CN111135159B

CN111135159B - Application of diterpene compound in preparation of tyrosinase inhibitor

Info

Publication number: CN111135159B
Application number: CN201911377046.4A
Authority: CN
Inventors: 徐巧林; 谭建文; 曾雷; 陈颖乐; 王颂
Original assignee: South China Agricultural University; Guangdong Academy of Forestry
Current assignee: South China Agricultural University; Guangdong Academy of Forestry
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2021-01-12
Anticipated expiration: 2039-12-27
Also published as: CN111135159A

Abstract

The invention discloses application of diterpene compounds in preparation of tyrosinase inhibitors. The chemical structure of the diterpene compound is 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid, which can be separated from plant materials such as south American wedeloa and the like, and the preparation process is easy to operate. The diterpene compound has obvious tyrosinase activity inhibition, high safety and rapid natural degradation in the environment. The diterpene compound has stable monomer, easy storage, obvious activity of inhibiting tyrosinase equivalent to that of kojic acid, no phenolic hydroxyl group, safer potential use performance, and better prospect, and can be further developed into tyrosinase inhibitor medicines or whitening cosmetics for effectively and safely preventing and treating melanin deposition.

Description

Application of diterpene compound in preparation of tyrosinase inhibitor

Technical Field

The invention particularly relates to application of a diterpene compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid or a medicinal salt thereof in preparing a tyrosinase inhibitor.

Background

Melanin is a major factor responsible for human skin, eye and hair pigmentation and is produced by epidermal melanocytes. Under normal physiological conditions, melanin pigmentation has a beneficial effect on photoprotection of human skin, can protect against harmful ultraviolet radiation damage, and has important evolutionary effects in camouflage and animal simulation. However, excessive secretion of melanin causes the appearance of skin diseases such as freckles, solar black spots (senile plaques), black spots of children, and inflammatory melanosis. Melanogenesis is a complex process involving a combination of enzymatic and chemical catalytic reactions, whereas tyrosinase is a key enzyme in the melanin biosynthesis pathway of melanocytes. The tyrosinase inhibitor can prevent or reduce the synthesis and deposition of melanin by inhibiting the activity of tyrosinase, can be used for treating skin diseases such as melasma caused by excessive melanin secretion and the like, and can realize the skin whitening function by inhibiting the tyrosinase to reduce the synthesis of the melanin, so that the effective tyrosinase inhibitor has important value application in the fields of medicines and cosmetics, and has wide application in the field of whitening cosmetics. It is statistical that about 15% of people worldwide buy whitening products, of which asia dominates. At present, most of whitening cosmetics or whitening agents in the market adopt tyrosinase inhibitors, such as Hydroquinone (HQ), arbutin, kojic acid and other tyrosinase inhibitors are used as whitening agents, but the tyrosinase inhibitors actually applied have certain defects respectively. For example, hydroquinone can mutate mammalian cells and also has adverse reactions including contact dermatitis, allergies, transient erythema, burning, tingling, vitiligo, petechiae nails, hypopigmentation and brown yellow; arbutin is not very stable chemically, can release hydroquinone to be decomposed into benzene-containing metabolites, and has potential toxicity to bone marrow; kojic acid, however, has carcinogenicity and instability during storage, and thus its use in cosmetics is limited. Therefore, there is an urgent need to develop novel tyrosinase inhibitors having pharmaceutical properties.

The diterpene compound 3 alpha-cinamoyloxy-ent-kaur-16-en-19-oic acid was isolated from other plants (Qiang et al, Helv. Chim. acta,2011,94, 817-one 823), which is also present in Wedelia trilobata plants. As a natural compound without a phenolic hydroxyl structure, no report that the compound has tyrosinase inhibitor activity is found at present. Our preliminary experimental analysis and review literature reports do not show that the compound has obvious cytotoxicity so far, and thus the compound has potential to be comprehensively good in use safety. The chemical structural formula of the diterpene compound is shown as the following formula (I),

disclosure of Invention

The invention aims to provide application of a diterpenoid compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid or a pharmaceutically acceptable salt thereof in preparing tyrosinase inhibitors.

In-vitro pharmacological experiments prove that the diterpenoid compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid provided by the invention has the obvious tyrosinase inhibiting activity, the tyrosinase inhibiting activity is equivalent to that of a positive control product kojic acid, and the diterpenoid compound has the potential to be developed and prepared into potential drug candidate molecules for preventing and/or treating physiological changes or diseases caused or related to tyrosinase. Among them, the physiological changes or diseases caused or associated with tyrosinase include, but are not limited to, skin diseases such as melasma caused by excessive melanin secretion, and also include the application in the field of beauty and whitening cosmetics.

The pharmaceutical salt of the diterpene compound can be converted into the corresponding diterpene compound molecule shown in the formula (I) under the using environment or physiological conditions, and the active ingredient for substantially inhibiting tyrosinase is the same as the compound molecule shown above, so that the diterpene compound belongs to the protection scope of the invention.

The diterpene compound molecule or the pharmaceutically acceptable salt thereof can be combined with common auxiliary materials or carriers in pharmacy or cosmetic production to prepare the medicine or the medicine composition or the beauty and whitening cosmetics which have the tyrosinase inhibitory activity of the compound molecule and can be used for preventing and treating diseases related to pigment deposition, such as black spot, melanoma and the like. The medicine or the pharmaceutical composition can be applied to external application of gel or paste by wiping, or can be prepared into dosage forms such as wettable powder, tablets, granules, capsules, oral liquid, dropping pills and the like, and can also be prepared into controlled release or sustained release dosage forms or nano preparations known in the pharmaceutical industry.

The application of the extract of plants such as wedelia trilobata and the like taking the diterpene compound molecules as effective components in preparing tyrosinase inhibitor medicines belongs to the protection scope of the invention because the diterpene compound molecules are substantial effective components.

The invention also provides a preparation method of the diterpenoid compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid, which is extracted and separated from a Wedelia trilobata plant.

Specifically, the preparation method comprises the following steps: pulverizing dried whole herb of Wedelia trilobata, soaking and extracting with 95% v/v ethanol at room temperature, mixing extractive solutions, and concentrating under reduced pressure to obtain alcohol-free extract; suspending the extract with appropriate amount of water, and extracting with petroleum ether for 4 times; concentrating under reduced pressure to obtain petroleum ether part; subjecting the petroleum ether extract to normal phase silica gel column chromatography, gradient eluting with petroleum ether-acetone at volume ratio of 100:0-0:100, detecting by TLC thin layer chromatography, and mixing fractions with the same main point to obtain P₁～P₁₂A total of 12 components; eluting the component P with petroleum ether-acetone at a volume ratio of 10:1₅Performing normal phase silica gel column chromatography, performing gradient elution by petroleum ether-ethyl acetate according to the volume ratio of 40: 1-1: 1, detecting by TLC, and combining fractions with the same main point to obtain P_5-1～P_5-5A total of 5 subcomponents; eluting with petroleum ether-ethyl acetate at a volume ratio of 5:2 to obtain subfraction P_5-3Separating and purifying by reversed-phase medium-pressure column chromatography, gradient eluting with methanol-water according to the volume ratio of 60: 40-100: 0, and then performing SAnd (3) performing ephadex LH-20 gel column chromatography, and eluting with chloroform/methanol at a constant gradient of a volume ratio of 1:4 to obtain the diterpene compound 3 alpha-cinamoyloxy-ent-kaur-16-en-19-oic acid.

The diterpene compound 3 alpha-cinnamyloxy-ent-kaur-16-en-19-oic acid with obvious tyrosinase activity inhibition is a natural non-phenol compound which has high safety and can be naturally degraded in the environment without residue, can be separated from plant materials such as south American wedelia and the like, and has easy operation in the preparation process. The diterpenoid compound has stable monomer and easy storage, the tyrosinase inhibitory activity of the diterpenoid compound is equivalent to that of the tyrosinase inhibitor kojic acid used in the market, and the compound is a non-phenolic hydroxyl compound, has safer potential use performance, is very likely to be further developed into a tyrosinase inhibitor medicine or a whitening cosmetic related to effective and safe prevention and/or treatment of melanin deposition, and has good prospect.

Drawings

FIG. 1 is a drawing showing that compound 3 α -Cinnamoyloxy-ent-kaur-16-en-19-oic acid (1)¹H NMR spectrum.

FIG. 2 is a diagram of the compound 3 α -Cinnamoyloxy-ent-kaur-16-en-19-oic acid (1)¹³C NMR spectrum.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications made to the invention in light of the spirit thereof are intended to be included within the scope of the present invention as claimed.

Example 1: preparation of diterpene compound 1 in south American wedelia chinensis plant

1.1 plant origin and identification

Whole plant of south American Wedelia chinensis (Wedelia trilobata (L.) Hitchc) is collected from south China plant park scientific research area of Chinese academy of sciences in 2011 at 9 months, and the material is identified by south China plant park Schenfuwu researchers.

1.2 extraction and separation

Pulverizing dried Wedelia trilobata whole plant (8kg), soaking in 95% v/v ethanol at room temperature, mixing extractive solutions, and concentrating under reduced pressure to obtain alcohol-free extract. Suspending the extract with appropriate amount of waterExtracting with petroleum ether for 4 times; after concentration under reduced pressure, a petroleum ether fraction (180g) was obtained. Subjecting the obtained petroleum ether extract (180g) to normal phase silica gel column chromatography (200-300 mesh, 1000 × 105mm i.d.), gradient eluting with petroleum ether-acetone (100:0-0:100, v/v, each 2.5L), detecting by TLC thin layer chromatography, and mixing fractions with the same main point to obtain P₁～P₁₂The total number of the components is 12. Fraction P eluted with Petroleum Ether-acetone (10:1, v/v)₅(18.8g), performing normal phase silica gel column chromatography (200-300 mesh, 800 × 45mm i.d.), gradient eluting with petroleum ether-ethyl acetate (40: 1-1: 1, v/v), and detecting by TLC to obtain P_5-1～P_5-5A total of 5 subcomponents. Eluting with petroleum ether-ethyl acetate at a volume ratio of 5:2 to obtain subfraction P_5-3(3.9g) was separated and purified by reverse phase medium pressure column chromatography, eluted with methanol-water (60: 40-100: 0, v/v, each1.0l) gradient, then chromatographed on Sephadex LH-20 gel column (1550 × 13.4mm i.d.) with chloroform/methanol (1:4, v/v) constant gradient to give compound 1(18.9 mg).

1.3 structural characterization of Compound 1

The obtained compound 1 is white powder with molecular formula C₂₉H₃₆O₄，ESI-MS m/z:487[M+K]⁺,471[M+Na]⁺,449[M+H]⁺,895[2M-H]^-,447[M-H]^-；¹H NMR(600MHz,CDCl₃):δ_H 7.70(1H,d,J＝16.0Hz,H-3'),7.52(2H,m,H-5',H-9'),7.37(3H,m,H-6',H-7',H-8'),6.48(1H,d,J＝16.0Hz,H-2'),4.82(1H,s,H-17a),4.76(1H,s,H-17b),4.71(1H,dd,J＝12.2,4.7Hz,H-3),1.34(3H,s,Me-18),1.08(3H,s,Me-20)；¹³C NMR(150MHz,CDCl₃):δ_C180.9(C-19),166.9(C-1'),155.5(C-16),145.2(C-3'),134.6(C-4'),130.4(C-7'),129.0(C-6', C-8'),128.3(C-5', C-9'),118.5(C-2'),103.5(C-17),79.0(C-3),56.6(C-5),55.3(C-9),48.9(C-15),48.2(C-4),44.1(C-8),43.9(C-13),41.1(C-14),39.7(C-7),39.6(C-10),38.9(C-1),33.2(C-12),24.3(C-2),24.0(C-18),21.7(C-6),18.7(C-2),15.5 (C-20). According to the comprehensive analysis of the spectrum data, the chemical structure of the compound 1 is analyzed to be 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid, and the structural formula is shown in the formula (I).

Example 2: tyrosinase inhibitory Activity assay for diterpene Compound 1

2.1 instruments and reagents

An experimental instrument: the microplate reader Genois microplate reader (Tecan GENios, Swizerland).

Reagent sample preparation: tyrosinase (sigma), levodopa (L-DOPA), Kojic acid (al addin), Na₂HPO₄，NaH₂PO₄(ii) a Diterpene compound 1 was prepared by the method of experimental example 1 above. Another 4 diterpene compounds used for comparative analysis, namely 2 beta, 16 alpha-Dihydroxy-ent-kauran-19-oic acid (2), ent-16 beta, 17-Dihydroxy-9(11) -kauren-19-oic acid (3), Pterokaurene L₃(4) And 3 alpha-Cinnamoyloxy pterokaurene L₃(5) The preparation of (A) was carried out using the literature methods which we have published [ Ren et al, Phytochemistry Letters,2015,11: 260-.

2.2 test methods

1. Preparing a medicine solution: preparing a compound to be detected and kojic acid into 10mg/mL solutions by dimethyl sulfoxide (DMSO) respectively; 67mmoL of phosphate buffer (prepared with ultra pure water); 46U/mL tyrosinase solution (prepared with phosphate buffer); 2.5mM L-DOPA (in phosphate buffer).

2. And (3) determining the tyrosinase median inhibitory concentration of the compound to be detected by a colorimetric method through a 96-hole cell culture plate. Firstly, diluting a sample solution to be detected by phosphate buffer according to a certain proportion (the amount of DMSO is less than 5% v/v), and adding 120 mu L of the sample solution to be detected into each hole to ensure that the final concentration of a sample to be detected is as follows: 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, 1.56. mu.g/mL, 0.78. mu.g/mL, 40. mu.L of tyrosinase solution (46U/mL) was added to the sample wells, the reaction was carried out at 23 ℃ for 10min, and finally 40. mu.L of substrate L-DOPA (2.5mM) was added. After reaction at 23 ℃ for 10min, the reaction mixture was finally measured at a wavelength of 475nm using a microplate reader. The positive control is kojic acid, and the negative control is a solution containing kojic acid and a sample dissolving solution in the same ratioDMSO phosphate buffer, all blanks were the same volume of phosphate buffer instead of enzyme solution, and each set of experiments was repeated three times. The formula for calculating the tyrosinase inhibition rate of the test compound is as follows: inhibition ratio (%) ═ (OD)_{negative control}–OD_{negative blank})–(OD_test–OD_{test blank})/(OD_{negative control}–OD_{negative blank}) X 100%. Wherein the half Inhibitory Concentration (IC) of the test compound on tyrosinase₅₀) From dose-response curves.

2.3 results of the experiment

In the experiment, the diterpenoid compound 1 and four diterpenoid compounds with similar structures, namely 2 beta, 16 alpha-Dihydroxy-ent-kauran-19-oic acid (2), ent-16 beta, 17-Dihydroxy-9(11) -kauren-19-oic acid (3) and Pterokaurene L₃(4) And 3 alpha-Cinnamoyloxy pterokaurene L₃(5) And positive control compound kojic acid, the tyrosinase inhibitory activity of which was compared, and the experimental results (as shown in table 1) revealed that only compound 1 of the five diterpene compounds exhibited significant inhibitory activity against tyrosinase and inhibitory activity (IC) against tyrosinase₅₀12.99 μ M) and a positive control, kojic acid (IC)₅₀12.55 μ M), showing that this compound 1 is a compound of this class of diterpene compounds that is specific in inhibiting tyrosinase.

TABLE 1 tyrosinase inhibitory Activity of diterpene Compound 1 and Compounds of similar Structure 2-5

2.4 conclusion of the experiment

The experimental result shows that the diterpenoid compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid provided by the invention has the activity of obviously inhibiting tyrosinase equivalent to that of kojic acid, is a non-phenolic hydroxyl compound, has safer potential use performance, is very likely to be further developed into a tyrosinase inhibitor medicine or whitening cosmetic related to effective and safe prevention and treatment of melanin deposition, and has potentially important application value.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. The application of a diterpene compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid or a medicinal salt thereof in preparing tyrosinase inhibitors is disclosed, wherein the structural formula of the diterpene compound is shown as the formula (I):

2. the use according to claim 1, wherein the diterpene compound 3 α -cinamoyloxy-ent-kaur-16-en-19-oic acid is isolated from a plant belonging to Wedelia trilobata or a tissue site thereof by extraction.

3. The use according to claim 2, wherein the diterpene compound 3 α -cinamoyloxy-ent-kaur-16-en-19-oic acid is prepared by the steps of: pulverizing dried whole herb of Wedelia trilobata, soaking and extracting with 95% v/v ethanol at room temperature, mixing extractive solutions, and concentrating under reduced pressure to obtain alcohol-free extract; suspending the extract with appropriate amount of water, and extracting with petroleum ether for 4 times; concentrating under reduced pressure to obtain petroleum ether part; subjecting the petroleum ether extract to normal phase silica gel column chromatography, gradient eluting with petroleum ether-acetone at volume ratio of 100:0-0:100, detecting by TLC thin layer chromatography, and mixing fractions with the same main point to obtain P₁～P₁₂A total of 12 components; eluting the component P with petroleum ether-acetone at a volume ratio of 10:1₅Performing normal phase silica gel column chromatography, performing gradient elution by petroleum ether-ethyl acetate according to the volume ratio of 40: 1-1: 1, detecting by TLC, and combining fractions with the same main point to obtain the final productTo P_5-1～P_5-5A total of 5 subcomponents; eluting with petroleum ether-ethyl acetate at a volume ratio of 5:2 to obtain subfraction P_5-3Separating and purifying by reverse phase medium pressure column chromatography, performing gradient elution by using methanol-water according to the volume ratio of 60: 40-100: 0, performing Sephadex LH-20 gel column chromatography, and performing constant gradient elution by using chloroform/methanol according to the volume ratio of 1:4 to obtain the diterpenoid compound 3 alpha-cinnamyl-ent-kaur-16-en-19-oic acid.