CN109953999B - Composition comprising Ganoderma polysaccharide and Polyporus polysaccharide with immunity enhancing effect - Google Patents
Composition comprising Ganoderma polysaccharide and Polyporus polysaccharide with immunity enhancing effect Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention provides a composition with an immunity enhancing effect, which consists of ganoderma lucidum polysaccharide and grifola polysaccharide, and application thereof. The polysaccharide composition contains ganoderan and grifola polysaccharide, wherein the ganoderan and grifola polysaccharide in the composition have compatibility, and the ganoderan and the grifola polysaccharide have synergistic effect, so that the NK cell activity of an organism and the function of macrophages are obviously improved, and the immunity of the organism is obviously enhanced.
Description
Technical Field
The invention belongs to the field of health care products, and particularly relates to a composition with an immune enhancement function, which consists of ganoderma lucidum polysaccharide and grifola polysaccharide, and more particularly relates to an edible fungus polysaccharide composition, application of the polysaccharide composition in preparing food, medicines or health care products, application of the polysaccharide composition in preparing a kit, and food, health care products or medicines with the immune enhancement function.
Background
The immune system is a defense system of the body against diseases and plays an important role in maintaining the internal environment and physiological balance of the body. When the immune function is low, the body is easy to fatigue, the sleep quality is poor, the appetite is reduced, and in addition, the low immune function is easy to induce infection and the like related to bacteria, viruses and fungi. The natural medicinal plants are utilized to develop natural medicine for enhancing the body immunity and have obvious advantages.
The edible fungi are popular food on dining tables and have rich nutritive value. Researches show that the edible fungi contain various functional components, are the dominant raw materials for researching and developing natural medicines or health care products, and have the characteristics of high efficiency, small toxic and side effects or no toxicity, difficult generation of drug resistance and the like. Research shows that bioactive substances extracted from edible fungi, such as polysaccharides, nucleosides, triterpenes and minerals, have antioxidant, anticancer, diabetes treating, antiallergic, cardiovascular protecting, antiviral, antibacterial, parasite killing, toxic substance removing, and liver protecting effects.
Ganoderma lucidum is a traditional edible and medicinal fungus, has been used for thousands of years as a medicine for promoting health and prolonging life in Asian countries such as China, Japan, etc., and the traditional Chinese medicine theory holds that the Ganoderma lucidum has the efficacies of tonifying qi, soothing the nerves, strengthening the body resistance and consolidating the constitution. The polysaccharide contained in ganoderma lucidum is one of the main active ingredients, and the ganoderma lucidum polysaccharide has wide pharmacological actions of resisting oxidation, resisting tumors, reducing blood sugar, reducing blood fat and the like. Ganoderan has become a common natural raw material for the research and development of health products and medicines due to its wide range of effects.
Polyporus umbellatus is also a fungus medicinal material used as food and medicine, and has thousands of years of use history as the traditional precious Chinese medicine in China. The theory of traditional Chinese medicine considers that the polyporus umbellatus has the effects of promoting diuresis and excreting dampness. The polysaccharides extracted from Polyporus umbellatus have antioxidant and antitumor effects. At present, grifola polysaccharide is prepared into injection and capsule, and is combined with anti-tumor chemotherapeutic drugs to enhance the curative effect of the chemotherapeutic drugs and reduce the toxic and side effects of the chemotherapeutic drugs.
Ganoderma lucidum and Polyporus umbellatus are edible mushrooms and traditional natural drugs with a long history of application, and are all recorded in Shen nong Ben Cao Jing. The superior products in Shennong Bencao Jing are usually used as monarch drugs, the Chinese traditional medicines are ministerial drugs, the lucid ganoderma is listed as the superior products, the lucid ganoderma has the effects of long-time eating, light weight and no aging, and the old-time immortal, the polyporus umbellatus is listed as the Chinese traditional medicines, and the lucid ganoderma has the characteristics of 'benefiting water passage, light weight and no aging after long-time taking'. The lucid ganoderma is used as a monarch drug to regulate the five internal organs of the body, invigorate qi and calm the nerves; polyporus umbellatus is used as ministerial drug for promoting diuresis. Meanwhile, both the lucid ganoderma and the polyporus umbellatus have the effect of prolonging life, and the lucid ganoderma and the polyporus umbellatus are compatible, so that the health-care tea has the characteristic of tonifying without damaging healthy energy. The record of the book on the ganoderma lucidum and the polyporus umbellatus provides a theoretical basis for the combination of the ganoderma lucidum and the polyporus umbellatus.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the inventor finds that after the ganoderma lucidum polysaccharide and the polyporus umbellatus polysaccharide are compatible, the prepared preparation can be effectively used for treating or improving the immune function of an organism, the composition which is prepared from the high-purity water-soluble polysaccharide extracted from the ganoderma lucidum and the polyporus umbellatus according to a certain proportion shows no toxicity in a mouse oral experiment, has a synergistic enhancement immune function, and can obviously improve the NK cell activity and the macrophage function of the organism.
Therefore, the invention provides an edible fungus polysaccharide composition. According to an embodiment of the invention, the polysaccharide composition comprises ganoderan and polyporus polysaccharide. As described above, the ganoderma lucidum polysaccharide and the polyporus umbellatus polysaccharide in the edible fungus polysaccharide composition according to the embodiment of the invention have a compatibility effect, and the two have a synergistic effect, so that the NK cell activity and the macrophage function of an organism are remarkably improved, and the immunity of the organism is remarkably enhanced.
According to an embodiment of the invention, the above polysaccharide composition further comprises at least one of the following additional technical features:
according to an embodiment of the present invention, the polysaccharide composition comprises 3 parts by weight of ganoderan and 1 part by weight of polyporus polysaccharide. The inventor finds that the synergistic effect of the ganoderma lucidum polysaccharide and the grifola polysaccharide is remarkable under the formula, and the effects of improving the NK cell activity of an organism and the function of macrophage and remarkably enhancing the immunity of the organism are more remarkable.
In a second aspect of the invention, the invention proposes the use of the polysaccharide composition as described above for the preparation of a food, pharmaceutical or nutraceutical product for enhancing the immunity of the body. The food, the medicine or the health care product prepared by the polysaccharide composition can obviously improve the immunity of the organism.
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
according to an embodiment of the present invention, the enhancing of the body immunity is achieved by at least one of the following means:
synergistically enhance macrophage function;
enhancing the delayed allergic reaction capability of the organism;
obviously improve the NK cell activity of the organism.
In a third aspect of the invention, the invention proposes the use of a polysaccharide composition as hereinbefore described in the preparation of a kit for use in at least one of: enhancing the function of macrophages; improving the activity of NK cells. The pharmaceutical composition provided by the embodiment of the invention can be used in scientific research experiments to improve the function of macrophages or improve the activity of NK cells, and has a remarkable effect.
In a fourth aspect of the invention, the invention features a method of enhancing macrophage function or increasing NK cell viability. According to an embodiment of the invention, the method comprises contacting the macrophages or NK cells to be treated with the polysaccharide composition as described previously. The inventor finds in experiments that when the macrophage or the NK cell is treated by the composition, the function of the macrophage or the activity of the NK cell can be obviously improved, and the lethality of the NK cell is enhanced.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to the embodiment of the invention, the cells to be treated are macrophages, and the concentration of the polysaccharide composition in the reaction system is 50-200 mug/mL. The inventor finds that when the macrophage is treated by the polysaccharide composition with the concentration of 50-200 mug/mL, such as 100 mug/mL or 200 mug/mL, the phagocytic capacity of the macrophage is remarkably enhanced, and after the macrophage is treated by the polysaccharide composition with the concentration of 50 mug/mL or 100 mug/mL, the macrophage releases NO remarkably.
In a fifth aspect of the present invention, the present invention provides a food, health product or pharmaceutical having an immune enhancing effect. According to an embodiment of the present invention, the food, health product or pharmaceutical product comprises the edible fungus polysaccharide composition as described above. The food, the health-care product or the medicine provided by the embodiment of the invention has no toxicity, and has an obvious effect on improving the immunity of the organism.
According to an embodiment of the present invention, the food, health product or medicine may further include at least one of the following additional technical features:
according to an embodiment of the present invention, the food, health product or pharmaceutical further comprises a food, health product or pharmaceutical acceptable auxiliary. The medicine and pharmaceutically acceptable adjuvant ingredients can be made into corresponding preparations, such as granules, tablets, capsules, injection and oral liquid. Thereby being beneficial to the storage or absorption of foods, health products or medicines by the organism and increasing the effective utilization rate of active ingredients.
Detailed Description
Example 1
(1) The composition of the ganoderma lucidum polysaccharide and the grifola polysaccharide can coordinate and enhance the phagocytic capability of a mouse macrophage system RAW 264.7.
Sample processing and testing method
Weighing water-soluble ganoderan and grifola polysaccharide with the purity of 98% 200 g respectively, dissolving in 50mL of cell culture medium, filtering with 0.22nm filter, preparing stock solution with the concentration of 4mg/mL, storing at 4 deg.C for a short period, and storing at-20 deg.C for a long period.
The macrophage cell line RAW264.7 was measured at 4X 104The number of the cells/well was plated on a 96-well plate, after 24 hours of cell culture, polysaccharide solutions (50, 100, 200. mu.g/mL) and lipopolysaccharide LPS (LPS) at different concentrations were added, and after further 24 hours of culture, the cells were treated according to the instructions provided by the neutral Red kit (Biyuntian Bio Inc.) and absorbance at 560nm was measured on a microplate reader for evaluation of phagocytic ability of macrophages.
Results of the experiment
As can be seen from Table 1, LPS (positive control) significantly enhanced the ability of macrophages to phagocytose neutral red, with a significant difference (P < 0.0001). Compared with the blank control group, both the ganoderan and the grifola polysaccharide can enhance the ability of macrophage to phagocytize neutral red, and are dose-dependent within a certain concentration range. The composition has a tendency to stimulate macrophage ability to phagocytose neutral red compared to ganoderan. The composition has an enhanced ability to stimulate phagocytosis of neutral red by macrophages compared to the grifola polysaccharide group, and is significant at a concentration of 100,200 μ g/mL (see table 1). In this experiment, when 3/4 parts of ganoderan with 1/4 parts of polyporus umbellatus were compared to the average OD value of the composition at each concentration, it was found that 3/4 parts of ganoderan with 1/4 parts of polyporus umbellatus were found to have a smaller average OD value than the composition (see table 2). The experiment shows that the ganoderma lucidum polysaccharide and the polyporus umbellatus polysaccharide can enhance the phagocytic capacity of macrophages, the composition consisting of the ganoderma lucidum polysaccharide and the polyporus umbellatus polysaccharide has a synergistic effect between the ganoderma lucidum polysaccharide and the polyporus umbellatus polysaccharide, the macrophage function can be enhanced synergistically, and the effect of enhancing the macrophage function is obviously greater than the sum of the effects of the single ganoderma lucidum polysaccharide and the single polyporus umbellatus polysaccharide with the same quality.
Table 1: effect of the composition on the phagocytic potency of the mouse macrophage line RAW264.7 neutral Red
Note: compared with the blank control group, the composition of the composition,*：P<0.05,**：P<0.01,***：P<0.001. in contrast to the same dosage of the composition,▲：P<0.05,▲▲：P<0.01。
table 2: 3/4 part of Ganoderma polysaccharide and 1/4 parts of Polyporus polysaccharide, and comparing the average OD value with that of the composition
(2) The combination of ganoderan and grifola polysaccharide can coordinate and enhance the capability of mouse macrophage system RAW264.7 to generate NO
The sample treatment and experiment method are the same as before, after the experiment is finished, cells are treated according to the instruction provided by the NO kit (Biyuntian biological company), a standard curve is made by using the standard substance provided by the kit, the absorbance at 560nm is detected on an enzyme labeling instrument, the relative release amount of NO in each group is calculated through the standard curve, and the experiment is used for evaluating the capability of macrophage for releasing NO.
Results of the experiment
As can be seen from Table 3, LPS (positive control) significantly enhanced the ability of macrophages to release NO, with a significant difference (P < 0.0001). Compared with the blank control group, both the ganoderan and the grifola polysaccharide can enhance the capability of macrophage to release NO, and are dose-dependent within a certain concentration range. The tendency of the composition to stimulate the ability of macrophages to release NO compared to ganoderan. The composition has an enhanced ability to stimulate NO release from macrophages compared to the grifolan group and is significant at a concentration of 50, 100 μ g/mL (P <0.05) (see table 3). In this experiment, when the average NO release of 3/4 parts of ganoderan +1/4 parts of polyporus umbellatus was compared to the average NO release of the composition at each concentration, it was found that 3/4 parts of ganoderan +1/4 parts of polyporus umbellatus found that the average NO release was less than the average NO release of the composition (see table 4). The experiment shows that the ganoderma lucidum polysaccharide and the grifola polysaccharide can enhance the capability of macrophage for releasing NO, the composition consisting of the ganoderma lucidum polysaccharide and the grifola polysaccharide has a synergistic effect between the ganoderma lucidum polysaccharide and the grifola polysaccharide, the function of the macrophage can be enhanced synergistically, and the effect of enhancing the function of the macrophage is obviously greater than the sum of the effects of single ganoderma lucidum polysaccharide and single grifola polysaccharide with the same quality.
Table 3: effect of the composition on the ability of the mouse macrophage line RAW264.7 to release NO
Note: compared with the blank control group, the composition of the composition,*：P<0.05,**：P<0.01,***：P<0.001. in contrast to the same dosage of the composition,▲：P<0.05。
table 4: 3/4 parts of ganoderan mean NO Release amount +1/4 parts of Polyporus umbellatus polysaccharide mean NO Release amount compared with composition mean NO Release amount
Example 2
Pharmacodynamic test of immunomodulatory effect of ganoderan and grifola polysaccharide composition on mice
1) Grouping and dose design
The composition (dissolved by ultrapure water) is prepared into low, medium and high three concentrations: 0.0036g/mL, 0.012g/mL, and 0.036 g/mL. The animals were divided into 12 animals per group of control, low, medium and high dose groups, and the gavage volume of the mice was 0.1mL/10 g.BW. The control group was gavaged with ultrapure water, and each group of mice was gavaged 1 time a day at 0.10mL/10g.BW for 30 days continuously. And (5) measuring each index after the intragastric administration is finished.
2) Experimental methods and results
The experimental method was performed with reference to the examination for enhancing immune function in "test and evaluation of health food" technical Specification (2003 edition). And SPSS software is adopted to carry out the single-factor analysis of variance experiment results.
(1) Effect of compositions on immune function of mouse cells
Mice were immunized intraperitoneally with 2% (v/v) SRBC, and each mouse was injected with 0.2mL (about 1X 10)8One SRBC). After 4 days, 20% (v/v) SRBC was injected subcutaneously at the plantar region of the left hind foot, 20. mu.L (about 1X 10) per mouse8Individual SRBC), left hind plantar thickness was measured at 24h post injection, three times at the same site, and averaged. The results are shown in Table 5.
Table 5: effect of compositions on mouse DTH
Note: compared with the solvent control group,*：P<0.05
as a result: compared with the control group, the thickness of the toes of the medium-dose group is obviously increased, and the difference is significant (P < 0.05).
(2) Effect of the composition on mouse monocyte-macrophage function
30 days after administration, mice were injected intraperitoneally with a 2% suspension of chicken red blood cells. At intervals of 30min, taking off cervical vertebrae to kill animals, injecting 2mL of physiological saline into abdominal cavity, gently kneading abdominal cavity of mice, cutting abdominal wall skin, sucking out 1mL of abdominal cavity washing liquid, and averagely dripping on 2 glass slides; placing into a wet box, and incubating at 37 deg.C for 30 min; rinsing with physiological saline. Air-drying, fixing with 1:1 acetone methanol solution, staining with 4% (v/v) Giemsa-phosphate buffer solution for 3min, rinsing with distilled water, air-drying, and observing under microscope for counting. The results are shown in Table 6.
Table 6: influence of composition on phagocytosis of chicken red blood cells by mouse abdominal cavity macrophages
Note: compared with the solvent control group,**：P<0.01. smear with no observed chicken red blood cells was removed.
As a result: compared with the control group, the abdominal cavity macrophages of the mice in the medium and high dose groups have obviously enhanced chicken erythrocyte phagocytosis functions (phagocytosis rate and phagocytosis index) compared with the solvent control group (P < 0.01).
(3) Measurement of NK cell Activity of composition on mouse
Taking spleen aseptically, and making into single cell suspension. After 2 times of filtration and Hanks liquid washing, the suspension is resuspended, the erythrocytes are lysed and Hanks liquid is added. Centrifuging to remove supernatant; resuspending complete culture medium, adjusting cell concentration to 5 × 106one/mL. Taking 100 mu L of each target cell (YAC-1 cell) and effector cell (the effective-target ratio is 50: 1), and adding into a 96-well culture plate; target cells are added into a natural release hole of the target cells and 100 mu L of culture solution respectively, and target cells and 1% NP40 are added into a maximum release hole of the target cells and 100 mu L of culture solution respectively; all the above items are provided with three parallel holes at 37 ℃ and 5% CO2Culturing for 4h, centrifuging a 96-well culture plate, taking 100 mu L of supernatant per well, placing the supernatant into the 96-well culture plate, simultaneously adding 100 mu L of LDH matrix solution, reacting for 5min, adding 30 mu L of 1mol/L HCl per well, and measuring the optical density value at 490nm of an enzyme labeling instrument. The results are shown in Table 7.
Table 7: effect of compositions on NK cell Activity in mice
Note: compared with the solvent control group,**：P<0.01
as a result: compared with a blank control group, the NK cell activity of the mice in the medium-dose group is obviously enhanced compared with that in the solvent control group, and no obvious change is seen in other dose groups.
3) Conclusion
After the mice are orally administered with the composition of 0.036, 0.12 and 0.36g/kg.BW for 30 days, the delayed allergic reaction capability of the mice in the medium-dose group is obviously enhanced compared with that of the mice in the solvent control group; the function (phagocytosis rate and phagocytosis index) of chicken red blood cells phagocytosed by abdominal cavity macrophages of the mice in the medium and high dose groups is obviously enhanced compared with that of a solvent control group; the NK cell activity of the mice in the medium-dose group is obviously enhanced compared with that in the solvent control group. The detection result shows that the composition has obvious enhancement effect on the phagocytic function of the macrophages of mice; has obvious enhancement effect on the activity of NK cells. The composition is suggested to have the function of enhancing immunity.
Example 3
Effect of Large dose of the composition on mice
1) Experimental methods
20 ICR mice were selected, half each. The composition was administered by oral gavage 2 times (4 hours apart) within 24 hours at 0.2ml/10g body weight. The composition was administered at a concentration of 4 g/ml. The dosage of the composition is 16.0g/kg body weight (according to a large number of documents, the effective dose of the mouse is usually about 0.1g/kg, which is equivalent to 160 times of the effective dose of the mouse). Animals were dosed after a 16 hour overnight fast, and subsequently observed for signs of intoxication, mortality and time to death, 1 time per day for 14 days. During the observation period, the body weight of the mice was weighed on days 1, 7 and 14 after the administration, respectively. At the end of the experiment, the animals were sacrificed and gross dissection was performed.
2) Results of the experiment
The composition is administered to mice at a weight of 16.0g/kg, and no obvious toxic reaction is observed in the animals within 14 days of observation period. The animals gained substantially normal body weight (see table 8). The animals were examined by gross dissection after sacrifice, and no obvious abnormality was observed in each organ. The results show that the oral acute toxicity LD50 of mice of both sexes is more than 16.0g/kg body weight. According to the grading standard of the acute toxicity of the health food, the composition belongs to the non-toxic grade in the oral acute toxicity of mice.
Table 8: effect of the composition on mouse body weight
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (6)
1. An edible fungus polysaccharide composition for enhancing body immunity is characterized by comprising 3 parts by weight of ganoderma lucidum polysaccharide and 1 part by weight of polyporus umbellatus polysaccharide.
2. Use of the polysaccharide composition of claim 1 in the preparation of a food, pharmaceutical or nutraceutical product for enhancing immune function.
3. The use according to claim 2, wherein said enhancing of body immunity is achieved by at least one of:
synergistically enhance macrophage function;
enhancing the delayed allergic reaction capability of the organism;
obviously improve the NK cell activity of the organism.
4. Use of the polysaccharide composition of claim 1 in the preparation of a kit for at least one of:
enhancing the function of macrophages;
improving the activity of NK cells.
5. A food, health product or pharmaceutical product having an immunopotentiating effect, comprising the polysaccharide composition of edible fungi according to claim 1.
6. The food, health product or pharmaceutical of claim 5, further comprising a food, health product or pharmaceutical acceptable adjuvant.
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| 六种多糖对小鼠免疫功能调节作用的比较;马兴铭等;《中药药理与临床》;20031231;第19卷(第4期);第14-15页 * |
| 毛头鬼伞多糖对体液免疫调节作用的研究;王岩;《万方学位论文》;20101222;第1-39页 * |
| 王岩.毛头鬼伞多糖对体液免疫调节作用的研究.《万方学位论文》.2010,第1-39页. * |
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