ood Chemistry $24 (2020) 125872 Contents lists available at ScienceDisect foe Food Chemistry journal homepage: www.clsevier.comjlocate/foodchem Studies on interactions of pentagalloyl glucose, ellagic acid and gallic acid |) with bovine serum albumin: A spectroscopic analysis a Liangliang Zhang", Yuchen Liu’, Xinyu Hu", Man Xu, Yongmei Wang" Key Lah. fas Bary an Mata Nang ings Province 210042 China eof heal nny of Foren Prec, CAR Nanjing 21042, Ch “Coinraton Cee of cient Proeing an Uatin of Fares Recarer, Naking Frey Unter, Nain 20042, Cha Keyword Fluorescence techniques an eizculardieoism (CD) were ulizeé to investigate the interactions of etypicl ash follotannin12.346-pena-O-galloy4-D-sleopyranose(2GG) and two simple phenolie compounds ellagie eid ne (EA) and gale aid (GA) with bovine rerum albumin (BSA). Fluorescence experiments showed that PGG and EA Galli ac ‘could strongly interact with BSA. The binding constants showed a pfl-depencent binding of phenolic acids by. Bhs ate SA The cD specraeveaed ha GA BA and POC as sgh eet onthe secondary Str of 8A a eo ‘concentration {50 uD. However, obvious alterations ofthe scondury structure of BSA were observed at high hore aes encentations (rm 100 ko 300 pt). The interaction of GA, FA and PGG a high concentration with A ‘aused an unfolding ofthe secondary structure of SA. BGG has a greater impact on the secondary structure of [BSA when compare with stall meleule compounds GA and EA 1. Introduction ‘Tannins are water-soluble polyphenols that present in many feats, cereal grains, beverages, and other vegetablederived foodstuts Tannins are considered nuteitionally undesirable because they pre ipitate proteins, inhibit digestive enzymes and affect the utilization of vitamins and minerals. Tannins can be divided into two condensed tannins (GI) and hydrolysable tannins (HT) Guang, Liv, Zhao, Hu, & ‘Wang, 2017). The structures of hydrolysable tannins are made up of polyol core (commonly D-glucose), whieh is esterified with gallie or hhexabydrory diphenic acid, Gallic acid can form chains of gale acid through depside bonds (causing gollotannins), or they can undergo ‘oxidative coupling reactions (causing ellagic acids and ellaitannins) (eller Harvey, 2001; Landete, 2011). A structurally well-defined gallotannin, is 1,2.34,6-penta-0-gallaylf-D.glicopyranose (PGG) (its ‘chemical structure is shown in Fig. 1A), which contained ina variety of ‘medicinal plants that exhibits @ variety of biological functions, in cluding antiviel, antitumor and antiinflammatory (Kiss, Filipel, & Czerwinska, 2010; Zhang, Liu, Hu, Wang, & Xu, 2018). PGG has the defining property of binding/preciptating proteins similar to tannins in general (Hagerman, Rice, & Richard, 1998; Dobreva etal, 2011; KatosSchwartz et al, 2018). Previous work has reported that tannin.protein associations are involved in the cross linking of protein molecules by tannins, which act as polydentate ligands, binding with proteins and peptides through hydrophobic in teraction and hydrogen bonding (Chen & Hagerman, 2004; Dobreva cal, 2014; Chai et al, 2015; Pattanayak, Basak, Sen, & Bhattacharyya 2017), In addition to the protein structure, interactions between pro- teins and tannins are also affeted by the relative eoncenteations of ‘annin and protein inthe solution, the solvent composition, the pH, and the presence of other substrater such as polysaccharides (Hagerman cecal, 1998; Chen & Hagerman, 2004; Chanphal & Tajmit-Riah, 2019), SSeram albumin is the most abundant protein in mammalian blood plasma, Tt ean bind with many intrinsic and extrinsic materials. The most important property of serum albumin is that it acts asa carver for many drugs to different molecular targets, It is responsible for the transport of metals, drugs, and various metabolites, including hor- ‘ones. Bovine serum albumin (BSA) is one of the most extensively utilized protein in laboratory practice and is used as @ human serum albumin (HSA) substitute in many experiments due to its structural homology with HSA (Jaldappagari, Balakrishnan, Hegde, Teradal, & Narayan, 2013; Sengupta, Pal, Mondal, & Bote, 2018) BSA Isa protein composed of 583 amino acid residues, containing two tryptophans ‘Trpl34 and Tep213, embedded in the fist IB and subdomain IA, re~ spectively. Trp134 is Tocated on the surface, and Trp213 is buried In a Bydrophobie pocket (Lis e¢ sl, 2019; Precups, Leonties, Nese, Sandu, & Popa, 2019) Though a number of works have been published in regard to * coresponding author a Key 1a, of Biomass Energy and Material, Nanjing, angst Province 210042, China mail ree: 2008509153 com CL Zan). op//do ong/10.1016/ foodchem.2020.126872 Received 10 January 2020 Received in revised form 10 Api! 2020; Accepted 18 Apel 2020 Avallable online 20 April 2020, (0308-81467 © 2020 Fleevier td All rights reservedFig. 1 Stuctres of (A) PGG, (B) elagie sd, and (©) alle sid. researches on the binding interaction between BSA and tannins Gravis, Papadopoulou, Muelle-HHarvey, Kissoon, & Green, 2003; Labieniee & Gabryelak, 2006; Dobreva et al, 2011; Dobreva et al 2014; Karonen, Oravita, Mueller-arvey, Salminen, & Green, 2015), ‘more information is necessary to understand how polyphenol structures atfet the protein-polyphenol interactions. Moreover, there has been ew detailed and quantitative studies on tannins-protein interactions using pure tannin compounds. Complexity of the tannin structures might be a reason but the erucial bottleneck forthe related research ght be the fact thatthe commercially available tannins are not pure tannin compounds (erually raw extracts of tanninrich plants) but mixtures of chemically defined tannins. Generally, those traditional ‘commercial tannins bear no strictly nomenclatures of chemistry and should not be regarded as pure tannin chemicals. This sty, a pure and ‘chemically defined tannin compound, PGG was used in this study. And two simple phenolic compounds ellagic acid (EA) and gallic acid (GA) were used for comparison purpose and GA could be regarded as the ‘monomer ofthe hydrolysable tannins. The binding interaction of PG, FEA, and GA with BSA were investigated by using Auorescence spec. troscopy and circular dichroism (CD) spectra, 2, Materials and methods 2.1. Reagents GA (purty > 999%), BA (purity > 998) and BSA without fatty acid were purchased from Sigma-Aldrich Co, (St. Louis, MO, USA) and used as received. PCG (purity > 99%) was purchased from Chengdu Purul Bio-Technigue Co, Ltd (Chengdu, China). Methanol, bydro chloric acid (HCD, sodium acetate (CHyCOON@'3H,0) and acetic acid (CH,COOH) were purchased from Nanjing Chemical Reagent Co, 12d (Nanjing, China) and were of analytical grade without further pur ication, Water war delivered by 2 Mill-Qavater purification system Mlilipore, Bedford, MA, USA) and used throughout the experiments “The chemical structures of GA, EA and PGG are shown in Fig. 2. ‘The stock solution of EA (0.25 mM) was prepared in a methanol/ water (1:1, v/v) slution and kept a 0", Working solutions of phenols were prepared by dissolving GA, EA or PGG in a methanol/water (I: '¥/¥) solution, The stock solution of BSA was prepared in 0.2 M sodium phosphate buffer (pH 7.4) or sodium acetate butfer (pH 3.0 and 5.0) land kept at 0 °C. The concentrations of BSA was determined spectro photometrically at 280 nm using an extinction coefficient of 42925 M7! cm”, The concentrations of PGG were determined spec: trophotometrically at 280 nm using an extinction coefcient of 54100 M7" em=! (Li & Hagerman, 2014), The concentrations of GA ‘were determined spectrophotometriclly at 263 nm using an extinction ‘coefficient of 6000 M7! em. The concentrations of EA sere de termined spectrophotometrcally at 260 nm using an extinction coeff Gent of 32.1 mL mg" em™ ‘red Chery 32 2020) 126872 2.2, Tryptophan fluorescence quenching Experiments were performed using a Lengguang F96 fluorescence spectrometer (Shanghai, China), withthe sample in I-em fluorescence cuvette. The emission spectra of tryptophan were obtained using an excitation wavelength at 280 nm. The emission spectra were set at range of 280-450 nm, The fluorescence speci of BSA were determined. atthe room temperature and both excitation and emission sits were set st 10 nm. Titration experiments were manually performed by using a micropipette forthe addition of phenolic acid solution to BSA solution at different pHs. The stock solution of BSA (0.1 mM) was further diluted 1 desired concentration (0.17 jIMD with buffer in the cuvette, The BSA solution was titrated with 10 pl additions ofthe 0.25 mM GA or EA, mixing by inversion after each addition. For PGG, the sample was ‘tented with 2 iL addicions of the 0.25 mM PGG, All tration experi= ‘ments were repeated independently at least three times. ‘Quenching can occur by diferent mechanisms, which usually clase sified as either dynamic quenching or static quenching. Dynamic ‘quenching results ffom collision between the fivorophore and a ‘quencher, while static quenching result fom non-fluorescent complex formation in the ground sate of the fluorophore. Dynamic and static ‘quenching can be distinguished by their differing dependence on tem= perature and viseosity. Dynamic quenching depends upon diffusion. For ‘dynamic quenching, the mechanism can be described by the Stemn- Volmer equation F/B = 1 + kyralQ) = 1 + Kel] o where fis the fluorescence intensities in the absence of quencher, Pis the fluorescence intensities in the presence of quencher (phenolic Acids); hy Is the quenching rate constant of the bimolecule, ty is the lifetime of the fluorophore in the absence of quencher; Ky is the dy ‘amic quenching constant; [Q] isthe quenchers concentration. The modified Stem-Volmer equation is used to calculate binding aifinities. yA OG, x KNX AAQD + Vf. @ Where AF = FF, Fy is the relative fluorescence intensities before the ‘dition ofthe quencher, Fis the relative fluorescence intensities after 1¢ addition of the quencher; fs the faction of fluorophore (protein) accessible to the quencher (phenolic acids); [Q] isthe concentration of ‘quencher; K, i the binding affinity. The values for f, and K, were calculated from the intercept and slope of the linear plot of Fy/AF ‘against 1/(Q), 23, Cirelar dichroism (CD) measurement CoD was determined on a J-1500 spectrometer (JASCO), Stock s0- lution of BSA (0.5 4M) was prepared by dissolving BSA in 0.2 M phosphate buffer (pH 7-4). The CD measurements of BSA inthe absence and presence of phenolic acids were collected from 190 nm to 400 am. {20°C ata scanning speed of 100 nm/min. The contents of three types of secondary structure (achelix, turn and random col) were analyzed by CCdpro software package (ling, Liu, Yan, Tan, & Chen, 2015). 3. Results and diseussion 5.1, Fluorescence quenching studies Effect of pH values on spectra of 0.17 JM BSA fluorescence are shown in Fig. 2A, Itwas demonstrated that postion of maximum and the spectrum shape of fuorescence of native BSA significantly depends fn pHin range of 3-7-4. At pH5.0 0r7.4, the emission spectrum of BSA. covers the range 300-450 am with the maximum at 344 nm. When the pH was decreased to 4.0, the emission spectra of BSA ranged from 300, fo 420 nm with a maximum at 332 nm, Thus, the maximum was1 zhong eo am wie i ong om) EM] + wre reciom ‘Food Chery 324 2020) 126872 Wonlah om Fo] sence Fig. 2. (A) fect of pl values on spetea of 0.17 uM BSA Auocescence. The quenching fect of PGG an BSA orescence intensity at phosphate buller pH 7.4 (50, and (0) 20; ) The ffcencies of quenching were compared at IT 3.0, 5.0, and 7-4 by plotting the relative forecence intensity (AF > 100/Fs)agsnat PGC concencation: () The Stecn-Volmer plot for BSA Nuoreeence quenching by PGG at pl 7.4, 5.0, and 30. ‘redshifted from 382 to 344 nm wien the pH increased from 3.0 to 50. “The effect of pH values on the conformation changes of BSA in aqueous solution was studied by Huai, He, Sheng, & Tao (1996). Their results demonstrated that there were diferent conformational states in BSA at ‘pH 5.0 and 9.0 and suggested an increase in sheet and turn structures thigh pH values. Effect of various concentrations of PGG, BA, and GA ‘on BSA intrinsic fluorescence has been studied. fig. 2B-D showed ‘quenching curves of native BSA in the presence of PGG at various pH Values. The intensity of the peaks decreased as the PGG concentration increased due to ite quenching of the tryptophan fluorophore in BSA. A small bathochromte shift (red-shift) of the peak maximum was observed asthe PGG concentration increased at pHl 3.0. This shift (0 a higher ‘wavelength (red-shift) in the emission spectra was associated with the polarity inerease of the local microenvironment of Trp 213 in BSA, indicating an unfolding of the secondary structure of the protein (Dobreva et si, 2011), These observations are in agreement with the ‘earlier reports fom tannin-BSA binding studies (Labienics & Gabryelak, 2006; Dobreva etal, 2012) Fluorescence quenching titration revealed that the efficacy of PGG- induced quenching showed a pH-dependent pattern (see Fig. 28). The results indicated that 0.83 yM PG quenched the intrinsic fluorescence ‘of BSA to 56.26% ofthe contol value at pH 3.0. Inthe presence ofthe same concentration of PGG, only 37.72% of the control Muorescence remained at pH 5.0. At the same concentration, PGG reduced the emission intensity t0 65.75% of the contol value when the pH was increased (0 7.4, The Stern-Volmer plots for the quenching of intrinsic ‘luorescence of BSA at all tested pH values were linear (Fi. 2F) and the Stern-Volmer quenching constants (K,) were obtained from the equa- sion and listed in Table 1 Fig. SAAC show the fluorescence quenching effect of EA with BSA; ig. SD-E show the Stern-Volmer plots of BSA Muorescence quenching Tablet Sern-Volmer constants describing BSA uorescencequenehlag by PGG, lage dor gli eid a pI 3.0, 5.0, and 7.4 ce nae m5 Pee W689 = 146 Tas 2 ae alnated as K, fom modiled SV plot Data are represented as a mean + 8D of thre repeat1 zhong eo ° ‘Food Chery 324 2020) 126872 rege 5. The quenching effec of ellagi acd on BSA fsrescence intensity at phosphate blfer pit (A) 7.4, (B) 5.0, and (©) .0;The concentrations of lage aid were 0, 0185, 167,25, 3:33, and 4.17 pM The Stern-Volmer plot fr SA toorescence guenching by elagic acid at pl (D174, 5.0, and () 30, nsec: the modified erm Volmer plot fr elagc acl quenching of BSA fiorescence at pH 30 induced by EA. At pl] 5.0 and 7.4, an inerease of EA concentration from 083 to 4.17 uM induced a linear reduction in the intensity of the uorescence emission, whereas at pH 3.0, the Stern-Volmer plots were nonlinear and this is consistent with the presence of multiple fluor. ‘ophores with different accessibilties to ellagic acid (Lakowicz, 1999), ‘This reslt is diferent from that of PGG mentioned above and this may be explained by the diferent binding modes for PGG and EA with BSA. At pH 3.0, a linear modified Stern-Volmer plot was observed for the [BSA fluorescence quenching by EA (see Fig. 3). Therefore, the Stem ‘Volmer constants for pH 5.0 and 7.4 were obtained by using the Stern Volmer equation and for plf 3.0 were obtained by using the modified ‘Ster-Volmer equation (See Table 1). These results showed that the binding interaction between BSA and FA was a strongly the pH-de pendent ‘Compared with EA and PGG, GA was found to be a very weak ‘quencher, ‘The results of Muoreseence spectra showed GA binding slightly changed the spectra of BSA fluorescence, and the decrease in Intensity of luorescence emission was small at high concentrations of, GA at all test pH values (see Supplemental Fig. 1A-C). The Stern Volmer plots of intrinsic fluorescence quenching of BSA by GA at all ‘ested pH values Were nonlinear. The binding constants of GA with BSA were calculated from a modified Stern-Volmer equation (see Supplemental Fig. 1D) and listed in Table 1, The eesults were consistent with very weak interactions between GA and BSA and this is in agreement with the previous studies (Labieniee & Gabryelak, 2008; Pripp, Vreeker, & Duyahoven, 2005), 3.2. Conformational changes monitored by CD spectroscopy To elucidate the effect of phenolic acids on the secondary structure OF BSA, the CD spectroscopy of BSA with various concentrations of GA, EA or PG were detected. Fig. 4 showed the CD spectra of 0.5 uM BSA, before and ater the adition of GA (from 50 uM to 500 uN), EA or POG (rom 50 yM to 100 4M), Detailed analysis of the results of GD spec- troscopy revealed that no change occurred with the addition of GA. ‘under concentration of 50 4M. At the same concentration, little change was observed with the addition of EA and PGG, However, the addition of higher concentration of GA (500 uD), EA or PGG (500 p30) caused an obvious change of BSA CD spectra. The negative bands at 208 nm and 222 nm could indicate the acelicl structure of the proteins (Jing ct. 2015). The contents of three types of secondary structure of BSA as shown in Table 2 were analyzed by Cdpro software package. With the gradual addition of phenolic acids to BSA solution, the helix content OF BSA decreased and turn and Random coil contents had the opposite‘red Chery 32 2020) 126872 oe: — 25 arash | ‘Bassai 2 FL ‘emeani Fig. 4. CD spectra of (A) GA + BSA, () EA + BSA, and (C) PGG + BSA system — i: | - a i o. ractions of secondary strctre of BSA in the absence and presence of elie acid (GA), lagi acid EA), and POG, Reseion sens Secondary rare oneat n ASA (| SoogMGA VOS)MBSA 528-04 22111 I= 15, TooyMteA'y O5yMESA S708 179.805 SIs 08 Too WMPGS 1 OSuMBSA 520208 229415 21 +13 ata are represented ata mean ‘SD of three repeat ‘tendency, which indicated thatthe interaction of GA, EA and PGG with BSA resulted in unfolding of the secondary steueture (Pan, Ye, Cai, ‘Wang, & Ca, 2012; Hing etal, 2015), ‘The content of echelix in the BSA decreased 2.95% in the presence ‘of EA at the concentration of 100 4M. The same concentration of PGG ‘caused 11.51% decrease of the content of avhelix in the BSA. The ‘contents of tr inthe BSA were increased 5.71% inthe presence of FA at the concentration of 100 uM. The same concentration of PGG caused 35.43% increase ofthe content of turn inthe BSA. The results indicated Ut PGG has a greater impact on the secondary structure of BSA when ‘compared with small molecule compounds GA and FA. The results showed that PGG had obvious influence on the amino acid residues of the main polypeptide chain and damaged the hydrogen bonding of BSA. Herein, the reslte were consistent with fluorescence spectra, 4, Conclusions In this paper, the interaction of GA, EA and PGG with BSA was, studied by fluorescence and CD spectroscopy. The experimental results indicate that PGG and EA can form a srong relationship with BSA and the binding constants of phenolic acids to BSA showed in a manne {dependent on pH values. The CD spectra revealed that GA, FA and PG has slight effect on the secondary structure of BSA at the low con- centration (50 jMD, which may have litle impact on the physiological function of BSA. However, obvious alterations ofthe secondary struc: ture of BSA were observed at high concentrations (from 100 uM to 500 ji), The interaction of GA, EA and PGG at high concentrations with BSA caused an unfolding of the secondary structure of BSA. The fuoreseence quenching of BSA and the alterations of the secondary structure after phenolic acids addition all might cause denaturation of protein, The results indicated that PGG has a greater impact on the secondary structure of BSA when compared with small molecule com= pounds GA and EA. Therefore, PGG has obvious effects onthe structure And funetion of BSA. Author contributions Liangliang Zhang-collected Muorescence and CD data, prepared, figures, drafted manuscript. Wrote final version of manuscript, quality control of data, analysis of data, developed context for study. ‘Yuchen Liv-ollected fluorescence data. Xinyu Hu-collected CD data ‘Man Xe-collected fvorescence titration data, Yongmei Wang-polished manuscript and provided insights into phenolies/B6A. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ. fence the work reported inthis paper. Acknowledgement This work was financially supported by the Key Lab of Biomass Energy and Material, Jiangsu Province (KLBEM) (JSBEM-S-202005) Wie thank the safe ofthe Central Laboratory at Jiangse Academy of Agricultural Sciences for their help in CD spectroscopy analysis.1 zhang eo Appendix A. Supplementary data ‘Supplementary data to this article can be found online at hisps// oi.org/10.1016/).£ondchem.2020.126872, rouatharyaniie ar ware reine hii: Sacre carci fahbary sty sed mechan Juma of Artal en aed Chay, 85033), 79817387. eneen bavne rerum bum snd, 23,4 pet-Oloy Deucpyranre Dy MAGDITOF WS. Jour of Areal an Foo! 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