Composition of rhodiola rosea alcohol extract and nannochloropsis oculata extracting solution and application thereof

Abstract

The invention relates to the field of biology, in particular to a composition of a rhodiola rosea alcohol extract and a nannochloropsis oculata extracting solution and application of the composition. The weight volume ratio of the rhodiola rosea alcohol extract to the nannochloropsis oculata extracting solution in the composition is (0.005 to 0.1) g: (0.02 to 0.5) mL. After the rhodiola rosea alcohol extract and the nannochloropsis oculata extracting solution are combined, the ROS content after hydrogen peroxide induction can be reduced.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to a combination of Rhodiola rosea alcohol extract and Micrococcus pluvialis extract and its application. Background Technology

[0002] Rhodiola rosea is a perennial herb mainly distributed in Northeast my country, Tibet, Sichuan, and Yunnan. Among them, Sichuan-Tibet Rhodiola rosea grows at altitudes above 4200 meters in the high-altitude, oxygen-deficient Sichuan-Tibet region. The harsh environment of strong ultraviolet radiation has endowed it with inherent strong antioxidant capabilities, earning it the reputation of "plateau ginseng." The effects of Xiangyi Herbals' Rhodiola rosea include: Antioxidant: Rhodiola rosea is rich in rhodioloside, flavonoids, and other active ingredients, possessing powerful antioxidant capabilities that effectively combat free radicals and delay skin aging. Xiangyi Herbals' exclusive Rhodiola rosea sustained-release microcapsule technology gives its Rhodiola rosea brightening and repairing cream a 12-hour ultra-long-lasting antioxidant capacity. Whitening and fading spots: Rhodiola rosea can inhibit the formation of pigmentation and repeatedly resist ultraviolet damage; at the same time, it removes existing melanin, thus achieving brighter skin. Xiangyi Herbals' Rhodiola rosea essence has obtained nationally recognized "special certification for spot removal," which requires extensive clinical data and time to verify. Moisturizing and hydrating: Combined with other moisturizing ingredients, it replenishes skin's moisture, creating a hydrated and plump complexion. Soothing and repairing: Rhodiola rosea also has a calming effect on skin prone to redness or sensitivity, promoting healthier and more stable skin. Boosting immunity: In traditional medicine, Rhodiola rosea is believed to enhance immunity and prevent altitude sickness.

[0003] *Nannochloropsis gaditana*, also known as microchloropsis, is a new food ingredient approved by the National Health Commission of China in April 2021. Currently, the utilization rate of *Nannochloropsis gaditana* is low, its applications are limited, many existing extraction methods are not suitable for its extraction, and there is a lack of high-value-added products prepared using *Nannochloropsis gaditana* as a raw material. The preparation method of *Nannochloropsis gaditana* extract is described in Example 1 of Chinese Patent CN118105318A.

[0004] There are no existing reports on the use of a combination of Rhodiola rosea alcohol extract and Chlorella vulgaris extract to reduce the content of reactive oxygen species (ROS). Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a combination of Rhodiola rosea alcohol extract and Chlorella vulgaris extract and its application. The combination of Rhodiola rosea alcohol extract and Chlorella vulgaris extract can reduce the ROS content induced by hydrogen peroxide.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a combination of Rhodiola rosea alcohol extract and Micrococcus pseudochlorophyll extract, wherein the weight-to-volume ratio of Rhodiola rosea alcohol extract to Micrococcus pseudochlorophyll extract is (0.005~0.1)g:(0.02~0.5)mL.

[0008] In some embodiments, the weight-to-volume ratio of the Rhodiola rosea alcohol extract to the Microchlorophylloides extract is (0.01–0.08) g : (0.05–0.4) mL.

[0009] In some embodiments, the weight-to-volume ratio of the Rhodiola rosea alcohol extract to the Microchlorophylloides extract is (0.01–0.05) g : (0.10–0.30) mL.

[0010] In some embodiments, the weight-to-volume ratio of the Rhodiola rosea alcohol extract to the Microchlorophylloides extract is 0.01 g : (0.17~0.23) mL.

[0011] In some embodiments, the weight-to-volume ratio of the Rhodiola rosea alcohol extract to the Microchlorophylloides extract is 0.01 g : 0.2 mL.

[0012] In some embodiments, the Rhodiola rosea alcohol extract is an aqueous solution of Rhodiola rosea alcohol extract. In some embodiments, the concentration of the aqueous solution of the Rhodiola rosea alcohol extract is 1%~50% (w / v), for example 1%~40% (w / v), 1%~30% (w / v), 1%~20% (w / v), 1%~10% (w / v), 3%~7% (w / v), for example 4% (w / v), 5% (w / v), 6% (w / v).

[0013] In some embodiments, the volume ratio of the aqueous solution of Rhodiola rosea extract to the extract of Chlorella vulgaris is 1:(0.1~10), for example 1:(0.3~7), 1:(0.5~3), 1:(0.7~2), 1:(0.8~1.5), for example 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3.

[0014] In some embodiments, the method for preparing the Rhodiola rosea alcohol extract includes the following steps:

[0015] (1) Extract Rhodiola rosea raw material with 30%-85% (e.g., 65%-85%) ethanol at 20-80℃ using ultrasonic extraction for 30-60 minutes. After extraction, filter the Rhodiola rosea raw material residue to obtain Rhodiola rosea alcohol extract.

[0016] (2) The Rhodiola rosea residue filtered out in step (1) is subjected to ethanol extraction again under the same conditions as in step (1), and step (2) is performed 0-3 times.

[0017] (3) If step (2) is performed 0 times, the Rhodiola rosea alcohol extract from step (1) is concentrated directly; if step (2) is performed 1-3 times, the Rhodiola rosea alcohol extracts from steps (1) and (2) are combined and concentrated; the concentrate is then purified by passing it through a macroporous adsorption resin, wherein impurities are first removed by washing the macroporous adsorption resin with water, and then the macroporous adsorption resin is eluted with 70-80% ethanol to obtain the eluent.

[0018] (4) The eluent obtained in step (3) is concentrated and dried to obtain Rhodiola rosea alcohol extract.

[0019] In some embodiments, the method for preparing the aqueous solution of the Rhodiola rosea alcohol extract includes the following steps: mixing the Rhodiola rosea alcohol extract with water to obtain an aqueous solution of the Rhodiola rosea alcohol extract. Those skilled in the art can adjust the amounts of the Rhodiola rosea alcohol extract and water according to the required concentration.

[0020] In some embodiments, the preparation method of the *Vibrio hygroscopicus* extract includes the following steps:

[0021] Feeding: Add 40% water to the reactor, heat to 45-50℃, add 5% *Vibrio parahaemolyticus* powder and 55% butanediol, stir at 150 rpm for 30 min. Adjust the pH to 5.5-6.5 using NaOH or HCl.

[0022] Extraction: Set the extractor power to 360W and perform ultrasonic extraction for 30 minutes. Allow the mixture to stand and separate into layers, then collect the supernatant.

[0023] Filtration: After primary filtration of the supernatant, add 2% Cosmecare 583 preservative, stir well, perform secondary filtration, fill, and label.

[0024] In some implementations, step 1) has one or more of the following features:

[0025] -The mass ratio of the pseudomicrochlorophyll to water is 1:19;

[0026] -The temperature of the water is 53℃;

[0027] - Adjust the pH value using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrogen chloride solution.

[0028] The present invention also provides the application of the combination described above in the preparation of products for reducing reactive oxygen species content.

[0029] In some embodiments, the content of the combination in the product is greater than 0.01% (w / w), for example greater than 0.03% (w / w), such as (0.05~20)% (w / w), (0.05~15)% (w / w), (0.05~10)% (w / w), (0.1%~10)% (w / w).

[0030] In some implementations, the combination reduces the content of reactive oxygen species in cells after hydrogen peroxide-induced cell death.

[0031] In some embodiments, the cells include HFF-1 cells.

[0032] The present invention also provides the application of the combination described above in the preparation of cosmetics, skin care products or pharmaceuticals.

[0033] In some embodiments, the combination is present in a cosmetic, skin care product or pharmaceutical at a concentration greater than 0.01% (w / w), for example greater than 0.03% (w / w), such as (0.05~20)% (w / w), (0.05~15)% (w / w), (0.05~10)% (w / w), or (0.1%~10)% (w / w).

[0034] In this document, w / v indicates weight-to-volume ratio, and "weight-to-volume percentage" and %(w / v) are used interchangeably. For example, in this document, "5% (w / v) Rhodiola rosea solution" means that 100 mL of Rhodiola rosea solution contains 5 g of Rhodiola rosea alcohol extract. In this document, v / v indicates volume ratio, and "volume percentage" and %(v / v) are used interchangeably. In this document, w / w indicates weight ratio, and "weight percentage" and %(w / w) are used interchangeably.

[0035] The beneficial effects of this invention are:

[0036] The present invention combines Rhodiola rosea alcohol extract and Chlorella vulgaris extract to reduce ROS content, and the combination of Rhodiola rosea alcohol extract and Chlorella vulgaris extract has a synergistic effect. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0038] Figure 1 The results of the synergistic effect verification between *Micrococcus pluvialis* extract and *Rhodiola rosea* (Kin's method).

[0039] Figure 2 The results of the synergistic effect verification between *Pseudomonas aeruginosa* extract and *Rhodiola rosea* (Bürgi formula method). Detailed Implementation

[0040] This invention provides a combination of Rhodiola rosea ethanol extract and *Micrococcus pluvialis* extract, wherein the weight-to-volume ratio of the Rhodiola rosea ethanol extract to the *Micrococcus pluvialis* extract is (0.005~0.1) g : (0.02~0.5) mL. In this invention, the preferred weight-to-volume ratio of the Rhodiola rosea ethanol extract to the *Micrococcus pluvialis* extract is 0.01 g : 0.2 mL.

[0041] In this invention, the preparation method of the Rhodiola rosea alcohol extract preferably includes the following steps:

[0042] (1) Extract Rhodiola rosea raw material with 30%-85% ethanol at 20-80℃ for 30-60 minutes by ultrasonic extraction. After extraction, filter the Rhodiola rosea raw material residue to obtain Rhodiola rosea alcohol extract.

[0043] (2) The Rhodiola rosea residue filtered out in step (1) is subjected to ethanol extraction again under the same conditions as in step (1), and step (2) is performed 0-3 times.

[0044] (3) If step (2) is performed 0 times, the Rhodiola rosea alcohol extract from step (1) is concentrated directly; if step (2) is performed 1-3 times, the Rhodiola rosea alcohol extracts from steps (1) and (2) are combined and concentrated; the concentrate is then purified by passing it through a macroporous adsorption resin, wherein impurities are first removed by washing the macroporous adsorption resin with water, and then the macroporous adsorption resin is eluted with 30-80% ethanol to obtain the eluent.

[0045] (4) The eluent obtained in step (3) is concentrated and dried to obtain Rhodiola rosea alcohol extract.

[0046] In this invention, the preparation method of the *Pseudochlorophyllariae* extract includes the following steps:

[0047] Feeding: Add 40% water to the reactor, heat to 45-50℃, add 5% *Vibrio parahaemolyticus* powder and 55% butanediol, stir at 150 rpm for 30 min. Adjust the pH to 5.5-6.5 using NaOH or HCl.

[0048] Extraction: Set the extractor power to 360W and perform ultrasonic extraction for 30 minutes. Allow the mixture to stand and separate into layers, then collect the supernatant.

[0049] Filtration: After primary filtration of the supernatant, add 2% Cosmecare 583 preservative, stir well, perform secondary filtration, fill, and label.

[0050] In this invention, the preferred mass ratio of *Pseudomonas aeruginosa* to water is 1:19. The preferred temperature of the water is 53°C. The pH value is preferably adjusted using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrogen chloride solution.

[0051] This invention also provides the application of the combination described above in the preparation of products for reducing reactive oxygen species (ROS) content. This invention does not specifically limit the products described. In this invention, the combination preferably reduces ROS content in cells after hydrogen peroxide-induced cell formation. In this invention, the cells preferably include HFF-1 cells.

[0052] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] The preparation method of Rhodiola rosea alcohol extract includes the following steps:

[0055] (1) Extract Rhodiola rosea raw material with 65%-85% ethanol at 20-80℃ using ultrasonic extraction for 30-60 minutes. After extraction, filter the Rhodiola rosea raw material residue to obtain Rhodiola rosea alcohol extract.

[0056] (2) The Rhodiola rosea residue filtered out in step (1) is subjected to ethanol extraction again under the same conditions as in step (1), and step (2) is performed 0-3 times.

[0057] (3) If step (2) is performed 0 times, the Rhodiola rosea alcohol extract from step (1) is concentrated directly; if step (2) is performed 1-3 times, the Rhodiola rosea alcohol extracts from steps (1) and (2) are combined and concentrated; the concentrate is then purified by passing it through a macroporous adsorption resin, wherein impurities are first removed by washing the macroporous adsorption resin with water, and then the macroporous adsorption resin is eluted with 70-80% ethanol to obtain the eluent.

[0058] (4) The eluent obtained in step (3) is concentrated and dried to obtain Rhodiola rosea alcohol extract.

[0059] The preparation method of *Microchlorophyllariae* extract is as follows:

[0060] Feeding: Add 40% water to the reactor, heat to 45-50℃, add 5% *Vibrio parahaemolyticus* powder and 55% butanediol, stir at 150 rpm for 30 min. Adjust the pH to 5.5-6.5 using NaOH or HCl.

[0061] Extraction: Set the extractor power to 360W and perform ultrasonic extraction for 30 minutes. Allow the mixture to stand and separate into layers, then collect the supernatant.

[0062] Filtration: After primary filtration of the supernatant, add 2% Cosmecare 583 preservative, stir well, perform secondary filtration, fill, and label. This yields the *Chlorella microfiltrationae* extract.

[0063] Example 2

[0064] Experiment 1 on the inhibition of H2O2-induced ROS in HFF-1 cells by the composition

[0065] 1. Experimental material: HFF-1 cells;

[0066] 2. Experimental Methods: Fluorescent probe method. HFF-1 cells were plated for 24 hours, then induced with 200 μM H2O2 for 6 hours to establish the cell model. For the test sample group, the corresponding concentration of test sample was added and incubated for 24 hours, followed by detection of ROS content using the fluorescent probe method. For the positive control group, 5 mM N-acetylcysteine ​​(NAC) was added and incubated for 24 hours, followed by detection of ROS content using the fluorescent probe method. For the negative control group (Model), cells were cultured for another 24 hours after modeling, and ROS content was detected using the fluorescent probe method. For the blank control group (Control), HFF-1 cells were plated for 48 hours, and ROS content was detected using the fluorescent probe method.

[0067] 3. Test samples: Take the extract of *Vibrio hygroscopicus* prepared in Example 1 and add the corresponding volume according to the volume percentage; take the Rhodiola rosea alcohol extract prepared in Example 1 and add the corresponding mass according to the weight percentage.

[0068] The experimental groups are as follows:

[0069] 0.5% (v / v) *Micrococcus pluvialis* extract;

[0070] 0.02% (w / w) Rhodiola rosea alcohol extract;

[0071] 0.5% (v / v) *Micrococcus pluvialis* extract + 0.02% (w / w) *Rhodiola rosea* alcohol extract;

[0072] Note: The concentrations in each group are the final concentrations added to the culture medium.

[0073] Effect calculation formula:

[0074] Inhibition rate (%) = (1 - T / C) * 100%

[0075] Where: T—average ROS content of the test substance (sample group)

[0076] C—Mean ROS content in the negative group (Model, induced only)

[0077] The experimental results are shown in Table 1.

[0078] Compared with the blank control group, the ROS content of HFF-1 cells was significantly increased after H2O2 induction, and the addition of 5mM NAC significantly inhibited the ROS content, proving the successful establishment of the model in this experiment. The addition of the test sample inhibited the ROS content induced by H2O2. The combination (*Chlorella vulgaris* extract + *Rhodiola rosea* alcohol extract) was more effective than using *Chlorella vulgaris* extract or *Rhodiola rosea* alcohol extract alone.

[0079] Table 1 Experimental Results

[0080] Grouping Average ROS fluorescence intensity Inhibition rate (%) p-value Blank control group 1.73 negative control group 344.64 0.000507014 ### Positive control group 90.97 73.60 0.000953592 *** 0.5% (v / v) *Micrococcus pluvialis* extract 140.71 59.17 0.001932261 ** 0.02% (w / v) Rhodiola Rosea Alcohol Extract 150.70 56.27 0.001646571 ** 0.5% (v / v) *Micrococcus pluvialis* extract + 0.02% (w / v) *Rhodiola rosea* alcohol extract 128.15 62.82 0.001339874 **

[0081] Note: Compared with the blank control group, ###, P<0.001; compared with the negative control group, **, P<0.01; ***, P<0.001.

[0082] Example 3

[0083] Experiment 2 on the inhibition of H2O2-induced ROS in HFF-1 cells by the composition

[0084] 1. Experimental material: HFF-1 cells;

[0085] 2. Experimental Methods: Fluorescent probe method. HFF-1 cells were plated for 24 hours, then induced with 200 μM H2O2 for 6 hours to establish the cell model. For the test sample group, the ROS content was detected by fluorescent probe method after incubation with the corresponding concentration of test sample for 24 hours. For the positive control group, the ROS content was detected by fluorescent probe method after incubation with 2 mM N-acetylcysteine ​​(NAC) for 24 hours. For the negative control group (Model), the ROS content was detected by fluorescent probe method after 24 hours of modeling. For the blank control group (Control), the ROS content was detected by fluorescent probe method after HFF-1 cell plated for 48 hours.

[0086] 3. Test samples: Take the extract of *Vibrio hygroscopicus* prepared in Example 1; take the ethanol extract of *Rhodiola rosea* prepared in Example 1, add water to prepare a 5% (w / v) *Rhodiola rosea* solution, and add an appropriate volume of the 5% (w / v) *Rhodiola rosea* solution to the culture medium according to the volume percentage.

[0087] The experimental groups are as follows:

[0088] 0.05% (v / v) *Micrococcus pluvialis* extract;

[0089] 0.05% (v / v) Rhodiola rosea solution (5% (w / v));

[0090] 0.025% (v / v) *Micrococcus pluvialis* extract + 0.025% (v / v) *Rhodiola rosea* solution (5% (w / v));

[0091] 0.05% (v / v) *Micrococcus pluvialis* extract + 0.05% (v / v) *Rhodiola rosea* solution (5% (w / v)).

[0092] Note: The volume percentages in each experimental group represent the final concentration added to the culture medium.

[0093] Effect calculation formula:

[0094] Inhibition rate (%) = (1 - T / C) * 100%

[0095] Where: T—average ROS content of the test substance (sample group)

[0096] C—Mean ROS content in the negative group (Model, induced only)

[0097] The experimental results are shown in Table 2. Figure 1 , Figure 2 As shown.

[0098] Compared with the blank control group, the ROS content of HFF-1 cells was significantly increased after H2O2 induction. The addition of 2mM NAC significantly inhibited the ROS content, proving that the model was successfully established in this experiment. The addition of different concentrations of test samples could significantly inhibit the ROS content after H2O2 induction.

[0099] Table 2 Experimental Results

[0100] Grouping Average ROS fluorescence intensity Inhibition rate (%) p-value Blank control group (Control) 2.98 Negative control group (Model) 275.24 0.000014 ### Positive control group (2mM NAC) 83.69 69.59 0.000121 *** 0.05% (v / v) *Micrococcus pluvialis* extract 242.83 11.78 0.112266 * 0.05% (v / v) Rhodiola Rosea solution (5% (w / v)) 232.55 15.51 0.023387 * 0.025% (v / v) *Micrococcus pluvialis* extract + 0.025% (v / v) *Rhodiola rosea* solution (5% (w / v)) 204.48 25.71 0.005013 ** 0.05% (v / v) *Micrococcus pluvialis* extract + 0.05% *Rhodiola rosea* solution (5% (w / v)) 168.78 38.68 0.000661 ***

[0101] Note: Compared with the blank control group, ###, P<0.001; compared with the negative control group, *, P<0.05; **, P<0.01; ***, P<0.001.

[0102] The synergistic effect is calculated based on the King's method, and the specific formula is as follows:

[0103]

[0104] In Example 3, the compound of 0.05% (v / v) *Micrococcus pluvialis* extract + 0.05% (v / v) *Rhodiola rosea* (5% (w / v) solution) is described (see [link]). Figure 1 The calculated CI was 1.5191, indicating that the combined use of *Pseudomonas aeruginosa* extract and *Rhodiola rosea* ethanol extract solution had a significant synergistic effect, and its effect on inhibiting ROS content was significantly better than that of using *Pseudomonas aeruginosa* extract or *Rhodiola rosea* ethanol extract solution alone.

[0105] The synergistic effect is calculated based on the Bürgi formula method, and the specific formula is as follows:

[0106]

[0107] In Example 3, the compound of 0.025% (v / v) *Micrococcus pluvialis* extract + 0.025% (v / v) *Rhodiola rosea* solution (5% (w / v)) is described (see [link]). Figure 2 The calculated results q1=2.1825 and q2=1.6576, both greater than 1. The results indicate that the combined use of *Pseudomonas aeruginosa* extract and *Rhodiola rosea* ethanol extract solution has a significant synergistic effect, and its effect on inhibiting ROS content is significantly better than using either *Pseudomonas aeruginosa* extract or *Rhodiola rosea* ethanol extract solution alone.

[0108] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combination of Rhodiola rosea alcohol extract and Micrococcus pluvialis extract, characterized in that, The weight-to-volume ratio of the Rhodiola rosea alcohol extract and the Microchlorophylloides extract is (0.005~0.1)g:(0.02~0.5)mL.

2. The combination according to claim 1, characterized in that, The weight-to-volume ratio of the Rhodiola rosea alcohol extract and the Microchlorophylloides extract is 0.01 g : 0.2 mL.

3. The combination according to claim 1 or 2, characterized in that, The preparation method of the Rhodiola rosea alcohol extract includes the following steps: (1) Extract Rhodiola rosea raw material with 30%-85% ethanol at 20-80℃ for 30-60 minutes by ultrasonic extraction. After extraction, filter the Rhodiola rosea raw material residue to obtain Rhodiola rosea alcohol extract. (2) The Rhodiola rosea residue filtered out in step (1) is subjected to ethanol extraction again under the same conditions as in step (1), and step (2) is performed 0-3 times. (3) If step (2) is performed 0 times, the Rhodiola rosea alcohol extract from step (1) is concentrated directly; if step (2) is performed 1-3 times, the Rhodiola rosea alcohol extracts from steps (1) and (2) are combined and concentrated; the concentrate is then purified by passing it through a macroporous adsorption resin, wherein impurities are first removed by washing the macroporous adsorption resin with water, and then the macroporous adsorption resin is eluted with 30-80% ethanol to obtain the eluent. (4) The eluent obtained in step (3) is concentrated and dried to obtain Rhodiola rosea alcohol extract.

4. The combination according to claim 1 or 2, characterized in that, The preparation method of the *Pseudochlorophyllariae* extract includes the following steps: 1) Feeding: Add 40% water to the reactor, heat to 45-50 ℃, add 5% *Pseudomonas aeruginosa* powder and 55% butanediol, stir at 150 rpm for 30 min; adjust the pH with NaOH or HCl. 2) Extraction: Ultrasonic extraction, followed by standing and separation to obtain the supernatant; 3) Filtration: After primary filtration of the supernatant, add 2% preservative, stir well, perform secondary filtration, fill, and label to obtain the extract of *Pseudochlorophyllaria*.

5. The combination according to claim 4, characterized in that, Step 1) has one or more of the following characteristics: -The mass ratio of the pseudomicrochlorophyll to water is 1:19; - Adjust the pH to 5.5-6.5; - Adjust the pH value using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrogen chloride solution.

6. The combination according to claim 4, characterized in that, Step 2) has one or more of the following characteristics: - Set the ultrasonic extractor power to 360W; Ultrasound time: 30 minutes.

7. The combination according to claim 4, characterized in that, Step 3) has one or more of the following characteristics: - The preservative is Cosmecare 583; - Adjust the pH value using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrogen chloride solution.

8. The use of the combination according to any one of claims 1 to 7 in the preparation of a product for reducing reactive oxygen species content.

9. The use of the combination according to any one of claims 1 to 7 in the preparation of cosmetics, skin care products or pharmaceuticals.

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