EXTRACTS

Technical Field

The present invention is related to the development of unique microemulsion formulations from standardized extracts in terms of malvin contents obtained from the flowers of the Malva sylvestris L. plant and the activity of these formulations against fatty liver.

The invention is for the discovery and development of a phytotherapeutic agent against fatty liver and is aimed at creating economic and domestic value in the relevant field of the pharmaceutical industry.

State of the Art (Background)

The fact that more than 5% of dietary liver histiocytes independent of alcohol consumption are filled with fat vacuoles is defined as “fatty liver disease”. Fatty liver and related diseases are common but still have no effective treatment. These disorders are also important health problems in Tiirkiye and bring about social and economic problems.

Non-alcoholic fatty liver disease is among the most common chronic metabolic disorders in developed countries. In recent years, it has been observed that the prevalence of non-alcoholic fatty liver disease has increased significantly worldwide. With the progressive outbreaks of obesity, nonalcoholic 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 notable. The presence of a significant number of plants widely used by the public has attracted many clinical and pharmaceutical studies to this field.

The article titled “A Review on Health Benefits of Malva sylvestris L. Nutritional Compounds for Metabolites, Antioxidants, and Anti-Inflammatory, Anticancer, and Antimicrobial Applications" emphasizes that extensive research has been conducted on the chemical compounds and pharmacological effects of M. sylvestris, a medicinal plant and functional food, and emphasizes the antimicrobial, hepatoprotective, anti-inflammatory and antioxidant properties of the plant in question. According to the aforementioned article, considering the hepatoprotective activity of M. sylveslris, the presence of antioxidant compounds confirmed by the literature in this plant is an important, rich, and natural resource that can be used in the prevention and treatment of liver problems, as it removes free radicals in the liver and especially helps to protect liver tissues (Mousavi et. al., 2021).

Patent document WO2021144446A1 is particularly related to the invention of novel compositions with natural ingredients for use in the treatment or prevention of hepatoprotection and hepatosteatosis. Said new compositions comprise the following:

(a) From a combination of Citrus bergami extract and Cynara cardunculus extract,

(b) Cynara cardunculus extract

(c) A combination of naringin and chlorogenic acid.

The combination of the invention is for use in the treatment or prevention of hepatosteatosis. The combination may therefore be administered as a prophylactic treatment to prevent the condition from developing or to treat the condition after it has already developed. The combination of Citrus bergamia extract containing naringin and Cynara cardunculus extract containing chlorogenic acid has been found to be beneficial for the treatment of hepatosteatosis, i.e., reducing or preventing fat accumulation in the liver.

Patent document US2012171312A1 relates to the field of utilization of the therapeutic properties of compositions extracted from naturally occurring substances, and, more particularly, to the invention of a substance and method for reducing hepatic triglycerides and increasing antioxidant levels. The present invention includes a method for reducing the concentration level of liver lipids present in mammals with non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver diseases (NAFLD) by administering an extract from the Inula viscosa plant to mammals in need thereof. The method comprises administering a therapeutically effective amount of an extract derived from the plant Inula viscosa to the mammal in need thereof to obtain one or more of the following changes in hepatic lipid levels of said mammal: (i) reduction of cholesterol and protein concentrations C, (ii) reduction of MDA level and (iii) reduction of triglyceride concentration. In the state-of-the-art, standardized Malva sylvestris flower extracts, which are made into a microemulsion formulation, are investigated by in vivo and in vitro methods in terms of their effect against fatty liver, and at this point, there is no study showing that the extract placed in both a natural agent and a carrier system gives good results against fatty liver, both macroscopically, biochemically, and histologically.

Brief Description and Objects of the Invention

With the present invention, a unique microemulsion formulation was designed against fatty liver disease based on natural raw material derived from Malva sylvestris extracts, and it was observed that these obtained microemulsion formulations had positive results macroscopically and histologically in cell culture and rats against fatty liver.

Natural resources have become very popular in the treatment of diseases. They have been included in the content of products obtained from plants and preparations used in the pharmaceutical field from the past to the present. In the invention, a unique microemulsion formulation was designed against fatty liver based on natural plant-based raw materials from Malva sylvestris extracts, which have been proven to have different effects on the liver in various studies and have been used safely among the public for centuries. With the invention

• Malva sylvestris flower extract is standardized over malvin,

• Considering the increasing demand for natural raw materials today, a product with a natural content was targeted in terms of the substance responsible for the effect,

• This extract was loaded into the developed original microemulsion formulation,

• The effect of the extract on fatty liver in vitro and the formulation has been demonstrated in rats.

Thus, it was aimed to develop natural phytotherapeutic agents against fatty liver, which is a common problem in human life, and to gain economic and domestic value in combating this situation.

Thanks to the phytotherapeutic preparation put forward, it is considered to obtain a result obtained from a plant grown in Tiirkiye that reduces foreign dependency. In addition, considering the obesity cases in the world, it is predicted that its use will be beneficial. This idea put forward in the study is thought to meet an up-to-date need from both a natural and domestic source. In this sense, this product, which is based on reliable traditional use in this sense, can be updated in the future by using different pharmaceutical forms and nanotechnological methods. Unlike various products with a similar effect, the fact that the invention is not of synthetic but of natural origin and is domestic makes the invention meaningful and different.

Definitions of Figures Describing the Invention

The figures and related descriptions required to better understand the invention are as follows.

Figure 1. Alkaline phosphatase levels (U/L) were measured from serum samples of the experimental groups. (NC: Normal diet control group, SC: Oily diet control group, MC: Empty carrier group, Ml : 75 mg/kg M. sylvestris extract group, M2: 150 mg/kg M. sylvestris extract group, M3: 300 mg/kg M. sylvestris extract group, M4: 450 mg/kg M. sylvestris extract group, SV : Simvastatin group)

Figure 2. Aspartate aminotransferase levels (U/L) were measured from serum samples of experimental groups. *p<0.05 (vs. SC group). Data are presented as mean ± standard error. (NC: Normal diet control group, SC: Oily diet control group, MC: Empty carrier group, Ml : 75 mg/kg M. sylvestris extract group, M2: 150 mg/kg M. sylvestris extract group, M3: 300 mg/kg M. sylvestris extract group, M4: 450 mg/kg M. sylvestris extract group, SV: Simvastatin group)

Figure 3. 1. Alanine aminotransferase levels (U/L) measured from serum samples of the experimental groups. **p<0.01 (relative to the SC group). Data are presented as mean ± standard error. (NC: Normal diet control group, SC: Oily diet control group, MC: Empty carrier group, Ml : 75 mg/kg M. sylvestris extract group, M2: 150 mg/kg M. sylvestris extract group, M3: 300 mg/kg M. sylvestris extract group, M4: 450 mg/kg M. sylvestris extract group, SV : Simvastatin group)

Figure 4. HDL levels were measured from serum samples of experimental groups (mg/dL). *p<0.05 (vs. SC group). Data are presented as mean ± standard error. (NC: Normal diet control group, SC: Oily diet control group, MC: Empty carrier group, Ml : 75 mg/kg M. sylvestris extract group, M2: 150 mg/kg M. sylvestris extract group, M3: 300 mg/kg M. sylvestris extract group, M4: 450 mg/kg M. sylvestris extract group, SV: Simvastatin group)

Figure 5. LDL levels were measured from serum samples of the experimental groups (mg/dl). *p<0.05 and ***p<0.001 (relative to the SC group). Data are presented as mean ± standard error. (NC: Normal diet control group, SC: Oily diet control group, MC: Empty carrier group, Ml : 75 mg/kg M. sylvestris extract group, M2: 150 mg/kg M. sylvestris extract group, M3: 300 mg/kg M. sylvestris extract group, M4: 450 mg/kg M. sylvestris extract group, SV: Simvastatin group)

Detailed Description of the Invention

In this detailed description, the process steps of developing the unique microemulsion formulations of the standardized extracts in terms of malvin contents obtained from the flowers of the Malva sylvestris plant and the fatty liver treatment activities of these formulations are described.

1. Formulation Development

In the preparation of microemulsion formulations, the surface-active agent/auxiliary surfaceactive agent (SAA/ASAA) ratios that give the most stable microemulsion region were determined by first using triangular phase diagrams. The titration method was used in the preparation of microemulsion formulations.

Soybean oil was used as the oil phase, the surface-active agent Span 80 and Cremophor EL, and propylene glycol and ethanol were used as the auxiliary surface-active agent. While formulations were being developed, SAA/ASAA (w/w) ratios were mixed in ratios of 1 : 1, 1 :2, 1 :3, 1 :4, and 1 :5. After the SAA/ASAA ratios were determined, the oil, SAA (Span 80: Cremophor EL) and ASAA (Propylene glycol: Ethanol) were mixed with a rotation speed of 150 rpm, while the formulations were titrated with distilled water to the point where transparency was impaired and the water consumption volumes where transparency was lost were determined. The amount of distilled water consumed at the point where the transparency of the system was impaired was calculated, and the oil, water, and SAA/ASAA ratios at this point were calculated. All formulation studies were performed at room temperature.

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

According to this optimum formulation, the microemulsion comprises 7.5%-45% by weight standardized M. sylvestris flower extract, 5%-25% by weight oil-phase soybean oil, 5%-25% by weight Sorbitan monooleate and l%-5% by weight Polyoxyl-35 castor oil mixture surface active agent, 0.001%-15% by weight propylene glycol and 0.001%-15% ethanol mixture auxiliary surface-active agent and 5-10% by weight water phase.

2. Characterization

The optimal formulations were studied at 0 min in terms of appearance, electrical conductivity, pH, refractive index, viscosity, droplet size, polydispersity index and zeta potential parameters as previously published.

The physical appearance of the prepared microemulsion formulations was examined and evaluated at room temperature (25°C ± 2°C). An electrical conductivity device is used to determine the type of microemulsion formulations prepared. The measurement was repeated three times. Optimum microemulsion formulations were centrifuged at 13,000 rpm at 25°C ± 2°C for 30 minutes. Measurements were repeated 3 times. The pH of the microemulsion formulations was measured using a pH meter at room temperature. Refractive index measurement of ideal microemulsion formulations was performed with a refractometer. The viscosity of the ideal microemulsion formulations was measured using a viscometer at room temperature of 25°C ± 2°C. These values were also used in the measurement of droplet size. The droplet sizes, poly dispersity index and zeta potentials of the ideal microemulsion formulations were measured.

3. Stability Optimum microemulsion formulations were prepared and evaluated for 6 months in a stability cabinet at 4°C ± 3°C, 25°C ± 2°C 60% ± 5% relative humidity and 40°C ± 2°C 65% ± 5% relative humidity. The formulations were evaluated at 3 and 6 months for appearance, viscosity, pH, zeta potential, droplet size, and poly dispersity index.

4. Non-alcoholic fatty liver model

Weight tracking of 64 Wistar Albino rats divided into 8 different groups was performed weekly. Table 1 shows the weight information recorded on the day when the feeding of the animals is started, and the experiment is terminated as the average weight of the experimental groups separately.

Table 1. Weight averages of animal groups (Weeks 0-7-15th (±SD)

According to the results of Table 1, it can be concluded from the liver index results that the formulation loaded with M. sylvestris extract does not prevent the increase in body mass but slows the increase in liver mass.

5. Evaluation of biochemical parameters from serum and liver samples Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured to evaluate serum lipids. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels were analyzed to assess liver function. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels were measured to evaluate antioxidant levels. Serum glucose and insulin levels were evaluated to assess insulin resistance. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels were measured in liver samples. For the rat groups given phytotherapeutic formulations loaded with M. sylvestris extract, 4 different doses were coded as Ml, M2, M3, and M4 (75, 150, 300, and 450 mg/kg, respectively) in the plots from low dose to high dose. While the group fed with the normal diet was coded as NC in the graphics, the control group fed with the fatty diet was coded as SC. The positive control simvastatin group was coded as SV. Specified as MC for carrier group. a) Alkaline phosphatase (ALP) measurements of serum samples

Alkaline phosphatase levels measured from serum samples are given in detail in the graphs in Figure 1.

In line with the results given in Figure 1, a decrease in ALP levels was observed in serum samples in all groups given extract compared to the fatty diet group (SC). There was a 2.5% decrease in ALP levels in the M. sylvestris carrier group (MC) compared to the fatty diet group. Compared to the carrier group, a decrease of approximately 21% was found in the groups given 300 and 450 mg/kg of M. sylvestris extract for ALP levels. In the simvastatin group, a 10.1% decrease in serum ALP levels was observed. b) Aspartate aminotransferase (AST) measurements of serum samples

Aspartate aminotransferase levels measured from serum samples are given in detail in the graphs in Figure 2.

In line with the results given in Figure 2, a decrease in AST levels was observed in serum samples in all groups given extract compared to the fatty diet group (SC). There was a 1.9% decrease in AST levels in the M. sylvestris carrier group (MC) compared to the fatty diet group. Compared to the carrier group, a decrease of approximately 31% (p<0.05) was found in the groups given 300 and 450 mg/kg of M. sylvestris extract for AST levels. c) Alanine aminotransferase (ALT) measurements of serum samples

The findings of alanine aminotransferase levels measured from serum samples are given in detail in the graphs in Figure 3.

In line with the results given in Figure 3, a decrease in ALT levels was observed in serum samples in all groups given extract compared to the fatty diet group (SC). There was a 7.1% decrease in ALT levels in the carrier group (MC) compared to the fatty diet group, while a 26.4% (p<0.01) decrease was found in the group given 450 mg/kg M. sylvestris extract compared to the carrier group. d) High-density lipoprotein (HDL) measurements of serum samples

The findings of HDL levels measured from serum samples are given in detail in the graphs in Figure 4.

In line with the results given in Figure 4, an increase was observed in HDL levels in serum samples in all groups given extract compared to the fatty diet group (SC). There was a 4.2% decrease in HDL levels in the group (MC) compared to the fatty diet group, while there was a 25.3% increase in the group given 300 mg/kg M. sylvestris extract compared to the carrier group. In the positive control simvastatin group, an increase of 30.4% (p<0.05) was observed in serum HDL levels compared to the HFD group. e) Low-density lipoprotein (LDL) measurements of serum samples

The findings of LDL levels measured from serum samples are given in detail in the graphs in Figure 5.

In line with the results given in Figure 5, a decrease was observed in LDL levels in serum samples in all groups given extract compared to the fatty food group (SC). No difference was observed in LDL levels in the carrier group (MC) compared to the fatty diet group, while a decrease of 24.1% (p<0.05) was found in the group given 450 mg/kg M. sylvestris extract compared to the carrier group. In the positive control simvastatin group, a 47.4% (p<0.001) decrease was observed in serum LDL levels compared to the HFD group.