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The Open Complementary Medicine Journal, 2009, 1, 84-91
Hypolipidemic Activity of Extracts from Eriobotrya japonica and Olea
europaea, Traditionally Used in the Greco-Arab Medicine in Maintaining
Healthy Fat Levels in the Blood
Omar Said1,2,*, Bashar Saad2, 3, Stephen Fulder1, Riyad Amin5, Eli Kassis4 and Khaled Khalil1 1 Antaki Center for Herbal Medicine Ltd. Kfar Kana 16930, P.O. Box 2205 Israel 2 Research and Development Regional Center - The Galilee Society, P.O. Box 437, Shefa Amr 20200, Israel; and Qasemi Research Center- Al-Qasemi Academic College, Baga Algharbiya, Israel 3 Faculty of Arts and Sciences, Arab American University Jenin, P.O. Box 240, Jenin, Palestine 4 Sprunk-Jansen, A/S Strandvejen 100, 2900 Hellerup Denmark 5 Department of Biochemstry and Molecular Biology, Alquds University - Palestine Abstract: Loquat (Eriobotrya japonica) and olive leaves (Olea europaea) were investigated for their safety, anti-oxidant,
and anti-hypercholesterolemic properties. Therefore, a fixed combination of dry extracts of Eriobotrya japonica and Olea
europaea was prepared into so called Cholevel tablets and tested for safety and efficacy in vitro and in vivo tests. No sign
of toxicity (LDH-release) was observed in cultured human fibroblasts incubated with increasing concentrations of
Cholevel. Similar results were seen in MTT assay in co-cultures of human hepatocytes and monocytes. In addition, a high
level of safety was seen in rats with an LD50 of 17.3 g/kg. The extent of Lipid peroxidation in isolated human LDL frac-
tions, rat liver homogenates, and ferrosulphate was substantial at low concentrations of Cholevel. Hypolipidemic proper-
ties were assessed in a double blinded- randomized clinical study carried out among 41 human volunteers with hyperlipi-
demia values. The volunteers were divided into three groups. They were asked to continue their usual diet and medica-
tions unchanged and were evaluated for efficacy and tolerability of Cholevel for 3 months. Group 1 included 12 of per-
sons who were at a fixed dose of statins therapy without fully responding to their medications they consumed Cholevel
tablets 1 x 3 daily. Group 2 included 20 volunteers who consumed only Cholevel tablets 1 x 3 daily. Group 3 (control
group) included 9 volunteers who consumed placebo tablets 1 x 3 daily. Cholevel was well tolerated by all subjects and no
side effects were reported. Cholesterol levels were significantly reduced in groups 1 and 2 by 24% and 14.3% after three
months of Cholevel consumption, respectively. Parallel reductions in both LDL and triglycerides levels and increments in
HDL levels were observed. Taken together, results demonstrate safety, tolerability and efficacy of Cholevel that seems to
have dual inhibition on both the absorption and production of cholesterol.
Keywords: Eriobotrya japonica, olea europaea, cholevel, cholesterol, Greco-Arab medicine.
Atherogenesis is a multifactor process that includes oxi- dative modification of LDL which triggers pathological Hypercholesterolemia is often associated with obesity, events through multiple pathways leading to atherosclerosis diabetes mellitus and hypertension, each and all contribute to . Research in recent years has been directed towards die- elevated cardiovascular mortality . There is a general con- tary antioxidants of plant-derived foods to normalize the sensus that these metabolic disorders share hyperinsulinemia augmented levels of cholesterol atherogenous fractions, and insulin resistance as a common link [2,3] leading to both mainly LDL, and of glucose in an attempt to reduce the car- micro- and macro-angiopathies . During the last two dec- ades, statins have been in clinical use as selective inhibitors of the key enzyme, Hydroxymethylglutaryl Coenzyme-A Greco-Arab Herbal Medicines: Based on scientific and
(HMG-CoA)-reductase that determines the rate of choles- traditional knowledge, there is little doubt that the concept of terol synthesis in hepatocytes. In population studies, statins Greco-Arab and Islamic herbal medicinal therapy has shown have been evidenced to reduce cholesterol levels by about remarkable success in healing acute as well as chronic dis- 25% and mortality due to myocardial infarction by about eases. According to recent surveys, there are about 350-450 medicinal plants in the Eastern region of the Mediterranean and about 230 medicinal plants in coastal Mediterranean region in Egypt. These plants are used by healers for the *Address correspondence to this author at the Antaki Center For Herbal Medicine and R&D Center, The Galilee Society, P.O.Box: 2205, Kufur- treatment and preventions of almost all types of human dis- kana 16930, Israel; Tel: +972-4-6412712/015; Fax: +972-4-6412713; eases, such as cancer; skin, respiratory, digestive, liver dis- E-mails: firstname.lastname@example.org; email@example.com 1876-391X/09
2009 Bentham Open
Hypolipidemic Activity of Extracts from Eriobotrya japonica
The Open Complementary Medicine Journal, 2009, Volume 1 85
eases; diabetes; and others. These plants are sold or traded in 1% glutamine, 100 U/mL penicillin, and 10 g/mL strepto- market places in the Mediterranean region or internationally. mycin. Cells were maintained in humidified atmosphere with In many cases plant extracts are prepared into a mixture 5% CO2 at 37°C. The medium of cells from both cell lines [9,10]. Several plant species have been investigated and bio- was changed twice a week. At 70-80% confluence, cells active ingredients extracted to treat various human diseases. were trypsinized and seeded in 96-well plates in cell density Plant parts used included leaves, flowers, stems, roots, seeds, of 1.5 x 104 HepG2 cells and 5 x 103 THP1 cells. Twenty four hours after cell seeding cells were exposed to various concentrations of the plant extracts in fresh serum-free me- Olive and Loquat: Recent data have evidenced anti-
atherogenic and antioxidant activities of extracts from leaves from both olive  and loquat . MTT Assay: The tetrazolium dye, MTT, is widely used
The safety of both herbs used in the present study is to assess the viability and/or the metabolic state of the cells documented by their use in traditional Greco-Arab medicine [28,29]. This colorimetric assay is based on the conversion through centuries [11,14]. The leaves of loquat are well of the yellow tetrazolium bromide (MTT) to the red forma- known and safe household remedy especially in the Far  zan derivative by mitochondrial succinate dehydrogenase in and Middle East . Extracts from these leaves have been viable cells. Twenty four hours after cell seeding, cells were reported to exhibit a significant hypoglycaemic effect both in incubated with varying concentrations Cholevel for 24 hours Italy [16-18] and Pakistan . They were also reported to at 37°C. Following the removal of the media from each well, have antiviral , antitumour effects  and to exhibit cells were washed in phosphate buffered saline. The cells cytotoxic effects against tumour cell lines but not against were then incubated in serum free DMEM to which MTT normal cells . Moreover, they have been evidenced as (0.5 mg/mL) was added to each well (100 L), and incubated potent natural antioxidants [13,22]. In all these scientific for a further four hours. Then the medium was removed and publications, no evidence of any adverse effects of loquat the cells were incubated for 15 minutes with 100L of acidic leaf extracts has been indicated. On the contrary, liver pro- isopropanol (0.08 N HCl) to dissolve the formazan crystals. tective effects of seed's extracts have been evidenced in ani- The absorbance of the MTT formazan was determined at 570 mals . As for safety of olive leaf extracts, it has been nm in an ELISA-reader. Viability was defined as the ratio widely documented both in Europe [4, 12, 24-27] and in the (expressed as a percentage) of absorbance of treated cells to Middle East  and their anti-atherogenic, antioxidant and antidiabetic properties have been evidenced both in Germany  and France . Lactate Dehydrogenase: In the Lactate dehydrogenase
(LDH) assay the leakage of the cytoplasm located enzyme The present study aimed at investigating the safety and LDH into the extracellular medium is measured. The pres- efficacy of a fixed mixture of both olive and loquat leaves. ence of the exclusively cytosolic enzyme, LDH, in the cell Safety studies were carried out in animals and in vitro whereas therapeutic efficacy was evaluated in human volun- culture medium is indicative of cell membrane damage teers. The study was undertaken in accord with legal and ethical requirements as well as current scientific standards as For the LDH assay, 1.5 x 104 HepG2 cells and 5 x 103 indicated by European Good Clinical Practice Guidelines THP1 were seeded per well of 96-microtiter plates. Twenty- four hour after cell seeding, cells were exposed to varying MATERIAL AND METHODS
concentrations of the Cholevel. After 24h of treatment, the supernatants were collected from each well. Cell monolayers Preparation of Cholevel: The leaves of loquat and olive
were then treated with a cell lysis solution for 30 minutes at were collected from the Galilee region, dried under shade room temperature to lyse. The cells and the lysate were col- and powdered to a fine grade as extracts by Antaki Ltd.- lected. LDH activity was measured in both the supernatants laboratories, Kfar Cana, Israel. Cholevel (310 mg/tablet) and the cell lysate fractions by using CytoTox 96 a nonra- were prepared at Karmat Micro Encapsulation laboratories, dioactive cytotoxicity assay kit (Promega, WI, USA) in ac- Kibbutz Ramot Menashe, Israel. Each tablet contained 98mg cordance with the manufacturer’s instruction. The intensity loquat, 56mg olive, 7 mg Vitamin C, 2 mg Vitamin E, and of the color is proportional to LDH activity. The absorbance is determined at 490 nm with 96-well plate ELISA reader. Safety Studies
The percent of LDH release from the cells was determined using the formula: LDH release = (Absorbance of the super- Cell Culture: The effects of Cholevel on cell viability
was assessed in a cell culture system using cells from the natant)/ (absorbance of the supernatant and cell lysate)*100. mouse fibroblast cell line (3T3), the human hepatoplastoma Determination of LD
cell line HepG2, and from the human monocyte cell line THP1. HepG2 cell line retains differentiated parenchymal Thirty-six male Sprague Dawley rats (average weight: functions of normal hepatocytes, including the expression of 148 ± 16g) were divided into 4 groups. Large single doses of P450 isoenzymes, thus permitting long-term studies to be Cholevel were placed directly into the stomach of each group performed. The cells from both cell lines were grown in Dul- and observed for 14 days to determine the LD50. Animal becco’s modified Eagle’s medium (DMEM) with a high study approval was given from the Faculty of medical glucose content (4.5 g/L) supplemented with 10% vol/vol inactivated fetal calf serum, 1% nonessential amino acids, 86 The Open Complementary Medicine Journal, 2009, Volume 1
Said et al.
Fig. (1). MTT Assay in co-culture of HepG2 & THP1 cells after an overnight incubation with 10 μg and 100 μg Cholevel/mL. The absor-
bance of the MTT formazan was determined at 570 nm in an ELISA reader. Cell viability was defined as the ratio (expressed as a percent-
age) of absorbance of treated cells to untreated cells. Values given represent the mean ± standard deviations of three independent experi-
ments carried out in triplicates.
general physician in five clinics in Galilee if they were moti-vated to take herbal supplements to maintain a healthy fat Rat liver homogenates and human blood LDL fractions level in the blood. Forty one human volunteers were chosen were used to assess antioxidant effects of Cholevel. carefully on the base of close hyperlipidemaia values and Oxidative stress leads to generation of reactive oxygen species (ROS) which play an important pathogenetic role in Group 1: Included 12 volunteers who were at a fixed
different disease-states. Lipid peroxidation has damaging dose of statins therapy but without therapeutically efficiency. effects on liver cell membrane. The extent of lipid peroxida- Volunteers continued their usual diet and medications un- tion was measured using a technique based on the thiobarbi- changed and consumed Cholevel tablets 1 x 3 daily for 3 turic acid reactive substances (TBARS) assay that detects malondialdehyde (MDA), an end product of peroxidative decomposition of polyionic fatty acids in in-vitro systems. Group 2: Included 20 volunteers who consumed only
To accurately quantify TBARS in the analytical procedure, Cholevel tablets 1 x 3 daily for 3 months. the protein was precipitated before the addition of thiobarbi- Group 3: Included 9 volunteers who consumed placebo
turic acid to the reaction, while the antioxidant butylated tablets 1 x 3 daily for 3 months. This group served as con- hydroxytoluene was added before the heating of samples. Rat Liver Homogenates: Rat liver homogenates were in-
After a thorough review of Cholevel-components, they cubated with 100 μM FeSO4 as ROS generating system  were asked to continue their daily activities and food habits and with various concentrations of the product. unchanged. An informed consent was obtained from each Human Blood LDL Oxidation: Blood samples were col-
subject who was given a free of charge box containing 90 lected, into vacutainer tubes containing EDTA (1 g/L blood), from normocholesterolemic adult volunteers, and centrifuged At baseline, fasting cholesterol levels were estimated for at 4°C (833 x g for 10 min) to isolate plasma. The LDL frac- every subject who was scheduled for the next visit on 4 tion was isolated by ultracentrifugation through a KBr dis- weeks and was asked to return the Cholevel box so that re- continuous gradient and collected as the fraction floating at a turned tablets could be counted as the only measure of pa- density of 1.019–1.063 kg/L (1, 2). A rapid filtration through tient compliance with the protocol. This was repeated during disposable desalting columns was needed to remove the each of the 3 consequent visits when fasting cholesterol lev- EDTA, and LDL protein was measured by the method of els were estimated and careful investigations of well being Lowry et al.,  on the same day using bovine serum albu- and of any adverse effect were undertaken. min as the standard. Filtered LDL was immediately used for oxidation experiments. Statistics
The Wilcoxon signed-rank test was used. Comparisons between groups were performed by the Wilcoxon rank-sum Selection of Volunteers and Clinical Protocol: Human
test. A 0.05 level of significance was set. Data obtained were volunteers were selected on the basis of routine visits to their expressed as mean ± standard error of mean (SEM). Hypolipidemic Activity of Extracts from Eriobotrya japonica
The Open Complementary Medicine Journal, 2009, Volume 1 87
Fig. (2). LDH-release in arbitrary units from cultured human fibroblasts at baseline (0) and during incubating the fibroblasts with 180 and
360 μg Cholevel/ml for 24, 48, and 72 hours. Values given represent the mean ± standard deviations of three independent experiments car-
ried out in triplicates.
Efficacy of Cholevel
Safety of Cholevel
Lipid Peroxidation: Lipid peroxidation induced by incu-
bating rat liver homogenates with ferrosulphate is expressed LD50 in Rats: An extremely high dose of the Cholevel
in Fig. (3A) as the extent of MDA production. The addition
(17.3 g/kg) was necessary to obtain the LD50 value in rats. of very low dose of the Cholevel (10 μg/ml) to the medium In Vitro Cytotoxicity Assessments: MTT and LDH as-
significantly reduced MDA-release from 0.88 ±0.10 to 0.62 says were carried out with fibroblast cell line (3T3) and co- ± 0.05 nm/mg protein (p ‹ 0.01). Higher concentrations of cultures of human hepatoplastoma cell line HepG2 and cells the Cholevel (50 μg/ml) further reduced MDA release to from the human monocyte cell line THP1. HepG2 cell line 0.32 ±0.03 nm/mg protein (p ‹ 0.001). No further antioxida- retains differentiated parenchymal functions of normal hepa- tive effects were seen by increasing Cholevel concentration tocytes, including the expression of P450 isoenzymes thus up to 100 μg/ml (Fig. 3A).
permitting long-term studies to be performed. LDL Oxidation: In vitro inhibition of human LDL oxida-
MTT Test: The metabolic activity can be evaluated by
tion by Cholevel in the presence of ferrosulphate is ex- measuring the activity of a mitochondrial enzyme succinate pressed in Fig. (3B) as the extent of MDA formation.
dehydrogenase using the MTT test. This test is widely used Cholevel at low concentrations of 50, 100 and 400 μg/ml in the in vitro evaluation of the toxicity of plant extracts. We significantly inhibited the LDL oxidation and reduced the applied the MTT test to evaluate the safety of Cholevel. levels of MDA from 21 ± 3 to 15 ± 2, 12 ± 1.7 and 8 ± 1.5 Cells were exposed to 10 μg Cholevel/mL and 100 μg Cholevel/mL of culture medium for 24h. No sign of any Clinical Studies: Forty one human volunteers were re-
negative effects were observed after treatment with the two cruited into the study, 12 of them were already on a fixed concentrations (Fig. 1).
dose of statins therapy. All 41 subjects were evaluated for LDH-Release Test: Membrane integrity can be evaluated
efficacy and tolerability during all scheduled visits as patient by measuring lactate dehydrogenase activity. Lactate dehy- compliance with the protocol was excellent. Cholevel was drogenase, an enzyme located in the cytoplasm, catalyses the well tolerated by all 32 subjects (groups 1+2) and no minor conversion of lactate and pyruvate. When lactate dehydro- or major adverse effect was noted by any subject. They all genase is found within the media on the cells, there are two tolerated Cholevel with other medications they were on es- possible causes: The first is cellular death and the second is a pecially those who were on statins therapy (group 1). In two 'leak' in a cell membrane. When cells are disrupted, the lac- of the 32 subjects, cholesterol levels were not elevated at tate dehydrogenase activity is elevated. Results obtained baseline and did not substantially change during the 3 indicate no significant changes of the LDH levels in the cul- ture medium after exposure to Cholevel at concentration up Group1 (Cholevel + Statins): the cholesterol levels were
to 360 μg/ml (Fig. 2).
significantly reduced within the first month of Cholevel con- 88 The Open Complementary Medicine Journal, 2009, Volume 1
Said et al.
Fig. (3). Effects of different concentrations (10, 50, and 100 μg/ml) of Cholevel upon malondialdehyde (MDA) release that reflects the ex-
tent of lipid peroxidation in rat liver cells incubated with 100 μM ferrosulphate (A) and in vitro inhibition of human LDL oxidation by
Cholevel in the presence of ferrosulphate (B). Values given represent the mean ± standard deviations of three independent experiments car-
ried out in triplicates.
sumption by 13% and by 18.3% and 24.2% in the two other 30.56 mg% at baseline to 206.42 ± 16.23 mg% at 3 months months of treatment respectively (Table 1). In group 1, cho-
(Table 1 and Table 4).
lesterol levels were 287.6 ± 17.9 mg% at baseline and de- Group 2 (Cholevel Only): cholesterol levels were 282.2
creased to 249.5 ± 13, 229.4 ± 14 and 217.7 ± 11 mg% at 1, ± 13.8 mg% at baseline and decreased to 268.3 ± 14, 254.6 ± 2 and3 months of Cholevel intake. These reductions are 12.8 and 241.7 ± 10.6 mg% at 1, 2 and 3 months of Cholevel highly significant. As shown in Table 1, LDL levels de-
intake (Table 2). As shown in Table 2, LDL levels decreased
creased from 169.83 ± 7.17 mg% at baseline to 136.5 ± 7.35 from 175.05 ± 5.79 mg% at baseline to 149.25 ± 5.53 mg% mg% at 3 months, whereas, HDL levels increased from at 3 months, whereas, HDL levels increased from 36.8 ± 40.08 ± 7.03 mg% to 48.83 ± 5.56 mg% at 3 months. These 5.33 mg% to 42.3 ± 5.43 mg% at 3 months. These changes changes were accompanied by significant reductions in the were accompanied by significant reductions in the levels of levels of triglycerides after the study 3 months from 258 ± triglycerides after the study 3 months from 272.4 ± 28.5 Hypolipidemic Activity of Extracts from Eriobotrya japonica
The Open Complementary Medicine Journal, 2009, Volume 1 89
Table 1 (Group 1). Fasting Lipid Levels in the 12 Subjects Using Statin Medications at Baseline and During Each of the 3 Months
Study While Consuming One Cholevel Tablet Three Times Daily. Values Given Represent the Mean in mg% ±
After 1 Month
After 2 Months
After 3 Months
Table 2 (group 2). Fasting Lipid Levels in the 20 Subjects at Baseline and During Each of the 3 Months Study While Consuming One
Cholevel Tablet Three Times Daily. Values Given Represent the Mean in mg% ± Standard Deviations
After 1 Month
After 2 Months
After 3 Months
Table 3 (group 3). Fasting Lipid Levels in the 9 Control Subjects at Baseline and During Each of the 3 Months Study While Consum-
ing One Placebo Tablet Three Times Daily. Values Given Represent the Mean in mg% ± Standard Deviations
After 1 Month
After 2 Months
After 3 Months
mg% at baseline to 235.95 ± 21.91 mg% at 3 months (Table Group 3 (Control): No significant changes were ob-
2 and Table 4).
served in the control group (Table 3 and Table 4).
90 The Open Complementary Medicine Journal, 2009, Volume 1
Said et al.
Table 4. Fasting Lipid Levels after 3 Months Study While Consuming in the Three Groups
fact that Cholevel was well tolerated by all studied subjects and no adverse effect could be traced. The results disclose that Cholevel is safe and well toler- ated by all 32 studied subjects and is therapeutically efficient Taken together, results obtained indicate safety and hy- as substantial and incremental reductions of cholesterol lev- polipidemic properties of Cholevel, consisting of extracts els were observed during each of the study 3 months. At 3 from Eriobotrya japonica and Olea europaea, traditionally months, baseline cholesterol levels in group 1 (Cholevel + used in the Greco-Arab medicine [9, 9,11,35,36]. statins) subjects were reduced by 24% which is comparable ACKNOWLEDGEMENTS
to that of 25% observed during simvastatin therapy . Vol-unteers who consumed Cholevel without statins, (group 2) The authors would like to thank Mr. Assad Dahamshe showed significant reduction in cholesterol levels (14.3%) and Mr. Bahaa Hadieh from Antaki Center for Herbal Medi- after three months of the clinical test. Combination therapy cine Ltd. Kfar Kana for preparing the figures. of Cholevel with statins is efficient and can be helpful to REFERENCES
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JUDICIAL DIALOGUE ON THE NEW YORK CONVENTION New Delhi THE CASUARINA HALL at THE INDIA HABITAT CENTRE Mr Shishir Dholakia - Chief Rapporteur Mr Harisankar K S - Co-Rapporteur Introduction This report summarises the proceedings and key conclusions of the ‘Judicial Dialogue on the New York Convention’ that took place in New Delhi on Saturday, 23rd November 2013. The