Evaluation of Caralluma edulis for its Potential Against Obesity, Atherosclerosis and Hypertension

Animals

Wistar albino rats (180-250 g) of either sex (3 ♀ and 3 ♂), approximately 8–10 weeks old, were employed in experimental work and Swiss albino mice (20-30 g) were used for the acute toxicity study. Animals were housed in the animal house of the Pharmacology research laboratory, department of Pharmacology, faculty of Pharmacy, the Islamia University of Bahawalpur (IUB). Animals were kept in standard polycarbonate cages under controlled temperature (23 ± 2°C), along with 55 ± 5% humidity and a 12 h light/dark cycle. Food and water were provided ad libitum. Animals were acclimatized to experimental conditions for 1 week before start of the study. The study protocols were approved by pharmacy animal ethics committee (PAEC) under reference number PAEC/2020/25.

Plant Material

The stems of C. edulis were collected from the Cholistan desert of Bahawalpur, Pakistan. After collection, the plant material was identified by a botanist and its specimen was preserved in the herbarium of Pharmacology research laboratory, department of Pharmacology, faculty of Pharmacy, IUB, Pakistan. Voucher number; CE-SM-02-18-209, was issued for future reference. The plant material was washed, dried in the shade and carefully screened to remove any extraneous material.

Preparation of Crude Extract

1.5 kg dried stem of C. edulis was ground into coarse powder and soaked in 70:30 methanol and distilled water solution for 3 days thrice. The soaked plant was filtered through a muslin cloth and then with Whatman grade 1 filter paper, residues were discarded, and the filtrate was subjected to the rotary evaporator (Heidolph, Laborota 400-efficient, Germany) under reduced pressure to prepare a concentrated semi-solid paste.^18,19 The thick semi-solid crude extract of C. edulis (CE. Cr) was weighed, labelled, and stored in a freezer for future use.

Phytochemical Analysis

CE.Cr was analyzed qualitatively for the presence of secondary metabolites like alkaloids, flavonoids, glycosides, tannins, anthraquinones, coumarins, saponins, polyphenols, carbohydrates, amino acids and proteins by standard procedures.²⁰

High-Performance Liquid Chromatographic Analysis

HPLC analysis was carried out on Shimadzu LC10-AT VP Liquid Chromatograph equipped with SIL-20A autosampler (Shimadzu Scientific Instruments, Kyoto, Japan) and SPD-10AV UV VIS Detector. A shim-pack CLC-ODS (C-18, 25 cm × 4.6 mm, 5 µm), maintained at room temperature, was used for separation. The mobile phase consisted of binary solvent system; that is, solvent A (water: acetic acid-94:6, PH = 2.2) and the solvent B (acetonitrile) with the following gradient elution (linear gradient v/v): 0–15 minutes, 85% A: 15% B, 15–30 minutes, 55% A: 45% B and 30-35 minutes 0%A: 100% B (equilibration). The flow rate was 1.0 mL/min and the detection wavelength was 280 nm.²¹

In Vitro Antioxidant Activity by DPPH Assay

The antioxidant potential of CE.Cr was measured in terms of free radical scavenging or hydrogen donating ability of stable 1, 1- diphenyl 2-picrylhydrazyl (DPPH) free radical according to the protocol described by Jabeen et al with slight modifications.²² 1 mL of 0.1 mM methanolic solution of DPPH was mixed with 1 mL of each CE.Cr methanolic solution of varying concentration (50-1000 μg/ml). L-Ascorbic acid solution was used as a reference standard, and a mixture of 1 mL DPPH and 1 mL methanol was used as control. The reaction was carried out in triplicate and all the solutions were incubated for 30 minutes and absorbance was measured at 517 nm using UV-Spectrophotometer. DPPH percent scavenging activity was calculated using the following formula

Ac is the Absorbance of control; whereas, As is the absorbance of sample

Normal Diet & High-Fat Diet

1 Kg normal diet was prepared by mixing 500 g of poultry feed, 350 g of choker and 150 g dry milk,⁹ obtained from the local market of Bahawalpur. The energy of a normal diet was calculated as 402 kcal/100 g, containing carbohydrate 54%, protein 22%, fat 10%, fibers 8%, minerals 2% and moisture 2%.

High-Fat Diet (HFD) consisted of fat 26%, carbohydrate 48%, and protein 20% with total energy value calculated as 510 kcal/100 g. 1% cholesterol and 0.5% cholic acid (Sigma Aldrich, USA) were mixed in the normal diet to induce hypercholesterolemia and butter was used as a source of fat. It is the most common method for developing hypercholesterolemia in the rat model; that is, 1% cholesterol to the normal diet is reported in 12.1% of the studies already published.^23-27

Sample Size Calculation

The sample size or the number of animals in each group was measured by using power analysis method. A simple calculation can be carried out manually with the help of formula and complex calculations power analysis-based software is available²⁸

where SD = standard deviation, d = difference between mean values and Z value was checked from Z-table.

Rat Model of Hypercholesterolemia

Albino rats were divided into five groups each comprising six animals. All the groups received tap water ad libitum and respective treatment for 28 days. Normal control group was given normal feed and the positive control group was fed on HFD. Both the groups were administered normal saline (1 mL/Kg/day, p.o.) by oral gavage feeding tubes. CE.Cr was given to the treatment groups, at doses of 100, 300 and 500 mg/Kg (p.o. per day), respectively, along with HFD. The standard control group was given atorvastatin (Pfizer, USA), at the dose of 5 mg/Kg/day; p.o. It is a synthetic HMG-CoA reductase inhibitor which lowers plasma cholesterol levels by inhibiting endogenous cholesterol synthesis. CE.Cr and atorvastatin were diluted in normal saline. After 28 days, all the animals were fasted overnight (for about 12-18 hours) before the induction of anesthesia. The aorta was dissected and blood samples were collected for the estimation of biochemical markers; that is, serum TC, LDL, HDL and TG levels.

Evaluation of Hypotensive Potential by Using Invasive Technique

CE.Cr was evaluated for its hypotensive effects by using invasive technique.³⁰ Animals were anesthetized and fixed in a supine position on a dissecting table. Temperature was maintained with the help of an overhead lamp. The trachea, right jugular vein and left carotid artery were exposed by a small mid-tracheal incision. The trachea was cannulated with 18-gauge polyethylene tubing to facilitate spontaneous respiration. The right jugular vein was cannulated with polyethylene tubing PE-50 for intravenous injection of drugs and CE.Cr solutions. The left carotid artery was also cannulated with polyethylene tubing; PE-50, filled with heparinized saline (60 IU/ml) and was connected to a pressure transducer (MLT0699 disposable BP transducer) coupled with PowerLab 4/30 and LabChart Pro software (AD Instruments, Australia) for blood pressure (BP) and heart rate (HR) recordings. A system calibration was performed with the help of a mercury manometer connected to the pressure transducer before the start of the first experiment every day. The exposed surface of the cannulated area was covered with a piece of cotton swab moistened with warm saline. Heparinized saline (0.1 mL) was injected into cannulated rat to prevent blood clotting. Acetylcholine (1 μg/Kg) and Nor-adrenaline (1 μg/Kg) were used to check the hypotensive and hypertensive responsiveness of each animal before administration of CE.Cr. After 15–20 minutes of equilibration, 0.1 mL of CE.Cr was injected, at doses of 1, 3, 10 and 30 mg/Kg intravenously, followed by 0.1 mL of saline flush. BP was returned to the resting level before every next dosing. Mean arterial blood pressure (MABP) was calculated by adding the values of DBP and one-third of the pulse width. Change in blood pressure was measured as the difference between the steady-state values before administration of the dose and the lowest reading after administration of each dose of the test substance.

Diuretic Activity

Rats (other than those used in the HFD model) were randomly divided into different groups. The control group received normal saline (10 mL/kg, i.p.). Another group of animals was given frusemide (10 mg/kg, i.p.) as standard diuretic. The treatment groups of animals were injected with doses of 100, 300 and 500 mg/kg of CE.Cr, intraperitoneally. Immediately after dosing, animals were individually housed in the metabolic cages (Techniplast, Italy) and the urine was collected for 6 hours. Total urine volume was noted. Sodium and potassium urinary concentrations were measured by using a clinical flame photometer (Model 410C, Sherwood, UK).³¹

Acute Toxicity Study

Acute toxicity test was performed to evaluate the toxic potential of CE.Cr by following the OECD guidelines.³² Swiss albino mice weighing 20–30 g were divided into different groups, comprising of five mice in each group (2♀ and 3♂). Mice were fasted overnight and received only water ad libitum. Normal control group received normal saline (10 mL/kg, p.o.) and other groups received CE.Cr, at doses of 0.5, 1, 3 and 5 g/kg; p.o. All the animals were observed critically for 2 hours and then at the interval of 60 minutes for the next 6 hours, for any type of behavioral changes and then mortality was noted for the next 14 days.

Statistical Analysis

The results were expressed as mean ± SEM for six animals in each group. The results were statistically analyzed, using one-way ANOVA followed by Tukey’s test. The data were computed using GraphPad Prism version 8 and P < 0.05 was considered significant.¹⁸

Phytochemical Analysis

CE.Cr was found to be rich in alkaloids, saponins, quinones, carbohydrates, flavonoids, tannins, phenols, terpenes, terpenoids and glycosides.

HPLC Analysis

The constituents found in CE.Cr include chlorogenic acid, caffeic acid, vanillic acid, p-coumaric acid, hydroxycinnamic acid, quinic acid, apigenin, quercetin-rhamno-di-hexoside, syringic acid, hyperoside, naringenin, quercetin-3-O-glucopyanoside and trans-ferulic acid (Figure 1A) which have already been reported for their therapeutic importance. The chromatogram was compared with that of standard (Figure 1B and Table 1).

Antioxidant Activity by DPPH

The present study suggested the remarkable antioxidant potential of CE.Cr. The results are shown in Figure 2. Antioxidant potential was increased in a graded manner. Maximum radical scavenging effects shown by CE.Cr was 78% which was comparable to that of ascorbic acid (89%), at the concentration of 1000 μg/mL.

Anti-Obesity Potential

Throughout the experimental period, change in body weight of the normal and positive control groups was found to be increased; that is, 43.00 ± 2.51 and 68.50 ± 1.19 g, respectively, and percent increase in body weight was also observed (Figures 3 and 4). A decrease in body weight was observed in treatment groups, receiving different doses of CE.Cr and atorvastatin. CE.Cr, at doses of 300 and 500 mg/kg, prevented the increase in body weight and results were highly significant (P < 0.001) as compared to the positive control group (Figure 3). Whereas, there was no statistical difference found in the food consumption among different experimental groups (P > 0.05).

Biochemical Markers

The serum levels of TC, TGs, LDL and HDL in experimental animals are shown in Figure 5. HFD increased serum TC levels in the positive control group and CE.Cr, at doses of 100, 300 and 500 mg/kg, reduced serum TC concentration. CE.Cr showed highly significant (P < .001) decrease in TC levels, at all doses, as compared to that of positive control group. HFD increased the levels of TGs in the positive control group which were reduced significantly in treatment groups, at all doses (100, 300 and 500 mg/kg). HFD decreased serum HDL levels in the positive control group and the levels were significantly (P < 0.01) increased, at doses of 300 and 500 mg/kg, as compared to the positive control group. CE.Cr exhibited a significant (P < 0.001) reduction in serum LDL levels, at doses of 100, 300, and 500 mg/kg.

Hypotensive Effects

CE.Cr, at doses of 1, 3, and 10 mg/kg did not show significant decrease in mean arterial blood pressure (MABP); that is, 130 ± 2.42, 129 ± 2.87 and 121 ± 3.12 mmHg, respectively. CE.Cr produced significant hypotensive effects, at the dose of 30 mg/kg, as shown in Figure 6 and Table 2.

Diuretic Activity

Urinary volume (ml/100 g/6 h) was measured in different groups of animals, and mean value was calculated as 1.10 ± 0.13 mL for the normal control group, 4.31 ± 0.05 mL for the standard group and CE.Cr, at doses of 100, 300, and 500 mg/kg showed an increase in urinary volume; that is, 2.58 ± 0.7, 3.38 ± 0.28 and 3.84 ± 0.45 mL, respectively. In addition to an increase in urinary volume, CE.Cr also enhanced the Na⁺ and K⁺ excretion. CE.Cr, at doses of 300 and 500 mg/kg, showed highly significant increase in the sodium and potassium ions excretion and results were comparable with those of the standard drug, frusemide (Table 3).

Anti-Atherosclerotic Potential

Histological studies of cross-sections of the aorta showed several foam cells in the positive control group with erosion of the endothelial membrane which was a sign of first-stage atherosclerosis. The positive control group was scored as 3 according to the severity grade (Table 4). The normal control group showed the uniform histological structure of the endothelial membrane. Experimental groups receiving different doses of CE.Cr showed restoration of normal endothelial cell morphology. Therefore, histological images showed the anti-atherosclerotic effects of CE.Cr, at doses of 300 and 500 mg/kg and were scored as 1, similar to that of the standard drug, atorvastatin (Figure 7).

Acute Toxicity Study

Acute toxicity assay showed no signs of toxicity and mortality and was found to be safe up to 5 g/kg and the results of highest dose (5 g/kg) were tabulated in Table 5.

Limitations

HMG-CoA reductase pathway for hypercholesterolemia while the ACE system, calcium channel, and autonomic nervous system for hypertension are the major limitations of this study.