A process for the preparation of acetylated raspberry ketone and uses thereof

Abstract

The present application relates to a preparation method of acetylated raspberry ketone and its use, wherein the preparation method realizes the hydroxyl acetylation of raspberry ketone by using acetic anhydride as an acetylation reagent under the conditions of a suitable catalyst and a solvent, and high-purity acetylated raspberry ketone is obtained. The present application has mild reaction conditions, does not require high temperature and high pressure, has high product purity, is simple to operate and is suitable for industrialized production. Moreover, the acetylated raspberry ketone is used for preparing an antimicrobial composition, and the effect is remarkable, and the microorganisms include listeria bacteria or salmonella typhimurium.

Description

Technical Field

[0001] This invention relates to the field of organic chemical synthesis technology, specifically to a method for preparing acetylated raspberry ketone and its uses. Background Technology

[0002] Raspberry ketone is a natural aromatic compound widely used in food, cosmetics, and pharmaceuticals. Its chemically modified product—acetylated raspberry ketone—is gaining increasing attention due to its potential applications in antioxidation, anti-inflammation, and the regulation of specific aromas. Currently, there are few methods for preparing acetylated raspberry ketone, and most processes suffer from harsh reaction conditions, low yields, and numerous byproducts. Therefore, it is necessary to develop an efficient and environmentally friendly synthetic method. Summary of the Invention

[0003] One of the objectives of this invention is to provide a simple and efficient method for preparing acetylated raspberry ketone.

[0004] A method for preparing acetylated raspberry ketone, the method comprising the following steps: 1) First, dissolve raspberry ketone in an inert solvent to obtain solution A, wherein the solid-liquid ratio of raspberry ketone to inert solvent is (8~10) mmol: (45~50) mL; 2) While stirring solution A at a temperature of 0~5℃, acetic anhydride is added to obtain solution B. The molar ratio of acetic anhydride to raspberry ketone added is (6~5):1. 3) Continue to add catalyst to solution B, stir twice, and react at 0~5℃ for 8~12h to obtain mixture A. The catalyst is anhydrous pyridine or BF3·Et2O, and the molar ratio of the added catalyst to raspberry ketone is (0.01~0.2):1. 4) After neutralizing, extracting, and purifying mixture A, the target product is obtained, wherein the target product includes 95.1%~97.5% by mass of acetylated raspberry ketone, and the chemical formula of the acetylated raspberry ketone is C. 12 H 14 O3. The remainder of the target product is unacetylated raspberry ketone.

[0005] The ratio of raspberry ketone to inert solvent in solution A must be appropriate. If the amount of raspberry ketone added is insufficient and the amount of inert solvent added is excessive, it will not only reduce the reaction rate, prolong the reaction time, and increase energy consumption, but also lead to an increase in side reactions, complicated subsequent processing, and product purity affected by impurities. Although the conversion rate may be high, the overall cost and purity are significantly inferior. Conversely, if the amount of raspberry ketone added is excessive and the amount of inert solvent added is insufficient, it will lead to uneven system, poor mass transfer, local overheating, an increase in side reactions, difficulty in product purification, reduced yield, waste of raw materials, and difficulty in separating the product from unreacted substrates, affecting both purity and economy.

[0006] The amount of acetic anhydride added must also be appropriate. If too little acetic anhydride is added, there will be defects such as low raw material conversion rate, decreased yield, and difficulty in separating unreacted substrates. Conversely, if too much acetic anhydride is added, side reactions will increase, post-processing will become more complicated, and residual impurities will affect product quality.

[0007] The temperature at which acetic anhydride is added in step 2) must also be appropriate. If the temperature is too high, side reactions will increase dramatically, impurities will increase, the risk of product decomposition will increase, the reaction will run away from control, and safety will decrease. Conversely, if the temperature is too low, the reaction rate will be extremely slow, the raw materials will not be completely converted, the raw materials will not dissolve and mix evenly, local reactions will be abnormal, and the catalyst activity will be inhibited.

[0008] Insufficient catalyst addition will lead to insufficient reaction kinetics, resulting in slow rate, low conversion rate and impure product. Conversely, excessive catalyst addition will cause side reactions, increase the difficulty of post-processing, and even contaminate the product.

[0009] Step 3) If the reaction temperature is too high: the core problem is a surge in side reactions and product decomposition, leading to a decrease in both purity and yield, and posing safety risks. If the reaction temperature is too low: the core problem is reaction stagnation, incomplete conversion of raw materials, low yield, and uneven mixing of the system. If the reaction time is too long: the core problem is product decomposition and impurity accumulation, leading to a decrease in yield and poor stability. If the reaction time is too short: the core problem is insufficient conversion of raw materials, low yield, and difficulty in separating the product from the unreacted raw materials.

[0010] Furthermore, the yield of the preparation method described in this application is 80%~90%.

[0011] Yield (%) = (Theoretical yield (g) / Actual yield (g)) × 100%; Parameter descriptions: Actual yield: refers to the mass (in grams) of the final pure acetylated raspberry ketone obtained after experimental separation and purification. It requires precise weighing after removing impurities through methods such as recrystallization and column chromatography.

[0012] Theoretical yield: refers to the maximum mass (in g) of acetylated raspberry ketone that can theoretically be produced assuming the reaction proceeds completely (100% of the raw materials are converted into products).

[0013] The calculation steps are as follows: Identify the limiting reagent in the reaction: that is, the substance with the smallest molar amount among the reactants (other reactants are in excess), and its consumption determines the theoretical yield.

[0014] For example, if raspberry ketone is used as the substrate and acetic anhydride as the acylation reagent, the following needs to be determined based on the molar amounts of both and the reaction equation (1:1 reaction): If the molar amount of raspberry ketone is less than that of acetic anhydride, then raspberry ketone is the limiting reagent. Based on the molar amount of the limiting reagent and the molar mass of acetylated raspberry ketone, the theoretical yield is calculated: Theoretical yield (g) = nlimiting reagent × Macetylated raspberry ketone. Where nlimiting reagent is the molar amount of the limiting reagent (unit: mol), and Macetylated raspberry ketone is the molar mass of acetylated raspberry ketone (unit: g / mol, which can be calculated from the molecular formula, such as C...). 12 H 14 The molar mass of O3 is 194 g / mol.

[0015] Furthermore, in the preparation method described in this application, the inert solvent is anhydrous ethanol or dichloromethane.

[0016] Furthermore, in the preparation method described in this application, the neutralization process in step 4) specifically includes: heating mixture A to room temperature and then adding a dilute alkali solution to carry out a neutralization reaction to obtain mixture B, in order to remove excess acetic anhydride. Mixture B does not contain acetic anhydride. The dilute alkali solution is a 5% NaHCO3 solution by mass; the molar ratio of NaHCO3 in the added dilute alkali solution to the acid in mixture A is (1.1~1.2):1.

[0017] Furthermore, in the preparation method described in this application, the extraction process in step 4) includes: placing mixture B in a separatory funnel to separate the organic layer, then drying the organic phase in the organic layer with anhydrous sodium sulfate; and then removing the solvent in the organic layer by a rotary evaporator to obtain the crude product.

[0018] Furthermore, in the preparation method described in this application, the purification in step 4) is specifically carried out by column chromatography or recrystallization.

[0019] Furthermore, in the preparation method described in this application, the stirring speed in step 2) is 200~220 rpm and the stirring time is 80~90 min.

[0020] Furthermore, in the preparation method described in this application, the stirring speed of the secondary stirring in step 3) is 300~350 rpm, and the stirring time is 110~120 min.

[0021] The present invention also provides a use of acetylated raspberry ketone.

[0022] One use of acetylated raspberry ketone based on any of the above, wherein the acetylated raspberry ketone is used to prepare an antimicrobial composition, the microorganism including Listeria or Salmonella typhimurium.

[0023] Furthermore, in the uses described in this application, the antimicrobial composition is an oral formulation.

[0024] Intracellular bacterial infection refers to a form of infection in which pathogens survive and proliferate after invading host cells, such as Listeria monocytogenes and Salmonella typhimurium. These bacteria survive by evading the host's immune response and inhibiting phagocytosis-lysosomal fusion. Traditional antibiotics have limited effectiveness in clearing intracellular bacteria because they have difficulty penetrating the host cell membrane.

[0025] Furthermore, the microorganisms described in this invention are Listeria monocytogenes or Salmonella typhimurium from mouse cells, and the mice are WT mice; the cells are Raw264.7 cell lines or L929 cell lines BMDM cells.

[0026] Compared with the prior art, the present invention has the following beneficial technical effects: On the one hand, this invention provides a simple and efficient method for preparing acetylated raspberry ketone. By using acetic anhydride as the acetylation reagent, the hydroxyl groups of raspberry ketone are acetylated under appropriate catalyst and solvent conditions to obtain high-purity acetylated raspberry ketone. The reaction conditions of this invention are mild, requiring no high temperature or high pressure; the product has high purity; the operation is simple, and it is suitable for industrial production.

[0027] On the other hand, this invention established mouse models of acute Listeria monocytogenes infection and Salmonella typhimurium infection, orally administered the acetylated raspberry ketone described in this invention, and monitored body weight. The results showed that acetylated raspberry ketone significantly reduced the number of bacteria within infected cells. The acetylated raspberry ketone (50 mg / kg) group showed significantly less body weight loss compared to the PBS group, indicating that acetylated raspberry ketone has a good inhibitory effect on intracellular bacteria, especially Listeria monocytogenes and Salmonella typhimurium. Attached Figure Description

[0028] Figure 1This is a graph showing the results of the bacterial infection and drug intervention experiment in Example 2 of the present invention; Figure 2 This is a graph showing the results of the bacterial infection and drug intervention experiment in Example 3 of the present invention; Figure 3 This is an experiment on the effect of drugs on body weight changes in an animal Listeria infection model described in Example 4 of the present invention. Figure 4 This experiment, described in Example 5 of the present invention, examines the effect of drugs on body weight changes in an animal model infected with Salmonella typhimurium. Detailed Implementation

[0029] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments are now described in detail with reference to the accompanying drawings. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the detailed embodiments, conventional conditions or conditions provided by the manufacturer shall apply.

[0030] In the following description, when referring to the accompanying drawings, the same numbers in different drawings denote the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. Specific implementation method one:

[0032] A method for preparing acetylated raspberry ketone, the method comprising the following steps: 1) First, dissolve raspberry ketone in an inert solvent to obtain solution A, wherein the solid-liquid ratio of raspberry ketone to inert solvent is (8~10) mmol: (45~50) mL; 2) While stirring solution A at a temperature of 0~5℃, acetic anhydride is added to obtain solution B. The molar ratio of acetic anhydride to raspberry ketone added is (6~5):1. 3) Continue to add catalyst to solution B, stir twice, and react at 0~5℃ for 8~12h to obtain mixture A. The catalyst is anhydrous pyridine or BF3·Et2O, and the molar ratio of the added catalyst to raspberry ketone is (0.01~0.2):1. 4) After neutralizing, extracting, and purifying mixture A, the target product is obtained, wherein the target product includes 95.1%~97.5% by mass of acetylated raspberry ketone, and the chemical formula of the acetylated raspberry ketone is C. 12 H 14 O3.

[0033] In other embodiments, the yield of the preparation method is 80% to 90%.

[0034] In other embodiments, the inert solvent is anhydrous ethanol or dichloromethane.

[0035] In other embodiments, the neutralization process in step 4) specifically includes: heating mixture A to room temperature and then adding a dilute alkali solution to carry out a neutralization reaction to obtain mixture B, so as to remove excess acetic anhydride; the dilute alkali solution is a 5% NaHCO3 solution by mass, and the molar ratio of NaHCO3 in the added dilute alkali solution to the acid in mixture A is (1.1~1.2):1.

[0036] In other embodiments, the extraction process in step 4) includes: placing mixture B in a separatory funnel to separate the organic layer, then drying the organic phase in the organic layer with anhydrous sodium sulfate; and then removing the solvent in the organic layer by a rotary evaporator to obtain the crude product.

[0037] In other embodiments, the purification in step 4) is specifically performed by column chromatography or recrystallization.

[0038] In other embodiments, the stirring speed in step 2) is 200~220 rpm and the stirring time is 80~90 min.

[0039] In other embodiments, the stirring speed of the secondary stirring in step 3) is 300~350 rpm, and the stirring time is 110~120 min. Specific Implementation Method Two:

[0041] The use of acetylated raspberry ketone prepared by the preparation method described in any of the above embodiments, wherein the acetylated raspberry ketone is used to prepare an antimicrobial composition, wherein the microorganism includes Listeria monocytogenes or Salmonella typhimurium.

[0042] In other embodiments, the antimicrobial composition is an oral formulation.

[0043] The present invention will be further described in detail below with reference to specific embodiments. Example 1:

[0044] A method for preparing acetylated raspberry ketone includes the following steps: 1) First, dissolve an appropriate amount of raspberry ketone in an inert solvent to obtain solution A, wherein the solid-liquid ratio of raspberry ketone to inert solvent is 10 mmol: 50 mL; 2) Add an appropriate amount of acetic anhydride while stirring at a temperature of 0~5℃ to obtain solution B. The molar ratio of acetic anhydride to raspberry ketone added is 5.5:1. 3) Continue to add an appropriate amount of catalyst to solution B, stir twice, and react at 0~5℃ for 8~12h to obtain mixture A. The catalyst is anhydrous pyridine or BF3·Et2O. 4) After neutralizing, extracting, and purifying mixture A, the target product is obtained, wherein the target product includes 95.1%~97.5% by mass of acetylated raspberry ketone, and the chemical formula of the acetylated raspberry ketone is C. 12 H 14 O3.

[0045] In this Example 1, the inert solvent is anhydrous ethanol. Mixture A is heated to room temperature and then neutralized with a dilute alkaline solution to obtain mixture B, thereby removing excess acetic anhydride; the dilute alkaline solution is a 5% (w / w) NaHCO3 solution. The molar ratio of the added NaHCO3 to the acid in mixture A is (1.1~1.2):1.

[0046] The extraction process in step 4) includes: placing mixture B in a separatory funnel to separate the organic layer, then drying the organic phase with anhydrous sodium sulfate; and then removing the solvent using a rotary evaporator to obtain the crude product. Purification is specifically performed by column chromatography or recrystallization.

[0047] The stirring speed described in step 2) is 200~220 rpm, and the stirring time is 80~90 min.

[0048] In step 3), the stirring speed for the second stirring is 300~350 rpm, and the stirring time is 110~120 min.

[0049] The yield of the preparation method is 80-90%. Example 2:

[0050] Using BMDM (bone marrow-derived macrophages) to study Listeria monocytogenes (a pathogenic bacterium) infection.

[0051] Intervention group: PBS group (black): treated with phosphate buffer (blank control).

[0052] AC-Raspberry Ketone group (red): treated with the target product from Example 1, which is acetyl Raspberry ketone group.

[0053] Observation indicator: The number of Listeria viable in cells / supernatant after 24 hours (measured as "log CFU of colony formation units"). like Figure 1 The results showed that the number of Listeria in the acetylated raspberry ketone group (lower Log CFU) was significantly less than that in the PBS group (p < 0.0001, statistically significant difference), indicating that acetylated raspberry ketone can effectively inhibit the survival / proliferation of Listeria in macrophages and may have anti-infection and immunomodulatory potential.

[0054] The initial bacterial count was 6.0–6.1 Log CFU, and after 24 hours, the Listeria count in the acetylated raspberry ketone group was 5.5–5.7 Log CFU, a decrease of 74.9%–80.1%. Example 3:

[0055] Infect Salmonella typhimurium (a common pathogen) with BMDM (bone marrow-derived macrophages).

[0056] Grouping: PBS group (blank control): treated with phosphate-buffered saline (PBS) AC-Raspberry Ketone group: treated with the target product in Example 1, resulting in the acetylated raspberry ketone group.

[0057] Observation: Survival of Salmonella typhimurium inside / outside macrophages after 24 hours (measured as "Log CFU, logarithmic colony-forming units"). like Figure 2 As shown, the conclusion is: The number of bacteria (lower Log CFU) in the acetylated raspberry ketone group was significantly less than that in the PBS group (p < 0.0005, statistically significant), indicating that acetylated raspberry ketone can effectively inhibit the survival / proliferation of Salmonella typhimurium in macrophages and has the potential to play a role in anti-infection and adjuvant immunity.

[0058] The initial bacterial count was 6.0–6.1 Log CFU, and after 24 hours, the count of Salmonella typhimurium in the acetylated raspberry ketone group was 5.3–5.5 Log CFU, a decrease of 74.9%–80.1%. Example 4:

[0059] Background: Animals were infected with Listeria monocytogenes (a pathogenic bacterium), and changes in body weight were observed after infection.

[0060] Grouping: PBS group (black dots, control): treated with phosphate buffer (simulating no drug administration).

[0061] AC-Raspberry Ketone group (yellow triangle): treated with acetylated raspberry ketone (50 mg / kg dose) prepared in Example 1.

[0062] Observation: Within 4 days after infection, the percentage of the animal's body weight relative to its "starting body weight" (% Starting Body Weight). The lower the value, the greater the weight loss.

[0063] like Figure 3 As shown, key results: after Listeria monocytogenes infection, the body weight of the PBS group continued to decrease (curve from 1.00 to about 0.80); the body weight of the acetylated raspberry ketone group decreased significantly less (curve was flatter, and the final body weight was higher), and the difference was statistically significant at some time points (such as 4 days) (p = 0.0200).

[0064] Conclusion: Listeria monocytogenes infection leads to significant weight loss in animals (a manifestation of disease-induced wasting), while acetylated raspberry ketone can "alleviate weight loss" by exerting its effects through anti-infection and disease state improvement, such as inhibiting bacterial proliferation and reducing inflammatory wasting. Example 5:

[0065] Experimental background: Animals were infected with Salmonella typhimurium (a pathogenic bacterium). The changes in body weight after infection were observed, and the effects of drug intervention were evaluated.

[0066] Grouping and intervention: PBS group (black line, control): treated with phosphate-buffered saline (simulating "no drug administration", representing the natural course of the disease) AC-Raspberry Ketone group (yellow line): treated with acetylated raspberry ketone (50 mg / kg dose) (to observe the effect of the drug on post-infection weight).

[0067] The observation indicator "% Starting Body Weight" is the percentage of body weight relative to the starting weight at the beginning of the experiment.

[0068] Results: PBS group (black line): The body weight continued to decrease after infection (from 1.000 to about 0.925), indicating that Salmonella typhimurium infection caused significant weight loss in the animals (a manifestation of disease consumption).

[0069] The acetylated raspberry ketone group (yellow line) showed an initial slight increase in body weight, followed by a stable increase (from approximately 1.000 to 1.050), with the difference from the PBS group becoming increasingly significant over time. 1.5 days: p = 0.1900 (small difference, close to significant); 2.5 days: p = 0.0450 (significant difference); 3.0 days: p = 0.0300 (significant difference); like Figure 4 As shown, the conclusion is that infection with Salmonella typhimurium causes a significant decrease in animal weight (disease-induced weight loss), while acetylated raspberry ketone can "reverse the trend of weight loss" and even cause a slight increase in weight. Example 6:

[0070] The only difference between Example 6 and Example 1 is that the solid-liquid ratio of raspberry ketone to inert solvent in solution A is 8 mmol: 50 mL, so the yield of the preparation method is only 81-86.5%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.5%~97.5%. Example 7:

[0071] The only difference between Example 7 and Example 1 is that the solid-liquid ratio of raspberry ketone to inert solvent in solution A is 10 mmol: 45 mL, so the yield of the preparation method is 80.5-88.6%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.5%~97.0%. Example 8:

[0072] The only difference between Example 8 and Example 1 is that the molar ratio of acetic anhydride to raspberry ketone added in step 2) is 6:1, so the yield of the preparation method is 80-90%, and the mass fraction of acetylated raspberry ketone in the final target product is 96.5%-97.5%. Example 9:

[0073] The only difference between Example 9 and Example 1 is that the molar ratio of acetic anhydride to raspberry ketone added in step 2) is 5:1, so the yield of the preparation method is 80-90%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.1%-96.5%.

[0074] Example 10: The only difference between Example 10 and Example 1 is that the temperature at which acetic anhydride is added in step 2) is 3°C, so the yield of the preparation method is only 84-90%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.1%-97.5%.

[0075] Example 11: The only difference between Example 11 and Example 1 is that the temperature at which acetic anhydride is added in step 2) is 5°C, the yield of the preparation method is 80-85%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.1%-97.5%.

[0076] Example 12: The only difference between Comparative Example 12 and Example 1 is that the amount of catalyst added in step 3) is 0.1 mmol, so the yield of the preparation method is only 80.5~86.2%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.1%~97.5%.

[0077] Example 13: The only difference between Comparative Example 13 and Example 1 is that the amount of catalyst added in step 3) is 2.0 mmol, so the yield of the preparation method is only 86.5~90%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.1%~97.5%.

[0078] Comparative Example 1: The only difference between Comparative Example 1 and Example 1 is that the solid-liquid ratio of raspberry ketone to inert solvent in solution A is 5 mmol: 50 mL, so the mass fraction of acetylated raspberry ketone in the final target product is only 80-85%.

[0079] Comparative Example 2: The only difference between Comparative Example 2 and Example 1 is that the solid-liquid ratio of raspberry ketone to inert solvent in solution A is 20 mmol: 45 mL, so the yield of the acetylated raspberry ketone preparation method is about 65-70%.

[0080] Comparative Example 3: The only difference between Comparative Example 3 and Example 1 is that the molar ratio of acetic anhydride to raspberry ketone added in step 2) is 3:1, so the mass fraction of acetylated raspberry ketone in the final target product is only 78%.

[0081] Comparative Example 4: The only difference between Comparative Example 4 and Example 1 is that the temperature at which acetic anhydride is added in step 2) is 10°C, which causes a dramatic increase in side reactions, and the yield of the preparation method is only 65-70%.

[0082] Comparative Example 5: The only difference between Comparative Example 5 and Example 1 is that the temperature at which acetic anhydride is added in step 2) is -10°C, so the yield of the preparation method is only 65-70%, and the mass fraction of acetylated raspberry ketone in the final target product is 95.1%-97.5%.

[0083] This invention has been described through the specific embodiments described above. Those skilled in the art should understand that various modifications and equivalent substitutions can be made to this invention without departing from its scope. Parts not described in detail in this specification are well-known to those skilled in the art. Furthermore, various modifications can be made to this invention for specific situations or circumstances without departing from its scope. Therefore, this invention is not limited to the specific embodiments disclosed, but should include all embodiments falling within its scope.

Claims

1. The use of an acetylated raspberry ketone, characterized in that, The acetylated raspberry ketone is used to prepare an antimicrobial composition, wherein the microorganism is an intracellular bacterium; the intracellular bacterium is Listeria monocytogenes or Salmonella typhimurium.

2. The use according to claim 1, characterized in that, The antimicrobial composition is an oral formulation.

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