2014 JOURNAL OF MEDICAL RESEARCHVol.1 No.1Vol.1Jul.–Dec. 2014ERA’S JOURNAL OF MEDICAL RESEARCHJul.–Dec. No.1ERA’S
HYPolIPIDEMIC ACTIVITY oF CASSIA
ToRA SEEDS IN HYPERlIPIDEMIC RATS
Vishnu Kumar1, Brijesh Rathore,1 Sharique Ahmad1 , Farzana Mahdi, 1, Ramesh Chander1,
Ashok Kumar Khanna 2, Jitendra Kumar Saxena 2 Abbas Ali Mahdi3 Pradyumn Singh4 and Raj Kumar Singh5
Department of Biochemistry & Pathology, Era’s Lucknow Medical College, Division of Biochemistry, Central Drug Research Institute and
Department of Biochemistry, ( KGMC ), Lucknow– 226003, India, Department of Biochemistry, Shri Guru Ram Rai Institute of Medical &
Health Sciences, Patel Nagar, Dehradun 248001. 4Department of Pathology CMC, Vellore. 5Department of Biochemistry,
Shri Guru Ram Rai Institute of Medical & Health Sciences, Patel Nagar, Dehradun 248001.
The hypolipidemic activity of Cassia tora (Chakvat, Chakunda) (Family: Caesalpiniaceae) seeds
extract has been studied in two hyperlipidemic models of rat. These are triton injected and Address for correspondence
. Vishnu Kumar Awasthi
cholesterol rich HFD fed model of hyperlipidemia. In triton WR–1339 induced hyperlipidemia, DrAssistant Professor
feeding with root extract (500 mg/ kg body wt/ day p.o. ) exerted lipid lowering effect as Department of Biochemistry
assessed by reversal of plasma levels of total cholesterol (TC), phospholipids (PL), triglyceride Address:Era’s Lucknow Medical
ollege & Hospital
(TG) and reactivation of Post Heparin Lipolytic Activity (PHLA) of plasma. The other model CSarfarazganj, Hardoi Road,
was fed with cholesterol rich HFD and seeds extract of Cassia tora (500 mg/ kg body wt/ day Lucknow–226003
p.o.) simultaneously for 30 days. This also caused lowering of lipid levels in plasma and liver Ph No : +91895358976
homogenate and reactivation of plasma post heparin lipolytic activity, hepatic total lipoprotein E–m
lipase activity. The hypolipidemic activity of Cassia tora seeds was compared with a standard
drug guggulipid (200 mg/ kg body wt/ day p.o.), a known lipid lowering drug in both models.
Key Words: Cassia tora seeds, Triton model of hyperlipidemia, Cholesterol rich HFD,
Hypolipidemic agent, PHLA, Hepatic LPL activity, Hepatic steatosis.
Cassia tora (Chakvat, Chakunda) Cassia tora is a plant of The plant is claimed to be effective against a variety of ailments
family Caesalpiniaceae. Chakvat grows throughout India in indigenous medicine such as in treatment of jaundice.
especially on way sides and waste places, on hills of low In Chinese medicine, it is highly valued for the treatment
elevations up to 1,800 m as well as in plains. Plant of Cassia of hyperlipidemia. Several polyherbal, formulations are
tora is a herbaceous foetid annual weed, almost an under available in Chinese market for preventing the formation
shrub, up to 90 cm in height; leaves pinnately compound, of atherosclerosis plaque (7). The aim of present work is to
rachis grooved with a conical gland between each of the two further evaluate the hypolipidemic activity of cassia tora
lowest pairs of leaflets, leaflets three pairs, obviate–oblong, which grows as a common weed in India.
membranous, base somewhat oblique, main nerves 8–10
pairs; flowers yellow, sub sessile pairs, in the axils of the Cardiovascular diseases are leading cause of death in both
leaves, the upper ones crowded, stamen serven, perfect and industrialized and developing nations. Disorders of lipid
three staminodes; fruits subtetragenous obliquely septate metabolism following oxidative stress are the prime risk
pods, 15–23 cm long, the sutures very broad, rhombohedral, factors for initiation and progression of these diseases
23–30 per pod. The leaves and seeds of these plants are (8). The known lipid lowering drugs such as fibrates,
used for medicinal purpose. The leaves and seeds are acrid, statins, bile acid sequestraints have many side effects in
thermogenic, laxative depurative, antiperiodic, liver tonic, patients (9). Therefore, the research and development
antihelmintic, cardiotonicEJMR and are useful in ringworm, of lipid lowering drugs from natural products are the
pruritis, leprosy, skin disease, hepatopathy, helminthiasis, best option and also are in great demand. In view of the
flatulence, dyspepsia, intermittent fevers, constipation, above considerations, the present study was designed to
ophthalmopathy, cough, bronchitis, cardiac disorders, investigate hypolipidemic activity of Cassia tora seeds in
haemorrhoids, antifungal, hypolipidemic, hepatoprotective, hyperlipidemic rats.
and hypotensive activities (1– 6).
67 2014 JOURNAL OF MEDICAL RESEARCHVol.1 No.1Vol.1Jul.–Dec. 2014ERA’S JOURNAL OF MEDICAL RESEARCHJul.–Dec. No.1ERA’S
MATERIAlS AND METHoDShyperlipidemia was produced by feeding with cholesterol rich
Preparation of root extractHFD for 30 days. Drugs were administered orally at the same
doses as above simultaneously with cholesterol rich HFD in
Cassia tora seeds were collected from local area of the drug treated groups. Control animals received the same
Lucknow and identified taxonomically by Department of amount of vehicle. At the end of experiment, rats were fasted
Pharmacology, Era’s Lucknow Medical College, Lucknow. overnight, anaesthetized with thiopentone solution (50mg/
A voucher specimen (CT–005/10) was also submitted. Seeds kg body wt/day i.p.), prepared in normal saline. Blood was
were crushed and dried under shade. The powder (500g) withdrawn from retro–orbital plexus using glass capillary in
was extracted with 95 % ethanol (10) in a soxhlet extractor EDTA coated tubes (3mg/ ml blood). There after animals
for 72 h, the extract was concentrated to dryness under were sacrificed liver was excised immediately washed with
reduced pressure and controlled temperature (50–60°C), cold 0.15 m KCl and kept it – 40 oC till analyses. Blood was
yielding 23g of reddish brown solid (crude extract). This was centrifuged and plasma was taken (10).
stored in refrigerator and used to investigate hypolipidemic
activity in rats. Guggulipid, a potent lipid lowering agent from Biochemical analysis of plasma and liver
Commephora mukul (Guggulipid) developed in Central Drug Plasma Post heparin lipolytic activity (PHLA) was assayed in
Research Institute, Lucknow, was used as a standard drug.plasma spectrophotometrically using Intralipid as artificial
Preparation of Cholesterol rich high fat dietsubstrate (11) plasma was diluted with normal saline in
a ratio of 1:3 and used for the analysis of total cholesterol
Deoxycholic acid (5g) was mixed thoroughly with 700g of TC (12), phospholipids PL (13) and triglyceride TG (14)
powdered rat Chow diet supplied by Ashirvad Industries, using standard enzymatic kits supplied by Merck India Ltd.
Chandigarh, India. Ingredient and nutrient composition Mumbai India. Liver was homogenized (10%w/v) in cold
of the normal rat diet was: casein 210; corn starch 440; 100mM phosphate buffer pH 7.2 and used for the assay of
sucrose 100; maltose dextrin 100; cellulose 50; soya bean oil total Lipolytic; the lipoprotein lipase (LPL) activity (11). The
50; vitamin mix 10 and minerals 35g/kg. Other Ingredients lipid extract of each homogenate prepared in a mixture of
included choline bitrate (2g/kg) and t–butyl hydroquinone CHCl3: CH3OH (2:1, v/v) was used for estimation of TC (12),
(0.008 g/kg). Proximate analysis of diet showed it contained PL (21) and TG (14). Plasma and tissue were also estimated
crude protein 21; crude fat 5; crude fiber 4; and ash 8%. for protein content (15).
Simultaneously Cholesterol (5g) was dissolved in 300g warm
coconut oil. This oil solution of Cholesterol was added slowly STATISTICAl ANAlYSIS
into powdered mixture to obtain homogeneous soft cake. One way analysis of variance (ANOVA–New man’s
This Cholesterol rich (HFD) was molded in shape of pellet of student t– test) was performed by comparison of values for
about 3g each (10).hyperlipidemic groups with control, hyperlipidemic and
Animalsdrug treated groups with hyperlipidemic All hypothesis
testing were two–tailed. P<0.05 was considered statistically
In vivo experiments were conducted as per guidelines significant and results were expressed as mean ± SD of six
provided by Animal Ethics Committee of Central Drug rats. The graph pad INSTAT 3.0 software carried out the
Research Institute, Lucknow, India. Male adult rats of Charles statistically analysis. (16).
foster strain (200–225g) bred in animal house of the Institute
were used. The animals were housed in polypropioline cages RESUlT S
and kept in uniform hygienic conditions, temperature 25–26 Effect of Cassia tora seeds extract in triton induced
°C, relative humidity 50–70% and 12/12 h light/dark cycle hyperlipidemia
(light from 8:00 a.m.to 8:00 p.m.) and provided with standard
rat pellet diet and water ad libitum (10).The data in Table 1 shows that in acute administration of
triton WR–1339 in rats caused marked increase in their
Triton and Cholesterol rich HFD induced hyperlipidemiaplasma levels of TC (2.86 fold), PL (2.76 fold) and TG (2.68
The rats were divided into four groups: control, hyperlipidemic, fold) following inhibition of PHLA by 26%.Treatment with
hyperlipidemic treated with Cassia tora seeds or guggulipid Cassia tora seeds extract exerted a decrease in these levels
(standard drug). Containing six animals in each group. In of TC (25%), PL (26%), TG (28.0 %) simultaneously with
the acute experiment to induce hyperlipidemia, triton WR–reactivation of PHLA by (22%). The hyperlipidemic action
1339 (Sigma Chemical Company, St.Louis, MO, USA) was of guggulipid (31–35 %) was comparatively higher to that of
administered (400 mg/ kg body wt/ day p.o.) by intraperitonial Cassia tora seeds extract.EJMR
injection. Cassia tora seeds extract and guggulipid were Effect of Cassia tora seeds extract in cholesterol rich HFD
macerated with aqueous gum accacia (0.2 % w/v) suspension induced hyperlipidemia
and fed orally at the doses of 500 and 200 mg/ kg body wt/
day p.o.), respectively, simultaneously with triton and blood In this model of hyperlipidemia (table–2), feeding with
was collected after 18 hrs (10). In the chronic experiment, cholesterol rich HFD in rats caused marked increase in their
89 2014 JOURNAL OF MEDICAL RESEARCHVol.1 No.1Vol.1Jul.–Dec. 2014ERA’S JOURNAL OF MEDICAL RESEARCHJul.–Dec. No.1ERA’S
plasma levels of TC (2.34 fold), PL (1.71 fold) and TG (2.18 is compared with control, cholesterol rich HFD and drug
fold) following inhibition of PHLA by (32%). Treatment with treated groups with cholesterol rich HFD. *P<0.05; **P<0.01;
Cassia tora seeds extract for 30 days, reversed these plasma ***P<0.001.
levels of TC (25%), Pl (21%) and TG (31%) simultaneously
with reactivation of PHLA by (21.0%). Feeding with Table 3: Effect of Cassia tora (seeds) extract and guggulipid
cholesterol rich HFD in rats also caused marked accumulation on liver lipid in Cholesterol Rich HFD induced
of TC (1.53 fold), PL (1.72 fold) and TG (1.57 fold) following hyperlipidemia.
diminution of LPL activity (37%) in their liver (Table–3). Experimental Tot a l Phospholipid Triglyceride lPl activity(µ mol
However, treatment with Cassia tora seeds extract decrease Schedulecholesterol (mg/dl)(mg/dl)Free Fatty Acid
in these levels of TC.(mg/dl)released/h/mg
Effect of Cassia tora seeds extract in cholesterol rich HFD Control6.75±0.30 22.43±2.71 5.44±0.60 79.85±4.82
diet induced steosis in liveCholesterol 10.33***±0.6238.58***±5.338.56***±1.0250.26***±2.72
(26%), PL (22%) and TG (29%) was observed following rich HFD (1.553 fold)(1.72 fold)(1.57 fold)(–37%)
reactivation of LPL activity (26%) in hyperlipidemic animals. treated
Guggulipid was more effective hypolipidemic to that of Cholesterol 7.67***±0.622940***±3.605.22***±0.7463.09*±3.75
Cassia tora seeds.richCassia HFD+ tora (–25.75%)(–22.29%)(–28.62%)(+25.52%)
Table 1: Effect of Cassia tora (seeds) extract and guggulipid (seeds)
on plasma lipids in triton induced hyperlipidemiaextract
ExperimentalTotal cholesterolPhospholipidTriglyceridePHlA rich HFD (–28.75%)(–26.20%)(–31.19%)(–29.30%)
Schedule(mg/dl)(mg/dl)(mg/dl)(n mol Free +Guggulipid
released Values are mean ± SD of 6 animals Values in the parenthesis
/h/l)indicate percent change, cholesterol rich HFD treated groups
Control82.46±3.33 90.66±8.69 87.52±6.11 16.86±0.95were compared with control cholesterol rich HFD and drug
Triton 235.89***±12.09 250.57***±24.86 234.60***±12.09 11.56**±1.34treated groups with cholesterol rich HFD. *P<0.05; **P<0.01;
treated(+2.86 fold) (2.76 fold) (2.68 fold) (–25.50 %)
Triton + 177.89***±8.69 185.88**±12.80 169.87***±6.14 14.11*±0.70
Cassia tora (–24.58%) (–25.81%) (–27.59%) (+22.56%)DISCUSSIoN
extractTriton WR–1339 acts as a surfactant and suppresses the
Triton + 159.14***±7.85 149.14***±8.23 153.13***±8.65 14.36*±0.68action of lipases to block the uptake of lipoproteins from
Guggulipid(–32.53%) (–32.49%) (–32.72%) (+24.22%)circulation by extra hepatic tissues, resulting into increased
Values are mean ± SD of 6 animals. Values in parenthesis blood lipid concentration (10, 17). The lipid lowering
indicate percent change. Triton treated group is compared effect of Cassia tora seeds extract may be due to an early
with control, triton and drug treated with triton *P<0.05; clearance of lipids from circulation in Triton model and it
**P<0.01; ***P<0.001.may be due to reactivation of lipolytic enzymes as evidenced
by increased PHLA. Investigations with cholesterol rich
Table 2: Effect of Cassia tora (seeds) extract and guggulipid HFD fed hyperlipidemic animals showed that seed extract
on plasma lipids in Cholesterol Rich HFD induced stimulates PHLA and hepatic LPL activity, both of which
hyperlipidemia.play a key role in lipid catabolism and their utilization in
ExperimentalTotal cholesterolPhospholipidTriglyceridePHlA ( n mol the body (18), leading to decrease in the level of plasma
Schedule(mg/dl)(mg/dl)(mg/dl)Free Fatty Acid and liver lipids in above models. It is reported that
released /h/l )hypolipidemic action of guggulsterone, the active principle
Control88.59±6.98 80.62±4.29 114.80±11.30 19.31±1.30of guggulipid, is mediated through activation of PHLA, LPL
Cholesterol 208.18***±22.10138.27***±24.86251.35***±24.5913.11***±1.18and LCAT, inhibition of hepatic cholesterol biosyntheses
rich HFD (2.34 fold)(1.71 fold)(2.18 fold)(–32%)and increased faecal bile acid excretion (19, 20, and 21). The
treatedsame mechanism may also interplay in the hypolipidemic
rich HFD + (–25.37%)( –21.26%)(–31.21%)(+21.12%)effect of Cassia tora seeds extract.
Cassia tora Here we have tested crude extract of Cassia tora seeds which
however, upon research and development, may produce a more
Cholesterol 153.39***±8.83100.30***±8.77169.92***±6.0916.33 *±1.64
rich HFD+ (–26.31%)(–27.46%)(–32.72%)(–24.56%)potent lipid lowering natural product or a pure compound
Guggulipidlike guggulipid/ guggulsterone from commephora mukul
Values are mean ± SD 0f 6 animals. Values in the parentheses (22).biological Further activity work in vivo on drug and metabolism in vitro Cassia and assessment tora seeds and the
indicate percent change. Cholesterol rich HFD treated group
its fraction is in progress to substantiate the present findings.
89 2014 JOURNAL OF MEDICAL RESEARCHVol.1 No.1Vol.1Jul.–Dec. 2014ERA’S JOURNAL OF MEDICAL RESEARCHJul.–Dec. No.1ERA’S
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