Preparation method of saussurea involucrata extract containing saussurea involucrata amide

Abstract

The preparation method comprises the following steps: S1, taking a fresh mirabilis jalapa plant as an extracted part, and extracting the mirabilis jalapa plant to obtain a mirabilis jalapa extract with high mirabilis jalapa amide; s2, mixing the extracted part with a bacillus subtilis solution; then placing for 24-48 hours, and sterilizing; s3, immersing the extracted part into alcohol for extraction, and taking an extracting solution part; concentrating under reduced pressure to obtain a concentrated solution; s4, extracting the concentrated solution by using organic ester to obtain an extracting solution; and S5, carrying out desolventizing treatment on the extracting solution to obtain the chrysanthemum indicum extract. The method comprises the following steps: pre-treating fresh euphorbia helioscopia plants by using a bacillus subtilis solution; and the yield of the vermilion amide in the extract can be obviously improved. According to the method, recyclable alcohols and organic esters are used as extraction solvents, extraction process parameters are optimized, and the extraction efficiency and the final effective component content are optimized.

Description

Technical Field

[0001] The invention belongs to the field of plant extraction, and in particular relates to a method for preparing a spilanthol extract containing high spilanthol amide. Background Art

[0002] Acmella oleracea, also known as longan chrysanthemum, is a species of the genus Acmella in the Asteraceae family. Its extract is called Acmella oleracea extract in the INCI system, and its English INCI name is Acmella oleracea EXTRACT. It is a dark brown to yellow oil-soluble liquid. Its plant extract standard substance is spilantholamide ((2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide, with a structural formula of Figure 1 shown).

[0003] Spinach extract is considered to have great potential both topically and internally due to its major lipophilic N-alkylamide (N-alkylamide, derived from isobutylamide). Alkylamide is a secondary metabolite with anti-inflammatory, immunomodulatory and cannabinoid effects; therefore, Spinach extract has multifaceted activities such as analgesia, antibacterial, stimulation of collagen production, hydration, antioxidant and anti-inflammatory.

[0004] At present, the artificial synthesis method for spilanthol has the problems of too long synthesis route and complicated operation; CN117510358A proposes a preparation method of spilanthol (2E, 6Z, 8E)-N-isobutyl-2,6,8-decatrienamide, which is simpler than the route proposed by Yoshihiko Ikeda et al. in 1986, but still requires 7 steps of reaction to synthesize the target product, which is still relatively cumbersome.

[0005] Moreover, in addition to spilanthol, there are other unknown active substances in spilanthol extract, so artificial synthesis and natural extraction are not mutually exclusive solutions; and the highest content of spilanthol known in spilanthol extract is 48wt%, which has great room for improvement. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a spilanthol extract containing high spilanthol amide, so as to effectively increase the content of spilanthol amide in the spilanthol (ACMELLA OLERACEA) extract.

[0007] In order to achieve the above object, the present invention provides a method for preparing a spilanthol extract containing high spilanthol amide, the method comprising:

[0008] S1. Take fresh Globenia odorifera plants as the extracted part;

[0009] S2, mixing the extracted part with the Bacillus subtilis solution; then leaving it for 24-48 hours for sterilization;

[0010] S3, immersing the extracted part in alcohol for extraction, taking the extract; and concentrating under reduced pressure to obtain a concentrated solution;

[0011] S4, extracting the concentrated solution with organic esters to obtain an extract;

[0012] S5, performing solvent removal treatment on the extract to obtain the spathodea chrysanthemum extract.

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

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

[0015] Preferably, in S2, after the sterilization treatment, a drying treatment is performed.

[0016] Preferably, in 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.

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

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

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

[0020] Preferably, in S4, the amount of organic ester added is at least 5 times the volume of the concentrated solution, and the number of extractions is at least 3 times.

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

[0022] The present invention also provides the use of the above-mentioned spilanthol extract containing high spilanthol in the preparation of cosmetics.

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

[0024] (1) The present invention found that the yield of spilanthol in the extract can be significantly increased by pre-treating fresh spilanthol plants with a Bacillus subtilis solution, while this effect is not achieved by using dried spilanthol plants as raw materials.

[0025] (2) The present invention uses recyclable alcohols and organic esters as extraction solvents, and optimizes the extraction process parameters, the extraction efficiency and the final content of effective ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0027] Figure 1 It is the structural formula of spilanthol. DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the present invention is further described below in conjunction with specific serial numbers, wherein the terms used in the serial numbers are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0029] In the present invention:

[0030] The globeflower plants were sourced from a local farm.

[0031] Bacillus subtilis was obtained from Jinan Xinran Chemical Co., Ltd., with a CFU of 1×10 11 .

[0032] EM bacteria were obtained from Jinan Xinran Chemical Co., Ltd., with a CFU of 1×10 11 .

[0033] Cellulase comes from Jianchen brand flagship store.

[0034] Example 1

[0035] The preparation method of the spatholobi extract comprises the following steps:

[0036] S1. Take the flowers, stems and leaves of fresh Globenia plants (harvested within 24 hours) as the extracted parts;

[0037] S2. Mix the extracted part with a 100-fold diluted Bacillus subtilis solution at a solid-liquid ratio of 1 mL / g, and then place it for 24 hours; take out the extracted part and sterilize it at a high temperature of more than 120° C., and then dry it to constant weight.

[0038] S3, immersing the extracted part in anhydrous ethanol for reflux extraction, each time for 3 hours, reflux three times; the amount of anhydrous ethanol added is 5 times that of the extracted part;

[0039] After the reflux extraction is completed, the solid part is discarded and the extract is taken; the concentrated solution is obtained by decompression and concentration until the volume does not change;

[0040] S4, extracting the concentrated solution with 5 times the volume of ethyl acetate three times to obtain an extract;

[0041] S5. Combine the three extracts, concentrate under reduced pressure, and recover the solvent to obtain an extract of spathodea japonica.

[0042] The difference between Example 2 and Example 1 is that:

[0043] In step S1, flowers, stems and leaves of fresh zinnia plants are taken and left for 7 days to serve as the extracted parts.

[0044] The difference between Example 3 and Example 1 is that:

[0045] In step S1, the dried flowers and stems of the zinnia plant are taken as the extracted parts.

[0046] The difference between Example 4 and Example 1 is that:

[0047] Step S2 is: mixing the extracted part with the 100-fold diluted cellulase solution at a solid-liquid ratio of 1 mL / g, and then leaving it for 24 hours; and then drying it to constant weight.

[0048] The difference between Example 5 and Example 1 is that:

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

[0050] The difference between Example 6 and Example 1 is that:

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

[0052] The difference between Example 7 and Example 1 is that:

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

[0054] The difference between Example 8 and Example 1 is that:

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

[0056] The difference between Example 9 and Example 1 is that:

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

[0058] The difference between Example 10 and Example 1 is that:

[0059] The solution in step S4 is acetone.

[0060] The difference between Example 11 and Example 1 is that:

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

[0062] The difference between Example 12 and Example 1 is that:

[0063] After step S3, step S4 is no longer performed.

[0064] The difference between Example 13 and Example 1 is that:

[0065] In step S2, step S3 is skipped and the process goes directly to step S4.

[0066] The difference between Example 13 and Example 1 is that:

[0067] In step S2, step S3 is performed first, then step S4 is performed, and finally step 5 is performed.

[0068] Determination of spilanthol content in a fused silica capillary column with a stationary phase of 35% phenyl-methylpolysiloxane, a column length of 30m, an inner diameter of 0.25mm, and a film thickness of 0.25μm.

[0069] The present invention adopts HPLC (high performance liquid chromatography) method to detect the content of spilanthol.

[0070] Entrust Chengdu Zhichun Bencao Biotechnology Co., Ltd. to conduct the test;

[0071] The chromatographic conditions are:

[0072] Chromatographic column: RP18 reverse phase separation column;

[0073] Injection volume: 10 μl;

[0074] Column box temperature: 40°C;

[0075] Mobile phase: acetonitrile / water (50 / 50)

[0076] Detection wavelength: 240nm.

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

[0078] Table 1 Detection results of spilanthol content

[0079] Sample name Content of spilanthol (%) 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 Embodiment 11 62.18 Example 12 41.12 Example 13 18.21 Embodiment 14 43.26 Acmefleur (commercially available) 48.01

[0080] As shown in Table 1:

[0081] As can be seen from the comparison of Example 1, Example 5, and Example 11, in the first step of the extraction of the present invention, whether methanol or ethanol is used; when ethyl acetate or ethyl valerate is used during extraction, better extraction effects can be achieved, and other alcohols or organic esters can achieve similar effects.

[0082] According to the record 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 spilanthol all contain spilanthol, so the present invention uses this part as the extraction part.

[0083] As can be seen from the comparison of Example 1, Example 2, Example 3, and Example 6, the pretreatment with Bacillus subtilis solution in the present invention can significantly increase the yield of spilanthol, but pretreatment after the spilanthol plants have been placed for 7 days or dried has no effect on increasing the yield. The present invention speculates that since spilanthol is a secondary metabolite of spilanthol, it must be produced when the plant still has physiological activities, and the physiological effect of Bacillus subtilis can stimulate spilanthol to produce spilanthol.

[0084] As can be seen from the comparison of Example 1, Example 4, and Example 7, the inventor tried to use cellulase and EM bacteria instead of Bacillus subtilis solution for pretreatment, but there was no effect of improving the yield. The inventor speculates that pathogens harmful to Asteraceae such as Downy Mildew, Puccinia chrysanthemi, Alternaria alternata, and Fusarium oxysporum can also achieve similar effects. However, considering that the use of such pathogens has the risk of biological leakage and the subsequent sterilization may be difficult and the subsequent application may be harmful to the human body, such experiments are eliminated.

[0085] As can be seen from the comparison of Example 1, Example 8, Example 9, Example 10, and Example 11; in the S3 extraction step, alcohols are the best choice; in step S4, only organic esters are used for extraction to further improve the yield; these effects are also 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 OC, SILVA PMS, SILVAS YS, 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, the yield is significantly different from that of the extraction using only alcohols; and the yield of first extracting with organic esters and then extracting with alcohols is no different from that of extracting with only alcohols. It can be seen that the order of solvents also has a significant effect on the yield.

[0087] It should be noted that the workflow described above is merely illustrative and does not limit the scope of protection of the present invention. In practical applications, technicians in this field can select part or all of them according to actual needs to achieve the purpose of the present embodiment, and no limitation is made here.

[0088] In addition, for technical details that are not described in detail in this embodiment, reference can be made to the parameter operation method provided in any embodiment of the present invention, and will not be repeated here.

[0089] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or system. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or system including the element.

[0090] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0091] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a spilanthol extract having high spilanthol amide, characterized in that: The method comprises: S1. Take fresh Globenia odorifera plants as the extracted part; S2, mixing the extracted part with the Bacillus subtilis solution; then leaving it for 24-48 hours for sterilization; S3, immersing the extracted part in alcohol for extraction, taking the extract; and concentrating under reduced pressure to obtain a concentrated solution; S4, extracting the concentrated solution with organic esters; S5, performing solvent removal treatment on the extract to obtain the spathodea chrysanthemum extract.

2. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In S1, at least one part of the flower, stem and leaf of a fresh agave violet plant is taken.

3. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In S2, the number of bacteria in the Bacillus subtilis solution is ≥ 1×10 9 CFU; material-liquid ratio ≥1mL / g.

4. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In S2, after the sterilization treatment, a drying treatment is performed.

5. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In 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.

6. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In S3, the concentrated solution is concentrated under reduced pressure until the volume of the concentrated solution no longer changes.

7. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In S3, the extraction method is reflux extraction, the number of times is at least 3 times; each time is at least 3 hours.

8. The method for preparing the spilanthol extract containing high spilantholamide according to claim 1, characterized in that: In the step S4, the amount of organic ester added is at least 5 times the volume of the concentrated solution, and the number of extractions is at least 3 times.

9. The method for preparing the spilanthol extract containing high spilanthol amide according to claim 1, characterized in that: The desolventizing treatment in S4 is concentration under reduced pressure; and the organic esters are ethyl acetate and / or ethyl valerate.

10. Use of the spilanthol extract containing high spilantholamide according to any one of claims 1 to 9 in the preparation of cosmetics.

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