Salvia plebeia extract, preparation method thereof and application of salvia plebeia extract in asthma treatment

Abstract

The invention discloses a salvia plebeia extract and a preparation method and application thereof in asthma treatment, and the preparation method comprises the following steps: (1) extraction: crushing salvia plebeia hay, mixing with an ethanol aqueous solution, performing ultrasonic-assisted extraction, filtering and concentrating to obtain a salvia plebeia extracting solution; and (2) drying: adding water to dilute the salvia plebeia extract obtained in the step (1), and freeze-drying to obtain the salvia plebeia extract. The preparation method of the salvia plebeia extract is simple, and rapid, efficient and energy-saving extraction of active substances of the salvia plebeia can be realized. The salvia plebeia extract disclosed by the invention has a relatively good scavenging effect on DPPH free radicals, and has a relatively good reducing capability on iron ions; the airway resistance of an ovalbumin-induced asthma model mouse can be reduced. The salvia plebeia extract prepared by the invention has no cytotoxicity and good safety, and can be used as an active component to be applied to related products such as asthma treatment drugs and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of lychee grass extracts, and particularly relates to a lychee grass extract, a preparation method thereof, and application thereof in the treatment of asthma. Background Art

[0002] Asthma is a common chronic inflammatory airway disease characterized by recurrent episodes of wheezing and shortness of breath, with or without chest tightness or cough. Its prevalence has been increasing year by year globally in recent years. Gene-environment interactions drive the onset and persistence of asthma, and multiple environmental factors, including biological and social factors, may play a significant role in the development of asthma. Traditional single-target chemical therapies are unlikely to fundamentally control asthma recurrences. Therefore, the development of natural plant extracts to pursue multi-target and causal treatments offers significant advantages for asthma control.

[0003] Salvia plebaia R. Brown, also known as snow primrose, is a member of the genus Salvia in the Lamiaceae family. First described in the Compendium of Materia Medica, it has a bitter and pungent flavor with a cooling nature. It enters the stomach, lung, and kidney meridians. Its leaves are opposite, with glandular dots on the back and short hairs on both sides. It is a biennial, upright herb, and the entire plant can be used as medicine. As a traditional Chinese medicinal ingredient, plebaia is used in traditional prescriptions and has shown excellent therapeutic effects for diarrhea, hemorrhoids, coughs, and other ailments.

[0004] Modern pharmacological research reports that lychee grass contains terpenes, phenylpropanoids, flavonoids, and polysaccharides. Its extracts have antioxidant, anti-inflammatory, hepatoprotective, antitussive and antiasthmatic, analgesic, antibacterial, and anti-tumor properties. Lychee grass is rich in rosmarinic acid, which has anti-asthma, antibacterial, antiviral, antitumor, antidepressant, and antianxiety properties and is widely used in pharmaceuticals and cosmetics. Flavonoids play an important role in anti-asthma, anti-inflammatory, anticancer, antiviral, antidiabetic, and antioxidant activities.

[0005] Therefore, how to efficiently extract the effective ingredients of Litchi Grass needs further research. Summary of the Invention

[0006] Purpose of the Invention: To address the challenges of the prior art, the present invention provides a Herba Lychieae extract, its preparation method, and its use in the treatment of asthma. The Herba Lychieae extract prepared by the present invention is non-cytotoxic and safe, and can be used as an active ingredient in asthma treatments and other related products.

[0007] Technical solution: In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a Herba Lychitae Extract, comprising the following steps:

[0009] (1) Extraction: crush the hay of Lychee Herba, mix it with ethanol and water solution, perform ultrasound-assisted extraction, filter, and concentrate to obtain Lychee Herba Extract;

[0010] (2) Drying: diluting the Herba Lychieae extract obtained in step (1) with water, and freeze-drying to obtain the Herba Lychieae extract.

[0011] As a specific implementation scheme, in step (1), the lychee grass hay is the aboveground part of lychee grass.

[0012] As a specific implementation scheme, in step (1), the lychee grass hay is crushed to a particle size between 5-5000 mesh.

[0013] As a specific embodiment, in step (1), the concentration of the ethanol aqueous solution is 20%-80%.

[0014] As a specific implementation scheme, in step (1), the mass ratio of the lychee grass to the ethanol aqueous solution is 1: (5-25).

[0015] As a specific implementation scheme, in step (1), the ultrasonic power used in the ultrasonic-assisted extraction is 10-1500W and the frequency is 10-60Hz; preferably, the ultrasonic power is 150-600W and the frequency is 20Hz.

[0016] As a specific implementation scheme, in step (1), the time of the ultrasound-assisted extraction is 20 min-60 min, and the temperature is 20-70°C; preferably, the time is 20 min and the temperature is 60°C.

[0017] In a second aspect, the present invention provides a Herba Lychieae extract, which is prepared by the above-mentioned preparation method.

[0018] In a third aspect, the present invention provides the use of the Herba Lychitae Extract in the preparation of a drug for treating asthma.

[0019] Beneficial Effects: The ultrasonic-assisted extraction technology of this invention, applied to the preparation of lychee grass extract, reduces the several hours required for conventional extraction to 5-30 minutes, reducing extraction time by 25-50% compared to conventional methods and increasing the extraction yield by over 50%. The process is simple to operate and achieves a high extraction yield, enabling industrial-scale production. Using a smaller reactor as the material container reduces noise during the extraction process without affecting the dissolution of the target substance, thereby protecting the health and safety of production workers.

[0020] In the preparation method of the lychee grass extract of the present invention, the rosmarinic acid extraction rate is higher than 5 mg / g relative to the original lychee grass dry powder raw material. (生药)The total flavonoid extraction rate is 6 mg / g (生药) The extract has good stability, and the contents of rosmarinic acid and total flavonoids between different batches are not much different, and the appearance properties of the extract are not much different. The prepared lychee grass extract has no lung cytotoxicity and has high safety. The prepared lychee grass extract has a good scavenging effect on DPPH free radicals and a strong reducing ability for iron ions. It has a good effect on reducing the airway resistance of ovalbumin-induced asthmatic mice and can be used as a therapeutic substance in the treatment of asthma and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result graph of rosmarinic acid extraction rate from lychee grass under different conditions.

[0022] Figure 2 This is the result graph of total flavonoids extraction rate from Lychee Herba under different conditions.

[0023] Figure 3 This is a graph showing the DPPH free radical scavenging ability of different concentrations of lychee grass extract solutions.

[0024] Figure 4 This is the result of measuring the antioxidant activity of different concentrations of lychee grass extract solutions using the FRAP method total antioxidant activity assay kit.

[0025] Figure 5 This figure shows the results of the cytotoxicity assay of the Herba Lychitae chinensis extract on RAW 264.7 macrophages.

[0026] Figure 6 This is a schematic diagram of the experimental process of using lychee herb extract to treat asthma in mice.

[0027] Figure 7 This is the result of measuring the airway resistance value of mice using the WBP whole body plethysmography system after the process of treating asthma mice with lychee grass extract was completed. DETAILED DESCRIPTION

[0028] The present invention will be further described by the following examples. These examples are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention. The invention will be further described below in conjunction with the accompanying drawings and examples.

[0029] Materials, reagents, and instruments

[0030] Materials: Litchi grass: purchased from Guangshuo Pharmaceutical Co., Ltd., Bozhou City, Anhui Province in April 2024

[0031] Reagents: Anhydrous ethanol (analytical grade) was from General-Reagent, methanol (chromatographic grade) was from Anhui Tiandi High Purity Solvent Co., Ltd., and formic acid (chromatographic grade) was from Aladdin.

[0032] instrument:

[0033] Magnetic stirring heater: MS-H-Pro DLAB

[0034] Rotary evaporator: N-1300, Tokyo Rika, Japan

[0035] Constant temperature water bath: OSB-2200, Tokyo Rika, Japan

[0036] Ultrasonic cleaner: KH3200 Kunshan Hechuang

[0037] High-speed centrifuge: TG16-WS Xiangli

[0038] Circulating water multi-purpose vacuum pump: SHZ-D Shanghai Xiniu Laber Instrument Co., Ltd.

[0039] Electric blast drying oven: Shanghai Yiheng

[0040] High performance liquid chromatograph: Agilent 1260 high performance liquid system, DAD detector.

[0041] Example 1

[0042] This embodiment provides an ultrasound-assisted preparation method for a lychee grass extract, the preparation method comprising:

[0043] The herb was crushed and placed in a reactor, an ultrasonic generator probe was installed, and then 60% ethanol was added at a mass-to-volume ratio of 1:20. Then, continuous countercurrent ultrasonic extraction was performed at 60°C, 20 kHz, and 150W for 30 minutes, and the filtrate was filtered to obtain a herb extract. The extract was concentrated and the solvent was replaced with methanol. The concentrate was analyzed by high performance liquid chromatography for rosmarinic acid content, and the rosmarinic acid extraction rate was 5.58 mg / g. The concentrate was taken and the total flavonoid content was determined by visible light spectrophotometry, and the total flavonoid extraction rate was 2.83 mg / g. (生药) The DPPH free radical scavenging rate was 52.75%. The concentrated extract was freeze-dried to obtain the Herba Lychitae Extract.

[0044] Example 2

[0045] This embodiment provides an ultrasound-assisted preparation method for a lychee grass extract, the preparation method comprising:

[0046] The herb was crushed and placed in a reactor, an ultrasonic generator probe was installed, and then 20% ethanol was added at a mass-to-volume ratio of 1:20. Then, continuous countercurrent ultrasonic extraction was performed at 60°C, 20 kHz, and 150W for 10 minutes, and the filtrate was filtered to obtain a herb extract. The extract was concentrated and the solvent was replaced with methanol. The concentrate was analyzed by high performance liquid chromatography for rosmarinic acid content, and the rosmarinic acid extraction rate was 6.48 mg / g. The concentrate was taken and the total flavonoid content was determined by visible light spectrophotometry, and the total flavonoid extraction rate was 3.72 mg / g. (生药) The DPPH free radical scavenging rate was 68.16%. The concentrated extract was freeze-dried to obtain the Herba Lychitae Extract.

[0047] Example 3

[0048] This embodiment provides an ultrasound-assisted preparation method for a lychee grass extract, the preparation method comprising:

[0049] The herb was crushed and placed in a reactor, an ultrasonic generator probe was installed, and then 60% ethanol was added at a mass-to-volume ratio of 1:10. Then, continuous countercurrent ultrasonic extraction was performed at 60°C, 20KHz, and 750W for 20 minutes, and the filtrate was filtered to obtain a herb extract. The extract was concentrated and the solvent was replaced with methanol. The concentrate was analyzed by high performance liquid chromatography for rosmarinic acid content, and the rosmarinic acid extraction rate was 5.11 mg / g. The concentrate was taken and the total flavonoid content was determined by visible light spectrophotometry, and the total flavonoid extraction rate was 6.18 mg / g. (生药) The DPPH free radical scavenging rate was 57.10%. The concentrated extract was freeze-dried to obtain the Herba Lychitae Extract.

[0050] Test Example 1

[0051] Analysis and detection of rosmarinic acid in lychee grass extract using high performance liquid chromatography

[0052] The rosmarinic acid in the extract of Litchi Herb was analyzed by high performance liquid chromatography. The extraction rate of rosmarinic acid was as follows: Figure 1 The chromatographic conditions are as follows:

[0053] Chromatographic column: GALAK EF-C18H, 5 μm, 4.6 × 250 mm

[0054] Mobile phase: methanol (A)-water (B, containing 0.5% formic acid); flow rate 1 ml / min

[0055] Injection volume: 20 μl

[0056] Detection wavelength: 190-400 nm, preferably 330 nm.

[0057] The test results are as shown in the instructions. Figure 1shown.

[0058] Test Example 2

[0059] Determination of total flavonoids content in extracts:

[0060] The total flavonoids content in the Herba Lychitae chinensis extract prepared by the present invention was determined by the sodium nitrite-aluminum nitrate method with rutin as a reference substance.

[0061] A rutin reference substance was prepared into a 1 mg / mL standard solution, and eight rutin solutions of different concentrations were prepared by a doubling dilution method as standard solutions; the lychee grass concentrate prepared in the preparation process of Examples 1-3 was diluted 100 times to prepare a test sample; 1.0 mL of the standard solution and the test sample were respectively taken into a 10 mL volumetric flask, 0.5 mL of a 5% sodium nitrite solution was respectively added, mixed, and allowed to stand for 6 minutes; 0.5 mL of a 1% aluminum nitrate solution was respectively added, mixed, and allowed to stand for 6 minutes; 4.0 mL of a 4% sodium hydroxide solution was respectively added, mixed, fixed to volume, and allowed to stand at room temperature for 15 minutes; 1 mL of the standard solution and the test sample solution were respectively taken into a 10 mL volumetric flask, and 95% ethanol solution was added to the fixed volume to serve as a blank control; the absorbance of each solution was tested at 510 nm, and a standard curve was drawn according to the absorbance value and the concentration of the rutin standard solution.

[0062] The obtained standard curve is Y=1.210X-0.00005488, R 2 =0.9994

[0063] The test results are as shown in the instructions. Figure 2 shown.

[0064] Test Example 3

[0065] Extract antioxidant capacity test:

[0066] A DPPH free radical scavenging experiment was used to calculate the DPPH free radical scavenging effect of the test sample through the change in absorbance level. The concentration of DPPH was prepared at 1 mg / mL; the lychee grass extract was prepared into a 1 mg / mL stock solution, and 11 test samples with different concentrations were obtained after dilution. The 96-well plate was divided into a sample group and a control group; the sample group and the DPPH solution were added in equal volumes of 1:1; the reaction was carried out at room temperature in the dark for 30 minutes, shaken and evenly mixed, and the absorbance of each point on the 96-well plate at 517 nm was measured. The absorbance of each group of samples was averaged by the duplicate wells to calculate the DPPH scavenging rate of each test sample. The DPPH scavenging rate calculation formula is as follows:

[0067] DPPH clearance rate (%) = [1-(A i -A j ) / A0]×100%

[0068] A0 is the absorbance of the control group without the solution of lychee extract, A i is the absorbance of the reaction between the lychee extract solution and the DPPH solution, A j is the absorbance of the background without DPPH free radicals.

[0069] The test results are as shown in the instructions. Figure 3 shown.

[0070] Test Example 4

[0071] The antioxidant capacity of the Herba Lychiae extract was determined using a FRAP total antioxidant activity assay kit provided by Shanghai MacLean Biochemical Technology Co., Ltd. The Herba Lychiae extract was prepared into a 1 mg / mL stock solution and diluted to obtain 11 test samples of different concentrations. The assay was performed according to the kit instructions. The assay results are shown in the attached instructions. Figure 4 shown.

[0072] Test Example 5

[0073] The extract of Litchi Herba was dissolved in DMEM solution at concentrations of 100, 200, 300, 400, and 500 μg / mL. Briefly, RAW 264.7 macrophages were cultured in DMEM medium containing 10% (v / v) PBS, 100 U / mL penicillin, and 100 U / mL streptomycin at 37°C and 5% CO2. Subsequently, 2×10 4 RAW 264.7 macrophages with a concentration of 100 μg / mL were transferred to a 96-well plate and incubated at 37°C in 5% CO2 for 24 hours. Then, 100 μL of different concentrations of lychee grass extract (100-500 μg / mL) were added to the wells and incubated for 24 hours. Then, 10 μL of CCK-8 solution was added to each well and incubated in the dark for 1 hour. The absorbance of the supernatant in each well was detected at 450 nm to analyze the toxicity of the lychee grass extract. DMEM culture medium without lychee grass extract was used as a control. The test results are shown in the attached instructions. Figure 5 shown.

[0074] Test Example 6

[0075] Ovalbumin-induced mice were used as an asthma model, and a model group, a high-dose oral group, and a low-dose oral group were set up. From day 0 to day 14, the mice were intraperitoneally injected with 200 μl of OVA sensitization solution every 7 days. The sensitization solution was prepared by 80 μg OVA, 1 mg aluminum hydroxide, and 200 μL PBS. From day 21 to day 27, 50 μl of OVA provocation solution was dripped into the nose every day. The provocation solution was prepared by 5 mg OVA and 100 μL PBS. From day 25 to day 27, the mice in the high-dose group were gavaged with 300 μL of lychee herb extract solution at a concentration of 250 mg / mL, and the mice in the low-dose group were gavaged with 150 μL of lychee herb extract solution at a concentration of 250 mg / mL. On day 28, the pulmonary airway resistance of the mice was measured using a WBP whole-body plethysmography system. The experimental process is as shown in the attached manual. Figure 5 The test results are shown in the appendix of the manual. Figure 6 shown.

[0076] The above describes the implementation of the present invention in detail with reference to specific embodiments. However, the present invention is not limited to the above implementation. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for preparing a lychee grass extract, characterized in that: The following steps are involved: (1) Extraction: crush the hay of Lychee Herba, mix it with ethanol and water solution, perform ultrasound-assisted extraction, filter, and concentrate to obtain Lychee Herba Extract; (2) Drying: diluting the Herba Lychieae extract obtained in step (1) with water, and freeze-drying to obtain the Herba Lychieae extract.

2. The method for preparing the Herba Lychitae chinensis extract according to claim 1, wherein: In step (1), the lychee grass hay is the aboveground part of lychee grass.

3. The method for preparing the Herba Lychitae chinensis extract according to claim 1, wherein: In step (1), the hay of Lychee chinensis is crushed to a particle size between 5 and 5000 meshes.

4. The method for preparing the Herba Lychitae chinensis extract according to claim 1, wherein: In step (1), the concentration of the ethanol aqueous solution is 20%-80%.

5. The method for preparing the Herba Lychitae chinensis extract according to claim 1, wherein: In step (1), the mass ratio of the lychee grass to the ethanol aqueous solution is 1: (5-25).

6. The method for preparing the Herba Lychitae chinensis extract according to claim 1, wherein: In step (1), the ultrasonic power used in the ultrasonic-assisted extraction is 10-1500 W and the frequency is 10-60 Hz; preferably, the ultrasonic power is 150-600 W and the frequency is 20 Hz.

7. The method for preparing the Herba Lychitae chinensis extract according to claim 1, wherein: In step (1), the ultrasonic-assisted extraction is carried out for 20 min to 60 min at a temperature of 20 to 70° C.; preferably, the extraction time is 20 min and the temperature is 60° C.

8. A Herba Lycheeae extract, characterized in that: The lychee grass extract is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the Herba Lychitae Extract according to claim 8 in the preparation of a drug for treating asthma.

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