1516 Regular Article Biol. Pharm. Bull. 35(9) 1516–1524 (2012) Vol. 35, No.

Meliacinolin: A Potent α-Glucosidase and α-Amylase Inhibitor Isolated

from Azadirachta indica Leaves and in Vivo Antidiabetic Property in
Streptozotocin-Nicotinamide-Induced Type 2 Diabetes in Mice
Rosa Martha Perez-Gutierrez*,a and Monica Damian-Guzmanb
a
Laboratorio de Investigación de Productos Naturales, Escuela Superior de Ingeniería Química e Industrias
Extractivas IPN; Av Instituto Politecnico S/N, Col Zacatenco, CP 07758, México D.F.: and b Departamento de
Tecnologia de Alimentos, Escuela Nacional de Ciencias Biologicas IPN; Carpio/Plan de Ayala S/N, Casco Santo
Tomas, CP 0734, México D.F. Received March 13, 2012; accepted June 7, 2012

In India, Azadirachta indica is typically known as ‘neem tree’ and its leaves has long been used in
the ayurvedic medical tradition as a treatment for diabetes mellitus. In-depth chromatographic investiga-
tion on chloroform extract resulted in identification of one new tetranortriterpenoid. Structural elucida-
tion was established on the basis of spectral data as 24,25,26,27-tetranor-apotirucalla-(apoeupha)-1α-
senecioyloxy-3α,7α-dihydroxy-14,20,22-trien-21,23-epoxy named by us as meliacinolin (1). The present study
investigated the effect hypoglycaemic, hypolipidemic, oxidative stress, insulin resistance, α-glucosidase and
α-amylase of 1 from A. indica. Diabetic rats were treated with 1 for 28 d and a set of biochemical parameters
were studied including: glucose level, total cholesterol, triglycerides, lipid peroxidation, liver and muscle gly-
cogen, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. We also looked into
liver function by determining glucose-6-phosphatase, glucokinase and hexokinase activities, and the effect on
insulin level. While in vitro inhibition of α-glucosidase and α-amylase enzyme activities were used as indices
of effect on glucose absorption. As a result we found that blood glucose level, serum biochemical parameters,
hepatic enzymes, thiobarbituric acid reactive substances, and insulin level were restored in streptozotocin
(STZ)-diabetic mice to normal levels with 1. Meliacinolin inhibited α-glucosidase and α-amylase activities.
We conclude that meliacinolin can efficiently inhibit insulin resistance, improvement of renal function, lipid
abnormalities, and oxidative stress, indicating that its therapeutic properties may be due to the interaction
of meliacinolin with multiple targets involved in diabetes pathogenesis. α-Glucosidase and α-amylase inhibi-
tors offer an effective strategy to lower the levels of post prandial hyperglycemia prevents the digestion of
carbohydrates.
Key words hypoglycaemic; hypolipidemic; insulin; neem

In India, Azadirachta indica is typically known as ‘neem
tree’ and its leaves has long been used in the ayurvedic medi-
cal tradition as a treatment for diabetes mellitus and peptic ul-
cers. Recalling the insecticidal properties of neem, researchers
began programs in the early 60’s to identify the active princi-
ples and screen them against major crop pests. In the last two
decades research on neem has been intensified and many agri-
cultural and medical properties of the tree were rediscovered.1)
There has been a tremendous interest in this plant as can be
evidenced by the voluminous work on its different parts have
been carried out by various groups, leading to the isolation
and structure elucidation of more than 135 compounds.2) A. in-
dica, belongs to the family of meliaceae.3) It is one of the most
useful medicinal plants.4) Blood glucose lowering activity of
A. indica seed oil and leaf extracts have been reported in vari-
ous models of diabetic animals.5–9) Ethanol extracts of neem
leaves have also been shown to demonstrate anti-lipid per-
oxidative, antihyperglycaemic and anti-hypercholesterolaemic
activities as well as reduce serum triglyceride level in diabetic
rat model.10) Chloroform extract produced attenuation of anti-
glycation non-enzimatic, increase level antioxidant enzymes,
glucose-6-phosphatase, glucokinase, hepatic glucogen and
insulin plasma levels.11) In other study aqueous extract of this
plant increased body weight gain and decreased blood glucose
in rats12) whereas supplementation with leaf powder (1 g daily)
in diabetic reduced symtoms of polydypsia, polyphagia and
The authors declare no conflict of interest.
* To whom correspondence should be addressed. e-mail: rmpg@prodigy.net.mx
headache.13) Oral administration of nimbidin demonstrated
significant hypoglycaemic effect in fasting rabbits.14) Azadi-
rachtolide possesses α-amylase and α-glucosidase inhibition,
anti-hyperglycemic and anti-lipidemic effect at dose of 50
and 100 mg/kg.15) In fact, this two tetranortriterpenoids are
known as active ingredients in A. indica that are in partly re-
sponsible for the decrease in blood glucose in animal models
of diabetes. The possible mechanisms underlying the hypo-
glycaemic activity of the aqueous leaf extract have also been
discussed indicated that A. indica leaf extract, in itself, was
found to have no action on peripheral utilization of glucose
or on hepatic glycogen however, increase insulin release in
rat pancreas.16) In this study we took advantage of the partial
protection exerted by suitable dosage of nicotinamide against
the beta-cytotoxic effect of streptozotocin (STZ) to create a
new experimental diabetic syndrome in adult mice that ap-
pears closer to non-insulin-dependent diabetes mellitus type
2 than other available animal models with regard to insulin
responsiveness to glucose and meliacinolin. In conclusion, this
novel diabetes type 2 syndrome with reduced pancreatic insu-
lin stores, which is similar to human diabetes type 2 in that it
has a significant response to glucose may provide a particu-
larly advantageous. Streptozotocin-induced severely diabetic
rats and streptozotocin-nicotinamide-induced mildly diabetic
mice were established to compare their characteristics and to
investigate the hypoglycaemic effects of antidiabetic drugs.17)
α-Glucosidase is an enzyme that plays a central role
in carbohydrate metabolism by hydrolysing the terminal
© 2012 The Pharmaceutical Society of Japan
September 2012
glycosidic bonds at the non-reducing end of saccharide poly-
mers to release α-glucose. Much attention has been given
to α-glucosidase in the pharmaceutical community because
inhibition of its catalytic activity leads to impaired glucose
absorption and a decrease in postprandial blood glucose
levels. Therefore, α-glucosidase is currently the preferred
target for the development of new antidiabetes agents and
some α-glucosidase inhibitors have been developed as clini-
cal drugs, such as acarbose and miglitol, these hypoglycemic
agents have their limitations and are known to produce serious
side effects.18) Pancreatic α-amylase is a key enzyme in the
digestive system and catalyses the initial step in hydrolysis
of starch to maltose and finally to glucose. Degradation of
this dietary starch proceeds rapidly and leads to elevated post
prandial hyperglycemia. It has been shown that activity of
human pancreatic α-amylase in the small intestine correlates
to an increase in post-prandial glucose levels, the control of
which is therefore an important aspect in treatment of diabe-
tes.19) Hence retardation of starch digestion by inhibition of
enzymes such as α-amylase would play a key role in the
control of diabetes. However, the discovery of specific high-
affinity inhibitors of pancreatic α-amylase for the development
of therapeutics has remained elusive. In the present study, we
extended the research on the antidiabetic effects of A. indica
to examine the possible actions of the new tetranortriterpe-
noid isolated from chloroform extract of A. indica on glucose
levels, lipid metabolism, oxidative stress, insulin resistance,
α-glucosidase, α-amylase.
MATERIALS AND METHODS
General Experimental Procedures IR spectra were
recorded on a Perkin-Elmer FTRI 1720X. 1H- and 13C-NMR
spectra were taken at a Bruker DRX-300 NMR spectrometer,
with UXNMR software package, was used for NMR experi-
ments; chemical shifts are reported in δ (ppm), downfield rela-
tive to tetramethylsilane (TMS) as an internal standard. The
NMR experiments were carried out using the conventional
pulse sequences as described in the literature. High resolution-
electron ionization (HR-EI)-MS were measured on a JEOL
HX 110 mass spectrometer. Precoated TLC silica gel 60 F254
aluminum sheets and Sephadex LH-20 from Sigma-Aldrich
(St. Louis, U.S.A.) were used. Column chromatography was
carried on Silica gel 60 (230–400 mesh, Merck Co., New
Jersey (U.S.A.), solvents used as eluents from Fermont (Cali-
fornia, U.S.A.).
Plant Material The aerial part of the plant A. indica was
collected in the month of August 2010 from Colima State
Colima. The plant material was taxonomically identified and
the voucher specimen was deposited in Herbarium of Escuela
Nacional de Ciencias Biologicas, IPN for further reference
(No. 7216).
Extraction, Isolation and Characterization of the A. in-
dica Tetranortriterpenoid-Derived Plant material was air
dried and the ground leaves (25 kg) was extracted twice with
chloroform each for 3 h. The plant extracts were combined and
evaporated in vacuo to generate a residue (1972 g residue). The
resulting extract was loaded onto a silica gel column chroma-
tography and eluted with chloroform–ethylic ether 4 : 0.5 and
7 fractions (F1–F7) have been obtained. These fractions were
then tested for hypoglycemic activity. Active fractions were
1517
pooled together according to their similarities provides by thin
layer chromatography analysis. The fraction F5 was the frac-
tion that showed hypoglycemic properties. F5 was subjected
to silica gel column chromatography eluted with chloroform–
ethyl acetate (4.5 : 1) to produce nine fractions (F4-1 to F4-9).
The active fraction F4-2 was subjected to chromatographed
over silica gel column using acetone–hexane 1 : 3 to yield five
subfractions (F42-1 to F42-5). The F42-5 fraction was further
purified by preparative plate using petroleum ether–ethyl ac-
etate 1 : 4 and visualized with UV at 254 nm. Fraction F425-4
was separated by Sephadex LH-20 using a gradient of CHCl3–
MeOH (from 10 : 1 to 5 : 1) to yield 1 (687 mg).
Animals The study was conducted in male mice, weigh-
ing about 20–25 g. Before and during the experiment, animals
were fed a standard laboratory diet (Mouse Chow 5015, Pu-
rina) with free access to water. Mice were procured from the
bioterio of Escuela Nacional de Ciencias Biologicas, and were
housed in microloan boxes in a controlled environment (tem-
perature 25±2°C). Animals were acclimatized for a period of
three days in their new environment before the initiation of
experiment. Litter in cages was renewed three times a week
to ensure hygiene and maximum comfort for animals. The
experiments reported in this study were can out following
the guidelines stated in Principles of Laboratory Animal Care
(NIH publication 85-23, revised 1985 and the Mexican Official
Normativity (Norma oficial Mexicana NOM-062-Z00-1999).
Food consumption and weight gain were measured daily.
Experimental Induction of Diabetes Type-2 diabetes
mellitus was induced in overnight fasted mice by administer-
ing a single dose of freshly prepared solution of streptozotocin
(STZ), 60 mg/kg body weight (b.w.) intraperitoneally (i.p.)) in
0.1 mol/L cold citrate buffer (pH 4.5), 15 min after the intra-
peritoneal administration of 120 mg/kg nicotinamide. The STZ
treated animals were allowed to drink 5% glucose solution
over night to overcome drug induced hypoglycemia. After 10 d
of development of diabetes, mice with moderate diabetes hav-
ing persistent glycosuria and hyperglycaemia (blood glucose
>250 mg/dL) were used for further experimentation.20)
Experimental Design Mice were divided into seven
groups, of six animals in each: (i) Group I normoglucemic
served as control and received 2% w/v Tween 80; (ii) Group
II served as diabetic control; (III) Groups III–V normal mice
for single dose of 1 at 10, 20 and 30 mg/kg; (IV) Groups
VI–VIII diabetic mice for single dose of 1 at 10, 20 and
30 mg/kg; Group IX diabetic mice administered with gliben-
clamide 4 mg/kg. Ten days after the STZ injection, animals of
groups III to VIII received 1 and glibenclamide, respectively.
Animals were fasted for 5 h prior to drug administration and
blood samples were obtained 0, 2 h, 4 h, 6 h, 8 h and 12 h af-
ter administration of test substances. In a parallel study four
groups (n=6) of diabetic rats were used to determine the
chronic effect of 1. After 4 weeks, the animals were fasted
overnight and autopsied under light ether anesthesia. All the
drugs solutions or vehicle were administered orally by gastric
intubations once daily at 9:00 a.m. for 28 d. At the end of the
experiment the effect each group was investigated by serum
determination of total cholesterol (TC), triglycerides (TG) and
high density lipoprotein (HDL)-cholesterol, using a commer-
cial Diagnostic Kit (Genzyme Diagnostics) and low density
lipoprotein (LDL)-cholesterol was calculated as the remaining
difference of total cholesterol and HDL. Blood glucose levels
1518
were measured employing the glucose oxidase–peroxidase
(GOD–POD) method.21) Lipid peroxidation, that is, thiobarbi-
turic acid reactive substances (TBARS) was estimated by the
method of Fraga et al.,22) and expressed as µM /g of liver and
kidney tissue.
Oral Glucose Tolerance Test Mice of each group were
orally administered meliacinolin at dose of 25 mg/kg body
weight on a daily basis for 28 d. At the end of the experiment,
an oral glucose tolerance test (OGTT) was performed to as-
sess the animals sensitivity to a high glucose load. Overnight-
fasted mice were fed orally 2 g glucose/kg b.w. Blood samples
were collected from the caudal vein from a small incision at
the end of the tail at 0 min (immediately after glucose load),
30, 60, 90 and 120 min after glucose administration.
Assay of Glycogen Content in Liver and Skeletal Muscle
and Glucose-6-phosphatase (G6Pase) and Glucokinase
(GK) Activities in Liver Mice were sacrificed by decapita-
tion, livers and kidneys were extracted. Frozen livers were
thawed, weighed and homogenized in Tris–HCl (5 mmol/L,
pH 7.4) buffer containing 2 mmol/L ethylenediaminetetra-
acetic acid (EDTA). Homogenates were centrifuged at 1000×g
for 15 min at 4°C. The supernatant was mixed with glucose-
6-phosphate dehydrogenase and reduced nicotinamide ad-
enine dinucleotide phosphate (NADPH) and the activity of
hexokinase was determined at 37°C for 3 min at 30 s intervals
at 340 nm.23) The hepatic glycogen content was measured ac-
cording to the anthrone–H2SO4 method.24) Briefly, liver tissue
(64–144 mg) was homogenized in five volumes of an ice-cold
30% KOH solution and the homogenate was placed in a boil-
ing water-bath (l00°C) for 20 min. The glycogen was re-dis-
solved in distilled water and treated with an anthrone reagent
(2 g anthrone/L of 95% (v/v) H 2SO4) and the absorbance was
measured at 620 nm.
The hepatic G6Pase activity was assayed by the method of
Baginski et al.25) Shortly, the glucose-6-phosphate in the liver
extract was converted into glucose and inorganic phosphate.
The inorganic phosphate liberated was determined with am-
monium molybdate; ascorbic acid was used as the reducing
agent. Excess molybdate was removed by the arsenite–citrate
reagent, so that it could no longer react with other phosphate
esters or with inorganic phosphate formed by acid hydrolysis
of the substrate. The amount of phosphate liberated per time
unit, determined as the blue phosphor–molybdous complex at
700 nm, was a measure of the glucose-6-phosphatase activ-
ity. The protein content of the liver extract was quantified by
Bradford reaction (Bio-Rad protein assay kit).26) The G6Pase
activity (mU) was expressed as mmol of phosphate released/
min/mg of protein.
GK activity was measured using a spectrophotometric
method as previously described by Panserat et al.27) Briefly,
liver tissues were homogenized and the supernatant obtained
by centrifugation was supplemented with 1 m M NADP, 5 m M
ATP, and l00 or 0.5 m M glucose at pH 7.5. The enzymatic
reaction was started by the addition of 0.2 unit of glucose-
6-phosphate dehydrogenase (EC 1.1.1.49) and incubated for
5 min at 37°C. NADPH generated by GK was measured using
a spectrophotometer at 340 nm. GK activity was estimated by
the standard method, i.e. subtracting the rate of NADPH for-
mation in the presence of 0.5 m M glucose from that obtained
in the presence of 100 m M glucose.28) Protein concentration
was quantified by Bradford and one unit of enzyme activity
Vol. 35, No. 9
(mU) was defined as mmol of substrate molecules converted
by 1 mg protein per minute. GK activity was estimated as
the difference in activity when samples were assayed at
100 mmol/L (GK plus hexokinase activity) and 0.5 mmol/L
glucose (hexokinase activity).
Measurements of Glutathione Reductase (GSH), Super-
oxide Dismutase (SOD), Catalase Activity (CAT), Glutathi-
one Peroxidase (GPx) and Lipid Peroxidation After 28 d
of treatment, mice were fasted overnight and were euthanized
by anesthesia. Antioxidant enzyme activities in the liver,
pancreas and kidney were assayed using commercial kits:
SOD assay kit Bioxytech SOD-525 for SOD activity (Oxis
International), catalase assay kit for CAT (Cayman Chemi-
cal), and GSH assay kit Bioxytech GR-340 for GR activity,
(Oxis International), GPx assay kit GPx-340 for GPx (Oxis
International) and lipid peroxidation using malondialdehyde
level by commercial kit (thiobarbituric acid-reactive substance
(TBARS) Assay Kit). In the pancreas the protein concentra-
tion was determined by the Bradford method as described in
the Bio-Rad protein assay kit.
Determination of Serum Insulin Level, and Pancreatic
Insulin Content Diabetic mice serum and pancreatic insulin
were measured by a Glazyme Insulin-EIA Test according to
the manufacturer’s instructions.29) Blood samples and pancreas
were taken for insulin determination. The level of insulin in
serum was expressed in µIU/mL.
α-Amylase Inhibition The known porcine pancre-
atic α-amylase (PPA) and acarbose were used for preliminary
screening of α-amylase inhibitors from the compound. The
inhibition assay was performed using the chromogenic DSA
method.30) The total assay mixture composed of 500 µL of
0.02 M sodium phosphate buffer (pH 6.9 containing 6 m M so-
dium chloride), 0.04 units of PPA solution, and 1 at concentra-
tion from 10–100 µg/mL were incubated at 37°C for 10 min.
After pre-incubation, 500 µL of 1% (v/v) starch solution in the
above buffer was added to each tube and incubated at 37°C
for 15 min. The reaction was terminated with 1.0 mL DNSA
reagent, placed in boiling water bath for 5 min, cooled to room
temperature, diluted, and the absorbance measured at 540 nm.
The control reaction representing 100% enzyme activity did
not contain any compound. One unit of enzyme activity is
defined as the amount of enzyme required to release one
micromole of maltose from starch per min under the assay
conditions. For the determination of the inhibitor concentra-
tion at which 50% inhibition of enzyme activity occurs (IC50),
the assay was performed as above except that the inhibitor/
compound concentrations were varied from 10–100 µg/mL.
Acarbose was used as a positive control at a concentration
range of 6.5–32.8 µg/mL. The IC50 values were determined
from plots of percent inhibition versus log inhibitor concentra-
tion and calculated by logarithmic regression analysis from
the mea n inhibitory values. The IC50 values were defined as
the concentration of the 1, containing the α-amylase inhibitor
that inhibited 50% of the PPA activity.
Inhibition of α-Glucosidase α-Glucosidase activity
was determined by incubating a solution (0.1 mL) of enzyme
preparation with Tris buffer, pH 8 (0.1 mL) containing increas-
ing concentration (5, 10, 20, 40, 80, 100 µg/mL) of the (0.1 mL)
inhibitor at 37°C for 60 min. The reaction mixture was heated
for 2 min in a boiling water bath to stop the reaction. The
amount of liberated glucose was measured by glucose oxidase
September 2012 1519

method. The absorbance was measured at 540 nm.31) To fur-
ther study the inhibitory property of 1 as an α-glucosidase
inhibitor, the concentration-dependent effect of 1 on the inhi-
bition of α-glucosidase was evaluated. Varying concentrations
of 1 were added to the α-glucosidase-catalysing reaction.
Statistical Analysis Values obtained were expressed as
mean±standard error mean (S.E.M.) and analyzed using the
statistical package for social sciences (SPSS) version 10 using
analysis of variance (ANOVA) followed by Dunnett’s test and
p<0.05 was considered to be significant.
RESULTS
Structural Elucidation of Tetranortriterpenoid
24,25,26,27-Tetranor-apotirucalla-(apoeupha)-1α-senecioyl-
oxy-3α,7α-dihydroxy-14,20,22-trien-21,23-epoxy (1). IR ν max
(CHCl3) cm−1: 3423 (OH), 1708 (ester), 1462 (C=C), 1140
and 1080 (ether linkage); HR-EI-MS (positive) m/z 510.2993
[M+H]+ Calcd for C31H42O6, 510.2981; electrospray ionization
(ESI)-MS-MS m/z 510 [M], (2), 477 [M−CH3−H2O] (5), 443
[C27H39O5] (2), 403 [C24H36O5] (2), 380 [C23H24O5] (100), 335
[C19H27O5] (3), 304 [C18H24O4] (3), 284 [C14H19O3 or C15H24O5],
(6), 236 [C17H16O] (6), 192 (17), 156 [C8H12O3] (1), 83 [C5H7O]
(3); 1H- and 13C-NMR (CDCl3) data, Table 1.
Compound 1 has a molecular formula on the basis of the
positive HR-EI-MS showing a ion peak at m/z 510.2993 (Calcd
for C31H42O6, 510.2981). The IR spectrum revealed absorp-
tion bands of hydroxyl (3423 cm−1), ester (1708 cm−1), ether
(1139, 1082 cm−1), and double bond (1462 cm−1) groups. The
13
C-NMR (distortionless enhancement by polarization transfer
(DEPT)) exhibited 31 carbon atoms assigned to six methyl
group, five methylenes, twelve methines and eight quaternary
carbon atoms one of which was carbonyl group. The tetran-
ortriterpenoid character of 1 was indicated by the presence of
four quaternary methyl singlets at δ H 0.93 (H-18), 0.95 (H-19),
1.12 (H-29), and 1.20 (H-30). The 1H-NMR spectrum further
showed two one-proton signals at δ H 5.32 (t, J=1.5 Hz) and δ H
2.46 (m) responding to H-15 and H-17, respectively. These sig-
nals and their corresponding carbons at δC 119.6 and δC 51.9,
respectively, along with the carbons at δC 147.3 (C-14) and
δC 34.5 (C-16) were suggestive of ring-D of 14,15 deoxy-ge-
dunin.32) H-15 of 1 appeared downfield as compared to those
of deoxy-gedunin and nimocinol which supported the hydroxy
substituent at C-7.33) Similarly, the double bond at C-14 re-
sulted in a downfield shift of H-7. Further, oxygen-bearing
methane signals at δC 79.6 and δ H 4.28 (d, J=3.0 Hz) displaced
an upfield methylene resonance indicating that a methylene
carbon was substituted by a hydroxyl group. The positioning

Table 1. 300 MHz 1H- and 13C-NMR Spectral Data of Compound 1

C/H No. δH J (Hz) δC HMBC correlations protons

1 4.12 t (J=3.0) 74.1 H-3, H-19

2 2.00 m 29.6 H-2
3 4.20 t (J=3.0) 75.2 H-2, H-28, H-29
4 — — 44.2 H-3, H-28, H-29
5 2.35 d (J=12.0) 39.1 H-28, H-29
6 3.98 dd (J=12.0, 3.0) 76.3 H-5, H-7
7 4.28 d (J=3.0) 79.6 H-5, H-6
8 — — 44.2 H-7, H-9, H-15
9 2.28 dd (J=9.5, 3.0) 38.5 H-5, H-7, H-11
10 — — 41.5 H-2, H-5, H-6, H-11
11 1.61 m 17.5 H-9
12 1.65 m 30.6 H-9, H-11, H-18
13 — — 36.4 H-15, H-18
14 — — 147.3 H-7, H-15, H-17, H-18
15 5.32 t (J=1.5) 119.6 H-17
16 2.30 m 34.5 H-15, H-17
17 2.46 m 36.9 H-15, H-18, H-21
18 0.93 s 19.8 H-17
19 0.95 s 14.3 H-5, H-9
20 — — 122.5 H-17, H-21, H-22
21 7.12 dd, (J=1.6, 0.9) 146.0 H-17, H-23
22 6.80 dd, (J=1.6, 0.9) 112.6 H-17, H-21
23 7.36 t (J=1.5) 128.7 H-17, H-21
24a 4.12 d (J=7.5) 77.6 H-3, H-5, H-29
24b 3.72 d (J=7.5) 77.6 H-3, H-5, H-29
25 1.12 s 22.1 H-3, H-5, H-28
26 1.20 s 30.7 H-7, H-9
Osen
1′ — — 171.7 H-2′, H-3
2′ 5.67 (br s) 128.3 H-4′, H-5′
3′ — — 123.7 H-2′, H-4′, H-5′
4′ 1.46 (br s) 27.5 H-2′
5′ 2.12 (br s) 21.0 H-2′
Senecioiloxi substituent (Osen).
1520 Vol. 35, No. 9

Fig. 1. 1
H–1H COSY — Correlations Detected for Meliacinolin
of the hydroxyl group at C-7 was based on observation of
the heteronuclear multiple bond connectivity (HMBC) cor-
relation of the proton at δ H 4.28 (1 H, d, J=3.0 Hz, H-7) and
the carbons at δC 39.1 (C-5), 44.2 (C-8), 38.5 (C-9), and 147.3
(C-14). One oxygenated methine signals appeared at δ H 4.20
(t, J=3.0 Hz) and δC 75.2, this implied that a hydroxyl group is
located at C-3. The assumption was confirmed by the correla-
tions of Me-29 to C-3 in de HMBC experiment. According to
the nuclear Overhauser effect spectroscopy (NOESY) correla-
tions of H3-19 to H3-29 and H3-29 to H-3. This supported that
the hydroxyl groups at C-3 and C-7 are axial. The 1H-NMR
spectrum (Table 1) exhibited three downfield signals typi-
cal of a furan side chain at δ H 7.12 (dd, J=1.6, 0.9 Hz, H-21),
6.80 (dd, J=1.6, 0.9 Hz, H-22), 7.36 (t, J=1.5 Hz, H-23). This
was supported by the presence of a pair of doublet bonds at
δC 122.5 (C-20), 146.0 (C-21), and 112.6 (C-22), 128.7 (C-23),
in the 13C-NMR spectrum (Table 1). These values for the
furan ring are comparable with those of deoxygedunin.32) In
ring A, a senecioyl substituent is present at C-1 (δ H-1 4.12, t,
J=3.0 Hz; δ H-2′ 5.67, br s; δ H-4′ 41.46, br s; δ H-5′ 2.12 br s; δC-1
74.1, δC-1′ 171.7, δC-2′ 128.3, δC-3′ 123.7δC-4′ 27.5, δC-5′ 21.0. The
interaction of C-1′ with H-1 in the HMBC plot displayed the
senecioyl moiety at C-1, which was also evident from the
mass fragment at m/z 83. Two one-proton double doublets at
δ H 3.98 (J=12.0, 3.0 Hz) and δ H 2.28 (J=9.5, 3.0 Hz) were at-
tributable to H-6 and H-9, respectively. The chemical shift and
multiplicity of H-6 suggested an oxygen function at C-6. The
oxygen at C-6 form an ether linkage between C-6 and C-28
(δ H-6 3.97, dd, J = 12.0, 3.0; δ H-28a 4.12 d, J=7.5 Hz; δ H-28b
3.72, d, J=7.5 Hz; δC-6 76.3, δC-28 77.6. HMBC data Table 1
allowed the connection of the moieties, in particular, cross-
peaks of Me-19, Me-25, and Me-18 indicated the structure
of ring A, including the placement of an senecioyloxy at C-1
and the hydroxyl at C-3, cross-peaks of H-23 (δ H 7.36) with
C-21 (δC 146.0) suggested the presence of an oxygen bridge
between C-23 and C-21, given rise to a furan ring. Interac-
tions of C-1′ with H-3 displayed the osen group at C-3, C-15
had a connectivity with H-17 while C-22 showed connectiv-
ity with H-17. Interactions in the NOESY spectrum showed
connectivity H-3 with H-6, H-19 and H-26. An interaction
H-17 with H-23; and H-17 with H-26. The α orientation of
the side chain could be deduced from the spatial interac-
tion of H-18 with H-22 and that of H-17 with H-26 in the
NOESY spectrum. 1H–1H correlation spectroscopy (COSY)
correlations are showed in the Fig. 1. Thus, the structure of
1 was determined as 24,25,26,27-tetranor-apotirucalla-(apo-
eupha)-1α-senecioyloxy-3α,7α-dihydroxy-14,20,22-trien-21,23-
epoxy named by us as meliacinolin.
Normal, STZ-Induced Changes of Glucose Levels, Body
Weight and Food and Water Intake Table 2 shows effect
of a single dose treatment on blood glucose determined at
various time intervals for 12 h. Treatment of 1 the mice with
doses of 10, 20 and 30 mg/kg, control drug: glibenclamide
4 mg/kg, significantly decreased blood glucose in diabetic
mice. The percentage of glucose reduction at 6 h was 22, 29
and 40%, respectively. Gibenclamide 4 mg/kg dose produced
41.0% of blood glucose reduction at 6 h. Maximum percent-
age of blood glucose reduction to normoglycemic rats was
achieved at 6 h with 30 mg/kg of 1 (57.33%). Gibenclamide
4 mg/kg dose produced 30.5% blood glucose reduction at 8 h
in no diabetic rats (Table 1).
In the chronic study, daily administration of 1 the body

Table 2. Effect of 1 on Fasting Blood Glucose Level in Diabetic Mice

At the time of Blood glucose levels (mg/dL) at different time intervals (h)
Group Dose (mg/kg)
grouping 2h 4h 6h 8h 12 h

Non-diabetic — 103.48±1.34 101.61±0.95 102.25±1.38 100.25±0.98 99.54±1.75 98.67±0.85

control
10 103.18±0.92 93.25±4.21a 86.31±1.95a 80.46±0.87a 85.22±3.51a 90.47±0.63a
Non-diabetic
20 102.35±3.45 85.43±5.38a 56.65±2.48a 51.06±1.54a 55.13±2.37a 66.47±1.39a
control+1
30 104.29±1.29 81.31±4.95a 49.61±2.38a 44.50±1.76a 52.56±2.85a 63.25±1.83a
Non-diabetic 4.0 99.56±5.19 89.49±5.22 82.67±4.39 74.13±4.73 69.21±3.89a 54.37±2.76a
control+GB
Diabetic control — 383.34±3.67 378.87±4.17 368.39±4.10 367.84±5.16 363.48±4.47 360.62±5.33
10 346.23±3.29 324.18±4.38b 311.68±4.80b 270.23±3.55b 274.19±2.76b 292.43±2.56b
Diabetic+1 20 369.68±2.81 328.43±3.43b 279.89±2.68b 262.14±5.89b 268.36±3.49b 276.52±4.24b
30 359.34±4.20 305.71±5.17b 258.48±3.36b 215.24±4.19b 219.87±1.87b 227.48±3.98b
Diabetic+GB 4.0 351.46±5.79 276.78±6.87b 205.19±2.59b 206.30±2.94b 212.57±1.98b 229.17±2.83b
Each values represent mean±S.D. (n=6). a p<0.05 compared to normal group (ANOVA) followed by Dunnett’s test. b p<0.05 compared to diabetic group (ANOVA) fol-
lowed by Dunnett’s test. Gibenclamide (GB).
September 2012 1521

weight and food, water intakes in control and experimental
animals are shown in Table 3; the induction of STZ-diabetes
resulted in elevated intake of both, significant decrease in
body weight during the 28 d was observed in the diabetic
control mice compared with control mice, showing no differ-
ence between initial and final values; however, administration
of 1 to diabetic mice increased body weight gain significantly
(from 22.4 to 29.9 g). Diabetic mice showed increase in food
and water intakes as compared to normal control mice but the
administration of meliacinolin led to a decrease in water and
food intake in experimental groups.
Oral Glucose Tolerance Test The effect of 1 on the
blood glucose tolerance was determined after treatment by
28 d. Postprandial blood glucose levels in mice show a sig-
nificant change after glucose loading, increasing rapidly in
all groups of diabetic mice within 30 min and remaining high
over the next 120 min in diabetic control mice. Table 4 shows
the changes in the levels of blood glucose in diabetic control
and experimental group after oral administration of glucose
(2 g/kg). Oral treatment (20 mg/kg) in diabetic control mice
with 1 showed significant (p<0.05) reduction in blood glucose
levels over 120 min compared to STZ-diabetic mice showed
47% decreased in glucose levels compared with STZ-diabetic
mice.
Determination of Serum Total Cholesterol, Triglyc-
erides, LDL-Cholesterol and TBARS Levels in Diabetic
Mice Liver and Kidney after Treatment with Meliacinolin
There was a significant elevation in serum triglycerides, total
cholesterol and TBARS levels in the liver and kidney of dia-
betic groups in comparison to control. Daily administration of
1 at a dose of 20 mg/kg to diabetic mice for 28 d significantly
reduced total cholesterol and triglycerides by 46.0% and 47%
respectively. TBARS level in diabetic mice also decreased
after treatment with the compound, a 39.0% in liver and 31%
in kidney. Administration of this tetranortriterpenoid results
a significant diminution of these parameters and the levels of
these parameters were resettled toward the control level (Table
5). While HDLc, a friendly lipoprotein, was decreased in
diabetic groups in respect to the control (Table 5). After 28 d
of administration of 1 there was a significant elevation of this
lipoprotein level in serum and was resettled toward the control
level. The results also demonstrate a significant control of se-
rum lipid profiles in the 1 treated diabetic mice.
Effect of 1 on G6Pase, GK, Glycogen and Hexokinase
(HK) Activity Table 6 shows the effect of 1 on G6Pase, GK,
HK activity and glycogen content of liver and skeletal muscle.
The administration of 1 increased the content of hepatic gly-
cogen, GK and HK in diabetic mice while G6Pase decreased.
The result showed that the hexokinase activity tended to be
reversed to normal values, while normal mice did not exhibit
any significant alteration. Furthermore, the attenuating effect
of this compound on experimental physiological symptoms of

Table 3. Effect on Physico-Metabolic Symptoms of Meliacinolin (1)

Body weight (g) Intake (g/d)

Group (mg/kg)
Initial Final Gain Food Water
a a a a
No-diabetic 23.7±2.7 39.5±6.3 15.8±5.2 21.6±3.3 40.8±5.6a
Diabetic 21.4±4.6c 23.2±5.8c 1.8±0.3c 30.8±2.9c 140.8±6.5c
1 (20 mg/kg) 22.4±5.5c 29.9±7.4b 7.5±2.7a 24.3±4.1c 100.2±7.0a
Each value represents mean±S.E.M. (n=10), ANOVA followed by multiple two-tail “t” test. In each vertical column, mean with different superscripts (a, b, c) differ from t′
each other significantly, p<0.05.

Table 4. Meliacinolin (1) on Oral Glucose Tolerance Test (OGTT) in Diabetic Rats

Blood glucose levels (mg/dL)

Groups
0 min 30 min 60 min 90 min 120 min
a a a
Normal 92.76±1.82 181.37±3.17 162.49±2.83 129.13±2.49 96.21±1.53a
Diabetic control 270.23±3.42 346.71±1.36b 412.86±5.21c 376.29±5.12c 351.63±3.71a
1 (20 mg/kg) 266.35±4.71 327.80±5.12b 247.36±4.71c 200.62±3.14c 141.29±1.45a
GB (4 mg/kg) 239.73±2.75 312.34±2.67b 221.43±3.28c 172.74±4.96c 138.56±2.53c
Values are mean±S.D. (n=6). ANOVA followed by multiple two tail “t” test. Line with (a, b, c) fixed within duration of measurement differ from each other significantly,
p>0.05.

Table 5. Effect of Meliacinolin (1) on Lipid Profile in STZ-Induced Diabetic Rats

Mean concentration (mg/g)±S.E.M.

Group (mg/kg) TBARS (µM/g)

Triglycerides (mg/dL) Total cholesterol (mg/dL)HDL-cholesterol (mg/dL)
Liver Kidney

Normal 92.26±4.86 130.10±6.71 68.24±5.21 0.98±0.006 1.7±0.03

Diabetic control 186.32±5.19a 246.32±2.74a 34.48±1.26a 1.62±0.05a 2.6±0.08a
1 (20 mg/kg) 90.87±4.19ab 130.68±5.82ab 58.53±2.85ab 0.99±0.08ab 1.8±0.05ab
GB 4 mg/kg 89.94±5.28ab 126.32±2.65ab 54.39±2.75ab 0.93±0.09ab 1.5±0.04ab
All values are expressed as mean±S.E.M., n=6 values. a p<0.05 when compared to normal control group, b p<0.01 when compared to diabetic control group, where the
significance was performed by one-way ANOVA followed by post hoc Dunnett’s test.
1522 Vol. 35, No. 9

Table 6. Effect of Meliacinolin (1) on Hepatic Glucose Regulation Enzyme Activities (G6Pase and GK), Hepatic Glycogen Content and Hexokinase
(HK) in Diabetic Mice

Glycogen (mg/g) HK activity (U/mg

Group (mg/kg) G6Pase activity (mU) GK activity (mU)
Liver Skeletal muscle protein)

Normal control 0.38±0.004 3.32±0.07 19.60±2.43 11.58±2.83 1.62±0.04

Diabetic control 0.69±0.006a 1.22±0.03a 9.25±1.65a 4.40±1.10a 1.30±0.02a
1 (20 mg/kg) 0.40±0.008ab 2.91±0.09ab d
17.86±1.98b d
10.84±2.38b d
1.59±0.05ab
GB 4 0.36±0.009b 3.11±0.07b 17.78±2.31b 11.01±1.98b 1.60±0.03ab
Each values represent mean±S.D. (n=6); ANOVA followed by multiple two tail “t” test. In each vertical column, mean with different superscripts (a, b) differ from each
other. All values are expressed as mean±S.D., n=6 values. Significant difference of diabetic control from normal control a p<0.001. Significant difference of treated groups
from diabetic control b p<0.01, c p<0.05. d p<0.01 when compared with glibenclamide 4 mg/kg treated group.

Table 7. Effect of Meliacinolin (1) on Antioxidant Enzyme Activities in Liver and Kidney in Diabetic Mice

Parameters Normal control Diabetic control Diabetic+1 (20 mg/kg) Diabetic+GB (4 mg/kg)
a b
SOD-Liver 7.51±1.67 3.82±0.76 6.75±0.50 6.80±0.41c
SOD-Kidney 13.79±2.43 7.16±1.81a 12.80±2.23b 13.025±1.57b
CAT-Liver 82.15±4.54 44.64±2.53a 69.75±4.21b 70.26±2.16b
CAT-Kidney 35.07±2.76 20.76±1.65a 33.41±2.47c 34.17±1.79b
GSH-Liver 47.21±2.80 21.80±1.97a 42.23±2.19b 42.38±2.28b
GSH-Kidney 24.18±5.36 5.79±2.06a 19.13±2.12b 19.27±3.58b
GPx-Liver 7.26±1.59 4.31±1.30a 5.72±4.37b 5.92±1.26b
GPx-Kidney 5.92±1.42 3.53±0.92a 4.76±2.48b 4.76±0.94b
All values are expressed as mean±S.E.M., n=6 values. a p<0.01 when compared to normal control group; b p<0.01 c p<0.05 compared to diabetic control group; where the
significance was performed by one-way ANOVA followed by post hoc Dunnett’s test. Glibenclamide (GB). The values are given in U/mg of protein.

Table 8. Effect of Meliacinolin (1) on Plasma Insulin Level and Pancre- Table 9. Inhibitory Effects of Meliacinolin (1) on α-Amylase and
atic Insulin Content in Diabetic Mice α-Glucosidase

Pancreatic insulin Concentration % Inhibition

Plasma insulin (µU/mL)
(µU/mL) (µg/mL)
Groups α-Amylase α-Glucosidase Acarbose
Before 1 adminis- After 1 adminis- After 1 adminis-
tration (0 h) tration (28 d) tration (28 d) 10 34.15±1.98 49.23±1.56 18.6±3.63
20 41.65±1.93 46.21±1.98 23.6±3.75
No-diabetic 3.51±0.78 3.60±0.21 25.78±4.12 40 54.78±2.65 57.30±3.67 28.7±3.21
control
60 68.53±2.87 69.48±2.93 39.16±2.54
Diabetic control 0.75±0.59 1.42±0.18a 14.87±3.76a
d
80 74.68±3.54 76.23±2.73 57.68±2.76
1 (20 mg/kg) 0.73±0.43 3.34±0.42b 22.41±1.84bc
100 82.49±3.17 85.48±3.90 68.39±1.96
Glibenclamide 0.74±0.61 3.46±0.27b 19.35±3.23bc
4 mg/kg
All values are expressed as mean±S.D., n=6 values. Plasma insulin values at 0 h Insulin Content Serum insulin level and pancreatic insulin
before drug administration are significantly different compared to respective days 28
after drug treatment. Significant difference of diabetic control from normal control content were significantly decreased in streptozotozin induced
a
c
p<0.001. Significant difference of treated groups from diabetic control b p<0.01, diabetic mice, as lower as (1.3 µIU/mL) in the STZ untreated
p<0.05. d p<0.01 when compared with glibenclamide 4 mg/kg treated group.
mice compared with the non-diabetic control group (3.1 µIU/
mL). Effects of 1 and glibenclamide for 28 d on elevation of
streptozotocin induced diabetes has been confirmed here by serum insulin and pancreatic insulin levels which were par-
the study of glucose-6-phosphatase activity in liver, as well allel with those anti-hyperglycemic effects in STZ-diabetic
as the quantification of glycogen in liver and skeletal muscle, mice. The effect of the tetranortriterpenoid on insulin is
which are the important indicators of diabetes mellitus. In the shown in Table 8.
present study, 1 had a beneficial effect in terms of peripheral Inhibition of α-Amylase and α-Glucosidase Incuba-
glucose utilization. tion of the α-amylase with the substrate led to generation
Effect of 1 on SOD, CAT, GSH and GPx in Hepatic and of maltose with the addition of 1 significantly inhibited in a
Renal Tissues The antioxidant effect of the 1 over tissue concentration-related manner (IC50: 46.74 µg/mL) as compared
oxidative markers was studied here (Table 7). Diabetic mice with acarbose with a IC50: 12.23 µg/mL. While α-glucosidase
showed a significant reduction in SOD, CAT, GSH and GPx in inhibitory effect was IC50: 32.18 µg/mL as compared with
hepatic and renal tissues indicating persistent oxidative stress. acarbose with a IC50: 78.54 µg/mL (Table 9).
In addition, administration of 1 have shown increased SOD,
GPx, and CAT activities after 28 d of STZ treatment in mice DISCUSSION
indicated that extract can reduce reactive oxygen free radicals
and improve the activities of the antioxidant enzymes. In view of enormous therapeutic and economic
Effect of 1 on Serum Insulin Level and Pancreatic importance34) attributed to Azadirachta indica (neem), studies
September 2012
undertaken by several groups of workers on its various parts
have led to the isolation of a host of new constituents.35) In
continuation of investigations on the terpenoidal constituents
of the leaves of neem one new tetracyclic triterpenoids meliac-
inolin (1) has been isolated from the chloroform leaves extract.
The results of the present study indicated that 1 was an ef-
fective drug against chronic STZ-induced diabetes and al so
in preventing the gain of food and drink intake induced as
a consequence of diabetes. A deficiency or insufficiency of
insulin secretion or insulin resistance in the diabetic state usu-
ally causes a decrease in body weight gain and increases in
food intake, water intake, In our experimental condition, the
STZ-induced diabetic rats also showed the same symptoms.
However, administration of 1 slightly improved these physico-
metabolic abnormalities.
Meliacinolin could reduce blood glucose via increased in-
sulin, as evidenced by STZ-treated mice (Table 8), which is
capable of modulating the pancreatic secretion. The results of
the OGTT study strongly suggest that 1 reduces postprandial
blood glucose levels. This postprandial hypoglycaemic effect
of 1 could be due to its ability to facilitate or enhance clear-
ance of postprandial blood glucose in mice. The improving
actions of 1 on serum levels of glucose and insulin were simi-
lar to those in STZ-diabetic mice. These results indicated that
our STZ-diabetic mice can be released insulin from pancreatic
β cells by both glibenclamide and 1.
Interestingly, there was decrease in the concentration of
serum triglyceride, total cholesterol and HDL-cholesterol. The
mechanism by which 1 induce reduction in lipid concentration
could be explained by stimulation of lipolysis and fatty acid
utilization and/o suppression of hepatic fatty acid synthesis
in mice.36) It is well known that insulin activates lipoprotein
lipase, that hydrolyzes triglycerides under normal conditions.
Destruction of β-cells leads to depletion of plasma insulin,
which results in hyperlipidemia and hypercholesterolemia
caused by derangement of metabolic abnormalities.37) We
proved that repeated administration of 1 has a beneficial effect
lowering hyperlipidemia associated with hyperglycemia.
In this study, 1 caused a marked increase of hepatic gly-
cogen content in STZ-induced diabetic mice, which indicates
that mice cloud decrease hepatic glucose production (HGP) by
increasing glycogen content. In addition, 1 decreased G6Pase
activity and increased GK activity in liver, which indicates
that there, could be an increase in hepatic glucose uptake
and decrease in hepatic glucose release. Therefore, this study
strongly suggests that meliacinolin enhances hypoglycemic
activity probably by reducing HGP via decreasing G6Pase
activity and increasing GK activity. The increased activity of
hexokinase can promote glycolysis and increase utilization of
glucose for energy production. The administration of meliac-
inolin to the diabetic mice increased the activity of hepatic
hexokinase causing an increase in glycolysis.
SOD, CAT, GSH and GPx are defensive agents against
reactive species (ROS). Decrease in the activity of these
enzymes in liver may indicate increased production of free
radicals. Activity of these enzymes particularly catalase and
glutathione peroxidase reached the non-diabetic level with 1.
Hepatic lipid peroxidation was also effectively inhibited by 1
indicating an anti-diabetic effects. Decreased activities of an-
tioxidant enzyrnes in kidney were reversed with meliacinolin.
TBARS are an index of endogenous lipid peroxidation and
1523
oxidative stress as an intensified free radical production, has
been shown under diabetic conditions.38) The administration
of 1 reduced the renal and hepatic TBA-reactive substance
level significantly. These results suggest that 1 may alleviate
oxidative stress associated with diabetic pathological condi-
tions through the inhibition of lipid peroxidation.
Digestion of starch by key gastrointestinal enzymes can
be retarded and this can benefit the diabetic patient. Release
of glucose from food sources is the main factor affecting
postprandial hyperglycemia.39) Our finding revealed that me-
liacinolin efficiently inhibited α-amylase suggesting that 1 is
a starch blocker. α-Glucosidase inhibitors retard the digestion
of carbohydrates to simpler carbohydrates and slows down the
absorption of the later in the small intestine thus preventing
high glucose concentration in the blood after a meal.40) Meli-
acinolin showed appreciable α-amylase and α-glucosidase in-
hibitory activities so this compound plays a role in the carbo-
hydrate metabolism, thereby causing a delay in carbohydrate
digestion and absorption.
The mechanism of action for the antidiabetic activity of 1
in streptozotocin-induced mildly diabetic type 2 (MD) mice.
Meliacinolin was orally administered to MD mice at a dose
of 20 mg/kg once a day for 28 d. Meliacinolin-treated MD
mice showed significant reduction in fasting blood glucose.
Assessment of plasma insulin and pancreatic insulin follow-
ing treatment with 1 revealed significant elevation in their
levels. Administration of 1 enhanced the activity of hepatic
hexokinase and suppressed glucose-6-phosphatase in diabetic
mice. A significant rise in glycogen content was also observed
in liver and muscles of MD mice. The findings of the study
suggest that the antidiabetic effect of meliacinolin could be
due to its insulinogenic action. In addition, impaired glucose
homeostasis was improved by 1 through amelioration in the
carbohydrate metabolic pathways. Thus, the extract may exert
an antidiabetic effect through pancreatic as well as extrapan-
creatic action such as the intestinal inhibition of glucose.
In conclusion, the results obtained demonstrated that meli-
acinolin acts as an antihyperglycemic agent in streptozotocin-
nicotinamide induced diabetes mice by reducing the severity
of oxidative stress and acuity of hyperglycemia induced by
STZ, through the improvement of hyperlipidemia, insulin
resistance, and antioxidant defense system. In part the anti-
diabetic action of meliacinolin, is attributed to the intestinal
inhibition of glucose absorption. Is for all this reasons that,
meliacinolin can be considered as a compound with a very in-
teresting and promising potential as an anti-diabetic agent, for
its hypoglycemic and insulin augmenting effect and inhibition
of intestinal α-gluciosidase and pancreatic α-amylase.
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