A method for the production of caffeoylputrescine and chlorogenic acid by lantern fruit hairy root induction and culture

Abstract

The present application relates to a kind of methods for producing coffee acyl butanediamine and chlorogenic acid by inducing and cultivating lantern fruit hairy root, by pre-culturing lantern fruit leaf explants, activating rhizobium, co-culturing rhizobium with lantern fruit explants, degerming culture of lantern fruit, identification of lantern fruit hairy root and screening of high-quality strain to obtain lantern fruit hairy root;Using extraction method and organic solvent extraction method, macroporous resin adsorption method is separated to obtain coffee acyl butanediamine and chlorogenic acid.The T-DNA of rhizobium Ri plasmid is introduced into lantern fruit to obtain hairy root, and the efficient induction system of lantern fruit hairy root is first established, the induction system of three kinds of rhizobium C58C1, R1601 and A4 is successfully established, and the relatively high and stable hairy root clone of chlorogenic acid and coffee acyl butanediamine is obtained, so as to facilitate the production of chlorogenic acid and coffee acyl butanediamine.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology engineering and relates to a method for inducing and culturing the hairy roots of Physalis to produce caffeoylbutanediamine and chlorogenic acid. Background Technology

[0002] Physalis peruviana L., a species of annual herb belonging to the genus Physalis Linn. of the family Solanaceae (Solanum Li), is used medicinally. The entire plant is valuable; its roots are rich in alkaloids, its stems and leaves are rich in phenols and vitamins, and its fruit is rich in vitamin C, carotenoids, minerals, and antioxidants, making it highly nutritious. Modern research indicates that Physalis peruviana contains various bioactive components, including phenols, flavonoids, tannins, alkaloids, anthocyanins, and carotenoids, possessing potential medicinal value, such as anti-inflammatory, antitussive, antipyretic, diuretic, detoxifying, and antitumor effects. Furthermore, it exhibits strong antioxidant activity, capable of scavenging reactive oxygen free radicals, thereby reducing oxidative damage and preventing cancer, thus showing broad market potential. However, due to the low content of medicinal active ingredients in Physalis peruviana, actual production faces numerous challenges, including difficulty in ensuring quality and high production costs. Therefore, the production of effective components from Physalis peruviana requires the development of new bioengineering technologies.

[0003] Due to the extremely complex chemical structures of the physiologically active substances in *Physalis alkekengi*, such as phenolic compounds and flavonoids, the yield of these secondary metabolites is low and unstable, despite the expanding application of plant cell culture technology, hindering industrial-scale production. In contrast, hairy roots formed by *Agrobacterium rhizogenes* infection offer advantages such as rapid growth, stable genetic traits, no need for additional techniques, and high content of plant secondary metabolites, making them an important raw material for the production of medicinal plant secondary metabolites. Existing research on *Physalis alkekengi* mainly focuses on cultivation, quality control, extraction of active ingredients, and pharmacological activity; research on the induction of hairy roots in *Physalis alkekengi* is rarely reported. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides a method for inducing and producing caffeoylbutyric acid and chlorogenic acid from the hairy roots of *Phyllostachys edulis*. This invention establishes a hairy root induction system for *Phyllostachys edulis*, including the selection of *Agrobacterium* and analysis of transformation conditions. The feasibility of using its hairy roots to produce pharmacologically active components such as caffeoylbutyric acid and chlorogenic acid is explored, aiming to provide a reference for further establishing an efficient production system of effective components from *Phyllostachys edulis* through hairy root culture.

[0005] This invention is achieved using the following technical solution: a method for inducing hairy roots in *Phyllostachys edulis*, the process of which is as follows:

[0006] Pre-culture of Physalis leaf explants: Cut leaves and tender stems of sterile Physalis explants; make incisions on the tender stems, keeping both ends of the cut smooth, and place them in 1 / 2 MS solid medium for dark culture for 2-3 days;

[0007] Co-culture of Agrobacterium rhizogenes with Physalis alkekengi: The pre-cultured Physalis alkekengi leaf explants were immersed in Agrobacterium rhizogenes 1 / 2 MS suspension for 5-15 min, and then transferred to 1 / 2 MS medium for co-culture and cultured in the dark at 25±2℃ for 2-6 days.

[0008] Sterilization culture of *Physalis alkekengi*: After co-culturing, *Physalis alkekengi* leaves were rinsed several times with sterile water and then blotted dry with sterile filter paper. The leaves were then placed on sterile medium (1 / 2 MS + 400–600 mg / L Cef) for 6–10 days. Afterward, they were transferred to 1 / 2 MS + 200–400 mg / L cefotaxime sodium (Cef). During this period, white hairy roots will gradually appear at the wound sites on the leaves. After 1–2 weeks, the white hairy roots were transferred to 1 / 2 MS + 50–300 mg / L cefotaxime sodium (Cef). After another 1–2 weeks, they were transferred to 1 / 2 MS + 50–300 mg / L cefotaxime sodium (Cef). After 2–3 weeks, when sterilization was basically complete, the leaves were transferred to antibiotic-free 1 / 2 MS solid medium. The culture conditions were 25±2℃, in the dark.

[0009] Screening of high-quality Physalis hairy root lines: Physalis hairy roots that have been thoroughly sterilized and identified by PCR were selected. Physalis hairy roots with good growth status were transferred to blank 1 / 2 MS culture medium and cultured at 110 rpm and 28±2℃ to obtain Physalis hairy roots.

[0010] Further optimization was performed to obtain sterile explants of *Phyllostachys edulis* as follows: *Phyllostachys edulis* seeds were soaked in water for 30 min–2 h, then disinfected with 75% alcohol for 20–30 s, rinsed 2–3 times with sterile water, and then disinfected with 4% hypochlorous acid solution for 2–4 min, followed by rinsing 5–6 times with sterile water. The seeds were inoculated onto a solid culture medium with the following formula: 1 / 2 MS solid medium, 30 g / L sucrose, 0–0.2 mg / L NAA and 0–0.2 mg / L IBA, with 7–8 g / L agar powder added. The mixture was then cultured in the dark at 28 ± 2℃ for 7–14 days, and then transferred to a light chamber with a light intensity of 2000 lx and a light duration of 12 hours / day to obtain sterile *Phyllostachys edulis* explants.

[0011] Further preferably, the preparation process of the Agrobacterium rhizogenes 1 / 2MS suspension includes:

[0012] Activation of Agrobacterium rhizogenes: Agrobacterium rhizogenes strains stored at -80℃ were evenly spread on YEB or LB solid medium containing antibiotics such as rifampicin, and cultured at 28℃ for 2-6 days. Single colonies were picked with an inoculation needle and inoculated into YEB or LB liquid medium containing Rif antibiotics. The culture was then incubated in the dark at 28℃ with shaking at 100-400 rpm for 20-26 hours. 10-500 μL of the activated bacterial solution was added to 10-50 mL of YEB (LB) liquid medium containing antibiotics such as Rif. The culture was then incubated in the dark at 28℃ with shaking at 100-400 rpm until the OD value reached 0.2-1.0.

[0013] Resuspension of Agrobacterium rhizogenes: The activated Agrobacterium rhizogenes was centrifuged at 1000-5000 rpm for 5-20 min at room temperature, and the bacterial cells were collected and resuspended in 1 / 2 MS liquid medium. The suspension was then incubated in the dark at 25-30℃ with shaking at 50-200 rpm for half an hour until the bacterial concentration reached OD600 = 0.2-1.2, thus obtaining the Agrobacterium rhizogenes 1 / 2 MS suspension.

[0014] Further optimization was performed on the PCR identification of the hairy roots of *Phyllostachys edulis*: Genomic DNA was extracted from the hairy roots of *Phyllostachys edulis* using a kit method. The positive control was the corresponding *Agrobacterium rhizogenes* engineered bacteria, and the negative control was *Phyllostachys edulis* leaves. The genes to be detected were rolB and rolC. PCR amplification was performed, and the products were separated by agarose gel electrophoresis and detected by ultraviolet light.

[0015] A method for inducing and culturing the hairy roots of *Phyllostachys edulis* to produce caffeoylbutyric acid and chlorogenic acid.

[0016] The methods for inducing and culturing the hairy roots of *Phyllostachys edulis* are the same as above.

[0017] Extraction of caffeoyl butyric acid and chlorogenic acid from the hairy roots of Physalis alkekengi: The hairy roots of Physalis alkekengi were dried and ground into powder; extracted four times with 2-3 times the amount of ethanol in a 50°C water bath, the extracts were combined and filtered; the filtrate was concentrated under reduced pressure at 50-60°C until alcohol-free; the concentrate was diluted with water and the pH was adjusted to 8-9, and then extracted four times with the same volume of chloroform. The aqueous phase was adsorbed onto a macroporous adsorption resin, washed with water, and then eluted with ethanol. The ethanol eluent was collected and concentrated under reduced pressure to recover caffeoyl butyric acid and chlorogenic acid.

[0018] Advantages of this invention: 1. In this invention, T-DNA of Agrobacterium rhizogenes Ri plasmid is introduced into Physalis alkekengi cells using transgenic technology to obtain hairy roots. By screening superior clones, a hairy root clone with relatively high and stable yields of chlorogenic acid and caffeoylbutyric acid diamine is obtained, providing a new, high-quality, and sustainable source of medicinal and food functional factors for Physalis alkekengi production, and laying the foundation for the industrial production of its physiologically active substances such as phenols and flavonoids.

[0019] 2. All processes in this system are within a controllable range and are not limited by natural conditions. It has essentially eliminated the impact of pesticides and other heavy metal pollution on product quality control, and has good social and economic benefits.

[0020] 3. By artificially regulating and optimizing culture conditions, the growth of hairy roots and the accumulation of secondary metabolites can be significantly improved. This technology is adjustable and does not add any hormones during the hairy root culture process, thus greatly reducing costs. Attached Figure Description

[0021] Figure 1 Photo of sterile seedlings of Physalis alkekengi.

[0022] Figure 2 Photographs of hairy roots induced by Agrobacterium rhizogenes C58C1.

[0023] Figure 3 Photographs of hairy roots induced by Agrobacterium rhizogenes R1601.

[0024] Figure 4 Photograph of hairy roots induced by Agrobacterium rhizogenes A4.

[0025] Figure 5 This is a photograph of the rapid growth of hairy roots on a solid culture medium.

[0026] Figure 6 This is a PCR detection image of the rol gene in hairy roots.

[0027] Figure 7 Photograph of the hairy roots of Physalis alkekengi grown in liquid culture.

[0028] Figure 8 This is a liquid phase diagram of the hairy roots of the lantern fruit.

[0029] Figure 9 This is the secondary mass spectrum of caffeoylbutanediamine.

[0030] Figure 10 This is a secondary mass spectrum of chlorogenic acid. Detailed Implementation

[0031] The present invention will be further explained in detail below with reference to the embodiments.

[0032] Obtaining sterile explants from *Phyllostachys edulis*: *Phyllostachys edulis* seeds were soaked in water for 30 min–2 h, then disinfected with 75% alcohol for 20–30 s, rinsed 2–3 times with sterile water, and then disinfected with 4% hypochlorous acid solution for 2–4 min, rinsing 5–6 times with sterile water. The seeds were inoculated onto a solid culture medium with the following formula: 1 / 2 MS solid medium, 30 g / L sucrose, 0–0.2 mg / L NAA and 0–0.2 mg / L IBA, with 7–8 g / L agar powder added. The seeds were cultured in the dark at 28±2℃ for 7–14 days, then transferred to a light chamber with a light intensity of 2000 lx and a light duration of 12 h / day to obtain sterile *Phyllostachys edulis* explants. Figure 1 As shown.

[0033] Agrobacterium rhizogenes was used to genetically transform Physalis to obtain Physalis hairy roots.

[0034] 1. Activation of Agrobacterium rhizogenes: Different types of Agrobacterium rhizogenes strains (model: C58C1 / A4 / R1601) stored at -80℃ were evenly spread on YEB(LB) solid medium containing antibiotics such as rifampicin and cultured at 28℃ for 2-6 days. Single colonies were picked with an inoculation needle and inoculated into YEB(LB) liquid medium (pH=7.0-7.4) containing antibiotics such as Rif. The culture was then incubated in the dark at 28℃ with shaking at 100-400 rpm for 20-26 hours. 10-500 μL of the activated bacterial solution was added to 10-50 mL of YEB(LB) liquid medium containing antibiotics such as Rif. The culture was then incubated in the dark at 28℃ with shaking at 100-400 rpm until the OD value reached 0.2-1.0.

[0035] 2. Resuspension of Agrobacterium rhizogenes: The activated Agrobacterium rhizogenes (C58C1 / A4 / R1601) was centrifuged at 1000-5000 rpm for 5-20 min at room temperature. The cells were collected and resuspended in 1 / 2 MS liquid medium. The cells were then incubated in the dark at 25-30℃ with shaking at 50-200 rpm for half an hour until the bacterial concentration reached OD600 = 0.2-1.2, thus obtaining the Agrobacterium rhizogenes 1 / 2 MS suspension for later use.

[0036] 3. Pre-culture of Physalis leaf explants: Cut leaves and tender stems from sterile Physalis explants, and cut the leaves into 1cm pieces. 2 The tender stems are 2-4 cm long. Make 2-3 incisions on the tender stems, keeping both ends smooth, and place them on 1 / 2 MS solid medium. Incubate in the dark at 25±2℃ for 2-3 days to allow the wounded areas to secrete more phenolic substances and improve the conversion rate.

[0037] 4. Co-culture of Agrobacterium rhizogenes and Physalis alkekengi: The pre-cultured Physalis alkekengi leaf explants were immersed in Agrobacterium rhizogenes 1 / 2 MS suspension for 5-15 min (gently shaken to ensure full contact between the Physalis alkekengi leaf explants and the bacterial suspension). The Physalis alkekengi leaf explants were then removed, and the residual bacterial suspension on the surface of the Physalis alkekengi leaf explants was blotted dry with sterile filter paper. They were then transferred to 1 / 2 MS medium for co-culture and cultured in the dark at 25±2℃ for 2-6 days.

[0038] 5. Sterilization Culture of Physalis: After co-culturing for 1-2 days, the Physalis leaves were rinsed several times with sterile water and then blotted dry with sterile filter paper. They were then placed on sterile medium (1 / 2 MS + 400-600 mg / L Cef) for sterilization. After 6-10 days, they were transferred to 1 / 2 MS + 200-400 mg / L cefotaxime sodium (Cef). During this period, white hairy roots will gradually appear at the wound sites on the Physalis leaves. After 1-2 weeks, the white hairy roots were transferred to 1 / 2 MS + 50-300 mg / L cefotaxime sodium (Cef), and after another 1-2 weeks, transferred to 1 / 2 MS + 50-300 mg / L cefotaxime sodium (Cef). After 2-3 weeks, when sterilization was basically complete, the leaves were transferred to antibiotic-free 1 / 2 MS solid medium. The culture conditions were 25±2℃ in the dark. The hairy root induction rate was 45%-85%.

[0039] 6. Screening of high-quality Physalis hairy root lines: Physalis hairy roots that have been thoroughly sterilized and identified by PCR were selected. Physalis hairy roots with good growth status were transferred to blank 1 / 2 MS culture medium and cultured at 110 rpm and 28±2℃ to obtain Physalis hairy roots.

[0040] PCR detection of the rol gene in the hairy roots of Physalis alkekengi:

[0041] 1. Genomic DNA was extracted from the hairy roots of Physalis alkekengi using a kit from Beijing TransGen Biotech Co., Ltd. The steps are as follows:

[0042] 1) Take 100-200mg of young, tender, hairy roots of the lantern fruit, add liquid nitrogen and grind into powder;

[0043] 2) Place the powdered Physalis hairy roots into a 1.5 ml Eppendorf centrifuge tube, add 250 μL LRB1 (Resuspension Buffer 1) and 15 μL RNase A, mix thoroughly, and place in a 55°C water bath for 15 min.

[0044] 3) Centrifuge at 12000 rpm for 5 min, and gently aspirate the supernatant into a clean centrifuge chamber;

[0045] 4) Add 100 μL of solution PB1 (precipitation buffer 1), mix thoroughly, incubate on ice for 5 min, and centrifuge at 12000 rpm for 5 min;

[0046] 5) Gently aspirate the supernatant into a clean centrifuge tube, add 375 μL of solution BB1 (Binding Buffer 1), mix thoroughly to obtain the mixture;

[0047] 6) Add all the mixture to the centrifuge column, centrifuge at 12000 rpm for 30 seconds, and discard the effluent;

[0048] 7) Add 500 μL of solution CB1 (Clean Buffer 1), incubate at 12000 rpm for 30 seconds, and discard the eluent;

[0049] 8) Add 500 μL of solution WB1 (wash buffer 1), incubate at 12000 rpm for 30 seconds, and discard the eluent;

[0050] 9) Repeat step 8;

[0051] 10) Centrifuge at 1200 rpm for 2 min to completely remove residual WB1;

[0052] 11) Place the centrifuge column in a clean centrifuge tube, add 100 μL of preheated EB (Elution Buffer) to the center of the centrifuge column (60-70℃ or deionized water (pH>7.0), let stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and elute the genomic DNA of the hairy roots of the lantern fruit).

[0053] 12) To obtain more genomic DNA from the hairy roots of the lantern fruit, a second elution was performed, repeating step 11, and then stored at -20℃ for later use.

[0054] 2. PCR detection of rolB and ro1C genes in the hairy roots of *Phyllostachys edulis*

[0055] The primers for rolB gene amplification were frolb(5'-GCTCTTGCAGTGCTAGATTT-3') and rrolb(5'-GAAGGTGCAAGCTACCTCTC-3');

[0056] The primers for rolC gene amplification were frolc(5'-CTCCTGACATCAAACTCGTC-3') and rrolc(5'-TGCTTCGAGTTATGGGTACA-3');

[0057] The reaction system consisted of (50 μL) components: 2 μL ddH2O2, 1 μL Template DNA, 1 μL Forward Primer (10 μM), and 1 μL Reverse Primer (10 μM). PCR SuperMix 25μL.

[0058] The PCR reaction conditions were: 94℃ for 5 min, 35 cycles (94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 90 sec), 72℃ for 10 min.

[0059] The positive control was the corresponding engineered *Agrobacterium rhizogenes* strain, and the *Phyllostachys edulis* leaves served as the negative control. PCR products were separated by agarose gel electrophoresis and detected by UV to determine the presence of amplified bands for rolB and rolC. The amplified band size for rolB was 423 bp, and for rolC it was 626 bp.

[0060] UPLC-ESI-MS detection of hairy roots of Phyllostachys edulis

[0061] 1. Sample preparation

[0062] Harvest the hairy roots of the lantern fruit, dry the sample at 60℃ to constant weight, crush and grind it, and pass it through a 40-60 mesh sieve to obtain the dried sample. Weigh about 0.1g of the dried sample and add 1mL of 70% ethanol. Extract by ultrasonication at 60-80℃ for 1h; centrifuge at 10000r-14000r at 25℃ for 10min and collect the supernatant.

[0063] 2. UPLC-ESI-MS detection

[0064] 1) Mass Spectrometer: Hybrid Quadrupole-TOF LC / MS / MS Mass Spectrometer: TripleTOF5600+ (AB Sciex).

[0065] 2) UPLC: LC30 SYSTEM (Shimadzu); Column: Shim-pack GIST C18 (2.1mm×75mm; 2μm); Mobile phase: acetonitrile-0.3% formic acid water (gradient elution); Elution gradient conditions: 0–10 min 5–7% (C), 10–18 min 7–15% (C), 18–43 min 15–40% (C), 43–45 min 40% (C), 45–46 min 40–5% (C), 46–50 min 5% (C); Column temperature: 40℃; Injection volume: 5μL; Flow rate: 0.3mL / min;

[0066] 3. UPLC-ESI-MS analysis revealed the presence of the primary fragment ion peak at m / z 249 [MH]. - 353 [MH] - Based on structural composition analysis and fragment ion analysis using secondary mass spectrometry, the two compounds were preliminarily identified as caffeoylbutanediamine and chlorogenic acid. Further confirmation of these two compounds was obtained through comparison with standards.

[0067] Detection of caffeoylbutyric acid and chlorogenic acid content in the hairy roots of Physalis alkekengi:

[0068] 1. Sample preparation: Harvest the hairy roots of Physalis alkekengi, dry the sample at 60℃ to constant weight, crush and grind, and pass through a 40-60 mesh sieve to obtain the dried sample. Weigh about 0.1g of the dried sample and add 1mL of 70% ethanol. Extract by ultrasonication at 60-80℃ for 1h; centrifuge at 10000r-14000r at 25℃ for 10min, and take the supernatant.

[0069] 2. Preparation of the reference standard curve: Weigh 1 mg of caffeoyl butyl diamine and chlorogenic acid standards respectively, dissolve them in 1 mL of methanol by sonication to prepare 1 mg / mL standard solutions of caffeoyl butyl diamine and chlorogenic acid. Filter a certain amount of the standard solution through a 0.22 μm microporous membrane and analyze by HPLC. Chromatographic conditions: High performance liquid chromatograph (Agilent 1260), Waters C1 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (C) – 0.3% formic acid water (A); elution gradient conditions: 0–10 min 3% (C), 10–18 min 3–15% (C), 18–43 min 15–40% (C), 43–45 min 40–90% (C), 45–55 min 90% (C), 55–56 min 90–3% (C), 56–60 min 3% (C). Column temperature: 40℃; injection volume: 3μL; flow rate: 1mL / min; different volumes of the same concentration were injected to obtain chromatograms and chromatographic parameters. Linear regression was performed on peak area and content, and a standard curve was plotted with content as the abscissa and reference peak area as the ordinate.

[0070] The hairy roots of *Phyllostachys edulis* were cultured in cycles of 20–28 days, with a fresh weight (FW) of 180–220 g / L and a dry weight (DW) of 14–20 g / L. HPLC analysis revealed caffeoylbutanediamine content of 200–1200 μg / g and chlorogenic acid content of approximately 200–1200 μg / g. Although current research indicates that the production of secondary metabolites from *Phyllostachys edulis* hairy roots still faces challenges such as low yields, affecting their practical application, the hairy roots exhibit rapid growth, and yields can be further increased through elicitor stimulation and other methods.

[0071] Extraction of caffeoyl butyric acid and chlorogenic acid from the hairy roots of Physalis alkekengi: The hairy roots of Physalis alkekengi were dried and ground into powder; extracted four times with 2-3 times the amount of ethanol in a 50°C water bath, and the extracts were combined and filtered using filter paper; the filtrate was concentrated under reduced pressure at 50-60°C until alcohol-free; the concentrate was diluted with water and the pH was adjusted to 8-9, and then extracted four times with the same volume of chloroform; the aqueous phase was adsorbed onto a macroporous adsorption resin, washed with water, and then eluted with ethanol, and the ethanol eluent was collected; the ethanol eluent was concentrated under reduced pressure to recover caffeoyl butyric acid and chlorogenic acid.

[0072] Example 1

[0073] Pre-culture of Physalis leaf explants: Leaves and tender stems of sterile Physalis explants were collected, with the leaves cut into 1cm pieces. 2 The tender stems are 2-4 cm long. Make 2-3 incisions on the tender stems, keeping both ends smooth, and place them on 1 / 2 MS solid medium. Incubate in the dark at 25±2℃ for 2 days to allow the wounded areas to secrete more phenolic substances and improve the conversion rate.

[0074] Co-culture of Agrobacterium rhizogenes and Physalis alkekengi: The pre-cultured Physalis alkekengi leaf explants were immersed in a 1 / 2 MS suspension containing activated C58C1 Agrobacterium rhizogenes for 10 min (gently shaken to ensure full contact between the Physalis alkekengi leaf explants and the bacterial suspension). The Physalis alkekengi leaf explants were then removed, and the residual bacterial suspension on the surface of the explants was blotted dry with sterile filter paper. They were then transferred to a 1 / 2 MS medium for co-culture and incubated in the dark at 25±2℃ for 2 days.

[0075] Sterilization culture of *Physalis alkekengi*: After co-culturing for 2 days, *Physalis alkekengi* leaves were rinsed several times with sterile water and then blotted dry with sterile filter paper. They were then placed on sterile medium (1 / 2 MS + 500 mg / L Cef) for sterilization. After 6 days, they were transferred to 1 / 2 MS + 300 mg / L cefotaxime sodium (Cef). During this period, white hairy roots would gradually appear at the wound sites on the *Physalis alkekengi* leaves. After 2 weeks, the white hairy roots were transferred to 1 / 2 MS + 100 mg / L cefotaxime sodium (Cef), and after another 2 weeks, transferred to 1 / 2 MS + 100 mg / L cefotaxime sodium (Cef). After 3 weeks, when sterilization was basically complete, the roots were transferred to antibiotic-free 1 / 2 MS solid medium. The culture conditions were 25 ± 2℃, in the dark. The resulting *Physalis alkekengi* hairy roots are shown below. Figure 2 As shown, Figure 2 The experimental procedure is recorded in Chinese characters (DLG: abbreviation for plant, C58C1: type of inducing strain, cultured under sterile conditions on 100 mg / L Cef). The induction rate of hairy roots in *Phyllostachys edulis* was 66.67%.

[0076] Screening of high-quality Physalis hairy root lines: Selected hairy roots that have been thoroughly sterilized and identified by PCR as Physalis hairy roots. The hairy roots in good growth condition were transferred to blank 1 / 2 MS medium and cultured at 110 rpm and 28±2℃.

[0077] UPLC-ESI-MS analysis revealed the presence of a primary fragment ion peak at m / z 249 [MH]. - 353 [MH] - Based on structural composition analysis and fragment ion analysis using secondary mass spectrometry, the two compounds were preliminarily identified as caffeoylbutanediamine and chlorogenic acid. Comparison with standards further confirmed the identities of these two compounds. HPLC analysis revealed that the content of caffeoylbutanediamine was 373.30 μg / g, and the content of chlorogenic acid was approximately 607.75 μg / g.

[0078] Extraction of caffeoyl butyl diamine and chlorogenic acid from the hairy roots of *Physalis alkekengi*: The hairy roots of *Physalis alkekengi* were dried and ground into powder. They were extracted four times with three times the volume of ethanol in a 50°C water bath. The extracts were combined and filtered using filter paper. The filtrate was concentrated under reduced pressure at 50°C until alcohol-free. The concentrate was diluted with water to adjust the pH to 8, and then extracted four times with an equal volume of chloroform. The aqueous phase was adsorbed onto a macroporous adsorption resin, washed with water, and then eluted with ethanol. The ethanol eluate was collected and concentrated under reduced pressure to obtain the products, which were then analyzed by HPLC. The obtained products were caffeoyl butyl diamine and chlorogenic acid.

[0079] Example 2

[0080] Pre-culture of Physalis leaf explants: Leaves and tender stems of sterile Physalis explants were collected, with the leaves cut into 1cm pieces. 2 The tender stems are 2-4 cm long. Make 2-3 incisions on the tender stems, keeping both ends smooth, and place them on 1 / 2 MS solid medium. Incubate in the dark at 25±2℃ for 2 days to allow the wounded areas to secrete more phenolic substances and improve the conversion rate.

[0081] Co-culture of Agrobacterium rhizogenes and Physalis alkekengi: The pre-cultured Physalis alkekengi leaf explants were immersed in a 1 / 2 MS suspension containing activated R1601 for 8 min (gently shaken to ensure full contact between the Physalis alkekengi leaf explants and the bacterial suspension). The Physalis alkekengi leaf explants were then removed, and the residual bacterial suspension on the surface of the Physalis alkekengi leaf explants was blotted dry with sterile filter paper. The explants were then transferred to a 1 / 2 MS co-culture medium and cultured in the dark at 25±2℃ for 2 days.

[0082] Sterilization culture of *Physalis alkekengi*: After co-culturing for 2 days, *Physalis alkekengi* leaves were rinsed several times with sterile water and then blotted dry with sterile filter paper. The leaves were then placed on sterile medium 1 / 2 MS + 500 mg / L Cef for sterilization. After 10 days, they were transferred to 1 / 2 MS + 300 mg / L cefotaxime sodium (Cef). During this period, white hairy roots will gradually appear at the wound sites on the *Physalis alkekengi* leaves. After 1 week, the white hairy roots were transferred to 1 / 2 MS + 100 mg / L cefotaxime sodium (Cef), and after another week, transferred to 1 / 2 MS + 50 mg / L cefotaxime sodium (Cef). After 3 weeks, when sterilization was basically completed, the leaves were transferred to antibiotic-free 1 / 2 MS medium. The resulting *Physalis alkekengi* hairy roots are shown in the image. Figure 3 As shown, Figure 3 The experimental procedure is recorded in Chinese (DLG: abbreviation for plant, R1601: type of inducing strain, cultured under sterile conditions at 300 mg / L Cef). Culture conditions were 25 ± 2℃, in the dark. The hairy root induction rate was 53.33%.

[0083] Screening of high-quality Physalis hairy root lines: Physalis hairy roots that have been thoroughly sterilized and identified by PCR were selected. Hairy roots with good growth were transferred to blank 1 / 2 MS medium and cultured at 110 rpm and 28±2℃.

[0084] UPLC-ESI-MS analysis revealed the presence of a primary fragment ion peak at m / z 249 [MH]. - 353 [MH] - Based on structural composition analysis and fragment ion analysis using secondary mass spectrometry, the two compounds were preliminarily identified as caffeoylbutanediamine and chlorogenic acid. Comparison with standards further confirmed the identities of these two compounds. HPLC analysis revealed that the content of caffeoylbutanediamine was 297.48 μg / g, and the content of chlorogenic acid was approximately 362.48 μg / g.

[0085] Extraction of caffeoyl butyl diamine and chlorogenic acid from the hairy roots of *Physalis alkekengi*: The hairy roots of *Physalis alkekengi* were dried and ground into powder. They were extracted four times with three times the volume of ethanol in a 50°C water bath. The extracts were combined and filtered using filter paper. The filtrate was concentrated under reduced pressure at 55°C until alcohol-free. The concentrate was diluted with water to adjust the pH to 8, and then extracted five times with an equal volume of chloroform. The aqueous phase was adsorbed onto a macroporous adsorption resin, washed with water, and then eluted with ethanol. The ethanol eluate was collected and concentrated under reduced pressure to obtain the products, which were then analyzed by HPLC. The obtained products were caffeoyl butyl diamine and chlorogenic acid.

[0086] Example 3

[0087] Pre-culture of Physalis leaf explants: Leaves and tender stems of sterile Physalis explants were collected, with the leaves cut into 1cm pieces. 2The tender stems are 2-4 cm long. Make 2-3 incisions on the tender stems, keeping both ends smooth, and place them on 1 / 2 MS solid medium. Incubate in the dark at 25±2℃ for 2 days to allow the wounded areas to secrete more phenolic substances and improve the conversion rate.

[0088] Co-culture of Agrobacterium rhizogenes and Physalis alkekengi: The pre-cultured Physalis alkekengi leaf explants were immersed in a 1 / 2 MS suspension containing activated A4 for 5 min (gently shaken to ensure full contact between the Physalis alkekengi leaf explants and the bacterial suspension). The Physalis alkekengi leaf explants were then removed, and the residual bacterial suspension on the surface of the Physalis alkekengi leaf explants was blotted dry with sterile filter paper. The explants were then transferred to a 1 / 2 MS co-culture medium and cultured in the dark at 25±2℃ for 2 days.

[0089] Sterilization culture of *Physalis alkekengi*: After co-culturing for 2 days, *Physalis alkekengi* leaves were rinsed several times with sterile water and then blotted dry with sterile filter paper. The leaves were then placed on sterile medium 1 / 2 MS + 500 mg / L Cef for sterilization. After 7 days, they were transferred to 1 / 2 MS + 300 mg / L cefotaxime sodium (Cef). During this period, white hairy roots will gradually appear at the wound sites on the *Physalis alkekengi* leaves. After 2 weeks, the white hairy roots were transferred to 1 / 2 MS + 50 mg / L cefotaxime sodium (Cef), and after another 2 weeks, transferred to 1 / 2 MS + 50 mg / L cefotaxime sodium (Cef). After 3 weeks, when sterilization was basically complete, the leaves were transferred to 1 / 2 MS medium without antibiotics. The resulting *Physalis alkekengi* hairy roots are shown in the image. Figure 4 As shown, Figure 4 The experimental procedure is recorded in Chinese (DLG: abbreviation for plant, A4: type of inducing strain, cultured under 300 mg / L Cef conditions). Culture conditions were 25±2℃, in the dark. The hairy root induction rate was 76.92%.

[0090] Screening of high-quality hairy root lines: Hairy roots that have been thoroughly sterilized and identified by PCR were selected. Hairy roots with good growth were transferred to blank 1 / 2 MS medium and cultured at 110 rpm and 28±2℃.

[0091] UPLC-ESI-MS analysis revealed the presence of a primary fragment ion peak at m / z 249 [MH]. - 353 [MH] - Based on structural composition analysis and fragment ion analysis using secondary mass spectrometry, the two compounds were preliminarily identified as caffeoylbutanediamine and chlorogenic acid. Comparison with standards further confirmed the identities of these two compounds. HPLC analysis revealed that the content of caffeoylbutanediamine was 371.45 μg / g, and the content of chlorogenic acid was approximately 370.50 μg / g.

[0092] Extraction of caffeoyl butyl diamine and chlorogenic acid from the hairy roots of *Physalis alkekengi*: The hairy roots of *Physalis alkekengi* were dried and ground into powder. They were extracted four times with three times the volume of ethanol in a 50°C water bath. The extracts were combined and filtered using filter paper. The filtrate was concentrated under reduced pressure at 60°C until alcohol-free. The concentrate was diluted with water to adjust the pH to 9, and then extracted six times with an equal volume of chloroform. The aqueous phase was adsorbed onto a macroporous adsorption resin, washed with water, and then eluted with ethanol. The ethanol eluate was collected and concentrated under reduced pressure to obtain the products, which were then analyzed by HPLC. The obtained products were caffeoyl butyl diamine and chlorogenic acid.

[0093] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or variations made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be included within the scope of protection of the present invention.

Claims

1. A method for inducing and culturing caffeoylbutyric acid and chlorogenic acid from the hairy roots of *Phyllostachys edulis*, characterized in that, Pre-culture of Physalis leaf explants: Cut leaves and tender stems of sterile Physalis explants; make incisions on the tender stems, keeping both ends of the cut smooth, and place them in 1 / 2 MS solid medium for dark culture for 2-3 days; Co-culture of Agrobacterium rhizogenes and Physalis alkekengi: The pre-cultured Physalis alkekengi leaf explants were immersed in Agrobacterium rhizogenes 1 / 2 MS suspension for 5-15 min, and then transferred to 1 / 2 MS medium for co-culture and cultured in the dark at 25±2℃ for 2-6 days. Sterilization culture of Physalis alkekengi: After co-culturing, the Physalis alkekengi leaves were rinsed several times with sterile water and then dried with sterile filter paper. They were then placed on sterile medium 1 / 2 MS + 400-600 mg / L cefotaxime sodium for sterilization. After 6-10 days, they were transferred to 1 / 2 MS + 200-400 mg / L cefotaxime sodium. During this period, white hairy roots will gradually appear at the wound sites on the Physalis alkekengi leaves. After 1-2 weeks, the white hairy roots were transferred to 1 / 2 MS + 50-300 mg / L cefotaxime sodium. After another 1-2 weeks, they were transferred to 1 / 2 MS + 50-300 mg / L cefotaxime sodium. After 2-3 weeks, when sterilization was basically complete, the leaves were transferred to antibiotic-free 1 / 2 MS solid medium. The culture conditions were 25±2℃, in the dark. Screening of superior lineages of *Phyllostachys edulis* hairy roots: Select *Phyllostachys edulis* hairy roots that have been thoroughly sterilized and identified by PCR. Transfer the *Phyllostachys edulis* hairy roots in good growth condition to blank 1 / 2 MS medium and culture them at 110 rpm and 28±2℃ to obtain *Phyllostachys edulis* hairy roots. Extraction of caffeoyl butyric acid and chlorogenic acid from the hairy roots of Physalis alkekengi: The hairy roots of Physalis alkekengi were dried, ground and pulverized; extracted 4 times with 2-3 times the amount of ethanol in a 50℃ water bath, the extracts were combined and filtered; The filtrate was concentrated under reduced pressure at 50-60℃ until alcohol-free; the concentrate was diluted with water and its pH was adjusted to 8-9, and then extracted four times with the same volume of chloroform. The aqueous phase was adsorbed onto a macroporous adsorption resin, washed with water, and then eluted with ethanol. The ethanol eluent was collected and concentrated under reduced pressure to recover caffeoylbutane and chlorogenic acid.

2. The method for inducing and culturing caffeoylbutane and chlorogenic acid from the hairy roots of *Phyllostachys edulis* according to claim 1, characterized in that, The method for obtaining sterile explants of *Phyllostachys edulis* is as follows: Soak *Phyllostachys edulis* seeds in water for 30 min to 2 h, then disinfect with 75% alcohol for 20 min to 30 s, rinse 2 to 3 times with sterile water, then disinfect with 4% sodium hypochlorite solution for 2 to 4 min, and rinse 5 to 6 times with sterile water. Inoculate onto a solid culture medium with the following formula: 1 / 2 MS solid medium, 30 g / L sucrose, 0–0.2 mg / L NAA and 0–0.2 mg / L IBA, with 7–8 g / L agar powder added. Incubate in the dark at 28 ± 2℃ for 7–14 days, then transfer to a light chamber with a light intensity of 200 nm. 0 lx The light exposure time was 12 hours / day, and sterile explants of Phyllostachys edulis were obtained.

3. The method for inducing and culturing caffeoylbutyric acid and producing caffeoylbutyric acid from the hairy roots of *Phyllostachys edulis* according to claim 1, characterized in that, The preparation process of the Agrobacterium rhizogenes 1 / 2MS suspension includes: Activation of Agrobacterium rhizogenes: Agrobacterium rhizogenes strains stored at -80℃ were evenly spread on YEB or LB solid medium containing Rif antibiotics and cultured at 28℃ for 2-6 days. Single colonies were picked with an inoculation needle and inoculated into YEB or LB liquid medium containing Rif antibiotics. The culture was then incubated in the dark at 28℃ with shaking at 100-400 rpm for 20-26 hours. 10-500 μL of the activated bacterial solution was added to 10-50 mL of YEB or LB liquid medium containing Rif antibiotics and incubated in the dark at 28℃ with shaking at 100-400 rpm until the OD600 reached 0.2-1.

0. Resuspension of Agrobacterium rhizogenes: The activated Agrobacterium rhizogenes was centrifuged at 1000-5000 rpm for 5-20 min at room temperature, and the bacterial cells were collected. The cells were resuspended in 1 / 2 MS liquid medium and incubated in the dark at 25-30℃ with shaking at 50-200 rpm for half an hour until the bacterial concentration reached OD600=0.2, thus obtaining the Agrobacterium rhizogenes 1 / 2 MS suspension.

4. The method for inducing and culturing caffeoylbutyric acid and chlorogenic acid from the hairy roots of *Phyllostachys edulis* according to claim 1, characterized in that, PCR identification of hairy roots of *Phyllostachys edulis*: Genomic DNA was extracted from hairy roots of *Phyllostachys edulis* using a kit method. The positive control was the corresponding *Agrobacterium rhizogenes* engineered bacteria, and the negative control was *Phyllostachys edulis* leaves. The genes to be detected were rolB and rolC. PCR amplification was performed, and the products were separated by agarose gel electrophoresis and detected by ultraviolet light.

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