A method for preparing soluble microneedles of Artemisia annua alkaloids

Abstract

This invention discloses a method for preparing a soluble microneedle formulation of *Artemisia segetalis* alkaloids. The method involves extracting *Artemisia segetalis* alkaloids from the herb, purifying the extract to obtain a purified alkaloid, and finally preparing soluble microneedles. The soluble microneedles showed a cumulative permeability of over 50% after 6 hours, and near-complete release after 24 hours. The drug release equation conformed to the Higuchi equation, and the cumulative permeability reached 306.79 µg / cm³. 2 .

Description

Technical Field

[0001] This invention relates to the field of extracting Artemisia selengensis, and in particular to a method for preparing soluble microneedles of Artemisia selengensis alkaloids. Background Technology

[0002] *Aconitum brachypodum* Diels, *Aconitum pendulum* Busch, and *Aconitum subrosullatum* H-M., all belonging to the genus *Aconitum* in the Ranunculaceae family, are dried tuberous roots that can dispel wind and dampness, reduce inflammation, and relieve pain. They are used to treat muscle, bone, and joint pain caused by wind-cold-dampness syndrome, with significant efficacy, but they are also highly toxic. Transdermal administration is a common route of administration for toxic traditional Chinese medicines. It is convenient, allows direct application to the treatment site, reduces the concentration of the drug entering the circulatory system, allows for immediate discontinuation of the drug, reduces the occurrence of toxic side effects, avoids the first-pass effect in the liver, and improves drug bioavailability. Therefore, preparing *Aconitum brachypodum* into a transdermal absorption formulation is a more ideal method of administration.

[0003] Microneedles (MNs) are transdermal drug delivery systems with arrays of needle tips at the micrometer scale. They are classified into solid microneedles, coated microneedles, hollow microneedles, soluble microneedles, and hydrogel microneedles. Dissolving microneedles (DMNs) are microneedles prepared using biodegradable polymeric materials as a matrix, offering advantages such as safety, effectiveness, and painlessness. After the needle penetrates the skin, the drug within the needle is released, and the matrix material degrades within the body, making it safe and non-toxic. Currently, most microneedles researched and prepared domestically and internationally are tip-loaded with drugs, resulting in low drug loading and the inability to sustain drug release for extended periods. Furthermore, existing literature does not report on the preparation of soluble microneedles from *Saussurea involucrata* alkaloids. This application studies the preparation process of soluble microneedles fully loaded with *Saussurea involucrata* alkaloids and examines their in vitro transdermal behavior, providing a reference for the development and utilization of transdermal drug delivery systems for *Saussurea involucrata*. Summary of the Invention

[0004] The purpose of this invention is to prepare a soluble microneedle containing the alkaloid of *Saussurea involucrata*. The method employs a centrifugal injection molding process to prepare the soluble microneedle. The microneedle has a clear and complete shape, good skin penetration performance, good mechanical strength, good permeability, and a high cumulative permeation rate.

[0005] The technical solution of the present invention is: a method for preparing soluble microneedles of Artemisia argyi alkaloids, wherein the method is to extract Artemisia argyi alkaloids from Artemisia argyi medicinal material to obtain Artemisia argyi alkaloid extract, then purify it to obtain Artemisia argyi alkaloid purified product, and finally prepare soluble microneedles.

[0006] The aforementioned method for preparing the soluble microneedles of *Saussurea involucrata* alkaloids is carried out according to the following steps:

[0007] (1) Take the medicinal material *Artemisia argyi*, crush it, add 6-12 times the amount of water and decoct it 2-4 times, each time for 0.5-1.5 hours. Filter the decoction and concentrate it over low heat until the raw herb content is 0.25-1.5 g / mL. -1 The medicinal solution yielded product A;

[0008] (2) Dissolve sodium chloride in 30-70% ethanol to prepare a sodium chloride-ethanol elution solution of 0.01-0.04 g / mL, i.e., product B;

[0009] (3) Take 001×7 type cation exchange resin with a diameter-to-height ratio of 1:5 to 1:9, take sample A, and load it at a volume of 2 to 10 BV and a flow rate of 1.0 to 2.0 mL / min. -1 After sample loading, wash with water to remove impurities at a flow rate of 1.0–2.0 mL / min. -1 Wash with water for 5-15 BV, then use product B at 0.5-1.0 mL / min. -1 Elution was performed at a flow rate of 15–25 BV for product B, and the eluent was collected to obtain product C.

[0010] (4) Take product C and adjust the pH to 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product is obtained. The dried product is extracted by reflux with 6-12 times the amount of anhydrous ethanol for 0.5-1.5 h. The extract is collected by filtration. The extract is concentrated and dried. Then it is extracted twice by reflux with chloroform. The first time, it is extracted with 6-10 times the amount of chloroform. The second time, it is extracted with 4-8 times the amount of chloroform. Each time, it is extracted for 1.0-2.0 h. The chloroform reflux extract is collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloid of *Saussurea involucrata*, i.e., product D, is obtained.

[0011] (5) Take 180-360 mg of polyvinylpyrrolidone K120 and 60-140 mg of chondroitin sulfate, add 2-3.5 ml of 30-50% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix, i.e. Product E.

[0012] (6) Take 15-25 mg of product D and add it to product E. Stir until product D is completely dissolved, inject it into the microneedle mold, centrifuge at 4000 r / min for 20 min, take it out and put it in the oven to dry, demold it, and you will get the soluble microneedles of Artemisia argyi alkaloid.

[0013] In step (1) above, take the medicinal material *Artemisia argyi*, crush it, add 12 times the amount of water and decoct it 3 times, 1 hour each time. Filter the decoction and concentrate it over low heat until the raw herb content is 0.25 g·mL. -1 The medicinal liquid was used to obtain product A.

[0014] In step (2) above, sodium chloride is dissolved in 50% ethanol to prepare a sodium chloride-ethanol solution of 0.02 g / mL, i.e., product B.

[0015] In step (3) above, 001×7 type cation exchange resin with a diameter-to-height ratio of 1:9 was used. Sample A was loaded at a volume of 5 BV and a flow rate of 2.0 mL / min. -1 After sample loading, wash with water to remove impurities at a flow rate of 2.0 mL / min. -1 Wash with water for 10 BV, then use product B at 0.5 mL / min. -1 The flow rate was used for elution, and the elution volume of product B was 20 BV. The eluent was collected to obtain product C.

[0016] In step (4) above, product C was adjusted to pH 6-7 with saturated sodium hydroxide, and ethanol was recovered by rotary evaporation to obtain dried product. The dried product was extracted by reflux with 10 times the amount of anhydrous ethanol for 1 hour. After the extract was concentrated and dried, it was extracted twice by reflux with chloroform, the first time with 8 times the amount of chloroform and the second time with 6 times the amount of chloroform, each time for 1.5 hours. The chloroform reflux extracts were collected by filtration and the chloroform was evaporated by rotary evaporation to obtain purified alkaloids of Artemisia argyi, namely product D.

[0017] In step (5) above, take 250-320 mg of polyvinylpyrrolidone K120 and 100-130 mg of chondroitin sulfate, add 2-3 ml of 35-45% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix, i.e., product E.

[0018] In step (5) above, 298 mg of polyvinylpyrrolidone K120 and 123 mg of chondroitin sulfate were taken and added to 2.4 ml of 40% ethanol. The mixture was stirred thoroughly in a water bath until it dissolved and became transparent, thus obtaining the microneedle matrix, namely product E.

[0019] In step (6) above, take 20 mg of product D, add it to product E, stir until product D is completely dissolved, inject it into the microneedle mold, centrifuge at 4000 r / min for 20 min, take it out and put it in an oven at 35℃ for 6 h, demold it, and you will get the soluble microneedles of Artemisia argyi alkaloid.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. Modern pharmacological studies have shown that the alkaloids in Artemisia selengensis have significant analgesic and anti-inflammatory effects, and the extraction and purification of Artemisia selengensis alkaloids have practical application value.

[0022] 2. The cumulative permeability of the alkaloid-soluble microneedles from *Saussurea involucrata* reached over 50% after 6 hours and was basically complete after 24 hours. After fitting, it conformed to the Higuchi equation, and the cumulative permeability reached 91.4%. The prepared *Saussurea involucrata* soluble microneedles have good mechanical strength, enabling effective transdermal drug delivery. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0024] Example 1:

[0025] (1) Extraction method of Artemisia selengensis:

[0026] Take the medicinal herb *Artemisia argyi*, crush it, add 12 times the amount of water, and decoct it three times, one hour each time. Filter the decoction and concentrate it over low heat until the raw herb content is 0.25 g / mL. -1 The medicinal liquid yielded an alkaloid extract of Artemisia argyi.

[0027] (2) Purification method of Saussurea involucrata alkaloids:

[0028] Dissolve sodium chloride in 50% ethanol to prepare a solution of 0.02 g / mL. -1 The sodium chloride-ethanol eluent;

[0029] A 001×7 type cation exchange resin with a diameter-to-height ratio of 1:9 was used to load alkaloid extracts of Artemisia selengensis at a loading volume of 5 BV and a flow rate of 2.0 mL / min. -1 After sample loading, wash with water to remove impurities at a flow rate of 2.0 mL / min. -1 Wash with water for 10 BV, then wash with water to remove impurities, and then use 0.02 g·mL. -1 The sodium chloride-ethanol eluent was dissolved at 0.5 mL / min. -1 Elution at a flow rate of 0.02 g / mL -1 The elution volume of the sodium chloride-ethanol eluent was 20 BV. The eluent was collected to obtain crude alkaloids of Artemisia argyi.

[0030] The crude alkaloids of *Saussurea involucrata* were adjusted to pH 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product was obtained. The dried product was extracted by reflux with 10 times the amount of anhydrous ethanol for 1 hour. The extract was concentrated and dried, and then extracted twice by reflux with chloroform, the first time with 8 times the amount of chloroform and the second time with 6 times the amount of chloroform, each time for 1.5 hours. The two chloroform reflux extracts were collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloids of *Saussurea involucrata* were obtained.

[0031] (3) Preparation method of microneedles containing alkaloids from Artemisia capillaris:

[0032] Take 298 mg of polyvinylpyrrolidone K120 and 123 mg of chondroitin sulfate, add 2.4 ml of 40% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix.

[0033] Take 20 mg of purified alkaloids from *Saussurea involucrata* and add it to the microneedle matrix. Stir until the purified alkaloids from *Saussurea involucrata* are completely dissolved. Pour the solution into a microneedle mold, centrifuge at 4000 r / min for 20 min, then remove and dry in an oven at 35℃ for 6 h. Demold to obtain soluble microneedles of *Saussurea involucrata* alkaloids.

[0034] Example 2:

[0035] (1) Extraction method of Artemisia selengensis:

[0036] Take the medicinal herb *Artemisia argyi*, crush it, add 10 times the amount of water, and decoct twice, 0.5 hours each time. Filter the decoction and concentrate it over low heat until the content of raw herb is 0.5 g / mL. -1 The medicinal liquid yielded an alkaloid extract of Artemisia argyi.

[0037] (2) Purification method of Saussurea involucrata alkaloids:

[0038] Dissolve sodium chloride in 30% ethanol to prepare a solution of 0.01 g·mL⁻¹. -1 The sodium chloride-ethanol eluent;

[0039] A 001×7 type cation exchange resin with a diameter-to-height ratio of 1:7 was used to load the alkaloid extract of Artemisia selengensis. The loading volume was 2 BV, and the loading flow rate was 1.0 mL·min. -1 After sample loading, wash with water to remove impurities at a flow rate of 1.0 mL / min. -1 Wash with water for 5 BV, then wash with water to remove impurities, and then use 0.01 g·mL. -1 The sodium chloride-ethanol eluent was dissolved at 0.5 mL / min. -1 Elution at a flow rate of 0.01 g / mL -1 The elution volume of the sodium chloride-ethanol eluent was 15 BV. The eluent was collected to obtain crude alkaloids of Artemisia argyi.

[0040] The crude alkaloids of *Saussurea involucrata* were adjusted to pH 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product was obtained. The dried product was extracted by reflux with 6 times the amount of anhydrous ethanol for 0.5 h. The extract was concentrated and dried, and then extracted twice by reflux with chloroform, the first time with 6 times the amount of chloroform and the second time with 4 times the amount of chloroform, each time for 1.0 h. The two chloroform reflux extracts were collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloids of *Saussurea involucrata* were obtained.

[0041] (3) Preparation method of microneedles containing alkaloids from Artemisia capillaris:

[0042] Take 180 mg of polyvinylpyrrolidone K120 and 60 mg of chondroitin sulfate, add 2.0 ml of 30% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix.

[0043] Take 20 mg of purified alkaloids from *Saussurea involucrata* and add it to the microneedle matrix. Stir until the purified alkaloids from *Saussurea involucrata* are completely dissolved. Pour the solution into a microneedle mold, centrifuge at 4000 r / min for 20 min, then remove and dry in an oven at 35℃ for 6 h. Demold to obtain soluble microneedles of *Saussurea involucrata* alkaloids.

[0044] Example 3:

[0045] (1) Extraction method of Artemisia selengensis:

[0046] Take the medicinal herb *Artemisia argyi*, crush it, add 6 times the amount of water and decoct it 4 times, 1.5 hours each time. Filter the decoction and concentrate it over low heat until the raw herb content is 0.25 g / mL. -1 The medicinal liquid yielded an alkaloid extract of Artemisia argyi.

[0047] (2) Purification method of Saussurea involucrata alkaloids:

[0048] Dissolve sodium chloride in 40% ethanol to prepare a solution of 0.02 g / mL. -1 The sodium chloride-ethanol eluent;

[0049] A 001×7 type cation exchange resin with a diameter-to-height ratio of 1:5 was used to load 10 BV of Artemisia annua alkaloid extract onto the resin at a flow rate of 1.5 mL / min. -1 After sample loading, wash with water to remove impurities at a flow rate of 1.5 mL / min. -1 Wash with water for 15 BV, then wash with water to remove impurities, and then use 0.02 g·mL. -1 The sodium chloride-ethanol eluent was dissolved at 1.0 mL / min. -1 Elution at a flow rate of 0.02 g / mL -1 The elution volume of the sodium chloride-ethanol eluent was 20 BV. The eluent was collected to obtain crude alkaloids of Artemisia argyi.

[0050] The crude alkaloids of *Saussurea involucrata* were adjusted to pH 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product was obtained. The dried product was extracted by reflux with 12 times the amount of anhydrous ethanol for 1.5 h. The extract was concentrated and dried, and then extracted twice by reflux with chloroform, the first time with 10 times the amount of chloroform and the second time with 8 times the amount of chloroform, each time for 2.0 h. The two chloroform reflux extracts were collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloids of *Saussurea involucrata* were obtained.

[0051] (3) Preparation method of microneedles containing alkaloids from Artemisia capillaris:

[0052] Take 360 ​​mg of polyvinylpyrrolidone K120 and 140 mg of chondroitin sulfate, add 3.5 ml of 50% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix.

[0053] Take 20 mg of purified alkaloids from *Saussurea involucrata* and add it to the microneedle matrix. Stir until the purified alkaloids from *Saussurea involucrata* are completely dissolved. Pour the solution into a microneedle mold, centrifuge at 4000 r / min for 20 min, then remove and dry in an oven at 35℃ for 6 h. Demold to obtain soluble microneedles of *Saussurea involucrata* alkaloids.

[0054] Example 4:

[0055] (1) Extraction method of Artemisia selengensis:

[0056] Take the medicinal herb *Artemisia argyi*, crush it, add 8 times the amount of water and decoct it three times, 0.5 hours each time. Filter the decoction and concentrate it over low heat until the raw herb content is 0.5 g / mL. -1 The medicinal liquid yielded an alkaloid extract of Artemisia argyi.

[0057] (2) Purification method of Saussurea involucrata alkaloids:

[0058] Dissolve sodium chloride in 70% ethanol to prepare a solution of 0.04 g / mL. -1 The sodium chloride-ethanol eluent;

[0059] A 001×7 type cation exchange resin with a diameter-to-height ratio of 1:9 was used to load 10 BV of Artemisia argyi alkaloid extract onto the resin at a flow rate of 2.0 mL / min. -1 After sample loading, wash with water to remove impurities at a flow rate of 2.0 mL / min. -1 Wash with water for 15 BV, then wash with water to remove impurities, and then use 0.01 g·mL. -1 The sodium chloride-ethanol eluent was dissolved at 1.0 mL / min. -1 Elution at a flow rate of 0.04 g / mL -1 The elution volume of the sodium chloride-ethanol eluent was 25 BV. The eluent was collected to obtain crude alkaloids of Artemisia argyi.

[0060] The crude alkaloids of *Saussurea involucrata* were adjusted to pH 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product was obtained. The dried product was extracted by reflux with 10 times the amount of anhydrous ethanol for 1.5 h. The extract was concentrated and dried, and then extracted twice by reflux with chloroform, the first time with 6 times the amount of chloroform and the second time with 8 times the amount of chloroform, each time for 1.0 h. The two chloroform reflux extracts were collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloids of *Saussurea involucrata* were obtained.

[0061] (3) Preparation method of microneedles containing alkaloids from Artemisia capillaris:

[0062] Take 298 mg of polyvinylpyrrolidone K120 and 123 mg of chondroitin sulfate, add 2.4 ml of 40% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix.

[0063] Take 20 mg of purified alkaloids from *Saussurea involucrata* and add it to the microneedle matrix. Stir until the purified alkaloids from *Saussurea involucrata* are completely dissolved. Pour the solution into a microneedle mold, centrifuge at 4000 r / min for 20 min, then remove and dry in an oven at 35℃ for 6 h. Demold to obtain soluble microneedles of *Saussurea involucrata* alkaloids.

[0064] Example 5:

[0065] (1) Extraction method of Artemisia selengensis:

[0066] Take the medicinal herb *Artemisia argyi*, crush it, add 10 times the amount of water, and decoct it four times, one hour each time. Filter the decoction and concentrate it over low heat until the raw herb content is 0.25 g / mL. -1 The medicinal liquid yielded an alkaloid extract of Artemisia argyi.

[0067] (2) Purification method of Saussurea involucrata alkaloids:

[0068] Dissolve sodium chloride in 50% ethanol to prepare a solution of 0.02 g / mL. -1 The sodium chloride-ethanol eluent;

[0069] A 001×7 type cation exchange resin with a diameter-to-height ratio of 1:9 was used to load the alkaloid extract of Artemisia selengensis. The loading volume was 2 BV, and the loading flow rate was 1.0 mL·min. -1 After sample loading, wash with water to remove impurities at a flow rate of 1.5 mL / min. -1 Wash with water for 5 BV, then wash with water to remove impurities, and then use 0.02 g·mL. -1 The sodium chloride-ethanol eluent was dissolved at 0.5 mL / min. -1 Elution at a flow rate of 0.02 g / mL -1 The elution volume of the sodium chloride-ethanol eluent was 15 BV. The eluent was collected to obtain crude alkaloids of Artemisia argyi.

[0070] The crude alkaloids of *Saussurea involucrata* were adjusted to pH 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product was obtained. The dried product was extracted by reflux with 12 times the amount of anhydrous ethanol for 1 hour. The extract was concentrated and dried, and then extracted twice by reflux with chloroform, the first time with 10 times the amount of chloroform and the second time with 4 times the amount of chloroform, each time for 1.5 hours. The two chloroform reflux extracts were collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloids of *Saussurea involucrata* were obtained.

[0071] (3) Preparation method of microneedles containing alkaloids from Artemisia capillaris:

[0072] Take 230 mg of polyvinylpyrrolidone K120 and 120 mg of chondroitin sulfate, add 2.5 ml of 40% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix.

[0073] Take 20 mg of purified alkaloids from *Saussurea involucrata* and add it to the microneedle matrix. Stir until the purified alkaloids from *Saussurea involucrata* are completely dissolved. Pour the solution into a microneedle mold, centrifuge at 4000 r / min for 20 min, then remove and dry in an oven at 35℃ for 6 h. Demold to obtain soluble microneedles of *Saussurea involucrata* alkaloids. Attached Figure Description

[0074] Figure 1 Leakage curve of cation exchange resin for citric acid;

[0075] Figure 2 Elution curve of citrinum on cation exchange resin;

[0076] Figure 3 Box-Behnken response surface and contour plot;

[0077] Figure 4 Morphology of microneedles containing alkaloids from *Saussurea involucrata* (A: digital camera image; B: microneedle array under SEM; C: appearance of a single microneedle);

[0078] Figure 5 Microneedle puncture experiment diagrams (A. Aluminum foil puncture; B. Trypan blue staining results of rat skin after microneedle puncture; C. Tissue section diagram of rat skin after microneedle puncture (×100));

[0079] Figure 6 Transdermal curves of drug-loaded microneedles and gels.

[0080] This invention has undergone extensive experimental research, and the results of this experimental research are as follows:

[0081] 1. Materials

[0082] 1.1 Instruments: PDMS negative mold (needle length 550μm, Zhejiang Taizhou Microchip Pharmaceutical Technology Co., Ltd.); TD5A-120 high-speed centrifuge (Changzhou Jintan Liangyou Instrument Co., Ltd.); 101-3AB electric heating drying oven (Tianjin Tester Instrument Co., Ltd.); SH-50N push-pull force gauge (Yueqing Aidebao Instrument); SZX7 stereomicroscope (Olympus); ion sputtering device (Hitachi High Technology Noca Co., Ltd., Japan, E-1045); scanning electron microscope (FEI, Inspect, USA); AE240 0.0001% electronic balance (Shanghai Mettler Instruments Co., Ltd.); Agilent 1260 high-performance liquid chromatograph; AUY220 0.0001% analytical balance (Shanghai Mettler Instruments Co., Ltd.); RQJ-12B transdermal drug diffusion assay instrument (Shanghai Huanghai Pharmaceutical Testing Instruments); pathological slide machine (Leica RM, Germany). 2016); Microtome (Japanese Yuba R35); Tissue spreader (Wuhan Junjie JK-6); Slides and coverslips (Jiangsu Shitai Experimental Equipment Co., Ltd.); Microscope (Olympus BX53).

[0083] 1.2 Drugs and Reagents: * **Artemisia capillaris reference standard (National Institutes for Food and Drug Control, batch number 110895-200404);** Methanol (Sinopharm Chemical Reagent Co., Ltd., batch number 20201224); **Acetonitrile (Sinopharm Chemical Reagent Co., Ltd., batch number 20200507);** Chloroform (Sinopharm Chemical Reagent Co., Ltd., 20200402); * **Artemisia capillaris** (purchased from Yunnan *Artemisia capillaris* Pharmaceutical Co., Ltd.), identified by Associate Professor Zhong Ke of Guizhou University of Traditional Chinese Medicine as the dried tuberous root of *Aconitum carmichaelii* var. *shortstalk*; *Artemisia capillaris* alkaloids (laboratory-prepared, alkaloid content >70%, calculated as artemisia capillaris A); **Polyvinylpyrrolidone K30 (PVPK30, Solarbio Biotechnology Co., Ltd., batch number 511D03)** 1) Polyvinylpyrrolidone K90 (PVPk90, Shanghai Yuanye Biotechnology Co., Ltd., batch number B01A10S84446); Polyvinylpyrrolidone K120 (PVPk120, Shanghai Yuanye Biotechnology Co., Ltd., batch number G13M11B113099); Chondroitin sulfate (CS, Xi'an Lavia Biotechnology Co., Ltd., batch number 20180222); Edible alcohol (Chongqing Jiangchuan Chemical Co., Ltd.); Hematoxylin (Sigma, H9627); Eosin Y (water-soluble) (71014544); Anhydrous ethanol (10009218); Xylene (10023418); Hydrochloric acid (10011018); Embedded paraffin (69019361); Neutral resin (10004160) were all purchased from Sinopharm Group.

[0084] 1.3 Experimental animals: SD rats, with a body weight of 250 - 280 g, animal production license number: SCXK(Xiang) 2019 - 0014, certificate number: 430726210100322185. All animal experiments followed the regulations of the Ethics Committee of Guizhou University of Traditional Chinese Medicine regarding the management and use of experimental animals, and complied with the 3R principle. Ethical review number: 20210046.

[0085] 2019 - 0014, certificate number: 430726210100322185. All animal experiments followed the regulations of the Ethics Committee of Guizhou University of Traditional Chinese Medicine regarding the management and use of experimental animals, and complied with the 3R principle. Ethical review number: 20210046.

[0086] 2. Study on the extraction process of Saussurea involucrata alkaloids

[0087] 2.1 Investigation on the pretreatment of Saussurea involucrata herbs before extraction

[0088] 2.1.1 Investigation on the soaking of herbs [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​The results in the table above show that the content of sucralose extracted from crushed medicinal materials is 0.35%, while the content of sucralose extracted from uncrushed medicinal materials is 0.28%. It is clear that the content of sucralose extracted from crushed medicinal materials is higher than that from uncrushed medicinal materials. Therefore, it is determined that the medicinal materials should be crushed.

[0099] 2.1.3 Investigation on the water absorption of medicinal materials

[0100] This experiment investigated the water absorption of medicinal materials, providing a reasonable basis for the first water addition ratio in orthogonal experiments.

[0101] Experimental method: 10.026g, 10.008g, and 10.057g of crushed *Artemisia argyi* were weighed and placed in three beakers, respectively. Water was added in volumes of 6, 8, and 10 times the volume of the beaker, respectively. The samples were filtered at the designed time points, and the volume of the filtrate was measured. The amount of water absorbed by the herb at that time point was the difference between the volume of water added and the volume of the filtrate. After the measurement, the filtrate was poured back into the original beaker for the next measurement. The results are shown in Table 3.

[0102] Table 3. Investigation of water absorption of medicinal materials (n=2)

[0103]

[0104] The water absorption data were statistically analyzed using a t-test. The differences in water absorption at different time points were not statistically significant, and the water absorption was approximately 0.3 times the weight of the medicinal material itself. The results indicate that the water absorption of *Saussurea involucrata* is very small, therefore, there is no need to consider increasing the amount of water added initially.

[0105] 2.2 Orthogonal Experiment to Optimize Extraction Process Parameters of Artemisia selengensis

[0106] The results of the single-factor study determined that the medicinal materials did not require soaking; instead, they were crushed and extracted using a water decoction method. The orthogonal experiment was then used to optimize the water decoction extraction conditions.

[0107] 2.2.1 Orthogonal Experimental Design

[0108] The medicinal material *Artemisia argyi* was extracted using a decoction method, and the optimal extraction process parameters were selected through orthogonal experiments.

[0109] Solvent usage (A), number of extractions (B), and extraction time (C) were selected as the factors to be investigated. Each factor was designed with three levels, using an L9(3) model. 4 An orthogonal array was used to design the experiment. The extraction process of *Saussurea involucrata* was optimized using the extraction rates of sucralose and total alkaloids as indicators. The factor levels are shown in Table 4.

[0110] Table 4 Factor Levels

[0111]

[0112] 2.2.2 Experimental Methods

[0113] Weigh 18 portions of *Artemisia selengensis* (10g each) and conduct experiments according to Table 4 (each experiment was performed in duplicate). Determine the contents of sucralose and total alkaloids, and calculate their extraction rates.

[0114] 2.2.3 Experimental Results and Analysis of Variance

[0115] The results of the orthogonal experiment are shown in Table 5, and the results of the analysis of variance are shown in Table 6.

[0116] Table 5. Results of the orthogonal experiment (n=2)

[0117]

[0118] Note: Overall score = (Snowflake extract rate / maximum snowflake extract rate × 0.5 + Snowflake total alkaloid extract rate / maximum snowflake total alkaloid extract rate × 0.5) × 100%

[0119] Table 6. Results of Analysis of Variance

[0120]

[0121]

[0122] Significance level: α = 0.05F 0.05 (2,2) = 19.00* indicates a significant difference.

[0123] As shown in the range values ​​in Table 5, the order of influence of each factor is B > A > C, meaning that the number of extractions has the greatest impact on the extraction of *Saussurea involucrata*, followed by the amount of water added, and finally the extraction time. Comparing the means, level 3 in factor A has the highest extraction rate, as does level 3 in factor B, and level 2 in factor C. The variance analysis results in Table 6 show that factors A and B have significant differences in their combined effects on the extraction rates of *Saussurea involucrata* and total alkaloids. Therefore, the extraction scheme should be A3B3C1. However, since the extraction rate at level 1 in factor C is much lower than that at level 2, and the 1-hour extraction time does not pose a time-consuming problem for water decoction, the optimal extraction scheme is ultimately determined to be A3B3C2, which involves decocting and extracting three times with 12 times the amount of water, each time for 1 hour.

[0124] 2.2.4 Validation test of extraction process

[0125] Three portions of the medicinal material, each weighing 10g, were weighed and subjected to a verification test according to the optimized extraction process. The selected extraction process was: 12 times the amount of water, decocted and extracted three times, each time for 1 hour. The experimental results are shown in Table 7.

[0126] Table 7. Verification Test Results

[0127]

[0128] As shown in Table 7 and Table 4, the optimal extraction process has a similar extraction rate for sucralose and total alkaloids to the optimal extraction process in the orthogonal experiment (Experiment 9), and the extraction rate of sucralose has good repeatability. Therefore, the optimal extraction process conditions selected by the orthogonal experiment are stable and feasible.

[0129] 2.3 Conclusions and Discussion

[0130] The extraction process parameters were determined as follows: three decoction extractions, one hour each time, and the solvent volume was 12 times the volume of water each time. This extraction process is stable and feasible.

[0131] 3. Study on the purification process of Artemisia selengensis alkaloids

[0132] 3.1 Screening of Resin Type

[0133] 3.1.1 Types and properties of resins

[0134] The types and properties of ion exchange resins are shown in Table 8.

[0135] Table 8. Types and properties of ion exchange resins

[0136]

[0137] 3.1.2 Resin Pretreatment

[0138] Pretreatment of ion exchange resin: Place an appropriate amount of cation exchange resin in a 3BV 10% NaCl solution and soak for 3 hours. Discard the brine, rinse with distilled water until nearly colorless, then soak the resin in 3BV 95% ethanol for 3 hours, and wash with distilled water until no alcohol odor remains. Use 3BV 1 mol·L⁻¹ ethanol... -1 Soak the resin in hydrochloric acid for 3 hours, discard the acid solution, wash with distilled water until nearly neutral, and then soak in 1 mol·L⁻¹ hydrochloric acid at 3 BV. -1 Soak in NaOH solution for 3 hours, discard the alkaline solution, and finally wash with distilled water until nearly neutral. Then, use 1 mol·L⁻¹ NaOH solution (3 BV). -1 Soaking in hydrochloric acid for 3 hours converts the resin to H+. + The sample is then washed with distilled water until it is nearly neutral, and then set aside.

[0139] 3.1.3 Preparation of sample loading solution

[0140] The optimal extraction process was used to extract *Artemisia selengensis*, specifically the water decoction method, with 12 times the amount of water added, and extraction was performed three times, one hour each time. The three extracts were combined and concentrated over a low flame until the crude drug content was 0.5 g / mL. -1 The medicinal solution is prepared using the following method:

[0141] (2) Preparation of loading solution for screening ion exchange resin type: The loading solution contains 0.5 g / mL of crude drug. -1 The pH of the drug solution was adjusted to 1.0 with hydrochloric acid at 2400 rpm. -1 Centrifuge for 30 minutes and collect the supernatant.

[0142] 3.1.4 Resin Type Screening Method

[0143] The adsorption and desorption of various resins were investigated using a static adsorption method.

[0144] Precisely measure 10 mL each of the pretreated ion exchange resins 001×4, 001×7, 001×1.1, and D001, and place them in separate Erlenmeyer flasks. Add 20 mL of sample loading solution to each flask and place them in a 25°C constant temperature water bath shaker at 100 rpm. -1 Shake for 4 hours, filter, collect the filtrate as the residual liquid, wash the resin 5 times with distilled water, 10 mL each time, filter, and combine the washing liquids as the washing solution.

[0145] Desorption method for ion exchange resin: Add 20 mL of 2 mol·L⁻¹ ion exchange resin. -1 Ammonia-ethanol (80% ethanol) was added to the above ion exchange resin, and the mixture was shaken for 4 hours. The solution was filtered, and the resin was washed with 30 mL of 95% ethanol. The washings and filtrates were combined and concentrated appropriately to remove the ammonia. 95% ethanol was added to a 50 mL volumetric flask to obtain the eluent. All residual solution, washings, and 5 mL of eluent were evaporated to dryness in an 80°C water bath. The residues were dissolved in methanol and diluted to a 50 mL volumetric flask. The solutions were filtered through a 0.45 μm microporous membrane, and the filtrates were used for analysis.

[0146] The content of artemisinin in *Saussurea involucrata* was determined by HPLC, and the adsorption rate and desorption rate of artemisinin by each resin were calculated according to the following formulas.

[0147] E%=(C0V0-C1V1-C2V2) / C0V0×100%

[0148] E'%=C3V3 / (C0V0-C1V1-C2V2)×100%

[0149] In the formula, E is the adsorption rate, E' is the desorption rate, C0V0 is the content of the target component in the loading solution, C1V1 is the content of the target component in the residual solution, C2V2 is the content of the target component in the washing solution, and C3V3 is the content of the target component in the desorption solution.

[0150] 3.2 Investigation of Ion Exchange Resin Purification Process

[0151] Ion exchange resin purification of alkaloids utilizes the exchange of alkaloid salt cations with the resin to separate them from other components and impurities, thus achieving purification. Literature review revealed that ammonia-ethanol solution and sodium chloride-ethanol solution are commonly used as eluents to elute alkaloids adsorbed on ion exchange resins, achieving good results.

[0152] 3.2.1 Examination of the concentration degree of the sample solution

[0153] The decoction extract of *Saussurea involucrata* (equivalent to 100.03g of medicinal material) was concentrated to a crude drug content of 0.5g / mL. -1 0.25 g·mL -1 The drug solutions were refrigerated at 4°C for 24 hours and then centrifuged. The precipitate was then treated with 0.001 mol·L⁻¹ water. -1 The solution was washed with hydrochloric acid, and the washing liquid was brought to a certain volume. The total alkaloid content in the washing liquid, i.e., the loss of total alkaloids, was determined by ultraviolet-visible spectrophotometry. The results are shown in Table 9.

[0154] Table 9. Investigation of sample drug concentration (n=2)

[0155]

[0156] The results in the table above show that the solution is concentrated to 0.25 g·mL. -1 The total alkaloid loss rate in the medicinal solution was 0.5 g·mL. -1 Small, 0.25 g·mL -1 The following concentrations are too dilute, resulting in a large workload in actual production. Furthermore, excessively diluted solutions will reduce the adsorption rate on the resin. Therefore, further dilution will not be considered, and the sample solution concentration is determined to be 0.25 g·mL. -1 .

[0157] 3.2.2 pH value of the sample solution

[0158] The extract of *Saussurea involucrata* was concentrated to 0.25 g / mL. -1 After refrigerating at 4℃ for 24 hours, centrifuge and collect the supernatant. Adjust the pH to 1.00, 2.00, 3.00, and 5.96 with hydrochloric acid (without pH adjustment). Take 40 mL of the above solution for dynamic adsorption at 1.0 mL / min. -1 Flow rate through 0.01×7 cation exchange resin (15mm ID, 20mL), wash with water 10 BV, and use 2 mol·L⁻¹ water. -1 Elute with ammonia and 60% ethanol for 10 BV, and collect the eluent. Observe the loading and elution, and determine the content of artemisinin in the drug solution before acidification, after acidification, and in the eluent. Calculate the artemisinin loss rate and eluent rate, and screen the optimal pH value of the loading solution. The results are shown in Table 10.

[0159] Table 10. pH value of the sampled drug solution (n=2)

[0160]

[0161] *Artemisia annua loss rate = Amount of artemisia annua reduction in the solution after acidification / Artemisia annua content in the solution before acidification

[0162] As shown in the table above, the elution rate was highest with a sample solution pH of 1.00, but the concentration of artemisinin in the solution was lowest, and the loss rate of artemisinin was significantly higher than the other solutions. There was no significant difference in elution rates among sample solutions with pH values ​​of 2.00, 3.00, and 5.96, but the loss rate of artemisinin was highest at pH 2.00 and lowest at pH 5.96. Therefore, the pH value of the sample solution should not be adjusted.

[0163] 3.2.3 Sample loading flow rate investigation

[0164] Take 20 mL of the treated cation exchange resin and pack it into a chromatography column of the same type (15 mm ID). Accurately pipette 40 mL of the sample solution and inject it at 0.5 mL / min. -1 1.0 mL·min -1 and 2.0 mL·min -1 After dynamic adsorption, wash with water for 10 BV (200 mL), then add 0.02 g·mL⁻¹. -1 NaCl-70% ethanol at 1 mL·min -1 Elution was performed at a rate of 10 BV. Residual liquid and eluent were collected, and leakage rate and resolution rate were calculated to select the optimal loading flow rate. The results are shown in Table 11.

[0165] Table 11. Sample loading flow rate investigation (n=2)

[0166]

[0167] As shown in the table above, the sample loading flow rate is 2.0 mL / min. -1 The highest resolution was achieved during this period, and there was no leakage; therefore, the sample loading flow rate was determined to be 2.0 mL / min. -1 .

[0168] 3.2.4 Investigation of Maximum Sample Loading Amount

[0169] Take 20 mL of the treated cation exchange resin and pack it into two chromatography columns (15 mm ID). Take the sample solution and spray it at 2 mL / min. -1 Samples were loaded at a flow rate of 1 BV and collected once. The content of artemisinin in the samples was determined, and a leakage curve was plotted. The results are shown in Table 12.

[0170] Table 12 Investigation of Maximum Sample Loading Amount (n=2)

[0171]

[0172] The leakage curves show that leakage occurred after the drug solution was loaded, but the leakage rate was very small before the loaded drug solution reached 5 BV, with a cumulative leakage rate of 0.28%. After loading 5 BV, the leakage rate increased, reaching 1.69% when the loaded drug solution reached 10 BV. Therefore, the maximum loading volume was determined to be 5 BV.

[0173] 3.2.5 Investigation of water washing flow rate and water washing volume

[0174] Take 20 mL of the treated cation exchange resin and pack it into two chromatography columns (15 mm ID). Take the sample solution and spray it at 2 mL / min. -1 Loading rate: 5 BV, with distilled water at 2 mL / min. -1 Wash with water at a flow rate of 1 BV, collecting one batch at a time. A portion was used for the Molish reaction test, and the other portion was used to determine the leakage of scleroderma by HPLC. The results showed that after washing with water for 10 BV, the Molish reaction was negative and no scleroderma leakage was observed. Therefore, a flow rate of 2 mL / min was determined. -1 A water wash with a flow rate of 10 BV is sufficient.

[0175] 3.2.6 Investigation of Sodium Chloride Concentration in Sodium Chloride Ethanol Eluent

[0176] Take 20 mL of the treated cation exchange resin and pack it into a chromatography column of the same type (15 mm ID). Accurately pipette 40 mL of the sample solution and spray at 2.0 mL / min. -1 Dynamic loading and adsorption at flow rate, followed by washing with water at 10 BV (2.0 mL·min). -1 ), respectively at 0.01 g·mL -1 NaCl 70% ethanol solution, 0.02 g / mL -1 NaCl-70% ethanol solution, 0.03 g / mL -1 NaCl-70% ethanol solution, 0.04 g·mL -1 NaCl-70% ethanol solution was administered at 1 mL / min. -1 Elution was performed at a flow rate of 10 BV. The eluent was collected and the content of sclerosing agent was determined by HPLC. The eluent rate was calculated, and the optimal sodium chloride concentration was determined. The results are shown in Table 13.

[0177] Table 13 Investigation of sodium chloride concentration in sodium chloride ethanol eluent (n=2)

[0178]

[0179]

[0180] The results in the table above show that the sodium chloride concentration is 0.02 g·mL. -1 Since succinate had the highest resolution, the sodium chloride concentration in the eluent was determined to be 0.02 g·mL. -1 .

[0181] 3.2.7 Investigation of ethanol concentration in sodium chloride ethanol eluent

[0182] Take 20 mL of the treated cation exchange resin and pack it into a chromatography column of the same type (15 mm ID). Accurately pipette 40 mL of the sample solution and spray at 2.0 mL / min. -1 Dynamic loading and adsorption at flow rate, followed by washing with water at 10 BV (2.0 mL·min). -1 The eluent was prepared to a concentration of 0.02 g / mL. -1 Sodium chloride - 0% ethanol, 0.02 g / mL -1 Sodium chloride-30% ethanol, 0.02 g / mL -1 Sodium chloride-50% ethanol, 0.02 g / mL -1 Sodium chloride-70% ethanol solution, at 1 mL / min -1 Elution was performed at a flow rate of 10 BV. The eluent was collected, the content of sclerotin was determined, the eluent rate was calculated, and the ethanol concentration was screened. The results are shown in Table 14.

[0183] Table 14 Investigation of ethanol concentration in sodium chloride ethanol eluent (n=2)

[0184]

[0185] 0.02 g·mL -1 Sodium chloride does not dissolve completely in ethanol with a concentration of 90% or higher, so it is not prepared in this solution. Experimental results show that 30% and 50% ethanol have the highest resolution rates, with little difference between them. However, considering the ease with which the eluent evaporates, 50% ethanol was chosen.

[0186] 3.2.8 Analysis of Flow Rate

[0187] Take 20 mL of the treated cation exchange resin and pack it into a chromatography column of the same type (15 mm ID). Accurately pipette 40 mL of the sample solution and spray at 2.0 mL / min. -1 Dynamic loading and adsorption at flow rate, followed by washing with water at 10 BV (2.0 mL·min). -1 ), 0.02 g·mL -1 Sodium chloride-50% ethanol was prepared at concentrations of 0.5, 1.0, and 2.0 mL / min. -1 Elution was performed at a flow rate of 10 BV. The eluent was collected, the content of sclerotin was determined, the eluent rate was calculated, and the optimal elution flow rate was selected. The results are shown in Table 15.

[0188] Table 15 Analytical Analysis of Flow Velocity (n=2)

[0189]

[0190] The experimental results show that 0.5 mL·min -1 Elution at the optimal flow rate yielded the highest resolution; therefore, the optimal elution flow rate was determined to be 0.5 mL / min. -1 .

[0191] 3.2.9 Analysis of liquid volume

[0192] Take 20 mL of the treated cation exchange resin and pack it into a chromatography column of the same type (15 mm ID). Then, spray at a rate of 2.0 mL / min. -1 5 BV of drug solution was loaded dynamically at a flow rate, followed by washing with 10 BV (2.0 mL / min) of water. -1 ), 0.02 g·mL -1 Sodium chloride-50% ethanol at 0.5 mL / min -1 Elution was performed at a flow rate of 1 BV, and the eluent was collected once. The content of sclerotin was determined, the cumulative elution rate of sclerotin was calculated, and the required volume of eluent was determined. The results are shown in Table 16.

[0193] Table 16 Analysis of Liquid Volume (n=2)

[0194]

[0195] From Table 16 and Figure 1 , Figure 2 It can be seen that the cumulative resolution of sclerosing agent increases with the increase of elution solvent. After the elution volume reaches 20 BV, the increase in cumulative resolution becomes smaller and smaller. Therefore, the elution volume of the elution solution is determined to be 20 BV.

[0196] 3.2.10 Examination of resin diameter-to-height ratio

[0197] 14 mL, 20 mL, and 24.5 mL of the treated resin were respectively packed into the same type of chromatography column (15 mm ID), making the diameter-to-height ratios 1:5, 1:7, and 1:9, respectively. The columns were then injected at 2 mL / min. -1 Loading rate: 5 BV; washing with water: 10 column volumes (2 mL / min) -1 ), 0.02 g·mL -1 Sodium chloride-50% ethanol at 0.5 mL / min -1 Elution was performed at a flow rate of 20 BV. The eluent was collected, the content of sclerosing agent was determined, the eluent rate was calculated, and the optimal diameter-to-height ratio of the resin was determined. The results are shown in Table 17.

[0198] Table 17 Resin Diameter-to-Height Ratio Investigation (n=2)

[0199]

[0200] The results in the table show that the resolution is highest when the diameter-to-height ratio is 1:9, so a diameter-to-height ratio of 1:9 is chosen.

[0201] 3.2.11 Verification Test

[0202] Take 24.5 mL of pretreated cation exchange resin (diameter-to-height ratio 1:9) and pack it into a chromatography column of the same type (15 mm ID). 0.25 g·mL -1 The sample solution was loaded at a rate of 2.0 mL / min. -1 Dynamic loading at a flow rate of 5 BV, followed by washing with water at 10 BV (2.0 mL / min). -1 ), 0.02 g·mL -1 Sodium chloride-50% ethanol at 0.5 mL / min -1 Elution was performed at a flow rate of 20 BV. The eluent was collected, the content of sclerotin was determined, and the eluent rate was calculated. Results are shown in 18.

[0203] Table 18 Verification Tests

[0204]

[0205] The results of the verification test show that the average resolution of the drug solution after passing through the cation exchange resin is 95.47% (RSD% = 0.78%), indicating that the purification process of the drug solution by the cation exchange resin is stable and feasible.

[0206] 3.3 Investigation of the desalting and refining process of the eluent

[0207] Using sodium chloride-ethanol solution as the eluent can solve the problems of resin poisoning and low eluent efficiency in other eluent methods. However, sodium chloride-ethanol solution also introduces sodium chloride salt impurities, so further desalting studies are needed to ensure the purity of the purified product.

[0208] 3.3.1 Investigation of desalting solvent after direct drying of the eluent

[0209] After the eluent was concentrated and dried, a certain amount (approximately 4.4 g) was taken and subjected to ultrasonic extraction using 95% ethanol, anhydrous ethanol, and chloroform as solvents, respectively. The extraction was performed three times: the first extraction was with 50 mL of solvent for 30 min, and the second and third extractions were with 25 mL of solvent for 15 min. The three extracts were combined, cooled, and filtered. The filtrate was concentrated and dried under reduced pressure to obtain the purified product. The total alkaloid content was determined by ultraviolet light, and the loss rate and purity after desalting were calculated. The results are shown in Table 19.

[0210] Table 19. Investigation of desalting solvents after direct drying of the eluent (n=2)

[0211]

[0212]

[0213] The results in the table above show that the purity of total alkali in the purified product after chloroform desalting is relatively high, but its loss rate is also the greatest. Further investigation can be conducted after treating the eluent with alkali.

[0214] 3.3.2 Desalting solvent test after neutralizing the eluent with alkali

[0215] The eluent was found to be slightly acidic, so it was considered to neutralize it with saturated sodium hydroxide solution (pH = 6-7) before concentration and drying. Ultrasonic desalination was then performed using anhydrous ethanol and chloroform. The total alkali loss rate and purity after desalination were calculated. The results are shown in Table 20.

[0216] Table 20: Investigation of desalting solvents after neutralization with alkali in the eluent (n=2)

[0217]

[0218] The results in the table above show that the purity of the purified product after desalting with anhydrous ethanol is still very low, but it can remove most of the salt. In contrast, the purified product obtained after desalting with chloroform has higher purity, and its loss rate is lower than before alkalization. Therefore, it is preferable to use anhydrous ethanol to remove most of the salt, followed by further purification with a small amount of chloroform.

[0219] 3.3.3 Examination of the degree of alkalinity of the analytical solution

[0220] The eluent was alkalized with saturated sodium hydroxide solution, and the pH of the eluent was adjusted to 6–7, 7–9, and 10–12. The effects of different degrees of alkalization on the loss rate and purity of total alkali in the snow were investigated. The optimal pH range of the eluent was selected, and the results are shown in Table 21.

[0221] Table 21 Analysis of the degree of alkalization of the solution (n=2)

[0222]

[0223] The results in the table above show that when the pH of the eluent was adjusted to 6-7 and 7-9 using high-concentration sodium hydroxide, there was no significant difference in the total alkaloid loss rate. However, when the pH was around 11, the total alkaloid loss rate increased significantly, possibly because excessive alkalinity destroys the alkaloids. Therefore, it is recommended to adjust the pH of the eluent to 6-7 using high-concentration sodium hydroxide.

[0224] 3.3.4 Investigation of desalination extraction methods

[0225] Considering that the ultrasonic method is not suitable for large-scale production, the reflux method was chosen to investigate its desalination effect. After the eluent was concentrated and dried, a certain amount (approximately 4.4 g) was extracted three times by reflux: the first time by refluxing with 50 mL of chloroform for 1 h, and the second and third times by refluxing with 25 mL of chloroform for 0.5 h. The three extracts were combined, cooled, filtered, and the filtrate was concentrated and dried under reduced pressure to obtain the purified product. The total alkali content was determined by ultraviolet light, and the loss rate and purity were calculated. The results compared with the ultrasonic method are shown in Table 22.

[0226] Table 22 Investigation of desalination extraction methods (n=2)

[0227]

[0228] As shown in the table above, the total alkali loss rate of the chloroform reflux method is significantly lower than that of the ultrasonic method, and the purity of the total alkali in the purified product is higher than that obtained by the ultrasonic method. Therefore, the reflux method is determined to be the desalination method.

[0229] 3.3.5 Preliminary Desalination Study of Ethanol

[0230] Due to the high toxicity of chloroform, to reduce the amount of chloroform used during desalination, it is considered to first remove a large amount of salt with ethanol and then further desalinate with a small amount of chloroform. To reduce the complexity of the process while ensuring a high extraction rate, a single-stage extraction method for desalination is proposed. The following is an investigation of the extraction rate of active ingredients after single-stage desalination with different solvent volumes.

[0231] A certain amount (approximately 4.4 g) of the dried eluent was extracted and desalted by reflux with 10, 20, and 30 times the volume of ethanol, respectively. Each solvent volume was repeated in duplicate, once for 1 hour. After extraction, the solution was filtered and brought to a specific volume to obtain the desalted sample solution. 5 ml of the eluent was accurately measured, evaporated to dryness, dissolved in methanol, and brought to a final volume of 10 ml to obtain the undesalted sample solution. Appropriate amounts of both the undesalted and desalted sample solutions were filtered through a 0.45 μm microporous membrane. The sucralose content was determined by HPLC, and the extraction rate of sucralose after desalting was calculated. The results are shown in Table 23.

[0232] Table 23 Preliminary investigation of ethanol desalination (n=2)

[0233]

[0234] The results in the table above show that after one round of reflux with ethanol for desalination, there was no significant difference in extraction rates among 10, 20, and 30 times the amount of ethanol. Furthermore, the lowest extraction rate for sucralose reached 92.84%, meaning that 3 / 4 of the salt could be removed, significantly reducing the amount of chloroform required. Therefore, it was determined that reflux extraction with 10 times the amount of anhydrous ethanol for 1 hour would be used, followed by concentration and drying of the extract, and further desalination with chloroform for further investigation.

[0235] 3.3.6 Orthogonal experiments were conducted to optimize the chloroform reflux desalination process conditions.

[0236] Using chloroform as the extraction solvent, solvent volume (A), number of extractions (B), and extraction time (C) were selected as the factors to be investigated. Each factor was designed with three levels, using an L9(3) model. 4 An orthogonal array was used to design the experiment. The factor level table is shown in Table 24.

[0237] Table 24 Factor Level Table

[0238]

[0239] After preliminary desalination with ethanol, the extract was concentrated and dried to obtain a dried product. A certain amount of this dried product was weighed, totaling 9 portions, and experiments were conducted according to the orthogonal experimental design table. The extraction process conditions for desalination by chloroform reflux were optimized using the total alkali extraction rate and the sucralose extraction rate as indicators. The results are shown in Table 25.

[0240] Table 25 Results of the orthogonal experiment

[0241]

[0242] Note: Overall score = (Snowflake extract rate / maximum snowflake extract rate × 0.5 + Snowflake total alkaloid extract rate / maximum snowflake total alkaloid extract rate × 0.5) × 100%

[0243] Table 26 Analysis of Variance Table

[0244]

[0245] Significance level: α = 0.05F 0.05 (2,2) = 19.00* indicates a significant difference.

[0246] The intuitive analysis table shows that the effects of the three factors on the extraction of sucralose and total alkaloids are B>C>A, indicating that the number of extractions has the greatest impact, followed by extraction time and solvent volume. The analysis of variance table shows that the number of extractions has a significant impact on the extraction rate of the target component, but the mean values ​​for levels 2 and 3 show little difference. Considering the workload and chloroform usage in large-scale production, two extractions are recommended. This ensures complete extraction while minimizing the impact on workload and chloroform usage; therefore, solvent volume and extraction time should be selected as A2 and C3, respectively. The orthogonal experimental results show that experiment 5 has the highest extraction rate, consistent with the optimal scheme. This is A2B2C3, which involves two chloroform reflux extractions, with 8 times the amount of chloroform for the first extraction and 6 times the amount for the second, each lasting 1.5 hours.

[0247] 3.3.7 Verification Test

[0248] Three 1g portions of the ethanol-desalted extract were weighed and extracted according to the optimized scheme A2B2C3, i.e., chloroform reflux extraction twice, the first time with 8 times the amount of chloroform and the second time with 6 times the amount of chloroform, each time for 1.5 hours. The content of sucralose in the extract was determined by HPLC, and the content of total alkaloids was determined by UV-Vis spectrophotometry. The extraction rates of both were calculated. The extract was then concentrated and dried to obtain a purified product. The purity of sucralose and the purity of total alkaloids in the purified product were determined. The results are shown in Table 27.

[0249] Table 27 Verification Tests

[0250]

[0251] The results above show that after desalination by chloroform reflux, the extraction rate of sucralose reached 85.32%, the extraction rate of total alkaloids reached 83.69%, the purity of sucralose in the purified product was 19.55%, and the purity of total alkaloids was 60.57%. This indicates that after preliminary desalination with ethanol, further desalination by chloroform reflux is effective and the process is stable and feasible.

[0252] 4. Study on the preparation process of soluble microneedles containing alkaloids from *Saussurea involucrata*

[0253] 4.1 Preparation of soluble microneedles

[0254] Soluble microneedles were prepared by centrifugation: an appropriate amount of matrix material was weighed, an appropriate amount of ethanol was added, and the mixture was stirred thoroughly in a water bath until it dissolved and became transparent. The drug was added and stirred until it was completely dissolved. The mixture was then injected into a microneedle mold and centrifuged at 4000 r / min for 20 min. After centrifugation, the microneedles were placed in an oven at 35℃ for 6 h and then demolded to obtain the desired microneedles.

[0255] 4.2 Screening of Soluble Microneedle Formulations

[0256] 4.2.1 Single-factor investigation

[0257] 4.2.1.1 Establishment of Evaluation Methods

[0258] Appearance: Visually inspect whether the microneedle backing is flat; observe the integrity of the microneedle array under a stereomicroscope.

[0259] Solubility: The solubility of microneedles was evaluated by the dissolution time of the microneedle tip. Measurement of microneedle tip dissolution time: The microneedle was inserted into plastic wrap, allowing the tip to fully penetrate the wrap. The plastic wrap was then placed over a beaker filled with water at 37°C. At 3 min, 5 min, 8 min, and 10 min, the microneedle was removed and the dissolution of the tip was observed under a stereomicroscope. The dissolution time of the microneedle tip was recorded.

[0260] Mechanical strength: The mechanical strength of microneedles was evaluated by the puncture rate in rat skin. The specific procedure was as follows: Healthy SD rats were euthanized by cervical dislocation and hair removal. The skin was peeled, subcutaneous fat removed, and the skin repeatedly rinsed with physiological saline. The skin was then preserved in physiological saline for later use. Before use, the skin surface was dried, and the skin was placed with the stratum corneum side facing upwards and fixed on a foam board. Microneedles were applied vertically to the skin with a pressure of 20N and held for 30 seconds. Immediately afterwards, the skin at the microneedle application site was stained with 1% trypan blue solution. After 30 seconds, excess dye was wiped away with a cotton swab, and the skin was cleaned with physiological saline. The number of puncture sites on the skin surface was observed and recorded. Puncture rate = (number of puncture sites on skin / number of microneedle arrays) * 100%. Number of microneedle arrays = 400 (number of arrays of the microneedle negative mold). The appearance, solubility, and mechanical strength of the microneedles were evaluated using the above method. The evaluation criteria are shown in Table 28.

[0261] Table 28 Evaluation Criteria

[0262]

[0263] 4.2.1.2 Screening of matrix materials

[0264] Literature review revealed that commonly used soluble microneedle materials mainly include various polymers and sugars. Polymers include biodegradable materials such as polyvinyl alcohol (PVA), hyaluronic acid (HA), polyvinylpyrrolidone (PVP), and chondroitin sulfate (CS). Commonly used sugars include trehalose, maltose, sucrose, and galactose. CS is a commonly used nutritional supplement in arthritis treatment, well-tolerated by patients, with few side effects and minimal drug interactions. Literature reports that CS can be used to prepare soluble microneedles, and preliminary experiments have shown that microneedles prepared by mixing CS and PVP in a certain ratio have good shape retention and a certain degree of puncture capability. Therefore, CS and PVP were selected as the microneedle matrix materials.

[0265] The higher the ethanol concentration, the greater the solubility of Artemisia annua alkaloids. Based on preliminary tests and considering the solubility of the solvent on the matrix and the drug, 40% ethanol was selected as the solvent.

[0266] PVP comes in different grades due to its varying molecular weight, such as PVPk30, PVPk90, and PVPk120. Different molecular weights result in different viscosities; the higher the molecular weight, the higher the viscosity. The properties of the microneedle matrix directly affect the morphology, solubility, and mechanical properties of the microneedles. Therefore, we investigated soluble microneedles prepared from three different grades of PVP, using morphology, solubility, and mechanical strength as indicators.

[0267] CS was weighed separately from PVPk30, PVPk90, and PVPk120, with 90 mg of CS and 270 mg of PVP. Each was then added to 2.5 ml of 40% ethanol, and soluble microneedles were prepared according to section "4.1". The appearance, solubility, and mechanical strength of the microneedles were evaluated using a comprehensive score to select the most suitable PVP. The results showed that the microneedles prepared from the mixture of CS and PVPk120 exhibited good appearance, rapid dissolution, and superior mechanical strength. The results are shown in Table 29.

[0268] Table 29 Screening Results of Matrix Material Types

[0269]

[0270] 4.2.1.3 Screening of Chondroitin Sulfate (CS) Dosage

[0271] Three portions of 270 mg PVPk120 were weighed and respectively mixed with 60 mg, 90 mg, and 120 mg of CS. 2.5 ml of 40% ethanol was added, and soluble microneedles were prepared according to section "4.1". The appearance, solubility, and mechanical strength of the microneedles were evaluated. The optimal amount of CS was selected. The results showed that the microneedles prepared with 120 mg of CS had the highest overall score; the results are detailed in Table 30.

[0272] Table 30: Screening Results of CS Dosage

[0273]

[0274] 4.2.1.4 Screening of PVP k120 dosage

[0275] Three portions of 120 mg CS were weighed and respectively mixed with 180 mg, 270 mg, and 360 mg of PVP k120. 2.5 ml of 40% ethanol was added, and soluble microneedles were prepared according to section "4.1". The morphology, solubility, and mechanical strength of the microneedles were evaluated. The optimal amount of PVP k120 was determined. Results showed that 270 mg of PVP k120 produced the best microneedles; details are shown in Table 31.

[0276] Table 31 Screening Results of PVP k120 Dosage

[0277]

[0278] 4.2.1.5 Screening of 40% Ethanol Dosage

[0279] Three portions of CS and PVPk120 were weighed, with 120 mg of CS and 270 mg of PVPk120 respectively. 2 ml, 2.5 ml, 3 ml, and 3.5 ml of 40% ethanol were added respectively, and soluble microneedles were prepared according to section "4.1". The morphology, solubility, and mechanical strength of the microneedles were evaluated. The optimal solvent dosage was selected. The results showed that the microneedles prepared with a solvent dosage of 2.5 ml received the best overall score, as detailed in Table 32.

[0280] Table 32 Investigation of Ethanol Dosage

[0281]

[0282] 4.2.1.6 Investigation of Dosage

[0283] Three portions of the screened matrix were weighed, and 15 mg, 20 mg, and 25 mg of purified *Saussurea involucrata* extract were added respectively. Soluble microneedles were prepared according to section "4.1". The appearance, solubility, and mechanical strength of the microneedles were evaluated. The maximum dosage was optimized. The results showed that when the dosage was 25 mg, precipitation occurred after centrifugation. The appearance, solubility, and mechanical strength were all good when the dosages were 15 mg and 20 mg. Therefore, the dosage was determined to be 20 mg. The results are detailed in Table 33.

[0284] Table 33 Investigation of Dosage

[0285]

[0286] 4.2.2 Optimizing Microneedle Formulations Using Box-Behnken Response Surface Methodology

[0287] Based on the results of the single-factor study, the dosage of CS (A), PVP k120 (B), and 40% ethanol (C) were selected as the factors of study, and the puncture rate was used as the evaluation index. The Box-Behnken response surface methodology was employed to optimize the microneedle formulation. Factors and levels are shown in Table 34, experimental design and results are shown in Table 35, and the results of the analysis of variance are shown in Table 36. The response surface plot and contour plot are shown in Table 37. Figure 3 .

[0288] Table 34: Factors and Levels of the Box-Behnken Response Surface Method.

[0289]

[0290] Table 35 Experimental Design and Results

[0291]

[0292] Table 36 Analysis of Variance in the Box-Behnken Experiment

[0293]

[0294] The Box Behnken design factors and experimental results, after being fitted using Design Expert 10, conform to a quadratic multinomial regression model: Y = 97.20 + 2.62*A + 7.50*B - 1.63*C - 1.50*AB - 1.75*AC - 4.00*BC - 12.48*A 2 -13.22*B 2 -7.48*C 2 The significance test results are shown in Table 8. The model's P < 0.0001 (highly significant), and the P = 0.2687 > 0.05 (not significant) for the lack-of-fit term, demonstrating that the quadratic multinomial regression model can well reflect the relationship between the overall score (Y) and the amounts of CS, PVP, and solvent. The model's coefficient of variation is 2.78, indicating model stability. The coefficient of determination and corrected coefficient of determination are 0.9853 and 0.9664, respectively, indicating that the model has a good fit.

[0295] The optimized microneedle formulation of *Saussurea involucrata* alkaloids, obtained using Design-Expert 10 software, consisted of 123.025 mg of CS, 297.794 mg of PVP k120, and 2.399 ml of 40% ethanol. Under these conditions, the predicted microneedle puncture rate was 98.954%. Considering practical considerations, the final microneedle formulation was determined to be: 123 mg of CS, 298 mg of PVP k120, and 2.4 ml of 40% ethanol.

[0296] 4.2.3 Verification Experiment

[0297] Three batches of soluble microneedles containing *Saussurea involucrata* alkaloids were prepared according to the optimal formulation selected by Box-Behnken response surface methodology, and the porosity of the microneedles was measured. The results showed that the porosity of the three batches of microneedles were 97%, 98%, and 100%, respectively, with an average of 98.3% and an average relative deviation of 1.3%. The measured values ​​were close to the predicted values, and the repeatability of the verification experiment was good.

[0298] 4.3 Characterization of soluble microneedles

[0299] 4.3.1 Morphological characteristics

[0300] The morphology of the microneedles prepared with the optimal formulation was photographed using a digital camera, and the results are as follows: Figure 4 As shown in (A), the morphology of the microneedles was observed using a scanning electron microscope, and the results are as follows. Figure 4 (B) Figure 4 As shown in (C). The prepared *Saussurea involucrata* alkaloid microneedles are square patches (2.79 cm²). 2It is yellow, with a smooth surface and a complete array of 400 needles with a spacing of 400μm. The microneedles are conical in shape, with a length of 550μm and a bottom diameter of 300μm.

[0301] 4.3.2 Puncture performance

[0302] The puncture performance of the microneedles was investigated using aluminum foil puncture and ex vivo mouse skin puncture tests.

[0303] Aluminum foil puncture test: Lay aluminum foil flat on foam, press the aluminum foil with a microneedle using your thumb for 30 seconds, then remove it and observe the surface of the aluminum foil. Holes should be clearly visible on the aluminum foil. The results are as follows: Figure 5 As shown in (A).

[0304] Ex vivo mouse skin puncture test: The puncture performance of the prepared microneedles was characterized according to the method for evaluating mechanical strength under section "4.2". The results are as follows: Figure 5 As shown in (B), pinhead-like blue marks were left on the surface of the rat's skin.

[0305] Histological examination of isolated rat skin via puncture: SD rats were anesthetized with chloral hydrate, and their abdomens were shaved and dried. Drug-loaded microneedles were applied to the shaved skin at a pressure of 20 N and held for 30 seconds before being removed. The punctured skin was immediately dissected within 1 minute and fixed in 4% paraformaldehyde for 24 hours. After dehydration, paraffin embedding, sectioning, drying, and HE staining, the puncture site on the isolated skin was observed by photographing. Results are as follows: Figure 5 As shown in (C), the soluble microneedles successfully pierced the stratum corneum and entered the epidermis, indicating that the microneedles broke through the barrier of the stratum corneum without reaching the nerve endings in the dermis, thus achieving a painless drug delivery effect. Microneedle puncture experiments showed that the microneedles could pierce aluminum foil and mouse skin, demonstrating good puncture performance.

[0306] 4.3.3 Determination of drug content in microneedles

[0307] Three tablets of the prepared Artemisia argyi soluble microneedle were accurately weighed and extracted with 80 times the amount of methanol by ultrasonic extraction for 30 min. After centrifugation, the insoluble matrix material was removed and the tablets were placed in a 10 mL volumetric flask. The volume was adjusted to 10 mL with methanol and filtered through a microporous membrane (0.22 μm). The initial filtrate was discarded and the subsequent filtrate was collected. The drug content was determined. Each tablet of Artemisia argyi soluble microneedle contained (0.94±0.025) mg of Artemisia argyi A.

[0308] 4.4 In vitro transdermal test

[0309] 4.4.1 In vitro skin penetration experiment of Saussurea involucrata alkaloid microneedles and gel

[0310] Preparation of Artemisia annua alkaloid gel ointment: The gel ointment formula is glycerin: pure water: NP-700: Al(OH)3: tartaric acid (0.6475:0.1712:0.1446:0.0163:0.0068), and the gel ointment contains 0.94 mg / g of artemisia annua alkaloid. Take 1g of gel ointment and spread it evenly on the backing layer, with the coating area being the same as the microneedle area.

[0311] Male SD rats were anesthetized with 10% chloral hydrate. After anesthesia, most of the fur in the affected area was shaved with a shaver, and the remaining fur was shaved off with an electric shaver. The skin was removed, and the adipose tissue and fascia were separated. The rats were repeatedly washed with physiological saline, dried with filter paper, wrapped in aluminum foil, and placed in a -80°C freezer. Before use, the rats were removed from the -80°C freezer and soaked in physiological saline for 30 minutes.

[0312] A modified Franz diffusion cell was used, with the gel-ointment group as a control, to investigate the in vitro transdermal performance of drug-loaded microneedles. A microneedle was placed on a prepared isolated rat skin and a force of 20 N was applied. After 1 minute, it was fixed with medical tape, ensuring the skin's integrity throughout the process. After treatment, the skin was fixed at the junction of the supply and receiving chambers, with the stratum corneum facing the supply chamber and the dermis in contact with the receiving chamber. 6.5 mL of physiological saline was injected into the receiving chamber, air bubbles were removed, and the receiving liquid surface was ensured to be in complete contact with the skin. Simultaneously, a separate group received the same amount of *Saussurea involucrata* alkaloid gel-ointment without any skin treatment, applied to the upper side of the stratum corneum. The microneedle group and the gel-ointment group were each in triplicate. After fixing the supply and receiving chambers, the diffusion cell was placed on a thermostatic magnetic stirrer in a 37°C water bath at a stirring speed of 300 rpm. -1 At 0, 1, 2, 4, 6, 8, 12, 18, and 24 hours after transdermal administration, 1.5 mL of the receiving fluid was placed in a 2 mL PVC tube, and an equal volume of physiological saline at the same temperature was added simultaneously. The sample solution was filtered through a 0.22 μm microporous membrane, and the drug content in the filtrate was determined. Data were processed using SPSS 26, and a cumulative drug permeation curve was plotted. The results are as follows: Figure 6 As shown, after 1 hour, the microneedle group had more drug permeation than the gel group, and the cumulative permeation rate was greater than 50% after 6 hours and reached 91.4% after 24 hours, while the cumulative permeation rate of the gel ointment control group was only 41.8%. The cumulative drug permeation of the drug-loaded microneedle group was significantly higher than that of the gel ointment group, indicating that the prepared Saussurea involucrata alkaloid microneedles can effectively puncture the skin and release drugs transdermally, and can also significantly improve the transdermal permeation effect of Saussurea involucrata alkaloids.

[0313] Calculation formula:

[0314] Cumulative penetration rate (%) = Qn / drug loading × 100%;

[0315] In the formula, Qn is the cumulative permeation of the drug per unit area at the nth sampling point (μg / cm²). 2 Cn is the drug concentration (μg / mL) in the sample solution received at the nth sampling point, Ci is the drug concentration (μg / mL) in the receiving solution measured at the i-th (i≤n-1) sampling point, V0 is the sampling volume (1.5mL), and S is the effective permeation area (2.8cm²) during drug permeation. 2 V is the volume of the receiving cell (6.5 mL).

[0316] 4.4.2 Data Processing

[0317] Model fitting was performed on the data from the above transdermal experiments, including zero-order equations, first-order equations, and Higuchi equations, as shown in Table 37. Based on the correlation coefficient R... 2 It can be seen that the penetration of *Saussurea involucrata* alkaloid microneedles conforms to the Higuchi equation, while the penetration of *Saussurea involucrata* alkaloid gel conforms to the zero-order equation. The steady-state transdermal rates Jss of the *Saussurea involucrata* alkaloid microneedle group and the gel group are 9.27 and 6.13, respectively, indicating that the permeability of the microneedles is stronger than that of the gel.

[0318] Table 37. Data Fitting Results of Transdermal Permeation Tests for Drug-Loaded Microneedles and Gels

[0319]

[0320] Note: Group A is the microneedle group; Group B is the gel / ointment group.

[0321] 4.5 Discussion

[0322] This experiment aims to achieve effective release and reduced toxicity of *Saussurea involucrata* alkaloids via transdermal administration using soluble microneedles. Currently, most microneedles developed domestically and internationally are tip-loaded, relying primarily on the needle tip to penetrate the stratum corneum and dissolve to release the drug. However, these microneedles have low drug loading capacity and cannot sustain drug release for extended periods. Literature review revealed that the channels opened by microneedles can be maintained for 4-6 hours, allowing for continued drug release along the backing layer. Therefore, this experiment aims to prepare fully drug-loaded microneedles, hoping to rapidly establish effective blood drug concentrations and maintain stability over a certain period.

[0323] In vitro skin penetration results showed that, compared with the gel / ointment group, the soluble microneedle group had faster penetration within 1 hour of administration, with a cumulative penetration rate of approximately 22%. This rate slowed down after 1 hour, reaching approximately 40% by 2 hours. The penetration rate then gradually decreased during the 2-8 hour and 8-24 hour periods, but remained relatively stable within these periods. The soluble microneedle group achieved a cumulative penetration rate exceeding 50% at 6 hours and was essentially fully released by 24 hours. The drug release equation conformed to the Higuchi equation, and the cumulative penetration reached 306.79 μg / cm³. 2 The experimental results show that after transdermal drug delivery using the prepared soluble microneedles, the drug in the microneedles can quickly penetrate the skin in a short time. After the drug is released from the needle tip, the backing layer continues to release the drug to maintain a certain drug concentration, thereby achieving a sustained therapeutic effect.

Claims

1. A method for preparing soluble microneedles of *Saussurea involucrata* alkaloids, characterized in that: The method for preparing the soluble microneedles of Artemisia argyi alkaloids is to extract Artemisia argyi alkaloids from Artemisia argyi medicinal material, then purify the extract to obtain purified Artemisia argyi alkaloids, and finally prepare soluble microneedles. The preparation method of the soluble microneedles of Artemisia annua alkaloids is carried out according to the following steps: (1) Take the medicinal material *Artemisia argyi*, crush it, add 6-12 times the amount of water and decoct it 2-4 times, each time for 0.5-1.5 hours. Filter the decoction and concentrate it over low heat until the raw drug content is 0.25-1.5 g·mL. -1 The medicinal solution yielded product A; (2) Dissolve sodium chloride in 30-70% ethanol to prepare a sodium chloride-ethanol solution with a concentration of 0.01-0.04 g / mL, i.e., product B; (3) Take 001×7 type cation exchange resin with a diameter-to-height ratio of 1:5 to 1:9, take product A for loading, the loading volume is 2 to 10 BV, and the loading flow rate is 1.0 to 2.0 mL·min -1 After sample loading, wash with water to remove impurities at a flow rate of 1.0–2.0 mL / min. -1 Wash with water for 5-15 BV, then use product B at 0.5-1.0 mL / min. -1 Elution was performed at a flow rate of 15–25 BV for product B, and the eluent was collected to obtain product C. (4) Take product C and adjust the pH to 6-7 with saturated sodium hydroxide. After rotary evaporation to recover ethanol, the dried product is obtained. The dried product is extracted by reflux with 6-12 times the amount of anhydrous ethanol for 0.5-1.5 h. The extract is collected by filtration. The extract is concentrated and dried. Then it is extracted twice by reflux with chloroform. The first time, 6-10 times the amount of chloroform is used, and the second time, 4-8 times the amount of chloroform is used. Each time, it is extracted for 1.0-2.0 h. The chloroform reflux extract is collected by filtration. After rotary evaporation to evaporate the chloroform, the purified alkaloid of *Saussurea involucrata*, i.e., product D, is obtained. (5) Take 180-360 mg of polyvinylpyrrolidone K120 and 60-140 mg of chondroitin sulfate, add 2-3.5 ml of 30-50% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix, i.e. Product E. (6) Take 15-25 mg of product D and add it to product E. Stir until product D is completely dissolved, inject it into the microneedle mold, centrifuge at 4000 r / min for 20 min, take it out and put it in the oven to dry, demold it, and you will get the soluble microneedles of Artemisia argyi alkaloid.

2. The method for preparing soluble microneedles of *Artemisia segetalis* alkaloids according to claim 1, characterized in that: In step (1), take the medicinal material *Artemisia argyi*, crush it, add 12 times the amount of water and decoct it 3 times, 1 hour each time. Filter the decoction and concentrate it over low heat until the raw herb content is 0.25 g·mL. -1 The medicinal liquid was used to obtain product A.

3. The method for preparing soluble microneedles of *Artemisia segetalis* alkaloids according to claim 1, characterized in that: In step (2), sodium chloride is dissolved in 50% ethanol to prepare a sodium chloride-ethanol solution of 0.02 g / mL, namely product B.

4. The method for preparing soluble microneedles of *Saussurea involucrata* alkaloids according to claim 1, characterized in that: In step (3), 001×7 type cation exchange resin with a diameter-to-height ratio of 1:9 is used. Sample A is loaded at a volume of 5 BV and a flow rate of 2.0 mL / min. -1 After sample loading, wash with water to remove impurities at a flow rate of 2.0 mL / min. -1 Wash with water for 10 BV, then use product B at 0.5 mL / min. -1 The flow rate was used for elution, and the elution volume of product B was 20 BV. The eluent was collected to obtain product C.

5. The method for preparing soluble microneedles of *Saussurea involucrata* alkaloids according to claim 1, characterized in that: In step (4), product C is adjusted to pH 6-7 with saturated sodium hydroxide, and ethanol is recovered by rotary evaporation to obtain dried product. The dried product is extracted by reflux with 10 times the amount of anhydrous ethanol for 1 hour. After the extract is concentrated and dried, it is extracted twice by reflux with chloroform, the first time with 8 times the amount of chloroform and the second time with 6 times the amount of chloroform, each time for 1.5 hours. The chloroform reflux extracts are collected by filtration and the chloroform is evaporated by rotary evaporation to obtain purified alkaloids of Artemisia argyi, namely product D.

6. The method for preparing soluble microneedles of *Saussurea involucrata* alkaloids according to claim 1, characterized in that: In step (5), take 250-320 mg of polyvinylpyrrolidone K120 and 100-130 mg of chondroitin sulfate, add 2-3 ml of 35-45% ethanol, and stir thoroughly in a water bath until dissolved and transparent to obtain the microneedle matrix, i.e., product E.

7. The method for preparing the soluble microneedles of *Saussurea involucrata* alkaloids according to claim 6, characterized in that: In step (5), 298 mg of polyvinylpyrrolidone K120 and 123 mg of chondroitin sulfate are taken and added to 2.4 ml of 40% ethanol. The mixture is stirred thoroughly in a water bath until it dissolves and becomes transparent, thus obtaining the microneedle matrix, i.e., product E.

8. The method for preparing soluble microneedles of *Saussurea involucrata* alkaloids according to claim 1, characterized in that: In step (6), 20 mg of product D is added to product E and stirred until product D is completely dissolved. The mixture is then injected into a microneedle mold, centrifuged at 4000 r / min for 20 min, and then placed in an oven at 35℃ for 6 h to dry. After demolding, soluble microneedles of alkaloids from *Artemisia argyi* are obtained.

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