Oriental Pharmacy and Experimental Medicine

1 23

Oriental Pharmacy and ExperimentalMedicine ISSN 1598-2386Volume 11Number 4 Orient Pharm Exp Med (2011)11:263-270DOI 10.1007/s13596-011-0031-5

Antihyperlipidemic activity of leaf and rootextracts of Premna esculenta (Roxb.) inPoloxamer-407 induced hyperlipidemicmice and rats

Zobaer A. Mahmud, Sitesh C. Bachar &Nazmul Qais

1 23

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RESEARCH ARTICLE

Antihyperlipidemic activity of leaf and root extractsof Premna esculenta (Roxb.) in Poloxamer-407 inducedhyperlipidemic mice and rats

Zobaer A. Mahmud & Sitesh C. Bachar & Nazmul Qais

Received: 9 July 2011 /Accepted: 25 August 2011 /Published online: 14 September 2011# Institute of Oriental Medicine, Kyung Hee University 2011

Abstract The ethanolic extracts of leaves and roots ofPremna esculenta (Roxb.) were evaluated for antihyperlipi-demic activity in poloxamer 407 induced hyperlipidemicrats and mice. The administration of the ethanolic extract ofleaves induced a significant (p<0.05) reduction in serumtotal cholesterol (TC), triglycerides (TG), low densitylipoprotein (LDL) and very low density lipoprotein (VLDL)levels as well as the atherogenic index compared to the P-407induced hyperlipidemic control mice after 24 h of treatment ata single dose of 500 mg/kg p.o. Both the leaf and root extractssignificantly (p<0.05) reduced the serum TC, TG, VLDLand LDL levels as well as the atherogenic index compared toP-407 induced hyperlipidemic control rats after 4 days ofpretreatment at a dose of 250 mg/kg/day p.o. The lipidlowering effects were compared with standard drug atorvas-tatin (50 mg/kg p.o.). Furthermore, both extracts showedpotential to moderate scavenging activity of superoxide,nitric oxide and DPPH free radicals. In DPPH radicalscavenging assay the IC50 values of the leaf and root extractswere found to be 425.79 and 20.26 μg/ml, respectively whileascorbic acid has the IC50 value 16.34 μg/ml. The IC50

values of the leaf and root extracts against superoxideradicals were found to be 109.57 and 366.2 μg/ml,respectively which were comparable to that of ascorbic acid(99.66 μg/ml). The findings of the investigation demonstrat-ed both antihyperlipidemic and antioxidant activities of the

leaf and root extracts of P. esculenta which could help inprevention of hyperlipidemia associated disorders.

Keywords Premna esculenta . Antihyperlipidemic activity .

Poloxamer 407 . Rats . Antioxidant activities . Ethanolicextracts

Introduction

Premna esculenta (Roxb) (Family: Verbenaceae, Vernacularname: Lelom pata (Bengali)) is an important medicinalplant claimed to have potential curative properties and havebeen traditionally used by tribal people of Chittagong HillTracts of Bangladesh in the treatment of gout, hook worminfestation, hysteria, jaundice (hepatocellular jaundice),leucorrhea, lipoma (tumor), edema, snake bite, stomachdisorder and ureterolithiasis (Uddin 2006).The plant is asmall, branching shrub and grows in shady, wet places inprimary rain forests of Bangladesh and India (Assam). Intraditional system of remedies the leaves of the plant areapplied for the treatment of arthritis and bacterial and fungalinfection. Roots, barks and leaves of the plant are used in thetreatment of lipoma/tumor. Roots are commonly used incombination with other plants in the curement of edema andjaundice (Uddin 2006). Leaves are used as an appetizer.

Currently available hypolipidemic drugs: statins,fibrates, bile acid sequestraints have been associated witha number of toxic side effects and devoid of antioxidantproperty (Chattopadhyaya et al. 1996). But herbal treatmentfor hyperlipidemia has no side effect and is relatively cheapand locally available (Srikanth and Muralidharan 2009).Moreover, the natural products having dual property ofantihyperlipidemic and antioxidant activities can bettermanage the hyperlipidemia associated disorders which areusually accompanied by increased oxidative stress. Various

Z. A. Mahmud :N. Qais (*)Department of Clinical Pharmacy and Pharmacology,Faculty of Pharmacy, University of Dhaka,Dhaka 1000, Bangladeshe-mail: [email protected]

S. C. BacharDepartment of Pharmaceutical Technology, Faculty of Pharmacy,University of Dhaka,Dhaka 1000, Bangladesh

Orient Pharm Exp Med (2011) 11:263–270DOI 10.1007/s13596-011-0031-5

Author's personal copy

indigenous plants are used for antihyperlipidemic effects inthe traditional system of medicine and recently there has beenfocus on the lipid lowering effect of medicinal plants. Someplants of Premna species such as Premna corymbosa, Premnatomentosa have been reported to have antihyperlipidemicactivity in animal model (Karthikeyan and Deepa 2008).

Although P. esculenta (Roxb) have various ethnopharma-cological uses, the plant yet have not been undergone anychemical or pharmacological investigation. Therefore, thepresent study was designed to investigate antihyperlipidemicactivity of ethanolic extracts of leaves and roots ofP. esculentain Poloxamer 407 induced hyperlipidemic rats and micemodel. The in vitro antioxidant activities of the plant extractswere also determined to explain the potential role in theprotection against oxidative degradation of body lipids.

Materials and methods

Chemicals and reagents

Poloxamer 407, also called Pluronic RF-127, was donatedby BASF Bangladesh Ltd. Atorvastatin at strength of40 mg in teblet form was obtained from BeximcoPharmaceuticals Ltd. Bangladesh. Total cholesterol (TC),total triglyceride (TG), high density lipoprotein cholesterol(HDL) measuring kits were obtained from Linear ChemicalsS.L. (Barcelona, Spain) and used according to manufacturer’sinstructions after precipitation techniques and modifiedenzymatic procedures to plasma samples (Allain et al. 1974;Bucolo and David 1973; Burstein et al. 1980; Finley et al.1978). DPPH, nitroblue tetrazolium, hydroxylamine hydro-chloride and Tween-80 were purchased from Sigma Aldrich.All other reagents and chemicals were of BDH and E Merckanalytical grade.

Plant material

The leaves and roots of P. esculenta were collected fromRangamati, Chittagong Hill Tracts, Bangladesh in September,2010 at the flowering stage. The plant samples were identifiedand authenticated by Bangladesh National Herbarium and avoucher specimen (accession No: DACB 35035) was depos-ited there for future reference. The plant parts, after cuttinginto small pieces, were sun dried for several days. The plantmaterials were then oven dried for 24 h for better grinding.The dried cutting pieces were pulverized by a mechanicalgrinder and stored into an air-tight container.

Extraction of the plant material and sample preparation

Pulverized powdered leaves (1.2 kg) and roots (1.0 Kg) ofthe plant were macerated with 6.0 and 5.0 L of absolute

ethanol, respectively at room temperature for 15 days withoccasional shaking. The ethanolic extracts of leaves androots were collected, filtered by cotton plug followed bywhatman filter paper (no. 1) and evaporated to dryness (45°C)under reduced pressure by rotary evaporator. The extracts andstandard drug atorvastatin were suspended in normal salineusing 1.0% Tween- 80.

Phytochemical screening

The freshly prepared crude ethanolic extracts of leaves androots were qualitatively tested for the presence of alkaloids,phenols, tannins, reducing sugar, flavonoids, steroids,terpenoids and saponins by using standard phytochemicalprocedures (Ghani 2003; Trease and Evans 1989).

Experimental animals

Swiss albino mice (weighing 35–40 g) and Long Evans rats(Rattus norvigicus) of either sex weighing 150–190 gmwere purchased from the animal resource branch ofInternational Centre for Diarrheal Diseases and Research,Bangladesh (ICDDR,B). The animals were housed 5 percage in a temperature controlled room (25±1°C), with alight/dark cycle of 12 h. For a week following their receipt,the animals were allowed free access to a standard rat chowdiet (ICDDR,B formulated) and fresh water ad libitumwhile they were acclimatizing to the environment. Duringthe experimentation all rats had free access to standard ratchow and water at all times unless otherwise stated in themethods section. At the start of the experiment, animalswere randomly distributed so that body weights, initial TG,TC and other parameters were similar in all the experimentalgroups. The design and performance of research studyinvolving mice and rats have been approved by the EthicalReview Committee, Faculty of Biological Science, Universityof Dhaka through the submission of a research protocol beforethe study.

Poloxamer 407 induced hyperlipidemic mice model

The antihyperlipidemic activity was evaluated according toYogendrasinh and Sunita (2010). Twenty swiss albino micewere divided into four groups of five mice each. The firstgroup (Group-I) served as normal control group andreceived only 1% Tween-80 in normal saline at a dose of5 mL/kg, p.o.. Based on the reported method (Johnston andPlamer 1993), the animals of Group II, III and IV weremade hyperlipidemic by an intraperitoneal injection of600 mg/kg body weight of poloxamer 407, followed by a6 h fasting. The poloxamer 407 was made at a finalconcentration of 30% (w/w) by dissolving the powder indistilled cool water; the solution was then kept refrigerated

264 Z.A. Mahmud et. al.


overnight to facilitate its dissolution. Needles and syringesused to administer P-407 were cooled prior to administra-tion to prevent P-407 gelation within the syringe. Two hoursafter the administration of P-407, the mice of Group III weretreated with atorvastatin at a single dose of 50 mg/kg p.o.,while group IV received a single dose (500 mg/kg p.o.) ofethanolic extract of leaves of P. esculenta.

Blood sampling

After 15 and 24 h of treatment, blood samples were collectedfrom retroorbital plexus under normal anesthesia with diethylether and incubated at room temperature for 30 min. Theblood samples were then centrifuged at 4000 rpm at 4°C for10 min, after which the serum was separated and storedat −40°C until to be used for biochemical tests.

Poloxamer 407 induced hyperlipidemic rat model

The antihyperlipidemic activity was evaluated according tothe method described by Samer (Samer et al. 2005). Twentyfive Long Evans rats were divided into five groups of fiverats each. A normal control (NC) group did not receive anytreatment apart from 1% Tween 80 in normal saline (5 mL/kg/day) by oral gavaging. A second group (P-407 control)was treated with 1% Tween-80 in normal saline p.o. at5 mL/kg body weight/day. In the third group rats wereadministered atorvastatin at a dose of 75 mg/kg/day. GroupIV and V received ethanolic extract of leaves and roots of P.esculenta, respectively at a dose of 250 mg/kg/day. Thevehicle (1% Tween 80 in normal saline) or test drugs wereadministered orally for 4 consecutive days. After 4 days,hyperlipidemia was induced in the rats of Group II to GroupV by injecting rats with Poloxamer 407 (600 mg/kg bodyweight) intraperitoneally 48 h prior to blood collection(Johnston and Plamer 1993).

At the end of the experimental period the rats wereanesthetized with diethyl ether and sacrificed bydecapitation. Blood was then collected from the neckwound and incubated at room temperature for 30 min.The blood samples were then centrifuged at 4000 rpmat 4°C for 10 min, after which the serum was separatedand stored at −40°C until to be used for biochemicaltests.

Biochemical estimations

The levels of triglycerides (TG), total cholesterol (TC) andhigh density lipoprotein cholesterol (HDL-C) in the serumwere determined by enzymatic colorimetric methods usingcommercial Kits (Linear Chemicals Ltd, Spain). Very lowdensity lipoprotein cholesterol (VLDL-C) was calculated asTG/5. Low density lipoprotein cholesterol (LDL-C) levels

were calculated using Friedewald’s formula (Friedewald etal. 1972):

LDL ¼ TC � HDL� TG

5

The atherogenic index (A.I.) was calculated using thefollowing formula:

ðA:I :Þ ¼ VLDLþ LDL

HDL

Statistical analysis

The results were expressed as mean±SEM. The data wereanalyzed using Kruskal-Wallis test followed by post hocpaired comparisons to determine the level of significance.Here the Kruskal-Wallis test was performed on all thegroups together. Then Group I and II were tested togetherfirst followed by testing Group III, IV and Veach separatelywith Group II using the Mann–Whitney U test to evaluatepair wise differences among the groups. A value of P<0.05was considered to be significant (Woodson 1987). Thestatistical analysis was carried out using the SPSS program(version 17.0).

Evaluation of antioxidant activity

DPPH radical scavenging activity

The free radical scavenging activities of the ethanolicextracts of leaves and roots of the plant on the stableradical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were esti-mated by the method of Brand-Williams (Brand-Williams etal. 1995). 2.0 mL of a methanol solution of the sample(extractives/ positive control) at different concentration(500 μg/mL to 0.977 μg/mL) were mixed with 3.0 mL ofa DPPH methanol solution (20 μg/mL). The reactionmixture was vortexed thoroughly and left in the dark atroom temperature for 30 min. The absorbance of themixture was measured spectrophotometrically at 517 nm.The ability of the plant extract to scavenge DPPH radicalwas calculated by the following equation:

Inhibitionð%Þ ¼ 1� As

Ao

� �� 100 ð1Þ

Where Ao is the absorbance of the control reaction(containing all reagents except the test material) and As isthe absorbance of the sample material. Then percentinhibitions were plotted against respective concentrations.IC50 values were calculated as the concentration of eachsample required to give 50% DPPH radical scavengingactivity from the graph. BHT (tert-butyl-1- hydroxytoluene)

Antihyperlipidemic activity of leaf and root extracts of P. esculenta 265


was used as positive control. The experiment was performedthrice and the result was expressed as mean±S.E.M inevery case.

Superoxide radical scavenging activity

Assay for the super oxide radical scavenging activity wasbased on the capacity of the sample to inhibit blueformazan formation by scavenging the superoxide radicalsgenerated in Hydroxylamine-sodium carbonate-EDTA-NBTsystem (Beauchamp 1971). Briefly, 1 mL of 50 mM sodiumcarbonate, 0.4 mL of 24 μM nitroblue tetrazolium and0.2 mL of 0.1 mM EDTA were taken in a test tube. Then100 μL of plant extract (conc. ranging from 1000 μg/mL to7.513 μg/mL) was added to the test tube. Reaction wasinitiated by adding 0.4 mL of 1 mM hydroxylaminehydrochloride. The reaction mixture was incubated for 5 minat ambient temperature and then the absorbance at 562 nmwasmeasured against an appropriate blank to determine thequantity of formazan generated. The percentage inhibition ofsuperoxide anion generation (I %) was calculated by formula(1). The experiment was performed thrice and the result wasexpressed as mean±S.E.M in every case.

Nitric oxide (NO·) radical scavenging activity

The method of Garrat (Garrat 1964) was adopted todetermine the nitric oxide radical scavenging activity ofethanolic extracts of roots and leaves of P. esculenta. Avolume of 2 mL of 10 mM sodium nitroprusside and0.5 mL of phosphate buffer saline (pH 7.4) were mixedwith 0.5 mL of plant extract and ascorbic acid (positivecontrol) individually at various concentrations (500 to3.9 μg/mL). The reaction mixture was incubated at 25°Cfor 150 min. Then, 0.5 mL of incubation solution waswithdrawn and mixed with 1 mL of sulfanilic acid reagent(0.33% in 20% glacial acetic acid) and allowed to stand for5 min at room temperature for completing diazotization.Then 1 mL of 0.1% w/v napthylethylenediamine dihydro-chloride was added, mixed well and the mixture wasincubated at room temperature for 30 min. The absorbanceof the mixture was taken at 540 nm. The amount of nitricoxide (NO·) radical inhibited by the extract was calculatedusing the formula (1). The experiment was performed thriceand the result was expressed as mean±S.E.M in every case.

Results

The ethanol extract of leaves and roots of P. esculenta wereevaluated for their antihyperlipidemic activity in P-407induced hyperlipidemic mice and rats as well as their freeradical scavenging activities against DPPH, superoxide andnitric oxide radicals. Phytochemical analyses revealed thepresence of steroid, terpenoid, phenols, tannin, reducing sugarin the ethanolic extract of leaves (Table 1) while the screeningtests indicated the presence of steroid, terpenoid, flavonoid,tannin and reducing sugar in the root extract of P. esculenta.

The effect of ethanolic extracts of leaves of P. esculentaon serum lipid profile in P-407 induced hyperlipidemic mice

The oral administration of the EtOH extract of leaves of P.esculenta (EEPEL) was found to be effective in significantlyreducing serum TG, LDL and VLDL (p<0.05) levels as wellas the atherogenic index (p<0.05) when compared to the P-407 induced hyperlipidemic control mice, after 15 and 24 hof treatment at a dose of 500 mg/kg p.o. (Tables 2 and 3).EEPEL also significantly reduced the serum TC levels (p<0.05) after 24 h of treatment and increased the serum HDL(p<0.05) levels after 15 h of treatment. These effects werecomparable to those of the reference standard, atorvastatin.

The effect of ethanolic extracts of leaves and rootsof P. esculenta on serum lipid profile in P-407 inducedhyperlipidemic rats

Both the EtOH extract of leaves (EEPEL) and roots(EEPER) of P. esculenta significantly (p<0.05) reducedserum TG and VLDL levels as well as the atherogenicindex when compared to the P-407 induced hyperlipidemiccontrol rats, after 4 days of pretreatment at a dose of250 mg/kg/day (Table 4). EEPER also significantly (p<0.05) lowered the serum TC levels. Though EEPELreduced serum TC and LDL levels and increased theHDL levels, the results were not found to be statisticallysignificant. The positive control atorvastatin also signifi-cantly (p<0.05) reduced TG, TC, VLDL and A.I. comparedto P-407 control group. From these findings it can bedemonstrated that the EtOH extract of leaves and roots ofthe plant have potential antihyperlipidemic activity whichare comparable to that of the standard drug atorvastatin.

Table 1 Result of chemical group test of the ethanol extracts of leaves (EEPEL) and roots (EEPER) of Premna esculenta

Extract Steroid Terpenoid Alkaloid Reducing sugar Phenols Tannin Flavonoid Saponin

EEPEL + + − + + + − −EEPER ++ ++ − + + + + −

(+): Present; (−): Absent



Tab

le2

Effectof

ethano

licextractof

leaves

ofPremna

esculenta(EEPEL)on

serum

lipid

profile

15hafterpo

loxamer

407–ind

uced

acutehy

perlipidem

iain

micea

Group

TC(m

mol/L)

TG

(mmol/L)

HDLb(m

mol/L)

LDL(m

mol/L)

VLDL(m

mol/L)

A.I.

Group

-I(N

ormal

control)

3.62

±0.42

3.19

±0.16

1.13

±0.09

2.05

±0.39

0.63

±0.03

2.42

±0.39

Group

-II(P-407

control)

18.39±0.63

*16

.93±0.75

*5.04

±0.20

*9.9±0.67

*3.38

±0.08

*2.67

±0.27

Group

-III(A

torvastatin

)13

.47±0.51

**(26.75

)9.45

±0.40

**(44.18

)7.68

±0.29

**(52.38

)3.9±0.56

**(60.60

)1.89

±0.08

**(44.10

)0.76

±0.08

**(71.53

)

Group

-IV

(EEPEL)

17.03±0.60

(7.39)

10.10±0.24

**(41.60

)6.58

±0.22

**(30.55

)8.42

±0.55

(14.94

)2.02

±0.05

**(40.23

)1.6±0.10

**(40.10

)

Allvalues

areexpressedas

mean±SEM

(n=5).*comparedto

norm

alcontrolgrou

p(p<0.05

),**

comparedwith

P-407

controlgrou

p(p<0.05

).Kruskal-W

allis

testfollo

wed

byMann–

Whitney

Utest.a

TC:totalcholesterol,TG:triglyceride,HDL:H

ighdensity

lipop

rotein,L

DL:low

density

lipop

rotein,V

LDL:v

erylowdensity

lipop

rotein,A

.I.:atherogenicindex.

Figures

inparenthesesare

thepercentage

redu

ctioncomparedto

P-407

controlgrou

p.bFigures

inparenthesesarethe%

increase

comparedto

P-407

controlgrou

p

Tab

le3

Effectof

ethano

licextractof

leaves

ofPremna

esculenta(EEPEL)on

serum

lipid

profile

24hafterpo

loxamer

407–ind

uced

acutehy

perlipidem

iain

micea

Group

TC(m

mol/L)

TG

(mmol/L)

HDLb(m

mol/L)

LDL(m

mol/L)

VLDL(m

mol/L)

A.I.

Group

-I(N

ormal

control)

4.21

±0.51

3.43

±0.13

1.18

±0.07

2.46

±0.44

0.68

±0.02

2.64

±0.32

Group

-II(P-407

control)

24.28±0.89

*17

.66±0.46

*5.05

±0.17

*15

.7±0.80

*3.52

±0.09

*3.82

±0.20

*

Group

-III(A

torvastatin

)14

.6±0.44

**(39.86

)13

.61±0.24

**(22.93

)7.00

±0.30

**(38.61

)4.87

±0.69

**(68.98

)2.72

±0.49

**(22.72

)1.1±0.12

**(71.20

)

Group

-IV

(EEPEL)

19.30±0.29

**(20.51

)14

.50±0.64

**(17.89

)5.30

±0.13

(4.95)

11.08±0.36

**(29.42

)2.92

±0.12

**(17.04

)2.65

±0.12

**(30.62

)

Allvalues

areexpressedas

mean±S.E.M

.(n=5).*comparedwith

norm

alcontrolg

roup

(p<0.05

),**

comparedwith

P-407

controlgrou

p(p<0.05

).Kruskal-W

allis

testfollo

wed

byMann–Whitney

Utest.a

TC:totalcholesterol,TG:triglyceride,H

DL:High-density

lipop

rotein,L

DL:low-density

lipop

rotein,V

LDL:very

low-density

lipop

rotein,A

.I.:atherogenicindex.

Figures

inparentheses

arethepercentage

redu

ctioncomparedto

P-407

controlgrou

p.bFigures


increase

with

respectto

P-407

controlgrou

p



In-vitro antioxidant activity

The DPPH, super oxide and nitric oxide radical scavengingactivity of ethanolic extracts of leaves and roots of the plantare shown in Figs. 1, 2 and 3, respectively and thecorresponding IC50 values are presented in Table 5.

Discussion

The P-407 treated rodent is a well established animal modelof dose dependent hyperlipidemia and atherosclerosis(Johnston 2004). P-407 has been utilized in the hyper-lipidemic model due to its convenience, reproducibility andthe lack of undesirable underlying pathological conditions(Wout et al. 1992).

Tab

le4

Effectof

ethano

licextractof

leaves

(EEPEL)androots(EEPER)of

P.esculentaon

serum

lipid

profile

inpo

loxamer

407–ind

uced

hyperlipidem

icratsa

Group

TC(m

mol/L)

TG

(mmol/L)

HDL(m

mol/L)b

LDL(m

mol/L)

VLDL(m

mol/L)

A.I.

Group

-I(N

ormal

control)

3.74

6±0.94

41.25

7±0.20

81.46

±0.20

92.03

±0.77

0.25

±0.04

11.53

±0.32

4

Group

-II(P-407

control)

12.51±1.7*

20.02±3.73

*3.78

±0.113*

4.86

±1.38

9*4±0.74

8*2.33

±0.39

*

Group

-III(A

torvastatin

)9.44

±1.4*

*(24.54

)5.84

±1.41

**(70.00

)4.99

±0.611*

*(32.00

)3.28

±1.64

(32.51

)1.17

±0.28

3**(70.75

)0.93

±0.49

**(60.00

)

Group

-IV

(EEPEL)

10.96±0.13

6(12.39

)8.46

±1.03

**(57.00

)4.64

±0.77

(22.75

)4.41

±0.89

(9.25)

1.69

±0.20

7**(57.75

)1.41

±0.39

**(39.48

)

Group

-V(EEPER)

10.69±0.54

**(14.54

)8.77

±1.56

**(56.19

)5.41

4±1.12

(43.22

)3.52

±1.38

(27.57

)1.75

±0.31

**(56.25

)1.06

±0.311*

*(54.50

)

Allvalues

areexpressedas

mean±S.E.M

.(n=5).*com

paredwith

norm

alcontrolgrou

p(p<0.05

),**

comparedwith

P-407

controlgrou

p(p<0.05

).Kruskal-W

allis

test

follo

wed

byMann–

Whitney

Utest.aTC:totalcholesterol,TG:triglyceride,HDL:High-density

lipop

rotein,LDL:low-density

lipop

rotein,VLDL:very

low-density

lipop

rotein,A.I.:atherogenicindex.

Figures

inparenthesesarethepercentage

redu

ctioncomparedto

P-407

controlgrou

p.bFigures


increase

with

respectto

P-407

controlgrou

p

Fig. 1 DPPH radical scavenging activity of ethanolic extracts ofleaves (EEPEL) and roots (EEPER) of P. esculenta at differentconcentrations. BHT: butylated hydroxyl toluene. The ethanolic rootextract exhibited potential DPPH radical scavenging activity compa-rable to that of standard BHT while the ethanolic leaf extract showedmoderate activity

Fig. 2 Super oxide radical scavenging activity of ethanolic extracts ofleaves (EEPEL) and roots (EEPER) of P. esculenta at differentconcentrations. ASA: ascorbic acid. The leaf and root extractssignificantly quenched the superoxide radical dose dependently



Increased serum cholesterol and triglycerides levels areimportant risk factors for atherosclerosis (Kannel et al.1979; John et al. 2005). Furthermore, the high serum levelsof LDL play a crucial role in the development ofatherosclerosis lesions that progress from fatty steaks toulcerated plaques following the accumulation of the LDL inthe extracellular sub-endothelial space of arteries (Ross1993). Thus, lowering the abnormally increased serumtriglycerides, total cholesterol and LDL levels reduces theincidence of atherosclerosis. In the present study, adminis-tration of both the ethanolic extract of leaves and roots ofthe plant significantly reduced the serum TG, TC, VLDLand LDL levels (Tables 3 and 4) and this implies that theextracts have beneficial effects on serum lipid profile byreducing the lipid levels. The A.I., the ratio of LDL toHDL, is commonly used as an index for atherosclerosis(Fki et al. 2005). The results showed that oral administra-tion of ethanolic extract of leaves and roots of the plantsignificantly reduced the atherogenic index (A.I.) comparedto P-407 induced hyperlipidemic control suggesting thepotential ability of the extracts in reducing the risk ofatherosclerosis.

Since the increase in triglycerides (TG) seen followingP-407 ip. injection results primarily from an inhibition of

TG degradation, where P-407 directly inhibits capillarylipoprotein lipase (LPL) responsible for plasma TG hydro-lysis (Johnston and Plamer 1993; Johnston 2004), theserum TG lowering effects of leaf and roots extracts of theplant can be attributed to the activation of lipoproteinlipase.

The mechanism responsible for the elevation of serumcholesterol following i.p. injection of poloxamer 407solution to rats may be due to stimulation of 3-hydroxy-3-methylglutaryl-Co-enzyme A (HMG-CoA) reductase activityin the liver by the poloxamer vehicle (Wout et al. 1992;Johnston 2004). Besides, Atorvastatin, used as positivecontrol in this study is a HMG-CoA reductase inhibitorwhich is the rate-limiting enzyme in the biosynthesis ofcholesterol. Thus, the cholesterol lowering effects of theextracts might be related partly to the decreased activity ofhepatic HMG CoA reductase.

Hyperlipidemia is associated with increased oxidativestress and overproduction of oxygen free radicals (Zalba etal. 2001). Elevated NO· radical induces oxidation of LDLwithin the arterial walls that plays a significant role in theinitiation and progression of atherosclerosis (Steinberg et al.1989; Parthasarthy et al. 1992; Tanner et al. 1999). Theresults of in vitro antioxidant activities of the plant extractsshowed that ethanolic extract of leaves and roots possesspotential scavenging activity of DPPH, superoxide and NO·

free radicals which may help in preventing the oxidativemodifications of various vital biomolecules, includinglipids (LDL), in hyperlipidemic subjects.

The observed antihyperlipidemic activity of the leaf androot extracts may be due to the presence of steroids,flavonoids (in root extract), phenols, tannins and triterpe-noids found in the preliminary phytochemical screening aspreviously it has been reported that steroids (Ghule et al.2009), terpenoids (Liu et al. 1987), flavonoids (Patel et al.2009) possesses antihyperlipidemic activity. According toprevious reports (Hideo et al. 1993; Marja et al. 1999; Ocket al. 2003) a highly positive relationship between phenoliccompounds and antioxidant activity appears to be the trendin many plant species. This activity is believed to be mainlydue to their redox properties (Zheng and Wang 2001),which play an important role in adsorbing and neutralizingfree radicals and quenching singlet and triplet oxygen. So,

Fig. 3 Nitric oxide radical scavenging activity of ethanolic extracts ofleaves (EEPEL) and roots (EEPER) of P. esculenta at differentconcentrations. ASA: ascorbic acid. Both the leaf and root extractssignificantly scavenged the nitric oxide radical in a dose dependentmanner. The scavenging activity of the root extract is more potent thanthe reference standard, ascorbic acid

Table 5 In-vitro antioxidant ac-tivity of ethanolic extract ofleaves (EEPEL) and roots(EEPER) of P. esculenta

aValues are expressed as mean±S.E.M. in three replicates.bpositive control used

Extract/compound Antioxidant activity (IC50 μg/mL)a

DPPH assay Superoxide radical assay Nitric oxide radical assay

EEPEL 425.79±3.42 109.57±0.65 525.67±5.61

EEPER 20.26±0.065 366.2±6.75 319.52±2.33

BHTb 16.34±0.40

Ascorbic acidb 99.66±1.82 355.96±0.051



the overall radical scavenging activity of leaves and roots ofthe plant might be attributed to its phenolic constituentssuch as tannin, flavonoids etc. which scavenged directlyDPPH and superoxide radicals by the hydrogen-donatingcapacity of their phenolic groups.

In summary, the present study demonstrated new propertiesof EtOH extract of leaves and roots of P. esculenta as a potentantihyperlipidemic and antioxidant agent. These resultssuggest that the plant extracts may be beneficial inpreventing atherosclerotic cardiovascular diseases and furtherinvestigations are warranted to examine the mechanism ofaction as well as to isolate the active constituents responsiblefor these activities.

Disclosure of conflict of interest There is no conflict of interestamong the authors.

References

Allain CC, Poon LS, Chan CS, Richmond W, Fu PC (1974)Enzymatic determination of total serum cholesterol. Clin Chem20:470–475

Beauchamp CF (1971) Super oxide dismutase: improved assay and anassay applicable to acrylamide gel. Anal Biochem 10:276

Brand-Williams W, Cuvelier ME, Berset C (1995) Use of a free radicalmethod to evaluate antioxidant activity. Lebensm Wiss Technol28:25–30

Bucolo G, David H (1973) Quantitative determination of serumtriglycerides by the use of enzymes. Clin Chem 19:476–482

Burstein M, Scholnick HR, Morfin R (1980) Rapid method for theisolation of lipoproteins from human serum by precipitation withpolyanions. Scand J Clin Lab Invest 40:560

Chattopadhyaya R, Pathak D, Jindal DP (1996) Antihyperlipidemicagents. A review. Indian drugs 33:85–97

Finley PR, Shifman RB, Williams RS, Lichti DA (1978) Cholesterolin high-density lipoprotein: use of Mg2+/dextran sulfate in itsenzymic measurement. Clin Chem 24:931–933

Fki I, Bouaziz M, Sahnoun Z, Sayadi S (2005) Hypocholesterolemiceffects of phenolic-rich extracts of Chemali olive cultivar in ratsfed a cholesterol-rich diet. Bioorg Med Chem 13:5362–5370

Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation of theconcentration of low- density lipoprotein cholesterol without theuse of preparative ultracentrifuge. Clin Chem 18:499–502

Garrat DC (1964) The quantitative analysis of drugs. Chapman andHall, Japan

Ghani A (2003) Medicinal plants of Bangladesh with chemicalconstituents and uses. Asiatic Society of Bangladesh, Dhaka

Ghule BV, Ghante MH, Saoji AN, Yeol PG (2009) Antihyperlipidemiceffect of the methanolic extract from Lagenaria siceraria Standfruit in hyperlipidemic rats. J Ethnopharmacol 124:333–337

Hideo O, Akio M, Yasuko Y, Mitsuru N, Yoshimasa T (1993)Antioxidant activity of tannins and flavonoids in Eucalyptusrostrata. Phytochemistry 33:557–561

John KS, Kathiravan MK, Somani RS, Shishoo CJ (2005) The biologyand chemistry of hyperlipidemia. Bioorg Med Chem 15:4674–4699

Johnston TP (2004) The P-407-induced murine model of dose-controlled hyperlipidemia and atherosclerosis. J CardiovascPharmacol 43:595–606

Johnston TP, Plamer WK (1993) Mechanism of poloxamer 407-induced hypertriglyeridemia in rat. Biochem Pharmacol46:1037–1042

Kannel WB, Castelli WP, Gordon T (1979) Cholesterol in predictionof atherosclerotic disease. New perspectives based on theFramingham study. Ann Intern Med 90:85–91

Karthikeyan M, Deepa MK (2008) Antihyperlipidemic activity ofPremna corymbosa in liver damaged Wistar Albino rats. J PharmRes 1:61–63

Liu J, Chen XF, Xia L, Geng XZ, Li ZS (1987) Effect of oleanolic acid onblood lipid metabolism in rats. Chinese Pharm Bull 4:14–15

Marja PK, Anu IH, Heikki JV, Jussi-Pekka R, Kalevi P, Tytti SK,Marina HJ (1999) Antioxidant activity of plant extracts containingphenolic compounds. J Agric Food Chem 47:3954–3962

Ock KC, Dae-Ok K, Chang YL (2003) Superoxide radical scavengingactivity of the major polyphenols in fresh plums. J Agric FoodChem 51:8067–8072

Parthasarthy S, Steinberg D, Seitztum JL (1992) The role of low-density lipoprotein in the pathogenesis of atherosclerosis. AnnRev Med 43:219–225

Patel DK, Patel KA, Patel UK, Thounaoja MC, Jadeja RN, AnsarullahPGS, Salunke SP, Devkar RV, Ramachandra AV (2009) Assess-ment of lipid lowering effect of Sida rhomboidea. Roxbmethanolic extract in experimentally induced hyperlipidemia. JYoung Pharma 1:233–238

Ross R (1993) The pathogenesis of athrosclerosis: a perspective forthe 1990s. Nature 362:801–809

Samer M, Fugen A, Neal MD, Basil DR (2005) Phytopreventiveantihyperlipidemic effects of Gynostemma pentaphyllum in rats. JPharm Pharmaceut Sci 8:507–515

Srikanth J, Muralidharan P (2009) Antihyperlipidemic activity ofSapindus emarginatus in Triton WR-1339 induced albino rats.Research J Pharm Tech 2:319–323

Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL (1989)Beyond cholesterol: modification of low-density lipoprotein thatincreases its atherogenecity. N Engl J Med 320:915–924

Tanner M, Bu X, Steimle J, Myers P (1999) The direct release of nitricoxide by gypenosides derived from the herb Gynostemmapentaphyllum. Nitric oxide 3:359–365

Trease GE, Evans WC (1989) A text book of pharmacognosy. BrailliarTindall Ltd., London

Uddin SN (2006) Traditional uses of ethnomedicinal plants of theChittagong Hill Tracts. Bangladesh National Herbarium, Dhaka

Woodson RF (1987) Statistical methods for the analysis of biochemicaldata. Wiley, Chichester

Wout ZG, Pec EA, Maggiore JA, Williams RH, Palicharla P, JohnstonTP (1992) Poloxamer-407 mediated changes in plasma choles-terol and triglycerides following intraperitoneal injection to rats. JParenter Sci Technol 46:192–200

Yogendrasinh BS, Sunita MJ (2010) Antihyperlipidemic activity ofClitoria ternatea and Vigna mungo in rats. Pharm Biol 48:915–923

Zalba G, San JG, Moreno MU, Fortuno A, Beaumont FJ, Diez J(2001) Oxidative stress in arterial hypertention. Role of NADPHoxidase. Hypertention 38:1395–1399

Zheng W, Wang SY (2001) Antioxidant activity and phenoliccompounds in selected herbs. J Agric Food Chem 49:5165–5170



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