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Amit Gupta et al, AJMPS, 2015, 3(1): 219–223ISSN: 2348-0165Asian Journal of Medical andPharmaceutical SciencesJournal Home Page: www.pharmaresearchlibrary.com/ajmpsResearch ArticleOpen AccessAnti-inflammatory activity of flavonoids from medicinal plants against hepatitis Bvaccine antigen on human peripheral blood mononuclear cellsAmit Gupta* and Sushama R ChaphalkarDepartment of Immunology, Vidya Pratishthan’s School of Biotechnology, Baramati, District Pune, Maharashtra IndiaABSTRACTThe objective of our study is to investigate the anti-inflammatory activity of the variable doses of flavonoids (6.25 – 25 mg)extracted from the leaves of Syzygium cumini, Acacia catechu and Azadirachta indica on human peripheral bloodmononuclear cells using specific antigen (i.e. hepatitis vaccine) is to estimate the nitric oxide production from cell culturesupernatant and also observed its cytotoxicity. In contrast, these flavonoids also measured its effect on lymphocytes,monocytes and granulocytes count on human whole blood using hepatitis vaccine as antigen which is determined throughflow cytometry. The results showed that these flavonoids extracted from these medicinal plants showed maximum inhibitionof nitric oxide and cytotoxicity at higher doses. Further, these flavonoids also showed its decline in the level of monocytescount at higher doses as compared to control. The data suggests that flavonoids extracted from these medicinal plants i.e.Syzygium cumini, Acacia catechu and Azadirachta indica showed anti-inflammatory activity.Keywords: Flavonoids, Syzygium cumini, Acacia catechu, Azadirachta indicaARTICLE INFOCONTENTS1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2192. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2203. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2214. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2225. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222Article History: Received 21 March 2015, Accepted 24 May 2015, Available Online 19 June 2015*Corresponding AuthorAmit GuptaDepartment of Immunology,Vidya Pratishthan’s School of BiotechnologyBaramati, Pune, Maharashtra IndiaManuscript ID: AJMPS2574PAPER-QR CODECitation: Amit Gupta, et al. Anti-inflammatory activity of flavonoids from medicinal plants against hepatitis B vaccine antigen on humanperipheral blood mononuclear cells. A. J. Med. Pharm, Sci., 2015, 3(1): 219-223.Copyright 2015 Amit Gupta, et al. This is an open-access article distributed under the terms of the Creative Commons AttributionLicense, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.Asian Journal of Medical and Pharmaceutical Sciences219
Amit Gupta et al, AJMPS, 2015, 3(1): 219–223ISSN: 2348-01651. IntroductionFlavonoids, one of the members or class of secondarymetabolites and it belongs to a group of polyphenoliccompounds and generally classified as flavones, flavanones,catechins and anthocyanins [1, 2, 3]. The number offlavonoids of different classes extracted from variousmedicinal plants has several immunopharmacologicalactivities [2] and possessed biochemical effects [3, 4].These flavonoids are normally present in fruits especiallycitrus (flavanones) and also found in onions, parsley,legumes, green tea etc. In addition, number of flavonoidsare also present in miscellaneous products i.e. anthocyanins(e.g. wine); flavans (e.g. apples and tea) and isoflavones(e.g. soya products). In contrast, these flavonoids can beeasily degraded or break down through enzyme action onlywhen the collected plant material is fresh or non-dried [ 3,4]. For flavonoid extraction, used the dry plant material andgenerally ground into a fine powder. For flavonoidextraction, polarity is an important criterion for itscharacterization i.e. less polar flavonoids in the form ofisoflavones/flavanones/methylated flavones/flavonols areextracted with chloroform/diethyl ether/ethyl acetate whileflavonoid glycosides and more polar aglycones areextracted with alcohols/ alcohol–water mixtures [4, 5]. Inthe view of their wide immunopharmacological and severalbiological actions, they seem to have great therapeuticpotential. Syzygium cumini (commonly known as Jamun orblack plum; family Myrtaceae) important medicinal plant invarious traditional systems of medicine. This medicinalplant is generally more effective and already reported forthe treatment of diabetes mellitus, inflammation, ulcers anddiarrhea and number of preclinical studies which is alreadydone and reported as chemopreventive; radioprotective andantineoplastic properties [6, 7, 8, 9]. Acacia catechu(commonly known as khair; family Mimosaceae),medicinal plant and is generally used for the treatment ofseveral diseases including fever, diarrhoea etc. Previously,Acacia catechu already reported as hypoglycaemic activityand also has hepatoprotective; antipyretic and digestiveproperties [10, 11, 12].Azadirachta indica (commonly known as neem; familyMeliaceae) is one of the important medicinal plant and iswidely in the Indian traditional system of medicine for itsdiverse medicinal properties. Its extracts/fraction(methanolic/ethanolic/chloroform) extracted from plants i.e.roots, stem and leaves have vast or numerousimmunopharmacological activities e.g. anti-viral [13];immunostimulatory [14, 15]; anti-malaria [16, 17] andcontrolling gastric hyperacidity and ulcer [18, 19]. Thepresent study compared the anti-inflammatory effect ofthese flavonoids of three most commonly used medicinalplants i.e. Syzygium cumini, Acacia catechu andAzadirachta indica against hepatitis B vaccine antigen onhuman peripheral blood mononuclear cells.2. Materials and Methods2.1. Collection of plant materialFresh leaves of these medicinal plants i.e. Syzygium cumini,Acacia catechu and Azadirachta indica were collected fromAsian Journal of Medical and Pharmaceutical Sciencesthe garden of Vidya Pratishthan’s School of Biotechnology(VSBT), Baramati (Pune), Maharashtra. Fresh harvestedplant leaves were washed with tap or distilled water andthen dried in a shady area.2.2. Extraction and qualitative analysis of flavonoidTo proceed the flavonoid extraction [20] from threemedicinal plants i.e. Syzygium cumini, Acacia catechu andAzadirachta indica. Weigh 5 g of fresh plant leavesmacerated with liquid nitrogen to prepare the finelygrounded plant powder and reflux with 50 ml of 80 %methanol for 2 h at 100 ºC. Afterwards, cool down thesolution containing plant material and filtered throughwhatman filter paper and collect the filtrate in separatingfunnel and then add ethyl acetate (10 ml) with distilledwater (20 ml), mix it properly. After 6 -8 h incubation,separating layers were observed i.e. collect only upper layeri.e. ethyl acetate and evaporate it and then dried the plantextracts. The dried extracts i.e. flavonoids settled at thebottom. For the estimation of flavonoid in medicinal plantsusing lead acetate test, small quantity of lead acetatesolution is added and yellow precipitation appears. Thisyellow colour precipitation showed the presence offlavonoid.2.3 Cytotoxicity assay of human peripheral bloodmononuclear cellsNon-infected EDTA blood samples (2 – 3 ml) of humanwere collected from Mangal Pathology laboratory,Baramati for immunological set of experiments. For thepreparation of human peripheral blood mononuclear cells(PBMC, 105 cells/ml) using Ficoll–Hypaque gradientcentrifugation and plated in 96 well flat bottom tissueculture plates and incubated in the presence of hepatitisvaccine (1 µg) along with serial dilutions of flavonoidsextracted from three medicinal plants i.e. Syzygium cumini,Acacia catechu and Azadirachta indica at 37 C for 24 h.After incubation, 96 well flat bottom plates werecentrifuged at 1500 rpm for 4 minutes and then thesupernatant (50 µl) was collected for the estimation of nitricoxide and then add equal volume of fresh medium. AddMTT solution (5 mg/ml, 10 µl) were added to each well andthen incubated for 4 h. Again, the 96 well flat bottom plateswere spinned or centrifuged at 1500 rpm for 4 minutes andthe supernatant was discarded. Add 100 μl of DMSOsolution to the formazon crystals and the absorbance wasevaluated in an ELISA reader at 570 nm [25]. Allexperiments were performed in triplicate.2.4. Determination of nitric oxide (NO) productionHuman peripheral blood mononuclear cells were incubatedfor 24 h with variable doses of flavonoids in the presence ofhepatitis vaccine antigen as mentioned above. After 24 hincubation using 50 μl of PBMC cell culture medium wasmixed with 50 μl of Griess reagent (1% sulfanilamide and0.1% naphthylethylenediamine dihydrochloride in presenceof 2.5% phosphoric acid) and incubated in 96 well flatbottom plates at room temperature for 10 minutes, and theabsorbance at 540 nm was measured in a microplate reader.The fresh culture medium (RPMI containing 10 % fetalbovine serum) was used as a blank. The quantity of nitrite220
Amit Gupta et al, AJMPS, 2015, 3(1): 219–223was determined from a sodium nitrite standard curve [21,22].2.5 Estimation of lymphocytes, monocytes andgranulocytes count using flow cytometryIn this experiment, 100 μl of human whole blood was takenin each flacon tube including hepatitis vaccine (1 µl). Addserial dilutions of flavonoids of various medicinal plants i.e.Syzygium cumini, Acacia catechu and Azadirachta indicaon human whole blood and then incubated the samples indark for 2 -3 h at 37 C carbon dioxide incubator.Subsequently, 2 ml of red cell lysis buffer/FACS lysingsolution were added to each falcon tube containing wholeblood at room temperature with gentle mixing followed byincubation for 10 min. The samples were spinned at 4000rpm at 4 C and the supernatant was aspirated and washedtwo times with phosphate buffered saline (PBS). After tenminutes centrifugation, pellet settled at the bottomdissolved in PBS and observed the cells through flowcytometer [23, 24].2.6. Statistical analysisAll values are mentioned as Mean S.E. Data isrepresented by One way ANOVA test (Boniferroni multiplecomparison test).3. Results and Discussion3.1 Cytotoxicity assayThe effect of variable doses of flavonoid on cytotoxicityassay as shown in Fig. 1. The results showed thatflavonoids showed cytotoxicity at higher doses (25 mg) ascompared to control.3.2 Estimation of nitric oxide (NO) productionThe effect of variable doses of flavonoid extracted from theleaves of Syzygium cumini, Acacia catechu and Azadirachtaindica on nitric oxide production were observed in the cellculture supernatant of human peripheral blood mononuclearcells as shown in Fig .2. The results showed that there wasa significantly dose related decreased in NO production ascompared to control.3.3. Estimation of blood counts using flow cytometryThe effect of variable doses of flavonoid extracted from theleaves of Syzygium cumini, Acacia catechu and Azadirachtaindica on human EDTA whole blood lymphocytes,monocytes and granulocytes count using hepatitis vaccineas antigen which is determined through flow cytometry asshown in Fig 3. At higher doses, there is dose dependentdecrease in monocytes count at higher doses as compared tocontrol.ISSN: 2348-0165as antigen is a matter of research interest for many studentsas well as scholars. Several studies related to thesemedicinal plants with respect to plant physiology,pharmacology, immunomodulatory activity (using specificas well as non-specific antigen on humoral and cellmediated immune response) etc have been done [26, 27].In the present study, we explored the effect of variabledoses of flavonoids (determined through qualitative basedassay using lead acetate test) on human peripheral bloodsamples using hepatitis vaccine as antigen. The quality offlavonoids is determined through qualitative based assaywhich represents the immunopharmacological activecomponents of the medicinal plants. The active componenti.e. flavonoids isolated from various medicinal plants andobserved its immunopharmacological parameters. One ofthe mediators for inducing the inflammation is nitric oxideand played an important role in mediating many aspects ofinflammatory responses. It can modulate the release ofvarious inflammatory mediators from a wide range of cellsparticipating in inflammatory responses (e.g., leukocytes,macrophages etc). It can modulate blood flow, adhesion ofleukocytes (white blood cells) to the vascular endotheliumand the activity of numerous enzymes [28, 29, 30], all ofwhich can have an impact on inflammatory responses. Theresults showed that these medicinal plants showedinhibitory activity of nitric oxide at higher doses in case ofcell culture supernatant of human peripheral bloodmononuclear cells treated with hepatitis vaccine as antigen.In addition, these medicinal plants showed cytotoxicity athigher doses. Generally, flow cytometry is commonly usagein preclinical, clinical and veterinary laboratories for theassessment of the immune status of healthy/diseasedanimals and human [24].However, the main application of this flow cytometry isimportant in immunopharmacological studies such ashematology, toxicology etc. In this study, the resultsshowed that the flavonoids extracted from the leaves ofthese medicinal plants i.e. Syzygium cumini, Acacia catechuand Azadirachta indica showed anti-inflammatory effect athigher doses, this is due to sudden loss of monocytes count.The results obtained
Amit Gupta et al, AJMPS, 2015, 3(1): 219–223 ISSN: 2348-0165 Asian Journal of Medical and Pharmaceutical Sciences 220 1. Introduction Flavonoids, one of the members or class of secondary
