DNJ EXTRACTION METHOD

Abstract

The invention relates to a two-stage energy-assisted extraction method.

Description

DNJ EXTRACTION METHOD Technical Field to Which the Invention Relates The invention primarily concerns organic substances within the scope of chemistry, food and / or pharmaceutical technology. a dual-stage energy-assisted system developed for obtaining separation processes It is related to the extraction method. Prior knowledge of the art related to the invention. 1-deoxynojirimicin (DNJ) is found in various plant species, primarily the mulberry plant. It is an α-glucosidase / sucrase inhibitor organic compound from the iminosugar class. DNJ source The plant species in question, considered as such, is the mulberry (a tree of the Morus genus belonging to the Moraceae family). (type of) plant; morning glory (Commelina communis) plant; Japanese lady's bell (Adenophora) Examples include the Triphylla plant and the hyacinth (Hyacinthus orientalis). DNJ's analysis of these plants includes various parts such as leaves, shoots, fruits, root bark / woody tissue. It is possible to obtain them from botanical regions. However, DNJ is only available from plant sources. It is not found in the sources; it can be obtained from silkworm (Bombyx mori) larvae / pulps. Various species such as Actinoplanes, Streptomyces, Bacillus, Flavobacterium, or Ganoderma lucidum. They can also be produced by organisms through microbial synthesis methods. The mulberry plant, belonging to the Moraceae family, is a species of tree in the genus Morus, and the white mulberry (Morus alba), It has various species such as black mulberry (Morus nigra) and red mulberry (Morus rubra). The fruit of the plant is widely consumed as food; the leaves of the plant in question... It is used in fruit production, color pigment extraction, and silkworm breeding, and as a sugar regulator. Due to its properties and antioxidant effects, it is an important resource in pharmaceutical applications. It is observed that the botanical section of the plant in question is the richest in DNJ. Because the secondary metabolite load is quite high, it is located in the shoot / apical leaf region. This is reported. However, as a result of the study conducted by Kimura et al. [1] DNJ is located in fruit, and especially in early / green stage fruit (MS-I), in 1976. A study has shown that DNJ can be isolated from the root bark. 1 In studies conducted on silkworms (Bombyx mori), silkworm larvae were found to be... DNJ appears to accumulate in tissues; some studies have shown that this accumulation... The amount was reported to be approximately 2.7 times greater than the amount contained in the leaf. This accumulation is due to the enzymatic defenses that the silkworm develops against the mulberry tree. adaptation (insensitive / low-sensitivity sucrase-trehalase phenotype to iminosugars) It was observed that DNJ and biomass were present in the 5th stage of development of the silkworm larvae in question. It has been observed that the rate can reach its peak. In studies specifically conducted on Commelina communis (morning flower), DNJ and in the aqueous fraction... It has been shown that iminosugars such as DMDP can be obtained; and in 70% EtOH fractions. It has been reported that flavonoid glycosides have been isolated. Similarly, different studies have been conducted. Studies have also shown that DNJ and iminosugars are present in Japanese lady's bell (Adenophora triphylla) plants and It also appears to be obtained from the hyacinth (Hyacinthus orientalis) plant. However, during the day Considering the flower and the studies conducted specifically on the plants mentioned above When considered, the DNJ level in the relevant plant species is higher than that of the mulberry plant (Morus). much lower and the amount of DNJ depends on tissue, harvest time and method selection It appears to vary. As mentioned above, DNJ is found in various plant sources and microorganisms. However, the natural extraction yield of the active substance in question, DNJ, is quite low. It is low and fluctuating. Therefore, in order to obtain DNJ, various methods have been described in the literature. The researcher has turned his attention to chemical synthesis. Examples of different chemical synthesis studies found in the literature include those by Braga et al. [2] A formal total synthesis from D-glucitol was achieved by Iida et al. [3] Synthesis of optically pure DNJ was achieved by Wang et al. [4] Development of a practical synthesis protocol for one of its derivatives and optimization of DNJ [5] A study on chemical synthesis pathways prepared by Iftikhar and colleagues. This can be done. Based on these studies, it has been shown that the chemical synthesis of DNJ is possible. And chemical synthesis appears to be a potential alternative to natural extraction methods. However, DNJ, which contains numerous chiral carbons in its structure, has the correct configuration. Complex guarding group strategies, asymmetric catalysis, and are necessary for the synthesis of the isomer. Diastereoselective steps are required. Furthermore, a protecting group is necessary throughout the synthesis process. various chemical transformations such as addition / removal, oxidation, reduction, cyclization, and amination. 2 Since the steps need to be carried out, the control of the multi-step reaction sequence and Therefore, overall synthesis becomes more difficult. The difficulty of synthesis stems from these steps. In addition, it should also be considered that there may be efficiency losses at each step. When this is the case, the total yield may remain below approximately 20-40%. It is observed that the organic solvents used in these synthesis steps are reactive. the amount of waste generated due to chemicals, preservatives and / or catalysts and the related Considering the cost of component or waste disposal, chemical DNJ synthesis It appears to have various disadvantages. Moreover, the synthesized DNJ or its derivatives, Biological activity (inhibitory capacity, bioavailability, and similar characteristics) of natural DNJ, Due to the potential for differences in toxicity and stability, the synthesized It is stated precisely that the components can be used as exact substitutes for natural DNJ. It is not possible. The large number of steps involved in these methods and their high costs This is because stereochemical control is challenging in chemical synthesis methods, and yield losses occur, the potential for posing an environmental threat, and chemical Differences in the biological activity of the components obtained as a result of synthesis lead to a more efficient natural approach. This leads to the investigation of the extraction approach, and the method in question is the subject of the invention. It plays an important role in its development. In conclusion, all the problems mentioned above make innovation in the relevant field imperative. has brought. Purposes and Brief Description of the Invention The main objective of the invention is to produce 1- in high yield using a natural extraction method. The goal is to obtain deoxynojirimicin (DNJ). Another objective of the invention is to improve the solvent used in the extraction of 1-deoxynojirimicin (DNJ). The goal is to ensure its recovery. Detailed Description of the Invention The invention relates to a method for obtaining a 1-deoxynojirimicin (DNJ)-rich extract. The method described in this invention allows for the production of DNJ, an efficient method known in the literature to be achieved with limited yield. The extraction of the substance is ensured, thanks to the method described in the invention, the relevant extraction is carried out. The efficiency of the method is being increased. 3 The raw materials that can be used in the method described in the invention are 1-deoxynojirimicin (DNJ). It is expected to be rich in terms of raw materials. In this context, the raw material in question is derived from different plants. These can be obtained from different species and botanical parts of these plant species. The plant species that can be preferred as raw material are preferably mulberry (Moraceae). (a tree species of the genus Morus belonging to the family) plant; morning glory (Commelina communis) plant; Japanese honeysuckle (Adenophora triphylla) plant; hyacinth (Hyacinthus orientalis) plant and There may be other plants with similar high DNJ content. If the aforementioned plant species are used as raw materials, i.e., as a source of DNJ, As mentioned above, the extraction method that is the subject of this invention is related to different botanical departments. It is possible to subject them to this. The botanical departments in question are preferably responsible for the plants mentioned. It can include leaves, shoots, fruit, and / or root bark / woody tissue. In the preferred application of the invention, mulberry plant is preferably used as the DNJ source, and The raw material in question serves as an example to illustrate the method described in the invention. This In this context, the plant in question is of a nature that would limit the method described in the invention. This should not be interpreted. However, the DNJ ratio found in the mulberry plant in question... It should not be overlooked that it is preferred because of its height compared to the plants mentioned above, the most The potential for the concentration of highly active substances in this particular plant should be considered. The raw material in question, for the method described in this invention, is preferably Morus alba, or white mulberry. Morus rubra, also known as red mulberry, or similar mulberry varieties with high DNJ content can be used. However, in the preferred application of the invention, the high DNJ content is particularly problematic. White or red mulberry varieties are preferred. The detailed description of the invention section... Furthermore, the explanations given regarding the method in question are based on the plant species mentioned. It is described. Furthermore, although the mulberry plant has a high DNJ ratio, this is also specific to that particular plant. Since the proportion of the active ingredient (DNJ) of the botanical part to be used varies, the invention The specific botanical section of the raw material to be subjected to the method is important. Contains botanical parts such as leaves, shoots, fruits, root bark / woody tissue. Considering the amount of DNJ, especially the DNJ located in the leaf section. Due to its higher level compared to other botany departments, the method in question is the subject of the invention. 4 In its preferred application, preferably the leaf and especially the apical, or young leaf section (MS- i) or exile is used. The fruit contains much less DNJ than the leaf, and its content increases as it ripens. Since the amount of DNJ found is known to decrease, the botanicals to be subjected to the method described in this invention If the section is fruit, the fruit in question must be in the early / green stage. is required. The invention describes a method involving the sequential performance of two extraction methods and the aforementioned It is improved by optimizing the extraction parameters. The first of these extraction processes is DNJ's process of extracting raw materials (preferably leaves). to ensure rapid uptake (from the matrix) into the solvent phase and to improve mass transfer is being carried out, and thanks to the first extraction method mentioned above. The process involves breaking down the cell walls of the raw material, thereby disrupting the matrix structure. The subject of the second extraction, which will feed the phytate obtained in the first extraction, is its purity. This is carried out with the aim of providing this effect by applying a mechanical cavitation effect. It increases mass transfer efficiency. The first extraction in question should preferably be done using MAE (microwave-assisted extraction), i.e., microwave. the first extraction is assisted by a first method, and the second extraction is preferably performed using UAE (Ultrasound Assisted Extraction). In other words, it is ultrasound-assisted extraction. Optimizing the extraction parameters on a method basis constitutes the subject of this invention. because microwave radiation will directly affect the efficiency of the method; its effects on cellular structure and its ability to increase cell permeability homogeneous heating will accelerate mass transfer during the initial extraction. This is achieved through the first extraction, and the yield is increased with the second extraction mentioned. Each Both extraction methods, especially maceration and Soxhlet extraction methods Compared to others, they are described as more environmentally friendly, or "green," extraction methods. It requires less solvent and energy, thus saving resources. This ensures that, at the same time, these extraction methods are highly sustainable. These are environmentally harmless methods; and in terms of efficiency and speed, they surpass traditional methods. They are exceeding. While maceration and Soxhlet extraction methods need to be carried out for 12–24 hours; An extraction using microwave-assisted extraction takes 2 minutes, ultrasound If an extraction is performed using assisted extraction, it is sufficient to continue for 33.52 minutes. It is seen. Furthermore, a study conducted revealed that mechanical extraction, a traditional extraction method, is effective. When the mixing method is used (control), a yield of 0.65% is obtained in 2 minutes. An optimized MAE method was found to provide 5,455% efficiency in the same time period. The specified conventional extraction method (control) extracts this powdered raw material. Mechanical processing is carried out after adding the raw material to a solvent selected appropriately for it. The process is carried out by mixing; in the study conducted, the solution in question was mixed at a speed of 600 rpm. mixed using a specific mixing process to prevent the solvent from evaporating. This was carried out at a controlled temperature and in a closed system. In this context, control The traditional extraction method mentioned, referred to as a method, uses a standard solid. It is described as liquid extraction; the method in question is to be carried out for 2 minutes. In this case, as stated above, a yield of 0.65% is obtained. In this context, the MAE method is 7.38 times more efficient than the aforementioned traditional extraction method. It appears to be effective. Similarly, a traditional extraction method (control) lasting 33 minutes... The yield obtained subsequently was 2.31%, while an optimized UAE method yielded the same result in the same time. It has been observed that the yield obtained as a result of continuing this process throughout the period is 7.44%. In this context, the UAE method is the traditional extraction method mentioned (control). When compared, it is seen that 3.2 times more efficiency is obtained with the UAE method. The yield stated here is the yield of the target compound (DNJ) extracted from the source from which the extract is obtained. It represents the quantity; the amount of extract obtained as a result of the method is the amount subjected to the method. It refers to the ratio to the amount of resources held. At the same time, as a result of the studies, it was found that the extract obtained from UAE contains the total phenolic content (TPC) compared to conventional extraction methods (control) It increased 4.2 times, and within the extract obtained as a result of MAE, it increased 1.25 times. It is seen. 6 In this context, in addition to the yield obtained within the scope of DNJ, the UAE and MAE methods were also considered. in the extraction processes carried out, bioactive components are also better preserved. It is seen. In this context, within the scope of the invention, in light of the above-mentioned research results, MAE and UAE A sequential extraction method incorporating these techniques is being developed; By combining the different advantages of the methods, a synergistic effect is achieved, and the invention... As a result of the method developed within this scope, functional food with anti-diabetic properties, food supplements or pharmaceutical products that can be used to manage blood sugar, A valuable extract enriched with DNJ is obtained. The raw material in question, preferably fresh or dried, is used in the method described in this invention. It can be provided. However, in the preferred application of the invention, the solvent used within the scope of the invention is mentioned. The subject is to be able to extract the active ingredient contained in the raw material with high efficiency. the particle size of the raw material in question and its surface area in relation to this particle size It needs to be increased. Therefore, in the preferred application of the invention, the aforementioned The raw material is preferably used in dried form. If fresh or moist raw materials are used, the invention developed within the scope of this document... The purity of the solvent used in the method may be compromised, as may its polarity and viscosity. Their properties may change. In this context, MAE and UAE may change as a result of high humidity. The energy efficiency of the methods, and consequently the method in question, decreases proportionally. The yield of the extract obtained within the scope of the invention is decreasing. In the first stage of the method described in the invention, microwave radiation is used. The polar molecules contained within the cells of the raw material are heated, and the heated molecules... The molecules evaporate, creating high pressure within the cell. This pressure then... by bursting the cell wall outwards, the raw material in question is released. It enables the release of the active substance into the solvent. In this context, the raw material in question... In the case of fresh leaves, the applied microwave power penetrates inside the cell, i.e., intracellularly. Instead of vaporizing molecules, it targets those located outside the cell or between cells, that is... Since it will affect interstitial molecules, there will be a loss in the microwave power in question. This will occur, and consequently, energy loss and decreased efficiency will result from this stage. 7 Similarly, UAE, by creating cavitation bubbles in the solvent, causes them to burst violently. becoming functional; mechanical shock waves obtained as a result of these explosions and Extraction occurs as a result of shear forces acting on the cell wall. This is accomplished in this context, using fresh and / or moist cells, i.e., cells with high tugor pressure. Due to its elastic structure, mechanical shock waves or shear waves applied to the cell wall their forces can be dampened and therefore, as seen in MAE, the power used can be reduced. It appears that losses may occur. This power loss, in turn, leads to a loss of efficiency. In the preferred application of the invention, the raw material in question may preferably be dried when used in the invention. The subject is communicated to the method. In this context, the raw material in the dried form, MAE inability to resist microwave radiation applied within the scope of the UAE This ensures that the material becomes more fragile and sensitive to the mechanical force applied within this scope. Furthermore, the aforementioned raw material should preferably be available whole, in pieces, or ground. It is possible to use it. However, in the preferred application of the invention, the said The raw material shall be subjected to the method described in the invention in such a way as to have a homogeneous particle size. Since the aim is to preserve it, the raw material in question is preferably ground and then processed using the method described in the invention. This is because the unit of power transmitted to the raw material within the scope of the invention is... Increasing productivity as a result of homogeneous distribution across the area and the method in question It is the shortening of the period during which it is active. The grinding process in question involves grinding the raw material preferably to a size of 40 – 60 mesh. It is concluded with the introduction of these particle sizes. The reason for choosing these particle sizes is... The aim is to optimize the efficiency to be obtained within the scope of the invention and at the same time to achieve high It is based on ensuring processability. In this context, the aforementioned 40-60 mesh size If the raw material being brought in is used; the aforementioned active ingredient will be added to the solvent. The transition is being accelerated, cavitation activity is taking place within the scope of UAE. The potential filtration process to be carried out on a method basis is being increased, targeting small particles. This prevents disruptions caused by large particles and / or potential loss of efficiency. The drop is eliminated. As mentioned above, the relevant particle size is 60. If the mesh size exceeds 250 µm, the amount obtained by the method described in the invention When the efficiency decreases and the particle size is small, the filtration of the relevant particles... and / or appears to cause blockages during steps such as filtration. 8 It is possible to supply the raw material in question to the method in question instantly. It is also possible to obtain it after it has been stored for a certain period of time. Therefore, the aforementioned If the extraction processes are not carried out instantly, the homogenized powder in question... he Imported raw materials should preferably be stored at -20°C. Furthermore, in order to preserve the raw material content during the aforementioned storage period, The raw material in question should preferably be stored protected from moisture and preferably from light. This In this way, the raw material in question is brought to room temperature when the analysis or extraction day arrives. It can be brought in and used, and the DNJ content in the raw material can be preserved. The first step of the method described in the invention, namely the initial extraction (MAE), is the extraction of the aforementioned raw material. the dissolution process is carried out by treating it with a solvent and the resulting solution This is achieved through the MAE process. In the preferred application of the invention, the solution in question is a component of the aforementioned raw material. The solvent should preferably have a liquid / solid ratio by volume of 5 – 100 (liquid) : 1 (solid), and especially 20 – 80. (liquid) : 1 (solid) (mL / g) is obtained by adding and dissolving (solution). is being done. The solvent in question is preferably a methanol-water, ethanol-water, acetone-water, or dichloromethane-water mixture. It is preferred in this way. In the preferred application of the invention, the aforementioned solvents are suitable for the aforementioned raw material. It is also possible to use different organic solvent-water mixtures, such as those mentioned. However, in the preferred application of the invention, the solvent in question is an ethanol / water combination; preferably an ethanol / water ratio of 10-30%, and especially 20-30%. is being done. In the preferred application of the invention, a 30% ethanol / water solution is used, in particular; The aforementioned solution ratio pertains to the solution in question, specifically in terms of the solubility of DNJ. It is defined as the optimum solvent concentration value specific to the optimized DNJ. 9 As a result of the studies, the 30% ethanol / water mixture is considered ideal as a solvent. If preferred, a 10% ethanol / water mixture increases efficiency depending on the conditions under which it is used. This is observed. Therefore, in the preferred application of the invention, as stated above, The preferred solvent is 30% ethanol / water. The solution in question should preferably be placed in a conventional microwave-assisted extraction device. It is nourished. The solution in question is preferably in the 915 MHz – 2.45 GHz range, and specifically 2.45 GHz microwave. It is fed into a microwave-assisted extraction device with frequency capacity, and this Frequency determines how many times per second the solvent molecules will vibrate. In the preferred application of the invention, the supplied solution should preferably be in the range of 300 – 800 W or It is available in the 560-640W range, and specifically at 600W. The solution in question, in the preferred application of the invention, preferably has a power of 5 – 20. It is subjected to for minutes or 7-13 minutes, and especially for 11 minutes. he Furthermore, the process in question, in the preferred application of the invention, preferably involves temperatures between 20 and 80°C. in the range of, and specifically, between, the temperature range of 45–65 °C or 70–80 °C. It is carried out at a certain temperature. The relevant temperature value applies to the extraction that will be carried out after the first extraction mentioned. in a way that is suitable for the temperature sensitivity of the active substance to be exposed, i.e., DNJ It has been determined that the first and second extractions will be carried out consecutively. This is determined by considering a combination of methods. In this way, long-term having a structure that may experience losses under prolonged and / or uncontrolled heat treatments DNJ protection has been ensured; efficiency is achieved on a methodological basis and is effective. The stability of the material is increased. In the preferred application of the invention, the solution in question is preferably subjected to a power of 600 W, preferably 11 It is subjected to a temperature range of 70-80 °C for several minutes. This ensures that the raw material... The cell walls are broken down, thus allowing DNJ to pass into the solvent. Efficiency is increased by obtaining optimum levels of DNJ. In the preferred application of the invention, the product subjected to MAE treatment is preferably placed in a cooling system. It is processed through a cooling process, after which the second extraction method is applied. It is being transmitted. The cooling process in question involves the spontaneous cooling of the mixture that has undergone the MAE process. or can be achieved by cooling. In the preferred application of the invention, the aforementioned The cooling process involves bringing the product obtained through the MAE process preferably to a temperature range of 0 – 50 °C. It is concluded with. The cellular structure of the MAE-treated product within the scope of the first extraction. The active ingredient has been broken down and any separation process that may be carried out will not affect its integrity. because it would cause a loss, in the preferred application of the invention, the amount obtained in the MAE process. The product is preferably not subjected to any centrifugation and / or filtration process, secondly It is preferably conveyed directly to the extraction process. In the example where the preferred application of the invention is used, 0.50 g of dry leaf sample is added to 40 ml preferably added to a 30% (v / v) ethanol / water solvent under the conditions specified above, It is processed at a microwave frequency of 2.45 GHz, preferably with temperature monitoring. The second extraction (UAE), which is the second step of the method described in the invention, is obtained in the first extraction. This is achieved by subjecting the resulting mixture to an ultrasonic extraction method. The mixture containing the DNJ component, which is to be purified by the second step of the method described in the invention, When a solution is added, the energy requirement increases within the scope of the method described in this invention. Since the added solvent needs to be evaporated, the process efficiency decreases and additional steps are required. Time is wasted due to the steps involved. Therefore, the preferred method for the invention is... In this application, the mixture in question is conveyed to the UAE process preferably without the addition of solvent. In order to expose the mixture in question to ultrasonic vibrations, the mixture should preferably be... It is fed into an ultrasonically assisted extraction device. The solution supplied via this device is preferably 120 – 500 W, 150 – 500 W or 120 – It is subject to power in the 240W range, and specifically 180W. 11 In the preferred application of the invention, the ultrasonic vibrations in question are preferably between 200 and 1500. seconds, 200-320 seconds or 600-1500 seconds and especially 260 seconds is being carried out. The reason for keeping the specified time and power parameters within this range is due to the implemented... To ensure that the cavitation effect is maintained at a level that preserves its effectiveness specific to the sample. transmitting sufficient force to rupture the cell wall of the aforementioned raw material and the solvent The aim is to provide access to the aforementioned active ingredient (DNJ). If the relevant power and time values ​​are exceeded, the bioactive components will degrade. its potential is increasing, there is a loss of the aforementioned active substance, and the method in question is... Its efficiency is decreasing. Furthermore, in order to structurally maintain the form of DNJ, which is a heat-sensitive component... Maintaining temperature control throughout the UAE is extremely important. Therefore, the aforementioned... During the process (UAE), the temperature is controlled / monitored, preferably via a thermostatically controlled bath. oo is being done; the temperature is preferably in the range of 45-50°C, and especially at 50°C. is being held. If the specified temperature values ​​and the duration of exposure to these temperatures are exceeded, DNJ stability decreases, leading to loss of active ingredient and reduced yield. is happening. The temperature parameters and time limitations specified within the scope of the invention method are specific. DNJ active ingredient is prone to degradation at high temperatures and long extraction times. It is specifically optimized to prevent the loss of the aforementioned active ingredient. They are defined as established conditions. In an alternative application of the invention, the UAE in question could use a conventional ultrasound device. This is carried out using pulse (intermittent) extraction, and the second extraction in question is preferably performed using pulse (intermittent) technology. It is performed in mode. The mentioned pulse (intermittent) mode is preferably in a 5-10 second on / 5-10 second off cycle. It is being implemented and serves as a means of improving thermal control. 12 In the preferred application of the invention, the product subjected to the aforementioned UAE process is i.e. In order to obtain the DNJ contained in the suspension, the product in question must preferably be... It needs to undergo a solid-liquid separation process. The solid-liquid separation process in question should preferably be carried out using centrifugation, filtration, or a centrifugation process. It is preferred as a combination of subsequent filtration processes. In the preferred application of the invention, the relevant solid-liquid separation process involves centrifugation and a combination of filtration processes, and especially the centrifugation process that follows It is preferred as a combination of filtration processes; the separation process in question is obtained This ensures the physical clarity of the resulting product and the ease of recovery of the pure phase. In the preferred application of the invention, the product in question is obtained as a result of the aforementioned UAE process. oo The temperature of the product being processed should preferably be in the range of 2 – 25 C or 4 – 25 C. Specifically, when the temperature reaches the range of 4 – 10 °C, it undergoes the aforementioned solid-liquid separation process. is being held. The centrifuge in question was used to separate the DNJ-rich supernatant. The process, in order to maintain the stability of the DNJ, preferably in the preferred application of the invention, should be 4 - In the temperature range of -25 °C and especially in the temperature range of 4 – 10 °C or 2 – 10 °C It is carried out within this period. Bringing the mentioned product to the specified temperature range constitutes the preferred method of the invention. In this application, the product in question should preferably undergo a cooling process or natural treatment. This is achieved by allowing it to cool down through various means. In the preferred application of the invention, the product subjected to the aforementioned UAE process After it cools down, the solid-liquid separation process is carried out. In the preferred application of the invention, the product subjected to the aforementioned UAE process is preferably... It is subjected to centrifugation, and then the centrifuged product is preferably... It undergoes a filtration process. The centrifugation process in question is preferably used to separate insoluble particles and collect the supernatant. This is carried out for the purpose of centrifugation, preferably for 5-25 minutes or 8- It takes minutes, and specifically 10 minutes. 13 In the preferred application of the invention, the centrifugal acceleration of the centrifugal process in question is preferably... It will be in the range of 6,000 – 12,000 g, 8,000 – 12,000 g, or 6,000 – 10,000 g. It is adjusted in this way. [6] The importance and function of this centrifugation process were determined by Sentís-Moré and colleagues. This has been investigated; the study in question revealed the efficiency of the relevant solid-liquid separation method. By doing so, low molecular weight DNJ / polyphenol fractions are obtained within the scope of the invention. It supports the approach towards its implementation. In the preferred application of the invention, the centrifugation of the mixture subjected to the UAE process is performed. After being subjected to centrifugation, the supernatant is preferably filtered. It is subjected to this process. The aforementioned filtration process preferably involves a filter media of 0.22 – 0.45 μm, and especially This is performed using a filter with a size of 0.22 μm; the filtering process mentioned here is as follows: If the resulting product is a liquid extract, it is defined as a DNJ extract. The parameters used in the aforementioned centrifugation and filtration processes are intended to preserve the DNJ content. optimized to prevent loss and ensure the removal of cellular particles. the solid-liquid separation of the suspension obtained after the aforementioned UAE process has been performed. It is designed to be carried out efficiently. The invention describes the application and optimization of the method steps shown in the method. Thanks to the parameters, the two energy-intensive steps (MAE and ) observed in the method described in the invention Despite sequential application (UAE), DNJ, a heat-sensitive active substance, was obtained. This method provides DNJ extraction yields of 90% and above. is reached. In this context, the method described in the invention involves only the first extraction and the second extraction. It should not be seen as a combination, but rather as a unique system that provides unpredictable performance specifically for DNJ. It should be classified as a method. Within this classification, the subject matter of the invention includes the method, raw material preparation, and the aforementioned Particle size determined within the scope of raw materials, solvent selection and ratio, temperature control, The type and sequence of process steps, the sequential transfer of energy on the same suspension. It is defined as a combination of various strategies, such as its application; the subject of the invention Thanks to the combination of these elements mentioned in the method, as stated above... 14 as a result, an unexpected increase in yield and purity of the active ingredient was achieved. is being done. As stated in the detailed description section where the invention is explained, the aforementioned The cellular structure of the raw material is weakened by the first extraction method mentioned, The mixture obtained in the first extraction mentioned is not subject to any intermediate process or solid-liquid mixture. It is sent directly to the second extraction process mentioned without undergoing any separation. Then, thanks to the aforementioned initial extraction process, the partially fragmented cell structure, In the second extraction method mentioned, direct exposure to cavitation effect. is left as is. Therefore, the cell breakdown obtained within the scope of the first extraction mentioned above. by combining the mechanical effect obtained within the scope of the second extraction mentioned with its effect DNJ can be obtained with high efficiency. The aforementioned extractions, carried out sequentially in a reactive medium, and the invention cover these processes. Thanks to the determined method parameters, it takes the same amount of time as traditional extraction methods. The method described in the invention, implemented within the scope of this study, achieves an efficiency of 90% or higher. The 55% yield level achieved with traditional extraction methods is exceeded. As mentioned above, the first example seen in the dual-stage energy-assisted method is the subject of this invention. Through extraction, the cell wall of the raw material is broken, followed by a second process. The yield obtained in the extraction process is increased, and the classical water extraction method seen in the literature is improved. The yield obtained with these extraction methods was exceeded when compared with these extraction methods. The method described in this invention achieves at least 1.5 times higher DNJ efficiency. The invention... This efficiency achieved within the scope of the invention relates to the amount of active substance obtained with the method described in the invention. This increase is within the scope of what was found; it is an achievement obtained in contrast to traditional extraction methods. It is defined as superiority. Thanks to the method implemented within the scope of the invention, especially the preferred method of the invention In this application, 98% of the aforementioned active ingredient is extracted and its purity is ensured. This is observed. In this context, the results obtained using traditional methods performed in the same time period... Considering the amount of the active ingredient mentioned above (55% extraction), As stated, the method described in the invention has superiority over the aforementioned traditional methods. It appears to have arrived. This efficiency increase is the result of optimized values ​​from a two-stage active energy-assisted approach. This stems from its use in a specific context; as mentioned in the first step. The raw material is processed by removing the moisture present within the cell, thus transforming the mentioned cell. While the first step involves detonation, the second step involves the fragmented cell structure. obtaining the aforementioned active substance by providing a mechanical effect through cavitation. This is ensured. 16 REFERENCES [1] Kimura, T., Nakagawa, K., Kubota, H., Kojima, Y., Goto, Y., Yamagishi, K., Oita, S., Oikawa, S., & Miyazawa, T. (2007). Food-grade mulberry powder enriched with 1- deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. Journal of Agricultural and Food Chemistry, 55(14), 5869–5874. https: / / doi.org / 10.1021 / jf062680g [2] Braga, R. M., & Kato, L. (2003). A formal total synthesis of deoxynojirimycin from D- glucitol Journal of the Brazilian Chemical Society, 14(5), 822–827. https: / / doi.org / 10.1590 / s0103-50532003000500019 [3] Iida , H. , Yamazaki , N. , & Kibayashi , C. (1987). Total synthesis of (+)-nojirimycin and (+)- 1-deoxynojirimycin. The Journal of Organic Chemistry, 52(15), 3337–3342. https: / / doi.org / 10.1021 / jo00391a031 [4] Wang , Jiajia , Zhao , Y. , Zhao , W. , Wang , P. , & Li , J. (2016). Total synthesis of n-butyl-1- deoxynojirimycin. Journal of Carbohydrate Chemistry, 35(8–9), 445–454. https: / / doi.org / 10.1080 / 07328303.2017.1330415 [5] Iftikhar , M. , Wang , L. , & Fang , Z. (2017). Synthesis of 1-deoxynojirimycin: Exploration of optimized conditions for reductive amidation and separation of Epimers. Journal of Chemical Research, 41(8), 460–464. https: / / doi.org / 10.3184 / 174751917x15000341607489 [6] Sentis-More , P. , Ortega-Olive , N. , Mas-Capdevila , A. , & Rosemary-Fabregat , M.-P. (2022). Impact of centrifugation and vacuum filtration step on yield and molecular weight distribution of protein hydrolysates from rapeseed and sunflower meals. LWT, 165, 113741. https: / / doi.org / 10.1016 / j.lwt.2022.113741 17

Claims

1. It is a DNJ extraction method, characterized by: i. The process of dissolution by treating a raw material with a solvent. to be carried out, ii. The resulting solution is subjected to microwave-assisted extraction at a constant temperature. subjection to iii. Ultrasound-assisted extraction of the mixture obtained as a result of microwave-assisted extraction subjected to a constant temperature during the extraction process, iv. Obtaining the liquid extract containing DNJ, It includes the steps of the process.

2. This is a method that conforms to Claim 1, and its characteristic is that the mentioned raw material is obtained from the mulberry plant. It can be derived from a morning glory plant, a Japanese bellflower plant, or a hyacinth plant.

3. A method that complies with claim 1 or 2, characterized by the fact that the mentioned raw material is fresh. or, after drying, adding it to the aforementioned solvent.

4. A suitable method that conforms to any of claims 1, 2 or 3, and whose characteristic is; the raw material, whether whole, crushed or ground, is added to the solvent mentioned. It is an addition.

5. A method that complies with any of the above requirements, and whose characteristic is; as mentioned After grinding the raw material to a size of 40-60 mesh, it is added to the aforementioned solvent. It is an addition.

6. This is a method that conforms to Claim 1, and its characteristic feature is that the solvent mentioned is ethanol / water.

7. A method that conforms to claim 6, characterized by the fact that the solvent in question is used in a ratio of 10–30 by volume. The ethanol that contains ethanol is water. 18 8. A method that conforms to any of claims 1, 6 or 7, and whose characteristic is; The raw material shall be mixed with the aforementioned solvent in such a way that the liquid / solid ratio is 5 – 100 : 1 (mL / g). It is an addition.

9. A method that complies with Claim 8, characterized by a liquid / solid ratio of 20–80 for the mentioned raw material. : 1 (mL / g) to be added to the mentioned solvent.

10. A method that complies with Claim 1, characterized by the fact that the mentioned solution operates at 0.915 – 2.45 GHz. subjected to microwave-assisted extraction process mentioned at microwave frequency It is an eclipse.

11. A method that conforms to either claim 1 or 10, and whose characteristic is that the solution in question... The aforementioned microwave-assisted extraction process is carried out within a power range of 300 – 800 W. It is an eclipse.

12. A method that conforms to any of claims 1, 10 or 11, and whose characteristic is; the solution to the aforementioned microwave-assisted extraction process in the power range of 560 – 640 W It is to be subjected to.

13. A method conforming to any of claims 10-12, the characteristic of which is that the mentioned solution The aforementioned microwave-assisted extraction process is applied for a period of 5-20 minutes. It is an eclipse.

14. A method conforming to any of claims 10–13, the characteristic of which is that the solution in question... The mentioned process involves subjecting the specimens to microwave-assisted extraction for 11 minutes.

15. A method conforming to any of claims 10–14, the characteristic of which is that the solution in question... he - Subjected to the aforementioned microwave-assisted extraction process at a temperature range of -80°C. It is an eclipse. he 16. This is a method that complies with claim 15, and its characteristic feature is that the mentioned solution is in the range of 70-80°C. This refers to subjecting the subject to a microwave-assisted extraction process. 19 17. This is a method that complies with Claim 1, and its characteristic feature is that it is obtained as a result of microwave-assisted extraction. he After the product is cooled to a maximum temperature range of 50°C, the aforementioned ultrasound... It is subjected to an assisted extraction process.

18. A method that complies with either claim 1 or 17, and is characterized by being microwave-assisted. The aforementioned mixture obtained as a result of extraction is in the power range of 120 – 500 W. This refers to the ultrasound-assisted extraction process.

19. A method that complies with any of claims 1, 17 or 18, and whose characteristic is microwave. The aforementioned mixture, obtained as a result of assisted extraction, is produced at a power of 180 W. This refers to the ultrasound-assisted extraction process.

20. A method that complies with any of claims 17–19, characterized by being microwave-assisted. The aforementioned mixture obtained as a result of the extraction is treated with the aforementioned ultrasound-assisted method. The extraction process involves subjecting the material to the test for a period ranging from 200 to 1,500 seconds.

21. A method that complies with any of claims 17-20, characterized by being microwave-assisted. The aforementioned mixture obtained as a result of the extraction is treated with the aforementioned ultrasound-assisted method. The extraction process involves subjecting the sample to a 260-second interval.

22. A method conforming to any of claims 17-21, the characteristic of which is that the mixture in question he The aforementioned ultrasound-assisted extraction process is subjected to a temperature range of 45-50°C. It is an eclipse.

23. A method suitable for any of the above requests, and its characteristic is; the aforementioned ultrasound Separation of solid-liquid from the product obtained as a result of assisted extraction. The process involves obtaining the aforementioned extract.

24. A method that complies with claim 23, and its characteristic is; the aforementioned ultrasound-assisted extraction. he After the resulting product cools to a temperature range of 2 – 25 C, the aforementioned It is subjected to a solid-liquid separation process.

25. A method that conforms to request 23 or 24, characterized by its ultrasound-assisted nature. he The product obtained as a result of extraction must be cooled to a temperature range of 4 – 10 C. then it undergoes the aforementioned solid-liquid separation process.

26. A method that conforms to either of claims 23-25, and whose characteristic is; centrifuge and / or The filtration process involves separating the solid and liquid components of the product in question.

27. A method that conforms to either of claims 23-25, and whose characteristic is centrifugation and filtration. The process involves separating the solid from the liquid of the product in question.

28. A method that complies with claim 27, and its characteristic feature is that after the aforementioned centrifugation process This involves carrying out the aforementioned filtration process.

29. A method suitable for either of the requests 26-28, characterized by the aforementioned ultrasound. The aforementioned product, obtained as a result of assisted extraction, has a weight range of 6,000 – 12,000 g. This involves subjecting the material to the centrifugation process mentioned, using centrifugal acceleration.

30. A method suitable for either of the requests 26-29, characterized by the aforementioned ultrasound. The aforementioned product, obtained as a result of assisted extraction, is left for 5-25 minutes. This refers to subjecting it to the centrifugation process.

31. A method that complies with either of points 26-28, and whose characteristic is the aforementioned filtering. The process is carried out using a filter with a size of 0.22 – 0.45 μm. 21