INTRODUCTION:

Atherosclerosis normally arises when the fatty, yellow-coloured plaques i.e. (atheroma's) grow up on the arterial walls, narrowing the arteries and inhibiting the flow of blood¹.

Hypercholesterolemia is a health problem that is strongly linked to cardiovascular disease (CVD), the most significant cause of mortality globally². CVD accounts for a large proportion of all deaths worldwide, with hypertension and hypercholesterolemia playing a significant role³. Total cholesterol levels and coronary artery disease have a progressive connection. According to the World Health Organization (WHO), hypercholesterolemia has become a more serious concern in recent years since it is linked to nearly half of all ischemic heart disease cases globally⁴. CVD is primarily caused by dyslipidemia, oxidative stress, inflammation, hypertension, and insulin resistance⁵. Epidemiological studies have shown that the occurrence of hypercholesterolemia is strongly linked to poor dietary habits, such as the over consumption of foods high in cholesterol and saturated fats, which leads to an increased prevalence of atherosclerosis⁶. Other risk factors for atherosclerosis include a family history of early cardiovascular disease, age, and smoking⁷.

Hypercholesterolemia is a lipid metabolic disorder characterised by an increase in triglyceride (TG), low-density lipoprotein (LDL), total cholesterol (TC), and reduction in high-density lipoprotein (HDL) levels in the blood⁵.Animal model research, epidemiological cohort studies, genetic investigations in polygenic and monogenic human illnesses, and randomised trials on LDL levels have all shown the significance of LDL as a primary cause of atherosclerosis⁸. LDL accounts for the vast majority of total cholesterol. Maintaining normal levels of TG and cholesterol is critical for lowering the risk of cardiovascular disease. Dyslipidemia is defined as abnormal lipid levels in the bloodstream, with a low level of "good" cholesterol designated as HDL, a high amount of "bad" cholesterol denoted as LDL, and/or higher levels of TG⁹.

Some medications are enzyme inhibitors, which have a role in the progression of many diseases. Concerns about synthetic enzyme inhibitors' toxicity and side effects have prompted a quest for novel, safe, and effective inhibitors, mostly from natural sources. The quest for natural products has been boosted by discovering novel medications that can be reduced to molecular type and/or control metabolism¹⁰. Therefore, any dietary and pharmaceutical intervention that improves or normalizes aberrant lipid metabolism may be effective for decreasing the risk of cardiovascular illnesses. There are now several medicines available to treat dyslipidemia. However, there is increased interest in the usage of herbal products¹¹.

Rhizophora apiculata is a medicinal plant that grows along the coast of Indonesia. According to previous research, the HPLC examination of mangrove Rhizophora apiculata bark extracts revealed that catechin was the most prevalent component of the flavonoid monomers¹². Another previous research has shown that the root bark of Rhizophora apiculata is a major source of phytosterols, which may provide possible support for using traditional medicine¹³. In addition to being a rich source of phytosterols, prior research has clearly demonstrated that Rhizophora apiculata's pyroligneous acid dichloromethane extract (CPAE) is also a potent antioxidant due to its high total phenolic content, superior free radical scavenging activity (significant antioxidant activities), and ferric reducing power¹⁴. The mangrove tannins are comparable to the synthetic standards and other commercial tannins assessed in terms of their significant reduction power, DPPH as well as ABTS free radical-scavenging capacities¹⁵. Research by Gurudeeban et all; showed that the ethanolic extract and dichloromethane fraction of Rhizophora apiculata leaves demonstrated antihyperglycemic effects¹⁶. This study aimed to examine the effects of Rhizophora apiculata barks (RAB) ethanolic extract on lipid metabolism, namely cholesterol, triglyceride, LDL, and HDL levels in rats given a high-cholesterol diet (HCD).

MATERIAL AND METHODS:

Rhizophora apiculata barks preparation:

Fresh Rhizophora apiculata barks were collected from East Lampung Regency, Lampung Province, Indonesia. The barks were washed under running water, rinsed with distilled water, and air dried for three days in an oven (Memmert^®) at 48^oC Laboratory of Department of Biochemistry Physiology and Molecular Biology (Faculty of Medicine, Universitas Lampung). Rhizophora apiculata barks were pulverised using an electric chopper (Miyako^®) into fine and homogeneous powder.

Extraction:

The maceration process was used to extract the bark of Rhizophora apiculata.The dried barks were ground in an electric grinder before being passed through sieve No.40¹⁷. Maceration was performed on the powdered materials¹⁸. Bark powder of 1500g was soaked in three litres of 96% ethanol for 72 hours before being swirled and renewed with 96% ethanol solvents every 24 hours¹⁹. The maserat was filtered using Whatman No. 1 filter paper, condensed with a rotary evaporator (Buchi R120) at 60⁰C, and concentrated with a water bath (Memmert®) at 50⁰C to evaporate the ethanol until it produced a thick extract with a consistent weight²⁰.

Animal treatments:

This study had an experimental design with a post-test-only control group. The study included 30 white male rats (Rattus norvegicus) of the Sprague Dawley strain. The inclusion criteria included white male rats, healthy, 2-3 months old, 200-250gram body weight, and with no congenital anatomical abnormalities. Mice that seemed unwell died during the treatment, or weighed less than 200-250grams following adaption were excluded. The entire rat population was acclimatised for seven days and given a standard meal twice daily. Mice were divided into six groups (n=5) after a week of acclimation. Group 1, as the control group, was fed a standard diet; group 2 was given the High-Cholesterol Diet (HCD); group 3 was given simvastatin 40 mg/kgbwt (p.o.); and groups 4, 5, and 6 were treatment groups given Rhizophora apiculata barks (RAB) extract 56.55mg/kgbwt (p.o.), RAB 28.28mg/kgbwt (p.o.), and RAB 14.14mg/kgbwt (p.o.) respectively. All treatments began on the same day and were carried out for 30 days. The Ethics Committee and ethical approval on Animal Use of Lampung University, Bandar Lampung, Indonesia, granted permission for the study (Protocol No: 438/UN26.18/PP.05.02.00/2021).

Analysis of Serum Animal:

Blood samples were collected from euthanized mice on the final day of the experiment. To separate serum from the blood sample, 2-3ml of rat blood was collected transcardially and centrifuged for 10 minutes at 2500 rpm. The sample cup is filled with 300-400L of serum for further biochemical analysis. Total Cholesterol, Serum Triglyceride (TG), Low-Density Lipoprotein (LDL), and High-Density Lipoprotein (HDL) were measured using their respective assay kits (ABX PENTRA®).

Statistical Analysis:

The results were presented as mean SD (standard deviation). A one-way analysis of variance was used to determine statistical significance (p< 0.01), followed by LSD post hoc test.

RESULTS AND DISCUSSION:

This study demonstrated that RAB effectively alleviated hyperlipidemia-related symptoms in the HCD mice model. Cholesterol feeding has frequently been used to raise blood cholesterol levels in animals in order to investigate hypercholesterolemia-related metabolic abnormalities¹⁸. Compared to the mice that treated HCD alone, the RAB administration during four-week (30-day) period resulted in significant reductions in blood TC, TG, LDL, and HDL levels.

Figure. 1: Average levels of Total Cholesterol of Rattus norvegicus (sparague dawley) given a high-cholesterol diet with different doses of Rhizophora apiculata Barks (RAB) extract. **p < 0.01 comparing HCD with control diet; #p < 0.01 comparing HCD + RAB with HCD alone; *p < 0.01 comparing HCD +SIM with HCD + RAB; ¤p< 0.01 comparing control with HCD + RAB. All data are mean ± SD (n = 5).

The effect of an ethanolic extract of Rhizophora apiculata barks on Total Cholesterol (TC) is displayed in (Fig. 1). HCD (alone) mice acquired considerably higher TC (71.40mg/dL) than control diet-fed animals (40.60 mg/dL; p<0.01). The ethanolic extract dropped the TC by 41.18% (42.00mg/dL) at a dosage of 56.55 mg/kgbwt, 35.57% (46.00mg/dL) at a dose of 28.28 mg/kgbwt, and 9.24% (64.80mg/dL) at an amount of 14.14mg/kgbwt. Serum TC concentrations in ethanolic extract of Rhizophora apiculata bark of groups 4 and 5 decreased after 30 days of therapy compared to the HCD group (p< 0.01). Cholesterol is a precursor and a part of cell membranes for steroid hormones and bile acids, which are synthesised and absorbed by body cells¹⁸. Lipoproteins, which are complexes of lipids and apolipoproteins, transport cholesterol in the blood²¹. Lipoproteins are divided into four categories: low-density lipoproteins (LDL), high-density lipoproteins (HDL), very low-density lipoproteins (VLDL), and chylomicrons. LDL is responsible for cholesterol transport to peripheral cells while HDL controls cholesterol uptake from cells. The four lipoprotein classes have distinct associations with coronary atherosclerosis²². Contributes of LDL-cholesterol (LDL-C) is to the formation of atherosclerotic plaques within the arterial intima and is strongly linked to coronary heart disease (CHD) and related mortality. Even if total cholesterol is within the normal range, an increase in LDL-C concentration indicates a high risk. HDL-C has a protective effect on plaque formation and is inversely related to CHD prevalence. Low HDL-C levels are an independent risk factor. Totalcholesterol (TC) levels are determined for screening purposes, but for a more accurate risk assessment, HDL-C and LDL-C levels must also be measured²³.

Figure. 2. Average levels of Triglycerides of Rattus norvegicus (sparague dawley) fed a high-cholesterol diet with different doses of Rhizophora apiculataBarks (RAB) extract.**p < 0.01 comparing HCD with control diet; #p < 0.01 comparing HCD + RAB with HCD alone; *p < 0.01 comparingHCD + SIM with HCD + RAB; ¤p< 0.01 comparing control with HCD + RAB.All data are mean ± SD (n = 5).

The impact of an ethanolic extract of Rhizophora apiculata barks on Triglyceride (TG) is depicted in (Fig. 2). HCD (alone) mice gained significantly more TG (134.80mg/dL) than control diet-fed animals (71mg/dL; p 0.01). The ethanolic extract reduced the TG by 45.85% (73.00mg/dL) at 56.55mg/kgbwt, 43.77% (75.80 mg/dL) at 28.28mg/kgbwt, and 27.74% (97.40 mg/dL) at 14.14mg/kgbwt. TG levels of all HCD + RAB groups decreased after 30 days of therapy compared to the HCD group (p<0.01). There is no difference in lowered TG levels between groups 4 and 5 after 30 days of treatment compared to the control group (p>0.01). Simvastatin, as a positive control, differed significantly from the control group (p>0.01). It may be determined that RAB has a more significant potential for lowering TG levels than Simvastatin.

Figure. 3: Average levels of LDLCholesterol of Rattus norvegicus (sparague dawley) fed a high-cholesterol diet with different doses of Rhizophora apiculata Barks(RAB) extract.**p< 0.01 comparing HCD with control diet; #p < 0.01 comparing HCD + RAB with HCD alone; *p < 0.01 comparing HCD +SIM with HCD + RAB All data are mean ±SD (n = 5).

The impact of an ethanolic extract of Rhizophora apiculata barks on average LDLlevels of Rattus norvegicus is expressed in Figure 3. HCD (alone) group gained significantly more LDL (21.24 mg/dL) than control diet-fed animals (13.88mg/dL; p 0.01). The ethanolic extract reduced the LDL by 26.08% (15.70mg/dL) at 56.55mg/kgbwt, 16.55% (17.73mg/dL) at 28.28mg/kgbwt, and 11.21% (18.86mg/dL) at 14.14 mg/kgbwt. After 30 days of treatment, LDL levels in all HCD + RAB groups were reduced relative to the HCD group (p<0.01). Regarding lowering LDL levels, Group 5 exhibits no significant difference from Simvastatin as a positive control (p>0.01). To lower LDL levels, an ethanolic extract of Rhizophora apiculata barks is equivalent to Simvastatin. LDL is the primary cholesterol transporter in the body, delivering cholesterol into circulation. These lipoproteins are controlled and removed by the liver via LDL receptors found on hepatocyte surfaces²⁴. A lack of LDL receptors causes an increase in LDL levels in the circulatory system. When LDL levels are too high, particles in the circulatory system reach the endothelium of artery walls, causing inflammation, smooth muscle injury, and the formation of lipid-rich plaques²⁵. Experiments in LDL receptor-deficient mice revealed that high LDL-C levels cause severe atherosclerosis²⁶. In contrast, animals lacking LDL-C have no ability to develop atherosclerosis regardless of risk factors for CHD or diet²⁷.

Figure 4: Average levels of HDL of Rattus norvegicus (Sparague dawley) fed a high-cholesterol diet with variety doses of Rhizophora apiculata Barks (RAB) extract. **p < 0.01 comparing HCD with control diet; #p < 0.01 comparing HCD + RAB with HCD alone; *p < 0.01 comparing HCD +SIM with HCD + RAB All data are mean ± SD (n = 5).

HDL is an abbreviation for high-density lipoprotein. Each bit of HDL cholesterol is a microscopic blob that consists of rim of lipoprotein surrounding a cholesterol center. Because HDL cholesterol particles are dense in comparison to other types of cholesterol particles, they are referred to as high-density cholesterol particles²⁸. The impact of an ethanolic extract of Rhizophora apiculata barks on HDL cholesterol is depicted in Figure 4. HCD (alone) mice lowered significantly more HDL cholesterol (14.93 mg/dL) than control diet-fed animals (20.95 mg/dL; p 0.01). The ethanolic extract increased the HDL cholesterol by 19.37% (17.82 mg/dL) at 56.55 mg/kgbwt, 15.12% (17.19 mg/dL) at 28.28 mg/kgbwt, and 13.21% (16.90 mg/dL) at 14.14 mg/kgbwt. HDL, or good cholesterol, has reversed the transport function. It transports cholesterol away from the body, including the coronary arteries, and deposits it in the liver²⁹. HDL-cholesterol levels are well known to play a protective role in coronary artery disease³⁰.

CONCLUSIONS:

RAB-treated animal groups (56.55; 28.28; and 14.14 mg/kg) had markedly lessened total cholesterol, triglyceride, LDL and increased HDL levels (p < 0.01) related to the HCD alone batch. These findings imply that the ethanolic extract of Rhizophora apiculata barks influences lipid metabolism which highlighting the extract's promising anti-hyperlipidemic effects and has potency as traditional medicine.

ACKNOWLEDGEMENTS:

This study was fully funded by the Research Innovation and Collaboration Programme of HETI Project Universitas Lampung and supported by Laboratory of Department of Biochemistry Physiology and Molecular Biology (Faculty of Medicine, Universitas Lampung).

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Received on 11.12.2022 Modified on 20.03.2023

Research J. Pharm. and Tech 2024; 17(1):396-400.

DOI: 10.52711/0974-360X.2024.00062