CN107661359A - A kind of paecilomycerol pupa base cordyceps sinensis composition of strengthen immunity anti-aging and its application - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicine and discloses a composition capable of enhancing immunity and anti-aging and an application thereof. An immunity-enhancing Paecilomyces pupae-based Cordyceps composition, which mainly consists of 20-70 parts of Paecilomyces pupae-based Cordyceps pupae, 20-40 parts of Paecilomyces pupae-based Cordyceps pupae and Paecilomyces fumigatus pupae Composed of 1-10 parts of Cordyceps base, preferably 50-70 parts of Paecilomyces pupaeum, 30-40 parts of Paecilomyces pupaeum and 2-5 parts of Paecilomyces pupaeum. composition. The aforementioned composition can be further made into powder, tablet, capsule or oral liquid. The composition of the invention is prepared from natural raw materials and has the functions of enhancing immunity and anti-aging.

Description

Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging and its application

technical field

The invention belongs to the field of traditional Chinese medicines, and relates to a Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging and an application thereof.

Background technique

Aging is a progressive process of functional and organic decline that occurs in all individuals over time. Aging, also known as aging, is an inevitable stage in the process of life activities. Immunity refers to a specific physiological response of the body to contact with "antigenic foreign bodies" or "foreign components". The immune system plays an important role in maintaining normal physiological functions of the body. When people's nutrient intake is insufficient (deficiency of protein, vitamins, trace elements, etc.), the body's resistance will decline, and the body with low immunity is prone to infection or cancer; Allergic reactions, autoimmune diseases, etc. Young and middle-aged people are under the dual pressure of work and life, excessive consumption and overdraft of physical fitness, low immunity, and premature aging of tissues and organs tend to be younger. People are constantly looking for ways to live a long and healthy life, improve immunity, overcome various diseases, and improve physical functions, so as to improve the quality of life. Therefore need to constantly develop the health care products with the same origin of medicine and food with anti-aging function.

Cordyceps sinensis (Berk.) Sacc.] is one of the "three treasures" in my country's medical treasure house, wild ginseng and velvet antler, and enjoys a very high reputation. The medicine has the functions of benefiting the essence, tonifying deficiency and impairment, protecting the lung, nourishing the kidney, hemostasis, and resolving phlegm. However, due to its high growth conditions, it is currently on the verge of extinction and is listed as a national key protected wild plant (level II). [2016] No. 21). Cordyceps sinensis urgently needs to find a suitable substitute.

Paecilomyces is an important group of entomogenous fungi, about 50 species have been recorded, many of which are important natural control factors for pests, and some are important materials for biological control of pests; some of them are important medicinal fungal resources Cordyceps The anamorph of Cordyceps, some species such as Paecilomyces cicadae P.cicadae (Miquel) Samson can also be directly used as medicine. P. farinosus (Holm ex Gray) Brown, P. umosoroseus (Wize) Brown&Smith and P. lilacinus (Thom.) Samaon have been registered in the former Soviet Union and the Philippines application.

The mycelium and metabolites of Paecilomyces fungi have certain medicinal value. The research in this area mainly focuses on Paecilomyces gourni, Paecilomyces tenipedes and Paecilomyces cicadae. Paecilomyces paecilomyces has pharmacological effects such as sedation, analgesia, memory improvement, and the rate of viable tumor cells; Mice have normal pressure resistance to hypoxia, analgesia and sedation, and are beneficial to the recovery of AIDS; Paecilomyces cicadae can improve lipid metabolism and protect organs and tissues. Anti-aging and antipyretic effects, and minimal toxicity, but less research on other Paecilomyces strains. And in the utilization of Paecilomyces at present, except that Paecilomyces militaris is inoculated into Cordyceps militaris, most of them stay on the development and utilization of mycelium.

In order to overcome the limitations of existing development and utilization, it is necessary to develop new drugs that can fundamentally treat the etiology and effectively improve disease symptoms.

Contents of the invention

The purpose of the present invention is to provide a Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging and its application.

Another object of the present invention is to provide a Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging and a preparation method thereof.

The purpose of the present invention can be achieved through the following technical solutions:

A Paecilomyces pulitaris-based Cordyceps composition for enhancing immunity and anti-aging, which mainly consists of 20-70 parts of Paecilomyces pupaeci-based Cordyceps, 20-40 parts of Paecilomyces pumilaris-based Cordyceps and rose-smoky Paecilomyces pupae The mold consists of 1 to 10 parts of Cordyceps pupae, preferably 50 to 70 parts of Paecilomyces pupae, 30 to 40 parts of Paecilomyces pupae, and 2 to 2 parts of Paecilomyces pupae. Makes up 5 servings.

Application of the Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging in the preparation of anti-aging drugs for enhancing immunity.

A preparation method of a Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging, comprising the following steps:

a. Dry and pulverize each component with a mesh size of not less than 20 mesh;

b. Weigh the pulverized powdery raw materials according to weight percentage to obtain the traditional Chinese medicine composition of the present invention evenly; wherein the weight percentage of the raw materials is composed of: 20-70 parts of Paecilomyces grunnii, Cordyceps militaris, P. Composed of 20-40 parts of Penicillium pupae-based Cordyceps and 1-10 parts of Paecilomyces pupae-based Cordyceps, preferably 50-70 parts of Paecilomyces pupae-based Cordyceps, 30 parts of Paecilomyces pupae-based Cordyceps ～40 parts and 2～5 parts of Paecilomyces fumigatus pupae-based Cordyceps.

c. The composition is further made into powder, tablet, capsule or oral liquid.

Wherein, the pulverized meshes of the three raw materials of Paecilomyces coronis, Paecilomyces pupae, and Paecilomyces pupae are 20-80 mesh.

The Paecilomyces militaris-based Cordyceps composition for enhancing immunity and anti-aging prepared according to the above method.

The Paecilomyces pulitaris-based Cordyceps composition for enhancing immunity and anti-aging is prepared from natural raw materials, and has functions such as enhancing immunity, anti-aging, relieving physical fatigue, and assisting in improving memory. The activity of the invention is better than that of single use of each raw material, and better than that of Cordyceps sinensis, and provides a new way for the development of immunity-enhancing anti-aging drugs.

Detailed ways

The present invention will be further elaborated below in conjunction with specific examples, and these examples are only for the purpose of illustration, and are not intended to limit the scope of the present invention. The experimental animals of the present invention were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army (production license number: SCXK (Army) 2007-004).

The comparison of different Paecilomyces strain inoculation effects of embodiment 1

Seven kinds of Paecilomyces strains with Lepidoptera as hosts (Paecilomyces, Paecilomyces chrysalis, Paecilomyces gourney, Paecilomyces annulus, Paecilomyces tenipedes, Paecilomyces cicadae, Paecilomyces) was the strain to be tested. Inoculate the tested strains on PDA medium respectively, culture at a constant temperature of 26°C for 14 days, cut out the agar sheet with bacteria with a sterilized puncher (diameter 1 cm), and place it in the 30 mL of 0.1% Tween-80 sterile water with glass beads was shaken for 30 minutes. After the conidia were completely dispersed and uniform, filtered with sterile gauze to obtain a spore suspension. The spore concentration was measured with a hemocytometer, and the spore production of each strain was calculated (three replicates were set for each strain), and finally the concentration of the spore suspension was diluted to 1×10 ⁷ spores/mL.

Use the injection method to inoculate on the ultra-clean workbench. Use a 1mL syringe to inject about 0.1mL of the bacterial solution into each silkworm chrysalis. The injection site was selected at the intersection of the rear of the wing and the third link. When inoculating, insert the needle of the syringe into the surface of the pupa about 1mm, then make the needle parallel to the pupa, advance it forward about 3mm, inject the bacterial solution, and quickly pull out the needle. liquid.

After inoculation, move to a constant temperature incubator to avoid light, and culture at 25°C for about one month. After the color change, it is cultivated by scattered light, kept at 18-22°C and humidity at 85%-90% until harvested.

Inoculate under sterile conditions in the inoculation box, and culture the mycelium in dark at 20-25°C and relative humidity of 85%-90%. After the hyphae are cultivated, light culture is carried out to induce the production of fruiting body primordium, and 1000 lx is illuminated (9.5-10) h every day. When a small amount of fruiting body primordia appeared, a 0.2 mm thick needle was used to prick holes for ventilation, and 5 holes were pricked in each bottle. After cultivating for 45 days, the top of the fruiting body expands and can be harvested.

The number of hardened cocoons after inoculation, the hardening rate, the weed rate and the average fresh weight of Cordyceps: the hardening rate refers to the ratio of the hardened silkworm chrysalis after silkworm chrysalis inoculation 3-5 days to the total number of inoculated silkworm chrysalis; The ratio of the total number of hardened silkworm chrysalis; the average fresh weight of Cordyceps refers to the ratio of the total fresh weight of silkworm chrysalis Cordyceps to the total number of silkworm chrysalis.

Table 1 Comparative study on inoculation effect of silkworm chrysalis inoculated with different strains

It can also be seen from the data in Table 1 that the Paecilomyces strain has a low hardening rate, and the quality of the fruiting body after the grass is low. Paecilomyces chrysalis and Paecilomyces fumigatus had hardening rates of 98% and 99.3% respectively after inoculation; the rate of weeding reached 96.7% and 98.6% respectively, and the fruiting body quality was also high. Except for Paecilomyces strains, the other six Paecilomyces strains had no significant difference in the three indicators of hardening rate, weeding rate and average fresh weight of Cordyceps.

Example 2 Comparative study on the effect of fruiting bodies of different silkworm chrysalis-Paecilomyces strains on the immune activity of blood-deficiency mice

Grouping and treatment of animals: ICR mice were randomly divided into normal group, model group, positive control (lentinan group), fruiting bodies after inoculation (Paecilomyces, Paecilomyces pupaecillum, Paecilomyces coronis, Paecilomyces annulus) Penicillium, Paecilomyces tenipedes, Paecilomyces cicadae, Paecilomyces fumigatus) single-dose groups, a total of 10 groups, 20 rats in each group. The mice in the test group were given intragastric administration (0.4ml) according to the dose, the mice in the normal group and the model group were given equal volume of normal saline at the same time, and the positive control group was given 0.4ml of lentinan aqueous solution, once a day, for 15 consecutive days . From the 12th day, the mice in the model group and the treatment group were intraperitoneally injected with cyclophosphamide 80 mg·kg ^-1 for 3 consecutive days, and the mice in the normal group were injected with an equal volume of normal saline at the same time.

Detection of indicators: 1.0 hours after the last administration on the 15th day, 10 rats in each group were injected with 10ml/kg body weight of Indian ink diluted 5 times with normal saline into the tail vein, and the time was immediately measured. At 2min (t1) and 12min (t2) after the injection, 20μl of blood was collected from the inner canthal venous plexus, added to 2ml of 0.1% Na ₂ CO ₃ solution and shaken evenly. The Na ₂ CO ₃ solution was used as the blank control, and a spectrophotometer was used to Measure the absorbance value (A) at a wavelength of 600nm. Immediately after the second blood collection, the mice were killed, and their body weight was weighed; the thymus, liver and spleen were taken out, and the blood stains on the surface of the organs were blotted with filter paper, and the wet weight was measured to calculate the spleen index and thymus index. Spleen index (mg/g)=spleen weight (mg)/body weight (g); thymus index (mg/g)=thymus weight (mg)/body weight (g). The other 10 mice were removed from the eyeballs to obtain blood, and the peripheral blood was measured. Calculate the phagocytosis index, calculate the clearance index K and corrected clearance index α according to the following formula.

K=(lgA1-lgA2)/(t2-t1); α=K ^1/3 ×body weight/(liver weight+spleen weight).

Where A is the absorbance value of the blood sample, and t1 and t2 are the blood collection time after ink injection.

As can be seen from Table 2, compared with the normal group, white blood cells (WBC), platelets (PLT) and hemoglobin (Hb) in the model group were significantly decreased (p<0.01), and red blood cells (RBC) were significantly decreased, suggesting that the ring Phosphoramide hematopoietic deficiency model was successful. Compared with the model group, the leukocytes, platelets and hemoglobin levels of mice in the Paecilomyces pumilaris, Paecilomyces pumilaris, Paecilomyces pupaeci, and Paecilomyces pumilaris groups The amount of red blood cells all increased significantly (p<0.01), and the number of red blood cells rebounded significantly, tending to normal, indicating that Paecilomyces pumilaris, Paecilomyces pupae, Cordyceps pupae, Paecilomyces pupae Both Paecilomyces chrysogenum and Cordyceps pupae can effectively improve the peripheral blood of mice with blood deficiency, and the effect on peripheral blood is equivalent. The amount of white blood cells, platelets and hemoglobin in the other silkworm chrysalis-Paecilomyces strain fruiting body groups had no significant difference from the model group (p>0.05).

Table 2 Comparison of the effect of fruiting bodies of different silkworm chrysalis-Paecilomyces strains on the peripheral blood of mice with blood deficiency ( n=20)

Note: Compared with the model group, *: p<0.05, **: p<0.01

It can be seen from Table 3 that the immune organ index of the mice in the model group was significantly lower than that of the normal group (p<0.01). The immune organ indexes of the mice in the Cordyceps, Paecilomyces finelegs, and Paecilomyces pumilaris groups were significantly increased (p<0.01); Compared with the model group, Paecilomyces militaris, Paecilomyces pupae, Cordyceps pupaecilium, Paecilomyces pupae The K value and α value of the mice in the Cordyceps-based group were significantly increased (p<0.01, p<0.05). In general, Paecilomyces pumilaris, Paecilomyces pulitaris, Paecilomyces pumilaris, and Paecilomyces pumilaris had different effects on the immune organ index and single The effect on phagocytosis of nuclear macrophages was comparable. The K value and α value of the other silkworm chrysalis-Paecilomyces strain fruiting body groups had no significant difference from the model group (p>0.05).

Table 3 Effects of fruiting bodies of different silkworm chrysalis-Paecilomyces strains on the immune organ index and phagocytic function of mononuclear macrophages in mice with anemia deficiency ( n=20)

Note: Compared with the model group, *: p<0.05, **: p<0.01

The results showed that Paecilomyces militaris Cordyceps militaris, Paecilomyces militaris Cordyceps militaris, Paecilomyces tenipedum and Paecilomyces pupaeci can treat the immune dysfunction of mice with blood deficiency. There was no significant difference, indicating that Paecilomyces pupaecillum, Paecilomyces gourni, Paecilomyces tenipedes and Paecilomyces fumigatus infected silkworm chrysalis can produce fruiting bodies with immunosuppressive activity. Strain research provides a reference.

The preparation of embodiment 3 immunosuppressive active Paecilomyces pulitaris composition

Dry and pulverize Paecilomyces pupae, Paecilomyces pupae, Paecilomyces pupae, and Paecilomyces pulverus, with a mesh size of not less than 20 meshes;

Sample 1: The pulverized powdery raw materials were weighed by weight percentage to obtain a traditional Chinese medicine composition evenly; wherein the weight percentage of the raw materials consisted of: 50 parts of Paecilomyces grunnii pupae, 50 parts of Paecilomyces tenipedes pupae Composed of 40 parts of Cordyceps base and 10 parts of Paecilomyces pupaeci;

Sample 2: The pulverized powdery raw materials were weighed by weight percentage to obtain a traditional Chinese medicine composition evenly; wherein the weight percentage of the raw materials consisted of: 70 parts of Paecilomyces grunidus pupae, 70 parts of Paecilomyces tenipedes pupae Composed of 20 parts of Cordyceps genus and 10 parts of Paecilomyces fumigatus pupae;

Sample 3: The pulverized powdery raw materials were weighed by weight percentage to obtain a traditional Chinese medicine composition evenly; wherein the weight percentage of the raw materials consisted of: 58 parts of Paecilomyces grunidus pupae, 58 parts of Paecilomyces tenipedes pupae Composed of 40 parts of Cordyceps base and 2 parts of Paecilomyces pupaeci;

Sample 4: The pulverized powdery raw materials were weighed by weight percentage to obtain a traditional Chinese medicine composition uniformly; wherein the weight percentage of the raw materials was composed of: 100 parts of Paecilomyces pumilaris pupae;

Sample 5: The pulverized powdery raw material was weighed according to the weight percentage to obtain a traditional Chinese medicine composition; wherein the weight percentage of the raw material was composed of: 100 parts of Paecilomyces gunini Cordyceps militaris;

Sample 6: The pulverized powdery raw material was weighed according to the weight percentage to obtain a traditional Chinese medicine composition; wherein the weight percentage of the raw material was composed of: 100 parts of Paecilomyces tenipedes Cordyceps militaris;

Sample 7: The pulverized powdery raw material was weighed by weight percentage to obtain a traditional Chinese medicine composition uniformly; wherein the weight percentage of the raw material was composed of: 100 parts of Paecilomyces fumigatus Cordyceps militaris;

Example 4 Immunosuppressive Activity Paecilomyces pulitaris Composition Active Establishment

With reference to the method in the Ministry of Health "Technical Specifications for Inspection and Evaluation of Health Food" (2003 edition), samples 1-7 prepared in Example 3 were used to measure the immune function of mice.

ConA-induced mouse spleen lymphocyte transformation assay (MTT method)

The samples were given to each group by gavage, once a day, for 30 consecutive days. On the 30th day of the experiment, aseptically remove the spleen from each mouse, put it in a small plate filled with an appropriate amount of sterile Hank's solution, and gently tear it up with tweezers to make a single-cell suspension, filter through a 200-mesh sieve, wash, and count , and finally adjust the cell concentration to 3×10 ⁶ ·mL ^-1 with RPMI1640 complete culture medium. Add the cell suspension into two wells of a 24-well culture plate, 1 mL per well, add 75 μL of ConA solution (equivalent to 7.5 μg·mL ^-1 ) to 1 well, and use the other well as a control, culture at 37°C, 5% CO ₂ Cultivate in the box for 72h. 4 hours before the end of the culture, 0.7 mL of the supernatant was gently aspirated from each well, and 0.7 mL of RPMI1640 culture solution without calf serum was added. At the same time, 50 μL·well of MTT (5 mg·mL ^{-1 )} was added, and the culture was continued for 4 hours. After culturing, add 1mL of acidic isopropanol to each well, and mix well by pipetting to completely dissolve the purple crystals. Optical density (OD) was measured at a wavelength of 570 nm. Finally, subtract the optical density value of the well without ConA from the optical density value of the well with ConA to represent the proliferation ability of lymphocytes.

Table 4 Effects of different samples on ConA -induced proliferation of mouse spleen lymphocytes and sheep erythrocytes induced delayed-type hypersensitivity (DTH) in mice ( x±s , n=10)

It can be seen from the data of the net proliferation value (expressed in OD570 value) of splenic lymphocytes stimulated by ConA in Table 4 that compared with the negative control group, samples 1-7 groups can all increase the proliferation ability of ConA-induced mouse splenic lymphocytes. The activity of samples 1-3 is better than that of samples 4-7, which is significantly different from the negative control group (p<0.05). It can be seen that the activity of the composition (sample 1-3) is better than that of a single fruiting body, and has the effect of stimulating spleen lymphocyte proliferation.

Delayed type hypersensitivity (DTH) test in mice induced by sheep erythrocytes (SRBC) (foot plantar thickening method)

The samples were given to each group by gavage, once a day, for 30 consecutive days. On the 25th day of the experiment, each mouse was injected intraperitoneally with 0.2 mL of 2% (v·v ^-1 ) packed sheep red blood cell (SRBC) suspension for immunization. 4 days after immunization, measure the thickness of the plantar part of the left hind foot, then inject 20% (v·v ^-1 ) SRBC subcutaneously at the measurement site, 20 μL per mouse (about 1×10 ⁸ SRBCs), and measure the left hind foot 24 hours after injection The thickness of the sole of the foot was measured 3 times at the same site, and the average value was taken.

As can be seen from Table 4, the sheep red blood cell (SRBC) induced mouse DTH (plantar thickening method) test showed that the samples 1-7 groups can improve the difference between the thickness difference of the left hind foot plantar part of the mouse at 24h and the thickness difference at 0h, and the difference is significant. (p<0.05), indicating that samples 1-7 can increase the degree of swelling of the soles of the mice induced by sheep red blood cells (SRBC), and have the effect of promoting delayed hypersensitivity. It can be seen that samples 1-7 can enhance the cellular immune function of mice.

Determination of mouse serum hemolysin (hemagglutination method)

The samples were given to each group by gavage, once a day, for 30 consecutive days. On the 25th day of the experiment, each mouse was injected intraperitoneally with 0.2 mL of 2% (v·v ^-1 ) packed sheep red blood cell (SRBC) suspension for immunization. After 5 days, the blood was taken and centrifuged, and the serum was collected. The serum was serially diluted with normal saline, incubated in a 37°C incubator for 3 hours, the degree of hemagglutination was observed, and the anti-volume was calculated.

Antibody producing cell detection (Jerne modified slide method)

The samples were given to each group by gavage, once a day, for 30 consecutive days. On the 25th day of the experiment, each mouse was injected intraperitoneally with 0.2 mL of 2% (v·v- ¹ ) packed sheep red blood cell (SRBC) suspension for immunization. After 5 days, the spleen was aseptically taken from each mouse, placed in a small plate containing an appropriate amount of sterile Hank's solution, and gently torn up with tweezers to make a single-cell suspension, filtered through a 200-mesh screen, washed, counted, and finally used RPMI1640 complete culture medium was used to adjust the cell concentration to 5×10 ⁶ ·mL ^-1 . Heat and dissolve the surface medium (1g agarose plus double distilled water to 100mL), put it in a water bath at 45°C to keep warm, mix it with an equal amount of Hank's solution of pH7, divide into small test tubes, 0.5mL per tube, and then add 50μL of 10% SRBC (v·v ^-1 , prepared with SA solution), 20 μL splenocyte suspension (5×10 ⁶ ·mL ^-1 ), mix quickly, pour on a glass slide brushed with agarose thin layer, and make parallel slices After the agar is solidified, place the slide horizontally on the slide rack, put it in a carbon dioxide incubator and incubate for 1-1.5h, then add complement (1:8) diluted with SA buffer into the groove of the slide rack , after continuing to incubate for 1-1.5h, count the number of hemolytic plaques.

Table 5 Effects of different samples on mouse antibody producing cells and serum hemolysin ( n=10)

It can be seen from Table 5 that there is no significant difference between the serum hemolysin anti-volume values of the mice in sample 1-7 groups and the negative control group (p>0.05). Compared with the negative control group, the sample 1-7 groups can increase the number of hemolytic plaques in the mice, and the difference is significant (p <0.05), and the sample 1-3 group is extremely significant (p <0.01). It shows that samples 1-7 have the effect of promoting the proliferation of antibody-producing cells, thereby helping to enhance the humoral immune function of mice.

Phagocytosis of chicken red blood cells by mouse peritoneal macrophages (semi-in vivo method)

The samples were given to each group by gavage, once a day, for 30 consecutive days. 30 minutes before the animals were sacrificed, each mouse was intraperitoneally injected with 1 mL of 20% (v·v ^-1 ) chicken erythrocyte suspension, and then injected with 2 mL of normal saline, and the peritoneal fluid drops were taken, incubated in a 38°C incubator for 30 minutes, fixed, and stained. Microscopic examination, 100 macrophages were counted, and the phagocytosis rate and phagocytosis index were calculated.

Table 6 Effects of different samples on mouse peritoneal macrophages phagocytosis of chicken red blood cells and carbon clearance test phagocytosis index ( n = 10)

It can be seen from Table 6 that the phagocytosis rate and phagocytosis index of mouse peritoneal macrophages phagocytizing chicken erythrocytes in sample 1-7 groups were not significantly different from those of the negative control group (p>0.05). Compared with the negative control group, there was no significant difference between the carbon clearance ability of the mice in the sample 1-7 group (p>0.05).

mouse carbon clearance test

The samples were given to each group by gavage, once a day, for 30 consecutive days. On the 30th day of the experiment, Indian ink was injected into the tail vein of each mouse, calculated as 0.1 mL per 10 g of body weight, and the time was immediately counted after the ink was injected. 2 min and 10 min after the injection of ink, 20 μL of blood was collected from the inner canthus venous plexus respectively, and added to 2 mL of 0.1% Na ₂ CO ₃ solution, and the optical density (OD) was measured at 600 nm wavelength with a 721 spectrophotometer to obtain Na ₂ CO ₃ solution was used as blank control. After the second blood collection, the mice were sacrificed immediately, and the liver and spleen were taken out, and the blood stains on the surface of the organs were blotted with filter paper, and weighed. Calculate the phagocytosis index.

NK cell activity assay (LDH assay)

The samples were given to each group by gavage, once a day, for 30 consecutive days. On the 30th day of the experiment, aseptically remove the spleen from each mouse, put it in a small plate filled with an appropriate amount of sterile Hank's solution, and gently tear it up with tweezers to make a single-cell suspension, filter through a 200-mesh sieve, wash, and count , and finally adjust the cell concentration to 2×10 ⁷ ·mL ^-1 with RPMI1640 complete culture medium. The YAC-1 cells (target cells) were subcultured 24 hours before the test, washed three times with Hank's solution before application, and the cell concentration was adjusted to 4×10 ⁵ ·mL ^-1 with RPMI1640 complete culture medium. Take 100 μL each of target cells and splenocyte suspension (effector cells) (effect-to-target ratio 50:1), add them into U-shaped 96-well culture plate, add 100 μL each of target cells and culture medium in the natural release holes of target cells, and maximize the release of target cells. Add 100 μL of target cells and 1% NP40 to each well; set up 3 duplicate wells for each of the above items, incubate in an incubator at 37°C and 5% CO ₂ for 4 hours, centrifuge, absorb 100 μL of supernatant from each well and place on a flat-bottomed 96-well culture plate At the same time, 100 μL of LDH matrix solution was added, reacted for 5 min, and 30 μL of 1 mol·L ^-1 HCl was added to each well to measure the optical density (OD) value at a wavelength of 490 nm to calculate the activity of NK cells.

The influence of table 7 different samples on mouse NK cell activity n=10)

As can be seen from the data in Table 7, compared with the negative control group, the sample 1-7 groups can all improve NK cell activity, and there is a significant difference (p＜0.05), wherein the composition (sample 1-3) has a very significant difference (p <0.01), the activity is better than single fruiting body.

The comprehensive table 4-7 experimental result shows that, in improving the proliferative ability of the mouse spleen lymphocyte induced by ConA and enhancing NK cell activity, composition (sample 1-3) is better than single fruiting body (Paecilomyces pumilaris pumilaris cordyceps , Paecilomyces gourney, Paecilomyces pupae, Cordyceps pupaecilium, Paecilomyces pupae, Cordyceps pupaeum, Paecilomyces pupaeum, Cordyceps pupae).

Example 5 Preparation of immunosuppressive activity Paecilomyces pulitaris composition capsule

After weighing 58 parts of Paecilomyces grunnii Cordyceps pupae, 40 parts of Paecilomyces tenipedes and 2 parts of Paecilomyces pupae (20 mesh) by weight percentage, mix them evenly After obtaining the traditional Chinese medicine composition, it is prepared into capsules according to conventional methods.

Example 6 Preparation of Immunosuppressive Active Paecilomyces pupaecilium Composition Powder

After 70 parts of pulverized Paecilomyces pumilaris, 20 parts of Paecilomyces pumilaris and 10 parts of Paecilomyces pumilaris (20 mesh) were weighed by weight percentage, they were uniformly mixed Afterwards, the traditional Chinese medicine composition is obtained, which is prepared into powder according to a conventional method.

Example 7 Preparation of Immunosuppressive Activity Paecilomyces pulitaris Composition Tablet

70 parts of pulverized Paecilomyces pumilaris, 20 parts of Paecilomyces tenipedes and 10 parts of Paecilomyces pupae (80 mesh) were weighed by weight percentage, and mixed evenly Obtain the Chinese medicine composition afterward, be prepared into tablet according to conventional method.

Embodiment 8 Preparation of oral liquid of Paecilomyces pulitaris composition with immunosuppressive activity

After 50 parts of pulverized Paecilomyces pumilaris Cordyceps pulveris, 40 parts of Paecilomyces pumilaris and 10 parts of Paecilomyces pumilaris (80 mesh) were weighed by weight percentage, evenly mixed After obtaining the traditional Chinese medicine composition, it is prepared into an oral liquid according to a conventional method.

Example 9 Immunosuppressive Activity Paecilomyces pupaecillum Composition Powder Anti-aging Activity Establishment

The powder prepared in embodiment 6 is carried out anti-aging activity

Experimental animals and grouping: 40 male Wistar rats, weighing 180-220g. Divided into normal group, model group, caloric restriction group, Paecilomyces pupaecillum composition powder sample group (the powder prepared in embodiment 6) four groups by random number table method, each group 10. Modeling and drug administration: Wistar rats started modeling after 1 week of adaptation to the environment. The model group, the caloric restriction group, and the powder sample group were injected with 1% D-galactose saline solution intraperitoneally, 1 time/d, 1ml/100g body weight/time, for 6 weeks to create an aging model, and the powder sample group was given pupa Paecilomyces composition powder sample 8g/kg/d (equivalent to the amount of raw medicinal materials) was gavaged, and the normal group, the model group, and the caloric restriction group were gavaged with the same amount of normal saline at the same time. During the modeling period, the body weight of the rats was weighed and recorded every 4 days, and the dosage was converted according to the new body weight. Observe and record the general state of the rats in each group during the administration of the model.

Acquisition and preservation of animal tissues: Fasting for 12 hours after the last administration. When collecting materials, use 20% urethane solution to anesthetize the experimental rats, fix the anesthetized experimental rats on a fixed board, take blood from the abdominal aorta, and then dissect it, carefully remove the liver and brain tissue of the rats with tweezers, Wash the blood on the surface with normal saline, cut the brain tissue into left and right halves, and cut out part of the liver tissue, put half of the brain tissue and part of the liver tissue in 10% formaldehyde solution for fixation, and separate the half of the brain tissue and part of the liver tissue Store in -80°C refrigerator for later use. Rat blood was centrifuged at 3500r/min at 4°C for 10min, the supernatant was taken, and the serum was aliquoted and stored in a -80°C refrigerator.

ELISA method to detect the effect of Paecilomyces pupaecomb composition powder on the content of lipid peroxide in the liver tissue and brain tissue of aging rats: Weigh an appropriate amount of rat liver tissue and brain tissue, add physiological saline and use a manual homogenizer to make it 10% tissue homogenate was centrifuged at 3500r/min at 4°C for 10min, the supernatant was taken, aliquoted, and placed in a -80°C refrigerator for later use. During the experiment, liver tissue homogenate and brain tissue homogenate were taken to measure the contents of SOD, T-AOC and MDA respectively, and the detection method was strictly operated according to the instructions of the kit.

SOD, MDA and T-AOC content ( n=10)

It can be seen from Table 8 that compared with the normal group, the SOD activity in the liver tissue of rats in the D-galactose-induced aging model group decreased significantly (P<0.05), the T-AOC activity also decreased significantly (P<0.05), and the MDA content significantly decreased. increased (P<0.05); the activities of SOD and T-AOC in the liver tissue of the caloric restriction group were also significantly lower than those of the model group, while the content of MDA was significantly higher than that of the model group. The Paecilomyces pupaecillum composition powder group is compared with the model group, the SOD activity and the T-AOC activity in the rat liver of the Paecilomyces pupaecomposition powder group are significantly increased (P<0.05), and the MDA content is significantly reduced (P<0.05). 0.05), the change was better than that of the caloric restriction group.

SOD, MDA and T-AOC content ( n=10)

It can be seen from Table 9 that compared with the normal control group, the SOD activity in the brain tissue of rats in the D-galactose-induced aging model group decreased significantly (P<0.01), the T-AOC activity also decreased significantly (P<0.01), and the MDA content Significantly increased (P<0.01); Compared with the model group, the caloric restriction group and the Paecilomyces pupaecillum composition powder group had a significantly increased SOD activity, a significantly increased T-AOC activity, and a significantly reduced MDA content (P<0.05). better than the caloric restriction group.

ELISA method was used to detect the effect of Paecilomyces pupaecomb composition powder on telomerase activity in the brain tissue of aged rats: Weighed an appropriate amount of rat brain tissue, added physiological saline and made 10% tissue homogenate with a manual homogenizer, and then used Centrifuge at 3500r/min for 10min, take the supernatant, aliquot, and store in -80°C refrigerator for later use. During the experiment, the rat brain tissue homogenate was taken to measure brain telomerase (TE) activity, and the detection method was strictly operated according to the instructions of the kit.

TE content in the rat brain tissue of table 10 ( n=10)

As shown in Table 10, compared with the model group, the telomerase activity of the normal control group was significantly increased (P<0.01); the TE activity of the caloric restriction group was also significantly increased; group significantly increased (P<0.01).

Claims (6)

1. a kind of paecilomycerol pupa base cordyceps sinensis composition of strengthen immunity anti-aging, by paecilomyces gunniliang bacterium pupa base cordyceps sinensis, carefully Pin paecilomycerol pupa base cordyceps sinensis and Paecilomyces Fumosoroseus pupa base cordyceps sinensis composition.

2. the paecilomycerol pupa base cordyceps sinensis composition of the strengthen immunity anti-aging described in claim 1, it is characterised in that its group Turn into：20~70 parts of paecilomyces gunniliang bacterium pupa base cordyceps sinensis, 20~40 parts of thin pin paecilomycerol pupa base cordyceps sinensis and paecilomyces fumosoroseus 1~10 part of composition of bacterium pupa base cordyceps sinensis, preferably by 50~70 parts of paecilomyces gunniliang bacterium pupa base cordyceps sinensis, thin pin paecilomycerol pupa base worm 2~5 parts of compositions of 30~40 parts of grass and Paecilomyces Fumosoroseus pupa base cordyceps sinensis.

3. the application in immunity antiaging agent, the function is strengthen immunity, anti-aging, alleviates physical fatigue, auxiliary Improve memory etc..

4. a kind of preparation method of the paecilomycerol pupa base cordyceps sinensis composition of strengthen immunity anti-aging, comprises the following steps：A. By each component crushed after being dried, mesh number is no less than 20 mesh；B. after the powder raw material after crushing is weighed by weight percentage uniformly Obtain Chinese medicine composition of the present invention；The percentage by weight of wherein described raw material forms：Paecilomyces gunniliang bacterium pupa base cordyceps sinensis 20 ~70 parts, thin 20~40 parts of pin paecilomycerol pupa base cordyceps sinensis forms for 1~10 part with Paecilomyces Fumosoroseus pupa base cordyceps sinensis, is preferably By 50~70 parts of paecilomyces gunniliang bacterium pupa base cordyceps sinensis, 30~40 parts of thin pin paecilomycerol pupa base cordyceps sinensis and Paecilomyces Fumosoroseus pupa 2~5 parts of compositions of base cordyceps sinensis；C. pulvis, tablet, capsule or oral liquid is further made in composition.

5. the preparation method of the paecilomycerol pupa base cordyceps sinensis composition of strengthen immunity anti-aging according to claim 4, It is characterized in that the paecilomyces gunniliang bacterium pupa base cordyceps sinensis, thin pin paecilomycerol pupa base cordyceps sinensis, Paecilomyces Fumosoroseus pupa base worm Feed powder broken mesh number in grassland is 20~80 mesh.

6. the preparation method of the paecilomycerol pupa base cordyceps sinensis composition of strengthen immunity anti-aging according to claim 4, Characterized in that, formulation method is the common process of the formulation.