INTRODUCTION:

Eclipta prostrata Linn. belongs to the family Asteraceae is a common plant, abundantly grown in tropical and sub tropical countries. It is popularly known as false daisy or Bhingaraj. The plant has been reported to contain phytosterols, β-amyrin, triterpines such as ecalbatin, echinocystic acid, and flavones such as luteolin and coumarin such as wedelolactone. Traditionally the plant is used as analgesic, antispasmodic, fungicidal, bactericidal and has a stimulant properties^1,2.

A global reliance on alternative system of medicine for chronic and acute ailments resulted in an intense area of research and discovery of a number of herbs with potential to curb diseases. Among them, ample number of herbs has been exploited for modulation of immune system from Ayurvedic formulation either alone or in combinations³. Environmental pollutants and dietary habits cause disturbances in immune activities and diet containing micronutrients and antioxidants are known to prevent these alterations⁴. The use of herbs as immunomodulators in the indigenous system of medicines, indeed, can modulate the body’s defense mechanism. The following active constituents of plant derivatives such as polysaccharides, lectins, peptides, flavonoids and tannins have been reported to modulate the immune system in different experimental models⁵. The present study was aimed to evaluate the immunomodulatory effect of Eclipta prostrata whole plant extract on albino rats.

METHODOLOGY:

Collection:

The whole plant of Eclipta prostrata was collected from the surrounding villages of Muramalla, East Godavari district.

Preparation of Methanolic whole plant Extract of Eclipta prostrata:

The dried whole plant was powdered, 500g of powder was subjected to extraction using Soxlet apparatus with various solvents like Methanol^6,7. The extract was evaporated in vacuum under reduced in a rotary vacuum evaporated until all the solvent had been removed to give a semi solid extract. The solvent was then removed under reduced pressure which will give a brown colored sticky residue. The extract was weighed with respect to dry powered material and stored in a desicator with anhydrous calcium chloride. The percentage yield of the extract was 5.2% w/w

Determination of Immunomodulatory Activity:

Experimental animals:

Albino Wistar rats weighing between 200-250g were used. Institutional Animal Ethics Committee approved the Experimental protocol; animals were maintained under standard conditions in an Animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). The animals were given pellet food (Lipton India Ltd, Mumbai, India) and water ad libitum.

Chemicals:

Levamisole (Vermisole-250mg), India. Leishmann’s stain, WBC diluting fluid, zinc sulphate and barium chloride and cyclophosphamide (Endoxan Injection).

Instruments:

Spectro photo meter, Nephelo turbidity meter, Differential white blood cell counter

Methods: Carbon Clearance Assay (Phagocytic activity):

To evaluate the phagocytic activity of the reticulo-endothelial system in-vivo, the Animals were divided in 4 groups each having 6 animals^8,9 Group 1-Vehicle (Distilled Water), Group 2- Low dose 250 mg/ kg,p. 0 Treated, Group. 3-High dose 500mg/ kg, p.o Treated, Group 4- Standard (levamisol) 50mg/kg. Oral Treated a carbon clearance test was performed after completion of the drug pretreatment. On day 14, the treated rats received an intravenous injection (tail vain) of carbon suspension (1:50 dilution of Indian ink, Camel) in a dose of 0.5mL/100 g body weight. Blood was withdrawn from the retro-orbital venous plexus before injection, immediately after injection and at 5 min and 15 min after injection of the carbon suspension. 0.05mL of blood was lysed with 4mL of 0.1% Na₂CO₃ and the optical density was measured spectrophotometrically at 650 nm wavelength. The phagocytic index K was calculated using the following equation11 log (OD0) - log (OD0)/t Where OD0 is the OD at 0 min and ODt is the OD at t min

Delayed Type Hypersensitivity Responses (Cell Mediated Immunity):

Rats were divided into four groups of six each similarly to carbon clearances assay were immunized on day 0 by i.p. administration of 0.5x109 SRBC/rat and challenged by a subcutaneous (subplantar region) administration of 0.025x 109 SRBC/mL into right hind foot pad and on day +14. All treatment with respectively administered orally from day -14 until day +13. The thickness of the right hind footpad was measured (before challenge) using vernier caliper on the 14th day Foot pad reaction was assessed after 24 hr. (on 15th day), in terms of increase in the thickness of footpad as a result of hypersensitivity reaction due to oedema, The footpad reaction was expressed as the difference in the thickness (m m.) between12.

Cyclophosphamide-Induced Myelosuppression:

Animals were divided into four groups of six animals each. Group I (Normal control group) and Group II (Cyclophosphamide-treated group) received the vehicle (d. water) for period of 13 days. Group III. Eclipta prostrata Low dose 250mg/kg Group IV. Eclipta prostrata High dose 250mg/kg respectively, p.o., daily for 13 days. The animals of groups II-, IV were injected with Cyclophosphamide (30 mg/kg, i.p.) on the 11th, 12th, and 13th day, 1 hr after the administration of the respective drug treatments. Blood samples were collected from retro orbital plexus on the 14th day of the experiment. Determination of total and differential white blood cells was carried out¹⁰.

Neutrophil Adhesion test:

Albino Wistar rats were treated orally with drugs or vehicle for 14 days. On day 14blood samples were collected from the retro-orbital plexus into heparinized vials and analyzed for DLC. After the initial counts, blood samples were incubated with 80mg/ml of nylonfibers for 10 min at 37 °C. The incubated blood samples were again analyzed for DLC. The difference in the neutrophil count before and after incubation of blood with nylon fibres was taken as a measure of neutrophil adhesion^11,12.

Effect on serum immunoglobulins:

Rats were divided into four groups depending on the treatment with the drug or the vehicle. The animals were treated with the drugs orally for 21 days. Six hours after the last dose of drug, blood was collected and the serum was used for estimation of immunoglobulin levels. Briefly, for each serum sample to be analyzed, a control tube containing 6 ml of distilled water and a test tube containing 6 ml of zinc sulphate solution were prepared. To each, 0.1 ml of serum was added from a pipette. They were inverted to enable complete mixing of the reagents and left to stand for 1 hr at room temperature. The first tube served as blank and the second tube was taken as sample. The turbidity developed was measured using a digital nephelo turbidity meter. The turbidity obtained (sample-blank) was compared with that obtained with standard barium sulphate (BaSO₄) solution. The standard BaSO4 solution was prepared by adding 3 ml of BaCl2 solution (1.15% w/v) to 97 ml of 0.2N sulphuric acid. The turbidity obtained with this solution was expressed as 20 zinc sulphate turbidity (ZST) units¹³.

RESULTS:

Cyclophosphamide Induced Neutropenia;

Administration of cyclophosphamide (30mg/kg ip) produced a decrease in neutrophil count. None of the treatments were effective in significantly preventing the neutropenia induced by cyclophosphamide. However, the percentage reductions in neutrophil count were less in all the treated groups compared to control (Table 1).

Fig;1 Evaluation of immunomodulatory activity of methanolic extract for Eclipta prostrata whole plant.

Carbon Clearance Assay:

Administration of Eclipta prostrata high dose (500 mg/kg p.o) increased the clearance of carbon particlesfrom blood as indicated by a significant increase in phagocytic index (p<0.05). The Eclipta prostrata high dose (250 mg/kg p.o) and levamisole (2.5mg/kg p.o) did not show any significant effect on the phagocytic index in the carbon clearance assay (Table 2).

Table 2. Effect of Eclipta prostrata on carbon clearance test:-

S. no.	Treatment	Dose (mg/kg p.o.)	Phagocytic index
1	Control	Saline 1ml/250g	0.0181±0.0037
2	Levamisole	2.5	0.0215±.0084
3	Lowdose	250	0.0175±0.0055
4	High dose	500	0.0461±0.0100

All values are mean ± SEM, n = 5-6. P<0.05 when compared to control group.

Fig;2 Evaluation of immunomodulatory activity of methanolic extract for Eclipta prostrata whole plant.

Neutrophil Adhesion Test:

Incubation of blood samples with nylon fibers produced a reduction in neutrophil count in all the treatment groups. The reduction in neutrophil count in blood samples from EHD treated group was significantly more compared to the control group (p<0.05). No significant change in the neutrophil adhesion was observed between ELD, levamisole and vehicle treated groups (Table 3).

Table 3. Effect of Eclipta prostrata on neutrophil adhesion in rats:-

S. no	treatment	Neutrophil (%)		Difference A-B
S. no	treatment	UB (A)	NFTB (B)	Difference A-B
1	Control	20.33±2.54	14.83±2.07	4.50±2.11
2	Levamisole	21.16±2.83	12.66±0.95	8.00±2.47
3	Low dose	23.00±2.25	14.00±1.86	8.00±1.63
4	High dose	26.83±3.42	10.50±1.54	13.66±.06

All values are mean ± SEM, n = 6, P<0.05 when compared to control group.

Fig;3 Evaluation of immunomodulatory activity of methanolic extract for Eclipta prostrata

whole plant.

Serum immunoglobulin levels:

Both EHD and ELD produced a significant increase the serum immunoglobulin levels. When compared to control. The EHD was more effective (p<0.01) than CLD (p<0.05) in increasing the immunoglobulin levels. Levamisole also produced a significant increase (p<0.01) in serum immunoglobulin levels (Table 4)

Table 4. Effect of Eclipta prostrata on serum immunoglobulins:-

S. No.	Treatment	Serum immunoglobulin levels (ZST units)
S. No.	Treatment	Range obtained	Mean ±SEM
1	Control	17.32 - 20.25	18.49±0.470
2	Levamisole	20.56 - 21.82	21.12±0.183^**
3	ELD	17.72 - 21.50	20.01±0.574^*
4	EHD	20.02 - 20.94	20.64±0.134^**

All values are mean ± SEM, n = 6.*P<0.05, **P<0.01 when compared to control group.

Fig4; Evaluation of immunomodulatory activity of methanolic extract for Eclipta prostrata whole plant

DISCUSSION:

In the present study, EHD (250/500 mg/kg p.o) showed immunostimulant activity. It prevented the mortality of mice challenged with Pasteurella multocida, increased phagocytic index in carbon clearance test; increased serum immunoglobulin level and also increased the antibody titre in indirect haemagglutination test. ELD (250/500) mg/kg p.o) showed only an increase in serum immunoglobulin levels. The results indicate that Eclipta prostrata at high dose increases both cell mediated and humoral immunity and at low dose shows effect only on humoral immunity. The immunomodulatory effect of Eclipta prostrata was evaluated using different animal models to determine its effect on various immunological responses. The carbon clearance assay was used to evaluate the effect on reticulo endothelial cell mediated phagocytosis^8,9. When ink containing colloidal carbon is injected intravenously, the macrophages engulf the carbon particles of the ink. Rate of clearance of (carbonparticles) ink from blood is known as phagocytic index. The EHD produced an increase in phagocytic index suggesting its effect on reticulo endothelial system.

Cyclophosphamide, nitrogen mustard with anti cancer property is known to cause myleosupression resulting in neutropenia. DLC before and after cyclophosphamide administration were determined to study the effect of Eclipta prostrata on haemopoesis. Iwas found that there was no significant change in reduction of neutrophils after treatment with both doses of Eclipta prostrata indicating that it does not have any effect on the haemopoeitic system in immune suppressed states. Cell adherence property of neutrophils is one of the earliest responses of both immunological and physical injury¹². In neutrophil adhesion test, cell adherence property of neutrophils was assessed in blood sample from different groups, by treating with nylon fibers to which the neutrophils adhere. Both doses of Eclipta prostrata and levamisole failed to show any significant effect on neutrophil adhesion property. In mice lethality test, all the treated groups except negative control were sensitized with HS vaccine to boast antibody production¹⁴. The vaccinated animals were challenged with 25 LD50 dose of Pasteurella multocida bacterial culture and observed for period of 72h for mortality. Eclipta prostrata at higher dose and levamisole showed protection against viral challenge. Estimation of immunoglobulin level is a direct measure of humoral immunity. Immunoglobulin levels were determined by using zinc sulphate turbidity test. This method is dependent on the correlation between the concentration of specific immunoglobulin present in the serum and the intensity of the zinc sulphate reaction developed¹³. The EHD, ELD and levamisole produced an increase in serum immunoglobulin levels. The humoral immunity involves interaction of B cells with the antigen and their subsequent proliferation and differentiation to antibody secreting plasma cells (Ose, 1968). In indirect haemagglutination test, SRBCs that act as specific antigens were injected subcutaneously which triggered production of specific antibodies that were estimated in serum samples of different treated groups. The mean antibody titer values of EHD treated and levamisole treated animals were significantly more compared to control confirming their effect on humoral immunity. The results indicate that Eclipta prostrata at high dose increases both cell mediated and humoral immunity and at low dose shows effect only on humoral immunity. Eclipta prostrata bark contains cinnamaldehyde, benzaldehyde, cuminaldehyde and terpenes⁶. Cinnaaldehyde is reported to inhibit lymphocyte proliferation and NF-kappa B stimulation^15,16. The exact constituent responsible for immunostimulant property of Eclipta prostrata is not known. Further work should be carried out to evaluate effect of terpenes present in Eclipta prostrata stem on the immune system. The results of the present study substantiate the belief that Eclipta prostrata is an immune system booster. To conclude, Eclipta prostrata bark possesses immunostimulant activity. In high dose, it stimulates both cellular and humoral arms of the immune response and the low dose stimulates only the production of antibodies.

CONCLUSION:

The Eclipta prostrata possesses immunomodulatory activity. The Eclipta prostrata whole plant increased the phagocytic index in carbon clearance test; prevented mortality induced by Eclipta prostrata by 17% in mice lethality test and prevented the cyclophosphamide induced neutropenia. The Eclipta prostrataalso affected the humoral immunity; it increased the levels of serum immunoglobulins significantly and produced a significant increase in antibody titre in indirect haemagglutination test. Eclipta prostrata low dose showed significant effect only in increasing the serum immunoglobulins level whereas it increased mortality in mice lethality test. Eclipta prostrata in high doses stimulated both cellular and humoral immunity and at low dose, increased only the non-specific serum immunoglobulin levels.

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Received on 18.03.2017 Modified on 01.05.2017

Res. J. Pharmacology & Pharmacodynamics.2017; 9(2): 41-45.

DOI: 10.5958/2321-5836.2017.00008.8

Treatment	Neutrophil count (%)		Reduction of neutrophil (%)
Treatment	Before CP treatment	After CP treatment	Reduction of neutrophil (%)
Control	24.33±1.647	12.17±1.302	52.19
Levamisole	26.33±3.018	9.83±0.792	40.98
Low dose250mg/kg	22.50±2.837	9.17±.600	44.31
Highdose500mg/kg	24.00±3.786	9.00±1.438	41.51