hepatotoxicity and cholestasis in rats induced by the sesquiterpene,...

J BIOCHEM MOLECULAR TOXICOLOGYVolume 15, Number 5, 2001

Hepatotoxicity and Cholestasis in Rats Induced by theSesquiterpene, 9-oxo-10,11-Dehydroageraphorone,Isolated from Eupatorium adenophorumRenu Bhardwaj,2 Ajay Singh,2 Om P. Sharma,1 Rajinder K. Dawra,1 Nitin P. Kurade,1

and Shashi B. Mahato3

1Biochemistry Laboratory, Indian Veterinary Research Institute, Regional Station, Palampur 176 061, HP, India; E-mail: [email protected] of Chemistry and Biochemistry, Himachal Pradesh Krishi Vishvavidyalaya, Palampur 176 062, HP, India3Indian Institute of Chemical Biology, Jadavpur, Calcutta 700 032, India

Received 27 March 2001; revised 26 June 2001; accepted 7 July 2001

ABSTRACT: Eupatorium adenophorum leaves causehepatotoxicity and cholestasis in rats. The hep-atotoxicant has been characterized as 9-oxo-10,11-dehydroageraphorone (ODA), a cadinene sesquiter-pene. Oral administration of ODA, mixed in feed torats, caused jaundice in 24 h. The liver of the intoxicatedanimals had focal areas of hepatocellular necrosis, pro-liferation, and dilation of bile ducts with degenerativechanges in the lining epithelium. There was markedincrease in the conjugated form of plasma bilirubinand in the activities of the enzymes glutamate ox-aloacetate transaminase, glutamate pyruvate transam-inase, alkaline phosphatase, lactate dehydrogenase,g-glutamyltranspeptidase, glutamate dehydrogenase,and 5′-nucleotidase. The histopathological lesions inliver and biochemical profile of marker enzymes showthat ODA induced hepatotoxicity and cholestasis inrats. This is the first report on the toxicity of a cadinenesesquiterpene in rats. C© 2001 John Wiley & Sons, Inc. JBiochem Mol Toxicol 15:279–286, 2001

KEYWORDS: Eupatorium adenophorum; Hepatotoxici-ty; Hepatotoxicant; Sesquiterpene; 9-oxo-10,11-Dehyd-roageraphorone

INTRODUCTION

Eupatorium adenophorum (syn. Ageratina adenophora,common name: Crofton Weed), a perennial weed, is na-tive to Mexico [1]. Now, the plant has encroached uponvast expanse of pastures in Australia, New Zealand,Hawaii, the Philippines, sub-Himalayan region ofIndia, Nepal, and China [1–3]. There is a lot of vari-ation in the susceptibility of different animal species to

Correspondence to: Om P. Sharma.c© 2001 John Wiley & Sons, Inc.

the noxiousness of E. adenophorum [1,4]. The plant fo-liage causes pulmonary toxicity in horses in Australia[4–6]. Mice administered freeze-dried E. adenophorumleaf powder had hepatic injury [7]. Experimental feed-ing of E. adenophorum samples from sub-Himalayan re-gion in India caused anorexia and photosensitizationin cattle [8]. E. adenophorum leaves and flowers col-lected from Numinbah Valley (Australia) fed to rats for52 days did not elicit any toxicity symptoms [4]. Con-trariwise, E. adenophorum leaf samples collected fromKangra Valley (India) and partially purified extractsfrom leaf samples mixed in the diet caused hepatotox-icity and cholestasis in rats [9,10]. We report here theisolation and chemical characterization of the hepato-toxicant and describe its hepatotoxicity in rats.

MATERIALS AND METHODS

Chemicals

Silica gel (60–120 mesh) for column chromatog-raphy, heparin (disodium salt), NADH (disodiumsalt), and 2-oxoglutaric acid were from Sisco ResearchLaboratories, Mumbai. Silica gel G was from E. Merck(India). L-g -glutamyl-p-nitroanilide was purchasedfrom Sigma Chemical Co., St. Louis, U.S.A. All otherchemicals were of analytical grade. The solvents forHPLC were of HPLC grade and were from S.D. FineChemicals, Mumbai, India.

Leaf Samples

Leaf samples of E . adenophorum were collected fromthe vicinity of Indian Veterinary Research Institute,Regional Station, Palampur. The samples were dried

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280 BHARDWAJ ET AL. Volume 15, Number 5, 2001

in a hot-air oven at 60◦C and a fine powder wasprepared. The powder was used for extraction of thehepatotoxicant.

Isolation and Purificationof E. adenophorum Toxin

Figure 1 shows the flow sheet of bioactivity-guidedpurification of the E. adenophorum hepatotoxicant. Ex-tracts and fractions at different steps were subjectedto TLC and bioassay in rats [9]. A comparison of theresults on bioassay and TLC revealed that the extractsand fractions positive for hepatotoxicity gave pink spoton detection with vanillin-sulfuric acid spray reagent.This helped tracking down the hepatotoxicant.

E. adenophorum leaf powder (100 g) was extractedwith 3 × 500 mL of methanol. The extract was mixedwith activated charcoal (25 g) and was left overnight.The mixture was filtered and the golden-yellow filtratewas dried at 40◦C in vacuo. The residue was mixedwith water (100 mL) and partitioned with ethyl acetate(3× 100 mL). The ethyl acetate layers were combinedand dried at 40◦C in vacuo. The residue was subjectedto column chromatography.

FIGURE 1. Flow sheet for the purification and characterization ofE. adenophorum hepatotoxicant.

Column Chromatography

The residue from the preceding step was dissolvedin dichloromethane (20 mL) and mixed with silica gel(20 g, 60–120 mesh). The solvent was removed in vacuoand the sample adsorbed on silica gel was loaded onsilica gel column (50 g, 60–120 mesh). Elution wasdone with chloroform (50 mL) followed by chloro-form/methanol (99:1) (775 mL). Fraction volume was25 mL. Aliquots (0.5 mL) of the fractions were dried un-der a stream of nitrogen and the residue was dissolvedin 2 mL of HPLC grade methanol. Aliquots (20 mL) weretaken for HPLC analysis.

The partially purified fractions (fractions 28–32,positive for the hepatotoxicant) from the first chromato-graphic step were pooled, the solvent was removedin vacuo at 40◦C. The residue was dissolved in 1 mLof chloroform and was applied on the silica gel col-umn (15 g, 60–120 mesh). The elution was done usingchloroform. Fraction volume was 10 mL. Elution of thehepatotoxicant started from fraction 59 (elution volume590 mL) onwards and continued till fraction 115 (elu-tion volume 1150 mL) which was pure as ascertainedby HPLC analysis. The fractions containing the hepa-totoxicant with 98% or above purity were pooled.

Thin Layer Chromatography

TLC was done on glass plates using silica gel G [11].The solvent system was benzene/chloroform (1:1). Thedetection was done by vanillin-sulfuric acid reagent[11]. The spray reagent was prepared by dissolvingwith cooling 0.5 g vanillin in 100 mL of H2SO4/ethanol(40:10). After spraying, the plates were heated at 110◦C.

High Performance Liquid Chromatography

HPLC was done using the Waters HPLC unit witha binary system of 510 and 515 solvent delivery sys-tems used in the isocratic mode, Novapak C18 (4m,4.6× 250 mm) column, Rheodyne injector, 490E multi-channel detector, and Millennium 2010 Chromatogra-phy Manager. Aliquots (0.5 mL) of the fractions underinvestigation were dried in a stream of nitrogen andredissolved in HPLC grade methanol. A 20 mL aliquotwas injected. The mobile phase was acetonitrile/water(40:60) at a flow rate of 1 mL/min. The monitoring wasdone at 240 nm and 280 nm.

Spectroscopy

For UV spectroscopy, a methanolic solution of thepure toxin (20 mg/mL in HPLC grade methanol) wasused. Spectrum was taken using Hitachi spectropho-tometer, model 150-20. The NMR spectra of the toxin

Volume 15, Number 5, 2001 HEPATOTOXICITY OF 9-OXO-10,11-DEHYDROAGERAPHORONE IN RATS 281

were recorded on a Bruker 300 DPX spectrometerusing TMS (tetramethylsilane) as internal standard.

Bioassays

Male Wistar rats (180± 10 g) were used for thebioassays during bioactivity-guided purification ofthe hepatotoxicant [9,10]. The animals were kept inmetabolic cages and were fasted overnight with waterprovided ad libitum. A portion of the sample for bioas-say dissolved in dichloromethane was mixed with ratfeed. The solvent was removed at 40◦C in vacuo. Thecontrol feed was processed similarly without the sam-ple of the hepatotoxicant. The animals in the test andcontrol group were offered the feed, with and with-out sample to be tested, respectively. For the bioassayof the pure putative toxin, the amount of pure toxinin the feed offered to animals in the test group wasequivalent to the dose of 200 mg/kg body weight. Eachanimal was offered 20 g feed. The animals were mon-itored for feed intake and general condition. The earpinnae of the animals in the test group were distinctlyyellow when observed at 24 h after start of feeding thediet containing the toxin. At this stage animals in boththe test and control group were euthanized, using di-ethyl ether. Blood was collected from the heart in hep-arinized vessels for the preparation of blood plasma[12]. Liver samples were collected for histopathologicalexamination [13]. Bilirubin was estimated in bloodplasma by the method of Malloy and Evelyn [14].Alkaline phosphatase (AP) (EC 3.1.3.1) was assayedby the method of Walter and Schuett [15]. Glutamateoxaloacetate transaminase (GOT) (EC 2.6.1.1) and glu-tamate pyruvate transaminase (GPT) (EC 2.6.1.2) wereassayed by the colorimetric methods [16]. Glutamatedehydrogenase (GLDH) (EC 1.4.1.2) was assayed bythe method of King [17]. Lactate dehydrogenase was as-sayed by the spectrophotometric method of Bergmeyerand Bernt [18]. g -Glutamyltranspeptidase (g -GT) (EC2.3.2.2) was assayed by the method of Szasz [19]. 5′-Nucleotidase (EC 3.1.3.5) was assayed by the methodof Gerlach and Hilby [20].

The data were compared by the Student’s t-testusing Sigmastat software (Jandel Scientific, USA).

RESULTS

A flow sheet of the bioactivity-guided purifica-tion protocol is given in Figure 1. The E. adenopho-rum leaves sample was extracted with methanol. Themethanolic extract after decolorization with activatedcharcoal was golden yellow. The decolorized samplewas dried in vacuo and dissolved in dichloromethane

for purification by column chromatography using silicagel. During elution different fractions were subjectedto TLC and pooled up on the basis of similarity of con-stituents. A TLC profile of pooled fractions is shown inFigure 2. A comparison of the TLC profile and bioas-says showed that the hepatotoxicity was due to the con-stituent that gave pink spot on spraying with vanillin-sulfuric acid and heating at 110◦C for 5 min. On furtherkeeping the sprayed plates at 110◦C for 15 min, the spotfor hepatotoxicant became steel grey in color. Fractions28–32 induced hepatotoxicity and gave single spot onTLC. An HPLC profile of fractions 28–32 is shown inFigure 3a. The major constituents in fractions 28–32 hada retention time of 12.7 min. In addition compounds Xand Y were present as minor constituents (Figure 3a).Compounds X and Y could not be resolved from themain constituent using TLC (Figure 2) and the puritycould be checked only by HPLC.

The compounds X and Y essentially coeluted withthe putative hepatotoxicant in the first column chro-matography step. So, the fractions with minimum im-purities and positive for the hepatotoxicity (fractions28–32) were dried in vacuo. The residue was subjectedto rechromatography. The elution was done using chlo-roform. After monitoring each fraction by TLC, puritywas ascertained by HPLC. It was observed that eluantchloroform resolved the compounds X and Y. Therewas a decrease in the amount of compound X withthe increase in fraction number. The fractions 105 on-wards (elution volume 1050 mL) had >98% purity forthe putative hepatotoxicant and were combined. Thesolvent was removed in vacuo. The sample obtainedwas viscous oil at room temperature. The pure sampleof the toxin was used for spectroscopic analysis andbioassays. An HPLC profile of pure the hepatotoxicantis shown in Figure 3b, c. During HPLC analysis, the

FIGURE 2. Diagrammatic sketch of the thin layer chromatogramof column chromayography fractions obtained for purifaction ofE. adenophorum hepatotoxicant. Solvent system: benzene/chloroform(1:1).


FIGURE 3. (a) HPLC profile of fractions (pooled) 28–30, obtainedafter column chromayography. The chromatogram shows the impu-rity of compounds X, Y, and other minor components. (b and c) HPLCprofile of pure hepatotoxicant (0.2 mg/mL in HPLC garde methanol)of E. adenophorum. Mobile phase: acetonitrile:water (40:60); injectionvolume: 20 mL; flow rate: 1 mL/min; detection: (a) 240 nm, (b) 240nm, and (c) 280 nm.

FIGURE 4. Proton NMR spectrum of E. adenophorum hepatotoxicant.(a) Normal form and (b) expanded form.

detector was set at 240 and 280 nm simultaneously.The compound had higher absorbance at 240 nm ascompared to 280 nm (Figures 3b and 3c). The usualimpurities of compound X and Y could be detectedonly at 240 nm and not at 280 nm. The channel ratio ofA240/A280 for the hepatotoxicant was 6.1.

The hepatotoxicant had a UVλmax at 247 nm and themolar extinction coefficient was 12,309. The 1H NMRspectrum (Figure 4) helped to identify an olefinic me-thine proton at δ 6.17, three olefinic methyls at 1.95 s,1.83 s, 1.66 s, and a secondary methyl at δ 0.97 dd. As-signment of the 13C NMR δ values (Table 1, Figure 5)were made with the help of distortionless enhance-ment by polarization transfer (DEPT) studies (Figures6a and 6b) and known chemical shifts rules [21,22].Thus, the hepatotoxicant was identified as 9-oxo-10,11-dehydroageraphorone (Figure 7), abbreviated as ODA,previously isolated by Bohlmann and Gupta [23]. It hasa molecular formula of C15H20O2 and molecular massof 232.

ODA was administered to rats as mixed in feed. Theanimals were examined at 24 h after the start of feed-ing. The test group animals were icteric, dull, and haircoat appeared rough and erect. The ear pinnae were yel-low. The urine was also yellow. The animals of both thegroups were euthanized using diethyl ether. Necropsyexamination of rats revealed yellowish discolorationof subcutaneous tissue and abdominal muscles in thetreated rats. The stomach revealed more retention offood in the treated rats as compared to the control an-imals. Liver in some of the treated rats revealed smallnecrotic foci throughout the parenchyma. Caecal con-tents were scanty in the treated rats. Histopathologi-cal examination of various organs except liver did notreveal any significant microscopic changes. The liversections of the treated rats revealed presence of small

TABLE 1. 13C NMR Chemical Shifts δ (± 0.1) of E. adenopho-rum Hepatotoxicant

Carbon No. Chemical Shift

1 40.52 197.03 135.04 145.55 41.96 42.77 28.28 49.59 202.0

10 133.211 143.012 22.213 21.914 19.115 14.6


FIGURE 5. 13C NMR spectrum of E. adenophorum hepatotoxicant.

focal areas of hepatocellular necrosis, proliferation, anddilation of bile ducts with degenerative changes in thelining epithelium (Figure 8). Plasma of the test groupanimals was yellow. It was used for the estimation ofbilirubin content and assay of AP, GOT, GPT, LDH,

FIGURE 6. (a) Dept90 and (b) Dept135 of E. adenophorum hepatotoxicant.

GLDH, g -GT, and 5′-nucleotidase. There was a markedincrease in conjugated form of bilirubin (Table 2). Theactivities of plasma enzymes AP, GOT, GPT, GLDH,g -GT, and 5′-nucleotidase were markedly increased(Table 2).


FIGURE 7. Chemical structure of E. adenophorum hepatotoxicant.

DISCUSSION

E. adenophorum hepatotoxicant has been identifiedas ODA, which is a cadinene sesquiterpene (Figure 7).This compound has been reported earlier by Bohlmanand Gupta [23] from the aerial parts of E. adenophorum.The compound in the E. adenophorum leaves samplescollected from Brookfield area near Brisbane, Queens-land, Australia, which induced hepatotoxicity in micewas also identified as ODA [3]. ODA was the majorconstituent in the mature leaves of E. adenophorum [24].ODA exhibited contact toxicity and growth retardingactivity against larvae of a noctuid species [24]. Duringpurification of E. adenophorum hepatotoxin novel HPLCprotocols have been developed, which provided baseline separation of the hepatotoxicant from common

FIGURE 8. Photomicrographs of liver (H.&E.). (a) Control (Magnifiaction: 400×). (b) For the group administered ODA. The liver showingproliferation (1) and dilatation (2) of bile ducts and focal areas of hepatocyte necrosis (3) (Magnification: 200×).

TABLE 2. Effect of ODA Toxicity on Plasma Bilirubin Levelsand the Activity of Plasma Enzymes in Blood Plasma of Rats

Control Test

Bilirubin (mg/100 mL plasma)Conjugated 0.21 ± 0.07 (8) 4.45 ± 1.6* (8)Unconjugated 0.37 ± 0.2 (8) 0.86 ± 0.3*** (8)Total 0.58 ± 0.1 (8) 5.31 ± 1.8* (8)

AP (U/L) 124.8 ± 36.8 (6) 356.1 ± 139.6** (7)GOT (U/L) 52.0 ± 11.1 (7) 103.7 ± 8.07* (7)GPT (U/L) 14.2 ± 5.2 (5) 81.0 ± 17.0* (6)Lactate dehydrogenase (U/L) 75.3 ± 21.5 (6) 645.6 ± 188.0* (6)GLDH (U/L) 2.09 ± 0.6 (6) 31.22 ± 3.5* (6)g -GT (U/L) 9.98 ± 5.0 (6) 86.79 ± 22.8* (6)5′-Nucleotidase (U/L) 1.32 ± 0.6 (6) 19.73 ± 0.4* (6)

Values are mean ± SD. The number of animals in each group is given inparentheses.

*P < 0.001, **P < 0.005, ***P < 0.01.

impurities like compounds X and Y (Figure 3). Simi-larly, the TLC protocol provided good separation of thehepatotoxicant from a large number of compounds ex-cept compounds X and Y (Figure 2). The detection usingvanillin-sulfuric acid spray with observation at 5 and15 min heating at 110◦C, also appeared fairly specificfor the hepatotoxicant.

The clinical picture of the animals in the test groupafter administration of ODA was similar to that ob-served earlier, when E. adenophorum leaf powder or


partially purified toxin was administered [9,10]. Theanimals in the test group exhibited typical symptomsof jaundice and photosensitization, when observed at24 h after ingestion of the experimental feed. Oelrichset al. [3] observed hepatotoxicity in mice on oral ad-ministration of ODA but they did not record jaundiceor cholestasis. Histopathological examination of livershowed lesions that are consistent with cholestasis andliver injury [25]. Earlier, hepatotoxicity has been ob-served in mice and rats on administration of freeze-dried E. adenophorum leaf powder [7,9]. There was amarked increase in the plasma bilirubin level, particu-larly of the conjugated type (Table 2). Increase in plasmaconjugated bilirubin level is a marker of cholestasis[26]. A similar increase in bilirubin level has been ob-served during hepatotoxicity and cholestasis in guineapigs induced by lantana toxins [27,28]. Elevated lev-els of LDH imply tissue damage and hepatic injury[29]. There was an increase in the activities of AP, GOT,and GPT in the animals of the test group (Table 2). Acomparable increase in these enzymes was observedin hepatotoxicity and cholestasis in rats induced by E.adenophorum leaf powder [9]. An increased g -GT ac-tivity implies cholestasis [26,30]. Similarly, elevation inthe activity of GLDH shows hepatic necrosis and ob-structive jaundice [29,30]. Increased 5′-nucleotidase ac-tivity indicates diseases of hepatobiliary system, e.g.cholestasis, biliary inflammation, and biliary obstruc-tion [20,29]. An increase in the activities of g -GT andGLDH has been observed in cholestasis in guinea pigson administration of the hepatotoxicant lantadene A[28]. In conclusion, the histopathological studies andalterations in biochemical parameters showed that E.adenophorum compound ODA induced cholestasis andhepatotoxicity in rats.

ACKNOWLEDGMENTS

This work was funded by the Indian VeterinaryResearch Institute. We thank the director of theinstitute for the facilities and the scientist in charge,Dr. B. Singh, for helpful discussions.

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hepatotoxicity and cholestasis in rats induced by the sesquiterpene,...

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