WO2024001714A1

WO2024001714A1 - Method for preparing high-content ganoderma triterpene extract

Info

Publication number: WO2024001714A1
Application number: PCT/CN2023/099176
Authority: WO
Inventors: 冯鹏; 周亚杰; 陈晓红; 沈建; 钱一帆
Original assignee: 南京中科药业有限公司; 中科健康产业集团股份有限公司
Priority date: 2022-06-29
Filing date: 2023-06-08
Publication date: 2024-01-04
Also published as: CN115040553A

Abstract

Disclosed is a method for preparing a high-content Ganoderma triterpene extract, comprising the following steps of: carrying out ultrasonic extraction on a dry Ganoderma powder with water 2-3 times, with 30-90 minutes each time, filtering, combining obtained filtrates, drying to obtain a Ganoderma aqueous extract solid, adding ethanol to the Ganoderma aqueous extract solid, carrying out ultrasonic extraction for 30-90 minutes, filtering, and drying an obtained filtrate at a low temperature to obtain the high-content Ganoderma triterpene extract. The present invention can save a large amount of ethanol, the extraction efficiency is high, and the obtained solid has a high content of Ganoderma triterpene.

Description

Preparation method of high-content Ganoderma lucidum triterpene extract

Technical field

The invention belongs to the field of Ganoderma lucidum processing, and specifically relates to a preparation method of a high-content Ganoderma lucidum triterpene extract.

Background technique

Ganoderma lucidum, known as "divine mushroom", "Huanyang grass" and "spiritual grass" by the ancients, has the effect of strengthening the body and strengthening the root. It is called top grade in "Shengnong's Materia Medica". Although it has no power to return to heaven, it is a top health-care product that "boils it for a hundred times and its taste is refreshing and fragrant. Drinking it can improve your eyesight, clear your brain, calm your heart, and strengthen your kidneys."

Modern scientific research shows that the main functional ingredient in Ganoderma lucidum is Ganoderma lucidum triterpenoids. It has been confirmed that hundreds of triterpenes exist in Ganoderma lucidum. Ganoderic acid is an important active ingredient of Ganoderma lucidum triterpenoids. The common one is Ganoderic acid A. , B, D, etc.

Ganoderma triterpenoids have a variety of pharmacological effects: 1) Therapeutic effect on kidney disease: Ganoderma triterpenes prevent the occurrence of renal cysts through the generation and extension of cyst tubules and the differentiation of epithelial cells. The active ingredients of Ganoderma lucidum triterpenoids are expected to become new drugs for the treatment of autosomal dominant polycystic kidney disease. 2) Anti-tumor cell effect: Studies have proven that ganoderic acid has a good anti-cancer effect. It can kill cancer cells through multiple pathways such as cytotoxicity, induction of apoptosis, and inhibition of metastasis to achieve anti-cancer purposes. 3) Anti-aging effect: Ganoderma triterpenoids have been shown to have antioxidant properties both in vitro and in vivo, reducing oxidative damage by directly scavenging free radicals generated in cells, including free radical scavengers and anti-cancer activities, and triterpenoids It also shows activity in scavenging superoxide anion radicals and hydroxyl radicals. 4) Hepatoprotective effect: Ganoderma acid extract in Ganoderma lucidum can enhance liver function, improve its detoxification effect, and can significantly reduce the increase in transaminase levels caused by carbon tetrachloride poisoning. In addition, ganoderic acid also has various pharmacological effects such as anti-inflammatory, lowering blood pressure, and lowering cholesterol. Therefore, the preparation of high-content Ganoderma lucidum triterpene extracts is of great significance for the future research and development of new drugs and other aspects.

At present, Ganoderma triterpenes are generally extracted directly with organic solvents, which consumes a large amount of organic solvents, and the content of Ganoderma triterpenes in the obtained extracts is low.

Contents of the invention

The object of the present invention is to overcome the above shortcomings and provide a preparation method for a high-content Ganoderma lucidum triterpene extract. The method is divided into two steps of extraction. In the first step, Ganoderma lucidum dry powder is extracted with water two to three times, and in the second step, Ganoderma lucidum dry powder is extracted with water. On the basis of the extract, the Ganoderma lucidum extract is extracted again with alcohol, and the obtained extract has high triterpenoid content and consumes less ethanol.

The purpose of the present invention is specifically achieved in the following ways:

A method for preparing a high-content Ganoderma lucidum triterpene extract. The method includes extracting Ganoderma lucidum dry powder by ultrasonic water for 2 to 3 times, each extraction time is 30 to 90 minutes, filtering, combining the obtained filtrate, and drying to obtain Ganoderma lucidum water. To extract the solid matter, add ethanol to the water-extracted solid matter of Ganoderma lucidum, conduct ultrasonic extraction, extract for 30 to 90 minutes, filter, and dry the obtained filtrate at low temperature to obtain it.

Preferably, water ultrasonic extraction is used twice. After the first extraction using water ultrasonic, it is filtered to obtain a first filtrate and a first filter residue; wherein, the amount of pure water added is: dry Ganoderma lucidum powder: volume of pure water = 1:10 to 1:30 (g/mL) , the ultrasonic power is 550~650W, the frequency is 30~50kHz, and the extraction time is 30~90min. The second time uses water ultrasonic extraction and filtration to obtain the second extraction filtrate and the second extraction filter residue; the amount of pure water added is: weight of the first extraction filter residue: pure water volume = 1:5 ~ 1:20 (g/mL), The ultrasonic power is 550~650W, the frequency is 30~50kHz, and the time is 30~90min.

The temperature used in the above-mentioned low-temperature drying is lower than 60°C.

The amount of ethanol added above is: weight of Ganoderma lucidum water-extracted solids: volume of ethanol = 1:10~1:30 (g/mL). The preferred ethanol concentration is 80% to 100%. Add ethanol for ultrasonic extraction, the ultrasonic power is 550-650W, and the frequency is 30-50kHz.

Compared with the prior art, the beneficial effects of the present invention are: the present invention performs alcohol extraction on the basis of 2-3 times of water extraction under specific conditions. The obtained extract has high triterpenoid content in Ganoderma lucidum and consumes less total ethanol. By adopting the ultrasonic extraction method, the extraction efficiency is significantly improved. The method of the present invention is particularly suitable for industrial production, significantly saves energy and reduces consumption, and improves product competitiveness.

Description of drawings

Figure 1 shows the chromatogram of the reference substance.

Figure 2 is the chromatogram of the high-content triterpene extract of Example 1

Figure 3 shows the chromatogram of the alcohol extract obtained by direct alcohol extraction.

Figure 4 is a chromatogram of the aqueous extract of Example 1

In Figure 1 to Figure 4, peaks a, b, c, d, e, f, g, h are: ganoderic acid G, ganoderic acid B, ganoderic acid E, ganoderic acid A, ganoderic acid A, ganoderic acid D , Ganoderma acid D, Ganoderma acid F.

Detailed ways

Specific implementations of the present invention will be described in further detail below with reference to examples.

Example 1

(1) Take 5kg Ganoderma lucidum fruit bodies and crush them to obtain 4.95kg Ganoderma lucidum dry powder.

(2) Take 50g of the above Ganoderma lucidum dry powder, add 750mL of pure water, conduct ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain a filtrate and a filter residue.

(3) Take the first filter residue and add 10 times (g/mL) water, conduct ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the second filter residue and second filter residue.

(4) Combine the two extraction filtrates and dry at 50°C to obtain Ganoderma lucidum water-extracted solids.

(5) Take the water-extracted solids of Ganoderma lucidum, add 15 times absolute ethanol (g/mL), perform ultrasonic extraction (600W, 40kHz, 30min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids, which is High content of Ganoderma triterpene extract.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 1.

Table 1 Extract weight and triterpene content of Ganoderma lucidum

Example 2

(1) Take 60g of Ganoderma lucidum dry powder from the same batch in Example 1, add 1200ml of pure water, conduct ultrasonic extraction (600W, 40kHz, 50min), and filter to obtain a filtrate and a filter residue.

(2) Take the first filter residue and add 8 times (g/mL) water, perform ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the second filter residue and second filter residue.

(3) Combine the two extraction filtrates and dry at 50°C to obtain water-extracted solids.

(4) Take the water-extracted solids, add 20 times (g/mL) absolute ethanol, perform ultrasonic extraction (600W, 40kHz, 40min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids, which is high content Ganoderma lucidum triterpene extract.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 2.

Table 2 Extract weight and triterpene content of Ganoderma lucidum

Example 3

(1) Take 100g of Ganoderma lucidum dry powder from the same batch in Example 1, add 2500ml of pure water, conduct ultrasonic extraction (600W, 40kHz, 60min), and filter to obtain a filtrate and a filter residue.

(2) Take the first filter residue and add 15 times (g/mL) water, perform ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the second filter residue and second filter liquid.

(4) Take the water-extracted solids and add 20 times (g/mL) absolute ethanol, perform ultrasonic extraction (600W, 40kHz, 60min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids, which is high content Ganoderma lucidum triterpene extract.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 3.

Table 3 Extract weight and triterpene content of Ganoderma lucidum

Comparative example 1

(1) Take 50g of Ganoderma dry powder from the same batch in Example 1, add 750ml of pure water, conduct ultrasonic extraction (600W, 40kHz, 60min), filter, and dry the supernatant at 50°C to obtain water-extracted solids.

(4) Take the water-extracted solids, add 15 times (g/mL) absolute ethanol, perform ultrasonic extraction (600W, 40kHz, 30min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids, which is high content Ganoderma lucidum triterpene extract.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 4.

Table 4 Extract weight and triterpene content of Ganoderma lucidum

Comparative example 2

(1) Take 50g of Ganoderma lucidum dry powder from the same batch as in Example 1, add 750ml of pure water, conduct ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain a filtrate and a filter residue.

(2) Take the first filter residue and add 10 times (g/mL) water, perform ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the second filter filtrate and second filter residue.

(3) Take the second extraction filter residue and add 10 times (g/mL) water, conduct ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the third extraction filtrate and third extraction filter residue.

(4) Combine the three extraction filtrates and dry at 50°C to obtain water-extracted solids.

(5) Take the water-extracted solids, add 15 times (g/mL) absolute ethanol, conduct ultrasonic extraction (600W, 40kHz, 30min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 5.

Table 5 Extract weight and triterpene content of Ganoderma lucidum

Comparative example 3

(1) Take 100g of Ganoderma lucidum dry powder from the same batch in Example 1, add 2500ml of absolute ethanol, conduct ultrasonic extraction (600W, 40kHz, 60min), and filter to obtain a filtrate and a filter residue.

(2) Take the first extraction filter residue, add 15 times (g/mL) absolute ethanol, conduct ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the second extraction filtrate and second extraction filter residue.

(3) Combine the two extraction filtrates and dry at 50°C to obtain alcohol-extracted solids.

The weight of alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 6.

Table 6 Extract weight and triterpene content of Ganoderma lucidum

Comparative example 4

(1) Take 100g of Ganoderma lucidum dry powder from the same batch in Example 1, add 500ml of pure water, conduct ultrasonic extraction (600W, 40kHz, 60min), and filter to obtain a filtrate and a filter residue.

(2) Take the first filter residue and add 2 times (g/mL) water, perform ultrasonic extraction (600W, 40kHz, 30min), and filter to obtain the second filter residue and second filter residue.

(4) Take the water-extracted solids, add 5 times (g/mL) absolute ethanol, perform ultrasonic extraction (600W, 40kHz, 60min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids, which is high content Ganoderma lucidum triterpene extract.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 7.

Table 7 Extract weight and triterpene content of Ganoderma lucidum

Comparative example 5

(4) Extract the solid matter with water, add 20 times (g/mL) absolute ethanol, conduct ultrasonic extraction (600W, 40kHz, 60min), and filter to obtain a filtrate and a filter residue.

(5) Take the first-extraction filter residue of step (4), add 15 times (g/mL) absolute ethanol, conduct ultrasonic extraction (600W, 40kHz, 60min), and filter to obtain the second-extraction filtrate and second-extraction filter residue.

(6) Combine the two alcohol extraction filtrates and dry them at 50°C to obtain alcohol extraction solids, which are high-content Ganoderma lucidum triterpene extracts.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 8.

Table 8 Extract weight and triterpene content of Ganoderma lucidum

Comparative example 6

(4) Take the water-extracted solids, add 20 times (g/mL) mass concentration of 60% ethanol, conduct ultrasonic extraction (600W, 40kHz, 60min), filter, and dry the supernatant at 50°C to obtain the alcohol-extracted solids, that is It is a high-content Ganoderma triterpene extract.

The weight of water-extracted solids and alcohol-extracted solids and the content of Ganoderma lucidum triterpenes are shown in Table 9.

Table 9 Extract weight and triterpene content of Ganoderma lucidum

Comparing Comparative Example 1 and Comparative Example 2 with Example 1, it can be seen that the number of water extractions has a significant impact on the solid yield. The weight of the solids obtained by two water extractions is 50% higher than the solid weight obtained by one water extraction. The final impact on The yield of solids extracted by alcohol, while the weight of solids obtained by three water extractions is only 10-15% higher than the weight of solids obtained by two water extractions. Therefore, considering the solids yield and the time and energy consumption of extraction, etc., Water extraction twice is the best process. From the comparison between Comparative Example 3 and Example 3, it can be seen that the direct alcohol extraction not only uses a large amount of ethanol, but also the content of Ganoderma lucidum triterpenes in the obtained alcohol-extracted solids is low. It is 5-6 times that of direct alcohol extraction. In addition, comparing Comparative Example 4 with Example 3, it can be seen that the amount of extraction solution also significantly affects the solid yield, and insufficient amount of extraction solution will cause insufficient extraction. From the comparison between Comparative Example 5 and Example 3, it can be seen that two alcohol extractions have almost no impact on the weight of the high-content Ganoderma lucidum triterpene extract finally obtained. Therefore, in order to save time and ethanol consumption, one alcohol extraction is sufficient. From the comparison between Comparative Example 6 and Example 3, it can be seen that the ethanol concentration has a certain impact on the yield and content of alcohol-extracted solids. If the ethanol concentration is too low, the alcohol-extracted solid yield will be significantly reduced, and the content of Ganoderma lucidum triterpenes will also decrease. reduced. In summary, the method of the present invention can fully extract the triterpenoid components in Ganoderma lucidum and obtain a high-content Ganoderma lucidum triterpenoid extract on the basis of saving a large amount of ethanol consumption.

The detection method of Ganoderma lucidum triterpenes is as follows (external standard one-point method):

1.1 Sample processing

Precisely weigh 100mg of the sample, place it in a 50mL volumetric flask, add 25ml of absolute ethanol accurately, shake well, ultrasonic for 10min (600W, 40kHz), cool to room temperature, use the starting ratio of mobile phase (0.05% formic acid aqueous solution: acetonitrile = 80 :20) Dilute to the mark, shake well, filter with 0.45μm organic filter membrane, take the remaining filtrate as the test solution.

1.2 Test conditions

Mobile phase: Phase A is 0.05% formic acid aqueous solution: acetonitrile (80:20); phase B is acetonitrile.

Column: Thermo C18 (100*2.1mm, 2.4μm) (S/N: 1005142X7)

Detection wavelength: 257nm

Flow rate: 0.4mL/min

Column temperature: 25℃

Injection volume: 10μL

gradient:

1.3 Preparation of reference substance

A total of 8 kinds of ganoderma acids were selected for the experiment, and the solvents of absolute ethanol: phase A = 1:1 were used to prepare corresponding concentrations. The actual concentrations are: Ganoderic acid G 5.39ug/mL, Ganoderic acid B 4.998ug/mL, Ganoderic acid E 20.776ug/mL, Ganoderic acid A 19.208ug/mL, Ganoderic acid A 5.096ug/mL, Ganoderic acid D 9.702 ug/mL, ganoderic acid D 9.702ug/mL, ganoderic acid F 20.188ug/mL.

1.4 Calculation method

In the formula: X--------The content of Ganoderma lucidum triterpenes in the sample, %;

S ₁ -------The corresponding peak area of the sample;

S ₂ -------The corresponding peak area of the reference substance;

ρ-------corresponding reference substance concentration, ug/mL;

V------sample constant volume, mL;

m-------The mass of the sample, g.

Claims

A method for preparing a high-content Ganoderma lucidum triterpene extract, which is characterized in that the method includes extracting Ganoderma lucidum dry powder by ultrasonic water for 2 to 3 times, each extraction time is 30 to 90 minutes, filtering, and combining the obtained filtrate and drying to obtain To obtain Ganoderma lucidum water-extracted solids, add ethanol to the Ganoderma lucidum water-extracted solids, conduct ultrasonic extraction, extract for 30 to 90 minutes, filter, and dry the obtained filtrate at low temperature.
The preparation method of high-content Ganoderma lucidum triterpene extract according to claim 1, characterized in that the water ultrasonic extraction is used twice.
The preparation method of high-content Ganoderma lucidum triterpene extract according to claim 2, characterized in that, after the first ultrasonic extraction with water, it is filtered to obtain a first filtrate and a first filter residue; wherein, pure water is added The amount is: Ganoderma lucidum dry powder: pure water volume = 1:10~1:30g/mL, ultrasonic power is 550~650W, frequency is 30~50kHz, and extraction time is 30~90min.
The preparation method of high-content Ganoderma lucidum triterpene extract according to claim 3, characterized in that the second time uses water ultrasonic extraction and filtration to obtain the second extraction filtrate and the second extraction filter residue; the amount of pure water added is: The weight of the first filter residue: pure water volume = 1:5~1:20g/mL, ultrasonic power is 550~650W, frequency is 30~50kHz, and time is 30~90min.
The method for preparing high-content Ganoderma lucidum triterpene extract according to claim 1, characterized in that the temperature used for the low-temperature drying is lower than 60°C.
The method for preparing high-content Ganoderma lucidum triterpene extract according to claim 1, characterized in that the amount of ethanol added is: weight of Ganoderma lucidum water-extracted solids: ethanol volume = 1:10 to 1:30g/mL.
The method for preparing high-content Ganoderma lucidum triterpene extract according to claim 1, wherein the ethanol mass concentration is 80% to 100%.
The preparation method of high-content Ganoderma lucidum triterpene extract according to claim 1, characterized in that ethanol is added for ultrasonic extraction, the ultrasonic power is 550-650W, and the frequency is 30-50kHz.