INTRODUCTION:

Immunology is the study that deals with the defence of the body against infectious agents (like bacteria, viruses, fungi and parasites) and other foreign substances, which enter into the body (1). The immune system is involved in the aetiology as well as pathophysiologic mechanism of many diseases. Modulation of the immune responses to alleviate various diseases has been of interest for many years (2). The immune system is the collection of cells, organs, tissues and molecules that functions to defend us against infectious microbes. The coordinated reaction of the immune system against infections (and other foreign substances) is known as the immune response.

Abnormalities of the immune system that result in defective immune responses make individuals susceptible to infections by viruses, bacteria, fungi and parasites. The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal, viral infections and from the growth of tumor cells. Many of these cell types have specialized functions. The cells of the immune system can engulf bacteria, kill parasites or tumour cells, or kill viral infected cells. Often, these cells depend on the T helper subset for activation signals in the form of secretions formally known as cytokines, lymphokines, or more specifically interleukins (3) The immune system is a part of body to detect the pathogen by using a specific receptor to produce immediately response by the activation of immune components cells, cytokines, and chemokines and also release of inflammatory mediator. In the innate immune the nature killer cell plays an important role to the defiance against virus-infected and malignant cell to destroy the abnormal cell (4). Medicinal plants which are used as immunomodulatory effect to provide alternative potential to conventional chemotherapy for a variety of diseases, especially in relation to host defence mechanism. The use of plant product like polysaccharides, lectins, peptides, flavonoids and tannins has been the immune response or immune system in various in-vitro models (5). Some of the plants acts as a immunomodulatory agents, which overcome the potential adverse effects of present immunomodulators. Salvia hispanica. L belongs to the family Laminaceae (6) was rich in fatty acids and dietary fibres (7). Hence this study aims on the evaluation of immunomodulatory activity of salvia hispanica.L seeds by in-vitro studies.

MATERIALS AND METHODS:

Procurement and Authentication:

The chia seeds were procured from the “Bon Appetit”, The organic and health food shop, Puducherry. It was identified and authentified by DR. T. Ganesan, Head and Associate professor, Kanchi Mamunivar, Center for Post Graduate Studies, Puducherry – 605008.

Extraction (8):

100g Coarse grinding chia seeds was packed in a Soxhlet and extracted with solvent hexane for the Period of 72 hours, after extraction, solvent free extract obtained by vacuum evaporation and packed in an airtight container for further studies. The extract was subjected for the phytochemical screening by using conventional methods.

Drugs and Chemicals:

All the chemical such as Nitro blue tetrazolium, Zinc sulphate, Congo red, RPMI medium, HEPES buffer, Hanks blank salt solution (HBSS), etc. as well as drugs were procured from Subra Scientifics, Puducherry.

Instruments and Apparatus:

Spectrophotometry, oil immersion microscope, Incubator, Magnetic stirrer.

Preliminary photochemical screening (9):

The preliminary phytochemicals screening was performed for identification of various phytoconstituents like carbohydrates, proteins, steroids, starch, cellulose, cholesterol, saponins, alkaloids, flavonoids, terpenoids, volatile oils, tannins, glycosides in hexane extracts of Salvia hispanica. L seeds and followed by identification of functional groups present in the extract analysed using Attenuated total reflectance (ATR) (Thermo Scientific). Immunomodulatory Activity

Lymphocyte isolation from the human blood (10):

Lymphocytes were isolated from blood of health human volunteer by ficoll method. Platelet cells were obtained from density gradient centrifugation (380 × g at 4 °C for 10 min), washed with PBS three time and adjusted to desired concentration in complete medium [RPMI 1640 supplemented with12 mM HEPES (pH 7.1)for further use. The cell number was counted with a haemocytometer by the trypan blue dye exclusion technique. Cell viability exceeded 95%.

Bactericidal activity (Phagocytosis) (11):

The lymphocyte suspension was incubated with bacterial suspension (Escherichia coli) at 37°C for 60 min for. The lymphocytes were lysed with sterile distilled water spread on agar plate and incubated at 37°C for 24 h. Bacterial suspension was spread in the control plate. Number of colony forming units (CFU) developed in control and test plates were counted.

Nitro-Blue-Tetrazolium (NBT) Test (13,15):

A suspension of leucocytes (5 x 10⁶/ml) was prepared in PBS solution and taken 500μl into 7 tubes represented as control, standard and different concentrations of test. Added a volume of 0.1 ml PBS in 1^st tube as control and 5μl ml of standard drug in 2^nd as standard and different concentrations (5,10,25,50 and 100 μl) of test samples in remaining test tubes. All the test tubes added with 5μl of bacterial suspension and 0.2 ml of freshly-prepared 0.15% NBT solution, incubated at 37°C for 20 min and centrifuged at 400g for 3-4 min to discard the supernatant. The cells were re-suspended in the small volume of PBS solution. A thin film was made with the drop on a slide, dried, fixed by heating, counterstained with dilute Carbol-fuchsin for 15 sec. The slide was washed under tap water, dried and focused under 100X oil immersion objective. 200 neutrophils were counted for the percentage of NBT-positive cells containing blue granules/lumps.

Phagocytosis of killed Candida albica (13):

a) Preparation of Candida albicans suspension:

The Candida albicans culture was incubated in Sabouraud broth overnight and then centrifuged to form a cell button at the bottom; repeated washing was made with sterile Hank’s Balanced Salt Solution (HBSS) by discarding the supernatant and centrifuged for the analysis of phagocytosis activity, the cell suspension of concentration 1 × 10⁸ was prepared from the final cell button was mixed with sterile HBSS and human serum in proportion of 4:1.

b) Slide preparation:

Human blood (0.2 ml) was obtained by finger prick method on a sterile glass slide, incubated at 37°C for 25 min to allow clotting and drained the slide slowly with sterile normal saline after removing the blood clot very gently, taking care not to wash the adhered neutrophils (invisible). The slide consisting of polymorphonuclear neutrophils (PMNs) was flooded with predetermined concentration of test sample and incubated at 37°C for 15 min. The PMNs were covered with Candida albicans suspension and incubated at 37°C for 1 hour. The slide was drained, fixed with methanol and stained with Giemsa stain.

c) Phagocytosis evaluation:

The mean number of Candida cells phagocytosed by PMNs on the slide was determined microscopically for 100 granulocytes using morphological criteria. This number was taken as phagocytic index (PI) and was compared with basal PI of control. This procedure was repeated for different concentrations (10, 20 and 50μl) of test samples. The Percentage stimulation of the Neutrophils as immunostimulation was calculated by using the equation, stimulation (%) = (PI of test – PI of control) / PI of control) ×100.

Phagocytosis of Congo red stained yeast cells (16):

Congo red stained yeast cells, which phagocytosed by the cells, used in this assay. First, yeast cells (Candida albicans) were stained with Congo red. 3 ml of 0.87% Congo red PBS was added to yeast cell suspension (1.5 g), allowed staining for 15 min at room temperature. Afterwards, thoroughly mixed with 7ml of distilled water, resulting solution was autoclaved for 15 min to kill and fix the yeast and stored at 4⁰C after several times washed with HBBSS. Prior to use, cells were resuspended at 4×10⁷ cells/ml in HBSS. Two hundred and fifty microliters of the leukocyte solutions were mixed with 500g/l of the Congo red stained and autoclaved yeast cell suspension (yeast cell/leukocyte, 40/1). The mixtures were incubated at room temperature for 60 min. Following incubation, 1ml ice-cold HBSS was added and 1ml of histopaque (1.077) was layered to the bottom of each sample tube by a syringe. The samples were centrifuged at 850 × g for 5 min to separate macrophages from free yeast cells. Macrophages were harvested and washed twice in HBSS. The cells were re-suspended in 1ml trypsin–EDTA solution (5 g/l trypsin and 2 g/l EDTA, Sigma) and incubated at 37⁰C overnight. The absorbance values were measured at 510 nm using trypsin–EDTA as blank.

RESULT AND DISCUSSION:

Preliminary Phytochemical Screening of HESSH:

From the preliminary phytochemical screening of the hexane extract of the seeds of Salvia hispanica revealed the presence of phytoconstituents such as Saponins, Terpenes, Sterols and Steroids, Proteins and amino acids, Fatty acids and fixed oils (Table-1). Several studies reveal the presence of Fixed oils and Fatty acids may exhibits the immunomodulatory activity, solubility test of the HESSH shows the presence of oils and fats.

Table-1: Identification of Phytoconstituents in Hexane Extract of Salvia hispanica L Seeds

Sl. No	Experiment	Inference
1.	Carbohydrates	-
2	Proteins and Amino acids	+
3	Glycosides	-
4.	Saponins	+
5.	Alkaloids	-
6.	Flavonoids	-
7.	Proteins	-
8	Terpenes	+
9	Phenolic compounds	-
10.	Tannins	-
11.	Sterols and Steroids	+
12.	Fats and Fixed oils	+

+ Positive - Negative

Identification of Functional Group Present in the HESSH:

Infrared spectroscopy of the HESSH revealed the presence of COOH, C=O,C=C,C-OH,CH3,CH2,CH (Phenyl),CH=CH functional groups in the range of 3010.95-7180.50 (Fig. 1, Tab-2), which were present in the essential fatty acids structures. These functional groups support the presence of fatty acids in HESSH.

Figure-1: IR Spectra of HESSH Oil

Table-2: Identification of functional group in the Hexane Extract of Seeds of Salvia hispanica.L (HESSH)

Range of peaks	Functional group	Obtained Range
3110-3000	C=C-H, Ph-H, CH3X, CH2X	3010.95
3300-2800	COOH	2923.43
1200-1000	C-OH	1021.25
2000-1650	CH(Phenyl),	1742.49
1950-1600	C=O	1742.49
1475-1450	CH2, CH3	1453.68
730-675	CH=CH	718.50

In-vitro Immunomodulatory activity:

Figure -2: Lymphocytes isolation from the Human blood

Phagocytic activity (Bactericidal activity) (11):

Phagocytosis of Hexane extract of seeds of Salvia hispanica.L showed that the colony count was more than 300 in control group whereas 40 in standard, 50-80 in test-1, 10-30 in test-2 and there was no more colony in test-3 (Tab-3). In vitro-data consistently report that alterations in macrophage membrane fatty acid composition are linked to an altered Phagocytic capacity, i.e. an increase in membrane unsaturated fatty acid content is associated with an increase in engulfment and killing rate. The mode of action of fatty acids seems to be the modulation of the physical nature of the macrophage plasma membrane (12).

Table-3: Phagocytosis activity of HESSH Oil by using Escherichia coli

Group	Treatment	No. of Colony Counts
Control	2µl of E.coli +PBS	>300
Standard	100µl of human lymphocytes+ 2 µl of E.coli + 2 µl of Standard drug (Ampicillin) +96 µl of PBS	40
Test 1	100µl of human lymphocytes + 2 µl of E.coli + 2 µl of HESSH+96 µl of PBS	50-80
Test 2	100µl of human lymphocytes + 2 µl of E.coli + 5µl of HESSH +93 µl of PBS	10-30
Test 3	100µl of human lymphocytes + 2 µl of E.coli + 10 µl of HESSH +96 µl of PBS	No colony

Lymphocytes are the one of the component of WBC cell its main role is to kill are engulfing foreign particles such as bacteria, pathogens, viruses, fungal by phagocytosis. From this mechanism, these results will have been concluded the colony count of bacterial suspension decreased in the 10 µl of test extract. The results showed the immunomodulatory activity of the extract by enhances the lymphocytes to engulf the bacterial cell by phagocytosis process may be presence of phytoconstituent like essential fatty acids.

Quantified the phagocytosis of Candida albicans by Hexane extract of seeds of Salvia hispanica. L as describe above, we incubated the Candida albicans suspension with different concentration of the test sample as 2µl, 5µl and 10µl and observed maximum decreased colonies at 10µl equalised with standard group when compared with 2µl, 5µl of test extracts as well as control groups (Tab-4).

Table-4: Phagocytosis activity of HESSH Oil by using Candida albicans

Group	Treatment	No. of Colony Counts
Control	2 µl of Candida albicans +PBS	250
Standard	100µl of human lymphocytes+ 2 µl of Candida albicans +2 µl of standard drug +96 µl of PBS	10
Test 1	100µl of human lymphocytes + 2 µl of Candida albicans+2 µl of oil+96 µl of PBS	150
Test 2	100µl of human lymphocytes + 2 µl of Candida albicans +5µl of oil+93 µl of PBS	19
Test 3	100µl of human lymphocytes + 2 µl of Candida albicans +10 µl of oil+96 µl of PBS	10

Nitro-Blue-Tetrazolium (NBT) Test:

NBT reduction assay is a qualitative test useful for monitoring the NBT activity for the identification of activators as well as inhibitors of the phagocyte system (13). Phagocytic activity of the test drug represents by WBCs turns into blue when NBT is added. In the present study, the intracellular killing of microorganisms by the WBCs revealed that incubation of different concentrations (5, 10, 25, 50 and 100µl) of test extract of Salvia hispanica. L and standard drug with leukocytes resulted in significant increase in percentage of NBT positive granules as compared to control (Tab-5). Increase in NBT positive granules represents the decreased in microorganism by phagocytic activity, the maximum bioactivity was observed at 100µl in dose dependent. Present results suggested that the capable of stimulating the immune function of macrophages as evidenced by an increase in NBT positive granules through decrease in number of microorganism at high concentration of test sample, however, the bactericidal activity of the phagocytes and metabolic activity of granulocytes or monocytes depends on oxygen it was indirectly represented by the NBT reduction test (13,10).

Table-5: NitroblueTetrazolium (NBT) Test of Hexane Extract of Seeds of Salvia hispanica. L

Samples	Concentration	No of NBT Stained Cells	% NBT Positive Granules
Control	-	7	7.50%
Standard	5 µl	37	18.50%
Test 1	5 µl	25	12.50%
Test 2	10 µl	38	14%
Test 3	25 µl	36	18%
Test 4	50 µl	38	19%
Test 5	100µl	44	22%

Phagocytosis of killed Candida albicans:

Phagocytosis of killed C. albicans assay is one of the methods for measuring phagocytosis which relies on the uptake of particles by phagocytes over a brief period (14). In this the number of particles phagocytosed by neutrophill cells was counted under the microscope. (20,21) Immunostimulation of the test drugs was increased with concentration in dose dependent manner; the maximum percentage of stimulation was 70 % found at the concentration of 50µl when compared to positive control (50 %), however, at the low concentration of 10µl and 20µl, the stimulation of phagocytic activity was 46 % and 56 %. The results were shown in Fig-3.

Figure-3: Phagocytosis Activity of Hexane Extract of Seeds of Salvia hispanica. L by killed Candida albicans

In this study, the preliminary phytochemical screening of hexane extract of seeds of Salvia hispanica. L revealed the presence of Proteins and Amino acids, Saponins, Terpenes, Sterols and Steroids, Fats and Fixed oils (20). Different studies suggested that the saponins are possess the different pharmacological activities such as anti-allergic, cytotoxic, antitumor, antiviral, immunomodulating, anti-hepatotoxic, antifungal activities and recently immunostimulating activity of saponins have been reported from Paris polyphyllaas well as Triphala which contains Tannins from Terminalia chebula, Terminalia belerica, and Emblica officialis (21). Steroids also involved in several pharmacological activities, recently c21 steroids from Cynanchum plants were focused on antitumor, antidepressant, antifungal, anti-tagging, Na(+)/K(+)-ATPase inhibitory, appetite suppressing and antiviral activities (17). Hence, the immunomodulatory action of the extracts could be attributed to the collective presence of steroids, saponins, and tannins.

Phagocytosis of Congo Red Stained Yeast Cells (15)

Phagocytosis the main function of WBC cells and the phagocytic activity (18,19) of blood leucocytes was determined spectrophotometrically involving the measurement of stained yeast cells with congo red that had been phagocytosed (internalization of yeast) by blood leukocyte (1 × 10⁶ cells ml⁻¹) (14).

Figure-4: Phagocytosis Activity of Hexane Extract of Seeds of Salvia hispanica. LCongo Red Stained Yeast Cells

In the present study, congo red stained yeast cells were decreased and the absorbance of the test samples was increased as 0.15, 0.26 and 0.31 with respectively from test-1 to test-3. However, the high concentration of test sample significantly resemble with the standard absorbance was 0.32. Fig-4. Hence, the presence of fatty acids in the test extract May supports the immunomodulatory activity of test sample through decreased Congo red stained yeast cell by the leukocytes.

CONCLUSION:

Salvia hispanica. L (Chia) is one of the traditional foods in central and southern America, due to the presence of PUFA fatty acids derived from ALA and antioxidants, these seeds possess significant anti-inflammatory and antioxidant properties and literature provides an array of pharmacological properties. In the present study, results from bactericidal activity, NBT test and phagocytosis activity strongly supports the immunomodulatory activity with the presence of steroids, fatty acids, Saponins, tannins etc. furthermore in-vivo studies should be conducted to supports its immunomodulatory activity.

Table-6: List of the Abbreviations

Short form	Abbreviation
WBC	White blood cell
NK	Natural killer cell
PMNs	Polymorphonuclearneutrophills
CFU	Colony forming unit
E.Coli	Escherichia coli
PBS	Phosphate buffer solution
HBSS	Hanks' Balanced Salt solution
IR	Infra-red
NBT	Nitro blue terazolium
HESSH	Hexane extract of seeds of Salvia hispanica
RPMI medium	Roswell Park Memorial Institute medium
HEPES buffer	(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) buffer
EDTA	Ethylenediaminetetraacetic acid
PI	Phagocytic index
Mm	Millimole
g/L	Gram per liter
µl	Microliter
%	Percentage
°c	Degree celusis
H	Hour
Min	Minutes
ATR	Attenuated total reflectance

ACKNOWLEDGEMENT:

I would like to extend my heartfelt thanks to my guide, Mr. J. Gopi Sudheer Kumar for providing constant encouragement and timely help in completion of this project. I would also like to thank my HOD, DR. S. Kavimani for his support, and valuable guidance with endless encouragement. I am highly indebted to my institution, Mother Theresa post graduate and research institute of health sciences, Puducherry. I am also grateful to Miss. S. Divya, Research scholar, Department of microbiology, Pondicherry University for providing timely help and for valuable guidance for the successful completion of my project without any hindrance. I would like to express my sincere thanks to DR. R. Vimalavathini for her valuable suggestions, and encouragement. Lastly I would like to thank my parents, friends and other staff in my department as well as other department for supporting me.

REFERENCES:

1. Venugopal Jayapal. Fundamentals of Medical Immunology.2007; 1stEd:4:18.

2. Sharma P. Charaka Samhita, Chikitsasthana. Chaukhamba Orientalia, Varanasi,1983;2.

3. Nagarathna PKM, Reena K, Sriram Reddy, Johnson Wesley. Review on Immunomodulation and Immunomodulatory Activity of Some Herbal Plants. Int. J. Pharm. Sci. Rev. Res., 22(1), Sep – Oct 2013; nᵒ 41:223-230.

4. Shivaprasad HN; Kharya MD; Rana; Mohan S. Pharmaceutical Biology, 2006;44:32–34.

5. British Pharmacopoeia, Department of Health, British Pharmacopoeia Commission, London. The Stationary Office: 1999.

6. Mohd Ali N, et al The promising future of chia, Salvia hispanica L. J Biomed Biotechnol; 2012.

7. Ayerza R and Coates W, “Omega-3 enriched eggs: the influence of dietary 𝛼-linolenic fatty acid source on egg production and composition,” Canadian Journal of Animal Science.2000; 81(3): 355–362.

8. Ofori-Boateng C., Teong L.K., Jitkang L. Comparative energy analyses of Jatropha curcas oil extraction methods: Solvent and mechanical extraction processes. Energy Conversion and Management, 2012;55: 164–171.

9. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. Nirali Prakashan, Pune.2006; 166, 181, 182, 186, 222, 347, 348,516, 590-592.

10. Sheikh Abid Alia, Aruna Bhatiab, Qazi Parvaiza, H. M. Bhatc, Sheikh Fayaz Ahmadd, Nishu Kherab and Refaz Ahmada “In vitro immunomodulatory study of different parts of Prunuscerasus L. (sour cherry) Plant” Asian Journal of Plant Science and Research, 2013, 3(3):35-43.

11. Mishra.T and Bhatia A J. ethanopharmacol.2010; 127,341-345.

12. Schumann J, Leichtle A, T hiery J, Fuhrmann H. Fatty acid and Peptide profiles in plasma membrane and membrane Rafts of PUFA supplemented RAW264.7 Macrophages. PLos ONE 2011: 6(8).

13. Wilkinson PC. Neutrophil leucocyte function test, Tech in Clin Immunol, Edited by Thomson RA, Blackwell Scientific Publication, Oxford, London, IInd ed.1981;273-93.

14. Daniel P.S, Basic and Clin. Immuno, 1987, 64-68.

15. Gooi C. A practical approach. ClinImmunol 1990:51-78.

16. Seeley PD, Gillespie D, and Weeks BA. A Simple technique for the rapid spectrophotometric determination of phagocytosis by fish macrophages.Mar.Environ.Res.1990;30.

17. Sanjeev Heroor, Arun Kumar Beknal, and Nitin Mahurkar “Immunomodulatory activity of methanolic extracts of fruits and bark of Ficus glomerata Roxb.in mice and on human neutrophils” Indian J Pharmacol. 2013 Mar-Apr; 45(2): 130–135.

18. Gu XJ, Hao DC “Recent advances in phytochemistry and pharmacology of C21 steroid constituents from Cynanchum plants.” Chin J Nat Med. 2016 May;14(5):321-34.

19. Walsh CJ, Leur CA. Comparative phagocytic and pinocytic activities of leukocytes from peripheral blood and lymphomyloid tissues of the nurse shark (Ginglymostomacirratum Bonaterre) and clear nose shate (Raja eglanteria Bosc).Fish andshellfish immunology;1998:(3);197-215.

20. Clamp AG, Ladha S, Clark DC, Grimble RF, Lund EK. The influence of dietary lipids on the composition and membrane fluidity of rat hepatocyteplasma membrane. Lipids 1997; 32:179–84.

21. Tiwary RK, Clandinin MT, Cinader B et al. Effect of high polyunsaturated fat diets on the composition of B cell and T cellmembranelipids.Nutr.Res.1987; 7: 489–98.

Received on 11.06.2017 Modified on 19.07.2017

Research J. Pharm. and Tech 2017; 10(12): 4255-4260.

DOI: 10.5958/0974-360X.2017.00779.X

c) Phagocytosis evaluation:

Hanks' Balanced Salt solution

Attenuated total reflectance