Metabolism of echitamine and plumbagin in rats

Transcription

1 J. Biosci., Vol. 3, Number 4, December 1981, pp Printed in India. Metabolism of echitamine and plumbagin in rats B. CHANDRASEKARAN and B. NAGARAJAN Microbiology Division, Cancer Institute, Madras MS received 23 February 1981; revised 10 July 1981 Abstract. When administered to rats, echitamine was absorbed rapidly from the tissues and was detected in circulation within 30 min. The drug level reached a maximum value by 2 h and then decreased steadily. The drug had completely disappeared from the blood in 6 h. The presence of echitamine was observed within 2 h in urine and the maximum amount of drug was excreted between 2 and 4 h. About 90% of the drug was excreted in urine in 24 h and the drug could not be detected in urine after 48 h. Along with echitamine, its metabolites were also detected in the urine. Plumbagin was not detected in blood upto 24 h when administered into rats. The drug was detected in urine within 4 h after administration; a major portion of the drug was excreted in urine by 24 h and traces of the drug were observed in the urine even after 48 h. Keywords. Echitamine; plumbagin; pharmacokinetics; urinary metabolites; plant extracts. Introduction During the past 25 years, chemotherapy has gained an important role in the treatment of tumours. Plants are a suitable source for they provide chemicals with novel structures. So far, many plant products have been found to be active antitumour agents both in animal tumour systems and human tumours (Hartwell, 1976). Preliminary studies with echitamine, an indole alkaloid, extracted from, Alstonia scholaris and plumbagin, a naphthoquinone derivative (Chandrasekaran and Nagarajan, unpublished observations; Krishnaswamy and Purushothaman, 1980) from Plumbago zeylanica were reported to have antitumour activity on rat fibrosarcoma. Simple and sensitive methods have been developed for the detection and quantitative estimation of echitamine and plumbagin in biological samples (Chandrasekaran et al., 1981). Information on the metabolism and subsequent excretion of these drugs is of particular importance, since serious and unexpected toxicity can arise from accumulation of a drug normally detoxified by the liver and/or excreted by the kidney in patients with compromised hepatic or renal function (Sieber et al., 1976). In this paper, we report the metabolic fate and excretory pattern of echitamine and plumbagin in rats. Materials and methods Echitamine and plumbagin were generous gifts from Dr K. K. Purushothaman of Captain Srinivasamurthi Research Institute, Madras. Echitamine used was in the 395

2 396 Chandrasekaran and Nagarajan form of echitamine chloride. Echitamine (2.8 mg/0.2 ml) or plumbagin (1.6 mg 0.2 ml) was injected intramuscularly in the thigh region to rats. A total number of 48 male Wistar rats (about 100 g) were injected with the drug and divided into 16 groups of 3 each. Blood samples were drawn into heparinized tubes from 3 rats at different time intervals from 0-24 h by heart puncture. Plasma was separated and used for estimation of drugs under study. Plasma samples containing high content of lipid were excluded for the estimation of drug since the removal of lipid from plasma sample resulted in loss of drug also. Urine samples were collected in metabolic cages from drug injected rats (3) at the intervals of 2, 4, 6, 24 and 48 h. Two ml of toluene was used as preservative for urine. Control urines were collected from the normal rats in a similar manner. Estimation of drugs in blood Echitamine: Plasma (0.5 ml) was deproteinized by adding 1 ml of 10% sodium tungstate followed by 1 ml of 0.66 Ν suphuric acid, the resultant protein-free supernatant adjusted to 20% NaOH concentration and extracted with 10 volumes of dioxan. The dioxan extract was dried in vacuo and dissolved 1 ml of water. The blue colour developed on the addition of 0.66 Ν phenol reagent (1 ml) (Folin and Ciocalteau, 1927) followed by 20% Na 2 CO 3 (1 ml) was read at 700 nm in a Carlzeiss JENA VSU 2 spectrophotometer, 15 min after addition of the reagents. The recovery was about 70%. Blood collected just prior to the administration of the drug (0 h) was treated as control. Plumbagin: Plasma (0.5 ml) was treated with 1 ml of 10% sodium tungstate, 1 ml of 0.66 Ν sulphuric acid and 1 ml of 10% NaOH. The mixture was heated at 80 C for 30 min, cooled to room temperature (30 C) and centrifuged (1000 g) in a Remi T 8 centrifuge. The clear supernatant was separated and the absorbance was measured at 500 nm. The recovery of the added drug was about 80%. Metabolic studies Echitamine: Pooled urine sample from 3 treated rats (100 ml) was concentrated in vacuo to about 3 ml, mixed with an equal quantity of 40% NaOH and extracted with six vol of dioxan. The dioxan layer was separated and dried in vacuo and the dried mass dissolved in 1 ml of water was chromatographed using a Whatman 3 MM paper in duplicate along with reference echitamine and control-urine extract using butanol: citric acid: water (87:0.48:13, v/w/v) and butanol: acetic acid: water (67:10:23, v/v) as solvent systems. The chromatograms were sprayed with 0.66 Ν phenol reagent followed by 20% Na 2 CO 3 or Dragendorff reagent (Clarke, 1974). Blue and orange colour were developed with phenol and Dragedorff reagents respectively on reaction with echitamine. Blank regions of the chromatograms similarly processed served as control. Plumbagin: The pooled urine sample (100 ml) from drug treated rats was concentrated in vacuo. The concentrated material was acidified to ph 4.0 and extracted with 6 vol of chloroform and concentrated to 0.5 ml. Chloroform

3 Metabolism of echitamine and plumbagin 397 extract (10 µl) was chromatographed on thin layer silica gels using hexanebenzene solvent system (1:1, v/v). Plumbagin was detected by exposing the plates either to iodine or bromine vapours. To the vacuum dried aliquots of chloroform extract (20-80 µl),1 ml of 10% NaOH was added followed by 2 ml water. The tubes were kept at 80 Cfor 30 min, cooled and the intensity of the colour developed was measured at 500 nm. Urine control and standards were studied in a similar manner. Results and discussion The presence of echitamine could be detected in blood within 30 min of its administration. The drug level steadily incrbased upto 2 h and then decreased gradually and at the end of 6 h, the drug could not be detected in blood (figure 1). Analysis of urine at different time intervals (2, 4, 6, 24 and 48 h) after administration of echitamine showed the presence of echitamine in all the samples. Chromatography of the dioxan extract from urine in butanol, citric acid solvent system showed the presence of echitamine alone (Rf, 0.8) at 2 and 4 h, but in subsequent samples, the presence of two compounds with Rf 0.25 and 0.1 were observed along with echitamine. Separation by butanol, acetic acid solvent Figure 1. Time course of appearance of echitamine in blood. Each rat was injected 2.8 mg of the drug intramuscularly and blood was collected at time intervals indicated in the figure by cardiac puncture and echitamine present was estimated.

4 398 Chandrasekaran and Nagarajan system showed only one compound (Rf, 0.7) which corresponded to automatic echitamine (table 1). The compounds with Rf value 0.25 and 0.1 were considered as metabolites of echitamine since none of the metabolites from control urine developed colour with Dragendorff reagent under the experimental conditions. Table 1. Paper chromatographic separation of echitamine and its metabolites. (a) Butanol: citric acid: water (87:0.48:13 v/w/v); (b) Butanol: acetic acid: water (67:10:23, v/v) solvent system. Thin layer chromatographic separation of plumbagin in (c) hexane: benzene (1:1 v/v) solvent system from urine extracts of rats after drug administration. * A and Β are metabolites of echitamine and the chemical nature of these compounds are yet to be characterized. N.D. Metabolites of echitamine A and Β were not detectable in this solvent system. However, the chemical structures of these compounds are yet to be characterized. Excretion of the drug was maximum between 2 and 4 h. Eighty per cent of the drug administered was excreted unchanged. About 90% of the drug and its metabolites were excreted on the 1st day and the remaining portion was excreted on the 2nd day. The drug was not detectable in the urine after 48 h. Excretion of echitamine at different time intervals has been shown in table 2. The excretory patterns of echitamine in normal rats and tumour-bearing rats were similar. Table 2. Excretion of echitamine and plumbagin in urine of rats. Values represent mean values from 6 rats, 3 in each set. Amount of echitamine injected 2.8 mg to each rat. Amount of plumbagin injected 1.6 mg to each rat.

5 Metabolism of echitamine and plumbagin 399 Even though phenol reagent is sensitive, it is not specific for the drug and its metabolites. Some of the normal metabolites from control urine were extracted into dioxan and developed blue colour with phenol reagent, whereas Dragendorff reagent was specific for the drug under the experimental conditions. However, the latter was not useful when the drug was present in lower concentrations. Administration of plumbagin into rats and subsequent collection of blood at different time intervals did not show its presence in any sample. It could be postulated that the drug might have been extensively distributed in body tissues with a very small level in the blood and thus its detection in the blood would not be possible with the current methods. Alternatively, there may also be an unidentified compound present in the blood (cellular and/or humoral component, nonfilterable by the kidneys) which could possibly interfere with the detection of plumbagin in the blood. Analysis of urine at different time intervals after administration of plumbagin showed the presence of plumbagin in all the samples except 2 h urine (table 2). Separation of a chloroform extract by thing layer chromatography showed the presence of one spot (Rf, 0.6) in benzene: hexane (1:1, v/v) solvent system which corresponded to authentic plumbagin (table 1). A considerable amount of plumbagin was lost during its extraction from urine. So the extraction method was modified by taking aliquots of urine collected from plumbagin-injected rats and 1 ml of 10% NaOH was added and the final volume made upto 3 ml with water; the colour developed had an absorption peak at 490 nm. This method is useful only if the concentration of plumbagin is above 5 µg in urine and it is free from turbidity. It was observed that none of the normal metabolites extracted into chloroform developed colour similar to plumbagin with 10% NaOH. The contents of duodenum and small intestines of rats were washed with normal saline and analyzed for plumbagin after administration of the drug using the extraction procedure described for urine. The presence of the drug was observed after 6 h. Attempts made to quantitate the drug were not successful due to the interference with other compounds present, but it was apparent that considerable amount of plumbagin was excreated in faeces. Echitamine is absorbed rapidly from the tissues into blood and cleared from the systemic blood in a short period. The injected drug is almost completely excreted in urine within 48 h. Similarly a major portion of plumbagin was excreted in urine and faeces within 48 h, questioning the direct lethal effect of echitamine on tumours On the other hand, the antitumour effect reported may be due to the triggering of some reaction by the parent compound. Experiments are under way to isolate the metabolic products when echitamine is incubated with liver slices in vitro. Acknowledgements Financial support from Central Council for Research (Siddha), Government of India is gratefully acknowledged.

6 400 Chandrasekaran and Nagarajan References Chandrasekaran, B., Vijayendran, R., Purushothaman, K. K. and Nagarajan, Β. (1981) Indian J. Biochem. Biophys., (in press). Clarke, E. G. C. (1974) Isolation and identification of drugs (London: British Pharmaceutical Press) vol. 1, p.799. Folin, O. and Ciocalteau, V. (1927) J. Biol. Chem., 73, 627. Hartwell, J. L. (1976) Cancer Treat, Rep., 60, Krishnaswamy, M. and Purushothaman, Κ. Κ. (1980) Indian J. Exp. Biol., 18, 876. Sieber, Μ., Mead, J. A. R. and Adamson, R. Η. (1970) Cancer Treat. Rep., 60, 1127.