Oxidation chemistry and biochemistry of indole and effect of its oxidation product in albino mice

Bioelectrochemistry and Bioenergetics 45 Ž1998. 47–53

Oxidation chemistry and biochemistry of indole and effect of its oxidation product in albino mice Rajendra N. Goyal ) , Neeraj Kumar, Naveen K. Singhal Department of Chemistry, UniÕersity of Roorkee, Roorkee-247 667, India Received 7 October 1997; revised 3 December 1997; accepted 3 December 1997

Abstract Electrochemical oxidation of Indole has been studied in phosphate buffers in the pH range 2.0–10.6 at pyrolytic graphite electrode. Below pH 6.0, the oxidation of Indole occurred in a single 1e step. The nature of the electrode reaction was found as EC in which charge transfer is followed by irreversible chemical step. A UV absorbing intermediate was noticed at around 235 nm which decayed in a pseudo first order reaction to give trimer as the major product. The trimer was characterized by m.p., 1 H NMR and mass spectrum and a tentative mechanism for its formation is also suggested. A comparison of toxicity of Indole and the oxidation product in albino mice was made by analyzing blood parameters after intracranial injection. The studies clearly suggested that the trimer is more toxic in comparison to Indole. q 1998 Elsevier Science S.A. Keywords: Indole; Electrooxidation; Toxicity

1. Introduction The biological and clinical significance of Indole and its derivatives have been widely reported in literature w1x. The electrochemical and enzymic oxidation of various Indole derivatives has attracted considerable attention in recent years due to reports of decrease in metabolites concentration of 5-hydroxytryptamine in Alzheimer’s patients w2,3x. Indole is one of the major products formed in marine organisms by the bacterial decomposition of proteins w4x. The electrochemical oxidation of Indole has been studied previously to some extent, however, most of the investigations were carried out in non-aqueous media. Haque w5x reported oxidation of Indole in acetonitrile and the formation of thin film at the electrode surface is reported. Several other workers also studied anodic oxidation of Indole and the formation of a black polymeric material is suggested w6–9x. In all these reports the product has not been characterised and results in non-aqueous media probably give little useful information concerning the biological oxidation of Indole. As Indole derivatives have been

)

Corresponding author.

0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 6 6 - X

found in central nervous system and claimed responsible for various physiological activities of human system, it was considered interesting to study the oxidation behaviour of simple Indole ŽI. for which not a single product of oxidation is known. The purpose of the present work was to study electrooxidation of Indole with the expectation that the results obtained may provide deep insights into the redox chemistry of naturally occurring Indoles. The electrochemical studies coupled with spectral techniques have been found useful in probing the oxidation chemistry and biochemistry of various biologically significant molecules w10,11x and hence are used in the present investigations. Effect of intracranial injection of a single dose of Indole and its oxidation product in albino mice indicated that the oxidation product Žtrimer. is much more toxic in comparison to starting compound.

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2. Experimental

Indole was obtained from Sigma, USA and was used as received. All experiments were carried out in phosphate buffers w12x of ionic strength 0.1 M and were prepared by mixing different volumes of NaH 2 PO4 P 2H 2 O Ž0.5 M.; Na 2 HPO4 P 2H 2 O Ž0.5 M. and Na 3 PO4 P 12H 2 O Ž0.05 M.. The equipments used for linear and cyclic sweep voltammetry, coulometry and controlled potential electrolysis were essentially same as have been reported earlier w13,14x. Pyrolytic graphite electrode Žarea ; 0.04 cm2 . was prepared in the laboratory by the method reported earlier w15x and was used as working electrode. Platinum wire and SCE were used as auxiliary and reference electrodes, respectively. Controlled potential electrolysis was carried out in a conventional H-cell using a home-made potentiostat. The pH during CPE showed a variation of about 0.2 pH and hence was maintained by adding drops of dilute NaOH solution. The value of n, number of electrons involved in the oxidation, were determined by connecting a coulometer in the series. Spectral changes during electrolysis were monitored in a 1.0 cm quartz cell using Beckman DU-6 spectrophotometer. For this purpose solution of Indole in a buffer of desired pH was oxidised at a potential 100 mV more positive than peak potential and the UV spectra were recorded by transferring small volume in 1 cm quartz cell at different time intervals. The kinetics of decay of UV-absorbing intermediate was monitored by turning off the potential when absorbance at the lmax 270 nm reached to ; 50%. The change in absorbances at selected wavelengths with time were then monitored. The stock solution of Indole Ž2 mM. was prepared in double distilled water. For recording voltammograms, 2.0 ml of the stock solution was mixed with 2.0 ml of buffer of desired pH. Nitrogen gas was passed for 8–10 min before recording the voltammograms. For the identification of oxidation products, about 10–12 mg of the Indole was oxidised at a large PGE plate Ž; 6 = 1 cm2 . in the buffer of desired pH at potential 100 mV more positive than peak I a . Nitrogen gas was continuously bubbled during electrolysis. The progress of electrolysis was monitored by recording cyclic voltammograms at different time intervals. When peak I a completely disappeared, the exhaustively electrolysed solution was removed from the cell and lyophilized. The brown coloured material obtained exhibited a single spot in TLC Ž R f ; 0.65; benzene–methanol 9:1.. The product was extracted with solvent ether Ž3 = 10 ml., lyophilized and the dark brown coloured material was characterized. The mass spectrum was recorded using a Jeol JMSD 300 mass spectrometer and the 1 H NMR was recorded using FX-100 Jeol NMR spectrometer. FT-IR spectrum of the product was recorded as KBr pellets using Perkin Elmer IR spectrophotometer.

3. Biological studies Male albino mice Ž25 " 2 g. obtained from the animal house of Indian Drugs and Pharmaceuticals, Rishikesh were used for the biochemical studies. The animals were housed five per cage and allowed access to food and water ad libitum. No animals were used in the studies until at least 7 days, following receipt from the supplier. Intracranial injections were made using 10 m l syringe ŽHamilton, USA. and a teflon stopper was placed on the needle so that the insertion depth in the left ventricle portion remained constant Ž4 mm. in all the injections. The toxicity of Indole ŽI. and trimer ŽVI. were initially determined by the LD50 values by the method of Dixon w16x. The compounds I and VI were administered in 5 m l of the isotonic saline Ž0.9% NaCl. containing 1 mgrml ascorbic acid and the effect of these compounds on blood parameters were determined after 24 h, 72 h and 1 week following administration. The animals were sacrificed at fixed times and the blood was collected. The blood with EDTA as anti-coagulant was used for the determination of Hb, Ht, RBC, WBC, ESR, BUN by well known methods of Oser w17x and Marsh et al. w18x. The uncoagulated blood was used to determine serum glucose, cholesterol, AST, ALT, alkaline phosphatase, acid phosphatase by the standard methods w17–19x. The control animals were treated with only isotonic saline containing 1 mgrml ascorbic acid under otherwise identical conditions.

4. Results and discussion Linear sweep voltammetry of Indole at sweep rate of 10 mV sy1 exhibited a well defined anodic peak ŽI a . in the pH range 2.0–10.5. The peak potential of the peak I a was practically constant in the pH range 2.0–6.0 and shifted to less positive potential with increase in pH ŽFig. 1.. The

Fig. 1. Variation of Ep with pH for electrooxidation peak I a of 1 mM Indole at the PGE. Sweep rate 10 mV sy1 .

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Fig. 4. Variation of the peak current function Ž i p r6Õ . with the logarithm of the voltage sweep for 1 mM Indole at pH 6.0 at PGE.

Fig. 2. Typical cyclic voltammograms observed for 1 mM Indole at PGE ŽA. pH 6.0; ŽB. pH 9.0 at a sweep rate of 100 mV sy1 .

linear dependence of the peak potential Ž Ep . on pH in the pH range 6.0–10.5 can be represented by the relation: Ep Ž pH 6.0–10.5 . s w 1.0 y 0.045 pH x V vs. SCE In cyclic sweep voltammetry compound ŽI. exhibited a sharp anodic peak I a when the sweep was initiated in the positive direction. In the reverse sweep, two overlapping cathodic peaks II c and III c were observed in the pH range 2.0–6.0. The Ep of the two peaks was so close that most of the times, they were merged to a single peak. At pH ) 6.0, the two reduction peaks clearly merged to a single peak. The peak potentials of peaks II c and III c were also dependent on pH and shifted to more negative potential with increase in pH. When the direction of sweep was further reversed, peaks II a and III a were observed, which formed quasi reversible couples with peaks II c and III c in the pH range 2.0–6.0. Some typical cyclic voltammograms of Indole are presented in Fig. 2.

The peak current for the oxidation peak ŽI a . was more or less constant in the entire pH range studied. However, the i p increased with increase in concentration of Indole. Fig. 3 presents the plot of peak current values observed at different concentrations of Indole. It was found that the peak current increased linearly up to about 2 mM concentration and attained more or less constant value at higher concentrations. This behaviour indicated adsorption of Indole at the electrode surface w20x. The adsorption complications were further confirmed by the dependence of peak current Ž i p . on sweep rate. The plot of i pr 'Õ vs. log Õ ŽFig. 4. indicated an increase in the peak current with the increase in sweep rate confirming the strong adsorption of the reactant at the surface of pyrolytic graphite electrode w21x. The ratios of peaks II crII a and III crIII a were found to be approximately 0.9 and 0.8 in the entire pH range and did not change significantly with increase in concentration of compound I. However, at concentrations ) 0.5 mM, peaks II c and III c merged even in the pH range 2.0–6.0. The Ep of peak I a was found to shift to more positive potentials with increase in sweep rate in the range 10 mV sy1 to 1000 mV sy1 . In the sweep range 10–100 mV sy1 the shift in Ep was 20–30 mV per 10-fold increase in the sweep range whereas the shift decreased to 8–10 mV at higher sweep rates. The plot of D Epr2rDlog Õ vs. log Õ

Table 1 Coulometric n-values observed for the electrooxidation of Indole at different pH at PGE

Fig. 3. Peak current versus concentration behavior for the voltammetric oxidation of Indole obtained at a PGE at sweep rate 100 mV sy1 at pH 6.0.

pH

Pot. mV vs. SCE

Exp.a n value

2.0 3.2 5.0 7.0 8.0 9.5 10.6

800 800 800 800 800 700 700

0.90 0.92 1.06 1.04 0.90 0.98 1.06

a

Average of at least three replicate determinations.

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R.N. Goyal et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 47–53

Fig. 5. Cyclic voltammetric changes observed during electrolysis of 1 mM Indole at pH 6.0. Curves were recorded at ŽA. 0 min ŽB. 15 min ŽC. 60 min and ŽD. 180 min of electrolysis.

was s- shaped and hence suggested the nature of the electrode reaction as EC in which charge transfer is followed by irreversible chemical reactions w22x. The value of ‘n’ number of electrons involved in the oxidation was determined using a coulometer and was found to be 1.0 " 0.1 in the entire pH range studied. The values of n obtained at different pH are presented in Table 1. Controlled potential electrolysis of 1 mM solution of Indole generally took about 3 h for complete oxidation corresponding to peak I a potentials. With the progress of electrolysis the colour of the solution changed from colourless to light yellow and finally to brown. The progress of electrolysis was monitored by recording cyclic voltammograms at different intervals of time. The cyclic voltammetric changes obtained at pH 6.0 with progress of electrolysis are presented in Fig. 5. Thus, curve A in Fig. 5 presents a cyclic voltammogram of Indole just before electrolysis and peaks I a , II c , III c and II a were clearly observed. With progress of electrolysis the peak current for peak I a systematically decreased Žcurves B–D., whereas peaks II c and III c merged to a single peak and increased. The oxidation peak I a completely disappeared at the end of the electrolysis Žcurve D.. This cyclic voltammogram did not change even when the electrolysed solution was left for several hours at room temperature.

5. Spectral studies The UV spectra of Indole ŽI. were recorded in the entire pH range Ž2.0–10.5. and l max at 220, 270, 276 nm were observed along with a shoulder at around 285 nm. Curve Ž1. in Fig. 6 exhibits the UV spectrum of 0.05 mM solution of Indole at pH 3.2 and lmax at 220, 270, 276 nm were noticed. Upon application of potential 100 mV more

Fig. 6. Spectral changes observed during electrooxidation of 0.05 mM of Indole at pH 3.2. Curves Ž1 to 4. were recorded at an interval of 5 min and Ž5–8. at an interval of 15 min. Curve 9 was recorded after 8 h of electrolysis Pot. 800 mV vs. SCE.

positive than that of peak I a the absorbance at 270, 276 and 285 nm, first increased Žcurve 2. and then a systematic decrease in absorbance was noticed Žcurves 3 to 8.. The exhaustively electrolysed solution of Indole exhibited l max at 201 and 244 nm. Thus, the l max of the product were shifted to shorter wavelengths Žcurve 9. in comparison to the starting material. The spectral changes during electrolysis were also monitored at pH 7.0 and the changes were similar to that observed at pH 3.2. The identical spectral changes indicated that products of electrooxidation of Indole ŽI. at pH 3.2 and 7.0 are same. In a separate experiment, the applied potential was switched to 0 volt after recording curve 5 in Fig. 6 and spectral changes were monitored. It was found that the absorbance in the region 230–245 nm systematically increased. Hence, it was concluded that a UV-absorbing intermediate is generated during the oxidation of Indole which absorbed in the wavelength region 230–245 nm. Table 2 Observed rate constants for the decomposition of UV-absorbing intermediate generated during electrooxidation of Indole at different pH pH

Pot. mV vs. SCE

l rnm

k r10y3 sy1

2.0 3.2 5.0 6.0 7.0 8.0 8.5 9.5 10.6

800 800 800 800 750 750 700 700 700

235 235 235 235 235 235 235 235 235

1.1 1.1 1.2 1.0 1.2 1.2 1.0 1.0 1.0

)Average of at least three replicate determinations.

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The kinetics of decomposition of the UV-absorbing intermediate was monitored at 235 nm. For this purpose a solution of Indole was electrooxidised and when the absorbance at lmax 270 nm reached 50% Žusually in 30 min., the potential was turned off. The change in absorbance with time was monitored and an exponential decay was observed. The values of the rate constant were calculated from the linear plots of log Ž A–A` . versus time. The values of k obtained at different pH are presented in Table 2.

6. Characterization of the product The product of electrooxidation of Indole ŽI. was characterized at pH 3.2 and 7.0. The brown coloured material obtained had a m.p. 758C. It gave prominent peaks in FT-IR spectrum at 3049 Žs.; 1616 Žm.; 1578 Žm.; 747 Žs.; 726 Žs. corresponding to aromatic C–H; 2920 Žm. corresponding to C5C; 3399 Žm. and 1246 Žs. for N–H Žsecondary.; 1353 Žs. and 1337 Žs. for C5N. The mass spectrum of the material exhibited a clear molecular ion peak at mre 349 Ž18.4%. and suggested the molar mass of the material as 349. Other prominent high mass peaks observed in the mass spectrum were at 348 Ž5.2%.; 347 Ž1.8%.; 279 Ž24.2%.; 233 Ž13.8%.; 223 Ž27.0%.; 219 Ž10%.; 195 Ž8.0%.; 117 Ž22.3%. and 116 Ž20.1%.. However, no attempt was made to explain the fragmentation pattern. The 1 H NMR spectrum of the product exhibited following signals, d s 7.14 Žs, 12H.; 7.9Žd, 1H.; 7.6Žd, 2H.; 7.5Žd, 2H. and 6.5Žd, 2H.. The molar mass of 349 clearly indicates that the product obtained is a trimer of Indole and corresponds to structure ŽVI.. The formation of trimer ŽVI. was further confirmed by recording its cyclic voltammogram. As compound ŽVI. possesses two reducible sites ŽC5N.; two closely overlapping reduction peaks were observed when the sweep was initiated in the negative direction with Ep at practically similar potentials as that observed for peaks II c and III c . The peak potentials of peaks II c and III c were so close that many time only a single peak was noticed. They were also dependent on the concentration of indole and often merged in a single peak at concentrations ) 0.5 mM in the pH range 2.0–6.0. One would expect that peaks II a , III a should be observed during CPE in the first positive going sweep. However, as the trimer VI formed in the solution is reducible due to the presence of C5N linkages, peaks II a , III a are observed only when peaks II c , III c are scanned.

7. Redox mechanism The results reported earlier suggest that electrooxidation of Indole in phosphate buffer proceeds in 1e process. The nature of the electrode reaction was found as EC in which charge transfer is followed by irreversible chemical reac-

Scheme 1. Tentative mechanism proposed for the oxidation of Indole at pH -6.0 and pH )6.0.

tions. The nature of Ep vs. pH plot suggested that as below pH 6.0, the peak potential was independent of pH, no protons are involved in the electrode reaction. Thus, at pH - 6.0, Indole is oxidized in 1e process to give cationic free radical which can exist is two tautomeric structures. The cationic free radical ŽII. readily combines with a similar species to give dimer ŽIII., which undergoes hydrolysis in a follow up chemical step. The attack of water molecule occurs at position 2, which is least electronegative due to positively charged nitrogen atom at position 1. The values of k observed for the decomposition of UV-absorbing intermediate during spectral studies represents the disappearance of species III ŽScheme 1.. Thus, the dimer ŽIII or VIII. is attacked by a molecule of water in a follow-up chemical step to ultimately give trimer ŽVI.. The increase in absorbance in the region 230–245 is due to the formation of trimer ŽVI. as confirmed by recording the UV spectrum of the isolated product in which a broad band at around 240 nm was observed. Position 2 of species ŽIV. becomes electropositive due to the presence of N and O and provides a space for attack of nucleophile. One more molecule of Indole then attacks through position 2, which is sufficiently nucleophilic w23x and species ŽV. is obtained. Removal of Hq from species ŽV. gives trimer ŽVI. having molar mass 349. It may be realized that the

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proposed reaction sequence for the formation of ŽVI. is one out of the several possible pathways. At pH ) 6.0 where Ep was dependent on pH indicated that protons are involved in the electrooxidation. Thus, 1e, Hq oxidation of ŽI. rapidly gives a free radical ŽVII. which on combining with similar species gives dimer of Indole ŽVIII. in which two units are joined at b position. Attack of water molecule on species VIII causes hydrolysis of C5N and nucleophilic attack of Indole then gives trimer ŽVI. in the steps similar to that proposed for pH - 6.0.

8. Biological studies The toxicity of the major product of oxidation Žcompound VI. and Indole were evaluated in albino mice. The LD50 value which caused the death of 50% animals within 24 h was determined for the compound VI and was found to be 300 " 2.0 m g Žmean " standard deviation on a log microgram scale.. The LD50 value for the Indole was found to be 720 " 2.0 m g. The behavioural effects evoked following administration of compounds I and VI typically lasted for only a short period of time. After the recovery from light ether anaesthesia Žtypically 3–4 min., the animals exhibited aggressive behaviour and started rapidly moving in the cage. After 30 min, all animals exhibited behaviour indistinguishable from that of controls.

9. Blood analysis Table 3 presents the various blood parameters after the treatment of mice with compounds I and VI. It was interesting to observe that intracranial injection of a single dose Ž300 m g. of Indole in albino mice did not alter most

of the haematological and biochemical parameters, except that glucose and serum cholesterol increased significantly. The increase in these parameters remained statistically significant even after 1 week of administration of compound I. The blood sugar level shows a condition identical to diabetes mellitus. The insulin secretion is thus inhibited and the entire carbohydrate metabolism is disturbed. The elevated glucose level of blood most probably results from the diminished permeability of cellular membrane from glucose in muscular and fatty tissues. Administration of patulin has also been found to cause increase in blood glucose levels by Devaraj et al. w24x in rats. One more possibility for significant increase in glucose level after administration of compound I even after 1 week is the retarded glucose oxidation in body cells and accelerated gluconeogensis in liver cells. Similar explanation has also been suggested for the increase in blood glucose levels by other workers w25x. The prolonged hyperglycaemia caused by intracranial injection of single dose of compound I may lead to glycosuria, increased osmotic pressure in blood and damage to nervous system. As the system was unable to utilise glucose for energy production, the cells use protein for it and hence the mice became weak. The cholesterol level was found to increase whereas urea and urea nitrogen in the blood serum were found to decrease after 1 day of administration of compound I ŽTable 3.. Such an observation indicates the possibility of nephritis with edema due to extrarenal disturbance of the lipid metabolism. Nephritis also causes non protein retention in the blood and hence urearurea nitrogen also decrease in the blood serum. The cholesterol level remained significantly increased even after 1 week whereas urea and urea nitrogen became normal on the 3rd day. The long time required to reach normal value of cholesterol may be due to the hormonal control of hypothyroidism condition

Table 3 Effects evoked by intracranial injection of Indole ŽI. and trimer ŽIV. on the blood parameters of albino mice Ž n s 6. Parameters

Control

Hb Žgr100 ml. 9.0 " 0.2 Ht Ž%. 36.0 " 1.0 RBC = 10 6 Žmm3 . 5.3 " 0.2 WBC = 10 3 Žmm3 . 10.0 " 0.4 ESR Žmmrh. 1.0 " 0.1 BT Žs. 130.0 " 2.0 CT Žs. 150.0 " 2.0 Glucose Žmgr100 ml. 110.0 " 1.0 AST ŽIUrml. 28.0 " 1.0 ALT ŽIUrml. 10.0 " 0.5 Alkaline phosphatase ŽKA. 3.0 " 0.2 Acid phosphatase ŽKA. 9.6 " 0.1 Cholesterol Žmgr100 ml. 120.0 " 4.0 Urea Žmgr100 ml. 43.1 " 1.0 BUN Žmgr100 ml. 20.0 " 1.0 ) p - 0.05.

Compound I

Compound VI

1 day

3 days

1 week

1 day

3 days

1 week

9.0 " 0.2 35.0 " 1.1 5.3 " 0.2 9.8 " 0.3 1.2 " 0.2 125.0 " 2.0 150.0 " 2.0 140.6 " 2.0) 29.0 " 2.0 10.0 " 0.4 2.8 " 0.2 9.2 " 0.2 168.2 " 4.0) 30.0 " 2.0 15.8 " 1.6)

9.0 " 0.2 35.0 " 1.0 5.3 " 0.1 9.8 " 0.3 1.0 " 0.2 128 " 0.2 150 " 2.0 138.0 " 2.0) 29.0 " 2.0 10.0 " 0.5 2.9 " 0.1 9.4 " 0.2 148 " 0.2) 40.2 " 2.0 20.0 " 1.5

9.2 " 0.2 35.0 " 1.1 5.0 " 0.2 9.8 " 0.3 1.0 " 0.2 128 " 0.2 150.0 " 2.0 130.0 " 1.8) 30.0 " 2.0 10.0 " 0.5 2.8 " 0.2 9.6 " 0.2 140 " 3.0) 41.0 " 2.0 20.0 " 1.5

7.6 " 0.1) 35.0 " 1.0) 4.5 " 0.2) 11.2 " 0.2) 2.0 " 0.1 128.0 " 0.2 152.0 " 3.0 195.6 " 2.0) 50.1 " 1.3) 5.0 " 0.2) 3.0 " 0.2 8.7 " 0.2 220 " 0.8) 30.6 " 4.0) 16.0 " 1.8)

8.8 " 0.2 36.0 " 1.0 5.2 " 0.2 10.0 " 0.3 1.0 " 0.1 128 " 2.0 150.0 " 2.0 161.4 " 3.0) 43.4 " 1.0) 8.0 " 0.5 3.2 " 0.2 9.0 " 0.3 270.1 " 3.0) 40.0 " 3.0 19.5 " 1.0

9.0 " 0.2 36.0 " 1.0 5.1 " 0.2 10.0 " 0.4 1.0 " 0.1 128.0 " 2.0 150.0 " 3.0 145.0 " 3.0) 30.0 " 2.0 10.2 " 0.5 3.0 " 0.2 9.0 " 0.3 260 " 3.0) 40.2 " 3.0 20.0 " 1.5

R.N. Goyal et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 47–53

which has been reported w26x to need longer times for becoming normal. In contrast to Indole, its oxidation product ŽVI. was found to be much more toxic. The significant decrease in Hb content after 24 h administration ŽTable 3. is most probably due to the intra-erythropoietic depletion as has been reported in albino rats and in birds w27,28x. The decrease in RBC and a significant increase in WBC after 1 day ŽTable 3. can be accounted for on the basis of removal of cellular debris of necrossed tissues as reported by McLay and Brawn w29x. The increase in ESR can be assigned to monoclonal blood protein disorders such as multiple myeloma or macroglobulinema w30x. Bleeding time and clotting time ŽCT. did not show significant change. An increase in AST values indicate hepatotoxic action of tissue damage and necrosis. The altered membrane permeability thus causes leakage of soluble tissue enzymes into the blood. A similar explanation for the increase in AST values has also been suggested by Rahman et al. w31x. The significant decrease in ALT, urea and BUN, value observed also indicate nephrosis condition which is further supported by the higher values of cholesterol in treated animals. Nephrosis has been reported to occur due to the degeneration of glomeruli w32x in the presence of low urea Žnitrogen waste. and higher values of cholesterol. The higher value of glucose levels even after 1 week strongly indicate hyperglycaemia condition. After 1 week of administration of compound ŽVI. glucose and cholesterol levels were found significantly higher in comparison to controls whereas all other parameter reached to the normal values. Thus, a comparison of blood parameters for compound I and VI clearly indicate that the trimer ŽVI. is more toxic than Indole.

Acknowledgements One of the authors ŽNK. is thankful to UGC, New Delhi for the award of Senior Research Fellowship.

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