Toxicology Letters, 37 (1987) 165-172 Elsevier

165

TXL 01799

ISOLATION AND PARTIAL PURIFICATION OF LANTANA (Lantana camara L.) TOXINS

(Lantana camara; hepatotoxicity; lantadene A; lantadene B; lantana toxins; guinea pigs)

O.P. SHARMA*, R.K. DAWRA and H.P.S. MAKKAR

Biochemistry Laboratory, Indian Veterinary Research Institute, Regional Station, Palampur (H.P.) 176 061 (India)

(Received 1 December 1986)

(Revision received 2 March 1987)

(Accepted 8 March 1987)

SUMMARY

A partially purified toxin fraction and lantadene A were obtained from Lantana camara L. leaves by

batch extraction, column chromatography and fractional crystallization. Toxicity was tested in guinea

pigs. The total number of chemical entities in the partially purified toxin preparation was 7, the 2 major

ones being lantadene A and lantadene B. Lantadene A was nontoxic in itself. Likewise, another fraction

containing lantadene A, lantadene B and 3 more components with higher polarity was found to be non-

toxic. The toxic component(s) are different from lantadene A/B but appear to resemble them very

closely.

INTRODUCTION

Lantana camara (common name lantana, bunchberry) is a hepatotoxic straggling shrub which causes heavy economic losses in livestock in lantana-infested localities [l-3]. Oral administration of lantana leaf powder or a tarry extract from leaves to guinea pigs elicited symptoms of lantana poisoning including photosensitization, cholestasis, hyperbilirubinemia and hepatomegaly [4,5]. Louw [6,7] isolated a triterpenoid compound, lantadene A (22&angeloyloxy-3-oxooleanolic acid) from lantana leaf powder which when given to a sheep orally (2 g) caused photosensitiza- tion and severe icterus. Brown et al. [8], however, observed that pure lantadene A

* To whom all correspondence should be addressed.

0378-4274/87/$ 03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

166

was not cholestatic and icterogenic in rabbits. Similarly, Seawright [4] found lan- tadene A to be nontoxic to guinea pigs and lambs. On the other hand, a partially purified extract of lantana leaves was found to be toxic to guinea pigs and lambs [4,9,10]. Subsequently, Hart et al. [l l] reported lantadene A to be toxic to sheep. We have previously established the suitability of the guinea pig as a laboratory animal model vis-a-vis ruminants which are field victims of lantana poisoning [ 121. In order to study the molecular mechanisms of lantana toxicity, it is imperative to understand the chemistry of lantana toxin(s). The above incongruous observations prompted us to purify and further investigate the toxic actions of lantadene A and other lantana leaf components.

MATERIALS AND METHODS

Preparation of partially purified lantana toxin(s) and Iantadene A Dry lantana leaf powder (200 g) was extracted up to the methanol/benzene parti-

tion step according to Barton et al. [ 131. We then removed the solvent by distillation in vacua and recovered the residue called fraction ‘P’ which has been found toxic to guinea pigs [lo]. Lantana toxin(s) were purified further by the following scheme:

Acetone extraction. Fraction ‘P’ from the above step was extracted with acetone. We then recovered the acetone-soluble fraction and removed the solvent in vacua. The residue (3.5 g) obtained was subjected to adsorption chromatography on silica gel and neutral alumina.

Chromatography on silica gel. Silica gel (50 g, 60-120 mesh) slurry was prepared in benzene and filled in a glass column to a bed size of 33 x 2.4 cm. The residue from the acetone fractionation step was dissolved in benzene and applied to the column. Elution was done with 2 bed volumes of benzene followed by chloroform/ethanol (98:2). Benzene elution removed some colored components while tarry polar com- ponents were not eluted at all. All the column effluent was dried in a boiling water bath followed by drying in vacua. The bulk components (1.7 g) which contained lan- tadene A as well were eluted with chloroform/ethanol (98:2). This fraction was toxic to guinea pigs (data not shown).

Chromatography on alumina, neutral (active). Neutral alumina column (80 g) slurry was prepared in chloroform and filled in a glass column to a bed size of 24 x 2.4 cm. The toxic residue from silica gel column chromatography was dissolved in chloroform and applied to the column. Elution was done with 2 bed volumes of chloroform followed by chloroform/ethanol (99:l) and methanol. The first two eluants, viz. chloroform and chloroform/ethanol (99: 1) removed some colored im- purities while the bulk components (1.2 g) were eluted with methanol. The methanol effluents contained lantadene A as well (Fig. 1). This fraction was called ‘AT’ (toxic fraction obtained by chromatography on neutral alumina) and was toxic to guinea pigs.

Fractional crystallization. A saturated solution of fraction ‘AT’ (2 g) was

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prepared in 100 ml hot methanol and left overnight at 0-4°C. Copious crystalliza- tion took place. We recovered the mother liquor and crystals separately. The crystalline part was subjected to one more crystallization step through methanol. Mother liquor was combined with the mother liquor from the first crystallization step and crystalline material was recovered. Solvent was removed from pooled mother liquor and the residue was called fraction ‘M’. Similarly, the crystalline material obtained after 2 crystallization steps was called fraction ‘C’.

Preparation of lantadene A. Fraction ‘C’ from the above was subjected to further repeated crystallization until the crystalline material obtained was chromatog- raphically pure. It was compared with a reference sample of lantadene A.

The correlation of the different fractions is shown below:

Fraction ‘M’ Lantana leaf powder-Fraction ‘AT’<

Fraction ‘C’

Lantadene A

Thin-layer chromatography Thin-layer chromatography was done on silica gel G plates all along the fractiona-

tion steps for monitoring separation and purification [14,15]. The chemical consti- tuents of fractions ‘AT’, ‘C’, ‘M’ were compared with reference lantadene A and lantadene B. The detection methods used were the Liebermann-Burchard reaction, charring by dichromate-sulfuric acid solution or primulin spray [IS, 161.

Chemicals Silica gel (60-120 mesh) was purchased from Sisco Research Laboratories, Bom-

bay, India. Neutral alumina (active) was from BDH, India. Solvents were redistilled before use. All the other reagents were of analytical grade. Reference samples of lantadene A and lantadene B were kindly provided by Prof. J.A. Lamberton, Divi- sion of Organic Chemistry, CSIRO, Melbourne, Australia.

Animals Thirty-two male guinea pigs obtained from the Indian Veterinary Research In-

stitute Stock Colony were starved overnight (water ad libitum). They were divided into 5 groups (A, B, C, D, E) at random. Animals in group A were administered orally, as a single dose, fraction ‘AT’ filled in gelatin capsules at a level of 125 mg/kg body weight. Animals in group B were similarly administered lantadene A in 3 equally divided doses on 3 consecutive days (a total of 1.2 g/kg body weight). Similarly, for testing toxicity of fraction ‘M’ (group C) and ‘C’ (group D), the animals were administered orally the respective fraction filled in gelatin capsules as a single dose at a level of 125 mg/kg body weight. The animals in the control group

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TABLE I

EFFECT OF ADMINISTRATION OF LANTADENE A AND PARTIALLY PURIFIED LANTANA

TOXIN(S) TO GUlNEA PIGS

Bilirubin values for groups B, C, D and E are expressed as mean f SD. Number of animals in each set

is given in parentheses.

Group Animal Clinical observations Bilirubin (m&100 ml)

No.

A: Fraction ‘AT’

Body weight (g)

(300-350, n=2) 1

2

(350-400, n = 6) 3

4

5

6

I

8

(400-450, n= 1) 9

B: Lantadene ‘A’

Body weight (g)

(300-350, n = 2)

(350-400, n = 1)

(400-450, n = 2)

C: Fraction ‘M’

Body weight (g)

(W-450, n = 7)

D: Fraction ‘C’

Body weight (g)

(400-450, n = 5)

Conju- Unconju- Total

gated sated

Icteric, photosensitive, moribund

after 48 h. Sacrificed

Icteric after 48 h, severely icteric,

sedated, moribund at 96 h.

Sacrificed

No symptoms until 96 h;

Severely icteric, photosensitive at

24 h. Sacrificed

Severely icteric, photosensitive at

24 h. Sacrificed

Symptoms appeared after 24 h,

severely icteric, photosensitive

at 60 h. Sacrificed

Slightly icteric at 48 h, severely

icteric, sedated at 96 h.

Sacrificed

lcteric, photosensitive at 48 h.

Sacrificed

Icteric, eyes sunken at 48 h,

severely icteric, photosensitive

at 72 h. Sacrificed

No symptoms of toxicity

No symptoms of toxicity

No symptoms of toxicity

6.36

0.53

0.10

3.40

3.05

4.10

5.40

6.27

10.42

0.14*

1.23 7.59

0.53 1.06

0.37 0.47

1.70 5.10

1.85 4.90

3.18 7.28

1.32 6.12

5.08 11.35

0.70 11.12

0.91 -I 1.05+

0.06 (5) NS 0.49 (5) NS 0.53 (5) NS

All animals sedated, severely

icteric, photosensitive within

48 h

5.24* 1.57 + 6.80+

1.06 (7)* 0.60 (7)* 1.80 (7)*

No symptoms of toxicity 0.17* 0.42 + 0.59+

0.05 (5) NS 0.06 (5) NS 0.10 (5) NS

(continued)

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TABLE I (continued) -.-.- --. Group Animal Clinical observations

No.

_. Bilirubin (mg/lOO ml)

Conju- Unconju- Total rrated Eated

E: None

Body weight (g) (300-350, n = 3) (400-450, n = 3)

Normal control animals 0.15* 0.65 f 0.84 + Normal control animals 0.13 (6) 0.39 (6) 0.44 (6)

*P<O.Of; NS, not significant.

(group E) were administered empty gelatin capsules. The animals in the fraction ‘AT’ and fraction “M’ groups became icteric, sedated, photosensitive and moribund within 24-96 h and were sacrificed. Blood was collected in heparinized vessels for preparation of plasma. The postmortem findings were compared with our previous observations to establish lantana intoxication IS]. The animals in group B and group D exhibited no symptoms of lantana toxicity [5] until the 4th day and were killed by decapitation along with the animals in the control group for the collection of blood samples and postmortem examination for toxicity. Plasma bilirubin was determined by the diazo reaction according to Malloy and Evelyn [17].

RESULTS AND DISCUSSION

The effect of oral administration of partially purified lantana toxin(s), viz. frac- tion ‘AT’, fraction ‘M’, fraction “C’ and lantadene A to guinea pigs is shown in Table I. Out of a total of 9 animals in group A, 3 became icteric and photosensitive at 24 h, 5 at 48 h and the remaining one animal did not exhibit any symptom until 96 h after the administration of fraction ‘AT’. Animals in all the 3 body weight groups were susceptible to the action of partially purified lantana toxin(s). Seven out of 8 intoxicated animals in group A had elevated bilirubin levels (5-l 1 mg/lOO ml). Animal No. 2 was icteric, sedated and moribund when sacrificed and the blood ex- amined for bilirubin content. However, its bilirubin content was in the normal range. This might be seen in light of the earlier observation that icterus and hyper- bilirubinemia do not always coincide in time [IS]. Animal No. 3 was agile until 96 h of observation and exhibited no clinical symptoms associated with lantana poison- ing [S]. The plasma bilirubin content in animal No. 3 was also in the normal range. Most of the bilirubin in the poisoned animals of group A was of the conjugated type, which agrees with our previous observations wherein lantana leaf powder had been used for toxicity studies [5, 121 and is indicative of obstructive jaundice 1181. Thus, the partially purified lantana toxin fraction obtained (fraction ‘AT’) simulates the toxic action induced in guinea pigs by lantana leaf powder. The animals in group B remained agile during experimental period of 96 h and exhibited no clinical symptoms characteristic of lantana toxicity [5]. There was no significant

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difference in bilirubin levels in animals of groups B and E, but the group average in the lantadene A group tended to be slightly higher. A thin-layer chromatography separation profile of ‘AT’ and lantadene A is shown in Fig. 1. In the ‘AT’ fraction a total of 4 chemical constituents could be detected, of which lantadene A and lan- tadene B were the major ones.

All the animals in the fraction ‘M’ group became sedated, icteric and photosen- sitive within 48 h after administration of the toxin preparation. The bilirubin levels, both conjugated and unconjugated, were significantly higher than the control values. Moreover, most of the increased bilirubin was of the conjugated form. On

SF.

Fig. 1. Diagrammatic sketch of thin-layer chromatogram of the toxin fraction ‘AT’ obtained after ad- sorption chromatography on neutral alumina (active). LA, lantadene A; sample size, 40 pg; solvent system, benzene/methanol/ethyl acetate (85:10:5); S.F., solvent front.

-2 nd -2nd

1 V5 ‘a 9@6

:t’ :!3’2

Z

t_ FractioriM’ B .

, ? Fmction’c’ I Fig. 2. Diagrammatic sketch of two-way thin-layer chromatogram of fractions ‘M’ and ‘C’. First direc- tion: benzene/methanol/ethyl acetate (85:lO:S). Second direction: petroleum ether (60~EO’C)/ benzene/ethyl acetate/acetic acid (40:80:28:2). Sample size, 40 pg.

the other hand, none of the animals in the fraction ‘C’ group (group D) exhibited any clinical symptoms of lantana toxicity and their bilirubin level was within the normal range. The comparative two-way thin-layer chromatographic profiles of fractions ‘M’ and ‘C’ are shown in Fig. 2. Two major components in both the frac- tions were lantadene A (compound 1) and lantadene B (compound 2). Fraction ‘C’ was richer in lantadene A but fraction ‘M’ was richer in lantadene B. Similarly, both the fractions contained components 3, 4 and 7 (Fig. 2) which were more polar than both lantadene A and B. Fraction ‘C’ was richer in component 3. The toxic fraction ‘M’ contained components 5 and 6 as well, which gave an intense pink fluorescence when the thin-layer plates sprayed with Liebermann-Burchard reagent were kept at 110°C for 5 min and observed at 366 nm. However, components 5 and 6 were not detectable in the nontoxic fraction ‘C’. The components 5 and 6 do not become resolved in the solvent system benzene/methanol/ethyl acetate (85: 10:5), but are un- masked in the solvent system petroleum ether (60-BO”C)/benzene/ethyl acetate/acetic acid (40:80:28:2) (Figs. 1 and 2).

Our observations as regards the nontoxic action of lantadene A agree with those of Seawright [4] wherein pure crystalline lantadene A was found to be nontoxic to guinea pigs and lambs, but they are incongruous with those of Louw [6] and Seawright and Hrdlicka [19] who observed lantana poisoning symptoms upon oral administration of lantadene A to lambs. Brown et al. [B] explained that the reported toxicity of lantadene A [6,19] was due to some minuscule impurities. However, later workers [ 1 I] again reported toxicity of lantadene A. Our studies corroborate the observations of Brown et al. [B] that pure lantadene A is not cholestatic and icterogenic when administered alone to animals. The toxic action seems to be con- fined to components 5 and/or 6 of the toxic fraction ‘M’ (Table I and Fig. 2). In view of their polarity and chromatographic mobility, components 5 and/or 6 can be appropriately described as ‘nonidentical twins vis-a-vis lantadene A and B’.

ACKNOWLEDGEMENTS

The authors are grateful to the Director, Indian Veterinary Research Institute for facilities. We thank the Scientist In Charge of the Research Centre for helpful discussions.

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