Powder formulation having a function of enhancing immunity and method for preparing the same

Abstract

The present disclosure relates to the field of health care products, disclosing a powder formulation made from inulin, GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS and stevioside. The raw materials of the powder formulation are all from natural Chinese herbal medicine without addition of excipient. In addition, dose required for the powder formulation is small; it is easy to be taken and can be dissolved in water; it is soluble in cold water and can be absorbed quickly. The method for preparing the powder formulation is simple and suitable for large-scale production. The powder formulation obtained has a good stability, long storage time, good taste. Experiments show that the powder formulation of the present disclosure can significantly enhance immunity, therefore can be used to prepare the health care foods having function on improving immunity.

Claims

1. A method of enhancing immunity, comprising administering a powder formulation to a subject in need thereof, wherein the powder formulation is made from inulin, GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS and stevioside, wherein the mass ratio of inulin, GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS and stevioside is (50 to 80):(10 to 30):(3 to 8):(2 to 8):(1 to 5):(1 to 5):(1 to 5):(0.5 to 1):(0.2 to 0.8):(0.2 to 0.8):(0.2 to 0.8):(0.2 to 0.8).

2. The method according to claim 1, wherein the mass ratio of inulin, GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS and stevioside is 50:16:8:8:5:5:5:1:0.5:0.5:0.5:0.5.

3. The method according to claim 1, wherein the mass ratio of inulin, GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS and stevioside is 80:10:3:2:1:1:1:0.5:0.5:0.4:0.3:0.3.

4. The method according to claim 1, wherein the mass ratio of inulin, GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS and stevioside is 65:20:5:3:1.5:1.5:1:1:0.5:0.5:0.5:0.5.

5. The method according to claim 1, wherein the powder formulation is in the form of health care food.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) In order to describe the technical solutions in the examples of the present disclosure or the conventional art more clearly, the accompanying drawings used in description of the embodiments or the prior art will be illustrated briefly.

(2) FIG. 1 shows the flow chart of preparing the health care powder formulation in the present disclosure.

(3) FIG. 2 shows the effect of the sample on the transformation of ConA-induced lymphocyte.

(4) FIG. 3 shows the effect of the sample on the generation of antibody.

(5) FIG. 4 shows the effect of the sample on the activity of NK cell.

(6) FIG. 5 shows the effect of the sample on the phagocytosis ratio and phagocytic index of mouse macrophage.

DETAILED DESCRIPTION

(7) The present disclosure discloses a powder formulation having a function of enhancing immunity and method for preparing the same. One of ordinary skill in the art can learn from the contents herein and improve the process parameters appropriately. In particular, it shall be noted that all the similar substitutions and modifications are apparent to one of ordinary skill in the art and are to be considered within the scope of the present invention. The method and product of the present invention have been described with preferred examples. It is apparent that one of the ordinary skill in the art can make change or modify the combination to the method and product of the present invention without departing from the spirit, scope and spirit of the invention, therefore realizing and applying the techniques of the present invention.

(8) In order to understand the present disclosure further, the technical solutions in the embodiments of the present disclosure will be described clearly and completely herein in conjunction with the examples of the present disclosure. Apparently, the described examples are only a part of the examples of the present disclosure, rather than all examples. Based on the examples in the present disclosure, all of other examples, made by one of ordinary skill in the art without any creative efforts, fall into the protection scope of the present disclosure.

(9) Without special illustration, all the reagents in the examples of the present disclosure are commercial products, which can be purchased on the market.

EXAMPLES

Example 1: Heath Care Powder Formulation of the Present Disclosure

(10) Formulation:

(11) TABLE-US-00001 Inulin 50 g GANODERMA 16 g COICIS SEMEN 8 g PORIA 8 g ATRACTYLODIS 5 g MACROCEPHALAE RHIZOMA CUSCUTAE SEMEN 5 g POLYGONATI RHIZOMA 5 g OPHIOPOGONIS RADIX 1 g GLYCYRRHIZAE RADIX ET 0.5 g RHIZOMA LYCII FRUCTUS 0.5 g SCHISANDRAE CHINENSIS 0.5 g FRUCTUS Stevioside 0.5 g
Preparation Method:

(12) GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS were put into an extracting tank and purified water 8 to 15 times the weight of the starting materials was added. Extraction was performed for 2 to 4 hours, followed by filtration. Purified water 10 to 20 times the weight of the starting materials was added again and extraction was performed for 2 to 5 hours, followed by filtration. The two filtrates were combined and concentrated. Inulin was added, and spray drying or belt drying or freeze drying was carried out to obtain a dry powder formulation of Chinese herbal medicine extract. Stevioside was added into the dry powder formulation of Chinese herbal medicine extract, followed by granulating, drying and sizing. The particle size of the product was controlled between 30-mesh and 100-mesh.

Example 2: Heath Care Powder Formulation of the Present Disclosure

(13) Formulation:

(14) TABLE-US-00002 Inulin 80 g GANODERMA 10 g COICIS SEMEN 3 g PORIA 2 g ATRACTYLODIS 1 g MACROCEPHALAE RHIZOMA CUSCUTAE SEMEN 1 g POLYGONATI RHIZOMA 1 g OPHIOPOGONIS RADIX 0.5 g GLYCYRRHIZAE RADIX ET 0.5 g RHIZOMA LYCII FRUCTUS 0.4 g SCHISANDRAE CHINENSIS 0.3 g FRUCTUS Stevioside 0.3 g
Preparation Method:

(15) GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS were put into extracting tank and purified water 8 to 15 times the weight of the starting materials was added. Extraction was performed for 2 to 4 hours, followed by filtration. Purified water 10 to 20 times the weight of the starting materials was added and extraction was performed for 2 to 5 hours, followed by filtration. The two filtrates were combined and concentrated. Inulin was added, and spray drying or belt drying or freeze drying was carried out to obtain a dry powder formulation of Chinese herbal medicine extract. Stevioside was added into the dry powder formulation of Chinese herbal medicine extract, followed by granulating, drying and sizing. The particle size of the product was controlled between 30-mesh and 100-mesh.

Example 3: Heath Care Powder Formulation of the Present Disclosure

(16) Formulation:

(17) TABLE-US-00003 Inulin 65 g GANODERMA 20 g COICIS SEMEN 5 g PORIA 3 g ATRACTYLODIS 15 g MACROCEPHALAE RHIZOMA CUSCUTAE SEMEN 15 g POLYGONATI RHIZOMA 1 g OPHIOPOGONIS RADIX 1 g GLYCYRRHIZAE RADIX ET 0.5 g RHIZOMA LYCII FRUCTUS 0.5 g SCHISANDRAE CHINENSIS 0.5 g FRUCTUS Stevioside 0.5 g
Preparation Method:

(18) GANODERMA, COICIS SEMEN, PORIA, ATRACTYLODIS MACROCEPHALAE RHIZOMA, CUSCUTAE SEMEN, POLYGONATI RHIZOMA, OPHIOPOGONIS RADIX, GLYCYRRHIZAE RADIX ET RHIZOMA, LYCII FRUCTUS, SCHISANDRAE CHINENSIS FRUCTUS were put into extracting tank and purified water 8 to 15 times the weight of the starting materials was added. Extraction was performed for 2 to 4 hours, followed by filtration. Purified water 10 to 20 times the weight of the starting materials was added and extraction was performed for 2 to 5 hours, followed by filtration. The two filtrates were combined and concentrated. Inulin was added, and spray drying or belt drying or freeze drying was carried out to obtain a dry powder formulation of Chinese herbal medicine extract. Stevioside was added into the dry powder formulation of Chinese herbal medicine extract, followed by granulating, drying and sizing. The particle size of the product was controlled between 30-mesh and 100-mesh.

Experimental Example 1: Immunity Enhancing Test

(19) The enhancement of immunity function is evaluated by four aspects including cellular immune function, humoral immune function, monocyte-macrophage function and NK cell activity. The test sample is regarded as having a function of enhancing immunity if any two aspects show positive results.

(20) 1. Test sample: the powder formulation prepared in Example 3.

(21) 2. Experimental animals: BALB/C inbred strain mouse, male, 18 to 22 g of each mouse. The mice were adoptive fed for a week and divided into groups (12 per group) randomly according to their body weights.

(22) 3. Grouping and time of administration of the test sample

(23) Test animals were divided into 4 groups, i.e., a control group (administrated with same amount of distilled water) and 3 treatment groups including low-dose group, medium-dose group and high-dose group, wherein the treatment groups were given 5 times (166.67 mg/kg), 10 times (333.33 mg/kg) and 30 times (1000 mg/kg) of the human recommended amount of the test sample, respectively, three repetitions per dose. The test sample was given for 30 days.

(24) 4. Experiment methods and results

(25) 4.1 Effect of the Test Sample on the Body Weight of Mice

(26) TABLE-US-00004 TABLE 1 Effect of the test sample on the body weight of mice Original Weight Final Weight Group (average (average (g/kg .Math. BW) standard deviation) standard deviation) Control Group 17.24 1.70 25.71 2.13 Low-dose Group 16.20 2.02 26.10 1.33 Medium-dose Group 18.77 1.94 26.19 1.36 High-dose Group 18.23 2.05 26.27 1.33

(27) Table 1 showed that 30 days after the administration of test sample at different doses orally, the weight of the mice in three treatment groups did not show significant differences compared with that of the control group (P>0.05), indicating that the test sample was safe.

(28) 4.2 Effect of the Test Sample on Spleen/Body Weight Ratio of Mice

(29) TABLE-US-00005 TABLE 2 Effect of the test sample on spleen/body weight ratio of mice Group Spleen/Weight (g/kg .Math. BW) ratio (%) P Control Group 0.33 0.02 Low-dose Group 0.34 0.08 0.282 Medium-dose Group 0.35 0.04 0.069 High-dose Group 0.39 0.12 0.084

(30) Table 2 showed that 30 days after the administration of test sample at different doses orally, the spleen/body weight ratio of three treatment groups did not have significant differences compared with that of the control group (P>0.05).

(31) 4.3 Transformation Experiment of ConA-Induced Mouse Lymphocyte Cells (MTT Method)

(32) (1) Preparation of spleen cells suspension. Spleen was removed under sterile condition and placed in Hanks solution. The spleen was snipped into pieces by a tweezers to prepare a single cell suspension, which was passed a 200-mesh screen. The cells were washed with Hanks solution twice through 10 min centrifugation (1000 rpm/min) each time. The cell suspension was suspended in the complete medium and the cell density was adjusted to 310.sup.6/ml after counting the number of viable cells by trypan blue staining.

(33) (2) Spleen cell proliferation test. Each of the cell suspension was divided into two parts and added into 24-well plate, 1 mL per well. ConA solution (final concentration of 7.5 g/mL) was added into one well, and the other well was set as the control. Cells were incubated in an incubator for 72 h. MTT method was used to measure the absorbance OD value of each well.

(34) The proliferation function of lymphocytes is calculated by subtracting the OD value of the control well from the well added ConA. The statistical results were shown in FIG. 2.

(35) The results in FIG. 2 showed that, 30 days after the administration of test sample at different doses orally, there was no significant differences in ConA-induced lymphocyte transformation between three treatment groups and the control group (P>0.05). The experiment result was negative.

(36) 4.4 Delayed-Type Hypersensitivity (DTH)

(37) Dinitrofluorobenzene-induced mouse DTH (ear swelling assay)

(38) (1) Preparation of DNFB solution (1% of dinitrofluorobenzene)

(39) (2) Sensitization: the fur on abdomen of each mouse was shaved by barium sulfate at an area about 33 cm.sup.2, 50 L DNFB solution was applied evenly to induce hypersensitivity.

(40) (3) Development and measure of DTH: 5 days later, DNFB solution was applied evenly on both sides of the right ear of the mice for stimulation. The mice were sacrificed by cervical dislocation 24 h later. Two ears were cut off, and a piece with diameter of 8 mm was taken by a puncher and weighted.

(41) Results: DTH level was determined by mass differences between the left ear and the right ear, which was shown in Table 3.

(42) TABLE-US-00006 TABLE 3 Effect of the test sample on mouse ear swelling Group Mass difference between left (g/kg .Math. BW) ear and right ear (mg ) P Control Group 17.09 3.49 Low-dose Group 16.19 2.49 0.54 Medium-dose Group 14.96 2.37 0.15 High-dose Group 14.37 3.13 0.10

(43) Table 3 shows that, there was no significant mass differences between left ear and right era between three treatment groups and the control group (P>0.05), therefore the result was negative.

(44) 4.5 Antibody-Producing Cell Test (Modified Jerne's Slide Method)

(45) (1) Preparation of spleen cells suspension. The mice were sacrificed by cervical dislocation 4 to 5 days after SRBC immunization. Spleen was removed and prepared into spleen cells suspension. The cell density was adjusted to 510.sup.6 cell/mL.

(46) (2) Plaque assay. The surface culture medium was dissolved, placed in 45 C. water bath and mixed with the same amount of 2 Hanks solution (pH of 7.2). The medium was divided into aliquot in test tubes, 0.5 mL per tube. 50 L of 10% SRBC and 20 L of spleen cells suspension were added into the tube, mixed homogenously and rapidly. The mixture was poured on a glass slide with a thin layer of agarose. Control slide was prepared in parallel. After solidification, the glass slides were placed (upside down) in a slide box and incubated for 1.5 h. The statistic results of hemolytic plaque were shown in FIG. 3.

(47) FIG. 3 showed that, 30 days after the administration of the test sample at different doses orally, the test sample increased the antibody production of the spleen cells in comparison with the control group. The plaque number of the high-dose group showed a significant difference (P<0.05) from that of the control group, so the experiment result was positive.

(48) 4.6 NK Cell Activity Assay (Lactate Dehydrogenase Assay)

(49) Passage of target cell (YAC-1 cell): before the experiments, target cells were subcultured and cell conditions were adjusted.

(50) The preparation method for spleen cells suspension was the same as that of 4.3. Cell viability was above 95% based on trypan blue staining. The density of the separated spleen cells suspension was adjusted to 210.sup.7 cell/mL.

(51) NK cell activity assay: 100 L of target cells and 100 L of effector cells (effector cells:target cells=50:1) were added into 96-well plate. Spontaneously releasing well contained 100 L of target cell and 100 L of culture medium, while maximally releasing well contained 100 L of 1% NP40 or 100 L of 2.5% Triton. Each experiment has three repetitions. The cells were incubated in an incubator for 4 hours. Thereafter, the culture plate was subjected to a 5 min low-speed centrifugation and 100 L of suspension from each well was transferred to a flat-bottom 96-well plate. OD value of each well at 490 nm was measured by LDH assay kit. NK cell activity was calculated by the average value of the three repeat wells in each group, and the results were shown in FIG. 4. Formula for calculating NK cell activity was as follows:
NK cell activity (%)=(OD value of reaction wellOD value of spontaneously releasing well)/(OD value of maximally releasing wellOD value of spontaneously releasing well)

(52) FIG. 4 showed that, 30 days after the administration of the test sample at different doses orally, compared with the control group, the NK cell activity of the low-dose group has no significant difference (P>0.05), but the NK cell activities of both the medium-dose group and the high-dose group showed significant differences (P<0.05). The experiment result was positive.

(53) 4.7 Phagocytosis Experiment of Mouse Abdominal Macrophage on Chicken Red Blood Cell (Drop-Slide Method)

(54) Activation of mouse macrophage. 4 days before the experiment, 0.2 mL of 2% sheep red blood cells were injected to the abdomen of each mouse. The mice were sacrificed by cervical dislocation. 4 mL of Hanks solution containing calf serum was injected into the abdomen of each mouse and the abdominal macrophages were washed out by gently massage of the abdomen 20 times. A small outlet was cut on the abdominal wall, 2 mL of abdominal washing liquid was sucked out by a dropper. 0.5 mL of abdominal washing liquid was pippetted to the tube containing 0.5 mL of 1% chicken red blood cell suspension and mixed evenly. 0.5 mL of the mixture was added into the agarose circle on a glass slide. The slide was incubated in an incubator for 20 min. The non-adhered cells were rinsed off by physiological saline as soon as the incubation finished. The cells were fixed by methanol for 1 min and stained by Giemsa for 15 min. Thereafter, the slide was rinsed and dried. The percentage of phagocytosis and phagocytic index were counted under a microscope with 40 magnification. The results were shown in FIG. 5. Number of macrophages was counted under oil immersion lens, and there were 100 cells were counted on each slide. The formula for calculating percentage of phagocytosis and phagocytic index were as follows:
Percentage of phagocytosis (%)=number of macrophage containing chicken red blood cell/number of counted macrophage100
Phagocytic index=total number of the swallowed chicken red blood cell/number of counted macrophage

(55) FIG. 5 showed that, 30 days after the administration of the test samples at different doses orally, compared with the control group, the percentage of phagocytosis and phagocytic index of low-dose group have no significant differences (P>0.05), but the percentage of phagocytosis and phagocytic index of both medium-dose group and high-dose group showed significant differences (P<0.05). The experiment result was positive.

(56) 5. Conclusion

(57) The experiment scheme of the present disclosure conforms to animal welfare principles and the experimental process conforms to the requirements of animal experiment. The data are in detail and reliable. The results are subjected to statistical analysis and give the following conclusions:

(58) 5.1 The results of mouse lymphocyte transformation experiment and delayed-type hypersensitivity reaction are negative, indicating that the result of cell immune function test is negative.

(59) 5.2 The result of antibody-producing cell test is positive, indicating that the result of humoral immune function test is positive.

(60) 5.3 The result of phagocytosis experiment of mouse abdominal macrophage on chicken red blood cell is positive, indicating that the result of monocyte-macrophage function test is positive.

(61) 5.4 The result of lactate dehydrogenase assay is positive, indicating that the result of NK cell activity is positive.

(62) In these four aspects, cell immune function, humoral immune function, monocyte-macrophage function and NK cell activity tests, the results of the present experiments show that the test sample significantly improves the indexes of antibody production, phagocytosis of abdominal macrophage on chicken red blood cell and NK cell activity, which show significant differences compared to the control group.

(63) In view of above, under conditions of present laboratory, after 30 days' administration of distilled water, a low-dose, medium-dose and high-dose of the powder formulation obtained in Example 3, respectively, to BALB/C mice orally, the test results show that the powder formulation obtained in Example 3 has a function of enhancing immunity.

(64) The powder formulation obtained in Example 1 and Example 2 has a similar effect as that of Example 3.