Rhizanamides and related products, preparation method, application and quality standard thereof

Abstract

The application provides a rhizoma smilacis glabrae amide alkaloid and related products, a preparation method, application and quality standard, and belongs to the technical field of active ingredients of traditional Chinese medicines. In the rhizoma smilacis glabrae amide alkaloid, the mass content of total alkaloids is 50.03%-55.58%, the mass content of wind amide is 22.28‰-31.10‰, and the mass content of wall grass base is 9.18%-13.95%. By mixing, stirring and reducing pressure concentration of the rhizoma smilacis glabrae extraction liquid with macroporous adsorption resin, the content and transfer rate of effective components in the rhizoma smilacis glabrae amide alkaloid are significantly improved, and animal experiments show that compared with the prior art, the rhizoma smilacis glabrae amide alkaloid product provided by the application has a better analgesic effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of active ingredients of traditional Chinese medicine, and relates to a piper wallichii amide alkaloid and related products, a preparation method, application and quality standard thereof. BACKGROUND

[0002] Pain is an unpleasant feeling, and its causes are complex and diverse. According to the form and characteristics of pain, it can be classified from the central, inflammation and course length. Pain relief means stopping pain. For cancer pain, the three-step ladder treatment method can be selected. Non-opioid analgesics such as ibuprofen and aspirin are preferred. When these drugs do not work, weak opioid analgesics or opioid analgesics are considered. Non-opioid analgesics have poor therapeutic effect on moderate to severe cancer pain. Although opioid drugs can exert analgesic effect, they have side effects such as psychological dependence. Therefore, it is urgent to find a new and safer analgesic substance, especially a non-opioid substance, which has become a bottleneck technical problem to be broken through.

[0003] Piper wallichii (Miq.) Hand.-Mazz. is also known as Nanteng (Kai Bao Ben Cao), Dingfu and Dinggongji (Bielu), Dinggongteng (Ben Cao Shiyi), Soushantuhu (Dian Nan Ben Cao), Fengteng (Gangmu), and Bayanxiang (Fenlei Caoyao Xing). It is the dried stem of Piper wallichii (Miq.) Hand.-Mazz. of Piperaceae. Piper wallichii contains pipeweed ketone, magnolia fat B, N-isobutyl dec-trans-2-trans-4-dienamide, Nantengsu, Shanjutone C, gellertreva, dihydro-piperaquine, longspike euphorbia epoxide, and flavonoids. Piper wallichii is pungent and sweet, warm, and belongs to the liver and kidney channels. It can dispel wind and dampness, strengthen the knees and waist, tonify the kidney and invigorate yang, relieve cough and asthma, and invigorate blood and relieve pain. It is used to treat rheumatic arthralgia, soreness of the waist and knees, impotence, cough and asthma, dysmenorrhea, and contusion and swelling. Modern medicine shows that Piper wallichii extract can treat coronary heart disease, angina pectoris and cerebral infarction.

[0004] Patent CN115770276A discloses a method for improving the efficacy and stability of rheumatic pain medicinal liquor, which uses 27 kinds of medicinal materials such as Piper wallichii, Ephedra, Citrus aurantium, Cinnamomum cassia, silkworm sand, Polygonatum sibiricum, Citrus reticulata, Magnolia officinalis, bitter almonds, Alisma orientale, Dioscorea opposita, Atractylodes lancea, Cortex Moutan, Chuanxiong, Atractylodes macrocephala, Angelica dahurica, Wood Anemone, Xanthium sibiricum, Notopterygium, Foeniculum vulgare, Gleditsia sinuata, Psoralea corylifolia, Rhizoma Cyperi, Commiphora myrrha, and Angelica sinensis as main raw materials, adopts twice white wine percolation method, and uses polyoxyethylene laurate, alkyl glycoside, nano-alumina and anhydrous ethanol as modifiers to treat polytetrafluoroethylene-based film, which effectively improves the efficacy and stability of the product. Then, egg white powder and bentonite are used as clarifying agents, which not only improves the clarification rate, but also further improves the stability of the product.

[0005] Patent CN113599484A discloses a traditional Chinese medicine composition for treating pain, its preparation method and application. The traditional Chinese medicine composition is made from the following raw medicinal materials by weight: 515 parts of stone vine, 515 parts of wall grass root, 110 parts of wax gourd root, and 110 parts of dried ginger. The traditional Chinese medicine composition inhibits the occurrence of inflammation by reducing the levels of inflammatory factors and tumor necrosis factor in the serum of acetic acid-induced pain model mice, thereby achieving the effect of pain relief.

[0006] Patent CN117442698A discloses the application of a traditional Chinese medicine composition in the preparation of a drug for external evil and pestilential qi. The traditional Chinese medicine composition is derived from the folk Chinese medicine inheritance compound Nanteng Zhitong prescription and is prepared from raw materials including stone vine, wall grass root, dried ginger, and wax gourd root. This technology first discovers that the traditional Chinese medicine composition has a significant and effective therapeutic effect on diseases caused by external evil and pestilential qi or their sequelae. At the same time, the traditional Chinese medicine composition has diverse target points, is not prone to drug resistance, and has few or no adverse reactions.

[0007] Currently, there are few reports on the purification of stone vine amide total alkaloids. The article "Stone Vine Analgesic Active Site Screening and Purification" (Journal of Chinese Patent Medicine, March 2023, Vol. 45, No. 3, pp. 1007-1011) describes the preparation method of the stone vine cation resin eluate fraction. This method is aimed at the separation of total alkaloids, but in fact, most of the alkaloids in stone vine exist in the form of amides, such as wind vine amide and wall grass base.

[0008] The chemical structure of wind vine amide is as follows:

[0009]

[0010] The chemical formula of wall grass base is as follows:

[0011]

[0012] Amide alkaloids have little basicity, and the adsorption force of cation exchange resin on stone vine amide alkaloids is easily eluted, which causes the mixing of other substances in the effective fraction, affecting the analgesic effect to some extent. Different fractions separated by cation exchange resin have analgesic effects on acetic acid-induced pain in mice. On the one hand, amide alkaloids have poor water solubility and exist in the form of precipitates in the sample solution, causing incomplete adsorption of the resin and loss of medicinal substances; on the other hand, the medicinal parts of stone vine vary in different regions. The cation exchange resin method uses dried stem branches with leaves or the whole plant. The leaves of stone vine do not have analgesic effect, as verified by pre- efficacy studies. The content of leaves in the raw material seriously affects the analgesic effect of the drug, which also leads to the failure to fully exploit the analgesic potential of the stone vine effective fraction.

[0013] In the article "HPLC Characteristic Spectrum Research and Amide Content Determination of Rhizoma Humuli", Journal of Chinese Medicinal Materials, April 2022, Vol. 45, No. 4, pp. 922-926, a HPLC characteristic spectrum of Rhizoma Humuli and its identification method are provided, and the components in Rhizoma Humuli are extracted and identified. However, this method fails to achieve more sufficient extraction of the components in Rhizoma Humuli.

[0014] In summary, the prior art fails to provide a better method for extracting Rhizoma Humuli amide alkaloids in Rhizoma Humuli, and also fails to provide a Rhizoma Humuli amide alkaloid product with better analgesic effect. SUMMARY

[0015] Therefore, in view of the problem that the prior art fails to provide a better method for extracting Rhizoma Humuli amide alkaloids in Rhizoma Humuli, the purpose of the present application is to provide a Rhizoma Humuli amide alkaloid and related products, preparation method, application, and quality standard.

[0016] To achieve the above-mentioned purpose of the application, in one aspect, the present application provides a Rhizoma Humuli amide alkaloid, wherein the mass content of total alkaloids is 50.05%-55.58%, the mass content of Fengtengamide is 22.28‰-31.10‰, and the mass content of Qiangcaobase is 9.18%-13.95%.

[0017] The UPLC fingerprint spectrum of the Rhizoma Humuli amide alkaloid includes fingerprint peak 1, fingerprint peak 6, fingerprint peak 7, fingerprint peak 9, and fingerprint peak 11, the position of the fingerprint peak 1 is 3.90-4.10 min, the position of the fingerprint peak 6 is 12.45-12.55 min, the position of the fingerprint peak 7 is 12.80-12.95 min, the position of the fingerprint peak 9 is 13.10-13.25 min, and the position of the fingerprint peak 11 is 15.30-15.45 min.

[0018] Preferably, the chromatographic conditions of the UPLC include:

[0019] Chromatographic column: C 18 Chromatographic column;

[0020] Water as mobile phase A and acetonitrile as mobile phase B, gradient elution scheme as follows:

[0021]

[0022]

[0023] Column temperature 35℃, flow rate 0.8mL / min, detection wavelength 260nm.

[0024] The percentage of the mobile phase A and the mobile phase B is volume percentage. For example, at the elution time of 0-4 min, the volume percentage of the mobile phase A is 83% and the volume percentage of the mobile phase B is 17%.

[0025] More preferably, and as an example of the present application, the C 18 The model of the chromatographic column is Poroshell 120EC-C 18 1.9 μm, 2.1 x 50 mm.

[0026] Preferably, the UPLC fingerprint of the Rhynchophyllamide alkaloids further comprises fingerprint peak 2, fingerprint peak 3, fingerprint peak 4, fingerprint peak 5, fingerprint peak 8 and fingerprint peak 10, the position of the fingerprint peak 2 is 5.85-6.05 min, the position of the fingerprint peak 3 is 8.85-8.95 min, the position of the fingerprint peak 4 is 9.30-9.50 min, the position of the fingerprint peak 5 is 12.15-12.30 min, the position of the fingerprint peak 8 is 13.00-13.05 min, and the position of the fingerprint peak 10 is 13.90-14.00 min.

[0027] Preferably, the component corresponding to the fingerprint peak 1 is purpureaenamide; the component corresponding to the fingerprint peak 6 is windamide; the component corresponding to the fingerprint peak 7 is Pipersintenamide; the component corresponding to the fingerprint peak 9 is wallenrootine; and the component corresponding to the fingerprint peak 11 is (2E, 4E)-N-(2-methylpropyl)-dodeca-2, 4-dienamide.

[0028] The chemical structural formula of the Pipersintenamide is

[0029]

[0030] Preferably, the peak area ratio of the fingerprint peak 1, the fingerprint peak 6, the fingerprint peak 7, the fingerprint peak 9 and the fingerprint peak 11 is 1.435-1.868: 1.053-1.470: 1.505-1.986: 10.950-16.640: 0.975-1.040.

[0031] More preferably, the peak area ratio of the fingerprint peak 1, the fingerprint peak 6, the fingerprint peak 7, the fingerprint peak 9 and the fingerprint peak 11 is 1.530-1.615: 1.185-1.470: 1.774-1.802: 12.012-16.640: 0.993-1.007.

[0032] Most preferably, and as an example of the present application, the peak area ratio of the fingerprint peak 1, the fingerprint peak 6, the fingerprint peak 7, the fingerprint peak 9 and the fingerprint peak 11 is 1.600:1.470:1.780:16.640:1.001.

[0033] In another aspect, the present application provides a preparation method of the berberine amide alkaloids, comprising the following steps:

[0034] S1, mixing the berberine with an ethanol aqueous solution, refluxing and extracting to obtain an extract;

[0035] S2, mixing the extract obtained in step S1 with a macroporous adsorption resin, stirring, and concentrating under reduced pressure until no alcohol smell is left to obtain a sample mixture;

[0036] S3, performing sample loading on the sample mixture obtained in step S2, standing, flowing out, eluting to obtain an eluate;

[0037] S4, concentrating the eluate obtained in step S3 under reduced pressure, and drying to obtain the berberine amide alkaloids.

[0038] Preferably, in step S1, the concentration of the ethanol aqueous solution is 70%-80%.

[0039] More preferably, and as an example of the present application, in step S1, the concentration of the ethanol aqueous solution is 70%-80%.

[0040] Preferably, in step S1, the mass ratio of the berberine to the ethanol aqueous solution is 1:10-20.

[0041] More preferably, and as an example of the present application, in step S1, the mass ratio of the berberine to the ethanol aqueous solution is 1:14.

[0042] Preferably, in step S1, the refluxing and extracting is performed 1-3 times.

[0043] More preferably, and as an example of the present application, in step S1, the refluxing and extracting is performed 2 times.

[0044] Preferably, in step S1, the refluxing and extracting is performed for 1-4 h each time.

[0045] More preferably, and as an example of the present application, in step S1, the refluxing and extracting is performed for 2 h each time.

[0046] Preferably, in step S2, the type of the macroporous resin is selected from HP-20, D101, S-8, NKA-9, DA201 and HP2MGL.

[0047] More preferably, in step S2, the type of macroporous resin is selected from HP-20, D101, S-8 and NKA-9.

[0048] More preferably, in step S2, the type of macroporous resin is selected from HP-20 and S-8.

[0049] Further preferably, in step S2, the type of macroporous resin is HP-20.

[0050] Preferably, the ratio of the weight of the Smilax riparia to the dry weight of the macroporous resin in step S1 is 1:0.4-1.2.

[0051] More preferably, the ratio of the weight of the Smilax riparia to the dry weight of the macroporous resin in step S1 is 1:0.6-1.

[0052] More preferably, the ratio of the weight of the Smilax riparia to the dry weight of the macroporous resin in step S1 is 1:0.8.

[0053] Preferably, in step S2, the stirring is for 10-30 min.

[0054] Preferably, in step S2, the reduced pressure concentration is performed at 40-60℃.

[0055] Preferably, in step S2, the macroporous adsorption resin is a pretreated macroporous adsorption resin.

[0056] More preferably, the pretreatment comprises the following steps:

[0057] Soaking in ethanol for 24 h, then soaking in 5% hydrochloric acid solution for 24 h, washing with water until neutral, then soaking in 5% NaOH solution for 24 h, washing with water until neutral, then filtering, and airing.

[0058] Preferably, in step S3, the standing is for 15-30 min.

[0059] More preferably, and as an example of the present application, in step S3, the standing is for 20 min.

[0060] Preferably, in step S3, the flow rate of the outflow is 2-4 BV / h.

[0061] The process flow of mixing the filtrate obtained by alcohol extraction with macroporous resin, removing alcohol by reduced pressure concentration, column loading, standing, and outflow in steps S2 and S3 of the present application combines the advantages of wet sample loading and dry sample loading, and can effectively solve the problem of column blockage by fat-soluble alkaloid components in Smilax riparia amide alkaloids.

[0062] More preferably, and as an example of the present application, in step S3, the flow rate is 2-4 BV / h.

[0063] Preferably, in step S3, the elution comprises the following steps:

[0064] S31, eluting 3-5 BV of water at a flow rate of 2-4 BV / h to obtain a water washing part liquid;

[0065] S32, eluting 3-5 BV of 25%-30% ethanol aqueous solution at a flow rate of 2-4 BV / h to obtain an alcohol washing part liquid;

[0066] S33, eluting 9-12 BV of 40-80% ethanol aqueous solution at a flow rate of 2-4 BV / h to obtain an eluate.

[0067] More preferably, and as an example of the present application, in step S31, 3 BV of water is eluted at a flow rate of 3 BV / h.

[0068] More preferably, and as an example of the present application, in step S32, 5 BV of 30% ethanol aqueous solution is eluted at a flow rate of 3 BV / h.

[0069] More preferably, in step S33, 9-12 BV of 70%-80% ethanol aqueous solution is eluted at a flow rate of 2-4 BV / h.

[0070] More preferably, in step S33, 9-12 BV of 70%-80% ethanol aqueous solution is eluted at a flow rate of 3 BV / h.

[0071] Further preferably, in step S33, 9-12 BV of 75% ethanol aqueous solution is eluted at a flow rate of 3 BV / h.

[0072] Further more preferably, and as an example of the present application, in step S33, 10 BV of 75% ethanol aqueous solution is eluted at a flow rate of 3 BV / h.

[0073] Preferably, in step S4, the vacuum concentration is performed at 40-60°C, and the drying is freeze drying.

[0074] In another aspect, the present application provides a water washing part of the berberine alkaloids prepared by the above preparation method, and the preparation method comprises the following steps: drying the water washing part liquid obtained in step S31 of the above preparation method to obtain the water washing part of the berberine alkaloids.

[0075] In another aspect, the present application provides an alcohol washing part of the berberine alkaloids prepared by the above preparation method, and the preparation method comprises the following steps: drying the alcohol washing part liquid obtained in step S32 of the above preparation method to obtain the alcohol washing part of the berberine alkaloids.

[0076] In another aspect, the present application provides the use of the above-mentioned securinamide alkaloids and / or the securinamide alkaloids prepared by the above-mentioned preparation method and / or the above-mentioned water-washed parts of securinamide alkaloids and / or the above-mentioned alcohol-washed parts of securinamide alkaloids in the preparation of analgesic products.

[0077] Preferably, the analgesia is for the treatment of traumatic pain, thermal pain and inflammatory pain and bone cancer pain.

[0078] More preferably, the analgesic product is an analgesic drug.

[0079] In another aspect, the present application provides an analgesic drug, the effective component of which comprises the above-mentioned securinamide alkaloids and / or the securinamide alkaloids prepared by the above-mentioned preparation method and / or the above-mentioned water-washed parts of securinamide alkaloids and / or the above-mentioned alcohol-washed parts of securinamide alkaloids.

[0080] The drug can be prepared into pills, capsules, granules, oral liquids, powders, tablets, lozenges and injections, etc. for different administration forms, and suitable drug carriers in the art can be selected.

[0081] The drug carriers used can be solid, liquid or gas. Examples of solid carriers include lactose, white clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Examples of liquid carriers include sugar syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0082] In the preparation of oral dosage forms of drugs, any convenient pharmaceutical medium can be used. For example, water, ethanol, oil, alcohol, flavoring agents, preservatives, coloring agents, etc. can be used to form oral liquid preparations, such as suspensions, elixirs and solutions; and carriers, such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, disintegrating agents, etc. can be used to form oral solid preparations, such as powders, capsules and tablets. Due to their ease of administration, tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers. Standard aqueous or non-aqueous techniques can be selected for coating tablets.

[0083] Tablet pharmaceuticals containing the ericosonamide alkaloids prepared according to the present application can be prepared by compression or molding. One or more accessory ingredients or adjuvants can optionally be used. Tablets can be prepared by compression of a free-flowing form of the active ingredient, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent, in a suitable machine. Molded tablets can be made directly from a mixture of the powdered compound in a suitable machine, using an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient. Each packet or capsule preferably contains from about 0.05 mg to about 5 g of the active ingredient. For example, a preparation intended to be administered to the human adult population by oral

[0084] Pharmaceuticals of the present application suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water or non-irritating physiologically acceptable diluents. The proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0085] Pharmaceuticals of the present application intended to be administered by injection as a pharmaceutical composition, can comprise pharmaceutically acceptable carriers and constituents, and can be formulated in conventional form: 0 either as a liquid or suspension in an aqueous or non-aqueous fluid, or as an emulsion, cream, or paste, or as a solid for reconstitution into a fluid. Such formulations will contain typically from about 0.5% to about 90% of active compound, usually between about 1% and about 50%, and usually between about 5% and about 20%. The active compound can be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like; and combinations thereof. If desired, buffering agents can be included, e.g., bicarbonates, phosphates, tris, or the like; and combinations thereof. The pH and ionic strength of the resulting composition can be adjusted with pH adjusting and ionic strength adjusting agents respectively. In some cases, the resulting compositions can be made into aerosol formulations to be administered by inhalation.

[0086] Pharmaceuticals of the present application can be in a form suitable for local administration, such as an aerosol, cream, ointment, lotion, powder, or the like. Also, the pharmaceuticals can be in a form suitable for use in a transdermal delivery device. The pharmaceuticals of the present application can be prepared for use in such formulations by conventional methods. For example, creams or ointments can be prepared by mixing the active ingredient with a hydrophilic material and water, and about 5% to about 10% by weight of the compound.

[0087] Pharmaceuticals of the present application can be in a form suitable for rectal administration wherein the carrier is a solid. The compound mixture can be compressed into tablets or pellets each containing a unit dosage amount of the active ingredient. Suitable carriers include cocoa butter and other materials commonly used in the art. The tablets or pellets can be formed by first forming a mixture containing the softened or melted carrier, and then cooling and shaping in a mold.

[0088] In addition to the carrier components described above, the above-mentioned drugs may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other excipients may be added, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants, and flavoring agents, etc., to make the formulation isotonic with the blood of the intended recipient. Components containing the styracil alkaloids of this invention can also be prepared in powder or concentrated form.

[0089] Preferably, the dosage form of the drug is selected from pills, capsules, granules, oral liquids, powders, tablets, lozenges, and injections.

[0090] Preferably, the drug is an oral medication.

[0091] On the other hand, the present invention provides the quality standards for the above-mentioned senna-type alkaloids, including the detection of total alkaloid content, the detection of senna-type alkaloid content, the detection of senna-type alkaloid content, and the UPLC fingerprint spectrum detection of senna-type alkaloids.

[0092] The detection method for the UPLC fingerprint of the alkaloids of the *Photinia serratifolia* class includes the following steps:

[0093] The alkaloids of the pine vine were dissolved in 75% methanol, sonicated to remove the solids, and injected into a liquid chromatograph for UPLC detection to obtain the UPLC fingerprint spectrum.

[0094] The quality of *Syngonium styracifolium* alkaloids was evaluated based on the obtained UPLC fingerprints, and the evaluation criteria included:

[0095] (1) The UPLC fingerprint spectrum includes fingerprint peak 1, fingerprint peak 6, fingerprint peak 7, fingerprint peak 9 and fingerprint peak 11. The position of fingerprint peak 1 is 3.90-4.10 min, the position of fingerprint peak 6 is 12.45-12.55 min, the position of fingerprint peak 7 is 12.80-12.95 min, the position of fingerprint peak 9 is 13.10-13.25 min, and the position of fingerprint peak 11 is 15.30-15.45 min;

[0096] (2) The UPLC fingerprint spectrum further comprises fingerprint peak 2, fingerprint peak 3, fingerprint peak 4, fingerprint peak 5, fingerprint peak 8 and fingerprint peak 10, the position of the fingerprint peak 2 is 5.85-6.05 min, the position of the fingerprint peak 3 is 8.85-8.95 min, the position of the fingerprint peak 4 is 9.30-9.50 min, the position of the fingerprint peak 5 is 12.15-12.30 min, the position of the fingerprint peak 8 is 13.00-13.05 min, and the position of the fingerprint peak 10 is 13.90-14.00 min.

[0097] Preferably, the standard for the total alkaloid content detection is that the total alkaloid mass content is ≥50.05%.

[0098] More preferably, the standard for the total alkaloid content detection is that the total alkaloid mass content is 50.05%-55.58%.

[0099] Preferably, the method for detecting the total alkaloid content comprises the following steps:

[0100] Preparation of a chromotropic acid concentrated sulfuric acid solution, preparation of a reference solution, obtaining of a standard curve, preparation of a freeze-dried powder sample solution, and ultraviolet-visible spectrum detection.

[0101] More preferably, the method for detecting the total alkaloid content comprises the following steps:

[0102] Preparation of a chromotropic acid concentrated sulfuric acid solution: 0.2 g of chromotropic acid powder is added to 200 mL of 90% concentrated sulfuric acid solution, stirred and dissolved to obtain a chromotropic acid concentrated sulfuric acid solution;

[0103] Obtaining of a standard curve: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of the reference solution are precisely pipetted into 10 mL glass test tubes with stoppers, 5 mL of the chromotropic acid concentrated sulfuric acid solution is precisely added, and after uniform suspension, it is placed in a boiling water bath for heating for 30 min, and then diluted to 10 mL with water, and the absorbance is measured at 485 nm, and a standard curve is drawn with the concentration as the abscissa and the absorbance as the ordinate;

[0104] Preparation of a freeze-dried powder sample solution: 10 mg of freeze-dried powder is precisely weighed and placed in a stoppered conical flask, and the weight is determined, 25 mL of 75% methanol is added and ultrasonically treated for 30 min, and then taken out and cooled to room temperature, and the lost weight is made up with 75% methanol, and then shaken and filtered, and the filtrate is the freeze-dried powder sample solution;

[0105] UV-Vis spectroscopy detection: Accurately pipette 0.2 mL of the lyophilized powder sample solution into a 10 mL stoppered glass test tube, accurately add 5 mL of chromotropic acid concentrated sulfuric acid solution, mix well, heat in a boiling water bath for 30 min, and dilute with water to 10 mL to obtain the test solution. Measure the absorbance and determine the total amide alkaloid content in the lyophilized powder according to the obtained standard curve.

[0106] Preferably, the standard for detecting the content of fentanyl is: fentanyl content ≥ 22.28‰.

[0107] More preferably, the standard for detecting the content of fentanyl is: the mass content of fentanyl is 22.28‰-31.10‰.

[0108] Preferably, the standard for detecting the content of wallwort alkaloids is: wallwort alkaloid content ≥ 9.18% by mass.

[0109] More preferably, the standard for detecting the content of wall-plant alkaloids is: the mass content of wall-plant alkaloids is 9.18%-13.95%.

[0110] Preferably, the methods for detecting the content of fentanyl amide and the content of wallwort alkaloid are liquid chromatography.

[0111] More preferably, the methods for detecting the content of fentanyl amide and the content of wallwort alkaloids include the following steps:

[0112] Take an appropriate amount of reference standard, weigh it accurately, and add an appropriate amount of methanol to prepare a reference standard solution of 20 μg / mL;

[0113] Accurately weigh approximately 10 mg of lyophilized powder and place it in a stoppered conical flask. Seal the flask tightly and weigh it again. Add 25 mL of 75% methanol and sonicate for 0 min. Remove the flask and cool it to room temperature. Make up the lost weight with 75% methanol, shake well, and filter. Take the filtrate, dilute it with methanol by 1:1, centrifuge at 10000 r / min for 5 min, and filter it through a 0.22 μm microporous filter to obtain the test solution.

[0114] Accurately pipette 5 μL of the reference solution and the test solution into the liquid chromatograph for liquid chromatography analysis to obtain the final product.

[0115] The standards for detecting total alkaloid content, fentanyl amide content, and wallwort alkaloid content mentioned above are all standards for the quality qualification of fentanyl amide alkaloids.

[0116] Preferably, the solid removal step includes filtration to obtain the filtrate and centrifugation to obtain the supernatant.

[0117] Preferably, the chromatographic conditions for the UPLC detection include:

[0118] Column: C 18Chromatographic column

[0119] The gradient elution scheme is as follows: water as mobile phase A, acetonitrile as mobile phase B:

[0120] Elution time / min Mobile phase A / % Mobile phase B / % 0-4 83 17 4-8.7 83-75 17-25 8.7-10.6 75-68 25-32 10.6-12 68-50 32-50 12-17 50 50 17-20 50-10 50-90

[0121] The column temperature is 35 DEG C, the flow rate is 0.8 mL / min, and the detection wavelength is 260 nm.

[0122] More preferably, the C 18 The model of the chromatographic column is Poroshell 120EC-C 18 1.9 mu m, 2.1 x 50 mm.

[0123] Preferably, the peak area ratio of the fingerprint peak 1, the fingerprint peak 6, the fingerprint peak 7, the fingerprint peak 9 and the fingerprint peak 11 is 1.435-1.868: 1.053-1.470: 1.505-1.986: 10.950-16.640: 0.975-1.040.

[0124] More preferably, the peak area ratio of the fingerprint peak 1, the fingerprint peak 6, the fingerprint peak 7, the fingerprint peak 9 and the fingerprint peak 11 is 1.530-1.615: 1.185-1.470: 1.774-1.802: 12.012-16.640: 0.993-1.007.

[0125] Compared with the prior art, the present application has the following beneficial effects:

[0126] (1) A preparation method of rhizocladine alkaloids is provided, by optimizing the preparation process such as the type of resin for purification, the ratio of crude drug to dry mass of resin and elution conditions, the total content of alkaloids, the content of effective components rhizocladine and wall grass alkaloids, the transfer rate and the product yield of rhizocladine alkaloids in rhizocladine alkaloids products are greatly improved, and the rhizocladine alkaloids in rhizocladine are more efficiently utilized for extracting rhizocladine alkaloids.

[0127] (2) The present application optimizes the loading mode of rhizocladine alkaloids macroporous resin, effectively overcomes the problem that lipid-soluble amide alkaloids are easy to block the chromatographic column during loading, and provides a train of thought for the purification of similar lipid-soluble substances by macroporous resin.

[0128] (3) The rhizocladine alkaloid preparation method provided by the present application is stable and easy to repeat, the inter-group standard deviation of parameters such as the total content of alkaloids, the content of effective components rhizocladine and wall grass alkaloids, the transfer rate and the product yield is small, and it is beneficial to popularization and application.

[0129] (4) The prepared eremophilamides in the preparation method have better analgesic function and can effectively relieve bone cancer pain of experimental mice.

[0130] (5) The application further provides a detection standard of eremophilamides, which fills the blank of the detection standard of eremophilamide products. BRIEF DESCRIPTION OF DRAWINGS

[0131] Figure 1 The figure is the UPLC fingerprint of eremophilamides in Example 1 and the control fingerprint of fingerprint peaks 1, 6, 7, 9 and 11.

[0132] Figure 2 The figure is the column chart of the Western Blot result of the influence of eremophilamides on the expression level of TPRV1 in the dorsal root ganglion of mice in the pharmacodynamic example 1.

[0133] In the formula, group N represents a blank group, group M represents a model group, group Y represents a positive control group, S-1 represents the water extraction part of eremophilamides prepared in Example 1, S-2 represents the alcohol extraction part of eremophilamides prepared in Example 1, and S-3 represents eremophilamides prepared in Example 1.

[0134] In A, ### represents P<0.05 between group N and group M, and ns represents no significant difference between group M and group Y; *** represents P<0.001 between group S-1, S-2, S-3 and group N, M and Y.

[0135] In B, *** represents P<0.001 between group S-1, S-2 and group S-3. DETAILED DESCRIPTION

[0136] The following non-limiting examples can make those skilled in the art more fully understand the present application, but do not limit the present application in any way. The following content is only an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application according to the disclosed content, and it should also belong to the scope of the present application.

[0137] The present application is further described below in the form of specific examples. The various chemical reagents used in the examples of the present application are obtained through conventional commercial channels unless otherwise specified. The content described below is the mass content unless otherwise specified. It is understood to be carried out at room temperature unless otherwise specified.

[0138] Example 1

[0139] The preparation of eremophilamides is as follows:

[0140] S1, take the stone south rattan, after crushing with 14 times the mass of 75% ethanol aqueous solution reflux extraction, repeated 2 times, extraction 2h, combined extract.

[0141] S2, pretreatment of HP-20 macroporous adsorption resin: soak in ethanol for 24h, then soak in 5% hydrochloric acid solution for 24h, wash with water to neutral, then soak in 5% NaOH solution for 24h, wash with water to neutral, filter, dry.

[0142] Take the appropriate volume of pretreated HP-20 macroporous adsorption resin, and the extract obtained in step S1 is mixed by stirring. The ratio of the mass of stone south rattan in step S1 to the dry mass of macroporous adsorption resin is 1:0.8. Stir for 20 min, mix thoroughly, concentrate at 40-60℃ under reduced pressure until no alcohol odor, get the sample mixture.

[0143] S3, the sample mixture obtained in step S2 is loaded into the column. Stand for 20 min, flow out at a flow rate of 2BV / h. Then elute

[0144] S31, elute with water at a flow rate of 3BV / h, 3BV, get water washing part liquid;

[0145] S32, elute with 30% ethanol aqueous solution at a flow rate of 3BV / h, 5BV, get alcohol washing part liquid;

[0146] S33, elute with 75% ethanol aqueous solution at a flow rate of 3BV / h, 10BV, get eluate.

[0147] S4, the eluate obtained in step S3 is concentrated under reduced pressure at 40-60℃, until the ratio of the mass of stone south rattan in step S1 to the volume of liquid is 0.1g:1mL. Freeze-drying, get stone south rattan amide alkaloids (freeze-dried powder).

[0148] The water washing part liquid obtained in step S31 is concentrated under reduced pressure, dried, get stone south rattan amide alkaloids water washing part.

[0149] The alcohol washing part liquid obtained in step S32 is concentrated under reduced pressure, dried, get stone south rattan amide alkaloids alcohol washing part.

[0150] The obtained stone south rattan amide alkaloids are characterized by UPLC fingerprint. The characterization process is as follows:

[0151] Accurately weigh approximately 10 mg of *Photinia serratifolia* alkaloids and place them in a stoppered conical flask. Seal the flask tightly and weigh again. Add 25 mL of 75% methanol and sonicate (40 kHz, 100 W) for 30 min. Remove and cool to room temperature. Make up the lost weight with 75% methanol, shake well, and filter. Take the filtrate, dilute it 1:1 with methanol, centrifuge at 10000 r / min for 5 min, and filter through a 0.22 μm microporous filter to obtain the test solution for single-component content determination. Accurately inject 5 μL of both the reference solution and the test solution into a high-performance liquid chromatograph (UPLC) for detection. The UPLC chromatographic conditions are as follows:

[0152] Column: Poroshell 120EC-C 18 1.9 μm, 2.1 × 50 mm column;

[0153] Using water as mobile phase A and acetonitrile as mobile phase B, the gradient elution scheme is as follows:

[0154] Elution time / min Mobile phase A / % Mobile phase B / % 0-4 83 17 4-8.7 83-75 17-25 8.7-10.6 75-68 25-32 10.6-12 68-50 32-50 12-17 50 50 17-20 50-10 50-90

[0155] The column temperature was 35℃, the flow rate was 0.8 mL / min, and the detection wavelength was 260 nm.

[0156] Characterization results as follows Figure 1 As shown.

[0157] The UPLC fingerprint of the alkaloids of *Syngonium stenoptera* includes fingerprint peaks 1, 6, 7, 9, and 11. Fingerprint peak 1 is located at 3.90-4.10 min, fingerprint peak 6 at 12.45-12.55 min, fingerprint peak 7 at 12.80-12.95 min, fingerprint peak 9 at 13.10-13.25 min, and fingerprint peak 11 at 15.30-15.45 min.

[0158] The peak area ratios of fingerprint peaks 1, 6, 7, 9, and 11 are as follows:

[0159] 1.600:1.470:1.780:16.64:1.001.

[0160] Among them, fingerprint peak 1 represents purpuric bisamide; fingerprint peak 6 represents fentanylamide; fingerprint peak 7 represents Pipersintenamide; fingerprint peak 9 represents fentanyl alkaloid; and fingerprint peak 11 represents (2E,4E)-N-(2-methylpropyl)-dodecyl-2,4-dieneamide.

[0161] The UPLC fingerprint spectrum further comprises fingerprint peak 2, fingerprint peak 3, fingerprint peak 4, fingerprint peak 5, fingerprint peak 8 and fingerprint peak 10, the position of the fingerprint peak 2 is 5.85-6.05 min, the position of the fingerprint peak 3 is 8.85-8.95 min, the position of the fingerprint peak 4 is 9.30-9.50 min, the position of the fingerprint peak 5 is 12.15-12.30 min, the position of the fingerprint peak 8 is 13.00-13.05 min, and the position of the fingerprint peak 10 is 13.90-14.00 min.

[0162] Example 2

[0163] Compared with Example 1, in step S1, 75% ethanol aqueous solution with 10 times the mass was used for reflux extraction, repeated 2 times, and the rest was the same.

[0164] Example 3

[0165] Compared with Example 1, in step S1, 75% ethanol aqueous solution with 20 times the mass was used for reflux extraction, repeated 2 times, and the rest was the same.

[0166] Comparative Example 1

[0167] Compared with Example 1, in step S1, 75% ethanol aqueous solution with 30 times the mass was used for reflux extraction, repeated 2 times, and the rest was the same.

[0168] Example 4

[0169] Compared with Example 1, in step S2, D101 macroporous adsorption resin was used, and the rest was the same.

[0170] Example 5

[0171] Compared with Example 1, in step S2, S-8 macroporous adsorption resin was used, and the rest was the same.

[0172] Example 6

[0173] Compared with Example 1, in step S2, NKA-9 macroporous adsorption resin was used, and the rest was the same.

[0174] Example 7

[0175] Compared with Example 1, in step S2, DA201 macroporous adsorption resin was used, and the rest was the same.

[0176] Example 8

[0177] Compared with Example 1, in step S2, HP2MGL macroporous adsorption resin was used, and the rest was the same.

[0178] Comparative Example 2

[0179] Compared with Example 1, in step S2, D001 macroporous strong acid cation resin was used instead, and the rest were the same.

[0180] Example 9

[0181] Compared with Example 1, in step S1, the mass ratio of stone bamboo to the dry mass of macroporous adsorption resin in step S2 was 1:0.6, and the rest were the same.

[0182] Example 10

[0183] Compared with Example 1, in step S1, the mass ratio of stone bamboo to the dry mass of macroporous adsorption resin in step S2 was 1:1, and the rest were the same.

[0184] Example 11

[0185] Compared with Example 1, in step S1, the mass ratio of stone bamboo to the dry mass of macroporous adsorption resin in step S2 was 1:0.4, and the rest were the same.

[0186] Example 12

[0187] Compared with Example 1, in step S1, the mass ratio of stone bamboo to the dry mass of macroporous adsorption resin in step S2 was 1:1.2, and the rest were the same.

[0188] Comparative Example 3

[0189] Compared with Example 1, in step S1, the mass ratio of stone bamboo to the dry mass of macroporous adsorption resin in step S2 was 1:0.35, and the rest were the same.

[0190] Example 13

[0191] Compared with Example 1, in step S3, it was replaced by standing for 15 min, and the rest were the same.

[0192] Example 14

[0193] Compared with Example 1, in step S3, it was replaced by standing for 30 min, and the rest were the same.

[0194] Comparative Example 4

[0195] Compared with Example 1, in step S3, it was replaced by standing for 24 h, and the rest were the same.

[0196] Example 15

[0197] Compared with Example 1, in step S32, it was replaced by eluting 5 BV of 25% ethanol aqueous solution at 3 BV / h, and the rest were the same.

[0198] Comparative Example 5

[0199] Compared with Example 1, in step S32, 5 BV of 65% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0200] Example 16

[0201] Compared with Example 1, in step S33, 10 BV of 70% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0202] Example 17

[0203] Compared with Example 1, in step S33, 10 BV of 80% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0204] Comparative Example 6

[0205] Compared with Example 1, in step S33, 10 BV of 95% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0206] Comparative Example 7

[0207] Compared with Example 1, in step S33, 10 BV of 50% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0208] Example 18

[0209] Compared with Example 1, in step S33, 9 BV of 75% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0210] Example 19

[0211] Compared with Example 1, in step S33, 12 BV of 75% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0212] Comparative Example 8

[0213] Compared with Example 1, in step S33, 8 BV of 75% ethanol aqueous solution was eluted at 3 BV / h, and the rest was the same.

[0214] Comparative Example 9

[0215] Compared with Example 1, in step S3, the step of standing for 20 min was omitted, and the rest was the same.

[0216] Comparative Example 10

[0217] According to the method for extracting effective components of Shishan Teng recorded in the article “Screening and Purification of Analgesic Active Part of Shishan Teng” (Chinese Patent Medicine Journal, March 2023, Vol. 45, No. 3, pp. 1007-1011), Shishan Teng was extracted. The specific steps are as follows:

[0218] Take an appropriate amount of Rubus corchorifolius S. Moore, crush and extract with 75% ethanol aqueous solution for 2 times, each time for 2 hours. Combine the extract, and concentrate under reduced pressure to remove the alcohol smell. Add water to the extract to make a suspension, and load the suspension onto a D001 macroporous cation exchange resin column. Take 2 BV of 0.4 g / mL sample solution, and flow through the column. Elute with 5 BV of 70% ethanol, at a flow rate of 2 BV / h. Collect the eluate, concentrate under reduced pressure to remove the alcohol smell, and freeze-dry to obtain the product.

[0219] Comparative Example 11

[0220] Compared with Example 1, step S2 is replaced by the following steps,

[0221] Pretreat the HP-20 macroporous adsorption resin by soaking in ethanol for 24 hours, then soaking in 5% hydrochloric acid solution for 24 hours, washing with water until neutral, then soaking in 5% NaOH solution for 24 hours, washing with water until neutral, and then filtering and air-drying.

[0222] Take an appropriate amount of the pretreated HP-20 macroporous adsorption resin, and load the resin into a column. Concentrate the extract obtained in step S1 under reduced pressure at 40-60°C until the alcohol smell is removed, and then load the sample mixture.

[0223] In step S3, flow the sample mixture through the column after loading the resin column. The remaining steps are the same as in Example 1.

[0224] Comparative Example 12

[0225] Compared with Example 1, in step S4, the temperature for concentration under reduced pressure is changed to 65-75°C, and the remaining steps are the same.

[0226] In Examples 1-19 and Comparative Examples 1-12, the peak area ratios of fingerprint peak 1, fingerprint peak 6, fingerprint peak 7, fingerprint peak 9, and fingerprint peak 11 are shown in the following table (the position of fingerprint peak 1 is 3.90-4.10 min, the position of fingerprint peak 6 is 12.45-12.55 min, the position of fingerprint peak 7 is 12.80-12.95 min, the position of fingerprint peak 9 is 13.10-13.25 min, and the position of fingerprint peak 11 is 15.30-15.45 min):

[0227]

[0228]

[0229] Effect Evaluation

[0230] 1. The powder yield of Rubus corchorifolius S. Moore amide alkaloids (freeze-dried powder) prepared in Examples 1-19 and Comparative Examples 1-12

[0231] 1.1 Calculation method

[0232] Powder yield = weight of freeze-dried powder ÷ dosage of kadsurae cortex x 1000 ‰.

[0233] 1.2 Experimental results

[0234] Each example and each comparative experiment was repeated 5 times, and the average value and standard deviation of the powder yield obtained each time were calculated, and the results are summarized in the table below:

[0235]

[0236]

[0237] 2. Total alkaloid content in kadsurae cortex amides alkaloids (freeze-dried powder) obtained from examples 1-19 and comparative examples 1-12

[0238] 2.1 Determination method

[0239] Preparation of chromotropic acid concentrated sulfuric acid solution: take a suitable amount of pure water, add concentrated sulfuric acid to prepare a 90% concentrated sulfuric acid solution. Add 0.2 g of chromotropic acid powder to 200 mL of 90% concentrated sulfuric acid solution, stir to dissolve, cool, and then seal in a brown bottle for storage. The chromotropic acid concentrated sulfuric acid solution is obtained.

[0240] Preparation of reference solution: take a suitable amount of wall grass alkaloid reference solution, add anhydrous ethanol to prepare a reference solution with a concentration of 0.2 mg / mL.

[0241] Obtaining of standard curve: precisely take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mL of the reference solution, and place it in a 10 mL glass test tube with a stopper. Precisely add 5 mL of chromotropic acid concentrated sulfuric acid solution, suspend uniformly, and then place in a boiling water bath for 30 min. After taking it out and cooling, slowly add water to the mark, suspend uniformly, and cool. Measure the absorbance at 485 nm. The concentration is the abscissa (X), and the absorbance is the ordinate (Y). Draw the standard curve. Wall grass alkaloid shows a good linear relationship with absorbance in the range of 20-140 μg / mL. The regression equation is Y=0.0016X+0.1267 (R2=0.9966).

[0242] Preparation of freeze-dried powder sample solution: take 10 mg of freeze-dried powder, precisely weigh, and place it in a stoppered conical flask. Seal the weight, add 25 mL of 75% methanol, and ultrasonic (40 kHz, 100 W) for 30 min. Take it out and cool to room temperature. Make up the lost weight with 75% methanol, shake uniformly, and then filter. Take the filtrate, which is the freeze-dried powder sample solution.

[0243] UV-visible spectrum detection: precisely pipette 0.2 mL of the freeze-dried powder sample solution into a 10 mL glass test tube with a stopper, precisely add 5 mL of the colorless acid concentrated sulfuric acid solution, mix uniformly, and then place in a boiling water bath for 30 min. After taking out and cooling, slowly add water to the mark, mix uniformly, and cool. The test solution is obtained. The absorbance is measured, and the total alkaloid content in the freeze-dried powder is determined by the standard curve method.

[0244] 2.2 Determination results

[0245] Each example and each comparative experiment was repeated 5 times, and the average value and standard deviation of the total alkaloid content in the freeze-dried powder obtained each time were calculated. The results are summarized in the following table:

[0246]

[0247]

[0248] 3. Content and transfer rate of fomesamidine and raddeanamine in the fomesamidine alkaloids (freeze-dried powder) obtained from examples 1-19 and comparative examples 1-12

[0249] 3.1 Determination method

[0250] Chromatographic conditions:

[0251] Chromatographic column: Poroshell 120EC-C18 (1.9 μm, 2.1 x 50 mm);

[0252] Water was used as mobile phase A and acetonitrile was used as mobile phase B. The gradient elution scheme is shown in the following table:

[0253] Elution time / min Mobile phase A / % Mobile phase B / % 0-4 83 17 4-8.7 83-75 17-25 8.7-10.6 75-68 25-32 10.6-12 68-50 32-50 12-17 50 50 17-20 50-10 50-90

[0254] Column temperature: 35°C, flow rate: 0.8 mL / min, detection wavelength: 260 nm.

[0255] Preparation of the reference solution: take an appropriate amount of raddeanamine and fomesamidine reference substances, accurately weigh and determine, and add an appropriate amount of methanol to prepare a 20 μg / mL reference solution.

[0256] Preparation of the test solution:

[0257] Alcohol extract sample: after dilution with methanol 5 times, centrifugation at 10000 r / min for 5 min, and passing through a 0.22 μm microporous filter, it is obtained.

[0258] Effluent and eluent sample: after dilution with methanol 1 times, centrifugation at 10000 r / min for 5 min, and passing through a 0.22 μm microporous filter, it is obtained.

[0259] Freeze-dried powder sample: about 10 mg of freeze-dried powder was precisely weighed, placed in a conical bottle with a plug, and the weight was determined. 25 mL of 75% methanol was added and ultrasonicated (40 kHz, 100 W) for 30 min. The sample was removed and cooled to room temperature. The lost weight was made up with 75% methanol, and the mixture was shaken and filtered. The filtrate was diluted with methanol to 1-fold, and centrifuged at 10,000 r / min for 5 min. The filtrate was obtained by passing through a 0.22 μm microporous filter. The single component content test sample solution was obtained.

[0260] Determination operation: 5 μL of the control sample solution and the test sample solution were precisely taken and injected into the liquid chromatograph, and the determination was completed.

[0261] Transfer rate = single component content in freeze-dried powder ÷ single component content in filtrate obtained in step S1 × 1000‰. The single component is windfengamide or wall grass base. The single component content is detected by a chromatographic method.

[0262] 3.2 Determination results

[0263] The preparation of rhynchosia nitida amide alkaloids in each example and comparative example was repeated 5 times. The average value and standard deviation of the content of windfengamide in the rhynchosia nitida amide alkaloids were obtained, and the experimental results are shown in the following table:

[0264]

[0265]

[0266] The preparation of rhynchosia nitida amide alkaloids in each example and comparative example was repeated 5 times. The average value and standard deviation of the content of wall grass base in the rhynchosia nitida amide alkaloids were obtained, and the experimental results are shown in the following table:

[0267]

[0268]

[0269] The preparation of rhynchosia nitida amide alkaloids in each example and comparative example was repeated 5 times. The average value and standard deviation of the transfer rate of windfengamide in the rhynchosia nitida amide alkaloids were obtained, and the experimental results are shown in the following table:

[0270]

[0271]

[0272] The preparation of rhynchosia nitida amide alkaloids in each example and comparative example was repeated 5 times. The average value and standard deviation of the transfer rate of wall grass base in the rhynchosia nitida amide alkaloids were obtained, and the experimental results are shown in the following table:

[0273]

[0274]

[0275] In summary, the preparation methods provided in Examples 1-19, compared to those in Comparative Examples 1-12, can achieve higher contents and transfer rates of fentanyl and fentanyl alkaloids.

[0276] Pharmacological Example 1: Inhibitory Effect of Photinia styrax alkaloids on acute pain and inflammatory pain in mice 1. Experimental materials

[0277] 1.1 Drugs and Reagents

[0278] The following reagents were prepared in the laboratory: *Photinia serratifolia*, ibuprofen tablets (batch number 2212627, Shandong Xinhua Pharmaceutical Co., Ltd.), Running Buffer and Transfer Buffer (prepared in the laboratory), 10% gel casting kit (Novizan; cat: E303; Lot: 071E2221LA), TBS (Biosharp; cat: BL602A), Tween-20 (Soleb: cat: T8220; Lot: 424E013), BSA (Biosharp: cat: BS114; Lot: 22215659), protein marker (Thermo Fisher Scientific: cat: 26616; Lot: 91295071), primary antibody TRPV1 (Bioworld: cat: BS 60454; Lot: CN89330), and primary antibody β-actin (Abclonal: cat: AC004; Lot: 3500100011).

[0279] 1.2 Instruments

[0280] Electrophoresis apparatus (Bio Rad, PowerPac Basic), semi-dry transfer apparatus (Bio Rad, TRANS-BLOT SD), imaging system (Tianneng, 500S), centrifuge (Eppendorf, Centrifuge 5417R), IL-1β kit (batch number E0408231494R), and PGE2 kit (batch number E0408231494R) were purchased from Nanjing Sains Biotechnology Co., Ltd.; KH-30E ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); YWLG-10A vacuum freeze dryer (Nanjing Yanwo Biotechnology Co., Ltd.); RT-6100 microplate reader (Shenzhen Leidu Life Science Co., Ltd.); and TGL16M refrigerated centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

[0281] 1.3 Animals

[0282] SPF grade ICR mice, half male and half female, body weight (20±2) g, purchased from Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd.

[0283] 2. Method and result

[0284] 2.1 Grouping

[0285] Randomly grouped (n=8). The specific grouping method is as follows:

[0286] Blank group: no pain model establishment, intragastrically administered with 0.5% sodium carboxymethyl cellulose physiological saline solution.

[0287] Model group: pain model establishment was performed, and 0.5% sodium carboxymethyl cellulose physiological saline solution was intragastrically administered.

[0288] Positive drug group: pain model establishment was performed, and ibuprofen solution (30 mg ibuprofen per kilogram of mouse body weight per day) was intragastrically administered.

[0289] Example 1 water washing part group: pain model establishment was performed, and the water washing part of the ericamiamide alkaloids obtained in Example 1 was intragastrically administered.

[0290] Example 1 alcohol washing part group: pain model establishment was performed, and the alcohol washing part of the ericamiamide alkaloids obtained in Example 1 was intragastrically administered.

[0291] Each example, comparative example group: pain model establishment was performed, and the ericamiamide alkaloids obtained in each example, comparative example were intragastrically administered.

[0292] The ericamiamide alkaloids water washing part administered to the Example 1 water washing part group, the ericamiamide alkaloids alcohol washing part administered to the Example 1 alcohol washing part group, and the ericamiamide alkaloids administered to each example, comparative example group were all intragastrically administered at a dose of 30 mg per kilogram of mouse body weight per day.

[0293] The base solution used for intragastrical administration in each of the above administration groups was 0.5% sodium carboxymethyl cellulose physiological saline solution.

[0294] 2.2 Experimental method

[0295] Drug administration, modeling and sampling: The mice were adaptively fed for 7 days, and after adaptive feeding, each group was continuously gavaged for 7 days, 3 days before modeling, and the fixed time was selected. According to the grasping method when injecting the sole, the mice were grasped to adapt to the injection posture. After the adaptation, 30 min after the last administration, 5% formalin solution (prepared from 0.9% normal saline solution) 1 μL / g was injected into the left foot of the mouse, 8 mice per group. The licking frequency and licking time of the acute pain phase within 5 min and the inflammatory pain phase of 10-60 min were observed after injection. After the observation, the mice in each group were taken blood from the eye socket, and the mice were sacrificed by removing the spine, and the dorsal root ganglion in the L4-6 joint of the mouse was taken.

[0296] Data processing: The data in this study were processed using Graphpad Prism 9.0 software, and the data were expressed as , and the comparison between multiple groups used one-way analysis of variance, and P<0.05 indicated that the difference was statistically significant.

[0297] 2.3 Experimental results

[0298] 2.3.1 Licking frequency and licking time of mice

[0299] The results of licking time and licking frequency of phase pain are summarized in the following table n=8):

[0300]

[0301]

[0302] (Note: compared with the model group, *P<0.05, ***P<0.001.)

[0303] From the above table, compared with the model group, each example group has a significant analgesic effect, which can significantly reduce the licking time (P<0.05) and licking frequency (P<0.001) of the phase of the pain mouse; the water washing part of example 1 reduces the licking frequency of the phase, but the licking time is still close to the model; the alcohol washing part of example 1, the positive drug ibuprofen group and each comparative example group have no significant analgesic effect on phase pain.

[0304] II Licking time and licking frequency of phase pain are summarized in the following table n=8):

[0305]

[0306]

[0307] (Note: compared with the model group, *P<0.05, **P<0.01, ***P<0.001.)

[0308] From the above table, compared with the model group, each example group has a significant analgesic effect, which can significantly reduce the licking time (P<0.01) and licking frequency (P<0.01) of the pain mouse phase; the positive drug group also has a significant analgesic effect, which can significantly reduce the licking time (P<0.01) and licking frequency (P<0.001) of the pain mouse phase; the rest of each group does not exert analgesic effect.

[0309] In summary, the analgesic effect of the rhynchosia minima amide alkaloids provided by each example is better than that of ibuprofen to a certain extent. Although the water washing part of the rhynchosia minima amide alkaloids provided by example 1 can reduce the licking frequency of the mouse phase, the licking time is not reduced. After analyzing the data one by one, it is found that this is due to the long single licking time of the mouse in this group. And the water washing part of the rhynchosia minima amide alkaloids provided by example 1 does not exert analgesic effect on the pain phase, which shows that the water washing part of the rhynchosia minima amide alkaloids provided by example 1 does not have analgesic effect on the acute pain and inflammatory pain induced by formalin. n=8):

[0310] The detection results of the serum IL-1β and PGE2 levels of the mice in each group are summarized in the following table:

[0311]

[0312]

[0313] (Note: compared with the blank control group, ###P<0.05; compared with the model control group, *P<0.05, **P<0.01, ***P<0.001)

[0314] From the above table, the water washing part of the rhynchosia minima amide alkaloids provided by example 1, the alcohol washing part of the rhynchosia minima amide alkaloids, and the rhynchosia minima amide alkaloids provided by each example can reduce inflammatory mediators to a certain extent. Among them, the inhibitory effect of the rhynchosia minima amide alkaloids provided by example 1 is the strongest (P<0.001), which is similar to the anti-inflammatory effect of the positive drug ibuprofen, indicating that the effective part of rhynchosia minima may exert a certain analgesic effect by inhibiting inflammatory factors in serum.

[0315] 2.4.3 Western Blot detection of dorsal root ganglion (DGR) TRPV1 level of mice

[0316] The TRPV1 level in the isolated dorsal root ganglion of mice was detected by Western Blot method, and the results are shown in Figure 2 Figure 2 ​It can be seen that, compared with the model group, the alkaloids of *Photinia serratifolia* provided in Example 1 had the strongest effect on TRPV1 expression level. Although the water-washed and alcohol-washed fractions of the alkaloids of *Photinia serratifolia* provided in Example 1 also affected TRPV1 expression level, their effect was far less than that of the alkaloids of *Photinia serratifolia*. The increase in TRPV1 level may be related to the warm nature of *Photinia serratifolia*.

[0317] 3. Conclusion

[0318] Based on the above results, the senna-based alkaloids provided in each example (especially Example 1) may exert a significant analgesic effect by acting on the TRPV1 pathway and inhibiting serum inflammatory factors. Pharmacological Example 2: The regulatory effect of senna-based alkaloids on the pain threshold in mice.

[0319] 1. Experimental Materials

[0320] 1.1 Drugs and Reagents

[0321] Lyophilized powder of effective parts of *Photinia serratifolia*, ibuprofen tablets (batch number 2212627, Shandong Xinhua Pharmaceutical Co., Ltd.), acetaminophen and tramadol tablets, cypermethrin reference standard (≥98%), cypermethrin (≥98%) and pipersintenamide (≥95%).

[0322] 1.2 Instruments

[0323] KH-30E ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.), YWLG-10A vacuum freeze dryer (Nanjing Yanwo Biotechnology Co., Ltd.), TGL16M refrigerated centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

[0324] 1.3 Animals

[0325] SPF-grade ICR mice, half male and half female, weighing (20±2)g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0326] 2. Methods and Results

[0327] 2.1 Grouping

[0328] Random grouping (n=8). The specific grouping method is as follows.

[0329] Model group: administered physiological saline solution containing 0.5% sodium carboxymethyl cellulose by gavage.

[0330] Ibuprofen group: Ibuprofen solution was administered by gavage (15 mg ibuprofen per kilogram of mouse body weight per day).

[0331] Amphetaminyl tramadol group: intragastrically administered amphetaminyl tramadol solution (55 mg of amphetaminyl tramadol per kilogram of mouse body weight per day).

[0332] Example 1 low-dose group: intragastrically administered the ericamiamide alkaloids prepared in Example 1 (9 mg of ericamiamide alkaloids per kilogram of mouse body weight per day).

[0333] Example 1 high-dose group: intragastrically administered the ericamiamide alkaloids prepared in Example 1 (36 mg of ericamiamide alkaloids per kilogram of mouse body weight per day).

[0334] Comparative Example 10 low-dose group: intragastrically administered the ericamiamide alkaloids prepared in Comparative Example 10 (9 mg of ericamiamide alkaloids per kilogram of mouse body weight per day).

[0335] Comparative Example 10 high-dose group: intragastrically administered the ericamiamide alkaloids prepared in Comparative Example 10 (36 mg of ericamiamide alkaloids per kilogram of mouse body weight per day).

[0336] Wallenrootine group: intragastrically administered wallenrootine (5 mg of wallenrootine per kilogram of mouse body weight per day).

[0337] Windamycin group: intragastrically administered windamycin (5 mg of windamycin per kilogram of mouse body weight per day).

[0338] Pipersintenamide group: intragastrically administered Pipersintenamide (5 mg of Pipersintenamide per kilogram of mouse body weight per day).

[0339] The base solution used in the above administration groups was a 0.5% sodium carboxymethyl cellulose solution in physiological saline.

[0340] 2.2 Experimental method

[0341] Hot water tail-flick test:

[0342] The instrument and mice were adjusted, the power was turned on, and the temperature of the water bath was controlled at 50±0.5. The tail end of the mouse was immersed in the constant-temperature water bath for 3 cm, and the time from the entry of the tail end into the water to the exit was measured. The measurement was repeated for 3 times with an interval of not less than 15 min, and the average of the 3 times was taken as the basic pain threshold of the mouse. Then the mice in each group were administered, and the administration was performed once a day for 3 consecutive days. On the fourth day, the administration was performed again as the last intragastric administration. The tail end of the mouse was immersed in the constant-temperature water bath for 3 cm at 30, 45 and 60 min after the last intragastric administration to measure the pain threshold; if there was still no response after 15 s, the mouse was taken out to avoid scalding, and the pain threshold was calculated as 15 s, which was recorded as the pain threshold after administration. The basic pain threshold and the pain threshold after administration were measured, and the pain threshold improvement rate was calculated according to the following formula.

[0343] Pain threshold improvement rate = (pain threshold after administration - pain threshold before administration) / basal pain threshold x 100%

[0344] Formalin-induced mouse biphasic pain experiment:

[0345] Three days before modeling, select a fixed time, according to the grip method when injecting the sole, grip the mouse, and make it adapt to the injection posture. Each group was given medicine continuously for seven days, once a day, and fasted but not watered 6 hours before the last administration. 30 minutes after the last administration, the blank group was not treated, and the other groups of mice were injected with 5% formalin solution (prepared from 0.9% saline solution) 1 μl / g in the left foot sole, 8 mice per group. The mouse behavior was observed and scored within 5 minutes of acute pain and 20-60 minutes of inflammatory pain after injection. The specific standards are shown in the table below:

[0346] Evaluation scale Evaluation criteria Class A Both feet on the ground, weight evenly distributed Class B Injection foot lightly on the ground Class C Injection foot raised Class D Licking and biting the injection foot

[0347] After injecting the formalin solution, the occurrence of corresponding rating behaviors was observed and recorded, and the observation was continued for 60 minutes. Among them, the 0-5 minutes is the acute pain period, called phase; 10-20 minutes is the interval; 2-60 minutes is the inflammatory pain period, called phase. The number of times of different rating behaviors in phase I and phase II was calculated respectively. The pain score was calculated according to the following formula:

[0348] Pain score = 1 x B + 2 x C + 3 x D

[0349] Where B is the number of times of B rating behavior; C is the number of times of C rating behavior; D is the number of times of D rating behavior.

[0350] Data processing: The data in this study were processed by Graphpad Prism 9.0 software, and the data were expressed as The comparison between multiple groups was analyzed by one-way ANOVA, and P<0.05 indicated that the difference was statistically significant.

[0351] 2.3 Experimental results

[0352] 2.3.1 Hot water tail flick test results

[0353] The results of hot water tail flick test are summarized in the table below n=8):

[0354]

[0355] (Note: compared with the model group, *P<0.05, **P<0.01, ***P<0.001.)

[0356] From the above table, it can be seen that the pain threshold improving effect of the stonebamides alkaloids provided in Example 1 is higher than that of the stonebamides alkaloids provided in Comparative Example 10 at the same dosage. After 30 min of administration, the stonebamides alkaloids provided in Example 1 can significantly improve the pain threshold of mice (P < 0.01), and the effect is slightly stronger than that of the positive drug control group ibuprofen and amitriptyl tramadol; 45 min after administration, amitriptyl tramadol has the most significant effect on improving the pain threshold of mice (P < 0.001), and the stonebamides alkaloids provided in Example 1 have a similar effect on improving the pain threshold of mice; 60 min after administration, the stonebamides alkaloids provided in Example 1 can significantly improve the pain threshold of mice (P < 0.01).

[0357] 2.4.2 Results of pain comprehensive score experiment of mice with formalin-induced biphasic pain

[0358] The results of the formalin-induced biphasic pain experiment of mice are summarized in the following table. n = 8.

[0359]

[0360]

[0361] (Note: compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the amitriptyl tramadol group, #P < 0.05.)

[0362] From the above table, it can be seen that the analgesic effect of the stonebamides alkaloids provided in Example 1 is stronger than that of the stonebamides alkaloids provided in Comparative Example 10. Compared with the model group, the amitriptyl tramadol and the low-dose group of the stonebamides alkaloids provided in Example 1 have similar analgesic effects, and can reduce the number of mice at each pain level (P < 0.01); compared with amitriptyl tramadol, the high-dose group of the stonebamides alkaloids provided in Example 1 has a more significant analgesic effect (P < 0.05); ibuprofen does not play a role in the pain of the phase. Compared with the model group, ibuprofen has a very significant analgesic effect (P < 0.001), and the low-dose group of the stonebamides alkaloids provided in Example 1 and amitriptyl tramadol also have a good analgesic effect, but the overall effect is not as good as ibuprofen; the high-dose group of the stonebamides alkaloids provided in Example 1 has a very significant analgesic effect, and the analgesic effect is similar to that of ibuprofen; compared with amitriptyl tramadol, the low-dose group of the stonebamides alkaloids provided in Example 1 has a more significant analgesic effect (P < 0.05) in phase II.

[0363] 3. Conclusion

[0364] The lyophilized alkaloids of Pipersintenamide prepared in Example 1 effectively retained their analgesic activity, and their content of active ingredients and analgesic effect were superior to those provided in Comparative Example 10. Ibuprofen is a commonly used antipyretic analgesic, while tramadol is used for cancer pain or chronic pain. The lyophilized alkaloids of Pipersintenamide prepared in Example 1 have similar or even better analgesic effects than these two, providing better analgesia for pain patients. Furthermore, compared to the three active ingredients (pipersintenamide, fenestrone, and pipersintenamide) in the lyophilized alkaloids, the lyophilized alkaloids of Pipersintenamide prepared in Example 1 can exert a better combined analgesic effect.

[0365] Example 20

[0366] The analgesic alkaloids prepared in Example 1 were purified to obtain various analgesic drugs with different components. The preparation method is as follows:

[0367] Take an appropriate amount of the pine alkaloids prepared in Example 1 and dissolve them completely in methanol to obtain a methanol solution. Mix the methanol solution with alumina at a weight ratio of pine alkaloids (lyophilized powder) to alumina of 1:1 and place the mixture in a rotary flask. Concentrate under reduced pressure at 40-60℃ until a dry powder is obtained. Pack the column using a wet method and load the sample (the sample being the pine alkaloids prepared in Example 1) using a dry method. Elute with a gradient of petroleum ether-ethyl acetate. Gradient elution program: petroleum ether, petroleum ether-ethyl acetate mixture (volume ratio 95:5→90:10→85:15→80:20→75:25→70:30→65:35→60:40→55:45→50:50→45:55→40:60→35:65→30:70→25:75→20:80→15:85→10:90→5:95), ethyl acetate. The volume of solution used for each gradient elution is 5 BV.

[0368] The elution fractions of petroleum ether:ethyl acetate = 90:10 (referred to as solution 1) and petroleum ether:ethyl acetate = 85:15 (referred to as solution 2) were separated by chromatography.

[0369] The methanol reconstituted solution of solution 1 was centrifuged at 10000 rpm for 10 minutes, the supernatant was taken, and was filtered through a 0.22 μm microporous filter and was subjected to preparative liquid phase chromatography using a Hanbon ODS preparative column, isocratic elution with acetonitrile: water (87:13), detection wavelength 260 nm, and flow rate 3.5 mL / min. The fractions containing wallenbergloline, and other fractions 1 were collected. The methanol reconstituted solution of solution 2 was centrifuged at 10000 rpm for 10 minutes, the supernatant was taken, and was filtered through a 0.22 μm microporous filter and was subjected to preparative liquid phase chromatography using a Hanbon ODS preparative column, isocratic elution with acetonitrile: water (75:25), detection wavelength 260 nm, and flow rate 3.5 mL / min. The fractions containing windtengamide, the fractions containing Pipersintenamide, and other fractions 2 were collected. The other fractions 1 and the other fractions 2 were mixed to obtain other fractions.

[0370] The fractions containing wallenbergloline, the fractions containing windtengamide, the fractions containing Pipersintenamide, and the other fractions were freeze-dried to obtain analgesic drug 1, analgesic drug 2, analgesic drug 3, and analgesic drug 4, respectively.

[0371] Pharmacodynamic Example 3: Pain relief experiment in a mouse model of bone cancer pain

[0372] 1. Experimental materials

[0373] 1.1 Drugs and reagents

[0374] Rhodotypos umbellatum, positive drug ibuprofen tablets (batch number 2212627, Shandong Xinhua Pharmaceutical Co., Ltd.), positive drug amitriptyline tramadol tablets, wallenbergloline, windtengamide, and Pipersintenamide.

[0375] 1.2 Instruments

[0376] KH-30E ultrasonic cleaner (Kunshan Huochuang Ultrasonic Instrument Co., Ltd.), YWLG-10A vacuum freeze dryer (Nanjing Yanwo Biological Technology Co., Ltd.), TGL16M refrigerated centrifuge (Shanghai Luxiang Instrument Co., Ltd.).

[0377] 1.3 Animals

[0378] SPF grade ICR mice, half male and half female, body weight (20±2) g, purchased from Beijing Vantoll Life-Science Experimental Animals Technology Co., Ltd.

[0379] 2. Methods and results

[0380] 2.1 Grouping

[0381] Randomized grouping (n=8), the specific grouping scheme is as follows:

[0382] Bone cancer pain group: establish bone cancer pain animal model (see "experimental method" part for specific process), and give intragastrically 0.5% carboxymethylcellulose sodium solution in normal saline.

[0383] Sham operation group: the difference from the bone cancer pain group is that the tumor cells are injected in situ instead of the same volume of normal saline, and the rest are the same.

[0384] Bone cancer pain + ibuprofen group: establish bone cancer pain animal model, and give intragastrically ibuprofen (daily dose of 30 mg / kg mouse body weight).

[0385] Bone cancer pain + amitriptyline tramadol group: establish bone cancer pain animal model, and give intragastrically amitriptyline tramadol (daily dose of 30 mg / kg mouse body weight).

[0386] Example 1 group: establish bone cancer pain animal model, and give intragastrically the ericamiamide alkaloids provided in example 1 (daily dose of 30 mg / kg mouse body weight).

[0387] Example 1 + analgesic drugs 1-3 group: establish bone cancer pain animal model, and give intragastrically the ericamiamide alkaloids provided in example 1 and analgesic drugs 1, 2 and 3 provided in example 20 (total daily dose of 30 mg / kg mouse body weight, the mass ratio of the ericamiamide alkaloids provided in example 1 and analgesic drugs 1, 2 and 3 provided in example 20 is 100:3:3.6:14).

[0388] Analgesic drugs 1-3 group: establish bone cancer pain animal model, and give intragastrically analgesic drugs 1, 2 and 3 provided in example 20 (total daily dose of 30 mg / kg mouse body weight, the mass ratio of analgesic drugs 1, 2 and 3 provided in example 20 is 3:3.6:14).

[0389] Analgesic drug 4 group: establish bone cancer pain animal model, and give intragastrically analgesic drug 4 provided in example 20 (daily dose of 30 mg / kg mouse body weight).

[0390] The base solution used in each intragastric administration group is 0.5% carboxymethylcellulose sodium solution in normal saline.

[0391] 2.2 Experimental method

[0392] Bone cancer model: use intelligent hot plate instrument as heat source, adjust temperature to control at 55±0.5, and select mice with pain threshold of 5-30s.

[0393] The method for measuring the pain threshold of mice by the hot plate method is as follows:

[0394] The mice were randomly divided into groups after modeling of bone cancer, placed on a constant temperature hot plate, measured continuously for 3 times, each time interval was not less than 15 min, and the time from the mouse being placed on the hot plate to jumping or licking the foot was recorded, and the average of 3 times was taken as the basic pain threshold of the mouse. Continuous administration for 7 days, once a day, after the last administration, the pain threshold of the mice was measured at 30, 45 and 60 min; if there was still no response after 60 s, the mice were taken out to avoid burns, and the pain threshold was calculated as 60 s.

[0395] After screening, the mice were anesthetized in a supine position, the left hind limb was disinfected after shaving, a 0.5-1.0 cm long skin incision was made at the knee joint along the direction of the rectus femoris tendon, the patella was carefully exposed, a disposable sterile 1 mL syringe needle was used to puncture and punch the patella along the long axis of the femur, then a 10 μL syringe was replaced to enter the femoral bone marrow cavity, and 10 μL of solution containing Lewis cells (3 x 10 5 After injection, the mice were sutured and disinfected, and then placed back in the cage after waking up.

[0396] The sham operation group was injected with 0.9% normal saline solution, and the rest was the same as the above treatment method.

[0397] Pain threshold improvement rate = (pain threshold after administration - basic pain threshold) / basic pain threshold x 100%

[0398] Data processing: Graphpad Prism 9.0 software was used for data processing in this study, and the data was represented by P<0.05 indicates that the difference is statistically significant.

[0399] 2.3 Experimental results

[0400] The results of the pain threshold improvement rate of the bone cancer pain model mice are as follows: n=8):

[0401]

[0402]

[0403] (Note: compared with the M group, *P<0.05, **P<0.01, ***P<0.001)

[0404] The results showed that, compared with the sham-operated group, bone cancer-induced pain hypersensitivity in mice was reduced, and the baseline pain threshold was lowered. Under the condition of no drug interference, the pain threshold did not change significantly, indicating that the model can evaluate the effect of drugs on cancer pain. As shown in the table above, ibuprofen has almost no analgesic effect on pain hypersensitivity induced by bone cancer. This indicates that non-opioid drugs are ineffective in relieving pain induced by bone cancer in mice. Weak opioids can significantly increase the pain threshold in mice and have a significant analgesic effect (P<0.01). The analgesic effect of pine amide alkaloids decreased after knocking out the three components, pine amide and pine amide, but they increased the pain threshold in mice with bone cancer (P<0.05). The analgesic effect of pine amide alkaloids was significantly increased when the three active ingredients were added (P<0.001). The three active ingredients also had a certain analgesic effect when used alone (P<0.01). This indicates that pine amide alkaloids mainly exert their significant analgesic effect through the combined action of these three active ingredients and other amide alkaloids with relatively low content.

[0405] 3. Conclusion

[0406] The amide alkaloids of *Pyrrosintenamide* have a significant analgesic effect on cancer pain. They work through alkaloids such as *Pyrrosintenamide*, *Pyrrosintenamide*, and other amide alkaloids. The analgesic effect is rapid onset, strong, and has a prominent sustained analgesic effect, making them of significant value.

[0407] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing acetamide alkaloids, characterized in that, Includes the following steps: S1. Mix the photinia serratifolia with an ethanol-water solution, reflux and extract to obtain the extract; S2. Mix the extract obtained in step S1 with macroporous adsorption resin, stir, and concentrate under reduced pressure until there is no alcohol odor to obtain the sample mixture. S3. Load the sample mixture obtained in step S2, let it stand for 15-30 minutes, let it flow out, elute, and obtain the eluent. S4. Concentrate the eluent obtained in step S3 under reduced pressure and dry it to obtain senna amide alkaloids. The total alkaloids in the *Sedum sarmentosum* alkaloids contain 50.03%-55.58% by mass, the *Sedum sarmentosum* alkaloids contain 22.28‰-31.10‰ by mass, and the *Sedum sarmentosum* alkaloids contain 9.18%-13.95% by mass. In step S1, the concentration of the ethanol-water solution is 70%-80%, and the mass ratio of the *Photinia serratifolia* to the ethanol-water solution is 1:10-20; the reflux extraction is performed 1-3 times, with each extraction lasting 1-4 hours. In step S2, the macroporous adsorption resin is selected from HP-20, D101, S-8, NKA-9, DA201 and HP2MGL; in step S2, the vacuum concentration is carried out at 40-60°C. The weight ratio of the *Photinia serratifolia* in step S1 to the dry weight ratio of the macroporous adsorption resin in step S2 is 1:0.4-1.2; In step S3, the elution includes the following steps: S31. Elute with water at a flow rate of 2-4 BV / h for 3-5 BV to obtain the water wash solution; S32. Elute with 25%-30% ethanol aqueous solution at a flow rate of 2-4 BV / h for 3-5 BV to obtain the alcohol-washed fraction. S33. Elute 9-12 BV with 70%-80% ethanol aqueous solution at a flow rate of 2-4 BV / h to obtain eluent; In step S4, the vacuum concentration is carried out at 40-60°C, and the drying is freeze drying.

2. The preparation method according to claim 1, characterized in that, The UPLC fingerprint of the *Syngonium styracifolium* alkaloids includes fingerprint peaks 1, 6, 7, 9, and 11. Fingerprint peak 1 is located at 3.90-4.10 min, fingerprint peak 6 at 12.45-12.55 min, fingerprint peak 7 at 12.80-12.95 min, fingerprint peak 9 at 13.10-13.25 min, and fingerprint peak 11 at 15.30-15.45 min.

3. The preparation method according to claim 2, characterized in that, The chromatographic conditions for the UPLC include: Column: C 18 Chromatographic column; Using water as mobile phase A and acetonitrile as mobile phase B, the gradient elution scheme is as follows: The column temperature was 35℃, the flow rate was 0.8 mL / min, and the detection wavelength was 260 nm.

4. The preparation method according to claim 2, characterized in that, The UPLC fingerprint of the *Syngonium styracifolium* alkaloids also includes fingerprint peaks 2, 3, 4, 5, 8, and 10. Fingerprint peak 2 is located at 5.85-6.05 min, fingerprint peak 3 at 8.85-8.95 min, fingerprint peak 4 at 9.30-9.50 min, fingerprint peak 5 at 12.15-12.30 min, fingerprint peak 8 at 13.00-13.05 min, and fingerprint peak 10 at 13.90-14.00 min.

5. The preparation method according to claim 2, characterized in that, The component corresponding to fingerprint peak 1 is purpuric diamide; The component corresponding to fingerprint peak 6 is fentanyl amide; The component corresponding to fingerprint peak 7 is Pipersintenamide; The component corresponding to fingerprint peak 9 is wallwort alkaloid; The component corresponding to fingerprint peak 11 is (2E,4E)-N-(2-methylpropyl)-dodecyl-2,4-dieneamide.

6. The preparation method according to claim 2, characterized in that, The ratio of the peak areas of fingerprint peak 1, fingerprint peak 6, fingerprint peak 7, fingerprint peak 9 and fingerprint peak 11 is 1.435-1.868: 1.053-1.470: 1.505-1.986: 10.950-16.640: 0.975-1.

040.

7. The alkaloids of the sennae amide class prepared by the preparation method according to any one of claims 1-6.

8. The use of the senna-based alkaloids prepared by the method according to any one of claims 1-6 in the preparation of analgesic products.

9. An analgesic drug, characterized in that, The active ingredient includes the senna-type alkaloids prepared by the preparation method described in any one of claims 1-6.

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