3.1 General

L-550 desktop low-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); AVANCE III 400 MHz, AVANCE III 500 MHz, AVANCE III 600 MHz and AVANCE III 800 MHz NMR spectrometers (Bruker GmbH)); V100 circular dichroic spectrometer (Applied Photophysics, UK); Autopol VI polarimeter (Rudolf, Germany); Shimadzu UV2401PC UV/VIS spectrophotometer (Shimadzu Corporation, Japan); Rotary evaporator (Heidolph AG of Germany); Agilent 1100 liquid chromatography (Agilent, USA), semi-prepared column ZORBAX SB-C18 (55 μm, 10 × 250 mm) (Agilent Corporation, USA); Thin layer chromatography silica gel, 200–300 mesh column chromatography silica gel (Qingdao Ocean Chemical Plant); Gel column chromatography Sephadex LH-20 (Pharmacia Corporation). The color developer is an ethanol solution of 10% H2SO4.

3.2 Plant material

The stem, collected in 2019 from Zhaotong City, Yunnan Province, was identified by Professor Liu Shoujin of Anhui University of Chinese Medicine as derived from the Gastrodia elata Bl.

3.3 Extraction and isolation

The dried flow branch (170 kg) of G. elata were re-extracted 3 times with 95% ethanol, each time for 2 h, the extraction solution was obtained, and then extracted 3 times with ethyl acetate and other volumes, and the ethyl acetate phase and aqueous phase were obtained after decompression and concentration. The ethyl acetate phase is detected by silica gel column chromatography (petroleum ether-acetone (elution of 100:1–0:1) gradient), TLC detection and merger of the same polarity part to obtain 8 components (Fr.1–Fr.8). Through TLC detection and color development results of color developers, select Fr.5. Fr.7 and Fr.8 LC–MS analysis was performed, and according to the analysis results, Fr.7 and Fr.8, were separated and purified. Drag Fr.7 (124 g) normal-phase silica gel column chromatographic section with petroleum ether/acetone as eluent and yielded 14 parts: Fr.7.1–Fr.7.14. Drag Fr. Part 7.10 was obtained by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), and then by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (methanol) to obtain Compound 7 (yellow powder, 10 mg). Drag Fr.7.11 Part is obtained by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), and then by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (methanol) to obtain compound 5 (yellow powder, 12 mg). Drag Fr.7.13 Part is obtained by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), and then subjected to repeated atmospheric pressure normal-phase column chromatography (200–300 mesh) and recrystallization to obtain compound 11 (yellow amorphous powder, 50 mg). Drag Fr.8 (743 g) Atmospheric pressure normal-phase silicone column chromatographic section with chloroform/acetone as eluent yielded 13 parts: Fr.8.1–Fr.8.13. Drag Fr.8.6 (26 g) Atmospheric pressure normal-phase silicone column chromatographic section with chloroform/acetone as eluent yielded 6 parts: Fr.8.6.1–Fr.8.6.6. Fr.8.6.6 (5.3 g) partially purified by Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1), repeated atmospheric pressure normal-phase column chromatography (200 to 300 mesh) and Sephadex LH-20 gel column chromatography, semi-prepared HPLC to obtain compounds 1 (yellow powder, 5 mg), 2 (yellow powder, 23 mg), 3 (yellow powder, 4 mg), 4 (yellow powder, 0.8 mg), 8 (yellow powder, 13 mg) and 12 (yellow powder, 22 mg). Drag Fr.8.8 Removed by MCI gives seven parts, Fr.8.8.1 to Fr.8.8.7. Fr.8.8.1 Compound 9 (black powder, 29 mg) was obtained by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (chloroform: methanol 1:1) and Sephadex LH-20 gel column chromatography (methanol), and further semi-prepared HPLC purification to give compound 6 (yellow powder, 3 mg). Drag Fr.8.8.2 Compound 10 (light yellow powder, 7 mg) and Compound 13 (black amorphous powder, 53 mg) was purified by repeated atmospheric pressure normal-phase column chromatography (200–300 mesh), Sephadex LH-20 gel column chromatography (200 to 300 mesh), Sephadex LH-20 gel column chromatography (methanol), and semi-prepared HPLC (Fig. 4).

Fig. 4

Flow chart of extraction and separation of the flow branch of Gastrodia elata

3.4 Spectroscopic data of the isolates3.4.1 Isogastrodinol (1)

Yellow powder; [α 26L] − 2.81 (c 0.077, MeOH); UV (MeOH) λmax (log ε): 195 (4.90), 221 (4.60), 279 (4.32), 377 (3.47) nm; IR (KBr) vmax 3413, 3021, 2924, 2852, 1631, 1614, 1594, 1151, 1542, 1446, 1422, 1394, 1331, 1236 cm−1; 1D NMR data see Table 1; HRESI-MS m/z 469.1662 [M–H]−, calcd for C29H25O6, 469.1657 (Additional file 1: Figs. 1–8).

3.4.2 Gastrodinol B (2)

Yellow powder; UV (MeOH) λmax (log ε): 201 (4.81), 224 (4.88), 316 (4.68), 366 (3.74), 441 (3.53) nm; IR (KBr) vmax 3395, 3066, 3023, 2920, 2850, 1719, 1669, 1612, 1514, 1469, 1443, 1384, 1264 cm−1; 1D NMR data see Table 1; HRESI-MS m/z 673.1695 [M + CF3COO]−, calcd for C35H28O7CF3CO2, 673.1691 (Additional file 1: Figs. 9–16).

3.4.3 Gastrodinol C (3)

Yellow powder; UV (MeOH) λmax (log ε): 201 (4.96), 226 (4.97), 277 (4.38), 319 (4.77) nm; IR (KBr) vmax 3432, 3027, 2971, 2920, 2851, 1713, 1668, 1613, 1514, 1484, 1442, 1417, 1383, 1359, 1261 cm−1; 1D NMR data see Table 1; HRESI-MS m/z 605.1812 [M + COOH]−, calcd for C35H28O7CO2H, 605.1817 (Additional file 1: Figs. 17–24).

3.4.4 Gastrodinol D (4)

Yellow powder; [α] 26L − 10.41 (c 0.078, MeOH); UV (MeOH) λmax (log ε): 195 (4.71), 225 (4.23), 313 (3.90), 382 (3.21) nm; IR (KBr) vmax 3425, 3023, 2922, 2852, 1732, 1679, 1613, 1598, 1514, 1441, 1428, 1383, 1258 cm−1; 1D NMR data see Table 1; HRESI-MS m/z 673.1685 [M + CF3COO]−, calcd for C35H28O7CF3CO2, 673.1691 (Additional file 1: Figs. 25–32).

3.4.5 Isotetrapterols A (5)

Yellow powder; [α] 20L − 20.35 (c 0.115, MeOH); UV (MeOH) λmax (log ε): 195 (4.55), 216 (4.66), 284 (4.34), 315 (416) nm; IR (KBr) vmax 3371, 3030, 2969, 2925, 2854, 1712, 1635, 1506, 1457, 1434, 1381, 1362, 1273 cm−1; 1D NMR data see Table 2; HRESI-MS m/z 417.1349 [M–H]−, calcd for C25H21O6, 417.1344 (Additional file 1: Figs. 33–40).

3.4.6 Morusinol B (6)

Yellow powder; [α] 25L − 23.00 (c 0.060, MeOH); UV (MeOH) λmax (log ε): 199 (4.60), 225 (4.32), 271 (4.49), 348 (3.69) nm; IR (KBr) vmax 3421, 3263, 2975, 2930, 1653, 1622, 1602, 1579, 1483, 1434, 1382, 1351, 1255 cm−1; 1D NMR data see Table 2; HRESI-MS m/z 453.1562 [M–H]−, calcd for C25H25O8, 453.1555 (Additional file 1: Figs. 41–48).

3.4.7 Cyclomorusinol hydroperoxide (7)

Yellow powder; [α] 20L − 18.10 (c 0.084, MeOH); UV (MeOH) λmax (log ε): 203 (4.31), 227 (3.82), 273 (4.02), 311 (3.58), 367 (3.14) nm; IR (KBr) vmax 3428, 3074, 2974, 2928, 2856, 1712, 1631, 1533, 1508, 1461, 1402, 1383, 1278 cm−1; 1D NMR data see Table 2; HRESI-MS m/z 467.1353 [M − H]−, calcd for C25H23O9, 467.1348 (Additional file 1: Figs. 49–56).

3.4.8 Benzylkuwanon C (8)

Yellow powder; UV (MeOH) λmax (log ε): 203 (5.05), 268 (4.65), 305 (4.29) nm; IR (KBr) vmax 3412, 3041, 2969, 2925, 2856, 1647, 1620, 1562, 1512, 1466, 1441, 1357, 1222 cm−1; 1D NMR data see Table 2; HRESI-MS m/z 527.2071 [M–H]−, calcd for C32H31O7, 527.2075 (Additional file 1: Figs. 57–64).

3.4.9 Benzylmulberrofuran G (9)

Black powder; [α] 20D  + 475.49 (c 0.142 MeOH); UV (MeOH) λmax (log ε): 198 (5.03), 223 (4.61), 284 (4.20), 314 (4.28) nm; IR (KBr) vmax 3425, 3069, 2924, 2853, 1712, 1622, 1563, 1511, 1488,1449,1414, 1364, 1257 cm−1; 1D NMR data see Table 3. HRESI-MS m/z 669.2126 [M + H]+, calcd for C41H33O9, 669.2119 (Additional file 1: Figs. 65–72).

3.4.10 gastrodiamide (10)

Light yellow powder; IR (KBr) vmax 3417, 3263, 3069, 3016, 2930, 2853, 1758, 1702, 1630, 1613, 1514, 1470, 1434, 1404, 1383, 1255 cm−1; 1D NMR data see Table 4. HRESI-MS m/z 797.2710 [M + H]+, calcd for C46H41N2O11, 797.2705 (Additional file 1: Figs. 73–79).

3.4.11 Cyclomulberrin (11)

Yellow amorphous powder; C25H24O6; 1H NMR (600 MHz, Methanol-d4) δH: 7.61 (d, J = 8.6 Hz, 1H, H-6′), 6.52 (dd, J = 8.6, 2.2 Hz, 1H, H-5′), 6.32 (d, J = 2.2 Hz, 1H, H-3′), 6.22 (s, 1H, H-6), 6.15 (d, J = 9.2 Hz, 1H, H-1′′′), 5.42 (d, J = 9.2 Hz, 1H, H-2′′′), 5.26 (t, J = 5.9 Hz, 1H, H-2′′), 3.58 (dd, J = 5.9, 15 Hz, 2H, H-1′′), 1.95 (s, 3H, H-4′′′), 1.84 (s, 3H, H-5′′′), 1.70 (s, 3H, H-5′′), 1.69 (s, 3H, H-4′′); 13C NMR (150 MHz, Methanol-d4) δC: 179.93 (C-4), 164.70 (C-8a), 162.87 (C-7), 160.94 (C-4′), 159.58 (C-2), 157.16 (C-2′), 155.72 (C-5), 139.79 (C-3′′′), 132.41 (C-3′′), 126.21 (C-6′), 123.79 (C-2′′), 122.56 (C-2′′′), 110.93 (C-5′), 110.04 (C-1′), 108.87 (C-3), 108.15 (C-4a), 105.59 (C-8), 105.05 (C-3′), 99.40 (C-6), 70.71 (C-1′′′), 25.90 (C-4′′, 5′′), 22.49 (C-1′′), 18.68 (C-4′′′), 18.17 (C-5′′′).

3.4.12 Kuwanon C (12)

Yellow amorphous powder; C25H26O6; 1H NMR (500 MHz, Methanol-d4) δH: 7.07 (d, J = 8.3 Hz, 1H, H-6′), 6.42 (d, J = 2.3 Hz, 1H, H-3′), 6.39 (dd, J = 8.3, 2.3 Hz, 1H, H-5′), 6.23 (s, 1H, H-6), 5.16 (m, 1H, H-2′′), 5.10 (m, 1H, H-2′′′), 3.32 (d, J = 7.1 Hz, 2H, H-1′′), 3.09 (d, J = 6.9 Hz, 2H, H-1′′′), 1.59 (s, 6H, H-4′′′, H-5′′′), 1.55 (s, 3H, H-5′′), 1.39 (s, 3H, H-4′′); 13C NMR (125 MHz, Methanol-d4) δC: 184.02 (C-4), 163.59 (C-7), 162.71 (C-4′), 161.82 (C-8a), 160.68 (C-2), 157.86 (C-2′), 157.07 (C-5), 132.60 (C-3′′′), 132.42 (C-6′), 132.01 (C-3′′), 123.43 (C-2′′), 122.97 (C-2′′′), 121.33 (C-3), 113.53 (C-1′), 107.84 (C-5′), 107.50 (C-8), 105.34 (C-4a), 103.73 (C-3′), 98.92 (C-6), 25.94 (C-4′′′), 25.84 (C-4′′), 24.83 (C-1′′′), 22.34 (C-1′′), 17.75 (C-5′′′), 17.65 (C-5′′).

3.4.13 Mulberrofuran G (13)

Black amorphous powder; C34H26O8; 1H NMR (400 MHz, Methanol-d4) δH: 7.34 (d, J = 8.4 Hz, 1H, H-4), 7.14 (d, J = 8.4 Hz, 1H, H-14′′), 7.09 (d, J = 8.4 Hz, 1H, H-20′′), 6.92 (s, 1H, H-7), 6.91 (d, J = 1.8 Hz, 2H, H-2′, H-6′), 6.82 (s, 1H, H-3), 6.73 (dd, J = 8.4, 2.1 Hz, 1H, H-5), 6.46 (dd, J = 8.4, 2.6 Hz, 1H, H-19′′), 6.40 (d, J = 4.7 Hz, 1H, H-2′′), 6.35 (d, J = 2.6 Hz, 1H, H-17′′), 6.33 (d, J = 2.4 Hz, 1H, H-11′′), 6.14 (dd, J = 8.6, 2.4 Hz, 1H, H-13′′), 3.34 – 3.23 (m, 2H, H-3′′, H-4′′), 2.95 (dd, J = 11.4, 5.5 Hz, 1H, H-5′′), 2.66 (dd, J = 17.0, 5.5 Hz, 1H, H-6′′a), 2.01 (dd, J = 17.0, 11.4 Hz, 1H, H-6′′b), 1.78 (s, 3H, H-7′′); 13C NMR (100 MHz, Methanol-d4) δC: 158.65 (C-10′′), 157.02 (C-18′′), 156.51 (C-12′′), 156.33 (C-6), 155.85 (C-2), 155.39 (C-5′), 154.34 (C-3′), 153.52 (C-16′′), 152.21 (C-7a), 132.52 (C-1′′), 130.14 (C-1′), 129.18 (C-14′′), 126.60 (C-20′′), 121.96 (C-2′′), 121.71 (C-3a), 120.60 (C-4), 116.89 (C-4′), 115.96 (C-15′′), 112.52 (C-9′′), 111.85 (C-5), 108.65 (C-19′′), 105.63 (C-13′′), 104.10 (C-3), 103.60 (C-6′), 103.15 (C-2′), 102.78 (C-17′′), 101.70 (C-8′′), 100.76 (C-11′′), 97.10 (C-7), 36.24 (C-3′′), 35.34 (C-6′′), 34.02 (C-5′′), 27.44 (C-4′′), 22.54 (C-7′′).

3.5 Compound anti-Staphylococcus aureus activity experiment

Prepare samples with DMSO as a solvent into a solution with an initial concentration of 20 mM; Take a 96-well culture plate, dilute the sample to be measured, and add bacterial solution to each well, and the final concentration is 5 × 105 CFU/mL; Incubate at 37 °C for 24 h, and the microplate reader determines the OD value at 625 nm. The experiment also set up a media blank control, a bacterial control and a vancomycin-positive drug control.

MIC50 (50% minimum inhibitory concentration) is calculated according to the Reed & Muench method.

3.6 Acetylcholinesterase inhibition activity screening results

Use phosphate buffer (0.1 M Na2HPO4 solution per 100 mL of phosphate buffer 94.7 mL; 0.1 M NaH2PO4 solution 5.3 mL, pH adjusted to 8.0) Dilute AChE into 0.1 U/mL working solution; Thioacetylcholine iodide and DTNB are formulated with phosphate buffer to form a 6.25 mM solution (working solution); The compound is diluted with DMSO to form a concentration gradient. The positive control is tacrine, diluted with DMSO to a concentration gradient; The negative control group (NC group) was a 2% DMSO solvent control. The reaction is carried out in a 96-well plate with plates plated by 200 μL/system with 3 replicates per sample; Detect the absorbance value of 405 nm every 30 s within 1 h after the addition of the color developer and substrate. Select the sample absorbance value when the average absorbance value of the NC group is about 1, calculate the average value of the compound absorbance value (compound measurement value—background value), and calculate the compound AChE inhibition rate according to (NC-compound absorbance value average)/NC*100%.