Characterization and interactions between piperine and ezetimibe in their Anti-hyperlipidemic efficacy using Biopharmaceutics and Pharmacokinetics

Abstract

Background

Piperine, a secondary metabolite, affects the antihyperlipidemic effect of Ezetimibe (EZ). Hyperlipidemia is one of the independent risk factors for cardiovascular disorders such as atherosclerosis. Antihyperlipidemic drugs are essential for reducing cardiovascular events and patient mortality. Our study aimed to improve the solubility of EZ, a lipid-lowering drug that belongs to BCS II and has low solubility. Piperine, a bioenhancer, can increase the bioavailability of other pharmaceuticals without modifying their fundamental characteristics or enhancing their efficacy. The objective of this study was to increase the bioavailability of EZ while also improving its potency and reducing its toxicity by using piperine as a bioenhancer. Therefore, rats were given piperine combined with EZ, and their antihyperlipidemic activity was assessed while fed a high-fat diet.

Method

The in vivo antihyperlipidemic effect of EZ with piperine was assessed at doses of 10 and 5–20 mg/kg b.w. The evaluation was conducted using propylthiouracil-induced and triton X-100-induced hyperlipidemia in rats. Give 400 mg/kg body weight of propylthiouracil along with piperine. Serum levels of total cholesterol (TC) (p < 0.01), triglycerides (TG) (p < 0.01), low-density lipoprotein (LDL) (p < 0.01), and very low-density lipoprotein (VLDL) (p < 0.01) all went up significantly. Additionally, it led to the induction of high-density lipoprotein (HDL) (p < 0.01). Administration of Triton X-100 via intraperitoneal injection at a single dose resulted in an elevation of lipid levels.

Results

Lower levels of high-density lipoprotein (LDL), total cholesterol (TC), triglycerides (TG), and very low-density lipoprotein (VLDL) were significantly reduced by EZ at 10 mg/kg b.w. and piperine at 20 mg/kg b.w., respectively (p < 0.01 and p < 0.05). Liver histology studies provided further evidence supporting the present findings. Areas of concentrated periportal lymphocytes and hepatocytes formed a cord pattern in rats with hyperlipidaemia. It seemed like the hepatocytes, periportal area, and centrilobular part of the liver were all normal in the group who had the treatment. An analysis of the EZ plasma drug concentration with time was carried out in a research. The medication’s most effective concentration (Cmax) was determined to be within 4 h after delivery, and The quantified concentration of the active medication was detectable in the bloodstream for 24 h.

Conclusion

In combination with piperine, EZ has demonstrated significant antioxidant and antihyperlipidemic effects. This indicates that EZ could be further utilised for treating hyperlipidemia and atherosclerosis due to its potential to boost the bioavailability and oral absorption of the drug.

Introduction

Approximately 2.6 million people die every year from hypercholesterolemia (hyperlipidemia), a condition defined by unusually high plasma cholesterol levels. This condition accounts for 4.5% of all fatalities worldwide. Atherosclerosis is characterized by increased levels of plasma triglycerides (> 200 mg/dL) and reduced levels of HDL (< 40 mg/dL) and increased LDL levels (> 190 mg/dL) [1]. Prolonged elevated cholesterol levels might increase the likelihood of developing certain cardiovascular illnesses. Typical anti-hyperlipidemic medications used to treat such risk factors include fibrates, bile acid-binding resins, and statins. Unfortunately, there is a long list of undesirable side effects linked to these medications, including problems with the esophagus, aches and pains in the muscles and joints, and elevated liver enzyme activity. The optimization of lipid-lowering treatment, particularly in cases of severe hypercholesterolemia, is ongoing due to concerns about side effects, statin tolerance, hereditary diseases, drug resistance, noncompliance, and other therapeutic issues. We need novel, safe, and effective therapeutic agents to decrease cholesterol while minimizing the adverse effects of conventional lipid-lowering medications [2]. A new strategy has emerged that involves the combination of nonstatin medicines with low-intensity statins rather than high-intensity statins [3]. This technique improves statin tolerance and prevents dropout. Hyperlipidemia—high serum and plasma lipid levels—is the main cause of mortality and disability globally. Most arterial, cerebrovascular, and cardiac illnesses link to high levels of cholesterol and lipids in the blood [4]. Previous studies predicted a shocking 25–30% rise in worldwide mortality by 2020; however, a mere 10% drop in cholesterol levels could prevent 30% fewer cardiovascular problems and deaths [5].

Ezetimibe (EZ), the newest successful hypercholesterolemia treatment after statins, lowers cholesterol [6]. You can use EZ alone or in conjunction with statins to treat primary hypercholesterolemia and reduce cholesterol. Tolerance and safety are its best qualities. If you stop the Niemann-Pick C1-like 1 protein from doing its job in the small intestine, cholesterol absorption stops, but not fat-soluble vitamin or mineral absorption [7]. Due to its lower pharmacokinetic interactions with cytochrome P450-metabolized drugs, EZ is less likely to injure muscles [8].

EZ belongs to BCS Class II because of its low solubility in water and strong tissue permeability [9]. This lipophilic molecule exhibits subject-to-subject variability, with a log p-value of 4.5 and an oral bioavailability ranging from 35 to 60%. Its rapid efflux via p-glycoprotein (P-Gp), low dissolving rate, and water solubility (0.00846 mg/mL) contribute to its uncertain bioavailability [10]. Many approaches have developed over the years to enhance the oral bioavailability of poorly soluble medications. Accordingly, there are several advantages to these treatments and hardly any disadvantages [11]. They argue that their key benefits are to reduce the unpredictability of oral medicinal molecules, increase oral bioavailability, and reduce favorable dietary effects [12].

The metabolic processes, including shikimate, acetate-malonate, and acetate-mevalonate, provide the bulk of the hallucinogenic compounds included in herbal medicines. Plants contain components such as alkaloids, peptides, polysaccharides (such as gums and mucilages), resins, essential oils, phenolic glycosides (like flavonoids, cyanogens, and glucosinolates), terpenoids (such as sesquiterpenes, steroids, carotenoids, and saponins), and volatile oils. Complexity worsens the negative interactions that can occur between therapeutic drugs [13]. Given the limited understanding and investigation of botanical-drug interactions, more research into herbal medicine is essential to analyze specific plant constituents and elucidate their interactions with food and pharmaceuticals. Thus, it is crucial to study botanicals for therapeutic uses and promote their responsible and safe use, considering their impact, effectiveness, and modes of operation. Consequently, a significant issue with the use of both herbs and conventional medications is the herb-drug interaction (HDI) [14]. Researchers are extensively studying the HDI of popular herbal remedies and extracts, both alone and in conjunction with medicines [15].

Ayurveda and other Indian medicine prioritize natural bioenhancers. These substances increase the bioavailability and biological activity of active drugs, even at moderate dosages. Bioenhancers lower dose and frequency while minimizing toxicity and adverse pharmacological effects. Bioenhancers increase the bioavailability and effectiveness of active medications, regardless of their pharmacological qualities. Bioenhancers improve oral absorption, metabolism, and drug molecule conversion [16]. Long pepper (Piper longum L.) and white pepper (Piper nigrum L.) both have high piperine content [17]. Despite its PIP-related pungency, people have long valued pepper for its therapeutic, preservative, aromatic, and spicy qualities [18]. According to recent studies, PIP has anti-inflammatory, immunomodulatory, anti-cancer, antispasmodic, anti-secretory, and anti-hyperlipidemic properties [19]. In pharmacokinetic investigations, PIP inhibits enzymes such as aryl hydroxylase, O-deethylase, cytochrome P450, UDP-glucuronosyl transferase, sulfotransferase, and CYP3A4 [20]. Piperine increases the bioavailability of rosuvastatin, peurarin, and docetaxel by blocking the actions of CYP3A4 and P-glycoprotein [21]. The supposed antihyperlipidemic potential of piperine is the reason why it is used in some herbal cardiotonic preparations [22]. These include Ridayarishta, Mahamrityunjaya rasa, Heart plus, Cardana, etc.

Multiple studies have looked at how piperine affects the metabolism and absorption of different drugs. Besides anti-inflammatory and hepatoprotective effects, piperine is an effective antioxidant. Furthermore, studies have shown that piperine enhances the absorption of pharmaceuticals such as curcumin and simvastatin, all while maintaining their efficacy [23]. Therefore, we evaluated piperine’s efficacy in increasing EZ bioavailability in this study without compromising hepatic function.

Materials and methods

Sava Healthcare Ltd of Pune, India provided the ezetimibe sample. Sava Healthcare Ltd. from Pune, India provided the internal standard diclofenac sodium, and Sigma Aldrich Chem. Co. from Mumbai, India provided 97% pure piperine. Sigma-Aldrich in India supplied the medications, chemicals, and biochemical test kits for the research. All solvents and compounds were analytical-grade for HPLC. Analytical techniques used water filtered using a 0.25-µm membrane and double-distilled.

Experimental animals

The research utilized Sprague-Dawley rats. The National Institute of Nutrition, a public health, nutrition, and translational research entity in Hyderabad, India, under the Indian Council of Medical Research, purchased 190–230 gram male rats from an animal source. The seller’s paperwork showed healthy, active rats. We set up our lab at 23 °C and maintained a 12:12 light: dark ratio. We kept the mice in polypropylene cages, providing them with an unrestricted supply of water and a pellet diet [24]. The Institutional Animal Ethics Committee (IAEC) authorized the use of animals in this study. The Society of Toxicology USP 1989 established standards for animal care and procedures, which guided the use of animals in toxicology. Jeeva Life Sciences’ Institutional IAEC, located in Uppal, Hyderabad, India (approval number: CPCSEA/IAEC/JLS/28/08/24/002). We carried out all procedures in accordance with CPCSEA guidelines.

Classifying and preprocessing

After a week of acclimatization, we randomly assigned two groups of five rats each to the experiment. Control animals on a regular diet were in Group 1. With regular pellet feed, Group 2 animals received a 10% fat, 2% cholesterol diet for 30 days. Documenting body weights before and after exams confirmed hypercholesterolemia start.

Therapeutic intervention and pharmacological delivery

Four-week trial pharmaceutical interventions: group 1 (Normal) served as the normal saline control; group 2 (HFD) (high fat diet) served as the disease control and did not receive any drug treatment; group 3 (EZ) served as the drug control and received EZ alone at a dose of 10 mg/kg/day; group 4 (PIP) received piperine alone at a dose of 5 mg/kg/day; group 5 (EZ-PIP-5) received EZ and piperine at doses of 10 mg/kg/day and 5 mg/ kg/day, respectively; group 6 (EZ-PIP-10) received EZ and piperine at doses of 10 mg/kg/day and 10 mg/kg/day, respectively; and group 7 (EZ-PIP-20) received EZ and piperine at doses of 10 mg/kg/day and 20 mg/kg/day, respectively [25]. The animals were euthanized by inhaling carbon dioxide (CO₂), a widely utilized and humane technique that causes a quick loss of consciousness and eventual death. The process usually entails putting the animals in a tight space and administering CO₂ at a rate that renders them unconscious in a matter of seconds.

Propylthiouracil induced hyperlipidemia

We administered propylthiouracil (PTU) at 110 mg/kg p.o.b. wt. and 0.01% PTU to the animals for 7 days to induce hyperlipidemia. On the eighth day, we administered the most effective drug orally to the animals [26]. We completely randomized the rats into seven groups, each containing six animals. Group I: Control (received normal saline).Group II consisted of hyperlipidemic rats that received PTU (110 mg/kg b. wt) for 1–8 days and received cholesterol (400 mg/kg b. wt) on the 8th day. Group III received PTU (10 mg/kg b. wt) for 1–8 days, 400 mg/kg b. wt of cholesterol on the 8th day, and 10 mg/kg b. wt of EZ alone on the 8th day. Group IV: PTU (10 mg/kg b. wt) Group V: PTU (10 mg/kg b. wt.) Group V: PTU (10 mg/kg b. wt.) 1–8 days + cholesterol (400 mg/kg b. wt.) on the 8th day + EZ (10 mg/kg b. wt.) + piperine (10 mg/kg b. wt.) on the 8th day. Group VI: PTU (10 mg/kg b. wt) 1–8 days + cholesterol (400 mg/kg b. wt) on the 8th day + EZ (10 mg/kg b. wt) on the 8th day + piperine (20 mg/kg b. wt) on the 8th day.Group VII consisted of hyperlipidemic rats who received PTU (110 mg/kg b. wt) for 1–8 days, cholesterol (400 mg/kg b. wt) on the 8th day, and simvastatin (110 mg/kg b. wt) on the 8th day. We measured the lipid levels on the 8th day using a cholesterol measurement kit; Table 2 presents the analyzed data.

Hyperlipidemic rat model caused by Triton

Each of the seven rat groups consisted of six rats. Group I administered regular saline to the control group. One example of a Category II therapy is intravenous administration of Triton X-100 at a dose of 100 mg/kg. The third group of subjects received either Triton X-100 (100 mg/kg body weight intraperitoneally) or EZ (10 mg/kg). The fourth category of medications includes EZ (10 mg/kg), Triton X-100 (100 mg/kg intraperitoneally), piperine (5 mg/kg), and Triton X-100. Triton X-100 (100 mg/kg intraperitoneally), EZ (10 mg/kg), and piperine (10 mg/kg) make up the trio that comprises the sixth regimen. Step 6: Introduce the mixture intraperitoneally by mixing 100 mg/kg Triton X-100 with 10 mg/kg EZ and 20 mg/kg piperine. Both Triton X-100 (100 mg/kg intraperitoneally) and Simvastatin (10 mg/kg) are part of Group VII [27]. The results of the cholesterol measurement kit indicate the lipid levels in the results section.

Assessment of serum lipid metrics

We drew and coagulated the blood from the abdominal aorta at room temperature. We extracted the serum after 10 min of centrifugation at 3000 rpm. We stored the samples at 4 °C to analyze lipid markers such as overall cholesterol (TC), triglyceride levels (TG), HDL, LDL, and VLDL [28].

Assessment of hepatic lipid and functional parameters

We meticulously extracted 0.5 g of rat livers and mixed them with 0.15 g of body mass per 1 mL of phosphate-buffered saline (PBS, pH 7.2). We separated the solids by centrifuging the mixture at 3000 rpm for 10 min, recovered the liquid phase, and kept it at 4 °C for liver enzyme tests. Enzyme assay kits were used [29].

Biochemical and histopathological studies

We assessed the concentrations of total lipids, total triglycerides, low-density lipoprotein, high-density lipoprotein, serum alanine aminotransferase, and total protein using standard diagnostic kits. During the experiment, we euthanized the animals and removed their livers following blood collection [30]. The liver tissue specimens underwent washing in phosphate-buffered saline (PBS), fixation in 10% neutral buffered formalin, dehydration in rising alcohol grades, and storage before embedding in paraffin for histological examination. H&E dyes were applied to paraffin-preserved ultrathin slices (5 μm thickness) after microtomy. We examined the slices using Olympus E-330 imaging equipment and a BX51 light microscope from Tokyo, Japan. Five slides each showed hepatotoxicity and liver damage.

Molecular docking studies

Molecular docking research determined the proper orientation of the ligand in protein active sites, thereby reducing false positives. AutoDock, an advanced molecular docking tool, docked the ligands used in this investigation to the active regions of proteins [31, 32]. We sourced the X-ray crystal structures of C-Reactive Protein (1B09) and human lanosterol 14alpha-demethylase (3LD6) from the Protein Data Bank (http://www.rcsb.org/pdb). To make the protein crystal structures dockable, we added hydrogen atoms, ordered the bonds, and removed any ions, water, or ligands. We used Marvin Sketch 5.11.4 to make the ligands, which include ezetimibe, piperine, and a conjugate of the two.

MTT cytotoxicity assay

To ascertain the cytotoxic effects of EZ, piperine, and Group VII, vero cells from Akkar Bitech Pune served as a control group. We employed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic activity of the compounds following just minor modifications [33]. We seeded 96-well plates with 3 × 10^5 cells/mL and incubated them at 37 °C for one day after adding 100 µL of cells to each well, ensuring a density of 5% CO2. We treated each test sample’s cells in triplicate at different concentrations (0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 g/mL). After washing the cell monolayers three times with sterile PBS, we added twenty microliters of a 5 mg/mL MTT stock solution to each well after 72 h. After incubating at 37 °C for 4 h, we removed the medium from the wells. To dissolve azan crystals, 200 µL of acidified isopropanol was used in each well. We produced isopropanol using a 0.073-milliliter solution of 0.04% hydrochloric acid in 100% isopropanol. We measured the 570 nm absorbance of the formazan solutions using a multiwell plate reader. Formula for calculating live cell percentages. An online software estimated this [34]. The medication concentration that inhibits 50% of cellular growth is determined by optical density (OD).

$$\% \; {\text{viability}} = \frac{{{\text{Mean OD Treated}}}}{{{\text{Mean OD Control}}}}$$

Estimation of EZ concentration in plasma

Configuration of HPLC system

The drug levels in the blood plasma were determined using reversed phase-HPLC. The HPLC system was manufactured by Shimadzu (Japan) and it was equipped with an LC-10 AT-VP system controller with an LC20AT pump and SPD-10 A ultraviolet (UV) detector. The system was coupled to a Phenomenex C18 analytical column (4.6 250 mm) and the packed particle size was 5 μm. A Rheodyne7725-I auto-injector was used to inject 25 µL of the plasma sample. Acetonitrile and 0.4% w/v phosphoric acid in distilled water were filtered through a 0.25-µm filter membrane and used at a ratio of 53:47. A constant flow rate and temperature were maintained at 1.2 mL/min and 22 °C, respectively, throughout the analysis. UV detection was performed at 232.5 nm, e column temperature was sustained at 22 °C to provide uniform separation and excellent resolution, as indicated by preliminary studies. Detection occurred at 254 nm, the peak absorbance for the target chemical [35].

Deproteination and plasma preparation

The pharmacokinetic study was carried out by analysing the plasma EZ levels in tyloxapol induced hyperlipidemic rats. The animals were made hyperlipidemic with intraperitoneal injection of tyloxapol. EZ and EZ along with piperine (10 mg/kg) were administered at a dose level of 10 mg/kg, orally in different set of animals by oral gavage. We administered the drugs using the above method on the last day of the study. We collected blood samples from the retro-orbital plexus at 30, 1, 2, 4, 12, 16, and 24 h following ether anesthesia. Next, mix 1 mL of blood with 50 µL of the freshly prepared 0.02% sodium EDTA solution in a centrifuge tube [36]. Samples remained at -20 °C until required. We blended the material with equal acetonitrile after centrifuging it for 10 min at 3000 rpm. We removed the organic layer using cellulose acetate filter sheets before introducing it into the HPLC apparatus for EZ measurement [37].

Comparative statistics

We utilized GraphPad Prism 10.0.2.0 on Windows 10 to analyze the data. We extracted the data from each iteration of the experimental process and presented it as the mean standard deviation. Using Dunnett’s test in both a one-way and a two-way variance analysis (ANOVA), we determined if there were significance levels. We considered P < 0.001 and P < 0.05 to be statistically significant.

Results

Impact of concurrent treatment of EZ and piperine weight increase

Fluctuations on body weight

The findings demonstrated that hyperlipidemia was effectively induced during 30 days, as the HFD group gained a significantly higher weight of 171.3 g compared to group-I (53.6 ± 6.94 g). The rat weights in the EZ group were around 65.29 ± 4.13 g, which was considerably lower than in group-I. A similar outcome was accomplished by PIP.Weights were normalised and did not differ from group-I when EZ was given with 5 mg/kg piperine. Table 1; Fig. 1 show that when the dosage of piperine was increased to 10 mg/kg and 20 mg/kg, respectively, a significant decrease in weight of -12.32 ± 4.65 g and − 7.13 ± 2.35 g was seen, in comparison to the initial weights.

Table 1 EZ & piperine affect rat weight gain

Fig. 1

Weight fluctuations in rats subjected to treatment drug and piperine with different concentrations

The standard deviation of rat weight with a 6-sample size is shown. An one-way ANOVA was performed to assess the data, and Dunnett’s test determined statistical significance. A notable difference was highlighted by *P < 0.001 in comparison to group-I. aLoss of weight is indicated by negative readings. A significant change is shown by bP < 0.001, when comparing the liver weight to the original body weight.

An increase in dietary carbohydrates and lipids has been linked to obesity [36]. A few studies have indicated that various HFD strains cause obesity in rats and mice [37]. It is true that certain rat strains, such A/J and C57BL, can become overweight being fed high-fat. Whenever given the exact same HFD, SWR rats showed no signs of becoming overweight.

Hepatic weight variations

Every rat’s liver was weighed, and its weight was later determined by dividing the difference between its starting and ending weights by 100 grammes. The first group showed notable increases in liver weight (10.24 ± 0.26 g) compared to the starting body weight and 5.34 ± 0.36 g compared to the end weight.Group I’s livers put on extra pounds and deposited more cholesterol and fat as a result. The other groups’ relative liver weights were within the normal range and did not change substantially from their starting and ending body weights, as seen in Fig. 2.

Fig. 2

Fluctuations in hepatic mass in relation to body mass in rats

Impact of concurrent treatment of EZ and piperine on lipid metrics

Lipid profile of serum

The high-fat diet raised serum lipid levels in rats. In contrast to normal rats, the HDF rats had a considerably higher TG content in their serum, reaching 246.31 ± 2.54mmol/L. Using piperine as a sole treatment did not appreciably reduce the high TG, and the value stayed at 182.43 ± 1.95mmol/L. In the EZ group, there was no discernible drop in TG levels.

We have established that atherosclerosis and myocardial infarction can result from elevated blood cholesterol and low-density lipoprotein levels. There is evidence that elevated serum triglycerides (TGs) contribute to the worsening of atherosclerosis [38]. Drugs that reduce oxygen Many studies show that TG, LDL, and cholesterol prevent heart disease [39]. The antiatherogenic phenols gallic acid and linoleic acid in green tea extracts lower blood triglycerides and cholesterol and increase energy expenditure, fat oxidation, and stools [40]. Nevertheless, the TG levels dropped to 182.43 ± 1.95, 167.94 ± 1.37, and 152.34 ± 1.29 mmol/L when piperine was administered with EZ at 5, 10, and 20 mg/kg, respectively. There were also noticeable shifts in the TC concentrations. The HDF group’s TC level was 312.64 ± 2.64 mmol/L, which was lower than the usual group. The simultaneous treatment of EZ and piperine at 10 and 20 mg/kg reduced TC levels significantly. Tables 2–4 shows that HDL levels increased following EZ treatment and simultaneously with piperine at all doses. Therefore, the efficacy of piperine therapy was enhanced when administered in conjunction with EZ. The lipid-lowering action of EZ was therefore significantly enhanced by the co-administration of piperine (Figs. 3–5).

Table 2 Effect of EZ and piperine serum lipid profile on high fat diet method

Table 3 Anti-hyperlipidemic activity for EZ heads on Propylthiouracil induced hyperlipidemic rats

Table 4 Anti-hyperlipidemic activity for EZ heads on Triton induced hyperlipidemic rat’s

Fig. 3

Effect of EZ and piperine serum lipid profile on High fat diet method

Fig. 4

Hyperlipidaemia treatment is effective of EZ in Propylthiouracil-induced hyperlipidemic rats

Fig. 5

Anti-hyperlipidemic activity for EZ heads on Triton induced hyperlipidemic rats

Mean ± SEM (n = 6) values are shown. After ANOVA, The statistical analysis was conducted using Dunnett’s test. We compared the outcomes to those of the control group, the hyperlipidemic control, and the standard (a = p ⋤ 0.01, b = p ≤ 0.05), with p-values ranging from 0.01 to 0.05 for the control group and 0.05 for hyperlipidemic control.

Abnormal lipid profiles are a common pathogenic outcome of diabetes, found in 40% of diabetics. Diabetes causes hyperglycaemia, hypercholesterolaemia, and hypertriglyceridemia due to organ dysfunction in the heart, arteries, kidneys, eyes, and neurones [41]. In hyperlipidaemia, oxidative stress, lipid peroxidation, and reactive oxygen species (ROS) may only develop in a state of excess total cholesterol, or TC, and high-density lipoprotein cholesterol (LDL). Moreover, DNA and mitochondrial membrane oxidation are the primary contributors to significant cellular damage resulting from elevated ROS levels [42,43,44]. Studies have shown that OS is essential for the development and progression of several human disorders, such as CVI, CVD, and DM [45,46,47].

Accordingly, natural flavonoids like piperine have demonstrated strong antioxidant capabilities, which can halt or slow the progression of diseased states caused by harmful situations. More than that, persistent hyperlipidaemia raises the odds of developing advanced heart diseases such atherosclerosis [48, 49]. Figure 5 shows the results of routine blood tests for total cholesterol (TC), total lipids (TG), low-density lipoprotein (LDL-C), and very low-density lipoprotein (VLDL-C) in rats that were put on a high-fat diet (HFD) to determine the effects of hyperlipidaemia.

These results are provided as Mean ± SEM with a sample size of 6. We then utilised Dunnett’s test for statistical analysis. Comparisons were made with the control group, hyperlipidemic control, and benchmarks. The significance levels were There are two outcomes: A and B, both with p-values less than or equal to 0.05.

Rats as controls and atorvastatin-treated rats were compared to the EZ and piperin treatments. In rats given 5, 10, or 20 mg piperin, total cholesterol fell similarly to atorvastatin (10 mg). With 10 mg EZ, total cholesterol dropped significantly. Supporting these findings is the observation that rats without hyperlipidaemia had lower levels of TG, HDL, LDL, and VLDL. When rats were given ezetimibe and piperine together, the high-fat diet had less of an impact on them. Due to HFD, there was a significant rise in blood total cholesterol, triglycerides, LDL, and VLDL and a significant fall in HDL (p 0.01). After EZ was given, total cholesterol, triglycerides, LDL, and VLDL levels significantly decreased (bp < 0.01). EZ therapy also significantly raised HDL values.

Examining lipid profile parameters changes caused by cholesterol intake and persistent high-fat diet consumption has helped determine the effects of pathophysiological alterations on blood homeostasis and EZ-piperin. We created hyperlipidaemia in rats by feeding them saturated fat and injecting propylthiouracil and triton X-100 orally. The injection of cholesterol raises plasma cholesterol levels. Triton X-100 and Propylthiouracil raised lipid levels and boosted duodenal cell cholesterol absorption into the bloodstream. Hyperlipidaemia was explored by measuring TC, TG, LDL, and HDL after a high-fat meal, Propylthiouracil, and Triton X-100 treatment developed it. Unsurprisingly, the control group’s cholesterol levels were significantly lower than those of the high-fat diet group. Total cholesterol levels are higher in hyperlipidaemia, according to the data. The results show that EZ-Piperine significantly decreased total cholesterol when compared to the group on a high-fat diet. The HFD group had greater total and low-density lipoprotein levels when contrasted with the negativity control group. Unlike the high-fat diet groups treated with Propylthiouracil and Triton X-100, EZ-piperine significantly reduced TG and LDL levels in this research. Curiously, as compared to animals treated with EZ alone, mice given EZ-piperine (20 mg) exhibited remarkable improvements in HDL, triglyceride, and cholesterol levels.

Hepatic lipid profile

Table 5 shows blood lipid levels matched liver tissue homogenate lipid characteristics (TC and TG). however, piperine had a larger action when compared to EZ alone. Lipids in the liver were unaffected by EZ’s ability to reduce blood lipid levels. Nevertheless, when compared to the individual medications, the combined effects of EZ and piperine were far more effective. For both TGs and TCs, the outcomes were comparable.

Table 5 Hepatic lipid profile effects of EZ and piperine using high fat diet method

Hepatic function test

HFF rats’ high lipid content in hepatic tissues may cause oxidative damage and large elevations in SGOT, SGPT, and ALP (Table 5). Both EZ and piperine significantly lowered these increases. The groups given medication or piperine alone had enzyme levels within the normal range despite a significant drop, indicating improved liver function. Taking both drugs together boosted liver function. Curiously, piperine dose-dependently increased hepatic activity, even at 10 mg/kg (Figs. 6–8).

Fig. 6

Hepatic lipid profile effects of EZ and piperine using PIU method

Fig. 7

Hepatic: lipid profile effects of EZ and piperine using PIU method

When there are six samples, the results are shown as the mean plus or minus the standard error of the mean. Statistical significance is determined when ap < 0.01 and bp < 0.05, in comparison to the normal control group. If cp. < 0.01 and dp < 0.05, then there is statistical significance when compared to the HFD control group.

Fig. 8

Hepatic lipid profile effects of EZ and piperine using Triton X-100 induce method

After being administered different dosages of EZ and piperine, rats that were treated with propylthiouracil (TRITON X-100), given a high-fat diet (HFD), or received no treatment whatsoever had their liver enzyme activity and total protein levels measured (Tables 6 and 7). The enzymatic activities of liver function (SGOT, SGPT, and ALP) were found to be elevated in rats that were subjected to a high-fat diet (HFD), administered propylthiouracil (PT), or caused hyperlipidaemia, according to the results. Results were similar across dosages in the combination EZ and piperine groups., and in the groups treated with either compound alone, demonstrating substantial and normalising effects. At doses two times greater than the level of HFD, Propylthiouracil, and Triton X-100 (10 mg), EZ and piperine (at 10 and 20 mg) were just as effective as HFD, Propylthiouracil, and Triton X-100. In addition, both the 10 mg and 20 mg doses of EZ and piperine had a notable impact; however, the ideal dosage for hyperlipidaemia was found to be 20 mg.

Table 6 Hepatic lipid profile effects of EZ and piperine using PIU method

Table 7 Hepatic lipid profile effects of EZ and piperine using Triton X-100 induce method

Interestingly, SGPT and ALP activities responded better to EZ with piperine’s lowering effect than to EZ alone or combinations below 20 mg. After p.o. therapy with group VII (10 and 20 mg/kg), SGOT and SGPT levels significantly lowered (cp. < 0.01). After treatment with EZ and piperine, ALP enzyme activity considerably lowered (dp < 0.05). EZ and piperine restored total protein levels in HDF-induced hyperlipidemic rats. Taking atorvastatin helped reduce hyperlipidemia-related levels of total protein, the SGOT, SGPT, and ALP in rats that were given a high-fat diet.

Histopathological examination

Figure 9 shows that hepatic tissue samples from rats with HFD-induced hyperlipidaemia were more disorganised, had fatty alterations, and condensed nuclei than those from rats without HFD, It showed a well-structured liver with a central vein, bile duct, and cord-shaped hepatocytes. Hepatocytes treated with EZ showed an improvement in cellular histoarchitectural shape, although the histopathology profile was unique from that of rats treated with piperine, which had condensed nuclei (Table 8).

Fig. 9

Hisopatholocal examination of HFD method Group-I to Group-VII

Table 8 Effect of EZ-piperine alone and combination on histopathological changes

NAFLD prevalence ranges from 10 to 35%. contingent upon the demographic surveys and diagnostic methodologies employed. However, out of all chronic liver illnesses, the United States has the highest prevalence rate at 75% [50]. Theoretically, by influencing metabolic and lipid profiles, high-fat diets (HFDs) may exacerbate non-alcoholic fatty liver disease (NAFLD) [51,52,53]. Biochemical and histological results showed hepatic mutilation in 90-day high-fat diet (HFD) rats. When treated with EZ and piperine, enzyme activity decreased temporarily, demonstrating that the two drugs protected against hyperlipidaemia. Piperine and cobmination EZ shown to have considerable antihyperlipidemic and hepatoprotective properties, as indicated by the reduced incidence of high-fat diet-induced NAFLD.

Cardiovascular disease can cause hyperlipidaemia, which is characterised by minimal and extremely low density lipoprotein cholesterol as well as high total cholesterol. WHO estimates that 40% of the global population suffers from hyperlipidaemia. This is concerning because high cholesterol is considered the leading cause of death (Organisation, 2019). Approximately 23.6% of adult Africans have dyslipidaemia, according to meta-analyses [54], whereas 31% of Asians are at high risk for hyperlipidaemia, according to other reports [55].

Fatty liver and myopathy are two of the several disorders that are more common in people with hyperlipidaemia. The impact of group VII on hepatic cells and skeletal muscle structure was investigated in this investigation. Histology confirms biochemical results in this research. Under the light microscope, liver tissue sections taken from the negative control group revealed a typical histological layout of the surrounding hepatocytes (A) and central vein (CV). In the positive control group, the liver portion had a few inflammatory cells, diffuse fatty change surrounding hepatocytes, a clogged cardiovascular system, enlarged sinusoids, and bile duct dilatation (B). Fewer fat vacuoles in hepatocytes, dilated sinusoids, minor congestion around the portal region, and modest bile duct dilatation (C) were seen in the liver tissues of animals treated with EZ solution. In contrast, animals given EZ and piperine exhibited a return to normal hepatic cord organisation around the portal vein and sinusoids. Pattern of hepatocytes known as mild cord. Mild haemorrhage and dilatation of the sinusoidal space. The Kupffer cells are operating normally (D). There was modest dilatation of the sinusoidal gaps and haemorrhage in the extrahepatic area (E), but the hepatocytes and periportal and centrilobular regions seemed normal. Periportal liver sinusoidal space dilatation was modest. (F), but otherwise the hepatocytes and periportal and centrilobular regions seemed normal. Hepatocyte cord pattern in a normal state. A few number of lymphocytes are present around the portal of entry. It seemed like the sinusoids and Kupffer cells were OK. A few number of lymphocytes are present around the portal of entry. G: No signs of fibrosis.

Molecular interaction studies

Two distinct receptors, human C-reactive protein (PDB ID: 1B09) and human lanosterol 14-alpha-demethylase (PDB ID: 3LD6), were analysed using molecular docking simulations to see how our formulations interacted with them. In order to better understand how our formulation interacts with these proteinsWe performed molecular docking experiments. To further our comprehension of the interaction between our formulation and these proteins, we performed molecular docking tests.

The molecular docking results demonstrate that all of the compounds had lower binding energies relative to the gold standard medication Atorvastatin (-9.2 Kcal/mole) when applied to the human C-reactive protein receptor. Ezetimibe has two hydrogen bonds with amino acid residues Lys114 and Glu88 and a dock score of -8.4 Kcal/mole when taken alone. In contrast, our phytochemical piperic acid has a dock score of just − 6.6 Kcal/mole, suggesting that it does not interact significantly with the receptor on its own. Thr41, Ala92, Thr90, and Tyr73 are the amino acids with which piperic acid forms hydrogen bonds (Tables 9 and 10). Ezetimibe and piperic acid, when formed into a conjugate, demonstrated promising outcomes. The conjugate docked with a binding score of -7.9 Kcal/mole, and it formed hydrogen bonds with Arg116 and Ser44 (Figs. 10 and 11). Accordingly, it appears that piperic acid will have a more favourable effect when combined with Ezetimibe rather than when used alone. In both cases, the hydrogen connection between piperic acid and Ser44 remains intact. However, when compared to being used alone, the way Ezetimibe interacts in conjugate formulation is completely different.

Table 9 Molecular docking studies interaction with 1B09

Table 10 Molecular docking studies Interaction with 3LD6

Fig. 10

A&B are E&F, B&C, and Docking models of ezetimibe and piperic acid with 1B09 protein A model for the docking of the 1B09 protein with ezetimibe and piperic acid

Fig. 11

A & B are The docking models of ezetimibe and piperic acid conjugate with 3LD6 protein are shown in E & F, alongside the docking models of piperic acid and 3LD6 protein in B & C

Molecular docking studies on the human C-reactive protein receptor revealed that, with the exception of piperic acid, all substances exhibited lower binding energies than the gold standard medication atorvastatin (-8.7 Kcal/mole). With three π-π bonds with amino acid residues Phe34, Tyr31, and Trp39, the medication Ezetimibe alone has a dock score of -10.6 Kcal/mole. However, with a dock score of just − 7.2 Kcal/mole, our phytochemical piperic acid has not demonstrated significant interaction with that receptor on its own. Piperic acid forms hydrogen bonds with the amino acid His89. Ezetimibe and piperic acid, when formed into a conjugate, demonstrated promising outcomes. Conjugate binding dock scores were − 11.5 Kcal/mole, and hydrogen bond interactions were observed with Arg82 and Phe34. Phe34, Tyr31, and Trp39 are amino acid residues that form π-π bonds with this compound.

MTT cytotoxicity assay

To confirm their safety, we evaluated EZ’s cytotoxic effects using the MTT test., an optimised combination of EZ and pipierine, and piperine on normal Vero cells after 72 h of treatment. In a manner that depends on the drug decreased the number of viable Vero cells compared to the control, with an IC50 value of 36.54 µg/mL (refer to Fig. 12 for details). Compared to the optimal combination of EZ and piperine, the medication suppressed cells more effectively at 259 µg/mL. According to experiments on typical Vero cells, EZ plus piperine was significantly safer than EZ alone [56].

Fig. 12

Drug’s effect on normal Vero cells, as opposed to untreated cells, as well as the optimised combination of EZ and pipierine, and piperine

The optimal outcomes from the pharmacological studies regarding in vitro characterization and cell survival were derived from an enhanced combination of EZ and piperine. Consequently, pharmacological research was undertaken on the optimized combination of EZ and piperine to elucidate the effect of the EZ solution on reducing cholesterol levels.

Pharmacokinetic studies

Enhancement of plasma concentration of EZ

We estimated the plasma concentrations of EZ at different times after administration, and Table 11 shows the results. The highest level of EZ in the plasma was 24.36 ± 1.36 µg/mL at 2 h, and it dropped by 50% after 4 h, which is in line with the T_1/2 and C_max values that had already been reported for EZ. EZ and piperine were given together in doses of 10 mg and 20 mg. Within 4.36 h, the peak plasma concentration of 159.86 ± 0.54 µg/mL was reached, which was 100% higher than when EZ was given alone. The desired plasma concentration continued for 24 h. There were no significant variations or increases in the concentration when the piperine dose exceeded 30 mg/kg. As a result, giving piperine and EZ together greatly increased the drug’s concentration in the blood and slowed its elimination, which is in line with the increased activity mentioned above. Figure 13 illustrates the changes in the plasma concentration of EZ over time and after its co-administration with piperine [57].

Table 11 Pharmacokinetic studies of pure EZ solution and combination of EZ and Piperine solution

Fig. 13

Concentrations of EZ solution, piperine, and the optimised EZ/piperine solution in the blood changing over time

Discussion

Almost half of all the tiny compounds that have been approved as medications this decade have come from natural sources. It has been proposed that the present method of drug development could benefit from a change from discovering new chemical entities to combining existing agents. Additionally, the main problems with medications are that they require greater dosages, have poor absorption, low bioavailability, and patients do not comply. The active chemical component of black pepper is piperine, which accounts for about 5–9% of the total. Its safer use as a spice led the FDA to classify it as a herb. It seems that the LD₅₀ of the human dose is much higher than the dosage of 15–20 mg/person/day that can be administered in divided doses of Piperine. For a number of xenobiotic agents, piperine can increase their efficacy while decreasing the frequency of dose. Because of its lower dosage and enhanced availability at the site of action, piperine decreases the side effects and toxicity of the medications when taken with the medication [15]. Interestingly, there were no discernible differences between Groups II and III, and the rats’ body weights recovered to normal when 5 mg/kg of piperine was combined with EZ. At this concentration, it appears that this dosage of piperine may not significantly impact weight reduction. Nevertheless, a notable decrease in weight growth of -12.32 ± 4.65 g and − 7.13 ± 2.35 g, respectively, was noted when the dosage of piperine was raised to 10 mg/kg and 20 mg/kg. Based on previous research on the thermogenic and metabolic properties of piperine, this dose-dependent effect indicates that taking larger dosages of the compound may increase its weight-lowering effects. Higher dosages of piperine had a more noticeable impact on body weight, demonstrating its potential as an adjuvant to EZ in weight management. It is possible that this weight loss is due to piperine’s capacity to raise thermogenesis, improve lipid metabolism, and decrease fat storage; however, additional research is needed to clarify the specific pathways involved. This study found that the onset of hyperlipidemia (high cholesterol, low HDL) and triglycerides, total cholesterol, low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) were all significantly increased in serum lipid levels of individuals following a high-fat diet (HFD). Combining piperine with EZ resulted in a dose-dependent reduction in triglyceride and total cholesterol levels, together with a significant increase in HDL, but piperine alone had no significant effect on lowering triglyceride levels. Research shows that at higher doses (10 and 20 mg/kg), piperine enhances EZ’s lipid-lowering effectiveness. The improved lipid profiles, particularly the reduced levels of triglycerides and low-density lipoprotein, support the notion that EZ-piperine combos may aid in the prevention of atherosclerosis and heart disease. As a cholesterol-lowering medication, EZ-piperine was just as effective as atorvastatin. That it shows great promise as a medication for the treatment of hyperlipidemia and related metabolic diseases is rather encouraging. Although EZ significantly reduced serum lipid levels when taken alone, the results of the hepatic lipid profile show that it had no discernible effect on hepatic lipids. Combining EZ with piperine improved the lipid-lowering effects in the liver by causing a greater reduction in triglycerides (TG) and total cholesterol (TC). Significant increases in SGOT, SGPT, and ALP levels were seen in hyperlipidemic rats treated with HFD, propylthiouracil, and Triton X-100, suggesting oxidative liver injury. When administered in combination, EZ and piperine significantly reduced these enzyme levels, indicating improved liver function. Piperine increased hepatic activity in a dose-dependent manner, particularly at higher dosages (10 and 20 mg/kg). When taken together, EZ and piperine were more effective than either medicine alone at these dosages in reducing liver enzyme levels and enhancing liver function. This study confirms that EZ and piperine are helpful in treating hyperlipidemia and reducing liver damage caused by lipid accumulation. The results were similar to those of atorvastatin.

Histological examination of liver tissues revealed significant structural abnormalities, including steatosis, architectural disarray, and nuclear condensation, in rats with high fat diets. A condition linked to hyperlipidemia, non-alcoholic fatty liver disease (NAFLD) is indicated by these changes. Although hepatic architecture was partially improved by EZ treatment, some hepatocytes still showed compacted nuclei. On the other hand, piperine may have hepatoprotective effects as rats given the drug had a very normal histological profile. The most significant improvement was observed when EZ and piperine were administered together. This was accompanied by a return to the normal position of the hepatic cord around the central vein, with minimal bleeding and only modest sinusoidal dilatation. This finding provides more evidence that EZ and piperine work together to protect the liver from NAFLD-related liver damage and lower hyperlipidemia. The findings and biochemical data indicate that the combination of EZ and piperine possesses significant hepatoprotective and antihyperlipidemic effects. This suggests they have potential as a treatment for hyperlipidemia and non-alcoholic fatty liver disease. Molecular docking simulations were employed to investigate how the medication formulations interacted with two main receptors: human lanosterol 14α-demethylase and human C-reactive protein (CRP). All compounds exhibited binding energies lower than the reference medicine, Atorvastatin (-9.2 Kcal/mole), according to the results for the human CRP receptor. By itself, ezetimibe had a dock score of -8.4 Kcal/mole, with stable contact indicated by hydrogen bonds formed with Lys114 and Glu88. Thr41, Ala92, Thr90, and Tyr73 were hydrogen-bonded to piperic acid, which had a docking score of -6.6 Kcal/mole and a lower binding affinity. Specifically, Ezetimibe conjugated with piperic acid enhanced the dock score to -7.9 Kcal/mole and formed more hydrogen bond interactions with Arg116 and Ser44, indicating a stronger binding affinity. The importance of this interaction in the conjugate was highlighted by the fact that the hydrogen bond with Ser44 was consistently observed in both the conjugated and free piperic acid situations. Based on the data, it appears that the binding affinity to the CRP receptor is improved when Ezetimibe and piperic acid are combined, with piperic acid showing more effectiveness than when used alone.

Lipid buildup in arterial walls is the complex process that leads to atherosclerosis. Atherosclerotic coronary artery disease (CAD) and other cardiovascular diseases are largely caused by hyperlipidemia, which is worsened by high-fat diets. In order to lower the risk of ischemic heart disorders, it is necessary to regulate cholesterol and lipid profiles, especially by raising HDL cholesterol and decreasing TC, LDL cholesterol, and TG levels. The plant-derived chemicals hesperetin and piperine have anti-inflammatory, neuroprotective, and antioxidant properties that make them an attractive treatment option for a range of medical issues, including cardiovascular illnesses. Unfortunately, their low solubility and poor absorption restrict their clinical application. To overcome this obstacle and increase the therapeutic potential of these chemicals, the current work set out to develop amorphous systems that would increase their bioavailability. The study demonstrated that the potent antihyperlipidemic medication Ezetimibe (EZ) had its action much increased when given with piperine, since piperine is a bio-enhancer. Piperine increased EZ bioavailability and absorption at levels as high as 20 mg/kg without negatively impacting liver function. It appears that treating hyperlipidemia and cardiovascular illnesses with EZ and piperine taken together can be a safe and effective way to maximize the therapeutic effects of both drugs by increasing their bioavailability and pharmacological efficacy.

Conclusion

Researchers found that ezetimibe’s (EZ) pharmacokinetics, liver function, lipid profiles, and weight management were all significantly affected when piperine and EZ were administered side by side. The combination of EZ and piperine, particularly at higher dosages of piperine (10 mg/kg and 20 mg/kg), considerably reduced weight gain in rats with hyperlipidemia. The levels of blood triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) were all significantly reduced by this combination, while levels of high-density lipoprotein (HDL) were increased. When combined with EZ, these effects were much stronger than when administered alone. Hepatoprotective benefits were demonstrated by histological findings and improvements in liver enzyme levels (SGOT, SGPT, ALP) when EZ and piperine were administered together. Rats with normal lipid profiles had more organized liver tissues and less damage than rats with hyperlipidemic livers. When piperine was added to EZ, its plasma concentration was considerably increased. The peak concentration of EZ was reached 4.36 h later, which is significantly later and greater than when EZ was delivered alone. Based on these results, it appears that piperine increases EZ’s bioavailability and keeps its effects going. Compared to EZ alone, the optimized combination of EZ and piperine had less cytotoxic effects on normal cells (Vero cells), suggesting a safer treatment profile. Ultimately, the synergy between EZ and piperine offers great potential as a treatment strategy for hyperlipidemia and related liver diseases, since it improves lipid control and liver function while simultaneously increasing EZ’s pharmacokinetics.