Hypolipidemic activity of Ceciwis ethanol extract on wistar rats induced by high fat in vivo ‎

Heru Nurcahyo¹*, Aldi Budi Riyanta¹, Rizki Febriyanti¹, Heri Sutanto², Wiwin Herdwiani³

¹Department of Pharmacy, Politeknik Harapan Bersana, Tegal, Indonesia. ²Department of Physics, Universitas Diponegoro, Semarang, Indonesia. ³Department of Pharmacy, Universitas Setia Budi, Surakarta, Indonesia.

Correspondence: Heru Nurcahyo, Department of Pharmacy, Politeknik Harapan Bersana, Tegal, Indonesia. [email protected]

ABSTRACT

In Indonesia, cruciferous cabbage is known as Ceciwis (Brassica oleracea L. var. capitata f. alba Alef), a vegetable that can be consumed as a diet from Tegal is one of the plants rich in flavonoids and phenol proposed has an antidyslipidemic ac-tivity. The purpose of this study is to see how Ceciwis affect the lipid profile of Wistar rats (Rattus norvegicus) fed a high-fat diet. Method: Rats divided into seven groups: the normal group, the negative group, positive group, and the treatment group with several doses of ethanol extract Brassica oleracea L. var. capitata f. alba Alef (EEB) 125, 250, 500, and 1000mg/KgBW. Each test group was given a high-fat diet of egg yolk 2 times a day 2 mL/200g (p.o), as well as PTU (Propylthiouracil) 0.01% during the 2-week induction period, then for 2 weeks the extract was dosed and took cholesterol measurements to determine the lipid profile with parameters of total cholesterol (TC), low density lipoprotein (LDL), and tryglyceride (TG). Results: EEB activity at a dose of 1000mg/KgBW significantly <0.05 decreased lipid profiles for TC, LDL, and TG almost equiva-lent to simvastatin positive control. Conclusion: EEB at a dose of 1000 mg/KW containing flavonoids and phenols significantly reduced the TC, LDL, and TG profiles‎.

Keywords: Ceciwis, Ethanol extract, Hypolipidemic, In vivo

Introduction  

Hypolipidemic is a decrease in triglycerides and cholesterol with an increase in high density lipoproteins. These efforts are used to decrease the activity of dyslipidemia which can interfere with the metabolic activity of carbohydrates, proteins and fats that cause various diseases [1]. Dyslipidemia activities can cause the risk of diseases such as heart disease, diabetes to the risk of death [2, 3]. Endothelial dysfunction becomes the beginning of atherosclerosis plaque deposits due to increased LDL in the blood vessels [4]. So it needs to be addressed by consuming plant extracts that have been proven hypolipidemia activity.

Large side effects are the cause of the increased use of herbal plants. Herbal plants were chosen because of the great potential in the territory of Indonesia. In addition, side effects are relatively small and cheaper [5].  Various plant extracts have been tested for hypolipidemia activity for the treatment of dyslipidemia. Ethanol extract and fraction from the seeds of Cassia tora L. Able to decrease total cholesterol by 71,25%, increase HDL by 19,18% and lower triglycerides by 38,46% [6]. Moringa oleifera Lam methanol extract is known to reduce the risk of LDL, VLDL, triglycerides, atherogenic index and increase HDL [7, 8]. Aqueous extract of Capparis spinosa L. (CS) at a concentration of 20 mg/ kg body weight was able to significantly lower plasma cholesterol through observation for 4 days in normal and diabetic rats [9]. Great potential for developed herbal plant extracts to be an option for hypolipidemic activity.

Ceciwis "cruciferous cabbage" (Brassica oleracea L. var. capitata f. alba Alef) has the potential to have hypolipidemia activity. Ceciwis contains volatiles such as phenolic aldehydes, alcohols, and ketones [10]. Red cabbage (Brassica oleracea L.) contains anthocyanin dyestuffs [11, 12]. In addition, Ceciwis is also known to contain flavonoids and flavonols that have antioxidant activity  [13-16]. The content of anthocyanins, flavonoids and flavonols has hypolipidemic activity [17]. For this reason, it is necessary to investigate the hypolipidemic activity of ceciwis extract and see the extent of cholesterol reduction in rats induced with high fat.

Hypolipidemic activity can be performed by inducing rats with high fat. Zhang et al., (2020) reported optimization of extraction of R [18]. Laevigatae fruit and hypolipidemia activity by inducing rats with it was obtained that R. laevigatae extract affected lipid metabolism and oxidative stress in a high-fat diet. Jensi, V. and Gopu (2018) investigated T. Arjuna extract with quercetin, gallocatechin, luteolin, terminoside a content against hypolipidemia activity in rats [19]. So that this induction method in rats can be carried out to investigate ceciwis extract (Brassica oleracea L. var. capitata f. alba Alef) against hypolipidemic activity.

Materials and Methods

Tools

Reflux extractor, Rotary vacuum evaporator (Rotavapor, Buchi®), analytical balance (Precisa®), measuring cup (pyrex), magsonde for oral administration of extracts and tools for the manufacture of liver histology preparations, namely: cutting board, scalpel knife, tweezers, tissue cassette, automatic processor machine, vacuum machine, blocking machine, freezer (-20°C).

Ingredients

Ethanol, Ceciwis (Brassica oleracea var. capitata f. alba alef), distilled water, quail eggs, PTU, biolabo reagent, physiological NaCl 0.9 percent.

Sample treatments

Ceciwis samples were collected from farmers in the Tegal Regency of Central Java, Indonesia. Preparation included wet sorting, washing, chopping, dry sorting, and chopping. The chopped ceciwis is then ready to be extracted using the reflux method.

Methods of extraction

Reflux extraction using 70% ethanol solvent and a sample: solvent ratio of 1:10, i.e. 100 grams of finely ground shallots in 1000 ml 70% ethanol. Reflux lasts for four to five hours with 70% alcohol solvent with a heating temperature of 50 degrees Celsius, then the liquid extract is evaporated with an evaporator at 50 degrees Celsius, and then transferred closer to a water bath at 50 degrees Celsius.

Test animal preparation

Male white rats (Rattus norvegicus) of the Wistar strain, aged 8–10 weeks, were used as the study's population. They were obtained from the Pharmacy of Politeknik Harapan Bersama Indonesia animal laboratory. The experimental animals were then acclimatized for seven days with conventional feed and aquadest drink, with observations made on their behavior and health conditions, and then weighed following the acclimatization period.

Research method

This research is a laboratory experimental study where the induction of wistar rats with high fat is exogenously and endogenously. Exogenously, a high fat diet feed with quail egg yolk as the main ingredient was used, while endogenously, PTU was used for 14 days. Hyperlipidemic conditions were created by monitoring blood lipid levels in wistar rats with levels ranging from 107 to 207 mg/dL. Furthermore, for the treatment and administration of wistar rats during a 14-day period, they were separated into 7 groups, each with 5 test animals:

1. The first group (K normal) was made up of normolipidemic rats fed a standard diet without action with a high-fat diet.
2. The second group (K-) was made up of hyperlipidemic rats fed a high-fat diet without drugs.
3. The third group (H1) was hyperlipidemic rats fed a high-fat diet and ethanol extract (Brassica oleracea var. capitata f. alba alef) 125mg/KgBW (EEB 125).
4. The fourth group (H2) was hyperlipidemic rats fed a high-fat diet and ethanol extract (Brassica oleracea var. capitata f. alba alef) 250mg/KgBW (EAB 250).
5. The fifth group (H3) was hyperlipidemic rats fed a high-fat diet and ethanol extract (Brassica oleracea var. capitata f. alba alef) 500 mg/KgBW (EAB 500).
6. The sixth group (H4) was hyperlipidemic rats fed a high-fat diet and ethanol extract (Brassica oleracea var. capitata f. alba alef) 1000 mg/KgBW (EAB 1000).

The seventh group (K+) was hyperlipidemic rats that were given a high-fat diet and given the hypolipidemic drug simvastatin at a dose of 1.8 mg/KgBW/day.

Investigating the lipid profile

On wistar rats, lipid profiles were examined by drawing blood through the caudalis vein with a scalpel, centrifuging the blood, and extracting the serum. The total cholesterol levels in the blood were assessed using the cholesterol oxidase peroxidase-4-aminophenazone (CHOD-PAP) method. The glycerol-3-phosphate oxidase, peroxidase, and chromogenic reaction with 4-amonophenazone (GPO-PAP) method was used to determine the amounts of triglycerides.

Research ethics

This research was conducted based on the ethics of animal research, where the ethics of research was obtained by the Semarang Health Polytechnic Ethics Commission with the number 0686/EA/KEPK/2022.

Results and Discussion

A small sample of ceciwis and a simple procedure wasused to conduct phytochemical screening to determine whether secondary metabolites are present, which are beneficial to the human beings [20]. The results of qualitative compound identification based on observations can be seen in Table 1.

The test results showed that the ethanolic extract of ceciwis contains  flavonoids and phenols. The results of a qualitative test were conducted on extracts  thought to have the most flavonoids and phenols compounds. It can be seen by the color change that occurs from clear yellow to red-orange and blue-black for identification of phenols. The higher the content of flavonoid and phenols compounds, the darker the color produced [21].

Flavonoids identical with quercetin has activity in inhibiting the work of the lipase enzyme, which in the body oil can be hydrolyzed into fatty acids. Quercetin has activity in inhibiting the work of the lipase enzyme, which in the body oil can be hydrolyzed into fatty acids. Through beta oxidation, fatty acids can also be transformed into acetyl CoA, which is a precursor to cholesterol. The presence of quercetin in shallots inhibits the growth of precursors, which leads to an increase in blood cholesterol levels. An increase in free radicals [22] can damage nucleic acids, proteins, and lipid membranes, potentially leading to cancer and liver damage. Furthermore, quercetin can lower the activity of the enzyme acyl-CoA cholesterol acyltransferase (ACAT), which regulates cholesterol absorption in the intestine as well as lipoprotein synthesis in the liver [23]. Flavonoids can also increase the lipase enzyme's breakdown of lipids, allowing fatty acids, monoglycerides, and cholesterol to be absorbed by intestinal mucosal cells while lipids are expelled with feces, resulting in lower cholesterol and triglyceride levels [24, 25].

Hyperlipidemia is a disease burden impressively associated with obesity and characterized by the presence of extraordinary fats in the blood serum. The lipid profile in our blood is generally expressed by the presence of fats in the blood, such as cholesterol and triglycerides [26].

Normal Total cholesterol (TC) levels in rats with total cholesterol values of above 107-207 mg/dL were compared to baseline in rats with total cholesterol values of above 107-207 mg/dL [27], and the treatment was given with various doses on days 14 to 28 while the lipid profile level of the rats was observed.

In addition to the standard tests above, tests were also carried out with mice Wistar hypercholesterolemia. With treatment using egg yolk and PTU for two weeks Serum average cholesterol level has increased from 65 ± 7.023 mg/dL to 214.508 ± 2.002 mg/dL, which means that you have hypercholesterolemia.

For the results of the Paired T-Test between the administration of high fat on day 14 and after administration of ethanol extract on day 28, sig was found to be 0.0001, which means that there is a relationship between the time of administration of ethanol extract with a decrease in cholesterol levels.

For the results of the Paired T-Test between before and after giving the extract, the value of sig found 0.0001 for a dose of EEB 1000, that the dose has a relationship between the time of administration of the ethanol extract with a decrease in LDL levels, other than that dose does not provide a significant reduction.

For the results of the Paired T-Test between the administration of high fat on day 14 and after administration of ethanol extract on day 28, sig was found to be 0.0001, which means that there is a relationship between the time of administration of ethanol extract with a decrease in TG levels.

Giving EEB for 14 days showed a good downward trend in lipid profile levels (TC, LDL, and TG), indicating specific hypolipidemic activity. Data on lipid levels acquired on day 14 compared to a decrease in lipid levels on days  28, indicating specific hypolipidemic activity. The data were analyzed using a paired t test to compare the negative control with the treatment to determine lipid profile activity; the sign (*) with a significant value of p value is significant (0.05) where there is a significant decrease in (A) total cholesterol lowering activity, (B) LDL lowering activity, and (C) triglyceride lowering activity.

EEB 1000 extract dose had a significant decrease for TC, LDL and TG, where the compound Phenolics increase cholesterol metabolism [28]. Several phenolic compounds such as b-sitosterol have been proven to increase cholesterol metabolism. Phenolics are one of the antioxidant groups [29]. Antioxidants have hypolipidemic activity because they are able to activate LDL-cholesterol receptors so that LDL-cholesterol levels in plasma decrease [4, 19]. flavonoids are able to reduce LDL, increase LDL density through receptors in the liver and are able to bind to apolipoprotein B. Additionally, flavonoids can inhibit the work of the enzyme 3-hydroxy 3-methylglutaril coenzyme A reductase (HMG Co-A reductase) to reduce trigliseride and to increase HDL [2, 5, 12].

Conclusion

Based on the results of research and discussion, then it can be concluded that EEB activity at a dose of 1000mg/KgBW significantly <0.05 decreased lipid profiles for TC, LDL, and TG almost equivalent to simvastatin positive control. Conclusion: EEB at a dose of 1000 containing flavonoids and phenols significantly reduced the TC, LDL, and TG profiles

Acknowledgments: This research no potential conflict of interest was reported by the authors, We would like to thank Direktorat Riset dan Pengabdian Masyarakat (DRPM) from Ministry of Research and Technology.

Conflict of interest: The researcher stated that there was no conflict of interest in the manuscript and the code of ethics was violated and was in accordance with the provisions.

Financial support: We would like to thank Direktorat Riset dan Pengabdian Masyarakat (DRPM) from Ministry of Research and Technology, Higher Education  for funding this research through the “PKPT grant scheme” program with number 6/065012/PPKM.PTV/SP2H/AK.04/022.

Ethics statement: The researcher stated that no code of ethics was violated and in accordance with the provisions.

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