Antihyperlipidemic Activity of Feronia limonia Leaves Extract against Triton X 100 and High Fat Induced Hyperlipidemic in Rats

 

Sravanthi P.1, Ramya Krishna P.S.2, Narahari N. Palei3, Sannidhi Nagarjuna2

1Neermala College of Pharmacy, Kadapa, Andhra Pradesh, India.

2Raghavendra Institute of Pharmaceutical Education and Research, Anantapur, Andhra Pradesh, India.

3 School of Pharmacy, The Neotia University, Sarisha, Diamond Harbour Road, West Bengal, India.

*Corresponding Author E-mail: narahari.palei@gmail.com

 

ABSTRACT:

Objective: The objective of this study was to investigate the antihyperlipidemic activity of Feronia limonia leaves extract in rats. Methods: Hyperlipidemia was induced in rats by feeding high fat diet (HFD) for four successive weeks and Triton X 100(400mg/kg, i.p.) for 24 hours. From 15th to 30th day, extract (250 and 500mg/kg, p.o) and atorvastatin (20mg/kg, p.o.) per se were administered 2 hours prior to feeding rats with high fat diet (HFD). Blood samples of all rats were collected and estimated biochemical parameters on the last day of treatment i.e., 30th day for HFD, and after 24 hours for of Triton X 100 induced hyperlipidaemia using standard diagnostic kits. White adipose tissue obtained from all rats were fixed in 10% formo-saline and analyzed for adipocyte size. Results and Discussion: HFD or Triton X 100 significantly increased serum total cholesterol, triglycerides, LDL-c, and decreased HDL-c concentration. The Extract (250mg/kg, p.o. and 500mg/kg, p.o.) significantly reversed HFD-induced and Triton X 100 induced hyperlipidemia. The adipose tissue of animals which were treated with MEFL showed significant and dose-related decrease in cell size when compared to animals of control group. Conclusions: It can be concluded that extact showed significant anti hyperlipidemic activity in rats and hence it could be a possible herbal medicine for the treatment of hyperlipidemia.

 

KEYWORDS: Feronia limonia, Antihyperlipidemic, HFD, Triton X 100, Biochemical parameters.

 

 


INTRODUCTION:

Plants are the source of various potential phytoconstituents and they have been used as an alternative to develop potential drugs for treatment of various diseases such as hyperlipidemia, inflammation, diabetes and cancer etc1. Natural products from plants are a rich source used for centuries to cure various diseases and disorders2. Feronia limonia is belonging the family of Rutaceae Swingle (syns. F. elephantum Correa) commonly known as wood-apple3. Different parts of Feronia limonia plant such as fruit, bark root, leaves have shown potential therapeutic properties. The extract of different parts of Feronia limonia has been studied for anticancer, anti-inflammatory, anthelmintic activity4.

 

 

Hyperlipidemia is a metabolic disorder associated with alteration occurring serum lipid profile due to increase of total cholesterol (TC), low density lipoprotein cholesterol (LDL-c), Very low-density lipoprotein (VLDL) and triglycerides (TGs) with decrease of high density lipoprotein cholesterol (HDL-c) in the blood circulation. Exercise and use of lipid-lowering diets and drugs are mainly used for treating hyperlipidemia5. HMG- CoA reductase inhibitors (statins) mainly used for treatment of hyperlipidemia. The use of herbal medicine becoming popular due to less toxic and free from side effects than synthetic ones. Because of these advantages of herbal medicine several works have been reported to possess antihyperlipidemic activity such as Allium sativum6, Albizia amara7, Aloe succotrina8, Nymphaea nouchali9, Emilia sonchifolia10, and Pistacia khinjuk11. Thus, the aim of our study was to investigate the antihyperlipidemic activity of Feronia limonia leaves extract against HFD and Triton X 100 induced hyperlipidemia in rats.

 

MATERIALS AND METHODS:

Plant collection:

Feronia limonia leaves were obtained from local area of Anantapur, Andhra Pradesh. Leaves were dried at room temperature and shade dried leaves were made into powder.

 

Authentication:

The leaves of Feronia limonia belonging to the family Rutaceae was collected from local area of Anantapur district (India) and was identified and authentified by Dr. J. Raveendra Reddy, M. Pharm., Ph.D., Department of Pharmacognosy, Raghavendra Institute of Pharmaceutical Education and Research, Anantapur and voucher specimen (RIPER-04/12) is conserved in Department of Pharmacology, Raghavendra Institute of Pharmaceutical Education and Research, Anantapur.

 

Methanolic extract of leaves of Feronia limonia (MEFL):

The leaves of Feronia limonia was extracted by maceration process by using methanol as solvent. In the maceration procedure a total amount of powdered leaves were macerated with methanol for three days with occasional stirring. The extract was filtered, concentrated and it was dried using rotary evaporator. The percentage yield obtained was found to be 15%.

 

Experimental animals:

Male Wistar rats of 180-200g were obtained from Raghavendra Enterprises, Bengaluru to carry out the antihyperlipidemic activity. The animals had free access to standard commercial diet and water ad libitum and were housed in cages under standard laboratory conditions i.e., 12:12 hour light/dark cycle at 25±20C.

 

Ethical approval:

The Institutional Animal Ethics Committee (878/ac/05/CPCSEA/004/2012) has approved the experimental protocol at Post Graduate Department of Pharmacology, Raghavendra Institute of Pharmaceutical Education and Research, Anantapur, Andhra Pradesh, India.

 

Acute oral toxicity:

The limit test for acute toxicity was carried out at 2000 mg/kg oral dose of Feronia limonia extract in group of three rats as per OECD 423 guidelines. The rats were observed continuously for 2h for behavioural, neurological and autonomic profiles and, after a period of 24h, 72 h, and 7days, for any lethality, moribund state or death12.

 

High fat diet (HFD) induced hyperlipidaemia13:

Normal: Distilled water, 10ml/kg

Negative control: High fat diet for 30 days

Standard: High fat diet for 30days + Atorvastatin 20mg/kg, p.o. for 30 days

Test – I: High fat diet for 30 days + MEFL 250mg/kg, p.o. for 30 days

Test – II: High fat diet for 30 days + MEFL 500mg/kg, p.o. for 30 days

 

Triton X 100 induced hyperlipidaemia14:

Normal : Distilled water, 10ml/kg

Negative control: Triton X - 400mg/kg, i.p. for 24 hours

Standard: Triton X - 400mg/kg for 24hours + Atorvastatin 20mg/kg, p.o. for 24 hours

Test – I: Triton X – 400mg/kg for 24hours + MEFL 250 mg/kg, p.o. for 24 hours

Test – II: Triton X – 400mg/kg for 24hours + MEFL 500mg/kg, p.o. for 24 hours

 

Biochemical parameters:

Blood collection and separation of serum:

At the termination of each model, blood samples of all rats were collected directly from the retro orbital sinus puncture under inhaled diethyl ether anaesthesia. Blood samples were collected into plain sample bottles and allowed to clot at room temperature for 4 h before they were centrifuged (Remi) at 10,000 × g at the same temperature for 20min for separating the sera. The following biochemical parameters were estimated using standard diagnostic kits (ERBA diagnostics, Mallaustr, Mannheim/Germany) on the last day of treatment i.e., 30th day incase of high-fat-diet induced hyperlipidaemia, and after 24hours incase of Triton X 100 induced hyperlipidaemia.

 

Histopathology of adipose tissue:

White adipose tissue obtained from all rats were fixed in 10% formo-saline and fixed. After fixation, they were stained with hematoxylin and eosin and analyzed for adipocyte size with an Eclipse TE2000U Inverted Microscope with twin CCD cameras (Nikon, Tokyo, Japan)15.

 

Statistical Analysis:

All data were presented as Mean±SEM. One way analysis of variance (ANOVA) followed by Tukey's test was applied to determine the significance difference among groups. The results were considered significant at P < 0.05.

 

RESULTS AND DISCUSSION:

Biochemical parameters:

Effect of MEFL on HFD induced hyperlipidemic model:

Animals treated with HFD alone for 30 days showed a significant (p<0.05) decrease in HDL-c and increase in LDL-c, triglycerides, cholesterol when compared to control group. Animals treated with atorvastatin (20 mg/kg, p.o.) and MEFL (500mg/kg, p.o.) per se significantly decreased serum total cholesterol, triglycerides, LDL-c, and increased HDL-c levels as compared to high fat diet control16. Animals receiving low dose of MEFL (250mg/kg, p.o.) for 30 days incase of HFD model caused a significant (p<0.05) and dose-related elevation in the serum HDL but effect was less as compared to animals receiving high dose (500mg/kg, p.o.) of MEFL. HDL inhibits the uptake of LDL-c by the arterial wall facilitates the transport of cholesterol from peripheral tissue to the liver, where its catabolised excreted from the body resulting lowering the LDL level17. The hypolipidemic activity of natural products can be correlated to the presence of flavonoids due to their properties of inhibiting cholesterol biosynthesis1,18. Yang Liu et al reported that hypolipidemic activity was observed may be due to presence of total triterpenoids in extract19. The results are depicted in Table 1.

 

Effect of MEFL on SGOT and SGPT:

Animals treated with HFD alone for 30 days caused a significant (p<0.001) increase in the serum SGOT, SGPT when compared to control group. Animals treated with atorvastatin (20mg/kg, p.o.) in case of HFD model for 30 days caused a significant (p<0.001) reduction in the serum SGOT, SGPT when compared to negative control group. Animals receiving high dose of MEFL (500mg/kg, p.o.) for 30 days incase of HFD model caused a significant (p<0.001) and dose-related reduction in the serum SGOT, SGPT when compared to low dose group and negative control group (Table 1).

 

Tissue parameters on HFD induced hyperlipidemic model:

Treatment with only HFD for 30 days showed significant (p<0.001) decrease in SOD, glutathione when compared to normal group. Treatment with atorvastatin (20mg/kg, p.o.) for 30 days showed significant increase (p<0.01) in superoxide dismutage (SOD), glutathione, catalage, and decrease in lipid peroxidation when compared to negative control group. Treatment with low dose (250 mg/kg, p.o.) and high dose (500mg/kg, p.o.) of MEFL for 30 days caused a significant increase (p<0.01) in SOD compared to negative control group however, high dose showed promising effect on SOD, glutathione, lipid peroxidation could be suggesting that, extract showed dose related effect on different tissue parameters. (Fig. 1).


 

Table 1: Effect of MEFL on HFD induced hyperlipidemic model

Group

Serum HDL-c

(mg/dL)

Serum LDL-c (mg/dL)

Serum triglyceride (mg/dL)

Serum total cholesterol (mg/dL)

SGOT

(U/L)

SGPT

(U/L)

Normal

43 ± 1.6

45 ± 1.1

80 ± 1.2

107 ± 2.2

32 ± 1.1

34 ± 2.7

Negative control

26 ± 1.7#

103 ± 0.7##

131 ± 1.7#

155 ± 1.4#

100 ± 2.0#

105 ± 2.1#

Standard

49 ± 2.2**

43 ± 1.5**

94 ± 1.3***

111 ± 1.3***

62 ± 2.0*

62 ± 1.9*

Test 1

43 ± 2.0*

75 ± 3.3**

121 ± 1.5*

143 ± 1.9**

43 ± 1.9*

60 ± 2.6*

Test 2

58 ± 3.9**

50 ± 2.7**

114 ± 1.5***

132 ± 1.4***

38 ± 3.0*

46 ± 2.3*

All values are expressed as mean ± S.E.M, (n=6) in each group; ## p<0.001 when compared to control; # p<0.05 when compared to Control ; ***p<0.001 when compared to negative control ; **p<0.05 when compared to negative control *p<0.1 when compared to negative control.

 

 

Fig. 1: Effect of MEFL on tissue parameters.

All values are expressed as mean ± S.E.M, (n=6) in each group; ## p<0.001 when compared to control; # p<0.05 when compared to Control; ***p<0.001 when compared to negative control; **p<0.05 when compared to negative control *p<0.1 when compared to negative control.

 


Histopathology of adipose tissue on HFD induced hyperlipidemic model:

The adipose tissue of animals which were treated with HFD alone for 30 days showed increase in cell size when compared to animals of normal group. The adipose tissue of animals which were treated with low dose (250 mg/kg, p.o.) and high dose (500 mg/kg, p.o.) of MEFL for 30 days showed significant and dose-related decrease in cell size when compared to animals of negative control group (Fig. 2).

 

 

                            (A)                                                    (B)

                          (C)                                                      (D)

Fig. 2: Adipose tissue of (A) Control; (B) Negative Control;

(C) HFD + MEFL(250mg/kg) treated;

(D) HFD + MEFL(500mg/kg)treated

 

Triton X-100 induced hyperlipidaemia model:

Effect of MEFL on Triton X-100 induced hyperlipidemic model:

Triton X-100 has been commonly used to induce the acute hyperlipidemia by blocking the removal of triglyceride and cholesterol in various animal models20. Animals treated with Triton X-100 (400mg/kg, i.p.) alone for 24 hours showed a significant (p<0.01) decrease in HDL-c, and increase in LDL-c, triglycerides, and cholesterol when compared to normal group and animals treated with atorvastatin (20mg/kg, p.o.) of Triton X-100 model for 24 hours caused a significant (p<0.001) elevation in the serum HDL-c and decrease in LDL-c, triglycerides, and cholesterol when compared to negative control group21. Animals receiving low dose of MEFL (250mg/kg, p.o.) for 24 hours in case of Triton X-100 model caused a significant (p<0.05) and dose-related elevation in the serum HDL-c when compared to negative control group. Animals receiving high dose of MEFL (500mg/kg, p.o.) for 24 hours in case of Triton X-100 model caused a significant (p<0.001) and dose-related elevation in the serum HDL-c and decrease in LDL-c, triglycerides, and cholesterol when compared to low dose group and negative control group (Table 2). It reduces the LDL-c, cholesterol, triglycerides and raise HDL-c level in serum may be due to the inhibition of lipid peroxidation processes22. From the results, it was observed that the extract a significantly lower plasma LDL-c cholesterol levels in the animals which likely to be cardio-protective potential of the extract23,24.

 

Table 2: Effect of MEFL on Triton -X induced hyperlipidemic model

Group

Serum HDL-c

(mg/dL)

Serum LDL-c (mg/dL)

Serum tri-glyceride (mg/dL)

Serum total cholesterol (mg/dL)

SGOT (U/L)

SGPT (U/L)

Normal

41 ±

1.8

47 ±

0.8

84 ±

1.4

105 ±

1.2

30 ± 2.1

35 ± 2.5

Negative control

23 ± 0.8#

107 ± 0.5##

138 ±

1.3#

158 ±

1.5#

110 ± 2.5#

113 ± 2.1#

Standard

42 ± 2.1**

51 ± 0.6**

100 ± 0.8****

114 ± 1.6****

63 ± 2.1*

65 ± 2.3*

Test 1

37 ± 1.2*

91 ± 2.2**

131 ±

1.5*

155 ±

1.2

54 ± 2.1*

63 ± 1.6*

Test 2

53 ± 3.2**

68 ± 1.7**

121 ± 1.1****

146 ± 1.7***

41 ± 2.3*

56 ± 2.3*

All values are expressed as mean ± S.E.M, (n=6) in each group;

## p<0.001 when compared to control; # p<0.05 when compared to control; ***p<0.001 when compared to negative control;

**p<0.05 when compared to negative control

*p<0.1 when compared to negative control.

 

Effect of MEFL on SGOT and SGPT:

Animals treated with Triton X-100 (400mg/kg, i.p.) alone for 24 hours caused a significant (p<0.001) elevation in the serum SGOT and SGPT, when compared to normal group. Animals treated with atorvastatin (20mg/kg, p.o.), low dose of MEFL (250 mg/kg, p.o.), and high dose of MEFL (500mg/kg, p.o.) in case of Triton X-100 model for 24 hours caused a significant (p<0.001) reduction in the serum SGOT, and SGPT when compared to negative control group. The Results of SGOT and SGPT are shown in Table 2.

 

Tissue parameters on Triton X-100 induced hyperlipidemic model:

Treatment with only Triton X-100 (400mg/kg, i.p.) for 24 hours showed significant (p<0.001) decrease in SOD, catalase, glutathione and increase in lipid peroxidation when compared to normal group. Treatment with atorvastatin (20mg/kg, p.o.) incase of Triton X-100 model for 24 hours showed significant (p<0.1) increase in SOD, catalase, glutathione and decrease in lipid peroxidation when compared to negative control group. Treatment with low dose (250mg/kg, p.o.) of MEFL for 24 hours incase of Triton X-100 model caused a significant (p<0.1) and dose-related increase in SOD, catalase, glutathione and decrease in lipid peroxidation when compared to negative control group. Treatment with high dose (500mg/kg, p.o.) of MEFL for 24 hours incase of Triton X-100 model caused a significant (p<0.05) and dose-related increase in SOD, catalase, glutathione and decrease in lipid peroxidation when compared to negative control group (Fig. 3).


 

 

Fig. 3: Effect of MEFL on tissue parameters.

All values are expressed as mean ± S.E.M, (n=6) in each group; ## p<0.001 when compared to control; # p<0.05 when compared to Control; ***p<0.001 when compared to negative control; **p<0.05 when compared to negative control *p<0.1 when compared to negative control.

 


Histopathology of adipose tissue on Triton X-100 induced hyperlipidemic model:

The adipose tissue of animals which were treated with Triton X-100 (400mg/kg, i.p.) alone for 24 hours showed increase in cell size when compared to animals of normal group. The adipose tissue of animals which were treated with atorvastatin (20mg/kg, p.o.) incase of Triton X-100 model for 24 hours showed significant decrease in cell size when compared to animals of negative control group. The adipose tissue of animals which were treated with low dose (250mg/kg, p.o.) and high dose (500 mg/kg, p.o.) of MEFL for 24 hours incase of Triton X-100 model showed significant and dose-related decrease in cell size when compared to animals of negative control group (Fig. 4).

 

                            (A)                                                    (B)

 

                          (C)                                                      (D)

Fig. 4: (A) Control; (B) Negative Control ; (C) Triton X 100 + MEFL (250mg/kg) treated; (D) Triton X 100 + MEFL (500mg/kg) treated.

CONCLUSION:

It can be concluded that ethanolic Feronia limonia leaves extract showed significant anti hyperlipidemic activity in rats and hence it could be a potential herbal medicine as adjuvant with existing therapy for the treatment of hyperlipidemia. Further studies are required to isolate the active constituents involved in antihyperlipidemic activity of Feronia limonia.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 06.08.2020           Modified on 17.06.2021

Accepted on 13.12.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(6):2407-2412.

DOI: 10.52711/0974-360X.2022.00400