Composition having synergistic effect and use thereof

Abstract

A composition having a synergistic effect and use thereof. The composition comprises a Saussurea laniceps callus extract and nicotinamide. The Saussurea laniceps callus extract can interact synergistically with nicotinamide, thereby promoting the removal of DPPH free radicals, inhibiting the production of ROS, and achieving synergistic antioxidation effect. This facilitates the effective development and utilization of the Saussurea laniceps extract and nicotinamide, demonstrating broad application prospects.

Description

A synergistic composition and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetics or food, and particularly relates to a synergistic composition and application thereof. BACKGROUND

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

[0003] Saussurea laniceps is a perennial herb of Saussurea DC. belonging to Compositae, and is a national second-class protected species. It mainly grows in alpine scree at an altitude of 3200-5280 meters and is a typical alpine plant. It is distributed in Sichuan, Yunnan and Tibet, China. Its leaves and capitula are covered with thick wool. The flowering and fruiting period is from August to October. Saussurea laniceps has high medicinal and ornamental value and is one of important Tibetan medicinal plant resources. Studies have shown that Saussurea laniceps contains rich flavonoids, polyphenols, polysaccharides and secondary metabolites, and has functions of scavenging free radicals, anti-aging and analgesia. Saussurea laniceps mainly reproduces by seeds, and at present, no one has successfully planted it. The predatory mining destroys the ecological balance and seriously affects its natural growth and reproduction. Therefore, using plant tissue culture technology to obtain a large amount of Saussurea laniceps callus is an effective way to develop rare and endangered species. The extract of Saussurea laniceps callus has certain antioxidant effect.

[0004] Nicotinamide (Niacinamide), 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, studies have confirmed that nicotinamide has antibacterial activity and can effectively inhibit bacteria and prevent the formation of biofilms. Nicotinamide can treat acne, reduce sebum, and also solve the problems of skin pigmentation and yellowing related to glycation. In addition, nicotinamide can also protect collagen, inhibit matrix-degrading enzymes or promote the generation of collagen and elastin, and help maintain the integrity of the extracellular matrix of skin cells.

[0005] It is a problem to be solved how to compound various active ingredients with skin care effects to achieve synergistic effect without increasing the amount of addition, so as to simplify the formula. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a composition with synergistic effect, which improves the DPPH free radical scavenging rate and inhibits the ROS generation rate, and has good antioxidant effect.

[0007] The technical scheme of the present application is a composition with synergistic effect, which contains an extract of callus of Chirita pinnata and nicotinamide.

[0008] The callus of Chirita pinnata is derived from an organ of Chirita pinnata, and the organ of Chirita pinnata includes at least one of roots, stems, leaves, flowers, fruits and seeds, and preferably at least one of roots, stems and leaves.

[0009] The callus of Chirita pinnata includes callus of Chirita pinnata and / or subcultured callus of Chirita pinnata.

[0010] The extract of callus of Chirita pinnata is a water extract and / or an alcohol extract of callus of Chirita pinnata, 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 callus of Chirita pinnata.

[0011] The mass ratio of nicotinamide to the extract of callus of Chirita pinnata 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 mode of the present application, 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 nicotinamide and the extract of callus of Chirita pinnata, the concentration of nicotinamide 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 more further preferably 0.4%-1%.

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

[0014] The preparation method of the above-mentioned cotton head snow rabbit callus extract comprises the following steps: extracting the cotton head snow rabbit callus with ethanol and / or water. Further, 60%-80% ethanol or water is used to extract the cotton head snow rabbit callus; further, 65%-75% ethanol or water is used to extract the cotton head snow rabbit callus; and further, water is used to extract the cotton head snow rabbit callus.

[0015] Further, the cotton head snow rabbit callus is immersed in ethanol and / or water for extraction, and the obtained extraction solution is subjected to centrifugal and / or filtration treatment, and then dried. Further, 60%-80% ethanol or water is used to extract the cotton head snow rabbit callus; further, 65%-75% ethanol or water is used to extract the cotton head snow rabbit callus; and further, water is used to extract the cotton head snow rabbit callus.

[0016] Before the extraction, the ethanol and / or water mixed with the cotton head snow rabbit callus is ground and crushed. Further, 60%-80% ethanol or water is selected, further 65%-75% ethanol or water is selected, and further water is selected.

[0017] The extraction solution is subjected to centrifugal treatment first, and then filtration treatment.

[0018] The drying treatment is freeze-drying.

[0019] The induction method of the cotton head snow rabbit callus comprises the following steps: cutting the organs of the cotton head snow rabbit, inoculating the organs into an induction medium, and inducing the callus. The organs are any one or more of roots, stems or leaves.

[0020] The MS medium is used as a basic medium for inducing and culturing the cotton head snow rabbit callus. Preferably, the medium further contains plant growth hormones and sucrose.

[0021] The induction medium uses the MS medium as a basic medium. Preferably, the induction medium further contains plant growth hormones; the plant growth hormones comprise 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 cotton head snow rabbit callus comprises the following steps: subculturing the cotton head snow rabbit callus for 10-35 days; and the medium for subculturing uses the MS medium as a basic medium.

[0023] Preferably, the medium further contains plant growth hormones; the plant growth hormones comprise 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 sucrose and / or 20-40 g / L potato powder.

[0025] The present application uses the extract of the snow rabbit callus and nicotinamide to promote the clearance of DPPH free radicals, inhibit the generation of ROS, and improve the antioxidant activity. The snow rabbit callus extract and nicotinamide composition with synergistic effect provided by the present application can be used for preparing a DPPH free radical clearance product, and / or a ROS generation inhibition product, and / or an antioxidant product.

[0026] The DPPH free radical clearance product, and / or the ROS generation inhibition product, and / or the antioxidant product provided by the present application contains the extract of the snow rabbit callus and nicotinamide.

[0027] The mass ratio of the nicotinamide to the extract of the snow rabbit callus is 1:0.001-25, preferably 1:0.002-20, further 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 mode of the present application, it is 1:0.05-2.5 or 1:2.5-5; more preferably 1:0.05-1 or 1:2.5-5.

[0028] The concentration of the nicotinamide in the product 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 more further preferably 0.4%-1%.

[0029] The concentration of the extract of the snow rabbit callus in the product is 0.01%-5%, preferably 0.1%-2.5%, and further preferably 0.25%-1%.

[0030] The DPPH free radical clearance product, and / or the ROS generation inhibition product, and / or the antioxidant product described in the present application can be an additive, and / or a cosmetic, and / or an oral cosmetic, and / or a health product, and / or a food, and / or a medicine, and the presentation state includes but is not limited to liquid, or solid, or oil, or powder, or milk, or paste, or cream, or gel, or aerosol, or powder mist.

[0031] As described above, the cosmetic refers to a daily chemical industry product applied to the human body surface such as skin, hair, nail, and lip by rubbing, spraying, or other similar methods for the purpose of cleaning, protecting, beautifying, and modifying. As a cosmetic, it can be classified into a cleansing type (for washing skin), a skin care type (such as cleansing cream, face wash, bath agent, hair wash, shaving cream, etc.), a base type (pre-treatment of face and hair before makeup, such as various creams, honey, makeup water, face mask, hair cream, hair gel, etc.), a beauty type (for beautifying face and hair, such as rouge, lipstick, eye shadow, hair dyeing and perming, hair style treatment, fixing, etc.), and a therapeutic type (a daily chemical product between medicine and cosmetic, such as cooling, deodorant, freckle-removing, sunscreen, body shaping, hair growing, hair removing, hair dyeing, insect repellent, olive essence, etc.), and can be classified into a liquid (such as face wash, bath liquid, hair wash, makeup water, perfume, skin cleansing water, makeup remover, essence, and original liquid), an emulsion (such as honey, milk, hair care cream, and essence cream), a cream (such as face cream, foundation cream, hair wash cream, concealer, hair cream, essence cream, and pre-makeup cream), a powder (such as face powder, body powder, loose powder, skin cleansing powder, and powder), a block (such as powder compact, cheek rouge, lipstick, and hair wax), and an oil (such as makeup remover, skin softener, hair softener, and essence oil).

[0032] As described above, as a health care product, it can be prepared in the form of powder, tablet, capsule, liquid preparation, gel, tea bag, beverage, and candy.

[0033] As described above, as a medicine, it can be classified into a solution type (a uniform dispersion system in which a medicine is dispersed in a molecular or ionic state (a diameter of a particle is less than 1 nm) in a dispersion medium, also called a low molecular solution, such as aromatic water, solution, syrup, glycerin, spirit, and injection), a colloidal solution type (a uniform dispersion system in which a high molecule (a diameter of a particle is 1 to 100 nm) is mainly dispersed in a dispersion medium, also called a high molecular solution, such as gum solution, collodion, and coating agent), an emulsion type (a non-uniform dispersion system in which an oil medicine or oil solution of a medicine is dispersed in a dispersion medium in the form of a droplet, such as oral emulsion, intravenous injection emulsion, and partial liniment), a suspension type (a non-uniform dispersion system in which a solid medicine is dispersed in a dispersion medium in the form of a particle, such as mixture, lotion, and suspension), a gas dispersion type (a dispersion system in which a liquid or solid medicine is dispersed in a gas dispersion medium in the form of a particle, such as aerosol), a micro particle dispersion type (a dispersion system in which a medicine is dispersed in the form of a particle of different sizes in a liquid or solid state, such as microsphere preparation, microcapsule preparation, and nanocapsule preparation), and a solid dispersion type (a dispersion system in which a solid medicine exists in the form of an aggregate, such as tablet, powder, granule, capsule, and pill).

[0034] The main advantages of the present application include: the present application finds that the extract of the callus of the rabbit-foot fern has a synergistic effect with nicotinamide, promotes the elimination of DPPH free radicals, inhibits the generation of ROS, and has good antioxidant effect. The present application provides a new path for the effective development and utilization of the extract of the callus of the rabbit-foot fern and nicotinamide, realizes the functional maximization of limited resources, and has a wide application prospect. DETAILED DESCRIPTION

[0035] Experimental principle

[0036]

Principle of DPPH free radical scavenging method

[0037]

Principle of rapid detection of intracellular reactive oxygen

[0038] Experimental method

[0039] I. Preparation of rabbit-foot fern extract

[0040] 1. Induction and subculture of rabbit-foot fern plant callus

[0041] In a sterile clean bench, the roots, stems or leaves of the rabbit-foot fern were cut into 0.5 cm-1.0 cm segments with a scalpel, and inoculated into a MS+6-BA 0.5 mg / L+NAA 0.3 mg / L+sucrose 30 g / L+Klucel 3.5 g / L medium formula to induce callus.

[0042] A portion of the rabbit-foot fern callus was transferred to a MS+6-BA 0.5 mg / L+NAA 0.5 mg / L+potato powder 30 g / L+sucrose 30 g / L+Klucel 3.5 g / L culture medium formula for subculture, and the rabbit-foot fern callus was harvested after 25-30 days, counted as "rabbit-foot fern callus", and stored frozen for later use.

[0043] Another portion of the P. gossypiphilus callus was transferred to a 1 / 2MS + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + gellan gum 3.5 g / L medium formula for subculture, and after 25-30 days, the P. gossypiphilus callus (non-normal passage) was harvested and frozen for later use.

[0044] 2. Preparation of P. gossypiphilus plant callus water extract or alcohol extract

[0045] The frozen P. gossypiphilus callus was thawed, ground into a homogenate, and then extracted for 3 hours after adding deionized water or 70% ethanol. The extract was then centrifuged at 8000 rpm-10000 rpm for 40 min-60 min, and the supernatant was obtained after centrifugation, filtered with a 0.45 uM filter, and the filtrate was obtained. The filtrate was freeze-dried at low temperature for 10-14 days, and the obtained powder was the P. gossypiphilus callus extract.

[0046] 3. Comparative cell culture of T. przewalskii

[0047] The comparative P. gossypiphilus plant was a T. przewalskii culture purchased from Ansebo.

[0048] II. Solution preparation

[0049] 1. Preparation of related solutions for DPPH free radical scavenging method

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

[0051] 4 mg of DPPH powder was taken in a 1.5 mL centrifuge tube, 100 mL of anhydrous ethanol was taken with a 10 mL pipette into a sample cup, and the DPPH powder was dissolved in the anhydrous ethanol in the sample cup and transferred to the sample cup. The sample cup was wrapped in tin paper to avoid light.

[0052] 1.2 Preparation of P. gossypiphilus callus extract solution

[0053] 10 mg of P. gossypiphilus extract powder was weighed and dissolved in 1 mL of deionized water to prepare a 10 mg / mL P. gossypiphilus callus extract solution, i.e., a 1% P. gossypiphilus callus extract solution.

[0054] 1.3 Preparation of nicotinamide solution

[0055] Take 10 mg, 20 mg, 50 mg, 100 mg respectively, dissolve in 1 mL deionized water, prepare 10 mg / mL, 20 mg / mL, 50 mg / mL, 100 mg / mL nicotinamide solution, which corresponds to 1%, 2%, 5%, 10% nicotinamide solution.

[0056] 1.4 Preparation of solution of L. japonica callus extract and nicotinamide composition

[0057] Prepare L. japonica and nicotinamide composition with different ratios, L. japonica callus extract concentration is 0.25%, 0.5% and 1%, and nicotinamide concentration is 0.1%, 1%, 2%, 5%, 10%; add to the reaction system according to different ratios.

[0058] 2. Preparation of solution related to rapid detection of intracellular reactive oxygen species

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

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

[0061] III. Experimental procedure

[0062] 1. Experimental procedure of DPPH free radical scavenging method

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

[0064] In the 96-well plate, according to the experimental sample table, add sample and DPPH solution or anhydrous ethanol respectively, make 3 parallel holes for each experimental group, place in the dark, use microplate constant temperature oscillator at 300 r / min, react at room temperature for 30 min. After the reaction is completed, detect the absorbance value at 517 nm by enzyme marker. The DPPH scavenging rate is calculated as follows: P = [1-(A s -A c ) / A< / s> b ]*100%

[0065] P - scavenging rate;

[0066] A s - absorbance of the mixture of test solution and DPPH solution;

[0067] A c - absorbance of the mixture of test solution and anhydrous ethanol solution;

[0068] A b - absorbance of the mixture of DPPH solution and sample solvent solution.

[0069] 2. Experimental Procedure for Rapid Detection of Intracellular Reactive Oxygen Species

[0070] 2.1 Cell Treatment

[0071] Based on 4.0×10 4 B16 cells were seeded into 96-well plates at a seeding rate of [number] cells / well and cultured for 24 h. The culture medium was aspirated, and the cells were washed once with DPBS. The test samples were added and cultured for 2 h, followed by two washes with DPBS. The prepared DCFH-DA working solution was added, and the plates were incubated at 37°C for 30 min. The DCFH-DA solution was removed, and the cells were washed twice with DPBS. The cell blank group was wrapped in aluminum foil, and the remaining groups were irradiated with 100 mJ UVB. Finally, the fluorescence values ​​were read using a microplate reader at Ex / Em = 485 nm / 535 nm.

[0072] 2.2 Data Processing

[0073] The ROS inhibition rate is calculated as follows:

[0074] Inhibition rate (%) = [mean value of control group - mean value of sample group / mean value of control group] * 100%

[0075] Table 1. DPPH scavenging rate of the combination of aqueous extract of "Saussurea involucrata callus" and nicotinamide.

[0076] Table 1 shows that the water extract of normal passaged tissue from Saussurea involucrata callus exhibits synergistic effects with nicotinamide, demonstrating good DPPH scavenging activity.

[0077] The basis for judging synergy here is that the scavenging efficiency of the composition is higher than the sum of the efficiency of the two individual samples.

[0078] Table 2. ROS inhibition rate of the combination of aqueous extract of "Snow Rabbit callus" and nicotinamide

[0079] Table 2 shows that the combination of the aqueous extract of normal passaged tissue of Saussurea involucrata callus and nicotinamide exhibits enhanced ROS scavenging activity to a certain extent, further demonstrating the synergistic activity between the aqueous extract of normal passaged tissue of Saussurea involucrata callus and nicotinamide.

[0080] Table 3. DPPH scavenging rate of nicotinamide compositions from *Saussurea involucrata* extract processed using different techniques.

[0081] The results in Table 3 show that the alcohol extract of the normal subculture tissue of the Saussurea laniceps callus and the nicotinamide exhibit a certain synergistic activity, but the degree is obviously lower than that of the combination of the water extract and the nicotinamide. The water extract of the abnormal subculture tissue of the Saussurea laniceps callus and the nicotinamide do not have the synergistic DPPH scavenging effect.

[0082] Table 4. DPPH scavenging rate of the nicotinamide combination of other Saussurea laniceps cultures (Saussurea laniceps cell culture, Ansenbo)

[0083] The results in Table 4 show that the commercially available Saussurea laniceps culture and the nicotinamide do not have the DPPH synergistic scavenging activity.

[0084] Table 5. Synergistic range definition

[0085] In summary, the Saussurea laniceps callus extract and the nicotinamide have the synergistic activity, which improves the DPPH scavenging rate and the ROS inhibition rate. The DPPH scavenging rate is even superior to that of the commercially available Saussurea laniceps extract or the DPPH scavenging rate of the combination thereof and the nicotinamide, especially the water extract of the normal subculture culture of the Saussurea laniceps callus. Therefore, the Saussurea laniceps callus extract and the nicotinamide combination of the present application synergistically scavenge DPPH, inhibit ROS generation, and exhibit excellent antioxidant activity.

Claims

1. A composition having a synergistic effect, characterized in that, An extract of a callus of a Chirita filipes and nicotinamide.

2. The composition of claim 1, wherein, The callus of the Chirita filipes is from an organ of the Chirita filipes, and the organ of the Chirita filipes includes at least one of a root, a stem, a leaf, a flower, a fruit, and a seed.

3. The composition of claim 1, wherein, The extract of the callus of the Chirita filipes is a water extract and / or an alcohol extract of the callus of the Chirita filipes.

4. The composition according to any one of claims 1 to 3, characterized in that, The callus of the Chirita filipes includes a callus of the Chirita filipes and / or a subcultured callus of the Chirita filipes.

5. The composition of claim 1, wherein, The mass ratio of the nicotinamide to the extract of the callus of the Chirita filipes is 1:0.001-25.

6. The composition of claim 5, wherein, The concentration of the nicotinamide is 0.01%-10%.

7. The composition of any one of claims 1-6, characterized in that, An MS medium is used as a basic medium for inducing and culturing the callus of the Chirita filipes.

8. The composition of any one of claims 1-7 for use in the manufacture of at least one of: (1) a product for scavenging DPPH radicals; (2) a product for inhibiting ROS generation; and (3) an antioxidant product.

9. At least one product characterized by, The composition of any one of claims 1-7 is used in: (1) a product for scavenging DPPH radicals; (2) a product for inhibiting ROS generation; and (3) an antioxidant product.

10. Use according to claim 8 or product according to claim 9, characterized in that, The product is at least one of an additive, a cosmetic, an oral cosmetic product, a health food, a food, and a pharmaceutical.

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