阅读说明：本技术 光保护性植物来源组合物 (Photoprotective plant-derived compositions ) 是由 肖莎娜·本·瓦利德 盖伊·科恩 于 2020-02-06 设计创作，主要内容包括：本文描述了一种从漆树叶子和/或萌芽果实中获得UV吸收材料,可选地为1,2,3,4,6-五没食子酰葡萄糖的方法,以及根据所述方法获得的UV吸收材料,和包括这种UV吸收材料的组合物。该方法包括将漆树叶子和/或萌芽果实与水混溶性有机溶剂接触,并除去水混溶性有机溶剂。本文所述的组合物,其中UV吸收材料的浓度为至少0.005mg/ml的防晒组合物,以及药物和/或化妆品组合物。(Described herein is a method of obtaining a UV absorbing material, optionally 1,2,3,4, 6-pentagalloylglucose, from sumac leaves and/or germinating fruits, as well as UV absorbing materials obtained according to said method, and compositions comprising such UV absorbing materials. The method comprises contacting the leaves and/or germinating fruits of the Lacca Pacifica with a water-miscible organic solvent and removing the water-miscible organic solvent. Compositions as described herein, wherein the concentration of UV absorbing material is at least 0.005mg/ml of a sunscreen composition, and pharmaceutical and/or cosmetic compositions.)

1. A sunscreen composition comprising a UV absorbing material and a dermatologically acceptable carrier, wherein the UV absorbing material is extracted from leaves and/or germinating fruits of sumac (Rhus spp.) and the concentration of said UV absorbing material in the composition is at least 0.005 mg/ml.

2. The composition of claim 1, wherein the concentration of the UV absorbing material in the composition is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 1 mm.

3. The composition as claimed in claim 1, wherein the Rhus verniciflua comprises Rhus occidentalis (Rhus coriaria).

4. The composition of any one of claims 1 to 3, wherein the UV absorbing material comprises at least one compound having a glucose moiety with a plurality of galloyl substituents.

5. The composition of claim 4, wherein the at least one compound comprises 1,2,3,4, 6-pentagalloylglucose.

6. The composition of any one of claims 1 to 5, wherein the UV absorbing material is obtained by a process comprising contacting the Lacca tree leaves and/or germinating fruits with a water miscible organic solvent and removing the water miscible organic solvent, thereby obtaining the UV absorbing material.

7. The composition of claim 6, wherein the water-miscible organic solvent comprises C_1-4An alcohol.

8. The composition of claim 7, wherein the water-miscible organic solvent comprises ethanol, acetone, and/or glycerol.

9. The composition of any one of claims 6 to 8, wherein the contacting comprises extraction achieved using a Soxhlet extractor.

10. The composition of any one of claims 6 to 9, wherein said contacting is achieved using a ratio of 5 to 40ml of said water miscible organic solvent per gram dry weight of said jatropha leaves and/or germinating fruit.

11. The composition of any one of claims 6 to 10, wherein the method further comprises partitioning the UV absorbing material in a polar solvent and a non-polar solvent, and collecting the fraction partitioned into the polar solvent.

12. The composition of claim 11, wherein the non-polar solvent comprises an alkane.

13. The composition of claim 12, wherein the alkane comprises hexane.

14. The composition of any one of claims 11 to 13, wherein the polar solvent comprises water.

15. The composition of any one of claims 11 to 14, wherein the method further comprises partitioning the fraction partitioned into the polar solvent into water and a water-immiscible polar organic solvent, and collecting the fraction partitioned into the water-immiscible polar organic solvent.

16. The composition of claim 15, wherein the water-immiscible polar organic solvent comprises an ester.

17. The composition of claim 16, wherein the ester comprises ethyl acetate.

18. The composition of any one of claims 15 to 17, wherein the method further comprises effecting crystallization of the UV absorbing material by contacting the fraction dispensed into the water-immiscible polar organic solvent with a solvent comprising water.

19. The composition of any one of claims 6 to 18, wherein the method further comprises purifying the UV absorbing material by column chromatography.

20. A sunscreen composition comprising 1,2,3,4, 6-pentagalloylglucose isolated from leaves and/or germinating fruits of sumac (Rhus spp.) and a dermatologically acceptable carrier, wherein said 1,2,3,4, 6-pentagalloylglucose is present in the composition at a concentration of at least 0.005 mg/ml.

21. The composition of claim 20, wherein the concentration of the UV absorbing material in the composition is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 1 mm.

22. A pharmaceutical or cosmetic composition comprises a UV absorbing material extracted from leaves and/or germinating fruits of Rhus verniciflua (Rhus spp.) and a dermatologically acceptable carrier.

23. The composition of claim 22, wherein the Rhus verniciflua comprises Rhus occidentalis (Rhus coriaria).

24. The composition of any one of claims 22 to 23, comprising at least one compound having a glucose moiety with multiple galloyl substituents.

25. The composition of claim 24, wherein the at least one compound comprises 1,2,3,4, 6-pentagalloylglucose.

26. The composition according to any one of claims 22 to 25, for use in the treatment of a condition selected from the group consisting of skin aging and wounds.

27. A composition as claimed in any one of claims 22 to 25 wherein the cosmetic composition is a skin rejuvenating composition and/or an exfoliating composition.

28. A method of obtaining a UV absorbing material, the method comprising contacting leaves and/or germinating fruits of sumac (Rhus spp.) with a water miscible organic solvent and removing said water miscible organic solvent, thereby obtaining said UV absorbing material.

29. The method of claim 28, wherein the water-miscible organic solvent comprises C_1-4An alcohol.

30. The method of claim 29, wherein the water-miscible organic solvent comprises ethanol, acetone, and/or glycerol.

31. The method of any one of claims 28 to 30, wherein the contacting comprises extraction achieved using a soxhlet extractor.

32. The method of any one of claims 28 to 31, wherein said contacting is achieved using a ratio of 5 to 40ml of said water miscible organic solvent per gram dry weight of said jatropha leaves and/or germinating fruit.

33. The method of any one of claims 28 to 32, further comprising partitioning the UV absorbing material in a polar solvent and a non-polar solvent, and collecting fractions partitioned into the polar solvent.

34. The method of claim 33, wherein the non-polar solvent comprises an alkane.

35. The method of claim 34, wherein the alkane comprises hexane.

36. The method of any one of claims 33 to 35, wherein the polar solvent comprises water.

37. The method of any one of claims 33 to 36, further comprising partitioning the fraction partitioned into the polar solvent into water and a water-immiscible polar organic solvent, and collecting the fraction partitioned into the water-immiscible polar organic solvent.

38. The method of claim 37, wherein the water-immiscible polar organic solvent comprises an ester.

39. The method of claim 38, wherein the ester comprises ethyl acetate.

40. The method of any one of claims 37 to 39, further comprising effecting crystallization of the UV-absorbing material by contacting the fraction dispensed into the water-immiscible polar organic solvent with a solvent comprising water.

41. The method of any one of claims 28 to 40, further comprising purifying the UV absorbing material by column chromatography.

42. A method according to any one of claims 28 to 41 wherein the Rhus verniciflua comprises Rhus occidentalis (Rhus coriaria).

43. The method of any one of claims 28 to 42, wherein the UV absorbing material comprises at least one compound having a glucose moiety with a plurality of galloyl substituents.

44. The method of claim 43, wherein the at least one compound comprises 1,2,3,4, 6-pentagalloylglucose.

45. The method of claim 44, wherein the purity of the 1,2,3,4, 6-pentagalloylglucose in the UV-absorbing material is at least 95 weight percent.

46. UV absorbing material obtained by the method according to any one of claims 28 to 45.

Technical field and background

The present invention, in some embodiments thereof, relates to photoprotection and, more particularly, but not exclusively, to UV blocking compositions obtainable from plant materials.

Chronic exposure to solar radiation, particularly chronic exposure to the ultraviolet b (uvb) region thereof, directly affects the epidermal surface and lining, which is associated with a variety of pathophysiological changes, including sunburn, erythema, modulation of immune responses, and increased extrinsic skin aging [ Matsurama & atherwamy, Toxicol Appl Pharmacol 2004,195: 298-; kraemer, Proc Natl Acad Sci USA 1997,94: 11-14; lavker et al, J Am Acad Dermatol 1995,32: 53-62; Portugal-Cohen et al, Exp Dermatol 2009,18:781-789 ]. Importantly, UVB can also damage skin and damage DNA of epidermal cells by inducing apoptosis, which is considered to be one of the major risk factors for the development of skin Cancer [ Tuorkey, Eur J Cancer Prev 2015,24: 430-.

Sunscreens are used to protect against solar radiation, not only as an important tool against skin cancer, but also to alleviate other skin diseases caused by solar radiation, such as aging, wrinkle formation, undesirable pigmentation and collagen loss [ Mancebo et al, Dermatol Clin 2014,32:427-428 ]. However, it has been reported that the commercial sunscreens currently used are capable of penetrating and penetrating the skin, reaching the circulatory system and causing systemic damage to the body [ Touitou & Godin, Clin Dermatol 2008,26: 375-. Skin treated with sunscreens and subsequently exposed to UV radiation shows paradoxical exacerbation of ROS (reactive oxygen species) [ Hanson et al, Free Radic Biol Med 2006,41: 1205-. This indicates both the ability of the sunscreen to penetrate the outermost skin layer and that certain sunscreens have a radical-philic effect. Furthermore, sunscreens and their degradation byproducts were detected in urine and breast milk of individuals using sunscreen-containing cosmetics [ Hayden et al, Lancet 1997,350: 863-one 864 ]. The ability of some sunscreens to bind and activate estrogen or thyroid receptors has also been reported [ Gilbert et al, Int J Cosmet Sci 2013,35:208-219 ].

Sunscreens exhibit significant toxicity to human users, but also pose an ecological and survival threat to corals and coral reefs, which are contaminated by the sunscreens on swimmers or carried in the wastewater [ Danovaro et al, Environ Health Persport 2008,116: 441-.

The term "lacquer tree" (meaning red in the Syrian language) refers to plants of the genus Rhus verniciflua (Rhus) and related genera in the family Anacardiaceae (Anacardiiae).

The Rhus coronaria (Rhus coriaria) is a particularly well-known species of Rhus verniciflua, usually 1 to 3 meters tall shrub or small tree, with odd-feathered leaves, 9 to 15 lobules, compact and erect panicle, small and green white flowers, and the fruit is drupes with long villi, red, 1 seed. The Rhus occidentalis is grown wildly in any type of deep and well-drained soil in the Mediterranean summer shrub communities and forests.

The main use of Rhus verniciflua is as a spice, which is popular in the middle east and is made from its sour fruit. Immature fruits and seeds are also consumed. In addition, Rhus verniciflua contains pigments and tannins for dyeing and tanning fine leather and for dyeing protein-based textile materials such as silk and wool [ Shabbir, J Animal Plant Sci 2012,22:505-]. Dyes of various colors can be prepared from different parts of the plant. In addition, the oil extracted from the seeds can be used to make candles. Sumac is reported to contain hydrolyzable tannins (including gallotannins), volatile oils, anthocyanins, gallic acid, and flavonoids such as myricetin, quercetin, and kaempferol [ Mavlyanov et al, Chem Nat Comp 1997,33: 209;&Koyuncu,Turkish J Med Sci 1994,20:11-13；el-Sissi et al.,Planta Med 1972,21:67-71；Mehrdad et al.,J AOAC Int 2009,92:1035-1043]。

rhus verniciflua is reported to be resistant to bacteria [ Adwan et Al, Asian Pac J Trop Med 2010, 266-.

U.S. patent application publication No. 2010/0215630 describes the use of extracts of fennel, astragalus, coriander, cinnamon, clove, dill, fenugreek, feverfew, kudzu, licorice, magnolia, marjoram, oregano, paprika, mint, popcorn tree, rosemary, sage, spearmint, scutellaria, john's wort, sumac, tarragon, thyme, and/or valerian to protect insect microbial preparations from ultraviolet radiation to maintain the efficacy of insect pathogens.

The term "tannin" refers to a polyphenol biomolecule capable of forming strong complexes with various molecules, typically through hydroxyl or carboxyl groups in the tannin.

Plant-derived tannins generally belong to one of two main classes: hydrolysable tannins, including a core carbohydrate esterified with phenolic groups (from gallic acid or ellagic acid); and condensed tannins, formed by the polycondensation of flavonoids. Hydrolysable tannins including gallic acid esters are also known as "gallotannins".

The term "tannic acid" includes tannins, which are polygalloyl esters of glucose or quinic acid. Commercially available tannic acid is usually extracted from Tara (Tara spinosa) pods, gallnut from chinese sumac (Rhus chinensis) or aleppo oak (Quercus infetoria), or tanned sumac (Rhus coriaria) leaves. The number of galloyl moieties will generally be in the range of 2 to 12, depending on the source, although the formula for tannic acid is generally described by convention as that for decagalloylglucose.

Over 500 molecules consisting of galloyl esters of glucose have been identified in more than 20 plant families, ranging from very simple 1-mono-galloyl- β -glucose (galloylglucose) (molecular weight 332Da) to complex polymers with molecular weights in excess of 4000 Da.

Rothman and Henningsen [ J Invest Dermatol 1947,9: 307-.

U.S. patent No. 4,104,368 describes a composition for providing conditioning and sun protection comprising a long chain quaternary ammonium salt and an acidic moiety that provides sun protection, such as p-aminobenzoic acid and its derivatives, salicylic acid and its derivatives, malonic acid and its derivatives, cinnamic acid and its derivatives, tannic and gallic acids, naphthol sulfonic acids and anthranilic acids.

Tannic acid is also included in the list of UV filters used in sunscreen formulations in U.S. patent nos. 5,169,624, 8,703,753, 9,737,472 and 10,064,797.

U.S. patent No. 10,111,821 describes phototherapy by applying electromagnetic radiation of a particular wavelength, using a filtered portion of the electromagnetic radiation spectrum of a topical composition. Various ultraviolet absorbing molecules are described, including pentagalloylglucose.

U.S. patent No. 7,776,915 describes a topical composition for improving the appearance of aged skin comprising a lipoid acid, carnitine and carnosine, and optionally other agents such as antioxidants, anti-glycation agents, collagen enhancers, mitochondrial resuscitation agents, thioredoxin, glutathione, NADH, anti-inflammatory agents, depigmentation agents, skin protection lipids and sunscreens. Wherein pentagalloylglucose is included in the long list of anti-inflammatory agents.

U.S. patent No. 4,741,915 describes the use of gallotannins such as pentagalloylglucose as antioxidants in foods and cosmetics such as shaving foam, aftershave lotion, body lotion, make-up removing lotion, face cream, sun block lotion, and facial mask.

Other background art includes Berardini et al [ Rapid Commun Mass Spectrum 2004,18: 2208-; chuarienthong et al [ Int J Cosmet Sci 2010,32:99-106 ]; cohen et al [ Negev, Dead Sea and Arava students 2015,7:66-74 ]; haddock et al [ J Chem Soc Perkin Trans 11982, 0: 2535-; nichols and Katiyar [ Arch Dermatol Res 2010,302:71-83 ]; ozer et al [ J Ethnopharmacol 2015,161:86-91 ]; schuch et al [ Free Rad Biol Med 2017,107:110-124 ]; schwack and Rudolph [ J Photochem Photobiol B Biol1995,28:229-234 ]; and Wineman et al [ J Herbal Med 2015,5: 199-.

Disclosure of Invention

According to an aspect of some embodiments of the present invention there is provided a composition comprising a material extracted from leaves and/or germinating fruits of Rhus verniciflua (Rhus spp.) wherein the material absorbs ultraviolet and/or blue light.

According to an aspect of some embodiments of the present invention there is provided a sunscreen composition comprising UV absorbing material and a dermatologically acceptable carrier, wherein the UV absorbing material is extracted from the leaves and/or germinating fruit of sumac (Rhus spp.) and the concentration of UV absorbing material in the composition is at least 0.005 mg/ml.

According to an aspect of some embodiments of the present invention there is provided a sunscreen composition comprising 1,2,3,4, 6-pentagalloylglucose isolated from the leaves and/or germinating fruits of Rhus verniciflua (Rhus spp.) and a dermatologically acceptable carrier, wherein the concentration of 1,2,3,4, 6-pentagalloylglucose in the composition is at least 0.005 mg/ml.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical or cosmetic composition comprising a UV absorbing material extracted from leaves and/or germinating fruits of sumac (Rhus spp.) and a dermatologically acceptable carrier.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical or cosmetic composition comprising 1,2,3,4, 6-pentagalloylglucose isolated from the leaves and/or germinating fruits of Rhus verniciflua (Rhus spp.) and a dermatologically acceptable carrier.

According to an aspect of some embodiments of the present invention, there is provided a method of obtaining a UV absorbing material, the method comprising contacting leaves and/or germinating fruits of sumac (Rhus spp.) with a water miscible organic solvent and removing the water miscible organic solvent, thereby obtaining the UV absorbing material.

According to an aspect of some embodiments of the present invention, there is provided a method of obtaining 1,2,3,4, 6-pentagalloyl glucose, the method comprising contacting leaves and/or germinating fruits of Rhus verniciflua (Rhus spp.) with a water-miscible organic solvent, removing the water-miscible organic solvent, thereby obtaining 1,2,3,4, 6-pentagalloyl glucose.

According to an aspect of some embodiments of the invention, there is provided a UV absorbing material obtained according to the method described herein according to any of the respective embodiments.

According to an aspect of some embodiments of the invention there is provided 1,2,3,4, 6-pentagalloylglucose obtained according to the method described herein according to any of the respective embodiments.

According to some of any of the embodiments described herein, the paint tree comprises cercidia (Rhus coriaria).

According to some of any of the embodiments described herein with respect to the composition, the concentration of the UV absorbing material in the composition is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 1 mm.

According to some of any of the embodiments described herein, the UV absorbing material comprises at least one compound having a glucose moiety with a plurality of galloyl substituents.

According to some of any of the embodiments described herein with respect to at least one compound having a glucose moiety with multiple galloyl substituents, the at least one compound comprises 1,2,3,4, 6-pentagalloyl glucose.

According to some of any of the embodiments described herein with respect to a UV absorbing material comprising 1,2,3,4, 6-pentagalloylglucose, the purity of the 1,2,3,4, 6-pentagalloylglucose in the UV absorbing material is at least 95 weight percent.

According to some of any of the embodiments described herein with respect to a pharmaceutical or cosmetic composition, the composition is for use in treating a condition selected from the group consisting of skin aging and a wound.

According to some of any of the embodiments described herein with respect to a cosmetic composition, the cosmetic composition is a skin rejuvenating composition and/or an exfoliating composition.

According to some of any of the embodiments described herein with respect to UV absorbing materials, the UV absorbing material may be obtained by a method comprising contacting leaves and/or germinating fruits of a lacquer tree with a water miscible organic solvent and removing the water miscible organic solvent, thereby obtaining the UV absorbing material.

According to some of any of the embodiments described herein with respect to the method, the water-miscible organic solvent comprises C_1-4An alcohol.

According to some of any of the embodiments described herein with respect to the method, the water-miscible organic solvent comprises ethanol, acetone, and/or glycerol.

According to some of any of the embodiments described herein with respect to the method, contacting the lacquer tree leaves and/or germinating fruits with the water miscible organic solvent comprises an extraction achieved using a soxhlet extractor.

According to some of any of the embodiments described herein with respect to the method, contacting the lacquer tree leaves and/or germinating fruits with the water miscible organic solvent is achieved using a ratio of 5 to 40ml of water miscible organic solvent per gram of dry weight of the lacquer tree leaves and/or germinating fruits.

According to some of any of the embodiments described herein with respect to the method, the method further comprises partitioning the UV absorbing material in a polar solvent and a non-polar solvent, and collecting the fraction partitioned into the polar solvent.

According to some of any of the embodiments described herein with respect to the method comprising partitioning in a polar solvent and a non-polar solvent, the non-polar solvent comprises an alkane.

According to some of any of the embodiments described herein with respect to a method comprising partitioning in a polar solvent and a non-polar solvent (comprising an alkane), the alkane comprises hexane.

According to some of any of the embodiments described herein with respect to the method comprising partitioning in a polar solvent and a non-polar solvent, the polar solvent comprises water.

According to some of any of the embodiments described herein with respect to the method comprising partitioning in a polar solvent and a non-polar solvent, the method further comprises partitioning the fraction partitioned into the polar solvent into water and a water-immiscible polar organic solvent, and collecting the fraction partitioned into the water-immiscible polar organic solvent.

According to some of any of the embodiments described herein with respect to the method comprising dispensing in water and a water-immiscible polar organic solvent, the water-immiscible polar organic solvent comprises an ester.

According to some of any of the embodiments described herein with respect to a method comprising partitioning in water and a water-immiscible polar organic solvent (including an ester), the ester comprises ethyl acetate.

According to some of any of the embodiments described herein with respect to the method comprising dispensing in water and a water-immiscible polar organic solvent, the method further comprises effecting crystallization of the UV absorbing material by contacting the fraction dispensed into the water-immiscible polar organic solvent with a solvent comprising water.

According to some of any of the embodiments described herein with respect to the method, the method further comprises purifying the UV absorbing material by column chromatography.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be necessarily limiting.

Drawings

Some embodiments of the invention are described herein by way of example only and with reference to the accompanying drawings. Referring now in specific detail to the drawings, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the embodiments of the present invention. In this regard, the description taken with the drawings make it apparent to those skilled in the art how the embodiments of the invention may be practiced.

In the figure:

FIG. 1 is a bar graph showing SPF values of ethanol extracts (0.1mg/ml) of shoots, twigs, leaves, germinating fruits, mature fruits and roots of Rhus coriander (Rhus coriaria) from different geographical locations (Israel).

FIG. 2 schematically depicts a method of extracting an exemplary UV-absorbing substance (SH-101) from leaves of Sicilaria occidentalis according to an exemplary embodiment of the present invention.

Figure 3 is a graph showing the SPF value and percent yield of material extracted from 50 grams of dry sumac leaf sub-powder as described in figure 2 as a function of the volume of ethanol (in ml) used to obtain the ethanol extract.

Fig. 4A to 4C show HPLC chromatograms (absorption at 290 nm) of an exemplary UV absorbing substance (SH-401) extracted from the leaves of a cisapricots tree according to some embodiments of the present invention (fig. 4A and 4B), and UV absorption spectra of the exemplary substance (fig. 4C) associated with the main peak of the HPLC chromatograms (the results shown represent 3 experiments).

FIG. 5 depicts the structure of an exemplary UV absorbing compound (pentagalloylglucose) extracted from leaves of Sicilaria occidentalis as determined by NMR analysis.

Fig. 6 is a bar graph showing SPF values (n ═ 3) at a concentration of 0.01mg/ml for an exemplary substance isolated from sumac tree (SH-401), crude sumac tree extract, and commercial UVB filter.

Fig. 7 is a bar graph showing SPF values of SH-401 (at a concentration of 0.01 mg/ml) as a function of the time of exposure of the SH-101 film to UV radiation (n ═ 3).

FIGS. 8A-8C show graphs showing exposure of cells to UVB radiation (350 mJ/cm)²) After 24 hours, the mixture is used in advance at a concentration of 0.02, 0.1, 0.2, 1 or 2. mu.g/cm²SH-401 or commercial SPF 30 composition (drEpidermal cell viability (determined by MTT method) of human skin explants after incubation with SH-401 in explants as a function of SH-401 (compound) concentration (fig. 8A), as well as apoptosis levels (fig. 8B) and TNF α (fig. 8C) (. p)<0.05 relative to the non-irradiated vehicle control (0); # p<0.05 relative to irradiated vehicle control (UV); n-3).

FIG. 9 is a bar graph showing the use of 0, 0.02, 0.1, 0.2, 1 or 2 μ g/cm with or without UVB radiation²In SH-101-treated skin explants, the level of ROS (reactive oxygen species) production (in arbitrary units) (n-3;. p) determined by DCFDA (dichlorofluorescein diacetate) determination<0.05 relative to UVB and 0. mu.g/cm²SH-101 treatment of).

FIG. 10 is a bar graph showing the use of 0, 0.02, 0.1, 0.2, 1 or 2 μ g/cm with or without UVB radiation²In SH-101-treated skin explants, the level of lipid peroxidation determined by ELISA assay as measured by MDA (malondialdehyde) concentration (n-3;. p)<0.05 relative to UVB and 0. mu.g/cm²SH-101 treatment of).

FIG. 11 is a bar graph showing the ability of SH-401 to scavenge active oxygen (measured in micromolar Trolox equivalent units) at concentrations of 0.1, 0.5 or 1 weight percent, as determined based on the color shift of DPPH (diphenylpicrylhydrazine) (0% SH-401 carrier as control; n-3;. p <0.05 vs. carrier control).

FIG. 12 shows UVB radiation (350 mJ/cm)²) Treating skin explants and/or 2. mu.g/cm²After incubation with SH-401 (compound) of (1), images of epidermal cells after analysis of COMET DNA fragmentation (after treatment of the skin, the epidermis was stripped and single cell preparations were obtained with EDTA; images represent 3 experiments; arrows indicate the "tails" of damaged cells).

FIG. 13 is a bar graph showing exposure to 0, 0.01, 0.02, 0.2, or 2 μ g/cm²After SH-401, at a concentration of 350mJ/cm²In isolated epidermal cells of UVB-irradiated or non-irradiated skin explants, the level of Cyclobutane Pyrimidine Dimer (CPD) determined by ELISA (n-3;. p)<0.05 vs. non-irradiatedVector control, # p<0.05 relative to irradiated vehicle control).

FIGS. 14A to 14D show a sample subjected to 350mJ/cm²Of (a) UVB radiation (fig. 14B and 14D) or a histological image of a skin explant not subjected to UVB radiation (fig. 14A and 14C) with SH-401 (fig. 14C and 14D) or with no SH-401 (fig. 14A and 14B) (control ═ control sample not exposed to SH-401 or UVB; arrows in fig. 14B emphasize changed features; all images represent triplicate samples).

FIGS. 15A and 15B present histograms showing the levels of procollagen (FIG. 15A) and MMP1 (FIG. 15B) determined by ELISA in skin explants subjected to UVB radiation and SH-101 at the indicated concentrations, or to radiation without SH-101 (UVB) (control: vehicle control sample not exposed to SH-401 or UVB;. p <0.05 vs. vehicle control;. p <0.05 vs. UVB only; n: 3).

Fig. 16 shows microscopic images of fused HaCaT cells with induced wounds (between two vertical lines) exposed to SH-101 or vehicle (ethanol) for 24 hours (control ═ control sample not exposed to SH-401 or vehicle; all images represent triplicate samples).

Detailed Description

The present invention, in some embodiments thereof, relates to photoprotection and, more particularly, but not exclusively, to UV blocking compositions obtainable from plant materials.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The present inventors have discovered that natural UV absorbing materials in sumac, which are particularly effective agents for blocking harmful UV radiation (e.g. sunscreens), surprisingly exhibit other beneficial effects on skin cells, such as anti-aging and wound healing effects.

While reducing the present invention to practice, the inventors have also discovered an effective method for isolating specific UV absorbing compounds, such as pentagalloylglucose.

Referring now to the drawings, FIG. 1 shows that the ethanol extract of the leaves and germinating fruit of Rhus verniciflua blocks harmful UV radiation to a greater extent than other parts of the plant.

FIG. 2 illustrates a method of extracting an exemplary UV-absorbing substance (SH-101) from the leaves of a lacquer tree according to an exemplary embodiment of the present invention. Fig. 4A to 4B show the purity and UV absorption of SH-401. Figure 3 shows the effect of ethanol volume on the extraction efficiency of UV absorbing materials.

FIG. 5 shows the structure of SH-401 (pentagalloylglucose) as determined by extensive spectral evidence. Figure 6 shows that SH-401 has better efficacy as a sunscreen (as measured by SPF values) than commercial sunscreens. Fig. 7 shows that SH-401 is stable after exposure to UV radiation.

Fig. 8A shows that SH-401 is non-toxic to human skin cells, and fig. 8B, fig. 8C, and fig. 14A to fig. 14D show that SH-401 protects human skin cells from UVB radiation. Fig. 9 and 10 show that SH-401 reduces ROS (reactive oxygen species) generation and lipid peroxidation in human skin. FIG. 11 shows that SH-401 is an antioxidant. Fig. 12 and 13 show that SH-401 reduces UV-induced DNA damage in skin cells. Fig. 15A-15B show that SH-401 reverses UV-induced collagen degradation in skin.

Composition (A):

according to an aspect of an embodiment of the present invention, there is provided a composition comprising a UV absorbing material and a dermatologically acceptable carrier, wherein the UV absorbing material is extracted from leaves of sumac and/or sprouted fruits.

Throughout this document, the term "UV absorption" refers to the absorption of electromagnetic radiation in at least a portion of the wavelengths in the range of 200nm to 500 nm. The sub-range of 400nm to 500nm is also referred to herein as "blue light" and for the sake of brevity, absorption of such wavelengths is included within the scope of the term "UV absorption".

In some of any of the respective embodiments described herein, the term "UV absorption" refers to absorption of at least a portion of wavelengths in the range of 280nm to 500 nm. In some such embodiments, the term "UV absorption" refers to absorption of at least a portion of wavelengths in the range of 280nm to 400nm, also referred to herein as "ultraviolet B" (UVB) (280nm to 320nm) and "ultraviolet a" (UVA) (320nm to 400 nm). In some such embodiments, the term "UV absorption" refers to absorption of at least a portion of wavelengths in the range of 280nm to 320nm (i.e., UVB). In some such embodiments, the term "UV absorption" refers to absorption of at least a portion of wavelengths in the range of 290nm to 320nm (i.e., UVB).

In some of any of the respective embodiments described herein, the term "UV absorption" refers to absorption of at least a portion of wavelengths in the range of 300nm to 500 nm. In some such embodiments, the term "UV absorption" refers to absorption of at least a portion of wavelengths in the range of 300nm to 400nm, which wavelengths are also referred to in the art as "near ultraviolet".

In some of any of the respective embodiments described herein, the term "UV absorption" refers to absorption of blue light, i.e., absorption of at least a portion of wavelengths in the range of 400nm to 500 nm. Materials that absorb such wavelengths may have a clearly visible color, such as yellow.

In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.005 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.01 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.02 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.05 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.1 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.2 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 0.5 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 1 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 2 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 5 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 10 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 20 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 50 mg/ml. In some embodiments, the concentration of the UV absorbing material in the composition is at least 100 mg/ml.

In some embodiments, the concentration of the UV-absorbing material in the composition is no more than 400mg/ml, optionally no more than 200mg/ml, optionally no more than 100mg/ml, optionally no more than 50mg/ml, optionally no more than 20mg/ml, and optionally no more than 10 mg/ml.

In some embodiments, the concentration of the UV-absorbing material in the composition ranges from about 0.05 to about 10mg/ml, or from about 10mg/ml to about 50mg/ml, or from about 50 to about 200mg/ml, or from about 100 to about 400mg/ml, including any intermediate values and subranges therebetween.

In some of any of the embodiments with respect to a composition, the composition is a sunscreen composition.

Herein, the phrase "sunscreen composition" refers to a composition that, when applied as a thin layer on the skin, at least partially blocks or shields UV radiation from the sun (typically radiation in the range of 290 to 320nm, and optionally in a broader wavelength range, e.g. radiation in the range of 290 to 400nm) by absorbing and/or reflecting the UV radiation. The sunscreen composition is optionally identified for use (e.g., in or on a packaging material in which the sunscreen composition is packaged) to block or shield UV radiation or sunlight in general, and/or to minimize sun-related damage (e.g., sunburn, cancer risk) (e.g., by topical application).

In some of any of the embodiments described herein, the concentration of the UV absorbing material in the composition is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 1 mm. In some embodiments, the concentration is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 3 mm. In some embodiments, the concentration is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 0.1 mm. In some embodiments, the concentration is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 0.03 mm. In some embodiments, the concentration is sufficient to absorb at least 90% of radiation having a wavelength of 300nm over a path length of 0.01 mm.

One skilled in the art will appreciate that (according to beer-lambert law) at least 90% of the absorption corresponds to an absorbance of at least 1 (and at least 99% of the absorption corresponds to an absorbance of at least 2, and at least 99.9% of the absorption corresponds to an absorbance of at least 3), where absorbance is the product of the attenuation coefficient (the intrinsic property of the material), the concentration, and the path length. Thus, an absorption of at least 90% over a path length of 1mm corresponds to a product of the attenuation coefficient and the concentration of at least 1mm^-1. This limitation therefore defines the concentration of a compound with a given absorption characteristic. In the case of mixtures of compounds that absorb radiation at 300nm (e.g., in the UV absorbing materials described herein), the sum of the products of the attenuation coefficient and the concentration of each compound is at least 1mm^-1。

Since a given path length (e.g., 1mm) defines the product of attenuation coefficient and concentration (e.g., at least 1mm)^-1) It will therefore be appreciated that the absorption of the material may be measured for different path lengths, for example over shorter path lengths in order to minimise scattering or absorption by other compounds in the composition. Thus, for example, an absorbance of at least 1 for a path length of 1mm may in practice optionally be determined as an absorbance of at least 10 (defined herein and in the art) for a path length of 10mm, an absorbance of 0.1 (defined herein and in the art) for a path length of 0.1mm, and so on.

In some of any of the embodiments described herein, the UV absorbing material comprises at least one compound having a plurality of galloyl (i.e., 3,4, 5-trihydroxybenzoyl) substituents, for example, 2 to 10 galloyl substituents, or 4 to 6 (e.g., 5) galloyl substituents. In some embodiments, at least one compound has a carbohydrate moiety, such as a glucose moiety, with a plurality of galloyl substituents. The carbohydrate moiety can optionally be a sugar (e.g., hexose) moiety, such as a D-glucose moiety, or a quinic acid moiety.

In some of any of the respective embodiments described herein, the galloyl substituent is connected to the other moiety via an ester bond, i.e., the galloyl group is connected to an oxygen atom of the other moiety.

The galloyl substituents may optionally each be attached to a backbone moiety (e.g., a carbohydrate moiety), or some galloyl substituents may optionally be attached directly to a backbone moiety and some galloyl substituents may be attached to other galloyl substituents. In some such embodiments, each galloyl substituent of the backbone moiety (e.g., glucose moiety) is itself in the form of a galloyl group, or a galloyl group substituted with a galloyl group (e.g., a 3, 4-dihydroxy-5- [ (3,4, 5-trihydroxybenzoyl) oxy ] benzoyl group). In some such embodiments, each galloyl substituent of the backbone moiety (e.g., glucose moiety) is itself in the form of a galloyl group.

As exemplified herein, 1,2,3,4, 6-pentagalloylglucose (including 5 galloyl moieties attached directly to different oxygen atoms of glucose) can be extracted from Rhus verniciflua at high efficiency.

In some of any of the respective embodiments, the concentration of 1,2,3,4, 6-pentagalloylglucose in the UV-absorbing material is at least 80 weight percent. In some embodiments, the concentration of 1,2,3,4, 6-pentagalloylglucose is at least 90 weight percent. In some embodiments, the concentration of 1,2,3,4, 6-pentagalloylglucose is at least 95 weight percent. In some embodiments, the concentration of 1,2,3,4, 6-pentagalloylglucose is at least 98 weight percent. In some embodiments, the concentration of 1,2,3,4, 6-pentagalloylglucose is at least 99 weight percent.

In some of any of the embodiments described herein, the UV absorbing material according to any of the respective embodiments described herein is 1,2,3,4, 6-pentagalloylglucose isolated from leaves of sumac and/or germinating fruits (e.g., according to any of the embodiments described herein with respect to isolating 1,2,3,4, 6-pentagalloylglucose from sumac). In some embodiments, the concentration of 1,2,3,4, 6-pentagalloylglucose is at least 0.005mg/ml (according to any of the respective embodiments described herein).

Throughout this document, the term "sumac" refers to Rhus spp, i.e., any plant belonging to the genus Rhus (Rhus). Examples of paint trees include, but are not limited to, Rhus chinensis (also known as Chinese sumac), Rhus delavayi, Rhus hypoleuca, Rhus punjabensis (also known as parajasens), Rhus taiitensis, Rhus sanwicensis, Rhus coriaria (also known as tanned, Siberian, or elm leaf paint), Rhus aromatic (also known as aromatic paint), Rhus copallinum (also known as winged or shiny paint), Rhus glabra (also known as glossy paint), rhus lancelolia (also known as Rhus verniciflua), Rhus michauxii (also known as Lacquer tree of Mijox), Rhus typhina (also known as Ceratoptera nigra), Rhus chloriphylla (also known as Rhus japonicas), Rhus integrifolia (also known as Citrinia lemonas), Rhus kearney (also known as Rhus Kernensis), Rhus microphylla (also known as Rhus desert or Rhus lobular Stokes), Rhus ovata (also known as Saururus chinensis), Rhus trilobata (also known as Rhus trilobata), Rhus virens (also known as Rhus verniciflua) and Russ muleri (also known as Mueller Lacquer). In an exemplary embodiment, the paint tree is Rhus coriaria (cisimi paint tree).

In some of any of the embodiments described herein, the UV absorbing material may be obtained by the method described herein, according to any of the respective embodiments.

The method comprises the following steps:

according to an aspect of an embodiment of the present invention, there is provided a method of obtaining a UV-absorbing material (e.g. a UV-absorbing material according to any of the respective embodiments described herein) from a sumac tree. The method comprises contacting the leaves and/or germinating fruits of the lacquer tree with a water miscible organic solvent (e.g. to obtain the UV absorbing material as an extract). In some embodiments, the method further comprises removing the water-miscible organic solvent, for example, by evaporation of the solvent.

The sumac leaves and/or germinating fruits are optionally dried prior to contact with the water miscible organic solvent, for example by exposure to air (optionally dry air) and/or by mild heating (e.g. at a temperature below 100 ℃, or below 75 ℃, or below 50 ℃).

In some of any of the respective embodiments described herein, the water-miscible organic solvent comprises an alcohol (optionally two or more alcohols), comprising one-OH group or more than one-OH group (e.g., ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, and/or glycerol); and/or ketones (e.g., acetone). In some embodiments, the water-miscible organic solvent includes a C1-4 alcohol (e.g., t-butanol, 1-propanol, isopropanol, ethanol, and/or methanol), acetone, and/or glycerol. According to some embodiments, ethanol is an exemplary water-miscible organic solvent.

In some of any of the respective embodiments described herein, contacting the lacquer tree leaves and/or germinating fruits with the water miscible organic solvent comprises extraction. In some such embodiments, the extraction is accomplished using a soxhlet extractor.

As used herein, the term "soxhlet extractor" refers to a device configured to recover solvent for extraction by contacting the solvent with a source (e.g., sumac) to extract the material, evaporating the solvent used to extract the material, and condensing the solvent vapor so that the vapor is returned to the contacting source, thereby further extracting the material. The term "soxhlet extractor" encompasses a variety of specific designs, including those also known in the art as "sichuan extractors" (Kumagawa extractors).

In the examples relating to soxhlet extractors, volatile water-miscible organic solvents are particularly suitable, for example solvents having a boiling point (at atmospheric pressure) of not more than 100 ℃.

In some of any of the respective embodiments described herein, the amount of water-miscible organic solvent contacted with the sumac is at least 5ml of solvent per gram (dry weight) of sumac, optionally at least 10ml of solvent per gram (dry weight) of sumac, and optionally at least 15ml of solvent per gram (dry weight) of sumac. In some such embodiments, the solvent comprises ethanol.

In some of any of the respective embodiments described herein, the amount of water-miscible organic solvent contacted with the sumac is no more than 40ml of solvent per gram (dry weight) of sumac, optionally no more than 30ml of solvent per gram (dry weight) of sumac, and optionally no more than about 20ml of solvent per gram (dry weight) of sumac. In some such embodiments, the solvent comprises ethanol.

In some of any of the respective embodiments described herein, the amount of water-miscible organic solvent contacted with the sumac is from 5 to 40ml of solvent per gram (dry weight) of sumac, and optionally from 10 to 30ml of solvent per gram (dry weight) of sumac. As exemplified herein, about 20ml of water miscible organic solvent per gram (dry weight) of Rhus verniciflua provide effective extraction. In some embodiments, the solvent comprises ethanol.

In some of any of the respective embodiments described herein, the method further comprises partitioning the UV-absorbing material (e.g., in the form of an extract obtained using a water-miscible organic solvent, according to any of the respective embodiments described herein) in a polar solvent and a non-polar solvent, and collecting the fraction partitioned into the polar solvent, e.g., to obtain a purer (more efficiently absorbing) UV-absorbing material.

In this context, the term "partitioning" refers to contacting a material with two immiscible liquids, for example, allowing different portions of the material to transfer (i.e., "partition") to different phases based on the different affinities and/or solubilities of the different components of the material in each of the two liquids. One or both phases may optionally be separated from the other phase, thereby collecting the portion that partitions into that phase.

In some of any of the respective embodiments described herein, the non-polar solvent comprises one or more hydrocarbons, and optionally one or more aliphatic hydrocarbons. In some embodiments, the non-polar solvent comprises one or more alkanes, for example, one or more alkanes having 5 to 16 carbon atoms. According to some embodiments, hexane is an exemplary non-polar solvent.

In some of any of the respective embodiments described herein, the polar solvent comprises water, for example at least 80 weight percent water, or at least 90 weight percent water, or at least 95 weight percent water, or at least 98 weight percent water, or at least 99 weight percent water. In an exemplary embodiment, the polar solvent consists essentially of water.

In some of any of the respective embodiments described herein, the non-polar solvent is an aliphatic hydrocarbon, and the polar solvent consists essentially of water.

Without being bound by any particular theory, it is believed that the non-polar solvent separates out a substantial amount of other plant derived substances, such as chlorophyll, from the UV absorbing material that partitions into the polar solvent. It is further believed that a suitable ratio of polar and non-polar solvents (e.g., as described herein) may increase the proportion of isolated plant-derived material and/or minimize the proportion of wasted UV absorbing material.

In some of any of the respective embodiments described herein, the ratio of non-polar solvent to water for dispensing is at least 0.05ml of solvent per ml of water, optionally at least 0.1ml of solvent per ml of water, optionally at least 0.15ml of solvent per ml of water, and optionally at least 0.25ml of solvent per ml of water. In some such embodiments, the solvent comprises hexane.

In some of any of the respective embodiments described herein, the ratio of non-polar solvent to water for dispensing is no more than 1ml of solvent per ml of water, optionally no more than 0.6ml of solvent per ml of water, optionally no more than 0.4ml of solvent per ml of water, and optionally no more than 0.25ml of solvent per ml of water. In some such embodiments, the solvent comprises hexane.

In some of any of the respective embodiments described herein, the ratio of non-polar solvent to water for dispensing is in the range of 0.05 to 1ml of solvent per ml of water, optionally in the range of 0.1 to 0.6ml of solvent per ml of water, optionally in the range of 0.15 to 0.4ml of solvent per ml of water, and optionally about 0.25ml of solvent per ml of water. In some such embodiments, the solvent comprises hexane.

In some of any of the respective embodiments described herein, the method further comprises dispensing the UV-absorbing material into water and a water-immiscible polar organic solvent, and collecting the fraction dispensed into the water-immiscible polar organic solvent, e.g., to obtain a purer (more efficiently absorbing) UV-absorbing material. According to any of the respective embodiments described herein, the UV absorbing material is optionally in the form of a fraction that partitions into a polar solvent (rather than a non-polar solvent).

Examples of water-immiscible polar organic solvents include, but are not limited to, esters such as C1-4 alkyl acetates (e.g., methyl acetate, ethyl acetate, isopropyl acetate, and butyl acetate) and propylene carbonate; alcohols such as 1-butanol, 2-butanol, isobutanol, pentanol, isopentanol, octanol, cyclohexanol; ketones, such as 2-butanone, methyl isobutyl ketone, acetophenone, cyclohexanone; aldehydes, such as furfural; amines, such as aniline; polar chlorinated solvents such as dichloromethane and 1, 2-dichloroethane; carbon disulfide; and nitro compounds such as nitromethane, nitropropane and nitrobenzene. According to some embodiments, ethyl acetate is an exemplary water-immiscible polar organic solvent.

In some of any of the respective embodiments described herein, the ratio of the water-immiscible polar organic solvent to water for dispensing is at least 0.3ml of solvent per ml of water, optionally at least 0.6ml of solvent per ml of water, optionally at least 1ml of solvent per ml of water, and optionally at least 1.5ml of solvent per ml of water. In some such embodiments, the solvent comprises ethyl acetate.

In some of any of the respective embodiments described herein, the ratio of the water-immiscible polar organic solvent to water for dispensing is no more than 7.5ml of solvent per ml of water, optionally no more than 4ml of solvent per ml of water, optionally no more than 2.5ml of solvent per ml of water, and optionally no more than 1.5ml of solvent per ml of water. In some such embodiments, the solvent comprises ethyl acetate.

In some of any of the respective embodiments described herein, the ratio of the water-immiscible polar organic solvent to water for dispensing is in the range of 0.3 to 7.5ml of solvent per ml of water, optionally in the range of 0.6 to 4ml of solvent per ml of water, optionally in the range of 1 to 2.5ml of solvent per ml of water, and optionally about 1.5ml of solvent per ml of water. In some such embodiments, the solvent comprises ethyl acetate.

In some of any of the respective embodiments described herein, the method further comprises effecting crystallization of the UV-absorbing material, e.g., to obtain a purer UV-absorbing material. Crystallization may alternatively be achieved by any suitable technique known in the art. In some embodiments, the crystallization is achieved by contacting the UV absorbing material (e.g., in the form of a fraction that partitions into a water-immiscible polar organic solvent according to any of the respective embodiments described herein) with a solvent comprising water.

The solvent comprising water may optionally be, for example, a mixture of water and one or more water-miscible organic solvents (according to any of the respective embodiments described herein). In some of any of the respective embodiments, the solvent comprising water comprises water and an alcohol, e.g., at a concentration in a range of about 20 to about 80 weight percent (e.g., alcohol: water weight ratio of 20: 80 to 80: 20 alcohol: water), or in a range of about 30 to about 60 weight percent (e.g., alcohol: water weight ratio of 30: 70 to 60: 40), or about 40 weight percent (e.g., alcohol: water weight ratio of 40: 60). Alcohol/water (40: 60 by weight) is an exemplary solvent including water for effecting crystallization.

After crystallization, the crystals of UV absorbing material may optionally be separated from the solvent by filtration and/or solvent evaporation. Thus, an isolated UV absorbing material of relatively high purity can be obtained.

In some of any of the respective embodiments described herein, the UV absorbing material obtained according to any of the respective embodiments described herein (optionally including crystallization as a previous step) is further purified by column chromatography, for example by identifying the fraction exiting the column with UV absorption. In some embodiments, the stationary phase is hydrophobic. In an exemplary embodiment, carbon chain (octadecyl or C18) bonded silica is used as the stationary phase. A gradient of aqueous solution (e.g., 0.1% trifluoroacetic acid) and acetonitrile is an exemplary mobile phase.

In some of any of the embodiments described herein with respect to the method for obtaining a UV absorbing material comprising 1,2,3,4, 6-pentagalloylglucose, the 1,2,3,4, 6-pentagalloylglucose in the UV absorbing material has a higher purity, e.g., at least 95 weight percent of the UV absorbing material. In some embodiments, the 1,2,3,4, 6-pentagalloylglucose is at least 98 weight percent pure. In some embodiments, the 1,2,3,4, 6-pentagalloylglucose is at least 99 weight percent pure. In some embodiments, the 1,2,3,4, 6-pentagalloylglucose is at least 99.5 weight percent pure. In some embodiments, the 1,2,3,4, 6-pentagalloylglucose is at least 99.8 weight percent pure.

According to an aspect of an embodiment of the invention, there is provided a UV absorbing material obtained according to the method described herein according to any of the respective embodiments.

The preparation and the application are as follows:

as described above, the UV absorbing material according to any of the embodiments presented herein may be used as part of a composition comprising a dermatologically acceptable carrier.

Such compositions may optionally be any composition for topical use.

As further mentioned above, the composition is optionally a sunscreen composition, i.e. intended for blocking or shielding UV radiation (e.g. in the sun). It will be appreciated that the sunscreen composition (according to any of the respective embodiments described herein) may optionally have a primary intended use (e.g., cosmetic and/or pharmaceutical use) other than as a sunscreen, wherein the sunscreen activity is an ancillary activity of the composition.

Alternatively or additionally, the composition (according to any of the respective embodiments described herein) is optionally a pharmaceutical composition and/or a cosmetic composition (e.g., a composition comprised in a cosmetic product). In some such embodiments, the compositions are identified for use in treating skin aging or wounds, for example, pharmaceutical compositions for promoting wound healing. In some embodiments, the cosmetic composition is a skin rejuvenating composition and/or an exfoliating composition, e.g., identified for skin rejuvenation or skin exfoliation.

Herein, the terms "cosmetic" and "cosmetic composition" and "cosmetic product" refer to a substance or product (article) for external use for aesthetic purposes. The cosmetic composition optionally includes a material that further exhibits pharmaceutical activity to facilitate providing a desired aesthetic effect.

Cosmetic compositions or products, in which the active ingredients described herein may be advantageously utilized, include, for example, cosmetics, gels, paints, eye shadows, lip gloss, lipstick, and the like.

As used herein, the terms "pharmaceutical" and "pharmaceutically" refer to any compound and/or composition that is intended to beneficially alter the condition and/or state of at least a portion of the body (e.g., skin), including cosmetically altering, for example, skin. It is to be understood that such a definition may be broader than the use of such terms by regulatory agencies, which may exclude, for example, cosmetic effects from the scope of the terms.

The effect of the pharmaceutical or cosmetic composition may optionally be associated with preventing damage induced by UV radiation, optionally but not necessarily mediated at least in part by reducing the amount of UV radiation reaching the skin.

Alternatively or additionally, the beneficial effects of the pharmaceutical or cosmetic composition may be associated with preventing damage induced by UV radiation by mechanisms other than UV radiation reduction (e.g., by anti-oxidative action), and/or with treating damage unrelated to UV radiation, such as wounds (e.g., by promoting wound healing unrelated to UV radiation).

According to a further aspect of embodiments of the present invention there is provided the use of a UV absorbing material derived from a sumac or a composition comprising a UV absorbing material derived from a sumac (according to any of the respective embodiments described herein) in the manufacture of a medicament, for example for the treatment of skin ageing or a wound.

According to a further aspect of an embodiment of the present invention there is provided a method of treating skin ageing and/or a wound in a subject in need thereof, the method comprising topically applying to the subject a composition comprising a UV-absorbing material derived from sumac (according to any of the respective embodiments described herein).

As used herein, the term "dermatologically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the applied compound when applied to the skin of an organism.

The compositions used in accordance with embodiments of the present invention may thus be formulated in conventional manner using one or more carriers comprising excipients and auxiliaries, which facilitate processing of the compounds described above into preparations which can be used cosmetically and/or pharmaceutically.

Herein, the term "excipient" refers to an inert substance added to the composition to further facilitate administration of the active ingredient, e.g., a UV absorbing material according to any of the respective embodiments described herein. Examples of suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Techniques for formulating and administering active ingredients can be found in "Remington's Pharmaceutical Sciences" macpress, easton, pa, latest edition, which is incorporated herein by reference.

According to various embodiments of the present invention, the compositions described herein may be manufactured by methods well known in the art, for example, by conventional mixing, dissolving, granulating, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes.

By selecting an appropriate carrier and optionally other ingredients that can be included in the composition, the compositions described herein can be formulated in any form suitable for topical administration, as detailed herein. Thus, the composition may be in the form of, for example, a cream, ointment, paste, gel, lotion, and/or soap.

Ointments are semi-solid formulations, usually based on vegetable oils (e.g., shea butter and/or cocoa butter), petrolatum or petroleum derivatives. As with the other carriers or excipients, the ointment base should be inert, stable, non-irritating, and non-sensitizing.

Lotions are formulations that can be applied to the skin without abrasion. Lotions are typically water or alcohol based liquid or semi-liquid formulations, such as oil-in-water emulsions. Lotions are generally preferred for treating large areas (e.g., as is often required for sunscreen compositions) due to the ease of applying more fluid compositions.

Creams are viscous liquid or semisolid emulsions, which may be oil-in-water or water-in-oil. Cream bases generally comprise an oil phase, an emulsifier and an aqueous phase. The oil phase, also referred to as the "lipophilic" phase, optionally includes petrolatum and/or fatty alcohols, such as cetyl or stearyl alcohol. The aqueous phase optionally comprises a humectant. The emulsifier in the cream formulation is optionally a nonionic, anionic, cationic or amphoteric surfactant.

As used herein, the term "emulsion" refers to a composition that includes liquids in two or more distinct phases (e.g., a hydrophilic phase and a lipophilic phase). Non-liquid materials (e.g., dispersed solids and/or gas bubbles) may also optionally be present.

As used herein and in the art, a "water-in-oil emulsion" is an emulsion characterized by an aqueous phase dispersed in a lipophilic phase.

As used herein and in the art, an "oil-in-water emulsion" is an emulsion characterized by a lipophilic phase dispersed in an aqueous phase.

Pastes are semisolid dosage forms, which, depending on the nature of the matrix, can be fatty pastes or pastes made from single-phase aqueous gels. The base in the fat paste is usually vaseline, hydrophilic vaseline, etc. Pastes made from single-phase aqueous gels generally contain carboxymethylcellulose or the like as a base.

Gel formulations are semi-solid, suspension type systems. The single-phase gel optionally comprises organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous; but also preferably comprises a non-aqueous solvent and optionally an oil. Preferred organic macromolecules (e.g. gelling agents) include cross-linked acrylic polymers, such as a family of carbomer polymers, for example carboxy polyolefins (carbopol olefins), which may be trade marksAre commercially available. Other types of preferred polymers in this context are hydrophilic polymers, such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose phthalate, methylcellulose; gums, e.g. tragacanthGums and xanthan gums; sodium alginate; and gelatin. To prepare a homogeneous gel, a dispersing agent such as alcohol or glycerin may be added, or the gelling agent may be dispersed by grinding, mechanical mixing or stirring, or a combination thereof.

Compositions formulated for topical application may optionally be present in a patch, swab, gauze and/or pad.

Skin patches and the like may include some or all of the following components: a composition to be administered (e.g., as described herein); a liner for protecting the patch during storage, optionally removed prior to use; an adhesive for adhering the different ingredients together and/or for adhering the patch to the skin; a backing to protect the patch from the external environment; and/or a membrane that controls the release of the drug to the skin.

According to alternative embodiments, the composition is stable (e.g., without substantial chemical change and/or phase separation) at room temperature (e.g., 20 ℃) for at least 2 weeks, alternatively at least 1 month, alternatively at least 2 months, alternatively at least 6 months, alternatively at least 1 year.

Pharmaceutical, cosmetic and sunscreen compositions suitable in the context of the embodiments of the present invention include compositions comprising an effective amount of the active ingredient to achieve the respective intended purpose.

Determining an effective amount for a given purpose is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The pharmaceutical or cosmetic composition to be administered and/or the amount of active ingredient in the pharmaceutical or cosmetic composition may depend on the subject being treated, the severity of the affliction, the mode of administration, the judgment of the physician prescribing the pharmaceutical composition, and the like.

The amount of sunscreen composition and/or active ingredient (e.g., UV absorbing material according to any of the respective embodiments described herein) to be applied may depend on the degree of sun protection, (e.g., affected by skin pigmentation) the subject's sensitivity to sun, and whether general protection is required (e.g., a moderate UV exposure may be required to induce tanning), among other factors.

The degree of UV blocking activity provided by the sunscreen composition (e.g., as a function of active ingredient concentration) may optionally be expressed according to techniques known in the art, e.g., quantitatively expressed as a Sun Protection Factor (SPF) value.

The composition according to any of the respective embodiments described herein may optionally further comprise other active ingredients, e.g. as described herein, suitable to provide the desired effect of the composition. Such other active ingredients may be, for example, sunscreens, UV absorbers, antioxidants, skin protectants and/or agents for the treatment of the conditions described herein.

For example, other active ingredients suitable for blocking UV radiation (e.g., for use in sunscreen compositions) include, but are not limited to, benzophenones (e.g., benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4, benzophenone-5, benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9, benzophenone-10, and diethylamino hydroxybenzoyl hexyl benzoate), p-aminobenzoic acid and derivatives thereof, such as N-alkyl substituted derivatives and/or esters thereof (e.g., isoamyl N-dimethyl-p-aminobenzoate and octyl N-dimethyl-p-aminobenzoate), avobenzone (avobenzone), bis-ethylethoxyphenol methoxyphenyl triazine (benotrizinol), dioctylphenol triazole (bisoctrizole), 3- (4-methylbenzylidene) -camphor, cresoltrazol trisiloxane, ecamsule (ecamsole), ethylhexyl triazone, menthyl anthranilate, octocrylene (octocrylene), diethylhexyl butamido triazone (isocitrinol), methoxycinnamate or derivatives (e.g., esters) thereof (e.g., isoamyl 4-methoxycinnamate, octyl 4-methoxycinnamate, and cinoxate), polysiloxane-15, salicylic acid and salts thereof (e.g., triethanolamine salicylate) or derivatives (e.g., esters) thereof (e.g., homosalate and octyl salicylate), and inorganic substances such as TiO₂And/or ZnO.

In some embodiments, the other active (UV blocking) ingredient is TiO₂And/or ZnO, for example, so that synthetic organic reagents may optionally be avoided.

Other active ingredients suitable for use in compositions for treating and/or promoting wound healing include, but are not limited to, skin soothing and/or healing agents, such as panthenol and derivatives thereof (e.g., ethyl panthenol), aloe vera, pantothenic acid and derivatives thereof, allantoin, bisabolol, and dipotassium glycyrrhizinate.

Antioxidants suitable for use as other active ingredients (e.g., for reducing UV-induced or non-UV-induced skin damage) in the compositions described herein include, but are not limited to, ascorbic acid, butylated hydroxyanisole, butylated hydroxytoluene, carotenes and carotenoids (e.g., alpha-carotene, beta-carotene, canthaxanthin, cryptoxanthin, lutein, lycopene, zeaxanthin and vitamin a), curcumin, eugenol, flavonoids (e.g., flavone, isoflavone, flavanol, flavonol, stilbenes, anthocyanin), glutathione, propyl gallate, t-butylhydroxyquinone, tocopherols (e.g., vitamin E), uric acid, and antioxidant enzymes (e.g., thioredoxin, catalase, and superoxide dismutase).

The compositions described herein may also include other components that are added, for example, to enrich the composition with fragrance and nutritional factors (e.g., skin or hair nutritional factors).

These components are selected, within the scope of sound medical judgment, to be suitable for topical use in humans without causing toxicity, incompatibility, instability, allergic response, and the like. In addition, such optional ingredients are useful so long as they do not unacceptably alter the benefits of the active ingredients of the present invention.

The CTFA cosmetic ingredient handbook, second edition (1992), describes a number of non-limiting cosmetic ingredients commonly used in the skin care industry that are suitable for use in the compositions of the present invention. Examples of these composition classes include: an abrasive; an absorbent; aesthetic components such as fragrances, pigments/colorants, essential oils, skin sensates, astringents, and the like (e.g., clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate); an anti-acne agent; an anti-caking agent; defoaming agents; an antibacterial agent; antioxidants (e.g., for preserving the composition, rather than for use as an active ingredient); a binder; a biological additive; a buffering agent; a filler; a chelating agent; a chemical additive; a colorant; cosmetic astringents; a cosmetic bactericide; a denaturant; a drug astringent; pain reliever for external use; film formers or materials, such as polymers, to aid in the film forming properties and substantivity of the composition (e.g., copolymers of eicosene and vinyl pyrrolidone); an opacifying agent; a pH adjusting agent; a propellant; a reducing agent; a sequestering agent; skin conditioning agents (e.g., moisturizers, including heterogeneous and occlusive); a skin treatment agent; a thickener; and vitamins and their derivatives.

Since the compositions according to some embodiments described herein are used in vivo, the compositions preferably have high purity and are substantially free of potentially harmful contaminants, such as at least National Food (NF) grade, typically at least analytical grade, and preferably at least pharmaceutical grade. If a given compound must be synthesized prior to use, such synthesis or subsequent purification should preferably result in a product that is substantially free of any potentially contaminating toxic substances that may have been used during the synthesis or purification process.

The compositions of the present invention, if desired, may be presented in a pack or dispenser device, such as an FDA (U.S. food and drug administration) approved kit, which may contain one or more unit dosage forms comprising the active ingredient. The package may, for example, comprise a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser device may also be contained by a notice associated with the container provided in a form prescribed by a governmental agency regulating the manufacture, use or sale of cosmetics and/or pharmaceuticals, which notice reflects approval by the agency of the form of the composition for human or veterinary administration. Such a notice, for example, may be a label approved by the U.S. food and drug administration for prescription drugs, or an approved product insert. Compositions according to any of the embodiments of the present invention can also be prepared, placed in an appropriate container, and labeled for treatment and/or protection of the skin (e.g., according to any of the embodiments described herein).

Thus, according to some embodiments of the invention, the pharmaceutical composition described herein is packaged in a packaging material and identified in print, in or on the packaging material, for use in treating a condition described herein in a subject in need thereof.

As used herein, the term "about" means ± 10%.

The terms "comprising," including, "" containing, "" having, "and variations thereof mean" including, but not limited to.

The term "consisting of … …" means "including and limited to".

The term "consisting essentially of … …" means that the composition, method, or structure may include other ingredients, steps, and/or portions, but provided that the other ingredients, steps, and/or portions do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of the present invention may be presented in a range format. It is to be understood that the description of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have explicitly disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, such as 1,2,3,4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is intended to include any number of the referenced number (fractional or integer) within the indicated range. The phrases "range between" a first indicated number and "a second indicated number" and "ranging from" the first indicated number "to" the second indicated number are used interchangeably herein and are intended to include the first and second indicated numbers and all fractions and integers therebetween.

As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmaceutical, biological, biochemical and medical arts.

As used herein, the term "treating" includes eliminating, substantially inhibiting, slowing or reversing the progression of a disorder, substantially ameliorating clinical or aesthetic symptoms of a disorder or substantially preventing the appearance of clinical or aesthetic symptoms of a disorder.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features of the invention that are described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiments are inoperable without those elements.

As described above and as claimed in the claims section below, support for experimentation is found in the following examples.

Examples of the invention

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.

Materials and methods

Materials:

DMEM (Dulbecco's modified Eagle's Medium supplemented with 100 units/ml penicillin and 100. mu.g/ml streptomycin) was purchased from Biological Industries (Israel).

DPPH (diphenylpicrylhydrazine) was purchased from Sigma Aldrich (Sigma Aldrich).

Ethanol was purchased from Mercury Scientific & Industrial Products Ltd.

Methanol was purchased from Mercury scientific Industrial products, Inc.

MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide salt) was purchased from Sigma Aldrich.

And (3) calculating a protection coefficient:

according to Dutra et al [ Braz J Pharma Sci 2004,40: 381-385-]Methods are described, using OptLab^TMThe X software (asanis), using mansure equation, absorbance spectra was used to calculate SPF (sun protection factor) values.

High performance liquid chromatography:

by distilling water in double distilled water: 95 parts of methanol: 5 the sample was diluted to a concentration of 0.01mg/ml in the mixture, 10. mu.l was loaded on an RP-18 column of a Wottech HPLC apparatus with a photodiode array detector, and an HPLC (high performance liquid chromatography) chromatogram was obtained using a gradient from a 96% aqueous solution of trifluoroacetic acid (0.1%) to 100% acetonitrile at a flow rate of 1 ml/min.

Testing of human skin explants:

after signing the informed consent, skin was obtained from a healthy 30 to 60 year old female admitted to abdominal cosmetic surgery. The skin was cut into 0.8X 0.8 square centimeter pieces using mechanical compression according to the method described by Portugal-Cohen et al Exp Dermatol 2011,20: 749-755. Skin explants were maintained in an air/liquid interface with the dermal side submerged in culture medium according to the methods described by Portugal-Cohen et al [ Exp Dermatol 2011,20:749-755] and Cohen et al [ Dead Sea and Arava students 2015,7:66-74 ]. Human skin explants were placed in 6-well plates containing DMEM (Dulbecco's modified Eagle's medium) supplemented with 100 units/ml penicillin and 100. mu.g/ml streptomycin, with the dermal side facing down and the epidermal side facing up in the medium. All samples were used after overnight recovery.

Example 1

UV blocking Capacity screening of plants

To study the UVB blocking of various plantsCharacteristically, traditional herbs growing in the vermilion-desert have been screened on a large scale. The leaves, twigs, shoots, fruits and roots of each test plant were collected, dried, ground and extracted in ethanol, water, chloroform or propylene glycol methyl ether acetate (PMA), respectively, using the soxhlet system. After evaporation of the solvent in vacuo, 0.1 g of the crude extract was resuspended in an equal volume of solvent using sonication and Cary without integrating sphere was used^TMA 60 dual beam spectrophotometer (agilent) was diluted to a fixed concentration to analyze the UV absorption spectra.

Preliminary results (data not shown) indicated that ethanol leaf extract exhibited the highest UV absorbance values in most of the plant species tested.

Table 1 summarizes the SPF (sun protection factor) values of the ethanol leaf extracts of various plants (determined as described above).

Table 1: sun Protection Factor (SPF) values (mean ± SEM, n ═ 3) of ethanol extracts (0.0833mg/ml) from various plant leaves from the vermilion desert

As shown in table 1, ethanol extracts of Rhus coriaria (Rhus coriaria) showed the highest SPF values in vitro (protection against UVB radiation) in the test plants so far.

Thus, further tests were carried out on extracts of tanned sumac. Extracts of different parts of the sumac are prepared from sumac in different cultivation areas.

As shown in fig. 1, the extracts of sumac leaves and germinating fruits are most effective in filtering UV light than the extracts of sumac branches, twigs, mature fruits or roots.

Example 2

Separation of UV-blocking substances from leaves of Lacquertree

From the various sumac leaf sub-extracts described in example 1 above, the most effective extracts (samples from the Kiryat Arba region) were further purified by subjecting them to various separation techniques. After each step, the fractions obtained were evaluated based on their UV absorbance values to separate the UV absorbing components, herein referred to as "SH-101". Purity was determined by HPLC according to the method described above.

The following general extraction methods were designed to maximize yield through laborious experimentation.

As shown in fig. 2, extraction 100 comprises, in a first step, obtaining a dry powder 10 of the leaves of the jatropha curcas, for example by grinding. The dry powder may optionally be obtained according to any suitable technique known in the art. After removing water and volatile compounds from the leaves of the Sicilaria occidentalis, the weight of the dry powder 10 may be about 35 + -5 weight percent of the weight of the undried leaves.

In the next step, the dry powder 10 is contacted with ethanol (optionally warm ethanol) (e.g., using a soxhlet extractor) and the ethanol is then evaporated to obtain an ethanol extract 20 of the leaves of the jatropha curcas.

The ratio (weight to volume) of dry powder 10 to ethanol is optionally about 1: 20(1 g dry powder 10 to 20ml ethanol). At this ratio, a yield of 39.64 ± 2.35% (weight of ethanol extract 20 relative to the weight of dry powder 10) was obtained.

As shown in fig. 3, the use of more than 1000ml of ethanol after extraction of 50 grams of dry jatropha leaf powder with different volumes of ethanol correlates to lower SPF values and higher yields (weight of extracted material), while the use of less than about 1000ml (especially less than about 400ml) correlates to similar SPF values and lower yields.

These results indicate that the use of more than about 20ml of ethanol per gram of dry powder 10 results in the extraction of a large amount of material that does not contribute to UV protection, while the use of less than about 20ml (especially less than about 8ml) of ethanol per gram of dry powder 10 results in a significant reduction in the extracted UV absorbing material.

In the next step, the ethanol extract 20 is suspended in distilled water 22, partitioned 24 with n-hexane/water, and the water extract obtained in step 24 is partitioned 26 with ethyl acetate to obtain an ethyl acetate extract 30. After partitioning with n-hexane/water 24, the green black solid can be extracted into the hexane phase.

Suspension in distilled water 22 and partition 24 is optionally achieved using a ratio of about 1 gram of ethanol extract 20 to about 20ml of water to about 5ml of hexane (e.g., about 50 grams of ethanol extract 20, about 1000ml of water, and about 250ml of hexane). Dispensing 26 is optionally accomplished using a ratio of 1ml of water to 1.5ml of ethyl acetate (e.g., about 1000ml of water and about 1500ml of ethyl acetate). Using these ratios, yields of 26.46 ± 3.78% (weight of ethyl acetate extract 30 relative to weight of ethanol extract 20) were obtained.

The ethyl acetate extract 30 is then crystallized 32 in a mixture of ethanol and water, optionally at a ratio of 40: 60 (ethanol: water), followed by filtration and solvent evaporation 34 to obtain a combined dry crystallization supernatant 40.

Crystallization 32 is optionally achieved at a ratio of extract 30 to solvent (ethanol/water) of about 0.1mg of extract 30 per ml of solvent. Using this ratio, a maximum yield of 34.8 ± 4.2% (weight of combined dry crystallization supernatant 40 relative to the weight of ethyl acetate extract 30) was obtained.

The combined dried crystalline supernatants 40 are optionally subjected to reverse phase column chromatography 42, optionally using a C18 column, eluting with an acetonitrile gradient of 4% to 25% aqueous 0.1% trifluoroacetic acid (TFA) in acetonitrile to obtain combined pure SH-40150. A chromatographic yield of 30.15 ± 2.34% (weight of SH-40150 relative to the weight of the combined dried crystallization supernatant 40) was obtained.

In an exemplary extraction using the general method described above, a total SH-401 yield of 0.452. + -. 0.082% (weight of SH-401 relative to the weight of the undried Rhus verniciflua leaves) was obtained.

This simple but elegant process results in high yields of the compound, the total cost of which is comparable to that of chemically related commercially available compounds. For example, by optimizing the environmental agricultural parameters of plants (e.g., using strains characterized by high yields of SH-101), additional yield increases can be obtained, thereby further reducing the overall cost of the material.

Example 3

Characterization of UV-blocking substances (SH-401) in Rhus verniciflua leaves

SH-101 is isolated from the leaves of Toxicodendron vernicifluum using, for example, the method described in example 2 above. The purified SH-101 is then characterized by HPLC and various spectroscopic techniques.

As shown in fig. 4A and 4B, a fraction with high purity product was obtained as determined by HPLC analysis (-99.79% purity by peak area%) (retention time 107.9 minutes).

As shown in FIG. 4C, the resulting fraction showed strong absorption in the relevant UVB region (290 to 320nm), with absorption peaks at 217nm and 279.5 nm.

The isolated compound was obtained as a white, mostly amorphous powder with a melting point in the range of 220 to 250 ℃.

SH-401 in FeCl₃Show an intense blue color in the presence of KIO₃In the presence of this, a light red color is displayed. These color reactions are similar to those reported for gallotannins [ Haddock et al, J Chem Soc Perkin Trans 11982, 0:2535-]。

Furthermore, the infrared (KBr) spectrum (not shown) was at 3950cm^-1And 1712cm^-1Shows absorption, consistent with the hydroxyl and carbonyl groups in the galloyl moiety, respectively.

In summary, the above color reaction and UV and IR spectra indicate that SH-401 is a gallotannin.

The purified fractions were further analyzed by mass spectrometry using a liquid chromatography-mass spectrometry (LC-MS) and high resolution mass spectrometry using a Q-TOF 6545 (high resolution) LC-MS (ESI/APCI/ASAP) mass spectrometer (agilent).

As shown in Table 2 below, the mass spectra obtained by LC-MS include peaks at about 939Da and about 469Da, corresponding to (M-H), respectively^-And (M-2H)^-2Ion, indicating that SH-401 has the formula C₄₁H₃₂O₂₆。

Table 2: liquid chromatography-mass spectrometry results of SH-101

Importantly, the molecular weight of SH-101 is more than three times that of conventional commercial UV absorbers, a factor that may reduce the potential for penetration into the skin and potentially systemic deleterious effects.

Furthermore, the mass spectrum obtained by Q-TOF mass spectrometry comprised an ion peak at m/z 939.09 (consistent with the LC-MS results described above), and also comprised an ion peak at m/z 168.46, corresponding to the mass of the gallate anion (169 Da); the ion peak at m/z 769.08, corresponding to the expected mass of gallic acid neutral loss (loss of 170 Da); and a set of ion peaks from m/z 331.07 to m/z 939.09 separated by a constant difference of 152 corresponding to the galloyl moiety (C)₇H₄O₄) The expected mass of.

The above results are consistent with the description of Berardini et al [ Rapid Commun Mass Spectrum 2004,18: 2208-.

Thus, the above results indicate that SH-401 comprises multiple galloyl moieties and has a molecular weight of about 940 Da; and indicates compounds comprising five galloyl groups and an additional moiety of molecular weight 180Da (e.g., hexoses, where SH-101 is C₄₁H₃₂O₂₆)。

SH-401 gallotannin has a structure of¹H-NMR and¹³C-NMR 1D spectral data (in CD)₃OD obtained at 700 MHz) and determined by DEPT, COSY, HMBC and HMQC 2D-NMR.

As detailed below, based on NMR data analysis, SH-401 was determined to have 1,2,3,4, 6-pentagalloyl- β -D-glucose (. beta. -D-glucopyranose, pentavalent (3,4, 5-trihydroxybenzoate), as shown in FIG. 5.¹H and¹³assignment summary of C chemical shiftsIn table 3 below. The above structure is also supported by the UV absorption, infrared and mass spectral data discussed above.

¹The H-NMR spectrum included five aromatic single peaks, in the spectral range between 6.5 and 7.15ppm, consistent with the presence of five aromatic moieties assigned to the five magnetic nonequivalent protons of galloyl groups in the molecule.

The second group of protons, represented by resonances occurring between 4.35 and 6.25ppm, is assigned to the seven carbon-linked protons of the glucopyranosyl moiety. In the sugar region, the spectrum shows five distinctly downward-shifted proton resonances. One is¹The H signal, a double peak at 6.23ppm, has a large coupling constant and can be attributed to glucose isomerate in the alpha and beta configurations. At 5.90ppm¹Triple peaks of H and three at 5.61ppm (d, J12 Hz) and 4.40ppm (dd, J12, 44Hz) and 5.58ppm¹The H signal is assigned to the H-2 and H-4 and H-6 glucose protons. The signals for these protons are significantly reduced compared to those in β -D-glucopyranose, indicating the position of the galloyl unit at these centers.

As shown in the HMBC experiments, the 2H signals for glucose methylene groups appear at 4.51 and 4.37. DEPT NMR data show that SH-401 contains only one methylene group (CH)₂) A group.

Table 3: by¹H-NMR and¹³correlation of chemical shifts observed by C-NMR with atoms of 1,2,3,4, 6-pentagalloylglucose.

As shown in the table 3 below, the following examples,¹H–¹the H homonuclear COSY experiment allows the identification of a coupled network of glucose moieties from H-1 to H-6 a/b. Furthermore, the observed correlation between the proton at 4.37ppm and the proton at 4.51ppm confirms the assignment of these protons to the glucose methylene (CH)₂) A group.

¹³C-NMR spectra at 93.89, 72.26, 69.88, 74.18, 74.50(5 isomeric C); 63.19 (glucose methylene C); 166.27 (gallate 1, C ═ O); 119.81, 110.69, 146.61, 146.61 (gallate 1, C); 167.08 (gallate 2, C ═ O); 120.32, 110.48, 146.43, 140.36 (gallate 2, C); 166.99 (gallate 3, C ═ O); 120.28, respectively; 110.53, respectively; 146.50, respectively; 140.41 (gallate 3, C); 167.36 (gallate 4, C ═ O); 120.44, respectively; 110.45, respectively; 146.34, respectively; 140.18, respectively; (gallate 4, C); 167.99, respectively; (gallate 6, C ═ O); 121.12, respectively; 110.40, respectively; 146.53, respectively; 140.06, respectively; (gallate 6, C) exhibits a signal (. delta.)_C)。

For detecting single bonds¹Hx¹³HMQC experiments on C correlation indicate that five carbon atoms (ortho to the galloyl moiety) are attached to hydrogen at chemical shifts of about 7.11, 7.05, 6.98, 6.95, and 6.90 ppm.

For detecting long range¹Hx¹³HMBC experiments with C correlation showed that protons with chemical shifts of about 7.05ppm were associated with carbon atoms at about 140, 122 and 110 ppm.

Taken together, these results indicate that the hydroxyl groups at C-1, C-2, C-3, C-4 and C-6 are gallated [ De Bruyn et al, Bull Soc Chim Belges 1977,86:259-265 ].

The above molecular weight is more than three times that of known commercial UV filters. This factor may reduce the potential for penetration into the skin and associated deleterious effects.

As shown in FIG. 6, at 0.01mg/ml, the purification process increased the SPF value of the obtained SH-401 by more than 10-fold relative to the SPF value of the crude extract, increasing from 0.67. + -. 0.09 for the crude Rhus occidentalis mixture to 9.09. + -. 0.72 for the purified SH-401 fraction.

This result indicates that the isolated agent is superior to the crude extract mixture as a sunscreen agent, confirming the importance of SH-401 as a UV absorbing component of Rhus verniciflua.

As further shown in FIG. 6, the SPF value of SH-401 (at 0.01 mg/ml) is at least as high as the SPF value of all tested commercial synthetic UV filters.

To evaluate the photostability, uniform pure SH-401 films were prepared on PMMA (polymethyl methacrylate) slides by depositing 2ml of SH-101 solution on the slides and evaporating the solvent. The samples were then exposed to UV radiation at different time intervals (ranging up to 25 hours). The glass plate was then immersed in a mixture of ethanol/water (2: 98% v/v) and the film was ultrasonically dissolved. The UV samples were then quantified by UV-visible absorption spectroscopy and SPF values were determined and plotted as a function of time according to the method described above.

As shown in fig. 7, SH-401 exhibited considerable stability to UV radiation with substantially no change in SPF value after 12 hours of exposure to UV radiation.

Taken together, the above results indicate that SH-401 has sufficient UV absorption and photostability to be effective as a sunscreen agent.

Example 4

Effect of SH-401 in Lacquertree leaves on human skin cells

To investigate the safety and efficacy of the active compound SH-401 (isolated as described in example 2), several experiments were performed on human skin explants according to the methods described in the materials and methods section above.

As shown in fig. 8A, external application of SH-401 did not compromise skin viability as determined by the MTT assay over the entire tested concentration range (up to maximum solubility). The results indicate that human skin is well-tolerated for SH-401.

As shown in FIGS. 8B and 8C, SH-401 attenuated UVB-induced epidermal apoptosis (FIG. 8B) and excessive TNF α secretion (FIG. 8C) as determined by the caspase-3 activation assay and ELISA assay, respectively. As further shown therein, the effect of SH-401 is comparable to that of the commercial preparation Ultrasol used as a control^TMSPF 30 formulations (dr. fisher) were comparable in efficacy.

One common feature of UVB-induced damage is ROS (reactive oxygen species) production, which increases DNA damage, amplifies lipid peroxidation production and blocks intracellular proteins and cell membranes [ Schuch et al, Free Rad Biol Med 2017,107:110-124 ]. Thus, the effect of SH-401 on UVB-induced ROS generation in skin cells was tested, which is believed to be a factor in counteracting the deleterious effects of UVB radiation. ROS generation following UVB irradiation was assessed in skin explants by DCFDA (dichlorofluorescein diacetate) assay and by measuring lipid peroxidation associated with ROS generation (by ELISA).

As shown in fig. 9, SH-401 attenuated UVB-induced ROS production in skin cells in a dose-dependent manner as determined by DCFDA assay.

Furthermore, as shown in fig. 10, SH-401 attenuated UVB-induced lipid peroxidation in skin cells in a dose-dependent manner.

The above results indicate that SH-401 attenuates the formation of ROS in skin cells caused by UVB radiation.

The DPPH (diphenylpicrylhydrazine) method was used to further evaluate antioxidant capacity. The color shift of DPPH in the presence of SH-401 and conversion to units of Trolox (6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acid) equivalent antioxidant capacity according to a calibration curve.

As shown in FIG. 11, SH-401 is a strong antioxidant, and a 1 weight percent solution exhibits about 2,310 micromolar Trolox Equivalent (TE) units per 100 grams.

These results indicate that the protection exhibited by SH-401 against UV radiation is mediated, at least in part, by the antioxidant properties of the compound.

In addition, OxiSelect was used according to the manufacturer's instructions^TMComet assay kit, DNA fragmentation of similarly processed samples was determined by Comet (single cell gel electrophoresis) analysis.

As shown in FIG. 12, SH-401 attenuated UVB-induced DNA damage as determined by COMET analysis.

As shown in FIG. 13, SH-401 reduces UVB-induced formation of Cyclobutane Pyrimidine Dimers (CPD), which are the major types of DNA mutations caused by UVB.

The effect of SH-401 on the cells was further assessed by histological examination of the treated skin explants.

As shown in fig. 14A-14D, UVB radiation induced the formation of pycnotic "sunburn cells" and the reduction of the epidermal layer (fig. 14B), and SH-401 reversed this deleterious effect of radiation.

These results indicate that SH-401 is effective in protecting human skin from UV radiation.

Example 5

Effect of SH-401 on skin aging and wound healing

By examining two important parameters in the extracellular matrix balance affecting skin aging and wrinkle formation: collagen synthesis after environmental damage; and the activity of matrix metalloproteinase-1 (MMP1), a key enzyme in collagen degradation, to evaluate the effect of SH-401 on skin aging.

As shown in fig. 15A and 15B, SH-401 enhanced collagen synthesis and reduced MMP1 activity in a dose-dependent manner following environmental injury (UVB radiation).

These results indicate that SH-401 exhibits anti-aging properties in skin.

The effect of SH-401 on skin wound healing was evaluated using an in vitro model of fused HaCaT cell wound closure.

As shown in FIG. 16, SH-401 enhanced wound closure in an in vitro model.

These results indicate that SH-401 promotes wound healing.

While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. They should not be construed as necessarily limiting insofar as chapter headings are used.

In addition, the entire contents of any priority document of the present application are incorporated herein by reference.