Composite plant extract beneficial to enhancing immunity as well as preparation method and application of composite plant extract

Abstract

The invention discloses a composite plant extract helpful for enhancing immunity and a preparation method and application thereof, the composite plant extract comprises the following raw materials by weight: 30-40 parts of a Poria cocos extract, 20-30 parts of a Radix Codonopsis extract, 20-30 parts of a honeysuckle extract, 5-10 parts of a Cornus officinalis extract, and 5-10 parts of a mulberry leaf extract. The composite plant extract is prepared by compounding a poria cocos extract, a codonopsis pilosula extract, a honeysuckle flower extract, a dogwood extract and a mulberry leaf extract, wherein chlorogenic acid in the honeysuckle flower extract can enhance the phagocytic ability of cells; a main component codonopsis pilosula polysaccharide in the codonopsis pilosula extract can improve indexes and responses of immune organs such as spleen, thymus, liver and intestinal lymph nodes, active substances such as polysaccharide, ursolic acid and iridoid glycoside in the dogwood extract also have a relatively good immunoregulation effect, and under the synergistic effect of the raw materials, the immunity of the human body is improved. And the composite plant extract can play a better role in enhancing immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of functional foods, and specifically relates to a composite plant extract that helps to enhance immunity, and a preparation method and application thereof. Background Art

[0002] Immunity is a self-defense mechanism of the human body. It is the body's ability to identify and eliminate foreign bodies that invade, deal with aging, damaged, dead, and degenerated cells, and identify and deal with mutant cells and virus-infected cells in the body. With the rapid development of society, people's dietary structure, living habits, and living environment have changed greatly. Staying up late for a long time, irregular diet, smoking, heavy drinking, lack of exercise, etc., have damaged our body's immunity. Immunity is the "firewall" of human health. Once cracks or gaps appear in this wall, the body will have a corresponding reaction. When the immune function is disordered or low, it will reduce the body's ability to fight infection, reduce the ability to identify and eliminate its own aging tissue cells, and reduce the ability to kill and eliminate abnormal mutant cells in the body, resulting in a decrease in the body's disease resistance and self-healing ability, which is related to the occurrence of many diseases.

[0003] A body with low immunity is prone to infection or cancer, and the most direct manifestation is that it is easy to get sick. Frequent illness increases the body's consumption, so there are generally symptoms such as physical weakness, malnutrition, mental depression, fatigue, decreased appetite, and sleep disorders. Getting sick, getting injections, and taking medicine have become commonplace. It takes a long time to recover from each illness, and it often recurs. In the long run, it will lead to physical and intellectual stunting, and it is also easy to induce major diseases. Most patients with low immunity take vitamins and minerals to improve their immunity, but the treatment effect is not good, and long-term use will produce sequelae.

[0004] Chinese patent application CN114617937A discloses a composition for improving immunity, and its preparation method and application. The raw materials of the composition are made of the following components in parts by weight: 30-40 parts of Codonopsis pilosula extract, 15-20 parts of Astragalus membranaceus extract, 8-10 parts of donkey-hide gelatin extract, 4-8 parts of Pueraria lobata extract, 10-12 parts of Polygonum multiflorum extract, 5-8 parts of Rehmannia glutinosa extract, 2-6 parts of Dendrobium officinale extract, 2-4 parts of Solanum nigrum extract, 3-5 parts of Licorice extract, and 8-10 parts of honey. The Codonopsis pilosula extract, Astragalus membranaceus extract, Donkey-hide gelatin extract, Pueraria lobata extract, Polygonum multiflorum extract, Rehmannia glutinosa extract, Dendrobium officinale extract, Solanum nigrum extract, Licorice extract and honey are uniformly mixed and prepared using natural plant extracts, which greatly reduces the side effects after taking the composition. After taking the composition, the immunity can be improved and the health of the human body can be promoted. Chinese patent application CN108815318A discloses a composition for enhancing immunity, and its preparation method and use. The composition comprises by weight: 20 parts of yellow mealworm, 1 part of gecko powder, 1 part of toad powder, 1 part of cat's claw grass, 1 part of three-leaf cliff creeper, and 5 parts of Li Wang grass. The invention also provides a preparation method and use of the composition. The method is: weigh each drug raw material in proportion, dry it, then grind and crush it separately and sieve it, then mix it evenly, and finally seal it in a bag. The composition is used to prepare oral granules for enhancing immunity. The invention has the characteristics of reasonable formulation, small dosage, significant effect, and no toxic side effects. Oral administration can effectively enhance immunity, is particularly suitable for cancer patients, and can also be used for anti-aging, treatment of three highs, etc. However, the efficacy of enhancing immunity cannot be seen from the description of the above patent, and the composition in the above patent uses more than ten kinds of raw materials, and the preparation is relatively complicated.

[0005] Therefore, it is of great significance to develop a composite plant extract that helps to enhance immunity in the art. Summary of the invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a composite plant extract that helps to enhance immunity, and a preparation method and application thereof. The composite plant extract is compounded with Poria cocos extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract. The raw materials used are all plants that are both medicinal and edible, with a wide range of sources, safe and healthy. Through the synergistic effect between the raw materials, the composite plant extract has excellent immunity enhancement effect.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0009] 30-40 parts of Poria cocos extract, 20-30 parts of Codonopsis pilosula extract, 20-30 parts of Honeysuckle extract, 5-10 parts of Cornus officinalis extract, and 5-10 parts of Mulberry leaf extract.

[0010] Preferably, a composite plant extract that helps to enhance immunity comprises the following raw materials by weight:

[0011] 30-35 parts of Poria cocos extract, 25-30 parts of Codonopsis pilosula extract, 25-30 parts of Honeysuckle extract, 5-7 parts of Cornus officinalis extract, and 5-8 parts of mulberry leaf extract.

[0012] In the present invention, Poria cocos is the dried sclerotium of the fungus Poria cocos of the Polyporaceae family, which often parasitizes the roots of Masson pine and Pinus densiflora. Poria cocos contains a variety of components such as triterpenes, polysaccharides, sterols, diterpenes, proteins, amino acids, organic acids and their esters, flavonoids and trace elements, among which polysaccharides and triterpenes are the main active ingredients for its drug effects. Polysaccharides are the main components in Poria cocos, which have a variety of health care effects such as immunomodulation, diuresis, anti-inflammatory, liver protection, and calmness, and have certain food and nutrition characteristics.

[0013] Codonopsis pilosula is a traditional Chinese medicine that can be used as both medicine and food. It mainly comes from the dried roots of Codonopsis pilosula, Codonopsis pilosula, Codonopsis tubulosa, Codonopsis pilosula, and Codonopsis pilosula of the Campanulaceae family. It has the effects of tonifying the middle, replenishing qi, and promoting the production of body fluids. It is mainly used to treat weak spleen and stomach, deficiency of both qi and blood, fatigue, poor appetite, thirst, chronic diarrhea, and prolapse of the anus.

[0014] Honeysuckle, also known as honeysuckle, contains a variety of plant active ingredients that are beneficial to the human body, such as organic acids, flavonoids, volatile oils, cyclopentane glycosides, etc. Among them, organic acid components can be separated into chlorogenic acid and isochlorogenic acid with heat-clearing, detoxifying and antibacterial effects. Flavonoid components, such as luteolin and honeysuckle glycosides, are often used as the main active substances with antiviral, anti-tumor and antioxidant activities. Volatile oil components have antibacterial, anti-inflammatory and hemostatic effects. Cyclopentane glycosides play a role in protecting the liver and gallbladder.

[0015] Cornus officinalis is the dried mature pulp of Cornus officinalis, a plant of the Cornaceae family. It is harvested when the peel turns red in late autumn and early winter, baked over a slow fire or slightly scalded in boiling water, and the core is removed in time and dried. Cornus officinalis tastes sour, astringent, and slightly warm, and it enters the liver and kidney meridians. Its effects are to nourish the liver and kidney, astringent essence, and consolidate the body. It is clinically used for internal heat and thirst, dizziness and tinnitus, soreness of the waist and knees, impotence and spermatorrhea, frequent urination, metrorrhagia, leukorrhea, sweating and collapse, etc. The main active ingredients in Codonopsis pilosula are flavonoids, alkaloids, sugars, saponins, steroids, amino acids, sesquiterpenes, etc. Codonopsis pilosula has multiple pharmacological effects such as enhancing immune function, anti-inflammatory, antioxidant, anti-aging, anti-tumor and antibacterial.

[0016] Mulberry leaf is the dried leaf of the mulberry plant of the Moraceae family. It is cold in nature and bitter in taste. It belongs to the lung and liver meridians. It has the effects of dispersing wind-heat, clearing heat and moistening dryness, clearing the liver and improving eyesight. It is used for wind-heat cold, lung heat and dry cough, dizziness and headache, red and blurred eyes. Mulberry leaf extract contains a variety of effective ingredients such as flavonoids, alkaloids, mulberry leaf polysaccharides and proteins, and also has pharmacological effects such as lowering blood sugar, anti-oxidation, anti-tumor, and lowering blood lipids.

[0017] Preferably, the method for preparing the honeysuckle extract comprises the following steps:

[0018] Epigallocatechin gallate is added to anhydrous ethanol, and stirred evenly to obtain an epigallocatechin gallate solution; γ-cyclodextrin is added to deionized water, and stirred evenly to obtain a γ-cyclodextrin solution; then the epigallocatechin gallate solution is added to the γ-cyclodextrin solution, and stirred at a constant temperature to obtain a mixed solution; honeysuckle pollen is added to the ethanol aqueous solution, and immersed at room temperature, and hot water of 70-80°C is added after the immersion is completed, and then caprylic decanoic acid glyceride and the mixed solution are added, and reduced pressure extraction is performed under ultrasonic conditions, and after the extraction is completed, it is filtered, and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0019] In the present invention, epigallocatechin gallate solution is first compounded with γ-cyclodextrin to form an inclusion solution, and then the inclusion solution is added during the extraction process of honeysuckle. The γ-cyclodextrin in the inclusion solution can improve the solubility of chlorogenic acid, increase the extraction rate and stability of chlorogenic acid, and epigallocatechin gallate can promote the body's absorption of chlorogenic acid, increase the bioavailability of chlorogenic acid, and play a synergistic role in enhancing immunity with the active substances in honeysuckle. At the same time, adding caprylic decanoic acid glyceride during the extraction process can improve the solubility and stability of chlorogenic acid, thereby improving the bioavailability of chlorogenic acid.

[0020] Preferably, the mass concentration of the epigallocatechin gallate solution is 1-2%, the mass concentration of the γ-cyclodextrin solution is 4-6%, the mass ratio of the epigallocatechin gallate solution and the γ-cyclodextrin solution is 20-30:40-60, the temperature of the constant temperature stirring is 35-40°C, and the time is 4-6h; the mass concentration of ethanol in the ethanol aqueous solution is 70-80%, the mass ratio of the honeysuckle pollen, ethanol aqueous solution, hot water, caprylic decanoic acid glyceride, and the mixed solution is 60-80:150-200:900-1200:1-2:60-90, the immersion time is 30-40min, the power of the ultrasound is 200-300W, the pressure of the reduced pressure extraction is 20-30kpa, and the time is 15-25min.

[0021] Preferably, the method for preparing the Cornus officinalis extract comprises the following steps:

[0022] Under light-proof conditions, beta-carotene is added to acetone, stirred evenly, then beta-cyclodextrin aqueous solution is added, stirred at a constant temperature, then rotary evaporation is performed to remove water and acetone, and the solid product is washed and dried to obtain composite cyclodextrin; cornus officinalis powder is added to water, then pectinase, cellulase and neutral protease are added to perform enzymolysis, after the enzymolysis is completed, ethanol and composite cyclodextrin are added to perform leaching, after the leaching is completed, filtering is performed, the filtrate is concentrated under reduced pressure to 1 / 4 of the volume, and freeze-dried to obtain cornus officinalis extract.

[0023] In the present invention, beta-carotene and beta-cyclodextrin are first mixed to form a complex, and then the cornus officinalis is enzymolyzed by a composite enzyme to dissolve active substances such as polysaccharides and ursolic acid in the cornus officinalis. Subsequently, the composite cyclodextrin is added and extracted with an ethanol aqueous solution to extract water-soluble substances and alcohol-soluble substances in the cornus officinalis, thereby improving the extraction rate of active substances in the cornus officinalis.

[0024] Preferably, the mass fraction of the β-cyclodextrin aqueous solution is 1-1.8%, the mass ratio of the β-carotene, acetone and β-cyclodextrin aqueous solution is 0.03-0.05:100-150:100-150, the temperature of the constant temperature stirring is 35-40°C, and the time is 8-12h; the mass ratio of the Cornus officinalis powder, water, pectinase, cellulase, neutral protease, ethanol and composite cyclodextrin is 50-70:600-800:1-2:1-2:0.5-1:200-300:2-4, the temperature of the enzymatic hydrolysis is 40-45°C, and the time is 2-3h; the temperature of the extraction is 50-60°C, and the time is 1-2h; the pressure of the reduced pressure concentration is 20-30kpa.

[0025] Preferably, the method for preparing the Poria cocos extract comprises the following steps:

[0026] The tuckahoe powder is degreased by reflux with ethanol and then added into deionized water, swelled at a certain temperature, and then filtered to obtain the pretreated tuckahoe powder; the pretreated tuckahoe powder is added into a sodium hydroxide solution, alkali extracted twice, the filtrates are combined, the pH is adjusted to 7, the filtrates are concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution is added for precipitation, the precipitate is washed, desalted and dried to obtain the tuckahoe extract.

[0027] Preferably, the mass ratio of the Poria powder and deionized water is 20-30:400-600, the swelling temperature is 40-50°C, and the time is 1-2h; the concentration of the sodium hydroxide solution is 0.5-0.8mol / L, the mass ratio of the pretreated Poria powder and sodium hydroxide solution is 10:200-300, the alkali extraction temperature is 20-30°C, and the time is 4-6h.

[0028] The present invention also protects a method for preparing the composite plant extract that helps to enhance immunity as described above, comprising the following steps:

[0029] Weigh the raw materials according to the formula, and evenly mix the Poria extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract to obtain the product.

[0030] The present invention also protects the use of the composite plant extract as described above that helps to enhance immunity in the preparation of food or medicine that helps to enhance immunity.

[0031] Preferably, the food is a health food or a functional food.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The composite plant extract that helps to enhance immunity provided by the present invention is compounded with honeysuckle extract, codonopsis extract, cornus extract, mulberry leaf extract, and poria extract. The chlorogenic acid in the honeysuckle extract can increase the content of anti-inflammatory factors in the body, reduce the content of pro-inflammatory factors, enhance the phagocytic ability of cells, and improve the body's immunity. The main component of the codonopsis extract, codonopsis polysaccharide, can improve the index and response of immune organs such as the spleen, thymus, liver and intestinal lymph nodes, maintain the balance of immune cells such as lymphocytes and macrophages, and give full play to the beneficial role of promoting and restoring the body's immune function; the polysaccharides, ursolic acid, iridoid glycosides and other active substances in the cornus extract also have good immunoregulatory effects; under the synergistic effect of the raw materials, the composite plant extract can play a better effect of enhancing immunity.

[0034] (2) The composite plant extract provided by the present invention is helpful for enhancing immunity. The honeysuckle extract is added. First, γ-cyclodextrin is reacted with epigallocatechin gallate to form an inclusion compound, which improves the water solubility of epigallocatechin gallate. Then, the active substances such as chlorogenic acid in the honeysuckle are extracted by combining the reduced pressure internal boiling method with the ultrasonic method. The chlorogenic acid in the honeysuckle can be extracted at a lower temperature and in a shorter time. At the same time, caprylic decanoic acid glyceride and a mixed solution are added during the extraction process. The epigallocatechin gallate inclusion compound in the mixed solution can improve the extraction of chlorogenic acid during the extraction process. The invention discloses a novel method for preparing the honeysuckle extract from the present invention, which can improve the extraction rate and stability of chlorogenic acid in the honeysuckle extract, reduce the decrease in the activity of the effective substances in the honeysuckle during the extraction process, and thus improve the effect of the active substances in the honeysuckle extract; the inventors of the present application unexpectedly discovered that the inclusion complex formed by γ-cyclodextrin and epigallocatechin gallate and then added to the honeysuckle extract during the extraction process can effectively improve the extraction rate and stability of chlorogenic acid in the honeysuckle extract, so that the obtained honeysuckle extract has a stronger effect of enhancing immunity; at the same time, the inclusion complex of epigallocatechin gallate can also promote the absorption of the active substances in the composition, enhance immunity with the active substances in the honeysuckle, and improve the bioavailability of the composition.

[0035] (3) The composite plant extract that helps to enhance immunity provided by the present invention comprises the following: the added cornus extract is extracted by a composite enzyme method and cyclodextrin-assisted extraction, so that the polysaccharides and ursolic acid in the cornus are extracted simultaneously, and the composite cyclodextrin added during the extraction process contains β-carotene, which can effectively avoid the reduction of the activity of the effective substances during the extraction process and improve the quality of the cornus extract; the added Poria extract is subjected to a degreasing treatment of the Poria powder to destroy the cell membrane structure, which is beneficial to the dissolution of the intracellular polysaccharides, and then subjected to alkali extraction. The pretreatment can improve the extraction rate of the alkali-soluble polysaccharide; the alkali-soluble polysaccharide in the Poria is extracted, and the polysaccharide is a linear β (1→3) glycosidic bond-linked glucan containing a relatively long β (1→6) glycosidic bond-linked branch, and is a long-chain macromolecular polysaccharide, which is combined with the active ingredients such as chlorogenic acid in honeysuckle and ursolic acid in cornus to form a complex, which helps the active ingredients to exert a synergistic effect, so that the composite plant extract has a better effect of enhancing immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The figure shows the effect of the plant extract containing honeysuckle of the present invention on the cellular immune function of mice; Figure a is the optical density difference, and Figure b is the plantar thickness. Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0037] Figure 2The figure shows the effect of the plant extract containing honeysuckle of the present invention on the humoral immune function of mice; Figure a shows the number of hemolytic plaques, and Figure b shows the number of anti-plasma. Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0038] Figure 3 The figure shows the effect of the plant extract containing honeysuckle on the function of mouse monocyte-macrophages; Figure a is the carbon clearance phagocytosis index, Figure b is the ability of peritoneal macrophages to phagocytose chicken red blood cells (phagocytosis rate), and Figure c is the phagocytosis index. Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05,

[0039] ##P<0.01.

[0040] Figure 4 The figure shows the effect of the plant extract containing honeysuckle of the present invention on the activity of mouse NK cells. Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] In the present invention, the Codonopsis pilosula extract was purchased from Hubei Haijia Biotechnology Co., Ltd., and is a water extract with a specification of 10:1; the mulberry leaf extract was purchased from Jiangsu Jiujia Biotechnology Co., Ltd., and is a water extract with a specification of 10:1.

[0043] Example 1

[0044] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0045] 35 parts of Poria cocos extract, 25 parts of Codonopsis pilosula extract, 25 parts of Honeysuckle extract, 7 parts of Cornus officinalis extract, and 8 parts of Morus alba extract.

[0046] The preparation method of the Poria cocos extract comprises the following steps:

[0047] 25g of Poria powder was degreased by reflux with ethanol, then added into 500g of deionized water, swelled at 45°C for 1.5h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 250g of 0.7mol / L sodium hydroxide solution, and alkali extracted twice at a temperature of 25°C for 5h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0048] The preparation method of the honeysuckle extract comprises the following steps:

[0049] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 1.5%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 5%; then 25g of epigallocatechin gallate solution is added to 50g of γ-cyclodextrin solution, and stirred at a constant temperature of 38°C for 5h to obtain a mixed solution; 70g of honeysuckle pollen is added to 180g of ethanol aqueous solution (the mass concentration of ethanol is 75%), and immersed for 35min at room temperature. After the immersion is completed, 1100g of hot water with a temperature of 75°C is added, and then 1.5g of caprylic decanoic acid glyceride and 75g of the mixed solution are added, and reduced pressure extraction is performed for 20min under the conditions of ultrasonic power of 250W and pressure of 25kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0050] The preparation method of the Cornus officinalis extract comprises the following steps:

[0051] Under light-proof conditions, 0.04 g of β-carotene is added to 130 g of acetone, stirred evenly, and then 130 g of a 1.5% mass% β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 38° C. for 10 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 60 g of cornus officinalis powder is added to 700 g of water, and then 1.5 g of pectinase, 1.5 g of cellulase and 0.8 g of neutral protease are added, and enzymolysis is performed at 43° C. for 2.5 hours. After the enzymolysis is completed, 250 g of anhydrous ethanol and 3 g of the composite cyclodextrin are added, and the mixture is extracted at 55° C. for 1.5 hours. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 25 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0052] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0053] Weigh the raw materials according to the formula, and evenly mix the Poria extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract to obtain the product.

[0054] Example 2

[0055] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0056] 30 parts of Poria cocos extract, 20 parts of Codonopsis pilosula extract, 20 parts of Honeysuckle extract, 5 parts of Cornus officinalis extract, and 5 parts of Morus alba extract.

[0057] The preparation method of the Poria cocos extract comprises the following steps:

[0058] 20g of Poria powder was degreased by reflux with ethanol, then added into 400g of deionized water, swelled at 40°C for 2h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 200g of 0.8mol / L sodium hydroxide solution, and alkali extracted twice, the temperature of alkali extraction was 20°C, the time was 6h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0059] The preparation method of the honeysuckle extract comprises the following steps:

[0060] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 1%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 4%; then 20g of epigallocatechin gallate solution is added to 40g of γ-cyclodextrin solution, and stirred at a constant temperature of 35°C for 6h to obtain a mixed solution; 60g of honeysuckle pollen is added to 150g of ethanol aqueous solution (the mass concentration of ethanol is 80%), and immersed at room temperature for 30min. After the immersion is completed, 900g of hot water with a temperature of 80°C is added, and then 1g of caprylic decanoic acid glyceride and 60g of the mixed solution are added, and reduced pressure extraction is performed for 15min under the conditions of ultrasonic power of 200W and pressure of 20kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0061] The preparation method of the Cornus officinalis extract comprises the following steps:

[0062] Under light-proof conditions, 0.03 g of β-carotene is added to 100 g of acetone, stirred evenly, and then 100 g of a 1% by mass β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 35° C. for 12 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 50 g of cornus officinalis powder is added to 600 g of water, and then 1 g of pectinase, 1 g of cellulase and 0.5 g of neutral protease are added, and enzymolysis is performed at 40° C. for 3 hours. After the enzymolysis is completed, 200 g of anhydrous ethanol and 2 g of the composite cyclodextrin are added, and the mixture is extracted at 50° C. for 2 hours. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 20 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0063] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0064] Weigh the raw materials according to the formula, and evenly mix the Poria extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract to obtain the product.

[0065] Example 3

[0066] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0067] 40 parts of Poria cocos extract, 30 parts of Codonopsis pilosula extract, 30 parts of Honeysuckle extract, 10 parts of Cornus officinalis extract, and 10 parts of Morus alba extract.

[0068] The preparation method of the Poria cocos extract comprises the following steps:

[0069] 30g of Poria powder was defatted by reflux with ethanol, then added into 600g of deionized water, swelled at 50°C for 1h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 300g of 0.5mol / L sodium hydroxide solution, and alkali extracted twice, the temperature of alkali extraction was 30°C, the time was 4h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0070] The preparation method of the honeysuckle extract comprises the following steps:

[0071] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 2%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 6%; then 30g of epigallocatechin gallate solution is added to 60g of γ-cyclodextrin solution, and stirred at a constant temperature of 40°C for 4h to obtain a mixed solution; 80g of honeysuckle pollen is added to 200g of ethanol aqueous solution (the mass concentration of ethanol is 70%), and immersed for 40min at room temperature. After the immersion is completed, 1200g of hot water with a temperature of 70°C is added, and then 2g of caprylic decanoic acid glyceride and 90g of the mixed solution are added, and reduced pressure extraction is carried out for 25min under the conditions of ultrasonic power of 300W and pressure of 30kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0072] The preparation method of the Cornus officinalis extract comprises the following steps:

[0073] Under light-proof conditions, 0.05 g of β-carotene is added to 150 g of acetone, stirred evenly, and then 150 g of a 1.8% β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 40° C. for 8 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 70 g of cornus officinalis powder is added to 800 g of water, and then 2 g of pectinase, 2 g of cellulase and 1 g of neutral protease are added, and enzymolysis is performed at 45° C. for 2 hours. After the enzymolysis is completed, 300 g of anhydrous ethanol and 4 g of the composite cyclodextrin are added, and the mixture is extracted at 60° C. for 1 hour. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 30 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0074] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0075] Weigh the raw materials according to the formula, and evenly mix the Poria extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract to obtain the product.

[0076] Comparative Example 1

[0077] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0078] 35 parts of Poria cocos extract, 25 parts of Codonopsis pilosula extract, 25 parts of Honeysuckle extract, 7 parts of Cornus officinalis extract, and 8 parts of Morus alba extract.

[0079] The preparation method of the Poria cocos extract comprises the following steps:

[0080] 25g of Poria powder was degreased by reflux with ethanol, then added into 500g of deionized water, swelled at 45°C for 1.5h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 250g of 0.7mol / L sodium hydroxide solution, and alkali extracted twice at a temperature of 25°C for 5h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0081] The preparation method of the honeysuckle extract comprises the following steps:

[0082] Add 70g of honeysuckle pollen to 180g of ethanol aqueous solution (the mass concentration of ethanol is 75%), soak at room temperature for 35 minutes, add 1100g of hot water at 75°C after soaking, then add 1.5g of caprylic decanoic acid glyceride, 0.375g of epigallocatechin gallate, and 2.5g of γ-cyclodextrin, and extract under reduced pressure for 20 minutes under the conditions of ultrasonic power of 250W and pressure of 25kPa. After extraction, filter and rotary evaporate the filtrate to obtain honeysuckle extract.

[0083] The preparation method of the Cornus officinalis extract comprises the following steps:

[0084] Under light-proof conditions, 0.04 g of β-carotene is added to 130 g of acetone, stirred evenly, and then 130 g of a 1.5% mass% β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 38° C. for 10 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 60 g of cornus officinalis powder is added to 700 g of water, and then 1.5 g of pectinase, 1.5 g of cellulase and 0.8 g of neutral protease are added, and enzymolysis is performed at 43° C. for 2.5 hours. After the enzymolysis is completed, 250 g of anhydrous ethanol and 3 g of the composite cyclodextrin are added, and the mixture is extracted at 55° C. for 1.5 hours. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 25 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0085] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0086] Weigh the raw materials according to the formula, and evenly mix the Poria extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract to obtain the product.

[0087] Compared with Example 1, in this comparative example, epigallocatechin gallate and γ-cyclodextrin are not included to form an inclusion complex, but the two are directly added to the honeysuckle extract.

[0088] Comparative Example 2

[0089] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0090] 35 parts of Poria cocos extract, 25 parts of honeysuckle extract, 7 parts of cornus officinalis extract, and 8 parts of mulberry leaf extract.

[0091] The preparation method of the Poria cocos extract comprises the following steps:

[0092] 25g of Poria powder was degreased by reflux with ethanol, then added into 500g of deionized water, swelled at 45°C for 1.5h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 250g of 0.7mol / L sodium hydroxide solution, and alkali extracted twice at a temperature of 25°C for 5h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0093] The preparation method of the honeysuckle extract comprises the following steps:

[0094] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 1.5%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 5%; then 25g of epigallocatechin gallate solution is added to 50g of γ-cyclodextrin solution, and stirred at a constant temperature of 38°C for 5h to obtain a mixed solution; 70g of honeysuckle pollen is added to 180g of ethanol aqueous solution (the mass concentration of ethanol is 75%), and immersed for 35min at room temperature. After the immersion is completed, 1100g of hot water with a temperature of 75°C is added, and then 1.5g of caprylic decanoic acid glyceride and 75g of the mixed solution are added, and reduced pressure extraction is performed for 20min under the conditions of ultrasonic power of 250W and pressure of 25kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0095] The preparation method of the Cornus officinalis extract comprises the following steps:

[0096] Under light-proof conditions, 0.04 g of β-carotene is added to 130 g of acetone, stirred evenly, and then 130 g of a 1.5% mass% β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 38° C. for 10 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 60 g of cornus officinalis powder is added to 700 g of water, and then 1.5 g of pectinase, 1.5 g of cellulase and 0.8 g of neutral protease are added, and enzymolysis is performed at 43° C. for 2.5 hours. After the enzymolysis is completed, 250 g of anhydrous ethanol and 3 g of the composite cyclodextrin are added, and the mixture is extracted at 55° C. for 1.5 hours. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 25 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0097] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0098] Weigh the raw materials according to the formula, and evenly mix the Poria cocos extract, the honeysuckle extract, the cornus officinalis extract, and the mulberry leaf extract to obtain the product.

[0099] Compared with Example 1, no Codonopsis pilosula extract was added in this comparative example.

[0100] Comparative Example 3

[0101] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0102] 35 parts of Poria cocos extract, 25 parts of Codonopsis pilosula extract, 25 parts of Honeysuckle extract, and 8 parts of Morus alba extract.

[0103] The preparation method of the Poria cocos extract comprises the following steps:

[0104] 25g of Poria powder was degreased by reflux with ethanol, then added into 500g of deionized water, swelled at 45°C for 1.5h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 250g of 0.7mol / L sodium hydroxide solution, and alkali extracted twice at a temperature of 25°C for 5h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0105] The preparation method of the honeysuckle extract comprises the following steps:

[0106] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 1.5%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 5%; then 25g of epigallocatechin gallate solution is added to 50g of γ-cyclodextrin solution, and stirred at a constant temperature of 38°C for 5h to obtain a mixed solution; 70g of honeysuckle pollen is added to 180g of ethanol aqueous solution (the mass concentration of ethanol is 75%), and immersed for 35min at room temperature. After the immersion is completed, 1100g of hot water with a temperature of 75°C is added, and then 1.5g of caprylic decanoic acid glyceride and 75g of the mixed solution are added, and reduced pressure extraction is performed for 20min under the conditions of ultrasonic power of 250W and pressure of 25kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0107] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0108] Weigh the raw materials according to the formula, and evenly mix the Poria cocos extract, Codonopsis pilosula extract, Honeysuckle extract and Mulberry leaf extract to obtain the product.

[0109] Compared with Example 1, this comparative example does not add the Cornus officinalis extract.

[0110] Comparative Example 4

[0111] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0112] 35 parts of Poria cocos extract, 25 parts of Codonopsis pilosula extract, 25 parts of Honeysuckle extract, and 7 parts of Cornus officinalis extract.

[0113] The preparation method of the Poria cocos extract comprises the following steps:

[0114] 25g of Poria powder was degreased by reflux with ethanol, then added into 500g of deionized water, swelled at 45°C for 1.5h, and then filtered to obtain pretreated Poria powder; 10g of pretreated Poria powder was added into 250g of 0.7mol / L sodium hydroxide solution, and alkali extracted twice at a temperature of 25°C for 5h, the filtrate was combined, the pH was adjusted to 7, the filtrate was concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution was added for precipitation, the precipitate was washed, desalted and dried to obtain Poria extract.

[0115] The preparation method of the honeysuckle extract comprises the following steps:

[0116] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 1.5%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 5%; then 25g of epigallocatechin gallate solution is added to 50g of γ-cyclodextrin solution, and stirred at a constant temperature of 38°C for 5h to obtain a mixed solution; 70g of honeysuckle pollen is added to 180g of ethanol aqueous solution (the mass concentration of ethanol is 75%), and immersed for 35min at room temperature. After the immersion is completed, 1100g of hot water with a temperature of 75°C is added, and then 1.5g of caprylic decanoic acid glyceride and 75g of the mixed solution are added, and reduced pressure extraction is performed for 20min under the conditions of ultrasonic power of 250W and pressure of 25kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0117] The preparation method of the Cornus officinalis extract comprises the following steps:

[0118] Under light-proof conditions, 0.04 g of β-carotene is added to 130 g of acetone, stirred evenly, and then 130 g of a 1.5% mass% β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 38° C. for 10 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 60 g of cornus officinalis powder is added to 700 g of water, and then 1.5 g of pectinase, 1.5 g of cellulase and 0.8 g of neutral protease are added, and enzymolysis is performed at 43° C. for 2.5 hours. After the enzymolysis is completed, 250 g of anhydrous ethanol and 3 g of the composite cyclodextrin are added, and the mixture is extracted at 55° C. for 1.5 hours. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 25 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0119] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0120] Weigh the raw materials according to the formula, and evenly mix the Poria cocos extract, Codonopsis pilosula extract, Honeysuckle extract and Cornus officinalis extract to obtain the product.

[0121] Compared with Example 1, no mulberry leaf extract was added in this comparative example.

[0122] Comparative Example 5

[0123] A composite plant extract that helps to enhance immunity, comprising the following raw materials by weight:

[0124] 25 parts of Codonopsis pilosula extract, 25 parts of honeysuckle extract, 7 parts of Cornus officinalis extract, and 8 parts of mulberry leaf extract.

[0125] Wherein, the preparation method of the honeysuckle extract comprises the following steps:

[0126] Epigallocatechin gallate is added to anhydrous ethanol, and stirred to obtain an epigallocatechin gallate solution with a mass concentration of 1.5%; γ-cyclodextrin is added to deionized water, and stirred to obtain a γ-cyclodextrin solution with a mass concentration of 5%; then 25g of epigallocatechin gallate solution is added to 50g of γ-cyclodextrin solution, and stirred at a constant temperature of 38°C for 5h to obtain a mixed solution; 70g of honeysuckle pollen is added to 180g of ethanol aqueous solution (the mass concentration of ethanol is 75%), and immersed for 35min at room temperature. After the immersion is completed, 1100g of hot water with a temperature of 75°C is added, and then 1.5g of caprylic decanoic acid glyceride and 75g of the mixed solution are added, and reduced pressure extraction is performed for 20min under the conditions of ultrasonic power of 250W and pressure of 25kpa. After the extraction is completed, it is filtered and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

[0127] The preparation method of the Cornus officinalis extract comprises the following steps:

[0128] Under light-proof conditions, 0.04 g of β-carotene is added to 130 g of acetone, stirred evenly, and then 130 g of a 1.5% mass% β-cyclodextrin aqueous solution is added, and the mixture is stirred at a constant temperature of 38° C. for 10 hours, followed by rotary evaporation to remove water and acetone, and the solid product is washed and dried to obtain a composite cyclodextrin; 60 g of cornus officinalis powder is added to 700 g of water, and then 1.5 g of pectinase, 1.5 g of cellulase and 0.8 g of neutral protease are added, and enzymolysis is performed at 43° C. for 2.5 hours. After the enzymolysis is completed, 250 g of anhydrous ethanol and 3 g of the composite cyclodextrin are added, and the mixture is extracted at 55° C. for 1.5 hours. After the extraction is completed, the mixture is filtered, and the filtrate is concentrated to 1 / 4 of the volume under reduced pressure at 25 kPa, and freeze-dried to obtain a cornus officinalis extract.

[0129] A method for preparing a composite plant extract that helps to enhance immunity comprises the following steps:

[0130] Weigh the raw materials according to the formula, and evenly mix the codonopsis pilosula extract, honeysuckle extract, cornus officinalis extract and mulberry leaf extract to obtain the product.

[0131] Compared with Example 1, no Poria cocos extract was added in this comparative example.

[0132] The extraction rates of the honeysuckle extracts in Examples 1-3 and Comparative Example 1 were calculated and the chlorogenic acid content was determined. The results are shown in Table 1 below:

[0133] Extraction rate = (weight of honeysuckle extract - weight of epigallocatechin gallate - weight of γ-cyclodextrin - weight of caprylic decanoate glyceride) / weight of honeysuckle pollen × 100%

[0134] Table 1 Extraction rate and chlorogenic acid content of honeysuckle extract

[0135]

[0136]

[0137] It can be seen from Table 1 above that the preparation method of the honeysuckle extract provided by the present invention can effectively increase the content of chlorogenic acid in the extract, make the chlorogenic acid in the honeysuckle extract have better stability, and improve the quality of the honeysuckle extract.

[0138] The plant extracts prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to mouse experiments to verify the effect of the plant extracts of the present invention in enhancing immunity, as follows:

[0139] Experimental animal grouping: 250 SPF grade Kunming male mice were randomly divided into 5 large groups of A, B, C, D and E, with 50 mice in each group. Each large group was further randomly divided into a blank group, a model group, an embodiment 1 group, an embodiment 2 group, an embodiment 3 group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, a comparative example 4 group, and a comparative example 5 group, with 5 mice in each group. SPF grade Kunming male mice, 6 to 8 weeks old, weighing 20 ± 2.0g. The experimental animals were raised in a barrier environment animal room with a temperature of 22 ° C, a humidity of 40-70%, 12h light and dark alternation, fed with standard feed, free drinking water and food, and adaptive feeding for 1 week. On the 1st to 3rd day, except for the blank group which was intraperitoneally injected with normal saline, the other groups of mice were intraperitoneally injected with 100 μL cyclophosphamide (50 mg / kg) to establish an immunocompromised mouse model; 3 days later, the blank group and the model group were gavaged with normal saline, the Example 1-3 group was gavaged with the plant extracts in Example 1-3, and the Comparative Example 1-5 group was gavaged with the plant extracts in Comparative Example 1-5, with a gavage dose of 300 mg / kg / day for 30 consecutive days. See Table 2 for specific grouping and experimental items of each group.

[0140] Table 2 Animal grouping

[0141]

[0142]

[0143] The determination of immune function indicators refers to the "Technical Specifications for Inspection and Evaluation of Health Foods" (2023 Edition). The determination of immune function mainly includes cellular immunity, humoral immunity, monocyte-macrophage function and NK cell activity. The method for determining the immune function of mice in this experiment is as follows.

[0144] 1. Cellular immune function assay

[0145] (1) Splenic lymphocyte transformation test (MTT method): On the 31st day of the experiment, mice in group B were killed and immersed in 75% ethanol for sterilization for 10 min. The spleen was removed under sterile conditions and placed in a plate containing an appropriate amount of sterile Hank's solution. The spleen was gently ground with tweezers to prepare a single cell suspension and counted. The cell concentration was adjusted to 3×10 cells / mL using RPMI1640 complete culture medium. 6 / mL. 1mL of cell suspension was added to each well of a 24-well culture plate, 75μL of ConA solution (equivalent to 7.5μg / mL) was added to three wells respectively, and the other three wells were used as controls. They were cultured in a 37°C, 5% CO2 incubator for 72h. 4h before the end of the culture, 0.7mL of supernatant was removed from each well, 0.7mL of RPMI1640 culture solution without calf serum was added, and 50μL / well of MTT (5mg / mL) was added at the same time, and the culture was continued for 4h. After the culture was completed, 1mL of acidic isopropanol was added to each well, and the mixture was blown and mixed to completely dissolve the purple crystals. The optical density (OD) was measured at 570nm. Finally, the optical density of the wells without Con A was subtracted from the optical density of the wells with Con A, and the difference represented the proliferation capacity of lymphocytes.

[0146] (2) Delayed hypersensitivity reaction test (plantar thickening method): On the 31st day of the experiment, each mouse in group C was immunized by intraperitoneal injection of 0.2 mL of 2% (v / v) packed sheep red blood cell (SRBC) suspension. 4 days after immunization, the thickness of the left hind paw was measured, and then 20% (v / v) SRBC was subcutaneously injected at the measurement site, 20 μL (about 1×10 8 SRBC), the thickness of the left hind foot plantar was measured 24 hours after injection, and the same part was measured three times to take the average value.

[0147] 2. Humoral immune function test

[0148] (1) Serum hemolysin determination (hemagglutination method): On the 31st day of the experiment, each mouse in group D was immunized by intraperitoneal injection of 0.2 mL of 2% (v / v) SRBC suspension. After 4 days, the eyeballs were removed and blood was collected in a centrifuge tube. The tube was placed for about 1 hour, and the coagulated blood was separated from the tube wall to allow the serum to be fully separated. The serum was collected by centrifugation at 2000 r / min for 10 minutes. The serum was diluted in multiples with physiological saline, and the serum of different dilutions was placed in a micro-hemagglutination plate, 100 μL per well, and then 100 μL of 0.5% (v / v) SRBC suspension was added. The plate was mixed, placed in a moist flat plate, covered, and incubated in a 37°C incubator for 3 hours for serum hemolysin determination. The hemagglutination degree was counted and the corresponding anti-volume number was calculated.

[0149] (2) Antibody-producing cell detection (Jerne modified slide method): On the 31st day of the experiment, each mouse in group D was immunized by intraperitoneal injection of 0.2 mL of 2% (v / v) SRBC suspension. Four days later, the mice were killed by cervical dislocation, and the spleen was removed and placed in a small dish containing an appropriate amount of sterile Hank's solution. The spleen was gently ground to make a cell suspension, which was filtered through a 200-mesh sieve and centrifuged (1000 r / min) for 10 min. The cells were washed twice with Hank's solution and finally suspended in 5 mL of RPMI1640 culture medium. The number of cells was counted and the cell concentration was adjusted to 5×10 6 After the surface culture medium (1g agarose plus double distilled water to 100mL) was heated and dissolved, it was placed in a 45℃ water bath for heat preservation, mixed with an equal amount of Hank's solution with a pH of 7.4 and twice the concentration, and dispensed into small test tubes, 0.5mL per tube, and then 50μL of 10% SRBC (v / v, prepared with SA buffer) and 20μL of spleen cell suspension (5×10 6 / mL), quickly mix, pour on the glass slide brushed with a thin layer of agarose, make parallel slices, wait for the agar to solidify, put the glass slide horizontally on the slide rack, put it in a carbon dioxide incubator for incubation for 1 hour, then add the complement diluted with SA buffer (1:8) into the groove of the slide rack, continue incubation for 1 hour, and count the number of hemolytic plaques.

[0150] 3. Monocyte-macrophage function assay

[0151] (1) Carbon powder removal experiment in mice: On the 31st day of the experiment, the mice in group A were weighed and injected with 10 mL / kg.BW of India ink (diluted 4.0 times with saline) through the tail vein of the mice according to their body weight. The timer was immediately started after the ink was injected. 2 minutes and 10 minutes after the injection of the ink, 20 μL of blood was collected from the medial canthal venous plexus, and quickly added to 2 mL of 0.1% Na2CO3 solution and shaken well. The Na2CO3 solution was used as a blank control, and the optical density (OD) was measured at a wavelength of 600 nm using an enzyme marker. The liver and spleen were taken after the mice were killed, and the blood stains on the surface of the organs were blotted with filter paper and weighed separately. The phagocytic index represents the carbon clearance ability of mice. The phagocytic index a was calculated according to the following formula. The results of the mouse body weight and organ / body weight ratio are shown in Table 3.

[0152]

[0153] Table 3 Effect of lymphoid organ / body weight ratio of mice in each group

[0154] Group Initial weight / g Final weight / g Thymus / body weight ratio (mg / g) Spleen / body weight ratio (mg / g) Blank Group 20.63±1.24 34.81±1.95 3.53±0.72 4.18±1.01 Model Group 20.92±1.36 29.83±1.58** 1.86±0.59** 2.45±0.52** Example 1 Group 20.28±1.13 <![CDATA[33.26±1.91 # ]]> <![CDATA[3.31±0.68 ## ]]> <![CDATA[3.93±0.81 ## ]]> Example 2 Group 20.47±1.22 <![CDATA[32.51±1.83 # ]]> <![CDATA[3.18±0.51 ## ]]> <![CDATA[3.68±0.63 ## ]]> Example 3 Group 20.19±1.31 <![CDATA[32.86±1.77 # ]]> <![CDATA[3.27±0.63 ## ]]> <![CDATA[3.79±0.68 ## ]]> Comparative Example 1 21.06±1.45 <![CDATA[32.35±1.63 # ]]> <![CDATA[2.93±0.44 # ]]> <![CDATA[3.34±0.47 # ]]> Comparative Example 2 20.73±1.34 <![CDATA[31.25±1.59 # ]]> 2.68±0.56 3.23±0.66 Comparative Example 3 20.31±1.19 <![CDATA[31.79±1.68 # ]]> 2.72±0.65 <![CDATA[3.41±0.72 # ]]> Comparative Example 4 20.05±1.03 <![CDATA[32.04±1.72 # ]]> <![CDATA[3.05±0.58 # ]]> <![CDATA[3.46±0.59 # <!-- 12 -->]]> Comparative Example 5 20.44±1.28 <![CDATA[32.96±1.64 # ]]> <![CDATA[2.99±0.61 # ]]> 3.29±0.75

[0155] Note: Compared with the blank group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01

[0156] As can be seen from Table 3 above, the body weight of each group increased, but the weight gain of the model group was significantly lower than that of the blank group, indicating that the immunosuppressed mice were modeled, and the weight gain of the Example 1-3 group was significantly higher than that of the model group (P<0.05), indicating that the plant extract containing honeysuckle prepared by the present invention has a certain effect on the weight recovery and growth of immunosuppressed mice, and there was no significant difference in the final body weight of the mice in the Example 1-3 group and the Comparative Example 1-5 group, indicating that the raw materials of the composition have little effect on the weight of the mice.

[0157] Thymus and spleen are important immune organs, and thymus index and spleen index can be calculated according to their development. The height of thymus index and spleen index can indirectly reflect the overall immune level of the body, which is of great significance in the evaluation of immune function. According to the thymus / body weight ratio and spleen / body weight ratio data, it can be seen that compared with the normal group, the thymus and spleen index of the model group mice are significantly reduced, with extremely significant (P < 0.01), indicating that immune function declines. And embodiment 1-3 group thymus, spleen index increase compared with the model group, with extremely significant difference (P < 0.01), illustrating that the plant extract prepared by the present invention can promote the development of thymus and spleen in immunocompromised mice, effectively alleviate the atrophy and damage of organs, increase the quality of immune organs, thereby improve the immune state of the body, and there is synergistic effect between the plant extract raw materials.

[0158] (2) Peritoneal macrophage phagocytosis of chicken red blood cells (semi-in vivo method): On the 31st day of the experiment, 1 mL of 20% (v / v) chicken red blood cell suspension was added to the peritoneal cavity of each mouse in group E. The mice were killed by cervical dislocation at intervals of 1 hour, fixed on a mouse board in a supine position, the abdominal wall skin was cut in the middle, and 2 mL of normal saline was injected into the peritoneal cavity. The mouse board was rotated for 1 minute. Then 1 mL of peritoneal washing fluid was aspirated and evenly dropped on 2 glass slides, placed in an enamel box with wet gauze, and incubated at 37°C for 30 minutes. After incubation, rinsed in normal saline to remove unattached cells. Dry, fix with 1:1 acetone methanol solution, stain with 4% (v / v) Giemsa-phosphate buffer, rinse with distilled water and dry. Observe 100 macrophages under an oil microscope, count the number of macrophages phagocytizing chicken red blood cells, the total number of chicken red blood cells phagocytized by macrophages and the degree of digestion, and calculate the phagocytosis rate and phagocytosis index according to the formula.

[0159]

[0160] 4. NK cell activity assay

[0161] Lactate dehydrogenase (LDH) method was used to measure NK cell activity: on the 30th day, the target YAC-1 cells were subcultured and the cell concentration was adjusted to 4×10 5 / mL, and on the 31st day, the mice in group B were killed and sterilized in 75% ethanol. The spleen was taken out with sterile forceps and ground with a sieve, filtered and centrifuged, and the supernatant was discarded. 0.5mL of sterile water was added to the cytoplasm for 20s, and 0.5mL of 2x Hank's solution and 8mL of Hank's solution were added after lysing the red blood cells. The cells were centrifuged at 1000r / min for 10min, and the red supernatant was discarded. The cells were resuspended with 1mL of RPMI1640 complete culture medium containing calf serum, diluted with 1% glacial acetic acid and counted, and the number of live cells was counted with trypan blue staining (should be above 95%). Finally, a single cell suspension of the spleen was made with RPMI1640 complete culture medium as effector cells, and the cell concentration was adjusted to 2×10 7 / mL. The ratio of target cells to effector cells is about 50:1. Take 100μL of target cell and effector cell solutions and add them to a U-shaped 96-well plate. Add 100μL of target cells and culture medium to the target cell natural release wells, and add 100μL of target cells and 1% NP40 to the target cell maximum release wells. Set up 3 parallel wells. After culturing in a 37℃, 5% CO2 incubator for 4h, centrifuge the 96-well culture plate at 1500r / min for 5min, take 100μL of supernatant from each well and spread it on a new 96-well plate, add 100μL of LDH matrix solution at the same time, react at room temperature for 10min, add 30μL of hydrochloric acid solution (1mol / L) to each well to terminate the reaction, and use an enzyme reader to measure the optical density at 490nm. Calculate the NK cell activity according to the following formula.

[0162]

[0163] When T lymphocytes are stimulated by ConA, proliferative reactions occur in the mother cells. Mitochondrial hydrolases in living cells, especially proliferating cells, can decompose MTT (a pale yellow azole nitrogen salt) into blue-purple crystals, whose optical density value can reflect the proliferation of cells. Figure 1 As can be seen in (a), compared with the blank group, the absorbance value of the spleen lymphocyte transformation experiment of the model group mice was significantly lower than that of the normal control group, indicating that the low-immunity mouse model was formed. The absorbance values ​​of spleen lymphocytes in the Example 1-3 group and the Comparative Example 1, 3, and 4 groups were all higher than those in the model group, with significant differences, indicating that the plant composition can improve cellular immune function, and the Example 1-3 groups have extremely significant differences, indicating that the raw materials of the plant composition prepared by the present invention have synergistic effects. Figure 1 As can be seen from (b), the degree of plantar thickening of mice in Example 1-3 groups and Comparative Examples 1, 3, 4, and 5 groups was higher than that in the model group, with significant differences, indicating that the plant composition can improve cellular immune function, and there is a very significant difference between Example 1-3 groups, indicating that the raw materials of the plant composition prepared by the present invention have a synergistic effect.

[0164] from Figure 2As can be seen in (a), compared with the blank group, the number of hemolytic plaques in the model group mice was significantly lower than that in the normal control group, while the absorbance values ​​of spleen lymphocytes in the Example 1-3 group and the Comparative Example 1, 4, and 5 groups were all higher than those in the model group, with significant differences, indicating that the plant composition can increase the number of hemolytic plaques and enhance humoral immune function, and the Example 1-3 groups have extremely significant differences, indicating that the raw materials of the plant composition prepared by the present invention have synergistic effects. Figure 2 As can be seen from (b), the antibody volume count of the model group mice was significantly lower than that of the normal control group, while the spleen lymphocyte absorbance values ​​of the Example 1-3 groups and the Comparative Examples 1, 4, and 5 groups were all higher than those of the model group, with significant differences, indicating that the plant composition can increase the antibody volume count and enhance the humoral immune function, and the Example 1-3 groups have extremely significant differences, indicating that the raw materials of the plant composition prepared by the present invention have a synergistic effect.

[0165] from Figure 3 As can be seen from (a), the carbon clearance phagocytosis index of the model group mice was significantly lower than that of the normal control group, while the spleen lymphocyte absorbance values ​​of the Example 1-3 group and the Comparative Example 1-5 group were higher than those of the model group, with significant differences (P < 0.05). Figure 3 As can be seen from (b), the phagocytic percentage of mice in the model group was significantly lower than that in the normal control group, while the absorbance values ​​of spleen lymphocytes in the Example 1-3 group and the Comparative Example 1-5 group were higher than those in the model group, with significant differences (P < 0.05). Figure 3 As can be seen from (c), the phagocytic index of the model group mice was significantly lower than that of the normal control group, while the phagocytic index of the Example 1-3 group and the Comparative Example 1-5 group was higher than that of the model group, with a very significant difference (P < 0.01), indicating that the plant extract prepared by the present invention has a certain effect of enhancing the nonspecific immunity of mice, but the synergistic effect between the compositions is not obvious, and the difference is not significant.

[0166] from Figure 4 It can be seen that compared with the blank group, the NK cell activity of the model group was significantly reduced (P < 0.01), indicating that the immunosuppressed mice were modeled; compared with the model group, the Example 1-3 groups had extremely significant differences (P < 0.01), indicating that the plant composition of the present invention has a significant improvement on the NK cell activity of mice, and the effect of the Example 1-3 groups is better than that of the comparative example 1-5, indicating that the raw materials of the plant composition of the present invention have a synergistic effect.

[0167] According to the results of the immunity enhancement function test in the Technical Specifications for Inspection and Evaluation of Health Foods, the plant extract containing honeysuckle prepared in the present invention has two positive experimental results in the cellular immune function determination project, which can be judged as positive for the cellular immune function determination result; the two experimental results in the humoral immune function determination project are both positive, which can be judged as positive for the humoral immune function determination result; more than one dose group result in the NK cell activity determination experiment is positive, which can be judged as positive for the NK cell activity result. And among the four aspects of cellular immune function, humoral immune function, monocyte-macrophage function, and NK cell activity, the results of three aspects of cellular immune function, humoral immune function, and NK cell activity are positive, which can be judged that the plant extract containing honeysuckle prepared in the present invention has the effect of helping to enhance immunity.

[0168] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite plant extract that helps enhance immunity, characterized in that: By weight, it includes the following raw materials: 30-40 parts of Poria cocos extract, 20-30 parts of Codonopsis pilosula extract, 20-30 parts of Honeysuckle extract, 5-10 parts of Cornus officinalis extract, and 5-10 parts of Mulberry leaf extract.

2. The composite plant extract according to claim 1, characterized in that By weight, it includes the following raw materials: 30-35 parts of Poria cocos extract, 25-30 parts of Codonopsis pilosula extract, 25-30 parts of Honeysuckle extract, 5-7 parts of Cornus officinalis extract, and 5-8 parts of mulberry leaf extract.

3. The composite plant extract according to claim 1, characterized in that The preparation method of the Poria cocos extract comprises the following steps: The tuckahoe powder is degreased by reflux with ethanol and then added into deionized water, swelled at a certain temperature, and then filtered to obtain the pretreated tuckahoe powder; the pretreated tuckahoe powder is added into a sodium hydroxide solution, alkali extracted twice, the filtrates are combined, the pH is adjusted to 7, the filtrates are concentrated under reduced pressure to 1 / 4 of the volume, 4 times the volume of 95% ethanol solution is added for precipitation, the precipitate is washed, desalted and dried to obtain the tuckahoe extract.

4. The composite plant extract according to claim 3, characterized in that The mass ratio of the Poria powder and deionized water is 20-30:400-600, the swelling temperature is 40-50°C, and the time is 1-2h; the concentration of the sodium hydroxide solution is 0.5-0.8mol / L, the mass ratio of the pretreated Poria powder and the sodium hydroxide solution is 10:200-300, the alkali extraction temperature is 20-30°C, and the time is 4-6h.

5. The composite plant extract according to claim 1, characterized in that The preparation method of the Cornus officinalis extract comprises the following steps: Under light-proof conditions, beta-carotene is added to acetone, stirred evenly, then beta-cyclodextrin aqueous solution is added, stirred at a constant temperature, then rotary evaporation is performed to remove water and acetone, and the solid product is washed and dried to obtain composite cyclodextrin; cornus officinalis powder is added to water, then pectinase, cellulase and neutral protease are added to perform enzymolysis, after the enzymolysis is completed, ethanol and composite cyclodextrin are added to perform leaching, after the leaching is completed, filtering is performed, the filtrate is concentrated under reduced pressure to 1 / 4 of the volume, and freeze-dried to obtain cornus officinalis extract.

6. The composite plant extract according to claim 5, characterized in that The mass fraction of the β-cyclodextrin aqueous solution is 1-1.8%, the mass ratio of the β-carotene, acetone and β-cyclodextrin aqueous solution is 0.03-0.05:100-150:100-150, the temperature of the constant temperature stirring is 35-40°C, and the time is 8-12h; the mass ratio of the cornus powder, water, pectinase, cellulase, neutral protease, ethanol and composite cyclodextrin is 50-70:600-800:1-2:1-2:0.5-1:200-300:2-4, the temperature of the enzymatic hydrolysis is 40-45°C, and the time is 2-3h; the temperature of the extraction is 50-60°C, and the time is 1-2h; the pressure of the reduced pressure concentration is 20-30kpa.

7. The composite plant extract according to claim 1, characterized in that The preparation method of the honeysuckle extract comprises the following steps: Epigallocatechin gallate is added to anhydrous ethanol, and stirred evenly to obtain an epigallocatechin gallate solution; γ-cyclodextrin is added to deionized water, and stirred evenly to obtain a γ-cyclodextrin solution; then the epigallocatechin gallate solution is added to the γ-cyclodextrin solution, and stirred at a constant temperature to obtain a mixed solution; honeysuckle pollen is added to the ethanol aqueous solution, and immersed at room temperature, and hot water of 70-80°C is added after the immersion is completed, and then caprylic decanoic acid glyceride and the mixed solution are added, and reduced pressure extraction is performed under ultrasonic conditions, and after the extraction is completed, it is filtered, and the filtrate is subjected to rotary evaporation to obtain a honeysuckle extract.

8. The composite plant extract according to claim 7, characterized in that The mass concentration of the epigallocatechin gallate solution is 1-2%, the mass concentration of the gamma-cyclodextrin solution is 4-6%, the mass ratio of the epigallocatechin gallate solution to the gamma-cyclodextrin solution is 20-30:40-60, the temperature of the constant temperature stirring is 35-40°C, and the time is 4-6h; the mass concentration of ethanol in the ethanol aqueous solution is 70-80%, the mass ratio of the honeysuckle pollen, the ethanol aqueous solution, the hot water, the caprylic decanoic acid glyceride, and the mixed solution is 60-80:150-200:900-1200:1-2:60-90, the immersion time is 30-40min, the power of the ultrasound is 200-300W, the pressure of the reduced pressure extraction is 20-30kpa, and the time is 15-25min.

9. A method for preparing a composite plant extract that helps enhance immunity as claimed in any one of claims 1 to 8, characterized in that: The following steps are involved: Weigh the raw materials according to the formula, and evenly mix the Poria extract, Codonopsis pilosula extract, Honeysuckle extract, Cornus officinalis extract, and Mulberry leaf extract to obtain the product.

10. Use of the composite plant extract according to any one of claims 1 to 8 that helps enhance immunity in food or medicine.

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