A pollen composition with whitening and anti-wrinkle effects, a preparation method, a product and applications thereof

Abstract

The application discloses a preparation method of a pollen composition with whitening and anti-wrinkle effects, a product and application thereof, and belongs to the technical field of compositions. The technical problem to be solved is to provide an active component with the double effects of whitening and anti-wrinkle, and the active component is extracted and the component is simple and stable. The technical solution is as follows: at least two or more of pine pollen, sunflower pollen, tea pollen and rose pollen are used as raw materials, the raw materials are physically crushed, then subjected to enzymolysis by using a composite cellulase, and then subjected to hot water extraction, so that whitening components and anti-wrinkle components are enriched, and the prepared pollen composition has excellent whitening effect and anti-wrinkle effect.

Description

Technical Field

[0001] This invention relates to the field of composition technology, specifically to a pollen composition with both whitening and anti-wrinkle effects, its preparation method, the product, and its application. Background Technology

[0002] With increasing environmental stressors (such as UV radiation and pollution) and changing lifestyles, skin aging and pigmentation have become global beauty and health issues. Consumers are increasingly demanding safe, effective, and multifunctional skincare products, with anti-wrinkle (improving elastin and collagen loss) and whitening (inhibiting melanin production) being two core directions in skincare product development. Compared to chemically synthesized active ingredients, natural plant extracts are gaining popularity in the market and among consumers due to their better safety, lower irritation, and rich bioactivity. Pollen, as a traditional nutritional supplement, is considered a highly promising natural cosmetic ingredient due to its rich nutritional components and bioactive substances.

[0003] Relevant patent documents retrieved:

[0004] This document, published in China (CN104862364A) on June 5, 2018, discloses a method for extracting oligopeptide compositions from rapeseed bee pollen and the oligopeptide compositions obtained by this method. The rapeseed bee pollen oligopeptide compositions provided by this invention have high bioavailability and are safer in terms of immunity; it also provides food, health product, or cosmetic compositions containing the rapeseed bee pollen oligopeptide compositions.

[0005] The document, published in China (CN111494294A) on August 7, 2020, discloses a bee pollen facial mask and its preparation method. The mask contains bee pollen enzymatic hydrolysate and a nutrient solution. The nutrient solution contains sea cucumber polysaccharide extract, ginseng extract, poria cocos extract, pearl powder, aloe vera, honey, and hyaluronic acid. The preparation method includes the following steps: (1) mixing bee pollen with water, breaking the cell wall, cooling to room temperature after a boiling water bath, and then adding protease for enzymatic hydrolysis; (2) centrifuging the bee pollen enzymatic hydrolysate, taking the supernatant, mixing it with the nutrient solution, and adding it to the facial mask paper. The resulting broken-cell bee pollen has a high yield and good quality. After enzymatic hydrolysis, the broken-cell bee pollen solution is homogeneous and has a unique aroma, making it convenient for use in various fields such as daily necessities, cosmetics, functional foods, and health products.

[0006] Relevant non-patent literature retrieved: The journal title is *Oral Care Products Industry*, the article title is *Research on Extraction Process of Pine Pollen Extract for Cosmetics*, Volume 32, Publication Date: October 2022. This article discloses an extraction process for Pine Pollen Extract. By analyzing and comparing the inhibition rate of pine pollen extract on IL-1β cytokines, the influence of different extraction processes on the anti-inflammatory efficacy of pine pollen was explored. The measured changes in IL-1β concentration showed that pine pollen extract has a significant effect on inhibiting IL-1β cytokines. Among them, the pine pollen raw material extracted by water extraction process significantly reduced the concentration of IL-1β cytokines, indicating that the pine pollen raw material extracted by water extraction process has better efficacy expression than that extracted by alcohol process.

[0007] Despite the potential of pollen, current technologies face four main bottlenecks when applying it to high-end cosmetics, particularly for achieving both whitening and anti-wrinkle effects: 1) Existing cell-wall breaking technologies are mostly geared towards the food or health supplement industry, aiming to improve nutrient absorption, and are not specifically optimized for the extraction and preservation of whitening and anti-wrinkle active ingredients required in cosmetics; 2) Most existing research focuses on using pollen as a general nutritional additive or utilizing only one of its properties (such as antioxidants), lacking a systematic approach to synergistically extract and retain whitening ingredients (such as tyrosinase inhibitors). 1) The core ingredients for anti-wrinkle (such as anti-oxidation and promoting collagen synthesis); 2) The combination of pollen extract with other chemical whitening agents (such as arbutin and niacinamide) or anti-wrinkle ingredients (such as retinol) is relatively common, but it deviates from the "all-natural" positioning and may increase the risk of skin allergies. Technical solutions that purely utilize the multi-component characteristics of pollen to achieve dual effects are relatively scarce; 3) How to stably add pollen active ingredients to different cosmetic formulations (such as serums, lotions, creams, and masks) and maintain their long-term activity and skin permeability is a technical challenge.

[0008] Therefore, there is an urgent need to develop a pollen composition and preparation method that has both whitening and anti-wrinkle effects. Summary of the Invention

[0009] Addressing the shortcomings of existing technologies, this invention aims to overcome their core deficiencies by providing a highly efficient, gentle method for pollen cell wall disruption and extraction that maximizes the retention of active ingredients. Through specific processes and systematic research, it synergistically enriches active components with both whitening and anti-wrinkle effects from specific pollen combinations. This is not a simple mixture, but ultimately provides an all-natural, synergistic, and highly bioavailable whitening and anti-wrinkle cosmetic solution.

[0010] In order to achieve the above objectives, the present invention aims to provide: A pollen composition with both whitening and anti-wrinkle effects, and related technologies thereof, to solve the technical problems in the prior art such as the complex extraction and compounding of active components with both whitening and anti-wrinkle effects, poor stability, or combinations thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as mixing and stirring, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The terms "optional / arbitrary" or "optionally / arbitrarily" refer to events or situations described subsequently that may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: The term "cell wall breaking" as used in this article refers to the process of breaking down cell walls through physical or biological techniques, making it easier for the body to absorb active ingredients.

[0016] The term "enzymatic hydrolysis" as used in this article refers to the decomposition process catalyzed by enzymes, which is the process of breaking down large molecular compounds into smaller molecular compounds.

[0017] The term "reduced pressure concentration" as used in this article refers to a concentration technique that lowers the boiling point of a liquid by reducing pressure, thereby evaporating the solvent at a lower temperature. Its core principle is to use a vacuum environment to allow the liquid to evaporate at a temperature below its boiling point under normal pressure, thus avoiding the damage of heat-sensitive substances by high temperatures.

[0018] The term "freeze-drying" as used in this article refers to a drying technology that removes moisture through freezing and vacuum sublimation. Its core principle is to freeze the water-containing material into solid ice, and then, under vacuum conditions, directly sublimate the ice into water vapor, thereby achieving drying.

[0019] In a first aspect, the present invention provides: a pollen composition having both whitening and anti-wrinkle effects, wherein the pollen composition is selected from at least two or more of pine pollen, sunflower pollen, camellia pollen and rose pollen.

[0020] Preferably, the pollen composition is a mixture of pine pollen and camellia pollen, or a mixture of pine pollen, camellia pollen and rose pollen, or a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen.

[0021] More preferably, the pollen is a mixture of pine pollen and camellia pollen in a mass ratio of 1-3:1; or the pollen is a mixture of pine pollen, camellia pollen and rose pollen in a mass ratio of 1-3:1:1; or the pollen is a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen in a mass ratio of 1-5:1:1:1.

[0022] Preferably, the mass ratio of pine pollen to camellia pollen is any value or range between 1 and 3:1, and can be selected from 1:1, 2:1 or 3:1; more preferably 1:1.

[0023] Preferably, the mass ratio of pine pollen, camellia pollen and rose pollen is any value or range between 1 and 3:1:1, and can be selected from 1:1:1, 2:1:1 or 3:1:1; more preferably 1:1:1.

[0024] Preferably, the mass ratio of pine pollen, sunflower pollen, camellia pollen and rose pollen is any point or range between 1 and 5:1:1:1, and can be selected from 1:1:1:1, 2:1:1:1, 3:1:1:1, 4:1:1:1 or 5:1:1:1. More preferably, the pollen is a mixture of pine pollen, sunflower pollen, camellia pollen, and rose pollen in a mass ratio of 1:1:1:1, 2:1:1:1, or 5:1:1:1.

[0025] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred option is that the pollen is selected from one or more of pine pollen, sunflower pollen, camellia pollen, and rose pollen. This technical solution, while addressing the technical problem of "antioxidant properties," further addresses the technical problem of "anti-wrinkle properties."

[0026] The second preferred option is a mixture of pine pollen, sunflower pollen, camellia pollen, and rose pollen in a mass ratio of 1-5:1:1:1. This technical solution, while addressing the technical issue of "antioxidant properties," further addresses the technical issues of "proliferation of photo-aged cells and enhancement of type I and type III collagen."

[0027] Secondly, the present invention provides a method for preparing the above-mentioned pollen composition.

[0028] The preparation method includes the following steps: Pollen cell wall disruption → formulation → preparation of enzymatic hydrolysis system → pH adjustment → enzymatic hydrolysis → inactivation → extraction → centrifugation → collection of supernatant → vacuum concentration → freeze drying.

[0029] The preparation method specifically includes the following steps: (1) Take pollen and break the cell wall to obtain pollen after cell wall breaking; (2) Add cellulase to the pollen after cell wall disruption, then add pure water to carry out enzymatic hydrolysis to obtain hydrolyzed pollen; (3) After the pollen is inactivated by enzymatic hydrolysis, the solutions are combined, centrifuged, the supernatant is collected, concentrated and dried to obtain the pollen composition.

[0030] The pollen is selected from at least two or more of pine pollen, sunflower pollen, camellia pollen, and rose pollen.

[0031] Preferably, the pollen is a mixture of pine pollen and camellia pollen, or a mixture of pine pollen, camellia pollen and rose pollen, or a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen.

[0032] More preferably, the pollen is a mixture of pine pollen and camellia pollen in a mass ratio of 1-3:1; or the pollen is a mixture of pine pollen, camellia pollen and rose pollen in a mass ratio of 1-3:1:1; or the pollen is a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen in a mass ratio of 1-5:1:1:1.

[0033] Preferably, the mass ratio of pine pollen to camellia pollen is any value or range between 1 and 3:1, and can be selected from 1:1, 2:1 or 3:1; more preferably 1:1.

[0034] Preferably, the mass ratio of pine pollen, camellia pollen and rose pollen is any value or range between 1 and 3:1:1, and can be selected from 1:1:1, 2:1:1 or 3:1:1; more preferably 1:1:1.

[0035] Preferably, the mass ratio of pine pollen, sunflower pollen, camellia pollen and rose pollen is any point or range between 1 and 5:1:1:1, and can be selected from 1:1:1:1, 2:1:1:1, 3:1:1:1, 4:1:1:1 or 5:1:1:1. More preferably, the pollen is a mixture of pine pollen, sunflower pollen, camellia pollen, and rose pollen in a mass ratio of 1:1:1:1, 2:1:1:1, or 5:1:1:1.

[0036] The amount of cellulase added is 0.5-1.0% of the substrate weight; preferably, the amount of cellulase added is 0.6-0.9% of the substrate weight; more preferably, the amount of cellulase added is 0.7-0.8% of the substrate weight. Preferably, the amount of cellulase added is any value or range between 0.5% and 1.0% of the substrate weight, and can be selected from 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. More preferably, the amount of cellulase added is 0.8% of the substrate weight.

[0037] The mass ratio of pollen to pure water in step (2) is 1:15-30; preferably 1:18-25; and even more preferably 1:20-22.

[0038] Preferably, the mass ratio of pollen to pure water is any value or range between 1:15 and 30, and can be selected from 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22; 1:23, 1:24 or 1:25.

[0039] More preferably, the mass ratio of pollen to pure water is 1:20.

[0040] The pH value of the pure water is 5.5-6.5; preferably 6.0-6.2. Preferably, the pH value of the pure water is any point or range between 5.5 and 6.5, and can be selected from 5.5, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5.

[0041] More preferably, the pH value of the pure water is 6.0.

[0042] The enzymatic hydrolysis temperature is 50-55℃; preferably 52-55℃. Preferably, the enzymatic hydrolysis temperature is any point or range between 50-55℃, and can be selected from 50℃, 51℃, 52℃, 53℃, 54℃ or 55℃.

[0043] More preferably, the enzymatic hydrolysis temperature is 52°C.

[0044] The enzymatic hydrolysis time is 4-6 hours; preferably 4.5-5.5 hours. Preferably, the enzymatic hydrolysis time is any value or range between 4 and 6 hours, and can be selected from 4 hours, 4.5 hours, 5 hours, 5.5 hours or 6 hours.

[0045] More preferably, the enzymatic hydrolysis time is 5°C.

[0046] The sterilization temperature is 85-95℃; preferably 90-95℃. Preferably, the sterilization temperature is any point or range between 85-95℃, and can be selected from 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃ or 95℃.

[0047] More preferably, the sterilization temperature is 90°C.

[0048] The sterilization time is 8-15 minutes; preferably 10-12 minutes. Preferably, the sterilization temperature is any point or range between 85-95°C, and can be selected from 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0049] More preferably, the sterilization time is 10 minutes.

[0050] The extraction method is either reflux extraction or ultrasonic extraction.

[0051] The extraction time for the reflux extraction is 1-2 hours; preferably 1 hour.

[0052] The ultrasonic extraction temperature is 80-90℃ for 1 hour; preferably, the ultrasonic extraction temperature is 85℃ for 1 hour.

[0053] The centrifugation speed is 4000-6000 rpm / min; preferably 4500-5500 rpm / min.

[0054] Preferably, the centrifugation speed is any point or range between 4000-6000 rpm / min, and can be selected from 4000 rpm / min, 4200 rpm / min, 4500 rpm / min, 4800 rpm / min, 5000 rpm / min, 5200 rpm / min, 5500 rpm / min, 5800 rpm / min or 6000 rpm / min.

[0055] More preferably, the centrifugation speed is 5000 rpm / min.

[0056] The centrifugation time is 8-15 minutes; preferably 10-12 minutes. Preferably, the centrifugation time is any point or range between 8 and 15 minutes, and can be selected from 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0057] More preferably, the centrifugation time is 10 minutes.

[0058] Thirdly, the present invention provides: the application of the above-mentioned pollen composition in the preparation of a cosmetic product, wherein the cosmetic product has both whitening and anti-wrinkle effects; the product is an oral or topical product. Fourthly, the present invention provides a product that has both whitening and anti-wrinkle effects, the product comprising the above-mentioned pollen composition and acceptable excipients in the product.

[0059] In this invention, Examples 1-3 at least support the protection range of enzyme addition amount, pollen to pure water mass ratio, pure water pH value, enzymatic hydrolysis temperature and time, sterilization temperature and time, centrifugation speed and time.

[0060] The "enzyme addition amount" mentioned above is derived from the corresponding technical features of 75mg, 120mg, 150mg, etc., in the foregoing explanation and / or Examples 1-3, summarized by the common feature "the amount of cellulase added is 0.5-1.0% of the substrate weight". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature cellulase and its addition amount, substantially equivalent technical means, and technical means that can replace cellulase and its addition amount based on the existing technical level within the scope of conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing cellulase with hemicellulase, pectinase, etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0061] The "mass ratio of pollen to pure water" mentioned herein is derived from the corresponding technical features 1:20, 1:15, 1:30, etc., in the foregoing explanation and / or Examples 1-3, summarized by the common feature "the mass ratio of pollen to pure water is 1:15-30". Therefore, those skilled in the art can reasonably infer that the technical features pollen, pure water, and their subordinate concepts, substantially equivalent technical means, and technical means that can replace pure water within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention. For example, replacing pure water with deionized water, purified water, etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0062] The "enzymatic hydrolysis temperature and time" mentioned above are derived from the corresponding technical features of 50℃, 55℃, 52℃ and 4h, 5h and 6h in the foregoing explanation and / or Examples 1-3, summarized by the common feature "the enzymatic hydrolysis temperature is 50-55℃ and the enzymatic hydrolysis time is 4-6 hours". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature temperature and time, the substantially equivalent technical means, and the technical means that can replace the enzymatic hydrolysis temperature and time based on the existing technical level within the scope of conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, if the enzymatic hydrolysis temperature is replaced with 51℃ or 54℃, or the enzymatic hydrolysis time is replaced with 4.2h or 4.6h, while other technical features remain unchanged, it still falls within the protection scope of this invention.

[0063] The "centrifugation speed and time" mentioned herein are derived from the corresponding technical features of 5000 rpm / min, 4000 rpm / min, 6000 rpm / min, and 10 min, 15 min, and 8 min in the foregoing explanation and / or Examples 1-3, summarized by the common feature "centrifugation speed is 4000-6000 rpm / min, and centrifugation time is 8-15 min". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical features temperature and time, substantially equivalent technical means, and technical means that can replace speed and time based on existing technology and conventional technical means and common knowledge should all fall within the scope of protection of this invention. For example, replacing the centrifugation speed with 5500 rpm / min or 5600 rpm / min, or replacing the centrifugation time with 9 min or 12 min, while keeping other technical features unchanged, still falls within the scope of protection of this invention.

[0064] Examples 4-11 of this invention at least support the protection scope of pollen species and mass ratio.

[0065] The "pollen species and mass ratio" mentioned herein is derived from the aforementioned explanation and / or the corresponding technical features in Examples 4-11, where the mass ratios of pine pollen, sunflower pollen, camellia pollen, and rose pollen are 1:1:1:1, 2:1:1:1, or 5:1:1:1, summarized by the common feature "the mass ratio of pollen to pure water is 1-5:1:1:1". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical features pollen, pure water, and mass ratio, the substantially equivalent technical means, and the technical means that can replace the pollen species and mass ratio based on existing technology and conventional technical means and common knowledge should all fall within the scope of protection of this invention. For example, replacing the pollen mass ratio with 3:1:1:1, 4:1:1:1, etc., while keeping other technical features unchanged, still falls within the scope of protection of this invention.

[0066] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in terms of anti-oxidation and anti-wrinkle.

[0067] According to experimental tests, when the pollen composition obtained by fermenting pine pollen, sunflower pollen, camellia pollen and rose pollen in combination is at a concentration of 5 mg / ml, the ROS level inhibition rate is increased to more than 20%; the maximum proliferation rate of photo-aged cells is increased to more than 40%; the maximum enhancement rate of type I collagen is increased to more than 20%; and the maximum enhancement rate of type I collagen is increased to more than 15%.

[0068] Furthermore, based on the present invention: Based on the comparison of Examples 1-11 and Comparative Examples 1-4, the present invention employs a combination of at least two or more of pine pollen, sunflower pollen, camellia pollen, and rose pollen to achieve new technical effects with better antioxidant and anti-wrinkle effects. The combined technical effect is superior to the sum of the effects of each individual technique.

[0069] In addition, the present invention employs the following methods in its implementation: (1) Extraction process combining enzymatic hydrolysis and hot water extraction This patented process involves physical crushing of pollen cells, followed by enzymatic hydrolysis using a complex cellulase, and then hot water extraction. This process does not result in a "hodgepodge" extract, but rather concentrates whitening and anti-wrinkle components. The whitening effect of the prepared pollen composition is comparable to that of glycyrrhizin, a gold standard whitening ingredient, and its anti-wrinkle effect is comparable to that of palmitoyl tripeptide-5, a high-end anti-aging ingredient.

[0070] (2) Optimal combination of raw materials Based on a series of studies and verifications, a powerful synergistic effect between different pollen varieties has been discovered. Most products on the market that simultaneously claim to whiten and anti-wrinkle effects are formulated by mixing extracts or chemical ingredients from different sources. However, the sole ingredient in this patent is pollen, which, through a specific process, becomes a complete "super ingredient" with dual benefits. In terms of anti-wrinkle effects, the composition exhibits strong antioxidant properties and promotes the synthesis of collagen and elastin in fibroblasts. In terms of whitening, the composition significantly inhibits melanin production and tyrosinase activity. Detailed Implementation

[0071] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0072] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0073] Example 1: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. Specifically, the steps include the following: Pine pollen and camellia pollen were separately cell-wall broken to obtain pollen concentrate, which was then mixed at a mass ratio of 1:1 to obtain a pollen composition. 15.0 g of the pollen composition was weighed and placed in an Erlenmeyer flask. 120 mg of cellulase was added at a ratio of 4000 U / g of material, and pure water with pH 6 was added at a material-to-liquid ratio of 1:20. After shaking well, the mixture was placed in a 52℃ constant temperature shaking water bath for 5 h of enzymatic hydrolysis. After hydrolysis, the enzyme was inactivated at 90℃ for 10 min, and then refluxed in a round-bottom flask for 1 h, maintaining a gentle boiling state throughout. After reflux extraction, the solutions were combined, centrifuged at 5000 rpm / min for 10 min, and the supernatant was collected. The supernatant was concentrated under reduced pressure and freeze-dried to obtain the pollen enzymatic extract.

[0074] Example 2: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. Specifically, the steps include the following: Pine pollen and camellia pollen were separately cell-wall broken to obtain pollen concentrate, which was then mixed at a mass ratio of 1:1 to obtain a pollen composition. 15.0 g of the pollen composition was weighed and placed in an Erlenmeyer flask. 75 mg of cellulase was added at a ratio of 4000 U / g of material, and pure water with a pH of 5.5 was added at a material-to-liquid ratio of 1:15. After shaking well, the mixture was placed in a 50℃ constant temperature shaking water bath for enzymatic hydrolysis for 6 h. After enzymatic hydrolysis, the enzyme was inactivated at 85℃ for 15 min, and then extracted by ultrasonication at 85℃ for 1 h in a round-bottom flask, maintaining a gentle boiling state throughout the extraction. After reflux extraction, the solutions were combined and centrifuged at 4000 rpm / min for 15 min. The supernatant was collected. The supernatant was concentrated under reduced pressure and freeze-dried to obtain the pollen enzymatic extract.

[0075] Example 3: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. Specifically, the steps include the following: Pine pollen and camellia pollen were separately cell-wall broken to obtain pollen concentrate, which was then mixed at a mass ratio of 1:1 to obtain a pollen composition. 15.0 g of the pollen composition was weighed and placed in an Erlenmeyer flask. 150 mg of cellulase was added at a ratio of 4000 U / g of material, and pure water with a pH of 6.5 was added at a material-to-liquid ratio of 1:30. After shaking well, the mixture was placed in a 55℃ constant temperature shaking water bath for enzymatic hydrolysis for 4 h. After hydrolysis, the enzyme was inactivated at 95℃ for 8 min, and then refluxed in a round-bottom flask for 1 h, maintaining a gentle boiling state throughout. After reflux extraction, the solutions were combined, centrifuged at 6000 rpm / min for 8 min, and the supernatant was collected. The supernatant was concentrated under reduced pressure and freeze-dried to obtain the pollen enzymatic extract.

[0076] Example 4: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with sunflower pollen and camellia pollen; otherwise, they are the same as in Example 1.

[0077] Example 5: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen and sunflower pollen, otherwise it is the same as Example 1.

[0078] Example 6: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen, sunflower pollen and camellia pollen, and mixed in a mass ratio of 1:1:1. Everything else is the same as in Example 1.

[0079] Example 7: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen, sunflower pollen and rose pollen, mixed in a mass ratio of 1:1:1, otherwise the same as Example 1.

[0080] Example 8: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen, camellia pollen and rose pollen, mixed in a mass ratio of 1:1:1, otherwise the same as Example 1.

[0081] Example 9: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen, sunflower pollen, camellia pollen and rose pollen, mixed in a mass ratio of 1:1:1:1, otherwise the same as Example 1.

[0082] Example 10: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen, sunflower pollen, camellia pollen and rose pollen, and mixed in a mass ratio of 2:1:1:1. Otherwise, it is the same as Example 1.

[0083] Example 11: A method for preparing a pollen composition with both whitening and anti-wrinkle effects. The only difference from Example 1 is that pine pollen and camellia pollen are replaced with pine pollen, sunflower pollen, camellia pollen and rose pollen, and mixed in a mass ratio of 5:1:1:1. Otherwise, it is the same as Example 1.

[0084] Comparative Example 1 The only difference from Example 1 is that only pine pollen is used; otherwise, it is the same as Example 1.

[0085] Comparative Example 2 The only difference from Example 1 is that only sunflower pollen is used; otherwise, it is the same as Example 1.

[0086] Comparative Example 3 The only difference from Example 1 is that only camellia pollen is used; otherwise, it is the same as Example 1.

[0087] Comparative Example 4 The only difference from Example 1 is that only rose pollen is used; otherwise, it is the same as Example 1.

[0088] Effect test: Example 1: Cellular antioxidant activity - Measurement of reactive oxygen species (ROS) levels HepG2 cell culture conditions: Incubation in DMEM low-glucose medium (hereinafter referred to as medium) containing 10% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 5% CO2.

[0089] Log-phase HepG2 cells were seeded into 24-well plates and cultured for 24 h. Three groups were established: a normal control group (with added culture medium), an oxidative stress model group (with culture medium containing 1.0 mM H2O2), and a sample group (with culture medium containing 1.0 mM H2O2 and the sample). Each group had three parallel wells. The old culture medium was discarded, and the cells were washed once with PBS. 100 μL of culture medium was added to each well in the normal control group, 100 μL of culture medium containing 1.0 mM H2O2 was added to each well in the model group, and 100 μL of culture medium containing different concentrations of the sample and 1.0 mM H2O2 was added to each well in the sample group. All wells were then incubated for another 24 h. Discard the culture medium, wash the cells twice with PBS, add 0.5 mL of 10 μmol / L 2',7' dichlorofluorescein bis(SZ) salt (DCFH-DA) to each well, incubate at 37°C in the dark for 20-30 min, then wash the cells three times with PBS buffer, add 0.5 mL of serum-free culture medium to each well, and measure the fluorescence intensity (F) using a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 525 nm). Discard the supernatant, wash the cells once with PBS, and perform subsequent operations on ice. Add 100 μL of lysis buffer (containing 1 mM PMSF) to each well to ensure sufficient contact between the lysis buffer and the cells. Pipette the cells off the plate and transfer them to a 1.5 mL EP tube. Centrifuge at 12000 rpm for 5 min, collect the supernatant (cell protein solution), transfer it to a new 1.5 mL EP tube, and determine the total protein content according to the Beyotime BCA assay kit (catalog number: P0010). If the protein concentration is high, dilute 2-4 times before measurement. The ROS level is expressed as the ratio of fluorescence intensity F to total protein content (denoted by "R"). The inhibition rate of ROS level is calculated using the following formula.

[0090] The inhibition rate of ROS level (%) = (R 模型组 -R 样品组 ) / R 模型组 100.

[0091] The test results are shown in Table 1 below.

[0092] Table 1

[0093] Note: "-" indicates no inhibitory effect. When the positive control (L-ascorbic acid) inhibited the ROS level by 20.82%, the concentration of the positive control group was 0.05 mg / ml. When the concentration exceeded 0.05 mg / ml, the inhibitory effect of L-ascorbic acid on ROS began to decrease. When the concentration exceeded 4 mg / ml, the cells showed more severe damage than the model group.

[0094] As shown in Table 1, the free radical scavenging ability of the enzymatic hydrolysates of each pollen combination was evaluated. The IC50 value for scavenging DPPH free radicals was determined using a chemical evaluation method; the lower the IC50 value, the higher the antioxidant capacity. The inhibition rate of reactive oxygen species (ROS) was determined using a cellular evaluation method, which better reflects physiological relevance; the higher the ROS inhibition rate, the stronger the cellular antioxidant capacity. According to the research results, among the enzymatic hydrolysates of single pollen, camellia pollen had the strongest antioxidant capacity, followed by pine pollen and sunflower pollen. Rose pollen had the weakest cellular antioxidant capacity. The antioxidant capacity was significantly enhanced after combination. The best effect was achieved by combining pine pollen:sunflower pollen:camellia pollen:rose pollen = 1:1:1:1, which is the pollen composition prepared in Example 9.

[0095] Example 2: Assay of Skin Fibroblast Proliferation HFF1 cell culture conditions: Incubation in DMEM high glucose medium (hereinafter referred to as medium) containing 15% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 5% CO2.

[0096] Logarithmic growth phase cells were seeded into 96-well plates and cultured for 24 h. Three groups were set up: a normal control group (culture medium), a model group (culture medium irradiated with UVB), a sample group (culture medium containing the test sample irradiated with UVB), and a positive control group (culture medium containing the positive control irradiated with UVB). Each group had three parallel wells. The appropriate culture medium was added to each well, the old medium was discarded, and the cells were washed once with PBS. Each well was then covered with PBS. The normal control group was covered with aluminum foil. The UVB radiation dose was 300 mJ / cm². 2 Irradiate for 20 min, discard PBS, and then add culture medium and culture medium containing the test sample, respectively. Incubate at 37℃ with 5% CO2 for 24 h. Prepare CCK8 incubation solution by adding 100 μl of CCK8 solution to 1 ml of culture medium according to the Beyotime kit instructions, mix well, discard the supernatant after incubation, wash once with PBS, add 110 μl of CCK8 incubation solution to each well, and incubate for 2 h. After incubation, measure OD450 and calculate cell proliferation rate using the following formula.

[0097] Cell proliferation rate (%) = (OD value of sample well - OD value of normal well) / OD value of normal well × 100.

[0098] The test results are shown in Table 2.

[0099] Table 2

[0100] Note: The effect of extracts from each pollen combination on the proliferation of photoaged cells increases with increasing concentration, but the effect of palmitoyl tripeptide on the proliferation of photoaged cells decreases with increasing concentration.

[0101] As shown in Table 2, except for rose pollen, the enzymatic hydrolysates of all pollen combinations exhibited strong proliferative effects on photoaged cells, with effects stronger than palmitoyl tripeptide-5 (a high-end anti-wrinkle ingredient used in cosmetics). Among single pollen, pine pollen hydrolysate showed the strongest proliferative effect, while sunflower and camellia pollen hydrolysates showed similar proliferative effects. Among pollen combinations, the hydrolysate obtained from a pine pollen:sunflower pollen:camellia pollen:rose pollen = 1:1:1:1 ratio was the strongest (i.e., the pollen composition prepared in Example 9). Rose pollen itself has relatively weak anti-wrinkle proliferative activity, but it provides excellent auxiliary effects, enhancing the overall anti-wrinkle activity of the combination.

[0102] Example 3: Type I collagen content in skin fibroblasts HFF1 cell culture conditions: Incubation in DMEM high glucose medium (hereinafter referred to as medium) containing 15% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 5% CO2.

[0103] HFF1 cells in the logarithmic growth phase were seeded into 24-well plates and cultured for 24 h. Three groups were set up: a normal control group (culture medium), a model group (culture medium irradiated with UVB), a sample group (culture medium containing the test sample irradiated with UVB), and a positive control group (culture medium containing the positive control irradiated with UVB). Three parallel wells were prepared for each group. The corresponding culture medium was added to each well, the old medium was discarded, and the cells were washed once with PBS. Each well was then covered with PBS. The normal control group was covered with aluminum foil. The UVB radiation dose was 300 mJ / cm². 2 Irradiate for 20 min, discard PBS, and then add culture medium and culture medium containing the test sample, respectively. Incubate at 37℃ with 5% CO2 for 24 h. Collect the supernatant from each well into a 1.5 ml centrifuge tube, centrifuge at 3000 rpm / min for 5 min, and collect the supernatant for analysis. Detect the type I collagen concentration in the supernatant of each well according to the instructions of the human type I collagen kit. "C" represents the collagen concentration. The formula for calculating the increase rate of type I collagen is as follows: Increase rate of type I collagen (%) = (C 样品 -C 模型 ) / C 模型 ×100 The test results are shown in Table 3.

[0104] Table 3

[0105] As shown in Table 3, except for rose pollen, the enzymatic hydrolysates of all pollen combinations showed an enhancing effect on type I collagen. Among single pollen, the enhancing effects of pine pollen and sunflower pollen hydrolysates were similar, while the enhancing effect of camellia pollen hydrolysate was weaker. Among the pollen combinations, the hydrolysate resulting from the combination of pine pollen:sunflower pollen:camellia pollen:rose pollen = 2:1:1:1 (i.e., the pollen composition prepared in Example 10) was the strongest, and the combined effect was significantly stronger than that of single pollen hydrolysates.

[0106] Example 4: Type III collagen content in fibroblasts HFF1 cell culture conditions: Incubation in DMEM high glucose medium (hereinafter referred to as medium) containing 15% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 5% CO2.

[0107] HFF1 cells in the logarithmic growth phase were seeded into 24-well plates and cultured for 24 h. Three groups were set up: a normal control group (culture medium), a model group (culture medium irradiated with UVB), a sample group (culture medium containing the test sample irradiated with UVB), and a positive control group (culture medium containing the positive control irradiated with UVB). Three parallel wells were prepared for each group. The corresponding culture medium was added to each well, the old medium was discarded, and the cells were washed once with PBS. Each well was then covered with PBS. The normal control group was covered with aluminum foil. The UVB radiation dose was 300 mJ / cm². 2 Irradiate for 20 min, discard PBS, and then add culture medium and culture medium containing the test sample, respectively. Incubate at 37℃ with 5% CO2 for 24 h. Collect the supernatant from each well into a 1.5 ml centrifuge tube, centrifuge at 3000 rpm / min for 5 min, and collect the supernatant for analysis. Detect the type III collagen concentration in the supernatant of each well according to the instructions of the human type III collagen kit. Let "C" represent collagen concentration. The formula for calculating the increase rate of type III collagen is as follows: Increase rate of type III collagen (%) = (C 样品 -C 模型 ) / C 模型 ×100 The test results are shown in Table 4.

[0108] Table 4

[0109] As shown in Table 4, except for rose pollen, the enzymatic hydrolysates of all pollen combinations showed an enhancing effect on type III collagen. Among single pollen, sunflower pollen hydrolysate showed the strongest enhancing effect, followed by camellia pollen hydrolysate and pine pollen hydrolysate. Among pollen combinations, the hydrolysate of the combination of pine pollen:sunflower pollen:camellia pollen:rose pollen = 2:1:1:1 (i.e., the pollen composition prepared in Example 10) was the strongest, and the combined effect was significantly stronger than that of single pollen hydrolysates.

[0110] Example 5: Elastin content in fibroblasts HFF1 cell culture conditions: Incubation in DMEM high glucose medium (hereinafter referred to as medium) containing 15% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 5% CO2.

[0111] HFF1 cells in the logarithmic growth phase were seeded into 24-well plates and cultured for 24 h. Three parallel wells were set up for each group: a normal control group (culture medium), a model group (culture medium irradiated with UVB), a sample group (culture medium containing the test sample irradiated with UVB), and a positive control group (culture medium containing the positive control irradiated with UVB). The corresponding culture medium was added to each well, the old medium was discarded, and the cells were washed once with PBS. Each well was then covered with PBS. The normal control group was covered with aluminum foil. The UVB radiation dose was 300 mJ / cm². 2 Irradiate for 20 min, discard PBS, and then add culture medium and culture medium containing the test sample, respectively. Incubate at 37℃ with 5% CO2 for 24 h. Collect the supernatant from each well into a 1.5 ml centrifuge tube, centrifuge at 3000 rpm / min for 5 min, and use the supernatant for analysis. Detect the elastin concentration in the supernatant of each well according to the elastin kit instructions. Let "C" represent the elastin concentration. The elastin enhancement rate is calculated using the following formula: Elastin increase rate (%) = (C 样品 -C 模型 ) / C 模型 ×100 The test results are shown in Table 5.

[0112] Table 5

[0113] As shown in Table 5, the enzymatic extracts from each pollen combination all exhibited a boosting effect on type III collagen, with most pollen hydrolysates showing a stronger boosting effect than palmitoyl tripeptide-5. Among single pollen sources, pine pollen hydrolysate showed the strongest boosting effect, followed by sunflower pollen hydrolysate and camellia pollen hydrolysate. In pollen combinations, the hydrolysates from combinations of pine pollen:sunflower pollen:camellia pollen:rose pollen = 1-5:1:1:1 all showed strong effects, with the combined effect significantly stronger than that of single pollen hydrolysates.

[0114] Example of effect 6: Inhibition of melanin production in melanocytes B16F10 cell culture conditions: Incubation in DMEM high glucose medium (hereinafter referred to as medium) containing 10% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 7% CO2.

[0115] Logarithmic growth phase cells were seeded into 12-well plates and cultured for 24 h. A normal control group (culture medium), a sample group (culture medium containing different concentrations of samples), and a positive control group (culture medium containing positive control) were set up, with three parallel wells in each group. The culture supernatant was discarded. Culture medium was added to the normal control wells, culture medium containing the samples was added to the sample wells, and culture medium containing the positive control was added to the positive control wells. Cells were cultured for 48 h. Cells were collected from the bottom using a pipette and transferred to 1.5 ml centrifuge tubes. The tubes were centrifuged for 5 min using a handheld centrifuge. Cells were washed twice with PBS, centrifuged for 5 min after each wash, and the supernatant was discarded. 1 ml of 1 M NaOH solution containing 10% DMSO was added to each well, and the tubes were incubated at 80°C for 1 h. After cooling to room temperature, the solution was mixed, and 200 μL of the solution was added to a 96-well plate. The OD value at 405 nm was measured using a multi-mode microplate reader. The melanin production inhibition rate was calculated as follows: Melanin production inhibition rate (%) = (OD 正常孔 -OD 样品孔 ) / OD 正常孔 100 The test results are shown in Table 6.

[0116] Table 6

[0117] As shown in Table 6, the enzymatic extracts from each pollen combination inhibited melanin production. Among single pollen samples, the enzymatic extract from rose pollen showed the strongest inhibitory effect, followed by sunflower pollen and pine pollen. Among the pollen combinations, the enzymatic extract from the combination of pine pollen:sunflower pollen:camellia pollen:rose pollen = 2:1:1:1 was the strongest, and the combined effect was significantly stronger than that of single pollen extracts.

[0118] Example of effect 7: Inhibitory effect on tyrosinase activity in melanocytes B16F10 cell culture conditions: Incubation in DMEM high glucose medium (hereinafter referred to as medium) containing 10% fetal bovine serum, and cultured in a cell culture incubator at 37°C with 7% CO2.

[0119] Logarithmic growth phase cells were seeded into 12-well plates and cultured for 24 h. A normal control group (culture medium), a sample group (culture medium containing different concentrations of samples), and a positive control group (culture medium containing positive control) were set up, with 3 parallel wells in each group. The culture supernatant was discarded. Culture medium was added to the normal control wells, culture medium containing the samples was added to the sample wells, and culture medium containing the positive control was added to the positive control wells. Cells were cultured for 48 h. Cells were collected from the bottom using a pipette and transferred to 1.5 ml centrifuge tubes. The tubes were centrifuged for 5 min using a handheld centrifuge. Cells were washed twice with PBS, centrifuged for 5 min after each wash, and the supernatant was discarded. 1 ml of 1% Triton X-100 solution was added to each tube for lysis. The tubes were quickly incubated at -80°C for 30 min, then thawed at room temperature for approximately 20 min. After mixing, the cells were incubated at 4°C for 20 min, centrifuged, and the supernatant was collected. 100 μl of the cell extract was transferred to a 96-well plate, and 100 μl of L-L-DOPA solution was added and mixed. The plate was incubated at 37°C for 1 h. The OD value at 475 nm was measured using a multi-mode microplate reader. The inhibition rate of cellular tyrosinase activity was calculated as follows: Tyrosinase activity inhibition rate (%) = (OD 正常孔 -OD 样品孔 ) / OD 正常孔 100 The test results are shown in Table 7.

[0120] Table 7

[0121] As shown in Table 7, the enzymatic extracts from all pollen combinations inhibited cellular tyrosinase activity. Among single pollen species, rose pollen and pine pollen showed the strongest inhibitory effects, both exceeding the positive control, glycyrrhizin. Glycyrrhizin is often referred to as "whitening gold" or "the Hermes of whitening," and its whitening effect is considered one of the most potent ingredients. Among pollen combinations, the enzymatic hydrolysate from a pine pollen:sunflower pollen:camellia pollen:rose pollen = 1:1:1:1 ratio was the strongest, with the combined effect significantly stronger than that of single pollen hydrolysates.

[0122] Based on the above tests, a powerful synergistic effect was discovered between different pollen varieties. Most products on the market that simultaneously claim to whiten and anti-wrinkle effects are formulated by mixing extracts or chemical ingredients from different sources. However, the sole raw material of this invention is pollen, which, through a specific process, becomes a complete "super ingredient" with dual benefits. In terms of anti-wrinkle effects, the composition exhibits strong antioxidant properties and promotes the synthesis of collagen and elastin in fibroblasts. In terms of whitening, the composition significantly inhibits melanin production and tyrosinase activity.

[0123] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A pollen composition possessing both whitening and anti-wrinkle effects, characterized in that: The pollen is selected from at least two or more of pine pollen, sunflower pollen, camellia pollen, and rose pollen.

2. The pollen composition according to claim 1, characterized in that: The pollen is a mixture of pine pollen and camellia pollen, or a mixture of pine pollen, camellia pollen and rose pollen, or a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen.

3. The pollen composition according to claim 1, characterized in that: The pollen is a mixture of pine pollen and camellia pollen in a mass ratio of 1-3:1, preferably 1:1; or the pollen is a mixture of pine pollen, camellia pollen and rose pollen in a mass ratio of 1-3:1:1, preferably 1:1:1; or the pollen is a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen in a mass ratio of 1-5:1:1:

1.

4. The pollen composition according to claim 3, characterized in that: The pollen is a mixture of pine pollen, sunflower pollen, camellia pollen and rose pollen in a mass ratio of 1:1:1:1, 2:1:1:1 or 5:1:1:

1.

5. A method for preparing the pollen composition according to any one of claims 1-4, characterized in that: Includes the following steps: (1) Take pollen and break the cell wall to obtain pollen after cell wall breaking; (2) Add cellulase to the pollen after cell wall disruption, then add pure water to carry out enzymatic hydrolysis to obtain hydrolyzed pollen; (3) After the pollen is inactivated by enzymatic hydrolysis, the solutions are combined, centrifuged, the supernatant is collected, concentrated and dried to obtain the pollen composition.

6. The preparation method according to claim 5, characterized in that: The amount of cellulase added is 0.5-1.0% of the substrate weight.

7. The preparation method according to claim 5, characterized in that: The amount of cellulase added is 0.8% of the substrate weight.

8. The preparation method according to claim 5, characterized in that: The pH value of pure water is 5.5-6.

5.

9. The preparation method according to claim 8, characterized in that: The pH value of pure water is 6.

0.

10. The preparation method according to claim 5, characterized in that: The enzymatic hydrolysis is performed at a temperature of 50-55℃ for 4-6 hours.

11. The preparation method according to claim 10, characterized in that: The enzymatic hydrolysis was performed at a temperature of 52°C for 5 hours.

12. The preparation method according to claim 5, characterized in that: The extraction is performed by reflux extraction or ultrasonic extraction; the extraction time for reflux extraction is 1-2 hours; the extraction temperature for ultrasonic extraction is 80-90℃ for 1 hour.

13. The preparation method according to claim 5, characterized in that: The centrifugation speed is 4000-6000 rpm / min and the centrifugation time is 8-15 min.

14. The preparation method according to claim 13, characterized in that: The centrifugation speed is 5000 rpm / min and the centrifugation time is 10 min.

15. The use of the pollen composition according to any one of claims 1-4 in the preparation of cosmetic products, characterized in that: The beauty product described is one that has both whitening and anti-wrinkle effects.

16. A product that combines whitening and anti-wrinkle effects, characterized in that: The product comprises the pollen composition according to any one of claims 1-4 and acceptable excipients in the product.

17. The product according to claim 16, characterized in that: The product in question is either an oral or topical product.

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