An extract of ganoderma applanatum and a method for extracting the same

Abstract

The application discloses a snow ganoderma extract and an extraction method thereof, and belongs to the technical field of biological extraction. The extraction method is a hot water ultrasonic enzymolysis method, a series of extraction parameters such as ultrasonic time, ultrasonic power, temperature, selection of a composite enzyme and proportioning of the use amount are optimized, and the yield and stability of the snow ganoderma extract are improved. The prepared snow ganoderma extract has high content and high-quality active ingredient polysaccharide, and has good repair and anti-light damage effects, and the industrial application value of the snow ganoderma is improved.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction technology, specifically relating to a Ganoderma lucidum extract and its extraction method. Background Technology

[0002] Snow Lingzhi, the whole herb of *Arenaria kansuensis* Maxim., a plant in the Caryophyllaceae family, mainly grows in Gansu, Qinghai, western Sichuan, Yunnan, and Tibet. It is an extremely valuable wild Tibetan medicinal resource. The herbal texts and medicinal effects of Snow Lingzhi are recorded in works such as *The Four Medical Classics*, *Jingzhu Materia Medica*, *Tibetan Materia Medica*, and *Secret Formulas and Supplements*. Snow Lingzhi, also known as Zaozhui or Wuxincai, uses the whole herb for medicinal purposes. It is bitter and cold in nature, and has the effects of clearing heat and benefiting the lungs, relieving cough, reducing swelling, lowering blood pressure, and promoting urination and relieving pain. It is commonly used to treat jaundice, gonorrhea, uterine diseases, pneumonia, and various lung diseases.

[0003] Snow Ganoderma lucidum is rich in polysaccharides, triterpenoids, flavonoids, sterols, triterpenoids, alkaloids, amino acids, vitamins, etc. Due to its unique bioactive components and medicinal value, it has become a subject of intense research by scholars at home and abroad. In recent years, the research on the chemical components and pharmacological activities of this medicinal material has become increasingly in-depth. For example, Wang Guojun et al. isolated eight flavonoid compounds from snow Ganoderma lucidum (Research on the Chemical Components and Anti-inflammatory Activities of Snow Ganoderma lucidum, West China Journal of Pharmaceutical Sciences, 2019, 34(3): 225-229); Liu Liyan et al. studied the bioactivity of alfalfa extract, the main component of snow Ganoderma lucidum, in anti-inflammatory, anti-allergic, anti-tumor, bone regeneration enhancement, anti-skin photoaging, and antimicrobial activity (Research Progress on the Bioactivity of Alfalfa Extract, Chinese Journal of Modern Applied Pharmacy, 2020, 37(6): 764-768). At present, the research and application of snow Ganoderma lucidum are mostly limited to simple pathological studies, while there is very little research on the extraction process of active components of snow Ganoderma lucidum. The fruiting body of *Ganoderma lucidum* is mainly composed of cellulose, hemicellulose, and lignin, with a compact structure. The main active ingredient, polysaccharides, is attached to the inner side of the cell wall. During extraction, high temperatures, acidic or alkaline conditions can cause the glycosidic bonds of the polysaccharides to break, thereby reducing their biological activity. Therefore, the extraction of *Ganoderma lucidum* polysaccharides is quite difficult. Traditional polysaccharide extraction methods mainly employ hot water extraction, acid extraction, and alkaline extraction. While acid or alkaline extraction can improve the extraction rate, it requires sophisticated equipment, easily damages the polysaccharide structure, and poses environmental pollution problems. Traditional hot water extraction is time-consuming, energy-intensive, and yields low active ingredient extraction rates. Therefore, improving the extraction yield of polysaccharides from *Ganoderma lucidum* extract while effectively preserving their high antioxidant activity is an urgent problem to be solved.

[0004] Chinese patent CN114601828B discloses a Ganoderma lucidum extract and its use in the preparation of anti-inflammatory drugs. The extract is obtained by separating and purifying the Ganoderma lucidum extract through column chromatography, chromatographic separation, concentration, and drying. Although this extract exhibits good anti-inflammatory properties, its extraction process is quite cumbersome. Chinese patent CN101258927A discloses that pulverizing Ganoderma lucidum and then repeatedly boiling it yields an extract containing varying amounts of total saponins. While preparations made from this extract can be used in health supplements to enhance human immunity, the boiling extraction method severely damages the content of the effective components in Ganoderma lucidum. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, the present invention first provides a method for extracting Ganoderma lucidum extract, the extraction method comprising the following steps:

[0006] S1. Dry and pulverize the whole Ganoderma lucidum herb;

[0007] S2. Mix the snow lingzhi powder and water, heat and ultrasonically extract to obtain a mixture, and adjust the pH of the mixture to 5-6.

[0008] S3. Add the compound enzyme to the mixture and hydrolyze it at a constant temperature. After the hydrolysis is complete, inactivate the enzyme and centrifuge to collect the supernatant.

[0009] S4. After purifying the supernatant with macroporous resin, concentrate it.

[0010] Preferably, the snow lingzhi includes, but is not limited to, at least one of Qinghai snow lingzhi, Tibetan snow lingzhi, and Gansu snow lingzhi.

[0011] Furthermore, the drying temperature in S1 is 50-70℃, and the particle size of the pulverized powder is 30-60 mesh.

[0012] Preferably, the drying temperature in S1 is 60±2℃, and the particle size of the pulverized powder is 40 mesh.

[0013] Furthermore, the mixing ratio of Ganoderma lucidum powder and water in S2 is 1:40-120 w / v, preferably 1:60-80 w / v.

[0014] Furthermore, the heating temperature in S2 is 40-60℃, preferably 50-55℃. Heating during the ultrasonic process is beneficial for enhancing the permeability of cell walls and membranes, facilitating the release and dissolution of polysaccharides, and enhancing the transfer and mixing of substances through the ultrasonic cavitation effect, thereby increasing the extraction rate of active ingredients. However, excessively high temperatures can damage heat-sensitive components, leading to polysaccharide deterioration or the release of other byproducts, affecting the extraction effect and reducing the stability of the extract.

[0015] Furthermore, the ultrasonic power ranges from 150 to 380W, with selectable values ​​such as 150W, 170W, 190W, 200W, 210W, 220W, 230W, 250W, 270W, 290W, 300W, 310W, 320W, 330W, 340W, 350W, 360W, 370W, and 380W.

[0016] Preferably, the ultrasonic power is 200-320W. The ultrasonic process mainly promotes polysaccharide extraction through cavitation, mechanical, and thermal effects. The ultrasonic power directly affects the intensity of these effects and thus the extraction efficiency. When the ultrasonic power is low, the cavitation effect is weak, resulting in fewer microbubbles with low collapse energy, leading to insufficient disruption of the Ganoderma lucidum cell wall, limited release of active ingredients, low mass transfer rate, and limited contact area between hot water and Ganoderma lucidum powder, requiring a longer extraction time. When the ultrasonic power is too high, the cavitation effect is too strong, and localized high temperatures can destroy some polysaccharides. Furthermore, cell debris increases, and impurities such as lipids and proteins dissolve more, affecting subsequent purification operations and the stability of the extract. In this invention, the ultrasonic power has a significant impact on the extraction effect; only by controlling the ultrasonic power parameter to 200-320W can the optimal extraction rate and efficiency be obtained.

[0017] Furthermore, the ultrasonic time is 15-40 minutes. The ultrasonic time needs to be coordinated with the hydrothermal temperature, ultrasonic power, and particle size of the Ganoderma lucidum powder. If the ultrasonic time is too short, firstly, the cavitation effect is insufficient and the cell wall is not broken down sufficiently, so as not to produce sufficient release of polysaccharides. Secondly, the components extracted in a short time are mainly small molecules such as free sugars and small molecule phenols, and the polysaccharide dissolution rate is low. If the ultrasonic time is too long, the polysaccharide structure may be sheared due to the thermal effect, thereby reducing its molecular weight. This will also increase the degree of dissolution of impurities and affect the purity and quality stability of the extract.

[0018] Furthermore, the pH of the mixture in S2 can be adjusted using at least one of HCl, NaOH, KOH, H3PO4, etc.

[0019] Furthermore, the complex enzyme in S3 is selected from at least two of cellulase, papain, neutral protease, and plant proteolytic enzyme.

[0020] Preferably, the complex enzyme is cellulase and papain.

[0021] More preferably, the cellulase has an enzyme activity ≥10000 U / g, and the papain has an enzyme activity ≥10000 U / g.

[0022] Furthermore, the mass ratio of cellulase to papain is 2-5:1, with options such as 2:1, 2.2:1, 2.5:1, 2.8:1, 3:1, 3.3:1, 3.5:1, 3.7:1, 3.9:1, 4:1, 4.3:1, 4.5:1, 4.7:1, and 5:1. Cellulase primarily breaks down cellulose and hemicellulose in the cell wall, disrupting the cell wall structure and promoting the release of intracellular active polysaccharides. However, residual intracellular proteins hinder polysaccharide dissolution. Papain can break down intracellular proteins (such as structural proteins), reducing the binding of proteins to polysaccharides and improving the latter's dissolution efficiency. By precisely controlling the relative content of both, their combined action enhances the release of Ganoderma lucidum polysaccharides, significantly improving extraction rate, purity, and retention rate of active ingredients, while reducing cost and the risk of side reactions.

[0023] In a preferred embodiment, the mass ratio of cellulase to papain is 3:1.

[0024] Furthermore, the amount of compound enzyme added is 1-5% of the mass of Ganoderma lucidum powder, preferably 2-3%, and more preferably 2.5%. Within a certain range, the extraction rate will significantly increase with the increase of the amount of compound enzyme added, but if it exceeds a certain limit, excessive compound enzyme will lead to enzyme aggregation or excessive decomposition of substrate, thereby affecting the extraction efficiency of polysaccharides; in addition, the above-mentioned amount of compound enzyme improves the biological activity of Ganoderma lucidum polysaccharides.

[0025] Furthermore, the temperature of the S3 isothermal enzymatic hydrolysis process is 45-55℃, more preferably 55℃. Enzymes exhibit peak activity within a specific temperature range; excessively high or low temperatures reduce their catalytic efficiency. Excessively high temperatures may lead to enzyme denaturation and inactivation, affecting the extraction rate; excessively low temperatures result in insufficient enzyme activity, slowing the reaction rate and decreasing the extraction rate. Therefore, selecting a suitable enzymatic hydrolysis temperature is a crucial step in the extraction process. This invention achieves the optimal extraction rate of Ganoderma lucidum polysaccharides only when the enzymatic hydrolysis temperature is controlled at 45-55℃, most preferably 50℃.

[0026] Furthermore, the enzymatic hydrolysis time is 10-40 min, preferably 15-20 min.

[0027] Furthermore, the macroporous resin can be selected from at least one of AB-8, D101, or HPD100. Papain and cellulase have a certain degree of hydrophobicity, making them suitable for removal with non-polar resins. D101 macroporous resin is a non-polar adsorption resin that can adsorb enzymes through hydrophobic interactions, making it suitable for preliminary purification. AB-8 is a weakly polar resin with adsorption performance between polar and non-polar resins, making it suitable for separating highly polar polysaccharides, especially those with a wide molecular weight distribution. It exhibits good adsorption selectivity and is suitable for fine purification, used for further purification of Ganoderma lucidum polysaccharides. HPD100 has a large specific surface area and hydrophobicity, making it suitable for adsorbing hydrophobic impurities. It can remove hydrophobic impurities such as proteins and lipids, has a large adsorption capacity, and is suitable for large-scale purification.

[0028] In one embodiment, the macroporous resin is AB-8, which has a more suitable specific surface area and pore size. The pore size directly affects whether impurity molecules can freely enter the resin for adsorption. If the pore size is too small, impurity molecules such as proteins may not be able to enter the resin smoothly, resulting in a decrease in adsorption efficiency. If the pore size is too large, although impurity molecules can enter freely, it may reduce the selectivity of the resin. The larger the specific surface area, the stronger the adsorption capacity of the resin is usually. However, if the specific surface area is too high and the pore size is too small, it may cause the adsorption rate of the resin to slow down, the desorption to be insufficiently concentrated, and the separation effect of impurities to be poor. The higher the porosity, the greater the adsorption capacity of the resin is usually, but it may also lead to a decrease in the mechanical strength of the resin. The strength and service life of the resin need to be considered.

[0029] Furthermore, the macroporous resin purification process includes the following steps:

[0030] (1) Pretreatment: Immerse the macroporous resin in 95wt% ethanol for 12-48h, and then rinse the resin column with 95wt% ethanol and deionized water respectively.

[0031] (2) Purification: Filter the supernatant and pass it into the resin column at a flow rate of 1-2 BV / h;

[0032] (3) Elution: Deionized water and 10-20 wt% ethanol are passed into the resin column for elution, followed by 50-70 wt% ethanol.

[0033] (4) Regeneration: 95wt% ethanol and deionized water are sequentially passed into the resin column for rinsing.

[0034] Furthermore, in step (1), the rinsing rate of ethanol and water on the resin column is 1-2 BV / h.

[0035] Preferably, in step (2), a 0.45 μm filter membrane is used for filtration to remove insoluble impurities; during the purification process, the effluent is collected and the polysaccharide content is detected until the resin adsorption is saturated (i.e., the polysaccharide concentration in the effluent increases significantly).

[0036] Further, in step (3), the impurities (such as pigments, proteins, etc.) adsorbed on the resin are removed first, and then the target polysaccharide adsorbed on the resin is eluted; the flow rates of deionized water, 10-20wt% ethanol and 95wt% ethanol are 1-2 BV / h.

[0037] Furthermore, in step (4), the flow rate of 95wt% ethanol and deionized water is 1-2 BV / h to remove residual polysaccharides and impurities.

[0038] Furthermore, the concentration method described in S4 includes, but is not limited to, at least one of dialysis, ethanol precipitation, and freeze-drying, preferably freeze-drying.

[0039] Furthermore, the freeze-drying temperature is below -40℃, and the freeze-drying time is 24-36 hours.

[0040] Secondly, the present invention also provides a snow fungus extract prepared according to the extraction method.

[0041] Furthermore, the Ganoderma lucidum extract can be used in the formulation of medicines and health products, as well as in the formulation of cosmetics with antioxidant, anti-inflammatory, anti-photodamage, and collagen-promoting effects.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The extraction method of the present invention uses a combination of complex enzymes with specific extraction parameters such as ultrasonic time, ultrasonic power, enzymatic hydrolysis temperature, etc., which can not only greatly improve the yield and purity of Ganoderma lucidum polysaccharide, but also effectively preserve the biological activity of Ganoderma lucidum polysaccharide, effectively increasing the added value and economic value of Ganoderma lucidum.

[0044] (2) The extraction method of the present invention is simple, time-saving and has low process cost.

[0045] (3) The snow lingzhi extract of the present invention has higher antioxidant, anti-inflammatory, anti-photodamage and collagen-promoting effects. Attached Figure Description

[0046] Figure 1 : Representative diagram of fluorescent staining area of ​​zebrafish embryos in different groups during the test of resistance to light damage. Detailed Implementation

[0047] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1

[0049] This embodiment provides a method for extracting Ganoderma lucidum extract, the extraction method comprising the following steps:

[0050] S1. Select, clean and dry the whole Ganoderma lucidum herb at 60℃, then pulverize it and pass it through a 40-mesh sieve to obtain Ganoderma lucidum powder;

[0051] S2. Mix the snow lingzhi powder and water at a ratio of 1:70w / v, heat to 50℃, and ultrasonically extract at 280W for 30 minutes to obtain a mixture. Adjust the pH of the mixture to 6.

[0052] S3. Add the compound enzyme to the mixture and hydrolyze at 50℃ for 20 min. After hydrolysis, incubate at 80℃ for 20 min to inactivate the enzyme. Centrifuge and collect the supernatant. The amount of compound enzyme used is 2.5% of the weight of the Ganoderma lucidum powder. The compound enzyme is a mixture of cellulase (enzyme activity of 10000 U / g) and papain (enzyme activity of 10000 U / g) in a mass ratio of 3:1.

[0053] S4. Purify using AB-8 macroporous resin, followed by freeze-drying (-40℃, 36h). The purification process is as follows:

[0054] (1) Pre-processing

[0055] Soaking: Soak the macroporous resin in 95% ethanol for 24 hours to fully swell and remove organic impurities.

[0056] Rinsing: Rinse the resin column with 95% ethanol at a flow rate of 1-2 BV / h until the effluent is colorless and transparent; then rinse the resin column with deionized water at a flow rate of 1-2 BV / h until the effluent has no ethanol odor.

[0057] (2) Purification

[0058] Filtration: The supernatant of S3 was filtered through a 0.45μm filter membrane to remove insoluble impurities.

[0059] Purification: Pass the filtrate into the resin column at a flow rate of 1-2 BV / h.

[0060] Monitoring: Collect the effluent and test the polysaccharide content until the resin adsorption is saturated (i.e., the polysaccharide concentration in the effluent increases significantly).

[0061] (3) Washing

[0062] Deionized water elution: Elute with deionized water at a flow rate of 1-2 BV / h to remove unadsorbed impurities.

[0063] Eluting with low-concentration ethanol: Elute with 20wt% ethanol at a flow rate of 1-2 BV / h, consuming 6 BV, to further remove polar impurities.

[0064] High-concentration ethanol elution: Elute with 60wt% ethanol at a flow rate of 1-2 BV / h, consuming 4 BV, and collect the eluent.

[0065] The polysaccharide content in the eluent was measured until elution was complete (the polysaccharide concentration in the eluent decreased significantly).

[0066] (4) Resin regeneration

[0067] Ethanol rinsing: Rinse the resin column with 95% ethanol at a flow rate of 1-2 BV / h to remove residual polysaccharides and impurities.

[0068] Rinse with deionized water: Rinse the resin column with deionized water at a flow rate of 1-2 BV / h until the effluent has no ethanol odor.

[0069] This embodiment also provides a snow fungus extract prepared according to the above extraction method.

[0070] Example 2

[0071] This embodiment provides a method for extracting Ganoderma lucidum extract, the extraction method comprising the following steps:

[0072] S1. Select, clean and dry the whole Ganoderma lucidum herb at 50℃, then pulverize it and pass it through a 60-mesh sieve to obtain Ganoderma lucidum powder;

[0073] S2. Mix Ganoderma lucidum powder and water at a ratio of 1:60 w / v, heat to 40℃, and ultrasonically extract at 320W for 15 minutes to obtain a mixture. Adjust the pH of the mixture to 6.

[0074] S3. Add the compound enzyme to the mixture and hydrolyze at 45℃ for 40 min. After hydrolysis, incubate at 80℃ for 20 min to inactivate the enzyme. Centrifuge and collect the supernatant. The amount of compound enzyme used is 5% of the weight of Ganoderma lucidum powder. The compound enzyme is a 2:1 mass ratio of cellulase (enzyme activity of 10000 U / g) and papain (enzyme activity of 10000 U / g).

[0075] S4. Purify using D101 macroporous resin, following the same procedure as in Example 1. After purification, freeze-dry (-40℃, 36h).

[0076] This embodiment also provides a snow fungus extract prepared according to the above extraction method.

[0077] Example 3

[0078] This embodiment provides a method for extracting Ganoderma lucidum extract, the extraction method comprising the following steps:

[0079] S1. Select, clean and dry the whole Ganoderma lucidum herb at 70℃, then pulverize it and pass it through a 30-mesh sieve to obtain Ganoderma lucidum powder;

[0080] S2. Mix Ganoderma lucidum powder and water at a ratio of 1:80 w / v, heat to 55°C, and ultrasonically extract at 200W for 40 minutes to obtain a mixture. Adjust the pH of the mixture to 6.

[0081] S3. Add the compound enzyme to the mixture and hydrolyze at 55℃ for 30 min. After hydrolysis, incubate at 80℃ for 20 min to inactivate the enzyme. Centrifuge and collect the supernatant. The amount of compound enzyme used is 1% of the weight of the Ganoderma lucidum powder. The compound enzyme is a mixture of cellulase (enzyme activity of 10000 U / g) and papain (enzyme activity of 10000 U / g) in a mass ratio of 5:1.

[0082] S4. Purify using HPD100 macroporous resin. The purification procedure is the same as in Example 1. After purification, freeze-dry (-40℃, 36h).

[0083] This embodiment also provides a snow fungus extract prepared according to the above extraction method.

[0084] Comparative Example 1

[0085] It is basically the same as Example 1, except that the ultrasonic power of S2 is adjusted to 400W.

[0086] Comparative Example 2

[0087] It is basically the same as Example 1, except that the ultrasonic extraction time of S2 is adjusted to 60 min.

[0088] Comparative Example 3

[0089] It is basically the same as Example 1, except that the enzymatic hydrolysis temperature of S3 is adjusted to 43°C.

[0090] Comparative Example 4

[0091] The basic formula is the same as Example 1, except that the snow lingzhi powder and water are mixed at a ratio of 1:40 w / v.

[0092] Comparative Example 5

[0093] The basic formula is the same as in Example 1, except that the compound enzyme is a combination of cellulase (enzyme activity of 10000 U / g) and pectinase (enzyme activity of 10000 U / g) in a mass ratio of 3:1.

[0094] Comparative Example 6

[0095] It is basically the same as Example 1, except that the amount of compound enzyme used is 10% of the weight of Ganoderma lucidum powder.

[0096] Performance testing methods and results:

[0097] I. Repair Efficacy Test

[0098] (1) This experiment intends to use Q-pCR to detect the gene expression levels of collagen and elastin to reflect the repair efficacy of the sample to be tested; Test sample: the original extract of Ganoderma lucidum after purification in step S4 and before freeze drying in Example 1.

[0099] (2) Zebrafish rearing conditions: In the breeding and special breeding system for adult wild zebrafish, the pH value range is 7.0-8.0, the conductivity range is 500-800μS / cm, the room temperature range is (26℃±1℃), the water temperature range is (28℃±1℃), and the photoperiod is 14 hours of light and 10 hours of darkness.

[0100] (3) Preparation of experimental embryos: On the afternoon of the day before collecting fish eggs, 3-18 month old male and female zebrafish that have already eaten are transferred to the mating tank and a partition is inserted. The next morning between 9:00 and 9:30, the partition in the middle of the mating tank is removed, and the embryos are collected 15 minutes after the partition is removed. The embryos are placed in a constant temperature incubator at (28±0.5℃) and embryo buffer is added. When the embryos reach 24 hpf, the normally developing embryos are selected for the experiment. Embryo buffer preparation: 15.75g NaCl + 0.9g NaHCO3 + 0.25g KCl + 0.53g CaCl2 + 4.5L ultrapure water, filtered using a vacuum filter and then autoclaved, and stored at room temperature.

[0101] (3) This experiment included one blank control group and three experimental groups. Six-well cell culture plates were used, with three wells in each group and 20 embryos per well. The blank control group was incubated with 6 mL of embryo buffer solution, experimental group one with 6 mL of 2 g / L test sample, experimental group two with 6 mL of 4 g / L test sample, and experimental group three with 6 mL of 8 g / L test sample. All embryos were cultured in a 28 ℃ ± 1 ℃ incubator until they reached 72 hpf. RNA extraction was performed, and real-time PCR data were collected. Gene expression levels in each zebrafish group were statistically analyzed and compared with the blank control group. GraphPad Prism 8 was used for statistical analysis. Differences between groups were analyzed using a T-test, and P < 0.05 was generally considered statistically significant. Tables 1-3 show the statistical results of the relative expression levels of zebrafish genes Collala, Colla2, and Elna, with P representing the data obtained from comparing the blank control group and the experimental group.

[0102] Table 1. Statistical results of relative expression levels of the zebrafish gene Collala.

[0103]

[0104] Table 2. Statistical results of relative expression levels of the zebrafish gene Colla2.

[0105]

[0106] Table 3. Statistical results of relative expression levels of the zebrafish gene Elna.

[0107]

[0108] Experimental conclusion: Under the experimental conditions, the concentrations of the test samples at 2 g / L, 4 g / L, and 8 g / L all promoted the expression levels of collagen (Col1a1a, Col1a2) and elastin (Elna) genes, indicating that the samples had a repairing effect.

[0109] II. Test of resistance to light damage

[0110] (1) Detection principle: A zebrafish embryo photodamage model was constructed, and the skin permeability was labeled using fluorescent staining reagents. The activity of the test sample in inhibiting skin permeability was evaluated by testing the red fluorescent area of ​​the zebrafish skin, thereby reflecting the photodamage repair efficacy of the test sample. Test sample: The original extract of Ganoderma lucidum after purification in step S4 and before freeze-drying in Example 1.

[0111] (2) Zebrafish rearing conditions: same as repair efficacy test.

[0112] (3) Preparation of experimental embryos: Same as the repair efficacy test.

[0113] (4) This experiment consisted of 1+1+1+3 groups, namely, blank group, model group, positive group, and sample group*3 (sample group). Wild-type zebrafish embryos of 48 hpf were collected and cultured in 6-well cell culture plates, with one well for each group. Each group consisted of 20 embryos. The blank group and model group were given 6 mL of embryo buffer, the positive group was given 6 mL of ceramide solution at a concentration of 300 μg / mL, and the sample groups I, II, and III were given 6 mL of test sample at concentrations of 2 g / L, 4 g / L, and 8 g / L, respectively. The samples were placed in a constant temperature incubator at 28℃±1℃ for 4 h before administration. Except for the blank group, the other groups were placed in a UV irradiation chamber and irradiated for 2.5 h. After treatment, the samples were stained, washed, and the fluorescence staining area of ​​each group of zebrafish was observed under an upright fluorescence microscope. The images were taken and recorded. A representative diagram of the fluorescence staining area of ​​zebrafish embryos is attached. Figure 1 The damage staining showed red fluorescence.

[0114] ImageJ image analysis software was used to count the fluorescent staining area of ​​zebrafish tails. GraphPad Prism 8 statistical analysis software was used to analyze the data, and differences between different groups were analyzed using the T-test. Generally, P < 0.05 was considered statistically significant. The statistical results of the relative fluorescent staining area of ​​zebrafish are shown in Table 4, where # represents data from the comparison between the blank group and the model group; * represents data from the comparison between the sample group and the model group.

[0115] Table 4

[0116]

[0117] Experimental conclusion: Under the experimental conditions, the test samples of different concentrations showed an inhibitory effect on light damage to zebrafish compared with the model group, indicating that the former has the effect of resisting light damage.

[0118] III. Snow Ganoderma lucidum polysaccharide extraction rate test:

[0119] Extraction rate = mass of polysaccharides in the extract / mass of Ganoderma lucidum raw material * 100%; results are shown in Table 5.

[0120] IV. Stability Testing: The snow lingzhi extracts obtained in the examples and comparative examples were subjected to heat resistance, cold resistance, high and low temperature cycling, and light stability tests.

[0121] Heat resistance test: Store at 45℃ for 30 consecutive days, and observe the appearance, color and odor of the extract every 7 days. If there is no change, the heat resistance test is considered to be qualified.

[0122] Cold resistance test: Store at -15℃ for 30 consecutive days, and observe the appearance, color and odor of the extract every 7 days. If there is no change, the cold resistance test is considered to be qualified.

[0123] High and low temperature cycling test: Store at 45℃ for 24 hours, then store at -5℃ for 24 hours. This is considered one cycle. Perform 5 consecutive cycles. If the appearance, color, and odor of the extract do not change, the high and low temperature cycling test is considered to be qualified.

[0124] Light resistance test: The extract is placed in the packaging and irradiated under a D65 UV light source (1d, 5d, 10d). If there is no change in the appearance, color and odor of the extract, the light resistance test is considered to be qualified.

[0125] Experiments show that the examples and comparative examples passed the heat resistance, cold resistance, and high and low temperature cycling tests, but differed in the light resistance test. The specific results are shown in Table 5.

[0126] Table 5

[0127]

[0128] As shown in Table 5, the extraction rates of Ganoderma lucidum polysaccharides by the methods in Examples 1-3 of this application are all relatively high, generally above 3.8%, and the Ganoderma lucidum extracts have passed the stability tests of heat resistance, cold resistance, high and low temperature cycling and light exposure.

[0129] The relatively high ultrasonic power tested in Comparative Example 1 may have caused local overheating of the internal environment, which may have broken the polysaccharide molecular chains and thus reduced the extraction rate of Ganoderma lucidum polysaccharides. Furthermore, local high temperature may have caused some components to be oxidized and deactivated, changed the molecular weight and structure of the effective components of the extract, and produced more oxidation products. The combined effect of the above factors directly led to the deterioration of the stability of the extract.

[0130] In Comparative Example 2, a longer ultrasonic time was tested. Although extending the ultrasonic time can promote the interaction between Ganoderma lucidum and the solvent and allow Ganoderma lucidum polysaccharides to dissolve fully, excessive ultrasonic treatment time can easily damage the structure of Ganoderma lucidum polysaccharides and reduce the yield of Ganoderma lucidum products. Similarly, as in Comparative Example 1, the stability of the extract also decreased.

[0131] Comparative Example 3 showed that lowering the enzymatic hydrolysis temperature significantly reduced the enzyme's reaction rate, and the yield was only half that at the optimal temperature within the same time frame.

[0132] Comparative Example 4 adjusted the solid-liquid ratio during the ultrasonic process. Ultrasonic extraction is very sensitive to changes in the solid-liquid ratio. Insufficient solvent will cause the target components to fail to dissolve or diffuse completely. Under the same extraction time, the yield is only 40% of that under optimal conditions.

[0133] Comparative Example 5 adjusted the type of compound enzyme, but the extraction rate of Ganoderma lucidum polysaccharide decreased, indicating that only a suitable compound enzyme can achieve the best dissolution effect of active ingredients.

[0134] Comparative Example 6 increased the amount of compound enzyme, but the yield of Ganoderma lucidum polysaccharide did not increase significantly, while the method cost increased significantly.

[0135] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0136] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting Ganoderma lucidum extract, characterized in that, The extraction method includes the following steps: S1. Dry and pulverize the whole Ganoderma lucidum herb; S2. Mix the snow lingzhi powder and water, heat and ultrasonically extract to obtain a mixture, and adjust the pH of the mixture to 5-6. S3. Add the compound enzyme to the mixture and hydrolyze it at a constant temperature. After the hydrolysis is complete, inactivate the enzyme and centrifuge to collect the supernatant. S4. After purifying the supernatant with macroporous resin, freeze-dry it. The purification process using macroporous resins includes the following steps: (1) Pretreatment: Immerse the macroporous resin in 95wt% ethanol for 12-48h, and then rinse the resin column with 95wt% ethanol and deionized water respectively. (2) Purification: Filter the supernatant and pass it into the resin column at a flow rate of 1-2 BV / h; (3) Elution: Deionized water and 10-20 wt% ethanol are passed into the resin column in sequence for elution, and then 50-70 wt% ethanol is passed into the column for elution. (4) Regeneration: 95wt% ethanol and deionized water are sequentially passed into the resin column for rinsing; The mixing ratio of Ganoderma lucidum powder and water in S2 is 1:40-120w / v; the ultrasonic power is 200-320W, and the ultrasonic time is 15-40min. The complex enzymes in S3 are cellulase and papain, with a mass ratio of 2-5:

1. The amount of complex enzymes added is 2-3% of the mass of the Ganoderma lucidum powder. The temperature of the constant temperature enzymatic hydrolysis process in S3 is 45-55℃, and the enzymatic hydrolysis time is 10-40 min. The macroporous resin is AB-8.

2. The Ganoderma lucidum extract prepared by the extraction method according to claim 1.

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