Process for the preparation of spilanthol extract of spilanthes

Abstract

The application provides a preparation method of spilanthol extract of spilanthol, and comprises the following steps: S1, taking fresh spilanthol plants as the extracted part; S2, mixing the extracted part with a bacillus subtilis solution, and then placing and sterilizing the mixture for 24-48 hours; S3, immersing the extracted part in alcohol to extract, taking part of the extract, reducing and concentrating the extract to obtain a concentrated solution; S4, extracting the concentrated solution with organic ester to obtain an extract solution; and S5, performing desolventizing treatment on the extract solution to obtain the spilanthol extract. The fresh spilanthol plants are pretreated with the bacillus subtilis solution, so that the yield of spilanthol in the extract can be significantly improved. The alcohol and the organic ester can be recycled as the extraction solvent, the process parameters of the extraction are optimized, and the extraction efficiency and the content of the effective component are optimized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant extraction, and particularly relates to a preparation method of Acmella extract with high spilanthol. BACKGROUND

[0002] Acmella oleracea, also known as guiyuan chrysanthemum, is a species of the genus Acmella in the family Asteraceae. Its extract has an INCI name of ACMELLA OLERACEA extract, an English INCI name of ACMELLA OLERACEA EXTRACT, is an oil-soluble liquid with a deep brown to yellow color, and a plant extract calibration substance of spilanthol ((2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide, a structure formula as shown in Figure 1 .

[0003] The Acmella extract is considered to have great potential both locally and internally due to its main lipophilic N-alkyl amide (N-alkyl amide from isobutyramide). Alkaloids are secondary metabolites with anti-inflammatory, immunomodulatory and cannabinoid effects; therefore, the Acmella extract has multiple activities of analgesia, antibiosis, stimulation of collagen production, hydration and moisturization, antioxidation and anti-inflammation.

[0004] Currently, the artificial synthesis method for spilanthol has the problems of a long synthesis route and complex operation. CN117510358A proposes a preparation method of spilanthol (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide. Although the method is relatively simple compared with the route proposed by Yoshihiko Ikeda et al. in 1986, it still needs 7 steps of reactions to synthesize the target product, and is still relatively complicated.

[0005] Moreover, the artificial synthesis and natural extraction are not mutually replaceable solutions because the Acmella extract contains other active substances that are not yet known in addition to spilanthol. The highest content of the known spilanthol in the Acmella extract is 48 wt%, and there is a great room for improvement. SUMMARY

[0006] The purpose of the present application is to propose a preparation method of Acmella extract with high spilanthol, which effectively increases the content of spilanthol in the ACMELLA OLERACEA extract.

[0007] In order to achieve the above purpose, the present application provides a preparation method of Acmella extract with high spilanthol, which comprises the following steps:

[0008] S1, taking a fresh Acmella plant as an extracted part;

[0009] S2, mixing the extracted part with Bacillus subtilis solution; then placing for 24-48h, sterilizing treatment;

[0010] S3, immersing the extracted part in alcohol for extraction, taking the extracted liquid part; reducing pressure concentration, obtaining concentrated liquid;

[0011] S4, extracting the concentrated liquid with organic ester, obtaining extracted liquid;

[0012] S5, carrying out desolventizing treatment on the extracted liquid, and obtaining spilanthes extract.

[0013] Preferably, in S1, at least one part of the fresh spilanthes plant flower, stem and leaf is taken.

[0014] Preferably, in S2, the number of bacteria in the Bacillus subtilis solution is ≥1×10 9 CFU; the ratio of material to liquid is ≥1mL / g.

[0015] Preferably, in S2, after sterilization treatment, drying treatment is carried out.

[0016] Preferably, in S3, the alcohol is methanol and / or ethanol; the alcohol is added in an amount of 3-5 times the volume of the raw material each time.

[0017] Preferably, in S3, the reducing pressure concentration is carried out until the volume of the concentrated liquid no longer changes.

[0018] Preferably, in S3, the extraction method is reflux extraction, and the number of times is at least 3; each time is at least 3h.

[0019] Preferably, in S4, the organic ester is ethyl acetate, ethyl propionate, ethyl valerate, etc.

[0020] Preferably, in S4, the addition amount of the organic ester is at least 5 times the volume of the concentrated liquid, and the number of extraction times is at least 3.

[0021] Preferably, the desolventizing treatment in S4 is reducing pressure concentration.

[0022] The application also provides the use of the aforementioned spilanthes extract with high spilanthes amide in the preparation of cosmetics.

[0023] The beneficial technical effects of the application are at least as follows:

[0024] (1) The application finds that using fresh spilanthes plants and using Bacillus subtilis solution for pretreatment can significantly improve the yield of spilanthes amide in the extract; and using dry spilanthes plants as raw materials does not have this effect.

[0025] (2) The application adopts recyclable alcohol and organic ester as the extraction solvent, optimizes the process parameters of extraction, optimizes the extraction efficiency and the content of the final effective component. BRIEF DESCRIPTION OF DRAWINGS

[0026] The application is further described by using the drawings, but the embodiments in the drawings do not constitute any limitation on the application, and other drawings can be obtained by the ordinary skilled in the art without creative labor according to the following drawings.

[0027] Figure 1 The structural formula of spilanthol amide. DETAILED DESCRIPTION

[0028] In order to better understand the application, the application is further described below in combination with specific serial numbers, wherein the terms used in the serial numbers are used to describe specific embodiments, and do not constitute a limitation on the protection scope of the application.

[0029] In the application:

[0030] The spilanthes plant comes from a local farm.

[0031] The bacillus subtilis comes from Jinan Xinran Chemical Co., Ltd., and the CFU is 1x10 11 .

[0032] The EM bacteria come from Jinan Xinran Chemical Co., Ltd., and the CFU is 1x10 11 .

[0033] The cellulase comes from the flagship store of Jianchen brand.

[0034] Example 1

[0035] The preparation method of spilanthes extract includes the following steps:

[0036] S1, taking fresh spilanthes plant flowers, stems and leaves (harvested within 24 hours) as the extracted part;

[0037] S2, mixing the extracted part with 100 times diluted bacillus subtilis solution, the solid-liquid ratio is 1 mL / g, then placing for 24 h; taking out the extracted part and sterilizing at a high temperature above 120 DEG C, then drying to constant weight.

[0038] S3, immersing the extracted part in anhydrous ethanol for reflux extraction, each time for 3 h, and refluxing for three times; the addition amount of anhydrous ethanol is 5 times of the extracted part;

[0039] After the reflux extraction is completed, the solid part is discarded, and the extraction liquid part is taken; the extraction liquid part is concentrated under reduced pressure until the volume no longer changes, and the concentrated liquid is obtained;

[0040] S4, the concentrated solution is extracted with 5 volumes of ethyl acetate for 3 times to obtain an extracted solution;

[0041] S5, the 3 extracted solutions are combined and concentrated under reduced pressure to recover the solvent to obtain a spilanthes acuta extract.

[0042] Example 2 differs from example 1 in that:

[0043] In step S1, fresh flowers, stems and leaves of spilanthes acuta plants are taken as the extracted part.

[0044] Example 3 differs from example 1 in that:

[0045] In step S1, dried flowers and stems of spilanthes acuta plants are taken as the extracted part.

[0046] Example 4 differs from example 1 in that:

[0047] Step S2 is: the extracted part is mixed with a cellulase solution diluted by 100 times, the ratio of material to liquid is 1 mL / g, and then placed for 24 h; and then dried to constant weight.

[0048] Example 5 differs from example 1 in that:

[0049] The placing time of step S2 is 48 h; and the solution of step S3 is methanol, and the amount used is 3 times the extracted part.

[0050] Example 6 differs from example 1 in that:

[0051] Step S2 is: the extracted part is dried to constant weight.

[0052] Example 7 differs from example 1 in that:

[0053] The solution of step S2 uses EM bacteria instead of bacillus subtilis, and is also diluted by 100 times.

[0054] Example 8 differs from example 1 in that:

[0055] The solution of step S3 is petroleum ether.

[0056] Example 9 differs from example 1 in that:

[0057] The solution of step S4 is petroleum ether.

[0058] Example 10 differs from example 1 in that:

[0059] The solution of step S4 is acetone.

[0060] Example 11 differs from example 1 in that:

[0061] The solution of step S4 is ethyl valerate.

[0062] Example 12 differs from example 1 in that:

[0063] Step S4 is not performed after step S3.

[0064] Example 13 differs from example 1 in that:

[0065] Step S4 is performed directly after step S2, skipping step S3.

[0066] Example 13 differs from example 1 in that:

[0067] Step S4 is performed after step S3, and step 5 is performed after step S4, after step S2.

[0068] The stationary phase is a 35% phenyl-methylpolysiloxane, 30 m long, 0.25 mm internal diameter, 0.25 μm film thickness, fused silica capillary column.

[0069] The content of spirodiclofen is detected by HPLC (high performance liquid chromatography).

[0070] Chengdu Zhi Chun Botanical Biotechnology Co., Ltd. is commissioned to detect;

[0071] The chromatographic conditions are as follows:

[0072] The chromatographic column is an RP18 reversed-phase separation column.

[0073] The injection volume is 10 μl.

[0074] The column oven temperature is 40°C.

[0075] The mobile phase is acetonitrile / water (50 / 50).

[0076] The detection wavelength is 240 nm.

[0077] The detection results are shown in Table 1 below.

[0078] Table 1: Detection results of spirodiclofen content

[0079] Sample name Spicamate content (%) Example 1 65.82 Example 2 55.31 Example 3 53.91 Example 4 54.84 Example 5 64.47 Example 6 52.67 Example 7 51.43 Example 8 33.28 Example 9 40.94 Example 10 42.31 Example 11 62.18 Example 12 41.12 Example 13 18.21 Example 14 43.26 Acmefleur (commercially available) 48.01

[0080] As the results of Table 1 show:

[0081] As can be seen from the comparison of example 1, example 5 and example 11, in the first step of extraction, whether methanol or ethanol is used, and whether ethyl acetate or ethyl valerate is used for extraction, good extraction results can be achieved, and similar results can be achieved using other alcohols or organic esters.

[0082] According to the records of "MONROE D, LUO R, TRAN K, et al. Lc-hrms and nmr analysis of lyophilized acmella oleracea capitula, leaves and stems [J]. The Natural Products Journal, 2016, 6: 116-125.", the flowers, leaves and stems of the acmella oleracea also contain spilantholamide, so the present application uses this part as the extraction part.

[0083] As can be known from the comparison of Example 1, Example 2, Example 3 and Example 6, the use of Bacillus subtilis solution for pretreatment can significantly improve the yield of spilantholamide, but pretreatment after the acmella oleracea plants are placed for 7 days or after drying does not have the effect of improving the yield; the present application speculates that spilantholamide is a secondary metabolite of acmella oleracea; it must be generated when the plant still has physiological activity; and the physiological effect of Bacillus subtilis can stimulate acmella oleracea to generate spilantholamide.

[0084] As can be known from the comparison of Example 1, Example 4, Example 7, the inventors tried to use cellulase and EM bacteria instead of Bacillus subtilis solution for pretreatment, but there was no effect of improving the yield. The inventors speculate that harmful fungi to the Asteraceae family such as Peronospora sp., Puccinia sp., Alternaria sp., Fusarium sp. can also have similar effects, but considering the risk of biological leakage when using such fungi, the difficulty of subsequent sterilization and the possible harm to the human body when used subsequently, such experiments are excluded.

[0085] As can be known from the comparison of Example 1, Example 8, Example 9, Example 10 and Example 11, in the S3 extraction step, alcohol is the best choice; only organic esters are used for extraction in step S4 to further improve the yield; such effects are consistent with the records in "RONDANELLI M, FOSSARI F, VECCHIO V, et al. Acmella oleracea for pain management [J]. Fitoterapia, 2020, 140: 104419." and "BALIEIRO O C, SILVA P M S, SILVAS Y S, et al. Analytical and preparative chromatographic approaches for extraction of spilanthol from acmella oleracea flowers [J]. Microchemical Journal, 2020, 157: 105035."

[0086] As can be seen from the comparison of Example 1, Example 12 and Example 13, although the use of a single organic ester for extraction also has a certain effect, there is a significant difference in yield compared with the use of only alcohol for extraction; and the yield of using organic ester for extraction first and then using alcohol for extraction is the same as that of using only alcohol for extraction, which can be known that the order of solvents also has a significant impact on the yield.

[0087] It should be noted that the above-described workflow is merely illustrative and does not limit the scope of protection of the present application. In actual application, a person skilled in the art can select part or all of them according to actual needs to achieve the purpose of the embodiment scheme, which is not limited here.

[0088] In addition, technical details not described in detail in the present embodiment can refer to the parameter operation method provided by any embodiment of the present application, which will not be repeated here.

[0089] It should be noted that in this document, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or system including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or system. Without more limitations, the element defined by the statement "including a" does not exclude the existence of other identical elements in the process, method, article or system including the element.

[0090] The above-mentioned embodiment numbers of the present application are only for description, not representing the advantages and disadvantages of the embodiments.

[0091] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields based on the content of the present application specification and drawings, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing a chamomile extract with high chamomile amide, characterized in that, The method includes: S1. Take fresh chrysanthemum plants as the part to be extracted; S2. Mix the extract with Bacillus subtilis solution; then let stand for 24-48 hours for sterilization. S3. Immerse the part to be extracted in an alcohol for extraction, and collect the extract; concentrate under reduced pressure to obtain a concentrated solution; S4. Extract the concentrate using organic esters; S5. The extract is desolventized to obtain chrysanthemum extract; In step S1, at least one part of the flowers, stems, and leaves of a fresh chrysanthemum plant is taken. In step S4, the amount of organic esters added is at least 5 times the volume of the concentrate, and the extraction is performed at least 3 times. The solvent removal process in S4 is vacuum concentration; the organic esters are ethyl acetate and / or ethyl valerate. In S2, the number of Bacillus subtilis in the solution is ≥1×10⁻⁶. 9 CFU; material-to-liquid ratio ≥1 mL / g; In step S2, after sterilization, a drying process is performed. In step S3, the alcohol is methanol and / or ethanol; the amount of alcohol added each time is 3-5 times the volume of the raw material. In step S3, the concentration is carried out under reduced pressure until the volume of the concentrated liquid no longer changes. In step S3, the extraction method is reflux extraction, performed at least three times, with each extraction lasting at least three hours.

2. The application of the high-hydrogenase extract of chrysanthemum obtained by the preparation method according to claim 1 in the preparation of cosmetics.

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