An areca catechu extract, a preparation method and application thereof

Abstract

The present application relates to a kind of areca seed extract and its preparation method and application, it relates to the technical field of medicine.The areca seed extract includes total alkaloid extract, total flavone extract, total alkaloid extract is after areca seed alkalization microwave extraction, extraction is obtained, total flavone extract is obtained using ionic liquid synergic microwave extraction for areca seed.The areca seed extract has better analgesic anti-inflammatory effect, for the inflammation / pain caused by physical damage, chemical damage, and the inflammation / pain caused by bacterial infection has better treatment effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a sago palm extract, its preparation method, and its application. Background Technology

[0002] Sago palm (Arengawesterhoutii Griffith), belonging to the genus Arengawesterhoutii in the family Arecaceae, is also known as southern coconut, sago palm, and face palm. It is a commonly used traditional medicine in southern my country, and is recorded in ancient herbal texts and floras of various regions. Sago palm fruit is the dried fruit of the sago palm plant. Its medicinal effects are recorded in the "Kaibao Materia Medica" and "Compendium of Materia Medica" as "treating stagnant blood," "breaking up stagnant blood and accumulated blood, and the juice can treat postpartum hematoma and various pains in women, as well as cold pain in the heart and stomach." It has the functions of promoting blood circulation, removing blood stasis, and eliminating food stagnation. It is often used to treat many diseases in women caused by blood stasis, such as irregular menstruation, menstrual disorders, lower abdominal distension and pain, postpartum abdominal pain due to blood stasis, lochia retention, food stagnation, indigestion, and cold pain in the stomach and abdomen.

[0003] Natural products are an important source of drugs, and finding innovative drugs from traditional medicinal materials has always been a crucial avenue for drug discovery. Continuously searching for more active components and key pharmacodynamic substances from traditional Chinese medicine resources for anti-inflammatory and analgesic purposes is one of the effective ways to further develop highly effective and low-toxicity anti-inflammatory and analgesic drugs. Therefore, further development and utilization of the effective components in Areca catechu is of great significance. Summary of the Invention

[0004] To address the above problems, the present invention provides a sago palm extract, which has good analgesic and anti-inflammatory effects and is effective in treating inflammation / pain caused by physical damage, chemical damage, and bacterial infection.

[0005] To achieve the above objectives, the present invention provides a sago palm extract, comprising a total alkaloid extract and a total flavonoid extract. The total alkaloid extract is obtained by microwave extraction and extraction of sago palm after alkalization, and the total flavonoid extract is obtained by microwave extraction of sago palm using ionic liquid synergistically.

[0006] The above method preserves the total alkaloids and flavonoids in sago palm to the greatest extent. The combined sago palm extract has good analgesic and anti-inflammatory effects and is effective in treating inflammation / pain caused by physical and chemical damage, as well as inflammation / pain caused by bacterial infection, especially inflammation / pain caused by oral ulcers.

[0007] The present invention also provides a method for preparing the areca nut extract, wherein the method for preparing the total alkaloid extract includes the following steps:

[0008] Pretreatment: Boil and wash the sago palms with alkaline solution, dry, spray with alkaline solution, and dry again;

[0009] Preparation of total alkaloid extract: The pretreated sago palms were alkalized, allowed to stand, and then extracted by microwave to obtain the total alkaloid extract.

[0010] The preparation method of the total flavonoid extract includes the following steps:

[0011] Pretreatment: Areca catechu was soaked in an ionic liquid;

[0012] Preparation of total flavonoid extract: The pretreated Areca catechu was extracted and purified using an ionic liquid-assisted microwave extraction method to obtain the total flavonoid extract.

[0013] Ionic liquids (ILs) are ionic compounds composed of inorganic anions and relatively large organic cations. They have relatively low charge density and weak electrostatic forces with oppositely charged ions, thus lowering their melting point. When these forces fall below a certain value, the substance becomes liquid at room temperature; hence, they are also called room-temperature ionic liquids. Compared to traditional organic solvents, ionic liquids possess unique physicochemical properties and have wide applications in catalysis, extraction, electrochemistry, and the synthesis of polymers and nanomaterials. Ionic liquids are composed of designable anions and cations, and variations in their structure and their interactions significantly influence their properties. Traditional methods for extracting flavonoids, such as alkaline extraction, alcohol extraction, and reflux extraction, are energy-intensive and yield low extraction rates. This study innovatively utilizes microwave-assisted ionic liquid extraction to extract flavonoids from Areca catechu.

[0014] In one embodiment, the method for preparing the total alkaloid extract includes the following steps:

[0015] Pretreatment: Boil and wash the sago palms with sodium bicarbonate solution, dry, spray with sodium hydroxide solution, and dry again;

[0016] Preparation of total alkaloid extract: The pretreated sago palms were alkalized with ammonia water, allowed to stand, and extracted with microwave at 380-350W power for 80-150s. A mixture of ethanol and methanol was used as an entrainer for supercritical fluid extraction to obtain the total alkaloid extract.

[0017] The preparation method of the total flavonoid extract includes the following steps:

[0018] Pretreatment: Soak the sago palms in a mixed solution of ethanol and 1-butyl-3-methylimidazolium bromide ionic liquid;

[0019] Preparation of total flavonoid extract: Pretreated Areca catechu was added to a mixed solution of ethanol and 1-butyl-3-methylimidazolium bromide ionic liquid, and extracted by microwave to obtain crude extract. The crude extract was purified by weakly basic anion exchange resin and macroporous adsorption resin to obtain total flavonoid extract.

[0020] Supercritical fluid extraction (SFE) is a novel separation technology that integrates extraction, separation, and concentration. It boasts significant advantages such as low energy consumption, no pollution, simple operation, high selectivity, and preservation of substance activity, thus finding wide application in the extraction of natural products. Microwave combined with CO2 supercritical extraction is used to extract alkaloids, yielding a high total alkaloid content.

[0021] Macroporous adsorption resins have a good separation and purification effect on active ingredients such as alkaloids, flavonoids, and saponins in single-herb Chinese medicine extracts. However, the components of Chinese medicine compound prescriptions are complex, and the physicochemical properties of their active ingredients or active parts vary greatly. Macroporous resins selectively adsorb different components, making it difficult to separate and purify all active ingredients of Chinese medicine with a single resin. Therefore, the inventors chose to use a weakly basic anion exchange resin in combination with macroporous adsorption resin to separate and purify active ingredients of Chinese medicine.

[0022] In one embodiment, the weakly basic anion exchange resin is Mitsubishi DIAION WA30LL weakly basic anion exchange resin.

[0023] The aforementioned weakly basic anion exchange resin has excellent mechanical properties and permeability.

[0024] In one embodiment, the method for preparing the total alkaloid extract includes the following steps:

[0025] Pretreatment: Boil and wash the sago palms in a sodium bicarbonate solution with a mass fraction of 5%-10% for 30-40 minutes at a boiling and washing temperature of 43-50℃, dry, spray with a sodium hydroxide solution with a mass fraction of 0.1-0.5%, and dry at 45-50℃;

[0026] Preparation of total alkaloid extract: Pretreated sago palms were alkalized with ammonia, allowed to stand, and extracted with microwave at 300-400W for 80-150s. Supercritical fluid extraction was performed using a mixture containing ethanol and methanol as an entrainer to obtain the total alkaloid extract. The volume fraction of ethanol in the mixture was 10-20%, and the volume fraction of methanol was 10-20%.

[0027] In one embodiment, the conditions for supercritical fluid extraction include: a pressure of 30-35 MPa, a temperature of 55-60°C, a time of 80-120 min, a carbon dioxide flow rate of 25-30 kg / h, and an extraction time of 1.5-2 h.

[0028] In one embodiment, the method for preparing the total flavonoid extract includes the following steps:

[0029] Pretreatment: Soak the sago palm fruit in a mixed solution of ethanol and 1-butyl-3-methylimidazolium bromide ionic liquid for 2-2.5 hours;

[0030] Preparation of total flavonoid extract: Pretreated Areca catechu was added to a mixed solution of ethanol and 1-butyl-3-methylimidazolium bromide ionic liquid and extracted with microwave at 600-650W for 10-15 min to obtain crude extract. The crude extract was eluted once with a weakly basic anion exchange resin and a macroporous adsorption resin, and then eluted a second time with a macroporous adsorption resin to obtain total flavonoid extract.

[0031] This study employs the latest liquid ion extraction method to obtain high-purity total flavonoids. A two-stage continuous elution process is used: first, impurities are retained on anion exchange resin using a mixture of weakly basic anion exchange resin and macroporous adsorption resin as packing material, resulting in a good impurity adsorption rate. Simultaneously, a second elution is performed using macroporous resin to obtain high-purity flavonoids.

[0032] In one embodiment, in the step of preparing the total flavonoid extract, the volume ratio of the 1-butyl-3-methylimidazolium bromide ionic liquid to the ethanol solution is 1:(1-1.5).

[0033] In one embodiment, in the first elution step, the volume ratio of the weakly basic anion exchange resin to the macroporous adsorption resin is 1:(1-1.5).

[0034] The present invention also provides the use of the areca nut extract in the preparation of medicaments for relieving inflammation and / or pain.

[0035] In one embodiment, the inflammation and / or pain includes inflammation and / or pain caused by infection, physical injury, chemical injury, and / or autoimmune response.

[0036] In one embodiment, the infection includes a bacterial infection.

[0037] The present invention also provides the use of the areca nut extract in the preparation of medicaments for the prevention and / or treatment of oral ulcers.

[0038] The present invention also provides a medicament for relieving inflammation and / or pain, comprising pharmaceutically acceptable excipients and the areca nut extract.

[0039] In one embodiment, the dosage form of the drug includes a topical preparation, an oral preparation, or an injectable preparation.

[0040] The present invention also provides a medicament for the prevention and / or treatment of oral ulcers, comprising pharmaceutically acceptable excipients and the areca nut extract.

[0041] In one embodiment, the dosage form of the drug includes tablets or films.

[0042] In one embodiment, the tablet comprises an oral patch, and the film comprises an oral film.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention discloses a sago palm extract, its preparation method, and its applications. This sago palm extract exhibits good analgesic and anti-inflammatory effects, showing good therapeutic efficacy for inflammation / pain caused by physical and chemical injuries, as well as inflammation / pain caused by bacterial infections. Based on ancient records and folk remedies, and building upon previous chemical composition and pharmacological experiments, this invention creatively discovers the local anti-inflammatory and analgesic effects of sago palm extract, representing an innovative research field. Different sago palm extracts are obtained through extraction and purification methods different from conventional methods. The active sites of sago palm extract that produce analgesic effects are studied using the hot plate test and formalin-induced analgesia method. Acute and chronic inflammation models of xylene-induced mouse ear swelling and egg white-induced rat paw edema are used to explore the active sites of sago palm extract that exert their anti-inflammatory effects. Anti-inflammatory and analgesic tests are used as evaluation indicators to examine the medicinal effects of different sago palm extracts and obtain the optimal extraction and purification process. Meanwhile, the characteristics of the sago palm extract were investigated, suitable matrices were screened, and formulation process research was conducted. Novel drug delivery formulations such as "thermosensitive gel," "gel," "plaster," and "emulsion" were used to prolong the retention time of the sago palm extract on the skin surface, thereby improving its anti-inflammatory and analgesic effects, which is an innovative study. Furthermore, by measuring the total flavonoid content and total alkaloid content, indicators for the quality evaluation of the sago palm extract were established. Attached Figure Description

[0045] Figure 1 This is the standard curve in Example 5. Detailed Implementation

[0046] To facilitate understanding of the present invention, a more complete description will be given below with reference to relevant embodiments. Preferred embodiments of the invention are shown in the embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the disclosure of the present invention will be achieved.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0048] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0049] Example 1

[0050] Preparation method of total alkaloid extract.

[0051] I. Preprocessing.

[0052] Weigh 1 kg of sago palm raw material and boil it in a 5% (w / w) NaHCO3 solution for 30 minutes at a temperature of 45°C. After boiling and washing, air-dry the sago palm. Spray the dried sago palm with a 0.1% (w / w) NaOH solution and dry it at 45°C. Obtain the dried product.

[0053] II. Preparation of total alkaloid extract.

[0054] Weigh 1 kg of pretreated areca nut, add an appropriate amount of ammonia water to alkalize it, and then spread it evenly. Let it stand for a period of time to allow the solution to fully penetrate into the cell tissue. Place the fully moistened sample into a microwave extractor and process it for 100 seconds at a microwave extraction power of 300W.

[0055] The microwave-extracted sample was loaded into an extraction vessel for supercritical fluid extraction. The process parameters were: pressure 30 MPa, temperature 55 °C, time 100 min, CO2 flow rate 25.0 kg / h, and a mixture containing 10% anhydrous ethanol and 10% methanol (i.e., the volume fraction of ethanol in the mixture was 10% and the volume fraction of methanol was 10%) as an entrainer. The extraction time was 1.5 h to obtain the alkaloid extract.

[0056] Example 2

[0057] Preparation method of total alkaloid extract.

[0058] Weigh 1 kg of areca nut raw material, add an appropriate amount of ammonia water to alkalize it, and then spread it evenly. Let it stand for a period of time to allow the solution to fully penetrate into the cell tissue. Place the fully moistened sample into a microwave extractor and process it for 100 seconds at a microwave extraction power of 300W.

[0059] The microwave-extracted sample was placed in an extraction vessel for supercritical fluid extraction. The process parameters were: pressure 30 MPa, temperature 55 °C, time 100 min, CO2 flow rate 25.0 kg / h, and a mixture containing 10% anhydrous ethanol and 10% methanol as an entrainer. The extraction time was 1.5 h to obtain the alkaloid extract.

[0060] Compared to Example 1, the difference is that there is no pretreatment step.

[0061] Example 3

[0062] I. Preprocessing.

[0063] Weigh 1 kg of sago palm raw material and boil it in 5% NaHCO3 solution for 30 minutes at a temperature of 45°C. After boiling, air-dry the sago palm. Spray the dried sago palm with 0.1% NaOH solution and dry at 45°C. Obtain the dried product.

[0064] II. Preparation of total alkaloid extract.

[0065] Weigh 1 kg of pretreated areca nut, add an appropriate amount of ammonia water to alkalize it, and then moisten it evenly. Spread it out and let it stand for a period of time to allow the solution to fully penetrate into the cell tissue. Place the sample into an extraction vessel for supercritical fluid extraction. The process parameters are: pressure 30 MPa, temperature 55℃, time 100 min, CO2 flow rate 25.0 kg / h, using a mixture containing 10% anhydrous ethanol and 10% methanol as the entrainer, and extraction time 1.5 h to obtain the alkaloid extract.

[0066] Compared with Example 1, the difference is that in the step of preparing the total alkaloid extract, microwave extraction was not performed first, and the pretreated sago palm was directly alkalized and then subjected to supercritical fluid extraction.

[0067] Example 4

[0068] I. Preprocessing.

[0069] Weigh 1 kg of sago palm raw material and boil it in 5% NaHCO3 solution for 30 minutes at a temperature of 45°C. After boiling, air-dry the sago palm. Spray the dried sago palm with 0.1% NaOH solution and dry at 45°C. Obtain the dried product.

[0070] II. Preparation of total alkaloid extract.

[0071] Weigh 1 kg of pretreated areca nut, add an appropriate amount of ammonia water to alkalize it, and then spread it evenly. Let it stand for a period of time to allow the solution to fully penetrate into the cell tissue. Place the fully moistened sample into a microwave extractor and process it for 100 seconds at a microwave extraction power of 300W.

[0072] The microwave-extracted sample was placed in an extraction vessel for supercritical fluid extraction. The process parameters were: pressure 30 MPa, temperature 55 °C, time 100 min, CO2 flow rate 25.0 kg / h, with a solution containing 10% anhydrous ethanol as the entrainer, and extraction time 1.5 h, to obtain the alkaloid extract.

[0073] The difference from Example 1 is that a mixture of ethanol and methanol was not used as an entrainer.

[0074] Example 5

[0075] The content of alkaloid compounds in the total alkaloid extracts prepared in Examples 1-4 was determined.

[0076] Determination method: The content of total alkaloids in sago palm was determined by acid dye colorimetric method.

[0077] I. Instruments and reagents.

[0078] Ultraviolet spectrophotometer, pH meter, ultrasonic cleaner, 0.001 g electronic balance, electric thermostatic water bath.

[0079] Arecoline hydrobromide; 0.8 mg / mL -1 Bromocresol green solution, 0.01 mol·L -1 All potassium hydroxide anhydrous ethanol solutions were of analytical grade.

[0080] Areca nut medicinal material.

[0081] Preparation of the reference solution:

[0082] Accurately weigh 125 mg of arecoline hydrobromide and place it in a 50 mL volumetric flask. Add pH 5.0 acetate-sodium acetate buffer solution (from 0.1 mol·L⁻¹). -1 Sodium acetate and 0.1 mol·L -1 The buffer solution was prepared with acetic acid and diluted to the mark to obtain the reference stock solution. 1 mL of the stock solution was accurately pipetted into a 50 mL volumetric flask, and the buffer solution was added to dilute to the mark to obtain the reference solution.

[0083] Preparation of the test solution:

[0084] Accurately weigh approximately 1.0000 g of areca nut powder (previously pulverized and sieved through a No. 3 sieve) and place it in a stoppered, ground-glass conical flask. Accurately add 6 mL of concentrated ammonia and soak for 2 minutes. Add 30 mL of diethyl ether and sonicate for 20 minutes. Separate the ether solution. Add 10 mL of diethyl ether to the residue and sonicate three times for 5 minutes each time. Combine the ether solutions. Finally, wash the conical flask 2-3 times with an appropriate amount of diethyl ether. Evaporate to approximately 10 mL in a 50°C water bath. Transfer to a separatory funnel and dilute with 0.01 mol·L⁻¹ water. -140 mL of sulfuric acid solution was extracted four times, with extraction amounts of 15, 10, 10, and 5 mL each time. The extracted acid and aqueous solutions were then placed in a 50 mL volumetric flask and treated with 1 mol·L⁻¹ sulfuric acid solution. -1 Adjust the pH to 5 with sodium acetate, shake well, and dilute to the mark with pH 5.0 acetate-sodium acetate buffer solution to obtain the test solution.

[0085] II. Plotting the standard curve.

[0086] Accurately pipette 0, 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL of the reference solution into separate separatory funnels. Accurately add 4 mL of pH 5.0 acetate-sodium acetate buffer and 2 mL of bromocresol green solution, and gently mix. Extract three times, adding 2, 3, and 5 mL of chloroform respectively, shaking for 2 min each time, and allowing to stand for 30 min. Separate the chloroform layer and place it in a dry volumetric flask pre-filled with 0.2 g of anhydrous sodium sulfate. Shake and allow to stand for 30 min. Accurately pipette 5 mL of the supernatant and add 1 mL of 0.01 mol·L⁻¹ potassium hydroxide anhydrous ethanol solution. Shake well and measure the absorbance at 618 nm. Plot a standard curve with arecoline amount as the x-axis (X) and absorbance as the y-axis (Y) to obtain the regression equation.

[0087] III. Determination of total alkaloid content.

[0088] Accurately pipette 1 mL of the test solution into a separatory funnel, and accurately add 4 mL of pH 5.0 acetate-sodium acetate buffer and 2 mL of bromocresol green solution. Gently mix. Extract three times, accurately adding 2, 3, and 5 mL of chloroform each time, shaking for 2 min each time, and allowing to stand for 30 min. Separate the chloroform layer and place it in a dry volumetric flask pre-filled with 0.2 g of anhydrous sodium sulfate. Shake and allow to stand for 30 min. Accurately pipette 5 mL of the supernatant and accurately add 0.01 mol·L⁻¹ ... -1 1 mL of anhydrous potassium hydroxide ethanol solution was shaken well and the absorbance was measured at a wavelength of 618 nm.

[0089] IV. Measurement Results.

[0090] The standard curve was measured as follows Figure 1 As shown in the table below, the data for plotting the standard curve is presented.

[0091] Table 1 Measurement Results

[0092]

[0093]

[0094] Table 2. Content of alkaloids in total alkaloid extracts from Examples 1-4

[0095] Example 1 1.41％ Example 2 0.65％ Example 3 0.81％ Example 4 1.24％

[0096] Example 6

[0097] Preparation method of total flavonoid extract.

[0098] I. Preprocessing.

[0099] Weigh 500g of raw sago palm and cut it into thick slices. Add a mixture of ethanol (95% by volume) and 1-butyl-3-methylimidazolium bromide ionic liquid (1 mol / L) with a mass ratio of 3 times that of the raw sago palm and soak for 2 hours. The volume ratio of ethanol to 1-butyl-3-methylimidazolium bromide ionic liquid in the mixture is 1:1.

[0100] II. Preparation of total flavonoid extract.

[0101] 1. Microwave extraction.

[0102] Accurately weigh 100g of pretreated Areca catechu sample and place it in a round-bottom flask. Add a stir bar, then add a 1000mL mixture of 1mol / L 1-butyl-3-methylimidazolium bromide ionic liquid and 70% ethanol solution (volume ratio 1:1) to the round-bottom flask. Place the flask in a microwave reactor, set the microwave power to 600 W, and the extraction time to 10 min for microwave-assisted extraction. After extraction, centrifuge and remove the filter residue to obtain crude Areca catechu extract.

[0103] 2. Purification.

[0104] (1) Single elution: Mitsubishi DIAION WA30LL weak base anion exchange resin and macroporous resin 941 were mixed at a volume ratio of 1:1 to obtain mixed resin.

[0105] Resin pretreatment: The anion exchange resin is regenerated using an alkali-acid-alkali process, ultimately converting the resin to the OH- form. The alkali is 1 mol / L NaOH, washed for 4 BV; the acid is 1 mol / L HCl, washed for 4 BV. After both acid and alkali washings, the resin is washed with deionized water until the pH reaches 6–8.

[0106] Resin loading onto the column: Secure the glass chromatography column and pack it using the wet method.

[0107] Column elution: The crude extract of Areca catechu was loaded onto the column. The loading conditions were 1.0 mg / ml (w / v) and 3 ml / min (v / v). The eluent was collected. Then, elution was performed with 30% ethanol (v / v), and the eluent was collected. The two eluents were combined, concentrated, and rotary evaporated until odorless to obtain crude pure Areca catechu extract 1.

[0108] (2) Secondary elution: Resin selection: Macroporous resin D17 was used as the adsorption resin. The macroporous resin was soaked in 95% ethanol for 12 hours by volume, packed into the column by wet method, washed with water until the effluent had no alcohol odor and did not become turbid when mixed with anhydrous ethanol, and then soaked in water for later use.

[0109] Resin loading: Load crude pure solution 1 from Areca catechu onto the resin. Allow static adsorption for 1 hour, then wash with water at a flow rate of 1.2 column volumes for 3 column volumes. Discard the eluent. Elute with 50% ethanol at a flow rate of 0.8-1.0 column volumes for 3 column volumes, collecting the eluent. Lyophilize to obtain lyophilized powder of purified Areca catechu flavonoids (i.e., total flavonoid extract).

[0110] Example 7

[0111] Total flavonoid extract was prepared using the traditional ethanol percolation method.

[0112] 1. Take 500g of Areca catechu and soak it in 95% ethanol (volume fraction) at twice its weight for 48 hours. Filter the solution and retain the filtrate. Repeat this process twice and combine the filtrates. Concentrate the filtrate to a suitable volume to obtain a crude extract of Areca catechu for later use.

[0113] II. Only one time the pillar is placed.

[0114] Resin selection: Macroporous resin D17 was used as the adsorption resin. The macroporous resin was soaked in 95% ethanol for 12 hours by volume, packed into a column using the wet method, washed with water until the effluent had no alcohol odor and did not become turbid when mixed with anhydrous ethanol, and then soaked in water for later use.

[0115] Load the crude extract of Areca catechu onto the sample and allow it to adsorb statically for 1 hour. Wash with water at a flow rate of 1.2 column volumes for 3 column volumes. Discard the eluent. Elute with 50% ethanol at a flow rate of 0.8-1.0 column volumes for 3 column volumes, collecting the eluent. Lyophilize to obtain a lyophilized powder of purified Areca catechu flavonoids.

[0116] Example 8

[0117] I. Extraction was performed using ethanol reflux extraction.

[0118] Take 500g of Areca catechu and soak it in 95% ethanol (volume fraction) with a mass twice the weight of the Areca catechu for 48 hours. Then extract it with ethanol (volume fraction eight times the weight of the Areca catechu) for 4 hours each time, for a total of two extractions. Combine the extracts and concentrate the filtrate to a suitable volume to obtain a crude extract of Areca catechu for later use.

[0119] II. Only one time the pillar is placed.

[0120] Resin selection: Macroporous resin D17 was used as the adsorption resin. The macroporous resin was soaked in 95% ethanol for 12 hours by volume, packed into a column using the wet method, washed with water until the effluent had no alcohol odor and did not become turbid when mixed with anhydrous ethanol, and then soaked in water for later use.

[0121] Load the crude extract of Areca catechu onto the sample and allow it to adsorb statically for 1 hour. Wash with water at a flow rate of 1.2 column volumes for 3 column volumes. Discard the eluent. Elute with 50% ethanol at a flow rate of 0.8-1.0 column volumes for 3 column volumes, collecting the eluent. Lyophilize to obtain a lyophilized powder of purified Areca catechu flavonoids.

[0122] Example 9

[0123] Preparation method of total flavonoid extract.

[0124] I. Microwave extraction.

[0125] Accurately weigh 100g of Areca catechu sample and place it in a round-bottom flask. Add a stir bar, then add a 1000mL mixture of 1mol / L 1-butyl-3-methylimidazolium bromide ionic liquid and 70% ethanol solution (volume ratio 1:1) to the round-bottom flask. Place the flask in a microwave reactor, set the microwave power to 600W, and the extraction time to 10min for microwave-assisted extraction. After extraction, centrifuge and remove the filter residue to obtain crude Areca catechu extract.

[0126] 2. Purification.

[0127] (1) Single elution: Mitsubishi DIAION WA30LL weak base anion exchange resin and macroporous resin 941 were mixed at a volume ratio of 1:1 to obtain mixed resin.

[0128] Resin pretreatment: The anion exchange resin is regenerated using an alkali-acid-alkali process, ultimately converting the resin to the OH- form. The alkali is 1 mol / L NaOH, washed for 4 BV; the acid is 1 mol / L HCl, washed for 4 BV. After both acid and alkali washings, the resin is washed with deionized water until the pH reaches 6–8.

[0129] Resin loading onto the column: Secure the glass chromatography column and pack it using the wet method.

[0130] Column elution: The crude extract of Areca catechu was loaded onto the column. The loading conditions were 1.0 mg / ml (w / v) and 3 ml / min (v / v). The eluent was collected. Then, elution was performed with 30% ethanol (v / v), and the eluent was collected. The two eluents were combined, concentrated, and rotary evaporated until odorless to obtain crude pure Areca catechu extract 1.

[0131] (2) Secondary elution: Resin selection: Macroporous resin D17 was used as the adsorption resin. The macroporous resin was soaked in 95% ethanol for 12 hours by volume, packed into the column by wet method, washed with water until the effluent had no alcohol odor and did not become turbid when mixed with anhydrous ethanol, and then soaked in water for later use.

[0132] Resin loading: Load crude pure solution 1 of Areca catechu onto the resin. Allow static adsorption for 1 hour, then wash with water at a flow rate of 1.2 column volumes for 3 column volumes. Discard the eluent. Elute with 50% ethanol at a flow rate of 0.8-1.0 column volumes for 3 column volumes, collecting the eluent. Lyophilize to obtain lyophilized powder of purified Areca catechu flavonoids.

[0133] The difference from Example 6 is that there is no pretreatment step, and microwave extraction uses a single ionic liquid as the extractant.

[0134] Example 10

[0135] Preparation method of total flavonoid extract.

[0136] I. Preprocessing.

[0137] Weigh 500g of raw sago palm and cut it into thick slices. Add a mixture of ethanol (95% by volume) and 1-butyl-3-methylimidazolium bromide ionic liquid (1mol / L) with a mass ratio of 3 times that of the raw sago palm and soak for 2 hours. The volume ratio of ethanol to 1-butyl-3-methylimidazolium bromide ionic liquid in the mixture is 1:1.

[0138] II. Preparation of total flavonoid extract.

[0139] 1. Microwave extraction.

[0140] Accurately weigh 100g of pretreated Areca catechu sample and place it in a round-bottom flask. Add a stir bar, then add a 1000mL mixture of 1mol / L 1-butyl-3-methylimidazolium bromide ionic liquid and 70% ethanol solution (volume ratio 1:1) to the round-bottom flask. Place the flask in a microwave reactor, set the microwave power to 600W, and the extraction time to 10min for microwave-assisted extraction. After extraction, centrifuge and remove the filter residue to obtain crude Areca catechu extract.

[0141] 2. Purification.

[0142] (1) Single elution: Mitsubishi DIAION WA30LL weak base anion exchange resin was selected.

[0143] Resin pretreatment: The anion exchange resin is regenerated using an alkali-acid-alkali process, ultimately converting the resin to the OH- form. The alkali is 1 mol / L NaOH, washed for 4 BV; the acid is 1 mol / L HCl, washed for 4 BV. After both acid and alkali washings, the resin is washed with deionized water until the pH reaches 6–8.

[0144] Resin loading onto the column: Secure the glass chromatography column and pack it using the wet method.

[0145] Column elution: The crude extract of Areca catechu was loaded onto the column. The loading conditions were 1.0 mg / ml (w / v) and 3 ml / min (v / v). The eluent was collected. Then, elution was performed with 30% ethanol (v / v), and the eluent was collected. The two eluents were combined, concentrated, and rotary evaporated until odorless to obtain crude pure Areca catechu extract 1.

[0146] (2) Secondary elution: Resin selection: Macroporous resin D17 was used as the adsorption resin. The macroporous resin was soaked in 95% ethanol for 12 hours by volume, packed into the column by wet method, washed with water until the effluent had no alcohol odor and did not become turbid when mixed with anhydrous ethanol, and then soaked in water for later use.

[0147] Resin loading: Load crude pure solution 1 of Areca catechu onto the resin. Allow static adsorption for 1 hour, then wash with water at a flow rate of 1.2 column volumes for 3 column volumes. Discard the eluent. Elute with 50% ethanol at a flow rate of 0.8-1.0 column volumes for 3 column volumes, collecting the eluent. Lyophilize to obtain lyophilized powder of purified Areca catechu flavonoids.

[0148] The difference from Example 6 is that a single resin is used for elution in one step.

[0149] Example 11

[0150] Preparation method of total flavonoid extract.

[0151] I. Preprocessing.

[0152] Weigh 500g of raw sago palm and cut it into thick slices. Add a mixture of ethanol (95% by volume) and 1-butyl-3-methylimidazolium bromide ionic liquid (1mol / L) with a mass ratio of 3 times that of the raw sago palm and soak for 2 hours. The volume ratio of ethanol to 1-butyl-3-methylimidazolium bromide ionic liquid in the mixture is 1:1.

[0153] II. Preparation of total flavonoid extract.

[0154] 1. Microwave extraction.

[0155] Accurately weigh 100g of pretreated Areca catechu sample and place it in a round-bottom flask. Add a stir bar, then add a 1000mL mixture of 1mol / L 1-butyl-3-methylimidazolium bromide ionic liquid and 70% ethanol solution (volume ratio 1:1) to the round-bottom flask. Place the flask in a microwave reactor, set the microwave power to 600W, and the extraction time to 10min for microwave-assisted extraction. After extraction, centrifuge and remove the filter residue to obtain crude Areca catechu extract.

[0156] 2. Purification.

[0157] (1) Single elution: Macroporous resin 941 was selected as the adsorption resin.

[0158] Resin pretreatment: The anion exchange resin is regenerated using an alkali-acid-alkali process, ultimately converting the resin to the OH- form. The alkali is 1 mol / L NaOH, washed for 4 BV; the acid is 1 mol / L HCl, washed for 4 BV. After both acid and alkali washings, the resin is washed with deionized water until the pH reaches 6–8.

[0159] Resin loading onto the column: Secure the glass chromatography column and pack it using the wet method.

[0160] Column elution: The crude extract of Areca catechu was loaded onto the column. The loading conditions were 1.0 mg / ml (w / v) and 3 ml / min (v / v). The eluent was collected. Then, elution was performed with 30% ethanol (v / v), and the eluent was collected. The two eluents were combined, concentrated, and rotary evaporated until odorless to obtain crude pure Areca catechu extract 1.

[0161] (2) Secondary elution: Resin selection: Macroporous resin D17 was used as the adsorption resin. The macroporous resin was soaked in 95% ethanol for 12 hours, packed into the column by wet method, washed with water until the effluent had no alcohol odor and did not become turbid when mixed with anhydrous ethanol, and then soaked in water for later use.

[0162] Resin loading: Load crude pure solution 1 of Areca catechu onto the resin. Allow static adsorption for 1 hour, then wash with water at a flow rate of 1.2 column volumes for 3 column volumes. Discard the eluent. Elute with 50% ethanol at a flow rate of 0.8-1.0 column volumes for 3 column volumes, collecting the eluent. Lyophilize to obtain lyophilized powder of purified Areca catechu flavonoids.

[0163] The difference from Example 6 is that a single resin is used for elution in one step.

[0164] Example 12

[0165] The content of flavonoids in the total flavonoid extracts prepared in Examples 6-11 was determined.

[0166] The determination method used rutin as a reference standard and 5% NaNO2, 10% Al(NO3)3, and 4% NaOH as colorimetric reagents. The total flavonoid content in the sample was determined by ultraviolet spectrophotometry at a wavelength of 510 nm.

[0167] I. Instruments and reagents.

[0168] Ultraviolet spectrophotometer (PerkinElmer, USA), electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.);

[0169] Rutin reference standard (Chengdu Ruifens Biotechnology Co., Ltd.), sodium nitrite (Tianjin Yongda Chemical Reagent Co., Ltd.), aluminum nitrate (Tianjin Kemio Chemical Reagent Co., Ltd.), sodium hydroxide (Xilong Scientific Co., Ltd.).

[0170] II. Preparation of reference solution.

[0171] Preparation of rutin reference solution: Accurately weigh 10.0 mg of rutin standard, dissolve it in 75% ethanol, and dilute to a 50 mL volumetric flask. Mix well to ensure the rutin is fully dissolved and evenly distributed, preparing a 0.2 mg / mL rutin reference solution. To maintain its stability, the prepared solution should be stored away from light at 4°C for subsequent use.

[0172] III. Draw the standard curve.

[0173] Accurately pipette 0.00, 2.00, 4.00, 6.00, 8.00, and 10.00 mL of 0.2 mg / mL rutin reference solution into 25 mL amber volumetric flasks. Add 1 mL of 5% NaNO₂ solution sequentially, mix well, and let stand for 6 min. Then add 1 mL of 10% Al(NO₃)₃ solution, mix well, and let stand for 6 min. Add 10 mL of 4% NaOH solution, and dilute to the mark with 75% ethanol. Mix well and let stand for 15 min to obtain a series of standard solutions. Measure the absorbance at 510 nm. Use the solution in the volumetric flask containing 0 mL of standard solution as a reference solution. Plot a standard curve based on the concentration of the standard solution and the corresponding absorbance.

[0174] IV. Ultraviolet spectrophotometry detection.

[0175] Accurately pipette 1 mL of the test solution into a 25 mL amber volumetric flask. Add 1 mL of 5% NaNO₂ solution sequentially, mix well, and let stand for 6 min. Then add 1 mL of 10% Al(NO₃)₃ solution, mix well, and let stand for 6 min. Add 10 mL of 4% NaOH solution, and dilute to the mark with 75% ethanol. Mix well, let stand for 15 min, and then measure the absorbance at 510 nm. Based on the measured absorbance, find the corresponding concentration data on a pre-constructed standard curve and calculate the total flavonoid content.

[0176] V. Measurement Results.

[0177] The results are shown in the table below.

[0178] Table 3. Content of flavonoids in total flavonoid extracts from Examples 6-11

[0179]

[0180]

[0181] Example 13

[0182] The total alkaloid extracts and total flavonoid extracts obtained in Examples 1 and 6 were used to prepare topical and oral preparations of different dosage forms.

[0183] I. Creams.

[0184] Take the total flavonoid extract and total alkaloid extract of Areca catechu (mass ratio 1:1) to form Areca catechu extract, which accounts for 1%-3% of the drug by mass. Dissolve in an appropriate amount of dimethyl sulfoxide and set aside. Take 10% by mass of white petrolatum, 8%-10% by mass of stearyl alcohol, 3%-5% by mass of liquid paraffin, and 1%-2% by mass of glyceryl monostearate, and heat in a water bath to melt them. The heating time in the water bath is about 10-15 minutes, and the temperature is controlled at 70-80℃. Add the prepared solution and shear for 5-10 seconds to obtain the oil phase.

[0185] Weigh and verify the following: sodium dodecyl sulfate (0.5%-1%), menthol (1%-2%), glycerin (5%-6%), ethylparaben (0.05%-1%), and an appropriate amount of water. Heat to dissolve, controlling the temperature at 70-80℃ (i.e., aqueous phase).

[0186] Slowly add the oil phase to the aqueous phase while shearing to emulsify. The emulsification time is about 20 minutes.

[0187] II. Tinctures / Sprays

[0188] The total flavonoid extract and total alkaloid extract of Areca catechu (mass ratio 1:1) are used to form Areca catechu extract, which accounts for 1%-3% of the total mass of the drug. The extract is dissolved in an appropriate volume fraction of 75% ethanol, and menthol (mass ratio 0.5%-1%) and glycerin (mass ratio 4%-5%) are added. After stirring and dissolving, 75% ethanol is added to make up to the final volume, and the mixture is filtered to obtain the final product.

[0189] III. Temperature-sensitive gel.

[0190] Accurately weigh poloxamer (1%-3% by mass), block polyether F-127 (15%-18% by mass), glycerin (3%-5% by mass), and propylene glycol (3%-5% by mass) and dissolve them in 50 ml of water for later use.

[0191] Take the total flavonoid extract and total alkaloid extract of Areca catechu (mass ratio 1:1) to form Areca catechu extract, the mass percentage of which is 1%-3% in the drug. Dissolve in 30ml of water, and after complete dissolution, add to the swollen poloxamer solution and mix well. Add purified water to make up to 100g to obtain the final product.

[0192] Thermosensitive gels are mostly prepared from water-soluble polymer materials and have a highly hydrophilic three-dimensional mesh structure. Compared with other traditional drug delivery systems, in-situ gels have significant advantages: ① By preparing drugs into in-situ gels and administering them through mucous membranes, they can bypass gastrointestinal digestion and first-pass elimination by the liver, resulting in a smaller effective dose and higher bioavailability; ② The physicochemical properties of the gel and the state of the drug in the body can be adjusted according to environmental changes to achieve timely and effective treatment; ③ They have strong affinity for mucous membrane tissues, long retention time, and good sustained-release or controlled-release effects; ④ They have good tissue compatibility, are easy to use, reduce the frequency of administration, and improve patient compliance; ⑤ They can be administered through multiple routes, such as transdermal, ocular, nasal, rectal, and injection, with a wide range of applications; In-situ gels can also be used for local drugs, systemic drugs, macromolecular drugs, cellular and tissue-based drugs, hydrophilic drugs, and hydrophobic drugs.

[0193] IV. External gel.

[0194] Weigh out Carbomer 940 (1%-3% by mass), and swell it in 50 ml of purified water at 65°C for later use;

[0195] Separately dissolve glycerol (5%-7% by mass), azone (0.5%-1% by mass), and sago palm extract (made by combining total flavonoid extract and total alkaloid extract of sago palm in a 1:1 ratio, with the sago palm extract accounting for 1%-3% of the drug by mass) in 30 ml, mix well, add to a fully swollen carbomer solution and mix, add ethylparaben (0.05%-0.1%), then add purified water to make up to 100 g, add an appropriate amount of triethanolamine, and stir until a gel is formed.

[0196] 5. Plaster.

[0197] Take glycerol (8%-12% by mass), sodium polyacrylate (2%-3% by mass), PVP (0.8%-1.2% by mass), tartaric acid (0.05%-0.2% by mass), aluminum hydroxyglycoside (0.1%-0.2% by mass), and azone (0.5%-1.2% by mass) and stir them evenly to form phase A;

[0198] Separately dissolve sago palm extract (made by combining total flavonoid extract and total alkaloid extract of sago palm in a mass ratio of 1:1, with the sago palm extract accounting for 1%-3% of the drug by mass) and menthol (0.05%-0.12% by mass) in 10 ml of 30% ethanol solution to form phase B;

[0199] Add 25ml of water to phase A, stir, and let it stand until it forms a gel. Heat at 80℃ for 10 minutes, stirring constantly to ensure complete swelling. Add phase B, stirring continuously to mix. If necessary, heat the mixture until it is homogeneous. Remove air bubbles in a vacuum and then coat it.

[0200] VI. Nano-micro adhesive patches.

[0201] 1. Oral patches are film-based medications that can be administered sublingually or through mucous membranes. Oral patches are small in size and, after being moistened by saliva, develop adhesive forces, thus adhering to the buccal membrane or under the tongue. The release of drugs through the oral mucosa or under the tongue by oral patches avoids the first-pass effect in the liver, thereby improving bioavailability.

[0202] The drug-eluting microneedle layer penetrates the oral mucosa without touching nerve tissue, releasing the drug as the carrier dissolves without causing pain. Compared to the stratum corneum of skin, the mucosal epithelium is not keratinized and has a thinner membrane, making it easier for microneedles to penetrate and facilitating drug absorption. The submucosa has abundant capillaries with high blood flow, enabling rapid absorption. Oral microneedle patches can improve the bioavailability of Areca catechu extract.

[0203] This patent first prepares a nano-suspension of sago palm extract (total flavonoid extract of sago palm: alkaloid extract (1:1) and then further incorporates the sago palm suspension into a hyaluronic acid hydrogel to achieve uniform drug distribution, thereby creating a hyaluronic acid microneedle patch carrying a water-soluble sago palm aqueous nanocrystal suspension.

[0204] 2. Preparation method: Weigh the following ingredients according to the mass ratio of sago palm extract: soybean lecithin: cholesterol 5:2:1, then add an appropriate amount of anhydrous ethanol to dissolve them, stir magnetically until completely dissolved, and use a microsyringe to quickly inject the mixed solution into purified water (containing T-80) to form a light yellow nano suspension solution.

[0205] Sodium hyaluronate was added to purified water and mixed with a magnetic stirrer to prepare a homogeneous gel-like solution with a concentration of 0.2 g / ml. The previously prepared sago palm nano-suspension was then added to the sodium hyaluronate solution, and the mixture was stirred until homogeneous. The mixed hydrogel was then spread evenly onto a polydimethylsiloxane (PDMS) microneedle mold. The mold was placed in a vacuum drying oven, and after vacuuming, it was placed in an electrically heated drying oven at 37°C for approximately 8 hours. After complete drying, the mold was demolded to obtain a hyaluronic acid microneedle patch containing a water-soluble sago palm aqueous nanocrystalline suspension.

[0206] 7. Oral medicated film.

[0207] 1. Fast Disintegrating Oral Films (FDOFs) are a novel dosage form that rapidly absorbs water in the oral cavity, adheres to the oral mucosa, then disintegrates to release the drug. The drug can disintegrate and dissolve within 30 seconds, without requiring chewing or administration with water. It has good palatability and is a new type of rapid drug release preparation.

[0208] 2. Preparation method: Using PVA and HPMC as film-forming materials at a ratio of 2:1, mix the film-forming materials with solvent water at a ratio of 1:8 to 1:10. Stir with a magnetic stirrer until completely dissolved, then let stand until all bubbles disappear. Use a disposable syringe to draw 2ml of PVA and 1ml of HPMC into a 50ml beaker, then accurately add 0.5ml of PEG400. Take the total flavonoid extract of arenga pinnata seed and alkaloid extract at a ratio of 1:1, place them in an evaporating dish, grind until uniform and free of bubbles, then pour into the beaker containing the film-forming materials and plasticizer. Stir for 2 hours at a speed of 100r / min with a magnetic stirrer to form a dark green uniform viscous liquid, pour the liquid onto a glass plate (5 layers of transparent tape with a thickness of about 250μm are pasted on both sides of the transparent glass plate to ensure the thickness of the formed film), spread the liquid with a film applicator, place in an oven at 50°C, take out after 12-15 minutes, peel off the film, and cut into finished film preparations of 2*2cm in size.

[0209] Experimental Example 1

[0210] In vitro analgesic and anti-inflammatory effect experiment of arenga pinnata seed extract.

[0211] I. Materials.

[0212] 1. Laboratory animals:

[0213] SPF grade ICR mice, both male and female, weighing 20-22g.

[0214] SPF grade SD rats, male, weighing 200-220g.

[0215] All laboratory animals are provided by the Laboratory Animal Center of Harbin Medical University, with the certificate number: SCXK(Hei)2024-002.

[0216] 2. Drugs and reagents:

[0217] Arenga pinnata seed extract; eggs (purchased from Beijing Hualian Supermarket); sodium chloride, ethanol, xylene and formalin are all domestic analytical pure reagents.

[0218] 3. Main instruments:

[0219] Intelligent hot plate apparatus (Chengdu Taimei Software Co., Ltd., RB-200); analytical balance (Mettler Toledo International Co., Ltd., Switzerland, ME1002 / 02)

[0220] II. Experimental Methods:

[0221] 1. Preparation of Areca catechu extract

[0222] A total alkaloid extract of Areca catechu was prepared according to Example 1, and a total flavonoid extract of Areca catechu was prepared according to Example 6.

[0223] 2. Grouping and Dosing

[0224] They were randomly divided into 7 groups, with 10 animals in each group. The groups are as follows:

[0225] Group 1: Group 1: Group 1: Group 1: Group 2: Group 3: Group 4: Group 5: Total Flavonoids from Group 6: Total Alkaloids from Group 6: (mass ratio 1:1)

[0226] Areca catechu group 2. Areca catechu total flavonoids group

[0227] Group 3 of Areca catechu. Total alkaloids of Areca catechu.

[0228] Four groups of Areca catechu: a group containing total flavonoids from Areca catechu and a group containing total alkaloids from Areca catechu (mass ratio 2:1).

[0229] Five groups of Areca catechu were included: a group containing total flavonoids from Areca catechu and a group containing total alkaloids from Areca catechu (mass ratio 1:2).

[0230] Blank control group (0.9% NaCl)

[0231] Model control group (model created according to requirements)

[0232] The components and dosages administered to each group in subsequent experiments were the same as in this experiment.

[0233] To facilitate application and ensure application quality, 10 μL was applied to mice and 40 μL to rats.

[0234] 3. Analgesic experiment of Areca catechu extract

[0235] 3.1 Hot Plate Method

[0236] (1) Screening: After female mice were acclimatized for 7 days, they were placed in a 55℃ constant temperature intelligent hot plate instrument and measured twice. The time required for the mouse to lick its hind paws after being placed on the hot plate was taken as the pain threshold of the mouse. The baseline pain threshold was calculated and animals that jumped were removed.

[0237] (2) Administration: Apply topically to the soles of both feet once a day for 3 consecutive days.

[0238] (3) Pain threshold measurement before administration: The pain threshold of each mouse was measured twice before administration, and the average of the two pain threshold measurements was taken as the pain threshold of the mouse before administration.

[0239] (4) Detection: 30 min, 60 min and 90 min after the last administration, the pain threshold of each group of mice was measured. If there was no response after 60s, the mice were removed immediately to avoid burns. The pain threshold was calculated based on 60s.

[0240] The results are shown in the table below. Compared with the blank control group, the latency period for licking the paws of mice in groups 1, 4, and 5 was significantly prolonged 30 minutes after administration (P<0.05). There were no differences among the groups at 60 minutes and 90 minutes after administration (P>0.05).

[0241] Table 4. Effects of Areca catechu extract on the latency period of hot plate induced paw licking in mice ( n=10)

[0242]

[0243] Note: Compared with the blank control group, * P<0.05.

[0244] 3.2 Formalin analgesia

[0245] (1) Grouping and administration: After 7 days of acclimatization, male mice were divided into 6 groups: Areca catechu group 1, Areca catechu group 2, Areca catechu group 3, Areca catechu group 4, Areca catechu group 5, and model control group. The drug was applied to the skin of the dorsal side of the right paw once a day for 3 consecutive days.

[0246] (2) Detection: 30 min after the last administration, 20 μL of 1% formalin solution was subcutaneously injected into the dorsal side of the right paw of the mouse. The total time the mouse spent licking its paw was recorded from 0 to 5 min and from 10 to 40 min. The inhibition rate was calculated.

[0247] Inhibition rate (%) = (Model group time - Drug group time) / Model group time × 100%

[0248] The results are shown in the table below. Compared with the model control group, the sago palm groups 1, 4 and 5 significantly inhibited the total time of the mouse paw licking response in both time phases (P<0.05).

[0249] Table 4. Effects of Areca catechu extract on formalin-induced paw-licking response in mice ( n=10)

[0250]

[0251]

[0252] Note: Compared with the model control group * P<0.05

[0253] 4. Anti-inflammatory experiment of sago palm extract.

[0254] 4.1 Mouse ear swelling method.

[0255] (1) Grouping and administration: After 7 days of acclimatization, male mice were randomly divided into 6 groups according to their body weight: Areca catechu group 1, Areca catechu group 2, Areca catechu group 3, Areca catechu group 4, Areca catechu group 5, and model control group. The drug was applied topically to the left auricle of the mice once a day for 3 consecutive days.

[0256] (2) Detection: 30 min after the last administration, 20 μL of xylene was applied to both sides of the left ear of the mouse. 2.5 h later, the mouse was sacrificed, and the left and right ears were punched out using an 8 mm punch, and the weights were measured using an analytical balance. The difference in weight between the left and right ear pieces was used as an indicator of the degree of swelling, and the intensity of the anti-inflammatory effect was expressed as the inhibition rate (%).

[0257]

[0258] The results are shown in the table below. Compared with the model control group, the Areca catechu groups 1, 4, and 5 significantly reduced the xylene-induced left ear weight in mice (P<0.05). The Areca catechu group 4 showed the strongest effect, with an ear swelling rate of only 20.55%.

[0259] Table 5. Effects of Areca catechu extract on xylene-induced ear swelling in mice ( n=10)

[0260]

[0261] Note: Compared with the model control group, ** P<0.01, * P<0.05.

[0262] 4.2 Rat paw edema method.

[0263] (1) Grouping and administration: Male SD rats were acclimatized for 7 days and then randomly divided into 7 groups according to body weight: Areca catechu group 1, Areca catechu group 2, Areca catechu group 3, Areca catechu group 4, Areca catechu group 5, model control group, and blank control group. The drug was applied topically to the right hind paw of each group of rats once a day for 3 consecutive days.

[0264] (2) Inflammation: 30 minutes after the drug was applied, except for the control group, 0.1 mL of 10% fresh egg white solution was injected subcutaneously.

[0265] (3) Detection: The ankle circumference was measured with a non-elastic soft measuring tape at 30 min and 1 h after inflammation. The swelling rate was calculated according to the following formula.

[0266]

[0267] The results are shown in the table below. Compared with the blank control group, the paw edema rate of rats in the model control group was significantly increased at all time points after inflammation (P<0.01). Compared with the model control group, the extracts of Areca catechu in groups 1, 4, and 5 could reduce the paw edema rate induced by egg white in rats.

[0268] Table 6. Effects of Areca catechu extract on egg white-induced paw edema in rats ( n=5)

[0269]

[0270] Note: Compared with the blank control group ## P<0.01 # P<0.05.

[0271] Experimental Example 2

[0272] I. Antibacterial experiment.

[0273] 1. Sample preparation: Dissolve the total flavonoid group of Areca catechu prepared in Example 6 and the total alkaloid group of Areca catechu prepared in Example 1 (mass ratio 1:1) in physiological saline, and dilute them into three aqueous extracts of the concentrations shown in the table below. Autoclave at 121℃ for 10 min and take each concentration.

[0274] 2. Bacterial suspension dilution: Using a sterile pipette tip, take 1 mL of the prepared original bacterial suspension and place it into a test tube containing 9 mL of sterile physiological saline. Shake thoroughly to disperse the bacteria evenly, obtaining 1×10⁻⁶ cells / mL. -1 Diluent; take 1 mL of the above diluent and dilute it using the same method to obtain 1×10 -2 The diluent was then diluted sequentially to a concentration of 1×10⁻⁶. -3 1×10 -4 1×10 -5 Diluent.

[0275] 3. Experimental group: Take 1×10⁻⁶ of the bacterial suspension from the dilution step. -5 Add 100 μL of diluted bacterial solution to the aqueous extract solutions of various concentrations from the sample preparation steps and mix well. Take 100 μL of the suspension of the above bacteria and Areca catechu extract, place it in a 4°C refrigerator and let it stand for 24 h, then spread it evenly on the surface of agar medium in a sterilized petri dish, let it stand for 15 min, and then incubate it upside down in a 37°C constant temperature incubator for 24 h.

[0276] 4. Blank group: Take 1×10⁻⁶ of the bacterial suspension from the dilution step. -5Add 100 μL of diluted bacterial solution to 0.5 mL of sterile physiological saline, mix well, take 100 μL and place it in a 4℃ refrigerator for 24 h, then spread it evenly on the surface of agar medium in a sterilized culture dish, let it stand for 15 min, and then place it in a 37℃ constant temperature incubator and invert it for 24 h.

[0277] 5. Inhibition rate: Inhibition rate % = [(Number of colonies in the blank group - Number of colonies in the experimental group) / Number of colonies in the blank group] × 100%.

[0278] 6. Results: As shown in the table below, the extract has a certain antibacterial ability at different concentrations, and the antibacterial ability increases with the increase of extract concentration.

[0279] Table 7. Inhibition rate of Areca catechu extract against three pathogenic bacteria (n=3)

[0280]

[0281] II. Pharmacodynamic studies.

[0282] 1. Experimental Methods: Forty rats (8-week-old SPF-grade male SD rats, weighing approximately 180–220 g) were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg) and fixed on the operating table. The labial mucosa of the SD rats was dried with sterile cotton balls and isolated from moisture with sterile cotton balls. A 3 mm × 3 mm square filter paper was placed in 50% glacial acetic acid for 5 seconds, then removed and immediately placed on the dried labial mucosa. After maintaining this for 30 seconds, the filter paper was removed, and the treated area was wiped with a sterile cotton ball containing physiological saline to remove residual acetic acid.

[0283] After calcination with glacial acetic acid for 48 hours, the above-mentioned oral ulcer model rats were randomly divided into 4 groups, with 10 rats in each group: 3 dosage groups and 1 blank control group. The 3 dosage groups were low-dose, medium-dose, and high-dose groups, respectively, prepared with distilled water to concentrations of 1.0 mg / mL, 2 mg / mL, and 4 mg / mL using the test substance example (total flavonoid extract of Areca catechu + total alkaloid extract of Areca catechu (mass ratio 1:1)). Rats in each group were administered the drug by gavage at a dose of 10 mL / kg BW daily, while the blank control group received an equal volume of distilled water.

[0284] 2. Detection indicators: 48 hours after acetic acid burning, ulcer formation was observed and recorded. Rats in each group continued treatment until the ulcers were completely healed. The number of days of treatment was recorded, and the ulcer healing time was calculated. The healing criterion was that the mucosal epithelium completely covered the ulcer depression again.

[0285] 3. Statistical Analysis: SPSS 12.0 software was used for statistical analysis of the data. Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and LSD method was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0286] 4. Results: The results are shown in the table below. After burning the labial mucosa with glacial acetic acid for 48 hours, all rats developed ulcers, which were characterized by central depression, pseudomembrane coverage, redness and swelling at the ulcer edges, strong pain response to stimulation, and regular round shape with a diameter of about 5-6 mm, indicating that the model was successful.

[0287] The average healing time of oral ulcers in the low, medium, and high dose sago palm extract treatment groups was significantly shorter than that in the blank control group, and the higher the dose, the shorter the healing time, indicating that sago palm extract has a good therapeutic effect on oral ulcers.

[0288] Table 8. Efficacy of Areca catechu extract in treating oral ulcers.

[0289] Blank control group 11.59±0.89 Low-dose sago palm extract <![CDATA[7.25±1.45 3) ]]> Medium-dose sago palm extract <![CDATA[6.68±2.47 3) ]]> High-dose sago palm extract <![CDATA[5.92±0.96 3) ]]>

[0290] Note: Compared with the blank control group: 1) P < 0.05, 2) P < 0.01, 3) P < 0.001.

[0291] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0292] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of sago palm extract in the preparation of a drug for inhibiting bacteria, Staphylococcus aureus or Candida albicans, wherein the sago palm extract comprises a total alkaloid extract and a total flavonoid extract, wherein the mass ratio of the total alkaloid extract to the total flavonoid extract is 1:1; The preparation method of the total alkaloid extract includes the following steps: Pretreatment: Boil and wash the sago palms in a sodium bicarbonate solution with a mass fraction of 5%-10% for 30-40 minutes at a boiling and washing temperature of 43-50℃, dry, spray with a sodium hydroxide solution with a mass fraction of 0.1-0.5%, and dry at 45-50℃; Preparation of total alkaloid extract: Pretreated sago palms were alkalized with ammonia, allowed to stand, and extracted with microwave at 300-400W for 80-150s. Supercritical fluid extraction was performed using a mixture containing ethanol and methanol as an entrainer to obtain the total alkaloid extract. The volume fraction of ethanol in the mixture was 10-20%, and the volume fraction of methanol was 10-20%. The preparation method of the total flavonoid extract includes the following steps: Pretreatment: Soak Areca catechu in a mixed solution of ethanol and 1-butyl-3-methylimidazolium bromide ionic liquid for 2-2.5 h; the volume ratio of the ethanol solution to the 1-butyl-3-methylimidazolium bromide ionic liquid is 1:1, the volume fraction of the ethanol solution is 95%, and the concentration of the 1-butyl-3-methylimidazolium bromide ionic liquid is 1 mol / L; Preparation of total flavonoid extract: Pretreated Areca catechu was added to a mixed solution of ethanol and 1-butyl-3-methylimidazolium bromide ionic liquid, and extracted with microwave at 600-650W for 10-15 min to obtain crude extract. The crude extract was eluted once with a mixed resin of weakly basic anion exchange resin and macroporous adsorption resin, and then eluted a second time with macroporous adsorption resin to obtain total flavonoid extract. The volume ratio of 1-butyl-3-methylimidazolium bromide ionic liquid to ethanol solution was 1:(1-1.5), the volume fraction of ethanol solution was 70%, the concentration of 1-butyl-3-methylimidazolium bromide ionic liquid was 1 mol / L, and the volume ratio of weakly basic anion exchange resin to macroporous adsorption resin was 1:(1-1.5). 1.5).

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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