TOXICOLOGYANDAPPLIEDPHARMACOLOGY36,22l-231(1976)

Teratological Phenolic

and Toxicological Compounds from

Studies Sokanum

of Alkaloidal and Tuberosum t.’

SHAKUNTALA CHAUBE AND CHESTER A. SWINYARD Institute of Rehabilitation Medicine, New York University Medical Center, New York, New York 10016 Received August 21,1975: accepted November 24,1975

Teratological and Toxicological Studies of Alkaloidal and Phenolic Compoundsfrom Solanum Tuberosum L. CHAUBE, S.AND SWINYARD, C. A. (1976)Toxicol. Appl. Pharmacol. 36,227-237.A comparativestudy of acute and chronic ip administration was made in pregnant and nonpregnant (female) rats of alkaloidal (a-chaconine,a-solaninetotal glycoalkaloidal extract (TGA-extract)) and phenolic compounds(chlorogenicand caffeic acid) from Solarium Tuberosum L. The acute LD50 and 95% confidence limits for a-chaconine,a-solanine,and the TGA-extract were 84 (65.6 107.5),67 (52.3%85.7), and 60 mg/kg (35.7-100.8).There wasno significant differencein their potency at the 95% confidencelimit. Chronic administration of a-chaconine,a-solanine,and the TGA-extract to nonpregnantrats for 2 days(40mglkgjday) and 8 days(20mg/kg/day) resultedin 40 and42% mortality, respectively.Eight daily injectionsof a-chaconine(5-20 mg/kg) on Days 5-12 or 2 (40 mg/kg) on Days 5 and 6 to pregnantrats resultedin maternal (40-66x) and fetal death (15-lOO%), whereasa-solanineand the TGA-extract administeredsimilarly were lethal only to the fetus (17-86%). Eight daily injections of chlorogenic(5-500 mg/kg) and caffeic acid (40-187.5mg/kg) on Days 5-12 of gestationdid not causematernalor fetal lethality. None of the compoundstestedproducedneuraltube defects, but a few 21-day-oldfetuseshad rib abnormalities. In 1972, a British geneticist (Renwick, 1972)hypothesized, on the basisof comparative epidemiological studies of late blight of potatoes and a serious birth defect known as spina bifida manifesta (myelomeningocele), that this birth defect wascausedby maternal consumption of potatoes blighted with the fungal organism Phytophrhora i&stuns. Although the embryogenesisof spinabifida in man is well known (Swinyard et&., 1973), the etiology of this birth defect is unknown. One epidemiological study (Clarke et al., 1973)and severalexperimental investigations (Poswillo et al., 1972,1973; Chaube et al., 1973; Swinyard and Chaube, 1973) failed to confirm the Renwick hypothesis. Potatoes contain known toxic alkaloids (cr-solanine, a-chaconine), phenolic compounds (chlorogenic and caffeic acids), and the coumarins (scopolin, umbelliferone, coumarin). The toxicity of cl-solaninehas been reported in man (Wilson, 1959) and animals (von Kiinig and Staffe, 1953; Gull, 1961; Kline et al., 1961; Patil et al., 1972; Swinyard and Chaube, 1973). The toxicity of chlorogenic and caffeic acids, which are 1Requestsfor reprintsshouldbe addressed to: C. A. Swinyard,M.D., Children’sHospitalat Stanford,520Willow Road,PaloAlto, California94304. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

22-l

228

CHAUBE AND SWlNYARD

present in a large number ofplants used as human food (Sondheimer, 1961) is unknown. Coumarin, whose derivatives are used clinically as potent anticoagulants, is toxic to man (Quenneville et al., 1959; Mahairas and Weingold, 1963 ; Saidi et al., 1965; Fillmore and McDevitt, 1970) and animals (Quick, 1946: Kraus et al., 1949). Ruddick et al. (1974) found that the coumarin, scopoletin, reduced litter size in rats, but was not teratogenic. Since it has been shown that the stress response of the potato to infection with an incompatible race ofPhytopht/zora infestansis a two- to threefold increase in a-solanine, a 25-fold increase in chlorogenic acid, and a IO- to 20-fold increase in coumarins (Ku& 1972), the objectives of this study are to evaluate the teratogenicity and toxicity of the alkaloidal and the phenolic compounds of Solanum TuberosumL.

METHODS

The glycoalkaloids were isolated from potato blossoms, collected by our staff, and from blossoms provided by the United States Department of Agriculture,2 by the method of Porter (1972) using the reactants in the following proportions. One thousand milliliters of 3 % acetic acid and 100 ml of concentrated NH,OH (pH 9-10) were used per 100 g of dried blossom powder to extract and precipitate the total glycoalkaloid content (TGA-extract). The precipitate was dried overnight at room temperature, redissolved in 250 ml of acetic acid, reprecipitated with 50 ml of NH,OH, digested over a steam bath, cooled overnight, and filtered. The residue was washed with 1% NH,OH and dried. The above procedure was then repeated once. The residue obtained was dissolved in 80 % ethanol (1 g/200 ml) by refluxing, filtered hot, evaporated to dryness (room temperature), suspended in 1% NH,OH (10 ml), and passed through a 50 x 4-cm column packed with silica gel and potato starch (Loev and Goodman, 1970). The column was washed with 500 ml of a 3 : 3 : 1 mixture of CHCI,-EtOH-I % NH, (Roensch and Schreiber, 1966) and fractions collected in 50-ml aliquots. The fractions were evaporated at room temperature, and the crystals obtained were redissolved in MeOH (1 mgiml). Five microliters were used for identification of the compounds by TLC. The silica gel plates (Eastman Kodak) were irrigated with 2 : 2 : I CHCI,-EtOH-I % NH, (Boll, 1962) organic phase and developed in the presence of iodine vapor (Munier, 1953). Alpha solanine and a-chaconine were identified against purified reference standards obtained from the USDA with Rf values of 0.42 and 0.50, respectively. TLC of the TGA-extract revealed three distinct spots which were identified as a-solanine, a-chaconine (the two major components) (Fig. I), and /3-chaconine (R, = 0.56). The extraction procedure yielded on the average 5 % TGA-extract, 0.33 % a-solanine, 0.67 % a-chaconine, and trace amounts of /?-chaconine (co.1 %) on a dry weight basis. Thus cc-solanine, a-chaconine, and /3-chaconine comprised about 20% of the TGAextract; the remaining 80% consisted of inorganic substances3 which were not quantitated. Chlorogenic and caffeic acids (Fig. 1) were obtained from the Sigma ChemicaI Company, St. Louis, Missouri. ’ Provided by E. A. Talley, Ph.D., Research Chemist Plant Products Laboratory, Northeastern Region Research Center, United States Department of Agriculture, Philadephia, Pennsylvania. 3 Dr. E. A. Talley, personal communication.

TOXICITY

OF POTATO

ALKALOIDS

AND

PHENOLS

229

All compounds were suspended in 0.5 % carboxymethyl cellulose (CMC) and made up fresh on each injection day. Animals. Nine-week-old female (359) and pregnant (174) Wistar rats weighing 210-230 g were obtained from Marland Farms, Hewitt, New Jersey, housed in individual cages, fed laboratory chow, and allowed water ad libitum. Pregnancy was confirmed by

ALPHA-CHACONINE R, = D-GhCOSe Rz = R3 = L-Rhamnose

ALPHA-SOLANINE RI = D-&kXtOSe RZ = D-Glucose R3 = L-Rhamnose

CAFFEIC

ACID

,

7

OH H

OOCCH=

/

CH

\

OH

-a-

H

OH

CHLORO&NIC FIG.

1. Chemical

ACID structures.

vaginal smears, and the day sperm was detected was designated Day zero. The animals were treated according to the following schedule : Acute toxicity studies. Nonpregnant female rats (204 animals) were given single ip injections of 10-100 mg/kg of cc-chaconine, 10-85 mg/kg of a-solanine, a combined dose of 5 and 10 mg/kg of a-solanine and a-chaconine, respectively; 10-60 mg/kg of TGA-extract, 400-4000 mg/kg of chlorogenic acid, 400-l 500 mg/kg of caffeic acid, or equal volumes of CMC. The animals were observed for evidence of toxicity, weighed 24 hr after injection and on alternate days thereafter for a total of 11 post-treatment days.

230

CHAUBE

AND

SWINYARD

The animals were sacrificed, an autopsy was then performed, and the tissues and organs were examined grossly and microscopically for evidence of pathological change. Chronic toxicity studies. Nonpregnant female rats (153 aminals) were given ip injections of 5-10 mg/kg of a-chaconine, cr-solanine, and TGA-extract, 60-500 mg/kg of chlorogenic acid, and 20-187.5 mg/kg of caffeic acid for 8 consecutive days; or 40 mg/kg of a-chaconine, a-solanine, and TGA-extract for 2 consecutive days. Control animals received equal volumes of CMC according to the same schedule. The animals were examined daily and killed 9 days after the last injection (total experimental period, 17 days). Teratogenic studies. One-hundred-and-sixty-eight pregnant animals received ip injections of varying amounts of a-chaconine (2.5-20 mg/kg), a-solanine (5-20 mg/kg). TGA-extract (5-20 mg/kg), chlorogenic acid (5-500 mg/kg), or caffeic acid (40-I 87.5 mg/kg) alone or in combination (5 mg/kg of cc-solanine, and 10 mg/kg of a-chaconine) on gestational Days 5-12; or 40 mg/kg or a-chaconine, a-solanine, and TGA-extract on gestational Days 5 and 6. Control animals received equal volumes of CMC according to the same schedule. The rats were observed daily for evidence of toxicity and an autopsy was performed on Day 21 of gestation. The number of resorption sites and dead fetuses were recorded. The live fetuses were removed, weighed, examined for external and visceral abnormalities, and fixed in 95 % ethanol for subsequent staining of the skeleton (Dawson, 1926). An autopsy was immediately performed on animals which died during treatment. Pieces of the liver, spleen, lung, kidney, uterus, and ovary were removed, fixed in neutral 10 y0 formalin, sectioned, stained with hematoxyline and eosin, and examined histologically. Mean LD50, 957; confidence limits, and potency ratios of the compounds were computed by the method of Litchfield and Wilcoxon (1949). Significance of differences in fetal weight and embryonic lethality between the control and experimental animals was assessedby the Student’s t test, and p values of 5 “/;,or less were regarded as significant. RESULTS Acute toxicity. Results of single ip injections of the alkaloidal and the phenolic compounds given to adult rats are summarized in Table 1. There was no lethality among the control animals. The alkaloids (a-chaconine, a-solanine, TGA-extract) did not differ significantly in their dose-related effects nor in their potency ratios. Although delayed death occurred at some doses, the majority of the animals died within 24 hr of treatment. External signs of toxicity included periorbital, nasal, and oral hemorrhage, and internally there was bloody ascitic fluid and pleuritic fluid. Chlorogenic and caffeic acids were nontoxic except at the highest doses used (4000 and 1500 mg/kg, respectively). Chronic toxicity. The results of repetitive treatment of nonpregnant rats with the alkaloidal and phenolic compounds are shown in Table 2. Lethality was only observed with 20 and 40 mg/kg of a-chaconine, and 40 mg/kg of a-solanine, and TGA-extract. External signs of toxicity were similar to those described for acute treatment. Sanguinous ascitic fluid was recovered from all animals that died, and from 47 % of those which

414’

loo

40 60 85

20

1/9d 318” 616’

0115

67.0 (52.3-85.7)

a-Solanine ___.___ Dose LD50 (mg/kg) D/T (mg/kg)

40 60

20

LD50 (m&3 60.0 5 (35.7-l 00.8) and 10O/6

3/9d 4/8d*e

O/8

Dose (mdkg) D/T

TGA-extract

a Female rats were given single ip injection and an autopsy was performed 9-18 days after treatment. b Dead/total. c 95 ok confidence limit. d Rats died 3-7 days after injection. e Rats died within 24 hr after injection.

50 75 85

84.0 (65.5-107.5) 3/11d 216’ 5/10@

o/22

LDSO bm/kg)

20

Dose @x/kg) D/Tb

a-Chaconine

4000

416”~~ 1500

518’9 c

400-2437 0,/16 400-1250 O/16 5-4000 O/30

a-Solanine and Chlorogenic Caffeic a-chaconine acid acid CMC --__. - Dose Dose Dose Dose bg/W D/T brig/kg) D/T bg/W D/T hdkd D/T

ACUTETOXICITYINTHERATOFTHEALKALOIDAL(C(-CHA~ONINE,~-SOLANINE,TGA-EXTRACT)ANDPHEN~LICCOMPOUNDS (CHLOROGENIC,CAFFEICACIDS)OFTHEPOTATO'

TABLE 1

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TOXICITY

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Chlorogenic

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O/6

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014

D/T --.__

acid

TGA-EXTRACT) POTATO'

Dose wdkddw (total) -~~

OFTHE

a-Solanine and a-chaconine ~ .-Dose w/k/day (total) D/T -_

ACIDS)

2

0 Female rats were given 8 consecutive ip injections and an autopsy was performed 9 days after the last injection. h Dead/total. c Rats died 3 days after last injection. d Rats died after 2, 5, or 7 injections. e Two consecutive injections.

(lzl)

(ix,

(6

TGA-extract ~______ ~Dose wdkgldw (total) D/T

JNTHERATOFTHEALKALOJDAL(G[-CHACONJNE,C&OLANINE, (CHLORCGENIC,CAFFEIC

a-Chaconine a-Solanine _.--. __ ~----. Dose Dose mdWdw mglkddw (total) D/T* (total) D/T ~-...-

CHRONJC

TABLE

187.5 (15W

(E)

20,40 (160, 320)

Dose w/Wdw (total)

016

016

019

D/T

Caffeic acid

ANDPHENOLICCOMPOIJNDS

S-500

Dose m/kg/day

CMC

O/38

D/T

2 6

$

z

2 $ E

Mean f-SD

5.1d 0.41

a Rats were given ip injections b Rats died after 2-7 injections. =p