Polyphenol-enriched lettuce composition for wound healing promotion and skin care

Abstract

This specification provides polyphenol-fortified lettuce powder containing particles of polyphenol-fortified lettuce or extract, the polyphenol-fortified lettuce containing 40 to 280 mg / g of polyphenols. The polyphenols include any combination of chlorogenic acid, neochlorogenic acid, chicoric acid, quercetin, quercetin derivatives, and anthocyanins. This disclosure also provides compositions and methods for using polyphenol-fortified lettuce powder to promote wound healing and treat inflammation and skin disorders.

Description

Background Art

[0001] Technical Field The present disclosure relates to novel compositions having therapeutic and cosmetic properties that promote wound healing and provide skin care effects.

[0002] Description of Related Art Although antibacterial therapy and symptomatic treatment have advanced significantly, wound treatment can still present significant challenges. Invasive infections, blunt trauma, burns from electrical or chemical accidents, radiation burns, and other similar injuries remain difficult to treat effectively and heal. Unfortunately, despite the introduction of clinically effective antibacterial agents, strains of bacteria resistant to such treatments are emerging rapidly, exacerbating the problem.

[0003] In aging countries such as the United States, significant challenges are faced by patients with lower leg ulcers. Lower leg ulcers are estimated to result in 2 million lost workdays in the United States. The cost of treating these refractory wounds can be incalculable, and the prevalence of lower body pressure ulcers (bedsores or decubitus ulcers) in the elderly population is estimated to be 3 - 11%. The morbidity and mortality associated with pressure ulcers are high, and the mortality rate of patients with pressure ulcers is four times that of patients without pressure ulcers. Furthermore, the mortality rate of septic elderly patients with pressure ulcers exceeds 50% in the hospital. Treatment costs can be high as they include not only intensive care but also adjunctive therapies such as anti - pressure devices, protective dressings, and skin treatments [Monika P, Chandraprabha MN, Rangarajan A, Waiker PV, Chidambara Murthy KN. Challenges in Healing Wound: Role of Complementary and Alternative Medicine. Front Nutr. 2022, 20; 8:791899].

[0004] Wound management presents significant challenges, particularly in diabetic patients where wounds are often considered untreatable. Diabetes is a common underlying condition for lower leg ulcers, and while various treatments exist, such as multilayer compression bandages, topical recombinant human platelet-derived growth factor, and human skin equivalents for skin grafts, these are expensive and unaffordable for many patients. In some cases, wounds may remain untreated, leading to amputation of the infected limb. Wound management in animals presents further challenges due to their susceptibility to environmental infections. Flies, especially myiasis-inducing flies such as Sarcophagidae and Calliphoridae, can worsen wounds and cause complications. Pouring creoline (a mixture of phenol and tar) into wounds is a common practice, but it damages tissue, is toxic, and provides only short-term protection from insects. Direct application of insecticides to maggot-infected wounds is also problematic due to the risk of animal poisoning and food chain contamination.

[0005] This specification provides novel formulations, compositions, and uses of polyphenol-enhanced red lettuce powder / extract that provide unexpectedly superior effects in promoting wound healing, addressing unmet needs in the art. [Overview of the Initiative]

[0006] The general object of this disclosure is to provide novel plant compositions for wound healing / skin care and methods for preparing / manufacturing the same. This disclosure introduces novel powder compositions comprising particles of polyphenol-fortified lettuce powder or extract, wherein the polyphenol-fortified lettuce exhibits increased production and concentration of at least one polyphenol compared to control lettuce. More specifically, this disclosure relates to polyphenol-fortified lettuce powder or extract having potent anti-inflammatory and wound-healing properties, wherein this powder or extract is highly effective in healing injuries of all nature in humans, including acute and chronic wounds, such as abrasions, burns, lacerations, vascular ulcers, diabetic ulcers, and pressure ulcers. This polyphenol-fortified lettuce powder / extract is also highly effective in treating wounds in mammals. The disclosed powder / extract and methods can be applied to the four basic tissues of the human / animal body (nerve, epithelium, muscle, and connective tissue). Furthermore, the disclosed powder / extract can also be used in cosmetics for skin care. Polyphenol-fortified lettuce powder / extract can be incorporated into various cosmetic formulations, including sunscreens, facial cleansers, face masks, hair conditioners and shampoos, and body washes, offering various skin benefits such as anti-aging, radiation protection, skin whitening, moisturizing, freckle removal, acne treatment, and hair regeneration promotion. The powder / extract can be designed for external / topical or oral use and possesses excellent wound healing, anti-inflammatory, and antioxidant properties. This powder / extract and method are particularly effective in treating wounds that are considered untreatable with conventional methods. The combination of natural ingredients in polyphenol-fortified lettuce powder / extract creates a powerful and effective action, resulting in a synergistic effect of the components.

[0007] These and other aspects of the present disclosure will become apparent by reference to the embodiments for carrying out the invention described below and the accompanying drawings. All references disclosed herein are incorporated herein by reference in whole as if each were incorporated individually. [Brief explanation of the drawing]

[0008] [Figure 1] Figures A-C show quercetin naturally present in red lettuce (Figure 1A), as well as its derivatives, quercetin-3-O-glucoside (Q3G) (Figure 1B) and quercetin-3-O-malonylglucoside (Q3MG) (Figure 1C), which are highly usable by the body after oral ingestion. [Figure 2] Examples of chlorogenic acids identified from red lettuce, such as 3-caffeoylquinic acid (neochlorogenic acid or 3-CQA) (Figure 2A) and 3,4-dicaffeoylquinic acid (3,4-diCQA) (Figure 2B), are shown. [Figure 3] This shows the chicory acid (CRA) content in red lettuce. [Figure 4] Exemplary anthocyanins such as cyanidin-3-O-glucoside (Figure 4A) and cyanidin-3-(6"-malonylglucoside) (Figure 4B) are shown. [Figure 5] This is a flowchart of a method for preparing an extract from polyphenol-fortified lettuce powder. [Figure 6] HPLC-UV chromatograms showing the enhancement of bioactive components by genomics-based technology, confirming the production of specific metabolites from red lettuce treated with an elicitor. (1) Untreated lettuce. (2) Treated lettuce. A: Chlorogenic acid (3-CQA), B: Chicoric acid (CRA), C: Quercetin-3-O-glucoside (Q3G), D: Quercetin-3-O-malonyl glucoside (Q3MG), E: 3,4-Dicaffeoylquinic acid (3,4-diCQA). [Figure 7] Figures A and B show that the production of chlorogenic acid and chicoric acid (Figure 7A), as well as more hydrophilic quercetin derivatives (Figure 7B), increased 3 to 9 times in red lettuce treated with a plant elicitor. Figure A illustrates the production of chlorogenic acid (3-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), and chicoric acid (CRA). Figure B illustrates the production of quercetin derivatives (Q3G and Q3MG). [Figure 8] This bar graph compares the changes in mouse body weight before and after the experiment in the polyphenol dressing treatment group (A-D) and the control group. [Figure 9] Photographs of the spleens of mice treated with polyphenol dressing (A-D) and the control group are shown. [Figure 10] The images show the wound size over time in mice treated with polyphenol dressing (A-D) and in the control group. [Figure 11A] The image shows a photograph of a tissue section of a wound in a control group of mice. [Figure 11B] The image shows a photograph of a tissue section of a wound in a mouse treated with polyphenols. [Figure 11C] The image shows a photograph of a tissue section of a wound in a mouse treated with polyphenols. [Figure 11D] The image shows a photograph of a tissue section of a wound in a mouse treated with polyphenols. [Figure 11E] The image shows a photograph of a tissue section of a wound in a mouse treated with polyphenols. [Figure 12] This bar graph shows the changes in TNF-α over time in mice treated with polyphenol dressing (A-D) and the control group. [Figure 13] This bar graph shows the changes in IL-6 levels over time in mice treated with polyphenol dressing (A-D) and the control group. [Figure 14] This bar graph shows the changes in IL-8 levels over time in mice treated with polyphenol dressing (A-D) and the control group. [Modes for carrying out the invention]

[0009] This specification presents examples relating to the use of high-polyphenol-fortified lettuce as a raw material, and to the resulting extracts and formulations. Such lettuce contains high levels of polyphenols such as quercetin derivatives, chlorogenic acid, chicoric acid, and anthocyanins. While not limited to theory, the polyphenols contained in such lettuce are thought to exhibit synergistic bioactivity for wound healing and skincare applications.

[0010] This specification presents powders containing particles of polyphenol-fortified lettuce powder or extract, wherein the polyphenol-fortified lettuce exhibits increased production and concentration of at least one polyphenol compared to control lettuce. In some embodiments, the polyphenol is at least one of chlorogenic acid, neochlorogenic acid, chicoric acid, quercetin, quercetin derivatives, anthocyanins, or any combination thereof. In some embodiments, the powder can be formulated for topical and / or oral administration. Furthermore, this disclosure provides wound dressing powders. This disclosure also provides hydrogels containing such powders. Similarly, this specification provides methods of treatment comprising administering such powders, and methods for preparing such powders.

[0011] This disclosure includes various embodiments that can be combined in different ways. The following description is provided to enumerate the elements and to illustrate some embodiments of this disclosure. These elements are enumerated together with the first embodiment. However, it should be understood that additional embodiments may be created by combining these embodiments in any way and in any number. The various examples and preferred embodiments described should not be construed as limiting this disclosure to only the expressly described systems, technologies, and uses. Furthermore, this description should be understood to support and encompass the description and claims of any various embodiments, systems, technologies, methods, devices, and uses, including any number of elements disclosed, each element individually, and all sorts of permutations and combinations of all elements in this application or subsequent applications.

[0012] One of the highly concentrated natural polyphenols found in red lettuce is quercetin derivatives. Quercetin is one of the most abundant flavonoids in food. Quercetin can be found in many plants and foods, including red wine, onions, green tea, apples, berries, Ginkgo biloba, St. John's wort, and American elder (Flavonoids, Micronutrient Information Center, Linus Pauling Institute, Oregon State University, 2015). Quercetin is associated with improved athletic performance, as well as reduced inflammation, and lower blood pressure and blood sugar levels. It may also possess neuroprotective, anti-allergic, and anti-cancer, antibacterial, and antiviral properties. Quercetin has been extensively studied in relation to wound healing, and its use is documented in numerous research papers covering various wound conditions affecting different organs [Polera N, Badolato M, Perri F, Carullo G, Aiello F. Quercetin and its Natural Sources in Wound Healing Management. Curr Med Chem. 2019;26(31):5825-5848]. For example, quercetin derivatives in Oxytropis falcata Bunge have been documented as a key active ingredient for treating knife wounds and inflammation. In mice, quercetin has been shown to promote skin wound healing by promoting fibroblast proliferation and migration, suppressing inflammation, and increasing growth factor expression. This effect is attributed to the activation of the Wnt / β-catenin signaling pathway and TERT [Yuhui Mi, Lei Zhong, Saijian Lu, Po Hu, Yang Pan, Xuelin Ma, Binghui Yan, Zhenhuan Wei, Guangming Yang, Quercetin promotes cutaneous wound healing in mice through Wnt / β-catenin signaling pathway, Journal of Ethnopharmacology, 290, 2022, 115066].These findings provide a foundation for a deeper understanding of the mechanism of action of O. falcata Bunge in the treatment of knife wounds and burns. Another study [Jia Fu, Jingjuan Huang, Man Lin, Tingting Xie, Tianhui You, Quercetin Promotes Diabetic Wound Healing via Switching Macrophages From M1 to M2 Polarization, Journal of Surgical Research, 246, 2020, 213-223] demonstrated that quercetin suppresses inflammatory responses and promotes the repair of diabetic wounds by modulating the switching of macrophage polarization from M1 to M2 phenotypes.

[0013] Another important bioactive component in polyphenol-fortified lettuce is chlorogenic acid. Chlorogenic acid (CGA, a phenolic compound found in human food) has been shown to have several health-enhancing properties, including antioxidant, anti-inflammatory, antibacterial, anti-diabetic, and lipid-lowering functions. Numerous reports indicate that CGA has beneficial effects on improving wound healing. For example, a recent report [Liu Song, Hao Yang, Di Liang, Di Chu, Leilei Yang, Meng Li, Bo Yang, Ying Shi, Zheng Chen, Zhuo Yu, Jianfeng Guo. "A chlorogenic acid-loaded hyaluronic acid-based hydrogel facilitates anti-inflammatory and pro-healing effects for diabetic wounds, Journal of Drug Delivery Science and Technology, 2022, 70, 103232] evaluated the ability of CGA to promote diabetic wound healing using in vitro and ex vivo experimental approaches. The results showed that CGA significantly achieved anti-inflammatory activity, re-epithelialization effect, and angiogenesis-promoting function. Subsequently, CGA was topically administered to full-thickness wounds in diabetic mice using a hyaluronic acid-based hydrogel in vivo. The resulting formulation (CGA-HA) was used in commercially available wound dressings (INTRASITE). Compared to Gel, it significantly improved the healing effect, accompanied by reduced inflammation, enhanced re-epithelialization and angiogenesis, and maturation of collagen reconstruction, demonstrating the potential of CGA-HA in clinical applications for diabetic wound healing.

[0014] Polyphenol-fortified lettuce is also rich in bioactive anthocyanins. Anthocyanins are water-soluble pigments belonging to the phenol group (Khoo et al., Food Nutr Res. 61(1), 2017). The pigments are in a glycosylated form. Anthocyanins, responsible for red, purple, and blue colors, are found in fruits and vegetables. Berries, currants, grapes, and some tropical fruits have high anthocyanin content. Red to purplish-blue leafy vegetables, grains, root vegetables, and tubers are edible vegetables that contain high levels of anthocyanins. Among anthocyanin pigments, cyanidin-3-glucoside is the major anthocyanin found in most plants. Anthocyanins possess anti-diabetic, anti-cancer, anti-inflammatory, antibacterial, and anti-obesity effects, as well as cardiovascular disease prevention effects (He et al., J Ethnopharmacol. 137(3) (2011): 1135-1142. In particular [Xu L, Choi TH, Kim S, Kim SH, Chang HW, Choe M, Kwon SY, Hur JA, Shin SC, Chung JI, Kang D, Zhang D. Anthocyans from black soybean seed coat enhance wound healing. Ann Plast Surg. 2013 Oct; 71(4): 415-20.]. It has been demonstrated that anthocyanins inhibit the translocation of nuclear factor-κB(p65) from the cytosol to the nucleus and prevent the phosphorylation of IκBα. Anthocyanins promote wound healing through cytoprotective effects, promotion of angiogenesis, and anti-inflammatory effects.In other research results [Palungwachira P, Tancharoen S, Phruksaniyom C, Klungsaeng S, Srichan R, Kikuchi K, Nararatwanchai T. Antioxidant and Anti-Inflammatory Properties of Anthocyanins Extracted from Oryza sativa L. in Primary Dermal Fibroblasts. Oxid Med Cell Longev. 2019 Jul 31;2019:2089817], it has been suggested that anthocyanins have anti-inflammatory properties and anti-aging potential by regulating the expression of type I collagen genes and suppressing H2O2-induced NF-κB activation in skin fibroblasts.

[0015] Regarding dressing materials, when considering topical use, particle size is an important factor for effective wound healing [see, for example, Patent Nos. TWI764332, TWI786786, TWM636367, TWI787696, TWI774168, TWI774055, TWI774046, TWI760999, TWI760961, TWI758625, TWM602560]. Appropriate particles can penetrate the skin more easily, reach deeper layers of tissue, and enable more efficient delivery of therapeutic agents to the wound site. In contrast, larger particles may be more difficult to penetrate the skin and may remain on the surface, having little effect on the underlying tissue. Therefore, it is important to consider particle size when designing topical wound healing treatments to ensure optimal therapeutic effects.

[0016] Wound Healing and Wound Molecular Biology Wound healing is a complex and dynamic process consisting of several consecutive steps, including induction of the inflammatory response, regeneration of parenchymal tissue, migration and proliferation of parenchymal tissue cells, production of extracellular matrix proteins, tissue remodeling, and increase in wound strength [Harding KG, Morris HL, Patel GK. Healing chronic wounds. BMJ. (2002) 324:160 - 3]. Wound healing involves multiple cell types, secreted growth factors, cytokines, extracellular matrix, and various enzymes. Platelets, neutrophils, monocytes, macrophages, fibroblasts, keratinocytes, endothelial cells, epithelial cells, and myofibroblasts are one of the various cell types involved in the wound healing process. Fibroblasts have long been recognized as important cells in wound healing because they play an important role in all three stages [Monika P, Waiker PV, Chandraprabha MN, Rangarajan A, Murthy KNC. Myofibroblast progeny in wound biology and wound healing studies. Wound Repair Regen. (2021) 29:531 - 47]. Several factors, such as microbial infection or biofilm formation, can affect the wound healing process. Furthermore, ischemia and reperfusion play an important role in damaged skin. Thomas et al. [Mustoe, Thomas A, O’Shaughnessy K, Kloeters O. Chronic wound pathogenesis, and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg. (2006) 117:35S - 41] discussed the molecular biology involved in ischemia and reperfusion, which are the main causes of the pathological state of wounds. Ischemia - reperfusion injury is characterized by a series of biochemical and cellular events that lead to extensive cell damage via pathways leading to activation of leukocytes and complement, oxidative stress, and microvascular dysfunction.

[0017] In normal wound healing, numerous molecular factors, including growth factors and receptors such as platelet-derived growth factor (PDGF) and transformed growth factor beta (TGF-β), cytokines such as fibroblast growth factor (FGF), tumor necrosis alpha (TNFα), and interleukin-1 (IL-1), and enzymes such as matrix metalloproteinases (MMPs), MMP tissue inhibitors, and seperinases, play important and overlapping roles [Blakytny R, Jude EB. Altered molecular mechanisms of diabetic foot ulcers. Int J Low Extrem Wounds. (2009) 8:95-104]. Furthermore, angiogenesis, which involves the development of capillary angioblasts that invade the extracellular matrix (ECM) stroma and form tubular structures, is important for wound healing [Honnegowda TM, Kumar P, Udupa EGP, Kumar S, Kumar U, Rao P. Role of angiogenesis and angiogenic factors in acute and chronic wound healing. Plast Aesthetic Res. (2015) 2:243-9]. Various angiogenic stimulants, such as vascular endothelial growth factor (VEGF), TGF-β, TNFα, PDGF, FGF, angiogenins, and angiopoietin-1, play crucial roles in different stages of angiogenesis, including initiation, amplification, proliferation, stabilization, and network formation [Honnegowda TM, Kumar P, Udupa EGP, Kumar S, Kumar U, Rao P. Role of angiogenesis and angiogenic factors in acute and chronic wound healing. Plast Aesthetic Res. (2015) 2:243-9). While the activation of these molecular factors is essential for wound healing, the complex series of interactions between these molecules can lead to wound persistence if inhibited. A deeper understanding of changes in the expression of these molecular factors and their effects on cellular function may provide valuable insights into potential intervention targets.

[0018] Wound healing in acute and chronic conditions Wounds can be broadly categorized into two types: acute and chronic. Acute wounds typically heal in an orderly and efficient manner, progressing through four distinct but overlapping stages: hemostasis, inflammation, proliferation, and reconstruction [Diegelmann R. Wound healing: an overview of acute, fibrotic, and delayed healing. Front Biosci. (2004) 9:283-9]. Acute wounds usually show clear signs of healing within 2 to 4 weeks, after going through the stages of inflammation, proliferation, and reconstruction. Conversely, chronic wounds may not follow a continuous healing process and may "stop" at a single stage, showing no signs of healing within 4 weeks [Swezey L. The difference between acute and chronic wounds (2019)].

[0019] During normal physiological processes such as wound healing (in the case of acute wounds), inflammatory cells assist in tissue repair by secreting cytokines and growth factors that are replenished at the site of injury and promote tissue remodeling and angiogenesis. However, in all chronic wounds, angiogenesis is impaired, leading to further tissue damage, chronic hypoxia, and impaired micronutrient delivery. Vascular diseases associated with diabetes include abnormal angiogenesis, decreased angiogenesis, and accelerated atherosclerosis, which can lead to coronary artery disease, peripheral vascular disease, and cerebrovascular disease [Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev. (2003) 23:117-45]. Vascular endothelial growth factor (VEGF) is an important angiogenesis-stimulating substance in wound healing, and studies have shown that VEGF levels are deficient in diabetic chronic wounds. The application of VEGF has been shown to stimulate the healing of chronic wounds in animal models [Howdieshell TR, Callaway D, Webb WL, Gaines MD, Procter CD, Sathyanarayana, et al. Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation. J Surg Res. (2001) 96:173-82] [Johnson KE, Wilgus TA. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Av Wound Care. (2014) 3:647-61].In contrast, circulating VEGF levels are elevated in patients with chronic venous congestive ulcers [Shoab SS, Scurr JH, Coleridge-Smith PD. Plasma VEGF as a marker of therapy in patients with chronic venous disease treated with oral micronized flavonoid fraction - a pilot study. Eur J Vasc Endovasc Surg. (1999) 18:334-8]. Since many factors regulate wound angiogenesis, it is important to understand the various dysregulated cellular and molecular events in angiogenesis that cause refractory chronic wounds.

[0020] The role of natural products in wound healing Natural products, including phytochemicals and naturally derived substances derived from plants, have recently attracted significant research interest due to their potential in wound healing. These substances have long been used for their anti-inflammatory, antioxidant, angiogenic, and cell synthesis-regulating properties. There is abundant evidence supporting the use of natural products and naturally derived substances in wound care, and therefore they are currently attracting considerable attention from researchers. Phytochemicals and naturally derived substances are attractive options because they contain a variety of chemicals that can promote wound healing in many ways. Medicinal plants have been shown to be effective in accelerating wound healing by treating wounds and fighting infections. According to recent research by Nigussie, Lawsonia inermis and Azadirachta indica are among the most studied plant species for wound healing, with the most common in vivo methods used in anti-inflammatory and wound healing assays being carrageenan-induced pectoral edema and excision / incision models, respectively [Nigussie D, Makonnen E, Tufa TB, Brewster M, Legesse BA, Fekadu A, et al. Systematic review of Ethiopian medicinal plants used for their anti-inflammatory and wound healing activities. J Ethnopharmacol. (2021) 276:114179]. The detailed roles of phytochemicals and naturally derived substances in wound healing research, including their uses, applications, mechanisms of action, and outcomes, have been widely demonstrated.

[0021] Another study has highlighted the synergistic effects of plant and naturally derived substances in wound healing, but has also pointed out the risk of side effects such as inflammation and allergic hypersensitivity [Sivamani RK, Ma BR, Wehrli LN, Mavericks E. Phytochemicals and naturally derived substances for wound healing. Adv Wound Care. (2012) 1:213-7]. On the other hand, recent studies have suggested that neem leaf extract may be a safe alternative to ordinary saline for washing foot ulcers, with no systemic complications reported [Jayalakshmi MS, Thenmozhi P, Vijayaraghavan R. Plant leaves extract irrigation on wound healing in diabetic foot ulcers. Evid Based Compl Altern Med. (2021) 2021:9924725]. It is important to note that phytochemicals and naturally derived substances may carry a high risk of contamination by infectious agents, and therefore proper sterilization and microbiological testing before use are essential. Key markers for evaluating the benefits of natural products in wound healing include hydroxyproline content, microscopic observation, and the physical appearance of the wound. For example, pomegranate peel extract in the form of a water-soluble gel has shown promising results in wound healing, as evidenced by these markers [Murthy KNC, Reddy KV, Vegas JM, Murthy UD. Study on wound healing activity of Punica granatum peel. J Med Food. (2004) 7:256-9]. Ongoing research in this area is demonstrating the stimulating potential of natural products for wound healing.

[0022] Polyphenols have attracted considerable attention for their potential in wound healing due to their ability to act as antioxidants, as well as their regenerative and antibacterial properties. [Dzialo, MA; Mierziak, J.; Korzun, U.; Preisner, M.; Szopa, J.; Kulma, A. The potential of plant phenolics in prevention and therapy of skin disorders. Int. J. Mol. Sci. 2016, 17, 160; Ghuman, S.; Ncube, B.; Finnie, J.; McGaw, L.; Njoya, EM; Coopoosamy, R.; Van Staden, J. Antioxidant, anti-inflammatory and wound healing properties of medicinal plant extracts used to treat wounds and dermatological disorders.S.Afr.J.Bot.2019,126,232-240;Guimaraes,I.;Baptista-Silva,S.;Pintado,M.;L.Oliveira,A.Polyphenols:A Promising Avenue in Therapeutic Solutions for Wound Care.Appl.Appl.Sci.2021,11,1230;Luque,GC;Moya,M.;Picchio,ML;Bagnarello,V.;Valerio,I.;Bolanos,J.;Vethencourt,M.;Gamboa,S.-H.;Tome,LC;Minari,RJ;Mecerreyes,D.Polyphenol Iongel Patches with Antimicrobial, Antioxidant and Anti-Inflammatory Properties.Polymers 2023,15,1076].These compounds are typically extracted from plants and marine organisms and, depending on the number of phenolic units and their bonding structure, are divided into two main subgroups: flavonoids (such as flavonols and anthocyanidins) and non-flavonoid compounds (such as phenolic acids, tannins, and lignans) [Liakos, I.; Rizzello, L.; Hajiali, H.; Brunetti, V.; Carzino, R.; Pompa, PP; Athanassiou, A.; Mele, E. Fibrous wound dressings encapsulating essential oils as natural antimicrobial agents. J. Mater. Chem. B 2015, 3, 1583-1589]. Polyphenols have been the focus of extensive research on their potential in medical and pharmaceutical applications, particularly in wound care [Dzialo, MA; Mierziak, J.; Korzun, U.; Preisner, M.; Szopa, J.; Kulma, A. The potential of plant phenolics in prevention and therapy of skin disorders. Int. J. Mol. Sci. 2016, 17, 160]. These compounds are known for their potent antioxidant activity, which promotes the neutralization of free radicals by donating electrons or hydrogen atoms and protects against reactive oxygen species (ROS). Furthermore, certain polyphenols have been shown to exhibit antibacterial activity against specific bacteria commonly found in infected chronic wounds [Ghuman, S.; Ncube, B.; Finnie, J.; McGaw, L.; Njoya, EM; Coopoosamy, R.; Van Staden, J. Antioxidant, anti-inflammatory and wound healing properties of medicinal plant extracts used to treat wounds and dermatological disorders. S.Afr.J.Bot.2019, 126, 232-240].Although the mechanism by which polyphenols exert their antibacterial effects is not yet fully understood, it is thought that polyphenols may function by degrading bacterial cell walls via the hydrophobic components of phenolic compounds, altering intracellular functions via hydrogen bonding, or modifying the structure and rigidity of the cell wall through interactions with the cell membrane [Dzialo, MA; Mierziak, J.; Korzun, U.; Preisner, M.; Szopa, J.; Kulma, A. The potential of plant phenolics in prevention and therapy of skin disorders. Int. J. Mol. Sci. 2016, 17, 160]. It has been found that increasing the lipophilicity of polyphenols enhances their antibacterial activity [Bouarab-Chibane, L.; Forquet, V.; Lanteri, P.; Clement, Y.; Leonard-Akkari, L.; Oulahal, N.; Degraeve, P.; Bordes, C. Antibacterial properties of polyphenols: Characterization and QSAR (Quantitative Structure-Activity Relationship) models. Front. Microbiol. 2019, 10, 829]. Furthermore, the antibacterial activity of polyphenols has been shown to be particularly effective against antibiotic-resistant strains such as methicillin-resistant S. aureus [Negut, I.; Grumezescu, V.; Grumezescu, A. Treatment strategies for infected wounds. Molecules 2018, 23, 2392].

[0023] definition As used herein, “wound” means a break in the continuity of the skin. The structure of the skin is complex, and wound biology is understood by knowing the factors that influence the local physiological environment. Many local conditions influence the occurrence, persistence, and healing of wounds. The skin consists of at least three layers: the epidermis, dermis, and subcutaneous tissue. A wound can be a break in the continuity of any one of the epidermis, dermis, and / or subcutaneous tissue, or any combination thereof. Examples of wounds include damage to living tissue caused by cutting, rupture, impact, or other physical destruction.

[0024] As used herein, "polyphenol" refers to an organic chemical substance containing multiple phenolic structural units. Major bioactive polyphenols found in lettuce include phenolic acid derivatives (e.g., hydroxybenzoic acid derivatives, hydroxyphenylacetic acid derivatives), coumarin derivatives (e.g., esculin, 6,7-dihydroxycoumarin), flavonoids (e.g., quercetin glycosides, kaempferol glycosides, luteolin glycosides, apigenin glycosides, flavanones, flavones, etc.), and anthocyanins (e.g., cyanidin glycosides, etc.). In preferred embodiments, the bioactive polyphenols disclosed herein include anthocyanins, chlorogenic acid, chicoric acid, dicaffeoylquinic acid, and quercetin derivatives (quercetin glycosides).

[0025] As used herein, "powder" refers to fine, dry particles produced by the grinding, crushing, or disintegration of a solid material.

[0026] As used herein, “particles” refers to small topical compositions having a set of chemical and physical properties. Generally, particles are about 0.01 μm to about 150 μm in width, length, and height, with one or more of these dimensions.

[0027] As used herein, "nano-sized particles" or "nanoparticles" are used interchangeably and refer to particles smaller than 0.1 μm.

[0028] As used herein, “us-stressor” and “elicitor” are used interchangeably and refer to various biological, physical, or chemical stressors that induce signaling pathways that enhance the bioactive compound content and quality characteristics of plant products. Us-stressors / elicitors can be classified as biomaterials and non-biological materials, examples of which are described in WO2022 / 183014. Plant hormones / plant growth regulators (e.g., salicylic acid (SA), jasmonate, etc.) are also considered us-stressors / elicitors. Us-stressors / elicitors of biological, chemical, or physical origin may enhance the agricultural / nutritional properties of plants by activating reactions that may include defense responses between them, leading to improvements in the functional quality of fruits and vegetables, for example. Plant growth regulators (PGRs) may be used as us-stressors / elicitors to stimulate the production of secondary metabolites in plants. Plant growth regulators may include naturally occurring hormonal substances (plant hormones) and their synthetic analogues.

[0029] Plant growth regulators (PGRs) are chemical substances used to modify plant growth, such as increasing branching, inhibiting shoot growth, increasing re-flowering, removing excess fruit, or altering fruit maturity. PGRs are sometimes also called plant hormones or elicitors. Examples of PGRs include auxins, cytokinins (CK), gibberellins (GA), ethylene, brassinosteroids, jasmonates (JA), strigolactones (SL), and salicylic acid (SA).

[0030] In this specification, "lettuce" refers to the plant of the species Lactuca sativa L. Lactuca sativa belongs to the tribe Cichorieae of the family Asteraceae (Compositae). Lettuce is closely related to chicory, sunflower, aster, dandelion, artichoke, and chrysanthemum. L. sativa is one of approximately 300 species in the genus Lactuca. A highly pleomorphic species, L. sativa is cultivated for its edible bulbs and leaves. As a crop, lettuce is commercially grown wherever environmental conditions allow for the production of profitable yields. Fresh lettuce is consumed almost exclusively as a fresh, raw food, and sometimes as a cooked vegetable. Lettuce is an increasingly popular crop. Lettuce consumption continues to increase worldwide. Due to this high demand, there are advantages to seeking increased polyphenol production in novel genetically modified lettuce. In particular, improved genetically modified lettuce that is stable, high-yielding, agrochemically sustainable, and has enhanced production of health-beneficial polyphenols is commercially viable, especially for human consumption.

[0031] "Lettuce plant" refers to the entire lettuce plant, whether immature or mature, and includes lettuce plants from which seeds, roots, or leaves have been removed. Seeds or embryos that produce this plant are also considered lettuce plants. Lettuce plants can be produced by sowing seeds directly into the ground (e.g., soil such as field soil) or by germinating seeds under controlled environmental conditions (e.g., a greenhouse) and then transplanting the seedlings into the field. See, for example, Gonai et al., J. of Exp. Bot., 55(394), 111-118, 2004; Louise Jackson et al., Acquaah, Principles of Plant Genetics and Breeding, 2007, Blackwell Publishing; and Jackson, Louise, et al., University of California, Publication 7216 (all of which are incorporated herein by reference).

[0032] "Lettuce cells" or "lettuce plant cells" refers to cells used herein, including lettuce plant cells, whether isolated, in tissue culture, or incorporated into a lettuce plant or lettuce plant part.

[0033] As used herein, “lettuce plant part” includes lettuce heads, lettuce leaves, parts of lettuce leaves, pollen, ovules, flowers, and the like. In another embodiment, the disclosure further relates to lettuce heads, lettuce leaves, parts of lettuce leaves, flowers, pollen, and ovules isolated from a lettuce plant.

[0034] The terms “subspecies” or “cultivar” refer to a group of plants belonging to the lowest known rank in a botanical taxonomy, defined by the expression of traits resulting from a given genotype or combination of genotypes, regardless of whether the conditions for granting breeder’s rights are fully met, and are considered a unit in that they are distinguished from any other group of plants by the expression of at least one of the aforementioned traits and are suitable for propagation in their original form.

[0035] "Plant" includes any part of a plant, preferably having the same (or very similar) genetic makeup as the plant from which it was obtained, such as the whole plant, or plant organs (such as harvested or unharvested leaves), plant cells, plant protoplasts, plant cell or tissue cultures capable of regenerating a whole plant, plant callus, plant cell aggregates, plant grafts, seedlings, undamaged plant cells within a plant, plant clones or micropropagations, or parts of a plant (such as harvested tissue or organs) (such as plant cuttings, vegetative propagation, embryos, pollen, ovules, flowers, leaves, heads, seeds, cloned plants, roots, stems, stalks, root tips, grafts, or any of these parts) or their derivatives. It also includes any developmental stage, such as seedlings, pre-rooting or post-rooting cuttings, mature and / or immature plants, or mature and / or immature leaves.

[0036] As used herein, a polynucleotide or polypeptide is “recombinant” if it is artificial or manipulated, or derived from an artificial or manipulated protein or nucleic acid. For example, a polynucleotide inserted into a vector or any other heterologous position, e.g., the genome of a recombinant organism, and not associated with a normally adjacent nucleotide sequence as it is found in nature, is a recombinant polynucleotide. A polypeptide expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Similarly, a polynucleotide sequence that does not occur in nature, e.g., a variant of a gene of natural origin, is recombinant.

[0037] As used herein, “heterogeneous” means a sequence derived from an alien species, or, if derived from the same species, a sequence whose composition and / or genomic locus has been substantially modified from its native form by intentional human intervention. For example, a promoter operably ligated to a heterogeneous polynucleotide may be derived from a different species than the one from which the polynucleotide originates, or, if derived from the same / similar species, one or both may be substantially modified from their original form and / or genomic locus, or the promoter may not be the original promoter for the polynucleotide to which it is operably ligated.

[0038] As used herein, “transgene” refers to a gene or gene product introduced into the genome of a lettuce plant by genetic engineering methods, such as transformation. Exemplary transgenes include cDNA (complementary DNA) segments, which are copies of mRNA (messenger RNA), and the gene itself, which is present in the original region of the genomic DNA. One example describes a DNA segment containing a gene sequence introduced into the genome of a lettuce plant or lettuce plant cell. This exogenous DNA segment may retain the ability to produce RNA or protein in the genetically modified lettuce plant, or it may alter the normal function of the genetic code in the genetically modified plant. Generally, transtransplant nucleic acids are incorporated into the germline of the plant. Transgenes can also describe any DNA sequence, whether it contains a gene coding sequence or is artificially constructed, that has been introduced into a lettuce plant or vector construct that was not previously found.

[0039] "Operatablely linked" is intended to mean a functional link between two or more elements. For example, an operatable link between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that enables the expression of the polynucleotide of interest. Operatablely linked elements may be continuous or discontinuous. When used to refer to the linking of coding regions of two proteins, operatablely linked means that the coding regions are within the same reading frame. A cassette may further contain at least one additional coding sequence / gene to be co-transformed into an organism. Alternatively, additional coding sequences / genes may be provided on multiple expression cassettes. Such an expression cassette comprises multiple restriction and / or recombination sites for the insertion of the coding polynucleotide of interest or its active variant or fragment, under the transcriptional control of a regulatory region (e.g., a promoter). An expression cassette may further contain a selectable marker gene.

[0040] An "expression cassette" refers to a polynucleotide encoding the polypeptide of interest operably ligated to at least one polynucleotide encoding an expression regulatory sequence. The expression cassette may include, in the 5'-3' direction of transcription, a transcription and translation initiation region (i.e., a promoter), a polynucleotide encoding the polypeptide of interest or an active variant or fragment thereof, and a transcription and translation termination region (i.e., a termination region) that functions in the plant. The regulatory regions (i.e., the promoter, transcription regulatory region, and translation termination region) and / or the polynucleotide or its active variant or fragment may be native / similar to or from the host cell. Alternatively, the regulatory regions and / or the polynucleotide or its active variant or fragment may be heterogeneous to or from the host cell.

[0041] The expression cassette may further contain a 5' leader sequence, which may act to enhance translation. Translational readers are known in the art, including picornavirus readers, e.g., EMCV readers (encephalomyocarditis 5' non-coding region) (Elroy-Stein et al. (1989) Proc.Natl.Acad.Sci.USA 86:6126-6130); Pochivirus readers, e.g., TEV readers (tobacco eczema virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV readers (maize mosaic virus) (Virology 154:9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); and non-translational readers from alfalfa mosaic virus coat protein mRNA (AMV RNA 4) (Jobling et al. (1987) Nature). This includes 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize fading spot virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385. See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968).

[0042] A “regulatory expression sequence” refers to a segment of a nucleic acid molecule that can increase or decrease the expression of a polypeptide encoded by an expression cassette. Examples of regulatory regions include promoters, transcriptional regulatory regions, and translation termination regions. Termination regions may be native with transcription initiation regions, with operably linked polynucleotides or their active variants or fragments, with plant hosts, or from a source other than promoters, polynucleotides or their active fragments or variants, plant hosts, or any combination thereof (i.e., exogenous or heterologous). Convenient termination regions, such as octopine synthase and nopalin synthase termination regions, are available from the Ti-plasmid of A. tumefaciens. Guerineau et al.(1991) Mol.Gen.Genet.262:141-144;Proudfoot (1991) Cell 64:671-674;Sanfacon et al.(1991) Genes Dev.5:141-149;Mogen et al.(1990) Plant Cell 2:1261-1272;Munroe et al. See also Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0043] A "control" provides a baseline for measuring changes in the phenotype of a subject. For example, a control for wound healing or closure may be an equivalent wound that did not receive the active ingredient that promotes wound healing. The control wound may be treated with a vehicle or dressing that does not contain the active ingredient that promotes wound healing. The control wound may be present in the same subject or in different but comparable subjects.

[0044] The "control lettuce" or "control lettuce cells" provide a baseline for measuring phenotypic changes in the lettuce plant or lettuce plant cells of interest, and may be any suitable lettuce plant or lettuce cell. Control lettuce or lettuce cells may include, for example, (a) wild-type or natural lettuce or lettuce cells, i.e., lettuce or lettuce cells with the same genotype as the starting material for the genetic modification resulting in the lettuce or lettuce cells of interest; (b) lettuce or lettuce cells with the same genotype as the starting material but transformed with a null construct (i.e., a construct that does not have a known effect on the trait of interest, such as a construct containing a marker gene); (c) lettuce or lettuce cells that are untransformed isolates from the progeny of the lettuce or lettuce cells of interest; (d) lettuce or lettuce cells that are genetically identical to the lettuce or lettuce cells but have not been exposed to the same treatment as the lettuce or lettuce cells of interest (e.g., us-stressor / elicitor treatment, herbicide treatment); or (e) the lettuce or lettuce cells of interest themselves under conditions in which the gene of interest is not expressed.

[0045] "Effective dose" or "therapeutic effective dose" may refer to the amount of therapeutic agent (e.g., lettuce extract, lettuce plant, or lettuce plant part as described herein) that provides a desired physiological change, such as wound healing. The desired physiological change may be, for example, increased wound closure, decreased wound-related inflammation, increased cell proliferation, and / or increased vascular tissue formation and / or regeneration.

[0046] In this specification, the term “about” means ±20% of the range, value, or structure indicated unless otherwise indicated. The term “essentially consisting of” limits the scope of the claims to specific materials or steps and those that do not substantially affect the basic and novel characteristics of the claimed embodiment. The terms “a” and “an” as used herein should be understood to mean “one or more” of the enumerated components. The use of options (e.g., “or”) should be understood to mean one of the options, both, or any combination thereof. Where used herein, the terms “include” and “have” are used synonymously, and these terms and their variations are intended to be construed as non-limiting. The term “comprise” means the presence of the described features, details, steps, or components stated in the claims, but not that it excludes the presence or addition of one or more other features, details, steps, components, or groups thereof.

[0047] The recombinant DNA, molecular cloning, and gene expression techniques used in this disclosure are known in the art and are described in reference to Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, New York, 2001, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD, 1999. All documents (e.g., patent publications) are incorporated herein by reference in their entirety.

[0048] Various modifications and variations of the products and methods described herein will be obvious to those skilled in the art without departing from the scope and spirit of this disclosure. Although this disclosure has been described in relation to certain preferred embodiments, it should be understood that the claimed disclosure should not be unduly limited to such specific embodiments. In fact, various modifications of the methods described for carrying out the invention that are obvious to those skilled in the art in chemistry, biology, or related fields are intended to be within the scope of the following claims.

[0049] Polyphenol-fortified powder In some embodiments, the disclosure provides powders containing particles of polyphenol-fortified lettuce powder or extract. Polyphenol-fortified lettuce exhibits increased production and concentration of at least one polyphenol compared to control lettuce. Representative bioactive components identified from such red lettuce include flavonoids, particularly their derivatives that are highly available to the body when orally ingested (quercetin-3-O-glucose and quercetin-3-O-malonyl glucoside) (Figures 1A-1C), chlorogenic acid: 3-caffeoylquinic acid (3-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA) (Figures 2A and 2B), chicory (Figure 3), and anthocyanins (e.g., cyanidin-3-O-glucoside and equivalents) (Figures 4A and 4B). In some embodiments, the polyphenol is at least one of chlorogenic acid, neochlorogenic acid, chicory, quercetin, quercetin derivatives, anthocyanins, or any combination thereof. In some embodiments, polyphenol-fortified lettuce exhibits increased production and concentration of chlorogenic acid, chicoric acid, quercetin derivatives, and anthocyanins. In some embodiments, chlorogenic acid includes 3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), or any combination thereof. In some embodiments, quercetin derivatives include quercetin-3-O-glucoside (Q3G), quercetin glucuronide, quercetin-3-O-malonylglucoside (Q3MG), or all of these. In some embodiments, the anthocyanins include cyanidin 3-galactoside, cyanidin-3-O-glucoside, cyanidin-3-6"-malonylglucoside, or any combination thereof. In some embodiments, the polyphenols include 4-CQA, neochlorogenic acid, chicoric acid, and cyanidin 3-galactoside. In some embodiments, the increase in polyphenol production is quantified by LC-MS. In some embodiments, the increase in polyphenol production is quantified by HPLC.A method for producing polyphenol-fortified lettuce is described in WO2022 / 183014, which is incorporated herein by reference in its entirety.

[0050] The compositions and methods disclosed herein are applicable to at least four tissues of the human and animal body and provide protection, repair, and treatment for the following purposes: epithelial tissue (e.g., wounds, skin disorders, and hair), muscle tissue (e.g., sarcopenia and muscle injury), connective tissue (e.g., repair and regeneration), and nerve tissue (e.g., peripheral neuropathy and nerve injury).

[0051] In certain embodiments, polyphenol-fortified lettuce was treated with at least one eu-stressor / elicitor, or its homologs, isomers, or derivatives, that increase the production of polyphenols in the lettuce. For example, the at least one eu-stressor / elicitor may be an abiotic eu-stressor / elicitor selected from indole-3-acetic acid (IAA), auxin, cytokinin (CK), gibberellin (GA), ethylene, brassinosteroid, jasmonate (JA), strigolactone (SL), salicylic acid (SA), arachidonic acid (AA), 5-aminolevulinic acid (5-ALA), oxalic acid, and any homologs, isomers, derivatives, synthetic analogs, or any combination or mixture thereof. In some embodiments, the polyphenol-fortified lettuce contains a 2- to 20-fold increase in polyphenol products compared to control lettuce, and optionally, a 3- to 9-fold increase in polyphenol products compared to control lettuce. In some embodiments, the concentrations of chlorogenic acid, quercetin derivatives, chicoric acid, and anthocyanins are increased by at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times compared to powder produced from control lettuce.

[0052] In some embodiments, the total polyphenol concentration in the polyphenol-fortified lettuce powder or its extract is about 40 to 280 mg / g. In some embodiments, the chlorogenic acid concentration is about 6 to 38 mg / g. In some embodiments, the chicoric acid concentration is about 4 to 45 mg / g. In some embodiments, the quercetin derivative concentration is about 18 to 175 mg / g. In some embodiments, the anthocyanin concentration is about 6 to 20 mg / g.

[0053] Particle size distribution (PSD) is a critical characteristic of pharmaceuticals of all preparation types, including solid oral formulations, semi-solids, aerosols, and sterile liquid products. Ball milling, conical milling, and hammer milling can be used to control particle size and shape, achieving a consistent, narrow PSD and resulting in high-quality products and efficient production systems. The choice of ball milling, conical milling, or hammer milling depends on several factors, including an evaluation of the type of product to be processed and the desired outcome.

[0054] In topical and transdermal drug delivery, particle size plays a crucial role in determining the pharmacological properties of the product. For example, if an API is suspended in a vehicle, such as a gel or cream, particle size interacts with the vehicle's solubility to determine the rate of dissolution. Particle size also influences the rate, depth, and pathway of absorption through the skin. Ultimately, selecting the appropriate particle size for a topical preparation can help maximize the drug's effect at the site of action and minimize the risk of systemic adverse reactions.

[0055] In some embodiments, the preferred particle size range for the powder is about 0.1 to about 150 μm. Clearly defined powders may have a size range of about 0.1 μm to about 10,000 μm. Suitable D50 and D90 values ​​for bioactive lettuce powder or extracts are defined and achieved in specific formulations. In some embodiments, the polyphenol-enhanced red lettuce powder includes nanoparticle powder. As used herein, the nanoparticles have a particle size distribution range of about 0.1 μm or less. In some embodiments, the polyphenol-enhanced red lettuce powder has a particle size distribution range of about 0.01 μm to about 0.1 μm. In some embodiments, the polyphenol-enhanced red lettuce powder has a particle size distribution range of about 0.05 μm to about 0.1 μm.

[0056] An exemplary method for defining the particle size of polyphenol-fortified lettuce powder or its extract is by mesh size. The term “mesh size” refers to the fineness or coarseness of a material, determined by the ability of particles to pass through a mesh. Mesh size is related to the mesh number and is generally defined as the number of mesh openings in a sieve screen within a 1-inch x 1-inch area. To determine the mesh number, one may, for example, count the number of openings in one linear inch of the screen. For example, a 4-mesh screen has four square openings across one inch of screen, and a 100-mesh screen has 100 openings per inch. As the mesh number increases, the size of the mesh openings decreases, resulting in smaller particle sizes that can be captured by the screen. The mesh size indicates the particle size of the material that can pass through the corresponding mesh openings. For example, a mesh size of 200 means that the material can pass through a sieve screen with 200 openings within a 1-inch x 1-inch area. Similarly, a larger mesh size indicates finer particle size, and a smaller mesh size indicates coarser particle size. Therefore, mesh size can be used to determine the fineness or coarseness of material particles, rather than directly measuring their size. However, it is important to note that mesh size is not an accurate measure of the mesh opening size, as screens can be made using various materials with different strand or wire thicknesses. The thickness of the strands used in the screen affects the size of the opening and, consequently, the particle size that can pass through.

[0057] By using different mesh sizes in the powder mixture, 3D proportional formulations can achieve the following functions: Firstly, combining different mesh sizes in the formulation increases the contact surface area between particles. This strengthens interparticle bonding and can improve the overall strength and integrity of the printed object. Secondly, including different mesh sizes improves air convection between powder particles during the printing process. This improved airflow helps reduce the risk of particle aggregation, ensures a uniform powder distribution, and consequently improves print quality and consistency. Thirdly, by adjusting the mesh size of the formulation, it is possible to control the adhesion or aggregation of powder particles. This can be beneficial in achieving the desired flowability of the powder during printing. Reducing particle adhesion can prevent clogging or aggregation, while increasing particle aggregation can improve interlayer adhesion and enhance the structural integrity of the powder.

[0058] The polyphenol-fortified lettuce powder or extract disclosed herein possesses effective anti-inflammatory and antioxidant properties. Therefore, it can be used in various cosmetic formulations, particularly sunscreens, facial cleansers, face masks, and conditioners. This incorporation may provide the skin with various benefits, such as anti-aging effects, protection from radiation, skin whitening, hydration, freckle reduction, acne treatment, and hair growth stimulation. Using the polyphenol-fortified lettuce powder or extract, different formulations such as powders, aqueous solutions, sprays, gels, oils, creams, surfactants, and ointments can be created for specific purposes in four major human tissues. For example, the polyphenol-fortified lettuce powder or extract may be added to soaps, shampoos, conditioners, hair dyes, body washes, and cleansers to enhance their functions. In some embodiments, the polyphenol-fortified lettuce powder or extract may be dissolved in surfactants, mixtures, or solvents. The disclosed polyphenol-fortified lettuce powder or extract may also provide antioxidant functionality. Polyphenol-fortified lettuce powder or its extract may also be mixed with powdered antibiotics to enhance their bactericidal and bacteriostatic effects. In other embodiments, polyphenol-fortified lettuce powder or its extract may be combined with collagen, seaweed extract, β-glucan, carboxymethylcellulose (CMC), and vitamin D3 to create a dressing that promotes wound healing. In other embodiments, the polyphenol-fortified lettuce powder or its extract may be combined with hyaluronic acid, collagen, and CMC to form a base for products that can be used for the treatment of skin disorders or skin care. In other embodiments, the polyphenol-fortified lettuce powder or its extract may be combined with antibacterial glycerin as an emulsion base. In other embodiments, the polyphenol-fortified lettuce powder or its extract may be combined with colloids and diluents for the protection and repair of skin and soft tissues. In yet another embodiment, the polyphenol-fortified lettuce powder or its extract may be combined with paraffin or glycerin for the protection and repair of skin and soft tissues.

[0059] The polyphenol-fortified lettuce powder or extracts disclosed herein may, for convenience, be administered in combination with appropriate excipients in a pharmaceutical composition containing a bioactive composition. Such pharmaceutical compositions may be prepared by methods well known in the art and may contain excipients. Such methods and components can be found in Remington's Pharmaceutical Sciences [Alfonso Gennaro et al., eds., Nov. 8, 2012; Lippincott, Williams & Wilkins, Baltimore, Md., 20” ed., 2000]. The formulations and compositions of the present invention may be administered topically, orally, or rectally.

[0060] For topical administration, formulations may contain polyphenol-fortified lettuce powder or an extract thereof with a high polyphenol concentration as the active ingredient. Compositions according to this disclosure can be conveniently formulated using one or more pharmaceutically acceptable carriers or excipients, which may be solid or liquid. Such compositions may take the form of, for example, ointments, lotions, creams, powders, liquids, gels, oils, surface tensioners, extracts, intravenous infusions (e.g., eye drops or ear drops), or sprays. The percentages of compositions and preparations may, of course, vary and may conveniently be between about 2% and about 100% of the weight of a given unit dosage form. The amount of the active compound in such therapeutically useful compositions is such that an effective dose level is obtained.

[0061] In some embodiments, the polyphenol-fortified lettuce powder or extract disclosed herein may be in the form of a cream or ointment. Ointments can typically be prepared by melting white soft paraffin (white petrolatum) incorporating any additives, such as surfactants and solvents, and blending it into a drug slurry in a minimal amount of liquid paraffin. The melt is then cooled under controlled conditions and stirred until solidification occurs. Creams can typically be prepared by mixing the oil phase of the ointment as a melt, as described above, with an aqueous phase containing appropriate oil-soluble and water-soluble surfactants, as well as the drug and appropriate antimicrobial preservatives, homogenizing to form a cream, and gently stirring until cooled.

[0062] Compositions containing polyphenol-fortified lettuce powder or extracts thereof as disclosed herein may generally contain additional excipients, such as preservatives (e.g., benzoic acid), emulsifiers (e.g., polysorbate 60), and viscosity improvers (e.g., cetostearyl alcohol).

[0063] Bioactive compositions can be mixed with soaps, shampoos, conditioners, hair dyes, body washes, and cleansing agents to enhance their functions. These compositions can also be mixed with antibiotics to enhance antibacterial activity. Furthermore, materials such as hyaluronic acid, collagen, alginates, β-glucans, carboxymethylcellulose (CMC), and vitamin D3 can be mixed to create dressings that promote wound healing.

[0064] Accordingly, in some embodiments, what is disclosed herein is a topical formulation comprising the above-mentioned polyphenol-fortified lettuce powder or an extract thereof and the following pharmaceutically acceptable carriers or excipients as described in the examples. In some embodiments, the polyphenol-fortified lettuce powder or an extract thereof is present in the topical formulation at a percentage of about 0.1% to 10%, about 0.5% to 5%, about 1% to 10%, about 5% to 20%, about 10% to 50%, or about 50% to 90% of the final weight of the topical formulation. In some embodiments, the pharmaceutically acceptable carriers or excipients are solid or liquid. In some embodiments, the topical formulation is in the form of an ointment, lotion, cream, powder, liquid, gel, hydrogel, oil, surface tension agent, drip, aerosol, emulsion, nanoemulsion, nanoliposome, gel, microencapsulation, paste, or foam. In certain embodiments, the topical formulation may be in the form of a sunscreen, facial cleanser, face mask, soap, shampoo, conditioner, body wash, or hair dye. In some embodiments, the topical formulation may further include a emollient, such as paraffin. In some embodiments, the topical formulation may further include an antimicrobial preservative. In some embodiments, the topical formulation may further include one or more emulsifiers and / or viscosity improvers.

[0065] In some embodiments, the topical formulations disclosed herein are, by weight percentage, 1-10% polyphenol-fortified lettuce powder or extract thereof, 5-15% alcohol, 5-15% emollient, 1-5% emulsifier, 5-15% propylene glycol and Antimicrobial preservatives of less than 1%, The remaining percentage of water to make it equal to 100%, Includes.

[0066] In some embodiments, wound dressing powders are provided herein, comprising the polyphenol-fortified lettuce powder or an extract thereof and the following pharmaceutically acceptable powdered carriers or excipients as described in the examples. Examples of pharmaceutically acceptable carriers or excipients include starch, cellulose, synthetic polymers, polysaccharides, chitosan, mineral powders, clay powders, or any combination thereof. In some embodiments, the starch is corn starch, potato starch, rice starch, or any combination thereof. In some embodiments, the clay is bentonite, kaolin, smectite, or any combination thereof. In some embodiments, the cellulose is microcrystalline cellulose, sodium carboxymethylcellulose (CMC), or any combination thereof. In some embodiments, the synthetic polymer is polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), or a combination thereof. In some embodiments, the polysaccharide is calcium alginate. In some embodiments, the mineral powder is zinc oxide powder.

[0067] In a particular embodiment, the wound dressing powder is i. Mineral powder containing 50% to 90% wt / wt, ii. Clay powder in a concentration of 5% to 15% wt / wt, iii. Polyphenol-fortified lettuce powder or extract containing 1% to 15% wt / wt, and optionally, iv. Essential oils, for example, lavender essential oil and Includes.

[0068] In some embodiments, the methods disclosed herein involve the delivery of nanoparticles mediated by hydrogels. Due to the inherent properties of hydrogels, they have the potential to enhance the bioavailability, solubility, and stability of polyphenol-fortified lettuce powder in medical applications. Furthermore, hydrogels can be modified with specific targeting ligands, thereby guiding the payload to the precise site of action [Khalvati, B.; Sheikhsaran, F.; Sharifzadeh, S.; Kalantari, T.; Behzad Behbahani, A.; Jamshidzadeh, A.; Dehshahri, A. Delivery of plasmid encoding interleukin-12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles. Artificial cells, nanomedicine, and biotechnology 2017, 45 (5), 1036-1044]. This targeted delivery approach can reduce side effects while increasing pharmacological activity at the target site [Sheikhsaran, F.; Sadeghpour, H.; Khalvati, B.; Entezar-Almahdi, E.; Dehshahri, A. Tetraidothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding IL-12 gene. Colloids Surf., B 2017, 150, 426-436]. Furthermore, stimulus-responsive hydrogels offer an opportunity to precisely control payload migration [McKenzie, M.; Betts, D.; Suh, A.; Bui, K.; Kim, LD; Cho, H. Hydrogel-based drug delivery systems for poorly water-soluble drugs. Molecules 2015, 20 (11), 20397-20408].Furthermore, using hydrogels as a delivery vehicle for polyphenol-fortified lettuce powder enables the application of the drug via different routes of administration, such as oral, topical, nasal, or parenteral.

[0069] Accordingly, in some embodiments, the Specified provides a hydrogel comprising a polyphenol-fortified lettuce powder or an extract thereof disclosed herein and a pharmaceutically acceptable hydrogel polymer. In some embodiments, the powder is present in a percentage of about 0.1% to 10%, about 0.5% to 5%, about 1% to 10%, about 5% to 20%, or about 10% to 50% of the final weight of the topical formulation. In some embodiments, the polyphenol-fortified lettuce powder or an extract thereof contains particles having a particle size distribution range of 0.1 μm to 150 μm. In some embodiments, the polyphenol-fortified lettuce powder or an extract thereof contains nanoparticles or consists essentially of nanoparticles. In some embodiments, the nanoparticles have a particle size distribution range of 0.1 μm or less, and optionally, the particle size distribution range is 0.01 μm to 0.1 μm, or 0.05 μm to 0.1 μm. In some embodiments, the hydrogel polymer is at least one of chitosan, gelatin, collagen, polysaccharide, starch, alginate, or agarose. In some embodiments, the hydrogel further comprises a polymer surfactant, such as Pluronic P123. In some embodiments, the hydrogel further comprises a targeted ligand that targets the site of action.

[0070] For oral therapeutic administration, the polyphenol-fortified lettuce powder or extracts disclosed herein may be combined with one or more excipients and may be used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and wafers. Such compositions and preparations should contain at least 1% of the active composition. For example, the polyphenol-fortified lettuce powder may be present in a percentage of about 0.1% to 10%, about 0.5% to 5%, about 1% to 10%, about 5% to 20%, or about 10% to 50% of the final volume of the oral formulation. Tablets, lozenges, pills, capsules, etc. may also contain the following: That is, binders such as tragacanth gum, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavor may be added. If the unit dosage form is a capsule, it may contain a liquid carrier such as vegetable oil or polyethylene glycol in addition to the above types of materials. Various other materials may be present as a coating or otherwise to change the physical form of the solid unit dosage form. For example, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar. The syrup or elixir may contain the active compound. Sucrose or fructose as a sweetener, methyl and propylparaben as preservatives, dyes and flavorings, e.g., cherry flavor or orange flavor. The active composition may also be incorporated into sustained-release preparations and devices.

[0071] The topical formulations of this disclosure may be absorbed via transmembrane pathways, including, for example, the sublingual gland, nasal mucosa, rectal mucosa, and / or the intestinal pathway. In preferred embodiments, the topical formulations may be applied topically to the oral cavity to treat localized disorders such as oral ulcers, periodontitis, and plaque.

[0072] In some embodiments, wound healing can be promoted by combining topical administration with topical oral administration and photodynamic therapy. For example, a topical formulation can be applied to the wound area, followed by oral administration of an oral formulation, and then the wound area can be irradiated with blue light having a wavelength in the range of 400 nm to 520 nm (peak wavelength 440 nm). This blue light emission can generate a large amount of reactive oxides (ROS) that promote wound sterilization. At the same time, polyphenols in the oral formulation can protect normal human tissue cells and avoid damage from free radicals caused by irradiation. In some embodiments, irradiating the wound area with red light in the range of 617 to 850 nm (peak wavelengths 660 nm and 720 nm, respectively) can produce a synergistic effect that promotes wound healing. Compared to blue light emission, red light emission can penetrate deeper into the subcutaneous tissue and stimulate cellular energy production, thereby promoting cell repair and regeneration and accelerating wound healing. Furthermore, the degree of inflammatory response can also be reduced by regulating macrophage M1 / M2 polarization.

[0073] Method for producing extract from polyphenol-fortified lettuce powder In some embodiments, the present disclosure provides an extract of polyphenol-fortified lettuce powder. As described above, this extract is a solid-form powder extract containing a high concentration of polyphenols. In some embodiments, the extract contains at least 40 mg / g of polyphenols. In some embodiments, the total polyphenol concentration contained in the polyphenol-fortified lettuce powder or its extract is about 40 to 280 mg / g. In some embodiments, the chlorogenic acid concentration is about 6 to 38 mg / g. In some embodiments, the chicoric acid concentration is about 4 to 45 mg / g. In some embodiments, the quercetin derivative concentration is about 18 to 175 mg / g. In some embodiments, the anthocyanin concentration is about 6 to 20 mg / g.

[0074] In some embodiments, methods for preparing extracts from polyphenol-fortified lettuce, which are formed from polyphenol-fortified lettuce powder, are disclosed herein. Figure 5 is a flowchart of Method 500 for preparing extracts from polyphenol-fortified lettuce according to some embodiments of the present disclosure. For additional embodiments of the Method, additional steps may be provided before, during, and after Method 500, and it should be understood that some of the steps described below may be replaced or omitted.

[0075] Referring to Figure 5, Method 500 includes step 502 of grinding polyphenol-fortified lettuce to form polyphenol-fortified lettuce powder. In some embodiments, lettuce leaves or any above-ground parts may first be dried, for example, by air drying or freeze-drying. The dried lettuce may then be cut into smaller pieces and then powdered using, for example, ball milling, conical milling, or hammer milling to form polyphenol-fortified lettuce powder. The polyphenol-fortified lettuce powder is then sorted by size according to the mesh size. In some embodiments, the polyphenol-fortified lettuce powder may have a particle size in the range of about 50 mesh to 1500 mesh, for example, about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 mesh. In some embodiments, the polyphenol-fortified lettuce powder may have a particle size in the range of 0.1 to 10,000 μm, for example, 0.1 to 150 μm.

[0076] In step 504 of Method 500, the polyphenol-fortified lettuce powder is subjected to extraction. In some embodiments, the extraction may be carried out using a solvent extraction method by immersing the polyphenol-fortified lettuce powder in an extraction solvent. In some embodiments, the extraction solvent may be water, alcohol, a mixture of water and alcohol, or a supercritical fluid. Examples of alcohols for extraction include, but are not limited to, ethanol or isopropanol. In some embodiments, the extraction solvent is water. In some embodiments, the extraction solvent is ethanol. Furthermore, the extraction solvent is a mixture of water and ethanol, with ethanol making up about 50% to about 90% of the mixture. In some embodiments, the extraction solvent is a supercritical fluid such as supercritical carbon dioxide (CO2). More polar extraction solvents, such as alcohol, water, or mixtures thereof, result in an extract containing a larger amount of polyphenols, while nonpolar extraction solvents such as supercritical CO2 produce an extract with a lower polyphenol content and a concentration of nonpolar compounds such as terpenes, carotenoids, and vitamins.

[0077] In some embodiments, the ratio of polyphenol-fortified lettuce powder to solvent (g / mL) may be in the range of approximately 1:10, 1:5, 2:5, 3:5, 4:5, or 1:1. In a particular embodiment, the ratio of polyphenol-fortified lettuce powder to solvent (g / mL) is 2:5.

[0078] Extraction can be carried out at temperatures in the range of approximately 15°C to 85°C, for example, approximately 15°C to 20°C, 20°C to 25°C, 20°C to 30°C, 30°C to 35°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 60°C to 65°C, 65°C to 70°C, 70°C to 75°C, 75°C to 80°C, or 80°C to 85°C.

[0079] The duration of the extraction process ranges from approximately 0.5 hours to approximately 24 hours, for example, approximately 0.5 hours to approximately 1 hour, approximately 1 hour to approximately 2 hours, approximately 2 hours to approximately 3 hours, approximately 3 hours to approximately 4 hours, approximately 4 hours to approximately 5 hours, approximately 5 hours to approximately 6 hours, approximately 6 hours to approximately 10 hours, approximately 10 hours to approximately 18 hours, or approximately 18 hours to approximately 24 hours.

[0080] Extraction may be carried out under pressurized or atmospheric conditions by shaking, stirring, vibrating, or a combination thereof. In some embodiments, extraction may be assisted by ultrasound. Ultrasound applies strong shear and stress to the polyphenol-fortified lettuce powder and the extraction solvent, which helps to accelerate the penetration of the extraction solvent into the polyphenol-fortified lettuce powder. Thus, ultrasound can improve extraction efficiency and shorten extraction time. The ultrasound may be pulsed. In some embodiments, the ultrasound may have a pulse duration of 100 ps to 1 ms, for example, 100 ps to 100 ns, 100 ns to 500 ns, 500 ns to 1 μs, 1 μs to 10 ns, 10 μs to 500 μs, or 500 μs to 1 ms; the pulse power may be in the range of 1 kW to 50 kW, for example, 1 kW to 5 kW, 5 kW to 10 kW, 10 kW to 20 kW, or 20 kW to 50 kW; and the frequency may be in the range of 100 kHz to 200 MHz, for example, 500 kHz to 25 MHz, 500 kHz to 200 MHz, 1 MHz to 5 MHz, 1 MHz to 7 MHz, 1 MHz to 10 MHz, 1 MHz to 20 MHz, 1 MHz to 25 MHz, 1 MHz to 30 MHz, 1 MHz to 200 MHz, 2 MHz to 5 MHz, 2 MHz to 10 MHz, or 2 MHz to 200 MHz. In some embodiments, ultrasound is generated using an ultrasonic transducer or an ultrasonic horn.

[0081] Once the extraction process is complete, the liquid portion is separated from the solid portion (also called "lettuce residue") by means of filtration or centrifugation. After drying, the lettuce residue may be mixed with magnesium calcium phytate for use as animal feed or plant fertilizer.

[0082] In some embodiments, finely crushed lettuce may be pre-mixed with the solvent to form a slurry before immersion in the extraction solvent. Suitable solvents may be water, ethanol, or a mixture thereof.

[0083] In step 506 of method 500, the liquid portion is dried, for example by freeze-drying, to remove the solvent and obtain a polyphenol-enhanced extract in powder form. Drying can be carried out at temperatures ranging from about 35°C to about 70°C, for example, in the range of about 35°C to about 40°C, about 40°C to about 45°C, about 45°C to about 50°C, about 50°C to about 55°C, about 55°C to about 60°C, about 60°C to about 65°C, or about 65°C to about 70°C. After drying, calcium magnesium phytate may be added to prevent aggregation of the extract and extend its shelf life.

[0084] In some embodiments, the extract can be mixed with lettuce residue to allow the active ingredients in the extract to adhere to the surface of the lettuce residue. When used internally, this mixture helps enhance the absorption of polyphenols by the intestinal tract due to the higher fiber content of the lettuce residue. When used externally, this mixture helps promote wound healing because the lettuce residue increases its breathability.

[0085] Method and Use In some embodiments, the foregoing describes a method for treating a wound, comprising administering to the wound site a therapeutically effective amount of polyphenol-fortified lettuce or its extract, a topical formulation, a wound dressing powder, a hydrogel, and / or an oral formulation as disclosed herein. In some embodiments, the wound includes acute wounds, skin infections, burn wounds, ulcers, chronic wounds, diabetic wounds, or refractory wounds.

[0086] Useful dosages can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art (see, for example, U.S. Patent No. 4,938,949).

[0087] In some embodiments, the foregoing provides a method for treating a skin disorder, the method comprising topically administering to the affected site a therapeutically effective amount of polyphenol-fortified lettuce or its extract, a topical formulation disclosed herein, a wound dressing powder disclosed herein, a hydrogel disclosed herein, and / or an oral formulation disclosed herein. Skin disorders generally refer to any skin-related diseases, such as inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral rash, herpes zoster, tinea, and cancers, such as basal cell carcinoma, squamous cell carcinoma, and melanoma. Skin disorders treated with the polyphenol-fortified lettuce or its extracts, topical formulations, wound dressing powders, hydrogels, and / or oral formulations disclosed herein include skin ulcers, bedsores, diabetic dermatitis, hypertrophic scars, keloid scars, telangiectasias (spider veins), skin atrophy, precancerous skin lesions, herpes, inflammatory acne, acne vulgaris, acne comedones or multiformes, nodular cystic acne, acne clusters, senile acne, sun-induced acne, occupational acne, etc. This includes, but is not limited to, any secondary acne, ichthyosis, ichthyosis-like diseases, Darier's disease, palmoplantar keratosis, leukoplakia and leukoplakia-like diseases, or lichen and lichen planus, psoriasis of the skin, mucous membranes or nails, psoriatic rheumatoid arthritis, cutaneous atopic dermatitis including eczema, dry skin, skin inflammation, flushing, solar erythema, actinic keratosis, cutaneous allergies and allergic or irritant contact dermatitis, atopic dermatitis, rosacea, hyperpigmentation, benign pigmented lesions (lentiformity, freckles, age spots, melasma), and aging skin.

[0088] Another aspect of this disclosure is a method for administering to a human or animal patient to provide an anti-inflammatory effect. For example, polyphenol-fortified lettuce powder or an extract thereof has been demonstrated to have anti-inflammatory effects both in vivo and in vitro. The tested samples showed efficacy in inhibiting NF-κB activity. Polyphenol-fortified lettuce powder or an extract thereof has also shown efficacy in reducing the levels or activity of various cytokines known to contribute to inflammatory diseases or diseases with significant inflammatory elements, as well as antioxidant efficacy.

[0089] In some embodiments, the herein is disclosed a method for treating an inflammatory condition or disease, the method comprising administering to a subject in need of treatment a therapeutically effective amount of the polyphenol-fortified lettuce or its extract, the topical formulations disclosed herein, the wound dressing powders disclosed herein, the hydrogels disclosed herein, and / or the oral formulations disclosed herein. In some embodiments, the production of inflammatory cytokines by immune cells is reduced compared to immune cells before treatment. In some embodiments, NF-kappa B activity is reduced. In some embodiments, the method comprises administering the composition to skin, e.g., epithelial tissue. In some embodiments, the method comprises administering the composition to muscle tissue. In some embodiments, the method comprises administering the composition to connective tissue. In some embodiments, the method comprises administering the composition to nerve tissue.

[0090] In some embodiments, the foregoing discloses a skin care method comprising administering to a subject a therapeutically effective amount of polyphenol-fortified lettuce or its extract as disclosed herein, a topical formulation as disclosed herein, a wound dressing powder as disclosed herein, a hydrogel as disclosed herein, and / or an oral formulation as disclosed herein.

[0091] In any of the embodiments disclosed herein, the subject may be a human, a non-human primate, a mouse, a rat, a gerbil, a rabbit, a dog, a cat, a horse, a cattle, a pig, a goat, a sheep, a donkey, a llama, an alpaca, a guinea pig, a mule, a deer, a buffalo, a chicken, a duck, a goose, or a turkey. Preferably, the subject is a human.

[0092] In some embodiments, methods for preparing nanoparticle powders are disclosed herein, i. Wash and dry at least one polyphenol-fortified red lettuce leaf, ii. To form polyphenol-fortified lettuce powder, mill at least one red lettuce leaf using a milling tool, iii. To remove particles larger than the sieve opening, the powder is passed through a sieve, iv. In order to reduce the particle size of the powder to a particle size distribution range of 0.1 μm or less, the powder is pulverized, Includes.

[0093] In some embodiments, grinding is performed using a ball mill, conical mill, hammer mill, or homogenizer. In some embodiments, the particle size distribution is 0.01 μm to 0.1 μm, or 0.05 μm to 0.1 μm. In some embodiments, the drying temperature is 40 to 45°C.

[0094] In some embodiments, this specification discloses a method for preparing wound dressing powder, which is: i. Mixing mineral powder and clay powder to form a powder mixture, ii. Adding the polyphenol-fortified lettuce powder or extract thereof disclosed herein to a powder mixture, iii. Blending the powder mixture, iv. Adding pharmaceutically acceptable excipients, carriers, or diluents to the powder mixture, Includes.

[0095] In some embodiments, the mineral powder contains zinc oxide. In some embodiments, the clay powder contains bentonite clay. In some embodiments, the method further comprises adding essential oil to the powder mixture and mixing the powder mixture in order to disperse the oil throughout the powder mixture.

[0096] In some embodiments, methods for preparing hydrogels are disclosed herein, i. To form a chitosan solution, the chitosan powder is dissolved in an acid solution, ii. Adding the polyphenol-fortified lettuce powder or extract thereof disclosed herein to a chitosan solution, iii. To prepare the gelatin solution, mix the gelatin powder with water, iv. Mixing a chitosan solution containing powder extract with a gelatin solution, v. Adding a crosslinking agent to a mixture of chitosan solution and gelatin solution, vi. To form a hydrogel, pour the mixture from v. into a hydrogel mold and allow the mixture to solidify. vii. Removing the hydrogel from the mold, viii. To promote crosslinking, heat is applied to the hydrogel, Includes.

[0097] In some embodiments, the acid solution contains 1% acetic acid. In some embodiments, the method further includes maintaining the pH of the mixture at about 7.0 by adding a NaOH solution when mixing the chitosan solution with the gelatin solution. In some embodiments, solidifying the mixture includes allowing the mixture to solidify at room temperature for 2-3 hours. In some embodiments, heating the hydrogel includes placing the hydrogel in a 50°C oven for 24 hours. In some embodiments, the crosslinking agent contains glutaraldehyde. In some embodiments, the method further includes adding a polymer surfactant to the gelatin solution. In some embodiments, the method further includes freeze-drying the hydrogel.

[0098] Those skilled in the art will be able to recognize further aspects and features of the invention by reading the following description of specific embodiments of the invention in conjunction with the accompanying drawings. The following examples are provided for illustrative purposes only and should not be considered limiting. The experimental procedures used in the examples are also described. The following examples further illustrate the invention but are not intended to limit the scope of the invention. [Examples]

[0099] Example 1 Enhancement of polyphenol production in red lettuce using a stressor / elicitor. This example demonstrates that polyphenol production in red leaf lettuce increases when treated with biological / abiotic use stressors / elicitors.

[0100] Plant material, growing conditions, and use stressor / elicitor treatments. Red lettuce plants (Lactuca sativa) were cultivated in an experimental greenhouse with an average photoperiod of 12 hours / day, a temperature of 25-28°C, and a relative humidity of 40-60%. The abiotic eu-stressor used was indole-3-acetic acid (IAA) at concentrations of 30, 60, and 120 mg / L. The eu-stressor was dissolved in deionized water (those insoluble in water were first dissolved in 1 mL of ethanol). Groups of samples were added to water containing only 1 mL of ethanol. An untreated control sample was added. On day 7 before harvest, the red lettuce was treated with the elicitor. Each experimental unit consisted of five lettuce plants randomly selected and assigned to one treatment. Each sample was treated by root absorption or foliar spraying, with each elicitor sprayed three times (approximately 1.70 mL). The lettuce samples were harvested at 50 days.

[0101] Extraction and quantification After extracting treated and untreated (control) red lettuce samples with 50% ethanol, the major polyphenols beneficial to health were characterized and quantified. Generally, 2 grams of sample (ground with liquid nitrogen) was mixed with 5 mL of ethanol, shaken at room temperature for 4 hours, and centrifuged at 5000 × g for 10 minutes (4°C). The supernatant was collected, filtered, and subjected to LC-MS / HPLC analysis.

[0102] result Enhanced polyphenol production was confirmed using LC / MS / UV (or HPLC).

[0103] As shown in Figure 6, HPLC chromatograms of the enhancement of bioactive components by genomics-based technology confirm the production of specific metabolites in red lettuce treated with biotope / abiotic stressors. Polyphenol 3-CQA, chicoric acid, 3,4-dicaffeoylquinic acid (3,4-diCQA), quercetin-3-O-glucoside (Q3G), and quercetin-3-O-malonylglucoside (Q3MG) are shown to be produced in treated lettuce compared to untreated lettuce control.

[0104] As shown in Figures 7A-7B, the production of chlorogenic acid (Figure 7A) and water-soluble quercetin derivatives (Figure 7B) increased 3 to 9 times in red lettuce treated with elicitors / plant growth regulators. Chlorogenic acid and its derivatives (3-CQA, chicoric acid, and 3,4-diCQA), as well as quercetin derivatives (Q3G and Q3MG), were improved in treated lettuce compared to untreated lettuce controls.

[0105] These results demonstrate that treatment with biotic / abiotic use stressors increases polyphenol production in vivo in red lettuce.

[0106] Example 2 Milling process for nano-sized polyphenol-fortified lettuce powder This specification provides a process for producing dried nano-sized polyphenol-fortified lettuce powder using milling technology.

[0107] Select fresh lettuce leaves and wash them thoroughly to remove all dirt and debris. Dry the leaves at 40-45°C.

[0108] The dried leaves are milled using a high-energy ball mill or other suitable milling equipment. The milling process should be carried out in a dry, cool environment to prevent the lettuce from becoming too hot and losing its bioactive components.

[0109] After milling, the lettuce powder is sifted through a fine mesh to remove large particles or impurities.

[0110] Further reduce the powder particle size to the desired nano-size range using a homogenizer or other suitable equipment. This may require passing the powder through the homogenizer or other equipment multiple times.

[0111] Finally, transfer the nano-sized lettuce powder to an airtight container and store it in a cool, dry place until ready for use.

[0112] It is important to note that specific milling processes can vary depending on the type of milling equipment used, the desired particle size range, and other factors.

[0113] As an example of the produced powder, a powder having clearly defined particle size and characteristics, as well as a 3D proportional formulation, can be obtained by combining polyphenol-fortified lettuce powder with artificial additives. For example, examples of artificial additives (e.g., preservatives) including the following may be added to the powder. (1) Active ingredients: Sodium benzoate; potassium sorbate; nitrates and nitrites; sulfites; propionic acid; sodium erythorbate; ascorbic acid. (2) Inertness: For better preservation, several inert additives can be added to the powder. These are, a) Anticoagulation agents: These are added to prevent food powders from clumping together by absorbing moisture and reducing the surface tension of the powder particles. Examples include silicon dioxide, calcium silicate, magnesium carbonate, and talc. b) Stabilizers: These are added to prevent the separation of components in the powder, such as oil and water. Examples include gum arabic, xanthan gum, and carrageenan. c) Emulsifiers: These are added to improve the mixing and dispersion of components in the powder, especially when there are components that are difficult to dissolve or blend. Examples include lecithin, as well as monoglycerides and diglycerides. d) Wetting agents: These are added to prevent the powder from drying out and hardening. Drying and hardening of the powder can shorten its shelf life. Examples include glycerol and propylene glycol.

[0114] Particle size characterization: (1) Particle Size Distribution (PSD): A key characteristic of pharmaceuticals of all preparation types, including solid oral formulations, semi-solids, aerosols, and sterile liquid products. A precisely controlled particle size distribution is extremely important in pharmaceutical development. (2) Another method for defining the particle size of polyphenol-fortified lettuce powder is by mesh size. Mesh size refers to a measurement of the number of meshes, which is a US standard used to determine the relationship between the size of the mesh openings and the size of particles that can pass through these openings. To determine the number of meshes, it is necessary to count the number of openings in one linear inch of the screen. For example, a 4-mesh screen has four square openings in the entire inch of screen, and a 100-mesh screen has 100 openings per inch. As the number of meshes increases, the size of the mesh openings decreases, resulting in smaller particle sizes that can be captured by the screen. However, it is important to note that mesh size is not an exact measurement of the mesh opening size, as screens can be made using various materials with different strand or wire thicknesses. The thickness of the strands used in the screen affects the size of the openings and, consequently, the size of particles that can pass through.

[0115] Mesh size may be used to determine the particle size of powder particles instead of directly measuring the particle size of the powder particles. Examples of defined ranges for mesh size are shown in Tables 1 and 2 below.

[0116] [Table 1]

[0117] [Table 2]

[0118] 3D proportional formulation (mixture of different pore sizes):

[0119] By using different pore sizes in the mixture, 3D proportional formulation can achieve the following functions: (1) Increased contact surface area: By using a combination of different mesh sizes in the formulation, it is possible to increase the contact surface area between particles. This strengthens the inter-particle bonding and can improve the overall strength and integrity of the printed object. (2) Improved air convection between powder particles: By including different mesh sizes, air convection between powder particles during the printing process is improved. This improved airflow helps reduce the risk of particle aggregation, ensures a uniform powder distribution, and as a result can improve print quality and consistency. (3) Control of particle adhesion and aggregation: By adjusting the mesh size of the formulation, it is possible to control the adhesion or aggregation of powder particles. This can be beneficial in achieving the desired flowability of the powder during printing. By reducing particle adhesion, clogging or aggregation can be prevented, while by increasing particle aggregation, interlayer adhesion can be improved, thereby improving the structural integrity of the powder.

[0120] Example 3 Preparation of extracts from polyphenol-fortified lettuce powder using SC-CO2 Polyphenol-fortified lettuce powder (400 mesh size) was placed in an extraction container. Even dispersion was confirmed. Supercritical carbon dioxide (SC-CO2) was added to the mixture, and SC-CO2 extraction of oils from the polyphenol-fortified lettuce powder was performed in an ultrasonic bath under extraction pressure of 300 Bar, temperature of 40°C, and CO2 flow rate of 2 mL / min. After extraction for 120 minutes, the resulting mixture was centrifuged. The resulting liquid portion was freeze-dried to obtain the extract in powder form. Magnesium calcium phytate was then added to the extract, and the mixture was stored at 4°C before use.

[0121] Example 4 Preparation of an extract from polyphenol-fortified lettuce powder using a mixture of ethanol and water. Polyphenol-fortified lettuce powder (400 mesh size) was placed in an extraction vessel. Polyphenol extraction from the polyphenol-fortified lettuce powder was performed at 0°C using an ultrasonic cleaner with ethanol / water (1:1) as the extraction solvent. After extraction for 30 minutes, the resulting mixture was centrifuged. The resulting liquid portion was evaporated by rotation at 40°C and then freeze-dried to obtain a powdered extract. Magnesium calcium phytate was then added to the extract, and the mixture was stored at 4°C before use.

[0122] Example 5 Preparation of extracts from polyphenol-fortified lettuce powder using water Polyphenol-fortified lettuce powder (400 mesh size) was placed in an extraction vessel. Polyphenol extraction from the polyphenol-fortified lettuce powder was carried out in an ultrasonic bath at 0°C using water as the extraction solvent. After extraction for 40 minutes, the resulting mixture was centrifuged. The resulting liquid portion was freeze-dried to obtain the extract in powder form. Magnesium calcium phytate was then added to the extract, and the mixture was stored at 4°C before use.

[0123] Example 6 Basic wound dressing powder formulations and preparation methods formulation: 80% zinc oxide powder, 10% bentonite clay powder, 5% polyphenol-fortified lettuce powder / extract (400 mesh size), and 5% lavender essential oil.

[0124] Preparation method: 1. Add 80% zinc oxide powder and 10% bentonite clay powder to a clean, dry container. 2. Mix the powders well until they are thoroughly blended. 3. Add 5% polyphenol-fortified lettuce powder / extract to the mixture and blend well. 4. Add 5% lavender essential oil to the mixture and mix until the oil is evenly dispersed throughout the powder. 5. Store the wound dressing powder in a clean, airtight container until ready for use.

[0125] Example 7 Polyphenol-fortified lettuce powder / extract preparation Polyphenol-fortified lettuce powder or its extract can be incorporated into ointments, lotions, creams, oils, etc. Table 3 shows examples of formulations using polyphenol-fortified lettuce powder or its extract, and corresponding weight ratios.

[0126] [Table 3]

[0127] Example 8 Preparation of Polyphenol-Fortified Lettuce Powder / Extract-Supported Hydrogels for Wound Healing: Anti-inflammatory and Antioxidant Effects A more specific aspect of this disclosure is the development of anti-inflammatory and antioxidant effects of polyphenol-enhanced lettuce powder / extract hydrogels for wound healing applications. For example, the combination of chitosan-Pluronic P123-polyphenol-enhanced lettuce powder / extract-gelatin appears to be a very promising option for the development of hydrogels for wound healing. Chitosan hydrogels have been found to be useful in wound healing, and they can be crosslinked with conjugate agents via their amine functional groups to form a three-dimensional hydrogel system. When polyphenol-enhanced lettuce powder / extract-supported chitosan nanoparticles (measured at 167-251 nm) were tested for transdermal application, they showed significant transdermal permeability, enhanced drug release, and high cell viability, all of which suggest that they may be an effective means of promoting wound healing. Furthermore, the swelling behavior of the double-supported hydrogel was found to be more than 1.2 times that of the hydrogel without gelatin, indicating that this system may increase water absorption when mixed with gelatin. Considering that gelatin can act as a cell adhesive and polyphenol-enhanced lettuce powder / extract has antioxidant properties that can aid in wound healing, the chitosan-P123 nanopolyphenol-enhanced lettuce powder / extract-gelatin system was found to exhibit superior wound dressing properties compared to single-supported hydrogels.

[0128] An example of a general protocol for preparing a chitosan-polyphenol-fortified lettuce powder / extract-supported hydrogel is as follows:

[0129] material Chitosan powder, Nano-sized polyphenol-fortified lettuce powder / extract, Crosslinking agent (e.g., glutaraldehyde), Pluronic P123, Gelatin powder, Sodium hydroxide, and Acetic acid.

[0130] Basic Procedure 1. Dissolve the chitosan powder in a 1% acetic acid solution and stir for several hours until the powder is completely dissolved. The concentration of chitosan can be adjusted based on the desired properties of the hydrogel. 2. Add the polyphenol-fortified lettuce powder / extract to the chitosan solution and stir until the powder is completely dispersed. The concentration of the polyphenol-fortified lettuce powder / extract can be adjusted based on the desired drug load. 3. In a separate beaker, dissolve the Pluronic P123 and gelatin powder in distilled water to prepare a gelatin solution. 4. Slowly add the chitosan-polyphenol-fortified lettuce powder / extract solution to the gelatin solution under constant stirring. Monitor the pH of the mixture and adjust it using NaOH, maintaining a pH of approximately 7.0. 5. A crosslinking agent (e.g., glutaraldehyde) is added to the mixture to promote the formation of a three-dimensional network structure. 6. Pour the mixture into a hydrogel mold and allow it to harden at room temperature for 2-3 hours. 7. Remove the hydrogel from the mold and place it in a 50°C oven for 24 hours to ensure complete crosslinking. 8. Finally, the hydrogel is freeze-dried to remove residual moisture and obtain a dried chitosan-polyphenol-enhanced lettuce powder / extract-supported hydrogel.

[0131] Example 10 Preparation of powder dressings to promote wound healing This provides a basic procedure for preparing a powder dressing for wound healing by combining polyphenol-fortified lettuce powder / extract with collagen, seaweed extract, β-glucan, carboxymethylcellulose (CMC), and vitamin D3. 1. Measure the desired amount of each ingredient based on the intended formulation and target use. 2. Mix the polyphenol-fortified powder, collagen, seaweed extract, β-glucan, CMC, and vitamin D3 in a dry mixer or similar mixing device. Blend the mixture thoroughly to ensure that the ingredients are uniform. 3. Optionally, if necessary to ensure the compatibility and stability of the components, adjust the pH of the mixture. 4. Once the powders are evenly blended, sift them through a fine sieve to remove any clumps or lumps. 5. After sieving the mixture, store it in a cool, dry place in an airtight container to prevent moisture and humidity from affecting the stability of the ingredients.

[0132] Example 11 In vivo evaluation of polyphenol dressings in wounded mice The effect of polyphenol dressings on promoting wound healing in injured mice was evaluated.

[0133] Laboratory Mice: The efficacy of in vivo wound healing was evaluated using BALB / c mice as the primary target animals. BALB / c laboratory mice were used because their uniform skin color minimizes the risk of pigmentation issues that could affect the interpretation of results. Fifteen BALB / c mice supplied by the National Center for Laboratory Animals were reared to 10 weeks of age before the start of the experiment. These mice were randomly divided into five groups for different treatments.

[0134] Materials and methods BALB / c mice, averaging approximately 28.5 grams at 10 weeks of age, were anesthetized with isoflurane gas (initially 3-4%, maintained at 1-2%). The mice's backs were shaved (covering a 2 x 4 cm area). A 1 x 1 cm wound was made on the back of each mouse using sterile scissors. Immediately after the wound was created, a dressing was applied to the wound. The dressing was applied to completely cover the wound. Throughout the experiment, a scale was placed next to the wound for photography, and the condition of the wound before and after each dressing application was recorded. Dressings were applied twice in total: once on day 0 and again on day 7.

[0135] Body weight was recorded before and after the experiment. Skin examinations were performed on days 0, 3, 5, 7, 10, and 14, and visual images were taken at each time point. Blood samples were taken before the second application of the dressing on day 7 to test for common inflammatory factors, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). Blood samples were taken again three days after the second application of the dressing on day 10 to reassess the levels of these three inflammatory factors, providing insights into the specific effects of the complex polyphenol dressing on the inflammatory response. Finally, on day 14, after euthanasia of the animals, wound samples were taken for tissue sectioning and analysis. In addition, photographs of the spleen were taken.

[0136] Table 4 provides the formulations of four polyphenol dressings used in the experiment on different groups of mice. The polyphenol dressing for group A mice was prepared from untreated lettuce powder. Group B mice were treated with a dressing prepared from an extract obtained from lettuce powder using ethanol / water as the extraction solvent. Group C mice were treated with a dressing prepared from an extract obtained from lettuce powder using supercritical CO2 as the extraction solvent. Group D mice were treated with a dressing prepared from a mixture of untreated powder and CMC. The results of mice treated with different polyphenol dressing formulations were compared to those of a control group of mice that did not receive any dressing.

[0137] [Table 4]

[0138] Table 5 shows the relative content of major bioactive polyphenols in various types of dressings. As shown in Figure 5, these extracts contain high levels of chicoriic acid compared to the initial state of lettuce powder. Furthermore, the polarity of the extraction solvent also affects the polyphenol content in the extract. Polar extraction solvents (e.g., aqueous ethanol) resulted in extracts containing high levels of extractable polyphenols such as chicoriic acid and quercetin derivatives.

[0139] [Table 5]

[0140] result Changes in spleen weight and macroscopic differences Figure 8 compares the difference in average body weight of mice before and after the experiment in each group (A-D and the control group). All groups of mice showed an increase in weight after the experiment, and there was no statistically significant difference in weight before and after the experiment. Furthermore, no significant difference in weight change was observed between groups A-D and the control group. Figure 9 shows photographs of the spleens of mice treated with polyphenol dressings (A-D) and the control group. The spleens of the control group mice were slightly larger than those of the mice treated with polyphenol dressings (A-D), indicating that the use of polyphenol dressings helps reduce wound inflammation.

[0141] Comparison of wound healing effects The wound healing effect of polyphenol dressings was evaluated by monitoring changes in wound size in mice treated with polyphenol dressings (AD) and in the control group during the treatment period. Figure 10 shows photographs of wound size over time in mice treated with polyphenol dressings (A-D) and in the control group. As shown in Figure 10, the control group mice had the slowest wound healing rate. On day 14, the control group mice had the largest wound size. In contrast, among the polyphenol dressing groups (A-D), group B mice showed the fastest healing rate and the smallest wound size. This effect may be due to the use of an aqueous alcohol extraction solvent (e.g., ethanol / water mixed solvent) which yields a higher extract content of polar polyphenols, including chicoriic acid and quercetin derivatives. Higher polar polyphenols act synergistically to enhance wound healing properties.

[0142] Comparison of wound tissue sections Wound tissue sections from mice treated with polyphenol dressings (A-D) and the control group were analyzed. Figures 11A-11E show photographs of wound tissue sections from mice treated with polyphenols and the control group. The control group mice had the slowest wound healing rate (Figure 11A). The tissue sections from the control group mice clearly showed excessive epithelialization and disordered cell arrangement. In contrast, as shown in Figures 11B-11E, mice treated with polyphenol dressings showed more orderly cell arrangement and more normal epithelialization.

[0143] Comparison of inflammatory cytokine levels in the blood

[0144] Common inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8), were measured using blood samples from mice collected before the second dressing application (day 7), 3 days after the second dressing application (day 10), and before sacrifice (day 14). Figure 12 is a bar graph showing the changes in TNF-α over time in mice treated with polyphenol dressing (A-D) and the control group. Figure 13 is a bar graph showing the changes in IL-6 over time in mice treated with polyphenol dressing (A-D) and the control group. Figure 14 is a bar graph showing the changes in IL-8 over time in mice treated with polyphenol dressing (A-D) and the control group. These data show that levels of inflammatory cytokines, specifically TNF-α, IL-6, and IL-8, were lower in mice treated with polyphenol-containing dressings (groups A-D) than in control mice. Furthermore, mice treated with extracts (groups B and C) showed superior anti-inflammatory effects compared to mice treated with the initial polyphenol powder (groups A and D). Additionally, comparing mice treated with the initial polyphenol powder, group D mice showed lower levels of IL-6 and IL-8 than group A mice. Overall, group B mice exhibited the most effective suppression of all measured inflammatory cytokines.

[0145] Further embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or enumerated in the application datasheet are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified as necessary to adopt the concepts of various patents, applications, and publications in order to provide further embodiments.

[0146] In light of the detailed description above, these and other modifications may be made to the embodiments. In general, the terms used in the following claims should not be construed as limiting the claims to any particular embodiment disclosed herein and in the claims, but rather as encompassing all possible embodiments, along with the full scope of the equivalent claims to which rights are granted. Thus, the claims are not limited by this disclosure. This application claims priority to U.S. Provisional Application No. 63 / 509,677, filed with the U.S. Patent and Trademark Office on 22 June 2023, the entire contents and disclosures thereof are incorporated herein by reference.

Claims

1. A powder comprising particles of polyphenol-fortified lettuce powder or extract, wherein the polyphenol-fortified lettuce powder or extract contains 40 to 280 mg / g of polyphenols, and the polyphenols include chlorogenic acid, neochlorogenic acid, chicoric acid, quercetin, quercetin derivatives, anthocyanins, and any combination thereof.

2. The powder according to claim 1, wherein the powder comprises chlorogenic acid in an amount in the range of about 6 to 38 mg / g, chicoric acid in an amount in the range of about 4 to 45 mg / g, quercetin derivative in an amount in the range of about 18 to 175 mg / g, and anthocyanin in an amount in the range of about 6 to 20 mg / g.

3. The powder according to claim 1 or 2, wherein the chlorogenic acid comprises 3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA), 5-O-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), or any combination thereof.

4. The quercetin derivative is the powder according to any one of claims 1 to 3, comprising quercetin-3-O-glucoside (Q3G), quercetin glucuronide, and quercetin-3-O-malonyl glucoside (Q3MG).

5. The powder according to any one of claims 1 to 4, wherein the anthocyanin comprises cyanidin 3-galactoside, cyanidin-3-O-glucoside, cyanidin-3-6"-malonylglucoside, or any combination thereof.

6. The powder according to any one of claims 1 to 5, wherein the polyphenol comprises 4-CQA, neochlorogenic acid, chicoric acid, and cyanidin 3-galactoside.

7. The powder according to any one of claims 1 to 6, wherein the polyphenol-enhanced lettuce powder or extract is obtained from polyphenol-enhanced lettuce obtained by treating control lettuce with at least one use stressor / elicitor that increases the production of polyphenols in the control lettuce, or homologs, isomers, or derivatives thereof.

8. The powder according to claim 7, wherein the at least one use stressor / elicitor is an abiotic use stressor or elicitor selected from indole-3-acetic acid (IAA), auxin, cytokinin (CK), gibberellin (GA), ethylene, brassinosteroid, jasmonate (JA), strigolactone (SL), salicylic acid (SA), arachidonic acid (AA), 5-aminolevulinic acid (5-ALA), oxalic acid, and any homologues, isomers, derivatives, synthetic analogs, or any combination or mixture thereof.

9. The powder according to any one of claims 7 to 8, wherein the polyphenol-fortified lettuce contains polyphenol products increased by 2 to 20 times compared to control lettuce, and optionally contains polyphenol products increased by 3 to 9 times compared to control lettuce.

10. The powder according to any one of claims 7 to 9, wherein the concentrations of the chlorogenic acid, the quercetin derivative, the chicoric acid, and the anthocyanin are increased by at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 times compared to the powder produced from the control lettuce.

11. The powder according to any one of claims 1 to 10, wherein the particles have a particle size distribution range of 0.1 μm to 150 μm.

12. The powder according to any one of claims 1 to 11, wherein the polyphenol-fortified lettuce powder comprises nanoparticle powder, and the particle size distribution range of the nanoparticle powder is 0.01 μm to 0.1 μm.

13. The powder according to claim 12, wherein the particles have a particle size distribution range of 0.05 μm to 0.1 μm.

14. The powder according to claim 13, wherein the particle size distribution range of the nanoparticle powder is 0.1 μm or less.

15. A topical formulation comprising the powder according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier or excipient.

16. A topical preparation for treating a skin disorder, wherein the topical preparation comprises a powder containing particles of polyphenol-fortified lettuce powder or extract, and the polyphenol-fortified lettuce powder or extract contains 40 to 280 mg / g of polyphenols.

17. The topical formulation according to claim 16, wherein the polyphenol comprises two or more of chlorogenic acid, chicoric acid, quercetin and its derivatives, and anthocyanins.

18. The topical formulation according to claim 16, wherein the skin disorder includes inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral rash, herpes zoster, tinea, or skin cancer.

19. The topical preparation according to any one of claims 15 to 18, wherein the powder is present in a percentage of about 0.1% to 10%, about 0.5% to 5%, about 1% to 10%, about 5% to 20%, about 10% to 50%, or about 50% to 90% based on the final weight of the topical preparation.

20. The topical formulation according to any one of claims 15 to 19, wherein the pharmaceutically acceptable carrier or excipient is solid or liquid.

21. The topical formulation according to any one of claims 15 to 20, wherein the topical formulation is in the form of an ointment, lotion, cream, powder, liquid, gel, hydrogel, oil, surface tension agent, drip, aerosol, emulsion, nanoemulsion, nanoliposome, gel, microencapsulation, paste, or foam.

22. The topical preparation according to any one of claims 15 to 21, wherein the topical preparation is in the form of a sunscreen, facial cleanser, face pack, soap, shampoo, conditioner, body wash, or hair dye.

23. A topical formulation according to any one of claims 15 to 22, further comprising a skin emollient.

24. The topical formulation according to claim 23, wherein the skin emollient comprises paraffin.

25. A topical preparation according to any one of claims 15 to 24, further comprising an antimicrobial preservative.

26. A topical formulation according to any one of claims 15 to 25, further comprising one or more emulsifiers and viscosity improvers.

27. The aforementioned topical preparation is, by weight percentage, 1-10% of the aforementioned powder, 5-15% alcohol, 5-15% emollient, 1-5% emulsifier, 5-15% propylene glycol, Antimicrobial preservatives of less than 1%, The remaining percentage of water to make it equal to 100%, A topical formulation according to any one of claims 15 to 26, including the above.

28. A wound dressing powder comprising the powder according to any one of claims 1 to 14 and a pharmaceutically acceptable powdered carrier or excipient.

29. The wound dressing powder according to claim 28, wherein the pharmaceutically acceptable carrier or excipient is starch, cellulose, synthetic polymer, polysaccharide, chitosan, mineral powder, clay powder, or any combination thereof.

30. i. The mineral powder contains 50% to 90% wt / wt, ii. The clay powder contains 5% to 15% wt / wt, iii. The powder contains 1% to 15% wt / wt. The wound dressing powder according to claim 29.

31. The wound dressing powder according to claim 29 or 30, wherein the mineral powder is zinc oxide powder.

32. The wound dressing powder according to any one of claims 29 to 31, wherein the clay powder comprises bentonite clay.

33. A wound dressing powder according to any one of claims 28 to 32, further comprising an essential oil.

34. The wound dressing powder according to claim 33, wherein the essential oil is lavender essential oil.

35. A hydrogel comprising the powder according to any one of claims 1 to 14 and a pharmaceutically acceptable hydrogel polymer.

36. The hydrogel according to claim 35, wherein the powder is present in a percentage of about 0.1% to 10%, about 0.5% to 5%, about 1% to 10%, about 5% to 20%, or about 10% to 50% based on the final weight of the topical formulation.

37. The hydrogel according to claim 35 or 36, wherein the powder has a particle size distribution range of 0.1 μm to 150 μm.

38. The hydrogel according to claim 35 or 36, wherein the powder comprises or is essentially composed of nanoparticles.

39. The hydrogel according to claim 38, wherein the nanoparticles have a particle size distribution range of 0.1 μm or less, and optionally the particle size distribution range is 0.01 μm to 0.1 μm or 0.05 μm to 0.1 μm.

40. The hydrogel according to any one of claims 35 to 39, wherein the hydrogel polymer is at least one of chitosan, gelatin, collagen, polysaccharide, starch, alginate, or agarose.

41. A hydrogel according to any one of claims 35 to 40, further comprising a polymer surfactant.

42. The hydrogel according to claim 41, wherein the polymer surfactant is Pluronic P123.

43. The hydrogel according to any one of claims 35 to 42, further comprising a targeted ligand that targets the site of action.

44. An oral formulation comprising the powder according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier, binder, excipient, disintegrant, lubricant, or any combination thereof.

45. The oral preparation according to claim 44, wherein the powder is present in a percentage of about 0.1% to 10%, about 0.5% to 5%, about 1% to 10%, about 5% to 20%, or about 10% to 50% of the final volume of the oral preparation.

46. The oral formulation according to claim 44 or 45, wherein the binder comprises tragacanth gum, acacia, corn starch, or gelatin.

47. The oral formulation according to any one of claims 44 to 46, wherein the excipient comprises dicalcium phosphate.

48. The oral formulation according to any one of claims 44 to 47, wherein the disintegrant comprises corn starch, potato starch, and alginic acid.

49. The oral formulation according to any one of claims 44 to 48, wherein the lubricant comprises magnesium stearate.

50. An oral formulation according to any one of claims 44 to 49, further comprising a sweetener containing one or more of sucrose, fructose, lactose, and aspartame.

51. An oral preparation according to any one of claims 44 to 50, further comprising a flavoring agent.

52. An oral formulation according to any one of claims 44 to 51, further comprising a food preservative.

53. The oral formulation according to any one of claims 44 to 52, wherein the powder is in the form of an ingestible tablet, buccal tablet, lozenge, capsule, pill, elixir, suspension, syrup, or wafer.

54. A method for treating a wound, comprising administering to the target wound site a therapeutically effective amount of the powder according to any one of claims 1 to 14, the topical preparation according to any one of claims 15 to 27, the wound dressing powder according to any one of claims 28 to 34, the hydrogel according to any one of claims 35 to 43, or the oral preparation according to any one of claims 44 to 53.

55. The method according to claim 54, wherein the wound includes an acute wound, a skin infection, a burn wound, an ulcer, a chronic wound, a diabetic wound, or a refractory wound.

56. A method for treating an inflammatory condition or disease, comprising administering to a subject in need of treatment a therapeutically effective amount of the powder according to any one of claims 1 to 14, the topical preparation according to any one of claims 15 to 27, the wound dressing powder according to any one of claims 28 to 34, the hydrogel according to any one of claims 35 to 43, or the oral preparation according to any one of claims 44 to 53.

57. The method according to claim 56, wherein the production of inflammatory cytokines by immune cells is reduced compared to immune cells before treatment.

58. The method according to claim 56 or 57, wherein NF-kappa B activity is reduced.

59. The method according to any one of claims 54 to 58, comprising administering the composition to epithelial tissue.

60. The method according to any one of claims 54 to 58, comprising administering the composition to muscle tissue.

61. The method according to any one of claims 54 to 58, comprising administering the composition to connective tissue.

62. The method according to any one of claims 54 to 58, comprising administering the composition to nerve tissue.

63. A method of skin care, comprising administering to a subject a therapeutically effective amount of the powder according to any one of claims 1 to 14, the topical preparation according to any one of claims 15 to 27, the wound dressing powder according to any one of claims 28 to 34, the hydrogel according to any one of claims 35 to 43, or the oral preparation according to any one of claims 44 to 53.

64. The method according to any one of claims 54 to 63, wherein the subject is a human, a non-human primate, a mouse, a rat, a gerbil, a rabbit, a dog, a cat, a horse, a cow, a pig, a goat, a sheep, a donkey, a llama, an alpaca, a guinea pig, a mule, a deer, a buffalo, a chicken, a duck, a goose, or a turkey.

65. A method for preparing wound dressing powder, i. Mixing mineral powder and clay powder to form a powder mixture, ii. Adding the powder according to any one of claims 1 to 14 to the powder mixture, iii. Blending the aforementioned powder mixture, iv. Adding a pharmaceutically acceptable excipient, carrier, or diluent to the powder mixture, The method, including the method described above.

66. The method according to claim 65, wherein the mineral powder contains zinc oxide.

67. The method according to claim 65 or 66, wherein the clay powder comprises bentonite clay.

68. The method according to any one of claims 65 to 67, further comprising adding essential oil to the powder mixture and mixing the powder mixture in order to disperse the oil throughout the powder mixture.

69. A method for preparing nanoparticle powder, i. Wash and dry at least one polyphenol-fortified lettuce leaf, ii. To form a powder, the at least one red lettuce leaf is ground using a milling tool, iii. In order to remove particles larger than the opening of the sieve, the powder is passed through the sieve, iv. In order to reduce the particle size of the powder to a particle size distribution range of 0.1 μm or less, the powder is pulverized, The method, including the method described above.

70. The method according to claim 69, wherein the grinding is performed using a ball mill, conical mill, hammer mill, or homogenizer.

71. The method according to claim 69 or 70, wherein the particle size distribution is 0.01 μm to 0.1 μm, or 0.05 μm to 0.1 μm.

72. The method according to any one of claims 69 to 71, wherein the drying temperature is 40 to 45°C.

73. A method for preparing a hydrogel, i. To form a chitosan solution, the chitosan powder is dissolved in an acid solution, ii. Adding the powder according to any one of claims 1 to 14 to the chitosan solution, iii. To make a gelatin solution, mix gelatin powder with water, iv. Mixing the chitosan solution containing the powder with the gelatin solution, v. Adding a crosslinking agent to the mixture of the chitosan solution and the gelatin solution, vi. In order to form a hydrogel, pour the mixture from v. into a hydrogel mold and allow the mixture to solidify, vii. Removing the hydrogel from the mold, viiii. To promote crosslinking, heat is applied to the hydrogel, The method, including the method described above.

74. The method according to claim 73, further comprising maintaining the pH of the mixture at approximately 7.0 by adding an NaOH solution when mixing the chitosan solution with the gelatin solution.

75. The method according to claim 73 or 74, wherein solidifying the mixture comprises solidifying the mixture at room temperature for 2 to 3 hours.

76. The method according to any one of claims 73 to 75, wherein the heating of the hydrogel includes placing the hydrogel in an oven at 50°C for 24 hours.

77. The method according to any one of claims 73 to 76, wherein the crosslinking agent comprises glutaraldehyde.

78. The method according to any one of claims 73 to 77, further comprising adding a polymer surfactant to the gelatin solution.

79. The method according to any one of claims 73 to 78, wherein the acid solution contains 1% acetic acid.

80. The method according to any one of claims 73 to 79, further comprising freeze-drying the hydrogel.

81. A method for treating a skin disorder, comprising administering to a subject a therapeutically effective amount of the powder described in any one of claims 1 to 14 or the topical preparation described in any one of claims 15 to 27.

82. The method according to claim 81, wherein the skin disorder includes inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral rash, herpes zoster, tinea, and skin cancer.

83. A method of treating wounds, Administer to the target wound site a therapeutically effective amount of the powder according to any one of claims 1 to 14, the topical preparation according to any one of claims 15 to 27, the wound dressing powder according to any one of claims 28 to 34, or the hydrogel according to any one of claims 35 to 43. The subject is orally administered a therapeutically effective amount of the oral formulation described in any one of claims 44 to 55. The aforementioned wound site is irradiated with blue light radiation, The wound site is irradiated with red light radiation, The method, including the method described above.

84. The method according to claim 83, wherein the blue light emission has a wavelength in the range of 400 nm to 520 nm.

85. The method according to any one of claims 83 to 84, wherein the red light emission has a wavelength in the range of 617 nm to 850 nm.

86. The method according to any one of claims 83 to 85, wherein the wound includes an acute wound, a skin infection, a burn wound, an ulcer, a chronic wound, a diabetic wound, or a refractory wound.

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