A purification process for compound Muniziqi macroporous resin

Abstract

This invention discloses a macroporous resin purification method for Compound Muniziqi Formula. The method includes the following steps: (1) macroporous resin pretreatment; (2) column packing; (3) sample loading; and (4) obtaining the total alkaloid fraction and / or the total saponin fraction; optionally, (5) obtaining the total flavonoid fraction. This invention utilizes the macroporous resin method to establish a process for simultaneously enriching alkaloids, flavonoids, and saponins in Compound Muniziqi Formula, and simultaneously preliminarily separating the total extract, total alkaloids, total flavonoids, and / or total saponin fraction; on the other hand, in pharmacodynamic studies, the anti-inflammatory and antioxidant activities of Compound Muniziqi Formula and its different fractions in vivo and in vitro are studied using cell models, thereby elucidating the pharmacodynamic material basis of Compound Muniziqi Formula and its alkaloids, flavonoids, and saponins, laying the foundation for the further development and expansion of the indications of Compound Muniziqi Formula.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a macroporous resin purification process of compound Fumiziqi recipe. BACKGROUND

[0002] The compound Fumiziqi recipe (FFMN) is derived from the famous medical book of Uygur nationality, "Kajibadingkadel", and is composed of 13 medicinal materials, i.e. Peganum harmala, Nigella glandulifera, Pimpinella anisum, Foeniculum vulgare, Matricaria recutita, Apium graveolen, Cichorium intybus, Cichorium intybus, Cymbopogon citratus, Dracocephalum moldavica, Glycyrrhizauralensis, Ocimum basilicum and Althaea rosea.

[0003] Macroporous adsorption resin is a kind of high polymer material. The structure of macroporous resin is porous and three-dimensional, and has selective adsorption function. Its advantages are relatively simple operation, small environmental pollution, good selectivity for different components, low use cost and renewable and reusable, etc. The main skeleton of macroporous resin is acrylate or styrene, and according to the different groups polymerized on the different skeletons, it is generally divided into three types of non-polar, weakly polar and polar. At present, macroporous resin with basic skeleton of styrene has been widely used in the separation and purification of active components of traditional Chinese medicine such as flavonoids, saponins and alkaloids. Macroporous resin mainly separates and purifies components according to the difference in polarity and the size of the pore diameter. Generally speaking, for the same resin, when the molecular weight of the separated components is close, the resin has strong adsorption force for components with small polarity and is not easy to be eluted; when the polarity of the separated components is similar, the components with small molecular weight are more easily adsorbed on the resin. At the same time, the appropriate sample loading flow rate also has a certain influence on the adsorption effect of macroporous resin.

[0004] There is no method for simultaneously enriching alkaloids, flavonoids and saponins in the compound Fumiziqi recipe (FFMN) in the prior art. SUMMARY

[0005] Based on this, the present application provides a macroporous resin purification process of compound Fumiziqi recipe, which comprises the following steps:

[0006] (1) Pretreatment of macroporous resin: the macroporous resin is sequentially soaked in an ethanol solution, washed and soaked in a hydrochloric acid solution, washed and soaked in a sodium hydroxide solution, and washed with water until the pH is neutral, to obtain pretreated macroporous resin;

[0007] (2) Column packing: the pretreated macroporous resin is packed into a column by wet method to obtain a first macroporous resin adsorption column and / or a second macroporous resin adsorption column;

[0008] (3) Sample loading: a proper amount of medicinal material is precisely weighed, sieved, and extracted by reflux extraction after immersion, and the filtrate is combined after filtration, concentrated under reduced pressure until there is no alcohol taste, and then water is added to a certain concentration of crude drug, and the supernatant is obtained by centrifugation, the sample loading solution is obtained, and the sample loading solution is passed through the first macroporous resin adsorption column at a certain flow rate so that the sample loading solution is adsorbed on the first macroporous resin adsorption column, and optionally, the unabsorbed sample loading solution is collected to obtain a leakage solution; and

[0009] (4) Obtaining total alkaloid fraction and / or total saponin fraction: after impurities are removed by passing a certain volume of 5%-15% ethanol through the first macroporous resin adsorption column at a certain flow rate, 25%-35% ethanol, 35%-45% ethanol and 65%-75% ethanol are sequentially used for elution at a certain flow rate, and the eluate is collected to obtain the total alkaloid fraction and / or the total saponin fraction;

[0010] Optionally, (5) obtaining total flavone fraction: the supernatant is passed through the second macroporous resin adsorption column at a certain flow rate after centrifugation so that the supernatant is adsorbed on the second macroporous resin adsorption column, and after impurities are removed by passing a certain volume of 5%-15% ethanol through the second macroporous resin adsorption column at a certain flow rate, 25%-35% ethanol is used for elution at a certain flow rate, and the eluate is collected to obtain the total flavone fraction.

[0011] Further, in step (1), the macroporous resin is H20 resin, H60 resin, HPD100 resin, LSA-5B resin, D101 resin or AB-8 resin.

[0012] Further, the model of the macroporous resin is D101 resin or H20 resin.

[0013] Further, the concentration of the ethanol solution is about 95%, and the soaking time of the ethanol solution is about 24 hours.

[0014] Further, the volume of the hydrochloric acid solution is about 2BV, the concentration of the hydrochloric acid solution is about 5%, and the soaking time of the hydrochloric acid solution is about 2 hours.

[0015] Further, the volume of the sodium hydroxide solution is about 2BV, the concentration of the sodium hydroxide solution is about 2%, and the soaking time of the sodium hydroxide solution is about 2 hours.

[0016] Further, the water is ultrapure water.

[0017] Further, in step (3), the medicinal materials include Khus-Khus seed, Nigella seed, Anise fruit, Anise root-bark, Chamomile, Celery root, Chicory seed, Chicory root, Citronella, Siberian Cockcomb seed, Licorice, Basil seed, and Hollyhock seed.

[0018] Further, the sieve is No. 1 sieve.

[0019] Further, the extraction is carried out by adding about 5 times the mass of the medicinal materials with about 70% ethanol.

[0020] Further, the extraction is carried out by adding about 8 times the mass of the medicinal materials with about 70% ethanol.

[0021] Further, the extraction is carried out for 2 times, each for about 1 hour.

[0022] Further, the concentration of the crude drug is about 0.3 g / mL.

[0023] Further, the centrifugation is carried out at a speed of 7000 r / min to 9000 r / min for 5 min to 15 min.

[0024] Further, the centrifugation is carried out at a speed of about 8000 r / min for about 10 min.

[0025] Further, the pH of the sample solution is 3 to 7.

[0026] Further, the pH of the sample solution is about 5.

[0027] Further, the mass concentration of the sample solution is 0.15 g / mL to 0.5 g / mL.

[0028] Further, the mass concentration of the sample solution is 0.3 g / mL.

[0029] Further, the flow rate of the sample solution is about 3 BV / h.

[0030] Further, the volume of the sample solution is 10 BV to 15 BV, for example, about 11 BV or about 14 BV.

[0031] Further, step (4) is replaced by the following step (4'): after impurities are removed by using a certain volume of 5% to 15% ethanol at a certain flow rate through the first macroporous resin adsorption column, elution is carried out by using 65% to 75% ethanol, and the eluate is collected to obtain the total extract of the compound Wuni Zhiqi recipe (i.e., total fraction).

[0032] Furthermore, in step (4) or step (4') or step (5), the flow rate is approximately 3 BV / h.

[0033] Furthermore, prior to this impurity removal, water of 3BV to 5BV, for example, about 4BV, is passed through the first macroporous resin adsorption column or the second macroporous resin adsorption column at a flow rate of about 3BV / h.

[0034] Furthermore, the concentration of ethanol in the impurity removal process is approximately 10%.

[0035] Furthermore, the volume of the ethanol in the impurity removal process is approximately 5 BV.

[0036] Further, elution was performed using approximately 30% ethanol, approximately 40% ethanol, and approximately 70% ethanol at a certain flow rate, and the eluent was collected to obtain the total alkaloid fraction and / or the total saponin fraction.

[0037] Furthermore, the fourth to tenth BVs of the eluent containing approximately 30% ethanol and the first BV of the eluent containing approximately 40% ethanol were combined as the total alkaloid fraction.

[0038] Furthermore, the fourth to ninth batches of the eluent containing approximately 40% ethanol and the first to third batches of the eluent containing approximately 70% ethanol were combined as the total saponin fraction.

[0039] Further, elution was performed using approximately 5 BV of approximately 30% ethanol, and the eluent was collected to obtain the total flavonoid fraction.

[0040] Further, elution was performed using approximately 5 BV of approximately 70% ethanol, and the eluent was collected to obtain the total extract of Compound Muniziqi Formula.

[0041] Furthermore, the centrifuge speed is 7000 r / min to 9000 r / min, and the centrifuge time is 5 min to 15 min.

[0042] Furthermore, the centrifuge speed is approximately 8000 r / min, and the centrifugation time is approximately 10 min.

[0043] According to another aspect of the present invention, a total alkaloid fraction prepared by the above method is provided.

[0044] According to another aspect of the present invention, a total saponin fraction prepared by the above method is provided.

[0045] According to another aspect of the present invention, a total flavonoid fraction prepared by the above method is provided.

[0046] According to another aspect of the present invention, a total extract of compound Muniziqi formula prepared by the above method is provided.

[0047] According to another aspect of the present invention, a pharmaceutical composition comprising the above-described total alkaloid fraction, the above-described total saponin fraction, and / or the above-described total flavonoid fraction is provided.

[0048] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0049] Furthermore, the excipient is selected from one or more of the following: dispersants, wetting agents, binders, diluents, retention aids, lubricants, slow-release agents, plasticizers, disintegrants, encapsulating agents, flavoring agents, light-blocking agents, and antioxidants.

[0050] Furthermore, the dosage form of the pharmaceutical composition is tablets, pills, capsules, powders, injections, films, lozenges, granules, or oral liquids.

[0051] The beneficial effects of this invention are:

[0052] This invention utilizes a macroporous resin method to establish a process for simultaneously enriching alkaloids, flavonoids, and saponins in Compound Muniziqi Formula, while also preliminarily separating total alkaloids, total flavonoids, and total saponins. Furthermore, in pharmacodynamic studies, cell models are used to investigate the anti-inflammatory and antioxidant activities of Compound Muniziqi Formula and its different fractions in vitro and in vivo, thereby elucidating the pharmacodynamic material basis of the anti-inflammatory and antioxidant effects of Compound Muniziqi Formula and its alkaloids, flavonoids, and saponins, laying the foundation for further development and expansion of the indications for Compound Muniziqi Formula. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0054] Figure 1 This is a schematic diagram of the leakage curves for HAL and total flavonoids. Where A: HAL leakage rate; B: total flavonoid (FLA) leakage rate.

[0055] Figure 2 This diagram illustrates the amount of solids removed by ultrapure water elution with different column volumes.

[0056] Figure 3 This is a schematic diagram of gradient elution curves for different ethanol concentrations. Where A represents the elution amount of total alkaloids (ALK); B represents the elution amount of total saponins (SAP); and C represents the elution amount of total flavonoids (FLA).

[0057] Figure 4This is a schematic diagram of the elution curves for 70% ethanol. Where A: elution curves for each component; B: cumulative elution rate for each component.

[0058] Figure 5 A flowchart for separating the ALK, FLA and SAP components of Compound Muniziqi Formula (FFMN).

[0059] Figure 6 This is a schematic diagram showing the effects of different components of Compound Muniziqi Formula (FFMN) on RAW264.7 cells.

[0060] Figure 7 This is a schematic diagram showing the effects of different components of Compound Muniziqi Formula (FFMN) on NO production in an LPS-induced RAW264.7 cell inflammation model. #### Compared with the control group, P<0.0001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

[0061] Figure 8 This is a schematic diagram showing the effects of different concentrations of H2O2 on PC12 cells.

[0062] Figure 9 This is a schematic diagram illustrating the effects of different components of Compound Muniziqi Formula (FFMN) on a H2O2-induced oxidative damage model in PC12 cells. Among them, #### Compared with the control group, P<0.0001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001; $$$ Compared with EXT, P < 0.0001 and $$$$ P < 0.00001.

[0063] Figure 10 This is a schematic diagram showing the effects of different components of Compound Muniziqi Formula (FFMN) on SOD activity. Among them, #### Compared with the control group, P<0.0001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001; $ Compared with EXT, P < 0.05 and $$$$ P < 0.00001. Detailed Implementation

[0064] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0065] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0066] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0067] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0068] In this invention, the terms "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "has," "contains," or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0069] As described in the background section, there is a lack of existing methods for simultaneously enriching alkaloids, flavonoids, and saponins in Compound Muniziqi Formula (FFMN). To address this issue, the present invention provides a macroporous resin purification method for Compound Muniziqi Formula, comprising the following steps:

[0070] (1) Macroporous resin pretreatment: The macroporous resin was soaked in ethanol solution, rinsed and soaked in hydrochloric acid solution, rinsed and soaked in sodium hydroxide solution, and rinsed with water until the pH was neutral to obtain pretreated macroporous resin.

[0071] (2) Column packing: The pretreated macroporous resin is wet-packed into a column to obtain a first macroporous resin adsorption column and / or a second macroporous resin adsorption column.

[0072] (3) Sample loading: Accurately weigh an appropriate amount of medicinal material, sieve, extract by reflux, filter, combine the filtrates, concentrate under reduced pressure until no alcohol odor remains, add water to a certain concentration of raw medicinal material, centrifuge and collect the supernatant to obtain a macroporous resin loading solution. Pass the loading solution through the first macroporous resin adsorption column at a certain flow rate so that the loading solution is adsorbed onto the first macroporous resin adsorption column. Optionally, collect the unadsorbed loading solution to obtain the leakage liquid; and

[0073] (4) Obtaining the total alkaloid fraction and / or the total saponin fraction: After removing impurities by passing a certain volume of 5% to 15% ethanol through the first macroporous resin adsorption column at a certain flow rate, elute by sequentially using 25% to 35% ethanol, 35% to 45% ethanol and 65% to 75% ethanol at a certain flow rate, collect the eluent to obtain the total alkaloid fraction and / or the total saponin fraction.

[0074] Optionally, (5) to obtain the total flavonoid fraction: After centrifuging the leaked liquid, take the supernatant and pass it through the second macroporous resin adsorption column at a certain flow rate so that the supernatant is adsorbed on the second macroporous resin adsorption column. After removing impurities by passing a certain volume of 5% to 15% ethanol through the second macroporous resin adsorption column at a certain flow rate, elute with 25% to 35% ethanol at a certain flow rate, collect the eluent, and obtain the total flavonoid fraction.

[0075] In this invention, when concentration, volume, time, rotation speed, pH, flow rate, pressure, proportion, equivalent, concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range "5 to 15" is disclosed, the described range should be interpreted as including ranges "5 to 15", "5 to 14", "5 to 13", "5 to 12", "5 to 11", "5 to 10", "5 to 9", "5 to 8", "5 to 7", "5 to 6", "6 to 15", "6 to 14", "6 to 13", "6 to 12", "6 to 11", "6 to 10", "6 to 9", "6 to 8", "6 to 7", "7 to 15", etc. When a range of values ​​is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range, and the technical effects of the present invention can be achieved within the aforementioned range.

[0076] Flavonoids, alkaloids, and saponins are the three main components of FFMN, forming the pharmacological basis for its therapeutic effects. Therefore, selecting appropriate methods to enrich these three components is fundamental to the secondary development of FFMN and is of great significance for promoting the secondary development of major Uyghur medicines and the research and development and industrial development of drugs in Xinjiang.

[0077] Although flavonoids are the most abundant component in FFMN as determined by UPLC-Q-TOF-MS, the analysis of the FFMN ethanol extract revealed low levels of monomeric flavonoids, making it difficult to quantify individual monomers. Therefore, a suitable method for enriching flavonoids is needed. Polyamide resin was initially considered as an enrichment method due to its strong adsorption properties for flavonoids, primarily through hydrogen bonding and van der Waals forces. Polyamide resin's advantage lies in its specific enrichment effect on flavonoids and its good removal of some non-flavonoid components. However, the research objective of this project is not only to enrich flavonoids; alkaloids and saponins in FFMN are also important "pharmacologically active ingredient groups" and cannot be discarded. Therefore, polyamide resin was not used to enrich total flavonoids. Instead, a macroporous resin with good enrichment effects on alkaloids, flavonoids, and saponins was selected to enrich the FFMN ethanol extract.

[0078] The acid-base precipitation method was also investigated as an enrichment method. This method has a good enrichment effect on alkaloids. An appropriate pH is beneficial to the hydrolysis of components, making them easier to be adsorbed by the resin and absorbed by the body. However, excessive acid can destroy the core structure of flavonoids and saponins, which is not conducive to the enrichment of flavonoids and saponins. Finally, a macroporous resin that has a good enrichment effect on flavonoids, saponins and alkaloids was selected.

[0079] In a preferred embodiment, in step (1), the macroporous resin is H20 resin, H60 resin, HPD100 resin, LSA-5B resin, D101 resin or AB-8 resin.

[0080] In a preferred embodiment, the macroporous resin is either D101 resin or H20 resin.

[0081] In a preferred embodiment, the concentration of the ethanol solution is about 95%, and the soaking time of the ethanol solution is about 24 hours.

[0082] In a preferred embodiment, the hydrochloric acid solution has a volume of about 2 BV, a concentration of about 5%, and a soaking time of about 2 hours.

[0083] In a preferred embodiment, the volume of the sodium hydroxide solution is about 2 BV, the concentration of the sodium hydroxide solution is about 2%, and the soaking time of the sodium hydroxide solution is about 2 hours.

[0084] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 95" includes ±5% of 95, or from 90.25 to 99.75; "about 24" includes ±5% of 24, or from 22.8 to 25.2; "about 2" includes ±5% of 2, or from 1.9 to 2.1; and "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0085] In a preferred embodiment, the water is ultrapure water.

[0086] In a preferred embodiment, in step (3), the medicinal materials include camel husk, black cumin seed, fennel fruit, fennel root bark, chamomile, celery root, chicory seed, chicory root, lemongrass, fragrant blue clover seed, licorice, basil seed and hollyhock seed.

[0087] In a preferred embodiment, the sieve is sieve No. 1.

[0088] In a preferred embodiment, the extraction is performed by adding about 70% ethanol, approximately 5 times the weight of the medicinal material, to extract the medicinal material.

[0089] In a preferred embodiment, the reflux extraction is performed by adding approximately 70% ethanol (approximately 8 times the weight of the medicinal material) to reflux the medicinal material.

[0090] In a preferred embodiment, the reflux extraction is performed twice, each time for approximately one hour.

[0091] In a preferred embodiment, the concentration of the crude drug is about 0.3 g / mL.

[0092] In a preferred embodiment, the centrifugation speed is 7000 r / min to 9000 r / min, and the centrifugation time is 5 min to 15 min.

[0093] In a preferred embodiment, the centrifugation speed is about 8000 r / min and the centrifugation time is about 10 min.

[0094] In a preferred embodiment, the pH of the loading solution is 3 to 7.

[0095] In a preferred embodiment, the pH of the loading solution is about 5.

[0096] The alkaloids, flavonoids, and saponins in FFMN exhibit certain differences in properties, which is particularly evident during pH testing. Similar to the results of the acid-extraction-alkali-precipitation method, the alkaloids in FFMN readily form a free state under weakly alkaline conditions, making them more easily adsorbed by the resin. Conversely, the flavonoids and saponins are easily hydrolyzed under acidic conditions, forming a free state that is also easily adsorbed by the resin. Therefore, to maximize the simultaneous enrichment of these three types of components, a trade-off must be made. Ultimately, after pH testing, pH=5 (i.e., the pH of the original solution) was selected. This ensures the maximum simultaneous enrichment of the three types of components without introducing other substances, reducing interference and facilitating subsequent industrial production.

[0097] In a preferred embodiment, the mass concentration of the loading solution is 0.15 g / mL to 0.5 g / mL.

[0098] In a preferred embodiment, the mass concentration of the loading solution is 0.3 g / mL.

[0099] In a preferred embodiment, the flow rate of the loading solution is approximately 3 BV / h.

[0100] In a preferred embodiment, the volume of the loading solution is 10 BV to 15 BV, for example, about 11 BV or about 14 BV.

[0101] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, “about 5” includes 5 ± 5%, or from 4.75 to 5.25; “about 70” includes 70 ± 5%, or from 66.5 to 73.5; “about 8” includes 8 ± 5%, or from 7.6 to 8.4; “about 1” includes 1 ± 5%, or from 0.95 to 1.05; “about 0.3” includes 0.3 ± 5%, or from 0.285 to 0.315; “about 8000” includes 8000 ± 5%, or from 7600 to 8400; “about 10” includes 10 ± 5%, or from 9.5 to 10.5; “about 3” includes 3 ± 5%, or from 2.85 to 3.15; “about 11” includes 11 ± 5%, or from 10.45 to 11.55; and “about 14” includes 14 ± 5%, or from 13.3 to 14.7.

[0102] In a preferred embodiment, step (4) is replaced with the following step (4'): after removing impurities by passing a certain volume of 5% to 15% ethanol through the first macroporous resin adsorption column at a certain flow rate, elution is performed by 65% ​​to 75% ethanol, and the eluent is collected to obtain the total extract of Compound Muniziqi Formula.

[0103] In a preferred embodiment, in step (4), step (4'), or step (5), the flow rate is approximately 3 BV / h.

[0104] In a preferred embodiment, prior to the impurity removal, 3BV to 5BV, for example, about 4BV, of water is passed through the first macroporous resin adsorption column or the second macroporous resin adsorption column at a flow rate of about 3BV / h.

[0105] In a preferred embodiment, the concentration of ethanol in the impurity removal process is about 10%.

[0106] In a preferred embodiment, the volume of ethanol in the impurity removal process is approximately 5 BV.

[0107] In a preferred embodiment, elution is performed using about 30% ethanol, about 40% ethanol, and about 70% ethanol at a certain flow rate, and the eluent is collected to obtain the total alkaloid fraction and / or the total saponin fraction.

[0108] In a preferred embodiment, the fourth to tenth BVs of the eluent containing about 30% ethanol and the first BV of the eluent containing about 40% ethanol are combined as the total alkaloid fraction.

[0109] In a preferred embodiment, the fourth to ninth batches of the eluent containing about 40% ethanol and the first to third batches of the eluent containing about 70% ethanol are combined as the total saponin fraction.

[0110] In a preferred embodiment, elution is performed using about 5 BV of about 30% ethanol, and the eluent is collected to obtain the total flavonoid fraction.

[0111] In a preferred embodiment, elution is performed using about 5 BV of about 70% ethanol, and the eluent is collected to obtain the total extract of Compound Muniziqi Formula.

[0112] In a preferred embodiment, the centrifugation speed is 7000 r / min to 9000 r / min, and the centrifugation time is 5 min to 15 min.

[0113] In a preferred embodiment, the centrifugation speed is about 8000 r / min and the centrifugation time is about 10 min.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 30" includes ±5% of 30, or from 28.5 to 31.5; "about 40" includes ±5% of 40, or from 38 to 42; "about 70" includes ±5% of 70, or from 66.5 to 73.5; "about 8000" includes ±5% of 8000, or from 7600 to 8400.

[0115] According to another aspect of the present invention, a total alkaloid fraction prepared by the above method is provided.

[0116] According to another aspect of the present invention, a total saponin fraction prepared by the above method is provided.

[0117] According to another aspect of the present invention, a total flavonoid fraction prepared by the above method is provided.

[0118] According to another aspect of the present invention, a total extract of compound Muniziqi formula prepared by the above method is provided.

[0119] According to another aspect of the present invention, a pharmaceutical composition comprising the above-described total alkaloid fraction, the above-described total saponin fraction, and / or the above-described total flavonoid fraction is provided.

[0120] In a preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0121] In a preferred embodiment, the pharmaceutical formulation of the present invention contains at least one pharmaceutically acceptable excipient in an amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.% relative to the weight of the pharmaceutical formulation.

[0122] In this invention, the term "pharmaceutically acceptable" means a substance, such as a carrier or diluent, that does not destroy the biological activity or properties of a compound and is relatively non-toxic, such that, when administered to an individual, it will not cause unwanted biological effects or interact with any of its constituent components in a harmful manner.

[0123] In this invention, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0124] In a preferred embodiment, the excipient is selected from one or more of the following: dispersants, wetting agents, binders, diluents, retention aids, lubricants, slow-release agents, plasticizers, disintegrants, encapsulating agents, flavoring agents, light-blocking agents, and antioxidants.

[0125] Those skilled in the art will know how to select specific chemical substances within the aforementioned excipient categories. For example, the dispersant may be selected from one or more of the following: croscarmellose sodium, starch glycolate sodium, and pregelatinized corn starch. The diluent may be selected from one or more of the following: powdered sugar, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate, and calcium carbonate. The sustained-release agent may be selected from one or more of the following: sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, hydroxyethyl cellulose, gum arabic, gelatin, and shellac. The wetting agent may be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, polysorbate esters such as polysorbate 80, cetyl alcohol, glyceryl fatty acid esters (such as triacetin, glyceryl monostearate and the like), polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, sodium lauryl sulfate, sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene ethers, benzalkonium chloride, polyoxyethylene castor oil, and sodium docusate. The flavoring agent may be selected from one or more of the following: sorbitol, glucose, mannose, sucrose, and lactose. The lubricant may be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid, and colloidal silica. The binder may be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol, and methylcellulose. The disintegrant may be selected from one or more of the following: carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose. The plasticizer may be dibutyl sebacate and / or various citrate esters. The antioxidant may be selected from one or more of the following: sodium bisulfite, sodium metabisulfite, sodium sulfite, and sodium thiosulfate.

[0126] These excipients are preferably drug-inert, or may have synergistic or enhancing effects to enhance the therapeutic activity of the drug composition. The above excipients are merely examples, and the excipients actually used in this invention are not limited to the above excipients. They can be adjusted according to the actual situation to achieve the effects of this invention.

[0127] In a preferred embodiment, the dosage form of the pharmaceutical composition is tablets, pills, capsules, powders, injections, films, lozenges, granules, or oral liquids.

[0128] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0130] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0131] Example

[0132] 1. Experimental apparatus

[0133] Agilent 1260 HPLC system, Agilent 1260 Infinity evaporative light scattering detector (Agilent Technologies, USA); Milli-Q pure water system (Millipore, MA, USA); Satorius BSA 124S-CW Electronic Analytical Balance (Beijing Sartorius Instrument Systems Co., Ltd.); TU-1901 Double-Beam UV-Vis Spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.); DHG-9053A Electric Heating Drying Oven, HWS26 Electric Heating Constant Temperature Water Bath (Shanghai-Heng Scientific Instruments Co., Ltd.); KQ-250DB CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); SHB-1000 Circulating Water Multipurpose Vacuum Pump (Shanghai Chengxian Instrument Equipment Co., Ltd.); Haier Medical 4℃ Refrigerator, Haier BC / BD-429H-20℃ Electric Freezer (Qingdao Haier Co., Ltd.); Electric Heating Constant Temperature Shaking Water Bath (Shanghai Jinghong Experimental Equipment Co., Ltd.); N-1000 Rotary Evaporator, OSB-2200 Water Bath, CCA-1112A Cooling Water Circulation Device. Micromass Quattro PremierXE tandem triple quadrupole mass spectrometer (Waters, Manchester, UK); ACQUITY UPLC HSS T3 column; CO2 cell incubator (Millipore, Germany); VARIOSKAN FLASH microplate reader (Thermo, USA); Olympus CKX41 microscope (Olympus, Japan); Thermo biosafety cabinet; SCIENTZ-IID ultrasonic cell disruptor; Haier DW-86L726G cryogenic storage box (Qingdao Haier Co., Ltd.).

[0134] 2. Experimental reagents and materials

[0135] Reference standards: HAL, HAR, rutin, and Sie.A were all obtained from the Shanghai Research Center for Standardization of Traditional Chinese Medicine (purity ≥98%); Glycyrrhizic acid (GA) was purchased from Chengdu Desite Biotechnology Co., Ltd. (batch number DST210620-060, purity ≥98%); Samples: HAL seeds (batch number Y2001032), fennel seeds (batch number Y1912001), fennel root bark (batch number Y2005056), and black cumin seeds (batch number Y200...). The following ingredients were supplied by Xinjiang Uygur Pharmaceutical Co., Ltd., and were identified as genuine products by Professor Wang Changhong of Shanghai University of Traditional Chinese Medicine: 9019), fragrant orchid seeds (batch number Y1911023), hollyhock seeds (batch number Y2003001), chamomile (batch number Y2005054), chicory root (batch number Y2010002), licorice (batch number Y2005050), celery root (batch number Y2009014), chicory seeds (batch number Y2001017), lemongrass (batch number Y2009033), and basil seeds (batch number Y1909015). Acetonitrile and glacial acetic acid were of chromatographic grade; other reagents were of analytical grade; and water was ultrapure water. Macroporous resins H20, H60, HPD100, LSA-5B, D101, and AB-8 were purchased from Tianjin Yunkai Resin Co., Ltd. DMEM (high glucose), RPMI 1640, fetal bovine serum (FBS), penicillin-dextrose antibodies, and phosphate-buffered saline (PBS) were all purchased from Gibco. Dimethyl sulfoxide (DMSO), H2O2, and LPS were all purchased from Sigma-Aldrich. Nitric oxide (NO) and superoxide dismutase (SOD) kits were purchased from Beyotime Biotechnology Co., Ltd. CCK-8 was purchased from Yisheng Biotechnology Co., Ltd. RAW264.7 cells were kindly provided by Professor Wang Shunchun's research group at Shanghai University of Traditional Chinese Medicine. PC12 cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd.

[0136] 3. Experiment on the purification process of compound Muniziqi formula macroporous resin

[0137] 3.1 Experimental Methods

[0138] 3.1.1 Preparation of macroporous resin loading solution

[0139] Weigh out 13 medicinal powders according to the prescription ratio, including 120g each of camel husk seed, fennel root bark, and fennel fruit, and 60g each of the remaining herbs. Pass the powders through a No. 1 sieve, add 5 times the weight of the herbs in 70% ethanol for thorough extraction, then add 8 times the weight of the herbs in 70% ethanol, reflux for 1 hour, and extract twice. Filter and combine the filtrates, concentrate under reduced pressure until no alcohol odor remains, and add ultrapure water to a crude herb concentration of 0.3g / mL. -1 The concentrate was heated at 8000 r·min -1 After centrifugation for 10 minutes, collect the supernatant to obtain the macroporous resin loading solution. Store at -20℃ for later use to prevent the solution from deteriorating.

[0140] 3.1.2 Pretreatment of macroporous resin

[0141] Six resins—H20, H60, HPD100, LSA-5B, D101, and AB-8—were soaked in 95% ethanol for 24 hours. They were then rinsed sequentially with 2 BV of HCl (5%) and NaOH (2%), and soaked for 2 hours each time. Subsequently, they were rinsed with ultrapure water until the pH was neutral. The resins were then stored in anhydrous ethanol for later use. Before use, the ethanol in the resins must be replaced with ultrapure water, and the rinsing solution should be free of any alcohol odor before sample adsorption.

[0142] 3.1.3 Static Adsorption Experiment

[0143] 3.1.3.1 Screening of Macroporous Resin Types

[0144] Add 1g of each of the six treated resins to a 100mL Erlenmeyer flask. Add 30mL of sample aqueous solution (0.3g crude drug·mL). -1 The resins were shaken at 30℃ for 24 hours. The weight was measured before shaking and after shaking to make up for any weight loss. The contents of HAL, HAR, GA, and Sie.A in the solution after adsorption equilibrium were determined by HPLC, and the total flavonoid content was determined by UV spectrophotometry. The adsorption rate and adsorption amount were calculated. Subsequently, the six resins after adsorption were added to conical flasks containing 30 mL of 90% ethanol, and shaken at 30℃ for 24 hours. The weight was measured before shaking and after shaking to make up for any weight loss. The supernatant was taken and the contents of each component were determined according to the above method. The desorption rate and desorption amount were calculated.

[0145] Adsorption capacity = (C0-C1)V / m; Adsorption rate = (C0-C1) / C0;

[0146] Desorption capacity = C2V / m; Desorption rate = C2 / (C0-C1).

[0147] Wherein, C0: the concentration of each component in the loading solution; C1: the concentration of each component in the solution after adsorption equilibrium; C2: the concentration of each component in the desorption solution; V: the volume of the adsorption solution and the desorption solution; m: the mass of the macroporous resin.

[0148] The HPLC determination was improved based on the following reference (Wei Yue, Cheng Juanjuan, Cheng Xuemei, et al. Study on quality control method of Uyghur medicine compound Muniziqi granules [J]. Journal of Pharmaceutical Analysis, 2017, 37(10):1799-1809.). The specific method was as follows: the chromatographic column was Diamonsil Plus C 18-A (250×4.6mm, 5μm); Acetonitrile (A)-ammonium acetate buffer (B) was selected as the mobile phase, the column temperature was 30℃, and the gradient elution program was 0–10 min: 19% A, 10–20 min: 19%–30% A, 20–35 min: 30% A; the flow rate was 1 mL·min -1 The ELSD evaporator and drift tube temperatures were both 50°C, the gas flow rate was 1.6, and the Gain value was set to 8.

[0149] The ultraviolet spectrophotometric determination was based on the following reference (Zhao Xiang, Huan Xiaohan, Cheng Xuemei, et al. Optimization of the alcohol extraction process of compound Muniziqi [J]. Chinese Traditional and Herbal Drugs, 2023, 45(03):907-910).

[0150] 3.1.3.2 Effect of pH value of sample loading solution on adsorption rate

[0151] Weigh 1g of D101 resin into a 100mL Erlenmeyer flask, and add 30mL of sample aqueous solution (0.3g crude drug·mL). -1 Adjust the pH to 1, 3, 5 (stock solution) and 9 respectively using 0.1M hydrochloric acid and 0.1M sodium hydroxide, and follow the steps in section "3.1.3.1" to calculate the adsorption rate.

[0152] 3.1.3.3 Effect of sample solution concentration on resin adsorption rate

[0153] In 100mL Erlenmeyer flasks, five portions of 1g of pretreated D101 resin were added, with crude drug concentrations of 0.067, 0.15, 0.3, 0.5, and 1.0g·mL at pH 5, respectively. -1 Take 30 mL of the test solution, shake and adsorb according to the steps in section “3.1.3.1”, determine the content of each component and calculate the static adsorption rate.

[0154] 3.1.4 Dynamic Adsorption Experiment

[0155] 3.1.4.1 Effect of sample loading volumetric flow rate on resin adsorption

[0156] Take approximately 25g (wet weight) of pretreated D101 resin and pack it into a column using a wet packing method (column volume approximately 40mL). Therefore, 1 BV is 40mL. Add 0.3g·mL -1 Sample solutions with pH 5 were prepared at concentrations of 1, 2, and 3 BV·h, respectively. -1 The sample was added to the resin column at a flow rate of 10 mL (1 BV), and a sample was collected for every 40 mL (1 BV). The sample volume and leakage ratio were plotted as the x and y axes to determine the appropriate sample loading flow rate and volume.

[0157] 3.1.4.2. Water washing volume assessment

[0158] The purpose of water washing is to remove large molecular impurities such as proteins and sugars. Therefore, the sample is loaded for adsorption according to the process parameters determined above, and then washed with ultrapure water at 3 BV·h. -1 Elution was performed at a flow rate of 10 BV. Eluent was collected every 2 BV. The mass of solids in the washing solution was determined to ascertain the washing volume.

[0159] 3.1.4.3 Investigation on impurity removal and elution ethanol volume fraction

[0160] After loading the sample according to the determined process parameters, it was eluted with 4 BV of ultrapure water to remove impurities, and then purified with 3 BV·h. -1 To determine the elution flow rate, elution was performed sequentially with 10%, 30%, 50%, 70%, and 95% ethanol (3BV). The eluates of each concentration were combined, and the elution volumes of alkaloids, flavonoids, and saponins were measured. The elution volumes of these three components in the ethanol eluates of different concentrations were calculated. A gradient elution curve was plotted with ethanol concentration on the x-axis and elution volume on the y-axis to determine the optimal elution volume fraction for impurity removal and elution.

[0161] 3.1.4.4 Investigation into the amount of elution ethanol used

[0162] After loading the sample according to the determined process parameters, first remove impurities with 5 BV of 10% ethanol, then elute with 70% ethanol for 5 BV, collecting one sample per 1 BV. Determine the content of alkaloids, flavonoids, and saponins to determine the amount of eluting ethanol to be used.

[0163] 3.1.5 Verification Test

[0164] Following the established process, 25g (wet weight) of pretreated D101 resin was packed into the column. The pH of the FFMN loading solution was 5, and the mass concentration was 0.3g·mL. -1 The loading and elution flow rates were both 3 BV·h. -1 The sample loading volume was 11 BV. After dynamic adsorption, macromolecular impurities were removed with 5 BV of 10% ethanol, and a total of 5 BV of 70% ethanol eluent was collected. The contents of alkaloids, flavonoids and saponins were determined, and the amount of extract was measured in triplicate.

[0165] 3.2 Experimental Results

[0166] 3.2.1 Static Adsorption Experiment

[0167] 3.2.1.1 Determination of Macroporous Resin Type

[0168] Based on the static adsorption data of six resins, D101 and H20 resins showed good adsorption and desorption effects for alkaloids, flavonoids, and saponins. D101 resin exhibited better adsorption and desorption of saponins than H20 resin, and better adsorption of alkaloids and flavonoids, but its desorption was slightly weaker. Considering factory production costs, the lower-priced and widely used D101 resin was selected for subsequent experiments. The static adsorption results are shown in Tables 1 and 2.

[0169] Table 1 Adsorption and desorption rates of different resin types

[0170]

[0171]

[0172] Among them, alkaloids: the sum of the contents of salsaponin and dehydrosalsaponin; saponins: the sum of the contents of glycyrrhizic acid and isoamycin; flavonoids: the total flavonoid content.

[0173] Table 2 Static adsorption and desorption capacities of different resin types

[0174]

[0175] 3.2.1.2 Determination of pH of the sample loading solution

[0176] At lower pH values, D101 resin exhibits good adsorption for flavonoids and saponins. As pH increases, the adsorption capacity for flavonoids and saponins weakens, but for alkaloids, the adsorption capacity increases with increasing pH. Considering the combined effects of pH on the three types of components, D101 resin shows good adsorption for all three types of components at pH = 5 (stock solution). Therefore, pH 5, the pH of the stock solution, was chosen as the pH of the loading solution. The effects of pH on the adsorption rate are shown in Table 3.

[0177] Table 3 Effect of pH on adsorption rate

[0178]

[0179]

[0180] 3.2.1.3 Determination of the mass concentration of the sample loading solution

[0181] When the concentration of the sample solution is 0.067 g·mL -1 Increase to 1 g·mL -1 At this time, the adsorption rates of alkaloids and saponins gradually decreased, while the adsorption of flavonoids initially increased with increasing sample concentration, reaching a peak when the sample concentration was >0.3 g·mL.-1 Subsequently, the adsorption rate began to decrease. Therefore, based on the experimental results and considering the manpower and time costs in the production process, the final selected sample solution concentration was 0.3 g·mL. -1 The effect of the sample concentration is shown in Table 4.

[0182] Table 4. Effect of sample solution concentration on adsorption rate

[0183] Mass concentration / (g mL -1 ) Total alkaloids / (%) Total flavonoids / (%) Total saponins / (%) 0.067 100 24.82 100 0.15 100 31.07 100 0.3 67.56 57.29 100 0.5 52.57 15.38 60.43 1 46.88 13.99 44.67

[0184] 3.2.2 Dynamic Adsorption Experiment

[0185] 3.2.2.1 Determination of the sample loading volume flow rate

[0186] Preliminary investigations determined that HAL was the first component to leak among HAL, HAR, Sie.A, and GA. Therefore, when the concentration of HAL in the effluent after adsorption by the macroporous resin reached 10% of the HAL concentration in the loading solution, the adsorption of total alkaloids and total saponins was considered saturated. Similarly, based on the HAL adsorption saturation standard, the loading amount for total flavonoid adsorption saturation was determined. The results showed that the loading flow rate had little effect on the alkaloid, flavonoid, and saponin components in FFMN; therefore, to save time, the highest loading flow rate, 3 BV·h, was selected. -1 When the flow rate is 3 BV·h -1 HAL leaked at 21 BV, while flavonoids began to leak at 11 BV. Therefore, the optimal loading flow rate was 3 BV·h. -1 The maximum loading volume was 11 BV. The leakage curves for HAL and total flavonoids are shown below. Figure 1 As shown.

[0187] 3.2.2.2 Determination of water washing volume

[0188] The washing results showed that a washing volume of 4 BV could remove most of the large molecular impurities; therefore, the washing volume was determined to be 4 BV. The solids removal results for different washing volumes are shown below. Figure 2 As shown.

[0189] 3.2.2.3 Determination of the volume fraction of eluted ethanol after impurity removal

[0190] The results show that 10% ethanol has almost no elution effect on the three types of components; therefore, 10% ethanol can be chosen to replace ultrapure water for impurity removal. In addition, 70% ethanol can elute almost all the target components; therefore, 70% ethanol was used as the elution solvent, and the elution curves for each component are shown below. Figure 3 As shown.

[0191] 3.2.2.4 Determination of the Amount of Ethanol Used for Elution

[0192] Elution experiments showed that the elution volume of each component gradually increased with the increase of the amount of eluting ethanol. When the elution volume was 5 BV, the cumulative elution rate of all three components was close to 100%. Therefore, the optimal amount of eluting ethanol was 5 BV. The elution curve for 70% ethanol is shown below. Figure 4 As shown.

[0193] 3.2.2.5 Optimal Process

[0194] The optimal process is to use a sample loading solution with a pH of 5 and a flow rate of 3 BV·h. -1 The mass concentration is 0.3 g·mL. -1 An aqueous solution of FFMN was loaded with a volume of 11 BV. After dynamic adsorption, impurities were removed with 5 BV of 10% ethanol, and finally with 5 BV of 70% ethanol at a concentration of 3 BV·h. -1 The solution is obtained by elution at a certain flow rate.

[0195] 3.2.2.6 Verification Test

[0196] The results of the verification experiment showed that the desorption rate of total alkaloids was 74.15%, the desorption rate of total saponins was 83.28%, and the desorption rate of total flavonoids was 92.2%. The yield of extract decreased from 18.08g before purification to 2.32g. The purity of total alkaloids increased by 5.80 times, the purity of total saponins increased by 6.52 times, and the purity of total flavonoids increased by 7.20 times (before enrichment by this optimal process, the purity of alkaloids, saponins, and flavonoids in the FFMN alcohol extract was 2.59%, 1.05%, and 10.75%, respectively; after enrichment by this optimal process, the purity of alkaloids, saponins, and flavonoids was 15.02%, 6.85%, and 77.38%, respectively). This proves that the purification process is stable, has good reproducibility, and has a good separation and purification effect on the total alkaloids, total flavonoids, and total saponins in compound Munizi. The results of the content of various components in the total fraction obtained by the above-mentioned optimal process "3.2.2.5" after enrichment with macroporous resin in the verification test are shown in Table 5.

[0197] Table 5. Content of various components in the total fraction after enrichment.

[0198]

[0199]

[0200] 4. Activity experiments of different parts of Compound Muniziqi Formula

[0201] 4.1 Experimental Methods

[0202] 4.1.1 Preparation of different parts of FFMN

[0203] According to "3.2.2.1", the optimal flow rate is 3 BV·h.-1 At loading volumes of 10 BV and 12 BV, the flavonoid leakage rates were 9% and 14%, respectively, while the HAL leakage rates at loading volumes of 21 BV and 22 BV were 9.6% and 13.2%, respectively. Therefore, flavonoids leak at 11 BV, while alkaloids leak at 21 BV. To minimize flavonoid leakage, the loading volume for the macroporous resin enrichment process was determined to be 11 BV. During the process of exploring the separation of different fractions, it was found that flavonoids and alkaloids have similar polarities and are intermixed. 10% ethanol could not elute either, while 30% ethanol could elute both simultaneously. When separating the flavonoid fraction, the content of alkaloids and saponins in the flavonoid fraction should be minimized as much as possible; the separation principle for other fractions is the same. Therefore, the total flavonoid fraction can be prepared according to the order of leakage of flavonoids and alkaloids. To enrich flavonoids as much as possible, the effluent after adsorption by macroporous resin (for example, when enriching FFMN ethanol extract, the flavonoids leaked in the 11 BV sample are considered flavonoids leaked within the allowable leakage range of the enrichment process and therefore not flavonoids in the final enriched site) should contain more flavonoids. Most total saponins are eluted at 40%-70% ethanol concentration. Therefore, when separating alkaloids, flavonoids, and saponins, the enrichment process of FFMN ethanol extract can be slightly modified, increasing the sample volume to 14 BV while keeping other sample loading conditions unchanged. The effluent containing flavonoids after adsorption by macroporous resin (without alkaloids and saponins) is collected for flavonoid enrichment. After adsorption of the sample solution, it was eluted sequentially with 10 BV of 30% ethanol, 9 BV of 40% ethanol, and 3 BV of 70% ethanol. The eluents were collected, and the contents of each component were determined. According to the results under "3.1.4.3", most of the flavonoid components could be eluted with 30% ethanol for 3 BV. Therefore, to minimize the flavonoid components in non-flavonoid fractions, the first 3 BV of 30% ethanol were discarded. Based on the content of each component in each BV, 30% 4-10 BV and 40% 1 BV were combined as alkaloid fractions, and 40% 4 BV-70% 3 BV were combined as saponin fractions. After the alkaloid and saponin fractions were rotated until no alcohol odor remained, they were frozen at -80℃ and then freeze-dried into lyophilized powder using a freeze dryer to obtain the total alkaloid and total saponin fractions, which were stored at -20℃. After centrifuging the collected flavonoid eluent, the sample solution was loaded for adsorption. Impurities were first removed with 5 BV of 10% ethanol, followed by elution with 30% ethanol. The eluent was collected and vortexed until no alcohol odor remained. It was then frozen at -80℃ and freeze-dried to obtain the total flavonoid fraction, which was stored at -20℃. The flowchart is shown below. Figure 5 As shown.

[0204] Take 0.98 g, 0.44 g, and 1.08 g of the lyophilized fractions of total extract, total alkaloids, and total flavonoids obtained from the FFMN macroporous resin enrichment process prepared above. Dissolve each fraction in 1 mL of DMSO, sonicate to dissolve, wrap in aluminum foil, and store at 4°C. Take 0.39 g of the lyophilized fraction of total saponins, dissolve in 500 μL of DMSO, sonicate, and store at 4°C. Take 10 μL of each of the DMSO solutions of total extract, total alkaloids, total flavonoids, and total saponins, add to 10 mL of DMEM medium, vortex to dissolve completely, and prepare concentrations of 980, 440, 1080, and 780 μg·mL, respectively. -1 The mother liquor should be stored away from light and kept in a refrigerator at 4°C for later use.

[0205] 4.1.2 Cell Culture

[0206] 4.1.2.1 Cell resuscitation

[0207] Remove cryovials containing RAW264.7 or PC12 cells from the -80℃ freezer, disinfect them with alcohol, and then transfer them to the cell culture room. Quickly place them in a 37℃ water bath, agitating constantly until completely thawed (this process should be rapid to prevent cell damage). After alcohol disinfection, transfer the cell suspension to a 15mL centrifuge tube, add 3mL of DMEM or RPMI 1640 complete medium, gently pipette to mix, and centrifuge to remove the supernatant. Then add 1mL of complete medium (containing 5% FBS and 1% antibiotics), pipette to mix, and transfer to a culture dish containing 6mL of DMEM or RPMI 1640 complete medium. Use a cross-hatching method to evenly distribute the cells in the dish and place it in an incubator for culture. Change the medium the next day to prevent residual DMSO from affecting cell growth.

[0208] 4.1.2.2 Cell passage

[0209] When the cell number reaches 80% of the culture dish, after aspirating the culture medium with a suction pump, PC12 cells should be washed twice with PBS preheated to 37°C. Then, 1 mL of 0.25% trypsin should be added, and digestion should continue for 2 minutes. Afterward, an appropriate amount of RPMI 1640 complete culture medium should be added to terminate the digestion, and the cells should be completely detached by pipetting. RAW264.7 cells should be washed twice with cold PBS, and then gently and repeatedly pipetted with pre-cooled PBS until the cells are completely detached. The detached cell suspension should be transferred to a 15 mL centrifuge tube as described in "3.2.3.1". After centrifugation at 800 rpm for 4 minutes, the supernatant should be aspirated with a suction pump, and the cells should be resuspended in 1 mL of the corresponding culture medium. An appropriate amount of the cell suspension should be transferred to a dish containing 7 mL of complete culture medium, shaken in a crosswise direction, and observed under a microscope for cell dispersion. The cells should then be placed in an incubator for further culture.

[0210] 4.1.2.3 Cytotoxicity Detection

[0211] In a 96-well plate, the density added to each well is 1 x 10⁻⁶. 5 ·mL -1 and 2 x 10 5 ·mL -1 100 μL of RAW264.7 and PC12 cell suspensions were used. A drug-free control group was included. The total fractions, total alkaloids, total saponins, and total flavonoids were enriched using macroporous resin at concentrations of 10, 20, 40, 50, 60, 70, 80, 90, and 100 μg / mL. -1 The cells were divided into two groups, each with six replicates. A blank control group containing only culture medium and no cells was also included. After culturing for 24 hours, 100 μL of complete culture medium was added to the blank and control groups, while 100 μL of culture medium containing different drug concentrations was added to each drug-treated group. The cells were then incubated for another 24 hours, after which the culture medium was discarded. 100 μL of prepared CCK-8 reagent (CCK-8:basal medium = 1:10) was added to each well (in the dark), and the cells were incubated together for 30 minutes. The absorbance (A) was measured at 450 nm, and cell viability was calculated. The concentration with a viability ≥ 80% was selected as the maximum drug concentration. The formula was: Viability = [(Experimental Group A - Blank Group A) / (Control Group A - Blank Group A)] × 100%.

[0212] 4.1.3. Different sites of FFMN inhibit LPS-induced inflammation in RAW264.7 cells.

[0213] The density added to each well of the 96-well plate is 1 x 10⁻⁶. 5 ·mL -1 100 μL of RAW264.7 cell suspension was cultured for 24 h, and then the culture medium was discarded. Based on the maximum drug concentration determined in "4.1.2.3", 100 μL of fresh culture medium containing different concentrations of different fractions of FFMN was added to each group. After 2 h of pretreatment, except for the blank group, all other groups were treated with LPS (final concentration, 2 μg / mL). -1 The samples were incubated together for 24 hours. The NO content in the supernatant of each group was determined by the Griess method. The absorbance of each well was read at a wavelength of 540 nm to calculate the NO content.

[0214] 4.1.4, PC12 Antioxidant Damage Test

[0215] 4.1.4.1 Establishment of the PC12 Oxidation Damage Model

[0216] The density added per well in a 96-well plate is 2 x 10⁻⁶. 5 ·mL -1100 μL of PC12 cell suspension was cultured for 24 h. Then, 100 μL of culture medium containing H2O2 concentrations of 0, 20, 50, 100, 200, 500, 800, 1000, 1500, and 2000 μM were added to each well. After 24 h of culture, cell viability was measured in each group. Cell survival rate at each concentration was calculated to determine the optimal H2O2 concentration for H2O2 modeling.

[0217] 4.1.4.2 Protective effect of different sites of FFMN against oxidative damage in PC12 cells

[0218] Following the maximum drug concentration determined in "4.1.2.3", 100 μL of fresh culture medium containing different concentrations of different fractions of FFMN was added to each well to pretreat PC12 cells for 2 hours, followed by stimulation with H2O2 (final concentration, 500 μM). After co-culturing with the drug for 24 hours, cell viability was determined using the CCK-8 assay.

[0219] 4.1.4.3 Superoxide dismutase (SOD) content in PC12 cells

[0220] The density added to each well of the 6-well plate is 1 x 10⁻⁶. 6 ·mL -1 PC12 cell suspension was prepared according to section "4.1.4.2". After drug pretreatment and H2O2 stimulation, the cells were digested with 400 μL of trypsin, and then 1 mL of RPMI 1640 complete culture medium was added to terminate the digestion. The cells were then transferred to 1.5 mL centrifuge tubes and centrifuged at 800 rpm. -1 Centrifuge, discard the supernatant, resuspend in PBS, and centrifuge at 800 rpm. -1 After centrifugation and disruption of cells using a cell sonicator, SOD activity was measured according to the kit instructions.

[0221] 4.2 Experimental Results

[0222] 4.2.1 Preparation results of FFMN at different sites

[0223] The purity of the total alkaloids in the initially separated FFMN alkaloid fraction was 30.2% (the mass of alkaloids in the alkaloid fraction was 942.56 mg, and the mass of the lyophilized powder was 3.12 g). This purity was 2.0 times higher than that obtained from the total alkaloid fraction after enrichment with macroporous resin using the optimal process described in "3.2.2.5" above, and 11.6 times higher than that from the FFMN alcohol extract. The purity of the total saponins in the total saponin fraction was 60.6% (the purity of saponins in the total saponin fraction was 60.6%). The total saponin content was 563.9 mg (930 mg of lyophilized powder). Compared with the total saponin obtained by the above-mentioned optimal process "3.2.2.5" after enrichment with macroporous resin, the purity of the total saponin was increased by 8.8 times, and compared with the total saponin in FFMN alcohol extract, the purity was increased by 57.7 times. The total flavonoid fraction contained only flavonoid components, and no alkaloids and saponins were detected in the HPLC chromatogram. The purity was 26.3% (1810 mg of total flavonoids in the flavonoid fraction, 6.88 g of lyophilized powder).

[0224] 4.2.2 Cytotoxicity Results

[0225] For RAW264.7 cells, the concentrations of total phytoliths, total alkaloids, total flavonoids, and total saponins exceeded 50, 60, 70, and 90 μg·mL, respectively. -1 At that time, the survival rate of RAW264.7 cells was less than 80%, indicating that the drug had begun to have toxic effects on the cells. Therefore, the concentrations corresponding to each group were taken as the maximum dosing concentrations.

[0226] For PC12 cells, 100 μg·mL -1 Different fractions of FFMN showed no toxicity to PC12 cell growth, therefore, they were chosen as the maximum therapeutic concentration. The effects of different fractions of FFMN on RAW264.7 cells are as follows: Figure 6 As shown.

[0227] 4.2.3 FFMN and its different sites inhibit LPS-induced NO production in RAW264.7 cells.

[0228] The NO release results showed that, compared with the control group, the NO production in the model group was significantly increased, proving the successful establishment of the LPS model. The FFMN main site, total flavonoids, and total saponins groups significantly inhibited NO production, demonstrating their significant anti-inflammatory activity. For the total alkaloids group, only the highest administered concentration (60 μg / mL) showed significant anti-inflammatory activity. -1 At the [specific concentration], there was a significant difference compared to the model group (P < 0.01), and none of the other concentrations could inhibit NO production (P > 0.05). The results of inhibiting NO release are as follows: Figure 7 As shown.

[0229] 4.2.4, PC12 Antioxidant Damage Test

[0230] 4.2.4.1 Establishment of the PC12 Oxidation Damage Model

[0231] As the concentration of H2O2 increased, the survival rate of PC12 cells continuously decreased. When the concentration of H2O2 was 500 μM, the viability of PC12 cells decreased by 54.2%. Therefore, 500 μM H2O2 was chosen as the concentration for establishing the oxidative damage model. The effects of different concentrations of H2O2 on PC12 cells are shown below. Figure 8 As shown.

[0232] 4.2.4.2 Protective effect of FFMN and its different fractions against oxidative damage in PC12 cells

[0233] The results of oxidative damage studies showed that the main FFMN fractions, total alkaloids, total flavonoids, and total saponins all had a protective effect against H2O2-induced oxidative damage. However, only the highest concentration of the total alkaloids fraction showed a significant difference from the model group. The results of the H2O2-induced oxidative damage model of PC12 cells by different FFMN fractions are as follows: Figure 9 As shown.

[0234] 4.2.4.3 Effects of FFMN and its different fractions on SOD production in H2O2-induced PC12 cells

[0235] Superoxide dismutase (SOD) primarily functions to scavenge superoxide free radicals in the body, and its activity level indirectly reflects the degree of cellular oxidative damage. Results showed that the total flavonoid and total saponin groups significantly increased SOD activity in PC12 cells, while the alkaloid group showed no significant difference compared to the model group. The effects of different FFMN fractions on SOD activity are shown below. Figure 10 As shown.

[0236] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for purifying a compound monomisqi macroporous resin, characterized in that, The method comprises the following steps: (1) macroporous resin pretreatment: D101 resin is sequentially soaked with an ethanol solution, washed and soaked with a hydrochloric acid solution, washed and soaked with a sodium hydroxide solution, and washed with water until the pH is neutral, to obtain pretreated D101 resin; (2) column packing: the pretreated D101 resin is packed into a column by wet method, to obtain a D101 resin adsorption column; (3) loading: a certain amount of medicinal materials is accurately weighed, sieved, and then extracted by reflux extraction after immersion, the filtrates are combined after filtration, concentrated under reduced pressure until there is no alcohol taste, water is added to a certain concentration of crude drug, and the supernatant is obtained by centrifugation, to obtain a macroporous resin loading solution, the pH of the loading solution is 4.75-5.25, the mass concentration of the loading solution is 0.3 g·mL -1 , and the volume of the loading solution is 13.3-14.7 BV, and the loading solution is passed through the D101 resin adsorption column at a flow rate of 2.85-3.15 BV·h -1 , so that the loading solution is adsorbed on the D101 resin adsorption column, and the unadsorbed loading solution is collected to obtain a leakage solution; and (4) obtaining the total alkaloid fraction and the total saponin fraction: after removing impurities by passing 4.75-5.25 BV of 9.5%-10.5% ethanol through the D101 resin adsorption column at a flow rate of 2.85-3.15 BV·h -1 , the 4th BV to the 10th BV of the eluate of 28.5%-31.5% ethanol and the 1st BV to the 3rd BV of the eluate of 66.5%-73.5% ethanol are combined as the total saponin fraction; -1 , the 4th BV to the 10th BV of the eluate of 28.5%-31.5% ethanol and the 1st BV to the 3rd BV of the eluate of 66.5%-73.5% ethanol are combined as the total saponin fraction; (5) obtaining total flavonoids fraction: after centrifugation of the leakage liquid, the supernatant is passed through the D101 resin adsorption column at a flow rate of 2.85-3.15 BV·h -1 After impurities are removed from the D101 resin adsorption column using 4.75-5.25 BV of 9.5%-10.5% ethanol at a flow rate of 2.85-3.15 BV·h -1 After impurities are removed from the D101 resin adsorption column using 4.75-5.25 BV of 9.5%-10.5% ethanol at a flow rate of 2.85-3.15 BV·h -1 The eluate is collected to obtain the total flavonoids fraction.

2. The method of claim 1, wherein, In step (1), the concentration of the ethanol solution is 90.25%-99.75%, and the soaking time of the ethanol solution is 22.8-25.2 hours.

3. The method of claim 1, wherein, In step (1), the volume of the hydrochloric acid solution is 1.9-2.1 BV, the concentration of the hydrochloric acid solution is 4.75%-5.25%, and the soaking time of the hydrochloric acid solution is 1.9-2.1 hours.

4. The method of claim 1, wherein, In step (1), the volume of the sodium hydroxide solution is 1.9-2.1 BV, the concentration of the sodium hydroxide solution is 1.9%-2.1%, and the soaking time of the sodium hydroxide solution is 1.9-2.1 hours.

5. The method of claim 1, wherein, In step (1), the water is ultrapure water.

6. The method of claim 1, wherein, In step (3), the medicinal materials include Khus-khus seeds, Nigella seeds, Aniseed fruits, Anise root barks, Chamomile, Celery root, Chicory seeds, Chicory roots, Citronella, Garlic mustard seeds, Licorice, Basil seeds and Hollyhock seeds.

7. The method of claim 1, wherein, In step (3), the sieve is a No. 1 sieve.

8. The method of claim 1, wherein, In step (3), the maceration is carried out by adding 4.75-5.25 times the mass of the medicinal materials with 66.5%-73.5% ethanol to macerate the medicinal materials.

9. The method of claim 1, wherein, In step (3), the reflux extraction is carried out by adding 7.6-8.4 times the mass of the medicinal materials with 66.5%-73.5% ethanol to reflux extract the medicinal materials.

10. The method of claim 1, wherein, In step (3), the reflux extraction is carried out for 2 times, each time for 0.95-1.05 hours.

11. The method of claim 1, wherein, In step (3), the concentration of the crude drug is 0.285-0.315 g / mL.

12. The method of claim 1, wherein, In step (3), the centrifugation is carried out at a speed of 7000-9000 r / min for 5-15 min.

13. The method of claim 12, wherein, The centrifugation is carried out at a speed of 7600-8400 r / min for 9.5-10.5 min.

14. The method of claim 1, wherein, Before the impurity removal, 3-5 BV of water is passed through the D101 resin adsorption column at a flow rate of 2.85-3.15 BV / h.

15. The method of claim 14, wherein, Before the impurity removal, 3.8-4.2 BV of water is passed through the D101 resin adsorption column at a flow rate of 2.85-3.15 BV / h.

16. The method of claim 1, wherein, In step (5), the centrifugation is carried out at a speed of 7000-9000 r / min for 5-15 min.

17. The method of claim 16, wherein, The centrifugation is carried out at a speed of 7600-8400 r / min for 9.5-10.5 min.

18. A total alkaloid fraction prepared by the method of any one of claims 1-17.

19. A total saponin fraction prepared by the method of any one of claims 1-17.

20. A total flavonoid fraction prepared by the method of any one of claims 1-17.

21. A pharmaceutical composition comprising the total alkaloid fraction of claim 18, the total saponin fraction of claim 19, or the total flavonoid fraction of claim 20.

22. The pharmaceutical composition of claim 21, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

23. The pharmaceutical composition of claim 22, wherein, The excipient is selected from one or more of the following: dispersing agents, wetting agents, binding agents, diluents, retention agents, lubricants, sustained release agents, plasticizers, disintegrating agents, inclusion agents, flavoring agents, opacifiers, and antioxidants.

24. The pharmaceutical composition of claim 21, wherein, The dosage form of the pharmaceutical composition is a tablet, a dripping pill, a capsule, a powder, an injection, a film, a lozenge, a granule, or an oral liquid.

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