Pyrrolizidine Alkaloids: Testing for Toxic Constituents of Comfrey John J. Vollmer, Noemi C. Steiner, Gitte Y. Larsen, and Krista M. Muirhead Mills College, Oakland, CA 94613 Russell J. Molyneux Western Regional Research Center, Agricultural Research Service, Albany, CA 94710 The use of herbs as medicine has increased dramatically in recent years. It is hased lareclv on a helief that the traditional and the natural must, inherently, be safer and healthier. However, historical use does not, by itself, assure safety. Many recent articles have reported the presence of natural toxins in medicinal herbs, and warnings have appeared about their use. With one specific example, comfrey, we would like to show that toxic constituents can be detected selectively by thin-layer chromatography and quantitatively by NMR spectroscopy. The experiment is suitable for iutroducine advanced undergraduates to natural nroducts chemistry. ft utilizes standarld equipment and p l a i t material that is readilv available. Manv students are familiar with the use of comfiey and the current popularity of medicinal herbs. This heightens their interest and helps make this experiment a valuable experience. The experiment can also be used as demonstration or case study for less advanced courses. Comfrey is known botanically as Symphytum officinale. I t is a perennial that grows up to four feet tall and has large, hairy leaves and small flowers. Comfrey is an herb that for centuries was used to aid healing of broken bones and ulcers (I). Modern reported applications also include the treatment of colds, arthritis, gall and kidney stones, gangrene, eye injury, headaches, gout, anemia, tuberculosis, cancer, and other illnesses (1-3). In a recent publication, comfrey was described as "being good for almost every ill of mankind" (3). Comfrey is often used as salad and vegetable and is readily available in health food stores as root or leaf or in capsule form. I t has even been recommended as an essential ingredient of a regular diet (3-5). However, there have been no medical studies of the effectiveness of comfrey and the evidence provided is only anecdotal (lc). In fact, the regular ingestion of comfrey is hazardous because it contains chemicals that are toxic and carcinogenic: unsaturated pyrrolizidine alkaloids' (6). A wide varietv. of . ~ l a n t tsh r o u ~ h o uthe t world have been found to contain pyrrolizidine aikaloids; over 200 different ~vrrolizidinealkaloids have been identified ( .7) . .Each con&.s of a fused bicyclic ring system in which the nitrogen is shared ( I ) , forming a tertiary alkaloid. Normally a hydroxymethylene group is attached at C-1 and a hydroxyl group a t C-7; one or both hydroxyl groups are commonly esterified (1). Frequently, di-esters contain a large ring of 11 to 13 atoms; riddelliine, 2, is a specific example. A double bond between C-1 and C-2 is a common feature and is prerequisite for toxicity.
Se\,eral unsuturatrd pyrrolizidine alkaloids have been isolated from comfrev and identified 18). . . Thev are a mixture of several closely related acyclic esters of the same diol, retronecine (3a). . . . but differ in the nature and stereochemistrv of the acyl groups. The major constituents are two mono-esters, lycopsamine (3b) and intermedine (3e), their acetyl derivatives, 7-acetyllycopsamine (3d) and l-acetylintermedine (3e), plus the di-ester symphytine (3f).
Name
R,
3a retronecine 3b 3c 36
38 31
H H
lycopsrnine intermedine 7-acelyilycopsamine 7-acatylintermedine symphytine
H
CH&O CH&O
Ii 111 I1 111
I
ii
H
0 H
CH,
-C
HF
HO
OH
-C
CHa HO
H
That pyrrolizidine alkaloids are toxic has been established in several ways. Several common range plants that contain pyrrolizidine alkaloids in high concentrations have long been known to cause the slow death, by severe liver damage, of horses and cattle (9,lO). Feeding experiments with range animals and rats have clearly established cause and effect (11).Manv cases of human ~oisoninebv" -~vrrolizidine alkaioids havebeen reported; t i e primary chronic effect is cirrhosis of the liver. Pvrrolizidine alkaloids are ineested most commonly by eatin; contilmin~tedcereal (121 , r by using medicinal herbs ( 1 ; ) ) . Excellent re\.iews of the human health hilzard from pyrn~lizidinealkaloids nrr availahlr (1.1). The toxicity ofcomfrey has also been estahliihed independently. ~ e e d i experiments n~ with rats have shown co&sively that comfrey is toxic to the liver and causes cancer of the liver (6a, 15). Human testing of comfrey by feeding experiments is clearly not acceptable, but in a recent case, a woman with severe liver damage was found to have con-
' Under federallaw. medicinal herbs are not considered to be drugs and F D A . approval s not needed. Therefore. studtes to determine the salety of heros are not requ red, as they are lor orugs. Volume 64
Number 12 December 1967
1027
sumed comfroy root capsules as a regular food supplement (16). In a similar case in New Zealand, a young man died from liver collapse after ingesting comfrey leaves on a regular basis (17). unsaturated pyrrolizidine alkaloids are toxic because they are converted to the corresvondin~nvrroles in the liver (7a,
RCOO
CHZOCOR
RCOO
achieved by spraying the TLC plate with hydrogen peroxide, thus forming the N-oxide, and then treating the plate with acetic anhydride followed by heating (21). Any saturated pyrroliaidine alkaloids present will not react. The second spray is p-dimethylaminohenzaldehyde in acidic solution. Known as Ehrlich's reagent, i t forms a colored complex with pyrroles. Only toxic pyrrolizidine alkaloids will become visible as intense purple spots:
CHtOCOR
RCOO
a
These pyrroles are very powerful alkylating agents. Apparently they react very rapidly with cell constituents and may destroy them, inhibiting liver function or causing irregular growth. The oxidation of 1,2-unsaturated pyrrolizidine alkaloids in the liver is brought about by mixed function oxidases (18). I t is interesting to note that these enzymes normally deactivate toxic chemicals, but in this case they actually produce the toxin. Saturated pyrrolizidine alkaloids do not undergo enzymatic oxidation.
RCOQ
CH,OCOR
CHIOCOR
M RCOO
CWO , COR
ALKALOID WRROLE
lsolatlon and ldentificallon of Pyrrollzldlne Alkaloids of Comfrey The toxic constituents of comfrey are easily isolated hecause, as alkaloids, they can he extracted with dilute aqueous acid. The toxic pyrrolizidine alkaloids of comfrey are unsaturated and therefore can be detected selectively by TLC, using specific sprays sequentially. The concentration of these alkaloids in a samnle of comfrev can he determined accurately with NMR spectroscopy by relating the integration of the sienal of the viuvl oroton to that of an external standard.
This TLC analysis is rapid and specific. I t is a vivid illustration of the presence of toxic constituents in comfrey. Clearly, this approach can also he used to test other plants. Toxic nvrrolizidine alkaloids have also been renorted in the following plants which are commonly availad~efrom health food stores or herb companies: borage (Borago officinalis) (221, coltsfoot (Tussilago farfara) (23), and life root plant (Senecio aureus) (24). Includine the analvsis of such vlants could enhance a i d extend the experiment.
Method of Extraction
Analysis by NMR
Pyrrolizidine alkaloids are soluble in methanol and in the normal isolation procedure they are initially extracted from dried, ground plant material with this solvent (19). After removal of the methanol the residue is treated with dilute aqueous acid which removes all alkaloids by protonation. Washing this acid fraction with ether removes nonbasic components which may he water-soluhle. In the plant,pyrrolizidine alkaloids normallv occur toeether with then Noxidr derivati\.es. Because these are soluble in mt,thanol and water, and insoluble in ether. the\. will ,r
