Introduction

The immune system is known to be involved in the etiology as well as pathophysiologic mechanism of various diseases. This problem can be overcome by boosting the immune system by the use of immunomodulatory drugs of natural or synthetic origin. Few drugs act as immunopotentiators and induce cytotoxicity leading to diseases and exert a variety of side effectsm¹ This has given an initiation  in the search for investigating natural resources showing immunomodulatory activity. Immunology is one of the area of biomedical research has great promises with regard to prevention and treatment of wide range of disorders.² The combination of phytosterols, terpenoids, minerals, fibre, phenolics and other antioxidants are probably reliable for these effects.³

Avicenniaofficinalisbelonging to Avicenniaceae is commonly available in coastal states of India which is mainly used in the treatment of rheumatism,asthma, skin disease, paralysis, ulcer and snake bites.⁴

Materials and Methods

Chemicals and Reagents

All the chemicals and reagents were purchased from SD fine chemicals, Sisco laboratories and are analytical grade.

Preparation of Extract

The plant was authentified by Dr.Jeyaraman, Botanist, Plant Research Centre, Tambaram, and the authentification no. was PARC/2015/3171.The coarsely powdered   Avicenniaofficinalis  leaves were  extracted with n-hexane, 70% hydroalcohol and ethyl acetate successively by hot percolation process  for 72hrs. The percentage yield were calculated. The phytochemical screening of the three extracts were performed as per standard protocol.Then the  extracts were subjected toinvitroimmunomodulation studies.

NitroblueTetrazolium Reduction Assay (NBT)

A suspension of leucocytes was prepared in 0.5ml of PBS solution , to it 0.1ml of phosphate buffer saline solution followed by 0.1ml of endotoxin was added. The different concentration (10,20,50,100,200,400,800ug/ml) of test samples used was taken in  3-7 test tubes .about 0.2ml of freshly prepared 0.15% NBT solution was added to each tube, incubate at 37 c for 20 mins. Thr resultant was centrifuged at 400mg for 3-4mins to discard the supernatant and  cells were resuspended in small volume of PBS solution and thin film was made with drop on slide and after drying, fixed by heating, counter strained with carbon fuschin for 15 seconds and the slide was washed under tap water, dried and focussed under 100x magnification oil immersion.200 Neutrophils were counted for the % of NBT positive  cells containing blue granules.⁵

Phagocytosis of Candida Albicans (Killed)

Preparation of Suspension of Candida Albicans

Candida albicans culture incubated in sabouraud broth overnight, centrifuged to form cell button and supernatant discarded.  The cell button was washed with Hank’s solution and centrifuged  repeatedly  for 3-4 times where final cell button was mixed with mixture of HBBS and  human serum in proportion of 4:1.⁶

Preparation of Slide

Sterile glass slide containing 0.2ml of fresh human blood by finger prick and incubated at 37 degree c for 25mins to  allowclotting.The blood clot was removed gently and drained slowly with sterile normal saline without washing off adhered neutrophil.The slide containing polymorphonuclear neutrophils (PMN) was flooded with  concentation of test sample and then incubated for 15mins at 37degree c. The slide was drained, fixed with methanol and was stained with geisma stain.

Candidacidal Assay

The same dilution and procedure was followed as phagocytosis and the pellets of assay tubes were suspended in 100ulMEM and incubated for 30mins at 37degree celsius.At the end of incubation period,0.25ml of 2.5% Na deoxycholate added to each test tube to lyse the leucocytes.0.25ml of 0.01% methylene blue added to each test tubes and mixed well.The tubes was centrifuged, supernatant was decanted and smears prepared on slides using resultant pellets.The % of candida cells was determined⁷

Results  and Discussion

Immunomodulatory agents of plant and animal origin enhance the immune responsiveness of the body against pathogens by activating the non-specific immune system. However, there is a need for systemic studies on medicinal plants to potentiate the therapeutic claims made regarding their clinical utility. The primary step in this study  wasauthentification of the plant which was done  by Dr.Jeyaraman ,Director PARC, West Tambaram. The percentage yield of hydroalcoholic extract, n-hexane and ethyl acetate extract was found to be 7.30 % w/w, 1.32 % w/wand 0.86% w/w respectively. The colour of these extract were greenishbrown, greenish brown and greenish yellow respectively.

Phytochemical Screening

The preliminary phytochemical screening showed the presence alkaloids, tannins, iridoidglycosides,triterpenoids, steroids in ethanolic extract, wheras n-hexane posses steroid, triterpenoids and ethylacetate fraction showed the presence of tannins , and iridoid glycosides.

Nitroblue Tetrazolium Assay

In-vitro NBT Assay

Nitrobluetetrazolium (NBT) assay was performed as one of the evaluation tests for the immune modulatory activity assessment. This semi-quantitative microscopic nitrobluetetrazolium (NBT) assay is used to determine the production of superoxide anion (O2 (-)) in various phagocytic cells. This assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitrobluetetrazolium (Y-NBT) by O2(-).^[8]Nitrobluetetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles.Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction.⁹

In NBT  assay, the 70% ethanolic extract and ethyl acetate extract  has suppressed  the neutrophils and so reduced formazan formation. The percentage  reduction of reduced neutrophils was found to be 21.32 , 25.98 for ethanolic and ethyl acetate extract  respectivelyand the results were mentioned in Table-1. Both the extracts showed effective suppressant action at  higher concentration level (800µg/ml) but the n-hexane showed least action compared to other two extracts. Thus the phytomolecule in the ethanolic extract and ethyl acetate has significant immunomodulation effect.

Phagocytosis Assay

The Polymorphonuclear neutrophils (PMN) and mononuclear phagocytes represent an important first line and effector function in the control of Candidaalbicans.¹⁰  This evaluation is based on the principle that the viable Candida albicansdo not stain supravitally with methylene blue.¹¹

In this assay, the phagocytosis effect on killed candida albicans mean particle numbers were found to be 2,1 for ethanolic extract and ethyl acetate extract respectively wheras n-hexane group  showed non significant effect. At lower concentration, immunosuppressant effect was least compared  with higher dose.The isolated compound stimulates the phagocytosis of killed candida albicans.the mean particle was found to be 5-6,4 and 4 for isolated compound at concentration of 1000,100,40ug/ml and when compared to pooled serum 6,4-5,6 at same concentration. At low concentration of 20 and 10ug/ml the stimulation of phagocytic activity is negligible. The phagocytic assay result mentioned in Table:2.

Candidacidal Assay

The engulfment mechanism of neutrophils is considered as main phagocytic action of an organism[^12]and the results were mentioned in Table-3. The ethanolic extract and ethyl acetate extract of Avicenniaofficinalishas significantly increases reduced neutrophils mean particle number of killed candida alicans and also increases the percentage of candidacidal  action  The percentage  of candidacidal activity was found to be 34.42% for pooled serum,32.34% for ethanolic extract, 24.42% for n-hexane and 14.26% for ethyl acetate extact has least action.Thus the ethanolic extract and ethyl acetate extract of Avicenniaofficinalis has significantly increases reduced neutrophils.

Table 1: Percentage of reduced neutrophils by Avicenniaofficinalis using Nitrobluetetrazolium assay

The data are expressed as mean percentage reduced neutrophils ± standard error mean. Significant difference from Positive Control by oneway ANOVA followed by Dunnet’s‘t’ test. (n = 4) p^**<0.01.P^*<0.5Phosphate Buffer Saline (Normal Control), Endotoxin-activated plasma (Positive Control)

Table 2: Particle number phagocytosis of killed Candida albicans after treatment with extracts of Avicennia officinalis

The dataswere expressed as mean particle number phagocytosed.

Table 3: percentage of Killed Candida after treatment with extracts of Avicennia officinalis leaves by Candidacidal Assay

The data are expressed as mean percentage of killed candida. Significant difference from Positive Control (Std.) by One Way ANOVA followed by Dunnet’s ‘t’ test. (n = 4); *p<0.5.

Conclusion

The immunomodulatory effect of hydroalcoholic extract of Avicenniaofficinalishas proved significantly based on the reports of invitro studies namely -NBT assay,Candidacidal assay and phagocytosis assay. The immunosuppressive activity may be due to presence of active biomolecules present in it.In future, the phytomolecule has to be isolated, characterized and finally formulated  for clinical trials to serve the society.

Acknowledgement

Our sincere thanks to our Chancellor for his encouragement to conduct research work in this institution and I would like to thank our Dean Dr.K.S.Lakshmi for her  constant support in research.

References

1. Krzystynia K., Tryphonas H., Fournier M.  Environmental Health Perspectives. 1995;103(9):17-22.
   CrossRef
2. Wagner H. (Ed). In.  Immunomodulatory agents from plants, Basel Birkhauser, Germany. 1999;1-35.
3. Ruxton C., Gardner E.,Walker D.   International Journal of Food Science and Nutrition. 2006;57:249-272.
   CrossRef
4. Choi H. S., Kim J. W., Cha Y. N., Kim C.   A quantitative nitrobluetetrazolium assay fordetermining intracellular superoxide anion production in phagocytic cells. J Immunoassay.Immunochem. 2006;27(1):31-44.
   CrossRef
5. Segal A. W., Levi A. J.  Cell damage and dye reduction in the quantitative nitrobluetetrazolium (NBT) test. Clin. Exp.Immunol. 1975;19(2):309-18.
6. Vonk A. G., Wieland C. W., Netea M. G., Kullberg B.  J.  Phagocytosis and intracellular killing of Candida albicansblastoconidia by neutrophils and macrophages a comparison of different microbiological test systems.J Microbiol Methods. 2002;49(1):55-62.
   CrossRef
7. Wilkinson P. C.  Neutrophil in Thomas R. A editor .techniques in clinical immunology,2nd edition london blackwell scientific publication. 1981.
8. Ponkshe C. A., Indap M. M.  in vivo and in vitro studies of Immunomodulatory activity of marine animal extract with reference to phagocytosis. Indian J ExpBiol. 2002;40:1399-402.
9. Lehrer R. I.,Cline M. J. Leukocytecandidacidal activity and resistance to systemic candidasis in patientswith cancer. 1971;27(5):1211-1217.
10. kulkarni S. K.  Biostatics in experimental Pharmacology. Handbook of ExpPharmacol, vallabh prakashan. 1999.
11. Konig P.,Hordvik N. L.,Kreutz C. The preventive effect duration of action of nedocroml sodium and cromolyn sodium on excercise induced asthma inn adults.J Allergy clinImmunol. 1987;79:64-80.
    CrossRef
12. Daniel P. S.,AbbaI T.,Tristram G. P.  Basic and clinical immunology;8th edition ,USA.Appleton and lange. 1994