International Journal of Applied and

Natural Sciences (IJANS)

ISSN(P): 2319-4014; ISSN(E): 2319-4022
Vol. 7, Issue 3, Apr - May 2018; 31-40
© IASET

NOVEL BITTER MELON (MOMORDICA CHARANTIA L.) AND OLIVE LEAVES

(OLEA EUROPAEA L.) PHYTOSOMES: PREPARATION AND ITS EVALUATION
FOR ANTI-HYPERGLYCEMIC ACTIVITIES BY ORAL GLUCOSE TOLERANCE TEST
(OGTT)

Bandar Hamadneh1, Hayder AL-Domi2 & Malik Haddadin3

1
Research Scholar, Human Nutrition and Dietetics, Department of Nutrition and Food Technology,
Faculty of Agriculture, the University of Jordan
2
Professor of Nutrition and Dietetics, Department of Nutrition and Food Technology,
Faculty of Agriculture, the University of Jordan
3
Associated Professor of Food Biotechnology, Department of Nutrition and Food Technology,
Faculty of Agriculture, the University of Jordan

ABSTRACT
Objectives

The objectives of this study was to prepare and investigate the phytosomes of crude bitter melon and olive leaves
extracts for anti-hyperglycemic activities in glucose-induced hyperglycemic rats.

Methods

Phytosomes were prepared by reacting one mole of the phenolic compounds of each extract with two moles of
Phosphatydil-choline. Bitter melon and olive leaves phytosomes (150 and 200 mg/kg) were evaluated for oral toxicity.

Results

In the oral toxicity study, no mortality was observed among the rats received the phytosomes at the single dose of
150, 200mg/kg. Hence one tenth of the phytosomes dose tested (20mg/kg). Moreover, it is evaluated for hypoglycemic
effects by oral glucose tolerance test in induced hyperglycemic rats. In the orally glucose induced hyperglycemic rats,
phytosomes were significantly reduced serum glucose levels at 60, 90 and120 minute (P<0.001) than it is extracts.
Most significant reduction was observed at 90 minute (26%). Glibenclamide was used as standard drug.

Conclusions

Our results indicated that the bitter melon and olive leaves phytosomes is more potent in anti-hyperglycemic
activities than extracts. Clearly, further studies are warranted to improve our understanding of the underlying
mechanisms.

KEYWORDS: Bitter Melon Extract, Olive Leaves Extract, OGTT, Phytosome, Phosphatydil-Choline

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32 Bandar Hamadneh, Hayder AL-Domi & Malik Haddadin

Article History
Received: 03 Apr 2018 | Revised: 09 Apr 2018 | Accepted: 13 Apr 2018

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder reaching pandemic proportions worldwide
[1, 2]. In 2014, WHO reported that the prevalence of DM in adults aged 18 or more was 9%, 90% of them with T 2 DM
[3]. More than 382 million individuals from all age groups is diagnosed with DM, which is estimated to reach about 592
million in 2035 [4]. Although, there are many available anti-diabetic drugs such as biguanides and sulphonylureas, yet they
have several side effects, including hypoglycemia, liver toxicity, dyslipidemia, and hypertension [5]. Whereas, the natural
plants are not only recommended to use from WHO, but also more cost-effective with lower side effects as compared to
chemical drugs [6, 7].

Alternative medicine has been used widely in order to treat T2DM by using natural compounds. Nonetheless,
limited evidence exists regarding the efficacy, safety, and mechanisms of action for the used natural compounds for the
treatment of diabetes [8]. Furthermore, abundant plants products are available in the market that is useful for the treatment
of metabolic disorder, including diabetes, nonetheless, these products are water-soluble, which can be easily destroyed by
gastric acids and gut enzymes leading to a limited bioavailability [9].

The olive tree (Olea europaea L.) is cultivated in several parts of the world, but the Mediterranean region is the
superior crop production area with approximately 98% of the world’s olive cultivation [10]. Olive leaves are well known in
folk medicine to treat hyperglycemia, hypertension, and infectious diseases, especially in Europe that attributed to its high
phenolic compounds [11]. Whereas, bitter melon (Momordica charantia L.) is a tropical medicinal plant cultivated for its
edible fruit. It's a rich in phenolic and saponin compounds, which are linked with higher antioxidant activity [12].
Bitter melon is a well-known agent to decrease blood glucose levels in Asia, Africa, and Latin America, and also its
extracts had been called insulin vegetable [5].

Phytosome technology is patented technology based on a combination of various plant's extracts or water-soluble
molecules to produce complex lipid molecules called phytosome [13]. Phytosome is highly absorbable and highly
bioavailable with greater clinical benefits and without affecting the nutrient safety of the phytosome [14].
Phytosome protects the component of the herbal extracts away from destruction through digestive secretions and gut
bacteria [15].Thus, phytosome technology is an effective method of delivery used for the improvement of various dietary
supplements activity against diseases [16]. However, to best of our knowledge, studies on providing olive leaves and bitter
melon extracts in the phytosome form to treat T2DM are scarce. Thus, the objective of the current study is to prepare crude
olive leaves and bitter melon phytosomes, and to evaluate its anti-hyperglycemic activities in glucose-induced
hyperglycemic rats by OGTT.

Impact Factor (JCC): 5.0273 NAAS Rating 3.73

Novel Bitter Melon (Momordica Charantia L.) and Olive Leaves (Olea Europaea L.) Phytosomes: Preparat ion 33
and its Evaluation for Anti-Hyperglycemic Activities by Oral Glucose Tolerance Test (OGTT)

MATERIALS AND METHODS

Reagents

Glibenclamide (Hikma Company, Jordan) D-glucose, ethanol (England), acetone (Lonover chemicals, England),
gallic acids (Sigma, USA. MW,170.12 g /mole). Phosphatidylcholine (Soyabean Lecithin Cargill, Belgium E 322.
MW, 677.93 g /mole).

Animals

This research was approved by the Department of Nutrition and Food Technology Committee for Animal
Experimentation/University of Jordan. 55 male Sprague-Dawley rats, weighing about 150-180 grams and 8 weeks of age
were kept at Animal Unit, Scholl of Agriculture, the University of Jordan, Jordan. Each rat was housed in a single
metabolically- ventilated plastic cage with a stainless steel wire mesh floor and front. The rats were kept under controlled
temperature (22±2ºC) and maintained at a 12:12 hours light: dark cycles (from 19:00 pm to 7:00 am) with free access to
water and standard laboratory chow for one week of acclimatization.

Preparation of Plants Material

On Apr 2016, olive (Olea europaea) leaves were randomly and directly collected from an olive tree
(Baladi variety) from a local farm in Irbid, Northen-Jordan. The Baladi olive trees were of about 20 years old, not irrigated
and no phytosanitary treatments had been utilized for the last10 years. Collected leaves were kept in plastic bags.
The leaves were washed with running water to remove impurities such as dust, then dried under shade at room temperature
and then crush using a mechanical blender (Moulinex Miller, France) and passed through mesh cloth to get leaves powder
and stored at 4◦C until use [17].

Fresh bitter melon fruits were purchased from the local market in Irbid, Northern-Jordan. The fruits were washed
with running water to remove impurities and then dried in an oven (WTC Binder, USA) at a temperature of 37 ◦C for 5
days and then crush using a mechanical blender (Moulinex Miller, France) and passed through mesh cloth to get plants
powder and stored at 4◦C until use [18].

Preparation of Olive Leaves and Bitter Melon Fruits Extracts

Fifty grams of each plant powder was extracted with 500 mL of 95% ethanol. The mixture was mixed on a rotary
shaker (New Brunswick Scientific, USA) for two hours at 180 rpm at room temperature 20 ◦C and then for 15 minutes in
an ultrasonic bath at 37 °C (Bandelin Electronic-RK-103 H, Germany). The mixture was filtered through Whatman no: 4
and then membrane filter (cellulose type filter, 0.45 µm). After filtration, the solvent was evaporated under vacuum using a
rotary evaporator (Büchi, RE 121, Switzerland) to remove solvent under reduced pressure at 38 °C and 120 rpm.
All the obtained crude extracts were then transferred to vials covered with aluminum foil and then stored in the dark at
room temperature to protect them from photo-oxidation [18, 19].

Total Phenolic, Flavonoids, and Tannins Content

The total phenols content of olive leaves and bitter melon fruits extract were determined by Folin-Ciocalteau
assay and were expressed as gallic acid equivalent/gram of extracts (mg GAE/G) [20]. Total Flavonoids was determined by
the aluminum chloride colorimetric method [20]. Total tannins were determined by Folin and Ciocalteu method [21].

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34 Bandar Hamadneh, Hayder AL-Domi & Malik Haddadin

Phytosome Preparation

Olive leaves and bitter melon phytosomes were prepared by adding 2 moles of phosphatidyl-choline for each one
mole of the total phenolic compounds of plant extracts independently. In brief, 2 moles of phosphatidyl-choline was
dissolved in a solution containing 200 ml of ethanol, and then one mole of polyphenolic compounds of plant extract was
added. The mixture was mixed on a rotary shaker (New Brunswick Scientific, USA) for two hours at 180 rpm at room
temperature. After that, ultrasonic bath (Bandelin Electronic-RK-103 H, Germany) was used to enhance the formation of
the phytosome structure at 37C for 15 minutes. The solvent was evaporated under vacuum using a rotary evaporator
(Büchi, RE 121, Switzerland) at 38 °C and 120 rpm. Finally, 50 ml of acetone was added to each mixture, and then left in
the dark at room temperature to precipitate phytosome structure [13, 22].

Microroscopic Scanning

Microscope digital camera (Nicon YS2-H, Japan) was used to confirm the formation of phytosome complex
between plant extracts and the phospholipids.

Acute Oral Toxicity Study

Oral toxicity test was performed according to the Organization of Economic Cooperation and Development
(OECD) guideline 425. A total of 25 male Sprague-Dawley rats were used. All rats fasted overnight, then divided into five
groups with five rats in each group. Group 1 stands as control group received distilled water. Groups (2 and 3) received a
single oral dose (150, 200mg/kg) of olive leaves phytosome respectively. Groups (4 and 5) received a single oral dose
(150 and 200mg/kg) of bitter melon fruits phytosome respectively. After the phytosomes administration, food withheld for
3-4 hours. Rats were observed post the dose at 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, and every day for 14 days for general toxic
signs, morphological behavior and mortality. One tenth of the dose was taken from the OGTT [23].

Oral Glucose Tolerance Test

A test was performed in overnight fasted normal rats. The experimental rats were divided into 6 groups of five
rats each. Fasted rats have then received D-glucose solution (2mg/kg/) orally. After thirty minutes, rats were treated as
follows: Group 1- stands for normal control group received distal water. Group 2 and 3 were received bitter melon
phytosome and extract at the dose of 20mg/kg. Group 4 standard control group received the glibenclamide drug at the dose
of 0.25mg/kg. Group 5 and 6 were received olive leaves phytosome and extract at a dose of 20mg/kg. The blood glucose
level was estimated before glucose administration (-30 min), 30 min later (0 min) and at 30, 60, 90 and 120 minutes after
administration of extracts, phytosome, and standard drug using a glucometer (ACCU-CHEK 68305 Mannheim, Germany)
[24].

STATISTICAL ANAYLSIS

Experimental values are expressed as mean ± SEM. One–Way Analysis of Variance (ANOVA) was used.
All Pair-wise comparisons were made using Duncan’s Multiple Range Test (DMRT) post-hoc test. Statistical significance
was considered to be indicated by a p-value (p<0.001).

Impact Factor (JCC): 5.0273 NAAS Rating 3.73

Novel Bitter Melon (Momordica Charantia L.) and Olive Leaves (Olea Europaea L.) Phytosomes: Preparat ion 35
and its Evaluation for Anti-Hyperglycemic Activities by Oral Glucose Tolerance Test (OGTT)

RESULTS
Total Phenol Content

Table 1 shows the total phenolics, flavonoids, and catechin of olive leaves extract and bitter melon extract.
The highest value was recorded in olive leaves extract.

Microscopic Scanning

A Microscopic scan showed that the phytosome is a complex cell- like structure in which phyto-constituents of
the plants extract present on the inner surface of the phytosome and enveloped by PC (Figure 1).

Acute Toxicity Study

No mortality was observed among the rats received olive leaves and bitter melon phytosomes at the single dose of
150 and 200mg/kg. Hence, one-tenth of the tested phytosome dose was selected (i.e., 20mg/kg).

OGTT

Hyperglycemia induction by intra-gastric gavage of glucose resulted in a one -fold increase in glucose levels of
the plasma by comparing groups at 0 minutes with that at -30 minutes (Table. 2). Olive leaves phytosome showed a
reduction of blood glucose levels of 10, 20, 26, and 9% at 30, 60, 90, 120 minutes respectively. Whereas, bitter melon
phytosome showed a reduction of 12, 14, 26, 10% at 30, 60, 90, 120 minutes respectively. Most significant reduction of
glucose levels was observed in the phytosomes of olive leaves and bitter melon and then for glibenclamide drug at 90
minutes.

DISCUSSIONS

The total phenolic compounds of olive leaves and bitter melon extracts were consistency with reported values
from different previous reports [25, 26]. Moreover, it is well known that the activities of the olive leaves and bitter melon
extracts in management chronic diseases, especially diabetes are due to the antioxidant activities of the phenolic
compounds.In this study, the quantitative phytochemicals of each extract responsible for the observed anti-hyperglycemic
activities have not been isolated or determined, and further work is now ongoing in our laboratory to determine the
phytochemicals content and bioavailability rate.

Microscopic scanning of the produced phytosomes showed a cell-like structure that was strongly in agreement
with previous studies [27, 28]. The cell-like the structure of the phytosome resulted from the action of
phosphatidyl-choline in which they bind to the phyto-constituents of herbal extract, and engulfing it to produce
microsphere which is called phytosome [27]. Therefore, using phosphatidy l-choline in the phytosome structure is to make
the structure highly similar to the cell membrane composition that facilitates and accelerate the absorption of the plants
material into blood circulation [29].

The OGTT is an important and very necessary parameter to determine the rate of the body ability to consume
glucose, and also gives information about the efficacy of a drug and plant extracts in acute hyperglycemia [24]. In this
study, olive leaves and bitter melon extracts provided in the phytosome form at the dose of 20mg/kg reported higher
glucose reduction of hyperglycemic rats in comparison to non- phytosome form. With regard to the hypoglycemic
activities of the tested plants, the olive leaves constituent responsible for these effects is oleuropein and hydroxytyrosol,
which are the most phenolic component [30], whereas bitter melon extract contents appear to have similarities with the
www.iaset.us editor@iaset.us
36 Bandar Hamadneh, Hayder AL-Domi & Malik Haddadin

structure to the animal insulin-like charantin [31, 32]. Glucose levels were reduced by phytosome form could be attributed
to the enhancement of the phenolic compounds of the extracts. In previous studies, the phytosome structure improves the
levels of polyphenolic constituents in the blood serum with at least 2-6 times more than non phytosome form. This is
actually attributed to the chemical bondages between polyphenol and phosphatidyl-choline molecules [33].

To our knowledge, the finding of this study constitutes the first experimental evidence of the reducing effect of
olive leaves and bitter melon phytosomes preparation on glycaemia in laboratory rodents. Moreover, potency and efficacy
of it may require further investigation.

CONCLUTIONS

Phytosome structure is a novel delivery system that overcomes the problems that are associated with the using of
plants extracts such as poor stability in gastric environment, herbal toxicity, poor pharmacological activity, physical and
chemical degradation, and high doses of ingredients that affect the safety and efficacy of the extracts. Olive leaves and
bitter melon extracts provided in a phytosome form resulted significant anti-hyperglycemic activities in the
glucose- induced hyperglycemic rats in compared to non-phytosome form. Further studies are required to monitor the
bioavailability of the active compounds in blood, and other metabolic factors related to the metabolic process of the
diabetes .

ACKNOWLEDGEMENTS

We acknowledge the Deanship of Academic Research of the University of Jordan for funding the study

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Novel Bitter Melon (Momordica Charantia L.) and Olive Leaves (Olea Europaea L.) Phytosomes: Preparat ion 39
and its Evaluation for Anti-Hyperglycemic Activities by Oral Glucose Tolerance Test (OGTT)

APPENDIES

Table 1: Total Polyphenols, Total Flavonoids, and Total Tannins Extracted from Olive Leaves/Bitter Melon
Total Polyphenols Total Flavonoids Total Tannins
Plant*
(mg GAE/g Extract) (mg Rutin /g Extract) (mg GAE /Extract)
Olive leaves extract 294 ± 6.1 18.18± 5.15 27.35± 10.3
Bitter melon extract 172± 7.45 13.46± 3.55 8.24± 4.6
*Values are represented as the mean of three replicates ± SEM

Table 2: Effect of Provide Olive Leaves and Bitter Melon Extracts and
Phytosomes on the Glucose-Induced Hyperglycemia in Rats
-30Minute 0 Minute 30 Minute 60 Minute
90 Minute 120 Minute
Blood Blood Blood
Treatment Blood Glucose Blood Glucose Blood Glucose
Glucose Glucose Glucose
(mg/dl) % (mg/dl) % (mg/dl) %
(mg/dl) ** (mg/dl) % (mg/dl) %
a a
142.8 ±1.20 - 136.8 ± 0.80 - 125.8 ± 0.58 112.6 ±1.75 a -
Control 76.2 ± 0.86 152.2 ± 0.86 a 76 a
9 6 -11 13
Bitter melon b b b 125.2±1.36 a 112.8 ±1.43 a -
74.2 ±1.16 144.8 ± 0.86 71 136.4 ±1.40 -8 129.2±1.28 -7
extract -4 12
Bitter melon b 133.8 ±1.20 b - 119.8 ±1.02 c - 93.4±1.36 b - 83.2 ±1.28 b -
74.0 ± 0.71 145.4 ±1.25 71
phytosome 12 14 26 10
Glibenclamide b 133.6 ±1.36 b - 111.2 ±1.66 d - 91.2 ±1.16 b 75.6 ±1.50 c -
74.6 ±1.33 145.2 ±1.11 71
drug 12 22 -20 16
Olive leaves b 141.6 ± 0.93 a - b 125.0 ±1.52 115.0 ±1.52 a -
76.8± 0.66 145.8 ±1.39 69 132.4 ±1.54 -9 a
extract 4 -7 10
Olive leaves b 135.0 ±1.22 - 115.0 ±1.70 - 89.0±3.18 b
b d
74.6±0.51 144.6 ±1.36 70 79.8 ±1.02 b -9
phytosome 10 20 -26
p-value 0.205 0.001* <0.001* <0.001* <0.001* <0.001*
*One-Way ANOVA, all Pair-wise comparisons were made using Duncan’s Multiple Range Test (DMRT) post-hoc test,
and is significant at p<0.001.
** Data are presented as mean ±SEM
%Degree of blood glucose levels reduction

Figure 1: Microscopic Feature of the Phytosome

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