THE EFFECTS OF SOLVENT POLARITY ON HYPOGLYCEMIC AND HYPOLIPIDEMIC ACTIVITIES OF PORTULACA OLERACEA AND ACHILLEA ERIOPHORA DC EXTRACTS

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THE EFFECTS OF SOLVENT POLARITY ON HYPOGLYCEMIC AND HYPOLIPIDEMIC ACTIVITIES OF PORTULACA OLERACEA AND ACHILLEA ERIOPHORA DC EXTRACTS

Abbas Ahmadi,

^1,*

Mohsen Khalili,

²

Atena Roghani,

¹

Adeleh Behi,

³

and Sommayeh Nazirzadeh

⁴

Original article submitted December 11, 2015.

The influence of solvent polarity for hydroethanol (a), chloroform (b), and carbon tetrachloride (c) on bioactive extraction contents ofPortulaca oleracea(II) andAchillea eriophoraDC (III) were evaluated by GC-MS analysis. The antidiabetic and antilipidemic properties of different extracts were investigated on streptozotocine-induced diabetic rats and compared to glibenclamide as well-known chemical drug for the treatment of diabetes. The results indicated that extracts IIa – IIIc, IIIa, and IIIb reduced blood serum glucose significantly on days 9 and especially 16 after the induction of diabetes. In addition, LDL cholesterol level was reduced markedly by using IIb, IIc and IIIc extracts. Serum cholesterol and TG were significantly decreased (especially by carbon tetrachloride and chloroform extracts) in IIc and IIIc animal groups compared to control and glibenclamide groups. These results can be related to more extracted sterols, fatty acids, polyols (or alcohols), phenols and flavonoids as well as antraquinones and terpenes by polar or non-polar organic solvents compared to aqueous extracts of these medicinal herbs.

Keywords: Portulaca oleracea; Achillea eriophora DC; solvent polarity, blood glucose; blood lipids;

streptozotocine-induced diabetes; rats.

1. INTRODUCTION

Several approaches are presently used in treatment of type 2 diabetes mellitus (T2DM) including sulfonylureas which stimulate insulin secretion from pancreatic islet cells, biguanides with ability to reduce hepatic gluconeogenesis, anda-glucosidase inhibitors preventing the digestion of car- bohydrates [1]. Unfortunately, all of these therapies have var- ious side effects, so that searching for safe compounds is essential to overcome these problems [2]. Recent studies have focused on the use of medicinal plants with wide range of natural and active components for treatments of hypergly- cemia and hyperlipidemia with ensuing development of car- diovascular disorders and the major causes of morbidity and mortality [3, 4].

Portulaca oleraceaL.(II) commonly known as purslane or khorfeh in Persian belongs to the Portulacaceae family. It is widely distributed in the tropical and subtropical areas of the world around the Mediterranean and tropical Asian countries. The plant has been used as a folk medicine in many countries and possesses a wide spectrum of pharmacological properties such as neuroprotective, antimicrobial, antidiabetic, antioxidant, anti-inflammatory, antiulcerogenic and anti- cancer activities which are associated with its diverse chemical constituents, including flavonoids, alkaloids, polysaccha- rides, fatty acids, terpenoids, sterols, proteins, vitamins and minerals [5, 6].

Achillea eriophoraDC (III) known as yarrow or bouma- daran in Persian is an endemic species of Achillea genus which grows in the south of Iran. It is a flowering plant in the family of Asteraceae which has been used in traditional medicine with several characteristic pharmacological effects such as gastroprotective, antibacterial, antioxidant, antiseptic, an- algesic, anti-inflammatory, antihypertensiv, antihyperlipide- mic and antioxidant activities [7 – 11]. The medicinal properties of Achillea plants are worldwide recognized and vari- ous biochemical compounds such as azulene, chamazulene, 1,8-cineole, camphor, flavonoids, sterols, tannins and alka- 1225

University, Karaj, Iran.

2Neurophysiology Research Center, Shahed University, Tehran, Iran.

3Department of Chemistry, Faculty of Medicinal Chemistry, Tehran Medi- cal Sciences, Islamic Azad University, Tehran, Iran.

4Department of Horticultural Science, Faculty of Agriculture, Karaj Branch, Islamic Azad University, Karaj, Iran.

*e-mail: [email protected]; [email protected]

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loids have been characterized in the aerial parts of this plant by phytochemical analyses [12 – 14].

In this research, because of mentioned significant hypoglycemic and hypolipidemic activities of these plants, effect of solvent polarity on the type and quantity of bioactive extracted components was investigated for three organic solvents with low to high polarities (carbon tetrachloride, 70%

ethanol – water, and dichloromethane). Glucose and lipid-lowering activities of extracts were evaluated in comparison to glibenclamide and control groups by well-known procedures [15].

2. EXPERIMENTAL CHEMICAL PART

2.1. General

Ethanol, carbon tetrachloride, dichloromethane, streptozotocine (STZ) and all other chemicals were purchased from Merck (Darmstadt, Germany) and Sigma-Al- drich (United States) chemical companies. Sixty four male Wistar rats, weighing 200 – 220g (Pasteur Institute, Iran) were used in pharmacological tests.

2.2. Essential Oil Analysis

Gas chromatography. Gas chromatographic analysis was carried out on an Aglient 7890N chromatograph coupled with Agilent MS 5975C Mode EI capillary column HP-5MS (30 m´0.25 mm i.d.; film thickness 0.25 µm) was used. The column operating conditions were as follows: 60°C for 2 min, then 7°C/min to 280°C. The carrier gas was helium at a flow rate of 1 mL/min. Samples (1mL) of diluted essential oils were injected manually.

Identification of components. The components of oil samples were identified by their retention time, retention in- dices relative to C₆-C₃₆ n-alkanes computer matching with Wiley/NIST library as well as comparison of their mass spectra with the authentic samples or with data already avail- able in the literature. The percentage composition of identified compounds was computed from the GC peak areas with- out any correction factors and was calculated in relative units.

2.3. Plant Material and Preparation of Hydroethanolic, Dichloromethane or Chloroform, and Carbon Tetrachloride Extracts

Aerial parts ofP. oleracea(II) andA. eriophora DC(III) were purchased from the local market of Tehran and authen- ticated by Prof. Gholamreza Amin (Department of Pharma- cology, Islamic Azad University, Pharmaceutical Sciences branch, Tehran, Iran), where voucher specimens were depos- ited in the herbarium (No: 1526-AUPF and PMP-320, respectively). The seeds and aerial parts of these plants were protected from direct sunlight, air-dried in the shade, finely powdered, and then extracted by maceration using mentioned solvents with different polarities: 70% ethanol – water

(a), dichloromethane or chloroform (b), and carbon tetrachloride (c) for 72 h separately (100 g of a powdered plant material was macerated in 500 mL solvent). The mixtures were filtered and concentrated to yield extracts used for GC-MS analysis (methanol solution: 1%, injection volume 1µL) and for antidiabetic and antilipidemic tests in rats. All extracts were standardized for analyzing according to the methods recommended by the World Health Organization (WHO) [15]. Glucose, cholesterol and triglycerides were measured using the corresponding biochemical kits (Zistshimi, Tehran, Iran).

3. EXPERIMENTAL PHARMACOLOGICAL PART

3.1. Animals

Initially, 64 adult male Wistar rats weighing 200 – 220g, (Pasteur Institute, Iran) with blood glucose under 150 mg/dL as non-diabetic animals were randomly selected and housed three to four per cage in a temperature-controlled colony room under 12 h light/dark cycle. Animals were given free access to water and standard laboratory rat chow (Pars Com- pany, Tehran, Iran). All the experiments were conducted between 11 a.m. and 4 p.m. under normal room light at 25^oC.

This study was carried out in line with the policies provided in the Guide for the Care and Use of Laboratory Animals (NIH) and those in the Research Council at Shahed Univer- sity of Medical Sciences (Tehran, Iran).

3.2. Serum Parameters Analysis

Diabetes was induced in rats by intraperitoneal (i.p.) injection of streptozotocine (STZ, Sigma, United States) at a dose of 70 mg/kg, dissolved in 0.1 M cold citrate buffer (pH 4.5) [15]. Animals were randomly divided into five groups;

Control: glibenclamide (Sigma-Aldrich Company, United States), 0.25 mg/kg, i.p); carbon tetrachloride, 70%

hydroethanol and chloroform extracts (250 mg/kg, i.p) [16].

The drugs were injected to animals on days 3 to 16 after STZ injection. Fasted STZ-treated rats with blood glucose con- centrations between 250 – 400 mg/dL were considered diabetic and used in this study.

Glucose assay. The serum glucose as the main parameter for efficiency of extracts was measured after the STZ injection on days 4, 9 and 16. For this purpose, a drop of tail vein blood was taken by using blood glucose monitoring system (EasyGluco, Infobia Co.) and then serum glucose was evaluated in each sample. Only animals with serum glucose content higher than 250 mg/dL were selected as diabetic for the following measurements.

Lipid profile measurement. Triglyceride, total cholesterol, LDL and HDL were determined on day 16 after STZ injection. To measure these serum parameters, for lipid profile analysis blood samples were collected from rat retroorbotal plexus. Serum triglyceride, total cholesterol and HDL cholesterol levels were spectrophotometrically measured by appropriate kits (Zistshimi, Tehran). LDL and a

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TABLE 1. Bioactive Compounds in Hydroethanolic Extracts ofP. oleracea(II) andA. eriophoraDC (III) Identified by GC-MS^*

No. R. T. Compound M. F. M. W.

%Area in A.eriophora

DC

%Area in

P. oleracea Family Pharmacological activity

1 11.47 4-Vinylphenol C8H8O 120.15 0.42 — Phenols Antioxidant [17],

Antidiabetic [18]

2 13.31 2-Methoxy-4-vinylphenol C9H10O2 150.17 2.75 0.99 Phenols Antioxidant [17], Antidiabetic [18]

3 20.65 Chamazulene C14H16 184.28 3.2 — Aromatic

derivative of azulene

Antioxidant and Antidiabetic [19, 20]

4 20.91 Myristic acid C14H28O2 228.37 0.31 — Saturated fatty

acid

5 20.94 Mome Inositol

((1R,2R,3S,4S,5R,6S)- cyclohexane-1,2,3,4,5,6-hexol)

C6H12O6 180.16 0.3 56.26 Polyols Antioxidant [23]

6 22.08 Phytol C20H40O 296.53 1.38 5.87 Alcohole Antioxidant and

Anti-inflammatory [24, 25]

7 23.11 Lidocaine C14H22N2O 234.34 0.49 — Amino acetamide local anesthetic,

anti-inflammatory [26]

8 23.86 Palmitic acid

(n-hexadecanoic acid)

C16H32O2 256.42 7.01 6.94 Fatty acid (saturated)

9 24.69 Inositol C6H12O6 180.16 0.3 — Polyols Antioxidant, [23]

10 26.17 Linoleic acid

(9,12-Octadecadienoic acid)

C18H32O2 280.45 1.98 — Fatty acid (polyunsaturated

omega-6)

11 26.27 Linolenic acid, methyl ester C19H32O2 292.45 5.33 — Fatty acid esters

12 26.28 7,10,13-Hexadecatrienoic acid, methyl ester

C17H28O2 264.40 — 1.48 polyunsaturated Fatty acid

13 26.51 Stearic acid C18H36O2 284.47 0.49 — Fatty acid

(saturated)

14 26.61 Linoleic acid ethyl ester C20H36O2 308.49 — 0.7 Fatty acid esters

15 26.67 Phenol,

2,6-dichloro-4-(1-methylpropyl)

C10H12Cl2O 219.10 0.35 — Phenol Antioxidant [17], Antidiabetic [18]

16 26.83 Achillin C15H18O3 246.30 5.75 — Sesquiterpene

lactone

Antidiabetic [27]

17 28.14 2-Methyl-Z,

Z-3,13-octadecadienol

C19H36O 280.48 0.77 — Unsaturated

Alcohole

Anti-inflammatory, antioxidant, antidiabetic and antilipidemic [28, 29]

18 29.88 4-Oxo-b-isodamascol C13H20O2 208.29 1.05 — Unsaturated

Alcohole

Anti-inflammatory, antioxidant, antidiabetic and antilipidemic [28, 29]

19 30.65 Palmitic acidb-monoglyceride (Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)eth

yl ester)

C19H38O4 330.5 1.55 — Fatty acid

esters

20 30.94 Norcinnamolaurine C17H17NO3 283.32 0.43 — benzylisoquino-

line alkaloids

Antioxidant and Antidiabetic[30]

21 31.62 Allylbenzene C9H10 118.17 3.77 — Phenylpropa-

noids

Antioxidant [31]

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very low-density lipoprotein (VLDL) cholesterol levels were calculated using the following formulas:

VLDL = Triglyceride/5;

LDL = Total cholesterol – HDL cholesterol – VLDL.

3.3. Statistical Analysis

Measurement data were tabulated as mean±S.E.M.

Comparisons were carried out using one way analysis of variances (ANOVA) followed by post-hoc Tukey test and p< 0.05 was used as the level of significance.

4. RESULTS

4.1. Chemistry

The title extracts were obtained by maceration of two medicinal plants in the mentioned organic solvents with low to high polarities. Results of GC-MS measurements for all extracts are summarized in Tables 1 – 6.

Hydroethanolic extract of P. oleracea (IIa). Polyols (total content: 56.26%), sterols (total content: 12.98%) and fatty acids (total content: 9.12%) with antioxidant, antidiabetic and anti-inflammatory activities were more than other components. Also, alcohols (total content: 5.87%) with antioxidant, antidiabetic, and anti-inflammatory activities in

this extract may be other reasons for mentioned pharmacological activities of this extract.

Chloroform extract ofP. oleracea (IIb). Sterols (total:

23.92%), alcohols (total contents: 23.27%), aromatic deriva-

%Area in A.eriophora

DC

%Area in

P. oleracea Family Pharmacological activity

22 35.48 Flavone,

5-hydroxy-4¢,7-dimethoxy-

C17H14O5 298.29 1.51 — Flavonoids Antidiabetic [32]

23 38.59 1,6,8-Trihyd-

roxy-2-isopropyl-3-methoxy- 9,10-anthraquinone

C18H16O6 328.31 28.58 — Antraquinone Antidiabetic [33, 34]

24 39 Norstictic acid C18H12O9 372.29 4.21 — Phenols Antioxidant [17],

Antidiabetic [18]

25 40.98 Stigmasterol C29H48O 412.7 1.38 — Phytosterols Antioxidant and

Antidiabetic [35]

26 42.34 b-sitrosterol C29H50O 414.71 0.54 12.98 Phytosterols Antioxidant and

Antidiabetic [35]

27 42.49 5-Hydroxy-3’,4’,6,7-tetramethox y flavone

C19H18O7 358.34 1.03 — Flavonoide Antidiabetic [32]

28 42.95 4H-1-Benzopyran-4-one, 2-(3,4-dimethoxyphenyl)-5-hydro

xy-3,6,7-trimethoxy-

29 44.43 Beta.-Amyrin C30H50O 426.72 1.1 — Alcohole Antihyperglycemic

and hypolipidemic [36]

30 47.46 Lupeol (Fagarasterol) C30H50O 426.73 0.8 — Sterols Antioxidant and

Antidiabetic [37]

* Upon the separation and identification by GC/MS technique; components were identified on the basis of retention time (R. T.) and spectral index using the NIST and WILEY library data for time interval 0.50 sec; startm/z50; endm/z600.

TABLE 2. Quantitative Analysis of Phytochemicals in Hydroethanolic Extracts of P. oleracea(II) and A. eriophoraDC (III) Identified by GC-MS

No. Phytochemical family

Percentage in extract ofP. oleracea(II)

Percentage in extract ofA. eriophora

DC.(III)

1 Sterols 12.98 2.72

2 Fatty acids 9.12 16.67

3 Phenols 0.99 7.73

4 Polyols 56.26 0.6

5 Saturated and unsaturated

Alcohols ^5.87 ^4.3

6 Flavonoids — 8.45

7 Antraquinones — 28.58

8 Sesquiterpene lactone — 5.75

9 Others 14.78 21.98

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TABLE 3. Bioactive Compounds in Chloroform Extracts ofP. oleracea(II) andA. eriophoraDC (III) Identified by GC-MS

%Area inA.

eriophoraDC.

(III)

%Area in P. olerace a(II)

Family Pharmacological Activity

1 7.45 1,8-Cineole C10H₁₈O 154.24 0.97 — Monoterpene Antidiabetic and anti in-

flammatory [38, 39]

2 9.05 Beta-Thujone C10H16O 152.24 1.46 — Monoterpene Antidiabetic and anti in-

flammatory [38, 39]

3 9.89 Camphor C10H16O 152.24 3.93 — Monoterpene Antidiabetic and anti in-

flammatory [38, 39]

4 10.34 Borneol C10H18O 154.24 0.22 – Monoterpene Antidiabetic and anti in-

flammatory [38, 39]

5 12.47 2,6-Dimethylocta-1,7-dien- 3,6-diol

C10H18O2 170.25 0.49 — Unsaturated

Alcohole

Anti-inflammatory, antioxidant, antidiabetic and antilipidemic [28, 29]

6 12.87 Carvacrol C10H14O 150.21 0.47 — Monoterpene Antidiabetic and anti in-

flammatory [38, 39]

7 13.33 2-Methoxy-4-vinylphenol C9H10O2 150.17 0.29 — Phenols Antioxidant [17], Antidiabetic [18]

8 19.15 3-Methy-

lene-bicyclo[3.2.1]oct- 6-en-8-ol

C9H12O 136.2 0.2 — Unsaturated

Alcohole

9 19.22 Methyl jasmonate C13H20O3 224.3 0.28 — Fatty acid esters Antioxidant and Antidiabetic [21, 22]

10 19.22 Safranal C10H14O 150.21 0.23 — Unsaturated Al-

dehyde

Antidiabetic [40]

11 20.64 Chamazulene C14H16 184.28 0.5 — aromatic deriv-

ative of azulene

12 20.92 Myristic acid C14H28O2 228.37 0.31 — saturated fatty

acid

13 22.08 Phytol C20H40O 296.53 7.21 23.27 Alcohole Antioxidant and Anti-in-

flammatory [24, 25]

14 23.85 Palmitic acid (n-hexadecanoic acid)

C16H32O 256.42 2.76 — Fatty acid (saturated)

16 26.18 Linoleic acid

(9,12-Octadecadienoic acid)

C18H32O2 280.45 0.56 — Fatty acid (polyunsaturated

omega-6)

17 26.20 1,2,3,10,11,12,12a,12b-Octahy droperylene

C20H20 260.37 7.88 — polycyclic aro-

matic hydrocar- bon,–

18 26.83 Achillin C15H18O3 246.30 5.75 — sesquiterpene

lactone

Antidiabetic [27]

19 29.29 Diphenylphosphinic acid C12H11O2P 218.19 2.98 — phosphinic acid,—

20 29.89 4-Oxo-b-isodamascol C13H20O2 208.29 1.27 — Unsaturated

Alcohole

22 31.95 Octadeca-3,13-dien-1-ol C18H34O 266.46 0.46 — Unsaturated Alcohole

23 33.78 1-Nonadecene C19H38 266.5 — 3.37 Alkens Anti-inflammatory [41]

24 35.48 Flavone,

5-hydroxy-4’,7-dimethoxy-

25 37.6 Nonadecane C19H40 268.518 — 29.06 Alkanes,—

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tives (total contents: 4.09%), flavonoids and phenols (total contents: 1.65%) with antidiabetic and anti-inflammatory activities were more than other components.

Carbon tetrachloride extract ofP. oleracea (IIc).

Sterols (total: 27.25%), alcohols (total contents:

12.37%), phenols and fatty acids (total contents: 2.15%) with

antidiabetic and anti-inflammatory activities were more than other components.

Hydroethanolic extract of A. eriophora DC (IIIa).

Antraquinones (total contents: 28.58%), fatty acids (total contents: 16.67%), phenols and flavonoids (total contents:

16.18%), polyols and alcohols (total contents: 4.9%) and

%Area inA.

eriophoraDC.

(III)

%Area in P. olerace a(II)

Family Pharmacological Activity

26 38.37 Vitamin E C29H50O2 430.70 — 2.43 Tocopherols Antioxidant, [42]

27 38.61 1,6,8-Trihyd-

roxy-2-isopropyl-3-methoxy- 9,10-anthraquinone

C18H16O6 328.31 25 — Antraquinone Antidiabetic [33, 34]

28 39.01 5-Hydroxy-3’,4’,6,7- tetramethoxy flavone

29 39.07 1,3-dimethyl-

4-azaphenanthrene

C15H13N 207.27 — 4.09 Polycyclic aro-

matic hydrocar- bon

—

30 40.28 Campesterol

(Ergost-5-en-3-ol)

C28H48O 400.68 0.34 — Phytosterol Antioxidant and Antidiabetic [35]

31 40.38 Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-

tetradecamethyl-

C14H42O6Si7 503.07 — 10.44 Si-compounds —

32 40.98 Stigmasterol C29H48O 412.7 1.38 — Phytosterols Antioxidant and

Antidiabetic [35]

34 42.96 4H-1-Benzopyran-4-one, 2-(3,4-dimethoxyphenyl)-5-

hydroxy-3,6,7-trimethoxy-

35 44.43 Lupeol (Fagarasterol) C30H50O 426.73 0.77 — Sterol Antioxidant and

Antidiabetic [37]

36 46.17 beta.-Amyrin acetate C32H52O2 468.75 0.75 — Ester of amyrin Antihyperglycemic and hypolipidemic [38]

37 47.67 b-sitrosterol C29H50O 414.71 0.52 23.92 Phytosterols Antioxidant and Antidiabetic [37]

38 48.06 Pyrocatechol, 3,5-di-tert-butyl- C14H22O2 222.32 — 1.65 Phenols Antioxidant [17], Antidiabetic [18]

%Area in extract of P. oleracea(II)

%Area in extract of A. eriophora

DC.(III)

1 Sterols 23.92 3.01

2 Fatty acids — 3.91

3 Flavonoids and Phenols 1.65 9.81

4 Nondihydropyridine s — —

5 Terpenes — 12.8

6 Saturated and Unsaturated Alcoholes 23.27 10.61

7 Antraquinone — 25

%Area in extract of P. oleracea(II)

%Area in extract of A. eriophora

DC.(III)

8 Aromatic derivatives 4.09 8.38

9 phosphinic acid — 2.98

10 Si-compounds 10.44 —

11 Alkanes 29.06 —

12 Alkenes 3.37 —

13 Tocopherol 2.43 —

14 Others 1.77 23.5

TABLE 4. Quantitative Analysis of Phytochemicals in Chloroform Extracts ofP. oleracea(II) andA. eriophoraDC (III) Identified by GC-MS

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TABLE 5. Bioactive Compounds in Carbon Tetrachloride Extracts ofP. oleracea(II) andA. eriophoraDC (III) Identified by GC-MS

No. R. T. Compound M. F. M. W.

%Area in A. eriophora

DC. (III)

%Area in P. oleracea

(II)

Family Pharmacological activity

1 9.89 Camphor C10H16O 152.24 1.21 — Monoterpene Antidiabetic and anti in-

flammatory [38, 39]

2 12.47 3,7-Dimethylocta- 1,7-dien-3,6-diol

C10H18O2 170.25 0.47 — Unsaturated

Alcohole

Anti-inflammatory, antioxidant, antidiabetic and antilipidemic [28, 29]

3 16.21 Butylated Hydroxyanisole C11H16O2 180.24 0.62 — Phenols Antioxidant [17], Antidiabetic [18]

4 16.96 4-Methyl-2,6-di-tert- butylphenol

C15H24O 220.35 1.34 — Phenols Antioxidant [17],

Antidiabetic [18]

5 20.64 Chamazulene C14H16 184.28 0.73 — Aromatic

derivative of azulene

Antioxidant and Antidiabetic [19. 20]

6 20.92 Myristic acid C14H28O2 228.37 0.44 — Saturated

fatty acid

7 22.08 Phytol C20H40O 296.53 4.91 5.07 Alcohole Antioxidant and Anti-in-

flammatory [24, 25]

8 22.59 4-t-butylbenzyl alcohol C11H16O 164.24 1.18 — Phenol Antioxidant [17], Antidiabetic [18]

9 23.84 Palmitic acid (n-hexadecanoic acid)

C16H32O 256.42 3.38 — Fatty acid

(saturated)

10 26 Arglabin C15H18O3 246.3 1.28 — Sesquiterpen

e lactone

Antidiabetic [27]

11 26.18 Linoleic acid (9,12-Octadecadienoic

acid)

C18H32O2 280.45 — 0.26 Fatty acid

(polyunsaturated omega-6)

12 26.6 7,10,13-Hexadecatrienoic acid, methyl ester

C17H28O2 264.40 — 0.23 Polyunsatu-

rated Fatty acid

13 26.82 Achillin C15H18O3 246.30 6.93 — Sesquiterpen

e lactone

Antidiabetic [27]

14 27.28 E-2-Tetradecen-1-ol C14H28O 212.37 1.32 — Unsaturated

Alcohole

15 29.11 Pyrocatechol, 3,5-di-tert-butyl-

C14H22O2 222.32 — 1.65 Phenols Antioxidant [17],

Antidiabetic [18]

16 33.78 1-Eicosanol C20H42O 298.56 — 3.77 Unsaturated

Alcohole

17 35.49 Flavone, 5-hydroxy-4’,7- dimethoxy-

18 37.44 Cyclobarbital C12H16N2O3 236.26 0.51 0.39 Barbiturate

derivative

Antidiabetic [43]

19 37.61 n-Eicosane C20H42 282.56 — 48.39 Alkanes,—

20 38.58 1,6,8-Trihyd- roxy-2-isopropyl-3-methoxy-9,10-anthraquinone

C18H16O6 328.31 31.1 — Antraquinon

e

Antidiabetic [33, 34]

21 39.00 5-Hydroxy-3¢,4¢,6,7- tetramethoxy flavone

22 38.37 Vitamin E C29H50O2 430.70 — 3.16 Tocopherols Antioxidant, [42]

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sesquiterpene lactones (total contents: 5.75%) with mentioned pharmacological activities were more than other components.

Chloroform extract of A. eriophora DC (IIIb).

Antraquinones (total contents: 25%), terpenes (total contents: 12.8%), phenols and flavonoids (total contents:

9.81%), alcohols (total contents: 10.61%), fatty acids (total contents: 3.91%), Sterols (total: 3.01%), aromatic derivatives (total contents: 8.38%) and phosphinic acid (total contents:

2.98%) with antidiabetic and anti-inflammatory activities were more than other components.

Carbon tetrachloride extract of A. eriophora DC (IIIc). Antraquinones (total contents: 31.1%), terpenes (total contents: 22.1%), phenols and flavonoids (total contents:

16.94%), alcohols (total contents: 7.45%), fatty acids (total contents: 3.82%), Sterols (total: 2.32%), tocopherol (total contents: 3.16%) and sesquiterpene lactone (total contents:

9.30%) with antidiabetic and anti-inflammatory activities were more than other components.

As was indicated for hydroethanolic (a) extracts, polyols and sterols in II and anthraquinone and fatty acids in III were more than other compounds. In chloroform (b) extract, sterols and alcohols in II, anthraquinone and terpenes in III were higher than other components. In carbon tetrachloride (c) extract, sterols and alcohols in II, anthraquinone and flavonoids in III were higher than other components.

4.2. Pharmacology

General considerations. Mortality (number of deaths), morbidity (defined as any abnormal condition or behavior due to a disorder), irritability (a condition of aggressiveness or increased response on handling) and other related abnormal states were observed in experimental animals. However, the motor coordination index (measured by Rota-rod appara- tus, Harvard, UK) did not indicate any significant differences between treated rats.

Blood serum glucose in hydroethanolic, chloroform and carbon tetrachloride extracts ofP. oleracea (II) and A. eriophora DC (III). The extracts of P. oleracea (IIa – IIc) could markedly decrease serum blood glucose on day 16 after STZ injection (Fig. 1A). Similarly, the extracts of A. eriophora DC (IIIa and especially IIIb) could have shown anti-hyperglycemic activity at 9 and 16^th days after STZ application (Fig. 1B).

No. R. T. Compound M. F. M. W.

%Area in A. eriophora

DC. (III)

(II)

Family Pharmacological activity

23 40.26 Campesterol

(Ergost-5-en-3-ol)

C28H48O 400.68 — 4.57 Phytosterol Antioxidant and

Antidiabetic [35]

24 40.28 b-sitrosterol C29H50O 414.71 0.41 17.3 Phytosterols Antioxidant and

Antidiabetic [35]

25 40.98 Stigmasterol C29H48O 412.7 0.95 5.38 Phytosterols Antioxidant and

Antidiabetic [35]

26 42.94 4H-1-Benzopyran-4-one, 2-(3,4-dimethoxyphenyl)-

5-hydroxy-3,6,7- trimethoxy-

27 44.45 Lupeol (Fagarasterol) C30H50O 426.73 0.96 — Sterol Antioxidant and

antidiabetic [37]

28 44.53 beta-Amyrin acetate C32H52O2 468.75 0.75 3.53 Ester of

amyrin

Antihyperglycemic and hypolipidemic [36]

29 46.15 Aristolone C15H22O 218.33 1.1 — Sesquiterpen

e lactone

Antidiabetic [27]

TABLE 6. Quantitative Analysis Phytochemicals in Carbon Tetra- chloride Extracts ofP. oleracea(II) andA. eriophoraDC (III) Iden- tified by GC-MS

(II)

%Area in A. eriophoraDC.

(III)

1 Sterols 27.25 2.32

2 Fatty acids 0.49 3.82

3 Phenols 1.65 3.14

4 Alkaloids — —

5 Saturated and unsaturated alcohols

12.37 7.45

6 Alkanes 48.39 —

7 Sesquiterpene lactone — 9.30

8 Antraquinone — 31.1

9 Flavonoides — 13.8

10 Tocopherols — 3.16

11 Others 9.85 25.91

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Blood serum triglyceride (TG), cholesterol (LDL and HDL) in hydroethanolic, chloroform, and carbon tetra- chloride extracts ofP. oleracea (II) and A. eriophora DC (III). As shown in Fig. 2B, carbon tetrachloride extract ofP.

oleracea(IIc) reduced total cholesterol in comparison with control rats (41.04%). In addition, IIb and IIc diminished LDL cholesterol by 55.88 and 51.96% which in comparison with control (102 ± 15) groups (p< 0.05). Moreover, IIc could diminish LDL level more than other extracts (Fig. 2C) (p< 0.05). In A. eriophora DC.(III), the results indicated that only IIIc could significantly diminish TG (Fig. 3A) and LDL (Fig. 3C) by 52.13 and 51.74 %, respectively (p< 0.05).

5. DISCUSSION

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, anti-diabetic and anti-lipidemic properties ofP. oleracea(II) andA. eriophora DC. (III), two natural health products recommended for diabete treatments in folk medicines in Iran, [5 – 10] were in-

vestigated in this research by three organic solvents with different polarities.

InP. oleraceaextracts (II), the results of the blood glucose level indicated that all extracts showed significant hypoglycemic activity compared to control groups in all times (carbon tetrachloride, chloroform and hydroethanolic extracts, respectively). In addition to hypoglycemic activity, these extracts could significantly diminish cholesterol and LDL levels and increased HDL/LDL ratio (especially in carbon tetrachloride extract) compared to control and glibenclamide groups. It seems that inP. oleraceaextract (II), more sterols and alcohols with anti-inflammatory, antioxidant, antidiabetic and antilipidemic activities in carbon tetrachloride and chloroform extracts may be reason of more pharmacological properties of these ones. In addition, polyols and sterols with mentioned activities in hydroethanolic extract is the reason of mentioned pharmacological effects of it, which can reconfirm more effective role of sterols and fatty acids compared to polyols for decreasing glucose and lipid profile levels.

InA. eriophora DC.(III), the results of the blood glucose level indicated that all extracts showed significant Fig. 1. Comparative effects of glibenclamide and hydroethanol (a), chloroform (b) and carbon tetrachloride (c) extracts ofP. oleracea(II, A) andA. eriophora DC(III, B) on blood serum glucose on days 4, 9 and 16 after STZ injection. However, both extracts could show noticeable anti-hyperglycemic activities but no significant blood glucose lowering activity between these extracts and glibenclamide animal groups. Bars show mean ± SEM serum glucose (n= 8) in each group; *P< 0.05 shows reliable difference from control group.

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Fig. 2. Comparative effects of glibenclamide and hydroethanol (A), chloroform (B) and carbon tetrachloride (C), extracts ofP. oleracea(II) on blood lipid profiles. Bars show mean ± SEM serum glucose (n= 8) in each group. *^,$P< 0.05 shows the difference from control and other solvent extracts, respectively.

Fig. 3. Comparative effects of glibenclamide and hydroethanol (A), chloroform (B) and carbon tetrachloride (C) extracts ofA. eriophoraDC.(III) on blood lipid profiles. Bars show mean±SEM serum glucose (n= 8) in each group. *P< 0.05 shows the difference from control animals.

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hypoglycemic activity compared to control and glibenclamide groups on days 9 and 16 after STZ application (especially in chloroform and hydroethanolic extracts). In addition, these extracts could significantly diminish TG and LDL levels and increased HDL/LDL ratio (especially in carbon tetrachloride extract) compared to control and glibenclamide groups, which may be concern to more antraquinones, terpenes, phenols and flavonoids in them.

Comparing the polarity of mentioned solvents, results re- vealed that carbon tetrachloride (non-polar and non-aqueous organic solvent) and chloroform (polar and non-aqueous organic solvent) could extracted mentioned effective phytochemicals better than hydroethanolic (polar and aqueous solvent) one which may be concern to more dissolving of these compounds in non-aqueous organic solvents (polar or non- polar) than aqueous one.

Anti-hyperglycemic effects of these plants are attributed to their ability to restore the function of pancreatic tissues by causing an increase in insulin output or inhibiting the intesti- nal absorption of glucose or to the facilitation of metabolites in insulin dependent processes [44 – 46]

Based on previous studies, fatty acids with insulin secretion and protecting â-cell from high glucose-induced apoptosis [47, 48], sterols with decreasing in cholesterol con- centration, increasing in insulin level and protective effect on pancreatic tissue [49, 50] and phenolic compounds with antioxidant anda-glucosidase inhibitor activities may be reason of antidiabetic and antilipidemic properties of these medicinal herbs [3, 51].

Thus, it seems that the presence of aforementioned phytochemicals with many corresponding pharmacological properties may be responsible for hypoglycemic and hypolipidemic activities in these herbal medicines.

CONFLICT OF INTEREST

The authors declare no conflict of interests.

ACKNOWLEDGEMENTS

This work was a research project of the Pharmaceutical Sciences Research Center, Pharmaceutical Sciences Branch, Islamic Azad University (Tehran, Iran), to which the authors would like to express their gratitude. They also appreciate Mojtaba Chaichi, EFL educator at Safir English Language Academy, for proofreading the initial draft of this article.

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