Hypolipidemic and Antioxidant Activity of Anthocephalus Indicus ( Kadam) Root Extract. Indian Journal of Biochemistry & Biophysics

8/11/2019 Hypolipidemic and Antioxidant Activity of Anthocephalus Indicus ( Kadam) Root Extract. Indian Journal of Biochemi

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Indian Journal of Biochemistry & BiophysicsVol. 47, April 2010, pp. 104-109

Hypolipidemic and antioxidant activity ofAnthocephalus indicus

(Kadam) root extract

Vishnu Kumar1, Farzana Mahdi

1, Ramesh Chander

1, Ranjana Singh

2, Abbas Ali Mahdi

2, Ashok Kumar Khanna

3,

Sanjay Bhatt4, Rajeev Singh Kushwaha

4, Kalbe Jawad

5, Jitendra Kumar Saxena

3and Raj Kumar Singh

4*

1Department of Biochemistry, Eras Lucknow Medical College & Hospital, Lucknow 2260012Department of Biochemistry, CSM Medical University, Lucknow 226003

3Division of Biochemistry, Central Drug Research Institute, Lucknow 2260034Department of Biochemistry, Shri Guru Ram Rai Institute of Medical & Health Sciences, Patel Nagar, Dehradun 248001

5Department of Biochemistry, UP Rural Institute of Medical Sciences & Research, Saifai, Etawah, UP 206301

Received 06 November 2009; revised 03 March 2010

The present study was carried out to explore the anti-diabetic, anti-dyslipoproteinemic and anti-oxidant activities ofAnthocephalus indicus root extract in alloxan-induced (150 mg/kg body wt.) diabetic rats. A marked increase in plasmalevels of glucose and lipid peroxides accompanied with an elevation in the lipids and apoprotein levels of serum very lowdensity lipoprotein (VLDL) and low density lipoprotein (LDL) following decrease in lipid and protein constituents of highdensity lipoprotein (HDL) were observed. The alterations in lipoprotein pattern was associated with inhibition of lipolytic

and antioxidant enzymes. Oral administration of root extract (500 mg/kg body wt.) for 30 days in dyslipidemic animalsresulted in significant decrease in plasma glucose, total cholesterol, phospholipids, triglyceride and lipid peroxides. Thedecrease of lipids and apoprotein levels of VLDL and LDL were followed by stimulation of plasma post-heparin lipolyticactivity and lecithin cholesterol acyltransferase as well as hepatic superoxide dismutase and catalase activities. Lipid andapoprotein levels of HDL were also recovered partially on treatment with root extract.

Keywords: Cholic acid, Deoxycholic acid, Lipid peroxides, Superoxide dismutase, Catalase, Lipoprotein lipase,Triglyceride lipase, Lecithin cholesterol acyltransferase.

Anthocephalus indicus (Family: Rubiaceae) iscommonly known as Kadam and its roots, bark,leaves and fruits have been used in Ayurvedic remedyfor skin diseases and metabolic disorders and alsomentioned in many other ancient Indian medical texts

to possess antidiarrheal, detoxifier, analgesic andaphrodisiac properties

1,2. The plant is found

abundantly throughout India, especially at lowaltitude and in humid places. In traditional system ofmedicine, warm aqueous extract of leaves has beenused to alleviate the pain, swelling and for cleansing

and healing of wounds and in treatment of

menorrhegia. The decoction of bark is effective indiarrhea, dysentery and colitis. The root extract ishelpful in urinary ailments like dysuria, calculi andglycosuria.

The heartwood and leaves contain 3 and 3

isomers of dihydrocadambine3,4

and stem bark

cadambagic acid, quinovic acid and sitosterol5.

A complex polysaccharide from flower and seeds has

also been isolated6. The above-mentioned compound

(cadambagic acid, quinovic acid, sitosterol, 3 and

3 isomers of dihydrocadambine) of the plant may be

responsible for its antidiarrheal, detoxifier, analgesicand aphrodisiac properties. Furthermore, fruit juice

augments the quality of breast milk of lactating

mothers and act as a lactodepurant7. Recently, we

reported that alcoholic extract ofA. indicusroot exerts

hypoglycemic activity in alloxan-induced diabetic rats

and hypolipidemic activity as well as anti-oxidant

activity8-10.

Diabetes mellitus (DM) is a chronic disease caused

by the inherited and/or acquired deficiency in

production of insulin by pancreas or by the

*Corresponding authorE-mail: [email protected]

Tel: +91-135-2522168 (0) ; Fax: +91-135-2522117.Abbreviations: CAT, catalase; DM, diabetes mellitus; FFA, freefatty acid; HDL, high density lipoprotein; LCAT, lecithincholesterol acyltransferase; LDL, low density lipoprotein; LPL,

lipoprotein lipase; LPL, lipoprotein lipase activity; LPO, lipidperoxides; PHLA, post-heparin lipolytic activity; PL,phospholipids; SOD, superoxide dismutase; TC, total cholesterol;TG, triglyceride; TGL, triglyceride lipase; VLDL, very low

density lipoprotein.


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KUMAR et al.: HYPOLIPIDEMIC AND ANTI-OXIDATIVE EFFECT OFANTHOCEPHALUS INDICUS ROOT 105

ineffectiveness of the insulin produced, which leads tohyperglycemia and at later stages lipid metabolism is

also affected11,12. DM in human and experimental

models also increased oxidative stress due topersistent and chronic hyperglycemia13

, and protein

glycation14

due to depletion of antioxidant defensesystem15, thus promotes de novo free radical

generation16. Current therapies used for controlling

diabetic complications are associated with several

side effects17

. Moreover, as synthetic antioxidants are

suspected to be carcinogenic18

, there is a need foreffective, safe and better oral hypoglycemic agents.

Herbal formulations are preferred due to lesser side

effects and their low cost. One of the etiologic factors

implicated in the development of diabetes and its

complications is the damage induced by free radicals.Thus, a drug having multi-fold properties such as anti-

diabetic, lipid lowering and antioxidant activities is in

great demand. Therefore, in the present study, an

attempt has been made to explorehypolipidemic and

antioxidant activities of Anthocephalus indicus

(Kadam) root extract.

Material and MethodsAlloxan monohydrate and standard drug

glibenclamide were procured from Sigma Chemical

Co., St. Louis, MO, USA.

Preparation of root extract

A. indicus (secondary and tertiary) roots were

collected from local area of Lucknow and identified

taxonomically by the Department of Pharmacology,

Eras Lucknow Medical College & Hospital,

Lucknow and a voucher specimen was also submitted

to Department of Pharmacology, Eras Lucknow

Medical College & Hospital, Lucknow (AI-001/06).

Roots were dried under shade and made into fine

powder using laboratory mill and powder (250 g) and

extracted thrice with 1500 ml portions of 95% ethyl

alcohol in a laboratory percolator at roomtemperature9. Time allowed for each extraction was

8 h. The root extract obtained after third extraction

was colorless. All the extracts were mixed (total

amount of solvent of 3 extractions were 1500 ml),alcohol was distilled out at reduced temperature

(20C) and reduced pressure (100 psi) in a rotorevaporator. The whole mass was then taken out in a

pre-weighed beaker and subjected to vacuum drying

for 6 h which yielded 16 g of crude extract, which

was used for in vivoand in vitrostudies.

Animals and treatment

Animal study was performed with the approval ofAnimal Care Committee of Division of Laboratory

Animal, Central Drug Research Institute, Lucknow,

India and confirmed to the guidelines for Care and

Use of Laboratory Animals of the Institute. Male

adult rats of Charles Foster strain weighing200-225 g bred in the animal house of the Institute

were used. The rats were divided into four groups

having six animals each as follows: Group 1:

Control rats; Group 2: Diabetic rats (on normal

saline); Group 3: Diabetic rats + A. indicus

(500 mg/kg b.w.)8-10

; Group 4: Diabetic rats +

Glibenclamide (600 g/kg b.w.)20. After 30 days of

feeding rats were fasted overnight and blood was

withdrawn from the retro-orbital plexus. A group of

normal rats without treatment with alloxan were alsoincluded to serve as control. These animals were

housed in polypropioline cages and kept in uniform

hygienic conditions, temperature 25-26C, relativehumidity 60-70% and 12/12 h light/dark cycle (light

from 08:00 to 20:00) and provided with standard

pellet diet (Lipton India Ltd.), and water ad libitum.

Diabetes was induced in rats by a singleintraperitonial injection of alloxan monohydrate

150 mg/kg b.w. in 18 animals. After 2 weeks, rats

with serum glucose level 280-367 mg/dl were taken

for the study19

.

Biochemical analysis

Serum from above rats was fractionated into very

low density lipoprotein (VLDL), low density

lipoprotein (LDL) and high density lipoprotein (HDL)

by polyanionic precipitation method21. Serum as well

as lipoproteins were analyzed for their total

cholesterol (TC)22

, triglyceride (TG)23

, phospholipids

(PL)24 and apoprotein25. Serum lipid peroxides

(LPO)26, free fatty acid (FFA)27, plasma protein28,

plasma lecithin cholesterol acyl transferase (LCAT)

activity29

and PHLA30

were also estimated.

Liver homogenized (10% w/v) in cold 1 Mphosphate buffer (pH 7.2) was used for the assay of

lipoprotein lipase (LPL)30 and triglyceride lipase

(TGL)31 activities. Liver homogenate 10% w/v in

0.15 M KCl was also used for the estimation of

superoxide dismutase (SOD)32

and catalase (CAT)33

.

The lipid extract of each homogenate was used for the

estimation of TC, PL and TG by above-mentioned

methods. The rats faeces spilling from all groups over

30 days were collected and processed for the

quantification of cholic and deoxycholic acid34

.


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INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL 2010106

Statistical analyses

One-way-analysis of variance (ANOVA-Newmans student test) was performed by

comparison of values for alloxan-treated group with

control, alloxan and drug-treated with alloxan only.

All hypothesis testing were two-tailed. P


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HDL-TC, PL, apoprotein (25% 3.86, 24% 6.84,27% 3.69 respectively; P


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INDIAN J. BIOCHEM. BIOPHYS., VOL. 47, APRIL 2010108

DiscussionAlloxan selectively damages the -cells and

suppresses the production of insulin in pancreas, thus

is used to induce diabetes in laboratory animals. Thisoccurs most likely because of selective uptake of the

alloxan, due to its structural similarity to glucose aswell as highly efficient uptake mechanism in -cells.

However, alloxan is not toxic to human -cells, even

in very high doses, probably due to differing glucose

uptake mechanisms in human and rodents36

.

In our experiment, we observed higher levels of

serum lipids in alloxan-treated diabetic rats. The levelof serum lipids is usually raised in diabetes and such

an elevation represents a risk factor for cardiovascular

diseases37

. Lipases play a significant role in

lipoprotein metabolism and decreased lipoproteinlipases activities in diabetes is main cause ofatherosclerosis38. In case of DM, PHLA, serum LPL,

HTGL, sciatic nerve LPL and adipose tissue LPL

activities have been found to decrease39. On the other

hand, pancreatic lipase and co-lipase activities

increase in pancreatic acinar tissues of diabetic rats40

.In the present study, activities of SOD and CAT were

found to decrease in alloxan-induced diabetic rats and

similar observations were reported by other workers41.

The treatment with A. indicus recovered the activity

of these antioxidant enzymes in alloxan-induced

diabetic rats.The abnormal high concentration of serum lipid in

diabetes is mainly due to the increase in the

mobilization of free fatty acid from the peripheral

depots, since insulin inhibits the hormone sensitive

lipase42

. On the other hand, glucagon, catecholamine

and other hormones enhance lipolysis. The marked

hyperlipidemia that characterizes the diabetic state

may, therefore, be regarded because of the

unregulated actions of lipolytic hormones on the fat

depots42

. In the present study, we also observed higher

levels of blood glucose, TC, TG, PL and FFA in

alloxan-induced diabetic rats. Similar results alsohave been reported by other workers in alloxan-

induced diabetic rats19

.

A. indicus root and glibenclamide both caused asignificant decrease in the plasma levels of TC, TG,

PL and FFA in alloxan-induced hyperglycemia. In

alloxan-induced diabetic rats,A. indicus increased the

level of HDL by stimulating the activity of LCAT,

which might contribute to the regulation of blood

lipids. LCAT play a key role in lipoprotein

metabolism and most of the lipoprotein changes are

the outcome of primary abnormality owing to thediseases related with lipid metabolism43. A. indicus

enhanced the excretion of bile acids through feces and

this contributed to regress the cholestesteosis in liverdamage.

In conclusion, the lipid lowering activity of

A. indicus might be due to inhibition of hepatic

cholesterol biosynthesis, activation of tissue lipases,

SOD, and CAT. These beneficial effects might be due

to bioactive compounds like typical alkaloids,

quinovich acid, cadambine and its derivatives presentin theroot

8-10.

AcknowledgementOne of us (V K) is grateful to Director, CDRI,

Lucknow for experimental support and Eras

Lucknow Medical college, Lucknow for financial

support.

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Hypolipidemic and Antioxidant Activity of Anthocephalus Indicus ( Kadam) Root Extract. Indian Journal of Biochemistry & Biophysics