Method for inducing anoectochilus formosanus to improve kinsenoside and adjust energy metabolites

Abstract

The invention provides a method for inducing anoectochilus formosanus to improve kinsenoside and regulate energy metabolites. The method comprises the following steps: taking Fujian anoectochilus formosanus subjected to tissue culture for 4 months as a material, hardening seedlings for 2-3 days, transplanting the seedlings to a porous plastic foam board, culturing for 15 days by adopting a Hoagland nutrient solution, and adding NaCl into the nutrient solution until the final concentration is 100 mmol. L <-1 >. And culturing for 4 days, taking anoectochilus formosanus, and measuring the content of kinsenoside of the anoectochilus formosanus, which is 3.25% and is 40.63 times that of untreated anoectochilus formosanus According to the method, the content of kinsenoside in anoectochilus formosanus can be greatly increased.

Description

Technical Field

[0001] The invention belongs to the field of agriculture and biotechnology, and in particular relates to a method for inducing Anoectochilus roxburghii to increase anoectochilus glycoside and regulating energy metabolites. Background Art

[0002] Golden thread lotus Anoectochilus roxburghii ) is a perennial herbaceous plant of the genus Anoectochilus in the Orchidaceae family, also known as golden thread orchid. It has pharmacological effects such as clearing heat and cooling blood, protecting the liver and kidneys, and can enhance the body's resistance. It has the reputation of "king of medicine". Anoectochilus roxburghii contains a variety of active ingredients such as anoectochilus glycosides, flavonoids, organic acids, steroids, etc. Among them, anoectochilus glycosides are the characteristic substances of Anoectochilus roxburghii and are the most important indicator for evaluating the quality of Anoectochilus roxburghii. Due to environmental damage and wanton cultivation, the number of wild Anoectochilus roxburghii has sharply decreased, and cultivated Anoectochilus roxburghii has become the main source. However, the content of active ingredients in cultivated Anoectochilus roxburghii is much lower than that in wild Anoectochilus roxburghii. The present invention found that under salt induction conditions, flavonoid compounds such as rutin, quercetin, kaempferol, and ergosterol, sitosterol, stigmasterol, etc. in Anoectochilus roxburghii and Anoectochilus roxburghii in Fujian and Taiwan all increased to varying degrees.

[0003] Anoectoside, chemical name 3(R)-3-β-D-glucopyranosyloxybutanolide, is a natural biologically active glycoside that has been shown to have significant pharmacological effects. At present, the synthesis of anoectoside is to use malic acid to synthesize 3-hydroxy-γ-butyrolactone, and then use β-D-glucose as a glycosylation agent and β-D-glycosidase as a catalyst to obtain anoectoside and goodyeroside A. Its synthesis is closely related to energy metabolism. Energy metabolism is the process of generating small molecule metabolites by producing energy ATP in anaerobic glycolysis and aerobic respiration of nutrients such as glucose and fatty acids. Plants release energy through ATP decomposition, participate in morphological structure, carbon and energy metabolism, nucleic acid regulation, signal transduction and other processes, and provide for plant growth and development, and resist biotic and abiotic stresses.

[0004] At present, the content of anoectochiloside in cultivated golden thread vine is much lower than that in wild golden thread vine, and the ability to increase the content of anoectochiloside in golden thread vine through fertilizers and other means is limited. Summary of the invention

[0005] The purpose of the present invention is to provide a method for inducing Anoectochilus roxburghii to increase anoectochiloside and regulating energy metabolites.

[0006] To achieve the above object, the present invention adopts the following technical solution: The present invention uses Anoectochilus fujianensis that has been tissue cultured for 4 months as the material. After 2-3 days of seedling training, the seedlings are transplanted onto a porous plastic foam board. After culturing with Hoagland nutrient solution for 15 days, NaCl is added to the nutrient solution to a final concentration of 100 mmol·L -1 After culturing for 4 days, the content of anoectochiloside in the roxburghii was determined, and the content was 3.25%, which was 40.63 times that of the untreated one.

[0007] Preferably, the culture conditions are: relative humidity of 60% to 70%, light 1000 Lux (12 h) / darkness (12 h), temperature 25°C (day) / 20°C (night).

[0008] The present invention has found that: 1. Salt induction can increase the content of anoectochiloside in Anoectochilus roxburghii; 2. After 4 days of salt induction, the content of anoectochiloside in Anoectochilus roxburghii was the highest.

[0009] 3. Salt induction can increase the content of anoectochiloside in Anoectochilus roxburghii, which is related to salt induction regulating the energy metabolites in Anoectochilus roxburghii and improving the anabolism of anoectochiloside in Anoectochilus roxburghii.

[0010] The advantages of the present invention are: The advantages of this solution over the prior art are reflected in the following aspects: first, unlike the previous method of using fertilizer to improve the quality of Anoectochilus roxburghii and thus increase the content of Anoectochilus roxburghii, this solution uses short-term low-concentration salt treatment. Second, this solution can significantly increase the content of Anoectochilus roxburghii in Anoectochilus roxburghii. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the content of anoectochiloside in Anoectochilus roxburghii under salt induction conditions; Figure 2 is the content of sugar phosphate in Anoectochilus roxburghii under salt treatment; Figure 3 is the content of phosphate compounds in Anoectochilus roxburghii under salt treatment conditions; Figure 4 is the content of organic acids and their derivatives in Anoectochilus roxburghii under salt treatment conditions; Figure 5 is the content of amino acids and their derivatives in Anoectochilus roxburghii under salt treatment conditions; Figure 6 It is the content of nucleic acid and its derivatives in Anoectochilus roxburghii under salt treatment conditions; Figure 7 This is a heat map of energy metabolites of Anoectochilus roxburghii. DETAILED DESCRIPTION

[0012] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0013] Example 1: Salt-induced treatment of Anoectochilus roxburghii The tissue culture of Anoectochilus fujianensis for 4 months was used as the material. After 2 days of seedling training, the seedlings were transplanted onto porous plastic foam boards and cultured with Hoagland nutrient solution. The relative humidity was 60%-70%, the light was 1000 Lux (12 h) / darkness (12 h), and the temperature was 25℃ (day) / 20℃ (night). After 15 days of hydroponic culture of Anoectochilus fujianensis, NaCl was added to the nutrient solution to a final concentration of 100mmol·L -1 Samples were collected at 0 (control) d, 0.5 d, 1 d, 2 d, 4 d, 8 d, 16 d and 32 d, placed in a drying oven at 60°C for drying, and the dried Anoectochilus roxburghii was ground into powder, passed through a 60-mesh sieve, 0.5 g was taken and placed in a stoppered conical flask, 7.5 mL of methanol was added, ultrasonicated for 1 h, and filtered with filter paper to obtain a methanol extract. The extract was evaporated to dryness, then shaken with 4 mL of water, extracted and defatted with an appropriate amount of chloroform, the aqueous layer was separated, the volume was fixed to 5 mL, filtered with a 0.22 uL filter membrane, and the filtrate was diluted 10 times to obtain the Anoectochilus roxburghii glycoside extracts of different treatment periods. Each sample had at least three biological replicates.

[0014] Example 2: Determination of Anoectochilus glycoside Accurately weigh 5 mg of roxburghii glycoside standard into a 50 mL volumetric flask and add methanol to prepare a 0.10 mg mL -1 Take the reference stock solution and dilute it with methanol to 0.02, 0.03, 0.04, 0.05, 0.08, and 0.09 mg·mL -1 Agilent 1200 high performance liquid chromatograph was used, and the chromatographic conditions of high performance liquid chromatography were referred to and improved. The chromatographic column used was C18 column, and the mobile phase was methanol-acetonitrile-water solution (10:5:85); the flow rate was 1.00 mL·min -1 ; Detection wavelength: 215 nm; Column temperature: 30°C; Sample volume: 10 uL, Detection of peak time and peak area of ​​anoectochilus glycoside standard, with concentration (mg·mL -1 ) is the horizontal axis ( X ), with the peak area integral value (m AU) as the ordinate ( Y), draw a standard curve. Take the 0d treated anoectin extract, inject it 5 times continuously, measure the RSD value of anoectin, determine the precision of the instrument, inject the anoectin extract of each treatment period, calculate the content of anoectin, and repeat the treatment 3 times for each sample. The calculation formula of anoectin content in the sample is as follows. Where: C is the anoectin concentration (mg·mL -1 ), V is the volume of aqueous solution, and m is the mass of sample (g).

[0015] The peak time of the standard anointing agent is between 2 and 3 min. With the concentration of anointing agent as the horizontal coordinate and the absorption peak area as the vertical coordinate, the regression equation is: y = 77.30 x -0.13, R 2 =0.9996, showing a good linear relationship. Under the above HPLC conditions, the anoectin extract of Anoectochilus roxburghii after induced treatment on day 0 was injected 6 times in succession. The relative standard deviation of each chromatographic peak was determined to be 0.15%, and the relative standard deviation of the peak area was 0.97%, indicating that the method has good accuracy.

[0016] Substitute the peak area of ​​each sample into the standard curve of anointing line glycoside to calculate the concentration of anointing line glycoside, and then substitute it into the formula to calculate the anointing line glycoside content ( Figure 1 ). The results showed that the content of anoectin in Anoectochilus roxburghii increased slowly when it was not induced by NaCl, and was 0.08%, 0.10% and 0.14% at 0, 4 and 32 days, respectively. After being induced by NaCl, the content of anoectin increased sharply, reaching the highest level (3.25%) at 4 days, which was 40.63 times that of the untreated state. After that, the content of anoectin decreased slowly, and was 2.16% at 32 days after salt induction, which was 27.00 times that of the untreated state.

[0017] Example 3: Detection of energy metabolite content Weigh 50 mg (± 2.50 mg) of the Anoectochilus roxburghii samples with different induction treatment times, add 500 μL of 70% methanol-water extract precooled at -20 °C, vortex for 3 min, and rotate at 12000 r·min at 4 °C. -1 Centrifuge for 10 min, aspirate 300 μL of the supernatant, let stand at -20 °C for 30 min, and then spin at 12000 r·min at 4 °C. -1Centrifuge for 10 min, take 200 μL of the supernatant and pass it through a protein precipitation plate and store it at -20 °C. Ultra-high performance liquid chromatography and tandem mass spectrometry were used, and the chromatographic column used was ACQUITY UPLCBEH Amide column (1.7 μm, 100 mm×2.1 mm), mobile phase: phase A, ultrapure water (10 mL ammonium acetate, 0.3% ammonia water); phase B, 90% acetonitrile / water (V / V); flow rate: 0.40 mL·min -1 ; Column temperature 40°C; injection volume 2 µL, draw standard curves of different energy substances, and then calculate the peak area and substance content of the test sample through the standard curve. The calculation formula is as follows. Where: c: sample concentration value (ng·mL -1 ); V: volume of solution used for sample extraction (μL); m: sample mass (mg).

[0018] Energy metabolism of organisms includes carbohydrate metabolism, amino acid metabolism, lipid metabolism and nucleic acid metabolism. Compared with the untreated group, the contents of 34 substances in Anoectochilus roxburghii were significantly different (P<0.01) and 9 substances were significantly different (P<0.05) at 4 days after treatment. The contents of 36 substances were significantly different (P<0.01) and 11 substances were significantly different (P<0.05) at 16 days after treatment. Among them, deoxycytidine, 2-phospho-D-glyceric acid and 3-phosphoglyceric acid had no significant difference under salt treatment.

[0019] Among the eight sugar phosphate products detected, glucose had the highest content, which was 1343400.49 ng / mg, and 6-phosphogluconic acid had the lowest content, which was 720.01 ng / mg ( Figure 2 The contents of glucose, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate decreased continuously at 4 and 16 days after salt treatment, while fructose 1,6-diphosphate, erythrose 4-phosphate, 6-phosphogluconic acid, and trehalose-6-phosphate increased continuously ( Figure 2 ).

[0020] Among the six triose phosphate compounds detected, the content of glycerol-3-phosphate (16790.62 ng·mg -1 ) was higher than the content of other triose phosphate compounds, and a valley value appeared at 4 days after salt treatment, and the content was lower than that of untreated and treated 16 days ( Figure 3 The contents of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate continued to increase after salt treatment ( Figure 3 BC in the middle). The content of glycerate-2-phosphate and glycerate-3-phosphate did not change significantly under the treatment conditions ( Figure 3The pyruvic acid content showed a continuous decrease under salt treatment ( Figure 3 Middle F).

[0021] When untreated, citric acid had the highest content among the nine organic acids and their derivatives detected in Anoectochilus roxburghii, which was 174227.95 ng·mg -1 ( Figure 4 A). The contents of aconitic acid and isocitric acid were 13760.83 and 1989.07 ng·mg respectively. -1 ( Figure 4 BC in the middle). The contents of α-ketoglutaric acid, succinic acid and fumaric acid were similar, which were 49294.60, 57586.76 and 53188.21 ng·mg respectively. -1 ( Figure 4 DF). Malic acid content is 111805.57ng·mg -1 ( Figure 4 In addition, the contents of itaconic acid and argininosuccinic acid were relatively low, at 12101.65 and 578.15 ng·mg, respectively. -1 ( Figure 4 The contents of citric acid, isocitric acid, succinic acid and argininosuccinic acid in organic acids and their derivatives showed an upward trend at 4d and 16d, while aconitic acid, α-ketoglutaric acid, fumaric acid and itaconic acid decreased ( Figure 4 ).

[0022] Among the 13 amino acids and their derivatives detected in Anoectochilus roxburghii, L-aspartic acid (2497031.32 ng·mg -1 ) had the highest content, followed by L-asparagine (307807.96 ng·mg -1 ), they and threonine (19254.9 ng·mg -1 ) are all oxaloacetate-derived amino acids. Under salt induction, the expression of amino acids is continuously downregulated ( Figure 5 AC). Glutamic acid (128929.53 ng·mg -1 ), glutamine (4704.18 ng·mg -1 ), ornithine (650.39 ng·mg -1 ) and lysine (9391.93 ng·mg -1 ) is an amino acid derived from α-ketoglutarate. Under salt induction, its expression is consistent with α-ketoglutarate and also shows a continuous decline ( Figure 5 DG). Citrulline (52.34 ng·mg -1 ) and arginine (27936.36ng·mg -1), which are the amino acids downstream of ornithine, under salt induction, the content change trend of these two amino acids is opposite to that of ornithine, and they are upregulated ( Figure 5 HI). Leucine (16150.78 ng·mg -1 ) and alanine (62402.57 ng·mg -1 ) is a pyruvate-derived amino acid, serine (25245.14 ng·mg -1 ) and tyrosine (34382.14 ng·mg -1 ) were glycerol-3-phospho-type and erythrose-4-phospho-type amino acids, respectively. Under salt induction, the contents of these amino acids showed a continuous decrease ( Figure 5 Chinese JM).

[0023] The contents of 15 substances in nucleic acids and their metabolites were detected. Among them, the contents of guanosine and uridine 5-monophosphate were 28197.61 and 15318.91 ng·mg, respectively. -1 , ranking the first two; the content of adenosine triphosphate was the lowest, only 140.84 ng·mg -1 Among the four deoxynucleotides, deoxythymidylate has the highest content (2380.74 ng·mg -1 ), the content of deoxycytidine was the lowest (177.93 ng·mg -1 ). When untreated, adenine (1908.21 ng·mg -1 ) and uracil (1908.21 ng·mg -1 ) is close to ( Figure 6 ). After salt induction, the contents of GDP, deoxythymidylic acid, deoxyadenylic acid, UDDP-N-acetylglucosamine, guanosine and ADP decreased continuously; the enrichment of ATP, deoxyuridine, uridine 5-monophosphate, inosine-1-phosphate, adenylic acid and adenine was affected by salt induction, and increased significantly at 4 days, but decreased to varying degrees at 16 days. Under salt induction, the contents of inosine and uracil decreased at 4 days, but increased to more than twice that of the untreated cells at 16 days ( Figure 6 ).

[0024] Example 4: Heat map of energy metabolites The obtained metabolite content data were processed by UV (unit variance scaling), and the mean of the processed data was set to 0 and the standard deviation was set to 1. The formula is as follows. In the formula: μ is the mean and σ is the standard deviation. The R software ComplexHeatmap package was used to draw a heat map, and a hierarchical cluster analysis (HCA) was performed on the accumulation of energy metabolites at different salt treatment times.

[0025] Z-value μ) / σ From the sample clustering results, it can be seen that most nucleotides and their metabolites are red when induced by salt for 4 days, and green when untreated and treated for 16 days, indicating that these substances have a peak at 4 days of salt induction, and the content is higher than that of untreated and salt-treated for 16 days. Amino acids, organic acids and their derivatives, and some phosphate sugar metabolites are red when untreated, and green after salt induction, indicating that their content is reduced; some phosphate sugars and phosphate compounds are green when untreated, and red after salt induction for 16 days, indicating that their content increases under salt induction ( Figure 7 ).

[0026] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for inducing Anoectochilus roxburghii to increase anoectochiloside and regulate energy metabolites, characterized in that: The following steps are involved: Anoectochilus fujianensis, which had been in tissue culture for 4 months, was used as the material. After 2-3 days of seedling training, the seedlings were transplanted onto porous plastic foam boards and cultured with Hoagland nutrient solution for 15 days. Then, NaCl was added to the nutrient solution to a final concentration of 100 mmol·L -1 , cultured for 4 days.

2. The method according to claim 1, characterized in that: Culture conditions: Relative humidity is 60%~70%.

3. The method according to claim 1, characterized in that Culture conditions: light 1000 Lux (12 h) / darkness (12 h).

4. The method according to claim 1, characterized in that: Culture conditions: temperature 25℃ (day) / 20℃ (night).

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