CN111978199A

CN111978199A - Purslane amides and uses thereof

Info

Publication number: CN111978199A
Application number: CN202010875796.0A
Authority: CN
Inventors: 滕景斌; 吴峰; 肖永堂
Original assignee: Fuzhou Melalyn Biotechnology Co ltd
Current assignee: Fuzhou Melalyn Biotechnology Co ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-11-24

Abstract

The invention relates to the technical field of cosmetics, in particular to a purslane amide compound and application thereof; the present invention makes it possible to prepare a cosmetic agent or a pharmaceutical agent by preparing a series of compounds having anti-inflammatory, anti-histamine, anti-oxidation and whitening effects, and the cosmetic agent and the pharmaceutical agent in the present invention are not particularly limited. For example, the skin softening lotion can be a washing-off type cosmetic such as a softening lotion, a nourishing lotion, a massage cream, a skin moistening lotion, a gel, a nourishing cream, a facial mask, jelly or shower gel shampoo and the like, and can also be a medicinal preparation product such as a cream, an ointment, a cream, a plaster or a spray and the like; it can be used as sensitive skin, and can be added into infant and children cosmetics for reducing irritation.

Description

Purslane amides and uses thereof

Technical Field

The invention relates to the technical field of cosmetics, in particular to a purslane amide and application thereof.

Background

Herba Portulacae is also called Changchi vegetable and herba Portulacae, and is a plant of Portulacaceae. Is widely distributed in temperate and tropical regions all over the world, and is used as a medicine and food dual-purpose plant in China and other countries.

Modern pharmacological research shows that the purslane has the effects of resisting bacteria, relaxing skeletal muscles, exciting uterus, reducing blood fat, reducing blood sugar, resisting inflammation, easing pain, promoting wound healing, resisting oxidation, resisting aging, protecting nerves and the like. The main chemical components of purslane comprise flavonoids, coumarins, terpenoids, steroids, alkaloids, amino acids, various pigments, minerals and the like. Wherein alkaloids are a major chemical component in purslane.

Inflammation is a vital pathological process in the human body that is itself an autoimmune response as a stimulus to foreign or foreign bodies. When this response is deregulated or over-responsive, it results in self-injury to the body, which becomes inflammatory. In these processes, proinflammatory factors such as TNF- α, IL-6, IL-1 β, etc. play an important role. Mast cells are distributed mainly in connective tissue and submucosa, and basophils are distributed mainly in peripheral blood. The surfaces of the two types of cells both express high-affinity IgEFc receptors, and the IgEFc receptors can be combined with Fc segments of IgE to enable organisms to be in a sensitized state. In the sensitization phase of type I hypersensitivity, when the same allergen reenters the body, the allergen specifically binds to IgEFab segments on the surface of sensitized target cells (namely, mast cells and basophils) to trigger the change of cell membranes of the target cells (namely, the mast cells and the basophils) so as to degranulate the target cells and synthesize new active mediators. These mediators act on the corresponding effector organs, causing pathological changes in the effector organs. The active mediators released after the activation of mast cells and basophils are mainly histamine, kininogenase, leukotriene, prostaglandin D2, platelet activating factor.

The mechanism of action of the antioxidant can be directly acting on free radicals or indirectly consuming substances which are easy to generate free radicals to prevent further reaction. The current research methods for free radical scavengers mainly belong to 2 types, one type is an in vitro model, and the other type is an in vivo model, wherein the DPPH method is the most common method in the in vitro model.

Tyrosinase has a number of characteristic catalytic activities, tyrosinase activity playing a major role in skin melanin deposition. The whitening agent has the function of whitening the skin by inhibiting the activity of tyrosinase or blocking the oxidation pathway of the tyrosine to generate melanin or reducing the expression of the tyrosinase, thereby reducing the generation of the melanin.

Therefore, aiming at the defects of the existing products, the problem to be solved is that a product with anti-inflammatory, anti-histamine, anti-oxidation and whitening effects is needed to be provided.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to synthesize a class of compounds, the compounds have the effects of resisting inflammation, histamine, oxidation and whitening, and can be used for preparing cosmetic reagents.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention proposes

A class of purslane amides has the following structural formula:

wherein R1, R2, R3 and R4 are H, OH or OCH₃；

An exemplary purslane amide has the following structural formula:

the invention also proposes the use of a compound of the above general formula 1 as a preparation of an anti-inflammatory cosmetic agent;

the invention also proposes the use of a compound of formula 1 as defined above as a cosmetic agent for the preparation of antihistamines;

the invention also proposes the use of the compounds of the general formula 1 as agents for preparing cosmetic agents against oxidation;

the invention also provides the application of the compound of the general formula 1 in preparing a whitening cosmetic reagent.

The invention has the beneficial effects that: the present invention provides a method for preparing a series of compounds having anti-inflammatory, anti-histamine, anti-oxidant and whitening functions, so that the obtained compounds can be used for preparing cosmetic or pharmaceutical agents, and the cosmetic and pharmaceutical agents of the present invention are not particularly limited. For example, the skin softening lotion can be a washing-off type cosmetic such as a softening lotion, a nourishing lotion, a massage cream, a skin moistening lotion, a gel, a nourishing cream, a facial mask, jelly or shower gel shampoo and the like, and can also be a medicinal preparation product such as a cream, an ointment, a cream, a plaster or a spray and the like; it can be used as sensitive skin, and can be added into infant and children cosmetics for reducing irritation.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of Portulamide 1 prepared in example 1 of the present invention;

FIG. 2 is a nuclear magnetic carbon spectrum of Portulamide 1 prepared in example 1 of the present invention;

FIG. 3 is a mass spectrum of Portulamide 1 prepared in example 1 of the present invention;

FIG. 4 is a nuclear magnetic hydrogen spectrum of Portulamide 2 prepared in example 2 of the present invention;

FIG. 5 is a nuclear magnetic carbon spectrum of Portulamide 2 prepared in example 2 of the present invention;

FIG. 6 is a mass spectrum of Portulamide 2 prepared in example 2 of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The most key concept of the invention is as follows: synthesizing a class of compounds, so that the compounds have the effects of resisting inflammation, histamine, oxidation and whitening, and can be used for preparing cosmetic reagents.

Referring to fig. 1 to 6, the present invention provides a purslane amide, which has the following structural formula:

wherein R1, R2, R3 and R4 are H, OH or OCH₃；

The structural formula of purslane amide is listed below.

The invention also proposes the use of a compound of the above general formula 1 as a preparation of an anti-inflammatory cosmetic agent; the invention also proposes the use of a compound of formula 1 as defined above as a cosmetic agent for the preparation of antihistamines;

Example 1

1. Synthesis of p-acetoxy phenylpropionic acid

50g of p-hydroxyphenylpropionic acid is added with 150ml of acetonitrile and stirred until the mixture is completely dissolved, 22.5g of sodium hydroxide is dissolved in 150ml of water and added into the reaction solution, 46.5g of acetic anhydride is dripped into the reaction solution, the dripping is completed, the mixture is stirred for 1h at room temperature, the TLC monitors the complete reaction, 6M hydrochloric acid acidification solution with pH value of 1 is added into the reaction solution, 150ml of ethyl acetate is added for extraction and delamination, the organic phase is washed with water until the organic phase is neutral, and the organic phase is concentrated until the organic phase is dry, so 59.2g of white solid product is obtained.

2. Condensation of

59g of p-acetoxyphenylpropionic acid, 39g of tyramine, 7.1g of DMAP, 300ml of tetrahydrofuran, 35.9g of DIC, 120ml of tetrahydrofuran, were added dropwise to the reaction mixture, stirred at room temperature for 1 hour, and the completion of the reaction was monitored by TLC.

3. Hydrolyzing and acidifying

Concentrating to remove tetrahydrofuran, adding 180ml of water and 180ml of absolute ethanol into the residue, adding sodium hydroxide to adjust the pH value to 13, heating to 50 ℃, hydrolyzing for 30min, cooling to room temperature, filtering to remove insoluble substances, concentrating the filtrate to remove ethanol, adding 6M hydrochloric acid into the residual water, acidifying until the pH value is 1, separating out a solid, filtering, washing a filter cake to be neutral by using water, drying at 85 ℃, adding 180ml of ethanol into the dried solid, heating to 70 ℃ to dissolve the dried solid, adding 3g (5%) of activated carbon to decolor for 30min, filtering to remove carbon, adding 180ml of water into the filtrate, cooling to 4-10 ℃, separating out the solid, filtering, and drying at 85 ℃ to obtain 60g of a white solid product.

Nuclear magnetic data for oleracein 1 prepared in example 1 are as follows:

nuclear magnetic hydrogen spectrum (400MHz, DMSO-d6): 9.20(s, 2H), 7.85(d, J ═ 4.0Hz, 1H), 6.97(m, 4H), 6.71(d, J ═ 8.0Hz, 4H), 3.22(t, J ═ 12.0Hz, 2H), 2.73(t, J ═ 12.0Hz, 2H), 2.58(t, J ═ 16.0Hz, 2H), 2.34(t, J ═ 12.0Hz, 2H).

Nuclear magnetic carbon spectrum (100MHz, DMSO-d6) 172.09, 156.07, 155.91, 131.85, 130.03, 129.96, 129.54, 115.58, 115.52, 41.09, 38.04, 34.89 and 30.88.

Example 2

50g tyramine, 73g dihydrocaffeic acid, 4.5g N, N' -dimethylamino pyridine is dissolved by 300ml anhydrous tetrahydrofuran, the temperature is reduced to 0 ℃, 50.6g diisopropyl carbodiimide is dissolved by 200ml anhydrous tetrahydrofuran, the mixture is dripped into the mixed solution at 0 ℃, after the dripping is finished, the temperature is raised to room temperature, the mixture is stirred for 20 hours, insoluble substances are removed by filtration, the tetrahydrofuran is removed by concentration, the residue is dissolved by 300ml1M sodium hydroxide, the insoluble substances are removed by filtration, the filtrate is acidified to pH 1 by 1M hydrochloric acid, white-like solid is separated out, the filtration is carried out, the filter cake is washed by water to be neutral, the solid is dried at 85 ℃, 92g product is obtained, and the yield: 83.8 percent.

The purslane amide 2 nuclear magnetic data obtained in example 2 were as follows:

nuclear magnetic hydrogen spectrum (400MHz, DMSO-d6): 6.94(d, J ═ 8.0Hz, 2H), 6.67(t, J ═ 12.0Hz, 2H), 6.63(s, 2H), 6.50(m, 1H), 3.28(t, J ═ 16.0Hz, 2H), 2.72(t, J ═ 16.0Hz, 2H), 2.60(t, J ═ 16.0Hz, 2H), 2.36(t, J ═ 16.0Hz, 2H).

Nuclear magnetic carbon spectrum (100MHz, DMSO-d6) 176.61, 160.73, 150.12, 148.44, 137.36, 134.74, 134.65, 123.92, 120.83, 120.56, 120.24, 45.75, 42.76, 39.61, 35.80.

Application example 1

The purslane amide 1 provided in the embodiment 1 of the application is applied to anti-allergy cream, and the formula table 1 shows that:

TABLE 1

The process flow comprises the following steps:

1. accurately weighing the phase A, putting the phase A into an emulsifying pot, heating to 85 ℃, and stirring for 1 hour under the condition of heat preservation;

2. accurately weighing phase B, heating to 85 deg.C, adding into phase A, homogenizing and stirring for 1 hr;

3. cooling to 50 deg.C, adding phase C into emulsifying pot, stirring for 10 min, and taking out.

Application example 2

The oleracein 2 provided in example 2 of the present application is applied to anti-allergy essence, and the formula table 2 shows:

TABLE 2

The process flow comprises the following steps:

2. accurately weighing phase B, dissolving completely, adding into phase A, homogenizing and stirring for 1 hr;

Firstly, anti-inflammatory activity test:

the invention discloses a test for inhibiting mouse ear swelling caused by dimethylbenzene by using purslane amide, which comprises the following steps: 50 male Kunming mice weighing 18-22 g were randomly divided into 5 groups of 10, each group consisting of test groups (1, 2, 3) including the preparation of purslane amide 2 in example 2 (dissolved in 10% DMSO to prepare test sample solutions of different concentrations (5mg/mL, 10mg/mL, 20 mg/mL), positive control group (aspirin dissolved in 10% DMSO to prepare test sample solutions of 10 mg/mL), blank group (10% DMSO), each group was administered by gavage at a dose of 20 mL. kg-1 for 1 time a day for 7 days continuously, 30min after the last administration, mice were treated with 20. mu.L of xylene for inflammation in the right ear, left ears were sacrificed after 2h, both ears were cut off, the double ears were cut with a punch of 9mm diameter and weighed, the difference between the weight of the right ear and the weight of the left ear was used as an evaluation criterion of inflammatory swelling degree, and the inhibition rate of ear swelling of each group of mice was calculated according to the following formula.

The inhibition rate of ear swelling of mice is%

TABLE 3 Effect of groups on xylene-induced ear swelling in mice (n, X. + -. s)

TABLE 3

Group of	n	Dosage form	Degree of ear swelling (mg)	Inhibition ratio (%)
					Blank group	10	200mg/kg	17.36±0.27*Δ	-
Positive control group	10	200mg/kg	6.34±1.1*	60.7
					Test set 1	10	200mg/kg	7.85±1.0*Δ	50.3
Test set 2	10	200mg/kg	6.81±0.74**	58.9
					Test group 3	10	200mg/kg	5.35±0.85**Δ	68.4

Note represents P <0.01 compared to the blank control group; represents P <0.01 compared to test group 1; Δ represents P <0.01 compared to test group 1.

The influence of each group on the mouse ear swelling caused by the dimethylbenzene is known from experimental results, and compared with a blank control group, each administration group shows stronger mouse ear swelling inhibition activity, which indicates that the oleracein 2 can significantly inhibit the mouse ear swelling caused by the dimethylbenzene.

Second, anti-Histamine Activity test

RBL-2H3 cells were cultured in a 24-well plate, washed twice with PBS, and added with 0.5ml of fresh DMEM medium containing 10% fetal bovine serum for further culture for 30 min. Each well was filled with 20 μ l of a purslane amide-containing solution (samples were dissolved in ethanol) at different concentrations as test group 1, test group 2, and test group 3, and the same volume of PBS was added as a normal control group, and 5 μ M dexamethasone was additionally added as a positive control. After 30min, C48/8020. mu.l with a final concentration of 10. mu.g/ml was added, and the culture was continued for 1 h. Mu.l of the cell culture supernatant was aspirated and added to a 96-well plate, followed by addition of 50. mu.l of a substrate solution (2mM of p-nitrobenzene-N-acetyl-. beta. -D-glucosamine, 0.2M citric acid, pH4.5) and reaction at 37 ℃ for 2 hours. The reaction was terminated by adding 100. mu.l of 1M Tris buffer (pH9), and the absorbance was measured at a wavelength of 405 nm. Accordingly, the cells were disrupted with 0.1% TritonX-100, centrifuged to remove the precipitate, and the cells were assayed for β -hexosaminidase in the same manner as described above. Protein quantification was performed per well of cells, balancing cell number differences. The content of beta-hexosaminidase in the cell supernatant and the cells was calculated according to the beta-hexosaminidase standard curve, respectively, and the release rate of beta-hexosaminidase was 100% of beta-hexosaminidase/(supernatant beta-hexosaminidase + intracellular beta-hexosaminidase) of the supernatant.

Data processing results are presented as mean ± standard deviation, 3 wells each, with results from 3 replicates.

RBL-2H3 cells were treated with either 0.1%, 0.5% and 1.0% concentration (by volume) of either Portulamide solution or dexamethasone (10. mu.M) to determine the effect on C48/80-induced cell degranulation, histamine and beta-hexosaminidase release, respectively, and the results are shown in Table 4

TABLE 4

Compared with the negative control group, # P <0.05, # P <0.01, # P <0.001, compared with the C48/80 group (model group), it can be seen from the results of table 4 above that the degranulation rate, the histamine release rate, and the β -hexosaminidase release rate were significantly reduced in both the purslane amide medium-dose and high-dose groups, compared with the C48/80 group, while the effects were comparable in the high-dose group and the dexamethasone group.

Third, antioxidant activity test

Determination of superoxide anion scavenging antioxidant capacity: chitosan, sodium hyaluronate and oleracein 1 prepared in example 1 were each tested for superoxide anion scavenging ability and compared (table 5): after the purslane amide 1 prepared in example 1 was freeze-dried in vacuum to a constant weight, solutions with concentrations of 0.2, 0.4, 1.6, and 3.2mg/mL were prepared with each of Tris-HCl buffer solutions (1.9155g of Tris +0.8mL of concentrated HCl, and water was added to a constant volume of 1000 mL). 1.5mL of sample solutions with different concentrations, 1.5mL of Tris-HCl buffer solution and 0.5mL of NADH (468. mu.M) are taken, then 0.5mL of PMS (60. mu.M) is added into the reaction solution, after the mixture is mixed in a test tube, the final concentrations of the samples are 0.1, 0.2, 0.8 and 1.6mg/mL, the mixture is kept still for 5min at room temperature, the absorbance is measured at 560nm, and the blank group of 0.5mL of Tris-HCl buffer solution replaces NADH (note: the samples to be measured are measured three times and averaged).

Superoxide anion scavenging capacity (%) [ (a blank-a sample)/a blank ] × 100

TABLE 5 antioxidant activity (%)

	0.1mg/mL	0.2mg/mL	0.8mg/mL	1.6mg/mL
					Chitosan	11.23	15.41	19.78	24.36
Hyaluronic acid sodium salt	22.14	25.36	27.16	29.45
					Purslane amide 1	47.54	56.54	63.27	71.39

The experimental results are as follows: the purslane amide synthesized by the invention has antioxidant activity, and has stronger antioxidant activity than chitosan and sodium hyaluronate.

Inhibition experiment of tyrosinase Activity

Experimental test group 1, test group 2 and test group 3 were prepared by adjusting the density of B16 melanocytes to 1X 105 melanocytes per mL using a cell culture medium containing 10% fetal calf serum, penicillin 100U/mL and streptomycin 100U/mLDMEM, inoculating 3mL cells per well in a 6-well cell culture plate, culturing at 37 ℃ under 5% CO2 for 24 hours, and then administering 1. mu.M α -MSH to each well, and administering 1mL of the anti-allergy essence of application example 2 (containing 0.5%, 1.0%, 2.0% of Portulamide 2 of example 2) at a concentration of 100. mu.g/mL. The control group was added with the same volume of culture medium as the composition, and the positive control group was given an equal volume of culture medium containing arbutin to a final concentration of 100. mu.g/ml. After 3 days of drug action, the supernatant was discarded and washed 2 times with PBS. Adding 180 mu L of PBS solution containing 1% TritonX-100 into each well, ultrasonically crushing in ice bath, adding 20 mu L of 10 mmol/L-DOPA into each well, incubating at 37 ℃ for 60min, centrifuging at 1500r/min for 5min, respectively taking 100 mu L, transferring to a 96-well cell culture plate, detecting the absorbance value at 492nm by using a microplate reader, and calculating the activity of intracellular tyrosinase.

Tyrosinase activity ═ (A/B). times.100%

A is the average absorbance of the drug group, and B is the average absorbance of the control group

TABLE 6

(n＝4，

) (x, P <0.01 vs control group; # and P <0.05 vs model group; #, P <0.01 vs model group)

As can be seen from the above table, the composition of the present invention can significantly reduce the tyrosinase activity in B16 cells, and the inhibition rate of test group 3 on tyrosinase is the highest.

In summary, the present invention provides a series of compounds having anti-inflammatory, anti-histamine, anti-oxidation and whitening effects, so that the obtained compounds can be used for preparing cosmetic or pharmaceutical agents, and the cosmetic and pharmaceutical agents in the present invention are not particularly limited. For example, the skin softening lotion can be a washing-off type cosmetic such as a softening lotion, a nourishing lotion, a massage cream, a skin moistening lotion, a gel, a nourishing cream, a facial mask, jelly or shower gel shampoo and the like, and can also be a medicinal preparation product such as a cream, an ointment, a cream, a plaster or a spray and the like; it can be used as sensitive skin, and can be added into infant and children cosmetics for reducing irritation.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims

1. Purslane amide, the structural formula of which is as follows:

wherein R1, R2, R3 and R4 are H, OH or OCH₃；

An exemplary purslane amide has the following structural formula:

2. use of the purslane amide of claim 1 as a preparation of an anti-inflammatory cosmetic agent.

3. Use of the purslane amide of claim 1 as a cosmetic agent for the preparation of antihistamines.

4. Use of the purslane amide of claim 1 as a cosmetic agent for the preparation of antioxidant.

5. Use of the purslane amide of claim 1 as a preparation of a whitening cosmetic agent.