Cultivation and harvesting method for increasing chicoric acid content of echinacea purpurea leaves

Abstract

The invention discloses a cultivation and harvesting method for improving the chicoric acid content of echinacea purpurea leaves, and the cultivation method comprises the following steps: applying an organic fertilizer, turning over, ridging in a field, and planting at a planting interval of 29-31 cm * 29-31 cm; in the plant growth process, hormone foliage spraying regulation and control are conducted twice, and the interval between two times of spraying is at least one week; the hormone is selected from paclobutrazol with the concentration of 1000 mg / L or 500 mg / L; in the later stage of the full-bloom stage each time, harvesting and harvesting overground parts, the harvesting time is 10 o'clock to 12 o'clock in the morning, and a distance is reserved at the base part; the content of chicoric acid in the harvested leaves is increased. According to the method, the content of chicoric acid in echinacea purpurea leaves is increased, growth is promoted, and plants are dwarf and strong by adjusting and controlling base fertilizer, planting spacing, harvesting in different months, harvesting in different time periods and hormone spraying in the cultivation process.

Description

Technical Field

[0001] The invention relates to a cultivation method of echinacea, and in particular to a cultivation and harvesting method for increasing the chicoric acid content in echinacea leaves. Background Art

[0002] The information provided in this section is for the purpose of generally presenting the background of the present disclosure. To the extent described in this section, the work of the presently named inventors and aspects of the description that may not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present disclosure.

[0003] Echinacea, also known as purple coneflower and purple coneflower, is a perennial herbaceous plant of the genus Echinacea in the family Asteraceae. It is native to North America and is a perennial flower with high ornamental and medicinal value. In recent years, due to its good immune-enhancing effect, Echinacea has been widely used in countries such as Germany, Canada, and the United States, becoming one of the top ten popular herbs in the international herbal medicine market.

[0004] The whole herb of Echinacea contains a secondary metabolite, cichoric acid, which has become an indicator component for evaluating the quality of Echinacea medicinal materials in the international market because of its effects on enhancing immune function, anti-inflammatory, inhibiting hyaluronidase, and protecting collagen III. In addition, Echinacea also has good ornamental value and is planted as an ornamental flower in many places, especially the Echinacea cultivars that are currently widely used in gardening.

[0005] However, there are two main problems in the cultivation of echinacea. First, the plants are tall and easily fall over after heavy rain and strong winds. Second, the content of chicoric acid needs to be improved. Therefore, if the growth of echinacea plants can be promoted and the content of effective ingredients in the leaves can be increased, then the ornamental and medicinal echinacea will definitely have an advantage over other flowers. For example, large-scale production can better serve rural revitalization, so it has great application value and promotion prospects. Summary of the invention

[0006] The purpose of the present invention is to provide a cultivation and harvesting method for increasing the chicoric acid content in echinacea leaves, which can increase the chicoric acid content in echinacea leaves, promote the growth of echinacea and make the plants short and strong.

[0007] In order to achieve the above-mentioned purpose, the present invention provides a cultivation and harvesting method for increasing the content of chicoric acid in echinacea leaves. The cultivation method comprises: applying organic fertilizer as base fertilizer and then tilling the field, making ridges in the field, and planting at a planting spacing of 29 to 31 cm × 29 to 31 cm; during the growth of the plants, spraying the leaves with hormones twice for regulation, with an interval of at least one week between the two sprayings; the hormone is paclobutrazol with a concentration of 1000 mg / L or 500 mg / L; harvesting is carried out separately in the late stage of each flowering period, harvesting the aboveground parts, the harvesting time is 10 to 12 o'clock in the morning, and leaving a distance at the base; the chicoric acid content in the harvested leaves is increased.

[0008] Preferably, the cultivation method further comprises: applying compound fertilizer once before and after spraying the hormone, and tilling and weeding.

[0009] Preferably, the compound fertilizer is applied at an amount of 22 kg per mu. More preferably, the compound fertilizer is 15-15-15 compound fertilizer, with total nutrients ≥ 45% and nitrate nitrogen ≥ 6%.

[0010] Preferably, the cultivation method further comprises: performing weeding and fertilizing between each harvest.

[0011] Preferably, after the first harvest, weeds around the base are removed after harvesting, compound fertilizer is sprinkled around the base, and compound fertilizer is applied again during the early flowering period, and the second harvest is carried out in the late second flowering period.

[0012] Preferably, the cultivation method further comprises: raising seedlings: sowing in a greenhouse, using peat as the substrate, sowing seeds in a flat tray, covering with a thin layer of peat, and transplanting the seedlings into a hole tray when the seedlings have 1 to 2 true leaves.

[0013] More preferably, the peat is Pinnacle peat with a size of 0 to 10 mm.

[0014] More preferably, the cultivation method further comprises: transplanting the seedlings from the hole tray to a pot.

[0015] Preferably, the field ridges have a width of 1.3m, a furrow width of 20cm, and a ridge height of 15cm; or / and, the soil of the field ridges is sandy loam with medium soil fertility and medium permeability; or / and, the amount of organic fertilizer is 800 kg / mu. More preferably, the organic matter content of the organic fertilizer is ≥45%.

[0016] Preferably, the base leaves 20 cm.

[0017] The cultivation and harvesting method for increasing the chicoric acid content in echinacea leaves of the present invention has the following advantages:

[0018] (1) The method of the present invention adopts the method of applying organic fertilizer first and then inorganic fertilizer, which makes full use of the characteristics of organic fertilizer having a long effective period and better effect after tillage;

[0019] (2) The method of the present invention increases the chicoric acid content of Echinacea leaves by regulating the base fertilizer, planting spacing, harvesting in different months, harvesting in different time periods, and hormone spraying during the cultivation process;

[0020] (3) The method of the present invention, through two sprayings of 1000 mg / L of paclobutrazol, prevents the excessive growth of echinacea from falling over, promotes the synthesis of chlorophyll in leaves, increases the content of chicoric acid in leaves, makes the echinacea plants short and strong, increases the number of flowers, and improves the overall ornamental value of the plants;

[0021] (4) The method of the present invention is simple and easy to implement, and can improve the dwarfing of the echinacea plant, prevent it from falling over, increase the number of flowers, and improve its ornamental value. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] It should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0024] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0025] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0026] In order to increase the chicoric acid content in Echinacea leaves, the inventors of the present invention conducted a series of studies and found that there are large differences in the chicoric acid content in the leaves of different Echinacea varieties, and in the cultivation process, base fertilizer, planting spacing, different months, different time periods and different hormones will affect the chicoric acid content in the leaves. Based on these influencing factors, the inventors explored a cultivation and harvesting method for increasing the chicoric acid content in Echinacea leaves. The cultivation method of the present invention can significantly increase the chicoric acid content in Echinacea leaves, improve the quality of Echinacea medicinal materials, and has great application value and promotion prospects.

[0027] The following is a detailed description of a cultivation and harvesting method for increasing the chicoric acid content in Echinacea leaves provided by the present invention through experimental examples and application examples.

[0028] Experimental Example 1 Comparison of chicoric acid content in leaves of different varieties of Echinacea

[0029] Under the same cultivation conditions, different Echinacea varieties were selected (see Table 1) to compare the chicoric acid content in their leaves. The results are shown in Table 1. It can be seen that the selected Echinacea variety 'Rainbow Wreath' is one of the varieties with a higher chicoric acid content in its leaves.

[0030] Table 1 Comparison of chicoric acid content (μg / g FW) in leaves of eight varieties of Echinacea

[0031]

[0032] Experimental Example 2 Comparison of conventional dosage of organic fertilizer and compound fertilizer as base fertilizer

[0033] The conventional dosage of organic fertilizer and compound fertilizer (800 kg / mu of organic fertilizer and 40 catties / mu of compound fertilizer) was selected as the base fertilizer for comparison, and 4 kinds of planting spacing were compared. The specific experimental methods are as follows:

[0034] Three-year-old double-petaled Echinacea 'Rainbow Wreath' seedlings with consistent growth were used, planted in the ground, raised ridges, and planted in April. The planting density of Echinacea was set at 4 different levels (A1: 25cm×25cm, A2: 30cm×30cm, A3: 35cm×35cm, A4: 40cm×40cm), and the base fertilizer used was conventional compound fertilizer B1 (Longzehui compound fertilizer 15-15-15 produced by Hubei Aotel Chemical Co., Ltd., total nutrients ≥45%, nitrate nitrogen ≥6%) and organic fertilizer B2 (refined organic fertilizer produced by Hangzhou Shengfeng Compound Fertilizer Factory, registration certificate: Zhejiang Agricultural Fertilizer (2018) No. 0058, organic matter ≥45%), with a total of 8 treatments. The chicoric acid content of leaves was measured in the late flowering period.

[0035] The results are shown in Table 2, which show that the chicoric acid content in leaves is higher when organic fertilizer is used as base fertilizer than compound fertilizer, and the chicoric acid content in leaves treated with a planting spacing of 30cm×30cm is the highest.

[0036] Table 2 Comparison of chicoric acid content (μg / g FW) in leaves of Echinacea with different basal fertilizers and planting spacing

[0037]

[0038] Experimental Example 3 Comparison of chicoric acid content in leaves in different months

[0039] The specific experimental method for comparing the chicoric acid content in leaves of Echinacea in different months is as follows:

[0040] For three-year-old double-petaled Echinacea 'Rainbow Wreath' ground-planted seedlings with consistent growth, the planting spacing was 30cm×30cm from May to October. From May to October, the chicoric acid content in the leaves was compared and determined every month (May 24, late flowering period, June 24, growing period, July 24, flowering period, August 24, late flowering period, September 24, and October 24, late flowering period).

[0041] The results are shown in Table 3. It can be seen that when the harvest time is the end of May and the end of August (just in the late flowering period), the chicoric acid content in the leaves is significantly higher than in other periods.

[0042] Table 3 Comparison of chicoric acid content (μg / g FW) in leaves of Echinacea detected in different months

[0043]

[0044] Experimental Example 4 Comparison of chicoric acid content in leaves harvested at different time periods

[0045] The chicoric acid content of leaves was compared by sampling and testing at different time periods of the same day. The specific experimental method is as follows:

[0046] In the same plot and under the same cultivation conditions, the planting spacing was 30cm×30cm. For the three-year-old double-petaled Echinacea 'Rainbow Wreath' field-planted seedlings with consistent growth, the above-ground parts were harvested at different time points (8:00 a.m., 10:00 a.m., 12:00 noon, 2:00 p.m., and 4:00 p.m.) on the same day in the late flowering period, and samples were taken to determine the chicoric acid content in the leaves.

[0047] The results are shown in Table 4. It can be seen that the chicoric acid content in the leaves is highest when the harvest time is 12 o'clock in the morning, followed by 10 o'clock. Considering the length of the harvest time, the best harvest time of the day is between 10 o'clock and 12 o'clock.

[0048] Table 4 Comparison of chicoric acid content (μg / g FW) in Echinacea leaves sampled at different time periods on the same day

[0049]

[0050] Experimental Example 5 Comparison of chicoric acid content in leaves of Echinacea sprayed with different hormones

[0051] The chicoric acid content of leaves of Echinacea sprayed with 4 hormones was compared with the control (clear water). The specific experimental method is as follows:

[0052] The test materials are the two-year-old double-petaled Echinacea 'Rainbow Wreath' seedlings with consistent growth. They are planted in the ground, raised in ridges, with a ridge width of 1.3m and a planting density of 0.3m×0.3m. The two-year-old Echinacea seedlings are planted for two weeks and then sprayed. They are sprayed once every other week for a total of 2 treatments. Each treatment has 50 plants, repeated 3 times, and a protective row is set between treatments.

[0053] A completely randomized block design was used. The four hormones were brassinolide, paclobutrazol, uniclobutrazol, and gibberellin. Three concentrations were set for each hormone, and each concentration was repeated three times. A clear water control (CK) was set. The hormone concentration configuration is shown in Table 5.

[0054] A unified survey and statistics were conducted in the late stage of flowering. Ten plants (with consistent growth) were selected from each treatment to measure indicators such as plant height, crown width, number of branches, flower height, leaf size, stem diameter, number of flowers, and flower size. Samples were also taken to measure the chlorophyll content, carotenoid content, and chicoric acid content in the leaves.

[0055] Extraction and determination of photosynthetic pigments: The ethanol method was used to determine the content of chlorophyll and carotenoids. Cut the fresh leaves after the veins are removed, cut into 2mm pieces, weigh 0.2g of the cut fresh sample, put it in a mortar, add 5mL of 95% ethanol, and grind it into a homogenate. Continue to whiten the tissue. Pour the extract into a funnel, filter it into a test tube with filter paper, rinse the mortar, pestle and residue several times with a small amount of ethanol, and finally pour it into the funnel together with the residue. Use a dropper to absorb ethanol and wash all the chloroplast pigments on the filter paper into the funnel until there is no green in the filter paper and residue. Finally, use 95% ethanol to make the volume 10mL and shake well. Add the chloroplast pigment extract to a 96-well plate, use 95% ethanol as a blank, and measure the absorbance at wavelengths of 665nm, 649nm, and 470nm. According to the measured absorbance values, use the following calculation formula to calculate the concentration of chlorophyll and carotenoids. The calculation formulas for chlorophyll a concentration (Ca) and chlorophyll b concentration (Cb) are:

[0056] Ca=13.95A 665 -6.88A 649

[0057] Cb=24.96A 649 -7.32A 665

[0058] The calculation formula of carotenoid Cx is: Cx = (1000A 470-2.05Ca-114.8Cb) / 245

[0059] The leaf chlorophyll and carotenoid content was calculated based on the concentration. The calculation formula is:

[0060] Chlorophyll or carotenoid content (mg / g) = (concentration*extract volume*dilution multiple) / sample fresh weight.

[0061] Determination of chicoric acid content in leaves: Extraction method: accurately weigh 0.1g of sample leaves, place in a 2mL centrifuge tube, add steel balls, add 1mL of pre-prepared 80% methanol (chromatographic grade) solution, seal, and grind into slurry. Ultrasonic extraction for 1h, overnight at 4℃, shake well, filter, and filter the filtrate with a 0.22μm microporous filter membrane to obtain the chicoric acid content. Use high performance liquid chromatography to determine the chicoric acid content.

[0062] Chromatographic conditions: mobile phase: acetonitrile-0.1% phosphoric acid water (18:82); column temperature: 40°C; flow rate: 1 mL / min; detection wavelength: 330 nm; injection volume: 10 μL.

[0063] Preparation of standard curve: Accurately weigh 0.0012g of chicoric acid reference substance and dilute with 1mL of methanol. Accurately pipette 10μL and add it to the liquid chromatograph, and measure the peak area according to the above chromatographic conditions (the points of the standard curve are: 3μg / mL, 10μg / mL, 30μg / mL, 60μg / mL, 200μg / mL, 300μg / mL, 500μg / mL, 700μg / mL, 800μg / mL). With the mass of chicoric acid as the horizontal coordinate (x) and the peak area as the vertical coordinate (y), draw the standard curve and calculate the regression equation.

[0064] Data processing and statistical analysis: One-way analysis of variance (P < 0.05 or P < 0.01) was used to test the differences in growth and flowering indicators, photosynthetic pigment content and chicoric acid content after different hormone treatments, and Duncan multiple comparisons were performed. SPSS software was used for data analysis.

[0065] The results are shown in Table 5. Spraying Echinacea with 1000 mg / L paclobutrazol increased the chicoric acid content in leaves by 85.21% compared with no spraying. Compared with other treatments, Echinacea plants grew shorter and stronger, and the number of flowers increased, which improved the overall ornamental value of the plants. At the same time, it promoted the synthesis of photosynthetic pigments in leaves, with obvious effects.

[0066] Table 5 Comparison of the effects of 4 hormone sprays on the growth of Echinacea and the content of chicoric acid in leaves

[0067]

[0068]

[0069] Note: The conventional control in the table is spraying with clean water.

[0070] Application Examples

[0071] A cultivation and harvesting method for increasing the chicoric acid content in Echinacea leaves, the method comprising:

[0072] (1) Variety selection: Choose the Echinacea variety 'Rainbow Wreath' which has a high content of chicoric acid;

[0073] (2) Seedling cultivation: Seedlings were sown in a greenhouse on November 9, with a substrate of 0-10 mm Peat moss. Seeds were sown on flat trays and covered with a thin layer of peat moss. Seedlings emerged after 6 days. When the seedlings had 1-2 true leaves, they were transplanted to 128-hole trays on December 8.

[0074] (3) Repotting: On February 10, repot in a 10 cm small pot;

[0075] (4) Ridge formation in the field: The experimental site was sandy loam with medium soil fertility and medium permeability. On March 18, 800 kg / mu of organic fertilizer (refined organic fertilizer produced by Hangzhou Shengfeng Compound Fertilizer Factory, registration certificate: Zhejiang Agricultural Fertilizer (2018) No. 0058, organic matter ≥ 45%) was applied and then ploughed. Ridge formation was carried out in the field with a width of 1.3 m, a furrow width of 20 cm, and a ridge height of 15 cm.

[0076] (5) Field planting: Planting in the field on March 24, with a planting spacing of 30 cm × 30 cm;

[0077] (6) Two hormone regulation: spray 1000 mg / L paclobutrazol on the leaves on April 27, and spray the same concentration on the leaves again on May 4;

[0078] (7) Application of inorganic fertilizer: Apply compound fertilizer once on April 7 and May 10, 22 kg per mu;

[0079] (8) Intertillage and weeding: Intertillage and weeding were carried out once on April 5 and May 8 respectively;

[0080] (9) First harvest: Harvest the above-ground parts between 10 am and 12 pm at the end of May, leaving 20 cm at the base;

[0081] (10) Weeding and fertilization: After harvesting, remove weeds around the base and sprinkle compound fertilizer (Longzehui compound fertilizer 15-15-15 produced by Hubei Aotel Chemical Co., Ltd., total nutrients ≥ 45%, nitrate nitrogen ≥ 6%) around the base at 22 kg / mu; apply compound fertilizer again on August 11, the initial flowering period, with the same amount as step (7);

[0082] (11) Second harvest: Harvest the above-ground parts at 10:00 am to 12:00 pm at the end of August during the late flowering period, leaving 20 cm at the base.

[0083] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for cultivating and harvesting Echinacea leaves to increase the chicoric acid content, characterized in that: The cultivation method comprises: After applying organic fertilizer as base fertilizer, the field is tilled and ridged, and the planting spacing is 29-31cm×29-31cm for planting; After the plants are planted and survive, in the early stage of plant growth, two foliar sprays of hormones are performed, with an interval of at least one week between the two sprays; the hormone selected is paclobutrazol at a concentration of 1000 mg / L or 500 mg / L; At the end of each flowering period, harvest the above-ground parts separately, from 10 to 12 a.m., leaving a distance at the base; The chicoric acid content in the harvested leaves is increased.

2. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 1, characterized in that: The cultivation method also comprises: Before and after spraying the hormone, apply compound fertilizer once, and perform tillage and weeding.

3. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 2, characterized in that: The amount of compound fertilizer used is 22 kilograms per mu.

4. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 1, characterized in that: The cultivation method also comprises: Between each harvest, weed and fertilize.

5. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 4, characterized in that: After the first harvest, remove the weeds around the base, sprinkle compound fertilizer around the base, apply compound fertilizer again during the early flowering period, and then harvest for the second time in the late second flowering period.

6. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 1, characterized in that: The cultivation method also comprises: Seedling cultivation: Use greenhouse sowing, the substrate is peat, the seeds are sown on a flat tray and covered with a thin layer of peat. When the seedlings have 1 to 2 true leaves, transplant them into the hole tray.

7. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 6, characterized in that: The peat is selected from Pinnacle peat with a specification of 0 to 10 mm.

8. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 6, characterized in that: The cultivation method also comprises: Transplant the seedlings from the hole tray into the pot.

9. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 1, characterized in that: The field is ridged, with a ridge width of 1.3m, a furrow width of 20cm, and a ridge height of 15cm; Or / and, the field is ridged, and the soil of the planting site is sandy loam with medium soil fertility and medium permeability; Or / and, the amount of organic fertilizer used is 800 kg / mu.

10. The method for cultivating and harvesting Echinacea leaves for increasing the content of chicoric acid according to claim 1, characterized in that: The base is left with 20 cm.

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