AU2021105119A4 - Drug for treating ovarian cancer, and preparation method and use thereof - Google Patents

Abstract

OF THE DISCLOSURE The present disclosure discloses a drug for treating ovarian cancer, and a preparation method and use thereof. The drug is prepared from natural autumnus Artemisia plants through extraction, preliminary filtration, fine filtration, sterilization, and lyophilization, and has a purity as high as 99.95% to 99.99% (mass percentage content). The present disclosure discovers a botanical active substance for treating ovarian cancer for the first time. It has been confirmed by a large number of experiments that an autumnus Artemisia alkaloid can lock and selectively kill ovarian cancer cells, with an inhibition rate on ovarian cancer as high as 75.8% to 89.5%, which is truly a natural botanical active substance for treating ovarian cancer with a remarkable curative effect and small side effects. Therefore, a drug with the autumnus Artemisia alkaloid as a medicinal active ingredient provided in the present disclosure is expected to become an anti-ovarian cancer drug with promising clinical application prospects. As the raw material for extracting the autumnus Artemisia alkaloid in the present disclosure is common and low in cost, the present disclosure can both alleviate the pain and reduce the economic burden for a patient. ABSTRACT DRAWING - FIG 1 -1/2 FIG. 1

Description

-1/2

FIG. 1

DRUG FOR TREATING OVARIAN CANCER, AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

[01] The present disclosure belongs to various disciplines of botany, medicine, pharmacy, pharmaceutics, etc. The present disclosure mainly relates to a botanical active substance, pharmaceutical preparation, preparation and rational use of a medicament, and other aspects, and particularly relates to an oral botanical preparation that includes an alkaloid extracted from autumnus Artemisia as a pharmaceutical active substance and is mainly used for treating ovarian cancer, and a preparation method thereof.

BACKGROUNDART

[02] Ovarian tumors:

[03] Ovarian tumors refer to tumors that occur on ovaries. Ovarian malignant tumors are also tumors with the highest mortality among gynecological malignancies. Great progress has been made in basic research and clinical diagnosis and treatment of ovarian malignant tumors in recent years, but unfortunately a 5-year survival rate is still not high, hovering at 30%.

[04] So far, the pathogeny of ovarian malignant tumors is unclear.

[05] Treatment of ovarian tumors:

[06] At present, in the treatment of tumors, there are two major treatment manners for ovarian tumors, including western medicine treatment and traditional Chinese medicine (TCM) treatment. Western medicine treatment generally refers to surgery, radiotherapy, chemotherapy, biotherapy, immunotherapy, etc.; and TCM treatment includes oral administration (prescriptions, Chinese patent medicine, and modern TCM extract), topical administration (pasting, dressing, and moxibustion), dietotherapy, etc.

[07] The development of safe and effective drugs (especially botanical drugs) for treating ovarian tumors has always been an important research topic for people.

[08] In addition, TCMs have a very complicated composition and include active ingredients, ineffective ingredients, and toxic ingredients. In order to improve the therapeutic effect of TCMs, it is necessary to extract the effective ingredients to the greatest extent and remove the ineffective and toxic ingredients. Therefore, developing an efficient extraction process for TCMs is particularly important for improving the inherent quality and clinical efficacy of TCM preparations. However, traditional extraction methods (such as decoction, supercritical CO extraction, reflux, dipping, etc.) show shortcomings such as large active ingredient loss, long extraction period, too-many procedures, and low extraction rate in terms of retaining effective ingredients and removing ineffective ingredients. Therefore, in order to improve a yield and purity for effective ingredients in TCMs, there is an urgent need for a new process for extracting effective ingredients from TCMs.

SUMMARY

[09] The present disclosure is intended to provide a method for extracting an alkaloid from autumnus Artemisia.

[10] The present disclosure provides a method for extracting an alkaloid from autumnus Artemisia, including the following steps:

[11] 1) drying and ultra-fine crushing native autumnus Artemisia plants to obtain an autumnus Artemisia powder with a particle size of 80 mesh to 120 mesh (37 m to 23 m);

[12] 2) extraction: mixing the autumnus Artemisia powder with extraction water at a volume ratio of 1:6 to obtain an extraction system; filtering extraction water by a reverse osmosis (RO) filter cartridge with an accuracy of 0.001 m to 0.0001 [m, and distilling and cooling to 15°C to °C to obtain distilled water; and diluting the extraction system with the distilled water, and subjecting a resulting mixture to dynamic stirring for 50 min to 60 min and then to ultrasonic treatment for 30 min to 50 min;

[13] 3) preliminary filtration at 15°C to 25°C: subjecting an obtained extract solution to cloth preliminary filtration, with a filtration pore size of 30 m;

[14] 4) fine filtration at 15°C to 25°C: filtering with a 0.45 m molecular membrane to obtain a filtrate;

[15] 5) sterilizing with a polymer membrane at 15°C to 25°C, where the polymer membrane has a filtration pore size of 0.45 m (preferably 0.22 [m); and

[16] 6) spray-drying a sterilized filtrate to obtain a powder, and mixing the powder with the distilled water obtained by the process of filtering extraction water by an RO filter cartridge (with an accuracy of 0.001 m to 0.0001 [m) and distilling and cooling to 15°C to 25°C at a volume ratio of 1:1; and lyophilizing a resulting mixture to obtain a powder, which is an autumnus Artemisia alkaloid.

[17] In step 1), an ultrasonic generator generates a signal of a specific frequency through a signal generator, and this specific frequency is the frequency of a transducer. Ultrasonic frequencies generally used in ultrasonic equipment are 20 KHz, 25 KHz, 28 KHz, 33 KHz, 40 KHz, and 60 KHz.

[18] In step 1), the autumnus Artemisia may be crushed with an ultrasonic cell crusher (product model: preferably GDC-TQ/ZS/3, Shandong Jining Sinobest Biological Machinery Co., Ltd.) under the following working parameters: ultrasonication/interval: 5 s/5 s, and preferably 3 s/3 s; number of cycles: 70 (total time: 10 min), and preferably 60 to 120 (total time: 6 min to 12 min); and power: ) 500 W (preferably 800 W).

[19] In step 2), the extraction may be conducted with an ultrasonic extraction machine in an intelligently-controlled continuous ultrasonic multifunctional extraction machine set (product model: preferably GDC-TQ/ZS/3, Shandong Jining Sinobest Biological Machinery Co., Ltd.); and the ultrasonic extraction machine may have: ultrasonic frequency: two frequency bands of 26 KHz and 70 KHz, each with a frequency adjustment range of± 3 KHz; ultrasonic power: 800 W, continuously adjustable; and extraction tube specification: diameter: 300 mm, length: 300 mm, and volume: > 20 L. In step 2), a transducer assembly may be mainly composed of a transducer and an amplitude transformer.

[20] In step 3), the equipment (preferably a product of Shandong Jining Sinobest Biological Machinery Co., Ltd.) where a noise generated during a working process can be effectively reduced to keep a laboratory quiet may be used for preliminary filtration, for example, filter: ED180x400; filter bag: polypropylene (PP); and ALY-500 device (product model: preferably Aoliyuan/ALY-500, Shanghai Aoliyuan Environmental Technology Co., Ltd.).

[21] In step 4), the fine filtration may be conducted with a formed filter material, where an original mixture passes through the filter material under the action of pressure, a filter residue is left on a tube wall, and afiltrate flows out through the filter material, thus achieving the purpose of filtration. The formed filter material may include: filter cloth, filter screen, filter disc, sintered filter tube, string wound filter cartridge, melt blown filter cartridge, etc. The fine filtration is conducted to remove suspended solids, some colloidal substances, fine particles, etc. The filter used for the fine filtration has the following advantages: high filtration accuracy, uniform filter cartridge pore size, small filtration resistance, large flux, strong retaining capacity, long service life, high cleanliness of filter cartridge material, no contamination to a filter medium, resistance to acid, alkali and other chemical solvents, high strength, high temperature resistance, uneasy deformation offilter cartridge, low price, low operating cost, easy cleaning, and replaceable filter cartridge. In step 4), the fine filtration may be conducted with an ALY-500 device (product model: preferably ALY-500, Shanghai Aoliyuan Environmental Technology Co., Ltd.) under the following working parameter: high filtration accuracy.

[22] The polymer membrane in step 5) may preferably be a product of Haining Yongsheng Membrane Filtration Equipment Manufacturing Co., Ltd, with a model of QJ50, which is a new filter that combines decolorization, decarbonization, insulation, deep filtration, semi-fine filtration, and high-flow fine filtration. A host machine is made of carbon steel, stainless steel, and strong acid and alkali resistant materials, and a wood machine is polished to 400 mesh internally and externally. The filter meets the Gmp standard, and is an adaptable multi-purpose filter device with excellent craftsmanship, safe and stable performance, corrosion resistance, durability, simple operation, flexibility, and energy efficiency. This step is conducted to remove suspended solids, some colloidal substances, and fine particles.

[23] In step 6), the lyophilizing may be conducted with a vacuum lyophilizer (product model: preferably GLZY-1BS (CIP + SIP), Shanghai Pudong Lyophilization Equipment Co., Ltd.). Working parameters: a lamellar cylinder is equipped with an SUS316L stainless steel bellow to protect a hydraulic rod and a product, with an on-line integrity detection function; a hydraulic system is equipped with a back pressure valve, which can achieve pressure relief for a cylinder chamber during steam sterilization to avoid damage; a plug hydraulic cylinder is equipped with an anti-slide device to prevent a lamella from sliding down during a lyophilization process; and during a CIP process, the lamella is dynamically cleaned according to a set value.

[24] In step 6), because the vacuum lyophilizing is conducted at a low temperature and a low pressure, and the water is directly sublimated, a product is imparted with many special properties. For example, a vacuum lyophilization technology can also achieve thorough dehydration for thermosensitive materials, and a lyophilized drug is very stable, which is convenient for long-term storage. Since the drying of a material is completed in a frozen state, compared with other drying methods, the lyophilization results in little change to a physical structure and a molecular structure of the material, whose structure and appearance are well retained. During a vacuum lyophilization process, there is no surface hardening on a material and a porous sponge-like structure is formed inside the material, so a dried material has excellent rehydration properties and can be restored to the state before drying in a short time. Because a lyophilization process is conducted at a very low temperature and the air is basically isolated, biological, chemical or physical changes in thermosensitive materials are effectively inhibited, and the high-active substances in a raw material and the color of a raw material are well retained.

[25] Vacuum lyophilizer (product model: preferably GLZY-1BS (CIP + SIP), Shanghai Pudong Lyophilization Equipment Co., Ltd.).

[26] Another objective of the present disclosure is to provide an alkaloid extracted from autumnus Artemisia by the method described above.

[27] The present disclosure also provides an oral botanical preparation that is mainly used for treating ovarian cancer with a remarkable curative effect and small side effects and includes the alkaloid extracted from autumnus Artemisia as a medicinal active substance.

[28] A medicinal active ingredient in the botanical drug provided by the present disclosure is the alkaloid extracted from autumnus Artemisia.

[29] If needed, one or more pharmaceutically acceptable adjuvants can be added to the above-mentioned drug. The adjuvants include a diluent, an excipient, a filler, an adhesive, a wetting agent, an absorption promoter, a surfactant, a lubricant, a stabilizer, and other conventional adjuvants in the pharmaceutical field. If necessary, a flavoring agent, a sweetening agent, and a pigment can also be added.

[30] The drug can be made into a tablet, a granule, a powder, a capsule, an oral liquid, and other pharmaceutically acceptable oral dosage forms, preferably a tablet, and more preferably an enteric-coated tablet. When an enteric-coated tablet is adopted, an added adjuvant can be any one or more from the group consisting of pharmaceutical dextrin, colloidal silicon dioxide, starch, and microcrystalline cellulose (MCC), and a part by weight ratio of the excipient to all pharmaceutical raw materials in the tablet can be 1:(40-50).

[31] The drug in an enteric-coated tablet may specifically include the following components: 40 to 50 parts (preferably 50 parts) of the autumnus Artemisia alkaloid and 1 to 2 parts (preferably 2 parts) of MCC.

[32] The drug tablet may have a coating made from a film coating material and preferably from water-soluble hydroxypropyl methylcellulose (HPMC); and the drug tablet may be white with the coating being removed.

[33] Usage and dosage of the drug: measurement standard: 250 mg/tablet; an adult takes three times a day, with one tablet each time; a juvenile or child takes at an amount half of that for an adult; and The drug can exhibit a preferred effect when taken on an empty stomach with warm water.

[34] The present disclosure provides a method for extracting an alkaloid from autumnus Artemisia. The alkaloid is extracted from natural autumnus Artemisia plants through extraction, preliminary filtration, fine filtration, sterilization, and lyophilization, and has a purity as high as 99.95% to 99.99% (mass percentage content). The present disclosure discovers a botanical active substance for treating ovarian cancer for the first time, namely, an autumnus Artemisia alkaloid, which is a water-soluble white crystalline monomer and has a yield of 18%. It has been confirmed by a large number of experiments that the autumnus Artemisia alkaloid can lock and selectively kill ovarian cancer cells, with an inhibition rate on ovarian cancer as high as 75.8% to 89.5%, which is truly a natural botanical active substance for treating ovarian cancer with a remarkable curative effect and small side effects. Therefore, a drug with the autumnus Artemisia alkaloid as a medicinal active ingredient provided in the present disclosure is expected to become an anti-ovarian cancer drug with promising clinical application prospects. As the raw material for extracting the autumnus Artemisia alkaloid in the present disclosure is common and low in cost, the present disclosure can both alleviate the pain and reduce the economic burden for a patient. In addition, the botanical drug with the autumnus Artemisia alkaloid as a medicinal active substance provided in the present disclosure also has the advantages of convenient administration and simple production process, which is convenient for popularization and application. The extraction process of the autumnus Artemisia alkaloid according to the present disclosure has the following characteristics:

[35] 1. The extraction water is filtered with an RO filter cartridge with a filtration accuracy of 0.001 m to 0.0001 m to remove bacteria and heavy metals in the water. The extraction water, after being filtered with the RO filter cartridge with a filtration accuracy of 0.001 m to 0.0001 [m, has high permeability, can achieve a high leaching rate (up to 99.9%) of active ingredients in a drug, and allows active ingredients in a drug to be 100% soluble and fully absorbed.

[36] 2. After the water is distilled and cooled to 15°C to 25°C, resulting distilled water is taken to dilute the extraction system, and a resulting mixture was dynamically stirred for 30 min to 60 min and then subjected to ultrasonic treatment for 30 min to 40 min, where a water temperature is always controlled at 15°C to 25°C throughout the process.

[37] 3. Studies have shown that most of active ingredients in an original medicine show poor efficacy after being extracted, and this is mainly because the traditional extraction requires heating concentration that will cause the loss of volatiles in a drug. Studies have confirmed that the medicinal volatiles can penetrate through an intestinal barrier, a cellular barrier, and a blood-brain barrier (BBB) in a few seconds at a small dosage, exhibiting quick onset. The extraction method of the present disclosure is conducted always at 15°C to 25°C, such that the volatiles in the drug can be completely locked and will not be lost. The low-temperature extraction method of the present disclosure involves no concentration and no heating and directly adopts lyophilization to obtain a powder after the extraction and filtration are completed, which cannot be achieved by traditional methods so far. In the low-temperature extraction method, after extraction at 15°C to 25°C, lyophilization is directly conducted to obtain a powder (there is no need to conduct heating concentration at 55°C to 65°C until a relative density is 1.1 to 1.15). Further studies have confirmed that an extract obtain by room-temperature extraction at 15°C to 25°C and then directly lyophilizing an extract product into a powder has high contents of medicinal active ingredients with high activity, retains the aboriginality and pure nature, and shows advantages such as strong efficacy, light color (with high activity and other benefits), low dosage, easy absorption, and quick onset.

[38] 4. Compared with the traditional extraction method, the present disclosure has the following advantages: Rapid extraction and separation can be achieved at room temperature without adding any solvent, which ensures the leaching out of almost all effective ingredients in a product, with high product purity, simple operation, less energy consumption, less time consumption, and low cost. Compared with a traditional extraction method, the present disclosure takes a shorter extraction time, leads to a higher yield, and requires no heating. Therefore, the present disclosure is a technological innovation in the field of TCM active ingredient extraction.

[39] The present disclosure is further described in detail below with reference to specific examples.

BRIEF DESCRIPTION OF DRAWINGS

[40] FIG. 1 shows microscopic examination result of the autumnus Artemisia alkaloid (white crystalline powder); and

[41] FIG. 2 shows an extraction process flow of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[42] Unless otherwise specified, the percentage concentration refers to a mass percentage concentration, a mass/volume (W/V, in unit of g/100 mL) percentage concentration, or a volume/volume percentage concentration.

[43] The manners for acquiring various biological materials described in the examples are provided only to achieve specific objectives of the present disclosure, and should not constitute a limitation on sources of the biological materials of the present disclosure. In fact, the biological materials used are extensively available, and any biological materials that can be acquired without violating laws and ethics can be used instead according to prompts in the examples.

[44] The examples are implemented on the premise of the technical solutions of the present disclosure and the detailed implementations and specific operation processes are provided. The examples will help understand the present disclosure, but the protection scope of the present disclosure is not limited to the following examples.

[45] Example 1. Extraction of an autumnus Artemisia alkaloid

[46] I. Raw material

[47] Autumn artemisia (Latin name: autumnus Artemisia), also known as Artemisia hedinii, Artemisia annua, or Artemisia absinthium, which is the whole plant of Artemisia annua in the Compositae family.

[48] II. Extraction of the autumnus Artemisia alkaloid

[49] A method for extracting the autumnus Artemisia alkaloid provided by the present disclosure included: raw material screening, crushing, extraction, preliminary filtration, fine filtration, sterilization, lyophilization, and product testing, and specifically included the following steps:

[50] 1. The autumnus Artemisia was screened according to a conventional method (standard screen size: optional feed size < 10 (mm); screen pore size: 1 (mm); screen material: woven wire mesh; material running track: three-dimensional vibration screening; applicable object: plastic, aluminum, zinc, and other metal ash, sand, and soil; brand: (preferably) Xinxiang Jiasheng Vibration Machinery Co., Ltd; and type: entirely new; production capacity: 1 (t/h); motor power: 1.1 (Kw))

[51] Model: preferably ZY-40HM

[52] Product of Guangzhou Zhiya Microwave Equipment Co., Ltd.

[53] A conventional drying method for TCMs includes: slicing, and sun-curing or shade-drying. Both the sun-curing method and the shade-drying method do not require special equipment, and have the advantages of economy, convenience, and low cost. However, the methods require a floor surface and are easily affected by weather and climate, and hygiene cannot be guaranteed. Microwave dryer is a new type of drying equipment. This type of drying equipment is not affected by the climate, and needs a shorter drying time and is more hygienic than natural drying, such that the above disadvantages of the conventional drying methods are completely avoided. A medicine dried by the microwave dryer can well retain the original medicinal ingredients and shows a nearly-natural shape and color. The microwave dryer can also achieve low-temperature drying, sterilization, and disinsection for pharmaceutical products.

[54] The autumnus Artemisia was dried until a moisture content was less than 10%, and then crushed into a powder with a particle size of 450 mesh to 600 mesh (32 m to 23 m) in strict accordance with the sanitary GMP standard.

[55] The autumnus Artemisia was crushed with an ultrasonic cell crusher (product model: preferably AIS99-IIDL, Shandong Jining Sinobest Biological Machinery Co., Ltd.) under the following working parameters: ultrasonication/interval: 5 s/5 s, and preferably 3 s/3 s; number of cycles: 70 (total 10 min), and preferably 60 to 120 (total time: 6 min to 12 min); and power: 500 W to 600 W (preferably 550 W).

[56] The principle of the ultrasonic cell crusher: the cavitation effect is produced in a liquid through strong ultrasound, thereby achieving ultrasonic treatment to animal and plant cells. That is, electrical energy is converted into sound energy through a transducer, the sound energy is transformed into dense small bubbles through a liquid medium, and these small bubbles burst rapidly to produce energy like small bombs, thus playing the role of crushing cells and other substances. The ultrasonic cell crusher can be used to crush a variety of animal and plant cells. Moreover, the ultrasonic cell crusher (vertical temperature-controlled type) can be optionally equipped with a low-temperature thermostatic bath, with a temperature control range of -20°C to 100°C, which is provided to ensure the high activity of an extract.

[57] Main purpose of the ultrasonic cell crusher:

[58] With the functions of crushing tissues, bacteria, viruses, spores, and other cell structures, homogenizing, emulsifying, mixing, degassing, disintegrating and dispersing, leaching and extracting, accelerating a reaction, and the like, the ultrasonic cell crusher is widely used in biology, medicine, chemistry, pharmacy, food, cosmetics, environmental protection, and other laboratory research and enterprise production.

[59] Working parameter setting of the ultrasonic cell crusher:

1) Time: An ultrasonic time should preferably not exceed 5 s each time, and an interval time should be greater than or equal to the ultrasonic time to facilitate heat emission. The time setting

should be based on the principle of short ultrasonic time and large number of cycles, which can

extend the life of an ultrasonic machine and probe. 2) Ultrasonic power: The ultrasonic power should not be too high to avoid splashing or

foaming of a sample. At a sample capacity of less than 10 mL, the power should be lower than 200

W, a 2 mm ultrasonic probe is adopted, and an amplitude transformer select switch on the back of a panel is adjusted to a corresponding position; at a sample capacity of 10 mL to 200 mL, the power

should be 200 W to 400 W, a 6 mm ultrasonic probe is adopted, and an amplitude transformer

select switch on the back of a panel is adjusted to a corresponding position; and at a sample capacity of more than 200 mL, the power should be 300 W to 600 W, a 10 mm ultrasonic probe is

adopted, and an amplitude transformer select switch on the back of a panel is adjusted to a

corresponding position. The power of a 2 mm small probe is prohibited to exceed 350 W. 3) Container selection: A beaker is selected according to the quantity of a sample, which is

conducive to the convection of a sample under ultrasound and improves the crushing efficiency. For

example, a 20 mL beaker is preferably adopted for treating 20 mL of a sample. For example, for 100 mL of an Escherichia coli (E. coli) sample, the parameters are set as

follows: ultrasonication/interval: 5 s/5 s; number of cycles: 70 (total time: 10 min); and power: 300

W (for reference only). At a sample volume of 500 mL, the power is set to 500 W to 800 W. The working parameters of using the ultrasonic cell crusher (AIS99-IIDL) to crush autumnus

Artemisia are illustrated by an example below: Total Container Interval time Ultrasonictime time Power Crushing rate Crsigbjc ievolume Cuhnrt (s) (s) (m.) (W) (mL)() autumnus 3 3 6 to 12 550 50 more than 92 Artemisia

[60] Particle size determination method

[61] A.1 Equipment

[62] A.1.1 Screening mesh (with a screening cover and a receiving box)

[63] A.1.1.1 Mesh: 80.

[64] A.1.2 Balance (scale division: 0.1 g).

[65] A.2 Instrument

[66] A.2.1 The screening mesh was placed above the receiving box.

[67] A.2.2 About 100 g of an extract powder (Mi, g) was weighed and added to the screening mesh, and then the screening mesh was covered with the screening cover.

[68] A.2.3 The screening mesh was gently moved right and left for 5 min, with the screening mesh always remaining horizontal.

[69] A.2.4 An extract powder in the receiving box was weighed (M 2 , g).

[70] A.3 Calculation M!2

[71] Passing rate= M X100

[72] Pack density determination method

[73] Pack density: bulk density and tap density.

[74] B.1 Equipment

[75] B.1.1 Balance (scale division: 0.1 g).

[76] B.1.2 Glass measuring cylinder (100 ml).

[77] B.2 Operating procedures

[78] B.2.1 Preparation of a measuring cylinder: a clean and dry measuring cylinder was taken and weighed (Mo, g).

[79] B.2.2 Determination of bulk density: a sample sieved through a 20-mesh screening mesh was loosely transferred into the measuring cylinder to 90 5 ml, and a total weight of the measuring cylinder and the sample was measured (Mi, g), with an accuracy of 0.1 g; and a surface of the sample was slightly flattened, and a volume of the sample was read (Vi, ml).

[80] B.2.3 Determination of tap density: the above measuring cylinder with the sample was placed on a table (which was covered with about 5 mm-thick rubber) and then allowed to freely fall onto the table from a height of about 2 cm; the above operation was repeated about 100 times, and a volume of a compacted sample was measured (Vo); the above operation was further repeated about times, and a volume of the sample was measured (V2, ml); and If a difference between Vo and V2 was less than 2 ml, a final volume was read (V2, ml), otherwise, the above operations were repeated until the condition was met.

[81] B.3 Calculation Mi-Mo

[82] Bulk density= x 100

[83] Tap density = Mi-Mo 100 V2

[84] Artemisia carvifolia content determination method

[85] C.1 Chromatographic conditions

[86] C.1.1 Chromatographic column: ZORBAX ExtendC-18 4.6x150mm 5 m

[87] C.1.2 Mobile phase: methanol: acetonitrile : water = 8:4:88

[88] C.1.3 Flow rate: 1 ml/min

[89] C.1.4 Detection wavelength: 276 nm

[90] C.1.5 Injection volume: 5 pl

[91] C.1.6 Column temperature: 30°C

[92] C.2 Sample preparation

[93] C.2.1 Preparation of a reference substance: an appropriate amount of an Artemisia carvifolia reference substance was accurately weighed and prepared into a standard sample at about 0.2 mg/ml.

[94] C.2.2 Preparation of a test substance: 50 mg of the test substance was accurately weighed and added to a 25 ml volumetric flask, an appropriate amount of water was added, and a resulting mixture was subjected to ultrasonic treatment for 30 min, then cooled, and diluted to 25 ml.

[95] C.3 Sample analysis

[96] C.3.1 Reference substance: 5 1 of a reference substance solution was accurately drawn for injection.

[97] C.3.2 Test substance analysis: an appropriate amount of a test substance solution was drawn with a 10 ml injection needle and filtered with a syringe filter, 5 1 was injected for analysis with an injection needle, and a sample content was calculated by the external standard method.

[98] C.4 Calculation method

[99] The external standard method was used to calculate a sample content:

[100] Sample content (%)= Cstandard samplexA tet samplex 2 5 X 100% A standardsample Xmtest sample

[101] where Cstandar sample- concentration of a standard sample,

[1021 Astandard sampie--peak area of a standard sample,

[1031 Atest sampie- peak area of a test sample, and

[1041 mtest sample- weight of a test sample.

[105] 2) Extraction at 15°C to 25°C: the autumnus Artemisia powder was mixed with extraction water at a volume ratio of 1:6 to obtain an extraction system; extraction water was filtered by an RO filter cartridge with an accuracy of 0.001 m to 0.0001 [m, then distilled, and cooled to 15°C to °C to obtain distilled water; and the extraction system was diluted with the distilled water, and a resulting mixture was subjected to dynamic stirring for 50 min to 60 min and then to ultrasonic treatment for 30 min to 50 min.

[106] The extraction was conducted with a production line shown in FIG. 2, and specifically involved the following equipment:

[107] Part 1: an intelligently-controlled continuous ultrasonic multifunctional extraction machine set

[108] equipment name: Intelligently-controlled continuous ultrasonic multifunctional extraction machine set

[109] model: GDC-TQ/ZS/3, Shandong Jining Sinobest Biological Machinery Co., Ltd.

[110] 1) Preliminary filtration at 15°C to 25°C: an obtained extract solution was subjected to cloth preliminary filtration, with a filtration pore size of 30 m; and

[111] the equipment in the technical solution of preliminary filtration production line was used for preliminary filtration.

[112] 2) Fine filtration at 15C to 25C: fine filtration was conducted with a 0.45 m molecular membrane to obtain a filtrate.

[113] 3) The filtrate was sterilized by filtration with a polymer membrane at 15°C to 25°C, where the polymer membrane had a filtration pore size of 0.45 m to 0.22 m (preferably 0.22 m).

[114] Specifically:

[115] The equipment in the technical solution of filtration sterilization production line was used for filtration sterilization.

[116] 4) A sterilized filtrate was spray-dried to obtain a powder, and the powder was mixed with the distilled water obtained by the process of filtering extraction water by an RO filter cartridge (with an accuracy of 0.001 m to 0.0001 jm) and distilling and cooling to 15°C to 25°C at a volume ratio of 1:1; and a resulting mixture was lyophilized to obtain a powder, which was an autumnus Artemisia alkaloid.

[117] The equipment in the technical solution of lyophilization production line was used for lyophilization.

[118] Structure and technical parameters of GLZY-1BS (CIP + SIP) vacuum lyophilizer (Shanghai Pudong Lyophilization Equipment Co., Ltd.):

Product structure Technical parameters Remarks Power supply (three-phase five-wire 380 V/50 HZ system) Power consumption 12 Kw Cooling water consumption > 3 m3 /h (T < 30°C/P = 0.15 Mpa to 0.2 Mpa Compressed air volume > 0.1 m 3/min (P = 0.5 Mpa to 0.8 Mpa) Washing water consumption > 100 L/min (T = 80°C/P > 0.4 Mpa to 0.5 Mpa Steam consumption for sterilization > 20 Kg/(P = 0.2 Mpa to 0.3 Mpa) Steam consumption for defrosting 5 Kg/batch (P > 0.2 Mpa to 0.3 MPa) Volume of the complete machine (about) 3300 x 1650 x 2600 (in accordance with (length x width x height) design) Structure type integral type (rectangular drying box

+ rectangular cooling trap) Weight of the complete machine 2100 kg (about)

[119] Performance parameters: 1 Design and manufacture standard GB150-1998 standard 2 Cabinet design pressure 0.16 Mpa/-0.1 Mpa 3 Cabinet design temperature 130 0 C 4 Cabinet vacuum leakage rate 5 x 10-3 Pa.m 3/sec

[120] Sensory indexes:

[121] The sensory indexes of autumnus Artemisia alkaloids with various specifications should meet the following requirements: Item Requirements Color pure white Smell bitter Taste bitter Morphology Uniform powder without visible foreign matters

[122] Physical and chemical indexes:

[123] The physical and chemical indexes should meet the following requirements: Item Index

Product name autumnus Artemisia alkaloid

Index component content pure powder

Particle size (passing rate on a 100-mesh screening mesh, %) > 99.99

Loss on drying (%) < 60.0

Residue on ignition (%) < 5.0

Heavy metal content (calculated based on Pb, mg/kg) < 1.0

Arsenic salt content (calculated based on As, mg/kg) < 0.5 Hexachlorocyclohexane (HCH) (mg/kg) < 0.01

Dichlorodiphenyltrichloroethane (DDT) (mg/kg) < 0.01 Pesticide residue Pentachloronitrobenzene (PCNB) (mg/kg) < 0.01

Aflatoxin B1 content (g/kg) <5

[124] Microbiological indexes:

[125] The microbiological indexes of products with different specifications should meet the following requirements: Item Index

Coliform (MPN/kg) Not detectable

Total bacterial count (cfu/g) < 1000 Pathogenic bacteria (Staphylococcus aureus (S. aureus), Notdetectable Salmonella, and Shigella) Mold and yeast counts (cfu/g) <50

Item Index

Bulk density 30 to 60 Pack density (g/100 ml) Tap density 51 to 80

[126] C5 Test methods

[127] 5.1 Sensory test:

[128] 5.2.1 An appropriate amount of a test sample was taken and placed on a white porcelain dish, and the color, smell, taste, organization, and foreign matter were observed with sensory organs.

[129] 5.2 Physical and chemical requirements:

[130] 5.2.1 Loss on drying:

[131] The loss on drying was determined according to a determination method for loss on drying in the GB/T 5009.3 Food Microbiological Inspection.

[132] 5.2.2 Residue on ignition:

[133] The residue on ignition was determined according to a method specified in Appendix IX J of Chinese Pharmacopoeia (2005 edition).

[134] 5.2.3 Heavy metal content:

[135] The heavy metal content was determined according to a determination method of lead in food in GB/T 5009.12.

[136] 5.2.4 Arsenic salt content:

[137] The arsenic salt content was determined according to a determination method of total arsenic in food in GB/T 5009.11.

[1381 5.2.5 HCH, DDT, and PCNB contents:

[139] The HCH, DDT, and PCNB contents were determined according to methods specified in GB/T 5009.146.

[140] 5.2.6 Aflatoxin BI content:

[141] The aflatoxin BI content was determined according to a method specified in GB/T 5009.22.

[142] 5.2.7 Index component content:

[143] The autumnus Artemisia alkaloid content was determined according to a test method specified in Appendix A.

[144] 5.3 Microbiological indexes:

[145] The microbiological indexes were determined according to a microbiological test method in the GB/T 17325 Food Hygiene Inspection Method.

[146] 6 Test rules

[147] 6.1 Batch:

[148] Products that can be uniformly mixed and have the same properties and quality to some extent are regarded as in the same batch.

[149] 6.2 Sampling:

[150] The sampling was conducted according to a sampling method for medicinal materials in Appendix IIA of Chinese Pharmacopoeia (2005 edition).

[151] 6.3 Test category:

[152] The test includes factory test and type test.

[153] 6.3.1 Factory test:

[154] 6.4 Test results of the autumnus Artemisia alkaloid

[155] Test result: autumnus Artemisia alkaloid test report from Beijing Physical and Chemical Analysis and Testing Center: Report: SB-438 HPLC Shimadzu LC-6AD Sample information: sample name: water-soluble autumnus Artemisia alkaloid sample No.: 110919-YFC-003 Sample type: unknown collection date: 2011-9-22-15:18:43

Sample bottle#: 15 processing date: 2011-9-27-9:51:43 Injection volume: 1 L data file: autumnus Artemisia 2.lcd Columnflowrate: 0.1mg/mL method file: Alkaloid-2.lcm Sample concentration: 1.0 mL/min sample pH: 7.0 Sample pretreatment method: (specified by customer): 10 mg of the sample was accurately weighed, dissolved in ultrapure water, and diluted to 1.0 mL. A sample solution was allowed to stand for 72 h, then filtered through a 0.22 m microporous membrane, and then tested on a machine. Sample chromatogram mAU 203732/autumnusArtemisia 100% 0.0 2.5 5.0 7.5 min Peak table Detector: PDA Chl 192 nm 4 nm Peak# Name: Retention time Area Height Area

% 1 autumnus Artemisia 2.373 33543 10208 100.000

[156] Example 2. Detection of the autumnus Artemisia alkaloid (medicinal active ingredient)

[157] According to the autumnus Artemisia alkaloid test report from Beijing Physical and Chemical Analysis and Testing Center,

[158] the alkaloid extracted from autumnus Artemisia was fully water-soluble and had a purity of 99.95% to 99.99% (mass percentage). Report: SB-438 HPLC Shimadzu LC-6AD Sample information: sample name: water-soluble autumnus Artemisia alkaloid with a water-soluble crystalline structure sample No.: 110919-YFC-003 Sample type: unknown collection date: 2011-9-22-15:18:43 Sample bottle#: 15 processing date: 2011-9-27-9:51:43 Injection volume: 1 L data file: autumnus Artemisia 2.lcd Columnflowrate: 0.1mg/mL method file: Alkaloid-2.lcm Sample concentration: 1.0 mL/min sample pH: 7.0 Sample pretreatment method: (specified by customer): 10 mg of the sample was accurately weighed, dissolved in ultrapure water, and diluted to 1.0 mL. A sample solution was allowed to stand for 72 h, then filtered through a 0.22 m microporous membrane, and then tested on a machine. Sample chromatogram mAU 203732/autumnusArtemisia 100% 0.0 2.5 5.0 7.5 min Peak table Detector: PDA Chl 192 nm 4 nm Peak# Name: Retention time Area Height Area %

1 autumnus Artemisia 2.373 33543 10208 100.000

[159] Study on the function of the autumnus Artemisia alkaloid

[160] 1) The autumnus Artemisia alkaloid is a botanical active substance for treating ovarian cancer. It has been confirmed by a large number of experiments that an autumnus Artemisia alkaloid can lock and selectively kill ovarian cancer cells, with an inhibition rate on ovarian cancer as high as 75.8% to 89.5%. With the outstanding curative effect of "high efficiency, quick onset, low toxicity", the autumnus Artemisia alkaloid is truly a natural botanical active substance for treating ovarian cancer with a remarkable curative effect and small side effects.

[161] 2) The autumnus Artemisia alkaloid can impact the proliferation of human breast cancer cells MCF-7: 24 h after administration of the alkaloid, the proliferation of MCF-7 cells is significantly inhibited, the cells are blocked at the Go + G 1 phase, and cells at the S phase are significantly reduced.

[162] 3) The study also shows that the autumnus Artemisia alkaloid leads to high lethality on human breast cancer cells HTB27, exhibits no obvious cytotoxic side effects on normal human breast cells HTB125, and can induce the apoptosis of breast cancer cells MCF-7.

[163] 4) It can be seen from experiments that autumnus Artemisia has an alkaloid content of 11.5%, while Artemisia carvifolia has an alkaloid content only of 2.5% and thus shows an inhibitory effect on tumor cells that is significantly weaker than that of autumnus Artemisia. This is because tender branches and leaves are collected. It is believed that the best harvest time of Artemisia carvifolia is from June to July when Artemisia carvifolia plants have the most luxuriant leaves, and after this period, a dry Artemisia carvifolia leaf yield per mu will decrease from 180 kg to 140 kg over time. Since autumnus Artemisia is harvested at the end of autumn, an alkaloid content in autumnus Artemisia is significantly higher than that (9%) in Artemisia carvifolia, which proves that the autumnus Artemisia alkaloid is a monomer compound capable of passing through 0.22 m filtration pores and has an anticancer effect superior to that of artemisinin (which is derived from small molecules and can quickly penetrate through the intestinal barrier, cellular barrier, and BBB).

[164] Function mechanism: The autumnus Artemisia alkaloid plays an anti-ovarian cancer effect mainly by inhibiting cell proliferation, inducing cell apoptosis, and inhibiting angiogenesis. In addition, the autumnus Artemisia alkaloid shows strong targetability to ovarian cancer cells, and no drug resistance or cross-resistance with traditional chemotherapeutic drugs occurs during the treatment. Therefore, the safe and low-toxic autumnus Artemisia alkaloid is expected to become an active ingredient for a new botanical drug against ovarian cancer with clinical value.

[165] Example 3. Preparation of an oral botanical preparation with the autumnus Artemisia alkaloid as a medicinal active ingredient

[166] Based on the study of the autumnus Artemisia alkaloid in Example 2, the autumnus Artemisia alkaloid was used as a medicinal active ingredient to prepare an oral botanical preparation mainly for treating ovarian cancer in this example. A preparation method was specifically as follows:

[167] 1. An original medicine: the autumnus Artemisia alkaloid, which is 100% water-soluble and fully absorbed in water, has a purity of 99.95% to 99.99% (mass percentage), and can be directly used as the main ingredient in a drug.

[168] 2. An oral preparation: the drug can be made into a tablet, a granule, a powder, a capsule, an oral liquid, and other pharmaceutically acceptable oral dosage forms, preferably a tablet, and more preferably an enteric-coated tablet.

[169] MCC is commonly used as a pharmaceutical adjuvant for the production of an enteric-coated tablet, which is a crystalline cellulose with a low degree of polymerization obtained by partial hydrolysis of cellulose. MCC has prominent compressibility and strong binding power, so a formed tablet has high hardness. Moreover, MCC can be used as a dry adhesive for direct tableting of powder. There are several specifications based on different particle sizes. The drug in a tablet may specifically include the following components: 40 to 50 parts (preferably 50 parts) of the autumnus Artemisia alkaloid and 1 to 2 parts (preferably 2 parts) of MCC.

[170] Specifications: Tablets with 0.5% MCC have a preferred disintegration effect, and a total weight of a tablet is generally not less than 300 mg (with 0.5 mg of MCC and 250 mg of autumnus Artemisia alkaloid), which can reduce a dosage deviation of the main ingredient, improve the compression molding effect of the drug, etc. A dry state is beneficial to tableting.

[171] The drug tablet may have a coating made from afilm coating material and preferably from water-soluble HPMC; and the drug tablet may be white with the coating being removed.

[172] 3. Functions and indications: the drug is mainly used for treating ovarian cancer. Particularly, the drug can lock the tumorigenesis and rapid development of ovarian malignant tumors.

[173] 4. Usage and dosage: measurement standard: 500 mg/tablet; an adult takes three times a day, with 10 tablets each time; a juvenile or child takes at an amount half of that for an adult; and The drug can exhibit a preferred effect when taken on an empty stomach with warm water.

[174] 5. Pharmacokinetic mechanism: The botanical drug of the present disclosure is disintegrated in gastrointestinal tracts and absorbed in the small intestine, with a disintegration time of 5 min to 10 min. The botanical drug is absorbed by the small intestinal mucosa and lymph and then enters into the circulatory system to function, and finally decomposed by the liver to become ineffective. About 7 h to 8 h after administration, the drug is completely excreted out of the body. The botanical drug has advantages such as quick onset, long duration, precise effect, no toxic side effects, and no drug dependence.

[175] An enteric-coated tablet is mainly dissolved in an intestinal tract and will not cause gastric ulcer due to no irritation to the stomach. An enteric-coated tablet is disintegrated and absorbed in an intestinal tract. The human stomach is in an acidic environment, while the intestinal tract is in an alkaline environment. An enteric-coated preparation is a dosage form used in the drug production to prevent gastric acid and insoluble substances in the stomach from affecting the efficacy of a drug. In the alkaline environment of an intestinal tract, the tablet coating is ruptured and disintegrated, and the active ingredient is absorbed to function.

[176] 6. No toxic side effects: no drug dependence.

[177] 7. Contraindication: the drug shall not be taken by women who are pregnant or breastfeeding.

[178] Example 4. Efficacy of the oral botanical preparation with the autumnus Artemisia alkaloid as a medicinal active ingredient

[179] Both in vitro and in vivo studies have shown that the oral botanical preparation has significant anti-ovarian cancer activity and low toxicity to normal ovarian cells, which may have promising therapeutic application prospects.

[180] The research team first exposed a human ovarian cancer cell line and a human ovarian cell line to five artemisinin derivatives to test the cytotoxic effect. Subsequently, the researchers primarily explored the anti-tumor activity and mechanism of artemisinin and main derivatives thereof on an ovarian cancer cell line when used alone or in combination with a traditional chemotherapeutic drug through in vitro studies and in vivo experiments in model animals.

[181] Studies have shown that, among the five artemisinin derivatives, the autumnus Artemisia alkaloid has the strongest anti-cancer activity and can effectively inhibit the growth of ovarian cancer cells when used alone or in combination with carboplatin. The anti-cancer effect is achieved through death receptors and mitochondrion-mediated and caspase-dependent apoptosis pathways.

[182] The autumnus Artemisia alkaloid is an active ingredient extracted from autumnus Artemisia, and main derivatives thereof include dihydroartemisinin, artemether, arteether, artesunate, etc. In recent years, the anti-cancer effects of artemisinin and derivatives thereof have received extensive attention. The study has confirmed that dihydroartemisinin can inhibit the growth of ovarian cancer cells, can inhibit the activity of tumors in model animals, and can also enhance the efficacy of carboplatin by promoting cell apoptosis. This result lays a foundation for future clinical research on the treatment of ovarian cancer with artemisinin derivatives.

[183] The autumnus Artemisia alkaloid and derivatives thereof can significantly inhibit the growth of liver cancer and ovarian cancer cells, and thus become preferred anti-tumor drugs.

[184] The LWZYS team started the research on the anti-tumor function and mechanism of autumnus Artemisia alkaloid compounds as early as 1999. The LWZYS team found through studies that Artemisia carvifolia has an alkaloid content of 2.5%; autumnus Artemisia has an alkaloid content of 11.5%; and the alkaloid and derivatives thereof can exhibit an anti-cancer activity against ovarian cancer and liver cancer and can specifically kill human liver cancer and ovarian cancer cell ) lines, but show no significant effect on normal cell lines of liver and ovary. It was also found from animal experiments that the autumnus Artemisia alkaloid, when used in combination with carboplatin (a chemotherapeutic drug for ovarian cancer), can significantly increase the killing effect on ovarian cancer cells; and the autumnus Artemisia alkaloid, when used in combination with gemcitabine (a chemotherapeutic drug for liver cancer), can significantly improve the killing effect on liver cancer cells.

[185] The autumnus Artemisia alkaloid compounds enable a new anti-tumor mechanism. Research results show that the autumnus Artemisia alkaloid can cause the shortage of iron in tumor cells, reduce the iron absorption, and interfere with the existing iron balance in cells. Tumor cells require more iron than normal cells due to rapid proliferation. Therefore, insufficient iron intake will inhibit the growth of tumor cells and further lead to cell apoptosis. The iron absorption of cells is mainly mediated by the transferrin receptor 1 on the cell surface. The autumnus Artemisia alkaloid compound can induce abnormal modification and endocytosis of the transferrin receptor 1 on the cell surface to reduce a content of transferrin receptor 1" on the cell surface, thereby inhibiting the iron absorption by cells and causing tumor cells to die from iron deficiency.

[186] The research result for the autumnus Artemisia alkaloid compound shows that a key protein of the metabolism pathway may serve as a potential therapeutic target for anti-tumor drugs, which is of guiding significance for the modification and optimization of artemisinin compounds and the development of iron-targeted drugs.

[187] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general

knowledge in the art, in Australia or any other country.

[188] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to

specify the presence of the stated features but not to preclude the presence or addition of further

features in various embodiments of the invention.

Claims (5)

WHAT IS CLAIMED IS:

1. A preparation method of a drug for treating ovarian cancer, comprising the following steps: 1) drying and crushing native autumnus Artemisia plants to obtain an autumnus Artemisia powder with a particle size of 80 mesh to 120 mesh (32 m to 23 m); 2) extraction: mixing the autumnus Artemisia powder with extraction water at a volume ratio of 1:6 to obtain an extraction system; filtering extraction water by a reverse osmosis (RO) filter cartridge with an accuracy of 0.001 m to 0.0001 [m, and distilling and cooling to 15°C to 25°C to obtain distilled water; and diluting the extraction system with the distilled water, and subjecting a resulting mixture to dynamic stirring for 50 min to 60 min and then to ultrasonic treatment for 30 min to 50 min; 3) preliminary filtration at 15°C to 25°C: subjecting an extract solution obtained under high filtration accuracy to cloth preliminary filtration, with a filtration pore size of 30 m; 4) fine filtration at 15°C to 25C: filtering with a 0.45 m molecular membrane to obtain a filtrate; 5) sterilizing with a polymer membrane at 15°C to 25°C, wherein the polymer membrane has a filtration pore size of 0.45 m to 0.22 m (preferably 0.22 [m); and 6) spray-drying a sterilized filtrate to obtain a powder, and mixing the powder with the distilled water obtained by the process of filtering extraction water by an RO filter cartridge (with an accuracy of 0.001 m to 0.0001 jm) and distilling and cooling to 15°C to 25°C at a volume ratio of 1:1; and lyophilizing a resulting mixture to obtain a powder, which is an autumnus Artemisia alkaloid.

2. The preparation method according to claim 1, wherein the autumnus Artemisia is crushed with an ultrasonic cell crusher (product model: preferably GDC-TQ/ZS/3, Shandong Jining Sinobest Biological Machinery Co., Ltd.) under the following working parameters: ultrasonication/interval: 5 s/5 s, and preferably 3 s/3 s; number of cycles: 70 (total time: 10 min), and preferably 60 to 120 (total time: 6 min to 12 min); and power: 500 W (preferably 800 W).

The preparation method according to claim 1, wherein the extraction in step 2) is conducted with an ultrasonic extraction machine in an intelligently-controlled continuous ultrasonic multifunctional extraction machine set (product model: preferably GDC-TQ/ZS/3, Shandong Jining Sinobest Biological Machinery Co., Ltd.); and the ultrasonic extraction machine has: ultrasonic frequency: two frequency bands of 26 KHz and 70 KHz, each with a frequency adjustment range of

±3 KHz; ultrasonic power: 800 W, continuously adjustable; and extraction tube specification: diameter: 300 mm, length: 300 mm, and volume: > 20 L; wherein the preliminary filtration in step 3) is conducted with an Aoliyuan/ALY-500 device (product model: preferably ALY-500, Shanghai Aoliyuan Environmental Technology Co., Ltd.) under the following working parameter: high filtration accuracy; wherein the fine filtration in step 4) is conducted with a QJ50 device (product model: preferably QJ50, Haining Yongsheng Membrane Filtration Equipment Manufacturing Co., Ltd.) under the following working parameter: high filtration accuracy; wherein the polymer membrane in step 5) is preferably a product from Haining Yongsheng Membrane Filtration Equipment Manufacturing Co., Ltd., with a model of QJ50; wherein the lyophilizing in step 6) is conducted with a vacuum lyophilizer (product model: preferably GLZY-1BS (CIP + SIP), Shanghai Pudong Lyophilization Equipment Co., Ltd.); and the vacuum lyophilizer is designed according to the following standards: GMP power supply (three-phase five-wire system): 380V/50HZ; power consumption: 12 Kw; cooling water consumption: > 0.1 m3/min (P = 0.5 Mpa to 0.8 Mpa); washing water consumption: > 100 L/min (T = 80°C/P > 0.4 Mpa to 0.5 Mpa); steam consumption for sterilization: > 20 Kg/(P = 0.2 Mpa to 0.3 Mpa); steam consumption for defrosting: 5 Kg/batch (pressure > 0.2 MPa to 0.3 MPa); volume of the complete machine (length x width x height): (about) 3,300 x 1,650 x 2,600; structure type: integral type (rectangular drying box + rectangular cooling trap); and weight of the complete machine (about): 2,100 kg.

3. An autumnus Artemisia alkaloid extracted by the method according to any one of claims 1 to 7, wherein the autumnus Artemisia alkaloid is fully water-soluble and has a purity of 99.95% to 99.99% (mass percentage).

4. A botanical drug mainly for treating ovarian cancer with a remarkable curative effect and small side effects, wherein the botanical drug comprises the autumnus Artemisia alkaloid according to claim 3 as a medicinal active ingredient.

5. The botanical drug according to claim 4, wherein the botanical drug can be made into a tablet, a granule, a powder, a capsule, an oral liquid, and other pharmaceutically acceptable oral dosage forms, preferably a tablet, and more preferably an enteric-coated tablet; and when an enteric-coated tablet is adopted, an added adjuvant can be any one or more from the group consisting of pharmaceutical dextrin, colloidal silicon dioxide, starch, and microcrystalline cellulose (MCC), and a part by weight ratio of the excipient to all pharmaceutical raw materials in the tablet can be 1:(l-50); wherein the botanical drug in an enteric-coated tablet may specifically comprise the following components: 40 to 50 parts (preferably 50 parts) of the autumnus Artemisia alkaloid and I to 2 parts (preferably 2 parts) of MCC; wherein the drug tablet has a coating made from a film coating material and preferably from water-soluble hydroxypropyl methylcellulose (HPMC); and the drug tablet is white with the coating being removed.

FIG. 1 －1/2－

－2/2－ 09 Aug 2021

Extraction process flow

Dry raw material

Wash

Feed

100% distilled water 2021105119

Reflux extraction for 2 h extraction Material-liquid ratio of 1:8 Extraction temperature of 50°C

Extract solution 1 Dreg

Reflux extraction for 2 h

Extract solution 2 Dreg

Reflux extraction for 2 h

Extract solution 3 Dreg

Vacuum filtration

Combine extract Recover filtrate Dreg solutions and filter

Static settlement with 8- Recover filtrate Recycle Dispose fold volume of water

Filter and concentrate a supernatant

Vacuum concentration

Extract Density p-1.1-1.20

lyophilization

Crush

Package

Label

Sampling test Product

Qualified Warehouse product

FIG. 2