obtained was stored in freezer at 80 1C until
tion of medicinal plants used traditionally in treating diabetes is further tests.
of growing interest (Kameswara and Appa, 2001). Therefore
considerable focus has been given to an intensive search for novel 2.5. Preliminary phytochemicals studies
type of antioxidants from numerous plant materials for manage-
ment of diabetes without any side effects. The extract was subjected to various phytochemicals tests to
Emblica officinalis Gaertn. belongs to the family Euphorbiaceae. determine the active constituents present in the crude hydro-
In traditional Indian medicine, all parts of the plant including the methanolic leaves extracts of Emblica officinalis Gaertn. (Okerulu
fruit, seed, leaves, root, bark and flowers are used in various and Ani, 2001).
herbal preparations. The plant is used in many forms. One of the
most popular is as a decoction and infusion of leaves and seeds. 2.6. Acute toxicity study
Traditional use of Emblica officinalis Gaertn. leaves is against cold,
in anemia, dysentery, fever, gravel, sores (agya ghao, rokoc ghao) Acute oral toxicity study was performed as per OECD (Orga-
(Dey, 1896). Decoctions of the leaves are used in the treatment of nization of Economic Cooperation and Development) 423 guide-
diabetes mellitus (Treadway, 1994) and chemical-free bactericidal line (Acute toxic class method). Healthy male albino wistar rats
mouthwash in the treatment of aphthae (Nadkarni and Nadkarni, were randomly divided into 6 groups with 6 animals in each
1999). An infusion of the leaves with fenugreek seed is given for group. The animals were kept fasting overnight providing only
chronic diarrhea (Jayaweera, 1980). Green fresh leaves combined water, after which the hydro-methanolic extract of Emblica
with curds have carminative and stomachic effect (Nadkarni and officinalis Gaertn. leaves was administered orally with increasing
Nadkarni, 1999). Leaves have been used for anti-inflammatory and doses (10, 50, 100, 500, 1000 and 2000 mg/kg) by intragastric
antipyretic treatments (Burkill, 1966). The milky juice of the leaves tube to determine the safe doses by up and down staircase
is a good application to sores (Treadway, 1994). Infusion of the method (Ghosh, 1984). The animals were observed continuously
leaves is applied to sore eyes (Drury, 1873). Emblica officinalis for 1 h, then frequently for 4 h and later at the end of 24 h for
Gaertn. leaves are rich in tannins, amlaic acid, astragalin, ellagic general behavioral, neurological and autonomic profile. Further,
acid, gallo-tannin, kaempferol, kaempferol-3-o-glucoside, phyllan- one group was administered high dose of Emblica officinalis
tidine, phyllantine and rutin (Thakur et al., 1989; Duke, 1992). Gaertn. leaves extract orally once daily for 15 days and observed
Despite a long traditional use of Emblica officinalis Gaertn. leaves in for any lethality and death (Turner, 1965).
diabetes, no systematic phytochemical and pharmacological work has
been carried out on this potential plant. So the aim of the present 2.7. Induction of experimental diabetes
investigation is to find out Emblica officinalis Gaertn leaves as an
alternative natural remedy for the treatment of diabetes. The animals were fasted overnight and diabetes was induced
by a single intraperitoneal injection of a freshly prepared solution
of streptozotocin (50 mg/kg b.w.) in 0.1 M citrate buffer (pH 4.5)
2. Material and methods (Hemalatha et al., 2004). The animals were allowed to drink 5%
glucose solution to overcome the drug induced hypoglycemia
2.1. Chemical used (Balasubramaian et al., 2004). On the third day of STZ-injection,
the rats were fasted for 6 h and blood was withdrawn by retro
Streptozotocin was purchased from Sigma-Aldrich, St. Louis, orbital puncture under light ether anesthesia. Rats with moderate
USA. Glibenclamide (standard drug) was a gift sample from diabetes having hyperglycemia (that is, with blood glucose of
Wokhardt Pvt. Ltd. All other commercial reagents used were of 250–400 mg/dl) were taken for the experiment (Burcelin et al.,
analytical grade. 1995).

2.2. Animals 2.8. Animal grouping

Male albino wistar rats aged 7–8 weeks (200–250 g) were used. In the experiment, a total of 42 rats (36 diabetic surviving rats
Animals were kept in animal house at an ambient temperature of and six normal rats) were used. The rats were divided into seven
25–30 1C and 45–55% relative humidity with a 12 h each of dark groups of six rats each: group-I, normal control (untreated) rats;
 P. Nain et al. / Journal of Ethnopharmacology 142 (2012) 65–71 67

group-II, diabetic control rats; group-III, diabetic rats given glib- 2.13. Statistical analysis
enclamide (1 mg/kg body weight); group-IV, diabetic rats given
Emblica officinalis Gaertn. leaves extract (100 mg/kg body weight), All the grouped data was statistically evaluated. Hypothesis
group-V, diabetic rats given Emblica officinalis Gaertn. leaves testing method included one-way analysis of various (ANOVA)
extract (200 mg/kg body weight); group-VI, diabetic rats given followed by Dunnette’s comparison tests. P-values of less than
Emblica officinalis Gaertn. leaves extract (300 mg/kg body weight); 0.05 were considered to indicate statistical significance. All the
group-VII, diabetic rats given Emblica officinalis Gaertn. leaves results were expressed as mean 7SEM for 6 animals in each
extract (400 mg/kg body weight). The experimentation was car- group.
ried out for 45 days, with oral administration of Emblica officinalis
Gaertn. leaves extract.
3. Results
2.9. Biochemical analysis
3.1. Preliminary phytochemistry of the plant extract
Rats were fasted overnight and the blood was withdrawn by
retro orbital puncture under light ether anesthesia on the 1st, Preliminary phytochemical analysis revealed that the plant
22nd and 45th day post induction to determine blood glucose and possessed phytoconstituents tannins, phenolic compounds, and
plasma insulin level. The change in body weight was observed flavonoids.
throughout treatment period in the experimental animals.
At the end of 45 days, the animals were deprived of food
overnight and sacrificed by cervical decapitation for biochemical 3.2. Acute toxicity studies
parameters (i.e. hemoglobin, glycosylated Hb, total protein, serum
creatinine, serum urea, SGOT, SGPT, alkaline phosphate and lipid Animals showed good tolerance to testing single doses of
profile) and antioxidant enzyme (SOD, CAT, GSH, GPx, LPO) hydro-methanolic extract of Emblica officinalis Gaertn. leaves in
estimation. Blood was collected from the heart in two different doses as high as 2 g/kg that were found to be non-lethal. Highest
tubes, i.e. one with anticoagulant for plasma, and another without dose of extract did not show any noticeable signs of toxicity and
anticoagulant for serum separation. Serum was separated by mortality after once daily administration orally for 15 days. So,
centrifugation 3500  g at 25 1C for 10 min. Fasting blood glucose the extract is safe for long term administration.
was estimated by O-toluidine method (Sasaki et al., 1972). Plasma
insulin level was assayed by the radio-immunoassay method. All 3.3. Effect on body weight
other biochemical tests were carried out in our lab by using
commercial kits obtained from Erba diagnostic Mannheim Gmbh, Decrease in body weight due to derangement of metabolic
Germany. pathways is a common feature in diabetes (Al-Shamaony et al.,
1994). But in the present study hydro-methanolic extract of
2.10. Oral glucose tolerance test Emblica officinalis Gaertn. leaves, to diabetic rats (Groups V and
VII) increased body weight significantly (p o0.05) which was
The rats were divided into four groups of 6 animals (n¼ 6) each. comparable to increase in the body weight of normal controls
Group I served as control and received distilled water. Group II served (Fig. 1).
as diabetic control and received distilled water. Group III served as
positive control, received glibenclamide (1 mg/kg b.w.). Group IV
received HMELEO 400 gm/kg orally. The rats were fasted for 18 h and 3.4. Effect on blood glucose and plasma insulin level
the test performed by oral administration of glucose (2 g/kg) to
diabetic and normal rats 30 min after dosing. Blood samples were Fasting blood glucose levels in the normal controls rats remained
collected from the retro-orbital plexus of the eye (under light unchanged during the course of the experiment. In diabetic groups,
ether anesthesia) immediately (0 h), 30, 60, 90, and 120 min after level of fasting blood glucose was significantly (po0.05) higher and
the glucose administration and the blood glucose levels were the plasma insulin level was significantly decreased as compared to
estimated. normal control group. On the other hand, administration of hydro-
methanolic leaves extract of Emblica officinalis Gaertn. for 45 days was
2.11. Preparation of liver and kidney homogenate found to lower the blood glucose and increase the insulin level

290
The liver and kidney were carefully removed, weighed and 1st day 30th day 45th day
washed in ice-cold saline to remove the blood. Then both were 270
sliced separately into pieces and homogenized with buffer con-
250
Body Weight(gms)

taining 0.25 M sucrose and 0.1 M TrisHCl buffer (pH 7.4). The
homogenate was centrifuged at 3000  g for 10 min at 0 1C in cold 230
centrifuge. The supernatant was separated and used for various 210
antioxidant enzyme estimations.
190

2.12. Assay of antioxidant enzyme 170

150
The levels of lipid peroxidation (LPO) in tissues were estimated
by the method of Okhawa et al. (1979). Superoxide dismutase 130
Group-VII
Group-IV

Group-VI
Group-V
Group-III
Group-II
Group-I

(SOD) was assayed by the method of Kakkar et al. (1984). The

activity of catalase (CAT) was determined by the method of
Sinha (1972). Glutathione peroxidase (GPx) was estimated by
the method of Rotruck et al. (1973). Reduced glutathione (GSH) Fig. 1. Effect of hydro-methanolic extract of leaves of Emblica officinalis Gaertn.
was estimated by the method of Ellman (1959). on body weight.
68 P. Nain et al. / Journal of Ethnopharmacology 142 (2012) 65–71

Table 1
Effect of HMELEO on blood sugar and plasma insulin level in streptozotocin induced diabetic rats.

Groups Blood sugar (mg/dL) Plasma insulin (mU/ml)

Initial On 22nd day On 45th day Initialn On 22nd day On 45th day

Groups-I 71.277 3.0 73.61 7 2.5 74.43 7 2.6 15.33 70.81 16.077 0.88 15.907 0.56
Groups-II 298.67 2.28 314.83 7 5.7 340.21 7 7.3 06.07 70.78 04.977 0.93 03.727 0.73
Groups-III 282.617 2.7 167.14 7 3.4n 79.33 7 2.5n 05.87 70.83 10.457 0.89n 14.707 0.81n
Groups-IV 283.217 3.9 249.27 7 4.3 202.25 7 5.7n 06.33 70.95 07.117 1.94 07.937 0.81n
Groups-V 290.617 3.2 224.31 7 4.8n 163.71 7 4.0n 07.06 71.15 08.877 2.09na 09.047 1.71n
Groups-VI 287.677 2.9 207.23 7 4.3na 137.42 7 3.1na 07.52 71.12 10.347 1.56na 11.787 1.30na
Groups-VII 279.177 2.6 179.21 7 4.1na 103.56 7 3.4na 06.87 71.17 09.717 0.90a 12.897 1.10na

Data are expressed as mean 7SEM, n¼ 6, initial means 3rd day (after 72 h) of stz injection.
n
p o 0.05 when experimental groups were compared with diabetic control.
a
p o 0.05 when experimental groups were compared with glibenclamide treated group.

Table 2
Effect of HMELEO on biochemical parameter in streptozotocin induced diabetic rats.

Parameter/Groups Groups-I Groups-II Groups-III Groups-IV Groups-V Groups-VI Groups-VII

Hemoglobin (mg/dL) 13.43 72.1 8.387 1.2 11.92 7 2.3 09.567 1.1n 10.197 1.9na 11.977 1.7na 12.467 1.6na
Glycosylated Hb (Hb%) 6.85 70.93 14.45 7 1.1 7.217 0.87 12.707 0.9 11.157 0.90n 9.707 0.83na 8.347 0.89na
Serum creatinine (mg/dL) 0.92 70.1 2.107 0.3 0.997 0.3n 1.727 0.2n 1.257 0.4n 1.107 0.3na 0.997 0.4na
Serum urea (mg/dL) 32.50 72.1 84.93 7 2.0 36.97 7 1.5n 78.307 2.3 67.087 2.1n 56.337 1.7na 42.157 2.5na
Total protein (g/dL) 7.6 71.3 5.17 1.4 7.27 1.5n 6.07 1.1n 6.57 1.3na 6.97 1.0na 7.47 1.5na
Alkaline phosphate (IU/L) 119.4 76.3 291.6 7 5.7 128.7 7 6.1n 242.37 6.4 191.27 5.8n 169.97 5.9na 135.67 7.0na
SGOT (IU/L) 19.14 73.0 45.27 7 3.3 20.29 7 3.9n 42.637 3.8 34.567 3.5n 27.787 4.2na 21.327 3.9na
SGPT (IU/L) 25.21 74.9 57.63 7 4.8 28.07 7 5.3n 51.487 5.7 42.347 5.1n 33.877 5.0na 26.347 5.2na
Total cholesterol (mg/dL) 128.9 75.7 259.8 7 6.1 154.4 7 6.6n 242.87 5.9 203.07 7 6.3n 170.37 4.4na 142.77 4.9na
Triglycerides (mg/dL) 81.1 72.9 179.7 7 3.2 90.37 2.5n 163.17 2.8 133.67 3.3n 112.47 3.5na 97.347 3.4na
HDL (mg/dL) 51.30 73.4 17.61 7 3.2 48.35 7 2.9n 22.727 3.1 30.547 3.2n 39.437 3.3na 48.817 4.1na

Data are expressed as mean 7SEM, n¼ 6.

n
p o 0.05 when experimental groups were compared with diabetic control.
a
p o 0.05 when experimental groups were compared with glibenclamide treated group.

Table 3
Effect of hydro-methanolic extract of Emblica officinalis Gaertn leaves on OGTT in stz-induced diabetic rats.

Group treatment (n¼6) Fasting blood glucose level (mg/dL) at (h)

0h 0.5 h 1h 1.5 h 2h

Group-I 97.25 7 0.64 125.30 7 1.67 134.597 1.42 142.67 71.78 150.31 7 1.59
Group-II 250.36 7 1.47 270.63 7 0.95 282.477 1.36 296.62 71.10 309.56 7 1.34
Group-III 253.20 7 1.29 267.837 1.81n 276.177 1.01n 289.31 71.65n 268.617 1.38n
Group-IV 255.477 1.64 266.987 1.37n 279.377 1.49n 298.76 71.37n 270.17 7 1.80n

n
p o 0.05, values are mean 7 SEM, n ¼6.

significantly (po0.05) in a dose dependent manner when compared 3.6. Effect of hydro-methanolic extract of Emblica officinalis Gaertn
with positive control groups (Table 1). leaves on oral glucose tolerance test (OGTT)

3.5. Effect on biochemical parameter The results from the study clearly indicated that the hydro-
methanolic extract of emblica officinalis Gaertn leaves (400 mg/kg)
In order to examine the effect of Emblica officinalis Gaertn. and glibenclamide (1 mg/kg) reduced the blood glucose level
supplementation on the regulation of biochemical parameters i.e. (hyperglycemia due to glucose load 2 g/kg p.o.) significantly
SGOT, SGPT and alkaline phosphate of the diabetic rats were to (p o0.05) after 120 min of oral administration, when compared
evaluate the hepatic function, while creatinine and urea concen- to diabetic control (Table 3).
tration were studied to assess the renal function. STZ induced
significant (po0.05) elevations in SGOT, SGPT, alkaline phos- 3.7. Antioxidant status in liver and kidney tissue
phate, serum creatinine and serum urea level when compared to
control group. However, administration of hydro-methanolic Oxidative stress assessment was performed by recording the
extract of Emblica officinalis Gaertn. leaves for 45 days signifi- activities of anti-oxidative enzymes i.e. catalase (CAT), Glutathione
cantly (p o0.05) reduced SGOT, SGPT and ALP level in diabetic peroxidase (GPx), superoxide dismutase (SOD), reduced glutathione
groups at dose dependant manner. On the other hand, treatment (GSH) and lipid peroxidation. The diabetic rats showed significant
with different doses of leaves extract of Emblica officinalis Gaertn. (po0.05) increase in TBARS along with significantly decreased level
significantly (po0.05) reduced serum creatinine and serum urea of antioxidant enzymes i.e. CAT, GPx, GSH and SOD in hepatic and
level and increase total protein when compared to those of renal tissues. Treatment with HMELEO significantly (po0.05)
diabetic groups (Table 2). increased CAT, GPx, GSH and SOD in hepatic and renal tissues in
 P. Nain et al. / Journal of Ethnopharmacology 142 (2012) 65–71 69

Table 4
Effect of HMELEO treatment for 45 days on superoxide dismutase, catalase, glutathione peroxidase, glutathione and lipid peroxidation in liver of control and experimental
groups of rats.

Groups Liver

SOD (units/mg protein) CAT (mmol/min/mg protein) GPx (mmol/min/mg protein) GSH (mM/100 g tissue) TBARS (mmol/100 g tissue)

Group-I 8.317 1.29 86.43 72.27 10.32 7 0.91 52.11 7 1.70 0.977 0.27
Group-II 3.777 0.17a 30.29 72.09a 5.44 7 0.89a 23.37 7 2.10a 2.037 0.19a
Group-III 6.237 0.25n 78.32 73.04n 10.01 7 1.24n 47.23 7 1.81n 0.907 0.22n
Group-IV 4.737 0.19 43.24 72.21n 07.03 7 1.02n 33.27 7 2.09 1.86 7 0.57
Group-V 5.807 0.20n 57.67 73.49n 08.50 7 1.05n 39.89 7 2.11n 1.507 0.51n
Group-VI 6.417 0.18n 69.14 73.05n 09.70 7 1.04n 45.36 7 2.83n 1.17 7 0.50n
Group-VII 7.727 0.21n 83.73 73.43n 10.50 7 1.06n 48.71 7 2.70n 0.917 0.32n

Values are given as mean 7 SEM, n ¼6.

a
p o 0.05 when diabetic control group were compared with normal control group.
n
p o 0.05 when experimental groups were compared with diabetic control.

Table 5
Effect of HMELEO treatment for 45 days on superoxide dismutase, catalase, glutathione peroxidase, glutathione and lipid peroxidation in kidney of control and
experimental groups of rats.

Groups Kidney

SOD (units/mg protein) CAT (mmol/min/mg protein) GPx (mmol/min/mg protein) GSH (mM/100 g tissue) TBARS (mmol/100 g tissue)

Group-I 14.71 70.31 72.29 72.9 13.06 7 1.02 36.71 7 1.58 1.52 7 0.21
Group-II 08.51 70.22a 51.73 73.0a 7.21 7 0.99a 27.29 7 1.61a 2.407 0.27a
Group-III 13.15 70.36n 71.14 72.7n 11.47 7 0.86n 33.41 7 1.93n 1.63 7 0.31n
Group-IV 09.99 70.51 58.09 72.8n 8.93 7 1.10 30.90 7 1.30 2.077 0.52
Group-V 11.09 70.67n 64.75 73.67n 10.52 7 1.54n 32.79 7 1.43n 1.81 7 0.63n
Group-VI 12.17 70.72n 69.34 73.26n 11.48 7 1.10n 33.70 7 1.38n 1.58 7 0.79n
Group-VII 13.10 70.50n 73.39 73.53n 12.007 1.31n 34.42 7 1.17n 1.43 7 0.92n

Values are given as mean 7 SEM, n ¼6.

a
p o 0.05 when diabetic control group were compared with normal control group.
n
p o 0.05 when experimental groups were compared with diabetic control.

diabetic rats. The increased level of TBARS was found to be reverted cells of pancreas resulting in increased secretion of large amount
back to near normal status after treatment of HMELEO (Tables 4 and of insulin which in turn brings down blood glucose level (Andrew,
5). The HMELEO was found to possess antioxidant effect in a dose 2000). From the results it is assumed that the leaves extract of
dependent manner. Emblica officinalis Gaertn. could be responsible for stimulation of
insulin and the observed restoration of metabolic activity.
Diabetes is associated with weight loss. The reversal of weight
4. Discussion loss in extract-treated diabetic group indicates that the restora-
tive effect of HMELEO may be by the reversal of gluconeogenesis
Medicinal plants are widely used by the population of devel- and glycogenolysis (Griesmacher et al., 1995). The decreased level
oping countries as alternative therapy. In India, hundreds of of total hemoglobin in diabetic rats is mainly due to the increased
plants are used traditionally for the management of diabetes formation of HbA1c. During diabetes mellitus, the excess glucose
mellitus. Unfortunately only a few of such Indian medicinal plants present in the blood reacts with hemoglobin to form HbA1c
have received scientific scrutiny. The present study was therefore (Koening et al., 1976). The amount of HbA1c increase is directly
designed to study the antioxidant potential and hypoglycemic proportional to the fasting blood glucose level (Al-yassin and
effect of Emblica officinalis Gaertn. leaves extract against strepto- Ibrahim, 1981). Administration of Emblica officinalis Gaertn. leaves
zotocin induced diabetic rats. The continuous treatment of HME- extract to diabetic rats reduced the glycosylation of hemoglobin
LEO for a period of 45 days produced a significant (p o0.05) by virtue of its normoglycaemic activity and thus increase the
decrease in blood glucose level in diabetic rats which is compar- levels of hemoglobin in diabetic rats. The concentrations of lipids,
able to that of standard and diabetic control group. such as cholesterol, triglycerides (TG) and HDL-C, were signifi-
An increase in blood glucose seen in the oral glucose tolerance cantly higher in diabetic rats than in the control group. A variety
test (OGTT) was significantly greater in the diabetic rats than in of derangements in metabolic and regulatory mechanisms, due to
the non-diabetic rats. The level of plasma insulin was increased insulin deficiency, are responsible for the observed accumulation
by glucose tolerance test in the non-diabetic rats, while it was not of lipids (Rajalingam et al., 1993).
changed in diabetic rats. Oral administration of HMELEO 400 mg/ The elevation of biomarker enzymes such as SGOT, SGPT, and
kg significantly improved the impaired glucose tolerance in the ALP was observed in diabetic control rats and indicates the hepa-
diabetic rats with change in plasma insulin level. Considering the tocellular damage (Jaeschkle et al., 2002). The present study also
above result, the hypoglycemic effect of the plant may involve its shows that injection of STZ induces hepatic damage that elevates
insulin-like action i.e., acting at peripheral level to increase intracellular enzymes, such as transaminases and alkaline phos-
cellular glucose uptake or increase glycogenesis. A number of phate. The diabetic complications such as increased gluconeogenesis
plants have been shown to exert hypoglycemic activity through and ketogenesis may be due to elevated transaminase activity
stimulation of insulin release (Prince and Menon, 2000) like (Ghosh and Suryawansi, 2001). The hepatic damage was restored
glibenclamide that is reported to enhance the activity of beta hepatocytes and the elevated transaminases were significantly
70 P. Nain et al. / Journal of Ethnopharmacology 142 (2012) 65–71

reduced by HMELEO extract. From this point of view Emblica result from radical-induced inactivation and glycation of the
officinalis Gaertn. leaves extracts may act as hepatoprotective agent. enzyme (Hodgson and Fridovich, 1975). Administration of Emblica
The diabetic hyperglycemia induces elevation of the serum level of officinalis Gaertn. leaves extract and glibenclamide increases the
urea and creatinine which was considered as significant markers of activities of GPx in the tissue of diabetic rats.
renal dysfunction (Almdal and Vilstrup, 1988). An increase in serum The recovery of antioxidant enzymes with HMELEO in treated
level of urea and creatinine levels in STZ-diabetic rats may indicate group has been supported here by the diminution in the level of
diminished ability of the kidney to filter these waste products from end products of lipid peroxidation as this is the good sensor for
the blood and excrete them in the urine. On the other hand, the the assessment of oxidative stress. The fruits of Emblica officinalis
results indicates that treatment of diabetic group with Emblica Gaertn. show antioxidant and antidiabetic activity (Sabu and
officinalis Gaertn. leaves extract significantly reduced serum urea Kuttan, 2002) due to the presence of polyphenols like ellagic
and creatinine level. Based on these findings, the extract of leaves of acid, gallic acid and ascorbic acid (Nampoothiri et al., 2011),
this plant may enhance the ability of the kidney to remove these tannins like emblicanin-A and emblicanin-B (Ghosal et al., 1996)
waste products from the blood, as indicated by a protective effect on and due to tannoids like kaempferol and quercetin (Bhattacharya
the kidney of diabetic rats. et al., 1999). The leaves of the plant also contain ellagic acid, gallic
Diabetes is strongly co-related with oxidative stress induction. acid, ascorbic acid, tannins emblicannin A and B (Thakur et al.,
Lipid peroxidation is one of the characteristic features of diabetes 1989), and tannoids like kaempferol, kaempferol-3-o-glucoside
mellitus. Measurement of plasma thiobarbituric acid reactive and rutin (Duke, 1992). On the basis of this evidence it is possible
substances (TBARS) was used as an index of lipid peroxidation that these activities of Emblica officinalis Gaertn. leaves are due to
and it helps to assess the extent of tissue damage (Gutteridge, the presence of the above said phytoconstituents.
1995). Several studies have reported an increase in TBARS and
hydroperoxides in plasma, liver and kidney in experimental
diabetes mellitus (Venkateswaran and Pari, 2002; Ananthan 5. Conclusion
et al., 2004). The result of the present study shows that Emblica
officinalis Gaertn. leaves extract significantly (p o0.05) decreases In conclusion, the present study showed that Emblica officinalis
TBARS level and reduces the risk of tissue damage. Gaertn. leaves extract possesses potent antioxidant activity,
Oxidative stress in diabetes is coupled to a decrease in the which may be responsible for its hypoglycemic property. From
antioxidant status, which can increase the deleterious effects of preliminary phytochemical analysis it was found that the major
free radicals. The SOD and CAT are the two major scavenging chemical constituents of the HMELEO were tannins, polyphenolic
enzymes that remove free radicals. Reduced activities of these compound and flavonoids so it is possible that the presence of
antioxidant enzyme in liver, kidney and pancreas tissues have tannins or flavonoids may be responsible for the observed anti-
been observed in diabetic rats and this activity may result in a diabetic activity and reduce oxidative stress. Further pharmaco-
number of deleterious effects due to accumulation of superoxide logical and biochemical investigations are underway to find out
anion (O) and hydrogen peroxide (H2O2), which in turn generate the active constituents responsible for antidiabetic activity and to
hydroxyl radicals (OH), resulting in initiation and propagation of elucidate its mechanism of action.
LPO. SOD protects from oxygen free radicals by catalyzing the
removal of superoxide radical, which damage the membrane and
biological structures. Catalase was shown to be responsible for Acknowledgment
the detoxification of H2O2, and protects the tissues from highly
reactive hydroxyl radicals (Mahboob et al., 2005). This decrease in We hereby acknowledge the Maharishi Markandeshwar Uni-
CAT activity could result from inactivation by glycation of enzyme versity for financial support.
(Yan and Harding, 1997). In the present study, extract treated
groups showed a significant increase in the hepatic and renal SOD
and CAT activities of the diabetic rats. This means that the References
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