Composition for calendula callus culture and application thereof

Abstract

The invention provides a composition for calendula callus culture and application thereof. The composition comprises an induction culture medium, a subculture medium and a suspension culture medium, the induction culture medium is prepared from the following components according to final concentration: 0.1 to 0.5 mg / L of 6-benayl aminopurine and 2 to 4 mg / L of naphthylacetic acid; the subculture medium is prepared from the following components according to final concentration: 0.1 to 0.5 mg / L of 6-benayl aminopurine and 2 to 4 mg / L of naphthylacetic acid; the suspension culture medium is prepared from the following components according to final concentration: 0.1 to 0.2 mg / L of 6-benayl aminopurine and 1 to 3 mg / L of naphthylacetic acid; the basic culture medium is at least one of an MS culture medium, a WPM culture medium and a DCR culture medium. By exploring a proper callus induction hormone combination and constructing a mature suspension culture system, the callus quality and the suspension cell proliferation efficiency are remarkably improved, and a reliable technical basis is provided for large-scale production of active ingredients of calendula.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture, in particular to a composition for callus culture of Calendula officinalis and application thereof. BACKGROUND

[0002] Calendula officinalis L. is an annual herbaceous plant of the genus Calendula in the Asteraceae family. Calendula officinalis extract, also known as calendula essence, is a natural ingredient widely used in skin care products. Its main effects on the skin include antioxidant, anti-inflammatory, calming and repairing. The main active ingredients of Calendula officinalis include flavonoids, triterpenoids, polyphenols, carotenoid compounds, etc. Calendula officinalis extract has anti-inflammatory and antibacterial effects, especially on staphylococcus and streptococcus. Based on the proliferation effect of Calendula officinalis extract on epidermal cells and dermal cells, the inhibition of elastase activity, combined with its excellent antioxidant properties, it can enhance skin activity, moisturizing, anti-inflammatory, antioxidant, anti-aging, and is widely used in the fields of medicine, cosmetics and functional food. However, its active ingredients depend on traditional planting extraction, which has the problems of long cycle and content affected by environmental fluctuations. Using plant tissue culture technology to produce callus cells and combining stable suspension culture technology is an ideal alternative for large-scale production, but the key is the lack of a mature callus tissue suspension cell culture system of Calendula officinalis. At present, there are few reports on the induction of callus tissue from Calendula officinalis leaves, and it is necessary to conduct in-depth research and explore Calendula officinalis extract with higher activity value in skin care. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a composition for callus culture of Calendula officinalis and application thereof, by exploring suitable hormone combinations for callus induction and constructing a mature suspension culture system, significantly improving callus quality and suspension cell proliferation efficiency, providing a reliable technical foundation for the value enhancement and large-scale production of Calendula officinalis active ingredients.

[0004] To achieve the above technical purposes, the technical solutions adopted by the present application are as follows:

[0005] In a first aspect, the present application provides a composition for callus culture of Gypsophila paniculata, which comprises an induction medium, a subculture medium and a suspension medium; wherein the induction medium comprises, in terms of final concentrations, 6-benzylaminopurine 0.1-0.5 mg / L and naphthalene acetic acid 2-4 mg / L; the subculture medium comprises, in terms of final concentrations, 6-benzylaminopurine 0.1-0.5 mg / L and naphthalene acetic acid 2-4 mg / L; and the suspension medium comprises, in terms of final concentrations, 6-benzylaminopurine 0.1-0.2 mg / L and naphthalene acetic acid 1-3 mg / L; and the basic medium used in the induction medium, the subculture medium and the suspension medium is at least one of MS medium, WPM medium and DCR medium.

[0006] Preferably, the basic medium is MS medium; and / or the induction medium, the subculture medium and the suspension medium further comprise sucrose 25-30 g / L and plant gum 3-5 g / L.

[0007] Further preferably, the induction medium comprises sucrose 25-26 g / L and plant gum 4-5 g / L; and / or the subculture medium comprises sucrose 25-26 g / L and plant gum 4-5 g / L; and / or the suspension medium comprises sucrose 29-30 g / L.

[0008] In a second aspect, the present application provides the use of the composition for callus culture of Gypsophila paniculata according to the first aspect in obtaining callus of Gypsophila paniculata.

[0009] In a third aspect, the present application provides a method for culturing callus of Gypsophila paniculata, which comprises the following steps:

[0010] Step 1: obtaining leaves of Gypsophila paniculata;

[0011] Step 2: inducing callus from the leaves of Gypsophila paniculata by using the induction medium in the composition according to claim 1 or 2;

[0012] Step 3: subculturing the callus in the subculture medium in the composition according to claim 1 or 2;

[0013] Step 4: suspending the subcultured callus in the suspension medium in the composition according to claim 1 or 2 to obtain callus culture of Gypsophila paniculata.

[0014] There are many ways to obtain leaves of Gypsophila paniculata in the above step 1, which are conventional operations, and the present application preferably provides a way to obtain leaves of Gypsophila paniculata, which comprises the following steps: sterilizing seeds of Gypsophila paniculata and then culturing the seeds to germination, and further culturing the germinated seeds to obtain leaves.

[0015] Optionally, one operation mode of the germination process is to culture the sterilized seeds in MS medium at a temperature of 26±2℃ and under light intensity of 2500-3000 Lux with a light cycle of 12h / d -1 .

[0016] In step 2, the leaf is usually cut into small pieces, such as about 0.5×0.5cm, for better obtaining and observing the callus. Then the small pieces of the leaf are added to the induction medium, and the pH is adjusted to be between 5.50 and 5.60, preferably between 5.80 and 5.90, and more preferably between 5.80 and 5.85, and the small pieces of the leaf are cultured at a temperature of 26±2℃ in the dark (completely avoiding light) for 15-30 days, preferably for 20-30 days.

[0017] In step 3, the culture conditions are the same as those in step 2, and the number of subcultures is not less than 2 times to obtain callus with uniform growth state and high activity, and the subculture is stopped when the callus is observed to grow stably. The number of subcultures is preferably 3 times.

[0018] In step 4, the suspension culture conditions include inoculation at a weight / volume ratio of 1-10:50, adjustment of the pH of the system to be between 5.50 and 5.60, preferably between 5.80 and 5.90, and more preferably between 5.80 and 5.85, and dark culture at a rotation speed of 80-150rpm for 20-40 days.

[0019] Preferably, the suspension culture further includes a pre-culture, and the pre-culture time is 15-25 days, preferably 20-22 days; and the suspension culture after the pre-culture is performed for 12-18 days, preferably 13-15 days; preferably, the cell inoculation amount is a weight / volume ratio of 1-5:50, and more preferably a weight / volume ratio of 1-3:50.

[0020] In a fourth aspect, the present application provides a callus of Calendula officinalis obtained by the culture method of the third aspect.

[0021] In a fifth aspect, the present application provides a callus extract of Calendula officinalis, wherein the callus extract is obtained from the callus of Calendula officinalis of the fourth aspect, and the callus extract contains flavonoids, triterpenoids and polyphenols, wherein the weight content of the flavonoids is not less than 0.2%, the weight content of the triterpenoids is not less than 0.4%, and the weight content of the polyphenols is not less than 0.4%.

[0022] In a sixth aspect, the present application provides a preparation method of the callus extract of Calendula officinalis of the fifth aspect, which comprises drying the callus of Calendula officinalis and then adding a solvent to extract the callus to obtain the callus extract of Calendula officinalis.

[0023] Preferably, the solvent includes, but is not limited to, at least one of organic alcohol, organic ether, organic ketone, organic ester, water, phosphate buffer.

[0024] Further preferably, the solvent is phosphate buffer, which contains Tween-80 with a final concentration of 0.0125wt%, 10mM NaH2PO4, and 150mM NaCl.

[0025] Preferably, the extraction process uses heating at 50-70℃ for 1-3h; or, uses heating at 50-70℃ combined with ultrasonic assistance for 20-40min.

[0026] Further preferably, the ultrasonic power is 100-300W, and the frequency is 30-50kHz.

[0027] In a seventh aspect, the present application provides use of the calendula callus extract of the sixth aspect in the preparation of a skin external use composition.

[0028] In an eighth aspect, the present application provides a skin external use composition, which includes the calendula callus extract of the sixth aspect.

[0029] Preferably, it further includes other active ingredients and / or ingredients that produce synergistic effects with the calendula callus extract of the sixth aspect.

[0030] In a ninth aspect, the present application provides at least one of the following 1) to 7) skin products, characterized in that it includes the skin external use composition of the eighth aspect:

[0031] 1) a skin product for repairing damaged cells;

[0032] 2) a skin product for eliminating inflammation;

[0033] 3) a skin product for acne removal;

[0034] 4) a skin product for bacteria removal;

[0035] 5) a skin product for oil control;

[0036] 6) a skin product for moisturizing;

[0037] 7) a skin product for anti-aging and wrinkle removal.

[0038] The skin product can also optionally contain, but is not limited to, various adjuvants such as surfactants, diluents, emulsifiers, thickening agents, dispersants, etc. Surfactants such as: cetyl stearyl olivate, sorbitan olivate, polysorbate-60, polysorbate-80, methyl glucose sesquistearate, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butoxy ether-26, PEG-4 polyglyceryl-2 stearate, etc. Diluents such as glycerin, dipropylene glycol, butylene glycol, etc. Emulsifiers such as: one or more of polyglyceryl-3 methyl glucose distearate, glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyceryl-10 dioleate, polyglyceryl-10 laurate, polyglyceryl-10 isostearate, polyglyceryl-10 oleate, polyglyceryl-10 diisostearate, polyglyceryl-6 laurate, polyglyceryl-6 myristate, sucrose stearate, sucrose polysstearate, etc. Thickening agents such as: one or more of carbomers, acrylates and their derivatives, xanthan gum, acacia gum, polyethylene glycol-14M, polyethylene glycol-90M, succinoglucan, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. high molecular polymers. Dispersants such as: gelatin, pectin, starch, polyvinyl alcohol, polyacrylic acid, etc.

[0039] The skin product can also optionally contain other active ingredients such as one or more of tocopherol (vitamin E), retinol, retinol palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin, ellagic acid, ubiquinone, lactoprotein, polypeptide, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyloyl phytosphingosine, concentrated birch sap, silymarin, silk fibroin, sodium tocopheryl phosphate, ribonucleic acid (RNA), dipeptide diaminobutyroyl hydrazide benzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide-7, VITIS VINIFERA (GRAPE) SEED EXTRACT, PTEROCARPUS MARSUPIUM (KINO TREE) BARK EXTRACT, CAMELLIA SINENSIS (GREEN TEA) POLYPHENOLS, WINE EXTRACT, APPLE SEED EXTRACT, FAGUS SYLVATICA (BEECH) BUD EXTRACT, HYDROLYZED ADANSONIA DIGITATA (BAOBAB) EXTRACT, ARTEMIA EXTRACT, IRIS FLORENTINA (IRIS) ROOT EXTRACT, HEPANTRINE, GINSENOSIDE, SALVIA MILTIORRHIZA (DANSHEN) EXTRACT, NICOTINAMIDE, URSOLIC ACID, SODIUM HYALURONATE, SODIUM ACETYLATED HYALURONATE, SODIUM HYDROLYZED HYALURONATE, LYCOPENE, COFFEA ARABICA (COFFEE) EXTRACT, DIPEPTIDE-2, LACTIC ACID, SUPER OXIDE DISMUTASE (SOD), OENOTHERA BIENNIS (EVENING PRIMROSE) OIL, CERAMIDE, DIPALMITOYL HYDROXYPROLINE, HYDROXYSTEARIC ACID, SALICYLIC ACID, ERGOTHIONE, LYPOHYDROXY ACID, CARNOSINE, DECARBOXYLASE CARNOSINE HCL, THIOCTIC ACID, ADENOSINE, GLYCOGEN, RESVERATROL, FERULIC ACID, SCHIZACHYRYUM MOUNTAIN FERMENT EXTRACT, LACTOBACILLUS FERMENT EXTRACT, etc.

[0040] The Calendula callus extract or the freeze-dried powder of the present application can be mixed with other pharmaceutical ingredients or cosmetic ingredients to obtain a pharmaceutical composition or a cosmetic composition according to any method known in the skin product industry. The other pharmaceutical ingredients or cosmetic ingredients are the ingredients commonly used in skin products as described above.

[0041] In addition, the skin product can be manufactured in various dosage forms such as a solution, a suspension, a paste, a cream, an emulsion, a gel, a powder, or a spray, etc. according to the need.

[0042] The present application has the following advantageous effects compared to the prior art.

[0043] (1) The application can significantly improve the yield and purity of target active ingredients (flavonoids, triterpenoids, polyphenols and the like) in the callus of the calendula by optimizing the culture system, and the component difference is controllable, and the RSD of the target active ingredients in the callus of the calendula prepared in 6 batches at different time periods is less than 5%, and the batch stability is far superior to planting and extraction.

[0044] (2) The application changes the calendula leaves (non-main harvesting part) into treasure, which meets the green circular economy, and the factory production is not affected by seasons and climate, and the safety of the downstream industry chain is ensured.

[0045] (3) The extract of the callus of the calendula has good effects in anti-inflammatory repair and oil control and the like through multi-target mechanism verification, and is expected to be developed into skin products with multiple effects such as repairing damaged cells, eliminating inflammation, removing acne, eliminating bacteria, resisting and controlling oil, moisturizing, anti-aging and wrinkle removal. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Table 1 is the callus induction result of the callus of the calendula in Example 1 of the application after adopting different hormone component proportions.

[0047] Figure 2 Table 2 is the callus induction result of the callus of the calendula in Example 1 of the application after adopting different basic culture media.

[0048] Figure 3 Table 3 is the subculture result of the callus of the calendula in Example 1 of the application after adopting different hormone component proportions.

[0049] Figure 4 Table 4 is the culture result of different plant cells in Example 2 of the application after adopting the same induction culture medium and subculture medium.

[0050] Figure 5 Table 5 is the repair effect of the extract of the callus of the calendula in Example 3 of the application.

[0051] Figure 6 Table 6 is the oil control effect of the extract of the callus of the calendula in Example 3 of the application.

[0052] Figure 7 Table 7 is the anti-inflammatory effect of the extract of the callus of the calendula in Example 3 of the application, wherein A to G respectively correspond to the expression amounts of the inflammation-related genes COX-2, IL-1a, IL-1β, IL-6, TNF-a, IL-10 and NOS-2. DETAILED DESCRIPTION

[0053] In the embodiment, the detection method of flavonoids, triterpenoids and polyphenols in the extract of the callus of the calendula refers to the high performance liquid chromatography method (general rule 0512), and the specific method is as follows:

[0054] 1) Polyphenols content (calculated as chlorogenic acid): C18-AQ HP column (1.9 μm, 2.1 x 100 mm) was used with acetonitrile: 0.1% phosphoric acid aqueous solution = 15:85 as mobile phase, flow rate 0.2 mL / min, column temperature 35°C, injection volume 2 μL, detection wavelength 375 nm. A certain amount of marigold callus extract was accurately pipetted, dissolved with mobile phase, and shaken to constant volume. The corresponding chromatographic peak area was determined and recorded according to the above chromatographic conditions, and the standard curve was plotted with the concentration of the reference solution as the abscissa and the peak area as the ordinate. The linear regression equation was calculated, and the concentration of chlorogenic acid in the test solution was calculated by the peak area external standard method through the standard curve regression equation.

[0055] Preparation of reference solution: 0.5 g of chlorogenic acid reference substance was accurately weighed, dissolved with mobile phase, and diluted to 10 mL. After shaking, a series of concentration reference solutions were obtained by gradient dilution.

[0056] 2) Triterpenoids content (calculated as oleanolic acid): C18-AQ HP column (1.9 μm, 2.1 x 100 mm) was used with acetonitrile: 0.1% phosphoric acid aqueous solution = 80:20 as mobile phase, flow rate 0.2 mL / min, column temperature 35°C, injection volume 2 μL, detection wavelength 210 nm. A certain amount of marigold callus extract was accurately pipetted, dissolved with mobile phase, and shaken to constant volume. The corresponding chromatographic peak area was determined and recorded according to the above chromatographic conditions, and the standard curve was plotted with the concentration of the reference solution as the abscissa and the peak area as the ordinate. The linear regression equation was calculated, and the concentration of oleanolic acid in the test solution was calculated by the peak area external standard method through the standard curve regression equation.

[0057] Preparation of reference solution: 0.5 g of oleanolic acid reference substance was accurately weighed, dissolved with mobile phase, and diluted to 10 mL. After shaking, a series of concentration reference solutions were obtained by gradient dilution.

[0058] 3) Flavonoids content (calculated as quercetin): C18-AQ HP column (1.9 μm, 2.1 x 100 mm) was used with acetonitrile: 0.1% phosphoric acid aqueous solution according to a specific gradient (acetonitrile 5%, 0.1% phosphoric acid aqueous solution 95% at 0 min; acetonitrile 20%, 0.1% phosphoric acid aqueous solution 80% at 1 min; acetonitrile 30%, 0.1% phosphoric acid aqueous solution 70% at 2 min; acetonitrile 45%, 0.1% phosphoric acid aqueous solution 55% at 8 min; acetonitrile 80%, 0.1% phosphoric acid aqueous solution 20% at 8.2 min; acetonitrile 80%, 0.1% phosphoric acid aqueous solution 20% at 9 min; acetonitrile 5%, 0.1% phosphoric acid aqueous solution 95% at 9.5 min; acetonitrile 5%, 0.1% phosphoric acid aqueous solution 95% at 15 min) elution, flow rate 0.2 mL / min, column temperature 40°C, injection volume 2 μL, detection wavelength 360 nm. A certain amount of calendula callus extract was accurately taken, diluted with water, shaken, and then determined according to the above chromatographic conditions to record the corresponding chromatographic peak area. The standard curve was drawn with the concentration of the reference solution as the abscissa and the peak area as the ordinate, and the linear regression equation was calculated. The peak area external standard method was used to calculate the concentration of quercetin in the test solution by the standard curve regression equation.

[0059] Preparation of reference solution: 0.5 g of quercetin reference substance was accurately weighed, dissolved in a small amount of methanol, and then diluted with water to 50 mL. After shaking, a series of concentration reference solutions were obtained by gradient dilution.

[0060] Further, in the description of the present application, it should be noted that, in the examples, no specific conditions are specified, and conventional conditions or manufacturer's recommended conditions are used. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market.

[0061] Unless otherwise defined, all professional terms used below have the same meaning as understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present application.

[0062] In order to facilitate the understanding of the present application, the present application will be described more fully and specifically below in conjunction with the drawings and examples, but the scope of protection of the present application is not limited to the following specific examples.

[0063] Example 1:

[0064] The present application provides an embodiment of calendula callus culture method, which is as follows:

[0065] (1) Obtaining of calendula sterile seedlings

[0066] The full and plump marigold seeds are surface sterilized by immersing in 75% ethanol solution for 30-60 seconds, then washed with sterile water for 3-5 times. The seeds are then deep sterilized by adding 2-3 drops of Tween 20 into 84 sterilization solution and slowly rotating for 20 minutes, and finally washed with sterile water for 5 times until the seeds are clean. The seeds are inoculated on MS medium and cultured in a plant incubator at a temperature of 26±2℃, light intensity of 2500-3000 Lux, and light cycle of 12h / d. 1 The seeds start to germinate after about 21 days, and the germinated seeds are inoculated in a glass culture bottle for culture. After the leaves are unfolded, the leaves are cut into small pieces of about 0.5x0.5 cm for standby use.

[0067] (2) Induction of callus

[0068] The treated leaf pieces are inoculated on callus induction medium, 9 leaf pieces are placed on each culture plate, and the medium in each culture plate is about 25 mL. The callus induction medium is MS medium added with 2,4-D 0-1 mg / L, 6-BA 0-0.5 mg / L, NAA 0-4 mg / L, sucrose 25 g / L, and plant gum 5 g / L, and the pH value is adjusted to 5.80-5.85. The inoculated medium is cultured in the dark at a temperature of 26±2℃ for 24 days to induce callus. The feeding and experimental results are shown in Figure 1 and Table 1.

[0069] Table 1: Results of callus induction

[0070] Serial number 6-BA (mg / L) NAA (mg / L) 2,4-D (mg / L) Experimental results 1 0.5 0.5 0.5 Callus appeared quickly browning 2 0.5 1 0 There were more callus often appeared, but browning was serious 3 0.5 2 0 There were more callus often appeared, slight browning 4 0.5 3 0 There were less callus appeared 5 0.5 4 0 There were more callus appeared, a small amount of browning 6 0.1 2 0 There were more callus appeared, and the color was normal 7 0 0 1 There were less callus appeared, and all browning 8 0 0 0.5 There was no callus appeared, leaves withered and died

[0071] From the data in Figure 1 and Table 1, the medium compositions corresponding to serial numbers 3-6, i.e. 6-BA 0.1-0.5 mg / L and NAA 2-4 mg / L, can normally induce callus, and the medium compositions of serial numbers 3, 5, and 6 have relatively good callus induction effects, and the most preferred is the medium composition of serial number 6.

[0072] Based on the induction medium of serial number 6 in Table 1, the induction effects of different basic media are further investigated, and the operation is the same as above. The different basic media are shown in Table 2.

[0073] Table 2: Induction results of different basic media

[0074]

[0075] In combination with Table 2 and Figure 2It is evident that only MS, DCR, and WPM as basic culture media can be used to obtain Calendula callus tissue, and MS is the most effective basic culture medium.

[0076] (3) Subculture of callus

[0077] Callus induction was performed using an induction medium composed of 0.1 mg / L 6-BA, 2 mg / L NAA, 25 g / L sucrose, and 5 g / L plant gum, with MS medium as the basal medium. Callus tissue from the induction plates was transferred to corresponding plates and subcultured using medium. Each plate contained 16–20 cell clusters, approximately 0.2 × 0.2 cm in size, with approximately 25 mL of medium per plate. Subculture was performed using MS medium as the basal medium, supplemented with 0.1–0.5 mg / L 6-BA, 2–4 mg / L NAA, 30 g / L sucrose, and 3 g / L plant gum, adjusting the pH to 5.80–5.85. Culture conditions were the same as the callus induction phase, with subculture every 20 days for a total of three subcultures to obtain callus tissue with uniform growth and high viability. Experimental Results Figure 3 As shown in Table 3.

[0078] Table 3 Results of callus subculture

[0079] Serial number 6-BA (mg / L) NAA (mg / L) Experimental results (subculture 3 times) 1 0.5 2 Cell proliferation was faster, and the particles were large 2 0.5 4 Cell proliferation was slow, and the particles were large 3 0.1 2 Cell proliferation was fast, and the particles were loose, which was beneficial to suspension culture

[0080] pass Figure 3 According to the data in Table 3, during the subculture stage, all three subculture culture media compositions could promote the proliferation of callus cells, but the third group showed the best effect.

[0081] (4) Establishment of suspension culture system

[0082] 1) Investigation of culture medium components

[0083] The subculture medium is composed of 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 30 g / L, plant gum 3 g / L, and MS as the basic medium. The stable callus after subculture is transferred into liquid medium for suspension culture, and the cell inoculation amount is 1.5 g / 50 mL culture system. The suspension culture medium is based on MS medium, and 6-BA and NAA shown in Table 4 are added, and sucrose 30 g / L is added, and the pH value is adjusted to 5.80-5.85. The domestication culture is carried out in a 250 mL conical flask for 21 days, and the culture condition is dark culture at a shaking bed speed of 100 r / min. After the granularity of the cells is small and basically uniform, the domesticated cells are inoculated into new identical culture liquid at the same inoculation amount for suspension culture for 14 days. The culture liquid is removed, the cell moisture is absorbed with a paper towel, the fresh weight of the cells is weighed, the best suspension culture composition and proportion are screened, and the results are shown in Table 4.

[0084] Table 4: Medium composition and results of suspension culture

[0085] Note: The culture time in Table 4 refers to the domestication culture time.

[0086] The data in Table 4 show that in the suspension stage, the three groups of suspension culture medium compositions can all make the callus cells proliferate, but the best one is the first group.

[0087] 2) Investigation of cell inoculation amount

[0088] The subculture medium is composed of 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 30 g / L, plant gum 3 g / L, and MS as the basic medium. The stable callus after subculture is transferred into liquid medium for suspension culture, and the cell inoculation amount is 1.5 g / 50 mL culture system. The suspension culture medium is based on MS medium, and 6-BA 0.1 mg and NAA 2 mg / L are added, and sucrose 30 g / L is added, and the pH value is adjusted to 5.80-5.85. The domestication culture is carried out in a 250 mL conical flask for 21 days, and the culture condition is dark culture at a shaking bed speed of 100 r / min. After the granularity of the cells is small and basically uniform, the domesticated cells are inoculated into new identical culture liquid at the same inoculation amount for suspension culture for 14 days. The culture liquid is removed, the cell moisture is absorbed with a paper towel, the fresh weight of the cells is weighed, the best suspension culture composition and proportion are screened, and the results are shown in Table 4.

[0089] Table 5: Experimental results of suspension culture inoculation amount

[0090]

[0091] The data in Table 5 show that, under the condition that the culture system is 50 mL, the cell inoculation amount can obtain good proliferation effect between 1 and 5, but when the cell addition amount is 1-3 g, the final obtained cell weight and the proliferation fold are relatively high, so the inoculation ratio in the process of suspension culture is controlled at 1.0-3.0 g / 50 mL of culture medium, and the optimal is 1.20 g / 50 mL of culture medium.

[0092] Example 2

[0093] Referring to the induction culture and subculture process in Example 1, the induction culture medium is composed of 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 25 g / L, plant gum 5 g / L, and MS as the basic medium; the subculture medium is composed of 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 30 g / L, plant gum 3 g / L, and MS as the basic medium; the induction and subculture of callus of calendula, radix sinopodophyllum, rice, radish and dendrobium candidum protocorm are carried out to investigate the proliferation and development of the callus in the induction culture medium and the subculture medium of the application. Among them, the induction culture medium is composed of MS medium as the basic medium, 6-benzylaminopurine (6-BA) 0.1 mg / L, naphthalene acetic acid (NAA) 2 mg / L, sucrose 25 g / L, plant gum 5 g / L, and the pH value is adjusted to 5.80-5.85; the subculture medium is composed of MS medium as the basic medium, 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 30 g / L, plant gum 3 g / L, and the pH value is adjusted to 5.80-5.85. The induction culture and subculture conditions are the same as in Example 1. The calendula callus used in this embodiment is the cell with serial number 5 in Table 4 of Example 1, and the radix sinopodophyllum callus, rice callus, radish callus and dendrobium candidum protocorm are all self-developed products of Guangdong Kepu Rui Biological Technology Co., Ltd.

[0094] The experimental results are shown in Table 6. Figure 4 and Table 6.

[0095] Table 6 shows the culture results of different plant cells

[0096] Serial number Different plant cells Experimental results 1 Calendula callus After subculture for 20 days, cell proliferation was maximized, and the cells were loose and normal in color 2 Callus of Marsdenia tenacissima After subculture for 30 days, callus had proliferation, but the color was greenish, and there was a trend of differentiation into buds 3 Rice callus After subculture for 30 days, callus had proliferation, but the color was whiteish, and the state was not good 4 Radish callus After subculture for 30 days, callus had proliferation, but the color was greenish, and there was a trend of differentiation into buds 5 Dendrobium candidum protocorm After subculture for 30 days, protocorm had no proliferation phenomenon

[0097] By Figure 4From the data of Table 6, it can be seen that, using the induction medium and subculture medium of the present application for induction and subculture of different plant cells, only the Gypsophila paniculata callus grows well, and the growth and development of the callus of Gypsophila paniculata, the rice callus, the radish callus, and the Dendrobium candidum protocorm are not good. Therefore, the induction medium and the subculture medium are only suitable for Gypsophila paniculata callus culture at present.

[0098] Example 3

[0099] Based on the results of Example 1, this example uses the preferred conditions to culture Gypsophila paniculata callus, and the obtained callus culture is further prepared into an extract, as follows:

[0100] (1) Gypsophila paniculata callus induction and culture

[0101] After the full Gypsophila paniculata seeds are selected and the seed coat is removed, the seeds are soaked in a 75% ethanol solution for 30-60 seconds for surface sterilization, and then washed with sterile water for 3-5 times. Then, the seeds are placed in 84 disinfectant solution with 2-3 drops of Tween 20 added, mixed, and slowly rotated for 20 minutes for deep sterilization, and finally washed with sterile water for 5 times until the seeds are clean. The seeds are inoculated on MS medium and placed in a plant incubator for culture, with a culture temperature of 26±2℃, a light intensity of 2500-3000 Lux, and a light cycle of 12h / d. -1 The seeds start to germinate after about 21 days, and the germinated seeds are inoculated in a glass culture bottle for culture. After the leaves are unfolded, the leaves are cut into small pieces of about 0.5×0.5 cm for standby use.

[0102] The treated leaf pieces are inoculated on callus induction medium, with 9 leaf pieces per culture plate and about 25 mL of medium per culture plate. The callus induction medium is MS medium with 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 25 g / L, and plant gum 5 g / L, with the pH value adjusted to 5.80-5.85. The inoculated medium is cultured in the dark at a temperature of 26±2℃ for 20-30 days to induce callus.

[0103] The callus appeared on the induction plate was transferred to the corresponding plate for subculture medium to culture, 16-20 cell masses were plated on each culture plate, each cell mass was about 0.2*0.2 cm in size, and about 25 mL of medium was contained in each culture plate. The subculture medium was based on MS medium, 6-BA 0.1 mg / L, NAA 2 mg / L, sucrose 30 g / L, and plant gum 3 g / L were added, and the pH value was adjusted to 5.80-5.85. The culture conditions were the same as those in the callus induction stage, and the subculture was performed once every 20 days, and the subculture was performed for 3 times in succession to obtain callus with uniform growth state and high activity.

[0104] The stable callus after subculture was transferred into a liquid medium for suspension culture, and the inoculation amount was 1.2 g / 50 mL, and 50 mL represented the total volume of the suspension culture system. The suspension culture medium was based on MS medium, 0.1 mg / L 6-BA, 2 mg / L NAA, and 30 g / L sucrose were added, and the pH value was adjusted to 5.80-5.85. The domestication culture was performed in a 250 mL conical flask for 21 days, the culture condition was 100 r / min of shaking bed rotation speed in the dark, and the particle size of the cells was small and substantially uniform. Then the domesticated cells were inoculated into new identical culture solution for suspension culture for 14 days.

[0105] According to the above method, more than 20 batches of callus of Calendula officinalis were prepared from 2024 to 2025, 6 batches of callus samples of Calendula officinalis prepared in different time periods were randomly selected, the callus extract of Calendula officinalis was prepared according to the extraction method of phosphate buffer as a solvent in the present example (2), and then the content of flavonoids, triterpenes and polyphenols was detected, as shown in Table 7, the RSD of flavonoids, triterpenes and polyphenols was 4.30%, 4.90% and 3.86% respectively, all less than 5%, indicating that the content fluctuation of active ingredients between batches was extremely small, and the batch stability was excellent. The RSD of traditional planting extraction is generally greater than 10%.

[0106] Table 7 Content of flavonoids, triterpenes and polyphenols in callus obtained in different time periods

[0107]

[0108] (2) Preparation of Calendula officinalis callus extract

[0109] (2-1) Sample preparation: the callus of Calendula officinalis obtained from the same batch was dried at 42°C until completely dehydrated to obtain dry powder callus, which was then ground and sieved through a 60-mesh sieve, and 100 g was evenly divided into 4 parts for standby.

[0110] (2-2) Extraction solvent: water, ethanol, acetone, phosphate buffer (containing 0.0125wt% Tween-80 + 10mM NaH2PO4+ 150mM NaCl).

[0111] (2-3) Extraction method: 4 equal parts of Calendula officinalis callus dry powder were added to the above-mentioned certain amount of extraction solvent according to the solid-liquid ratio of 1:20 (w / v) and vortexed. Ultrasonic-assisted extraction was carried out in a 60°C water bath (ultrasonic power 200W, frequency 40kHz) for 30min. After the extraction was completed, the sample was cooled to room temperature, made up to the mark with the extraction solvent, centrifuged at 4000r / min for 10min, and the supernatant was taken and filtered (0.22μm filter membrane) to obtain the Calendula officinalis callus extract. Then, HPLC detection was performed, and the same sample was independently repeated for 3 times of HPLC detection. The extraction results of different solvents are shown in Table 8.

[0112] Table 8 Extraction results of different solvents

[0113] Extraction solvent Flavonoid content mg Polyphenol content mg Triterpenoid content mg Water 20.1 22.5 12.5 Ethanol 32.5 30.1 14.9 Acetone 27.5 27.2 24.8 Phosphate buffer 22.8 45.3 22.9

[0114] From Table 8, the extraction effects of the four solvents of ethanol, acetone, phosphate buffer on the three active ingredients are better than water. Considering the factors of precise pH control of phosphate to ensure the stability of active ingredients and the higher biological safety of phosphate than ethanol / acetone, etc., the phosphate buffer is finally selected as the extraction solvent. In addition, in the HPLC content determination test of multiple batches, the flavonoids (calculated by quercetin) in the Calendula officinalis callus extract obtained by using the phosphate buffer were in the content range of 0.9-1.1mg / g. The triterpenoids (calculated by oleanolic acid) were in the content range of 0.8-1.1mg / g. The polyphenols (calculated by chlorogenic acid) were in the content range of 1.7-2.0mg / g.

[0115] Example 4:

[0116] This example verifies the efficacy of the Calendula officinalis callus extract obtained according to Example 3, as follows:

[0117] (1) Repair efficacy of different amounts of Calendula officinalis callus extract on HaCaT cells

[0118] Take commercially available human immortalized keratinocytes HaCaT to 80-90% confluence, well-conditioned T25 bottle HaCaT cells, remove the old culture medium, wash once with 1xPBS, add 1 mL of 0.25% trypsin to the culture bottle and place in the incubator for 7-8 min, then add 3 mL of DMEM complete medium (containing 10% FBS) to terminate digestion, repeatedly blow to make a cell suspension, centrifuge at 200xg for 5 min, resuspend and count to adjust the cell density to 3x10 6 cells / mL; insert the scratch insert into the 24-well plate (source: Kuzhenguo, item number: CR211-2), inoculate the cell suspension into the insert well, 100 μL per well (i.e. 3x10 5 cells per well), incubate in a 37°C, 5% CO2 incubator for 24 h; remove the insert, gently wash the remaining in the well with PBS, add 1 mL of Calendula callus extract diluted with DMEM basic medium to each well of the sample group (the final concentration in the system ranges from 0% to 0.25%), add DMEM medium containing 10% FBS (fetal bovine serum) to the control group, set 2 replicate wells for each group, take pictures under a 10x microscope (1-3 pictures can be taken per well), record as 0h; continue to culture for 24 h, then take pictures under the same conditions, measure the area of the scratch area using Image J software, and calculate the cell migration rate according to the formula "cell migration rate = [(0h scratch area-24h scratch area) / 0h scratch area]x100%".

[0119] Figure 5 The cell migration rate of HaCaT cells after 24h with different amounts of Calendula callus extract is given, and the results show that the addition of 0.25% or more Calendula callus extract for 24h can achieve a keratinocyte repair effect of more than 47%.

[0120] (2) Oil control effect of Calendula callus extract with different amounts of addition

[0121] The inhibition rate of the sample on 5a-reductase is detected by using human 5a-reductase ELISA kit by double antibody sandwich method, and the specific steps are as follows: the required strip is taken out from the aluminum foil bag balanced at room temperature for 20 min, and the remaining strip is sealed with a self-sealing bag and put back into the 4℃ environment; set blank control group, negative control group, positive control group and sample group, each group has 3 parallel holes, the blank control group does not add any reagent, and the rest of the groups add 25μL of 20U / mL 5a-reductase standard product to each hole, then add water to the negative control group, add 0.5mg / mL finasteride to the positive control group, and add the sample to the sample group (the addition amount is 0.5% and 5% of the mass percentage in the system), the addition amount is 25μL, and it is placed in a 37℃ constant temperature incubator for incubation for 60 min; then the liquid is discarded, and the water paper is wiped dry, each hole is filled with washing liquid, and it is left for 1 min, then the washing liquid is shaken off and the water paper is wiped dry, and the plate is washed repeatedly for 5 times; except for the blank control group, 100μL of horseradish peroxidase (HRP) labeled detection antibody is added to each hole of the rest of the groups, the reaction hole is sealed with sealing film, and it is incubated in a 37℃ constant temperature incubator for 60 min; the liquid is discarded again, the water paper is wiped dry, each hole is filled with washing liquid, and it is left for 1 min, then the washing liquid is shaken off and the water paper is wiped dry, and the plate is washed repeatedly for 5 times; then 100μL of color developing liquid A and B mixed liquid is added to each hole, and it is incubated at 37℃ for 15 min in the dark; finally, 50μL of termination liquid is added to each hole, and the OD value of each hole is measured at 450nm wavelength within 15 min, and the inhibition rate is calculated according to the formula “5a-reductase inhibition rate (%) = [1-(sample group / positive control group mean value-blank control group mean value) / (negative control group mean value-blank control group mean value)]x100%”, the sample group / positive control group is the OD value of the sample solution in the reaction hole of (5a-reductase+sample / positive control+HRP labeled antibody+color developing liquid A, B+termination liquid), the negative control group is the OD value of the sample solution in the hole of (5a-reductase+sample diluent+HRP labeled antibody+color developing liquid A, B+termination liquid), and the blank control group is the OD value of the sample solution in the hole of (color developing liquid A, B+termination liquid). The test results are shown in Tables 9, 10 and Figure 6

[0122] Table 9 Test results of sample group with concentration of 0.5%

[0123] Table 10 Test results of sample group with concentration of 5%

[0124]

[0125]

[0126] ​The results show that the inhibition rate of 5α-reductase of the Calendula officinalis callus extract is 35.7% and 61.2% at the concentrations of 0.5% and 5%, respectively, p<0.05, and the Calendula officinalis callus extract has a strong oil control effect when the dosage reaches 5%.

[0127] (3) Anti-inflammatory effect

[0128] Further investigate the inhibitory effect of the Calendula officinalis callus extract on the expression of inflammation-related genes of RAW264.7 cells. Commercially available RAW264.7 cells (mouse monocyte macrophage leukemia cells) are cultured in DMEM medium containing 10% FBS (fetal bovine serum) (DMEM and FBS are purchased from Gibco). The blank group (DMEM basic medium), the model group (DMEM basic medium + 2 μg / mL LPS (lipopolysaccharide), final concentration, the same hereinafter), the positive control (80 μM dexamethasone + 2 μg / mL LPS), and the sample group (DMEM basic medium is diluted to the required test concentration in turn + 2 μg / mL LPS) are set. RAW264.7 cells are resuspended with fresh complete culture medium (DMEM basic medium + 10% FBS) and diluted with cell culture medium to a seeding density of 50% at 24 h after inoculation. Inoculate into a 6-well plate. After incubation in an incubator for 24 h, discard the culture medium in the 6-well plate, add 0.015% or 0.05% mass concentration of Calendula officinalis callus extract to the basic culture medium in the sample group, add 80 μM dexamethasone to the basic culture medium in the positive control group, and add only the basic culture medium to the blank group, 1 mL per well. After administration, place in a CO2 incubator for 1 h, then add 1 ml of basic medium to the blank group and 1 ml of 2 μg / mL LPS to the model group for 6 h of stimulation. Extract total RNA with TRIzol, detect RNA purity and concentration with NanoDrop, synthesize cDNA and perform PCR amplification, confirm the target gene band by agarose gel electrophoresis, and perform Real-Time PCR quantitative detection with cDNA to analyze the effect of the sample on the expression of inflammation-related genes. The process is entrusted to a professional company that undertakes related detection services.

[0129] The test results are as follows Figure 7As shown, the expression amounts of inflammation-related genes COX-2, IL-1a, IL-1β, IL-6, TNF-a, IL-10, NOS-2 were about 74%, 96%, 96%, 88%, 88%, 92%, 102% of the model group respectively at a sample concentration of 0.015%, which had a certain inhibitory effect on COX-2, IL-6, TNF-a, and had a significant difference compared with the model group, and had no obvious inhibitory effect on IL-1a, IL-1β, IL-10, NOS-2; at 0.05%, the expression amounts of inflammation-related genes COX-2, IL-1a, IL-1β, IL-6, TNF-a, IL-10, NOS-2 were about 73%, 69%, 90%, 84%, 85%, 84%, 80% of the model group, which had a certain inhibitory effect on COX-2, IL-1a, IL-6, TNF-a, IL-10, NOS-2, and had a significant difference compared with the model group, and had no obvious inhibitory effect on IL-1β. Therefore, the callus of calendula has a certain inhibitory effect on inflammation.

[0130] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A composition for culturing calendula callus, characterized in that, The composition comprises an induction medium, a subculture medium, and a suspension medium; wherein, the induction medium comprises, according to a final concentration, 0.1–0.5 mg / L of 6-benzylaminopurine and 2–4 mg / L of naphthaleneacetic acid; the subculture medium comprises, according to a final concentration, 0.1–0.5 mg / L of 6-benzylaminopurine and 2–4 mg / L of naphthaleneacetic acid; the suspension medium comprises, according to a final concentration, 0.1–0.2 mg / L of 6-benzylaminopurine and 1–3 mg / L of naphthaleneacetic acid; the basic culture medium used for the induction medium, the subculture medium, and the suspension medium is at least one of MS medium, WPM medium, and DCR medium.

2. The composition for culturing calendula callus according to claim 1, characterized in that, The basic culture medium is MS medium; and / or, the induction medium, the subculture medium, and the suspension medium further include: 25-30 g / L sucrose and 3-5 g / L plant gum.

3. The use of the composition for culturing calendula callus according to claim 1 or 2 in obtaining calendula callus.

4. A method for culturing calendula callus, characterized in that, The steps include the following: Step 1: Obtain marigold leaves; Step 2: Callus tissue is induced from calendula leaves using the induction medium in the composition described in claim 1 or 2. Step 3: Subculture the callus tissue in the subculture medium of the composition described in claim 1 or 2; Step 4: The subcultured callus tissue is suspended in the suspension culture medium of the composition described in claim 1 or 2 to obtain calendula callus tissue culture.

5. The cultivation method according to claim 4, characterized in that, In step 1, the method for obtaining marigold leaves includes: disinfecting marigold seeds and culturing them until germination, then further culturing the germinated seeds until leaves are formed; and / or, Step 2 includes: processing the marigold leaves into small pieces, adding them to the induction culture medium described in claim 2 or 3, adjusting the pH, and culturing them at a temperature of 26±2℃ in the dark for 15-30 days; and / or, In step 3, the conditions for subculture are the same as those for the induction culture in step 2, and the number of subcultures is no less than 2; and / or, In step 4, the suspension culture conditions include: inoculating at a weight-to-volume ratio of 1 to 10:50, adjusting the pH of the system, and culturing in the dark at a rotation speed of 80 to 150 rpm for 20 to 40 days.

6. Calendula callus obtained by the culture method according to claim 4 or 5.

7. A calendula callus extract, characterized in that, The extract is obtained from the calendula callus tissue described in claim 6. The extract contains at least flavonoids, triterpenoids, and polyphenols, wherein the weight content of flavonoids is not less than 0.2%, the weight content of triterpenoids is not less than 0.4%, and the weight content of polyphenols is not less than 0.4%.

8. The method for preparing the calendula callus extract according to claim 7, characterized in that, include: After drying the calendula callus tissue, a solvent was added for extraction to obtain the calendula callus tissue extract.

9. The preparation method according to claim 8, characterized in that, The solvent includes, but is not limited to, at least one of organic alcohols, organic ethers, organic ketones, organic esters, water, and phosphate buffer.

10. The use of the calendula callus extract according to claim 7 in the preparation of a topical skin composition.

11. A topical skin composition, characterized in that, include: The calendula callus extract according to claim 7.

12. The topical skin composition according to claim 11, characterized in that, Also includes: Other active ingredients and / or ingredients that produce synergistic effects with the calendula callus extract as described in claim 7.

13. At least one skin product according to 1) to 7) below, characterized in that, Including the topical skin composition as described in claim 11 or 12: 1) Skin products that repair damaged cells; 2) Skin products that reduce inflammation; 3) Acne-fighting skin products; 4) Antibacterial skin products; 5) Oil-controlling skin products; 6) Moisturizing skin products; 7) Anti-aging and wrinkle-reducing skin products.

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