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Chemoprotective activity of an extract of Phyllanthus amarus against cyclophosphamide induced toxicity in mice.

Abstract

The effect of 75% methanolic extract of the plant Phyllanthus amarus (P. amarus) was studied against cyclophosphamide (CTX) induced toxicity in mice. Administration of CTX (25 mg/kg b.wt, i.p.) for 14 days produced significant myelosuppression as seen from the decreased WBC count and bone marrow cellularity. Administration of P. amarus extract at doses 250 and 750 mg/kg b.wt significantly reduced the myelosuppression and improved the WBC count, bone marrow cellularity as well as the number of maturing monocytes. CTX treatment also reduced the activity of glutathione system and increased the activity of phase I enzyme that metabolize CTX to its toxic side products. P. amarus administration was found to decrease the activity of phase I enzyme. Administration of P. amarus also increased the cellular glutathione (GSH) and glutathione-S-transferase (GST), thereby decreasing the effect of toxic metabolites of CTX on the cells. Administration of P. amarus did not reduce the tumor reducing activity of CTX. In fact, there was a synergistic action of CTX and P. amarus in reducing the solid tumors in mice. Results indicated that administration of P. amarus can significantly reduce the toxic side effects of CTX and is not interfering with the antitumor efficiency of CTX.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Chemo protection; Cyclophosphamide; Phyllanthus amarus; Toxicity; Antioxidant

Introduction

Cyclophosphamide (CTX) is one the most frequently used alkylating anti-neoplastic drug for the treatment of breast cancer, lymphomas, childhood tumors, and many solid tumors (Colvin, 1997). The most severe dose-limiting toxicity of CTX is fulminant cardiac toxicity. Other toxic side effects of CTX are hematopoietic depression, hemorrhagic cystitis, gonadal dysfunction, alopecia, nausea, gastrointestinal toxicity, renal toxicity, antidiuresis and vomiting (Slavin et al., 1975).

Recently there is an increase in interest in the search of potential drugs of plant origin that are capable of minimizing the toxicity induced by chemotherapy to normal cells with out compromising its anti-neoplastic activity. Traditional system of Indian medicine extensively uses the plant derived compounds and formulations to modulate the immune system of the host. These herbal formulations were found to be either less toxic or non-toxic.

Phyllanthus amarus Schum & Thonn (Kizharnelli in local language), family Euphorbiaceae, is used in Ayurvedic system of medicine to combat many liver disorders (Sane et al., 1995; Calixto et al., 1998) and is a well known anti-viral agent (Thyagarajan et al., 1990). Use of P. amarus in cancer prevention and treatment has been studied extensively in our laboratory (Rajeshkumar and Kuttan, 2000; Rajeshkumar et al., 2002). We had also shown the effect of P. amarus as a radio protective agent in mice exposed to lethal dose of [gamma]-radiation (Harikumar and Kuttan, 2004). The present study was aimed to investigate the use of P. amarus as a chemoprotective agent against CTX and the effect of administration P. amarus on solid tumor bearing mice treated with CTX.

Materials and methods

Cyclophosphamide (Ledoxan[R], Batch No. 2155) was obtained from Dabur India Ltd., New Delhi, Nicotinamide adenine dinucleotide phosphate reduced (NADPH), GSH, 5-5'dithiobis (2-nitrobenzoic acid) (DTNB), and 1-chloro-2,4-dinitrobenzene (CDNB) were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Bovine serum albumin (BSA) was obtained from E-Merck, Germany. Harris haematoxylin was purchased from Qualigens Chemicals, Mumbai. Dalton's lymphoma ascites (DLA) cells were originally obtained from Cancer Institute, Madras and propagated as transplantable ascites tumors in BALB/c mice. All other chemicals used in the present study were of analytical reagent grade.

Preparation of the plant extract

Aerial parts (stem and leaves) of authenticated P. amarus were collected from Thrissur district of Kerala State and were dried at 45[degrees]C. A voucher specimen of the plant was identified and kept in the herbarium (voucher no. Eup-9) of Amala Ayurvedic Hospital and Research Centre. Dried parts of P. amarus were powdered and extracted (100 g) twice with five times (500 ml) volume of 75% methanol by stirring overnight at the room temperature. The solution was then centrifuged at 2500 rpm to separate the supernatant and the supernatant was evaporated to dryness at 50[degrees]C using a rotary evaporator under reduced pressure. The yield of the preparation was 7.9%. Portion of the extract was reconstituted in distilled water before the experiment.

Animals

Inbred old male BALB/c mice (6-8 weeks) weighing 25-30 g were purchased from National Institute of Nutrition, Hyderabad. They were kept in well-ventilated cages under standard conditions at room temperature, pressure and humidity. The animals were provided with normal mouse chow (Sai Durga Feeds and Foods, Banglore, India) and water ad libitum. All animal experiments conducted during the study after getting prior permission and followed the guidelines of Institutional Animal Ethics Committee (IAEC).

Determination of the effect P. amarus on heamatological changes after CTX administration

24 mice were used for this experiment. Animals were randomly divided into four groups, six animals in each group. Group I was kept as normal animals. Groups II-IV received CTX at a dose of 25 mg/kg b.wt (i.p.) every day for 14 days. This dosage has been earlier shown to produce severe myelosuppression in mice (Praveenkumar et al., 1995). Oral administration of P. amarus (750 and 250 mg/kg b.wt, respectively) was started for groups III and IV began 5 days prior to CTX administration and continued for another 30 days. Body weights of all animals were recorded 1 day prior to CTX administration and every third day after CTX administration.

Blood was collected from the caudal vein into heparinised tubes 1 day prior to CTX administration and every third day thereafter and following parameters were checked. (a) Total WBC count (haemocytometer method): briefly 20 [micro]l of heparinised blood was mixed with 380 [micro]l of diluting fluid (100 ml of diluting fluid contain 1.5 ml glacial acetic acid, 1 ml 10% acetic acid and remaining distilled water) and counted on a haemocytometer (Nelson and Morris, 1984). (b) Differential count and (c) haemoglobin (cyanomethhemoglobin method).

Determination of the effect P. amarus on bone marrow cellularity and [alpha]-esterase activity after CTX administration

Forty-eight animals were randomly divided into four groups as given above having 12 animals in each group. Treatments for each group were same as described. On 3rd, 9th, 12th, and 30th day after CTX administration three animals from each group was sacrificed by cervical dislocation.

The bone marrow cells from both femurs were flushed into phosphate buffered saline containing 2% bovine calf serum. The number of bone marrow cells was determined using a haemocytometer and expressed as total live cells (x [10.sup.6])/femur. Bone marrow cells from the above preparation were immediately smeared on a clean slide and stained as per the method of Bancroft and Cook (1984) to determine the presence [alpha]-esterase activity, which was expressed as number of positive cells/4000 cells.

Determination of the effect P. amarus on phases I and II enzyme levels after CTX administration

Livers from above animals were excised quickly and washed thoroughly with ice-cold saline (0.89%) and kept in -70[degrees]C till further analysis. On the day of analysis 10% homogenate of liver tissue was made in ice-cold Tris buffer (0.1 M, pH 7.4) and the homogenate was centrifuged at 4[degrees]C at 12000 rpm for 30 min. The supernatant was used in the analysis. Total protein content was estimated by the method of Lowry et al. (1951) using BSA as the standard. The activity of phase I enzyme aniline hydroxylase activity was estimated by the modified method of Mazel (1971) and molar extinction co-efficient (284 n[M.sup.-1] [cm.sup.-1]) was used for calculating the activity. The activity of phase II enzyme GST activity was assayed by the method of Habig et al. (1974) and activity calculated using molar extinction coefficient of the product, which was 9.6 n[M.sup.-1] [cm.sup.-1]. GSH levels were assayed by the method of Moron et al. (1979) using DTNB and intensity of yellow colour formed was measured at 412 nm using a spectrophotometer.

Effect of administration of P. amarus on solid tumor reducing potential of CTX

Set of 42 mice was randomly divided into six groups with seven animals in each group.
Group I Control (untreated)
Group II CTX alone
Group III CTX + P. amarus 750 mg/kg b.wt p.o.
Group IV CTX + P. amarus 250 mg/kg b.wt p.o.
Group V P. amarus 750 mg/kg b.wt p.o.
Group VI P. amarus 250 mg/kg b.wt p.o.


Dalton's lymphoma ascites tumor (DLA) cells (1 X [10.sup.6] cells/animal) were injected subcutaneously to the right hind limb of each animal. Treatment of P. amarus and CTX started on the same day. CTX was administered at a dose of 10 mg/kg b.wt (i.p.) for 14 days and P. amarus administration continued for another 30 days. This lowered dose was used for the experiment to get only 50% reduction of tumor volume. The radii of developing tumor were measured using Vernier calipers for 30 days and tumor volume was calculated using the formula V = 4/3[pi][r.sub.1.sup.2][r.sub.2], were [r.sub.1] and [r.sub.2] denotes the radii of tumor at two different planes.

Statistical analysis

Data was expressed as mean [+ or -] standard deviation (SD). Significance levels for comparison of differences were determined using one-way ANOVA followed by Dunnet's test. Mean of the treated groups were compared with that of CTX alone group and p value [less than or equal to] 0.005 was considered to be significant.

Results

There was no significant change in the body weight of animals treated with CTX and CTX along with P. amarus treatment (data not shown).

Effect of P. amarus extract on heamatological parameters of CTX administered animals

CTX administration significantly reduced the total WBC count in mice (Fig. 1). Myelosuppression as seen from the WBC count was observed through out the period of CTX administration. P. amarus treated animals showed an increase in their WBC levels from day 12 onwards. On day 18 the CTX alone treated group showed a count of 3525 [+ or -] 294 while it was 5866 [+ or -] 265 in animals treated with CTX and P. amarus at a concentration of 750 mg/kg b.wt indicating that the levels of WBC was significantly elevated after P. amarus treatment. Differential count and haemoglobin content did not show any significant variation (data not shown) in both treated and untreated animals.

Fig. 2 shows the effect P. amarus administration on the bone marrow cellularity levels. Animals had a significantly lowered the bone marrow cellularity during CTX administration. But the continued administration of P. amarus increased the levels of bone marrow cellularity. On day 12 bone marrow cellularity was 6.63 [+ or -] 0.64 X [10.sup.6] cells for CTX alone treated animals where as animals treated with P. amarus (750 mg/kg b.wt) had an increased value of 11.16 [+ or -] 1.85 (p < 0.005) clearly indicating that bone marrow cell proliferation is significantly increased after P. amarus treatment.

The effect P. amarus treatment had a significant effect on the [alpha]-esterase activity of maturing monocytes. Levels of [alpha]-esterase positive cells were significantly decreased after CTX administration. P. amarus treatment elevated the number of [alpha]-esterase positive cells. On day 12 P. amarus treated group had 587.66 [+ or -] 18.61 (p < 0.005) positive cells/4000 cells while CTX alone administered group had only 205 [+ or -] 31.22 positive cells (Table 1).

Effect of P. amarus extract on the levels of phases I and II enzymes in CTX treated animals

We have also studied the effect of P. amarus extract in phases I and II enzymes of animals treated with CTX. Phase I enzymes are involved in the activation of CTX where as phase II enzymes produces the detoxification of CTX derived metabolites. The effect of P. amarus administration on the levels of phase I enzyme aniline hydroxylase is shown in Table 2. Administration of CTX elevated the levels of the enzyme significantly. P. amarus treatment lowered the levels of aniline hydroxylase as evident from lowered enzyme activity. On day 12 activity of P. amarus (750 mg/kg, wt) treated group was 0.168 [+ or -] 0.016 (p < 0.005) while CTX alone treated group showed a value of 0.208 [+ or -] 0.001.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

CTX administration was also found to decrease the levels of GSH (Table 3). Continuous administration of P. amarus elevated the levels of GSH in treated groups. On day 12 CTX alone treated animals had lowered GSH level of 6.69 [+ or -] 1.18 nmol/ml as compared to the GSH level of 14.40 [+ or -] 1.31 in P. amarus (750 mg/kg b.wt) treated animals (p < 0.005).

The activity of the phase II enzyme GST is shown in Table 4. On day 12 animals administered with P. amarus showed a significant activity of 467.97 [+ or -] 17.91 (p < 0.005) while CTX alone treated animals showed an activity of 298.60 [+ or -] 21.39 indicating that GST activity was significantly elevated after P. amarus administration.

Effect of P. amarus on solid tumor reduction in animals produced by CTX

The effect of P. amarus on solid tumor reduction in animals treated with CTX is given in Fig. 3. The administered dose of CTX was found to be effective in controlling tumor growth. The administration of P. amarus did not have any inhibitory effect on CTX mediated tumor reduction. On the other hand P. amarus administration produced a synergistic effect on tumor growth inhibition. P. amarus administered at dose of 750 mg/kg b.wt on CTX treated animals had lowest tumor volume among all the groups.

Discussion

Alkylating agents were among the first compounds to be identified useful in cancer chemotherapy. All the alkylating agents have a common property of dissociating a positive charged, electrophilic alkyl group capable of attacking negatively charged electron rich, nucleophilic sites on most of the biological molecules. The chemotherapeutic usefulness of alkylating agents derives from their ability to form a variety of DNA adducts that sufficiently alter DNA structure or function or both so as to have a cytotoxic effect on the cells. Many of them undergo a very complex activation process before it can generate reactive intermediates. Initial activation reaction of CTX carried out by microsomal oxidation system in liver produces 4-hydroxy CTX, a cytotoxic metabolite, which diffuses from hepatocytes into plasma and distributed throughout the body. 4-hydroxy CTX is then further converted to other cytotoxic metabolites such as acrolein and phosphoramide mustard (Berger, 1993; Grochow, 1996). Phosphoramide mustard is known to cause myelosuppression. In the present study the myelosuppression caused by CTX is effectively prevented by P. amarus. The P. amarus treated animals also showed enhanced levels of bone marrow cellularity, which indicate that P. amarus stimulate the heamatopoetic system and this observation is further supported by the increased number of [alpha]-esterase positive cells, a marker of maturing monocytes.

[FIGURE 3 OMITTED]

P. amarus was found to suppress the activity of phase I enzyme aniline hydroxylase. Phase I enzymes play a role in the activation of CTX. P. amarus was also found to significantly elevate the activity of phase II enzyme GST and levels of the one of the major cellular nonenzymatic antioxidant GSH, which is involved in the detoxification of toxic electrophilic xenobiotics, hydrogen peroxide and free radicals. The metabolism of CTX in the body produces highly reactive electrophiles and the decreased value of GSH in CTX treated group is probably due to the electrophilic burden on the cells and also due to the formation of acrolein, which is known to deplete GSH content and DNA alkylation (McDiarmid et al., 1991). Treatment with P. amarus reduces the electrophilic burden and thereby increases GSH levels. Elevated GSH levels in the cells further conjugate with the electrophiles and decrease their toxicity. The liver cytosolic detoxifying enzyme GST is also involved in the removal of toxic metabolites (Dirven et al., 1994; Gul et al., 2000; Rana et al., 2002). GST level has been shown to be increased by P. amarus treatment making cells more effective with respect to detoxification of toxic metabolites. P. amarus has been shown to be a good antioxidant in vitro as well as in vivo and a powerful scavenger of oxygen radicals (Joy and Kuttan, 1995).

The solid tumor model clearly demonstrated that P. amarus does not interfere the antitumor efficacy of CTX, on the other hand administration of P. amarus on CTX treated animals had a synergistic action on the inhibition of tumor growth.

P. amarus contain several active ingredients. Presence of variety of tannins, several lignans like phyllanthin and hypophyllanthin, polyphenols, flavinoids such as quercetin, astragalin and some ellgitannins like catechin and epigallocatechin were isolated from P. amarus (Foo, 1995). Many plant phenolic compounds show excellent antioxidant activities. Although the exact mechanism of action P. amarus is not clear, the combined action of extract is manifested as a sum total of interactions between different ingredients. The degree of chemo protection will depend on the interactions of the ingredients singly or collectively with the cytotoxic agents.

In conclusion P. amarus exert its chemo protection by detoxification of CTX derived toxic metabolites, enhancing the recovery and repair process, accelerating the hematopoietic recovery and down regulating the activity of phase I enzyme and up regulating the activity of phase II enzymes and GSH. Moreover scavenging the free radicals or reactive metabolites by P. amarus with out affecting the antitumor efficacy of CTX would be beneficial to the host as well as will enhance the efficiency of the treatment. P. amarus has been found to inhibit the proliferation of cancer cells during the process of carcinogenesis. P. amarus extract was also found to produce significant protection against radiation. Hence P. amarus could act both as a chemo protector, tumor growth inhibiter and as an efficient radio protector. A detailed clinical study to fully exploit the potential of P. amarus in cancer is highly warranted.

Acknowledgements

Authors are thankful to Ms. Santha Bai, Dept. Statistics, College of Veterinary animal Sciences, Thrissur for statistical analysis.

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K.B.H. Kumar, R. Kuttan*

Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala State, India

Received 20 November 2003; accepted 10 March 2004

*Corresponding author. Tel.: +91 487 2307950; fax: +91 487 2307868.

E-mail address: amalaresearch@rediffmail.com (R. Kuttan).
Table 1. Effect of P. amarus treatment on the number of [alpha]-esterase
positive cells (positive cells/4000 cells) of CTX administered animals

Group 3rd day 9th day

Normal 941.00 [+ or -] 20.66 948.66 [+ or -] 24.78
CTX alone 258.33 [+ or -] 17.55 189.00 [+ or -] 11.53
CTX + P. amarus 483.00 [+ or -] 20.95* 527.66 [+ or -] 18.61*
 750 mg/kg b.wt
CTX + P. amarus 410.66 [+ or -] 19.21* 451.66 [+ or -] 24.66*
 250 mg/kg b.wt

Group 12th day 30th day

Normal 916.66 [+ or -] 25.16 965.66 [+ or -] 29.14
CTX alone 205.00 [+ or -] 31.22 637.00 [+ or -] 33.51
CTX + P. amarus 593.33 [+ or -] 16.50* 878.66 [+ or -] 22.27*
 750 mg/kg b.wt
CTX + P. amarus 469.33 [+ or -] 23.15* 843.33 [+ or -] 42.24*
 250 mg/kg b.wt

Values are expressed as mean [+ or -] SD. Statistical significance of
the treatment was done using one-way ANOVA followed by Dunnet's test.
*p [less than or equal to] 0.005 against CTX alone group (Dunnet's
test).

Table 2. Effect of P. amarus treatment on the aniline hydroxylase levels
([micro]mol/min/mg protein) of CTX administered animals

Group 3rd day 9th day

Normal 0.220 [+ or -] 0.02 0.211 [+ or -] 0.01
CTX alone 0.266 [+ or -] 0.10 0.240 [+ or -] 0.02
CTX + P. amarus 0.188 [+ or -] 0.01* 0.218 [+ or -] 0.004*
 750 mg/kg b.wt
CTX + P. amarus 0.182 [+ or -] 0.01* 0.213 [+ or -] 0.01*
 250 mg/kg b.wt

Group 12th day 30th day

Normal 0.216 [+ or -] 0.01 0.195 [+ or -] 0.02
CTX alone 0.208 [+ or -] 0.006 0.240 [+ or -] 0.10
CTX + P. amarus 0.168 [+ or -] 0.02* 0.195 [+ or -] 0.20
 750 mg/kg b.wt
CTX + P. amarus 0.182 [+ or -] 0.02* 0.208 [+ or -] 0.003
 250 mg/kg b.wt

Values are expressed as mean [+ or -] SD. Statistical significance of
the treatment was done using one-way ANOVA followed by Dunnet's test.
*p[less than or equal to] 0.005 against CTX alone group (Dunnet's test).

Table 3. Effect of P. amarus treatment on the liver GSH levels (nmol/mg
protein) of CTX administered animals

Group 3rd day 9th day

Normal 9.23 [+ or -] 1.12 9.47 [+ or -] 0.57
CTX alone 6.21 [+ or -] 0.62 6.61 [+ or -] 1.57
CTX + P. amarus 14.91 [+ or -] 1.96* 18.86 [+ or -] 1.71*
 750 mg/kg b.wt
CTX + P. amarus 12.65 [+ or -] 1.32* 14.62 [+ or -] 1.52*
 250 mg/kg b.wt

Group 12th day 30th day

Normal 11.56 [+ or -] 1.45 8.11 [+ or -] 0.96
CTX alone 6.69 [+ or -] 1.18 10.70 [+ or -] 1.51
CTX + P. amarus 14.40 [+ or -] 1.31* 12.02 [+ or -] 3.20
 750 mg/kg b.wt
CTX + P. amarus 13.70 [+ or -] 1.12* 10.46 [+ or -] 1.29
 250 mg/kg b.wt

Values are expressed as mean [+ or -] SD. Statistical significance of
the treatment was done using one-way ANOVA followed by Dunnet's test.
*p [less than or equal to] 0.005 against CTX alone group (Dunnet's
test).

Table 4. Effect of P. amarus treatment on the liver GST (nmoles of
CDNB-GSH conjugate formed/min/mg protein) levels of CTX administered
animals

Group 3rd day 9th day

Normal 243.85 [+ or -] 18.21 212.43 [+ or -] 24.10
CTX alone 339.25 [+ or -] 15.34 324.49 [+ or -] 21.40
CTX + P. amarus 459.17 [+ or -] 37.95 543.53 [+ or -] 37.15*
 750 mg/kg b.wt
CTX + P. amarus 315.73 [+ or -] 40.52 419.78 [+ or -] 20.09
 250 mg/kg b.wt

Group 12th day 30th day

Normal 282.05 [+ or -] 27.89 249.23 [+ or -] 46.48
CTX alone 298.60 [+ or -] 21.39 309.95 [+ or -] 30.04
CTX + P. amarus 467.97 [+ or -] 17.31* 289.46 [+ or -] 26.36
 750 mg/kg b.wt
CTX + P. amarus 360.28 [+ or -] 20.57** 263.21 [+ or -] 6.25
 250 mg/kg b.wt

Values are expressed as mean [+ or -] SD. Statistical significance of
the treatment was done using one-way ANOVA followed by Dunnet's test.
*p [less than or equal to] 0.005 against CTX alone group (Dunnet's
test). **p [less than or equal to] 0.01.
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Author:Kumar, K.B.H.; Kuttan, R.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Jun 1, 2005
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