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Protective effect of Cassia occidentalis L. on cyclophosphamide-induced suppression of humoral immunity in mice
Journal of Ethnopharmacology 75 (2001) 13 – 18 www.elsevier.com/locate/jethpharm
Protective effect of Cassia occidentalis L. on cyclophosphamide-induced suppression of humoral immunity in mice Bilal Bin-Hafeez, Iqbal Ahmad, Rizwanul Haque, S. Raisuddin * Department of Medical Elementology & Toxicology, Jamia Hamdard (Hamdard Uni6ersity), New Delhi, 110 062, India Received 20 October 2000; accepted 3 November 2000
Abstract Cassia occidentalis L. (Kasaundi) is a widely used medicinal plant. Earlier, we have shown that it possesses antimutagenic activity against benzo[a]pyrene (BaP) and cyclophosphamide (CP)-induced mutagenicity in mice. In this study, we investigated if this plant could also provide protection against CP-induced immunosuppression in animal models. Swiss albino male mice were treated per os with the aqueous extract of C. occidentalis (100 mg/kg, body weight (b.w.)) for 14 days. Cyclophosphamide was given intraperitoneally in a single dose of 50 mg/kg b.w. Body weight, relative organ weight, lymphoid organ cellularity, hemagglutination titre (HT), plaque forming cell (PFC) assay and quantitative hemolysis of SRBC (QHS) were studied in these animals. CP, as expected, showed suppressive effects on lymphoid organ weight and cellularity and other parameters of humoral immunity. Plant extract treatment itself produced no toxicity. The administration of plant extract to CP-exposed animals resulted in improved humoral responses. C. occidentalis treatment significantly (PB0.01) enhanced PFC response in CP-treated animals. In QHS assay, also C. occidentalis showed protection in CP-treated animals. Bone marrow cell counts, which were reduced in CP-treated animals, were reversed significantly (pB 0.01) to normal levels in CP + plant extract group animals. In our earlier study, we found that C. occidentalis modulated hepatic drug metabolizing enzymes. It is suggested that by a similar mechanism, it may be influencing the hematotoxic and immunotoxic responses of cyclophosphamide. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cassia occidentalis; Cyclophosphamide; Immunosuppression; Immunomodulation; Cancer chemotherapy
1. Introduction The concept that modulation of immune responses to alleviate the diseases has existed in ancient systems of medicine, Ayurveda (Indian system of medicine) and Unani-Tib (Greco-Arab system of medicine; Said, 1969). Plants have been extensively used as a source of medicine in these systems to promote health and to maintain body’s resistance against infection by potentiating immunity, re-establishing body equilibrium and conditioning of the body tissues (Savnur, 1950; Bhagwandash, 1978). Cassia occidentalis L. (Kasaundi) of family leguminaceae is extensively used in the indige-
* Corresponding author. Tel.: +91-11-6089688 ext. 217; fax: +9111-6088874. E-mail address: [email protected] (S. Raisuddin).
nous and folk-lore medicine systems for a variety of purposes (Chopra et al., 1980; Nadkarni, 1982). In Unani medicine, it is used as an antidote of poisons, blood purifier, expectorant, anti-inflammatory agent and a remedy for the treatment of liver diseases (Kabiruddin, 1951). The ethanolic extract of leaves of this plant was found to possess the anti-hepatotoxic activity against carbon tetrachloride- and thioacetamide-induced liver damage (Saraf et al., 1994). The anti-inflammatory activity of its constituents isolated from roots and stem has also been reported (Kuo et al., 1996). Recently, a group of researchers from our university has shown its hepatoprotective effects against ethyl alcohol- and paracetamol-induced hepatotoxicity in rats (Jafri et al., 1999). We have also shown that the aqueous extract of C. occidentalis possessed anti-mutagenic activity against benzo[a]pyrene (BaP) and cyclophosphamide (CP)-in-
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 3 8 2 - 2
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B. Bin-Hafeez et al. / Journal of Ethnopharmacology 75 (2001) 13–18
duced mutagenicity in mice (Sharma et al., 1999). Later, we demonstrated that C. occidentalis inhibits CP-induced mutagenicity in Ames test (Sharma et al., 2000a). Immunosuppression particularly of humoral immunity is a common consequence of long-term CPchemotherapy in cancer patients (Zaidi et al., 1990). Reduction of immunosuppressive effects may thus become beneficial to patients undergoing CP-chemotherapy. In this study, we, therefore, assessed the protective effect of C. occidentalis against cyclophosphamide-induced suppression of humoral immunity in mice. 2. Materials and methods
2.1. Plant extract Total aqueous extract of C. occidentalis in semi-solid form was purchased from Plant Extract Division of M/S. Saiba Industries Pvt. Ltd., Mumbai. A certificate of its authenticity was provided along with the extract by their expert taxonomist.
2.1.1. Chemical analysis of extract Water content of C. occidentalis extract was determined by the Karl Fischer apparatus (Orian, Switzerland). The water content of plant extract was found to be 1.328% (w/w). For development of thin layer chromatography (TLC) fingerprints, extract (10 g) was suspended in 100 ml volume of three different solvents viz., ethanol (90%), chloroform (containing 2% v/v of ethanol as preservative) and petroleum ether (60 – 80°C). The extraction was done by continuous orbital shaking for 24 h. The extracted material along with solvent was filtered through Whatman No. 1 filter paper. The TLC was done for each extract on a precoated silica gel 60 F254 plates (E. Merck, 0.20 mm thickness) using different mobile phases. Toluene –formic acid (95:5) was used as a mobile phase for the petroleum ether extract, while the chloroform and ethanolic extract were run using toluene – ethyl formate –formic acid (5:4:1) as the mobile phase. Chromatograms were viewed under long wavelength UV light (366 nm) and photographed using a Reprostar Chromatography Documentation Apparatus (Camag, Switzerland). The Rf values for different spots were calculated. The fingerprint chromatograms are shown in Fig. 1. Details of the fingerprint analysis are given in Table 1.
2.3. Animals Study was conducted on Swiss albino male mice (30 –35 g). Female guinea pigs (250 g) were used for the preparation of complement for plaque forming cell (PFC) assay. Animals were provided by the Central Animal House Facility of the University. The animals were bred and maintained under standard laboratory conditions: temperature (2592°C) and photoperiod of 12 h. Commercial pellet diet and water were given ad libitum.
2.4. Dosage CP and plant extract were suspended in normal saline. Plant extract (100 mg/kg body) was administered by gavage (per os) and cyclophosphamide (50 mg/kg) was injected intraperitoneally (i.p.). The dose volume was 0.2 ml. Control animals received same volume of normal saline. The CP and plant extract doses and dose schedules are based on our own published report (Sharma et al., 1999) and that of De et al. (1998).
2.5. Body weight, lymphoid organ weight and cellularity Animals were divided into four groups (I–IV). Each group comprised of a minimum of six animals. Group I (control) received normal saline, group II (CP) animals were injected with a single dose of CP on the twelfth day of initiation of experiment, group III (plant extract) animals were administered with plant extract for 14 consecutive days and group IV (plant extract+ CP) animals were given plant extract treatment for 14 days with a single injection of CP on twelfth day. The animals were sacrificed by cervical dislocation 24 h
2.2. Chemicals CP, Hank’s balanced salt solution (HBSS), phosphate buffer saline (PBS) and RPMI-1640 medium were purchased from Sigma Chemical Co., St Louis, MO and petroleum jelly was obtained from Hi-Media Labs, Mumbai.
Fig. 1. Fingerprint TLC chromatograms of C. occidentalis under UV light. A, B and C are chromatograms developed from ethanol, chloroform and petroleum ether extracts, respectively. For A and B toluene– ethyl formate– formic acid (5:4:1) was used as a mobile phase while for C toluene– formic acid (95:5) was used.
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Table 1 Detail of fingerprint chromatograms of C. occidentalis extract Extract
Solvent system
No. of spots
Ethanolic extract (chromatogram A in Fig. 1)
Toluene–ethyl formate–formic 19 acid (5:4:1)
Chloroform extract (chromatogram B in Fig. 1)
Toluene–ethyl formate–formic 19 acid (5:4:1)
Rf values 0.05, 0.55, 0.98 0.10, 0.60, 0.96 0.05, 0.68,
0.08, 0.09, 0.11, 0.16, 0.22, 0.25, 0.40, 0.49, 0.57, 0.61, 0.66, 0.72, 0.77, 0.83, 0.88, 0.91, 0.13, 0.18, 0.25, 0.29, 0.39, 0.48, 0.53, 0.56, 0.63, 0.67, 0.68, 0.72, 0.75, 0.81, 0.86, 0.89,
Petroleum ether extract (chromatogram C Toluene–formic acid (95:5) in Fig. 1)
12
after the last dose. Body weight gain (percentage) and relative organ weight (organ weight/100 g of body weight) of kidney, liver, spleen and thymus were determined for each animal. Single cell suspensions were prepared in RPMI-1640 medium from bone marrow (from femur), spleen and thymus and cell counts were made using Neubauer chamber (Raisuddin et al., 1990).
Paul, MN). The chambers were loaded with a known volume of assay mixture, sealed with petroleum jelly and incubated at 37°C for 1 h. The plaques were counted under a light microscope (Olympus BX50) and expressed as PFC per 106 spleen cells.
2.6. Assessment of humoral immune functions Four groups of animals were made as described above. Animals of all the groups were challenged with 0.2 ml of 10% sheep red blood cells (SRBC), i.p. on the tenth day of initiation of experiment. The following parameters of humoral immunity were studied in those animals.
2.6.1. Hemagglutination titer (HT) assay It was performed using procedure of Mungantiwar et al. (1999). Blood was collected from the retro-orbital plexus of each mouse for serum preparation. Serial two-fold dilution of serum samples was made in 50 ml of PBS (pH 7.2) in 96-well microtiter plates and mixed with 50 ml of 1% SRBC suspension in PBS. After mixing, plates were kept at room temperature for 2 h. The value of antibody titer was assigned to the highest serum dilution showing visible haemagglutination. 2.6.2. PFC assay The PFC assay was performed using the method of Raisuddin et al. (1991). In brief, 0.2 ml of 10% SRBC prepared in normal saline was injected to animals, i.p. The animals were sacrificed on the fifth day of immunization. The spleen was removed, cleaned free of extraneous tissues and a single cell suspension of 106 cells/ml was prepared from it in RPMI-1640 medium. For PFC assay, the SRBC were prepared at a cell density of 5× 108 cells/ml in PBS. One milliliter of SRBC in medium along with 0.5 ml of diluted guinea pig complement (1 ml of serum + 9 ml of normal saline) was added to 1 ml of spleen suspension. Cunningham chambers were prepared using glass slide, coverslips and doubled-sided tape (Scotch Brand, St.
0.07, 0.11, 0.16, 0.20, 0.25, 0.29, 0.37, 0.62, 0.83, 0.88
2.6.3. Quantitati6e hemolysis of SRBC (QHS) assay QHS assay was performed using the methods of Simpson and Gozzo (1978) with some modification. Spleens were removed and a cell suspension of 10× 106/ml was prepared in PBS. One milliliter of 0.2% SRBC and 1 ml of 10% guinea pig serum were mixed with cell suspension and incubated for 1 h at 37°C. After centrifugation at 3000 rpm for 3 min, optical density of the supernatant was measured at 413 nm using spectrophotometer (Shimadzu UV-1201). 2.7. Statistical analysis Data were statistically analyzed using Student’s t-test to determine significant differences in data of various groups; p values less than 0.5 were considered significant. The values are expressed as means+ SE.
3. Results
3.1. Effect of plant extract on body weight, organ weight and cellularity None of the groups showed mortality. No significant body weight gain differences were recorded in various groups of animals. As regards relative organ weight, CP treatment caused a significant (PB 0.001) reduction in the spleen weight when compared with control (normal saline treated group I) animals (Table 2). Interestingly, the plant extract also caused spleen weight reduction (PB 0.01). No significant recovery of spleen weight was observed in the plant extract+ CP (group IV) animals. CP treatment also induced a significant reduction in the relative organ weight of thymus (PB0.05). Plant extract had no adverse effect on the thymus weight and had no protective effect on CP-induced reduction of
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Table 2 Effect of C. occidentalis and cyclophosphamide treatment on the relative organ weight of micea Group
I (control) II (CP) III (plant extract) IV (plant extract +CP)
Relative organ weight (means 9 S.E.) in g
Table 4 Effect of C. occidentalis on CP-induced suppression of antibody titre in mice Group
Spleen
Thymus
Liver
Kidney
0.7219 0.05 0.2469 0.02b 0.49790.009c
0.159 90.007 0.089 90.012d 0.210 9 0.016
5.13 9 0.25 5.12 90.22 4.92 9 0.22
1.042 9 0.060 1.025 9 0.020 1.027 9 0.020
0.3069 0.03
0.089 9 0.008
4.41 90.13
1.164 9 0.100
Values are means 9 S.E. g of five mice. PB0.001 when compared with group I. c PB0.01 when compared with group I. d PB0.05 when compared with group I (significantly different). a
b
thymus weight. No significant reduction was observed in case of relative organ weights of other organ viz., kidney and liver of the animals of different groups. CP treatment resulted in a decrease in cellularity of bone marrow, spleen (PB 0.001) and thymus (P B0.001) as compared to control (group 1) animals (Table 3). The plant extract also caused significant depletion of splenic cell counts (PB 0.01) but increased bone marrow cell counts in a significant manner (P B 0.001). The plant extract showed protective effects on cellularity of bone marrow (p B 0.05), spleen (P B0.01) and thymus (PB 0.01) when compared with CP-treated animals.
3.2. Effect of plant extract on CP-induced suppression of humoral immunity parameters A single dose of CP (50 mg/kg b.w.) induced a significant reduction in all the parameters of humoral immunity, antibody titer (Table 4), PFC assay (Fig. 2) and QHS assay (Fig. 3) in mice. The reduction was
I (control) II (CP) III (plant extract) IV(plant extract+CP)
Titer level 1:2048 1:4 1:4096 1:256
significant (P B0.001) when compared with control animals (group I). The C. occidentalis extract showed a stimulatory effect in the PFC assay (PB 0.01). When compared with CP-treated animals (group II), animals receiving plant extract treatment along with CP (group IV) showed significant recovery in the antibody titre, in the PFC assay (PB 0.001) and in the QHS assay (PB 0.05).
4. Discussion Cassia occidentalis treatment to animals protected them from CP-induced suppression of humoral immunity. Animals also showed improvement in CP-induced immunopathy in different organs. Though the plant extract showed no suppressive effect on any of the parameters of humoral immunity, it reduced spleen weight and spleen cellularity. We do not have an exact explanation for this selective toxicity to spleen at this juncture. C. occidentalis is not only reported for its medicinal value but some reports also showed toxic effects in various species (Martin et al., 1981; Marrero et al., 1998). The PFC response of plant extract treated animals showed no adverse effect. In fact, all the
Table 3 Effect of C. occidentalis treatment on CP-induced depletion of cellularity of lymphoid organsa Groups
Cellularity (means 9S.E.)×106 Spleen
I (control) 342.83 924.45 II (CP) 53.30 95.32b III (plant 237.33 9 14.77c extract) IV (plant 79.50 92.79d extract+CP)
Bone marrow
Thymus
20.12 92.80 14.569 2.80 36.109 2.26c
65.62 9 7.10 14.409 1.41b 51.7193.69
25.09 1.41d
28.269 3.01d
Values are means 9 S.E.×106of six mice. b PB0.001 when compared with group I. c PB0.01 when compared with group I values. d PB0.01 when compared with group II (significantly different). a
Fig. 2. Effect of C. occidentalis extract (100 mg/kg) on CP-induced suppression of humoral immunity as assessed by plaque forming cell assay. Data are means 9 S.E. of six animals. * P B0.01 and ** PB 0.001 when compared with control group animals, *** PB 0.01 when compared with values of CP-treated animals (significantly different).
B. Bin-Hafeez et al. / Journal of Ethnopharmacology 75 (2001) 13–18
Fig. 3. Effect of C. occidentalis extract (100 mg/kg) on CP-induced suppression of humoral immunity as assessed by quantitative hemolysis of SRBC assay. Data are means 9 S.E. of six animals. * P B0.05 when compared with values of CP-treated animals, ** P B0.001 when compared with control group animals (significantly different).
parameters of humoral immunity that were suppressed as a result of CP treatment showed recovery in the animals treated with C. occidentalis extract. Our studies have shown that the total aqueous extract of C. occidentalis possesses antimutagenic activity against BaP and CP (Sharma et al., 1999). Using three doses (50 mg, 100 mg and 250 mg/kg b.w.) in mice, we found that 100 and 250 mg/kg showed almost same level of antimutagenic effect. In that study, we also showed that C. occidentalis had a modulatory effect on hepatic activation and disposition mechanisms; it decreased cytochrome P450 contents (phase I metabolism) and increased glutathione S-transferase (GST) activity (phase II metabolism). This was helpful in reducing the activation of toxicant to reactive metabolites on the one hand and facilitating quick disposition of toxicants and their metabolites on the other hand. CP also undergoes cytochrome P450-mediated activation by phase I enzymes and GST-mediated detoxification phase (Torkelson et al., 1974). Two of the metabolites of CP have been identified as acrolein and phosphoramide mustard (Domeyer and Sladek, 1980). Although useful in the chemotherapeutic treatment of various forms of cancers, CP toxicity has been of major concern limiting its use. Besides being a potent immunosuppressant and mutagen, CP is also reported to induce bladder cancer (McCann et al., 1975; Seino et al., 1978). In the Ames test, it requires metabolic activation and this strengthens our hypothesis that by modulating activation and detoxification mechanisms CP toxicity including the immunotoxicity could be reduced to a great extent. Some of the chemical constituents isolated from C. occidentalis include anthraquinones, fatty oils, flavonoids, gallactomannan, polysaccharides and tan-
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nins (Lal and Gupta, 1973, 1974; Kudav and Kulkarni, 1974; Singh and Singh, 1985; Gupta et al., 1995). Many of these constituents isolated from other plant sources possess immunomodulatory activity (Wang et al., 1991; Hase et al., 1996; Middleton, 1998). Most of the studies concerning the evaluation of immunomodulatory activities of the plants have been undertaken utilizing the crude extracts (Sohni and Bhatt, 1996; Ziauddin et al., 1996; Dhuley, 1997). In some studies, a combination of various herbs or herbs in combination of minerals have been used taking into consideration Ayurvedic, Chinese, Japanese, Korean or Unani traditional formulations (Komatsu et al., 1986; Yamaguchi, 1992; Nakai et al., 1993; Sohni and Bhatt, 1996; De et al., 1998; Bajaj et al., 1999). Though it may be rational to use single plant or its single constituent, it has been a general experience that the total plant extract shows more efficacy versus single constituent (Dhir et al., 1990, 1991; Sharma et al., 2000b). The findings of present study are significant in the direction of developing safer strategies for cancer treatment.
Acknowledgements The financial support received from the Central Council for Research in Unani Medicine (CCRUM) and the University Grants Commission (UGC) for this study is gratefully acknowledged. We thank Dr M.S. Alam, Department of Chemistry for his expert help in chemical analysis of the extract. Assistance of Mr. Razi Ahmad is also acknowledged.
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Protective effect of Cassia occidentalis L. on cyclophosphamide-induced suppression of humoral immunity in mice Bilal Bin-Hafeez, Iqbal Ahmad, Rizwanul Haque, S. Raisuddin * Department of Medical Elementology & Toxicology, Jamia Hamdard (Hamdard Uni6ersity), New Delhi, 110 062, India Received 20 October 2000; accepted 3 November 2000
Abstract Cassia occidentalis L. (Kasaundi) is a widely used medicinal plant. Earlier, we have shown that it possesses antimutagenic activity against benzo[a]pyrene (BaP) and cyclophosphamide (CP)-induced mutagenicity in mice. In this study, we investigated if this plant could also provide protection against CP-induced immunosuppression in animal models. Swiss albino male mice were treated per os with the aqueous extract of C. occidentalis (100 mg/kg, body weight (b.w.)) for 14 days. Cyclophosphamide was given intraperitoneally in a single dose of 50 mg/kg b.w. Body weight, relative organ weight, lymphoid organ cellularity, hemagglutination titre (HT), plaque forming cell (PFC) assay and quantitative hemolysis of SRBC (QHS) were studied in these animals. CP, as expected, showed suppressive effects on lymphoid organ weight and cellularity and other parameters of humoral immunity. Plant extract treatment itself produced no toxicity. The administration of plant extract to CP-exposed animals resulted in improved humoral responses. C. occidentalis treatment significantly (PB0.01) enhanced PFC response in CP-treated animals. In QHS assay, also C. occidentalis showed protection in CP-treated animals. Bone marrow cell counts, which were reduced in CP-treated animals, were reversed significantly (pB 0.01) to normal levels in CP + plant extract group animals. In our earlier study, we found that C. occidentalis modulated hepatic drug metabolizing enzymes. It is suggested that by a similar mechanism, it may be influencing the hematotoxic and immunotoxic responses of cyclophosphamide. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cassia occidentalis; Cyclophosphamide; Immunosuppression; Immunomodulation; Cancer chemotherapy
1. Introduction The concept that modulation of immune responses to alleviate the diseases has existed in ancient systems of medicine, Ayurveda (Indian system of medicine) and Unani-Tib (Greco-Arab system of medicine; Said, 1969). Plants have been extensively used as a source of medicine in these systems to promote health and to maintain body’s resistance against infection by potentiating immunity, re-establishing body equilibrium and conditioning of the body tissues (Savnur, 1950; Bhagwandash, 1978). Cassia occidentalis L. (Kasaundi) of family leguminaceae is extensively used in the indige-
* Corresponding author. Tel.: +91-11-6089688 ext. 217; fax: +9111-6088874. E-mail address: [email protected] (S. Raisuddin).
nous and folk-lore medicine systems for a variety of purposes (Chopra et al., 1980; Nadkarni, 1982). In Unani medicine, it is used as an antidote of poisons, blood purifier, expectorant, anti-inflammatory agent and a remedy for the treatment of liver diseases (Kabiruddin, 1951). The ethanolic extract of leaves of this plant was found to possess the anti-hepatotoxic activity against carbon tetrachloride- and thioacetamide-induced liver damage (Saraf et al., 1994). The anti-inflammatory activity of its constituents isolated from roots and stem has also been reported (Kuo et al., 1996). Recently, a group of researchers from our university has shown its hepatoprotective effects against ethyl alcohol- and paracetamol-induced hepatotoxicity in rats (Jafri et al., 1999). We have also shown that the aqueous extract of C. occidentalis possessed anti-mutagenic activity against benzo[a]pyrene (BaP) and cyclophosphamide (CP)-in-
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duced mutagenicity in mice (Sharma et al., 1999). Later, we demonstrated that C. occidentalis inhibits CP-induced mutagenicity in Ames test (Sharma et al., 2000a). Immunosuppression particularly of humoral immunity is a common consequence of long-term CPchemotherapy in cancer patients (Zaidi et al., 1990). Reduction of immunosuppressive effects may thus become beneficial to patients undergoing CP-chemotherapy. In this study, we, therefore, assessed the protective effect of C. occidentalis against cyclophosphamide-induced suppression of humoral immunity in mice. 2. Materials and methods
2.1. Plant extract Total aqueous extract of C. occidentalis in semi-solid form was purchased from Plant Extract Division of M/S. Saiba Industries Pvt. Ltd., Mumbai. A certificate of its authenticity was provided along with the extract by their expert taxonomist.
2.1.1. Chemical analysis of extract Water content of C. occidentalis extract was determined by the Karl Fischer apparatus (Orian, Switzerland). The water content of plant extract was found to be 1.328% (w/w). For development of thin layer chromatography (TLC) fingerprints, extract (10 g) was suspended in 100 ml volume of three different solvents viz., ethanol (90%), chloroform (containing 2% v/v of ethanol as preservative) and petroleum ether (60 – 80°C). The extraction was done by continuous orbital shaking for 24 h. The extracted material along with solvent was filtered through Whatman No. 1 filter paper. The TLC was done for each extract on a precoated silica gel 60 F254 plates (E. Merck, 0.20 mm thickness) using different mobile phases. Toluene –formic acid (95:5) was used as a mobile phase for the petroleum ether extract, while the chloroform and ethanolic extract were run using toluene – ethyl formate –formic acid (5:4:1) as the mobile phase. Chromatograms were viewed under long wavelength UV light (366 nm) and photographed using a Reprostar Chromatography Documentation Apparatus (Camag, Switzerland). The Rf values for different spots were calculated. The fingerprint chromatograms are shown in Fig. 1. Details of the fingerprint analysis are given in Table 1.
2.3. Animals Study was conducted on Swiss albino male mice (30 –35 g). Female guinea pigs (250 g) were used for the preparation of complement for plaque forming cell (PFC) assay. Animals were provided by the Central Animal House Facility of the University. The animals were bred and maintained under standard laboratory conditions: temperature (2592°C) and photoperiod of 12 h. Commercial pellet diet and water were given ad libitum.
2.4. Dosage CP and plant extract were suspended in normal saline. Plant extract (100 mg/kg body) was administered by gavage (per os) and cyclophosphamide (50 mg/kg) was injected intraperitoneally (i.p.). The dose volume was 0.2 ml. Control animals received same volume of normal saline. The CP and plant extract doses and dose schedules are based on our own published report (Sharma et al., 1999) and that of De et al. (1998).
2.5. Body weight, lymphoid organ weight and cellularity Animals were divided into four groups (I–IV). Each group comprised of a minimum of six animals. Group I (control) received normal saline, group II (CP) animals were injected with a single dose of CP on the twelfth day of initiation of experiment, group III (plant extract) animals were administered with plant extract for 14 consecutive days and group IV (plant extract+ CP) animals were given plant extract treatment for 14 days with a single injection of CP on twelfth day. The animals were sacrificed by cervical dislocation 24 h
2.2. Chemicals CP, Hank’s balanced salt solution (HBSS), phosphate buffer saline (PBS) and RPMI-1640 medium were purchased from Sigma Chemical Co., St Louis, MO and petroleum jelly was obtained from Hi-Media Labs, Mumbai.
Fig. 1. Fingerprint TLC chromatograms of C. occidentalis under UV light. A, B and C are chromatograms developed from ethanol, chloroform and petroleum ether extracts, respectively. For A and B toluene– ethyl formate– formic acid (5:4:1) was used as a mobile phase while for C toluene– formic acid (95:5) was used.
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Table 1 Detail of fingerprint chromatograms of C. occidentalis extract Extract
Solvent system
No. of spots
Ethanolic extract (chromatogram A in Fig. 1)
Toluene–ethyl formate–formic 19 acid (5:4:1)
Chloroform extract (chromatogram B in Fig. 1)
Toluene–ethyl formate–formic 19 acid (5:4:1)
Rf values 0.05, 0.55, 0.98 0.10, 0.60, 0.96 0.05, 0.68,
0.08, 0.09, 0.11, 0.16, 0.22, 0.25, 0.40, 0.49, 0.57, 0.61, 0.66, 0.72, 0.77, 0.83, 0.88, 0.91, 0.13, 0.18, 0.25, 0.29, 0.39, 0.48, 0.53, 0.56, 0.63, 0.67, 0.68, 0.72, 0.75, 0.81, 0.86, 0.89,
Petroleum ether extract (chromatogram C Toluene–formic acid (95:5) in Fig. 1)
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after the last dose. Body weight gain (percentage) and relative organ weight (organ weight/100 g of body weight) of kidney, liver, spleen and thymus were determined for each animal. Single cell suspensions were prepared in RPMI-1640 medium from bone marrow (from femur), spleen and thymus and cell counts were made using Neubauer chamber (Raisuddin et al., 1990).
Paul, MN). The chambers were loaded with a known volume of assay mixture, sealed with petroleum jelly and incubated at 37°C for 1 h. The plaques were counted under a light microscope (Olympus BX50) and expressed as PFC per 106 spleen cells.
2.6. Assessment of humoral immune functions Four groups of animals were made as described above. Animals of all the groups were challenged with 0.2 ml of 10% sheep red blood cells (SRBC), i.p. on the tenth day of initiation of experiment. The following parameters of humoral immunity were studied in those animals.
2.6.1. Hemagglutination titer (HT) assay It was performed using procedure of Mungantiwar et al. (1999). Blood was collected from the retro-orbital plexus of each mouse for serum preparation. Serial two-fold dilution of serum samples was made in 50 ml of PBS (pH 7.2) in 96-well microtiter plates and mixed with 50 ml of 1% SRBC suspension in PBS. After mixing, plates were kept at room temperature for 2 h. The value of antibody titer was assigned to the highest serum dilution showing visible haemagglutination. 2.6.2. PFC assay The PFC assay was performed using the method of Raisuddin et al. (1991). In brief, 0.2 ml of 10% SRBC prepared in normal saline was injected to animals, i.p. The animals were sacrificed on the fifth day of immunization. The spleen was removed, cleaned free of extraneous tissues and a single cell suspension of 106 cells/ml was prepared from it in RPMI-1640 medium. For PFC assay, the SRBC were prepared at a cell density of 5× 108 cells/ml in PBS. One milliliter of SRBC in medium along with 0.5 ml of diluted guinea pig complement (1 ml of serum + 9 ml of normal saline) was added to 1 ml of spleen suspension. Cunningham chambers were prepared using glass slide, coverslips and doubled-sided tape (Scotch Brand, St.
0.07, 0.11, 0.16, 0.20, 0.25, 0.29, 0.37, 0.62, 0.83, 0.88
2.6.3. Quantitati6e hemolysis of SRBC (QHS) assay QHS assay was performed using the methods of Simpson and Gozzo (1978) with some modification. Spleens were removed and a cell suspension of 10× 106/ml was prepared in PBS. One milliliter of 0.2% SRBC and 1 ml of 10% guinea pig serum were mixed with cell suspension and incubated for 1 h at 37°C. After centrifugation at 3000 rpm for 3 min, optical density of the supernatant was measured at 413 nm using spectrophotometer (Shimadzu UV-1201). 2.7. Statistical analysis Data were statistically analyzed using Student’s t-test to determine significant differences in data of various groups; p values less than 0.5 were considered significant. The values are expressed as means+ SE.
3. Results
3.1. Effect of plant extract on body weight, organ weight and cellularity None of the groups showed mortality. No significant body weight gain differences were recorded in various groups of animals. As regards relative organ weight, CP treatment caused a significant (PB 0.001) reduction in the spleen weight when compared with control (normal saline treated group I) animals (Table 2). Interestingly, the plant extract also caused spleen weight reduction (PB 0.01). No significant recovery of spleen weight was observed in the plant extract+ CP (group IV) animals. CP treatment also induced a significant reduction in the relative organ weight of thymus (PB0.05). Plant extract had no adverse effect on the thymus weight and had no protective effect on CP-induced reduction of
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Table 2 Effect of C. occidentalis and cyclophosphamide treatment on the relative organ weight of micea Group
I (control) II (CP) III (plant extract) IV (plant extract +CP)
Relative organ weight (means 9 S.E.) in g
Table 4 Effect of C. occidentalis on CP-induced suppression of antibody titre in mice Group
Spleen
Thymus
Liver
Kidney
0.7219 0.05 0.2469 0.02b 0.49790.009c
0.159 90.007 0.089 90.012d 0.210 9 0.016
5.13 9 0.25 5.12 90.22 4.92 9 0.22
1.042 9 0.060 1.025 9 0.020 1.027 9 0.020
0.3069 0.03
0.089 9 0.008
4.41 90.13
1.164 9 0.100
Values are means 9 S.E. g of five mice. PB0.001 when compared with group I. c PB0.01 when compared with group I. d PB0.05 when compared with group I (significantly different). a
b
thymus weight. No significant reduction was observed in case of relative organ weights of other organ viz., kidney and liver of the animals of different groups. CP treatment resulted in a decrease in cellularity of bone marrow, spleen (PB 0.001) and thymus (P B0.001) as compared to control (group 1) animals (Table 3). The plant extract also caused significant depletion of splenic cell counts (PB 0.01) but increased bone marrow cell counts in a significant manner (P B 0.001). The plant extract showed protective effects on cellularity of bone marrow (p B 0.05), spleen (P B0.01) and thymus (PB 0.01) when compared with CP-treated animals.
3.2. Effect of plant extract on CP-induced suppression of humoral immunity parameters A single dose of CP (50 mg/kg b.w.) induced a significant reduction in all the parameters of humoral immunity, antibody titer (Table 4), PFC assay (Fig. 2) and QHS assay (Fig. 3) in mice. The reduction was
I (control) II (CP) III (plant extract) IV(plant extract+CP)
Titer level 1:2048 1:4 1:4096 1:256
significant (P B0.001) when compared with control animals (group I). The C. occidentalis extract showed a stimulatory effect in the PFC assay (PB 0.01). When compared with CP-treated animals (group II), animals receiving plant extract treatment along with CP (group IV) showed significant recovery in the antibody titre, in the PFC assay (PB 0.001) and in the QHS assay (PB 0.05).
4. Discussion Cassia occidentalis treatment to animals protected them from CP-induced suppression of humoral immunity. Animals also showed improvement in CP-induced immunopathy in different organs. Though the plant extract showed no suppressive effect on any of the parameters of humoral immunity, it reduced spleen weight and spleen cellularity. We do not have an exact explanation for this selective toxicity to spleen at this juncture. C. occidentalis is not only reported for its medicinal value but some reports also showed toxic effects in various species (Martin et al., 1981; Marrero et al., 1998). The PFC response of plant extract treated animals showed no adverse effect. In fact, all the
Table 3 Effect of C. occidentalis treatment on CP-induced depletion of cellularity of lymphoid organsa Groups
Cellularity (means 9S.E.)×106 Spleen
I (control) 342.83 924.45 II (CP) 53.30 95.32b III (plant 237.33 9 14.77c extract) IV (plant 79.50 92.79d extract+CP)
Bone marrow
Thymus
20.12 92.80 14.569 2.80 36.109 2.26c
65.62 9 7.10 14.409 1.41b 51.7193.69
25.09 1.41d
28.269 3.01d
Values are means 9 S.E.×106of six mice. b PB0.001 when compared with group I. c PB0.01 when compared with group I values. d PB0.01 when compared with group II (significantly different). a
Fig. 2. Effect of C. occidentalis extract (100 mg/kg) on CP-induced suppression of humoral immunity as assessed by plaque forming cell assay. Data are means 9 S.E. of six animals. * P B0.01 and ** PB 0.001 when compared with control group animals, *** PB 0.01 when compared with values of CP-treated animals (significantly different).
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Fig. 3. Effect of C. occidentalis extract (100 mg/kg) on CP-induced suppression of humoral immunity as assessed by quantitative hemolysis of SRBC assay. Data are means 9 S.E. of six animals. * P B0.05 when compared with values of CP-treated animals, ** P B0.001 when compared with control group animals (significantly different).
parameters of humoral immunity that were suppressed as a result of CP treatment showed recovery in the animals treated with C. occidentalis extract. Our studies have shown that the total aqueous extract of C. occidentalis possesses antimutagenic activity against BaP and CP (Sharma et al., 1999). Using three doses (50 mg, 100 mg and 250 mg/kg b.w.) in mice, we found that 100 and 250 mg/kg showed almost same level of antimutagenic effect. In that study, we also showed that C. occidentalis had a modulatory effect on hepatic activation and disposition mechanisms; it decreased cytochrome P450 contents (phase I metabolism) and increased glutathione S-transferase (GST) activity (phase II metabolism). This was helpful in reducing the activation of toxicant to reactive metabolites on the one hand and facilitating quick disposition of toxicants and their metabolites on the other hand. CP also undergoes cytochrome P450-mediated activation by phase I enzymes and GST-mediated detoxification phase (Torkelson et al., 1974). Two of the metabolites of CP have been identified as acrolein and phosphoramide mustard (Domeyer and Sladek, 1980). Although useful in the chemotherapeutic treatment of various forms of cancers, CP toxicity has been of major concern limiting its use. Besides being a potent immunosuppressant and mutagen, CP is also reported to induce bladder cancer (McCann et al., 1975; Seino et al., 1978). In the Ames test, it requires metabolic activation and this strengthens our hypothesis that by modulating activation and detoxification mechanisms CP toxicity including the immunotoxicity could be reduced to a great extent. Some of the chemical constituents isolated from C. occidentalis include anthraquinones, fatty oils, flavonoids, gallactomannan, polysaccharides and tan-
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nins (Lal and Gupta, 1973, 1974; Kudav and Kulkarni, 1974; Singh and Singh, 1985; Gupta et al., 1995). Many of these constituents isolated from other plant sources possess immunomodulatory activity (Wang et al., 1991; Hase et al., 1996; Middleton, 1998). Most of the studies concerning the evaluation of immunomodulatory activities of the plants have been undertaken utilizing the crude extracts (Sohni and Bhatt, 1996; Ziauddin et al., 1996; Dhuley, 1997). In some studies, a combination of various herbs or herbs in combination of minerals have been used taking into consideration Ayurvedic, Chinese, Japanese, Korean or Unani traditional formulations (Komatsu et al., 1986; Yamaguchi, 1992; Nakai et al., 1993; Sohni and Bhatt, 1996; De et al., 1998; Bajaj et al., 1999). Though it may be rational to use single plant or its single constituent, it has been a general experience that the total plant extract shows more efficacy versus single constituent (Dhir et al., 1990, 1991; Sharma et al., 2000b). The findings of present study are significant in the direction of developing safer strategies for cancer treatment.
Acknowledgements The financial support received from the Central Council for Research in Unani Medicine (CCRUM) and the University Grants Commission (UGC) for this study is gratefully acknowledged. We thank Dr M.S. Alam, Department of Chemistry for his expert help in chemical analysis of the extract. Assistance of Mr. Razi Ahmad is also acknowledged.
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