Cytotoxic activity of Vitex trifolia purpurea extracts.

Abstract

Defatted 85% crude hot aqueous methanol extract of Vitex trifolia purpurea (AME) successively extracted with, chloroform and ethyl acetate. Cytotoxicity of (AME), chloroform methanol extract (CE), ethyl acetate methanol extract (EE) and the residue obtained from methanol extract after successive extraction (RME) have been evaluated on brine shrimp (Artemia salina) and Hep-G2 cell lines as well. In brine shrimp lethality bioassay the results revealed that the (RME) is the most potent one with LC50 value 173 microg/ml while LC50 values of (AME), (CE) and (EE) was 180, 199 and 286 microg/ml, respectively. As well as the results of cytotoxic assay against Hep-G2 cell lines are in full agreement with previous results, with IC50 values 6, 10.7, 20.8 and 65.8 microg/ml for (RME), (AME), (CE) and (EE), respectively.

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Journal of the Egyptian Society of Parasitology, Vol. 42, No. 2, August 2012
J. Egypt .Soc. Parasitol., 42 (2), 2012: 431– 442
AMYLIN LEVEL AND GASTRIC EMPTYING IN OBESE CHILDREN:
BEFORE AND AFTER WEIGHT LOSS
By
OMNIA F. EL-RASHEIDY1, DINA A. AMIN1, HALA A. AHMED2,
HESHAM EL MASRY3AND ZEINAB M. MONTASER4
Departments of Pediatric1, Clinical Pathology2and Internal Medicine3, Fac-
ulty of Medicine Ain Shams University1,2,3, Cairo 11566 and Health Insur-
ance Organization, Ministry of Health4,Cairo, Egypt.
Abstract
Amylin is a neuroendocrine hormone that inhibits food intake and gastric empty-
ing in animal studies. Its role in obese human beings is still controversial. We
aimed to estimate the fasting and postprandial amylin levels in obese children be-
fore and after weight loss [through weight loss intervention program (WLIP)] and
to relate these levels to the lipid profile, homeostasis model assessment of insulin
resistance (HOMA-IR) and ultrasonography gastric half emptying time (t1/2). This
study was conducted initially on 20 obese children, 10 of them only continued 6
months follow up strategy on WLIP. Ten lean children served as controls. All chil-
dren were subjected initially and on follow up to dietetic history taking, anthropo-
metric measurements and laboratory assessment oflipid profile, HOMA-IR, fasting
and postprandial amylin levels and ultrasonographygastric half emptying time
(t1/2). The results showed that IR, significantly increased in fasting and postpran-
dial amylin levels and significant delay in gastric emptying among obese children.
After WLIP completion, IR was reversible, fasting and postprandial amylin levels
decreased and gastric emptyingsignificantly improved yet still significantly delayed
than controls. There was significant positive correlation between fasting amylin
levels and body mass index %, fasting insulin and HOMA-IR.
Key words: Obesity, obese children, amylin, insulin and gastric emptying.
Introduction
Overweight and obesity in children
have become a worldwide problem, not
more limited to high-income countries;
among developing countries, the Mid-
dle East has one of the highest rates of
childhood obesity (Kelishadi, 2007).
Salazar-Martinez et al. (2006) reported
that the overall prevalence of over-
weight and obesity in Egypt was 12.1
& 6.2%, respectively and that the most
consistent correlates of body mass in-
dex were age and rural residence.
Amylin is a 37 amino acid polypeptide,
which is co-localized and co-secreted
with insulin by pancreatic beta cells. It
has multiple actions, including the in-
hibition of gastric emptying and the
secretion of glucagon and insulin, li-
pase, and amylase (Cooper et al, 1987;

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Young and Denaro, 1998; Reda et al,
2002;Cancello et al, 2004).
In rodents, intra-peritoneal injection
of amylin inhibits gastric emptying
(Young et al, 1995; 1996; Gedulin and
Young, 1998)with a potency that is at
least 10 times greater than CCK-8, gas-
tric inhibitory peptide, and glucagon-
like peptide-1, peptides thought to be
physiological inhibitors of gastric emp-
tying (Young et al, 1996).
In humans, few studies demonstrated
that amylin concentrations correlate
with the degree of overweight and that
the basal amylin concentrations are
higher in obese than in lean human
subjects (Kong et al, 1997; Reda et al,
2002; Reinehr et al, 2007).
The study aimed to estimate the fast-
ing and postprandial amylin levels in
obese children before and after WLIP
and to relate these levels to the lipid
profile, HOMA-IR and ultrasonogra-
phy gastric half emptying time (t1/2).
Subjects, Materials and Methods
The study was a pilot prospective
study on 20 obese children recruited
from the Endocrinology Clinic of 6th
October Hospital, Health Insurance
Organization. Obesity was defined as
body mass index (BMI) at or above the
95thpercentile for children of the same
age and sex, based on the 2000 Centers
for Disease Control and Prevention
Growth Charts for the United States
and expert committee (Flegal et al,
2002). They were 7 males and 13 fe-
males with ages ranging between 4-12
years and a mean age of 7.81±2.31
years. Ten apparently healthy lean
children were selected cross matching
the patient’s group age, sex, pubertal
stage and socioeconomic status. They
were 6 males and 4 females with ages
ranging between 4-12 years and a mean
age of 8.20±2.83 years.
Children with any condition or medi-
cation that may affect the body weight,
lipid profile or glucose metabolism
(e.g. diabetes, premature adrenarche,
syndromal obesity, endocrinal and/or
metabolic diseases) were excluded.
All obese children were followed up
for six months, participating in a
WLIP. This outpatient intervention
program was based on physical exer-
cise, nutrition education and behavior
therapy including the child and his or
her family. The nutritional course
aimed to decrease the total caloric in-
take to the normal references for age.
The diet contained 30% fat, 15% pro-
teins and 55% carbohydrates including
5% sugar.
The local ethical committee approved
the study, and consents were obtained
from the parents of the included chil-
dren. Detailed medical history was tak-
en with special emphasis on age of on-
set of obesity, the socioeconomic stan-
dard (SES) of the family (Park and
Park, 1979), physical activities (dura-
tion of continuous physical exercise
(hours/week) and family history of
obesity, diabetes, hypertension, dys-
lipideamia or cardiovascular diseases.
Thorough clinical examination was
done for each enrolled case with spe-
cial emphasis on signs of insulin re-
sistance such as acanthosis nigricans,
skin tags and hirsutism.

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Dietetic history: 24 hour recall was
obtained before and after WLIP laying
stress on their appetite, food intake,
feeling of satiety, eating habits while
watching television and meal content.
24 hour recall was interpreted and cal-
culated in the Institute of Nutrition acc-
ording to WHO/FAO/UNU (1989) and
WHO/FAO (2002).
Measurements: a-Height and weight
were measured using a wall-mounted
stadiometer and a calibrated weight
scale, respectively, wearing underwear
only. Body-mass index (BMI) was cal-
culated by using the formula: BMI =
weight (kg)divided
Weight, height and BMI were plotted
against the percentiles to obtain the
percent from the median for age ac-
cording to Ogden et al. (2002), b- Mid-
arm circumference (MAC) was meas-
ured at the midarm point (midway be-
tween the tip of acromial process and
the epicondyle of the humerus). The
measuring was the mean of two read-
ings taken to the nearest millimeter. c-
Skin fold thickness (SFT) was meas-
ured using the halpender caliber. Read-
ings were approximated to the neatest
millimeters. MAC and SFT were plot-
ted against the percentiles to obtain the
percent from the median for age (Fri-
sancho, 1981).d-Waist circumference:
The child was wearing light clothing
for this measurement. The subject
stood erect with abdomen relaxed,
arms at sides, and feet together. The
measurement was taken at the narrow-
est part of the torso, above the level of
the navel. e- Hips circumference: The
child stood erect with arms aside and
feet together. The tape is positioned at
height (m2).
the maximum circumference of the
buttocks. f- Waist hip ratio: Waist cir-
cumference / Hips circumference.
Sampling: Five ml of venous blood
were withdrawn under complete asep-
tic condition from all children after an
over-night fast. One part of the blood
was collected in plain tube where the
separated serum was stored in aliquot
at -70C till the assessment of serum
insulin, lipid profile and fasting blood
glucose. The other part of the blood
was collected in EDTA tube and the
separated plasma was stored in aliquot
at -70C till the assay of serum amylin.
The previous sampling procedure was
repeated 2-hour postprandial for the
estimation of serum insulin and plasma
amylin.
Analytical methods: a- Fasting blood
sugar, serum cholesterol, triglycerides
and HDL-C were assayed on Synchron
CX9 auto-analyzer using a quantitative
enzymatic colorimetric method.
LDL-C was calculated by Fried-Wald
Formula. c- Serum insulin was assayed
by a commercially available sandwich
ELISA kit (DRG International Inc.,
USA). e- Plasma amylin was assayed
by a commercially available sandwich
ELISA kit (DRG International Inc.,
USA). f- The rate of change of amylin
% and insulin % was calculated as:
(amylin and insulin % after-amylin and
insulin % before) / amylin and insu-
lin% before x100 g- homeostasis model
assessment of the insulin resistance
(HOMA-IR) was calculated equation:
HOMA-IR =fasting glucose (mg/dL)x
fasting insulin (IU/ml) divided by 405.
b-

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Gastric emptying: Obese children and
controls were examined by ultrasonog-
raphy to assess the gastric half empty-
ing time (t1/2) by measuring the
changes in the gastric antrum cross sec-
tional area (Bolondi et al, 1985). The
examination was done after a 12 hours
fasting by using a real-time ultrasonog-
raphy scanner (Pie Medical-260 Cor-
vus) with a 3.5-5 MHz convex linear
assay transducer. The measurements
were taken by the same examiner and
were repeated three times to make sure
of its reliability. The probe was posi-
tioned at the level of the transpyloric
plane for simultaneous visualization of
the antrum, the superior mesenteric
vein, and the aorta. The measurements
of the antrum were always taken from
the outer profile of the wall because the
lumen of the wall is narrow in fasting
patients and the inner side of the wall is
difficult to outline. The cross-sectional
area of the antrum was automatically
calculated using the area ellipse of
theultrasound apparatus and was also
Table 1: Calorie and nutrient content of test meals administered for ultrasonogra-
phymeasurement of gastric-emptying time:
calculated for confirmation by the fol-
lowing formula:
constant 3.14, A the longitudinal diam-
eter of the gastricantrum and B the an-
troposterior diameter of the gastric an-
trum (Bolondi et al, 1985). The meas-
urements of the antrum were taken in
sitting position before the administra-
tion of a mixed solid±liquid meal
(Tab.1). The study reached the half-
emptying time (t 1/2) of the stomach
volume which equals to largest antral
surface area+ fasting antral surface area
divided by 2.Ultrasonographicgastric
half emptying time (t 1/2) was chosen
in the present study as it was found to
be reliable and offered a valid method
in clinical practice compared to scinti-
graphic measurement whether with
liquid (Marzioet al, 1989; Irvine et al,
1993) or semisolid meals (Darwiche et
al, 2003), which proved tobe safe, rela-
tively cheap, easy to perform anddid
not require intubation or exposure to
irradiation (Bolondi et al, 1985).
AB/4, whereisa
VariablesAge (years)
3-9
60.0
55.0
0
212.5
17.2
68.8
20.5
184.0
71.3
285.0
537.8
9-16
100.0
55.0
5.0
127.0
20.2
80.8
24.7
223.3
86.0
344.0
647.1
Bread (g)
Processed meat (g)
Butter (g)
Fruit juice (ml)
Protein (g)
kcal
Lipids (g)
kcal
Carbohydrates
kcal
Total calories
After completion of the WLIP, obese
children who completed the follow up
strategy and showed significant weight
reduction (n=10) were reassessed using

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the previously mentioned clinical, radi-
ological and laboratory parameters.
Statistical analysis: Statistical Pack-
age for Special Science (SPSS) soft-
ware computer program version 13 was
used for data entry and analysis. Data
were described using mean ± standard
deviation (SD) and frequencies accord-
ing if they are quantitative or qualita-
tive respectively. Student t test of two
independent samples was used to com-
pare quantitative variables. Paired t or
Wilcoxon signed ranks tests were used
to compare variables before and after
WLIP. The multiple regression analysis
was also performed to determine effect
of various factors on a dependent vari-
able. Pearson's and Spearman's correla-
tion test were used for correlating the
normal and non-parametric variables
respectively. A probability p=<0.05
was considered significant. Also, the
graphic presentation was done (Daniel,
1995).
Results
The age, gender and the SES were
comparable among the studied groups.
Twenty obese children agreed to partic-
ipate in the study, only ten showed sig-
nificant weight reduction.
The anthropometric measurements
were significantly higher in obese chil-
dren compared to controls (p< 0.001)
(Tab. 2). After participating in the
WLIP, these values significantly de-
creased in children with significant
weight reduction (p< 0.05), yet it did
not reach the control values (Tab.3).
Lipid profile was within normal ref-
erence range for age and sex in both
obese children and controls. On the
other hand, the HOMA-IR index was
significantly higher in obese children
compared to controls (p<0.05) (Tab. 2),
however, after participating in WLIP,
HOMA-IR decreased in children with
significant weight reduction with no
statisticalsignificant difference as
compared to controls (p>0.05). Inter-
estingly, both fasting and postprandial
amylin levels were significantly higher
in obese children compared to controls
(p < 0.05) (Tab. 2). After participating
in WLIP, the fasting amylin levels de-
creased (p>0.05) and postprandial amy-
linlevelssignificantly
(p<0.05) in children with significant
weight reduction but without statistical
significant difference compared to con-
trols (p>0.05) (Tab. 3).
The mean values of half emptying
time t (1/2) was significantly delayed
in obese children compared to controls
(p<0.001) (Tab.2). The values showed
significant improvement after partici-
pating in the WLIP (p< 0.001), howev-
er, still they were significantly delayed
than controls (p< 0.01) (Tab. 3).
Before WLIP fasting amylin levels
were significantly positively correlated
with BMI % (r=0.45, p<0.05), fasting
insulin (r=0.98, p<0.001) and HOMA-
IR (r=0.94, p<0.001) (fig. 1). Also, the
rate of change of amylin % was signifi-
cantly positively correlated with rate of
change of insulin% (r=0.975, p<0.001)
(fig. 2). In linear regression analysis of
fasting amylin as a dependent variable
in relation to triglycerides, total choles-
terol, fasting glucose, fasting insulin
and BMI %, the fasting amylin was
significantly correlated to fasting insu-
lin (t=9.21, p<0.001).
decreased