OF TYPE II DIABETES

Field of Invention

The invention relates to a food additive with the attribute to reduce the risk of type II diabetes that can be used as a promoter and supplement, and the preparation method thereof.

Prior Art

Turkey has a wide variety of plant species due to its geographical location and climatic characteristics. The role of medicinal plants in the treatment of diseases and protection of health as therapeutic alternatives is rapidly progressing worldwide and in our country. It is known that plants are very important for human health and that they have been cherished in the history of humanity from the earliest times to the present day at the point of healing diseases.

Due to its ecological conditions, our country is the homeland of many plant species. In Turkey, which has a great richness with over 11 thousand plant diversity, medicinal and aromatic plants are an important part of this richness. From the export of medicinal and aromatic plants, of which tens of varieties are subject to trade, Turkey earns more than 300 million dollars. In our country, the number of species and varieties collected from nature and the number of plants traded in the domestic market is about 350.

• About 100 of these plants are exported.

• Although it changes over the years, 20 kinds of medicinal and aromatic plants are cultivated on an area of 1.3 million decares in Turkey.

• In terms of production amount, black tea, red pepper, opium poppy, cumin, mint, thyme, oil rose, and anise take place on the top.

• 53 of these plants are consumed as natural herbal tea.

Medicinal and aromatic plants are used as medicine in traditional and modern medicine for the prevention, improvement, and maintenance of health. They are also used as nutritional supplements, herbal tea, flavour, and condiment in nutrition. In addition to being used in perfumery, body care products in perfumery and cosmetics, it finds a wide area of use in different branches of industry as brighteners and even insecticides. The dried parts of roots, leaves, bark, flowers, seeds, and fruits of these plants are used.

Medicinal and aromatic plants are used in almost every field, and they are considered as biological, cultural and industrial resources. According to the research, it is known that the demand for these resources has increased considerably in recent years and it is predicted that it will continue to increase in the future. The main reason for this is that these plants have a versatile effect compared to the synthetically obtained active substances in the field of medicine and health, and they do not have side effects.

As in every sector, the use of plants for therapeutic purposes varies according to the development level of the countries. In developing countries, 80% of the population benefits from herbal products for therapeutic purposes. In some countries of regions such as Asia, Africa and the Middle East, this rate rises to 95%. In developed countries, this rate is less, and it is 40-50% in Germany, 42% in the USA, 48% in Australia and 49% in France. (Titz, 2004, p: 72-80) However, the most important trade centres of medicinal plants are also located in Germany, USA, Japan, and England. It is predicted by the World Health Organization that treatments with herbs will increase worldwide in the coming years. In general, medicinal and aromatic plants are traded mostly in dried form, then as essential oil and fresh plants.

About 90-95% of people with type 2 diabetes worldwide are characterized by high and/or fluctuating blood sugar levels due to insulin resistance. Obesity and lack of proper lifestyle and especially nutritional characteristics can cause intensely increased blood glucose concentration and subsequently vascular disorders including high blood cholesterol and blood pressure.

The Gl (Glycaemic index) consists of carbohydrates determined from various foods. Some foods can raise blood sugar quickly, while others can raise it slightly. The invention relates to staple foods, some of which contain simple sugars such as glucose, and some contain complex foods such as modified and resistant starches, fibres, etc. These components can be elevated to lower glucose synthesis, which is related to the complex nature of the extracted polysaccharides, except for starch, which is simply broken down by the enzymes mentioned above. Carbs with a Gl estimate of less than 40 are classified in low-glycaemic index foods such as pumpkin bread, pasta, legumes, and parboiled rice. Carbs with a Gl between 40 and 70, such as porridge, belong to the middle zone, and those with a Gl estimate of over 70, such as popcorn, chocolate, and wafers, can be considered high glycaemic index carbohydrates. Despite acarbose, miglitol and voglibose (a-glucosidase and a- amylase inhibitors) used to reduce postprandial hyperglycaemia in diabetics, side effects and discomforts such as stomach discomfort, diarrhoea, bloating, and nausea have been observed. For this reason, many studies have been carried out to obtain natural substances with insulin-like effects in starchy foods, and it has been proven that diets rich in polyphenols reduce blood sugar by inhibiting the activity of some digestive enzymes. The inhibition effects of the mentioned polyphenols, especially associated with extraction and purification, decreased the glycaemic index in foods containing high carbohydrates. Polyphenol components can affect key enzymes involved in starch breakdown and subsequently improve glycaemic control through inhibition of amylolytic enzymes.

The fact that we have already reached the diabetes incidence figures predicted by WHO for 2030 in our country in these years has revealed the necessity of making critical moves regarding diabetes. The fact that nearly 25% of the country's health expenditures are spent on diabetes and related diseases reveals the fact that diabetes is very important in terms of the burden it brings to the country's economy as well as the health of our citizens. In this sense, there is a need for effective measures for the prevention and control of diabetes in the provision of Primary Preventive Health Services, early diagnosis and appropriate treatment, and awareness raising about the prevention of diabetes-related diseases and complications.

The International Diabetes Federation (IDF) estimates that 382 million adults worldwide have diabetes. The prevalence of diabetes has reached epidemic proportions and is expected to reach 592 million by 2035. Currently, it is estimated that there are more than 7 million diabetic patients in Turkey. Only 11 % (800 thousand people) of diagnosed diabetes patients can achieve the targeted treatment results and lead a life without complications. In 2009, the IDF estimated that Turkey's diabetes-related health expenditures would reach $6.5 billion by 2030. However, this figure was already reached in 2010. When diabetes causes complications, treatment and healthcare costs can be more than 5 times that of diabetes treatment alone. Current IDF statistics for 2013 show that the average annual cost of treatment for a person with diabetes in Turkey is $866 (IDF, 2013).

Over the past 15 years, the number of diabetics in Turkey has nearly tripled, rising from 2.5 million in 1998 to nearly 7 million in 2013. Two population-based diabetes studies conducted in Turkey in 1998 and 2010 show that diabetes in the adult population increased by 90% in this period and continues to increase. Turkey carries almost 13% of the diabetes burden in Europe due to the high number of diabetes patients. In addition to these statistics, approximately 3.7 million people in Turkey have impaired glucose tolerance (IGT), which is called the precursor of type II diabetes. According to IDF projections, the number of individuals with diabetes will reach approximately 12 million by 2035.

Studies have shown that the risk of diabetes can be prevented or delayed by 44-58% with healthy lifestyle changes, including nutrition. Therefore, by consuming food additives that have the attribute of reducing the risk of type II diabetes and foods produced with the addition of these additives, it will help to reduce the risk of diabetes and the costs spent for the treatment of this disease, and indirectly contribute to the country's economy.

Since it is possible to reduce the risk of diabetes by 44-58% with changes such as healthy diet and lifestyle, it is necessary to enrich the foods with daily consumption habits with functional food additives that have the attribute of reducing the risk of type-ll diabetes.

Black pine bark, which has a wide distribution in Turkey, constitutes 13-21 % of the tree's dry matter by weight. The bark is separated from the wood after the tree is cut and is released as waste. The bark is used in areas such as the leather industry and biofuel production. However, obtaining the valuable components found in the bark and using them in the food industry is not known in the art. Brief Description and Aims of the Invention

The primary purpose of the invention is to produce valuable, functional food additives with high added value for the food industry from Black Pine (Pinus nigra) bark, which is a waste of the forest industry, and to prove its antidiabetic, antioxidant, and anticancer activity.

The aim of the invention is to obtain a functional food additive with antidiabetic, anticancer and antioxidant properties, containing powdered bioactive components extracted from the bark of Black Pine (Pinus nigra). With the invention, a food additive with high added value, functional properties and reducing the risk of type-ll diabetes was prepared for the food industry from Black Pine bark, which is not used in the food industry in a short time and at low temperatures.

Another aim of the invention is to provide opportunities for use in the food industry and to reveal new/high value-added usage areas of Black Pine (Pinus nigra) industrial bark, which is generally considered as waste in the production of forest products or used in areas with low added value. Considering the predicted increase in the number of patients with diabetes in Turkey, enrichment of foods with daily consumption habits with functional food additives (bioactively rich soluble tree bark extract), which has the attribute of reducing the risk of type II diabetes, can help reduce the risk of this disease.

Another aim of the invention is to contribute to the prevention of environmental pollution by evaluating the wastes left to the environment within the scope of the "ZERO WASTE" approach.

The invention is a new approach to incorporate the potential of natural products in food formulation, and to control the blood glucose concentration through inhibition of a-glucosidase although there are some pharmacological procedures to control the diabetic patient's blood glucose concentration and deliver insulin-like effects, and it is important in terms of food-health relationship.

By enriching the foods with daily consumption habits with functional food additives (bioactive rich water-soluble tree bark extracts) that have the feature of reducing the risk of type-ll diabetes, the invention allows; (i) to help reduce the risk of disease, and

(ii) production of functional food additives and supplementary food additives

(iii) to obtain new products with high added value for the food industry from larch shells, which are widely distributed in terms of the country's economy.

Black pine bark extracts obtained by using ultrasound, which is one of the new extraction methods, reduced blood glucose levels at lower doses against acarbose used in the treatment of type-ll diabetes. It is also supportive with its immune- boosting antioxidant properties.

With the invention, it has been shown that the extracts obtained by the extraction of bioactive components from black pine bark, which is a waste of the forest industry, are used as an immune-enhancing antioxidant-rich functional food additive that is effective in reducing the risk of type-ll diabetes.

Food additives and auxiliaries are widely used in the production of foods, improving and regulating their properties, adapting them to industrial production techniques, increasing their added value by extending their shelf life and protecting the sensory properties of final food products. These substances, which constitute an important cost item among the inputs of the food industry, are largely met by imports since they do not have sufficient production in our country. It is important to develop, produce and improve food additives and auxiliaries to reduce the foreign dependency of our country.

In line with consumer demands, the development of additives and auxiliaries obtained from natural sources instead of chemical/synthetic additives is rapidly gaining importance, and therefore, the present invention is important in terms of carrying out industrial studies.

Description of the Drawings

Figure 1: Flow chart of preparation method of functional food additive that reduces the risk of TYPE II diabetes from Black Pine (Pinus nigra) bark.

Figure 2: Comparison of cell viability results in CaCo-2, HEK and MiaPaCa-2 cells applied with black pine bark extracts. Detailed Description of the Invention

The invention is a functional food additive that reduces the risk of TYPE II diabetes, containing bioactive components extracted from the bark of black pine (Pinus nigra). The food additive can be in liquid or powder form.

Black pine (Pinus nigra) is a species of pine from the pine family (Pinaceae). In the invention, an ultrasonic assisted extraction system, which is one of the modem extraction techniques, was used for the extraction of functional bioactive substances from black pine bark, and the antidiabetic-anticancer-antioxidant activity of bioactive substances supporting the in vitro biological food-health relationship was determined. The black pine bark required in the invention was obtained from Isparta-Egirdir region.

With the invention, it has been determined that water-soluble bioactive substances obtained from black pine (Pinus nigra) bark are effective in the regulation of blood sugar level due to their inhibitory effect on the a-D-glucosidase enzyme.

After most of the trees that are considered as wood in the forest industry are cut, the bark is stripped from the wood body or used as a waste to a large extent, either as fuel or used as raw material in relatively different industries (biofuel production, leather making, etc.). Looking at the composition of the bark, it has been found that it contains valuable components that can be of great importance in food science and technology. The fact that bark contains higher levels of phenolic substances and antioxidants compared to fruit/vegetable pulp, which is evaluated as food waste, constituted the starting point of the invention and it was thought that it could be used as a different industrial waste that could be evaluated for the food industry.

A functional additive with the ability to lower blood sugar was obtained by in vitro tests produced from bark extracts prepared within the scope of the invention.

The flow diagram of the preparation of the functional food additive that reduces the risk of TYPE II diabetes is shown in Figure 1 .

Preparation of functional food additive that reduces the risk of TYPE II diabetes comprises the process steps of:

■ Supplying of black pine bark and grinding it to a particle size of less than 1 mm,

■ Extracting the functionally effective bioactive components from black pine bark in alcohokwater mixture by ultrasonic extraction method from ground bark under optimized conditions at 20-40 °C, extraction time 3-9 minutes and ultrasonic power 50-150 W in ultrasonic extraction with response surface method, and

■ Drying the extracts in a lyophilizer (freeze dryer), removing the alcohol in them and turning them into powder.

The antidiabetic activity of the extracts obtained using the conditions optimized by ultrasonic extraction (temperature 20-40 °C, extraction time 6-9 minutes and ultrasonic power 50-150 W) is determined. Extracts with high antidiabetic activity are characterized by antioxidant, antimicrobial and anticancer activity and phenolic acid composition in HPLC. Finally, bark extracts are added to functional food products and their effect on lowering the glycaemic index is determined. For example, by adding water-soluble lyophilized powdered black pine bark extract at certain concentrations to white bread and pasta, the glycaemic index was lowered compared to control white bread.

The black pine bark to be used as an example is obtained from the trees with the most uniform form in the region where the trees form a forest, according to the TAPP I T 257 cm-02 standard just before the analysis period. The samples were quickly taken to the laboratory after cutting the tree and cut into as small pieces as possible by preliminary shredding. The samples dried in the Freeze-dryer (lyophilizer, freeze- drying) device were ground in a laboratory type Wiley mill with a particle size of less than 1 mm.

In Table 1 , Table 2 and Table 3, 17 different points were studied by adjusting the extraction temperature at 20-40 °C, extraction time 3-9 minutes, and ultrasonic power 50-150 W. The extraction conditions predicted for maximum yield were determined as 34.33°C, 6.89 min and 98.45 W. By entering these points, analysis results and the data given in Table 2 into the program, the program responds as 34.33°C, 6.89 min and 98.45 W as the extraction conditions predicted for maximum yield, according to the R2 values given in Table 3. In the extraction of bioactive components from black pine bark, 80% methanokwater was used as a solvent. The bark:solvent ratio was determined as 1 :10 (m:v) by preliminary experiments. Extraction processes were carried out with ultrasonic assistance, and in the optimization study, the experimental design obtained by the Surface Centred Composite Design of the 3-factor (extraction time, extraction temperature and ultrasound amplitude) and 3-level (low, medium and high) Response Surface Method (RSM) and shown in Table 1 was utilized. In the present study, Design Expert Version 7.0.0 (Stat-Ease Corporation, Minneapolis, MN, USA) software was used to create a Box Behnken Design (BBD). BBD was used to evaluate the effect of independent variables, i.e. extraction temperature (X1 , 20-40 °C), extraction time (X2, 3-9 min) and ultrasonic power on yield (X3, 50-150 W) were determined. The levels of process variables were determined based on preliminary experiments in the laboratory and the relevant literature search. The experimental design, coded and uncoded as 17 random trials with 3 repetitions and 3 factors, is given in Table 1 .

In response, yield and total phenolic content, total tannin content and antioxidant methods FRAP (Ferric Antioxidant Power) and ABTS (2,2'-azinobis-(3- ethylbenzothiazoline-6-sulfonate) were evaluated and the obtained values are given in Table 2. In 17 different samples, the point at which all responses were maximum was determined with the help of the program as the optimum condition of extraction.

The extraction conditions predicted for maximum yield were determined as 34.33°C, 6.89 mins and 98.45 W. In a short time of 6.89 minutes, an extract rich in antioxidants and phenolics, with antimicrobial, anticancer and antidiabetic effects, was obtained and proved by the results of the experiment. A new food additive with high added value has been introduced to the food industry with the attribute of reducing the risk of type-ll diabetes of the black pine bark extract, which is rich in functionality. As a result, it can be concluded that black pine (Pinus nigra) bark extract can strengthen the antioxidant status with its important phenolic component content, prevent postprandial hyperglycaemia by means of its a-glucosidase inhibition activities, and can be used as a reducer of the risk of type II diabetes. Table 1 Ultrasonic extraction experimental design prepared with Surface Centred Composite Design (RSM)

X1 , X2 and X3 model parameters are extraction temperature, extraction time and ultrasonic power, respectively. Table 2 Extraction yield and bioactive properties of black pine (Pinus nigra) bark samples

GAE: gallic acid equivalents; CE: catechin equivalents; TEAC: Trolox equivalents antioxidant The estimated model equations and regression coefficient values for black pine (Pinus nigra) bark samples are given in Table 3. R ² values are significant, and the experimental design was carried out successfully. P value is < 0.05 and the model is significant. Even with the increase in temperature and time, the efficiency has increased, but the effect of time is more effective than temperature. At the point where the extraction temperature is 30°C, the yield is at its highest as the power and time increase. Power and duration are more effective than temperature.

Table 3 Estimated model equations and regression coefficients value for black pine (Pinus nigra) bark samples

Table 4: Mineral content of extract from black pine bark (ultrasound maceration extraction comparative)

There is no study in the literature on the mineral profile of black pine bark extract. The amount of minerals found in the ultrasonication-assisted extracts of black pine bark is higher than the water bath extract.

Mineral profile of Black Pine Bark is higher in ultrasonic extraction considering both extraction methods. It is seen that the mineral with the highest amount in black pine is sodium, followed by potassium and calcium (Table 4).

When Table 5 is examined, the major component in black pine bark is a-Pinene and it is 27.2%. The main components in black pine bark are a-pinene, [3 -pinene, limonene, [3-Caryophyllene and germacren D. It supports its antimicrobial effect with its rich volatile aroma profile. Antimicrobial results are given in Table 7. When the antimicrobial results were examined, it was found that ultrasonic extraction of black pine bark was more effective against pathogenic microorganisms than maceration samples. A low concentration proves that it has an antimicrobial effect at a lower concentration. The best inhibition was against S. typhimurium with 0.350 mg/mL. Table 5: Aroma Profile Analysis Results of Black Pine Extract by GC Table 6: Phenolic acid profile of black pine bark extracts (ultrasound maceration extraction comparative)

The phenolic acid profile is given in Table 6. Ultrasonic extraction, chosen as the extraction method, was compared with classical maceration (water bath) extraction. When the values are examined, ultrasound extraction results are better than water bath extraction. In terms of phenolic acid, syringic 15.4019±0.01 (pg/g), elagic 54.2575±0.01 (pg/g) were found, but it was not determined in the maceration extract. A total of 14 phenolic acids, caffeic, syringic, rans4hydroxycinnamic, rutin, ferulic, trans3hydroxy, dcoumaric, myricetin, quercetin, chyrsin, elagic, m-coumaric, gallic and protocatechic, were determined in the black pine bark extract. Ultrasound power helped in the release of phenolics. The highest amount of phenolic acid was m- coumaric acid, and it was found as 104.7304±0.02 (pg/g). Ultrasound extraction of black pine bark is richer in phenolic acid profile than water bath. The presence of rutin phenolic acid with antidiabetic effect in the structure (Table 6) made black pine bark reduce the risk of type-ll diabetes and have antidiabetic effects. Suggested mechanisms for the antihyperglycemic effect of Rutin include decreased carbohydrate absorption from the small intestine, inhibition of tissue gluconeogenesis, increased tissue glucose uptake, stimulation of insulin secretion from beta cells, and protection of the islet of Langerhans against degeneration. Rutin also reduces the formation of sorbitol, reactive oxygen species, enhanced glycation end product precursors and inflammatory cytokines. These effects are thought to be responsible for the protective effect of rutin against hyperglycaemia and dyslipidemia- induced nephropathy, neuropathy, liver damage and cardiovascular disorders (Ghorbani 2017).

Its ability to reduce the risk of type-ll diabetes and its antidiabetic effect, and the results of a-glucosidase enzyme inhibition are given in Table 8. In Table 8, the ultrasonic method used in black pine bark extract was compared with classical maceration and it was proven that black pine bark extract was a better antidiabetic than acarbose used in the treatment of type-ll diabetes. The ICso value is the concentration of inhibitor that inhibits 50% of the enzyme, and a low ICso indicates a high inhibition value.

In Table 8, while the ICso value of acarbose used in the treatment of TYPE-II diabetes was 244.63 ± 1 .23, the ICso value of black pine bark extract obtained by ultrasonic extraction is 145.35±0.23, and the ICso value of black pine bark extract obtained by maceration is 160.34 ± 0.76. In other words, Black Pine bark extract was able to inhibit the a-glucosidase enzyme 50% by using a lower concentration than acarbose. These results show that black pine bark extract has a better antidiabetic effect than acarbose used in the treatment of TYPE-11 diabetes. Also, black pine bark extracts obtained by ultrasonic extraction (34.33°C, 6.89 mins and 98.45 W), which was chosen as the extraction method, gave better results than extracts obtained by maceration, and these were a better antidiabetic and reduced blood glucose levels at lower doses.

Table 7. Antimicrobial activity of black pine bark extracts (ultrasound maceration extraction comparative)

Table 8: Antidiabetic effect of black pine bark, inhibition of a-glucosidase enzyme

Its alpha-amylase inhibition activity and alpha-glucosidase inhibition activity against Acarbose, which is a synthetic drug and an a-glucosidase inhibitor, frequently used in the treatment of diabetes, was in vitro determined by preliminary trials. Black Pine bark is a more effective antidiabetic agent compared Acarbose. The results of the experiment showed that the extracts obtained from black pine bark by ultrasonic extraction were a better antidiabetic agent than the classical water bath extracts and at the same time were more effective than acarbose.

Figure 2 shows the anticancer effect of black pine bark extracts on intestinal and gastric cancer cells in comparison with the positive control healthy cells.

While a weak cytotoxic effect of black pine extracts was observed on human healthy epithelial cells (HECK 293) only at a dose of 1000 pg/mL, when compared with the control group, it was determined that it decreased cell viability in a dose-dependent manner on CACO-2 (human epithelial colorectal adenocarcinoma) cells and therefore had cytotoxic effects and it was anticarcinogenic (p<0.05).

In CaCo-2 cells, it was observed that black pine bark extracts significantly reduced cell viability at all doses above 1 pg/mL. When the cell viability values of each dose in CACO-2 and HEK cells were compared, a significant (P<0.05) difference was found in all experimental groups except 1 and 10 pg/mL doses (Figure 2). The ICso value of black pine bark on the CaCo-2 (intestinal cancer) cell line was found to be 440 pg/mL. In determining the anticancer activity of tree bark extracts, MiaPaCa-2 cells were more effective on gastric cancer cells. The ICso value of black pine bark on MiaPaCa-2 cell line was found to be 390 pg/mL.