Cordyceps sobolifera spore water extract as well as preparation method and application thereof

Abstract

The invention discloses a cordyceps sobolifera spore water extract as well as a preparation method and application thereof, and belongs to the technical field of natural product extraction. The preparation method specifically comprises the following steps: (1) adding cordyceps sobolifera spore powder into a solvent, and mixing; and (2) heating to boiling, cooling to room temperature, centrifuging, collecting supernate, concentrating and filtering to obtain the traditional Chinese medicine composition. According to the method, the cordyceps sobolifera spore water extract is prepared by adopting an optimized water boiling method, and the method has the advantages of remarkable operation simplicity and convenience, high extraction efficiency and high purity. In the extraction process, it is ensured that effective substances are completely released, and the biological activity of the effective substances is completely reserved, so that the finally obtained water extract has wide application potential in the fields of medicine, cosmetics, healthy food and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and more particularly to a water extract of Cicadae Convolvulus spores and a preparation method and application thereof. Background Art

[0002] Cicada fungus (Cordyceps cicadae), commonly known as giant caterpillar fungus, is a fascinating organism with both "animal" and "plant" morphological characteristics. Its roots are the bodies of cicada larvae, and its flowers grow from the heads of a single or 2-3 cicada larvae, about an inch long, branching and blooming from the top. Its pollen is creamy yellow and is called "cicada fungus spore powder," or simply "seed," which has the function of reproduction. Cicada fungus spore powder grows from the end of the spore stalk bundle that grows from the sclerotium after the cicada fungus is fully fungalized. Cicada fungus spore powder is gradually transformed from the active ingredients of the sclerotium and spore stalk bundle, and it concentrates the active essence of the cicada fungus. It is the most active ingredient and the most effective part of the medicinal cicada fungus. Cicada fungus spore powder is rich in polysaccharides, mannitol, adenosine, cordycepin and other active ingredients. It has extremely high nutritional value and has the effects of regulating immunity (enhancing the body's resistance by activating immune cells), anti-oxidation and anti-aging (eliminating free radicals and delaying cell aging), neuroprotection (protecting the nervous system through adenosine receptor activation), anti-inflammatory and anti-tumor (inhibiting inflammatory responses and enhancing the killing effect on cancer cells).

[0003] However, the current methods for extracting active ingredients from cicada fungus spore powder have problems such as easy loss of effective ingredients, complex operations, and low extraction efficiency, which limit its further application.

[0004] Therefore, how to develop an efficient and simple method for extracting the contents of Cordyceps sinensis spore powder is a problem that those skilled in the art urgently need to solve. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a water extract of Cicadae Convolvulus spores and a preparation method and application thereof, so as to solve the deficiencies in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing an aqueous extract of cicada convolvulus spores comprises the following steps:

[0008] (1) adding cicada conidia spore powder to a solvent and mixing to obtain a cicada conidia spore suspension;

[0009] (2) The cicada fungus spore suspension is heated to boiling, cooled to room temperature, centrifuged, the supernatant is collected, concentrated, and filtered to obtain the cicada fungus spore water extract.

[0010] Furthermore, in the above step (1), the solvent is ultrapure water.

[0011] Furthermore, in the above step (1), the usage ratio of Cicadae Convolvulus spore powder and water is 1 g:100 mL.

[0012] Furthermore, in the above step (2), the heating method is direct fire heating or induction cooker heating.

[0013] Furthermore, in the above step (2), the boiling time is 30 minutes.

[0014] Furthermore, in the above step (2), the centrifugal speed is 3000 rpm and the time is 5 min.

[0015] Furthermore, in the above step (2), the concentration method is microwave or rotary evaporation; furthermore, the power of the microwave is 300W.

[0016] A further beneficial effect of the above method is that microwave or rotary evaporation can efficiently concentrate the water extract of Cicadae Convolvulus spores, quickly remove the solvent, and is suitable for solvent-sensitive compounds, which can help laboratories quickly and accurately process a large number of samples and improve work efficiency.

[0017] Furthermore, in the above step (2), the filtration method is to filter and sterilize the supernatant using a 0.22 μm microporous filter membrane.

[0018] A further beneficial effect of the above method is that filtration sterilization can effectively remove microorganisms in the water extract of Cicadae Convolvulus spores, thereby ensuring the sterility and safety of the product.

[0019] The present invention also claims protection for an aqueous extract of cicada fungus spores prepared by the above preparation method.

[0020] The present invention also claims protection for the use of an aqueous extract of cicada fungus spores prepared by the above preparation method in the preparation of medicines, cosmetics and foods (especially health products).

[0021] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. Easy to operate: The water boiling method is used to extract the active ingredients in the cicada fungus spores, which does not require complicated instruments and equipment and the operation process is simple and easy.

[0023] 2. High extraction efficiency: By optimizing the boiling conditions (such as heating method, time, etc.), efficient extraction of polysaccharides and nucleosides in cicada fungus spores can be achieved.

[0024] 3. High purity of the extract: Centrifugal separation and filtration sterilization steps can effectively remove impurities and microorganisms in the extract, ensuring the purity of the extract.

[0025] 4. Wide application: It contains the active ingredients in cicada fungus spore powder, and the content of effective substances is equivalent to that of the original cicada fungus spore powder. It can be widely used in pharmaceuticals, cosmetics, functional foods and other fields, and has broad market prospects.

[0026] In summary, the present invention utilizes an optimized boiling method to prepare an aqueous extract of Cicadae convolvulus spores, demonstrating remarkable operational simplicity, high extraction efficiency, and high purity. During the extraction process, the effective substances are fully released and their biological activity is fully preserved, resulting in a final aqueous extract with broad potential applications in medicine, cosmetics, health foods, and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a chromatogram showing the content of nucleoside substances in the aqueous extract of Cicadae Convolvulus spores in Example 1;

[0028] Figure 2 The exact content of nucleoside substances in the water extract of Cicadae Convolvulus spores in Example 1;

[0029] Figure 3 The effect of the aqueous extract of Cicadae Convolvulus spores on the immune organ indexes of immunosuppressive model mice in Example 1 (n=10, x±s); wherein, A: spleen index (mg / g), B: liver index (mg / g), C: thymus index (mg / g); Note: * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001; the immune organ development index of the treatment group was significantly improved compared with the control group (p<0.05);

[0030] Figure 4 This is the effect of the aqueous extract of Cicadae Convolvulus spores on blood cells in immunosuppressive model mice in Example 1; wherein, A: hemoglobin, B: lymphocytes, C: erythrocytes, D: leukocytes; Note: compared with the blank group, # indicates p < 0.05, ## indicates p < 0.01; compared with the model group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; mean ± SE. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of 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 making any creative efforts are within the scope of protection of the present invention.

[0032] Example 1

[0033] The preparation method of the water extract of Cicadae Convolvulus spores specifically comprises the following steps:

[0034] (1) Weigh 10.0 g of Cicadae Constrictae spore powder, add it to 1 L of ultrapure water, and mix to obtain a Cicadae Constrictae spore suspension;

[0035] (2) The cicada fungus spore suspension was transferred to a beaker, heated to boiling using an induction cooker with a power of 600 W and maintained for 30 min, quickly cooled to room temperature, centrifuged at a speed of 3000 rpm for 5 min, and the supernatant was collected. The suspension was concentrated to 50 mL using a microwave with a power of 300 W, and the supernatant was filtered and sterilized using a 0.22 μm microporous filter membrane to obtain the cicada fungus spore water extract, which was stored at -20°C for later use.

[0036] Performance Testing

[0037] 1. Detection of nucleoside substances in the aqueous extract of Cicadae convolvulus spores

[0038] 1. Regression equation

[0039] Accurately weigh 1.0 mg of uridine, inosine, guanosine, adenosine, cordycepin and N 6 2-(2-Hydroxyethyl)adenosine was added to a 10 mL centrifuge tube, and 5 mL of a mixture of acetonitrile and water (5:95, mobile phase) was added and dissolved by ultrasonication. Then, standard solutions of 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 mg / mL were prepared. 6 The mass concentration of -(2-hydroxyethyl)adenosine standard solution is the X-axis, the peak area is the Y-axis, and the regression equation is obtained.

[0040] 2. Chromatographic conditions

[0041] A Thermo Fisher Ultimate 3000 HPLC system was used, with an Agilent Eclipse Plus C8 column (4.6 mm x 250 mm x 5 μm) and a DAD detector. The mobile phase consisted of double-distilled water (A) and methanol (B), with a gradient elution: 5-5% (B) from 0 to 4 min; then 5-10% (B) from 4.0 to 10.0 min; 10-40% (B) from 10.0 to 15.0 min; 40-80% (B) from 15.0 to 20.0 min; 80→100% (B) from 20.0 to 23.0 min; 100-5% (B) from 23.0 to 23.1 min; and 5-5% (B) from 23.1 to 28 min. The flow rate was 1 mL / min, the injection volume was 20 μL, and the column temperature was 25°C.

[0042] 3. Sample testing

[0043] The relative abundance of nucleosides in each sample was assessed by determining the peak area of each nucleoside component relative to a nucleoside standard used as an external control. The retention time of the individual nucleoside standard peaks was identical to that of the nucleoside standard mixture. Therefore, a nucleoside standard mixture can be used in place of individual nucleoside standards for injection. Each sample was analyzed in triplicate.

[0044] HPLC analysis of nucleoside standards uses the chromatographic peak area of each standard as the ordinate and the concentration of the nucleoside standard solution as the abscissa to determine the regression equation and correlation coefficient. Within the linear range, the peak area of the standard shows a good linear relationship with the concentration of the standard. Therefore, the nucleosides in the sample can be quantitatively analyzed based on the chromatographic peak area.

[0045] 4. Experimental Results

[0046] The results are as follows Figure 1 、 Figure 2 shown.

[0047] Depend on Figure 1 、 Figure 2 The final analysis showed that the uridine content in the water extract of cicada fungus spores in Example 1 was 80.175 μg / g, the inosine content was 94.885 μg / g, the guanosine content was 738.93 μg / g, the adenosine content was 2513.95 μg / g, the cordycepin content was 81.175 μg / g, and the N 6 The content of -(2-hydroxyethyl)adenosine is 574.39 μg / g.

[0048] 2. Restorative Effects of Cicadae Convolvulus Spores Water Extract on the Immune System of ICR Mice

[0049] Seventy ICR mice were divided into seven groups, including a normal group, low-, medium-, and high-dose groups of Cicadae Convolvulus spore water extract, a levamisole hydrochloride group, a Poria polysaccharide oral solution group, and a cyclophosphamide model group. The mice were gavaged according to the doses in Table 1.

[0050] Table 1 Groups and dosages

[0051]

[0052]

[0053] 1. Experimental Procedure

[0054] The immunosuppressive model was established by intraperitoneal injection of cyclophosphamide. Except for the normal group, the other groups were injected with cyclophosphamide (80 mg kg -1 ·d -1 The mice in the Cicadae Constricta spore water extract group were orally gavaged with the Cicadae Constricta spore water extract of Example 1 according to the dosage in Table 1 for 21 consecutive days.

[0055] 2. Measurement indicators

[0056] The weight changes, immune organ indexes, carbon particle clearance index and blood routine indicators in the mice were monitored.

[0057] 3. Experimental Results

[0058] The results are shown in Tables 2, 3 and Figure 3 、 Figure 4 shown.

[0059] Table 2 Effects of the aqueous extract of Cicadae Convolvulus spores in Example 1 on the body weight and organ indexes of immunosuppressed mice

[0060]

[0061] Table 3 Effect of Example 1 Cicadae Convolvulus Spore Water Extract on the Phagocytic Function of Macrophages in Experimental Mice

[0062]

[0063] From Table 2 and Figure 3 It can be seen that the water extract of Cicadae Convolvulus spores can increase the body weight growth rate and immune organ index of immunosuppressed mice.

[0064] As shown in Table 3, the carbon clearance index showed that the water extract of Cicadae convolvulus spores enhanced the phagocytic ability of mouse monocytes and macrophages.

[0065] The water extract of Cicadae Convolvulus spores has a good repairing effect on the immune system of ICR mice.

[0066] Depend on Figure 4 It can be seen that routine blood tests showed that the water extract of cicada fungus spores can help restore the hemoglobin content and red blood cell count of immunosuppressed mice, which increased significantly; the number of lymphocytes can also be restored as the concentration of the water extract increases; and the number of white blood cells also increased significantly.

[0067] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an aqueous extract of Cicadae Contagiosum spores, characterized in that: The specific steps include: (1) adding cicada conidia spore powder to a solvent and mixing to obtain a cicada conidia spore suspension; (2) heating the Cicadae Contagiosum spore suspension to boiling, cooling to room temperature, centrifuging, collecting the supernatant, concentrating, and filtering to obtain the Cicadae Contagiosum spore water extract.

2. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (1), the solvent is ultrapure water.

3. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (1), the usage ratio of the Cicadae Convolvulus spore powder and water is 1g:100mL.

4. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (2), the heating method is direct fire heating or electromagnetic cooker heating.

5. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (2), the boiling time is 30 minutes.

6. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (2), the centrifugal speed is 3000 rpm and the time is 5 minutes.

7. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (2), the concentration method is microwave or rotary evaporation; the power of the microwave is 300W.

8. The method for preparing a water extract of Cicadae Contagiosum spores according to claim 1, wherein: In step (2), the filtration method is to filter and sterilize the supernatant using a 0.22 μm microporous filter membrane.

9. An aqueous extract of Cicadae Contagiosum spores obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the water extract of Cicadae Convolvulus spores obtained by the method according to any one of claims 1 to 8 in the preparation of medicines, cosmetics and foods.

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