INTRODUCTION:

Hyperlipidemia is a metabolic disorder characterized by increased levels of total cholesterol, triglycerides, low-density lipoprotein (LDL), and decreased high-density lipoprotein (HDL), which is caused by a long-term high-fat diet (HFD)¹. Excess fat can cause metabolic syndrome due to obesity². Furthermore, hyperlipidemia is provoked by several factors like genetics, diets, and lack of exercise. Besides causing hyperlipidemia, high-fat foods can induce oxidative stress, thereby increasing the production of Reactive Oxygen Species (ROS) in the blood and increasing the production of endogen antioxidants³.

In addition, hyperlipidemia and oxidative stress have been associated with disease progressions such as cardiovascular disorders, degenerative diseases, and metabolism diseases with various complications⁴. Oxidative stress occurs due to an imbalance between oxidation and antioxidants. Free radicals or oxidants can increase lipid peroxidation, which will decompose into malondialdehyde (MDA) in the blood⁵. Increasing lipid peroxidation can be detected by measuring concentrations of MDA⁶, Superoxide dismutase (SOD)⁷, and Glutathione (GSH)⁸. The antioxidant properties of honey and garlic have been widely known due to their phenolic and flavonoid contents. Previous research showed the combination of gelam honey and ginger significantly decreased SOD, catalase activity (CAT), and MDA levels⁹. While GSH levels and GSH/GSSG ratio were significantly increased in STZ-induced diabetic rats compared to control rats. In addition, another research revealed that oral supplementation of royal jelly and honey in rats decreased MDA, CAT, SOD, and glutathione peroxidase (GPx) compared to the control group. However, they did not show any hypoglycemic potential¹⁰.

Based on previous research states that fermentation technology is generally easy to decompose biologically and produces a higher nutritional content¹¹. In addition, research on fermented honey is still not widely developed, especially in hyperlipidemic rats. Therefore, this study aimed to examine the total phenolic and flavonoid content of fermented honey and garlic, then test its antioxidant properties in hyperlipidemia rats.

MATERIALS AND METHODS:

Materials:

The forest honey used for the fermentation process with single garlic is forest honey from honey farmers in Kediri, East Java. The reagents used were Folin Ciocalteu, Na2CO3, Gallic acid, AlCl3, ascorbic acid, quercetin, DPPH (2,2-diphenyl-2-picrylhydrazyl; Sigma-Aldrich), TCA (thichloroacetic acid; Sigma- Aldrich), TBA (thiobarbituric acid). ; Sigma-Aldrich), DTNB (5.5'-dithio-bis-(2-nitrobenzoic acid); Sigma- Aldrich) is an analytical grade.

Instrumentation:

The instrumentation used in this research include Easy Touch GCU (Glucose, cholesterol, and Uric Acid), Semi Automatic Photometer SINNOWA BS-3000P, Spectrophotometer UV-Vis Thermo Scientific Genesys 840-208100.

Procedure:

Determination of Total Phenolic Content (TPC):

Determination of Maximum Wavelength:

This procedur was describe by Chandra, 202¹². Put 300 ul of gallic acid 30ppm into a test tube then add 1.5ml of Folin Ciocalteu, vortex the tube and let stand for 3 minutes. 1.2ml of 7.5% Na2CO3 solution was added to the mixture, then vortexed and incubated for 30minutes at room temperature. The absorbance is measured at a wavelength of 400-800nm

Preparation of Gallic Acid Standard Curve:

Gallic acid solutions were made with concentrations of 15, 20, 30, 40, 50, 60, and 70ppm. Read the absorbance at the maximum wavelength then make a calibration curve of the relationship between the gallic acid concentration (ug/ml) as the x-axis and absorbance as the y-axis.

Determination Samples

Dissolve 10mg of each sample into 10ml of distilled water, pipette 300ul of sample then add 1.5ml of Folin Ciocalteu reagent, vortex the sample, and let stand for 3 minutes. 1.2ml of 7.5% Na2CO3 solution was added to the mixture, then allowed to stand at room temperature for 30 minutes. Measure the absorbance at the maximum wavelength. Calculate the TPC level using the following formula:

TPC: C.V.fp

Information :

TPC : Total Phenolic Content

C : Phenolic Concentration ( value x)

V : Volume of extract used

Fp : dilution factor

G : Weight of the sample used.

Determination Total Flavonoid Content (TFC):

Determination of Maximum Wavelength:

^{0.5ml of quercetin standard solution was
taken, then reacted with 0.10ml of 10% AlCl}3^{. Add 0.10ml}of 1M sodium acetate solution and 2.80ml of distilled water. Measure the absorption in the wavelength range of 400-500nm. The wavelength that shows the high absorption value is the maximum wavelength

Preparation of Quercetin Standard Curve:

A total of 0.02g of quercetin was dissolved with pro-analytical ethanol up to 10ml (mother solution 2000 ug/ml). The standard working solution of quercetin 100 ug/ml was prepared by diluting the mother liquor, 1ml of the mother liquor was taken and then diluted with ethanol p.a to 20ml. Prepare a series of standard solutions with concentrations of 20, 30, 40, 50, 60, and 70g/ml. An amount of 0.5ml of each standard solution was added 0.10ml of 10% AlCl3. Add 0.10ml of 1M sodium acetate and 2.80ml of distilled water. The absorbance of the solution was measured at the maximum wavelength.

Determination Samples:

The blank solution was made with 0.5ml of ethanol p.a. Then 0.10ml of 10% AlCl₃ was added, 0.10ml of 1M sodium acetate was added, then 2.80 aquadest was added. Each absorbance measurement was compared with a blank. Determination of the sample is done by dissolving 0.2g of the sample with ethanol p.a in 10 ml volumetric flask. Pipette 0.5ml of the sample solution into a test tube. Add 0.10ml of 10% AlCl3, 0.10 ml of 1 M sodium acetate and 2.80ml of distilled water. Absorption is measured at the maximum wavelength.

TFC : C.V.fp

Information:

TFC : Total Flavonoid Content

C : Flavonoid Concentration ( value x)

V : Volume of extract used

Fp : dilution factor

g : Weight of the sample used.

Antioxidant Activity with DPPH:

Determination of the maximum wavelength of DPPH

1ml of DPPH solution was added to the test tube. Add methanol until the volume is 5ml, then homogenize and incubate at 37oC for 30 minute. Measure the solution using a UV-Vis spectrophotometer in the wavelength range of 500-600nm and the wavelength with the maximum absorption is determined.

Preparation of ascorbic acid standard curve:

Ascorbic acid standard solution was prepared with a series of concentrations of 2ppm, 4ppm, 6ppm 8ppm and 10ppm as much as 25ml in methanol at each concentration using a measuring flask. Read the absorbance at the maximum wavelength then make a calibration curve of the relationship between the gallic acid concentration (ug/ml) as the x-axis and absorbance as the y-axis

Preparation of blanko:

1ml of DPPH solution was put into a test tube and added methanol until the volume became 5ml, then covered with aluminum foil. The mixture was homogenized and incubated in a dark room at 37^oC for 30 minute. Measure the blank solution against a UV-Vis spectrophotometer with a wavelength of 525nm.

Measurement of the antioxidant activity of fermented honey:

Pipette samples of fermented honey with a concentration of 500ppm as much as 0.1ml; 0.2ml ; 0.4ml; 0.8ml; and 1.6ml to obtain concentrations of 10 ppm, 20 ppm, 40 ppm, 80ppm and 160ppm. Add 1ml of DPPH at each concentration. Add methanol to a volume of 5ml. Homogenize the solution and incubate in a dark room at 37^oC for 30 minutes. The absorbance of each sample was measured using a UV-Vis spectrophotometer at the maximum wavelength. The absorbance results that have been obtained are then carried out calculations to determine the IC50 value¹³.

Animal Treatment:

A total of 30 white rats were divided into 6 groups were adapted in the Veterinary Medicine Laboratory of Universitas Nahdlatul Ulama Surabaya for 7 days. The cage is optimized at room temperature with the help of indirect sunlight. The experimental animal treatment group was grouped as follows:

K+ : Positive control of high fat diet

K– : Negative control without treatment

P0: White rat sample (Rattus norvegicus) induced by Propilthiouracyl for 14 days. P1: Treatment of 1 group of rats given FHG 0.2gr/kgBB/day

P2: Treatment of 2 groups of rats given FHG 0.5 gr/kgBB/day P3: Treatment of 3 groups of rats given FHG 1 gr/kgBB/day P4: Standard drug simvastatin 0.18 mg/day/200gBW Determination SOD, MDA and GSH

Determination Superoxide Dismutase (SOD):

SOD levels were measured using the colorimetric method with an Elisa reader according to the EnzyChromTM Superoxide Dismutase Assay Kit Catalog# ESOD – 100 procedure at a wavelength of 450 nm. Determination Malondialdehyde (MDA).

Measurement of MDA levels was first carried out by taking 3ml of blood samples from the hearts of rats and then put them in a blood tube (EDTA). After that, centrifugation was carried out at 3000rpm for 10 minutes to obtain blood plasma to be used in determining MDA levels (if the sample is not examined immediately, it can be stored at a temperature of -20°C for a maximum of 1 month until the examination is carried out). 100L of rat blood plasma was put in a test tube, 1mL of 7.5% TCA and 2mL of 0.67% TBA were added. The mixture was heated in a water bath or water bath at 95ºC for 15minutes and cooled at room temperature for 10minutes. The mixture was centrifuged at 5000rpm for 10 minutes and the pink supernatant was transferred to a cuvette. The sample was then measured its absorbance with a UV-Vis spectrophotometer at the maximum wavelength ( max = 532nm).

Determination Glutathione (GSH):

Measurement of Glutathione (GSH) levels begins with making a standard curve to determine the value of Glutathione (GSH), by: Making a standard solution of Glutathione by dissolving 2mg/ml in 0.1M phosphate buffer pH 8.0. From the standard solution, 0 L, 5 L, 10 L, 15 L, 20 L, 25 L were taken, each of which was put in a test tube. Add 0.1M phosphate buffer solution pH 8.0 added to each tube until the volume becomes 9 ml. Enter 1ml of 5% TCA solution added into each tube, shaken or vortexed until homogeneous. From each tube 4 ml was taken and 0.05ml of DTNB reagent was added. The remaining solution from each tube was used for blanks. Then the standard absorbance was measured using a spectrophotometer at a wavelength of 412nm.

In this study, the Ellman method was used to measure GSH levels. 50µL of plasma sample was inserted into the tube. After that, 200µL of 5% TCA was added and vortexed until cloudy. 1,750 L of phosphate buffer pH 8 was added. Then centrifugation was carried out at 3000 rpm for 5 minutes and 800 L of supernatant was taken and 10 L of DTNB was added and incubated for 1 hour. Then the absorbance was measured using a spectrophotometer at 412nm.

RESULT:

Result of Total Phenolic Content (TPC):

TPC examinations were carried out using the Folin-Ciocalteu method, with gallic acid as a standard. The measurement of gallic acid absorbance was carried out using a maximum wavelength of 405nm and absorbance was measured in various concentrations at 15, 20, 30, 50, 60, and 70ppm. The calibration curve of gallic acid can be seen in Figure 1. TPC determination of the fermented honey sample was carried out by extrapolating sample absorbance to the standard curve and resulted in 6.4mg QE/g.

Figure 1: Calibration curve of Gallic Acid

Determination Total Flavonoid Content (TFC):

TFC was determined using the colorimetric method employing quercetin as a standard. The absorbance measurement of quercetin was measured using a maximum wavelength of 433 nm and the absorbance was measured in various concentrations at 15, 30, 40, 60, and 70 ppm. The calibration curve is obtained in Figure 2. TFC determination of the sample was carried out by extrapolating sample absorbance to the standard curve and resulted in 4.7 mg QE/g.

Figure 2: Callibration curve of Quercetin

Result of Antioxidant Activity with DPPH:

The results of the standard curve of the relationship between concentration and % inhibition of fermented honey-garlic were obtained the equation is y = 1.3662x + 8.8171 with R² = 0.9333 as depicted in Figure 3.

Figure 3: Correlation of % inhibition and concentration in fermented honey-garlic

Based on the above equation, calculations were carried out to determine the value of x or IC₅₀ in the fermented honey sample which was then obtained at 49.34 g/mL which means the IC₅₀ value < 50 ppm, it shows that the fermented honey sample has antioxidant activity which is included in the very strong category.

Result of Cholesterol Levels:

Based on the results of the study showed that the average cholesterol levels in hyperlipidemic rats decreased in all treatment groups after giving fermented honey. The results can be shown in table 1 below.

Table 1: Cholesterol Levels after treatment Fermented Honey-Garlic

Groups	Average of Cholesterol Levels ± Std. Dev	P Value
K (-)	50,00 ± 13,92
K (+)	136.75 ± 9.25
P (1)	51,25 ± 10,40
		0.000 (P<0.05)
P (2)	44,75 ± 13,501
P (3)	41,00 ± 5,09
Std	48,75 ± 8,92

If the results of cholesterol levels are depicted in the form of a graph, it can be seen in the figure 3 below.

Figure 4: Cholesterol levels Chart

Based on table 1 and figure 3 known that the average total cholesterol level after administration of fermented honey in the K- group was 50,00 ± 13,92 U/ml; the K+ group was 136.75 ± 9.25 U/ml; the P1 group was 51,25 ± 10,40 U/ml; the P2 group was 44,75 ± 13,501 U/ml; the P3 group was 41,00 ± 5,09 U/ml; the standard drug group was 48,75 ± 8,92 U/ml. Based on the results of statistical analysis using one way ANOVA obtained a P value of 0.000 (P <0.05) which indicates that the administration of honey-garlic fermentation has a significant effect on cholesterol levels in tthe treatment group.

Result of SOD Levels:

Based on the results of the study, it was found that the levels of SOD in the sample group were presented in table 2 and the figure 4 below.

Table 2: SOD Levels after treatment Fermented Honey-Garlic

Groups	Average of SOD Levels ± Std. Dev	P Value
K (-)	1,658 ± 0,282
K (+)	0,345 ± 0,196
P (1)	1,235 ± 0,216
		0.000 (P<0.05)
P (2)	1,239 ± 0,106
P (3)	0,424 ± 0,173
Std	0,483 ± 0,092

If the results of SOD levels are depicted in the form of a graph, it can be seen in the figure 4 below.

Figure 5: SOD Levels Chart

Based on Figure 5.6, it is known that the highest mean serum SOD level is the negative control, which is 1.658 ±0.282 U/ml. Meanwhile, the lowest serum SOD level was the positive control, which was 0.345±

0.196U/ml. In the treatment group, the serum SOD level closest to the negative control was P2 which was 1.239±0.106U/ml, followed by P1 of 1.235±0.216 U/ml, P4 of 0.483±0.092 U/ml, and P3 of 0.424±0.173 U/ml. Based on the results of statistical analysis using one way ANOVA obtained a P value of 0.000(P <0.05) which indicates that the administration of honey-garlic fermentation has a significant effect on SOD levels in the treatment group.

Determination of MDA Levels:

Based on the results of the determination of MDA levels in the treatment group, the data obtained in the following table 3 and figure 5:

Table 3: SOD Levels after treatment Fermented Honey-Garlic

Groups	Average of MDA Levels ± Std. Dev	P Value
K (-)	0,744 ± 0,742
K (+)	1,008 ± 0,405
P1	0,759 ± 0,298
		0.646(P>0.05)
P2	0,745 ± 0,367
P3	0,466 ± 0,228
STD	0,847 ± 0,746

If the results of MDA levels are depicted in the form of a graph, it can be seen in the figure 5 below.

Figure 6. MDA levels chart

From the data above, it shows that the average MDA level in group K (+) is much higher than the group given fermented honey-garlic therapy. Based on the above data, it is known that the MDA levels in the K- group were 0.744 ± 0.742, the K+ group with MDA levels of 1.008 ± 0.405, the P1 group was 0.759 ± 0.298, the P2 group was 0.745 ± 0.367, the P3 group was 0.466 ± 0.228 and the standard group was 0.466 ± 0.228. 0.847 ± 0.746.

Determination of GSH Levels:

Based on the results of the examination of GSH levels, it can be seen in the following table.

Tabel 4: Glutathione (GSH) Levels

Groups	Average of GSH levels ± Sd	P Value
K-	44,47 ± 15,38
K+	36,43 ± 25,53
P1	41,36 ± 15,01
		0.985 (P>0.05)
P2	44,84 ± 15,73
P3	52,89 ± 22,84
Std	45,43 ± 12,63

If the results of GSH levels are depicted in the form of a graph, it can be seen in the figure 6 below.

Figure 7: GSH levels chart

From Figure 6, the average GSH level in the K- group is 44.47M/ml. The P1 treatment group was

41.63M/ml, the P2 group was 44.84M/ml, the P3 group was 52.89M/ml, and the Std group was 45.43M/ml. Meanwhile, in the K+ group or positive control, the value was 36.43M/ml.

DISCUSSION:

This study begins with the determination of total phenolic content (TPC) and flavonoid content (TFC) levels. Phenolic compounds are compounds that have one or more hydroxyl groups attached to an aromatic ring. Phenolic compounds are formed from the metabolic pathway of shikimic acid and phenyl propanoid. Phenolic compounds from plants have several effects, namely antioxidant, anti-inflammatory, antiproliferative, antimutagenic, antimicrobial, anticarcinogenic, and prevention of heart disease. Examples of phenolic compounds are phenolic acids, flavonoids, condensed tannins, coumarins and alkyl resorcinols. The largest group of phenolic compounds are flavonoids. The classification of flavonoids is based on the presence of a heterocyclic ring substituent containing oxygen and the difference in the distribution of the hydroxyl group on the C3 atom (Figure 3). The difference in the C3 atom is what determines the properties, properties and groups of flavonoids. Flavonoids can be distinguished based on the diversity in the C3 chain, namely: flavonols, flavones, isoflavones, flavanones, flavanonols, catechins, leukoanthocyanidins, anthocyanins, chalcones, and aurons¹⁴.

In determining the phenolic content in the standard sample, gallic acid was used colorimetrically with the colorimetric method using the Folin-Cioacalteu method. The basic principle for this method is the oxidation of phenolic-hydroxyl groups. Folin-Ciocalteu reagent oxidizes phenolics and reduces heteropoly acids to a molybdeum-tungsten (Mo-W) complex. During the reaction, the phenolic-hydroxyl group will react with the Folin-Ciocalteu reagent to form a blue phosphotungstate -phosphomolybdate complex¹⁵.

Figure 8. Reaction of phenolic compounds with Folin-Ciocalteu reagent

The determination of total flavonoid content was carried out using the Chang method. The principle of this method is the complexation reaction between flavonoid compounds with AlCl₃ reagent to form aluminum- flavonoid complex compounds, in the form of a yellow solution so that it can be measured with a visible spectrophotometer. This chelate complex is formed on the keto group and the hydroxyl group of the flavonoid¹⁶.

Based on the result of measurement antioxidant activity with DPPH, the value of IC50 in fermented honey-garlic sample obtained at 49.34 g/mL which means the IC50 value < 50ppm, it shows that the fermented honey sample has a strong antioxidant activity. As a key source of antioxidants including glucose oxidase, catalase, ascorbic acid, phenolic compounds, carotenoids, organic acids, amino acids, and proteins, honey holds a prominent position in terms of nutrition¹⁷. Besides that, alliin (S-allyl- cysteine sulfoxide), an organosulfur, has been shown to be a significant antioxidant component in single garlic.

The treatment of white rats to achieve cholesterol levels was carried out by providing standard feed accompanied by standard drug propylthioracil (PTU). PTU induction is able to increase cholesterol levels endogenously, namely by reducing thyroid hormone synthesis. PTU works as an antithyroid which inhibits thyroid cells in mice to produce thyroid hormone by suppressing metabolism at the LDL-receptor, so that LDL- cholesterol levels will increase¹⁸. Normal levels of cholesterol in rats are 40 – 130mg/dl. The results can be seen in table 1. From the data it can be seen that there is a significant differences in the average cholesterol levels in each treatment group.

Honey contains more than 150 polyphenolic compounds containing flavonoids, phenolic acids, catechins, and cinnamic acid derivatives which are compounds that function as antioxidants. The flavonoid compounds are the largest group in phenolic compounds¹⁹. The antioxidant activity in honey was mainly due to these two compounds because there was a strong correlation between antioxidant activity with phenolic and flavonoid compounds. The antioxidant capacity of phenolic compounds will suppress the bad effects of free radicals on the body. Under conditions of hyperlipidemia in obese rats, it can induce glucose auto-oxidation and protein glycation which leads to the formation of reactive oxygen species (ROS)²⁰. Apart from honey, antioxidant content is also obtained from the active compounds contained in garlic, where garlic is a supporting ingredient in the manufacture of fermented honey in this study. The efficacy of garlic (Allium sativum) has been widely studied as a therapeutic ingredient ranging from antibacterial, antiviral, antifungal, antithrombotic, antibiotic, anticancer, antioxidant, immunomodulatory, anti-inflammatory, and hypoglycemic effects²¹. Garlic (Allium sativum) has been used as an herbal remedy for thousands of years because of the active organosulfur compounds present in garlic. The active organosulfur compounds in garlic are flavonoids, adenosine, ahoene, and alliin. When raw garlic is destroyed, there will be a cell breakdown process from allicin to Allylthiosulfate which plays an important role in the process of reducing lipids, anticancer, and antioxidants²². These conditions will increase the burden on the antioxidant enzyme system, namely superoxide dismutase (SOD), which is characterized by a decrease in the activity of the enzyme. Superoxide dismutase (SOD) is a primary antioxidant whose job is to reduce superoxide radicals and convert them into hydrogen peroxide. The ability of phenolic compounds as antioxidants is supported by the ability to donate electrons or hydrogen atoms from the –OH group²³. After donating electrons, phenol compounds themselves will experience instability, but can be neutralized through electron resonance by the benzene ring. The electron donating mechanism is a primary antioxidant mechanism similar to the action of the SOD enzyme which also acts as an endogenous primary antioxidant²⁴.

The mechanism of decreasing cholesterol and MDA levels in the administration of honey is thought to be due to the antioxidant activity contained in honey. In the fermented honey there is also a single garlic which has antioxidant compounds that can help reduce MDA levels. MDA is an aldehyde compound derived from lipoperoxidation and produces additional covalent substances that can damage proteins, and their accumulation will also damage tissues²⁵. Oxidative damage to proteins can occur through indirect mechanisms involving the production of aldehydes derived from lipoperoxidation such as MDA and 4-hydroxynonenal (HNE). MDA fluctuations indicate oxidative damage to proteins that allow uptake of metals and ROS. Reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide, and hydroxyl, nitric oxide, and peroxynitrite radicals are crucial in oxidative stress associated with the etiology of numerous serious diseases²⁶. High levels of MDA in the body can be reduced through antioxidant activity that can fight free radicals. Allium sativum Lin. (single garlic) contains more than one hundred secondary and biologically beneficial metabolites, such as antioxidants²⁷. One of the antioxidants in single garlic, namely flavonoids and organo sulfur compounds are chemical components contained in single garlic and work as antioxidants. Organo sulfur compounds are also able to reduce lipid levels in the blood, and work as antithrombotic, blood anticoagulation, antihypertensive, anticancer, antioxidant, and antimicrobial²⁸. Antioxidants are compounds that might guard against the harm that unstable molecules called free radicals do to cells. Cancer may result from free radical damage. Free radicals interact and are stabilized by antioxidants, which may limit some of the harm they may otherwise do²⁹. By giving one of the free radicals' electrons, antioxidants neutralize them, ending the electron-electron reaction³⁰.

Flavonoids and polyphenols found in plants also increase GSH production through their influence on the expression of substrates required for glutathione synthesis such as CT cystine antiporter, gamma- glutamylcysteine synthetase and glutathione synthase. Previous study also said that in the group of rats given a single garlic extract with ethanol, the results showed that GSH levels increased compared to controls³¹.

CONCLUSION:

Administration of FHG could significantly decrease cholesterol level and increase SOD levels but there was no significant change in MDA and GSH levels. The reduction of oxidative stress in rats treated with FGH can be through modulation of antioxidant activity. This antioxidant activity is also supported by the high content of phenolic and flavonoid compounds contained in FHG.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Universitas Nahdlatul Ulama Surabaya for their financial and facilities support.

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Received on 06.09.2022 Modified on 07.12.2022

Research J. Pharm. and Tech 2023; 16(9):4085-4092.

DOI: 10.52711/0974-360X.2023.00669