Polyphenol-rich lettuce compositions for enhanced wound healing and skin care

Abstract

Provided herein is a polyphenol-rich lettuce powder comprising particles of a polyphenol-rich lettuce or extract wherein the polyphenol-rich lettuce comprises from 40 to 280 mg / g of polyphenol. The polyphenols include any combination of chlorogenic acid, neochlorogenic acid, chicoric acid, quercetin, quercetin derivatives, and anthocyanins. The present disclosure also provides compositions and methods for promoting wound healing and treating inflammation and skin disorders using polyphenol-rich lettuce powder.

Description

background Technical Field

[0002] The present disclosure relates to novel compositions having therapeutic and cosmetic properties for promoting wound healing and providing skin care benefits.

[0003] Description of Related Technology

[0004] Although significant advances have been made in antimicrobial therapy and supportive measures, wound care still faces significant challenges. Invasive infections, blunt trauma, burns caused by electrical or chemical accidents, radiation burns, and other similar injuries remain difficult to effectively treat and heal. Unfortunately, despite the introduction of clinically effective antimicrobial agents, bacterial strains that are resistant to such treatments have rapidly emerged, further exacerbating the problem.

[0005] Countries with aging populations, such as the United States, face significant challenges with patients suffering from leg ulcers. It is estimated that leg ulcers cause the loss of 2 million workdays in the United States. The cost of treating these non-healing wounds can be huge, and the prevalence of pressure ulcers (pressure sores or bedsores) on the lower body in the elderly population is estimated to be between 3 and 11%. The morbidity and mortality associated with pressure ulcers are significant, with the mortality rate of patients with pressure ulcers being four times that of patients without pressure ulcers. In addition, septic elderly patients with pressure ulcers have an in-hospital mortality rate of more than 50%. Due to the inclusion of intensive care care and adjuvant therapies (such as anti-pressure devices, protective dressings, and skin treatments), treatment costs can be expensive [Monika P, Chandraprabha MN, Rangarajan A, Waiker PV, ChidambaraMurthy KN. Challenges in Healing Wound: Role of Complementary and Alternative Medicine. Front Nutr. 2022, 20; 8: 791899].

[0006] Wound management faces significant challenges, especially in diabetes, where wounds are generally considered unhealable. Diabetes is a common underlying condition for leg ulcers, and although there are a variety of therapies that help wound healing (such as multilayer compression bandages, local recombinant human platelet-derived growth factor, and human skin equivalents for skin grafts), they may be expensive and unaffordable for many patients. In some cases, wounds may not be treated, resulting in amputation of infected limbs. Managing wounds in animals faces additional challenges because they are more susceptible to environmental infection. Flies (especially myiasigenic flies, such as sarcophagidae and calliphoridae) can aggravate wounds and cause complications. Pouring creolin (a mixture of phenol and tar) on wounds is a common practice, but it is tissue damaging, toxic, and only provides short-term insect protection. Due to the risk of animal poisoning and food chain contamination, the use of insecticides directly on wounds infected with maggots is also problematic.

[0007] Provided herein are novel formulations, compositions and uses of polyphenol-rich red leaf lettuce powders / extracts that provide unexpected and superior effects in promoting wound healing, which addresses an unmet need in the art. Summary of the invention

[0008] The overall purpose of the present disclosure is to provide new botanical compositions for wound healing / skin care and methods for preparing / manufacturing the same. The present disclosure introduces new powder compositions comprising particles of polyphenol-rich lettuce powder or extracts, wherein the polyphenol-rich lettuce has an increased yield and concentration of at least one polyphenol relative to a control lettuce. More specifically, it relates to polyphenol-rich lettuce powders or extracts thereof having potent anti-inflammatory and wound healing properties, which are quite effective in healing external wounds of any nature in humans, including acute wounds and chronic wounds, such as abrasion wounds, burn wounds, laceration wounds, vascular ulcers, diabetic ulcers, and pressure injuries, etc. The polyphenol-rich lettuce powder / extract is also quite effective in treating wounds in mammals. The disclosed powder / extract and method can be applied to the four basic tissues of the human / animal body (nervous tissue, epithelial tissue, muscle tissue, and connective tissue). In addition, the disclosed powder / extract can also be used in cosmetic products for skin care. The lettuce powder / extract rich in polyphenols can be introduced into various cosmetic preparations, such as sunscreens, facial cleansers, facial masks, conditioners and shampoos, shower gels, etc., to provide a series of benefits to the skin (including anti-aging, radiation protection, skin whitening, moisturizing, spot removal, acne treatment and hair regeneration promotion). The powder / extract can be designed for external / topical use or oral use, and has excellent wound healing and anti-inflammatory and antioxidant properties. The powder / extract and method are particularly effective for treating wounds that are considered unhealable using traditional methods. The combination of naturally occurring components in the lettuce powder / extract rich in polyphenols produces a potent and powerful effect, resulting in a synergistic effect of the components.

[0009] These and other aspects of the disclosure will become apparent upon reference to the following detailed description and accompanying drawings.All references disclosed herein are incorporated by reference in their entirety as if each were individually incorporated. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A to 1C The quercetin naturally present in red leaf lettuce ( Figure 1A ) and its highly orally bioavailable derivative quercetin-3-O-glucoside (Q3G) ( Figure 1B ) and quercetin-3-O-malonylglucoside (Q3MG) ( Figure 1C ).

[0012] FIG2 shows examples of chlorogenic acids such as 3-caffeoylquinic acid (neochlorogenic acid or 3-CQA) identified from red leaf lettuce ( Figure 2A ) and 3,4-dicaffeoylquinic acid (3,4-diCQA) ( Figure 2B ).

[0013] Figure 3 Showing chicoric acid (CRA) in red leaf lettuce.

[0014] FIG. 4 shows examples of anthocyanins, such as cyanidin-3-O-glucoside ( Figure 4A ) and cyanidin-3-(6"-malonylglucoside)( Figure 4B ).

[0015] Figure 5 is a flow chart of a method for preparing an extract from polyphenol-rich lettuce powder.

[0016] Figure 6 HPLC-UV chromatograms showing bioactive components enhanced by genomics-based technology, confirming the production of specific metabolites in red leaf lettuce treated with elicitors. (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).

[0017] Fig. 7A and 7B It was shown that chlorogenic acid and chicoric acid ( Fig. 7A ) and more hydrophilic quercetin derivatives ( Figure 7B ) production increased by 3 to 9 times. Fig. 7A The production of chlorogenic acid (3-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), and chicoric acid (CRA) is depicted. Figure 7B The production of quercetin derivatives (Q3G and Q3MG) is depicted.

[0018] Figure 8 is a bar graph comparing the changes in body weight of mice in the polyphenol dressing-treated groups (A to D) and the control group before and after the experiment.

[0019] Fig. 9 Photographs of spleens of mice in the polyphenol dressing-treated group (A to D) and the control group are shown.

[0020] Fig.10 Photographs illustrating the changes in wound size over time in mice in the polyphenol dressing-treated group (A to D) and the control group are shown.

[0021] Figures 11A to 11E Photographs illustrating wound tissue sections of mice in the polyphenol-treated and control groups are shown.

[0022] Fig.12 is a bar graph showing the changes in TNF-α over time in mice in the polyphenol dressing-treated groups (A to D) and the control group.

[0023] Fig.13 is a bar graph showing the changes in IL-6 over time in mice of the polyphenol dressing-treated groups (A to D) and the control group.

[0024] Fig.14 is a bar graph showing the changes in IL-8 over time in mice treated with polyphenol dressings (AD) and control groups.

[0025] Details

[0026] Examples related to the use of highly polyphenol-rich lettuce as a raw material and subsequent extracts and preparations thereof are provided herein. Such lettuce contains high levels of polyphenols, such as quercetin derivatives, chlorogenic acid, chicoric acid, and anthocyanins. Without wishing to be bound by theory, it is believed that the polyphenols in such lettuce exhibit synergistic bioactivity for wound healing and skin care applications.

[0027] Provided herein is a powder comprising particles of a polyphenol-rich lettuce powder or extract, wherein the polyphenol-rich lettuce has an increased yield and concentration of at least one polyphenol relative to a control lettuce. In some embodiments, the polyphenol is at least one of chlorogenic acid, neochlorogenic acid, chicoric acid, quercetin, a quercetin derivative, anthocyanin, or any combination thereof. In some instances, the powder can be formulated for topical and / or oral administration. In addition, the present disclosure provides a wound dressing powder. The present disclosure also provides a hydrogel comprising the powder. Also provided herein is a method of treating comprising administering such a powder, and a method of preparing such a powder.

[0028] The present disclosure includes multiple aspects that can be combined in different ways. The following description is provided to list the elements and describe some embodiments of the present disclosure. These elements are listed together with the initial embodiment; however, it should be understood that these embodiments can be combined in any way and in any number to produce other embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present disclosure to only the systems, technologies and applications clearly described. In addition, this description should be understood to support and cover the description and claims of all various embodiments, systems, technologies, methods, devices and applications, which have any number of disclosed elements, have each element alone, and also have any and all various permutations and combinations of all elements in this application and any subsequent application.

[0029] One of the highly abundant natural polyphenols in red leaf lettuce is a quercetin derivative. Quercetin is one of the most abundant dietary flavonoids. Quercetin can be found in many plants and foods, such as red wine, onions, green tea, apples, berries, Ginkgo biloba, St. John's wort, American elder, etc. (Flavonoids, Micronutrient Information Center, Linus Pauling Institute, Oregon State University, 2015). Quercetin is related to improving athletic performance and reducing inflammation, blood pressure and blood sugar levels. It can also have brain protection, anti-allergic and anti-cancer, anti-bacterial and anti-viral properties. Quercetin has been extensively studied in wound healing, and its use has been described in many research papers, covering a range of wound conditions affecting various organs [Polerà 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 the main active ingredients in treating cuts and inflammation. In mice, quercetin has been shown to enhance the proliferation and migration of fibroblasts, inhibit inflammation, and increase the expression of growth factors, thereby promoting skin wound healing. 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 basis for a deeper understanding of the mechanism of action of Oxytropis falciformis in the treatment of cut 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 inhibits inflammatory response by regulating the polarization of macrophages from M1 to M2 phenotype, thereby accelerating diabetic wound repair.

[0030] Another important class of bioactive components in polyphenol-rich lettuce is chlorogenic acid. Chlorogenic acid (CGA, a phenolic compound found in human dietary products) has been shown to have a variety of health-improving properties, including antioxidant, anti-inflammatory, antimicrobial, antidiabetic and lipid-lowering functions. Numerous reports have demonstrated that CGA has a beneficial effect on improving wound repair. For example, in 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. "Achlorogenic 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], in vitro and ex vivo experimental methods were used to evaluate the ability of CGA to promote diabetic wound healing. Therefore, CGA significantly achieved anti-inflammatory activity, re-epithelializing effect and pro-angiogenic function. Hyaluronic acid-based hydrogels were then used in vivo for topical administration of CGA to full-thickness wounds in diabetic mice. Compared with commercial wound dressings (INTRASITE The resulting formulation (CGA-HA) significantly improved the healing effect, accompanied by reduced inflammation, enhanced re-epithelialization and angiogenesis, and mature collagen remodeling, demonstrating the potential of CGA-HA for clinical application in diabetic wound healing.

[0031] The polyphenol-rich lettuce raw material also contains a large amount of biologically active anthocyanins. Anthocyanins are colored water-soluble pigments belonging to the phenolic group (Khoo et al., Food Nutr Res. 61 (1), 2017). The pigment is in a glycosylated form. Fruits and vegetables contain anthocyanins that cause the colors red, purple and blue. Berries, currants, grapes and some tropical fruits have high anthocyanin content. Red to purple-blue leafy vegetables, cereals, roots and tubers are edible vegetables containing high levels of anthocyanins. Among anthocyanin pigments, cyanidin-3-glucoside is the main anthocyanin found in most plants. Anthocyanins have anti-diabetic, anti-cancer, anti-inflammatory, anti-microbial and anti-obesity effects, as well as preventing cardiovascular disease (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. Anthocyanins from black soybean seed coat enhance wound healing. Ann Plast Surg. 2013 Oct; 71(4): 415-20], anthocyanins have been shown to inhibit the translocation of nuclear factor-κB (p65) from the cytosol to the nucleus and prevent IκBα phosphorylation. Anthocyanins enhance wound healing through cytoprotective effects, enhanced angiogenesis, and anti-inflammatory effects. Other findings [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] suggest that anthocyanins have anti-inflammatory properties and anti-aging potential in skin fibroblasts by regulating type I collagen gene expression and inhibiting H2O2-induced NF-κB activation.

[0032] In terms of dressing materials, particle size is an important factor in effective wound healing when considering topical use [e.g., see Patents Nos. TWI764332, TWI786786, TWM636367, TWI787696, TWI774168, TWI774055, TWI774046, TWI760999, TWI760961, TWI758625, TWM602560]. Suitable particles can more easily penetrate the skin and reach deeper tissues, allowing more effective delivery of therapeutic agents to the wound site. In contrast, larger particles may have more difficulty penetrating the skin and may stay on the surface, providing little benefit to the underlying tissue. Therefore, when designing a topical wound healing treatment, it is important to consider particle size to ensure optimal therapeutic effect.

[0033] Wound healing and wound molecular biology

[0034] Wound healing is a complex and dynamic process consisting of several sequential steps, including the induction of inflammatory responses, regeneration of parenchymal tissue, migration and proliferation of parenchymal tissue cells, production of extracellular matrix proteins, tissue remodeling, and increased wound strength [Harding KG, Morris HL, Patel GK. Healing chronic wounds. BMJ. (2002) 324: 160-3]. Wound healing involves a variety of cell types, secreted growth factors, cytokines, extracellular matrix, and various enzymes. Platelets, neutrophils, monocytes, macrophages, fibroblasts, keratinocytes, endothelial cells, epithelial cells, and myofibroblasts are the various cell types involved in the wound healing process. Fibroblasts have long been considered 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: 53147]. Various factors such as microbial infection or biofilm formation may affect the wound healing process. In addition, 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 is a major contributor to wound pathology. Ischemia-reperfusion injury is characterized by a series of biochemical and cellular events that cause extensive cellular damage through pathways leading to leukocyte and complement activation, oxidative stress, and microvasculature dysfunction.

[0035] Many molecular factors play important and overlapping roles in normal wound healing, including growth factors and receptors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), cytokines (such as fibroblast growth factor (FGF), tumor necrosis α (TNFα) and interleukin-1 (IL-1)) and enzymes (such as matrix metalloproteinases (MMPs), tissue inhibitors of MMPs and seperinases) [Blakytny R, Jude EB. Altered molecular mechanisms of diabetic foot ulcers. Int J Low Extrem Wounds. (2009) 8: 95104]. In addition, angiogenesis involves the development of capillary sprouts that invade the extracellular matrix (ECM) matrix and form tubular structures, which 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) 2:2439]. Various angiogenic stimulators, such as vascular endothelial growth factor (VEGF), TGF-β, TNFα, PDGF, FGF, angiogenin, and angiopoietin-1, play important 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: 2439]. The activation of these molecular factors is essential for wound healing, and the complex interaction groups between these molecules, if disrupted, may lead to the persistence of wounds. A deeper understanding of the changes in the expression of these molecular factors and their effects on cellular function can provide valuable insights into potential intervention targets. Wound healing in acute and chronic conditions

[0036] There are two main types of wounds: acute wounds and chronic wounds. Acute wounds usually heal in an orderly and efficient manner, progressing through four different but overlapping stages: hemostasis, inflammation, proliferation and remodeling [Diegelmann R.Wound healing: an overview of acute, fibrotic, and delayed healing.Front Biosci. (2004) 9: 2839]. Within 2-4 weeks, acute wounds usually show obvious signs of healing as they pass through the inflammation, proliferation and remodeling stages. In contrast, chronic wounds do not follow a continuous healing stage and may become "stuck" in a single stage and fail to show signs of healing within 4 weeks [Swezey L.The difference between acute and chronic wounds (2019)].

[0037] During normal physiological processes, such as wound healing (as in the case of acute wounds), inflammatory cells are recruited to the site of injury and assist in tissue repair by secreting cytokines and growth factors that promote tissue remodeling and angiogenesis. However, angiogenesis is impaired in all chronic wounds, leading to further tissue damage and causing chronic hypoxia and impaired micronutrient delivery. Vascular lesions associated with diabetes include abnormal angiogenesis, reduced angiogenesis, and accelerated atherosclerosis, which may 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: 11745]. Vascular endothelial growth factor (VEGF) is an important angiogenesis stimulator in wound healing, and studies have shown that VEGF levels in chronic wounds of diabetes are insufficient. Administration 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, patients with chronic venous stasis ulcers have elevated levels of VEGF in their circulation [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-apilot study. Eur J Vasc Endovasc Surg. (1999) 18:334-8].Because many factors regulate wound angiogenesis, it is important to understand the various dysregulated cellular and molecular events in angiogenesis that lead to non-healing chronic wounds.

[0038] The role of natural products in wound treatment

[0039] Natural products, including plant-derived phytochemicals and naturally derived substances, have recently received significant research attention for their potential in wound healing. These substances have been used for a long time due to their anti-inflammatory, antioxidant, angiogenic and cell synthesis regulating properties. There is a large amount of evidence to support the use of natural products and naturally derived substances in wound care, which is why they are currently receiving a lot of attention from researchers. Phytochemicals and naturally derived substances contain a variety of chemicals that can enhance wound healing in a variety of ways, making them an attractive option. Plants with medicinal properties have been found to be effective in treating wounds and fighting infections, thereby accelerating wound healing. According to a recent study by Nigussie, henna (Lawsonia inermis) and neem (Azadirachta indica) are the most studied plant species for wound healing, and the most common in vivo techniques for anti-inflammatory and wound healing assays are carrageenan-induced paw edema and excision and incisional wound 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 role of phytochemicals and naturally derived substances in wound healing research, including their uses, applications, mechanisms of action, and outcomes, has been extensively demonstrated.

[0040] Another study highlighted the synergistic effects of plants and naturally derived substances in wound healing, but also pointed out the risk of side effects such as irritation and allergic hypersensitivity [Sivamani RK, Ma BR, Wehrli LN, Maverakis E. Phytochemicals and naturally derived substances for wound healing. Adv Wound Care. (2012) 1: 213-7]. However, a recent study has shown that neem leaf extract can be a safe alternative to saline for foot ulcer irrigation, 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 worth noting that phytochemicals and naturally derived substances may have a higher risk of contamination with infectious agents, and therefore proper sterilization and microbiological testing before use is essential. Key markers for evaluating the benefits of natural products in wound healing include hydroxyproline content, microscopic observations, 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 demonstrated by these markers [Murthy KNC, Reddy KV, Veigas JM, Murthy UD. Study on wound healing activity of Punica granatum peel. J Med Food. (2004) 7: 256-9]. Ongoing research in this area demonstrates the exciting potential of natural products for wound healing.

[0041] Polyphenols have gained significant attention for their potential in wound healing due to their ability to act as antioxidants as well as their regenerative and antimicrobial properties.[ M.A.;Mierziak,J.;Korzun,U.;Preisner,M.;Szopa,J.;Kulma,A.The potential of plant phenolics in prevention and therapy of skindisorders.Int.J.Mol.Sci.2016,17,160;Ghuman,S.;Ncube,B.;Finnie,J.;McGaw,L.;Njoya,E.M.;Coopoosamy,R.;Van Staden,J.Antioxidant,anti-inflammatory and woundhealing properties of medicinal plant extracts used to treat wounds anddermatological disorders.S.Afr.J.Bot.2019,126,232-240; I.;Baptista-Silva,S.;Pintado,M.;L.Oliveira,A.Polyphenols:A Promising Avenue inTherapeutic Solutions for Wound Care.Appl.Sci.2021,11,1230;Luque,G.C.;Moya,M.;Picchio,M.L.;Bagnarello,V.;Valerio,I.; J.; Vethencourt, M.; Gamboa, S.-H.; Tomé, LC; Minari, RJ; Mecerreyes, D. Polyphenol Iongel Patches with Antimicrobial, Antioxidant and Anti-Inflammatory Properties. Polymers 2023, 15, 1076]. These compounds are usually extracted from plants and marine organisms and are divided into two main subgroups based on the number and bonding structure of their phenolic units: flavonoid compounds (such as flavonols and anthocyanins) 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 become the focus of extensive research due to their potential in medical and pharmaceutical applications, especially in wound care [ MA;Mierziak, J.;Korzun, U.;Preisner, M.;Szopa, J.;Kulma, A.The potential of plantphenolics in prevention and therapy of skin disorders.Int.J.Mol.Sci.2016,17,160]. These compounds are known for their potent antioxidant activity, which helps neutralize free radicals by donating electrons or hydrogen atoms and provides protection against reactive oxygen species (ROS). In addition, certain polyphenols have been shown to exhibit antimicrobial 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 mechanisms by which polyphenols exert their antimicrobial effects are not fully understood, it is believed that they may act by breaking down bacterial cell walls through the hydrophobic components of phenolic compounds, by altering intracellular functions through hydrogen bonding, or by altering cell wall structure and rigidity through interactions with cell membranes [ 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 lipophilic nature of polyphenols enhances their antimicrobial activity [Bouarab-Chibane, L.;Forquet, V.;Lantéri, P.;Clément, Y.;Léonard-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]. In addition, the antimicrobial potential of polyphenols has been shown to be particularly effective against antibiotic-resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (S. aureus) [Negut, I.; Grumezescu, V.; Grumezescu, AM. Treatment strategies for infected wounds. Molecules 2018, 23, 2392].

[0042] definition

[0043] As used herein, a "wound" is a break in the continuity of the skin. The structure of the skin is complex, and wound biology is understood by understanding the factors that affect the local physiological environment. Many local conditions can affect 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 or any combination of the epidermis, dermis and / or subcutaneous tissue. Examples of wounds include living tissue damage caused by incisions, lacerations, impacts or other physical damage.

[0044] As used herein, "polyphenol" refers to an organic chemical substance containing more than one phenol structural unit. The main biologically active polyphenols 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, flavonoids, etc.) and anthocyanins (e.g., anthocyanidin glycosides, etc.). In a preferred embodiment, the biologically active polyphenols disclosed herein include anthocyanins, chlorogenic acid, chicoric acid, dicaffeoylquinic acid and quercetin derivatives (quercetin glycosides).

[0045] As used herein, "powder" refers to fine dry particles produced by grinding, crushing or disintegrating a solid substance.

[0046] As used herein, a "particle" refers to a small topical composition of matter having a set of chemical and physical properties. Typically, one or more of a particle's width, length, and height is from about 0.01 μm to about 150 μm.

[0047] As used herein, "nanosized particles" or "nanoparticles" are used interchangeably and refer to particles smaller than 0.1 μm.

[0048] As used herein, "eustressor" and "inducer" are used interchangeably and refer to various biological, physical or chemical stressors that trigger signal transduction pathways to cause plant products to have higher bioactive compound content and quality attributes. Eustressors / inducers can be divided into biological substances and non-biological substances, examples of which are described in WO2022 / 183014. Plant hormones / plant growth regulators (e.g., salicylic acid (SA), jasmonates, etc.) are also considered to be eustressors / inducers. Eustressors / inducers of biological, chemical or physical origin can increase plant agronomic / nutritional properties due to the activation of reactions that may include defense responses, resulting in, for example, improved functional quality of fruits and vegetables. Plant growth regulators (PGRs) can be used as eustressors / inducers to stimulate the production of plant secondary metabolites. Plant growth regulators can include naturally occurring hormone substances (plant hormones) and their synthetic analogs.

[0049] "Plant growth regulators" (PGRs) refer to chemicals used to alter plant growth, such as increasing branching, inhibiting branch growth, increasing re-flowering, removing excess fruit, or altering fruit maturity. PGRs may also be called plant hormones or inducers. PGRs may include auxins, cytokinins (CK), gibberellins (GA), ethylene, rapeseed steroids, jasmonates (JA), strigolactones (SL), and salicylic acid (SA).

[0050] "Lettuce" refers to the plant of species lettuce (Lactuca sativa L.) herein. Lettuce is the Cichorieae tribe of the Asteraceae (Compositae) family. Lettuce is related to chicory, sunflower, aster, dandelion, artichoke and chrysanthemum. Lettuce (L.sativa) is one of about 300 species of the genus Lactuca. As a highly polymorphic species, lettuce is planted for its edible head and leaves. As a crop, lettuce is commercially grown in any place where environmental conditions allow for economically viable yields. Fresh lettuce is almost completely eaten as a fresh raw product, and occasionally eaten as a cooked vegetable. Lettuce is an increasingly popular crop. Global lettuce consumption continues to increase. Due to its high demand, it is beneficial to seek to increase the polyphenol production of new transgenic lettuce. In particular, the improved transgenic lettuce with improved healthy polyphenol production is stable, high-yield and agronomically sustainable, and will be particularly commercially viable for human consumption.

[0051] "Lettuce plant" refers to an immature or mature whole lettuce plant, including a lettuce plant from which seeds, roots or leaves have been removed. The seeds or embryos that will produce the plant are also considered to be lettuce plants. Lettuce plants can be produced by sowing directly in the ground (e.g., soil, such as soil on a field), or by germinating the seeds in controlled environmental conditions (e.g., a greenhouse) and then transplanting the seedlings into the field. See, e.g., 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.

[0052] "Lettuce cell" or "lettuce plant cell" refers to a cell as used herein, including lettuce plant cells, whether isolated, in tissue culture, or introduced into a lettuce plant or lettuce plant part.

[0053] As used herein, "lettuce plant parts" include lettuce heads, lettuce leaves, parts of lettuce leaves, pollen, ovules, flowers, etc. In another embodiment, the present disclosure also relates to lettuce heads, lettuce leaves, parts of lettuce leaves, flowers, pollen and ovules isolated from lettuce plants.

[0054] The term "variety" or "cultivar" means a grouping of plants within a single botanical taxonomic unit of the lowest known rank, which grouping can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, whether or not the conditions for the grant of breeders' rights are fully met, which grouping is distinguishable from any other grouping of plants by the expression of at least one of said characteristics and which is regarded as a unit in terms of its suitability for propagation without alteration.

[0055] "Plant" includes whole plants or any part thereof, such as plant organs (e.g., harvested or unharvested leaves, etc.), plant cells, plant protoplasts, plant cells or tissue cultures that can regenerate whole plants, plant callus, plant cell masses, plant grafts, seedlings, intact plant cells in plants, plant clones or micropropagations, or parts of plants (e.g., harvested tissues or organs), such as plant cuttings, vegetative propagation, embryos, pollen, ovules, flowers, leaves, heads, seeds, cloned plants, roots, stems, petioles, root tips, grafts, parts of any of these and the like, or derivatives thereof, preferably having the same genetic composition (or very similar genetic composition) as the plant from which it was obtained. Also included are any developmental stages, such as seedlings, cuttings before or after rooting, mature and / or immature plants, or mature and / or immature leaves.

[0056] As used herein, a polynucleotide or polypeptide is "recombinant" when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous location (e.g., in the genome of a recombinant organism) so that it is not associated with the nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A polypeptide expressed in vitro or in vivo by a recombinant polynucleotide is an example of a recombinant polypeptide. Similarly, polynucleotide sequences that do not occur in nature, such as variants of naturally occurring genes, are recombinant.

[0057] As used herein, "heterologous" refers to a sequence that originates from a foreign species, or, if from the same species, has been substantially altered from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same / similar species, one or both are substantially altered from their original form and / or genomic locus, or the promoter is not the native promoter of the operably linked polynucleotide.

[0058] As used herein, "transgenic" refers to genes or genetic material that are transferred to the genome of a lettuce plant, for example, by genetic engineering methods, such as by transformation. Exemplary transgenics include cDNA (complementary DNA) fragments, which are copies of mRNA (messenger RNA), and the genes themselves that are located in the original region of their genomic DNA. In one example, a DNA fragment containing a gene sequence that is introduced into the genome of a lettuce plant or a lettuce plant cell is described. This non-natural DNA fragment may retain the ability to produce RNA or protein in a transgenic lettuce plant, or it may change the normal function of the genetic code of a transgenic plant. Typically, the nucleic acid transferred is introduced into the reproductive system of a plant. Transgenic can also describe any DNA sequence, whether or not it contains a gene coding sequence or it is artificially constructed, which has been introduced into a lettuce plant or a vector construct in which it has not been found before.

[0059] "Operably connected" is intended to represent the functional connection between two or more elements. For example, the operable connection between the target polynucleotide and the regulatory sequence (i.e., promoter) is a functional connection that allows the expression of the target polynucleotide. The elements that are operably connected can be continuous or discontinuous. When used to refer to the connection of two protein coding regions, "operably connected" means that the coding region is in the same reading frame. The box can also contain at least one other coding sequence / gene to be co-transformed into the organism. Alternatively, other (multiple) coding sequences / genes can be provided on multiple expression cassettes. Such expression cassettes are provided with multiple restriction sites and / or recombination sites for inserting the target coding polynucleotide or its active variant or fragment to be subject to transcriptional regulation of the regulatory region (e.g., promoter). The expression cassette can also contain a selectable marker gene.

[0060] "Expression cassette" refers to a polynucleotide encoding a target polypeptide, which is operably connected to at least one polynucleotide encoding an expression control sequence. The expression cassette can include a transcription and translation initiation region (i.e., a promoter), a polynucleotide encoding a target polypeptide or its active variant or fragment, and a transcription and translation termination region (i.e., termination region) that has a function in plants in the 5' to 3' direction of transcription. The regulatory region (i.e., promoter, transcription regulatory region, and translation termination region) and / or polynucleotide or its active variant or fragment can be homologous / similar to the host cell or to each other. Alternatively, the regulatory region and / or polynucleotide or its active variant or fragment can be heterologous to the host cell or to each other.

[0061] The expression cassette may also contain a 5' leader sequence. Such a leader sequence may play a role in enhancing translation. Translation leader sequences are known in the art and include: picornavirus leader sequences, such as the EMCV leader sequence (encephalomyocarditis 5' non-coding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); Potato virus Y leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al. (1995) Gene 165 (2): 233-238), MDMV leader sequence (maize dwarf mosaic virus) (Virology 154: 9-20) and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); non-translated leader sequence from alfalfa mosaic virus coat protein mRNA (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, Cech (Liss, New York, ed.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385. See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0062] "Expression control sequence" refers to a nucleic acid molecule fragment that can increase or decrease the expression of a polypeptide encoded by an expression cassette. Examples of expression control regions include promoters, transcriptional regulatory regions, and translational termination regions. The termination region may be homologous to the transcriptional initiation region, may be homologous to an operably linked polynucleotide or an active variant or fragment thereof, may be homologous to a plant host, or may be derived from another source (i.e., foreign or heterologous) that is different from the promoter, polynucleotide or active fragment or variant thereof, plant host, or any combination thereof. Convenient termination regions may be obtained from the Ti plastid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also 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. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0063] A "control" provides a reference point for measuring phenotypic changes in a subject. For example, a control for wound healing or closure may be a comparable wound that has not received an active ingredient that promotes wound healing. A control wound may be treated with a vehicle or dressing that does not contain an active ingredient that promotes wound healing. A control wound may be present on the same subject or on a different but comparable subject.

[0064] A "control lettuce" or "control lettuce cell" provides a reference point for measuring phenotypic changes in a target lettuce plant or lettuce plant cell, and can be any suitable lettuce plant or lettuce cell. A control lettuce or lettuce cell can include, for example: (a) a wild-type or native lettuce or lettuce cell, i.e., a starting material having the same genotype as the genetically altered starting material from which the target lettuce or lettuce cell was generated; (b) a lettuce or lettuce cell having the same genotype as the starting material but having been transformed with an ineffective construct (i.e., with a construct that has no known effect on the target trait, such as a construct comprising a marker gene); (c) a lettuce or lettuce cell that is an untransformed segregant in a progeny of the target lettuce or lettuce cell; (d) a lettuce or lettuce cell that is genetically identical to the lettuce or lettuce cell but has not been exposed to the same treatment as the target lettuce or lettuce cell (e.g., benign stressor / inducer treatment, herbicide treatment); or (e) the target lettuce or lettuce cell itself under conditions where the target gene is not expressed.

[0065] An "effective amount" or "therapeutically effective amount" can refer to an amount of a therapeutic agent (e.g., a lettuce extract, lettuce plant, or lettuce plant part described herein) that provides a desired physiological change, such as wound healing. The desired physiological change can be, for example, increased wound closure, decreased inflammation associated with a wound, increased cell proliferation, and / or increased formation and / or regeneration of vascular tissue.

[0066] In this specification, unless otherwise stated, the term "about" means +20% of the indicated range, value or structure. The term "consisting essentially of ... " limits the scope of the claim to the specified materials or steps and those materials or steps that do not substantially affect the basic and novel features of the claimed embodiment. It should be understood that, as used herein, the terms "one" and "a" refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two or any combination of the alternatives. As used herein, the terms "including" and "having" are used synonymously, and the term and its variants are intended to be interpreted as non-restrictive. The term "comprising" indicates the presence of the features, integers, steps or components mentioned in the claim, but it does not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0067] The recombinant DNA, molecular cloning and gene expression techniques used in the present disclosure are known in the art and are described in references such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 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.

[0068] Without departing from the scope and spirit of the present disclosure, various modifications and variations of the products and methods of the present disclosure will be apparent to those skilled in the art. Although the present disclosure has been described in conjunction with specific preferred embodiments, it should be understood that the disclosure claimed for protection should not be unduly limited to such specific embodiments. In fact, various modifications of the described patterns that are apparent to chemistry, biology or related field technicians for implementing the present disclosure are intended to fall within the scope of the appended claims.

[0069] Polyphenol-rich powder

[0070] In some embodiments, the present disclosure provides a powder comprising particles of a polyphenol-enriched lettuce powder or extract. The polyphenol-enriched lettuce has an increased yield and concentration of at least one polyphenol relative to a control lettuce. Representative bioactive components identified from such red leaf lettuce include flavonoids, particularly its highly orally bioavailable derivatives (quercetin-3-O-glucoside and quercetin-3-O-malonyl glucoside) ( Figures 1A-1C ), chlorogenic acid: 3-caffeoylquinic acid (3-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA) ( Figure 2A and 2B )、Chicoric acid( Figure 3 ) and anthocyanins (e.g., cyanidin-3-O-glucoside and equivalents) ( Figure 4A and 4B ). 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, polyphenol-rich lettuce has increased yields and concentrations 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-malonyl glucoside (Q3MG), or all of them. In some embodiments, anthocyanins include cyanidin 3-galactoside, cyanidin-3-O-glucoside, cyanidin-3-6"-malonyl glucoside, or any combination thereof. In some embodiments, polyphenols include 4-CQA, neochlorogenic acid, chicoric acid, and cyanidin 3-galactoside. In some embodiments, the increased polyphenol production is quantified by LC-MS. In some embodiments, the increased polyphenol production is quantified by HPLC. Methods for producing polyphenol-rich lettuce are described in WO 2022 / 183014, which is incorporated herein by reference in its entirety.

[0071] The compositions and methods disclosed herein are applicable to at least four tissues of the human and animal body to 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 neural tissue (e.g., peripheral neuropathy and nerve injury).

[0072] In certain embodiments, the polyphenol-rich lettuce is treated with at least one benign stressor / inducer or a homologue, isomer or derivative thereof that increases polyphenol production in lettuce. For example, at least one benign stressor / inducer may be an abiotic benign stressor / inducer selected from the group consisting of indole-3-acetic acid (IAA), auxin, cytokinin (CK), gibberellin (GA), ethylene, rapeseed steroids, jasmonates (JA), strigolactones (SL), salicylic acid (SA), arachidonic acid (AA), 5-aminolevulinic acid (5-ALA), oxalic acid and any homologue or isomer or derivative, synthetic analogue or any combination or mixture thereof. In some embodiments, the polyphenol-rich lettuce comprises a 2-fold to 20-fold increase in polyphenol production compared to the control lettuce, optionally a 3-fold to 9-fold increase in polyphenol production compared to the control lettuce. In some embodiments, the concentration of chlorogenic acid, quercetin derivatives, chicoric acid, and anthocyanins is increased by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold compared to a powder produced from a control lettuce.

[0073] In some embodiments, the polyphenol-rich lettuce powder or extract thereof contains a total polyphenol concentration of about 40-280 mg / g. In some embodiments, the concentration of chlorogenic acid is about 6-38 mg / g. In some embodiments, the concentration of chicoric acid is about 4-45 mg / g. In some embodiments, the concentration of quercetin derivatives is about 18-175 mg / g. In some embodiments, the concentration of anthocyanins is about 6-20 mg / g.

[0074] Particle size distribution (PSD) is an important characteristic of pharmaceutical products for all types of formulations, including solid oral pharmaceutical products, semisolids, aerosols, and sterile liquid products. Ball mills, cone mills, and hammer mills can be used to adjust particle size and shape and achieve a consistent, tight particle size distribution, resulting in high-quality products and efficient manufacturing systems. The choice of whether to use a ball mill, cone mill, or hammer mill will depend on several factors, including an evaluation of the type of product that needs to be processed and the desired results.

[0075] In topical and transdermal drug delivery, particle size plays an important role in determining the pharmacological properties of the product. For example, when the API is suspended in a vehicle such as a gel or cream, the particle size interacts with the solubility of the vehicle to determine the dissolution rate. Particle size also affects the rate, depth, and path of absorption through the skin. Ultimately, the choice of particle size in a topical formulation can help maximize the efficacy of the drug at the site of action and minimize the risk of systemic adverse reactions.

[0076] In some embodiments, the preferred particle size range of the powder is from about 0.1 to about 150 μm. Although the size range of the well-defined powder can be from about 0.1 μm to about 10,000 μm. The appropriate D50, D90 of the bioactive lettuce powder or its extract will be defined and achieved in the specific formulation. In some embodiments, the red leaf lettuce powder rich in polyphenols includes nanoparticle powder. As used herein, nanoparticles have a particle size distribution range of about 0.1 μm or less. In some embodiments, the red leaf lettuce powder rich in polyphenols has a particle size distribution range of about 0.01 μm to about 0.1 μm. In some embodiments, the red leaf lettuce powder rich in polyphenols has a particle size distribution range of about 0.05 μm to about 0.1 μm.

[0077] An exemplary method of defining the particle size of the polyphenol-rich lettuce powder or extract thereof is by mesh size. The term "mesh size" refers to the fineness or coarseness of a material determined by the ability of a particle to pass through a mesh. Mesh size is related to mesh size and is generally defined as the number of mesh openings within a 1 inch x 1 inch area in a screen. For example, to determine the mesh size, the number of openings in a one-inch length of the screen can be calculated. For example, a 4-mesh screen has four square openings on a one-inch screen, while a 100-mesh screen has 100 openings per inch. As the mesh size increases, the opening size of the screen decreases, resulting in a smaller particle size that can be captured by the screen. The mesh size indicates the particle size of the material that can pass through the corresponding mesh opening. For example, a mesh size of 200 indicates that the material can pass through a screen with 200 openings within a 1 inch x 1 inch area. Similarly, as the mesh size increases, the finer the indicated particle size, while the smaller the mesh size, the coarser the indicated particle size. Therefore, the mesh size can be used to determine the fineness or coarseness of the particle, rather than directly measuring the size of the material particle. Note however that mesh size is not an exact measure of the size of the mesh opening, as the screen can be made using different materials that may have different strand or wire thicknesses. The thickness of the strands used in the screen will affect the size of the opening, and thus the size of the particles that can pass through.

[0078] By using different mesh sizes in the powder mixture, 3D ratio formulations can achieve the following functions. First, using a combination of different mesh sizes in the formulation allows for an increase in the contact surface area between particles. This can enhance inter-particle bonding and improve the overall strength and integrity of the printed object. Second, the inclusion of different mesh sizes can promote improved air convection between powder particles during the printing process. This improved airflow can help reduce the risk of particle agglomeration and ensure uniform powder distribution, thereby achieving better print quality and consistency. Third, by adjusting the mesh size in the formulation, the adhesive or cohesive properties of the powder particles can be controlled. This can be beneficial in achieving the desired flowability of the powder during printing. Reducing particle adhesion can prevent blocking or agglomeration, while increasing particle cohesion can enhance interlayer adhesion and improve the structural integrity of the powder.

[0079] The polyphenol-rich lettuce powder or its extract disclosed herein has powerful anti-inflammatory and antioxidant properties. Therefore, it can be used in different cosmetic preparations, such as sunscreens, facial cleansers, facial masks and hair conditioners. This introduction can provide various advantages to the skin, such as anti-aging effects, protection from radiation, skin whitening, moisturizing, reducing freckles, acne treatment and stimulating hair growth. The polyphenol-rich lettuce powder or its extract can be used to produce different preparations, such as powders, aqueous solutions, sprays, gels, oil bases, creams, surfactants and ointments, for specific purposes in four major human tissues. For example, the polyphenol-rich lettuce powder or its extract can be added to soap, shampoo, hair conditioner, hair dye, shower gel and cleaning products to enhance its function. In some instances, the polyphenol-rich lettuce powder or its extract can be dissolved in a surfactant, mixture or solvent. The disclosed polyphenol-rich lettuce powder or its extract can also provide antioxidant function. The polyphenol-rich lettuce powder or its extract can also be mixed with powdered antibiotics to enhance its bactericidal and antibacterial effects. In other embodiments, lettuce powder or its extract rich in polyphenols can be combined with collagen, algae extract, beta-glucan, carboxymethyl cellulose (CMC) and vitamin D3 to produce a dressing that promotes wound healing. In other embodiments, lettuce powder or its extract rich in polyphenols can be combined with hyaluronic acid, collagen and CMC to form a matrix that can be used to treat skin disorders or for skin care products. In other embodiments, lettuce powder or its extract rich in polyphenols can be combined with glycerol that reduces bacteria as an emulsion matrix. In other embodiments, lettuce powder or its extract rich in polyphenols can be combined with colloids and diluents for protecting and repairing skin and soft tissue. In yet other embodiments, lettuce powder or its extract rich in polyphenols can be combined with paraffin or glycerol for protecting and repairing skin and soft tissue.

[0080] The polyphenol-rich lettuce powder or extract thereof disclosed herein can be conveniently administered as a pharmaceutical composition containing a bioactive composition in combination with a suitable excipient. Such pharmaceutical compositions can be prepared by methods well known in the art and contain excipients well known in the art. Such methods and compositions can be found in Remington's Pharmaceutical Sciences [Alfonso Gennaro et al., ed., Lippincott, Williams & Wilkins, Baltimore, Md., 20th edition, 2000, November 8, 2012]. The preparations and compositions of the present invention can be administered topically, orally or rectally.

[0081] For topical application, the preparation can contain lettuce powder or its extract rich in polyphenols with high polyphenol concentration as active ingredient. Compositions according to the present disclosure can be conveniently prepared using one or more pharmaceutically acceptable carriers or excipients, which can be solid or liquid. Such compositions can take the form of, for example, ointments, lotions, creams, powders, liquids, gels, oils, surface tension agents, extracts, drops (for example eye drops or ear drops) or sprays. Of course, the percentage of compositions and preparations can vary and can be conveniently between about 2% to about 100% of a given unit dosage form weight. The amount of the active compound in such a therapeutically useful composition makes it possible to obtain an effective dosage level.

[0082] In some embodiments, the lettuce powder or extract rich in polyphenols disclosed herein can be in the form of a cream or ointment. Ointment can be prepared by melting white soft paraffin (white petrolatum), introducing any additives (e.g., surfactants and solvents) and incorporating a drug slurry in a minimum amount of liquid paraffin. The melt is then cooled and stirred under controlled conditions until solidification occurs. Creams can be prepared by combining the oil phase of the ointment as a melt with suitable oil-soluble and water-soluble surfactants and the aqueous phase containing medicine and suitable antimicrobial preservatives as described above, homogenizing to form a cream and gently stirring until cooling.

[0083] Compositions containing the polyphenol-enriched lettuce powder or extracts thereof as disclosed herein may typically contain additional excipients, for example preservatives (such as benzoic acid), emulsifiers (such as polysorbates, e.g., polysorbate 60), and viscosity enhancers (such as cetearyl alcohol).

[0084] Bioactive compositions can be mixed into soaps, shampoos, conditioners, hair dyes, shower gels and cleaning products to enhance their function. The compositions can be mixed with antibiotics to enhance antimicrobial activity. In addition, it can be mixed with materials such as hyaluronic acid, collagen, alginate, beta-glucan, carboxymethyl cellulose (CMC) and vitamin D3 to make dressings that promote wound healing properties.

[0085] Thus, in some embodiments, disclosed herein are topical formulations comprising polyphenol-rich lettuce powder or extracts thereof as described above and in the examples below and a pharmaceutically acceptable carrier or excipient. In some embodiments, the polyphenol-rich lettuce powder or extracts 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 carrier or excipient is a 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, drops, aerosol, emulsion, nanoemulsion, nanoliposome, gel, microcapsule, paste, or foam. In certain embodiments, the topical formulation is in the form of a sunscreen, a facial cleanser, a mask, a soap, a shampoo, a conditioner, a shower gel, or a hair dye. In some embodiments, the topical formulation further comprises an emollient, such as paraffin. In some embodiments, the topical formulation further comprises an antimicrobial preservative. In some embodiments, the topical formulation further comprises one or more of an emulsifier and / or a viscosity enhancer.

[0086] In some embodiments, the topical formulations disclosed herein comprise the following in % weight / weight:

[0087] 1 to 10% polyphenol-rich lettuce powder or extract thereof;

[0088] 5 to 15% alcohol;

[0089] 5 to 15% emollient;

[0090] 1 to 5% emulsifier;

[0091] 5 to 15 percent propylene glycol;

[0092] Less than 1% antimicrobial preservatives; and

[0093] The remaining percentage of water equals 100%.

[0094] In some embodiments, wound dressing powder is provided herein, which comprises lettuce powder or its extract rich in polyphenols described above and in the examples below and a powdered pharmaceutically acceptable carrier or excipient. 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, starch is corn starch, potato starch, rice starch, or any combination thereof. In some embodiments, clay is bentonite, kaolin, montmorillonite, or any combination thereof. In some embodiments, cellulose is microcrystalline cellulose, sodium carboxymethyl cellulose (CMC), or any combination thereof. In some embodiments, synthetic polymers are polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), or a combination thereof. In some embodiments, polysaccharides are calcium alginate. In some embodiments, mineral powder is zinc oxide powder.

[0095] In certain embodiments, the wound dressing powder comprises:

[0096] i. 50% to 90% weight / weight mineral powder,

[0097] ii. 5% to 15% w / w clay powder; and

[0098] iii. 1% to 15% w / w polyphenol-rich lettuce powder or extract thereof; and optionally

[0099] iv. Essential oils, for example, lavender essential oil.

[0100] In some embodiments, disclosed herein are methods for delivering nanoparticles mediated by hydrogels. Due to the unique properties of hydrogels, they have the potential to enhance the bioavailability, solubility, and stability of polyphenol-rich lettuce powder for medical applications. In addition, hydrogels can be modified with specific targeting ligands that guide the payload to the precise site of action [Khalvati, B.; Sheikhsaran, F.; Sharifzadeh, S.; Kalantari, T.; BehzadBehbahani, 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 method can reduce side effects while increasing pharmacological activity at the target site [Sheikhsaran, F.; Sadeghpour, H.; Khalvati, B.; Entzar-Almahdi, E.; Dehshahri, A. Tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptormediated delivery of the plasmid encoding IL-12 gene. Colloids Surf., B 2017, 150, 426-436]. In addition, stimuli-responsive hydrogels provide the opportunity to precisely control payload transfer [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]. The use of hydrogel as a delivery vehicle for the polyphenol-rich lettuce powder also allows for administration of the drug via different routes of administration, such as oral, topical, nasal, or parenteral.

[0101] Thus, in some embodiments, provided herein is a hydrogel comprising a polyphenol-rich lettuce powder or extract thereof disclosed herein and a pharmaceutically acceptable hydrogel polymer. In some embodiments, the powder is present in a percentage of about 0.1%-10%, about 0.5%-5%, about 1%-10%, about 5%-20%, or about 10%-50% of the final weight of the topical formulation. In some embodiments, the polyphenol-rich lettuce powder or extract thereof comprises particles having a size distribution range of 0.1 μm to 150 μm. In some embodiments, the polyphenol-rich lettuce powder or extract thereof comprises nanoparticles or consists essentially of nanoparticles. In some embodiments, the size distribution range of the nanoparticles is 0.1 μm or less, optionally with a size distribution range of 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, polysaccharides, starch, alginate, or agarose. In some embodiments, the hydrogel further comprises a polymeric surfactant, for example, pluronic P 123. In some embodiments, the hydrogel further comprises a targeting ligand that targets the site of action.

[0102] For oral therapeutic administration, the polyphenol-rich lettuce powder or extract thereof disclosed herein can be combined with one or more excipients and used in the form of ingestible tablets, lozenges, troches, capsules, elixirs, suspensions, syrups, and wafers. Such compositions and formulations should contain at least 1% of the active composition. For example, the polyphenol-rich lettuce powder can be present in a percentage of about 0.1%-10%, about 0.5%-5%, about 1%-10%, about 5%-20%, or about 10%-50% of the final volume of the oral formulation. Tablets, lozenges, pills, capsules, etc. may also contain the following: binders, such as tragacanth, gum arabic, corn starch or gelatin; excipients, such as calcium hydrogen phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose or aspartame, or flavoring agents, such as mint, wintergreen oil or cherry flavor, may also be added. When 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-mentioned types of materials. Various other materials may exist as coatings or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. Syrups or elixirs may contain active compounds. Sucrose or fructose are used as sweeteners, methylparaben and propylparaben are used as preservatives, dyes and flavors (such as cherry or orange flavors). The active compositions may also be incorporated into sustained-release preparations and devices.

[0103] The topical formulations of the present disclosure can be absorbed through transmembrane routes, including, for example, sublingual, nasal mucosa, rectal mucosa and / or intestinal routes. In a preferred embodiment, the topical formulations can be topically applied in the oral cavity for the treatment of local disorders such as oral ulcers, periodontitis and dental plaque.

[0104] In some embodiments, wound healing can be promoted by combining topical administration with topical oral administration and photodynamic therapy. For example, after the topical preparation is applied to the wound area and the oral preparation is orally administered, the wound area can be irradiated with blue light having a wavelength range of 400nm to 520nm (peak wavelength is 440nm). The blue light radiation can produce a large amount of reactive oxygen species (ROS) to promote wound sterilization. At the same time, the polyphenols in the oral preparation can protect normal human tissue cells and avoid free radical damage caused by radiation. In some embodiments, irradiating the wound area with red light having a wavelength range of 617 to 850nm (peak wavelengths are 660nm and 720nm, respectively) can produce a synergistic effect to promote wound healing. Compared to blue light radiation, red light radiation can penetrate deeper into the subcutaneous tissue to stimulate cell energy production, thereby promoting cell repair and regeneration and accelerating wound healing. In addition, by regulating macrophage M1 / M2 polarization, the degree of inflammatory response can also be reduced.

[0105] Method for preparing extract from lettuce powder rich in polyphenols

[0106] In some embodiments, the present disclosure provides an extract of lettuce powder rich in polyphenols. As described above, the extract is a powdered extract in solid form, 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-rich lettuce powder or its extract is about 40 to 280 mg / g. In some embodiments, the concentration of chlorogenic acid is about 6 to 38 mg / g. In some embodiments, the concentration of chicoric acid is about 4 to 45 mg / g. In some embodiments, the concentration of quercetin derivatives is about 18 to 175 mg / g. In some embodiments, the concentration of anthocyanins is about 6 to 20 mg / g.

[0107] In some embodiments, disclosed herein are methods of preparing an extract from polyphenol-rich lettuce from which a polyphenol-rich lettuce powder is formed. Figure 5 is a flow chart of a method 500 for preparing an extract from polyphenol-rich lettuce according to some embodiments of the present disclosure. It should be understood that for additional embodiments of the method, additional steps may be provided before, during, and after the method 500, and some of the steps described below may be replaced or eliminated.

[0108] refer to Figure 5 , method 500 includes step 502, wherein the polyphenol-rich lettuce is ground to form a polyphenol-rich lettuce powder. In some embodiments, the leaves or any above-ground parts of the lettuce can be first dried, for example, by air drying or freeze drying. Next, the dried lettuce can be cut into smaller pieces and then pulverized, for example, using a ball mill, a conical mill, or a hammer mill, to form a polyphenol-rich lettuce powder. The size of the polyphenol-rich lettuce powder is then classified according to the size of the sieve. In some embodiments, the polyphenol-rich lettuce powder can have a particle size ranging from 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-rich lettuce powder can have a particle size ranging from 0.1 to 10,000 μm, for example, 0.1 to 150 μm.

[0109] In step 504 of method 500, the lettuce powder rich in polyphenols is extracted. In some embodiments, a solvent extraction method can be used to extract the lettuce powder rich in polyphenols by immersing it in an extraction solvent. In some embodiments, the extraction solvent can be water, alcohol, a mixture of water and alcohol, or a supercritical fluid. Examples of alcohols used for extraction include, but are not limited to, ethanol or isopropanol. In some embodiments, the extraction solvent is water. In other embodiments, the extraction solvent is ethanol. In addition, the extraction solvent is a mixture of water and ethanol, wherein ethanol accounts for about 50% to about 90% of the mixture. In some embodiments, the extraction solvent is a supercritical fluid, such as supercritical carbon dioxide (CO2). Extraction solvents with greater polarity (e.g., alcohol, water, or a mixture thereof) produce extracts containing higher amounts of polyphenols, while non-polar extraction solvents such as supercritical CO2 produce extracts with lower polyphenol content and rich in non-polar compounds (such as terpenes, carotenoids, vitamins, etc.).

[0110] In some embodiments, the ratio of polyphenol-rich lettuce powder to solvent (g / mL) can range from about 1:10, 1:5, 2:5, 3:5, 4:5, or 1: 1. In certain embodiments, the ratio of polyphenol-rich lettuce powder to solvent (g / mL) is 2:5.

[0111] The extraction can be carried out at a temperature ranging from about 15°C to about 85°C, such as, for example, from about 15°C to about 20°C, from about 20°C to about 25°C, from about 20°C to about 30°C, from about 30°C to about 35°C, from about 40°C to about 45°C, from about 45°C to about 50°C, from about 50°C to about 55°C, from about 55°C to about 60°C, from about 60°C to about 65°C, from about 65°C to about 70°C, from about 70°C to about 75°C, from about 75°C to about 80°C, or from about 80°C to about 85°C.

[0112] The duration of the extraction process can range from about 0.5 hours to about 24 hours, for example, from about 0.5 hours to about 1 hour, from about 1 hour to about 2 hours, from about 2 hours to about 3 hours, from about 3 hours to about 4 hours, from about 4 hours to about 5 hours, from about 5 hours to about 6 hours, from about 6 hours to about 10 hours, from about 10 hours to about 18 hours, or from about 18 hours to about 24 hours.

[0113] The extraction can be carried out under pressure or atmospheric conditions with agitation, stirring, shaking or a combination thereof. In some embodiments, the extraction can be assisted by ultrasound. Ultrasound applies strong shearing forces and stresses to the polyphenol-rich lettuce powder and the extraction solvent, which helps to accelerate the penetration of the extraction solvent into the polyphenol-rich lettuce powder. Therefore, ultrasound can improve the extraction efficiency and shorten the extraction time. The ultrasound can be pulsed. In some embodiments, ultrasound can have a pulse duration ranging from 100ps to 1ms, for example, 100ps to 100ns, 100ns to 500ns, 500ns to 1μs, 1μs to 10ns, 10μs to 500μs, or 500μs to 1ms; a pulse power ranging from 1kW to 50kW, for example, 1kW to 5kW, 5kW to 10kW, 10kW to 20kW, or 20kW to 50kW; and a frequency ranging from 100kHz to 200MHz, for example, 500kHz to 25MHz, 500kHz to 200MHz, 1MHz to 5MHz, 1MHz to 7MHz, 1MHz to 10MHz, 1MHz to 20MHz, 1MHz to 25MHz, 1MHz to 30MHz, 1MHz to 200MHz, 2MHz to 5MHz, 2MHz to 10MHz, or 2MHz to 200MHz. In some embodiments, ultrasound is generated using an ultrasonic transducer or an ultrasonic horn.

[0114] Once the extraction process is complete, the liquid portion is separated from the solid portion (also referred to as "lettuce residue"), for example, by filtration or centrifugation. After drying, the lettuce residue can be mixed with calcium magnesium phytate for use as animal feed or plant fertilizer.

[0115] In some embodiments, the comminuted lettuce may be pre-mixed with a solvent to form a slurry before being immersed in the extraction solvent. Suitable solvents may be water, ethanol, or a mixture thereof.

[0116] In step 506 of method 500, the liquid portion is dried, for example by freeze drying, to remove the solvent, thereby obtaining a polyphenol-rich extract in the form of a powder. Drying can be performed at a temperature ranging from about 35° C. to about 70° C., such as, for example, 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 can be added to prevent caking and extend the shelf life of the extract.

[0117] In some embodiments, the extract can be mixed with lettuce residues so that the active ingredients in the extract can adhere to the surface of the lettuce residues. When used internally, the mixture helps to enhance intestinal absorption of polyphenols because of the high fiber content in lettuce residues. On the other hand, when used externally, the mixture helps to promote wound healing because of the increased ventilation provided by lettuce residues.

[0118] Methods and uses

[0119] In some embodiments, disclosed herein is a method for treating a wound, the method comprising applying a therapeutically effective amount of a polyphenol-rich lettuce powder disclosed herein or an extract thereof, a topical formulation disclosed herein, a wound dressing powder disclosed herein, a hydrogel disclosed herein, and / or an oral formulation disclosed herein to a wound site of a subject. In some embodiments, the wound comprises an acute wound, a skin infection, a burn wound, an ulcer, a chronic wound, a diabetic wound, or a non-healing wound.

[0120] Useful dosages can be determined by comparing in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art (eg, see US Pat. No. 4,938.949).

[0121] In some embodiments, provided herein is a method for treating a skin disorder, the method comprising topically applying to the subject a therapeutically effective amount of a polyphenol-rich lettuce powder or extract thereof disclosed herein, a topical formulation disclosed herein, a wound dressing powder disclosed herein, a hydrogel disclosed herein, and / or an oral formulation disclosed herein to the site of the disorder. Skin disorders generally refer to any skin-related disease, for example, inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral rashes, herpes zoster, ringworm, and cancers (such as basal cell carcinoma, squamous cell carcinoma, and melanoma). Skin disorders treated with the polyphenol-rich lettuce powder or extract thereof, topical formulations, wound dressing powders, hydrogels, and / or oral formulations disclosed herein include, but are not limited to, skin ulcers, bedsores, diabetic skin sores, hypertrophic scars, keloids, telangiectasias (spider veins), skin atrophy, precancerous skin lesions, herpes, inflammatory acne, acne vulgaris, comedonic or polymorphic acne, nodulocystic acne, conglobate acne, senile acne, and secondary acne (such as solar, drug-induced, or occupational acne), ichthyosis, ichthyosis-like conditions, Darier's disease, palmoplantar keratoses, vitiligo and vitiligo-like conditions or lichen and lichen planus, ungual psoriasis of the skin, mucous membranes, or nails, psoriatic rheumatism, cutaneous atopy, and dermatitis. atopy) (including eczema, dry skin, skin inflammation, flushing, solar erythema, actinic keratosis, skin allergies and allergic or irritant contact dermatitis, atopic dermatitis, rosacea, pigmentation, benign pigmented lesions (lentigines, freckles, brown spots, melasma)) and skin aging.

[0122] Another aspect of the present disclosure is a method for administering to a human or animal patient to provide an anti-inflammatory effect. For example, polyphenol-rich lettuce powder or extracts thereof have shown anti-inflammatory effects in vivo and in vitro. The samples tested showed efficacy in inhibiting NF-kB activity. Polyphenol-rich lettuce powder or extracts thereof also showed efficacy in antioxidant effects, as well as reducing the levels or activities of multiple cytokines known to cause inflammatory diseases or diseases with significant inflammatory components.

[0123] Therefore, disclosed herein is a method for treating an inflammatory condition or disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polyphenol-rich lettuce powder disclosed herein or an extract thereof, a topical formulation disclosed herein, a wound dressing powder disclosed herein, a hydrogel disclosed herein, and / or an oral formulation disclosed herein. In some embodiments, the inflammatory cytokines produced by immune cells are reduced compared to immune cells before treatment. In some embodiments, NF-κB activity is reduced. In some embodiments, the method comprises applying the composition to the skin, for example, epithelial tissue. In some embodiments, the method comprises applying the composition to muscle tissue. In some embodiments, the method comprises applying the composition to connective tissue. In some embodiments, the method comprises applying the composition to neural tissue.

[0124] In some embodiments, disclosed herein is a method of skin care, comprising administering to a subject a therapeutically effective amount of a polyphenol-rich lettuce powder or an extract thereof disclosed herein, a topical formulation disclosed herein, a wound dressing powder disclosed herein, a hydrogel disclosed herein, and / or an oral formulation disclosed herein.

[0125] In any of the embodiments disclosed herein, the subject can be a human, non-human primate, mouse, rat, gerbil, rabbit, dog, cat, horse, cow, pig, goat, sheep, donkey, llama, alpaca, guinea pig, mule, deer, buffalo, chicken, duck, goose or turkey. Preferably, the subject is a human.

[0126] In some embodiments, disclosed herein is a method of preparing a nanoparticle powder, the method comprising:

[0127] i. washing and drying at least one polyphenol-rich red leaf lettuce leaf;

[0128] ii. grinding at least one red leaf lettuce leaf using a grinding tool to form a polyphenol-rich lettuce powder;

[0129] iii. sieving the powder to remove particles larger than the sieve openings;

[0130] iv. Grinding the powder to reduce the particle size of the powder to a particle size distribution range of 0.1 μm or less.

[0131] In some embodiments, the grinding is performed with a ball mill, a cone mill, a hammer mill, or a 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.

[0132] In some embodiments, disclosed herein is a method of preparing a wound dressing powder, the method comprising:

[0133] i. mixing the mineral powder and the clay powder to form a powder mixture;

[0134] ii. adding the polyphenol-rich lettuce powder or extract thereof disclosed herein to the powder mixture;

[0135] iii. blending the powder mixture; and

[0136] iv. Adding a pharmaceutically acceptable excipient, carrier or diluent to the powder mixture.

[0137] In some embodiments, the mineral powder comprises zinc oxide.In some embodiments, the clay powder comprises bentonite clay.In some embodiments, the method further comprises adding an essential oil to the powder mixture and mixing the powder mixture to distribute the oil throughout the powder mixture.

[0138] In some embodiments, disclosed herein is a method of preparing a hydrogel, the method comprising:

[0139] i. dissolving chitosan powder in an acid solution to form a chitosan solution;

[0140] ii. adding the polyphenol-rich lettuce powder or extract thereof disclosed herein to the chitosan solution;

[0141] iii. mixing gelatin powder in water to produce a gelatin solution;

[0142] iv. mixing the chitosan solution containing the powdered extract with the gelatin solution;

[0143] v. adding a cross-linking agent to the mixture of the chitosan solution and the gelatin solution;

[0144] vi. pouring the mixture of v. into a hydrogel mold, and allowing the mixture to solidify to form a hydrogel;

[0145] vii. removing the hydrogel from the mold; and

[0146] viii. Applying heat to the hydrogel to promote cross-linking.

[0147] In some embodiments, the acid solution comprises 1% acetic acid. In some embodiments, the method further comprises 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 comprises solidifying the mixture at room temperature for 2 to 3 hours. In some embodiments, applying heat to the hydrogel comprises placing the hydrogel in an oven at 50° C. for 24 hours. In some embodiments, the cross-linking agent comprises glutaraldehyde. In some embodiments, the method further comprises adding a polymer surfactant to the gelatin solution. In some embodiments, the method further comprises freeze drying the hydrogel.

[0148] After reading the description of the following specific embodiments of the present invention in conjunction with the accompanying drawings, those skilled in the art will be able to recognize other aspects and features of the present invention. The following examples are provided for illustrative purposes only and should not be considered as limiting. The experimental procedures used in the examples are also described. The following examples provide further description of the present invention, but are not intended to limit the scope of the invention. Example

[0149] Example 1

[0150] Enhanced polyphenol production in red leaf lettuce using benign stressors / inducers

[0151] This example demonstrates that polyphenol production in red leaf lettuce is increased when treated with biotic / abiotic benign stressors / inducers.

[0152] Plant materials, growth conditions and benign stressor / inducer treatments

[0153] Red variety lettuce plants (Lactuca sativa) were grown in a laboratory greenhouse at 25-28°C, 40-60% relative humidity, with an average photoperiod of 12h / day. The abiotic benign stressors used were 30, 60 and 120mg / L of indole-3-acetic acid (IAA). The benign stressors were dissolved in deionized water (non-aqueous soluble benign stressors were pre-dissolved in 1mL of ethanol). A set of samples and water containing only 1mL of ethanol were added. Untreated control samples were added. Inducer treatment was applied to red leaf lettuce on the 7th day before harvest. Each experimental unit consisted of five randomly selected lettuces and was assigned to one treatment. Each sample was treated by rooting absorption or foliar aspersion, and each inducer (about 1.70mL) was sprayed 3 times. Lettuce samples were harvested at the 50th day.

[0154] Extraction and quantification

[0155] The main health-promoting polyphenols in treated and untreated (control) red leaf lettuce were characterized and quantified after extracting the samples with 50% ethanol. Typically, two grams of sample (ground with liquid nitrogen) were 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.

[0156] result

[0157] The increased production of polyphenols was confirmed using LC / MS / UV (or HPLC).

[0158] like Figure 6 As shown in, the production of specific metabolites in red leaf lettuce treated with biotic / abiotic benign stressors was confirmed by HPLC chromatograms of bioactive components enhanced by genomics technology. The production of polyphenols 3-CQA, chicoric acid, 3,4-dicaffeoylquinic acid (3,4-diCQA), quercetin-3-O-glucoside (Q3G), and quercetin-3-O-malonyl glucoside (Q3MG) in the treated lettuce was increased compared to the untreated lettuce control.

[0159] like Figures 7A to 7B As shown in , in red leaf lettuce treated with inducers / plant growth regulators, chlorogenic acid ( Fig. 7A ) and water-soluble quercetin derivatives ( Figure 7B ) increased by 3 to 9 times. Chlorogenic acid and derivatives (3-CQA, chicoric acid and 3,4-diCQA) and quercetin derivatives (Q3G and Q3MG) showed increased yields in the treated lettuce compared to the untreated lettuce control.

[0160] These results demonstrate that treatment with biotic / abiotic benign stressors can increase in vivo polyphenol production in red leaf lettuce.

[0161] Example 2

[0162] Grinding process of nano-sized polyphenol-rich lettuce powder

[0163] This article provides a method for preparing nano-sized dried polyphenol-rich lettuce powder using grinding technology:

[0164] Select fresh lettuce leaves and wash them thoroughly to remove any dirt or debris. Dry the leaves at 40 to 45°C.

[0165] Grind the dried leaves using a high energy ball mill or other suitable grinding equipment. The grinding process should be done in a dry and cool environment to prevent the lettuce from becoming overheated and losing its bioactive components.

[0166] After grinding, the lettuce powder is sieved through a fine mesh screen to remove any large particles or impurities.

[0167] Use a homogenizer or other suitable equipment to further reduce the particle size of the powder to the desired nanometer size range. This may require multiple passes through the homogenizer or other equipment.

[0168] Finally, the nano-sized lettuce powder is packaged in airtight containers and stored in a cool, dry place until ready for use.

[0169] It should be noted that the specific grinding process may vary depending on the type of grinding equipment used, the desired particle size range, and other factors.

[0170] As an example of a prepared powder, the powder may include polyphenol-rich lettuce powder combined with artificial additives and having a well-defined particle size and characterization, and a 3D ratio formulation. Examples of artificial additives (e.g., preservatives) that may be added to the powder include, for example:

[0171] (1) Non-inert: sodium benzoate; potassium sorbate; nitrates and nitrites; sulfites; propionic acid; sodium isoascorbate; ascorbic acid.

[0172] (2) Inertness: A variety of inert additives can be added to the powder for better preservation. These include:

[0173] a) Anti-caking agents: Anti-caking agents are added to prevent food powders from clumping by absorbing moisture and reducing the surface tension of the powder particles. Examples include silicon dioxide, calcium silicate, magnesium carbonate and talc;

[0174] b) Stabilizers: Stabilizers are added to prevent separation of ingredients in the powder, such as oil and water. Examples include gum arabic, xanthan gum, and carrageenan.

[0175] c) Emulsifiers: Emulsifiers are added to improve mixing and dispersion of ingredients in powders, especially when there are components that do not dissolve or blend well. Examples include lecithin and mono- and di-glycerides.

[0176] d) Humectants: Humectants are added to prevent the powder from drying out and hardening, which would reduce its shelf life. Examples include glycerin and propylene glycol.

[0177] Characterization of particle size:

[0178] (1) Particle size distribution (PSD): An important characteristic of drug products in all types of dosage forms, including solid oral drug products, semisolids, aerosols, and sterile liquid products. Strict control of particle size distribution is very important for drug development.

[0179] (2) Another way to define the particle size of the polyphenol-rich lettuce powder is by mesh size. Mesh size refers to the measurement of mesh size, which is a U.S. standard used to determine the relationship between the size of the openings in a sieve and the particle size that can pass through those openings. In order to determine the mesh size, the number of openings in a one-inch length of the sieve must be counted. For example, a 4-mesh sieve has four square openings in a one-inch sieve, while a 100-mesh sieve has 100 openings per inch. As the mesh size increases, the size of the openings in the sieve decreases, resulting in a smaller particle size that can be captured by the sieve. However, it is worth noting that mesh size is not an exact measure of the size of the sieve openings, as the sieve can be made using different materials with different strand or wire thicknesses. The thickness of the strands used in the sieve will affect the size of the openings, and thereby the particle size that can pass through.

[0180] Mesh size can be used to determine the particle size of powder particles rather than directly measuring their diameter. Example defined ranges for mesh size are shown in Tables 1 and 2 below.

[0181] Table 1. Example range of mesh sizes (mesh size 2 to 90)

[0182]

[0183]

[0184] Table 2. Example range of mesh sizes (mesh size 10 to 10000)

[0185]

[0186]

[0187] 3D ratio formula (mixture of different pore sizes):

[0188] By using different pore sizes in the mix, 3D Ratio formulations can achieve the following:

[0189] (1) Increased contact surface area: Using a combination of different mesh sizes in the formulation allows for increased contact surface area between particles. This can enhance inter-particle bonding and improve the overall strength and integrity of the printed object.

[0190] (2) Improved air convection between powder particles: Including different mesh sizes will promote better air convection between powder particles during the printing process. This improved airflow will help reduce the risk of particle agglomeration and ensure uniform powder distribution, thereby achieving better print quality and consistency.

[0191] (3) Controlling particle adhesion and cohesion: By adjusting the mesh size in the formulation, the adhesion or cohesion properties of the powder particles can be controlled. This can be beneficial in achieving the desired flowability of the powder during printing. Reducing particle adhesion can prevent blocking or agglomeration, while increasing particle cohesion can enhance interlayer adhesion and improve the structural integrity of the powder.

[0192] Example 3

[0193] Preparation of extracts from polyphenol-rich lettuce powder using SC-CO2

[0194] The lettuce powder (400 mesh size) rich in polyphenols is placed in an extraction container. Ensure that it is evenly distributed. Supercritical carbon dioxide (SC-CO2) is added to the mixture, and the SC-CO2 extraction of oil is carried out from the lettuce powder rich in polyphenols in an ultrasonic bath with an extraction pressure of 300 bar, a temperature of 40°C and a CO2 flow rate of 2mL / min. After extraction for 120 minutes, the resulting mixture is centrifuged. The resulting liquid portion is freeze-dried to provide an extract in powder form. Then calcium magnesium phytate is added to the extract, and the mixture is stored at 4°C before use.

[0195] Example 4

[0196] Preparation of extract from polyphenol-rich lettuce powder using a mixture of ethanol and water

[0197] The polyphenol-rich lettuce powder (400 mesh size) was placed in an extraction vessel. The polyphenols were extracted from the polyphenol-rich lettuce powder in an ultrasonic bath at a temperature of 0°C using ethanol / water (1:1 ratio) as the extraction solvent. After extraction for 30 minutes, the resulting mixture was centrifuged. The resulting liquid portion was rotary evaporated at 40°C and then freeze-dried to provide an extract in powder form. Calcium magnesium phytate was then added to the extract and the mixture was stored at 4°C before use.

[0198] Example 5

[0199] Preparation of extract from polyphenol-rich lettuce powder using water

[0200] The polyphenol-rich lettuce powder (400 mesh size) was placed in an extraction vessel. The polyphenols were extracted from the polyphenol-rich lettuce powder in an ultrasonic bath at a temperature of 0°C using water as the extraction solvent. After 40 minutes of extraction, the resulting mixture was centrifuged. The resulting liquid portion was freeze-dried to provide an extract in powder form. Calcium magnesium phytate was then added to the extract and the mixture was stored at 4°C before use.

[0201] Example 6

[0202] Basic wound dressing powder preparation and preparation method

[0203] preparation:

[0204] 80% zinc oxide powder;

[0205] 10% bentonite clay powder;

[0206] 5% polyphenol-rich lettuce powder / extract (400 mesh size); and

[0207] 5% lavender essential oil.

[0208] Preparation method:

[0209] 1. In a clean and dry container, add 80% zinc oxide powder and 10% bentonite clay powder.

[0210] 2. Mix the powder thoroughly until well blended.

[0211] 3. Add 5% polyphenol-rich lettuce powder / extract to the mixture and blend thoroughly.

[0212] 4. Add 5% lavender essential oil to the mixture and mix until the oil is evenly distributed in the powder.

[0213] 5. Store the wound dressing powder in a clean, airtight container until ready to use.

[0214] Example 7

[0215] Polyphenol-rich lettuce powder / extract preparation

[0216] The polyphenol-rich lettuce powder or extract thereof can be formulated into ointments, lotions, creams, oils, etc. Table 3 shows exemplary formulations of the polyphenol-rich lettuce powder or extract thereof and corresponding weight / weight examples.

[0217] Table 3. Examples of polyphenol-rich lettuce powder / extract formulations

[0218] Element %w / w Polyphenol-rich lettuce powder / extract 5 Cetearyl Alcohol 10 White soft paraffin 10 Polysorbate 60 2.5 Propylene glycol 10 benzoic acid 0.20 Purified water Up to 100.00

[0219] Example 8

[0220] Formulation of anti-inflammatory and antioxidant effects of polyphenol-rich lettuce powder / extract-loaded hydrogel for wound healing

[0221] The more specific aspect of the present disclosure is to develop the anti-inflammatory and antioxidant effects of the lettuce powder / extract hydrogel rich in polyphenols in wound healing applications.For example, the combination of chitosan-Pluronic 123-rich in polyphenols lettuce powder / extract-gelatin seems to be a very promising selection for the development of hydrogels intended for wound healing.It has been found that chitosan hydrogel can be used for wound healing, and it can be cross-linked with a binding agent by its amine functional group, to form a three-dimensional hydrogel system.When the chitosan nanoparticles (measured between 167-251nm) of the lettuce powder / extract rich in polyphenols to load were tested for transdermal application, they demonstrated significant transdermal permeability, enhanced drug release and high cell viability, all of which indicate that they may be effective ways to promote wound healing.In addition, it was found that the swelling behavior of the double-loaded hydrogel was more than 1.2 times that of gelatin-free hydrogel, which proves that the system has the potential to increase water absorption when mixed with gelatin. Given that gelatin can act as a cell glue and polyphenol-rich lettuce powder / extract has antioxidant properties that aid in wound healing, the chitosan-P123 nano-polyphenol-rich lettuce powder / extract-gelatin system was found to exhibit superior wound dressing properties when compared to single-loaded hydrogels.

[0222] The general protocol for preparing chitosan-polyphenol-rich lettuce powder / extract loaded hydrogels is exemplified as follows:

[0223] Material

[0224] Chitosan powder;

[0225] Nano-sized polyphenol-rich lettuce powder / extract;

[0226] Cross-linking agents (e.g., glutaraldehyde);

[0227] Pluronic P123;

[0228] Gelatin powder;

[0229] Sodium hydroxide; and

[0230] Acetic acid.

[0231] General Procedures

[0232] 1. Dissolve chitosan powder in 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.

[0233] 2. Add the polyphenol-rich lettuce powder / extract to the chitosan solution and stir until the powder is fully dispersed. The concentration of the polyphenol-rich lettuce powder / extract can be adjusted based on the desired drug payload.

[0234] 3. In separate beakers, dissolve Pluronic P123 and gelatin powder in distilled water to create gelatin solution.

[0235] 4. Slowly add the chitosan-polyphenol-rich lettuce powder / extract solution to the gelatin solution while stirring constantly. The pH of the mixture should be monitored and adjusted using NaOH to maintain the pH around 7.0.

[0236] 5. A cross-linking agent (eg, glutaraldehyde) is added to the mixture to promote the formation of a three-dimensional network structure.

[0237] 6. Pour the mixture into a hydrogel mold and allow it to solidify at room temperature for 2-3 hours.

[0238] 7. The hydrogel was removed from the mold and placed in a 50°C oven for 24 hours to ensure complete cross-linking.

[0239] 8. Finally, the hydrogel was freeze dried to remove any residual water and obtain dry chitosan-polyphenol-rich lettuce powder / extract loaded hydrogel.

[0240] Example 10

[0241] Preparation of powder dressing for promoting wound healing

[0242] A general procedure is provided for preparing a powder dressing for wound healing by combining polyphenol-rich lettuce powder / extract with collagen, algae extract, β-glucan, carboxymethylcellulose (CMC) and vitamin D3.

[0243] 1. Based on the intended formulation and target application, measure out the desired amount of each ingredient.

[0244] 2. Combine the polyphenol-rich powder, collagen, algae extract, beta-glucan, CMC and vitamin D3 in a dry blender or similar mixing device. Blend the mixture thoroughly to ensure even distribution of the ingredients.

[0245] 3. Optionally, adjust the pH of the mixture if necessary to ensure compatibility and stability of the ingredients.

[0246] 4. After the powders are blended uniformly, sieve them through a fine mesh screen to remove any lumps or agglomerates.

[0247] 5. Once the mixture is sieved, store it in an airtight container in a cool, dry place to prevent moisture and humidity from affecting the stability of the ingredients.

[0248] Embodiment 11

[0249] In vivo evaluation of polyphenol dressings on injured mice

[0250] The efficacy of polyphenol dressings in promoting wound healing was evaluated in injured mice.

[0251] Experimental mice: BALB / c mice were used as the primary target animals for the evaluation of in vivo wound healing efficacy. BALB / c laboratory mice were used because of their uniform skin color, which minimizes pigmentation issues that potentially affect the interpretation of the results. Fifteen BALB / c mice (obtained from the National Laboratory Animal Center) were raised until 10 weeks of age before the start of the experiment. These mice were randomly divided into five groups for different treatments.

[0252] Materials and methods

[0253] BALB / c mice at 10 weeks of age and averaging about 28.5 grams were anesthetized with isoflurane gas (initial 3-4%, maintained at 1-2%). Their backs were shaved (covering an area of ​​2x4 cm). Sterile scissors were used to create a 1x1 cm wound on the back of each mouse. A dressing was applied to the wound area immediately after the wound area was created. The dressing was applied to completely cover the wound area. During the experimental period, the wound condition was recorded before and after each dressing was applied, and a scale placed next to the wound was used for taking pictures. The dressing was applied twice, once on the 0th day, and once again on the 7th day.

[0254] Body weight was recorded before and after the experiment. Skin examinations were performed on days 0, 3, 5, 7, 10, and 14, with visual images taken at each time point. Blood samples were collected before the second dressing application on day 7 to detect common inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). Blood samples were collected again three days after the second dressing application on day 10 to re-evaluate the levels of these three inflammatory factors, providing insights into any specific effects of the composite polyphenol dressing on the inflammatory response. Finally, on day 14, after the animals were euthanized, wound samples were collected for tissue sectioning and analysis. In addition, photos of the spleen were also taken.

[0255] Table 4 provides the formulations of four polyphenol dressings used on different groups of mice in the experiment. The polyphenol dressings for mice in group A were made from the original lettuce powder. Mice in group B were treated with a dressing made from an extract obtained from lettuce powder using ethanol / water as the extraction solvent. Mice in group C were treated with a dressing made from an extract obtained from lettuce powder using supercritical CO2 as the extraction solvent. Mice in group D were treated with a dressing made from a mixture of the original powder and CMC. The results of mice treated with the different polyphenol dressing formulations were compared with a control group of mice that were not given a dressing.

[0256] Table 4. Polyphenol dressing preparations and experimental conditions

[0257]

[0258] Table 5 provides the relative contents of the main bioactive polyphenols in different types of dressings. Figure 5 As shown in , the extracts contained higher levels of chicoric acid compared to the original lettuce powder. In addition, the polarity of the extraction solvent also affects the polyphenol content in the extract. Polar extraction solvents (e.g., aqueous ethanol) provide extracts containing higher levels of extractable polyphenols (such as chicoric acid and quercetin derivatives).

[0259] Table 5. Polyphenol content in lettuce powder and lettuce powder extract

[0260]

[0261]

[0262] result

[0263] Weight changes and macroscopic differences of the spleen

[0264] exist Figure 8 The average weight difference of mice in each group (A to D and control) before and after the experiment was compared. It can be observed that mice in all groups showed an increase in weight after the experiment, and there was no statistically significant difference in their weight before and after the experiment. In addition, there was no significant difference in the weight change of mice in the AD group and the control group.

[0265] Fig. 9 Photos of spleens of mice in the polyphenol dressing-treated group (A to D) and the control group are shown. Compared to the spleens of mice in the polyphenol dressing-treated group (AD), the control group mice had slightly larger spleens, indicating that the use of polyphenol dressings helped reduce wound inflammation.

[0266] Comparison of wound healing effects

[0267] The wound healing effect of polyphenol dressings was evaluated by monitoring the changes in wound size in the polyphenol dressing-treated group (AD) and the control group during the treatment period. Fig.10 Photographs showing the changes in wound size over time in mice treated with polyphenol dressings (AD) and control groups. Fig.10 As shown in Figure 1, the control group mice exhibited the slowest wound healing rate. On day 14, the control group mice had the largest wound size. In contrast, among the polyphenol dressing treated groups (AD), group B mice showed the fastest healing rate and had the smallest wound size. This effect may be attributed to the use of alcohol-aqueous extraction solvents (e.g., ethanol / water mixed solvents), which produce extracts with higher polar polyphenols (including chicoric acid and quercetin derivatives). Polyphenols with higher polarity act synergistically to enhance wound healing properties.

[0268] Comparison of wound tissue sections

[0269] Wound tissue sections from mice in the polyphenol dressing-treated groups (A to D) and the control group were analyzed. Figures 11A to 11E Photographs illustrating wound tissue sections from mice treated with polyphenols and those in the control group are shown. As can be seen, the control group mice exhibited the slowest wound healing rate ( Fig.11A ). Tissue sections from control mice showed excessive epithelialization and disorganized cell arrangement. In contrast, Figures 11B to 11E As shown in , mice whose wounds were treated with polyphenol dressings showed more orderly cell arrangement and more normal epithelial formation.

[0270] Comparison of inflammatory cytokine levels in blood

[0271] Common inflammatory cytokines, including tumor necrosis factor-α (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), three days after the second dressing application (day 10), and before sacrifice (day 14). Fig.12 is a bar graph showing the changes in TNF-α over time in mice treated with polyphenol dressings (AD) and control groups. Fig.13 is a bar graph showing the changes in IL-6 over time in mice treated with polyphenol dressings (AD) and control groups. Fig.14 is a bar graph showing the changes in IL-8 over time in mice treated with polyphenol dressings (AD) and control groups. The data show that for TNF-α, IL-6 and IL-8, the levels of inflammatory cytokines in mice treated with dressings containing polyphenols (AD group) were lower than those in the control group mice. In addition, mice treated with the extracts (Groups B and C) showed significantly better anti-inflammatory effects than mice treated with the original polyphenol powder (Groups A and D). In addition, when comparing mice treated with the original polyphenol powder, mice in Group D showed lower levels of IL-6 and IL-8 than mice in Group A. Overall, mice in Group B showed the most effective inhibition of all inflammatory cytokines measured.

[0272] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to provide additional embodiments using the concepts of various patents, applications, and publications.

[0273] Based on the above description, these and other changes can be made to the embodiments. Generally, in the appended claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted as including all possible embodiments and the full scope of equivalent schemes enjoyed by such claims. Therefore, the claims are not limited by the content of this disclosure. This application requests priority to U.S. Provisional Application No. 63 / 509,677 filed in the U.S. Patent Office on June 22, 2023, the entire contents and disclosures of which are incorporated herein by reference.

Claims

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

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

3. The powder of 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 powder of any one of claims 1 to 3, wherein the quercetin derivative comprises one or more of quercetin-3-O-glucoside (Q3G), quercetin glucuronide, and quercetin-3-O-malonyl glucoside (Q3MG).

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

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

7. The powder of any one of claims 1 to 6, wherein the polyphenol-enriched lettuce powder or extract is derived from polyphenol-enriched lettuce obtained by treating control lettuce with at least one benign stressor / inducer or a homologue, isomer or derivative thereof that increases polyphenol production in control lettuce.

8. The powder of claim 7, wherein the at least one benign stressor / inducer is an abiotic benign stressor or inducer selected from the group consisting of indole-3-acetic acid (IAA), auxins, cytokinins (CK), gibberellins (GA), ethylene, rapeseed steroids, jasmonates (JA), strigolactones (SL), salicylic acid (SA), arachidonic acid (AA), 5-aminolevulinic acid (5-ALA), oxalic acid and any homologues or isomers or derivatives, synthetic analogues or any combination or mixture thereof.

9. The powder of any one of claims 7 to 8, wherein the polyphenol-enriched lettuce comprises a 2-fold to 20-fold increased polyphenol yield compared to the control lettuce, optionally a 3-fold to 9-fold increased polyphenol yield compared to the control lettuce.

10. The powder of any one of claims 7 to 9, wherein the concentration of chlorogenic acid, quercetin derivatives, chicoric acid, and anthocyanins is increased by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold compared to a powder produced from the control lettuce.

11. The powder of 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 of any one of claims 1 to 11, wherein the polyphenol-enriched lettuce powder comprises nanoparticle powder, wherein the nanoparticle powder has a particle size distribution ranging from 0.01 μm to 0.1 μm.

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

14. The powder of claim 13, wherein the nanoparticle powder has a particle size distribution range of 0.1 μm or less.

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

16. A topical formulation for treating skin disorders, the topical formulation comprising a powder comprising particles of polyphenol-rich lettuce powder or extract, wherein the polyphenol-rich lettuce powder or extract comprises 40 to 280 mg / g of polyphenols.

17. The topical formulation of claim 16, wherein the polyphenols include two or more of chlorogenic acid, chicoric acid, quercetin and its derivatives, and anthocyanins.

18. The topical formulation of claim 16, wherein the skin disorder comprises inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral rash, shingles, ringworm, or skin cancer.

19. The topical formulation of 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% of the final weight of the topical formulation.

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

21. The topical formulation of 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, drops, aerosol, emulsion, nanoemulsion, nanoliposome, gel, microcapsule, paste or foam.

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

23. The topical formulation of any one of claims 15 to 22 further comprising an emollient.

24. The topical formulation of claim 23, wherein the emollient comprises paraffin.

25. The topical formulation of any one of claims 15 to 24, further comprising an antimicrobial preservative.

26. The topical formulation of any one of claims 15 to 25, further comprising one or more of an emulsifier and a viscosity enhancer.

27. The topical formulation of any one of claims 15 to 26, wherein the topical formulation comprises the following in % weight / weight: 1 to 10% of said powder; 5 to 15% alcohol; 5 to 15% emollient; 1 to 5% emulsifier; 5 to 15 percent propylene glycol; Less than 1% antimicrobial preservatives; and The remaining percentage of water equals 100%.

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

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

30. The wound dressing powder of claim 29, wherein i. the mineral powder comprises 50% to 90% weight / weight, ii. the clay powder comprises 5% to 15% w / w; and iii. The powder comprises 1% to 15% w / w.

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

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

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

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

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

36. The hydrogel of 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% of the final weight of the topical formulation.

37. The hydrogel of claim 35 or 36, wherein the powder comprises particles having a size distribution ranging from 0.1 μm to 150 μm.

38. The hydrogel of claim 35 or 36, wherein the powder comprises or consists essentially of nanoparticles.

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

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

41. The hydrogel of any one of claims 35 to 40, further comprising a polymeric surfactant.

42. The hydrogel of claim 41, wherein the polymeric surfactant is Pluronic P123.

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

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

45. The oral formulation of 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 formulation.

46. ​​The oral formulation of claim 44 or 45, wherein the binder comprises gum tragacanth, gum arabic, corn starch, or gelatin.

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

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

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

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

51. The oral formulation of any one of claims 44 to 50, further comprising a flavoring agent.

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

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

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

55. The method of claim 54, wherein the wound comprises an acute wound, a skin infection, a burn wound, an ulcer, a chronic wound, a diabetic wound, or a non-healing wound.

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

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

58. The method of claim 56 or 57, wherein NF-κB activity is reduced.

59. The method of any one of claims 54 to 58, wherein the method comprises administering the composition to an epithelial tissue.

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

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

62. The method of any one of claims 54 to 58, wherein the method comprises administering the composition to neural tissue.

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

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

65. A method of preparing a wound dressing powder, the method comprising: i. mixing the mineral powder and the 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 powder mixture; and iv. Adding a pharmaceutically acceptable excipient, carrier or diluent to the powder mixture.

66. The method of claim 65, wherein the mineral powder comprises zinc oxide.

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

68. The method of any one of claims 65 to 67, further comprising adding an essential oil to the powder mixture and mixing the powder mixture to distribute the oil throughout the powder mixture.

69. A method for preparing nanoparticle powder, the method comprising: i. washing and drying at least one polyphenol-rich lettuce leaf; ii. grinding the at least one red leaf lettuce leaf using a grinding tool to form a powder; iii. sieving the powder to remove particles larger than the sieve opening; and iv. grinding the powder to reduce the particle size of the powder to a particle size distribution range of 0.1 μm or less.

70. The method of claim 69, wherein the grinding is performed using a ball mill, a cone mill, a hammer mill, or a homogenizer.

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

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

73. A method for preparing a hydrogel, the method comprising: i. dissolving chitosan powder in an acid solution to form a chitosan solution; ii. adding the powder according to any one of claims 1 to 14 to the chitosan solution; iii. mixing gelatin powder in water to produce a gelatin solution; iv. mixing the chitosan solution comprising the powder with the gelatin solution; v. adding a cross-linking agent to the mixture of the chitosan solution and the gelatin solution; vi. pouring the mixture of v. into a hydrogel mold and allowing the mixture to solidify to form a hydrogel; vii. removing the hydrogel from the mold; and viii. applying heat to the hydrogel to promote cross-linking.

74. The method of claim 73, further comprising maintaining the pH of the mixture at about 7.0 by adding NaOH solution while mixing the chitosan solution with the gelatin solution.

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

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

77. The method of any one of claims 73 to 76, wherein the cross-linking agent comprises glutaraldehyde.

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

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

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

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

82. The method of claim 81, wherein the skin disorder comprises inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral rash, shingles, ringworm, skin cancer.

83. A method of treating a wound, the method comprising: administering a therapeutically effective amount of the powder of any one of claims 1 to 14, the topical formulation of any one of claims 15 to 27, the wound dressing powder of any one of claims 28 to 34, or the hydrogel of any one of claims 35 to 43 to a wound site of a subject; Orally administering to a subject a therapeutically effective amount of the oral formulation of any one of claims 44 to 55; irradiating the wound site with blue light radiation; and The wound site is irradiated with red radiation.

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

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

86. The method of any one of claims 83 to 85, wherein the wound comprises an acute wound, a skin infection, a burn wound, an ulcer, a chronic wound, a diabetic wound, or a non-healing wound.

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