Tremorgenic Indole Diterpenes from Ipomoea asarifolia and Ipomoea

Jul 16, 2018 - Dale R. Gardner*† , Kevin D. Welch† , Stephen T. Lee† , Daniel Cook† , and Franklin Riet-Correa‡§. † Poisonous Plant Resea...
0 downloads 0 Views 970KB Size
Download PDF
Note Cite This: J. Nat. Prod. 2018, 81, 1682−1686

pubs.acs.org/jnp

Tremorgenic Indole Diterpenes from Ipomoea asarifolia and Ipomoea muelleri and the Identification of 6,7-Dehydro-11-hydroxy12,13-epoxyterpendole A Dale R. Gardner,*,† Kevin D. Welch,† Stephen T. Lee,† Daniel Cook,† and Franklin Riet-Correa‡,§

Downloaded via UNIV OF SUSSEX on July 27, 2018 at 06:46:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 E. 1400 N., Logan, Utah 84341, United States ‡ National Institute of Agricultural Research (INIA), La Estanzuela, Colonia, CR 70.000, Uruguay § Veterinary Hospital, Federal University of Campina Grande, Patos, Paraiba 58429-900, Brazil S Supporting Information *

ABSTRACT: Indole diterpene alkaloids have been isolated from Ipomoea asarifolia and I. muelleri and are associated with a tremorgenic syndrome in livestock. To better characterize the tremorgenic activity of the major indole diterpene alkaloids in these two plants, terpendole K (1), 6,7-dehydroterpendole A (2), 11-hydroxy-12,13-epoxyterpendole K (3), terpendole C (5), paxilline (6), and a new compound, 6,7-dehydro-11-hydroxy12,13-epoxyterpendole A (4), were isolated and evaluated for tremorgenic activity in a mouse model. Compounds 1, 2, 5, and 6 all showed similar and significant signs of tremorgenic activity. In contrast, the 11-hydroxy-12,13-epoxy compounds, 3 and 4, showed no significant tremorgenic activity.

T

epoxide isomer of 2 and subsequently test the tremorgenic activity of the purified compounds in a mouse bioassay. The crude mixture of indole diterpene alkaloids was extracted and isolated from I. asarifolia seed material using solvent extraction, column chromatography, and solid-phase extraction procedures, as previously described.1 Partially purified compounds were then isolated by preparative HPLC, also as previously described. Final purification of the individual compounds was completed using semipreparative HPLC, resulting in sufficient quantities of the known indole diterpenes 1−3 and 5 for biological testing. The identities of these known indole diterpenes were confirmed by comparison of their HPLC-MS and NMR data to previously identified compounds.1 Compound 4, the unknown isomer of 2, gave a confirmed molecular formula of C32H39NO6, based on the (+)-HRESIMS (m/z 534.28513) of the protonated molecule. Comparison of the 1H and 13C NMR data (1H, 13C-DEPT, HSQC, and HMBC) of 4 with those of 2 and 3 showed key similarities with both compounds. The epoxy-isoprenyl substituent was evident by comparison of the 1H and 13C NMR data with those of 1 and 3 with the loss of the two olefinic carbon resonances (C-33 and C-34) at δ 121.8 and 140.3 and the appearance of two oxygen-bearing carbons at δ 63.0 and 58.1,

he analysis of indole diterpenes from two Ipomoea species, I. asarifolia and I. muelleri, was recently reported.1 A number of different indole diterpene alkaloids, including two new alkaloids, were confirmed as present in I. asarifolia and reported for the first time in I. muelleri. It is noted that the indole diterpene alkaloids are derived from a Clavicipitaceous symbiotic endophyte, Periglandula species, associated with ergot-alkaloid-containing Ipomoea species.2,3 It has been proposed that the indole alkaloid content in the two Ipomoea species is responsible for the tremorgenic syndrome observed in livestock grazing these species.4−11 Indole diterpenes such as terpendole C (5), terpendole M, and paxilline (6) have been shown previously to induce a tremorgenic syndrome in mice treated with these pure compounds, while other indole diterpenes lack such tremorgenic activity.3,12,13 Terpendole C (5) was previously found to be present in both I. asarifolia and I. muelleri and paxilline only in I. asarifolia.1 However, the major alkaloids observed in these two species included terpendole K (1), 6,7-dehydroterpendole A (2), a new isomer of 1, in which the 11,12-epoxide was shifted to the 12,13-position and identified as 11-hydroxy12,13-epoxyterpendole K (3), and an unknown isomer of 2 that was hypothesized to be a similar but unknown 12,13epoxide compound (4). No bioactivity data have been reported for 1−4; therefore, the objective of this study was to isolate sufficient quantities of these major indole diterpenes to be used to confirm the structure of the proposed 12,13© 2018 American Chemical Society and American Society of Pharmacognosy

Received: March 26, 2018 Published: July 16, 2018 1682

DOI: 10.1021/acs.jnatprod.8b00257 J. Nat. Prod. 2018, 81, 1682−1686

Journal of Natural Products

Note

other time points, there were no visual tremors observed in these mice. Mice were placed into a tremor monitor chamber 30 min postinjection. The mice dosed with 2 had significantly elevated movement energy compared to all other treatment groups across all frequencies (Figure 2). These data highlight the severity of the tremors experienced by the mice at this time point. Mice dosed with 1 and 5 had more severe tremors than the control and mice treated with 3, 4, and 6, but not as severe as mice dosed with 2. There was no difference in the movement energy of the mice treated with 3 or 4 compared to controls, indicating that the mice were not experiencing any tremors at this time point. The biological data obtained from dosing 1−6 into the mice again confirmed the tremorgenic activity of 5 and 6 and established the tremorgenic activity of 1 and 2, with the latter being the most tremorgenic compound using this model. The lack of tremorgenic activity of 3 and 4 indicates the importance of the 13-hydroxy substituent in the structure of the tremorgenic compounds. As terpendole K (1) and 6,7dehydroerpendole A (2) are the major indole diterepenes present in I. asarifolia and I. muelleri, the current data clearly support the hypothesis that such indole alkaloids are responsible for the tremorgenic syndrome observed in livestock grazing these species.

with the corresponding upfield shift of H-33 from δ 5.47 in 1 to δ 2.92 in 4 and identical to that in 2. The remaining resonances were essentially unchanged from those of 3, with the key changes in the chemical shifts and J couplings of H-5, H-6, H-9, H-10, and H-11, as previously described for 3. Therefore, the structure was assigned as 6,7-dehydro-11hydroxy-12,13-epoxyterpendole A (4), a new indole diterpene. Compounds 1−6 were tested for tremorgenic activity in an established mouse bioassay.14,15 Each indole diterpene compound was dosed intraperitoneally at 8 mg/kg. The tremorgenic activity was assessed for 120 min postinjection. Mice were observed for visual signs of tremors at 15, 30, 60, and 120 min postinjection. There were moderate to severe visually observable tremors in all mice dosed with compounds 1, 2, 5, and 6 at 15, 30, and 60 min postdosing (Figure 1). However, by 120 min these visual tremors were no longer observed in most mice. In contrast, the mice dosed with 3 or 4 showed only very minor tremors at 15 min postinjection. At all

■

EXPERIMENTAL SECTION

General Experimental Procedures. Paxilline (6) was obtained from Tocris Bioscience (Bristol, UK). Terpendole C (5) was obtained from BioVision Inc. (Milpitas, CA, USA) along with 16 mg of 5 isolated as described below for biological testing. All solvents were of analytical grade or HPLC grade for preparative HPLC. The optical rotation was obtained using an Autopol IV (Rudolph Research Analytical, Flanders, NJ, USA). NMR spectroscopic data (1H, 13C) were acquired using an Avance III HD spectrometer (500 MHz for 1 H, 125 MHz for 13C) (Bruker Biospin, Billerica, MA, USA) using solutions in chloroform-D (99.8 atom % D) (δH 7.24, δC 77.23). NMR data were analyzed using Top Spin software, version 3.2

Figure 1. Visual tremor score of mice dosed intraperitoneally with various indole diterpenes (1−6). The data represent the means ± standard deviation of the visual score for 5 or 6 mice per group. A score of 0 = no visually observable tremors, 1 = slight tremors, 2 = moderate tremors, and 3 = severe tremors. Tabulated data are available in Table S1 (Supporting Information). 1683

DOI: 10.1021/acs.jnatprod.8b00257 J. Nat. Prod. 2018, 81, 1682−1686

Journal of Natural Products

Note

Figure 2. Effect of indole diterpenes (1−6) on muscle tremors in mice. Mice were dosed intraperitoneally at 8 mg/kg and placed into the tremor monitor chamber 30 min postinjection. Mice were evaluated individually for 64 s. Data represent the means of the FFT movement energy magnitude, expressed in mV across all 64 Hz for the entire 64 s analysis. (Bruker). For high-resolution mass spectrometric analysis, a dilute solution (∼10 μg/mL in 50:50 methanol−0.1% formic acid) of the isolated compound was injected (5 μL) into a flow of 50:50 methanol−0.1% formic acid (0.3 mL/min) using an Ultimate 3000 HPLC (Thermo Scientific, San Jose, CA, USA) with no column and connected to a heated electrospray source of an Exactive Plus Orbitrap mass spectrometer (Thermo Scientific) calibrated as per the manufacturer’s instructions and with a scan range of 100−800 Da, resolution 70 000, microscans 1, sheath gas flow 35, auxiliary gas flow 10, spray voltage 4 kV, capillary temperature 320 °C, S lens RF field 55, and auxiliary gas temperature 300 °C. Preparative HPLC was performed using a Prep LC2000 preparative chromatography system (Waters Corp., Milford, MA, USA). The column used was a 150 × 21.2 mm i.d., 10 μm, Luna C18(2), with a 50 × 21.2 mm i.d. guard column of the same material (Phenomenex, Torrance, CA, USA). The eluent was monitored by a UV/vis detector (Waters Corp.) at λ 280 nm. The column was eluted with a gradient flow at a rate of 20 mL/min. The mobile phase program was 0.1% formic acid−acetonitrile, 80:20 v/v, for 1 min followed by a linear gradient to a composition of 100% acetonitrile at 40 min. Fractions (20 mL) were collected at 1 min intervals. Aliquots from each fraction were analyzed for the presence of indole diterpenes by HPLC-HRMS, as previously described.1 Semipreparative HPLC was performed using an Agilent 1260 Infinity HPLC system comprising a quaternary pump, a multiwavelength UV/vis detector, and a fraction collector (Agilent Technologies, Santa Clara, CA, USA). Sample aliquots (150 μL) were injected manually onto a Rainin Microsorb C18 (10 × 250 mm) dynamic axial compression HPLC column (Rainin Instrument Co., Woburn, MA, USA) with a guard column of the same phase. The column was eluted with a gradient mixture of acetonitrile (A) and 0.1% formic acid (B) at a flow rate of 3.0 mL/min starting with 20% A for 2 min, followed by a linear increase to 100% A at 30 min, remaining at 100% A until 35 min, then a return to 20% A by 39 min, and a final 6 min equilibration time before the next injection. Fractions were collected by UV detection at 280 nm, and all fractions of interest analyzed for the presence of indole diterpenes by HPLCHRMS, as previously described.1 Fractions of interest were evaporated to dryness using a Savant SPD 2010 Speed Vac concentrator (Thermo Fisher Scientific, Asheville, NC, USA). Plant Material. Ipomoea asarifolia (Desr.) Roem & Schult. seeds were collected near the veterinary hospital of the University of

Campina Grande, Campus of Patos in the city of Patos, Paraiba, Brazil (S 7°04′02″, W 37°16′53″) (voucher no. 21226 (PEL)). Extraction and Isolation. The solvent extraction and chromatography procedures (column chromatography, solid-phase extraction, and preparative HPLC) were repeated as described previously1 using a new collection of I. asarifolia seeds (340 g) to give four main fractions (fractions A−D) containing the target indole diterpenes as follows: fraction A (24 mg, unknown isomer of 2 major); fraction B (39 mg, mixture of 2 and 3); fraction C (27 mg, 1 major); fraction D (27 mg, 5 major). Fractions A−D were subjected to semipreparative HPLC as described above (General Experimental Procedures) to yield purified alkaloids 1 (16 mg), 2 (4 mg), 3 (5.9 mg), and 5 (16 mg). The compounds were identified by comparison of 1H NMR and HPLC-HRMS data as previously described.1 Fraction A, from prepHPLC, was also subjected to additional purification by semipreparative HPLC to give 4 (7.8 mg), for which the structure was determined by (+)HRESIMS and 1D and 2D NMR. 6,7-Dehydro-11-hydroxy-12,13-epoxyterpendole A (4): white, amorphous solid; [α]21D −11.6 (c 0.0017 CHCl3); 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Table 1; (+)HRESIMS, m/z 534.28513 (M + H)+ (calcd for C32H40NO6 534.28501). Testing for Tremorgenic Activity. Female Swiss Webster mice (8 weeks old) were purchased from Charles River Kingston (Stone Ridge, NY, USA). Mice were acclimated for 3 to 4 days with free access to a commercially pelleted rodent chow (Teklad rodent diet (w) 8604) and tap water before beginning experiments. Mice were housed under a controlled temperature (20−22 °C) environment with a 12:12 h light:dark cycle. All procedures were conducted under veterinary supervision and were approved by the Utah State University Institutional Animal Care and Use Committee (USU IACUC #2244). A 2 mg/mL solution of each test compound was prepared by dissolving the test compounds in DMSO−water (9:1). Each compound was injected intraperitoneally at a dose of 8 mg/kg in 5 or 6 mice per compound. Control mice were dosed with a similar volume of DMSO−water (9:1). There were no tremors observed in any of the control mice. Evaluations of the tremorgenic activity of each compound were made at 15, 30, 60, and 120 min postdosing. Visual Tremor Score. Mice were assessed for visual signs of tremorgenic activity by placing the mouse on an outstretched finger for 30 s at 15, 30, 60, and 120 min postinjection.14 Visual tremors 1684

DOI: 10.1021/acs.jnatprod.8b00257 J. Nat. Prod. 2018, 81, 1682−1686

Journal of Natural Products

■

Table 1. 1H and 13C NMR Spectroscopic Data (500 and 125 MHz, CDCl3) for 6,7-Dehydro-11-hydroxy-12,13epoxyterpendole A (4), Isolated from Ipomoea asarifolia Seeds position

δC, type

1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 31 33 34 35 36

NH 149.1, C 50.3, C 40.6, C 30.1, CH2 101.7, CH 146.4, C 69.5, CH 72.6, CH 63.2, CH 58.6, C 73.8, C 29.3, CH2 22.3, CH2 48.7, CH 27.4, CH2 118.7, C 125.2, C 118.8, CH 120.0, CH 121.1, CH 111.8, CH 140.2, C 15.0, CH3 19.3, CH3 74.8, C 28.3, CH3 17.0, CH3 94.9, CH 63.0, CH 58.1, C 19.8, CH3 24.8, CH3

*Tel: 435-752-2941. Fax: 435-753-5681. E-mail: dale. [email protected].

δH (J in Hz)

ORCID

Dale R. Gardner: 0000-0003-3218-6893 Kevin D. Welch: 0000-0002-5552-4894 Stephen T. Lee: 0000-0002-0597-8353 Daniel Cook: 0000-0001-8568-113X Franklin Riet-Correa: 0000-0001-5738-7785

2.71, brd (15.1), 1.77, dd (15.2, 8.2) 5.17, dd (8.0, 2.4)

Notes

The authors declare no competing financial interest.

■

4.14, d (10.4) 3.69, dd (10.2, 3.2) 4.13, d (3.0)

1.55, m dq (14.1, 4.3), 1.88, m m dd (13.5, 6.5), 2.46, dd (13.5, 10.5)

7.44, 7.07, 7.09, 7.29,

m m m m

ACKNOWLEDGMENTS We thank C. Hailes and S. Larsen for technical assistance and D. Pessoa (Federal University of Campina Grande) with assistance in collection of Ipomoea asarifolia seeds. Utah State University and the National Science Foundation (CHE1429195) are thanked for the use of and funding for the Bruker 500 MHz NMR instrument. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

■

s s d (6.3) d (6.3)

1.35, s 1.31, s

were rated on a scale of 0−3, with 0 = normal activity (i.e., no tremors), 1 = slight tremors, 2 = moderate tremors, and 3 = severe tremors. Tremor Monitor. Tremorgenic activity was assessed using a tremor monitor system (San Diego Instruments, San Diego, CA, USA). The tremor monitor differentiates tremor events from normal ambulatory movements.15 The monitor uses an ultrasensitive movement sensor to record continuous movement waveforms from 1 to 64 Hz. Mice were placed into the tremor monitor for 64 s at 30 min postinjection. The software provided data for the movement energy in mV at each frequency expressed as the fast Fourier transform (FFT) for the magnitude at each of the 64 frequencies.

■

REFERENCES

(1) Lee, S. T.; Gardner, D. R.; Cook, D. J. Agric. Food Chem. 2017, 65, 5266−5277. (2) Schardl, C. L.; Young, C. A.; Hesse, U.; Amyotte, S. G.; Andreeva, K.; Calie, P. J.; Fleetwood, D. J.; Haws, D. C.; Morre, N.; Oeser, B.; Panaccione, D. G.; Schweri, K. K.; Voisey, C. R.; Farman, M. L.; Jaromczyk, J. W.; Roe, B. A.; O’Sullivan, D. M.; Scott, B.; Tudzynski, P.; Zhiqiang, A.; Arnaoudova, E. G.; Bullock, C. T.; Charlton, N. D.; Chen, L.; Cox, M.; Dinkins, R. D.; Florea, S.; Glenn, A. E.; Gordon, A.; Guldener, U.; Harris, D. R.; Hollin, W.; Jaromczyk, J.; Johnson, R. D.; Khan, A. K.; Leistner, E.; Leuchtmann, A.; Li, C.; Liu, J. G.; Liu, J.; Mace, W.; Machado, C.; Nagabhyru, P.; Pan, J.; Schmid, J.; Sugawara, K.; Steiner, U.; Takach, J. E.; Tanaka, E.; Webb, J. S.; Wilson, E. V.; Wiseman, J. L.; Yoshida, R.; Zeng, Z. PLoS Genet. 2013, 9 (2), e1003323. (3) Panaccione, D. G.; Beaulieu, W. T.; Cook, D. Funct. Ecol. 2014, 28, 299−314. (4) Everist, S. L. In Poisonous Plants of Australia; Angus & Robertson: Sydney, 1974; pp 199−204. (5) Medeiros, R. M. T.; Barbosa, R. C.; Riet-Correa, F.; Lima, E. F.; Tabosa, I. M.; de Barros, S. S.; Gardner, D. R.; Molyneux, R. J. Toxicon 2003, 41, 933−935. (6) Dobereiner, J.; Tokarnia, C. H.; Canella, C. F. C. Inst. Biol. 1960, 3, 39−57. (7) Araújo, J. A. S.; Riet-Correa, F.; Medeiros, R. M. T.; Soares, M. P.; Oliveira, D. M.; Carvalho, F. K. L. Pesq. Vet. Bras. 2008, 28, 488− 494. (8) Tortelli, F. P.; Barbosa, J. D.; Oliveira, C. M. C.; Duarte, M. D.; Cerqueira, V. D.; Oliveira, C. A.; Riet Correa, F.; Riet Correa, G. Pesq. Vet. Bras. 2008, 28, 622−626. (9) Gardner, C. A.; Bennetts, H. W. In The Toxic Plants of Western Australia; Western Australian Newspapers Ltd: Perth, Australia, 1956; p 157. (10) Gardiner, M. R.; Royce, R.; Oldroyd, B. Br. Vet. J. 1965, 121, 272−277. (11) Dorling, P. R.; Colegate, S. M.; Allen, J. G.; Nickels, R.; Mitchell, A. A.; Main, D. C.; Madin, B. In Poisonous Plants and Related Toxins; CABI Publishing: Wallingford, UK, 2004; pp 140−145. (12) Gatenby, W. A.; Munday-Finch, S. C.; Wilkins, A. L.; Miles, C. O. J. Agric. Food Chem. 1999, 47, 1092−1097.

1.10, s 1.06, s 1.43, 1.30, 4.69, 2.92,

AUTHOR INFORMATION

Corresponding Author

7.69, brs

2.22, 1.96, 2.94, 2.79,

Note

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00257. Tabulated means and standard deviations of the observed visual tremor scores and selected MS and NMR spectra of compound 4 (PDF) 1685

DOI: 10.1021/acs.jnatprod.8b00257 J. Nat. Prod. 2018, 81, 1682−1686

Journal of Natural Products

Note

(13) Munday-Finch, S. C.; Wilkins, A. L.; Miles, C. O.; Tomoda, H.; Omura, S. J. Agric. Food Chem. 1999, 45, 199−204. (14) Miles, C. O.; Wilkins, A. L.; Gallagher, R. T.; Hawkes, A. D.; Munday, S. C.; Towers, N. R. J. Agric. Food Chem. 1992, 40, 234−238. (15) Welch, K. D.; Pfister, J. A.; Lima, F. G.; Green, B. T.; Gardner, D. R. Toxicol. Appl. Pharmacol. 2013, 266, 366−374.

1686

DOI: 10.1021/acs.jnatprod.8b00257 J. Nat. Prod. 2018, 81, 1682−1686