A composition for simultaneously promoting production of collagen type i and collagen type iii, use thereof, and food containing the same

Abstract

The first aspect of the present application provides a composition for simultaneously promoting the production of collagen type I and collagen type III, comprising pyrroloquinoline quinone disodium salt and ergothioneine, and the mass ratio of pyrroloquinoline quinone disodium salt to ergothioneine is 1-2:0.01-0.05.The composition can simultaneously promote the production of collagen type I and collagen type III in the skin, and can improve the elasticity of human skin and reduce wrinkles.

Description

Technical Field

[0001] This invention belongs to the field of functional composition technology, specifically to a composition that simultaneously promotes the production of type I and type III collagen, its uses, and food products containing the same composition. Background Technology

[0002] Human skin contains over 28 types of collagen, differentiated by tissue location, physiological function, and molecular structure. Type I and Type III collagen are the most abundant in the skin, accounting for approximately 80% and 20% of the total adult skin volume, respectively. Type III collagen makes up as much as 80% of infant skin, hence it is also known as "infant collagen." Type I collagen, a major component of adult skin, is part of collagen fibers and provides excellent support, playing a crucial role in supporting skin cells. A deficiency in Type I collagen leads to sagging and depressions in the skin. Type III collagen, distributed around Type I collagen, promotes cell proliferation, increases cell activity, and helps the body repair aging and damaged skin. A deficiency in Type III collagen results in skin that is no longer soft, smooth, or elastic.

[0003] In other words, the levels of type I and type III collagen directly affect the youthfulness of the skin. Therefore, the key to anti-aging skin is to supplement type I and type III collagen.

[0004] Type I collagen is relatively easy to supplement because the human dermis has the ability to produce type I collagen. Of course, this ability to produce collagen weakens with age. Currently, most oral functional foods on the market mainly use vitamin C and collagen peptides to promote the production of type I collagen.

[0005] Compared to type I collagen, type III collagen is relatively difficult to replenish because the human body has limited ability to produce it automatically or passively. Therefore, many current studies and patents often focus only on promoting the production and replenishment of type I collagen, while neglecting the importance of promoting the production of type III collagen.

[0006] Therefore, how to simultaneously promote the production of type I and type III collagen to further enhance the skin's anti-aging ability is an urgent problem to be solved. Summary of the Invention

[0007] To address the problems and shortcomings of existing technologies, this invention provides a composition that simultaneously promotes the production of type I and type III collagen, its uses, and food products containing the composition. This composition can simultaneously promote the production of type I and type III collagen in the skin, thereby improving skin elasticity and reducing wrinkles.

[0008] According to a first aspect of the present invention, a composition that simultaneously promotes the production of type I and type III collagen is provided, comprising disodium pyrroloquinoline quinone and ergothioneine; wherein the mass ratio of disodium pyrroloquinoline quinone and ergothioneine is 1 to 2: 0.01 to 0.05.

[0009] Pyrroloquinoline quinone disodium salt (PQQ) can inhibit the expression of skin aging markers MMP-1 and MMP-3, thereby protecting dermal fibroblasts, reducing the degradation of the dermal extracellular matrix, and thus reducing collagen loss. Ergothioneine can also inhibit the expression of MMP-1, a skin aging marker. Therefore, ergothioneine can work synergistically with PQQ to enhance the inhibition of MMP expression. Furthermore, these two substances exhibit a significant synergistic effect in a specific ratio, showing a stronger inhibitory effect on MMP compared to either PQQ alone or ergothioneine alone, thus strengthening the protection of dermal fibroblasts and further reducing their degradation. In addition, PQQ can promote mitochondrial biogenesis, while ergothioneine can target mitochondrial ROS production and protect mitochondrial DNA from damage via the OTCN1 transporter. The synergistic effect of both leads to enhanced mitochondrial function, increased ATP synthesis, and further promotes the production of type I and type III collagen by dermal fibroblasts. This increases the amount of type I and type III collagen produced by the skin itself, thereby effectively replenishing type I and type III collagen in the skin, improving skin elasticity, and reducing wrinkles.

[0010] Preferably, the mass ratio of disodium pyrroloquinoline quinone to ergothione is 2:0.01–0.05. At this mass ratio, disodium pyrroloquinoline quinone and ergothione can exert a better synergistic effect, further inhibiting the expression of matrix metalloproteinases, a marker of skin aging, and increasing the production rate of type I and type III collagen.

[0011] Preferably, it also includes *Hydrangea macrophylla*, and the mass ratio of disodium pyrroloquinoline quinone, ergothioneine, and *Hydrangea macrophylla* is 1–2:0.01–0.05:5.

[0012] The most important active ingredient in *Hydrangea macrophylla* is β-glucan. β-glucan possesses immunomodulatory, antibacterial, antioxidant, anti-aging, anti-inflammatory, anti-tumor, hypoglycemic, and hypolipidemic effects. β-glucan can also be recognized by receptors on the membranes of immune-active cells and activate these cells, increasing epidermal growth factor (EGF) in aging and wrinkled skin, thus promoting collagen and elastin production, improving skin appearance and reducing wrinkles. Therefore, *Hydrangea macrophylla* can simultaneously promote fibroblast proliferation and collagen synthesis, scavenge free radicals, combat UV damage, and enhance immune protection in skin anti-aging. At specific ratios, the active ingredients in *Hydrangea macrophylla* can further synergistically promote the production of type I and type III collagen in the skin with pyrroloquinoline quinone disodium salt and ergothioneine. However, if the proportion of *Hydrangea macrophylla* is too high, it will limit the effectiveness of pyrroloquinoline quinone disodium salt and ergothioneine in inhibiting the expression of matrix metalloproteinases, a marker of skin aging, and will not effectively increase the production of type I and type III collagen. However, if the proportion of *Hydrangea macrophylla* is too low, the synergistic effect of the composition on further inhibiting the expression of matrix metalloproteinases is not significant enough, and thus it cannot effectively improve the production of type I and type III collagen.

[0013] Preferably, the mass ratio of pyrroloquinoline quinone disodium salt, ergothioneine, and *Hydrangea macrophylla* is 2:0.05:5. At this ratio, the synergistic effect of the three is most pronounced, achieving the best effect in inhibiting matrix metalloproteinase expression and promoting the highest rate of type I and type III collagen production.

[0014] Preferably, the ergothioneine is derived from the extract of Pleurotus ostreatus, and the mass percentage of ergothioneine in the Pleurotus ostreatus extract is 0.8-1.2%.

[0015] Preferably, disodium pyrroloquinoline quinone is prepared by the following methods: Disodium pyrroloquinoline quinone can be obtained through two different processes. One is a chemical synthesis process, using ethyl 6-amino-5-methoxy-1H-indole-2-carboxylate and dimethyl 2-oxopentenedioate as raw materials, through coupling cyclization reaction, vacuum filtration, crystallization, solid-liquid separation extraction, mixing, vacuum drying, sieving, and packaging. The other method uses *Methylobacterium glutamicum* as a starter culture, prepared in shake flasks, and then proceeds with seed culture, fermentation culture, membrane filtration, chromatography, salting-out precipitation, redissolution crystallization precipitation, and drying and pulverization.

[0016] Preferably, the extract of Pleurotus ostreatus is obtained by the following pretreatment: water extraction, filtration, desolvation, mixing, and packaging.

[0017] Preferably, the *Hydrangea macrophylla* is prepared by the following pretreatment: pretreatment, extraction, vacuum concentration, spray drying, sieving and mixing, and packaging to produce *Hydrangea macrophylla* powder.

[0018] According to a second aspect of the present invention, a composition that simultaneously promotes the production of type I and type III collagen is used in promoting the production of type I and type III collagen by dermal fibroblasts. The composition provided by the present invention, as verified by cell experiments, can effectively promote the production of type I and type III collagen by dermal fibroblasts and increase the production rate of type I and type III collagen.

[0019] According to a third aspect of the invention, a food product is provided comprising the above-described composition that simultaneously promotes the production of type I and type III collagen.

[0020] Preferably, the above-mentioned food can be prepared by adding excipients to the above-mentioned composition, the excipients including at least one of maltitol, sodium bicarbonate, sodium carbonate, sorbitol, citric acid, flavoring, fruit juice, and thickener.

[0021] Preferably, the above-mentioned food can be oral liquid, liquid beverage, solid beverage, soft capsule, hard capsule, tablet and gummies, etc.

[0022] According to a fourth aspect of the present invention, there is provided the use of the above-mentioned food in promoting the generation of type I and type III collagen by skin fibroblasts.

[0023] In summary, the composition provided by this invention, containing pyrroquinoline quinone disodium salt (PQQ) and ergothioneine, can effectively inhibit the expression of skin aging markers matrix metalloproteinases MMP-1 and / or MMP-3. Furthermore, these two substances, in a specific ratio, can further exhibit a synergistic effect, further promoting the inhibitory effect of the composition on matrix metalloproteinase expression. This effectively protects dermal fibroblasts, promotes the production of type I and type III collagen by fibroblasts, and increases the production rate of type I and type III collagen. Therefore, the composition of this invention can effectively replenish type I and type III collagen in the skin, improve skin elasticity, and reduce wrinkles. The further addition of *Hydrangea macrophylla* to the composition can further promote the production of type I and type III collagen, further increasing the content of type I and type III collagen in the skin, making the skin more elastic. Attached Figure Description

[0024] Figure 1 The content of type I collagen in HFF-1 cells in the embodiments and comparative examples of this invention.

[0025] Figure 2 The content of type III collagen in HFF-1 cells in the embodiments and comparative examples of this invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0027] Example 1

[0028] The composition of this embodiment is obtained by mixing 2 parts by weight of disodium pyrroloquinoline quinone and 1 part by weight of extract of Pleurotus ostreatus (powder).

[0029] In this embodiment, the disodium pyrroloquinoline quinone was prepared by the following method: using *Bacillus thymolica* as the inoculum, the inoculum was prepared by shaking flask culture, followed by seed culture, fermentation culture, membrane filtration, chromatography, salting out precipitation, redissolution and crystallization precipitation, and drying and pulverization.

[0030] Golden-topped Pleurotus ostreatus extract was obtained through the following pretreatment: water extraction, filtration, solvent removal, mixing, and packaging. The extract contains 1 wt% ergothioneine.

[0031] Example 2

[0032] The composition of this embodiment is obtained by mixing 2 parts by weight of disodium pyrroloquinoline quinone and 5 parts by weight of extract of Pleurotus ostreatus (powder).

[0033] In this embodiment, the preparation or processing method of pyrroloquinoline quinone disodium salt and Pleurotus ostreatus extract is the same as in Example 1.

[0034] Example 3

[0035] The composition of this embodiment is obtained by mixing 1 part of disodium pyrroloquinoline quinone and 5 parts of Pleurotus ostreatus extract (powder) according to the weight proportions.

[0036] In this embodiment, the preparation methods of pyrroloquinoline quinone disodium salt, Pleurotus ostreatus extract, and Hydrangea macrophylla are the same as in Example 1.

[0037] Example 4

[0038] The composition of this embodiment is obtained by mixing 2 parts by weight of disodium pyrroloquinoline quinone, 1 part by weight of Pleurotus ostreatus extract (powder), and 5 parts by weight of Hydrangea macrophylla.

[0039] In this embodiment, the preparation or processing method of pyrroloquinoline quinone disodium salt and Pleurotus ostreatus extract is the same as in Example 1.

[0040] The *Hydrangea macrophylla* fungus undergoes the following pretreatment processes: pretreatment, extraction, vacuum concentration, spray drying, sieving and mixing, and packaging to produce *Hydrangea macrophylla* powder.

[0041] Example 5

[0042] The composition of this embodiment is obtained by mixing 1 part of disodium pyrroloquinoline quinone, 5 parts of Pleurotus ostreatus extract (powder), and 5 parts of Hydrangea macrophylla according to the weight proportions.

[0043] In this embodiment, the preparation or processing methods of pyrroloquinoline quinone disodium salt, Pleurotus ostreatus extract, and Hydrangea macrophylla are the same as in Example 4.

[0044] Example 6

[0045] The composition of this embodiment is obtained by mixing 2 parts by weight of disodium pyrroloquinoline quinone, 5 parts by weight of Pleurotus ostreatus extract (powder), and 5 parts by weight of Hydrangea macrophylla.

[0046] In this embodiment, the preparation or processing methods of pyrroloquinoline quinone disodium salt, Pleurotus ostreatus extract, and Hydrangea macrophylla are the same as in Example 4.

[0047] Comparative Example 1

[0048] The composition of this comparative example was obtained by mixing 1 part by weight of disodium pyrroloquinoline quinone and 10 parts by weight of Pleurotus ostreatus extract (powder).

[0049] In this comparative example, the preparation or processing methods of pyrroloquinoline quinone disodium salt and Pleurotus ostreatus extract are the same as in Example 1.

[0050] Comparative Example 2

[0051] Based on parts by weight, the composition in this comparative example contains only 1 part of disodium pyrroloquinoline quinone.

[0052] In this embodiment, the preparation or processing method of pyrroloquinoline quinone disodium salt is the same as in Example 1.

[0053] Comparative Example 3

[0054] Based on parts by weight, the composition in this comparative example contains only 1 part of Pleurotus ostreatus extract.

[0055] In this embodiment, the preparation or processing method of the extract of Pleurotus ostreatus is the same as that in Example 1.

[0056] Comparative Example 4

[0057] Based on parts by weight, the composition in this comparative example contains only 1 part of *Hydrangea macrophylla*.

[0058] In this embodiment, the preparation or processing method of *Hydrangea hydrangea* is the same as in Example 4.

[0059] Comparative Example 5

[0060] The comparative composition was obtained by mixing 1 part by weight of Pleurotus ostreatus extract and 1 part by weight of Hydrangea macrophylla.

[0061] In this comparative example, the preparation or processing methods of the extracts of Pleurotus ostreatus and Hydrangea macrophylla are the same as in Example 1.

[0062] Comparative Example 6

[0063] The composition of this comparative example was obtained by mixing 2 parts by weight of disodium pyrroloquinoline quinone and 5 parts by weight of *Hydrangea macrophylla*.

[0064] In this comparative example, the preparation or treatment methods of pyrroloquinoline quinone disodium salt and *Hydrangea rubra* are the same as in Example 1.

[0065] Comparative Example 7

[0066] The composition of this comparative example was obtained by mixing 5 parts by weight of disodium pyrroloquinoline quinone, 1 part by weight of Pleurotus ostreatus extract, and 5 parts by weight of Hydrangea macrophylla.

[0067] In this comparative example, the preparation or processing methods of pyrroloquinoline quinone disodium salt, Pleurotus ostreatus extract, and Hydrangea macrophylla are the same as in Example 1.

[0068] Comparative Example 8

[0069] The composition of this comparative example was obtained by mixing 1 part by weight of disodium pyrroloquinoline quinone and 10 parts by weight of extract of Pleurotus ostreatus.

[0070] In this comparative example, the preparation or treatment methods of pyrroloquinoline quinone disodium salt and *Hydrangea rubra* are the same as in Example 1.

[0071] Comparative Example 9

[0072] The composition of this comparative example was obtained by mixing 5 parts by weight of disodium pyrroloquinoline quinone and 1 part by weight of extract of Pleurotus ostreatus.

[0073] In this comparative example, the preparation or processing methods of pyrroloquinoline quinone disodium salt and Pleurotus ostreatus extract are the same as in Example 1.

[0074] Test case

[0075] 1. Experimental Construction Method

[0076] HFF-1 cell elasticity experiments were performed on the compositions obtained in all the above examples and comparative examples, as follows:

[0077] Reagents: DEME medium, fetal bovine serum, penicillin-streptomycin antibiotics, 0.25% trypsin-EDTA digestion solution, PBS solution, type I collagen (Col I) enzyme-linked immunosorbent assay kit, type III collagen (Col III) enzyme-linked immunosorbent assay kit.

[0078] Instruments: Centrifuge, microscope, multi-functional microplate reader, 24-well plate, incubator, cell culture consumables

[0079] Experimental methods: ① HFF-1 cells were cultured in DMEM medium containing 15% fetal bovine serum at 37°C and 5% CO2 in a cell culture incubator. HFF-1 cells in the logarithmic growth phase were seeded at 1×10⁵ cells / well into 24-well plates, 500 μL per well, and incubated at 37°C for 24 h with 5% CO2.

[0080] ② Discard the culture medium and wash once with PBS; add 200 μL of PBS to each well. The experiment was divided into a control group, a model group, and different combination groups. The control group did not receive UVA irradiation, while the model group and combination groups were treated with 10 J / cm2 UVA irradiation. After irradiation, the control group and model group continued to be cultured in DMEM basal medium, while the combination treatment groups were added with 300 μL of each of the different combination solutions at a concentration of 50 μg / mL, and incubated in a cell culture incubator for 24 h.

[0081] ③ Place the well plate in a -80℃ freezer and freeze-thaw it three times, collecting all the liquid in the well plate. Centrifuge at 5000 rpm for 20 min at 4℃ and collect the supernatant. Check the content of type I collagen (Col I) and type III collagen (Col III) in the collected solution according to the enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Biological Products Institute, catalog numbers: H142-1-1 and H144-1-1).

[0082] The production promotion rate of type I and type III collagen was calculated based on the content results of type I and type III collagen. The calculation method is as follows:

[0083]

[0084] 2. Experimental Results

[0085] The relevant formulations of the compositions obtained in all the above embodiments and comparative examples, as well as the data such as the formation rates of type I and type III collagen, are shown in Table 1.

[0086] Table 1 summarizes the relevant formulations of the compositions obtained in the examples and comparative examples, as well as data on the promotion rates of type I and type III collagen.

[0087]

[0088] As can be seen from Table 1, the composition prepared by using disodium pyrroloquinoline quinone and ergothioneine (actually an extract of Pleurotus ostreatus) in a specific ratio can effectively promote the production of both type I and type III collagen. Referring to Examples 1-6, the production rate of type I collagen reached 59.52% or higher, and the production rate of type III collagen reached 55.74% or higher.

[0089] In Comparative Examples 1 and 7–9, the mass ratio of disodium pyrroloquinoline quinone to ergothione was not within the range of 1–2:0.01–0.05, indicating a reduced synergistic effect between the two substances and a significant decrease in the production rates of type I and type III collagen. Comparative Examples 3–5, which did not contain disodium pyrroloquinoline quinone, and Comparative Examples 2 and 6, which did not contain ergothione, also showed a significant decrease in the production rates of type I and type III collagen. This suggests that disodium pyrroloquinoline quinone and ergothione have a certain synergistic effect when used together, and that when these two substances are used in combination, they are more conducive to promoting the production of type I and type III collagen.

[0090] Further comparisons were made with Examples 1, 4-6. Examples 4-6 also introduced *Hydrangea macrophylla*, which further improved the composition's ability to promote the production of type I and type III collagen. This is because the active ingredients in *Hydrangea macrophylla* can further synergistically promote the production of type I and type III collagen in the skin with pyrroloquinoline quinone disodium salt and ergothioneine.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A composition that simultaneously promotes the production of type I and type III collagen, characterized in that: The composition comprises disodium pyrroloquinoline quinone and extract of Pleurotus ostreatus, wherein the mass ratio of disodium pyrroloquinoline quinone to ergothioneine in the composition is 1-2:0.01-0.

05. Alternatively, the composition comprises disodium pyrroloquinoline quinone, extract of Pleurotus ostreatus, and Hydrangea macrophylla, wherein the mass ratio of the disodium pyrroloquinoline quinone, ergothioneine, and Hydrangea macrophylla in the composition is 1-2:0.01-0.05:

5. The ergothioneine is derived from the extract of Pleurotus ostreatus, and the ergothioneine accounts for 0.8-1.2% of the mass of the extract.

2. The composition according to claim 1 that simultaneously promotes the production of type I and type III collagen, characterized in that: When the composition comprises the disodium pyrroloquinoline quinone and the extract of Pleurotus ostreatus, the mass ratio of the disodium pyrroloquinoline quinone to ergothioneine is 2:0.01 to 0.

05.

3. The composition according to claim 1 that simultaneously promotes the production of type I and type III collagen, characterized in that: When the composition comprises the disodium pyrroloquinoline quinone, the extract of Pleurotus ostreatus, and the physalis fungus, the mass ratio of the disodium pyrroloquinoline quinone, ergothioneine, and the physalis fungus is 2:0.05:

5.

4. A food product, characterized in that: Includes the composition that simultaneously promotes the production of type I and type III collagen as described in any one of claims 1 to 3.

Images