Immunopharmacological screening of aqueous root extract of Santalum album

Gupta A and Chaphalkar SR Journal of HerbMed Pharmacology, Volume 5, Number 1, January 2016 http://www.herbmedpharmacol.com 8 Actually, this plant is being exploited for various purposes especially sandalwood oil is obtained mostly by steam distillation of its root and heartwood (8,9) with a number of medicinal uses e.g. treatment of many other ailments like diarrhea along with bleeding, intrinsic hemorrhage, inflamed hemorrhoids (piles), eye infections, inflammation of the umbilicus, hiccoughs initial phase of pox, and urticaria (10,11). The current study was focused on the immunopharmacological activity of Santalum album on human PBMCs against Hepatitis B surface antigen (HBsAg) and NDV. Materials and methods Assemblage and composition of plant material The fresh roots of Santalum albumin were collected from the garden of Vidya Pratishthan’s in the morning, between January and February 2015 in Baramati region, District Pune, Maharashtra, India. Afterwards, the fresh roots were washed in tap water and then with distilled water to remove dust and finally dried in a shady area. The plant roots were macerated with liquid nitrogen to prepare a fine powder. This powder was used for subsequent immunopharmacological assays. Phytochemical screening and extraction Different qualitative and quantitative based assays were carried out in order to determine the presence of secondary metabolites. Qualitative based assays revealed aqueous root extracts of Santalum album and the presence of Saponin (foam test); terpenoids (acetic anhydride test) and flavonoids (alkaline reagent test). Collection of NDV samples NDV Samples of suspected birds were collected under the programme of Biovillage scheme, Vidya Pratishthan’s School of Biotechnology. Specific pathogen free chicken eggs were purchased from Venkys India Ltd. The allantoic cavity route of embryonated (9-11 day old) chicken eggs was used for isolation and propagation of NDV from field samples (5). These eggs were observed in dark (using candle) at regular intervals and bigger sized embryos (air cell and area without blood vessels) selected for inoculation. After disinfection of egg shells with spirit, 0.2 ml of supernatant was inoculated at 45° angle into embryonated chicken eggs. Embryo motility was observed every 10 hours by candling. After the death of embryos, amnio-allantoic fluid was harvested and checked for presence of virus. This was determined through haemagglutination test (128 HA titre value). PBMC proliferation assay and nitric oxide production using HBsAg and NDV EDTA anti-coagulant human blood samples were collected from Mangal Pathology Laboratory, Maharashtra, India and scrutinized at the VSBT, Baramati, Maharashtra, India, between January to February 2015. PBMCs were separated by means of density gradient centrifugation and cultured with variable doses of aqueous root extracts (0.5-30 mg/ml, 50 μl) of Santalum album along with or without HBsAg (20 μg/ml, 10 μl) and NDV (1:80 dilution, 10 μl). Incubate 96-well plates for 48 hours at 37°C. HBsAg and NDV used as standard for these studies. Centrifuging the plates was done at 2500 rpm for 10 minutes at 4°C with collecting the supernatant for the estimation of nitric oxide production through Griess reagent. Afterwards, fresh complete medium was added into the 96-well plates. Again, incubating the plates for another 4 hours along with (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium-bromide; 5 mg/ml, 10 μl) continued. After incubation, the plates were suddenly centrifuged with discarding the supernatant, collecting the pellet and finally dispersing in dimethyl sulphoxide (DMSO) solution. The optical density was measured at 570 nm (12,13). After preincubation of PBMC (105 cells/ml) with or without HBsAg and NDV for 48 hours the supernatant (as mentioned above) was collected for estimation of nitric oxide production. Briefly, 50 μl of untreated and treated PBMC cell culture medium along with HBsAg/NDV was assorted with 50 μl of Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride [NED] in 2.5% phosphoric acid). For this experiment, Roswell Park Memorial Institute medium (RPMI) containing 10% fetal bovine serum was used as a blank followed by incubation of 96-well plates at room temperature for 10-15 minutes. After incubation, absorbance at 540 nm was measured in a microplate reader (14,15). Estimation of CD14 monocyte surface marker by flow cytometry In another set of experiments, human PBMCs were cultured with serial dilutions of aqueous root extracts of Santalum album along with or without HBsAg (20 μg/ml, 10 μl)/NDV (1:80 dilution, 10 μl) for 48 hours in 96-well plates. After incubation, samples of the PBMCs treated with or without HBsAg/NDV were collected and stained with CD14 FITC (3 μl) monoclonal antibody. The samples were then incubated, lysed and washed with phosphate buffered saline (PBS). Finally, the samples were prepared for flow cytometric analysis. The resulting stained cell pellet was resuspended in 2000 μl of PBS and run on a FACS Calibur flow cytometer (16,17). Statistical analysis All values were mentioned as mean ± SE. Data was represented by one-way analysis of variance (ANOVA) test (Bonferroni multiple comparison test). Results PBMC proliferation assay The effect of variable doses of aqueous root extract of Santalum album on PBMC proliferation assay with or without HBsAg and NDV using MTT is shown in Figure 1. The Immunopharmacological screening of Santalum album Journal of HerbMed Pharmacology, Volume 5, Number 1, January 2016 http://www.herbmedpharmacol.com 9 aqueous extract showed a dose-dependent decrease in the proliferation assay with or without HBsAg/NDV as compared to control. Also, there was a significant enhancement of PBMC proliferation as compared to control. Nitric oxide estimation The observed effect of variable doses of aqueous root extract in the cell culture medium of PBMC along with or without HBsAg/NDV is shown in Figure 2. The aqueous root extract showed a significant decrease in nitric oxide production at higher doses with or without HBsAg/NDV as compared to control. There was a significant enhancement of nitric oxide production as compared to control. CD14 monocyte surface marker The effect of aqueous root extract of CD14 monocyte surface marker on PBMC with or without HBsAg/NDV is shown in Figure 3. There was a significant decrease in CD14 monocyte surface marker at higher doses as compared to control. Discussion The prime objective of our study was to investigate the aqueous root extract of Santalum album for in vitro proliferation, nitric oxide production and CD14 monocyte surface marker on human PBMC using HBsAg and NDV, in order to validate the potential use of this aqueous root extract as a tool in screening for anti-inflammatory and anti-microbial effects on the immune system. As per the literature survey, there are a number of immunopharmacological studies related to medicinal plants which have already been done using non specific antigens on humoral and cell mediated immune response (18). These studies are a matter of great interest for many immunopharmacologists. For instance, one of the medicinal plant namely, Santalum album shows anti-inflammatory (reduction in HBsAg) as well as anti-microbial (inhibitory effect of NDV) activities at higher doses as compared to 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Control HBsAg O D at 5 70 n m Control 0.5 1 10 30