Anti-trichomonal, biochemical and toxicological activities of methanolic extract and some carbazole alkaloids isolated from the leaves of Murraya koenigii growing in Nigeria.

Abstract

The methanolic extract of Murraya koenigii leaf was screened for toxicological and biochemical effects on rats because of the folkloric uses as an anti-dysentery and anti-diabetes. The extract was moderately toxic (L[D.sub.50] = 316.23 mg/kg body weight) to rats and had appreciable effect on the liver and kidney at higher doses leading to liver inflammation. It had little or no effect on haematology and relative organ weight of lungs, heart and spleen. Acute doses ([greater than or equal to] 500 mg/kg) reduced significantly serum globulin, albumin, urea, glucose, total protein, aspartate transaminase (AST), and increased cholesterol and alanine transaminase (ALT) indicating hepatic injury. However, chronic administration for 14 days gave a significant (p<0.05) reduction in the serum cholesterol, glucose, urea, bilirubin, ALT and AST showing that the plant has hypoglycaemic and hepatoprotective effects after prolonged use. The activity demonstrated by some of the isolated carbazole alkaloids and their derivatives against Trichomonas gallinae confirmed that the anti-trichomonal activity of the leaf may be due to its carbazole alkaloids. The order of activity was [C.sub.18]>[C.sub.23]>[C.sub.13]. Girinimbine and girinimbilol with I[C.sub.50] values of 1.08 and 1.20 [micro]g/ml were the most active. Acetylation of girinimbilol and mahanimbilol improved their activities to 0.60 and 1.08 [micro]g/ml.

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Keywords: Anti-trichomonal; Anti-diabetic; Biochemical properties; Carbazole alkaloids; Murraya koenigii

Introduction

Murraya koenigii (L.) Spreng. (Rutaceae) is a spicy ancient Indian medicinal plant native to Indo-China but grown mostly in the tropics for the medicinal and flavourant properties of the leaves (Dastur, 1970; Gupta and Nigam, 1971; Stone, 1985). It is commonly called Curry leaf Tree (Chakraborty et al., 1965) and volatile oils and their numerous constituents have been reported from the leaf (Dutt, 1958; Nigam and Purohit, 1961; Macleod and Pieris, 1982; Wong and Tie, 1993; Onayade and Adebajo, 2000; Rana et al., 2004). The leaves are used as tonic, febrifuge, stomachic, antivomiting and eaten raw for curing dysentery and diarrhoea. The leaf, stem and root are used externally in skin eruptions and bites of venomous animals while the bark and root are used as stimulant (Chakraborty et al., 1965; Das et al., 1965; Dastur, 1970; Gupta and Nigam, 1971; Nutan et al., 1998). Other uses are as carminative, hypotensive, hypoglycaemic, anti-periodic and anti-fungal (Gupta and Nigam, 1971; Nutan et al., 1998).

[FIGURE 1 OMITTED]

Carbazole alkaloids, monomeric and recently binary carbazoles as the major components, (Fiebig et al., 1985; Atta-ur-Rahman et al., 1988; Hegnauer, 1990; Chakraborty and Roy, 1991; Ito et al., 1993; Reisch et al., 1992, 1994a; Bhattacharyya et al., 1994; Adebajo, 1997; Chakrabarty et al., 1997; Nutan et al., 1998) and simple-, furo- and pyrano-coumarins (Gupta and Nigam, 1971; Bhattacharyya and Chakraborty, 1984; Reisch et al., 1994b, c; Adebajo, 1997; Adebajo et al., 1997; Adebajo and Reisch, 2000) have been reported from the various parts of the plant. Anti-oxidant, -tumour, -microbial, -inflammatory, -trypanocidal and mosquitocidal activities have been indicated for some of these alkaloids (Das et al., 1965; Fiebig et al., 1985; Chakrabarty et al., 1997; Nutan et al., 1998; Ramsewak et al., 1999; Itoigawa et al., 2000; Nakatani, 2000; Adewunmi et al., 2001). Anti-microbial (Nutan et al., 1998), anti-tumuor (Fiebig et al., 1985; Chakrabarty et al., 1997), [alpha]-amylase inhibitory (Bawden et al., 2002), anti-oxidative (Tachibana et al., 2001), cytotoxic, depressant, anti-trichomonal (Nutan et al., 1998; Adebajo et al., 2004), anti-hypertensive, -treponemal, -spasmodic and -amoebic (Bhakuni et al., 1969; Kong et al., 1986), and anti-diabetic (Naraya and Sastry, 1975) activities have been given for the extracts. Antioxidative (Khan et al., 1997), hypoglycaemic (Iyer and Mani, 1990; Khan et al., 1995a; Yadav et al., 2002), hypocholesteromeia (Khan et al., 1996a, b) properties on powdered leaf have also been reported.

Since the extract has been shown to have anti-trichomonal activity (Adebajo et al., 2004), we were therefore interested in determining the contribution to this activity of some carbazole alkaloids isolated from the plant. The leaf is freely eaten and with the abundant pharmacological reports on M. koenigii leaves, its potential toxicity was investigated by examining the toxicological and biochemical effects of some doses of the leaf methanolic extract in rats and on some selected organs (Fig. 1).

Materials and methods

Plant material, isolation and derivitisation of constituents

M. koenigii was identified, collected and the constituents were isolated as earlier reported (Adebajo et al., 2004). The derivatives were also prepared as earlier given (Reisch et al., 1994a). The identity of the active carbazole alkaloids and their derivatives was confirmed by the comparison of their m.p., UV, NMR, MS and other spectroscopic data with those in the literature (Fiebig et al., 1985; Atta-ur-Rahman et al., 1988; Chakraborty and Roy, 1991; Ito et al., 1993; Reisch et al., 1994a; Adebajo et al., 2004). The cold MeOH extract of the leaves collected in May 2003 from the same tree was used for the biochemical studies. Voucher specimen FHI 105244 of the plant was deposited in the Herbarium of the Forestry Research Institute of Nigeria, Ibadan.

Animals

Swiss albino rats weighing 145-163 g were purchased from the Animal House, Department of Physiology, University of Ibadan and kept in the Department of Biochemistry laboratory, Federal University of Technology, Akure, Nigeria to acclimatise. The rats were housed and maintained under standard environmental conditions, fed with commercial grower mash (Ladokun Feeds, Ibadan, Nigeria) with free access to water. Principles of laboratory animal care (NIH Publication no.85-23) were followed in this study.

Determination of acute toxicity

This was done using Lorke's (1983) methods. Four groups of three rats were each fed orally with single doses of 200, 500, 1000 and 2000 mg/kg body weight, respectively and observed for 72 h for clinical signs and mortality. They were thereafter sacrificed and blood and organs were harvested for the determination of some enzyme markers and other biochemical indices. The acute dose is the geometric mean of the product of the dose A that produced 100% mortality or clinical signs in one group and the dose B that produced 0% mortality in another group. If there was any inconsistency, then probit log analysis was applied. Therefore, the acute toxicity was determined as L[D.sub.50] = [square root of (A x B)] = [square root of (500 x 200)] = 316.23 mg/kg body weight.

Sub-chronic and haematological studies

Four groups of five albino rats each were given 9% saline (control group), 250, 350 and 450 mg/kg body weight of the extracts (experimental groups), respectively, for 14 days based on the calculated L[D.sub.50] value above. The rats were sacrificed and blood and organs were harvested for haematological and biochemical (enzyme assay) examinations. The packed cell volume (PCV) and red blood cells (RBC) values were obtained visually and manually (Baker et al., 1998).

Assay for serum enzyme activities and other biochemical indices

Animals were anaesthetised with light chloroform 24 h after the last treatment and blood drawn by cardiac puncture. Serum was separated by centrifugation (3000 rev/min for 15 min) and aspartate transaminase (AST) and alanine transaminase (ALT) were estimated at the wavelength of 490 nm using Reitman and Frankel's (1957) method. Bilirubin concentration was calculated following the method of Doumas et al. (1973). Total protein was determined using biuret method (Peters, 1968). Urea was estimated using Bethelot-Searcy's method (Searcy et al., 1967) while alkaline phosphatase was done using phenolphthalein monophosphate method (Babson et al., 1966). The contents of glucose and cholesterol were obtained using enzymatic GOD-PAP (Trinder, 1969) and CHOD-PAP (Zoppi and Fellini, 1976) methods, respectively. Lastly, the albumin and globulin values were estimated using BCG method (Doumas and Biggs, 1972).

Anti-trichomonal test

Trichomonas gallinae isolated from the pigeon was dropped into a test tube of normal saline. The solution was distributed into test tubes of Ringer's egg-serum culture for enteric protozoan and incubated at 37 [degrees]C for growth. Stock solutions of the isolates and their derivatives, and Metronidazole (Flagyl[R], Aventis Pharma) in DMSO at the concentration of 20, 20 and 8 mg/ml, respectively, were made. Serial dilutions to 0.00, 1.953, 3.906, 7.8125, 15.625, 31.25, 62.5 and 125 [micro]g/ml for the isolates and their derivatives, and 0.00, 0.1562, 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0 and 30.0 [micro]g/ml for metronidazole with the fluid nutrient solution were used as the test agents. A 50 [micro]l of each test agent and 150 [micro]l of the nutrient solution were pipetted into the microwells and incubated in the steam incubator at 37 [degrees]C for 24 and 48 h. The number of organisms per millilitre in each well for 0, 24 and 48 h were counted using the microscope. The experiments were done in triplicates (Narcisi and Secor, 1996).

Statistics analysis

Statistical evaluation of whole means were carried out by one-way analysis of variance (ANOVA), followed by Duncan new Multiple Range test and student t-test. Statistical significance was accepted at p[less than or equal to]0.05, 0.01.

Results and discussion

With the exception of girinimbilyl acetate, all the carbazole alkaloids isolated from M. koenigii showed greater activity at 48 h than at 24 h probably due to increased contact time of the compounds with the organisms. Although they were less active than the standard drug Metronidazole (Flagyl[R]), the activity observed indicated that the carbazoles may be the active anti-trichomonal constituents responsible for this eth-nomedicinal use of the plant (Table 1). An I[C.sub.50]>30 [micro]g/ml has been reported for the M. koenigii leaf methanol extract (Adebajo et al., 2004); thus the isolates were probably not acting in synergism.

The two [C.sub.13] carbazoles tested were the least active and the presence of two carbonyl moieties in murraya-quinone-A (8) may have aided its activity over that of murrayanine (7). Replacement of a terminal methyl group at ring D of girinimbine (2) with an isohept-2-enyl moiety as in mahanimbine (1) led to a 57% and 51% reduction in activity at 24 and 48 h, respectively, in (1). A similar replacement in girinimbilol (5) and mahanimbilol (4) gave 70% and 51% reduction in the activity, respectively (Table 1).

Introduction of a 7-OH group into (1) as in mahanine (6) reduced the activity of (6) by about 29% and 27% at 24 and 48 h, respectively. Incorporation of a 6-OMe group into the carbazole skeleton as in koenimbine (3) significantly reduced the I[C.sub.50] by about 71% and 30% at 24 and 48 h, respectively, when compared with that of (2). The opening of the pyran ring as in (5) compared to (2) with a pyran ring led to a reduction in activity of (5) by 10% and 18% at 24 and 48 h, respectively. Similarly, a reduction of 38% and 18% in (4) compared to (1) was also observed. The replacement of the H of the 2-OH of (5) by an acetyl group in girinimbilyl acetate (10) gave a 50% and 41% increase in the activity of (10) at 24 and 48 h, respectively. Similarly, a respective increase of 73% and 60% was observed in mahanimbilyl acetate (9) over (4). Moreover, methylation of the 2-OH in (4) gave 67% and 54% increase at 24 and 48 h, respectively, in mahanimbilol methyl (12). In addition, the etherification of the 2-OH of (4) with an alkyl rest to give the novel pentacarbazole bicyclomahanimbiline (11) gave an increased activity of 55% and 51% at 24 and 48 h, respectively, of (11) (Table 1). Generally therefore, the activities of the carbazoles were shown to be reduced by the introduction of an oxygen atom on the carbazole nucleus. However, this reduction in activity could be reversed by the replacement of the hydrogen atom of the hydroxyl group by an alkyl or alkoxy group, especially acetyl and methyl rests.

The L[D.sub.50] of 316.23 mg/kg calculated from the acute administration of the extract showed that it is moderately toxic (Rodricks, 1992; Nakatani, 2000). The decrease (p<0.05) in weight (Table 2) could be due to decrease in appetite (food intake) which probably arose from the decrease in food efficiency ratio. This result is however contrary to the report of an insignificant body weight change when a 10% Curry leaf was administered to rats for 90 days (Khan et al., 1995a, 1996a). The significant changes in kidney, spleen, liver, heart and lung weights (Tables 3 and 4) could be as a result of chemical injury induced by the administered extract to the cells and tissues of these organs and therefore explain the possible toxicity of the plant.

At 250 mg/kg less than the lethal dose, the extract gave a significant (p<0.01) haematological effects reducing the PCV and RBC counts (Table 5). These are less than the reference values in animals (Mitruka and Rawnsley, 1977). However, feeding of rats with whole Curry leaf at doses equal to normal human intake was reported not to have any adverse effect on haemoglobin, RBC, WBC, total and differential counts of blood (Khan et al., 1995b).

The extent of hepatocellular injury is assessed by the increased serum levels of ALP, AST and ALT. These damages could be acute or chronic, reversible or irreversible (Padma et al., 1998; Mandal et al., 2000; Bhakta et al., 2001; Janbaz et al., 2002). Administration of the acute doses of the extract gave significant (p<0.05, 0.01) decrease in urea, total protein, albumin, albumin/globulin ratio, glucose and AST levels, increase in ALT and cholesterol levels especially, at 1000 and 2000 mg/kg (Table 6). However, the maximal decrease in the levels of glucose, urea and globulin was at the lower doses of 200 and 500 mg/kg (Table 6). The increase in ALP under chronic administration may confirm the liver damage (Table 7). Bain (2003) reported that acute hepatocellular injury tends to result in greater ALT elevation while chronic hepatic injury shows mild elevation of ALT activity. The ALT activity shown in Tables 6 and 7 may therefore indicate more hepatic injury in acute than in sub-chronic administration of the extract, leading to increase in liver weight and high toxicity. Chronic administration decreased the AST, ALT, glucose, urea, bilirubin and cholesterol levels (Tables 6 and 7) and could therefore serve to protect the liver, especially at 250 mg/kg. Based on reduced ALT, AST, ALP and bilirubin activities in the blood of rats, Cassia fistula leaf extract has been suggested to possess hepatoprotective activity (Bhakta et al., 2001).

Reduced serum proteins and blood urea (Tables 6 and 7) confirmed the diseased liver state of the rats, typified by liver inflammation leading to a depression of hepatic protein synthesis (Grant and Kachman, 1976; Griffith, 1979; Bickerton et al., 1996). The significant (p<0.01) reduction of estimated total cholesterol and urea in sub-chronic feeding of the extract (Table 7), may further indicate the relative safety of the sub-chronic administration of the M. koenigii. Moreover, a reduction (p<0.05) of bilirubin at sub-chronic administration (Table 7) may indicate a reduction in breakdown of RBC and buttress the fact that M. koenigii can serve as a liver protective agent. Hence, it should be safe for patients with hyperbilirubinaemia (Toohey, 1958). Globulins are major group of proteins (antibodies) in the body directed specifically to fight infections. The decrease observed in acute and the non-change in chronic administrations of the values of globulin, total protein and albumin levels (Tables 6 and 7) might imply that the chronic administration of the plant does not reduce the power of the subjects to fight infections and inflammation of essential organs, such as liver.

This is the first report on the effects of the acute administration of M. koenigii leaf on the organs, blood constituents/activities (Tables 3 and 6) and there was a strong indication of hepatic injury in the acute while chronic administration served to protect the liver and the integrity of other organs (Tables 3, 4, 6 and 7). The serum protein and cholesterol levels in rats fed chronically with Curry leaf methanol extract for 14 days (Table 7) are in agreement with those reported for the same constituents in rats and non-insulin-dependent diabetes mellitus (NIDDM) patients fed with diets of whole leaf for a period of 15-90 days (Iyer and Mani, 1990; Khan et al., 1995a, 1996a, b). However, the levels of other blood constituents found (Table 7) differed from those earlier reported (Khan et al., 1995a). These differences may be due to the powdered Curry leaf used (Iyer and Mani, 1990; Khan et al., 1995a, 1996a, b) while the methanol extract was employed in this present study. Elements, especially trace elements, present in the powdered leaf may account for the safety of the powdered plant when ingested for a long time (Obiajunwa et al., 2002). The plant has been suggested as a dietary additive (Grover et al., 2003).

At normal human dietary intake, hypoglycemic activity of the extract was reported in test animals (Khan et al., 1995a; Dahanukar et al., 2000) and hence a good medicinal plant that could be used in diabetes, hypercholesterol and hepatobiliary cases. Previous in vitro and in vivo reports (Naraya and Sastry, 1975; Iyer and Mani, 1990; Khan et al., 1995a, b; Kar et al., 1999; Bawden et al., 2002; Yadav et al., 2002; Adebajo et al., 2004; Grover et al., 2003) support our present result of significant (p<0.01) hypoglycaemic action of the methanol extract at 72 h and 14 days after acute and sub-chronic administrations, respectively (Tables 6 and 7). This present study demonstrates that Curry leaf exhibits a slow hypoglycaemic action, hence the inactivity earlier reported when blood glucose level was determined at 3 and 4h as well as the failure to stimulate insulin release from the INS-1 cells in the 90 min experiment (Kar et al., 1999; Adebajo et al., 2004). Hence, the apparent conflict in reports by various authors on the hypoglycaemic activity of M. koenigii appears to be resolved. The early onset of hypoglycaemic activity (3 days) found with the extract (Table 6) compared with 15-90 days for the powdered leaf (Iyer and Mani, 1990; Yadav et al., 2002; Grover et al., 2003) may suggest that the extract is more potent and should therefore be taken as a drug. Since most of the effects of this extract were more pronounced at high doses, excessive intake of the plant extract should be avoided bearing in mind these various effects on the body especially, the liver injury.

Conclusion

The anti-trichomonal activity of the plant extract was due to its carbazole alkaloids which may not act synergistically. This study also shows that acute administration of M. koenigii could be more dangerous than chronic administration, which could be primarily due to hepatic injuries. However, chronic administration of the extract had hepatoprotective activity. The hypoglycaemic activity of the plant was also shown to be slow in action taking more than 72 h before manifestation with the extract. The anti-diabetic, anti-trichomonal, and hepatoprotective activities of M. koenigii and also its moderate toxic effects on essential organs in acute form, may indicate that while the powdered leaf could be taken as diet adjuvant (food drug), the extract, however, should be used with great caution and be treated as a drug.

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A.C. Adebajo (a,*), O.F. Ayoola (b), E.O. Iwalewa (c), A.A. Akindahunsi (b), N.O.A. Omisore (c), C.O. Adewunmi (d), T.K. Adenowo (e)

(a) Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria

(b) Department of Biochemistry, Federal University of Technology, Akure, Nigeria

(c) Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria

(d) Drug Research and Production Unit, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria

(e) Department of Anatomy and Cell Biology, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

*Corresponding author. Tel.: [phone].

E-mail address: [e-mail] (A.C. Adebajo).

Table 1. The I[C.sub.50] values of isolated carbazole alkaloids from
Murraya koenigii and their derivatives against Trichomonas (Trichomonas
gallinae) parasites

Carbazole alkaloid tested              I[C.sub.50] ([micro]g/ml)
                           Class (no.
Name                       of carbon)  At 24 h  At 48 h

Mahanimbine (1)            [C.sub.23]  2.50     1.70
Girinimbine (2)            [C.sub.18]  1.08     0.84
Koenimbine (3)             [C.sub.18]  3.75     1.20
Mahanimbilol (4)           [C.sub.23]  4.00     2.08
Girinimbilol (5)           [C.sub.18]  1.20     1.02
Mahanine (6)               [C.sub.23]  3.50     2.33
Murrayanine (7)            [C.sub.15]  4.60     4.00
Murrayaquinone-A (8)       [C.sub.15]  3.90     3.00
Mahanimbilyl acetate (9)   [C.sub.23]  1.08     0.84
Girinimbilyl acetate (10)  [C.sub.18]  0.60     0.60
Bicyclomahanimbiline (11)  [C.sub.23]  1.82     1.02
Mahanimbilol methyl (12)   [C.sub.23]  1.32     0.96
Flagyl                                 0.14     0.12

Table 2. Effects of Murraya koenigii leaf methanol extract on the body
weights of rats during subchronic administration for 14 days

Doses      Body weight at day 1  Body weight at day 8

Control    148.2[+ or -]1.29     140.5[+ or -]1.12
250 mg/kg  149.2[+ or -]2.14     136.0[+ or -]3.26
350 mg/kg  154.6[+ or -]3.70     150.0[+ or -]3.17
450 mg/kg  151.0[+ or -]1.65     134.0[+ or -]2.32

Doses      Body weight at day 14  Body weight loss (days 1-14)

Control    135.3[+ or -]1.29      12.7[+ or -]2.67
250 mg/kg  127.2[+ or -]4.69      22.0[+ or -]4.01*
350 mg/kg  135.7[+ or -]2.88      18.9[+ or -]2.28
450 mg/kg  121.3[+ or -]3.83      29.7[+ or -]7.28*

Values are mean[+ or -]SEM; *p<0.05.

Table 3. Relative weights of selected organs 72 h after administration
of a single (acute) dose of Murraya koenigii leaf methanol extract

            Relative organ weight
Doses       Kidney              Liver               Heart

Control     0.53[+ or -]0.03    3.81[+ or -]0.04    0.37[+ or -]0.02
200 mg/kg   0.40[+ or -]0.02    3.65[+ or -]0.07    0.40[+ or -]0.00
500 mg/kg   0.64[+ or -]0.02**  5.08[+ or -]0.05**  0.30[+ or -]0.00
1000 mg/kg  0.51[+ or -]0.02    5.77[+ or -]0.13**  0.40[+ or -]0.02
2000 mg/kg  0.63[+ or -]0.00**  5.80[+ or -]0.05**  0.40[+ or -]0.00

            Relative organ weight
Doses       Spleen              Lung

Control     0.58[+ or -]0.02    0.83[+ or -]0.02
200 mg/kg   0.40[+ or -]0.01    0.58[+ or -]0.01**
500 mg/kg   0.82[+ or -]0.02**  0.63[+ or -]0.02**
1000 mg/kg  0.54[+ or -]0.00    0.50[+ or -]0.01**
2000 mg/kg  0.62[+ or -]0.04**  0.63[+ or -]0.00**

Values are mean[+ or -]SEM; **p<0.01.

Table 4. Relative weights of selected organs after 14 days of subchronic
administration of Murraya koenigii leaf methanol extract

           Relative organ weight
Doses      Kidney              Liver               Heart

Control    0.53[+ or -]0.03    3.81[+ or -]0.04    0.37[+ or -]0.02
250 mg/kg  0.71[+ or -]0.01**  4.70[+ or -]0.18**  0.40[+ or -]0.00
350 mg/kg  0.72[+ or -]0.02**  4.70[+ or -]0.03**  0.33[+ or -]0.01*
450 mg/kg  0.57[+ or -]0.00    4.74[+ or -]0.06**  0.30[+ or -]0.03*

           Relative organ weight
Doses      Spleen              Lung

Control    0.58[+ or -]0.02    0.83[+ or -]0.02
250 mg/kg  0.80[+ or -]0.03**  0.80[+ or -]0.06
350 mg/kg  0.58[+ or -]0.04    0.76[+ or -]0.04
450 mg/kg  0.50[+ or -]0.02*   0.80[+ or -]0.04

Values are mean[+ or -]SEM; *p<0.05; **p<0.01.

Table 5. Effects of Murraya koenigii leaf methanol extract on
haematological indices of rats during subchronic administration for 14
days

           Haematology (average)
Doses      PCV (%)             RBC (x[10.sup.6])

Control    33.3[+ or -]1.70    4.4[+ or -]0.37
250 mg/kg  24.0[+ or -]0.83**  2.7[+ or -]0.10**
350 mg/kg  30.8[+ or -]1.24    4.4[+ or -]0.36
450 mg/kg  36.4[+ or -]3.54    4.9[+ or -]0.57

Values are mean[+ or -]SEM; **p<0.01.

Table 6. Effects of Murraya koenigii leaf methanol extract on the serum
chemistry of rats 72 h after administration of a single (acute) dose

Single Doses  ALP (IU/l)         AST (IU/l)          ALT (IU/l)

Control       104.2[+ or -]3.35  69.4[+ or -]0.94     34.0[+ or -]0.94
200 mg/kg      98.8[+ or -]3.17  67.4[+ or -]4.06     56.6[+ or -]2.23**
500 mg/kg     112.0[+ or -]3.13  66.7[+ or -]1.25    100.6[+ or -]0.54**
1000 mg/kg     94.0[+ or -]5.04  60.0[+ or -]1.74**   72.6[+ or -]2.23**
2000 mg/kg     93.2[+ or -]4.82  59.0[+ or -]0.85**   72.8[+ or -]2.23**

              Albumin            Glucose              Urea
Single Doses  (mg/100 ml)        (mg/100 ml)          (mg/100 ml)

Control       3.4[+ or -]0.06    115.5[+ or -]4.20    61.4[+ or -]1.50
200 mg/kg     3.8[+ or -]0.09    101.6[+ or -]0.92**  36.6[+ or -]0.49**
500 mg/kg     2.7[+ or -]0.09**  106.4[+ or -]1.74*   28.3[+ or -]2.32**
1000 mg/kg    2.3[+ or -]0.18**  105.6[+ or -]1.96*   36.0[+ or -]2.01**
2000 mg/kg    2.3[+ or -]0.13**  103.8[+ or -]2.14*   36.0[+ or -]2.32**

              Total protein                        Globulin
Single Doses  (mg/100 ml)        Bilirubin         (mg/100 ml)

Control       7.7[+ or -]0.13    0.32[+ or -]0.06  4.2[+ or -]0.08
200 mg/kg     8.5[+ or -]0.09**  0.30[+ or -]0.02  4.7[+ or -]0.09**
500 mg/kg     6.7[+ or -]0.27**  0.40[+ or -]0.01  3.8[+ or -]0.09**
1000 mg/kg    6.4[+ or -]0.13**  0.30[+ or -]0.02  4.1[+ or -]0.09
2000 mg/kg    6.3[+ or -]0.09**  0.30[+ or -]0.04  4.0[+ or -]0.09

                                 Cholesterol
Single Doses  A-G                (mg/100 ml)

Control       0.80[+ or -]0.00    99.6[+ or -]2.9
200 mg/kg     0.76[+ or -]0.03   136.0[+ or -]1.7**
500 mg/kg     0.76[+ or -]0.03   129.0[+ or -]1.5**
1000 mg/kg    0.63[+ or -]0.06*  107.0[+ or -]2.9*
2000 mg/kg    0.67[+ or -]0.05*  110.4[+ or -]1.2**

Key: ALP = Alkaline phosphatase; AST = Aspartate transaminase;
ALT = Alanine amino transaminase; A-G = Albumin/globulin ratio.
Values are mean[+ or -]SEM; * p<0.05; **p<0.01.

Table 7. Effects of Murraya koenigii leaf methanol extract on the serum
chemistry of rats during subchronic administration for 14 days

Doses      ALP (IU/l)           AST (IU/l)          ALT (IU/l)

Control    100.6[+ or -]2.10    67.4[+ or -]2.23    30.0[+ or -]1.20
250 mg/kg  120.0[+ or -]1.74**  49.8[+ or -]0.67**  19.2[+ or -]0.45**
350 mg/kg  105.4[+ or -]2.99*   49.8[+ or -]0.85**  25.2[+ or -]1.52*
450 mg/kg  107.0[+ or -]0.71*   51.4[+ or -]2.90**  26.4[+ or -]1.52*

           Albumin           Glucose              Urea
Doses      (mg/100 ml)       (mg/100 ml)          (mg/100 ml)

Control    3.36[+ or -]0.12  115.0[+ or -]3.04    62.4[+ or -]2.59
250 mg/kg  3.54[+ or -]0.18  112.0[+ or -]2.68    32.0[+ or -]2.05**
350 mg/kg  3.12[+ or -]0.18  109.2[+ or -]3.66    63.8[+ or -]1.70
450 mg/kg  3.38[+ or -]0.18  101.0[+ or -]2.77**  23.6[+ or -]1.07**

           Total protein                       Globulin
Doses      (mg/100 ml)      Bilirubin          (mg/100 ml)

Control    7.6[+ or -]0.13  0.28[+ or -]0.02   4.16[+ or -]0.12
250 mg/kg  8.0[+ or -]0.18  0.24[+ or -]0.05   4.68[+ or -]0.16
350 mg/kg  7.9[+ or -]0.27  0.30[+ or -]0.01   4.18[+ or -]0.18
450 mg/kg  8.0[+ or -]0.13  0.18[+ or -]0.03*  4.42[+ or -]0.05

                             Cholesterol
Doses      A-G               (mg/100 ml)

Control    0.71[+ or -]0.00  99.2[+ or -]2.2
250 mg/kg  0.96[+ or -]0.02  99.8[+ or -]1.9
350 mg/kg  0.73[+ or -]0.02  95.0[+ or -]1.7
450 mg/kg  0.70[+ or -]0.02  75.2[+ or -]2.1**

Key: ALP = Alkaline phosphatase; AST = Aspartate transaminase; ALT =
Alanine amino transaminase; A-G = albumin/globulin ratio.
Values are mean[+ or -]SEM; *p<0.05; **p<0.01.