Journal of Ethnopharmacology 145 (2013) 363372

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Journal of Ethnopharmacology

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Anti-diabetic and anti-cataract effects of Chromolaena odorata Linn., in streptozotocin-

induced diabetic rats
M. Onkaramurthy, V.P. Veerapur n, B.S. Thippeswamy, T.N. Madhusudana Reddy,
Hunasagi Rayappa, S. Badami
Sree Siddaganga College of Pharmacy, Tumkur, Karnataka 572 102, India
article info abstract

Article history:
Received 1
May 2012
Received in
revised form 19
October 2012
Accepted 14
November 2012
Available online 23
November 2012
Keywords:
Chromolaen
a odorata
Streptoz
otocin
Diabetes
Cataract
Homeostatic model
assessment Insulin
tolerance test
Glucose uptake
Ethnopharmacological relevance: Chromolaena odorata Linn., is used in traditional Indian medicine in the
treatment of diabetes and eye problems.
Aim of the study: The present study was designed to investigate the effect of the ethanol extract
Chromolaena odorata leaves (ACO) in streptozotocin (STZ; 45 mg/kg, i.v) induced diabetes and cataract in
rats.

Materials and methods: Different doses of ACO (200 and 400 mg/kg) was administered once daily for eight
weeks to STZ-induced diabetic rats. To know the mechanism of action of title plant, AUC glucose, AUCinsulin,
Homeostatic Model Assessment (HOMA), insulin tolerance test (ITT) and glucose uptake by rat hemi-
diaphragms were carried out. Further, cataract score was taken once in a week upto eight weeks and opacity
index was measured. HPLC fingerprinting profiling of ACO was also carried out.
Results: Administration of ACO exhibited significant reduction in glucose, HOMA, lipid profiles and
significantly improved glucose and insulin tolerance, glycogen content, glucose uptake by skeletal muscle,
serum insulin and HDL-c levels. In addition, ACO also decreased oxidative stress by improving endogenous
antioxidants. Further, treatment of ACO showed significantly reduced onset and extent of cataract.

Conclusion: The present data suggested that the treatment of ACO reversed the STZ-induced diabetes and
cataract in rats. The observed beneficial effects may be mediated by interacting with multiple targets
operating in diabetes mellitus and its complication. Taken together, this study provided the scientific
evidence for the traditional use of Chromolaena odorata.

& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
 Diabetes mellitus is a heterogeneous metabolic disorder char-acterized by high levels of blood glucose
with disturbances of carbohydrate, lipid and protein metabolism resulting from defects in insulin secretion,
insulin action or both. Insulin deficiency and/ or insulin resistance is associated with the pathogenesis of
diabetic dyslipidemia and micro/macrovascular complications (Akpan et al., 2007). Diabetes mellitus is
possibly the worlds

largest growing metabolic disorder, and as the knowledge on the heterogeneity of this disorder is advanced,
the need for more appropriate therapy increases (Baily and Flatt, 1986). In spite of
the availability of various antihyperglycemic agents, diabetes and its secondary complications continue to
be a major problem in the world population. Medicinal plants and their bioactive constitu-ents are used for
the treatment of diabetes throughout the world

n
Corresponding author. Tel.: 91 816 227 3331; fax: 91 816 2252 792. E-mail address:
veeresh36@gmail.com (V.P. Veerapur).
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.11.023
and popularized as neutraceutical. Many indigenous medicinal plants have been found to be useful to
successfully manage diabetes (Subramoniam et al., 1996; Mukherjee et al., 1997). In addition,
many of the currently available drugs have been derived directly or indirectly from plant source. Even the
discovery of the widely used hypoglycemic drug metformin came from the traditional approach of using
Galega officinalis (Akpan et al., 2007).
The commonly encountered acute and late diabetic complica-tions are already responsible for major
causes of morbidity, disability and premature deaths in Asian countries. The underlying causes attributed to
hyperglycemia ultimately result in oxidative stress, alterations in enzyme activities, protein glycosylation
and several structural changes (Akpan et al., 2007). Prolonged exposure
to uncontrolled chronic hyperglycemia in diabetes can lead to various complications in the eye including
cataract and retinopathy. Cataract, characterized by cloudiness or opacification of the eye lens, is the leading
cause of blindness all over the world. In view of the widespread prevalence of diabetes in developing
countries like India, diabetic cataract may impact in the management of blindness (Suryanarayana et al.,
2007). At present, the cure for cataract is still
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Ethnopharmacology 145 (2013) 363372
surgery. However, surgery is not equally available to all, and where it is available, it does not produce equal
outcomes (Zhang et al., 2008).

Chromolaena odorata Linn. was commonly called as Siam weed or Christmas bush, a herb usually grow
as invasive weed or plant traditionally used in wound dressing, antispasmodic, antiprotozoal, antifungal,
antitrypanosomal, antibacterial, antihypertensive, anti-inflammatory, astringent, diuretic and hepatotropic
agent (Afolabi et al., 2007). The leaves of Chromolaena odorata has been reported to contain alkaloids,
saponins, tannins, flavonols (tamarixetin and kaemferide), flavonones (eupatilin), chalcones, phenolic acids
such as ferulic acid and protocatechuic acid (Phan et al., 2001). Tamar-ixetin and kaempferide, which are 4-
methyl ethers of quercetin and kaempferol, respectively. Quercetin and kaempferol have been known to be
potent anti-oxidant, anti-inflammatory and anti-diabetic agents. Furthermore, ferulic acid and
protocatechuic acid are known to ameliorate STZ-induced toxicity in diabetic rats (Balasubashini et al.,
2004; Harini and Pugalendi, 2010).

Chromolaena odorata Linn., is traditionally used to manage diabetes and in eye problems (Duke et al.,
1929; Pullaiah and Naidu, 2003). Due to the lack of scientific reports of these traditional claims of
Chromolaena odorata, we thought it worth-while to investigate the antidiabetic activity of ethanol extract of
title plant in STZ-induced diabetes along with prevention or delay in STZ-induced cataractogenesis in rats.
We also carried out HPLC fingerprinting analysis to know the possible chemoprofile/s.

2. Materials and methods

2.1. Chemicals

Thiobarbituric acid (TBA), 5,50-dithio-bis-2-nitrobenzoic acid (DTNB, Ellmans reagent) trichloroacetic

acid (TCA) Streptozotocin (STZ), bovine serum albumin (BSA), were procured from Himedia, Mumbai,
India. Protocatechuic acid was obtained as gift sample from Natural Remedies, Bangalore. All chemicals
used were of analytical grade and all solutions were prepared fresh.

2.2. Plant material and extraction

The leaves of Chromolaena odorata Linn., were collected around Siddapura, Bhadravathi taluk Shimoga
dist, Karnataka, in the month of June, 2010. The plant was identified and authenticated by Professor K.
Siddappa, HOD, Department of Botany, Sree Siddaganga College of Arts, Science, and Commerce for Boys
and a voucher specimen [SSCP11PC0007] of the plant was kept in the college herbarium.

The shade-dried, coarsely powdered leaves (2 kg) was extracted exhaustively with ethanol by cold
maceration for seven days with occasional replacement of solvent and the extract thus obtained was
concentrated to a small volume under vacuum using a rotary evaporator (Buchi rotavapor, Switzerland).
Then extract was eva-porated to dryness in a vacuum desiccator (J.R. Industrial Corpora-tion, Mumbai,
India) and stored in air tight container. The percentage yield of the extract (ACO) was found to be 4.4%
w/w. The dried extract (ACO) was subjected to various chemical tests to detect the different class of
phytoconstituents.

2.3. HPLC fingerprinting analysis of ACO

The HPLC finger printing profile of ACO was carried out using Shimadzu HPLC system (Kyoto, Japan)
equipped with dual pump LC-20AD binary system, photodiode array (PDA) detector SPD-M20A, Merck
RP C18 column (I.D. 4.6 250 mm, 5 mm). ACO (1 mg/ml) was dissolved in methanol were injected in
triplicate in to HPLC. Gradient
elution was performed using water containing 0.1% formic acid and acetonitrile at a total flow rate and
injection volume were 1.0 ml/min and 20 ml, respectively. Initially, elution was started with a gradient of
10% acetonitrile changing to 70% in 25 min and finally to 10% in 35 min. The chromatograms at 254 nm
were analyzed and compared.

2.4. Animals

Wistar rats of either sex weighing 150250 g were used for the study. The animals were obtained from the
inbred animal colony of central animal house, Sree Siddaganga College of Pharmacy, Tumkur. The animals
were maintained under controlled condi-tions of temperature (2372 1C), humidity (5075%) and 12-h light
dark cycles. The animals were randomized into experimen-tal and control groups and housed three in
sanitized polypropy-lene cages containing sterile paddy husk as bedding. They had free assessed to standard
pellets as basal diet and water ad libitum. Animals were habituated to laboratory conditions for 48 h prior to
experimental protocol to minimize if any of non-specific stress. All the studies conducted were approved by
the Institutional Animal Ethical Committee (IAEC) of Sree Sidd-ganga College of Pharmacy, Tumkur,
Karnataka (SSCPT/IAEC.clear/ 80/0910). According to prescribed guidelines of Committee for the
Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

2.5. Determination of acute toxicity

Healthy female Wistar rats, starved overnight (12 h), were divided into six groups of two each and were
orally fed with increasing doses (10, 30, 100, 300, 1000 and 2000 mg/kg) of ACO were studied to determine
the safe doses by up and down staircase method. The animals were observed continuously for 1 h, then
frequently for 4 h and later at the end of 24 h. After administration of the extracts, Irwin test was conducted,
where the animals were observed for behavioral changes. Further, animals were observed daily for 30 days,
and mortality was recorded (Ghosh, 1984). To know the multiple dose toxicity of ACO (200 and 400
mg/kg), normal rats were fed once daily for 15 days and observed for incidences of mortality for a period of
30 days.

2.6. Effect of ACO in standardized STZ-induced diabetic rat model

2.6.1. Induction of diabetes mellitus

Diabetic condition was induced in Wistar rats by single intravenous injection of STZ (45 mg/kg) [In
house standardized optimum dose] after overnight fasting for 12 h. Rats showing SG level 4300 mg/dl
seven days after STZ administration were considered diabetic and included in the study (Habbu et al.,
2010). Diabetic rats were randomized into different groups based on their SG levels.

2.6.2. Experimental design single-dose one-day study

The experimental rats were divided into five groups of six rats each treated as Group 1: normal control
(NC) received 1% CMC; Group 2: diabetic control (DC) received 1% CMC; Group 3: DC rats treated with
ACO (200 mg/kg, p.o.); Group 4: DC rats treated with ACO (400 mg/kg, p.o.); Group 5: DC rats treated
with glibencla-mide [GLB] (10 mg/kg, p.o.)

Blood samples were collected at 0, 2, 4 and 6 h after extract/ GLB administration. SG was estimated by
the enzymatic glucose oxidase method. Percentage reduction in glycemia was calculated with respect to the
initial (0 h) level according to: Percentage
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reduction in glycemia[(Gi Gt)XGi] 100; Where Gi is initial glycemia and Gt is glycemia at 2, 4, and 6 h
(Veerapur et al., 2010).

2.6.3. Experimental design for multiple-dose eight weeks study

The above groups of animals were further treated with respective doses of ACO and GLB for eight
consecutive weeks in order to evaluate the chronic effect of extract/GLB treatment on hyperglycemia.
Whereas, GLB (0.5 mg/kg, p.o./day) was adminis-tered for 8th weeks. Percentage reduction in glycemia
was calculated with respect to the initial (0 day) level according to percentage reduction in glycemia[(G i
Gt)XGi] 100; Where Gi is initial glycemia and Gt is the glycemia value at 1, 2, 3, 4, 5, 6, 7 and 8th week
(Veerapur et al., 2010).

2.6.4. Oral glucose tolerance test (OGTT)

On 5th week, glucose tolerance of various groups was estimated by a simple OGTT. Glucose (2 g/kg) was
administered to 12 h fasted rats and blood samples were collected from the retro-orbital plexus at 0 (before
glucose load), 30, 60 and 120 min after glucose administration. SG was estimated by the enzymatic glucose
oxidase method. The results were expressed as integrated area under curve for glucose (AUC glucose), which
was calculated by trapezoid rule. Also serum insulin was estimated 0 (before glucose load), 30 and 60 min
after glucose administration. Serum insulin (SI) was esti-mated by radioimmunoassay method using the kit
from CIS BIO INTERNATIONAL, France. The results were expressed as integrated area under curve for
insulin (AUCinsulin), which was calculated by trapezoid rule (Vishwakarma et al., 2003).

2.6.5. Insulin tolerance test (ITT)

Insulin tolerance test is a measure of the extent of peripheral utilization of glucose. On 7th week, insulin
(2 IU/kg, i.p.) was administered to six h-fasted rats. Blood samples were collected from the retro-orbital
plexus at 0 (just before insulin load), 15, 30 and 45 min after insulin injection. SG was estimated by the
enzymatic glucose oxidase method (Nair et al., 2008). The results were expressed as integrated area under
curve for glucose (AUCglucose), which was calculated by trapezoid rule (Vishwakarma et al., 2003).

2.6.6. Cataract study

Cataract study was simultaneously carried out with diabetic study in above mentioned model. The
intensity of cataract was scored once in a week for eight consecutive weeks. The cataract scores of ACO
treated groups were compared with normal group and diabetic control group. The cataract scores were
determined using slit lamp microscope. Progression and maturation of lenti-cular opacity was graded into
five stages as Stage 0clear lenses and no vacuoles present; Stage 1vacuoles cover approximately one-
half of the surface of the anterior pole forming a sub capsular cataract; Stage2some vacuoles have
disappeared and the cortex exhibits a hazy opacity; Stage 3a hazy cortex remains and dense nuclear
opacity is present; Stage 4a mature cataract is observed as a dense opacity in both cortex and nucleus
(Suryanarayana et al., 2007). Opacity index was calculated to quantitatively evaluate the degree of lens
opacity (Vats et al., 2004) by the following formula:

Opacity index Number of eyes in each stage Stage of the eye Total number of eyes

2.6.7. Estimation of biochemical parameters

At the end of the treatment schedule, blood samples were collected from retro-orbital plexus. Serum was
separated and triglyceride (STG), total cholesterol (STC) and HDL-cholesterol
(HDL-c) were analyzed by semi-autoanalyser (Qualigen, Mumbai) using diagnostic reagent kit (ERBA
diagnostics Mannheim GMBH, Germany). HOMA as a measure of insulin resistance was calcu-lated by the
following formula (Veerapur et al., 2012).

insulin mU=ml glucose mmol=l 22:5

VLDL-cholesterol (VLDL-c) and LDL-cholesterol (LDL-c) in serum were calculated as per Friedewalds
equation (Friedewald et al., 1972).

VLDL-c Triglyceride
5
LDL-c Total cholesterol Triglyceride HDL-c 5
The markers of dyslipidemia such as TC/HDL-c and LDL-c/HDL-c ratios were also calculated.

2.6.8. Glucose uptake by isolated hemi-diaphragm of diabetic rats

2.6.8.1. Isolation of diaphragm. After collection of blood for biochemical estimations, experimental rats
were sacrificed by cervical dislocation. The diaphragms were dissected out quickly with minimal trauma
and divided into two equal halves. The hemi-diaphragms were then rinsed in cold Tyrode solution (without
glucose) to remove any blood clots. Two diaphragms of five animals (10 diaphragms) in each group were
used for the study.

2.6.8.2. Treatment protocol. Six sets of six graduated test tubes each, were grouped as follows, Group 1: 2
ml Tyrode solution with 0.2 g% glucose (Diabetic control); Group 2: 2 ml Tyrode solution with 0.2 g%
glucoseinsulin (Nova Nordisk) 0.62 ml of 0.4 U/ml solution (insulin-treated group); Group 3: 2 ml of
Tyrode solution with 0.2 g% glucoseACO (50, mg/ml); Group 4: 2 ml of Tyrode solution with 0.2 g%
glucoseACO (100 mg/ml); Group 5: 2 ml of Tyrode solution with 0.2 g% glucoseinsulin (Nova Nordisk)
0.62 ml of 0.4 U/ml solutionACO (50 mg/ml); Group 6: 2 ml of Tyrode solution with 0.2 g%
glucoseinsulin (Nova Nordisk) 0.62 ml of 0.4 U/ml solutionACO (100 mg/ml)

The experiment was carried out as mentioned earlier (Ghosh et al., 2004) with modification. Briefly, the
hemi-diaphragms were placed in test tubes containing 1 ml of Tyrode solution and incubated for 15 min at
37 1C, bubbled with oxygen and shaking. The different concentration of extract/insulin and 0.2 g% w/v
glucose were added in respective test tubes and the volumes were made up to 4 ml with Tyrode solution.
Immediately, the initial glucose concentration was estimated by glucose-oxidase method. Then incubated at
37 1C for 30 min bubbled with oxygen. Glucose uptake per gram of tissue was calculated as the difference
between the initial and final glucose content in the incubated medium.

2.6.9. Endogenous antioxidant status

After collection of diaphragms, liver was perfused with saline; whole liver was dissected out. A 10%
homogenate of the livers of different groups were prepared with ice cold saline-EDTA and protein content
was determined. The homogenate was further subjected to the estimation of non-enzymatic (reduced
glutathione and total thiols) and enzymatic antioxidants (catalase) using standardized protocols quoted in
our previous publication. Lipid peroxidation levels of the liver homogenates were also determined
(Prabhakar et al., 2006).
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Ethnopharmacology 145 (2013) 363372
2.6.10. Liver glycogen
Glycogen is the primary short term energy storage molecule in animals. In diabetes condition, liver
glycogen is degraded and gluconeogenesis is increased while glucose utilization is inhibited. In addition,
glucose-6-phosphatase activity is increases, while hexokinase and glycogen synthase activity decreases in
the liver. As a result of this, the liver continues to produce glucose even with severe hyperglycaemia without
able to convert glucose to glyco-gen. Liver glycogen was estimated by Carrol et al. (1956). Briefly, 10%
liver homogenate was prepared with 5% TCA, centrifuged and filtered. To 1 ml of trichloroacetic acid
filtrate, 5 ml of 95% ethanol was added and thoroughly mixed. The tubes were stand overnight at room
temperature and the tubes were centrifuged at 3000 r.p.m. for 15 min. The clear liquid is gently decanted
and residue was dissolved by addition of 2 ml of distilled water. A reagent blank was prepared by pipetting 2
ml of water into a clean centrifuge tube. A standard was prepared by pipetting 2 ml of standard glucose
solution, containing 0.1 mg of glucose, into a similar tube. At this point 10 ml of anthrone reagent were
delivered into each tube with shaking and kept under running cold tap water. After all tubes have reached
room temperature, immersed in a boiling water bath for 15 min and cooled to room temperature. The
contents of each tube were read at 620 nm after adjusting the calorimeter with the reagent blank. The
content of glycogen was calculated from following equation and expressed as mg per 100 g of tissue.

D Volume of
U extract

D
S 0:1 g of tissue 100
0:9 mg of glycogen=100 g of tissue
where, DUOptical density of the unknown, DS Optical density of the standard, 0.1mg. of glucose in 2
ml. of standard solution, 0.9factor for converting glucose value to glycogen value.
2.7. Statistical analysis

The data were expressed as mean7S.E.M. Statistical compar-isons were performed by One-way ANOVA
followed by Tukeys post hoc test using GraphPad Prism Version 5.0 (San Diego, CA).

3. Results

3.1. Preliminary phytochemical investigation

Preliminary phytochemical tests of ethanol extract of Chromolaena odorata shows the presence of
alkaloids, glycosides, phytosterols, terpenoids, flavonoids, saponins and tannins.

3.2. HPLC fingerprinting analysis of ACO

The HPLC fingerprint profile standardized with marker served as a standard for comparison in the
subsequent preparation of ACO. HPLC chromatogram was found to contain constituents eluting between
2.85.0 min and 5.628.0 min with major peaks at 11.8 min. The presence of protocatechuic acid in ACO at
RT 8.68 min was confirmed by comparing its retention time and UV spectra with that of the reference
standard protocatechuic acid (Fig. 1).

3.3. Acute oral toxicity studies

 Animals showed good tolerance to single doses of ACO in doses as high as 2 g/kg and were non-lethal.
Therefore, 200 and 400 mg/kg of ACO were selected for the present study. Further, administration of both
the doses of ACO for 15 days did not produce any noticeable
Fig. 1. HPLC fingerprinting profile of ethanol extract of Chromolaena odorata [ACO] showing the presence
of Protocatechuic acid. Inset: Structure of Protocatechuic acid (3,4-dihydroxyl benzoic acid).

Fig. 2. Effect of ethanol extract of Chromolaena odorata [ACO] on serum glucose levels in STZ-induced
diabetic rats [Single-dose one-day study]. Bar graph represents the percentage reduction in glycemia with
respect to the initial (0 h) level. Each value represents mean7S.E.M. (n6). aPo0.05; bP o0.01; cPo0.001
compared to diabetic control (DC) of the same time interval (one-way ANOVA followed by Tukeys post-
test).

signs of toxicity (behavioral changes) and mortality during 30 days study period.

3.4. Effect of ACO in standardized STZ-induced diabetic rats

3.4.1. Single-dose one-day study

A single dose of ACO (200 and 400 mg/kg) showed significant reduction in SG levels at different time
intervals (2, 4, and 6 h) compared to basal levels (0 h). The maximum percent reduction in glycemia
(58.84% and 38.23%) was observed at sixth hour post treatment of ACO (400 mg/kg) and GLB,
respectively. Further-more, the treatment with ACO and GLB showed significant (Po0.01; Po0.001)
reduction in SG levels at different time intervals when compared to diabetic control (Fig. 2). These data
suggested that, ACO exhibited comparable reduction in glycemia to standard GLB.

3.4.2. Multiple-dose eight weeks study

Repeated administration of both the doses of ACO for eight weeks, showed significant (Po0.05; Po0.01;
Po0.001) reduc-tion in SG levels at all tested time intervals compared to respective basal values (0 day). On
8th weeks, both the doses of ACO and GLB showed significantly (Po0.001) greater percentage reduction in
glycemia (58.49%, 64.26% and 61.86%, respectively) compared to diabetic control (Fig. 3).
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(2013) 363372 367
Fig. 3. Effect of ethanol extract of Chromolaena odorata [ACO] on serum glucose levels in STZ-induced
diabetic rats [Multiple-dose eight-week studies]. Bar graph represents the percentage reduction in glycaemia
with respect to the initial (0 day) level. Each value represents mean7S.E.M. (n56). aP o0.05; bP o0.01, cP
o0.001 compared to diabetic control (DC) of the same time interval (one-way ANOVA followed by Tukeys
post-test).
3.4.3. Oral glucose tolerance test (OGTT)
On 5th week, intra gastric administration of glucose (2 g/kg) did not produced significant change in SG
level of normal control rats and AUC for the 120 min interval was not altered. The diabetic rats exhibited
significant elevation in fasting SG (at time zero) and showed significant impairment in glucose tolerance to
exogenously administered glucose compared to normal rats (Fig. 4a). Further, treatment of tested doses of
ACO exhibited significant (Po0.001) reduction in SG level over the period of 120 min compared to diabetic
control group (Fig. 4a).

Integrated area under the glucose curve over 120 min (AUC glucose) of diabetic group was significantly
higher (Po0.001) compared to normal control. Treatment with ACO and GLB produced a signifi-cantly
(Po0.001) decreased AUCglucose compared to diabetic control (Fig. 4b). Furthermore, estimation of AUC
values indicated that, treatment with ACO (200 and 400 mg/kg) and GLB (10 mg/kg) exhibited 47.16%,
57.38% and 57.20% decrease in SG levels, respec-tively compared to diabetic rats (Fig. 4b).

Exogenous administration of glucose (2 g/kg) stimulated the release of higher levels of insulin in normal
control rats, whereas glucose load was ineffective in stimulating the release of insulin in diabetic rats,
suggesting that these diabetic rats resembled severe diabetic (type I) condition in which a maximum
pancreatic

Fig. 4. Effect of five weeks treatment with ethanol extract of Chromolaena odorata [ACO] on oral glucose
tolerance in STZ-induced diabetic rats. [a] Serum glucose levels were measured prior to, and after p.o.
administration of glucose alone (2 g/kg body weight), or in combination with ACO or glibenclamide (GLB).
[b] Area under curve for glucose (AUC glucose) values for 0120 min post glucose load. G1: Normal control;
G2: Diabetic control (DC); G3: DCACO (200 mg/kg); G4: DC ACO (400 mg/kg); G5: DCGLB (10
mg/kg). Data represent the mean7S.E.M. (n6). cPo0.001 compared with diabetic control (one way ANOVA
followed by Tukeys post-test).
Fig. 5. Effect of five weeks treatment with ethanol extract of Chromolaena odorata [ACO] on serum insulin
(SI) levels in STZ-induced diabetic rats subjected to oral glucose tolerance test. [a] Serum insulin levels
were measured prior to, and after p.o. administration of glucose alone (2 g/kg body weight), or in
combination with ACO or glibenclamide (GLB). [b] Incremental AUC insulin values for 060 min post
glucose load. G1: Normal control; G2: diabetic control (DC); G3: DCACO (200 mg/kg); G4: DCACO
(400 mg/kg); G5: DCGLB (10 mg/kg). Data represent the mean7S.E.M. (n4). aPo0.05; c Po0.001
compared with diabetic control (one way ANOVA followed by Tukeys post-test).
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damage occurred. Whereas, treatment with different doses of ACO enhanced the glucose stimulated insulin
release from pan-creatic b-cells and this response was comparable with GLB treated diabetic rats (Fig. 5A).

Integrated area under the insulin curve over 60 min (AUC insulin) of diabetic rats was significantly lower
(Po0.001) compared to normal control (20857170.7 mU/ml/min). Further, the integrated AUCinsulin for the
treatment of ACO (200 and 400 mg/kg) and GLB (10 mg/kg) was found to be 16547139.7, 1778767.50 and
1620737.25 mU/ml/min, respectively compared to untreated dia-betic rats (1268739.92 mU/ml/min) (Fig.
5b).

3.4.4. Blood glucose, serum insulin and HOMA

On 5th week, diabetic rats exhibited significant (Po0.001) hyperglycemia (530.7710.49; SG levels rose to
between 490 to 560 mg/dl). The HOMA values (degree of insulin resistance) of diabetic rats (34.7572.68)
were significantly (Po0.001) higher than normal control rats (8.5671.41) suggested that, the diabetic rats
were under insulin resistance condition that is peripheral utilization of glucose was compromised (Table 1).

Oral administration of different doses of ACO and GLB to diabetic rats showed significant (Po0.001;
Po0.01; P o0.05) reduction in SG and HOMA values. Even though ACO treatment resulted in increase in SI
levels but effect was insignificant compared to diabetic rats (Table 1).

Table 1
Effect of five weeks treatment with ethanol extract of Chromolaena odorata [ACO] on serum glucose [SG],
serum insulin [SI] and homeostatic model assessment [HOMA] levels in STZ-induced diabetic rats.
Treatment SG SI
groups (mg/dl) (mU/ml) HOMA

Normal 99. 71.594 34.5075.1 71.41

control 80 c 8 8.56 c
Diabetic 53 26.5072.1
control 0.7 710.49 0 34.7572.68
ACO (200 28 33.7573.6
mg/kg) 0.2 78.41c 1 22.98 72.0a
ACO (400 24 34.5070.5
715.81 72.18
mg/kg) 9.2 c 0 20.56 b
GLB (0.5 25 715.46 28.5072.0 72.84
mg/kg) 2.0 c 2 17.73 c
16
F 2 1.46 17.19
4,3 4,2
df 0 4,20 0
Data represent the mean7S.E.M.
a
P o0.05. b Po0.01.
c
Po0.001 as compared with diabetic rats (one way ANOVA followed by Tukeys post-test).
3.4.5. Insulin tolerance test (ITT)
From the insulin tolerance test, it is possible to know the extent of peripheral utilization of glucose. In
contrast to estab-lished reports, at the end of 7th week, administration of insulin (2 IU/kg) to severe diabetic
rats did not exhibit a marked fall in SG levels suggested that, these diabetic rats were not able utilize the
exogenously administered insulin to reduce the glucose indicated that these animals experienced insulin
resistance. This observa-tion is consistent with HOMA values of diabetic rats. Taken together, we may
conclude that the diabetic rats were experience marginal loss of insulin sensitivity in diabetic rats, even
though these diabetic rats were in type 1 diabetic condition (as evident by lower insulin levels at different
time points after glucose challenge). Further, seven weeks treatment with ACO and GLB showed significant
(Po0.001) reduction in SG level at 0 to 45 min compared to diabetic control (Fig. 6a).

Integrated area under the glucose curve over 45 min (AUC glucose) of diabetic control was significantly
(Po0.001) higher compared to normal rats. Treatment with ACO and GLB produced a significantly
(Po0.001) decreased AUCglucose compared to diabetic control (Fig. 6b).

Further estimation of AUC values indicated that, there is 85.60% reduced level of SG in normal control
animals when compared to diabetic rats. Treatment with different doses of ACO to diabetic rats showed
62.39% and 70.11% reduction in SG levels respectively compared to diabetic control (Fig. 6b). This
observed effect of extract is comparable with GLB treated group (62%).

3.4.6. Cataract study

Staging of cataract and opacity index in various experimental groups is given in Table 2. Slit lamp
examination showed that lenses of normal control rats were in stage 0 throughout the duration of
experimental period. On the other hand, the onset of cataract was observed on 4th week in diabetic rats and
showed varying degree of catarectogenic changes (Suryanarayana et al., 2007). This is as evident by
increase in opacity index from 1.09 on the 4th week to 2.76 on the 6th week followed by maximum
opacification of 3.7 on 8th week (Fig. 7). The onset of cataractogenisis was started on 5th and 6th week,
respectively in case of ACO (200 and 400 mg/kg, respectively) and also opacity index in these groups were
much less in comparison to diabetic control group. Treatment of ACO to diabetic rats resulted in two to
three fold decrease in extent of cataract as evident by decrease in opacity index. By the end of 6th week
50% of diabetic animals were in 4th stage of cataract whereas, treatment of both the doses of ACO did not
exhibit 4th stage of

Fig. 6. Effect of seven weeks treatment with ethanol extract of Chromolaena odorata [ACO] on insulin
tolerance in STZ-induced diabetic rats. [a] Serum glucose levels were measured prior to, and after p.o.
administration of insulin alone (2 U/kg body weight), or in combination with ACO or glibenclamide (GLB).
[b] Area under curve for glucose (AUCglucose) values for 030 min post insulin injection. G1: Normal control;
G2: diabetic control (DC); G3: DCACO (200 mg/kg); G4: DC ACO (400 mg/kg); G5: DCGLB (10
mg/kg). Data represent the mean7S.E.M. (n5). cP o0.001 compared with diabetic control (one way
ANOVA followed by Tukeys post-test).
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M. Onkaramurthy et al. / Journal of Ethnopharmacology 145

(2013) 363372 369

Table 2
Effect of eight weeks treatment with ethanol extract of Chromolaena odorata [ACO] on incidence of
cataract and opacity index in STZ-induced diabetic rats.
Opacity
Groups 0 stage 1 stage 2 stage 3 stage 4 stage index
% Incidence of cataract on 4th
week
Normal control 100 0 0 0 0 0
Diabetic control
[DC] 0 91.66 8.33 0 0 1.09
DCACO (200
mg/kg) 100 0 0 0 0 0
DCACO (400
mg/kg) 100 0 0 0 0 0
% Incidence of cataract on 5th
week
Normal control 100 0 0 0 0 0
Diabetic control
[DC] 0 3.3 16.6 50 0 2.18
DCACO (200
mg/kg) 91.66 8.33 0 0 0 0.08
DCACO (400
mg/kg) 100 0 0 0 0 0
% Incidence of cataract on 6th
week
Normal control 100 0 0 0 0 0
Diabetic control
[DC] 0 20 20 10 50 2.76
DCACO (200
mg/kg) 80 10 10 0 0 0.3
DCACO (400
mg/kg) 91.33 8.33 0 0 0 0.08
% Incidence of cataract on 7th
week
Normal control 100 0 0 0 0 0
Diabetic control
[DC] 0 0 10 10 80 3.7
DCACO (200
mg/kg) 12.5 37.5 37.5 12.5 0 1.2
DCACO (400
mg/kg) 60 40 0 0 0 0.7
% Incidence of cataract on 8th
week
Normal control 100 0 0 0 0 0
Diabetic control
[DC] 0 0 0 16.66 83.33 3.7
DCACO (200
mg/kg) 0 50 37.5 12.5 0 1.2
DCACO (400
mg/kg) 40 50 10 0 0 0.7
 Fig. 7. Representative photographs showing the effect of eight weeks treatment with ethanol extract of
Chromolaena odorata [ACO] in prevention of cataract.

mature cataract by the end of 8th week reveals the preventive effect of the title plant (Table 2).

3.4.7. Estimation of lipid parameter

At the end of the study, lipid levels such as STG, STC, VLDL-c and LDL-c levels were significantly
(Po0.001; Po0.05) increased whereas HDL-c was decreased in diabetic rats compared to normal
rats (Table 3). The markers of dyslipidemia such as TC/HDL-c and LDL-c/HDL-c ratios were significantly
(Po0.001) elevated in the diabetic group. Both the dose of ACO treatment exhibited signifi-cant reduction
(Po0.001) in all tested lipid parameters and restor-ing them to near-normal values (Table 3). Whereas HDL-
c was significantly (Po0.05; Po0.01) increased with ACO treatment. GLB administration showed a
significant recovery in lipid profile in diabetic animals.
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370 M. Onkaramurthy et al. / Journal of Ethnopharmacology 145 (2013) 363372

Table 3
Effect of eight weeks treatment with ethanol extract of Chromolaena odorata [ACO] on lipid profile and
liver glycogen in STZ-induced diabetic rats.
DCACO DCACO DCGLB (0.5
Normal Diabetic
Parameters control control [DC] (200 mg/kg) (400 mg/kg) mg/kg) F df
70.2 c 137. 94.0 85.7 90.7 35.5 4,2
STG (mg/dl) 6 73.52 8 74.97 873.78c 2 73.7c 0 73.31c 3 5
63.4 c 131. 75.6 67.1 79.6 32.9 4,2
STC (mg/dl) 4 72.67 7 74.37 373.72c 474.93c 074.57c 5 5
32.1 a 13.1 33.4 34.6 31.4 4,2
HDL-c (mg/dl) 0 71.34 7 70.84 075.07a 873.76b 574.94a 4.71 5
14.0 c 27.5 18.8 17.1 18.1 35.5 4,2
VLDL-c (mg/dl) 5 70.70 7 70.99 270.75c 470.74c 470.66c 3 5
18.5 c 91.0 26.7 15.3 32.0 15.2 4,2
LDL-c (mg/dl) 9 72.9 0 74.21 177.57c 277.86c 078.28c 4 5
33.4 4,2
TC/HDL-c ratio 2.08 70.15 9.17 71.03
c
2.7670.56c 2.1270.45c 2.9870.49c 1 5
10.1 34.4 4,2
LDL-c/HDL-c ratio 0.62 70.19c 4 70.815 1.0870.43c 0.5970.36c 1.3170.39c 1 5
Liver glycogen 33.9 5.60 2.51 11.4 25.7 20.4 14.6 4,2
(mg/100 g of tissue) 0 73c 9 70.18 071.63 473.70c 073.08b 2 5
Each value represent mean7S.E.M. (n 5).
a
P
o0.05.
b

Po0.0
1.
c
Po0.001 compared to diabetic control. One way ANOVA followed by Tukeys post test.

Table 4
Effect of eight weeks treatment with ethanol extract of Chromolaena odorata [ACO] on endogenous
enzymatic and non-enzymatic antioxidant levels in STZ-induced diabetic rats.

CAT
(U/
Groups GSH (nmoles/g) TT (nmoles/g) LPO (nmoles/g) mg)

71.7 715.8 76.

1.6 54. b
Normal control 12.03c 132.5 7b 8 70.26c 76 9
Diabetic control 70.7 9. 71.
[DC] 3.26 39.827 7.05 9.271.25 64 08
DCACO (200 70.9b a b a
mg/kg) 712.6 43. 76. b
9.435 c 114.4 4b 4.5870.62c 36 02
D ACO (400 11.
C mg/kg) 82
137 722.7 52.
12.5571.38c .8 1b 2.1470.44c 79 7 b
DCGLB (0.5
mg/kg) 70.8 50. 72.
12.237 136.8720.35 2.1370.39 71 22
6.2 7.6
F 14.56 1 20.79 3
4,2 4,2 4,2 4,2
df 5 5 5 5

Each value represent mean7 S.E.M. (n5).

a
P
o0.05.
b

Po0.0
1.
 c
Po0.001 compared to diabetic control. One way ANOVA followed by Tukeys post test.

3.4.8. Liver glycogen

The mobilization of glucose into liver was significantly (Po0.001) decreased in diabetic animals when
compared to normal animals (Table 3). There was a significant increase in glycogen content of liver
(Po0.001; Po0.01) with ACO (400 mg/kg) and GLB treatment as compared to diabetic control, whereas in
ACO (200 mg/kg) there was increase in glycogen content of liver but statistically insignificant.

3.4.9. Endogenous enzymatic and non-enzymatic antioxidant levels

Non-enzymatic antioxidants such as reduced glutathione (GSH) and total thiols were significantly
(Po0.001) decreased, whereas LPO levels were significantly increased in untreated diabetic rats compared
to normal rats. However, ACO (200 and 400 mg/kg) and GLB treatment markedly ameliorated the dele-
terious effect of STZ. Different doses of ACO and GLB treated diabetic rats showed significant (Po0.05;
Po0.01; Po0.001) increase in GSH, total thiols and reduced TBARS compared to untreated diabetic rats
(Table 4).

Enzymatic antioxidant catalase was significantly (Po0.01) decreased in diabetic rats compared to normal
rats. ACO and GLB treated groups showed significant (Po0.05; P o0.01) increase levels in catalase level
compared to diabetic rats (Table 4).

3.4.10. Glucose uptake by isolated hemi-diaphragm of diabetic rats

Insulin (62 mU/ml) caused stimulation of glucose uptake lead-ing to three fold increase compared to
diabetic control values. ACO showed concentration dependent stimulation of glucose uptake by hemi-
diaphragm. ACO at 50 and 100 mg/ml increased the glucose uptake significantly (Po0.001) when compared
to diabetic control values (Fig. 8). The treatment of ACO (50 and
Fig. 8. Effect of different concentration of ethanol extract of Chromolaena odorata [ACO] on glucose uptake
by isolated hemi-diaphragm of diabetic rats. Bar graph represents the glucose uptake (mg/g) of tissue/30
min. G1: Diabetic control; G2: insulin (62 mU/ml); G3: ACO (50 mg/ml); G4: ACO (100 mg/ml); G5:
Insulin (62 mU/ ml)ACO (50 mg/ml); G6: insulin (62 mU/ml)ACO (100 mg/ml). Each value represents
mean7S.E.M (n5).aP o0.05; bPo0.01 and cP o0.001 vs. diabetic control. dP o0.05 vs. ACO (50 mg/ml).

100 mg/ml) plus insulin showed a significant (Po0.05; Po0.01) increase in the glucose uptake when
compared with diabetic alone groups indicating the synergistic effect of ACO with insulin. However, these
values are insignificant compare to insulin alone values.
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M. Onkaramurthy et al. / Journal of Ethnopharmacology 145

(2013) 363372 371
4. Discussion

Proper standardization and validation of diabetic model is very essential to know whether experimental
animals are in type I or type II diabetic or in both conditions. Carefully selection of experimental parameters
is handy to know the same. Such experiments will help to elucidate the possible mode of action of test
compound. It is well known that STZ administration causes rapid destruction of pancreatic b-cells and
model is resembles more of type-I diabetic condition (Habbu et al., 2010). In contrary,

we proved in the present study that treatment of STZ to rats resulted in both type I and type II diabetic
conditions and also it is dose dependent. The administration of STZ (45 mg/kg, i.v) to rats exhibited sever
or type I diabetic condition as evident by insulin estimation at different type points after oral glucose load.
In parallel, these diabetic rats showed higher HOMA values (measure of insulin resistance) and failed to
respond to exogen-ously administered insulin stimulated glucose utilization indicat-ing that in these rats
insulin sensitivity is compromised.

Administration of ACO exhibited significant reduction in serum glucose levels in both single-dose one
day and multiple-dose eight week STZ-induced diabetic studies. These experimen-tal protocols substantiate
the antidiabetic activity of title plant. Exogenously administered glucose (2 g/kg) to diabetic animals
exhibited higher glucose levels with increased AUC. In addition, the external feeding of glucose failed to
stimulate b-cell to release insulin as evident by lower insulin levels at different time point and AUC for
insulin. These data suggested that these diabetic rats resembling type-I or severe diabetic conditions in
which a maximum pancreatic b-cell damage occurred. Treatment of ACO exhibited improved glucose
tolerance and also increases in insulin levels in response to exogenously administered glucose. The efficacy
of ACO was comparable to standard glibenclamide, and could be mediated by improving the glycemic
control mechanisms and insulin secretion from remnant pancreatic b-cells and/or extra pancreatic pathways
may be in act.

The HOMA values (a marker of insulin resistance) of diabetic animals are higher than normal control.
These observations suggest that diabetic animals experience insulin resistance condition (i.e. peripheral
utilization of glucose was compromised). To confirm this observation, insulin tolerance test (ITT) was done
which is a measure of the extent of peripheral utilization of glucose, where SG levels were measured
following insulin challenge (2 IU/kg, i.p). In contrast to established reports, severe (type I) diabetic rats
subjected to insulin challenge did not exhibit a marked fall in SG levels suggested that, these diabetic rats
were not able to utilize the exogenously administered insulin to reduce SG levels. This contrary observation
may be due to marginal loss of insulin sensitivity in diabetic rats, even though these diabetic rats were in
type I diabetic condition (as evident by lower insulin levels at different time points of post glucose
challenge).

DM is often linked with altered lipid metabolism. It is well known that insulin activates enzyme
lipoprotein lipase, which hydrolyzes triglyceride under normal condition (Diwanjee et al.,
2009). The impairment of insulin secretion results in enhanced
metabolism of lipids from the adipose tissue to the plasma (Ananthan et al., 2004). It has been demonstrated
that insulin
deficiency in diabetes leads to a variety of disruption in metabolic and regulatory processes, which in turn
lead to accumulation of lipids (Goldberg, 1981). In the present study, the above-
mentioned changes in the lipid profile of diabetic animals were well documented. Treatment with the ACO
resulted in significant attenuation in serum TG, TC, VLDL-c and LDL-c. These effects might partly be due
to the insulin stimulatory effect of ACO and low secretion of cholesterol biosynthesis enzymes. The conver-
sion of glucose to glycogen in the liver cells is dependent on the
extracellular glucose concentration and the availability of insulin. The regulation of glycogen metabolism in
vivo occurs by the enzymes glycogen synthase and glycogen phosphorylase. The reduced glycogen store in
the diabetic rats has been attrib-uted to the reduced activity of glycogen synthase and increased activity of
glycogen phosphorylase. This is probably due to lack of insulin in the diabetic state, which results in the
inactivation of the glycogen synthetase systems (Shirwaikar et al., 2004). In the

present study, there was a significant decrease in the liver glycogen in diabetic rats. Treatment with ACO
significantly increased the glycogen levels of the diabetic animals and this may be because of the
reactivation of glycogen synthase system.
To know the effect of the ACO on insulin resistance and peripheral utilization of glucose in diabetic rats,
we performed insulin tolerance test with externally administered insulin. ACO treated rats showed a reduced
insulin resistance and increased peripheral glucose utilization. The latter effect was further confirmed with
rat hemi-diaphragm preparation, where the addition of ACO showed a significant increase in the glucose
uptake with ACO at both dose showing a synergistic effect with insulin when compared to diabetic alone
group.

STZ-induced diabetes is associated with increased oxidative stress resulting in diminished levels of
enzymatic (catalase) and non-enzymatic antioxidants (total thiols and reduced glutathione). Hyperglycemia
is a well known cause for elevation of free radical levels which can lead to increased lipid peroxidation
(Balasubashini
et al., 2004) and decreased levels of GSH, Catalse. Following
treatment with ACO, the enzymatic and non-enzymatic antioxidant activity was restored to near normal
levels that could be attributed to the strong antioxidant property of the title plant.
Cataract remains the major cause of blindness worldwide. Altera-tions in the aldose reductase and polyol
pathways are implicated to play a major role in the pathogenesis of development of cataract, in addition to
non-enzymatic glycation (Saito et al., 2008). Through the
polyol pathway, glucose is converted to sorbitol by aldose reductase, which is the rate-limiting enzyme.
Sorbitols and other polyols accumulate intracellularily, leading to osmotic damage and swelling. Further the
involvement of oxidative stress in cataract has been well investigated and is suggested to be an important
factor for the lens opacification and damage to the eyes (Javadzadeh et al., 2009). STZ
administration has been reported to produce lens opacity from the 3rd or 4th week of injection and peaks to
severity in the mid 6th and 8th week (Suryanarayana et al., 2007).
The cataract study was conducted along with the antidiabetic study and the cataract severity scores were
recorded each week up to eight consecutive weeks. The appearance of cataract in the diabetic animals was
augmented on the end of 3rd week. The diabetic animals exhibited an opacification of the lens and the
severity increased with time up to the end of the study. In the ACO treated animals, lens opacification was
observed only from 5th week in 200 mg/kg treated group, which further delayed by one week by its higher
dose. This effect was found to be constant up to the 8th week indicating the protective effect of the extract
against diabetic cataract dose dependently. The preventive action of ACO against the development of
cataract might be attributed to its potential antidiabetic and antioxidant activities.

The chemical composition and medicinal uses of Chromolaena odorata extract have been reported widely.
The title plant has been reported to contain alkaloids, saponins, tannins, phlobatan-nins, anthraquinones,
steroids, terpenoids, flavonoids and cardiac glycosides (Afolabi et al., 2007). Ling et al. (2007) reported the
presence of eleven flavonoids as the major group of compound and correlates the presence of these
flavonoids in the ethanol extract of title plant with antioxidant and anti-inflammatory activity . Further
protocatechuic acid is known to ameliorate STZ-induced toxicity in diabetic rats (Harini and Pugalendi,
2010).
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372 M. Onkaramurthy et al. / Journal of

Ethnopharmacology 145 (2013) 363372
5. Conclusion

The present study reports for the first time to our knowledge that Chromolaena odorata ethanol extract
possesses antidiabetic activity with prevention of cataract formation. Observed bene-ficial effects may be
attributed to bioactive flavonoids. Further-more, it could also result from synergizing action of a
combination of other biomarkers acting by interaction with multiple targets of diabetes. Taken together, the
present study provides the scientific evidence to justify the traditional value of the title plant.

Acknowledgments

We are gratefully acknowledging the financial support by the Vision Group on Science and Technology,
Government of Karnataka under the programme for Establishment of Centre of Excellence in Herbal Drug
Development (VGST/PRMG/CESEM-1/2009-10/199).

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