Lycium barbarum root polyphenol derivative composition, preparation method and application thereof

Abstract

The present invention relates to a wolfberry root extract, a wolfberry root polyphenol derivative and a preparation method thereof having anti-inflammatory and antipruritic effects. The present invention also relates to the application of a composition of the wolfberry root extract and the wolfberry root polyphenol derivative in anti-inflammatory and antipruritic effects. The wolfberry root extract disclosed in the present invention is mainly composed of polyphenol derivatives, and the wolfberry root extract has excellent anti-inflammatory and antipruritic effects. The polyphenol derivatives in the wolfberry root extract show excellent synergistic effects and have important application value in anti-inflammatory and antipruritic effects. The wolfberry root extract disclosed in the present invention and its preparation can be widely used as raw materials for cosmetics or medicines with anti-inflammatory and antipruritic effects.

Description

Technical Field

[0001] The invention relates to a wolfberry root extract, a wolfberry root polyphenol derivative composition, a preparation method thereof and applications of the same in anti-inflammatory and antipruritic cosmetics or medicines. Background Art

[0002] Atopic dermatitis (AD), also known as atopic eczema, is a chronic, recurrent, inflammatory skin disease characterized by repeated attacks and prolonged course, and patients often experience severe itching. At different ages, patients often also have other atopic diseases such as allergic rhinitis and asthma, so it is considered a systemic disease that requires long-term management as a chronic disease. The increase in the prevalence of AD in my country is later than that in Western developed countries, Japan, and South Korea, but it has grown rapidly in the past 10 years. In 2014, using the diagnostic criteria of clinicians, the prevalence of AD in children aged 1-7 years in 12 cities in my country reached 12.94%, and the prevalence of AD in infants aged 1-12 months reached 30.48%.

[0003] The treatment goal of atopic dermatitis is to relieve or eliminate clinical symptoms, eliminate inducing and / or aggravating factors, reduce and prevent recurrence, and improve the quality of life of patients. The clinical treatment of AD is divided into basic treatment, topical drug treatment, symptomatic treatment of pruritus, allergen-specific immunotherapy, and traditional Chinese medicine treatment. Among them, topical drug treatment mainly mentions the use of topical glucocorticoids and calcineurin inhibitors. Although they can temporarily relieve related symptoms, they cannot change the reality that the majority of patients live with the disease. At the same time, there are also a series of limitations and side effects such as easy recurrence of symptoms, large individual differences in effects, and unstable long-term efficacy.

[0004] Traditional Chinese medicine has great advantages and a long history in the treatment of AD. It has unparalleled superiority over Western medicine in relieving AD itching and skin lesions, reducing relapses, prolonging the remission period, and improving the quality of life of patients. Clinical reports show that some Chinese herbal extracts, such as compound glycyrrhizic acid tablets, tripterygium wilfordii tablets, and tripterygium wilfordii polyglycosides, can be used to control symptoms during acute attacks.

[0005] Cortex Lycii is obtained from the dried root bark of Lycium chinense Mill. It is a traditional Chinese medicinal material with a long history of application in my country. Cortex Lycii is rich in chemical components such as alkaloids, organic acids and their esters, glycosides, sterols, anthraquinones, coumarins, flavonoids, lycium barbarum, amines, etc., and has the effects of beauty and skin care, immunomodulation, anti-inflammatory and soothing. Among them, the immunomodulatory and anti-inflammatory and soothing effects are particularly prominent, but the anti-inflammatory, soothing and antipruritic activities of the active substances contained in the root of Lycium barbarum are rarely studied and reported.

[0006] Chinese patent 201911229462.X discloses a method for extracting polyphenols from Lycium barbarum and a Lycium barbarum polyphenol extract, the method comprising: drying the Lycium barbarum to a moisture content of no more than 0.6%, crushing it through a 60-mesh sieve to obtain Lycium barbarum powder, and sealing it in a dark place; mixing ammonium sulfate, ethanol solution and Lycium barbarum powder to form a mixed solution system, and after ultrasonic extraction, filtering the extract, standing and stratifying, taking the upper phase to obtain a Lycium barbarum polyphenol extract. However, the patent neither determines the structure of Lycium barbarum polyphenols nor the use of such polyphenols in cosmetics and medicines.

[0007] Therefore, it is necessary to separate and extract the anti-inflammatory and antipruritic active ingredients of wolfberry root based on the activity tracking method, to obtain wolfberry root polyphenol derivatives and their compositions with better anti-inflammatory and antipruritic activities, so as to develop anti-itching, anti-inflammatory and soothing cosmetics or drugs with important application value. Summary of the invention

[0008] One of the objects of the present invention is to provide a wolfberry root extract, a wolfberry root polyphenol derivative and a preparation method thereof prepared by an activity (analysis) tracking method; another object of the present invention is to provide the use of a wolfberry root extract and a wolfberry root polyphenol derivative composition in the preparation of anti-inflammatory and antipruritic cosmetics or medicines.

[0009] On the one hand, in order to achieve the above-mentioned purpose of the invention, the present invention provides one of the wolfberry root polyphenol derivatives shown in Formula I to Formula V in the following structural formula prepared from wolfberry root by an activity tracking method:

[0010]

[0011]

[0012] On the other hand, in order to achieve the above-mentioned purpose of the invention, the present invention also provides a method for preparing a wolfberry root extract and the wolfberry root polyphenol derivatives, wherein the wolfberry root polyphenol derivatives are prepared on the basis of the wolfberry root extract. In the present invention, the wolfberry root extract is prepared by a method comprising the following steps:

[0013] (1) Crush fresh wolfberry roots;

[0014] (2) The crushed wolfberry root was extracted by diafiltration (leakage) with ethanol-water;

[0015] (3) combining the extracts obtained in the reduced pressure concentration step (2) to obtain an extract;

[0016] (4) dissolving the extract obtained in step (3) in ethanol, and mixing and stirring with silica gel to obtain a mixture;

[0017] (5) rotary evaporating the mixture obtained in step (4) at a temperature of 35-45° C. in a rotary evaporator to remove ethanol and obtain an extract-silica gel adsorption mixture;

[0018] (6) separating the extract-silica gel adsorption mixture obtained in step (5) by silica gel column chromatography, and performing gradient elution with a petroleum ether-ethyl acetate mixed solvent to obtain several fractions;

[0019] (7) constructing a 2,4-dinitrochlorobenzene (DNCB)-induced normal human dermal fibroblast (NHDF) injury model, and evaluating the activity of each fraction obtained in step (6);

[0020] (8) separating the fraction with the best protective activity in step (7) by using a gel column and performing gradient elution with chloroform-methanol to obtain several fractions;

[0021] (9) evaluating the protective activity of each fraction obtained in step (8) based on the DNCB-induced NHDF cell injury model;

[0022] Wherein, the fraction with the best protective activity in step (9) is the wolfberry root extract of the present invention.

[0023] Furthermore, the present invention prepares the above-mentioned wolfberry root polyphenol derivatives based on the preparation of the above-mentioned wolfberry root extract, and the method comprises:

[0024] A. providing the wolfberry root extract;

[0025] B. Separate and purify the wolfberry root extract in step A by preparative HPLC to obtain a wolfberry root polyphenol derivative-1 shown in formula I, a wolfberry root polyphenol derivative-2 shown in formula II, a wolfberry root polyphenol derivative-3 shown in formula III, a wolfberry root polyphenol derivative-4 shown in formula IV, and a wolfberry root polyphenol derivative-5 shown in formula V.

[0026] In the present invention, the wolfberry root polyphenol derivatives and wolfberry root extracts are extracted and separated based on a normal human skin fibroblast (NHDF) injury model induced by 2,4-dinitrochlorobenzene (DNCB) by adopting an activity evaluation tracking method.

[0027] In the present invention, the activity mentioned generally refers to the ability to enhance the anti-inflammatory and anti-oxidative activities of skin fibroblasts.

[0028] More specifically, the wolfberry root extract of the present invention is an active component of wolfberry root separated based on the DNCB-induced NHDF cell inflammation model and activity tracking method, and its preparation process includes:

[0029] Fresh wolfberry roots are crushed, extracted by ethanol-water immersion, and the extract is concentrated to obtain an extract;

[0030] Based on the DNCB-induced NHDF cell inflammation model, the protective activity of different concentrations of ethanol-lycium barbarum root extract was evaluated;

[0031] The wolfberry root extract with the best protective activity is dissolved in ethanol and mixed with silica gel; the mixture is rotary evaporated in a rotary evaporator at a temperature of 35-45° C. to remove the ethanol and obtain an extract-silica gel adsorption mixture;

[0032] The extract-silica gel adsorption mixture was separated by silica gel column chromatography and eluted with a mixed solvent of petroleum ether-ethyl acetate to obtain Fr.1-15;

[0033] The protective activity of Fr.1-15 was evaluated based on the DNCB-induced NHDF cell inflammation model;

[0034] Fr.11 with the best protective activity was separated by gel column and gradient eluted with chloroform-methanol mixed solvent to obtain Fr.11-1-Fr.11-15;

[0035] The protective activity of Fr.11-1-Fr.11-15 was evaluated based on the DNCB-induced NHDF cell inflammation model;

[0036] Among them, the fraction Fr.11-5 component with the best protective activity is determined to be the wolfberry root extract of the present invention.

[0037] Furthermore, the compound with the highest content in Fr.11-5 with the best protective activity was separated and purified by preparative HPLC (methanol-water) to obtain compounds identified as wolfberry root polyphenol derivative-1 shown in formula I, wolfberry root polyphenol derivative-2 shown in formula II, wolfberry root polyphenol derivative-3 shown in formula III, wolfberry root polyphenol derivative-4 shown in formula IV, and wolfberry root polyphenol derivative-5 shown in formula V.

[0038] In the above step (2), the ethanol-water used for the leakage extraction of wolfberry root can be ethanol-water (ethanol: water-V:V) containing 15%-75% ethanol, preferably ethanol-water (ethanol: water-V:V) containing 20%-60% ethanol, and more preferably ethanol-water (ethanol: water-V:V) containing 25%-35% ethanol. For example, the ethanol-water used for the leakage extraction of wolfberry root is ethanol-water (ethanol: water-V:V) containing 30% ethanol.

[0039] In the above step (4), the ethanol used to dissolve the extract is preferably pure ethanol, and the silica gel used to mix with the extract ethanol solution is preferably 80-100 mesh silica gel.

[0040] In the above step (5), the temperature of the rotary evaporation is preferably 38-42°C, such as 40°C.

[0041] In the above step (6), the extract-silica gel adsorption mixture is separated by silica gel column chromatography, and the silica gel can be 200-300 mesh or 300-400 mesh, preferably 200-300 mesh silica gel.

[0042] In the above step (8), the fraction with the best protective activity (Fr.11) is separated by gel column chromatography, and eluted with a gradient of chloroform-methanol (100:10→10:100), preferably with a gradient elution of chloroform-methanol (100:50→50:100).

[0043] In the above step B, the fraction with the best protective activity (Fr.11-5) was separated and purified by preparative HPLC (methanol-water) to obtain compounds represented by Formula I to Formula V.

[0044] In the present invention, wolfberry root extract and wolfberry root polyphenol derivatives can be separated from wolfberry roots based on an activity tracking method, which provides a new approach for the preparation of wolfberry root extract and wolfberry root polyphenol derivatives.

[0045] On the other hand, in order to achieve the above-mentioned purpose of the invention, the present invention also provides a composition of wolfberry root polyphenol derivatives for anti-inflammatory and antipruritic effects, wherein the composition comprises at least one of the wolfberry root polyphenol derivatives shown in Formula I to Formula V.

[0046] Preferably, the composition of wolfberry root polyphenol derivatives of the present invention comprises wolfberry root polyphenol derivatives shown in Formula I to Formula V, and the mass ratio of each wolfberry root polyphenol derivative shown in Formula I to Formula V is 1:(1-100):(1-100):(1-100):(1-100). From the results of the evaluation experiment, there is a synergistic effect between the wolfberry root polyphenol derivatives in the wolfberry root extract, which can better enhance the anti-inflammatory activity of NHDF cells.

[0047] More preferably, in the wolfberry root polyphenol derivative composition of the present invention, the mass ratio of the wolfberry root polyphenol derivatives of Formula I to Formula V is 1:(1-50):(1-50):(1-50):(1-50). For example, in the wolfberry root polyphenol derivative composition of the present invention, the mass ratio of the wolfberry root polyphenol derivative compounds shown in Formula I to Formula V is approximately 1:4.5:13.5:8:3, but this is not a strictly enforced ratio and can fluctuate to a certain extent.

[0048] On the other hand, in order to achieve the above-mentioned purpose of the invention, the present invention also provides the use of the above-mentioned wolfberry root extract and the composition of wolfberry root polyphenol derivatives in the preparation of anti-inflammatory and antipruritic cosmetics or medicines.

[0049] In the application of the present invention, the application can be achieved by increasing the viability of NHDF cells, inhibiting the ROS content in NHDF cells, inhibiting the secretion of inflammatory cytokines in NHDF cells, inhibiting the activation of NLRP3 inflammasomes in NHDF cells, and / or inhibiting the activation of NF-κB signaling pathway in NHDF cells.

[0050] In the application of the present invention, the anti-inflammatory and soothing cosmetics or medicines may be external preparations in the form of sprays, mousses, ointments, emulsions, or liquid preparations.

[0051] In the application of the present invention, the inflammation involved in anti-inflammatory treatment refers to skin inflammation, including xeroderma, dermatitis, urticaria, eczema, and superficial fungal infection.

[0052] In the application of the present invention, the anti-inflammatory and antipruritic medicine can be a compound preparation, which includes auxiliary materials and the above-mentioned wolfberry root extract and / or wolfberry root polyphenol derivative composition.

[0053] In the application of the present invention, the composition of wolfberry root extract and / or wolfberry root polyphenol derivatives can be used as an additive for cosmetics or skin care products, and added into cosmetics or skin care products as an effective ingredient for anti-inflammatory and antipruritic effects.

[0054] The composition of the wolfberry root extract or wolfberry root polyphenol derivative of the present invention can inhibit the excessive expression of inflammatory factors in NHDF cells induced by DCNB, inhibit the oxidative damage of NHDF cells induced by DCNB, and exert anti-inflammatory protective activity.

[0055] The composition of the wolfberry root extract or wolfberry root polyphenol derivative of the present invention can inhibit the skin itching of skin itching model mice, reduce the number of scratching times of mice; inhibit the overexpression of inflammatory factors in skin itching model mice, and finally exert anti-inflammatory and antipruritic activities.

[0056] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0057] (1) The anti-inflammatory and antipruritic activities of the wolfberry root extract of the present invention are stronger than those of the common commercially available wolfberry root extract;

[0058] (2) The wolfberry root extract of the present invention can significantly reduce the overexpression of inflammatory cytokines in skin pruritus model mice and exert anti-inflammatory and antipruritic activity;

[0059] (3) The wolfberry root extract of the present invention can significantly reduce the number of scratching times of skin pruritus model mice and alleviate skin damage.

[0060] The technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments and accompanying drawings. It will be appreciated by those skilled in the art that the specific embodiments described herein are only embodiments of a part of the present invention, rather than all embodiments. Based on the spirit of the present invention, a person of ordinary skill in the art may make corresponding substitutions, changes, modifications or improvements without making creative work, but these substitutions, changes, modifications or improvements still fall within the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a derivative of Lycium barbarum root polyphenol-1 1 H-NMR spectrum;

[0062] Figure 2 It is a derivative of Lycium barbarum root polyphenol-1 13 C-NMR spectrum;

[0063] Figure 3 This is the HR-ESI-MS spectrum of Lycium barbarum root polyphenol derivative-1;

[0064] Figure 4 It is a derivative of Lycium barbarum root polyphenol-2 1 H-NMR spectrum;

[0065] Figure 5 It is a derivative of Lycium barbarum root polyphenol-2 13 C-NMR spectrum;

[0066] Figure 6 This is the HR-ESI-MS spectrum of Lycium barbarum root polyphenol derivative-2;

[0067] Figure 7 It is a derivative of Lycium barbarum root polyphenol-3 1 H-NMR spectrum;

[0068] Figure 8 It is a derivative of Lycium barbarum root polyphenol-3 13 C-NMR spectrum;

[0069] Fig. 9 This is the HR-ESI-MS spectrum of Lycium barbarum root polyphenol derivative-3;

[0070] Fig.10 It is a derivative of Lycium barbarum root polyphenol-4 1 H-NMR spectrum;

[0071] Fig.11 It is a derivative of Lycium barbarum root polyphenol-4 13 C-NMR spectrum;

[0072] Fig.12This is the HR-ESI-MS spectrum of Lycium barbarum root polyphenol derivative-4;

[0073] Fig.13 It is a derivative of Lycium barbarum root polyphenol-5 1 H-NMR spectrum;

[0074] Fig.14 It is a derivative of Lycium barbarum root polyphenol-5 13 C-NMR spectrum;

[0075] Fig.15 This is the HR-ESI-MS spectrum of Lycium barbarum root polyphenol derivative-5;

[0076] Fig.16 Schematic diagram of wolfberry root extract enhancing NHDF cell viability and inhibiting excessive ROS production;

[0077] Fig.17 This is a schematic diagram of the regulation of inflammatory cytokine secretion in NHDF cells by wolfberry root extract;

[0078] Fig.18 It is a schematic diagram of the inhibition of DCNB-induced activation of NLRP3 inflammasome and NF-κB signaling pathway in NHDF cells by wolfberry root extract;

[0079] Fig.19 Schematic diagram of the regulation of DCNB-induced inflammatory factor secretion in mice by wolfberry root extract. DETAILED DESCRIPTION

[0080] Example 1 Preparation of Lycium barbarum root extract

[0081] 1 kg of fresh wolfberry root was crushed and extracted with 30% ethanol (10 L) for 24 h, and the extracts were combined and concentrated under reduced pressure to obtain 164 g of extract.

[0082] The extract (50 g) was dissolved in 100 mL of ethanol, and the dissolved substance was mixed with 100 g of 80-100 mesh silica gel and stirred evenly. The mixture was evaporated at 40°C in a rotary evaporator to remove the ethanol to obtain an extract-silica gel adsorption mixture. The extract-silica gel adsorption mixture was separated by chromatography on a silica gel column (200-300 mesh), and gradient eluted with a mixed solvent of petroleum ether-ethyl acetate (100:30→30:100) to obtain Fr.1-15.

[0083] The protective activity of Fr.1-15 was evaluated based on the DNCB-induced NHDF cell inflammation model.

[0084] Fr.11 with the best protective activity was separated by gel column and eluted with chloroform-methanol (100:50→50:100) gradient to obtain Fr.11-1-Fr.11-15.

[0085] The protective activity of Fr.11-1-Fr.11-15 was evaluated based on the DNCB-induced NHDF cell inflammation model.

[0086] The Fr.11-5 component with the best protective activity is the wolfberry root extract of the present invention.

[0087] Example 2 Separation and identification of wolfberry root polyphenol derivatives

[0088] The Fr.11-5 component with the best protective activity was separated by preparative HPLC to obtain:

[0089] The specific conditions of preparative HPLC were as follows: RP-HPLC (Agilent 1200 HPLC) was separated on a Zorbax, SB C-18 column (9.4×250 mm, 5 μm). The elution solvent system consisted of water-trifluoroacetic acid (solvent A; 100:0.1, v / v) and acetonitrile-trifluoroacetic acid (solvent B; 100:0.1, v / v). Use gradient elution of 30% to 75% solvent B, at a flow rate of 5.0 mL / min for 35 min, and set the detection wavelength to 300 nm; collect the eluate corresponding to the chromatographic peak at 11.96 min, concentrate and dry to obtain the wolfberry root polyphenol derivative-1 with a structure shown in formula I; collect the eluate corresponding to the chromatographic peak at 12.16 min, concentrate and dry to obtain the wolfberry root polyphenol derivative-2 with a structure shown in formula II; collect the eluate corresponding to the chromatographic peak at 12.54 min, concentrate and dry to obtain the wolfberry root polyphenol derivative-3 with a structure shown in formula III; collect the eluate corresponding to the chromatographic peak at 12.68 min, concentrate and dry to obtain the wolfberry root polyphenol derivative-4 with a structure shown in formula IV; collect the eluate corresponding to the chromatographic peak at 12.77 min, concentrate and dry to obtain the wolfberry root polyphenol derivative-5 with a structure shown in formula V; wherein:

[0090] The structural analysis of the wolfberry root polyphenol derivative-1 of the structure shown in formula I is as follows:

[0091] See also Figure 1-Figure 3 The compound is light yellow oil, HR-ESI-MS m / z 338.0992[M+Na] + (calcdfor C 18 H 21 NO4Na, 338.1379), combined with the carbon and hydrogen nuclear magnetic resonance data spectra of the compound, the molecular formula of the compound was determined to be C 18 H 21 NO4, the degree of unsaturation is 9 and the relative molecular mass is 315.

[0092] observe 1Characteristic signals in the H NMR (300MHz, MeOH-d4) spectrum, there are 10 groups of hydrogen signals in the compound. There is a group of characteristic ABX spin system aromatic proton signals [δH6.66 (1H, s, H-5), 6.76 (1H, d, J = 4.8Hz, H-8), 6.61 (1H, d, J = 1.8Hz, H-9)], indicating that the compound contains a 1,3,4-trisubstituted benzene ring; AA'BB' spin system characteristic aromatic proton signal group [δH 6.68 (2H, d, J = 1.8Hz, H-5', 7'), 6.92 (2H, d, J = 8.4Hz, H-4', 8')], indicating that the structure contains a 1,4-disubstituted benzene ring. The four methylene proton signals are [δH 2.79 (2H, t, J = 7.5 Hz, H-3), 2.59 (2H, t, J = 7.5 Hz, H-2'), 2.39 (2H, d, J = 6.6, 15 Hz, H-2), 3.31 (2H, d, J = 8.4, 10 Hz, H-1')] and one methoxy proton signal [δH 3.82 (3H, s, 6-OCH3)]. 13 C NMR and DEPT spectra show that the compound contains 16 carbon signals, of which [δC 174.8 (C-1)] is the carbonyl carbon signal, a group of symmetrical 1,4-disubstituted phenyl aromatic carbon signals are [δC 130.7 (C-4', 8'), 116.1 (C-5', 7'], a group of aromatic carbon signals [δC 133.7 (C-4), 145.9 (C-6), 148.9 (C-7)] are the signals on 1,3,4-trisubstituted phenyl, one methoxy carbon signal [δC 56.3 (6-OCH3)], and four methylene carbon signals [δC 32.6 (C-3), 35.7 (C-2'), 39.4 (C-2), 42.3 (C-1')]. Its C-H NMR data are shown in Table 1.

[0093] Table 1 Lycium barbarum root polyphenol derivatives-1 1 HNMR and 13 CNMR Data

[0094]

[0095] Based on the above analysis, the compound structure was identified as the compound represented by Formula I and named as Lycium barbarum root polyphenol derivative-1 in the present invention.

[0096] The structural analysis of the wolfberry root polyphenol derivative-2 of the structure shown in formula II is as follows:

[0097] See also Figure 4-Figure 6 The compound is yellow oil, HR-ESI-MS m / z 302.1285[M+H] + (calcd for C17 H 20 NO4,302.1393), the molecular formula of the compound is determined to be C 17 H 20 NO4, the degree of unsaturation is 9 and the relative molecular mass is 301. 1 H NMR (600 MHz, MeOH-d4) spectrum showed that there were 9 groups of hydrogen signals in the compound, including a group of characteristic aromatic proton signals of the ABX spin system [δH 6.63 (1H, s, H-2), 6.67 (1H, d, J = 8.4 Hz, H-5), 6.51 (1H, d, J = 8.4 Hz, H-6)], indicating that the structure contained a 1,3,4-trisubstituted benzene ring; and a group of characteristic aromatic proton signals of the AA'BB' spin system [δH 6.95 (2H, d, J = 8.4 Hz, H-2', 6'), 6.69 (2H, d, J = 9 Hz, H-3', 5')], indicating that the compound contained a 1,4-disubstituted benzene ring. In the high field region, four methylene signals were found [δH 2.73 (2H, t, J = 7.8 Hz, H-7), 2.37 (2H, t, J = 7.8 Hz, H-8), 2.61 (2H, d, J = 7.8, 15 Hz, H-7'), 3.28 (2H, d, J = 7.8 Hz, H-8')]. 13 C NMR and DEPT spectra revealed that the compound contained 15 carbon signals, including a carbonyl carbon signal [δC 175.4 (C-9)], a group of symmetrical 1,4-disubstituted phenyl aromatic carbon signals [δC 130.7 (C-2', 6'), 116.2 (C-3', 5'], a group of 1,3,4-trisubstituted phenyl aromatic carbon signals [δC 116.5 (C-2), 116.3 (C-5), 120.6 (C-6)], and four methylene carbon signals [δC 32.5 (C-7), 39.4 (C-8), 35.7 (C-7'), 42.3 (C-8')]. Its C-H NMR data are shown in Table 2.

[0098] Table 2 Lycium barbarum root polyphenol derivatives-2 1 HNMR and 13 CNMR Data

[0099]

[0100] Based on the above analysis, the compound structure was identified as the compound represented by Formula II and named as Lycium barbarum root polyphenol derivative-2 in the present invention.

[0101] The structural analysis of the wolfberry root polyphenol derivative-3 of the structure shown in formula III is as follows:

[0102] See also Figure 7-Figure 9The compound is a brown solid, HR-ESI-MS m / z 314.1380[M+H] + (calcd for C 18 H 20 NO4,314.1393), the molecular formula of the compound is determined to be C 18 H 20 NO4, the degree of unsaturation is 10 and the relative molecular mass is 313.

[0103] observe 1 The characteristic signals in the H NMR (300 MHz, MeOH-d4) spectrum include 10 groups of hydrogen signals in total. There are two trans double bond proton signals [δH 5.81(1H,d,J=12.6Hz,H-2'),6.61(1H,d,J=12.6Hz,H-3')]; it contains a group of characteristic aromatic proton signals of the ABX spin system [δH 7.36(1H,d,J=2.1Hz,H-4),6.74(1H,d,J=5.1Hz,H-7),6.93(1H,d,J=8.1Hz,H-8)], indicating that there is a 1,3,4-trisubstituted benzene ring in the structure; there is a group of characteristic proton signals of the AA'BB' spin system [δH 6.99(2H,d,J=6.3Hz,H-5',9'),6.69(2H,d,J=6.6Hz,H-6',8')], indicating that the compound contains a 1,4-disubstituted benzene ring. In the high field region, two methylene signals [δH 2.69 (2H, t, J = 8.1, 15 Hz, H-2), 3.35 (2H, br.s, H-1)] and one methoxy signal [δH 3.83 (3H, s, 7'-OCH3)] were found.

[0104] Combination 13 C NMR and DEPT spectra revealed that the compound contained 16 carbon signals, including [δC 170.3(C-1')] for carbonyl carbon, two double bond carbon signals [δC 114.1(C-2'), 138.2(C-3')], a group of symmetrical 1,4-disubstituted phenyl aromatic carbon signals [δC 130.7(C-5',9'), 116.2(C-6',8')], a group of 1,3,4-trisubstituted phenyl aromatic carbon signals [δC 115.9(C-4), 142.0(C-7), 124.8(C-8)], two methylene carbon signals [δC 42.3(C-1), 35.2(C-2)], and one methoxy carbon signal [δC 56.4(7'-OCH3)]. Its NMR data are shown in Table 3.

[0105] Table 3 Lycium barbarum root polyphenol derivatives-3 1 HNMR and 13CNMR Data

[0106]

[0107] Based on the above analysis, the compound structure was identified as the compound represented by formula III and named as wolfberry root polyphenol derivative-3 in the present invention.

[0108] The structural analysis of the wolfberry root polyphenol derivative-4 of the structure shown in formula IV is as follows:

[0109] See also Figure 10-12 The compound is white powder, HR-ESI-MS m / z 318.1351[M+H] + (calcdfor C 17 H 20 NO5,318.1342), the molecular formula of the compound is determined to be C 17 H 19 NO5, unsaturation is 9, relative molecular mass is 317. 1 The characteristic signals provided by the H NMR (600MHz, MeOH-d4) spectrum show that there are 10 groups of hydrogen signals in the compound. The compound has two groups of characteristic ABX spin system aromatic proton signals [δH 6.63 (1H, s, H-2), 6.67 (1H, d, J = 9.6 Hz, H-5), 6.50 (1H, d, J = 7.8 Hz, H-6)] and [δH 6.62 (1H, s, H-2'), 6.66 (1H, d, J = 7.8 Hz, H-5'), 6.46 (1H, d, J = 8.4 Hz, H-6')], indicating that there are two 1,3,4-trisubstituted benzene rings in the structure. In the high field region, four methylene signals were found [δH 2.73 (2H, t, J = 7.8 Hz, H-7), 2.56 (2H, t, J = 7.8 Hz, H-7'), 2.37 (2H, t, J = 6.6 Hz, H-8), 3.28 (2H, t, J = 3.6 Hz, H-8')]. 13 C NMR and DEPT spectra show that the compound contains 15 carbon signals, including [δC 175.4 (C-9)] for carbonyl carbon signal, two groups of 1,3,4-trisubstituted phenyl aromatic carbon signals [δC 116.3 (C-2), 116.8 (C-5), 121.1 (C-6)] and [δC 116.3 (C-2'), 116.5 (C-5'), 120.6 (C-6')], and four methylene carbon signals [δC 32.5 (C-7), 36.0 (C-7'), 39.5 (C-8), 42.3 (C-8')]. Its C-H NMR data are shown in Table 4.

[0110] Table 4 Lycium barbarum root polyphenol derivatives-4 1HNMR and 13 CNMR Data

[0111]

[0112] Based on the above analysis, the compound structure was identified as the compound represented by Formula IV and named as Lycium barbarum root polyphenol derivative-4 in the present invention.

[0113] The structural analysis of the wolfberry root polyphenol derivative-5 of the structure shown in formula V is as follows:

[0114] See also Figure 13-Figure 15 The compound is yellow oil. HR-ES I-MS m / z 366.1329[M+Na] + (calcdfor C 19 H 21 NO5Na,366.1318), the molecular formula of the compound is determined to be C 19 H 21 NO5, the degree of unsaturation is 10 and the relative molecular mass is 343. 1 The characteristic signals in the H NMR (300MHz, DMSO-d6) spectrum revealed that there were 8 groups of hydrogen signals in compound 11. In the low field region, two trans double bond proton signals [δH 6.53 (1H, d, J = 15.6 Hz, H-2), 7.28 (1H, d, J = 13.5 Hz, H-3)] were observed for the compound; one group of characteristic aromatic proton signals [δH 6.84 (2H, s, H-5)] indicated the presence of a 1,3,4,5-tetrasubstituted benzene ring in the structure; and one group of AA'BB' spin system characteristic aromatic proton signals [δH 7.01 (2H, d, J = 8.7 Hz, H-2', 6'), 6.68 (2H, d, J = 8.7 Hz, H-3', 5')] indicated the presence of a 1,4-disubstituted benzene ring in the compound. In the high field region, two methylene signals [δH 2.64 (2H, t, J = 7.5 Hz, H-β), 3.28 (2H, m, H-α)] and two methoxy signals [δH 3.78 (6H, s, 6,8-OCH3)] were found. 13C NMR and DEPT spectra revealed that the compound contained 12 carbon signals, among which [δC 165.5 (C-1)] was a carbonyl carbon signal, a group of aromatic carbon signals [δC 125.3 (C-4), 148.1 (C-6), 129.5 (C-7), 148.1 (C-8)] were 1,3,4,5-tetrasubstituted phenyl, a group of aromatic carbon signals [δC 129.4 (C-2', 6'), 115.1 (C-3', 5'] were 1,4-disubstituted phenyl, two double bond carbon signals [δC 119.4 (C-2), 139.2 (C-3)], two methylene carbon signals [δC 41.5 (C-α), 34.4 (C-β)], and two methoxy signals [δC 56.0 (6,8-OCH3)]. Its C-H NMR data are shown in Table 5.

[0115] Table 5 Lycium barbarum root polyphenol derivatives-5 1 HNMR and 13 CNMR Data

[0116]

[0117] Based on the above analysis, the compound structure was identified as the compound represented by Formula V and named as Lycium barbarum root polyphenol derivative-5 in the present invention.

[0118] Effect Experimental Example 1 Evaluation of Anti-inflammatory Activity of Lycium Barbarum Root Extract

[0119] 5×10 3 NHDF cells / well were seeded in 96-well plates and cultured overnight. DCNB solution was added to a final concentration of 10 μM. After continued culture for 12 h, the cells were collected and washed twice with cold PBS. New culture medium containing wolfberry root polyphenol derivative-1, wolfberry root polyphenol derivative-2, wolfberry root polyphenol derivative-3, wolfberry root polyphenol derivative-4, wolfberry root polyphenol derivative-5, wolfberry root extract or commercially available wolfberry root extract was added to a final concentration of 1.0 mg / mL and cultured for 12 h. Cells were collected and cell activity was detected using the CCK 8 kit.

[0120] ②, 5×10 3 NHDF cells / well were inoculated in 96-well plates and cultured overnight. DCNB solution was added to a final concentration of 10 μM. After continued culture for 12 h, the cells were collected and washed twice with cold PBS. New culture medium containing wolfberry root polyphenol derivative-4 or wolfberry root extract or commercially available wolfberry root extract was added to a final concentration of 1.0 mg / mL and cultured for 12 h. Cells and supernatants were collected, and ELISA kits were used to detect changes in the secretion of cellular inflammatory factors, changes in intracellular ROS content, and western blotting to detect changes in related proteins.

[0121] Experimental results ( Fig.16 A) shows that compared with the blank control group (cell viability, 100%), the viability of NHDF cells in the DCNB group decreased by 23.5%, while the cell viability of the wolfberry root extract, wolfberry root polyphenol derivative-1, wolfberry root polyphenol derivative-2, wolfberry root polyphenol derivative-3, wolfberry root polyphenol derivative-4, and wolfberry root polyphenol derivative-5 groups were all higher than that of the DCNB group; indicating that wolfberry root polyphenol derivative-1, wolfberry root polyphenol derivative-2, wolfberry root polyphenol derivative-3, wolfberry root polyphenol derivative-4, wolfberry root polyphenol derivative-5 and wolfberry root extract have a repair effect on DCNB-induced cell damage and have a certain protective activity on NHDF cells. Further analysis of the experimental results found that under the same concentration conditions, the protective activity of wolfberry root extract is stronger than that of wolfberry root polyphenol derivatives and stronger than that of commercially available wolfberry root extract.

[0122] Intracellular reactive oxygen species are important factors that induce cellular oxidative stress and cellular oxidative damage. We used Lycium barbarum root polyphenol derivative-4, which has the best protective activity, as a representative compound to continue studying the potential molecular mechanism of Lycium barbarum root polyphenol derivatives and Lycium barbarum root extracts in exerting protective activity. Fig.16 B shows that the intracellular ROS content of NHDF cells in the DCNB group increased by 341% (p<0.01), indicating that DCNB exposure induced excessive production of ROS in NHDF cells, induced cell oxidative damage, and reduced NHDF cell viability. Compared with the DNCB group, the intracellular ROS content of NHDF cells in the wolfberry root polyphenol derivative-4, wolfberry root extract, and commercial wolfberry root extract groups decreased. Compared with the DCNB group, the intracellular ROS content of NHDF cells in the wolfberry root polyphenol derivative-4, wolfberry root extract, and commercial wolfberry root extract groups decreased by 39.9% (p<0.05), 61.7% (p<0.01), and 16.8% (p<0.05), respectively.

[0123] Inflammatory cytokines are an important component of the body's immunity. Among them, pro-inflammatory cytokines can aggravate the inflammatory response, mainly including IL-1β, IL-2, IL-6, IL-8, IL-13, TNF-α, etc.; while anti-inflammatory cytokines can inhibit the inflammatory response, mainly including IL-4, IL-10, etc.

[0124] The experimental results are as follows Fig.17 A, C, D, F, G, and H showed that the secretion of inflammatory cytokines IL-1β, IL-6, IL-8, IL-13, IL-18, and TNF-α in the DNCB group was significantly increased, while the secretion of anti-inflammatory cytokines IL-4 and IL-10 ( Fig.17B, E) secretion was significantly reduced. We speculate that DCNB induces inflammatory response in NHDF cells, causing severe inflammatory damage to NHDF cells, which in turn leads to reduced NHDF cell viability. The wolfberry root extract significantly inhibited the excessive secretion of inflammatory factors IL-1β, IL-6, IL-13, IL-18, and TNF-α in NHDF cells induced by DNCB, and also effectively alleviated the inhibition of DNCB-induced secretion of anti-inflammatory cytokine IL-10 in NHDF cells. The regulatory activity of wolfberry root extract on DNCB-induced NHDF cell inflammatory factor secretion disorder is superior to wolfberry root polyphenol derivative-4 and commercially available wolfberry root extract under the same concentration conditions. We speculate that the wolfberry root polyphenol derivatives in the wolfberry root extract have a synergistic effect, which can better play a role in improving the anti-inflammatory activity of NHDF cells.

[0125] Innate immunity plays a vital role in clearing foreign pathogens and guiding the body to produce effective adaptive immune responses. Innate immunity recognizes the conserved structure of pathogens through pattern recognition receptors, and then activates downstream signaling pathways to cause inflammatory responses. For example, NOD-like receptors (NLRs): a class of intracellular sensing molecules. After activation, NOD-like receptors form a huge protein complex, the "inflammasome".

[0126] The experimental results are as follows Fig.18 A, B, and C showed that the expression of NLRP3, ASC-1, and Caspase-1 proteins in NHDF cells in the DCNB exposure group increased significantly. Western blot experimental results showed that DCNB exposure activated the NLRP3 inflammasome of NHDF cells, and the activation of NLRP3 inflammasome further induced the secretion of inflammatory cytokines and aggravated the inflammatory damage of NHDF cells in the DCNB group. After treatment with wolfberry root extract, the increase in the expression of NLRP3, ASC-1, and caspase-1 proteins in NHDF cells induced by DCNB was significantly inhibited, and the activation of NLRP3 inflammasome was inhibited. We believe that the anti-inflammatory effect of wolfberry root extract and the protection of NHDF cell activity may be achieved by inhibiting the activation of NLRP3 inflammasome in NHDF cells induced by DCNB.

[0127] NF-κB is a nuclear transcription factor that can regulate target genes such as immune-related receptors, cytokines (such as IL-1, IL-2, IL-6, IL-12 and TNF-α), inflammatory factors (IL-8, MIP-1α, MCP1) and adhesion molecules (ICAM, VCAM and E-selectin), and plays an important role in regulating immune cell activation, cell apoptosis and inflammatory response.

[0128] The experimental results are as follows Fig.18D, E, and F show that DCNB significantly increased p65 and NF-κB proteins in NHDF cells; at the same time, the IκBα protein content in NHDF cells in the DCNB group was significantly reduced. The experimental results show that DCNB exposure activates the NF-κB signaling pathway in NHDF cells. After treatment with wolfberry root extract, the high expression of p65 and NF-κB proteins in NHDF cells induced by DCNB was significantly inhibited, and the degradation of IκBα protein was also significantly inhibited. We speculate that the anti-inflammatory activity of wolfberry root extract and the inhibition of DCNB-induced NF-κB signaling pathway activation in NHDF cells are also one of the potential molecular mechanisms. The experimental results in this part also show that the inhibitory activity of wolfberry root extract on DCNB-induced NF-κB signaling pathway activation in NHDF cells is stronger than that of wolfberry root polyphenol derivative-4 and commercially available wolfberry root extract. It is further verified that wolfberry root polyphenol derivatives can exert their protective activity through synergistic effects.

[0129] Effect Experimental Example 2 Evaluation of Anti-inflammatory and Antipruritic Activities of Lycium barbarum Root Extract

[0130] Kunming mice, SPF grade, male, weighing (21±2) g, were kept in a constant temperature environment, with a temperature of (20±2) ℃, a humidity of 50%, and a light rhythm of 12h:12h. They were allowed to eat and drink freely, and were adaptively fed for 1 week. The mice were randomly divided into 5 groups, 8 mice in each group, namely, the normal group, the skin itching model group, the wolfberry root extract group, the wolfberry root polyphenol derivative-4 group, and the commercial wolfberry root extract group. After the experiment started, except for the normal group, after shaving, 200 μL of 2% DNCB solution was applied to the back skin of the mice on a 1×1 cm patch for 1 week, and 200 μL of 0.2% DNCB solution was used to stimulate again twice a week.

[0131] Starting from the 21st day, mice in each treatment group were given 1 mg / cm 2 The drug was applied once in the evening every day for 14 consecutive days (the normal group and the skin pruritus model group were given the same dose of distilled water every day). During the experiment, the hair on the back of the neck of the mice was shaved to keep the back of the neck hairless. 30 minutes after the drug was applied on the 28th day, the scratching performance of the mice was recorded; the eyeballs were removed to collect blood, centrifuged at 3000r / min for 10 minutes, and serum was collected. The ELISA method was used to detect the levels of serum IL-1β, IL-6, IL-10, IL-13, TNF-α and IL-18.

[0132] Table 6 Effect of Lycium barbarum root extract on the latency time and frequency of skin itching in mice

[0133]

[0134] Compared with the blank group ## p<0.01; compared with DCNB group, * p<0.05,** p<0.01.

[0135] Among them, analyzing the experimental results in Table 6, compared with the blank control group, the latency of skin pruritus in the model group mice was significantly shortened, and the number of skin pruritus increased significantly. After treatment with wolfberry root extract, the latency of skin pruritus in mice was effectively prolonged (p<0.01), and the number of skin pruritus in mice was significantly reduced (p<0.01); at the same time, the latency of skin pruritus in the wolfberry root extract group mice was most significantly prolonged; the number of skin pruritus in mice was also most significantly reduced, and the effect was better than wolfberry root polyphenol derivative-4 and commercial wolfberry root extract under the same dosage conditions.

[0136] Effects of Lycium barbarum root extract on inflammatory factors in mice with skin itching ( Fig.19 ) showed that compared with the blank group, the secretion of IL-1β, IL-6, IL-13, TNF-α and IL-18 in the itch model group mice was significantly increased, while the secretion of IL-10 was significantly reduced. This shows that DCNB exposure induced severe inflammatory response in mice, which in turn caused inflammatory damage to the mice. After treatment with wolfberry root extract, compared with the model group, the content of IL-1β, IL-6, TNF-α and IL-18 in the wolfberry root extract group was significantly reduced, and the content of IL-10 was increased. Further analysis of experimental data showed that the inhibitory activity of wolfberry root extract on excessive secretion of IL-1β, IL-6, TNF-α and IL-18 induced by DCNB exposure, and the inhibitory activity on the expression and secretion of IL-10 induced by DCNB exposure were higher than those of wolfberry root polyphenol derivative-4 and commercially available wolfberry root extract under the same concentration conditions. This part of the data once again verified that wolfberry root polyphenol derivatives can exert anti-inflammatory activity through synergistic effects.

Claims

1. A composition of wolfberry root polyphenol derivatives for anti-inflammatory and antipruritic effects, the composition of wolfberry root polyphenol derivatives comprising wolfberry root polyphenol derivatives as shown in the following formulas I to V, and the mass ratio of each wolfberry root polyphenol derivative as shown in formulas I to V is controlled to be 1:(1-50):(1-50):(1-50):(1-50):

2. The composition of wolfberry root polyphenol derivatives according to claim 1, wherein The mass ratio of the wolfberry root polyphenol derivatives represented by Formula I to Formula V is controlled to be 1:4.5:13.5:8:

3.

3. The composition of wolfberry root polyphenol derivatives according to claim 1, wherein: The composition of wolfberry root polyphenol derivatives is a wolfberry root extract prepared by a method comprising the following steps: (1) Crush fresh wolfberry roots; (2) extracting the ground wolfberry root by diafiltration with ethanol-water; (3) combining the extracts obtained in the reduced pressure concentration step (2) to obtain an extract; (4) dissolving the extract obtained in step (3) in ethanol, and mixing and stirring with silica gel to obtain a mixture; (5) rotary evaporating the mixture obtained in step (4) at a temperature of 35-45° C. in a rotary evaporator to remove ethanol and obtain an extract-silica gel adsorption mixture; (6) separating the extract-silica gel adsorption mixture obtained in step (5) by silica gel column chromatography, and performing gradient elution with a petroleum ether-ethyl acetate mixed solvent to obtain several fractions; (7) constructing a 2,4-dinitrochlorobenzene (DNCB)-induced normal human dermal fibroblast (NHDF) injury model, and evaluating the activity of each fraction obtained in step (6); (8) separating the fraction with the best protective activity in step (7) by using a gel column and performing gradient elution with chloroform-methanol to obtain several fractions; (9) evaluating the protective activity of each fraction obtained in step (8) based on the DNCB-induced NHDF cell injury model; Wherein, the fraction with the best protective activity in step (9) is the wolfberry root extract.

4. Use of the composition of wolfberry root polyphenol derivatives as claimed in any one of claims 1 to 3 in the preparation of anti-inflammatory and antipruritic cosmetics or medicines.

5. The use according to claim 4, wherein: The anti-inflammatory and antipruritic cosmetics or medicines are external preparations in the form of sprays, mousses, ointments or emulsions.

6. The use according to claim 4, wherein: The inflammation involved in anti-inflammatory is skin inflammation, including dry skin, dermatitis, urticaria, eczema, and superficial fungal infections.

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