A light treatment method for increasing the content of 6'-o-coffea acyl arbutin in mature leaves of vaccinium uliginosum

Abstract

This invention discloses a light treatment method for increasing the content of 6′-O-caffeoylarbutin (CA) in mature leaves of *Vaccinium bracteatum*. The method involves treating *Vaccinium bracteatum* plants under 70%-80% full light intensity for 60-90 days. This light intensity is achieved by attenuating natural full light through a shading net. The mature leaves are the 2nd to 4th fully unfolded healthy leaves at the top of the plant. This invention reveals for the first time the regulatory mechanism of light intensity on CA content. After 60 days of treatment, the CA content in mature leaves reached 2.470±0.100% (dry weight), significantly higher than other light treatment groups (P<0.05). The CA synthesis gene... VdAT3 The relative expression level was increased to more than 1.17 times that of the control group; the maximum photochemical efficiency of PSⅡ, Fv / Fm, remained stable between 0.74 and 0.79, without causing light stress to the plants. This invention is simple to operate, low in cost, and suitable for the large-scale production of high CA content raw materials in the artificial domestication and cultivation of Citrus camphora.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation and secondary metabolite regulation technology, specifically relating to a light treatment method that increases the content of 6′-O-caffeoylarbutin (CA) in mature leaves of *Vaccinium bracteatum* by regulating light intensity. Background Technology

[0002] Camphor leaf blueberry ( Vaccinium dunalianum Vaccinium wightii (also known as Blueberry) is a plant belonging to the genus Vaccinium in the Ericaceae family. Its leaf buds are used to make "Sparrow's Beak Tea," a beverage consumed for centuries in the Yi ethnic region of Yunnan. Studies have shown that Vaccinium wightii is rich in three highly active secondary metabolites: 6′-O-caffeoylarbutin (CA), arbutin, and chlorogenic acid. Among these, 6′-O-caffeoylarbutin (CA, molecular formula C...) is particularly important. 21 H 22 O9 is a caffeoylated derivative of arbutin, and its structural formula is as follows: .

[0003] CA exhibits significant tyrosinase inhibitory and antioxidant activities. Melanin inhibition experiments using a zebrafish model showed that, at the same concentration, CA's melanin-inhibiting activity was approximately twice that of arbutin, while its cytotoxicity was only half that of arbutin. Therefore, CA has extremely high development value in fields such as skin-whitening cosmetics and pharmaceuticals.

[0004] However, previous research by our group found that when wild blueberry plants with high CA content (up to 23% dry weight of leaf buds) from Wuding County, Yunnan Province, were introduced to Kunming, the CA content significantly decreased to 1.89% of the dry weight, a reduction of over 90%. Drought stress and temperature treatments could not explain this phenomenon, and changes in light intensity were presumed to be the key factor. There are also no reports on the effect of light intensity on CA accumulation in blueberry leaves. Therefore, developing a light treatment method that can significantly increase CA content in blueberry leaves has significant industrial application value. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the purpose of this invention is to provide a light treatment method that increases the CA content in mature leaves of *Vaccinium bracteatum* by precisely controlling light intensity, thereby solving the technical problem of a significant decrease in CA content after the introduction and cultivation of *Vaccinium bracteatum*, and providing technical support for the artificial domestication and industrial raw material production of *Vaccinium bracteatum*.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for increasing the content of 6′-O-caffeoylarbutin (CA) in mature leaves of *Vaccinium camphora* includes the following steps: The camphor-leaved blueberry plants were placed under 70%~80% full light intensity for light treatment, which lasted for 60~90 days. The total light intensity is achieved by attenuating the natural total light intensity through a shade net; The mature leaves are the 2nd to 4th fully unfolded healthy leaves at the top of the blueberry plant.

[0007] Furthermore, the camphor-leaved blueberry plant is a tissue-cultured seedling or a cutting.

[0008] Furthermore, after the light treatment, the CA content in the mature leaves of the camphor blueberry reaches more than 2.3% by dry weight, preferably 2.470±0.100%.

[0009] Furthermore, during the light treatment process, the maximum photochemical efficiency (Fv / Fm) of the PSⅡ of the blueberry leaves remained stable between 0.74 and 0.79, indicating that the light treatment did not cause light stress to the blueberry.

[0010] Furthermore, during the light treatment process, the CA synthesis-related genes in the leaves of *Vaccinium bracteatum* are... VdAT3 The relative expression level was upregulated to more than 1.17 times that of the control group.

[0011] The present invention provides a method for preparing camphor leaf blueberry leaf raw material, wherein mature camphor leaf blueberry leaves processed by the method are harvested, dried and crushed to obtain camphor leaf blueberry leaf raw material.

[0012] Furthermore, the drying process involves drying at 45°C to a constant weight.

[0013] The present invention has the following beneficial effects: 1. This invention is the first to systematically study the effects of different light intensities (100%, 75%, 50%, 25% full light) and different treatment times (20d, 40d, 60d) on CA content in mature leaves of Vaccinium bracteatum, filling a research gap in this field and providing a theoretical basis for the artificial domestication and cultivation of Vaccinium bracteatum.

[0014] 2. This invention places the blueberry plants under 75% full light intensity for no less than 60 days, and the CA content in the mature leaves reaches 2.470±0.100% (dry weight), which restores and stabilizes the CA content by more than 30%, effectively solving the industry pain point of a sharp drop in CA content after introduction.

[0015] 3. This invention analyzes CA synthesis-related genes using qPCR technology. VdAT3 The expression pattern. Results showed that under 60d × 75% full illumination, VdAT3 The relative gene expression level was 1.172 times that of the control group.P <0.05, significantly higher than the 100% full illumination (0.481 times), 50% full illumination (1.052 times), and 25% full illumination (0.025 times) treatment groups.

[0016] 4. The embodiments of the present invention show that 75% full light intensity does not cause light stress to the camphor leaf blueberry, and the plant can grow healthily. This overcomes the technical defects of some secondary metabolite induction methods in the prior art, which are often accompanied by plant growth inhibition (such as high temperature treatment, which can increase the content of some compounds but seriously affects plant growth). It achieves the synergy between the accumulation of high-value products and healthy plant growth.

[0017] 5. This invention can be achieved simply by setting up a shade net (25% shading rate) to control the light intensity. It does not require complicated equipment or chemical treatment, has low investment cost, is easy to operate, and is suitable for the large-scale promotion of artificial domestication and cultivation of blueberry. It provides technical support for promoting the industrial application of blueberry in the fields of whitening cosmetics and functional foods. Attached Figure Description

[0018] Figure 1 This is the standard curve of CA in Embodiment 2 of the present invention; Figure 2 This is a graph showing the effect of different light intensity treatments on the content of 6′-O-caffeoylarbutin (CA) in mature leaves of *Vaccinium bracteatum* in Example 3 of this invention. Figure 3 Genes related to CA synthesis in mature leaves of *Vaccinium bracteatum* under different light intensity treatments in Example 5 of this invention. VdAT3 Relative expression level plot; Figure 4 This is a graph showing the effect of different light intensity treatments on the maximum photochemical efficiency (Fv / Fm) of PSⅡ in the leaves of *Vaccinium camphora* in Example 6 of the present invention. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0020] Example 1: Method for treating camphor-leaved blueberry leaves with light

[0021] This embodiment provides a light treatment method to increase the content of 6′-O-caffeoylarbutin (CA) in mature leaves of *Vaccinium bracteatum*, specifically including the following steps: 1. Materials The test material was a 4-year-old camphor-leaved blueberry cultivated by the research group. Vaccinium dunalianumThe plants included cuttings and tissue-cultured seedlings. The tissue-cultured seedlings were obtained by using wild blueberry stem segments with buds as explants, followed by rapid propagation and rooting. The plants were potted and grown in a greenhouse at the Kunming Institute of Botany, Chinese Academy of Sciences experimental base.

[0022] 2. Experimental Methods Four light intensity gradients were set up: 100% full light intensity, 75% full light intensity, 50% full light intensity, and 25% full light intensity. Different light intensities were achieved by constructing shade nets with different knitting densities to attenuate natural full sunlight. The light intensity values ​​of each treatment group were measured and calibrated using a photometer. Each treatment group consisted of 42 plants, with untreated plants (0 days) serving as a control.

[0023] Based on the above four light intensity gradients, four processing times were set: 0d, 20d, 40d, and 60d.

[0024] 3. Sampling Method Samples were taken at 0, 20, 40, and 60 days after treatment. For each treatment group, 5-7 plants were randomly selected, and samples were taken from the 2nd-4th fully expanded, healthy, mature leaves at the top of each plant. The leaf samples were then mixed to serve as the representative sample for that treatment group, with three biological replicates. Immediately after collection, samples were flash-frozen in liquid nitrogen and stored at -80°C for subsequent CA content determination and gene expression analysis.

[0025] 4. Specific steps of the light treatment method (1) The blueberry plants were transferred to the outdoor experimental base and the natural light intensity was reduced to 75%, 50% and 25% of the full light intensity by using shade nets (with unshaded natural light as 100% full light intensity). The light intensity of each treatment group was monitored and calibrated regularly by a photometer. (2) Continuous light treatment for 60 days; (3) During the treatment period, water regularly to maintain normal plant growth and record plant growth status; (4) At 0d, 20d, 40d and 60d, the 2nd to 4th fully unfolded healthy mature leaves at the top of the blueberry plant were collected for subsequent analysis.

[0026] Example 2: HPLC method for detecting CA content in blueberry leaves

[0027] 1. Sample pretreatment The collected camphor and blueberry leaf samples were chopped, dried at 45℃ to constant weight, ground in a mortar at room temperature, and passed through a 40-mesh sieve. 0.1 g of the sample powder was accurately weighed and placed in a 2 mL centrifuge tube. 1.6 mL of 73% methanol extraction solution was added at a liquid-to-solid ratio of 16:1. The mixture was ultrasonically extracted for 19 min in an ultrasonic cleaner, centrifuged, and the supernatant was collected. The extraction was repeated four times. The extracts were combined, mixed thoroughly, and set aside.

[0028] 2. HPLC detection conditions Chromatographic column: Thermo BDS HYPERSIL C18 (250 mm × 4.6 mm, 5 μm); mobile phase A: water (containing 1% glacial acetic acid), mobile phase B: chromatographically pure methanol; gradient elution program as follows: 0-5 min, A 95%, B 5%; 5-10 min, A linearly decreases from 95% to 85%, B increases from 5% to 15%; 10-50 min, A linearly decreases from 85% to 35%, B increases from 15% to 65%; 50-55 min, A linearly decreases from 35% to 5%, B increases from 65% to 95%; 55-70 min, A 5%, B 95%. Detection wavelength: 280 nm; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL.

[0029] 3. Establishment of the standard curve Accurately weigh 10.000 mg of 6′-O-caffeoylarbutin (CA) standard (purity ≥95%, isolated and purified from the leaf buds of *Vaccinium bracteatum*), and dilute to 10 mL in a volumetric flask with methanol to prepare a 1.0 mg / mL CA standard stock solution. Inject 1, 2, 4, 8, 16, 25, and 32 μL of the solution, respectively, and measure the HPLC peak areas of CA as follows: 1 1 1181372.493 2 2 2516561.853 4 4 4933897.479 8 8 10113784.39 16 16 19966100.15 25 25 31064079.52 32 32 38619520.08 A standard curve was plotted with the injection volume (μg) on ​​the x-axis and the peak area on the y-axis, and the detection wavelength at 280 nm, as shown below. Figure 1 As shown, the standard curve equation for CA is: y = 1217656x + 177361 (R² = 0.9995), indicating that CA has a good linear relationship within the detection range.

[0030] 4. Content Calculation Substitute the HPLC peak area of ​​the sample extract into the CA standard curve equation to calculate the mass of CA in the sample, and then calculate the dry weight percentage (%) of CA based on the dry weight of the sample.

[0031] Example 3: Effects of different light intensities on CA content in mature leaves of *Vaccinium bracteatum*.

[0032] The camphor-leaved blueberry plants were subjected to light treatment according to the method in Example 1, and the CA content in mature leaves of each treatment group was detected according to the method in Example 2. The results are as follows: Figure 2 As shown in Table 1.

[0033] Table 1. Effects of different light intensities on CA content (dry weight%) in mature leaves of *Vaccinium bracteatum*.

[0034] Note: Data in the table are mean ± standard deviation (n=3); different lowercase letters in the same row indicate significant differences. P <0.05).

[0035] Depend on Figure 2 As shown in Table 1, light intensity and duration of illumination significantly affected the CA content in mature leaves of *Vaccinium bracteatum*. At 60 days of treatment, the CA content in the 75% full light intensity treatment group was as high as 2.470 ± 0.100% (dry weight), significantly higher than that in the 100% full light intensity treatment group (1.445%), the 50% full light intensity treatment group (1.677%), and the 25% full light intensity treatment group (1.175%). P <0.05).

[0036] Of particular note is that the CA content in the 75% full light intensity treatment group reached its highest value at 60 days, a significant increase compared to 40 days (1.685%), exhibiting a counter-trend growth characteristic. In contrast, the CA content in the other three light intensity treatment groups (100%, 50%, and 25%) all showed a decreasing trend from 40 to 60 days (from 2.850% to 1.445%, from 2.918% to 1.677%, and from 3.662% to 1.175%, respectively). This indicates that the combination of 75% full light intensity × 60 days has a unique, non-linear synergistic effect in promoting CA accumulation.

[0037] Example 4: Effect of light treatment time on CA content in mature leaves of *Vaccinium camphora*

[0038] The blueberry plants were placed under 75% full light intensity for 20 days, 40 days and 60 days respectively. The CA content in mature leaves was detected according to the method in Example 2. The results are shown in Table 2.

[0039] Table 2. Effects of different treatment times under 75% full light intensity on CA content (dry weight%) in mature leaves of *Vaccinium camphora*.

[0040] Table 2 shows that under 75% full light intensity, the CA content exhibits a non-monotonic trend of "first decreasing and then increasing" over time: it was 2.510% at 20 days, decreased to 1.685% at 40 days, and then increased back to 2.470% at 60 days. This result indicates that a treatment time of at least 60 days is a necessary condition for achieving significant CA accumulation, and although the 40-day treatment time is shorter, the effect is actually worse.

[0041] Example 5: Effects of 75% full light intensity treatment on key genes in CA synthesis for 60 days VdAT3 The impact of expression This embodiment verifies the effect of light treatment on CA synthesis at the molecular level. Mature leaf samples of *Vaccinium bracteatum* from each treatment group in Example 3 were collected, total RNA was extracted, reverse transcribed into cDNA, and the glyceraldehyde-3-phosphate dehydrogenase gene VdGAPDH was used as an internal control. Quantitative real-time PCR (qPCR) was then used to detect genes related to CA synthesis. VdAT3 The relative expression level. VdAT3 The qPCR primer sequences for the gene are: upstream primer 5′-ACCGAGGTTTCCGATGTACG-3′, downstream primer 5′-TTGACCCATCCCACAACCAC-3′.

[0042] qPCR reaction system (20 μL): EvaGreen 2×qPCR MasterMix 10 μL, forward and reverse primers 0.6 μL each, internal control gene forward primer F 0.6 μL, internal control gene reverse primer R 0.6 μL, cDNA template 2 μL, ddH2O to 20 μL. Reaction program: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 56℃ annealing and extension for 30 s, for a total of 40 cycles. 2 -△△CT Method calculation VdAT3 The relative expression levels of genes, the results are as follows: Figure 3 As shown in Table 3.

[0043] Table 3. Camphor-leaved blueberry leaves treated with different light intensities VdAT3 Gene expression level

[0044] Note: Different letters in the same row indicate significant differences in the values ​​of that row (P<0.05). As can be seen from the above, the 60d×75% full illumination intensity treatment group VdAT3 The relative gene expression level was 1.172 ± 0.329 times that of the control group. P <0.05, significantly higher than the 100% full illumination (0.481 times), 50% full illumination (1.052 times), and 25% full illumination (0.025 times) treatment groups. VdAT3Gene expression levels peaked at 60 days under 75% full light intensity, highly consistent with the highest CA content (2.470%) under the same conditions. This indicates that the 75% full light intensity × 60 days treatment upregulated CA synthesis-related genes. VdAT3 The transcriptional level of CA promoted its accumulation in mature leaves of *Vaccinium camphora*. This molecular-level evidence provides intrinsic mechanistic support for the technical effects of this invention.

[0045] Example 6: Effects of light treatment on photosynthetic physiological indicators of camphor blueberry leaves

[0046] The light treatment was carried out according to the method of Example 1. The saturated photosynthetic rate (Pn) and PSⅡ maximum photochemical efficiency (Fv / Fm) of the camphor and blueberry leaves of each treatment group were measured using a portable photosynthesis measurement system Li-COR6400.

[0047] The saturation photosynthetic rate (Pn) is shown in Table 4: Table 4. Saturated photosynthetic rate (μmol·m⁻²·s⁻¹) of blueberry leaves under different light intensities for different durations

[0048] As shown in Table 4, the saturated photosynthetic rate Pn of *Vaccinium camphora* leaves under 50% full light intensity was highest at 20d, 40d, and 60d (3.795, 5.215, and 3.522 μmol·m⁻²·s⁻¹, respectively), indicating that 50% full light intensity is the most suitable light intensity for the growth of *Vaccinium camphora* plants, and the plants have the strongest photosynthetic capacity under this light intensity.

[0049] (2) Maximum photochemical efficiency of PSⅡ (Fv / Fm) Table 5. Photosynthetic parameters (Fv / Fm) of *Vaccinium camphora* leaves at different light intensities and durations.

[0050] From Table 5 and Figure 4 It can be seen that there were no significant differences in the Fv / Fm values ​​of blueberry leaves in each treatment group at 20d, 40d, and 60d. P <0.05), stable between 0.76 and 0.78. Fv / Fm is an important indicator reflecting the degree of photoinhibition in plants. Under normal conditions, this value is usually stable between 0.75 and 0.85, only decreasing significantly under stress. In this experiment, the Fv / Fm values ​​of all treatment groups remained stable, indicating that 75% full light intensity did not constitute photostress for *Vaccinium camphora*, and the plants could grow healthily. This result further demonstrates that the light treatment conditions used in this invention promote CA accumulation without sacrificing normal plant growth, exhibiting good practicality and safety.

[0051] Example 7: Large-scale production method of mature leaves of camphor-leaf blueberry with increased CA content

[0052] This embodiment provides a method for large-scale production of camphor blueberry leaves with high CA content.

[0053] Transplant the tissue-cultured and acclimatized seedlings of *Vaccinium bracteatum* to the field using conventional methods. Construct a 25% shade net (i.e., 75% full light intensity) over the field. Maintain a distance of 50-80 cm between the shade net and the top of the plant to ensure good ventilation. Perform standard water and fertilizer management. Harvest the 2nd-4th fully unfolded, healthy, mature leaves from the top of the plant 60 days after treatment.

[0054] HPLC analysis showed that the CA content in the mature leaves of the harvested blueberry was consistently above 2.3% (dry weight), which can be used for subsequent CA extraction and preparation of cosmetic raw materials. This method is simple to operate and low in cost, requiring only a one-time installation of a shade net, without the need for complex equipment or chemical treatments, making it suitable for large-scale promotion of the artificial domestication and cultivation of blueberry.

[0055] Comparative Example 1 (100% full light intensity × 60 days)

[0056] Following the method in Example 1, the blueberry plants were treated under 100% full light intensity for 60 days, and the CA content in mature leaves was detected according to the method in Example 2. The results showed that the CA content was only 1.445 ± 0.102% (dry weight), significantly lower than that in Example 3 of this invention (75% full light intensity × 60 days treatment group, 2.470%). P <0.05).

[0057] Comparative Example 2 (50% full light intensity × 60 days)

[0058] When blueberry plants were treated under 50% full light intensity for 60 days, the CA content in mature leaves was only 1.677±0.073% (dry weight), significantly lower than that in Example 3 of this invention (2.470%). P <0.05).

[0059] Comparative Example 3 (25% full light intensity × 60 days)

[0060] When blueberry plants were treated under 25% full light intensity for 60 days, the CA content in mature leaves was only 1.175±0.120% (dry weight), significantly lower than that in Example 3 of this invention (2.470%). P <0.05).

[0061] Comparative Example 4 (75% full light intensity × 40 days)

[0062] When blueberry plants were treated under 75% full light intensity for 40 days, the CA content in mature leaves was only 1.685±0.090% (dry weight), significantly lower than that in the 60-day treatment group (2.470%). P The concentration of CA was <0.05%, and lower than that of the 20-day treatment group (2.510%). This indicates that under 75% full light intensity, the CA content did not increase monotonically over time; in fact, it decreased after 40 days, and only reached significant accumulation again after ≥60 days of treatment.

[0063] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for phototreatment to increase the content of 6′-O-caffeoylarbutin in mature leaves of *Vaccinium bracteatum*, characterized in that, Includes the following steps: The camphor-leaved blueberry plants were placed under 70%~80% full light intensity for light treatment, which lasted for 60~90 days. The total light intensity is achieved by attenuating the natural total light intensity through a shade net; The mature leaves are the 2nd to 4th fully unfolded healthy leaves at the top of the blueberry plant.

2. The method according to claim 1, characterized in that, The camphor-leaved blueberry plants are tissue-cultured seedlings or cuttings.

3. The method according to claim 1, characterized in that, After the light treatment, the CA content in the mature leaves of the camphor blueberry reached more than 2.3% of the dry weight.

4. The method according to claim 1, characterized in that, During the light treatment process, the blueberry plants were in a normal growth state, and the maximum photochemical efficiency of PSⅡ in their leaves, Fv / Fm, remained stable between 0.74 and 0.

79.

5. The method according to claim 1, characterized in that, During the light treatment process, CA synthesis-related genes were found in the leaves of *Vaccinium bracteatum*. VdAT3 The relative expression level was upregulated to more than 1.17 times that of the control group.

6. A method for preparing camphor-leaf blueberry leaf raw material, characterized in that, Mature leaves of camphor blueberry processed by any one of claims 1-5 are harvested, dried and crushed to obtain camphor blueberry leaf raw material.

7. The preparation method according to claim 6, characterized in that, The drying process involves drying at 45°C to a constant weight.

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