Anti-hyperlipidemic Effect of Berberis lycium Royle Root Bark Extract in Alloxanized Swiss Albino Mice

| Hyperlipidemia is a condition in which blood is overloaded with lipids. This condition is challenging for living beings. There are many ways to solve this problem but herbal medicines take on paramount importance in this regard. The purpose of the current research was to inspect the effects of an aqueous extract of Berberis lycium Royle root bark on lipid profiles of Swiss albino mice in which diabetic phenotype was induced by alloxan. A single injection of Alloxan (150 mg/kg) was applied intravenously to induce diabetic phenotype. Alloxan induction raised the overall level of triglycerides, low density- lipids (LDLs), and cholesterol and reduced the level of high density lipids (HDLs). Oral administration of 200 mg/kg aqueous extract of B. lycium Royle root bark for 28 days rescued all these changes. This study shows that the root bark extract of B. lycium Royle has antihyperlipidemic properties. Novelty Statement | This study reports that root bark extract of Berberis lycium Royle has very strong antihyperlipidemic properties.


Introduction
H yperlipidemia is a situation in which there is an overload of lipids, mostly cholesterol as well as triglycerides in the blood. There are many causes of hyperlipidemia, one of which is diabetes. Abnormal conditions of insulin production and insulin action are responsible for a metabolic disorder known as diabetes mellitus which results in the disturbance of lipid metabolism. Diabetes mellitus results in dysfunction, long term damage, and failure of numerous organs ( Joshi et al., 2015).

Ethical statement
All experimentations were carried out in agreement with the University guidelines and International laws for the maintenance of experimental animals as mentioned in (Ali et al., , 2020aHussain et al., 2020;Ara et al., 2020;Khan et al., 2019;Mumtaz et al., 2019;Mughal et al., 2019;Dar et al., 2019). The Committee of the Faculty of Science, Government College University, Lahore, Pakistan has approved all procedures used in the experiments included in this study.

Collection of medicinal plants
Berberis lycium Royle plants were collected from Chinari, District Jhelum valley, Azad Kashmir, Pakistan. An Ethnobotanist from the Department of Botany, Faculty of Science, University of Azad Jammu and Kashmir Muzaffarabad, Pakistan identified the plants. The dust from the plants was removed by rinsing them with water. These plants were placed in the shade to dry, after that they were ground into a fine powder. Shade dried plant parts were crushed into a fine powder.

Preparation of plant extract
Ten-grams of shade dried plant material was boiled in 100 ml of distilled water. This solution was filtered through filter paper (Whatman No. 1) and the solution was concentrated with a rotary evaporator to obtain the extracts. The plant extract was dried in a vacuum oven (Vacucell, Einrichtungen GmbH) at 40°C for 24 hours.

Animal selection and grouping
Twenty-four male Swiss albino mice, with average age eight weeks and 36.6±1.5 g body weight were obtained from the GC University, Lahore. Before starting the experimental procedure, all mice were housed together for seven days. Then the experimental animals were randomly assigned to one of four different groups. The first group (four animals) did not receive any treatment and this group was designated as the normal control group. A single intraperitoneal injection of alloxan (one injection contained 150 mg/kg) was given to 20 animals to induce diabetes. Animals that showed glucose levels that were higher than 150 mg/dL were further categorized into three additional groups: Diabetic control group (6 animals); Diabetic+BLR extract group (6 animals) were diabetic mice that received a daily oral dose of BLR extract of 200 mg/kg for 28 consecutive days; Diabetic + glibenclamide group (6 animals) were diabetic phenotypic mice that received an oral dose of glibenclamide 10 mg/kg g for 28 consecutive days. The diagrammatic scheme of the experimental design is shown in Figure 1.

Animal management
Four polypropylene cages were used to house mice assigned to the different groups. Mice belonging to one group were housed in the same cage. Four or six mice were housed in each cage. Pure drinking water and Standard laboratory pelleted feed were given to animals ad libitum. The experimental room temperature was set at 22±3°C. A 12 h dark/ 12 h light cycle was maintained and 70% was the upper limit for humidity in the room. The oral dose of glibenclamide was administered using a gavage oral feeding tube.

Body weight measurement
The body weight of the mice in all groups was measured using a digital balance. The body weight of the mice was monitored from the beginning until the end of the study at seven-day intervals.

Induction of experimental diabetic phenotype
A single intraperitoneal dose of alloxan was administered to induce diabetic phenotype in the experimental animals, using a dose of 150 mg/kg in 0.9% saline. Freshly prepared alloxan was injected into twenty mice. Subsequently, the blood glucose levels of fasting mice were assessed and the diabetic group consisted of mice that exhibited a blood glucose level higher than 150 mg/dL.

Assessment of glucose levels
The tail vein of the mice was used to collect the blood samples. Fasting blood glucose was estimated with a One Touch Basic Glucometer after a 6-hour fasting period on the 0, 7 th , 14 th , 21 st and 28 th days.

Analysis of lipid profiles
Extraction of lipids An approach proposed by Friedewald et al. (1972) was employed to perform serum lipids extraction.

Estimation of total cholesterol
The enzymatic colorimetric method proposed by Allain et al. (1974) enzymatic colorimetric method was used to determine cholesterol level.
The reaction sequence was as follows:

Procedures
Samples of mice serum, standards and the reagent blank were preincubated for 5 minutes at 37 °C. Samples (10 μL) or standard (10 μL) and reagent blank (1,000 μL) were pipetted into cuvettes and mixed carefully by inversion. The cuvettes were put into the holder and a stopwatch was started to assess the time. The absorbance of the sample, standard and the reagent blank was measured at 400 nm wavelength within 60 minutes. As the final point, the absorbance of the sample (ΔA sample) and the standard (ΔA standard) against the reagent blank were calculated.

Estimation of triglycerides
The enzymatic colorimetric method published by Hearne and Fraser (1981) enzymatic colorimetric method was used to determine triglyceride (TG) levels.
The reaction sequence was as follows: To examine serum of the mice, the samples and the standard reagents are prepared for use on an automated analyzer. The enzymatic test was carried out at a wavelength (540 nm), a 1 cm optical path, at 37 o C and the measurements were compared against a reagent blank.

Procedure
The samples, standards and blank were preincubated for 5 minutes at 37 °C. The reagent blank (1,000 μL) and samples (10 μL) or standards (10 μL) were placed into cuvettes and mixed gently by inversion. The cuvettes were inserted into the cell holder and a stopwatch was used to assess the time. The absorbance of the samples, standards and blank was measured at 540 nm. Finally, the absorbance of the sample (ΔA sample) and the standards.
(ΔA standard) against the reagent blank were calculated.

Estimation of high density lipoprotein (HDL) cholesterol
The VLDL and LDL from serum were precipitated by phosphotungstate in the presence of magnesium ions. After elimination by centrifugation, the clear supernatant comprising the high density lipoproteins (HDL)-fraction and the cholesterol content was confirmed enzymatically according to methods mentioned in Seigler and Wu (1981).
The reactions were as follows: For this research, mouse serum was sampled and standard reagents were used on an automated analyzer. The enzymatic assay was carried out at 593 nm wavelength, 1 cm optical path, 37 °C temperature and measurements done against the reagent blank.

Procedure for precipitation
100 μL of reagent and 10μL of the samples were pipetted into a centrifuge tube, mixed well, allowed to stand for 5 minutes at 37°C and centrifuged at 3000 rpm for 20 minutes. The supernatant (sample) was collected for HDL test.

Procedure for determination of HDL
The reagent blank, samples and calibrator were preincubated for 5 minutes at 37°C. The reagent blank (10 μL distilled water and 750 μL enzymes) and samples (10 μL samples and 750 μL enzymes) or calibrator (10 μL calibrator and 750 μL enzymes) were pipetted into cuvettes and mixed gently by inversion. The cuvettes were inserted into the cell holder and a stopwatch was used to assess the time. The absorbance of the samples, standards and the reagent blank was measured at 593 nm after 5 minutes. Finally, the absorbance of the samples (ΔA sample) and the calibrator (ΔA standard) against the reagent blank were calculated.

Estimation of low density lipoprotein (LDL) cholesterol
Indirect method TC, TG and HDL are measured and LDL cholesterol is calculated from the primary measurements by use of the empirical Freidewald Formula equation:

Statistical data analysis
Data were presented as mean±standard error of the mean. Shapiro-Wilk test was used to check the distribution of the data. Parametric tests were used on normally distributed data and then examined statistically via one-way ANOVA, followed by "Dunnett's Multiple Comparison Test", to find any substantial variances among the group means. GraphPad Prism version 5.0 for Windows (Graph Pad Software, San Diego, CA, USA) was used for analyses. Values of P ≤ 0.05 were considered as significant.

Effect on body weight
Alloxan caused a gradual decrease in body weight. When the alloxan-treated mice were administered the extract and glibenclamide, the alloxan-induced changes in body weight were rescued (Table 1).

Effect on glucose level
Alloxan caused a gradual increase in blood glucose levels. When the alloxan-treated mice were administered the extract and glibenclamide, the alloxan-induced phenotypes in blood glucose were rescued (Table 2).

Effect on lipid profile
Total cholesterol In this study, intraperitoneal administration of a single dose of alloxan (150 mg /kg body weight) caused a highly significant increase in the overall cholesterol level of the alloxan-treated group (185.6±5) as compared to control (91.76±3). When extract and glibenclamide were given to alloxan-treated mice they caused a significant decrease in total cholesterol level (alloxan-treated+extract: 116.14±2; alloxan-treated+ glibenclamide: 99.43±4) as compared to the alloxan-treated group (185.6±5) ( Table 3).

Triglycerides
Intraperitoneal administration of a single dose of alloxan (150mg /kg body weight) caused a highly significant increase in the triglycerides level of the alloxan-treated groups (174.62±6) as compared to control (90.16±3). When extract and glibenclamide were given to the alloxan-treated mice they caused a significant decrease in the triglycerides level (alloxan-treated +extract: 130.98±3; alloxan-treated+ glibenclamide: 111.68±8) as compared to the alloxan-treated group (174.62±6) ( Table 3).

HDL-C
Intraperitoneal administration of a single dose of alloxan (150mg /kg body weight) caused a highly significant decrease in the HDL-C levels in the alloxan-treated group (23.38±3) as compared to controls (45.96±3). When the extract and glibenclamide were given to alloxan-treated mice they caused no significant change in HDL-C level (alloxan-treated + extract: 42.54±0.8; alloxan-treated+ glibenclamide: 41.16±1.8) as compared to the alloxantreated group (45.96±3) ( Table 3).

LDL-C
Intraperitoneal administration of a single dose of alloxan (150mg /kg body weight) caused a highly significant increase in the LDL-C levels in alloxan-treated (127.296±6) mice as compared to controls (27.708±4).

VLDL-C
Intraperitoneal administration of a single dose of alloxan (150 mg /kg body weight) caused a highly significant increase in the VLDL-C level of the alloxan-treated group (34.924±1.1) as compared to control (18.032±0.5). When the extract and glibenclamide were given to the alloxantreated mice they caused a significant decrease in VLDL-C level (alloxan-treated +extract: 26.196±0.6; alloxan-treated + glibenclamide: 22.336±1.6) as compared to the alloxantreated group (34.924±1.1) ( Table 3).
The present study showed that an aqueous extract of B. lyceum Royle root bark rescued the changes made by alloxan in Swiss albino mice. The present study revealed that B. lycium Royle root bark extract has an antihyperlipidemic effect in alloxan-treated Swiss albino mice.
The current results revealed a highly significant increase in plasma glucose, cholesterol, LDL and triglycerides in the alloxan-treated mice while a reduction in body weight and HDL level was seen. The same results were seen in Wistar rats, in which diabetes was induced by alloxan-induction (Luka and Tijjani, 2013;Bako et al., 2014). The level of TG, cholesterol and LDL in the plasma was decreased after treatment with the plant extract, while the level of HDL was increased. Similar results were reported in alloxan-induced diabetic rabbits (Wojtowicz et al., 2007). These results are in agreement with the current research. B. lycium Royel root bark powder significantly lessened the total LDL, triglycerides and cholesterol of treated mice as compared to alloxan-treated ones.   Oral administration of 200 mg/kg and 600mg/kg extract fruit of Berberis lycium for six weeks resulted in a significant reduction in total cholesterol, triglyceride, and LDLs levels. Berberis lycium treatment increased the levels of HDLs. Oral administration of 200 mg/kg and 600mg/ kg extract fruit of Berberis lycium for six weeks resulted in a significant reduction in total cholesterol, triglyceride, and LDLs levels. Berberis lycium treatment increased the levels of HDLs.
Oral administration of Berberis lycium fruit extract (200 mg/kg and 600mg/kg) for six weeks caused a significant elevation in the levels of HDLs while, a significant decline in LDLs, triglyceride and total cholesterol were found in the serum of diabetic phenotypic mice (Furman et al., 2020). Belwal et al. (2020) also have reported that the ethanolic as well as the aqueous extract of Berberis aristata showed better antidiabetic activity through eliciting decreased total cholesterol and elevated HDL-C levels, in diabetic and hyperglycemic patients leaf extracts of B. lycium lessened the lipid profile (Hussain et al., 2017). Many plant extracts lower the serum lipids especially total triglycerides, cholesterol, and level of low density lipoprotein, which are the therapeutic properties to combat atherosclerosis which is one of the main problems of diabetes (Luka and Tijjani, 2013). Different atheromatous problems e.g., ischemic heart diseases are persistently present in diabetic patients (Assmann et al., 1997). In diabetic patients, the atheromatous disease is due to the decrease in the level of high-density protein (Rang and Urban, 1995). It was discovered that treatment with B. lycium Royle root extract caused a decrease in the levels of LDL and an increase in level of HDL that can save diabetic patients from athermanous disease.
Berberis lycium Royle possesses berberine, berbamine, chinabine, karakoramine, palmatine balauchistanamine, gilgitine, jhelumine, punjabine, sindamine, chinabine acetic acid, maleic acid, ascorbic acid (Khare, 2004). Its rook bark contains active components such as isoquinoline alkaloid that is responsible for its antihyperlipidemic effects in alloxan induced diabetic phenotypic mice (Saeed, 1976;Birdsall and Kelly, 1997;Chand et al., 2007). Critical valuation exposed that pharmacological studies are deficient in the documentation of active ingredients responsible for pharmacological actions. Therefore, a further investigation aimed towards the identification and isolation of active constituents in future studies is suggested.

Conclusions and Recommendations
Current research proved that B. lycium Royle root bark extract contained antihyperlipidemic potential in alloxan-treated mice. It significantly increased the level of HDL while decreased the raised level of total LDL, triglycerides, cholesterol, in alloxan-induced diabetic mice.