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Preliminary immunomodulatory activities of methanol extracts of Eclipta alba and Centella asiatica.

Summary

An attempt has been made to assess the immunomodulatory activity of methanol extracts of whole plant of E. alba (1.6% wedelolactone) and C. asiatica (0.18% of asiaticoside) at five dose levels (dose-response relationship) ranging from 100 to 500 mg/kg body wt. using carbon clearance, antibody titer and cyclophosphamide immunosuppression parameters. In the case of E. alba, the phagocytic index and antibody titer increased significantly and the F ratios of the phagocytic index and WBC count were also significant. Regression analysis showed linearity in patterns of the dose-response relationship, greatest in the case of the phagocytic index, moderate in the WBC count and lowest in the antibody titer. For C. asiatica, significant increases in the phagocytic index and total WBC count were observed and the F ratio of the phagocytic index was also significant. Regressed values revealed maximum linearity in the case of the phagocytic index, moderate linearity in the total WBC count and lowest linearity in the antibody response.

Key words: antibody titer, carbon clearance test, C. asiatica, E. alba

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Introduction

Some plants are believed to promote positive health and maintain organic resistance against infection by establishing body equilibrium. It is tempting to speculate that the restorative and rejuvenating power of these herbs may be due to their action on the immune system. The concept of immunomodulation relates to non-specific activation of the function and efficiency of macrophages, granulocytes, complement, natural killer cells, and lymphocytes, and also to the production of various effector molecules generated by activated cells (Para immunity). It is expected that these non-specific effects give protection against different pathogens including bacteria, fungi, viruses, etc., and constitute an alternative to conventional chemotherapy (Atal et al. 1986).

Eclipta alba (Family Asteraceae) grows in tropical and subtropical countries at an altitude of up to 2000 meters. In India, it is called "Bhringraj". In traditional medicine, whole dried herb powder is used in liver disorders, especially jaundice (Chopra et al. 1966). The herb contains wedelolactone and dimethylwedelolactone as coumestan derivatives, the first one having been designated as the major anti-hepatotoxic compound of E. alba (Wong et al. 1988). This plant has also been reported to possess antinociceptive, anti-inflammatory and bronchodialator activities, due to the coumarin compounds (Leal et al. 2000). Recently, a triterpenoidal saponin, "eclalbatin", has also been isolated from this plant (Upadhyay et al. 2001).

Centella asiatica (Fam: Apiaceae) is a perennial creeper, growing abundantly in moist areas and distributed widely in tropical and subtropical countries. In India, it is called "Mandukaparani" and used in folk medicine for leprosy, lumps, syphilis, and tuberculosis and to improve mental function (Kartnig, 1988), and is also used in rasayanas (Chopra et al. 1958). Asiaticoside and its derivatives were isolated and reported to possess anti-leprotic activity (Boiteau et al. 1949) and also to elevate the anti-oxidant level in healing wounds (Shukla et al. 1999a, 1999b). A recent report reveals that an aqueous extract is effective in cognitive functions (Veerendrakumar and Gupta, 2002) and has an effect on the endogenous catalase enzyme. The reticuloendothelial-stimulating activity of the alcoholic extract of C. asiatica (DiCarlo et al. 1964) and an increase in the antibody titer and cell-mediated response (DTH) at 100 mg/kg body wt. of dried powder of C. asiatica have been reported (Patil et al. 1998). It was also reported that the aqueous extract of the whole plant produced an effect on classical and alternative human complement systems in an in vitro model (Labadie et al. 1989). It is used commercially as antiarthritis herb in the West. The selected plants form an important component of the traditional system of medicine, with a variety of bioactivities. The present preliminary studies were, therefore, aimed to assess these plants' immunomodulatory potential using suitable in vivo models.

Materials and Methods

Preparation of methanol extracts of E. alba and C. asiatica

The fresh whole herbs E. alba and C. asiatica were procured from a local vendor, and were authenticated by comparison with herbarium specimens of the Botany Department, The M. S. University of Baroda, India. These were washed, dried under shade, sieved through mesh no. 45 and subjected to cold maceration using methanol in order to restrict the degradation of bioactive molecules. The solvent was evaporated to dryness in vacuo. The contents of wedelolactone (1.6%) and asiaticoside (0.18%) in the respective extracts were determined according to reported methods (Zafar and Sagar, 1999; Inamdar et al. 1996). The methanol extracts of both plants were then suspended in distilled water with 1% SCMC (sodiumcarboxymethylcellulose), and used for further investigations.

Animal strain used

Swiss Albino mice of either sex, weighing 17-25 g each, were used. They were acclimatized to laboratory conditions with pellet feeding and water ad libitum.

Preparation of 20 v/v SRBC suspension

The blood was collected from a healthy sheep from the Baroda municipal slaughterhouse, India, in a mixture of 0.49% EDTA and 0.9% of sodium chloride solution. It was preserved at a temperature of 2-8[degrees]C. On the day of immunization, the blood sample was centrifuged at 5000 rpm for 10 min and then washed three times, to remove plasma, with 0.9% sodium chloride solution. The SRBC (20% v/v) suspension was then prepared in 0.9% sodium chloride solution.

Carbon clearance test

Mice were divided into 2 groups, each containing 10 animals. Group I (control) was given 1.0% SCMC in water (0.3 ml/mouse) for 5 days. Group II (treated) was given the test sample orally for 5 days. At the end of five days, after 48 h, mice were injected via the tail vein with carbon ink suspension (10 [micro]l/gm body wt.) (Pelican AG, Germany). Blood samples were drawn (in EDTA solution 5 [micro]l) from the retro-orbital vein at 0 and 15 min, a 25-[micro]l sample was mixed with 0.1% sodium carbonate solution (2 ml) and its absorbance at 660 nm was determined. The phagocytic index K was calculated using the following equation: K = (Lo[g.sub.e] O[D.sub.1]-Lo[g.sub.e] O[D.sub.2])/15, where O[D.sub.1] and O[D.sub.2] are the optical densities at 0 and 15 min, respectively. Results were expressed as the arithmetic mean [+ or -] S.E.M. of five mice.

Cyclophosphamide-induced myelosuppression assay

Animals were divided into 2 groups 6 animals each. Animals in the treated group were given the test sample daily for 13 days. Positive control and negative control group animals received 1.0% SCMC in water (0.3 ml/mouse, orally, daily for 13 days). On days 11, 12 and 13, all the animals except in the negative control group were given cyclophosphamide solution orally, 1 h after the administration of extract. Blood samples were collected on day 14 and the total white blood cell (WBC) count was determined.

Humoral antibody titer

Mice were divided into two groups containing six animals each and were then immunized with 20% SRBC (0.1 ml) intraperitonially. Group I (control) was given 1% SCMC in water (0.3 ml/mouse, orally) for seven days. Blood samples were collected from individual animals by retro-orbital puncture on day 8 and were centrifuged at 2500 rpm for 10 min to separate the serum. Two-fold dilution of 50 [micro]l sera (heat inactivated at 56 [degrees]C for 30 min) was performed in RPMI-1640 medium. Serial dilution (taking 50 [micro]l of the aliquot) was performed in 50 [micro]l RPMI-1640 medium into 96 well micro-titre plates. The fresh, SRBC (1.0%; 25 [micro]l) suspension was dispensed into each well and mixed thoroughly. The plates were then incubated at room temperature for 2 h and examined for button formation. The reciprocal of the dilution, just before the button formation, was observed and titer values were calculated. Group II (treated) was given test sample for seven days. The experiment was performed on day 7 as for the control group.

Results and Discussion

The use of immunostimulants, particularly as adjuvant to chemotherapy, to control and prevention of infection holds great promise (Chatterjee et al. 1998). Significant interest has now been generated in research on bioactive molecules from plant drugs designated as immunomodulatory agents in alternative systems of medicine (Lee et al. 1995). Compounds like polysaccharides, saponins, phenols and alkaloids (Ingolfsdottir et al. 1994) have been tested for their utility as both biological and chemical markers. Wedelolactone and asiaticoside are the major constituents of E. alba and C. asiatica, respectively, and therefore used as therapeutically active marker compounds. The methanol extracts (containing these markers) of both the plants were tested in the present study to determine their possible effect on immune function at five dose levels.

E. alba

Statistical analysis (t-test) wasused to determine statistically significant differences between the control and test groups. Phagocytic index was increased (p < 0.01) from the 100 to the 400 mg/kg/body wt. dose as compared to the control value. A further increase was observed at the 500 mg/kg/body wt. dose (p < 0.001) (Table 1). The antibody titer (p < 0.01) value at the 100 mg/kg/body wt. dose was increased, due to perhaps the stimulation of B-lymphocytes for the production of antibodies (Table 1), but remained unchanged up to a 500 mg/kg/body wt. dose. The WBC count was low at the 100 mg/kg/body wt. dose, on a par with the cyclophosphamide group, but at between 200 and 500 mg/kg/body wt. doses, the count increased significantly (p < 0.001). These results followed a pattern similar to those reported for Aloe vera, which contains phenolics having anti-inflammatory potential, and which also showed B-cell stimulation to a significant extent (Hartwell, 1969).

The exertion of an immune response needs a certain threshold dose, beyond which it does not show enhancement, and it has also been reported that immunostimulating substances could exhibit suppressive effects (Wagner and Jurcic, 1991). The above phenomenon was very noticeable in the case of antibody titer and WBC counts.

The t-test was further employed to compare the difference of significance levels between selected groups. Values of the phagocytic index between 100 and 500 mg/kg/body wt. doses showed significant difference (p < 0.05), while the HA titer values did not. In the case of the WBC count, the values were significantly different from each other, when the 100 mg/kg/body wt. dose was compared with higher dose levels (100 and 200; 300; 400; 500) (p < 0.001).

One-way ANOVA was applied to assess the overall significance among the groups. The F ratio values of the phagocytic index were 66.27 and 347.68 for the WBC count (p < 0.001). The F ratio of the HA titer values was, however, not significant.

Regression analysis of dose-response ([R.sup.2]) of the individual parameters was also carried out to assess linearity. The hierarchy was determined as follows: phagocytic index (0.67) > WBC count (0.49) > Antibody titer (0.32), as per the trend line analysis, which has suggested that the dose-response relationship is directly proportional; more in the case of phagocytic index, moderate in case of WBC count and least in the antibody titer.

Based on the significant values obtained, the methanol extract of E. alba has scored a value of 17 (p < 0.05; 1; p < 0.01; 2; p < 0.001:3). The probable reason is the presence of phenolic wedelolactone (1.6%). The result obtained in the present study was in agreement with reports that plant phenolics and saponins are effective as immunomodulators (Lee et al. 1995).

C. asiatica

The methanol extract of C. asiatica (0.18% asiaticoside) exhibited an increase (p < 0.05) in the phagocytic index at dose of 100 and 200 mg/kg/body wt., which further changed (p < 0.01) at 300 and 400 mg/kg/body wt. and was maximal (p < 0.001) at the 500 mg/kg/body wt. dose (Table 1). These results show similarity to those already reported for T. cordifolia, having a significant activity in kuffer cell function and the phagocytic index (Nagarkatti et al. 1994). In the case of antibody production, no significant rise was seen (Table 1), which reveals that the drug perhaps has no effect on B-lymphocyte activity. In one of the earlier reports on saponins of P. pseudogensing (Dua et al. 1989) and andrographolide diterpenes of A. paniculata (Puri et al. 1993), however, a rise in the antibody titer against sheep red blood cells was mentioned. The methanol extract of C. asiatica (0.18% asiaticoside) did not increase the titer values, perhaps due to the fact that asiaticoside is a triterpenoidal saponin. The value of the total WBC count, however, increased (p < 0.05) at the dose of 500 mg/kg/body wt. (Table 1).

When the 't' test was applied between the selected groups, a significant difference in the values of the phagocytic index was observed between 100 and 200; between 100 and 400; and between 100 and 500 mg/kg/body wt. doses at a level of p < 0.05. The difference in the values for the HA titer and WBC count among selected groups was not significant.

The F ratio value of the phagocytic index was determined to be 82.72 (p < 0.001). The F ratio values of antibody titer and WBC count were, however, not significant.

Regression analysis on the individual dose-response relationships followed a pattern; phagocytic index (0.97) > WBC count (0.83) > Antibody titer (0.57) as per the trend lines, suggesting that the values of the phagocytic index had maximum linearity, followed by total WBC count and the antibody response. The methanol extract of C. asiatica scored 11 points and suppressed humoral response.

On the whole, the results of the above studies on both the plants indicate preliminarily that they hold promise as immunomodulatory candidates, and provide a scope for further detailed investigation on the fractions/individual constituents.
Table 1. Results obtained from different immunomodulatory assays.

Dose Control 100
(mg/kg/
body wt.)

E. alba
PI 0.086 [+ or -] 0.015 0.148 [+ or -] 0.016 (b)
AT 149 [+ or -] 62 384 [+ or -] 67 (b)
WBC 3.8 [+ or -] 0.35 3.476 [+ or -] 0.35
C. asiatica
PI 0.086 [+ or -] 0.015 0.135 [+ or -] 0.016 (a)
AT 149 [+ or -] 62 203 [+ or -] 76
WBC 3.8 [+ or -] 0.35 3.476 [+ or -] 0.62

Dose 200 300
(mg/kg/
body wt.)

E. alba
PI 0.148 [+ or -] 0.018 (b) 0.149 [+ or -] 0.019 (b)
AT 395 [+ or -] 67 (b) 378 [+ or -] 69 (b)
WBC 6.29 [+ or -] 0.51 (c) 6.514 [+ or -] 0.48 (c)
C. asiatica
PI 0.138 [+ or -] 0.015 (a) 0.153 [+ or -] 0.018 (b)
AT 212 [+ or -] 70 201 [+ or -] 85
WBC 3.72 [+ or -] 0.47 3.89 [+ or -] 0.57

Dose 400 500
(mg/kg/
body wt.)

E. alba
PI 0.153 [+ or -] 0.013 (b) 0.170 [+ or -] 0.011 (c)
AT 400 [+ or -] 68 (b) 399 [+ or -] 67 (b)
WBC 6.24 [+ or -] 0.45 (c) 6.34 [+ or -] 0.55 (c)
C. asiatica
PI 0.160 [+ or -] 0.019 (b) 0.168 [+ or -] 0.017 (c)
AT 215 [+ or -] 100 272 [+ or -] 88
WBC 400 [+ or -] 0.51 4.89 [+ or -] 0.55 (a)

(a): p < 0.05; (b): p < 0.01; (c): p < 0.001.
PI: Phagocytic index; AT: Antibody titer: WBC: Total white blood cell
count (thousand X [mm.sup.3])


Acknowledgement

The authors are thankful to the CSIR, New Delhi, for providing financial assistance, and to Dr. P. K. Inamdar, Hoechst India, Ltd., and M/s Natural remedies, Bangalore, for providing gift samples of asiaticoside and wedelolactone.

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M. G. Jayathirtha and S. H. Mishra

Pharmacy Department, The M.S. University of Baroda, Kalabhavan, Vadodara 390 001, India

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S. H. Mishra, Pharmacy Department, The M.S. University of Baroda, Kalabhavan, Vadodara 390 001, India

e-mail: shmishra48@rediffmail.com
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Author:Jayathirtha, M.G.; Mishra, S.H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Apr 1, 2004
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