WO2023121610A1 - Development of original microemulsion formulations effective against fatty liver from standardized salvia triloba extracts - Google Patents

Abstract

The present invention relates to the development of original microemulsion formulations of extracts obtained from the leaves of the Salvia fruticosa plant, which is standardized in terms of salvigenin and rosmarinic acid contents, and the therapeutic activity of these formulations in fatty liver.

Description

DEVELOPMENT OF ORIGINAL MICROEMULSION FORMULATIONS

EFFECTIVE AGAINST FATTY LIVER FROM STANDARDIZED SALVIA TRILOBA EXTRACTS

Technical Field

This invention relates to original microemulsion formulations loaded with extracts of Salvia triloba (Salvia fruticosa) effective against fatty liver.

The State of the Art of the Invention (Prior Art)

In the state of the art, it is defined as "fatty liver disease" when more than 5% of liver histocytes are filled with fat vacuoles due to nutrition, and independent of alcohol consumption. As is known, fatty liver and related disorders are common, but still don’t have effective treatments. The said disease is one of the important health problems in Turkey and brings with it social and economic problems. In recent years, it has been observed that the prevalence of the non-alcoholic fatty liver disease has increased significantly worldwide. With the advancing obesity epidemic, non-alcoholic fatty liver disease has become the most common cause of chronic liver disease in adults and children. Therefore, the clinical and economic burden of the disease is remarkable. However, there is a significant number of herbs that are widely used among the public, and natural sources are becoming very popular in the treatment of diseases. They have been included in the products obtained from plants and in the preparations used in the pharmaceutical field from the past to the present.

There are many ethnob otani cal and experimental studies with Salvia fruticosa. In previous studies, it has been determined that this plant exhibits various activities such as antimicrobial, antiviral, antioxidant, anticholinesterase, anti-inflammatory, and cytotoxic activities.

The study by RosaliaTacconi etc. relates to the demonstration of hypoglycemic activity of the plant Salvia fruticosa (Salvia triloba). This study strongly suggests that S. fruticosa treatment produces hypoglycemia mainly by reducing intestinal absorption of glucose. Microemulsions are thermodynamically stable, low viscosity, transparent and self-forming systems with droplet sizes between 5-140 nm. Microemulsions, like conventional emulsions, can be of S/Y or Y/S type. However, they differ from conventional emulsions. The surfactant and surfactant mixture consists of co-surfactant, oil, and water phases.

Brief Description and Objects of the Invention

The primary object of the invention is to obtain new natural and herbal products that can be effective for the treatment of fatty liver.

The object of the invention is to prepare the active ingredient Salvia fruticosa extracts and the carrier microemulsion formulation containing this extract.

Another object of the invention is to develop phytotherapeutic agents of natural origin against fatty liver, which is a frequently encountered problem in human life, and to gain economic and domestic value in the fight against this condition. Thanks to the resulting phytotherapeutic preparation, it is aimed to obtain a result from a plant grown in Turkey, and reduce foreign dependency.

With the invention, standardized Salvia fruticosa extracts, which were turned into microemulsion formulations, were investigated in terms of their effect against fatty liver in rats in a fatty liver model formed by feeding with in vivo fatty diet and with in vitro cell culture methods. At this point, the extract placed in both a natural agent and a carrier system gave good results both macroscopically and histologically against fatty liver.

With the invention, an herbal and original microemulsion formulation originating from natural raw materials was designed from Salvia fruticosa extracts, which have proven antihyperglycemic and antihyperlipidemic effects on the liver and have been used safely among the public for centuries.

With the invention, Salvia fruticosa extract was standardized over salvigenin and rosmarinic acid. The content of rosmarinic acid and salvigenin, the major secondary metabolites of Salvia fruticosa extract, was determined by the HPLC method. The amount of rosmarinic acid in the extract was calculated as 1,36% and the amount of salvigenin as 3,68%.

Considering the increasing demand for natural raw materials, a product with completely natural ingredients has been aimed and Salvia fruticosa extract has been loaded into the original microemulsion formulation developed.

The effect of the extract against fatty liver in rats was demonstrated in vitro as loaded into a microemulsion formulation.

With the invention, it is thought that a current need can be met from both a natural and a local source. There are several products that have a similar effect. However, the fact that these products are synthetic or non-local makes this invention meaningful and different.

Description of the Figures Describing the Invention

Figure 1: Alkaline phosphatase levels (U/L) measured from serum samples of groups given S. fruticosa extract. *p<0.05 and **p<0.01 (based on SC group). Data are given as mean ± standard error. (NC: Normal diet control group, SC: Fatty diet control group, SaC: Blank carrier group for S. fruticosa, Sal : 100 mg/kg S. fruticosa extract group, Sa2: 200 mg/kg S. fruticosa extract group, Sa3 : 400 mg/kg S. fruticosa extract group, SV : Simvastatin group)

Figure 2: Aspartate aminotransferase levels (U/L) measured from serum samples of groups given S. fruticosa extract. *p<0.05 (based on SC group). Data are given as mean ± standard error. (NC: Normal diet control group, SC: Fatty diet control group, SaC: Blank carrier group for S. fruticosa, Sal : 100 mg/kg S. fruticosa extract group, Sa2: 200 mg/kg S. fruticosa extract group, Sa3: 400 mg/kg S. fruticosa extract group, SV: Simvastatin group)

Figure 3: Alanine aminotransferase levels (U/L) measured from serum samples of groups given S. fruticosa (B) extract. ** p<0.01 (based on SC group). Data are given as mean ± standard error. (NC: Normal diet control group, SC: Fatty diet control group, SaC: Blank carrier group for S. fruticosa, Sal : 100 mg/kg S. fruticosa extract group, Sa2: 200 mg/kg S. fruticosa extract group, Sa3 : 400 mg/kg S. fruticosa extract group, SV : Simvastatin group) Figure 4: HDL levels (mg/dl) measured from serum samples of groups given S. fruticosa extract. *p<0.05 and **p<0.01 (based on SC group). Data are given as mean ± standard error. (NC: Normal diet control group, SC: Fatty diet control group, SaC: Blank carrier group for S. fruticosa, Sal : 100 mg/kg S. fruticosa extract group, Sa2: 200 mg/kg S. fruticosa extract group, Sa3: 400 mg/kg S. fruticosa extract group, SV: Simvastatin group)

Figure 5: LDL levels (mg/dl) measured from serum samples of groups given S. fruticosa extract. *p<0.05 and ***p<0.001 (based on SC group). Data are given as mean ± standard error. (NC: Normal diet control group, SC: Fatty diet control group, SaC: Blank carrier group for S. fruticosa, Sal : 100 mg/kg S. fruticosa extract group, Sa2: 200 mg/kg S. fruticosa extract group, Sa3: 400 mg/kg S. fruticosa extract group, SV: Simvastatin group)

Detailed Description of the Invention

The microemulsion formulation effective against fatty liver comprises;

• The extract of Salvia fruticosa comprising rosmarinic acid and salvigenin as the active ingredient, and

• At least one water phase, at least one oil phase, at least one surfactant, and at least one cosurfactant.

In the invention, cinnamon oil was preferred as the oil phase, Tween 80 as the surfactant, and propylene glycol as the co-surfactant.

The extract of S. fruticosa in the microemulsion formulation of the invention contains 1.4% rosmarinic acid and 3.7% salvigenin according to HPLC analysis.

The water phase and oil phase are used for the solubility of substances, and the surfactant and co-surfactant were used for homogeneous dispersion of the oily and aqueous phases.

Within the scope of the invention, new delivery systems have been developed for the application of S. fruticosa extract to animals and for ease of dosing, and microemulsions have been selected for this purpose. Microemulsion formulations obtained with the invention gave positive macroscopic and histological results against fatty liver in cell culture and on rats.

Extraction and Standardization of the Herbal Material

S. fruticosa (1000 g) leaves ground in the range of 200-300 mesh were weighed and extracted with ethanol (1 L) for 1 hour. After filtration through Whatman No:l filter paper, concentrated by rotary evaporator. The extraction process was repeated 3 times in total. For HPTLC and HPLC analyzes, stock sample test solutions were prepared in methanol at a concentration of 50 mg/mL.

Preparation of Microemulsion Formulations

In the formulation, cinnamon bark oil containing safrole is used for the oil phase, Tween 80 as a surfactant, and propylene glycol as an auxiliary surfactant (co-surfactant). When the formulations were being developed, Surfactant/co- surfactant (s/cs) (w/w) ratios were 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 2: 1, 3: 1, 4: 1, 5: 1. Triangle Phase Diagram Analysis Software was used to generate the phase diagrams.

Using the data obtained during the formulation study, triangular phase diagrams were drawn for each s/cs ratio with the help of a computer program and optimum microemulsion formulations were developed by calculating the center of gravity of the region with the highest microemulsion area. The optimum formulation was prepared by using this center of gravity.

The optimum microemulsion formulation contains 20-30% water phase, 50-60% surfactant + co-surfactant, and 10-20% oil phase by weight. Surfactant-co-surfactant was mixed in a ratio of 4: 1. Standardized S. fruticosa extract at different concentrations was mixed and loaded into new microemulsion (M) systems with a magnetic stirrer and used in in vivo studies.

The results of the characterization studies of the formulations (viscosity, pH, zeta potential, droplet size, poly dispersity index) are shown in Table 1. The droplet size of the blank and extract-loaded microemulsion ranged from 0.401 to 1.365 nm with low PDI values. An almost neutral surface charge was observed for the formulations.

The pH values of the formulations vary between 6.26-6.99, approaching the pH values of normal skin compatible with the oral mucosa tissue. The viscosity of the blank microemulsion was determined as 613 cP. When the S. fruticosa ethanol extract was loaded into the microemulsion, the viscosity value was determined as 666 cP. The conductivity of the microemulsions was found to be between 9 and 15 pS/cm. The zeta potential values of the microemulsions were determined as -0.00884-(-0.000224) mV. The refraction index was measured and found to be between 1.44498 and 1.4444 ± 0.002.

Table 1. Characterization findings and standard deviation values of the formulations (± SD)

Stability Studies

For the control of the stability of the prepared microemulsion formulations, the microemulsion formulations were evaluated for 6 months in a stability cabinet at 4 ± 3°C, 25 ± 2°C 60 ± 5% relative humidity, and 40 ± 2°C 65 ± 5% relative humidity. Formulations were evaluated in terms of appearance, viscosity, pH, zeta potential, droplet size, and poly dispersity index at 3 and 6 months. The ideal microemulsion formulations were centrifuged at 13000 rpm for 30 minutes at 25 ± 2 °C with a centrifuge device (Eppendorf Centrifuge 5415R). It was investigated whether there was any phase separation in the microemulsion formulations at the end of centrifugation. Stability studies on clarity and separation suggested that the formulations were optimal, showing stability under any temperature and time frame. Stability tests were carried out at 4 ± 1°C, 25 ± 1°C, and 40°C for 6 months. After 6 months of stability testing, it was determined that there was no significant change in clarity, pH, droplet size, and viscosity. The microemulsions had isotropic transparent dispersions and no phase separation was observed after centrifugation. This demonstrated the good physical stability of the tested microemulsions.

Cytotoxicity

Cytotoxic concentrations of S. fruticosa extract on HepG2 cells were determined in vitro by the MTT method. The non-toxic concentration for S. fruticosa leaves was determined as 0.25 mg/mL. With the results obtained, the rest of the experiments were run in cell culture at a concentration of 0.25 mg/mL. The results obtained in the present invention determined that the ethanol extract of S. fruticosa was cytotoxic to liver cancer HepG2 cells at concentrations above 0.25 mg/mL.

In vitro Liver Fattening Model

The effects of the extracts determined within the scope of the invention were evaluated in vitro, and the extracts that were determined to be bioactive in the experiments performed according to the results obtained as a result of these studies were investigated in in vivo studies. Within the scope of in vitro studies, cytotoxic values were determined in the HepG2 cell line, which will be studied primarily. Lipid peroxidation and glutathione experiments were carried out in the HepG2 cell line, in which a fattening model was created. Then, the fat cells in the medium were detected by fluorescence microscopy before and after the extract treatment with Nil Red dye. ALT AST enzyme levels in the cell medium were also measured with commercial kits.

Lipid peroxidation (MDA) assay The determination of MDA, which is a lipid peroxidation indicator, is based on the principle that thiobarbituric acid (TBA) reacts with MDA to give a chromophoric compound that can be measured at a wavelength of 532 nm. Tetramethoxypropane (TMP) was used as a standard and the results were given in nmol/g (Daraie et al., 2012).

In human liver hepatocellular carcinoma cells (HepG2), an increase in MDA levels was observed when treated with Oleic acid, Palmitic acid, and their combinations compared to a blank medium. According to these results, it is seen that the extracts reduce the MDA level induced by oleic acid and palmitic acid.

Results of lipid peroxidation levels were compared with resveratrol used as a positive control. While resveratrol decreased the MDA level (nmol/g protein) in the cell medium by 31,8%, S. fruticosa ethanol extract caused a 27,3% decrease, which is close to the resveratrol positive control.

Glutathione (GSH) assay

Glutathione is a thiol that plays a critical role in cellular defense against oxidative stress in tissues and cells. DTNB (Ellman's reagent) reacts with thiol groups, and disulfide bonds in its structure are broken. The resulting TNB- transforms into TNB-2 in a neutral or alkaline medium and gives a yellow chromophoric group (Sedlak and Lindsey, 1968). Glutathione, which is a low molecular weight thiol was also measured based on this principle.

The data obtained as a result of the evaluation of GSH levels were compared with resveratrol used as a positive control. In human liver hepatocellular carcinoma cells (HepG2), a decrease in GSH levels was observed when treated with Oleic acid, Palmitic acid, and their combinations compared to a blank medium. The extract increased the GSH levels in the medium.

GSH levels were compared with resveratrol as a positive control. According to the results, oleic acid, palmitic acid, and their combinations decreased GSH levels and led to a significant increase in oxidative stress. 10 pM resveratrol increased the GSH level (pmol/g protein) in cell culture by 27,9%, while the S. fruticosa ethanol extract decreased the GSH level by 18,5% compared to the OAIOO pM+ PA 200 pM group.

Detection of fat cells by Nil Red staining

Nil Red is a fluorescent and lipophilic dye that is used to stain fat cells (Izdebska et al., 2010). Steatosis status in the cell culture medium was determined by fluorescence microscopy. Detection of oil droplets in the environment was also detected with Nil Red dye.

Based on the fluorescent microscopy images, less amount of fat droplets were detected in the cell media treated with S. fruticosa.

Determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities

The Enzyme-Linked Immune Assay (ELISA) test shows the reaction between an antigen (a specific protein of the microorganism) and an antibody (a protein-like molecule produced against the antigen) and measures the enzyme activity calorimetrically. ALT and AST are hepatic enzymes found in the cytoplasm of cells. The increase in ALT and AST levels in liver cell damage has been used as an indicator of liver damage (She et al., 2016).

In the cell culture study, AST and ALT levels were measured with ELISA kits according to the manufacturer's instructions. 20 pL of cell supernatant was used for measurements (Human AST ELISA Kit /ab263881 and Human ALT ELISA Kit/ab234578).

In human liver hepatocellular carcinoma cells (HepG2), an increase in AST and ALT enzyme levels was observed when treated with Oleic acid, Palmitic acid, and their combinations, compared to a blank medium. For the ALT assay, the results obtained from S. fruticosa extracts were found to be effective at 53,5%.

In vivo Fatty Liver

Non-alcoholic fatty liver model Weight monitoring of Wistar albino rats was done weekly. The chart which includes the weight information of the animals recorded at the beginning of feeding-beginning of gavage and on the day when the experiment is terminated, in the form of the weight average of the experimental groups separately, is given in Table 2.

Table 2. The average weight of animal groups (Weeks 0, 7, and 15) (± SS)

According to the results obtained from the average of the weights, after week 7 of gavage, a slowdown in body mass increase was observed in the group given S. fruticosa extract, and even a decrease in body mass indexes despite the continued fatty diet of the animals.

After the rats were sacrificed by decapitation under anesthesia, the weights of the livers removed from each rat were recorded by weighing. Starting from this, the liver index was calculated.

From the liver index results, it is understood that S. fruticosa extract caused a decrease in the body mass of rats, but did not affect the decrease in liver mass.

Evaluation of biochemical parameters from serum and liver samples To evaluate serum lipids, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured. ALT, AST, and alkaline phosphatase (ALP) levels were evaluated to evaluate liver function; and SOD, GSH-Px, and malondialdehyde (MDA) levels to evaluate antioxidant levels. Serum glucose and insulin levels were measured to evaluate insulin resistance. SOD, GSH-Px, malondialdehyde (MDA), and triglyceride levels were measured in liver samples. In the rats given S. fruticosa in the experimental groups, 3 different doses were coded as Sal, Sa2, and Sa3 (100, 200, and 400 mg/kg, respectively) from low to high dose. While the group fed with a normal diet was coded as NC in the graphics, the control group fed with a fatty diet was coded as SC. The positive control simvastatin group was coded as SV. Carrier groups are indicated as SaC for the carrier of the group given S. fruticosa.

Glutathione peroxidase (GSH-Px) measurements of liver and serum samples

GSH-Px levels measured from liver and serum samples for the group given S. fruticosa extract relative to the fatty diet group (SC) in the groups given S. fruticosa ethanol extract in both serum and liver samples, GSH-Px levels increased. A decrease of 1,1% was observed in serum glutathione level in the group given the carrier (SaC) compared to the fatty liver group. Compared to the carrier group, the serum GSH-Px levels increased by 41,5% (p<0.001) in the group given 400 mg/kg of S. fruticosa extract. For liver GSH-Px level, there was no difference between the carrier group and the fatty liver group, while an increase of 19,4% ( <0.01) in liver GSH-Px levels was detected in the group given 400 mg/kg S. fruticosa extract.

Super oxide dismutase (SOD) measurements of liver and serum samples

An increase in SOD levels was observed in both serum and liver samples in the groups given S. fruticosa ethanol extract compared to the fatty-fed group (SC). Compared to the Fatty Nutrition Diet (YBD) group, there was a 20,6% increase in serum SOD levels in the carrier group (SaC). Compared to the carrier group, the 400 mg/kg dose increased by 41,4% (p <0.05) for serum SOD levels, while the liver SOD level increased by 23,6% (p<0.05) in the group given 200 mg/kg extract. Malondialdehyde (MDA) measurements of liver and serum samples

A decrease in malondialdehyde levels was observed in both serum and liver samples in all groups given S. fruticosa ethanol extract compared to the fatty diet group (SC). Compared to the YBD group, there was a 9,2% decrease in serum MDA levels and an 11,9% decrease in liver MDA levels in the carrier group (SaC). Compared to the carrier group, the 200 mg/kg dose showed a 48,6% (p<0.01) decrease for serum MDA levels, while a 43,7% (p<0.01) decrease was found in the liver MDA level at the 400 mg/kg dose.

Triglyceride (TG) measurements of liver and serum samples

A decrease in triglyceride levels in both serum and liver samples was observed in all groups given S. fruticosa ethanol extract compared to the fatty-fed group (SC). Compared to the YBD group, there was a 2,6% increase in serum triglyceride levels and a 7,2% decrease in liver triglyceride levels in the carrier group (SaC). Compared to the carrier group, a decrease of 26,3% (p<0.05) and 34,4% (p<0.01) was detected in the group given 200 mg/kg extract for serum and liver triglyceride levels, respectively. In the simvastatin group, a decrease of 45,2% and 45,4% was observed for serum and liver triglyceride levels, respectively.

Alkaline phosphatase (ALP) measurements of serum samples

In line with the results given in Figure 1, a decrease in ALP levels was observed in serum samples in all groups given the extract compared to the fatty-fed group (SC). Compared to the YBD group, there was a 2,5% decrease in ALP levels in the M. sylvestris carrier group (MC) and a 10,8% decrease in ALP levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 30,9% (p<0.01) decrease in serum ALP level was detected in the group given 400 mg/kg S. fruticosa extract for ALP levels. In the simvastatin group, a 10,1% decrease was observed in serum ALP levels.

Aspartate aminotransferase (AST) measurements of serum samples

In line with the results given in Figure 2, a decrease in AST levels was observed in serum samples in all groups given the extract compared to the fatty-fed group (SC). Compared to the YBD group, a 6,7% decrease was observed in AST levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 5,6% decrease in serum AST level was detected in the group given 400 mg/kg S. fruticosa extract for AST levels.

Glucose measurements of serum samples

A decrease in glucose levels was observed in serum samples in all groups given the extract compared to the fatty-fed group (SC). Compared to the YBD group, a 2,6% increase in glucose levels was observed in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 21,8% (p<0.01) decrease in serum glucose level was detected in the group given 400 mg/kg S. fruticosa extract for glucose levels.

Alanine aminotransferase (ALT) measurements of serum samples

In line with the results given in Figure 3, a decrease in ALT levels was observed in serum samples in all groups given the extract compared to the fatty-fed group (SC). Compared to the YBD group, a 6,4% increase was observed in ALT levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 19,4% decrease in serum ALT level was detected in the group given 400 mg/kg S. fruticosa extract for ALT levels.

High-density lipoprotein (HDL) measurements of serum samples

In line with the results given in Figure 4, an increase in HDL levels was observed in serum samples in all groups given the extract compared to the YBD group. Compared to the YBD group, there was a 14,1% decrease in HDL levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 40,7% (p<0.01) increase in serum HDL level was detected in the group given 400 mg/kg S. fruticosa extract for HDL levels. An increase of 30,4% (p<0.05) in serum HDL levels was observed in the positive control simvastatin group compared to the YBD group.

Low-density lipoprotein (LDL) measurements of serum samples In line with the results given in Figure 5, a decrease in LDL levels was observed in serum samples in all groups given the extract compared to the YBD group. Compared to the YBD group, there was a 10,8% decrease in LDL levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 22,9% (p<0.05) increase in serum LDL level was detected in the group given 400 mg/kg S. fruticosa extract for LDL levels. An increase of 47,4% (p<0.001) in serum LDL levels was observed in the positive control simvastatin group compared to the YBD group.

Total cholesterol measurements of serum samples

A decrease in total cholesterol levels was observed in serum samples in all groups given the extract compared to the YBD group. Compared to the YBD group, there was a 4,9% decrease in total cholesterol levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a decrease of 27,1% ( <0.05) was found in the serum total cholesterol level in the group given 400 mg/kg S. fruticosa extract for total cholesterol levels. A 46,3% (/?<0.01 ) decrease in serum total cholesterol levels was observed in the positive control simvastatin group compared to the YBD group.

Insulin measurements of serum samples

A decrease in insulin levels was observed in serum samples in all groups given the extract compared to the YBD group. Compared to the YBD group, there was an 8,7% decrease in insulin levels in the S. fruticosa carrier group (SaC). Compared to the carrier group, a 38,2% ( <0.01) decrease in serum insulin level was detected in the group given 200 mg/kg S. fruticosa extract for insulin levels.

Histopathology

Hematoxylin-Eosin (HE) dye was applied in 5 pm sections in paraffin blocks prepared from liver lobes fixed in 10% formol solution and the results of the images made with Nikon Eclipse Ni research microscope were evaluated by scoring.

Ballooning in hepatocytes Findings such as fatty liver in nonalcoholic steatohepatitis and ballooning in hepatocytes are observed as in alcoholic liver disease.

A decrease in balloon cell density was observed in liver tissues in all groups given the extract compared to the YBD group. Compared to the YBD group, there was a 32,4% decrease in balloon cell density in the liver tissues in the S. fruticosa carrier group (SaC). Compared to the carrier group, there was a 53,2% ( <0.05) decrease in the balloon cell density level observed in the liver tissue in the group given 400 mg/kg S. fruticosa extract for balloon cell density.

Lobular inflammation

In nonalcoholic fatty liver disease, the known level of simple fattening may also be accompanied by mild lobular inflammation without ballooning. A decrease in the intensity of lobular inflammation in liver tissues was observed in all groups given the extract compared to the YBD group. Compared to the YBD group, a 16,6% decrease was observed in the lobular inflammation levels in the liver tissues in the S. fruticosa carrier group (SaC). Compared to the carrier group, a decrease of approximately 60% (p<0.05) was detected in the lobular inflammation level in the liver tissue in the groups given 200 and 400 mg/kg S. fruticosa extract.

Macro steatosis and micro steatosis

In nonalcoholic fatty liver disease, steatosis can be detected in macro and micro-vesicles. A decrease in macro-steatosis density was observed in liver tissues in all extract groups compared to the YBD group. Compared to the YBD group, In the S. fruticosa carrier group (SaC), a decrease of 24,8% was observed in the macro-steatosis levels in the liver tissues. Compared to the carrier group, a decrease of 58,7% ( <0.05) was found in the macro steatosis level observed in the liver tissue in the group given 400 mg/kg S. fruticosa extract for macro steatosis levels. A decrease in micro steatosis density was observed in liver tissues in all extract groups compared to the YBD group. No change in micro steatosis was observed in the S. fruticosa carrier group (SaC) compared to the YBD group. Compared to the carrier group, a 64,3% decrease was found in the micro steatosis level observed in the liver tissue in the groups given 200 and 400 mg/kg S. fruticosa extract for micro steatosis levels.

Claims

1. A microemulsion formulation effective against fatty liver, characterized in that it comprises;

• Extract of Salvia fruticosa leaves containing rosmarinic acid and salvigenin metabolites as active ingredient, and

• At least one water phase, at least one oil phase, at least one surfactant, and at least one co-surfactant.

2. A microemulsion formulation according to Claim 1, characterized in that the extract of Salvia fruticosa leaves comprises 1.4% rosmarinic acid and 3.7% salvigenin.

3. A microemulsion formulation according to Claim 1, characterized in that it comprises 20-30% of the water phase by weight, 10-20% of the oil phase by weight, and 50-60% of surfactant by weight, and co-surfactant mixture.

4. A microemulsion formulation according to Claim 1, characterized in that the concentration of Salvia fruticosa leave extract is at most 0.25 mg/mL.

5. A microemulsion formulation according to Claim 1, characterized in that the surfactant is Tween 80.

6. A microemulsion formulation according to Claim 1, characterized in that the cosurfactant is propylene glycol.

7. A microemulsion formulation according to Claim 1, characterized in that the oil phase comprises cinnamon oil.

8. A microemulsion formulation according to Claim 1, characterized in that it is used in the treatment of fatty liver.