A method for preparing leek seed extract and its application

Abstract

This invention discloses a method for preparing leek seed extract and its applications. The method includes two-phase solvent extraction, crystallization, liquid-phase preparation of active ingredient A, gel column preparation of active ingredient B, and low-temperature drying. The preparation process of this invention is simple and efficient, and the obtained leek seed extract is light in color and has a strong effect on promoting osteoblast proliferation and differentiation. It can be applied in the fields of pharmaceuticals, food, and cosmetics related to the prevention of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of functional natural product extract preparation technology, specifically to a method for preparing leek seed extract and its application. Background Technology

[0002] Osteoporosis is a common chronic skeletal disease caused by bone tissue damage exceeding the body's ability to regenerate and repair. Currently, estrogen replacement therapy and teriparatide are commonly used to prevent and treat osteoporosis, but these methods all exhibit certain side effects. While the more widely accepted approach of combining vitamin D with calcium supplementation is effective for some patients, excessive intake can increase the risk of kidney stones and cardiovascular disease. Modern research indicates that extracts from certain medicinal and edible plants and animals can significantly promote bone cell proliferation and differentiation, effectively preventing osteoporosis, and offering advantages such as wide availability, high bioavailability, and safety with no side effects. Examples include collagen peptides, pigeon pea leaf extract, and soybean isoflavone extract.

[0003] Leek seeds are the dried, mature seeds of the leek plant (Allium tuberosum Rottl. ex Spreng.), belonging to the Liliaceae family. Modern research shows that leek seeds are rich in nucleosides, saponins, flavonoids, and polysaccharides, among which nucleosides and saponins have significant effects in enhancing immune function, sexual function, and anti-stress. Currently, products made from leek seeds mainly focus on tonifying yang and replenishing deficiency, and improving immunity; there are few reports on leek seed extracts developed for the purpose of preventing osteoporosis. Therefore, this invention utilizes biphasic solvent extraction, crystallization, preparative liquid chromatography, and gel column chromatography for specific purification and retention of active ingredients to prepare leek seed extract. Experiments have confirmed that the leek seed extract prepared by this invention has a good effect on promoting osteoblast proliferation and differentiation, and can be applied in the fields of pharmaceuticals, foods, and cosmetics related to the prevention of osteoporosis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing leek seed extract. The technical solution adopted by this invention to solve its technical problem is as follows:

[0005] A method for preparing leek seed extract includes the following steps:

[0006] Step 1, Two-phase solvent extraction: First, coarsely crush and sieve the leek seeds to obtain crushed leek seed powder. Then, place the leek seed powder and ammonium sulfate in an extraction container at a certain ratio and use 15-25 times the amount of two-phase solvent for low-temperature stirring extraction. Filter, collect the extract, and let it stand to obtain the separated extract. Collect the upper extract and concentrate it to a paste state to obtain sample 1. Collect the lower extract and concentrate it to 1 / (10-20) of the original volume to obtain sample 2.

[0007] Step 2, crystallization: Add ethanol solution to sample 1, heat and stir to dissolve for 0.5-1.5 h, crystallize at room temperature, collect the precipitate, add pure water to dissolve and filter through a 0.45 μm filter membrane, collect the permeate;

[0008] Step 3: Preparation of active ingredient A by liquid chromatography: Pour the permeate into a preparative liquid chromatograph for gradient elution, collect the fractions eluted over a specific time period, and dry at low temperature to obtain active ingredient A;

[0009] Step 4: Preparation of active ingredient B by gel column: Sample 2 is loaded into a resin column packed with DEAE Seplife A25 packing material, and then eluted sequentially with sodium chloride solutions of 0, 0.1, 0.3, and 0.5 mol / L. The eluted portion of 0.3 mol / L sodium chloride solution is collected, dialyzed through a dialysis bag, and then used for later use to obtain a solution containing active ingredient B.

[0010] Step 5, Low-temperature drying: Dissolve active ingredient A obtained in step 3 in a solution containing active ingredient B, mix well, react at low temperature for 2-6 hours, and then dry at low temperature to obtain leek seed extract.

[0011] Preferably, in step one, the mass ratio of leek seed powder to ammonium sulfate is 1:5-10, and the two-phase solvent is composed of pure water: tert-butanol = 1:1.5-3.

[0012] Preferably, in step one, the extraction temperature is 30-40℃, the extraction time is 1.5-2.5h, and the standing time is 12-24h.

[0013] Preferably, in step two, the mass-to-volume ratio of the extract to the ethanol solution is 1:3-5, the volume fraction of the ethanol solution is 70-95%, the heating temperature is 80-95℃, and the crystallization time at room temperature is 12-24h.

[0014] Preferably, in step three, the mobile phase used in the prepared liquid chromatograph is methanol-water, the elution flow rate is 15-25 mL / min, and the detection wavelength is 190-600 nm.

[0015] Preferably, in step three, the elution gradient of the mobile phase is as follows:

[0016] Time (min) Acetonitrile or methanol (%) water(%) 0～8 6 94 8～20 6→18 94→82 20～45 18→27 82→73 45～70 27→55 73→45 .

[0017] Preferably, in step three, the specific time period for collecting the fraction using the liquid chromatograph is 40.0-44.0 min.

[0018] Preferably, in step three, the main component of active ingredient A is hyperoside, with a purity of ≥99%, and this active ingredient has a significant effect on promoting bone cell proliferation and differentiation.

[0019] Preferably, in step four, the sample loading flow rate for sample 2 is 1-3 BV / h, and the molecular weight specification of the dialysis bag is 8000-10000 Da.

[0020] Preferably, in step four, the solution containing active ingredient B mainly contains high-purity leek seed polysaccharide with a purity of ≥90%. It does not promote bone cell proliferation and differentiation on its own, but when combined with active ingredient A in step three, it can significantly enhance the ability of active ingredient A to promote bone cell proliferation and differentiation.

[0021] Preferably, in step five, the low-temperature reaction temperature is 40-60℃ and the low-temperature reaction time is 2-6h.

[0022] Preferably, in step five, the low-temperature drying temperature is 50-60℃.

[0023] Another objective of this invention is to provide the leek seed extract for use in pharmaceuticals, food, and cosmetics related to the prevention of osteoporosis.

[0024] Preferably, the obtained leek seed extract has a significant effect on promoting osteoblast proliferation and preventing osteoporosis.

[0025] The beneficial effects of this invention are:

[0026] 1. The preparation process of this invention is simple and efficient. The two components, active ingredient A and active ingredient B, in the leek seed extract have high purity and synergistic effect.

[0027] 2. The leek seed extract prepared by this invention has been shown by cell activity tests to have a good effect on promoting osteoblast proliferation and differentiation, and can be applied in the fields of medicines, food and cosmetics related to the prevention of osteoporosis. Attached Figure Description

[0028] Figure 1 The image shows the prepared liquid chromatogram of the permeate in Example 1.

[0029] Figure 2 The effects of each embodiment and comparative example on the relative proliferation rate of human osteoblasts.

[0030] Figure 3 ALP staining images for each embodiment and comparative example. Detailed Implementation

[0031] Example 1:

[0032] This embodiment provides a method for preparing leek seed extract, the specific steps of which are as follows:

[0033] (1) First, the leek seeds were coarsely crushed and sieved to obtain 0.5 kg of crushed leek seed powder. Then, 0.5 kg of leek seed powder and 2.5 kg of ammonium sulfate were placed in an extraction container and extracted at 30°C with 45 L of two-phase solvent (18 L of pure water and 27 L of tert-butanol) for 1.5 h. The mixture was filtered, the extract was collected, and the mixture was allowed to stand for 12 h to obtain a layered extract. The upper extract was collected and concentrated to a paste state to obtain 98 g of sample 1. The lower extract was collected and concentrated to 1 / 20 of the original volume to obtain 0.59 L of sample 2. 294 mL of 70% ethanol solution was added to sample 1 and heated to 80°C and stirred to dissolve for 0.5 h. After dissolution, the mixture was crystallized at room temperature for 12 h. 18 g of precipitate was collected, dissolved in pure water and filtered through a 0.45 μm filter membrane. The permeate was collected.

[0034] (2) Pour the permeate into a preparative liquid chromatograph for gradient elution, see [link to relevant documentation]. Figure 1 As shown, the mobile phase used in the preparative liquid chromatograph was methanol-water, the elution flow rate was 15 mL / min, the detection wavelength was 282 nm, and the mobile phase elution gradient was:

[0035] Time (min) Acetonitrile or methanol (%) water(%) 0～8 6 94 8～20 6→18 94→82 20～45 18→27 82→73 45～70 27→55 73→45

[0036] The fraction eluented during the period of 40-43.8 min was collected and dried at low temperature to obtain 2g of active ingredient A. Then, sample 2 was loaded into a resin column packed with DEAE Seplife A25 packing material at a flow rate of 1 BV / h, and then eluted sequentially with sodium chloride solutions of 0, 0.1, 0.3, and 0.5 mol / L. The eluted fraction with 0.3 mol / L sodium chloride solution was collected, dialyzed using a dialysis bag with a molecular weight of 8000 Da, and then used for later use, to obtain 20 mL of solution containing active ingredient B. Finally, active ingredient A was dissolved in the solution containing active ingredient B, mixed well, and reacted at low temperature at 40℃ for 2 h, and then dried at low temperature to obtain 3.1g of leek seed extract.

[0037] Example 2:

[0038] This embodiment provides a method for preparing leek seed extract, the specific steps of which are as follows:

[0039] (1) First, the leek seeds were coarsely crushed and sieved to obtain 0.5 kg of crushed leek seed powder. Then, 0.5 kg of leek seed powder and 4 kg of ammonium sulfate were placed in an extraction container and extracted at 35°C with 90 L of two-phase solvent (30 L of pure water and 60 L of tert-butanol) for 2.0 h. After filtration, the extract was collected and allowed to stand for 18 h to obtain a layered extract. The upper extract was collected and concentrated to a paste state to obtain 101 g of sample 1. The lower extract was collected and concentrated to 1 / 15 of the original volume to obtain 1.8 L of sample 2. 404 mL of 85% ethanol solution was added to sample 1 and heated to 90°C and stirred to dissolve for 1.0 h. After dissolution, the sample was crystallized at room temperature for 18 h. 21 g of precipitate was collected, dissolved in pure water and filtered through a 0.45 μm filter membrane. The permeate was collected.

[0040] (2) The permeate was poured into a preparative liquid chromatograph for gradient elution. The mobile phase used in the preparative liquid chromatograph was methanol-water, the elution flow rate was 20 mL / min, the detection wavelength was 282 nm, and the elution gradient of the mobile phase was:

[0041] Time (min) Acetonitrile or methanol (%) water(%) 0～8 6 94 8～20 6→18 94→82 20～45 18→27 82→73 45～70 27→55 73→45

[0042] The fraction eluent from 40.1 to 43.9 min was collected and dried at low temperature to obtain 2.1 g of active ingredient A. Sample 2 was then loaded onto a resin column packed with DEAE Seplife A25 packing material at a flow rate of 1.5 BV / h. It was then eluted sequentially with sodium chloride solutions of 0, 0.1, 0.3, and 0.5 mol / L. The 0.3 mol / L sodium chloride eluent fraction was collected and dialyzed using a dialysis bag with a molecular weight of 9000 Da, yielding 21 mL of a solution containing active ingredient B. Finally, active ingredient A was dissolved in the solution containing active ingredient B, mixed thoroughly, and reacted at 50℃ for 4 h, followed by low-temperature drying to obtain 3.3 g of leek seed extract.

[0043] Example 3:

[0044] This embodiment provides a method for preparing leek seed extract, the specific steps of which are as follows:

[0045] (1) First, the leek seeds were coarsely crushed and sieved to obtain 0.5 kg of crushed leek seed powder. Then, 0.5 kg of leek seed powder and 5 kg of ammonium sulfate were placed in an extraction container and extracted at 40°C with 137.5 L of a two-phase solvent (34.4 L of pure water and 103.1 L of tert-butanol) for 2.5 h. The mixture was filtered, the extract was collected, and the mixture was allowed to stand for 24 h to obtain a layered extract. The upper layer of the extract was collected and concentrated to a paste state to obtain 103 g of sample 1. The lower layer of the extract was collected and concentrated to 1 / 10 of the original volume to obtain 3.10 L of sample 2. 515 mL of 95% ethanol solution was added to sample 1 and heated to 95°C and stirred to dissolve for 1.5 h. After dissolution, the mixture was crystallized at room temperature for 24 h. 23 g of precipitate was collected, dissolved in pure water, and filtered through a 0.45 μm filter membrane. The permeate was collected.

[0046] (2) The permeate was poured into a preparative liquid chromatograph for gradient elution. The mobile phase used in the preparative liquid chromatograph was methanol-water, the elution flow rate was 25 mL / min, the detection wavelength was 282 nm, and the elution gradient of the mobile phase was:

[0047] Time (min) Acetonitrile or methanol (%) water(%) 0～8 6 94 8～20 6→18 94→82 20～45 18→27 82→73 45～70 27→55 73→45

[0048] The fraction eluent from the period of 40.2-44 min was collected and dried at low temperature to obtain 2.2 g of active ingredient A. Sample 2 was then loaded into a resin column packed with DEAE Seplife A25 packing material at a flow rate of 3 BV / h. It was then eluted sequentially with sodium chloride solutions of 0, 0.1, 0.3, and 0.5 mol / L. The 0.3 mol / L sodium chloride solution eluent was collected and dialyzed using a dialysis bag with a molecular weight of 10000 Da, yielding 19 mL of solution containing active ingredient B. Finally, active ingredient A was dissolved in the solution containing active ingredient B, mixed thoroughly, and reacted at 60℃ for 6 h, followed by low-temperature drying to obtain 3.35 g of leek seed extract.

[0049] Comparative Example 1:

[0050] This comparative example provides a method for preparing leek seed extract, the specific steps of which are as follows:

[0051] (1) First, the leek seeds were coarsely crushed and sieved to obtain 0.5 kg of crushed leek seed powder. Then, 0.5 kg of leek seed powder and 4 kg of ammonium sulfate were placed in an extraction container and extracted at 35°C with 90 L of two-phase solvent (30 L of pure water and 60 L of tert-butanol) for 2.0 h. The mixture was filtered, the extract was collected, and the mixture was allowed to stand for 18 h to obtain a layered extract. The upper extract was collected and concentrated to a paste state to obtain 101 g of sample 1. 404 mL of 85% ethanol solution was added to sample 1 and the mixture was heated to 90°C and stirred to dissolve for 1.0 h. After dissolution, the mixture was crystallized at room temperature for 18 h. 21 g of precipitate was collected, dissolved in pure water and filtered through a 0.45 μm filter membrane. The permeate was collected.

[0052] (2) The permeate was poured into a preparative liquid chromatograph for gradient elution. The mobile phase used in the preparative liquid chromatograph was methanol-water, the elution flow rate was 20 mL / min, the detection wavelength was 282 nm, and the elution gradient of the mobile phase was:

[0053] Time (min) Acetonitrile or methanol (%) water(%) 0～8 6 94 8～20 6→18 94→82 20～45 18→27 82→73 45～70 27→55 73→45

[0054] The fraction that flowed out during the period of 40.1-43.9 min was collected and dried at low temperature to obtain 2.1 g of active ingredient A sample.

[0055] Comparative Example 2:

[0056] This comparative example provides a method for preparing leek seed extract, the specific steps of which are as follows:

[0057] First, 0.5 kg of crushed leek seed powder was obtained by coarsely crushing and sieving leek seed powder. Then, 0.5 kg of leek seed powder and 5 kg of ammonium sulfate were placed in an extraction container and extracted at 40°C with 137.5 L of a two-phase solvent (34.4 L of pure water and 103.1 L of tert-butanol) for 2.5 h. After filtration, the extract was collected and allowed to stand for 24 h to obtain a layered extract. The lower layer of extract was collected and concentrated to 1 / 10 of the original volume to obtain 3.10 L of sample 2. Sample 2 was then loaded into a resin column packed with DEAE Seplife A25 packing material at a flow rate of 3 BV / h. It was then eluted sequentially with sodium chloride solutions of 0, 0.1, 0.3, and 0.5 mol / L. The 0.3 mol / L sodium chloride solution eluent was collected and dialyzed using a dialysis bag with a molecular weight of 10000 Da to obtain 19 mL of solution containing active ingredient B. The solution was then freeze-dried under vacuum to obtain the sample.

[0058] Comparative Example 3:

[0059] A calcium gluconate solution sample (calcium content 15 mg / mL), which has been shown to promote the proliferation and differentiation of human osteoblasts, was set up as Comparative Example 3.

[0060] Experimental Example 1:

[0061] Cell experiments were conducted on the samples from Examples 1-3 and Comparative Examples 1-3 to detect whether the above samples had the effect of promoting the proliferation and differentiation of human osteoblasts, thereby demonstrating whether the leek seed extract obtained in this invention can prevent osteoporosis.

[0062] Main reagents and instruments: Alkaline phosphatase (ALP) detection kit (Sigma Co., Ltd.); Human osteoblasts (Beijing Anolun Biotechnology Co., Ltd.); Alkaline phosphatase (ALP) staining kit (Beijing Ita Biotechnology Co., Ltd.)

[0063] Synergy H1 multi-functional microplate reader (Biotek, USA); AB135-S analytical balance (Mettler-Toledo); RM2016 pathological slider (Leica Instruments, Shanghai); Mshot MSX2 imaging system (Mingmei Optoelectronic Technology, Guangzhou).

[0064] ① MTT assay for the relative proliferation rate of human osteoblasts

[0065] First, passaged human osteoblasts were digested with EDTA, then centrifuged at 2000 rpm for 10 min. The resulting cell suspension was then seeded onto 96-well cell culture plates. After culturing for 20 h, samples at a concentration of 30 μg / mL (calcium gluconate solution containing 15 mg / mL calcium) were added. Cells treated with culture medium without samples served as a control group, and PBS buffer served as a blank group. Culture continued, and every 5 days after sample addition, 15 μL of MTT solution was added to each well of the 96-well cell culture plate. After culturing for another 5 h, the absorbance of the plates was measured at 450 nm using a microplate reader. The relative proliferation rate of human osteoblasts was calculated using the following formula:

[0066]

[0067] Experimental results: Figure 2 This is a trend graph showing the influence of each embodiment and comparative example on the relative proliferation rate of human osteoblasts. Analysis of the results in the graph shows that, except for Comparative Example 2, all other sample groups exhibited a proliferative effect on human osteoblasts. Overall, the order of relative proliferative capacity of each sample group on human osteoblasts is: Example 3 > Example 1 > Example 2 > Comparative Example 1 > Comparative Example 3 > Comparative Example 2. This indicates that the leek seed extract prepared in this invention has a significantly higher proliferative and differentiation-promoting effect on human osteoblasts than single active ingredient A and calcium gluconate solution. Furthermore, the graph also shows that active ingredient B has no proliferative effect on human osteoblasts, but the combination of active ingredient B and active ingredient A can significantly enhance the proliferative capacity of active ingredient A in promoting human osteoblast proliferation.

[0068] ②ALP staining experiment

[0069] Human osteoblasts in the logarithmic growth phase were seeded onto sterile cell culture plates and cultured for a period of time. When the cells adhered and grew, samples from each example and comparative example (sample concentration 30 μg / mL, of which the calcium content in the calcium gluconate solution was 15 mg / mL) were added. After culturing in a cell incubator for another 96 h, ALP staining was performed using a staining kit, and the staining results were observed.

[0070] Experimental results: Alkaline phosphatase (ALP) is a marker product produced during the early differentiation of human osteoblasts, and it represents the degree of bone calcification. Figure 3 The figure reflects the ALP staining of samples from different examples and comparative examples 96 hours after sample addition. As shown in the figure, all sample groups from the examples and comparative examples showed positive reactions after interaction with human osteoblasts. Among them, the reaction intensity of Examples 1-3 was significantly higher than that of Comparative Examples 1-3, which indicates that the leek seed extract prepared in this invention has a strong effect on promoting human osteoblast differentiation.

[0071] Based on the above data analysis results, the leek seed extract prepared in the embodiments of the present invention has a strong effect on promoting the proliferation and differentiation of human osteoblasts, and can be applied in the fields of pharmaceuticals, foods, and cosmetics related to the prevention of osteoporosis. In summary, the above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not cover all aspects of the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention is determined by the scope defined in the claims.

Claims

1. A method for preparing a semen foeniculi extract for preventing osteoporosis, characterized by, Comprising the following steps: Step one, two-phase solvent extraction: first, the crude Allium tuberosum seed is crushed and sieved to obtain broken Allium tuberosum seed powder, then the Allium tuberosum seed powder and ammonium sulfate are placed in an extraction container according to a mass ratio of 1:5-10, and 15-25 times the amount of two-phase solvent is used for low-temperature stirring extraction, filtration, collection of the extract, and standing to obtain layered extract; Collect the upper layer of the extract and concentrate it to the extract state to obtain sample 1, collect the lower layer of the extract and concentrate it to 1 / 10-20 of the original volume to obtain sample 2; the two-phase solvent is composed of pure water: t-butyl alcohol = 1:1.5-3; the extraction temperature is 30-40 ℃, the extraction time is 1.5-2.5 h, and the standing time is 12-24 h; Step two, crystallization: add an ethanol solution to sample 1 and stir to dissolve at an elevated temperature for 0.5-1.5 h, then crystallize at room temperature, collect the precipitate, dissolve it in pure water, and pass it through a 0.45 μm filter membrane to collect the permeate; Step three, preparation of active ingredient A: pour the permeate into a preparative liquid chromatograph for gradient elution, collect the fraction eluted at 40.0-44.0 min, and dry it at low temperature to obtain active ingredient A; The mobile phase used in the preparative liquid chromatograph is methanol-water, the elution flow rate is 15-25 mL / min, and the detection wavelength is 190-600 nm; The elution gradient of the mobile phase is as follows: 0-8 min, methanol accounts for 6%, and water accounts for 94%; 8-20 min, methanol accounts for 6-18%, and water accounts for 94-82%; 20-45 min, methanol accounts for 18-27%, and water accounts for 82-73%; 45-70 min, methanol accounts for 27-55%, and water accounts for 73-45%; Step four, preparation of active ingredient B by gel column: sample 2 is loaded onto a resin column filled with DEAE Seplife A25 filler, then eluted with 0, 0.1, 0.3, and 0.5 mol / L sodium chloride solutions in sequence, and the elution part of the 0.3 mol / L sodium chloride solution is collected, dialyzed, and then used, i.e. a solution containing active ingredient B is obtained; Step five, low-temperature drying: active ingredient A obtained in step three is dissolved in the solution containing active ingredient B, mixed, then reacted at 40-60 ℃ for 2-6 h, and then dried at 50-60 ℃ to obtain Allium tuberosum seed extract.

2. The production method according to claim 1, characterized by, In step two, the mass-volume ratio of the extract to the ethanol solution is 1:3-5, the volume fraction of the ethanol solution is 70-95%, the elevated temperature is 80-95 ℃, and the crystallization time at room temperature is 12-24 h.

3. The preparation method according to claim 1, characterized in that, In step four, the sample 2 loading flow rate is 1-3 BV / h, and the molecular weight specification of the dialysis bag is 8000-10000 Da.

4. Use of the Allium tuberosum seed extract prepared by the method of any one of claims 1-3 in the preparation of a drug for preventing osteoporosis.

Images