Tribonema extract with anti-ultraviolet function and application thereof

Abstract

The invention relates to application of tribonema in preparation of an ultraviolet absorbent, and a tribonema extract and a skin care composition with an anti-ultraviolet function. The invention finds that the tribonema extract has a very wide ultraviolet absorption spectrum, has a plurality of absorption peaks between 280-400 nm, and almost covers all UVA and UVB wavebands. On the basis, the preparation steps of the tribonema extract are optimized, and the tribonema extract capable of preventing cells cultured in vitro from being damaged by ultraviolet rays and the skin cream with the sunscreen function are obtained.

Description

Technical Field

[0001] This invention belongs to the field of microalgae industry, specifically relating to extracts of *Hygrophytes lutea* with anti-ultraviolet function and their applications. Background Technology

[0002] Ultraviolet (UV) radiation is a major environmental factor leading to various skin diseases, including skin cancer, photoaging, and immunosuppression. Traditional sunscreens are mainly divided into two categories: physical sunscreens and chemical sunscreens. Physical sunscreens work by reflecting and scattering UV rays, while chemical sunscreens provide protection by absorbing UV rays and converting them into heat.

[0003] While traditional physical sunscreens (zinc oxide, titanium dioxide) and chemical sunscreens (methoxycinnamates, benzophenones) are relatively mature in terms of protective efficacy, they still face many challenges in terms of safety, environmental impact, and user experience. Therefore, microalgae-based bio-sunscreen ingredients have attracted much attention due to their excellent UV absorption capacity, antioxidant activity, and biocompatibility, opening up a new path for the green development of sunscreen products.

[0004] Microalgae-based bio-sunscreens are a new type of sunscreen product that has emerged in recent years. They utilize natural active ingredients extracted from microalgae to achieve UV protection. These products represent an important trend in the development of sunscreen technology towards natural and green methods.

[0005] However, there is currently a lack of microalgae suitable for preparing UV-protective skincare products, which limits the development of microalgae-based UV-protective skincare products.

[0006] *Hylocereus fulva*, a representative filamentous microalga of the Xanthophyceae class, is abundant globally. Compared to single-celled microalgae, its larger cell structure makes it more resistant to changes in its growth environment, and it is easier to harvest through gravity sedimentation and filtration, making it suitable for large-scale production and thus reducing commercial production costs. Compared to some red algae, it grows faster and has a shorter cultivation cycle, allowing for large-scale production in relatively small spaces, unrestricted by geographical or climatic conditions. *Hylocereus fulva* also absorbs carbon dioxide during cultivation, possessing a carbon sequestration function, which aligns with the requirements of sustainable development. Currently, there are no reports on its function in ultraviolet (UV) protection. Summary of the Invention

[0007] To address the above problems, this invention provides the application of *Phyllostachys edulis* in the preparation of ultraviolet absorbers.

[0008] The present invention also provides a method for preparing a extract of *Phyllostachys edulis*, comprising the step of extracting *Phyllostachys edulis* cells using an organic solvent.

[0009] In one specific implementation, the method includes the following steps:

[0010] S1: Add the *Xanthophyllus* cells to ethanol to obtain an algae-ethanol mixture;

[0011] S2: Extract the algae-ethanol mixture;

[0012] S3: After solid-liquid separation, take the supernatant, which is the extract of *Xanthophyllaria*.

[0013] In one specific implementation, in S2, the extraction time is 1-3 hours;

[0014] The extraction temperature is 35-60℃.

[0015] In one specific implementation, the ratio of *Hygrophytes lucida* to ethanol in S1 is 1:50 to 1:200.

[0016] In one specific implementation, step S3 further includes steps such as decolorizing and removing impurities from the supernatant.

[0017] The present invention also provides an extract of *Hygrophytes macrantha* prepared by the above method.

[0018] The present invention also provides an ultraviolet absorber comprising *Phyllostachys edulis* cells and / or *Phyllostachys edulis* extract.

[0019] The present invention also provides a composition having anti-ultraviolet function, comprising the above-mentioned ultraviolet absorber.

[0020] In one specific embodiment, the extract includes the *Xanthophyllus* extract, and one or more combinations of the following components: glycerin, dimethicone, cetearyl alcohol, xanthan gum, vitamin E, tetrahydrodiferoylmethane, phenoxyethanol, ethylhexylglycerin, and citric acid.

[0021] This invention discovered that the extract of *Hylocereus undatus* possesses a broad ultraviolet absorption spectrum, with multiple absorption peaks between 280 and 400 nm, covering almost the entire UVA and UVB bands. Based on this, this invention optimizes the preparation steps of the *Hylocereus undatus* extract, obtaining an extract capable of protecting in vitro cultured cells from ultraviolet damage, as well as a skin cream with sun protection function. Attached Figure Description

[0022] Figure 1 These are the UV full-band scanning spectra of various microalgae extracts.

[0023] Figure 2 To investigate the effects of different feeding methods on the synthesis of ultraviolet-absorbing substances in *Phyllostachys edulis*.

[0024] Figure 3 The effect of activated carbon on the decolorization effect of extracts from *Hygrophorus spp.*

[0025] Figure 4 The effect of extraction time on the extraction efficiency of ultraviolet-absorbing substances from *Phyllostachys edulis*.

[0026] Figure 5 To investigate the effect of extraction temperature on the extraction efficiency of ultraviolet-absorbing substances from *Phyllostachys edulis*.

[0027] Figure 6 The effect of extraction times on the extraction efficiency of UV-absorbing substances from *Phyllostachys edulis*.

[0028] Figure 7 Full-band scanning of extracts of *Hygrophytes maculatus* from a 150L runway pool.

[0029] Figure 8 The stability of the extract of *Hygrophytes macrantha* under ultraviolet light.

[0030] Figure 9 The effect of the material-to-liquid ratio on the extraction efficiency of ultraviolet-absorbing substances from *Phyllostachys edulis*.

[0031] Figure 10 This refers to the protective effect of *Hygrophytes maculatus* extract on cells.

[0032] Figure 11 The results show the anti-aging efficacy of extracts from *Hygrophytes macrantha*.

[0033] Figure 12 The UV absorption spectra of different yellow algae are shown. Detailed Implementation

[0034] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0035] 1. Bubble column culture of *Pyrrosia lingua*, *Porphyra yezoensis*, *Spirulina*, and *Ulva prolifera*:

[0036] Using *Hylocereus undatus*, *Porphyra yezoensis*, and *Spirulina* as seed algae, 400 mL of the corresponding liquid culture medium was added to each algae in a bubble column reactor. Each algae species was precisely inoculated at a rate of 1 g / L and placed in a constant temperature environment of 25℃ with aeration for 3 days. After cultivation, 10 mg of biomass was collected from each algae strain. 10 mg of *Ulva prolifera* sample was obtained from dried *Ulva prolifera* powder preserved in the laboratory from the Qingdao summer seaside. Microalgae extracts were prepared using the following method, and absorption spectra from 250 to 450 nm were recorded using a UV-Vis spectrophotometer.

[0037] Weigh 10.0 mg of microalgae biomass into a centrifuge tube, add 3.0 mL of 20% (v / v) ethanol solution, and vortex for 1 min to thoroughly mix the sample. Place the centrifuge tube in a 45℃ water bath and extract for 2 h (gently agitate once every 30 min to ensure complete extraction). After extraction, place the sample in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm and 4℃ for 25 min. Carefully aspirate the supernatant into a new centrifuge tube, avoiding aspiration of precipitate. Slowly add 3 volumes of anhydrous ethanol to the supernatant to adjust the final ethanol concentration to 80% (v / v). Freeze the mixture at -20℃ for 2 h, then centrifuge at 12000 rpm and 4℃ for 10 min. Collect the supernatant, filter it through a 0.22 μm organic phase filter membrane, and perform a full-band UV-Vis scan.

[0038] Experimental results are as follows Figure 1 As shown, the extracts of *Porphyra yezoensis* and *Spirulina* did not show any absorption peaks between 300-400 nm, while the extract of *Ulva prolifera* only had a narrow single absorption peak around 300 nm. In contrast, *Hydrocotyle vulgaris* had multiple absorption peaks between 280-400 nm, indicating that the extract of *Hydrocotyle vulgaris* has a very broad ultraviolet absorption spectrum, covering almost all UVA and UVB bands.

[0039] 2. Effects of different feeding methods on the synthesis of ultraviolet-absorbing substances in *Phyllostachys edulis*

[0040] Yellow-haired algae were used as the test algae species and cultured on BG11 medium to the logarithmic growth phase.

[0041] Three identical bubble column photobioreactors were used, each containing 450 mL of BG11 culture medium. They were labeled as ① (control group), ② (partial nutrient supplementation group), and ③ (complete nutrient supplementation group), and seed culture was inoculated at a rate of 1 g / L.

[0042] The culture temperature was (25±0.5)℃, and sterile air filtered through a 0.22 μm filter membrane was introduced at a rate of 0.3-0.5 L / min. Light was maintained throughout the process to ensure the consistency of the culture environment for each group.

[0043] At 72 hours (day 3), each group was treated as follows:

[0044] ① No nutrients are added; the original culture system is maintained.

[0045] ②: Precisely supplement sodium nitrate (final concentration consistent with the initial concentration of BG11 medium, i.e., 1.5 g / L) and dipotassium hydrogen phosphate (final concentration consistent with the initial concentration of BG11 medium, i.e., 0.04 g / L), and shake quickly to fully dissolve the nutrients;

[0046] ③: According to the standard formula of BG11 medium, accurately supplement all nutrient components (the final concentration of each component is restored to the initial level), shake well and continue culturing.

[0047] The culture period lasted 144 hours (6 days). Starting from the 0h after inoculation (initial time), samples were taken daily, with each group taking 20-30 mL of samples each time. The obtained cell culture medium was filtered through a 0.45µm filter membrane and then dried with hot air for 6 hours to constant weight. 10.0 mg of dried *Hylocereus undatus* was accurately weighed and extracted using the *Hylocereus undatus* extract extraction method. The extract was scanned in the full wavelength range of 200-800 nm using a UV-Vis spectrophotometer, and the absorption spectrum data at 250-450 nm were recorded.

[0048] The results are as follows Figure 2 As shown, in control group ①, the content of anti-UV active ingredients in the extract of *Hylocereus undatus* showed an increasing trend followed by a decreasing trend, reaching its maximum after 120 h of culture. In group ② (partial nutrient salt group), the content of anti-UV active ingredients decreased sharply within 24 h after nutrient supplementation, followed by a significant increasing trend, also reaching its maximum after a total culture time of 120 h. In group ③ (complete nutrient salt group), the content of anti-UV active ingredients also showed a sharp decrease within 24 h after nutrient supplementation, followed by an increasing trend, but did not reach the levels of control group ① and group ② (partial nutrient salt group) after a total culture time of 120 h.

[0049] 3. Decolorization using activated carbon

[0050] Weigh 10.0 mg of *Xanthomonas macrantha* and place it in a centrifuge tube. Add 3.0 mL of 20% (v / v) ethanol solution and vortex for 1 min to thoroughly mix the sample. Place the centrifuge tube in a 45℃ water bath for extraction (gently agitate once every 30 min to ensure complete extraction), setting time gradients of 1 h, 2 h, 3 h, and 4 h. After extraction, place the sample in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm and 4℃ for 25 min. Carefully aspirate 2.5 mL of the supernatant into a new centrifuge tube, avoiding aspiration of precipitate. Slowly add 3 times the volume of anhydrous ethanol to the supernatant to adjust the final ethanol concentration of the system to 80% (v / v); place the mixture in a -20℃ freezer for 2 h, then centrifuge at 12000 rpm and 4℃ for 10 min, collect the supernatant, filter it through a 0.22 μm organic phase filter membrane, and add 0 mg, 1 mg, 5 mg, 10 mg, 15 mg, and 20 mg of activated carbon powder respectively for decolorization treatment. After decolorization, filter it through a 0.22 μm organic phase filter membrane and perform UV-Vis full-band scanning with a UV spectrophotometer to record the absorption spectrum data in the 250-450 nm range.

[0051] The results are as follows Figure 3 As shown, activated carbon at concentrations of 5 mg / 10 mL or higher can effectively elute pigments from *Hylocereus undatus* extract, and 5 mg / 10 mL activated carbon has little effect on the absorption peak at 300-400 nm.

[0052] 4. Effect of extraction time on the extraction efficiency of UV-absorbing substances from *Phyllostachys edulis*

[0053] Weigh 10.0 mg of *Xanthomonas macrantha* and place it in a centrifuge tube. Add 3.0 mL of 20% (v / v) ethanol solution and vortex for 1 min to thoroughly mix the sample. Place the centrifuge tube in a 45℃ water bath for extraction (gently agitate once every 30 min to ensure complete extraction), setting time gradients of 1 h, 2 h, 3 h, and 4 h. After extraction, place the sample in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm and 4℃ for 25 min. Carefully aspirate the supernatant into a new centrifuge tube, avoiding aspiration of precipitate. Slowly add 3 times the volume of anhydrous ethanol to the supernatant to adjust the final ethanol concentration of the system to 80% (v / v); place the mixture in a -20℃ freezer for 2 hours, then centrifuge at 12000 rpm and 4℃ for 10 minutes, collect the supernatant, filter it through a 0.22μm organic phase filter membrane, and perform a UV-Vis full-band scan using a UV spectrophotometer to record the absorption spectrum data in the 250-450 nm range.

[0054] The results are as follows Figure 4 As shown, the absorption peak of the extract of *Hylocereus undatus* at 300-400 nm showed a trend of first increasing and then decreasing with the extension of extraction time. Extracts containing ultraviolet-absorbing substances could be effectively extracted in 1-3 hours, with 2 hours being the optimal extraction time.

[0055] 5. Effect of extraction temperature on the extraction efficiency of ultraviolet-absorbing substances from *Phyllostachys edulis*

[0056] Weigh 10.0 mg of *Xanthomonas macrantha* and place it in a centrifuge tube. Add 3.0 mL of 20% (v / v) ethanol solution and vortex for 1 min to thoroughly mix the sample. Place the centrifuge tube in a constant temperature water bath at 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃ for 2 h of extraction (gently agitate once every 30 min to ensure complete extraction). After extraction, place the sample in a high-speed refrigerated centrifuge and centrifuge at 12000 rpm and 4℃ for 25 min. Carefully aspirate the supernatant into a new centrifuge tube, avoiding aspiration of precipitate. Slowly add 3 times the volume of anhydrous ethanol to the supernatant to adjust the final ethanol concentration of the system to 80% (v / v); place the mixture in a -20℃ freezer for 2 h, then centrifuge at 12000 rpm and 4℃ for 10 min, collect the supernatant, filter it through a 0.22 μm organic phase filter membrane, and perform a UV-Vis full-band scan using a UV spectrophotometer to record the absorption spectrum data in the 250-450 nm range.

[0057] The results are as follows Figure 5 As shown, the absorption peak of the extract of *Hygrophytes maculatus* at 300 nm-400 nm showed a trend of first increasing and then decreasing with the increase of extraction temperature. Extracts containing ultraviolet-absorbing substances could be effectively extracted at 35-60℃, with 45℃ being the optimal extraction time.

[0058] 6. Production of UV-absorbing substances from *Phyllostachys edulis* cultured in a 10L plate reactor.

[0059] A 10L plate reactor was used, and 10L of BG11 medium was added. Yellow filamentous algae were inoculated at an inoculation rate of 1g / L and placed in a constant temperature environment of 25℃ for 7 days with aeration. After the culture was completed, the yellow filamentous algae sludge was collected, and the extract was extracted using the yellow filamentous algae extract method and then freeze-dried under vacuum. The freeze-dried sample was accurately weighed.

[0060] The results are as follows Figure 6 As shown, a total of 50.7 g of *Hygrophytes globosum* was obtained from the culture of *Hygrophytes globosum* in a 10L plate reactor. The algal sludge was extracted five times. The fifth extraction contained virtually no ultraviolet-absorbing substances. After freeze-drying the extract, a total of 4.62 g of *Hygrophytes globosum* extract was obtained, accounting for 9.1% of the dry weight of the algae.

[0061] 7. Full-spectrum scanning of extracts from *Hygrophytes globosum* cultured in a large-scale outdoor 150L racetrack pool.

[0062] Yellow-haired algae extract was cultured in an outdoor 150L running track pool using BG11 medium in an open culture environment for 7 days. After culture, algal sludge was collected and extracted according to the extraction method for yellow-haired algae extract. The results are as follows: Figure 7 As shown, the ultraviolet-absorbing components in the extract of *Hylocereus undatus* remain stable during large-scale cultivation.

[0063] 8. Photostability test of *Phyllostachys edulis* extract

[0064] Take 10 mL of the extract of *Hylocereus undatus* and place it under a UV lamp. After 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, perform a UV-Vis full-band scan using a UV spectrophotometer and record the absorption spectrum data in the range of 250-450 nm.

[0065] Experiments such as Figure 8 As shown, ultraviolet absorbers with absorption peaks in the 350-450 nm range remained stable within 3 hours, while ultraviolet absorbers with absorption peaks in the 300-350 nm range remained stable within 2 hours.

[0066] 9. Effect of material-to-liquid ratio on the extraction efficiency of UV-absorbing substances from *Phyllostachys edulis*

[0067] Weigh 10.0 mg of *Xanthomonas macrantha* and place it in a centrifuge tube. Add 20% (v / v) ethanol solution sequentially at material-to-liquid ratios of 1:10, 1:50, 1:100, 1:150, 1:200, 1:250, and 1:300. Vortex for 1 min to ensure thorough mixing. Place the centrifuge tube in a 45°C water bath for extraction (gently agitate every 30 min to ensure complete extraction) for 2 h. After extraction, centrifuge the sample in a high-speed refrigerated centrifuge at 12000 rpm and 4°C for 25 min. Carefully transfer the supernatant to a new centrifuge tube, avoiding aspiration of precipitate. Slowly add 3 times the volume of anhydrous ethanol to the supernatant to adjust the final ethanol concentration of the system to 80% (v / v); place the mixture in a -20℃ freezer for 2 h, then centrifuge at 12000 rpm and 4℃ for 10 min, collect the supernatant, filter it through a 0.22 μm organic phase filter membrane, and perform a UV-Vis full-band scan using a UV spectrophotometer to record the absorption spectra at characteristic peaks of 306 and 320 nm.

[0068] The results are as follows Figure 9 As shown, extracts containing ultraviolet-absorbing substances can be effectively extracted at material-to-liquid ratios ranging from 1:50 to 1:200, with the best extraction effect observed at a material-to-liquid ratio of 1:100.

[0069] 10. Cellular Experiments with Yellow-haired Algae Extract

[0070] Accurately weigh 1.00 g of the lyophilized extract of *Hygrophytes flavomarginata* and dissolve it in 10 mL of sterile water to prepare a 10% solution for cell experiments. The method is as follows:

[0071] (1) Select human skin fibroblasts or keratinocytes and passage them to the logarithmic growth phase;

[0072] (2) Inoculated into 96-well plates at a density of 5 × 10⁻⁶. 4 -1×10 5 Individuals / cm², incubate overnight until 80%-90% confluence;

[0073] (3) Add 0.2% of the *Xanthophyllaria* extract sample and treat for 24 hours.

[0074] (4) Use a UV lamp box or a dedicated UV irradiation instrument, select the UVA (320-400nm) or UVB (280-320nm) band; set the irradiation dose (e.g., UVA 10-20J / cm², UVB 0.5-1.0J / cm²), and the irradiation time is 5-30 minutes;

[0075] (5) Add CCK-8 reagent and incubate for 2-3 hours, then detect cell viability and evaluate the protective efficacy of the extract against ultraviolet radiation.

[0076] The results are as follows Figure 10 As shown, a 0.2% concentration of *Hygrophytes maculatus* extract can effectively protect cells from the effects of ultraviolet radiation, with a cell survival rate of up to 93.4%.

[0077] 11. Anti-aging efficacy test of *Hylocereus undatus* extract

[0078] ① Control group: Weigh 0.50 g of ectoine and dissolve it in 10 mL of glycerol to prepare a 5% solution.

[0079] ② Experimental group: Weigh 0.50 g of the freeze-dried extract of *Hygrophytes macrantha* and dissolve it in 10 mL of glycerol to prepare a 5% solution.

[0080] The results are as follows Figure 11 As shown, the SPF of the experimental group was 1.29, which was higher than that of the control group (1.06), indicating that the extract of *Hymenopterus xanthipes* has a good protective effect against ultraviolet radiation.

[0081] 12. Absorption of ultraviolet light by different yellow algae

[0082] Eleven strains of *Xanthomonas flavomarginata* already existing in our laboratory were cultured, and extracts were obtained by ethanol extraction. The absorption of ultraviolet light was then tested, and the results are as follows: Figure 12 As shown, all *Phyllostachys glomeratus* species exhibit ultraviolet absorption peaks. This indicates that the UV-resistant function is not limited to a single *Phyllostachys glomeratus* strain, but is a common phenomenon among this species.

[0083] 13. Formulation of a bio-sunscreen lotion based on *Hylocereus undatus* extract.

[0084] 100g of sunscreen contains the following ingredients: 66.2g deionized water; 2-10g yellow algae extract; 5-15.0g glycerin; 1-4.0g dimethicone; 0.5-2.0g glyceryl stearate; 0.2-1.2g cetearyl alcohol; 0-0.15g xanthan gum; 0-0.5g vitamin E; 0-0.05g tetrahydrodiferoylmethane; 0.1-0.8g phenoxyethanol & ethylhexylglycerin; 0-0.1g citric acid.

[0085] The above components were mixed and then emulsified using conventional emulsification methods to prepare an emulsion. Experimental results showed that the emulsion had a sun protection effect.

[0086] Although the above experiments used extracts obtained by immersing *Phyllostachys edulis* in ethanol to test their UV protection, extracts with UV protection can also be obtained using other solvents such as methanol or aqueous acetic acid. Similar effects can also be achieved using *Phyllostachys edulis* cells or lysate.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of *Phyllostachys edulis* in the preparation of ultraviolet absorbers.

2. A method for preparing extracts of *Hylocereus undatus*, characterized in that, This includes the step of extracting Xanthophyllus cells using organic solvents.

3. The method according to claim 2, characterized in that, Includes the following steps: S1: Add the *Xanthophyllus* cells to ethanol to obtain an algae-ethanol mixture; S2: Extract the algae-ethanol mixture; S3: After solid-liquid separation, take the supernatant, which is the extract of *Xanthophyllaria*.

4. The method according to claim 3, characterized in that, In S2, the extraction time is 1-3 hours; The extraction temperature is 35-60℃.

5. The method according to claim 3, characterized in that, In S1, the ratio of *Hygrophytes macrantha* to ethanol is 1:50 to 1:

200.

6. The method according to claim 3, characterized in that, S3 also includes a step of decolorizing and removing impurities from the supernatant.

7. An extract of *Phyllostachys edulis*, characterized in that, It is prepared by the method of any one of claims 2-6.

8. A UV absorber, characterized in that, Includes *Hygrophytes lucida* cells and / or *Hygrophytes lucida* extract.

9. A skincare composition with anti-ultraviolet function, characterized in that, Includes the ultraviolet absorber as described in claim 8.

10. The skincare composition according to claim 9, characterized in that, It includes the aforementioned *Phyllostachys edulis* extract, and one or more of the following ingredients in combination: glycerin, dimethicone, dimethicone, cetearyl alcohol, xanthan gum, vitamin E, tetrahydrodiferoylmethane, phenoxyethanol, ethylhexylglycerin, and citric acid.

Images