Lettuce extract and its use in reducing uric acid

Abstract

The present application relates to a kind of natural extract technical field, specifically to a kind of lettuce extract and its application in reducing uric acid.The preparation method comprises the following steps: (1) lettuce leaf freeze-drying, after being pulverized, obtain lettuce freeze-dried powder;(2) in the condition that inert gas is continuously imported, in the environment without oxygen, lettuce freeze-dried powder is mixed with pure water, stirring is made to uniformly disperse powder, heating is boiled, and polyphenol oxidase is completely denatured and inactivated;(3) after enzyme inactivation is finished, in the oxygen-free environment, stop heating and make temperature drop to room temperature, and extract by stirring;(4) filter in the oxygen-free environment, freeze the filtrate, and freeze-drying is obtained, to obtain lettuce extract dry powder.The present application adopts freeze-dried raw material pretreatment, oxygen-free boiling enzyme inactivation, nitrogen protection extraction, low-temperature freeze-drying integrated combination process, and the technical difficulties that chrysanthemum acid is prone to enzymatic oxidation, chemical oxidation and thermal degradation in the existing conventional extraction process are solved.

Description

Technical Field

[0001] This invention relates to the field of natural extract technology, specifically to a lettuce extract and its application in lowering uric acid. Background Technology

[0002] Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder. It is an important biochemical basis for gout attacks and is closely related to hypertension, chronic kidney disease, and metabolic syndrome. Currently, commonly used uric acid-lowering drugs mainly include xanthine oxidase inhibitors (such as allopurinol and febuxostat) and uricosuric drugs (such as benzbromarone). However, long-term use of allopurinol may cause severe hypersensitivity syndrome, febuxostat has been warned of the risk of adverse cardiovascular events, and benzbromarone is restricted in use in some countries and regions due to its hepatotoxicity. Therefore, developing uric acid-lowering active ingredients from natural products with higher safety and fewer side effects has an urgent clinical need and important application value.

[0003] lettuce( Lactuca sativa L . Lettuce (Lactuca sativa) is a plant belonging to the genus Lactuca in the family Asteraceae, and is an important vegetable widely cultivated and consumed globally. Chicoric acid is one of the most abundant characteristic phenolic acids in lettuce. Its chemical structure contains caffeoyl and tartaric acid groups, exhibiting strong antioxidant and anti-inflammatory physiological activities. However, the catechol structure in the chicoric acid molecule makes it extremely sensitive to oxygen, readily undergoing oxidative degradation during extraction, concentration, and drying, leading to a significant decrease in content and a marked reduction in biological activity. This chemical instability presents considerable challenges to the extraction and activity research of chicoric acid in lettuce.

[0004] In terms of extraction technology, existing technologies for extracting phenolic acids from lettuce and similar plants mostly employ conventional water extraction or alcohol extraction methods. These methods are typically carried out under open or ordinary aerobic conditions. Dissolved oxygen in the extraction solvent and air introduced during crushing and stirring both cause severe oxidative degradation of chicoric acid. Simultaneously, polyphenol oxidases in lettuce tissues retain catalytic activity during the initial stages of raw material crushing, and under aerobic conditions, this further accelerates the enzymatic oxidation and loss of chicoric acid. In subsequent processing, conventional hot air drying or vacuum concentration also leads to secondary degradation and destruction of chicoric acid in the extract due to thermal effects and residual oxygen. These technological deficiencies result in low actual retention and activity of chicoric acid in lettuce extracts obtained by existing methods.

[0005] Among the known reports of medicinal activity, existing technologies disclose a variety of natural plant extracts with the potential to lower uric acid, such as celery seed extract and chicory root extract. However, as a common vegetable, lettuce extracts have long been considered only as a source of nutrients such as dietary fiber, and their medicinal value in lowering uric acid has not been recognized or disclosed in existing technologies.

[0006] In summary, there is an urgent need in the field to develop a method that can effectively inhibit the oxidative degradation of chicoric acid during the extraction process, in order to obtain lettuce extracts with high chicoric acid retention and high uric acid-lowering activity, thereby realizing new medicinal applications. This invention is a solution proposed to address the aforementioned technical problems.

[0007] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0008] This invention relates to the field of natural extract technology, specifically to a lettuce extract and its application in lowering uric acid.

[0009] To address the aforementioned technical problems, one objective of this invention is to provide a method for preparing lettuce extract, comprising the following steps: (1) Lettuce leaves were freeze-dried and then pulverized to obtain freeze-dried lettuce powder; (2) Under the condition of continuous inert gas and oxygen-free environment, the freeze-dried lettuce powder is mixed with pure water, stirred to disperse the powder evenly, and heated to boiling to completely denature and deactivate the polyphenol oxidase. (3) After enzyme inactivation, stop heating and allow the temperature to drop to room temperature in an anaerobic environment, then stir and extract. (4) Filter under anaerobic conditions, freeze the filtrate and freeze dry to obtain lettuce extract powder.

[0010] According to a preferred embodiment, in step (1), the sample environment pressure during freeze-drying is 0~100 Pa, the sample environment temperature is 20~30℃, and the freeze-drying duration is 12~48 h. Preferably, the sample environment pressure is 0, 10, 20, 30, 50, 70, 100 Pa or any value within this range. The sample environment temperature is 20, 25, 30℃ or any value within this range. The freeze-drying duration is 12, 24, 48 h or any value within this range.

[0011] According to a preferred embodiment, in step (2), the freeze-dried lettuce powder is mixed with water at a mass ratio of 1:40 to 100. Preferably, the freeze-dried lettuce powder is mixed with water at a mass ratio of 1:40, 50, 60, 70, 80, 90, 100, or any value within this range.

[0012] According to a preferred embodiment, in step (2), the inert gas is nitrogen.

[0013] According to a preferred embodiment, in step (3), the stirring extraction time is 1 to 2 hours. Preferably, the stirring extraction time is 1 or 2 hours, or any time within this range.

[0014] According to a preferred embodiment, in step (2), the heating to completely denature and inactivate the polyphenol oxidase is: heating at 80~100℃ for 5~15 min. Preferably, heating at 80℃ for 5, 10 or 15 min. Heating at 90℃ for 5, 10 or 15 min. Heating at 100℃ for 5, 10 or 15 min.

[0015] One of the objectives of this invention is to provide a lettuce extract prepared based on the above-described method for preparing lettuce extract.

[0016] One of the objectives of this invention is to provide the use of the above-mentioned lettuce extract in the preparation of a medicament for treating hyperuricemia.

[0017] One of the objectives of this invention is to provide a composition comprising lettuce extract prepared by the above-described method for preparing lettuce extract and excipients.

[0018] According to a preferred embodiment, the dosage form of the composition is tablets, capsules, granules, oral liquids, or solid beverages.

[0019] The beneficial effects of this technical solution are: 1. This invention adopts an integrated process of freeze-dried raw material pretreatment, anaerobic boiling to inactivate enzymes, nitrogen-protected extraction, and low-temperature freeze-drying. It specifically solves the technical problem that chicoric acid is prone to enzymatic oxidation, chemical oxidation, and thermal degradation in existing conventional extraction processes. It blocks the loss of chicoric acid from the whole process and multiple dimensions, and significantly improves the chicoric acid content and extraction recovery rate in the final product.

[0020] Freeze-drying is used to process fresh lettuce leaves. The low-temperature freeze-drying process can completely remove the internal moisture of the raw materials. At the same time, it can effectively inhibit the biological activity of endogenous polyphenol oxidase in lettuce from the source, avoid the enzymatic oxidation and decomposition of chicoric acid during the storage and pre-treatment stages of raw materials, and build a solid first line of defense for the retention of chicoric acid.

[0021] Meanwhile, the nitrogen-protected anaerobic boiling enzyme inactivation process is used to boil the lettuce freeze-dried powder solution at a constant temperature for at least 5 minutes in an anaerobic environment, which can completely denature and inactivate the residual polyphenol oxidase, eliminate the degradation and damage of chicoric acid by enzymatic oxidation during the extraction reaction stage, and eliminate the hidden danger of enzymatic hydrolysis loss.

[0022] In addition, the nitrogen-protected closed extraction system can completely eliminate dissolved oxygen in the extraction solvent, while isolating the outside air from contact with the extract, blocking the chemical oxidation pathway of chicoric acid, and solving the problem of severe oxidation loss in conventional open extraction.

[0023] Finally, the terminal uses a freeze-drying dehydration process, which is low-temperature and heat-free throughout the entire process. This completely avoids the high-temperature thermal degradation caused by traditional hot air drying and reduced-pressure heating concentration processes, while also avoiding secondary oxidation reactions caused by heating, thus maximizing the preservation of the activity and content of chicoric acid in the extract.

[0024] 2. The extraction process of this invention uses only pure water as the sole extraction solvent throughout the entire process, eliminating the use of various organic solvents such as ethanol and methanol. This fundamentally eliminates a series of problems such as organic solvent residue, solvent volatilization pollution, and waste liquid treatment. The entire extraction process involves no toxic or harmful substances and produces no pollutants, making the process green, environmentally friendly, clean, and safe. Furthermore, the solvent-free final product has high purity and good safety, fully complying with the production standards and access requirements for food, health food, and functional dietary ingredients. It has a wide range of applications and possesses extremely high market value.

[0025] 3. This invention is the first to clearly verify that the lettuce extract prepared by the above-mentioned proprietary process possesses excellent uric acid-lowering bioactivity. It breaks through the existing understanding that lettuce is merely an edible vegetable with no clear efficacy in intervening in hyperuricemia, thus clarifying a new application direction for lettuce in the intervention of hyperuricemia and the development of functional uric acid-lowering products. Animal model experiments using potassium oxonate-induced hyperuricemia demonstrated that, after gavage administration of the lettuce extract prepared in this invention to hyperuricemic rats / mice, the serum uric acid levels of the experimental animals were significantly and effectively reduced compared to the untreated model group. This clearly confirms the uric acid-lowering pharmacological activity of the extract, providing a new raw material selection and technical solution for the natural and safe intervention of hyperuricemia.

[0026] The raw material used in this invention is common edible lettuce, which has clear medicinal and edible properties, with no contraindications or potential safety risks. The core active ingredient, chicoric acid, is a natural plant phenolic acid with good biocompatibility and high human tolerance. Furthermore, this invention has been verified through systematic animal safety experiments. Within the effective dosage range, the experimental animals maintained good growth throughout the process, with no abnormalities in weight gain, daily diet, behavior, mental state, or other physiological indicators. No acute or chronic toxic side effects were observed. The overall product is safe, reliable, and highly tolerable, unlike chemical uric acid-lowering drugs which can cause liver and kidney damage and significant side effects. It can be used long-term in the development of functional foods and health products, meeting the high uric acid intervention needs of various population groups. Attached Figure Description

[0027] Figure 1The graphs show the serum uric acid levels in normal mice, hyperuricemia-inducing mice, allopurinol, lettuce extract prepared in Example 1, and chicoric acid after feeding them to hyperuricemia-inducing mice. The normal control group received normal diet and water, with no modeling treatment. The model group received potassium oxonate to induce a hyperuricemia model, with no intervention. The allopurinol group consisted of potassium oxonate and allopurinol. The chicoric acid group consisted of potassium oxonate and chicoric acid. The lettuce extract group consisted of potassium oxonate and lettuce extract. Figure 2 The graph shows the change in body weight over time of mice after feeding normal mice, hyperuricemic mice, allopurinol, lettuce extract prepared in Example 1, and chicoric acid to hyperuricemic mice. Figure 3 XIC diagram of chicoric acid standard; Figure 4 MS chromatogram of chicoric acid standard; Figure 5 MS / MS chromatogram of chicoric acid standard; Figure 6 XIC chromatogram of lettuce extract from Example 1; Figure 7 MS image of lettuce extract from Example 1; Figure 8 The image shows the MS / MS chromatogram of the lettuce extract from Example 1. Detailed Implementation

[0028] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0030] Example 1 This embodiment relates to a lettuce extract. Step (1): Raw material preparation Fresh lettuce leaves were placed in a freeze dryer and freeze-dried at 25°C and 20 Pa for 48 hours. After removal, the leaves were pulverized to obtain freeze-dried lettuce powder, which was then placed in an extraction container.

[0031] Step (2): Establishment of an anaerobic environment Nitrogen gas was continuously introduced into the extraction container containing freeze-dried lettuce powder and the container containing water for at least 10-20 minutes to purge the air from the container and establish and maintain an anaerobic environment.

[0032] Step (3): Enzyme inactivation Under continuous nitrogen purging, freeze-dried lettuce powder and pure water were mixed at a ratio of 1:100 (w / w), stirred to disperse the powder evenly, heated to boiling, and kept at a gentle boil for 10 minutes to completely denature and inactivate polyphenol oxidase.

[0033] Step (4): Anaerobic extraction After enzyme inactivation, stop heating and allow the system temperature to drop to room temperature. Continue to maintain an anaerobic, sealed environment under nitrogen protection and continue stirring for 2 hours.

[0034] Step (5): Anaerobic filtration After extraction, the mixture is filtered under nitrogen protection, the filtrate is collected, and the filter residue is discarded.

[0035] Step (6): Freeze-drying The obtained filtrate was dispensed into 2.5 mL EP tubes, flash-frozen with liquid nitrogen, and then freeze-dried in a rotary vacuum dryer at 30°C, 10 Pa vacuum, and 1500 rpm for 24 hours to obtain lettuce extract powder.

[0036] Each tube of dry powder is reconstituted with 200 μL of pure water and can then be used for mouse feeding experiments. Additionally, 800 μL of methanol solution is quickly added to the reconstituted solution, mixed well, centrifuged to remove the precipitate, and the supernatant is collected and diluted 100-fold again for mass spectrometry detection of chicoric acid content. Figure 6 , Figure 7 , Figure 8 The test results showed that the chicoric acid content was 10 mg chicoric acid / g dry weight of lettuce. Based on an oral gavage dose of 200 μL per mouse per day, the chicoric acid intake per kilogram of mouse was 10 mg.

[0037] Example 2 This embodiment relates to a lettuce extract.

[0038] Step (1): Raw material preparation Fresh lettuce leaves were placed in a freeze dryer and freeze-dried at 25°C and 0 Pa for 48 hours. After removal, the leaves were pulverized to obtain freeze-dried lettuce powder, which was then placed in an extraction container.

[0039] Step (2): Establishment of an anaerobic environment Nitrogen gas was continuously introduced into the extraction container containing freeze-dried lettuce powder and the container containing water for at least 10-20 minutes to purge the air from the container and establish and maintain an anaerobic environment.

[0040] Step (3): Enzyme inactivation Under continuous nitrogen purging, freeze-dried lettuce powder and pure water were mixed at a ratio of 1:40 (w / w), stirred to disperse the powder evenly, and heated to 90°C for 15 minutes to completely denature and inactivate polyphenol oxidase.

[0041] Step (4): Anaerobic extraction After enzyme inactivation, stop heating and allow the system temperature to drop to room temperature. Continue to maintain an anaerobic, sealed environment under nitrogen protection and continue stirring for 1 hour.

[0042] Step (5): Anaerobic filtration After extraction, the solution is filtered in an anaerobic operating table under nitrogen protection, the filtrate is collected, and the filter residue is discarded.

[0043] Step (6): Freeze-drying The obtained filtrate was dispensed into 5 mL EP tubes, 2 mL of which was then rapidly frozen in liquid nitrogen and placed in a rotary vacuum dryer. The tubes were freeze-dried at 30°C, 1 Pa vacuum, and 1500 rpm for 20 hours to obtain lettuce extract powder. Each tube of powder was reconstituted with 200 μL of pure water. 800 μL of methanol solution was quickly added to the reconstituted solution, mixed thoroughly, centrifuged to remove the precipitate, and the supernatant was collected and diluted 100-fold again for mass spectrometry analysis of chicoric acid content. The results showed a chicoric acid content of 17 mg chicoric acid / g dry lettuce weight.

[0044] Example 3 This embodiment is basically the same as Embodiment 1, except that the sample environment pressure during freeze-drying is 100 Pa and the ambient temperature is 20℃.

[0045] Example 4 This embodiment is basically the same as Embodiment 1, except that the sample is freeze-dried at an ambient temperature of 30°C.

[0046] Example 5 This embodiment is basically the same as embodiment 1, except that in step (3), the temperature is heated to 80°C and held for 5 minutes.

[0047] Test case A rat model of hyperuricemia induced by potassium oxonate was established. Male C57BL / 6J mice were used and acclimatized for 7 days under an ambient temperature of 22–25°C, relative humidity of 50%–60%, and a 12-hour light-dark cycle with free access to food and water. They were randomly divided into a normal control group, a model group, an allopurinol group (allopurinol, 5 mg / kg / day), a lettuce extract group (chicoric acid content 10 mg / kg / day), and a chicoric acid group (30 mg / kg / day, as shown in the original text). Figure 3 , Figure 4 , Figure 5 Each group was administered the drug once daily by gavage, with a volume of 200 μL each time, for 12 consecutive days. The normal control group did not undergo modeling or drug administration. The other groups were injected intraperitoneally with potassium oxonate (300 mg / kg / day) 1 hour before each administration to establish the model. Blood samples were taken to measure serum uric acid levels after the last administration.

[0048] The results showed that, compared with the model group, the serum uric acid levels of mice in the allopurinol group, lettuce extract group, and chicoric acid group all decreased. During the experiment, no abnormalities were observed in the weight, diet, or behavior of the animals in any group.

[0049] The experimental results showed that, compared with the control group (30 mg / kg / day of chicoric acid), the lettuce extract with a chicoric acid content of 10 mg / kg / day exhibited a superior uric acid-lowering effect. This result indicates that a plant extract system with a low chicoric acid content can still achieve a better uric acid-lowering intervention effect. These results demonstrate that the lettuce extract prepared by the method of this invention has the effect of lowering blood uric acid at an effective uric acid-lowering level.

[0050] Example 6 This embodiment relates to a lettuce extract in tablet form.

[0051] Take 995 g of lettuce extract obtained in Example 1 and 5 g of magnesium stearate, mix them evenly, compress them directly into tablets, and make 1000 tablets, each tablet containing 1 g and each tablet containing 995 mg of lettuce extract.

[0052] Example 7 This embodiment relates to a lettuce extract in capsule form.

[0053] Take 500 g of the lettuce extract obtained in Example 1 and fill it directly into hard capsules without adding any excipients to make 1000 capsules, each containing 500 mg of lettuce extract (or fill up to the upper limit according to the capsule shell capacity).

[0054] Example 8 This embodiment relates to a lettuce extract based on a solid beverage state.

[0055] The lettuce extract obtained in Example 1 was directly packaged without any excipients. Each 1 g bag contained 1000 mg of lettuce extract.

[0056] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention, all of which fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification is illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a lettuce extract, characterized in that, It includes the following steps: (1) Lettuce leaves were freeze-dried and then pulverized to obtain freeze-dried lettuce powder; (2) Under the condition of continuous inert gas and oxygen-free environment, the freeze-dried lettuce powder is mixed with pure water, stirred to disperse the powder evenly, and heated to completely denature and deactivate the polyphenol oxidase. (3) After enzyme inactivation, stop heating and allow the temperature to drop to room temperature in an anaerobic environment, then stir and extract. (4) Filter under anaerobic conditions, freeze the filtrate and freeze dry to obtain lettuce extract powder.

2. The preparation method according to claim 1, characterized in that, In steps (1) and (4), the sample environment pressure during freeze-drying is 0~100 Pa, the sample environment temperature is 20~30℃, and the freeze-drying duration is 12~48 h.

3. The preparation method according to claim 1, characterized in that, In step (2), the freeze-dried lettuce powder is mixed with water at a mass ratio of 1:40~100.

4. The preparation method according to claim 1, characterized in that, In step (2), the inert gas is nitrogen.

5. The preparation method according to claim 1, characterized in that, In step (2), the heating to completely denature and inactivate polyphenol oxidase is: heating at 80~100℃ for 5~15 min.

6. The preparation method according to claim 1, characterized in that, In step (3), the stirring extraction time is 1~2 h.

7. A lettuce extract, characterized in that, The lettuce extract is prepared based on the lettuce extract preparation method according to any one of claims 1 to 6.

8. Use of the lettuce extract of claim 7 in the preparation of a medicament for treating hyperuricemia.

9. A composition, characterized in that, It includes lettuce extract and excipients prepared by the method for preparing lettuce extract according to any one of claims 1 to 6.

10. The composition according to claim 9, characterized in that, The composition is available in the form of tablets, capsules, granules, oral liquids, or solid beverages.

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