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

Abstract

Provided herein are polyphenol-enriched lettuce powders comprising particles of a polyphenol-enriched lettuce or extract, wherein the polyphenol-enriched lettuce comprises from 40-280 mg / g of polyphenols. The polyphenols include any combination of a chlorogenic acid, a neochlorogenic acid, a chicoric acid, a quercetin, a quercetin derivative, and an anthocyanin. The disclosure also provides compositions and methods of using the polyphenol-enriched lettuce powders to promote wound healing and treat inflammation and skin disorders.

Description

POLYPHENOL-ENRICHED LETTUCE COMPOSITIONS FOR ENHANCED WOUND HEALING AND SKIN CAREBACKGROUNDTechnical Field

[0001] The present disclosure pertains to novel compositions that possess therapeutic and cosmetic properties for promoting wound healing and providing skincare benefits.Description of the Related Art

[0002] Although there have been significant advancements in anti-microbial treatment and supportive measures, wound treatment continues to present significant challenges. Invasive infections, blunt injuries, burns caused by electrical or chemical accidents, radiation burns, and other similar injuries remain difficult to effectively treat and cure. Unfortunately, despite the introduction of clinically effective anti-microbial agents, there has been a rapid emergence of bacteria strains that are resistant to such treatments, further exacerbating the issue.

[0003] Countries with aging populations, such as the United States, face significant challenges posed by patients with leg ulcers. It is estimated that leg ulcers result in the loss of 2 million workdays in the US. The cost of treating these non-healing wounds can be tremendous, and the prevalence of pressure ulcers (pressure sores or bedsores) of the lower body in the elderly population is estimated to be between 3-11%. The morbidity and mortality associated with pressure ulcers is significant, with the death rate in patients with pressure ulcers being four times greater than those without. Additionally, septic elderly patients with pressure ulcers have a hospital mortality rate exceeding 50%. The treatment costs can be expensive due to the inclusion of intensive nursing care, as well as adjunctive therapies such as anti-pressure devices, protective dressings, and skin treatments [Monika P, Chandraprabha MN, Rangarajan A, Waiker PV, Chidambara Murthy KN. Challenges in Healing Wound: Role of Complementary and Alternative Medicine. Front Nutr. 2022, 20; 8:791899],

[0004] Wound management presents significant challenges, particularly in diabetics, where wounds are often considered incurable. Diabetes is a common underlying condition in leg ulcers, and while various therapies exist to aid wound healing, such as multi-layer compression bandages, topical recombinant human platelet-derived growth factor, and human skin equivalents for skin grafting, they can be expensive and prohibitive for many patients. In some cases,wounds may remain untreated, leading to the amputation of infected limbs. Managing wounds in animals presents additional challenges, as they are more susceptible to environmental infestations. Flies, especially myiasigenic flies such as sarcophagidae and calliphoridae, can exacerbate wounds and lead to complications. Pouring creolin (a mixture of phenols and tar) on the wound is a common practice, but it is tissue-damaging, toxic, and provides only short-term insect protection. Direct use of insecticides on maggot-infected wounds is also problematic due to the risk of animal intoxication and contamination of the food chain.

[0005] Provided herein are novel formulations, compositions, and uses of powders / extracts of polyphenol -enriched red lettuces that provide unexpected and superior effects in promoting wound healing which provides an unmet need in the art.BRIEF SUMMARY

[0006] It is a general objective of the disclosure to provide a novel plant composition for wound healing / skin care and its methods of preparation / manufacture. The present disclosure introduces a new powder composition that includes particles of a polyphenol-enriched lettuce powder or extract, the polyphenol-enriched lettuce having increased production and concentration of at least one polyphenol relative to a control lettuce. More specifically, it relates to a polyphenol-enriched lettuce powder or its extract having potent anti-inflammatory and wound healing properties, which is quite effective in curing external wounds of any nature in humans, including acute wounds and chronic wounds such as abrasion wound, burn wound, laceration wound, vascular ulcer, diabetic ulcer and pressure injury etc. The polyphenol- enriched lettuce powder / extract is also quite effective in treating wounds in mammal animals. The disclosed powders / extracts and methods may be applicable to the four fundamental tissues (nervous, epithelial, muscle, and connective) of the human / animal bodies. Furthermore, the disclosed powders / extracts can also be used in cosmetic products for skin care. The polyphenol- enriched lettuce powders / extracts can be incorporated into a variety of cosmetic formulations, such as sunscreens, facial cleansers, facial masks, hair conditioners and shampoo, body wash, among others, offering a range of benefits for the skin, including anti-aging, radiation protection, skin whitening, moisturizing, freckle removal, acne treatment, and promotion of hair regeneration. The powder / extract may be designed for extemal / topical use or oral use and has exceptional wound-healing and anti-inflammatory and antioxidant properties. The powders / extracts and methods are particularly effective in treating wounds that are considered incurable using traditional methods. The combination of naturally-occurring components in thepolyphenol-enriched lettuce powder / extract creates a potent and powerful action, resulting in the synergistic effect of the components.

[0007] These and other aspects of the present disclosure will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figs. 1 A-1C show the naturally occurring quercetin (Fig. 1 A) in red lettuce and its highly oral-bioavailable derivatives, quercetin-3-O-glucoside (Q3G) (Fig. IB), and quercetin-3- O-malonylglucoside (Q3MG) (Fig. 1C).

[0009] Fig. 2 shows examples of chlorogenic acids identified from red lettuce, such as 3- caffeoylquinic acid (neochl orogenic acid or 3-CQA) (Fig. 2A) and 3,4-dicaffeoylquinic acid (3,4-diCQA) (Fig. 2B).

[0010] Fig. 3 shows chicoric acid (CRA) in red lettuce.

[0011] Fig. 4 shows example anthocyanins, such as cyanidin-3-O-glucoside (Fig. 4A) and cyanidin-3-(6”-malonylglucoside) (Fig. 4B).

[0012] Fig. 5 is a flowchart of a method for making an extract from a polyphenol- enriched lettuce powder.

[0013] Fig. 6 shows HPLC-UV chromatograms of bioactive components enhancement by genomics-based technologies confirming production of specific metabolites from red lettuce treated with elicitors. (1.) Non-treated lettuce. (2.) treated lettuce.: A: Chlorogenic acid (3- CQA); B: Chicoric acid (CRA); C: Quercetin-3-O-glucoside (Q3G); D: Quercetin-3-O- malonylglucoside (Q3MG); E: 3,4-Dicaffeoylquinic acid (3,4-diCQA).

[0014] Figs. 7A and 7B show the production of chlorogenic acids and chicoric acid (Fig. 7A) and the more hydrophilic quercetin derivatives (Fig. 7B) were increased by 3- to 9-fold in red lettuce treated with plant elicitors. Fig. 7A depicts production of chlorogenic acid (3-CQA,), 3,4-dicaffeoylquinic acid (3,4-diCQA), and chicoric acid (CRA). Fig. 7B depicts production of quercetin derivatives (Q3G and Q3MG).

[0015] Fig. 8 is a bar graph comparing the weight change of mice before and after the experiment for mice in polyphenol dressing-treated groups (A-D) and control group.

[0016] Fig. 9 shows photographs of the spleens of mice in polyphenol dressing-treated groups (A-D) and control group.

[0017] Fig. 10 shows photographs illustrating wound sizes of mice in polyphenol dressing-treated groups (A-D) and control group over time.

[0018] Figs. 11 A-l IE show photographs illustrating tissue sections of wounds in mice from polyphenol-treated groups and control group.

[0019] Fig. 12 is a bar graph showing TNF-a changes in mice of polyphenol dressing- treated groups (A-D) and control group over time.

[0020] Fig. 13 is a bar graph showing IL-6 changes in mice of polyphenol dressing- treated groups (A-D) and control group over time.

[0021] Fig. 14 is a bar graph showing IL-8 changes in the mice of the polyphenol dressing-treated groups (A-D) and the control group over time.DETAILED DESCRIPTION

[0022] Presented herein are examples related to the use of highly polyphenol-enriched lettuce as the raw material and subsequent extracts and formulations thereof. Such lettuce contains a high content of polyphenols such as quercetin derivatives, chlorogenic acids, chicoric acid, and anthocyanins. Without wishing to be limited by theory, it is believed that polyphenols in such lettuce show a synergistic biological activity for wound healing and skin care applications.

[0023] Presented herein are powders that comprise particles of a polyphenol-enriched lettuce powder or extract, the polyphenol-enriched lettuce having increased production and concentration of at least one polyphenol relative to control lettuce. In some embodiments, the polyphenol is at least one of a chlorogenic acid, a neochlorogenic acid, a chicoric acid, a quercetin, a quercetin derivative, an anthocyanin, or any combination thereof. In some examples, the powder may be formulated for topical administration and / or oral administration. Additionally, the present disclosure provides a wound dressing powder. The disclosure also provides a hydrogel comprising the powder. Also provided herein are methods of treatment comprising administering such powders, and methods of preparing such powders.

[0024] The present disclosure includes a variety of aspects, which may be combined in different ways. The following descriptions are provided to list elements and describe some of the embodiments of the present disclosure. These elements are listed with initial embodiments; however, it should be understood that these embodiments may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only theexplicitly described systems, techniques, and applications. Further, this description should be understood to support and encompass descriptions and claims of all the various embodiments, systems, techniques, methods, devices, and applications with any number of the disclosed elements, with each element alone, and also with any and all various permutations and combinations of all elements in this or any subsequent application.

[0025] One of the highly-enriched natural polyphenols in red lettuce are quercetin derivatives. 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, and others (Flavonoids, Micronutrient Information Center, Linus Pauling Institute, Oregon State University, 2015). Quercetin has been linked to improved exercise performance and reduced inflammation, blood pressure and blood sugar levels. It may also have brain-protective, anti-allergy, and anticancer, antibacterial, and antiviral properties. Quercetin has been extensively studied in the context of wound healing and its use has been described in numerous research papers covering a range of wound conditions affecting various organs [Pol era 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, it has been documented that the quercetin derivatives in Oxytropis falcata Bunge are main active components to treat knife wounds 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 cutaneous wound healing. This effect is attributed to the activation of the Wnt / p-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 / p-catenin signaling pathway, Journal of Ethnopharmacology, 290, 2022,115066], These findings provide a foundation for a deeper understanding of the mechanism of action of O. falcata Bunge in the treatment of knife wounds and burns. Another study [Jia Fu, Jingjuan Huang, Man Lin, Tingting Xie, Tianhui You, Quercetin Promotes Diabetic Wound Healing via Switching Macrophages From Ml to M2 Polarization, Journal of Surgical Research, 246, 2020, 213-223] demonstrated that quercetin inhibits inflammatory reactions via modulating macrophage polarization switching from Ml to M2 phenotype, thereby accelerating the diabetic wound repair.

[0026] Another type of important biologically active component in polyphenol-enriched lettuce is chlorogenic acids. Chlorogenic acids (CGAs, phenolic compounds found in human dietary products) have demonstrated several health-improving properties, including anti-oxidant,anti-inflammatory, anti-microbial, anti-diabetic, and lipid-lowing functions. Numerous reports have demonstrated that CGAs have beneficial effects in 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. “A chlorogenic acid-loaded hyaluronic acid-based hydrogel facilitates anti-inflammatory and pro-healing effects for diabetic wounds, Journal of Drug Delivery Science and Technology, 2022, 70, 103232], the capacity of CGA for promoting diabetic wound healing was assessed using in vitro and ex vivo experimental approaches. Consequently, CGA significantly achieved the anti-inflammatory activity, re-epithelializing effect, and pro-angiogenic function. A hyaluronic acid-based hydrogel was subsequently used in vivo for local administration of CGA to the full-thickness wounds of diabetic mice. The resulting formulation (CGA-HA) significantly improved the healing effects relative to a commercial wound dressing (INTRASITE Gel), which was accompanied with the reduction of inflammation, enhancement of re-epithelialization and angiogenesis, and maturation of collagen remodeling, demonstrating the potential of CGA-HA in the clinical application for diabetic wound healing.

[0027] Polyphenol-enriched lettuce raw material also contains a high 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 pigments are in glycosylated forms. Anthocyanins responsible for the colors, red, purple, and blue, are in fruits and vegetables. Berries, currants, grapes, and some tropical fruits have high anthocyanins content. Red to purplish blue-colored leafy vegetables, grains, roots, and tubers are the edible vegetables that contain a high level of anthocyanins. Among the anthocyanin pigments, cyanidin-3- glucoside is the major anthocyanin found in most of the plants. Anthocyanins possess antidiabetic, anticancer, anti-inflammatory, antimicrobial, and anti-obesity effects, as well as prevention of cardiovascular diseases (He et al., J Ethnopharmacol 3rl(3') (2011): 1135-1142. Particularly [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.], it has been demonstrated that anthocyanins inhibit the translocation of nuclear factor-xB (p65) from cytosol to nucleus and prevent the phosphorylation of IxBa. Anthocyanins enhance wound healing through a cytoprotective effect, enhancement of angiogenesis, and an anti-inflammatory effect. 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 antiaging potential by modulating type I collagen gene expression and suppressing FFCh-induced NF- / cB activation in skin fibroblasts.

[0028] In terms of dressing materials, the size of particles is an important factor for effective wound healing when considering topical use [For examples, see Pat. No. TWI764332, TWI786786, TWM636367, TWI787696, TWI774168, TWI774055, TWI774046, TWI760999, TWI760961, TWI758625, TWM602560], Suitable particles can penetrate the skin more easily and reach deeper layers of tissue, allowing for more efficient delivery of therapeutic agents to the wound site. In contrast, larger particles may be more difficult to penetrate the skin and may remain on the surface, providing little benefit to the underlying tissue. Therefore, it is important to consider particle size when designing topical wound healing treatments to ensure optimal therapeutic effects.Wound Healing and Wound Molecular Biology

[0029] Wound healing is a complex and dynamic process consisting of several sequential steps, including induction of the inflammatory response, regeneration of parenchymal tissue, migration and proliferation of parenchymal tissue cells, production of extracellular matrix proteins, tissue remodeling, and wound strength gain [Harding KG, Morris HL, Patel GK. Healing chronic wounds. BMJ. (2002) 324: 160-3], Multiple cell types, secreted growth factors, cytokines, the extracellular matrix, and various enzymes are involved in the healing of a wound. Platelets, neutrophils, monocytes, macrophages, fibroblasts, keratinocytes, endothelial cells, epithelial cells, and myofibroblasts are among the various cell types involved in the wound healing process. Fibroblasts have long been recognized as important cells in wound healing because they play a significant role in all three phases [Monika P, Waiker PV, Chandraprabha MN, Rangarajan A, Murthy KNC. Myofibroblast progeny in wound biology and wound healing studies. Wound Repair Regen. (2021) 29:531-47], Several factors, such as microbial infection or biofilm formation, can affect the wound healing process. Additionally, ischemia and reperfusion play an important role in injured 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 :35 S— 41 ] discussed the molecular biology involved in ischemia and reperfusion, which are major contributors to the pathological conditions of a wound. Ischemia-reperfusion injury is characterized by a sequence ofbiochemical and cellular events that cause extensive cell damage through pathways leading to leukocyte and complement activation, oxidative stress, and microvasculature dysfunction

[0030] Numerous molecular factors play important and overlapping roles in normal wound healing, including growth factors and receptors like platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-B), cytokines such as fibroblast growth factor (FGF), tumor necrosis alpha (TNFa), and interleukin-1 (IL-1), and enzymes like matrix metalloproteinases (MMPs), tissue inhibitors of MMPs, and seperinase [Blakytny R, Jude EB. Altered molecular mechanisms of diabetic foot ulcers. Int J Low Extrem Wounds. (2009) 8:95- 104], Additionally, angiogenesis, which involves the development of capillary sprouts that invade the extracellular matrix (ECM) stroma and form tube-like structures, is important for wound healing [Honnegowda TM, Kumar P, Udupa EGP, Kumar S, Kumar U, Rao P. Role of angiogenesis and angiogenic factors in acute and chronic wound healing. Plast Aesthetic Res. (2015) 2:243-9], Various angiogenic stimulators like vascular endothelial growth factor (VEGF), TGF-B, TNFa, PDGF, FGF, angiogenin, and angiopoietin-1 play significant roles in different stages of angiogenesis, including initiation, amplification, proliferation, stabilization, and network formation [Honnegowda TM, Kumar P, Udupa EGP, Kumar S, Kumar U, Rao P. Role of angiogenesis and angiogenic factors in acute and chronic wound healing. Plast Aesthetic Res. (2015) 2:243-9). The activation of these molecular factors is essential for wound healing, while a complex set of interactions between these molecules may cause the persistence of a wound if disrupted. A deeper understanding of altered expression of these molecular factors and their effects on cellular function could provide valuable insights into potential targets for intervention.Wound Healing in Acute and Chronic Conditions

[0031] There are two main categories of wounds: acute and chronic. Acute wounds typically heal in an orderly and efficient manner, progressing through four distinct but overlapping phases: hemostasis, inflammation, proliferation, and remodeling [Diegelmann R. Wound healing: an overview of acute, fibrotic, and delayed healing. Front Biosci. (2004) 9:283- 9], Within 2-4 weeks, acute wounds usually exhibit clear indications of healing as they pass through the stages of inflammation, proliferation, and remodeling. Conversely, chronic wounds do not follow the sequential stages of healing and may become “stuck” in a single phase, failing to display evidence of healing within 4 weeks [Swezey L. The difference between acute and chronic wounds (2019)].

[0032] 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 resulting in chronic hypoxia and impaired micronutrient delivery. Vasculopathies associated with diabetes include abnormal blood vessel formation, decreased angiogenesis, and accelerated atherosclerosis, which can lead to coronary artery disease, peripheral vascular disease, and cerebrovascular disease [Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev. (2003) 23 : 117-45], Vascular endothelial growth factor (VEGF) is an important angiogenic stimulator in wound healing, and studies have shown that diabetic chronic wounds have deficient VEGF levels. The application of VEGF has been shown to stimulate 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 SurgRes. (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, chronic venous stasis ulcer patients 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 - a pilot study. Eur J Vase Endovasc Surg. (1999) 18:334- 8], As many factors regulate wound angiogenesis, it is important to understand various dysregulated cellular and molecular events in angiogenesis that are responsible for non-healing chronic wounds.Role of Natural Products in Wound Treatment

[0033] Natural products, including phytochemicals derived from plants and naturally derived substances, have recently gained significant research interest 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-modulating properties. There is a wealth of evidence supporting 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 many ways, making them an attractive option. Plants with medicinal properties have been found to be effective in treating wounds and fighting infections, thus accelerating wound healing.According to a recent study by Nigussie, Lawsonia inermis and Azadirachta indica were the most studied plant species for wound healing, and the most common in vivo techniques used for the anti-inflammatory and wound healing assays were carrageenan-induced paw edema and excision and incision 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 roles of phytochemicals and naturally derived substances in wound healing studies, including their uses, applications, mechanisms of action, and outcomes, have been extensively demonstrated.

[0034] Another study highlights the synergistic effect of plant and naturally derived substances in wound healing, but also notes 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 suggested that neem leaf extract may be a safe alternative to normal 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 important to note that phytochemicals and naturally derived substances may have a higher risk of contamination with infectious agents, and thus proper sterilization and microbial testing are essential prior to use. Key markers for assessing the benefits of natural products in wound healing include content of hydroxyproline, microscopic observation, and physical appearance of the wound. For instance, pomegranate peel extract in the form of a water-soluble gel has shown promising results in wound healing, as evidenced by these markers [Murthy KNC, Reddy KV, Veigas JM, Murthy UD. Study on wound healing activity of Punica granatum peel. JMedFood. (2004) 7:256- 9], Ongoing studies in this area demonstrate the exciting potential of natural products for wound healing.

[0035] Polyphenols have garnered significant attention for their potential in wound healing due to their ability to act as antioxidants, as well as their regenerative and antimicrobial properties [Dzialo, M.A.; 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, 77, 160; Ghuman, S.; Ncube, B.; Finnie, J.; McGaw, L.; Njoya, E.M.; 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, 726, 232-240; Guimaraes, I.; Baptista-Silva, S.; Pintado, M.; L. Oliveira, A. Polyphenols: A Promising Avenue in TherapeuticSolutions for Wound Care. AppL Sci. 2021, 77, 1230; Luque, G.C.; Moya, M.; Picchio, M.L.; Bagnarello, V.; Valerio, I.; Bolanos, J.; Vethencourt, M.; Gamboa, S.-H.; Tome, L.C.; Minari, R.J.; Mecerreyes, D. Polyphenol longel Patches with Antimicrobial, Antioxidant and Anti- Inflammatory Properties. Polymers 2023, 75, 1076], These compounds are typically extracted from plants and marine organisms and are divided into two primary subgroups: flavonoids (such as flavonols and anthocyanidins) and non-flavonoid compounds (such as phenolic acids, tannins, and lignans), depending on the number and binding structure of their phenol units [Liakos, I.; Rizzello, L.; Hajiali, H.; Brunetti, V.; Carzino, R.; Pompa, P.P.; Athanassiou, A.; Mele, E. Fibrous wound dressings encapsulating essential oils as natural antimicrobial agents. J. Mater. Chem. B 2015, 3, 1583-1589], Polyphenols have been the focus of extensive research for their potential in medical and pharmaceutical applications, particularly in wound care [Dzialo, M.A.; 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, 77, 160], These compounds are known for their potent antioxidant activity, which helps to neutralize free radicals by donating an electron or hydrogen atom and provides protection against reactive oxygen species (ROS). Additionally, 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, E.M.; 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], While the mechanisms by which polyphenols exert their antimicrobial effects are not yet fully understood, it is believed that they may work by disintegrating the bacterial cell wall through the hydrophobic components of phenolic compounds, altering intracellular functions through hydrogen bonding, or modifying the cell wall structure and rigidity through interactions with the cell membrane [Dzialo, M.A.; 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, 77, 160], Increasing the lipophilic nature of polyphenols has been found to enhance their antimicrobial activity [Bouarab- Chibane, L.; Forquet, V.; Lanteri, P.; Clement, Y.; Leonard-Akkari, L.; Oulahal, N.; Degraeve, P.; Bordes, C. Antibacterial properties of polyphenols: Characterization and QSAR (Quantitative Structure-Activity Relationship) models. Front. Microbiol. 2019, 10, 829], Furthermore, the antimicrobial potential of polyphenols has been shown to be particularly effective against antibiotic-resistant strains of bacteria, such as methicillin-resistant S. aureus [Negut, I.;Grumezescu, V.; Grumezescu, A.M. Treatment strategies for infected wounds. Molecules 2018, 23, 2392],Definitions

[0036] 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 knowing the factors influencing the local physiological environment. Many local conditions influence wound occurrence, persistence, and healing. Skin is made up of at least three layers: the epidermis, the dermis, and the hypodermis. A wound can be a breakage in the continuity of any one of or any combination of the epidermis, the dermis, and / or the hypodermis. Examples of a wound include injury to living tissue caused by a cut, laceration, impact, or other physical disruption.

[0037] “Polyphenols” as used herein refer to organic chemicals that include more than one phenol structural units. The main bioactive polyphenols in lettuce include: phenolic acid derivatives (e.g., hydroxybenzoic acid derivatives, hydroxyphenylacetic acid derivatives), coumarin derivatives (e.g., aesculin, 6,7-Dihydroxycoumarin), flavonoids (e.g., quercetin glycosides, kaempferol glycosides, luteolin glycosides, apigenin glycosides, flavanones, flavones, and the likes), and anthocyanins (e.g., cyanidin glycosides and the likes). In preferred embodiments, the bioactive polyphenols disclosed herein include anthocyanins, chlorogenic acid, chicoric acid, dicaffeoylquinic acid, and quercetin derivatives (quercetin glycosides).

[0038] “Powder” as used herein refers to fine, dry particles produced by the grinding, crushing, or disintegration of a solid substance.

[0039] “Particle” as described herein refers to a small localized composition of matter that has a set of chemical and physical properties. Generally, a particle is about 0.01 pm to about 150 pm in one or more of width, length, and height.

[0040] As used herein “nano-sized particle” or a “nanoparticle” are used interchangeably and refer to a particle that is less than 0.1 pm.

[0041] As used herein “eustressor” and “elicitor” are used interchangeably and refer to various biological, physical or chemical stressful factors that trigger the signaling pathways leading to a higher bioactive compounds content and quality attributes of plant products. Eustressors / elicitors can be classified as biotic and abiotic substances, examples of which are described in WO 2022 / 183014. Plant hormones / plant growth regulators e.g., salicylic acid (SA), jasmonates, etc.) are also considered as eustressors / elicitors. Eustressors / elicitors of biological, chemical, or physical origin may increase plant agronomic / nutrition traits due to theactivation of responses that could include defense responses among them, leading to an increase of functional quality of, e.g., fruits and vegetables. Plant growth regulators (PGRs) can be used as eustressors / elicitors to stimulate the production of plant secondary metabolites. Plant growth regulators can include hormonal substances of natural occurrence (phytohormones) as well their synthetic analogues.

[0042] “Plant growth regulators” (PGRs) refer to chemicals used to modify plant growth such as increasing branching, suppressing shoot growth, increasing return bloom, removing excess fruit, or altering fruit maturity. PGRs may also be referred to as plant hormones or elicitors. PGRs may include auxins, cytokinins (CKs), gibberellins (GAs), ethylene, brassinosteroids, jasmonates (JAs), strigolactones (SLs), and salicylic acid (SA).

[0043] “Lettuce” refers herein to plants of the species Lactuca sativa L. Lactuca sativa is in the Cichorieae tribe of the Asteraceae (Compositae) family. Lettuce is related to chicory, sunflower, aster, dandelion, artichoke, and chrysanthemum. L. sativa is one of about 300 species in the genus Lactuca. As a highly polymorphic species, L. sativa is grown for its edible head and leaves. As a crop, lettuce is grown commercially anywhere environmental conditions permit the production of an economically viable yield. Fresh lettuce is consumed nearly exclusively as a fresh, raw product and occasionally as a cooked vegetable. Lettuce is an increasingly popular crop. Lettuce consumption continues to increase worldwide. Due to its high demands, there are benefits to seeking an increase in the production of polyphenols for new transgenic lettuces. In particular, improved transgenic lettuce with enhanced production of healthy polyphenols that are stable, high yielding, and agronomically sustainable will particularly be commercially viable for human consumption.

[0044] “Lettuce plant” refers to an immature or mature whole lettuce plant, including a lettuce plant from which seed, roots or leaves have been removed. Seed or embryo that will produce the plant is also considered to be the lettuce plant. Lettuce plants can be produced by seeding directly in the ground (e.g., soil such as soil on a field) or by germinating the seeds in a controlled environment condition (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 which are all herewith incorporated by reference.

[0045] “Lettuce cell” or “lettuce plant cell” refers to a cell as used herein which includes a lettuce plant cell, whether isolated, in tissue culture, or incorporated in a lettuce plant or lettuce plant part.

[0046] “Lettuce plant parts” as used herein includes lettuce heads, lettuce leaves, parts of lettuce leaves, pollen, ovules, flowers, and the like. In another embodiment, the present disclosure is further directed to lettuce heads, lettuce leaves, parts of lettuce leaves, flowers, pollen, and ovules isolated from lettuce plants.

[0047] The term “variety” or “cultivar” means a plant grouping within a single botanical taxon of the lowest known rank, which grouping, irrespective of whether the conditions for the grant of a breeder’s right are fully met, can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, distinguished from any other plant grouping by the expression of at least one of the said characteristics and considered as a unit with regard to its suitability for being propagated unchanged.

[0048] “Plant” includes the whole plant or any parts such as plant organs (e.g., harvested or non-harvested leaves, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, plant callus, plant cell clumps, plant transplants, seedlings, plant cells that are intact in plants, plant clones or micropropagations, or parts of plants (e.g., harvested tissues or organs), such as plant cuttings, vegetative propagations, embryos, pollen, ovules, flowers, leaves, heads, seeds, clonally propagated plants, roots, stems, stalks, root tips, grafts, parts of any of these and the like, or derivatives thereof, preferably having the same genetic make-up (or very similar genetic make-up) as the plant from which it is obtained. Also any developmental stage is included, such as seedlings, cuttings prior or after rooting, mature and / or immature plants or mature and / or immature leaves.

[0049] 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 a genome of a recombinant organism, such that it is not associated with 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 from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example, a variant of a naturally occurring gene is recombinant.

[0050] As used herein, “heterologous” in reference to a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form incomposition 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 / analogous species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0051] “Transgene” as used herein refers to a gene or genetic transferred into the genome of a lettuce plant, for example by genetic engineering methods, such as by transformation. Exemplary transgenes include cDNA (complementary DNA) segment, which is a copy of mRNA (messenger RNA), and the gene itself residing in its original region of genomic DNA. In one example, describes a segment of DNA containing a gene sequence that is introduced into the genome of a lettuce plant or lettuce plant cell. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic lettuce plant, or it may alter the normal function of the transgenic plant’s genetic code. In general, the transferred nucleic acid is incorporated into the plant’s germ line. Transgene can also describe any DNA sequence, regardless of whether it contains a gene coding sequence or it has been artificially constructed, which has been introduced into a lettuce plant or vector construct in which it was previously not found.

[0052] “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (z.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or noncontiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional coding sequence / gene to be co-transformed into the organism. Alternatively, the additional coding sequences / gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of a coding polynucleotide of interest or active variant or fragment thereof to be under the transcriptional regulation of the regulatory regions (e.g., promoter). The expression cassette may additionally contain selectable marker genes.

[0053] “Expression cassette” refers a polynucleotide encoding a polypeptide of interest operably linked to at least one polynucleotide encoding an expression control sequence. The expression cassette can include in the 5 '-3' direction of transcription, a transcriptional and translational initiation region (z.e., a promoter), polynucleotide encoding a polypeptide of interestor active variant or fragment thereof, and a transcriptional and translational termination region (z.e., termination region) functional in plants. The regulatory regions (z.e., promoters, transcriptional regulatory regions, and translational termination regions) and / or the polynucleotide or active variant or fragment thereof may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or the polynucleotide of or active variant or fragment thereof may be heterologous to the host cell or to each other.

[0054] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (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, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et rz / .(1991) Virology 81 :382-385. See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0055] “Expression control sequence” refers to a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of a polypeptide encoded by the expression cassette. Examples of expression control regions include promoters, transcriptional regulatory regions, and translational termination regions. The termination region may be native with the transcriptional initiation region, may be native with the operably linked polynucleotide or active variant or fragment thereof, may be native with the plant host, or may be derived from another source (z.e., foreign or heterologous) to the promoter, the polynucleotide or active fragment or variant thereof, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid 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.

[0056] A “control” provides a reference point for measuring changes in phenotype of a subject. For example, a control for wound healing or closure can be a comparable wound that does not received an active ingredient that promotes wound healing. The control wound may receive treatment with a vehicle or dressing that does not contain the active ingredient that promotes wound healing. A control wound may be present on the same subject or on a different, yet comparable subject.

[0057] A “control lettuce” or “control lettuce cell” provides a reference point for measuring changes in phenotype of the subject lettuce plant or lettuce plant cell, and may be any suitable lettuce plant or lettuce cell. A control lettuce or lettuce cell may comprise, for example: (a) a wild-type or native lettuce or lettuce cell, z.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject lettuce or lettuce cell; (b) a lettuce or lettuce cell of the same genotype as the starting material but which has been transformed with a null construct (z.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a lettuce or lettuce cell which is a non-transformed segregant among progeny of a subject lettuce or lettuce cell; (d) a lettuce or lettuce cell which is genetically identical to the lettuce or lettuce cell but which is not exposed to the same treatment (e.g., eustressor / elicitor treatment, herbicide treatment) as the subject lettuce or lettuce cell; or (e) the subject lettuce or lettuce cell itself, under conditions in which the gene of interest is not expressed.

[0058] An “effective amount” or a “therapeutically effective amount” may refer to an amount of 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 may be, for example, an increase in wound closure, a decrease in inflammation associated with a wound, an increase in cell proliferation, and / or an increase the formation and / or regeneration of vascular tissue.

[0059] In the present description, the term “about” means + 20% of the indicated range, value, or structure, unless otherwise indicated. The term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed embodiment. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include” and “have” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.The term “comprise” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0060] Recombinant DNA, molecular cloning, and gene expression techniques used in the present disclosure are known in the art and described in references, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, New York, 2001, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD, 1999. All documents (e.g., patent publications) are herein incorporated by reference in their entirety.

[0061] Various modifications and variations of the described products and methods of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with specific preferred embodiments, it should be understood that the present disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present disclosure which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.Polyphenol-Enriched Powders

[0062] In some embodiments, the present disclosure provides a powder comprising particles of a polyphenol-enriched lettuce powder or extract. The polyphenol-enriched lettuce has increased production and concentration of at least one polyphenol relative to control lettuce. Representative bioactive components identified from such red lettuce include flavonoids, especially their highly oral-bioavailable derivatives (quercetin-3-O-glucose and quercetin-3-O- malonylglucoside) (Figs. 1A-1C), chlorogenic acids: 3-caffeoylquinic acid (3-CQA), 3,4- Dicaffeoylquinic acid (3,4-diCQA) (Figs. 2A and 2B), chicoric acid (Fig. 3), and anthocyanins (e.g., Cyanidin-3-O-glucoside and equivalents) (Fig. 4A and 4B). In some embodiments, the polyphenol is at least one of a chlorogenic acid, a neochlorogenic acid, a chicoric acid, a quercetin, a quercetin derivative, an anthocyanin, or any combination thereof. In some embodiments, the polyphenol-enriched lettuce has an increased production and concentration of chlorogenic acid, chicoric acid, quercetin derivatives, and anthocyanins. In some embodiments, 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 anycombination thereof. In some embodiments, the quercetin derivative comprises quercetin-3-O- glucoside (Q3G), quercetin glucuronide, quercetin-3-O-malonylglucoside (Q3MG), or all of them. In some embodiments, the anthocyanin comprises cyanidin 3 -galactoside, cyanidin-3-O- glucoside, cyanidin-3-6”-malonylglucoside, or any combination thereof. In some embodiments, the polyphenol comprises 4-CQA, neochlorogenic acid, chicoric acid, and cyanidin 3- galactoside. In some embodiments, the increased production of polyphenols is quantified by LC- MS. In some embodiments, the increased production of polyphenols is quantified by HPLC. Methods for producing polyphenol-enriched lettuce are described in WO 2022 / 183014, incorporated by reference herein in entirety.

[0063] The compositions and methods disclosed herein are applicable to at least four tissues of the human and animal bodies, providing protection, repair, and treatment for the following purposes: epithelial tissue (e.g., wounds, skin disorders, and hair); muscle tissue (e.g., sarcopenia and muscle damage); connective tissue (e.g., repair and regeneration); and nervous tissue (e.g., peripheral neuropathy and nerve damage).

[0064] In certain embodiments, the polyphenol-enriched lettuce was treated with at least one eustressor / elicitor, or a homologue, isomer, or derivative thereof, that increases the production of polyphenols in lettuce. For example, the at least one eustressor / elicitor can be an abiotic eustressor / elicitor selected from: indole-3 -acetic acid (IAA), auxins, cytokinins (CKs), gibberellins (GAs), ethylene, brassinosteroids, jasmonates (J As), strigolactones (SLs), salicylic acid (SA), arachidonic acid (AA), 5-aminolevumic acid (5-ALA), oxalic acid, and any homologues or isomers or derivatives, synthetic analogues, or any combination or mixture thereof. In some embodiments, the polyphenol-enriched lettuce comprises a 2-fold to 20-fold increased production of polyphenols compared to the control lettuce, optionally a 3 -fold to 9-fold increased production of polyphenols compared to the control lettuce. In some embodiments, the concentrations of chlorogenic acid, quercetin derivatives, chicoric acid, and anthocyanins are increased by at least 3-fold, at least 4-fold, at least 5-fold, 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.

[0065] In some embodiments, the polyphenol-enriched lettuce powder or its extract contains a total polyphenol concentration is 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 the quercetinderivatives is about 18-175 mg / g. In some embodiments, the concentration of anthocyanins is about 6-20 mg / g.

[0066] Particle size distribution (PSD) is an important characteristic of pharmaceutical products across all types of preparation, including solid oral drug products, semi-solids, aerosols, and sterile liquid products. Ball milling, conical milling, and hammer milling can be used to regulate particle size and shape, and achieve consistent tight particle size distributions, leading to quality products and efficient production systems. The choice of whether to use ball milling, conical milling or hammer milling will be determined by several factors including an evaluation of the type of product that is needed to be processed, and the results desired.

[0067] In topical and transdermal drug delivery, particle size plays an important role in determining the pharmacologic properties of the product. For example, when the API is suspended in a vehicle, like a gel or cream, particle size interacts with the solubility of the vehicle to determine the dissolution rate. Particle size also affects the rate, depth, and pathway of absorption through the skin. Ultimately, the choice of particle size in topical preparations can help to maximize drug efficacy at the site of action and minimize the risk of systemic adverse reactions.

[0068] In some embodiments, the preferred particle size range of the powder is about 0.1 to about 150 pm. Although a well-defined powder could range in size from about 0.1 pm to about 10,000 pm. Suitable D50, D90 for the biologically active lettuce powder or its extract will be defined and achieved in specific formulation. In some embodiments, the polyphenol-enriched red lettuce powder comprises a nanoparticle powder. As used herein, a nanoparticle is having a particle distribution size range of about 0.1 pm or less. In some embodiments, the polyphenol- enriched red lettuce powder has a particle distribution size range of about 0.01 pm to about 0.1 pm. In some embodiments, the polyphenol-enriched red lettuce powder has a particle distribution size range is about 0.05 pm to about 0.1 pm.

[0069] An exemplary method to define the particle size of a polyphenol-enriched lettuce powder or its extract is by mesh size. The term “mesh size” refers to the fineness or coarseness of a material as determined by the ability of a particle to pass through a mesh. Mesh size is related to mesh number, and is commonly defined as the number of mesh openings in a sieve screen within a 1-inch by 1-inch area. To determine the mesh number, for example, one may count the number of openings in one linear inch of the screen. For instance, a 4-mesh screen has four square openings across one inch of the screen, while a 100-mesh screen has 100 openings per inch. As the mesh number increases, the size of the openings in the mesh decreases,resulting in smaller particle sizes that can be captured by the screen. The mesh size indicates the particle size of a material that can pass through the corresponding mesh opening. For example, a mesh size of 200 means that the material can pass through a sieve screen with 200 openings within a 1-inch by 1-inch area. Similarly, as the mesh size increases, it indicates a finer particle size, while a smaller mesh size indicates a coarser particle size. Thus, mesh sizes may be used to determine the fineness or coarseness of particles, rather than directly measuring the size of the material particles. However, it is important to note that the mesh size is not an exact measurement of the mesh opening size, as screens can be made using different materials with different strand or wire thicknesses. The thickness of the strands used in the screen affects the size of the openings, and therefore the particle sizes that can pass through.

[0070] By using different mesh sizes in a powder mixture, the 3D proportional formula can achieve the following functionalities. First, using a combination of different mesh sizes in the formula allows for increased contact surface area between the particles. This can enhance the inter-particle bonding and improve the overall strength and integrity of the printed object. Second, the inclusion of different mesh sizes promotes improved air convection between the powder particles during the printing process. This improved airflow can help in reducing the risk of particle agglomeration and ensure uniform powder distribution, resulting in better print quality and consistency. Third, by adjusting the mesh sizes in the formula, it is possible to control the adhesive or cohesive properties of the powder particles. This can be beneficial in achieving the desired flowability of the powder during printing. Lowering the particle adhesion can prevent clogging or clumping, while increasing the particle cohesion can enhance the adhesion between layers, improving the structural integrity of the powder.

[0071] The polyphenol-enriched lettuce powder or its extract as disclosed herein, possesses powerful anti-inflammatory and antioxidant properties. Therefore, it can be utilized in different cosmetic formulations, such as sunscreens, facial cleansers, facial masks, and conditioners, among others. This incorporation can provide various advantages for the skin, such as anti-aging effects, protection against radiation, skin lightening, hydration, reduction of freckles, acne treatment, and stimulation of hair growth. The polyphenol-enriched lettuce powder or its extract may be used to create different formulations such as powder, aqueous solution, spray, gel, oil-based, cream, surfactant, and ointment for specific purposes in the four major human tissues. For example, the polyphenol-enriched lettuce powder or its extract may be added to soap, shampoo, conditioner, hair dye, body wash, and cleaning products to enhance their functionality. In some examples, the polyphenol-enriched lettuce powder or its extract maybe dissolved in surfactants, mixtures, or solvents. The disclosed polyphenol-enriched lettuce powder or its extract may also provide antioxidant functionality. The polyphenol-enriched lettuce powder or its extract may also be mixed with powdered antibiotics to enhance their sterilization and bacteriostatic effects. In other embodiments, the polyphenol-enriched lettuce powder or its extract may be combined with collagen, seaweed extract, P-glucan, carboxymethyl cellulose (CMC), and vitamin D3 to create dressings that promote wound healing. In other embodiments, the polyphenol-enriched lettuce powder or its extract may be combined with hyaluronic acid, collagen, and CMC to form a base of products usable for treating skin disorder or for skin care. In other embodiments, the polyphenol-enriched lettuce powder or its extract may be combined with bacteria-reduced glycerin as an emulsion base. In other embodiments, the polyphenol-enriched lettuce powder or its extract may be combined with colloids and diluents for protection and repair of skin and soft tissue. In yet other embodiments, the polyphenol-enriched lettuce powder or its extract may be combined with paraffin or glycerin for protection and repair of skin and soft tissue.

[0072] The disclosed polyphenol-enriched lettuce powder or its extract herein may conveniently be administered in a pharmaceutical composition containing biologically active compositions in combination with a suitable excipient. Such pharmaceutical compositions can be prepared by methods and contain excipients which are well known in the art. Such methods and ingredients may be found in Remington’s Pharmaceutical Sciences [Alfonso Gennaro et al., eds., Nov. 8, 2012 Lippincott, Williams & Wilkins, Baltimore, Md., 20” ed., 2000], Formulations and compositions of the present invention can be administered topically, orally, or rectally.

[0073] For topical administration, formulations may contain polyphenol-enriched lettuce powder or its extract with high polyphenol concentration as active ingredients. Compositions according to the disclosure can conveniently be formulated using one or more pharmaceutically acceptable carriers or excipients, which may be a solid or liquid. Such compositions may take the form of, for example, ointments, lotions, creams, powders, liquids, gels, oils, surface tension agents, extracts, drops (e.g. eye, or ear drops) or sprays. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 100% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0074] In some embodiments, the polyphenol-enriched lettuce powders or extract disclosed herein may be in the form of creams or ointments. Ointments may normally beprepared by melting white soft paraffin, (white petrolatum) incorporating any additives e.g., surfactants and solvents, and blending in a slurry of the drug in a minimum quantity of liquid paraffin. The melt is then cooled under controlled conditions and stirred until solidification occurs. Creams may normally be prepared by combining the oily phase of an ointment as a melt as described above, with suitable oil and water-soluble surfactants and an aqueous phase containing the drug and suitable anti-microbial preservatives, homogenizing to form the cream and stirring gently until cool.

[0075] Compositions containing the polyphenol-enriched lettuce powder or its extract as disclosed herein may generally contain additional excipients, for example preservatives (such as benzoic acid), emulsifying agents (such as polysorbates, e.g. polysorbate 60), and viscosity enhancing agents (such as cetostearyl alcohol).

[0076] The biologically active compositions can be mixed into soap, shampoo, conditioner, hair dye, body wash, and cleaning products to enhance their functions. The composition can be mixed with antibiotics to enhance antimicrobial activities. Additionally, it can be mixed materials such as hyaluronic acid, collagen, alginate, P-glucan, carboxymethyl cellulose (CMC), and vitamin D3 to make dressings that promote wound healing properties.

[0077] Accordingly, in some embodiments, disclosed herein is a topical formulation comprising the polyphenol-enriched lettuce powder or its extract described above and, in the examples, below, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the polyphenol-enriched lettuce powder or its extract is present in the topical formulation at a percentage to the final weight of the topical formulation of about 0.1 %- 10%, about 0.5%-5%, about 1%- 10%, about 5%-20%, about 10%-50%, or about 50%-90%. In some embodiments, the pharmaceutically acceptable carrier or excipient is a solid or a liquid. In some embodiments, the topical formulation is in the form of an ointment, a lotion, a cream, a powder, a liquid, a gel, a hydrogel, an oil, a surface tension agent, a drop, an aerosol, an emulsion, nano-emulsion, nanoliposome, gel, micro-encapsulation, paste, or foam. In certain embodiments, the topical formulation is in the form of a sunscreen, a facial cleanser, a facial mask, a soap, a shampoo, a conditioner, body wash, or a hair dye. In some embodiments, the topical formulation further comprises an emollient, for example, paraffin. In some embodiments, the topical formulation further comprises an anti-microbial preservative. In some embodiments, the topical formulation further comprises one or more of an emulsifying agent and / or a viscosity enhancing agent.

[0078] In some embodiments, the topical formulation disclosed herein comprises by % weight / weight:1-10% of the polyphenol-enriched lettuce powder or its extract;5-15% of an alcohol;5-15% of an emollient;1-5% of an emulsifying agent;5-15% of propylene glycol; less than 1% of an anti-microbial preservative; and a remaining percentage of water to equal 100%.

[0079] In some embodiments, provided here in a wound dressing powder comprising the polyphenol-enriched lettuce powder or its extract described above and, in the examples, below, and a powdered pharmaceutically acceptable carrier or excipient. Examples of a pharmaceutically acceptable carrier or excipient include a starch, a cellulose, a synthetic polymer, a polysaccharide, a chitosan, a mineral powder, a clay powder, or any combination thereof. In some embodiments, the starch is a cornstarch, potato starch, rice starch, or any combination thereof. In some embodiments, the clay is bentonite, kaolin, smectite, or any combination thereof. In some embodiments, the cellulose is microcrystalline cellulose, sodium carboxymethylcellulose (CMC), or any combination thereof. In some embodiments, the synthetic polymer is a polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), or a combination thereof. In some embodiments, the polysaccharide is calcium alginate. In some embodiments, the mineral powder is zinc oxide powder.

[0080] In certain embodiments the wound dressing powder comprises: i. 50%-90% wt / wt mineral powder, ii. 5%-l 5% wt / wt clay powder; and iii. 1%-15% wt / wt polyphenol-enriched lettuce powder or its extract; and, optionally iv. an essential oil, e.g., Lavender essential oil.

[0081] In some embodiments, disclosed herein are the nanoparticle delivery methods mediated by a hydrogel. Due to the unique properties of hydrogels, they have the potential to enhance the bioavailability, solubility, and stability of the polyphenol-enriched lettuce powder for medical applications. Moreover, hydrogels can be decorated with specific targeting ligands, which guide the payload to the precise site of action [Khalvati, B.; Sheikhsaran, F.; Sharifzadeh, S.; Kalantari, T.; Behzad Behbahani, A.; Jamshidzadeh, A.; Dehshahri, A. Delivery of plasmid encoding interleukin- 12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles. Artificial cells, nanomedicine, and biotechnology 2017, 45 (5), 1036-1044], Thistargeted delivery approach can reduce side effects while increasing pharmacological activity at the target site [Sheikhsaran, F.; Sadeghpour, H.; Khalvati, B.; Entezar-Almahdi, E.; Dehshahri, A. Tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding IL-12 gene. Colloids Surf., B 2017, 150, 426-436], Additionally, stimuli-responsive hydrogels offer the opportunity to precisely control the transfer of the payload [McKenzie, M.; Betts, D.; Suh, A.; Bui, K.; Kim, L. D.; Cho, H. Hydrogel-based drug delivery systems for poorly water-soluble drugs. Molecules 2015, 20 (11), 20397-20408], The use of hydrogels as a delivery vehicle for the polyphenol-enriched lettuce powder also allows for the application of the drug through different routes of administration, such as oral, topical, nasal, or parenteral.

[0082] Accordingly, in some embodiments provided herein is a hydrogel comprising the polyphenol-enriched lettuce powder or its extract disclosed herein and a pharmaceutically acceptable hydrogel polymer. In some embodiments, the powder is present at a percentage to the final weight of the topical formulation of about 0.1%-10%, about 0.5%-5%, about 1%-10%, about 5%-20%, or about 10%-50%. In some embodiments, the polyphenol-enriched lettuce powder or its extract comprises particles having a particle distribution size range of 0.1 pm to 150 pm. In some embodiments, the polyphenol-enriched lettuce powder or its extract comprises or consists essentially of nanoparticles. In some embodiments, the nanoparticles have a particle distribution size range is 0.1 pm or less, optionally the particle distribution size range is 0.01 pm to 0.1 pm, or 0.05 pm to 0.1 pm. In some embodiments, the hydrogel polymer is at least one of a chitosan, gelatin, collagen, polysaccharide, starch, alginate, or agarose. In some embodiments, the hydrogel further comprises a polymer surfactant, for example, pluronic P123. In some embodiments, the hydrogel further comprises a targeting ligand that targets a site of action.

[0083] For oral therapeutic administration, the polyphenol-enriched lettuce powder or its extract disclosed herein may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. Such compositions and preparations should contain at least 1% of active compositions. For example, the polyphenol-enriched lettuce powder may be present at a percentage to the final volume of the oral formulation of about 0. l%-10%, about 0.5%-5%, about l%-10%, about 5%-20%, or about 10%-50%. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactoseor aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or Sugar and the like. A syrup or elixir may contain the active compound. Sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. The active composition may also be incorporated into sustained-release preparations and devices.

[0084] The topical formulations of the present disclosure can be absorbed via a transmembrane route including, for example, sublingual, nasal mucosal, rectal mucosal, and / or intestinal routes. In a preferred embodiment, the topically formulation may be applied locally in the oral cavity, for treating localized disorders such as oral ulcers, periodontitis, and plaque.

[0085] In some embodiments, wound healing may be facilitated by combining topical administration with localized oral administration and photodynamic therapy. For example, after applying the topical formulation to the wound area and orally administering the oral formulation, the wound area may be illuminated with blue light having wavelengths ranging from 400 nm to 520 nm (with a peak wavelength of 440 nm). This blue light radiation can produce a large amount of reactive oxides (ROS) to facilitate wound sterilization. At the same time, the polyphenols in the oral formulation can protect normal human tissue cell and avoid free radical damage caused by the irradiation. In some embodiments, illuminating the wound area with red light having wavelength ranging from 617 to 850 nm (with peak wavelengths of 660 nm and 720, 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. Additionally, through the regulation of Macrophage M1 / M2 polarization, the degree of inflammatory response can also be reduced.Methods of Making Extracts from Polyphenol-Enriched Lettuce Powder

[0086] In some embodiments, the present disclosure provides an extract of a polyphenol- enriched lettuce powder. 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 polyphenol-enriched lettuce powder orits extract contains a total polyphenol concentration is about 40-280 mg / g. In some embodiments, the concentration of chi orogenic 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 the quercetin derivatives is about 18-175 mg / g. In some embodiments, the concentration of anthocyanins is about 6-20 mg / g.

[0087] In some embodiments, disclose herein is a method of making an extract from a polyphenol-enriched lettuce from which the polyphenol-enriched lettuce powder is formed. FIG. 5 is a flowchart of a method 500 for preparing the extract from the polyphenol-enriched lettuce, in accordance with some embodiments of the present disclosure. It is understood that additional steps can be provided before, during, and after method 500, and some of the steps described below can be replaced or eliminated, for additional embodiments of the method.

[0088] Referring to FIG. 5, the method 500 includes step 502, in which the polyphenol- enriched lettuce is ground to form a polyphenol-enriched lettuce powder. In some embodiments, leaves or any above-ground parts of the lettuce may be first dried for example, by air drying or freeze drying. Next, the dried lettuce may be chopped into smaller pieces and then pulverized, for example, using ball milling, conical milling or hammer milling to form the polyphenol- enriched lettuce powder. The size of the polyphenol-enriched lettuce powder is then classified according to the mesh size. In some embodiments, the polyphenol-enriched lettuce powder may 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-enriched lettuce powder may have a particle size ranging from 0.1 to 10, 000 pm, for example, from 0.1 to 150 pm.

[0089] In step 504 of the method 500, the polyphenol-enriched lettuce powder is subjected to extraction. In some embodiments, the extraction may be carried out using a solvent extraction method by immersing the polyphenol-enriched lettuce powder into an extraction solvent. In some embodiments, the extraction solvent may be water, alcohol, a mixture of water and alcohol, or a supercritical fluid. Examples of alcohols for the 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. Additionally, the extraction solvent is a mixture of water and ethanol, with ethanol comprising about 50% to about 90% of the mixture. In some embodiments, the extraction solvent is a supercritical fluid, such as supercritical carbon dioxide (CO2). More polar extraction solvents, for example, alcohol, water, or their mixtures, yield extracts containing higher amounts of polyphenols, while non-polar extraction solvents likesupercritical CO2 produce extracts with less polyphenol content and enriched with non-poplar compounds such as terpenes, carotenoids, vitamins, and so on.

[0090] In some embodiments, the ratio of polyphenol-enriched 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-enriched lettuce powder to solvent (g / mL) is 2:5.

[0091] The extraction may be carried out at temperatures 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.

[0092] The duration of the extraction process may 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 hour to about 3 hours, from about 3 hour to about 4 hours, from about 4 hour to about 5 hours, from about 5 hour to about 6 hours, from about 6 hour to about 10 hours, from about 10 hour to about 18 hours, or from about 18 hour to about 24 hours.

[0093] The extraction may be carried out with agitation, stirring, shaking, or a combination thereof, under pressure or atmospheric conditions. In some embodiments, the extraction may be assisted by ultrasound. Ultrasound applies intense shear forces and stresses to the polyphenol-enriched lettuce powder and extraction solvent, which helps accelerate the penetration of the extraction solvent into the polyphenol-enriched lettuce powder. Ultrasound thus can improve extraction efficiency and shorten extraction time. The ultrasound may be pulsed. In some embodiments, the ultrasound may have a pulse duration ranging from 100 ps to 1 ms, for example, from 100 ps to 100 ns, from 100 ns to 500 ns, from 500 ns to 1 ps, from 1 ps to 10 ns, from 10 ps to 500 ps, or from 500 ps to 1 ms; a pulse power ranging from 1 kW to 50 kW, for example, from 1 kW to 5 kW, from 5 kW to 10 kW, from 10 kW to 20 kW, or from 20 kW to 50 kW; and a frequency ranging from 100 kHz to 200 MHz, for example, from 500 kHz to 25 MHz, from 500 kHz to 200 MHz, from 1 MHz to 5 MHz, from 1 MHz to 7 MHz, from 1 MHz to 10 MHz, from 1 MHz to 20 MHz, from 1 MHz to 25 MHz, from 1 MHz to 30 MHz, from 1 MHz to 200 MHz, from 2 MHz to 5 MHz, from 2 MHz to 10 MHz, or from 2 MHz to 200 MHz. In some embodiments, the ultrasound is generated using an ultrasonic transducer or ultrasonic horn.

[0094] Once the extraction process is completed, 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 may be mixed with calcium magnesium phytate for use as animal feeds or plant fertilizers.

[0095] In some embodiments, before immersing into the extraction solvent, the pulverized lettuce may be pre-mixed with a solvent to form a slurry. The suitable solvent may be water, ethanol, or a mixture thereof.

[0096] In step 506 of the method 500, the liquid portion is dried, for example, by freeze drying to remove the solvent, thus obtaining the polyphenol-enriched extract in powder form. The drying may be carried out at temperatures ranging from about 35 °C to about 70 °C, such as, for example, from about 35 °C to about 40 °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, or from about 65 °C to about 70 °C. After drying, calcium magnesium phytate may be added to prevent clumping and extend the shelf life of the extract.

[0097] In some embodiments, the extract may be mixed with the lettuce residue so that the active ingredients in the extract can be attached to the surface of the lettuce residue. When used internally, the mixture helps enhance the absorption of polyphenols by the intestinal tract due to the higher fiber content in the lettuce residue. On the other hand, when used externally, the mixture helps promote wound healing due to increased ventilation provided by the lettuce residue.Methods and Uses

[0098] In some embodiments, disclosed herein is a method of treating a wound, the method comprising administering to a wound site of a subject a therapeutically effective amount of the polyphenol-enriched lettuce powder or its extract disclosed herein, the topical formulations disclosed herein, the wound dressing powders disclosed herein, the hydrogels disclosed herein, and / or the oral formulation disclosed herein. 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.

[0099] Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art (for example, see U.S. Pat. No. 4,938.949).

[0100] In some embodiments, provided herein is a method of treating a skin disorder, the method comprising administering topically to the disorder site of a subject a therapeutically effective amount of the polyphenol-enriched lettuce powder or its extract disclosed herein, the topical formulations disclosed herein, the wound dressing powders disclosed herein, the hydrogels disclosed herein, and / or the oral formulations disclosed herein. The skin disorder generally refers to any skin-associated disease, for example, inflammation, rash, dermatitis, atopic dermatitis, eczema, psoriasis, dandruff, acne, cellulitis, rosacea, warts, seborrheic keratosis, actinic keratosis, tinea versicolor, viral exantham, shingles, ringworm, and cancer, such as basal cell carcinoma, squamous cell carcinoma, and melanoma. Skin disorders that are treated with the polyphenol-enriched lettuce powder or its extract, the topical formulations, the wound dressing powders, the hydrogels, and / or the oral formulations disclosed herein include, but are not limited to, skin ulcers, bedsores, diabetic skin sores, hypertrophic scars, keloid scars, telangiectasia (spider veins), skin atrophy, premalignant skin lesions, herpes, inflammatory acne, acne vulgaris, comedonic or polymorphic acne, nodulocystic acne, acne conglobata, senile acne and secondary acne such as solar, drug or occupational acne, ichthyoses, ichthyosiform conditions, Darier's disease, palmoplantar keratoderma, leukoplakia and leucoplakiform conditions or lichen and lichen planus, cutaneous, mucosal or ungual psoriases, psoriatic rheumatism, cutaneous atopy including eczema, dry skin, inflammation of the skin, red flushes, solar skin erythema, actinic keratosis, skin allergies and allergic or irritant contact dermatitis, atopic dermatitis, rosacea, hyperpigmentation, benign pigmented lesions (lentigines, freckles, brown spots, melasma), and aged skin.

[0101] Another aspect of the disclosure is a method for administering to a human or animal patient to provide an anti-inflammatory effect. For example, polyphenol-enriched lettuce powder or its extract has demonstrated anti-inflammatory effects both in vivo and in vitro. Samples tested demonstrated efficacy for inhibiting NF-kB activity. The polyphenol-enriched lettuce powder or its extract has also demonstrated the efficacy of antioxidant effect, as well as decreasing the levels or activity of a variety of cytokines known to contribute to inflammatory disease or diseases having a significant inflammatory component.

[0102] Accordingly, disclosed herein is 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 polyphenol-enriched lettuce powder or its extract disclosed herein, the topical formulations disclosed herein, the wound dressing powders disclosed herein, the hydrogels disclosed herein, and / or the oral formulations disclosed herein. In some embodiments,the production of inflammatory cytokines by immune cells is reduced compared to immune cells prior to treatment. In some embodiments, NF-kappaB activity is reduced. In some embodiments, the method comprises administering the composition to the skin, e.g., epithelial tissue. In some embodiments, the method comprises administering the composition to muscle tissue. In some embodiments, the method comprises administering the composition to connective tissue. In some embodiments, the method comprises administering the composition to nervous tissue.

[0103] In some embodiments, disclosed herein is a method of skincare, the method comprising administering to subject a therapeutically effective amount of the polyphenol- enriched lettuce powder or its extract disclosed herein, the topical formulations disclosed herein, the wound dressing powders disclosed herein, the hydrogels disclosed herein, and / or the oral formulations disclosed herein.

[0104] In any of the embodiments disclosed herein, the subject can be a human, a nonhuman primate, a mouse, a rat, a gerbil, a rabbit, a dog, a cat, a horse, a cow, a pig, a goat, a sheep, a donkey, a llama, an alpaca, a guinea pig, a mule, a deer, a buffalo, a chicken, a duck, a goose, or a turkey. Preferably, the subject is a human.

[0105] In some embodiments, disclosed herein is a method of preparing a nanoparticle powder, the method comprising: i. washing and drying at least one polyphenol-enriched red lettuce leaf; ii. milling the at least one red lettuce leaf using a milling tool to form a polyphenol-enriched lettuce powder; iii. passing the powder through a sieve to remove particles larger than the openings of the sieve; iv. grinding the powder to reduce the particle size of the powder to a particle distribution size range of 0.1 pm or less.

[0106] In some embodiments, the grinding is performed with a ball mill, a conical mill, a hammer mill, or a homogenizer. In some embodiments, the particle size distribution is 0.01 pm to 0.1 pm or 0.05 pm to 0.1 pm. In some embodiments, the drying temperature is 40-45°C.

[0107] In some embodiments, disclosed herein is a method of preparing a wound dressing powder, the method comprising: i. mixing a mineral powder and a clay powder to form a powder mixture; ii. adding polyphenol-enriched lettuce powder or its extract disclosed herein to the powder mixture;iii. blending the powder mixture; and iv. adding a pharmaceutically acceptable excipient, carrier, or diluent to the powder mixture.

[0108] 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.

[0109] In some embodiments, disclosed herein is a method of preparing a hydrogel, the method comprising: i. dissolving a chitosan powder in an acid solution to form a chitosan solution; ii. adding the polyphenol-enriched lettuce powder or its extract disclosed herein to the chitosan solution; iii. mixing a gelatin powder in water to create a gelatin solution; iv. mixing the chitosan solution comprising powdered extract with the gelatin solution; v. adding a crosslinking agent to the mixture of the chitosan solution and the gelatin solution; vi. pouring the mixture of v. into a hydrogel mold and setting the mixture to form a hydrogel; vii. removing the hydrogel from the mold; and viii. applying heat to the hydrogel to promote crosslinking.

[0110] In some embodiments, the acid solution comprises 1% acetic acid. In some embodiments, the method further comprises maintaining a pH of approximately 7.0 of the mixture when mixing the chitosan solution with the gelatin solution by adding a solution of NaOH. In some embodiments, setting the mixture comprises setting the mixture at room temperature for 2-3 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 crosslinking 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.

[0111] Upon reading the following description of specific embodiments of the invention in conjunction with the accompanying figures, those skilled in the art will be able to recognizeadditional aspects and features of the present invention. The following examples are provided for illustrative purposes only and should not be considered limiting. Experimental procedures used in the examples are also described. The following examples provide a further description of the invention, but are not intended to limit the scope of the invention.EXAMPLESEXAMPLE 1ENHANCEMENT OF POLYPHENOL PRODUCTION IN RED LETTUCE USING EUSTRESSOR / ELICITORS

[0112] This example demonstrates the increased production of polyphenols in red leaf lettuce when treated with biotic / abiotic eustressors / elicitors.Plant Materials, Growth Conditions and Eustressor / Elicitor Treatments

[0113] Lettuce plants (Lactuca sativa) of red varieties were grown in a lab greenhouse with an average photoperiod of 12 h / day, at 25-28°C, 40-60% relative humidity. Abiotic eustressor used were indole-3 -acetic acid (IAA), at 30, 60, and 120 mg / L. The eustressors were dissolved in deionized water (non-water dissolved ones were previously dissolved in 1 mL of ethanol). A group of samples and water with only 1 mL of ethanol were added. Control samples with no treatment were added. Elicitor treatments were applied on the 7th preharvest day on red lettuces. Each experimental unit consisted of five lettuces randomly selected and assigned to one treatment. Each sample was treated by rooting absorption or foliar aspersion, with 3 sprays of each elicitor (approximately 1.70 mL). Lettuce samples were harvested at 50 d.Extraction and Quantification

[0114] Major health beneficial polyphenols were characterized and quantified in treated and untreated (control) red lettuces after extracting samples with 50% of ethanol. Generally, two grams of the sample (grounded with liquid nitrogen) was mixed with 5 mL of ethanol, shaken for 4 hours at room temperature, and centrifuged at 5000*g for 10 min (4°C). The supernatant was collected, filtered, and subjected to LC-MS / HPLC analysis.Results

[0115] The enhanced production of polyphenols was confirmed using LC / MS / UV (or HPLC).

[0116] As shown in Fig. 6, the HPLC chromatograms of bioactive components enhancement by genomics-based technologies confirm the production of specific metabolites in red lettuce treated with biotic / abiotic eustressors. Polyphenols 3-CQA, chicoric acid, 3,4- dicaffeoylquinic acid (3,4-diCQA), Quercetin-3-O-glucoside (Q3G), Quercetin-3-O- malonylglucoside (Q3MG), show enhanced production in treated lettuce compared to nontreated lettuce control.

[0117] As shown in Figs. 7A-7B, the production of chlorogenic acids (Fig. 7A) and the water-soluble quercetin derivatives (Fig. 7B) were increased by 3- to 9-fold in red lettuce treated with elicitors / plant growth regulators. Chlorogenic acid and derivatives (3-CQA, chicoric acid, and 3,4-diCQA) and quercetin derivatives (Q3G and Q3MG) show enhanced production in treated lettuce compared to non-treated lettuce control.

[0118] These results demonstrate that treatment with biotic / abiotic eustressors increases in vivo polyphenol production in red lettuce.EXAMPLE 2MILLING PROCESS OF NANO-SIZED POLYPHENOL-ENRICHED LETTUCE POWDER

[0119] Provided herein is a process for producing dry nano-sized polyphenol-enriched lettuce powder using a milling technique:

[0120] Select fresh lettuce leaves and wash them thoroughly to remove any dirt or debris. Dry the leaves at 40-45°C.

[0121] Mill the dried leaves using a high-energy ball mill or other suitable milling equipment. The milling process should be conducted in a dry and cool environment to prevent the lettuce from becoming too hot and losing its biologically active ingredients.

[0122] After milling, sieve the lettuce powder through a fine mesh to remove any large particles or impurities.

[0123] Use a homogenizer or other suitable equipment to further reduce the particle size of the powder to the desired nano-size range. This may require multiple passes through the homogenizer or other equipment.

[0124] Finally, package the nano-sized lettuce powder in airtight containers and store them in a cool, dry place until ready to use.

[0125] It is important to note that the specific milling process may vary depending on the type of milling equipment used, the desired particle size range, and other factors.

[0126] As an example of the produced powder, the powder may include polyphenol- enriched lettuce powder combined with artificial additives, and have well-defined particle sizes and characterizations, as well as a 3D proportional formulation. Examples of artificial additives (e.g. preservatives), may be added to the powder, including as examples:(1) Non-inert: Sodium benzoate; Potassium sorbate; Nitrates and Nitrites; Sulfites; Propionic acid; Sodium erythorbate; Ascorbic acid.(2) Inert: Several inert additives can be added to the powder for better preservation. These include: a) Anticaking agents: These are added to prevent the clumping of food powders by absorbing moisture and reducing the surface tension of the powder particles. Examples include silicon dioxide, calcium silicate, magnesium carbonate, and talc; b) Stabilizers: These are added to prevent the separation of ingredients in the powder, such as oil and water. Examples include gum Arabic, xanthan gum, and carrageenan. c) Emulsifiers: These are added to improve the mixing and dispersion of ingredients in the powder, especially when there are components that do not easily dissolve or blend. Examples include lecithin and mono- and diglycerides. d) Humectants: These are added to prevent the powder from drying out and becoming hard, which can reduce its shelf life. Examples include glycerol and propylene glycol.

[0127] Characterization of particle sizes:(1) Particle size distribution (PSD): An important characteristic of pharmaceutical products across all types of preparation, including solid oral drug products, semisolids, aerosols, and sterile liquid products. Tightly controlled particle size distributions are highly important for pharmaceutical drug development.(2) Another method to define the particle size of polyphenol-enriched lettuce powder is by mesh size. Mesh size refers to the measurement of the mesh number, which is a US standard used to determine the relationship between the size of the openings in a mesh and the size of particles that can pass through these openings. To determine the mesh number, one must count the number of openings in one linear inch of the screen. For instance, a 4-mesh screen has four square openings across one inch of the screen, while a 100-mesh screen has 100openings per inch. As the mesh number increases, the size of the openings in the mesh decreases, resulting in smaller particle sizes that can be captured by the screen. However, it is important to note that the mesh size is not an exact measurement of the mesh opening size, as screens can be made using different materials with different strands or wire thicknesses. The thickness of the strands used in the screen affects the size of the openings, and therefore the particle sizes that can pass through.

[0128] Mesh size may be used to determine the particle size of powder particles instead of directly measuring their diameter. An example definition range of mesh sizes is as follows in Table 1 and Table 2.Table 1. Example range of mesh sizes (Mesh size 2-90)Table 2. Example range of mesh sizes (Mesh size 100-10000)

[0129] 3D Proportional Formulation (Mixtures of different pore sizes):

[0130] By using different pore sizes in the mixture, the 3D proportional formula can achieve the following functionalities:(1) Increase Contact Surface Area: Using a combination of different mesh sizes in the formula allows for increased contact surface area between the particles. This can enhance the inter-particle bonding and improve the overall strength and integrity of the printed object.(2) Improve Air Convection between Powder Particles: The inclusion of different mesh sizes promotes better air convection between the powder particles during the printing process. This improved airflow can help in reducing the risk of particle agglomeration and ensure uniform powder distribution, resulting in better print quality and consistency.(3) Control Particle Adhesion and Cohesion: By adjusting the mesh sizes in the formula, it is possible to control the adhesive or cohesive properties of the powder particles. This can be beneficial in achieving the desired flowability of the powder during printing. Lowering the particle adhesion can prevent clogging or clumping, while increasing the particle cohesion can enhance the adhesion between layers, improving the structural integrity of the powder.EXAMPLE 3PREPARATION OF EXTRACT FROM POLYPHENOL-ENRICHED LETTUCE POWDER USING SC-CO2

[0131] The polyphenol-enriched lettuce powder (400 mesh size) was placed in the extraction vessel. Make sure it is evenly distributed. Supercritical carbon dioxide (SC-CO2) was added into the mixture and SC-CO2 extraction of oil from the polyphenol-enriched lettuce powder was performed in an ultrasonic bath at an extraction pressure of 300 Bar, a temperature of 40 °C, and CO2 flow rate at 2 mL / min. After extracting for 120 min, the obtained mixture was centrifuged. The resulting liquid portion was freeze-dried to provide the extract in powder form. Calcium magnesium phytate was then added to the extract and the mixture was stored at 4 °C before use.EXAMPLE 4PREPARATION OF EXTRACT FROM POLYPHENOL-ENRICHED LETTUCE POWDER USING ETHANOL AND WATER MIXTURE

[0132] The polyphenol-enriched lettuce powder (400 mesh size) was placed in the extraction vessel. Extraction of polyphenols from the polyphenol-enriched lettuce powder was performed using ethanol / water (1 : 1 ratio) as extraction solvent in an ultrasonic bath at a temperature of 0 °C. After extracting for 30 min, the obtained mixture was centrifuged. The resulting liquid portion was rota-evaporated at 40 °C and then freeze-dried to provide the extract in powder form. Calcium magnesium phytate was then added to the extract and the mixture was stored at 4 °C before use.EXAMPLE 5PREPARATION OF EXTRACT FROM POLYPHENOL-ENRICHED LETTUCE POWDER USING WATER

[0133] The polyphenol-enriched lettuce powder (400 mesh size) was placed in the extraction vessel. Extraction of polyphenols from the polyphenol-enriched lettuce powder was performed using water as an extraction solvent in an ultrasonic bath at a temperature of 0 °C. After extracting for 40 min, the obtained mixture was centrifuged. The resulting liquid portion was freeze-dried to provide the extract in powder form. Calcium magnesium phytate was then added to the extract and the mixture was stored at 4 °C before use.EXAMPLE 6BASIC WOUND DRESSING POWDERING FORMULATION AND PREPARATION METHOD

[0134] Formulation:80% Zinc oxide powder;10% Bentonite clay powder;5% polyphenol-enriched lettuce powder / extract (400 mesh size); and 5% Lavender essential Oil.

[0135] Preparation Method:1. In a clean and dry container, add 80% zinc oxide powder and 10% Bentonite clay powder.2. Mix the powders thoroughly until well blended.3. Add 5% polyphenol-enriched lettuce powder / extract to the mixture and blend well.4. Add 5% Lavender essential oil to the mixture and mix until the oil is evenly distributed throughout the powder.5. Store the wound dressing powder in a clean, airtight container until ready to use.EXAMPLE 7POLYPHENOL-ENRICHED LETTUCE POWDER / EXTRACT FORMULATION

[0136] The polyphenol-enriched lettuce powder or its extract can be formulated into ointments, lotions, creams, oils, etc. Table 3 shows an example formulation for the polyphenol- enriched lettuce powder or its extract and corresponding weight per-weight examples.Table 3. Example polyphenol-enriched lettuce powder / extract formulationEXAMPLE 8PREPARATION OF ANTI-INFLAMMATORY AND ANTIOXIDANT EFFECTS OF THE POLYPHENOL- ENRICHED LETTUCE POWDER / EXTRACT LOADED HYDROGELS FOR WOUND HEALING

[0137] A more specific aspect of the disclosure is the development of anti-inflammatory and antioxidant effects of the polyphenol-enriched lettuce powder / extract hydrogels in wound healing applications. For example, the combination of chitosan-pluronic P123- polyphenol- enriched lettuce powder / extract-gelatin appears to be a highly promising option for the development of hydrogels that are intended for wound healing. Chitosan hydrogel has been found to be useful for wound healing, and it can be cross-linked with a conjugating agent via its amine functional groups to form a three-dimensional hydrogel system. When polyphenol- enriched lettuce powder / extract-loaded chitosan nanoparticles (measuring between 167-251 nm)were tested for transdermal application, they showed significant transdermal permeability, enhanced drug release, and high cell viability, all of which suggest that they could be an effective means of promoting wound healing. Additionally, the swelling behavior of the dual-loaded hydrogel was found to be over 1.2 times that of the gelatin-free hydrogel, which demonstrates the potential for this system to increase water absorption when mixed with gelatin. Given that gelatin can act as a cell glue and the polyphenol-enriched lettuce powder / extract possesses antioxidant properties that can aid in wound healing, the chitosan-P123 nano polyphenol- enriched lettuce powder / extract-gelatin system was found to exhibit superior wound dressing properties when compared to single-loaded hydrogel.

[0138] An example general protocol for preparing chitosan- the polyphenol-enriched lettuce powder / extract loaded hydrogel is as follows:

[0139] MaterialsChitosan powder;Nano-sized polyphenol-enriched lettuce powder / extract;Cross-linking agent (e.g. glutaraldehyde);Pluronic Pl 23;Gelatin powder;Sodium hydroxide; and Acetic acid.

[0140] General Procedure1. Dissolve chitosan powder in a solution of 1% acetic acid and stir for several hours until the powder is completely dissolved. The concentration of chitosan can be adjusted based on the desired properties of the hydrogel.2. Add polyphenol-enriched lettuce powder / extract to the chitosan solution and stir until the powder is completely dispersed. The concentration of the polyphenol-enriched lettuce powder / extract can be adjusted based on the desired amount of drug loading.3. In a separate beaker, dissolve Pluronic P123 and gelatin powder in distilled water to create a gelatin solution.4. Slowly add the chitosan-polyphenol-enriched lettuce powder / / extract solution to the gelatin solution with constant stirring. The pH of the mixture should be monitored and adjusted using NaOH to maintain a pH of around 7.0.5. Add the crosslinking agent (e.g. glutaraldehyde) to the mixture to facilitate the formation of a three-dimensional network structure.6. Pour the mixture into a hydrogel mold and allow it to set at room temperature for 2-3 hours.7. Remove the hydrogel from the mold and place it in an oven at 50°C for 24 hours to ensure complete crosslinking.8. Finally, freeze dry the hydrogel to remove any residual water and obtain a dry chitosan-polyphenol-enriched lettuce powder / extract loaded hydrogel.EXAMPLE 10PREPARATION OF A POWDER DRESSING FOR THE PROMOTION OF WOUND HEALING

[0141] Provided is a general procedure for preparing a powder dressing for wound healing by combining a polyphenol-enriched lettuce powder / extract with collagen, seaweed extract, P-glucan, carboxymethyl cellulose (CMC), and vitamin D3.1. Measure out the desired amounts of each ingredient, based on the intended formulation and target application.2. Mix the polyphenol-enriched powder, collagen, seaweed extract, P-glucan, CMC, and vitamin D3 in a dry blending mixer or a similar mixing device. Blend the mixture thoroughly to ensure even distribution of the ingredients.3. Optionally, adjust the pH of the mixture if necessary to ensure compatibility and stability of the ingredients.4. After the powder is uniformly blended, sieve it through a fine mesh sieve to remove any lumps or clumps.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.EXAMPLE 11IN VIVO EVALUATION OF POLYPHENOL DRESSINGS ON WOUNDED MICE

[0142] The effects of polyphenol dressings in promoting wound healing in wounded mice were evaluated.

[0143] Mice for Experiment: The evaluation of wound healing efficacy in vivo was conducted using BALB / c mice as the primary target animal. BALB / c laboratory mice were used due to their uniform skin color, which minimizes pigmentation issues potentially affecting the interpretation of results. Fifteen BALB / c mice, sourced from the National Laboratory AnimalCenter, were raised until 10 weeks of age before starting the experiment. These mice were randomly divided into five groups for different treatments.Materials and Methods

[0144] The BALB / c mice, at 10 weeks of age and averaging approximately 28.5 grams, were anesthetized with isoflurane gas (initially 3-4%, maintained at 1-2%). Their backs were shaved (covering an area of 2x4 cm). A sterile pair of scissors was used to create a 1x1 cm wound on the back of each mouse. Dressings were immediately applied to the wound areas after they were created. The dressings were applied to cover the wound area completely. During the experimental period, wound conditions were recorded before and after each dressing application, using a scale placed next to the wound for photographing. The dressings were applied twice, once on day 0 and again on day 7.

[0145] Body weight was recorded before and after the experiment. Skin inspections were conducted 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 test for common inflammatory factors, including tumor necrosis factor-alpha (TNF-a), 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 reevaluate the levels of these three inflammatory factors, providing insights into any specific effects of the compound polyphenol dressing on inflammatory responses. Finally, on day 14, after euthanizing animals, wound samples were collected for tissue sectioning and analysis. Additionally, photographs of the spleen were taken.

[0146] Table 4 provides formulations of four polyphenol dressings used on different groups of mice in the experiment. The polyphenol dressings for Group A mice were made from pristine lettuce powder. Group B mice were treated with dressings made from an extract obtained from lettuce powder using ethanol / water as the extraction solvent. Group C mice were treated with dressings made from an extract obtained from lettuce powder using supercritical CO2 as the extraction solvent. Group D mice were treated with dressings made from a mixture of pristine powder and CMC. The results from the mice treated with different polyphenol dressing formulations were compared with mice in a control group where no dressings were applied.Table 4. Polyphenol Dressing Formulations and Experimental ConditionsTable 5 provides relative contents of major bioactive polyphenols in different types of dressings. As shown in FIG. 5, the extracts contain a higher level of chicoric acid compared to the pristine lettuce powder. Additionally, the polarity of the extraction solvent also affects the contents of the polyphenols in the extract. A polar extraction solvent (e.g., aqueous ethanol) afforded an extract containing higher levels of extractable polyphenols, such as chicoric acid and quercetin derivates.Table 5. Polyphenol Contents in Lettuce Powder and Lettuce Powder ExtractResults

[0147] Weight Changes and Macroscopic Differences in SpleensThe average weight differences of mice before and after the experiment for each group (A-D and control) are compared in FIG. 8. It can be observed that the mice in all groups show an increase in weight after the experiment, and there are no statistically significant differences in their weights before and after the experiment. Additionally, there are no significant differences in weight changes among mice in the A-D and control groups.Fig. 9 shows photographs of the spleens of mice in the polyphenol dressing-treated groups (A-D) and the control group. The mice in the control group have slightly larger spleens compared to the spleens of the mice in the polyphenol dressing-treated groups (A-D), indicating that the use of polyphenol dressings helps to reduce wound inflammation.

[0148] Compari son of W ound Healing EffectsThe wound healing effects of the polyphenol dressings were evaluated by monitoring the wound size changes of the mice in the polyphenol dressing-treated groups (A-D) and the control group over the treatment period. Fig. 10 shows photographs illustrating the wound sizes of the mice in the polyphenol dressing-treated groups (A-D) and the control group over time. As shown in Fig. 10, the mice in the control group exhibit the slowest wound healing rate. On day 14, the mice in the control group have the largest wound sizes. In contrast, among the polyphenol dressing-treated groups (A-D), the mice in group B show the fastest healing rate and have the smallest wound sizes. This effect may be attributed to the use of aqueous alcoholic extraction solvents (e.g., ethanol / water mixed solvent), which yield extracts with higher contents of polar polyphenols, including chicoric acid and quercetin derivatives. The polyphenols with higher polarities work synergistically to enhance wound healing properties.

[0149] Comparison of Wound Tissue SectionsThe wound tissue sections of mice from polyphenol dressing-treated groups (A- D) and control groups were analyzed. Figs. 11 A-l IE show photographs illustrating the tissue sections of wounds in mice from the polyphenol-treated groups and the control group. It can be seen that the mice in the control group exhibit the slowest wound healing rate (Fig. 11 A). The tissue sections of the mice in the control group reveal excessive epithelialization and disorganized cell arrangement. In contrast, as shown in Figs. 1 IB-1 IE, the mice whose wounds were treated with polyphenol dressings show more orderly cell arrangement and more normal epithelialization.

[0150] Comparison of Inflammatory Cytokine Levels in Blood

[0151] Common inflammatory cytokines, including tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), and interleukin-8 (IL-8), were measured using blood samples from the mice taken 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 TNF-a changes in the mice of the polyphenol dressing-treated groups (A-D) and the control group over time. Fig. 13 is a bar graph showing IL-6 changes in the mice in the polyphenol dressing-treated groups (A-D) and the control group over time. Fig. 14 is a bar graph showing IL-8 changes inthe mice of the polyphenol dressing-treated groups (A-D) and the control group over time. The data indicate that for TNF-a, IL-6, and IL-8, the levels of inflammatory cytokines in the mice treated with polyphenol-containing dressings (Groups A-D) are lower than those in the mice of the control group. Furthermore, it is evident that the mice treated with extracts (groups B and C) exhibit better anti-inflammatory effects than the mice treated with pristine polyphenol powder (groups A and D). Additionally, when comparing the mice treated with pristine polyphenol powder, the mice in group D show lower IL-6 and IL-8 levels than the mice in group A. Overall, the mice in group B demonstrate the most effective suppression for all measured inflammatory cytokines.

[0152] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0153] These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. The present application claims priority to U.S. Provisional Application No. 63 / 509,677, filed on June 22, 2023, in the United States Patent Office, the entire contents and disclosure of which are incorporated herein by reference.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A powder comprising particles of a polyphenol-enriched lettuce powder or extract, wherein the polyphenol-enriched lettuce powder or extract comprises from 40-280 mg / g of polyphenols, the polyphenols comprising a chlorogenic acid, a neochl orogenic acid, a chicoric acid, a quercetin, a quercetin derivative, an anthocyanin, and any combination thereof.

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

3. The powder of claim 1 or 2, wherein the chlorogenic acid comprises 3-0- 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 1-3, wherein the quercetin derivative comprises one or more of quercetin-3-O-glucoside (Q3G), quercetin glucuronide, and quercetin-3-O- malonylglucoside (Q3MG).

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

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

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

8. The powder of claim 7, wherein the at least one eustressor / elicitor is an abiotic eustressor or elicitor selected from: indole-3 -acetic acid (IAA), auxins, cytokinins (CKs), gibberellins (GAs), ethylene, brassinosteroids, jasmonates (J As), strigolactones (SLs), salicylic acid (SA), arachidonic acid (AA), 5-aminolevumic 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 7-8, wherein the polyphenol-enriched lettuce comprises a 2-fold to 20-fold increased production of polyphenols compared to the control lettuce, optionally a 3-fold to 9-fold increased production of polyphenols compared to the control lettuce.

10. The powder of any one of claims 7-9, wherein the concentrations of chi orogenic acid, quercetin derivatives, chicoric acid, and anthocyanins are increased by at least 3 -fold, at least 4-fold, at least 5-fold, 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 1-10, wherein the particles have a particle distribution size range of 0.1 pm to 150 pm.

12. The powder of any one of claims 1-11, wherein the polyphenol-enriched lettuce powder comprises a nanoparticle powder, wherein the particle distribution size range of the nanoparticle powder is 0.01 pm to 0.1 pm.

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

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

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

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

17. The topical formulation of claim 16, wherein the polyphenols comprise two or more of a chlorogenic acid, a chicoric acid, a quercetin and a derivative thereof, and an anthocyanin.

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 exanthem, shingles, ringworm, or skin cancers.

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

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

21. The topical formulation of any one of claims 15-20, wherein the topical formulation is in the form of an ointment, a lotion, a cream, a powder, a liquid, a gel, a hydrogel, an oil, a surface tension agent, a drop, an aerosol, an emulsion, nano-emulsion, nano-liposome, gel, micro-encapsulation, paste, or foam.

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

23. The topical formulation of any one of claims 15-22, 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 15-24, further comprising an antimicrobial preservative.

26. The topical formulation of any one of claims 15-25, further comprising one or more of an emulsifying agent and a viscosity enhancing agent.

27. The topical formulation of any one of claims 15-26, wherein the topical formulation comprises by % weight / weight:1-10% of the powder;5-15% of an alcohol;5-15% of an emollient;1-5% of an emulsifying agent;5-15% of propylene glycol; less than 1% of an anti-microbial preservative; and a remaining percentage of water to equal 100%.

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

29. The wound dressing powder of claim 28, wherein the pharmaceutically acceptable carrier or excipient is a starch, a cellulose, a synthetic polymer, a polysaccharide, a 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%-90% wt / wt, ii. the clay powder comprises 5%-l 5% wt / wt; and iii. the powder comprises 1%-15% wt / wt.

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 29-31, wherein the clay powder comprises bentonite clay.

33. The wound dressing powder of any one of claims 28-32, 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 1-14 and a pharmaceutically acceptable hydrogel polymer.

36. The hydrogel of claim 35, wherein the powder is present at a percentage to the final weight of the topical formulation of about 0. l%-10%, about 0.5%-5%, about l%-10%, about 5%-20%, or about 10%-50%.

37. The hydrogel of claim 35 or 36, wherein the powder comprises particles having a particle distribution size range of 0.1 pm to 150 pm.

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 particle distribution size range 0.1 pm or less, optionally the particle distribution size range is from 0.01 pm to 0.1 pm, or from 0.05 pm to 0.1 pm.

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

41. The hydrogel of any one of claims 35-40, further comprising a polymer surfactant.

42. The hydrogel of claim 41, wherein the polymer surfactant is pluronic P123.

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

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

45. The oral formulation of claim 44, wherein the powder is present at a percentage to the final volume of the oral formulation of about 0. l%-10%, about 0.5%-5%, about l%-10%, about 5%-20%, or about 10%-50%.

46. The oral formulation of claim 44 or 45, wherein the binder comprises gum tragacanth, acacia, com starch, or gelatin.

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

48. The oral formulation of any one of claims 44-47, wherein the disintegrating agent comprises com starch, potato starch, and alginic acid.

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

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

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

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

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

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

55. The method of claim 54, wherein the wound comprises an acute wound, a skin infection, a bum 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 1-14, the topical formulation of any one of claims 15-27, the wound dressing powder of any one of claims 28-34, the hydrogel of any one of claims 35-43, or the oral formulation of any one of claims 44-53.

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

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

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

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

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

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

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

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

65. A method of preparing a wound dressing powder, the method comprising: i. mixing a mineral powder and a clay powder to form a powder mixture; ii. adding the powder of any one of claims 1-14 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 65-67, 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 of preparing a nanoparticle powder, the method comprising: i. washing and drying at least one polyphenol-enriched lettuce leaf;ii. milling the at least one red lettuce leaf using a milling tool to form a powder; iii. passing the powder through a sieve to remove particles larger than the openings of the sieve; and iv. grinding the powder to reduce a particle size of the powder to a particle distribution size range of 0.1 pm or less.

70. The method of claim 69, where the grinding is performed with a ball mill, a conical mill, a hammer mill, or a homogenizer.

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

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

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

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

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

76. The method of any one of claims 73-75, 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 73-76, wherein the crosslinking agent comprises glutaraldehyde.

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

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

80. The method of any one of claims 73-79, 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 1-14 or the topical formulation of any one of claims 15-27.

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 exanthem, shingles, ringworm., skin cancers.

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

84. The method of claim 83, wherein the blue light radiation has a wavelength ranging from 400 nm to 520 nm.

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

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

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