Effect of Chloroform and Aqueous Basic Fraction of Ethanolic Extract from Apium graveolens l. In...

Volume 8, Issue 1 2011 Article 30

Journal of Complementary andIntegrative Medicine

Effect of Chloroform and Aqueous BasicFraction of Ethanolic Extract from Apiumgraveolens l. In Experimentally-Induced

Hyperlipidemia in Rats

Deepa Iyer, VNS Institute of Pharmacy and Research CentreU.K. Patil, People's Institute of Pharmacy & Research

Centre

Recommended Citation:Iyer, Deepa and Patil, U.K. (2011) "Effect of Chloroform and Aqueous Basic Fraction ofEthanolic Extract from Apium graveolens l. In Experimentally-Induced Hyperlipidemia in Rats,"Journal of Complementary and Integrative Medicine: Vol. 8: Iss. 1, Article 30.DOI: 10.2202/1553-3840.1529

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Effect of Chloroform and Aqueous BasicFraction of Ethanolic Extract from Apiumgraveolens l. In Experimentally-Induced

Hyperlipidemia in RatsDeepa Iyer and U.K. Patil

AbstractThe present study was undertaken to explore the antihyperlipidemic effect of ethanolic extract

of seeds of Apium graveolens L. and its chloroform and aqueous basic fraction in olive oilinduced hyperlipidemic rats. The antihyperlipidemic activity of Apium graveolens was comparedwith a standard drug Atrovastatin (50mg/kg). The study involved phytochemical screening andchromatographic studies of extract and fractions. The ethanolic extract and its fractions wereadministered orally at doses of 200 and 400 mg/kg body weight in rats. Olive oil (5ml/kg oraldose) was administered 30 min after treatment. Blood was collected by ocular puncture 2 and 4 hafter olive oil treatment and centrifuged at 3000 rpm for 15-20 min. Serum samples were furthersubjected to biochemical analysis. The study dose dependently inhibited the total cholesterol (TC)triglycerides (TG), low density lipoproteins (LDL) level, and significantly increased high densitylipoprotein (HDL) level. Phytochemical screening revealed the presence of terpenoid, tannin,alkaloid, glycoside, flavanoid and sterols. UV λmax was found to be 206 nm with a melting pointof 137-138°C for the isolated component. The antihyperlipidemic effect was evaluated in olive oilloaded rats. Acute treatment caused stimulatory effect on HDL level and inhibition in TC and TGelevation induced by olive oil.

KEYWORDS: Anti-hyperlipidemic effect; Olive oil; total cholesterols; HDL, UV

Author Notes: Send correspondence to U.K. Patil, [email protected].

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1. Introduction Hyperlipidemia is an elevation of lipids in the bloodstream. These lipids include cholesterol, cholesterol esters, phospholipids and triglycerides. They are transported in the blood as part of large molecules called lipoproteins. Hyperlipidemia is a general term, it could be either high cholesterol in the blood (hypercholesterolemia), high triglycerides in the blood (hypertriglyceridemia) or it could be both. Elevated plasma lipid levels, mainly total cholesterol (TC), triglyerides (TG) and LDL along with decrease in HDL are known to cause hyperlipidemia which is core in initiation and progression of arteriosclerosis impasse. Therefore prime consideration in therapy for hyperlipidemia and arteriosclerosis is to enervate the elevated plasma level of TC, TG and LDL along with increase in HDL lipid levels. Apium graveolens is a plant species in the family- Apiaceae is commonly known as celery. It is also used as a flavouring agent in curries and chutneys. It was reported to exhibit anti- inflammatory activity1.Celery was also found to have anticarcinogenic and antiproliferation activities 2. Many other activities like hepatoprotective 3, antioxidant 4, gastroprotective 5 have been reported. Celery oil is dominated by terpenes, mostly limonene (70 to 80%) and the sesquiterpenes β-selinene (10%) and humulene; but its characteristic fragrance is caused by phthalides, coumarins, flavanoids 6-8. 2. Material and methods 2.1 Plant material The seeds of Apium graveolens L. were collected From Bhopal (M.P.), India and were identified and authenticated by Dr. Zia ul Hassan, Assistant professor, Department of Botany, Saifia College of Science & Education, Bhopal. A voucher specimen no.175/Bot/Safia/2010 is deposited in the herbarium of botany department. 2.2 Extraction and fractionation The dried drug was coarsely powdered and then exhaustively extracted with 90% ethanol in Soxhlet apparatus. The ethanolic extract so obtained was freed of solvent under vacuum to get 92 g (13.14% yield) of dark greenish brown mass. The solvent free ethanolic extract was acidified by 2M H2SO4 further dissolved and extracted with chloroform. Two layers i.e. chloroform and aqueous acid layers were obtained. The chloroform layer was separated and allowed to

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Iyer and Patil: Anti-Hyperlipidemic Activity of A.graveolens Fractions

Published by De Gruyter, 2011



evaporate. The aqueous acid layer was further basified by ammonium hydroxide and extracted with alcohol. Ethanolic extract, chloroform fraction and aqueous basic fraction were thus obtained.

2.3 Phytochemical profiling

Qualitative chemical test were performed to assess the presence of various phytoconstituents. The preliminary phytochemical screening revealed the presence of tannin, flavanoids, alkaloid and glycoside in crude ethanolic extract of A. graveolens. Chloroform fraction revealed the presence of terpenoids and sterols while aqueous basic fraction revealed the presence of flavanoids, alkaloid and glycosides.

2.4 Chromatographic studies

TLC of ethanolic extract and its fractions were carried out using various solvent systems. A mixture of Pet. Ether: Chloroform: methanol (7:1:2) gave the best resolution. Flash chromatography (Buchi controller C-610) was done for the extract mass and its chloroform fraction. The column was eluted with petroleum ether, chloroform and methanol successively in order of increasing polarity. Elution of the column was done with CHCl3: Methanol (99:1) with a view to isolate individual component. Melting point of 137-138°C was recorded for the component and UV λmax (MeOH) was recorded as 206 nm.

2.5 Screening for hypolipidemic activity

Screening for hypolipidemic activity was carried out in olive oil-loaded albino rats of either sex weighing 100-120 gm.

2.6 Preparation of test material

Ethanolic extract and its fractions were suspended in distilled water plus Polyoxyethylenesorbiton Mono-oleate (Tween 80).

2.7 Animal Model

The Swiss albino rats were selected and housed in polypropylene cages maintained under controlled conditions. The animals were fed with pellet food and water ad libitum. The animals fasted for 12-14 h before experimentation but it

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Journal of Complementary and Integrative Medicine, Vol. 8 [2011], Iss. 1, Art. 30

DOI: 10.2202/1553-3840.1529



was allowed free access to water. Rats of either sex, 6 - 8 weeks old and weighing 100-120 g, were taken for the experiments. 2.8 Measurement of biochemical parameters Fasted rats were divided into nine groups of six rats each. Group I served as vehicle control. Group II was kept as hyperlipidemic and administered with Olive oil only. Animals of Group III received atrovastatin at the oral dose of 50 mg/kg. Group IV and V were treated with ethanolic extract at the oral dose of 200mg/kg and 400mg/kg and group VI and VII were treated with chloroform fraction at the oral dose of 200mg/kg and 400mg/kg. Group VIII and IX were treated with aqueous basic fraction at the oral dose of 200mg/kg and 400mg/kg. Olive oil (5ml/kg oral dose) was administered 30 min after treatment9. The blood samples were withdrawn by ocular puncture 2 and 4 h after olive oil treatment and transferred directly into centrifuge tubes and allowed to clot at room temperature for 20-25 min and centrifuged for 15-20 min at 3000 rpm. The supernatant clear serum thus obtained was transferred carefully with the help pf micropipette into small test tubes for estimation. The serum concentration of total cholesterol, HDL and triglyceride were measured by standard procedure using auto- analyzer 10. 2.9 Histopathological study Histopathological study of rat liver of all the four groups was also performed. Tissue sections of 3μm thickness were stained with Erhlich’s hematoxylin and eosin and examined by light microscopy. 2.10 Statistical analysis Statistical evaluation of the data was done by Student‘t’ test. (Graph PAD Instat software, Kyplot).A value of p<0.05 was considered to be significant. 3. Results and Discussion The preliminary phytochemical screening revealed the presence of tannin, flavanoids, alkaloid and glycoside in crude ethanolic extract of A. graveolens. Chloroform fraction revealed the presence of terpenoids and sterols while aqueous basic fraction revealed the presence of flavanoids, alkaloid and glycosides. Melting point of 137-138°C was recorded for the component and UV λmax (MeOH) was recorded as 206 nm for the component.

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Ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 200mg/kg decreased serum level of total cholesterol by 32.54, 37.09 and 30.66 % respectively. On the other hand ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 400mg/kg decreased serum level of total cholesterol by 36.18, 39.35 and 31.41% respectively. Ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 200mg/kg increased the serum HDL cholesterol level by 8.47, 11.2 and 7.3%. On the other hand ethanolic extract, chloroform fraction and aqueous basic fraction and fraction at dose of 400mg/kg increased the serum HDL cholesterol level by 12.66, 14.71 and 9.05% respectively. Ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 200mg/kg decreased serum level of triglyceride level by 34.58, 41.73 and 32.56% respectively. On the other hand ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 400mg/kg decreased serum level of triglyceride level by 43.88, 50.25 and 33.97% respectively. The reduction in LDL cholesterol level by ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 200mg/kg were 67.41, 70.04 and 65.84 %respectively. On the other hand ethanolic extract, chloroform fraction and aqueous basic fraction at dose of 400mg/kg decreased LDL cholesterol by 71.93, 75.48 and 67.38%. LCAT plays a key role in the incorporation of free cholesterol into HDL and transferring it back to VLDL and LDL which are taken back later in liver cells 11.The possible mechanism of lipid lowering activity is may be due to enhancement of the activity of lecithin acyl transferase (LCAT) and inhibition of the action of hepatic TG- lipase on HDL12. As is evident from Fig. 1, the histopathological alterations viz. steatosis, sinusoidal dilatation and congestion, Kupfer cell hyperplasia or portal triaditis were not present in normal rat liver. Histopathology of liver in hyperlipidemic rats (Fig. 2) showed microvesicular steatosis along with sinusoidal dilatation, congestion and Kupfer cell hyperplasia. Liver histopathology (Fig. 3) of treated with Triton and Standard showed only mild sinusoidal congestion. Portal tracts and overall hepatic architecture were within normal limits. Rats treated with A. graveolens extract and its chloroform fraction (Fig. 4 and 5) showed mild feathery degenerative changes and mild vascular congestion but no steatosis. The overall hepatic architecture was maintained. The acute treatment with A. graveolens ethanolic extract and its chloroform fraction caused significantly inhibitory effects both on total cholesterol (TC) and triglyceride level (TG), compared to aqueous basic fraction, after olive oil administration. The maximum inhibitory effect on serum TG and TC level was observed with 400 mg/kg chloroform fraction. The drug and its fraction showed protective action as it slightly increased the HDL cholesterol level.

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DOI: 10.2202/1553-3840.1529



4. Conclusion The present investigation may be quite useful as this drug is highly valued as traditional system of medicine and can be used to control the state of hyperlipidemia. Complete treatment of various types of hyperlipidemia is possible by herbal drugs. Various herbal remedies including one or more herbal drugs not only decrease the serum levels of TC, TG and LDL cholesterol but also increase the serum level of beneficial HDL cholesterol.

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Effect of ethanolic extract, CHCl3 fraction and aqueous basic fraction of Apium graveolens on total cholesterol level (mg/dl) in olive oil induced

hyperlipidemic model (Table-1)

Group Initial Triglyceride level after 2 h

Triglyceride level after 4 h

Control 63.30± 0.24 65.19 ± 0.35 66.54±0.68

Hyperlipidemic 66.69 ± 0.16 87.50 ± 0.28 99.83 ±0.22

Atrovastatin (50mg/kg) 68.64 ± 1.70 87.56 ± 1.58 51.19±1.02

A. graveolens ethanolic extract

(200mg/kg) 69 .83±0.36 82.92± 0.52 63.72±.0.54b


(400mg/kg) 69.95±0.71 84.83± 0.91 55.83±0.35a

A. graveolens CHCl3fraction (200mg/kg)

70.24± 0.10 85.58± 1.12 60.32±0.58a

A. graveolens CHCl3fraction (400mg/kg) 69.52±0.50 87.37±1.36 53.41±0.43a

A. graveolens aq. basic

fraction(200mg/kg) 63.14±0.33 83.97± 1.25 69.27±1.76


fraction(400mg/kg) 67.56±0.82 85.38± 0.94 68.13±0.73

Total cholesterol concentrations are estimated by standard method. Values are expressed as mean ± S.E.M for six animals in each group. a: p<0.01 b : p<0.05 compared with hyperlipidemic group

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DOI: 10.2202/1553-3840.1529



Effect of ethanolic extract, CHCl3 fraction and aqueous basic fraction of Apium graveolens on triglyceride level (mg/dl) in olive oil induced

hyperlipidemic model (Table-2)

Group

Initial

Triglyceride level

after 2 h

Triglyceride

level after 4 h

Control

49.53± 1.48

49.25 ± 1.65

49.42±1.70

Hyperlipidemic

48.85 ± 1.64

69.70 ± 1.58

86.77 ±1.72

Atrovastatin (50mg/kg)

49.13 ± 1.70

64.58 ± 2.26

50.72±2.52

A. graveolens ethanolic extract (200mg/kg)

44.43±2.36

54.48±2.42

56.72±.1.41a

A. graveolens ethanolic extract (400mg/kg)

46.13±1.84

55.85±2.27

54.95±1.32b

A. graveolens CHCl3 fraction (200mg/kg)

45.76± 2.40

55.60± 2.85

52.26±1.39b


47.46±2.50

56.93±2.02

50.44±1.96a

A. graveolens aq. basic fraction(200mg/kg)

45.47±1.20

58.54±0.72

57.13±0.15

A. graveolens aq. basic fraction(400mg/kg)

43.22±1.08

57.52±1.21

57.60±0.84

Triglyceride concentrations are estimated by standard method. Values are

expressed as mean ± S.E.M. for six animals in each group. a: p<0.01 b : p<0.05 compared with hyperlipidemic group

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Effect of ethanolic extract, CHCl3 fraction and aqueous basic fraction of Apium graveolens on HDL level (mg/dl) in olive oil induced hyperlipidemic

model (Table-3)

Group Initial HDL level after 2 h

HDL level after 4 h

Control 32.34± 1.36 32.36 ± 1.11 32.86±1.34

Hyperlipidemic 32.20 ± 1.58 42.53 ± 1.24 43.02 ±1.49

Atrovastatin (50mg/kg) 33.45±1.63 44.43 ± 1.43 54.96.± 1.01


(200mg/kg) 34.20± 1.98 43.58±1.57 45.40±.1.71b


(400mg/kg) 33.69±2.11 42.36±1.49 47.35±1.52b

A. graveolens CHCl3fraction (200mg/kg)

33.30± 1.74 46.46± 2.09 49.83±1.42a

A. graveolens CHCl3fraction (400mg/kg) 33.46± 1.72 44.35± 1.92 50.44± 1.96a


fraction(200mg/kg) 33.45±1.10 41.63± 0.48 41.96±1.11


fraction(400mg/kg) 32.13± 1.24 41.55± 1.09 42.68±1.27

HDL concentrations are estimated by standard method. Values are expressed as mean ± S.E.M. for six animals in each group.

a: p<0.01 b : p<0.05 compared with hyperlipidemic group

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DOI: 10.2202/1553-3840.1529



Effect of ethanolic extract, CHCl3 fraction and aqueous basic fraction of Apium graveolens on LDL level (mg/dl) in olive oil induced hyperlipidemic

model (Table-4)

Group

Initial

LDL level after 2

h

LDL level after

4 h

Control

33.20± 1.36

33.31 ± 1.25

32.97±1.49

Hyperlipidemic

33.85 ± 1.85

50.88 ± 1.66

58.73 ±1.74

Atrovastatin (50mg/kg)

34.62±1.56

56.22 ± 1.42

35.23± 1.20


(200mg/kg)

34.31±2.13

51.16±1.72

44.75±.1.73b


(400mg/kg)

33.42±1.85

52.44±1.60

41.38±1.58b


32.30± 1.64

54.33± 2.32

42.35±1.63b


32.56± 1.77

55.23±2.15

39.44±2.05a


fraction(200mg/kg)

32.42±0.27

57.83±0.69

52.10±0.86


fraction(400mg/kg)

31.22±0.60

58.54±0.73

53.43±0.39

LDL concentrations are estimated by standard method. Values are expressed as

mean ± S.E.M. for six animals in each group. a: p<0.01 b : p<0.05 compared with hyperlipidemic group

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1-Control group

2- Hyperlipidemic group

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3- Standard treated group

4- A. graveolens ethanolic extract treated group (400mg/kg)

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5- A. graveolens chloroform fraction treated group (400mg/kg)

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DOI: 10.2202/1553-3840.1529



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(8) Chatterjee A. Treatise of Indian Medicinal Plants. Council for Scientific and Industrial Research, India, 1990, pp. 327.

(9) Tatiana S, Claus T, Reginaldo G, Silvio A, Eliane N, Rozangela C and Jane B. Antihyperlipidemic effect of Casearia sylvestris methanolic extract. Fitoterapia. (2008) 79: 465-467.

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Effect of Chloroform and Aqueous Basic Fraction of Ethanolic Extract from Apium graveolens l. In...

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