Effect of Zizyphus Jujuba Fruits on Dyslipidemia in Obese Adolescents: a Triple-masked Randomized Controlled Clinical Trial

Abstract

This study aimed to assess the effects of Ziziphus jujuba (ZJ) fruit on controlling dyslipidemia in obese adolescents.
This triple-blind randomized placebo-controlled clinical trial comprised 86 obese adolescents aged 12-18 years with dyslipidemia, i.e. serum low density lipoprotein-cholesterol (LDL-C) or total cholesterol (TC) or triglycerides (TG) equal or more than the age- and gender-specific 95th percentile or high density lipoprotein- cholesterol (HDL-C) less than 5th percentile. They were randomly assigned into two groups of equal number Both groups received similar recommendations for dietary and physical activity habits. The case group received 5 grams of ZJ fruit powder three times a day for one month and controls took the same amount of a placebo powder. Fasting blood sugar, TC, LDL-C, HDL-C and TG were measured at the beginning and at the end of the trial. Data were analyzed using General linear method (multivariate) test.
Overall, 70 participants (51% boys, mean age of 14 +/- 2) completed the trial. The two groups studied did not differ in terms of age, gender, weight and body mass index (BMI). After the trial, serum TC decreased significantly (19 +/- 37 mg/dl in controls vs. 170 +/- 29 mg/dl in cases, P = 0.007) reduction. The corresponding figure was also significant for LDL-C (114 +/- 38 mg/dl vs. 104 +/- 22 mg/dl, respectively, P = 0.004). The changes in BMI and other lipids were not significant.
This study suggest that ZJ's fruits is generally well tolerated and may have potential favorable effects on serum lipid profile. While healthy lifestyle is the mainstay of controlling childhood obesity, this nutraceutical may be considered as a complementary treatment. Key words: Zizyphus, dyslipidemia, adolescence, obesity.

Full text

156
Med Arh. 2013 Jun; 67(3): 156-159 • ORIGINAL PAPER
Effect of Zizyphus Jujuba Fruits on Dyslipidemia in Obese Adolescents: a Triple-masked Randomized Controlled Clinical Trial
DOI: 10.5455/medarh.2013.67.156-159
Med Arh. 2013 Jun; 67(3): 156-159
Received: January 28th 2013 | Accepted: March 25th 2013
CONFLICT OF INTEREST: NONE DECLARED
ORIGINAL PAPER
Effect of Zizyphus Jujuba Fruits on Dyslipidemia
in Obese Adolescents: a Triple-masked
Randomized Controlled Clinical Trial
Ali Mohammad Sabzghabaee
1
, Iman Khayam
2
, Roya Kelishadi
3
, Alireza Ghannadi
4
, Rasool Soltani
5
,Shirinsadat Badri
5
, Shahin Shirani
6
Isfahan Clinical Toxicology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
1
Child Growth and Development Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
2
Isfahan Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
3
Department of Pharmacognosy, Isfahan University of Medical Sciences, Isfahan, Iran
4
Department of Clinical Pharmacy and Pharmacy Practice, Isfahan University of Medical Sciences, Isfahan, Iran
5
Department of Cardiology, Isfahan University of Medical Sciences, Isfahan, Iran
6
O
bjective: is study aimed to assess the eects of Ziziphus jujuba (ZJ) fruit on controlling dyslipidemia
in obese adolescents. Methods: is triple-blind randomized placebo-controlled clinical trial com-
prised 86 obese adolescents aged 12-18 years with dyslipidemia, i.e. serum low density lipoprotein- cho-
lesterol (LDL-C) or total cholesterol (TC) or triglycerides (TG) equal or more than the age- and gender-specic
95th percentile or high density lipoprotein- cholesterol (HDL-C) less than 5th percentile. ey were randomly
assigned into two groups of equal number. Both groups received similar recommendations for dietary and
physical activity habits. e case group received 5 grams of ZJ fruit powder three times a day for one month
and controls took the same amount of a placebo powder. Fasting blood sugar, TC, LDL-C, HDL-C and TG
were measured at the beginning and at the end of the trial. Data were analyzed using General linear method
(multivariate) test. Findings: Overall, 70 participants (51% boys, mean age of 14±2) completed the trial. e
two groups studied did not dier in terms of age, gender, weight and body mass index (BMI). After the trial,
serum TC decreased signicantly (195±37 mg/dl in controls vs. 170±29 mg/dl in cases, P=0.007) reduction. e
corresponding gure was also signicant for LDL-C (114±38 mg/dl vs. 104±22 mg/dl, respectively, P= 0.004).
e changes in BMI and other lipids were not signicant. Conclusion: is study suggest that ZJ`s fruits is
generally well tolerated and may have potential favorable eects on serum lipid prole. While healthy lifestyle
is the mainstay of controlling childhood obesity, this nutraceutical may be considered as a complementary
treatment. Key words: Zizyphus, dyslipidemia, adolescence, obesity.
Corresponding author: Iman Khayam, MD. Child Growth and Development Research Center. Isfahan University of
Medical Sciences, Isfahan, Iran. E-mail: dr.khayam@gmail.com
1. INTRODUCTION
Chronic non-communicable dis-
eases are the main cause of mortality
worldwide (1, 2, 3). Both behavioral and
biological factors related to chronic dis-
eases persist from childhood to adult-
hood. Risk factors such as dyslipidemia,
obesity and high blood pressure track
from childhood to adulthood (4, 5).
Atherosclerosis origins in childhood,
and remains asymptomatic for many
years. erefore, measuring some re-
lated biomarkers which are expressed
in the early stages of atherosclerosis
can play an important role in primor-
dial and primary prevention of chronic
disease (2, 3).
Lifestyle modication is the main-
stay of prevention and control of risk
factors of chronic diseases, and using
chemical medication is not without
side eects, notably in the pediatric
age group. Herbal plants contain con-
siderable amount of complex materials
that can have potential pharmacologi-
cal therapeutic eects, and can be con-
sidered as a complimentary treatment
of such risk factors as obesity and dys-
lipidemia. Traditional medicine is still
widely practiced, and is well accepted
by populations of various countries (6).
Common jujube, Zizyphus jujuba
mill. (ZJ) is a plant native to Asia and
southern Europe, which belongs to
the Rhanmaceae family and has sweet
date-like red eatable fruits. e fruits
are cooling, digestible, tonic, aphrodi-
siac and laxative. ey also remove bil-
iousness, burning sensations, thirst and
vomiting (4, 5). e ZJ fruits contain
polysaccharides, amino acids, avo-
noids, polyphenols, mucilage and pec-
tin, a water-soluble ber. Major charac-
teristic constituents are Triterpene-like
Alphitolic, Betulinic, Maslinic, oleano-
lic and saponins (4,6). ere are sev-
eral experimental and human studies
indicating the ecacy and safety of ZJ
fruits on various health disorders (5).
Controlling childhood obesity and
its complications as dyslipidemia solely
by lifestyle modication is usually di-
cult in the pediatric age group, thus us-
ing a safe complimentary medication

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Effect of Zizyphus Jujuba Fruits on Dyslipidemia in Obese Adolescents: a Triple-masked Randomized Controlled Clinical Trial
may be of help. In this study, the e-
cacy and safety of ZJ fruit was assessed
for its possible eects on dyslipidemia
in adolescents.
2. MATERIALS AND
METHODS
This triple-masked placebo-con-
trolled clinical trial which was con-
ducted from July 2011 to June 2012 in
Isfahan Cardiovascular Research Insti-
tute, aliated to the Isfahan University
of Medical Sciences, Isfahan, Iran.
After providing detailed oral in-
formation about the study, oral assent
was obtained from participants and
written consent from their parents.
e study protocol was in accordance
with the Declaration of Helsinki and
was approved ethically by the board of
human studies at the Isfahan Univer-
sity of Medical Sciences (Registration
code: 388591). e study was also reg-
istered in the Iranian clinical trial reg-
istry, with the registration number of
IRCT: 201109092306n1).
e eligibility criteria consisted of
being aged 12-18 years, having a body
mass index (BMI) equal or more than
the age - and gender-specic 95th per-
centile, which is conrmed to be ap-
propriate for Iranian children and ad-
olescents, and having dyslipidemia. e
latter was dened as serum total cho-
lesterol (TC) or low density lipoprotein
- cholesterol (LDL-C) or triglycerides
(TG) equal or more than the age - and
gender-specic 95th percentile or high
density lipoprotein- cholesterol (HDL-
C) lower than 5th percentile (7). ose
individuals with syndromal appear-
ance, any chronic disease or receiving
any medication were not included to
the study (Figure 1) (8, 9).
Participants were selected by con-
venient sampling from among individ-
uals referred to the above mentioned
department. All of them received rec-
ommendations for healthy eating habits
and regular physical activity. en they
were randomly assigned to two groups
by using simple randomization sam-
pling method. We recommended the
case group to use one sealed sachet of
ZJ powder (5 grams) three times a day
for one month and the placebo group
were asked to take equal amounts of a
placebo powder in identical package for
the same time period.
e sample size was calculated as 35
in each groups (cases and controls) to
have a 90% chance of detecting a dier-
ence in mean cholesterol change of 10
mg/dl at the 5% level of signicance, as-
suming the standard deviation of total
cholesterol as 15.47mg/dl from a previ-
ous study (10).
Preparing dosage form
is process was done in the re-
search laboratories of the School of
Pharmacy and Pharmaceutical sciences
at the Isfahan University of Medical
Sciences. Plant material was collected
from Kouhpayeh district located in Is-
fahan province (Iran). It was taxonom-
ically identied by an academic herbal
botanist.
Dry fruits of ZJ plant were frozen
at -18C and then powdered with an
ordinary mechanical grinder. Then,
the seeds of ZJ fruits were withdrawn,
and nally the powder was packed in
5-gram sachets.
Measurement of pectin and total
phenol in the ZJ fruits
Ethanol 96° and distilled water were
used for the extraction (11). We used
the Water house and coworkers method
(12) for determining the total phenol
content of the pulp of ZJ. Figure-2 shows
the steps for determining the amount
of pectin in 10g ZJ powder.
The placebo was matched to the
study drug for size, color, and taste
and contained microcrystalline cellu-
lose. Parents were asked to contact our
clinic two weeks after the beginning of
the trial and to report the compliance of
their children for using the medication,
as well as any side eect of the medica-
tion used. We followed the children’s
compliance by regular phone calls. e
ndings of those participants who did
not take their medication regularly were
excluded from the statistical analysis.
Physical examination and labo-
ratory tests
Weight and height were measured
by calibrated instruments and under
standard protocols. e body mass in-
dex (BMI) was calculated by dividing
weight by the height squared (kg/m
2
).
Participants were instructed to fast for
12 hours before blood sampling and
their compliance for fasting was con-
firmed by interviewing them before
sampling. Five milliliter of blood sam-
ples were collected from ante-cubital
vein. Fasting blood sugar (FBS), TC, TG,
LDL-C, and HDL-C were measured by
standard enzymatic methods by stan-
dard kits (Pars Azmoun, Iran) using
a fully automatic auto-analyzer (Hit-
achi 902, Japan All measurements of
the baseline survey were repeated with
the same method at the end of the trial.
Statistical analysis
Statistical calculations were per-
formed by the Predictive Analytic
Software (PASW) Statistical Package
ver.18.0.1 (Chicago, USA), by using (K-
S) test, Chi-Square test, independent
sample T-test, and General linear model
(GLM). All data passed the Kolmogrov-
Smirnov test to assess their normal dis-
tribution. Dierences of the two groups
for the quantitative data of lipid prole
were analyzed by Independent Sample
T-Test and nominal demographic data
were analyzed using Chi-Square test.
As shown in Table 1, at the baseline
survey, mean FBS have signicant dif-
ferences between two groups, therefore
it was considered as a covariant factors
in the nal analysis performed by GLM
test. P-value of less than 0.05 was con-
sidered as statistically signicant.
3. RESULTS
Overall, 70 of 86 individuals (81
(93%) completed the trial according to
the study protocol. e main reasons
for the attrition were taking sachets
irregularly, low desire to consume sa-
chets, using chemical drugs during the
study period, and irregular follow up.
Demographic parameters
e baseline characteristics of the
subjects study participants in ZJ and
placebo group are shown in Table1.
e mean age of students in ZJ group
was13.7 ± 2 years and 14.3 ± 2 years in
placebo group. Before starting the trial,
there was no signicant dierence be-
tween the two groups in terms of age,
gender ratio, weight and BMI; however,
as illustrated in Table 1, the FBS factor
has noticeably dierent values among
them (p<0.05). erefore FBS was con-
sidered as a covariant factor in the -
nal analysis.
Table 2 illustrates the laboratory
ndings at the end of the trial in both

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Effect of Zizyphus Jujuba Fruits on Dyslipidemia in Obese Adolescents: a Triple-masked Randomized Controlled Clinical Trial
groups. P-values were calculated by
General Linear Model (Multivariate)
test. GLM multivariate test showed that
A after 1 month administration of pow-
dered ZJ fruits sachets, LDL-C, TC and
FBS levels had signicant dierences
but other measured parameters vari-
ables showed no change compared to
the placebo group.
4. DISCUSSION
is trial, which to the best of our
knowledge is the rst of its kind in the
pediatric age group, revealed that daily
consumption of 15g ZJ fruits for one
month can decrease the serum levels
of TC and LDL-C in dyslipidemic ad-
olescents.
Regarding that the pulp of ZJ has a
considerable amount of sugar, i.e. fruc-
tose, galactose and glucose, the impact
of this fruit on FBS was also evaluated
and revealed a small but significant
change of it in the limited time period
of the study.
e formation of foam-cells in hu-
man macrophages has an important
role in the initial origination of ath-
erosclerosis so that prevention of these
cells formation is considered to have a
key role in its prevention or treatment
(13, 14). Fujiwara et al. found that the
pulps and seeds of ZJ have a notice-
able inhibitory eect on the formation
of foam-cells in comparison with fty
other plants. is eect was attributed
to tri-terpenoids such as Oleanonic
acid, Pomolic acid, Pomonic acid and
tri-terpenoids containing a carboxylic
acid at C-28 (15). Pectin, a kind of sol-
uble ber (SF), is a crucial structural
compound found in the cell wall of all
fruits and vegetables, which has a con-
siderable positive impact on the blood
cholesterol level (16, 17, 18). In the cur-
rent trial, individuals in the drug group
consumed 15g of ZJ powder contain-
ing 300 mg pectin every day. ere are
three theories justifying the inuence
of pectin on cholesterol level: 1. increas-
ing fecal bile acid excretion which is
the consequence of preventing bile acid
re-absorption from small intestine; 2.
Slowing down of the digestion of glu-
cose and other macro-nutrients absorp-
tion by pectin, which results in a de-
creased insulin level since there is a cor-
relation between the falling of choles-
terol synthesis and blood insulin level;
and 3. the production of short chain
fatty acid (SCFA) from SF in the caecum
and colon which is mediated by the an-
aerobic bacteria fermentation process
and can deplete plasma cholesterol by
inhibiting hepatic cholesterol metabo-
lism (19). e intake of polyphenol-rich
foods has cardio-protective eects be-
cause of their antioxidant, vasodilator,
anti-inammatory, anti-brotic and an-
tiapoptotic characteristics (20, 21). Ac-
cording to the Folin-ciucalteo method
(12), subjects took 165mg of polyphe-
nols in each day of the study. Antiox-
idants modify the function of veins
by decreasing the thickness of intima
layer and media carotid
layer, which may decline
the risk of cardiovascu-
lar diseases (22, 23). Pro-
cyanidin B2, Epicatechin
and Quercetin are some
of the flavonoids exist-
ing in the fruits of ZJ (6).
Study limitations
and strengths:
e main limitation
of this trial is that we
did not measure any bio-
chemical factor for as-
sessment of the com-
pliance of participants.
Moreover, the duration
of the trial was 4 weeks,
and by longer follow up,
we might reach to more
favorable changes in
other serum lipids. e
strengths of this trial are its novelty in
the pediatric age group and its triple-
masked design.
5. CONCLUSION
It is concluded that not only the
daily use of 15g of ZJ fruits for 1 month
is almost well tolerated in teenagers,
but also has positive eects on reduc-
ing serum TC and LDL-C, which are
important factors for the prevention
or treatment of developing atheroscle-
rosis from childhood. Further studies
with other daily doses of ZJ and longer
time period of usage with larger sample
size is recommended.
No(%) of participant-
stients
Mean (SD) Cholesterol (mg/dl)
Triglycerides
(mg/dl)
Fasting
Blood Sugar
(mg/dl)
Male
Boys
Female
Girls
Age( yr)
BMI (kg/
m2)
Total HDL LDL
Placebo
Group
17 (47) 19 (51) 14.3 (2)
23.6
(4.5)
198.1
(37.5)
41 (9.8) 136 (52) 162.6 (54.6) 91.8 (11.2)
ZJ Group 19 (55) 15 (45) 13.7 (2)
24.4
(3.7)
190.7
(36)
44 (9.7) 114 (38.5) 168 (70) 83.1 (7.7)
P-value 0.759 0.276 0.452 0.413 0.211 0.052 0.723 <0.001
Table1. Demographic andlaboratory parameters Baseline characteristics of participants the
subjectsat the beginning of studyin ZJand placebo group.
Mean (SD)
BMI (kg/
m
2
)
Cholesterol (mg/dl)
Triglycerides
(mg/dl)
FBS
(mg/dl)
Total HDL LDL
Placebo 24.1 (4.5) 195.4 (37) 42.7 (9.7) 114.9 (38.6) 157.5 (47.9) 89.9 (10)
ZJ`s fruits 23.7 (3.5) 170 (29.4) 39.6 (11.8) 104.7 (22.4) 132.2 (49.7) 84.3 (7.6)
P-value 0.386 0.007 0.3 0.004 0.071 0.008
*: Data are presented as mean (SD)
Table 2. Body mass index MI and Llaboratory ndings *at the end of the trial in both groups studied.
Firgure 1. Consort diagram of the study

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Effect of Zizyphus Jujuba Fruits on Dyslipidemia in Obese Adolescents: a Triple-masked Randomized Controlled Clinical Trial
Acknowledgm ents
is study was a PharmD
thesis project which was nan-
cially supported by the Vice-
chancellery for research and
technology at the Isfahan Uni-
versity of Medical Sciences. Au-
thors would like to acknowledge
Dr. Samani, Dr. Sharian and
Mrs. Zare for their help in pa-
tient selection and Mr. Akocha-
kian for his contribution to the
collection of herbal plant and
the laboratory department of
the Isfahan Cardiovascular Research institute for
their assistance. We also thank Dr. Ziba Farajza-
degan for her kind help for the statistical analy-
sis of our data.
REFERENCES
1. Chadjichristos CE, Kwak, BR. Connexins:
new genes in atherosclerosis. Ann Med.
2007; 39(6): 402-411.
2. Kelishadi R. Childhood obesity today’s and
tomorrow’s health challenge. Indian Pedi-
atr. 2008; 45(6): 451-452.
3. Medina-Ruiz A. Athero-
sclerosis prevention starts in
childhood: the obesity epi-
demic. Bol Asoc Med P R. 2011;
103(4): 72-76.
4. C ho p d a M , M a h a ja n
R. Phyto-Pharmacology of
Ziziphus jujuba Mill. A plant
review. Pharmacognosy Re-
views. 2009; 3(6): 320-329.
5. Naftali T. et al., Ziziphus
jujuba extract for the treatment
of chronic idiopathic constipa-
tion: a controlled clinical trial.
Digestion. 2008; 78(4): 224-228.
6. Choi SH. et al., Distribu-
tion of free amino acids, avo-
noids, total phenolics, and an-
tioxidative activities of Jujube
(Ziziphus jujuba) fruits and
seeds harvested from plants
grown in Korea. J Agric Food
Chem. 2011; 59(12): 6594-604.
7. Kliegman RM, Stanton B,
Geme JS, Schor N, Behrman
RE. Nelson Textbook of Pediat-
rics: Expert Consult Premium
Edition - Enhanced Online Fea-
tures and Print: Elsevier - Health Sciences
Division, 2004.
8. Davidson MH. et al., A multicenter, ran-
domized, double-blind clinical trial com-
paring the low-density lipoprotein cho-
lesterol-lowering ability of lovastatin 10,
20, and 40 mg/d with uvastatin 20 and 40
mg/d. Clin er. 2003; 25(11): 2738-2753.
9. Shidfar F. et al., Eect of folate supplemen-
tation on serum homocysteine and plasma
total antioxidant capacity in hypercholes-
terolemic adults under lovastatin treatment:
a double-blind randomized controlled clin-
ical trial. Arch Med Res. 2009; 40(5): 380-
386.
10. Kelishadi R. et al., Association of changes in
oxidative and proinammatory states with
changes in vascular function after a life-
style modication trial among obese chil-
dren. Clin Chem. 2008; 54(1): 147-153.
11. Iranian herbal pharmacopeia Committee.
Iranian herbal pharmacopeia.1st ed. Teh-
ran: Iranian Ministry of Health & Medical
Education Publications. 2002: 550-557.
12. Darabi K, Abdel-Wahab O, Dzik WH. Cur-
rent usage of intravenous immune globulin
and the rationale behind it: e Massachu-
setts General Hospital data and a review
of the literature. Transfusion. 2006; 46(5):
741-753.
13. Dushkin MI. Macrophage/Foam cell is an
attribute of inammation: mechanisms of
formation and functional role. Biochemis-
try (Mosc). 2012; 77(4): 327-338.
14. Yuan Y, Li P, Ye J. Lipid homeostasis and
the formation of macrophage-derived foam
cells in atherosclerosis. Protein Cell. 2012;
3(3): 173-181.
15. Fujiwara Y. et al., Triterpenoids isolated
from Zizyphus jujuba inhibit foam cell for-
mation in macrophages. J Agric Food Chem.
2011; 59(9): 4544-4552.
16. Marounek M. et al., Eect of pectin and
amidated pectin on cholesterol homeosta-
sis and cecal metabolism in rats fed a high-
cholesterol diet. Physiol Res. 2007; 56(4):
433-442.
17. akur BR, Singh RK, Handa AK. Chem-
istry and uses of pectin - a review. Crit Rev
Food Sci Nutr. 1997; 37(1): 47-73.
18. Brouns F. et al. Cholesterol-lowering prop-
erties of dierent pectin types in mildly hy-
per-cholesterolemic men and women. Eur J
Clin Nutr. 2012; 66(5): 591-599.
19. Gunness P, Gidley MJ. Mechanisms under-
lying the cholesterol-lowering properties of
soluble dietary bre polysaccharides. Food
Funct. 2010; 1(2): 149-155.
20. Lecour S, Lamont KT. Natural Polyphe-
nols and Cardioprotection. Mini Rev Med
Chem. 2011: 28.
21. Scoditti E. et al., Mediterranean diet poly-
phenols reduce inammatory angiogene-
sis through MMP-9 and COX-2 inhibition
in human vascular endothelial cells: A po-
tentially protective mechanism in athero-
sclerotic vascular disease and cancer. Arch
Biochem Biophys, 2012.
22. Hashemi M. et al., Eects of Antioxidant-
rich Juices on Flow-mediated Dilation of the
Brachial Artery of Adolescents with Meta-
bolic Syndrome. JIMS. 2009; 27(92): 37-44.
23. Riccioni G. et al. Carotenoids and vitamins
C and e in the prevention of cardiovascular
disease. Int J Vitam Nutr Res. 2012; 82(1):
15-26.
Figure 2. Practical steps for determing the amount of pectin in ZJ powder
Figure3. Laboratory findings of participants who consumed 5 grams of Zizyphus
jujuba fruit per day before and after the study
Figure 3. Laboratory parameters ndings of ZJ participants
usingwho consumed 5 grams Zizyphusof Zizyphus jujuba fruit per
day before and after the study