Ocimum basilicum hairy root extract as well as preparation method and application thereof

Abstract

The invention provides a basil hairy root extract and a preparation method and application thereof, the basil hairy root extract at least contains polyphenol and flavone, the polyphenol content is not less than 35 mg / g, and the flavone content is not less than 120 mg / g. The preparation method comprises the following steps: infecting basil callus cells with agrobacterium tumefaciens, and culturing hairy roots through an artificial culture medium; and drying the obtained basil hairy roots, and adding a solvent to extract active ingredients, thereby obtaining the basil hairy root extract. According to the method, the sweet basil callus cells are infected by agrobacterium tumefaciens, and the hairy roots are cultured by an artificial culture medium, so that efficient culture of the sweet basil hairy roots is realized, and theoretical support and technical guarantee are provided for deep development of sweet basil resources and development of novel health-care and skin-care products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant active ingredient extraction, and particularly relates to a Laurus nobilis hairy root extract and a preparation method and application thereof. BACKGROUND

[0002] Laurus nobilis is an important spice and medicinal plant, and its roots, stems, leaves and flowers all have important pharmacological effects. With the increasing demand for the development and utilization of natural plant resources, plant tissue culture technology is increasingly widely used. Agrobacterium rhizogenes is a gram-negative aerobic bacterium widely distributed in soil, which has the characteristics of inducing plants to produce hairy roots. By infecting plant callus cells, cell mutation and formation of hairy roots can be induced. At present, plant cell culture technology has been widely used in food, medicine and cosmetics and other fields. Through plant tissue culture, a large number of plant cells or callus can be cultured under controllable conditions to provide enzyme preparations, pharmaceutical products and additives and other related raw materials for human beings. However, how to use plant cell tissue culture technology to extract secondary metabolites from Laurus nobilis plant cells or callus to improve the utilization efficiency and sustainable development of medicinal resources is still a problem to be solved. In the prior art, Laurus nobilis extract is mainly used in traditional Chinese medicine for food digestion, wind dispelling, stomach strengthening, toothache treatment and halitosis treatment. Its application in skin care, hair care and skin care is less studied, especially the mechanism of action in anti-inflammatory and anti-aging aspects has not been fully explored. In addition, the correlation between the related parameters in the preparation process of Laurus nobilis extract and the active ingredients obtained needs to be further clarified. Therefore, it is urgent to develop a new preparation method of Laurus nobilis hairy root extract to obtain a natural plant extract with anti-inflammatory and anti-aging effects, and to provide theoretical support for the in-depth development of Laurus nobilis resources and the research and development of new cosmetic raw materials. SUMMARY

[0003] Therefore, the present application aims to explore the correlation between the preparation process of Laurus nobilis extract and its experimental application effect, and the new application scenarios of Laurus nobilis extract, and to provide a Laurus nobilis hairy root extract and a preparation method and application thereof.

[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 Laurus nobilis hairy root extract, which contains at least polyphenols and flavonoids; the content of polyphenols in the extract is not less than 35 mg / g, and the content of flavonoids is not less than 120 mg / g.

[0006] According to the multi-batch test of the present application, the content of polyphenols in the obtained Laurus nobilis hairy root extract can reach more than 42 mg / g, and the content of flavonoids can reach more than 135 mg / g.

[0007] In a second aspect, the present invention provides a method for preparing the basil hairy root extract described in the first aspect, comprising the following steps:

[0008] Step 1: Infect basil callus cells with Agrobacterium tumefaciens and culture hairy roots using artificial culture medium;

[0009] Step 2: After drying the basil hairy roots obtained in Step 1, add solvent to extract the active ingredients to obtain basil hairy root extract.

[0010] Preferably, step 1 includes:

[0011] Step 1): After sterilizing the basil seeds, add them to a seed germination medium and culture them under light until they germinate; continue to culture the germinated seeds until the seedlings grow to 2-3 cm.

[0012] Step 2): Take a stem segment of the seedling, immerse the lower morphological end of the stem segment in cultured Agrobacterium tumefaciens solution, and insert the upper morphological end of the stem segment into the rooting medium to induce root development until hairy roots grow.

[0013] Step 3) Subculture the basil hairy roots obtained in Step 2).

[0014] In step 1), the seed disinfection process is a routine operation. The seeds can be soaked in a 75% ethanol solution for 30-60 seconds for surface disinfection, then rinsed with sterile water 3-5 times; then the seeds are placed in 84 disinfectant for deep disinfection, and finally rinsed with sterile water several times until the seeds are clean.

[0015] Optionally, in step 1), the seed germination process can be carried out by inoculating the seeds onto MS medium for light culture at a temperature of 26±2℃, a light intensity of 2500-3000 Lux, a photoperiod of 10-12 h / d, and the seeds begin to germinate after 2-3 days of culture.

[0016] Preferably, in step 2), the method for obtaining the cultured Agrobacterium tumefaciens bacterial solution includes:

[0017] Step ①: Add Agrobacterium to LB culture medium containing 20-25 μg / mL rifampin and incubate at 28±1℃ and 150-200 rpm for 20-30 h;

[0018] Step ②: Take an appropriate amount of Agrobacterium tumefaciens bacterial solution obtained in step ① and measure its OD. 600 Value, and set the OD 600 With a value of 0.1, add LB culture medium containing 20-25 μg / mL rifampicin to the calculated volume of Agrobacterium bacterial culture, and incubate at 28±1℃ and 150-200 rpm for 20-30 h;

[0019] Step 3) adding Acetosyringone (AS) into the Agrobacterium liquid obtained in step 2) to a final concentration of 190-210 μM, and culturing at 28±1℃, 150-200 rpm for 1-3 h;

[0020] Step 4) performing solid-liquid separation on the Agrobacterium liquid obtained in step 3), and suspending the Agrobacterium in the MSG / AS culture solution by adding the MSG / AS culture solution into the solid; taking an appropriate amount of the Agrobacterium liquid to measure the OD value, adjusting the OD value to 0.8-1, and adding the MSG / AS culture solution into the Agrobacterium liquid with the calculated volume to obtain the Agrobacterium liquid for use in the subsequent step; 600 600

[0021] The MSG / AS culture solution has a composition with a final concentration of 4.4 g / L MS, 30 g / L sucrose, 10 g / L glucose, and 200 μM AS, and the MSG is a basic culture medium.

[0022] Preferably, in step 4), the solid-liquid separation is performed by centrifugation at 4000-8000 g for 5-15 min.

[0023] Preferably, in step 2), the rooting culture medium has a composition with a final concentration of 25-30 g / L sucrose, 3-5 g / L plant glue, and 1 / 2 MS as a basic culture medium; and / or the rooting induction conditions include a temperature of 26±2℃, dark conditions, and a time period of 18-25 days.

[0024] Preferably, in step 3), the subculture medium has a composition with a final concentration of 25-30 g / L sucrose, 3-5 g / L plant glue, and 1 / 2 MS as a basic culture medium; and / or the subculture conditions include a temperature of 26±2℃, dark conditions, and a time period of 18-20 days for subculture once, and a time period of 2 or more times, preferably 3-5 times, for continuous subculture.

[0025] Preferably, the solvent is selected from at least one of ethanol, ethyl acetate, purified water, and phosphate buffer.

[0026] Further preferably, the solvent is PBS buffer containing NaCl and Tween 80.

[0027] More preferably, the buffer has a composition with a final concentration of 0.0003%-0.0007% Tween 80, 40-60 mM NaH2PO4, 180-220 mM NaCl, and a pH of 5.0-7.0.

[0028] In step 2), the extraction method is a conventional step, and methods such as water bath heating and ultrasonic assistance can be used. The extraction temperature is not suitable to be too high, and is generally controlled below 70℃ to prevent some active ingredients from being oxidized or inactivated. The extraction time is 1-5 h. ​​

[0029] Preferably, the solvent in step 2 includes, but is not limited to, organic alcohol, organic ester, organic ether, water, phosphate buffer.

[0030] In a third aspect, the present application provides use of the extract of Mentha haplocalyx Hance hairy root according to the first aspect or the extract of Mentha haplocalyx Hance hairy root obtained by the preparation method according to the second aspect in the preparation of a skin external composition.

[0031] In a fourth aspect, the present application provides a skin external composition comprising the extract of Mentha haplocalyx Hance hairy root according to the first aspect or the extract of Mentha haplocalyx Hance hairy root obtained by the preparation method according to the second aspect.

[0032] In a fifth aspect, the present application provides a hair loss prevention composition comprising the extract of Mentha haplocalyx Hance hairy root according to the first aspect or the extract of Mentha haplocalyx Hance hairy root obtained by the preparation method according to the second aspect.

[0033] In a sixth aspect, the present application provides a hair follicle anti-inflammatory composition comprising the extract of Mentha haplocalyx Hance hairy root according to the first aspect or the extract of Mentha haplocalyx Hance hairy root obtained by the preparation method according to the second aspect.

[0034] In the above-mentioned compositions, other active ingredients and ingredients that produce synergistic effects with the extract of Mentha haplocalyx Hance hairy root according to the first aspect or the extract of Mentha haplocalyx Hance hairy root obtained by the preparation method according to the second aspect can also be included.

[0035] In a seventh aspect, the present application provides at least one of the following 1) to 3) skin products comprising the skin external composition according to the fourth aspect:

[0036] 1) a skin product for eliminating inflammation;

[0037] 2) a skin product for anti-aging and wrinkle removal;

[0038] 3) a skin product for whitening and / or freckle removal.

[0039] In a ninth aspect, the present application provides a hair follicle anti-inflammatory product comprising the hair follicle anti-inflammatory composition according to the sixth aspect.

[0040] The skin product or the anti-shedding product or the anti-inflammatory product for hair follicle can also optionally contain, but not limited to, various adjuvants such as surfactants, diluents, emulsifiers, thickening agents, dispersants, etc. Surfactants such as: cetyl stearyl alcohol olivate, sorbitan olivate, polysorbate-60, polysorbate-80, methyl glucose sesquistearate, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butanol polyether-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 polystearate, 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.

[0041] The skin product or anti-hair loss product or anti-inflammatory product of hair follicle can also optionally contain other active ingredients such as one or more of the following: tocopherol (vitamin E), retinol, retinol palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, gosiplatin, tetrahydrocurcumin, ellagic acid, ubiquinone, whey protein, polypeptide, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyloyl phytosphingosine, concentrated birch sap, silymarin, silk fibroin protein, 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 seed extract, PTEROCARPUS MARSUPIUM bark extract, CAMELLIA SINENSIS polyphenols, grape wine extract, apple seed extract, FAGUS SYLVATICA bud extract, hydrolyzed ADANSONIA DIGITATA extract, ARTEMIA extract, IRIS FLORENTINA root extract, hesperidin, ginsenoside, SALVIA MILTIORRHIZA extract, nicotinamide, ursolic acid, sodium hyaluronate, acetylated sodium hyaluronate, hydrolyzed sodium hyaluronate, lycopene, COFFEA ARABICA extract, dipeptide-2, lactic acid, superoxide dismutase (SOD), OENOTHERA BIENNIS oil, ceramide, dipalmitoyl hydroxyproline, hydroxystearic acid, salicylic acid, ergothioneine, lysophosphatidylcholine, carnosine, decarboxycarnosine HCL, thioctic acid, adenosine, glycogen, resveratrol, ferulic acid, Schizochytrium limneticum fermentate, lactic acid bacteria fermentate, and the like.

[0042] The basil hairy roots 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 or anti-hair loss product or anti-inflammatory product of hair follicle industry. The other pharmaceutical ingredients or cosmetic ingredients are as described above and are commonly used in the relevant products.

[0043] In addition, the skin product or anti-hair loss product or anti-inflammatory product of hair follicle can be formulated into various dosage forms such as solution, suspension, paste, cream, emulsion, gel, powder or spray, etc. according to the needs.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] Firstly, the application realizes efficient culture of L. vulgare hairy roots by infecting L. vulgare callus cells with Agrobacterium rhizogenes and combining artificial medium culture of the hairy roots, provides a new way for deep development of L. vulgare resources, and improves the utilization efficiency of plant resources.

[0046] Secondly, the application extracts high-concentration active substances by using a water extraction process, combines UV absorbance test analysis of total polyphenol and total flavonoid yield, and establishes a systematic extraction process parameter control method, thereby solving the problem of needing to optimize extraction conditions such as extraction temperature and extraction time in the prior art.

[0047] Thirdly, the L. vulgare hairy root extract prepared by the application has a significant anti-inflammatory effect, can effectively inhibit the SA-β-Gal positive cell rate of hair follicle stem cells under the stimulation of DHT, and reduce the expression of inflammation-related genes TNF-α, COX-2, IL-1α, IL 10 and NOS-2, wherein the expression inhibition rates of TNF-α, IL-10 and NOS-2 are close to or lower than those of the positive control group dexamethasone, and the L. vulgare hairy root extract has a good anti-inflammatory effect, and the research finding provides a new idea for the application of the L. vulgare extract in the field of scalp care.

[0048] Fourthly, the application uses fibroblasts (HSF) as an anti-aging test cell model, proves that the L. vulgare hairy root extract can effectively promote the expression of various anti-aging genes such as elastin, fibronectin, collagen I and III, and MMp9 protein, and has a significant anti-aging effect when the addition amount is 0.15%, and the expression levels of anti-aging related genes Elastin, Fibronectin 1, Collagen III and MMP3, and MMP9 are significantly improved. The research confirms that the L. vulgare hairy root extract has the effects of prolonging cell life and enhancing skin barrier function.

[0049] Fifthly, the application effectively combines plant synthetic biology and plant cell culture technology, establishes a preparation method of the L. vulgare hairy root extract, and provides theoretical support and technical support for deep development of L. vulgare resources and development of new health care and skin care products. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 It is a comparison diagram of polyphenol content (A) and flavonoid content (B) in the L. vulgare hairy root extract and the L. vulgare leaf extract in Example 1 of the application.

[0051] Figure 2 It is an anti-inflammatory effect of the L. vulgare hairy root extract in Example 2 of the application, wherein A-F respectively represent the expression levels of inflammation-related genes COX-2, IL-1α, IL-1β, NOS-2, TNF-α and IL-10.

[0052] Figure 3Anti-aging effect of the basil hairy root extract in Example 2 of the present application, wherein A ~ E represent the expression levels of the anti-aging related genes Elastin, Collagen III, MMP-9, Fibronectin 1 and MMP-3, respectively.

[0053] Figure 4 Electron microscope observation results (A) and SA-β-Gal positive cell analysis results (B) of the human-derived mammary gland cells in Example 2 of the present application.

[0054] Figure 5 TNF-α gene expression level (A) and IL-10 gene expression level detection results (B) in Example 2 of the present application.

[0055] Figure 6 Inhibition results of the basil hairy root extract on tyrosinase in Example 2 of the present application.

[0056] Figure 7 DPPH radical scavenging effect of the basil hairy root extract in Example 2 of the present application. DETAILED DESCRIPTION

[0057] In the description of the present application, it should be noted that, in the examples, the specific conditions not mentioned are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0058] Unless otherwise defined, all the professional terms used below have the same meaning as generally understood by those skilled in the art. The professional terms used in this text are only for the purpose of describing the specific embodiments, and are not intended to limit the protection scope of the present application.

[0059] 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 protection scope of the present application is not limited to the following specific examples.

[0060] Example 1:

[0061] The present application provides an embodiment for preparing the basil hairy root extract, which is specifically as follows:

[0062] Step 1: Select plump basil seeds and soak them in a 75% ethanol solution for 30-60 seconds for surface disinfection. Then rinse them 3-5 times with sterile water. Next, disinfect the seeds with 84 disinfectant solution. Finally, rinse them 5 times with sterile water until the seeds are clean. Inoculate the seeds onto MS medium and place them in a plant culture incubator for photoculture at a temperature of 26±2℃, a light intensity of 2500-3000 Lux, and a photoperiod of 12h / d. Seeds begin to germinate in about 2 days. Germinated seeds are then transferred to glass culture bottles for cultivation. Once seedlings reach 2-3 cm in height, stem segments are taken for induction of hairy roots using Agrobacterium rooting agents. The lower morphological end of the stem segment is inoculated with cultured Agrobacterium solution, while the upper morphological end is inserted into a rooting medium prepared with 1 / 2 MS + 30 g / L sucrose + 3 g / L plant gum. The medium is then placed in a plant incubator at 26 ± 2℃ in the dark for root induction. Hairy roots emerge after about 21 days of induction. The basil hairy roots are then added to a subculture medium (1 / 2 MS + 25 g / L sucrose + 5 g / L plant gum) and subcultured at 26 ± 2℃ in the dark. Subculture is repeated every 20 days for a total of 3 subcultures to obtain basil hairy roots.

[0063] The method for obtaining cultured Agrobacterium tumefaciens bacterial suspension is as follows:

[0064] 1) Add Agrobacterium to 5 mL of LB culture medium containing 25 μg / mL rifampicin and incubate at 28℃ and 180 rpm for the next day.

[0065] 2) Take an appropriate amount of Agrobacterium tumefaciens bacterial solution, dilute it, and measure the OD value. 600 Value. Set the starting OD. 600 The value was set to 0.1, and the calculated bacterial culture volume was added to a conical flask containing 30 mL of LB culture medium with 25 μg / mL rifampicin. The flask was then incubated at 28°C and 180 rpm for the next day.

[0066] 3) Add Acetosyringone (AS) to the bacterial culture that has been cultured overnight to a final concentration of 200 μM and incubate at 28°C and 180 rpm for 2 hours.

[0067] 4) Pour the Agrobacterium tumefaciens culture into a 50 mL centrifuge tube, centrifuge at 6000 g for 10 minutes, and remove the supernatant; add 10 mL of MSG / AS culture medium to suspend the Agrobacterium tumefaciens, then take an appropriate amount of Agrobacterium tumefaciens culture to dilute and measure the OD. 600 Value; Adjust OD 600 The value is 0.8-1, and the calculated bacterial culture is added to a 50mL centrifuge tube. The volume is then increased to 20mL with MSG / AS culture medium (4.4g / L MS, 30g / L sucrose, 10g / L glucose, 200μM AS, MSG as the basic medium).

[0068] Step 2, dry the basil hairy roots, then add solvent extraction, to obtain the basil hairy roots extract.

[0069] The active ingredient extraction solvent used is PBS buffer containing NaCl and Tween 80, with a final concentration composition of: 0.0005wt% Tween 80 + 50mM NaH2PO4+ 200mM NaCl, PH 5.5. Dry the basil hairy roots and solvent according to the weight volume ratio (g / mL) is 1:20, extraction conditions: 60℃ water bath 1h.

[0070] This example also provides a method for obtaining basil leaf extract: purchase basil leaf medicinal materials from pharmacies, add the same solvent as the dried basil hairy roots for extraction, and the same amount, extraction conditions, etc. to obtain the basil leaf extract.

[0071] Test the total polyphenol and total flavonoid content in the basil hairy root extract and basil leaf extract, wherein,

[0072] 1. The total polyphenol detection method is:

[0073] The sample is determined by absorbance, and the sample relative gallic acid equivalent concentration is obtained according to the gallic acid standard sample calibration curve.

[0074] (1) Experimental principle: In an alkaline solution, tungsten molybdate can quantitatively oxidize polyphenol compounds, and itself is reduced (W 6+ becomes W 5+ ) to generate blue compounds, and the depth of blue is proportional to the number of phenolic groups, and the maximum absorption wavelength is 765nm, which can be used with gallic acid as a correction standard to quantify the total polyphenol content in the sample.

[0075] (2) Experimental instruments: spectrophotometer, centrifuge.

[0076] (3) Determination procedure

[0077] (3.1) Preparation of samples

[0078] Preparation of gallic acid standard solution: Prepare gallic acid standard solution with RO water to 20, 40, 60, 80, 100μg / mL for standby.

[0079] Preparation of test solution: Take an appropriate amount of basil hairy root extract and basil leaf extract and dilute 10 times with RO water for standby.

[0080] Preparation of reaction solution: Prepare 90mg / mL Na2CO3 solution and 50% (v / v) Folin phenol solution with RO water.

[0081] (3.2) Reaction and determination

[0082] Take 200 μL of 90 mg / mL Na2CO3 solution in a microcentrifuge tube, respectively add 100 μL of gallic acid standard solution, sample (reaction solution), blank group and mix well, then add 100 μL of 50% Folin phenol solution and mix well. After placing in a 45°C water bath for 30 min, centrifuge and take 200 μL of supernatant, use an enzyme marker to detect the absorbance value at 750 nm.

[0083] (3.3) Calculation

[0084] The absorbance value of the detected solution is plotted against the concentration, and the linear regression equation (y=ax+b) and the correlation coefficient (R2) are obtained. The measured absorbance value of the sample is calculated by interpolation to obtain the relative gallic acid equivalent concentration of the sample (gallic acid equivalent, GAE).

[0085] 2, Total flavone detection method:

[0086] The sample is determined by absorbance, and the relative rutin equivalent concentration of the sample is obtained according to the rutin standard detection line.

[0087] (1) Experimental principle: In the presence of neutral or weak alkali and sodium nitrite, flavonoids and aluminum salt form chelate, and after adding sodium hydroxide solution, it shows red orange color, and has an absorption peak at 500 nm wavelength and meets the Beer's law of quantitative analysis, generally with rutin standard for quantitative analysis. (Color development occurs at the ortho-substituted catechol hydroxyl site of flavonol components, and flavonol components without ortho-substituted catechol hydroxyl group do not show color when added to the above reagent).

[0088] (2) Experimental instruments: spectrophotometer, centrifuge.

[0089] (3) Determination procedure

[0090] (3.1) Preparation of sample

[0091] Preparation of test sample solution: Take the basil hairy root extract and basil leaf extract and dilute them 10 times with RO water for standby.

[0092] Preparation of rutin standard solution: Prepare the rutin standard to 20, 40, 60, 80, 100, 200 μg / mL for standby.

[0093] Preparation of reaction solution: Prepare 60% ethanol, 5% NaNO2, 10% Al(OH)3·9H2O, 4% NaOH solution, and the aforementioned percentages are mass percentage concentrations.

[0094] (3.2) Reaction and determination

[0095] Take 0.24 mL each of the rutin standard solution and the test solution (3 replicates), place them in a 2 mL centrifuge tube, add 40 μL of 5% NaNO2 solution, mix well, let stand for 6 min, add 40 μL of 10% Al(OH)3·9H2O, mix well, let stand for 6 min, add 0.4 mL of 4% NaOH solution, then add 0.28 mL of 60% ethanol (total volume 1 mL), mix well, let stand for 15 min, pipette 200 μL of the mixed liquid into a 96-well plate, and measure the absorbance at 500 nm using an ELISA reader.

[0096] (3.3) Calculation

[0097] Plot the absorbance values ​​of the tested solution against their concentrations, and obtain the linear regression equation (y = ax + b) and the correlation coefficient (R²). Calculate the relative rutin equivalent concentration (rutin equivalent) of the sample by interpolation based on the absorbance values ​​measured in the sample.

[0098] Test results as follows Figure 1 As shown, the polyphenol content in this batch of basil (fragrant) hairy root extract was 5.13 times that of basil leaf extract, and the flavonoid content was 5.37 times that of basil leaf extract. Furthermore, in multiple batches of basil hairy root extract obtained according to the method of Example 1 of this invention, the detected polyphenol content was not less than 42 mg / g, and remained stable between 42 and 55 mg / g; the flavonoid content was not less than 135 mg / g, and remained stable between 135 and 155 mg / g.

[0099] Example 2:

[0100] In this embodiment, the basil hairy root extract was obtained using the method of Example 1, and then its anti-inflammatory, anti-aging, and anti-hair loss effects were determined.

[0101] (1) Anti-inflammatory effects

[0102] COX-2 is prostaglandin-endoperoxide synthase 2 (Cydoxygenase-2, COX-2), also known as cyclooxygenase 2, an enzyme that is rapidly expressed by mononuclear macrophages, fibroblasts and other cells after the body is stimulated by an inflammatory factor, known as an induced enzyme, is one of the key enzymes that cause inflammatory reactions, can promote inflammatory reactions, leading to tissue damage. NOS-2 is a subtype of nitric oxide synthase, which can produce nitric oxide (NO). In macrophages, NO mediates tumor and bactericidal effects, participates in inflammatory reactions, and can enhance the synthesis of pro-inflammatory mediators such as IL-6 and IL-8. Interleukin-1 is a cytokine of the chemokine family, also known as hematopoietin 1, IL-1α is mainly produced by activated macrophages and neutrophils, epithelial cells and endothelial cells, and is abundant in the epidermis of normal humans. Pro-inflammatory cytokines mainly stimulate the expression of inflammation and autoimmune disease-related genes. There are two subtypes of interleukin-1 (IL-1α and IL-1β), of which IL-1β is the most common secreted form, produced by mononuclear / macrophages, lymphocytes, when macrophages are stimulated (such as LPS), can produce IL-1, cause granulocyte degranulation, mediate the process of tissue damage, and attract inflammatory cells into the lesion site, thereby causing damage to the body. Interleukin 10 is a subfamily of class II cytokines, also known as human cytokine synthesis inhibitory factor, an anti-inflammatory cytokine that can enhance B cell survival, proliferation and antibody production. IL-10 can block the activity of NF-κB and participate in the regulation of the JAK-STAT signaling pathway. Tumor necrosis factor (TNF) is one of the strongest inflammatory mediators in the body, with very strong inflammatory damage. It is mainly secreted by mononuclear macrophages and lymphocytes under the stimulation of inflammatory factors such as endotoxins. TNF-α can activate the MAPKs and NF-κB pathways through specific cell membrane connection receptors to change cells, and can promote the occurrence of inflammatory cascade reactions, induce the production of new inflammatory mediators or cytokines.

[0103] The basil hairy root extract was obtained according to the method of Example 1, a yellow-brown liquid, and tested for anti-inflammatory effect, Figure 2The expression levels of TNF-α, COX-2, IL-1α, IL-1β, IL-10, NOS-2, and other inflammatory factors in macrophages after the action of the Ocimum basilicum hairy root extract determined by using the real-time fluorescent quantitative polymerase chain reaction technology are given, wherein the blank control is without any component, the negative control is added with lipopolysaccharide (LPS), and the positive control is added with dexamethasone. The specific operation of the test includes: after the RAW264.7 cells are placed in a 6-well plate and cultured for 16 h, the DMEM culture solution containing different concentrations of Ocimum basilicum hairy root extract samples (0.15%, 0.3%, v / v) is replaced, the DMEM culture medium (i.e. the Ocimum basilicum hairy root extract is dissolved in the cell culture solution according to the volume percentage concentration), and an appropriate amount of LPS is added after 1 h for induction. After 6 h, the total RNA is extracted by TRIzon, and the RNA purity and concentration are detected by using NanoDrop. Then, the cDNA is synthesized and PCR amplification is performed, and the agarose gel electrophoresis analysis confirms the target gene band. Finally, the cDNA is used for Real-Time PCR quantitative detection, and the q-PCR reaction system (total 20 μL system) is: upstream primer: 1 μL, downstream primer: 1 μL, template: 1 μL, ddH2O: 7 μL qPCR enzyme (Mix enzyme): 10 μL, reaction condition: 95℃ 30 s, (95℃ 5 s, 60℃ 30 s) × 40 cycles, 65℃ 5 s, 95℃ 5 s. The influence of the tested sample on the expression of inflammation-related genes is obtained, and the process is entrusted to a professional company for related detection services.

[0104] By Figure 2 It can be seen from the results that the Ocimum basilicum hairy root extract plays a role in inhibiting the inflammatory response by reducing the expression of inflammation-related genes TNF-α, COX-2, IL-1α, IL 10, NOS-2, i.e. has a significant anti-inflammatory effect. The expression inhibition rates of TNF-α, IL-10, and NOS-2 are close to or lower than the positive control group dexamethasone, and have a better anti-inflammatory effect.

[0105] (2) Anti-aging effect

[0106] Elastin is an insoluble, large-molecular weight fibrous protein in ECM, which together with a microfibrillar structural glycoprotein forms the elastic fiber in connective tissue. Elastin not only maintains the elasticity of the tissue, as a major component of ECM, plays a role in cell adhesion and support, it and other ECM components such as collagen together maintain the stability of the tissue structure. The sharp decline in elastin biosynthesis is a direct cause of skin aging, and substances that stimulate elastin gene expression are beneficial in improving skin aging. Fibronectin 1 is an extracellular matrix glycoprotein encoded by the FN1 gene, which is expressed on the cell surface and in plasma. Fibronectin is mainly synthesized by fibroblasts and endothelial cells. Fibronectin is involved in many important functions, such as wound healing; cell adhesion; blood clotting; cell differentiation and migration; maintenance of cytoskeleton. Collagen type III is the main structural protein in the dermis, synthesized in fibroblasts, and normal skin is mainly composed of collagen types I and III. Collagen is the main insoluble fibrous protein in ECM and connective tissue. Matrix metalloproteinase-3 (MMP-3) has a wide substrate specificity and can degrade a large number of ECM proteins, such as fibronectin, laminin, elastin, collagen (types I, II, III), and proteoglycans. The main function of MMP-3 is to activate the proenzyme form (pro MMPs) of MMPS in ECM, such as collagenase, gelatinase B, and stromelysin. Matrix metalloproteinase 9 (MMP-9), also known as 92kDa collagenase type IV, 92kDa gelatinase, or gelatinase B (GELB), is a matrix protein belonging to the zinc metalloproteinase family of enzymes, involved in extracellular matrix degradation, tissue remodeling, wound healing, and maintaining ECM integrity.

[0107] The basil hairy root extract and (1) used for anti-aging testing are the same batch of samples, Figure 3 PCR technology (operation, conditions, etc. are the same as Figure 2 , primers are shown in Table 1, this experiment sets a blank control, also the same Figure 2The expression levels of anti-aging related genes Elastin, Fibronectin 1, Collagen III and MMP3, MMP9 in fibroblasts after the action of the Ocimum basilicum hairy root extract determined. Compared with the blank group (100%), when the Ocimum basilicum (Lanxiang) hairy root filtrate was added at a concentration of 0.05%, the expression levels of the anti-aging related genes Elastin, Collagen III, MMP-9, Fibronectin 1 and MMP-3 were 146%, 187%, 148%, 137% and 103%, respectively; when the Ocimum basilicum (Lanxiang) hairy root filtrate was added at a concentration of 0.15%, the expression levels of the genes were as high as 211%, 249%, 193%, 142% and 138%, respectively, indicating that the Ocimum basilicum (Lanxiang) hairy root filtrate can regulate the expression levels of elastin, fibronectin, collagen III and MMP9 proteins, has a significant anti-aging effect on tissue remodeling.

[0108] (3) Anti-hair loss effect

[0109] It has been shown that the aging of hair follicle stem cells can cause the hair follicle itself to gradually atrophy, leading to hair thinning and shedding. Therefore, inhibiting the aging of hair follicle stem cells can delay hair loss. β-galactosidase (SA-β-Gal) is a commonly used cell aging marker that exhibits increased activity during cell aging. By detecting the activity level of SA-β-Gal in cells, the degree of cell aging can be evaluated. This test is based on a DHT-stimulated human hair papilla cell model, and detects the SAβ-Gal positive cell rate in human hair papilla cells and the changes in TNF-α and IL-10 gene expression levels by SAβ-Gal staining method, to evaluate whether the test sample has an anti-hair loss effect. The Ocimum basilicum hairy root extract was obtained according to the method of Example 1, and the anti-hair loss effect test was performed as follows:

[0110] 1. Test materials

[0111] Test system: human hair papilla cells.

[0112] Main reagents: high-sugar DMEM culture medium (Ponsai), fetal bovine serum (Gibco), PBS (Gibco), trypsin (Gibco), β-galactosidase staining kit (Bi Yun Tian).

[0113] Main equipment: CO2 incubator (Thermo, 160i), biological safety cabinet (Sun Neng Antai, BSC-1604IIA2), inverted fluorescence microscope (Keynsh BZ-X810), enzyme label instrument (Tecan, Spark), floor half-body ultraviolet treatment instrument (Sigma High, SS-03AB), Q-PCR instrument (Summit, ABI7500).

[0114] 2. Experimental method:

[0115] 2.1 Cytotoxicity

[0116] (2.1.1) Cell Seeding: Cells were seeded into 96-well plates at a seeding density of 8 x 10 3 The 96-well plates were incubated in an incubator (37°C, 5% CO2) overnight.

[0117] (2.1.2) Experimental Grouping: The experiment was set up with a blank control group, a negative control group, a positive control group, and a sample group. In the sample group, 8 concentration gradients were set up for each sample, and 3 replicate wells were set up for each concentration gradient.

[0118] (2.1.3) Liquid Preparation: Sample working solutions of different concentrations were prepared according to the test concentration settings, as shown in Table 2.

[0119] Table 2 Test Concentration Setting Table

[0120]

[0121]

[0122] (2.1.4) Drug Administration: When the plating rate of cells in the 96-well plates reached 40% to 60%, drug administration was performed. The negative control group was added with 200 μL of culture medium containing 10% PBS per well; the positive control group was added with 200 μL of culture medium containing 10% DMSO per well; the sample group was added with 200 μL of culture medium containing the corresponding concentration of sample per well; the blank control group was not inoculated with cells and was only added with 200 μL of cell culture medium (this culture medium was a commercially available general culture medium, as described below). After drug administration was completed, the 96-well plates were placed in an incubator (37°C, 5% CO2) for culture.

[0123] (2.1.5) Detection: After 24 h of incubation and culture, the supernatant was discarded, MTT working solution (0.5 mg / mL) was added, and incubation was performed at 37°C in the dark for 4 h. After incubation was completed, the supernatant was discarded, 100 μL of DMSO was added to each well, and the OD value was read at 490 nm.

[0124] (2.1.6) Cell Viability Calculation: The calculation was performed according to the formula. The calculation results are shown in Table 3.

[0125]

[0126] Table 3 MTT Detection Results

[0127]

[0128] According to the MTT results, the sample basil hairy root extract did not exhibit cytotoxicity to human-derived mammary epithelial cells within the concentration range of 1.0000% (V / V).

[0129] 2.2 SA-β-Gal test results

[0130] 1) Cell inoculation: Cells were inoculated into 24-well plates at an appropriate seeding density (8 x 10 4 cells / well) and incubated in an incubator (37°C, 5% CO2) overnight.

[0131] 2) Experimental grouping: The experiment was set up with a blank control group, a negative control group, a positive control group, and a sample group. Three concentration gradients were set up in the sample group.

[0132] 3) Liquid preparation: The test concentration setting table 4 was used to prepare working solutions of different concentrations of the test substance. The blank control group was added with 1 mL of cell culture solution per well; the negative control group was added with 1 mL of cell culture solution per well; the positive control group was added with 1 mL of 0.2% minoxidil per well; and the sample group was added with 1 mL of culture solution containing the corresponding concentration of the test substance per well.

[0133] 4) Dosing: When the plating rate of the cells in the 24-well plate reached 40%-60%, 1 mL of culture medium containing different concentrations of samples was added according to table 4.

[0134] Table 4 Test design

[0135]

[0136] 3) Staining: Staining was performed according to the instructions of the β-galactosidase staining kit.

[0137] 4) Result analysis: SA-β-Gal positive cells were quantitatively analyzed using ImageProPlus software, and the results are shown in table 5 and Figure 4 .

[0138] Table 5 SA-β-Gal positive cell rate

[0139] Group Positive cell rate (%) SD p-value Inhibition rate (%) Blank control (BC) 9.58 0.02 / / Negative control (NC) 44.42 0.02 0.000## / Positive control (PC) 15.24 0.03 0.000** / Basil hairy root extract - 0.25% 31.81 0.03 0.004** 28.39 Basil hairy root extract - 0.50% 20.97 0.02 0.000** 52.79 Basil hairy root extract - 1.00% 14.42 0.00 0.000** 67.54

[0140] Note: When statistical analysis was performed using the t-test two-tailed test method, the significance of the sample group compared with the blank group was represented by *, p-value < 0.05 was represented by *, and p-value < 0.01 was represented by **.

[0141] Figure 4For SA-β-Gal staining results, the blue color in Figure A represents SA-β-Gal in human hair papilla cells, and the more blue color represents the more senescent cells. In Figure B, based on the DHT-stimulated human hair papilla cell model, the basil (lanxangica) hairy root filtrate significantly inhibited the SA-β-Gal positive cell rate of human hair papilla cells at concentrations of 0.25%, 0.50%, and 1.00% (V / V), which were 31.81%, 20.97%, and 14.42%, respectively. There was a statistically significant difference compared with the NC group (p<0.01), and the effect was consistent with that of the positive control group minoxidil, indicating that the basil (lanxangica) hairy root filtrate had an anti-hair loss effect.

[0142] 2.3 Related gene expression

[0143] 1) Cell inoculation: inoculate cells into a 24-well plate at an appropriate inoculation density (8 x 104 / well), and incubate in an incubator (37°C, 5% CO2) overnight.

[0144] 2) Experimental grouping: set up a blank control group, a negative control group, a positive control group, and a sample group. Set up three concentration gradients for the sample group.

[0145] 3) Liquid preparation: prepare the working solution of the test substance at different concentrations according to Table 4.

[0146] 4) Drug administration: when the plating rate of cells in the 24-well plate reaches 40%-60%, administer the drug. The blank control group is added with 1 mL of cell culture medium per well; the negative control group is added with 1 mL of cell culture medium per well; the positive control group is added with 1 mL of 0.2% minoxidil per well; and the sample group is added with 1 mL of culture medium containing the corresponding concentration of the test substance per well.

[0147] 5) Cell collection: after 24 hours of incubation, add 0.5 mL of lysis buffer to each well after the culture ends, and place it at room temperature for 5 minutes to allow it to be fully lysed. Transfer it to a 1.5 mL RNase-free Eppendorf tube, extract RNA according to the operating procedures of the commercially available RNA extraction kit, and follow the subsequent operations of Figure 2 qRT-PCR detection of TNF-α and IL-10 genes.

[0148] The results are as follows Figure 5As shown in Tables 6-7, the TNF-α gene expression of the human-derived mammary papilla cells was significantly reduced (p<0.01) by 79%, 67% and 55% at the concentrations of 0.25%, 0.50% and 1.00% (V / V) of the sample basil (Ocimum basilicum L.) hairy root filtrate, respectively, and there was a significant difference with the negative control and the same trend as the positive control, indicating that the sample had a very significant anti-inflammatory effect. In addition, the IL-10 gene expression of the human-derived mammary papilla cells was significantly increased (p<0.01) by 84%, 134% and 24% at the concentrations of 0.25%, 0.50% and 1.00% (V / V) of the sample basil (Ocimum basilicum L.) hairy root filtrate, respectively, and there was a significant difference with the negative control and the same trend as the positive control, indicating that the sample had a very significant anti-inflammatory effect.

[0149] Table 6: Summary of TNF-α gene relative expression

[0150]

[0151] Note: When using t-test method for statistical analysis, the significance of the NC group compared with the BC group is represented by #, p-value<0.05 is represented by #, and p-value<0.01 is represented by ##. The significance of the sample group and the PC group compared with the NC group is represented by *, p-value<0.05 is represented by *, and p-value<0.01 is represented by **.

[0152] Table 7: Summary of IL-10 gene relative expression

[0153] Group Mean SD p-value Blank control (BC) 1.00 0.0 / / Negative control (NC) 0.31 0.03 0.000## / Positive control (PC) 2.53 0.23 0.000** / Basil hairy root extract - 0.25% 0.84 0.09 0.001** 62.42 Basil hairy root extract - 0.50% 1.34 0.17 0.001** 76.43 Basil hairy root extract - 1.00% 2.40 0.11 0.000** 86.86

[0154] Note: When using t-test method for statistical analysis, the significance of the sample group and the PC group compared with the BC group is represented by *, p-value<0.05 is represented by *, and p-value<0.01 is represented by **.

[0155] (4) Melanin inhibition effect

[0156] Tyrosinase is an oxidase and a rate-limiting enzyme for the regulation of melanin production, directly affecting the synthesis of melanin. Tyrosinase has important physiological functions in organisms. At the same time, it is also related to the occurrence of diseases such as excessive deposition of melanin in human freckles, brown spots and the like. The present example evaluates the tyrosinase inhibition effect of Ocimum basilicum hairy root extract and Ocimum basilicum leaf extract. The test principle is as follows: the synthesis of melanin is affected by tyrosinase, which can catalyze the hydroxylation of L-tyrosine to dopa, and its oxidation forms dopaquinone (red), which finally reacts to form melanin. Test substances with tyrosinase activity inhibition can slow down the conversion of dopa to dopaquinone. By measuring the absorbance of dopaquinone at 475 nm, the change in absorbance is used to evaluate the inhibition effect of the test substance on tyrosinase activity. The Ocimum basilicum hairy root extract was obtained according to the method of Example 1, and the melanin inhibition test was carried out as follows:

[0157] 1) Required drugs and reagents

[0158] The required drugs are: anhydrous potassium dihydrogen phosphate (98%), disodium hydrogen phosphate (99%), tyrosinase, L-tyrosine, and kojic acid.

[0159] Reagent preparation:

[0160] ① 0.1 mol / L PBS buffer (pH = 6.8): weigh 3.402 g of potassium dihydrogen phosphate and 3.549 g of disodium hydrogen phosphate, add 400 mL of RO water, stir to dissolve, adjust the pH to 6.8 with HCl or NaOH, add RO water to 500 mL, mix well, and reserve.

[0161] ② 200 U / mL tyrosinase solution: take 1 tube of tyrosinase, add 990 μL of PBS buffer, and you get 200 U / mL tyrosinase solution, which is prepared immediately (tyrosinase has been divided into 200 U / tube (10 μL) and stored in a -20°C refrigerator).

[0162] ③ 200 μg / mL tyrosine solution: weigh 20 mg of L-tyrosine powder, dissolve in 100 mL of PBS buffer, and ultrasonically shake until completely dissolved, and prepare immediately.

[0163] ④ 100 μg / mL kojic acid solution: weigh 10 mg of kojic acid powder, dissolve in 100 mL of PBS buffer, and you get 100 μg / mL kojic acid solution.

[0164] ⑤ Kojic acid dilution: dilute the 100 μg / mL kojic acid solution with PBS buffer to a series of concentration gradients of 1, 2, 4, 8, 16, 32, and 64 μg / mL.

[0165] ⑥ Sample solution: take 1 mL of sample solution, and dilute it to 1 / 2, 1 / 3, 1 / 4, 1 / 5, and 1 / 10 concentration gradients in sequence.

[0166] 2) Experimental Procedure

[0167] A 96-well microplate was configured with solvent background wells (S0), solvent reaction wells (S), sample background wells (A0), and sample reaction wells (A). The S0 group consisted of solvent background wells, with neither L-tyrosine substrate solution nor sample solution added; the S group consisted of solvent reaction wells, with L-tyrosine substrate solution added but no sample solution added; the A0 group consisted of sample background wells, with neither L-tyrosine substrate solution nor sample solution added; and the A group consisted of sample reaction wells, with both L-tyrosine substrate solution and sample solution added. Each well was run in triplicate. The reagent loading is shown in Table 8.

[0168] Table 8. Feeding details for each group

[0169]

[0170] After the reaction was completed, the sample was immediately placed in an ELISA reader and the absorbance was measured at a wavelength of 475 nm.

[0171] 3) Calculation

[0172] Tyrosinase activity inhibition rate: Where: Y—Tyrosinase activity inhibition rate; A—Average absorbance of sample reaction wells; A0—Average absorbance of sample background wells; S—Average absorbance of solvent reaction wells; S0—Average absorbance of solvent background wells.

[0173] Experimental results are as follows Figure 6 As shown, the tyrosinase inhibition rates of the basil (fragrant basil) hairy root filtrate at dilutions of 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, and 100% were 18.37%, 35.58%, 45.69%, 68.54%, 77.28%, and 89.26%, respectively, while the basil leaf extract showed an inhibition rate of 0.1% at the corresponding concentrations. This indicates that the tyrosinase inhibition rate of the basil (fragrant basil) hairy root filtrate is higher than that of the basil leaf extract.

[0174] (5) Antioxidant effects

[0175] Skin is the first barrier of the human body, and it is the first tissue that can reflect the aging of the body. There are many factors that cause skin aging, in addition to genetic factors, oxidative stress from the external environment such as ultraviolet rays, smog, etc. can cause cell oxidation and thus produce free radicals, accelerate skin aging, and even lead to the occurrence of skin cancer. The present embodiment evaluates the DPPH free radical scavenging effect of Ocimum basilicum hairy root extract and Ocimum basilicum leaf extract. The experimental principle is as follows: 1,1-diphenyl-2-trinitrobenzene hydrazine (abbreviated as DPPH) is a stable long-lived free radical, and its alcohol solution is dark purple, with strong absorption at 517 nm. When there is a free radical scavenger, DPPH free radicals are scavenged, making the solution color lighter and the absorbance lower. Within a certain range, the change in absorbance is proportional to the degree of free radical scavenging. The ability of the test substance to scavenge DPPH free radicals can be evaluated by the change in absorbance. The Ocimum basilicum hairy root extract was obtained according to the method of Example 1, and the antioxidant efficacy test was carried out as follows:

[0176] 1) Reagent preparation

[0177] ① 0.12mg / mL DPPH stock solution: weigh 1,1-diphenyl-2-trinitrobenzene hydrazine 12mg and add 100mL 95% ethanol, stir to dissolve.

[0178] ② L-ascorbic acid (Vc) gradient dilution solution: weigh 0.05g L-ascorbic acid powder and dissolve in 10mL 95% ethanol solution to obtain 5mg / mL Vc solution for use. Dilute the 5mg / mL Vc solution with 95% ethanol to obtain a series of concentration gradients of 1mg / mL, 0.3mg / mL, 0.2mg / mL, 0.05mg / mL, 0.01mg / mL.

[0179] ③ Sample solution: take 1mL sample solution and dilute it by 0, 5, 10, 15, 20, 25 times (i.e. add 2.4%, 4%, 5%, 6%, 10%, 20%).

[0180] 2) Experimental operation

[0181] Add the sample to a 1.5mL centrifuge tube according to the requirements of Table 9, vortex to mix, and react at room temperature for 40min in the dark. Take 200μL of each reaction solution into a 96-well plate (repeat three times), and measure the absorbance at a wavelength of 517nm. Label them as A, S, S0, A0, respectively.

[0182] Reagent name (μL) A - Blank tube S - Sample tube [S0 - sample background] [A0 - positive control] Sample solution 0 50 50 0 VC 0 0 0 50 RO water (or 95% ethanol solution) 50 0 950 0 DPPH ethanol solution 950 950 0 950 Total (μL) 1000 1000 1000 1000

[0183] 3) Calculation formula

[0184] DPPH scavenging activity of positive control:

[0185]

[0186] DPPH scavenging activity calculation of the sample:

[0187]

[0188] Where A is the absorbance value of the blank control group; A0 is the absorbance value of the positive control group; S is the absorbance value of the extract sample reaction; and S0 is the background absorbance value of the sample.

[0189] A positive control test needs to be added to each batch of experiments. The IC50 of the positive control L-ascorbic acid (the concentration of the sample to achieve 50% clearance) should be between 0.076 mg / mL and 0.228 mg / mL for the experimental system to be considered effective. The SD (Standard Deviation) value of each parallel well is ≤3% for the parallel test to be considered effective.

[0190] like Figure 7 As shown, the DPPH free radical scavenging rate of basil (fragrant basil) hairy root filtrate was 23.03%, 28.93%, 34.77%, 51.23%, 79.83%, and 89.1% when the addition amount was 2.4%, 4%, 5%, 6%, 10%, and 20%, respectively. At the same addition amount, the DPPH free radical scavenging rate of basil leaf extract was 2.93%, 3.15%, 2.78%, 4.04%, 7.32%, and 19.25%. The DPPH free radical scavenging effect of basil (fragrant basil) hairy root filtrate was higher than that of basil leaf extract.

[0191] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A Solatium indicum hairy root extract, characterized in that, The extract contains at least polyphenols and flavonoids, wherein the polyphenol content is not less than 35 mg / g, and the flavonoid content is not less than 120 mg / g.

2. The method for preparing the extract of Mentha spicata L. hairy roots according to claim 1, characterized in that, The method comprises the following steps: Step 1, infecting the basil callus cells with Agrobacterium rhizogenes, and culturing the hairy roots by using an artificial medium; Step 2, drying the hairy roots obtained in step 1, and then adding a solvent to extract active ingredients to obtain a basil hairy root extract.

3. The production method according to claim 2, characterized by, The step 1 comprises: Step 1), disinfecting the basil seeds, and then adding a seed germination culture medium to culture the seeds under light until the seeds germinate; and continuing to culture the germinated seeds until the seedlings grow to 2-3 cm; Step 2), taking the stem segments of the seedlings, dipping the morphological lower end of the stem segments into the cultured Agrobacterium liquid, and inserting the morphological upper end of the stem segments into a rooting culture medium to induce rooting until the hairy roots grow out; Step 3), subculturing the hairy roots obtained in step 2).

4. The production method according to claim 2, characterized by, In the step 2, the solvent includes but is not limited to organic alcohols, organic esters, organic ethers, water, and phosphate buffer.

5. Use of the basil hairy root extract of claim 1 or the basil hairy root extract obtained by the preparation method of any one of claims 2-4 in the preparation of a skin external composition.

6. A topical skin composition, characterized in that, Comprise: The basil hairy root extract of claim 1 or the basil hairy root extract obtained by the preparation method of any one of claims 2-4.

7. A detangling composition characterized in that, Comprise: The basil hairy root extract of claim 1 or the basil hairy root extract obtained by the preparation method of any one of claims 2-4.

8. A hair follicle anti-inflammatory composition characterized in that, Comprise: The basil hairy root extract of claim 1 or the basil hairy root extract obtained by the preparation method of any one of claims 2-4.

9. The composition according to any one of claims 6 to 8, characterized in that, Comprise:

10. At least one skin product among 1) to 3) below, characterized in that, The basil hairy root extract of claim 1 or the basil hairy root extract obtained by the preparation method of any one of claims 2-4. Further comprise: other active ingredients and ingredients that produce synergistic effects with the basil hairy root extract of claim 1 or the basil hairy root extract obtained by the preparation method of any one of claims 2-4. The skin external composition of claim 6 comprises: 1) skin products for eliminating inflammation; 11. A detangling product characterized in that, 2) skin products for anti-aging and wrinkle removal; 12. A hair follicle anti-inflammatory product characterized in that, 3) skin products for whitening and / or removing spots. The anti-hair loss composition of claim 7. The hair follicle anti-inflammatory composition of claim 8.

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