Method for promoting formation of melanin on epidermis of black bamboo poles

Abstract

The method for promoting formation of melanin on the skin of the black bamboo poles comprises the following steps that 1, black bamboos with the consistent growth vigor are selected, floating ash on the surfaces of the black bamboos is wiped away for use, and gauze and a plurality of materials for wrapping are prepared; 2) preparing a H2O2 solution with a certain concentration on site; (3) the H2O2 solution is smeared on the surface of the green rod of the black bamboo through gauze, the smeared part is wrapped with a wrapping material, the wrapping material is taken down after acting for a period of time, and the wrapping material is naturally aired; 4, the operation in the step 3 is repeated until the skin of the black bamboo stalk is turned into purple black.According to the method, the green black bamboo stalk is treated with the 1% H2O2 solution, formation of melanin on the skin of the black bamboo stalk can be accelerated, the black bamboo stalk color forming time is shortened to about 40 days from original about 120 days, the ornamental period of the black bamboo can be advanced, and the industrial value of the black bamboo can be increased.

Description

Technical Field

[0001] This invention belongs to the field of agricultural planting technology, and specifically relates to a method for promoting the formation of melanin in the epidermis of purple bamboo stalks. Background Technology

[0002] Bamboo is an important non-timber forest plant belonging to the Bambusoideae subfamily of the Poaceae family. Bamboo easily undergoes variation during natural growth and artificial cultivation, resulting in a rich variety of excellent and rare varieties with diverse leaf shapes, leaf colors, and culm shapes and colors, such as the flowering bamboo (Phyllostachys edulis). Phyllostachys. edulis cv. Tao Kiang), Flowering Stem Early Bamboo ( P. praecox f. viridisulcata ), Purple Bamboo P. nigra These varieties, including purple bamboo (also known as purple bamboo), possess significant ornamental and commercial value. Among them, purple bamboo is renowned for its purplish-black culms and is one of the most famous ornamental bamboo species in classical Chinese gardens, still widely used in landscaping and courtyard planting. Purple bamboo also has important practical applications, being widely used in the production of exquisite bamboo carvings and bamboo flutes.

[0003] From a biological perspective, purple bamboo is a type of golden bamboo (Phyllostachys pubescens). P. nigra var. henonis The purple bamboo (Phyllostachys edulis) is a natural culm color variant. Compared to the green Phyllostachys edulis, the purple bamboo undergoes a unique stage of culm color development, transitioning from green to purplish-black. Based on the time it takes for the culm color to form, it can be roughly divided into "one-year purple," "two-year purple," and "three-year purple." Melanin is a large class of pigments ranging from dark brown to black, commonly found in plants, animals, and microorganisms, and has important biological functions. Plant melanin can be structurally divided into allomelanin and anthocyanin. Allomelanin is typically a nitrogen-free quinone polymer formed from the oxidation of phenolic substances. Protoplasmic oxidase (POD) is a highly active oxidoreductase widely found in animals, plants, and microorganisms. It can remove both phenolic compounds and peroxides. POD catalyzes the oxidation of phenolic substances by peroxides, leading to tissue browning. For example, POD can catalyze the formation of quinones from diphenols in the presence of H2O2. PPO is the main enzyme causing enzymatic browning. It primarily catalyzes the oxidation and hydroxylation of endogenous phenolic substances under aerobic conditions to form ortho-dihydroxyphenols, which then undergo dehydration and oxidation to quinones. Quinones are highly reactive and can further polymerize or undergo oxidative polymerization with amino acids, proteins, and other substances to generate higher molecular weight melanin or brown pigments.

[0004] The main pigment in the epidermis of purple bamboo culms is melanin. However, even for the "one-year purple" variety, it takes at least 10 months under natural conditions for it to fully turn purplish-black from the time of shoot emergence. The "two-year purple" and "three-year purple" varieties require even longer, which greatly limits the application of purple bamboo in landscaping and handicrafts. Current research indicates that low temperature is the main environmental factor promoting the color formation of purple bamboo culms, but no specific reagent treatment has been developed to promote this color formation. Therefore, inventing a method to promote the color formation of purple bamboo culms using external H2O2 is of great significance for shortening the color formation cycle and accelerating its application in the bamboo industry. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a method for promoting melanin synthesis in the epidermis of purple bamboo stalks by applying H2O2 externally, thereby ensuring the formation of purple bamboo stalk color while significantly shortening the time required for color formation, thus solving the problem of long color formation time in purple bamboo stalks.

[0006] This invention is achieved through the following technical solutions: A method for promoting melanin formation in the epidermis of purple bamboo stalks includes the following steps: 1) Select purple bamboo that grows uniformly, wipe off the surface dust and set aside. Prepare gauze and other materials for wrapping. 2) Prepare a fresh H2O2 solution of a certain concentration; 3) Apply H2O2 solution to the surface of the green stem of purple bamboo with gauze, wrap the applied area with wrapping material, remove the wrapping material after a period of time, and let it air dry naturally; 4) Repeat step 3) until the skin of the bamboo stalk turns purplish-black.

[0007] Further, in step 1), the purple bamboo with uniform growth is specifically selected from the new bamboo with mature green stalks that is growing in the current year.

[0008] Furthermore, in step 1), the material used for wrapping is aluminum foil.

[0009] Furthermore, in step 2), the H2O2 solution of a certain concentration refers to a 1% v / v H2O2 solution.

[0010] Furthermore, in step 3), the area to be wrapped and applied is treated once in the morning and once in the afternoon each day, with each wrapping session lasting 30-60 minutes.

[0011] This invention treats green purple bamboo stalks with a 1% H2O2 solution, which accelerates the formation of melanin in the epidermis of the purple bamboo stalks, shortening the color formation time from about 120 days to about 40 days. This helps to advance the viewing period of purple bamboo and increase its industrial value. Attached Figure Description

[0012] Figure 1 Phenotypic observation of purple bamboo after treatment with different concentrations of H2O2; Figure 2 Phenotypic changes of *Purple Bamboo* after treatment with 1% H2O2 solution; Figure 3 Statistics on the color formation cycle of purple bamboo stalks; Figure 4 To determine the chlorophyll content in the epidermis of purple bamboo culms under different treatments; Figure 5 To determine the melanin content in the epidermis of purple bamboo stalks under different treatments; Figure 6 The reaction of soluble melanin in the epidermis of purple bamboo stalks after H2O2 treatment; Figure 7 This is the precipitation reaction of melanin from the epidermis of purple bamboo stalks after H2O2 treatment; Figure 8 UV-Vis absorption spectroscopy analysis of melanin in the epidermis of purple bamboo stalks after H2O2 treatment; Figure 9 Infrared spectral analysis of melanin in the epidermis of purple bamboo stalks after H2O2 treatment. Detailed Implementation

[0013] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.

[0014] Example 1: Changes in the phenotype of purple bamboo after treatment with H2O2 solutions of different concentrations Select healthy, mature, green bamboo shoots from the current year as experimental material, with at least three biological replicates for each treatment. Using RO water as a control, prepare three different concentration gradients of H2O2 solutions (0.1%, 0.5%, and 1%). Apply these solutions to the surface of the bamboo shoots, immediately wrap them in aluminum foil to retain moisture, and remove the foil after 30 minutes to allow them to air dry. Prepare the solutions every two days to ensure their effectiveness. After approximately one month of treatment, observe the color change of the bamboo culms, and measure ΔL, Δa, and Δb using a colorimeter. "L" represents lightness, ranging from 0 to 100, where 0 represents black and 100 represents white. "A" represents red-green, with "+a" representing red and "-a" representing green. "b" represents yellow-blue, with "+b" representing yellow and "-b" representing blue.

[0015] The processing result is as follows Figure 1 Results A showed that, compared with the control, the color of the purple bamboo stalks deepened with increasing H2O2 solution concentration, with the most purplish-black spots appearing on the surface of the purple bamboo treated with 1% H2O2 solution. Figure 1The color difference measurement results (B) showed that, compared with the control and treatments with 0.1% and 0.5% H2O2 solutions, the ΔL and Δb values ​​were lowest and the Δa value was highest after treatment with 1% H2O2 solution. This indicates that the brightness and yellow-blue hue of the purple bamboo stalk surface decreased, while the red hue increased after treatment with 1% H2O2 solution. In summary, compared with other concentrations, 1% H2O2 solution had the most significant effect on promoting the color formation of purple bamboo stalks; therefore, 1% H2O2 solution was selected for subsequent experiments.

[0016] Example 2: Phenotypic changes of purple bamboo after treatment with 1% H2O2 solution 1. Material preparation Select mature, green bamboo shoots of the current year with uniform and healthy growth as experimental materials, and conduct at least 7 biological replicates for each treatment.

[0017] 2. Reagent treatment of stem epidermis (1) Prepare a fresh 1% H2O2 solution and replace the 1% H2O2 solution every two days on average; (2) Apply 1% H2O2 solution to the surface of the purple bamboo using gauze, and immediately wrap it with tin foil; (3) Remove the foil 30 minutes after application and let it air dry naturally; (4) Treat once in the morning and once in the afternoon each day; (5) Continue treatment for 38 days, observe the color change of the purple bamboo culms and take photos to record the sampling.

[0018] As a control, RO water was used for the same treatment.

[0019] The processing result is as follows Figure 2 As shown, the green bamboo culms of purple bamboo gradually darkened in color after treatment with 1% H2O2 solution, while the bamboo culms of the control group treated with RO water showed no significant color change.

[0020] 3. Statistics on the color formation cycle of purple bamboo stalks The time required for culm color formation in naturally grown purple bamboo during the same period was observed and recorded, and seven biological replicates were statistically analyzed. Results are as follows: Figure 3 As shown, starting from the first day of treatment, it takes about 120 days for the color of naturally grown purple bamboo stalks to completely turn purplish-black, while the color formation time of bamboo stalks is shortened to about 40 days after treatment with 1% H2O2 solution.

[0021] Example 3: Determination of pigment content in the epidermis of purple bamboo stalks under different treatments 1. Determination of chlorophyll content in the epidermis of purple bamboo culms under different treatments Weigh 0.05 g of thoroughly ground fresh tissue sample, add 25 mg of CaCO3 and 5 mL of 80% acetone, and extract in the dark for 3 h, shaking and mixing 2-3 times during the process. If the precipitate does not turn completely white after 3 h, the extraction time can be extended. Take the supernatant and measure the absorbance at 663 nm and 645 nm, and record them as A663 and A645, respectively.

[0022] Total chlorophyll content (mg / g fresh weight) = (20.29 × A645 + 8.05 × A663) × V_extracted × D ÷ m ÷ 1000 = 0.01 × (20.29 × A645 + 8.05 × A663) × D ÷ m V_extract: Extraction volume, 5 mL; D: Dilution factor; m: Sample mass, g.

[0023] The results are as follows Figure 4 As shown, the chlorophyll content in the epidermis of the purple bamboo stalks in the control group was significantly higher than that in the 1% H2O2 solution treatment.

[0024] 2. Determination of melanin content in the epidermis of purple bamboo stalks under different treatments.

[0025] Weigh 1 g of fresh bamboo stalk epidermal powder treated with 1% H2O2 solution, add 10 mL of 1 mol / L sodium hydroxide solution, and extract in a 95℃ water bath for 1 h. Centrifuge at 10,000 rpm for 10 min, retain the supernatant, and repeat the extraction 2-3 times. Collect the supernatant in the same container, adjust the pH of the supernatant to 2 with 6 mol / L hydrochloric acid solution, and let it stand overnight at room temperature to allow melanin to fully separate. Centrifuge the suspension at 10,000 rpm for 10 min, and wash the precipitate with ddH2O until neutral. Then wash with chloroform, anhydrous ethanol, and ethyl acetate in sequence to remove impurities. Finally, the precipitate is dried to obtain pigment powder, which is stored at -20℃.

[0026] The powder was dissolved in 0.5 mol / L NaOH, and its absorbance (A) was measured at 220 nm using a UV-26001 ultraviolet spectrophotometer. 220nm ), with A 220nm g -1 Dry weight indicates melanin content. Results are as follows: Figure 5 As shown, the melanin content in the epidermis of purple bamboo stalks treated with 1% H2O2 solution was significantly higher than that in the control group.

[0027] Example 4: Solubility and precipitation reactions of melanin in the epidermis of purple bamboo stalks after H2O2 treatment 1. Solubility reaction and precipitation reaction of melanin in the epidermis of purple bamboo stalks after H2O2 treatment. (1) Solubility reaction of melanin in the epidermis of purple bamboo stalks after H2O2 treatment The solubility of the pigment extract from the epidermis of *Phyllostachys edulis* culms treated with 1% H2O2 solution was analyzed using ddH2O (pH 6.8), NaOH (pH 8.0), acetonitrile, anhydrous ethanol, DMSO, and chloroform. 1.0 mg of the *Phyllostachys edulis* pigment extract was mixed with 1.0 mL of 1 mol / L NaOH solvent in a 1.5 mL test tube and reacted in a metal bath at 25°C for 4 h, inverting the mixture several times during the reaction. The mixture was then centrifuged at 10,000 rpm for 5 min, and the results were photographed. The results are shown below. Figure 6 As shown: The pigment extract from the epidermis of purple bamboo stalks treated with 1% H2O2 solution is insoluble in ddH2O, acetonitrile, anhydrous ethanol, and chloroform, slightly soluble in DMSO, and completely soluble in NaOH solution.

[0028] (2) Precipitation reaction of melanin in the epidermis of purple bamboo stalks after H2O2 treatment Weigh an appropriate amount of the pigment extract from the epidermis of *Phyllostachys edulis* treated with 1% H2O2 solution, and dissolve it in 1 mol / L NaOH to prepare a melanin solution (5 mg / mL). Prepare 10% ferric chloride and 6 mol / L hydrochloric acid solutions to analyze the precipitation reaction of melanin from *Phyllostachys edulis* treated with 1% H2O2 solution, and use 30% H2O2 to analyze the oxidation reaction of melanin. Add 125 μL of 10% FeCl3, 80 μL of 1mol / L HCl, and 125 μL of 30% H2O2 to 500 μL of the pigment extract solution from the epidermis of *Phyllostachys edulis* treated with 1% H2O2 solution, respectively. React at room temperature for 30 min, then centrifuge at 10000 rpm for 5 min. The results are as follows: Figure 7 As shown: The pigment extract solution of purple bamboo stalk epidermis treated with 1% H2O2 solution reacts with 10% ferric chloride and 6 mol / L hydrochloric acid to form a precipitate. The solution color becomes lighter after being mixed with 30% H2O2.

[0029] The results of the soluble reaction and precipitation reaction of the pigment extract from the epidermis of purple bamboo stalks after treatment with 1% H2O2 solution are consistent with the previously reported characteristics that melanin is insoluble in water and most organic reagents, but readily soluble in alkali. They are also consistent with the soluble reaction and precipitation reaction results of melanin in purple bamboo under natural conditions that have been determined by our research group.

[0030] Example 5: Spectral analysis of melanin in the epidermis of purple bamboo stalks after H2O2 treatment 1. UV-Vis absorption spectral analysis of melanin in the epidermis of purple bamboo stalks after H2O2 treatment Pigment extract from the epidermis of *Phyllostachys edulis* stalks, treated with 1 mg of 1% H₂O₂ solution, was dissolved in 1 mL of 0.5 mol / L NaOH. The absorbance was measured using a UV-26001 spectrophotometer (Shimadzu, Japan) in the wavelength range of 200 nm–700 nm. The results are as follows: Figure 8 As shown, the purple bamboo pigment extract solution treated with 1% H2O2 solution showed maximum absorption peaks at 220 nm and 285 nm, which are the same as the positions of the maximum absorption peak of purple bamboo melanin under natural conditions that have been determined by our research group.

[0031] 2. Infrared spectral analysis of melanin in the epidermis of purple bamboo stalks after H2O2 treatment Pigment extract from the epidermis of *Phyllostachys edulis* culms treated with 10 mg of 1% H₂O₂ solution was dissolved in KBr and measured using a ThermoScientific™ Nicolet™ iS™ 5 FT-IR spectrometer, with a measurement range of 4000–600 cm⁻¹. -1 The optical resolution is 4 cm. -1 The result is as follows Figure 9 As shown: Our research group has determined that the pigment extract from the epidermis of *Phyllostachys edulis* culms treated with 1% H2O2 solution is most effective at 3300-3400 cm⁻¹. -1 There is a relatively strong -OH and -NH2 absorption peak at all locations; in the 2800-2940 cm⁻¹ range... -1 There are two relatively weak absorption peaks at 1651 cm⁻¹, which are the absorption peaks of saturated hydrocarbon groups (CH) in aliphatic hydrocarbons; at 1651 cm⁻¹... -1 A strong benzene ring absorption peak is observed at all locations; a strong CO absorption peak is observed at 1403 cm⁻¹. This indicates that the characteristic absorption region of the pigment extract from the epidermis of *Phyllostachys edulis* culms treated with 1% H₂O₂ solution is basically consistent with the characteristic absorption region of melanin from *Phyllostachys edulis* culms under natural conditions.

Claims

1. A method for promoting melanin formation in the epidermis of purple bamboo stalks, characterized in that, Includes the following steps: 1) Select purple bamboo that grows uniformly, wipe off the surface dust and set aside. Prepare gauze and other materials for wrapping. 2) Prepare a fresh H2O2 solution of a certain concentration; 3) Apply H2O2 solution to the surface of the green stem of purple bamboo with gauze, wrap the applied area with wrapping material, remove the wrapping material after a period of time, and let it air dry naturally; 4) Repeat step 3) until the skin of the bamboo stalk turns purplish-black.

2. The method for promoting melanin formation in the epidermis of purple bamboo stalks as described in claim 1, characterized in that, Step 1) Select purple bamboo with uniform growth, specifically the new bamboo with mature green stems from the current year.

3. The method for promoting melanin formation in the epidermis of purple bamboo stalks as described in claim 1, characterized in that, In step 1), aluminum foil is used as the wrapping material.

4. The method for promoting melanin formation in the epidermis of purple bamboo stalks as described in claim 1, characterized in that, In step 2), a certain concentration of H2O2 solution refers to a 1% v / v H2O2 solution.

5. The method for promoting melanin formation in the epidermis of purple bamboo stalks as described in claim 1, characterized in that, In step 3), the area to be wrapped and applied should be treated once in the morning and once in the afternoon each day, with each wrapping time lasting 30-60 minutes.

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