A method for the combined extraction of cyperine and trehalose from Selaginella tamariscina

Abstract

This invention discloses a method for the combined extraction of cyperine and trehalose from Selaginella tamariscina. Using Selaginella tamariscina as raw material, this method combines the extraction of cyperine and high-purity trehalose, improving the purity of the final cyperine product. The trehalose yield is between 0.2% and 2.0%. Through solvent extraction, water washing purification, and crystallization, a pure trehalose product with a purity greater than 95.0% can be prepared. The process is environmentally friendly, generates no solid waste, and utilizes the widely available resources of Selaginella tamariscina, providing a cheap and readily available source for the preparation of trehalose-related drugs. This method is of significant importance for the comprehensive utilization and in-depth development of Selaginella tamariscina.

Description

Technical Field

[0001] This invention relates to a process for extracting cyperus flavonoids and trehalose from Selaginella tamariscina. Background Technology

[0002] Trehalose is a non-reducing disaccharide, also known as trehalose or mushroom sugar. It plays a role in stabilizing biological macromolecules, protecting the molecular structure of organisms in harsh environments from damage, and maintaining the biological characteristics of living organisms. Since its discovery by scientists in 1832, trehalose has attracted many scholars to conduct research and exploration due to its unique biological functions and mechanisms of action.

[0003] Currently, the main methods for preparing trehalose are as follows:

[0004] (1) Microbial extraction method: This is the traditional method of trehalose production, which uses lactic acid bacteria, yeast, mold and other trehalose-containing bacteria as the extraction source.

[0005] (2) Fermentation method: This method involves culturing microorganisms under specific conditions (pH, temperature, osmotic pressure, etc.) on a certain substrate, producing trehalose through biological fermentation, which is then extracted and refined from the culture medium. The disadvantages of this method are that the microorganisms have stringent requirements for fermentation conditions, the conversion rate is low, the fermentation broth has a complex composition, and the extraction and purification of trehalose are difficult.

[0006] (3) Chemical Synthesis: The chemical synthesis of trehalose mainly involves an ethylene oxide addition reaction between 2,3,4,6-tetraacetyl glucose and 3,4,6-triacetyl-1,2-dehydro-D-glucose. This method uses large amounts of organic solvents and suffers from complex synthetic routes, low yields, high synthesis difficulty, and separation challenges. Therefore, it is currently still in the research stage and has not yet been industrialized.

[0007] (4) Enzymatic Conversion Method: Glucose, sucrose, maltose, or starch can be used as substrates and converted into trehalose through the action of relevant enzymes. This method is the most widely used trehalose preparation method in the prior art. For example, patent CN202210362640.1 provides a trehalose production method based on immobilized trehalose synthase technology. Although the enzymatic conversion method has the characteristics of high specificity, speed, and mildness, enzymes are unstable and have low reproducibility, thus posing risks such as low conversion rate.

[0008] (5) Genetic engineering method: Genes that produce trehalose or convert glucose into trehalose are introduced into plants such as beets, potatoes, and tomatoes through a bacterium to create transgenic plants with the ability to produce trehalose.

[0009] In summary, for a long time, the main sources of trehalose in industrial production have been lactic acid bacteria, yeast, molds and other trehalose-containing microorganisms, enzyme synthesis, and genetically modified plants. There have been no reports of trehalose being produced from natural plant sources.

[0010] Selaginella is a plant belonging to the genus Selaginella in the family Selaginellaceae of the phylum Seraphim. There are about 700 species worldwide, and it is found in most provinces of my country. It was first recorded in the "Shennong's Classic of Materia Medica". The "Pharmacopoeia of the People's Republic of China" lists Selaginella as the dried whole herb of Selaginella tamariscina (Beauv.) Spring or Selaginella pulvinata (HooK. et Grev.) Maxim., both belonging to the family Selaginellaceae.

[0011] Selaginella tamariscina is rich in flavonoids, sterols, alkaloids, phenols, and trehalose. Currently, the development and application of Selaginella tamariscina extracts mainly focus on flavonoids and phenols, such as cypermethrin, phenylpropanol, and alkynylphenol. However, in existing technologies, when extracting and purifying plants containing glycosides and flavonoids, only a specific product with high content or high added value is usually extracted. For example, current processing techniques for Selaginella tamariscina mainly involve extracting cypermethrin, without addressing industrially feasible methods for separating and purifying sugars and flavonoids during the extraction process. Summary of the Invention

[0012] The purpose of this invention is to develop an industrially feasible process for extracting high-purity trehalose from waste materials used in the extraction of flavonoids from Selaginella tamariscina, thereby improving the comprehensive utilization of Selaginella tamariscina and further developing its applications.

[0013] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0014] A method for the combined extraction of cyperine and trehalose from Selaginella tamariscina includes the following steps:

[0015] Step S1: After the Selaginella tamariscina is pulverized, a solvent is added for extraction. The extract is collected and concentrated to obtain a crude extract of Selaginella tamariscina. The extraction method can be reflux extraction, or it can also be cold maceration extraction, ultrasonic extraction, or percolation extraction. Preferably, the solvent is composed of one or more solvents selected from water, lower alcohols, acetonitrile, or acetone. The lower alcohols refer to ethanol, methanol, etc.

[0016] Step S2: Add purified water to the crude extract of Selaginella tamariscina to dissolve it, filter it, and collect the filtrate and residue of the crude extract.

[0017] Step S3: The crude extract residue of Selaginella tamariscina from S2 is dissolved in ethanol, filtered, and the filtrate is loaded onto a macroporous adsorption resin column for separation and purification to obtain Taxodium tamariscina flavonoids.

[0018] The crude extract of Selaginella tamariscina from S2 was concentrated and dried to obtain trehalose extract.

[0019] Step S4: Add an ethanol aqueous solution with a volume fraction of 60-95% to the trehalose extract obtained in S3 and dissolve it at 60-80℃. Filter, keep the filtrate warm for 10-60 min, crystallize at 0-40℃ for 12 h, filter, and collect the crude trehalose crystals.

[0020] Step S5: Dissolve the crude trehalose crystals obtained in S4 by adding an ethanol aqueous solution with a volume fraction of 60-95% at 60-80℃, filter, keep the filtrate warm for 10-60 min, crystallize at 0-40℃ for 12 h, filter, and collect the high-purity trehalose crystals.

[0021] The mass-to-volume ratio of the crude trehalose crystals to the ethanol aqueous solution is 1:8-20 g / ml.

[0022] Specifically, the Selaginella is one of the following: Selaginella tamariscina, Selaginella pulvinata, Selaginella sinensis, and Selaginella moellendorffii.

[0023] Preferably, the Selaginella is Selaginella tamariscina or Selaginella pulvinata.

[0024] The inventors discovered that, using the same extraction process, the trehalose extracted from Selaginella tamariscina and Selaginella pulvinata has a higher purity, reaching over 90%.

[0025] Furthermore, the selaginella is Selaginella tamariscina, and the trehalose prepared from it can reach a purity of over 99%.

[0026] Preferably, the ratio of solvent volume to medicinal material mass in S1 is mL:g = (40-2):1, more preferably (15-2):1. The solvent is an aqueous ethanol solution with a volume concentration of 80-100%, preferably an aqueous ethanol solution with a volume concentration of 85-100%. More specifically, the solvent is an aqueous ethanol solution with a volume concentration of 95%.

[0027] Specifically, in step S4, the volume fraction of the ethanol-water solution is 60-95%. The mass-to-volume ratio of trehalose extract to the ethanol-water solution is 1:8-20 g / ml; preferably, the trehalose extract is heated to 60-80°C before adding the ethanol-water solution. Preferably, crystallization in S4 occurs at 4°C.

[0028] The beneficial effects achieved by this invention are:

[0029] 1. This invention uses Selaginella tamariscina as raw material and combines the extraction of cyperus tinctoria flavonoids and high-purity trehalose. This not only improves the purity of the final product of cyperus tinctoria flavonoids, but also avoids the waste of valuable trehalose in Selaginella tamariscina. The yield of trehalose is between 0.2% and 2.0%, and the purity of the product is greater than 95.0%, thereby improving the bioavailability of Selaginella tamariscina.

[0030] 2. The preparation method of this invention uses only solvent extraction. The solvent used is safe to operate, easy to remove from the product, and avoids solvent residue. A pure product with a content greater than 95.0% can be prepared by solvent extraction, water washing purification, and crystallization. No activated carbon treatment or resin purification is required. The preparation process is environmentally friendly, does not generate solid waste, is easy to scale up, and is simple.

[0031] 3. The raw materials described in this invention are Selaginella tamariscina, Selaginella stauntoniana, Selaginella sinensis, Selaginella moellendorffii, Selaginella pulvinata, and Selaginella doederleinii, etc., which are widely available and provide an inexpensive and readily available source for the preparation of trehalose-related drugs. This has significant implications for the comprehensive utilization and in-depth development of Selaginella. Detailed Implementation

[0032] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0033] Example 1

[0034] A method for the combined extraction of cypress flavonoids and trehalose from Selaginella tamariscina:

[0035] 1 kg of Selaginella tamariscina was crushed and mixed with 10 L of 85% ethanol aqueous solution. The mixture was refluxed at 85 °C for 2 h and extracted twice. The two extracts were combined and concentrated to obtain 193.1 g of crude Selaginella tamariscina extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected.

[0036] Add 100 mL of ethanol to the filter residue, stir to dissolve, filter, and load the filtrate onto an AB-8 macroporous adsorption resin column. Elute with 40% (v / v) ethanol aqueous solution for 3 column volumes, then elute with 70% (v / v) ethanol aqueous solution for 4 column volumes. Combine the 4 column volumes of fractions eluted with 70% (v / v) ethanol aqueous solution, and recover the solvent under reduced pressure to dryness to obtain 5.8 g of the flavonoid-enriched fraction of Taxodium spp., with a flavonoid content of 58.9%.

[0037] The filtrate was concentrated and dried to obtain 26g of trehalose extract from Selaginella tamariscina, with a trehalose content of 58.7%. 260mL of a 70% (v / v) ethanol aqueous solution was added at 70℃ to dissolve the extract. After filtration, the filtrate was kept at this temperature for 10 min and then crystallized at 4℃. The crystals were collected to obtain crude crystals. 260mL of a 70% (v / v) ethanol aqueous solution was added at 70℃ to dissolve the crude crystals. After keeping the solution at this temperature for 10 min, it was crystallized at 4℃ to obtain 8g of trehalose crystals with a content of 99.7%, yielding a yield of 0.8% (yield = final product / weight of medicinal material).

[0038] Example 2

[0039] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0040] 1 kg of Selaginella pulvinata was crushed and mixed with 10 L of 85% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 h and extracted twice. The two extracts were combined and concentrated to obtain 205.1 g of crude Selaginella pulvinata extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected.

[0041] The filter residue was processed in the same manner as in Example 1, yielding 7.6g of the flavonoid-rich fraction of Taxus chinensis with a content of 51.6%.

[0042] The filtrate was collected, concentrated, and dried to obtain 37g of trehalose extract from Selaginella pulvinata, with a trehalose content of 50.8%. 370mL of 70% ethanol aqueous solution was added at 70℃ to dissolve the extract. After filtration, the filtrate was kept at this temperature for 10min and then crystallized at 4℃. The crystals were collected, and 370mL of 70% ethanol aqueous solution was added at 70℃ to dissolve the crystals. After keeping the temperature at this temperature for 10min, the crystals were crystallized at 4℃ to obtain 11g of trehalose crystals with a content of 95.5%, yielding a yield of 1.1%.

[0043] Example 3

[0044] A method for the combined extraction of cypermethrin and trehalose from Selaginella sinensis:

[0045] 1 kg of Selaginella sinensis was crushed and mixed with 10 L of 85% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 h and extracted twice. The two extracts were combined and concentrated to obtain 199.2 g of crude Selaginella sinensis extract. 1 L of purified water was added to the crude extract to dissolve it. The mixture was filtered, and the filtrate and residue were collected. The residue was used to extract cypermethrin flavonoids, following the same procedure as in Example 1. The filtrate was collected, concentrated, and dried to obtain 21 g of trehalose extract of Selaginella sinensis, with a trehalose content of 48.7%.

[0046] Example 4

[0047] A method for the combined extraction of cypress flavonoids and trehalose from Selaginella moellendorffii:

[0048] 1 kg of *Selaginella moellendorffii* was crushed and mixed with 10 L of 85% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 h and extracted twice. The two extracts were combined and concentrated to obtain 216.8 g of crude *Selaginella moellendorffii* extract. 1 L of purified water was added to the crude extract to dissolve it. The mixture was filtered, and the filtrate and residue were collected. The residue was used to extract cypermethrin flavonoids, following the same procedure as in Example 1. The filtrate was collected, concentrated, and dried to obtain 31 g of trehalose extract of *Selaginella moellendorffii*, with a trehalose content of 40.1%.

[0049] Example 5

[0050] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0051] 1 kg of *Selaginella pulvinata* was crushed and mixed with 20 L of 85% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 h and extracted twice. The two extracts were combined and concentrated to obtain 247.8 g of crude extract of *Selaginella pulvinata*. 1 L of purified water was added to the crude extract to dissolve it. The mixture was filtered, and the filtrate and residue were collected. The residue was used to extract flavonoids from *Selaginella pulvinata*, following the same procedure as in Example 1. The filtrate was collected, concentrated, and dried to obtain 31 g of trehalose extract of *Selaginella pulvinata*, with a trehalose content of 51.2%. Dissolve the trehalose in 310 mL of 70% ethanol aqueous solution at 70℃, filter, filter the filtrate, keep the filtrate warm for 10 min, and then crystallize at 4℃. Collect the crystals, dissolve them in 310 mL of 70% ethanol aqueous solution at 70℃, keep the filtrate warm for 10 min, and then crystallize at 4℃ to obtain 11.5 g of trehalose crystals with a purity of 94.1%, with a yield of 1.15%.

[0052] When the volume of the extraction solvent is increased from 10L to 20L, the extract volume increases, the yield increases, but the purity decreases due to the increase in impurities.

[0053] Example 6

[0054] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0055] 1 kg of *Selaginella pulvinata* was crushed and mixed with 5 L of 85% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 hours, and extracted twice. The extracts were combined and concentrated to obtain 155.6 g of crude *Selaginella pulvinata* extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected. The residue was used to extract flavonoids from *Taxodium spp.*, following the same procedure as in Example 1. The filtrate was collected, concentrated, and dried to obtain 23 g of *Selaginella pulvinata* extract. Trehalose extract of Selaginella pulvinata, with a trehalose content of 55.3%, was dissolved in 230 mL of 70% (v / v) ethanol aqueous solution at 70°C. After filtration, the filtrate was kept at 4°C for 10 min and then crystallized. The crystals were collected, dissolved in 230 mL of 70% (v / v) ethanol aqueous solution at 70°C, kept at 10 min, and then crystallized at 4°C to obtain 8.12 g of trehalose crystals with a content of 97.5%, yielding a yield of 0.812%.

[0056] When the extraction solvent volume is reduced from 10L to 5L, the yield decreases, but the purity increases because there are fewer impurities.

[0057] Example 7

[0058] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0059] 1 kg of *Selaginella pulvinata* was crushed and mixed with 10 L of 50% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 hours, and the extraction was repeated twice. The extracts were combined and concentrated to obtain 236.6 g of crude *Selaginella pulvinata* extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected. The residue was used to extract paclitaxel flavonoids, following the same procedure as in Example 1. The filtrate was concentrated and dried to obtain 5 g of crude extract. 3g of trehalose extract from *Selaginella pulvinata*, with a trehalose content of 31.3%, was dissolved in 530mL of 70% ethanol aqueous solution at 70℃. After filtration, the filtrate was kept at this temperature for 10min and then crystallized at 4℃. The crystals were collected, dissolved in 530mL of 70% ethanol aqueous solution at 70℃, kept at this temperature for 10min, and then crystallized at 4℃ to obtain 10.24g of trehalose crystals with a content of 68.8%.

[0060] When the concentration of ethanol used as a solvent decreases to 50%, a large amount of protein and flavonoids are extracted, and the water-soluble components increase. Therefore, the purity of trehalose extract and trehalose prepared by secondary crystallization both decrease significantly.

[0061] Example 8

[0062] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0063] 1 kg of *Selaginella pulvinata* was crushed and mixed with 10 L of 100% ethanol aqueous solution as solvent. The mixture was refluxed at 85°C for 2 hours, and the extraction was repeated twice. The extracts were combined and concentrated to obtain 228.1 g of crude *Selaginella pulvinata* extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected. The residue was used to extract paclitaxel flavonoids, following the same procedure as in Example 1. The filtrate was concentrated and dried to obtain... 29.0 g of trehalose extract from *Selaginella pulvinata*, with a trehalose content of 53.1%, was dissolved in 290 mL of 70% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept at this temperature for 10 min and then crystallized at 4°C. The crystals were collected, dissolved in 290 mL of 70% ethanol aqueous solution at 70°C, kept at this temperature for 10 min, and then crystallized at 4°C to obtain 10.9 g of trehalose crystals with a content of 96.7%.

[0064] Example 9

[0065] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0066] 1 kg of *Selaginella pulvinata* was crushed and mixed with 10 L of 95% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 hours, and the extraction was repeated twice. The extracts were combined and concentrated to obtain 226.5 g of crude *Selaginella pulvinata* extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected. The residue was used to extract paclitaxel flavonoids, following the same procedure as in Example 1. The filtrate was used for concentration and drying. 30g of trehalose extract from *Selaginella pulvinata* was obtained, with a trehalose content of 51.5%. 300mL of 70% ethanol aqueous solution was added at 70℃ to dissolve the extract. The solution was filtered, and the filtrate was kept at this temperature for 10min. Crystallization was then carried out at 4℃. The crystals were collected, and 300mL of 70% ethanol aqueous solution was added at 70℃ to dissolve the crystals. After keeping the solution at this temperature for 10min, crystallization was carried out at 4℃ to obtain 10.3g of trehalose crystals with a content of 97.8%.

[0067] Example 10

[0068] A method for the combined extraction of cyperine and trehalose from Selaginella pulvinata:

[0069] 1 kg of *Selaginella pulvinata* was crushed and mixed with 10 L of 70% ethanol aqueous solution. The mixture was refluxed at 85°C for 2 hours, and the extraction was repeated twice. The extracts were combined and concentrated to obtain 229.8 g of crude *Selaginella pulvinata* extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate and residue were collected. The residue was used to extract paclitaxel flavonoids, following the same procedure as in Example 1. The filtrate was used for concentration and drying. 41g of trehalose extract of Selaginella pulvinata was obtained, with a trehalose content of 40.2%. 410mL of 70% ethanol aqueous solution was added at 70℃ to dissolve the extract. After filtration, the filtrate was kept at this temperature for 10min and then crystallized at 4℃. The crystals were collected, and 410mL of 70% ethanol aqueous solution was added at 70℃ to dissolve the crystals. After keeping this temperature for 10min, the crystals were crystallized at 4℃ to obtain 11.7g of trehalose crystals with a content of 69.5%.

[0070] When the concentration of ethanol used as a solvent decreases to 70%, a large amount of protein and flavonoids are extracted, and the water-soluble components increase. Therefore, the purity of trehalose extract and trehalose prepared by secondary crystallization both decrease significantly.

[0071] Example 11

[0072] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was dissolved in 260ml of 95% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept warm for 10min and then crystallized at 4°C. The crystals were collected, and 260ml of 95% ethanol aqueous solution was added to dissolve the crystals at 70°C. After keeping warm for 10min, the crystals were crystallized at 4°C to obtain 9.1g of trehalose crystals with a content of 95.8%.

[0073] Trehalose extract is mainly water-soluble and alcohol-insoluble. If the ethanol concentration during crystallization is too high, a large number of water-insoluble impurities will precipitate along with the trehalose during the crystallization process, which will reduce the purity of the final product.

[0074] Example 12

[0075] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was dissolved in 260mL of 80% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept warm for 10min and then crystallized at 4°C. The crystals were collected, and 260mL of 80% ethanol aqueous solution was added to dissolve the crystals at 70°C. After keeping warm for 10min, the crystals were crystallized at 4°C to obtain 8.9g of trehalose crystals with a content of 97.6%.

[0076] Example 13

[0077] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was taken and dissolved in 260mL of 60% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept at 4°C for 10min and then crystallized. The crystals were collected and dissolved in 260mL of 60% ethanol aqueous solution at 70°C. After keeping at 70°C for 10min, the crystals were crystallized at 4°C to obtain 5.6g of trehalose crystals with a purity of 98.8%.

[0078] Trehalose extract is mainly water-soluble and alcohol-insoluble. If the purity of the ethanol in the crystallization is too low, a large amount of protein and flavonoids will be extracted, increasing the water-soluble components and thus reducing the purity of the final product.

[0079] Example 14

[0080] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was taken and dissolved in 520mL of 70% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept warm for 10min and then crystallized at 4°C. The crystals were collected and dissolved in 520mL of 90% ethanol aqueous solution at 70°C. After keeping warm for 10min, the crystals were crystallized at 4°C to obtain 4.7g of trehalose crystals with a content of 98.3%.

[0081] With more crystallization solvent, more impurities are dissolved, resulting in fewer precipitated impurities during crystallization, a lower yield, and a higher purity final product.

[0082] Example 15

[0083] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was taken and dissolved in 130mL of 70% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept warm for 10min and then crystallized at 4°C. The crystals were collected and dissolved in 130mL of 90% ethanol aqueous solution at 70°C. After keeping warm for 10min, the crystals were crystallized at 4°C to obtain 12.5g of trehalose crystals with a content of 87.6%.

[0084] Trehalose extract is mainly water-soluble and alcohol-insoluble. If too little ethanol is used for crystallization, a large number of impurities will precipitate along with the trehalose during the crystallization process, increasing the yield and decreasing the purity of the final product.

[0085] Example 16

[0086] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was taken and dissolved in 260mL of 70% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept at 20°C for 10min and then crystallized. The crystals were collected and dissolved in 260mL of 90% ethanol aqueous solution at 70°C. After keeping at 20°C for 10min, the crystals were crystallized at 20°C to obtain 7g of trehalose crystals with a purity of 99.8%.

[0087] Example 17

[0088] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was dissolved in 260mL of 70% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept warm for 10min and then crystallized at 40°C. The crystals were collected, and 260mL of 90% ethanol aqueous solution was added at 70°C to dissolve the crystals. After keeping warm for 10min, the crystals were crystallized at 40°C to obtain 5.6g of trehalose crystals with a purity of 99.8%.

[0089] Example 18

[0090] According to Example 1, trehalose extract of Selaginella tamariscina was prepared. 26g of trehalose extract was taken and dissolved in 260mL of 70% ethanol aqueous solution at 70°C. After filtration, the filtrate was kept at 0°C for 10min and then crystallized. The crystals were collected and dissolved in 260mL of 90% ethanol aqueous solution at 70°C. After keeping at 70°C for 10min, the crystals were crystallized at 0°C to obtain 8.6g of trehalose crystals with a purity of 99.0%.

[0091] Comparative Example 1

[0092] Crude extract of Selaginella tamariscina was prepared according to Example 1. 100 mL of ethanol was added to the crude extract and stirred to dissolve. The mixture was filtered, and the filtrate was loaded onto an AB-8 macroporous adsorption resin column. Three column volumes were eluted with 40% (v / v) ethanol aqueous solution, followed by four column volumes eluted with 70% (v / v) ethanol aqueous solution at pH 7. The four column volumes eluted with 70% (v / v) ethanol aqueous solution were combined, and the solvent was recovered under reduced pressure to dryness, yielding 8.9 g of the tamariscina-enriched fraction, with a tamariscina-enriched fraction content of 35.6%.

[0093] Comparative Example 2

[0094] Cassava leaves contain trehalose, and the inventors attempted to extract trehalose from cassava leaves using the same method.

[0095] Compared with Example 1, 1 kg of cassava leaves were treated and mixed with 10 L of 85% ethanol aqueous solution as solvent. The mixture was refluxed at 85°C for 2 hours, and extracted twice. The two extracts were combined, and the extract was concentrated to obtain 288 g of crude cassava leaf extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate was collected, concentrated, and dried to obtain 89 g of trehalose extract from cassava leaves, with a trehalose content of 0.29%. 890 mL of anhydrous ethanol was added at 70°C to dissolve the extract, and the mixture was kept at this temperature for 10 min. The extract was then cooled at 4°C to precipitate the solid, which was collected. 445 mL of anhydrous ethanol was added at 70°C to dissolve the solid, and the mixture was kept at this temperature for 10 min. Crystallization was then carried out at 4°C to obtain 9 g of grayish-white solid trehalose crystals with a trehalose content of 1.7%. Compared with Example 1, the trehalose prepared using the process of this invention with cassava leaves as the raw material has a lower yield and lower purity. The inventors speculate that it was caused by other water-soluble components present in cassava leaves.

[0096] Comparative Example 3

[0097] Compared to Example 1, purified water was used instead of extraction solvent, while other conditions remained unchanged. 1 kg of *Selaginella tamariscina* was crushed and mixed with 10 L of purified water (volume fraction equal to the solvent). The mixture was refluxed at 85°C for 2 hours, and extracted twice. The two extracts were combined, and the extract was concentrated to obtain 223.3 g of crude *Selaginella tamariscina* extract. 1 L of purified water was added to the crude extract to dissolve it, and the mixture was filtered. The filtrate was collected, concentrated, and dried to obtain 198 g of trehalose extract from *Selaginella tamariscina* with a trehalose content of 3.7%. 260 mL of 70% ethanol aqueous solution was added at 70°C to dissolve the extract, and the mixture was filtered. After maintaining the filtrate at this temperature for 10 minutes, crystallization was carried out at 4°C. The crystals were collected, and 260 mL of 70% ethanol aqueous solution was added at 70°C to dissolve the crystals. After maintaining this temperature for 10 minutes, crystallization was carried out at 4°C to obtain 23 g of trehalose crystals with a content of 8.5%. Compared with Example 1, when the extraction solvent is purified water, the trehalose prepared using the process of the present invention has poor purity.

[0098] Except for the different Selaginella species used, the operations in Examples 1-4 were the same. It can be seen that the Selaginella tamariscina seaweed extract in Example 1 had the highest trehalose yield, reaching 58.7%, and the highest purity of the trehalose obtained, ultimately yielding 8g of trehalose with a purity of up to 99.7%; 5.8g of the Taxodium tamariscina flavonoid-rich fraction was also obtained, with a Taxodium tamariscina flavonoid content of 58.9%.

[0099] In Example 2, *Selaginella pulvinata* yielded the highest amount, with a yield of 53%, ultimately producing 11g of trehalose with a purity of 95.50%. In Example 2, *Taxus cusia* biflavonoids were present in 7.6g, representing a purity of 51.6%.

[0100] Comparative Example 1 only extracted cypermethrin, ultimately yielding 8.9g of the cypermethrin-enriched fraction with a cypermethrin content of 35.6%. Example 1 ultimately yielded 5.8g of the cypermethrin-enriched fraction with a cypermethrin content of 58.9%. The comparison between Example 1 and Comparative Example 1 shows that the extraction method of the present invention can significantly improve the purity of cypermethrin in the cypermethrin-enriched fraction, reducing the difficulty of subsequent extraction. Furthermore, the yield of cypermethrin in Example 1 is not significantly different from that in Comparative Example 1. This demonstrates that the extraction method of the present invention can obtain high-purity trehalose while significantly increasing the cypermethrin content without reducing the cypermethrin yield, achieving a true co-extraction of trehalose and cypermethrin.

[0101] The difference between Examples 2, 5, and 6 lies in the amount of solvent added. A comparison of the three examples reveals that increasing the solvent volume from 10L to 20L increases the yield, but reduces purity due to increased impurities. Conversely, decreasing the solvent volume from 10L to 5L decreases the yield, but increases purity due to fewer impurities. The ethanol concentration in the extraction solvent decreases to 50%, resulting in the extraction of large amounts of protein and flavonoids, and an increase in water-soluble components. Therefore, the purity of trehalose extract and the trehalose prepared by secondary crystallization both decrease significantly. Thus, the choice of solvent volume has a significant impact on the performance of the final product.

[0102] A comparison of Examples 2 and 7-10 revealed that when the ethanol concentration in the extraction solvent decreased to 50% or 70%, a large amount of protein and flavonoids were extracted, and the water-soluble components increased. Therefore, the purity of trehalose extract and trehalose prepared by secondary crystallization decreased significantly.

[0103] The comparison between Example 1 and Examples 11-13 illustrates that the main components of trehalose extract are water-soluble. These components have relatively poor solubility in alcohol compared to water. If the ethanol concentration during crystallization is too high, a large number of impurities will precipitate along with the trehalose during the crystallization process, which will reduce the purity of the final product.

[0104] The comparison between Examples 1 and 14-15 illustrates that with more crystallization solvent, more impurities are dissolved, resulting in fewer precipitated impurities during crystallization, a lower yield, and a higher purity final product. Trehalose extract is primarily water-soluble and alcohol-insoluble. If too little ethanol is used during crystallization, a large amount of impurities will precipitate along with the trehalose, increasing the yield but decreasing the purity of the final product.

[0105] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the combined extraction of cyperine and trehalose from Selaginella tamariscina, characterized in that, Includes the following steps: Step S1: After the Selaginella tamariscina is pulverized, a solvent is added for extraction, the extract is collected, and the extract is concentrated to obtain crude Selaginella tamariscina extract; the solvent is any one or more of ethanol, methanol, acetonitrile, or acetone aqueous solution; Step S2: Add purified water to the crude extract of Selaginella tamariscina to dissolve it, filter it, and collect the filtrate and residue of the crude extract. Step S3: The crude extract residue of Selaginella in S2 is dissolved in ethanol and filtered. The filtrate is then loaded onto a macroporous adsorption resin column for separation and purification to obtain Taxodium biflavonoids. The crude extract filtrate of Selaginella in S2 is concentrated and dried to obtain trehalose extract. Step S4: Add an ethanol aqueous solution with a volume fraction of 60-95% to the trehalose extract obtained in S3 and dissolve it at 60-80℃. Filter, keep the filtrate warm for 10-60 min, crystallize at 0-40℃ for 12 h, filter, and collect the crude trehalose crystals. Step S5: Dissolve the crude trehalose crystals obtained in S4 by adding an aqueous ethanol solution with a volume fraction of 60-95% at 60-80℃, filter, keep the filtrate warm for 10-60 min, crystallize at 0-40℃ for 12 h, filter, and obtain trehalose crystals.

2. The method for co-extracting cyperine and trehalose from Selaginella tamariscina as described in claim 1, characterized in that, The extraction method for S1 is any one of reflux extraction, cold soaking extraction, ultrasonic extraction, or percolation extraction.

3. The method for co-extracting cyperine and trehalose from Selaginella tamariscina as described in claim 1, characterized in that, The Selaginella mentioned is any one or more of Selaginella tamariscina, Selaginella pulvinata, Selaginella sinensis, or Selaginella moellendorffii.

4. The method for co-extracting cyperine and trehalose from Selaginella tamariscina as described in claim 1, characterized in that, In S1, the solvent is an aqueous ethanol solution with a volume concentration of 80-100%, and the ratio of solvent volume to the mass of medicinal material is (40-2) mL: 1 g.

5. The method for co-extracting cypress flavonoids and trehalose from Selaginella tamariscina as described in claim 4, characterized in that, In S1, the solvent is an aqueous ethanol solution with a volume concentration of 85% to 100%, and the ratio of solvent volume to the mass of medicinal material is (15 to 2) mL: 1 g.

6. The method for co-extracting cyperine and trehalose from Selaginella tamariscina as described in claim 1, characterized in that, In S4, the volume fraction of the ethanol-water solution is 60-95%, and the mass-volume ratio of trehalose extract to ethanol-water solution is 1g:(8-20)ml.

7. The method for co-extracting cyperus flavonoids and trehalose from Selaginella tamariscina as described in claim 1 or 6, wherein the trehalose extract in step S4 is heated to 60-80°C and then an aqueous ethanol solution is added.

8. The method for co-extracting cyperine and trehalose from Selaginella tamariscina as described in claim 1, characterized in that, The mass-to-volume ratio of crude trehalose crystals to aqueous ethanol solution in S5 is 1 g: (8-20) ml.

9. The method for co-extracting cyperine and trehalose from Selaginella tamariscina as described in claim 1, characterized in that, Step S1: After crushing S. tamariscina or S. pulvinata, add 85% to 100% ethanol aqueous solution. The ratio of solvent volume to herb mass is (15 to 2) mL: 1 g. Reflux at 85℃ for 2 hours. Extract twice, combine the two extracts, and concentrate to obtain crude S. tamariscina extract. Step S2: Add purified water to the crude extract of Selaginella tamariscina to dissolve it, filter it, and collect the filtrate and residue of the crude extract. Step S3: The crude extract residue of Selaginella in S2 is dissolved in ethanol and filtered. The filtrate is then loaded onto a macroporous adsorption resin column for separation and purification to obtain Taxodium biflavonoids. The crude extract filtrate of Selaginella in S2 is concentrated and dried to obtain trehalose extract. Step S4: Add an ethanol aqueous solution with a volume fraction of 60-95% to the trehalose extract obtained in S3 and dissolve it at 60-80℃. Filter, keep the filtrate warm for 10-60 min, crystallize at 0-40℃ for 12 h, filter, and collect the crude trehalose crystals. Step S5: Dissolve the crude trehalose crystals obtained in S4 by adding an ethanol aqueous solution with a volume fraction of 60-95% at 60-80℃ and at 70℃. Filter the solution, keep the filtrate warm for 10-60 min, and then crystallize it at 4℃ for 12 h. Filter the solution to obtain trehalose crystals.