1

Investigation on the conditions of hyaluronic acid extraction from rooster combs by protein precipitants
M.T. Chen & X.Y. Lin
Department of Bioindustry Technology, Da Yeh University 112 Shan Jeau Rd , Da Tsuen, !hanghua, Tai"an #1# $%&mail' michen(mail dyu edu t" bstract )any pharmaceutical *+ products have ,een prepared from um,ilical cord, synovial fluid, s-in and rooster com,s as "ell as micro,ial fermentation +t present, most of *+ is prepared from rooster com,s The *+ e.tract has ,een purified using precipitant to remove impurities and o,tain desira,le purity of product Because of the precipitant used in this purification procedure is a high to.ic organic solvent&&&chloroform and higher cost, the aim of this study is to search for other protein precipitants to replace of chloroform, and to investigate on the effect of different precipitants on the yield and /uality of *+ e.tracted from rooster com,s Introduction )any pharmaceutical *+ products have ,een prepared from um,ilical cord, synovial fluid, s-in and rooster com,s as "ell as micro,ial fermentation +t present, *+ is prepared from rooster com,s most commonly The process of the *+ e.tracts has ,een purified using precipitant to remove impurities and o,tain desira,le purity of product Because of the precipitant used in this purification procedure is a high to.ic organic solvent&&&&chloroform and higher cost, the aim of this study is to search for other protein precipitants to replace of chloroform, and to investigate on the effect of different precipitants on the yield and /uality of *+ e.tracted from rooster com,s Materials and methods Rooster com,s "ere collected from the local poultry slaughterhouse and placed at 0 for preparing HA extraction procedure: The procedure descri,ed ,y Bala1s 213435 are modified to e.tract crude *+ from com,s The com,s are cut into pieces ,y 2mm .#mm and soa-ed in ethanol'chloroform solution 2com,s'ethanol'chloroform617'07'15 to dehydrate and remove impurities such as ,lood The mi.tures are centrifuged at 8777rpm, 1#min 2*ermle 9:2:5 to o,tain the crude *+ e.tracts The crude *+ is purified ,y three different procedures2 see flo" chart 5 modified from the methods of Bala1s 213435 and ;imrod et al 213885 1<cetyl&pyridinium chloride2!=!5, 177< he.ane, 1< ammonium sulfate are used as precipitants to compare "ith the deproteini1ing efficient of chloroform +nd the crude *+ e.tract is >ust ad>usted p* to isolectric point to precipitate proteins as the control sample Hexuronic acid assay: =urity of crude *+ e.tract is e.pressed as the content of glucuronic acid of *+ The method of Bitter et al 213?25 is used to determine the content of glucuronic acid + modified colorimetric method is used for the estimation of ;&acetylamino sugars @uantification of *+ confirmed ,y using *=A! assay according to the method descri,ed ,y Jamal et al 227725 The dried *+ is dissolved in

2 concentrated sulfuric acid and ad>usted to p* # "ith ;aB* for *=A! analysis Viscosity measurement: 7 1mgCml of *+ solution sample is used to measure its viscosity ,y Broo-field DD&% viscometer !esults and discussion Yield% of crude HA extract: Eigure 1 is sho"ed the effect of precipitants on the yield< of crude *+ e.tract Ft indicated that the yield< of the control is the highest among the treatments, then follo"ed ,y !=!, 2;*052SB0, !hloroform and *e.ane in descending order !=! is one of /uaternary ammonium compound possesses positive charge, therefore it can ,ind * + "hich possesses negative charge to form a comple. compound precipitated under lo" salt concentration 2lo" ionic strength5 Therefore, !=! can efficiently ,ind "ith * + and resulting in a higher yield< than other deproteini1ing agents +gain, although, chloroform can efficiently deproteini1e during e.traction process, ,ut it can not ,ind "ith "ater solu,le * + due to its lipophilic property Thus, its yield< can not ,e higher than !=!

1 0.8 0.632 yi el d( % ) 0.6

A. 1%CPC B. 100%Hexane C. 100%CHCl3 D. Cont ol E.1%!H4("#4)2

0.661 0.575

0.447 0.4 0.2 0 A B C D E 0.277

"igure #. %ffect of deproteini1ing agents on yield< of crude *+ e.tracts Effect protein precipitants on purity of crude HA extracts: Eigure 2 it can ,e found that the purity of crude *+ e.tracts from chloroform precipitation is the highest among all samples, then follo"ed ,y the control, !=!, *e.ane and 2;*052SB0 in descending order The control sample is prepared "ithout deproteini1ing agent, and only ad>ust the p* to isoelectric point to remove the impurities, though its yield< is lo"er than that of chloroform ,ut still higher than the products prepared "ith other precipitants Fn order to confirm the purity measured ,y glucuronic acid content, *=A! is also employed Erom the *=Achromatography 2Eigure :5, it indicated that sample of chloroform has highest purity, and then follo"ed ,y the control, !=! and 2;*052SB0, in descending order They are 7 14?, 7 101, 7 1:8 and 7 178 mgCml ,respectively The repression e/uation y62%G7 ?.&##4? 4, R 267 330: The trends in the purity of the products from different precipitants are in the agreement "ith the measurement of glucuronic acid 2Eigure 25

10 8.4 8 $i %&o%i t y( &'%) 6 4 2 0 A B C D 5.04 7.6

A. Cont ol B. 100%He xa ne

5.86 D. 1%(!H4)2"#4
E. 1%CPC

C. 100%CHCl3

3.3

"igure $. %ffect of deproteini1ing agents on the purity of crude *+ e.tracts Viscosity of crude HA extracts: Eigure 0 indicated that the viscosity of the crude *+ e.tract from *e.ane is the highest among all treatments, the chloroform, the control, 2;* 052SB0, !=! The lo"est in viscosity of e.tracts "ith 2;* 052SB0, and !=! is due to the polymer chain contracting firmly from an opening structure "hen salts presented in the e.tract solution Thus, the salt concentration is higher the viscosity is lo"er This finding may ,e caused ,y salt ions and densed electrons interaction 2=iasrci- et al , 133#5

a. 0.2 mg/mlHA b. Control c. 1%CPC d. 100% CHCl3 e. 1% NH4(SO4)2

"igure %. The purity of crude *+ e.tracts form different deproteini1ing agents comfirmed ,y *=A!
0.4 0.317 Con& o( )l*&* onola&tone ( +),+- ) 0.3 0.201 0.2 0.143 0.125

A. Cont ol B. 100%Hexane C. 100%CHCl3 D. 1%!H4("#4)2 E. 1%CPC

0.16

0.1

0 A B C D E

analysis

0 "igure &. %ffect of deproteini1ing agents on viscosity of crude *+ e.tracts

# Conclusions 1<!=! and 1< 2;*052SB0, "ere used as deproteini1ing agents or precipitants to remove the proteins and other impurities e.isted in crude *+ solution in this e.periment *ighest yield< "as o,tained ,ecause of these t"o deproteini1ing agents possess positive charge in the solution "hich can ,ind *+ But purity "as lo"er due to their ,inding to *+ firmly, then thus lo"ering the viscosity of the products The purity and yield< prepared ,y using !hloroform as a deproteini1ing agent "ere higher, ,ut it has higher to.ic and more polluted to environment Fn addition, "e used concentrated sulfuric acid to dissolve the sample for the content of *+ determination ,y *=A!, it "as more efficient and lo"er cost than the en1ymatic method Ft suggested that it is a convenient method for *+ purity analysis !eferences Bala1s, % + 1343 Ultrapure hyaluronic acid and use US =at 010134: Bitter, T and )uir, * ) 13?2 + modified uronic acid car,a1ole reaction +nal Biochem 0'::7 +Eraser, J R % , Aaurent, T ! and Aaurent 1334 *yaluronan' its nature, distri,ution, functions and turnover J Fnt )ed , 202'24&:: Jamal, + + , Yahya )eratani, Reinhard * * ;eu,ert 2772 ;e" approaches for /uantifying hyaluronic acid in pharmaceutical semisolid formulations using *=A! and !9% J =harmaceutical and Biomedical analysis :7, 31:&313