Method for extracting tyrosinase inhibitory active component from waste tobacco leaves and application of tyrosinase inhibitory active component

Abstract

The invention belongs to the technical field of extraction and separation of secondary metabolism active components of herbaceous plants, and particularly relates to a preparation method for preparing tyrosinase inhibitory active components by taking waste tobacco leaves as raw materials and application. According to the invention, waste tobacco leaves are taken as a raw material, and five cembranean diterpenoid components are obtained through extraction, extraction, separation and purification, wherein the five cembranean diterpenoid components comprise two known compounds (1S, 2E, 4S, 6E, 8S and 11R)-2, 6, 12 (20)-cembraneriene-4, 8, 11-triol (compound 1) and 6-O-methyl-(1S, 2E, 4S, 6R, 7E and 11E)-2, 7, 11-cembraneriene-4-ol (compound 2); the compound A is a compound A, and the compound B is a compound C. The compound C is a compound 3, the compound E is a compound 4, and the compound V is a compound 5. Five components of the tobacco prepared by the method have a remarkable inhibition effect on tyrosinase, the chemical structure has tyrosinase inhibition activity which is reported for the first time, and the tobacco can be used as a novel tyrosinase inhibitor.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and separation of active components of herbaceous plant secondary metabolism, and specifically relates to a method for extracting tyrosinase inhibitory active components using waste tobacco leaves as raw materials and application thereof. Background Art

[0002] Tyrosinase (EC 1.14.18.1, Tyrosinase, TYR), also known as polyphenol oxidase, aged cheese enzyme, etc., is a complex multi-subunit ketone oxidase widely distributed in nature, and plays a vital role in the formation of melanin in organisms. In the biosynthesis of melanin, tyrosinase is a key enzyme, which acts on dopa to form dopaquinone, which spontaneously undergoes a series of reactions and finally forms melanin. Melanin has a certain protective effect on the human body under appropriate quantity and distribution, but excessive accumulation will endanger human health, such as skin melanin deposition, skin cancer, endocrine disorders, liver disease, uterine disease, etc. Therefore, suitable tyrosinase inhibitors are of great significance in preventing and treating the occurrence of the above diseases. In addition, tyrosinase inhibitors are believed to have a wide range of applications in the fields of cosmetic whitening agents and food preservatives. The separation of natural anti-tyrosinase active substances from plant metabolites has attracted more and more attention, mainly because plant-derived inhibitors have less toxicity and better bioavailability, and are particularly suitable for application in the fields of pharmaceuticals, food and cosmetics.

[0003] Tobacco (Nicotiana tabacum L.) is an annual or limited perennial herbaceous plant of the genus Nicotiana L in the family Solanaceae. my country is the world's largest tobacco producer, mainly planting flue-cured tobacco, which is distributed in more than 20 provinces. Tobacco can be made into cigarettes, dry tobacco, cigars, etc. for human consumption, which brings many harms to humans and is even called "poisonous weeds". However, as a medicinal plant with a long history, its medical value cannot be erased because of its harmfulness. In the process of harvesting, transporting and making cigarettes of high-quality tobacco leaves, about 25% of tobacco leaves and defective products are not used and discarded. These discarded tobacco leaves are often discarded or burned on the spot. After burning, tobacco produces more than 7,000 chemical components, hundreds of which are harmful substances, including at least 79 known carcinogens, including polonium-210, polycyclic aromatic hydrocarbons, N-nitrosoamines, aromatic amines, formaldehyde, etc. These carcinogens will cause gene mutations after being inhaled into the human body, and then lead to the occurrence of malignant tumors. In addition, harmful gases such as nitric oxide, hydrogen sulfide and ammonia produced by combustion can cause serious damage to the respiratory system. Researchers have also found heavy metals and radioactive substances in tobacco smoke, which not only causes waste of agricultural by-products and pollutes the environment, but also causes great harm to the human body. Therefore, strengthening the comprehensive utilization of discarded tobacco leaves, developing high-value-added products, and reducing the waste of agricultural by-products can not only turn waste into treasure, but also drive ecological, economic and social benefits. According to literature records, thousands of years ago, tobacco was regarded as a "panacea" in the Americas and was used to treat various diseases. In the 17th century, medical scientist Ni Zhumo wrote in "Compendium of Materia Medica": "Tobacco is a medicine that can clear the nine orifices, protect against the cold of frost, dew, wind and rain, and ward off the evil spirits of mountain poisons and ghosts. Children who eat it can kill malaria, and women who eat it can eliminate lumps and swellings. All diseases caused by cold stagnation can be cured by smoking it." "National Compendium of Chinese Herbal Medicines" records that tobacco has the effects of reducing swelling, detoxification, and killing insects. It is mainly used for boils, tinea capitis, white ringworm, snake bites, etc. It can also treat neck carbuncle, back carbuncle, wind phlegm, and goose knee. Modern pharmacological studies have shown that tobacco leaf compounds have significant pharmacological activities in anti-inflammatory and bactericidal, anti-hepatitis B virus, and anti-tumor, but so far, there are few reports on the research of tobacco tyrosinase inhibitory active ingredients. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting a novel tyrosinase inhibitory active component which is simple to operate, short in cycle, low in cost, suitable for large-scale production, can reduce environmental pollution caused by agricultural waste and can achieve high-value utilization of waste.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for extracting tyrosinase inhibitory active ingredients from waste tobacco leaves, comprising the steps of:

[0007] (1) After the discarded tobacco leaves are dried and crushed, they are extracted with 95% industrial ethanol at a temperature of 75° C. to 85° C. for 3 times, each time for 3 to 4 hours, the extracts are combined, filtered, and the filtrate is concentrated under reduced pressure to 2% to 5% of the original volume to obtain a concentrate of the tobacco leaf crude extract;

[0008] (2) adding water 5 times the volume of the concentrate, stirring to dissolve, sequentially adding an equal volume of petroleum ether to the concentrate for extraction 3 times to obtain a petroleum ether extraction phase, adding an equal volume of ethyl acetate to the concentrate to the remaining phase of the petroleum ether extraction for extraction 3 times to obtain an ethyl acetate extraction phase, adding an equal volume of n-butanol to the concentrate to the remaining phase of the ethyl acetate extraction for extraction 3 times to obtain an n-butanol extraction phase; concentrating the ethyl acetate extraction phase under reduced pressure to obtain an ethyl acetate extract of tobacco leaves;

[0009] The ethyl acetate extract of tobacco leaves was dissolved in a mixed solvent and then passed through a normal pressure silica gel column, and the mobile phase was gradient eluted with petroleum ether:ethyl acetate in a volume ratio of 20:1 to 1:1, and 6 fractions Z1 to Z6 were obtained by TLC analysis;

[0010] Fraction Z3 was eluted with MCI small pore resin gel using 50%, 70%, and 90% acetone:water as mobile phases to separate and obtain three subfractions Fr.1-Fr.3;

[0011] Fr.2 firstly used Sephadex LH-20CC for gel column chromatography with methanol as eluent to obtain 4 sub-fractions, and then purified the sub-fractions by cyclic preparative high performance liquid chromatography to obtain compounds 1-5;

[0012] Compounds 1-5 were used 1 H-NMR, 13 C-NMR, 2D-NMR, MS, X-ray, IR and UV modern spectral analysis instruments were used to analyze and identify the structures of the compounds, which were: (1S,2E,4S,6E,8S,11R)-2,6,12(20)-cembratriene-4,8,11-triol, 6-O-methyl-(1S,2E,4S,6R,7E,11E)-2,7,1l-cembratriene-4-ol, Cembraneine C, Cembraneine E and Cembraneine V.

[0013] The five chemical components prepared by the present invention are all light in color and high in purity (measured by HPLC, the purity is all ≥95%).

[0014] The above compounds were analyzed and found to contain compounds 1-5, which are: 2 known compounds (1S, 2E, 4S, 6E, 8S, 11R)-2, 6, 12(20)-cembratriene-4, 8, 11-triol and 6-O-methyl-(1S, 2E, 4S, 6R, 7E, 11E)-2, 7, 1l-cembratriene-4-ol; 3 new compounds Cembraneine C, Cembraneine E and Cembraneine V; the chemical structures of the 5 compounds are shown in the attached figure. Figure 1 shown.

[0015] The tyrosinase inhibitory activity of the components prepared from tobacco leaves was evaluated by spectrophotometry. The results showed that the 5 monomeric chemical components prepared from tobacco leaves, namely compounds 1 to 5, had a half-maximum concentration IC of tyrosinase inhibition. 50 The values ​​were 0.225mM, 0.117mM, 0.175mM, 0.203mM, and 0.115mM, respectively, while the half-maximum concentration IC of kojic acid in the positive control group inhibited tyrosinase. 50 The value is 0.279 mM. This fully demonstrates that compounds 1 to 5 prepared from tobacco leaves have significant inhibitory activity against tyrosinase and can be used to prepare tyrosinase inhibitors.

[0016] The beneficial technical effect of the present invention is that the present invention uses waste tobacco leaves as raw materials, and obtains five components through extraction, extraction, separation and purification: two known compounds (1S, 2E, 4S, 6E, 8S, 11R)-2, 6, 12 (20)-cembratriene-4, 8, 11-triol and 6-O-methyl-(1S, 2E, 4S, 6R, 7E, 11E)-2, 7, 1l-cembratriene-4-ol; three new compounds Cembraneine C, Cembraneine E and Cembraneine V. The five components prepared by the present invention have high purity, and the preparation method has the characteristics of simple operation, short cycle, low cost, can reduce environmental pollution caused by waste tobacco leaves and can realize high-value utilization of waste. The five components of tobacco leaves prepared by the present invention have a significant inhibitory effect on tyrosinase, which is the first report that such chemical structure has tyrosinase inhibitory activity, and can be used as a new type of tyrosinase inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 The diagram shows the structures of tyrosinase inhibitory active ingredients 1 to 5 prepared from tobacco leaves.

[0019] Figure 2 The present invention is a flow chart for preparing tyrosinase inhibitory active ingredients 1 to 5 in tobacco leaves.

[0020] Figure 3 The IC values ​​of 1 to 5 of the tyrosinase inhibitory active ingredients prepared from tobacco leaves 50 Value comparison chart. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Drugs and reagents

[0023] Waste tobacco leaves were collected from the dried leaves of tobacco variety Yunyan 87 in Tianma Village, Gulou Office, Pingba District, Anshun City, Guizhou Province (provided by Guizhou Tobacco Science Research Institute, Guiyang City, Guizhou Province). Tyrosinase (batch number J28IS220073) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and L-dopa (batch number 9YRN1RKK), disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Anhui Zesheng Technology Co., Ltd. HPLC-grade acetonitrile, methanol, and water were purchased from Adamas Reagent Co., Ltd., Shanghai, China. All analytical-grade solvents including methanol (MeOH), ethanol (EtOH), acetone (AC), chloroform (TCM), dichloromethane (DCM), ethyl acetate (EtOAc), and petroleum ether (PE) were from Anhui Senruis Energy Chemical Reagent Co., Ltd.

[0024] Example 1

[0025] like Figure 2 As shown, the preparation of tyrosinase inhibitory active ingredients from waste tobacco leaves

[0026] (1) After the discarded tobacco leaves are dried and crushed, they are extracted with 95% industrial ethanol at a temperature of 75° C. to 85° C. for 3 times, each time for 3 to 4 hours, the extracts are combined, filtered, and the filtrate is concentrated under reduced pressure to 2% to 5% of the original volume to obtain a concentrate of the tobacco leaf crude extract;

[0027] (2) adding water 5 times the volume of the concentrate, stirring to dissolve, sequentially adding an equal volume of petroleum ether to the concentrate for extraction 3 times to obtain a petroleum ether extraction phase, adding an equal volume of ethyl acetate to the concentrate to the remaining phase of the petroleum ether extraction for extraction 3 times to obtain an ethyl acetate extraction phase, adding an equal volume of n-butanol to the concentrate to the remaining phase of the ethyl acetate extraction for extraction 3 times to obtain an n-butanol extraction phase; concentrating the ethyl acetate extraction phase under reduced pressure to obtain an ethyl acetate extract of tobacco leaves;

[0028] The ethyl acetate extract of tobacco leaves was dissolved in a mixed solvent and then passed through a normal pressure silica gel column, and the mobile phase was gradient eluted with petroleum ether:ethyl acetate in a volume ratio of 20:1 to 1:1, and 6 fractions Z1 to Z6 were obtained by TLC analysis;

[0029] Fraction Z3 was eluted with MCI small pore resin gel using 50%, 70%, and 90% acetone:water as mobile phases to separate and obtain three subfractions Fr.1-Fr.3;

[0030] Fr.2 firstly used Sephadex LH-20CC for gel column chromatography with methanol as eluent to obtain 4 sub-fractions, and then purified the sub-fractions by cyclic preparative high performance liquid chromatography to obtain compounds 1-5;

[0031] Example 2 Tyrosinase inhibitory activity of tobacco leaf components prepared

[0032] The tyrosinase inhibitory activity of ingredients prepared from tobacco leaves was determined spectrophotometrically.

[0033] 40 μL of samples with concentrations of 2500, 1000, 500, 250, and 125 μg / mL (R) were mixed with 40 μL of mushroom tyrosinase (162.5 U / mL) on a 96-well plate. After incubation at 37°C for 10 minutes, 120 μL of L-DOPA (2.08 mM) was added to the 96-well plate. The mixture was incubated at 37°C for 25 minutes, and the absorbance at 475 nm was measured using a microplate reader (Varioskan LUX, Thermo, Waltham, MA, USA). Sodium phosphate buffer (PBS, 67 mM, pH = 6.8) was used instead of tyrosinase as the sample blank group (R0). The sample replaced with PBS was set as the negative control (C), and the mixture of PBS (80 μL) and L-DOPA (40 μL) was set as the negative blank (C0). Kojicacid, which has a strong inhibitory activity against tyrosinase, was used as the control group of the experiment. The tyrosinase inhibitory activity of five monomer compounds prepared from tobacco leaves was determined three times independently and expressed as IC 50 The tyrosinase inhibitory activity was calculated as follows:

[0034] Inhibition rate (%) = 100 - [(R-R0) / (C-C0)] 100

[0035] The experimental results showed that the half-maximum concentration IC of the five monomer compounds 1 to 5 prepared from tobacco leaves for inhibiting tyrosinase activity was 50 The values ​​were 0.225mM, 0.117mM, 0.175mM, 0.203mM, and 0.115mM, respectively, while the half-maximum concentration IC of kojicacid in the positive control group to inhibit tyrosinase 50 The value is 0.279mM, such as Figure 3 This fully demonstrates that compounds 1 to 5 prepared from tobacco leaves have significant inhibitory activity against tyrosinase and can be used to prepare tyrosinase inhibitors.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for extracting tyrosinase inhibitory active ingredients from waste tobacco leaves, characterized in that the steps include: (1) After the discarded tobacco leaves are dried and crushed, they are extracted with 95% industrial ethanol at a temperature of 75° C. to 85° C. for 3 times, each time for 3 to 4 hours, the extracts are combined, filtered, and the filtrate is concentrated under reduced pressure to 2% to 5% of the original volume to obtain a concentrate of the tobacco leaf crude extract; (2) adding water 5 times the volume of the concentrate, stirring to dissolve, sequentially adding an equal volume of petroleum ether to the concentrate for extraction 3 times to obtain a petroleum ether extraction phase, adding an equal volume of ethyl acetate to the concentrate to the remaining phase of the petroleum ether extraction for extraction 3 times to obtain an ethyl acetate extraction phase, adding an equal volume of n-butanol to the concentrate to the remaining phase of the ethyl acetate extraction for extraction 3 times to obtain an n-butanol extraction phase; concentrating the ethyl acetate extraction phase under reduced pressure to obtain an ethyl acetate extract of tobacco leaves; The ethyl acetate extract of tobacco leaves was dissolved in a mixed solvent and then passed through a normal pressure silica gel column, and the mobile phase was gradient eluted with petroleum ether:ethyl acetate in a volume ratio of 20:1 to 1:1, and 6 fractions Z1 to Z6 were obtained by TLC analysis; Fraction Z3 was eluted with MCI small pore resin gel using 50%, 70%, and 90% acetone:water as mobile phases to separate and obtain three subfractions Fr.1-Fr.3; Fr.2 firstly used Sephadex LH-20CC for gel column chromatography with methanol as eluent to obtain 4 sub-fractions, and then purified the sub-fractions by cyclic preparative high performance liquid chromatography to obtain compounds 1-5; Compounds 1-5 were used 1 H-NMR, 13 C-NMR, 2D-NMR, MS, X-ray, IR and UV modern spectral analysis instruments were used to analyze and identify the structures of the compounds, which were: (1S,2E,4S,6E,8S,11R)-2,6,12(20)-cembratriene-4,8,11-triol, 6-O-methyl-(1S,2E,4S,6R,7E,11E)-2,7,1l-cembratriene-4-ol, Cembraneine C, Cembraneine E and Cembraneine V.

2. Use of the compound prepared by the method of claim 1 in the preparation of tyrosinase inhibitors.

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