Preparation method and application of jasmine leaf cell extract

Abstract

The invention provides a preparation method and application of a jasmine leaf cell extract, and belongs to the technical field of biology. The method comprises the following steps: (1) sterilizing jasmine leaves, and inoculating the jasmine leaves into an induction culture medium to form calluses; (2) continuously culturing the formed calluses for 2-6 generations, selecting loose granular cell clusters, putting the loose granular cell clusters into a liquid culture medium, and culturing to obtain jasmine suspension cells; (3) filtering the jasmine suspension cells, and freeze-drying to obtain jasmine cell powder; and (4) carrying out enzymolysis on the jasmine cell powder, introducing hydrogen by adopting a bubbling method, collecting liquid, and freeze-drying to obtain the jasmine leaf cell extract. According to the invention, the plant cell culture technology is utilized to obtain the cells with high content of effective components, and the effective components of the obtained cells are extracted to obtain the product with high activity of the effective components. The obtained jasmine flower leaf cell extract is added into cosmetics and has the beautifying effects of whitening, resisting oxidation, resisting aging and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of a jasmine leaf cell extract and application thereof. Background Art

[0002] Jasmine is a plant of the genus Jasmine in the family Oleaceae. It is cultivated in large quantities in Hengzhou, Guangxi Zhuang Autonomous Region, Jiangsu, Sichuan, Fujian, Yunnan and other places. Its roots, leaves and flowers all have medicinal value. According to the Compendium of Materia Medica and Chinese Materia Medica, jasmine flowers are pungent, sweet and cool in nature, and have the effects of regulating qi and harmonizing the middle. Decoction has the effect of treating diarrhea and abdominal pain, and decoction for washing eyes can treat conjunctivitis; jasmine leaves are pungent and cool in nature, belong to the lung and stomach meridians, have the effects of clearing away heat and relieving symptoms, reducing swelling and relieving pain, and are often used to treat exogenous fever, abdominal distension and diarrhea, poisonous insect stings and other diseases; jasmine roots are bitter, cool and poisonous, have the effects of anesthesia, analgesia and pain relief, can be used to treat injuries and tendon pain, can be ground and mixed with cooked egg yolk to treat caries, and can be ground and taken with water to treat insomnia.

[0003] At present, the main methods for extracting effective ingredients from jasmine leaf cells are: organic solvent extraction, ultrasonic extraction, microwave extraction, supercritical extraction, etc. However, the organic solvent extraction method requires the use of more organic solvents, the process is cumbersome, and the operation safety is poor. Even if the organic solvent reflux method is used to reduce the use of organic solvents, the activity and yield of the obtained product are not ideal. Although the ultrasonic extraction method can use ultrasonic vibration to break the cell wall and cell membrane without using organic solvents, so that the effective ingredients are released from the cells, thereby achieving the purpose of extracting the effective ingredients, the ultrasonic extraction technology has requirements for the content of effective substances. If the content of the extracted effective ingredients is too low, the extraction effect will be affected, and due to the effect of ultrasound, some substances may undergo chemical changes during the extraction process, affecting the quality of the extract. Utilize microwave extraction to extract effective ingredients, because cell absorbs microwave energy, makes cell internal temperature rise rapidly, makes cell internal pressure exceed the ability that cell wall can bear, causes cell rupture, and then makes effective ingredients flow out, thereby reaches the purpose of obtaining effective ingredients at lower temperature, but because microwave required time is short, operation control difficulty is large, and polyphenols in effective ingredients lose seriously, therefore, can affect the effect of effective ingredients. Supercritical extraction has the dual function of extraction and separation, and process flow is simple, extraction efficiency is high, extraction product quality is good, and there is no environmental pollution, but because this method has low productivity, therefore, is not suitable for industrial production use. Therefore, a method for extracting jasmine leaf cells that can be suitable for industrial production is urgently needed. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method of a jasmine leaf cell extract and its application. The present invention utilizes plant cell culture technology to produce cells with high content of active ingredients through large-scale culture to obtain a large number of jasmine cells, and finally obtains a product with high active ingredient activity by extracting the active ingredients from the obtained jasmine cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a jasmine leaf cell extract, comprising the following steps:

[0007] (1) sterilizing leaves of jasmine and inoculating them into an induction medium to form callus;

[0008] (2) continuously culturing the formed callus tissue for 2 to 6 generations, selecting loose granular cell clusters and placing them in a liquid culture medium for culturing to obtain jasmine suspension cells;

[0009] (3) filtering the jasmine cell suspension, freeze-drying, and obtaining jasmine cell powder;

[0010] (4) After enzymatic hydrolysis of jasmine cell powder, hydrogen gas is introduced by bubbling method, and the liquid is collected and freeze-dried to obtain jasmine leaf cell extract.

[0011] Preferably, the induction medium comprises MS+0.5-3 mg / L 6-BA+1-4 mg / L NAA+50-100 mg / L inositol+20-35 g / L maltose+2-8 g / L agar powder.

[0012] Preferably, the culture temperature of the callus is 20-30° C., and the culture time is 30-40 days.

[0013] Preferably, the liquid culture medium comprises MS+0.5-2 mg / L 6-BA+1-4 mg / L NAA+50-100 mg / L inositol+10-50 mg / L fungal polysaccharide+50-200 μmol / L methyl jasmonate.

[0014] Preferably, the fungal polysaccharide includes any one of yeast polysaccharide, lentinan, ganoderma lucidum polysaccharide and pachyman polysaccharide.

[0015] Preferably, the enzymes used for the enzymolysis include cellulase and pectinase in a mass ratio of 1:1 to 5.

[0016] Preferably, the enzymatic hydrolysis temperature is 40-60° C., and the enzymatic hydrolysis time is 1-3 h.

[0017] Preferably, the timing of introducing hydrogen is after the enzymatic hydrolysis is completed, at a flow rate of 30 to 60 mL / min, and the injection time is 30 to 90 min.

[0018] The present invention also provides application of the jasmine leaf cell extract obtained by the preparation method in preparing cosmetics.

[0019] Preferably, the jasmine leaf cell extract is added to the cosmetic in an amount of 10 to 1500 μg / mL.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a preparation method of jasmine leaf cell extract and its application. The present invention uses jasmine leaves as culture materials to obtain jasmine callus with strong and stable differentiation and transgeneration ability, vigorous growth, and stable and uniform state. The callus obtained by the present invention significantly improves the content of effective ingredients in the obtained cells. The present invention uses freeze-drying technology to pretreat cells, and then extracts the effective ingredients in the cells through enzymatic reaction and hydrogen introduction. It is found that the activity of the effective ingredients of the jasmine leaf cell extract obtained by the method of the present invention is also significantly improved. The jasmine leaf cell extract obtained by the present application is tested for its antioxidant, anti-aging and whitening effects, and it is found that the jasmine leaf cell extract obtained by the present invention has good antioxidant, anti-aging and whitening effects. Adding it to cosmetics has beauty effects such as whitening, antioxidant and anti-aging. DETAILED DESCRIPTION

[0021] The present invention provides a method for preparing a jasmine leaf cell extract, comprising the following steps:

[0022] (1) sterilizing leaves of jasmine and inoculating them into an induction medium to form callus;

[0023] (2) continuously culturing the formed callus tissue for 2 to 6 generations, selecting loose granular cell clusters and placing them in a liquid culture medium for culturing to obtain jasmine suspension cells;

[0024] (3) filtering the jasmine cell suspension, freeze-drying, and obtaining jasmine cell powder;

[0025] (4) After enzymatic hydrolysis of jasmine cell powder, hydrogen gas is introduced by bubbling method, and the liquid is collected and freeze-dried to obtain jasmine leaf cell extract.

[0026] In the present invention, jasmine leaves are sterilized and then inoculated into an induction culture medium to form callus tissue; healthy young leaves of jasmine are selected, washed 2 to 6 times with 65% to 90% ethanol, and then incubated with 0.05% to 0.2% HgCl 2The solution is soaked for 2 to 10 minutes, and the sterile water is rinsed 3 to 5 times to obtain the sterilized leaves, and the leaves are cut into 0.3 to 1 cm × 0.3 to 1 cm under sterile conditions, and inoculated into the induction medium for callus culture; the concentration of the ethanol is preferably 70% to 80%, more preferably 75%, the number of ethanol rinses is preferably 3 to 5 times, more preferably 4 times, and the HgCl 2 The concentration of the solution is preferably 0.08-0.15%, more preferably 0.1%; the HgCl 2 The immersion time of the solution is preferably 4 to 8 minutes, more preferably 5 minutes; the size of the leaf is preferably 0.4 to 0.7 cm × 0.4 to 0.7 cm, more preferably 0.5 cm × 0.5 cm; the induction medium comprises MS + 0.5 to 3 mg / L 6-BA + 1 to 4 mg / L NAA + 50 to 100 mg / L inositol + 20 to 35 g / L maltose + 2 to 8 g / L agar powder, preferably MS + 1 to 2 mg / L 6-BA+1.5-3mg / LNAA+60-80mg / L inositol+25-33g / L maltose+4-7g / L agar powder, and more preferably MS+1.5mg / L6-BA+2mg / LNAA+70mg / L inositol+30g / L maltose+6g / L agar powder; the culture temperature of the callus tissue is 20-30°C, preferably 23-28°C, and more preferably 25°C; the culture time is 30-40d, preferably 33-38d, and more preferably 35d.

[0027] In the present invention, the formed callus is continuously cultured for 2 to 6 generations, loose granular cell clusters are selected and placed in a liquid culture medium for culture, and jasmine suspension cells are obtained; under sterile conditions, cell clusters with good cell morphology on the callus are picked and inoculated in a new induction culture medium for continuous culture, loose granular cell clusters in the callus that have been continuously cultured for 2 to 6 generations are selected and placed in a liquid culture medium, and cultured for 20 to 30 days at a speed of 50 to 150 r / min and a temperature of 20 to 30° C. to obtain jasmine suspension cells; the number of generations of continuous culture of the callus is preferably 3 to 5 generations, and more preferably 4 generations; the liquid culture medium comprises MS+0.5 to 2 mg / L 6-BA+1 to 4 mg / L NAA+50 to 100 mg / L inositol+10 to 50 mg / L fungal polysaccharide+50 to 200 μmol / L methyl jasmonate, preferably MS+0.8 to 1.5 mg / L 6-BA+1.5 to 3 mg / L NAA+60-80mg / L inositol+20-40mg / L fungal polysaccharide+100-180μmol / L methyl jasmonate, and more preferably MS+1.2mg / L 6-BA+2mg / LNAA+70mg / L inositol+30mg / L fungal polysaccharide+150μmol / L methyl jasmonate; the fungal polysaccharide includes any one of yeast polysaccharide, shiitake polysaccharide, ganoderma polysaccharide, and tuckahoe polysaccharide, and is preferably yeast polysaccharide; the rotation speed is preferably 80-120r / min, and more preferably 100r / min, the temperature of the jasmine suspension cell culture is preferably 23-28°C, and more preferably 25°C, and the time of the jasmine suspension cell culture is preferably 22-28d, and more preferably 25d.

[0028] In the present invention, jasmine suspension cells are filtered, freeze-dried, and jasmine cell powder is obtained; the present invention has no special limitation on the filtering method, and a conventional method can be used, and sterile water is used to rinse 3 to 5 times, the culture medium is removed, and freeze-dried to obtain freeze-dried jasmine cell powder. The present invention adopts freeze-drying to dry the obtained jasmine cells, which can maximize the activity of the effective components of the jasmine cells, and use a rapid freezing method to achieve preliminary destruction of the jasmine cells, laying a foundation for the subsequent extraction of effective components.

[0029] In the present invention, after enzymolysis of jasmine cell powder, hydrogen is introduced by bubbling method, liquid is collected, and lyophilization is performed to obtain jasmine leaf cell extract; water is added at a mass volume ratio of 1g:3-8mL of jasmine cell powder and water, enzyme is added at 0.005‰-0.1‰ of the mass of jasmine cell powder, and after enzymolysis at 40-60°C for 1-3h, hydrogen is injected at a flow rate of 30-60mL / min by bubbling method, the injection time is 30-90min, centrifugation is performed at a speed of 4000-6000r / min for 3-10min, liquid is collected, and lyophilization is performed to obtain jasmine leaf cell extract; the mass volume ratio of jasmine cell powder to water is preferably 1g:4-6mL, more preferably 1g:5mL, the amount of enzyme added is preferably 0.008‰-0.05‰, more preferably 0.01‰, and the enzyme used for enzymolysis is a mixed enzyme prepared by cellulase and pectinase in a mass ratio of 1:1-5, The mass ratio of cellulase to pectinase is preferably 1:2-3, and more preferably 1:2.5; the present invention utilizes the combined use of cellulase and pectinase to destroy the cell wall while disintegrating the plant cell wall and the intercellular layer, so that the effective ingredients are more easily released from the cell; the temperature of the enzymolysis is preferably 45-55°C, and more preferably 50°C; the time of the enzymolysis is preferably 1.5-2.5h, and more preferably 2h; the bubbling method is to cool the enzymolysis solution to room temperature after the enzymolysis is completed, and then pass a hydrogen tube into the enzymolysis solution to pass hydrogen; the flow rate of the hydrogen is preferably 40-55mL / min, and more preferably 50mL / min, and the injection time is preferably 40-70min, and more preferably 50min; the rotation speed is preferably 4500-5500r / min, and more preferably 5000r / min, and the centrifugation time is preferably 4-6min, and more preferably 5min. The present invention utilizes the non-polarity of hydrogen and its ability to easily penetrate biological membranes and tissues, and introduces hydrogen into the enzymatic hydrolyzate, thereby achieving the extraction of effective ingredients while protecting the activity of the effective ingredients.

[0030] The present invention also provides application of the jasmine leaf cell extract obtained by the preparation method in preparing cosmetics.

[0031] In the present invention, the amount of the jasmine leaf cell extract added to the cosmetics is 10 to 1500 μg / mL.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] To ensure that sufficient jasmine cell powder can be obtained for subsequent experiments, in all embodiments and comparative examples of the present invention, at least three groups of parallel experiments were performed simultaneously when preparing jasmine suspension cells, and the obtained suspension cells were mixed to prepare jasmine cell powder.

[0034] Example 1

[0035] (1) Select healthy young leaves of jasmine, rinse them four times with 75% ethanol, and then rinse them with 0.1% HgCl 2 The leaves were soaked in the solution for 5 min, rinsed with sterile water for 5 times, and the sterilized leaves were cut into 0.5 cm × 0.5 cm pieces under sterile conditions, inoculated into MS induction medium containing 1.5 mg / L 6-BA, 2 mg / L NAA, 70 mg / L inositol, 30 g / L maltose, and 6 g / L agar powder, and cultured at 25 ± 2 ° C in the dark for 35 days to obtain callus tissue;

[0036] (2) Under sterile conditions, cell clumps with good cell morphology on the callus were picked and inoculated into a new induction medium for culture. 5 g of loose granular cell clumps from the callus cultured for four generations were selected and placed in 100 mL of MS liquid medium containing 1.2 mg / L 6-BA, 2 mg / L NAA, 70 mg / L inositol, 30 mg / L fungal polysaccharide (yeast polysaccharide), and 150 μmol / L methyl jasmonate. The cells were cultured at a speed of 100 r / min and 25±2° C. in the dark for 25 days to obtain jasmine suspension cells.

[0037] (3) vacuum filtration of the jasmine suspended cells to remove the liquid culture medium, washing with sterile water 4 times to remove excess culture medium, and freeze-drying to obtain freeze-dried jasmine cell powder;

[0038] (4) Weigh 5 g of jasmine cell powder and add 25 mL of water, add 0.05 mg of enzyme (cellulase: pectinase = 1:2.5, both purchased from Guangzhou Huayu Biotechnology Co., Ltd.), and perform enzymolysis at 50°C for 2 h. Then, use the bubbling method to inject hydrogen at a flow rate of 50 mL / min for 50 min, centrifuge at a speed of 5000 r / min for 5 min, collect the liquid, and freeze-dry to obtain jasmine leaf cell extract.

[0039] Example 2

[0040] (1) Select healthy young leaves of jasmine, rinse them six times with 65% ethanol, and then rinse them with 0.2% HgCl 2The leaves were soaked in the solution for 4 min, rinsed with sterile water for 5 times, and the sterilized leaves were cut into 1 cm × 1 cm pieces under sterile conditions, inoculated into MS induction medium containing 3 mg / L 6-BA, 4 mg / L NAA, 50 mg / L inositol, 35 g / L maltose, and 8 g / L agar powder, and cultured at 25 ± 2 ° C in the dark for 30 days to obtain callus tissue;

[0041] (2) Under sterile conditions, cell clumps with good cell morphology on the callus were picked and inoculated into a new induction medium for culture. 5 g of loose granular cell clumps from callus cultured for 6 generations were selected and placed in 100 mL of MS liquid medium containing 2 mg / L 6-BA, 4 mg / L NAA, 50 mg / L inositol, 10 mg / L fungal polysaccharide (yeast polysaccharide), and 100 μmol / L methyl jasmonate. The cells were cultured at a speed of 100 r / min and 25±2° C. in the dark for 22 days to obtain jasmine suspension cells.

[0042] (3) vacuum filtration of the jasmine suspended cells to remove the liquid culture medium, washing with sterile water 4 times to remove excess culture medium, and freeze-drying to obtain freeze-dried jasmine cell powder;

[0043] (4) Weigh 5 g of jasmine cell powder and add 15 mL of water, add 0.025 mg of enzyme (cellulase: pectinase = 1:1, both purchased from Guangzhou Huayu Biotechnology Co., Ltd.), and perform enzymolysis at 50°C for 3 h. Then, use the bubbling method to inject hydrogen at a flow rate of 30 mL / min for 90 min, centrifuge at a speed of 5000 r / min for 5 min, collect the liquid, and freeze-dry to obtain jasmine leaf cell extract.

[0044] Example 3

[0045] (1) Select healthy young leaves of jasmine, rinse them three times with 95% ethanol, and then rinse them with 0.05% HgCl 2 The leaves were soaked in the solution for 10 min, rinsed with sterile water for 5 times, and the sterilized leaves were cut into 0.3 cm × 0.3 cm pieces under sterile conditions, inoculated into MS induction medium containing 0.5 mg / L 6-BA, 1 mg / L NAA, 100 mg / L inositol, 20 g / L maltose, and 4 g / L agar powder, and cultured at 25 ± 2 ° C in the dark for 40 days to obtain callus tissue;

[0046] (2) Under sterile conditions, cell clumps with good cell morphology on the callus were picked and inoculated into a new induction medium for culture. 5 g of loose granular cell clumps from the callus cultured for three generations were selected and placed in 100 mL of MS liquid medium containing 0.5 mg / L 6-BA, 1 mg / L NAA, 100 mg / L inositol, 50 mg / L fungal polysaccharide (yeast polysaccharide), and 180 μmol / L methyl jasmonate. The cells were cultured at a speed of 100 r / min and 25±2° C. in the dark for 28 days to obtain jasmine suspension cells.

[0047] (3) vacuum filtration of the jasmine suspended cells to remove the liquid culture medium, washing with sterile water 4 times to remove excess culture medium, and freeze-drying to obtain freeze-dried jasmine cell powder;

[0048] (4) Weigh 5 g of jasmine cell powder and add 40 mL of water, add 0.5 mg of enzyme (cellulase: pectinase = 1:5, both purchased from Guangzhou Huayu Biotechnology Co., Ltd.), and perform enzymolysis at 50°C for 1 h. Then, use the bubbling method to inject hydrogen at a flow rate of 60 mL / min for 30 min, centrifuge at a speed of 5000 r / min for 5 min, collect the liquid, and freeze-dry to obtain jasmine leaf cell extract.

[0049] Example 4

[0050] (1) Select healthy young leaves of jasmine, rinse them four times with 75% ethanol, and then rinse them with 0.1% HgCl 2 The leaves were soaked in the solution for 5 min, rinsed with sterile water for 5 times, and the sterilized leaves were cut into 0.5 cm × 0.5 cm pieces under sterile conditions, inoculated into MS induction medium containing 3 mg / L 6-BA, 4 mg / L NAA, 50 mg / L inositol, 35 g / L maltose, and 8 g / L agar powder, and cultured at 25 ± 2 °C in the dark for 40 days to obtain callus tissue;

[0051] (2) Under sterile conditions, cell clumps with good cell morphology on the callus were picked and inoculated into a new induction medium for culture. 5 g of loose granular cell clumps from the callus cultured for four generations were selected and placed in 100 mL of MS liquid medium containing 0.5 mg / L 6-BA, 1 mg / L NAA, 100 mg / L inositol, 50 mg / L fungal polysaccharide (yeast polysaccharide), and 180 μmol / L methyl jasmonate. The cells were cultured at a speed of 100 r / min and 25±2° C. in the dark for 28 days to obtain jasmine suspension cells.

[0052] (3) vacuum filtration of the jasmine suspended cells to remove the liquid culture medium, washing with sterile water 4 times to remove excess culture medium, and freeze-drying to obtain freeze-dried jasmine cell powder;

[0053] (4) Weigh 5 g of jasmine cell powder and add 25 mL of water, add 0.05 mg of enzyme (cellulase: pectinase = 1:2.5, both purchased from Guangzhou Huayu Biotechnology Co., Ltd.), and perform enzymolysis at 50°C for 2 h. Then, use the bubbling method to inject hydrogen at a flow rate of 50 mL / min for 50 min, centrifuge at a speed of 5000 r / min for 5 min, collect the liquid, and freeze-dry to obtain jasmine leaf cell extract.

[0054] Example 5

[0055] (1) Select healthy young leaves of jasmine, rinse them four times with 75% ethanol, and then rinse them with 0.1% HgCl 2 The leaves were soaked in the solution for 5 min, rinsed with sterile water for 5 times, and the sterilized leaves were cut into 0.5 cm × 0.5 cm pieces under sterile conditions, inoculated into MS induction medium containing 0.5 mg / L 6-BA, 1 mg / L NAA, 100 mg / L inositol, 20 g / L maltose, and 4 g / L agar powder, and cultured at 25 ± 2 ° C in the dark for 40 days to obtain callus tissue;

[0056] (2) Under sterile conditions, cell clumps with good cell morphology on the callus were picked and inoculated into a new induction medium for culture. 5 g of loose granular cell clumps from the callus cultured for four generations were selected and placed in 100 mL of MS liquid medium containing 2 mg / L 6-BA, 4 mg / L NAA, 50 mg / L inositol, 10 mg / L fungal polysaccharide (yeast polysaccharide), and 100 μmol / L methyl jasmonate. The cells were cultured at a speed of 100 r / min and 25±2° C. in the dark for 22 days to obtain jasmine suspension cells.

[0057] (3) vacuum filtration of the jasmine suspended cells to remove the liquid culture medium, washing with sterile water 4 times to remove excess culture medium, and freeze-drying to obtain freeze-dried jasmine cell powder;

[0058] (4) Weigh 5 g of jasmine cell powder and add 25 mL of water, add 0.05 mg of enzyme (cellulase: pectinase = 1:2.5, both purchased from Guangzhou Huayu Biotechnology Co., Ltd.), and perform enzymolysis at 50°C for 2 h. Then, use the bubbling method to inject hydrogen at a flow rate of 50 mL / min for 50 min, centrifuge at a speed of 5000 r / min for 5 min, collect the liquid, and freeze-dry to obtain jasmine leaf cell extract.

[0059] Comparative Example 1

[0060] The callus culture was performed on young leaves of jasmine in the same manner as in Example 1, except that maltose was replaced by sucrose and fructose, respectively.

[0061] Comparative Example 2

[0062] The callus tissue of young leaves of jasmine was cultured in the same manner as in Example 1, except that 6-BA in the induction medium was replaced by thiadiazole phenylurea (TDZ), 2-isopentenyl adenine (2-ip), and kinetin (KT), respectively.

[0063] Comparative Example 3

[0064] The callus culture was performed on young leaves of jasmine in the same manner as in Example 1, except that NAA in the induction medium was replaced by 2,4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), and indoleacetic acid (IAA), respectively.

[0065] Comparative Example 4

[0066] The callus culture was carried out on the young leaves of jasmine in the same manner as in Example 1, except that the inositol in the induction medium was replaced by coconut milk.

[0067] Comparative Example 5

[0068] The callus culture was carried out on the young leaves of jasmine in the same manner as in Example 1, except that the inositol in the induction medium was removed.

[0069] Comparative Example 6

[0070] The callus tissue of young leaves of jasmine was cultured and the jasmine leaf cell extract was obtained in the same manner as in Example 1, except that the induction medium used was: MS+0.3mg / L6-BA+0.5mg / LNAA+30mg / L inositol+30g / L maltose+6g / L agar powder.

[0071] Comparative Example 7

[0072] The callus tissue of young leaves of jasmine was cultured and the jasmine leaf cell extract was obtained in the same manner as in Example 1, except that the induction medium used was: MS+3.5mg / L6-BA+4.5mg / LNAA+130mg / L inositol+30g / L maltose+6g / L agar powder.

[0073] Comparative Example 8

[0074] The jasmine suspension cells were cultured in the same manner as in Example 1, except that the fungal polysaccharide in the liquid culture medium was removed.

[0075] Comparative Example 9

[0076] The jasmine suspension cells were cultured in the same manner as in Example 1, except that methyl jasmonate was removed from the liquid culture medium.

[0077] Comparative Example 10

[0078] Jasmine suspension cells were cultured and jasmine leaf cell extracts were obtained in the same manner as in Example 1, except that the liquid culture medium used was: MS + 0.3 mg / L 6-BA + 0.5 mg / L NAA + 30 mg / L inositol + 5 mg / L fungal polysaccharide + 40 μmol / L methyl jasmonate.

[0079] Comparative Example 11

[0080] Jasmine suspension cells were cultured and jasmine leaf cell extracts were obtained in the same manner as in Example 1, except that the liquid culture medium used was: MS + 2.5 mg / L 6-BA + 4.5 mg / L NAA + 130 mg / L inositol + 70 mg / L fungal polysaccharides + 220 μmol / L methyl jasmonate.

[0081] Comparative Example 12

[0082] The jasmine suspension cells were dried and the jasmine leaf cell extract was obtained in the same manner as in Example 1, except that freeze drying was replaced by drying at 40° C. for 40 h to obtain jasmine cell powder.

[0083] Comparative Example 13

[0084] The jasmine leaf cell extract was obtained in the same manner as in Example 1, except that the jasmine cells from which the excess culture medium was removed were directly subjected to enzymatic hydrolysis.

[0085] Comparative Example 14

[0086] The jasmine leaf cell extract was obtained in the same manner as in Example 1, except that, without enzymatic hydrolysis, the freeze-dried jasmine cells were directly subjected to hydrogen bubbling to extract the effective ingredients in the jasmine leaf cells, that is, freeze-dried jasmine cell powder was weighed and water 5 times its mass was added, mixed, hydrogen was introduced, the liquid was collected, and the jasmine leaf cell extract was obtained by freeze-drying.

[0087] Comparative Example 15

[0088] The jasmine leaf cell extract was obtained in the same manner as in Example 1, except that the jasmine cells from which excess culture medium was removed were directly subjected to a bubbling method to introduce hydrogen to extract the effective components in the jasmine leaf cells, that is, jasmine cells were weighed and water 5 times the mass of the water was added, mixed, hydrogen was introduced, the liquid was collected, and lyophilized to obtain the jasmine leaf cell extract.

[0089] Comparative Example 16

[0090] The jasmine leaf cell extract was obtained in the same manner as in Example 1, except that the liquid after enzymatic hydrolysis was directly collected to obtain the jasmine leaf cell extract.

[0091] Comparative Example 17

[0092] The jasmine leaf cell extract was obtained in the same manner as in Example 1, except that the jasmine cells from which the excess culture medium was removed were directly subjected to enzymatic hydrolysis, and the liquid after enzymatic hydrolysis was collected and freeze-dried to obtain the jasmine leaf cell extract.

[0093] Experimental Example 1

[0094] The callus tissues obtained in Examples 1 to 5 of the present invention and Comparative Examples 1 to 7 were observed and counted. The results are shown in Table 1.

[0095] Callus rate (%) = number of blocks induced to form dense callus tissue / number of inoculations × 100%;

[0096] Browning rate (%) = number of browned callus pieces / number of inoculated pieces × 100%.

[0097] Table 1 Effects of different components on callus healing rate and browning rate

[0098]

[0099]

[0100] As shown in Table 1, the callus induction rate of Examples 1 to 5 is higher than that of Comparative Examples 1 to 7, and the browning rate obtained in Comparative Examples 1 to 7 is higher than that of Examples 1 to 5. It can be seen from this that the induction effect of the induction medium in the present application on callus tissue is better than that of other hormone combinations, and the excessive or too low dosage of 6-BA, NAA and inositol will reduce the callus induction rate and increase the browning rate, which is not conducive to obtaining the ideal induction efficiency. When inositol is replaced with coconut milk that can promote callus growth, its callus induction rate is lower than that of the embodiment, and the browning rate is higher than that of the embodiment, indicating that the growth of callus tissue is jointly affected by multiple elements, and the change of any one element will affect the callus induction rate and browning rate of callus tissue.

[0101] Experimental Example 2

[0102] The suspension cells obtained in Examples 1 to 5 of the present invention and Comparative Examples 6 to 11 were observed and counted. The results are shown in Table 2.

[0103] Proliferation rate (%) = (fresh weight after proliferation - fresh weight before proliferation) / fresh weight before proliferation × 100%,

[0104] The fresh weight test method is: vacuum filter the cell suspension to remove the liquid culture medium, rinse with sterile water 4 times, and weigh the fresh weight after filtering and draining.

[0105] Table 2 Effects of different treatment groups on the proliferation rate of suspension cells

[0106] Example 1 Example 2 Example 3 Example 4 Example 5 Proliferation rate / % 375.12 356.24 368.71 349.96 355.75 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Comparative Example 10 Comparative Example 11 Proliferation rate / % 306.37 311.84 166.43 148.09 90.31 86.63

[0107] As shown in Table 2, the proliferation rate of the suspended cells in Examples 1 to 5 is higher than that in Comparative Examples 6 to 11. When fungal polysaccharides or methyl jasmonate are not used, the suspended cells proliferate slowly and are not suitable for the culture of jasmine suspension cells. When the concentration of fungal polysaccharides or methyl jasmonate is too high or too low, the suspended cells will also proliferate slowly. Compared with the results of Comparative Example 6 and Comparative Example 7, it can be seen that the obtained callus tissue is different, which also affects the subsequent proliferation of suspended cells, indicating that the proliferation of suspended cells is affected by multiple factors, and the change of any factor will affect the proliferation of suspended cells. The jasmine suspension cells obtained by the present invention have good dispersibility and high proliferation rate.

[0108] Experimental Example 3

[0109] The yields of the jasmine leaf cell extracts obtained in Examples 1 to 5 of the present invention and Comparative Examples 12 to 17 were calculated, and the results are shown in Table 3.

[0110] Yield (%) = jasmine leaf cell extract / jasmine cell powder × 100%.

[0111] Table 3 Effect of different treatment groups on the yield of jasmine leaf cell extract

[0112] Example 1 Example 2 Example 3 Example 4 Example 5 Yield / % 21.3 20.9 20.8 21.1 20.7 Comparative Example 12 Comparative Example 13 Comparative Example 14 Comparative Example 15 Comparative Example 16 Comparative Example 17 Yield / % 12.3 11.7 15.5 9.8 16.1 10.1

[0113] As shown in Table 3, the yields of Examples 1 to 5 are higher than those of Comparative Examples 12 to 17, indicating that the method of the present invention can more effectively extract the active ingredients from cells, and changes in the extraction method will also affect the extraction effect.

[0114] Experimental Example 4

[0115] 1. DPPH clearance rate

[0116] Using Examples 1 to 5 and Comparative Examples 6 to 17 as samples, 0.1 g of jasmine leaf cell extract was respectively taken and placed in 100 mL of anhydrous ethanol, and sample solutions were obtained after the samples were completely dissolved.

[0117] Preparation of DPPH ethanol solution: Accurately weigh 3.94 mg DPPH, add anhydrous ethanol to dissolve and dilute to 100 mL volumetric flask to prepare 0.1 mM DPPH solution, and store at low temperature (0-4°C) away from light.

[0118] 1 mL of sample solution was mixed with 3 mL of DPPH solution as the experimental group; DPPH solution was used as the control group and water and ethanol were used as the blank group. The absorbance was measured at 517 nm and the clearance rate was calculated. The results are shown in Table 4. The calculation formula is as follows:

[0119] DPPH clearance rate (%) = [A 对照 -(A 实验 -A 空白 )] / A 对照 ×100%.

[0120] 2. Hydroxyl radical (OH) clearance rate

[0121] According to the method described in the document "Spectrophotometric Determination of the Scavenging Effect of Two Traditional Chinese Medicines on Hydroxyl Free Radicals" (Mao Shaochun, Journal of Yunnan University (Natural Science Edition), 2006, (S1): 259-260.), the samples obtained in Examples 1 to 5 and Comparative Examples 6 to 17 were prepared into solutions with a concentration of 1 mg / mL, 1 mL of each solution was taken as the sample solution, and 1.0 mL of 7.5 mmol o-phenanthroline solution, 5.0 mL of pH 7.4 PBS, and 1.0 mL of FeSO were added to a 25 mL colorimetric tube. 4 solution, 1 mL sample solution, 1.0 mL H 2 O 2 The application liquid was supplemented with distilled water to a volume of 25.00 mL, left for 60 min, and the absorbance was measured at 510 nm. The hydroxyl radical scavenging rate was calculated according to the method described in the above literature. The results are shown in Table 4.

[0122] 3.ABTS + Clearance

[0123] According to the method described in the literature "Study on the Enzymatic Extraction Process of Chicken Collagen Peptide and Its Free Radical Scavenging Ability" (Hu Weiwei et al., Food Industry, Vol. 35, No. 2, 2014, pp. 144-119), the superoxide anion radical scavenging rate was determined by the pyrogallol method. The corresponding absorbance was measured at a wavelength of 325nm, and VC was used as a positive control. According to the method described in the aforementioned literature, ABTS was calculated. + The clearance rate and results are shown in Table 4.

[0124] Table 4 Free radical scavenging ability of different treatment groups

[0125]

[0126]

[0127] As shown in Table 4, the free radical scavenging ability of the extracts obtained in Examples 1 to 5 is higher than that of Comparative Examples 6 to 17, and significantly higher than that of Comparative Examples 8 to 11, while the free radical scavenging rate is similar to that of other Comparative Examples. The reason for this is that culturing jasmine suspension cells is a critical period for obtaining active ingredients, so changing the conditions for culturing jasmine suspension cells has an important impact on active ingredients. The free radical scavenging ability of Comparative Example 12 is slightly lower than that of other treatment groups except Comparative Examples 8 to 11. The reason for this may be that the drying conditions affect the active ingredients in the extract.

[0128] Experimental Example 5

[0129] The maximum safe concentration of the jasmine leaf cell extract prepared in Example 1 was determined by MTT method.

[0130] Cell plating: The density of mouse RAW264.7 macrophages (purchased from Wuhan Pronocell Life Science Co., Ltd.) was adjusted to 1×10 5 cell / mL, 200 μL (1×10 4 Cells / well) were inoculated in a 96-well plate, and basal culture medium was added around the 96-well plate to prevent edge effect. A zero adjustment group (200 μL basal culture medium without cells) was set up and incubated in an incubator (37°C, 5% CO 2 ) were cultivated.

[0131] Sample preparation: Use DMEM basal medium to prepare a solution with a concentration of 1.5 mg / mL of jasmine leaf cell extract, filter and sterilize with a 0.22 μm filter membrane, and take out and dilute with DMEM medium to the concentrations of 0.75 mg / mL, 0.375 mg / mL, 0.188 mg / mL, 0.094 mg / mL and 0.047 mg / mL of the sample solution before use.

[0132] MTT: Accurately weigh an appropriate amount of MTT powder, add PBS to dissolve and prepare a concentration of 5 mg / mL, filter with a 0.22 μm filter membrane, and store at 4°C in the dark for future use. The shelf life is 15 days.

[0133] Administration: After 24 hours of culture, the 96-well plate was removed, the old culture medium was discarded, and the plate was arranged according to 3 replicates per group and per concentration; 200 μL DMEM basal culture medium was added to each well of the zero adjustment group and the blank control group, and 200 μL of the sample solution of different concentrations was added to the experimental group, and then returned to the incubator for culture (37°C, 5% CO 2 ).

[0134] MTT assay for cell viability: 24 h after drug treatment, take out the 96-well plate, add 20 μL MTT (5 mg / mL) to each well, return to the incubator and continue culturing for 4 h, then discard the liquid in the wells, add 150 μL DMSO to each well again, shake for 10 min, and measure the absorbance (OD value) at a wavelength of 560 nm.

[0135] Experimental result processing: Cell viability % = (OD value of experimental group - OD value of zero adjustment well) / (OD value of blank control group - OD value of zero adjustment group) × 100%. When the cell viability is ≥ 90%, the corresponding sample concentration is the maximum safe concentration. The test results are shown in Table 5.

[0136] Table 5 Experimental results of the effect of jasmine leaf cell extract on macrophage activity

[0137]

[0138] As shown in Table 5, when the concentration of jasmine leaf cell extract was 1.5 mg / mL, the cell viability was 89.76%, indicating that the maximum safe concentration of cells was 1.5 mg / mL.

[0139] Experimental Example 6

[0140] Accurately weigh 0.00362g of tyrosinase powder (Sigma), dilute to 50mL with pH6.8 PBS phosphate buffer solution, prepare concentrations of 10, 50, 100, 200, 500, 1000, 1500μg / mL respectively. The sample test solution obtained in Example 1 is added according to Table 6, and then placed in a 37°C water bath for 10min; then tyrosinase is added to tubes A and C in sequence, with an interval of 30s between each enzyme addition, and vortexed for 15s to mix immediately after enzyme addition, and reacted for 15min. After the reaction tube returns to room temperature, the ultraviolet spectrophotometer measures the absorbance at 475nm to detect the inhibition rate of the sample on tyrosinase. The results are shown in Table 7, and the calculation formula of the inhibition rate of the sample on tyrosinase is as follows.

[0141] Inhibition rate (%) = [(AB) - (CD)] / (AB) × 100%,

[0142] Among them, A is the absorbance value of the blank sample with enzyme system; B is the absorbance value of the blank sample without enzyme system; C is the absorbance value of the sample group with enzyme system; D is the absorbance value of the sample group without enzyme system.

[0143] Table 6 Tyrosine detection reagents

[0144] Unit (μL) A B C D L-Tyrosine 800 800 800 800 sample 0 0 800 800 PBS 1500 2000 700 1200 Tyrosinase 500 0 500 0 Total volume 2800 2800 2800 2800

[0145] Table 7 Inhibition rate of tyrosinase at different concentrations

[0146] Concentration / (μg / mL) 10 50 100 200 500 1000 1500 Inhibition rate / % 20.26 51.46 93.42 100 100 100 100

[0147] It can be seen from Table 7 that the jasmine leaf cell extract obtained by the present invention has the effect of inhibiting tyrosinase, indicating that the jasmine leaf cell extract has a whitening effect, and the whitening effect can be achieved when a lower concentration is used.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a jasmine leaf cell extract, characterized in that: The following steps are involved: (1) sterilizing leaves of jasmine and inoculating them into an induction medium to form callus; (2) continuously culturing the formed callus tissue for 2 to 6 generations, selecting loose granular cell clusters and placing them in a liquid culture medium for culturing to obtain jasmine suspension cells; (3) filtering the jasmine suspended cells, freeze-drying, and obtaining jasmine cell powder; (4) After enzymatic hydrolysis of jasmine cell powder, hydrogen gas is introduced by bubbling method, and the liquid is collected and freeze-dried to obtain jasmine leaf cell extract.

2. The preparation method according to claim 1, characterized in that: The induction culture medium comprises MS+0.5-3 mg / L 6-BA+1-4 mg / L NAA+50-100 mg / L inositol+20-35 g / L maltose+2-8 g / L agar powder.

3. The preparation method according to claim 1, characterized in that: The culture temperature of the callus tissue is 20-30° C., and the culture time is 30-40 days.

4. The preparation method according to claim 1, characterized in that: The liquid culture medium comprises MS+0.5-2 mg / L 6-BA+1-4 mg / L NAA+50-100 mg / L inositol+10-50 mg / L fungal polysaccharide+50-200 μmol / L methyl jasmonate.

5. The preparation method according to claim 4, characterized in that: The fungal polysaccharide includes any one of yeast polysaccharide, lentinan, ganoderma lucidum polysaccharide and tuckahoe polysaccharide.

6. The preparation method according to claim 1, characterized in that: The enzymes used for the enzymolysis include cellulase and pectinase in a mass ratio of 1:1-5.

7. The preparation method according to claim 1, characterized in that: The enzymolysis temperature is 40-60° C., and the enzymolysis time is 1-3 hours.

8. The preparation method according to claim 1, characterized in that: The timing of introducing hydrogen is after the enzymatic hydrolysis is completed, and hydrogen is injected at a flow rate of 30 to 60 mL / min for a time of 30 to 90 minutes.

9. Use of the jasmine leaf cell extract obtained according to the preparation method according to any one of claims 1 to 8 in the preparation of cosmetics.

10. The use according to claim 10, characterized in that: The amount of the jasmine leaf cell extract added to the cosmetics is 10 to 1500 μg / mL.

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