Int J Pharm Pharm Sci, Vol 12, Issue 9, 29-35Original Article

STUDY OF THE ANTI-HYPERLIPIDEMIC EFFECT OF THE COMBINED ADMINISTRATION OF THREE NATURAL EXTRACTS IN A POLOXAMER-407 HYPERLIPIDEMIC MODEL AND THEIR LC-ESI-MS/MS2 AND HPLC PROFILING

S. A. MINA1*, A. M. ABD EL-MAKSOUD2, H. S. MOHAMMED3, M. A. FOUAD4

1*Pharmacognosy Department, Faculty of Pharmacy, Helwan University, 2Nutritional Requirements and Growth Department, National Nutrition Institute, 3HPLC Unit, Central Laboratory, 4Herbal Lab, Food Hygiene Department, National Nutrition Institute
Email: suzanmina@yahoo.com

Received: 20 May 2020, Revised and Accepted: 20 Jul 2020

ABSTRACT

Objective: Dyslipidaemia is considered a high-risk factor for inducing atherosclerosis and cardiovascular diseases (CVDs). This study aims to investigate the anti-hyperlipidemic effect of the co-administration of the ethanol extracts of both ginger (root and rhizome) and leek (leaves and bulbs) in addition to the aqueous extract of gum arabic. 

Methods: Rats were divided into eight groups: Hyperlipidaemia was induced in rats by a single intraperitoneal injection of Poloxamer 407 (P-407) [1 g/kg], negative control [saline injected], hyperlipidemic control [P-407 injected], positive control [Atorvastatin 70 mg/kg], groups four, five and six received ginger extract (400 mg/kg), leek extract (500 mg/kg) and gum arabic aqueous extract (7.5 g/kg) respectively and groups seven and eight received a co-administration of ginger, leek and gum arabic extracts at doses A and B respectively. Lipid profile was monitored. The profiling of all the tested extracts was performed by LC-ESI/MS and HPLC.

Results: A significant anti-hyperlipidemic activity (P<0.05) was seen for group eight among all the tested groups producing ≈54%, 72%, 50% and 72% decrease in the measured parameters total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) respectively. An overall of 56 and 45 compounds were tentatively identified in the ethanol extracts of ginger and leek, respectively. Galactose and arabinose sugars were found to be the major saccharides in gum arabic and glucuronic acid was the major polyuronide part.

Conclusion: the co-administration of a group of natural extracts in the given concentration proved to be more effective than the use of synthetic drugs or the use of a single component. 

Keywords: Hyperlipidaemia, Ginger, Gum arabic, Leek, Poloxamer 407

© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i9.38385. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps.

INTRODUCTION 

Dyslipidaemia can be defined as the elevation of cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C) serum levels while maintaining low serum levels of high-density lipoprotein cholesterol (HDL-C). This imbalance is considered a high-risk factor for inducing atherosclerosis and CVDs, which are considered the most common cause of death in both western and eastern countries [1]. As per the recommendation of the National Cholesterol Education Program, individuals with blood cholesterol levels above 240 mg/dl are considered hyper-cholesterolemic [2]. Many people achieve healthy levels by eating a balanced diet and through other aspects of their lifestyle including quitting smoking, exercise. However, some require medication to prevent additional health problems.

Traditionally, natural remedies were used to overcome such an imbalance in lipids metabolism and are claimed to be useful in controlling hyperlipidaemia and associated pathologies [3]. According to Hasani-Ranjbar et al. [4], About 53 clinical trials were performed to review the efficacy of several natural sources in the management of hyperlipidaemia, twenty-two plants of which were proven to show a significant decrease in total cholesterol and LDL cholesterol. Plants such as Ginger [Zingiber officinale R.], family Zingiberaceae, Leek, Allium ampeloprasum var. porrum (L.) also known as A. porrum, family Alliaceae (Liliaceae), and the edible dried gummy exudate gum arabic are all known to possess anti-hyperlipidemic activities in addition to other diverse biological effects which have been reported for this three species, such as anti-inflammatory, antitumor, antioxidant, hypotensive, prebiotic and ant-diabetic activities [5-10].

Synergy is a process in which the combined effect of some substances cooperates to reach a greater effect than the sum of their separate effects. Previous trials for the use of combined herbal therapy was reported for cancer treatment [11], hyperlipidemia [12] also a combination of herbal and synthetic drugs was under investigation for the treatment of diabetes [13, 14] as a mean to enhance therapeutic effects, reduce side effects and overcome drug resistance.

In the present work, an evaluation of the anti-hyperlipidemic effect of the co-administration of the ethanol extracts of both ginger (root and rhizome) and leek (leaves and bulb) and the aqueous solution of gum arabic versus Atorvastatin in a P-407 induced hyperlipidemia model in rats was performed. Examination of the active constituents of the tested extracts was done using liquid chromatography-electrospray ionization-mass spectroscopy/mass-mass spectroscopy (LC-ESI-MS/MS2) analysis and quantitative high-performance liquid chromatography (HPLC) analysis of the aqueous solution of gum arabic. 

MATERIALS AND METHODS 

Chemicals and reagents

Ator® (Atorvastatin) 40 mg tablets (EIPICO Company [Egyptian international pharmaceutical industries company–Egypt). Tablets were crushed into powder, dissolved in distilled water (120 mg/25 ml). Poloxamer 407, [Sigma-Aldrich Chemical Co. The USA]. P-407 was prepared by dissolving 8 g/100 ml saline for injection and then refrigerated overnight to facilitate its dissolution [15]. 70% ethanol (El Gomhoria Company).

Plant materials

Both dried entire ginger root and rhizome [Zingiber officinale R.] and Gum Arabic (Acacia senegal) were purchased from local Harazz commercial stores of Medicinal Plants, Cairo-Egypt. Leek (Allium porrum) plant leaves and bulbs were collected from Nahia region, Giza city, Egypt during May and June 2015. All used plants were botanically identified and authenticated by Dr. Abdul Halim Abdul Majali Mohammed, Head of Flora Research and Plant Classification department, Agricultural Research Centre. Three voucher specimens (Zof-152/2017 for ginger, Apo-153/2017 for leek and Gar-154/2017 for gum) were deposited in the herbarium of Pharmacognosy department, Faculty of Pharmacy, Helwan University. 

Extracts preparation

One Kg of dried ginger (root and rhizome) and one Kg of leaves and bulbs of leek were air-dried and ground to powder using a clean mill and mortar. They were separately macerated in (2LX3) 70% ethanol and left to stand 72 h with shaking until complete extraction. After filtration, the alcoholic extracts were concentrated under reduced pressure at 45 °C. The yields of the alcoholic extracts were 12.7%, 28.8% for ginger and leek, respectively. Aqueous solution of gum arabic was freshly prepared by dissolving 15 g of gum arabic in 50 ml distilled water to obtain 30% solution.

LC/MS analysis

LC/MS analysis was performed on triple stage quadruple mass spectrometer, TSQ Quantum Access MAX, Thermo Scientific, New York, USA, equipped with electrospray ionization (ESI) operated in the positive ionization mode (60 ev). Identification of the content of the extract was carried on the Accela U-HPLC system, which was composed of Accela 1250 quaternary pump and Accela open autosampler, New York, USA (operated at 25 oC). Hypersil Gold column (C18 bonded ultrapure silica-based column) 50 mm × 2.0 mm (1.9 μm), Thermo-scientific, New York, USA. Isocratic elution using fresh prepared Acetonitrile (A): 0.2% formic acid aqueous solution (B) (90:10) at room temperature. Flow rate (250 μl/min). X-calibur software version 2.2 was used to control all parameters of UPLC and MS and analysis of the obtained data.

Sugar analysis after hydrolysis of gum arabic by HPLC

Galactose: arabinose: rhamnose ratios in gum arabic extract was determined using HPLC. Aliquots of 0.03-0.05 g of the gum were weighed out accurately into tarred 15 cm3 stoppered Pyrex test tubes and 10 cm3 of 4% w/w sulphuric acid added to each. The tubes were placed in a water bath at 100 °C for 4 h and were then reweighed and made up to the original weight by the addition of distilled water. The solutions were neutralized by adding 2 g BaCO3 and shaking overnight. The filtered (0.45 urn) hydrolysates were analysed using the Perkin-Elmer Series 10 HPLC system equipped with a 25 x 0.46 cm SS Amino column (phase separations). The sample (60 µdm) was injected onto the column using 80:20 acetonitrile: water eluent at 22 °C and at a flow rate of 0.8 cm3/min. The retention times of the monosaccharide components were monitored using a Millipore Waters differential refractometer R401 and the proportions of each were determined by integration of the peaks. All analytes were compared with injected standards separately and calculated according to the following equation:

Animals

Forty Sprague-Dawley Albino adult male rats weighing 130-180 g, purchased from the animal house of National Research Centre, were used. The animals were housed in standard metal cages in an air-conditioned room at 22±3 °C, 55±5% humidity for 12 h [light and dark cycles were maintained]. The animals were left for an initial adaptation period of 7 d and were supplied with a standard pellet diet and water ad libitum. The experiment was carried for 4 d. All animal procedures were performed in accordance with the recommendation of the national institute of health guide for care and use of laboratory animals NIH guide, publication No 85-23: (revised1985) US department of health, education and welfare, specific national laws were applied and all experiments were examined by the appropriate ethics committees, approval No: 2017/FPHU150.

Experimental design and induction of hyperlipidemia

Rats were randomly divided into 8 groups, each comprising 8 rats. All rats were subjected to a six hours fasting. Animals of the first group were kept as negative control injected intraperitoneal [i. p.] with saline. Rats of the remaining seven groups were given 1g/kgbw (bodyweight) of Poloxamer-407 to induce hyperlipidaemia in a single dose on day one [14]. Group 2 was kept as hyperlipidemic control. Two hours later, group three were given atorvastatin (70 mg/kgbw) [16] and was considered as positive control. Similarly, group 4, 5 and 6 were administered 400 mg/kgbw of the ethanol extract of ginger [17], 500 mg/kgbw of the ethanol extract of leek [18] and 7.5 g/kgbw of the aqueous solution of gum arabic [19] respectively. As for group 7, it was co-administered dose A of the tested extracts, (ethanol extract of ginger (200 mg/kgbw), leek (250 mg/kgbw) and 30% aqueous solution of gum arabic 3.75 g/kgbw). While group eight was co-administered dose B (ethanol extract of ginger 400 mg/kgbw, leek 500 mg/kgbw and 30% aqueous solution of gum Arabic 7.5 g/kgbw). This was repeated once daily for 3 consecutive days by oral gavage [16].

Collection of blood samples

At the end of the experimental period (4 d) and after 12 h of fasting, the rats were anesthetized with isoflurane and oxygen then sacrificed. Blood was collected from the hepatic portal vein in centrifuge tubes and were incubated at room temperature for 30 min. Serum was obtained by centrifugation of blood samples [4000 rpm/15 min]. All samples were stored at-20 °C until further analysis. Handling procedures were conducted in accordance with the Institutional Ethics Committee and in accordance with the recommendations for the proper care and use of laboratory animals (NIH publication no. 85-23, revised 1985).

Biochemical analysis

Serum total cholesterol and triglycerides were estimated by enzymatic methods of CHOD-PAP and GPO-Trinder method [20]. Estimation of (HDL-C) was done by precipitation method [17]. All parameters were analysed by Spectrophotometric with quantitative diagnostic commercial kits. Serum concentrations of very low-density lipoprotein-cholesterol (VLDL-C) and Low-density lipoprotein-Cholesterol (LDL-C) were calculated using Friedewald's formula [21].

Statistical analysis

Data were expressed as means±standard error of means [SEM]. Assessment of the results was performed using a one-way analysis of variance [ANOVA] followed by Dunnett as post hoc test using SPSS program, version 16. The level of significance was set at P<0.05.

RESULTS AND DISCUSSION

Anti-hyperlipidemic effect

The concept of the use of a herbal combination to treat several diseases was the subject of many studies throughout the years [21-23], in the majority of which the combined administration of crude plants or their extracts proved to be beneficial in their intended action having the added value of being of natural sources and minimal side effects when compared to synthetic medications. Results illustrated in (table 1) and (fig. 1) showed that administration of P-407 induced significant elevation (P<0.05) in serum levels of TC by ≈ 4 folds, TG by ≈ 8 folds, HDL-C by ≈ 1.7 fold, LDL-C levels by ≈ 6 folds and VLDL-C levels by ≈ 8 folds as compared to a negative control group. The results also proved that group 8 showed a more effective anti-hyperlipidemic effect than group 7 and all the individually administered extracts. Administration of dose B of the tested combination (group 8) showed a significant (P<0.05) decrease in TG and LDL-C when compared to both a positive control and the anti-hyperlipidemic control group. It led to a more effective decrease in the lipid profile in all the measured parameters ranging from 10 to 15% more than that caused by atorvastatin reference. In addition to a significant difference seen in the TC and LDL level compared to the hyperlipidemic model. Although the administration of each component individually showed a promising anti-hyperlipidemic effect especially in case of TG and VLDL, the co-administered doses in group 8 surpassed the effect of each component delivered individually causing 54%, 72.5%, 51.2% and 72.5% reduction in TC, TG, LDL-C and VLDL-C levels respectively when compared to a positive control group. Moreover, TG, VLDL-C levels of this group were significantly decreased by 25.9 % and 25.9 %, respectively surpassing the effect of atorvastatin used as a positive standard drug. This may be explained by a synergistic effect of all the included components acting by a multi mechanism of action, including decreased intestinal fat absorption and/or decreased lipid biosynthesis and/or enhanced cholesterol elimination of ginger [17, 24]. In addition to the effect of sulphur amino acid compounds known to lower all the measured markers in the lipid profile [25]. The viscosity of the fermentable dietary fibers in gum arabic also contributes by producing an acidic PH during its fermentation process in the intestine to promote lipid excretion in stool [26]. Based on these results, such combination can be used alone as prophylactic management to hyperlipidemia as phytopharmaceutical, or as a booster to known anti-hyperlipidemic drugs, Where treating dyslipidaemia requires lifelong use of anti-hyperlipidemic drugs.

Table 1: Effect of different extracts of edible parts of ginger, leek and GA and their combinations versus atorvastatin on lipid pattern in P-407 induced hyperlipidemic rats

Groups

Parameter

 Negative control Hyperlipidemic control Atorvastatin Ginger leek Gum arabic Dose A Dose B
TC 90.8±3.5 390.3±24.2 227.5±13.9ab 230.4±18.4ᵃᵇ 245.3±24.1ᵃᵇ 238.2±27.3ᵃᵇ 277.7±6.4ᵃᵇ 179.3±13.6ᵃᵇ
TG 65.1±5.5 509.4±34.9 189.1±11.6ᵃᵇ 156.5±14.8ᵃᵇ 176.8±19.2ᵃᵇ 169.9±8.8ᵃᵇ 208±8.5 ᵃᵇ 140.1±11.4abc
HDL-C 42.8±1.9 73.3±3.9 52.2±4.6b 69.5±3.2ac 75.8±3.6ac 85.2±4.7abc 42±4.8 b 45.7±3.6b
LDL-C 35±3.2 215.1±21.4 137.5±9ᵃᵇ 129.6±15.2ᵃᵇ 134.1±22.6ᵃᵇ 119±28.9ᵃᵇ 194.1±8.9ac 105.6±9.5ᵃᵇ
VLDL-C 13±1.1 101.8±6.9 37.8±2.3ᵃᵇ 31.3±2.9ᵃᵇ 35.3±3.8ᵃᵇ 33.9±1.8ᵃᵇ 41.6±1.7ᵃᵇ 28±2.3abc

TC: total cholesterol, TG: triglyceride, HDL-C: high-density lipoprotein-cholesterol, LDL-C: low-density lipoprotein-cholesterol, VLDL-C: Very low-density lipoprotein-Cholesterol, all parameters were measured in (mg/dl) Results are expressed as mean±SEM N=8 animals in each group, aSignificant difference from the negative control group at P<0.05, bSignificant difference from the hyperlipidemic control group at P<0.05, cSignificant difference from Atorvastatin standard Control group at P<0.05

Table 2: Tentative identification of components detected in Zingiber officinale R. extract using LC/MS analysis

Peak

no.

R. T*

(min)

(-)ESI-MS

(m/z)

(+)ESI-MS

(m/z)

 MWt** Identified compounds Ref.
1 0.74 N. D 203 202 ɑ-Curcumene [29]
3 6.81 N. D 153 152 Neral [29]
4 7.84 195 N. D 196 Zingerol [30]
5 8.38 377 N. D 378 12-Gingerol [31]
6 8.51 N. D 197 196 3-(3',4'-Dihydroxy-5'-methoxyphenyl)-1-propanal [30]
7 10.52 389 N. D 390 5-hydroxy-1-(3,4-dihydroxy-5-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-3-heptanone [32]
8 10.81 389 N. D 390 3-acetoxy-5-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane [32]
9 11.49 405 N. D 406 3,5-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)heptane [32]
10 11.74 285 287 286 Kaempferol [33-35]
11 11.97 N. D 137 136 β-Phellandrene [29]
12 12.85 357 N. D 358 Matairesinol [36]
14 13.71 431 N. D 432 3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane [32]
15 13.89 505 N. D 506 3,5-diacetoxy-7-(3,4-dihydroxy-5-methoxyphenyl)-1-(4-hydroxy-3,5-dimethoxyphenyl)heptane [32]
16 14.09 355 379 356 1,7-bis-(4'-Hydroxy-3'-methoxyphenyl)-4-heptene-3-one [37]
17 14.76 401 N. D 402

2E-Geranial acetal of

4-Gingerdiol

 [30]
18 445 N. D 446

3,5-diacetoxy-7-(3,4-dihydroxyphenyl)-1-(4-hydroxy-

3-methoxyphenyl)heptane

 [32]
19 15.28 293 317 294 6-Gingerol [30]
20 15.79 359.3 N. D 360 12-Shogaol [31]
21 15.92 459 N. D 460 meso and (3S,5S)-3,5-Diacetoxy-1,7-bis-(4'-hydroxy-3'-methoxyphenyl)heptane [30]
25 16.78 N. D 219 218 (±)-(E)-Nuciferol [29]
26 16.88 375 N. D 376 12-Gingerdione [30]
28 17.68 275 277 276 6-Shogaol [37, 38]
33 18.69 387 N. D 388 1,7-bis-(4'-Hydroxy-3'-methoxyphenyl)-5-methoxyheptan-3-one [30]
34 18.95 N. D 403 402 12-gingediol [38]
35 19.43 289 291 290 Methyl 6-shogaol [37]
36 19.76 N. D 319 318 Methyl (E)-8-shogaol [30]
37 19.88 N. D 335 334 10-Paradol [30]
38 20.25 265 N. D 266 4-gingerol [38]
39 20.34 349 N. D 350 10-gingerol [38]
40 20.60 N. D 327 326 Methyl Icosanoate [36]
41 20.72 N. D 373 372 1,7-bis-(4'-Hydroxy-3'-methoxyphenyl)-3,5-heptadione [30]
42 21.09 295 N. D 296 6-gingerdiol [37, 38]
43 21.26 415 N. D 416 1-(4'-Hydroxy-3'-methoxyphenyl)-2-nonadecen-1-one [30]
44 21.26 N. D 205 204 ɑ-Zingiberene [29]
46 21.62 N. D 205 204 β-sesquiphellandrene [29]
49 22.11 N. D 387 386 1-(4'-Hydroxy-3',5'-dimethoxyphenyl)-7-(4'-hydroxy-3'-methoxyphenyl)-4-hepten-3-one [30]
51 22.51 N. D 333 332 10-Shogaol [30]
52 22.69 293 N. D 294 1-Hydroxy-6-paradol [30]
60 24.16 277 279 278 6-Paradol [37]
61 24.28 N. D 377 376 3,5-dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane [32]
63 24.61 N. D 205 204 α-Farnesene [29]
64 25.01 N. D 353 352 10-Gingerdiol [37]
65 25.19 279 N. D 280 6-Dihydroparadol [37]
66 25.28 N. D 353 352 5-Acetoxy-7-gingerdiol [37]
67 25.58 N. D 309 308 Methyl 6-gingerol [30]
71 26.26 N. D 409 408 Diacetoxy-8-gingerdiol [30]
73 26.50 N. D 409 408 10-Gingerdiol, cyclic methyl ortho ester [30]
76 26.88 N. D 307 306 8-Paradol [30]
79 27.35 N. D 381 380 Diacetoxy-6-gingerdiol [37]
80 28.06 N. D 309 308 Acetoxy-4-gingerol [30]
81 28.34 N. D 437 436 3,5-dihydroxy-1,7-bis (4-hydroxy-3,5-dimethoxyphenyl)heptane [32]
82 28.48 N. D 297 296 6-Gingerdiol [37]
84 28.79 N. D 311 310 5-Acetoxy-4-gingerdiol [30]
85 29.10 N. D 371 370 dihydrocurcumin [32]
93 30.89 N. D 223 222 β-Selinenol (β-Eudesmol) [29]
95 31.12 N. D 223 222 α-Trans-sesquicyclogeraniol [29]

**MWt. = molecular weight, *RT= retention time

LC-ESI-MS/MS analysis

An overall of 56 compounds was tentatively identified in the ethanol extracts of ginger (root and rhizome) (table 2). Four of the major constituents were subjected to further MS2 fragmentation (table 3), showing the characteristic fragmentation pattern of gingerol related compounds with a base peak at m/z 137 correspondings to the benzylic cleavage product associated with the 4'-hydroxy-3'-methoxy benzyl cation in all four compounds. 6-gingerol being the major constituent at m/z 295 [M+H]+.

Fig. 1: Percentage change of TC, TG, HDL-C, LDL-C and VLDL-C for all tested samples against atorvastatin control group in P-407 induced hyperlipidemic rats, Dose A =ethanol extract of ginger 200 mg/kg bw, leek 250 mg/kg bw and 30% aqueous solution of gum arabic 3.75 g/kg bw, Dose B= ethanol extract of ginger 400 mg/kg bw, leek 500 mg/kg bw and 30% aqueous solution of gum Arabic 7.5 g/kg bw, Results are expressed as mean±SEM n=8

Table 3: MS2 fragmentation pattern of four major constituents of Zingiber officinale R

Cpd No Peak No. Name (-)ESI-MS(m/z) (+)ESI-MS(m/z) MWt Fragmentation m/z Reference or/Mass bank N0
1 19 6-Gingerol 293 295 294 194, 177, 277, 137 [27] ID: CO000213
2 28 6-Shogaol 275 277 276 137,159,179 [27]
3 51 10-Shogaol N. D 333 332 137,123 [27]
4 65 6-Dihydroparadol 279 N. D 280 279, 138 [28]

MWt. = molecular weight, ND= not detected, cpd= compound

Table 4: Tentative identification of components detected Allium ampeloprasum var. porrum (L.) using LC/MS analysis

Peak

no.

 R. T* (min) (-)ESI-MS (m/z) (+)ESI-MS (m/z) MWt** Compounds Identified Ref.
1 0.75 N. D 175 174 Arginine [39]
2 0.83 149 N. D 150 Dipropyl disulfide [40]
3 1.03 150 N. D 151 Methiin [41]
4 1.35 130 132 130 leucine [39]
5 2.09 164 166 165 Ethiin [41]
7 4.12 181 N. D 182 Dipropyl trisulfide [40]
8 7.20 431 N. D 432 Apigenin-7-glucoside [40]
9 8.46 N. D 197 196 Hydroferulic acid [42]
10 8.64 112.9 N. D 114 Diallyl sulfide [40]
11 9.48 463 N. D 464 quercetin-3-glucoside [43; 44]
12 9.62 178 N. D 179 propiin [41]
13 10.32 N. D 284 283 N-p-coumaroyltyramine [45]
14 10.48 447 449 448 Astragaline [42; 44]
16 11.74 285 287 286 Luteolin [42; 45]
17 12.13 N. D 181 180 Caffeic acid [42; 44]
19 13.24 285 287 286 kaempferol [42; 45]
21 18.24 N. D 273 272 Naringenin [42; 44]
22 18.69 N. D 343 342 sucrose [39]
24 20.11 N. D 317 316 Isorhamnetin, Rhamnetin [17]
26 20.39 N. D 257 256 palmitic acid [40]
27 20.72 N. D 355 354 Chlorogenic acid [44]
28 21.26 295 297 296 Phytol [40]
29 21.46 577 N. D 578 Apigenin-7-O-neohespiroside [39]
32 22.44 N. D 537 536 kampherol dervatives [39]
33 22.61 579 N. D 580 Naringin [47]
38 23.81 459 N. D 460 (25R)-5α-spirostane-3β,6β-diol-2,12-dione (Porrigenin C) [46, 47]
40 24.02 N. D 225 224 Sinapenic acid [42, 44]
41 24.16 277 279 278 ɑ-linolenic acid [48]
42 24.40 N. D 499 498 Kaempferol hexose [39]
46 25.17 279 N. D 280 linoleic acid [48]
47 25.44 N. D 303 302 Quercetin [42; 44]
48 25.54 N. D 303 302 Hesperetin [42]
52 26.52 N. D 314 313 N-feruloyltyramine [46]
57 28.29 N. D 271 270 Apigenin [44]
59 28.58 N. D 285 284 Acacetin [46]
61 29.02 N. D 536 535 Kaempferol deoxyhexose [39]
63 29.47 N. D 611 610 Rutin [44]
64 29.85 N. D 684 683 Kaempferol deoxyhexose [39]
69 30.60 N. D 505 504 kestose [39]
70 30.68 N. D 535 534 Kaempferol malonyl hexose [39]
71 30.79 N. D 603 602 Kaempferol derivative [39]
73 31.15 197 199 198 Syringic acid [44]

**MWt. = molecular weight. *RT= retention time

Table 5: MS2 Fragmentation pattern of four major constituents of Allium ampeloprasum var. porrum (L.)

Cpd No.

Peak

No.

 Name

(-)ESI-MS

(m/z)

(+)ESI-MS

(m/z)

 MWt Fragmentation m/z Reference/Mass bank ID No
1 5 Ethiin 164 166 165 28,42,77,79,103,118,120 ID: HMDB0029432
2 11 Quercetin glucoside 463 N. D 464 151,243,255, 271, 299,300,301 ID: PR100677
3 14 astragaline 447 449 448 107,121,153,165,287 PR100243
4 19 kaempferol 285 287 286 107,121,133,153,165, 181,269,287 ID: PB004123

MWt. = molecular weight, ND= not detected, Results of HPLC quantitative analysis of sugars after hydrolysis of the aqueous solution of gum arabic tabulated in (table 6). The major constituents were found to be galactose, arabinose and glucouronic acid.

Table 6: Sugars and uronic acids analysed by HPLC in GA after hydrolysis

Peak no. Saccharides RT *(min) (%) Saccharides after hydrolysis
1 Glucuronic acid 5.141 6.60
2 Stachyose 5.442 0.20
3 Galacturonic 5.624 0.06
4 Sucrose 6.372 0.04
5 Glucose 7.425 0.63
6 Galactose 8.719 19.36
7 Arabinose 10.430 15.76
8 Manitol 14.267 0.006
9 Sorbitol 18.726 0.004

*RT= retention time

As for the leek ethanol extract, 45 compounds were tentatively identified (table 4) and four of its major constituents were further fragmented by MS2. It is the first record for MS2 analysis of ethiin detected as the major component in leek extract (table 5). It showed the protonated molecular ion at m/z 166 [M+H]+. The characteristic ion fragment at m/z 103 was detected due to loss of both -COOH and -NH2 groups and rearranging of the parent protonated molecule.

CONCLUSION 

It can be concluded that the co-administration of a group of natural extracts with each other in certain concentrations proved to be more effective than the use of synthetic drugs or the use of a single component. This may be due to the multi-mechanism of action of the extracts and/or they may be having less adverse effects. Such a combination can be used as prophylactic management or as a booster to known anti-hyperlipidemic drugs. 

FUNDING

Nil

AUTHORS CONTRIBUTIONS

S. A. Mina and A. M. Abd EL-Maksoud conceived and planned the experiments. H. S. Mohammed and M. A. Fouad carried out the experiments and contributed to sample preparation. S. A. Mina, A. M. Abd EL-Maksoudand H. S. Mohammed contributed to the interpretation of the results. S. A. Mina took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript.

CONFLICT OF INTERESTS

Declared none

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