CN109674039B - Method for synthesizing salidroside and icariside D2 through biotransformation and application - Google Patents
Method for synthesizing salidroside and icariside D2 through biotransformation and application Download PDFInfo
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- CN109674039B CN109674039B CN201811409983.9A CN201811409983A CN109674039B CN 109674039 B CN109674039 B CN 109674039B CN 201811409983 A CN201811409983 A CN 201811409983A CN 109674039 B CN109674039 B CN 109674039B
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- salidroside
- icariside
- motherwort
- hydroxyphenylethanol
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- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 title claims abstract description 113
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 title claims abstract description 113
- 238000000034 method Methods 0.000 title claims abstract description 31
- 230000036983 biotransformation Effects 0.000 title abstract description 11
- 230000002194 synthesizing effect Effects 0.000 title abstract description 8
- OJDSCNUKKOKOQJ-UHFFFAOYSA-N Icariside D2 Natural products C1=CC(CCO)=CC=C1OC1C(O)C(O)C(O)C(CO)O1 OJDSCNUKKOKOQJ-UHFFFAOYSA-N 0.000 title description 2
- OJDSCNUKKOKOQJ-RKQHYHRCSA-N Icariside D2 Chemical compound C1=CC(CCO)=CC=C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 OJDSCNUKKOKOQJ-RKQHYHRCSA-N 0.000 title description 2
- 239000000725 suspension Substances 0.000 claims abstract description 104
- IYCPMVXIUPYNHI-UHFFFAOYSA-N Icariside I Natural products C1=CC(OC)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 IYCPMVXIUPYNHI-UHFFFAOYSA-N 0.000 claims abstract description 86
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- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims abstract description 63
- 238000006243 chemical reaction Methods 0.000 claims abstract description 54
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- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims 1
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- 229930024421 Adenine Natural products 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
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- 231100000989 no adverse effect Toxicity 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/533—Leonurus (motherwort)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Hair brushRelates to the technical field of biotransformation, and discloses a method for synthesizing salidroside and icariside D by biotransformation 2 The method comprises the steps of adding p-hydroxyphenylethanol into a culture solution containing motherwort suspension cells, and converting for a certain period of time to ensure that the motherwort suspension cells contain salidroside and icariside D 2 The culture solution also contains salidroside and icariside D 2 . The invention directly adds the p-hydroxyphenylethanol into the culture solution containing the motherwort suspension cells, converts the p-hydroxyphenylethanol into salidroside and icariside D 2 The loss of the p-hydroxyphenylethanol is greatly reduced, the conversion rate is high, the time required by culturing the motherwort suspension cells is short, the motherwort suspension cells are easy to obtain, and the operation is very simple, convenient and safe.
Description
Technical Field
The invention relates to the technical field of biotransformation, in particular to a method for synthesizing salidroside and icariside D by biotransformation 2 The method and the application thereof.
Background
Salidroside (salidroside) is the most important active ingredient of plants in Rhodiola of Crassulaceae (Crassulaceae), and has various specific effects of resisting fatigue, enhancing immunity, protecting heart and blood vessels, resisting anoxia, aging, protecting nerve cells, protecting liver, resisting oxidation, resisting tumor, resisting radiation and the like. Has extremely important application value in the aspects of military affairs, spaceflight, sports, health care medicine and the like. In recent years, there have been increasing varieties of products such as pharmaceuticals, beverages, foods, and cosmetics produced using them as main raw materials, and demand for salidroside has been increasing.
The plants of the rhodiola genus are the main sources for extracting salidroside, but the wild resources are deficient, and the content of salidroside is very low, such as the most commonly used rhodiola sachalinensis and rhodiola crenulata, the content of salidroside in the plants is only 0.5-0.8%. Although the current artificial cultivation is advanced, the rhodiola seeds have poor maturity and low germination rate and need to grow in high-altitude mountainous areas with proper environment, so that the cost of the artificially cultivated rhodiola is high, the content of active ingredients is low, and the large supply of the rhodiola raw materials is difficult to ensure.
The biosynthesis of salidroside in nature takes tyrosol (i.e. p-hydroxyphenylethanol) as a substrate, and is catalyzed by glucosyltransferase in plants to transfer a glucosyl group to the tyrosol to form the salidroside. However, most plants in nature cannot synthesize tyrosol, so that salidroside is not contained.
Salidroside is a glycoside formed by combining glucose and tyrosol by glycosidic bond, and tyrosol is its aglycone and can be synthesized by enzyme method. It has been reported that salidroside can be synthesized using beta-glycosidases extracted from plant seeds, such as beta-glycosidase from apple seeds, and that glycosylation of 4-substituted benzyl alcohols and salidroside with glucose in a single phase water-dioxane medium results in glycoside yields of about 13.1 to 23.1%, with salidroside at about 15.8%. However, the main function of beta-glycosidase in organisms is to catalyze the hydrolysis of glycosidic bonds, the reverse reaction is utilized for synthesizing salidroside, the reaction is limited by thermodynamic control and reaction balance, and the yield is low.
Salidroside can also be synthesized by a chemical method, but because the chemical synthesis process needs multi-step protection and deprotection measures, the process is complex and high in cost, byproducts are more and difficult to separate, and the environmental hazard is large, large-scale production is not available.
Although the prior art is concerned with the addition of specific materials to the water, soil or leaves in which the plants are growing,the salidroside is generated through biotransformation, but the addition method has the disadvantages of high raw material loss, incapability of absorbing and utilizing most of raw materials, and low salidroside conversion rate. Icariside D 2 The content of rhodiola root is lower than that of salidroside, and no synthetic method is reported.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a simple, convenient and efficient method for synthesizing salidroside and icariside D through biotransformation 2 The method of (1).
In order to achieve the aim, the invention provides a method for synthesizing salidroside and icariside D by biotransformation 2 The method comprises the steps of adding p-hydroxyphenylethanol into a culture solution containing motherwort suspension cells, and converting for a certain period of time to ensure that the motherwort suspension cells contain salidroside and icariside D 2 The culture solution also contains salidroside and icariside D 2 。
The initial concentration of the p-hydroxyphenylethanol is 0.1-30 mmol/L; the initial concentration of the p-hydroxyphenylethanol is preferably 0.5-10 mmol/L; the initial concentration of the p-hydroxyphenylethanol is more preferably 1 to 5mmol/L.
The conversion time is 1-720 h; the conversion time is preferably 5 to 160 hours; the conversion time is more preferably 20 to 65 hours.
The adding frequency of the p-hydroxyphenylethanol is 1 to 15; the addition frequency of the p-hydroxyphenylethanol is preferably 1 to 8 times; the number of addition of the p-hydroxyphenylethanol is more preferably 1 to 4.
When the adding frequency of the p-hydroxyphenylethanol is more than 1 time, the time interval between two adjacent times is 24-240 hours; the time interval between two adjacent times is preferably 24-120 h; the time interval between two adjacent times is further preferably 48 to 72 hours.
The p-hydroxyphenylethanol is prepared by converting any one or the combination of at least two of p-hydroxyphenylethylamine, p-hydroxyphenylacetic acid and p-hydroxyphenylacetaldehyde in the motherwort suspension cells. After being added into the motherwort suspension cell culture solution, the p-hydroxyphenylethylamine, the p-hydroxyphenylacetic acid and the p-hydroxyphenylacetaldehyde are absorbed by motherwort suspension cells and can be converted into p-hydroxyphenylethanol to participate in subsequent reactions under the action of related enzymes.
The invention also provides a medicine containing salidroside and icariside D 2 The application of herba Leonuri suspension cell or culture solution containing salidroside and icariside D obtained by the above method 2 The suspension cell or culture solution of herba Leonuri can be used as salidroside or icariside D 2 And a composition containing salidroside and icariside D 2 The herba Leonuri suspension cells containing salidroside and icariside D obtained by the above method 2 The motherwort suspension cells can be used for preparing food, health food, food additives or medicines.
The invention directly adds the p-hydroxyphenylethanol into the culture solution containing the motherwort suspension cells, converts the p-hydroxyphenylethanol into salidroside and icariside D 2 The loss of the p-hydroxyphenylethanol is greatly reduced, the conversion rate is high, the time required by culturing the motherwort suspension cells is short, the motherwort suspension cells are easy to obtain, and the operation is very simple, convenient and safe. The medicine containing salidroside and icariside D obtained by the method of the invention 2 The herba Leonuri suspension cells and/or culture solution can be used as salidroside and icariside D 2 The extract raw material contains salidroside and icariside D obtained by the method of the invention 2 The herba Leonuri suspension cells can also be made into food, health food, food additive or medicine.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention providesFor synthesizing salidroside and icariside D by biotransformation 2 The method comprises the steps of adding p-hydroxyphenylethanol into a culture solution containing motherwort suspension cells, and converting for a certain period of time to ensure that the motherwort suspension cells contain salidroside and icariside D 2 The culture solution also contains salidroside and icariside D 2 。
Wherein the initial culture solution does not contain salidroside and icariside D 2 However, as the cells die, they lyse and burst, and salidroside and icariside D in the cells 2 Can be released into culture solution, and therefore, the culture solution also contains salidroside and icariside D 2 。
The present invention will be described in detail below by way of examples.
Example 1
1. Culturing motherwort suspension cells: 5g of the purified and vigorous leonurus loose callus was inoculated into a 250mL shake flask containing 100mL of culture medium (MS medium +0.5 mg/L2, 4-dichlorophenoxyacetic acid +2.0 mg/L6-benzylamino adenine). The new solution, old solution =3, was changed every 5 days. In the early stage of culture, large cell clusters are removed each time when the culture medium is changed. Culturing for a period of time, after sufficient small cell clusters exist, filtering the suspended cell liquid through a 50-mesh screen during liquid changing, harvesting filtrate, standing for a moment until cells sink, discarding the upper liquid according to the ratio of the new liquid to the old liquid of 3. Repeatedly sieving with a 50-mesh sieve, and culturing for a period of time to establish a herba Leonuri suspension cell line. After which the suspension was intermittently screened through a 50 mesh screen to maintain the suspension cells in dispersion.
Inoculating 5g/100mL of motherwort suspension cell seed liquid, and culturing at 25 ℃ under the conditions of a rotation speed of 110r/min and illumination of 12 h/day to obtain a motherwort suspension cell transformation system.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 3mmol/L, and harvesting every 12h after adding for 12 times in total.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): respectively taking suspension cell culture fluid of herba Leonuri of this example and blank group, vacuum filtering, storing the filtrate at-80 deg.C, washing cell residue with water for 3 times, adding 10 times of 70% methanol, and thermally refluxing at 80 deg.C for 1 hr. Cooling, adding the weight loss, and filtering with 0.45 μm filter membrane; the culture solution, i.e. filtrate, is directly thawed and then passed through 0.45 μm filter membrane to obtain 13 groups of test sample solutions. Taking salidroside standard, adding 50% methanol to obtain solution containing 0.05mg per 1mL as standard solution. Octadecylsilane chemically bonded silica is used as a filling agent; methanol-water (12; the detection wavelength was 275nm. The number of theoretical plates is not less than 3000 calculated according to salidroside peak. Precisely sucking 10 μ L of each of 13 groups of sample solution and standard solution, injecting into liquid chromatograph, measuring, and calculating conversion rate.
As can be seen from the detection results, the conversion system of the suspension cells of motherwort obtained in the example has the highest conversion rate of 60h, the total conversion rate of salidroside (58.92% in cells + 4.15% in culture solution) is 63.07%, and icariside D 2 The total conversion (6.69% in cells + 1.97% in culture) was 8.66%;12h is the lowest, the total conversion rate of salidroside (intracellular 14.48% + culture solution 0.10%) is 14.58%, icariside D 2 The total conversion (intracellular 3.02% + culture broth 0.12) was 3.14%;144h, the total conversion rate of salidroside (24.91% in cell + 10.35% in culture solution) is 35.26%, and icariside D 2 The total conversion (4.12% in cells + 1.12% in culture) was 5.24%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
Example 2
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell conversion system to enable the initial concentration to be 5mmol/L, respectively harvesting 24h and 48h after the addition, and harvesting for 2 times totally to obtain the final product.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the detection results, the total conversion rate of salidroside (29.78% in cells + 5.71% in culture solution) in the suspension cell transformation system of leonurus japonicus for 24h is 35.49%, and icariside D 2 The total conversion (5.03% + culture medium 1.02% in cells) was 6.05%; the total conversion rate of salidroside (35.39% in cells + 7.25% in culture solution) is 42.64% in 48h, and icariside D 2 The total conversion (5.76% + culture medium 1.97%) was 7.73%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
Example 3
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 7mmol/L, respectively harvesting 1h and 48h after the addition, and harvesting for 2 times in total.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate of salidroside (3.40% in cells + 1.09% in culture solution) in the suspension cell transformation system of motherwort obtained in this example is 4.49% for 1h, and icariside D 2 The total conversion (intracellular 1.08% + culture medium 0.76%) was 1.84%; the total conversion rate of salidroside (26.08% in cells + 9.76%) is 35.84% in 48h, and icariside D 2 The total conversion (intracellular 4.62% + culture medium 3.09%) was 7.71%, while no salidroside and icariside D were detected in suspension cells of motherwort herb obtained from blank group 2 。
Example 4
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 1mmol/L, respectively harvesting 36h and 120h after the addition, and harvesting for 2 times in total to obtain the compound.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate of salidroside (32.50% in cells + 0.56% in culture solution) for 36h in the suspension cell conversion system of leonurus obtained in this example is 33.06%, and icariside D 2 The total conversion (5.67% in cells + 0.15% culture medium) was 5.82%;120h total conversion rate of salidroside (26.50% in cells + 5.37%) is 31.87%, and icariside D 2 The total conversion (intracellular 5.02% + culture solution 1.12) was 6.14%, while salidroside and icariside D were not detected in the suspension cells of motherwort obtained from the blank group 2 。
Example 5
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into a motherwort suspension cell transformation system to enable the initial concentration to be 2mmol/L, adding p-hydroxyphenylethanol for 2 times in total, wherein the time interval between two adjacent times is 240 hours, and harvesting after 300 hours of the first addition.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the tests, the total conversion rate of salidroside (41.6% in cells + 10.5% in culture solution) in the suspension cell transformation system of leonurus japonicus obtained in this example was 52.1%, and icariside D was 2 The total conversion (7.3% + culture medium 1.5%) was 8.8%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
Example 6
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 6mmol/L, adding p-hydroxyphenylethanol for 4 times totally, wherein the time interval between every two adjacent times is 120h, and harvesting the mixture after 420h after the first addition.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate of salidroside (37.2% in cells + 9.2% in culture solution) in the suspension cell transformation system of leonurus japonicus obtained in this example is 46.4%, and icariside D 2 The total conversion (6.2% + culture medium 1.2%) was 7.4%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
Example 7
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into a motherwort suspension cell transformation system to enable the initial concentration to be 8mmol/L, adding p-hydroxyphenylethanol for 3 times totally, wherein the time interval between every two adjacent times is 24 hours, and harvesting 100 hours after the first addition.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 The detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate (35.2% + culture medium 7.3%) of salidroside in the suspension cell transformation system of leonurus japonicus obtained in this example is 42.5%, and icariside D 2 The total conversion (4.5% + culture medium 0.9%) was 5.4%, while the suspension cells of motherwort obtained from the blank group did not haveDetecting salidroside and icariside D 2 。
Example 8
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 10mmol/L, adding p-hydroxyphenylethanol for 2 times in total, wherein the time interval between two adjacent times is 24 hours, and harvesting 80 hours after the first addition.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate of salidroside (31.0% in cells + 3.2% in culture solution) in the suspension cell conversion system of leonurus obtained in this example was 34.2%, and icariside D was 2 The total conversion (intracellular 3.9% + culture medium 0.7%) was 4.6%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
Example 9
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to make the initial concentration of the aqueous solution be 20mmol/L, and harvesting 5h after the addition.
A blank group was also set, and the blank group was different from this example only in that the aqueous solution of p-hydroxyphenylethanol was not added to the blank group.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate of salidroside (3.6% in cells + 0.6% in culture solution) in the suspension cell conversion system of leonurus obtained in this example was 4.2%, and icariside D was obtained 2 The total conversion (1.7% + 0.2% of culture medium) was 1.9%, and no red color was detected in the suspension cells of motherwort obtained from the blank groupSedum glycosides and icariside D 2 。
Example 10
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethanol aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 30mmol/L, and harvesting 1h after the addition.
A blank was also set, and the blank was different from this example only in that no aqueous solution of p-hydroxyphenylethanol was added to the blank.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the tests, the total conversion rate of salidroside (intracellular 3.5% + culture solution 0.0%) in the suspension cell transformation system of leonurus obtained in this example was 3.5%, and icariside D 2 The total conversion (1.3% + culture medium 0.3%) was 1.6%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
Example 11
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a p-hydroxyphenylethylamine aqueous solution into the motherwort suspension cell transformation system to enable the initial concentration to be 0.5mmol/L, adding the p-hydroxyphenylethylamine 8 times in total, wherein the time interval between every two adjacent times is 72 hours, and harvesting 580 hours after the first addition.
A blank set was also set, which differed from this example only in that the blank set did not contain an aqueous solution of p-hydroxyphenylethylamine.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the detection, the total conversion rate of salidroside (9.1% in cells + 1.1% in culture solution) in the suspension cell transformation system of leonurus japonicus obtained in this example was 10.2%, and icariside D 2 The total conversion (1.4% + culture medium 0.7%) was 2.1%, and no salidroside was detected in the suspension cells of motherwort obtained from the blank groupAnd icariside D 2 。
Example 12
1. Culturing motherwort suspension cells: the cultivation method was the same as in example 1.
The transformation method comprises the following steps: adding a mixed aqueous solution of p-hydroxyphenylacetic acid and p-hydroxyphenylacetaldehyde into a motherwort suspension cell transformation system to ensure that the initial concentrations of the p-hydroxyphenylacetic acid and the p-hydroxyphenylacetaldehyde are both 0.1mmol/L, adding for 15 times in total, wherein the time interval between two adjacent times is 48h, and harvesting 720h after the first addition to obtain the compound.
A blank group was also provided, and the blank group was different from this example only in that the blank group was not added with the mixed aqueous solution of p-hydroxyphenylacetic acid and p-hydroxyphenylacetaldehyde.
2. Salidroside and icariside D 2 Detection of (2): the detection method was the same as in example 1.
As can be seen from the results of the tests, the total conversion rate of salidroside (10.9% in cells + 1.5% in culture solution) in the suspension cell transformation system of leonurus japonicus obtained in this example was 12.4%, and icariside D was obtained 2 The total conversion (intracellular 1.7% + culture medium 0.8%) was 2.5%, while salidroside and icariside D were not detected in suspension cells of motherwort obtained from the blank group 2 。
The specific parameters of the above examples are shown in the following table:
as can be seen from the data in the table, salidroside and icariside D are synthesized by biotransformation of the present invention 2 The method has simple and safe operation, and the p-hydroxyphenylethanol is directly added into the motherwort suspension cells, so that the p-hydroxyphenylethanol can be directly absorbed and converted by the motherwort suspension cells, and the loss of the p-hydroxyphenylethanol can be greatly reduced, thereby effectively extracting the p-hydroxyphenylethanolHigh salidroside and icariside D 2 The conversion of (a).
The invention also provides a medicine containing salidroside and icariside D 2 The application of herba Leonuri suspension cell or culture solution containing salidroside and icariside D obtained by the above method 2 The suspension cell or culture solution of herba Leonuri can be used as salidroside or icariside D 2 And a composition containing salidroside and icariside D 2 The herba Leonuri suspension cells containing salidroside and icariside D obtained by the above method 2 The motherwort suspension cells can be used for preparing food, health food, food additives or medicines.
Example 13: extraction of salidroside
The suspension cells of the motherwort obtained in example 1 were used as a raw material for salidroside extraction.
The extraction steps are as follows: taking a motherwort suspension cell culture solution, carrying out suction filtration, removing filtrate, filtering residues, namely motherwort suspension cells, taking 10Kg, adding 4 times of water, decocting for 1h, filtering, concentrating the filtrate to have a relative density of 1.25, cooling, extracting for 2 times by using n-butanol with the same volume, combining n-butanol extract, concentrating under reduced pressure until no alcohol smell exists, diluting by adding 3 times of water, passing through an AB-8 macroporous adsorption resin column at 1BV (bed volume)/h, washing by using 3 times of bed volume of water, discarding, then washing by using 2 times of bed volume of 10% ethanol, discarding, eluting by using 5 times of bed volume of 20% ethanol, collecting eluent, concentrating under reduced pressure until dryness to obtain a crude extract, adding a proper amount of methanol for dissolving, adding 3 times of silica gel according to the weight of the crude extract, uniformly mixing, adding a 20 times of bed volume of methanol-chloroform (2%) solution, eluting, collecting, evaporating under reduced pressure to dryness, crystallizing by using 10 times of absolute ethanol at a temperature below 10 ℃, carrying out centrifugal separation on crystal slurry, washing by using absolute ethanol, drying to obtain rhodioside; the salidroside with the purity of 99.1 percent is obtained, and the yield is 26 percent.
Example 14: preparation of health food containing salidroside
The suspension cells of motherwort obtained in example 1 were used as a raw material for preparing a health drink.
Taking herba leonuri suspension cell culture solution, carrying out suction filtration, removing filtrate, filtering residues, namely herba leonuri suspension cells, taking 10Kg, adding 4 times of water, decocting for 1h, filtering decoction, adding 2% of gelatin into filtrate, mixing uniformly, standing overnight, filtering, adding 5 times of water into filtrate for dilution, adding 12% of white granulated sugar, 0.02% of ascorbic acid, 0.12% of citric acid, 0.15% of caramel color and 0.15% of essence, sterilizing, subpackaging and preparing into the beverage.
The obtained beverage has the effects of relieving sports fatigue, enhancing immunity, improving anoxia tolerance, resisting oxidation, and protecting liver.
Example 15: preparation of food additive containing salidroside
The suspension cells of motherwort obtained in example 1 were used as a raw material for preparing food additives
Taking herba Leonuri suspension cell culture solution, vacuum filtering, removing filtrate, collecting residue to obtain herba Leonuri suspension cells, collecting 10Kg, adding 4 times of water, decocting for 1h, filtering decoction, adding 2% gelatin into filtrate, mixing, standing overnight, filtering, concentrating filtrate into soft extract, vacuum drying at 60 deg.C to obtain dry extract, and pulverizing to obtain food additive.
When making bread, the additive is added into wheat flour by 2%, so that the bread has no adverse effect on the sensory quality of the bread, has special plant faint scent taste, contains salidroside functional factors, and has health promotion function of relieving fatigue.
Example 16: preparation of medicine containing salidroside
The suspension cells of motherwort obtained in example 1 were used as raw materials for preparing medicines.
Taking herba Leonuri suspension cell culture fluid, vacuum filtering, removing filtrate, filtering residue to obtain herba Leonuri suspension cells, taking 10Kg, adding 4 times of water, decocting in water for 1h, concentrating the filtrate into soft extract, vacuum drying at 60 deg.C to obtain dry extract, pulverizing, collecting dry extract powder 300g, adding medicinal starch 50g, and making into tablet by conventional pharmaceutical process.
The obtained tablet can be used for treating and preventing altitude sickness, or treating coronary heart disease, cerebral infarction, senile dementia, etc.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. It will be apparent to those skilled in the art that several modifications or improvements can be made within the technical spirit of the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A bioconversion synthesis method of salidroside and icariside D 2 The method is characterized in that the culture solution containing motherwort suspension cells is added with p-hydroxyphenylethanol, and the culture solution is converted for a certain period of time to ensure that the motherwort suspension cells contain salidroside and icariside D 2 The culture solution also contains salidroside and icariside D 2 ；
The initial concentration of the p-hydroxyphenylethanol is 2-6 mmol/L, the adding times of the p-hydroxyphenylethanol are 1-4 times, the conversion time is 48-420 h, and the culture solution containing the motherwort suspension cells is obtained by culturing motherwort suspension cell seed solution;
the motherwort suspension cell seed solution is obtained by inoculating loose calluses of motherwort into a culture solution and intermittently culturing the calluses through a 50-mesh screen, wherein the culture solution is an MS culture medium containing 0.5mg/L of 2, 4-dichlorophenoxyacetic acid and 2.0mg/L of 6-benzylaminopurine.
2. The bioconversion synthesis of salidroside and icariside D according to claim 1 2 The method is characterized in that the initial concentration of the p-hydroxyphenylethanol is 2-3 mmol/L.
3. The bioconversion synthesis of salidroside and icariside D according to claim 1 2 The method of (1), wherein the conversion time is 60 to 300 hours.
4. The bioconversion synthesis of salidroside and icariside D according to claim 1 2 Method of (1), whichIs characterized in that the adding frequency of the p-hydroxyphenylethanol is 1 to 2.
5. The bioconversion synthesis of salidroside and icariside D according to claim 1 2 The method of (1), wherein when the number of addition of the p-hydroxyphenylethanol is greater than 1, the time interval between two adjacent additions is 120 to 240 hours.
6. The bioconversion synthesis of salidroside and icariside D according to any one of claims 1 to 5 2 The method of (1), wherein the p-hydroxyphenylethanol is obtained by converting any one or a combination of at least two of p-hydroxyphenylethylamine, p-hydroxyphenylacetic acid and p-hydroxyphenylacetaldehyde in the suspension cells of motherwort herb.
7. A medicinal composition containing salidroside and icariside D 2 The use of suspension cells or culture solution of leonurus japonicus, which is characterized in that the suspension cells or culture solution containing salidroside and icariside D obtained by the method of any one of claims 1 to 6 2 The suspension cell or culture solution of herba Leonuri can be used as salidroside or icariside D 2 The extraction raw material of (1).
8. A medicinal composition containing salidroside and icariside D 2 The use of suspension cells of motherwort, which is characterized by comprising salidroside and icariside D obtained by the method of any one of claims 1 to 6 2 The motherwort suspension cells can be used for preparing food, health food, food additives or medicines.
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