# Lipid Lowering Effect ofPunica granatum L. Peel in High Lipid Diet Fed Male Rats

Authors: Alireza Sadeghipour; Maryam Eidi; Ali Ilchizadeh Kavgani; Reza Ghahramani; Saleh Shahabzadeh; Ali Anissian
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

Many herbal medicines have been recommended for the treatment of dyslipidemia. The antilipidemic effect of hydroethanolic extract of pomegranate peel (Punica granatum L.) was investigated in high lipid diet fed male rats. Intraperitoneally administration of pomegranate peel extract (50, 100, 200, and 300 mg/kg body weight) for 23 days on the levels of serum cholesterol, triglycerides, LDL, HDL, alkaline phosphatase (AP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in high lipid diet fed male rats was evaluated. Treatment of pomegranate extract decreased body weight in treated rats, significantly. Administration of the plant extract significantly decreased serum total cholesterol, triglycerides, LDL-C, alkaline phosphatise, AST, and ALT levels, whereas it increased serum HDL-C in high lipid diet fed rats in comparison to saline control group. Also, histopathological study showed that treatment of pomegranate peel extract attenuates liver damage in high lipid diet fed rats in comparison to saline group. It is concluded that the plant should be considered as an excellent candidate for future studies on dyslipidemia.


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## Body

## 1. Introduction

Dyslipidemia is generally characterized by elevated levels of total cholesterol, triglycerides, low density lipoprotein cholesterol, and decreased levels of high density lipoprotein cholesterol [1]. Dyslipidemia as an independent preventable risk factor of coronary heart disease has been shown to increase the risk of cardiovascular mortality [2–7]. Therefore, the study on the various indicators and risk factors of dyslipidemia appears to be significant in future health outcomes.Punica granatum Linn. (Punicaceae) is a shrub or small tree and considered to be a native of Iran and Afghanistan. It is also found growing wild in the warm valleys and outer hills of the Himalayas [8]. The pomegranate fruit consists of the peel, seeds, and the arils. The peel makes up about 50% of the fruit, whereas the arils and seeds make up 40% and 10%, respectively. The peel is rich in many compounds such as phenolics, flavonoids, ellagitannins and proanthocyanidin compounds, complex polysaccharides, and many minerals including potassium, nitrogen, calcium, magnesium, phosphorus, and sodium [9].The different parts of pomegranate (Punica granatum L.) have been known as a reservoir of bioactive compounds with potential biological activities. Pomegranate decreased the dyslipidemia of obesity and cardiovascular risk factors [10]. Antiparasitic, antimicrobial, and antioxidant activities of pomegranate leaves extracts were reported [11–13]. Several papers were reported on the ability of pomegranate leaves extracts to fight obesity [14], cancer, and other human diseases [15].It is reported that 6-week treatment with pomegranate flower extract ameliorated fatty liver, reflected by diminishment of relative and total hepatic triglyceride contents and fatty droplet deposit in the livers of Zucker diabetic fatty rats [16].In traditional Chinese medicine, different pomegranate extracts and preparations including the bark, root, and juice of the fruit, especially the dried peels, have been used to treat many conditions [10].The aim of the present study was to investigate the antihyperlipidemic effects of pomegranate extract peel in high lipid diet fed male rats.


## 2. Materials and Methods

### 2.1. Plant Material

FreshPunica granatum L. peels were collected from Saveh area (October 2013). Voucher specimens (Farabi Herbarium number GUE 7321) were authenticated by Associate Professor Ali Mazooji, Department of Biology, Faculty of Biology, Islamic Azad University. The plant material was dried under shade and powdered using Ultra-Torax. The powder (60 g) was extracted with 300 mL aqueous 80% ethanol in a Soxhlet apparatus for 72 hours. The extract was filtered and concentrated to dryness under reduced pressure in a rotary evaporator at 40–50°C yielding 15.3% (w/w) plant extract. The extract yield was 19%. The obtained pomegranate alcoholic extract was stored at −20°C until usage. Plant extract was suspended in saline (doses 50, 100, 200, and 300 mg/kg body weight) prior to intraperitoneal administration to the experimental animals.


### 2.2. Experimental Animals and Induction of Hyperlipidemia

Male Wistar rats initially weighing 200 to 250 g purchased from the Pasteur Institute (Karaj, Iran) were used in the experiments. The diet was purchased from Pars-Dam food service, Tehran, Iran. The animal room was maintained at 22°C ± 2°C with timed lighting on from 7 AM to 19 PM and relative air humidity of 40% to 60%. Each animal was used once only. The animal protocol was approved by the Ethics Committee of Islamic Azad University, Tehran, Iran, and conforms to the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals, Iran, and also to international guidelines. Accordingly, five rats were housed per cage of size 50 cm × 23 cm. Hyperlipidemia was induced by feeding 10% lipid supplemented in the basal diet. The basal diet contained (g% of final diet), casein 15.0, sucrose 68.3, hydrogenated coconut oil 10.0, cellulose 2.0, salt mixture 4.0, vitamin mixture 0.5, and choline chloride 0.2. The animals were distributed into six groups each containing 8 rats. The control group was fed on the basal diet and given waterad libitum. Extract was dissolved in saline and administered intraperitoneally (i.p.) for 23 days. Animals in the control group received only 0.5 mL saline as vehicle.Experimental groups were as follows: Group 1: normal control, fed on basal diet; Group 2: untreated control, fed on 10% lipid diet and given saline 0.5 mL/rat (i.p.); Groups 3, 4, 5, and 6, fed on 10% lipid in diet and administered extract at doses 50, 100, 200, and 300 mg/kg/day (i.p.).The initial body weights of all the animals in each group were measured. After 23 days, the rats were fasted for 12 h and their final body weights were determined. Then, rats were fasted overnight, and blood samples were drawn from heart under light ether anaesthesia. The animals were removed after blood collection. Serum cholesterol, triglyceride, LDL, HDL, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined by kit (Parsazmoon Company, Iran).


### 2.3. Histopathological Studies in the Liver

For qualitative analysis of liver histology, the tissue samples were fixed for 48 h in 10% formalin-saline and dehydrated by passing successfully in different mixtures of ethyl alcohol-water, cleaned in xylene, and embedded in paraffin. Sections of the tissue were prepared by using a rotary microtome and stained with haematoxylin and eosin dye, which was mounted in a neutral deparaffinated xylene medium for microscopic observations. Histological damage including fatty change in hepatocyte, dilation of sinozoid, and congestion in high lipid diet fed. Each damage is given 1 score.


### 2.4. Statistical Analysis

Statistical analyses and representations were performed in Microsoft Excel. All data was analyzed by one-way ANOVA and presented as the mean value ± S.E.M. of eight rats (n
=
8). The results of the lipid fed untreated control group were compared to normal control group and those of extract treated groups were compared to untreated control group. P values were checked at three levels of significance, namely, 0.05, 0.01, and 0.001. P value less than 0.05 was considered “significant” and P less than 0.01 and 0.001 as “highly significant.”


## 2.1. Plant Material

FreshPunica granatum L. peels were collected from Saveh area (October 2013). Voucher specimens (Farabi Herbarium number GUE 7321) were authenticated by Associate Professor Ali Mazooji, Department of Biology, Faculty of Biology, Islamic Azad University. The plant material was dried under shade and powdered using Ultra-Torax. The powder (60 g) was extracted with 300 mL aqueous 80% ethanol in a Soxhlet apparatus for 72 hours. The extract was filtered and concentrated to dryness under reduced pressure in a rotary evaporator at 40–50°C yielding 15.3% (w/w) plant extract. The extract yield was 19%. The obtained pomegranate alcoholic extract was stored at −20°C until usage. Plant extract was suspended in saline (doses 50, 100, 200, and 300 mg/kg body weight) prior to intraperitoneal administration to the experimental animals.


## 2.2. Experimental Animals and Induction of Hyperlipidemia

Male Wistar rats initially weighing 200 to 250 g purchased from the Pasteur Institute (Karaj, Iran) were used in the experiments. The diet was purchased from Pars-Dam food service, Tehran, Iran. The animal room was maintained at 22°C ± 2°C with timed lighting on from 7 AM to 19 PM and relative air humidity of 40% to 60%. Each animal was used once only. The animal protocol was approved by the Ethics Committee of Islamic Azad University, Tehran, Iran, and conforms to the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals, Iran, and also to international guidelines. Accordingly, five rats were housed per cage of size 50 cm × 23 cm. Hyperlipidemia was induced by feeding 10% lipid supplemented in the basal diet. The basal diet contained (g% of final diet), casein 15.0, sucrose 68.3, hydrogenated coconut oil 10.0, cellulose 2.0, salt mixture 4.0, vitamin mixture 0.5, and choline chloride 0.2. The animals were distributed into six groups each containing 8 rats. The control group was fed on the basal diet and given waterad libitum. Extract was dissolved in saline and administered intraperitoneally (i.p.) for 23 days. Animals in the control group received only 0.5 mL saline as vehicle.Experimental groups were as follows: Group 1: normal control, fed on basal diet; Group 2: untreated control, fed on 10% lipid diet and given saline 0.5 mL/rat (i.p.); Groups 3, 4, 5, and 6, fed on 10% lipid in diet and administered extract at doses 50, 100, 200, and 300 mg/kg/day (i.p.).The initial body weights of all the animals in each group were measured. After 23 days, the rats were fasted for 12 h and their final body weights were determined. Then, rats were fasted overnight, and blood samples were drawn from heart under light ether anaesthesia. The animals were removed after blood collection. Serum cholesterol, triglyceride, LDL, HDL, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined by kit (Parsazmoon Company, Iran).


## 2.3. Histopathological Studies in the Liver

For qualitative analysis of liver histology, the tissue samples were fixed for 48 h in 10% formalin-saline and dehydrated by passing successfully in different mixtures of ethyl alcohol-water, cleaned in xylene, and embedded in paraffin. Sections of the tissue were prepared by using a rotary microtome and stained with haematoxylin and eosin dye, which was mounted in a neutral deparaffinated xylene medium for microscopic observations. Histological damage including fatty change in hepatocyte, dilation of sinozoid, and congestion in high lipid diet fed. Each damage is given 1 score.


## 2.4. Statistical Analysis

Statistical analyses and representations were performed in Microsoft Excel. All data was analyzed by one-way ANOVA and presented as the mean value ± S.E.M. of eight rats (n
=
8). The results of the lipid fed untreated control group were compared to normal control group and those of extract treated groups were compared to untreated control group. P values were checked at three levels of significance, namely, 0.05, 0.01, and 0.001. P value less than 0.05 was considered “significant” and P less than 0.01 and 0.001 as “highly significant.”


## 3. Results

### 3.1. General Improvement in the Hyperlipidemic State

Changes in initial and final body weights in control and experimental groups are shown in Figure1. The results showed treatment of extract decreased final body weight elevations in comparison to control saline group (P
<
0.01).Figure 1
Effect of i.p. administration of pomegranate peel hydroethanolic extract at doses of 50, 100, 200, and 300 mg/kg body wt on body weight in high lipid diet fed rats. Each column represents mean ± SEM for 8 rats. Control saline group was administrated with saline as vehicle.P
*
<
0.05, P
*
*
<
0.01, P
*
*
*
<
0.001 different from initial body weight in each group.The results showed treatment of extract decreased liver and kidney coefficients (liver weight/body weight and kidney weight/body weight) in comparison to control saline group, insignificantly. The administration of the pomegranate peel extract (50, 100, 200, and 300 mg/kg body wt) significantly decreased serum triglycerides, cholesterol, LDL, AP, ALT, and AST levels, while increasing serum HDL level in high lipid diet fed rats compared with saline group (Table1).Table 1
Effect of i.p. administration of hydroethanolic extract ofPunica granatum peel at doses 50, 100, 200, and 300 mg/kg on liver and kidney coefficients, serum parameters, and histopathological damage of liver in high lipid diet fed rats.
Parameters
Control
Saline
Extract (mg/kg)
50
100
200
300
Liver coefficient
0.028
0.042
0.037
0.035
0.039
0.039
Kidney coefficient
0.0027
0.0036
0.0033
0.0033+
0.0035
0.0036
Triglycerides (mg/dL)
146 ± 21
475 ± 11***
381 ± 23
325 ± 43
302 ± 31
210 ± 27+++
Cholesterol (mg/dL)
73 ± 8
110 ± 11***
87 ± 9++
82 ± 5++
80 ± 9++
81 ± 7++
LDL (mg/dL)
92 ± 6
321 ± 11**
209 ± 23
145 ± 29+
79 ± 8++
61 ± 7+++
HDL (mg/dL)
98 ± 9
28 ± 3*
89 ± 11
128 ± 5+++
179 ± 18+++
185 ± 20+++
AST (UI/L)
1234 ± 34
1538 ± 47***
1219 ± 39+++
1232 ± 71+++
1170 ± 49+++
1233 ± 36+++
ALT (UI/L)
1267 ± 56
1553 ± 32***
1130 ± 44+++
1246 ± 51+++
1233 ± 68+++
1290 ± 54+++
AP (UI/L)
983 ± 21
1362  ± 71***
1311 ± 39
1248 ± 48
1100 ± 59++
1049 ± 78+++
Histopathological damage of liver
0 ± 0
1.5 ± 0.11*
0.5 ± 0.23
0.4 ± 0.23
0.17 ± 0.31+
0.09 ± 0.27+
*
P
<
0.05, *
*
P
<
0.01, *
*
*
P
<
0.001 different from control group.
+
P
<
0.05, +
+
P
<
0.01, +
+
+
P
<
0.001 different from saline group.Histopathological study shows that the administration of the pomegranate peel extract (50, 100, 200, and 300 mg/kg body wt) significantly decreased histopathological damage of liver including fatty change in hepatocyte, dilation of sinusoid, and congestion (Figure2) in high lipid diet fed rats compared with saline group (Table 1).Histopathology of liver tissue in high lipid diet and normal diet fed rats (hematoxylin-eosin). (a) Control liver tissue (×100), (b) congestion damage in high lipid diet fed rats (×100), (c) dilation of sinusoid (×400), and (d) fatty change in hepatocyte (×100).
(a)
(b)
(c)
(d)


## 3.1. General Improvement in the Hyperlipidemic State

Changes in initial and final body weights in control and experimental groups are shown in Figure1. The results showed treatment of extract decreased final body weight elevations in comparison to control saline group (P
<
0.01).Figure 1
Effect of i.p. administration of pomegranate peel hydroethanolic extract at doses of 50, 100, 200, and 300 mg/kg body wt on body weight in high lipid diet fed rats. Each column represents mean ± SEM for 8 rats. Control saline group was administrated with saline as vehicle.P
*
<
0.05, P
*
*
<
0.01, P
*
*
*
<
0.001 different from initial body weight in each group.The results showed treatment of extract decreased liver and kidney coefficients (liver weight/body weight and kidney weight/body weight) in comparison to control saline group, insignificantly. The administration of the pomegranate peel extract (50, 100, 200, and 300 mg/kg body wt) significantly decreased serum triglycerides, cholesterol, LDL, AP, ALT, and AST levels, while increasing serum HDL level in high lipid diet fed rats compared with saline group (Table1).Table 1
Effect of i.p. administration of hydroethanolic extract ofPunica granatum peel at doses 50, 100, 200, and 300 mg/kg on liver and kidney coefficients, serum parameters, and histopathological damage of liver in high lipid diet fed rats.
Parameters
Control
Saline
Extract (mg/kg)
50
100
200
300
Liver coefficient
0.028
0.042
0.037
0.035
0.039
0.039
Kidney coefficient
0.0027
0.0036
0.0033
0.0033+
0.0035
0.0036
Triglycerides (mg/dL)
146 ± 21
475 ± 11***
381 ± 23
325 ± 43
302 ± 31
210 ± 27+++
Cholesterol (mg/dL)
73 ± 8
110 ± 11***
87 ± 9++
82 ± 5++
80 ± 9++
81 ± 7++
LDL (mg/dL)
92 ± 6
321 ± 11**
209 ± 23
145 ± 29+
79 ± 8++
61 ± 7+++
HDL (mg/dL)
98 ± 9
28 ± 3*
89 ± 11
128 ± 5+++
179 ± 18+++
185 ± 20+++
AST (UI/L)
1234 ± 34
1538 ± 47***
1219 ± 39+++
1232 ± 71+++
1170 ± 49+++
1233 ± 36+++
ALT (UI/L)
1267 ± 56
1553 ± 32***
1130 ± 44+++
1246 ± 51+++
1233 ± 68+++
1290 ± 54+++
AP (UI/L)
983 ± 21
1362  ± 71***
1311 ± 39
1248 ± 48
1100 ± 59++
1049 ± 78+++
Histopathological damage of liver
0 ± 0
1.5 ± 0.11*
0.5 ± 0.23
0.4 ± 0.23
0.17 ± 0.31+
0.09 ± 0.27+
*
P
<
0.05, *
*
P
<
0.01, *
*
*
P
<
0.001 different from control group.
+
P
<
0.05, +
+
P
<
0.01, +
+
+
P
<
0.001 different from saline group.Histopathological study shows that the administration of the pomegranate peel extract (50, 100, 200, and 300 mg/kg body wt) significantly decreased histopathological damage of liver including fatty change in hepatocyte, dilation of sinusoid, and congestion (Figure2) in high lipid diet fed rats compared with saline group (Table 1).Histopathology of liver tissue in high lipid diet and normal diet fed rats (hematoxylin-eosin). (a) Control liver tissue (×100), (b) congestion damage in high lipid diet fed rats (×100), (c) dilation of sinusoid (×400), and (d) fatty change in hepatocyte (×100).
(a)
(b)
(c)
(d)


## 4. Discussion

Dyslipidemia is a multifactorial and polygenic disorder resulting from an interaction between an individual’s genetic background and multiple environmental factors including behavioural and social risk factors [10].Fruits are rich sources of vitamins, minerals, and biologically active compounds. However, very often they are consumed without the peels despite the fact that some fruit peels are rich in polyphenolic compounds, flavonoids, ascorbic acid, and other biologically active components that have positive influence on health [2, 17].Our results demonstrated that administration of hydroethanolic extract fromPunica granatum peel showed marked antihyperlipidemic effects in high lipid diet fed rats. Pomegranate extract decreased serum cholesterol, triglycerides, LDL, ALT, AST, and AP, while increasing serum HDL levels in high lipid diet fed rats in comparison to saline treated rats. Also, the extract attenuated liver damage including fatty change in hepatocyte, dilation of sinusoid, and congestion in high lipid diet fed rats compared with saline group.In agreement, it is reported that the different parts of pomegranate (Punica granatum L.) have been known as a reservoir of bioactive compounds with potential biological activities. Pomegranate, especially the leaves of pomegranate, decreased the dyslipidemia of obesity and cardiovascular risk factors [10]. The ability of pomegranate leaves extracts to fight obesity is shown [14].On the other hand, pomegranate flower has been demonstrated to ameliorate hyperlipidemia and decrease excess cardiac lipid accumulation in Zuker diabetic fatty rats [18] and to attenuate atherosclerosis in apolipoprotein E deficient mice [19]. Moreover, oleanolic acid and ursolic acid, two of the active components contained in pomegranate flower [20], have been long-recognized to have antihyperlipidemic properties [21]. Gallic acid, another important component in pomegranate flower [20], has been demonstrated to improve high fat diet induced hyperlipidemia and fatty liver in mice [22].Also, Parmar and Kar reported pomegranate peel extract ameliorated biochemical and histopathologic alterations induced by the atherogenic diet [23]. The protective role of the fruit peel could be related to its flavonoids and polyphenolic contents, which possess antioxidative activity [24]. Moreover, the juice ofP. granatum is also known to prevent atherosclerosis, which further supports its antiatherogenic potential [25].It is reported that addition of pomegranate juice to simvastatin in a macrophage cell culture model system improves the statin ability to inhibit cellular cholesterol biosynthesis and to protect the cells from oxidative stress. These effects could be related to the antioxidant hydrolyzable tannin punicalagin and to the phytosterolβ-sitosterol, which are both present in pomegranate [26]. Moreover, phytosterols of pomegranate consumption decreased serum cholesterol levels in dyslipidemic patients, as well as their cardiovascular risk [27, 28].As a result, it may be concluded that pomegranate peel seeds extract possesses antilipidemic activities in high lipid diet fed rats and that the pomegranate peel extract may be of use as an antidyslipidemic agent. It is concluded that the plant should be considered as an excellent candidate for future studies on dyslipidemia. In addition, further comprehensive pharmacologic investigations, including experimental chronic studies, should be carried out.



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*Source: 432650-2014-09-10.xml*