Pollen-based compositions and extracts for stimulating hyaluronic acid production

Abstract

The present invention relates to a pollen-based composition or an extract from a pollen-based composition, wherein the composition or extract stimulates the production of hyaluronic acid (HA). The composition or extract comprises at least glucose-6-phosphate as a substrate or precursor for hyaluronic acid (HA) production. The compositions or extracts of the present invention further comprise other small molecular weight bioactive molecules and / or compounds selected from the group comprising flavonoids, flavonols, flavones, alkaloids, aglycones, glycosides, lipids, fatty acids, amino acids, anthranilic acids and isomers thereof, spermidine and isomers thereof, a caffeoyl spermidine compound; and hydroxycinnamic acid. The invention also relates to formulations comprising a pollen-based composition or extract useful for the treatment of a variety of conditions, including skin problems and skin aging as well as symptoms of climacteric genitourinary syndrome (GSM), the symptoms of the climacteric genitourinary syndrome (GSM) include urinary incontinence, vaginal dryness, vaginal atrophy, irritation, pruritus, rhagadia, thinning, redness, inflammation and a decline in sexual desire, sexual function, sexual response and / or sexual pleasure.

Description

Technical Field

[0001] The present invention relates to pollen compositions or extracts, as well as pharmaceutical preparations or compositions and cosmetic preparations or compositions containing the same. The compositions and extracts of the present invention are useful for treating and preventing skin-related conditions, particularly improving skin moisture, increasing skin firmness and elasticity, improving skin barrier function, improving skin healing, treating skin atrophy (including vaginal atrophy), rebalancing the skin microbiome, and treating and preventing skin aging. The compositions and extracts of the present invention are also useful for preventing and treating symptoms of GSM (genitourinary syndrome of menopause), including urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation, and decreased libido, sexual function, sexual response, and / or sexual pleasure. Background Art

[0002] Human skin aging is a complex biological process caused by a combination of endogenous (innate) and exogenous (environmental) factors. Intrinsic skin aging is influenced by hormonal changes that occur with age, and it is well established that decreased estrogen and androgen production leads to collagen degradation, dry skin, loss of elasticity, wrinkling, epidermal atrophy, and genitourinary syndrome of menopause (GSM), also known as vaginal atrophy or vulvovaginal atrophy. Extrinsic aging is the result of exposure to external environmental factors, namely ultraviolet (UV) radiation.

[0003] Photoaging, general aging, and skin atrophy (particularly GSM) are all characterized by a decrease in collagen, elastin, and hyaluronic acid, which affect skin structure and hydration. In the case of GSM, the loss of hyaluronic acid in vaginal tissue leads to many GSM symptoms, including urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation, and a decrease in libido, sexual function, sexual response, and / or sexual pleasure.

[0004] A key molecule involved in skin hydration is hyaluronan or hyaluronic acid (HA), a non-sulfate glycosaminoglycan (GAG) with a unique ability to bind and retain water molecules. HA is widely distributed in connective tissue, epithelial tissue, skin tissue, and neural tissue, and is a major component of the extracellular matrix. At least 50% of the HA present in the human body is distributed in the skin, that is, in the gaps between the epithelial cells and the extracellular matrix of the dermis.

[0005] HA is also present in all tissues and fluids of the body, including the eyes, synovial fluid, bone tissue, heart valves, lungs, aorta, prostate, penile tissue, and vaginal tissue. HA has multiple functions in the body, including hydration, joint lubrication, regulation of tissue repair and wound healing, activation of inflammatory cells to enhance immune response, and providing a framework for angiogenesis and fibroblast migration.

[0006] HA is synthesized by specific membrane-bound enzymes called HA synthetases (HAS). These enzymes synthesize HA on the inner surface of the cytoplasmic membrane, and then HA is squeezed out into the extracellular space through a pore-like structure. There are three mammalian enzymes, HAS-1, HAS-2, and HAS-3, which exhibit different enzymatic properties and synthesize HA chains of various lengths.

[0007] It is known that HA in the skin is depleted over time and is therefore a major cause of skin aging, as the epithelium loses key molecules responsible for binding and retaining water molecules, which leads to a loss of skin moisture. In the dermis, the main aging-related change is an increase in HA's affinity for tissue structure and a simultaneous loss of HA's extractability. This leads to a progressive cross-linking of collagen and a steady loss of collagen extractability with age. All of this leads to dehydration, atrophy, and loss of elasticity, which are the manifestations of aging skin and body tissues (including vaginal tissue).

[0008] Over the past decade, significant research has been conducted to identify methods to increase HA production in the skin in order to improve the condition of the skin and overcome the signs of aging. These approaches include the development of injectable dermal fillers (containing cross-linked HA) and topical skin preparations (containing HA molecules). While injectable treatments are becoming more popular, such treatments remain invasive and expensive and may have adverse side effects. Topical HA preparations are generally less effective than injectable preparations, but have the advantages of being minimally invasive, less expensive, and having fewer side effects. Therefore, minimally invasive treatments (such as topical cosmetics) are often the desired first choice of protection.

[0009] However, many topical HA formulations contain HA molecules that are actually too large to pass through the outer layers of the skin and are therefore less effective in treating the signs of aging, simply hydrating the surface layers of the skin. Studies have found that in order to effectively cross the skin barrier, HA molecules need to have a molecular weight of less than 500 kDa.

[0010] Other topical formulations containing active ingredients such as retinol and bakuchiol are well known and are also marketed for their anti-aging benefits. Retinoids work by triggering an inflammatory or stress response in skin cells, leading to apoptosis. However, when dosed above physiological levels, retinol, like bakuchiol, can cause necrosis. Essentially, these active ingredients trigger apoptotic and / or necrotic cell death, thereby stimulating cell renewal and turnover by increasing the production of collagen and HA. However, because these ingredients essentially trigger skin cell death and keep skin cells in a state of constant stress response, the long-term effects of continued use of these types of irritating ingredients are not yet clear.

[0011] There is a growing demand for more effective, non-invasive and gentler anti-aging products containing active ingredients that can be delivered to the target site in sufficient concentration to exert a specific biochemical mechanism of action in the target cells or skin tissue, where applicable.

[0012] There is a growing market demand for products that can more effectively prevent and treat GSM symptoms, many of which are associated with the loss of HA during aging. These symptoms include urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation, and decreased sexual function, response, and desire.

[0013] One object of the present invention is to provide a pollen composition and / or extract and / or a preparation or composition containing the same to address the above needs, or at least provide the public with a useful choice. Summary of the Invention

[0014] In a first broad aspect, the present invention relates to a pollen composition or an extract therefrom, wherein the composition or extract comprises at least glucose-6-phosphate as a substrate or precursor for the production of hyaluronic acid (HA). Preferably, the amount of glucose-6-phosphate present in the composition or extract is at least 0.05% by weight, more preferably in the range of about 0.05% to 5.0% by weight.

[0015] The pollen composition or extract of the present invention preferably further comprises other small molecular weight bioactive molecules and / or compounds selected from the group consisting of flavonoids, flavonols, flavones, alkaloids, aglycones, glycosides, lipids, fatty acids, amino acids, enzymes (including hexokinase, glucokinase, protease, peptidase), magnesium and other elements, anthranilic acid and its isomers, spermidine and its isomers, caffeoyl spermidine compounds, feruloyl spermidine compounds and hydroxycinnamic acid.

[0016] The pollen composition or extract preferably comprises one or more spermidine isomers and / or caffeoylspermidine compounds and / or feruloylspermidine compounds, and / or compounds that are cinnamic acid conjugates of spermidine.

[0017] The pollen composition or extract preferably comprises a compound selected from the group comprising tricaffeine spermidine (N1, N5, N10-tricaffeine spermidine), triferulinospermidine (N1, N5, N10-triferulinospermidine) and dicaffeoyl-p-coumarinospermidine.

[0018] The pollen composition or extract preferably comprises one or more compounds selected from the group comprising hydroxycinnamic acids. Preferably, the hydroxycinnamic acid is selected from the group comprising dicaffeoylquinic acid, dicaffeoyltartaric acid and isomers thereof.

[0019] The pollen composition or extract preferably comprises one or more flavonoids, flavonols and / or flavones. Preferably, the flavonoids, flavonols and / or flavones are selected from the group consisting of alkaloids, glycosides and aglycones, the latter including isorhamnetin, rhamnetin, quercetin (tricetin), kaempferol.

[0020] The pollen composition or extract preferably further comprises one or more lipids. Preferably, the lipids are selected from the group consisting of fatty acids, including myristic acid, palmitic acid, linoleic acid, and stearic acid. Preferably, the composition or extract further comprises a lipid selected from the group consisting of phospholipids, cholesterol, and glycolipids. It is believed that the lipids and / or fatty acids act as carriers by encapsulating and protecting other bioactive compounds in the composition or extract.

[0021] As used herein, the term "extract of the present invention" or "extract from the composition" or similar expressions refers specifically to a concentrated version or concentrated extract of the pollen composition of the present invention, wherein the extract comprises a more concentrated composition of the aforementioned beneficial bioactive components.

[0022] The pollen composition of the present invention can be concentrated by methods known in the art, such as centrifugation, membrane filtration, ultrafiltration, and adsorption.

[0023] The present invention also relates to a formulation comprising a pollen composition or extract and one or more carriers and / or excipients. Preferably, the formulation is a topical formulation or an oral formulation (such as a capsule or tablet). Preferably, the topical formulation comprises between about 1% and 10% by weight of the pollen composition or extract. More preferably, the topical formulation comprises between about 1% and 4% by weight of the pollen composition or extract. The oral formulation of the present invention may comprise a higher content of the pollen composition or extract, for example at least 20% by weight, and more preferably between about 20% and 100% by weight.

[0024] In another aspect, the present invention relates to a method of stimulating HA production comprising administering to a subject a therapeutically effective amount of a formulation comprising a pollen-based composition or an extract therefrom. The stimulating effects of HA provide multiple health benefits, not only in terms of treating skin conditions and anti-aging, but also in terms of eye health, hair health, connective tissue and joint health, vaginal health, and other conditions in which HA plays an important role.

[0025] In another aspect, the present invention relates to a method of treating or preventing vaginal atrophy comprising administering to an individual in need of such treatment a therapeutically effective amount of a formulation comprising a pollen-based composition or an extract from a pollen-based composition.

[0026] In another aspect, the present invention relates to a method of treating or preventing one or more symptoms of GSM, the method comprising administering to an individual in need of such treatment a therapeutically effective amount of a formulation comprising a pollen-based composition or an extract from a pollen-based composition.

[0027] In another aspect, the present invention relates to the use of a pollen composition or extract in the manufacture of a medicament for preventing and / or treating one or more symptoms of genitourinary syndrome of menopause (GSM), including urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation, and decreased sexual desire, sexual function, sexual response and / or sexual pleasure.

[0028] In another aspect, the present invention relates to a method for preparing a pollen composition comprising at least glucose-6-phosphate, the method comprising the following steps:

[0029] a. mixing one or more of raw bee pollen, raw honey and propolis with coconut water, coconut oil and / or coconut milk to form a mixture;

[0030] b. incubating the mixture anaerobically at a temperature between about 25°C and 65°C for about 12 hours to 96 hours;

[0031] c. freezing and / or lyophilizing the obtained fermentation composition.

[0032] In another aspect, the present invention relates to a method for preparing an extract from a pollen composition, wherein the extract comprises at least glucose-6-phosphate, the method comprising the following steps:

[0033] a. mixing one or more of raw bee pollen, raw honey and propolis with coconut water, coconut oil and / or coconut milk to form a mixture;

[0034] b. incubating the mixture anaerobically at a temperature between about 25°C and 65°C for about 12 hours to 96 hours;

[0035] c. concentrating the resulting fermentation composition;

[0036] d. Freezing and / or lyophilizing the concentrated composition.

[0037] Preferably, the concentration step is performed by centrifugation and the resulting supernatant is collected for freezing and / or lyophilization.

[0038] Preferably, the composition is frozen for up to 8 weeks and then lyophilized, and the lyophilized composition is ground into a powder to produce a lyophilized powder composition that can be incorporated into a variety of product forms.

[0039] In another aspect, the present invention relates to a method of treating or preventing a condition associated with decreased hyaluronan production or degradation of hyaluronan, the method comprising administering to a subject a therapeutically effective amount of a formulation comprising a composition or extract as described herein.

[0040] In another aspect, the present invention relates to the use of a composition or extract as described herein in the manufacture of a medicament for the treatment or prevention of a condition selected from the group comprising: skin diseases, skin aging, genitourinary syndrome of menopause (GSM), dry eyes, connective tissue and joint diseases, and other conditions that may benefit from stimulation or stabilization of hyaluronic acid (HA), genitourinary syndrome of menopause including urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation, and decreased libido, sexual function, sexual response and / or sexual pleasure. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A flow chart outlining the steps for making the composition or extract of the present invention.

[0042] Figure 2 is a photograph of the extract of the present invention after the centrifugation step.

[0043] Figure 3 Images are SDS-PAGE protein analyses of samples collected at various time points during the incubation step.

[0044] Figure 4 for Figure 3 SDS-PAGE image of the sample, showing the visible protein bands and their corresponding molecular weights.

[0045] Figure 5 RP-UHPLC (reverse-phase ultra-high performance liquid chromatography) and negative ion electrospray ionization (ESI) analyses of the pollen composition prepared in Example 1 are shown.

[0046] Figure 6This is a comparative chart showing the efficacy of the pollen composition of the present invention prepared in Example 1 in stimulating HA production in human keratinocytes.

[0047] Figure 7 (a) to Figure 7 (c) are photographs showing the growth of skin cells obtained during an efficacy study measuring the levels of cell proliferation and differentiation of the compositions and extracts of the present invention.

[0048] Figure 8 (a) to Figure 8 (f) is passed Figure 5 UV chromatograms of original fractions 5 to 10 of the pollen composition identified by RP-UHPLC analysis.

[0049] Figure 9 and Figure 10 (a) to Figure 10 (d) To pass Figure 5 UV chromatograms of original fractions 25 to 30 of the pollen composition identified by RP-UHPLC analysis in FIG.

[0050] Figure 11 The figure shows the PrestoBlue analysis of the effects of the extract (MYR) of the present invention and its subfractions on keratinocytes (compared with the control group).

[0051] Figure 12 and Figure 13 Shows the Figure 11 Some further UV chromatographic analysis of the more active subfractions was performed.

[0052] Figures 14 to 17 is a graph showing test results of lip balm formulations containing the extract of the present invention.

[0053] Figures 18 to 21 The present invention is a graph showing the test results of vaginal cream preparations containing the extract of the present invention. DETAILED DESCRIPTION

[0054] The following provides a detailed description of one or more embodiments of the present invention, in conjunction with the accompanying tables and drawings that illustrate the principles of the present invention. Therefore, this detailed description sets forth the present invention by way of example only and not by way of limitation. This description will clearly enable those skilled in the art to make and use the present invention, and sets forth several embodiments, adaptations, variations, alternatives, and uses of the present invention, including the best mode presently believed by the inventors to carry out the invention. It should be clearly understood that routine changes and adjustments may be made to the present invention as described above, and such changes and adjustments are fully within the spirit and scope of the present invention.

[0055] Raw bee pollen contains a variety of beneficial vitamins, minerals, nutrients, proteins, amino acids, lipids, fatty acids, enzymes, antioxidants, carotenoids, and bioflavonoids. The exact composition of bee pollen varies depending on the plant source and the season in which it was collected. Numerous studies have reported that raw bee pollen may have various health benefits, including antibacterial, antimicrobial, anti-inflammatory, and antioxidant benefits.

[0056] Raw bee pollen is naturally granular, with a hard outer shell of sporopollenin that is highly resistant to chemical degradation. Furthermore, pollen contains proteins on its surface that are known to trigger allergies. Therefore, it is desirable to "open" the pollen grains to access or release the beneficial components within, but this process is difficult.

[0057] In previous research, the inventors developed a unique method for treating pollen grains to stimulate their natural germination and naturally and gently release their contents. This "biological" method for stimulating pollen grains is described in patent document WO2015 / 028892, which is incorporated herein by reference. A two-stage process for producing pollen-based extract materials is also described: it involves using biological methods to open and / or germinate pollen grains, and then reacting the germinated pollen with one or more honeycomb components (such as, for example, beeswax, honey, propolis), other components (such as glycerin), and enzyme components to obtain extracts and compositions containing the extracts that are beneficial for treating skin diseases (such as eczema, psoriasis, and acne).

[0058] Since producing the extracts and compositions disclosed in WO2015 / 028892, the inventors have conducted further research and development. In particular, research has focused on developing novel extracts and compositions with enhanced bioactivity while reducing non-essential or non-beneficial components (such as macromolecules that are not fully absorbed by the skin and large proteins known to be allergens). Subsequent research and development has focused on optimizing the ingredients and processes for producing pollen-based compositions and extracts, as well as identifying and concentrating the most beneficial components to produce improved pollen-based extracts and compositions containing them.

[0059] Therefore, the inventors have developed an improved process for preparing fermented pollen-based compositions or extracts that have demonstrated unexpected properties and additional advantages. Specifically, the inventors unexpectedly discovered that pollen-based extracts and compositions can stimulate hyaluronic acid production through a unique mechanism of action that is different from the strong effects of anti-aging ingredients such as retinol and bakuchiol and is more gentle.

[0060] The inventors discovered that the improved extracts and compositions of the present invention can stimulate keratinocytes to produce hyaluronic acid and induce cell proliferation and differentiation. Furthermore, the extracts and compositions of the present invention have antibacterial effects against Propionibacterium acnes, Staphylococcus aureus, and Candida albicans (thrush). These factors are closely related to GSM and its associated symptoms, which include vaginal atrophy, vaginal dryness, burning, itching, irritation, redness, inflammation, decreased tissue turgor, an imbalanced microbiome (leading to an increased risk of UTIs and candidiasis), and decreased libido and sexual function. Therefore, the inventors discovered that the extracts and compositions of the present invention have significant efficacy in preventing and treating GSM and its associated symptoms.

[0061] Manufacturing method

[0062] The pollen composition of the present invention is produced using two main components, namely "honeycomb components" and "coconut components". Honeycomb components include raw bee pollen, raw honey and propolis. Honeycomb components can be obtained from any hive, but are preferably collected from hives in the Southern Alps region of New Zealand with beech forests. Preferably, the raw bee pollen is subjected to a cold shock before use, because the cold shock activates the apoptosis reversal or protective response of plant cells, thereby enhancing the beneficial bioactive components obtained in the resulting composition. Honey can be any type of pure raw honey or a mixture of raw honey. Importantly, the honey must not be pasteurized honey. Some examples of suitable honey include nectar honey, manuka honey, rewariwa honey, clover honey, beech honey and pohutukawa honey. Preferably, raw propolis is used, or alternatively, propolis can be prepared as an aqueous or isopropyl alcohol extract by mixing 25 g of propolis with 50 mL of isopropyl alcohol or water in a light-proof, sealed glass container and incubating at 28° C. with gentle rotation for 28 days. Raw propolis can be added to the mixture at the beginning of the process, or alternatively, propolis in isopropyl alcohol form can be added at the end of the incubation period.

[0063] The coconut component comprises one or more in coconut water, coconut oil and / or coconut milk. The coconut component can be obtained by extracting from fresh coconut, or commercially available coconut water, coconut milk and coconut oil can be used. Coconut water can promote the pollen grains to open or sprout naturally and gently, which can retain useful bioactive components. Coconut oil is used to promote anaerobic fermentation (optional if using a closed container), and can also provide a source of fatty acids, which are considered to help the bioactive components (such as flavonoids) in the extract to be transported to the skin.

[0064] The pollen composition and the extract derived from the pollen composition of the present invention are preferably dry powder compositions, and more preferably freeze-dried powder compositions.

[0065] Reference Figure 1The freeze-dried pollen composition can be prepared by the following steps. First, the selected honeycomb component is mixed with one or more coconut components (101). The mixture is placed in an anaerobically incubated (102) at a temperature between about 25°C and 65°C for about 12 hours to 96 hours. Preferably, the incubation is carried out at a temperature of about 37°C because this temperature is the optimum temperature for enzyme activity and will accelerate the reaction process so that the reaction can be completed in about 48 hours. Alternatively, the mixture is anaerobically incubated at a temperature of about 25°C for a longer period of time. It is believed that a longer incubation period is associated with an increased yield of isomers of the key components of the extract. However, it is important not to allow the incubation period to be too long to avoid the production of ethanol and degradation of the isomers. If a shorter incubation period of about 12 hours is used, the incubation temperature should be higher (for example, raised to about 65°C), which will significantly accelerate the reaction process.

[0066] If not added at the beginning of the process, propolis can be added after the incubation period. The resulting composition can then be immediately frozen (103) and placed in a freezer for a maximum period of 8 weeks.

[0067] Alternatively, and preferably, the resulting composition may be centrifuged (104) to separate the components of the composition and produce a more concentrated extract. The centrifugation step is performed using methods known in the art. It is important that the key components of the composition are retained during the centrifugation process. The inventors have adopted a process of bioactivity-guided separation to ensure that the key beneficial bioactive components of the composition (as described above) are retained in the supernatant phase. In the following example, the speed is set to 9600 rpm, the flow rate is set to 400 liters / hour, and the discharge of the heavy phase is set to 200 seconds. This design is based on a centrifuge manufactured by Alfa Laval, but the centrifuge parameters can obviously be adjusted as needed to suit different equipment and can be adjusted as needed to meet different applications. The supernatant phase is then collected (105) and can be frozen (103) or not. The collected supernatant has a gel-like texture and is as follows Figure 2 The collected composition is preferably initially frozen in a blast freezer for up to 48 hours. Thereafter, it is preferably frozen at -18°C for up to 8 weeks to reduce the microbial load. The longer the composition is frozen, the lower the microbial count. If the composition is not frozen immediately, the microbial count may be higher.

[0068] Alternatively, the collected supernatant may be dried, preferably by lyophilization, without the aforementioned freezing step. Lyophilization parameters may vary according to the knowledge of the skilled artisan and the desired application of the composition or extract.

[0069] If the composition or extract from the composition has been frozen, the composition or extract is thawed (106) and then dried by methods known in the art, preferably lyophilized (107). The resulting lyophilized composition can be ground into a powder or further processed by methods known in the art. The composition or extract of the present invention can be incorporated into a variety of product forms, including topical compositions or oral compositions (such as capsules, tablets) as described below.

[0070] If the product is frozen and then lyophilized according to the above process, the compositions and extracts of the present invention have very low microbial counts (<10 cfu). However, if the composition or extract is used in a pharmaceutical grade product, where a lower microbial count is desired, a further sterilization step may be performed as known in the art.

[0071] It is contemplated that the compositions or extracts of the present invention may be subjected to further processing steps by methods known in the art to produce further purified and / or more concentrated extracts, wherein the bioactive components that confer specific beneficial effects and / or mechanisms of action are isolated and concentrated. These purified or concentrated extracts may then be further included in a variety of formulations or compositions to deliver specific products with specific mechanisms of action and / or to meet specific needs.

[0072] Protein content of the composition

[0073] The inventors unexpectedly discovered that the compositions of the present invention can be prepared without the addition of any additional enzymes or enzyme-containing ingredients. It has been found that the addition of enzymes or enzyme-containing ingredients (such as pineapple extract) inhibits, rather than promotes, the breakdown of protein in the composition. However, surprisingly, it was found that when the incubation period was extended to 48 to 72 hours, no protein remained in the composition. All protein in the composition was completely broken down into pure amino acids.

[0074] During the incubation period, six aliquots were collected at various time points to allow analysis of protein / peptide content. Samples were collected at T = 0; T = 24 hours; T = 48 hours; T = 72 hours; T = 96 hours; and T = 196 hours. Samples were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0075] Each of the six liquid samples was first diluted 10-fold with water and then diluted with sample buffer (Bolt TM LDS, Invitrogen TM ) and then heated at 70°C for 10 minutes. The treated samples (15 μL each) were then loaded onto two Bis-Tris gels (NuPAGE TM 4-12%, Invitrogen TMThe gel was visualized after Coomassie blue staining, and images were collected and analyzed on a Gel doc system (Essential V2, UVITEC, Cambridge).

[0076] The PAGE images were analyzed and the molecular weights of the major protein bands in each sample were recorded. Figure 3 Shown are the imaging results of protein bands in the samples. Figure 4 Provided Figure 3 Molecular weights of the visible protein bands in the samples shown. These figures show a steady decrease in protein concentration from T = 0 to T = 48 hours, followed by complete disappearance of the bands at T = 72 hours, with no discernible protein bands. This confirms that complete protein degradation occurred during the incubation period between 48 and 72 hours.

[0077] The inventors believe that the germination and mild fermentation process releases enzymes including proteases, peptidases, hexokinase and glucokinase from the honeycomb components (i.e., pollen grains and honey), including enzymes secreted by Bacillus subtilis present in the honeycomb components, and that these enzymes are capable of breaking down protein components and producing glucose-6-phosphate, a precursor for HA production.

[0078] The above method can be further understood with reference to the following examples.

[0079] Example 1 - Preparation of Composition

[0080] Coconut water and raw bee pollen are mixed with coconut oil, honeydew, and raw propolis. All ingredients are incubated at 37°C for 72 hours. The resulting composition needs to be frozen for up to 8 weeks to stabilize its composition. After freezing, the composition is thawed, freeze-dried, and ground into a powder.

[0081] Example 2 - Preparation of concentrated extract

[0082] Coconut water and raw bee pollen were mixed with coconut oil, honeydew, and raw propolis. All ingredients were incubated at 37°C for 72 hours. The resulting composition was centrifuged in an Alfa Laval centrifuge set at 9,600 rpm. The flow rate was set to 400 liters per hour, and the heavy phase was discharged at 200 seconds. The supernatant was then collected and frozen for up to eight weeks to stabilize its composition. After freezing, the composition was thawed, freeze-dried, and ground into a powder.

[0083] Example 3: Preparation of concentrated extract

[0084] Coconut water and raw bee pollen are mixed with coconut oil, honeydew, and raw propolis. All ingredients are incubated at room temperature (25°C) for up to 96 hours. The resulting composition is centrifuged. The centrifuge used is manufactured by Alfa Laval and is set at 9600 rpm. The flow rate is set to 400 liters per hour, and the discharge of the heavy phase is set to 200 seconds. The supernatant is then collected and frozen for 8 weeks to stabilize its composition. After freezing, the composition is thawed, then freeze-dried and ground into a powder.

[0085] Example 4: Preparation of concentrated extract

[0086] Coconut water and raw bee pollen were mixed with coconut oil, honeydew, and raw propolis. All ingredients were incubated at approximately 65°C for 12 hours. The resulting composition was centrifuged. The centrifuge used was manufactured by Alfa Laval and set to 9,600 rpm. The flow rate was set to 400 liters per hour, and the heavy phase discharge was set to 200 seconds. The supernatant was then collected and frozen for up to eight weeks to stabilize its composition. After freezing, the composition was thawed, then freeze-dried and ground into a powder. The higher incubation temperature allowed the reaction to complete in a shorter period of time, but did not affect the protein content of the extract, as it was found to still contain no protein.

[0087] Antimicrobial benefits of the composition

[0088] The antibacterial activity of the composition of the present invention prepared according to Example 1 was tested by culturing the test strains Propionibacterium acnes and Staphylococcus aureus on agar plates and challenging the test strains with the composition. The results are shown in the following table.

[0089]

[0090]

[0091]

[0092] NB. The logarithmic difference has been adjusted from 0.5 to the next integer.

[0093] Chemical analysis of compositions and extracts

[0094] Figure 5 HPLC analysis of a composition of the invention prepared according to Example 1 is shown.

[0095] In the analysis of the concentrated extracts prepared by the methods described in Examples 2 to 4, the same HPLC profiles were observed.

[0096] After HPLC analysis, the following subfractions (27 in total) of the 60 fractions were collected and further analyzed using activity-tracking separation to identify the key bioactive components in the extract:

[0097] Table 1. Based on Figure 5 UV chromatogram of the collected fractions for activity tracking separation

[0098] Merged fraction number Original fraction number 1 1、2 2 3 3 4 4 5 5 6、7 6 8、9、10 7 11、12、13 8 14、15、16 9 17、18 10 19、20 11 21、22 12 23、24 13 25、26 14 27、28 15 29、30 16 31、32 17 33、34 18 35、36 19 37、38 20 39、40 21 41、42 22 43、44 23 45、46、47 24 48、49 25 50、51、52 26 53、54、55 27 56、57、58

[0099] Activity tracking separation

[0100] The efficacy of the compositions and extracts of the present invention on HA production in skin cells and their proliferative activity on skin cells were evaluated by a series of bioassays.

[0101] HA bioassay

[0102] Keratinocytes (skin cells) were grown in tissue culture monolayers overnight and then treated for 4 hours with the following test substances:

[0103] a. the composition of the present invention prepared according to Example 1 (overall composition),

[0104] b. the concentrated extract of the present invention (extract) prepared by Example 2,

[0105] c. Each of the 27 subfractions identified in Table 1 above,

[0106] d. Retinol (as a positive control), and

[0107] e. Blank control (untreated) (medium only).

[0108] The cells were then harvested, lysed, and analyzed using the Perkin Elmer Elisa assay (also known as the AlphaLISA HA assay) to measure HA production in skin cells after 4 hours. The skin cells used were S140 keratinocytes P2 derived from a single human donor. Retinol was 1 million IU of vitamin A palmitate / retinyl palmitate / naturally derived retinol. Required materials include: 10 μl of cell culture supernatant; 1 x 96-well plate; 500 assay points (48 wells for each assay, including standards); and triplicates for each sample.

[0109] Method steps:

[0110] a. Dissolve the test substance in DMSO. Leave at room temperature overnight to dissolve, then transfer to the refrigerator for later use.

[0111] b. 5000 keratinocytes per well in 200 μl of Kelch medium were seeded into all required wells;

[0112] c. Incubate at 37°C / 5% CO2 for 24 hours;

[0113] d. Serially dilute the test substance in DMSO so that all samples receive the same amount of DMSO regardless of the concentration of the test substance;

[0114] e. Remove all culture medium and replace with 200 μl Kelch medium containing appropriate concentrations of each test substance (including DMSO and retinol (diluted with DMSO)) placed in a 96-well plate;

[0115] f. Incubate at 37°C / 5% CO2 for 4 hours;

[0116] g. Take images 4 hours after treatment to assess the visible effects of the treatment;

[0117] h. Remove all cell culture supernatant and store at -20°C until ready for HA assay;

[0118] i. Perform HA assay on test substances.

[0119] result:

[0120] Figure 6 The present invention is a graph showing the results of HA assays for the overall composition of the present invention. It was surprisingly found that skin cells treated with the composition of the present invention produced more HA than skin cells from a control sample, and produced more HA than skin cells treated with retinol, which is widely considered by experts and dermatologists to be an effective active product that works at a deep cellular level to reduce cellular aging by promoting the production of collagen, elastin, sebum, and HA. Furthermore, the results show that the composition of the present invention promoted the growth of fibroblasts and scaffolds, such as Figure 7 As shown in the photograph shown in (b).

[0121] Depend on Figure 7 As can be seen from the photograph in (c), the concentrated extract of the present invention prepared by the method of Example 2 exhibits higher biological activity than the composition of the present invention, and exhibits excellent effects on the growth of fibroblasts and scaffolds.

[0122] Among the subfractions tested, subfraction 4, subfraction 5, subfraction 6, subfraction 13, subfraction 14 and subfraction 15 showed the most significant activity effects, so these fractions were selected for subsequent activity tracking separation to identify the important bioactive components in the compositions and extracts of the present invention that are most likely to stimulate HA production.

[0123] The HA bioassay showed that the activity of the composition and the extract was concentrated in two regions in the chromatogram. To avoid confusion, the pooled fractions tested in Table 1 are referred to as test fractions, while the collected fractions are referred to as original fractions.

[0124] These areas include:

[0125] Area 1: Test the activity of fractions 4(+), 5(++) and 6(+++). (Original fraction 5 to original fraction 10)

[0126] Area 2: Test the activity of fractions 13(+), 14(++) and 15(+++). (Original fraction 25 to original fraction 30)

[0127] Figures 8 to 10 UV chromatograms of original fractions 5 to 10 and original fractions 25 to 30 are shown.

[0128] Area 1 - such as Figure 8 (a)~ Figure 8 As shown in (f)

[0129] This zone of chromatogram is relative polarity, just in time eluting after main peak.The LCMS analysis of original fraction illustrates, and fraction 5 is similar to fraction 6, and hardly detects the signal of MS peak at RT after 3 minutes.Fraction 7~fraction 10 illustrates the reduction level of these early stage peaks, and the zone after 3 minutes illustrates the small peak among the TIC.Because fraction 9 and fraction 10 are the most active (test fraction 6), therefore activity may be relevant with the peak of eluting after 3 minutes.The MS data in this zone of chromatogram are complicated.There is negative ion peak at 326amu place, and it may be relevant with activity.

[0130] Area 2 - such as Figure 10 (a)~ Figure 10 As shown in (d)

[0131] This region corresponds to the approximately 15-minute elution time in a preparative HPLC run. The least active fractions (test fraction 13) are raw fractions 25 and 26, which are just before the main UV peak; the next most active fractions are raw fractions 27 and 28, which were collected from the main UV peak; and the most active fractions (29 and 30) are just at the tail end of the main UV peak. These fractions are more suitable for LCMS analysis. The two fractions 27 and 28 are primarily composed of caffeine-spermidine compounds. These compounds exhibit three peaks (isomers), with the first isomer being significantly less abundant. The mass spectra of these compounds show distinct strong ions at 630 (negative) and 632 (positive).

[0132] The most active fractions, 29 and 30, showed greater complexity. These two fractions were also quite similar to each other. There was evidence of some caffeoyl spermidine compounds (negative MS 630) and a late-eluting peak with a negative MS 614. This may be another spermine compound with a mixture of caffeoyl and coumaroyl acylation (missing one oxygen atom). In fraction 29, the spectrum and MS data indicated the presence of two isomers of this spermidine compound, while in fraction 30, the late-eluting peak was more prominent.

[0133] Fractions 29 and 30 also showed the presence of several other peaks of interest. In fraction 29, there was a single MS peak with a MW of 300 (301 negative, 303 positive), while in fraction 30, two additional MS peaks appeared, with MWs of 332 (331 negative, 333 positive) and 380 (379 negative, 381 positive). UV data suggested that these peaks likely originated from flavonoids, although their spectra overlapped to some extent with spermidine compounds. It is important to note that the molecular weights of these compounds indicate that they are not glycosylated.

[0134] Other target compounds in fractions 25 to 30 include, but are not limited to, anthranilic acid, dicaffeoylquinic acid and dicaffeoyltartaric acid.

[0135] Since the major activities were associated with fractions 29 and 30, it is likely that minor flavonoids and isomeric dicaffeoylquinic acids and dicaffeoyl tartaric acids are the more important bioactive substances contributing to the beneficial effects of the composition and extracts therefrom, rather than spermidine compounds.

[0136] However, it is noteworthy that raw fractions 1 to 4 contain very small water-soluble molecules and compounds that are lost or eluted in the early stages of chromatography and are therefore not included in the activity tracking separation. The inventors believe that these fractions contain hexokinase, glucokinase, glucose-6-phosphate, and other small water-soluble molecules (such as magnesium) that are important for the biological activity of the compositions and extracts of the present invention because they provide building blocks for HA production. When prepared by the above-described method, these small water-soluble molecules and compounds are well retained in the compositions and concentrated extracts of the present invention.

[0137] Keratinocyte proliferation bioassay

[0138] This concentrated extract was further analyzed using activity-tracking fractionation to determine which compounds in the extract may be key components responsible for the cell growth and proliferation observed in the HA bioassay.

[0139] The concentrated extract was separated based on polarity to produce 27 further subfractions, labeled as follows: 8-8, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 9-27, C1, C2, C3, C4, C5, C6, C7, C8, L1, L2, L3, LP23, LP25, LP30, LP36, LP37 and LP38.

[0140] The efficacy of the concentrated extract (labeled MYR) and each subfraction was then tested by treating keratinocytes and assessing any changes in keratinocyte proliferation and differentiation and / or changes in morphology. Compounds such as retinol and retinal are known to induce these characteristics, so keratinocytes were also treated with these compounds as positive controls.

[0141] Sample preparation:

[0142] The concentrated extracts of the present invention were prepared for testing by preparing a stock solution concentration of 100 mg / ml, dissolving the extract, and then transferring the precipitate-free supernatant to a new test tube. Subfractions were prepared by adding 100 μl of DMSO to each supplied test tube. Assuming each test tube contained 55 mg of material, the initial stock solution concentration was 550 mg / ml. The subfractions were then diluted with DMSO to a 100 mg / ml working solution.

[0143] method:

[0144] 1. Seed 5000 keratinocytes per well in 200 μl of Kelch+SB+KGF medium into all required wells of two 96-well plates (0.75 M in 30 ml medium or 0.5 M in 20 ml medium).

[0145] 2. Add DF10 medium along the edge of the plate.

[0146] 3. Incubate overnight at 37°C / 5% CO2.

[0147] 4. Add 1 μl of 100 mg / ml working stock of all treatments to 249 μl of Kelch+SB+KGF medium for cell treatment, bringing the final concentration to 400 μg / ml. Perform treatments in 96-well flat-bottom plates and image the culture media 24 hours later.

[0148] 5. Add 1 μl of DMSO to 249 μl of Kelch+SB+KGF medium for cell treatment to a final concentration of 400 μg / ml.

[0149] 6. Add 1 μl of CM5 to 249 μl of Kelch+SB+KGF medium for cell treatment to a final concentration of 1.5 μg / ml.

[0150] 7. Add 1 μl of 0.1 mM retinol to 999 μl of Kelch+SB+KGF medium for cell treatment to a final concentration of 0.1 μM / ml. 250 μl is added to each well under these treatment conditions.

[0151] 8. Add medium DF10 along the edge of the plate.

[0152] 9. Place the extract and subfractions in Kelch medium at 37°C for 24 hours to ensure that the extract does not aggregate in the cell culture medium. Check for clumping; if present, do not use.

[0153] 10. Add 1 μl of 13 mg / ml retinal to 249 μl of Kelch+SB+KGF medium for cell treatment to a final concentration of 52 μg / ml.

[0154] 11. Add 1 μl of 13 mg / ml glycerol medium control to 249 μl of Kelch+SB+KGF medium for cell treatment to a final concentration of 52 μg / ml.

[0155] 12. Remove all culture medium from the keratinocytes and replace with 200 μl of Kelch medium containing each treatment in a 96-well plate. A multichannel plate should be used at this time.

[0156] 13. Add DF10 medium along the edge of the plate.

[0157] 14. Incubate at 37°C / 5% CO2.

[0158] 15. Wells were imaged after 24 hours to assess the visible effects of the treatment.

[0159] PrestoBlue assay method:

[0160] 1. Remove all culture medium.

[0161] 2. Add 150 μl of PrestoBlue and Kelch+SB+KGF medium mixed in a ratio of 1:9 to each well.

[0162] 3. Incubate at 37°C / 5% CO2.

[0163] 4. Read the plate on a SpectraMax ID3 after 1.5-2 hours.

[0164] The above experiments were repeated eight times, and all samples were performed in triplicate.

[0165] Statistical analysis and data graphing were performed using GraphPad Prism 10.0. Figure 11 A summary of the bioassay results is shown. Figure 11 All data in Figure 5 are based on single wells subtracted from the mean of background PrestoBlue signal in the absence of cells.

[0166] result:

[0167] like Figure 11 As shown, compared with the positive control (retinol and retinal), the concentrated extract of the present invention (labeled as MYR) showed stronger keratinocyte proliferation and differentiation.

[0168] Several subfractions showed significant increases in keratinocyte proliferation and differentiation when compared to the DMSO only control group, and when compared to the positive control group, namely subfractions 8-8 to 8-16, 9-27, C3, C4, C5, L1, L2, L3.

[0169] Analysis of active fractions

[0170] Further UV chromatography analysis was performed on some of the most active subfractions, and the results were as follows: Figure 12 and Figure 13 shown.

[0171] about Figure 12 , the chromatogram shows that for fraction D8-11, two UV peaks and two corresponding main mass spectrum peaks are observed. The earlier peak is broader and may represent more than one compound. The main mass of the two peaks is 630 in negative mode and 632 in positive mode. This is almost certainly tricaffeine spermidine (MW 631). Comparison of fraction D8-11 with fractions D8-12 and D8-10 shows that the activity is associated with the later of the two peaks. The later tricaffeine spermidine peak is very sharp, which indicates that this may represent the trans isomer of the compound. The earlier peak may be a mixture of cis isomers and trans isomers.

[0172] Below the earlier peak, there is evidence for two flavonoids, with MS peaks at 310 and 331. The UV spectrum also shows signs of other compounds, not just caffeoyl compounds, at this peak, and these are most likely other flavonoids.

[0173] like Figure 13As shown, the chromatogram indicates that the activity associated with the L2 fraction is unrelated to the tricaffeoylspermidine compound, as the L3 fraction is enriched in these compounds. Further analysis of the L2 fraction revealed the presence of a pair of positive MS 674 peaks, which, after MS / MS (tandem mass spectrometry) analysis, were likely triferulospermidine (i.e., the o-methyl analog of the two peaks in fractions D8-11). In the L fraction, the minor flavonoids were effectively separated from the major flavonoids (i.e., spermine). The higher activity of the L fraction further suggests that the stimulation of HA production, cell proliferation, and differentiation in skin cells is not solely due to spermidine compounds. Other compounds present in the L fraction include dicaffeoylquinic acid and quercetin.

[0174] The activity tracking and separation analysis of the composition and concentrated extract of the present invention clearly showed that the observed therapeutic effects (i.e., increased HA production and enhanced skin cell proliferation and differentiation) were not caused by a single compound.

[0175] Surprisingly, the most beneficial components of the compositions and extracts of the present invention are not proteins, polypeptides or peptidoglycans.

[0176] The inventors believe that the following classes of compounds are present in the compositions and extracts of the present invention and are primarily responsible for the biological activities of the compositions and extracts and for the beneficial effects observed when formulations containing the compositions and extracts as active ingredients are used:

[0177] Spermidine isomers, caffeoyl spermidine compounds, feruloyl spermidine compounds, and compounds that are cinnamic acid conjugates of spermidine, including tricaffeoyl spermidine (N1, N5, N10 tricaffeoyl spermidine), triferuloyl spermidine (N1, N5, N10 triferuloyl spermidine), and dicaffeoyl-p-coumaryl spermidine;

[0178] Hydroxycinnamic acids, including dicaffeoylquinic acid, dicaffeoyltartaric acid and their isomers;

[0179] Flavonoids (flavonols and / or flavones), including alkaloids, glycosides, and aglycones (such as isorhamnetin, rhamnetin, quercetin, and kaempferol);

[0180] Water-soluble small molecules and compounds include: glucose-6-phosphate, hexokinase, glucokinase, proteases, peptidases, amino acids, magnesium and other elements, anthranilic acid and its isomers, etc.

[0181] Lipids, i.e. fatty acids including myristic acid, palmitic acid, linoleic acid, stearic acid and phospholipids, cholesterol and glycolipids.

[0182] The inventors believe that the lipids and / or fatty acids in the extract act as carriers by encapsulating and protecting other bioactive compounds in the extract;

[0183] Although the composition was prepared without the addition of any enzyme-containing ingredients, it still exhibited enzymatic activity in the presence of protein degradation, leading to an analysis of the enzymes present. This revealed that, in addition to the proteases and peptidases shown in the protein gel to be responsible for protein digestion within 72 hours, hexokinase and glucokinase were also identified. These enzymes are known to be present in pollen, bee saliva, and Bacillus subtilis.

[0184] Hyaluronic acid is phosphorylated by glucose through hexokinase to produce the main precursor of HA (i.e., glucose-6-phosphate). Therefore, the inventors believe that the process of coconut water inducing pollen germination will release hexokinase, and these enzymes will then phosphorylate glucose to glucose-6-phosphate. As a result, the HA synthesis pathway can be divided into two independent pathways for synthesizing the two major components of HA (i.e., glucuronic acid and N-acetylglucosamine). The inventors believe that the above-mentioned other components (including flavonoids, hydroxycinnamic acid and spermidine compounds) present in the composition or extract of the present invention will activate one or both pathways, thereby inducing hyaluronic acid synthesis and cell proliferation and differentiation.

[0185] Without being bound by theory, the inventors believe that the unique combination of components present in the compositions and extracts of the present invention results in stimulation of HA production and / or stabilization of HA levels in the skin's extracellular matrix. It is believed that these components, by providing the building blocks for HA production, act synergistically or harmoniously to provide a beneficial effect. The compositions or extracts themselves do not contain HA, but rather contain the components necessary to stimulate HA production. Thus, the compositions or extracts of the present invention provide a gentle and effective method for treating conditions associated with HA loss.

[0186] It is envisioned that isolated and concentrated extracts containing one or more important bioactive components, alone or in combination with other bioactive components, could be produced and then used in a variety of product formulations for more specific and targeted applications.

[0187] preparation

[0188] The composition or extract of the present invention can be incorporated into various pharmaceutical, cosmeceutical or nutritional preparations or compositions comprising one or more carriers and / or suitable excipients.

[0189] In a preferred embodiment of the present invention, the composition or the extract thereof is incorporated into a topical preparation in an amount ranging from about 1% by weight to about 10% by weight. Preferably, the composition or the extract is incorporated into such a product in an amount ranging from about 1% by weight to about 4% by weight.

[0190] According to certain embodiments, various topical cosmetic or pharmaceutical compositions can be provided in the form of ointments, creams, lotions, gels, or other transdermal delivery systems.

[0191] As used herein, an ointment refers to a semisolid preparation comprising an ointment base having one or more active ingredients incorporated or blended (i.e., melted together with the other components of the formulation and cooled with continuous stirring to form a solidified preparation). The ointment base can be in the form of an oily or hydrocarbon base (e.g., petrolatum or a petrolatum / wax combination); an absorbent base that allows the incorporation of an aqueous solution to form a water-in-oil emulsion (e.g., hydrophilic petrolatum), or an absorbent base that allows the incorporation of an additional amount of aqueous solution (e.g., lanolin); a water-removable base that can be diluted with water or an aqueous solution as an oil-in-water emulsion (e.g., hydrophilic ointment, USP); or a water-soluble base that does not contain an oily component (e.g., a polyethylene glycol (PEG) formulation that combines PEG with an average molecular weight of less than 600 with PEG with an average molecular weight of greater than 1000).

[0192] As used herein, a cream refers to a semisolid formulation containing the pollen composition or extract of the present invention dissolved or dispersed in a water-in-oil emulsion or an oil-in-water emulsion or in another type of water-washable matrix. Generally, creams are distinguished from ointments by the ease with which they can be applied / spread on a surface (such as the skin) and the ease with which they can be removed from the treated surface.

[0193] As used herein, lotion refers to a suspension of solid materials in an aqueous medium. Lotions generally have a non-greasy nature and have increased spreadability over large areas of skin compared to ointments, creams, and gels.

[0194] As used herein, gel refers to a semi-solid system comprising a dispersion of small molecules and / or macromolecules in an aqueous liquid medium, which is rendered gel-like by the addition of a gelling agent. Suitable gelling agents include, but are not limited to, synthetic polymers (such as carbomer polymers), cellulose derivatives (such as carboxymethyl cellulose and / or hydroxypropyl methylcellulose), and natural gums (such as tragacanth gum, carrageenan, etc.). The gel preparation may be in the form of a single-phase gel in which the active ingredient or pharmaceutical ingredient is uniformly dispersed throughout the liquid medium without visible boundaries, or in the form of a two-phase gel in which flocculants or small particles of the active ingredient or pharmaceutical ingredient are dispersed in the liquid medium.

[0195] Transdermal preparations can be formed from ointments, creams, or gels in combination with penetration enhancers and are intended for systemic delivery of active or medicinal ingredients. Penetration enhancers include, for example, dimethyl sulfoxide, ethanol, propylene glycol, glycerol, PEG, urea, dimethylacetamide, sodium lauryl sulfate, poloxamers, Spans, Tweens, lecithin, and / or terpenes.

[0196] Other semisolid formulations suitable for cosmetic and / or topical pharmaceutical compositions include pastes (preparations containing a higher proportion of solid material making them more viscous than ointments) and glycerol gelatin (plastic masses containing gelatin, glycerin, water and the active / pharmaceutical ingredient).

[0197] In addition, we are considering developing shampoo or other types of cleansing products. We are also considering developing topical sprays, including sprays suitable for single or multiple applications, such as for the skin, back, neck, arms, legs and torso.

[0198] For oral administration, pollen compositions or extracts derived from pollen compositions, or solid formulations thereof (such as lyophilized powders) can be combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules, or other suitable dosage forms. For example, the composition or extract can be combined with at least one excipient (such as a filler, binder, humectant, disintegrant, solution retarders, absorption enhancer, wetting agent, adsorbent, or lubricant). Other useful excipients include magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starch, carboxymethyl cellulose, microcrystalline cellulose, silica gel, gelatin, and silicon dioxide. It is also possible that oral formulations (such as capsules and tablets) can also include other beneficial bioactive components, such as collagen, probiotics, prebiotics, vitamins, minerals, or other components that can provide additional health benefits. In oral formulations, the amount of the composition or extract included in the formulation can be significantly higher. For example, an oral capsule formulation can contain the composition or extract in an amount of at least 20% by weight, or more preferably at least 50% by weight. It is also contemplated to produce oral capsule formulations containing 100% of the composition or extract of the present invention without any excipients.

[0199] Formulation Examples

[0200] Example 1 (external skin cream)

[0201] A topical skin cream formulation was prepared as follows:

[0202] water 61.89％ Bitter almond (sweet almond) oil 25.00％ Compositions or extracts of the present invention 4.00％ Cetearyl Alcohol 2.00％ Glyceryl Stearate SE 2.00％ Cetearyl Olivate 1.80％ Sorbitan Olivate 1.20％ Sodium stearoyl glutamate 1.00％ Benzyl alcohol 0.70％ Xanthan gum 0.20％ Tocopheryl acetate 0.10％ Dehydroacetic acid 0.06％ citric acid 0.05％

[0203] Example 2 (Vaginal Cream)

[0204] In another embodiment, a topical vaginal cream formulation is prepared as follows:

[0205] Water BP 67.98％ Compositions or extracts of the present invention 4.00％ Cetearyl Alcohol BP 10.00％ Caprylic-Capric Triglyceride USP 10.00％ Polysorbate 60 USP-NF / EP 3.00％ Sorbitan Stearate USP-NF / EP 2.00％ Cetyl Palmitate USP-NF 2.00％ Phenoxyethanol 1.00％ Rose Geranium Oil 0.02％

[0206] Example 3 (Vaginal Cream)

[0207] In another embodiment, a topical vaginal cream formulation is prepared as follows:

[0208]

[0209] Example 3 (Capsule Preparation)

[0210] In another embodiment, the following capsule formulation is prepared:

[0211] Element Wt(%) Compositions or extracts of the present invention 56％ Lactobacillus acidophilus 11.8％ Bifidobacterium 10B / g powder 11.8％ Excipients 20.1％

[0212] Example 4 (Capsule Preparation)

[0213] In another embodiment, the following capsule formulation is prepared:

[0214] Element Wt(%) Compositions or extracts of the present invention 50％ collagen 45％ Excipients 5％

[0215] Example 5 (Lip Balm)

[0216] In another embodiment, the following lip balm formulation was prepared. The formulation was prepared by first melting beeswax, oil, and shea butter, adding fragrance after the mixture was slightly cooled, and finally adding an antioxidant (vitamin E), and then thoroughly mixing the mixture and filling it into a suitable container and letting it stand to set (without a lid to prevent condensation).

[0217] Element Wt(%) Compositions or extracts of the present invention 3.00％ Jojoba oil 34.00％ almond oil 30.00％ beeswax 20.00％ Shea butter 5.00％ Argan oil 4.00％ vanilla essence 3.00％ Vitamin E 1.00％

[0218] Preparation efficacy research

[0219] Lip balm test

[0220] A trial was conducted by individually supervised university students to determine the efficacy of the lip balm formulation of Example 4. The subjects in this trial included 21 postmenopausal women whose lips tended to be dry, chapped, thin, and pale. The subjects had previously used a standard lip balm product two to three times a day for at least three months. This was to ensure that any benefits of the product were attributable to the active ingredient in the product (the extract of the present invention) and not to the base ingredients present in most standard lip balm formulations. The subjects were asked to complete a survey over a four-week period in which they were asked to enter a score from 1 to 5 (1 being the lowest) relating to whether any beneficial changes had occurred in four different parameters: hydration, plumpness, chapped lips, and color of the lips.

[0221] The data from this trial are Figures 9 to 12The data showed that the majority of subjects in the trial noted improvements in each of the four parameters tested. After analyzing the data and excluding subjects who reported a score of 5 for a parameter at the beginning of the trial, the results showed that the vast majority of subjects (approximately 90%) experienced improvements in hydration. Approximately 60% of subjects noted improvements in plumpness, approximately 77% noted improvements in chapped skin, and approximately 60% noted improvements in color. These improvements were observed after two weeks of use of the lip balm formulation containing the extract of the present invention.

[0222] Given that the skin structure of the lips is similar to vaginal skin tissue, it is expected that formulations comprising the extracts of the present invention will produce similar benefits when used to treat symptoms of vaginal atrophy, including vaginal dryness, irritation, redness, and inflammation.

[0223] Vaginal cream test

[0224] Given that the compositions and extracts of the present invention were found to increase hyaluronic acid in keratinocytes and to be beneficial for lip treatment, the vaginal cream formulation of Example 2 was initially tested for safety, potential efficacy, and tolerability before clinical trials were conducted.

[0225] This study was conducted independently by supervised university students. The trial involved 10 postmenopausal women referred by their gynecologists. Each participant was asked to apply the vaginal cream to the vulva and vaginal entrance at night for two weeks. During these two weeks, participants were asked to complete a survey in which they were asked to enter a score on a scale of 1 to 4 or 5 (1 for no symptoms, 4 or 5 for severe symptoms) for four different parameters: vaginal redness, itching, dryness, and thinning / cracks. The survey results and any comments or feedback were sent directly to the gynecologist.

[0226] The test data are as follows Figures 13 to 16 The data showed that the majority of subjects in the trial recorded improvements in each of the four parameters tested.

[0227] Redness is reported on a scale of 1 to 4. Figure 13 This indicates that 50% of subjects reported improvement within two weeks. However, 3 / 10 subjects did not report redness at Week 0, suggesting no potential for improvement. One subject reported initially being asymptomatic but developed redness in Week 1, which resolved by Week 2. One subject continued to experience moderate redness. Excluding subjects who initially reported a score of 1, 5 / 6, or 83.33%, of subjects reported improvement in redness in the vaginal area.

[0228] Itch was reported on a 1 to 5 point scale, where 1 indicated no itching and 5 indicated severe itching. Figure 14It shows that 80% of the subjects reported an improvement in pruritus. One subject reported no pruritus initially, so after excluding this case, the data shows 8 / 9 or 88.89% improvement.

[0229] Dryness was recorded on a 1 to 5 point scale, where 1 indicated no dryness and 5 indicated severe dryness. Figure 15 All subjects or dryness were shown to improve, with 10 / 10 or 100% of the subjects scoring a decrease of at least 1.

[0230] Thinning / cracking was reported on a scale of 1 to 5, with 1 being normal and 5 being severe. Figure 16 This shows that 8 / 10 or 80% of the subjects reported improvement. One subject reported no thinning or cracking at week 0, so after excluding this case, 8 / 9 or 88.89% of the subjects reported improvement.

[0231] Although the survey did not include questions about bladder strength or urinary incontinence, 6 out of 10 participants provided feedback, either via the survey comment section or directly to their gynecologists, stating that they noticed less bladder leakage and that their bladders seemed stronger while using the product.

[0232] Four out of ten subjects also reported improved physiological responses during sexual activity and orgasmic intensity. Similar responses are likely to be seen in men, given that penile tissue also experiences HA degradation during aging. Therefore, the compositions and extracts of the present invention could be used on the penis or orally by men to increase HA production, thereby treating or improving male sexual response.

[0233] These preliminary results indicate that the compositions and extracts of the present invention can be used to treat vaginal atrophy and GSM symptoms. A double-blind placebo-controlled clinical trial of the formulation of the present invention is about to be conducted to verify the efficacy of the formulation in treating GSM in postmenopausal women.

[0234] Advantages

[0235] The pollen compositions and extracts therefrom have several potentially achievable advantages. The pollen compositions and extracts therefrom are natural, mild formulations. Compared to existing products (such as retinol and bakuchiol), they work by a different, milder mechanism of action, which induces apoptosis and triggers an inflammatory / stress response in skin cells. Therefore, the extracts and compositions of the present invention provide a milder, longer-lasting and stable increase in HA levels. The extracts and compositions of the present invention are safe compositions that do not contain allergenic proteins. The formulations of the present invention are easy to incorporate into a variety of product forms. The formulations are effective for treating a variety of skin conditions, including skin aging and GSM symptoms, including urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation and loss of libido, sexual function, sexual response and / or sexual pleasure. It is expected that the formulations of the present invention can also be used to treat other conditions in which stimulating HA production or stabilizing HA is important or helpful.

Claims

1. A pollen composition or an extract derived from a pollen composition, wherein the composition or extract comprises at least glucose-6-phosphate as a substrate or precursor for hyaluronic acid (HA) production.

2. The composition or extract of claim 1, wherein the composition or extract further comprises one or more of hexokinase, glucokinase, protease, peptidase, and magnesium and other elements.

3. The composition or extract according to any one of the preceding claims, wherein the composition or extract further comprises one or more small molecular weight water-soluble molecules and / or compounds selected from the group comprising anthranilic acid and its isomers.

4. The composition or extract according to any one of the preceding claims, wherein the composition or extract further comprises one or more compounds selected from the group comprising flavonoids, flavonols, flavones, alkaloids, aglycones, glycosides, lipids, fatty acids, spermidine and its isomers, caffeoylspermidines, feruloylspermidines and other compounds that are cinnamic acid conjugates of spermidine and hydroxycinnamic acids.

5. The composition or extract of claim 4, wherein the caffeoylspermidine compound is selected from one or more of the group consisting of tricaffeoylspermidine (N1, N5, N10-tricaffeoylspermidine), triferulospermidine (N1, N5, N10-triferulospermidine) and dicaffeoyl-p-coumarinoylspermidine.

6. The composition or extract of claim 4, wherein the hydroxycinnamic acid is selected from the group comprising dicaffeoylquinic acid and dicaffeoyltartaric acid and isomers thereof.

7. The composition or extract according to claim 4, wherein The flavonoid, the flavonol and / or the flavonoid are selected from the group comprising isorhamnetin, rhamnetin, quercetin and kaempferol.

8. The composition or extract of claim 4, wherein the lipid is selected from the group consisting of fatty acids, phospholipids, cholesterol, and glycolipids.

9. The composition or extract of claim 8, wherein the fatty acid is selected from the group comprising myristic acid, palmitic acid, linoleic acid and stearic acid.

10. The composition of any one of the preceding claims, wherein the composition is prepared by a process comprising at least the following steps: a. mixing one or more of raw bee pollen, raw honey and raw propolis with coconut water, coconut oil and / or coconut milk to form a mixture; b. incubating the mixture anaerobically at a temperature between about 25°C and 65°C for about 12 hours to 96 hours; c. freezing and / or lyophilizing the obtained fermentation composition.

11. The extract according to any one of the preceding claims, wherein the extract is prepared by the method of claim 10 with the additional step of concentrating the resulting fermentation composition before step (c).

12. The extract of claim 11, wherein the concentration step comprises centrifugation and collecting the supernatant for freezing and / or lyophilization.

13. The composition or extract according to any one of the preceding claims, wherein the composition or extract is a lyophilized powder composition or extract.

14. A preparation comprising the pollen composition or extract according to any one of the preceding claims and one or more carriers and / or excipients.

15. The preparation according to claim 14, wherein the preparation is an external preparation comprising about 1% to 10% by weight of the pollen composition or extract.

16. The preparation according to claim 14, wherein the preparation is an oral preparation comprising at least 20% by weight of the pollen composition or extract.

17. The formulation of any one of claims 14 to 16, wherein the formulation is used to treat or prevent one or more symptoms of genitourinary syndrome of menopause (GSM), including urinary incontinence, vaginal atrophy, vaginal dryness, irritation, itching, inflammation, lack of sexual response and lack of libido.

18. A method of stimulating the production of hyaluronic acid (HA) in a human subject, the method comprising administering to the subject a therapeutically effective amount of the formulation of claim 14.

19. A method of treating or preventing a condition associated with decreased hyaluronic acid production or degradation of hyaluronic acid, the method comprising administering to the subject a therapeutically effective amount of the formulation of claim 14.

20. Use of a composition or extract according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment or prevention of a condition selected from the group comprising skin diseases, skin aging, genitourinary syndrome of menopause (GSM), dry eyes, connective tissue and joint diseases, and other conditions that may benefit from stimulation or stabilization of hyaluronic acid (HA), said genitourinary syndrome of menopause (GSM) including urinary incontinence, vaginal dryness, vaginal atrophy, irritation, itching, cracking, thinning, redness, inflammation and reduction in libido, sexual function, sexual response and / or sexual pleasure.

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