Process for enriching and refining ganoderic acid in ganoderma lucidum sporocarp

Abstract

The invention discloses a process for enriching and refining ganoderic acid in ganoderma lucidum sporocarp, which comprises the following steps of: (1) crushing and sieving the ganoderma lucidum sporocarp, enriching ganoderic acid by adopting a Soxhlet extraction method, centrifuging and freeze-drying to obtain a crude ganoderic acid extract; (2) adding an extracting agent into the ganoderic acid crude extract for extraction, concentrating the obtained extracting solution by using a rotary evaporator, and freeze-drying to obtain a ganoderic acid crude product; and (3) adding the ganoderic acid crude product obtained in the step (2) into ethanol for anti-solvent crystallization, and freeze-drying the obtained solid to obtain a ganoderic acid finished product. The purity of the extracted ganoderic acid is high (the liquid phase purity is gt, 98%), the conversion rate can reach 90% or above, the commodity value is improved, anti-solvent crystallization is used, the extracted ganoderic acid product is white crystals, the ultraviolet transmittance is increased to 91% from 20%, the ganoderic acid product can be stably stored in wine, and the appearance vision of a wine solution is not affected.

Description

Technical Field

[0001] The invention belongs to the field of active component extraction technology, and specifically relates to an enrichment, refining and decolorization process of ganoderic acid in ganoderma lucidum fruiting bodies. Background Art

[0002] Ganoderma lucidum is rich in a variety of active ingredients, each with different pharmacological effects. Triterpenes are one of the main active ingredients in Ganoderma lucidum. Ganoderic acid is a triterpenoid substance, and more than 100 species have been isolated, such as ganoderic acid A, B, C, D, E, F, G, I, L, MA, MD, MG, etc. Most ganoderic acids are tetracyclic triterpenes, which are highly oxidized lanostanes.

[0003] Ganoderic acid can inhibit the release of cell histamine, enhance the functions of various organs of the digestive system, and also has the effects of lowering blood lipids, lowering blood pressure, protecting the liver, and regulating. Among them, Ganoderic acid A is the main component of liver protection, and Ganoderic acid D is the main component of anti-cancer drugs. Ganoderic acid has the above-mentioned positive effects, so the content of Ganoderic acid has become one of the important indicators of Ganoderma lucidum and its products. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a process for enriching and refining ganoderic acid in ganoderma lucidum fruiting bodies in view of the deficiencies in the prior art.

[0005] In order to solve the above technical problems, the present invention discloses a process for enriching and refining ganoderic acid in ganoderma lucidum fruiting bodies, comprising the following steps:

[0006] (1) crushing and sieving the ganoderma lucidum fruiting body, enriching ganoderic acid by Soxhlet extraction with an organic solvent, centrifuging and freeze-drying to obtain a crude ganoderic acid extract;

[0007] (2) adding an extractant to the crude extract of Ganoderma lucidum for extraction, and then concentrating the obtained extract using a rotary evaporator and freeze-drying it to obtain a crude ganoderic acid extract;

[0008] (3) adding the crude ganoderic acid extract obtained in step (2) into ethanol for anti-solvent crystallization, and freeze-drying the obtained solid to obtain a ganoderic acid finished product.

[0009] Preferably, in step (1), the crushed Ganoderma lucidum fruiting bodies are passed through a 60-mesh sieve.

[0010] The centrifugal speed of step (1) is 8000-10000 rpm / min, and the time is 5-10 min. Preferably, the centrifugal speed is 10000 rpm / min, and the time is 5-10 min.

[0011] In step (1), the organic solvent used in the Soxhlet extraction method is any one of ethyl acetate, ethanol and methanol; the extraction temperature of the Soxhlet extraction method is 85-100° C., and the extraction time is 3h-8h.

[0012] In step (2), the extractant is ethanol with a volume ratio of 70% to 90%.

[0013] In step (2), the mass ratio of the extractant to the crude extract of Ganoderma lucidum is 1:50-1:300, the extraction time is 2-7 hours, and the extraction temperature is 25-40°C.

[0014] In step (2), the extraction is any one of ultrasonic extraction, Soxhlet extraction and thermal reflux extraction.

[0015] In step (3), each gram of ganoderic acid is added into 50 mL to 200 mL of ethanol having a volume ratio of 70% to 90%, preferably each gram of ganoderic acid is added into 50 mL to 200 mL of ethanol having a volume ratio of 70%.

[0016] In step (3), the anti-solvent of the anti-solvent crystallization is water.

[0017] Preferably, the rotation speed during the anti-solvent crystallization process is 100-300 rpm / min, the temperature is 30-40° C., and the flow rate is 1 mL / min-0.5 mL / min.

[0018] The centrifugal speed of step (1) is 10000 rpm / min, and the time is 5 to 10 min.

[0019] The ganoderic acid product prepared by the above preparation method is also within the protection scope of this application.

[0020] The present invention further proposes the application of the ganoderma lucidum product in the preparation of health wine.

[0021] The ganoderic acid product prepared by the present invention can be stably stored in 52° or 42° wine.

[0022] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0023] (1) After the experiment is scaled up, the extraction effect is still good and the product purity can be maintained.

[0024] (2) Using anti-solvent crystallization, the extracted ganoderic acid product is white crystals, and the UV transmittance is increased from 20% to 91%, which does not affect the visual appearance of the wine solution;

[0025] (3) The extracted ganoderic acid has a high purity (liquid phase purity>98%), and the conversion rate can reach more than 90%, which improves the commodity value;

[0026] (4) The extracted ganoderic acid has good stability when added to wine and can bring considerable social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the HPLC spectrum after ultrasonic extraction in Example 1;

[0028] Figure 2 The HPLC after Soxhlet extraction in Example 1;

[0029] Figure 3 This is the HPLC spectrum after methanol extraction in Example 2;

[0030] Figure 4 The HPLC after 6 hours of 70% ethanol extraction in Example 2;

[0031] Figure 5 The HPLC after 70% ethanol extraction for 3 hours in Example 3;

[0032] Figure 6 This is the HPLC spectrum of Example 4 with a solid-liquid ratio of 1:200 after extraction for 3 hours;

[0033] Figure 7 This is the color comparison of the product before and after anti-solvent crystallization;

[0034] Figure 8 The stability of ganoderic acid crystals in 52° wine at different solid-liquid ratios. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0036] The ganoderma lucidum fruiting bodies used in the following examples were purchased from Jiangsu Hongshou Bioengineering Co., Ltd. The purity of ganoderma lucidum was detected by high performance liquid chromatography (Agilent), the polysaccharide content was detected by sulfuric acid-phenol method (ultraviolet liquid chromatography, Japan Shimadzu UV2600), the total triterpene content was detected by vanillin-perchloric acid method (ultraviolet liquid chromatography, Japan Shimadzu UV2600), and the concentration was concentrated by rotary evaporator (Buchi Laboratory Equipment Trading (Shanghai) Co., Ltd.). The centrifugal conditions were a speed of 10000rmp / min and a time of 10min. The polysaccharide content in the sample ganoderma lucidum powder was 45%, the total triterpene content was 9.2%, the ganoderma lucidum A content was 1.3%, and the ganoderma lucidum D content was 1.1%. The calculation formula for each substance is as follows:

[0037]

[0038] Embodiment 1:

[0039] The fruiting bodies of Ganoderma lucidum were crushed, passed through a 60-mesh sieve, and ganoderic acid was enriched with ethyl acetate. Specifically, 2 g of the fruiting bodies were dissolved in 100 mL of ethyl acetate and extracted by Soxhlet extraction at 100°C for 6 hours, centrifuged, and freeze-dried. The centrifugation conditions were a speed of 10,000 rpm and a time of 10 minutes to obtain a crude extract of ganoderic acid. 2 g of the crude extract of ganoderic acid was added to 200 mL of methanol, and ultrasonic extraction or Soxhlet extraction (ultrasonic frequency of 220 W, temperature of 100°C; Soxhlet extraction temperature of 100°C) was performed for 6 hours, centrifuged, and the supernatant was taken for detection of ganoderic acid purity, polysaccharide content, and total triterpene content.

[0040] Experimental results:

[0041] The HPLC spectrum after ultrasonic extraction is shown in Figure 1 , HPLC after Soxhlet extraction Figure 2 The effects of Soxhlet extraction and ultrasonic extraction on various evaluation indicators are shown in Table 1.

[0042] Table 1 Effects of different extraction methods on the extraction of ganoderic acid

[0043]

[0044]

[0045] As shown in Table 1, Soxhlet extraction has a better extraction effect on ganoderic acid A and D than ultrasonic extraction. The extraction amount of ganoderic acid A and D reached 0.668% and 0.314% respectively. At the same time, the sugar content decreased significantly, and the extraction was more complete. Figure 1 It can be seen that there is a large impurity peak at 6 minutes with more impurities, 15-30 minutes are the peaks of various ganoderic acids, and there are also some impurity peaks between 50-60 minutes. After Soxhlet extraction, the intensity of the impurity peaks at 50-60 minutes has been significantly weakened, and some impurity peaks have even disappeared, which improves the purity of ganoderic acid. The purity of ganoderic acid A+D extracted by ultrasound is 28.58%, and the purity of total triterpenes is 57.17%; the purity of ganoderic acid A+D extracted by Soxhlet is 31.38%, and the purity of total triterpenes is 74.89%.

[0046] Embodiment 2:

[0047] The fruiting bodies of Ganoderma lucidum were crushed, passed through a 60-mesh sieve, and ganoderic acid was enriched with ethyl acetate. Specifically, 2 g of the fruiting bodies were dissolved in 100 mL of ethyl acetate and extracted at 100°C for 6 hours using the Soxhlet extraction method, centrifuged, and freeze-dried to obtain a crude extract of ganoderic acid. 2 g of the crude extract of ganoderic acid was added to 200 mL of 70% ethanol or methanol, and Soxhlet extraction was performed for 6 hours at an extraction temperature of 100°C. The supernatant of the extract was taken for ganoderic acid content and polysaccharide content, and then the extract was concentrated and freeze-dried.

[0048] Experimental results:

[0049] The HPLC spectrum after methanol extraction is shown in Figure 2 , HPLC after 70% ethanol extraction Figure 3 The effects of different extraction solvents on various evaluation indicators are shown in Table 2.

[0050] Table 2 Extraction effects of different extraction solvents

[0051] type Methanol 70% ethanol Ganoderic acid A content (%) 0.668 0.82 Ganoderic acid D content (%) 0.314 0.44 Total triterpenoid content (%) 6.89 8.7 Polysaccharide content (%) 1.11 0.87

[0052] As shown in Table 2, 70% ethanol has a better effect in extracting total triterpenoids of ganoderic acid, the contents of ganoderic acid A and D are increased, and the sugar content can be reduced to below 1%. Figure 3 It can be seen that the impurity peak at 50-60min disappeared and the purity was improved. The purity of ganoderic acid A+D extracted by methanol was 31.38%, and the purity of total triterpenes was 74.81%; the purity of ganoderic acid A+D extracted by 70% ethanol was 46.28%, and the purity of total triterpenes was 94.57%.

[0053] Example 3

[0054] The fruiting bodies of Ganoderma lucidum were crushed, passed through a 60-mesh sieve, and ganoderic acid was enriched with ethyl acetate. Specifically, 2 g of the fruiting bodies were dissolved in 100 mL of ethyl acetate and extracted at 100° C. for 6 h using the Soxhlet extraction method, centrifuged, and freeze-dried to obtain a crude ganoderic acid extract. 2 g of the crude ganoderic acid extract was added to 200 mL of 70% ethanol, and Soxhlet extraction was performed for 3 h and 6 h at a temperature of 100° C. The supernatant of the extract was taken for ganoderic acid content, ganoderic acid purity, and polysaccharide content, and then the extract was concentrated and freeze-dried.

[0055] Experimental results:

[0056] The HPLC spectrum after 6h extraction with 70% ethanol is shown in Figure 3 , HPLC analysis after 70% ethanol extraction for 3 h Figure 4 ,The influence of extraction time on various evaluation indicators is shown in Table 3.

[0057] Table 3 Effect of extraction time on solvent extraction effect

[0058] type Extraction 6h Extraction 3h Ganoderic acid A content (%) 0.82 1.27 Ganoderic acid D content (%) 0.44 1.07 Total triterpenoid content (%) 8.7 9.1 Polysaccharide content (%) 0.87 0.71

[0059] As can be seen from Table 3, when the extraction duration is 3 h, while the sugar content remains below 1%, the contents of ganoderic acids A and D increase to 1.27% and 1.07% respectively. From Figure 4 it can be seen that when extracting for 3 h, the intensity of impurity peaks between 0 min and 10 min weakens, and almost all the impurity peaks after 30 min disappear. When extracting for 6 h, the purity of ganoderic acids A + D is 46.28%, and the purity of total triterpenoids is 94.57%; when extracting for 3 h, the purity of ganoderic acids A + D is 62.69%, and the purity of total triterpenoids is 98.91%.

[0060] Example 4:

[0061] The Ganoderma lucidum fruiting body was crushed, passed through a 60-mesh sieve, and ganoderic acids were enriched with ethyl acetate. Specifically, 2 g of the fruiting body was dissolved in 100 mL of ethyl acetate and extracted for 6 h at 100 °C using Soxhlet extraction, centrifuged, and freeze-dried to obtain a crude extract of ganoderic acids. 1 g, 2 g, and 4 g of the crude extract of ganoderic acids were respectively added to 200 mL of 70% ethanol, and Soxhlet extraction was carried out for 3 h at a temperature of 100 °C. The supernatant of the extract was taken for the determination of the content of ganoderic acids and the content of polysaccharides, and then the extract was concentrated and freeze-dried.

[0062] Experimental results:

[0063] The high-performance liquid chromatography (HPLC) chromatogram of extraction for 3 h with a solid-liquid ratio of 1:100 is as Figure 4 shown, the HPLC chromatogram of extraction for 3 h with a solid-liquid ratio of 1:50 is as Figure 5 shown, the HPLC chromatogram of extraction for 3 h with a solid-liquid ratio of 1:200 is as Figure 6 shown, and the effects of different solid-liquid ratios on various evaluation indexes are shown in Table 4.

[0064] Table 4 Effects of different solid-liquid ratios on the extraction agent

[0065] type Solid-liquid ratio 1:50 Solid-liquid ratio 1:100 Solid-liquid ratio 1:200 Ganoderic acid A content (%) 0.87 1.27 0.88 Ganoderic acid D content (%) 0.87 1.07 0.58 Total triterpenoid content (%) 7.75 9.1 7.5 Polysaccharide content (%) 0.89 0.71 1.44

[0066] From Table 4 and Figure 4 、 Figure 5 、 Figure 6 it can be seen that after extraction with different solid-liquid ratios, the purities are all relatively high. However, when the solid-liquid ratio is 1:100 (2 g of the crude extract of ganoderic acids and 200 mL of 70% ethanol), the contents of ganoderic acids A and D are higher. When the solid-liquid ratio is 1:50, the purity of ganoderic acids A + D is 54.92%, and the purity of total triterpenoids is 84.24%; when the solid-liquid ratio is 1:100, the purity of ganoderic acids A + D is 62.69%, and the purity of total triterpenoids is 98.91%; when the solid-liquid ratio is 1:200, the purity of ganoderic acids A + D is 52.32%, and the purity of total triterpenoids is 81.52%.

[0067] Embodiment 5:

[0068] The fruiting bodies of Ganoderma lucidum were crushed, passed through a 60-mesh sieve, and ganoderic acid was enriched with ethyl acetate. Specifically, 2 g of the fruiting bodies were dissolved in 100 mL of ethyl acetate and extracted at 100 ° C for 6 h using the Soxhlet extraction method, centrifuged, and freeze-dried to obtain a crude extract of ganoderic acid. 2 g of the crude extract of ganoderic acid was added to 200 mL of 70% ethanol, and Soxhlet extraction was performed for 3 h at a temperature of 100 ° C. The supernatant of the extract was taken for ganoderic acid content, ganoderic acid purity, and polysaccharide content. The extract was then concentrated, freeze-dried, and finally crystallized with water as an antisolvent at a speed of 200 rmp / min, a temperature of 25 ° C, and a flow rate of 0.3 mL / min. 0.1 g of the crystals before antisolvent crystallization and the crystals after antisolvent crystallization were added to 10 mL of 70% ethanol, and the transmittance was detected at 430 nm using an ultraviolet spectrophotometer.

[0069] Experimental results:

[0070] Figure 7 The color comparison of the product before and after anti-solvent crystallization. The UV results show that the UV transmittance increased from 20% to 91%. Figure 7 It can be seen that after anti-solvent crystallization, the color changes significantly, which shows that the anti-solvent crystallization process is conducive to decolorization. At the same time, after anti-solvent crystallization, the purity of ganoderic acid A+D is 62.32%, and the purity of total triterpenes is 98.43%.

[0071] Embodiment 6:

[0072] The ganoderma lucidum fruiting body was crushed, passed through a 60-mesh sieve, and ethyl acetate was used to enrich the ganoderma lucidum acid. Specifically, 2g of the fruiting body was dissolved in 100mL of ethyl acetate and extracted at 100°C for 6h using the Soxhlet extraction method, centrifuged, and freeze-dried to obtain a crude extract of ganoderma lucidum acid. 40g of the crude extract of ganoderma lucidum acid was added to 4000mL of 70% ethanol, and Soxhlet extraction was performed for 3h. The supernatant of the extract was taken for ganoderma lucidum acid content, ganoderma lucidum acid purity, and polysaccharide content, and then the extract was concentrated and freeze-dried. Finally, the anti-solvent was crystallized with water (the specific conditions were the same as in Example 5), and the transmittance was tested at a maximum absorption wavelength of 430nm using an ultraviolet spectrophotometer, and the crystal was dissolved in 70% ethanol to detect the product transmittance. Repeat the above steps and make ten batches. Calculate the conversion rate of each batch, the calculation formula is as follows, and the results are shown in Table 5.

[0073]

[0074] Experimental results:

[0075] Table 5 Conversion rate of different batches

[0076] batch Conversion rate (%) First batch 95.9 Second batch 96.6 The third batch 98.7 The fourth batch 99.1 The fifth batch 98.2 The sixth batch 98.1 The seventh batch 97.1 The eighth batch 97.3 Ninth batch 97.6 The tenth batch 98.2 average value 97.68

[0077] As can be seen from Table 5, the conversion rate of each batch exceeds 95%, and the average value has reached 97.68%, which has a good commercial prospect.

[0078] Example 7

[0079] 300 mg, 500 mg and 1000 mg of the high-purity ganoderic acid (crystal) product prepared in Example 6 were respectively put into 50 mL of 52° wine to observe its stability.

[0080] Experimental results:

[0081] Stability Figure 8 .pass Figure 8 It can be seen that the Ganoderma lucidic acid after secondary purification has good stability in 52° wine at different solid-liquid ratios.

[0082] The present invention provides a thought and method for the enrichment and purification of ganoderic acid in ganoderma lucidum fruiting bodies. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. A process for enriching and refining ganoderic acid from ganoderma lucidum fruiting bodies, characterized in that: The steps include: (1) crushing and sieving the fruiting bodies of Ganoderma lucidum, enriching ganoderic acid by Soxhlet extraction, centrifuging and freeze-drying to obtain a crude ganoderic acid extract; (2) adding an extractant to the crude ganoderic acid extract for extraction, concentrating the obtained extract using a rotary evaporator and then freeze-drying it to obtain a crude ganoderic acid product; (3) The crude ganoderic acid product obtained in step (2) is added to ethanol for anti-solvent crystallization, and the obtained solid is freeze-dried to obtain a ganoderic acid product.

2. The process according to claim 1, characterized in that In step (1), the crushed Ganoderma lucidum fruiting bodies are passed through a 60-mesh sieve.

3. The process according to claim 1, characterized in that The centrifugal speed of step (1) is 8000-10000 rpm / min, and the time is 5-10 min.

4. The process according to claim 1, characterized in that In step (1), the organic solvent used in the Soxhlet extraction method is any one of ethyl acetate, ethanol and methanol; the extraction temperature of the Soxhlet extraction method is 85-100° C., and the extraction time is 3h-8h.

5. The process according to claim 1, characterized in that In step (2), the extractant is 70% to 90% ethanol by volume; the mass ratio of the extractant to the crude extract of Ganoderma lucidum is 1:50 to 1:300, the extraction time is 2 to 7 hours, and the extraction temperature is 25 to 40°C.

6. The process according to claim 1, characterized in that In step (2), the extraction is any one of ultrasonic extraction, Soxhlet extraction and thermal reflux extraction.

7. The process according to claim 1, characterized in that In step (3), each gram of ganoderic acid is added into 50 mL to 200 mL of ethanol having a volume ratio of 70% to 90%.

8. The process according to claim 1, characterized in that In step (3), the anti-solvent of the anti-solvent crystallization is water; the rotation speed during the anti-solvent crystallization process is 100-300 rpm / min, the temperature is 30-40°C; and the flow rate is 1 mL / min-0.5 mL / min.

9. The ganoderic acid product prepared by the process according to any one of claims 1 to 8.

10. Use of the ganoderma product according to claim 9 in preparing health wine.

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