Journal of Functional Foods 22 (2016) 547–555

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Antidiabetic effect of new chromane isolated

from Dillenia indica L. leaves in streptozotocin
induced diabetic rats

Navpreet Kaur, Lalit Kishore, Randhir Singh *

M.M. College of Pharmacy, Maharishi Markandeshwar University, Mullana, Ambala, Haryana 133207, India

A R T I C L E I N F O A B S T R A C T

Article history: The chemical constituents of Dillenia indica were isolated together with evaluation of their
Received 6 December 2015 antioxidant and antidiabetic activity. Alcoholic extract of D. indica (DAE) was subjected to
Received in revised form 8 February column chromatography for the isolation of compounds and it yielded a new chromane 3,5,7,-
2016 trihydroxy-2-(4-hydroxybenzyl)-chroman-4-one. The structure was determined by 1H, 13C-
Accepted 10 February 2016 NMR and ESIMS. DAE (100, 200 and 400 mg/kg) and chromane (5 and 10 mg/kg) were
Available online administered to streptozotocin (STZ) (50 mg/kg) induced diabetic rats to assess their effect
on fasting blood glucose, serum insulin and lipid levels. Oral administration of chromane
Keywords: and DAE significantly attenuated the elevated fasting blood glucose, lipid levels and oxi-
Antioxidant activity dative stress. The results indicated that DAE and chromane isolated from D. indica possessed
Antidiabetic activity antioxidant and antidiabetic activity and could be a therapeutic agent for regulating several
Chromane pharmacological targets for management of diabetes.
Dillenia indica © 2016 Published by Elsevier Ltd.

quercetin, isorhamnetin and naringenin have been isolated

1. Introduction
Diabetes mellitus is a major challenge for healthcare systems
worldwide and strongly associated with several health risk
factors (Ogden, Carroll, & Flegal, 2003). Chronic hyperglycaemia
is the main cause for the development of diabetic complica-
tions, such as nephropathy, neuropathy, retinopathy and
cardiovascular disorders (Singh, Kaur, Kishore, & Gupta, 2013).
Dillenia indica (D. indica) Linn. (Family: Dilleniaceae) grows in
moist and evergreen forests in India. It has been grown in
gardens for its handsome foliage and attractive flower as an
ornamental plant. The fruit is said to possess tonic laxative
properties and used for relieving abdominal pain. The bark and
leaves are astringent (Kumar, Mallick, Vedasiromoni, & Pal, 2010;
Sastri, 2003). Phytochemical investigation has shown the pres-
ence of triterpenoids and flavonoids. Flavonoids like kaempferol,
(Banerji, Majumdar, & Dutta, 1975). Triterpenes like betulin,
betulinic acid, betulinaldehyde have been isolated from stem
extract of the plant (Haque et al., 2008; Parvin, Rahman, Islam,
& Rashid, 2009). Dillenia indica is a medicinal plant used for treat-
ing diabetes mellitus and related symptoms (Taraka et al., 2011).
Leaf extract of the plant possesses antidiabetic activity owing
to the insulin-mimetic effect of the extract. It also showed
favourable effects on histopathological changes of pancreas,
liver and kidney (Kumar, Kumar, & Prakash, 2011a, 2011b). A
functional food provides preventive or curative function against
one or more diseases, in addition to its adequate nutritional
benefits (Roberfroid, 2007). Dietary measures form an impor-
tant part of antidiabetic treatment (Benhaddou-Andaloussi et al.,
2008). In addition, there is a need for research in the field of
food product development and technology to create new func-
tional food products rich in bioactive compounds for people

* Corresponding author. M.M. College of Pharmacy, Maharishi Markandeshwar University, Mullana, Ambala, Haryana 133207, India. Tel.:
+91 9896029234; fax: +91 8059930172.
E-mail addresses: dahiya_rsd@rediffmail.com; randhirsingh.dahiya@gmail.com (R. Singh).
http://dx.doi.org/10.1016/j.jff.2016.02.016
1756-4646/© 2016 Published by Elsevier Ltd.
548 Journal of Functional Foods 22 (2016) 547–555
suffering from various metabolic syndrome like diabetes.
However, there is a particular need for education on healthy
diet and for interventions that show potential improvement
in the quality of a patient’s life. In the current work, a novel
compound was isolated from D. indica and subjected to in vitro
antioxidant activity and antidiabetic activity in comparison with
crude alcohol extract of D. indica (DAE). To the best of our knowl-
edge, it is the first report on the antioxidant and antidiabetic
activity of the purified fraction of D. indica.
2. Materials and methods
2.1. General
Melting point was recorded on Perfit melting point appara-
tus, Tamil Nadu, India. IR spectra were recorded on Perkin–
Elmer Spectrum RZX FT-IR spectrometer (KBr discs) (Boston,
MA, USA). NMR spectra were obtained from Bruker, AVANCE
II 400, NMR spectrometer (Billerica, MA, USA) (400 MHz for 1H
and 100 MHz for 13C NMR, TMS as internal standard). MS was
recorded on LC-MS Q-TOF micro mass spectrometer (LC-MS),
Waters mass spectrometer (Milford, MA, USA). TLC (0.5 mm thick
layer) was carried out on silica gel G (Finar, India Pvt. Ltd.) and
spots were visualised by spraying with 5% H2SO4.
2.2. Plant material
Leaves of Dillenia indica L. were procured from Kurukshetra Uni-
versity, Kurukshetra, India. Taxonomic identification was done
by Dr. Sunita Garg, Head, Raw Materials Herbarium & Museum,
Delhi (RHMD), NISCAIR, New Delhi, India. A voucher speci-
men (NISCAIR/RHMD/Consult/2013/2352-132-2) of the plant has
been deposited in Departmental Herbarium for future records.
2.3. Extraction and isolation of the active compound
Powdered leaves (500 g) were extracted sequentially with pe-
troleum ether, chloroform, alcohol and hydro-alcohol (40%)
using Soxhlet apparatus. DAE was filtered and concentrated
under reduced pressure at 40 °C to yield black brown residue.
The dried alcoholic extract (5 g) was then subjected to column
chromatography (silica gel packed column, Molychem 100-
200 mesh, 160 g) by pre-adsorbing with silica gel (10 g). Extract
was eluted using chloroform (100%) and the mixture of chlo-
roform and methanol up to 5%. The fractions (100 ml each)
obtained from the column were collected and combined on
monitoring TLC. Seventy five fractions were obtained. Frac-
tion 65–71 yielded a compound, 3,5,7,-trihydroxy-2-(4-
hydroxybenzyl)-chroman-4-one (chromane).
2.4. In vitro antioxidant activity
2.4.1. 2,2-diphenyl-1-picrylhydrazyl scavenging activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching ability was
measured by the method proposed by Shimada, Fujikawa,
Yahara, and Nakamura (1992). Freshly prepared DPPH solu-
tion (1.0 ml; 0.1 mM in methanol) was mixed with 3.0 ml of DAE
(1–10 µg/ml) and chromane (1–5 µg/ml). The mixture was in-
cubated at 25 °C for 30 min in the dark, and the absorbance
of reaction liquid was measured at 517 nm. Ascorbic acid was
used as the positive control. The percentage scavenging radical
was calculated using the following equation:
A0 − At
% Inhibition = × 100
A0
where A0 was the absorbance of control (blank without sample)
and At was the absorbance in the presence of sample. All the
tests were performed in triplicate and graph was plotted with
mean values.
2.4.2. Hydrogen peroxide scavenging activity
The activity was determined according to the method of Ruch,
Cheng, and Klauning (1989). An aliquot of 40 mM H2O2 solu-
tion (0.6 ml) was mixed with various concentrations of DAE (10–
320 µg/ml) and Chromane (10–50 µg/ml). To the mixture, 2.4 ml
of phosphate buffer (0.1 M, pH 7.4) was added and shaken vig-
orously and incubated at room temperature for 10 min. Then,
the absorbance of the reaction mixture was determined at
230 nm. Ascorbic acid was used as the positive control. The H2O2
scavenging activity was calculated as follows:
⎛ A − A2 ⎞
% Inhibition = 1 − ⎜ 1 × 100
⎝ A 0 ⎟⎠
where A0 is the absorbance of the control (water instead of
sample), A1 is the absorbance of the sample and A2 is the ab-
sorbance of the sample only (phosphate buffer instead of H2O2
solution).
2.4.3. Superoxide radical scavenging activity
The activity was measured by the reduction of nitroblue
tetrazolium reagent (NBT) by the method as described by
Shukla, Mehta, Bajpai, and Shukla (2009). The method is based
on generation of superoxide radical (O2−) by autoxidation
of hydroxylamine hydrochloride in the presence of NBT,
which gets reduced to nitrite. Nitrite in the presence of
ethylenediaminetetraacetic acid (EDTA) gives a colour that was
measured at 560 nm. Different concentrations of DAE (10–
320 µg/ml) and chromane (10–50 µg/ml) were taken in a test
tube. To this, reaction mixture consisting of 1 ml of (50 mM)
sodium carbonate, 0.4 ml of (24 mM) NBT and 0.2 ml of 0.1 mM
EDTA solutions was added to the test tube and immediate
reading was taken at 560 nm. After incubating the reaction
mixture at 25 °C for 15 min, about 0.4 ml of (1 mM) of hydrox-
ylamine hydrochloride was added to initiate the reaction and
reduction of NBT was measured at 560 nm. Ascorbic acid was
used as the positive control. Decreased absorbance of the re-
action mixture indicates increased superoxide anion scavenging
activity. The percentage of inhibition was calculated accord-
ing to the following equation:
A0 − At
% Inhibition = × 100
A0
where A0 was the absorbance of the control (blank, without
sample) and At was the absorbance in the presence of the
sample. All the tests were performed in triplicate and the graph
 Journal of Functional Foods 22 (2016) 547–555
was plotted with the mean values.
2.4.4. Reducing power assay
The Fe3+-reducing power of DAE and Chromane was deter-
mined according to the method of Oyaizu (1986). Different
concentrations of DAE (10–320 µg/ml) and Chromane (10–50 µg/
ml) (2.5 ml) were mixed with 2.5 ml of 0.2 M sodium phosphate
buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide and in-
cubated at 50 °C for 20 min. After incubation, 2.5 ml of 10%
trichloroacetic acid (w/v) was added and the mixture centri-
fuged at 67 × g for 8 min. The supernatant (5 ml) was mixed
with 5 ml of distilled water and 1 ml of 0.1% of ferric chlo-
ride, and the absorbance was measured spectrophotometrically
at 700 nm. The assay was carried out in triplicate and the results
were expressed as mean values ± standard deviations. The
sample concentration providing 0.5 of absorbance (EC50) was
calculated from the graph plotted between absorbance at
700 nm against sample concentration. Ascorbic acid was used
as a standard.
2.5. Animals
Adult male Wistar rats (250–300 g) were obtained from Central
Animal Facility, NIPER, Mohali. They were housed in standard
environmental conditions maintained at 23 ± 2 °C with 12 h
light–dark cycle. Animals were fed with standard rodent diet
and water ad libitum. Experimental protocol was approved by
Institutional Animal Ethical Committee (IAEC) and the experi-
ments were performed according to the guidelines of CPCSEA
(MMCP/IAEC/13/11).
2.6. Chemicals
Streptozotocin (STZ) was purchased from Sigma-Aldrich (Mil-
waukee, WI, USA) and Nicotinamide from Finar India Ltd. All
the other chemicals used were of analytical grade. Diagnos-
tic kits for the biochemical estimation were obtained from
Reckon Diagnostics, India.
2.7. Induction of diabetes
Diabetes was induced by a single intraperitoneal injection of
STZ (50 mg/kg) freshly prepared in 0.1 M citrate buffer (pH 4.5).
After 7 days of STZ-administration, the fasting blood glucose
level of each rat was measured. Animals with fasting serum
glucose more than 250 mg/dl were selected for the study.
2.8. Experimental design
Animals were divided into eight groups and each group con-
sists of six rats. Group 1 consisted of normal control, Group 2
consisted of diabetic control rats, Groups 3–5 consisted of dia-
betic rats treated with DAE 100, 200 and 400 mg/kg respectively,
Group 6 consisted of diabetic rats treated with Chromane 5 mg/
kg, Group 7 consisted of diabetic rats treated with Chromane
10 mg/kg, and Group 8 consisted of diabetic rats treated with
Glimepiride 10 mg/kg. Different doses of the DAE and Chromane
were administered orally for 21 days. Rats were fasted over-
night and blood samples were collected from retro-orbital plexus
under ether anaesthesia. Biochemical estimation was done by
549
measuring fasting blood glucose level, serum insulin level, and
lipid profile (total cholesterol, triacylglycerol, HDL levels) by using
commercially available kits of Reckon Diagnostics Pvt. Ltd. Body
weight was measured before the induction of diabetes and
during the experimental period. Animals were sacrificed at the
end of the study and kidneys were obtained and stored at −70 °C
for biochemical estimation.
2.9. Biochemical analysis
Kidney homogenate was used to estimate thiobarbituric acid
reactive substances (TBARS) (Ohkawa et al., 1979) and the level
of antioxidant enzymes, namely superoxide dismutase (SOD)
and reduced glutathione (GSH) (Beutler et al., 1963; Wang et al.,
1998).
2.10. Histopathology
Kidneys were obtained from the animals and fixed in 10%
neutral buffered formalin solution, dehydrated in ethanol and
embedded in paraffin. Sections of 5 µm thickness were pre-
pared using a rotary microtome and stained with haematoxylin
and eosin (H&E) dye for histopathological examination.
2.11. Statistical analysis
Statistical analysis was performed using Graphpad Prism 6.
Values were expressed as mean ± SEM and one way analysis
of variance (ANOVA) was used for statistical analysis. ANOVA
was followed by Tukey’s as post hoc multiple comparison test.
The results were considered significant if p ≤ 0.05.
3. Results
3.1. Identification of compound
Chromane was obtained as off white amorphous powder with
M.P. 280–290 °C. IR maxυKBr spectrum exhibited hydroxyl group
at 3279 cm−1, carbonyl group at 1629 cm−1 and C-H stretch at
2702–3055 cm−1. The structure of the Chromane was deter-
mined using 1H NMR, 13C NMR, MS and by comparison of data
with literature (Atun, Arianingrum, Sulistyowati, & Aznam, 2013)
(Table 1). This is the first report for the compound 3,5,7-
Trihydroxy-2-(4-hydroxy-benzyl)-chroman-4-one. The 1H NMR
spectra of Chromane showed one singlet of two hydrogen at
δ 5.88 suggesting the presence of a 5,7,9,10-tetrasubstituted aro-
matic ring. Additionally, it showed two doublets of two hydrogen
each at δ 6.79 (8.52 Hz) and δ 7.32 (8.52 Hz), suggesting the pres-
ence of another aromatic ring with 1,4 substitution pattern.
One double doublet at δ 3.26 (17.08 Hz) for two hydrogen was
assigned to vicinal hydrogens at position ‘2’ and ‘a’, suggest-
ing the presence of an oxygenated group attached to a
methylene bridge. Two double doublet at δ 2.68 (17.68 and
3.04 Hz) and δ 5.44 (12.8 and 2.8 Hz) confirmed the presence
of oxyalkyl protons. Four proton signals for hydroxyl groups
were observed at δ 8.03, 12.15, 9.59 and 10.85. 13C NMR data in-
dicated six non-hydrogenated aromatic carbons including four
oxygenated at δ 163.7, δ 159.4, 197.6 (carbonyl carbon) and δ
550 Journal of Functional Foods 22 (2016) 547–555

310.3 [M+] (22), 220.2 (100), 91.1 (5) the molecular formula C16H14O6
Table 1 – 1H NMR and 13C NMR values of Chromane.
was deduced to Chromane. These results suggested that the
1 13
Proton H NMR Carbon C NMR compound was a chromane with substituted four hydroxyl
2 3.26 2 78.39 groups. Therefore, the compound is structurally 3,5,7-trihydroxy-
3 5.45 3 94.94 2-(4-hydroxybenzyl)-chroman-4-one (Fig. 1).
6 5.88 4 196.36
8 5.88 5 163.45
3.2. In vitro antioxidant activity
a 3.26,2.68 6 99.49
2′ 7.32 7 166.64
3′ 6.79 8 95.75 In the present study several methods have been used to assess
5′ 6.79 9 162.91 the antioxidant capacity of the DAE and Chromane. Scaveng-
6′ 7.32 10 101.72 ing of DPPH, hydrogen peroxide and superoxide radicals can
3-OH 8.03 a 41.94 alleviate oxidative stress and reduce the generation of reac-
5-OH 12.15 1’ 128.82
tive carbonyl compounds (Wu, Huang, Lin, & Yen, 2011). The
7-OH 9.59 2’ 128.31
DPPH radical is considered to be a model for a lipophilic radical.
4′-OH 10.85 3’ 115.12
4’ 157.69 A chain in lipophilic radicals is initiated by the lipid auto-
5’ 115.12 oxidation (Ingold, Bowry, Stocker, & Walling, 1993). DAE (1–
6’ 128.31 10 µg/ml) and chromane (1–5 µg/ml) showed significant
scavenging activity against DPPH radical with increasing con-
centrations. Effect of DAE and new chromane moiety was
157.6, besides signals of four aromatic hydrogenated carbons compared with ascorbic acid. The IC50 values of DAE, Chromane
in ring A, where two of them, those at δ 128.31 and δ 115.12, and ascorbic acid were found to be 2.98, 2.36 and 1.43 µg/ml
corresponded to two CH each and two hydrogenated carbons respectively (Table 2). Hydrogen peroxide via ∙OH acts as a mes-
in Ring B at δ 99.49 and δ 95.75. In the aliphatic region of spec- senger in the synthesis and inactivation of several inflammatory
trum, signals were observed at δ 78.39 (C), δ 94.94 (C) and δ 41.94 mediators (Jayaprakasha, Rao, & Sakariah, 2004). DAE and
(CH2) compatible with a substituted pyran group with a hy- Chromane were found to possess potent hydrogen peroxide
droxyl at C-3. Based on these data and EIMS (m/z, rel. int. %) scavenging activity. The IC50 values of DAE, Chromane and ascor-
bic acid were found to be 228.69, 23.06 and 80 µg/ml respectively
(Table 2). Reducing power assay is generally associated with
O OH the presence of reductones in the sample, which have been
shown to exert antioxidant action by breaking the free radical
5' 4 5
HO HO chain by donating a hydrogen atom (Barros, Ferreira, Queiros,
10
6' 6 Ferreira, & Baptista, 2007). EC50 (effective concentration at which
4' 3
the absorbance is 0.5) was calculated from the calibration curve
1' 7 and found to be 111 µg/ml for DAE, 26.95 µg/ml for Chromane
3' 2 9 and 21.42 µg/ml for ascorbic acid (Table 2). Superoxide is a re-
2' a O OH
1 8 active oxygen species, which can cause damage to the cells
and DNA leading to various diseases. Superoxide radicals were
3,5,7-Trihydroxy-2-(4'-hydroxy-benzyl)-chroman-4-one generated by auto-oxidation of hydroxylamine in the pres-
ence of NBT (Shukla et al., 2009). The decrease of absorbance
Fig. 1 – Structure of compound at 560 nm with antioxidants indicates the consumption of su-
[3,5,7-Trihydroxy-2-(4-hydroxy-benzyl)-chroman-4-one]. peroxide anion in the reaction mixture. DAE and Chromane

Table 2 – In vitro antioxidant activity of DAE and chromane.

Conc. DPPH scavenging Conc. Hydrogen peroxide Superoxide dismutase Reducing
(µg/ml) activity (µg/ml) scavenging activity scavenging activity power activity
DAE
1 36.99 ± 0.58 10 26.31 ± 0.66 32.14 ± 1.04 0.39 ± 0.01
2 47.97 ± 0.58 20 30.04 ± 1.52 38.90 ± 0.04 0.41 ± 0.02
4 57.80 ± 0.58 40 34.64 ± 0.38 47.77 ± 0.08 0.45 ± 0.02
6 63.87 ± 0.58 80 43.85 ± 1.52 55.91 ± 0.56 0.50 ± 0.08
8 70.90 ± 0.17 160 48.90 ± 0.38 67.96 ± 0.93 0.61 ± 0.02
10 78.90 ± 0.58 320 54.16 ± 2.11 87.33 ± 0.51 0.78 ± 0.07
IC50 2.98 µg/ml IC50 228.69 µg/ml 75.09 µg/ml EC50 = 111 µg/ml
Chromane
1 33.05 ± 0.03 10 33.11 ± 0.02 34.65 ± 0.01 0.169 ± 0.01
2 47.66 ± 0.01 20 47.56 ± 0.02 46.56 ± 0.02 0.376 ± 0.03
3 59.14 ± 0.01 30 59.27 ± 0.01 58.36 ± 0.02 0.578 ± 0.02
4 66.87 ± 0.01 40 66.92 ± 0.01 68.76 ± 0.02 0.794 ± 0.04
5 78.34 ± 0.01 50 78.37 ± 0.01 75.66 ± 0.03 0.998 ± 0.01
IC50 2.36 µg/ml IC50 23.61 µg/ml 23.49 µg/ml EC50 = 26.95 µg/ml
 Journal of Functional Foods 22 (2016) 547–555 551

had significant activity against superoxide radicals in a dose-

10.03 ± 0.23b*c†
dependent manner. IC50 of ascorbic acid was found to be

9.29 ± 0.06b*
10.81 ± 0.18b*
13.2 ± 0.18b*
9.08 ± 0.30b*

12.95 ± 0.18b*
5.65 ± 0.17a*
15.51 ± 0.23
27.96 µg/ml, and those of DAE and Chromane were 75.09 and

28th day
23.49 µg/ml respectively (Table 2).

Serum insulin (µIU/ml)

Each group (N = 6) represents mean ± SEM. Data were analysed by using one way ANOVA followed by Tukey’s multiple test: avs control, bvs diabetic control, cvs Chromane 5 mg/kg.
3.3. Effect of DAE and chromane on fasting blood glucose,
body weight and serum insulin

8.00 ± 0.40a*
15.07 ± 0.08

7.29 ± 0.06
7.31 ± 0.13
7.42 ± 0.14
7.53 ± 0.38
8.16 ± 0.49
9.00 ± 0.31
Administration of different doses of DAE (100, 200 and 400 mg/

7th day
kg) and chromane (5 and 10 mg/kg) for 21 days produced
significant attenuation in elevated fasting blood glucose level
as compared to diabetic control rats. Glimepiride (10 mg/kg)
treatment also resulted in significant reduction of blood glucose

245.83 ± 1.48b*
252 ± 2.45b*
260.33 ± 2.68b*
239.33 ± 1.73b*
244.50 ± 1.65b*
251.33 ± 2.02b*
214.00 ± 3.39a*
level in rats. Animals with similar body weight were selected

293.00 ± 5.28
for the study. Body weight and serum insulin were signifi-

28th day
cantly reduced in diabetic control rats as compared to normal
control animals. Oral administration of DAE (100, 200 and
400 mg/kg) and chromane (5 and 10 mg/kg) in experimental rats
significantly increased body weight and serum insulin in com-
parison to diabetic control rats (Table 3).

237.33 ± 2.08b†

239.83 ± 1.84b#
242 ± 2.45b*

244.00 ± 1.47b*
224.00 ± 3.39a*

245.17 ± 1.2b*
294.33 ± 4.58

235.67 ± 1.83
Table 3 – Effect of DAE and chromane on fasting blood glucose, body weight and serum insulin in diabetic-rats.

14th day
3.4. Effect of DAE and chromane on serum lipid profile

Administration of STZ also produced dyslipidaemia in the form

Body weight (gm)

of elevated serum TC, TG level, and reduced HDL level. DAE (100,
200 and 400 mg/kg) and 5 and 10 mg/kg of chromane pro-

230.17 ± 2.74a*
duced dose-dependent attenuation in TC as compared to

293.70 ± 5.24

231.67 ± 2.70
233 ± 2.79
225.5 ± 3.55
231.67 ± 1.71
227.17 ± 2.89
233.50 ± 1.73
diabetic rats. Similarly TG level was also found to be reduced 7th day
(123.12 ± 3.09 and 112.78 ± 2.23 mg/dl) after administration of
5 and 10 mg/kg chromane, respectively, as compared to dia-
betic control rats, whereas DAE (100, 200 and 400 mg/kg)
reduced the level of TG to 119.65 ± 1.926, 112.63 ± 1.440 and
103.59 ± 1.453 mg/dl respectively. Moreover, serum HDL-c level

242.86 ± 4.11b*c*
249.83 ± 2.926b*
227 ± 2.041b*
208.33 ± 2.575b*
271.71 ± 3.38b*

209.12 ± 3.80b*
449.75 ± 4.02a*
96.55 ± 1.43

reduced significantly in diabetic control rats, and the groups

28th day

treated with DAE (100, 200 and 400 mg/kg) and chromane (5
and 10 mg/kg) increased the level of HDL-c in a dose-dependent
manner (Table 4).

3.5. Effect of DAE and chromane on level of antioxidant

281.87 ± 3.98b*c#
248.33 ± 3.182b*

enzymes and TBARS

271.17 ± 2.76b*
260.5 ± 3.98b*

299.32 ± 2.80b*

252.63 ± 2.99b*
410.31 ± 3.71a*
94.58 ± 2.16
Fasting blood glucose (mg/dl)
14th day

Oxidative stress ensues with the reduction of antioxidant

enzymes and increased lipid peroxidation. The level of anti-
oxidant enzymes (SOD and GSH) reduced significantly in the
kidney of diabetic rats. Treatment with DAE 100, 200 and 400 mg/
kg and 5 and 10 mg/kg of chromane significantly increased the
324.82 ± 2.73a*
302.83 ± 4.941
312.17 ± 4.118
315.67 ± 6.267
92.35 ± 2.10

315.76 ± 3.52
317.32 ± 3.03
318.15 ± 3.67

level of antioxidant enzymes, viz. SOD and GSH, compared to

diabetic rats. The level of TBARS (markers of lipid peroxidation)
7th day

was found to be elevated in diabetic rats as compared to normal

control group, whereas administration of DAE and chromane
* p < 0.001, #p < 0.01, †p < 0.05.

significantly reduced the level of TBARS in comparison to dia-

betic rats (Table 5).
Glimepiride 10 mg/kg
Chromane 10 mg/kg
Chromane 5 mg/kg

3.6. Histopathological
Diabetic control
DAE 100 mg/kg
DAE 200 mg/kg
DAE 400 mg/kg

Induction of diabetes leads to structural alterations in the

Groups

Normal

kidneys of diabetic animals. Renal tissue of diabetic rats also

showed glomeruli with mesangiocapillary proliferation. Ad-
ministration of DAE, chromane and glimepiride to the
552 Journal of Functional Foods 22 (2016) 547–555

experimental animals significantly improved the structural ab-

39.78 ± 0.74b*c#
42.16 ± 0.876b*
47 ± 0.686b*
49.16 ± 0.913b*
normalities in the kidney (Fig. 2).

35.95 ± 0.70b*

46.95 ± 0.62b*
27.33 ± 0.65a*
54.83 ± 0.85
28th day
4. Discussion

Each group (N = 6) represents mean ± SEM. Data were analysed by using one way ANOVA followed by Tukey’s multiple test: avs control, bvs diabetic control, cvs Chromane 5 mg/kg.
Drug regimens currently available for the management of dia-

38.61 ± 0.65b*c#
35.33 ± 0.335b*
37.66 ± 0.958b*
39.16 ± 0.479b*
29.06 ± 0.41b*

34.45 ± 0.97b*

39.91 ± 0.65b*
betes mellitus have certain drawbacks that encourage research
54.07 ± 0.53
14th day

for safer and effective antidiabetic agents (Singh et al., 2013).

Herbal medicinal agents are efficacious and safer to be used
for the management of diabetes (Farnsworth, Akerele, & Bingel,
1985). The aim of the present study was to evaluate the
31.36 ± 0.78a*

antioxidant and antidiabetic effect of D. indica alcohol extract

HDL (mg/dl)

30.293 ± 1.021
31.66 ± 1.483
55.18 ± 1.07

29.44 ± 1.63

32.86 ± 0.90
31.53 ± 0.76
31.18 ± 0.79
(DAE) and its isolated compound [3,5,7-trihydroxy-2-(4-
7th day

hydroxybenzyl)-chroman-4-one], against STZ-induced diabetic

rats. The experimental diabetic model used in this study mim-
icked type II diabetes since low dose of STZ (50 mg/kg) destroyed
half a population of pancreatic beta cells (Aybar, Sanchez Riera,
119.65 ± 1.926b*
112.63 ± 1.440b*
103.59 ± 1.453b*
123.12 ± 3.09b*
112.78 ± 2.23b*
105.95 ± 4.32b*
160.12 ± 4.15a*

Grau, & Sanchez, 2002). There were residual beta cells that se-
98.79 ± 2.01

creted insufficient insulin causing type II diabetic model (Gomes,

28th day

Vedasiromoni, Das, Sharma, & Ganguly, 2001). It produces

diabetes via its cytotoxic effects on pancreatic β-cells (Kim
et al., 2003). The cytotoxic action of STZ in rats produces
hyperglycaemia and oxidative stress. Excessive production of
126.25 ± 1.086b*
118.74 ± 0.919b*
109.81 ± 0.873b*
130.95 ± 3.09b#
123.78 ± 3.02b*
114.45 ± 2.49b*
148.12 ± 2.67a#

reactive oxygen species ensues oxidative stress, which plays

98.96 ± 1.24

a key role in the development of diabetes and its related com-

14th day

plications (Singh, Kishore, & Kaur, 2014). The present study

demonstrates that DAE and chromane exhibit potential in vitro
antioxidant activity, suggesting its potential in ameliorating oxi-
dative stress induced by diabetes.
138.45 ± 3.12a*
144.36 ± 4.090
142.11 ± 1.793
141.27 ± 1.497
99.13 ± 3.22

139.28 ± 2.04
138.78 ± 2.55
139.28 ± 2.87

The increased levels of fasting blood glucose in STZ-

TG (mg/dl)

induced diabetic rats were lowered by the administration of

7th day

DAE and its isolated compound (chromane) in dose- and time-

dependent manner. The reduced glucose levels might be
Table 4 – Effect of DAE and chromane on TC, TG and HDL in diabetic-rats.

suggested by insulin-like effect on peripheral tissues by either

promoting glucose uptake metabolism by inhibiting hepatic glu-
106.83 ± 1.281b*
99.17 ± 2.295b*
89.92 ± 2.298b*
107.62 ± 2.53b*
100.28 ± 2.86b*
96.12 ± 2.65b*
145.12 ± 3.34a*
88.46 ± 2.97

coneogenesis, or by absorption of glucose into the muscle and

28th day

adipose tissues, through the stimulation of a regeneration

process and revitalisation of the remaining beta cells (Kaur,
Kishore, & Singh, 2015).
3,5,7-trihydroxy-2-(4-hydroxybenzyl)-chroman-4-one is
structurally related to α-Tocopherol. Supplementation
115.33 ± 1.412b*
112.83 ± 2.937b*
108.67 ± 2.664b*
115.78 ± 1.65b*
111.28 ± 1.99b*
104.95 ± 2.02b*
135.12 ± 2.51a*
88.13 ± 2.28

of α-Tocopherol (Vitamin E) to diabetic rats attenuated

14th day

hyperglycaemia and neuroprotective effects on myenteric

neurons (Roldi et al., 2009). Troglitazone, thiazolidinediones,
structurally contains chromane moiety, sensitises tissues to
insulin action and is a promising agent in the treatment of type
2 diabetes. It may be considered as a valuable alternative in
121.78 ± 1.79a*
120.83 ± 2.015

123 ± 2.041
88.63 ± 2.76

124.17 ± 2.41

124.28 ± 1.73
122.78 ± 1.35
121.45 ± 1.23
TC (mg/dl)

insulin-resistant diabetic patients, who appear to be the best

7th day

responders to the drug (Scheen & Lefebvre, 1999). The pos-

sible mechanism of action of chromane might be that it
* p < 0.001, #p < 0.01, †p < 0.05.

stimulated the beta islets to secrete insulin and also reduced

insulin resistance.
Glimepiride 10 mg/kg
Chromane 10 mg/kg

STZ-induced diabetes is associated with the characteris-

Chromane 5 mg/kg

tic loss of body weight, which is possibly due to increased

Diabetic control
DAE 100 mg/kg
DAE 200 mg/kg
DAE 400 mg/kg

muscle wasting and protein catabolism. Oral administration

of DAE and chromane improved the body weight in compari-
Groups

Normal

son to diabetic control rats (Swanston-Fiatt, Day, Bailey, & Flatt,

1990). An increase in the body weight of diabetic rats might
be due to an improvement in insulin secretion and glycaemia.
 Journal of Functional Foods 22 (2016) 547–555 553

Table 5 – Effect of DAE and chromane on SOD, GSH and TBARS in kidney of diabetic-rats.
Groups SOD (U/mg protein) GSH (µM/mg protein) TBARS (nmol/mg protein)
Normal 3.87 ± 0.08 63.84 ± 1.79 0.55 ± 0.02
Diabetic control 1.24 ± 0.03a* 35.27 ± 0.44a* 2.30 ± 0.05a*
DAE 100 mg/kg 1.71 ± 0.03b* 40.91 ± 0.51b# 2.14 ± 0.01b†
DAE 200 mg/kg 2.09 ± 0.02b* 50.75 ± 0.35b* 1.96 ± 0.03b*
DAE 400 mg/kg 3.07 ± 0.06b* 58.67 ± 0.39b* 1.61 ± 0.02b*
Chromane 5 mg/kg 3.41 ± 0.03b* 39.91 ± 0.65 2.12 ± 0.04b†
Chromane 10 mg/kg 2.31 ± 0.04b*c* 45.25 ± 1.56b*c† 1.82 ± 0.04b*c*
Glimepiride 10 mg/kg 1.82 ± 0.02b* 54.84 ± 0.75b* 1.16 ± 0.03b*
Each group (N = 6) represents mean ± SEM. Data were analysed by using one way ANOVA followed by Tukey’s multiple test: avs control, bvs dia-
betic control, cvs Chromane 5 mg/kg.
* p < 0.001, #p < 0.01, †p < 0.05.

Lipid abnormalities, including increased levels of TC and TG
and decreased HDL-c level, are commonly associated with dia-
betes and predispose diabetic patient to atherosclerosis
and other cardiovascular complications like coronary heart
disease (Thomas & Karalliedde, 2015). Similarly, in the present
study, a positive correlation between hyperglycaemia and
dyslipidaemia was observed. Serum TC and TG levels were
found to be significantly elevated, whereas HDL levels de-
creased. This dyslipidaemia occurs due to increased breakdown
of lipid and free fatty acids from peripheral stores (Maria et al.,
2012). Administration of DAE and chromane significantly
reduced TC and TG levels. Moreover, DAE and chromane ef-
fectively increased the level of serum HDL-c.
Oxidative stress is defined as an imbalance between the
pro-oxidants and antioxidant defence system of the body as
a result of steady state reactive oxygen species. Oxidative
stress has recently been shown to be responsible, at least in
part, for pancreatic β-cell dysfunction caused by glucose
toxicity in hyperglycaemia (Atalay & Laaksonen, 2002).
STZ treatment causes significant increase in TBARS but
decreases antioxidant enzymes, such as glutathione peroxi-
dase and superoxide dismutase activities, when compared
with diabetic control animals in experiments (Gul, Laaksonen,
Atalay, Vider, & Hannien, 2002). Administration of different
doses of DAE and chromane ameliorated the oxidative
stress.

Fig. 2 – Histopathological changes in kidney of normal and treated rats (H&E × 100): (A) normal, (B) diabetic control, (C)
standard, (D) DAE 100 mg/kg, (E) DAE 200 mg/kg, (F) DAE 400 mg/kg, (G) Chromane 5 mg/kg treated, and (H) Chromane
10 mg/kg.
554 Journal of Functional Foods 22 (2016) 547–555
Diabetes is characterised by a series of renal structure ab-
normality, including basement membrane thickening, mesangial
expansion, glomerulosclerosis and tubulointerstitial fibrosis
(Abe et al., 2011). In the present study, histopathological ob-
servations of kidney showed glomeruli with mesangiocapillary
proliferation, along with amelioration of haemodynamic pa-
rameters of kidney. Administration of DAE and chromane
reversed these structural alterations in the kidney of experi-
mental animals due to antihyperglycaemic and anti-oxidant
activities.
5. Conclusion
A purified chromane was successfully obtained by column chro-
matography and some basic chemical analysis was done. 3,5,7-
trihydroxy-2-(4-hydroxybenzyl)-chroman-4-one and DAE
exhibited high scavenging activity towards superoxide anion,
hydroxyl, and DPPH radicals, along with significant reducing
power activity. Leaves of D. indica represent a good candidate
for alternative and/or complementary medicine in the man-
agement of diabetes mellitus, since they exhibited beneficial
effects on the fasting blood glucose levels and associated bio-
chemical parameters of STZ-induced diabetic animals. All these
experimental results made the chemical basis for the ben-
efits of D. indica towards antidiabetic activity and lay the
foundation for further research on underlying mechanisms.
Acknowledgement
Financial assistance (F. No. SB/FT/LS-359/2012) from the De-
partment of Science and Technology, New Delhi, Government
of India is highly acknowledged.
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