A Production Method for Extracts Obtained from Black Mulberry (Morus nigra) Fruit Through Controlled Extraction and Formulation that Shows Antiviral Activity Against SARS-CoV-2.
Patent Information
- Application Number
- TR202601801
- Authority / Receiving Office
- TR · TR
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-06
- Publication Date
- 2026-06-22
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Abstract
Description
1 TARIFF Obtained from Black Mulberry (Morus nigra) Fruit through Controlled Extraction and Formulation. Production Method of Extracts Showing Antiviral Activity Against SARS-CoV-2 Technical Area This invention enables the production of extracts obtained from the fruit of the black mulberry (Morus nigra) and these 5 The bioactive components found in the extracts have antiviral activity against SARS-CoV-2. This relates to controlled extraction and formulation methods that enable this demonstration. Previous Technique Black mulberry (Morus nigra) fruit has traditionally been used to support the immune system in folk medicine. It is widely preferred among them. However, this method of use is 10 against SARS-CoV-2. There is no direct evidence that it provides a specific antiviral effect, and immunity... preventing or eliminating viral infections by supporting the system alone This is not considered a sufficient approach from that perspective, because in traditional usage... Mulberry fruit; molasses, jam, marmalade obtained mostly through prolonged heat treatment, Because it is consumed in extract and syrup form or as dried fruit, 15 during these processes. The high temperatures and processing conditions applied affect the fruit's natural bioactive component structure. This leads to significant changes. Therefore, these products are generally not well-known among the public. It is used as an immune booster, especially in a direct and targeted manner. There is no known specific antiviral effect against SARS-CoV-2. Furthermore, conventional methods... No controlled extraction process is applied to mulberry fruit in use. 20 And the quantity consumed cannot be standardized. This situation makes it impossible to determine what quantity of fruit or product is consumed. This creates uncertainty regarding whether its consumption can have a biological effect and potential antiviral properties. This makes it difficult to reliably evaluate the event. Therefore, traditional methods of use, extract composition, effective dosage range and antiviral activity are controlled. It is not standardized with scientific data. 25 Rubus species are included in publication WO / 2022 / 060304 in the known state of the art. Herbal compositions containing Artemisia annua have been identified. However, this document does not include black mulberry (Morus Targeted extraction processes of (nigra) fruit extracts and SARS-CoV-2 specific No method for producing it for antiviral purposes has been described. In the document by Thabti et al. (2020), extracts obtained from Morus spp. species were found to be effective against coronavirus 30 Its antiviral effects on viruses such as [name of virus] have been studied, and antiviral activity has been reported. 2 However, the extraction methods used are general in nature, and the technical optimization of the extract is not essential. It does not offer an approach to addressing this. Furthermore, the study does not directly address SARS-CoV-2. It is not unique. Document WO 2021 / 044448 A1 describes an oral contraceptive containing black mulberry (Morus nigra) fruit extract. The washing solution has been identified and the extract in question has antibacterial and antiviral effects. 5 It has been stated that this can be demonstrated. However, the document does not contain verifiable information regarding extract production. There are no parameters or systematic optimization approach involved. However, a cell culture-based antiviral evaluation against SARS-CoV-2. It does not include. Document CN 111298034 A describes 10 plants, including mulberry leaves. The effects of the compounds on various viruses, including SARS-CoV-2, have been studied, and they are antiviral. It has been stated that activity can be demonstrated. However, the document does not mention checking the extract composition or A feasible production method for determining the dose-response relationship. It is not defined. Furthermore, the study is not specific to mulberry fruit and is a multi-component plant study. It is based on mixtures. 15 In the document by Tsai et al. (2021), the antiviral effects of various herbal mixtures were examined. These compounds have been shown to be effective against coronaviruses. However, a solution for controlling or technically optimizing extraction processes It is not presented. Furthermore, the study is neither specific to SARS-CoV-2 nor based on mulberry fruit. The document by Kwong et al. (2020) provides a general overview of herbal approaches used against SARS-CoV-2. It addresses this issue in detail but does not offer a technical production method. Furthermore, it does not involve a specific application based on black mulberry (Morus nigra) fruit. The study published in the Egyptian Journal of Chemistry (2025) on Morus nigra It appears that their extracts have antiviral potential against SARS-CoV-2. However, the study in question was obtained by the applicant previously (October 2022) 25 While generally consistent with experimental findings, controlled extraction determination of parameters, bioactive content control, extract stabilization and cytotoxicity A systematic approach based on the combined evaluation of (CC50) and antiviral efficacy (IC50) data. It does not offer a production approach. Furthermore, in previous studies conducted by the applicant, it was shown that mulberry fruit has a 30% yield. After undergoing glass bead homogenization and mechanical crushing processes, it is thermally treated. 3 The antiviral effect of formulations obtained by processing (boiling) has been evaluated. and with this approach, the level of antiviral inhibition achieved is approximately 25%. It has been determined that this situation is based solely on the homogenization and heat treatment steps. traditional preparation methods have limited capabilities in increasing antiviral efficacy, and The subject matter reveals that the event was predictable within the current technical framework. 5 It places. In this context, academic studies, patent documents and the applicant's When preliminary studies are evaluated together, the presence of antiviral activity is generally considered. It is understood that this is known in this sense. However, this activity is achieved with controlled extraction parameters. increasing the content level of bioactive components, controlling the content level of extracts, stabilizing the extracts 10 and combined evaluation of cytotoxicity (CC50) and antiviral efficacy (IC50) data. a systematic technique for determining biocompatible and effective dose ranges It appears that no solution has been found. Therefore, the applicant sought to enhance antiviral efficacy and optimize biocompatible dose ranges. In order to achieve this, it is possible to preserve and selectively obtain bioactive components. They have developed a different and systematic technical approach based on controlled extraction. The application in question seeks to achieve low cytotoxicity by optimizing the parameters together. This enables the production of extracts that possess and exhibit high antiviral activity. in terms of not being directly derived from existing technology and for experts in the technical field A unique technical solution is offered that is clearly unpredictable. Furthermore, the developed production 20 The approach ensures that the resulting extracts are stable, reproducible, and applicable on an industrial scale. by ensuring its quality, pharmaceutical, biotechnological and functional product development It is applicable in these fields and offers a significant technical advantage. The Main Purpose of the Invention The main objective of this invention is to achieve controlled 25-month processing of extracts obtained from black mulberry (Morus nigra) fruit. Bioactive compounds that exhibit antiviral activity by enabling their production through extraction methods. The aim is to protect the components and increase their effectiveness. The invention also includes the bioactive components found within the said extracts. It exhibits antiviral effects against SARS-CoV-2, enabling it to suppress viral replication. The goal is to develop a controlled and repeatable production method. 30 4 However, naturally derived antiviral compositions are stable, standardizable, and extraction and that makes it possible to obtain it in a way that can be applied on an industrial scale. The goal is to present formulation processes. In addition, by preventing the loss of biological activity in the production process of plant-derived extracts. The goal is to enable the development of reliable and highly effective antiviral products. 5 In this context, based on bioactive components derived from mulberry fruit, existing techniques A natural product with enhanced antiviral efficacy through controlled production and dose determination approach, not involved in the production process. Our goal is to offer solutions. Technical Advantages Provided by the Invention Thanks to the controlled extraction methods developed within the scope of the invention, black mulberry (Morus 10 The degradation of bioactive components found in the fruit of the Nigra (Acetra Nigra) is prevented during the production process, and This ensures the high-yield production of active substances exhibiting antiviral activity. The developed method allows for the control of extraction parameters, thus surpassing existing methods. from simple infusion or random solvent use approaches included in the techniques In contrast, it offers the possibility of repeatable, standardizable and controllable production. 15 It offers. Extracts obtained by optimizing both extraction and formulation processes Stability is increased and the loss of biological activity is minimized. Furthermore, the invention includes the combined analysis of cytotoxicity (CC50) and antiviral efficacy (IC50) data. By evaluating it, 20 that preserve cellular biocompatibility and maximize antiviral efficacy This makes it possible to determine the optimal dose ranges. In this respect, the invention is not limited to demonstrating antiviral activity, but also includes the aforementioned a holistic technique that enables the controlled, measurable, and systematic increase of effectiveness It offers an approach. The production method in question has been successfully implemented on a laboratory scale, and 25 were used. Extraction and concentration steps can be scaled up with industrial equipment. It is of a qualitative nature. Thanks to this, the method is used in pharmaceutical preparations, herbal antiviral products, and functional drugs. It provides an infrastructure suitable for the mass production of food ingredients. In conclusion, the invention relates to the use of mulberry fruit-derived extracts for antiviral purposes. compared to existing techniques in terms of efficiency, reliability and industrial applicability It offers significant technical advantages. Brief Description of the Invention The replication of the SARS-CoV-2 virus, infection carried out in cell culture medium 5 It is related to the processes. Within the scope of the invention subject to the application, from the black mulberry (Morus nigra) fruit The resulting extracts are prepared under controlled extraction parameters, and the obtained The antiviral efficacy of the extracts is being evaluated in cell culture systems. Extracts were administered at different concentration ranges to test cytotoxicity (CC50) and antiviral properties. The efficacy (IC50) values are determined; by evaluating these data together, 10 biocompatible species are identified. and effective dosage ranges are determined. This method allows viral replication to be inhibited while preserving the cellular biocompatibility of the extracts. Concentration ranges that exert a suppressive effect are determined, and antiviral activity is assessed. This is demonstrated in a controlled, measurable, and repeatable manner. FIGURES 15 I. Production and Extraction Process Figure 1. Flowchart of the black mulberry extract production process. II. In Vitro Cytotoxicity Analyses Figure 2. Cell viability levels (low, medium, high, and very high) from the cytotoxicity test. Schematic diagram showing the classification 20 Figure 3. Experimental view of the cell culture plate for the cytotoxicity test. Figure 4. Cell culture plate layout diagram for the cytotoxicity test. Figure 5. Cell viability (%) curve of log10 concentration of mulberry extract #1. Figure 6. Cell viability (%) curve of log10 concentration of mulberry extract #2. Figure 7. Cell viability (%) curve as a function of log10 concentration of mulberry extract no. 3. Figure 8. Cell viability (%) curve of log10 concentration of mulberry extract #4. 6 Figure 9. Cell viability (%) curve of log10 concentration of mulberry extract #5. III. In Vitro Antiviral Efficacy Analyses Figure 10. Experimental view of the cell culture plate for the antiviral inhibition test (1,2 and 3). (Examples numbered) Figure 11. Cell culture plate layout diagram for antiviral inhibition test 5 Figure 12. Log10 concentration-dependent antiviral inhibition (%) curve of extract #1. Figure 13. Log10 concentration-dependent antiviral inhibition (%) curve of extract #2. Figure 14. Log10 concentration-dependent antiviral inhibition (%) curve of extract #3. Figure 15. Experimental view of the cell culture plate for the antiviral inhibition test (4 and 5). (Examples numbered) 10 Figure 16. Cell culture plate layout diagram for samples 4 and 5. Figure 17. Log10 concentration-dependent antiviral inhibition (%) curve of extract #4. Figure 18. Log10 concentration-dependent antiviral inhibition (%) curve of extract #5. Explanations of the symbols used in the figures: 1. Low cell viability (high cytotoxicity / significant cell damage) 15 2. Moderate cell viability (partial cellular involvement) 3. High cell viability (low cytotoxicity / biocompatible range) 4. Very high cell viability (maximum biocompatibility / cellular integrity) (protection) 5. Peak of maximum cell viability (optimum biocompatibility region) 20 6. Maximum antiviral inhibition zone (range of activity at which significant suppression occurs) 7. Cytotoxicity cell culture plate dated 10.10.2022. 8. Antiviral cell culture plate dated 15.10.2022. Detailed Description of the Invention This invention was developed using controlled extraction methods from black mulberry (Morus nigra) fruit. the production of the obtained extracts and the bioactive substances contained within those extracts 7 the components are formulated to exhibit antiviral activity against SARS-CoV-2 It relates to a production method aimed at... The method developed within the scope of the invention ensures the preservation of bioactive components during the production process. providing a highly effective, stable, reproducible and biocompatible antiviral extract. This makes it possible to obtain their compositions. 5 The defined extraction method includes the type of solvent used, extraction time, temperature, and It is based on the controlled regulation of parameters such as operating conditions, Efficient extraction of bioactive components through optimization of parameters. and this helps to increase antiviral efficacy. This method involves 10 different organic solvents, extraction times, and temperature ranges. While feasible, these variations make it difficult to obtain the bioactive components and under control in a way that will not prevent the demonstration of antiviral efficacy. is being carried out. Industrial Applicability The production of black mulberry (Morus nigra) fruit extracts developed within the scope of this invention took 15 years. Controlled extraction method for the food, pharmaceutical and cosmetic industries. It is feasible. The method includes lyophilization, extraction with an organic solvent, filtration, and solvent removal. It includes steps that can be applied on an industrial scale to existing production lines. It can be easily integrated. 20 The obtained extracts are used in the development of products exhibiting antiviral activity, and as supplements. in foods, functional beverages, pharmaceutical preparations and topical formulations available. Furthermore, the extract's low cytotoxicity and high antiviral efficacy indicate that the product is safe. This supports its suitability for industrial use. 25 In this respect, the invention is a method suitable for repeatable, scalable, and economical production. It offers. 8 Production Process of Extract Obtained from Black Mulberry Fruit The black mulberry (Morus nigra) fruit samples used in the invention were taken from different parts of Türkiye. They are sourced from geographical regions, and measures are taken to prevent the degradation of bioactive components. For this purpose, it is stored under low temperature conditions. Prior to extraction, fruit samples weighed in specified quantities were placed in sealed containers. 5 It undergoes lyophilization (freeze-drying) and this process reduces its water content. By removing these components, the stability of the bioactive ingredients is increased. The dry material obtained after the lyophilization process is appropriately ground to create a homogeneous mixture. is prepared and then, under controlled conditions, the selected organic solvent is added. The extraction process is being carried out. 10 The extraction process takes place in a light-protected environment and under specific time and temperature parameters. This is carried out under the supervision of a solvent, and in this way, bioactive components exhibiting antiviral activity are processed. The transition to the next phase is effectively ensured. Yield values of the obtained extracts were determined by lyophilization and mass extraction. These calculations take into account the changes, and the results regarding these values are given below in 15 This is presented in the table. Table 1. Comparison of yields at equal starting amounts in the mulberry extract production process. Example Number Province, District, Village Lyophilization After Relative Remaining Amount Extraction Relative Afterwards Yield Number 1 example Mersin-Mut- Çamlıca Intermediate level Intermediate level Number 2 example Ankara-Akyurt- Bugduz Low level Very low level Number 3 example Elazığ-Harput High level Very high level Number 4 example Izmir-Tire- Acrobat High level Intermediate level number example Hatay-Belen- Coal pit High level Intermediate level 9 The differences in yield obtained are decisive in determining the biological efficacy of the extracts. this could be the case and highlight the technical importance of controlling the extraction parameters. It is considered that he placed it there. After the extraction is complete, the resulting mixture is passed through fine-mesh filters. Solid-liquid separation is achieved by filtration. The solvent phase in the resulting filtrate is 5 It is removed under controlled temperature and rotation conditions using a rotary evaporator, and This process results in a concentrated extract. The obtained extracts are placed in sealed containers for storage under appropriate conditions, and It is then used in antiviral analyses. Thanks to this method, the bioactive components found in mulberry fruit are obtained with high yield. This ensures that structural defects that may occur during the production process are minimized and stable. This enables the preparation of reproducible antiviral extract compositions. Phytochemical (HPLC) Analysis Process The phytochemical properties of black mulberry (Morus nigra) fruit extracts obtained within the scope of this invention. Its composition was analyzed using high-performance liquid chromatography (HPLC). 15 The analyses were performed using an HPLC system with a diode array detector (DAD) and a reverse-phase C18 column. Efficient separation of bioactive components was achieved using this method. The mobile phase system consists of an aqueous phase and an organic solvent phase, with flow rate and column configuration. High resolution accuracy was achieved by adjusting the temperature under controlled conditions. Analyses The identification of compounds was carried out at specified wavelengths and reference standards 20 This was done comparatively. During sample preparation, specific amounts are taken from the extracts and mixed with a suitable solvent. Dissolved in the solution, filtered through fine-mesh filters, suitable for chromatographic analysis. The samples were prepared and analyzed at a constant concentration, and each sample was... Quantitative accuracy was ensured by measuring in multiple replicates. 25 This analysis method allows us to identify the phenolic compounds found in mulberry extracts and Organic acids were reliably and reproducibly determined, and the resulting phytochemical profile was obtained. This has been evaluated in conjunction with antiviral efficacy data. HPLC method of black mulberry (Morus nigra L.) fruit extracts obtained from five different regions The phenolic compound and organic acid contents (mg / g) determined are given in the table below. 30 It is presented. Table 2. Determination of Morus nigra L. extracts obtained from five different regions by HPLC. Phenolic compound and organic acid content (mg / g) Compound Mersin Mut (mg / g) Ankara Akyurt (mg / g) Elazığ Harput (mg / g) Izmir Tire (mg / g) Hatay Belen (mg / g) Ascorbic acid 0 1.21 0 0 0 Caffeic acid 0.07 0.05 0.05 0.03 0.09 Catechinhydra te 0 0 0 0.675 0.095 Catechin 1.88 0 0 0 0 Citric acid 59.27 64.75 79.60 119.80 104.60 Coumarin 0.04 0.145 0.095 0 0 Ellagic acid 0.18 0.54 0.11 0.08 0 Ferulic acid 0,555 1,5525 0,3275 0 0 Fumaric acid 0,15 0,05 0,01 0,02 0,03 Gallic acid 1,1325 2,5825 0,75 0 0 Gentisic acid 0 0,1475 0 0 0 Hydroxybenzo ic acid 0,0275 0,0275 0,0875 0 0 Isocitric acid 10,20 11,05 13,61 20,52 18,45 Isoquercitrin 0,34 1,55 0,13 0,05 0,11 Chlorogenic acid 0 0 0 1,3225 0 Malic acid 64,90 34,25 41,17 48,92 35,42 Naringin 0,035 0,2325 0,225 0 0,045 Neohesperidin 0,11 0 0 0 0 11 o-Coumaric acid 0,175 0,025 0,1025 0 0 p-Coumaric acid 0 0,09 0 0 0,13 Propionic acid 1,87 0,83 ND 0,62 ND Protocatechuic acid 0,0475 0,035 0,0475 0 0 Quercetin 0 0,0525 0 0,13 0,12 Quinic acid 114.10 60.40 0.72 171.85 65.02 Resveratrol 0 0.04 0 0 0.14 Rosmarinic acid 0 0 0 0 0.1025 Routine 0 0.05 0 0 0.1775 Salicylic acid 0.05 0.05 0 0 0 Shikimic acid 0.14 0.11 0.06 0.12 0.12 Succinic acid 14.77 11.85 6.97 12.60 9.75 Tannic acid 14.79 5.93 13.95 12.61 9.75 trance- Cinnamic acid 0 0.03 0 0.21 0 trans-Ferulic acid 0 0 0 0.08 0.08 trance- Cinnamic acid 0 0.0675 0 0 0 Vanillic acid 0.0925 0 0 0 0 Vanillin 0 0 0 0.175 0.0675 Evaluation of Phytochemical Analysis Results This table shows the phenolic content of Morus nigra L. fruit samples obtained from different geographical regions. It enables the quantitative comparison of compound and organic acid compositions. The data obtained show regional differences in the bioactive component content of mulberry extracts. 5 It demonstrates that it is able to show this. 12 In particular, organic acids such as citric acid, malic acid, isocitric acid, and quinic acid were present in all samples. found in high concentrations, whereas phenolic compounds are among the examples It has been determined that there is variability. In the İzmir (Tire) sample, quinic acid (171.85 mg / g) and citric acid... Components such as acid (119.80 mg / g) in higher concentrations compared to other regions The fact that this particular sample stands out in terms of antiviral efficacy is one of the five reasons for its existence. It is considered a determining factor. The data obtained in this context show that the antiviral efficacy of mulberry extracts is only present in the body. It turns out that it is not only based on content but is also directly related to content composition. and certain bioactive components play a critical role in antiviral activity. This shows that he can play. 10 The presented HPLC data reveal the content profile of the production method developed within the scope of the invention. It has been shown that it enables the production of extracts whose biological activity has been identified and supported. and that the method in question is controlled, unlike random herbal mixtures, It technically offers a repeatable and standardizable production approach. It supports. 15 The phytochemical content profile obtained in this context is specific to the extracts subject to the invention. It forms a chemical fingerprint and exhibits antiviral activity. analytical characterization of extracts, ensuring quality control and similar processes. This makes it possible to distinguish it from other products. In this respect, HPLC data not only provide analytical characterization, but also the same 20 By revealing the relationship between antiviral activity and chemical composition, the technical aspects of the invention were also discussed. It directly supports its effect. In Vitro Cytotoxicity Analyses The cytotoxic effects of black mulberry (Morus nigra L.) based extracts developed within the scope of this invention, The results were evaluated using the MTT colorimetric method in cell culture medium. 25 In the study; Mersin (sample 1), Ankara (sample 2), Elazığ (sample 3), İzmir (sample 4) A total of five different extracts were examined, including (sample no. 5) and Hatay (sample no. 5). Test substances were applied to cell culture medium at different concentrations, and incubation was performed. At the end of the period, cellular metabolic activity was determined by the MTT method using an ELISA reader. The measured absorbance values were compared with cell control groups for each concentration (30). Percentage of cell viability has been calculated. 13 Cell viability percentage is the absorbance obtained from the wells to which the tested extract was applied. by comparing the absorbance values of the cells to the absorbance values of the control group containing only cells The obtained values are expressed as percentages, and the cytotoxic effect of the extract is calculated accordingly. It was evaluated based on values. Cell Viability (%) = Example A A cell control × 100 5 The concentration at which the extract causes 50% cell viability (CC50), dose-response curve. This was determined using logarithmic concentrations of cell viability. plotted against the values and corresponding to 50% vitality on the curve The concentration value has been calculated. Based on the data obtained, non-cytotoxic concentration ranges were determined and 10 The cytotoxicity profiles of the relevant extracts have been determined. In this context, each extract... Half-maximum cytotoxic concentration (CC50) values have been calculated for antiviral Only biocompatible dose ranges were used in the analyses. Cell viability classification based on cytotoxicity assessments is shown in Figure 2, for experimental cells. The culture plate layout is presented in Figure 3 and the plate arrangement in Figure 4. 15 The graphs show the change in cell viability (%) depending on the log10 concentration of the extracts. This information is given in Figures 5–9. Cell culture plate images and equipment obtained at the end of the experimental incubation process. The results are presented visually to support the verifiability of the analyses. This In this context, the cell culture plate image from the cytotoxicity experiment dated 10.10.2022 is related to point 20. The figures are shown as experimental references. Additionally, ELISA for cytotoxicity testing is also included. Reader raw absorbance (OD) values are presented in Table 3. 14 Table 3. ELISA reader crude absorbance (OD) values for cytotoxicity test. Dose Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Cell control 1 / 1 1.0068 1.1794 0.3118 0.3229 1.2232 1.2312 1.1545 1.0735 1.1045 1.0630 1.1667 1.0704 1 / 2 1.2391 1.2818 0.2029 0.1028 1.1576 1.2634 1.1420 1.1273 1.1899 1.2195 1.1442 1.1333 1 / 4 1.4056 1.3411 0.9595 0.9551 1.2036 1.1957 1.2089 1.1677 1.2372 1.2626 1.1734 1.1462 1 / 8 1.3791 1.2946 1.1025 1.0713 1.2280 1.2587 1.1569 1.2374 1.2317 1.2635 1.1422 1.2006 1 / 16 1.3167 1.2872 1.2762 1.2790 1.2124 1.2362 1.1805 1.2686 1.2706 1.2307 1.1807 1.2270 1 / 32 1.2386 1.2418 1.2620 1.3495 1.1950 1.2201 1.2035 1.2319 1.2051 1.2057 1.1725 1.2238 1 / 64 1.1809 1.1981 1.3103 1.3051 1.2167 1.1895 1.2348 1.2594 1.2557 1.2937 1.2457 1.2148 1 / 128 0.9606 1.0803 1.1602 1.1775 1.0788 1.1401 1.1398 1.0370 1.1701 1.1293 1.1841 1.0825 The data presented in this table relate to technical repeat measurements performed for each extract. It shows the average optical density (OD) values. These data are used for cell control. Used in percentage cell viability calculations by comparison with the group. 5 15 Table 5. Average optical density (OD) values for cytotoxicity test. Dose 1 example Number 2 example Number 3 example Number 4 example number example Cell control 1 / 1 1.0931 0.3174 1.2272 1.1140 1.0838 1.1186 1 / 2 1.2605 0.1529 1.2105 1.1347 1.2047 1.1388 1 / 4 1.3734 0.9573 1.1997 1.1883 1.2499 1.1598 1 / 8 1.3369 1.0869 1.2434 1.1972 1.2476 1.1714 1 / 16 1.3020 1.2776 1.2243 1.2246 1.2507 1.2039 1 / 32 1.2402 1.3058 1.2076 1.2177 1.2054 1.1982 1 / 64 1.1895 1.3077 1.2031 1.2471 1.2747 1.2303 1 / 128 1.0205 1.1689 1.1095 1.0884 1.1497 1.1333 Average values are obtained from at least two technical replicate measurements performed for each sample. It was calculated by taking the arithmetic mean. The obtained raw absorbance data were normalized by comparing them with cell control values. This has been done to ensure that the effects of the extracts on cellular metabolic activity are reliable. The data have been evaluated in a reproducible manner. The data show that some, especially at high concentrations... Although a decrease in cell viability was observed in the extracts, overall a wide range of This indicates that biocompatibility is maintained within the concentration range. The dilution scheme and plate layout used in cytotoxicity analyses are shown in Figure 4. This is shown. The dose-concentration relationship used in this context is given in Table 6. 16 Table 6. Dose dilution plan and concentration equivalents. Plate Number Dose Concentration Log10 Concentration (µg / mL) 1 1 / 1 8 0.903 1000 2 1 / 2 4 0.602 500 3 1 / 4 2 0.301 250 4 1 / 8 1 0 125 1 / 16 0.5 -0.301 62.5 6 1 / 32 0.25 -0.602 31.25 7 1 / 64 0.125 -0.903 15.625 8 1 / 128 0.0625 -1.204 7.8125 Cytotoxicity profiles and calculated half-maximum values of different mulberry extract samples. Cytotoxic concentration (CC50) values are presented in Table 7. Table 7. Cytotoxicity (CC50) assessment 5 Example CC50 Cytotoxic Interval Safe Interval Mersin > 8 (not determined in the test range) No All doses Ankara ≈ 3–4 µg / mL ≥4 µg / Ml ≤2 µg / mL Elazığ > 8 (not determined in the test range) None All doses Izmir > 8 (not determined within the test range) No All doses Hatay > 8 (not determined in the test range) No All doses The results obtained show that the extracts, except for the Ankara sample, were within the tested concentration range. It does not show a significant cytotoxic effect and has a wide range of biocompatibility. This reveals the following. In the Ankara example, however, cells above certain concentrations... A decrease in viability was observed, indicating that the cytotoxic threshold value for the extract in question was exceeded. This shows that it is lower. 10 17 In this context, when determining the concentration ranges to be used in antiviral analyses, only Biocompatible dose levels have been considered, thus minimizing cytotoxicity-related effects of antiviral drugs. The ability to influence the results has been prevented. Concentration-dependent changes in cell viability (%) and log10 of different mulberry extracts. Concentration-dependent dose-response curves are shown in Figures 5–9, and the data in question are presented in 5. The numerical results are presented in Table 8. Table 8. Cell viability (%) results. Concentration Log10 No. 1 example Myrtle Number 2 example Ankara Number 3 example Elazığ Number 4 example Izmir number example Hatay 8 0.903 93.48 27.14 100 95.27 92.68 4 0.602 100 13.07 100 97.04 100 2 0.301 100 81.87 100 100 100 1 0 100 92.95 100 100 100 0.5 -0.301 100 100 100 100 100 0.25 -0.602 100 100 100 100 100 0.125 -0.903 100 100 100 100 100 0.0625 -1.204 87.27 99.96 94.88 93.08 98.32 The results obtained show that black mulberry extracts are effective against cellular damage over a wide concentration range. that it maintains its biocompatibility and that cytotoxic effects generally remain at a limited level. 10 It shows. In particular, the concentration ranges tested for extracts 1, 3, 4 and 5 were large. In the majority of these extracts, high levels of cell viability (≥90%) are maintained, indicating low levels of cell viability in these extracts. It reveals that it has a cytotoxicity profile. In contrast, extract number 2... At high concentrations of this substance, a significant decrease in cell viability was observed, and this situation is mentioned in 15 This indicates that there is a narrower biocompatible dose range for the extract. 18 Based on these findings, only non-cytotoxic agents should be considered in antiviral efficacy assessments. Concentration ranges were taken into account, thus ensuring that the antiviral effect is balanced with cellular toxicity. This has ensured that it is presented independently. This approach ensures the biological efficacy of the extracts developed within the scope of this invention at a safe dose. It allows optimization within these ranges, and cytotoxicity (CC50) and antiviral 5 a systematic and repeatable approach based on the combined evaluation of activity (IC50) data It presents an analysis method. In Vitro Antiviral Inhibition Analyses The invention involves the development of black mulberry (Morus nigra L.) based extracts against SARS-CoV-2. Antiviral effects were evaluated in vitro in cell culture medium. 10 In this context, those deemed suitable for use in antiviral tests based on cytotoxicity analysis results. Mersin (sample 1), Ankara (sample 2), Elazığ (sample 3), İzmir (sample 4) and A total of five different extracts, including Hatay (sample number 5), were examined comparatively. Test agents were applied to cells infected with SARS-CoV-2 at different dilutions, and The effect of viral replication on cell viability at the end of the incubation period MTT colorimetric 15 This was determined using the method. The obtained absorbance values were analyzed using virus control and cell control. Antiviral inhibition levels were calculated by comparing the groups. Antiviral efficacy was determined through testing. In order to determine the protective effect of the extract on virus-infected cells The inhibition percentage was calculated as the ratio of the absorbance value of the sample group to the virus control and This was obtained by normalizing the cells relative to control groups. In this context, antiviral 20 The inhibition percentage was calculated using the following equation. Inhibition (%) = (𝐴example−𝐴virus control) (A cell control - A virus control) × 100 Considering the biocompatible concentration ranges determined as a result of cytotoxicity analyses Only safe doses were used in antiviral tests. In this context, each extract... The highest non-cytotoxic concentration for antiviral assays starting dose (1 / 1) 25 This was accepted as the standard, and subsequently, series dilutions (1 / 2, 1 / 4, 1 / 8, etc.) were applied. For example, in the cytotoxicity analysis of extract number 2, cell viability was found to be at an acceptable level. It was determined that the highest concentration at which it was preserved was obtained at a 1 / 8 dilution, and this dose In antiviral assays, the initial concentration was accepted as (1 / 1). Similarly 19 Dose dilution plan based on biocompatible dose ranges for other extracts as well. It has been created. This approach allows for the evaluation of antiviral efficacy independently of cytotoxic efficacy. This has been ensured and it has been possible to demonstrate biological efficacy within safe dose ranges. It is well-known. 5 Cell culture plate images from antiviral inhibition experiments are shown in Figures 10 and 15. The plate layout arrangements are shown in Figures 11 and 16. The log10 of the extracts Dose-response curves showing the concentration-dependent change in antiviral inhibition (%) are shown in Figure 1. This is presented in Figures 12–14 and 17–18. In this context, cell line, virus application conditions, incubation period, dose interval and control 10 By standardizing the groups, comparable experimental conditions were ensured for all samples. Experimental accuracy and reliability of results are assessed using positive and negative control groups. It has been supported. This method combines cytotoxicity (CC50) and antiviral efficacy (IC50) data. It offers a systematic dose optimization approach based on evaluation, antiviral 15 ensuring that the efficacy is maximized within safe and biocompatible concentration ranges. It creates a unique technical solution. Dose dilution schedule and corresponding concentration used in antiviral inhibition testing. The values are given in Table 9. 25 Table 9. Dose dilution plan used in antiviral inhibition testing. Plate Number Dose Concentration Log10 Concentration (µg / mL) 1 1 / 1 8 0.903 1000 2 ½ 4 0.602 500 3 ¼ 2 0.301 250 4 1 / 8 1 0 125 1 / 16 0.5 -0.301 62.5 6 1 / 32 0.25 -0.602 31.25 7 1 / 64 0.125 -0.903 15.625 8 1 / 128 0.0625 -1.204 7.8125 Dose distribution and layout of a cell culture plate treated with SARS-CoV-2. This is shown schematically in Figure 11. According to this layout, different extracts... The samples were evaluated in multiple replicates; also, virus control and cell control groups were included. They were located in separate wells. Antiviral inhibition of samples 1, 2 and 3 is 5 Cell culture plate images related to the tests are shown in Figure 10 as experimental results. It is presented. 15 21 Table 10. Raw absorbance values of samples 1, 2 and 3 for antiviral inhibition test. Dose Sample 1 Sample 2 Sample 3 Virus control Cell control 1 / 1 0.5147 0.6111 0.5443 1.1683 1.2258 1.2253 0.5244 0.5443 0.4650 0.38530 0.8503 0.7667 1 / 2 0.5169 0.5368 0.4990 0.7293 0.8464 0.8565 0.4883 0.5029 0.4756 0.3497 0.8381 0.8800 1 / 4 0.4767 0.5407 0.4637 0.6567 0.6168 0.6290 0.4921 0.4677 0.4898 0.3540 0.8351 0.8956 1 / 8 0.4891 0.4889 0.4878 0.5177 0.5475 0.5528 0.5040 0.4781 0.4600 0.3637 0.8736 0.8899 1 / 16 0.5119 0.5298 0.5079 0.5130 0.5101 0.5116 0.4666 0.4934 0.4637 0.3609 0.8905 0.9320 1 / 32 0.5318 0.5654 0.4920 0.5095 0.5144 0.5055 0.4857 0.4767 0.4595 0.3815 0.9093 0.9780 1 / 64 0.5303 0.5479 0.4947 0.5490 0.5486 0.5095 0.5112 0.5169 0.4413 0.3908 0.9440 0.9610 1 / 128 0.5479 0.5720 0.5246 0.5778 0.5325 0.5407 0.5184 0.5296 0.5113 0.4354 0.9973 0.8611 9 Table 11 shows that an increase in cellular metabolic activity was observed at some concentrations. Table 11. Mean (MD) values of antiviral inhibition tests for samples 1, 2, and 3. Dose Sample 1 Sample 2 Sample 3 Virus control Cell control 1 / 1 0.5567 1.2065 0.5113 0.3853 0.8086 1 / 2 0.5176 0.8107 0.4889 0.3497 0.8591 1 / 4 0.4938 0.6342 0.4832 0.3541 0.8654 1 / 8 0.4886 0.5394 0.4807 0.3637 0.8818 1 / 16 0.5165 0.5116 0.4746 0.3609 0.9113 1 / 32 0.5298 0.5098 0.4740 0.3816 0.9437 1 / 64 0.5244 0.5357 0.4898 0.3908 0.9525 1 / 128 0.5482 0.5504 0.5198 0.4354 0.9293 22 Table 12. Raw absorbance values of samples 4 and 5 from the antiviral inhibition test. Dose Sample 4 Sample 5 Control group Virus control Cell control 1 / 1 0.5821 0.586 0.5998 0.5633 0.5275 0.5583 0.6939 0.6201 0.5893 0.2989 0.8886 0.774 ½ 0.5768 0.5453 0.5482 0.5458 0.5201 0.4698 0.7172 0.7218 0.6405 0.2974 0.7958 0.8473 ¼ 0.5775 0.5579 0.5283 0.5102 0.504 0.4951 0.8189 0.839 0.7912 0.3376 0.8921 0.8619 1 / 8 0.5804 0.5281 0.4826 0.5196 0.4962 0.4932 0.6507 0.6524 0.5748 0.3522 0.7065 0.9302 1 / 16 0.5437 0.5002 0.4516 0.5065 0.4663 0.4531 0.5236 0.5312 0.4635 0.4089 0.8688 0.9235 1 / 32 0.5787 0.5123 0.4798 0.4988 0.519 0.4687 0.5164 0.4855 0.4804 0.3812 1.0067 1.0541 1 / 64 0.5846 0.5359 0.5461 0.5201 0.4932 0.5222 0.4787 0.5284 0.4765 0.4208 0.9529 0.9527 1 / 128 0.6747 0.5639 0.6019 0.5681 0.5606 0.5671 0.5986 0.5192 0.5371 0.4399 0.831 0.7439 Table 13. Mean (MD) values of antiviral inhibition tests for samples 4 and 5. Dose Sample 4 Sample 5 Control group Virus control Cell control 1 / 1 0.5893 0.5497 0.6344 0.2989 0.8313 1 / 2 0.5568 0.5119 0.6932 0.2974 0.8216 1 / 4 0.5546 0.5031 0.8164 0.3376 0.8770 1 / 8 0.5304 0.5030 0.6259 0.3522 0.8184 1 / 16 0.4985 0.4753 0.5061 0.4089 0.8962 1 / 32 0.5236 0.4955 0.4941 0.3812 1.0304 1 / 64 0.5555 0.5118 0.4945 0.4208 0.9528 1 / 128 0.6135 0.5653 0.5516 0.4399 0.7875 Note: Raw absorbance data were normalized using virus control and cell control references. Antiviral inhibition percentages were calculated. The results obtained were compared. It has been evaluated as follows: 5 23 Selectivity index (SI) to determine the therapeutic safety range of the extract. The SI value was calculated as the ratio of cytotoxicity (CC50) and antiviral efficacy (IC50) values. It has been determined that high SI values indicate that the antiviral efficacy of the extract is relative to cytotoxicity. This shows that it is more powerful in comparison. 𝑆𝐼 = 𝐶𝐶50 𝐼𝐶50 CC50, IC50 and selectivity index (SI) values of black mulberry extract samples are given in Table 14. It has been given. Table 14. CC50, IC50 and selectivity index (SI) values. Example CC50 (µg / mL) IC50 (µg / mL) SI Maximum inhibition 1 – Mersin > 8.0 ≈ 0.6 > 13 High 2 – Ankara ≈ 2.9 ≈ 0.5 ≈ 5.8 Very high 3 – Elazığ > 8.0 ≈ 0.5 > 13 High 4 – Izmir > 8.0 ≈ 0.8 > 10 Medium-high – Hatay > 8.0 ≈ 1.0 > 8 Medium The results obtained show that mulberry extracts are non-cytotoxic within the concentration range of 10 It has been shown to provide significant antiviral inhibition, particularly based on the selectivity index (SI). The high values indicate a correlation between the antiviral efficacy of the extracts and their cellular safety profile. It reveals that it offers a balanced and optimized relationship. In this context, extracts 1 and 3 have high CC50 and low IC50 values. Having high SI values indicates that these samples are both safe and effective antiviral 15 This shows that extract number 2 stands out as a key agent. In contrast, extract number 2 has a lower level. Due to its CC50 value, it has a narrower biocompatible dose range, but it is low. It has been determined that it exhibits strong antiviral activity thanks to its IC50 value. This means that it has maximum... the importance of evaluating antiviral efficacy at non-cytotoxic concentrations It reveals. 20 Extracts 4 and 5, on the other hand, have antiviral properties over a wide range of non-cytotoxic concentrations. It was determined that it showed inhibition. In these samples, the antiviral activity levels were 1 and 3. Although more limited compared to the numbered extracts, biocompatibility is maintained. It has been observed that both together exhibit a stable antiviral profile. Especially extract number 4. It provides moderate to high levels of inhibition within specific concentration ranges, number 5 25 24 The extract, however, exhibited a more balanced but relatively lower level of antiviral effect. It has been determined. These findings suggest that the antiviral efficacy of the extracts depends on differences in their phytochemical content. This shows that it can vary, and the controlled extraction and dosage developed within the scope of the invention This clearly supports the technical effectiveness of the optimization approach. In this respect, the aforementioned 5 the method maximizes antiviral efficacy within safe and biocompatible concentration ranges It offers a systematic and feasible technical solution that enables this. Logarithmic concentration-dependent SARS-CoV-2 antiviral inhibition of black mulberry extracts. The profiles are shown in Figures 12–14 and Figures 17–18. Concentration-dependent cell viability. The numerical data revealing the relationship between antiviral inhibition and the drug are given in Table 16. 10 Table 16. Cell viability (%) of black mulberry extracts at different concentrations in antiviral test. results Concentration Log10 Mersin (%) Ankara (%) Elazığ (%) İzmir (%) Hatay (%) 8 0.903 60.04 99.90 54.65 63.91 59.21 4 0.602 55.41 90.14 52.01 60.05 54.73 2 0.301 52.58 44.86 51.33 59.79 53.69 1 0 51.97 60.72 51.04 56.92 53.68 0.5 -0.301 55.28 54.69 50.31 53.15 50.4 0.25 -0.602 56.85 54.49 50.24 56.12 52.79 0.125 -0.903 56.20 57.55 52.12 59.90 54.73 0.0625 -1.204 59.03 59.29 55.67 66.77 61.06 The results show that the test substances have the capacity to prevent virus-induced cellular damage. It shows that it varies depending on the concentration. Cell viability data and antiviral 15 When the inhibition results are evaluated together, the antiviral efficacy of the extracts is certain. It appears to be optimized within certain concentration ranges. In this context, extract number 2 has high antiviral properties at certain biocompatible concentrations. It exhibited inhibition; in contrast, extracts 1 and 3 showed a more balanced antiviral effect. It has been determined that extracts 4 and 5 exhibit a high selectivity index. Stable over a wider concentration range, but with relatively lower levels of antiviral activity. It has been observed that he / she exhibited a profile. These findings suggest that antiviral efficacy is determined not only by the maximum inhibition value, but also by This indicates that biocompatibility and dosage range should be evaluated together, 5 technical significance of the controlled dose optimization approach developed within the scope of the invention It supports. In previous formulations, only mechanical crushing, homogenization, and heat treatment were used. The level of antiviral inhibition in products obtained through methods based on these steps is approximately... It has been observed that it remains at around 25%. In contrast, within the scope of the present invention, 10 The extracts obtained through the developed controlled extraction and dose optimization approach, Significantly higher antiviral efficacy in non-cytotoxic concentration ranges. It has been shown that viral replication is significantly suppressed at certain concentrations. This situation indicates that the developed method has concrete and effective capabilities to increase antiviral efficacy. It clearly demonstrates that it has provided a measurable technical improvement. 15 The maximum antiviral inhibition zone shown in the figures indicates the viral inhibition of black mulberry extracts. it suppresses proliferation at the highest level and this effect is at a non-cytotoxic concentration. It refers to the region where it emerged within the range. This situation is developed within the scope of the invention. The extracts not only exhibit antiviral effects but also demonstrate biocompatibility. It demonstrates that it can be safely implemented within its borders. 20 Cell culture plate images and measurement results related to antiviral efficacy experiments are shown in Figure 1. The data is presented in detail in the range of 10–18, and the data obtained show that antiviral efficacy clearly reveals its variation depending on concentration and its maximum inhibition regions. It places. Invention Step 25 In current techniques, antiviral properties of black mulberry (Morus nigra L.) and similar plant materials are being investigated. Although there are various studies on its potential, these studies are generally in their raw form. the focus is on the biological effects of the extracts and the systematic optimization of antiviral efficacy It appears that no technical solution has been offered for this purpose. In this context, the existing technique; It is based on the direct use of plant extracts and the controlled acquisition of active ingredients. 30 to standardize the extract composition and to determine cytotoxicity and antiviral efficacy. 26 It does not include a holistic approach to optimizing them by evaluating them together. Therefore, it has high antiviral efficacy and at the same time does not cause cellular toxicity. There should be clear information regarding the production parameters and dosage ranges required to obtain the extract. A repeatable and applicable teaching method emerges within the scope of the existing technique. It is not included. 5 In contrast, the method developed within the scope of the invention subject to the application is obtained from mulberry fruit. the preparation of the obtained extracts under controlled extraction parameters, bioactive selective acquisition of components and their cytotoxicity (CC50) and antiviral efficacy (IC50) By evaluating the data together, the biocompatible and effective dose range is determined. It offers a systematic technical approach that enables its determination. 10 With this approach, antiviral efficacy is no longer merely an observed characteristic; extraction conditions and dosage parameters can be controlled and optimized. It is transformed into a technical output that can be obtained in a repeatable manner. In this respect, the invention, Unlike existing techniques, it allows for a quantitative increase in antiviral efficacy and biocompatibility. It allows for maximization within its limits. 15 Furthermore, the phytochemical content profile determined from the obtained HPLC analysis data indicates antiviral properties. It reveals the relationship between the activity and specific bioactive components and the aforementioned that the extracts are chemically identifiable and can be standardized This shows that the invention not only offers a biological effect, but also... a production method that is analytically verifiable and applicable on an industrial scale in time 20 It shows that it provides. In conclusion, the invention subject to the application concerns the controlled extraction parameters of antiviral activity and reproducible and cytotoxicity-based dose optimization allows for increased dose. It offers a standardizable production method. This technical approach differs from existing technology. 25 that cannot be directly derived and are clearly predictable from the perspective of a technical expert. It is a non-existent solution and meets the inventive step criterion. 27 Evaluation of Preliminary Studies In the preliminary studies carried out by the applicant, the black mulberry (Morus nigra L.) fruit was found. subjected to mechanical crushing and homogenization processes (including glass bead homogenization) Formulations obtained by holding and then applying heat treatment (boiling) have antiviral properties. The effects have been evaluated. In this approach, despite the fragmentation of the fruit matrix and the application of heat treatment, the following is obtained: It was determined that the level of antiviral inhibition achieved remained at approximately 25%. The situation is based solely on mechanical crushing, homogenization, and heat treatment steps. This clearly shows that the methods have limitations in terms of increasing antiviral efficacy. 10 In this context, it provides high antiviral efficacy while also having low cellular toxicity. a more efficient and systematic method for obtaining an extract that does not produce It has been understood that there is a need for it. In contrast, the controlled extraction method developed within the scope of the present invention obtains The extracts obtained were significantly more concentrated in non-cytotoxic concentration ranges. Experimental data have shown that it exhibits high antiviral efficacy. In the present invention, extracts are prepared through specific and controlled process steps, and these Extracts are primarily evaluated using in vitro cytotoxicity analyses. These analyses... As a result, biocompatible dose ranges that preserve cell viability are determined by considering CC50 values. these are determined, and then antiviral efficacy analyses are performed only at these safe concentrations. 20 This is carried out via IC50 data. Antiviral efficacy is evaluated using IC50 data. By analyzing these two parameters together, the appropriate range of effects is systematically determined. This approach makes it possible to achieve high antiviral efficacy with low cytotoxicity within the same system. that they can be achieved together and that this effect can be obtained in a controlled and repeatable manner This has been demonstrated. This situation represents a technical effect that is difficult to directly predict from the current technology. 25 is doing. Furthermore, the evaluations revealed that without any extraction process... Direct consumption of mulberry fruit results in higher cytotoxicity; Extracts obtained by controlled extraction methods have better biocompatibility. and it was determined that it exhibited an antiviral activity profile. This finding suggests that the extraction process was only 30 minutes long. 28 not only is there a preparation step, but the biological activity of the product is also directly affected. This reveals that there is a critical technical element that affects the process. By evaluating the obtained CC50 and IC50 data together, it was determined that the extracts are safe and Effective usage ranges were determined and the selectivity index (SI = CC50 / IC50) was calculated. The relationship between antiviral efficacy and cellular safety has been quantitatively established. This 5 The values obtained within this scope are presented in Table 15. A general overview of the antiviral potential of herbal extracts from a technical expert. While it is expected that it will have information, antiviral efficacy will be assessed based on cytotoxicity data. by systematically optimizing in this way and using these two parameters together to find the appropriate one. Determining the range of effect does not appear to be directly derived from the current technique. 10 In conclusion, the candidate invention in question concerns not only the use of a plant extract; Controlled extraction of the extract, determination of a safe dosage range, and antiviral It offers a holistic technical approach that involves quantitatively optimizing the efficiency. the approach cannot be directly derived from existing technology and is understood by a person specializing in the technical field It is a solution that is not clearly foreseeable and meets the inventive step criterion of 15. It meets the needs. The proposed invention not only demonstrates antiviral efficacy but also cytotoxicity. Biocompatibility is determined by evaluating both (CC50) and antiviral efficacy (IC50) data together. systematically optimizing the dosage range and controlling this effectiveness through extraction. It offers a production method that obtains repeatable results with its parameters.” 20 Data Evaluation Half-maximum cytotoxic concentration (CC50) obtained from cytotoxicity analyses. half-maximum inhibitory concentration (IC50) determined from antiviral tests with these values The biocompatibility and antiviral efficacy profiles of the extracts were determined by evaluating the data together. This has been demonstrated. Within this scope, the selectivity index (SI = CC50 / IC50) was calculated for each of the 25 The safe and effective usage ranges of the extract have been quantitatively determined. The raw data obtained were normalized by referencing cell control and virus control groups. and thus between different concentrations and different extract samples Comparability has been established. This approach ensures that antiviral efficacy is compared to cellular toxicity. This allows for independent evaluation. 30 29 Furthermore, the experimental results were supported by technical replications, and the repeatability of the measurements was established. and its reliability has been verified. This allows for statistical and analytical analysis of the data obtained. It has been shown to be consistent. The data evaluation approach carried out within this scope focuses solely on antiviral efficacy. not limited to determination; it also addresses biocompatibility, efficacy and dose relationship together. 5 It offers a holistic analytical method. In this respect, this approach allows for the analysis of extracts. to optimize to show maximum antiviral efficacy within safe dose ranges It stands out as a systematic technical evaluation method that provides... 15 25
Claims
REQUESTS Main (Independent) Claim: Antiviral activity from black mulberry (Morus nigra) fruit. It is a method for the production of extracts, using mulberry fruit material at low temperatures. preservation under these conditions, and lyophilization of the preserved material. drying, grinding the dried material to make it homogenous, and the resulting material 5 with at least one organic solvent, under specified parameters of time, temperature and solvent ratio. subjected to controlled extraction, the mixture obtained after extraction by subjecting it to a filtration process, removing the solvent phase under vacuum Obtaining a concentrated extract and testing the cytotoxicity (CC50) of the obtained extract. By evaluating antiviral efficacy (IC50) data together, a biocompatible dose of 10 was determined. This includes steps to determine the range and the non-cytotoxic nature of antiviral activity. A production method characterized by optimization within a specific concentration range. Dependent Claims 2. The method is according to Claim 1, and the mulberry fruit material is stored between −20 °C and +4 °C. A method characterized by its preservation. 15 3. Method according to claim 1 or 2, where the lyophilization process is carried out at low pressure and low temperature. A method characterized by being carried out under specific conditions.
4. The method is in accordance with claims 1–3, and the organic solvent used is ethanol, methanol, water or A method characterized by selecting from mixtures of these.
5. The method is according to claims 1–4, and the extraction process is carried out at a temperature range of 20–60 °C and 1–96 20 A method characterized by being performed over a period of hours.
6. The method is according to claims 1–5, and the extraction process is carried out in a light-protected environment. a method characterized by its execution.
7. Method according to claims 1–6, where the filtration process results in a pore diameter of 0.45 µm or smaller. This is achieved by microfiltration or vacuum filtration using filters that have 25 The method being characterized.
8. The method is according to claims 1–7, where the solvent phase is evaporated under vacuum using a rotary evaporator. A method characterized by removal. 31 9. The method is based on claims 1–8, and the resulting extract is treated with a carrier to improve stability. substances characterized by being formulated with solvents or preservative components method.
10. The method is according to claims 1–9, and the extract is in liquid, semi-solid or lyophilized dry form. A method characterized by its acquisition. 5 User Request 11. Extracts obtained according to claims 1–10 were tested against SARS-CoV-2 in cell culture medium. Its use as an antiviral agent to suppress its multiplication. 15 25