Compound with plant growth regulation effect and preparation method and application thereof

Abstract

The application provides a compound with plant growth regulation effect and a preparation method and application thereof. White peony alkaloids G and H are isolated and identified from traditional Chinese medicine white peony for the first time; the structure is novel; a butenolide structure which is same as that in a plant signal molecule strigolactone, trimethyl butenolide and the like is contained in the parent nucleus structure; however, the natural content of the two white peony alkaloids is extremely low, so the white peony alkaloids G and H are synthesized by using a chemical method, and a derivative is obtained. Research finds that the white peony alkaloids and the derivative of the application exhibit a significant promoting effect on the germination of dicotyledonous crop seeds and monocotyledonous crop seeds at a low concentration, and exhibit an inhibiting effect at a high concentration. It is indicated that the white peony alkaloids and the derivative above can be a new plant hormone-like molecule, and can play a regulation effect on the growth of plants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to compounds that regulate plant growth, their preparation methods, and applications. Background Technology

[0002] Angelica dahurica is a commonly used traditional Chinese medicine in clinical practice, possessing the effects of relieving exterior syndromes and dispelling cold, dispelling wind and relieving pain, clearing nasal passages, drying dampness and stopping leukorrhea, and reducing swelling and draining pus. Its pharmacological effects are extensive, including anti-inflammatory, analgesic, antispasmodic, antibacterial, antioxidant, and antitumor properties. Its main chemical components include volatile oils, coumarins, polysaccharides, amino acids, alkaloids, and trace elements.

[0003] The efficacy of Angelica dahurica alkaloids has been reported before. For example, the literature (Wang Jianxin, Ma Cuihua, Inhibitory effect of Angelica dahurica alkaloids on HeLa cells, Chinese Journal of Gerontology, 2014, Vol. 34, No. 7) and the literature (Li Kun, Li Qingwang, Liu Jing, Study on antitumor activity of Angelica dahurica alkaloids on U14 cervical cancer in mice, Journal of Yanshan University, 2012, Vol. 36, No. 1) have both disclosed that Angelica dahurica alkaloids have an inhibitory effect on cervical cancer.

[0004] There are currently no reports on the alkaloids of Angelica dahurica having plant hormone activity. Summary of the Invention

[0005] The purpose of this invention is to provide three compounds with plant growth regulation functions, their preparation methods, and applications.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] This invention first provides a compound with plant growth regulating function, characterized in that its structural formula is shown in formula (I), (II) or (III):

[0008]

[0009] Of the three compounds mentioned above, compounds with structural formulas (I) and (II) are the first to be isolated and identified from the traditional Chinese medicine Angelica dahurica, and are named Angelica dahurica alkaloids G and H, respectively. Their structures are novel, with the parent nucleus containing the same butenolactone structure as plant signal molecules such as strigolactone and trimethylbutenolactone. However, the natural content of these two Angelica dahurica alkaloids is extremely low. Therefore, this invention synthesized the two Angelica dahurica alkaloids using chemical methods and obtained an Angelica dahurica alkaloid derivative, the structural formula of which is shown in formula (III).

[0010] Studies have found that the aforementioned angelica alkaloids and their derivatives significantly promoted the germination of seeds in both dicotyledonous and monocotyledonous crops at low concentrations, while exhibiting an inhibitory effect at high concentrations. This suggests that the aforementioned angelica alkaloids and their derivatives may represent a new class of plant hormone-like molecules capable of regulating plant growth.

[0011] Based on this, the present invention also provides the application of the above-mentioned compounds with plant growth regulation function in the preparation of plant growth regulation reagents; the plant growth regulation reagents include seed germination promoting reagents or seed germination inhibiting reagents.

[0012] Meanwhile, the present invention also provides a seed germination promoting agent, wherein the effective component of the seed germination promoting agent includes at least one of the above three compounds with plant growth regulating effects, and the content is not more than 100 μM; and a seed germination inhibiting agent, wherein the effective component of the seed germination inhibiting agent includes at least one of the above three compounds with plant growth regulating effects, and the content is not less than 1000 μM.

[0013] As mentioned above, Angelica alkaloids G and H were isolated and identified from Angelica dahurica. The isolation method includes the following steps:

[0014] (1) Heat Angelica dahurica under reflux to extract the extract, and concentrate the extract to obtain a paste;

[0015] (2) After suspending the extract in water, extract it sequentially with petroleum ether, ethyl acetate and n-butanol. Concentrate the n-butanol extract under reduced pressure to obtain the n-butanol extract.

[0016] (3) Gradient elution of n-butanol extract was performed using macroporous resin and ethanol to obtain a fraction eluted with 20% ethanol.

[0017] (4) Gradient elution of the 20% ethanol eluent was performed using a silica gel column and an ethyl acetate-methanol mixed eluent. The ethyl acetate-methanol eluent was obtained under the elution of the ethyl acetate-methanol mixed eluent with a volume ratio of 15:1.

[0018] (5) The ethyl acetate-methanol elution fraction was purified by semi-preparative HPLC using an acetonitrile-water mixed eluent to obtain the above-mentioned Angelica dahurica alkaloids G and H.

[0019] Preferably, in step (5), isocratic elution is performed sequentially using an acetonitrile-water mixed eluent with a volume ratio of 20:80 and an acetonitrile-water mixed eluent with a volume ratio of 30:70.

[0020] In addition, the present invention also provides a chemical synthesis method for the above-mentioned Angelica dahurica alkaloids or their derivatives, the chemical synthesis method comprising the following steps:

[0021] (a) 4-hydroxyisoleucine was dissolved in sulfuric acid solution to obtain a 4-hydroxyisoleucine solution; sodium nitrite was slowly added dropwise to the 4-hydroxyisoleucine solution at 0°C, and the reaction was carried out in an ice bath; after the reaction was completed, compound 2 was obtained by extraction, drying, and vacuum concentration.

[0022] (b) Compound 2 was dissolved in dichloromethane, and equal masses of molecular sieve and silica gel were added. Pyridinium chlorochromate was dissolved in anhydrous dichloromethane at 0°C and then added dropwise to the reaction system. After the reaction was completed, compound 3 was obtained by filtration, drying, concentration and purification.

[0023] (c) 4-hydroxyisoleucine was dissolved in dilute hydrochloric acid solution and reacted at room temperature; after the reaction was completed, the solution was concentrated under vacuum to obtain compound 4;

[0024] (d) Compound 4 was dissolved in anhydrous ethanol, and compound 3 and activated 4A molecular sieve were added. The reaction was carried out at 80°C. After the reaction was completed, the solvent was recovered, extracted, dried, vacuum concentrated and purified to obtain compound 5 and compound 7.

[0025] Among them, compound 5 is Angelica alkaloid G, and compound 7 is Angelica alkaloid derivative.

[0026] (e) Compound 4 was racemized in the presence of sodium methoxide to obtain compound 4'. Compound 3 and activated 4A molecular sieve were added to compound 4' and reacted at 80°C. After the reaction was completed, the solvent was recovered, extracted, dried, vacuum concentrated and purified to obtain compound 6 and compound 7.

[0027] Among them, compound 6 is Angelica alkaloid H, and compound 7 is Angelica alkaloid derivative.

[0028] Compared with the prior art, the technical effects of the present invention are reflected in:

[0029] This invention marks the first isolation and identification of angelica alkaloids G and H from the traditional Chinese medicine Angelica dahurica. Their structures are novel, containing a butenolactone structure in the parent nucleus, identical to that found in plant signaling molecules such as strigolactone and trimethylbutenolactone. However, the natural abundance of these two angelica alkaloids is extremely low. Therefore, this invention synthesized angelica alkaloids G and H using chemical methods and obtained a derivative. Studies have shown that the angelica alkaloids and their derivative exhibit significant promoting effects on the germination of both dicotyledonous and monocotyledonous crop seeds at low concentrations, while showing inhibitory effects at high concentrations. This suggests that the aforementioned angelica alkaloids and their derivative may represent a new class of plant hormone-like molecules capable of regulating plant growth. Attached Figure Description

[0030] Figure 1The structure of Angelica dahurica alkaloid G of this invention is shown below;

[0031] Figure 2 The structure of Angelica dahurica alkaloid H in this invention is shown.

[0032] Figure 3 The present invention discloses the chemical synthesis route of Angelica dahurica alkaloids and their derivatives;

[0033] Figure 4 The structure of the Angelica dahurica alkaloid derivative (compound 7) of the present invention is shown below;

[0034] Figure 5 Line graph showing the effects of Angelica dahurica alkaloids G, H, compound 7, and nordihydroguaiaric acid of the present invention on Arabidopsis thaliana seed germination.

[0035] Wherein, Time represents drug treatment time, Germination rate represents germination rate, CK represents blank control, NDGA represents nordihydroguaiaric acid, G represents Angelica dahurica alkaloid G, H represents Angelica dahurica alkaloid H, and 7 represents compound 7, the same below.

[0036] Figure 6 Line graph showing the effects of Angelica dahurica alkaloids G, H, and compound 7 of the present invention on tobacco seed germination at 1 μM.

[0037] Figure 7 Line graph showing the effects of Angelica dahurica alkaloids G, H, and compound 7 of the present invention on tobacco seed germination at 10 μM.

[0038] Figure 8 This is a line graph showing the effect of Angelica dahurica alkaloid H of the present invention on soybean seed germination;

[0039] Figure 9 Line graph showing the effect of gibberellin on soybean seed germination;

[0040] GA3 represents gibberellin, and the same applies below;

[0041] Figure 10 This is a line graph showing the effect of Angelica dahurica alkaloid H of the present invention on peanut seed germination;

[0042] Figure 11 This is a line graph showing the effect of gibberellin on peanut seed germination.

[0043] Figure 12 Line graph showing the effect of Angelica dahurica alkaloid H and gibberellin on mung bean seed germination according to the present invention;

[0044] Figure 13 This is a line graph showing the effect of Angelica dahurica alkaloid H of the present invention on wheat seed germination;

[0045] Figure 14Line graph showing the effect of Angelica dahurica alkaloid G and compound 7 of the present invention on wheat seed germination;

[0046] Figure 15 This is a line graph showing the effect of gibberellin on wheat seed germination.

[0047] Figure 16 This is a line graph showing the effect of Angelica dahurica alkaloid G of the present invention on maize seed germination;

[0048] Figure 17 This is a line graph showing the effect of Angelica dahurica alkaloid H of the present invention on maize seed germination;

[0049] Figure 18 This is a line graph showing the effect of compound 7 of the present invention on maize seed germination;

[0050] Figure 19 This is a line graph showing the effect of gibberellin on maize seed germination.

[0051] Figure 20 This is a line graph showing the effect of Angelica dahurica alkaloid G of the present invention on rice seed germination;

[0052] Figure 21 This is a line graph showing the effect of Angelica dahurica alkaloid H on rice seed germination according to the present invention.

[0053] Figure 22 This is a line graph showing the effect of compound 7 of the present invention on rice seed germination;

[0054] Figure 23 This is a line graph showing the effect of gibberellin on rice seed germination. Detailed Implementation

[0055] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0056] The instruments and materials used in the embodiments of the present invention include:

[0057] Autopol IV fully automated polarimeter (Rudolf, USA); High-performance liquid chromatography-ion trap time-of-flight mass spectrometry system: UFLC SIL-20AC autosampler, CTO-20AC column oven, SPD M20A UV detector, LC-20ADXR pump, IT-TOF-MS equipped with ESI ion source (Shimadzu, Japan); Varian 500 nuclear magnetic resonance spectrometer (Varian, USA); Shimadzu LC-20AT semi-preparative high-performance liquid chromatograph (diode array detector, binary high-pressure gradient pump, Labsolution workstation, Shimadzu, Japan); Waters 2998 semi-preparative high-performance liquid chromatograph (Waters, USA); ODS semi-preparative column (SunFire™ C18, 150mm × 10mm, 5μm); Sephadex LH-20 packing material (Amerasham Biosciences, Sweden); ODS column packing material (Li-Chroprep). RP-C18 (40-63μm, Merck, Germany); D101 macroporous resin, silica gel for column chromatography (200-300 mesh), and GF254 silica gel pre-prepared plates for thin-layer chromatography were all produced by Qingdao Ocean Chemical Plant.

[0058] The Angelica dahurica used in the experiment was purchased in Bozhou City, Anhui Province in December 2013. It was identified by Professor Tu Pengfei of Peking University as the dried root of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook.f., a plant of the Apiaceae family.

[0059] Example 1: Isolation and Identification of Angelica Dahurica Alkaloids G and H

[0060] 1. Separation of Angelica dahurica alkaloids G and H

[0061] In this embodiment, two angelica alkaloids were isolated from angelica dahurica. The specific isolation and identification steps are as follows:

[0062] (1) Heat Angelica dahurica under reflux to extract the extract, and concentrate the extract to obtain a paste;

[0063] Specifically, 29.6 kg of dried Angelica dahurica was added to 80% ethanol (3×180L, 2h each time) and heated and refluxed three times for 2h each time. After concentration under reduced pressure, 1.5 kg of extract was obtained.

[0064] (2) After suspending the extract in water, extract it sequentially with petroleum ether, ethyl acetate and n-butanol. Concentrate the n-butanol extract under reduced pressure to obtain the n-butanol extract.

[0065] Specifically, after suspending the extract in water, it was extracted sequentially with petroleum ether (4 times × 4L), ethyl acetate (4 times × 4L) and n-butanol (4 times × 4L), and the solvent was recovered under reduced pressure to obtain 805g of n-butanol extract.

[0066] (3) Gradient elution of n-butanol extract was performed using macroporous resin and ethanol to obtain a fraction eluted with 20% ethanol.

[0067] Specifically, the n-butanol extract was eluted with 100%, 80%, and 20% ethanol using a macroporous resin to obtain the 100% ethanol eluent Fr.A, the 80% ethanol eluent Fr.B, and the 20% ethanol eluent Fr.C, respectively.

[0068] (4) Gradient elution of the 20% ethanol eluent was performed using a silica gel column and an ethyl acetate-methanol mixed eluent. The ethyl acetate-methanol eluent was obtained under the elution of the ethyl acetate-methanol mixed eluent with a volume ratio of 15:1.

[0069] Specifically, the 20% ethanol elution fraction Fr.C was eluted onto a silica gel column with an ethyl acetate-methanol gradient (15:1→1:1) to obtain 20 fractions Fr.C1 to C20. Fr.C1 (10.1 g) was used for subsequent experimental steps.

[0070] (5) The ethyl acetate-methanol elution fraction was purified by semi-preparative HPLC using an acetonitrile-water mixed eluent to obtain two Angelica dahurica alkaloids;

[0071] Specifically, Fr.C1 (10.1 g) was purified by semi-preparative HPLC separation (acetonitrile-water, 20:80) to obtain 12 fractions Fr.C1A to C1L. Fr.C1H (10 mg) was purified by semi-preparative HPLC separation (acetonitrile-water, 30:70) with isocratic elution to obtain Angelica dahurica alkaloid G (3.1 mg, t R =19min), Angelica dahurica alkaloid H (0.8mg, tR=20min).

[0072] 2. Identification of Angelica dahurica alkaloids G and H

[0073] Among them, Angelica dahurica alkaloid G is a colorless oily liquid; specific rotation [α] 2 D 5 +55 (c 0.1, MeOH); UVλ m M a e x OH (logε):220(0.85),266(3.00); IR(KBr)ν max:1746,1677,1329,1232,1174,1045cm -1 ; 1 H NMR (500MHz, CDCl3) δ4.73(q,J=6.0Hz,1H),4.31(d,J=12.0Hz,1H),4.07(dq,J=9.5,6.0Hz,1H),3.63(s ,1H),1.95-1.89(m,1H),1.87(s,3H),1.39(d,6.0Hz,3H),1.35(d,J=6.5Hz,3H),1.20(d,J=6.5Hz,3H). 13 C NMR (126MHz, CDCl3) δ 176.3, 170.3, 131.5, 128.5, 79.6, 78.5, 59.9, 46.4, 18.8, 18.5, 13.9, 10.5; HRESIMS in positive ion mode m / z 240.1231 [M+H]+ (theoretical value 240.1230).

[0074] The structural formula of Angelica dahurica alkaloid G is as follows: Figure 1 As shown.

[0075] Angelica dahurica alkaloid H is a colorless oily liquid; specific rotation [α] 2 D 5 –49 (c 0.1, MeOH); UVλ H Me m O a x (logε):238(0.99),265(1.35); IR(KBr)ν max :1747,1678,1330,1234,1176,1070,1023cm -1 ; 1 HNMR (500MHz, CDCl3) δ4.76(q,J=7.0Hz,1H),4.72(d,J=7.5Hz,1H),4.33(dq,J=6.5Hz,J=2.5Hz,1H),3.66( s,1H),2.60-2.54(m,1H),1.93(s,3H),1.43(d,J=6.5Hz,3H),1.38(d,J=7.0Hz,3H),1.05(d,J=7.0Hz,3H). 13C NMR (126MHz, CDCl3) δ 175.8, 170.2, 130.9, 128.4, 81.6, 78.6, 55.2, 41.0, 20.1, 18.9, 13.1, 10.5; HRESIMS in positive ion mode m / z 240.1234 [M+H]+ (theoretical value 240.1230).

[0076] The structural formula of Angelica dahurica alkaloid H is as follows: Figure 2 As shown.

[0077] Example 2: Chemical Synthesis and Identification of Angelica Dahurica Alkaloids and Their Derivatives

[0078] 1. Chemical Synthesis of Angelica dahurica Alkaloids and Their Derivatives

[0079] To obtain sufficient quantities of Angelica dahurica alkaloids for subsequent research, this embodiment provides a chemical synthesis method for Angelica dahurica alkaloids and their derivatives. The synthetic route of this method is as follows: Figure 3 As shown, it includes the following steps:

[0080] (a) 1.0 g (7.7 mmol) of 4-hydroxyisoleucine (compound 1) was dissolved in 1.5 g (15.4 mmol) of sulfuric acid solution (1.0 M) to obtain a 4-hydroxyisoleucine solution; 3.2 g (46.2 mmol) of sodium nitrite (2.0 mL) was slowly added dropwise to the 4-hydroxyisoleucine solution at 0 °C, and the reaction was carried out in an ice bath for 3 h; after the reaction was completed, ethyl acetate was added, and the mixture was extracted with saturated sodium carbonate solution (10 mL), dried with Na2SO4, and concentrated under vacuum to obtain compound 2;

[0081] (b) 20.13 g (1.0 mmol) of compound was dissolved in 10.0 mL of dichloromethane, and an equal mass of molecular sieve (water-free) and silica gel (dispersing the reaction system) was added. 431.1 mg (2.0 mmol) of pyridinium chlorochromate (PCC) was dissolved in anhydrous dichloromethane and added dropwise to the reaction system at 0 °C. After the reaction was completed, diatomaceous earth was added, followed by ethyl acetate filtration. The mixture was back-extracted three times with water, and the organic layer was dried with Na2SO4. The mixture was concentrated under vacuum and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain compound 3.

[0082] (c) 1.29 g (10.0 mmol) of 4-hydroxyisoleucine was dissolved in 55 mL (12.0 mmol) of 5% dilute hydrochloric acid solution and stirred at room temperature; after the reaction was completed, the solution was concentrated under vacuum to obtain compound 4.

[0083] (d) Compound 4 (1.0 mmol) was dissolved in 5 mL of anhydrous ethanol, and compound 3 and activated 4A molecular sieve were added. The mixture was stirred at 80 °C. After the reaction was completed, the ethanol was evaporated, ethyl acetate was added for extraction, the mixture was dried with Na2SO4, concentrated under vacuum, and separated by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain compound 5 and compound 7.

[0084] (e) Compound 4 was racemized in the presence of sodium methoxide to obtain compound 4'. Compound 3 and activated 4A molecular sieve were added to compound 4' (1.0 mmol), and the reaction was carried out at 80 °C. After the reaction was completed, the solvent was evaporated, ethyl acetate was added for extraction, the mixture was dried with Na2SO4, concentrated under vacuum, and separated by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain compound 6 and compound 7.

[0085] 2. Physicochemical constants and NMR data of intermediates, angelica alkaloids and their derivatives

[0086] Compounds 2, 3, 5, 6 and 7 were analyzed.

[0087] (1) Compound 2: colorless oily liquid; specific rotation [α] 2 D 5 –74(c 0.1,MeOH); IR(KBr)ν max :3422, 2977, 1773, 1385, 1210, 1134, 1058, 1003cm -1 ; 1 H NMR (400MHz, CDCl3) δ4.46 (d, J = 7.2Hz, 1H), 4.38 (qd, J = 6.44.4Hz, 1H), 2.97 (s, 1H), 2.32-2.24 (m, 1H), 1.40 (d, J = 6.4Hz, 3H), 1.11 (d, J = 7.2Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 177.3, 82.1, 70.0, 41.5, 19.7, 11.1. Positive ion mode HRESIMS m / z 131.0701 [M+H] + (Molecular formula C6H) 10 O3, theoretical value is 131.0703).

[0088] (2) Compound 3: colorless oily liquid; specific rotation [α] 2 D 5 –20(c 0.1,MeOH); IR(KBr)ν max:1685,1401,1145,1053,808cm -1 ; 1 H NMR (500MHz, CDCl3) δ5.60(s,1H),4.84(s,1H),1.91(s,3H),1.43(s,3H). 13 C NMR (126MHz, CDCl3) δ 170.2, 136.9, 132.2, 78.2, 18.6, 9.3. HRESIMS in positive ion mode m / z 129.0543 (molecular formula C6H8O3, theoretical value 129.0546).

[0089] (3) Compound 5: a colorless oily substance; 1 H NMR (400MHz, CDCl3) δ4.75(q,J=6.4Hz,1H),4.30(d,J=12.0Hz,1H),4.07(dq,J=9.6,6.0Hz,1H),3.52(s, 1H), 1.96-1.86 (m, 1H), 1.90 (s, 3H), 1.42 (d, J = 6.0Hz, 3H), 1.37 (d, J = 6.4Hz, 3H), 1.23 (d, J = 6.4Hz, 3H). 13 C NMR (101MHz, CDCl3) δ176.3,170.3,131.6,128.5,79.7,78.6,60.0,46.6,18.7,18.7,14.0,10.6.HRESIMS m / z 240.1231[M+H] + (calcd for C 12 H 17 The NMR data of compound 5 (NO4, 240.1230) are consistent with those of Angelica dahurica alkaloid G.

[0090] (4) Compound 6: a colorless oily substance. 1 H NMR (600MHz, CDCl3) δ4.75(q,J=6.6Hz,1H),4.70(d,J=7.2Hz,1H),4.30(dq,J=6.6,2.4Hz,1H),3.74(s, 1H),2.48-2.43(m,1H),1.90(s,3H),1.39(d,J=6.6Hz,3H),1.34(d,J=6.6Hz,3H),1.02(d,J=7.2Hz,3H). 13C NMR (151MHz, CDCl3) δ175.8,170.1,130.7,128.1,81.5,78.6,55.0,40.8,20.0,18.7,12.9,10.4.HRESIMS m / z 240.1234[M+H] + (calcd for C 12 H 17 The NMR data of compound 6 (NO4, 240.1230) are consistent with those of Angelica dahurica alkaloid H.

[0091] (5) Compound 7: Yellow oily liquid; specific rotation [α] 2 D 5 -15 (c 0.1, MeOH); UVλ H Me m O a x (logε):237(0.94),285(1.947); IR(KBr)ν max :1751,1683,1329,1236,1165,1064cm -1 ; 1 HNMR (400MHz, CDCl3) δ5.09 (s, 1H), 4.88 (q, J = 6.8Hz, 2H), 1.89 (s, 6H), 1.46 (dd, J = 6.7, 1.4Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 170.3, 141.0, 125.4, 78.7, 18.7, 11.1. Positive ion mode HRESIMS m / z 238.1066 [M+H] + (Theoretical value is 238.1070).

[0092] The structure of compound 7 is shown in [reference needed]. Figure 4 This is the Angelica alkaloid derivative of the present invention.

[0093] Example 3: Bioactivity analysis of Angelica dahurica alkaloids

[0094] 1. Effects of Angelica dahurica alkaloids on the seed germination of Arabidopsis thaliana and tobacco

[0095] Prepare a 10.0 mM / mL solution of the test drug and nordihydroguaiaric acid (NDGA) using DMSO and filter through a membrane. Weigh 2.15 g of MS salt and 15 g of sucrose, dissolve them in 950 mL of distilled water, adjust the pH to 5.8 with NaOH, and then add distilled water to a final volume of 1 L. Dispense the solution into 300 mL Erlenmeyer flasks, 180 mL per flask, and add 1.44 g of agar to each flask. Sterilize at 120 °C and transfer to a clean bench at 90 °C. After the culture medium has cooled slightly, add the corresponding volume of the test drug solution, mix thoroughly, pour the mixture onto a plate, cap it, and place it in a clean bench for later use. Place Arabidopsis thaliana and tobacco seeds in 1.5 mL EP tubes, add 1 mL of 75% ethanol for 30 seconds (timed), remove the ethanol, and wash with 1 mL of sterile water for 1 min. After absorbing the water, add 1 mL of 2% sodium hypochlorite solution for disinfection for 10 minutes. After absorbing the disinfectant, rinse with sterile water for 1 minute each time, repeating 6 times. The last rinse is placed in water for later use. Sow the disinfected seeds in petri dishes, 30 seeds per treatment, 3 replicates, for a total of 90 seeds. Seal the dishes with two layers of sealing film. Observe the surface of the petri dishes for water droplets; if present, lightly ignite them to prevent interference with observation. Place in a 22±1℃ climate chamber (16 hours light exposure) to observe the effects of Angelica dahurica alkaloids G, H, and compound 7 on the germination of Arabidopsis thaliana and tobacco seeds.

[0096] (1) Effects of Angelica dahurica alkaloids G, H and compound 7 on Arabidopsis thaliana seed germination

[0097] The results are shown in Table 1 and Figure 5 As shown.

[0098] Table 1. Effects of Angelica dahurica alkaloids G, H and compound 7 on Arabidopsis thaliana seed germination.

[0099]

[0100] From Table 1 and Figure 5 It was evident at 16h, 21h, and 24h that the germination rate of Arabidopsis thaliana was significantly higher than that of the blank control group (CK) under the treatment of 10μM Angelica dahurica alkaloids G and H and compound 7. The promoting effect was most pronounced at 21h, with the germination rates of the Angelica dahurica alkaloids G and H and compound 7 treatment groups increasing by 53.63%, 37.62%, and 49.89% respectively compared to the blank control group (CK). At 16h, the germination rates of Arabidopsis thaliana seeds treated with Angelica dahurica alkaloids G and compound 7 were 40.58±1.45% and 43.84±4.94%, respectively, higher than the germination rate of the positive control group NDGA (39.29±7.26%).

[0101] (2) Effects of Angelica alkaloids G, H and compound 7 on tobacco seed germination

[0102] The effects of Angelica dahurica alkaloids G, H, and compound 7 on the germination of tobacco seeds were observed, and the results are shown in Tables 2 and 3. Figure 6 , Figure 7 As shown.

[0103] Table 2. Effects of Angelica dahurica alkaloids G, H, and compound 7 on tobacco seed germination (concentration: 1 μM)

[0104]

[0105] Table 3. Effects of Angelica dahurica alkaloids G, H, and compound 7 on tobacco seed germination (concentration: 10 μM)

[0106]

[0107] From Table 2, Table 3 and Figure 6 , Figure 7 It was observed that at 40h, 44h, 48h, 56h, and 68h, the germination rate of tobacco under the influence of 1 μM Angelica dahurica alkaloid H increased by 27.78%, 33.33%, 53.33%, 52.22%, and 21.11% compared to the control group, respectively. Under the influence of 10 μM Angelica dahurica alkaloid H, the germination rate of tobacco increased by 32.22%, 45.55%, 55.55%, 56.67%, and 23.33% compared to the control group, significantly higher than the seed germination rate of the positive control group NDGA. Angelica dahurica alkaloid G and compound 7 (1 μM, 10 μM) could promote tobacco germination, with germination rates close to those of the positive control group NDGA.

[0108] 2. Effects of Angelica dahurica alkaloids on seed germination of peanuts, mung beans, soybeans, corn, rice, and wheat.

[0109] Select seeds of uniform size, disinfect them with 75% ethanol, remove the ethanol, and then rinse with 500 mL of sterile water. After removing the water, disinfect them with 1% sodium hypochlorite solution for 10 minutes, rinsing with sterile water for 1 minute each time, repeating 6 times. Air-dry the disinfected seeds for later use. Soak the seeds in different concentrations (soaking time as per literature). After soaking and drying, place the seeds evenly and neatly into 90 mm diameter petri dishes lined with double-layered filter paper. Each treatment contains 50 seeds, with 3 replicates, for a total of 150 seeds. Germinate at 25℃ under 16 hours of light. Add water regularly each day from the date of placement to keep the germination bed moist. Germination is defined as when the radicle length exceeds half the seed length, calculated from the sowing date. Record the number of germinating seeds daily and calculate the seed germination index.

[0110] (1) Effect of Angelica dahurica alkaloid H on soybean seed germination

[0111] The results are shown in Table 4 and Figure 8 , Figure 9 .

[0112] Table 4. Effects of Angelica dahurica alkaloid H on soybean seed germination.

[0113]

[0114] From Table 4 and Figure 8 , Figure 9 It can be seen that angelica alkaloid H (50, 100, and 200 μM) promoted the germination rate of soybean seeds three days before sowing. Angelica alkaloid H (100 μM) was more effective than the positive control GA3 (100 μM). After treatment with angelica alkaloid H (100 μM), the germination rates of soybeans in the first three days were 21.11±1.11%, 54.44±4.84%, and 71.11±2.94%, respectively, while the germination rates of soybeans in the positive control GA3 group (100 μM) were 7.78±1.11%, 52.22±4.84%, and 63.33±1.92%. Compared with the control group, angelica alkaloid H significantly promoted the growth of soybean radicles, with a better effect than the positive control GA3.

[0115] (2) Effect of Angelica dahurica alkaloid H on peanut seed germination

[0116] The results are shown in Table 5 and Figure 10 , Figure 11 .

[0117] Table 5. Effects of Angelica dahurica alkaloid H on peanut seed germination.

[0118]

[0119] From Table 5 and Figure 10 , Figure 11 It can be seen that the alkaloid H (50, 100, 200 μM) of Angelica dahurica on the 3rd and 4th day after sowing promoted the germination rate of peanut seeds, but the effect was not obvious.

[0120] (3) Effect of Angelica dahurica alkaloid H on mung bean seed germination

[0121] The results are shown in Table 6 and Figure 12 .

[0122] Table 6. Effects of Angelica dahurica alkaloid H on mung bean seed germination.

[0123]

[0124] From Table 6 and Figure 12 It can be seen that on the first day after sowing, Angelica alkaloid H (50 and 200 μM) had an inhibitory effect on the germination rate of mung bean seeds, while there was no significant effect at 100 μM.

[0125] (4) Effects of Angelica dahurica alkaloids G and H and compound 7 on wheat seed germination

[0126] The results are shown in Tables 7, 8, and 9. Figure 13 , Figure 14 , Figure 15 .

[0127] Table 7. Effects of Angelica dahurica alkaloids G and compound 7 on wheat seed germination.

[0128]

[0129] Table 8. Effects of Angelica dahurica alkaloid H on wheat seed germination.

[0130]

[0131] Table 9. Effects of GA3 on wheat seed germination

[0132]

[0133] From Tables 7, 8, and 9 Figure 13 , Figure 14 , Figure 15 It is evident that Angelica alkaloids G (10 μM) and H (10 μM, 50 μM) have a weak promoting effect on wheat seed germination, compound 7 has no significant effect, and Angelica alkaloids H (1000 μM, 500 μM, 200 μM, 100 μM) have a significant inhibitory effect on wheat seed germination.

[0134] (5) Effects of Angelica dahurica alkaloids G and H and compound 7 on maize seed germination

[0135] The results are shown in Tables 10, 11, 12, 13, and 14. Figure 16 , Figure 17 , Figure 18 , Figure 19 .

[0136] Table 10 Effects of Angelica dahurica alkaloid G on maize seed germination.

[0137]

[0138] Table 11 Effects of Angelica dahurica alkaloid H on maize seed germination

[0139]

[0140] Table 12 Effects of Compound 7 on Maize Seed Germination

[0141]

[0142] Table 13 Effects of GA3 on maize seed germination

[0143]

[0144] From Table 10, Table 11, Table 12, Table 13 and Figure 16 , Figure 17 , Figure 18 , Figure 19 It can be seen that on the first and second days after sowing, Angelica alkaloids G, H, and compound 7 (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) all significantly promoted the germination rate of maize seeds. The optimal concentrations for their effects were determined by Angelica alkaloids G (0.1 μM), Angelica alkaloids H (0.1 μM, 1 μM), and compound 7 (1 μM, 10 μM).

[0145] On day 1, the germination rate of maize seeds treated with Angelica alkaloid G (0.1 μM) reached 56.00±2.31%, while the germination rates of maize seeds treated with Angelica alkaloid H (0.1 μM, 1 μM) reached 70.67±1.33% and 62.67% respectively, and the germination rates of maize seeds treated with compound 7 (1 μM, 10 μM) reached 64.00±4.00% and 68.00±2.31% respectively. In contrast, the germination rate of the control group was 20.00±2.31%. Notably, the germination-promoting effect of Angelica alkaloid H (0.1 μM, 1 μM) on maize seeds was similar to that of GA3 (10 μM) (maize germination rate 69.33±4.81%).

[0146] On day 2, the germination rate of maize seeds treated with Angelica alkaloid G (0.1 μM) reached 85.33 ± 2.67%, those treated with Angelica alkaloid H (0.1 μM, 1 μM) reached 88.00 ± 2.31% and 93.33 ± 1.33% respectively, and those treated with compound 7 (1 μM, 10 μM) reached 80.00 ± 2.31% and 78.67 ± 2.67% respectively, while the germination rate of the control group was 53.33 ± 1.33%. Furthermore, Angelica alkaloid G, H, and compound 7 (1000 μM) significantly delayed maize seed germination.

[0147] (6) Effects of Angelica dahurica alkaloids G and H and compound 7 on rice seed germination

[0148] The results are shown in Tables 14, 15, 16, 17, and 18. Figure 20 , Figure 21 , Figure 22 , Figure 23 .

[0149] Table 14 Effects of Angelica dahurica alkaloid G on rice seed germination

[0150]

[0151] Table 15 Effects of Angelica dahurica alkaloid H on rice seed germination

[0152]

[0153] Table 16 Effects of compound 7 on rice seed germination

[0154]

[0155] Table 17 Effects of GA3 on rice seed germination

[0156]

[0157] From Tables 14, 15, 16, 17 and Figure 20 , Figure 21 , Figure 22 , Figure 23 It can be seen that two days before sowing, Angelica alkaloids G (1 μM), H (1 μM), and compound 7 (0.1 μM) can significantly promote the germination of rice seeds.

[0158] On day 1, the germination rate of rice seeds treated with Angelica dahurica alkaloid G (0.1 μM) reached 36.67 ± 1.76%, the germination rate of rice seeds treated with Angelica dahurica alkaloid H (1 μM) reached 46.67 ± 1.33%, and the germination rate of rice seeds treated with compound 7 (0.1 μM) reached 58.67 ± 2.91%, while the germination rate of the control group was 12 ± 2.31%. Angelica dahurica alkaloid H (1 μM) had a higher germination-promoting effect on rice seeds than GA3 (1 μM) (germination rate 33.33 ± 1.76); compound 7 (0.1 μM) had a higher germination-promoting effect on rice seeds than GA3 (0.1 μM) (germination rate 22.00 ± 2.00).

[0159] On day 2, the germination rate of rice seeds treated with Angelica dahurica alkaloid G (1 μM) reached 77.33 ± 0.67%, the germination rate of seeds treated with Angelica dahurica alkaloid H (1 μM) reached 86 ± 3.46%, and the germination rate of seeds treated with compound 7 (0.1 μM) reached 78.67 ± 1.33%, while the germination rate of the control group was 42 ± 4.16%. Furthermore, Angelica dahurica alkaloids G, H, and compound 7 (1000 μM) significantly delayed rice seed germination.

[0160] The above studies show that Angelica dahurica alkaloids G, H, and compound 7 significantly promote seed germination in Arabidopsis thaliana and tobacco at low concentrations; they also regulate the germination of dicotyledonous crops (soybeans, peanuts, soybeans) and monocotyledonous crops (rice, corn, wheat): at extremely low concentrations (0.01 μM, 0.1 μM, 1 μM, 10 μM), they significantly promote the germination of rice and corn seeds, while at high concentrations (1000 μM), they inhibit the germination of rice, corn, and wheat seeds, suggesting that Angelica dahurica alkaloids G and H may be a new class of plant hormone-like molecules. This research holds promise for discovering novel plant hormone-like molecules and applying them to the high-yield cultivation of medicinal plants or key crops, possessing significant academic value and application prospects.

Claims

1. A compound having a plant growth regulating effect, characterized in that, The structural formula is shown in formula (Ⅰ), (Ⅱ) or (Ⅲ): 。 2. The application of the compound with plant growth regulation function as described in claim 1 in the preparation of plant growth regulation reagents.

3. The application as described in claim 2, characterized in that, The plant growth regulating agents mentioned include seed germination promoting agents or seed germination inhibiting agents.

4. The application as described in claim 3, characterized in that, The content of the compound with plant growth regulation function in the seed germination promoting agent is less than 100 μM.

5. The application as described in claim 3, characterized in that, The seed germination inhibitor contained a compound with plant growth regulation function at a concentration of not less than 1000 μM.

6. A seed germination promoting agent, characterized in that, The active ingredient includes at least one of compounds having the structural formulas shown in formulas (I), (II), and (III): ； And the content does not exceed 100μM.

7. A seed germination inhibitor, characterized in that the active ingredient comprises at least one compound having the structural formulas shown in formulas (I), (II), and (III): ； And the content is not less than 1000μM.

8. The method for preparing the compound with plant growth regulating effect as described in claim 1, characterized in that, Includes the following steps: (1) Heat Angelica dahurica under reflux to extract the extract, and concentrate the extract to obtain a paste; (2) After suspending the extract in water, it was extracted with petroleum ether, ethyl acetate and n-butanol in sequence. The n-butanol extract was concentrated under reduced pressure to obtain the n-butanol extract. (3) The n-butanol extract was eluted with a gradient of macroporous resin and ethanol to obtain a fraction eluted with 20% ethanol; (4) Gradient elution of the 20% ethanol eluent was performed using a silica gel column and an ethyl acetate-methanol mixed eluent. The ethyl acetate-methanol eluent was obtained under the elution of the ethyl acetate-methanol mixed eluent with a volume ratio of 15:

1. (5) The ethyl acetate-methanol elution fraction was purified by semi-preparative HPLC using an acetonitrile-water mixed eluent to obtain compounds with structural formulas (Ⅰ) and (Ⅱ) that have plant growth regulatory effects.

9. The preparation method according to claim 8, characterized in that, In step (5), isocratic elution is performed sequentially using an acetonitrile-water mixed eluent with a volume ratio of 20:80 and an acetonitrile-water mixed eluent with a volume ratio of 30:

70.

10. The method for preparing the compound with plant growth regulating effect as described in claim 1, characterized in that, Includes the following steps: (a) 4-hydroxyisoleucine was dissolved in sulfuric acid solution to obtain a 4-hydroxyisoleucine solution; sodium nitrite was slowly added dropwise to the 4-hydroxyisoleucine solution at 0°C and the reaction was carried out in an ice bath; after the reaction was completed, compound 2 was obtained by extraction, drying and vacuum concentration. The structural formula of compound 2 is as follows: ; (b) Compound 2 was dissolved in dichloromethane, and an equal mass of molecular sieve and silica gel was added. Pyridinium chlorochromate was dissolved in anhydrous dichloromethane at 0°C and then added dropwise to the reaction system. After the reaction was completed, compound 3 was obtained by filtration, drying, concentration and purification. The structural formula of compound 3 is as follows: ; (c) Dissolve 4-hydroxyisoleucine in dilute hydrochloric acid solution and react at room temperature; after the reaction is completed, concentrate under vacuum to obtain compound 4; The structural formula of compound 4 is as follows: ; (d) Compound 4 was dissolved in anhydrous ethanol, and compound 3 and activated 4A molecular sieve were added. The reaction was carried out at 80°C. After the reaction was completed, the solvent was recovered, extracted, dried, vacuum concentrated and purified to obtain compounds with structural formulas (Ⅰ) and (Ⅲ) that have plant growth regulation effects. (e) Compound 4 was racemized in the presence of sodium methoxide to obtain compound 4'. Compound 3 and activated 4A molecular sieve were added to compound 4' and reacted at 80°C. After the reaction was completed, the compounds with plant growth regulation function were obtained by solvent recovery, extraction, drying, vacuum concentration and purification. The structural formula of compound 4' is as follows: .

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