... being effectively tried in a variety of pathophysiological states (Shashi et al., 1998). ..... PUFA from peroxidation (Kamal-Eldin and Andersson, 1997). Data about the ...... Rizk, A. M., Hammouda F. M. and Hussein L. 1982. 21 st. International ...
When the Lord created the world and people to live in itan enterprise which, according to modern science, took a very long time- I could well imagine that He reasoned with Himself as follows: “If I make everything predictable, these humans beings whom I have endowed with pretty good brains, will undoubtedly learn to predict everything and they will thereupon have no motive to do anything at all, because they will recognize that the future is totally determined and cannot be influenced by any human action. On the other hand, if I make everything unpredictable, they will gradually discover that there is no rational basis for any decision whatsoever and, as in the first case, they will thereupon have no motive to do anything at all. Neither scheme would make sense. I must therefore create a mixture of the two. Let some things be predictable and let others unpredictable. They will then, amongst many other things, have the very important task of finding out which is which” Schumacker E. F., Small is beautiful (1973). Published by Hartley and Marks, Vancouver, Canada.
1
2
PREFACE Wild medicinal plants contain a variety of chemical compounds that may benefit the host plant. These compounds may protect the plant from herbivores, attract pollinators or prevent competitive germination within a plant’s growing space. Several of the more than 5,000 identified alkaloids - organic compounds that have alkaline properties - that are found in angiosperm (flowering plants) plant families include caffeine, nicotine, morphine and quinine. In contrast, glycosides, chemical compounds with one or more sugar molecules, are found in plants like Ginseng, Almonds and Foxglove. The present book deals with the characterization of bioactive lipids (fatty acids, sterols and tocopherols) from the Pulicaria incise, Diplotaxis harra, Avicennia marina grown wild in Egypt. In addition the main goal of the present study is to evaluate the possible role of administrating P. incisa, D. harra and A. marina as hypocholesterolemic agents by using male albino rats.
Authors particularly grateful to Prof. Dr. Saed Soliman El-Saadany, Dr. Mohamed Mostafa Afify Amer, and all academic staff at Biochemistry Department, Faculty of Agriculture, Zagazig University, for the never-ending enthusiasm, for never of failing to share lifelong experience, give advice, inspire and care.
DR. MOHAMED FAWZY RAMADAN HASSANIEN & WALEED ABD EL-GLEEL JANUARY 2009
3
4
TABLE OF CONTENTS Preface
3
1. Introduction
7
2. Materials and methods
15
2.1 Materials
15
2.2 Methods
15
2.2.1 Determination of approximate chemical composition
15
2.2.2 Gas chromatography (GC) analysis of fatty acid methyl esters
16
2.2.3 Gas chromatography (GC) analysis of sterols (ST)
17
2.2.4 Normal-phase high performance liquid chromatography (NP-HPLC) 18 separation, identification and quantification of tocopherols 2.2.5 Antioxidant activity of methanolic extract
19
2.2.6 Radical scavenging activity (RSA) of methanolic extract
20
2.2.7 Experimental animals and diet composition
20
2.2.8 Blood sampling
22
2.2.9 Plasma lipid assay
22
2.2.10 Liver functions assay and serum glucose
23
2.2.11 Kideny functions assay
24
2.2.12 Statistical analysis
24
3. Results and discussion
25
3.1 Chemical Composition, lipids profile and antioxidant properties of 25 wild plants 3.1.1 Fatty acid composition
26
3.1.2 Sterols (ST) composition
28
3.1.3 Tocopherol composition
29
3.1.4 Antioxidant activity and antiradical action of plant extract
30
3.2 Impact of Pulicaria incisa, Diplotaxis harra and Avicennia marina as 31
5
hypocholesterolemic agents 3.2.1 Impact on body weight
32
3.2.2 Impact on lipid parameters, liver and kidney function
33
4. Conclusions
43
References
45
List of Dr. Mohamed Fawzy Ramadan Hassanien publications
55
6
Wild Medicinal Plants as Hypocholesterolemic Agents 1. Introduction The progress of civilization, intake of great amounts of fatty diets, lack of exercise, smoking habits and mental stress participate in hypercholesterolemia and the resultant arteriopathies. Although these mentioned exogenous factors play an important role in increasing the incidence of these biochemical phenomena, yet others significant endogenous conditions participate in its etiology. Idiopathic hypercholesterolemia is a term employed to signify the increase in cholesterol haematological levels without pinpointing its actual pathogenesis. Hypercholesterolemia is a clinical syndrome associated
with
an
abnormally
high
plasma
cholesterol
concentration that increased total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) and elevated high-density lipoprotein cholesterol (HDL). It is well known also that current implicates LDL otherwise known as the “bad” cholesterol as being especially “bad” when it becomes oxidized. Many drugs and some natural supplements with results that vary considerably are used in an effort to lower LDL levels (Sheehan, 2001). The excessive uses of synthetic chemicals have posed great danger to human health and ecosystem in recent years and there has been an upsurge in the clinical use of indigenous drugs. Such herbal plants, originally used in the traditional system of medicine, are now being effectively tried in a variety of pathophysiological states (Shashi et al., 1998). Nonspecific mechanisms like restoration of normal physiological milieu and generalized increase in resistance against infections are
7
proposed and the role of immune system in these drug effects was suggested (Shu, 1998).
Medicinal plants are a therapeutic resource much used by the traditional population of the world specifically for health care. Beneficial effects of plant based medicines and other plant based products
are
information
being is
rediscovered.
leading
to
Moreover,
the
Ethnobotanical
discovery
of
novel
phytopharmaceuticals and other phytoproducts. Nevertheless, validation and utilization as a phytopharmaceuticals product need much basic and applied research in order to put this resource on the same level as the patented pharmaceutical products so achieving acceptation by the medical system, and satisfying the requisite of efficacy, safety and quality.
World-wide interest in the use of medicinal and aromatic plants is increasing. Studying indigenous folk-medicinal plants is vital because such plants are fully adapted to local environments and to conditions compared with any introduced species. Bioactive compounds and extracts isolated from such plants used in herbal medicine have been and are still the centre of interest. Such plants are now being effectively tried in a variety of pathophysiological states. Beneficial effects of plant based medicines and other plant based products are being rediscovered. Medicinal plants can be used in different forms: (i) as a material for extraction of bioactives or for extraction of abundant but inactive compounds, or (ii) as traditional preparations (Amer et al., 2007).
8
Pulicaria incisa is a wild plant found in North Sinai (Egypt). The herb is used by the natives of some upper Egyptian areas in the form of a decoction, sweetened with sugar as a substitute for tea (Nabiel, 2003; Amer et al., 2007). The plant has pleasant aromatic smell and imparted for its essential oil which is partly responsible for its use in preparing the tonic drink. Karim et al. (1992) reported that Pulicaria incisa was used as a tonic and a tea substitute, antispasmodic, hypoglycemic and as an ingredient of a local perfume in the Sudan. The plant is used also in traditional medicine for treating heart diseases by Bedouins (Mansour et al., 1990; Nabiel, 2003). Tariq et al. (1987) analyzed Pulicaria incisa from different regions of Saudi Arabia and mentioned the presence of flavonoids and tannins. Aerial parts of Pulicaria undulata contain 0.33-1.4 % essential oil which had a characteristic aroma and exhibited activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa (Mossa et al., 1987; Rustaiyan et al., 1991; Elegami et al., 1994; EL-Kamali et al., 1998). Diplotaxis harra (Forssk.) Boiss (Brassicaceae) is a common annual species in the desert of Egypt. The plant is spreading whitehispid with thick, woody root and found in sandy stony desert valleys (Täckholm, 1974; Hegazy, 2001). The plant extracts contain high levels of arachidonic and palmitic acids, nonadecane, stigmasterol and β-sitosterol. The volatile constituents of Diplotaxis harra showed high activity against yeasts than Gram positive, Gram negative bacteria and fungi. The nonmethylated fatty acids of the herb showed also strong activity against Gram positive and Gram
9
negative bacteria than yeasts and fungi (Hashem and Saleh, 1999). Avicennia marina (Forssk) Vierh (family Verbenaceae) is a saltexcreting type of mangrove. The bark, leaves and fruits of Avicennia marina are used in traditional medicine to treat skin diseases (Fauvel et al., 1993). Chemical composition as well as the profile of alkaloids, flavonoids, tannins and saponins of the plants from India, Pakistan and Egypt was previously reported (Rizk, 1986; Khatib et al., 1987; Sharaf et al., 2000). Butanol extract exhibited a superior antimicrobial activity against both gram-positive and -negative bacteria as well as good antifungal activity (Mahasneh, 2002). Sitosterol and stigmasterol were the two major components. The monobasic acids composition of the fresh leaves ranged from C12 to C32 with 16:0, 18:1, 18:2 and 18:3ω3 present as predominant acids (Wannigama et al., 1981). Pulicaria incisa, Diplotaxis harra and Avicennia marina are important medical plants that have many medical properties and edible applications. This activity may originate from its contents of active components which may act as antioxidants, attack free radical, improve lipid pattern and organs function. The present communication deals with the characterization of lipid profile (fatty acids, sterols and tocopherols) from the Pulicaria incise, Diplotaxis harra, Avicennia marina grown wild in Egypt. We analyzed the whole plant to obtain informative profile of bioactive lipid compounds and antioxidants which will serve as a basis for further detailed chemical investigation and nutritional evaluation of these
10
plants. In addition the main goal of the present study is to evaluate the possible role of administrating P. incisa, D. harra and A. marina as hypocholesterolemic agents by using male albino rats.
11
Pulicaria incisa
12
Diplotaxis harra
13
Avicennia marina
14
2. Materials and methods 2.1 Materials
Whole plants of Pulicaria incise, Diplotaxis harra (Forssk.) and Avicennia marina (Täckholm, 1974) were obtained in May 2003 from North Sinai (Egypt). The plants were identified by herbarium of Desert Research Institute, Ministry of Agriculture (Egypt). Fresh plant samples were air dried at room temperature and finely grounded. Standards used for sterols (ST) characterization were purchased from Supelco (Bellefonte, PA, USA). Standards used for vitamin E (α-, β-, γ- and δ-tocopherol) were purchased from Merck (Darmstadt,
Germany).
1,1-diphenyl-2-picrylhydrazyl
(DPPH,
approximately 90%) was from Sigma (St. Louis, Mo, USA). Starch, cotton seed oil and buffalo fat were purchased from local market (Zagazig, Egypt). Casein was obtained from Edwic (Egypt). Cholesterol was obtained from Prolab (France). Bile salts (gall bladder extract) were obtained from slaughtered cow. Adult male’s albino rats (90±10 g) were obtained from Faculty of Veterinary Medicine, Zagazig University (Egypt). Reagents and chemicals used were of the highest purity available. 2.2 Methods 2.2.1 Determination of approximate chemical composition
Moisture, ash and total protein and crude fiber were determined according to AOAC (1995). For total lipid (TL)
15
determination, 10 grams of the plant sample was extracted with nhexane for 24 hours in a Soxhelt apparatus. Total carbohydrates were estimated (as glucose) after acid hydrolysis and determined using phenol/sulphuric acid reagent according to the method of Dubois et al. (1960). 2.2.2 Gas chromatography (GC) analysis of fatty acid methyl esters
Fatty acids were transesterified into methyl esters (FAMEs) using
N-trimethylsulfoniumhydroxide
(Macherey-Nagel,
Düren,
Germany) according to the procedure reported by Arens et al. (1994). FAMEs were identified on a Shimadzu GC-14A equipped with flame ionization detector (FID) and C-R4AX chromatopac integrator (Kyoto, Japan). The flow rate of the carrier gas helium was 0.6 mL/min and the split value with a ratio of 1:40. A sample of 1µL was injected on a 30 m x 0.25 mm x 0.2 µm film thickness Supelco SPTM-2380 (Bellefonte, PA, USA) capillary column. The injector and FID temperature was set at 250 °C. The initial column temperature was 100 °C programmed by 5 °C/min until 175 °C and kept 10 min at 175 °C, then 8 °C/min until 220 °C and kept 10 min at 220 °C. A comparison between the retention times of the samples with those of authentic standard mixture (Sigma, St. Louis, MO, USA; 99% purity specific for GLC), run on the same column under the same conditions, was made to facilitate identification.
16
2.2.3 Gas chromatography (GC) analysis of sterols (ST) Separation of ST was performed after saponification of the total lipids (TL) without derivatization according to Ramadan and Moersel (2003). TL (250 mg) were refluxed with 5 mL ethanolic potassium hydroxide solution (6%, w/v) and a few anti-bumping granules for 60 min. The unsaponifiables were firstly extracted 3times with 10 mL of petroleum ether, the extracts were combined and washed 3-times with 10 mL of neutral ethanol/water (1:1, v/v) and then dried overnight with anhydrous sodium sulphate. The extract was evaporated in a rotary evaporator at 25 °C under reduced pressure, and then ether was completely evaporated under nitrogen. GLC analyses of unsaponifiable residues were carried out using a Mega Series (HRGC 5160, Carlo Erba Strumentazione; Milan, Italy) equipped with FID. The following parameters were performed: DB 5 column (J&W scientific; Falsom, CA, USA) packed with 5% phenylmethylpolysiloxan, 30 m length, 0.25 mm i.d., 1.0 µm film thickness; carrier gas (helium) flow 38 mL/min (split-splitless injection was used). Detector and injector were set at 280 °C. The oven temperature was kept constant at 310 °C and the injected volume was 2 µL. The repeatability of the analytical procedure was tested and the relative standard deviation of three repeated analyses of a single sample was 0.05) was found among the experiments. The statistical analysis of results was calculated according to Bailey (1994).
24
3. Results and discussion 3.1 Chemical Composition, lipids profile and antioxidant properties of wild plants
Ethnobotanical information is leading to the discovery of phytopharmaceuticals
and
other
bioactive
phytoproducts.
Nevertheless, validation and utilization as a phytopharmaceuticals product need much basic and applied research in order to put this resource on the same level as the patented pharmaceutical products so achieving acceptation by the medical system, and satisfying the requisite of efficacy, safety and quality. Pulicaria incise, Diplotaxis harra and Avicennia marina are important medical plants that have many medical and edible applications. The whole dried plants were analyzed for chemical composition. In Pulicaria incise the percentages of moisture, total protein, total lipids, total carbohydrate, crude fiber and ash were found to be 7.45, 11.5, 0.44, 41.6, 25.8 and 13.1, respectively. In Diplotaxis harra the percentages
of
moisture,
total
protein,
total
lipids,
total
carbohydrate, crude fiber and ash were found to be 9.56, 7.84, 1.22, 41.8, 15.6 and 24.6, respectively. The percentages of moisture, total protein, total lipids, total carbohydrate, crude fiber and ash in Avicennia marina were 6.05, 1.80, 1.22, 52.48, 16.3 and 16.8, respectively.
25
3.1.1 Fatty acid composition
Total lipids (TL) extracted from Pulicaria incise, Diplotaxis harra and Avicennia marina were 0.44 g/100 g, 1.22 g/100 g and 1.8 g/100 g of dry matter, respectively. Fatty acid profiles of TL is presented in Table 2.
Table 2. Fatty acids composition of Pulicaria incise, Diplotaxis harra and Avicennia marina Pulicaria incise
Diplotaxis harra Relative content %
Avicennia marina
C12:0
0.52 ± 0.01
0.39 ± 0.01
0.66 ± 0.01
C14:0
1.56 ± 0.03
10.3 ± 0.35
4.55 ± 0.35
C16:0
20.7 ± 0.35
0.86 ± 0.03
36.3 ± 0.03
C16:1
2.12 ± 0.03
4.79 ± 0.03
1.20 ± 0.03
C18:0
5.16 ± 0.09
24.9 ± 0.09
5.17 ± 0.09
C18:1
19.3 ± 0.40
26.0 ± 0.40
21.3 ± 0.40
C18:2
37.1 ± 0.56
10.53 ± 0.56
12.5 ± 0.56
C18:3
0.98 ± 0.01
0.39 ± 0.01
2.99 ± 0.01
C20:0
7.80 ± 0.11
ND
ND
C22:0
0.77 ± 0.09
0.44 ± 0.01
0.35 ± 0.02
C20:2
ND a
2.07 ± 0.02
0.44 ± 0.02
C22:2
ND
16.4± 0.09
ND
C24:0
0.84 ± 0.05
0.47 ± 0.02
12.9 ± 0.09
C24:1
3.15 ± 0.15
2.46 ± 0.15
1.64 ± 0.04
Fatty acid
a
not detected.
Results are given as the average of triplicate determinations ± standard deviation.
26
According to the results shown in the table twelve fatty acids were identified in Pulicaria incisa, wherein the analysis of FAMEs gave the proportion of linoleic followed by palmitic and oleic as the major fatty acids, which comprising together more than 77% of total identified FAMEs. Aurand et al. (1987) mentioned that the nutritional value of linoleic acid was due to its metabolism at tissue levels which produces the hormone-like prostaglandin. The activity of these prostaglandins includes lowering of blood pressure and contraction of smooth muscle. Oleic followed by stearic acids were the major fatty acids identified in Diplotaxis harra, which together accounted for more than 50 % of total FAMEs. High level of palmitic acid was also detected in lipidic extract and these results are in line with Hashem and Saleh (1999). In Avicennia marina, palmitic followed by oleic were the major fatty acids, which comprising together ca. 55 % of FAMEs. A striking feature of the Pulicaria incise, Diplotaxis harra and Avicennia marina lipids was the relative high level of polyunsaturated fatty acids (PUFA). Fatty acid profile of Pulicaria incise, Diplotaxis harra and Avicennia marina evinces the lipids as a good source of the nutritionally essential fatty acids. Moreover, interest in the PUFA as health-promoting nutrients has expanded dramatically in recent years. A rapidly growing literature illustrates the benefits of PUFA, in alleviating cardiovascular, inflammatory,
heart
diseases,
atherosclerosis,
autoimmune
disorder, diabetes and other diseases (Finley and Shahidi, 2001; Riemersma, 2001). The fatty acid composition and high amount of unsaturated fatty acids makes oil of Pulicaria incise, Diplotaxis harra and Avicennia marina a special herbs suitable for nutritional application.
27
3.1.2 Sterols (ST) composition
Pulicaria incise, Diplotaxis harra and Avicennia marina crude lipids
are
characterized
by
a
relatively
high
amount
of
unsaponifiables (77 g/kg, 60 g/kg, and 120 g/kg TL, respectively). Five compounds were postulated (Table 3) in all plants. In Pulicaria incise the sterol marker was campesterol which comprised ca. 38.2% of the total ST content. The next major components were stigmasterol followed by β-sitosterol which accounted for ca. 50% of the total ST. Other components, e.g., ∆7-Avenasterol and ∆5avenasterol were presented at a lower levels and equal amounts (ca. 5% of total ST). β-Sitosterol comprised ca. 50 % of the total ST content of Diplotaxis harra. The next major components were stigmasterol followed by ∆5-Avenasterol which comprising together ca. 64% of total ST. Campesterol was the main ST in Avicennia marina (ca. 80% of total ST) and the next major components were
β-sitosterol and ∆7-Avenasterol. ∆5-avenasterol and stigmasterol were presented at a lower levels and equal amounts (ca. 3.33% and 0.89%, respectively, of total ST). Among the different plant sterols,
β-sitosterol has been most intensively investigated with respect to its physiological effects in man. Many beneficial effects have been shown for the β-sitosterol (Yang et al., 2001).
28
Table 3. Phytosterols and tocopherols profile (g/kg TL) of Pulicaria incise, Diplotaxis harra and Avicennia marina
a
Pulicaria incise
Diplotaxis harra
Avicennia marina
Stigmasterol
12.9 ± 0.26
19.58 ± 0.27
1.40 ± 0.02
β-sitosterol
12.2 ± 0.33
50.71 ± 0.35
7.73 ± 0.09
∆5-Avensterol
2.95 ± 0.09
17.35 ± 0.09
3.33 ± 0.03
Campesterol ∆7-Avensterol
19.1 ± 0.39
7.69 ± 0.30
80.2 ± 0.30
2.76 ± 0.10
4.67 ± 0. 08
7.34 ± 0.08
α- tocopherol γ- tocopherol δ- tocopherol
0.006 ± 0.00 0.004 ± 0.00
ND a ND
ND ND
1.99 ± 0.07
1.2 ± 0.07
2.9 ± 0.12
not detected.
Results are given as the average of triplicate determinations ± standard deviation.
Phytosterols, in general, are of interest due to their antioxidant activity and impact on health. Recently, phytosterols have been added to vegetable oils as an example of a successful functional food (Ntanios, 2001). This type of products is now available and has been scientifically proven to lower blood LDL-cholesterol by around 10-15% as part of a healthy diet.
3.1.3 Tocopherol composition
Tocopherols are the major lipid-soluble, membrane-localized antioxidants in humans. Deficiency of these compounds affects
29
many tissues in mammalian and bird models. Vitamin E deficiency in man causes defects in the developing nervous system of children and hemolysis in man. Eipdemiologic studies suggest that people with lower vitamin E and other antioxidant intake and plasma levels may be at increased risk for certain types of cancer and for atherosclerosis (Gey et al., 1991; Rimm et al., 1993). It is also suggested that supplementation with antioxidants may decrease the risk of these and other degenerative processes (Kallio et al., 2002). Tocopherols in vegetable oils, moreover, are believed to protect PUFA from peroxidation (Kamal-Eldin and Andersson, 1997). Data about the qualitative and quantitative profile of vitamins E in Pulicaria incise, Diplotaxis harra and Avicennia marina are summarized in Table 3. Tocopherols were estimated in high levels in the plants. In Pulicaria incise, δ-tocopherol constituted ca. 99.9% of the total analytes and the rest being α-and γ-tocopherol. δTocopherol was the only detectable component in Diplotaxis harra (1.2 g/kg TL) and Avicennia marina (2.9 g/kg TL).
3.1.4 Antioxidant activity and antiradical action of plant extract
Antioxidants have many biological activities, in addition to their direct quenching of radicals or acting as redox molecules in reducing reaction. Thiocyanate antioxidant assays was carried out for 6 days using synthetic and natural antioxidants dissolved in methanol. The antioxidiative activity of the synthetic antioxidant (BHT) was the highest (70%), while the antioxidant activity of methanolic BHA was 66 % and the lowest value was for ascorbic
30
acid (32.2%). Pulicaria incise, Diplotaxis harra and Avicennia marina greatly inhibit the oxidation of linoleic acid (67.4%, 66.3% and 40.5%, respectively) as compared to the blank. The tests expressing antioxidant potency can be categorized into two groups: assays for radical scavenging ability and assays that test the ability to inhibit lipid oxidation under accelerated conditions. However, the model of scavenging stable free radicals is widely used to evaluate the antioxidant properties in a relatively short time, as compared to other methods (Schwarz et al., 2000). After 10 min incubation of TL from Pulicaria incise, Diplotaxis harra and Avicennia marina the results of radical scavenging activity (RSA)
calculated as
percentages of inhibition were 79.6%, 79.7 and 66.6%, respectively. After 30 min incubation percentages of inhibition were 80.9%, 80.1% and 66.9% for TL from Pulicaria incise, Diplotaxis harra and Avicennia marina, respectively. Percentages of inhibition reached 81.2%, 80.4% and 67% for TL from Pulicaria incise, Diplotaxis harra and Avicennia marina, respectively. These results indicated that Pulicaria incise, Diplotaxis harra and Avicennia marina had a strong antiradical activities and high levels of antioxidants that can act rapidly (during the first 10 min) toward free radicals. 3.2 Impact of Pulicaria incisa, Diplotaxis harra and Avicennia marina as hypocholesterolemic agents
The excessive uses of synthetic chemicals have posed great danger to human health and ecosystem in recent years. There has been an upsurge in the clinical use of indigenous drugs. Such herbal plants, originally used in the traditional system of medicine,
31
are now being effectively tried in a variety of pathophysiological states. So that plants have always played a major role in the treatment of human and animal diseases. Medicinal plants can be used in different forms; (a) as a material for extraction of active compounds or abundant but inactive compounds, (b) as extracts and (C) as traditional preparations or as such. In this study the studied plants were used as such to study their roles as hypocholesterolemic agents. 3.2.1 Impact on body weight
Rats fed on high fat diet (Table 4) showed the highest daily gain in body weight 1.84 ± 0.03 g, while rats fed on D. harra diet showed a highly lower daily gain in body weight -0.54 ± 0.04 g. Addition of P. incisa to high fat diet in 8% increased significantly body weight gain from 37.3 ± 1.21 g in negative control to 68.0 ± 3.14 g after 9 weeks. In addition, daily gain in body weight increased in the same period from 0.57 ± 0.02 g in negative control to 1.04 ± 0.05 g. The same trend was nearly shown for rats fed on A. marina, where the body weight gain was increased from 37.3 ± 1.21 g to 44.5 ± 3.91 g and daily gain in body weight from 0.57 ± 0.02 g to 0.66 ± 0.03 g. D. harra followed an opposite trend, where gain in body weight and daily gain in body weight was decreased from 37.3 ± 1.21 g to –34.8 ± 2.80 g and from 0.57 ± 0.02 g to -0.54 ± 0.04 g, respectively. It's clear that the addition of such studied plants has a beneficial effect in daily gain in body weight and it may be due to its content of polyphenols antioxidants which has a
32
hypocholesterolimic effect (Hashem and Saleh, 1999; Rizk et al., 1986).
Table 4. Body weight gain of rats feed on basal diet (negative control), hypercholesterolemic diet (positive control), hypercholesterolemic diets supplemented with Pulicaria incise, Diplotaxis harra and Avicennia marina
Treatment Negative control Positive control Pulicaria incise group Diplotaxis harra group Avicennia marina group
Initial body weight
Final body weight
Body weight gain
Daily body weight increase
82
119
37.3
0.57
86
205
119
1.84
86
155
68.0
1.04
89
54
-34.8
-0.54
91
139
44.5
0.66
3.2.2 Impact on lipid parameters, liver and kidney functions
The amount of lipids as well as liver and kidneys functions in negative control were given the arbitrary value 100. The increase and decrease in lipids parameters as well as liver and kidney functions in positive control (high fat diet) and diets supplemented with the studied plants were related to 100. TL showed an increase from 400 ± 7.06 mg/dl in negative control to 1551 ± 43.7 mg/dl in positive control. By given the value 100 to negative control, the
33
increase in TL was ca. 387% for positive control. On other hand, TG and TC were also increased by ca. 559 % and 258%, respectively as compared with negative control (Table 5); all these values are highly significant.
Table 5. Effect of feeding on hypercholesterolemic diet supplemented with different medicinal wild plants for nine weeks period on total lipids, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol
Total lipids
Total cholesterol
mg/dl
%
400
100
Positive control
1551
Pulicaria incise group
Type of lipid pattern
Triglycerides
HDLcholesterol
LDLcholesterol
VLDLcholesterol
mg/dl
%
mg/dl
%
mg/dl
%
mg/dl
%
mg/dl
%
148
100
48
100
96
100
42
100
9.61
100
387
384
258
269
559
49
51
284
664
53
561
633
158
202
135
109
227
82.
85.8
97.
227
21.
227
Diplotaxis harra group
395
98.7
121
81.8
58.9
122
58.1
60.4
57.0
133
11.7
122
Avicennia marina group
663
165
217
146
117
244
78.5
81.6
117
274
23.4
244
Negative control
The impact of P. incisa, D. harra and A. marina administration for nine weeks on lipid pattern is recorded in Table 5. The levels of TL, TG and TC were highly significant for all treatments expect D. harra group which was non significant and the groups were
34
decreased from ca. 387% to 158% and 165% for TL, decreased from 559% to 227%, 122% and 244% for TG and decreased from 258% to 135%, 81% and 146% for TC after nine weeks of feeding on the studied wild plants. Our results agreed with those of Lizuka et al. (1998) who found that Dia-saiko-Hu-Tang (traditional herb medicine)
has
atheromatous
inhibitory plaque
effects
on
the
development
formation
in
spontaneous
of
familial
hypercholesterolemia model in rabbits.
HDL showed a decrease from 100% in negative control to 51.5% in positive control. On other hand, LDL and VLDL were increased by ca. 664% and 561%, respectively compared with negative
control;
all
these
values
are
highly
significant.
Administration of P. incisa, D. harra and A. marina for nine weeks decreased significantly the levels of LDL and VLDL from 664% to 227%, 133% and 274% for LDL and from 561% to 227%, 122% and 244% for VLDL, respectively. On the other hand, there was an increase from 51.5% to 85.8%, 60.4% and 81.6% for HDL, respectively. Our results agreed with those of Crouse et al. (1999) who found that soy isoflavones are believed to be soy’s main cholesterol-lowering ingredients. Soy preparations containing high amounts of isoflavones effectively lowered TC and LDL. On the other side, Rodriguez et al. (2002) found that vitamins C and E have an antioxidants properties and its supplementation have a clear beneficial effect in hypercholesterolemia-derived vascular dysfunction.
35
Data in Table 6 reveal the effect of administration P. incisa, D. harra
and
A.
marina
on
atherogenic
index,
risk
ratio,
chylomicronaemia, HDL/PC* and TG/HDL of hypercholesterolemic rats. The risk ratio which equal to the ratio of HDL/LDL cholesterol is a good indication for hypercholesterolemia. This ratio was 100% by negative control as compared with positive control 1302%. It decreased to 261%, 223% and 339% for P. incisa, D. harra and A. marina, respectively. These results are highly significant and in agreement with those of Newman et al. (1992) who found significant lowering of LDL, while HDL increased in chickens fed rice bran diet. Ghali et al. (2000) found that the addition of rice bran to hypercholesterolemic rats can improve the risk ratio. Atherogenic index which is considered as the predictor of atherogenesis was calculated after the experimental period to asses the effect P. incisa, D. harra and A. marina supplementations on the hypercholesterolemic rats. The atherogenic index was 100% for the negative control group and it was increased to 1268 % for the positive control group, while that were 266%, 218% and 333% for P. incisa, D. harra and A. marina, respectively. Once again it can be concluded that supplementation with studied wild plants is very important to rats fed on hypercholesterolemic diet to prevent free radicals formation and consequently atherogenesis. These results are agreed with those of Montano et al. (1998) and Shige et al. (1998) who mentioned that the role of vitamin C in decreasing plasma LDL might be due to the increase of receptor numbers. However
vitamin
E
suppress
the
acyl-CoA
cholesterol
acyltransferase activity in the cell lysate with no effect on hydrolysis
36
of cholesterol ester, which resulted in reducing the uptake of modified LDL and less cholesterol esterification in macrophage.
Table 6. Effect of feeding on hypercholesterolemic diet supplemented with the studied medicinal wild plants on the parameters of atherogenic index, risk ratio, chylomicronaemia ratio, HDL-Cholesterol/pooled lipoprotein cholesterol (PC*) and TG/HDL- cholesterol
Atherogenic index
Treatment
Chylomicronaemia
Risk ratio
%
HDL/ PC* ratio
ratio
%
%
TG / HDL ratio
%
%
Negative control
0.54
100
0.44
100
3.10
100
0.647
100.0 0
0.499
100
Positive control
6.85
1268
5.73
1302
1.42
45.8
0.128
19.78
5.445
1091
Pulicaria incise group Diplotaxis harra group Avicennia marina group
1.44
266
1.17
260.9
1.85
59.6
0.409
63.21
1.324
265
1.18
218
0.98
222.7
2.07
66.7
0.458
70.79
1.014
203
1.80
333
1.49
338.6
1.77
57.0
0.357
55.18
1.496
299
Atherogenic index= VLDL-Cholesterol + LDL-Cholesterol / HDL-Cholesterol, Risk ratio = HDL-Cholesterol / LDL-Cholesterol, Chylomicronaemia ratio = Serum Total Cholesterol / Triglycerides, PC*= VLDL-cholesterol + LDL-Cholesterol + HDL-Cholesterol
Chylomicronaemia (WHO Type I lipoprotein lipase deficiency) a rare disease, is due to deficiency of extrahepatic lipoprotein lipase. TC levels may be very high and the serum at 4 ºC shows an upper floating
layer
of
chylomicrons
above
a
clear
layer.
The
Chylomicronaemia ratio is usually less than 0.2 (normally above 3).
37
The Chylomicronaemia ratio was 3.10 ± 0.11 for the negative control group and it was decreased to 1.42 ± 0.04 for the positive control group, while it were 1.85 ± 0.06, 2.07 ± 0.24 and 1.77 ± 0.04, for P. incisa, D. harra and A. marina, respectively. So, we can say that negative or positive groups and all treatment groups were out of its range and not have that disease. This result was agreed with those of Gray and Howorth (1983). The ratio of HDL/pooled lipoprotein cholesterol (HDL/PC* where PC* = VLDL+LDL+HDL) was calculated. It was 0.647 ± 0.004 for the negative control group and it was decreased to 0.128 ± 0.008 for the positive control group, while it were 0.409 ± 0.015, 0.458 ± 0.004 and 0.357 ± 0.009, for P. incisa, D. harra and A. marina, respectively. They were highly significant affected by the treatments compared with the control group. This result was agreed with those of Kestin et al. (1990), Kahlon et al. (1996) and Ghali et al. (2000) who reported that the ratio of HDL to PC* was highly significantly increased.
The TG/HDL ratio was 0.49 ± 0.02 for the negative control group and it was increased to 5.44 ± 0.36 for the positive control group, while it were 1.32 ± 0.08, 1.01 ± 0.08 and 1.49 ± 0.05 for the studied wild plants, respectively. This result was agreed with those of Ghali et al. (2000) who found that the addition of rice bran to hypercholesterolemic
rats
can
improve
respectively for the studied wild plants.
38
the
ratio
TG/HDL
Table 7 shows the effect of administrating P. incisa, D. harra and A. marina diets on the liver function of hypercholesterolemic rats. Hypercholesterolemia was characterized by a significant increase in serum GPT. The increase was 226% in positive control group, while it was 147%, 147% and 153% for the studied plants (P. incisa, D. harra and A. marina), respectively. The increase in serum GPT activity indicates liver cell necrosis and hepatic injury. Treatment with P. incisa, D. harra and A. marina induced a decrease in the high activity of serum GPT and the levels were decreased compared to the positive control group after nine weeks administration. Also, hypercholesterolemia was characterized by an increase in serum GOT. The increase of serum GOT activity was more specific for cardiac injury. The increase of serum GOT were 260% in positive control group, while it was 107%, 142% and 187% for P. incisa, D. harra and A. marina, respectively. Percentage of serum GOT/GPT ratio in Table 7 refers to high ratio in positive control and A. marina groups that were 115% and 122%, respectively while they were 73.3% and 97.5% for P. incise and D. harra, respectively. Serum GOT, GPT and GOT/GPT ratio were high significant as shown as Table 7 and the data were agreed with those Hawcroft (1978) and Daher et al. (2006).
Bilirubin is an end product of heme degradation in mammals (Maines, 1988). Total serum bilirubin for the positive control group was 227% where it was 227%, 181% and 184% for P. incisa, D. harra and A. marina, respectively. This result was agreed with Neuzil and Stocker (1993) who decided that, bilirubin, in vitro, is a
39
powerful scavenger of peroxyl radicals and singlet oxygen perhaps the jaundice of premature babies has some physiological role provided it does not occur to excess. Bilirubin bound to albumin can protect both the protein and albumin-bound fatty acids against freeradical damage. However, there is little direct evidence that bilirubin is an important antioxidants in vivo. Table 7. Serum contents of total bilirubin, serum protein fractions and transaminase activities in rats fed on basal diet, high fat diet and diet supplemented with Pulicaria incisa , Diplotaxis harra and Avicennia marina SGOT
SGPT
SGOT/ SGPT
Total bilirubin
Total
Albumin
Globulin
protein
Globulin
ratio
Treatment
u/l
%
u/l
%
Negative control
6.75
100
5.60
100
Positive control
17.6 1
260
12.
Pulicaria incisa
7.27
107
9.64
12.6
Albumin/ ratio
%
gm/ dl
%
gm /dl
%
gm/ dl
%
gm/d l
%
%
1.20
100
0.33
100
7.13
100
4.64
100
2.49
100
1.86
100
226
1.39
115
0.75
227
9.59
134
5.33
114
4.4.2
171
1.21
65.0
8.26
147
0.88
73.3
0.75
227
7.74
108
3.64
78.4
4.10
164
0.91
48.9
142
8.26
147
1.17
97.5
0.39
181
4.28
60
2.20
47.4
2.08
85.5
1.06
56.9
187
8.60
153
1.47
122
0.61
184
8.27
115
3.66
78.8
4.61
185
0.79
42.4
group Diplotaxis harra
group Avicennia marina
group
Hypercholesterolemic state was accompanied by a high significant increase in total serum protein, albumin and globulin (Table 7). It was 134%, 114% and 171%, respectively. There was a
40
remarkable increase at total group treated with P. incisa and A. marina. The values were 108% and 115% for total protein, respectively and were 164% and 185% for globulin, respectively. Serum total protein and globulin were decreased to 60.0% and 85.5% for the group treated with D. harra, respectively. Albumin content was increased by 114% relative to negative control, while there were decreases in its values with all treated group and it was 78.4%, 47.4% and 78.8%, respectively. There were a high significant
decrease
in
serum
albumin,
globulin
and
albumin/globulin ratio and non significant decrease in globulin with the group treated with D. harra and this decrease may be due to the starvation status, which caused from the bad taste of this plant for rats (Strove, 1989).
On the other side, hypercholesterolemic state was accompanied by a high significant increase in serum glucose (Table 8), wherein the level was 127% for the positive control and it was 92.6%, 93.5% and 92.3% for the studied plants, respectively. This result was agreed with Labib et al. (1991) who reported that the increase of serum glucose might be due to impaired function of liver which is reported to play an important role in the regulation of serum glucose. Alternatively, it may be due to the lowered circulating insulin level caused by the high level of serum glucose.
41
Table 8. Serum glucose, urea and uric acid in rats fed normal diet, high fat diet and high fat diet supplemented with Pulicaria incisa, Diplotaxis harra and Avicennia marina Glucose mg/dl %
Urea mg/dl %
Uric acid mg/dl %
Negative control
88.2
100
18.3
100
5.84
100
Positive control
112
127
32.6
177
6.69
114
Pulicaria incisa group
81.8
92.6
29.7
162
5.71
97.7
Diplotaxis harra group
82.5
93.5
35.2
192
6.15
105
Avicennia marina group
81.5
92.3
29.3
159
5.77
98.8
Investigation Treatments
Serum uric acid (Table 8) was 114% for positive control and decreased to 97.7%, 105% and 98.8% for P. incisa, D. harra and A. marina, respectively. Blood urea was increased to 177% for positive control and it was 162%, 192% and 159% for P. incisa, D. harra and A. marina, respectively. High significant increase in D. harra (192%) was noticed. This significant increase may be due to starvation status caused by D. harra administration, which resulted from the bad taste of that plant.
42
4. Conclusions The trend towards natural ingredients and products promoting health is likely to increase. Improved knowledge on the composition, analysis and properties of Pulicaria incise, Diplotaxis harra and Avicennia marina would assist in efforts for functional applications of these plants. Data about Pulicaria incise, Diplotaxis harra and Avicennia marina are very few. On the other hand, there are not reports in literature about detailed composition of Pulicaria incise, Diplotaxis harra and Avicennia marina lipids. It could be concluded that the Pulicaria incise, Diplotaxis harra and Avicennia marina are a good source of essential fatty acids and lipid-soluble bioactives. The high linoleic acid content makes the oils nutritionally valuable. Tocopherols and sterols at the level estimated may be of nutritional importance in the application of the oil. Pulicaria incise, Diplotaxis harra and Avicennia marina could be nutritionally considered as a non-traditional supply for pharmaceutical industries and edible purposes.
Improves knowledge of the biological effect of wild medicinal plants would assist in the efforts to achieve the curing effect of these plants. Obtained data on the role of P. incisa, D. harra and A. marina as a hypocholesterolemic agent is of importance. P. incisa and A. marina plants can play a hypocholesterolemic role against hypercholesterolemic disease in concentration of 8% in diet. D. harra may work the same effect but in low dose. The possible role of these plants may be from its contents of active components which may play as antioxidants, attack free radical, improve lipid
43
pattern and organs function. Thus, these medical plants may consider as a new non-conventional supply for the pharmaceutical industries and for edible purposes.
44
REFERENCES Amer MMA, Ramadan MF, Abd El-Gleel W. 2007. Impact of Pulicaria incisa, Diplotaxis harra and Avicennia marina as hypocholesterolemic agent. Deutsche Lebensmittel Rundschau 103, 320-327. AOAC. 1995. Official Methods of Analysis. Association of Official Analytical Chemists, AOAC, Washington D.C. Arens M, Schulte E, Weber K. 1994. Fettsäuremethylester, Umesterung
mit
Trimthylsulfoniumhydroxide
(Schnellverfahren). Fat Sci. Technol. 96, 67-68. Aurand L W, Wood AF, Wells MR. 1987. Food composition and analysis. AVI Book, Van Nostrand Reinhold Co, New York (USA). Bailey NTJ. 1994. In statistical methods in biology, 3rd Ed. Cambridge University Press (UK). Bandaranayake WM. 1998. Traditional and medical uses of mangroves. Mangroves and Salt Marshes 2, 133-148. Bouls L. 1995. Flora of Egypt checklist. Al-Hadara Publishing. Cairo. Egypt: 479. Bucolo G., David H. 1973. Quantitative determination of serum triglycerides by use of enzyme. Clinic. Chem. 19, 476-482. Crouse JR, Morgan T, Terry JG. 1999. A randomized trial comparing the effect of casein with that of soy protein containing
varying
amounts
45
of
isoflavones
on
plasma
concentrations of lipids and lipoproteins. Arch. Intern. Med. 159, 2070-2076. Daher CF, Baroody FG, Baroody MG. 2006. Effect of Urtica dioica extract intake upon blood lipid profile in the rates. Fitoterapia 77, 183-188. Doumas BT, Watson WA, Biggs HG. 1971. Albumin standards and the measurement of serum albumin with bromocresol green. Clinic. Chem. Acta. 31, 87-96. Dubois MA, Geddes WF, Smith F. 1960. The carbohydrates of the gramineas. A quantitive study of carbohydrate of wheat germ. Cereal Chem. 37, 557-561. Elegami AAB, Ishag KE, Mahmoud EN, Alfutuh IMA, Karim EIA. 1994. Insecticidal activity of Pulicaria undulata oil. Fitoterapia 65, 82-83. EL-Kamali HH, Ahmed AH, Mohammed AS, Yahia AAM, EL-Tayeb I, Ali AA. 1998. Antibacterial properties of essential oils from Nigella sativa seeds, Cymbogeon citrates leaves and Pulicaria undulate aerial parts. Fitoterapia 69, 77-78. Estadella D, Oyama LM, Dâmaso AR, Ribeiro EB, Oller DNC. 2004. Effect of palatable hyperlipidic diet on lipid metabolism of sedentary and exercised rats. Nutrition 20, 218-224 Fauvel MT, Gleye JT, Fouraste I. 1993. Phenylpropanoid glycosides from Avicennia marina. Planta Med. 59, 389. Finley JW, Shahidi F. 2001. The chemistry, processing and health benefits of highly unsaturated fatty acids: an overview, In
46
Omega-3 Fatty Acids, Chemistry, Nutrition and Health Effects, John WJ, F. Shahidi F (ed), American Chemical Society: Washington DC; pp. 1-13. Friedewald T, William I, Robert L, Donald FS. 1972. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinic. Chem. 18, 499-502. Gey KF, Puska P, Jordan P, Moser UK. 1991. Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Amer. J. Clini.l Nut. 53, 3245-3265. Ghali Y, Laila R, Moustafa AA, Wasef MM, Soheir Abd El- Rahman N. 2000. Cholesterol-Lowering effect of various rice bran diets on hypercholesterolemic rats. Egyptian J. Biochem. 18, 213228. Gochman N, Schmitz JM. 1971. Automated determination of uric acid with use of uricase-peroxidase system. Clinic. Chem. 17, 1154-1159. Gray CH, Howorth PJN. 1983. "Clinical chemical pathology" Ninth edition, The English language book society and Edward Arnold (publishers) Ltd. P. 144. Haraguchi
H,
Hashimoto
K,
Yagi
A.
1992.
Antioxidative
substances in leaves of Polygonum hydropiper. J. Agric. Food Chem. 40, 271-291.
47
Hashem FA, Saleh MM. 1999. Antimicrobial components of some cruciferae plants (Diplotaxis harra Forsk. and Erucaria microcarpa Boiss.). Phytother. Res. 13, 329-32. Hawcroft, D. 1978. "Diagnostic enzymology". John Wiley and Sons, New York. Hegazy AK. 2001. Reproduction diversity and survival of the potential
annual
Diplotaxis
(Forssk.)
harra
Boiss
(Brassicaceae) in Egypt. Ecography 24, 403-412. Hegsted D. M., Mills R. C., Elvehjem C. A. and Hart E. B. 1941. Choline in the nutrition of chicks. J. Biol. Chem. 138: 459-466. Henry, R.J. 1964. Clinical Chemistry, Harber and Row Publisher, New York. p. 181. Kahlon, T. S., Chow M. M., Hudson C.A. and Sayre R. N. 1996. Cholesterol-lowering
by
rice
bran
and
rice
bran
oil
unsaponifiable matter in hamsters. Cereal Chem. 73(1): 69-74. Kallio H, Yang B, Peippo P, Tahvonen R, Pan R. 2002. Triacylglycerols,
glycerophospholipids,
tocopherols
and
tocotrienols in berries and seeds of two subspecies (ssp. sinensis and mongolica) of Sea buckthorn (Hippophaë rhamnoides). J. Agric. Food Chem. 50, 3004-3009. Kamal-Eldin A, Andersson R. 1997. A multivariate study of the correlation
between
tocopherol
content
and
fatty
acid
composition in vegetable oils. J. Amer. Oil Chem. Soc. 74, 375380.
48
Karim EI, Ishag KE, Elegami AAB, Mahmoud EN, Abu-AL-Futuh I. 1992. D-Carvotanacetone from Pulicaria undulate. Fitoterapia 63, 281. Kestin M, Moss R, Clifton PM, Nestel PJ.
1990. Comparative
effects of three cereal brans on plasma lipids, blood pressure and glucose metabolism in mildly hypercholesterolemic men. Am. J. Clinic. Nutr. 52, 661. Khatib G, Amed NF, Usmani SS, Usmani TH. 1987. Effect of seasonal variations on chemical composition of mangrove vegetation of Karachi. Pakistan. J. Sci. Ind. Res. 30, 294-297. Kikuchi H, Hayakawa N, Onodera S, Matsubara E, Yasuda O, Chonan R, Ishikawa F. 1998. Effect of soy milk and bifidobacterium fermented soy milk on lipid metabolism in aged ovariectomized
rats.
Bioscience,
Biotechnology
and
Biochemistry 62, 1688-1692. Labib S, Sitohy M, El-Massry R, El-Saadany S. 1991. Lipid metabolism in rats fed either butter or butter products as the fat source. Grasas y aceites 42: 255-260. Lee JC, Kim HR, Jange YS. 2002. Antioxidant property of an ethanol extract of the stem of Opuntia ficus indica var. Saboten. J. Agric. Food Chem. 50, 6490-6496. Lizuka A, Osamu TI, Fumihiko Y, Bunsho M, Sakae A, Yasuhiro K, Kazuo K, Akiyo M, Hiroshige I. 1998. Inhibitory effects of Daisaiko-to
(Da-Chai-Hu-Tang)
atherosclerotic
lesions
in
on
the
Kurosawa
progression and
of
Kusanagi-
hypercholesterolemic rabbits. J. Ethnopharmacology 63, 209218.
49
Mahasneh AM. 2002. Screening of some indigenous Qatari medicinal plants for antimicrobial activity. Phytother. Res. 16, 751-753. Maines MD. 1988. Heme oxygenase: function, multiplicity, regulatory mechanisms and clinical application. FASEB J. 2, 2557. Mansour RMA, Ahmed AA, Melek FR, Saleh NAM. 1990. The flavonoids of Pulicaria incisa. Fitoterapia 61, 186-187. Meleka MGB, Wafai ZAM, Fatehi AA. 2002. MSc. Thesis, Institute of African Research and Studies, Cairo University, Cairo, Egypt. Montano CE, Fernandez ML, McNamara DJ. 1998. Regulation of apolipoprotein B-containing lipoproteins by vitamin C level and dietary and fat saturation in guinea pigs. Metabolism Clinic. Exper. 47, 883-891. Mossa JS, Hifnawy MS, AL-Yahya MA, AL-Meshal IA, Mekkawi AG. 1987. Aromatic plants of Saudi Arabia part 8, GC/Ms analysis of essential oils of Pulicaria Arabica and Pulicaria undulate. Int. J. Crude Drug Res. 25, 113-119. Nabiel
AMS.
2003.
Global
Phytochemistry:
the
Egyptian
Experience. Phytochemistry 63, 239-241. Naito HK, Kaplan A. 1984a. Cholesterol. Clinic. Chem. The CV Mosby Co. St Louis. Toronto. Princeton; 437:1194-11206. Naito HK, Kaplan A. 1984b. High-density lipoprotein (HDL) cholesterol
Clinic. Chem. The CV. Mosby Co. St Louis.
Toronto. Princeton; 437:1207-1213.
50
Neuzil J, Stocker R. 1993. Bilirubin attenuates radical-mediated damage to serum albumin. FEBS Lett. 331, 281. Newman RK, Betsehart AA, Newman CW, Hofer PJ. 1992. Effect of full-fat or defatted rice bran on serum cholesterol. Plant Foods Human Nutr. 42, 37-43. Ntanios F. 2001. Plant sterol-ester-enriched spreads as an example of a new functional food. Eur J. Lipid Sci. Technol. 103, 102-106. Patton CG, Crouch SR. 1977. Enzymatic determination of urea. Anal. Chem. 49, 464-469. Ramadan MF, Moersel JT. 2003. Oil Goldenberry (Physalis peruviana L.). J. Agric. Food Chem. 51, 969-974. Ramadan MF, Mörsel JT. 2002. Direct isocratic normal phase assay of fat-soluble vitamins and β-carotene in oilseeds. Eur. Food Res. Technol. 214, 521-527. Reinhold, J.G. 1953. Standard Methods in Clinical Chemistry, Editor Reiner, M., Volume I, Academic Press, New York, P. 88. Reitman S, Frankel S. 1957. Determination of serum glutamate oxaloacetate and glutamate pyruvate transamin-ases. Am. J. Cilnic. Path. 28, 56- 60. Riemersma RA. 2001. The demise of the n-6 to n-3 fatty acid ratio? A Dossier. Eur J. Lipid Sci. Technol. 103: 372-373. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett, WC. 1993. Vitamin E consumption and the risk of coronary heart disease in men. The New England Journal of Medicine 328,1450-1456.
51
Rizk AM. 1986. The phytochemistry of the flora of Qatar. Scientific and applied research center, Qatar University, pp 83-84. Rizk, A. M., Hammouda F. M. and Hussein L. 1982. 21st International Horticultural Congress (Hamburg, Germany), Abstract collection, 2148. Rodriguez AJ, Andres G, Ezequiel E, Beatriz N, Maitane P, Roberto
SB,
Jose
AP,
Diego
M.
2002.
Dietary
supplementation with vitamins C and E prevents down regulation
of
endothelial
NOS
expression
in
hypercholesterolemia in vivo and in vitro. Atherosclerosis 165, 33-40. Rustaiyan A, Habibi Z, Saberi M, Jakupovic J. 1991. A norguaianolide and a glaucolide-like eudesmanolide from Pulicaria undulata. Phytochemistry 30, 2405- 2406. Schwarz K, Bretelsen G, Nissen LR, Gardner PT, Heinonen MI, Hopia A, Huynh-Ba T, Lambelet P, McPhail D, Skibsted LH, Tijburg L. 2000. Investigation of plant extracts for the protection of processed foods against lipid oxidation. Comparison of antioxidant assays based on radical scavenging lipid oxidation and analysis of the principal antioxidant compounds. Eur. Food Res. Technol. 21, 319-328. Shaker KH, Elgama MH, Seifert K. 2001. Iridoids from Avicennia marina. Z. Naturforsch 56,: 965-8. Sharaf M, El-Ansari MA, Saleh NAM. 2000. New flavonoids from Aicennia marina. Fitoterapia 71, 274-277
52
Shashi GPK, Gupta MKR, Somvanshi AS, Vijjan VK. 1998. Toxicity of azadirachtin (neem) based formulation in rates. Indian J. Environ. Toxicol. 8, 77- 80. Sheehan A. 2001. Cholesterol Update: Oxidized and Nonoxidized LDL cholesterol, Do We Have to Worry about Both? Dynamic. Chiropractic. Volume 19, issue 11. Shige H, Ishikawa T, Suzukawa M, Nishiwaki M, Nakajima K, Ito T, Higashi K, Ayaori M, Yonemura A, Nestel P, Nakamura H. 1998. Vitamin E reduces cholesterol esterification and uptake of acetylated low-density lipoprotein in macrophages. Lipids 33, 1169-1175. Shu YZ. 1998. Recent natural products based drug development: A pharmaceutical industry perspective. J. Nat. Prod. 61, 10531071. Strove EA. 1989. Biochemistry, Mir Publishers, Moscow. Täckholm V. 1974. Students flora of Egypt (second edition), Cairo University. Beirut, pp. 479. Tariq
M,
Mossa JS,
AL-Meshal
IA,
AL-Badr
AA.
1987.
Phytochemical and biological screening of Saudi medicinal plants. Part 10. A study on Saudi plants of family Compositae. Int. J. Drug Res. 25,17-25. Trinder P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem. 6, 24-27.
53
Vangronsveld J, Clijsters H. 1994. Mechanisms of metal tolerance in plants. Evolutionary Aspects. CRC Press, Boca Raton, FL, pp. 179-193. Wannigama GP, Volkman FTG, Nichols PD, Johns RB. 1981. A comparison of lipid components of the fresh and dead leaves and pneummatophores of the mangrove Avicennia marina. Phytochemistry 20, 659-666 Yang B, Karlsson RM, Oksman PH, Kallio HP. 2001. Phytosterols in
sea
buckthorn
(Hippophaë
rhamnoides
L.)
berries:
Identification and effects of different origins and harvesting times. J. Agric. Food Chem., 49, 5620-5629.
54
List of Dr. Mohamed F. Ramadan Hassanien publications Publications in the international peer-reviewed journals 1- Mahmoud Z. Sitohy, Salah M. Labib, Said S. El-Saadany, and Mohamed F. Ramadan (2000) Optimizing the conditions for starch dry
phosphorylation
with
sodium
mono-
and
dihydrogen
orthophosphate under heat and vacuum. Starch/Staerke 52 (4): 95100. 2- Mahmoud Z. Sitohy, Said S. El-Saadany, Salah M. Labib, and Mohamed F. Ramadan (2000) Physicochemical properties of different types of starch phosphate monoesters. Starch/Staerke 52 (4): 101-105. 3- Mahmoud Z. Sitohy, and Mohamed F. Ramadan (2001) Granular properties of starch phosphate monoesters. Starch/Staerke 53 (1): 27-34. 4- Mahmoud Z. Sitohy, and Mohamed F. Ramadan (2001) Degradability of different phosphorylated starches and thermoplastic films prepared from corn starch phosphomonoesters. Starch/Staerke 53 (7): 317-322. 5- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2002) Neutral lipid classes of black cumin (Nigella sativa L.) seed oils. European Food Research and Technology 214 (3): 202-206. 6- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2002) Direct isocratic normal-phase assay of fat-soluble vitamins and betacarotene in oilseeds. European Food Research and Technology 214 (6): 521-527. 7- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2002) Proximate neutral lipid composition of niger (Guizotia abyssinica Cass.) seed. Czech Journal of Food Sciences 20 (3): 98-104.
55
8- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2002) Characterization of phospholipid composition of black cumin (Nigella sative L.) seed oil. Nahrung/Food 46: 240-244. 9- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2002) Oil Composition of coriander (Coriandrum sativum L.) fruit-seeds. European Food Research and Technology 215: 204-209. 10- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Lipid profile of prickly pear pulp fractions. Journal of Food, Agriculture and Environment 1: 66-70 (invited contribution). 11- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Analysis of glycolipids from black cumin (Nigella sative L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) oilseeds. Food Chemistry 80: 197-204. 12- Mohamed F. Ramadan Hassanien, and Joerg-Thomas Moersel (2003) Das Physalisbeerenoel: Eine neuentdeckte Quelle an essentiellen
Fettsaeuren,
Phytosterolen
und
antioxidativen
Vitaminen. Fluessiges-Obst 7: 398-402. 13- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Oil goldenberry (Physalis perviana L.). Journal of Agricultural and Food Chemistry 51 (4): 969-974. 14- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Oil cactus pear (Opuntia ficus-indica L.). Food Chemistry 82 (3): 339345. 15- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Determination of lipid classes and fatty acid profile of niger (Guizotia abyssinica Cass.) seed oil. Phytochemical Analysis 14 (6): 366-370. 16- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Recovered lipids from prickly pear [(Opuntia ficus-indica (L.) Mill)] peel: a good source of polyunsaturated fatty acids, natural antioxidant vitamins and sterols. Food Chemistry 83 (3): 447-456.
56
17- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2003) Phospholipid composition of niger (Guizotia abyssinica Cass.) seed oil. Food Science and Technology/Lebensmittel-Wissenschaft und Technologie 36: 373-376. 18- Mohamed F. Ramadan Hassanien, and Joerg-Thomas Moersel (2003) Agro-waste products from prickly pear fruit processing as a source of oil. Fruit Processing 4: 242-248. 19- Mohamed F. Ramadan, Lothar W. Kroh and Joerg-Thomas Moersel (2003) Radical scavenging activity of black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) crude seed oils and oil Fractions. Journal of Agricultural and Food Chemistry 51: 6961-6969. 20- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2004) Goldenberry: a noval fruit source of fat soluble bioactives. INFORM 15 (2): 130-131. 21- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2004) Oxidative stability of black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) upon stripping. European Journal of Lipid Science and Technology 106 (1): 35-43. 22- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2004) Antiradical performance of some common and nontraditional vegetable oils. INFORM 15 (8): 553-555. 23- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2005) Cape gooseberry, a golden fruit of golden future. Fruit-Processing 6: 396400. 24- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2006) Mowrah butter: Nature's novel fat. INFORM 17:124-126.
57
25- Mohamed F. Ramadan, Sharanabasappa G., Seetharam Y. N., Seshagiri M. and Joerg-Thomas Moersel (2006) Characterisation of fatty acids and bioactive compounds of Kachnar (Bauhinia purpurea L.) seed oil. Food Chemistry 98 (2): 359-365. 26- Mohamed F. Ramadan, Sharanabasappa G., Seetharam Y. N., Seshagiri M. and Joerg-Thomas Moersel (2006) Profile and levels of fatty acids and bioactive constituents in mahua butter from fruitseeds of Buttercup tree [Madhuca longifolia (Koenig)]. European Food Research and Technology 222: 710-718. 27-
Ayman
M.
Gomaa
and
Mohamed
F.
Ramadan
(2006)
Characterisation of fatty acids and bioactive lipid compounds of Cistanche
phelypaea.
Electronic
Journal
of
Environmental,
Agricultural and Food Chemistry 5: 1306-1312. 28- Rafaat El-Sanhoty, Tamer Shahwan and Mohamed F. Ramadan (2006) Application of artificial neural networks to develop a classification model between genetically modified maize (Bt-176) and conventional maize by applying lipid analysis data. Journal of Food Composition and Analysis 19: 628-636. 29- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2006) Screening of the Antiradical Action of Vegetable Oils. Journal of Food Composition and Analysis 19: 838-842. 30- Rafaat El-Sanhoty, Afaf Desoky Abd El-Maged and Mohamed F. Ramadan (2006) Safety assessment of genetically modified potato Spunta: Degradation of DNA in gastrointestinal track and carry over to rat organs. Journal of Food Biochemistry 30: 556-578. 31- Abd El-Rahman M. Sulieman, Attya El-Makhzangy and Mohamed F. Ramadan (2006) Antiradical performance and physicochemical characteristics of vegetable oils upon frying of French fries: A preliminary comparative study. Journal of Food Lipids 13:259-276.
58
32- Mohamed F. Ramadan, Mohamed M. A. Amer and Abd El-Rahman M. Sulieman (2006). Correlation between physicochemical analysis and radical scavenging activity of vegetable oil blends as affected by frying of French fries. European Journal of Lipid Science and Technology 108: 670-678. 33- Amira E. A. El-Hanafy, Mohamed F. Ramadan, Mohamed H. Ahmed, Hany E. Showky (2006). Changes in fatty acid composition, cholesterol contents and quality attributes of bolti (Tilapia nilotica) fingerlings in relation to dietary lipid levels and sources in feeding regime. Deutsche Lebensmittel Rundschau 102: 518-522. 34- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2007). Impact of enzymatic treatment on chemical composition, physicochemical properties and radical scavenging activity of goldenberry (Physalis peruviana L.) juice. Journal of the Science of Food and Agriculture 87:452-460. 35- Mohamed F. Ramadan and Joerg Thomas Moersel (2007). Kachnar seed oil, INFORM 18: 13-15. 36- Mohamed F. Ramadan, Rawya Zayed and Hesham El-Shamy (2007) Screening of bioactive lipids and radical scavenging potential of some solanaceae plants. Food Chemistry, 103: 885-890. 37- Mohamed F. Ramadan (2007) Monitoring deep frying oils, INFORM 18:139-141. 38- Mohamed M. A. Amer, Mohamed F. Ramadan and W. Abd El-Gleel (2007). Impact of Pulicaria incisa, Diplotaxis harra and Avicennia marina as hypocholesterolemic agent. Deutsche Lebensmittel Rundschau 103: 320-327 39- Mohamed F. Ramadan, Mahmoud Z. Sitohy, Joerg T. Moersel (2008)
Solvent
and
enzyme-aided
aqueous
extraction
of
goldenberry (Physalis peruviana L.) pomace oil: Impact of
59
processing on composition and quality of oil and meal. European Food Research and Technology, 226: 1445-1458. 40- Mohamed F. Ramadan (2008) Quercetin increases antioxidant activity of soy lecithin in a triolein model system. LWT-Food Science and Technology 41: 581-587. 41- Mohamed F. Ramadan, Mohsen M. S. Asker, Zeinab K. Ibrahim (2008) Functional bioactive compounds and biological activities of Spirulina platensis lipids. Czech Journal of Food Science, 26: 211222. 42- Attya El-Makhzangy, Abd El-Rahman M. Sulieman and Mohamed F. Ramadan (2008). Darkening of green olives by rapid alkaline oxidation. Journal of Food Processing and Preservation, 32: 586599. 43- Mohamed F. Ramadan (2008) Total antioxidant potential of juices, hot drinks and beverages consumed in Egypt screened by DPPH in vitro assay. Grasas y Aceites, 59: 254-259. 44- Mohamed F. Ramadan, Mohamed M. A. Amer and Ahmed Awad (2008) Coriander (Coriandrum sativum L.) seed oil improves plasma lipid profile in rats fed diet containing cholesterol. European Food Research and Technology, 227: 1173-1182. 45- Mohamed F. Ramadan, Hany E. Showky and Abd El-Rahman M. Sulieman (2008) Comparison between the effect of γ-irradiation and roasting on the profile and antioxidant activity of wheat germ lipid. Grasas y Aceites, 59: 166-173. 46- Ayman M. Helmy and Mohamed F. Ramadan (2009) Agronomic performance and chemical response of sunflower (Helianthus annuus L.) to some organic nitrogen sources and conventional nitrogen fertilizers under sandy soil conditions. Grasas y Aceites, 60: 55-67.
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47- Mohamed F. Ramadan, Mohamed M. A. Amer and Ahmed Awad (2009) Changes of lipid profile by dietary vegetable oil blends in rats with
hypercholesterolemia.
Food
Science
and
Technology
International, in press. 48- Mohamed F. Ramadan and Mohsen M. S. Asker (2009) Antimicrobical and antivirial impact of novel quercetin-enriched lecithin. Journal of Food Biochemistry, in press. 49- Mohamed F. Ramadan, and Joerg-Thomas Moersel (2009) Oil Extractability from Enzymatically-treated Goldenberry (Physalis peruviana
L.)
Pomace:
Range
of
Operational
Variables.
International Journal of Food Science and Technology, in press. 50- Mohsen M. S. Asker, Mohamed F. Ramadan, Samir K. Abd El-Aal and Ebtsam M. M. El-Kady (2009) Characterization of trehalose synthase from Corynebacterium nitrilophilus NRC. World Journal of Microbiology and Biotechnology, in press.
61
Books, book chapters and reviews 1- Mohamed Fawzy Ramadan-Hassanien (2004) Investigation on lipid composition and functional properties of some Exotic oilseeds (Untersuchung
zur
Zusammensetzung
und
der
funktionalen
Eigenschaften der Lipide einger exotischer Oelsaaten), ISBN 38325-0525-3, Logos Verlag Berlin, Berlin, Germany. 2- Mohamed F. Ramadan (2007) Nutritional value, functional properties and nutraceutical applications of Black cumin (Nigella sativa L.) Oilseeds: An Overview. International Journal of Food Science and Technology 42: 1208-1218. 3- Mohamed F. Ramadan-Hassanien (2008) Starch Phosphorylation for Functional and Industrial Applications. Edited by M.F.RamadanHassanien. ISBN: 978-3-639-10872-9, VDM Verlag Dr. Müller, Saarbruecken, Germany. 4- Mohamed F. Ramadan-Hassanien (2008) Goldenberry: Golden fruit of golden future. Edited by M.F.Ramadan-Hassanien. ISBN: 978-38364-9489-2, VDM Verlag Dr. Müller, Saarbruecken, Germany. 5- Mohamed F. Ramadan (2009) Niger seed oil (Guizotia abyssinica Cass.). In: Gourmet and health-promoting oils. Edited by Afaf Kamal-Eldin and Robert A. Moreau, AOCS press (USA), in press.
62
Lectures and oral presentations in international conferences 1- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Analysis and composition of some exotic seed oils. German Food Chemsits’ Society Conference. Regionalverbandstagung (Nordost Deutschand) der Lebensmittelchemische Gesellschaft (Fachgruppe in der GDCh), Schwerin (Germany), 19 April, 2002. Abstract in Lebensmittelchemie Journal, 2002, 56(4) 85. 2-
Mohamed
F.
Ramadan,
and
Joerg-Thomas
Zusammensetzung der Lipide von Kaktusfeigen.
Moersel.
Zur
German Food
Chemsits’ Society Conference. Regionalverbandstagung (Nordost Deutschand) der Lebensmittelchemische Gesellschaft (Fachgruppe in der GDCh), Potsdam (Germany), 11 April, 2003 (Lecture in German). 3- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Lipid classes, sterols and tocopherols of clack cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) seed oils. 25th World Congress and Exhibition of ISF (International Society for Fat Research), Bordeaux (France), 12-15 Oct., 2003. 4- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Antiradical action and oxidative stability of clack cumin, coriander and niger crude seed oils and their fractions. 3rd Euro Fed Lipid Congress and Expo, Edinburgh (Scotland), 5-8 September, 2004. 5- Mohamed F. Ramadan. Goldenberry as a source of novel functional foods and drinks. Functional foods: scientific foundation and opportunities for the agro-food sector, Zaragoza (Spain), 3-7 April, 2006. 6- Rafaat El-Sanhoty, Mohamed F. Ramadan and Klaus Werner Boegl. Detect methods of genetically modified (GMO) foods and feeds. First International Conference and Exhibition: Food and Tourism, an approach to the world of tomorrow, Cairo (Egypt) 1-3 March, 2006.
63
7- Mohamed F. Ramadan, Rafaat El-Sanhoty and Joerg-Thomas Moersel. Goldenberry: Agolden fruit of golden future. First International Conference and Exhibition: Food and Tourism, an approach to the world of tomorrow, Cairo (Egypt) 1-3 March, 2006. 8- Mohamed F. Ramadan. Fast antiradical test for monitoring deep-fried oils. 5th Euro Fed Lipid Congress and Expo, Gothenburg (Sweden), 1619 September, 2007.
64
Posters and abstracts in international conferences 1- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Anaylse der Neutrallipide und Phospholipide aus Koriander (Coriandrum sativum L.). German Food Chemsits’ Society Conference. 30 Deutscher Lebensmittelchemikertag und Jahreshauptversammlung der GDCh, Braunschweig (Germany), 10-12 Sep., 2001 (Poster in German). Abstract in Lebensmittelchemie Journal, 2002, 56(2) 34-35. 2- Mohamed F. Ramadan, and Mahmoud Z. Sitohy. Degradability of different phosphorylated starches and thermoplastic films prepared from corn starch phosphomonoesters. German Food Chemsits’ Society Conference. 30 Deutscher Lebensmittelchemikertag und Jahreshauptversammlung der GDCh, Braunschweig (Germany), 10-12 Sep., 2001. Abstract in Lebensmittelchemie Journal, 2002, 56(2) 34. 3- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Analysis of phospholipids from African oilseeds. 24th World Congress and Exhibition of ISF (International Society for Fat Research), Berlin (Germany), 16-20 Sep., 2001. 4- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Phytosterols and antioxidants vitamins from goldenberry (Physalis peruviana L.) fruit oils. A Fresh Look at Antioxidants: Food Applications, Nutrition & Health. International Conference organized by the SCI Oils and Fats Group, Fitzwilliam College, Cambridge (UK), 14-16 April, 2002. 5- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Direct isocratic normal-phase assay of fat-soluble vitamins and beta-carotene in oilseeds. German Food Chemsits’ Society Conference. 31 Deutscher Lebensmittelchemikertag und Jahreshauptversammlung der GDCh, Frankfurt/Main (Germany), 9-11 Sep., 2002.
65
Abstract in Lebensmittelchemie Journal, 2003, 57(1) 5. 6- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Oil cactus pear (Opuntia ficus-indica L.). German Food Chemsits’ Society Conference. 32 Deutscher Lebensmittelchemikertag und Jahreshauptversammlung der GDCh, München (Germany), 6-11 Oct., 2003. 7- Mohamed F. Ramadan, and Joerg-Thomas Moersel. HPLC/UV Analysis of glycolipids from black cumin (Nigella sative L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) oilseeds. German Food Chemsits’ Society Conference. 32 Deutscher Lebensmittelchemikertag und Jahreshauptversammlung der GDCh, München (Germany), 6-11 Oct., 2003. 8- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Oil goldenberry (Physalis perviana L.). 25th World Congress and Exhibition of ISF (International Society for Fat Research), Bordeaux (France), 12-15 Oct., 2003. 9- Mohamed F. Ramadan, and Joerg-Thomas Moersel. Oil prickly pear [(Opuntia ficus-indica (L.) Mill]. 3rd euro Fed Lipid Congress and Expo, Edinburgh (Scotland), 5-8 September, 2004. 10- Mohamed F. Ramadan. Oil Recovery from Enzymatically-treated Goldenberry (Physalis peruviana L.) Pomace: Range of Operational Variables. 5th Euro Fed Lipid Congress and Expo, Gothenburg (Sweden), 16-19 September, 2007.
66
