Preparation method of alkaloid composition in coptis chinensis and fructus evodiae

Abstract

The invention provides a preparation method of an alkaloid composition in coptis chinensis and evodia rutaecarpa, main active substance alkaloids in coptis chinensis and evodia rutaecarpa are respectively enriched in combination with modern extraction and purification technologies, the purpose of enriching a large amount of alkaloids can be achieved, the content of limonin and other components is greatly reduced, the quality of the obtained product is more stable and controllable, and the preparation method is suitable for industrial production. Meanwhile, the drug effect of the traditional Chinese medicine is reserved. The whole preparation process is simple and convenient in flow, safe in reagent, small in usage amount, low in energy consumption and suitable for large-scale production, and the final product is stable in quality and high in safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a preparation method of a traditional Chinese medicine composition for treating bile reflux gastritis. BACKGROUND

[0002] Zuojinfang was created by Zhu Danxi, a famous physician in the Jin Dynasty. The prescription is excellent, with Coptis and Evodia rutaecarpa in a special ratio of 6:1. It has the effects of clearing liver fire and reducing adverse to stop vomiting, and is used to treat rib pain, indigestion, acid regurgitation, vomiting, bitter mouth, red tongue, yellow fur, and stringy pulse.

[0003] Coptis is the dried rhizome of Coptis chinensis Franch., Coptis deltoidea C.Y. Cheng et Hsiao or Coptis teeta Wall. of the Ranunculaceae family. The Coptis used in the present application is Coptis chinensis Franch. Modern pharmacological studies have shown that the main chemical components of Coptis are alkaloids, including berberine, coptisine, jatrorrhizine, palmatine, and jatrorrhizine, which have the effects of reducing blood sugar, antibacterial, antioxidant, anti-inflammatory, and anti-tumor.

[0004] Evodia rutaecarpa is the dried nearly mature fruit of Evodia rutaecarpa (Juss) Benth. of the Rutaceae family, which can warm the middle and dispel cold, and soothe the liver and relieve pain. It is used for treating headache, abdominal cold pain, vomiting and diarrhea, dysmenorrhea, and hypertension. The main chemical components are alkaloids, bitter principles, volatile oils, and flavonoids. Alkaloids are the main active components, and the main effects are analgesic and anti-inflammatory, anti-ulcer, anti-tumor, and protection of the cardiovascular system. The main clinical effects are analgesic and anti-emetic. Related studies have shown that water extract, alcohol extract, and volatile oil components of Evodia rutaecarpa can cause different degrees of liver damage in animals, which may be related to excessive use and incorrect compatibility of the drug.

[0005] At present, there are various extraction and purification methods for Coptis alkaloids, but most of them have the problems of complex process, large amount of organic solvent, low yield, high energy consumption, etc. The content of alkaloids in Evodia rutaecarpa is low, and the extraction and purification process is complex, which is not easy to be scaled up for industrial production.

[0006] The patent (CN 101429197 A) invented an extraction process technology for the co-production of coptis alkaloids using coptis chinensis root fibers as raw materials. The process flow mainly includes coptis chinensis root fibers - soaking in an appropriate amount of 0.05-0.1% sulfuric acid - neutralization and precipitation to remove impurities - cation separation column - step-by-step elution - group decolorization - solvent recovery - distribution precipitation and crystallization - drying - jatrorrhizine monomer, berberine monomer, and coptis chinensis complex alkaloids, achieving a berberine and jatrorrhizine content of more than 90%, and a coptis chinensis complex alkaloid content of more than 50%. However, the process is complex and has a long cycle. In addition, an ion exchange column is used, and there is no subsequent metal cation removal process, which may cause metal ions to exceed the standard and is not suitable for large-scale production.

[0007] Patent (CN 105853023 A) reports a method for extracting alkaloids from Coptis chinensis. The main steps involve desorption of Coptis chinensis powder in a desorption solution, addition of water, vacuum extraction, filtration, and concentration and drying of the filtrate to obtain a Coptis chinensis alkaloid extract. This method, which uses vacuum extraction, is suitable for small-batch production and has high energy consumption. The resulting Coptis chinensis alkaloid content is relatively low, ranging from 30% to 46%.

[0008] Patent (CN 108853227 A) discloses a method for preparing a Coptidis alkaloid extract: Coptidis chinensis is ground and placed in a stoppered conical flask. A 50% acetonitrile-hydrochloric acid mixture is added, the flask is sealed, and ultrasonic extraction is performed 2-4 times, each for 40-50 minutes. The extracts are combined and concentrated under reduced pressure to obtain an extract, which is then dissolved in a 1% hydrochloric acid solution. The extract is filtered, and the filtrate is alkalized with ammonia water. The extract is extracted 3-5 times with chloroform. The extracts are combined and concentrated under reduced pressure. The extract is separated by alkaline silica gel column chromatography using a chloroform-methanol gradient elution. The elution fraction is collected at a 15:1 ratio and concentrated. This method is only suitable for small-scale laboratory tests. It is complex and uses the organic solvent chloroform, which is not recommended for use in the Chinese Pharmacopoeia and is highly hazardous.

[0009] Patent (CN 101224250 A) describes a method for preparing Evodia rutaecarpa alkaloids through ultrasonic-enhanced extraction: Evodia rutaecarpa is ground into a coarse powder, placed in an ultrasonic extractor, and soaked in ethanol. Ultrasonic extraction is then performed, followed by filtration, centrifugation, concentration, solvent recovery, and drying to obtain the Evodia rutaecarpa alkaloids. This method yields alkaloids with low evodiamine and rutaecarpine content of 0.6%-0.8%, making it suitable only for small-scale sample preparation and insufficient for large-scale production.

[0010] Patent (CN 105497230 A) reports an extraction method of evodia effective components, mainly evodia is soaked with water, reflux extraction twice, filter while hot, filter liquor is concentrated under reduced pressure or freeze-dried into powder, knead in boiling water, supernatant is filtered through HPD-100 macroporous adsorption resin, eluted with 10% ethanol, 30% ethanol, 50% ethanol and 95% ethanol gradient, concentrated under reduced pressure, combine 30% and 50% ethanol elution parts, put in 500D and 100D dialysis bag, place in double distilled water beaker, stand at 4℃ for 48 hours, collect the liquid between 100D and 500D, and get it. The evodia alkaloid yield obtained by this method is low, and it is not easy to operate. The risk of kneading in boiling water is high. The citral content in the obtained alkaloid is high, and currently there are related toxicology studies focusing on citral, which finds that it has certain cytotoxicity.

[0011] Patent CN 113975326 A reports an extraction and purification method of coptis and evodia, mainly using ethanol aqueous solution to extract coptis and evodia respectively, using styrene macroporous adsorption resin for purification, and obtaining alkaloid extract after drying under reduced pressure respectively. The two kinds of alkaloid extracts have obvious prevention and treatment effects on gastric mucosa of reflux esophagitis model rats after being used together. We find that in the resin column purification stage of the process, precipitate and turbidity easily appear in the sample solution, sometimes causing resin column blockage, making separation difficult, and the cost is high, which is not suitable for industrial mass production.

[0012] There are many reported extraction and purification methods of coptis alkaloids, but they all have certain problems, are suitable for small batch preparation but not easy to match mass production, and cannot meet the process preparation demand, so it is urgent to develop a simple process to meet the needs of industrial mass production. SUMMARY

[0013] Under the premise of following the matching ratio of coptis: evodia 6:1, the present application provides a preparation method of coptis and evodia alkaloid composition, which is simple in process, low in energy consumption and suitable for large-scale industrial production.

[0014] The present application provides a preparation method of coptis and evodia alkaloid composition, which includes coptis alkaloid preparation and evodia alkaloid preparation.

[0015] The coptis alkaloid preparation method includes extraction step and purification step:

[0016] Take coptis, crush, make all pass through 8mm sieve, 10 times amount of 70% ethanol is heated to reflux and extract twice, 1 hour each time, filter, combine, concentrate under reduced pressure to 0.5g crude drug per milliliter, take supernatant. The filtrate is loaded on a pretreated macroporous adsorption resin column, eluted with ethanol, and the combined eluate is concentrated under reduced pressure and dried.

[0017] The Coptis chinensis is selected from medicinal materials or decoction pieces, preferably decoction pieces.

[0018] Preferably, Coptis chinensis is taken, broken to pass through an 8 mm sieve, 10 times the amount of 70% ethanol is added, heated to reflux and extracted for 2 hours each time, filtered, the extract is combined, ethanol is recovered under reduced pressure and concentrated to 0.5 g of crude drug per ml, centrifuged, and the filtrate is obtained. The filtrate is loaded onto a pretreated macroporous adsorption resin column with a diameter-height ratio of 1:6-10, adsorbed for 10-16 hours, first eluted with water at a flow rate of 1-3 times the column volume per hour, the water eluate is discarded, 40% ethanol is used for desorption at a flow rate of 1-3 times the column volume per hour, eluted for 4 times the column volume, the desorption liquid is collected, concentrated under reduced pressure, dried, and the dry extract is obtained by crushing.

[0019] Most preferably, Coptis chinensis decoction pieces are taken, broken to pass through an 8 mm sieve, 10 times the amount of 70% ethanol is added, heated to reflux and extracted for 2 hours each time, filtered, the extract is combined, ethanol is recovered under reduced pressure and concentrated to 0.5 g of crude drug per ml, centrifuged on a plate centrifuge or a tubular centrifuge, and the filtrate is obtained. The filtrate is added to a D101 macroporous adsorption resin column that has been pretreated at a maximum loading amount of 0.5 times the column volume, with a diameter-height ratio of 1:8, loaded at a flow rate of 1 times the column volume per hour, adsorbed for 12 hours, first eluted with water at a flow rate of 2 times the column volume per hour, the water eluate is discarded, 40% ethanol is used for desorption at a flow rate of 2 times the column volume per hour, eluted for 4 times the column volume, the desorption liquid is collected, concentrated under reduced pressure, dried, and the dry extract is obtained by crushing.

[0020] The evodia alkaloid preparation method comprises an extraction step and a purification step:

[0021] Evodia fruit is added to ethanol, reflux extracted, the extract is filtered through filter cloth, ethanol is recovered, and a concentrated liquid is obtained. Water is added to a content of 5%-15% of alcohol, allowed to stand and settle, centrifuged, the precipitate is taken, dried, and the dry extract is obtained by crushing.

[0022] The evodia fruit is selected from medicinal materials or decoction pieces, preferably decoction pieces.

[0023] Preferably, evodia fruit is taken, 8 times the amount of 70% ethanol is added, heated to reflux and extracted for 3 hours each time, the extract is filtered, combined, concentrated to a content of 30%-50% of alcohol, cooled to room temperature, water is added to a content of 5%-15% of alcohol, allowed to stand and settle, centrifuged, the precipitate is taken, dried, and the dry extract is obtained by crushing.

[0024] Most preferably, evodia fruit decoction pieces are taken, 8 times the amount of 70% ethanol is added, heated to reflux and extracted for 3 hours each time, the extract is filtered, combined, concentrated to a content of 40% of alcohol under reduced pressure, cooled to room temperature, water is added to a content of 10% of alcohol, allowed to stand and settle at 4°C-25°C for 8-24 hours, centrifuged on a plate centrifuge or a tubular centrifuge, the precipitate is taken, dried under reduced pressure, and the dry extract is obtained by crushing.

[0025] Advantages of the present application

[0026] The present application combines modern extraction and purification techniques to enrich alkaloids, the main active substances in Coptis and Evodia, in the two medicinal materials. The preparation method of the present application has been optimized in detail, and the quality of alkaloids in the extracts from the two medicinal materials is more stable. In particular, the purification process of Evodia alkaloid extract, compared with the prior art, discards the traditional macroporous resin purification and enrichment method, and selects a simple alcohol extraction and water precipitation purification process to achieve the purpose of enriching a large amount of alkaloids, greatly reduces the content of limonin components, and the quality is more stable and controllable, while retaining its efficacy. The whole preparation process is simple, the reagent is safe, the use amount is small, the energy consumption is low, it is suitable for mass production, the quality of the final product is stable, and the safety is high.

[0027] The ancient prescription Zuojinfang is used, and a special compatibility ratio of 6:1 of Coptis to Evodia is investigated. The composition has obvious improvement effect on the pathological changes of the gastric mucosa of the reflux gastritis model rats in the dose range of 0.3g-2.2g of crude drug / kg, which is verified by efficacy test. Compared with the original prescription, most of the non-alkaloid components are removed, and the dosage is also reduced. DETAILED DESCRIPTION

[0028] Example 1 Investigation of key parameters in the preparation of Coptis alkaloids

[0029] 1.1 Investigation of treatment methods of macroporous adsorption resin sample solution

[0030] Macroporous adsorption resin is a kind of high molecular adsorption resin without exchange groups and with macroporous structure, which has good macroporous network structure and large specific surface area, and can selectively adsorb organic matter in aqueous solution by physical adsorption. In order to make the Coptis extract pass through the macroporous adsorption resin more smoothly, the extract needs to be pretreated to a certain extent to prevent the resin from being blocked, reduce efficiency and product quality.

[0031] Take Coptis, crush it so that it can pass through an 8mm sieve, add 10 times the amount of 70% ethanol, heat and reflux for 2 times, 1 hour each time, filter the extract, combine and concentrate under reduced pressure to 0.5g of crude drug / ml, and then pass through a 100 mesh sieve and centrifuge. Take the total amount of 6 alkaloids such as Coptisine as the investigation index, and observe the properties of the treated liquid to optimize the pretreatment method.

[0032] Table 1 Optimization of treatment methods of macroporous adsorption resin sample solution

[0033]

[0034] Conclusion: Screen filter and centrifugal two kinds of processing methods, the impact of alkaloid components is not big, filtration has a small amount of sediment, centrifugal method without sediment, consider the later on macroporous adsorption resin, reduce the impact on the resin column, comprehensive analysis of the preferred centrifugal method for Coptis pretreatment.

[0035] 1.2 Resin diameter height ratio investigation

[0036] Resin column diameter height ratio has a great influence on the adsorption effect, so the resin column diameter height ratio is optimized. Take the treated D101 type macroporous adsorption resin, respectively, fill the diameter height ratio of 1:6 (column volume 300ml, D=4cm), 1:8 (column volume 400ml, D=4cm), 1:10 (column volume 500ml, D=4cm) macroporous resin column, standby; Take the concentration of 0.5g crude drug / ml of concentrated solution, centrifugal, respectively, take 600ml, 800ml and 1000ml, with 1 times column volume / hour flow rate through each macroporous adsorption resin for adsorption, collect the sample effluent after adsorption, detect the total amount of 6 kinds of alkaloids.

[0037] Table 2 Resin column diameter height ratio investigation results

[0038]

[0039]

[0040] Resin column diameter height ratio has a certain influence on the adsorption rate of Coptis alkaloids, when the diameter height ratio is 1:6, the column effect is reduced, the sample solution is not fully adsorbed, and part of it is leaked, when the diameter height ratio is 1:10, the wall effect is relatively increased, and the adsorption efficiency is not as good as when the diameter height ratio is 1:8, therefore, the optimal resin column diameter height ratio is 1:8.

[0041] 1.3 Sample flow rate investigation

[0042] Suitable sample flow rate can promote the adsorption of macroporous adsorption resin. Therefore, the sample flow rate is optimized.

[0043] Take the concentration of 0.5g crude drug / ml of Coptis concentrated solution, centrifugal, add D101 type macroporous adsorption resin column with diameter height ratio of 1:8, respectively, with 0.5, 1, 2 times column volume / hour flow rate for dynamic adsorption, collect the effluent, take the content of 6 kinds of alkaloids as index, calculate the total amount, the results are shown in Table 12.

[0044] Table 3 Sample flow rate investigation results

[0045]

[0046] The results show that the loading flow rate is 2 times the column volume per hour, and the alkaloids may not have enough time to be adsorbed due to the too fast flow rate, and the leakage amount is slightly high. When the adsorption flow rate is 0.5 BV / h or 1 BV / h, the saturated adsorption amount of the alkaloids on the D101 macroporous resin is relatively large. Considering the time efficiency, the preferred adsorption flow rate is 1 BV / h.

[0047] 1.4 Maximum loading amount investigation

[0048] To fully utilize the resin resources, the loading amount is investigated. The concentration of the Coptis concentrated solution is 0.5 g crude drug / ml, and the solution is centrifuged. The solution is passed through the treated D101 macroporous adsorption resin column (diameter-height ratio 1:8) at a speed of 1 column volume per hour, and the effluent is collected in batches. Each 0.25 column volume of the loading solution is collected once, and a total of 8 times of collection is performed. The effluent is sampled to determine the indicator components, and the content of the six alkaloids is calculated.

[0049] Table 4 Content of alkaloids in each effluent.

[0050]

[0051]

[0052] The results show that a trace amount of alkaloids leaks out from the third portion, and the preferred maximum loading amount is 0.5 column volume of the loading solution to maximize the retention of alkaloids.

[0053] 1.5 Coptis adsorption time investigation

[0054] The appropriate adsorption time is helpful for the resin to better adsorb the alkaloids of Coptis. Four portions of the Coptis concentrated solution with a concentration of 0.5 g crude drug / ml are centrifuged, and passed through the treated four D101 macroporous adsorption resin columns (diameter-height ratio 1:8) at a speed of 1 column volume per hour. After being adsorbed for 4, 8, 12, and 16 hours, respectively, the columns are first washed with 2 column volumes of water at a flow rate of 2 column volumes per hour. The water washing effluent is collected, and the indicator components are determined. Then, the columns are desorbed with 4 column volumes of 95% ethanol at a flow rate of 2 column volumes per hour. The desorption effluent is collected, and the total amount of the six alkaloids is determined. The desorption amount and the transfer rate in the desorption effluent are calculated.

[0055] Table 5 Results of adsorption time investigation

[0056]

[0057] The results show that the adsorption time of 4 and 8 hours is relatively short, and the resin cannot fully adsorb the alkaloids. The amount of effluent with water is slightly more. The adsorption amount of 12 and 16 hours is basically the same. Considering the elution efficiency, the preferred adsorption time is 12 hours.

[0058] 1.6 Investigation of the flow rate of the impurity-removing solvent

[0059] After the sample is loaded, some strong polar impurities remain in the resin column, affecting the enrichment effect of the desorption solution. Therefore, before desorbing the active ingredients, water is used to remove impurities. Take 3 portions of Coptis concentrated solution with a concentration of 0.5 g crude drug / ml, centrifuge, and pass through 3 treated D101 macroporous adsorption resin columns (diameter-height ratio 1:8) at a speed of 1 column volume per hour, respectively. After standing for 12 hours, wash with water, elute at a flow rate of 1, 2, and 3 column volumes per hour, respectively, elute 2 column volumes, collect the eluate, take samples, and determine the total amount of 6 alkaloids.

[0060] Table 6 Investigation results of the water washing flow rate

[0061]

[0062] From the above results, it can be seen that the water washing flow rate is 1 column volume per hour, the alkaloid content in the water washed dry powder is higher, and the loss is larger. The eluate at a flow rate of 3 column volumes per hour has less loss, but the impurity removal effect is slightly worse. After comprehensive comparison, the water washing flow rate of 2 column volumes per hour is preferred, with less loss of alkaloids and better impurity removal effect.

[0063] 1.7 Investigation of the amount of impurity-removing solvent

[0064] Some components in Coptis alkaloids have good water solubility, and the appropriate amount of impurity removal can remove non-alkaloids while retaining alkaloids to the greatest extent. Take 3 portions of Coptis concentrated solution with a concentration of 0.5 g crude drug / ml, centrifuge, and pass through 3 treated D101 macroporous adsorption resin columns (diameter-height ratio 1:8) at a speed of 1 column volume per hour, respectively. After standing for 12 hours, wash with water at a speed of 2 column volumes per hour, collect the water washings in batches, each time collecting 1 column volume, collect 4 times, and detect the total amount of 6 alkaloids.

[0065] Table 7 Investigation results of the water amount of impurity-removing agent

[0066]

[0067] With the increase of the elution times, the solid content continuously decreases. After eluting 2 column volumes, the solid content decreases significantly. To minimize the loss of alkaloids, elution of 2 column volumes is preferred.

[0068] 1.8 Investigation of the flow rate of the desorption solution

[0069] The flow rate of the eluent has a certain degree of influence on the amount of alkaloids eluted, so the elution flow rate was investigated. Three portions of the Coptis chinensis sample were loaded into three treated macroporous resin columns, and after 12 hours of standing, the columns were washed with water, the water was discarded, and 40% ethanol was used for desorption at a flow rate of 1, 2, or 3 times the column volume per hour. Four times the column volume was eluted, the eluent was collected, and the total amount of the six alkaloids was detected.

[0070] Table 8 Investigation results of the elution flow rate of Coptis chinensis

[0071]

[0072] From the above results, it can be seen that when the desorption flow rate is 3 times the column volume per hour, the alkaloid content in the eluent is low, and the desorption rate is low. When the desorption flow rate is 1 or 2 times the column volume per hour, the alkaloid content in the eluent is high and does not differ much. Considering the elution efficiency, the flow rate of 2 times the column volume per hour is preferred for desorption.

[0073] 1.9 Investigation of the amount of eluent

[0074] The Coptis chinensis sample was loaded into a treated macroporous resin column, and after 12 hours of standing, the column was eluted with the optimal amount of water, the water was discarded, and 40% ethanol was used for desorption. Six times the column volume was eluted, and the eluent was collected in portions, with each portion being 1 times the column volume. The total amount of the six alkaloids was detected.

[0075] Table 9 Investigation results of the amount of ethanol

[0076]

[0077] From the above results, it can be seen that when the amount of eluent is 4 times the column volume, the alkaloid desorption rate can reach 76.74%, and the amount of alkaloids in the eluent collected after 4 times is already very low. Considering the production cost, the amount of eluent is preferably 4 times the column volume.

[0078] In summary, the preferred preparation method for Coptis chinensis alkaloids is as follows. Coptis chinensis is taken, crushed to pass through an 8 mm sieve, and 10 times the amount of 70% ethanol is added. The mixture is heated to reflux and extracted twice, with each extraction lasting 1 hour. The filtrate is combined, the ethanol is recovered under reduced pressure, and the volume is concentrated to 0.5 g of crude drug per ml. The filtrate is centrifuged using a plate centrifuge or a tubular centrifuge. The maximum loading amount of 0.5 times the column volume is added to a treated D101 macroporous adsorption resin column, and the diameter-height ratio is 1:8. The loading flow rate is 1 times the column volume per hour, and the adsorption time is 12 hours. The column is eluted with water at a flow rate of 2 times the column volume per hour, and the water is discarded. The column is then desorbed with 40% ethanol at a flow rate of 2 times the column volume per hour, and 4 times the column volume is eluted. The eluent is collected, concentrated under reduced pressure, and dried to obtain a dry extract, which is pulverized to obtain the product.

[0079] Example 2 Investigation of key parameters in the preparation of evodia alkaloids

[0080] The process preferably proceeds as follows:

[0081] 2.1 Concentration temperature investigation of extract

[0082] The concentration temperature can have an impact on the final state of the extract, so the concentration temperature is optimized as a parameter.

[0083] Take the Evodia Rumpus decoction pieces, add 8 times the amount of 70% ethanol and heat to reflux extraction 3 times, 1 hour each time, filter the extract, combine, and divide into 3 parts, respectively at 50°C, 60°C and 70°C under reduced pressure to concentrate to 30%-50% alcohol content (50°C, the same below), then add water to 10% alcohol content (alcohol metering, the same below), room temperature overnight (16h), centrifugal take the precipitate, reduced pressure drying (65°C), detection content.

[0084] Table 10 Concentration temperature investigation results

[0085]

[0086] According to the results, the final dry powder state is not much different at each temperature, and the content of each component is slightly different, so as to get as much alkaloid as possible and reduce limonin, the concentration at 60°C is preferred.

[0087] 2.2 Investigation of alcohol content after adding water

[0088] After adding water, water precipitation is carried out, and the final alcohol content directly affects the yield and content of alkaloids, so the alcohol content at the end point is investigated.

[0089] Take 3 parts of Evodia Rumpus extract, concentrate to 30%-50% alcohol content at 60°C under reduced pressure, then add water to 5%, 10% and 15% alcohol content respectively, room temperature overnight (16h), centrifugal take the precipitate, reduced pressure drying (65°C), detection content.

[0090] Table 11 Investigation of alcohol content after adding water

[0091]

[0092] According to the results, the alkaloid content is not much different at each alcohol content, so the alcohol content is selected to be 5%-15% alcohol content.

[0093] 2.3 Investigation of standing settlement time

[0094] The settlement time can affect the yield and content of alkaloids, so the settlement time is explored.

[0095] Take 3 parts of extract, concentrate to 30%-50% alcohol content at 60°C under reduced pressure, then add water to 10% alcohol content, respectively room temperature for 8h, 16h and 24h, then centrifugal take the precipitate, reduced pressure drying (65°C), detection content.

[0096] Table 12 Results of standing settlement time investigation

[0097]

[0098] The length of water standing time has little effect on the dry powder state and the content of each component. There is little difference between 8-24 hours of settlement time. The water standing time can be selected as 8-24 hours.

[0099] 2.4 Investigation of standing settlement temperature

[0100] The settlement temperature can affect the content of alkaloids and limonin in the precipitate. Therefore, the effect of settlement temperature is investigated.

[0101] Three portions of the extract are concentrated under reduced pressure at 60°C to an alcohol content of 30%-50%, then water is added to an alcohol content of 10%. The mixtures are respectively left to stand at 4°C, 10°C and 25°C for 16 hours, then centrifuged to obtain the precipitate, which is dried under reduced pressure (65°C) and the content is detected.

[0102] Table 13 Results of standing settlement temperature investigation

[0103]

[0104]

[0105] The water standing settlement temperature has little effect on the dry powder state and the content of each component. Therefore, the settlement temperature is selected as 4°C-25°C.

[0106] In summary, the preferred extraction and purification method of evodia alkaloids is as follows: evodia is taken, 8 times the amount of 70% ethanol is added, heated and refluxed to extract 3 times, each time for 1 hour. The extract is filtered, combined, concentrated under reduced pressure to an alcohol content of 30%-50% (measured at 50°C), cooled to room temperature, then water is added to an alcohol content of 10% (measured by alcohol), left to stand and settle at 25°C for 8-24 hours, centrifuged by a flat plate centrifuge or a tubular centrifuge, the precipitate is taken, dried under reduced pressure, dry paste is obtained, and then it is crushed to obtain the product.

[0107] Example 3

[0108] Take Huanglian 21.5 kg, broken so that all through 8 mm screen, add 10 times the amount of 70% ethanol, heated reflux extraction 2 times, 1 hour each time, filter the extract, combined, reduced pressure recovery of ethanol and concentrated to 0.5 g crude drug / ml, centrifugation, get filtrate about 43 L, take filtrate 21.5 L added to the treated macroporous resin column (resin column and water exchange after the ratio of 1:8, 1 times the column volume is 43 L, i.e. 1 BV = 43 L, the same below), take the remaining filtrate added to the treated macroporous resin column 2 (resin column and water exchange after the ratio of 1:8), the flow rate control in 1 BV / h, after the completion of the sample adsorption 12 h, first with water wash elution 2 BV, elution flow rate 2 BV / h, discard the water wash, 40% ethanol desorption, desorption flow rate 2 BV / h, elution 4 BV, collect desorption liquid, reduced pressure concentration, drying, dry extract, crushing, obtained. UPLC detection of coptisine, epiberberine, jatrorrhizine, columbamine, berberine and palmatine content, and calculate the total amount, while testing moisture and extract rate, the results are as follows.

[0109] Table 14 Example 3 results

[0110]

[0111] Example 4

[0112] Take Huanglian 21.5 kg, broken so that all through 8 mm screen, add 10 times the amount of 70% ethanol, heated reflux extraction 2 times, 1 hour each time, filter the extract, combined, reduced pressure recovery of ethanol and concentrated to 0.5 g crude drug / ml, centrifugation, get filtrate about 43 L, take filtrate 21.5 L added to the treated macroporous resin column (resin column and water exchange after the ratio of 1:8, 1 times the column volume is 43 L, i.e. 1 BV = 43 L, the same below), take the remaining filtrate added to the treated macroporous resin column 2 (resin column and water exchange after the ratio of 1:8), the flow rate control in 1 BV / h, after the completion of the sample adsorption 12 h, first with water wash elution 2 BV, elution flow rate 2 BV / h, discard the water wash, 40% ethanol desorption, desorption flow rate 2 BV / h, elution 4 BV, collect desorption liquid, reduced pressure concentration, drying, dry extract, crushing, obtained. UPLC detection of coptisine, epiberberine, jatrorrhizine, columbamine, berberine and palmatine content, and calculate the total amount, while testing moisture and extract rate, the results are as follows.

[0113] Table 15 Example 4 results

[0114]

[0115] Example 5

[0116] Take Huanglian decoction pieces 21.5 kg, broken so that all through 8 mm screen, plus 10 times the amount of 70% ethanol, heated reflux extraction 2 times, 1 hour each time, extract the filtrate, combined, reduced pressure recovery of ethanol to 0.5 g crude drug / ml, centrifugal, get filtrate about 43, take filtrate 21.5 L added to the treated 1 macroporous resin column (resin column and water exchange after the ratio of 1:6, 1 times the column volume is 43 L, i.e. 1 BV = 43 L, same below), then take 21.5 L filtrate added to the treated 2 macroporous resin column (resin column and water exchange after the ratio of 1:6), the flow rate of loading control at 0.5 BV / h, after loading adsorption 4 h, first with water wash elution 1 BV, elution flow rate 1 BV / h, discard the water wash, 40% ethanol desorption, desorption flow rate 1 BV / h, elution 2 BV, collect desorption liquid, reduced pressure concentration, drying, dry extract, crushing, i.e. get. UPLC detection of berberine, epiberberine, jatrorrhizine, columbamine, berbamine and palmatine content, and calculate the total amount, while testing moisture and extract rate, the results are as follows.

[0117] Table 16 Example 5 results

[0118]

[0119] Example 6

[0120] Take Huanglian decoction pieces 21.5 kg, broken so that all through 8 mm screen, plus 10 times the amount of 70% ethanol, heated reflux extraction 2 times, 1 hour each time, extract the filtrate, combined, reduced pressure recovery of ethanol to 0.5 g crude drug / ml, centrifugal, get filtrate about 43, take filtrate 21.5 L added to the treated 1 macroporous resin column (resin column and water exchange after the ratio of 1:6, 1 times the column volume is 43 L, i.e. 1 BV = 43 L, same below), then take 21.5 L filtrate added to the treated 2 macroporous resin column (resin column and water exchange after the ratio of 1:6), the flow rate of loading control at 0.5 BV / h, after loading adsorption 4 h, first with water wash elution 1 BV, elution flow rate 1 BV / h, discard the water wash, 40% ethanol desorption, desorption flow rate 1 BV / h, elution 2 BV, collect desorption liquid, reduced pressure concentration, drying, dry extract, crushing, i.e. get. UPLC detection of berberine, epiberberine, jatrorrhizine, columbamine, berbamine and palmatine content, and calculate the total amount, while testing moisture and extract rate, the results are as follows.

[0121] Table 17 Example 6 results

[0122]

[0123] Example 7

[0124] Take 3.58 kg of Evodia Rutaecarpa slice, add 8 times the amount of 70% ethanol, heat and reflux extraction 3 times, 1 hour each time, filter the extract, and combine. Concentrate at 60°C under reduced pressure to 40% alcohol content (measured by alcohol at 50°C), cool to room temperature, add water to 10% alcohol content (measured by alcohol), and stand at 10°C for 16 hours. Centrifuge, take the precipitate, dry at 60°C under reduced pressure, grind, and obtain the product. UPLC detects the contents of dehydroevodiamine, evodiamine and rutaecarpin, calculates the total amount, and detects the content of limonin, the moisture content of the product and the extract yield, with the following results.

[0125] Table 18 Example 7 results

[0126]

[0127] Example 8

[0128] Take 3.58 kg of Evodia Rutaecarpa slice, add 8 times the amount of 70% ethanol, heat and reflux extraction 3 times, 1 hour each time, filter the extract, and combine. Concentrate at 60°C under reduced pressure to 40% alcohol content (measured by alcohol at 50°C), cool to room temperature, add water to 10% alcohol content (measured by alcohol), and stand at 10°C for 16 hours. Centrifuge, take the precipitate, dry at 60°C under reduced pressure, grind, and obtain the product. UPLC detects the contents of dehydroevodiamine, evodiamine and rutaecarpin, calculates the total amount, and detects the content of limonin, the moisture content of the product and the extract yield, with the following results.

[0129] Table 19 Example 8 results

[0130]

[0131] Example 9

[0132] Take 3.58 kg of Evodia Rutaecarpa slice, add 8 times the amount of 70% ethanol, heat and reflux extraction 3 times, 1 hour each time, filter the extract, and combine. Concentrate at 60°C under reduced pressure to 40% alcohol content (measured by alcohol at 50°C), cool to room temperature, add water to 10% alcohol content (measured by alcohol), and stand at 10°C for 16 hours. Centrifuge, take the precipitate, dry at 60°C under reduced pressure, grind, and obtain the product. UPLC detects the contents of dehydroevodiamine, evodiamine and rutaecarpin, calculates the total amount, and detects the content of limonin, the moisture content of the product and the extract yield, with the following results.

[0133] Table 20 Example 9 results

[0134]

[0135] Example 10

[0136] Pharmacodynamic experiment

[0137] ① Sample preparation

[0138] Preparation of the Coptis and Evodia alkaloid composition: the Coptis total alkaloid extract prepared in Example 3 and the Evodia alkaloid extract prepared in Example 6 were mixed in a ratio of 6:1 of crude drugs, respectively, to obtain the Coptis and Evodia alkaloid composition.

[0139] 2. Experimental method

[0140] Ten rats were randomly selected as the blank control group, and the rest of the animals were subjected to modeling. The rats in the modeling group were given 15 mL / kg of the reflux fluid by gavage twice a day, with an interval of 4 hours each time, for 7 consecutive weeks. The modeling rats were randomly divided into 7 groups, namely the model group, the positive drug aluminum magnesium carbonate group (0.6 g / kg), the Coptis and Evodia alkaloid composition 2 times equivalent dose group (2.2 g of crude drugs / kg, the recommended dose of Coptis and Evodia in humans is 12 g of crude drugs per day, which is equivalent to 1.1 g of crude drugs / kg in rats), the equivalent dose group (1.1 g of crude drugs / kg), the 1 / 2 equivalent dose group (0.6 g of crude drugs / kg), the 1 / 4 equivalent dose group (0.3 g of crude drugs / kg), and the 1 / 8 equivalent dose group (0.15 g of crude drugs / kg), with 10 rats in each group (9 rats in the 1 / 4 equivalent dose group and the 1 / 8 equivalent dose group), half male and half female. Each group was given the reflux fluid by gavage in the morning for continuous modeling, with a volume of 15 mL / Kg. Four hours after modeling each day, the corresponding drugs were administered to each group, and the animals in the blank control group and the model group were given 0.5% Tween 80 by gavage, and the animals in the rest of the groups were given the corresponding test products and positive drugs by gavage, with a volume of 10 mL / kg, for 14 consecutive days.

[0141] One hour after the last administration, the animals were anesthetized and bled, the stomach was cut along the greater curvature, the contents were washed out, the wet weight of the stomach was measured, and the gastric coefficient was calculated. The stomach was taken for histopathological examination.

[0142] 3. Experimental results

[0143] a. Gastric coefficient

[0144] Compared with the blank control group, the gastric coefficient of the rats in the model group was significantly increased (P<0.05), and there was no statistically significant difference in the gastric coefficient of the rats in the positive drug group and the test product groups compared with the model group. The results are shown in Table 20.

[0145] Table 21 Effect of the Coptis and Evodia alkaloid composition on the gastric coefficient of the reflux esophagitis model rats

[0146]

[0147] Note: Compared with the blank control group, ##P<0.01.

[0148] Compared with the blank control group, the stomach coefficient of the model group was significantly increased (P<0.05), and compared with the model group, the stomach coefficient of the positive drug group and the test product groups had no statistical difference.

[0149] b. Gastric histopathology examination

[0150]

Macroscopic observation

[0151] After the abdominal cavity of the animal was dissected, the stomach was taken, the outer membrane of the stomach was observed, the stomach was cut along the greater curvature, and the contents were washed away with physiological saline. The stomach mucosa of the blank control group had no abnormal changes in size, texture, and color, and the mucosa had no abnormal changes. Compared with the blank control group, the outer membrane of the stomach of the model group had no obvious changes in size, texture, and color, and the mucosa had no obvious changes.

[0152]

Microscopic observation

[0153] The blank control group had no obvious inflammatory cell infiltration in the gastric mucosa, and no increase in gastric mucous cells was observed. The model group had inflammatory cell infiltration in the gastric mucosa and submucosa, and an increase in mucus cells. Compared with the blank control group, the model group had a significantly increased degree of lesions (P<0.01). Compared with the model group, the positive drug aluminum magnesium carbonate group, the Huanglian Wumeiyu purified mixture ointment 2 times equivalent, and the equivalent dose group had a significantly reduced degree of inflammatory cell infiltration or gastric mucous cell increase in the gastric mucosa (P<0.05 or P<0.01). The Huanglian Wumeiyu purified mixture ointment 1 / 2 equivalent and 1 / 4 equivalent dose groups also had a certain effect on the degree of inflammatory cell infiltration or gastric mucous cell increase in the gastric mucosa. The results are shown in Table 22.

[0154] Table 22 Classification criteria for microscopic observation of gastric tissue

[0155]

[0156] Table 23 Effect of the test product on the degree of gastric lesions in the reflux gastritis model rats

[0157]

[0158]

[0159] Note: Compared with the blank group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0160] Compared with the blank control group, the model group animals had inflammatory cell infiltration in the gastric mucosa and submucosa, increased mucus cells, and significantly increased lesion degree (P<0.01). Compared with the model group, the positive drug aluminum magnesium carbonate group, the Huanglian Wuzhuyu purified mixture of ointment powder 2 times equivalent, equivalent dose group animals had significantly reduced inflammatory cell infiltration in the gastric mucosa or increased mucus cells in the gastric glands (P<0.05 or P≤0.01), and the Huanglian Wuzhuyu purified mixture of ointment powder 1 / 2 equivalent and 1 / 4 equivalent dose groups also had certain effects on the inflammatory cell infiltration in the gastric mucosa or the increased mucus cells in the gastric glands

[0161] ④Experimental conclusion

[0162] The Huanglian Wuzhuyu alkaloid composition has a significant improvement effect on the pathological changes of the gastric mucosa of the reflux gastritis model rats, and the effective dose is 0.3-2.2 g of crude drug / kg.

Claims

1. A method for preparing evodiamine, characterized by, Evodia fruit is added into ethanol, extracted by refluxing, the extract is filtered, ethanol is recovered, and a concentrated solution is obtained; water is added to reach an alcohol content of 5%-15%, and the mixture is allowed to stand and settle, centrifuged, and the precipitate is taken out and dried to obtain dry extract, which is crushed to obtain the product.

2. The method for preparing an Evodia rutaecarpa alkaloid according to claim 1, wherein: Evodia fruit is taken, 8 times the amount of 70% ethanol is added, and the mixture is extracted by refluxing for 3 times, 1 hour each time, the extract is filtered, combined, concentrated to an alcohol content of 30%-50%, cooled to room temperature, and then water is added to reach an alcohol content of 5%-15%, and the mixture is allowed to stand and settle, centrifuged, and the precipitate is taken out and dried to obtain dry extract, which is crushed to obtain the product.

3. The method for preparing an Evodia rutaecarpa alkaloid according to claim 1 or 2, wherein: Evodia fruit is taken, 8 times the amount of 70% ethanol is added, and the mixture is extracted by refluxing for 3 times, 1 hour each time, the extract is filtered, combined, concentrated to an alcohol content of 30%-50%, cooled to room temperature, and then water is added to reach an alcohol content of 5%-15%, and the mixture is allowed to stand and settle, centrifuged, and the precipitate is taken out and dried to obtain dry extract, which is crushed to obtain the product.

4. A method of preparing a combination of alkaloids of Coptidis Rhizoma and alkaloids of Evodiae Fructus, characterized in that, The method comprises the following steps: Preparation of alkaloids of Coptis: Coptis is taken, crushed to pass through an 8 mm sieve, and extracted by refluxing with 10 times the amount of 70% ethanol for 2 times, 1 hour each time, filtered, combined, and concentrated under reduced pressure to 0.5 g of crude drug per milliliter, and the supernatant is taken; the filtrate is loaded onto a pretreated macroporous adsorption resin column, eluted with ethanol, and the eluate is combined, concentrated under reduced pressure, and dried; the Coptis is selected from medicinal materials or decoction pieces; The preparation of alkaloids of Evodia fruit is as described in any one of claims 1-3.

5. The method for preparing a composition of coptis chinensis alkaloids and evodia rutaecarpa alkaloids according to claim 4, wherein: The preparation of alkaloids of Coptis comprises the following steps: Coptis is taken, crushed to pass through an 8 mm sieve, and extracted by refluxing with 10 times the amount of 70% ethanol for 2 times, 1 hour each time, filtered, combined, and concentrated under reduced pressure to 0.5 g of crude drug per milliliter, and the supernatant is taken; the filtrate is loaded onto a pretreated macroporous adsorption resin column, eluted with ethanol, and the eluate is combined, concentrated under reduced pressure, and dried; the Coptis is selected from medicinal materials or decoction pieces; Coptis is taken, crushed to pass through an 8 mm sieve, and extracted by refluxing with 10 times the amount of 70% ethanol for 2 times, 1 hour each time, filtered, combined, and concentrated under reduced pressure to 0.5 g of crude drug per milliliter, and the supernatant is taken; the filtrate is loaded onto a pretreated macroporous adsorption resin column, eluted with ethanol, and the eluate is combined, concentrated under reduced pressure, and dried; the Coptis is selected from medicinal materials or decoction pieces; 6. The method for preparing a composition of coptis chinensis alkaloids and evodia rutaecarpa alkaloids according to claim 5, wherein: The preparation of alkaloids of Coptis comprises the following steps: Coptis is taken, crushed to pass through an 8 mm sieve, and extracted by refluxing with 10 times the amount of 70% ethanol for 2 times, 1 hour each time, filtered, combined, and concentrated under reduced pressure to 0.5 g of crude drug per milliliter, and the supernatant is taken; the filtrate is loaded onto a pretreated macroporous adsorption resin column, eluted with ethanol, and the eluate is combined, concentrated under reduced pressure, and dried; the Coptis is selected from medicinal materials or decoction pieces; 7. The method for preparing a composition of coptis chinensis alkaloids and evodia rutaecarpa alkaloids according to claim 6, wherein: The diameter-height ratio of the resin column is 1:

8.

8. The method of claim 6, wherein the preparation of the combination of the alkaloids of Coptidis Rhizoma and the alkaloids of Evodiae Fructus is characterized by, The loading flow rate is 1 column volume per hour, and the adsorption time is 12 hours, and the elution flow rate is 2 column volumes per hour.

9. The method of claim 6, wherein the preparation of the combination of the alkaloids of Coptidis Rhizoma and the alkaloids of Evodiae Fructus is characterized by, The elution flow rate is 2 column volumes per hour, and the desorption flow rate is 2 column volumes per hour.

10. A pharmaceutical composition comprising alkaloids of Coptidis Rhizoma and alkaloids of Evodiae Fructus; the alkaloids of Coptidis Rhizoma are prepared by the method of any one of claims 4-9, the alkaloids of Evodiae Fructus are prepared by the method of any one of claims 1-3; the alkaloids of Coptidis Rhizoma and the alkaloids of Evodiae Fructus are mixed in a ratio of 6:1 by weight of crude medicinal materials.

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