Honey, Bees, and Hepatotoxic Alkaloid Echimidine

Experimental E. plantagineum grown in Connecticut was extracted with MeOH and after

removal of MeOH, the resulting oleoresin was defatted by partitioning between hexane-MeOH-water (10:9:1). The lower phase was partially concentrated and diluted with water. The pH was adjusted to 2 by adding H2SO4 and zinc dust was added to reduce the N-oxides during stirring. After 20 hr the reaction mixture was made alkaline with Na2CO3 and the alkaloids were extracted with ethyl acetate. This extract was again partitioned agains acidic water and the aqueous layer was re-extracted after addition of Na2CO3. This extract was evaporated to produce a crude mixture of pyrrolizidine alkaloids. Echimidine was separated from other alkaloids by Centrifugal Partition Chromatography (FCPC Kromaton A100, Rousellet Robatel, Fr.) using chloroform as the mobile phase and citrate buffer at pH 5.6 as the stationary phase. Analytical HPLCs were performed on a Kinetex EVO C18, 5µm, 100°A, 150 x 4.6 mm, and preparative on a Kinetex EVO C18, 5µm, 100°A, 150 x 21.2 mm column (Phenomenex). The mobile phase buffer was the same as for the preparative HPLC. The samples were eluted from the analytical column on a gradient from 5% acetonitrile to 16% acetonitrile, followed by a column wash with 29% acetonitrile. The flow rate was 1.0 ml/minute. The UV absorbance signal on the diode array detector was monitored at 210 nm.The mobile phase contained a buffer of 32 mM lithium phosphate, adjusted to pH 7.2 with phosphoric acid. The column was equilibrated in 32 mM lithium phosphate, pH 7.2/acetonitrile (9:1) and run in a gradient from 10% to 20% MeCN over 14.4 min at 20 ml/min. For preparative runs, samples of 60 mg were injected in DMF/methanol/water/acetic acid (1:1:1:0.003) to a concentration of 100 mg/ml. Fig. 2b shows how the cuts were determined. The total of 1.3 grams of CPC-fractionated material was loaded in 23 preparative HPLC runs.

Jan A. Glinski1, Marta Dudek2, Peter Kinkade1, Vitold B. Glinski1, Sławomir Kaźmierski3, Joao Calixto3. 1Planta Analytica LLC, 39 Rose St. Danbury, CT 06810, 2Dept. of Physical Chemistry, Medical University of Warsaw, 1 Banacha St, Warsaw Poland, 3Centre of Molecular and Macromolecular Studies, PAN, Sienkiewicza 112, Łódź, Poland, 4João B. Calixto, CIEnP, Av. Luiz Boiteux Piazza, 1302- Cachoeira do Bom Jesus, 88056-000- Florianópolis – S.C., Brazil

.

Echium plantagineum

Abstract Echimidine (1) is the main pyrrolizidine alkaloid of Echium plantagineum L, (Jane’s

Salvation, Patersons Curse), endemic to Australia[1]. The plant is a great attractant for bees and as a result echimidine finds its way into honey. Because of its hepatotoxicity, echimidine became a target of new EU regulation requiring testing of imported honey. Under normal RP HPLC conditions echimidine produces always a sharp peak. Only recently, we discovered that this single peak represents actually a mixture of three alkaloids. Using relatively new “core-shell” RP HPLC column (Kinetex EVO C18, Phenomenex) in a buffer system we resolved “echimidine” into two well separated peaks. An NMR analysis proved that later eluting peak belonged indeed to echimidine, while the earlier peak contained two, largely unresolved alkaloids echihumiline (2)[2,3] (major) and hydroxymyoscorpine (3)[2,4] (minor). Each of them hasbeen isolated before from other plants. All these alkaloids are isomeric C20H31NO7 and produce in MS a single MH+, signal at m/e 398, in and their NMR spectra are similar, which explains why they have not been detected before. Examination of several of echimidine samples derived from different plant collections revealed that the contribution of the early peak varied from 13% to 43%. Considering that these alkaloids may contribute unequally to the hepatotoxicity, effects of both, purified echimidine and the mixture of echihumiline and hydroxymyoscorpinewere compared in rat hepatocytes.

1. Claude C. J. Culvenor , John A. Edgar , Leslie W. Smith, J. Agric. Food Chem., 1981, 29 (5), 958–960, Pyrrolizidine alkaloids in honey from Echium plantagineum L.

2. El-Shazly, A, T. Sarg, A. Ateya, A. Abdel Aziz, and S. El-Dahmy, L. Witte, M. Wink, J. Nat. Prod. 1996, 59, 310-313. Pyrrolizidine Alkaloids from Echium setosum and Echium vulgare

3. Kretsi, O., Aligiannis, N., Skaltsonnis, A. L. and Chinou, I.B., Helvetica Chimica Acta 2003, 86, 3136-3140, Pyrrolizidine Alkaloids from Echium setosum and Echium vulgare.

4. Roeder E, Rengel B, Phytochemistry 1990 29, 690-693, Pyrrolizidine Alkaloids from Lithospermum erytrhorhizon.5. El-Shazly, A. and M. Wink, Diversity 2014, 6, 188-282, Diversity of Pyrrolizidine Alkaloids in the Boraginaceae. Structures,

Distribution, and Biological Properties, (review)

Literature

Background Pyrrolizidine alkaloids (PAs) are wide-spread in nature, occurring in about 3 % of

flowering plants.[5] An important biological feature of PAs is their hepatotoxicity, which in chronic form may not be readily recognizable, however may lead to irreversible liver damage and even death. Interestingly, many plants producing PAs attract bees., In the countries such as Argentina, Australia, and New Zealand, which are major producers and exporters of honey the valleys are blanketed by the plants belonging to the genus Echium. A presence of PAs in honey has been documented, however the questions what levels of PAs are dangerous remains open. There are currently several research programs underway initiated by the honey exporting as well as importing countries (European Union) aimed at determination of toxic levels. For most of the studies, echimidine was chosen as the specific toxic alkaloid.

Conclusions1. We have proven that the RP HPLC peak of “echimidine” derived from E. plantagineum consists of

three alkaloids: echimidine (1), echihumiline (2), and hydroxymyoscorpine (3). We believe that the alkaloids 2 and 3 have been missed so far because being isobaric are not differentiated by MS. Also, as a result of significant structural similarity most of the 1H NMR signals are overlapping and thus, signals of 2 and 3, which are at lower concentration can be easily overlooked.

2. On analytical scale as well as on preparative scale the separation of pure 1 and a peak consisting of partially unresolved 2 and 3 was achieved using Kinetex EVO C-18 core-shell technology (Phenomenex).

3. We did not have success in resolving of 1-3 using several RP HPLC columns representing different bonding chemistry.

4. In E. Plantagineum, the ratio of (1) to its isomers (2,3) is not constant throughout the vegetation season and the measurements of hepatoxicity for “echimidine” will vary as a result. We have initiated a comparative study to establish the relative potencies of (1) versus the mixture of (2,3).

(1) Echimidine

OO

N

H

CH3

O

O

CH3

H

CH3

CH3CH3OH

OH

OH

H

12

35

6 78

910

11

12

13

14

15

16

17

1819

2021

angelyl

(3) Hydroxymyoscorpine

OO

N

H

CH3

O

O

HCH3

CH3

CH3CH3OH

OH

OH

H

12

35

6 78

910

11

12

13

14

15

16

17

1819

20

21

senecionyl

(2) Echihumiline

OO

N

H

CH3

O

O

CH3H

CH3

CH3CH3OH

OH

OH

H

12

35

6 78

910

11

12

13

14

15

16

17

1819

20

21

tiglyl

1,2,3

Fig.1 Typical RP HPLC trace of “echimidine” comprising, in fact, 1,2 and 3.

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

0 5 10 15

mAu

min.

2,3

1

Fig.2A Kinetex analytical EVO C18 column resolves “echimidine” into 2 peaks

0

50

100

150

200

250

0 2 4 6 8 10 12 14 16

Vo

ltag

e (

mv)

min

12

3

Fig.2B Kinetex preparative EVO C18 column resolves “echimidine” into 2 peaks

Fig.3A Overlay of 13CNMR signals of pure echimidine(1) and a mixture of (2,3) as shown in Fig.2B.

Fig.3B Overlay of 1HNMR signals of pure echimidine(1) and a mixture of (2,3) as shown in Fig.2B.