ANTIMICROBIAL AND PHYTOCHEMICAL ANALYSIS OF LEAF EXTRACT OF MEDICINAL FRUIT PLANTS

Vol 9, Issue 4, 2016 Online - 2455-3891 Print - 0974-2441 Reserh Artile ANTIMICROBIAL AND PHYTOCHEMICAL ANALYSIS OF LEAF EXTRACT OF MEDICINAL FRUIT PLANTS ABSTRACT RINKY BISHT, SHEETAL CHANYAL, PAVAN KUMAR AGRAWAL* Deprtment of Biotehnology, G.B. Pnt Engineering College, Ghururi, Puri, Grhwl, Uttrkhn, Ini. Emil: p_k_grwl@reiffmil.om Reeive: 19 Mrh 2016, Revise n Aepte: 12 April 2016 Ojetive: This stuy ojetive ws to esrie the in vitro ntimiroil n ntifungl tivity of ethyl ette extrts from leves of ppy, pomegrnte, nn, n guv. The present investigtion showe tht leves extrt of fruits plnts re goo soure of iotive ompouns whih hve some ethnomeiinl pplitions were sreene for their ntiteril tivity ginst teril pthogen of humn. Methos: A totl of four plnt extrts were use in this stuy to exmine their ntimiroil properties n phytohemil nlysis. The ntimiroil tivity ws evlute for rue ethyl ette extrts ginst humn pthogen Esherihi oli, Billus sutilis, Stphyloous ureus, n Pseuomons eruginos using n gr iffusion ssy. Phytohemil nlysis tests for the sreening n ientifition of iotive hemil onstituents of extrts of the fruits lef ws performe. This stuy ws lso onute to etermine the totl phenols present in lef extrt of fruit plnts. Results: The guv leves rue extrt showe minimum inhiitory onentrtion of 3.75 mg/ml for B. sutilis n P. eruginos, whih showe its effiy s potent ntimiroil. The phytohemil nlysis of the extrts revele the presene of iotive ompoun suh s sponins, lklois, flvonois, terpenois, rohyrtes, n tnnins. The ethyl ette extrt of nn proue the highest zone of inhiition 23 mm for B. sutilis. This stuy showe tht Puni grntum lef is goo soure of phenoli ompouns. Conlusion: This stuy onlues tht these fruit leves re potentil soure for iotive metolites n my e use in phrmeutil inustry. On the sis of the present fining lef extrt of fruits possess, the pilities of eing goo nite in the serh for nturl ntimiroil gent ginst infetions n/or iseses use y humn pthogens. Keywors: Leves extrt, Fruits, Antimiroil tivities, Antifungl tivities, Minimum inhiitory onentrtion, Phytohemils nlysis. INTRODUCTION Diseses re the mjor use of eth in the eveloping ountries n ounts to 50% of it. Antimiroil gents re essentilly importnt in reuing the glol uren of infetious iseses. However, s resistnt pthogens evelop n spre, the effetiveness of the ntiiotis is iminishe. This type of teril resistne to the ntimiroil gents poses very serious thret to puli helth, n for ll kins of ntiiotis, inluing the mjor lst-resort rugs, the frequenies of the resistne re inresing worlwie [1]. Bteril resistne to ntiiotis inreses mortlity likelihoo of hospitliztion n length of sty in the hospitl [2]. Reently, there hs een lot of ttention fouse on prouing meiines n prouts tht re nturl. Severl fruits n fruit extrts, s well s rrowroot te extrt n ffeine, hve een foun to exhiit ntimiroil tivity ginst Esherihi oli O157:H7 [3]. This suggests tht plnts whih mnifest reltively high levels of ntimiroil tion my e soures of ompouns tht n e use to inhiit the growth of fororne pthogens. The teril ells oul e kille y the rupture of ell wlls n memrnes n y the irregulr isruption of the intrellulr mtrix when trete with plnt extrts [4]. Plnt-se ntimiroils represent vst untppe soure. The use of the plnt extrt for meiinl tretment hs eome populr when people relize tht the effetive life spn of ntiioti is limite n over-presription n misuse of tritionl ntiiotis re using miroil resistne [5]. Tritionlly use meiinl plnts proue vriety of ompouns of known therpeuti properties [6]. All meiinl plnts proue importnt seonry metolites suh s terpenois, flvonois, polyphenols, hlorophylls, n etlins. Among these, phenoli ompouns re onsiere to e the hief plnt onstituent euse of its pity to exhiit ntioxint, nti-nerous, n ntiinflmmtory properties [7]. In reent yers, ntimiroil properties of the meiinl plnts re eing inresingly reporte from ifferent prts of the worl [8]. At present, nerly 30% or more of the moern phrmologil rugs re erive iretly or iniretly from plnts n their extrts ominte in homeopthi or yurvei meiines [9]. Consiering the vst potentility of plnts s soures for ntimiroil rugs, this stuy ime to etet the ntiteril tivities of some nturl plnt extrts n investigte the effet of some ommeril ntiiotis ginst multirug resistnt humn linil teril isoltes. Mus umint, ommonly known s nn plnt, is vstly eing onsume ross the worl. It is known for mny phrmologil tivities n reports show tht nn leves ontin lrge mount of phenoli ompouns espeilly polyphenol oxise whih is use in the tretment of Prkinson s isorer [10]. The pomegrnte (Puni grntum) n nient, mystil, n highly istintive fruit elong to Puniee fmily. Chemil onstituents of the lef extrt of P. grntum re lmost similr to those of the fruit or see, e.g., ellgi i, tnnins (punilin n punifolin), n flvones glyosies inluing luteol in n pigenin. In previous stuy, it is reporte tht the ntiieti n ntihyperlipiemi effets of ethnoli extrt of P. grntum in lloxn-inue ieti rts [11]. Ppy (Cri ppy L.) is memer of Criee Fmily. The ppy is espeilly suseptile to prsites, pests, n iseses. This fussy

Bisht n Agrwl Asin J Phrm Clin Res, Vol 9, Issue 4, 2016, 131-136 plnt nees lot of wter ut must hve goo ringe n it ers most fruit in light, porous, slightly ii soils tht re rih in orgni mtter. It hs een use to tret igestive prolems n intestinl worms s well s wrts, sinusitis, ezem, utneous tuerules n hrness of the skin. Green fruits re use to tret high loo pressure, rounworm infetion, yspepsi, onstiption, menorrhe, skin isese, generl eility, n genitourinry isorers [12]. The tritionl use of C. ppy leves in the tretment of vrious ilments inluing urinry trt infetions informe the nee for this stuy, whih ws ime t evluting the ntiteril tivity of the leves ginst some gents of urinry trt infetion. Guv (Psiium gujv) in meiine, extrts of leves re use to tret gstroenteritis, vomiting, irrhe, ysentery, wouns, ulers, toothhe, oughs, sore throt, inflme gums, n numer of other onitions. Guv leves ontin essentil oil with the min omponents eing α-pinene, β-pinene, limonene, menthol, terpenyl ette, isopropyl lohol, longiylene, ryophyllene, β-isolene, ryophyllene oxie, β-opnene, frnesene, humulene, selinene, rinene, n urumene [13]. This stuy ws rrie for sreening the ntiteril tivity of four fruits plnts use for the linil tretment y lol ommunities ginst some pthogeni teril strins. METHODS Survey n olletion of plnt mterils The fresh n helthy leves of nn, pomegrnte, ppy n guv were ollete from ifferent prts of Ghururi, Puri, Grhwl, Uttrkhn, Ini. Leves smples were rought to the lortory n thoroughly wshe in istille wter. Eh plnt mteril ws lele, numere n informtion regring te of olletion, meiinl use of plnt leves n lotion from were ollete reore. Plnt mterils were ientifie n ontminte prtiles were remove. They were ir-rie n groun into fine power using mortr n pestle in the lortory [14]. Preprtion of leves extrts The leves extrt of totl four fruits plnt were ir rie, hoppe n gin she rie for 14 ys t room temperture n then groune lef power using griner for ese of the extrtion of tive ompouns. The powere plnt mteril (15 g) ws pke into soxhlet pprtus n extrte up to 4 hrs with ethyl ette (250 ml) t 60-80 C for eftting [15]. The extrt ws filtere, n the solvent ws evporte uner reue pressure using rotry vuum evportor t 65 C. These purifie extrts were then store t 4 C until further use. Antimiroil ssy Bse on their linil n phrmologil importne, Billus sutilis, E. oli, Stphyloous ureus, n Pseuomons eruginos were hosen for evluting ntiteril tivity. These teril strins otine from Institute of Miroil Tehnology, Chnigrh, n fungl isoltes from Forest Reserh Institute, Dehrun. The teril stok ultures were inute t 37 C for 24 hrs on nutrient gr n fungl pthogen Peniillium oxlium n Alternri lternte on potto extrose gr (PDA) t 27 C for 24 hrs followe y storge t 4 C until further use. A totl of four plnt extrts were use in this stuy to sreen their ntimiroil tivity. Antiteril suseptiility testing ws one using the well-iffusion metho. The plnt extrts were teste on Mueller-Hinton Agr pltes to etet the presene of ntiteril tivity. All pltes were inoulte with the test terium, sterile otton sw ws ippe into the suspension, rotte severl times, n presse firmly on the insie wll of the tue ove the flui level removing exess inoulum. The surfe of the gr plte ws streke over the entire sterile gr surfe rotting the plte to ensure n even istriution of inoulums. After spreing the pltes with teri, 5 mm imeter wells were punhe into the meium using sterile orer. The pltes re llowe 3-5 minutes to ry the exess moisture. 50 μl liquots of eh test extrt were ispense into eh well-fter the inoultion of the pltes with teri. For eh teril strin, ontrols were mintine where pure solvents were use inste of the extrt. The pltes re sele with prfilm, lele, n ple in n inutor set to 37 C. After 24 hrs of inution, eh plte ws exmine for inhiition zones. A ruler ws use to mesure the inhiition zones in mm. Determintion of minimum inhiitory onentrtion (MIC) MIC ws etermine fter ntiteril tivity of the plnt lef rue extrts y the stnr metho with minor moifition. Muller- Hinton Broth ws me n sterilize using utolve. 1.0 ml of the prepre roth ws ispense into the test tues lele from 1 to 5 using sterile syringe n neele. A stok solution ontining 60 mg/ml of the extrt ws prepre. Then, 1.0 ml of the solution ws ispense into the tue 1. Susequently, from tue 1 solution ws serilly trnsferre until tue 5-1.0 ml of the solution ws isre from it. Tue 6 ws use s ontrol for sterility of the meium n tue 7 for viility of the orgnisms. An overnight ulture of eh of the test isoltes ws prepre in sterile nutrient roth. 1 ml inoulum ws trnsferre into eh tue from tue 1 to tue 7 with exeption of 6, to whih nother sterile roth ws e. The finl onentrtion of the extrt in eh of the test tues numere fter ilution 60, 30, 15, 7.5, 3.75 mg/ml ws inute t 37 C for 24 hrs n exmine for growth. The test tue in whih growth file to our ws the MIC of the ulture. Antifungl tivity ssy PDA meium with 25% onentrtion of the solvent extrts of the test fruit plnts ws prepre. Aout 15 ml of the meium ws poure into eh Petri plte n llowe to soliify. 5 mm is of 7-y-ol ulture of the pthogeni fungi ws ple t the enter of the Petri pltes n inute t 27±2 C for 7 ys. After inution, the olony imeter ws mesure in millimeter. PDA meium without the solvent extrt serve s ontrol. The fungitoxiity of the extrts in terms of perentge inhiition of myelil growth ws lulte y using the formul % Inhiition = -t/ 100 Where = Averge inrese in myelil growth in ontrol, t = Averge inrese in myeli growth in tretment [16]. Phytohemil sreening of fruits leves rue extrts Phytohemil nlysis tests for the sreening n ientifition of iotive hemil onstituents of extrts of the fruits lef were performe with the stnr methos with moifitions [17]. Test for sponins Aout 1 ml liquots of the vrious plnt lef extrts were omine with 5 ml wter whih is t 60 C then shken for 2 minutes s sponins re known to proess frothing tivity, the volume of froth proue in this experiment ws oserve. Test for phenoli ompouns A vlue of 1 ml of test solution ws trete with 10% ethnoli ferri hlorie. Phenoli ompouns were onsiere present when olor hnges to rk green or luish lk. Test for tnnins The plnt lef rue extrt ws trete with loholi 0.1% ferri hlorie regent. A luish lk olor will pper. Test for terpenois The plnt lef rue extrt ws trete with 2 ml hloroform n 3 ml sulfuri i refully. Reish rown preipitte will pper if terpenois re present. 132

Bisht n Agrwl Asin J Phrm Clin Res, Vol 9, Issue 4, 2016, 131-136 Test for flvonois The plnt lef rue extrt ws trete with rops of 20% NOH. Yellow ppers whih eome olorless on ing ilute HCl. Test for lklois The plnt lef rue extrt ws evporte to ryness in oiling wter th. The resiue ws issolve in 2 NHCl. The mixture ws filtere n the filtrte ws trete with equl mount of Wgner s regent. The retion shows the pperne of rown preipitte inites the presene of respetive lklois. Test for rohyrtes Extrts were issolve iniviully in 5 ml istille wter n filtere. The filtrtes were use for the etetion of rohyrtes. Few rops of Beneit s regent ws e to the test solution n oile on wter th. The formtion of reish rown preipitte inites the presene of sugrs. Depening on the onentrtion of the reuing sugr, the mount n olor of the preipitte proue vrie. A positive Beneit s test ppers green, yellow, ornge, or re. Determintion of totl phenoli ompouns The onentrtion of phenolis in leve extrts ws etermine using spetrophotometri metho [18]. The retion mixture ws prepre y mixing 0.5 ml of queous solution of extrt, 2.5 ml of 10% Folin- Ciolteu s regent issolve in wter n 2.5 ml of 7.5% of soium i ronte (NHCO 3 ). The smples were therefter inute in thermostt t 45 C for 45 minutes. The sorne ws etermine using spetrophotometer t 765 nm. The smples were prepre in triplite for eh nlysis n the men vlue of sorne ws otine. The sme proeure ws repete for the stnr solution of glli i (0.010-0.060 mg/ml) n the lirtion line ws onstrue. Fruits lef extrts were prepre in ethyl ette t onentrtion of 0.06 g/ml. Bse on the mesure sorne, the onentrtion of phenolis ws re (mg/ml) from the lirtion line, then the ontent of phenolis in extrts ws expresse in terms of mg/g of the extrts s glli i [19]. Fig. 1: Antiteril tivity shown y leves extrts of fruits plnts on pthogens ginst Esherihi oli (): Bnn (): Guv (): Ppy (): Pomegrnte RESULTS Antiteril tivity ws ssesse y nlyzing the imeter of the zone of inhiition n the ethyl ette extrt of leves from four fruit plnts of meiinl vlue. In the se of P. eruginos n B. sutilis isoltes, the highest zone of inhiition ws 23 mm whih ws exhiite y Mus spientum. The lef extrt of P. gujv exhiite tivity ginst P. eruginos n B. sutilis (20 mm) isoltes s well s ginst E. oli n S. ureus (15 mm n 21 mm) isoltes (Tle 1, Figs. 1-4). M. spientum is n ntimiroil gent ginst infetions n/or iseses use y P. eruginos n S. ureus. MIC All leves extrts showing potent ntiteril tivity ws further etermine for their MIC (Tle 2). The extrts hve shown MIC rnge from 30 to 3.75 mg/ml for ginst P. eruginos, E. oli, S. ureus n B. sutilis. Lef extrt of nn showe MIC of 7.5 mg/ml for E. oli, wheres Guv showe MIC of 3.75 mg/ml for P. eruginos whih showe its effiy s potent ntimiroil. The ethyl ette extrt of ppy hs shown mximum MIC of 3.75 mg/ml for E. oli, wheres pomegrnte shows MIC of 3.75 mg/ml for P. eruginos. Antifungl tivity ssy Antifungl tivities were performe ginst the two fungl strin inluing P. oxlium (Fig. 5) n A. lternte (Fig. 6). The inhiition (%) of leves extrt ginst fungl pthogen is shown in Tle 3. The est tivities re nn ethyl ette ginst P. oxlium with inhiition of 40%, whih on further hemil investigtion will le to isoltion of ntifungl hemils. Phytohemil Sreening of lef extrt Photohemil nlysis of plnt lef extrts hs shown the presene of lmost ll the photohemil (Tle 4). Fig. 2: Antiteril tivity shown y leves extrts of fruits plnts on pthogens ginst Pseuomons eruginos (): Bnn (): Guv (): Ppy (): Pomegrnte Tle 1: Antimiroil tivity of meiinl plnt lef ethyl ette extrt ginst selete teri S. No. Plnt Men ler zonl imeter ( in mm) P. erugins E. oli S. ureus B. sutilis 1 Bnn 22 11 21 23 2 Guv 20 15 21 20 3 Ppy 10 10 15 12 4 Pomegrnte 12 15 15 20 P. erugins: Pseuomons erugins, E. oli: Esherihi oli, S. ureus: Stphyloous ureus, B. sutilis: Billus sutilis Tle 2: Minimum inhiitory onentrtion of the rue ethyl ette extrt of plnt leves Plnt extrt Minimum Inhiitory onentrtion (mg/ml) P. erugins E. oli S. ureus B. sutilis Bnn lef 30 7.5 15 15 Guv lef 3.75 25 15 3.75 Ppy lef 7.5 3.75 7.5 30 Pomegrnte lef 3.75 7.5 15 7.5 P. erugins: Pseuomons erugins, E. oli: Esherihi oli, S. ureus: Stphyloous ureus, B. sutilis: Billus sutilis 133

Bisht n Agrwl Asin J Phrm Clin Res, Vol 9, Issue 4, 2016, 131-136 Tle 3: Antifungl tivity of the rue ethyl ette extrt of plnt leves S. No. Leves extrt Perentge of Inhiition Peniillium oxlium 1 Bnn lef extrt 40 10 2 Guv lef extrt 25 14 3 Ppy lef extrt 25 20 4 Pomegrnte lef extrt 15 10 Alternri lternte Tle 4: Phytohemils present/sent in plnt extrts Phyto onstituents Bnn lef extrt Guv lef extrt Ppy lef extrt Sponins + + + + Tnnins + + + Flvonois + + + Alklois + + + + Crohyrte + + + + Terepnoi + + + Phenoli ompoun + + + + Pomegrnte lef extrt (+): Inites the presene of seonry metolite, ( ): Inites the sene of seonry metolite Fig. 4: Antiteril tivity shown y leves extrts of fruits plnts on pthogens ginst Billus sutilis (): Bnn (): Guv (): Ppy (): Pomegrnte Fig. 5: Antifungl tivity shown y rue extrts of meiinl fruit plnts ginst fungl pthogen Peniillium oxlium (): Control (): Bnn (): Guv (): Ppy (e): Pomegrnte e Fig. 3: Antiteril tivity shown y leves extrts of fruits plnts on pthogens ginst Stphyloous ureus (): Bnn (): Guv (): Ppy () Pomegrnte Determintion of totl phenol The mount of totl phenol ws etermine with the Folin-Ciolteu regent. The lirtion urve showe linerity for glli i in the rnge of 3.75-30 mg/ml, with orreltion oeffiient (R 2 ) of 0.9992 (Figs. 6-8). Leves of pomegrnte ontine the highest ontent of phenolis (104±2.0 mg/g), followe y nn (92.54±1.2 mg/g), guv (79.8±4.6 mg/g) n ppy (65.7±2.4 mg/g) (Fig. 7). DISCUSSION Guv lef extrt foun goo ntimiroil tivity ginst nine ifferent strins of S. ureus [20]. The ntiteril tivity of guv lef extrt ws teste ginst ne eveloping orgnisms [21]. The ntimiroil effets of pomegrnte were previously stuie. Inee, it is reporte tht the rk, leves, flowers n fruits of pomegrnte re wiely use s phytotherpeuti gents in Brzil [22]. Antiteril tivity of P. grntum L. stem, queous n ifferent solvent extrts of stem of this plnt were evlute for ntiteril tivity ginst Grm-positive (Stphyloous epiermiis n Billus megtrium) Fig. 6: Antifungl tivity shown y rue extrts of meiinl fruit plnts ginst fungl pthogen Alternri lternt (): Control (): Bnn (): Guv (): Ppy (e): Pomegrnte n Grm-negtive (Proteus morgnii, Enteroter erogens n Alligenes felis) teri of ATCC strin [23]. However, the plnts iffere in their ntimiroil tivity ginst test orgnism t vrious onentrtions. It is lso reporte tht Grm-negtive teri re more suseptile to the extrts of ppy lef n stem [24]. Aoring to report the queous extrt from leves of Mus prisivr e 134

Bisht n Agrwl Asin J Phrm Clin Res, Vol 9, Issue 4, 2016, 131-136 ntimiroil, ntimlril, heptoprotetive effet, nti-oxint, ntillergi, nti-glyemi, nti-inflmmtory, woun heling, nlgesi n mny more [31]. The phytohemil nlysis of the rue ethyl ette extrts of plnt lef hs shown the presene of lmost ll the phytohemils in C. ppy leves showe the presene of lklois, whih re quite proly n importnt element in efense ginst plnt pthogens [32]. Fig. 7: Stnr lirtion urve of glli i t onentrtions of 5, 10, 20, 30, 40, 50, 60 μg/ml. Spetrophotometri etetion ws t 765 nm All leves extrt hs shown the presene of phenol s iotive metolite. The ntioxint tivity of the phenoli ompouns is minly ue to their reox properties, whih n ply n importnt role in soring n neutrlizing free rils, quenhing singlet n triplet oxygen or eomposing peroxies. It is reporte tht the ontent of thyrotoxi perioi prlysis in the peel of Chinese pomegrnte ws 249.4±17.2 mg/g tnni i equivlents/g fresh weight [33]. Previous reports showing tht the phenoli ompoun ontent of wter extrt of guv lef extrt ws higher thn in pure ethnol n pure methnol extrts [34,35]. It is foun tht exts of M. spientum vr. Sylvester is ontine signifint mount of phenols, flvonois, n pronthoyniins [36]. Phenoli ontents re prtilly responsile for ntimiroil tivity of fruits leves, orroorting the relevne of fruit leves s helthy limentry prouts s soure of ntioxint n multi-resistnt teri rug sustnes. It is reporte tht the leves of plntin nn (M. spientum vr. prisi) n e use y the trils of Western Ghts in Ini for nging uts, listers n ulers [37]. CONCLUSION Fig. 8: Totl phenoli ontent of fruit lef extrts etermine y the Folin-Ciolteu ssy n lulte s glli i equivlent in mg. Results re the verge of triplites±stnr evition spientum exhiite tivity ginst pthogeni teri suh s Stphyloous n Pseuomons speies [25]. All tive leves extrts showing potent ntiteril tivity were further etermine for their MIC y tue ilution tehnique ginst P. eruginos, E. oli, S. ureus n B. sutilis. The MIC of P. grntum queous lef extrt in the se of B. sutilis, S. ureus n Slmonell typhi were foun to e 0.36 mg/ml, 0.13 mg/ml n 0.13 mg/ml respetively [26]. Ethnol extrt of Guv leves shows highest MIC of 40 μl ws ginst E. oli [27]. Using queous extrts of C. ppy lef n sees of mture ppy fruit (2:10 weight/v) t 10:4 v/v proportion in PDA reporte no inhiition of C. gloeosporioies fter 7 ys inution ompre to ontrol [28]. Three extrts (ethnol, petroleum ether n etone) showe exellent ntifungl tivity ginst oth Aspergillus terreus n Peniillium solitum. A host of plnt extrts inluing ethnoli extrts of pomegrnte showe ntifungl tivity ginst Cni lins [29]. The previous stuy emonstrte tht the etone extrts of leves of ove plnts showe moerte to strong tivity ginst Aspergillus niger, Fusrium sp., n C. lins [30]. Chemil nlysis ws rrie out on the plnt lef extrts to etermine the presene of hemil omponents s prospetive soure for meiinl n inustril use. Their presene is n initor tht they n e exploite s preursors in the evelopment n vnement of syntheti rugs. The tive metolites ontin hemil groups suh s phenols, flvonois, terpenois, lklois, tnnins, rohyrtes, n sponins. Guv lef extrts ontin flvonois, tnnins, α-pinene, limonene, phenoli ompouns, sponins n mny other importnt ompouns, n these ompouns show very useful n importnt properties suh s nti-irrhel, ntiteril, ntiner, The finings of this investigtion suggest tht the ethyl ette extrtion ws suitle to ientify the vrious phytohemils of meiinl plnts n they supporte y mny investigtion. The rue ethyl ette extrt of fruit plnt showe signifint ntimiroil tivity ginst test strins. This inite the gret potentil of these plnt extrts s effetive ntimiroil gents tht n e use s single or in omintion in meiines. 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