Application of nepeta hemsleyana extract in treating hypertension and preparation method thereof

Abstract

The invention relates to the technical field of medicines, in particular to a nepeta hemsleyana supercritical extract for inhibiting angiotensin converting enzyme activity and a preparation method thereof, and the preparation method comprises the following steps: S1, weighing a nepeta hemsleyana medicinal material, and crushing into powder to obtain nepeta hemsleyana medicinal material powder; and S2, carrying out supercritical extraction on the nepeta hemsleyana medicinal material powder. And S3, finishing extraction, and collecting a product from a discharge port to obtain the nepeta hemsleyana supercritical extract. The method is simple and practical in process flow, low in toxicity, environment-friendly, suitable for industrial production and wide in application prospect.

Description

[0001] This application is a divisional application of the invention patent with application number 202311189411.5, entitled "A Supercritical Extract of Nepeta cataria that Inhibits Angiotensin-Converting Enzyme Activity and Its Preparation Method", filed on September 15, 2023. Technical Field

[0002] This invention relates to the field of pharmaceutical technology, specifically to a supercritical extract of *Nepeta cataria* that inhibits angiotensin-converting enzyme activity and its preparation method. Background Technology

[0003] Hypertension is one of the most common cardiovascular diseases, significantly impacting human health and affecting organs such as the brain, kidneys, and heart. Research indicates that angiotensin-converting enzyme (ACE), a dipeptide carboxypeptidase, catalyzes the cleavage of angiotensin I into the potent vasoconstrictor angiotensin II. It also inactivates bradykinin, a vasodilator, playing a crucial role in raising blood pressure. ACE inhibitors reduce ACE activity, decreasing angiotensin II production and thus weakening its mediated vasoconstriction and pressor effects, ultimately leading to vasodilation and lowering of blood pressure. Based on this well-defined pathophysiological mechanism, ACE inhibitors have been approved by the FDA and recommended in clinical guidelines for use alone or in combination for hypertension treatment, making them one of the most fundamental and effective treatment methods in current hypertension management. Currently, the main drugs used to treat hypertension are ACE inhibitors (ACEIs), such as captopril, enalapril, and lisinopril. While these drugs can effectively inhibit ACE activity and lower blood pressure, they have significant side effects. Long-term use can lead to excessive blood pressure reduction, urinary tract infections, cough, taste disturbances, and angioedema. Therefore, screening and developing novel, safe, naturally occurring ACE inhibitors remains a research hotspot in the field of antihypertensive pharmaceuticals.

[0004] Tibetan catnip, the whole herb of *Nepeta angustifolia*, a plant belonging to the genus *Nepeta* in the family Lamiaceae, is a traditional and commonly used medicinal material unique to Tibet. It possesses pharmacological effects such as clearing the mind and relieving spasms, reducing swelling and relieving pain. It is mainly used to treat coma, convulsions, stroke, epilepsy, hemorrhagic cerebral infarction, traumatic swelling and pain, etc., with good clinical efficacy. Meanwhile, studies have shown that the volatile oil of Tibetan catnip has good hypoxia tolerance and anti-fatigue effects. To date, there have been no reports of supercritical fluid extracts of Tibetan catnip inhibiting ACE activity. Summary of the Invention

[0005] The purpose of this invention is to provide a supercritical extract of *Nepeta cataria* that inhibits angiotensin-converting enzyme activity, and to provide a preparation process for this supercritical extract of *Nepeta cataria*.

[0006] The following technical solution is adopted: An application of a supercritical extract of *Nepeta cataria* for the preparation of drugs to treat or prevent hypertension.

[0007] Preferably, it is used to prepare a drug that inhibits angiotensin-converting enzyme.

[0008] A method for preparing the supercritical extract of *Nepeta cataria* as described above includes the following steps: S1. Weigh out the Tibetan thorn herb and grind it into powder to obtain Tibetan thorn herb powder; S2. Supercritical extraction is performed on the powdered Tibetan thorn herb. S3. End the extraction and collect the product from the outlet to obtain the supercritical extract of *Nepeta cataria*.

[0009] Preferably, in step S2, the supercritical extraction includes supercritical carbon dioxide extraction.

[0010] Preferably, in step S2, the specific operation of supercritical extraction includes: loading the Tibetan thorn herb powder into the extraction tank of the supercritical equipment, turning on the equipment to heat it, setting the equipment parameters, and after the temperature rises to the set temperature, setting the carbon dioxide flow rate to the maximum, and pumping carbon dioxide to the set pressure for extraction.

[0011] Preferably, in step S2, the equipment parameters specifically include: extraction pressure of 20-30 MPa, extraction temperature of 35-45 degrees Celsius, separation pressure of 6-8 MPa, and extraction time of 60-100 minutes.

[0012] Preferably, step S1 specifically includes: first pulverizing the Tibetan thorn herb into coarse powder using a pulverizer, and then pulverizing it into fine powder using a wall-breaking machine to obtain the Tibetan thorn herb powder.

[0013] Preferably, in step S3, the nepeta lactone content of the product is 25% to 35%.

[0014] Through experimental research, this invention has found that the supercritical fluid extract of *Nepeta cataria* exhibits a good ACE inhibitory effect. Simultaneously, it was discovered that nepeta lactone in the *Nepeta cataria* extract also has a good ACE inhibitory effect. Therefore, the preparation process of *Nepeta cataria* was studied using nepeta lactone content as an evaluation index, and the optimal preparation process was determined. However, in fact, the ACE inhibitory effect in the obtained *Nepeta cataria* extract is not solely contributed by nepeta lactone; other active substances are also present, warranting further in-depth research.

[0015] Preferably, step S3 further includes filtering and drying the product to obtain a supercritical extract of *Nepeta cataria*.

[0016] Before the product is dried, the approximate content of nepeta lactone can be estimated by means such as liquid phase data calculation, and the process conditions can be optimized accordingly.

[0017] A supercritical extract of *Nepeta cataria* obtained by the method described above.

[0018] Compared with the prior art, implementing the present invention has the following beneficial effects: (1) This invention is the first to discover that the supercritical extract of Nepeta cataria has a significant angiotensin-converting enzyme inhibitory effect.

[0019] (2) This invention discovers for the first time a new use of supercritical extract of Nepeta cataria as a drug: it can be used to prepare drugs for lowering blood pressure.

[0020] (3) The present invention provides a method for preparing supercritical extract of *Nepeta cataria* that inhibits angiotensin-converting enzyme activity. The *Nepeta cataria* medicinal material is pulverized and cell wall broken in advance, so that the tissue structure of the treated *Nepeta cataria* becomes loose, which is more conducive to improving the extraction yield of *Nepeta cataria* components by supercritical carbon dioxide fluid.

[0021] (4) The present invention provides a method for preparing supercritical extract of Nepeta cataria that inhibits angiotensin-converting enzyme activity. By adopting steps such as crushing and breaking the cell wall and supercritical fluid extraction, the process is simple, practical, low-toxic and environmentally friendly, and can be applied to industrial production with broad application prospects. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments are provided to further describe the invention in detail, enabling those skilled in the art to better understand and implement it. However, the embodiments are not intended to limit the invention. Modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of the invention are all within the scope of this invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0023] Example 1 An extraction process for supercritical fluid extract of *Nepeta cataria* that can inhibit angiotensin-converting enzyme activity is described below: (1) 245.22 grams of Tibetan thorn herb were weighed and crushed into coarse powder in a DFY-500 swing-type high-speed pulverizer, and then crushed into fine powder in a BL2147 Chinese medicine wall-breaking machine.

[0024] (2) Put the powdered Tibetan thorn herb into the extraction tank of the HA230-65-07-CC supercritical equipment, turn on the equipment to heat up, set the extraction pressure to 20 MPa, the extraction temperature to 45 degrees Celsius, and the separation pressure to 7 MPa. After the temperature rises to the set temperature, set the carbon dioxide flow rate to the maximum, pump carbon dioxide into the set pressure, and maintain constant temperature and pressure for 100 minutes of circulating extraction.

[0025] (3) The content of the quality control component nepeta lactone in the extract was determined to be 35% by high performance liquid chromatography with external standard method. The extraction was then stopped, the product was collected from the outlet, filtered and dried to obtain 10.87 g of supercritical extract of Nepeta cataria.

[0026] Example 2 The inhibitory effect of supercritical fluid extract of *Vitex negundo* on enzyme activity was detected by an angiotensin-converting enzyme inhibition assay. The specific steps included: (1) Weigh 19.07 g of borax and 12.37 g of boric acid, dissolve them in 1 liter of ultrapure water, measure 325 ml of boric acid solution and 175 ml of borax solution, mix them well, adjust the pH to 8.3 with hydrochloric acid or sodium hydroxide aqueous solution, add 17.532 g of sodium chloride, and make up to 1 liter to prepare borate buffer solution.

[0027] (2) Weigh a certain amount of hippuryl-histidine-leucine and add it to borate buffer to prepare a solution of 5 mmol / L; add 10 mL of borate buffer to a reagent bottle containing 1 unit of angiotensin-converting enzyme, shake to dissolve it completely, and prepare angiotensin-converting enzyme solution.

[0028] (3) Weigh a certain amount of captopril and add it to borate buffer to prepare a solution of 100 micrograms per milliliter, which will be used as a positive control. Weigh a certain amount of supercritical extract of Nepeta cataria and add it to borate buffer to prepare a test solution of 100 micrograms per milliliter.

[0029] (4) Transfer 100 μL of the test solution and 100 μL of hippuryl-histidine-leucine solution, stir and mix evenly, and place in a 37°C water bath for 5 minutes.

[0030] (5) Add 10 μL of angiotensin-converting enzyme solution, react at a constant temperature of 37°C for 30 minutes, and add 150 μL of 1 mol / L hydrochloric acid solution to terminate the reaction; add 1.5 mL of ice-cold ethyl acetate, vortex to mix evenly, centrifuge at 3500 rpm for 5 minutes, and then take 1 mL of the ester layer into another clean test tube and dry at 85°C for 1 h.

[0031] (6) Add 3 ml of ultrapure water and check the absorbance at 228 nm wavelength using a UV spectrophotometer.

[0032] The calculation formula is as follows: Angiotensin-converting enzyme inhibition rate = (Model group - Sample group) / (Model group - Negative control group) × 100%. Detection results: Absorbance value of the negative control group was 0.1167, the model group was 0.5170, the captopril group was 0.2390, and the supercritical extract of *Schizonepeta tenuifolia* was 0.1990. Data analysis showed that the inhibition rate of captopril was 69.44%, and the inhibition rate of supercritical extract of *Schizonepeta tenuifolia* was 81.68%.

[0033] Example 3 An extraction process for supercritical fluid extract of *Nepeta cataria* that can inhibit angiotensin-converting enzyme activity is described below: (1) 247.81 grams of Tibetan thorn herb were weighed and crushed into coarse powder in a DFY-500 swing-type high-speed pulverizer, and then crushed into fine powder in a BL2147 Chinese medicine wall-breaking machine.

[0034] (2) Put the powdered Tibetan thorn herb into the extraction tank of the HA230-65-07-CC supercritical equipment, turn on the equipment to heat up, set the extraction pressure to 28 MPa, the extraction temperature to 35 degrees Celsius, and the separation pressure to 7 MPa. After the temperature rises to the set temperature, set the carbon dioxide flow rate to the maximum, pump carbon dioxide into the set pressure, and maintain constant temperature and pressure for 60 minutes of circulating extraction.

[0035] (3) The content of the quality control component nepeta lactone in the extract was determined to be 25% by high performance liquid chromatography with external standard method. The extraction was then stopped, the product was collected from the outlet, filtered and dried to obtain 8.69 g of supercritical extract of Nepeta cataria.

[0036] Example 4 The inhibitory effect of supercritical fluid extract of *Vitex negundo* on enzyme activity was detected by an angiotensin-converting enzyme inhibition assay. The specific steps included: (1) Weigh 19.07 g of borax and 12.37 g of boric acid, dissolve them in 1 liter of ultrapure water, measure 325 ml of boric acid solution and 175 ml of borax solution, mix them well, adjust the pH to 8.3 with hydrochloric acid or sodium hydroxide aqueous solution, add 17.532 g of sodium chloride, and make up to 1 liter to prepare borate buffer solution.

[0037] (2) Weigh a certain amount of hippuryl-histidine-leucine and add it to borate buffer to prepare a solution of 5 mmol / L; add 10 mL of borate buffer to a reagent bottle containing 1 unit of angiotensin-converting enzyme, shake to dissolve it completely, and prepare angiotensin-converting enzyme solution.

[0038] (3) Weigh a certain amount of captopril and add it to borate buffer to prepare a solution of 100 micrograms per milliliter, which will be used as a positive control. Weigh a certain amount of supercritical extract of Nepeta cataria and add it to borate buffer to prepare a test solution of 100 micrograms per milliliter.

[0039] (4) Transfer 100 μL of the test solution and 100 μL of hippuryl-histidine-leucine solution, stir and mix evenly, and place in a 37°C water bath for 5 minutes.

[0040] (5) Add 10 μL of angiotensin-converting enzyme solution, react at a constant temperature of 37°C for 30 minutes, and add 150 μL of 1 mol / L hydrochloric acid solution to terminate the reaction; add 1.5 mL of ice-cold ethyl acetate, vortex to mix evenly, centrifuge at 3500 rpm for 5 minutes, and then take 1 mL of the ester layer into another clean test tube and dry at 85°C for 1 h.

[0041] (6) Add 3 ml of ultrapure water and check the absorbance at 228 nm using a UV spectrophotometer. The calculation formula is as follows: Angiotensin-converting enzyme inhibition rate = (Model group - Sample group) / (Model group - Negative control group) × 100%. Detection results: Absorbance value of the negative control group was 0.1167, the model group was 0.5170, the captopril group was 0.2410, and the supercritical extract of *Schizonepeta tenuifolia* was 0.2000. Data analysis showed that the inhibition rate of captopril was 68.94%, and the inhibition rate of supercritical extract of *Schizonepeta tenuifolia* was 79.19%.

[0042] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An application of a supercritical extract of *Nepeta cataria*, characterized in that, Used to prepare drugs for treating hypertension.

2. The application of the supercritical extract of *Nepeta cataria* as described in claim 1, characterized in that, Used to prepare drugs that inhibit angiotensin-converting enzyme.

3. A method for preparing the supercritical extract of *Nepeta cataria* as described in claim 1, characterized in that, Includes the following steps: S1. Weigh out the Tibetan thorn herb and grind it into powder to obtain Tibetan thorn herb powder; S2. The powdered Tibetan thorn herb is subjected to supercritical carbon dioxide extraction. S3. End the extraction and collect the product from the outlet to obtain the supercritical extract of *Nepeta cataria*.

4. The method for preparing the supercritical extract of *Nepeta cataria* as described in claim 2, characterized in that, In step S2, the specific operation of supercritical extraction includes: loading the Tibetan thorn herb powder into the extraction tank of the supercritical equipment, turning on the equipment to heat it, setting the equipment parameters, and after the temperature rises to the set temperature, setting the carbon dioxide flow rate to the maximum, and pumping carbon dioxide to the set pressure for extraction.

5. The method for preparing the supercritical extract of *Nepeta cataria* as described in claim 4, characterized in that, In step S2, the specific equipment parameters include: extraction pressure of 20-30 MPa, extraction temperature of 35-45 degrees Celsius, separation pressure of 6-8 MPa, and extraction time of 60-100 minutes.

6. The method for preparing the supercritical extract of *Nepeta cataria* as described in claim 3, characterized in that, The specific operation of step S1 includes: first crushing the Tibetan thorn herb into coarse powder using a pulverizer, and then crushing it into fine powder using a wall-breaking machine to obtain the Tibetan thorn herb powder.

7. The method for preparing the supercritical extract of *Nepeta cataria* as described in claim 3, characterized in that, In step S3, the product contains 25% to 35% nepeta lactone.

8. The method for preparing the supercritical extract of *Nepeta cataria* as described in claim 3, characterized in that, Step S3 also includes filtering and drying the product to obtain a supercritical extract of *Nepeta cataria*.

9. A supercritical extract of *Nepeta cataria* obtained by the method for preparing supercritical extract of *Nepeta cataria* as described in claim 3.