Chemomodulatory efficacy of Basil leaf (Ocimum basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen-induced skin and forestomach papillomagenesis.
Basil or sweet basil (Ocimum basilicum) is cultivated throughout India and is known for its medicinal value. The effects of doses of 200 and 400 mg/kg body weight of hydroalcoholic extract (80% ethanol, 20% water) of the fresh leaves of Ocimum basilicum on xenobiotic metabolizing Phase I and Phase II enzymes, antioxidant enzymes, Glutathione content, Lactate dehydrogenase and lipid peroxidation in the liver of 8-9 weeks old Swiss albino mice were examined. Furthermore, the anticarcinogenic potential of basil leaf extract was studied, using the model of Benzo(a)pyrene-induced forestomach and 7,12 dimethyl benz(a)anthracene (DMBA)-initiated skin papillomagenesis. The hepatic glutathione S-transferase and DT-diaphorase specific activities were elevated above basal level by basil leaf treatment (from p < 0.005 to p < 0.001). Basil leaf extract was very effective in elevating antioxidant enzyme response by increasing significantly the hepatic glutathione reductase (GR) (p < 0.005), superoxide dismutase (SOD) (p < 0.05), and catalase activities (p < 0.005). Reduced glutathione (GSH), the major intracellular antioxidant, showed a significant elevation in the liver (p < 0.005) and also in all the extrahepatic organs (from p < 0.05 to p < 0.005). In the forestomach, kidney and lung, glutathione S-transferase and DT-diaphorase levels were augmented significantly, varying from p < 0.01 to p < 0.001. There were significant decreases in lipid peroxidation and lactate dehydrogenase activity. Chemopreventive response was evident from the reduced tumor burden (the average number of papillomas/mouse, p < 0.005 to p < 0.001), as well as from the reduced percentage of tumor bearing-animals. Basil leaf, as deduced from the results, augmented mainly the Phase II enzyme activity that is associated with detoxification of xenobiotics, while inhibiting the Phase I enzyme activity. There was an induction in antioxidant level that correlates with the significant reduction of lipid peroxidation and lactate dehydrogenase formation. Moreover, Basil leaf extract was highly effective in inhibiting carcinogen-induced tumor incidence in both the tumor models at peri-initiational level.
Key words: Chemoprevention, basil leaf (Ocimum basilicum), xenobiotic-metabolizing enzymes, skin and forestomach papillomagenesis, antioxidant defense
Although still in its infancy, the new science of chemoprevention has been established as an important approach to control malignancy. In recent years it has been shown that a large number of naturally occurring compounds from vegetables and fruits are effective as chemopreventive agents. An advantage of diet-derived products for cancer prevention is that they also have apparent benefit in other chronic diseases (Chemoprevention working group 1999; Sporn and Suh, 2000; Hakama, 1998; Kellof, 2000). These agents are expected to act by preventing activation of carcinogens or by increasing detoxification, or by blocking the interaction of the ultimate carcinogen with cellular macromoles, or by suppressing the clonal expression of neoplastic cell (Wattenberg, 1985; Tanaka, 1994; Morse and Stoner 1996; Dasgupta et al. 2001). Such substances maybe useful in the chemoprevention of cancer in humans (Kelloff et al. 2000; Murakami et al. 1996; Pezzutto 1995; Ren and Liece, 1997).
Ocimum basilicum (family Labiatae), employed traditionally as folklore remedy for a wide spectrum of ailments, is also incorporated into a number of herbal medicinal preparations in India. It is used for treating cold and coughs. There are many varieties of Ocimum sp. and numerous laboratory studies have shown various protective effects including radiation protection (Uma Devi et al. 2000), chemopreventive activity (Prakash and Gupta, 2000), anti-inflammatory activity (Klem et al. 2000), a nervous system stimulant effect (Maity et al. 2000), bactericidal activity (Koga et al. 1999), modulatory effect on glutathione and antioxidant enzymes (Devi and Ganasoundari, 1999), anti-ulcer activity (Singh, 1999), antidiarrheal effects (Offiah and Chikwendu, 1999), and blood-sugar lowering (Chattopadhyay, 1999), among others.
The inducibility of drug-metabolizing enzymes is one of the reliable biochemical markers to assess the chemopreventive potential of a test compound (Prochaska and Fernandes, 1993; Banerjee and Rao, 1995; Shijun et al. 2000). In the present study, an 80% ethanolic extract of basil leaf was used to evaluate these enzymes in the livers of mice, because the liver plays the pivotal role of carrying out various important reactions (Omura and Takasue, 1970). Antioxidant enzymes are the counterparts of oxidative damage that protect the cell (Shijun et al. 2000). The levels of hepatic antioxidant defense enzymes, comprising superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were therefore monitored. In addition to reduced glutathione (GSH), the intracellular antioxidant level was also evaluated. Lipid peroxidation and lactate dehydrogenase were measured in the microsome and cytosolic fractions, respectively, to evaluate free-radical-formation and cell-damage parameters. Furthermore the influence of basil leaf extract in increasing the detoxifying capabilities of extrahepatic organs such as the lung, kidney, and forestomach, has been examined.
Skin serves as a barrier against the deleterious effects of environmental factors. The most frequent skin cancers are melanomas, basal-cell cancers and squamous-cell cancers. For more than 50 years, the multistage model of carcinogenesis in mouse skin has provided a conceptual framework within which to study the carcinogenic process in tissues of epithelial origin. Stomach cancer is one of the most common forms of cancer in the world. Therefore, the modulatory influence of basil leaf on DMBA-induced skin and benzo(a)pyrene-induced forestomach papillomagenesis at the peri-initiational level was also evaluated. [B(a)P], employed in initiating
stomach cancer, is the prototypical and best-characterized member of the polycyclic aromatic hydrocarbon (PAH) family of chemical carcinogens, which are widespread in the environment and are suspected human carcinogens. So it becomes very important to identify compounds which can interfere with B(a)P-induced carcinogenesis.
Materials and Methods
Benzo(a)pyrene [B(a)P], 7,12-dimethylbenzanthracene (DMBA), 1-Chloro-2,4-dinitrobenzene (CDNB), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), oxidized glutathione (GSSG), pyrogallol, 2,6-dichlorophenolindophenol (DCPIP), potassium ferricyanide, triton X-100, ethylenediamine tetracetic acid (EDTA), bovine serum albumin (BSA), sodium pyruvate, thiobarbituric acid (TBA), reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The rest of the chemicals utilized were obtained from local firms (India) and were of the highest purity grade.
Modulator: Fresh basil leaf (Ocimum basilicum) was obtained from the nursery of the School of Life Sciences, JNU campus, where it was specially grown for this particular research purpose. The leaves were rinsed with water and blotted dry. Material of known weight was Soxheleted using 80% hydroalcoholic solvent (80% ethanol, 20% double distilled water) three times. Finally, the extract was lyophilized and stored at 4 [degrees]C.
Random-bred Swiss albino mice (8-9 weeks old) were used for this study. They were maintained in our airconditioned animal facility (Jawaharlal Nehru University, New Delhi) with a 12-h light/dark cycle, and provided (unless otherwise stated) with standard food pellets and tap water ad libitum. All animals were cared for according to the "Principles of Laboratory Animal care" of the National Institutes of Health (NIH, USA) and under strict adherence with the Indian Animal Ethics Committee (IAEC) guidelines.
Three separate, independent experiments were carried out to delineate specific objectives of the study as mentioned earlier; whereas Experiment I was limited to studying the inducibility of detoxifying parameters in hepatic and extrahepatic organs, Experiments II and III were aimed at evaluating the putative efficacy of the modulator in chemoprevention in the mouse skin and forestomach tumorigenesis models.
Modulation of hepatic and extrahepatic carcinogen-metabolizing and antioxidant enzymes.
Animals were assorted randomly into the following groups:
* Group--I (n = 8): Animals were put on a normal diet and treated with 50ml of an emulsion of peanut oil and double-distilled water (ratio 4:1 ml) used as vehicle for feeding the modulator, by oral gavage daily for 15 days; this group of animals served as a negative control.
* Group--II (n = 8): Animals were put on a normal diet and treated daily with 200 mg/kg body wt. of lyophilized basil leaf extract, which was dissolved in an emulsion of peanut oil and double-distilled water (ratio 4:1 ml), and was given to the mice (50 ml/mouse/day) by oral gavage for 15 days.
* Group--III (n = 8): Animals were put on a normal diet and treated daily with 400 mg/kg body wt. of lyophilized basil leaf extract, which was dissolved in an emulsion of peanut oil and double-distilled water (ratio 4:1 ml), and was given to the mice (50 ml/mouse/day) by oral gavage for 15 days.
Preparation of homogenates, cytosol and microsome fractions
Animals were sacrificed by cervical dislocation and the entire liver was then perfused immediately with cold 0.9% NaCl and thereafter carefully removed, trimmed free of extraneous tissue and rinsed in chilled 0.15 M Tris-KCl buffer (0.15 M KCl + 10 mM Tris-HCL, pH 7.4). The liver was then blotted dry, weighed quickly and homogenized in ice-cold 0.15 M Tris-KCl buffer (pH 7.4) to yield 10% (w/v) homogenate. An aliquot of this homogenate (0.5 ml) was used to assay reduced glutathione levels, while the remainder was centrifuged at 10,000 rpm for 20 min. The resultant supernatant was transferred into pre-cooled ultracentrifugation tubes and centrifuged at 105,000 X g for 60 min in a Beckman ultracentrifuge (Model--L870M). The supernatant (cytosol fraction), after discarding any floating lipid layer and appropriate dilution, was used for the assay of glutathione S-transferase, DT-diaphorase, lactate dehydrogenase and antioxidant enzymes, whereas the pellet representing microsomes was suspended in homogenizing buffer and used for assaying cytochrome [P.sub.450], cytochrome [b.sub.5], cytochrome [P.sub.450] reductase, cytochrome [b.sub.5] reductase and lipid peroxidation.
The lung, kidney and forestomach were removed carefully, along with the liver, trimmed free of extraneous tissue and rinsed in chilled 0.15 M Tris-KCl (pH 7.4). The lung was cut into small pieces. The stomach was opened longitudinally, the forestomach was separated from the glandular stomach and cleaned of all its contents by flushing with and changing the buffer 5-6 times. The lung, kidney and forestomach were then blotted dry, weighed quickly and homogenized in icecold 0.15 M Tris-KCl buffer (pH 7.4) to yield a 10% (w/v) homogenate. An 0.5-ml aliquot of this homogenate was used to assay reduced glutathione. The rest of the homogenate was centrifuged at 15,000 X g for 30 min at 4 [degrees]C; the resulting supernatant obtained was used to assay glutathione S-transferase (GST) and DT-diaphorase enzymes.
* Cytochrome [P.sub.450] and cytochrome [b.sub.5]: Cytochrome [P.sub.450] was determined using carbon monoxide difference spectra. Both cytochrome [P.sub.450] and cytochrome [b.sub.5] content were assayed in the microsomal suspension according the method of Omura and Sato (1964), using absorption coefficients of 91 and 185 c[m.sup.2] [M.sup.-1] [m.sup.-1], respectively.
* NADPH-cytochrome [P.sub.450] reductase and NADH cytochrome [b.sub.5] reductase: An assay of NADPH-cytochrome [P.sub.450] reductase was performed according to the method of Omura and Takesue (1970), with some modifications, measuring the rate of oxidation of NADPH at 340 nm. The reaction mixture contained 0.3 M potassium phosphate buffer (pH 7.5), 0.1 mM NADPH, 0.2 mM potassium ferricyanide and the microsomal preparation in a final volume of 1 ml. The reaction started at 25 [degrees]C by addition of NADPH. The enzyme activity was calculated using the extinction coefficient 6.22 m[M.sup.-1] c[m.sup.-1]. One unit of enzyme activity is defined as that causing the oxidation of one mole of NADPH per minute.
NADH-cytochrome [b.sub.5] reductase was assayed according to the method of Mihara and Sato (1972), measuring the rate of reduction of potassium ferricyanide at 420 nm by NADH. The reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.5), 0.1 mM NADH, 1 mM potassium ferricyanide and microsomal preparation in a final volume of 1 ml. The reaction was started at 25 [degrees]C by addition of NADH. The enzyme activity was calculated using an extinction coefficient of 1.02 m[M.sup.-1] c[m.sup.-1]. One unit of enzyme activity is defined as that causing the reduction of one mole of ferricyanide per minute.
* Glutathione S-transferase: The cytosolic and supernatant of extrahepatic glutathione S-transferase activity were determined spectrophotometrically at 37 [degrees]C according to the procedure of Habig et al. (1974). The reaction mixture (3 ml) contained 1.7 ml of 100 mM phosphate buffer (pH 6.5), 0.1 ml of 30 mM CDNB and 0.1 ml of 30 mM of reduced glutathione. After preincubating the reaction mixture at 37 [degrees]C for 5 min, the reaction was started by the addition of 0.1 ml diluted cytosol and the absorbance was followed for 5 min at 340 nm. The reaction mixture without the enzyme was used as blank. The specific activity of glutathione S-transferase is expressed as mmoles of GSH-CDNB conjugate formed/min/mg protein using an extinction coefficient of 9.6 m[M.sup.-1] c[m.sup.-1].
* DT-diaphorase: DT-diaphorase activity was measured as described by Ernest et al. (1962), with NADH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) as the electron acceptor at 600 nm. The activity was calculated using an extinction coefficient of 21 m[M.sup.-1] c[m.sup.-1]. One unit of enzyme activity has been defined as the amount of enzyme required to reduce one mole of DCPIP per min.
* Reduced glutathione: Reduced glutathione was estimated as the total non-protein sulphydryl group following the method described by Moron et al. (1979). Homogenates were immediately precipitated using 0.1 ml 25% trichloroacetic acid and the precipitate was removed following centrifugation. Free -SH groups were assayed in a total volume of 3 ml by adding 2 ml of 0.6 mM DTNB prepared in 0.2 M sodium phosphate buffer (pH 8.0), to 0.1 ml of the supernatant and absorbance was read at 412 nm using a Shimadzu UV-160 spectrometer. GSH was used as a standard to calculate mmole -SH content/g tissue.
* Glutathione reductase: Glutathione reductase was determined following the procedure described by Carlberg and Mannervick (1985). The reaction mixture (final volume 1 ml) contained 0.2 M sodium phosphate buffer (pH 7.0), 2 mM EDTA, 1 mM oxidized glutathione (GSSG) and 0.2 mM NADPH. The reaction was started by adding 25 1 cytosol and the enzyme activity was measured indirectly by monitoring the oxidation of NADPH following a decrease in OD/min for a minimum of 3 min at 340 nm. One unit of enzyme activity has been defined as nmoles NADPH consumed/min/mg protein based on an extinction coefficient of 6.22 m[M.sup.-1] c[m.sup.-1].
* Glutathione peroxidase: Glutathione peroxidase activity was measured using the coupled assay method as described by Paglia and Valentine (1967). Briefly, 1 ml of the reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.24 U/ml yeast glutathione reductase, 0.3 mM glutathione (reduced), 0.2 mM NADPH, 1.5 mM [H.sub.2][O.sub.2] and cytosol sample. The reaction was initiated by adding NADPH and its oxidation was monitored at 340 nm by observing the decrease in OD/min for 3 min. One unit of enzyme activity has been defined as nmoles of NADPH consumed/min/mg protein based on an extinction coefficient of 6.22 m[M.sup.-1] c[m.sup.-1].
* Catalase: Catalase was estimated at 240 nm by monitoring the disappearance of [H.sub.2][O.sub.2] as described by Aebi (1984). The reaction mixture (1 ml, vol.) contained 0.02 ml of suitably diluted cytosol in phosphate buffer (50 mM, pH 7.0) and 0.1 ml of 30 mM [H.sub.2][O.sub.2] in phosphate buffer. Catalase enzyme activity has been expressed as moles of [H.sub.2][O.sub.2] reduced/min/mg protein.
* Superoxide dismutase: Superoxide dismutase was assayed using the technique of Marklund and Marklund (1974), which involves inhibition of pyrogallol autoxidation at pH 8.0. A single unit of enzyme was defined as the quantity of superoxide dismutase required to produce 50% inhibition of autooxidation.
* Lipid peroxidation: Lipid peroxidation in the microsomes was estimated spectrophotometrically using the thiobarbituric acid reactive substances (TBARS) method, as described by Varshney and Kale (1990), and is expressed in terms of malondialdehyde (MDA) formed per mg protein. In brief, 0.4 ml of microsomal sample was mixed with 1.6 ml of 0.15 M Tris KCl buffer, to which 0.5 ml 30% TCA was added. Then 0.5 ml of 52 mM TBA was added and placed in a water bath for 45 min at 80 [degrees]C, cooled in ice and centrifuged at room temperature for 10 min at 3,000 rpm. The absorbance of the clear supernatant was measured against the blank of distilled water at 538.1 nm in a spectrophotometer (Hitachi U-2000).
* Lactate dehydrogenase: lactate dehydrogenase was assayed by measuring the rate of oxidation of NADH at 340 nm, according to the method of Bergmeyer and Bernt (1971). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.5), 0.5 mM sodium pyruvate, 0.1 mM NADH and the required amount of cytosolic fraction in a final volume of 1 ml. The reaction was started at 25 [degrees]C by addition of NADH and the rate of oxidation of NADH was measured at 340 nm. The enzyme activity was calculated using an extinction coefficient of 6.22 m[M.sup.-1] c[m.sup.-1]. One unit of enzyme activity is defined as that which causes the oxidation of one mole of NADH per min.
* Protein determination: Protein was determined following the method of Lowry et al. (1951) Using bovine serum albumin (BSA) as a standard at 660 nm.
Influence of Ocimum basilicum on DMBA-induced mouse skin papillomagenesis.
Preparations of chemicals and modulator
DMBA was dissolved at a concentration of 0.05 mg in 0.05 ml acetone. Basil leaf suspension was prepared in a mixture of acetone and double-distilled water (ratio 1:1 ml) and was administered topically to mice at two different dose levels, 150 and 300 mg/kg body wt./day.
DMBA-induced skin papillomagenesis was studied in Swiss albino mice as described by Yasukawa et al. (1995), with some modifications. The hairs on the dorsum (2 cm diameter) of the mice were clipped off three days before the application of the chemicals, and animals in the resting phase of the hair growth cycle were selected for the experiment. The animals were sorted into the following groups:
* Group I (n = 17): Animals were treated topically (0.1 ml) for 21 days with the mixture of acetone + double-distilled water (Ratio 1:1 ml) which was used as vehicle for suspending the modulator. On day 14, a single dose of DMBA (0.05 mg/0.05 ml acetone) was applied on the shaven area. Two weeks after the carcinogen application, 0.05 ml of 1% croton oil in acetone was applied twice a week until termination of the experiment. This group of mice served as the positive control group.
* Group II (n = 17): All animals in this group were treated topically (0.1 ml) with basil leaf extract suspended in the control vehicle at a dose of 150 mg/kg body wt for 21 days. On day 14, DMBA (0.05 mg/0.05 ml acetone) was applied topically to these animals in the shaven area (after a minimum gap of 6 h after treatment with the modulator) followed by croton oil treatment as given to Group I mice.
* Group III (n = 17): All animals in this group were treated topically (0.1 ml) with a high dose (300 mg/kg body wt) of basil leaf extract suspended in the control vehicle for 21 days. On day 14, DMBA (0.05 mg/0.05 ml acetone) was applied topically to these animals in the shaven area (after a minimum gap of 6 h after treatment with the modulator) followed by croton oil treatment as given to Group I mice.
Animals were weighed initially, and then weekly, and finally at autopsy. Papillomas appearing in the shaven area were recorded at weekly intervals and papillomas >1 mm in diameter were included in data analysis only if they persisted for 2 weeks or more. Animals were sacrificed 20 weeks after commencement of the treatments.
Influence of Ocimum basilicum on benzo(a)pyrene induced mouse fore-stomach papillomagenesis was studied as described by Azuine and Bhide (1992). This is a modification of a method described originally by Wattenberg et al. (1980).
Preparations of chemicals and modulator
Benzo(a)pyrene [B(a)P] was prepared in peanut oil and the concentration adjusted to 1 mg B(a)P/0.1 ml of peanut oil. Lyophilized Basil leaf extract emulsion was prepared in the mixture of peanut oil and double-distilled water (ratio 4:1 ml) at two dose levels, 200 and 400 mg/kg body wt.
The animals were sorted into the following groups:
* Group I (n = 20): Animals were given a mixture of peanut oil and double-distilled water (ratio 4:1 ml) for eight weeks through oral gavage, used as vehicle for feeding the modulator. Two weeks after initiation of vehicle treatment, each mouse received 1 mg B(a)P twice weekly for four weeks. This group of mice served as positive control group.
* Group II (n = 20): Animals were given the basil leaf extract, suspended in the control vehicle at a dose of 200 mg/kg body wt/day, starting two weeks before, during and after the carcinogen treatment (given as 1 mg B(a)P twice weekly for four weeks) as given to Group I animals.
* Group III (n = 20): Animals were given basil leaf extract, suspended in the control vehicle at a dose of 400 mg/kg body wt/day, starting two weeks before, during and after the carcinogen treatment as given to Group II animals.
After calculating the mean and the standard deviation, the Mann-Whitney Rank Sum test was performed to obtain the significant difference among groups. A value of p < 0.05 was considered significant.
Hepatic studies: Body and organ weight and general observations
Body weight and relative liver weight at the termination of the experiment have been summarized in Table 1. There is no significant difference in either the weight gain profile or terminal body weight, and the corresponding values of low and high doses were comparable to the control. The oral and topical administration of basil leaf extract did not cause any apparent changes in clinical signs such as survivability, or any gross visible changes attributable to toxicity in the liver, lung or kidney of mice.
The activity of LDH was diminished in both doses: the low dose by 0.66 fold (p < 0.005), and the high dose by 0.75 fold (p < 0.005) as compared to control group.
Lipid peroxidation (LP), measured as formation of MDA production, showed significant inhibition at both dose levels with basil leaf treatment. LP was reduced by 0.80 fold (p < 0.01) at low dose and by 0.74 fold (p < 0.005) at high dose, as compared to the control group.
Phase I enzymes
The major phase I enzymes, cytochrome [P.sub.450] and cytochrome [b.sub.5] work as an important cofactor component of mono-oxygenase enzymatic activity. Cytochrome [P.sub.450] was reduced significantly, by 0.85 fold (p < 0.05) at low dose and by 0.81 fold (p < 0.005) at high dose following the basil leaf treatment. The low-dose treatment revealed no significant alterations from its basal constitutive activities in the case of Cyt [b.sub.5], although the high dose treatment caused a significant decrease of 0.78 fold (p < 0.005).
Cytochrome [P.sub.450] reductase and cytochrome [b.sub.5] reductase
These enzymes were determined in hepatic microsomes. Relative to control, the low and high dose decreased the specific activity of cytochrome [b.sub.5] reductase by 0.83 fold (p < 0.001) and 0.62 fold (p < 0.001), respectively. In the case of cytochrome [P.sub.450] reductase, decrease of 0.73 fold (p < 0.005) and 0.62 fold (p < 0.001) for the low and high doses, as compared to control, was determined.
Phase II enzymes
Specific activities of both phase II enzymes studied, glutathione S-transferase (GST) and DT-diaphorase (DTD), showed significant increases at both dose levels of treatment with basil leaf extract, with respect to the control group.
Relative to the level in untreated control animals, glutathione S-transferase enzyme activity was enhanced by 1.4 fold (p < 0.005) and 1.6 fold (p < 0.001) in the low-dose and high-dose groups respectively.
Specific DTD activity was increased by 1.29 fold (p < 0.001) and 1.42 fold (p < 0.001) at low and high doses, respectively, compared to control.
The hepatic superoxide dismutase activity in extract-treated mice was increased by 1.11 fold (p < 0.05) and 1.14 fold (p < 0.05) in the low and high doses, respectively as compared to control.
Both doses of basil leaf extract investigated, augmented the specific activity of glutathione peroxidase enzyme significantly, relative to control basal levels. At low-dose treatment, an induction of 1.22 fold (p < 0.005), and at high-dose treatment an induction of 1.4 fold (p < 0.005), was evident.
Catalase activity was increased, at the low and high doses, by 1.56 fold (p < 0.005) and 1.58 fold (p < 0.005), respectively relative to control.
Both doses of basil leaf extract investigated, increased glutathione reductase activity in the liver cytosol of mice over that of the control animals by 1.16 fold (p < 0.005, Group I vs. Group II) and by 1.28 fold (p < 0.005, in Group I vs. Group III).
The level of GSH, the non-enzymatic antioxidant protein was enhanced in both basil-leaf-extract treated groups. At low dose administration, the basal reduced glutathione was increased by 2.08 fold (p < 0.005), whereas the higher dose induced a 2.0 fold (p < 0.005) increase in reduced glutathione concentration.
Results of the analysis of cytosolic protein from different experimental groups showed no significant statistical difference in basil leaf treatment. There was, however, a significant increase caused by high-dose of basil leaf treatment (1.57 fold, p < 0.005) in the microsomal protein value.
Pre-treatment with basil leaf extract caused a significant and dose dependent increase in the reduced glutathione level in all the extrahepatic organs examined. In the forestomach, the activity of GSH was increased by 1.23 fold at the low dose and by 1.3 fold (p < 0.05) at the high dose. In the kidney, we found an increase of 1.12 (p < 0.005) and 1.36 (p < 0.005) folds at the lower and higher doses, respectively. Lung GSH activity was augmented by 1.72 fold (p < 0.01) at the low dose and by 1.74 fold (p < 0.005) at the high dose, as compared with the control group.
DTD-specific enzyme activity evaluated in extrahepatic organs revealed a significant increase with basil leaf extract treatment. Relative to control animals, the specific enzyme activity was augmented by 1.68 and 1.63 folds in the forestomach following low-dose and high- dose treatments respectively. Increases of 1.8 (p < 0.005) and 1.73 folds (p < 0.005) for low-dose and high-dose treatments, respectively, were found in the kidney. In the lung, an induction of 1.23 fold (p < 0.005) was noted relative to the control group at both dose levels.
The specific activity of glutathione S-transferase as determined in the extrahepatic supernatant of the experimental animals revealed a significant increase above the basal level of activity in the respective organs investigated. In the forestomach, GST activity was increased 1.45 fold (p < 0.005) in Group I (vs. Group II) and 1.6 fold (p < 0.005) in Group I (vs. Group III). The kidneys of the treated mice showed increases of 1.48 fold (p < 0.005) and 1.44 fold (p < 0.01) relative to control group, at the low and high doses respectively. The lung, compared to control, showed increases of 1.42 (p < 0.005) and 1.48 folds (p < 0.005) for the low and high doses, respectively.
Mouse skin papillomagenesis experiment
Table 5 and Fig. 1 are depict the results of the skin papillomagenesis obtained via treatment with test material during the peri-initiational period. No adverse effect on body weight gain during the observation period was noticeable. Moreover, no evidence of development of spontaneous tumors, including skin lesions, has been encountered in our colony of Swiss mice. In Group I, 93.75% of animals treated with DMBA and croton oil developed skin papillomas, and the average number of tumors per mouse (tumor burden) was 6.53.98. The tumor burden was reduced to 2.692.0 (p < 0.01) and 1.4121.37 (p < 0.001) at low and high doses, respectively. In the control group, 93.75% of the animals treated with DMBA developed skin tumors. Among animals treated with low and high doses of basil leaf extract, 81.25% (12.50% reduction) and 75% (18.75% reduction) developed skin tumors.
Mouse forestomach papillomagenesis experiment
Table 6 shows the result obtained with basil leaf extract supplementation on benzo(a)pyrene-induced forestomach papillomagenesis. No difference was noticeable in the weight gain profile of animals treated with either doses of basil leaf extract, or in the positive control group of mice. 95% of the control animals developed forestomach papilloma with benzo(a)pyrene treatment, whereas only 76.19% (18.81% reduction) and 61.9% (33.1% reduction) of animals developed stomach tumors in the high-dose and low-dose basil-leaf treated groups, respectively.
The mean number of papilloma/mouse in this group of animals was 3.7061.58. In contrast, among animals pre-treated and post-treated with 200 and 400 mg/body wt of basil leaf extract, the mean number decreased to 1.52 [+ or -] 1.32 (p < 0.005) for low-dose and 1.158 [+ or -] 1.21 (p < 0.001) for high-dose treatment, respectively.
[FIGURE 1 OMITTED]
Ocimum basilicum, a well-known traditional medicinal plant on the Indian subcontinent, is incorporated in a number of herbal preparations for the treatment of various ailments. Many forms of Ocimum sanctum and Ocimum basilicum (holly basil) such as the ethanolic extract, flavonoids, seed oil, phenolic compounds, root extract, leaf extract, aqueous extract, fixed oil, fresh leaf paste and leaves in the diet, have been studied and have shown promising results for bone marrow radioprotection (Ganasoundri et al. 1998), chemoprotection (Prashar et al. 1994), Hypoglycemic activity (Chattopadhyay, 1993), and immunostimulant effects (Godhwani et al. 1998). Ocimum basilicum, the subject of our study, also has shown anti HIV I activity (Yamasaki et al. 1998), and anti-ulcer activity (Singh, 1999), among other effects which motivated us to plan a wide-spectrum study with this particular species of the Labiatae, for further evaluation of its potential.
Results obtained from the present study show that basil leaf extract administration to mice affects liver enzyme activities, as well as lipid peroxidation and lactate-dehydrogenase-related changes, which correlate with attenuating the risk of chemical carcinogenesis.
The cytochrome [P.sub.450] system refers to an enzyme super family that metabolize lipophilic compounds to more polar products, which are then acted upon by phase II enzymes, further increasing their polarity and assisting in their excretion (Henderson et al. 2000). Basil leaf extract pre-treatment was ineffective in elevating the basal level activity of hepatic phase I enzymes, rather inhibiting its activity significantly, while hepatic phase II enzymes were found to be elevated.
Phase II detoxifying enzymes detoxify carcinogens either by destroying their reactive centers or by conjugating them with endogenous ligands, facilitating their excretion. Mounting evidence has indicated that the induction of Phase II detoxification enzymes by numerous compounds and food phytochemicals results in protection against toxicity and chemical carcinogenesis, especially during the initiation phase. Among the phase II detoxification enzymes, GSTs are a family of enzymes that catalyze the conjugation of reactive chemicals with GSH and play a major role in protecting cells (Yoshimasa et al. 2000). There is evidence that GST activity is elevated in cell lines resistant to chemotherapeutic agents (Abraham and Singh, 1999). DT-diaphorase is a flavoprotein that catalyzes the two-electron reduction of quinones and other nitrogen oxides. This reaction prevents one-electron redox cycling of these groups, thereby preventing the formation of DNA-damaging reactive oxygen species. Reduction of quinones and nitrogen oxide might also make them available for conjugation with UDP-glucoric acid, facilitating their excretion (Begleiter et al. 1997; Talalay et al. 1995; Benson et al. 1980). Basil leaf-extract treatment increased the activities of both of these phase II enzymes in the liver and in all the extrahepatic organs examined (forestomach, lung and kidney in the present experiment).
With reference to antioxidant enzyme status in the liver, almost all the antioxidant-related enzymes, including glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase, were found to be elevated above the control basal values. Superoxide dismutase (SOD) plays an important role in the antioxidant enzyme defense system. SODs convert superoxide radicals into hydrogen peroxide (Shijun et al. 2000). Because the activity of SOD was induced in mice by the basil leaf extract treatment, the generation of reactive oxygen species is inhibited and the dismutation of super oxide radicals may well be accelerated by the catalyzing role of SOD. Catalase, the activity of which has also been augmented by basil treatment, helps in removing the hydrogen peroxide produced by the action of SOD. Indeed, SOD activity, along with that of catalase, explains the significant decrease in lipid peroxidation, which is an indicator of oxidative stress that persists in the cell. Glutathione reductase (GR) is another major antioxidant enzyme that catalyzes the NADPH-dependent reduction of glutathione disulfide to glutathione, thus maintaining GSH levels in the cell (Lee et al. 1995). Significant elevation of the activity of GR following basil leaf extract treatment was evident, thereby helping the cell to maintain the basal level of GSH, which is important for many other GSH-dependent detoxification reactions. Reduced glutathione (GSH) participates in spontaneous scavenging of electrophiles or free radicals and in reactions catalyzed by enzymes like G[P.sub.X] and GST (Meister, 1994; Coles and Ketterer, 1990; Hartman and Shankel, 1990; Hayes and McLellan, 1999). GSH activity was increased above basal level in the liver, as well as in all the extrahepatic organs investigated. The activity of lactate dehydrogenase, which is an indicator of cell damage, has decreased significantly at both the dose levels used for the present experiment, implying a cytoprotective effect exerted by basil leaf extract.
Because cancer has become the second major cause of mortality after cardiovascular diseases, it has become increasingly important to identify the naturally occuring/synthetic compounds that can interfere with and reduce the process of carcinogenesis. There are reports that a few substances can inhibit this process (Katiyer et al. 1993; Badary et al. 1999).
Various approaches to cancer chemoprevention exist. For example, the inducer of GSTs has received much attention as a potential chemopreventive agent, because the ability to induce GST is a property found in many chemopreventive agents, ameliorating toxicity and carcinogenicity (Wilkinson and Clapper, 1997; Kenster, 1995; Hayes and Pulford, 1995; Hu and Singh, 1997; Coles and Ketterer, 1990). Moreover many naturally-occurring chemopreventive agents have been reported to convert DNA-damaging entities into excretable metabolites through induction of GST. Another approach/mechanism of chemoprevention is the elevation of antioxidant defense that can combat the oxidative stress produced by reactive oxygen species (ROS), which often leads to mutation and cancer. It is suggested that the anticarcinogenic activity exerted by the chemopreventive agents occurs either by inhibiting the activation of B(a)P and/or by enhancing the detoxification of (+) anti BaPDE, which is the activated form (ultimate carcinogen) of benzo(a)pyrene.
The present investigation has demonstrated clearly that Basil leaf can be used as a potential cancer chemopreventive agent by virtue of its efficacy in inducing drug detoxification enzymes such as GST and DTD, as well as in blocking carcinogen-activating phase I enzymes (Henderson et al. 2000). It also protects against oxidative stress via the elevation of antioxidative defense enzyme, while significantly reducing the specific activity of LDH and the level of lipid peroxidation. All these effects considered together might result in significant reduction of DMBA-induced and B(a)P-induced papillomagenesis in Swiss albino mice at the peri-initiational period. Since Ocimum Basilicum has shown no toxic effect at the tested doses, it could well be applied in cancer chemoprevention, to reduce the risk of cancer.
Table 1. Modulatory influence of the two investigative doses of basil leaf extract (Ocimum basilicum) on weight gain profiles, protein levels and toxicity related parameters. Groups & Treatment Body weight (g) Initial Final Control 29.3 [+ or -] 84.10 31.25 [+ or -] 2.60 Vehicle (100) (100) 200 mg/kg body wt. 32.88 [+ or -] 3.39 34.12 [+ or -] 2.69 of Basil extract (111.9) (109.18) 400 mg/kg body wt. 30.0 [+ or -] 1.85 31.87 [+ or -] 1.88 of Basil extract (102.1) (101.98) Groups & Treatment Liver wt. X 100/ LDH  Final body wt. Control 6.20 [+ or -] 0.83 2.83 [+ or -] 0.12 Vehicle (100) (100) 200 mg/kg body wt. 4.92 [+ or -] 1.32 (a) 1.88 [+ or -] 0.28 (c) of Basil extract (79.35) (66.43) 400 mg/kg body wt. 4.88 [+ or -] 0.65 (c) 2.12 [+ or -] 0.17 (c) of Basil extract (78.70) (74.91) Groups & Treatment Protein (mg/ml) Microsome Cytosol Control 7.53 [+ or -] 1.72 11.60 [+ or -] 1.52 Vehicle (100) (100) 200 mg/kg body wt. 9.0 [+ or -] 1.64 12.58 [+ or -] 1.84 of Basil extract (119.52) (108.44) 400 mg/kg body wt. 11.86 [+ or -] 2.36 (c) 11.47 [+ or -] 1.69 of Basil extract (157.5) (98.87) Values are expressed as mean SD of 6-8 animals. Values in parentheses represent relative changes in parameters assessed (i.e., levels of parameter assessed in livers of mice receiving test substance relative to those of control mice). (a) (p < 0.05) and (c) (p < 0.005) represent significant changes as against vehicle-treated negative control  mole/mg protein. Abbreviations: LDH -- Lactate dehydrogenase. Treatment duration: 15 days Table 2. Modulatory influence of two different doses of Basil leaf extract (Ocimum basilicum) on mice hepatic phase I and phase II drug-metabolizing enzyme levels. Groups Cyt P450  Cyt b5  Control vehicle 0.472 [+ or -] 0.035 0.242 [+ or -] 0.021 (100) (100) 200 mg/kg body wt 0.402 [+ or -] 0.038 (a) 0.235 [+ or -] 0.018 Basil leaf (85.17) (97.10) 400 mg/kg body wt 0.383 [+ or -] 0.016 (c) 0.177 [+ or -] 0.025 (c) Basil leaf (81.14) (78.57) Groups Cyt P450 R  Cyt b5 R  Control vehicle 0.253 [+ or -] 0.036 4.95 [+ or -] 0.36 (100) (100) 200 mg/kg body wt 0.184 [+ or -] 0.007 (c) 4.11 [+ or -] 0.28 (d) Basil leaf (72.72) (83.0) 400 mg/kg body wt 0.157 [+ or -] 0.021 (d) 3.06 [+ or -] 0.58 (d) Basil leaf (62.0) (61.81) Groups GST  DTD  Control vehicle 2.50 [+ or -] 0.35 0.014 [+ or -] 0.001 (100) (100) 200 mg/kg body wt 3.5 [+ or -] 0.46 (c) 0.018 [+ or -] 0.001 (d) Basil leaf (140) (128.57) 400 mg/kg body wt 4.0 [+ or -] 0.58 (d) 0.020 [+ or -] 0.0017 (d) Basil leaf (160) (142.85) Values are expressed as mean SD of 6-8 animals. Values in parentheses represent relative change in parameters assessed (i.e., levels of activity in livers of mice receiving test substance relative to those in control mice). (a) (p < 0.05), (c) (p < 0.005) and (d) (p < 0.001) represent significant changes as against control.  nmole/mg protein,  [micro]mole of NADPH oxidized/min/mg protein,  [micro]mole of NADH oxidized/min/mg protein,  [micro]mole CDNB-GSH conjugate formed/min/mg protein,  [micro]mole of DCPIP reduced/min/mg protein. Abbreviations: Cyt P450 -- cytochrome P450; Cyt b5 -- cytochrome b5; Cyt P450 R -- cytochrome P450 reductase; Cyt b5 R -- cytochrome b5 reductase; GST -- glutathione S-transferase; DTD -- DT-diaphorase. Treatment duration: 15 days. Table 3. Modulatory influence of two different doses of Basil leaf extract (Ocimum basilicum) on mice hepatic-antioxidant related parameters and lipid peroxidation. Groups GSH  SOD  Control vehicle 2.56 [+ or -] 0.51 5.05 [+ or -] 0.44 (100) (100) 200mg/kg body wt 5.33 [+ or -] 0.77 (c) 5.64 [+ or -] 0.34 (a) Basil leaf (208.2) (111.68) 400mg/kg body wt 5.14 [+ or -] 0.85 (c) 5.76 [+ or -] 0.45 (a) Basil leaf (200) (114.05) Groups CAT  GPx  Control vehicle 56.46 [+ or -] 9.36 15.97 [+ or -] 1.13 (100) (100) 200mg/kg body wt 88.40 [+ or -] 6.56 (c) 19.51 [+ or -] 1.48 (c) Basil leaf (156.57) (122.16) 400mg/kg body wt 89.36 [+ or -] 6.59 (c) 22.42 [+ or -] 1.76 (c) Basil leaf (158.27) (140.38) Groups GR  LP  Control vehicle 30.0 [+ or -] 2.30 0.933 [+ or -] 0.072 (100) (100) 200mg/kg body wt 34.82 [+ or -] 1.43 (c) 0.753 [+ or -] 0.11 (b) Basil leaf (116.06) (80.70) 400mg/kg body wt 38.37 [+ or -] 1.63 (c) 0.697 [+ or -] 0.041 (c) Basil leaf (127.9) (74.70) Values are expressed as mean SD of 6-8 animals. Values in parentheses represent relative change in parameters assessed (i.e., levels of activity in livers of mice receiving test substance related to those in control mice) (a) (p < 0.05), (b) (p < 0.01) and (c) (p < 0.005) represent significant changes as against control  nmole GSH/g tissue,  specific activity expressed as mole/mg protein,  [micro]mole [H.sub.2][O.sub.2], consumed/min/mg protein,  nmole of NADPH consumed/min/mg protein,  nmole malondialdehyde formed/mg protein. Abbreviations: GSH -- reduced glutathione; GPX -- glutathione peroxidase; GR -- glutathione reductase; SOD -- superoxide dismutase; CAT -- catalase; LP -- lipid peroxidation; LDH -- lactate dehydrogenase Treatment duration: 15 days. Table 4. Modulatory influence of Basil leaf extract (Ocimum basilicum) on detoxifying and antioxidant enzyme profiles in extrahepatic organs of mouse. Groups & Treatment Organ wt. X 100/ GSH  Final body wt. Lung: Control vehicle 1.04[+ or -]0.29 0.83[+ or -]0.14 (100) (100) Basil extract 0.95[+ or -]0.14 1.43[+ or -]0.23 (b) (200 mg/kg body wt.) (91.34) (172.28) Basil extract 0.97[+ or -]0.18 1.45[+ or -]0.27 (c) (400 mg/kg body wt.) (93.26) (174.69) Kidney: Control vehicle 2.12[+ or -]0.38 0.92[+ or -]0.18 (100) (100) Basil extract 2.12[+ or -]0.28 1.29[+ or -]0.14 (c) (200 mg/kg body wt.) (100) (112.82) Basil extract 2.17[+ or -]0.22 1.49[+ or -]0.25 (c) (400 mg/kg body wt.) (102.35) (136.75) Fore-Stomach: Control vehicle 0.22[+ or -]0.043 0.78[+ or -]0.14 (100) (100) Basil extract 0.21[+ or -]0.058 0.96[+ or -]0.162 (200 mg/kg body wt.) (95.45) (123.07) Basil extract 0.21[+ or -]0.031 1.02[+ or -]0.11 (a) (400 mg/kg body wt.) (95.45) (130.76) Groups & Treatment GST  DTD  Lung: Control vehicle 0.45[+ or -]0.022 0.013[+ or -]0.0004 (100) (100) Basil extract 0.64[+ or -]0.094 (c) 0.016[+ or -]0.0012 (c) (200 mg/kg body wt.) (142.22) (123.07) Basil extract 0.67[+ or -]0.06 (c) 0.016[+ or -]0.0016 (c) (400 mg/kg body wt.) (148.88) (123.07) Kidney: Control vehicle 0.54[+ or -]0.085 0.015[+ or -]0.0024 (100) (100) Basil extract 0.80[+ or -]0.079 (c) 0.027[+ or -]0.0051 (c) (200 mg/kg body wt.) (148.14) (180) Basil extract 0.78[+ or -]0.10 (b) 0.026[+ or -]0.0027 (c) (400 mg/kg body wt.) (143.58) (173.33) Fore-Stomach: Control vehicle 0.33[+ or -]0.024 0.019[+ or -]0.0016 (100) (100) Basil extract 0.48[+ or -]0.034 (c) 0.032[+ or -]0.004 (c) (200 mg/kg body wt.) (145.45) (168.42) Basil extract 0.53[+ or -]0.055 (c) 0.031[+ or -]0.0045 (d) (400 mg/kg body wt.) (160.60) (163.15) Groups & Treatment Protein (mg/ml) Lung: Control vehicle 5.96[+ or -]0.80 (100) Basil extract 5.92[+ or -]0.71 (200 mg/kg body wt.) (99.32) Basil extract 5.88[+ or -]0.71 (400 mg/kg body wt.) (98.65) Kidney: Control vehicle 6.70[+ or -]1.24 (100) Basil extract 6.07[+ or -]1.36 (200 mg/kg body wt.) (90.59) Basil extract 5.90[+ or -]0.56 (400 mg/kg body wt.) (88.05) Fore-Stomach: Control vehicle 5.20[+ or -]0.65 (100) Basil extract 4.02[+ or -]0.95 (200 mg/kg body wt.) (77.30) Basil extract 4.29[+ or -]1.02 (400 mg/kg body wt.) (82.5) Values are expressed as mean SD of 6-8 animals. Values in parentheses represent relative change in parameters assessed (i.e., levels of activity in extrahepatic organs of mice receiving test substancerelative to those in extrahepatic organs of control mice). (a) (p < 0.05), (b) (p < 0.01), (c) (p < 0.005) and (d) (p < 0.001) represent significant changes as against control..  nmole GSH/g tissue,  [micro]mole CDNB-GSH conjugate formed/min/mg protein,  [micro]mole of DCPIP reduced/min/mg protein. Abbreviations: GSH -- reduced glutathione; GST -- glutathione S-transferase and DTD -- DT-diaphorase. Treatment duration: 15 days Table 5. Effect of two different doses of Basil leaf extract (Ocimum basilicum) on DMBA- induced skin papillomagenesis in mice. Groups & Treatment Body weight (mg) Initial Final Only vehicle + DMBA + Croton oil. 30.0[+ or -]1.64 32.11[+ or -]1.11 Basil leaf extract 31.06[+ or -]1.16 34.22[+ or -]1.22 (150 mg/kg body wt) + DMBA + Croton oil. Basil leaf extract 31.56[+ or -]1.65 34.40[+ or -]1.38 (300 mg/kg body wt) + DMBA + Croton oil. Groups & Treatment Total tumor Tumor Burden incidence(%) (tumor/mouse) Only vehicle + DMBA + Croton oil. 93.75 6.50[+ or -]3.98 Basil leaf extract 81.25 2.69[+ or -]2.0 (b) (150 mg/kg body wt) + DMBA + Croton oil. Basil leaf extract 75 1.412[+ or -]1.37 (d) (300 mg/kg body wt) + DMBA + Croton oil. Values are expressed as mean SD of 15-17 animals (b) (p < 0.01) and (d) (p < 0.001) represent significant changes as against control. Table 6. Effect of two different doses of Basil leaf extract (Ocimum basilicum) on benzo(a)pyrene-induced forestomach papillomagenesis in mice. Groups & Treatment Body weight (mg) Initial Final Benzo(a)pyrene + (only 24.0[+ or -]123 26.0[+ or -]3.06 vehicle) Benzo(a)pyrene + Basil leaf 24.5[+ or -]1.37 28.14[+ or -]1.79 extract (200 mg/kg body wt.) Benzo(a)pyrene + Basil leaf 25.09[+ or -]1.30 31.0[+ or -]3.0 extract (400 mg/kg body wt.) Groups & Treatment Total tumor Tumor Burden incidence(%) (tumor/mouse) Benzo(a)pyrene + (only 95 3.706[+ or -]2.56 vehicle) Benzo(a)pyrene + Basil leaf 76.19 1.524[+ or -]1.32 (c) extract (200 mg/kg body wt.) Benzo(a)pyrene + Basil leaf 61.90 1.158[+ or -]1.21 (d) extract (400 mg/kg body wt.) Values are expressed as mean SD of 18-20 animals. (d) (p < 0.001), (c) (p < 0.005), represent significant changes as against control
Trisha Dasgupta is thankful to the Indian Council for Cultural Relationship (ICCR). Government of India for providing the Ph.D scholarship. We also thank Dr. Banerjee for his invaluable suggestions and guidance to carry out this work.
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T. Dasgupta, A. R. Rao, and P. K. Yadava
Cancer Biology and Applied Molecular Biology Laboratories, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
A. R. Rao, Cancer Biology and Applied Molecular Biology Laboratories, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
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|Author:||Dasgupta, T.; Rao, A.R.; Yadava, P.K.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Feb 1, 2004|
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|Have you taken your Holy basil today?|
|Herbafrost's frozen Purple and Thai Basil increasingly make European Cuisine scene.|