Effects of Hypericum perforatum on serum lipid vascular systems in mice

Zena S. Hamed1, Rana R. Abed2, Mohammed S. Almashhadany3 and Marwan M. Merkhan1

1Department of Pharmacology and Toxicology, College of Pharmacy, 2Department of Chemistry, College of Education of Pure Science, University of Mosul, 3Assalam Teaching Hospital, Nineveh Health Directorate, Mosul, Iraq

zenasattam@uomosul.edu.iq, 0000-0003-1425-0265

ranaalbustani@uomosul.edu.iq, 0000-0001-8530-1045

dralmashhadany68@gmail.com, 0000-0002-4397-3799

marwanmerkhan@uomosul.edu.iq, 0000-0002-1694-2540

Abstract

Herbal products are increasingly used against lipid to attenuate its negative impact on vasculature system. However, there are uncertainty regarding the best administration approaches and preparation of herbal product to be used due to variation in absorption rate and extent with subsequent impaction on the lipid levels. The aim of the present study was comparing the pharmacological and analytical aspects of soaked versus powder form of Hypericum perforatum; a well-known anti-hyperlipidemic herbal product, using atorvastatin drug as a reference drug for comparison in addition to controlling groups (on/off lipid diet) handled as an additional control group. To conduct this research, the hyperlipidemia mice models were created by exposing the mice to a fatty diet and using H. perforatum and atorvastatin for evaluation. The plant extracts were also analyzed for active constituents. The results indicate that the plant analysis detected the presence of different antihyperlipidemic agents and the plant has effectively reduced plasma lipid parameters in exposed mice compared to control group; especially when taken forms of the plant was used. The study concluded that; soaked H. perforatum extract shown an effective reduction of lipid parameters and analysis of which revealed the presence of herbal active constituent which might be utilized in industrial pharmacy for new drug innovation.

Keywords: H. perforatum, Chromatography, Lipid, Atherosclerosis, Atorvastatin                                                                           

تأثیرات العرن المثقوب على دهون مصل الدم فی الأوعیة الدمویة للفئران

زینة سطام حمد، رنا رمزی عبد، محمد سالم المشهدانی و مروان محمد میرخان

1فرع الأدویة والسموم، کلیة الصیدلة، 2قسم الکیمیاء، کلیة التربیة للعلوم الصرفة، جامعة الموصل، 3مستشفى السلام التعلیمی، مدیریة صحة نینوى، 4فرع الأدویة والسموم، کلیة الصیدلة، جامعة الموصل، الموصل، العراق

الخلاصة

تستخدم المنتجات العشبیة على نحو متزاید ضد الدهون لتخفیض تأثیرها السلبی على نظام الأوعیة الدمویة. ومع ذلک، هناک عدم إثبات بالدلیل القاطع بشأن أفضل طریقة لإعطاء المستخلصات العشبیة وتحضیر المنتجات العشبیة لاستخدامها بسبب الاختلاف فی معدل وکمیة الامتصاص والتأثیر اللاحق على مستویات الدهون. وکان الهدف من هذه الدراسة هو مقارنة الجوانب الدوائیة والتحلیلیة للنقیع مقابل شکل مسحوق من العرن المثقوب ویعرف أیضا فی الغرب باسم نبتة القدیس یوحنا المثقبة؛ منتج عشبی معروف مضاد للفرط فی الدهون، باستخدام عقار أتورفاستاتین کدواء مرجعی للمقارنة بالإضافة إلى مجموعات التحکم (یتناول / لا یتناول الطعام الدهنی) التی یتم التعامل معها کمجموعات مراقبة إضافیة. لإجراء هذا البحث، تم إنشاء نموذج فرط الدهون لدى الفئران المختبریة عن طریق تعریض الفئران لنظام غذائی دهنی واستخدام عشبة القدیس یوحنا وأتورفاستاتین للتقییم. کما تم تحلیل مستخلص عشبة القدیس یوحنا للتأکد من وجود المادة الفعالة. وتشیر النتائج إلى أن تحلیل النبات کشف وجود عوامل مختلفة مضادة لفرط الدهون والعشبة قد خفضت بشکل فعال مؤشرات الدهون فی البلازما فی الفئران المعرضة للعشبة بالمقارنة مع مجموعة التحکم; وخصوصا عندما تم استخدام شکل النقیع المائی من النبات. وخلصت الدراسة إلى أن استخدام علیقة عشبة القدیس یوحنا أظهرت انخفاضا فعالا من المعلمات الدهنیة والتحلیل الذی کشف عن وجود مواد عشبیة فعالة التی یمکن استخدامها فی الصیدلة الصناعیة لابتکار عقارات جدیدة.

Introduction

The vast majority of clinical research has been done aimed at discovering drug treatments to avoid the atherosclerosis formation cycle. The use of herbal and natural medicines increased commonly because of fewer side effects and easier availability (1). The Clusiaceae is a family of about 400 flowering plant species and Hypericum perforatum (H. perforatum) is one genus of them. Different species of H. perforatum occur worldwide with a diverse geographic range including North America, Europe, Asia, Russia, India, and China (2). Hyperforin, hypericin, pseudohypericin, flavonoids, and tannins are considered the main active constituents. H. perforatum has several pharmacological activities including antipyretic, analgesic, antispasmodic, antiviral, and antimicrobial activities and its role in the treatment of psychological and neurological disorders such as depression and migraine (3).

In the health insurance industry, efforts have long been made to find a solution for preventing or treating atherosclerosis by managing elevated lipid levels which play an important role in the formation of atheroma plaque (4). Unfortunately, none of these attempts have been fully effective, and the concerns about the safety of prescription drugs have generated a growing desire for patients to use natural cholesterol-lowering products. Also, the scientists are searching for cholesterol-lowering and antiatherogenic activities of various plant products and their active ingredients in order to prevent or regulate atherosclerosis (5,6). Which is a complex inflammatory disease (7,8). And significant risk factors for atherosclerosis are cholesterol and triglyceride; this plays an important role in initiating coronary artery disease and atheroma plaque formation (9). The etiology of these disorders is complicated by oxidative stress. It has been discovered that hyperlipidemia induces oxidative stress, which causes cellular damage (10). Since atheroma plaque in diseased arteries usually contains inflammatory cells, many people now assume there is a connection between the inflammatory process and coronary atherosclerosis (11).

The effect of different forms of H. perforatum (powder and soaked) on cardiovascular disorders were explored in this research. And we have used atorvastatin, a chemical drug in the prevention and treatment of atherosclerosis, to equate its effect with that of HP. Also in this study, the lipid-lowering and antioxidative properties of H. perforatum were investigated in terms of lipid profiles and histologically in mice fed a high-fat diet (12).

Materials and methods

Preparation of extracts

The collected plant was dried at room temperature for about 14 days. Then, this was grinded and kept in a dark glass bottle before extraction. The natural products were extracted from the stalk of the H. perforatum plant using the continuous soxhlet apparatus method. It is a technique in which different polar solvents are used sequentially to extract the active ingredients from plants and obtaining pure ore free of impurities. Three types of different polarity solvents have been tested petroleum ether, chloroform and ethanol, the separation process was carried out at different temperatures according to the boiling point of each solvent provided that it does not exceed 80ºC. The sample was soaked for 24 hours in continuous soxhlet apparatus, as 250 ml of solvent was used for every 20 gm of the ground dried sample, the extraction process lasted 24 hours for each type of solvent until the color of the solvent disappeared, after obtaining the crude extract, it was filtered by filter paper and then evaporated to half its volume by ( Rotary Vacuum Evaporator) to obtain concentrated extracts it was placed in tightly closed dark bottles and kept in the refrigerator until the subsequent operations.

High performance liquid chromatography (HPLC)

A weighted quantity of extract was dissolved in HPLC grade methanol for HPLC analysis. The sample was analyzed by HPLC, Quaternary Gradient Pump, Auto sampler model: (S5200), Detector: UV S2340 and column oven. The mobile phase was A and B. In A phase Methanol : DW : Acetic acid at ratio 85:13:2, while B Methanol : DW : Acetic acid at ratio 25:70:5. The column is C18-ODS 25cm*4.6mm and detector UV 280-360 nm at flow rate 1 ml/min (13).

Gas chromatography (GC)

Sample was analyzed by using Gas chromatography device, model (Shimadzu 2010), Japan, using an ionized flame detector and using a capillary separation column in lengths 0.25mm*0.25um*30m where the temperature of the injection area and the detector were 330-280ºC respectively. Whereas, the temperature of the separation column was gradually starting from 120-280ºC at a rate of 8 degrees/min). Inert nitrogen gas was used as a carrier gas at a rate of 100KPa (14).

Experimental groups

A healthy adult albino Swiss mice weighing 20-30 g of both sex was randomly divided into five experimental groups of five mice each. Group 1, negative control group, control diet + water ad libitum. Group 2, positive control group, fatty diet + water with 1% H₂O₂. Group 3, hyperlipidemia group, control diet 50% + powder form of H. perforatum plant 25% + forage wheat 25% with water ad libitum. Group 4, hyperlipidemia group, control diet + soaked of H. perforatum taken orally. Group 5, hyperlipidemia group, control diet + water ad libitum + Atorvastatin drug 7mg/kg orally. The dried plant was grinded to produce the powder, and the soaked solution was produced by soaking 30g of the powder of H. perforatum plant overnight in boiling water 2L then filtered. Normal saline as a solvent for preparation of drug suspensions, 20 mg of atorvastatin tablet dissolved in 10ml of normal saline. Then the mice were administered 0.2 ml of it by intra-gastric gavage once per day for 30 consecutive days.

Induction of hyperlipidemia

Hyperlipidemia was induced by feeding the mice a fatty diet containing: wheat flour 34%, barley flour 20%, corn flour 25%, fatty milk powder 10%, animal protein 10%, salt 1% and vegetable oil and water for kneading with 1% water with H₂O₂ for a period of three months (15). After 12 weeks, the triggered hyperlipidemia demonstrated itself in the mice fed a fatty diet as a significant rise in mean triglyceride (TG), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels (P<0.05) when compared to those fed a control diet containing: animal protein 10%, soybean cake 20%, forage yellow corn 55%, forage wheat 14%, salt 0.5%, lime 0.5% and tap water.

Measuring the serum lipids

The lipid profile parameters were measured three times on separate days, first at baseline and then at the twelfth week and at the end of the sixteenth week. Blood samples after 12 h of fasting were collected from the vein of the eye of the mice. The serum was obtained by centrifuging the blood samples at 3000 rpm for 15 min then the lipid profile parameters were measured using special kits (Biolabo, Zenith Lab Co., Ltd). Atherosclerosis index (AI) was calculated according to the following formula: AI=TC/HDL-C (16).

Evaluation of aortic atherosclerosis

At the end of the experiment, the cervical dislocation was used to sacrifice all of the animals for histopathological research. After incising the chest wall, the aorta was cut and removed, then rinsed with ordinary saline solution before being stored in 10% formalin for the next stage. Harris's Hematoxylin-Eosin (HE) procedure was used to dye aortic tissue cuts.

Statistical analysis

Differences between groups were statistically evaluated by one-way ANOVA, and the differences between the means of groups were differentiated by the Least Significant Difference (LSD) test. All data is provided as mean ± standard deviation (mean±SD). P

Results

Chromatographic analysis

The key goal of this part was to use GC and HPLC to evaluate the chromatograms of standard chemical compounds that are commonly present in medicinal plant samples by using more than one mobile phase. These chromatography fingerprints of standard compounds could be used as benchmarks for reference when analyzing unknown compounds in any plant sample, both qualitatively and quantitatively. To measure the consistency and quantities of the various chemical constituents found in each herbal sample should be according to these criteria. Providing a good indicator of its potential therapeutic effectiveness. The HPLC and GC chromatogram of H. perforatum plant extract sowed in Figure 1 and the retention times of extracts constituent’s peaks showed in (Table 1).

Figure 1: Analysis profile of H. perforatum plant extract. HPLC (A) and GC (B).

Table 1: Retention time of H. perforatum extract constituent’s peaks by HPLC and GC

Biochemical factors

Mice exposed to a fatty diet and 1% H₂O₂ in drinking water for three months had significantly elevated levels of TC, TG, and LDL-C, contrasted with the negative control group and hyperlipidemic groups that administer powder and soaked forms of H. perforatum plant in daily doses orally and this elevated lipid profile then was significantly reduced (Figure 2). After one month of therapy in groups treated with powder and soaked forms of H. perforatum plant and with atorvastatin, serum HDL-C concentrations increased significantly 65.66±4.16, 81.33±3.21, 57.00±2.00 mg/dl respectively, compared to the negative and positive control 54.33±4.04 and 31.33±1.52 mg/dl groups respectively. HDL-C in the group taken soaked form was increased significantly more than in the group taken powdered form. While the soaked form of the H. perforatum plant group significantly reduced serum TC, TG and LDL-C concentration in comparison with the powdered form of the plant (Figure 2).

Figure 2: Plant extract reduced plasma lipid profile in experimental animal comparing soaked versus powder form versus atorvastatin in comparison to positive and negative control. Data displayed as mean ± standard deviation, * mean significant at P<0.05.

Histopathological study

A representative image of atherosclerotic alterations in the aortas from the five groups (Figure 3). The aortas of mice in group 1 on a regular diet had no atherosclerotic plaques. On a fatty diet and water with 1% H₂O₂ for 90 days, the grade of fatty streak formation in the aorta of group 2 was significantly raised. The amount of atherosclerosis was significantly reduced in the mice on powder form of H. perforatum plant with regular diet in group 3 and in group 4 mice on H. perforatum plant soaks taken orally with regular food for 30 days respectively, compared to the animals in group 2, and mice in group 5 treated with atorvastatin drug showed a lowering in the amount of atherosclerosis in contrast to group 2. The group has taken soaked form of H. perforatum plant shown a better reduction in the amount of atherosclerosis in contrast with the group taken powder form of H. perforatum plant with regular diet.

Figure 3: A representative image for mice aorta showing different histopathological changes using H&E stain, E (100X) others (400X). Arrows indicate thickness of layers (↔), endothelium (→) and smooth muscle cells (→). A: photomicrograph of mice aorta of negative control group shows normal architecture representing by the thickness of layers (↔), endothelium (→) and smooth muscle cells (→). B: photomicrograph of mice aorta of positive control group shows thickening of layers (↔), the present foam cells in intimal and medial layers (→) and hypertrophy of smooth muscle fibers (→).C: photomicrograph of mice aorta of positive control group shows a projection of intimal to the lumen as atherosclerotic plaque (↔), presence of foam cells in intimal and medial layers (→) and hypertrophy of smooth muscle fibers (→). D: photomicrograph of mice aorta of atorvastatin drug shows a projection of intima to the lumen as atherosclerotic plaque (↔), presence of foam cells in intimal and medial layers (→) and hypertrophy of smooth muscle cells with the presence of inflammatory cells and necrosis (→). E: photomicrograph of mice aorta of H. perforatum plant group shows normal architecture representing by the thickness of layers (↔) and smooth muscle cells (→) with presence of few foam cells (→). F: photomicrograph of mice aorta of the group treated with soaked form of the plant shows normal architecture representing by the thickness of layers (↔) and smooth muscle fibers (→) with presence of few foam cells (→). G: photomicrograph of mice aorta of the group treated with powdered form of the plant shows normal architecture representing by the thickness of layers (↔) and smooth muscle fibers (→) with presence of few foam cells (→).

Discussion

The present study confirmed that the dried plant of H. perforatum has a positive effect on overall measured parameters that reflect the etiology of cardiovascular diseases. This outcome has been confirmed by biochemical and histopathological studies compared to control groups. Moreover, analysis of the plant extract confirmed that the extract contains various biomolecules; some of which are well-known for their cardiovascular actions. Furthermore, the study also proved that the soaked form of the plant has induced a better effect than the dried powdered form. Nevertheless, in comparison to atorvastatin, the plant shown improved lipid profile weather dried or soaked. The positive effect of different H. species has been reported with different outcomes supporting our findings (3).

Also, the study mentioned that analysis of the extract of H. perforatum shown presence of various bioactive molecules. Analysis by HPLC revealed the presence of various extract constituents compared to standard and represented by various retention peaks (17). HPLC peaks matched the retention time of catechine, ferulic acid, quercetin, rutin, kaempferol, and apigenin while the retention peaks of GC matched only low molecular weight, volatile compounds: formic acid, acetic acid, propionic acid, butyric acid, and valeric acid (13).

This work reported that soaked form of the dried plant H. perforatum has induced a remarkable lipid parameters modulation represented by favorable reduction of LDL,TC and TG alongside elevation of HDL in comparison to the dried powdered form of the plant or atorvastatin group (18). Despite that, the dried powder is shown favorably better antihyperlipidemic effect than atorvastatin (19). In line with our study, the results of Zou et al. (20), investigation showed that H. perforatum has a strong hypolipidemic effect. It has the potential to lower blood TC, TG, and LDL-C levels while increasing blood HDL-C levels, Zou et al. (20), also confirmed that H. perforatum reduced malondialdehyde (MDA) levels in the blood and liver whereas dramatically increasing Superoxide dismutase (SOD) activity in both serum and liver, as well as catalase (CAT) activity in the liver (20). H. perforatum extract significantly decreased low density lipoprotein cholesterol, cholesterol, triglyceride, C-reactive protein (CRP), MDA as well as apolipoprotein B (apoB), atherosclerosis index, and increased apoA and high density lipoprotein in rabbits. This extract also confined the atherosclerotic lesions, according to histopathological findings to Asgary et al. (21). Moghaddam et al. (22), reported that LDL-C was dramatically lowered, and TC was also greatly reduced. The levels of TG and HDL-C did not alter significantly. However, MDA, aspartate aminotransferase, and alanine aminotransferase were all dramatically reduced (22). The histopathological studies are represented by tissue slices of mice aorta at the end of the study to further confirm the outcome (18,23). Fatty diet has induced atherosclerotic changes compared to the control group on a regular diet. These atherosclerotic changes approximately absent in mice treated by plants in soaked or dried form with slightly better outcomes with soaked form (24). On the other hand, atorvastatin shown greatly reduced atherosclerotic changes compared to a positive non-treated control group with slightly better outcomes obtained with plant preparation compared to atorvastatin (11).

Conclusion

The study concluded that the plant H. perforatum in dried or soaked form reduced lipid parameters compared to control groups (with/without fatty diet) and the soaked form showed better hypolipidemic effects than dried form. Both dried and soaked form has shown a better or comparable effect to atorvastatin; a well-known hypolipidemic agents. The hypolipidemic effect of the plant H. perforatum was confirmed through histopathology study. Analysis of the extract of the plant revealed the presence of effective biomolecules; some of which could be utilized for biosynthesis of new antihyperlipidemic drugs.

Acknowledgements

The authors are very grateful to the University of Mosul, College of Veterinary Medicine for the facilities provided, which helped in improving the quality of this work. The authors also thank the staff in the science and technology lab in Baghdad for their kind help with chromatography measurements.

Conflict of interests

The authors declare no conflict of interest