A new formula of Guiqi decoction for invigorating qi, generating blood and enhancing immunity and its preparation method

Abstract

The present invention provides a new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity and a preparation method thereof, which belongs to the field of medicine and food technology; the preparation method of the new formula of Guiqi Decoction comprises the following steps: extracting the formula amount of Angelica sinensis with water to obtain an extract; centrifuging the obtained extract; obtaining an Angelica sinensis extract; extracting the formula amount of Astragalus and Lycium barbarum with water, filtering, centrifuging, and obtaining an aqueous extract; concentrating the obtained aqueous extract, adding ethanol to precipitate, centrifuging, taking the supernatant, separating ethanol, and obtaining a mixed extract; mixing the obtained Angelica sinensis extract and the obtained mixed extract, preparing the crude drug concentration, and obtaining the new formula of Guiqi Decoction. The new formula of Guiqi Decoction contains 8-12 parts of Astragalus, 2-6 parts of Angelica sinensis, and 2-6 parts of Lycium barbarum by weight. Compared with the prior art, the new formula of Guiqi Decoction of the present invention is more concise, and the new formula of Guiqi Decoction prepared by the preparation method has a better effect of enhancing immunity and is not easy to cause getting angry.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and food, and relates to a new formula of Guiqi Decoction capable of invigorating qi, generating blood and enhancing immunity, and a preparation method thereof. Background Art

[0002] Due to the fast pace of life, high work pressure, lack of sleep and other reasons, modern people are in a sub-healthy state all year round, with low immunity and chronic fatigue as the main symptoms. The sub-healthy state has fatigue, decreased vitality and reduced immunity as the main clinical manifestations, and the proportion of sub-healthy patients in the population is increasing year by year.

[0003] Immunity is a common term in modern medicine, usually referred to as immunity. It is the body's ability to identify "self" and "non-self", eliminate and remove harmful foreign substances that invade the human body, and maintain a normal and healthy body state. The concept similar to "immunity" in traditional Chinese medicine is "qi". The "Inner Canon" says "positive qi is stored inside, and evil cannot interfere", pointing out that when the body's qi is sufficient, pathogenic factors will not easily invade the body and cause illness. The basic theory of modern traditional Chinese medicine proposes that Wei Qi flows through the skin and fur, helping the skin to protect the body and enabling the skin to play a barrier role against external evil. If Wei Qi is weak, the skin is loose and the pores are open, then external evil can easily invade and cause illness. Therefore, it is said: "When the weak evil enters the human body, it starts from the skin. If the skin is loose, the pores will open. If the pores are open, the evil will enter from the hair and enter the human body, and then enter the deep" ("Lingshu·The Beginning of All Diseases"). This explains the relationship between "qi" and the body's immunity. "Zheng Qi" and "Wei Qi" contain the two functions of "self-stabilization" and "defense" in modern immunology. Wang Ang, a doctor in the Qing Dynasty, wrote in the book "Yi Fang Ji Jie" that "If a person's vital energy is strong, external evil cannot harm him. The true energy must be weak first, and the blood and defense system must be empty, so the evil can take advantage of the weakness to enter... If the person is affected by the six external evils, he should also treat his qi first." Therefore, if you want to enhance immunity and improve the body's ability to resist external infection, you should first replenish your vital energy.

[0004] Clinically, drugs that increase immunity are divided into two categories: Western medicine (chemical drugs, biological products) and traditional Chinese medicine. Western medicine mainly includes thymosin enteric-coated tablets, pidotimod oral solution, human albumin, immunoglobulin, thymopentin, etc. Traditional Chinese medicine mainly uses medicinal flavors such as replenishing qi and blood, promoting blood circulation and qi circulation. Injectable immunoglobulins are mainly used to enable the human body to quickly acquire the corresponding active immune ability, but the effect period is short, it cannot be used for long-term improvement, and it is prone to adverse reactions such as allergies. Traditional Chinese medicine formulas take effect slowly, but they can improve the body's immunity in a long-term and stable manner, and unlike a single specific immunoglobulin, they can enable the body to resist a variety of pathogens.

[0005] CN110093224A discloses a production formula of health wine, which includes distilled wine and Chinese medicinal raw materials. The distilled wine is prepared from wheat, corn, sorghum, black rice, buckwheat, oats, highland barley and dried sweet potatoes, and the Chinese medicinal raw materials include ginseng, pilose antler, angelica, wolfberry, jujube, tortoise shell, Ligustrum lucidum, ganoderma lucidum, honey, kudzu root, polygonatum, astragalus, cordyceps sinensis, longan meat, eucommia, salvia miltiorrhiza, chuanxiong, rehmannia root, crystal sugar and deer whip, which can significantly improve human immunity, enhance metabolism mechanism and improve human vitality. However, it uses multiple Chinese medicines, the preparation method is relatively complicated, and it contains alcohol, which is not conducive to use in different groups of people.

[0006] CN115779038A discloses a Chinese medicine for improving immunity, belonging to the technical field of Chinese medicine. The Chinese medicine is composed of the following raw materials in parts by weight: 3-8 parts by weight of ginseng, 30-60 parts by weight of astragalus, 5-10 parts by weight of angelica, 10-15 parts by weight of polygonatum, 1-6 parts by weight of cinnamon, 15-20 parts by weight of wolfberry, 10-15 parts by weight of longan pulp, 8-13 parts by weight of jujube, 10-15 parts by weight of Morinda officinalis and 8-13 parts by weight of tangerine peel. The invention adopts a pure Chinese medicine formula to combine medicinal materials with different effects and different flavors, which is helpful to enhance immunity. When the raw materials in each group by weight are distributed and used in coordination with each other, the overall compatibility of the medicine is mild, and its effect and efficacy can be further enhanced, thereby effectively improving the overall applicability and effectiveness of the Chinese medicine, making it easy for the elderly, infants, pregnant women and people with long-term illness and infirmity to take, without the need for other flavoring additives, with a sweet and sour taste, and convenient for food supplement. However, the Chinese medicine formula provided by the invention also contains a variety of medicinal materials, and the Chinese medicine preparation method provided at the same time does not undergo extraction, and the effect is poor.

[0007] Modern people suffer from "getting angry" due to the fast pace of life, high work pressure, lack of sleep and unreasonable diet, which leads to loss of body fluids, retention of water and moisture, excessive heat and evil fire generated from within. Many Chinese medicines or health products that can improve immunity contain warm herbs such as ginseng and astragalus, which can easily cause or aggravate the symptoms of "getting angry". In hot weather, the symptoms will be more serious, which is not conducive to the long-term use of health products or Chinese medicines or their promotion among people of all ages and physical conditions, and it is difficult to continuously play the role of improving immunity.

[0008] Therefore, how to provide a new formula of Guiqi Decoction that is more concise, reasonably purifies the effective ingredients of medicinal materials, and can be used to effectively improve immunity without causing internal heat is one of the key issues studied by technical personnel in this field. Summary of the invention

[0009] In view of the problems in the prior art such as insufficient extraction of medicinal materials and the susceptibility of warm medicinal materials to aggravate the symptoms of getting angry, the present invention provides a new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity and a preparation method thereof.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] Preferably, the astragalus includes but is not limited to raw astragalus and roasted astragalus.

[0012] Preferably, the angelica includes but is not limited to raw angelica, wine-cooked angelica, stir-fried angelica, angelica head, angelica body, angelica tail and whole angelica.

[0013] Preferably, the wolfberries include but are not limited to wolfberries, fried wolfberries and salted wolfberries.

[0014] The present invention also provides a method for preparing the above-mentioned new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity, comprising the following steps:

[0015] (1) extracting the formulated amount of Angelica sinensis with water to obtain an extract;

[0016] (2) centrifuging the extract obtained in step (1) to obtain an angelica extract;

[0017] (3) extracting the formula amount of astragalus and wolfberry with water, filtering, and centrifuging to obtain a water extract;

[0018] (4) concentrating the aqueous extract obtained in step (3), adding ethanol for precipitation, centrifuging, taking the supernatant, separating the ethanol, and obtaining a mixed extract;

[0019] (5) The angelica extract obtained in step (2) and the mixed extract obtained in step (4) are mixed to adjust the raw drug concentration to obtain the new formula of Guiqi Decoction.

[0020] Preferably, the new formula of Guiqi Decoction is composed of the following components in parts by weight:

[0021] 8-12 parts of Astragalus, 2-6 parts of Chinese Angelica and 2-6 parts of Lycium barbarum.

[0022] Further preferably, the new formula of Guiqi Decoction is composed of the following components in parts by weight:

[0023] 9-11 parts of Astragalus, 2-4 parts of Chinese Angelica and 2-4 parts of Lycium barbarum.

[0024] Most preferably, the new formula of Guiqi Decoction consists of the following components in parts by weight:

[0025] 10 parts of Astragalus, 2 parts of Chinese Angelica and 2 parts of Lycium barbarum.

[0026] Preferably, the amount of water added in step (1) is 6-12 times the total mass of the Angelica sinensis in the formula.

[0027] Further preferably, the amount of water added is 6-10 times the total mass of Angelica sinensis in the formula.

[0028] Most preferably, the amount of water added is 8-10 times the total mass of Angelica sinensis in the formula.

[0029] Preferably, the extraction in step (1) is performed by immersion extraction at 60-80°C.

[0030] Further preferably, the extraction by immersion method at 60-80° C. is performed 2-3 times.

[0031] Preferably, after the extraction in step (1) is completed, the extracted liquid is filtered.

[0032] Preferably, the centrifugal speed in step (2) is 4000-15000 rpm.

[0033] More preferably, the centrifugal speed is 8000-15000 rpm.

[0034] More preferably, the centrifugal speed is 10000-15000 rpm.

[0035] Still more preferably, the centrifugal speed is 12000-15000 rpm.

[0036] Most preferably, the centrifugal speed is 15000 rpm.

[0037] Preferably, the amount of water added in step (3) is: 5-10 times the total mass of astragalus and wolfberry as the formula amount.

[0038] Further preferably, the amount of water added is: 5-7 times the total mass of astragalus and wolfberry as the formula amount.

[0039] Preferably, the extraction in step (3) is performed by immersion extraction at 60-80°C for 3.5-5h.

[0040] Preferably, the centrifugal speed in step (3) is 4000-15000 rpm, and the centrifugal time is 5-20 min.

[0041] More preferably, the centrifugal speed is 4000-10000 rpm, and the centrifugal time is 8-20 min.

[0042] Most preferably, the centrifugal speed is 6000 rpm and the centrifugal time is 15 min.

[0043] Preferably, the concentration in step (4) is: concentration to 10-20% of the original volume.

[0044] Further preferably, the concentration is: concentration to 12-18% of the original volume.

[0045] Most preferably, the concentration is: concentrated to 14-16% of the original volume.

[0046] Preferably, the ethanol in step (4) is ethanol with a volume fraction of 90-95%.

[0047] Further preferably, the amount of ethanol added is 4-6 times the volume of the concentrated liquid in step (4).

[0048] Preferably, the alcohol precipitation in step (4) is: stirring evenly and placing at 5-7°C for 12-18h.

[0049] More preferably, the alcohol precipitation is: stirring evenly and placing at 5-7°C for 15-17h.

[0050] Most preferably, the alcohol precipitation is: stirring evenly and placing at 6°C for 16 hours.

[0051] Preferably, the centrifugal speed in step (4) is 4000-5000 rpm, and the centrifugal time is 5-8 min.

[0052] Preferably, the mixing in step (5) is as follows: the concentration ratio of the raw herbs of astragalus, wolfberry and angelica in the solution is controlled to be consistent with the mass ratio of astragalus, wolfberry and angelica in the formula.

[0053] Preferably, the crude drug concentration prepared in step (5) is: water is added to control the crude drug concentration to 0.010-0.020 g / mL.

[0054] More preferably, the concentration of the crude drug is controlled to be 0.013-0.017 g / mL by adding water.

[0055] Most preferably, the concentration of the crude drug is controlled to be 0.015 g / mL by adding water.

[0056] The present invention also provides a new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity, which is prepared by the method for preparing the new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity.

[0057] The present invention also provides the use of the new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity prepared by the preparation method thereof in the production of health products for enhancing immunity.

[0058] The present invention also provides a health product for enhancing immunity, which comprises the new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity prepared by the preparation method of the new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity.

[0059] The medicinal materials and effects adopted by the present invention are as follows:

[0060] Lycium barbarum L. is the mature fruit of Lycium barbarum L., a plant of the Solanaceae family. It has the effects of nourishing the liver and kidneys, improving intelligence and vision. It is mainly used to treat liver and kidney yin deficiency and premature aging.

[0061] Astragalus is the root of Astragalus memeranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the genus Astragalus in the Leguminosae family. It has the effects of strengthening the spleen and replenishing the middle, raising yang and lifting the sunken, strengthening the body's defense system, promoting urination, and removing toxins and promoting muscle growth. It is mainly used to treat spleen qi deficiency syndrome, lung qi deficiency syndrome, qi deficiency and spontaneous sweating syndrome, qi and blood deficiency, sores that are difficult to heal or ulcers that are difficult to heal for a long time, etc.

[0062] Angelica sinensis is the root of Angelica sinensis (Oliv) Diels. of the Apiaceae family. It has the effects of nourishing blood and regulating menstruation, promoting blood circulation and relieving pain, and moistening the intestines and relieving constipation. It is mainly used to treat chlorosis due to blood deficiency, irregular menstruation, amenorrhea, dysmenorrhea, blood deficiency, blood stasis, pain caused by cold blood, injuries caused by falls, rheumatism, carbuncle, ulcer, dry intestine and constipation, etc.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) Astragalus and Angelica are combined to generate Qi and blood, and the TCM principle that Qi helps blood flow and blood helps Qi to grow is applied to nourish the vital energy. At the same time, wolfberry is used to replenish the essence and marrow, nourish the kidney yin, and replenish the blood. Together, they play the role of replenishing Qi, generating blood, and enhancing immunity.

[0065] (2) The present invention uses only three medicinal materials, namely, wolfberry, astragalus and angelica, and obtains a new formula of Guiqi Decoction by water extraction-water extraction and alcohol precipitation, finely separating impurities and adjusting the extraction method of the effective ingredients of the medicinal materials. The new formula of Guiqi Decoction can effectively improve immunity and is not easy to cause internal heat.

[0066] Compared with the prior art, the new formula of Guiqi Decoction of the present invention has simpler components, and the medicinal materials are fully extracted to separate impurities and the method of extracting the effective ingredients of the medicinal materials is adjusted. The new formula of Guiqi Decoction has a better effect in enhancing immunity and is not easy to cause internal heat. DETAILED DESCRIPTION

[0067] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0068] In a specific embodiment of the present invention, the test method performs functional evaluation in accordance with the requirements of the "Health Food Function Test and Evaluation Method (2022 Edition)" issued by the State Administration for Market Regulation. The health food function test and evaluation method helps to enhance immunity: if the results are positive in any two of the four aspects of cellular immune function, humoral immune function, monocyte-macrophage function, and NK cell activity, it can be determined that the test sample has the effect of helping to enhance immunity.

[0069] In order to fully highlight the effectiveness and efficiency of the composition preparation method adopted in the present invention and take into account the "3R principle of experimental animals", in the present invention, only the various indicators of mice are examined with the same composition dose, rather than the three dose groups required by the "Health Food Function Test and Evaluation Method (2022 Edition)", and the number of experimental animals in each group is reduced from 10 to 8.

[0070] Example 1 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0071] (1) Amount of raw materials

[0072] Take 200g of Astragalus, 40g of Angelica, and 40g of Lycium barbarum.

[0073] (2) Angelica Extract

[0074] Take angelica, grind it into powder, add 8 times the weight of purified water, extract it by immersion method at 70°C for 3 times, each time for 2 hours, filter, combine the filtrate, centrifuge at 15000rpm to remove the precipitate, and take the supernatant to obtain the angelica extract.

[0075] (3) Astragalus and Lycium barbarum extract

[0076] Take astragalus and wolfberry, grind into powder, add purified water 8 times the total mass of astragalus and wolfberry, extract by immersion method at 70°C for 3.5 hours; filter, centrifuge at 10000rpm to remove precipitation for 6 minutes; then take the supernatant and concentrate it to 15% of the original volume of the supernatant. Add 95% ethanol with a volume fraction of 5 times the volume of the concentrated liquid, keep warm in an incubator at 6°C for 15 hours; centrifuge at 4000rpm for 8 minutes, take the supernatant, separate the ethanol by rotary evaporation, and obtain a mixed extract.

[0077] (4) Preparation of a new formula for Guiqi Decoction

[0078] The angelica extract obtained in step (2) is mixed with the mixed extract obtained in step (3), and purified water is added to prepare the raw drug concentration to 0.015 g / mL to obtain a new formula of Guiqi Decoction.

[0079] Example 2 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0080] (1) Amount of raw materials

[0081] Take 240g of Astragalus, 120g of Angelica, and 120g of Lycium barbarum.

[0082] (2) Angelica Extract

[0083] Take angelica, grind it into powder, add 12 times the mass of purified water, extract it twice by immersion method at 60°C, each time for 2 hours, filter, combine the filtrate, centrifuge at 4000rpm to remove the precipitate, and take the supernatant to obtain the angelica extract.

[0084] (3) Astragalus and Lycium barbarum extract

[0085] Take astragalus and wolfberry, grind into powder, add purified water 10 times the total mass of astragalus and wolfberry, extract by immersion method at 80°C for 3.5 hours; filter, centrifuge at 15000rpm to remove precipitation for 5 minutes; then take the supernatant and concentrate it to 20% of the original volume of the supernatant. Add 90% ethanol with a volume fraction of 5 times the volume of the concentrated liquid, keep warm in an incubator at 7°C for 18 hours; centrifuge at 5000rpm for 5 minutes, take the supernatant, separate the ethanol by rotary evaporation, and obtain a mixed extract.

[0086] (4) Preparation of a new formula for Guiqi Decoction

[0087] The angelica extract obtained in step (2) is mixed with the mixed extract obtained in step (3), and purified water is added to prepare the raw drug concentration to 0.020 g / mL to obtain a new formula of Guiqi Decoction.

[0088] Example 3 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0089] (1) Amount of raw materials

[0090] Take 160g of Astragalus, 40g of Angelica, and 40g of Lycium barbarum.

[0091] (2) Angelica Extract

[0092] Take angelica, grind it into powder, add 6 times the mass of purified water, extract it twice by immersion method at 60°C, each time for 2 hours, filter, combine the filtrate, centrifuge at 8000rpm to remove the precipitate, and take the supernatant to obtain the angelica extract.

[0093] (3) Astragalus and Lycium barbarum extract

[0094] Take astragalus and wolfberry, grind into powder, add purified water 5 times the total mass of astragalus and wolfberry, extract by immersion method at 60℃ for 5h; filter, centrifuge at 4000rpm to remove precipitation for 20min; then take the supernatant and concentrate it to 10% of the original volume of the supernatant. Add 93% ethanol with a volume fraction of 4 times the volume of the concentrated liquid, keep warm in an incubator at 5℃ for 12h; centrifuge at 4500rpm for 6min, take the supernatant, separate the ethanol by rotary evaporation, and obtain a mixed extract.

[0095] (4) Preparation of a new formula for Guiqi Decoction

[0096] The angelica extract obtained in step (2) is mixed with the mixed extract obtained in step (3), and purified water is added to prepare the raw drug concentration to 0.010 g / mL to obtain a new formula of Guiqi Decoction.

[0097] Example 4 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0098] (1) Amount of raw materials

[0099] Take 220g of Astragalus, 60g of Angelica, and 100g of Lycium barbarum.

[0100] (2) Angelica Extract

[0101] Take angelica, grind it into powder, add 8 times the mass of purified water, extract it by immersion method at 80°C for 3 times, each time for 2 hours, filter, combine the filtrate, centrifuge at 15000rpm to remove the precipitate, and take the supernatant to obtain the angelica extract.

[0102] (3) Astragalus and Lycium barbarum extract

[0103] Take astragalus and wolfberry, grind into powder, add purified water 8 times the total mass of astragalus and wolfberry, extract by immersion method at 70°C for 3.5 hours; filter, centrifuge at 10000rpm to remove precipitation for 6 minutes; then take the supernatant and concentrate it to 15% of the original volume of the supernatant. Add 95% ethanol with a volume fraction of 5 times the volume of the concentrated liquid, keep warm in an incubator at 6°C for 15 hours; centrifuge at 4000rpm for 8 minutes, take the supernatant, separate the ethanol by rotary evaporation, and obtain a mixed extract.

[0104] (4) Preparation of a new formula for Guiqi Decoction

[0105] The angelica extract obtained in step (2) is mixed with the mixed extract obtained in step (3), and purified water is added to prepare the raw drug concentration to 0.015 g / mL to obtain a new formula of Guiqi Decoction.

[0106] Comparative Example 1 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0107] The difference between this comparative example and Example 1 is that the dosage of astragalus, angelica and wolfberry is different, specifically: 260g of astragalus, 20g of angelica and 140g of wolfberry.

[0108] Comparative Example 2: A method for preparing a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0109] The difference between this comparative example and Example 1 is that the dosage of astragalus, angelica and wolfberry is different, specifically: 140g of astragalus, 140g of angelica, and 140g of wolfberry.

[0110] Comparative Example 3 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0111] The difference between this comparative example and Example 1 is that the method of extracting the drug is different, specifically:

[0112] (1) Amount of raw materials

[0113] Take 220g of Astragalus, 60g of Angelica, and 100g of Lycium barbarum.

[0114] (2) Extraction of new formula of Guiqi Decoction

[0115] Take astragalus, wolfberry and angelica, grind into powder, add purified water 8 times the total mass of astragalus and wolfberry, extract by immersion method at 70°C for 3.5 hours; filter, centrifuge at 10000rpm to remove precipitation for 6 minutes; then take the supernatant and concentrate it to 15% of the original volume of the supernatant. Add 95% ethanol with a volume fraction of 5 times the volume of the concentrated liquid, keep warm in an incubator at 6°C for 15 hours; centrifuge at 4000rpm for 8 minutes, take the supernatant, separate the ethanol by rotary evaporation, and obtain a mixed extract.

[0116] (3) Preparation of a new formula for Guiqi Decoction

[0117] Purified water was added to the mixed extract obtained in (2) to prepare the crude drug concentration to 0.015 g / mL to obtain a new formula of Guiqi Decoction. Comparative Example 4 Preparation method of a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0118] The difference between this comparative example and Example 1 is that the method of extracting the drug is different, specifically:

[0119] (1) Amount of raw materials

[0120] Take 220g of Astragalus, 60g of Angelica, and 100g of Lycium barbarum.

[0121] (2) Extraction of new formula of Guiqi Decoction

[0122] Take astragalus, wolfberry and angelica, grind into powder, add 8 times the mass of purified water, extract twice by immersion method at 70°C, each time for 2 hours, filter, combine the filtrate, centrifuge at 15000rpm to remove the precipitate, and take the supernatant to obtain a mixed extract.

[0123] (3) Preparation of a new formula for Guiqi Decoction

[0124] Purified water was added to the mixed extract obtained in (2) to prepare the crude drug concentration to 0.015 g / mL to obtain a new formula of Guiqi Decoction. Comparative Example 5 A method for preparing a new formula of Guiqi Decoction for replenishing qi, generating blood and enhancing immunity

[0125] The difference between this comparative example and Example 1 is that the method of extracting the drug is different, specifically:

[0126] (1) Amount of raw materials

[0127] Take 220g of Astragalus, 60g of Angelica, and 100g of Lycium barbarum.

[0128] (2) Angelica Extract

[0129] Take angelica, grind it into powder, add purified water 8 times the total mass of angelica, extract it by immersion method at 70°C for 3.5 hours; filter, centrifuge at 10000rpm to remove precipitation for 6 minutes; then take the supernatant and concentrate it to 15% of the original volume of the supernatant. Add 95% ethanol with a volume fraction of 5 times the volume of the concentrated liquid, keep it warm in an incubator at 6°C for 15 hours; centrifuge at 4000rpm for 8 minutes, take the supernatant, separate the ethanol by rotary evaporation, and obtain the angelica extract.

[0130] (3) Astragalus and Lycium barbarum extract

[0131] Take astragalus and wolfberry, grind them into powder, add purified water 8 times the total mass of astragalus and wolfberry, extract by immersion method at 70°C for 3 times, each time for 2 hours, filter, combine the filtrate, centrifuge at 15000rpm to remove the precipitate, and take the supernatant to obtain a mixed extract.

[0132] (4) Preparation of a new formula for Guiqi Decoction

[0133] The angelica extract obtained in step (2) is mixed with the mixed extract obtained in step (3), and purified water is added to prepare the raw drug concentration to 0.015 g / mL to obtain a new formula of Guiqi Decoction.

[0134] Experimental Example 1: The efficacy of a new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity in improving immunity

[0135] (1) Experimental animals:

[0136] 296 SPF male BALB / c mice, 6-8 weeks old, 18-22 g, were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Zhejiang) 2022-0005, animal certificate number: 20221205Abbbbb0100999400.

[0137] (2) New formula of Guiqi Decoction for intragastric administration:

[0138] The new formula of Guiqi Decoction prepared in Examples 1-4 and Comparative Examples 1-5 was concentrated to a relative crude drug concentration of 0.19 g / mL.

[0139] (3) Animal grouping:

[0140] BALB / c mice were randomly divided into a normal group, and then 10 groups of Examples 1-4 and Comparative Examples 1-5 were set according to each example and comparative example, with 8 mice in each group. The mice in the normal group were gavaged with distilled water, and the other groups were gavaged with the new formula of Guiqi Decoction prepared in the corresponding example / comparative example, with a volume of 10 mL / kg, that is, the amount of crude drug in each group of Examples 1-4 and Comparative Examples 1-5 was 1.9 g / kg, once a day, and continuously administered for 30 days.

[0141] If the control group fails to meet the requirements in the first three experiments, that is, it does not meet the requirements of the "Health Food Function Test and Evaluation Method (2022 Edition)", the fourth experiment will not be carried out.

[0142] (4) Efficacy experiment:

[0143] ①ConA-induced mouse spleen lymphocyte proliferation ability assay - cellular immune function experimental method:

[0144] The mice were killed, the spleen was removed aseptically, ground, sieved, and spleen cell suspension was prepared. The cell number was adjusted to 3×10 6 Each spleen cell suspension was added to two wells of a 24-well culture plate, 1 mL per well, and 75 μL ConA solution (equivalent to 7.5 μg mL -1 ), and the other well was used as a control and cultured in a 5% CO2, 37°C CO2 incubator for 72 h. 4 h before the end of the culture, 0.7 mL of supernatant was gently aspirated from each well, and 0.7 mL of RPMI1640 culture medium without calf serum was added, and MTT (5 mg mL -1 )50μL / well, continue to culture for 4h. After the culture is completed, add 1mL of acidic isopropanol to each well, mix by pipetting, and completely dissolve the purple crystals. Then dispense into 96-well culture plates, make 3 parallel wells in each well, and use a microplate reader to measure the optical density value at a wavelength of 570nm.

[0145] The optical density value of the well with ConA added minus the optical density value of the well without ConA added represents the proliferation ability of lymphocytes. If the optical density difference of the test sample group is significantly higher than that of the control group, the experimental result can be judged to be positive.

[0146] Table 1 Effect of the new formula of Guiqi Decoction on the proliferation of mouse spleen lymphocytes induced by ConA ( n=10)

[0147]

[0148] Note: Compared with the normal group, * P<0.05, ** P<0.01.

[0149] The proliferation capacity of spleen lymphocytes of mice treated with the new formula of Guiqi Decoction provided by Examples 1-4 of the present invention was significantly enhanced (P<0.01).

[0150] ② NK cell activity assay experimental method:

[0151] 24 h before the experiment, the target cells (YAC-1 cells) were subcultured and the cell concentration was adjusted to 4×10 5 The mice were killed, the spleens were removed aseptically, and spleen cell suspension was prepared as effector cells. The cell concentration was adjusted to 2×10 7 100 μL of target cells and effector cells (50:1 effector-target ratio) were added to a U-shaped 96-well culture plate; 100 μL of target cells and culture medium were added to the target cell natural release well, and 100 μL of target cells and 1% NP40 were added to the target cell maximum release well; three parallel wells were set for each of the above items, and cultured in a 37°C, 5% CO2 incubator for 4 hours, and then the 96-well culture plate was rotated at 1500 r·min -1 Centrifuge for 5 minutes, aspirate 100 μL of supernatant from each well and place it in a flat-bottom 96-well culture plate. Add 100 μL of LDH matrix solution and react for 3-10 minutes depending on the room temperature. Add 1 mol·L -1 Add 30 μL of HCl and measure the optical density (OD) at 490 nm using a microplate reader.

[0152] The NK cell activity is calculated according to the following formula. If the NK cell activity of the test sample group is significantly higher than that of the control group, the experimental result can be determined to be positive.

[0153]

[0154] Table 2 Effect of the new formula of Guiqi Decoction on NK cell activity ( n=10)

[0155]

[0156] Note: Compared with the normal group, * P<0.05, ** P<0.01.

[0157] The NK cell activity of mice using the new formula of Guiqi Decoction provided in Examples 1-4 of the present invention was significantly enhanced (P<0.01).

[0158] ③ Serum hemolysin (half hemolysis value) determination - humoral immune function experimental method:

[0159] On the 26th day after gavage, 0.2 mL of 2% (v / v) SRBC was injected intraperitoneally for immunization. On the 30th day, 2 hours after administration, blood was collected from the mouse orbits, and serum was collected by centrifugation. Sample wells and blank control wells were set up. Sample wells: serum was diluted 200 times with SA buffer, and 50 μL of diluted serum was added to each well; blank control wells: 50 μL of SA buffer was added to each well, and then 10%

[0160] (v / v) SRBC 25 μL, complement 50 μL (diluted with SA solution at 1:8), placed in a 37°C constant temperature incubator for 30 min, terminated the reaction in an ice bath, and rotated at 1500 r·min -1 Centrifuge horizontally for 10 minutes, take 50 μL of the supernatant from each sample well and blank control well and add it to another 96-well culture plate, add 150 μL of Dulbecco's reagent. At the same time, set up half of the hemolysis wells and add 10% (v / v)

[0161] 12.5 μL of SRBC was added to Dulbecco's reagent to make up to 200 μL. The mixture was thoroughly mixed with an oscillator and allowed to stand for 10 min. The optical density of each well was measured at 540 nm using an ELISA reader.

[0162] The amount of hemolysin is expressed as the half hemolytic value (HC 50 ) means that the HC of the tested sample group is calculated according to the following formula. 50 Significantly higher than the control group HC 50 , the test result can be judged as positive.

[0163]

[0164] Table 3 Effect of the new formula of Guiqi Decoction on the median hemolysis value of mice ( n=10)

[0165]

[0166] Note: Compared with the normal group, *P<0.05, **P<0.01.

[0167] The median hemolysis value of mice treated with the new formula of Guiqi Decoction provided in Examples 1-4 of the present invention was significantly increased (P<0.05, 0.01)

[0168] ④Peritoneal macrophage phagocytosis of chicken red blood cells - monocyte-macrophage function experimental method:

[0169] On the 26th day after gavage, 0.2mL 2% (v / v) SRBC was injected intraperitoneally for immunization. Two hours after administration on the 30th day, the mice were killed by cervical dislocation, and 4mL / mouse of Hank's solution with calf serum was injected intraperitoneally. The abdomen was gently massaged 20 times to fully wash out the peritoneal macrophages, and then a small incision was made in the abdominal wall, and 2mL of peritoneal washing fluid was sucked into a test tube with a rubber-tipped pipette. 0.5mL of peritoneal washing fluid was sucked into a test tube containing 0.5mL 1% chicken blood red blood cell suspension with a 1mL pipette and mixed. 0.5mL of the mixed solution was sucked with a syringe (with a large needle) and added to the agar circle of the glass slide. Place in an incubator at 37℃ for 20 minutes. After the incubation, the non-adherent cells were quickly washed away with physiological saline, fixed in methanol solution for 1 minute, and stained with Giemsa solution for 15 minutes. Rinse with distilled water, dry, and count the phagocytic rate and phagocytic index under a 40× microscope.

[0170] The phagocytic rate is the percentage of macrophages that phagocytize chicken red blood cells among every 100 macrophages; the phagocytic index is the average number of chicken red blood cells phagocytized by each macrophage. The phagocytic rate and phagocytic index of the test sample group are significantly higher than those of the control group, which can be judged as positive for this test result.

[0171] Table 4 Effect of the new formula of Guiqi Decoction on the phagocytic rate and phagocytic index of mice ( n=10)

[0172]

[0173]

[0174] Note: Compared with the normal group, * P<0.05, ** P<0.01.

[0175] The phagocytic rate and phagocytic index of mice treated with the new Guiqi Decoction formula provided in Examples 1-4 of the present invention were significantly increased (P<0.05, 0.01).

[0176] (5) Conclusion

[0177] ① Cellular immune function

[0178] Mouse spleen lymphocyte transformation experiment: After 30 days of administration, the proliferation ability of spleen lymphocytes in mice in the new Guiqi Decoction formula groups of Examples 1-4 was significantly enhanced (P<0.01).

[0179] ②Humoral immune function

[0180] Determination of serum hemolysin level: After 30 days of administration, the median hemolysis value of mice in the new Guiqi Decoction group of Example 1-4 was significantly increased (P<0.05, 0.01).

[0181] ③Mononuclear-macrophage function assay

[0182] Experiment on phagocytosis of chicken red blood cells by mouse peritoneal macrophages: After 30 days of administration, the phagocytic rate and phagocytic index of mice in the new Guiqi Decoction group of Example 1-4 were significantly increased (P<0.05, 0.01).

[0183] ④NK cell activity assay

[0184] The NK cell activity of mice in the new Guiqi Decoction group of Example 1-4 was significantly enhanced (P<0.01).

[0185] In summary, the new formulation of Guiqi Decoction of the present invention has positive results in cellular immune function test, humoral immune function test, monocyte-macrophage function test, and NK cell activity test. According to the requirements of the "Health Food Function Test and Evaluation Method (2022 Edition)" issued by the State Administration for Market Regulation, the new formulation of Guiqi Decoction has the function of enhancing immunity.

[0186] However, the new formula of Guiqi Decoction prepared in Comparative Examples 1 and 2 did not show the effect of improving immunity in accordance with the requirements of the "Health Food Function Test and Evaluation Method (2022 Edition)" due to the use of medicinal materials not provided by the present invention. Comparative Examples 3-4 are due to the failure to use the extraction method provided by the present invention for extraction, and the medicinal material components in each medicinal material cannot be well extracted. Specifically, in Comparative Example 3, the effective ingredients are soaked in water, and some effective ingredients and impurities are separated by alcohol precipitation, resulting in the loss of angelica effective ingredients without further separation of effective ingredients and impurities, which significantly reduces the extraction effect, while Comparative Example 4 uses a conventional water soaking method that often relies on a longer soaking time, resulting in changes in effective ingredients, and ultimately resulting in poor drug efficacy; while Comparative Example 5 separates the effective ingredients of angelica, and extracts astragalus and wolfberry for a long time, ultimately resulting in poor drug efficacy.

[0187] Experimental Example 2 Experiment on the effect of a new formula of Guiqi Decoction on the indigestion (serum inflammatory factors)

[0188] Huang et al. characterized the effect of longan extract on "getting angry" by using serum levels of TNF-α and IL-6 in their work on Excessive Intake of Longan Arillus Alters gutHomeostasis and Aggravates Colitis in Mice. The results showed that longan destroyed the intestinal homeostasis of mice, and the results provided a scientific basis for the abnormal body state ("getting angry") induced by excessive longan by destroying intestinal homeostasis. Therefore, in this experimental example, we used serum levels of TNF-α and IL-6 to test the effect of the new formula of Guiqi Decoction of the present invention on getting angry.

[0189] The ELISA kit mouse tumor necrosis factor α (TNF-α) ELISA KIT (CSB-E04741m, Wuhan Huamei Bioengineering Co., Ltd.) was used for operation according to the instructions of the ELISA kit. The absorbance value (OD value) was detected by an enzyme reader, and the TNF-α and IL-6 concentrations in mouse serum were calculated from the standard curve.

[0190] The serum of mice sacrificed in Experiment 1 (4) was tested for the concentration of TNF-α and IL-6. The results are shown in Table 5:

[0191] Table 5 TNF-α and IL-6 concentrations in mouse serum

[0192]

[0193] Note: Compared with the normal group, * P<0.05, ** P<0.01.

[0194] As shown in Table 5, in the mouse serum, the TNF-α (P<0.05) and IL-6 (P<0.05) levels of the mice in Comparative Examples 1, 2, 4 and 5 were significantly higher than those in the normal group, especially Comparative Examples 4 and 5 showed extremely significant differences (P<0.01); the IL-6 (P<0.05) content of the mice in Comparative Example 2 was significantly higher than that in the normal group, indicating that the use of the dosage of the components of the new formulation of Guiqi Decoction not provided by the present invention and the method for preparing the new formulation of Guiqi Decoction cannot avoid the significant increase of serum inflammatory factors TNF-α and / or IL-6; and the TNF-α and IL-6 levels of the mice using the new formulation of Guiqi Decoction of Examples 1-5 of the present invention did not change significantly compared with the normal group. The results show that the dosage of each component of the new formulation of Guiqi Decoction provided by the present invention combined with the extraction method provided by the present invention can effectively avoid the increase in serum inflammatory factor levels caused by the new formulation of Guiqi Decoction. This may be because the present invention controls the special water extraction and alcohol precipitation conditions of astragalus and wolfberry to separate some polysaccharides and proteins, retains most of the polysaccharides while significantly reducing the increase in inflammatory factors caused by special types of polysaccharides, thereby effectively preventing "getting angry".

[0195] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a new formula of Guiqi Decoction for invigorating qi, generating blood and enhancing immunity, characterized in that: The new formula of Guiqi Decoction is prepared from the following components by weight: 8-12 parts of Astragalus, 2-6 parts of Angelica and 2-6 parts of Lycium barbarum; The preparation method comprises the following steps: (1) extracting the formulated amount of angelica sinensis with water to obtain an extract; (2) centrifuging the extract obtained in step (1) to obtain an angelica extract; (3) extracting the formulated amount of astragalus and wolfberry with water, filtering, and centrifuging to obtain a water extract; (4) concentrating the aqueous extract obtained in step (3), adding ethanol for precipitation, centrifuging, taking the supernatant, separating the ethanol, and obtaining a mixed extract; (5) mixing the angelica extract obtained in step (2) and the mixed extract obtained in step (4) to prepare the crude drug concentration; The concentration in step (4) is: concentrated to 10-20% of the original volume; the ethanol in step (4) is ethanol with a volume fraction of 90-95%; the amount of ethanol added is 4-6 times the volume of the liquid after concentration in step (4); The alcohol precipitation in step (4) is as follows: stirring evenly and placing at 5-7°C for 12-18h; The concentration of the prepared crude drug in step (5) is as follows: water is added to control the crude drug concentration to be 0.010-0.020 g / mL.

2. The preparation method according to claim 1, characterized in that: The amount of water added in step (1) is 6-12 times the total mass of the angelica sinensis in the formula.

3. The preparation method according to claim 1, characterized in that: The extraction in step (3) is: extraction by immersion method at 60-80°C.

4. The preparation method according to claim 1, characterized in that: The mixing in step (5) is as follows: the concentration ratio of the raw materials of astragalus, wolfberry and angelica in the solution is controlled to be consistent with the mass ratio of astragalus, wolfberry and angelica in the formula.

5. A new formula of Guiqi Decoction prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the new formula of Guiqi Decoction prepared by the preparation method according to any one of claims 1 to 4 in preparing health products for enhancing immunity.

7. A health product for enhancing immunity, comprising the new formula of Guiqi Decoction prepared by the preparation method according to any one of claims 1 to 4.

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