CN116270632B - A herba Achillea Wilsonianae alkaloid composition with detoxified effect and its preparation method - Google Patents

Abstract

The invention relates to the technical field of biological medicine, in particular to an alpine yarrow herb alkaloid composition on detoxified snow and a preparation method thereof, wherein the alkaloid composition has good analgesic effect, large safety dose, easy quality control, simple preparation method, favorable industrial production and good application value, and can be prepared into any pharmaceutically acceptable dosage form for treating various pains in clinic. Meanwhile, on the basis of deep research on chemical components of the alpine yarrow herb on snow, the invention utilizes monoester type alkaloids and alcohol amine type alkaloids with high polarity to be easily dissolved in an acid solution, and the diester type alkaloids with low polarity are more easily extracted by an organic solvent, so that the monoester type alkaloids, the alcohol amine type alkaloids and the diester type alkaloids can be separated, the preparation process is simple, the quality control is easy, the preparation method is simple and convenient, the industrial production is facilitated, and the invention has good application value.

Description

A herba Achillea Wilsonianae alkaloid composition with detoxified effect and its preparation method

Technical Field

The invention relates to the technical field of biological medicines, in particular to a detoxified alpine yarrow herb alkaloid composition and a preparation method thereof.

Background

Herba Achillea Wilsonianae (Aconitum brachypodum Diels) belonging to genus Aconitum of Ranunculaceae, is rich in aconite diterpenoid alkaloids, and is commonly used for treating rheumatalgia, traumatic injury, and pain. The preparation of the alpine yarrow herb patent medicine is firstly received in the medicine standard of Yunnan province (1974 edition) and is received in the pharmacopoeia of the people's republic of China in 1977, and the preparation of the alpine yarrow herb tablet, the alpine yarrow herb total alkaloid injection and the like are available at present.

The caraway on snow belongs to a strictly controlled highly toxic drug, and the toxicity of the caraway mainly comes from esterification of 8 th and 14 th positions in aconitine structures to form a diester structure, so that diterpenoid alkaloids show high activity, but the safety window is narrow, namely the effective dose and the poisoning dose range are relatively close, and poisoning events easily occur in the use process. The other active ingredient in the artemisia rupestris is monoester alkaloid or alcohol amine alkaloid with low toxicity, namely 8-position and 14-position are not esterified or only one is esterified, although the activity of the active ingredient is reduced, the toxicity of the active ingredient is greatly reduced, namely the safety window of the active ingredient is very large, and drug poisoning accidents can not occur in the use process. The diester-type toxic alkaloids in the alpine yarrow herb are removed as much as possible, the monoester-type or alcohol-amine-type alkaloids are reserved, the monoester-type or alcohol-amine-type alkaloids are purified and refined, the influence of the diester-type alkaloids on the safety medication is removed as much as possible, the key and hot problems of the medicinal value of the alpine yarrow herb are reasonably developed and utilized, but no effective preparation and refining means exist in the prior art, and the improvement is needed.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide the detoxification artemisia rupestris alkaloid composition and the preparation method thereof, and the detoxification artemisia rupestris alkaloid composition and the preparation method thereof can effectively solve the problems in the prior art.

In order to achieve the technical effects, the invention adopts the following technical scheme:

In a first aspect, the invention provides a detoxified alpine yarrow herb alkaloid composition, which comprises a first component and a second component, wherein the first component is a diester diterpenoid alkaloid, and the mass of the first component is not higher than 10% of the mass of the detoxified alpine yarrow herb alkaloid composition.

Further, the second component includes monoester-type diterpenoid alkaloids and alcohol-amine-type diterpenoid alkaloids.

Further, the detoxified alpine yarrow herb alkaloid composition also comprises pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable auxiliary materials are selected from one or more of starch, beta-cyclodextrin, carbomer, microcrystalline cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, calcium carboxymethyl cellulose, polyethylene glycol (PEG), sodium carboxymethyl cellulose, methylcellulose, ethylcellulose, mannitol, sodium lauryl sulfate, sodium croscarmellose, lactose, polyvinylpyrrolidone (PVP), crosslinked polyvinylpyrrolidone, magnesium stearate, magnesium trisilicate, talcum powder, micro-powder silica gel, aspartame, sweet orange essence, sodium bicarbonate and/or sodium carbonate.

Further, the detoxified alpine yarrow herb alkaloid composition is prepared into any one of injection, oral preparation or external preparation.

Preferably, the composition is prepared into any one of injection, pill, tablet, paste, powder, decoction, granule, capsule and dripping pill.

In a second aspect, the invention also provides an application of the above-mentioned first aspect of the detoxified alpine yarrow herb alkaloid composition in preparing analgesic drugs.

In a third aspect, the present invention also provides a method for preparing the detoxified alpine yarrow herb alkaloid composition provided in the first aspect, comprising the steps of:

s1: pulverizing radix Aconiti Brachypodi, and sieving to obtain powder;

S2: wetting the medicinal powder by adding 3-5% sulfuric acid solution, adding into a percolating barrel, performing percolating extraction by using 1-2% sulfuric acid solution as an extracting agent, combining percolates, and filtering the percolates to obtain an extracting solution;

s3: first extraction:

Extracting the extracting solution for 3-6 times by adopting an extracting agent, wherein the extracting agent with the volume of 1/6-1/3 of the extracting solution is used for each extraction, and a water phase is reserved after the extraction;

s4: and (3) extracting for the second time:

Regulating the pH of the water phase to 8-10 by adopting an alkaline aqueous solution, extracting for 3-6 times by using an extracting agent, wherein the volume of the extracting agent is 1/6-1/3 of that of the water phase for each extraction, combining the obtained organic phases, concentrating under reduced pressure, and drying to obtain a crude product of the detoxified alpine yarrow herb alkaloid composition;

S5: purifying the crude product of the detoxified artemisia rupestris alkaloid composition to obtain the detoxified artemisia rupestris alkaloid composition.

Further, the extractant is any one or a combination of more of dichloromethane, chloroform, toluene and ethyl acetate.

Preferably, the extractant is chloroform.

Preferably, the powder of the medicinal material in S1 is sieved by a 40-mesh sieve.

Preferably, the dosage of the extractant in the S2 is 10-15 times (V/W) of the medicinal material powder.

Further, the alkaline aqueous solution in S4 is any one of sodium hydroxide, sodium carbonate, and potassium hydroxide, and is preferably sodium hydroxide.

Further, S5 is specifically:

Dissolving crude product of herba Achillea Wilsonianae alkaloid composition with ethanol, loading onto basic alumina chromatographic column, eluting with ethanol, collecting eluate containing alkaloid, concentrating under reduced pressure to form soft extract, and drying the soft extract to obtain herba Achillea Wilsonianae alkaloid composition.

Preferably, the dosage of the alkaline alumina chromatographic column is 9-15 times of the mass of the crude product of the alpine yarrow herb alkaloid composition on the detoxified snow.

Compared with the prior art, the invention has the beneficial effects that:

On the basis of intensive research on chemical components of the artemisia rupestris, the invention utilizes monoester type alkaloids and alcohol amine type alkaloids with high polarity to be easily dissolved in an acid solution, and diester type alkaloids with low polarity to be extracted by an organic solvent, so that the monoester type alkaloids, the alcohol amine type alkaloids and the diester type alkaloids can be separated. The invention provides a detoxified alpine yarrow herb alkaloid composition which has good analgesic effect, large safety dosage, easy quality control, simple preparation method, and good application value, is beneficial to industrial production, and can be prepared into any pharmaceutically acceptable dosage form for treating various pains in clinic.

Detailed Description

The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents or apparatus used, unless specifically stated otherwise, are those commercially available and the specific conditions in the examples are as per conventional conditions or as recommended by the manufacturer.

Example 1

This example is a first preparation example of the present invention, and the purpose of this example is to prepare a detoxified alpine yarrowia herb alkaloid composition, which mainly includes monoester alkaloid and alcohol amine alkaloid, and its preparation method is as follows:

S1: 50kg of the artemisia rupestris medicinal material is crushed and sieved by a 40-mesh sieve to obtain medicinal material powder;

s2: moistening the medicinal powder with 3% sulfuric acid (50L), adding into a percolating barrel, percolating with 10 times (500L) 1% sulfuric acid solution as extractant at flow rate of 0.5L/min, and mixing percolates; filtering to obtain extractive solution;

S3: extracting the extractive solution with chloroform for 5 times (100L each time), mixing the chloroform extractive solutions, concentrating under reduced pressure, and drying to obtain deep brown toxic alkaloid crude product (127.7 g);

S4: the pH of the water phase is adjusted to 8.5-9.5 by using 1% sodium hydroxide solution, and the water phase is extracted by using chloroform for 5 times (100L each time), the chloroform extracts are combined, concentrated under reduced pressure and dried to obtain the crude product (358.2 g) of the deep tan detoxified radix aconiti brachycarpa alkaloid composition.

S5: the obtained crude product of toxic alkaloid (127.7 g) is dissolved by 1.3L of ethanol, the solution is added on a 10 times amount (1277 g) alkaline alumina chromatographic column, the elution is carried out by ethanol, the eluent (22.1L) containing the alkaloid is collected, the concentration is reduced to thick paste, and the thick paste is dried, thus obtaining 95g of the pale brown yellow toxic detoxified alpine yarrow herb alkaloid composition (mainly diester type alkaloid).

Dissolving crude product (358.2 g) of the obtained detoxified herba Achillea Wilsonianae alkaloid composition with 4L ethanol, adding to 10 times (3582 g) of alkaline alumina chromatographic column, eluting with ethanol, collecting alkaloid-containing eluate (40.7L), concentrating under reduced pressure to soft extract, and drying to obtain 285.5g of light brown yellow detoxified herba Achillea Wilsonianae alkaloid composition (identified as monoester alkaloid and alcohol amine alkaloid).

Example 2

The present embodiment is a second preparation embodiment of the present invention, and the purpose of the present embodiment is to prepare a detoxified alpine yarrowia herb alkaloid composition, which mainly includes monoester alkaloid and alcohol amine alkaloid, and its preparation method is as follows:

S1: 50kg of the artemisia rupestris medicinal material is crushed and sieved by a 40-mesh sieve to obtain medicinal material powder;

S2: moistening the medicinal powder with 4% sulfuric acid (50L), adding into a percolating barrel, percolating with 12 times (600L) 1% sulfuric acid solution as extractant at flow rate of 0.5L/min, and mixing percolates; filtering to obtain extractive solution;

s3: extracting the extractive solution with chloroform for 5 times (100L each time), mixing the chloroform extractive solutions, concentrating under reduced pressure, and drying to obtain deep brown toxic alkaloid crude product (129.1 g);

S4: the pH of the water phase is adjusted to 8.5-9.5 by using 1% sodium hydroxide solution, and the water phase is extracted by using chloroform for 5 times (100L each time), the chloroform extracts are combined, concentrated under reduced pressure and dried to obtain the crude product (357.3 g) of the deep tan detoxified radix aconiti brachycarpa alkaloid composition.

S5: the obtained crude toxic alkaloid (129.1 g) is dissolved by 1.3L ethanol, the solution is added on a 10 times (1291 g) alkaline alumina chromatographic column, the elution is carried out by ethanol, the eluent (23.4L) containing the alkaloid is collected, the concentration is reduced to thick paste, and the thick paste is dried, thus obtaining 95g of the pale brown yellow toxic detoxified alpine yarrow herb alkaloid composition (mainly diester type alkaloid).

Dissolving crude product (357.3 g) of the obtained detoxified herba Achillea Wilsonianae alkaloid composition with 4L ethanol, adding to 10 times (3573 g) of alkaline alumina chromatographic column, eluting with ethanol, collecting eluate (42.1L) containing alkaloid, concentrating under reduced pressure to soft extract, and drying to obtain 290.4g of light brown yellow detoxified herba Achillea Wilsonianae alkaloid composition (with monoester type alkaloid and alcohol amine type alkaloid as main components).

Example 3

The embodiment is an animal experiment embodiment of the invention, and specifically comprises the following steps:

1. experimental animals and materials

Experimental animals: SPF grade kunming mice were used, supplied by the university of kunming medical university laboratory animal school, to produce license numbers: SCXK (Yunnan) K2020-0004, 18-22 g weight, male and female, and adapted to be fed for 3-5 days under experimental conditions.

A test material comprising:

Sample: the detoxified alpine yarrow herb alkaloid composition (the detoxified alpine yarrow herb alkaloid composition prepared in example 1 is taken and hereinafter referred to as the composition of the invention) and the toxic alpine yarrow herb alkaloid (the crude toxic alkaloid prepared in example 1 is taken and hereinafter referred to as the toxic alkaloid);

the positive medicine is prepared from aconitine:

Taking aconitine, toxic alkaloid and the composition of the invention respectively, adding concentrated hydrochloric acid-absolute ethanol solution (concentrated hydrochloric acid: absolute ethanol=1:20) for dissolution, and then adding 0.9% sodium chloride injection for preparing the solution with the required concentration.

2 Experimental methods

2.1 Half-lethal dose experiment

Through pre-experiments, aconitine, toxic alkaloids and the composition are administrated by intramuscular injection with the maximum concentration and the maximum volume, spontaneous movement of mice is reduced after 5min, respiratory distress, prone immobility and convulsion are caused, death begins to occur after 15min, and animal mortality is 100%. The dosing concentration was adjusted for pre-trials to give a dose Dm of 100% mice dead and a dose Dn of 0% mice dead, between which 5 dose groups were set, with 12 blank groups each. After administration, the mice were observed 1 time each day in the morning and afternoon, and the mice were continuously observed for 14 days, symptoms and death conditions of all mice were recorded, necropsy was performed on mice that died during the experimental period and mice that were sacrificed after the experimental period, and pathological changes of each organ were observed. The mouse LD50 and 95% confidence interval were calculated by Bliss method.

The acute toxicity experiment result of the mice shows that the main viscera are observed in an anatomical way after 14 days of administration, and no obvious abnormality is seen in the surviving administration group mice and the control group mice. The Bliss method is adopted to calculate that the LD50 value of aconitine mice intramuscular injection is 0.32mg/kg, and the 95% confidence interval is 0.26-0.42mg/kg; the LD50 value of intramuscular injection of toxic alkaloid mice is 5.68mg/kg, and the 95% confidence interval is 4.21-7.74mg/kg.

2.2 Maximum dose experiment

The maximum dose was determined by pre-experiments in which the composition had no death in the animals after intramuscular administration at the maximum concentration and maximum volume, and the LD50 and maximum tolerated dose in mice could not be determined. The Kunming mice were taken, each half of the male and female, and were divided into 2 groups, a composition group and a blank group, each group being 12. The composition was given intramuscular corresponding solutions and the blank control was given intramuscular blank solutions according to the maximum dose determined by the pre-experiment. Following dosing, 14 days of continuous observation was followed during normal diet. Symptoms of all mice were recorded, feed intake and animal body weight were measured at regular time, and after the experiment was completed, the animals were dissected and observed for pathological changes of various organs.

The administration is continuously carried out for 14 days, the poisoning symptoms and death conditions of the mice are not found, and the normal physiological characteristics of the mice such as autonomous activity, hair color, ingestion and the like are not found. After 14 days, the main viscera are observed through dissection, and no obvious abnormality is seen in the mice of the administration group and the mice of the control group. The final maximum intramuscular injection dose of the mice in the composition group is 212mg/kg.

2.3 Analgesic experiments

2.3.1 Acetic acid writhing experiment in mice

The Kunming mice were selected from the group consisting of male and female mice, randomly grouped, blank control group, positive control group (aspirin group), aconitine group, toxic alkaloid group, and composition group with low, medium, and high dosage, each group comprising 12 mice. Each group of mice was dosed at 0.1mL/10g volume, with a blank control group intramuscular injection of a blank solvent system; aspirin group mice were intraperitoneally injected with an aspirin solution at 600mg/kg body weight; the aconitine groups with low, medium and high dosages are respectively injected into the muscle of 0.01, 0.02 and 0.03 mg/kg; the low, medium and high dosage groups of the toxic alkaloid are respectively injected into the muscle according to 0.07, 0.14 and 0.21 mg/kg; the low, medium and high dose groups of the composition were administered by intramuscular injection at 5.94, 11.88 and 17.82mg/kg, respectively. The mice in each group were given 30min, 0.2 mL/mouse was intraperitoneally injected with 0.6% glacial acetic acid, the number of twists of the mice in 20min was recorded, and the percentage of pain inhibition in each group was calculated. Percent pain inhibition = (number of vehicle control-number of vehicle administration)/number of vehicle control x 100%.

Compared with the blank control group, the administration groups can reduce the twisting times of mice, wherein aconitine groups have high, medium and low dosage groups, the total and medium dosage groups have inhibiting effect on the twisting of mice caused by glacial acetic acid, and the difference has statistical significance (P is less than 0.05), and the experimental results are shown in table 1.

Table 1 influence of each drug on pain caused by acetic acid in mice (x.+ -. S)

Note that: ^*P＜0.05,^**P＜0.01,^*** P < 0.001 compared with the blank group

2.3.2 Mice Hot plate pain test

Female mice of Kunming species were taken and placed in a hot plate at 55.+ -. 0.5 ℃ to thermally stimulate the feet of the mice to produce a pain response (licking hind paw), the pain threshold of the mice (time to produce a pain response, i.e., licking hind paw) was measured, and qualified female mice were selected from among those that licked hind paw within 5s and those that did not lick hind paw or evade paw within 30 s. The eligible mice were randomly grouped into a blank control group, a positive control group (morphine control group), a toxic alkaloid group, a low, medium, and high dose group of the composition, and 12 mice per group. Two pain threshold values were measured before administration of each group of mice, and the average value was taken as the basal pain threshold value. All groups of mice are administrated by intramuscular injection with the volume of 0.1mL/10g, morphine control group is administrated by morphine solution according to the weight of 10mg/kg, and low, medium and high dosage groups of toxic alkaloids are administrated by intramuscular injection according to the volumes of 0.07, 0.14 and 0.21mg/kg respectively; the low, medium and high dose groups of the composition were administered by intramuscular injection at 5.94, 11.88 and 17.82mg/kg, respectively. The pain threshold was measured 1 time each 15min, 30min, 60min, 90min, 120min, 150min after administration. If the mice still do not respond after being placed in the hot plate for 60 seconds, the mice are taken out, the pain threshold is calculated by 60 seconds, and the improvement percentage of the pain threshold of each group is calculated. Percent improvement in pain threshold = (post-dose pain threshold-basal pain threshold)/basal pain threshold x 100%.

Compared with the basic pain threshold of the group, the pain threshold (P < 0.05) of mice pain caused by a hot plate can be obviously prolonged after the administration of the group with high total alkali and medium dosage for 15min, the pain threshold of each tested drug is in a decreasing trend along with the prolonged time after the administration of the group with 90min, and the experimental results are shown in table 2.

Table 2 influence of each drug on pain-causing action of mouse hotplate (x.+ -. S)

Note that: ^*P<0.05,^**P<0.01,^*** P <0.001 compared to the basal pain threshold of the group

In conclusion, the medicine of the detoxified artemisia rupestris alkaloid composition provided by the invention has the advantages of definite main components, low toxicity, good analgesic effect and good application value.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.

Claims (6)

1. An alpine yarrow herb alkaloid composition on detoxified snow, which is characterized in that: the composition comprises a first component and a second component, wherein the first component is diester diterpenoid alkaloids, and the mass of the first component is not higher than 10% of the mass of the detoxification artemisia rupestris alkaloid composition; the second component is monoester diterpenoid alkaloids and alcohol amine diterpenoid alkaloids; the preparation method of the composition comprises the following steps:

s1: pulverizing radix Aconiti Brachypodi, and sieving to obtain powder;

s2: wetting the medicinal powder by adding 3-5% sulfuric acid solution, adding into a percolating barrel, performing percolating extraction by using 1-2% sulfuric acid solution as an extracting agent, combining percolates, and filtering the percolates to obtain an extracting solution;

s3: first extraction:

extracting the extracting solution for 3-6 times by adopting an extracting agent, wherein the extracting agent with the volume of 1/6-1/3 of the extracting solution is used for each extraction, and a water phase is reserved after the extraction;

s4: and (3) extracting for the second time:

Regulating the pH of the water phase to 8-10 by adopting an alkaline aqueous solution, extracting for 3-6 times by using an extracting agent, wherein the volume of the extracting agent is 1/6-1/3 of that of the water phase for each extraction, combining the obtained organic phases, concentrating under reduced pressure, and drying to obtain a crude product of the detoxified alpine yarrow herb alkaloid composition;

S5: purifying the crude product of the detoxified artemisia rupestris alkaloid composition to obtain the detoxified artemisia rupestris alkaloid composition; the method comprises the following steps:

Dissolving crude product of herba Achillea Wilsonianae alkaloid composition with ethanol, loading onto basic alumina chromatographic column, eluting with ethanol, collecting eluate containing alkaloid, concentrating under reduced pressure to form soft extract, and drying the soft extract to obtain herba Achillea Wilsonianae alkaloid composition.

2. A detoxified alpine yarrow herb alkaloid composition of claim 1, wherein: the extractant is chloroform.

3. A detoxified alpine yarrow herb alkaloid composition of claim 1, wherein: the detoxified artemisia rupestris alkaloid composition also comprises pharmaceutically acceptable auxiliary materials.

4. A detoxified alpine yarrow herb alkaloid composition of claim 3, wherein: the pharmaceutically acceptable auxiliary materials are selected from one or more of starch, beta-cyclodextrin, carbomer, microcrystalline cellulose, hydroxypropyl methyl cellulose, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium, polyethylene glycol, sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, mannitol, sodium dodecyl sulfate, crosslinked sodium carboxymethyl cellulose, lactose, polyvinylpyrrolidone, crosslinked polyvinylpyrrolidone, magnesium stearate, magnesium trisilicate, talcum powder, micro-powder silica gel, aspartame, sweet orange essence, sodium bicarbonate and/or sodium carbonate.

5. A detoxified alpine yarrow herb alkaloid composition of claim 4, wherein: the detoxified alpine yarrow herb alkaloid composition is prepared into any one of injection, oral administration or external use.

6. Use of a detoxified alpine yarrow herb alkaloid composition according to any one of claims 1 to 5 in the preparation of analgesic drugs.