To reduce the rate of mortality, it is therapeutically recommended to undergo diet control or/and drug therapy to lower lipid levels within the normal range. Allopathic hyperlipidemia drugs are available at large scale in the market but the side effects and contraindications of these drugs have marred their popularity (6). The herbal hypolipidemics have gained importance to fill the lacunae created by the allopathic drugs. Management of hyperlipidemia without any side effects is still a challenge to the medical system. Although many efficacious lipid lowering drugs exist, none is effective for all lipoprotein disorders, and all such agents are associated with some adverse effects. Plant products are frequently considered to be less toxic and more free from side effects than synthetic ones. A number of plants have been found to be useful in hyperlipidemia and been identified as hypolipidemics in Ayurveda (7). Paspalum scrobiculatum is one plant which has been shown to have hypolipidemic property in previous experiments on alloxan induced diabetes in rats (8). Though these studies show hypolipidemic activity of Paspalum scrobiculatum in diabetic rats, there are no reports available on its antihyperlipidemic activity in non diabetic rats.

The aim of the present study was to examine the possible antihyperlipidemic activities of hydroalcoholic extract of the grains of Paspalum scrobiculatum in high fat induced hyperlipidemia

MATERIALS AND METHODS:

Grains of Paspalum scrobiculatum were collected from the Kodo millet fields in Podili, Andhra Pradesh in the month of December 2012, identified and authenticated by Dr.M.V.C Gowda Project Co-ordinater, AICRP on Small Millets, ICAR, UAS, GKVK, Bangalore, Karnataka, India.

Preparation of Grains Extract

The fresh grains were collected, cleaned and shade dried at room temperature. The dried grains were coarse powdered by using grinder. The coarse powder was packed in Soxhlet column and then extracted with 70% hydro-alcohol (75-80^oC). Thereafter, the extract was concentrated using rotary flash evaporator (50^o C) (9).

Preliminary phytochemical qualitative analysis

The extract of Paspalum scrobiculatum grains was prepared and subjected to qualitative test for identication of various plant constituents (10).

Animals

Albino wistar rats weighing 150-220g were procured from Biogen, Bangalore. They were maintained in the animal house of Gautham College of Pharmacy. Animals were maintained under controlled condition of temperature at 27^o ± 2^oC and 12-hr. light-dark cycles. They were housed in polypropylene cages and had a free access to standard pellets (Amruth) and water ad libitum.

All the studies conducted were approved by the Institutional Animal Ethical Committee (IAEC) of Gautham College of Pharmacy, Bangalore (REF-IAEC/03/06/2012-13) according to prescribed guidelines of Committee for the Purpose of Control and Supervision of experiments on Animals (Reg No: 491/01/c/CPCSEA), Govt. of India.

Determination of acute oral toxicity (LD50)

Female Albino rats of weighing 160-220g were used for the study. They were nulliparous and non-pregnant. These were acclimatized to laboratory condition for one week prior to start of dosing.

Preparation of dose

Hydro-alcoholic extract of Paspalum scrobiculatum (HAPS) was dissolved in suitable solvent, to prepare a dose of 2000 mg/kg. The doses were selected according to the OECD guideline no. 425.

Procedure

The procedure was divided into two phases. Phase I (observation made on day one) and Phase II (observed the animals for next 14 days of drug administration). Two sets of healthy female rats (each set of 3 rats) were used for this experiment. First set of animals were divided into three groups, each of one in a group. Animals were fasted overnight with water ad libitum. Animals received a single dose of 2000 mg/kg, b.w. p.o. was selected for the test, as the test item was a source from herb. After administration of extract, food was withheld for 3-4 hrs. If the animal dies, conduct the main test to determine the LD₅₀. If the animal survives, dose four additional animals sequentially so that a total of five animals are tested. However, if three animals die, the limit test is terminated and the main test is performed. The LD₅₀is greater than 2000 mg/kg, b.w. p.o, if three or more animals survive. If an animal unexpectedly dies late in the study, and there are other survivors, it is appropriate to stop dosing and observe all animals to see if other animals will also die during a similar observation period. Late deaths should be counted the same as other deaths. The same procedure was repeated with another set of animals to nullify the errors (11).

Evaluation ofantihyperlipidemic activity (12)

Preparation of extract dose:

Accurately 6 gm of HAPS was weighed and suspended in 30 ml of distilled water using tween 80 and thus formed suspension is sonicated 10 min at medium vibration to obtain uniform suspension. Each ml of the suspension contained 5 mg/ml.

High fat induced hyperlipidemia¹⁷

Experimental animals

Albino wistar rats weighing 160-220g were divided into six groups of six in each group.

Materials and methods

High fat composition: Commercially available edible dalda (vanaspathy) and culinary grade coconut oil were obtained from local market. The high fat diet (HFD) was prepared by homogenously mixing dalda and coconut oil in the ratio of 3:2 w/w.

Induction of hyperlipidemia

Group I animals were administered with 10ml distilled water per kg body weight orally once daily for a period of four weeks by oral gavaging technique and served as negative control. For the Group II, III, IV, V, and VI in addition to normal diet and water prepared high fat diet was administered by gavaging to induce hyperlipidemia. HFD was gavaged at the dose rate of 10ml per kg body weight to each animal orally daily for a period of four weeks.

Treatment protocol

Once the hyperlipidemia was induced between 0 to 4^th week of the experiment, from the beginning of fifth week to the end of the eighth week, the HAPS treatment was carried out.

Group-I: Distilled water was administered and served as negative control.

Group-II: Distilled water was administered and served as positive control

Group-III: Standard drug (Fenofibrate 200mg/kg, b.w. p.o.) was administered.

Group-IV: HAPS was administered at a dose rate of 200mg/kg, b.w. p.o. body weight.

Group-V: HAPS was administered at a dose rate of 400mg/kg, b.w. p.o. body weight.

Group-VI: HAPS was administered at a dose rate of 600mg/kg, b.w. p.o. body weight.

After the completion of eighth week i.e., 56 days, on 57^th day blood was collected for the estimation of biochemical parameters. Before collection of blood the animals were kept overnight fasting.

Parameters studied for this test were body temperature, bodyweights, average feed intake, weights of liver, adipose tissue, blood glucose, total cholesterol, HDL, LDL, VLDL, triglycerides, SGOT, SGPT, atherogenic index.

Collection of blood and serum samples

At the end of the experiment, blood was collected by cardiac puncture from each rat under mild ether anaesthesia. The blood samples were used for the estimation of glucose levels and remaining was allowed to clot for 30 min at room temperature and they were centrifuged at 3000 rpm for 10 minutes. The serum was used for the study of biochemical parameters.

Collection of tissue

The animals were scarified and the Liver was collected, stored in formalin solution and was used for the histopathological study.

Physical parameters

Determination of body weight

Body weight of the all animals in each group of HFD induced hyperlipidemia method was determined on the 0^th,7^th, 14^th, 21^st, 28^th, 35^th, 42^nd, 49^th, and 56^th day of the experiment period. Differences in weights were observed.

Determination of average feed intake

During the experimental period, feed intake, of rats were measured daily during 56 days. The amount of diet ingested was calculated as the difference between the weight of feed that remained in the foodbin (Da) and the amount placed one day before (Db). These data were then used to calculate a daily average feed intake (gm) according to the formula:

where 6 correspond to the animals number in each cage.

Determination of weights of liver, adipose tissue.

Animals were sacrificed and livers, adipose tissues were isolated, washed with saline and weighed by using an electronic balance.

Biochemical Parameters

Biochemical parameters (triglycerides (TG), total cholesterol (TC), HDLc) were estimated by using Swemed diagnostic kits. Low density lipoprotein(LDL) and very low denisty lipoprotein (VLDL) values were calculated using Friedewald's formula as given below.

VLDL=TG/5

LDL=TC-(HDL+VLDL)

Atherogenic index (AI) were calcualted by the Friedwald formula which is given below AI=TC-HDL/HDL

Statistical analysis

The values are expressed as Mean ± SEM. The data was analysed by using one way ANOVA followed by Dunnett’s test using Graph Pad Prism software. Statistical significance was set at P ≤ 0.05.

RESULTS:

Preparation of extracts and the percentage yield:

Extraction of grains of Paspalum scrobiculatum was carried out by using the soxhlet apparatus with hydro alcoholic solvent (70%v/v alcohol) and the percentage yield of the extract is given below.

Table 1: Percentage yield of HAPS.

Sl. No	Extracts	Yield in gm	Percentage yield
1.	Hydroalcoholic	75	1.87%

Phytochemical constituents present in HAPS:

There is a presence of Carbohydrates, flavonoids, tannins and saponins in HAPS

Effect of HFD on body weights in HFD induced hyperlipidemic rats.

Groups	Treatment	Body Weights (g)
Groups	Treatment	Day 0	Day 7	Day 14	Day 21	Day 28
I	Vehicle control	161.0±0.36	168.7±1.99	178.3±2.01	193.2±2.56	199.5±1.56
II	HFD (10ml/kg, b.w.)	163.5±0.34	174.3±2.74^ns	193.7±2.47^***	231.2±2.79^***	270.8±2.67^***
III	HFD (10ml/kg, b.w.)	175.8±0.47	187.3±2.55^***	204.8±3.04^***	239.7±2.64^***	285.5±2.23^***
IV	HFD (10ml/kg, b.w.)	178.0±0.96	192.2±1.88^***	210.5±2.11^***	244.3±2.81^***	288.0±1.63^***
V	HFD (10ml/kg, b.w.)	172.7±0.80	184.2±2.73^***	203.2±1.88^***	237.5±2.17^***	286.7±3.09^***
VI	HFD (10ml/kg, b.w.)	175.7±0.66	188.7±2.59^***	215.2±3.00^***	240.3±3.08^***	283.2±1.70^***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test.

Where, *** P<0.001, ns is non significant.

All groups are compared with Group I (vehicle control).

Effect of HAPS on body weights in HFD induced hyperlipidemic rats.

Groups	Treatment	Body weights (g)
Groups	Treatment	Day 28	Day 35	Day 42	Day 49	Day 56
I	Vehicle control	199.5±1.56	219..3±2.26	235.3±2.26	252.3±2.26	270.3±2.26
II	HFD	270.8±2.67^a***	275.3±1.83a^***	283.7±3.15^a***	294.2±3.42^a***	309.2±3.37^a***
III	HFD+fenofibrate (200mg/kg, b.w.)	285.5±2.23^b***	278.7±2.30^bns	267.2±2.90^b**	242.7±2.66^b***	201.2±3.36^b***
IV	HFD+HAPS (200mg/kg, b.w.)	288.0±1.63^b***	284.0±2.42^bns	279.0±3.22^bns	271.3±2.30^b***	261.2±3.54^b***
V	HFD+HAPS (400mg/kg, b.w.)	286.7±3.09^b***	281.0±2.79^bns	271.8±2.98^b*	256.8±3.04^b***	234.8±4.36^b***
VI	HFD+HAPS (600mg/kg, b.w.)	283.2±1.70^b***	279.5±3.03^bns	267.5±2.88^b**	245.5±3.27^b***	206.8±2.63^b***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test.

Where, *** P<0.001, **P<0.01, *P<0.05, ns is non significant.^a is compared with Group I (vehicle control), ^b is compared with Group II (HFD).

Acute oral toxicity studies (LD₅₀)

Animals administered with single dose of HAPS (2000 mg/kg b.w.) showed no mortality nor any sign of toxicity in any of the animals. Thus three different doses were selected for the present study – 200 mg/kg, b.w., 400 mg/kg, b.w. and 600 mg/kg, b.w.

Effect of HAPS on HFD induced hyperlipidemic rats

A study of 56 days antihyperlipidemia was done in HFD induced hyperlipidemic rats with HAPS and the results were tabulated below.

Effect of HAPS on physical parameters of HFD induced hyperlipidemic rats

i. Body weight

During 56 days of hyperlipidemia induction and treatment, the body weights of animals were monitored for every 7 days starting from day 0. The values were tabulated in table below. In first 28 days i.e., from day 0 to day 28, animals in the Groups II to VI increased their weights with extreme significance (P<0.001) when compared to normal control group.

From day 29 to day 56 animals in the Groups II to VI were treated with Fenofibrate 200mg/kg, b.w., HAPS (200, 400and 600 mg/kg, b.w. p.o) shows significant decrease (P<0.001) in body weight.

ii. Average feed intake

Quantities of feed consumed by rats in group I were more when compared to remaining groups, this was because rats were fed with extra HFD for 1 to 28 days during hyerlipidemia induction. The quantities of feed intake were the same in all groups, although body weight gain differed significantly between control groups, standard and HAPS treated groups. This difference was probably due to the different doses of HAPS extracts seems to exert a protective effect against overweight in HAPS treated group as compared to HFD control group. The values of average feed intake were tabulated in table below.

Effect of HAPS on feed intake in HFD induced hyperlipidemic rats.

Groups	Treatment	Avg feed intake 1-28 days	Avg feed intake 29-56 days
I	Vehicle control	10.56±0.40	11.27±0.46
II	HFD	6.95±0.36	9.94±0.50
III	HFD+fenofibrate (200mg/kg, b.w.)	7.87±0.30	6.26±0.61
IV	HFD+HAPS (200mg/kg, b.w.)	8.03±0.35	9.54±0.52
V	HFD+HAPS (400mg/kg, b.w.)	7.49±0.26	10.06±0.51
VI	HFD+HAPS (600mg/kg, b.w.)	7.07±0.32	10.67±0.46

iii. Different organ weights

Weights of different organs like liver, adipose tissue were observed in HFD induced hyperlipidemic rats. The weights of these organs were increased slightly in HFD control group compared to normal control group, group of fenofibrate showed significant decrease (P<0.01) in liver weight and extremely significant decrease (P<0.001) in adipose tissue weight, 200mg/kg, b.w. p.o HAPS showed significant decrease (P<0.01) in liver and adipose tissue weight where as groups of 400mg/kg and 600mg/kg, b.w. p.o HAPS showed significant decrease (P<0.001) compared to HFD control group. The values of these weights were tabulated in table below.

Effect of HAPS on organ weights in HFD induced hyperlipidemic rats.

Groups	Treatment	Liver weight (g)	Adipose tissue weight (g)
I	Vehicle control	1.94±0.03	0.40±0.03
II	HFD	3.41±0.03^a***	0.76±0.03^a***
III	HFD+fenofibrate (200mg/kg, b.w.)	3.00±0.01^b**	0.52±0.02^b***
IV	HFD+HAPS (200mg/kg, b.w.)	3.07±0.08^b**	0.64±0.01^b**
V	HFD+HAPS (400mg/kg, b.w.)	2.91±0.06^b***	0.60±0.01^b***
VI	HFD+HAPS (600mg/kg, b.w.)	2.80±0.10^b***	0.55±0.01^b***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test.

Where, *** P<0.001, ** P<0.01.

^a is compared with Group I (vehicle control), ^b is compared with Group II (HFD).

Blood glucose levels

After 56 days of hyperlipidemia induction and treatment with HAPS in HFD induced hyperlipidemic rats blood glucose levels were monitored and the values were tabulated in table below.

Animals in HFD group exhibited very significant increase (P<0.001) in blood glucose levels compared to normal control group.

Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w) showed very significant decrease (P<0.001) in blood glucose level. Fenofibrate decreased blood glucose levels very significantly (P<0.001).

Serum lipid profile

The lipid profile was evaluated by estimating triglycerides (TG), total cholesterol (Tc), HDL-Cholesterol (HDL-c), LDL-Cholesterol (LDL-c) and VLDL-Cholesterol (VLDL-c) in normal and hyperlipidemic animals. The values were tabulated in table below.

Animals in HFD group exhibited very significant increase (P<0.001) in triglycerides levels compared to normal control group. Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant reduction (P<0.001) in triglycerides levels.

Animals in HFD group exhibited very significant increase (P<0.001) in total cholesterol levels compared to normal control group. Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant decrease (P<0.001) in total cholesterol levels.

Animals in HFD group exhibited very significant decrease (P<0.001) in HDLc levels compared to normal control group. Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant increase (P<0.001) in HDL-c levels.

Animals in HFD group exhibited very significant increase (P<0.001) in LDLc levels compared to normal control group. Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant decrease (P<0.001) in LDL-c levels.

Animals in HFD group exhibited very significant increase (P<0.001) in VLDLc levels compared to normal control group. Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant decrease (P<0.001) in VLDL-c levels.

Fenofibrate treated group showed potent antihyperlipidemic activity and showed decrease in triglyceride, total cholesterol, LDLc, VLDLc levels, increase in HDLc levels with extreme significance (P<0.001) compared to HFD control group.

Effect of HAPS on serum glucose levels in HFD induced hyperlipidemic rats.

Groups	Treatment	Week	Glucose
I	Vehicle control	4	67.50±2.32
I	Vehicle control	8	77.17±1.90
II	HFD	4	138.20±2.08^a***
II	HFD	8	125.30±3.15^b***
III	HFD	4	145.50±1.87^a***
III	HFD+Fenofibrate (200mg/kg, b.w.)	8	87.83±1.85^c***
IV	HFD	4	131.00±1.87^a***
IV	HFD+HAPS (200mg/kg, b.w.)	8	102.2±1.47^c***
V	HFD	4	139.20±1.35^a***
V	HFD+HAPS (400mg/kg, b.w.)	8	92.50±0.76^c***
VI	HFD	4	143.20±2.65^a***
VI	HFD+HAPS (600mg/kg, b.w.)	8	87.81±2.37^c***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test.

Where*** P<0.001.^a is compared with vehicle control (week 4), ^b is compared with vehicle control (week 8), ^c is compared with HFD (week 8).

Effect of HAPS on lipid profile in HFD induced hyperlipidemic rats.

Groups	Treatment	Week	Serum lipid profile mg/dl
Groups	Treatment	Week	TG	TC	HDL-C	LDL-C	VLDL-C
I	Vehicle control	4	64.19±3.01	77.17±3.74	24.17±1.24	40.16±3.53	12.84±0.60
I	Vehicle control	8	63.50±2.56	86.67±3.21	26.17±1.22	47.30±3.11	12.70±0.51
II	HFD	4	196.70±1.43^a***	255.20±3.16^a***	15.00±1.23^a***	200.80±2.88^a***	39.33±0.28^a***
II	HFD	8	183.30±2.45^b***	248.80±3.07^b***	17.17±1.07^b***	195.80±3.15^b***	36.67±0.49^b***
III	HFD	4	200.50±3.05^a***	242.0±2.28^a***	13.50±1.17^a***	188.40±2.64^a***	40.10±0.61^a***
III	HFD+fenofibrate200mg/kg	8	70.76±2.24^c***	89.50±3.56^c***	40.67±1.40^c***	34.61±4.69^c***	14.15±0.44^c***
IV	HFD	4	192.30±1.45^a***	230.2±2.61^a***	16.00±1.06^a***	175.70±3.31^a***	38.47±0.29^a***
IV	HFD+HAPS 200mg/kg	8	127.00±2.8^c***	146.8±2.58^c***	24.00±1.15^c**	98.43±2.80^c***	25.40±0.56^c***
V	HFD	4	202.00±1.528^a***	238.0±2.68^a***	14.83±1.51^a***	182.80±1.24^a***	40.40±0.30^a***
V	HFD+HAPS 400mg/kg	8	103.80±1.35^c***	108.50±3.65^c***	29.50±1.52^c***	57.73±4.00^c***	20.77±0.27^c***
VI	HFD	4	208.50±3.25^a***	248.7±3.23^a***	15.67±1.58^a***	191.30±2.79^a***	41.70±0.65^a***
VI	HFD+HAPS 600mg/kg	8	96.17±3.08^c***	98.17±2.92^c***	35.33±1.49^c***	44.77±3.86^c***	19.23±0.61^c***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test

Where, *** P<0.001, ^a is compared with vehicle control (week 4),

^b is compared with vehicle control (week 8)

Effect of HAPS on SGOT and SGPT in HFD induced hyperlipidemic rats.

Groups	Treatment	Week	SGOT	SGPT
I	Vehicle control	4	153.20±2.83	58.17±1.88
I	Vehicle control	8	163.2±2.58	59.02±2.10
II	HFD	4	235.00±2.97a^***	126.0±1.75^a***
II	HFD	8	211.2±1.99^b***	108.5±2.10^b***
III	HFD	4	232.20±2.12^a***	129.7±1.62^a***
III	HFD+fenofibrate (200mg/kg, b.w.)	8	253.5±2.39^c***	148.3±2.603^c***
IV	HFD	4	236.8±1.57^a***	124.7±1.89^a***
IV	HFD+HAPS (200mg/kg, b.w.)	8	191.2±3.14^c***	97.67±2.01^c**
V	HFD	4	233.2±2.442^a***	127.5±1.64^a***
V	HFD+HAPS (400mg/kg, b.w.)	8	182.2±2.21^c***	89.44±1.87^c***
VI	HFD	4	238.00±2.65^a***	133.8±2.33^a***
VI	HFD+HAPS (600mg/kg, b.w.)	8	177.7±2.67^c***	84.42±2.66^c***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test.

Where, *** P<0.001, **P<0.01.

^a is compared with vehicle control (week 4), ^b is compared with vehicle control (week 8), ^c is compared with HFD (week 8).

SGOT

Animals in HFD group exhibited very significant increase (P<0.001) in SGOT levels compared to normal control group.

Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant decrease (P<0.001) in SGOT levels. The values are showed in table below.

SGPT

Animals in HFD group exhibited very significant increase (P<0.001) in SGPT levels compared to normal control group.

Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed significant decrease (P<0.001) in SGPT levels. The values are showed in table..

Fenofibrate group showed significant increase (P<0.001) in SGOT and SGPT levels compared to HFD group.

Atherogenic index

Atherogenic index values were calculated in HFD induced hyperlipidemic rats and the values were tabulated in table below.

Animals in HFD group exhibited very significant increase (P<0.001) in Atherogenic index value compared to normal control group.

Hyperlipidemic animals treated with HAPS (200, 400 and 600mg/kg, b.w.) showed extremely significant decrease (P<0.001) in Atherogenic index.

Effect of HAPS on Atherogenic index in HFD induced hyperlipidemic rats.

Groups	Treatment	Atherogenic index
I	Vehicle water	2.26±0.16
II	HFD	14.10±0.74^a***
III	HFD+fenofibrate (200mg/kg, b.w.)	1.22±0.13^b***
IV	HFD+HAPS (200mg/kg, b.w.)	5.39±0.14^b***
V	HFD+HAPS (400mg/kg, b.w.)	2.62±0.15^b***
VI	HFD+HAPS (600mg/kg, b.w.)	1.88±0.13^b***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test.

Where, *** P<0.001.

^a is compared with Group I (vehicle control), ^b is compared with Group II (HFD).

DISCUSSION:

It has been well established that nutrition plays an important role in the etiology of hyperlipidimias and atherosclerosis It was demonstrated by body weights, feed intake, organ weights, lipid profile. The body weights during induction period of hyperlipidemia were increased with extreme significance, and up on treatment with HAPS, body weights were decreased. During the total experiment period feed intake by animals was quite similar. So feed intake did not affect the body weight reduction in animals and it might be due to the HAPS administration. Different organ weights were also observed which showed significant decrease in weights compared to HFD control.

The high fat diet (HFD) was prepared by homogenously mixing dalda and coconut oil in the ratio of 3:2w/w. Extreme body weights gained by the animals were the first change observed in HFD treated rats, when compared to normal control rats. It is well known that, hyperlipidemia is associated with increased adipose tissue accumulation in the body. When these HFD groups were further treated with various doses of HAPS (200, 400, 600 mg/kg, b.w.), decrease in body weight is noticed. High dose showed same reduction as that of standard drug fenofibrate. This decrease in body weights may be due to increased production of thyroxine increasing metabolic activity (12), supression of the apetite,increase of body's metabolism,interference with the body's ability to absorb specific nutrients in food (13).

It is reported that feeding high fat diet in rats leads to increase in weight of liver. The above facts can be verified by the data obtained in expeimental animals fed with HFD showed increase in the weight of the liver and also a significant increase in the weight of the adipose tissue. This fact again establishes that the HAPS has a definite influence in body fat metabolism. Feeding the rats with HFD showed a significant increase in the glucose levels. This may be due to a high fat intake does appear to contribute to insulin resistance due to the effect of free fatty acids on peripheral insulin resistance and glucose tolerance (14).Fat cells produce leptin, resistin and adiponectin. Leptin is normally released after a meal and dampens apetite. Resistin and adiponectin both affect cells response to insulin. (Too much resistin may cause insulin resistance; too little adiponectin may do the same) (15).High levels of blood circulating triglycerides interfere with insulin action due to its receptor(16) .It has become clear that excess fat disrupts the normal balance and functioning of these hormones, thereby contributing to insulin resistance and setting the stage for diabetes.

When the animals are treated with fenofibrate, there is significant decrease in glucose levels compared to control. This may be due to reduction in body fat content may lead to improvement of insulin sensitivity increases the expression of adiponectin receptor 1 in adipose tissue which may enhance adiponectin insulin-sensitizing effects despite no change in circulating adiponectin levels (17)^.

When the animals are treated with HAPS (200, 400, 600 mg/kg, b.w.) showed a significant decrease in blood glucose levels. This may be due to improvement of physiological action of insulin action and prevention of insulin resistance by increasing insulin receptor binding (18).

The significant increase in total cholesterol and triglyceride level in HFD fed animal can be attributed to increase in both de novo synthesis and intestinal absorption of cholesterol (19)^.Cholesterol feeding alone however does not affect the serum TG level. It is assumed that a high level of saturated fat in addition to cholesterol is required to significantly elevate serum TG level in rat model (20). When the rats are feed with HFD, there is a significant increase in triglyceride levels when compared to normal control. This may be due to absorption from the intake food, fat and liver cells synthesize and store triglycerides.

When the animals are treated with fenofibrate, there is a significant decrease in the levels of triglycerides when compare to the normal control. This may be due to increased triglyceride-rich lipoprotein (TRL) lipolysis (21), induction of hepatic fatty acid (FA) uptake and reduction of hepatic triglyceride production (22). When the rats are treated with HAPS 200, 400 and 600mg/kg, b.w., there is a significance decrease in serum triglyceride levels, this may be due to adipose tissue releases free fatty acids that drive the production of triglycerides in the liver. HAPS may block the release of free fatty acids from adipose tissue.

Diet containing saturated fatty acids increases the activity of HMGCoA reductase, the rate–determining enzyme in cholesterol biosynthesis; this may be due to higher availability of acetyl CoA, which stimulated the cholesterogenesis rate(23). Moreover, this could be associated with a down regulation in LDL receptors by the cholesterol and saturated fatty acids in the diet, which could also explain the elevation of serum LDL-C levels either by changing hepatic LDLR (LDL-receptor) activity, the LDL-C production rate or both (24).

The activity of cholesteryl ester transfer protein (CETP), a key enzyme in reverse cholesterol transport and HDL metabolism increase in high fat diet and mediates the transfer of cholesteryl esters from HDL-C to triglyceride-rich particles in exchange for triglycerides. This leads to increased plasma concentrations of TGs & decreased concentrations of HDL-C (25). LCAT enzyme is involved in the transesterification of cholesterol, the maturation of HDL and the flux of cholesterol from cell membranes into HDL (26).

The activity of the enzyme tends to decrease in diet-induced hypercholesterolemia. The increase in the concentrations of LDL and VLDL observed are mainly due to the dietary carbohydrates and cholesterol (27). Most of the cholesterol in the mature lesion originates from circulating LDL particles, the circulating LDL particles cross the endothelium into the intimal of blood vessels. In their native form they are unfavourable for uptake into intimal macrophages and most return to the circulation. However, some particles may be oxidized by local cells possibly facilitated by the presences of transition metal ions and binding to proteoglycans. After oxidative modification the LDL particles are rapidly taken up into macrophages via the scavenger receptor. Subsequent loading with cholesteryl esters forms so called foam cells, which could be responsible for the initiation of atherosclerosis (28).

When the animals are treated with fenofibrate, there is a significance decrease in cholesterol levels compare to normal control. The biochemical estimations shown that the drug HAPS increased the protective HDLc level and decreased the atherogenic, LDL and VLDL levels. This may be due to increased removal of LDL particles (29), reduction in neutral lipid (cholesteryl ester and triglyceride) exchange between VLDL and HDL (30), increase in HDL production and stimulation of reverse cholesterol transport (31).

When the animals are treated with HAPS (200, 400 and 600mg/kg, b.w.), there is a significant decrease in the Total cholesterol, HDL cholesterol and LDL cholesterol compared to normal control. The biochemical estimations shown that HAPS increased the protective HDL-C level and decreased the atherogenic, LDL and VLDL levels. This may be due to increase of HDL-C, which is attributed to the mobilization of cholesterol from peripheral cells to the liver by the action of Lecithin Cholesterol O-acyltransferase (LCAT) (32). The increased HDL-C facilitates the transport of TG or cholesterol from serum to liver by a pathway termed ‘reverse cholesterol transport’ where it is catabolised and excreted out of the body (33). Due to the activity of LCAT and inhibition of the action of TG-lipase on HDL, which may contribute for a rapid catabolism of blood lipids through extra hepatic tissues (34). Increase excretion of fecal bile components that results in increased total bile output and subsequent increase of conversion of cholesterol to bile acids and salts , prevention of accumulation of lipids in liver (35). Some components of HAPS may compete with cholesterol binding sites or interfere with cholesterol bio synthesis in liver, thereby reducing blood cholesterol level (36). Increasing in HDLc indicating that components in HAPS may involved in mobilizing cholesterol from extra hepatic tissue to liver where it is catabolised. Increased stimulation of bile acid synthesis may lead to an increased utilisation of cellular free cholesterol and thus help in reduction of cholesterol. Flavonoids present in HAPS may augment the activity of lecithin acyl tranferase (LCAT), which regulates blood lipids. These results established the idea that the hypolipidemic effect of HAPS in vivo was might be associated with flavonoids enriched in HAPS since flavonoids could inhibit HMG CoA reductase activity (37). Liver enzymes such as SGOT and SGPT are considered to be biochemical markers for assessing liver function. SGOT and SGPT levels are increased when there is damage to the liver or liver inflammation. Hepatic dysfunction was evidenced by an elevation of these serum marker enzymes after experimental induction of hyperlipidemia. When the rats are treated with HFD, there is a significance increase in levels of SGOT and SGPT. Increase in SGOT and SGPT levels may be due to excess fat deposition may lead to hepatic damage, physical stress due to handling of animals (38). whereas there is no significant decrease in SGOT and SGPT levels in animals treated with fenofibrate, HAPS (200, 400, 600 mg/kg, b.w.) in HFD model.

ACKNOWLEDGMENT:

The author sincerely thanks to guide Mr. Bandenawaz Ramadurg and Mrs. Saraswathi C.D. for rendering their suggestions and helping them in each and every step of completing this research paper successfully.

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Received on 28.10.2013

Modified on 18.11.2013

Accepted on 21.11.2013

Research J. Pharmacology and Pharmacodynamics. 5(6): November –December 2013, 362-370