Preparation method and application of fingered citron extract with anti-aging effect

Abstract

The invention discloses a preparation method and application of a fingered citron extract with an anti-aging effect, and relates to the technical field of biomedical materials.The preparation method comprises the following steps that fingered citron is smashed and then added into an ethanol solution, then ultrasonic extraction is conducted, and fingered citron extract freeze-dried powder is obtained through filtering, rotary evaporation concentration and freeze drying in sequence; dissolving the fingered citron extract freeze-dried powder in ultrapure water, then performing irradiation treatment, and then sequentially performing centrifugation, filtration and freeze drying to obtain the fingered citron extract with the anti-aging effect. The invention also discloses the fingered citron extract with the anti-aging effect prepared by the method. According to the method, the anti-aging, anti-inflammatory, anti-oxidation, anti-melanogenesis and anti-heat apoptosis effects of the fingered citron extract are improved through the irradiation effect, and the fingered citron extract has high application value in the aspect of preparation of cosmetics, medicines and health care products.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a preparation method and application of a bergamot extract with anti-aging effects. Background Art

[0002] Buddha's hand is a variety of citron plant of the Rutaceae family. It is native to India and is cultivated in Europe, North America and Southeast Asia. Most of my country's Buddha's hand resources are cultivated. They are distributed in Guangdong, Chongqing, Sichuan and Zhejiang. With the development of the Buddha's hand industry, Buddha's hand cultivation has been introduced to various parts of my country. There are now a large number of Buddha's hand planting bases. Sichuan is one of the largest production areas of Buddha's hand in my country.

[0003] As a plant with both medicinal and edible properties, bergamot is widely used in food, medicine, and daily chemicals. Modern research has found that bergamot extracts contain a variety of bioactive ingredients, mainly including volatile oils, flavonoids, polysaccharides and other compounds, with antioxidant, anti-inflammatory, anti-tumor, lipid regulation, anti-anxiety and depression, and antibacterial effects, and have huge development potential and diverse application prospects. In addition to the food and pharmaceutical industries, the functional application of bergamot essential oil in the beauty, perfume and daily necessities industries is also another breakthrough in the research and development of bergamot products, with multiple pharmacological effects such as anti-inflammatory, antibacterial and antioxidant. As research gradually deepens, more and more effects are discovered. Modern research results have found a variety of new activities in addition to traditional effects, but at this stage, there are still major deficiencies and gaps in research on it, which limits the development of bergamot deep-processing products. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for preparing a bergamot extract with anti-aging effect and its application. The method enhances the anti-inflammatory, antioxidant, anti-aging, anti-melanin production and anti-thermal apoptosis effects of the bergamot extract through the radiation effect, and has great application value in the preparation of cosmetics, medicines and health products.

[0005] The technical solution of the present invention to solve the above technical problem is as follows: a method for preparing a bergamot extract with anti-aging effect is provided, comprising the following steps:

[0006] (1) crushing the bergamot and adding it to an ethanol solution, then ultrasonically extracting it, and then filtering, rotary evaporating and freeze-drying it in sequence to obtain a freeze-dried powder of the bergamot extract;

[0007] (2) dissolving the freeze-dried powder of the bergamot extract obtained in step (1) in ultrapure water, then irradiating it, and then centrifuging, filtering and freeze-drying it in sequence to obtain a bergamot extract with anti-aging effects.

[0008] Furthermore, the bergamot has a moisture content of less than 10% and is crushed into 10-200 mesh powder.

[0009] Furthermore, in step (1), the concentration of the ethanol solution is 30%-70%; the mass volume ratio of bergamot to ethanol solution is 1g:5-20mL.

[0010] Furthermore, in step (1), ultrasonic extraction is performed for 0.5-1.5 h.

[0011] Furthermore, in step (1), the bergamot can be extracted multiple times using an ethanol solution, and then the extracts are combined.

[0012] Furthermore, in step (2), the mass volume ratio of the freeze-dried powder of bergamot extract and ultrapure water is 10-100 g:1 L.

[0013] Further, in step (2), using X-ray, 60 Irradiation is performed by Co-γ rays or 10MeV high-energy electron beams; the irradiation dose is 10-50kGy.

[0014] Furthermore, in step (2), centrifugation is performed at 4000-6000 rpm for 10-30 min.

[0015] Furthermore, in step (2), filtration is performed using a 0.45 μm filter membrane.

[0016] The invention also discloses the bergamot extract with anti-aging effect prepared by the preparation method of the bergamot extract with anti-aging effect.

[0017] The invention also discloses application of the bergamot extract with anti-aging effect in preparing anti-aging products.

[0018] Furthermore, the above-mentioned products are medicines, cosmetics, foods or health products.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] 1. The present invention uses an irradiation dose of 10-50 kGy to irradiate the bergamot extract, and then removes impurities such as mixts and cellulose by centrifugation and membrane filtration, and then obtains the bergamot extract with high anti-aging activity by freeze-drying.

[0021] 2. The bergamot extract obtained by irradiation processing in the present invention can not only inhibit the expression levels of inflammatory factors IL-6, IL-1β and NO, improve LPS-induced cellular inflammation, and reduce the levels of reactive oxygen, malondialdehyde and lactate dehydrogenase, and increase the content of superoxide dismutase and catalase related to antioxidant, but also inhibit the expression of DHICA oxidase and dopachrome tautomerase related to melanin production, and significantly improve cell apoptosis caused by heat stress.

[0022] 3. The main active substances of the bergamot extract in the present invention are phenols (such as bergamot glycosides), volatile oils (limonene, terpinene, etc.) and polysaccharides, and the anti-inflammatory, antioxidant, anti-aging, anti-melanin production and anti-thermal apoptosis effects are enhanced through the radiation effect. It has significant anti-aging function and has great application value in the preparation of cosmetics, medicines and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the results of a cell viability test;

[0024] Figure 2 This is the NO release level test result diagram;

[0025] Figure 3 This is the result of TNF-α expression detection;

[0026] Figure 4 This is the result of IL-1β expression detection;

[0027] Figure 5 It is a color comparison chart of the supernatant of each treatment group;

[0028] Figure 6 This is a graph showing the results of the detection of tyrosinase, TRP-1, TRP-2 and HSC70 expression levels;

[0029] Figure 7 This is the result of ROS fluorescence staining detection of HaCaT cells;

[0030] Figure 8 This is the result of superoxide dismutase (SOD) activity detection in HaCaT cells.

[0031] Fig. 9 Figure 2 shows the results of the catalase activity test in HaCaT cells.

[0032] Fig.10 Figure 2 shows the results of the detection of malondialdehyde (MDA) content in HaCaT cells.

[0033] Fig.11 Figure 2 shows the results of the detection of lactate dehydrogenase (LDH) release in HaCaT cells.

[0034] Fig.12 This is the result diagram of the effect of bergamot extract on heat resistance;

[0035] Fig.13 This is the result of HO-1 expression detection. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0037] Example 1

[0038] A bergamot extract with anti-aging effect, the preparation method of which comprises the following steps:

[0039] (1) After the bergamot is crushed, it is added to a 50% ethanol solution at a mass volume ratio of 1 g: 10 mL, and then ultrasonically extracted for 1 hour. After filtering, the filter residue is added to the ethanol solution for repeated extraction, and the filtrate is combined, and then concentrated by rotary evaporation and freeze-dried to obtain a freeze-dried powder of the bergamot extract;

[0040] (2) Dissolve the freeze-dried powder of the bergamot extract obtained in step (1) in ultrapure water at a mass volume ratio of 60 g:1 L. 60 The extract was irradiated with Co-γ rays at a dose of 30 kGy, then centrifuged at 5000 rpm for 20 min, filtered using a 0.45 μm filter membrane, and then freeze-dried to obtain a bergamot extract with anti-aging effects.

[0041] Example 2

[0042] A bergamot extract with anti-aging effect, the preparation method of which comprises the following steps:

[0043] (1) Crush the bergamot and add it to a 30% ethanol solution at a mass volume ratio of 1 g:5 mL, then perform ultrasonic extraction for 0.5 h, filter and add the residue to the ethanol solution for repeated extraction, combine the filtrate, and then perform rotary evaporation concentration and freeze drying to obtain freeze-dried powder of the bergamot extract;

[0044] (2) dissolving the bergamot extract freeze-dried powder obtained in step (1) in ultrapure water at a mass volume ratio of 10 g:1 L, irradiating the powder with a 10 MeV high-energy electron beam at a dose of 50 kGy, centrifuging at 4000 rpm for 10 min, filtering with a 0.45 μm filter membrane, and freeze-drying to obtain a bergamot extract with anti-aging effects.

[0045] Example 3

[0046] A bergamot extract with anti-aging effect, the preparation method of which comprises the following steps:

[0047] (1) After the bergamot is crushed, it is added to a 70% ethanol solution at a mass volume ratio of 1 g: 20 mL, and then ultrasonically extracted for 1.5 hours. After filtering, the filter residue is added to the ethanol solution for repeated extraction, and the filtrate is combined, and then concentrated by rotary evaporation and freeze-dried to obtain a freeze-dried powder of the bergamot extract;

[0048] (2) dissolving the freeze-dried powder of the bergamot extract obtained in step (1) in ultrapure water at a mass volume ratio of 100 g:1 L, irradiating the powder with X-rays at a dose of 10 kGy, centrifuging at 6000 rpm for 30 min, filtering the powder with a 0.45 μm filter membrane, and freeze-drying the powder to obtain a bergamot extract with anti-aging effects.

[0049] Example 4

[0050] A bergamot extract with anti-aging effect, the preparation method of which comprises the following steps:

[0051] (1) After the bergamot is crushed, it is added to a 50% ethanol solution at a mass volume ratio of 1 g: 10 mL, and then ultrasonically extracted for 1 hour. After filtering, the filter residue is added to the ethanol solution for repeated extraction, and the filtrate is combined, and then concentrated by rotary evaporation and freeze-dried to obtain a freeze-dried powder of the bergamot extract;

[0052] (2) Dissolve the freeze-dried powder of the bergamot extract obtained in step (1) in ultrapure water at a mass volume ratio of 60 g:1 L. 60 The extract was irradiated with Co-γ rays at a dose of 50 kGy, then centrifuged at 5000 rpm for 20 min, filtered using a 0.45 μm filter membrane, and then freeze-dried to obtain a bergamot extract with anti-aging effects.

[0053] Example 5

[0054] A lotion with skin improving effect, the preparation method of which is as follows:

[0055] 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of polyol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of methyl cyanoacetate, 0.01 g of antioxidant and 0.01 g of pigment are mixed and dissolved; 0.05 g of the bergamot extract with anti-aging effect obtained in Example 1 and 5 g of glycerol are dissolved in 85.33 g of pure water, and then the mixture is added and stirred to obtain a lotion with skin improvement effect.

[0056] Example 6

[0057] A water / oil-based skin-improving emulsion, the preparation method of which comprises the following steps:

[0058] 1.2g of ethanol, 10g of squalane, 2g of vaseline, 0.2g of ethyl paraben, 1g of glycerol monoacetate, 1g of polyoxyethylene monooleate and 0.1g of fragrance were heated and dissolved at 70°C; 0.05g of the bergamot extract with anti-aging effect obtained in Example 1, 5g of dipropylene glycol, 2g of polyethylene glycol 1500 and 0.2g of triethanolamine were heated and dissolved in 76.2g of purified water; the two were mixed and emulsified, and then cooled to obtain a water / oil-based emulsion with skin improvement effect. .

[0059] Example 7

[0060] A serum with skin-improving effect, the preparation method of which comprises the following steps:

[0061] 5 g of 95% ethanol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitosan, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E acetate, 0.2 g of sodium glycyrrhizinate, 0.1 g of parabens, 1 g of the bergamot extract with anti-aging effect obtained in Example 1 and an appropriate amount of pigment are mixed to obtain an essence with skin improvement effect.

[0062] Example 8

[0063] A hydrogel with skin improvement effect, the preparation method of which comprises the following steps:

[0064] 5 g of the bergamot extract with anti-aging effect obtained in Example 1 was added to a solution of 1 g of xanthan gum, 1 g of bean gum and 0.5 g of carrageenan, mixed evenly, and purified water was added to a total mass of 100 g to obtain a hydrogel with skin improvement effect.

[0065] Test Example 1

[0066] (1) RAW246.7 macrophages were exposed to 350 mM H2O2 and co-cultured with bergamot extracts with anti-aging effects prepared by irradiation at different doses (10-50 kGy). The cell survival rate was detected. The results were as follows: Figure 1 The results showed that the bergamot extract prepared by the method improved the cell survival rate under oxidative stress.

[0067] (2) BV2 brain glial cells were co-cultured with bergamot extracts with anti-aging effects obtained by irradiation at different doses (10-50 kGy), and inflammatory induction was verified by 1 μg / mL lipopolysaccharide (LPS). After the experiment, the NO release level and the expression levels of TNF-α and IL-1β were detected. The results were as follows: Figure 2-4As shown in the figure, * indicates significant difference from the blank group p < 0.05, ** indicates significant difference from the blank group p < 0.01, # indicates significant difference from the control group p < 0.05, ## indicates significant difference from the control group p < 0.01.

[0068] Depend on Figure 2-4 It can be seen that the bergamot extract prepared by this method can reduce LPS-induced BV2 brain glial cell inflammation by regulating the expression of inflammatory factors IL-6 and IL-1β and reducing the level of NO. The results show that the irradiated bergamot extract has a better inhibitory effect on inflammation.

[0069] Test Example 2

[0070] Referring to Example 1, bergamot extracts with anti-aging effects obtained by irradiation at different doses (10-50 kGy) were co-cultured with B16-F10 cells in the presence of α-MSH for 48 h to test the anti-melanin production ability of the irradiated bergamot extracts.

[0071] (1) The cell cultures of each treatment group were centrifuged and Figure 5 α-MSH can promote melanin production. In the treatment group with bergamot extract, the color of the cell supernatant became lighter with the increase of irradiation dose. The results showed that the bergamot extract prepared by this method has a good anti-melanin production effect.

[0072] (2) The expression levels of tyrosinase, DHICA oxidase (TRP-1), dopachrome isomerase (TRP-2) and HSC70 were as follows: Figure 6 shown.

[0073] Depend on Figure 6 It can be seen that after irradiation treatment, bergamot extract can better reduce the expression of DHICA oxidase (TRP-1) and dopachrome tautomerase (TRP-2), and with the increase of irradiation dose, it has a better inhibitory effect on the production of melanin.

[0074] Test Example 3

[0075] (1) Human immortalized keratinocytes HaCaT were induced to oxidative senescence by hydrogen peroxide, and the cells were co-cultured with bergamot extracts at different irradiation doses. ROS were detected by fluorescence staining. The results were as follows: Figure 7 The detection results of superoxide dismutase (SOD), catalase, malondialdehyde (MDA) and lactate dehydrogenase (LDH) are shown in Figure 8-11 shown.

[0076] Depend on Figure 7-11It can be seen that the bergamot extract prepared by the method of the present invention can reduce the expression of ROS, malondialdehyde (MDA) and lactate dehydrogenase (LDH), and can increase the levels of antioxidant-related superoxide dismutase (SOD) and catalase, indicating that it can improve aging-related oxidative stress.

[0077] (2) Fig.12 As shown, A is a heat-induced cell migration staining diagram, and B is a heat-induced cell migration number comparison diagram. The figure shows the effect of the irradiated bergamot extract of the present invention on the tolerance and migration number of HaCaT keratinocytes at a high ambient temperature of 42°C. The results show that the 50kGy irradiated bergamot extract can inhibit heat-induced cell apoptosis.

[0078] (3) Fig.13 As shown, the bergamot extract prepared by the method of the present invention can better promote the expression of HO-1, thereby more effectively improving various aging indicators.

[0079] Test Example 4

[0080] This test example is a test of skin elasticity, moisturizing and improvement of skin wrinkles of a cream composition containing a bergamot extract.

[0081] (1) Preparation of cream composition containing bergamot extract

[0082] A cosmetic containing the bergamot extract obtained in Example 4 was prepared to evaluate the efficacy of elasticity restoration, moisturizing and skin density improvement in humans. The cosmetic used for efficacy evaluation was in the form of a cream, and its ingredients were shown in Table 1. First, the group b recorded in Table 1 was mixed evenly and heated and stored at 70°C, and then the group b ingredients were added to the group a, and after preliminary stirring and emulsification, they were uniformly emulsified with a homogenizer, and then gradually cooled to prepare a cream.

[0083] Table 1 Formula of bergamot cream

[0084]

[0085] (2) Evaluation of the effect of bergamot cream on skin improvement

[0086] 21 subjects (women aged 30-50 years old) applied the above-mentioned bergamot cream twice a day for 2 consecutive months. After the 2-month period, the effects of improving epidermal elasticity, medium- and long-term moisturizing and skin density were measured at three time points: before use, 2 weeks after use, and 4 weeks after use. Among them, elasticity recovery was to examine epidermal elasticity, medium- and long-term moisturizing was to detect the water content of the stratum corneum, and skin density was to test the density of the dermis. The results are shown in Tables 2-4 respectively.

[0087] Table 2 Measurement results of epidermal elasticity

[0088]

[0089]

[0090] As shown in Table 2, the subjects who used the cream containing 50 kGy bergamot extract had a decrease in epidermal elasticity of 8.67% after 2 weeks of using the test bergamot cream, and an increase of 94% after 4 weeks of use, resulting in a statistically significant increase in epidermal elasticity compared to before the test. Therefore, the cream containing 50 kGy bergamot extract helps improve epidermal elasticity.

[0091] Table 3 Measurement results of water content in stratum corneum

[0092]

[0093] As shown in Table 3, the amount of epidermal moisture in the subjects who used the cream containing 50 kGy bergamot extract increased by 6.16% 2 weeks after the end of the experiment and by 13.64% 4 weeks after the end of the experiment, and the amount of water in the stratum corneum increased statistically significantly compared with before the test. Therefore, the bergamot cream containing 50 kGy bergamot extract helps to improve the moisture content in the stratum corneum.

[0094] Table 4 Dermis density test results

[0095]

[0096] As shown in Table 4, the skin density of the subjects who used the cream containing 50 kGy bergamot extract increased by 9.54% after 2 weeks of testing and increased by 18.78% after 4 weeks of testing, which was a statistically significant increase compared with before the test. Therefore, the bergamot cream containing 50 kGy bergamot extract helps to improve skin density.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a bergamot extract having an anti-aging effect, characterized in that: The following steps are involved: (1) crushing the bergamot and adding it to an ethanol solution, then ultrasonically extracting it, and then filtering, rotary evaporating and freeze-drying it in sequence to obtain a freeze-dried powder of the bergamot extract; (2) dissolving the freeze-dried powder of the bergamot extract obtained in step (1) in ultrapure water, then irradiating it, and then centrifuging, filtering and freeze-drying it in sequence to obtain a bergamot extract with anti-aging effects.

2. The method for preparing the bergamot extract with anti-aging effect as claimed in claim 1, characterized in that: In step (1), the concentration of the ethanol solution is 30%-70%; the mass volume ratio of the bergamot to the ethanol solution is 1g:5-20mL.

3. The method for preparing the bergamot extract with anti-aging effect as claimed in claim 1, characterized in that: In step (1), ultrasonic extraction is performed for 0.5-1.5 h.

4. The method for preparing the bergamot extract with anti-aging effect as claimed in claim 1, characterized in that: In step (2), the mass volume ratio of the freeze-dried powder of the bergamot extract and ultrapure water is 10-30 g:1 L.

5. The method for preparing the bergamot extract with anti-aging effect as claimed in claim 1, characterized in that: In step (2), X-ray, 60 Co-γ ray or 10MeV high energy electron beam irradiation; the irradiation dose is 10-50kGy.

6. The method for preparing the bergamot extract with anti-aging effect as claimed in claim 1, characterized in that: In step (2), centrifuge at 4000-6000 rpm for 10-30 min.

7. The method for preparing the bergamot extract with anti-aging effect as claimed in claim 1, characterized in that: In step (2), 0.45 μm filter membrane is used for filtration.

8. The bergamot extract with anti-aging effect obtained by the preparation method of the bergamot extract with anti-aging effect according to any one of claims 1 to 7.

9. Use of the bergamot extract with anti-aging effect according to claim 8 in the preparation of anti-aging products.

10. The use according to claim 9, characterized in that: The product is a medicine, cosmetic, food or health product.

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