Preparation method and application of lotus germ active matter

Abstract

The invention relates to the field of daily chemical products, in particular to a preparation method and application of a lotus germ active matter. The content of schaftoside in the lotus germ active matter is larger than or equal to 1.5 wt%, the content of methyl liensinine is larger than or equal to 3 wt%, and the chlorophyll concentration is smaller than or equal to 200 micrograms / g. According to the method, chlorophyll is selectively removed through two times of supercritical extraction, so that the problem of chlorophyll residues in the product is effectively improved, and after two times of supercritical extraction and entrainer and solvent extraction, the content of target components such as neferine is unexpectedly not influenced; according to the method disclosed by the invention, chlorophyll can be specifically and selectively removed without basically influencing the content of neensinine. The prepared lotus germ oil can inhibit / reduce secretion of inflammatory factors TNF-alpha and IL-6, so that the lotus germ oil has anti-inflammatory and soothing effects; the prepared lotus germ active matter inhibits secretion of ROS (reactive oxygen species) by improving ATP (adenosine triphosphate) synthesis, so that the effect of resisting skin aging is achieved, and the application direction of the lotus germ extract in cosmetics is widened.

Description

Technical Field

[0001] The invention relates to the field of daily chemical products, and in particular to a preparation method and application of lotus germ active substances. Background Art

[0002] Lotus germ Plumula Nelumbinis is the green germ and radicle of the mature seeds of the lotus plant Nelumbo nucifera Gaertn. of the Nymphaeaceae family. It is mainly produced in Hunan, Hubei, Jiangxi, Jiangsu, Zhejiang and other places. It is a kind of medicinal material with important medicinal value in my country. In folk medicine, people use lotus germ to treat irritability, insomnia, spermatorrhea, thirst, high blood pressure and other symptoms. Phytochemical research shows that lotus germ is rich in oil, alkaloids, and flavonoid active ingredients.

[0003] Studies have shown that lotus germ oil is rich in phytosterols and sterol esters, and the content is much higher than that of wheat germ oil, rice bran oil, corn germ oil, etc., which is relatively rare in other plants. Phytosterols and their esters not only have the functions of lowering serum cholesterol concentration and preventing cardiovascular and cerebrovascular diseases, but also have physiological functions such as anti-inflammatory and analgesic, anti-oxidation, anti-tumor, skin care and beauty, and anti-aging, and are known as the "key to life". Supercritical CO2, as a new type of extraction technology, is particularly suitable for the extraction of fat-soluble components. With its advantages of being green, environmentally friendly and free of solvent residues, it is gradually used in the extraction of fat-soluble chemical components in the daily chemical and pharmaceutical industries.

[0004] Mitochondrial dysfunction is one of the twelve major signs of cell aging. Its function is essential to maintaining the health of cells, and its functional degradation will lead to aging-related. Mitochondria are the power source of cells. With the increase of age, the factors that cause mitochondrial dysfunction mainly include the accumulation of mitochondrial DNA mutations, protein homeostasis defects that lead to instability of respiratory chain complexes, reduced organelle renewal, and changes in mitochondrial dynamics. The most direct manifestation of changes in mitochondrial dynamics is the decrease in mitochondrial membrane potential and the reduction in ATP (adenosine triphosphate) synthesis, which ultimately leads to cell death. In short, mitochondrial damage causes a decrease in the expression of ATP content. The level of ATP can reflect the functional state of mitochondria, and the decrease in ATP indicates the decline of body function.

[0005] Lotus germ contains rich flavonoids and alkaloids active ingredients. The flavonoids are represented by carbonyl flavonoids such as schaftoside, and the alkaloids are represented by methyl neferine. As the core secondary metabolites in lotus germ, they are used as indicator active ingredients to prepare lotus germ actives, and then the effect of lotus germ actives on improving mitochondrial dysfunction is evaluated from the synthesis of ATP. They are also used as active ingredients in skin care products. There is currently no relevant research report.

[0006] Therefore, the present invention is in urgent need of developing the application of lotus germ active ingredients in daily chemical products. Summary of the invention

[0007] The present invention aims to provide application of lotus germ active matter in daily chemical products, and specifically relates to a preparation method and application of the lotus germ active matter.

[0008] In a first aspect of the present invention, a lotus germ active is provided, wherein the lotus germ active has a schaftoside content of ≥1.5wt%, a neferine content of ≥3wt%, and a chlorophyll concentration of ≤200 μg / g.

[0009] In another preferred embodiment, the lotus germ active ingredient contains schaftoside at 1.5wt%-4wt%, neferine at 3wt%-5wt%, and chlorophyll concentration ≤160 μg / g.

[0010] In another preferred embodiment, the lotus germ active ingredient is in the form of freeze-dried powder.

[0011] In another preferred embodiment, the lotus germ active ingredient has a schaftoside content of 2-3wt%, a neferine content of 3-4wt%, and a chlorophyll concentration of ≤180 μg / g, based on dry weight.

[0012] In another preferred embodiment, the lotus germ active ingredient is prepared by the following method: (1) performing supercritical CO2 extraction on the raw lotus embryo to obtain a first extract and a first raffinate; (2) adding an entrainer to the first raffinate of step (1) and performing supercritical CO2 extraction to obtain a second extract containing chlorophyll and a second raffinate dechlorophyllized; (3) taking the dechlorophyllized second extract residue in step (2), and extracting it in ethanol under reflux for 1-3 times to obtain an extract; wherein the volume ratio of the dechlorophyllized second extract residue to ethanol is 1:7-11; (4) The extract obtained in step (3) is concentrated and dried to obtain the lotus germ active ingredient.

[0013] In another preferred embodiment, the water content of the raw lotus germ is 3-5 wt %.

[0014] In another preferred embodiment, the raw material lotus germ is crushed or un-crushed, preferably crushed lotus germ coarse powder, and the lotus germ coarse powder has a particle size of 10-30 meshes.

[0015] In another preferred embodiment, the first extract is lotus germ oil.

[0016] In another preferred embodiment, the extraction pressure of supercritical CO2 extraction in step (1) is 30-40 MPa.

[0017] In another preferred embodiment, the extraction temperature of the supercritical CO2 extraction in step (1) is 45-55°C.

[0018] In another preferred embodiment, the CO2 flow rate of the supercritical CO2 extraction in step (1) is 70-90 L / h.

[0019] In another preferred embodiment, the extraction time of the supercritical CO2 extraction in step (1) is 1.5-2.5h.

[0020] In another preferred embodiment, in step (2), the entrainer is ethanol.

[0021] In another preferred embodiment, the concentration of the entrainer ethanol in step (2) is 85-99 v / v%.

[0022] In another preferred embodiment, the extraction pressure of supercritical CO2 extraction in step (2) is 30-40 MPa.

[0023] In another preferred embodiment, the extraction temperature of the supercritical CO2 extraction in step (2) is 45-55°C.

[0024] In another preferred embodiment, the CO2 flow rate of the supercritical CO2 extraction in step (2) is 70-90 L / h.

[0025] In another preferred embodiment, the extraction time of the supercritical CO2 extraction in step (2) is 0.5-1.5h.

[0026] In another preferred embodiment, the volume ratio of CO2 to entrainer in step (2) is 30-50:1.

[0027] In another preferred embodiment, the entrainer flow rate in step (2) is 1 to 4 L / h.

[0028] In another preferred embodiment, the concentration of ethanol in step (3) is 70-90 v / v%.

[0029] In another preferred embodiment, the extraction is performed 1-3 times in step (3).

[0030] In another preferred embodiment, the extraction in step (3) comprises the steps of: performing a first hot reflux extraction in ethanol, with the extraction material-liquid volume ratio being 1:9-11, the heating temperature being 70-90° C., and the extraction time being 1.5-2.5 h, to obtain a first reflux extract; The extraction residue is subjected to a second hot reflux extraction in ethanol, the extraction material-liquid volume ratio is 1:7-9, the heating temperature is 70-90°C, and the heating time is 0.5-1.5h; and a second reflux extract is obtained; The first reflux extract and the second reflux extract are combined and filtered to obtain an extract.

[0031] In another preferred embodiment, the concentration in step (4) is performed by a conventional concentration method, such as concentration under reduced pressure.

[0032] In another preferred embodiment, the drying in step (4) is performed by a conventional drying method, such as vacuum freeze drying.

[0033] In a second aspect of the present invention, a method for preparing lotus germ active ingredients is provided, the method comprising the steps of: (1) providing a raw material of lotus germ to be subjected to supercritical CO2 extraction in an extraction kettle to obtain a first extract of lotus germ oil and a first raffinate; (2) adding an entrainer to the first raffinate of step (1) to perform supercritical CO2 extraction to obtain a second extract containing chlorophyll and a second raffinate dechlorophyllized; (3) taking the dechlorophyllized second extract residue in step (2), and extracting it in ethanol under reflux for 1-3 times to obtain an extract; wherein the volume ratio of the dechlorophyllized second extract residue to ethanol is 1:7-11; (4) The extract obtained in step (3) is concentrated and dried to obtain lotus germ active ingredients.

[0034] In another preferred embodiment, the water content of the raw lotus germ is 3-5 wt %.

[0035] In another preferred embodiment, the raw material lotus germ is crushed or un-crushed, preferably crushed lotus germ coarse powder, and the lotus germ coarse powder has a particle size of 10-30 meshes.

[0036] In another preferred embodiment, the extraction pressure of the supercritical CO2 extraction in step (1) is 30-40 MPa.

[0037] In another preferred embodiment, the extraction temperature of the supercritical CO2 extraction in step (1) is 45-55°C.

[0038] In another preferred embodiment, the CO2 flow rate of the supercritical CO2 extraction in step (1) is 70-90 L / h.

[0039] In another preferred embodiment, the extraction time of the supercritical CO2 extraction in step (1) is 1.5-2.5h.

[0040] In another preferred embodiment, in the step (2), the entrainer is ethanol.

[0041] In another preferred embodiment, the concentration of the entrainer ethanol in step (2) is 85-99 v / v%.

[0042] In another preferred embodiment, the extraction pressure of the supercritical CO2 extraction in step (2) is 15-40 MPa.

[0043] In another preferred embodiment, the extraction pressure of the supercritical CO2 extraction in step (2) is 20-35 MPa.

[0044] In another preferred embodiment, the extraction temperature of the supercritical CO2 extraction in step (2) is 45-55°C.

[0045] In another preferred embodiment, the volume ratio of the entrainer to CO2 in step (2) is 0.02-0.05:1.

[0046] In another preferred embodiment, in step (2), the flow rate of the entrainer is 1-4 L / h.

[0047] In another preferred embodiment, the supercritical CO 22 The extraction time is 0.5-1.5h.

[0048] In another preferred embodiment, the concentration of ethanol in step (3) is 70-90 v / v%.

[0049] In another preferred embodiment, the extraction is performed 1-3 times in step (3).

[0050] In another preferred embodiment, the extraction in step (3) comprises the steps of: performing a first hot reflux extraction in ethanol, with the extraction material-liquid volume ratio being 1:9-11, the heating temperature being 70-90° C., and the extraction time being 1.5-2.5 h, to obtain a first reflux extract; The extraction residue is subjected to a second hot reflux extraction in ethanol, the extraction material-liquid volume ratio is 1:7-9, the heating temperature is 70-90°C, and the heating time is 0.5-1.5h; and a second reflux extract is obtained; The first reflux extract and the second reflux extract are combined and filtered to obtain an extract.

[0051] In another preferred embodiment, the concentration in step (4) is performed by a conventional concentration method, such as concentration under reduced pressure.

[0052] In another preferred embodiment, the drying in step (4) is performed by a conventional drying method, such as vacuum freeze drying.

[0053] In another preferred embodiment, in the step (1), the method further comprises: performing supercritical CO2 extraction on the lotus germ in an extraction kettle, and releasing the extract, namely lotus germ oil, from a separation kettle.

[0054] In another preferred embodiment, the mass percentage of phytosterols in the lotus germ oil is ≥4%, calculated as β-sitosterol.

[0055] In another preferred embodiment, the mass percentage of phytosterols in the lotus germ oil is 5-7%, calculated as β-sitosterol.

[0056] In a third aspect of the present invention, a lotus germ oil is provided, wherein the lotus germ oil is prepared by the following method: Providing lotus germ as raw material, and subjecting it to supercritical CO2 extraction to obtain lotus germ oil as an extract; The extraction pressure of the supercritical extraction is 20-35Mpa; the extraction temperature is 45-55°C; the CO2 flow rate is 70-90L / h, and the extraction time is 1-5h.

[0057] In another preferred embodiment, the raw material lotus germ contains about 3-5 wt % water.

[0058] In another preferred embodiment, the raw material lotus germ is crushed or un-crushed, preferably crushed lotus germ coarse powder, and the lotus germ coarse powder has a particle size of 10-30 meshes.

[0059] In another preferred embodiment, the mass percentage of phytosterols in the lotus germ oil is ≥4%, calculated as β-sitosterol.

[0060] In another preferred embodiment, the mass percentage of phytosterols in the lotus germ oil is 5-7%, calculated as β-sitosterol.

[0061] In another preferred embodiment, the lotus germ oil has one or more characteristics selected from the following group: (a) Inhibit the secretion of NF-α; (b) inhibiting the secretion of IL-6; or a combination thereof.

[0062] In the fourth aspect of the present invention, there is provided a use of the lotus germ active ingredient as described in the first aspect of the present invention for preparing an anti-inflammatory and anti-aging composition.

[0063] In another preferred embodiment, the composition further comprises the lotus germ oil as described above.

[0064] In another preferred embodiment, the composition has one or more characteristics selected from the following group; (a) Increase ATP synthesis; (b) inhibiting the secretion of ROS; (c) inhibiting the secretion of NF-α, IL-6; or a combination thereof.

[0065] In another preferred embodiment, the composition inhibits / reduces the secretion of inflammatory factors TNF-α and IL-6, thereby having anti-inflammatory and soothing effects.

[0066] In another preferred embodiment, the inhibition rate of the composition on NF-α is ≥40%.

[0067] In another preferred embodiment, the inhibition rate of IL-6 of the composition is ≥35%.

[0068] In another preferred embodiment, the composition enhances ATP synthesis, thereby playing an anti-aging role for the skin.

[0069] In another preferred embodiment, the composition increases ATP synthesis by ≥40%.

[0070] In another preferred embodiment, the composition inhibits the secretion of ROS.

[0071] In another preferred embodiment, the composition is a cosmetic.

[0072] In another preferred embodiment, the cosmetics include: facial care, body care, after-sun care, including cream, lotion, essence, facial mask, spray, gel, and paste.

[0073] In another preferred embodiment, the cosmetic further contains additional ingredients that can be used for makeup.

[0074] In a fifth aspect of the present invention, a composition is provided, comprising: (1) the lotus germ active ingredient as described in the first aspect of the present invention; and (2) additional ingredients that can be used in daily chemical products.

[0075] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The HPLC detection spectrum of β-sitosterol is shown. Figure 2 The HPLC detection spectrum of neferine and schaftoside is shown, wherein Figure 2 A in the figure is the HPLC detection spectrum of neferine, Figure 2 B in the figure is the HPLC detection spectrum of schaftoside.

[0077] Figure 3 The results of the test on the anti-inflammatory effect of lotus germ oil were shown. Figure 3 A in the figure is the effect of lotus germ oil on the viability of Raw264.7 cells. Figure 3 B in the figure is the effect of lotus germ oil on IL-6 secretion. Figure 3 C in it represents the effect of lotus germ oil on TNF-α secretion.

[0078] Figure 4The results of the test on the efficacy of lotus germ active ingredients in enhancing ATP are shown. Figure 4 A in the figure is the effect of lotus germ active ingredients on fibroblast viability. Figure 4 B in the figure is the effect of lotus germ active ingredients on microscopic imaging of fibroblasts. Figure 4 C in the figure represents the effect of lotus germ active ingredients on ATP synthesis in fibroblasts.

[0079] Figure 5 The results show that lotus germ active ingredients can reduce UVB-induced cell ROS secretion. Figure 5 A in the figure is the effect of lotus germ active ingredients on the activity of Hacat cells. Figure 5 B in the figure represents the effect of lotus germ active ingredients on ROS secretion.

[0080] Figure 6 The flowchart of the preparation process of the lotus germ active ingredient described in the present application is shown. DETAILED DESCRIPTION

[0081] After extensive and in-depth research and a large number of experimental screenings, the inventor unexpectedly discovered for the first time that removing the chlorophyll in the raw material before extraction and then extracting it with a solvent can effectively improve the problem of chlorophyll residue in the product, especially after two supercritical extractions and entrainer solvent extractions, the content of the target component neferine is unexpectedly not affected. The method of the present invention can specifically and selectively remove chlorophyll without substantially affecting the content of neferine. In addition, the lotus germ active substance prepared by the present invention can inhibit / reduce the secretion of inflammatory factors TNF-α and IL-6, thereby having anti-inflammatory and soothing effects; by enhancing ATP synthesis and inhibiting the secretion of ROS, it can play an anti-aging role on the skin, broadening the application direction of lotus germ extract in cosmetics. On this basis, the inventor completed the present invention.

[0082] Neferine Neferine (Nef) is a bis-benzylisoquinoline alkaloid extracted from the green embryo (lotus core) of the mature seeds of the lotus (Nelumbo nuci fera Gaertn), a plant of the Nymphaeaceae family.

[0083]

[0084] Currently, the commonly used methods for extracting methyl nephrine from lotus seed core are: acid-base method, semi-bionic extraction and alcohol precipitation method, acid percolation extraction method, ion exchange resin method, membrane separation method, etc.

[0085] For example, the extraction method disclosed in CN 105949121 A is extraction with 40% to 90% alcohol solution. After the extract is concentrated, it is subjected to acid dissolution and alkali precipitation, and then purified by macroporous resin. The method has complicated steps and uses acid and alkali solvents, which is easy to pollute the environment and difficult to produce safely.

[0086] Other methods of extracting total alkaloids from lotus seeds are also prone to problems with chlorophyll and oil residues.

[0087] The method described in the present application uses supercritical CO2 extraction (95% ethanol as an entrainer) to remove chlorophyll and residual oil from the raw material, avoiding the use of acid and alkali in the process, and the process is more environmentally friendly.

[0088] Schaftoside

[0089] Schaftoside is a flavonoid compound that is widely present in many Chinese herbal medicines (such as Eleusine indica) and has a wide range of activities, such as choleretic, antibacterial, and anti-inflammatory.

[0090] Compared with the prior art, the present invention has the following beneficial effects: 1. In the present invention, the chlorophyll in the raw material is removed before extraction and then subjected to solvent extraction, thereby effectively improving the problem of chlorophyll residue in the product. In particular, after two supercritical extractions and entrainer solvent extractions, the content of the target component neferine is unexpectedly not affected, that is, the method of the present invention can specifically and selectively remove chlorophyll without substantially affecting the content of neferine.

[0091] 2. In the lotus germ active substance prepared by the present invention, the content of schaftoside is greater than 1.5wt%, the content of neferine is greater than 3wt%, and the chlorophyll concentration is less than <200μg / g.

[0092] 3. The lotus germ oil prepared by the present invention can inhibit / reduce the secretion of inflammatory factors TNF-α and IL-6, thereby having anti-inflammatory and soothing effects.

[0093] 4. The lotus germ active substance prepared by the present invention can enhance ATP synthesis and inhibit ROS secretion, thereby playing an anti-aging role for the skin.

[0094] 5. In the present invention, lotus germ active ingredients are extracted through a green and environmentally friendly preparation process, broadening the application direction of lotus germ extract in cosmetics.

[0095] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0096] General implementation method Supercritical CO2 extraction of lotus germ Weigh 2kg of dried lotus germ (containing about 3-5wt% water), crush it into coarse powder (about 10-30 mesh), add it to a basket, and put it into an extraction kettle for extraction. The extraction pressure is shown in Table 1 in Comparative Example 1, the CO2 flow rate is 80L / h, and the extraction is 2h. The separation kettle releases the first extract, which is the lotus germ oil of the present invention, and the first raffinate is retained in the extraction axe. In the presence of an optional entrainer (such as 95% ethanol), the first raffinate is extracted again by supercritical CO2, the extraction pressure is shown in Table 1 in Comparative Example 1, the temperature is 50°C, the CO2 flow rate is 80L / h, and the entrainer flow rate is 2L / h. The extract is released from the separation kettle, which is the second extract containing chlorophyll, and the raffinate is taken out from the extraction axe, which is the second raffinate of dechlorophyll. The second raffinate of dechlorophyll is subjected to solvent extraction, and the extraction solvent is shown in Table 3 in Comparative Example 3. The extract is concentrated and freeze-dried to obtain the lotus germ active substance of the present invention.

[0097] Comparative experiment 1: Effects of different extraction conditions on the extraction of β-sitosterol and chlorophyll from lotus germ In this embodiment, different extraction conditions are used to extract lotus germs, and the optimal extraction conditions are evaluated by testing the chlorophyll a concentration and β-sitosterol content in the obtained extracts.

[0098] Chlorophyll test method: accurately weigh 0.1-0.5g of germ oil under each extraction condition, add cyclohexane to dissolve, dilute to 10ml, shake well, filter with 0.45 μm filter membrane, and take the filtrate to obtain each sample. Detect the absorbance value of each sample at 750nm, 664nm, 647nm, and 630nm wavelengths, and calculate the concentration of chlorophyll a in each sample according to the following formula:

[0099] ρ1 = 11.85 x (A 664 -A 750 )- 1.54 x (A 647 -A 750 )- 0.08 x (A 630 -A 750 ) Where: ρ1——mass concentration of chlorophyll a in the sample, mg / L; A 664 ——The absorbance value of the sample at a wavelength of 664nm; A 647 ——The absorbance value of the sample at a wavelength of 647nm; A 630 ——The absorbance value of the sample at a wavelength of 630nm; A 750 ——The absorbance value of the sample at a wavelength of 750nm; The mass concentration of chlorophyll a in the sample (μg / g) is calculated according to the following formula: c = (ρ1·10) / m Where: c——mass concentration of chlorophyll a in the sample, μg / g ρ1——mass concentration of chlorophyll a in the sample, mg / L; m——sample mass, g β-sitosterol test method: accurately weigh 50 mg of germ oil under each extraction condition, add 2.5 mL of 2.5 mol / L KOH ethanol solution, react in a 90°C water bath for 1 h, shake once every 10 min, add 2.5 ml of saturated NaCl solution after cooling, add cyclohexane to extract twice, 10 ml each time, collect the cyclohexane part, concentrate and dry, add methanol to dissolve, make up to 10 ml, filter with 0.45 μm microporous filter membrane, take the filtrate to obtain each test solution, and determine the β-sitosterol content in each sample by HPLC. The β-sitosterol HPLC detection spectrum is shown as follows: Figure 1 The test results of chlorophyll a concentration, β-sitosterol content and extract yield in lotus germ oil under different extraction conditions are shown in Table 1.

[0100] Table 1: Chlorophyll a concentration, β-sitosterol content and extract yield in extracts under different extraction conditions

[0101] From the comparative experiments 1-1 and 1-2, the β-sitosterol content in the extracts, the extract yield, and the chlorophyll concentration in the extracts, it can be seen that when the extraction pressure is 35 MPa, the β-sitosterol content in the obtained extract is higher. Therefore, 35 MPa can be selected as the optimal extraction pressure for the process in the future.

[0102] From the comparison of the chlorophyll content in the extracts of the experimental groups 1-3 and 1-4 after adding the entrainer, it can be seen that after adding the entrainer, the chlorophyll concentration in the extract is significantly increased. Therefore, adding the entrainer for secondary extraction under a certain extraction pressure is helpful to further extract the chlorophyll in the residue. According to the experimental data, 20-35Mpa can be preferably used as the process extraction pressure, and 95% ethanol is preferably used as the entrainer to extract the chlorophyll in the raw material.

[0103] Comparative experiment 2: Chlorophyll content in the residue under different extraction conditions Accurately weigh 1g of the residue (crushed to 40 mesh) under each extraction condition, accurately add 25ml of anhydrous ethanol, seal, ultrasonically extract (power 250W, frequency 33kHz) for 35 minutes, cool, weigh again, make up the lost weight with anhydrous ethanol, shake well, filter with 0.45 μm filter membrane, and take the filtrate to obtain each sample. Detect the absorbance value of each sample at wavelengths of 750nm, 664nm, 647nm, and 630nm, and calculate according to the mass concentration (μg / g) of chlorophyll a in the sample in comparative experiment 1. The test results of chlorophyll a concentration in the residue under different extraction conditions are shown in Table 2.

[0104] Table 2: Chlorophyll a concentration in the residue under different extraction conditions

[0105] From the data in Table 2, it can be seen that after adding ethanol as an entrainer for extraction, the chlorophyll content in the residue is significantly reduced, indicating that when ethanol is used as an entrainer, the overall efficiency of chlorophyll extraction is improved.

[0106] Comparative experiment 3: Effects of different extraction solvents on the extraction of schaftoside and neferine in the residue Weigh 4 portions of the extract residues in experimental groups 1-4 in comparative experiment 1 (the content of schaftoside was 0.59%, the content of neferine was 1.00%), 30 g each, and perform hot reflux extraction with water, 30% ethanol, 60% ethanol, and 80% ethanol as solvents, respectively. The extraction was performed twice. The solid-liquid ratio of the first reflux extraction was 1:10, the water bath temperature was 93°C, and the extraction time was 2 h; the solid-liquid ratio of the second reflux extraction was 1:8, the water bath temperature was 93°C, and the extraction time was 1 h; the two extracts were combined, concentrated, filtered, and dried to obtain each extract. The content of schaftoside and neferine in each extract was detected by HPLC. The HPLC detection spectrum of neferine and schaftoside is shown as follows Figure 2 The results of the test on the contents of schaftoside and neferine in each extract and the transfer rate after extraction with different solvents are shown in Table 3.

[0107] Table 3: Contents of schaftoside and neferine in extracts under different extraction solvents

[0108] Note: Transfer rate: target component mass in extract / target component mass in raw material × 100%; From the data in the table, the contents of schaftoside and neferine in the extracts under various extraction conditions were compared. Under the 80% ethanol extraction condition, the yields of schaftoside and neferine were the highest, so 80% ethanol extraction was selected as the optimal extraction solvent.

[0109] Comparative experiment 4: Using 80% ethanol as the extraction solvent, extract the residues under different extraction conditions and compare the contents of schaftoside and neferine in each extract Weigh the extracts under different extraction conditions, 30g each, and perform hot reflux extraction with 80% ethanol as solvent. Extract twice. The solid-liquid ratio of the first reflux extraction is 1:10, the water bath temperature is 93°C, and the extraction time is 2h; the solid-liquid ratio of the second reflux extraction is 1:8, the water bath temperature is 93°C, and the extraction time is 1h; the two extracts are combined, concentrated, and dried to obtain each extract. The content of schaftoside and neferine in each extract, as well as the concentration of chlorophyll a in each extract, are detected by HPLC. The test results of schaftoside, neferine and chlorophyll a concentration in 80% ethanol extracts of the extracts under different extraction conditions are shown in Table 4.

[0110] Table 4: Contents of schaftoside, neferine and chlorophyll a in the extracts after extraction of the raffinate under different extraction conditions

[0111] As can be seen from the table data, before extraction, the chlorophyll in the raw material is removed after two supercritical extractions, and then after solvent extraction, the chlorophyll concentration in the obtained extract is significantly reduced. The content of chlorophyll a in the extract obtained by directly extracting with 80% ethanol without two supercritical extractions is 865.60 μg / g, and the content of chlorophyll a in the extract obtained by extracting with 80% ethanol after two supercritical extractions is reduced to 155.77 μg / g, which is 17.9% of the original, effectively improving the problem of chlorophyll residue in the product. In addition, in the present invention, especially after two supercritical extractions and entrainer solvent extraction, the content of neferine, the target component, is unexpectedly not affected, that is, the method of the present invention can specifically selectively remove chlorophyll without substantially affecting the content of neferine.

[0112] Example 1: Preparation of lotus germ active ingredients of the present invention (1) Preparation of lotus germ oil by supercritical CO2 extraction: Weigh 2 kg of dried lotus germ (containing about 3-5 wt% water), crush to coarse powder (about 10-30 mesh), add to a basket, put into an extraction kettle for extraction, set the pressure to 35 MPa, the temperature to 50°C, the CO2 flow rate to 80 L / h, and extract for 2 hours. Release the extract from the separation kettle to obtain lotus germ oil.

[0113] (2) Supercritical CO2 extraction of chlorophyll: After the extraction of lotus germ oil is completed, 95% ethanol is added as an entrainer, the pressure is 35 MPa, the temperature is 50°C, the CO2 flow rate is 80 L / h, the entrainer flow rate is 2 L / h, the extraction is carried out for 1 hour, the extract is released from the separation kettle, and vacuum distillation is performed to obtain the chlorophyll-containing extract (oil) and the dechlorophyllized second extract residue.

[0114] (3) Preparation of lotus germ active ingredients: Take the second chlorophyll-free extract in step (2), extract it twice with 80% ethanol under hot reflux, the solid-liquid ratio of the first reflux extraction is 1:10, the heating temperature is 80°C, and the extraction time is 2 h; the solid-liquid ratio of the second reflux extraction is 1:8, the heating temperature is 80°C, and the heating time is 1 h; filter to obtain an extract.

[0115] (4) Concentration: The extract obtained in step (3) is concentrated under reduced pressure at a temperature of 55 to 60° C. and a vacuum degree of -0.08 MPa to 1 times the amount of the feed, thereby obtaining a lotus germ extract concentrate.

[0116] (5) Freeze drying: The lotus germ extract is freeze dried in a vacuum freeze dryer for more than 24 hours. The freezing temperature of the freeze drying is (-70°C to -50°C) and the vacuum degree is less than 20 Pa. 440 g of lotus germ active freeze dried powder is obtained. After HPLC detection, the content of schaftoside is 2.05% and the content of neferine is 3.31%. Specifically, the preparation process flow chart of the lotus germ active material of the present application is as follows: Figure 6 shown.

[0117] Example 2: Efficacy Test 1. Test on the anti-inflammatory effect of lotus germ oil Sample group: The lotus germ oil prepared in Example 1 was used to prepare test samples of different concentrations using a basic culture medium.

[0118] Blank control group: select basal culture medium.

[0119] Positive control group: Dexamethasone (PC) was prepared at 100 μg / ml in basal medium.

[0120] 1.1 Main reagents and instruments DMEM high-glucose culture medium, fetal bovine serum, MTT, PBS, dexamethasone, pipette, microplate reader, inverted microscope, clean bench, ultrapure water machine, CO2 constant temperature incubator, ultra-low temperature freezing storage box, etc.

[0121] 1.2 Determination of maximum safe concentration by MTT method Raw264.7 cells growing in logarithmic phase were cultured at 8×10 4The cells were inoculated at a density of 1.5 cell / ml in a 96-well plate, and a blank control group was set up. After culturing for 24 h at 37°C and 5% CO2, different concentrations of the test substances were added to the cells as the sample group, and the culture medium was used to replace the sample as the negative control group. After culturing for 24 h, 20 μL of MTT solution was added to each well, and the culture was continued for 4 h. The liquid in the well was discarded, and 150 μL of DMSO solution was added to each well. The plates were oscillated at room temperature for 10 min, and the absorbance was measured at 490 nm.

[0122] According to: Cell viability % = (OD value of sample group - OD value of blank group) / (OD value of negative control group - OD value of blank group) × 100%. Calculate the cell viability at each concentration.

[0123] 1.3 Test steps Raw264.7 cells in the logarithmic growth phase were cultured in a plate at 37°C and 5% CO2 for 24 hours, and then different concentrations of the test substances were added. A negative control group and a positive control group were set up. LPS stimulation was added 1 hour later. The cells were returned to the incubator and incubated for 24 hours. The cell supernatants corresponding to each sample group were collected, and the concentrations of TNF-α and IL-6 in the cell supernatants were determined using ELISA kits.

[0124] 1.4 Experimental Results 1.4.1 MTT assay to determine cell viability, such as Figure 3 As shown in A, sample concentration ≤ 0.195 μg / ml had no significant effect on the viability of Raw264.7 cells.

[0125] 1.4.2 ELISA method was used to determine the inhibitory effect of lotus germ oil on TNF-α and IL-6. Figure 3 B and Figure 3 As shown in Figure C, compared with the blank control group (BC group), the secretion of TNF-α and IL-6 increased significantly after LPS stimulation (NC group) (###P<0.001), the IL-6 concentration in the positive control group (PC group) and the experimental group was significantly lower than that in the NC group, and the positive control dexamethasone (100μg / ml) significantly inhibited the secretion of TNF-α and IL-6 (***P<0.001). 0.195μg / ml and 0.04875μg / ml lotus germ oil can significantly inhibit / reduce the secretion of TNF-α and IL-6, and the inhibition rates of IL-6 are 38.37% and 42.62%, respectively, and the inhibition rates of TNF-α are 43.71% and 42.22%, respectively, indicating that the lotus germ oil of the present invention has certain anti-inflammatory and soothing effects.

[0126] 2. Lotus germ active ingredients enhance mitochondrial ATP efficacy test Sample group: The lotus germ active ingredient freeze-dried powder prepared in Example 1 was used to prepare test samples of different concentrations using basic culture medium.

[0127] Blank control group: select basal culture medium.

[0128] Positive control group: PC (Synephrine) was prepared into different concentrations using basal culture medium.

[0129] 2.1 Main reagents and instruments ATP bioluminescence analysis kit CLS II, MEM culture medium, fetal bovine serum, thiazolyl blue (MTT), PBS (pH7.4), pipette, microplate reader, inverted microscope, clean bench, ultrapure water machine, CO2 constant temperature incubator, ultra-low temperature freezer storage box, etc.

[0130] 2.2 Determination of maximum safe concentration by MTT method Mouse fibroblasts growing in logarithmic phase were cultured at 8×10 4 The cells were inoculated at a density of 1.5 cell / ml in a 96-well plate, and a blank control group was set up. After culturing at 37°C and 5% CO2 for 24 h, different concentrations of the test substances were added to the cells as the sample group, and the culture medium was used to replace the sample as the negative control group. After culturing for 24 h, 20 μL of MTT solution was added to each well, and the culture was continued for 4 h. The liquid in the well was discarded, and 150 μL of DMSO solution was added to each well. The plates were oscillated at room temperature for 10 min, and the absorbance was measured at 490 nm.

[0131] According to: Cell viability % = (OD value of sample group - OD value of blank group) / (OD value of negative control group - OD value of blank group) × 100%. Calculate the cell viability at each concentration.

[0132] 2.3 Test steps Mouse fibroblasts in the logarithmic growth phase were inoculated in a well plate and cultured at 37°C and 5% CO2 for 24 hours. Then, different concentrations of the test substances were added, and a blank control group and a positive control group were set up. After returning to the incubator and incubating for 24 hours, the ATP fluorescence intensity test was performed according to the manufacturer's instructions of the ATP bioluminescence assay kit CLS II.

[0133] 2.4 Experimental Results 2.4.1 MTT assay to determine cell viability, such as Figure 4 As shown in A, sample concentrations ≤ 500 μg / ml had no significant effect on the viability of mouse fibroblasts.

[0134] 2.4.2 ATP bioluminescence assay kit was used to determine the effect of lotus germ active ingredient freeze-dried powder on ATP production. Figure 4As shown in Figure B, after using different concentrations of lotus germ active ingredients and PC, the cell state and phase difference effects were significantly higher than those of the BC group. Figure 4 As shown in Figure C, compared with the blank control group (BC group), the PC group can significantly increase the expression of ATP (###P<0.001), indicating that this test model is effective. The lotus germ active substance freeze-dried powder at a concentration of 500μg / mL and 125μg / ml has a significant promoting effect on ATP synthesis, and the increase rates are 75% and 45% respectively compared with the PC group. This shows that the lotus germ active substance of the present application can increase ATP synthesis, which is beneficial to maintaining cell vitality and has a certain anti-aging effect.

[0135] 3. Experiment on reducing ROS secretion in cells induced by UVB irradiation by active ingredients from lotus germ Sample group: The lotus germ active ingredient freeze-dried powder prepared in Example 1 was used to prepare test samples of different concentrations using basic culture medium.

[0136] Blank control group: select basal culture medium.

[0137] Positive control group: Vitamin E (PC) was prepared into 200 μg / ml using basal culture medium.

[0138] 3.1 Main reagents and instruments MEM culture medium, fetal bovine serum, MTT, PBS (PH7.4), vitamin E, pipette, microplate reader, inverted microscope, clean bench, ultrapure water machine, CO2 constant temperature incubator, ultra-low temperature freezing storage box, etc.

[0139] 3.2. Determination of maximum safe concentration by MTT method Human keratinocytes (Hacat) growing in logarithmic phase were cultured at 8 × 10 4 The cells were inoculated at a density of 1.5 cell / ml in a 96-well plate, and a blank control group was set up. After culturing at 37°C and 5% CO2 for 24 h, different concentrations of the test substances were added to the cells as the sample group, and the culture medium was used to replace the sample as the negative control group. After culturing for 24 h, 20 μL of MTT solution was added to each well, and the culture was continued for 4 h. The liquid in the well was discarded, and 150 μL of DMSO solution was added to each well. The plates were oscillated at room temperature for 10 min, and the absorbance was measured at 490 nm.

[0140] According to: Cell viability % = (OD value of sample group - OD value of blank group) / (OD value of negative control group - OD value of blank group) × 100%. Calculate the cell viability at each concentration.

[0141] 3.3 Test steps Hacat cells in the logarithmic growth phase were inoculated into well plates and cultured at 37°C and 5% CO2 for 24 h. Then, different concentrations of the test substances were added, and negative and positive control groups were set up. After 1 h, the test substances were irradiated with 50 mJ / cm 2 After being stimulated by UVB, the cells were returned to the incubator for 24 h and the fluorescence intensity of the cells in each test group was detected.

[0142] 3.4 Experimental Results 3.4.1 MTT assay to determine cell viability, such as Figure 5 As shown in A, sample concentration ≤ 0.5 mg / ml had no significant effect on the viability of Hacat cells.

[0143] 3.4.2 DCFH-DA fluorescence method was used to determine the inhibitory effect of lotus germ active ingredient freeze-dried powder on ROS secretion. Figure 5 As shown in Figure B, compared with the blank control group (BC group), the secretion of ROS after UVB stimulation (NC group) increased significantly (###P<0.001), and the PC group could significantly inhibit the expression of ROS (###P<0.001), indicating that the stimulation conditions were effective. The lotus germ active substance freeze-dried powder at concentrations of 0.5 mg / mL and 0.125 mg / mL had a significant inhibitory effect on the ROS secreted by Hacat cells induced by UVB, with inhibition rates of 26.91% and 4.74%, respectively. This shows that the lotus germ active substance freeze-dried powder of the present invention can reduce the level of cellular active oxygen and has a certain anti-aging effect.

[0144] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A lotus germ active ingredient, characterized in that: The lotus germ active ingredient has a schaftoside content of ≥1.5wt%, a neferine content of ≥3wt%, and a chlorophyll concentration of ≤200μg / g.

2. The lotus germ active ingredient according to claim 1, characterized in that The lotus germ active substance is prepared by the following method: (1) performing supercritical CO2 extraction on the raw lotus embryo to obtain a first extract and a first raffinate; (2) adding an entrainer to the first raffinate of step (1) and performing supercritical CO2 extraction to obtain a second extract containing chlorophyll and a second raffinate dechlorophyllized; (3) taking the dechlorophyllized second extract residue in step (2), and refluxing and extracting it in ethanol for 1-3 times to obtain an extract; wherein the volume ratio of the dechlorophyllized second extract residue to ethanol is 1:7-11; (4) The extract obtained in step (3) is concentrated and dried to obtain the lotus germ active ingredient.

3. A method for preparing lotus germ active ingredients, characterized in that: The method comprises the steps of: (1) providing a raw material of lotus germ to be subjected to supercritical CO2 extraction in an extraction kettle to obtain a first extract of lotus germ oil and a first raffinate; (2) adding an entrainer to the first raffinate of step (1) to perform supercritical CO2 extraction to obtain a second extract containing chlorophyll and a second raffinate dechlorophyllized; (3) taking the dechlorophyllized second extract residue in step (2), and refluxing and extracting it in ethanol for 1-3 times to obtain an extract; wherein the volume ratio of the dechlorophyllized second extract residue to ethanol is 1:7-11; (4) The extract obtained in step (3) is concentrated and dried to obtain lotus germ active ingredients.

4. The method according to claim 3, characterized in that The extraction pressure of the supercritical CO2 extraction in step (1) is 30-40Mpa.

5. The method according to claim 3, characterized in that In the step (2), the entrainer is ethanol.

6. The method according to claim 3, characterized in that In step (2), the volume ratio of the entrainer to CO2 is 0.02-0.05:

1.

7. The method according to claim 3, characterized in that The extraction in step (3) comprises the steps of: performing a first hot reflux extraction in ethanol, with the extraction material-liquid volume ratio being 1:9-11, the heating temperature being 70-90° C., and the extraction time being 1.5-2.5 h, to obtain a first reflux extract; The extraction residue is subjected to a second hot reflux extraction in ethanol, the extraction material-liquid volume ratio is 1:7-9, the heating temperature is 70-90°C, and the heating time is 0.5-1.5h; and a second reflux extract is obtained; The first reflux extract and the second reflux extract are combined and filtered to obtain an extract.

8. A lotus germ oil, characterized in that The lotus germ oil is prepared by the following method: Providing lotus germ as raw material, and subjecting it to supercritical CO2 extraction to obtain lotus germ oil as an extract; The extraction pressure of the supercritical extraction is 20-35Mpa; the extraction temperature is 45-55°C; the CO2 flow rate is 70-90L / h, and the extraction time is 1-5h.

9. The use of the lotus germ active ingredient according to claim 1, characterized in that: Used for preparing anti-inflammatory and anti-aging compositions.

10. A composition, characterized in that The composition comprises: (1) the lotus germ active ingredient as claimed in claim 1; and (2) additional ingredients that can be used in daily chemical products.

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