A preparation method of active oxygen responsive radix glabrae extracellular vesicles

Abstract

This invention discloses a method for preparing extracellular vesicles of *Adenophora stricta* with reactive oxygen species responsiveness, mainly relating to the field of traditional Chinese medicine. The method includes the following steps: S1. Adding PBS to *Adenophora stricta* and extracting juice, then filtering to obtain a filtrate; S2. Centrifuging the filtrate and collecting the supernatant; S3. Centrifuging the supernatant again, collecting the precipitate and resuspending it; separating and resuspending to obtain an extracellular vesicle solution of *Adenophora stricta*; S4. Preparing a thioketal solution, adding a catalyst to obtain an activated thioketal solution; S5. Preparing a thioketal-resveratrol product; S6. Preparing purified Fuc-TK-Res, i.e., an amphiphilic carrier material; S7. Preparing the nano-formulation FTR@Cur; S8. Co-incubating *Adenophora stricta* extracellular vesicles with FTR@Cur at room temperature. The beneficial effects of this invention are as follows: ROS-responsive nanocarriers are constructed by fucoidan, thioester, and resveratrol, and curcumin is encapsulated to synergistically treat DSS-induced ulcerative colitis. Drug delivery in vivo is achieved through extracellular vesicles of Glehnia littoralis, which can avoid degradation by the gastric acid environment.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically a method for preparing extracellular vesicles of Glehnia littoralis with reactive oxygen species responsiveness. Background Technology

[0002] Traditional Chinese medicine is considered a treasure of my country's historical and cultural heritage and one of its greatest achievements. Thousands of years ago, our ancestors discovered the medicinal properties of many plants, using them to prevent and treat various diseases, thus giving birth to Chinese medicine. With the updating and development of separation techniques and advanced drug mechanism evaluation methods, more and more plants have been proven to have medicinal value, their active ingredients have been further isolated and purified, and their mechanisms of action have been further verified. Further research on the active ingredients of natural products has revealed that natural active ingredients have unique therapeutic potential for ulcerative colitis. Among them:

[0003] North American ginseng, the root of the umbelliferous plant *Glechoma hederacea*, is mainly distributed in Shandong, Liaoning, and Jiangsu provinces. It is a traditional medicinal herb from Laiyang, Shandong, and is listed in the *Compendium of Materia Medica* as one of the "Five Ginsengs" along with four other types. Rich in various bioactive components, it is a common resource used both as food and medicine. Coumarins, polysaccharides, phenolic acids, volatile oils, and lignins have been extracted from North American ginseng, thus exhibiting anti-inflammatory, anti-cancer, antioxidant, and immunomodulatory effects, which are significant for disease prevention and treatment.

[0004] Currently, most commercially available North American ginseng products are dried, with few reports on the extracellular vesicles of fresh North American ginseng. When North American ginseng is consumed directly for the treatment of ulcerative colitis, its active ingredients are partially degraded after gastrointestinal digestion, preventing it from fully exerting its efficacy. Furthermore, the existing anti-inflammatory drug components suffer from drawbacks such as hydrophobicity and low bioavailability, severely hindering their clinical application. Therefore, there is an urgent need for a novel drug delivery method to broaden the application of drug components and effectively treat DSS-induced colitis. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing extracellular vesicles of Glehnia littoralis with reactive oxygen species (ROS). This method constructs ROS-responsive nanocarriers using fucoidan, thioester, and resveratrol, and uses curcumin to synergistically treat DSS-induced ulcerative colitis. The drug is delivered in vivo through the extracellular vesicles of Glehnia littoralis, avoiding degradation by the gastric acid environment.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] A method for preparing extracellular vesicles of Glehnia littoralis with reactive oxygen species responsiveness includes the following steps:

[0008] S1. Add PBS to the North American ginseng according to the ratio of North American ginseng:PBS=1:3 (g / mL), extract the juice of North American ginseng, and filter it through a gauze to obtain the filtrate;

[0009] S2. Transfer the filtrate obtained in step S1 to a centrifuge tube, centrifuge at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant.

[0010] S3. Centrifuge the supernatant obtained in step S2 for another 2 hours, take the precipitate, and resuspend it in PBS; use sucrose density gradient centrifugation to separate the vesicle mixture of different particle sizes, and then resuspend it in PBS to obtain the extracellular vesicle solution of Glehnia littoralis.

[0011] S4. Dissolve an appropriate amount of thioacetal in an appropriate amount of organic solvent to obtain a thioacetal solution; weigh an appropriate amount of catalyst EDC and DMAP and dissolve them in an appropriate amount of organic solvent. After complete dissolution, mix them with the previously obtained thioacetal solution and react at a temperature of 35-45℃ for 2-3 hours to obtain an activated thioacetal solution.

[0012] S5. Weigh an appropriate amount of resveratrol, dissolve it in an appropriate amount of organic solvent, and after complete dissolution, add the mixed solution to the activated thioacetate solution obtained in step S4. React at room temperature of 25°C for 24 hours to obtain the thioacetate-resveratrol (TK-Res) product, which can be used for subsequent experiments.

[0013] S6. Dissolve a certain amount of EDC and DMAP in an appropriate amount of organic solvent, add them to the TK-Res solution obtained in step S5, and activate the carboxyl group at the other end of the thioacetate at a certain temperature; weigh a certain amount of fucose, dissolve it in an appropriate amount of solvent, add it to the activated TK-Res solution, react at room temperature of 25°C for 24 hours, and then dialyze and freeze dry to obtain purified Fuc-TK-Res, i.e., amphiphilic carrier material;

[0014] S7. Dissolve the amphiphilic carrier material obtained in step S6 to obtain a blank carrier solution; prepare a certain amount of curcumin (Cur) solution, mix the blank carrier solution with the curcumin solution, and after sonication and dialysis, obtain the ROS-sensitive nano-formulation FTR@Cur. Centrifuge and freeze-dry the supernatant for subsequent experiments.

[0015] S8. The extracellular vesicles of *Adenophora stricta* obtained in step S3 are co-incubated with the FTL@Cur prepared in step S7 at room temperature for 6 hours to obtain an extracellular vesicle of *Adenophora stricta* with reactive oxygen species (ROS) responsiveness.

[0016] Furthermore, in step S4, the molar ratio of thioacetate to EDC and DMAP is 1:1.5.

[0017] Furthermore, in step S5, the molar ratio of thioacetal to resveratrol is 1:1.

[0018] Furthermore, in step S6, the molar ratio of fucoidan to TK-Res is 1.5:1.

[0019] Furthermore, in step S7, the weight ratio of the amphiphilic carrier material to curcumin is 10:1.

[0020] Furthermore, the organic solvents used to dissolve the thioketone solution, EDC solution, and DMAP solution, as well as the organic solvents used to dissolve the resveratrol solution and the curcumin solution, are all anhydrous DMSO, and the solvent used to dissolve the fucoidan solution is anhydrous formamide.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. By co-incubating extracellular vesicles of Glehnia littoralis with ROS-responsive nano-formulation, a reactive oxygen species-responsive extracellular vesicle of Glehnia littoralis was prepared. The delivery of nanoparticles using extracellular vesicles of Glehnia littoralis can avoid the degradation effect of gastric acid environment after direct consumption, thus improving the therapeutic effect.

[0023] 2. By preparing a nano-formulation of fucoidan and resveratrol and loading curcumin into it, the drug is then delivered to the lesion site via extracellular vesicles of Glehnia littoralis. The drug can specifically accumulate at the site of inflammation, improving drug water solubility, increasing bioavailability, and treating ulcerative colitis more effectively.

[0024] 3. Elevated levels of reactive oxygen species (ROS) are commonly associated with various diseases, including cancer, inflammation, and neurodegenerative diseases. Based on the high ROS levels characteristic of inflammatory sites, researchers have explored various ROS-responsive compounds, one of which is thioketal (TK). In a high ROS environment, TK breaks down, releasing the drug and exerting a therapeutic effect. Considering the microenvironmental characteristics of inflammatory sites, this invention utilizes the specific cleavage of disulfide bonds at high ROS inflammatory sites to achieve specific drug release at the inflammatory site.

[0025] 4. Prepare an FTR blank carrier and load curcumin to solve the key problems of poor solubility and low bioavailability of natural Chinese medicinal materials. On this basis, resveratrol and curcumin can exert a synergistic therapeutic effect. Attached Figure Description

[0026] Appendix Figure 1 This is a synthesis route diagram of the amphiphilic carrier material of the present invention.

[0027] Appendix Figure 2The amphiphilic carrier material of the present invention 1 H-NMR.

[0028] Appendix Figure 3 This is a diagram of the nano-formulation of the present invention.

[0029] Appendix Figure 4 This is a particle size diagram of the nano-formulation of the present invention.

[0030] Appendix Figure 5 This is the potential diagram of the nano-formulation of the present invention.

[0031] Appendix Figure 6 This is a transmission electron microscope image of the nano-formulation of the present invention.

[0032] Appendix Figure 7 This is a transmission electron microscope image of the extracellular vesicles of Glehnia littoralis and the nano-formulation after co-incubation.

[0033] Appendix Figure 8 This is an in vitro release diagram of FTR@Cur of the present invention.

[0034] Appendix Figure 9 This is a comparative experiment on the cytotoxicity of the blank vector (FTR) of the present invention at 24h and 48h.

[0035] Appendix Figure 10 This is a comparative cytotoxicity test of the drug-loaded carrier and the free drug over 24 hours according to the present invention.

[0036] Appendix Figure 11 This is a comparative cytotoxicity test of the drug-loaded carrier and the free drug after 48 hours.

[0037] Appendix Figure 12 This is the RAW264.7 concentration-dependent uptake experiment of the present invention. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0039] Natural plants offer numerous benefits and are therefore receiving increasing attention in biomedical research. One component of plants—plant exosome-derived vesicles—are nanoscale vesicles secreted by plant cells. These vesicles are rich in bioactive substances and play a crucial role in intercellular communication, information transmission, and maintaining homeostasis. They also hold promise for treating diseases; their vesicle structure makes them suitable carriers for drug delivery and allows for large-scale industrial production.

[0040] Resveratrol (Res) is a natural polyphenol compound found in grapes and red wine. Besides its cardioprotective effects, it also possesses various antioxidant, antiplatelet aggregation, antitumor, and anti-inflammatory properties. Due to its anti-inflammatory characteristics, resveratrol may offer therapeutic benefits for many different diseases, including colitis, arthritis, and pancreatitis. Studies have shown that resveratrol has a protective effect against ulcerative colitis induced by sodium dextran sulfate (DSS). However, its hydrophobicity and low bioavailability severely hinder its clinical application.

[0041] Fucoidan (Fuc) is a highly bioactive seaweed substance with many physiological activities, mainly composed of fucose and sulfated polysaccharide groups. It possesses various biological activities such as anti-inflammatory, antioxidant, and antiviral properties. Therefore, purified fuc from brown seaweed holds great potential for the treatment and prevention of various diseases.

[0042] Example 1: A method for preparing extracellular vesicles of Glehnia littoralis with reactive oxygen species, comprising the following steps: 1. Preparation of extracellular vesicles of Glehnia littoralis:

[0043] (1) Add PBS to ginseng at a ratio of 1:3 (g / mL) of North American ginseng:PBS, extract the juice of North American ginseng, and filter it through a gauze to obtain the filtrate;

[0044] (2) Transfer the filtrate to a 50 mL centrifuge tube and centrifuge at 4 °C for 10 min at 1000 g, 20 min at 3000 g, and 60 min at 3000 g. Discard the bottom precipitate and take the supernatant.

[0045] (3) Centrifuge the supernatant obtained in step (2) at 150,000g for 2 hours, collect the precipitate, and resuspend it in PBS. Use sucrose density gradient centrifugation to separate vesicle mixtures of different particle sizes. Prepare 8%, 30%, 45%, and 60% sucrose PBS solutions respectively. First, add 2 mL of resuspension to an ultracentrifuge tube, then add 2.5 mL of 8% sucrose solution to the bottom using a fine needle. Then, add the same amounts of 30%, 45%, and 60% sucrose PBS solutions sequentially using the same method. Finally, add resuspension to the top layer up to 2 mm from the top of the centrifuge tube. After centrifugation at 150,000g for 2 hours, collect the bands of the mixture with sucrose densities between 30% and 45%, add an equal amount of PBS for resuspension, centrifuge at 100,000g for 1 hour, wash away the sucrose, collect the precipitate, and resuspend it in PBS to obtain the extracellular vesicle solution of *Adenophora stricta*.

[0046] 2. Preparation of ROS-responsive nanocarriers:

[0047] Thiocetyl was selected as a highly ROS-sensitive bond and dissolved in anhydrous dimethyl sulfoxide to obtain a thioacetyl solution. EDC and DMAP were dissolved separately in anhydrous dimethyl sulfoxide, and their solutions were mixed with the thioacetyl solution. The mixture was stirred at 40°C for 3 hours. After the reaction was completed, an activated thioacetyl solution was obtained for subsequent reactions. The molar ratio of thioacetyl to EDC and DMAP was 1:1.5. Resveratrol was dissolved in an appropriate amount of anhydrous dimethyl sulfoxide to obtain a resveratrol solution, which was added to the above reaction mixture. The mixture was stirred at room temperature for 24 hours to obtain the preliminary product thioacetyl-resveratrol. TK-Res solution was used for subsequent experiments, in which the molar ratio of thioacetate to resveratrol was 1:1; appropriate amounts of EDC and DMAP were weighed and dissolved in anhydrous dimethyl sulfoxide, added to the TK-Res solution, and stirred at 40°C for 3 hours to activate the carboxyl group at the other end of the thioacetate; a certain amount of fucoidan was weighed and dissolved in formamide to obtain a fucoidan solution, which was added to the above reaction and stirred at room temperature for 24 hours, followed by dialyzing for 36 hours using a dialysis bag with a molecular weight cutoff of 2000 Da; after dialysis, the supernatant was centrifuged and lyophilized to obtain FTR, i.e., an amphiphilic carrier material, the structural formula of which is shown below. 1 The structure was characterized by H-NMR, such as Figure 2 As shown.

[0048] .

[0049] 3. Preparation of ROS-sensitive nanoformulations (FTR@Cur):

[0050] The amphiphilic carrier material obtained above was dissolved in anhydrous dimethyl sulfoxide at a concentration of 5 mg / mL to obtain a blank carrier solution. A curcumin solution was prepared using anhydrous dimethyl sulfoxide at a concentration of 1 mg / mL. The blank carrier solution and curcumin solution were mixed and sonicated for ten minutes (the weight ratio of carrier material to curcumin was 10:1). The mixture was then dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 2000 Da. After dialysis, the supernatant was collected by centrifugation, filtered through a 0.8 μm microporous membrane, and lyophilized to obtain the ROS-sensitive nano-formulation.

[0051] 4. The extracellular vesicles of Glehnia littoralis obtained above are incubated with the FTL@Cur prepared above at room temperature for 6 hours to obtain an extracellular vesicle of Glehnia littoralis with reactive oxygen species (ROS) responsiveness.

[0052] Example 2: Characterization and performance experiments of ROS-sensitive nanoformulations:

[0053] 1. Determination of drug loading and encapsulation efficiency in nano-formulations:

[0054] The nano-formulation prepared in Example 1 is as follows Figure 3As shown, the encapsulation efficiency and drug loading of FTR@cCur were determined by high-performance liquid chromatography (HPLC). 1 mL of the prepared nano-formulation was demulsified with methanol, diluted to the desired concentration, and filtered through a 0.22 μm microporous membrane. The absorbance at 425 nm was measured by ultraviolet spectrophotometry. The drug loading and encapsulation efficiency of the nano-formulation were calculated using the following formula:

[0055] Drug loading (%) = (Drug content in the formulation / Total mass of the formulation) × 100%;

[0056] Encapsulation efficiency (%) = (Drug content in the formulation / Initial amount of drug added) × 100%.

[0057] In Implementation Case 1 of this application, the drug loading of curcumin micelles is 7.47%, and the encapsulation rate is 82.73%.

[0058] In this invention, the weight ratio of polymer micelle carrier material to curcumin is 10:1, which is important for this invention.

[0059] 2. FTR@Cur particle size and potential;

[0060] The prepared FTR@Cur was filtered through a 0.80 μm microporous membrane, and then the particle size and potential of FTR@Cur were measured using a particle size analyzer. The experimental results are as follows. Figure 4 , 5 As shown.

[0061] After passing through a 0.8 μm microporous membrane, the particle size is uniform, with a diameter of 131.5 nm ± 1.24 nm. The potential is -23.2 ± 0.76.

[0062] 3. Electron microscopic examination of FTR@Cur and FTR@Cur encapsulated in extracellular vesicles of Glehnia littoralis:

[0063] The prepared FTR@Cur was fixed and stained with copper mesh, and its morphology was photographed using a transmission electron microscope as shown in the figure. Figure 6 As shown.

[0064] 4. In vitro release experiment of nano-formulation:

[0065] First, the prepared FTR@Cur was concentrated using an ultrafiltration tube. Then, 1 mL of the concentrated FTR@Cur was precisely measured and transferred to identical dialysis bags (2000 Da), and divided into two groups (n=3). These dialysis bags were placed into 50 mL centrifuge tubes, and 45 mL of PBS containing 0.5% Tween 80 (pH 7.4) was added to each tube. The different groups contained different concentrations of H2O2, namely 0 mM H2O2, 0.5 mM H2O2, and 1 mM H2O2.

[0066] All centrifuge tubes were then placed in a constant-temperature shaking water bath at 37°C (100 rpm) and continuously shaken to simulate the human microenvironment. At pre-set time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, and 72 hours, 1 mL of the release medium sample was taken from each tube, and 1 mL of PBS in the same state was added to each centrifuge tube. The PBS was stored at 4°C for later use. After all samples were collected at all time points, all samples were filtered through a 0.22 μm aqueous filter membrane, and the amount of curcumin in each group of samples was determined by HPLC. The cumulative release rate was calculated, and the results showed that the cumulative release rate of 1 mL of MH2O2 was 77.93% at 72 hours. The experimental results are as follows. Figure 8 As shown.

[0067] 5. Cytotoxicity evaluation of blank vector and free drug:

[0068] The cytotoxicity of the nanoformulation was evaluated using the MTT assay to determine the culture medium solutions of the active pharmaceutical ingredient (API) and the blank carrier. Results showed that the FTR blank micelles caused minimal damage to RAW264.7 cells. After 48 hours of culture, the cell viability remained above 60% at a FTR blank micelle concentration of 200 μg / mL. This demonstrates that the FTR blank nanomicelles exhibit very low cytotoxicity and almost no toxicity to nerve cells. The experimental results are as follows: Figure 9 As shown.

[0069] We also verified the toxicity of free curcumin to RAW264.7 cells. Our MTT assay showed that when the concentration of free curcumin reached 20 μg / mL, treating RAW264.7 cells for 24 hours resulted in a cell viability rate of over 75%. The results are as follows: Figure 10 As shown in the attached figure. After treating RAW264.7 cells for 48 hours, the cell viability was found to be 65.8%. Figure 11 As shown in the figure. The two experiments above fully demonstrate that the nanocarriers prepared by the method of the present invention have good safety.

[0070] 7. Study on cellular uptake and distribution of nano-formulations:

[0071] We used laser confocal microscopy to observe the intracellular distribution of nano-formulations after endocytosis in RAW264.7 cells. Curcumin (Cur) was used as a fluorescent dye, and our prepared nanomicelles encapsulated curcumin. The phagocytosis by RAW264.7 cells was observed with increasing concentration and compared with free curcumin. The results showed that the cellular fluorescence intensity increased with increasing uptake time of different formulations. This indicates that the cellular uptake behavior of Free Cur and FTR@Cur by RAW264.7 cells is time-dependent, and that cells have a higher uptake of FTR@Cur. This demonstrates that the nanocarrier micelles have better bioavailability and can achieve better therapeutic effects. Experimental results are as follows: Figure 12 As shown.

[0072] In summary, this invention addresses the shortcomings of natural product active ingredients, such as their inability to effectively accumulate at the site of inflammation and their inability to specifically release drugs. It prepares the active ingredients of natural products into nanoparticles and loads them with natural drug active ingredients, improving their poor solubility and low bioavailability, and enhancing drug stability. Furthermore, the delivery of nanoparticles via extracellular vesicles of *Adenophora stricta* avoids degradation by the acidic environment of the stomach. In addition, the ROS-sensitive nanoparticle formulation constructed in this invention achieves specific drug release at the site of inflammation; improving drug bioavailability while avoiding systemic adverse reactions, resulting in more efficient treatment of ulcerative colitis.

Claims

1. A method for preparing extracellular vesicles of *Adenophora stricta* with reactive oxygen species responsiveness, characterized in that: Includes the following steps: S1. Add PBS to the North American ginseng according to the ratio of North American ginseng:PBS=1:3 (g / mL), extract the juice of North American ginseng, and filter it through a gauze to obtain the filtrate; S2. Transfer the filtrate obtained in step S1 to a centrifuge tube, centrifuge at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant. S3. Centrifuge the supernatant obtained in step S2 for another 2 hours, take the precipitate, and resuspend it in PBS; use sucrose density gradient centrifugation to separate the vesicle mixture of different particle sizes, and then resuspend it in PBS to obtain the extracellular vesicle solution of Glehnia littoralis. S4. Dissolve an appropriate amount of thioketane in an appropriate amount of organic solvent to obtain a thioketane solution; weigh an appropriate amount of catalyst EDC and DMAP and dissolve them in an appropriate amount of organic solvent. After complete dissolution, mix them with the previously obtained thioketane solution and react at a temperature of 35-45℃ for 2-3 hours to obtain an activated thioketane solution. S5. Weigh an appropriate amount of resveratrol, dissolve it in an appropriate amount of organic solvent, and after complete dissolution, add the mixed solution to the activated thioacetate solution obtained in step S4. React at room temperature of 25°C for 24 hours to obtain the thioacetate-resveratrol (TK-Res) product, which can be used for subsequent experiments. S6. Dissolve a certain amount of EDC and DMAP in an appropriate amount of organic solvent, add them to the TK-Res solution obtained in step S5, and activate the carboxyl group at the other end of the thioacetate at a certain temperature; weigh a certain amount of fucose, dissolve it in an appropriate amount of solvent, add it to the activated TK-Res solution, react at room temperature of 25°C for 24 hours, and then dialyze and freeze dry to obtain purified Fuc-TK-Res, i.e., amphiphilic carrier material; S7. Dissolve the amphiphilic carrier material obtained in step S6 to obtain a blank carrier solution; prepare a certain amount of curcumin (Cur) solution, mix the blank carrier solution with the curcumin solution, and after sonication and dialysis, obtain the ROS-sensitive nano-formulation FTR@Cur. Centrifuge and take the supernatant for freeze-drying and storage for subsequent experiments. The weight ratio of the amphiphilic carrier material to curcumin is 10:

1. S8. The extracellular vesicles of *Adenophora stricta* obtained in step S3 are co-incubated with the FTL@Cur prepared in step S7 at room temperature for 6 hours to obtain an extracellular vesicle of *Adenophora stricta* with reactive oxygen species (ROS) responsiveness.

2. The method for preparing extracellular vesicles of *Adenophora stricta* with reactive oxygen species responsiveness according to claim 1, characterized in that: In step S4, the molar ratio of thioacetal to EDC and DMAP is 1:1.

5.

3. The method for preparing extracellular vesicles of *Adenophora stricta* with reactive oxygen species responsiveness according to claim 1, characterized in that: In step S5, the molar ratio of thioacetal to resveratrol is 1:

1.

4. The method for preparing extracellular vesicles of *Adenophora stricta* with reactive oxygen species responsiveness according to claim 1, characterized in that: In step S6, the molar ratio of fucoidan to TK-Res is 1.5:

1.

5. The method for preparing extracellular vesicles of *Adenophora stricta* with reactive oxygen species responsiveness according to claim 1, characterized in that: The organic solvents used to dissolve the thioketone solution, EDC solution, and DMAP solution, as well as the organic solvents used to dissolve the resveratrol solution and the curcumin solution, are all anhydrous DMSO, and the solvent used to dissolve the fucoidan solution is anhydrous formamide.

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