CN113144020A

CN113144020A - Traditional Chinese medicine composition for treating arthralgia, preparation method thereof and traditional Chinese medicine preparation

Info

Publication number: CN113144020A
Application number: CN202110515862.8A
Authority: CN
Inventors: 李平; 王兴旺; 朱一强
Original assignee: Hainan Spider King Biotechnology Co ltd
Current assignee: Hainan Spider King Biotechnology Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2021-07-23

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating arthralgia, a preparation method thereof and a traditional Chinese medicine preparation. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-10 parts of spider freeze-dried powder, 10-30 parts of radix angelicae pubescentis, 10-30 parts of notopterygium root, 10-20 parts of radix clematidis, 1-10 parts of asarum, 15-25 parts of pawpaw, 5-15 parts of pseudo-ginseng, 5-20 parts of safflower carthamus, 10-20 parts of ligusticum wallichii, 10-20 parts of angelica sinensis, 1-5 parts of cinnamon oil, 15-25 parts of menthol and 1-10 parts of borneol. The traditional Chinese medicine composition has scientific compatibility, can be used for treating various arthralgia syndromes (rheumatism, gout, bone pain and the like), and has remarkable curative effect; the preparation method of the traditional Chinese medicine composition can extract active ingredients to the maximum extent, and ensures the curative effect of the composition.

Description

Traditional Chinese medicine composition for treating arthralgia, preparation method thereof and traditional Chinese medicine preparation

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating arthralgia, a preparation method thereof and a traditional Chinese medicine preparation.

Background

After thousands of years of inheritance, the spider is regarded as a "strong warrior totem" by the ancestors of Li nationality in Hainan, and is endowed with the original worship of mystery and strength. The spiders extinguish wind and relieve spasm, relieve swelling and detoxify, dispel wind and remove dampness, and dredge collaterals and relieve pain, have extremely high medicinal value and long history of being used as medicines, and have obvious effect of treating various rheumatic arthralgia.

The spiders of Wuzhishan are used as local medicinal materials in Hainan province, can promote blood circulation to remove blood stasis, diminish inflammation, remove toxicity and relieve pain, can treat various diseases such as rheumatic ostealgia, oblique middle wind gap and the like when used, and are recorded in various counties, medical records and the like, wherein Shansheng is doubly big. People in Li nationality in Hainan have the effects of dispelling wind and removing dampness by eating roasted spiders or soaking spiders in wine and the like in the ancient way, and strengthening the body; spiders are also used as monarch drugs in the local Li doctor in Wuzhishan mountain for treating gout, rheumatism, joint swelling and pain, neck and waist soreness and other diseases, and the curative effect is highly approved by local patients; modern dao Huang Xiong, who wen Yi in generations, especially excels in treating joint wind (local red, swollen, painful, movement disorder of joints, etc.) by using cutworm (spider in five fingers) as a medicine and soaking in wine, is deeply trusted by patients and is valued as dao Huang Xiong (dao Yao Wang); the Chenpejia physician of Chongshan health institute in Wuzhishan city takes the spider powder as the monarch drug and mainly treats carbuncle and furuncle; the military medical science clozendon (the deputy group of Li medicine general survey in Hainan province) in China's liberation army 162 hospital treats acute and chronic otitis media with spider (cutworm); the externally applied rheumatism tincture prescription of professor of Hualiangchi of famous Chinese medicine in China is based on folk prescriptions of Li nationality in Wuzhishan area, and is mainly used for treating swelling pain, inconvenient movement and the like caused by gout, rheumatism, rheumatoid and neck and lumbar diseases by taking spider powder as a monarch drug, so that the externally applied rheumatism tincture prescription is a finishing and improvement on the traditional Li medical prescriptions. At present, the spider powder traditional Chinese medicine preparation with a remarkable effect of treating rheumatic arthralgia still needs to be prepared.

Disclosure of Invention

In view of the above, the invention provides a traditional Chinese medicine composition for treating arthralgia, a preparation method thereof and a traditional Chinese medicine preparation. The Chinese medicinal composition can be used for treating various arthralgia syndromes (rheumatism, gout, bone pain and the like) and has a remarkable curative effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a traditional Chinese medicine composition for treating arthralgia, which is prepared from the following raw materials in parts by weight:

1-10 parts of spider freeze-dried powder, 10-30 parts of radix angelicae pubescentis, 10-30 parts of notopterygium root, 10-20 parts of radix clematidis, 1-10 parts of asarum, 15-25 parts of pawpaw, 5-15 parts of pseudo-ginseng, 5-20 parts of safflower carthamus, 10-20 parts of ligusticum wallichii, 10-20 parts of angelica sinensis, 1-5 parts of cinnamon oil, 15-25 parts of menthol and 1-10 parts of borneol.

At present, the Li medicine spider has scarce resources, the proved recipe is mainly transmitted orally, and the scattering folks are not systematic, and the research foundation of a scientific system is not provided. The invention provides a pain relieving preparation containing dao-drug spider for treating various arthralgia syndromes (rheumatism, gout, ostealgia and the like) and a preparation method thereof, wherein the pain relieving preparation is prepared by matching other traditional Chinese medicinal materials such as radix angelicae pubescentis, notopterygium root and the like according to the principle of monarch, minister, assistant and guide synergistic promotion of traditional Chinese medicines by applying the current research means on the basis of improvement of folk prescription arrangement of the dao-drug spider.

The monarch drug in the prescription is spider and pubescent angelica root. Spider-animals, being good at circulating up and down, can not only dispel wind and remove dampness, dredge collaterals and relieve pain, but also counteract poison with poison, thereby removing toxicity and killing parasites from outside to inside. The pubescent angelica root, radix angelicae pubescentis, pungent and warm, fragrant and dry, good in nature and capable of moving downward, and mainly dispersing latent wind and cold-dampness in the interior to relieve arthralgia and pain, the two medicines are matched, although the medicine properties are slightly different, the functions are similar, and are mutually restricted, so that the medicine is very good and safe to use, and has the effects of dispelling wind, removing dampness, dredging collaterals and relieving pain, so the medicine is the monarch medicine in the formula.

The ministerial drugs of the prescription are notopterygium root, clematis root, asarum and pawpaw. Notopterygium root, rhizoma Et radix Notopterygii, being pungent, warm, fragrant and dry, has ascending and dispersing properties, and can dispel wind-cold and dampness, and relieve arthralgia and alleviate pain. Clematis chinensis, radix Clematidis, with pungent and warm properties, moves vigorously and well and passes through twelve meridians, is the essential herb for treating arthralgia due to wind-dampness. Chaenomeles fruit, sour in flavor, enters liver, warm in nature and not dry, excels in relaxing tendons and activating collaterals to relieve spasm, so it is the essential herb for chronic arthralgia due to wind-damp and spasm of tendons and vessels. Asarum herb is a pungent and warm product, and it can move with fragrance and penetrate the exterior and interior, and has strong cold-dispelling and pain-relieving effects. The four drugs listed above strengthen the effect of the monarch drug from different angles, so they are ministerial drugs.

The adjuvant drugs of the prescription are pseudo-ginseng, szechuan lovage rhizome, Chinese angelica and safflower. Pseudo-ginseng is sweet in taste and warm in nature, good at stopping bleeding, good at removing blood stasis and long-lasting in relieving pain, has outstanding drug effect, has the characteristics of stopping bleeding without remaining blood stasis and not damaging vital qi by removing blood stasis, is good as a blood-syndrome-causing medicine and is an essential medicine for traumatology. Chuan Xiong is pungent and warm in property, and can activate blood, move qi and alleviate pain, so it is a "qi-flowing herb in blood". The angelica has sweet and heavy flavor, can enrich the blood, has good functions of enriching the blood, promoting blood circulation and relieving pain, can tonify the middle-jiao and tonify the middle-jiao, and is good for qi and medicine in blood. Safflower has the functions of dredging the channels and activating collaterals, clearing and activating the channels and collaterals, dissipating blood stasis and resolving masses, relieving swelling and pain, and activating blood and nourishing blood. The combination of the four medicines has three meanings: firstly, the rheumatism is not cured for a long time, so that the deficiency of qi and blood and the malnutrition of tendons and vessels are caused, and the blood-nourishing and blood-activating products are matched to give consideration to the evil and the body resistance and enhance the pathogenic force, and secondly, the blood-activating and qi-moving products are matched to promote blood circulation and promote qi circulation, so that the qi circulation can promote blood circulation, and the blood circulation can promote wind dispersion, so that the effects of treating wind first and treating blood and self-extinguishing blood circulation can be achieved, and the effects of relieving swelling and pain can be achieved, so that the traditional Chinese medicine is used as an adjuvant medicine in the formula.

The prescription has the guiding drugs of borneol, menthol and cinnamon oil. Borneol is pungent and fragrant, goes up and down completely, is ubiquitous, and has the characteristics of dispersing and refreshing, removing nausea and promoting tissue regeneration. Cinnamon oil is pure yang in nature, fragrant and transparent, and has the functions of warming and activating yang qi and promoting qi and blood growth when being matched with a small amount of cinnamon oil. Menthol is pungent and cool in flavor, has faint scent, can pass through the interior and exterior, and can relieve depression, stagnation and penetrate to the triple energizer. The three medicines are used together, can directly reach the disease focus through transdermal absorption, and can also use the cool and moist properties of borneol and peppermint oil to relieve the pungent, warm, fragrant and dry properties of pubescent angelica root, notopterygium root, clematis root, asarum and the like, so that the medicine is safe to use, protects the skin, can eliminate swelling and pain through the transdermal property, and is used as a guiding medicine in the formula.

Preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight:

1.5-6 parts of spider freeze-dried powder, 15-24 parts of radix angelicae pubescentis, 15-24 parts of notopterygium root, 15-18 parts of radix clematidis, 4-10 parts of asarum, 20 parts of pawpaw, 9-12 parts of pseudo-ginseng, 8-15 parts of safflower carthamus, 15-18 parts of ligusticum wallichii, 5-18 parts of angelica sinensis, 2-4 parts of cinnamon oil, 20 parts of menthol and 5 parts of borneol.

3 parts of spider freeze-dried powder, 18 parts of radix angelicae pubescentis, 18 parts of notopterygium root, 18 parts of radix clematidis, 6 parts of asarum, 20 parts of pawpaw, 9 parts of pseudo-ginseng, 12 parts of safflower, 18 parts of ligusticum wallichii, 18 parts of angelica sinensis, 2 parts of cinnamon oil, 20 parts of menthol and 5 parts of borneol.

The invention also provides a preparation method of the traditional Chinese medicine composition, which comprises the following steps:

the method comprises the following steps: mixing radix angelicae pubescentis, angelica sinensis, ligusticum wallichii, notopterygium root and asarum, adding water with the weight 5-7 times that of the mixture, soaking for 20-40 minutes, and heating and extracting for 2-4 hours by adopting a steam method to obtain volatile oil, volatile oil water liquid and medicine residues;

mixing the dregs, pawpaw, clematis root, pseudo-ginseng and safflower, adding 50% -90% ethanol water solution with the weight being 8-12 times of that of the mixture, and carrying out heating reflux extraction for 1-3 times, wherein the heating reflux extraction time for each time is 1-2 hours, so as to obtain an ethanol extract;

mixing the volatile oil water solution and the alcohol extract, concentrating until the density is 1.03-1.09 (60 ℃), adding ethanol until the alcohol content is 60-80%, and standing for 12-48 hours; taking the supernatant, adding borneol and menthol to obtain a first mixture;

step two: adding 30-70% ethanol aqueous solution which is 30-50 times of the weight of the spider freeze-dried powder into the spider freeze-dried powder, and performing cold-leaching extraction for 1-3 times, wherein the time of cold-leaching extraction for each time is 24-120 hours, so as to obtain a spider extracting solution;

step three: dissolving the volatile oil and cinnamon oil in ethanol with the weight of 4-6 times of that of the volatile oil and cinnamon oil to obtain a second mixture;

step IV: mixing the first mixture, the spider extract and the second mixture, standing at 0-4 ℃ for 12-48 hours, and filtering to obtain the traditional Chinese medicine composition.

Preferably, in step (i): mixing radix Angelicae Pubescentis, radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, Notopterygii rhizoma, and herba asari, adding 6 times of water, soaking for 30 min, and extracting by steam heating for 4 hr to obtain volatile oil, volatile oil water solution and residue;

mixing the residues, fructus Chaenomelis, radix Clematidis, Notoginseng radix and Carthami flos, adding 10 times of 70% ethanol water solution, heating and reflux extracting for 2 times, wherein the time for each heating and reflux extraction is 1.5 hr to obtain ethanol extractive solution;

mixing the volatile oil water solution and the ethanol extract, concentrating to density of 1.03(60 deg.C), adding ethanol until the ethanol content is 70%, and standing for 24 hr.

Preferably, in step two: adding 30% ethanol aqueous solution 40 times the weight of the spider freeze-dried powder, and cold-soaking and extracting for 2 times, wherein the cold-soaking extraction time is 72 hours each time.

Preferably, in step (iv): mixing the first mixture, the spider extract and the second mixture, and standing at 0-4 ℃ for 12-18 hours.

Preferably, the crushing degree of the radix angelicae pubescentis, the angelica sinensis and the ligusticum wallichii is 0-0.8 cm.

Preferably, notopterygium root and asarum are prepared into decoction pieces.

The invention also provides application of the traditional Chinese medicine composition in preparing a traditional Chinese medicine preparation for treating arthralgia.

In embodiments provided herein, the arthromyodynia is one of rheumatism, gout, or bone pain. The present invention is not limited thereto, and those skilled in the art will recognize types of arthralgia syndrome as falling within the scope of the present invention.

The invention also provides a traditional Chinese medicine preparation for treating arthralgia, which comprises the traditional Chinese medicine composition.

Preferably, the alcohol concentration of the Chinese medicinal preparation is 50-70%.

Preferably, the alcohol concentration of the Chinese medicinal preparation is 55-65%.

Preferably, the pH value of the Chinese medicinal preparation is 5.0-6.0.

The invention provides a traditional Chinese medicine composition for treating arthralgia, a preparation method thereof and a traditional Chinese medicine preparation. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-10 parts of spider freeze-dried powder, 10-30 parts of radix angelicae pubescentis, 10-30 parts of notopterygium root, 10-20 parts of radix clematidis, 1-10 parts of asarum, 15-25 parts of pawpaw, 5-15 parts of pseudo-ginseng, 5-20 parts of safflower carthamus, 10-20 parts of ligusticum wallichii, 10-20 parts of angelica sinensis, 1-5 parts of cinnamon oil, 15-25 parts of menthol and 1-10 parts of borneol. The invention has the technical effects that:

the traditional Chinese medicine composition has scientific compatibility, can be used for treating various arthralgia syndromes (rheumatism, gout, bone pain and the like), and has remarkable curative effect;

the preparation method of the traditional Chinese medicine composition can extract active ingredients to the maximum extent, and ensures the curative effect of the composition.

Drawings

FIG. 1 shows the effect of different extraction times on the extraction rate of volatile oil from Notopterygium incisum;

FIG. 2 shows the effect of different extraction times on the extraction rate of volatile oil from Asarum sieboldii;

FIG. 3 shows the effect of different extraction times on the extraction rate of volatile oil from Heracleum hemsleyanum;

FIG. 4 shows the effect of different extraction times on the extraction yield of volatile oil from Angelica sinensis;

FIG. 5 shows the effect of different extraction times on the extraction rate of volatile oil from Ligusticum chuanxiong Hort;

FIG. 6 shows that the color of ninhydrin is developed after spider extract is developed twice with n-butanol-water-glacial acetic acid (8: 1: 1); 1: water extraction; 2: extracting with 30% ethanol; 3: extracting with 50% ethanol; 4: extracting with 70% ethanol; 5: extracting with 90% ethanol.

Detailed Description

The invention discloses a traditional Chinese medicine composition for treating arthralgia, a preparation method thereof and a traditional Chinese medicine preparation, and a person skilled in the art can realize the treatment by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The raw materials, reagents and instruments used in the present invention are commercially available.

The standard of medicinal materials is as follows: the spider medicinal materials in the prescription only have Hainan medicinal material standards, and other medicinal materials have 2015 edition Chinese pharmacopoeia standards. All the medicinal materials meet the standard through detection.

The invention is further illustrated by the following examples:

example 1

The prescription (I):

(II) preparation method:

pulverizing radix Angelicae Pubescentis, radix Angelicae sinensis, and rhizoma Ligustici Chuanxiong into coarse powder, adding 6 times of water into five medicinal materials including Notopterygii rhizoma and herba asari, soaking for 0.5 hr, extracting volatile oil for 4 hr, and collecting volatile oil and volatile oil water solution; adding fructus Chaenomelis, radix Clematidis, Notoginseng radix (coarse powder) and Carthami flos into the residue, adding 10 times of 70% ethanol solution, heating and extracting for 2 times, each time for 1.5 hr, and collecting ethanol extractive solution; mixing the volatile oil water solution and the alcohol extract, concentrating to a density of about 1.03g/mL (60 ℃), adding ethanol until the alcohol content is 70%, and standing for 24 hours; taking the supernatant, adding borneol and menthol, and moderately shaking for complete dissolution;

② adding 40 times of 30 percent ethanol into the spider freeze-dried powder for cold-soaking and extracting for 2 times, each time for 72 hours, and merging the spider extract;

dissolving the volatile oil and the cinnamon oil by using a proper amount of ethanol;

fourthly, the three groups of solutions are combined, refrigerated, kept overnight, filtered, and the filtrate is added with water to reach the constant volume of 1000mL, thus obtaining the product.

Test example 1 examination of extraction Process

Process for extracting volatile oil

1. Single-factor investigation of single-ingredient medicinal material

1.1 Single factor investigation of Notopterygium incisum medicinal materials

1.1.1 investigating the degree of pulverization

Pulverizing Notopterygii rhizoma decoction pieces to coarse powder, and medium powder respectively. Weighing 100g of notopterygium root decoction pieces, 100g of notopterygium root coarse powder and 100g of notopterygium root medium powder, respectively adding 5 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, heating and extracting volatile oil for 5 hours by adopting a water vapor method, collecting the volatile oil, determining the volume of the volatile oil and calculating the extraction rate of the volatile oil, wherein the result is as follows:

TABLE 1

Degree of pulverization of medicinal materials	Decoction pieces	Coarse powder	Coarse powder	Medium powder
					Extraction ratio of volatile oil (%)	2.10	1.80	1.50	1.20

The above results show that the notopterygium root is prepared by extracting volatile oil from decoction pieces.

1.1.2 investigating the amount of water added

According to the research results, taking 4 groups of notopterygium root decoction pieces, each group is 100g, adding 3, 5, 7 and 9 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, respectively, extracting volatile oil by heating by adopting a steam method, collecting the volatile oil and calculating the extraction rate of the volatile oil:

TABLE 2

Adding water (times)	3	5	7	9
					Extraction ratio of volatile oil (%)	-	2.20	2.0	2.0

The above results indicate that 3 times of water can not completely wet the medicinal materials, and the medicinal materials will be burned in the extraction process. 5 times of water is used for extracting volatile oil, and the extraction rate is the maximum.

1.1.3 investigation of soaking time

According to the research results, 4 groups of notopterygium root decoction pieces are taken, 100g of each group is added with 5 times of water, the decoction pieces are soaked for 0 hour (immediately heated after being added with water), 0.5 hour, 1 hour and 2 hours respectively, the volatile oil is extracted by heating by adopting a steam method according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, the volatile oil is collected, and the extraction rate of the volatile oil is calculated:

TABLE 3

Soaking time (h)	0	0.5	1	2
					Extraction ratio of volatile oil (%)	2.4	2.1	2.0	1.2

The results show that the medicinal materials can be used for extracting the volatile oil without soaking, and the extraction rate is the maximum.

1.1.4 investigation of extraction time

According to the research results, referring to the volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, 100g of notopterygium root decoction pieces are taken, 5 times of water is added, then the decoction pieces are immediately heated to extract the volatile oil, and the volume of the volatile oil is read and the extraction rate of the volatile oil is calculated after 0.5h, 1h, 2h, 3h, 4h, 5h, 6h and 7h from the beginning of boiling:

TABLE 4

Extraction time (h)	0.5	1	2	3	4	5	6	7
									Extraction ratio of volatile oil (%)	1.0	1.60	2.20	2.60	2.60	2.60	2.60	2.60

The volatile oil amount is basically stable in 3 hours, and the oil amount is not increased any more. By making a scattergram of the effect of extraction time and volatile oil extraction rate, the results show that 3 hours is the "inflection point" of the experiment, and thus the optimal extraction time is 3 hours (fig. 1).

1.2 Single factor investigation of Asarum herb

1.2.1 investigating the degree of pulverization

Pulverizing herba asari decoction pieces to coarse powder, and medium powder, respectively. Weighing 100g of asarum decoction pieces, 100g of middlings powder and 100g of medium powder, respectively adding 5 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia, heating and extracting volatile oil for 5 hours by adopting a water vapor method, collecting the volatile oil, determining the volume of the volatile oil and calculating the extraction rate of the volatile oil, wherein the result is as follows:

TABLE 5

Degree of pulverization of medicinal materials	Decoction pieces	Coarse powder	Coarse powder	Medium powder
					Extraction ratio of volatile oil (%)	2.10	1.90	1.60	0.3

The above results show that the asarum is obtained by extracting the volatile oil from the decoction pieces.

1.2.2 investigating the amount of water added

According to the research results, taking 4 groups of notopterygium root decoction pieces, wherein each group is 50g, respectively adding 3 times, 5 times, 7 times and 9 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, heating and extracting volatile oil by adopting a water vapor method, collecting the volatile oil and calculating the extraction rate of the volatile oil:

TABLE 6

Adding water (times)	3	5	7	9
					Extraction ratio of volatile oil (%)	1.70	2.10	0.9	0.8

The above results show that the extraction rate is the maximum when 5 times of water is used for extracting volatile oil.

1.2.3 investigation of soaking time

According to the research results, taking 4 groups of asarum decoction pieces, each group of 50g, adding 5 times of water, soaking for 0 hour (immediately heating after adding water), 0.5 hour, 1 hour and 2 hours respectively, extracting volatile oil by heating by adopting a steam method according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, collecting the volatile oil and calculating the extraction rate of the volatile oil:

TABLE 7

Soaking time (h)	0	0.5	1	2
					Extraction ratio of volatile oil (%)	2.2	2.2	1.3	0.2

1.2.3 examining extraction time

According to the research results, referring to the volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, 50g of asarum decoction pieces are taken, 5 times of water is added, then the volatile oil is immediately heated and extracted, the volume of the volatile oil is read and the extraction rate of the volatile oil is calculated from boiling by 0.5h, 1h, 2h, 3h, 4h, 5h, 6h and 7 h:

TABLE 8

The extraction rate of the volatile oil in the first 1 hour is high, the volatile oil is basically stable in 3 hours, and the amount of the volatile oil is not increased any more. By making a scattergram of the effect of extraction time and volatile oil extraction rate, the results show that 3 hours is the "inflection point" of the experiment, and thus the optimal extraction time is 3 hours (fig. 2).

1.3 Single factor investigation of radix Angelicae Pubescentis medicinal materials

1.3.1 investigating the degree of pulverization

Pulverizing radix Angelicae Pubescentis decoction pieces to coarse powder, and medium powder, respectively. Weighing 100g of notopterygium root decoction pieces, 100g of middlings powder and 100g of medium powder, respectively adding 5 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia, heating and extracting volatile oil for 5 hours by adopting a water vapor method, collecting the volatile oil, determining the volume of the volatile oil and calculating the extraction rate of the volatile oil, wherein the result is as follows:

TABLE 9

Degree of pulverization of medicinal materials	Decoction pieces	Coarse powder	Coarse powder	Medium powder
					Extraction ratio of volatile oil (%)	0.25	0.3	0.2	0.1

The results show that the radix angelicae pubescentis contains a small amount of volatile oil, the difference between the decoction pieces and the coarsest powder is not large, and the coarsest powder is tentatively the optimal extraction particle size.

1.3.2 investigating the amount of water added

According to the research results, taking 4 groups of coarse powder of the pubescent angelica root, wherein each group is 100g, respectively adding 3 times, 5 times, 7 times and 9 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, heating and extracting volatile oil by adopting a water vapor method, collecting the volatile oil and calculating the extraction rate of the volatile oil:

watch 10

Adding water (times)	3	5	7	9
					Extraction ratio of volatile oil (%)	-	0.3	0.25	0.3

1.3.3 investigation of soaking time

According to the research results, 4 groups of coarse powder of the radix angelicae pubescentis are taken, 100g of each group is added with 5 times of water, the mixture is soaked for 0 hour (immediately heated after being added with water), 0.5 hour, 1 hour and 2 hours respectively, the volatile oil is extracted by heating through a steam method according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, the volatile oil is collected, and the extraction rate of the volatile oil is calculated:

TABLE 11

Soaking time (h)	0	0.5	1	2
					Extraction ratio of volatile oil (%)	0.3	0.25	0.25	0.2

1.3.4 investigation of extraction time

According to the research results, referring to the volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, 100g of notopterygium root decoction pieces are taken, 5 times of water is added, then the decoction pieces are immediately heated to extract the volatile oil, the volume of the volatile oil is read and the extraction rate of the volatile oil is calculated after 0.5h, 1h, 2h, 3h, 4h, 5h, 6h and 7h from the beginning of boiling:

TABLE 12

Extraction time (h)	0.5	1	2	3	4	5	6	7
									Extraction ratio of volatile oil (%)	0.1	0.1	0.2	0.25	0.30	0.3	0.3	0.3

The radix angelicae pubescentis contains less volatile oil, is basically stable in 4 hours, and the volatile oil amount is not increased any more. By making a scattergram of the effect of extraction time and volatile oil extraction rate, the results show that 4 hours is the "inflection point" of the experiment, and thus the optimal extraction time is 4 hours (fig. 3).

1.4 Single factor investigation of Angelica sinensis

1.4.1 investigating the degree of pulverization

The angelica medicinal material is soft and difficult to crush the Chinese angelica powder, so the angelica decoction pieces are respectively crushed into coarse powder and coarse powder. Weighing 50g of angelica decoction pieces, 50g of middlings and 50g of middlings, adding 5 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia, extracting volatile oil for 5 hours by heating by adopting a steam method, collecting the volatile oil, determining the volume of the volatile oil, and calculating the extraction rate of the volatile oil, wherein the result is as follows:

watch 13

Degree of pulverization of medicinal materials	Decoction pieces	Coarse powder	Coarse powder
				Extraction ratio of volatile oil (%)	0.5	0.6	0.6

The angelica is easy to be boiled during the decoction process, which affects the next alcohol extraction. The results show that the angelica contains a small amount of volatile oil, so the angelica decoction pieces are used for extracting the volatile oil.

1.4.2 investigating the amount of water added

According to the research results, taking 4 groups of angelica decoction pieces, 50g of each group, respectively adding 3 times, 5 times, 7 times and 9 times of water according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, extracting volatile oil by heating by adopting a steam method, collecting the volatile oil and calculating the extraction rate of the volatile oil:

TABLE 14

Adding water (times)	3	5	7	9
					Extraction ratio of volatile oil (%)	-	0.60	0.60	0.50

1.4.3 investigation of soaking time

According to the research results, 4 groups of angelica decoction pieces are taken, 50g of each group is added with 5 times of water, the mixture is soaked for 0 hour (immediately heated after being added with water), 0.5 hour, 1 hour and 2 hours respectively, the volatile oil is extracted by heating by adopting a steam method according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, the volatile oil is collected, and the extraction rate of the volatile oil is calculated:

watch 15

Soaking time (h)	0	0.5	1	2
					Extraction ratio of volatile oil (%)	0.7	0.4	0.4	0.4

1.4.4 investigating extraction time

According to the research results, referring to the volatile oil determination method in appendix 2204 of Chinese pharmacopoeia appendix, 100g of angelica decoction pieces are taken, 5 times of water is added, then the volatile oil is immediately heated and extracted, the volume of the volatile oil is read and the extraction rate of the volatile oil is calculated after 0.5h, 1h, 2h, 3h, 4h, 5h, 6h and 7h from the beginning of boiling:

TABLE 16

Extraction time (h)	0.5	1	2	3	4	5	6	7
									Extraction ratio of volatile oil (%)	0.4	0.3	0.45	0.55	0.60	0.60	0.60	0.60

The volatile oil is basically stable after being extracted for 4 hours, and the volatile oil amount is not increased any more. By making a scattergram of the effect of extraction time and volatile oil extraction rate, the results show that 3 hours is the "inflection point" of the experiment, and thus the optimal extraction time is 4 hours (fig. 4).

1.5 Single factor investigation of Ligusticum wallichii medicinal materials

1.5.1 investigating the degree of pulverization

Pulverizing rhizoma Ligustici Chuanxiong decoction pieces to coarse powder, and medium powder, respectively. Weighing 100g of decoction pieces, 100g of middlings powder and 100g of medium powder, respectively adding 5 times of water according to a volatile oil determination method in appendix 2204 of Chinese pharmacopoeia, heating and extracting volatile oil for 5 hours by adopting a water vapor method, collecting the volatile oil, determining the volume of the volatile oil and calculating the extraction rate of the volatile oil, wherein the results are as follows:

TABLE 17

Degree of pulverization of medicinal materials	Decoction pieces	Coarse powder	Coarse powder	Medium powder
					Extraction ratio of volatile oil (%)	0.15	0.35	0.35	0.25

The above results indicate that the rhizoma Ligustici Chuanxiong contains little volatile oil. The rhizoma ligustici wallichii decoction pieces are hard in material, so that the volatile oil can be extracted to the maximum extent by crushing to coarse powder.

1.5.2 investigating the amount of water added

According to the research results, taking 4 groups of the coarse powder of the ligusticum wallichii, wherein each group is 100g, respectively adding 3 times, 5 times, 7 times and 9 times of water according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, heating and extracting volatile oil by adopting a steam method, collecting the volatile oil and calculating the extraction rate of the volatile oil:

watch 18

Adding water (times)	3	5	7	9
					Extraction ratio of volatile oil (%)	0.2	0.5	0.45	0.45

1.5.3 investigation of soaking time

According to the research results, 4 groups of the coarse powder of the hemlock parsley are taken, 100g of each group is added with 5 times of water, the mixture is soaked for 0 hour (immediately heated after being added with water), 0.5 hour, 1 hour and 2 hours respectively, the volatile oil is extracted by heating by adopting a steam method according to a volatile oil determination method in Chinese pharmacopoeia appendix 2204, the volatile oil is collected, and the extraction rate of the volatile oil is calculated:

watch 19

Soaking time (h)	0	0.5	1	2
					Extraction ratio of volatile oil (%)	0.5	0.40	2.00.30	0.25

1.5.4 examining extraction time

watch 20

Extraction time (h)	0.5	1	2	3	4	5	6	7
									Extraction ratio of volatile oil (%)	0.05	0.1	0.25	0.4	0.5	0.5	0.5	0.5

The volatile oil is basically stable after being extracted for 4 hours, and the volatile oil amount is not increased any more. By making a scattergram of the effect of extraction time and volatile oil extraction rate, the results showed that 4 hours was the "inflection point" of the experiment, and thus the optimal extraction time was 4 hours (fig. 5).

2. Orthogonal test for extracting volatile oil from mixed medicinal materials

2.1 factors and levels

Because the volatile oil extraction influencing factors mutually influence, the volatile oil is extracted from the medicinal materials containing the volatile oil in the prescription by adopting an orthogonal test method. According to the single-factor investigation result, the volatile oil extracted from the mixed medicinal materials is not investigated on the extraction granularity any more, and the optimal extraction granularity of the medicinal materials in unit is referred to. According to the single-factor investigation result of the single medicinal material, the following factor level table is designed:

TABLE 21

Level of factor	Adding water (times)	Soaking time (h)	Extraction time (h)
				1	5	0	2
2	6	0.5	3
				3	7	1	4

2.2 orthogonal test

According to the factor levels, an L9(33) orthogonal test table is designed, the volatile oil is extracted from the tests 1 to 9 respectively, and the extraction rate of the volatile oil is calculated as follows:

TABLE 22

Comparing K value by visual analysis, the larger K value indicates that the level has the greatest influence on the factor, therefore, the optimal extraction process is A2B2C3, namely adding 6 times of water, soaking for 0.5 hour, and heating and extracting for 4 hours. Analysis of variance: none of the three factors has significant influence.

2.3 validation test

In order to verify the stability of the volatile oil extraction process, the preferred extraction process is verified three times. Weighing the formula amount of radix angelicae pubescentis, asarum, notopterygium root, angelica and ligusticum wallichii, adding 6 times of water, soaking for 0.5 hour, extracting volatile oil for 4 hours, wherein the volumes of the volatile oil for three times are 0.65mL, 0.65mL and 0.68mL respectively, and the RSD is less than 2%, and the result shows that the process is stable and feasible.

2.4 study of volatile oil and water solution

In order to master the extraction conditions of other effective components of five medicinal materials in the volatile oil extraction process, the volatile oil and the water solution thereof are specially subjected to index component content detection, and the result is as follows:

TABLE 23

Index component	Essential oils	Water solution
			Osthole	17.5％	0
Decursin	0	35.4％
			Asarone
	0	0

Detecting the content of the volatile oil index components: the result shows that the volatile oil contains osthole, no asafetida and no decursin. The detection result of the index component content of the volatile oil water solution shows that the volatile oil water solution contains a large amount of decursin, so that the water solution part cannot be discarded. Because the analgesic components of the prescription, such as decursin, asaricin and the like, belong to fat-soluble components, alcohol extraction is needed for further complete extraction.

(II) alcohol extraction process

1. Single factor investigation

1.1 examine the extraction method of effective ingredients

Heating reflux extraction: weighing the medicinal materials according to the prescription, extracting volatile oil according to the volatile oil extraction process, adding the clematis root, the safflower, the pseudo-ginseng (coarse powder) and the pawpaw according to the prescription into medicine dregs, adding a proper amount of 95% ethanol to ensure that the ethanol content is 70%, extracting by 8 times of volume of solvent, heating and refluxing for 2 times, collecting an extraction solution, recovering the ethanol, adding the volatile oil water solution, concentrating, and fixing the volume of the solution to 100 mL.

Percolation extraction: air drying the residue, adding radix Clematidis, Carthami flos, Notoginseng radix and fructus Chaenomelis, wetting with 70% ethanol solution, loading into column, soaking for 72 hr, percolating at percolating speed of 1mL/min, and percolating with volume the same as that of the reflux extraction. Collecting percolate, recovering ethanol, and concentrating the solution to 100 mL.

The content of the index components of the two extraction modes is measured by an HPLC method, and the result of calculating the transfer rate (%) of each index component is as follows:

watch 24

Comparing the two groups of data, the result shows that the heating reflux extraction has high index component content and time efficiency far higher than that of percolation extraction, so that the heating reflux extraction is adopted to extract the effective substances in the prescription.

1.2 investigating the concentration of the extracted alcohol

Weighing the medicinal materials according to the formula, extracting volatile oil according to the volatile oil extraction process, adding the radix clematidis, the safflower, the pseudo-ginseng (coarse powder) and the pawpaw according to the formula into medicine dregs, adding a proper amount of 95% ethanol to ensure that the ethanol content is 30%, 50%, 70%, 90% and 8 times of the volume of the solvent for extraction, heating and refluxing for 2 times, collecting an extraction solution each time for 2 hours, recovering the ethanol, adding a volatile oil water solution, concentrating, and fixing the volume of the solution to 100 mL.

TABLE 25

The content detection of the index components with two groups of data abnormity is shown by looking up documents and tests:

after the isoimperatorin in notopterygium root is heated to extract volatile oil, the content is reduced from 1.44% to 0.443%, because the isoimperatorin undergoes Maifang rearrangement under acidic conditions in the heating reflux process, and other coumarin components with similar structures are converted;

the document reports that decursin is a coumarin component and has an analgesic effect, so that the transfer rate of decursin is adopted as one of monitoring indexes in the subsequent extraction process.

The asarum herb has asarin transferring rate over 100% and the reason is that during heating reflux, methyl eugenol and other toxic components are converted into asarin, and the longer the heating time is, the more the conversion is, so that the asarin transferring rate is over 100%.

By comprehensively comparing and analyzing the transfer rates of the index components, the transfer rates of all indexes are considered, and 70% ethanol is selected as the optimal extraction concentration preliminarily.

1.3 alcohol extraction solvent multiple investigation

Heating reflux extraction: weighing the medicinal materials according to the formula, extracting volatile oil according to the volatile oil extraction process, adding the clematis root, the safflower, the pseudo-ginseng (coarse powder) and the pawpaw according to the formula into the medicine dregs, respectively adding 70% ethanol solutions 6 times, 8 times, 10 times and 12 times, heating and refluxing for 2 times, collecting the extraction solution after 2 hours each time, recovering ethanol, adding the volatile oil water solution, concentrating, and fixing the volume of the solution to 100 mL.

watch 26

Content of osthole in volatile oil liquid

Through the index component data analysis, the transfer rates of all indexes are integrated, so that 10 times of the index transfer rates are selected as extraction times to investigate subsequent extraction factors.

1.4 investigation of extraction time

Heating reflux extraction: weighing the medicinal materials according to the formula, extracting volatile oil according to the volatile oil extraction process, adding the radix clematidis, the safflower, the pseudo-ginseng (coarse powder) and the pawpaw according to the formula into medicine dregs, adding 10 times of 70% ethanol solution, respectively heating and refluxing for extraction twice for 1 hour, 2 hours, 3 hours and 4 hours each time, collecting the extraction solution, recovering ethanol, adding the volatile oil water solution, concentrating, and fixing the volume of the solution to 100 mL.

watch 27

The result of examining the extraction time shows that the transfer rate of the index components has no obvious change after 2-4 hours of extraction, but is obviously improved compared with 1 hour, so 2 hours are selected as the optimal extraction time.

1.5 examination of extraction frequency

Extracting volatile oil, collecting oil liquid, and refrigerating water liquid for later use. Adding radix Clematidis, Carthami flos, Notoginseng radix and fructus Chaenomelis into the residue, adding 10 times of 70% ethanol (230 mL of water in the residue, calculated), heating and reflux-extracting for 2 hr for 1, 2, and 3 times respectively, collecting extractive solution, recovering ethanol, mixing volatile oil water solutions, concentrating, and diluting to volume of 200 mL.

Watch 28

Compared and analyzed with the research data, the content of the index components extracted for 2 times and 3 times is obviously higher than that extracted for 1 time, and the extraction times of 2 times and 3 times are not obviously changed, so that the extraction times of 2 times are taken as the optimal extraction times due to the consideration of energy conservation and economic factors.

1.6 investigating the particle size of the extracted pseudo-ginseng

The pseudo-ginseng belongs to an expensive medicinal material, is hard and is difficult to extract effective components without being crushed, so that the most coarse powder is adopted in the single-factor investigation process, but the extraction transfer rate does not reach more than 80 percent, and the grinding particle size of the pseudo-ginseng is specially investigated.

By referring to the above alcohol extraction factors, the ginsenoside Rg1 is detected by inspecting the particle sizes of the coarse powder, the coarse powder and the medium powder of the panax notoginseng, and the results are as follows:

watch 29

Degree of pulverization	Transfer rate of ginsenoside Rg1
		Coarse powder	64.82
Coarse powder	64.31
		Medium powder	64.49

The results show that the transfer rates of the three grinding particle sizes are basically close to each other, and the grinding degree of the pseudo-ginseng has no obvious influence on the extraction transfer rate. Therefore, during the extraction process of the prescription, the pseudo-ginseng is only needed to be properly crushed.

2. Alcohol extraction process based on orthogonal test

2.1 factors and levels

The main factors influencing the alcohol extraction process include alcohol concentration, feed-liquid ratio, extraction time and extraction frequency. Designing the following factor level table according to the single factor investigation result:

watch 30

2.2 orthogonal test

Based on the above factor level, L9 (4) was designed³) Orthogonal test table, respectively extracting effective components under test 1-9 conditions, recovering ethanol, adding volatile oil water solution, concentrating to 100mL, and performing index component transfer rate analysis.

Watch 31

2.3 Multi-index weight in alcohol extraction Process

In order to solve the problem of 'considering the index component transfer rate' under different conditions, an analytic hierarchy process is adopted to set weight for each index component, and the process quality is evaluated by 'total score'.

According to the compatibility rule of the monarch, minister, assistant and guide medicines and the strength and weakness of the pharmacological action of each component in the compound, the experiment quantifies the cream yield and the dissolution amount of the index components as weight indexes, namely 6 indexes are divided into 4 levels, and the priority sequence of each index is determined: cnidium lactone > decursin > asafetida saponin > ginsenoside Rg1 ═ hydroxysafflor yellow A > ointment yield, a judgment priority matrix for pairwise comparison is constructed, relative scores among various indexes are given, and a priority matrix for index component comparison is constructed.

Table 32 index pairwise comparison judgment priority matrix

Target	Radix Angelicae Pubescentis	Notopterygium root	Herba asari	Safflower	Notoginseng radix	Rate of paste discharge
							Radix Angelicae Pubescentis	1	2	2	3	3	4
Notopterygium root	1/2	1	1	2	2	3
							Herba asari	1/2	1	1	2	2	3
Safflower	1/3	1/2	1/2	1	1	2
							Notoginseng radix	1/3	1/2	1/2	1	1	2
Rate of paste discharge	1/4	1/3	1/3	1/2	1/2	1

Calculating initial weight coefficients of osthole, decursin, asaricin, ginsenoside Rg1, hydroxysafflor yellow A and cream yield by AHP method according to the scoring results of the above tables, and calculating the initial weight coefficients according to the formula

The weights are normalized and the weight coefficients are calculated as 0.3315, 0.1952, 0.1952, 0.1074, 0.1074, 0.0633, respectively.

And CR is CI/RI as an index for judging whether the obtained weight coefficient is reasonable or not. CR is 0.00735/1.24 is 0.00593 < 0.1, meets and satisfies the requirement of consistency. Therefore, the weight of each index component is reasonable and feasible.

2.4 weight-based analytical orthogonal test

According to orthogonal test L9 (4)³) The test was carried out, the content of each index was measured by HPLC, the transfer rate was calculated, and the total score (M) of 0.3315 osthole transfer rate +0.1952 nodakenin transfer rate +0.1952 pinocembrin transfer rate +0.1074 hydroxysafflor yellow a transfer rate +0.1074 ginsenoside Rg1 transfer rate +0.0633 cream output rate was calculated for each of 9 orthogonal test sets by the above-identified weights, and the results were as follows:

watch 33

The 9 sets of orthogonal test data are visually analyzed, and the results are as follows:

watch 34

According to the visual analysis result, the maximum value of each K value is taken as the optimal value of the orthogonal test, so the optimal extraction process is A2B2C2D3, namely 10 times of 70% ethanol is added for extraction, and the extraction is carried out for three times, wherein each time is 1.5 hours.

According to the above results, taking the minimum R value as an error term, and performing variance analysis, the results are as follows:

watch 35

Factors of the fact	Squared deviation	Degree of freedom	F ratio	Critical value of F	Significance of
						Alcohol concentration	195.052	2	4.405	19.000	0.078
Ratio of material to liquid	44.281	2	1.000	19.000	0.059
						Extraction time	211.040	2	4.766	19.000	0.069
Number of times of extraction	631.361	2	14.258	19.000	0.070

The above results indicate that each factor has no significant influence.

2.5 reconfirmation of extraction times

The orthogonal test result of the alcohol extraction process is different from the single-factor investigation result, and the extraction times are confirmed again.

Extracting volatile oil, collecting oil liquid, and refrigerating water liquid for later use. Adding radix Clematidis, Carthami flos, Notoginseng radix and fructus Chaenomelis into the residue, adding 10 times of 70% ethanol (230 mL of water in the residue, calculated), heating and reflux extracting for 1.5 hr for 1 time, 2 times, 3 times and 4 times respectively, collecting extractive solution, recovering ethanol, mixing volatile oil water solutions, concentrating, and diluting to volume of 200 mL.

Watch 36

The extraction times are 2 times and 3 and 4 times without obvious change (RSD is 1.38%), and the extraction times are determined to be 2 times in consideration of large-scale production energy conservation and production cost in the future. Therefore, the alcohol extraction process is defined as: 10 times the volume of 70% ethanol, heat extracted twice, each for 1.5 hours.

2.6 validation test

Extracting volatile oil, collecting oil liquid, and refrigerating water liquid for later use. Adding radix Clematidis, Carthami flos, Notoginseng radix and fructus Chaenomelis into the residue, adding 10 times of 70% ethanol (230 mL of water in the residue, calculated), heating and refluxing twice, extracting for 1.5 hr, collecting extractive solution, recovering ethanol, mixing volatile oil water solutions, concentrating, and diluting to 200 mL. Three sets of experiments were performed in parallel.

Watch 37

Three groups of verification test results show that the RSD is 2.32 percent, which indicates that the process is stable and feasible.

3 concentration and alcohol precipitation

3.1 concentration mode

And (3) concentrating under normal pressure: extracting volatile oil, collecting oil liquid, and refrigerating water liquid for later use. Adding radix Clematidis, Carthami flos, Notoginseng radix and fructus Chaenomelis into the residue, adding 10 times of 70% ethanol (230 mL of water in the residue, calculated), heating and refluxing twice, extracting for 1.5 hr, collecting extractive solution, recovering ethanol, mixing volatile oil water solutions, and concentrating to 200mL under normal pressure.

And (3) concentrating under reduced pressure: extracting volatile oil, collecting oil liquid, and refrigerating water liquid for later use. Adding radix Clematidis, Carthami flos, Notoginseng radix and fructus Chaenomelis into the residue, adding 10 times of 70% ethanol (230 mL of water in the residue, calculated), heating and refluxing twice, extracting for 1.5 hr, collecting extractive solution, recovering ethanol, mixing volatile oil water solutions, and concentrating under reduced pressure (vacuum degree close to 0.1MP) to 200 mL.

The two concentration modes are respectively subjected to index component detection, and the results are as follows:

watch 38

The above results show that there is no significant difference between the index components, and the total evaluation score RSD of the analytic hierarchy process is 0.65% (< 2%), so reduced pressure concentration is adopted.

Process for extracting effective components from Aranea

According to the research of literature data, the main active ingredients of the spiders are biological proteins or polypeptides, and the active ingredients are extracted by cold soaking in view of the changeability of the ingredients under high temperature and high alcohol. In the process, ninhydrin special reaction is adopted for qualitative determination, and the absorbance is measured by an ultraviolet spectrophotometry for quantitative determination.

1. Ninhydrin reaction

Under the conditions of heating and weak acid, amino acid or peptide reacts with ninhydrin to produce purplish blue (brown product formed with asparagine. reaction with proline or hydroxyproline to produce (bright) yellow) compound, corresponding aldehyde and carbon dioxide.

The ninhydrin reaction is specific, so that the components extracted from the spider extract can be identified as amino acids or polypeptides through the reaction.

The above TLC results can be preliminarily concluded in two ways:

the effective components of spider are mainly amino acids, small molecular polypeptide and protein.

② TLC spectrogram has most 2-4 spots, so 30-70% ethanol is adopted to extract the components (figure 6).

2. Single factor investigation

2.1 investigating the extraction mode

According to the method of appendix 2201 of Chinese pharmacopoeia 2015 edition, 1G of spider freeze-dried powder is weighed, the following three extraction modes are respectively adopted, 25 times of 50% ethanol solution is added for extraction (heating reflux is carried out for 1 hour, ultrasonic treatment is carried out for 1 hour, cold immersion extraction is carried out for 24 hours), filtration is carried out, the extracting solution is dissolved in 100mL, the ethanol content is 60% ethanol, standing is carried out for 24 hours, filtration is carried out, 1mL of extracting solution is taken, 4mL of Coomassie brilliant blue G-250 solution is added, and the protein polypeptide content is measured by adopting an external standard method.

Watch 39

Extraction method	Content of Polypeptides (%)	Extract (%)
			Heating reflux extraction	2.48％	12.46
Cold leaching extraction	1.11％	9.71
			Ultrasonic extraction	0.99％	8.69

The heating reflux extraction method is abandoned because a great amount of flocculent precipitates appear after the heating reflux extraction liquid is cooled, and the possibility of destroying the living activity exists. Compared with cold soaking extraction and ultrasonic extraction, under the condition of the same extraction volume, the cold soaking is more suitable by combining the actual situation of mass production.

2.2 examination of alcohol extraction concentration

Refer to the Chinese pharmacopoeia 2015 edition appendix 2201 method. Weighing five groups of freeze-dried spider powder, wherein each group is 1G, respectively extracting with 25 times of water, 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol for 24 hours by cold immersion, filtering, dissolving the extract in 100mL, adding 60% ethanol, standing for 24 hours, filtering, taking 1mL of the extract, adding 4mL of Coomassie brilliant blue G-250 solution, and determining the content of protein polypeptide by an external standard method.

Watch 40

The water extract has putrefactive odor, and is discarded in consideration of actual conditions later, and no corresponding data is tested. By examining the extraction concentration, in combination with the TLC results, 30% ethanol was finally selected as the optimal extraction alcohol concentration.

2.3 investigating alcohol extraction times

Refer to the Chinese pharmacopoeia 2015 edition appendix 2201 method. Weighing four groups of freeze-dried spider powder, wherein each group is 1G, cold soaking and extracting with 25 times of 30% ethanol solution, 50 times of 30% ethanol solution, 75 times of 30% ethanol solution and 100 times of 30% ethanol solution for 24 hours, filtering, dissolving the extract in 100mL, adding 60% ethanol, standing for 24 hours, filtering, taking 1mL of the extract, adding 4mL of Coomassie brilliant blue G-250 solution, and determining the content of protein polypeptide by adopting an external standard method.

Table 41

Extraction method	Content of Polypeptides (%)	Extract (%)
			25 times of	1.89％	12.46
50 times of	2.47％	16.66
			75 times of	2.54％	17.05
100 times of	2.49％	16.70

By examining the extraction times and integrating the extraction cost and other factors, 50 times of 30% ethanol is finally selected as the optimal extraction alcohol concentration.

2.4 investigation of extraction time

Refer to the Chinese pharmacopoeia 2015 edition appendix 2201 method. Weighing four groups of freeze-dried spider powder, wherein each group is 1G, adding 50 times of 30% ethanol solution, respectively performing cold soaking extraction for 24 hours, 48 hours, 72 hours and 96 hours, filtering, dissolving the extract in 100mL of ethanol containing 60% ethanol, standing for 24 hours, filtering, taking 1mL of the extract, adding 4mL of Coomassie brilliant blue G-250 solution, and determining the content of protein polypeptide by adopting an external standard method.

Watch 42

Extraction time	Content of Polypeptides (%)	Extract (%)
			24 hours	2.16％	11.36
48 hours	2.34％	13.41
			72 hours	2.66％	15.11
96 hours	2.60％	15.23

Finally 72 hours were selected as the best extraction time by examining the extraction time.

3. Quadrature test

3.1 factors and levels

The main factors influencing the extraction process of the effective components of the spiders comprise alcohol concentration, feed-liquid ratio, cold-leaching extraction time and extraction times. Designing the following factor level table according to the single factor investigation result:

watch 43

Level of factor	Alcohol concentration	Multiple of	Time	Number of times
					1	30％	25	24 hours	1
2	50％	50	72 hours	2
					3	70％	75	120 hours	3

3.2 orthogonal test

Based on the above factor level, L9 (4) was designed³) Orthogonal test table, respectively extracting effective components under test 1-9 conditions, filtering, dissolving the extractive solution in 100mL of 60% ethanol, standing for 24 hr, filtering, collecting 1mL of the extractive solution, adding 4mL of Coomassie brilliant blue G-250 solution, and measuring protein polypeptide content by external standard method.

Watch 44

The content of the protein polypeptide is visually analyzed, and the results are as follows:

TABLE 45

K1	2.690	2.189	2.234	1.956
					K2	2.220	2.631	2.586	2.730
K3	2.446	2.527	2.527	2.661
					Extreme difference	0.480	0.442	0.352	0.774

And performing K value analysis on each factor to obtain an optimal extraction process, and performing variance analysis to show that no significant influence exists.

The extract content was visually analyzed, and the results were as follows:

TABLE 46

K1	21.930	19.173	19.157	18.958
					K2	18.925	20.632	20.285	20.552
K3	18.075	19.130	19.493	19.425
					Extreme difference	3.861	1.502	1.128	1.594

Combining the above results, the optimal extraction process is 50 times of 30% ethanol, and the extraction is performed twice, each time for 72 hours. Variance showed no significant effect.

3.3 reconfirming the multiple of the extraction solution

The result obtained by the orthogonal test is basically the same as the result of a single factor, and the spider freeze-dried powder is a powdery substance with large volume and light weight, so that the spider freeze-dried powder is not completely immersed in 20 times of volume. From the actual situation of mass production, the experiment again confirmed the extraction multiple.

Weighing three groups of freeze-dried spider powder, wherein each group is 1G, adding 30-time, 40-time and 50-time 30% ethanol solutions, respectively performing cold soaking extraction twice for 72 hours each time, filtering, dissolving the extract in 100mL, standing for 24 hours, filtering, taking 1mL of the extract, adding 4mL of Coomassie brilliant blue G-250 solution, and determining the content of protein polypeptide by adopting an external standard method. The results are as follows:

watch 47

Extraction method	Content of Polypeptides (%)	Extract (%)
			30 times of	2.94％	20.02
40 times of	3.11％	20.36
			50 times of	3.18％	20.44

The three groups of results show that the volume of 40 times is almost the same as that of 50 times, the aim of complete extraction is achieved, and the effective components of the spider can be completely extracted by 40 times of volume under the consideration of economic cost and other factors. Therefore, the optimal extraction process of the effective components of the spider is 40 times of 30% ethanol, and the effective components are extracted twice, each time for 72 hours.

3.4 validation test

Weighing three groups of freeze-dried spider powder, wherein each group is 1G, adding 40 times of 30% ethanol solution, respectively cold-soaking and extracting for two times, each time for 72 hours, filtering, fixing the extracting solution in 100mL, adding 60% ethanol, standing for 24 hours, filtering, taking 1mL of extracting solution, adding 4mL of Coomassie brilliant blue G-250 solution, and determining the content of protein polypeptide by adopting an external standard method. Three sets of experiments were performed in parallel, with the following results:

watch 48

Extraction method	Content of Polypeptides (%)	Extract (%)
			Verification 1	2.93％	19.390
Authentication 2	2.95％	19.23
			Authentication 3	2.93％	19.90

Three groups of verification tests RSD are less than 2%, and the results show that the process is stable and feasible.

(IV) preparation purification process

1. Alcohol precipitation process

The compound is prepared into a spray with a certain alcohol concentration, in the volatile oil extraction process, a volatile oil water solution contains components with large polarity, and a pre-test result shows that the concentrated solution is easy to precipitate under the condition of a certain alcohol concentration, so that the concentrated solution is necessary to be subjected to alcohol precipitation to ensure the stability and the clarity of a preparation. Because the factors influencing alcohol precipitation comprise sample concentration degree, alcohol concentration and alcohol precipitation time, an orthogonal test is designed to purify the prescription.

1.1 factors and levels

Three different concentration degrees (total crude drug mass/concentration volume) of 0.75g/mL, 1.00g/mL and 1.50g/mL are examined, and corresponding densities of 1.034, 1.054 and 1.089 are measured respectively.

Watch 49

Factors and levels	Concentration purity (g/mL)	Alcohol concentration	Time	Blank space
						1	0.75	60％	12h	-
2	1.00	70％	24h	-
					3	1.50	80％	48h	-

1.2 orthogonal test

Carrying out orthogonal tests according to the conditions, carrying out alcohol precipitation tests 1-9 respectively, taking the supernatant to be 1000mL, and carrying out index component detection, wherein the results are as follows:

watch 50

1.3 visual analysis and analysis of variance

And (3) carrying out visual analysis on the orthogonal test result:

watch 51

Factors and levels	Purity of concentration	Alcohol concentration	Time	Blank space
						K1	88.173	80.353	83.477	85.190	K1
K2	85.370	85.017	81.000	81.200	K2
						K3	74.033	82.207	81.000	81.187	K3
R1	14.140	4.664	2.477	4.003	R1

The most preferred purification process is A1B2C2, i.e., concentrated to 0.75g/mL (density 1.03), alcohol added to 70%, and left for 12 hours. Analysis of variance showed that the three factors had no significant effect.

1.3 validation test

Three verification tests were performed according to the above purification process, with the following results:

table 52

The results of three verification tests show that the results are stable (RSD is 1.67% < 2%).

1.4 separate alcohol precipitation of volatile oil water solution

In view of the fact that the precipitation part in the purification process mainly comes from the volatile oil water solution, the volatile oil water solution is independently precipitated by alcohol, namely the water solution is concentrated to a proper amount, a certain amount of ethanol is added to ensure that the alcohol content is 70 percent, and the supernatant is taken after the night (more than 12 hours). Concentrating the ethanol extractive solution, evaporating to dryness, dissolving in 70% ethanol, mixing the above ethanol extractive solutions, filtering, diluting to 1000mL, and measuring various index components.

Watch 53

Comparing the data with the verification test data, the result shows that the total evaluation score of the analytic hierarchy process is slightly smaller than that of the verification test, and the index components are respectively compared, and the difference of the safflower and the other components is not obvious.

Besides, when the alcohol extract is dissolved, precipitation can occur, and one-step filtration is added. Combining the above factors, the purification process combines the water extract with the alcohol extract, concentrates to a certain density, and then carries out alcohol precipitation purification.

2. Alcohol concentration of the formulation

2.1 menthol solubility

The menthol is dissolved by 30 percent, 50 percent, 70 percent and 95 percent ethanol for investigating the solubility and the stability of the menthol, and the result shows that the menthol can be quickly dissolved in 70 percent and 95 percent ethanol solution and does not precipitate crystals after being placed for a plurality of days at normal temperature.

2.2 solubility of volatile oils

The volatile oil and cinnamon oil are dissolved by 30%, 50%, 70% and 95% ethanol, and the result shows that the volatile oil can not be completely dissolved in the 30% ethanol, oil drops appear on the bottom layer of the solution, a small amount of oil drops exist in the 50% ethanol, and the 70% and 95% ethanol solutions can be completely dissolved, can not be layered after being placed for a plurality of days, and can be kept clear. The experiment shows that the alcohol concentration of the preparation should be more than 50% of ethanol.

2.3 formulation alcohol concentration

The analgesic components of the preparation mainly comprise coumarins, saponins and flavonoids, which can be easily dissolved in ethanol solution, so that the concentration of the preparation is investigated, and the appropriate alcohol concentration of the preparation is researched to ensure the stability and the clarity of the preparation.

Watch 54

Alcohol concentration	Osthole	Decursin	Hydroxy safflower yellow A
				The alcohol concentration is 30%	12.35	93.25	61.64
The alcohol concentration is 50%	47.86	93.47	59.00
				The alcohol concentration is 70%	61.26	102.71	62.83
The alcohol concentration is 90%	56.09	97.37	31.22

Content of osthole in undetected volatile oil

According to the four indexes detected, the alcohol concentration of 30% has a large influence on the content of the active ingredients of the osthole of the radix angelicae pubescentis in the prescription, the alcohol concentration of 90% has a large influence on the water-soluble ingredient hydroxy carthamus tinctorius yellow A in the carthamus tinctorius, and the alcohol concentration of the preparation is proper to be 50% -70% by combining the menthol solubility research, the volatile oil solubility and the preparation volatile oil stability research results.

To further study the alcohol concentration range, the contents of the respective index components and the clarity of the preparation were examined under the conditions of comparing the preparations with 50% ethanol, 55% ethanol, 60% ethanol, 65% ethanol and 70% ethanol to confirm the concentration of the preparation.

Watch 55

The results are subjected to hierarchical analysis, and the total score under each alcohol concentration is calculated by combining the weight of each index, and the results show that five groups of data have no obvious difference, the RSD is 1.81% < 2%, but the alcohol concentration range is only 55% -65% considering that the volatile oil can not be completely dissolved under the condition of a 50% ethanol preparation and that the 70% ethanol preparation influences the skin comfort level.

2.4 Cold storage

A preparation with alcohol concentration of 60% is prepared according to an extraction process and a purification process, flocculent precipitate appears after the preparation is placed, and the reason is analyzed, wherein the main reason is that spider macromolecular polypeptide is unstable in 60% ethanol, so that macromolecular protein and polypeptide components are removed to the maximum extent when the preparation is placed under a refrigeration condition.

2.4.1 examination of Placement time

Watch 56

2.5 pH of the formulation

The pH value is an important influence factor on the stability of the preparation, so that different pH values are considered for the stability of the preparation. According to the above extraction process, a preparation with alcohol concentration of 60% is prepared, and its pH value is measured to be 5.47.

Comparing the pH values of the mixture at 5.0, 5.5, 6.0, 6.5 and 7.0, the mixture was kept in the shade for 1 day, 7 days and 30 days, respectively, and the results were as follows:

watch 57

Time	5.0	5.5	6.0	6.5	7.0
						1 day	Clarification	Clarification	Clarification	A small amount of precipitate	A small amount of precipitate
7 days	Clarification	Clarification	Clarification	With precipitation	With precipitation
						30 days	Clarification	Clarification	Clarification	With precipitation	With precipitation

2.6 complete preparation Process

The complete preparation process of the prescription is obtained by combining the research results:

the thirteen medicines of the prescription are that pubescent angelica root, angelica and Szechuan lovage rhizome are crushed into coarse powder, five medicinal materials of incised notopterygium rhizome and asarum are added with 6 times of water to extract volatile oil for 3 hours, and the volatile oil water solution are collected for later use. Adding fructus Chaenomelis, radix Clematidis, Notoginseng radix (coarse powder) and Carthami flos into the residue, adding 10 times of 70% ethanol solution, heating and extracting for 2 times, each time for 1.5 hr, and collecting the ethanol extractive solution. Mixing the volatile oil water solution and the ethanol extract, concentrating to density of about 1.03g/mL (60 deg.C), adding ethanol until ethanol content is 70%, standing for 24 hr, collecting supernatant, adding Borneolum Syntheticum and Mentholum, and shaking moderately to dissolve completely. Adding 40 times of 30% ethanol into the spider lyophilized powder, cold-soaking and extracting for 2 times, each time for 72 hr, and mixing the spider extractive solutions. Dissolving the volatile oil and oleum Cinnamomi with appropriate amount of ethanol. Mixing the three solutions, refrigerating, standing overnight, filtering, and adding water into the filtrate to a volume of 1000 mL.

The characteristics are as follows: the content is a yellow-brown to tan clear liquid; has special fragrance, slightly bitter and spicy taste.

Test example 2

The product of the scheme is clinically tested and researched in a certain traditional Chinese medicine institute in Hainan province. 240 clinical patients with arthromyodynia (rheumatism, rheumatoid, osteoarthritis or chronic joint pain) were selected and divided into 12 groups of 20 each, 10 men and women, each aged 40-65 years, and subjected to 15-day test.

The evaluation criteria are as follows:

remarkably: the discomfort such as pain and swelling at the focus part is obviously relieved and basically disappears;

the method has the following advantages: the discomfort such as pain and swelling at the focus part still exists, and the symptoms are relieved;

and (4) invalidation: the same or even aggravation as before treatment.

The experimental groups were as follows:

test group 1: the experimental sample of the embodiment 1 is selected, and is applied to the affected part 3-4 times a day in a proper amount, and then is massaged for 1-2 minutes to promote the penetration and absorption of the liquid medicine. (3 g of spider freeze-dried powder, 18g of radix angelicae pubescentis, 18g of notopterygium root, 18g of radix clematidis, 6g of asarum, 20g of pawpaw, 9g of pseudo-ginseng, 12g of safflower, 18g of ligusticum wallichii, 18g of angelica sinensis, 2mL of cinnamon oil (medicinal), 20g of menthol (medicinal) and 5g of borneol)

Test group 2: 1.5g of spider freeze-dried powder, 15g of radix angelicae pubescentis, 15g of notopterygium root, 15g of radix clematidis, 4g of asarum, 20g of pawpaw, 9g of pseudo-ginseng, 8g of safflower, 15g of ligusticum wallichii, 5g of angelica sinensis, 2mL of cinnamon oil (medicinal), 20g of menthol (medicinal) and 5g of borneol. The preparation and use are the same as those of test group 1.

Test group 3: 6g of spider freeze-dried powder, 24g of radix angelicae pubescentis, 24g of notopterygium root, 18g of radix clematidis, 10g of asarum, 20g of pawpaw, 12g of pseudo-ginseng, 15g of safflower, 18g of ligusticum wallichii, 18g of angelica sinensis, 4mL of cinnamon oil (medicinal), 20g of menthol (medicinal) and 5g of borneol. The preparation and use are the same as those of test group 1.

Comparative group 1: the preparation method of other medicinal materials is the same as that of test group 1 in the absence of spider freeze-dried powder.

Comparative group 2: the amount of the pubescent angelica root and the notopterygium root is respectively reduced to 12g, and other preparation methods are the same as those of the test group 1.

Comparative group 3: the amount of the pubescent angelica root and the notopterygium root is respectively increased to 24g, and other preparation methods are the same as those of the test group 1.

Comparative group 4: the preparation method of other herbs without asarum is the same as that of test group 1.

Comparative group 5: the preparation method of other medicinal materials is the same as that of test group 1 except for radix Notoginseng.

Comparative group 6: the amount of Carthami flos is reduced to 6g, and the preparation method of other medicinal materials is the same as that of test group 1.

Comparative group 7: the amount of radix Angelicae sinensis is reduced to 9g, and the preparation method of other medicinal materials is the same as that of test group 1.

Control group 1: the commercially available detumescence and acesodyne tincture (flower red) is selected, wiped for 3-4 times every day, and massaged properly for 1-2 minutes to promote the penetration and absorption of the liquid medicine.

Control group 2: no treatment was done.

The clinical effects of the specific test are as follows:

TABLE 58 clinical trial effects

Sequence of	Group of	Is remarkable in that	Is effective	Invalidation	Effective rate%
						1	Test group 1	7	11	2	90
2	Test group 2	4	12	4	80
						3	Test group 3	10	8	2	90
4	Comparative group 1	4	8	8	60
						5	Comparative group 2	5	9	6	70
6	Comparative group 3	9	9	2	90
						7	Comparative group 4	5	9	6	70
8	Comparative group 5	3	7	10	50
						9	Comparative group 6	4	8	8	60
10	Comparative group 7	5	9	6	70
						11	Control group 1	3	10	7	65
12	Control group 2	0	3	17	15

As shown in the clinical effects of the clinical trial results in Table 59, the experimental effect of the method is significant, and the effects of the experimental groups 1 to 3 are significant due to the control group (commercially available swelling and pain relieving tincture). Meanwhile, the experimental group 3 increases the amount of the medicinal materials, the clinical effect is not significantly improved, and the experimental group 2 reduces the amount of the medicinal materials, and the clinical effect is significantly reduced, but still higher than that of the control group 1.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The traditional Chinese medicine composition for treating the arthralgia is characterized by being prepared from the following raw materials in parts by weight:

2. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw materials in parts by weight:

3. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw materials in parts by weight:

4. A method for preparing the traditional Chinese medicine composition of any one of claims 1 to 3, which is characterized by comprising the following steps:

the method comprises the following steps: mixing radix angelicae pubescentis, angelica sinensis, ligusticum wallichii, notopterygium root and asarum, adding water with the weight 5-7 times that of the mixture, soaking for 20-40 minutes, and heating and extracting for 2-4 hours by adopting a steam method to obtain volatile oil, volatile oil water solution and medicine residues;

5. The process according to claim 4, wherein in step (i): mixing radix Angelicae Pubescentis, radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, Notopterygii rhizoma, and herba asari, adding 6 times of water, soaking for 30 min, and extracting by steam heating for 4 hr to obtain volatile oil, volatile oil water solution and residue;

6. The method according to claim 4, wherein the step (II) comprises: adding 30% ethanol aqueous solution 40 times the weight of the spider freeze-dried powder, and performing cold-leaching extraction for 2 times, wherein the time for cold-leaching extraction for each time is 72 hours;

in the fourth step: mixing the first mixture, the spider extract and the second mixture, and standing at 0-4 ℃ for 12-18 hours.

7. The method of claim 4, wherein the degree of pulverization of the pubescent angelica root, the angelica sinensis and the ligusticum wallichii is 0-0.8 cm, and the notopterygium root and the asarum are decoction pieces.

8. Use of the Chinese medicinal composition of any one of claims 1 to 3 in the preparation of a Chinese medicinal preparation for treating arthromyodynia.

9. The use of claim 8, wherein the arthromyodynia is one of rheumatism, gout, or bone pain.

10. A Chinese medicinal preparation for treating arthralgia, which is characterized by comprising the Chinese medicinal composition of any one of claims 1 to 3, wherein the alcohol concentration of the Chinese medicinal preparation is 50-70%, and the pH value is 5.0-6.0.