ARTICLE IN PRESS

Food and Chemical Toxicology xxx (2010) xxx–xxx

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Antihyperglycemic activity and inhibition of advanced glycation end product

formation by Cuminum cyminum in streptozotocin induced diabetic rats
A.G. Jagtap 1, P.B. Patil *
Department of Pharmacology, Bombay College of Pharmacy, Kalina, Santacruz (East), Mumbai 400068, Maharashtra, India

a r t i c l e i n f o a b s t r a c t

Article history: Cuminum cyminum is widely used as a spice in many countries. The aim of the present study was to inves-
Received 12 November 2009 tigate the effect of methanolic extract of seeds of C. cyminum (CC) on diabetes, oxidative stress and for-
Accepted 30 April 2010 mation of advanced glycated end products (AGE) and obtain comparison with glibenclamide. In vitro
Available online xxxx
studies indicated that CC inhibited free radicals and AGE formation. Treatment of streptozotocin-diabetic
rats with CC and glibenclamide for 28 days caused a reduction in blood glucose, glycosylated hemoglobin,
Keywords: creatinine, blood urea nitrogen and improved serum insulin and glycogen (liver and skeletal muscle) con-
Cuminum cyminum
tent when compared to diabetic control rats. Significant reduction in renal oxidative stress and AGE was
Streptozotocin
Antidiabetic
observed with CC when compared to diabetic control and glibenclamide. CC and glibenclamide improved
Oxidative stress antioxidant status in kidney and pancreas of diabetic rats. Diabetic rats showed increase in rat tail tendon
Glycation collagen, glycated collagen, collagen linked fluorescence and reduction in pepsin digestion. Treatment
with CC significantly improved these parameters when compared to diabetic control and glibenclamide
group. Though the antidiabetic effect of CC was comparable to glibenclamide it had better effect in con-
trolling oxidative stress and inhibiting the AGE formation, which are implicated in the pathogenesis of
diabetic microvascular complications.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction in the glomerular and tubulointerstitial compartments and struc-

Diabetes mellitus is a heterogeneous metabolic disorder charac-
terized by hyperglycemia resulting from defective insulin secre-
tion, resistance to insulin action or both. The evolution of
numerous long term complications of diabetes mellitus correlates
with the severity and duration of hyperglycemia (Thévenod,
2008). Hyperglycemia is still considered the principal cause of dia-
betic complications. The polyol pathway, modification of protein
kinase C activity, formation of advanced glycated end products
(AGE) and oxidative stress are widely studied to explain the poten-
tial mechanisms by which hyperglycemia can result in diabetic
complications (Cumbie and Hermayer, 2007). AGE accumulation
Abbreviations: AGE, advanced glycated end products; AR, aldose reductase; BUN,
blood urea nitrogen; BSA, bovine serum albumin; CAT, catalase; DCCT, diabetes
control and complications trial; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ECM, extra-
cellular matrix; Glib, glibenclamide; GHb, glycosylated hemoglobin; MDA, mal-
ondialdehyde; CC, methanolic extract of seeds of Cuminum cyminum; PMS–NADH,
phenazine methosulfate–nicotinamide adenine dinucleotide; GSH, reduced gluta-
thione; STZ, streptozotocin; SOD, superoxide dismutase; TBA, thiobarbituric acid;
TBARS, thiobarbituric acid reactive substances.
* Corresponding author. Tel.: +91 9820463195; fax: +91 022 266770871.
E-mail addresses: jagtaparti@gmail.com (A.G. Jagtap), poonam_patil12@rediff-
mail.com (P.B. Patil).
1
Tel.: +91 022 266771027; fax: +91 022 266770871.
tural alterations of extracellular matrix (ECM) protein correlates
with the severity of diabetic nephropathy. In the presence of
AGE, glomerular podocytes upregulate the expression of RAGE
(receptors for AGE) triggering multiple intracellular signal trans-
duction cascades contributing to renal injury (Goh and Cooper,
2008). Collagen, a long lived protein, is highly susceptible to ad-
vanced glycation in vivo. Since collagen is an important constituent
of most of the tissues that are affected during diabetes, modifica-
tion of this protein plays a critical role in the complications of dia-
betes. Oxygen radicals formed during glucose oxidation and
glycated protein oxidation may be involved directly in the forma-
tion of AGEs and collagen cross-linking (Jakuš and Rietbrock, 2004).
The hallmark, Diabetes Control and Complications Trial (DCCT)
showed that, near-normalization of blood glucose by intensive
insulin therapy reduced the risk of development of diabetic com-
plications. However, intensive insulin therapy carries a high risk
of side effects, especially occurrence of severe hypoglycemia. Thus,
a large number of people still are prone to develop vascular com-
plications, and additional pharmacological approaches to prevent
these complications are warranted (Watkins, 2003).
Antioxidants and free radical scavengers are known to inhibit
the formation of AGEs and the cross-linking of collagen (Laurean
et al., 2006). Dietary measures and traditional plant therapies, as

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.04.048

Please cite this article in press as: Jagtap, A.G., Patil, P.B. Antihyperglycemic activity and inhibition of advanced glycation end product formation by Cumi-
num cyminum in streptozotocin induced diabetic rats. Food Chem. Toxicol. (2010), doi:10.1016/j.fct.2010.04.048
 ARTICLE IN PRESS
2 A.G. Jagtap, P.B. Patil / Food and Chemical Toxicology xxx (2010) xxx–xxx
prescribed by Ayurvedic and other indigenous systems of medicine
are commonly used in India. Cuminum cyminum Linn. commonly
known as Jeera, is consumed in large quantities by Indians. Litera-
ture survey showed limited evidence of hypoglycemic effect of C.
cyminum (Surya et al., 2002; Roman, 1995). Recently cuminalde-
hyde derived from C. cyminum seed oil showed aldose reductase
(AR) and alpha glucosidase inhibitory activity in vitro (Lee, 2005).
If dietary intervention could reduce the ensuing diabetes and its
complication it would prove a useful adjunct to antidiabetic ther-
apy. The present study was undertaken to confirm the antihyper-
glycemic potential of methanolic extract of seeds of C. cyminum
and explore its role in the modulation of diabetic complications
in vivo.
2. Materials and methods
2.1. Animals
Adult Wistar rats (190–210 g) of either sex were housed in standard polypro-
pylene cages with wire mesh top and maintained at 23 ± 2 °C with 12-h light–dark
cycle. Animals were fed with commercially available standard rodent pellet diet
and water ad libitum. Study protocols were approved by Institutional Animal Ethics
Committee and experiments were performed in accordance with CPCSEA
guidelines.
2.2. Extract preparation and toxicity study
Seeds of C. cyminum were procured and authenticated by Dr. Aashish S. Phadke,
M.D. (Ayur), an ayurvedic clinician in Mumbai. Seeds were powdered in a grinder
and extracted with methanol in a soxhlet assembly for 48 h. The methanolic extract
(CC) was further air dried to obtain a semisolid mass of 10–12% w/w, stored at 4–
8 °C and suspended in 0.5% w/v sodium CMC before use. Acute oral toxicity study
was performed as per OECD 423.
2.3. Chemicals
Streptozotocin (STZ) was obtained from Sisco Research Laboratories, India.
Diagnostic kits for the estimation of glucose and creatinine was obtained from Erba
Diagnostics, Germany. Insulin kit was obtained from Board of Radiation and Isotope
Technology (BRIT), India. All the other chemicals used were of analytical grade.
2.4. Phytochemistry
Qualitative phytochemical evaluation was performed as described in Khandel-
wal (2004). Phenol and flavonoid content was estimated in methanolic solution
of CC (0.2 mg/ml) by Folin Ciocalteu reagent (Singleton et al., 1999) and aluminum
chloride (AlCl3) (Zhishen et al., 1999) respectively. HPTLC analysis of CC was per-
formed using Camag Scanner III with WINCATS (Ver-1.1.3) data processor on pre-
coated silica gel 60 F254 TLC using toluene, ethyl acetate, methanol, distilled
water and glacial acetic acid (3:3:3:1:1) as solvent system (Shirke et al., 2008).
Peaks were observed in UV/VIS, photo-documented and respective Rf values noted.
2.5. In vitro radical scavenging assay
Solution of CC (10 mg/ml) was prepared in dimethyl sulphoxide. Ability of the
extract to scavenge superoxides generated in a non-enzymatic phenazine metho-
sulfate–nicotinamide adenine dinucleotide (PMS–NADH) system (Nishimiki et al.,
1972) was studied at different concentrations (10–1600 lg/ml). Ability to scavenge
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (Blois, 1958) was studied in CC
(1 mg/ml) prepared in 95% ethanol at various concentrations (10–320 lg/ml). Con-
centration required for 50% inhibition of formation of superoxides and loss of DPPH
color was determined graphically.
2.6. In vitro bovine serum albumin (BSA) glycation study
D-Glucose (200 mM) was incubated with BSA (10 mg/ml) in phosphate buffer
(50 mM, pH 7.4) containing 0.02% (w/v) sodium azide and different concentrations
of CC (200–1400 lg/ml) at 37 °C for 7 days (Sakai et al., 2002). Fluorescence was
measured at an excitation/emission wavelength of 370/440 nm and 335/385 nm
to quantify AGE and pentosidine compounds respectively.
Please cite this article in press as: Jagtap, A.G., Patil, P.B. Antihyperglycemic activity and inhibition of advanced glycation end product formation by Cumi-
num cyminum in streptozotocin induced diabetic rats. Food Chem. Toxicol. (2010), doi:10.1016/j.fct.2010.04.048
2.7. Induction of diabetes
Experimental diabetes was induced in overnight fasted rats by a single intra-
peritoneal (i.p) injection of freshly prepared streptozotocin, (STZ, 50 mg/kg body
weight) in 0.1 M chilled citrate buffer (pH 4.5). Fourteen days after STZ administra-
tion, rats with fasting serum glucose more than 250 mg/dl were selected to com-
pose the diabetic control and treatment groups. Total of 54 rats (30 diabetic and
24 normal rats) were used. Rats were divided into nine groups of six rats each.
Group I – normal control; Group II – diabetic control; Group III, IV and V consisted
of diabetic rats treated with CC at a dose of 200, 400 and 600 mg/kg respectively;
Group VI – diabetic rats receiving glibenclamide (Glib) 10 mg/kg. Extract control
groups namely, group VII, VIII and IX comprised of normal rats treated with CC at
a dose of 200, 400 and 600 mg/kg respectively. CC and Glib was administered orally
in 0.5% sodium CMC once daily for 28 days.
At the end of 28 days of treatment body weight was recorded. Blood was col-
lected from overnight fasted rats for estimation of serum glucose, insulin, glycosyl-
ated hemoglobin (GHb), blood urea nitrogen (BUN) and creatinine. GHb was
estimated in the blood homolysate. Animals were sacrificed and skeletal muscle, li-
ver, pancreas, kidneys and tail was removed and stored at -70 °C until use.
2.8. Preparation of homogenate
Kidney and pancreas were homogenized in 100 mM of Tris–HCl buffer pH 7.4 in
Teflon homogenizer. Thiobarbituric acid reactive substances (TBARS) were esti-
mated in a part of the homogenate and the remaining was centrifuged at 8000g
for 20 min at 4 °C. Supernatant used for the estimation of catalase (CAT), superoxide
dismutase (SOD) and reduced glutathione (GSH). Protein content was determined
by biuret method (Gernal et al., 1949).
2.9. Biochemical analysis
Serum glucose and creatinine was estimated by GOD/POD and Jafe’s method
respectively using standard commercial kit. Serum insulin was determined by RIA
insulin kit (BRIT, India) at BARC. GHb was determined by reaction with thiobarbitu-
ric acid (Sheikh et al., 2004). BUN was determined by reaction with diacetyl mon-
oxime (Godkar and Godkar, 2006). Glycogen was estimated in the liver and
skeletal muscle by phenol–sulfuric acid method (Good et al., 1993). Measurement
of malondialdehyde (MDA) by thiobarbituric acid (TBA) reactivity is the most
widely used method for assessing lipid peroxidation. The determination of thiobar-
bituric acid reactive substances (TBARS) in the pancreas and kidneys was performed
by the method of Ohkawa et al. (1979).
2.10. Estimation of antioxidants
CAT was determined in tissue supernatant by method of Aebi (1974). The
change in optical density at 240 nm per unit time was taken as a measure of cata-
lase activity. SOD was estimated using the principle of inhibition of epinephrine
autooxidation by the method of Mishra and Fridovich (1972). A unit activity of
SOD is defined as 50% inhibition of epinephrine auto oxidation per min. Total
non-protein sulphydryl content, an indirect measure of reduced glutathione was
determined in the tissue supernatant by the method of Sedlak and Lindsay (1968).
2.11. Renal AGE
Renal AGE content was determined by method described by Nakagawa et al.
(2003). Kidney was minced and delipidated by shaking gently with mixture of chlo-
roform and methanol (2:1 v/v) overnight. The delipidated tissue was homogenized
in 0.1 N NaOH, followed by centrifugation at 8000g for 15 min at 4 °C. The amount
of AGE in the supernatant was measured in a spectroflurometer at an excitation/
emission wavelength of 370/440 nm against 0.1 N NaOH blank. BSA preparation
(1 mg/ml in 0.1 N NaOH) was used as a reference, and its fluorescent intensity
was defined as 1 arbitrary unit (AU). The fluorescence intensities of the samples
were measured and expressed as arbitrary units (AU)/mg protein.
2.12. Estimation of parameters in rat tail tendon
Tendons separated from tails of experimental rats were washed thoroughly in
saline at 4 °C. Acid hydrolysis of the tendons was carried out at 121 °C for 4 h.
Hydroxyproline was estimated in hydrolysed tendon collagen samples (Woessner,
1968). Collagen content in the rat tail tendon was expressed in mg collagen/
100 mg tissue assuming that collagen weighs 7.46 times hydroxyproline. The extent
of collagen glycation in tail tendon was assessed by phenol–sulfuric acid method
(Rao and Pattabiraman, 1989) and expressed as, lg of glucose/mg collagen. Clean
tendons (50 mg, wet weight) were digested in freshly prepared pepsin solution
(1 mg/ml in 0.5 M acetic acid, 5 ml) for 24 h at 37 °C to determine the amount of
pepsin soluble collagen (Golub et al., 1978). The samples of pepsin-digested colla-
gen (0.25 ml) were mixed with 2.75 ml of 200 mM phosphate buffer (pH 7.5).
 ARTICLE IN PRESS

A.G. Jagtap, P.B. Patil / Food and Chemical Toxicology xxx (2010) xxx–xxx 3

Collagen linked fluorescence was quantified at 365 nm excitation and 416 nm emis- DPPH radical scavenging
sion, relative to the standard quinine sulfate solution (1 lg/ml) and expressed as a 80
AU/mg collagen (Stefek et al., 2000). 72.54
70 59.37

2.13. Statistical analysis 60

% Inhibition
50 42.64
One way analysis of variance (ANOVA) was used for multiple comparisons.
When ANOVA showed significant difference (p < 0.001), post hoc analysis was per- 40 32.73
formed with Tukey’s multiple comparison test (Graphpad PRISM 4). P < 0.05 was
considered statistically significant. 30
20.28
2013.77
3. Results 107.81

0
3.1. Acute oral toxicity study 0 50 100 150 200 250 300 350 400 450 500 550 600 650
Conc µg/ml
Single oral dose of CC 2000 mg/kg body weight did not cause
any morbidity or mortality in rats during the 14 days of observa-
tion period and was found to be safe.
b 80 Superoxide scavenging
70 69.49

3.2. Phytochemistry 60
53.05

% Inhibition
Qualitative analysis of CC showed the presence of essential oils, 50
phenolics, steroids and flavonoids. The total phenolic and flavonoid 40
content in CC was expressed in quercetin equivalent, QE and was 37.09

found to be 11.73 and 4.4 QE, lg/ml respectively. HPTLC was per- 30 21.84
formed to determine flavonoid glycosides. A bunch of two peaks
20 14.27
related to luteoline and some closely related glycosides were iden- 17.424
tified. The combined area of two peaks was used for the estimation 10 12.1
9.71
of total flavonoids and was found to be 51.87% w/w.
0
0 200 400 600 800 1000 1200 1400 1600 1800
3.3. Radical scavenging Concentration (µg/ml)

Superoxide anions were generated in vitro in a non-enzymatic Fig. 1. Effect of CC on radical scavenging. (a) Free radical scavenging effect of
PMS–NADH system through the reaction of PMS, NADH, and oxy- various concentrations of CC. Results are mean ± SD; n = 3. (b) Superoxide
scavenging effect of various concentrations of CC. Results are mean ± SD; n = 3.
gen. CC caused decrease in absorbance at 560 nm indicating the
consumption of O2 in the reaction mixture. In the presence of
antioxidants, DPPH free radical becomes paired off and the absorp-
dose dependent effect on all these parameters. Diabetes caused
tion at 517 nm vanishes, resulting in decolorization which is stoi-
significant reduced in glycogen stores in the liver and skeletal mus-
chiometric with respect to the number of electrons taken up. CC
cle. Treatment with CC and glibenclamide significantly increased
(10–1600 lg/ml) caused scavenging of DPPH free radicals
these levels as compared to diabetic control (Table 3). Effect of gli-
(Fig. 1a) and superoxides (Fig. 1b) in concentration dependent
benclamide and CC 400, 600 mg/kg on skeletal muscle glycogen
manner. IC50 value of CC for inhibition of DPPH and superoxide
was comparable to normal control. Extract control rats were com-
was found to be 230 and 1120 lg/ml respectively.
parable to normal control.

3.4. In vitro BSA glycation

Fluorescence intensity of glycated collagen was significantly
higher after incubation with glucose. CC (200–1400 lg/ml) inhib-
ited the glycation of BSA and subsequent formation of fluorescent
glycation products in a concentration dependent manner (Fig. 2).
IC50 value of CC for inhibition of AGE and pentosidine compounds
was found to be 1170 and 320 lg/ml respectively.
3.5. Effect of CC in streptozotocin induced diabetic rats
Streptozotocin induced diabetic rats showed symptoms of poly-
uria, polydipsia, and polyphagia (data not shown). Significant
reduction in body weight was observed. STZ-induced diabetes sig-
nificantly increased serum glucose and GHb and reduced insulin
levels as compared to normal control rats (Table 1). Treatment
with CC and glibenclamide significantly increased body weight
and serum insulin and reduced serum glucose compared to normal
control. Reduction in kidney function was indicated by elevated
levels of BUN and creatinine (Table 2). Treatment with CC and gli-
benclamide significantly reduced these levels as compared to dia-
betic group. The effect of CC600 mg/kg demonstrated better effect
than glibenclamide in normalizing kidney function. CC showed
3.6. Effect of CC on TBARS and antioxidants
Reduction in antioxidants was observed in the pancreas (Table 4)
and kidneys (Table 5) of diabetic rats. Treatment with CC and gliben-
clamide significantly increased the activity of antioxidant enzymes,
viz SOD and CAT compared to diabetic control. GSH was also found
to increase after treatment with CC and glibenclamide. Oxidative
stress as indicated by reduction in antioxidant enzymes and
increase in lipid peroxidation (Table 6) was observed in pancreas
and kidneys of diabetic rats. Treatment with CC and glibenclamide
significantly reduced the formation of TBARS. Extract control rats
showed levels similar to normal control.
3.7. Effect on renal AGE
Diabetes caused a five fold increase in the renal AGE content
compared to the normal rats (Fig. 3). Treatment with CC and gli-
benclamide showed significant reduction in AGE compared to dia-
betic control. Effect of CC 600 mg/kg was significantly (p < 0.001)
better than glibenclamide and comparable with normal rats. Ex-
tract control rats did not cause any change in renal AGE when com-
pared with normal control.

Please cite this article in press as: Jagtap, A.G., Patil, P.B. Antihyperglycemic activity and inhibition of advanced glycation end product formation by Cumi-
num cyminum in streptozotocin induced diabetic rats. Food Chem. Toxicol. (2010), doi:10.1016/j.fct.2010.04.048
 ARTICLE IN PRESS

4 A.G. Jagtap, P.B. Patil / Food and Chemical Toxicology xxx (2010) xxx–xxx

100
AGE
90 84.98
PEN 86.09
83.93
79.98
80 75.24 76.85
72.27 74.11
67.54
70
58.3
% Inhibition 60
52.3

50 44.97

40
30.9
28.5
30 25.02 26.1 27.4
24.9
20.6

20 13.7

10 4.3
0.6
0
200 300 400 500 600 700 800 900 1000 1200 1400

AGE 0.6468 4.2803 13.743 20.563 24.87 26.141 27.444 28.536 30.913 52.331 58.293
PEN 25.019 44.97 67.544 72.274 74.105 75.243 76.854 79.982 83.927 84.975 86.095
µg/ml

Fig. 2. Inhibitory effect of various concentrations of CC on the formation of AGE and pentosidine compounds in BSA-glucose model. Results are mean ± SD; n = 3.

Table 1
Effect of treatment on body weight, serum glucose, insulin and GHb.

Groups Body weight (g) Serum glucose (mg/dl) Serum insulin lIU/ml GHb (%)
Normal control 249.07 ± 14.70 78.84 ± 8.2 30.74 ± 2.01 6.822 ± 0.34
Diabetic control 140.95 ± 10.16a 284.2 ± 10.56a 11.6 ± 1.38a 15.39 ± 1.422a
Diabetic + CC (200 mg/kg) 169.63 ± 6.74ab 186.6 ± 6.99ab 18.71 ± 0.33ab 12.08 ± 1.12ab
Diabetic + CC (400 mg/kg) 181.58 ± 5.41ab 132.9 ± 7.64ab 20.4 ± 0.41ab 8.56 ± 0.45cb
Diabetic + CC (600 mg/kg) 210.00 ± 10.0ab 114.8 ± 4.86ab 22.77 ± 1.74abg 8.389 ± 0.29cb
Diabetic + Glib (10 mg/kg) 194.43 ± 10.75ab 109.3 ± 4.7ab 18.83 ± 1.15ab 8.10 ± 0.23cb
Normal + CC (200 mg/kg) 247.28 ± 6.99 74.02 ± 3.72 28.63 ± 0.61 7.02 ± 0.25
Normal + CC (400 mg/kg) 249.08 ± 15.44 74.11 ± 3.59 30.65 ± 1.59 6.77 ± 0.31
Normal + CC (600 mg/kg) 252.22 ± 6.96 72.77 ± 4.1 29.7 ± 1.06 6.99 ± 0.23

Data expressed as mean ± SD; n = 6.

a
p < 0.001 vs. normal control.
b
p < 0.001 vs. diabetic control.
c
p < 0.05 vs. normal control.
g
p < 0.05 vs. glibenclamide.

Table 2 Table 3
Effect of treatment on creatinine and BUN. Effect of treatment on skeletal muscle& liver glycogen.

Groups BUN (mg/dl) Creatinine (mg/dl) Groups Liver glycogen Skeletal glycogen
(mg/g tissue) (mg/g tissue)
Normal control 17.75 ± 1.13 0.5151 ± 0.02
Diabetic control 50.55 ± 2.96a 0.689 ± 0.019a Normal control 56.36 ± 3.43 4.523 ± 0.19
Diabetic + CC (200 mg/kg) 33.44 ± 2.77ab 0.6089 ± 0.04ab Diabetic control 21.02 ± 2.91a 0.8447 ± 0.16a
Diabetic + CC (400 mg/kg) 27.44 ± 1.73ab 0.5566 ± 0.021b Diabetic + CC (200 mg/kg) 32.93 ± 5.23ab 3.55 ± 0.28ab
Diabetic + CC (600 mg/kg) 17.89 ± 0.83bg 0.5273 ± 0.036bg Diabetic + CC (400 mg/kg) 34.94 ± 0.69ab 4.24 ± 0.151b
Diabetic + Glib (10 mg/kg) 26.6 ± 1.84ab 0.603 ± 0.012ab Diabetic + CC (600 mg/kg) 42.71 ± 1.95ab 4.38 ± 0.26b
Normal + CC (200 mg/kg) 17.82 ± 1.12 0.5369 ± 0.012 Diabetic + Glib (10 mg/kg) 41.05 ± 4.51ab 4.28 ± 0.25b
Normal + CC (400 mg/kg) 17.6 ± 0.563 0.5012 ± 0.017 Normal + CC (200 mg/kg) 54.17 ± 2.87 4.52 ± 0.34
Normal + CC (600 mg/kg) 18.04 ± 1.02 0.531 ± 0.033 Normal + CC (400 mg/kg) 59.46 ± 2.01 4.36 ± 0.23
Normal + CC (600 mg/kg) 57.31 ± 3.70 4.42 ± 0.24
Data expressed as mean ± SD; n = 6
a
p < 0.001 vs. normal control. Data expressed as mean ± SD; n = 6.
b a
p < 0.001 vs. diabetic control. p < 0.001 vs. normal control.
g b
p < 0.05 vs. glibenclamide. p < 0.001 vs. diabetic control.

collagen glycation and collagen linked fluorescence and reduction

3.8. Effect on rat tail tendon parameters in pepsin soluble collagen (Table 7). Treatment with CC and gli-
benclamide reversed these effects indicating a potential role in
Collagen was measured in rat tail tendon after acid hydrolysis. preserving collagen structure and function. Effect of CC 600 mg/
Diabetic rats showed a significant increase in tail tendon collagen, kg was significantly (p < 0.001) better than glibenclamide and

Please cite this article in press as: Jagtap, A.G., Patil, P.B. Antihyperglycemic activity and inhibition of advanced glycation end product formation by Cumi-
num cyminum in streptozotocin induced diabetic rats. Food Chem. Toxicol. (2010), doi:10.1016/j.fct.2010.04.048
 ARTICLE IN PRESS

A.G. Jagtap, P.B. Patil / Food and Chemical Toxicology xxx (2010) xxx–xxx 5

Table 4 Table 6
Effect of treatment on antioxidants in Pancreas. Effect of treatment on lipid peroxidation (TBARS).

Groups CAT (U/mg SOD (U/mg GSH (lM/mg Groups Pancreas TBARS (nmol Kidney TBARS (nmol
protein) protein) protein) MDA/mg protein) MDA/mg protein)
Normal control 65.9 ± 4.04 3.22 ± 0.44 71.78 ± 4.12 Normal control 0.487 ± 0.05 0.633 ± 0.06
Diabetic control 35.65 ± 1.78a 0.50 ± 0.01a 46.13 ± 1.61a Diabetic control 2.359 ± 0.39a 3.7 ± 0.34a
Diabetic + CC (200 mg/kg) 52.53 ± 4.22ab 1.01 ± 0.13ad 50.87 ± 4.18a Diabetic + CC (200 mg/kg) 1.95 ± 0.18ab 2.93 ± 0.29ab
Diabetic + CC (400 mg/kg) 60.71 ± 1.11b 1.38 ± 0.03ab 55.75 ± 1.95ab Diabetic + CC (400 mg/kg) 0.86 ± 0.11cb 1.14 ± 0.21ab
Diabetic + CC (600 mg/kg) 64.95 ± 6.83bg 1.58 ± 0.20abg 63.54 ± 2.07cb Diabetic + CC (600 mg/kg) 0.72 ± 0.02bg 0.7 ± 0.06bg
Diabetic + Glib (10 mg/kg) 49.61 ± 3.37ab 3.19 ± 0.2b 65.43 ± 5.05ab Diabetic + Glib (10 mg/kg) 1.05 ± 0.11ab 1.56 ± 0.14ab
Normal + CC (200 mg/kg) 66.29 ± 4.58 3.18 ± 0.2 71.88 ± 3.45 Normal + CC (200 mg/kg) 0.47 ± 0.06 0.63 ± 0.05
Normal + CC (400 mg/kg) 66.03 ± 3.86 3.3 ± 0.15 72.97 ± 2.21 Normal + CC (400 mg/kg) 0.49 ± 0.04 0.63 ± 0.07
Normal + CC (600 mg/kg) 64.21 ± 3.3 3.3 ± 0.16 70.82 ± 4.07 Normal + CC (600 mg/kg) 0.491 ± 0.03 0.63 ± 0.07

Data expressed as mean ± SD; n = 6. Data expressed as mean ± SD; n = 6.

a a
p < 0.001 vs. normal control. p < 0.001 vs. normal control.
b b
p < 0.001 vs. diabetic control. p < 0.001 vs. diabetic control.
c c
p < 0.05 vs. normal control. p < 0.05 vs. normal control.
d g
p < 0.05 vs. diabetic control. p < 0.05 vs. glibenclamide.
g
p < 0.001 vs. glibenclamide.

Table 5 4. Discussion
Effect of treatment on antioxidants in kidney.

Groups CAT (U/mg SOD (U/mg GSH (lM/mg STZ-induced diabetes is the most widely used animal model of
protein) protein) protein) human diabetes mellitus. Diabetes arises from irreversible destruc-
Normal control 581.2 ± 12.2 2.69 ± 0.25 70.81 ± 1.89 tion of pancreatic b cells causing reduction in insulin secretion.
Diabetic control 340.0 ± 7.40a 1.23 ± 0.07a 41.13 ± 2.61a Diabetic rats showed symptoms of polyuria, polyphagia and poly-
Diabetic + CC (200 mg/kg) 496.1 ± 27.29ab 1.66 ± 0.12a 46.43 ± 0.93ad dipsia which were reversed on treatment with CC and glibencla-
Diabetic + CC (400 mg/kg) 500.0 ± 15.51ab 2.54 ± 0.12b 50.50 ± 1.84ab
mide (data not shown). Treatment with CC and glibenclamide
Diabetic + CC (600 mg/kg) 548.8 ± 14.87cbg 2.69 ± 0.12bg 67.15 ± 0.83bg
Diabetic + Glib (10 mg/kg) 518.7 ± 8.17ab 2.18 ± 0.13cb 55.91 ± 2.86ab reduced elevated blood glucose and increased serum insulin and
Normal + CC (200 mg/kg) 577.0 ± 10.13 2.60 ± 0.29 71.09 ± 3.39 glycogen content. Sulphonyl ureas such as glibenclamide stimulate
Normal + CC (400 mg/kg) 573.8 ± 14.84 2.69 ± 0.35 70.93 ± 2.20 insulin secretion from pancreatic b cells principally by inhibiting
Normal + CC (600 mg/kg) 568.8 ± 20.12 2.66 ± 0.45 71.61 ± 2.13 ATP-sensitive K+ channels (Watkins 2003). CC may bring about
Data expressed as mean ± SD; n = 6. reduction in blood glucose through stimulation of surviving b cells
a
p < 0.001 vs. normal control. to produce more insulin. Protection of pancreas on treatment with
b
p < 0.001 vs. diabetic control. CC was observed as indicated by reduction in lipid peroxidation
c
p < 0.05 vs. normal control.
d
p < 0.05 vs. diabetic control.
and increase in antioxidants like superoxide dismutase, catalase
g
p < 0.001 vs. glibenclamide. and reduced glutathione. It is known that antioxidant treatment
suppressed apoptosis and exerted beneficial effect on pancreatic
b cells (Kaneto et al., 1976). In response to enhanced insulin levels,
the glycogen content in skeletal muscle and liver was found to in-
comparable with normal rats. Extract control rats did not cause crease. Thus antihyperglycemic effect of CC may be due to protec-
any change in tendon collagen parameters when compared to tion of surviving pancreatic b cells, increase in insulin secretion
normal control. and glycogen storage.

a Effect of CC on Renal AGE

12
10.09

10

ab
8
AU/mg protein

6.99
ab
5.56
6 ab
5.12

4
2.25 bg 2.37
2.25 2.25 2.27
2

0
Control DC D+CC200 D+CC400 D+CC 600 D+Glib CC200 CC400 CC600

Data expressed as mean ± SD; n = 6;

p < 0.001 vs a normal control, b diabetic control, g glibenclamide

Fig. 3. Effect on treatment on renal AGE.

Please cite this article in press as: Jagtap, A.G., Patil, P.B. Antihyperglycemic activity and inhibition of advanced glycation end product formation by Cumi-
num cyminum in streptozotocin induced diabetic rats. Food Chem. Toxicol. (2010), doi:10.1016/j.fct.2010.04.048
 ARTICLE IN PRESS
6 A.G. Jagtap, P.B. Patil / Food and Chemical Toxicology xxx (2010) xxx–xxx
Table 7
Effect of treatment of tail tendon collagen.
Groups Total collagen (mg/100 mg Pepsin-digested collagen (mg/100 mg
tendon) tendon)
Normal control 35.57 ± 3.68 3.58 ± 0.38
Diabetic control 73.32 ± 5.13a 1.59 ± 0.12a
Diabetic + CC (200 mg/kg) 52.89 ± 3.41ab 2.79 ± 0.21ab
Diabetic + CC (400 mg/kg) 45.51 ± 4.39ab 3.42 ± 0.23b
Diabetic + CC (600 mg/kg) 37.22 ± 1.95bh 3.39 ± 0.25bg
Diabetic + Glib (10 mg/kg) 45.55 ± 2.89ab 2.23 ± 0.16ab
Normal + CC (200 mg/kg) 35.02 ± 1.31 3.61 ± 0.19
Normal + CC (400 mg/kg) 34.2 ± 4.59 3.60 ± 0.34
Normal + CC (600 mg/kg) 35.02 ± 3.24 3.59 ± 0.29
Data expressed as mean ± SD; n = 6.
a
p < 0.001 vs. normal control.
b
p < 0.001 vs. diabetic control.
c
p < 0.05 vs. normal control.
g
p < 0.001 vs. glibenclamide.
h
p < 0.05 vs. glibenclamide.
Oxidative stress occurs when production of oxidants or reactive
oxygen species (ROS) exceeds local antioxidant capacity. ROS in-
clude free radicals such as superoxide (O2 ), hydroxyl (OH), and
peroxyl (RO2 ) and nonradical species such as hydrogen peroxide
(H2O2). Free radicals are formed disproportionately in diabetes by
glucose oxidation, non-enzymatic glycation of proteins, and the
subsequent oxidative degradation of glycated proteins. Abnormally
high levels of free radicals and the simultaneous decline of antiox-
idant defense mechanisms can lead to damage of cellular organelles
and enzymes, increased lipid peroxidation, and development of
insulin resistance (Andrea et al., 2004). ‘‘Unifying hypothesis” sug-
gest that the initiator of hyperglycemia induced diabetic nephrop-
athy is due to excess generation of mitochondrial superoxides,
which then leads to activation of four major biochemical pathways,
including increase AGE formation, activation of protein kinase C, in-
creased flux through polyol pathway and hexosamine pathway
(Brownlee, 2005). Diabetic rats showed increased kidney lipid per-
oxidation (TBARS), reduction in antioxidant defense and increase in
renal AGE. This is in agreement with other authors who reported a
significant increase in renal AGE formation in STZ-induced diabetic
rats (Nakagawa et al., 2003). Renal damage was evident with in-
crease in serum creatinine and BUN indicating reduced creatinine
and urea clearance. A decrease in renal function also increases cir-
culating AGE concentrations by reduced clearance as well as in-
creased formation. AGEs are involved in structural changes in
diabetic nephropathy such as glomerulosclerosis, interstitial fibro-
sis and tubular atrophy consequently affecting kidney functions
(Wendt et al., 2003). Treatment with CC and glibenclamide reduced
oxidative stress by increasing antioxidant defense and reducing
free radical induced lipid peroxidation. Increased oxidative stress
was associated with increased formation of renal AGE in diabetic
rats which was reduced on treatment with extract and glibencla-
mide. Effect of CC 600 mg/kg on reducing oxidative stress and renal
AGE was significantly (p < 0.05) better than glibenclamide. This ef-
fect was reflected by improved creatinine and BUN levels in diabetic
rats treated with CC 600 mg/kg. CC 600 mg/kg improved renal func-
tion to near normal and was significantly better than glibenclamide.
In vitro effect of CC on inhibition of BSA glycation and scaveng-
ing free radicals and superoxides correlated with the effects ob-
served in vivo. Antioxidant property of many plant extracts have
been attributed to their phenolic content (Amin and Razieh,
2007). Phenolic content of CC was found to be QE 11.73 lg/ml.
Rutin, a common dietary flavonoid is an established antioxidant.
Rutin was also found to inhibit glycation product formation in-
duced by glucose glycation of collagen I in vitro suggesting the
important role of flavonoids in AGE inhibition (Daniel et al.,
Please cite this article in press as: Jagtap, A.G., Patil, P.B. Antihyperglycemic activity and inhibition of advanced glycation end product formation by Cumi-
num cyminum in streptozotocin induced diabetic rats. Food Chem. Toxicol. (2010), doi:10.1016/j.fct.2010.04.048
Collagen glycation (lg glucose/mg Fluorescence (AU/mg
collagen) collagen)
2.91 ± 0.42 2.76 ± 0.45
13.97 ± 2.16a 25.21 ± 3.26a
7.06 ± 0.58ab 17.38 ± 1.01ab
4.67 ± 0.63cb 9.36 ± 0.65ab
3.35 ± 0.86bg 5.06 ± 0.38bg
7.58 ± 0.75 ab 16.1 ± 1.76ab
3.12 ± 0.51 2.49 ± 0.31
3.17 ± 0.58 2.53 ± 0.37
3.21 ± 0.47 2.50 ± 0.39
2006). CC was found to contain 51.87% w/w flavonoids, which
may be responsible for its antiglycation property.
Advanced glycation occurs over a prolonged period, affecting
long lived protein such as type IV collagen in structural
components of connective tissue matrix and basement membrane
(Figarola et al., 2003). STZ-induced diabetes mellitus characterized
by hyperglycemia caused a significant increase in rat tail tendon
collagen, glycated collagen, collagen linked fluorescence and
reduction in pepsin-digested collagen. Excessive collagen can re-
sult from an imbalance between its synthesis and degradation by
interstitial collagenases. Collagenous proteins are especially ex-
posed to glycation because they contain several lysine, hydroxyl ly-
sine and arginine residues with free amino groups, have a very
slow turnover rate and are exposed to ambient levels of glucose
(Subramanian et al., 2003). Glycation of collagen leads to formation
of AGE and an increase in collagen linked fluorescence. Glycation of
collagen interferes with the activation of metalloproteinases, the
enzymes responsible for collagen degradation (Kuzuya et al.,
2001). Inter and intra molecular cross links with collagen are
formed as a result of glycation which are responsible for resistance
to pepsin digestion. Treatment with CC and glibenclamide reversed
these parameters with respect to diabetic control. Reduction in col-
lagen may be attributed to the significant decrease in blood glu-
cose and consequent decrease in non-enzymatic glycation and
deposition of collagen in diabetic rats treated with CC and gliben-
clamide. Treatment with extract and glibenclamide significantly
reduced the levels of collagen linked fluorescence. CC 400 and
600 mg/kg significantly inhibited accumulation of AGE compounds
compared to glibenclamide which is in agreement with in vitro BSA
glycation study. Extract and glibenclamide improved the solubility
pattern with a relative increase in pepsin soluble collagen. These
changes indicated a reduction in cross-linking of collagen proteins
in CC and glibenclamide treated diabetic rats. Though the glycemic
control achieved by CC 600 mg/kg was comparable with conven-
tionally used glibenclamide, its effect on inhibition of oxidative
stress, collagen glycation and formation of advanced glycation
end products was significantly better than glibenclamide.
Dietary measures form an important part of antidiabetic treat-
ment. Seeds of C. cyminum are used in daily food preparation in
many Asian countries. If dietary intervention could reduce the ensu-
ing diabetic complications it can be a powerful adjunct to conven-
tional antidiabetic treatment. Seeds of C. cyminum contain many
important phytoconstituents like essential oils, phenolic
compounds, steroids and flavonoids. This study shows that CC has
an ability to reduce hyperglycemia, oxidative stress, and formation
of AGE and thus may serve as an adjunct to conventional antidia-
 ARTICLE IN PRESS
A.G. Jagtap, P.B. Patil / Food and Chemical Toxicology xxx (2010) xxx–xxx
betic treatment which may help in attenuating diabetic
complications.
Conflict of interest
The authors declare that there are no conflicts of interest.
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