Composition with synergistic effect and application thereof

Abstract

The invention belongs to the field of cosmetics or food, and particularly relates to a composition with a synergistic effect and application thereof. The composition with the synergistic effect contains the snow rabbit callus extract and the nicotinamide, the snow rabbit callus extract and the nicotinamide can achieve the synergistic effect, removal of DPPH free radicals is promoted, ROS generation is inhibited, the composition has the synergistic anti-oxidation effect, and the composition is free of toxic and side effects and free of toxic and side effects. A new path is provided for effective development and utilization of the extract of the callus of the snow rabbit and nicotinamide, function maximization of limited resources is achieved, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics or food, and particularly relates to a composition with synergistic effects and its applications. Background Art

[0002] With the continuous development of plant extraction and discovery technologies, more and more active molecules or active sites have been discovered. However, these discovered active molecules or active sites have not been well utilized, and their functions have not been maximally developed to achieve effective and sustainable utilization of resources.

[0003] Saussurea laniceps, a perennial herb of the genus Saussurea (Compositae), is a national second-class protected species. It mainly grows in alpine scree slopes at an altitude of 3200 - 5280 meters and is an extremely typical alpine plant, distributed in Sichuan, Yunnan, Tibet and other places in China. Its leaves and capitula are covered with thick wool, and the flowering and fruiting periods are from August to October. Saussurea laniceps has extremely high medicinal and ornamental values and is one of the important Tibetan medicine plant resources. Research shows that Saussurea laniceps contains rich flavonoids, polyphenols, polysaccharides and secondary metabolites, and has functions such as scavenging free radicals, anti-aging and analgesia. Saussurea laniceps mainly reproduces by seeds, and no one has successfully cultivated it so far. Predatory digging destroys the ecological balance and seriously affects its natural growth and reproduction. Therefore, using plant tissue culture technology to obtain a large amount of callus of Saussurea laniceps is an effective way to develop rare and endangered species. The extract of the callus of Saussurea laniceps has certain antioxidant effects.

[0004] Nicotinamide, also known as niacinamide, is an amide compound of nicotinic acid. Nicotinamide has good skin tolerance and is crucial for NAD+ synthesis, PARP and Sirtuin regulation. In addition, research has confirmed that nicotinamide has antibacterial activity, can effectively inhibit bacteria and prevent the formation of biofilms. Nicotinamide can treat acne, reduce sebum, and also solve skin pigmentation and yellowing problems related to glycation. In addition, nicotinamide can also protect collagen, inhibit matrix degrading enzymes or promote the production of collagen and elastin, helping to maintain the integrity of the extracellular matrix of skin cells.

[0005] How to compound various active ingredients with skin care effects to make them act synergistically, so as to achieve better skin care effects without increasing their addition amounts, thereby simplifying the formula, is a problem that needs to be solved. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a composition with a synergistic effect. The components in the composition act synergistically to increase the scavenging rate of DPPH free radicals and inhibit the generation rate of ROS, thus playing a good antioxidant role.

[0007] The technical solution of the present invention is a composition with a synergistic effect, containing an extract of Saussurea laniceps callus and niacinamide. The extract of Saussurea laniceps callus and niacinamide act synergistically to promote the scavenging of DPPH free radicals and inhibit the production of ROS, and can enhance the antioxidant effect.

[0008] The Saussurea laniceps callus comes from the organs of Saussurea laniceps, and the organs of Saussurea laniceps include at least one of root, stem, leaf, flower, fruit, and seed, preferably at least one of root, stem, and leaf.

[0009] The Saussurea laniceps callus includes Saussurea laniceps callus and / or subcultured Saussurea laniceps callus.

[0010] The extract of Saussurea laniceps callus is a water extract and / or an alcohol extract of Saussurea laniceps callus, preferably a water extract or a 60%-80% ethanol extract, further preferably a water extract or a 65%-75% ethanol extract, and preferably a water extract of Saussurea laniceps callus.

[0011] The mass ratio of niacinamide to the extract of Saussurea laniceps callus is 1:0.001-25, preferably 1:0.002-20, further preferably 1:0.005-15, further preferably 1:0.01-10, further preferably 1:0.05-10, further preferably 1:0.05-5. In a preferred embodiment of the present invention, it is 1:0.05-2.5 or 1:2.5-5; more preferably 1:0.05-1 or 1:2.5-5.

[0012] In the composition containing niacinamide and the extract of Saussurea laniceps callus, the concentration of niacinamide is 0.01%-10%, preferably 0.05%-10%, further preferably 0.1%-10%, further preferably 0.4%-4%, further preferably 0.4%-2.5%, further preferably 0.4%-2%, and even more preferably 0.4%-1%.

[0013] The concentration of the extract of Saussurea laniceps callus in the composition is 0.01%-5%, preferably 0.1%-2.5%; further preferably 0.25%-1%.

[0014] The preparation method of the above-mentioned Saussurea laniceps callus extract of the present invention comprises the steps of: extracting the Saussurea laniceps callus with ethanol and / or water. Further, the Saussurea laniceps callus is extracted with 60%-80% ethanol or water; further, the Saussurea laniceps callus is extracted with 65%-75% ethanol or water; furthermore, the Saussurea laniceps callus is extracted with water.

[0015] Further, the Saussurea laniceps callus is placed in ethanol and / or water for leaching, the leaching solution obtained by leaching is subjected to centrifugation and / or filtration treatment, and then drying treatment. Further, the Saussurea laniceps callus is leached with 60%-80% ethanol or water; further, the Saussurea laniceps callus is leached with 65%-75% ethanol or water; furthermore, the Saussurea laniceps callus is leached with water.

[0016] Before the leaching, the ethanol and / or water mixed with the Saussurea laniceps callus are ground and crushed. Further, the ethanol and / or water is selected from 60%-80% ethanol or water, further selected from 65%-75% ethanol or water, and furthermore selected from water.

[0017] The leaching solution is first subjected to centrifugation treatment and then filtration treatment.

[0018] The drying treatment is freeze-drying.

[0019] The induction method of the Saussurea laniceps callus is: cutting the organs of the Saussurea laniceps and inoculating them into an induction medium to induce callus. The organs are any one or more of roots, stems or leaves.

[0020] Using MS medium as the basal medium to induce and culture the Saussurea laniceps callus. Preferably, the medium also contains plant growth hormones and sucrose.

[0021] The induction medium uses MS medium as the basal medium. Preferably, the induction medium also contains plant growth hormones; the plant growth hormones include at least one of 6-BA and NAA. Preferably, the content of 6-BA is 0.4-0.6 mg / L and the content of NAA is 0.2-0.4 mg / L.

[0022] The culture method of the Saussurea laniceps callus is that the Saussurea laniceps callus is subcultured for 10-35 days; the medium for subculture uses MS medium as the basal medium.

[0023] Preferably, it also contains plant growth hormones; the plant growth hormones include at least one of 6-BA and NAA. Preferably, the content of 6-BA is 0.4-0.6 mg / L and the content of NAA is 0.4-0.6 mg / L.

[0024] Preferably, it also contains 20 - 40 g / L of sucrose and / or 20 - 40 g / L of potato powder.

[0025] The present invention uses the extract of Saussurea laniceps callus in synergistic action with niacinamide to promote the scavenging of DPPH free radicals, inhibit the generation of ROS, and improve its antioxidant activity. The composition of the extract of Saussurea laniceps callus and niacinamide with synergistic action provided by the present invention can be used to prepare products for scavenging DPPH free radicals, and / or products for inhibiting the generation of ROS, and / or products for antioxidation.

[0026] The products for scavenging DPPH free radicals, and / or products for inhibiting the generation of ROS, and / or products for antioxidation provided by the present invention contain the extract of Saussurea laniceps callus and niacinamide.

[0027] The mass ratio of the niacinamide to the extract of Saussurea laniceps callus is 1:0.001 - 25, preferably 1:0.002 - 20, further preferably 1:0.005 - 15, still further preferably 1:0.01 - 10, still further preferably 1:0.05 - 10, still further preferably 1:0.05 - 5. In a preferred embodiment of the present invention, it is 1:0.05 - 2.5 or 1:2.5 - 5; more preferably 1:0.05 - 1 or 1:2.5 - 5.

[0028] In the product, the concentration of niacinamide is 0.01% - 10%, preferably 0.05% - 10%, further preferably 0.1% - 10%, still further preferably 0.4% - 4%, still further preferably 0.4% - 2.5%, still further preferably 0.4% - 2%, and even more preferably 0.4% - 1%.

[0029] In the said product, the concentration of the extract of Saussurea laniceps callus is 0.01% - 5%, preferably 0.1% - 2.5%; further preferably 0.25% - 1%.

[0030] The products for scavenging DPPH free radicals, and / or products for inhibiting the generation of ROS, and / or products for antioxidation according to the present invention can be additives, and / or cosmetics, and / or oral beauty products, and / or health products, and / or foods, and / or pharmaceuticals, and the presented states include but are not limited to liquid, or solid, or oily, or powdery, or milky, or paste - like, or cream - like, or gel - like, or aerosol - like, or powder - mist - like.

[0031] As described above, cosmetics refer to daily chemical industrial products applied to the human body surface such as skin, hair, nails, and lips by means of rubbing, spraying or other similar methods for the purpose of cleaning, protecting, beautifying, and modifying. When used as cosmetics, they can be classified into cleansing cosmetics (used to clean the skin), skin care cosmetics (such as cleansing creams, facial cleansers, bath agents, hair shampoos and conditioners, shaving creams, etc.), basic cosmetics (basic treatment of the face and hair before makeup, such as various creams, lotions, toners, facial masks, hair creams, hair gels and other hair styling products), beauty cosmetics (used for beautifying the face and hair, such as rouge, lipsticks, eyeshadows, hair dyeing, perming, hairstyling and fixing products), and therapeutic cosmetics (daily chemical products between drugs and cosmetics, such as cooling, deodorizing, freckle-removing, sunscreen, body-building, hair-growing, hair-removing, hair-dyeing, insect-repellent, olive essence, etc.). According to their external forms, they can be divided into liquids (such as facial cleansers, bath liquids, hair shampoos, toners, perfumes, skin fresheners, makeup removers, essence liquids, stock solutions, etc.), lotions (such as lotions, milks, hair conditioners, essence lotions), creams and ointments (such as moisturizing creams, foundation creams, hair creams, concealers, hair straightening creams, essence creams, makeup primers), powders (such as face powders, talcum powders, loose powders, cleansing powders, rice powders), solids (such as powder compacts, blush powders, lipsticks, hair waxes), and oils (such as makeup remover oils, emollient oils, hair conditioners, essence oils).

[0032] As described above, when used as health products, they can be prepared into powders, tablets, capsules, liquid preparations, gels, etc., and can also be prepared into tea bags, beverages, candies, etc.

[0033] As described above, when used as drugs, they can be classified into solution type (a uniform dispersion system in which drugs are dispersed in the dispersion medium in the form of molecules or ions (the diameter of the particles is less than 1nm), also known as low molecular solution, such as aromatic waters, solutions, syrups, glycerins, spirits, injections, etc.), colloidal solution type (a uniform dispersion system mainly formed by macromolecules (the diameter of the particles is 1 - 100nm) dispersed in the dispersion medium, also known as macromolecular solution, such as mucilage, collodion, film-forming agents, etc.), emulsion type (a non-uniform dispersion system in which oily drugs or drug oil solutions are dispersed in the dispersion medium in the form of droplets, such as oral emulsions, intravenous emulsions, some liniments, etc. Suspension type: a non-uniform dispersion system in which solid drugs are dispersed in the dispersion medium in the form of fine particles, such as mixtures, lotions, suspensions, etc.), gas dispersion type (a dispersion system in which liquid or solid drugs are dispersed in the gas dispersion medium in the form of fine particles, such as aerosols), particulate dispersion type (drugs are dispersed in liquid or solid states in the form of different-sized particles, such as microsphere preparations, microcapsule preparations, nanocapsule preparations, etc.), and solid dispersion type (a dispersion system in which solid drugs exist in the form of aggregates, such as tablets, powders, granules, capsules, pills, etc.).

[0034] The main advantages of the present invention include: The present invention discovers the synergistic effect between the extract of Saussurea laniceps callus and niacinamide, promotes the scavenging of DPPH free radicals, inhibits the generation of ROS, and has good antioxidant effects. It provides a new path for the effective development and utilization of the extract of Saussurea laniceps callus and niacinamide, realizes the maximization of the functions of limited resources, and has broad application prospects. Detailed implementation manners

[0035] Experimental principle

[0036]

Principle of DPPH free radical scavenging method

[0037]

Principle of intracellular reactive oxygen species rapid detection method

[0038] Experimental methods

[0039] I. Preparation of Saussurea laniceps extract

[0040] 1. Induction and subculture of Saussurea laniceps plant callus

[0041] In a sterile laminar flow hood, use a scalpel to cut the roots, stems or leaves of Saussurea laniceps into segments with a length of 0.5 cm - 1.0 cm, and inoculate them into the medium formula of MS + 6-BA 0.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L to induce callus.

[0042] Transfer a part of the Saussurea laniceps callus to the medium formula of MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + potato powder 30 g / L + sucrose 30 g / L + gellan gum 3.5 g / L for subculture. After 25 - 30 days, harvest the Saussurea laniceps callus, designated as "Saussurea laniceps callus", and store it frozen for later use.

[0043] Transfer another part of the callus of Saussurea laniceps to the medium formula of 1 / 2MS + 6-BA 0.5mg / L + NAA 0.5mg / L + sucrose 30g / L + gellan gum 3.5g / L for subculture. After 25 - 30 days, harvest the callus of Saussurea laniceps (abnormal passage), designated as "Saussurea laniceps callus - 2", and store it frozen for later use.

[0044] 2. Preparation of aqueous extract or alcohol extract of Saussurea laniceps plant callus

[0045] Take the frozen - stored callus of Saussurea laniceps for thawing, add deionized water or 70% ethanol, then grind and homogenize it for 10 - 20 min, and extract for 3 hours. Then centrifuge the extracted liquid at 8000 rpm - 10000 rpm for 40 min - 60 min. After centrifugation, take the supernatant, filter it through a 0.45 uM filter membrane, and take the filtrate. Freeze - dry the filtrate at low temperature for 10 - 14 days, and the obtained powder is the extract of Saussurea laniceps callus.

[0046] 3. Comparison with Saussurea involucrata cell culture

[0047] The Saussurea involucrata plant for comparison is the Saussurea involucrata culture purchased from Ansabo.

[0048] II. Solution preparation

[0049] 1. Solution preparation related to DPPH free - radical scavenging method

[0050] 1.1 Preparation of 0.1 mmol / L DPPH solution

[0051] Take 4 mg of DPPH powder into a 1.5 mL centrifuge tube. Use a 10 mL pipette to transfer 100 mL of absolute ethanol to a sampling cup. Dissolve the weighed DPPH powder with the absolute ethanol in the sampling cup and transfer it to the sampling cup. Cover the sampling cup with tin foil for light protection.

[0052] 1.2 Preparation of Saussurea laniceps callus extract solution

[0053] Weigh 10 mg of Saussurea laniceps extract powder and dissolve it in 1 mL of deionized water to prepare a 10 mg / mL Saussurea laniceps callus extract solution, that is, 1% Saussurea laniceps callus extract solution.

[0054] 1.3 Preparation of nicotinamide solution

[0055] Weigh 10 mg, 20 mg, 50 mg, and 100 mg respectively and dissolve them in 1 mL of deionized water to prepare 10 mg / mL, 20 mg / mL, 50 mg / mL, and 100 mg / mL nicotinamide solutions, that is, 1%, 2%, 5%, and 10% nicotinamide solutions respectively.

[0056] 1.4 Preparation of the solution of the callus extract of Saussurea laniceps and nicotinamide composition

[0057] Prepare Saussurea laniceps and nicotinamide compositions at different ratios. The concentrations of the callus extract of Saussurea laniceps are 0.25%, 0.5% and 1% respectively, and the concentrations of nicotinamide are 0.1%, 1%, 2%, 5% and 10%; add them to the reaction system according to different ratios.

[0058] 2. Preparation of related solutions for the rapid detection method of intracellular reactive oxygen species

[0059] 2.1 Preparation of the DCFH-DA probe working solution

[0060] Dilute the original probe with the culture medium at a ratio of 1:1000 to obtain a working concentration of 1 μM.

[0061] III. Experimental procedures

[0062] 1. Experimental procedures for the DPPH free radical scavenging method

[0063] The experimental groups include a blank group, a negative control group, a positive control group, and a sample group.

[0064] Blank group Negative control group Positive control group Test sample group Sample 10 μL water 10 μL sample solvent 0.1 mg / mL L-ascorbic acid 10 μL DPPH solution 0 μL 140 μL 140 μL 140 μL Absolute ethanol 140 μL 0 μL 0 μL 0 μL Total 150 μL 150 μL 150 μL 150 μL

[0065] In a 96-well plate, add samples and DPPH solution or absolute ethanol according to the experimental sample addition table. Each experimental group has 3 parallel wells. Place it in the dark and react at room temperature for 30 min with a microplate constant temperature oscillator at 300 r / min. After the reaction is completed, measure the absorbance at 517 nm with an enzyme-linked immunosorbent assay reader. The DPPH scavenging rate is calculated as follows:

[0066] P = [1 - (A s - A c ) / A b * 100%

[0067] P - Scavenging rate;

[0068] A s - Absorbance of the mixture of the solution to be measured and the DPPH solution;

[0069] A c - Absorbance of the mixture of the solution to be measured and the absolute ethanol solution;

[0070] A b . - Absorbance of the mixture of the DPPH solution and the sample solvent solution.

[0071] 2. Experimental procedures for the rapid detection method of intracellular reactive oxygen species

[0072] 2.1 Cell treatment

[0073] Inoculate B16 cells into a 96-well plate at an inoculation density of 4.0×10 4 cells / well and culture for 24 h. Aspirate the culture medium, wash once with DPBS, add the sample to be tested and culture for 2 h, then wash twice with DPBS. Add the prepared DCFH-DA working solution and incubate in a 37 °C incubator for 30 min. Remove the DCFH-DA, wash twice with DPBS. Wrap the cell blank group with tin foil and irradiate the remaining groups with 100 mJ UVA. Finally, read the fluorescence value at Ex / Em = 485 nm / 535 nm using a microplate reader.

[0074] 2.2 Data processing

[0075] The calculation method of ROS inhibition rate is as follows:

[0076] Inhibition rate (%) = [(Average value of control group - Average value of sample group) / Average value of control group]×100%

[0077] Table 1. DPPH scavenging rate of the composition prepared from the water extract of "Saussurea laniceps callus" and nicotinamide

[0078]

[0079]

[0080] The results in Table 1 show that there is a synergistic effect between the water extract of the normal subcultured tissue of Saussurea laniceps callus and nicotinamide, and it has good DPPH scavenging activity.

[0081] The basis for judging synergy here is that the scavenging efficiency of the composition is higher than the sum of the two single sample groups.

[0082] Table 2. ROS inhibition rate of the composition prepared from the water extract of "Saussurea laniceps callus" and nicotinamide

[0083]

[0084] The results in Table 2 show that the composition prepared from the water extract of the normal subcultured tissue of Saussurea laniceps callus and nicotinamide exhibits enhanced ROS scavenging activity to a certain extent, which again proves the synergistic activity between the water extract of the normal subcultured tissue of Saussurea laniceps callus and nicotinamide.

[0085] Table 3. DPPH scavenging rate of the nicotinamide composition of Saussurea laniceps extracts with different processes

[0086]

[0087]

[0088] Table 3 results showed that there was a certain synergistic activity between the alcohol extract of the normal subcultured tissue of Saussurea laniceps callus and nicotinamide, but the degree was significantly lower than that of the combination of the water extract and nicotinamide. There was no synergistic effect in scavenging DPPH between the water extract of the abnormal subcultured tissue of Saussurea laniceps callus and nicotinamide.

[0089] Table 4. DPPH scavenging rate of nicotinamide compositions of other Saussurea cultures (Saussurea involucrata cell culture, Ansabo)

[0090]

[0091] Table 4 results showed that there was no DPPH synergistic scavenging activity between the commercially available Saussurea involucrata culture and nicotinamide.

[0092] Table 5 Definition of synergistic range

[0093]

[0094] In summary, there was a synergistic activity between the extract of Saussurea laniceps callus and nicotinamide in the present invention. This synergistic activity improved the DPPH scavenging rate and the inhibition rate of ROS. Its DPPH scavenging rate was even better than that of the commercially available Saussurea involucrata extract or the DPPH scavenging rate of its combination with nicotinamide. The effect of the water extract of the normal subcultured culture of Saussurea laniceps callus was the best. Therefore, the combination of the extract of Saussurea laniceps callus and nicotinamide in the present invention synergistically scavenged DPPH and inhibited the generation of ROS, showing excellent antioxidant activity.

Claims

1. A synergistic composition, characterized in that: Contains extract of snow hare callus and niacinamide.

2. The composition according to claim 1, characterized in that The snow rabbit callus tissue is derived from the organs of the snow rabbit, and the organs of the snow rabbit include at least one of roots, stems, leaves, flowers, fruits and seeds.

3. The composition according to claim 1, characterized in that The extract of the callus tissue of the snow rabbit is a water extract and / or an alcohol extract of the callus tissue of the snow rabbit.

4. The composition according to any one of claims 1 to 3, characterized in that The snow rabbit callus tissue comprises the snow rabbit callus tissue and / or the snow rabbit callus tissue that has been subcultured.

5. The composition according to claim 1, characterized in that The mass ratio of the nicotinamide to the extract of the snow rabbit callus is 1:0.001-25.

6. The composition according to claim 5, characterized in that The concentration of niacinamide is 0.01%-10%.

7. The composition according to any one of claims 1 to 6, characterized in that MS medium was used as the basal medium to induce and culture callus tissue of Snow Rabbit.

8. The composition according to any one of claims 1 to 7 is used to prepare at least one of the following products: (1) Products for scavenging DPPH free radicals; (2) Products for inhibiting ROS generation; (3) Antioxidant products.

9. At least one of the following products, characterized in that: A composition comprising any one of claims 1 to 7: (1) Products for scavenging DPPH free radicals; (2) Products for inhibiting ROS generation; (3) Antioxidant products.

10. The use according to claim 8 or the product according to claim 9, characterized in that: The product is at least one of an additive, a cosmetic, an oral beauty product, a health product, a food, and a medicine.

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