Study On Anti-allergic Compounds From Some Selective Brown Seaweed

Food allergy has becoming the serious threat in the world for which the search of aneffective and potent anti-allergic drug is the demand of time.Keeping in view of thepotentiality of seaweeds,the ethanol extracts of some selected sargassum species wereunder taken for the in vitro and in vivo study of antiallergenicity.In the interest ofidentification of most potent anti-allergic compound,a stepwise isolation process wasoptimized through macroporous resin in combination with sephadex LH-20 columnchromatography followed by High Performance Liquid Chromatography.Afterhaving isolation of active compounds,MS-TOF and NMR were used for theiridentification and characterization.Ethanol extracts of brown seaweeds from Pakistan and China were isolated andcompared for their antiallergenic activities.They included Sargassum tenerrimum (ST)and Sargassum cervicorne (SC) from Pakistan,and Sargassum graminifolium turn(SG),Sargassum thunbergii (STH),and Laminaria japonica (LJ) from China.Theethanol extracts of these brown seaweeds were optimized at 85% (v/v) ethanol for themaximum yield of phlorotannin,an inhibitor against hyaluronidase.Totalphlorotannins contained in the crude extracts were measured as 1.71% (SG),0.74%(STH),0.97% (LJ),3.30% (SC),and 5.06% (ST).The 50% inhibitory concentrations(IC₅₀) of Pakistani SC and ST were 109.5 and 21μg/ml,respectively,lower thanthose of Chinese SG,STH,and LJ (134,269,and 148μg/ml,respectively).Anantiallergic drug,disodiumcromoglycate (DSCG),had an IC₅₀=39μg/ml,and anatural inhibitor of hyaluronidase,catechin,had an IC₅₀=20μg/ml.The IC₅₀ of STextract was found similar to that of catechin (21 vs 20μg/ml) and lower than that ofDSCG (21 vs 39μg/ml).This suggests that ST is a potent inhibitor of hyaluronidase,indicating a promising future development of natural antiallergic medicines orfunctional foods.Since the crude algae extracts of ST,SC,and SG were found more potent ofanti-allergic activity in vitro in comparison to other brown seaweeds used in this study,these were undertaken for in vivo study of their antiallergenicity through passivecutanous anaphylaxis (PCA) and active cutanous anaphylaxis (ACA) in femaleBALB/c mice.Intraperitonial administration of these ethanol extracts inhibit mousePCA and ACA in a dose dependent manner using ovalbumin (OVA) and shrimpallergen as triggering agents to induce allergenicity over mice.The extract of STcontaining phlorotannin has been found most active over the suppression of PCAtriggered by OVA and shrimp with IC₅₀ values of 25.64 mg/kg and 40.98 mg/kgrespectively and an efficacy comparable to that of an antiallergic drugdisodiumchromoglycate (DSCG).Similarly,ST inhibits ACA triggered by ova andshrimp allergen in the mouse,with 50% suppression at 25.5 mg/kg and 43.53 mg/kgrespectively.The results presented here show that these extracts are activc on the studied models among which ethanol extract of ST was the most potent,leadingtowards the promising development of a new class of anti-allergic drugs.Crude Polyphenol from the ethanol extracts of sargassum tenerrimum (ST) withpotent antiallergic effects was then untaken to optimize separation process throughcolumn chromatography.In this section of study,the adsorption and desorptioncharacteristics of three widely used adsorbents macroporous resin,silica gel,andPVPP (polyvinylpolypyrrolidone) respectively,are critically evaluated and studied forthe optimization of preparative separation of polyphenols.Static adsorption anddesorption experiments on these adsorbents showed that macroporous resin has thebest adsorption and desorption capability among the all.Dynamic adsorption anddesorption experiments on macroporous resin packed column were conducted toestablish the optimum parameters as:concentration of extract solution (4 timesdiluted),pH value (6-7),adsorption speed (3 BV),concentration of ethanol (80 %),eluting speed (3 BV),eluting volume (7 BV).The chromatographic process sooptimized has given a purity of 62.43 % from the crude polyphenols and this work isa promising basis for the large scale preparation of bioactive polyphenols upon furtherscaling up tests.As the purity of polyphenol was not too high from the previous separation and alsoanti-allergic compounds were unknown,a method was established to isolate theanti-allergic compounds from the crude extract of Sargassum tenerrimum though stepwise separation of column chromatography of macrporous resin in combination withsephadex LH-20 followed by high performance liquid chromatography.During thisstudy,isocratic and gradient elutions were run in different conditions in order tooptimize the process for solvent system,flow rate and other effective parameters.Antihyaluronidase activity and total polyphenol were checked in each step ofseparation.The gradient elution of ethanol/water gave two distinct separate peakfractions of high antiallergic activities (IC₅₀ values of 4.23μg/mL and 8.28μg/mLrespectively) in two separate experiments using different fractions obtained fromsepahdex LH-20 chromatography.The fractions were subjected for identificationthrough MS-TOF and for characterization through NMR.Two compounds were isolated from the crude extract of sargassum tenerrimum.These are identified as phlorotannins probably eckol and bieckol possess theantiallergic activity of IC₅₀ values of 4.23μg/ml and 8.28μg/ml.