Study on Anti-allergic Compounds from some Selective Brown Seaweed

摘要

Food allergy has becoming the serious threat in the world for which the search of an effective and potent anti-allergic drug is the demand of time. Keeping in view of the potentiality of seaweeds, the ethanol extracts of some selected sargassum species were under taken for the in vitro and in vivo study of antiallergenicity. In the interest of identification of most potent anti-allergic compound, a stepwise isolation process was optimized through macroporous resin in combination with sephadex LH-20 column chromatography followed by High Performance Liquid Chromatography. After having isolation of active compounds, MS-TOF and NMR were used for their identification and characterization. Ethanol extracts of brown seaweeds from Pakistan and China were isolated and compared 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. The ethanol extracts of these brown seaweeds were optimized at 85％(v/v)ethanol for the maximum 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 (IC<,50>) of Pakistani SC and ST were 109.5 and 21μg/ml, respectively, lower thanthose of Chinese SG, STH, and LJ (134, 269, and 148 μg/ml, respectively). An antiallergic drug, disodiumcromoglycate (DSCG), had an IC<,50> =39 μg/ml, and a natural inhibitor of hyaluronidase, catechin, had an IC<,50> =20 μg/ml. The IC<,50> of STextract was found similar to that of catechin (21 vs 20 μg/ml) and lower than that of DSCG (21 vs 39 μg/ml). This suggests that ST is a potent inhibitor of hyaluronidase, indicating a promising future development of natural antiallergic medicines or functional foods. Since the crude algae extracts of ST, SC, and SG were found more potent of anti-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 passive cutanous anaphylaxis (PCA) and active cutanous anaphylaxis (ACA) in female BALB/c mice. Intraperitonial administration of these ethanol extracts inhibit mouse PCA and ACA in a dose dependent manner using ovalbumin (OVA) and shrimp allergen as triggering agents to induce allergenicity over mice. The extract of ST containing phlorotannin has been found most active over the suppression of PCA triggered by OVA and shrimp with IC<,50> values of 25.64 mg/kg and 40.98 mg/kgrespectively and an efficacy comparable to that of an antiallergic drug disodiumchromoglycate (DSCG). Similarly, ST inhibits ACA triggered by ova and shrimp 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 active on the studied models among which ethanol extract of ST was the most potent, leading towards the promising development of a new class of anti-allergic drugs. Crude Polyphenol from the ethanol extracts of sargassum tenerrimum (ST) with potent antiallergic effects was then untaken to optimize separation process through column chromatography. In this section of study, the adsorption and desorption characteristics of three widely used adsorbents macroporous resin, silica gel, and PVPP respectively, are critically evaluated and studied for the optimization of preparative separation of polyphenols. Static adsorption and desorption experiments on these adsorbents showed that macroporous resin has thebest adsorption and desorption capability among the all. Dynamic adsorption and desorption experiments on macroporous resin packed column were conducted to establish 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 so optimized 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 further scaling up tests. As the purity of polyphenol was not too high from the previous separation and also anti-allergic compounds were unknown, a method was established to isolate the anti-allergic compounds from the crude extract of Sargassum tenerrimum though stepwise separation of column chromatography of macrporous resin in combination with sephadex LH-20 followed by high performance liquid chromatography. During this study, 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 of separation. The gradient elusion of ethanollwater gave two distinct separate peak fractions of high antiallergic activities (IC<,50> values of 4.23μg/ml and 8.28μg/ml respectively) in two separate experiments using different fractions obtained from sepahdex LH-20 chromatography. The fractions were subjected for identification through 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 the antiallergic activity of IC<,50> values of 4.23 μg/ml and 8.28μg/ml.

目录

英文文摘

声明

0.0 Introduction

0.1 General introduction

0.2Lliterature review

0.3Objectives

0.4 Routes for study

Chapter1 Anti-allergic extracts of ethaol extracts from brown seaweeds

1.1 INTRODUCTION

1.2 MATERIALS AND METHODS

1.2.1 Algae

1.2.3 Chemicals

1.1.4 Extraction

1.2.5 Total phlorotannin content

1.2.6 Antihyaluronidase activity

1.3 Statistic analysis

1.4 RESULTS

2.6 DISCUSSION

1.7 Conclusion

Chapter 2 In vivo study of antiallergenicity of ethanol extracts from sargassum tenerrimum,sargassum cervicone and sargassum graminifolium turn

2.1 Introduction

2.2 Materials and methods

2.2.1 Algae

2.2.2 Chemicals

2.2.3 Extraction

2.2.4 Total phlorotannin content

2.2.5 Animals

2.2.6 Antigens

2.2.7 Antibodies production

2.2.8 Mouse belly passive cutaneous anaphylaxis(PCA)

2.2.9 Mouse belly active cutaneous anaphylaxis(ACA)

2.3 Statistic

2.4 Results

3.5 Discussion

3.6 Conclusion

Chapter 3 Optimizatin of preparative separation and purification of total polyphenols from sargassum tenerrimum by column chromatography

3.1 Introduction:

3.2 Materiais and methods:

3.3.2.1 Reagents and materials

3.2.2 Apparatus

3.2.3 Experimental procedures

3.3 Results and Discussions

3.4 Conclusion

Chapter#4 Optimization of separation technique for the polyphenolic antiallergenic compounds

4.1 Introduction

4.2 Experimental design

4.3 Materials and methods

4.3.1 Isocratic elution through MPR column chromatography(experiment#1)

4.3.2 Gradient elution through MPR column chromatography(Experiment2)

4.3.3 Isocratic elution through sephadex LH-20 column chromatography(Experiment#3)

4.3.4 Optimization for the isolation of active compounds through HPLC

4.3.5 Identification and characterization

4.4 Results

4.4.1 Isocratic elution through MPR column chromatography

4.4.2 Gradient elution through MPR column chromatography

4.4.3 Isocratic elution through sephadex LH-20 column chromatography

4.4.4 Gradient elution through High Performance Liquid chromatography (HPLC) for fraction 6-10 obtained from sephadex LH-20 chromatography

4.4.5 Isolations of active compounds thro ugh stepwise separation using sephadex LH-20 and HPLC without under going MPR

4.4.6 Gradient elution through High Performance Liquid chromatography (HPLC)for fraction 11-13 obtained from sepadex LH-20 chomatography

4.4.7 Identification and characterization of antiallergic compounds

4.5 Discussion

4.6 Conclusion

5.0 Conclusion of the whole thesis

6.0 Further study

List of publications

References

Acknowledgements