Antihyperlipidemic Effect of Syrian Mesquite ( Prosopis farcta ) Root in High Cholesterol Diet–Fed Rabbits

Abstract

Prosopis farcta root has been proposed as an efficacious natural drug for cardiovascular disorders in traditional medicine. The present study evaluates the efficacy of aqueous extract of Prosopis farcta root on experimental atherosclerosis development in rabbits with high cholesterol diet–induced hypercholesterolemia. Serum lipid parameters were significantly increased in the high cholesterol diet groups in comparison with the normal control group (P < .050). Histopathological findings revealed that atheromatous plaques were formed in both thoracic and abdominal aorta of hypercholestrolemic rabbits. Treatment with Prosopis farcta root significantly reduced total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, and very low density lipoprotein levels compared to high cholesterol diet rabbits (P < .050). This finding may reflect a reduction of chest pain or the beneficial effects of this plant root extract on cardiovascular health. The present study can serve as a basis for future investigations on the other effects of this plant on cardiovascular health.

Atherosclerosis is a cause for more than 50% of cardiovascular deaths in developed and developing countries. ¹ In Iran, 36% of deaths are related to atherosclerosis diseases, particularly due to myocardial infarction. ²

Prosopis is a genus of flowering plants in the Fabaceae family. Species of Prosopis are often spiny trees, 2 to 3 m or taller or small shrubs, well adapted to warm weather and drought. Their wood is usually hard, dense, and durable. The fruits are pods and may contain large concentrations of sugar. These plants are located commonly in the United States, North Africa, Southwestern Asia, and West to Middle East. ^3,4 Prosopis farcta, commonly known as Syrian mesquite, is native to Asia and distributed from India to Iran. This plant has acceptable capacity to grow under drought condition and is an invasive weed that is difficult to control. ⁵ Species of this genus have several functions. They have been utilized for gum, paint, cordage, ⁶ as dietary supplements for feeding ruminants, ⁷ as well as medicinal purposes. Beans and leaves of Prosopis farcta have been used for treatment of some diseases and disorders in traditional medicine including diabetes, inflammatory diseases, wounds and skin disorder, prostate disorders, measles, urinary diseases, diarrhea, and colds. ^{8 –10} Also, Prosopis farcta can be used to reduce cardiac or chest pain and for the management of cardiovascular disorders. ¹ To our knowledge, there is no academic investigation illustrating the therapeutic effect of this medicinal plant in rabbit model of cardiovascular disorders. The present study was conducted to investigate the efficacy of Prosopis farcta root extract in an experimental model of hypercholesterolemia and atherosclerosis.

Materials and Methods

Plant Material and Extract Preparation

Roots of Prosopis farcta were collected from Kermanshah province, west of Iran, and kept in standard conditions. The root parts of Prosopis farcta were powdered after drying. A total of 100 grams of plant powder was added to 1000 mL boiling water and mixed for 15 minutes. The whole content of the mixture was first filtered through an ordinary filter paper, and the filtrate was then passed through a No.1 Whatman filtering paper. The solution was transferred into a rotary evaporator for removing surplus water and about 80% of water was removed. The final solution was kept in a water bath at 30°C.

Animals and Experimental Design

Thirty-two young male NZW rabbits obtained from Pasteur Institute of Tehran and weighing about 1.5 to 2 kg were utilized. After arrival in the laboratory, they were kept under standard conditions of temperature (23 ± 1°C), relative humidity (55 ± 10%), 12-hour dark and 12-hour light cycle, and were fed with ground laboratory Chow 5321 (Ralston Purina Co, St Louis, MO). Ethical rules of the investigation on animals were considered carefully, and the ethic committees for animal study accepted the protocol of the present study. They were then randomly divided into 4 groups. The first group as control was fed by standard pellet and other groups were received 2% cholesterol amounts daily. ^11,12 NZW rabbits fed with high cholesterol diet were treated daily by distilled water, simvastatin (0.6 mg/kg), ¹³ or Prosopis farcta root extracts (500 mg/kg/day) orally by gavage for 30 days.

Biochemical Analysis

Blood samples were taken from the marginal ear vein of unanesthetized animals after the adaptation period (day 0) and at the end of treatment (day 30) with the simvastatin and plant extracts.

Total cholesterol, triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low density lipoprotein (VLDL) of serum were measured enzymatically using a quantification kit (Roche Diagnostics, Mannheim, Germany). The formula VLDL = TGs/5 was utilized for VLDL measurement.

Microscopic Assessment of Atherosclerotic Damage

In order to perform microscopic evaluation, rabbits were killed by chloroform (overdose) at the end of the investigation and their aortas were separated up to diaphragm. The aortas were then divided into the proximal, middle, and distal segments. The fixed segments in formalin 10% were embedded in paraffin and stained with hematoxylin and eosin. The histopathological assessment was performed by one who was blind to the treated groups. All sections were evaluated microscopically for fatty streak, foam cells, and extracellular lipid core, indicating atheromatous plaque factors. ^12,13

Statistical Analysis

Statistical analysis was performed using the SPSS statistical package version 16.0. The analysis of the variance appropriate for the design was carried out to detect the significance of the differences (P < .05) between the treatment and control groups. Duncan’s multiple range test was also performed to compare the significant difference between groups. Difference from the control was considered significant. All the values presented in this article are expressed as the means ± standard error of the mean.

Results

The results obtained from the current study demonstrated that feeding the rabbits with high cholesterol diet for 30 days resulted in a remarkable increase in serum lipid parameters including TG, total cholesterol, LDL, and VLDL. Daily administration of the standard drug, simvastatin, leads to reduction of TG, total cholesterol, LDL, HDL, and VLDL levels significantly in comparison with a high cholesterol diet (P < .05). Likewise, the results indicate that serum TG, total cholesterol, LDL, HDL, and VLDL levels decreased in the rabbits treated with Prosopis farcta root extract 500 mg/kg/day in a significant manner compared to a high cholesterol diet (P < .05). Alteration in total cholesterol, TG, HDL, LDL, and VLDL values of rabbits with hypercholesterolemia treated with simvastatin and plant root extracts is illustrated in Table 1.

Table 1.

Plasma Lipid Profiles in High Cholesterol Diet (HCD)–Fed Rabbits Treated With Prosopis farcta Root Extract for 30 Days^a.

Groups	TG (mg/dL)	Cholesterol (mg/dL)	LDL (mg/dL)	HDL (mg/dL)	VLDL (mg/dL)
Control-fed + vehicle	63.83 ± 6.94	32.5 ± 7.66	11.16 ± 3.86	21.833 ± 11.37	16.7 ± 7.04
HCD-fed + vehicle	125.25 ± 12.31b	1886.83 ± 170.39b	316.5 ± 99.45b	437.5 ± 59.01b	72.8 ± 26.57b
HCD-fed + simvastatin (0.6 mg/kg)	76.66 ± 10.13c	240.33 ± 30.26b,c	168.5 ± 50.05b,c	109.16 ± 21.59b,c	17.43 ± 8.67c
HCD-fed + Prosopis farcta extract	72.16 ± 14.02c	207.16 ± 39.44b,c	195.2 ± 40.62b,c	123.83 ± 23.82b,c	18.65 ± 6.28c

Abbreviations: HCD, high cholesterol diet; TG, triglyceride; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very low density lipoprotein.

^aResults are expressed as mean ± standard error of the mean.

^bSignificantly different from the normal group at P < .05.

^cSignificantly different from the HCD-fed + vehicle group at P < .05.

Histopathological findings revealed that atheromatous plaques were formed in both thoracic and abdominal aorta in hypercholestrolemic rabbits induced by the high cholesterol diet. The typical plaques were composed of fatty streaks with lipid-laden macrophages (foam cells) and smooth muscle cells. More severe and advanced lesions showing extensive necrotic area, calcification, and even cartilage metaplasia were also observed. ¹⁴ Thoracic aorta expressed more severe lesions with necrotic centers compared to abdominal aorta (Figure 1).

Figure 1.

The aortic cross-section in the rabbits fed with high cholesterol diet. The aortic wall in the thoracic area showed severe plaques composed of fatty streaks with lipid-laden macrophages (foam cells) and smooth muscle cells, as well as necrotic area with calcification (arrows). Coronary stenosis by hematoxylin-eosin staining.

The simvastatin-treated group showed mild atheromatic lesions in abdominal aorta with smaller plaques (Figure 2), whereas the plant root extract group demonstrated more profound plaques in abdominal aorta. Microscopic evaluations showed that the closer to the thoracic aorta the more complicated lesions with necrotic area and fibrous cap formation in plant extract treated group (Figure 3).

Figure 2.

The aortic cross-section in the high cholesterol diet–fed rabbits treated with simvastatin. The aortic wall showed mild atheromatic lesions in abdominal aorta with smaller plaques. Coronary stenosis by hematoxylin-eosin staining.

Figure 3.

The aortic cross-section in the high cholesterol diet–fed rabbits treated with Prosopis farcta root extract. The aortic wall near abdominal aorta showed more profound plaques in comparison with the simvastatin group. Coronary stenosis by hematoxylin-eosin staining.

Discussion

The current investigation demonstrated that treatment with Prosopis farcta root extract can significantly reduce serum levels of total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol (P < .050; Table 1). However, microscopic studies of aortic wall demonstrated that consumption of the plant root extract in hypercholestrolemic rabbits has no significant effects on decreasing the size of atherosclerotic plaques after being formed in aortic wall, and it might help the plaques to stabilize.

Rabbits as the most widely used animals in atherosclerosis researches have undergone various study designs with different lesional results. This animal exhibits hypercholesterolemia within a few days of administration of a high cholesterol diet. Rabbit is also more prone to atherosclerosis due to high cholesterol diets than are mice and rats. In rabbits, atherosclerotic lesions are distributed predominantly in aortic arch and thoracic aorta, whereas in humans, plaques are more abundant in abdominal aorta. Diets with more than 2% cholesterol can cause hypercholesterolemia and atherosclerotic lesions in short duration. It has been shown that the extent of damage caused by atherosclerosis is proportional to the amount of cholesterol consumed. ^15,16

Natural remedies have long been used for the management of different diseases and pathologic conditions. ^{17 –19} Composition of diet has an important role in controlling cholesterol levels in blood. Singh et al and others have reported that plant-based diets as therapeutic applications, if they are recognized to have minimal or no side effects, can have an beneficial impact on health. ^20,21 It has been known that atheromatous plaque rupture is the major clinical complication in atherosclerosis. Dietary natural supplements with lipid lowering effects have been shown to reduce macrophage penetration and help stabilize the plaque. ^20,21

Prosopis farcta has long been used as a therapeutic agent in order to reduce cardiac or chest pain and for managing cardiovascular disorder in Iran. Some reports showed dose-dependent and endothelium-dependent relaxation effects of Prosopis farcta on thoracic aorta of mice. ^1,2 Alcoholic extract of Prosopis farcta leaves reduced blood pressure in vivo and augmented contraction of heart in in vitro experiments. ¹ Prosopis farcta beans extract has demonstrated protective effects against acetaminophen-induced hepatotoxicity in an animal model. ²² Also, according to the study of Mollashahi et al, ²³ Prosopis farcta’s pod aqueous and ethanol extracts possess neuroprotective effect on rats. The findings of Nitesh and Desai showed that Prosopis julifora leaves have antihyperlipidemic effect and reverse the hypercholesterolemic conditions in hypercholesterolemic male Albino rats. ²⁴

Oxidative process plays a crucial role in the development of atherosclerosis, and it is suggested that establishment of oxidative-mediated modification of LDL-C is considered as the initial stage in atherogenesis. In addition, Regnström et al showed that susceptibility of LDL-C to oxidation is associated with the severity of atherosclerotic lesions. ²⁵ Accumulative body of evidence has suggested that suppression of oxidative stress and lipid oxidation possesses an essential contribution on prevention and remission of atherosclerotic damage. ²⁶ Various extracts obtained from Prosopis farcta have demonstrated remarkable antioxidant properties using high-performance liquid chromatography. ²⁷ The antioxidant activity of Prosopis farcta has a significant role in improving serum lipid parameters in hypercholesterolemic rabbits. Phytochemical investigations have demonstrated that flavonoid components are the main active constituents of this plant. ²⁸ Accumulating body of evidence has confirmed the remarkable antihyperlipidemic and antiatherosclerotic potential of flavonoids; thus, it is suggested that these components are the agents responsible for the therapeutic effects of Prosopis farcta in cardiovascular disorders. ²⁹

Conclusion

Overall, treatment with Prosopis farcta root significantly improves serum lipid profiles including total cholesterol, HDL, TG, LDL, and VLDL levels in hypercholesterolemic rabbits, compared to high cholesterol diet–fed control rabbits. This finding may reflect reduction of chest pain or the beneficial effects of this plant root extract on cardiovascular health. However, histopathological findings revealed that reduction in the size of atherosclerotic plaques in aortic wall of the plant-treated group are not remarkable, compared to a high cholesterol diet. Further experimental studies evaluating the preventive effect of this plant on atherosclerosis are needed in order to confirm the long history of traditional use in cardiac diseases. This study can serve as a basis for future investigations on the other effects of this plant on cardiovascular health. Also, phytochemical studies are suggested in order to identify active components of this plant responsible for its therapeutic effect in cardiovascular diseases. Well-designed randomized clinical trials evaluating the efficacy and safety profile of this natural drug in patients are required.