Chrysanthemum extract and preparation method thereof

Abstract

The invention relates to the field of plant extraction, and particularly discloses a preparation method of a millettia indica extract, the millettia indica extract obtained by the preparation method comprises the steps of pretreatment, extraction, purification and the like contains millettia indica amide with higher purity, and in the extraction step, the millettia indica amide is added into the millettia indica extract to obtain the millettia indica extract. According to the method, the eutecticevaporate solvent is used for extracting the millettia in a low-temperature environment under the assistance of ultrasonic waves, so that the heat-sensitive millettia amide in the extract is effectively protected; and through the special eutectic solvent, the mirabilis jalapa amide can be better dissolved out, and on the basis, the mirabilis jalapa amide with relatively high purity can be obtained in the mirabilis jalapa extract through simple re-dissolution of an organic solvent.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a method for preparing a spathodea chrysanthemum extract. Background Art

[0002] Spinachamide has multiple functions. It can inhibit the transmission of nerve impulse signals, relax facial muscles, reduce dynamic wrinkles, and achieve anti-wrinkle and anti-aging effects. It also has antioxidant and anti-inflammatory properties, which can protect the skin from damage by free radicals and reduce inflammation by inhibiting inflammatory factors (such as TNF-α and COX-2). In addition, spilanthol also exhibits certain antibacterial and antifungal activities, as well as analgesic and muscle tension relief effects.

[0003] At present, the market demand for spilanthol with the above-mentioned effects is constantly increasing. The synthetic preparation method has advantages in terms of raw material availability and production scale-up. For example, patent CN101910116A sequentially treats β-ketoester through reduction reaction, sulfonation reaction, elimination, Wittig reaction and amidation reaction, providing a method for preparing spilanthol in large quantities without using expensive reagents. For example, patent application CN117510358A uses cheap and readily available ethyl 4-bromobutyrate as raw material, and synthesizes spilanthol through seven steps including quaternary phosphonium salt reaction, Wittig reaction, Wittig-horner reaction, dehydration condensation, etc.

[0004] Although the above-mentioned synthetic preparation method can provide spilanthol in large quantities, there are still problems that need to be solved, such as the complexity of the synthetic route and the high cost of reagents. As people are increasingly aware of the pursuit of natural, green, healthy and safe, although spilanthol extracted from plants is popular due to its natural properties and green background, it also faces the challenges of low extraction and purification. Therefore, there is an urgent need for an extraction process of spilanthol with high extraction and simple purification. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention extracts spilanthol by using an ultrasound-assisted low eutectic solvent under a low temperature environment, thereby effectively protecting the thermosensitive spilanthol in the extract; and through a specially prepared low eutectic solvent, spilanthol can be better dissolved, showing good selectivity for spilanthol.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a method for preparing a schisandra chinensis extract, which specifically comprises the following steps:

[0008] (1) Pretreatment: Wash the spathodea chrysanthemum, freeze-dry it for 20 to 60 hours, and crush and sieve it to obtain spathodea chrysanthemum powder;

[0009] (2) Extraction: Mix the spatholobi powder with a low eutectic solvent, stir at 0-20° C. for 50-200 min, and then centrifuge to separate the residue and the supernatant, extract the residue repeatedly for 1-3 times, and mix the obtained supernatants to obtain a crude extract;

[0010] (3) Purification: The crude extract is subjected to rotary evaporation, and then an organic solvent at 40-60°C is added and mixed, and the temperature is lowered by 1-8°C to remove the first part of the precipitated substance, and the temperature is further lowered by 1-8°C to remove the second part of the precipitated substance. Finally, the remaining liquid is stored at -30--10°C overnight, the upper layer of liquid is discarded, the lower layer of solid is collected, washed, and freeze-dried to obtain the spatholobi extract.

[0011] In some embodiments, in step (1), the sieving particle size is 40-100 mesh.

[0012] The invention controls the particle size of the sieved particles, thereby ensuring the extraction rate and reducing the rotation speed of the subsequent centrifugal operation, reducing the stratification of the supernatant and avoiding the early precipitation of the effective components.

[0013] In some embodiments, in step (2), the stirring further requires ultrasonic oscillation for 2 to 8 minutes every 10 to 20 minutes, and the ultrasonic frequency is 40 to 80 khz.

[0014] The spilanthol extract contains amino acids, triterpenes, stigmasterol, beeswax alcohol and alkaloids, and is particularly rich in N-alkylamides. Among these N-alkylamides, Spilantho (hereinafter referred to as spilanthol) is the most abundant and stable amide. The currently commonly used method for extracting spilanthol is to extract it from plants using methanol, ethanol, supercritical carbon dioxide or n-hexane, and then purify it by preparative thin layer chromatography and / or high performance liquid chromatography. The main disadvantage of this method is that the purification efficiency is low. The present invention uses an ultrasonic-assisted low eutectic solvent to extract spilanthol, wherein the ultrasonic extraction performed at a low temperature can significantly shorten the extraction time, improve the leaching rate and efficiency, and is beneficial to protecting the heat-sensitive component spilanthol.

[0015] In some embodiments, in step (2), the mass ratio of the spilanthus powder to the low eutectic solvent is 1:(5-15).

[0016] In some embodiments, in step (2), the preparation method of the deep eutectic solvent is as follows:

[0017] Silver trifluoromethanesulfonate, glucose and citric acid are mixed, water is added and stirred at 40-100° C. until transparent and uniform to obtain a concentrated solution, and then the concentrated solution is diluted with water to 20-60v%, thereby obtaining a low eutectic solvent.

[0018] The present invention adopts silver trifluoromethanesulfonate and glucose as hydrogen bond acceptors and citric acid as hydrogen bond donor, and provides a low eutectic solvent with low cytotoxicity and high biocompatibility, wherein glucose and citric acid provide polarity matching that of spilanthol, and spilanthol has an amide with relatively high polarity and a fatty acyl group with relatively low polarity, so that spilanthol can be better dissolved.

[0019] The low eutectic solvent also showed good selectivity for spilanthol during the extraction process. Combined with the molecular structure of spilanthol, which is an olefin N-alkylamide with an isobutyl side chain, the high selectivity may be achieved by the double bond complexation of silver ions and olefins. On the basis of high selectivity, the subsequent purification process relies on the poor solubility of spilanthol, so that spilanthol with high purity can be obtained in the spilanthol extract by simply re-dissolving it in an organic solvent.

[0020] In some embodiments, the molar ratio of glucose to citric acid is (0.8-1.5):1.

[0021] In some embodiments, the molar ratio of silver trifluoromethanesulfonate to glucose is (0.01-0.05):1.

[0022] The invention controls the ratio of hydrogen bond acceptors to hydrogen bond donors, avoids high polarity caused by excessive hydrogen bond donors, enhances the permeability of the low eutectic solvent to spathiphyllum, and increases the dissolution ratio of the active substance.

[0023] In some embodiments, in step (2), the centrifugal speed is 2000-4000 rpm, and the time is 10-30 min.

[0024] The invention effectively separates spathiphyllum powder through specially selected centrifugal conditions and avoids early stratification of the low eutectic solvent.

[0025] In some embodiments, in step (3), the organic solvent comprises 50-80 wt % of petroleum ether and 20-50 wt % of ethyl acetate.

[0026] The present invention remixes the crude extract after rotary evaporation with a hot organic solvent, first removes a portion of the non-spilanthol amide alkylamide by gradient cooling, and then freezes the remaining liquid overnight. The high-content spilanthol in the extract will precipitate at the bottom of the container, and the organic solvent exists in the form of a supernatant. The two parts of the substance are clearly layered and will not mix even if slightly shaken, and the supernatant can be directly poured off. The present invention uses a specifically formulated organic solvent to better dissolve substances other than spilanthol in the spilanthol extract, thereby improving the purity of spilanthol in the extract.

[0027] The second aspect of the present invention provides the spilanthol extract obtained by the above preparation method, wherein the purity of spilanthol amide is ≥75%.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention provides a method for preparing a spilanthol extract through the steps of pretreatment, extraction and purification. In the extraction step, the heat-sensitive spilanthol is extracted by low-temperature assisted low-eutectic solvent using ultrasonic waves, which shortens the extraction time and improves the leaching rate and efficiency.

[0030] 2. The present invention adopts silver trifluoromethanesulfonate and glucose as hydrogen bond acceptors and citric acid as hydrogen bond donors to form a low eutectic solvent, which provides a polarity matching that of spilanthol, so that spilanthol can be dissolved better; in addition, the double bond complexation of the silver ions in the low eutectic solvent and the olefin of spilanthol also shows good selectivity for spilanthol.

[0031] 3. Based on high selectivity, the present invention utilizes the poor solubility of spilanthol to dissolve substances other than spilanthol in the spilanthol extract in the purification step by using a mixed organic solvent of petroleum ether and ethyl acetate, and improves the purity of spilanthol in the extract by gradient cooling. DETAILED DESCRIPTION

[0032] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following examples are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Without departing from the spirit or scope of the present invention, other combinations and various improvements within the concept of the present invention may be made. It is worth noting that the raw materials used in the following preparation examples and examples, unless otherwise specified, may be from any commercially available manufacturer.

[0033] Preparation Example 1

[0034] The preparation steps of deep eutectic solvent A are as follows:

[0035] 0.02 mol of silver trifluoromethanesulfonate, 1.1 mol of glucose and 1 mol of citric acid were mixed, 3 mol of water was added and stirred at 60°C until transparent and uniform to obtain a concentrated solution, and then the concentrated solution was diluted with water to 30v%, thereby obtaining a low eutectic solvent A.

[0036] Preparation Example 2

[0037] The preparation steps of deep eutectic solvent B are as follows:

[0038] 1.1 mol of glucose and 1 mol of citric acid were mixed, 3 mol of water were added and stirred at 60° C. until transparent and uniform to obtain a concentrated solution, and then the concentrated solution was diluted with water to 30 v %, thereby obtaining a low eutectic solvent B.

[0039] Preparation Example 3

[0040] The preparation steps of deep eutectic solvent C are as follows:

[0041] The difference between this preparation example and preparation example 1 is that the amount of citric acid used is 1.5 mol.

[0042] Preparation Example 4

[0043] The preparation steps of deep eutectic solvent D are as follows:

[0044] The difference between this preparation example and preparation example 1 is that the amount of citric acid used is 0.7 mol.

[0045] Preparation Example 6

[0046] The preparation steps of deep eutectic solvent E are as follows:

[0047] The difference between this preparation example and preparation example 1 is that the amount of silver trifluoromethanesulfonate used is 0.005 mol.

[0048] Preparation Example 7

[0049] The preparation steps of deep eutectic solvent F are as follows:

[0050] The difference between this preparation example and preparation example 1 is that the amount of silver trifluoromethanesulfonate used is 0.055 mol.

[0051] Example 1

[0052] A method for preparing a spatholobi extract comprises the following steps:

[0053] (1) Pretreatment: Wash the Globenia rubra, freeze-dry it for 40 hours, and grind it through a 60-mesh sieve to obtain Globenia rubra powder;

[0054] (2) Extraction: Mix the spatholobi powder and the low eutectic solvent A at a mass ratio of 1:10, stir at 10°C for 120 min, and use ultrasonic vibration for 5 min every 15 min during the stirring process. The ultrasonic frequency is 60 kHz, and then centrifuge at 3000 rpm for 20 min to separate the residue and the supernatant. Repeat the extraction of the residue twice, and mix the obtained supernatants to obtain a crude extract;

[0055] (3) Purification: The crude extract was subjected to rotary evaporation, and then 4 times the mass of a 50°C organic solvent was added, wherein the organic solvent contained 70 wt% of petroleum ether and 30 wt% of ethyl acetate, and the temperature was lowered by 5°C to remove the first part of the precipitated substance, and the temperature was further lowered by 8°C to remove the second part of the precipitated substance. Finally, the remaining liquid was kept at -20°C overnight, the upper layer of liquid was poured off, the lower layer of solid was collected, washed with anhydrous ethanol, and freeze-dried to obtain the spathiphyllum extract.

[0056] Example 2

[0057] A method for preparing a spatholobi extract comprises the following steps:

[0058] (1) Pretreatment: Wash the Globenia rubra, freeze-dry it for 20 hours, and grind it through a 40-mesh sieve to obtain Globenia rubra powder;

[0059] (2) Extraction: Mix Globenia powder and low eutectic solvent A in a mass ratio of 1:5, stir at 20°C for 50 min, use ultrasonic vibration for 3 min every 10 min during the stirring process, and the ultrasonic frequency is 80 khz, then centrifuge at 4000 rpm for 10 min to separate the residue and the supernatant, extract the residue repeatedly once, and mix the obtained supernatants to obtain a crude extract;

[0060] (3) Purification: The crude extract was subjected to rotary evaporation, and then 4 times the mass of a 40°C organic solvent was added, wherein the organic solvent contained 50 wt% of petroleum ether and 50 wt% of ethyl acetate, and the temperature was lowered by 8°C to remove the first part of the precipitated substance, and the temperature was further lowered by 5°C to remove the second part of the precipitated substance. Finally, the remaining liquid was kept at -30°C overnight, the upper layer of liquid was poured off, the lower layer of solid was collected, washed with anhydrous ethanol, and freeze-dried to obtain the spathiphyllum extract.

[0061] Example 3

[0062] A method for preparing a spatholobi extract comprises the following steps:

[0063] (1) Pretreatment: Wash the Globenia rubra, freeze-dry it for 60 hours, and grind it through a 100-mesh sieve to obtain Globenia rubra powder;

[0064] (2) Extraction: Mix the spatholobi powder and the low eutectic solvent A at a mass ratio of 1:15, stir at 0°C for 200 min, and use ultrasonic vibration for 8 min every 20 min during the stirring process. The ultrasonic frequency is 40 kHz, and then centrifuge at a speed of 2000 rpm for 30 min to separate the residue and the supernatant. Repeat the extraction of the residue for 3 times, and mix the obtained supernatants to obtain a crude extract;

[0065] (3) Purification: The crude extract was subjected to rotary evaporation, and then 4 times the mass of a 60°C organic solvent was added, wherein the organic solvent contained 80 wt% of petroleum ether and 20 wt% of ethyl acetate, and the temperature was lowered by 5°C to remove the first part of the precipitated substance, and the temperature was further lowered by 5°C to remove the second part of the precipitated substance, and finally the remaining liquid was kept at -10°C overnight, the upper layer of liquid was poured off, the lower layer of solid was collected, washed with anhydrous ethanol, and freeze-dried to obtain the spathiphyllum extract.

[0066] Example 4

[0067] This embodiment provides a method for preparing an extract of spilionella, and the specific implementation method is the same as that of Example 1, except that the low eutectic solvent A in step (2) is replaced by an equal amount of low eutectic solvent B.

[0068] Example 5

[0069] This embodiment provides a method for preparing an extract of spilionella, and the specific implementation method is the same as that of Example 1, except that the low eutectic solvent A in step (2) is replaced by an equal amount of low eutectic solvent C.

[0070] Example 6

[0071] This embodiment provides a method for preparing an extract of spilionella, and the specific implementation method is the same as that of Example 1, except that the low eutectic solvent A in step (2) is replaced by an equal amount of low eutectic solvent D.

[0072] Example 7

[0073] This embodiment provides a method for preparing an extract of spilionella, and the specific implementation method is the same as that of Example 1, except that the low eutectic solvent A in step (2) is replaced by an equal amount of low eutectic solvent E.

[0074] Example 8

[0075] This embodiment provides a method for preparing an extract of spilionella, and the specific implementation method is the same as that of Example 1, except that the low eutectic solvent A in step (2) is replaced by an equal amount of low eutectic solvent F.

[0076] Example 9

[0077] This embodiment provides a method for preparing a spatholobi extract, and the specific implementation method is the same as that of Example 1, except that:

[0078] (3) Purification: The crude extract was subjected to rotary evaporation, and then 4 times the mass of 50°C petroleum ether was added and mixed, and the temperature was lowered by 5°C to remove the first part of the precipitated substance, and the temperature was further lowered by 8°C to remove the second part of the precipitated substance. Finally, the remaining liquid was kept at -20°C overnight, the upper layer of liquid was poured off, and the lower layer of solid was collected, washed with anhydrous ethanol, and freeze-dried to obtain the spatholobi extract.

[0079] Example 10

[0080] This embodiment provides a method for preparing an extract of spatholobi. The specific implementation method is the same as that of Embodiment 1, except that: (3) Purification: The crude extract is subjected to rotary evaporation, and then 4 times the mass of 50°C ethyl acetate is added and mixed, the temperature is lowered by 5°C to remove the first part of the precipitated substance, and the temperature is further lowered by 8°C to remove the second part of the precipitated substance. Finally, the remaining liquid is kept at -20°C overnight, the upper layer of liquid is poured off, the lower layer of solid is collected, washed with anhydrous ethanol, and freeze-dried to obtain the spatholobi extract.

[0081] Embodiment 11

[0082] This embodiment provides a method for preparing a spatholobi extract, and the specific implementation method is the same as that of Example 1, except that:

[0083] (2) Extraction: Mix spathiphyllum powder and low eutectic solvent A in a mass ratio of 1:10, stir at 10°C for 120 min, and then centrifuge at 3000 rpm for 20 min to separate the residue and the supernatant. Repeat the extraction of the residue twice, and mix the obtained supernatants to obtain a crude extract.

[0084] Comparative Example 1

[0085] This comparative example provides a method for preparing an extract of Spinach spathulata. The specific implementation method is the same as that of Example 1, except that the low eutectic solvent A in step (2) is replaced by an equal amount of anhydrous ethanol.

[0086] Comparative Example 2

[0087] This comparative example provides a method for preparing a spilionella extract, and the specific implementation method is the same as that of Example 1, except that:

[0088] (3) Purification: The crude extract was subjected to rotary evaporation, and then 4 times the mass of a 50°C organic solvent was added and mixed, wherein the organic solvent contained 70 wt% of petroleum ether and 30 wt% of ethyl acetate, and the mixture was kept at -20°C overnight. The upper layer of liquid was poured off, and the lower layer of solid was collected, washed with anhydrous ethanol, and freeze-dried to obtain the spathodea extract.

[0089] Performance Testing:

[0090] 1. Liquid phase detection purity:

[0091] Using 1 kg of spilanthol as raw material, extraction was performed according to Examples 1 to 11 and Comparative Examples 1 to 2, and the spilanthol extracts obtained were tested by high performance liquid chromatography to obtain the purity and content of spilanthol amide therein, with the detection wavelength being 233 nm.

[0092] 2. Security Testing

[0093] Since glucose and citric acid are non-toxic to the human body, this test only uses high performance liquid chromatography to test whether the spilion extracts of Examples 1 to 11 and Comparative Examples 1 to 2 contain silver trifluoromethanesulfonate. The above results are shown in Table 1.

[0094] Table 1 Performance test results

[0095]

[0096] It can be seen from the data in Table 1 that no silver trifluoromethanesulfonate was detected in the spilionnae extracts of Examples 1 to 11 and Comparative Examples 1 to 2, indicating that the substance can be effectively removed in the purification step; and the amount of spilionnae amide extracted in the spilionnae extracts of Examples 1 to 3 is large, and the purity is higher than 94%, indicating that the extraction method of the present invention does not require thin layer chromatography purification or high performance liquid chromatography purification, and can selectively extract high-purity spilionnae amide from spilionnae.

[0097] Compared with Example 1, the low eutectic solvent used in Example 4 lacks silver trifluoromethanesulfonate. Due to the lack of double bond complexation between silver ions and olefins, the low eutectic solvent cannot selectively dissolve spilanthol, resulting in reduced purity and content of spilanthol; combined with Examples 7-8, it can be seen that changes in the amount of silver trifluoromethanesulfonate will also affect the purity and content of spilanthol. Examples 5-6 change the content of citric acid in the low eutectic solvent, which may change the polarity matching between the low eutectic solvent and spilanthol, and also reduce the purity and content of spilanthol.

[0098] It can be seen from Examples 1 and 9-10 that the mixed organic solvent of petroleum ether and ethyl acetate can better dissolve substances other than spilanthol in the spilanthol extract, thereby improving the purity of spilanthol. Compared with Example 1, Example 11 does not use ultrasound-assisted extraction, which also leads to a decrease in the purity and content of spilanthol. It can be seen from Example 1 and Comparative Example 1 that the low eutectic solvent used in the present invention significantly improves the purity and content of spilanthol in the extract compared to anhydrous ethanol. In combination with Example 1 and Comparative Example 2, it can be seen that the gradient cooling in the purification step can remove a portion of the non-spilanthol alkylamides, thereby improving the purity of spilanthol.

[0099] The embodiments and comparative examples described above do not impose any form of limitation on the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a spatholobi extract, characterized in that: The specific steps include: (1) Pretreatment: Wash the spathodea chrysanthemum, freeze-dry it for 20 to 60 hours, and crush and sieve it to obtain spathodea chrysanthemum powder; (2) Extraction: Mix the spatholobi powder with a low eutectic solvent, stir at 0-20° C. for 50-200 min, and then centrifuge to separate the residue and the supernatant, extract the residue repeatedly for 1-3 times, and mix the obtained supernatants to obtain a crude extract; (3) Purification: The crude extract is subjected to rotary evaporation, and then an organic solvent at 40-60°C is added and mixed, and the temperature is lowered by 1-8°C to remove the first part of the precipitated substance, and the temperature is further lowered by 1-8°C to remove the second part of the precipitated substance. Finally, the remaining liquid is stored at -30--10°C overnight, the upper layer of liquid is discarded, the lower layer of solid is collected, washed, and freeze-dried to obtain the spatholobi extract.

2. The method for preparing the Spinach extract according to claim 1, characterized in that: In step (1), the sieving particle size is 40 to 100 meshes.

3. The method for preparing the Spinach extract according to claim 1, characterized in that: In step (2), the stirring also needs to be oscillated with ultrasound for 2 to 8 minutes every 10 to 20 minutes, and the ultrasound frequency is 40 to 80 khz.

4. The method for preparing the Spinach extract according to claim 1, characterized in that: In step (2), the mass ratio of the spilanthus powder to the low eutectic solvent is 1:(5-15).

5. The method for preparing the Spinach extract according to claim 1, characterized in that: In step (2), the preparation method of the deep eutectic solvent is as follows: Silver trifluoromethanesulfonate, glucose and citric acid are mixed, water is added and stirred at 40-100° C. until transparent and uniform to obtain a concentrated solution, and then the concentrated solution is diluted with water to 20-60v%, thereby obtaining a low eutectic solvent.

6. The method for preparing the Spinach extract according to claim 5, characterized in that: The molar ratio of glucose to citric acid is (0.8-1.5):

1.

7. The method for preparing the Spinach extract according to claim 5, characterized in that: The molar ratio of silver trifluoromethanesulfonate to glucose is (0.01-0.05):

1.

8. The method for preparing the Spinach extract according to claim 1, characterized in that: In step (2), the centrifugal speed is 2000-4000 rpm and the time is 10-30 min.

9. The method for preparing the Spinach extract according to claim 1, characterized in that: In step (3), the organic solvent comprises 50-80 wt % of petroleum ether and 20-50 wt % of ethyl acetate.

10. An extract of Spinach obtained by the preparation method according to any one of claims 1 to 9, characterized in that: The purity of spilanthol in spilanthol extract is ≥75%.

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