Pomace cream and its use

Abstract

The application provides a cream containing enzyme-modified pomace polysaccharide and application thereof in treating skin diseases such as psoriasis. The application uses edible enzyme xylanase to modify pomace polysaccharide and components such as naringin and naringenin, and provides good antioxidant property, treatment activity and emulsification stability; compared with hormone drugs, the application is safer and more reliable, and can be used for daily long-term use.

Description

Technical Field

[0001] This application pertains to the fields of cosmetics and sugar engineering technology. Specifically, this application provides a cream containing grapefruit peel polysaccharide and its application in treating skin diseases such as psoriasis. Background Technology

[0002] Many intractable skin diseases, such as psoriasis, have complex mechanisms of development (related to multiple factors including infection and autoimmunity), and neither traditional Chinese medicine nor Western medicine has specific treatments. Long-term use of plant polysaccharides, polyphenols, or peptides with antioxidant, antibacterial, and immunomodulatory functions (such as naringin and naringenin) is a good option for treating and controlling the condition. Modifying polysaccharides through chemical, physical, or biological means is an effective way to further enhance their effects and even endow them with new functions. Enzymatic modification is a common method for improving the molecular structure and properties of polysaccharides. Treatment with certain hydrolytic enzymes not only reduces the molecular weight but also alters the spatial structure of polysaccharide molecules, such as the triple helix structure, thereby affecting the physiological activity of the polysaccharide.

[0003] Grapefruit is a widely consumed and processed fruit, but its peel contains a large amount of fiber and is bitter, having no nutritional value. Currently, it can only be used to process a few products such as flavorings and grapefruit tea. The polysaccharides and other components in grapefruit peel have significant antioxidant, antibacterial, immunomodulatory, and even anti-tumor functions, making it a valuable plant polysaccharide resource awaiting development. Summary of the Invention

[0004] In response to the above situation, the applicant attempted to use enzyme-modified grapefruit peel polysaccharide to prepare a cream containing a high concentration of active ingredients for the treatment of skin diseases such as psoriasis.

[0005] On the one hand, this application provides a grapefruit peel cream containing grapefruit peel polysaccharides.

[0006] On the other hand, this application provides the use of grapefruit peel polysaccharide in the preparation of cream-based pharmaceutical formulations for treating psoriasis.

[0007] Furthermore, the cream also contains naringin and naringenin.

[0008] Further, the grapefruit peel polysaccharide is prepared by the following method: grapefruit peel is dried, pulverized, and passed through a 100-mesh sieve; deionized water is added at a mass / volume ratio of 1:30 to adjust the pH to 5.0; extraction is carried out at 100 degrees Celsius for 2 hours; after cooling, it is filtered; 3 times the volume of ethanol is added to precipitate; the precipitate is collected by centrifugation, concentrated, and freeze-dried.

[0009] Furthermore, the grapefruit peel polysaccharide is modified by polygalacturonase or xylanase.

[0010] Furthermore, the enzyme-modified grapefruit peel polysaccharide was prepared according to the following method:

[0011] (1) Prepare grapefruit peel polysaccharide concentration of 1-10 mg / mL;

[0012] (2) Add xylanase to 100-300 U / mL, pH 56.0-8.0, and treat at 40-60 degrees Celsius for 1-4 hours; or add polygalacturonase to 50-100 U / mL, pH 5.0-7.0, and treat at 40-60 degrees Celsius for 1-4 hours.

[0013] (3) Inactivate enzymes; concentrate after cooling and freeze dry.

[0014] Furthermore, in step (1), the concentration of grapefruit peel polysaccharide is prepared to be 5 mg / mL;

[0015] Further, in step (2), xylanase was added to 200 U / mL, and the mixture was treated at pH 7.0 and 40 degrees Celsius for 3 hours.

[0016] Furthermore, the cream is prepared using the following raw materials: 50 parts deionized water, 0.2 parts triethanolamine, 8 parts stearic acid, 5 parts glyceryl stearate, 5 parts petrolatum, 15 parts glycerin, 15 parts liquid paraffin, 0.2 parts fragrance, and 8 parts grapefruit peel polysaccharide.

[0017] Further, the cream is prepared by the following method: stearic acid, glyceryl monostearate, petrolatum, and liquid paraffin are heated to 70 degrees Celsius and mixed to form the oil phase; glycerin and triethanolamine are added to 35 parts of deionized water and heated to 70 degrees Celsius to form the aqueous phase; the oil phase is slowly added to the aqueous phase while stirring to emulsify; the temperature is lowered to 40 degrees Celsius to dissolve the grapefruit peel polysaccharide and fragrance in 15 parts of deionized water; stirring is continued until the cream is formed at room temperature.

[0018] In addition, this application also provides a method for preparing the above-mentioned grapefruit peel polysaccharide.

[0019] The raw materials for preparing pomelo peel polysaccharides in this application are not limited to the Shatin pomelo used in the examples; other pomelo varieties, such as Wenzhou pomelo and Thai pomelo, can also be used. Pomelo peel polysaccharides can be prepared by oneself using known methods or commercially available finished products can be sold. Those skilled in the art can appropriately adjust the extraction and enzymatic modification methods according to the different pomelo peel polysaccharides.

[0020] This application may also include other components from grapefruit peel, such as naringin and naringenin, which can be extracted and prepared in the same process as grapefruit peel polysaccharide, or extracted separately or purchased commercially; their functions, such as anti-inflammatory, immunomodulatory, and antioxidant effects, are well known to those skilled in the art.

[0021] The enzymes described in this application do not have specific manufacturers or dosage details. Within common dosage ranges, those skilled in the art can calculate and adjust the dosage accordingly.

[0022] In addition to psoriasis, the cream of this application can be used for the treatment or daily care of a variety of skin diseases, including but not limited to psoriasis, dermatitis, acne, pityriasis rosea, urticaria, tinea, etc. Attached Figure Description

[0023] Figure 1 The effects of pectinase (A), α-amylase (B), β-amylase (C), polygalacturonase (D), pectin esterase (E), pectin lyase (F), arabinofuranase (G), xylanase (H), and untreated grapefruit peel polysaccharide (I) on the survival of HaCat cells, a psoriasis cell model (J is the control without polysaccharide added only to the culture medium, n=5).

[0024] Figure 2 The effects of pectinase (A), α-amylase (B), β-amylase (C), polygalacturonase (D), pectin esterase (E), pectin lyase (F), arabinofuranase (G), xylanase (H), and untreated grapefruit peel polysaccharide (I) on DPPH and superoxide anion radical scavenging rate (average of three trials).

[0025] Figure 3 Emulsification stability (average of three trials) of pectinase (A), polygalacturonase (D), pectin esterase (E), xylanase (H) modified grapefruit peel polysaccharide and untreated grapefruit peel polysaccharide (I). Detailed Implementation

[0026] The following embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of this application in any way. The scope of protection of this application is defined only by the claims. This application only shows some representative experimental data. Due to space limitations, some data, especially the early process of exploring parameters such as concentration, dosage, pH, and temperature, have not been fully shown.

[0027] Example 1: Materials and Basic Experimental Methods

[0028] Extraction method of grapefruit peel polysaccharides

[0029] The grapefruit peel polysaccharide shown in the examples was prepared in-house, using peeled Shatin grapefruit from the same batch sold by the brand / distributor.

[0030] Based on preliminary exploration, the preparation method is as follows: Grapefruit peel is dried, pulverized, and passed through a 100-mesh sieve; deionized water is added at a material-to-liquid ratio of 1:30 (mass / volume) to adjust the pH to 5.0; extraction is performed at 100 degrees Celsius for 2 hours; after cooling, the mixture is filtered; three times its volume of ethanol is added to precipitate the polysaccharide; the precipitate is collected by centrifugation, concentrated, and freeze-dried. Based on mass calculations, the yield of grapefruit peel polysaccharides is approximately 12%.

[0031] Subsequently, the applicant purchased two commercially available grapefruit peel polysaccharides (Shaanxi Hana Biotechnology and Lanzhou Waterles Biotechnology) and processed them using the preferred method described in the examples, achieving similar results.

[0032] Preparation and use of psoriasis cell models

[0033] The method was improved from "The Effects of Hot Blood-Activating and Detoxifying Compound (QHJD) on TNF-α-induced HaCat Cell Proliferation and Apoptosis". *Chinese Journal of Experimental Traditional Medical Formulae*, 2016, Vol. 22, No. 4; based on the rapid proliferation of psoriasis-like epidermal cells induced by TNF-α.

[0034] Immortalized human epidermal cells, HaCat cells, were used to prepare a psoriasis cell model (HaCat cells and special culture medium were purchased from Wuhan Shangen Biotechnology Co., Ltd., and the cell culture and passage methods were carried out in accordance with the operating instructions provided by the company).

[0035] HaCat cells in the logarithmic growth phase were washed with PBS and digested with 0.25% trypsin for 5 minutes. Culture medium was added to stop the digestion and the cells were aspirated to form a cell suspension. The cells were centrifuged, washed, and resuspended in culture medium. The resuspended cells were seeded into the wells of a 96-well plate at a density of 200 μL per well, approximately 5000 cells.

[0036] The wells were divided into blank, control, and test drug groups. After culturing for 24 hours in 96-well plates, the HaCat cells in the control and test drug groups were replaced with medium containing TNF-α (40 ng / mL), while the blank group was cultured in medium without TNF-α. After 24 hours, the medium containing the test drug was replaced with medium containing the test drug, while the blank and control groups were cultured in medium without TNF-α and the test drug. After 24 hours of culture, the cell viability / survival rate was detected by CCK-8 assay (CLPBIO CCK-8 kit, performed according to the instructions provided). The relative survival rates of the control and test drug groups were calculated with the cell viability of the blank group being 100%.

[0037] Preparation and use of mouse models of psoriasis

[0038] The method is an improvement on "Hesperidin inhibits keratinocyte proliferation and imiquimod-induced psoriasis-like dermatitis via the IRS-1 / ERK1 / 2 pathway, LifeScience, 2019, Vol. 219"; based on imiquimod / IMQ-induced psoriasis-like skin problems:

[0039] Healthy BALB / C mice, especially those without obvious skin problems after examination, had their backs prepared into 2cm×2cm sections. The prepared mice were then divided into a control group and a model group. The control group and the model group were treated with petroleum jelly cream and 5% IMO cream once a day, respectively. After one week, the skin condition was observed. The presence of wrinkles, keratinization, redness, and white scales compared to the control group indicated successful modeling.

[0040] The following criteria were used to group patients, ensuring that the initial scores of each group were basically consistent. Drug administration was administered for 7 consecutive days, with the cream applied to the prepared skin area once daily (gently massaging for 1 minute), followed by thorough washing off with purified water 1 hour later (to avoid possible irritation and allergic reactions). Scoring was performed before administration (day 0) and 24 hours after the last administration (day 7). Scoring referenced the PASI criteria for psoriasis. Since the model area was already determined, only four criteria were evaluated: skin lesions, scaling, plaque thickening, and erythema.

[0041] Table 1 Scoring criteria for mouse psoriasis model (referencing PASI)

[0042]

[0043] DPPH scavenging rate determination and superoxide anion radical scavenging rate determination

[0044] The DPPH scavenging rate and superoxide anion radical scavenging rate of a 0.5 mg / mL grapefruit peel polysaccharide solution were determined using commercially available DPPH scavenging rate assay kits (Shanghai Yanqi Biotechnology) and superoxide anion radical scavenging rate assay kits (Haisepesen Biotechnology).

[0045] Emulsion stability testing

[0046] Prepare a 1% grapefruit peel polysaccharide PBS buffer solution (pH 7.0, 0.02M); mix 10 mL of the above solution with 2 mL of vegetable oil (commercially available blended oil) and stir homogenously for 3 minutes to form an emulsion; take 0.1 mL from the bottom half of the container and disperse it in 10 mL of 0.1% SDS solution, and measure the absorbance at 500 nm using a spectrophotometer (A1); after the emulsion has stood for 15 minutes, take 0.1 mL from the bottom half of the container and disperse it in 10 mL of 0.1% SDS solution, and measure the absorbance at 500 nm using a spectrophotometer (A2); emulsion stability is expressed as A2 / A1×100%.

[0047] Cream formulation and preparation method

[0048] Cream formula (by weight): 50 parts deionized water, 0.2 parts triethanolamine, 8 parts stearic acid, 5 parts glyceryl stearate, 5 parts petrolatum, 15 parts glycerin, 15 parts liquid paraffin, 0.2 parts fragrance, and 8 parts grapefruit peel polysaccharide.

[0049] Preparation method: Stearic acid, glyceryl monostearate, petrolatum, and liquid paraffin are heated to 70 degrees Celsius and mixed to form the oil phase; glycerin and triethanolamine are added to 35 parts of deionized water and heated to 70 degrees Celsius to form the aqueous phase; the oil phase is slowly added to the aqueous phase while stirring to emulsify; the mixture is cooled to 40 degrees Celsius and has been pre-dissolved in grapefruit peel polysaccharide and fragrance added to 15 parts of deionized water; stirring is continued until room temperature to form a cream.

[0050] Example 2: Enzymatic Modification Experiment of Grapefruit Peel Polysaccharide

[0051] The following enzyme modification experiments were conducted using the same batch of grapefruit peel polysaccharides prepared as described in Example 1 (condition exploration experiments are not shown): All enzymes used in Example 2 were food-grade.

[0052] Prepare a polysaccharide concentration of 5 mg / mL. Based on previous experiments, add pectinase, α-amylase, and β-amylase to a concentration of 100 U / mL, and treat at pH 6.0 and 40°C for 2 hours; or add polygalacturonase, pectin esterase, and pectin lyase to a concentration of 80 U / mL, and treat at pH 6.0 and 40°C for 2 hours; or add arabinofuranase and xylanase to a concentration of 200 U / mL, and treat at pH 7.0 and 40°C for 3 hours. After treatment, inactivate the enzymes at 80°C for 3 minutes; after cooling, reconcentrate and freeze-dry.

[0053] Example 3: Effects of enzyme modification on the antioxidant effect of grapefruit peel polysaccharide and its efficacy in treating a psoriasis cell model.

[0054] The effect of grapefruit peel polysaccharide on a psoriasis cell model was determined using the method described in Example 1. Based on previous observations of culture medium viscosity, a polysaccharide concentration of 20 mg / mL was selected in the culture medium. The results are as follows: Figure 1 As shown, grapefruit peel polysaccharides can effectively inhibit the growth of HaCat cells. Among the enzyme-modified polysaccharides, except for arabinofuranylase which is basically ineffective, the modification of other enzymes can improve the inhibitory effect compared with unmodified polysaccharides, especially pectinase and polygalacturonase.

[0055] The antioxidant effects of grapefruit peel polysaccharides and enzyme-modified grapefruit peel polysaccharides were determined using the method described in Example 1, and the results are as follows: Figure 2 As shown, compared with unmodified polysaccharides, modification with several other enzymes can improve the antioxidant effect of polysaccharides.

[0056] Compared to liquid daily necessities such as shampoo, shower gel, and hand sanitizer, creams with higher concentrations of active ingredients and longer retention on the skin are a better choice for maximizing the therapeutic effects of grapefruit peel polysaccharides. The applicant subsequently used the method in Example 1 to prepare creams using pectinase-modified grapefruit peel polysaccharides and polygalacturonase-modified grapefruit peel polysaccharides. However, during preparation, the applicant found that the creams prepared from these two polysaccharides had poor stability, especially at concentrations above 5%. After about a week of refrigerated storage in the laboratory, visible liquid precipitation and uneven distribution easily occurred. This situation seriously affected the application scope of grapefruit peel polysaccharides.

[0057] Example 4: Study on the emulsification stability of enzyme-modified grapefruit peel polysaccharide

[0058] Literature review revealed that grapefruit peel polysaccharides exhibit good stability under acidic conditions, but poor emulsification stability under neutral or alkaline conditions, which are suitable for psoriasis creams. Therefore, the applicant expanded the selection range of enzymatically modified polysaccharides to seek enzymatic modification methods with better emulsification stability, with the following results: Figure 3 As shown, consistent with previous findings, pectinase-modified and polygalacturonase-modified grapefruit peel polysaccharides exhibited poor emulsification stability (lower than the unmodified form), while xylanase-modified grapefruit peel polysaccharides performed exceptionally well in this regard. Therefore, they were used to prepare creams.

[0059] After the cream was prepared, no visually visible instability was observed during normal laboratory storage at room temperature or under refrigeration (the standard pharmaceutical stability test has not yet been completed), indicating that this polysaccharide is suitable for preparing a cream.

[0060] Example 5: Therapeutic effect of grapefruit peel polysaccharide on a mouse model of psoriasis

[0061] Cream products containing no grapefruit peel polysaccharide, xylanase-modified grapefruit peel polysaccharide, and unmodified grapefruit peel polysaccharide were prepared according to the description in Example 1. Then, a mouse model of psoriasis was tested according to the protocol described in Example 1. The results are shown in Table 2.

[0062] Table 2. Scores of the mouse psoriasis model after 7 days of treatment (n=10 per group, all surviving).

[0063] Average score on day 0 Average score on day 7 blank 12.5 9.7 Xylanase-modified grapefruit peel polysaccharide 13.1 3.2 Unmodified grapefruit peel polysaccharides 12.7 6.4

[0064] The results showed that although the antioxidant activity and cell model performance were not as good as enzyme-modified polysaccharides such as pectinase, the xylanase-modified, emulsified, and stable polysaccharides could be used to prepare high-concentration creams, and they also achieved better results than unmodified polysaccharides in mouse model experiments. A horizontal comparison with the scoring results of a shower gel application submitted on the same day also showed better results (if subsequent evidence shows no significant irritation, and based on actual usage, the effect is expected to be further enhanced by not washing off the cream after use). In addition, creams also have the advantages of small size and weight, portability, and ease of use.

[0065] Subsequent trials with actual psoriasis patients also demonstrated the good effects of the cream, with over 80% of patients who used it daily experiencing relief within one month.

Claims

1. The application of enzyme-modified grapefruit peel polysaccharide in the preparation of an ointment formulation for treating psoriasis, wherein the enzyme-modified grapefruit peel polysaccharide is prepared according to the following method: (1) Prepare a grapefruit peel polysaccharide solution with a concentration of 5 mg / mL; (2) Add xylanase to 200 U / mL and treat at pH 7.0 and 40 degrees Celsius for 3 hours; (3) Inactivate the enzyme, cool, concentrate and freeze dry to obtain enzyme-modified grapefruit peel polysaccharide; The grapefruit peel polysaccharide was prepared by the following method: grapefruit peel was dried, pulverized, and passed through a 100-mesh sieve; deionized water was added at a mass / volume ratio of 1:30 to adjust the pH to 5.0; extraction was carried out at 100 degrees Celsius for 2 hours; after cooling, it was filtered; 3 times the volume of ethanol was added to precipitate the polysaccharide; the precipitate was collected by centrifugation, concentrated, and freeze-dried to obtain grapefruit peel polysaccharide.

2. The application according to claim 1, wherein the cream dosage form is prepared using the following raw materials in parts by weight: 50 parts deionized water, 0.2 parts triethanolamine, 8 parts stearic acid, 5 parts glyceryl stearate, 5 parts petrolatum, 15 parts glycerin, 15 parts liquid paraffin, 0.2 parts fragrance, and 8 parts enzyme-modified grapefruit peel polysaccharide.

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