Journal of Chemical and Pharmaceutical Research, 2013, 5(2): Research Article

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1 Aville online Journl of Chemil nd Phrmeutil Reserh, 213, 5(2): Reserh Artile ISSN : CODEN(USA) : JCPRC5 In vitro ntioxidtive nd inhiitory tions of phenoli extrt of some tropil green lefy vegetles on key enzymes linked to type 2 dietes nd hypertension 1 J. A. Sliu.* nd 2 G. Ooh. 1 Deprtment of Biohemistry, Adekunle Ajsin University, Akung Akoko, Ondo Stte 2 Biohemistry Deprtment, Federl University of Tehnology, Akure, Ondo Stte ABSTRACT Polyphenolis found in vegetles exhiit wide rnge of iologil tivities s result of their ntioxidnt properties. This study sought to determine the ntioxidtive nd inhiitory tions of phenoli extrt of some tropil green lefy vegetles ( Telfri oidentlis,celoi rgenti, Oimum grtiimum, Strutium sprejnophor, Corhorus olitorius nd Tlinum tringulre) on key enzymes linked with type 2 dietes nd hypertension. Free polyphenols of these vegetles were extrted y using 8% etone. The phenoli ontent, flvonoid ontent, ferri reduing pity, totl ntioxidnt pity nd lipid peroxidtion in pnres were susequently determined. Enzyme inhiition ssys of α mylse, α-gluosidse nd ngiotensin I- onverting enzyme were lso ssessed. The results of the study reveled tht the free polyphenoli extrt of Corhorus olitorius hs the highest flvonoid nd phenoli ontents (2.9mg/1g nd 1.79mg/g respetively) ompred to others. All the free phenoli extrts of these vegetles demonstrted strong ABTS-free rdil svenging ilities. The ferri reduing pity is highest in Strutium sprejnophor. Oimum grtiimum hs the lowest mlondidelhyde produed.in the lipid peroxidtion of pnres. There ws mild inhiitory tivity of the vegetle extrts on α mylse. However, Corhorus olitorius hs the highest inhiitory tivity (9.95%). All the vegetles tested showed strong inhiition ginst α-gluosidse nd ngiotensin-1-onverting enzymes. However, Corhorus olitorius still showed the strongest inhiition. Keywords: Polyphenolis, vegetles, α mylse, α-gluosidse, ngiotensin I- onverting enzyme, dietes, hypertension. INTRODUCTION Type 2 dietes mellitus (T2DM) hs een reognized s one of the most ommon metoli disorders nd the world prevlene of dietes mong dults (ged2 79yers) is 6.4%, ffeting 285million dults, in 21, nd will inrese to7.7%, nd 439million dults y 23 [1]. Hyperglyemi, ondition hrterized y n norml postprndil inrese of lood gluose level, hs een linked to the onset of type 2 dietes nd ssoited oxidtion-linked vsulr omplitions. Hyperglyemi is the most ommon feture of this disorder. Drugs involved in dietes mngement inlude α- mylse nd α-gluosidse inhiitors, whih re orl nti-dieti drugs used for dietes mellitus type 2 nd work y preventing the digestion of omplex rohydrtes.[2,3]. 148

2 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): Hene, inhiition of these enzyme systems redues the rte of digestion of omplex rohydrtes resulting in redued gluose sorption euse the rohydrtes re not roken down into gluose moleules. However, high inhiition of pnreti α-mylse ould result in the norml teril fermenttion of undigested strh in the olon, nd therefore mild α-mylse inhiitory tivity is useful [4]. Therefore, nturl inhiitors from dietry plnts re useful s they hve lower inhiitory tivity ginst α-mylse nd stronger inhiitory tivity ginst α - gluosidse nd n e used s effetive therpy for postprndil hyperglyemi with miniml side effets [5]. Free rdils hve een implited in the development nd omplitions of dietes in numer of wys; the white lood ell prodution of retive oxygen speies medites the immune destrution of the et ells in the islets of lngerhns in the pnres [6]. Furthermore, normlities in trnsition metl metolism re postulted to result in the estlishment of dietes [7]. Dietes-ssoited hyperglyemi uses intrellulr oxidtive stress, whih ontriutes to vsulr dysfuntion [8]. Phenolis hve een shown to hve high ntioxidnt tivity nd ertin therpeuti properties, inluding ntidieti nd ntihypertensive tivity [5]. Fruits nd vegetles re the mjor soure of dietry polyphenoli ntioxidnts. Vegetles ontin ompounds tht re vlule ntioxidnt nd protetnts, the min protetive tion of vegetles hve een ttriuted to the presene of ntioxidnts, espeilly ntioxidnt vitmins inluding sori id, α-toopherol, β-rotene nd phenolis [9, 1, 11 ]. However, numerous studies hve onlusively shown tht the mjority of the ntioxidnt tivity my e from ompound suh s flvonoids, isoflvone, flvones, nthoynin, tehin nd isotehin rther thn vitmin C, E nd β rotene [1,11]. Severl green lefy vegetles ound in tropil Afri tht is utilized s either ondiments or spies in humn diets. These vegetles ould e hrvested t ll stges in the proess of growth, nd ould e fed upon in fresh, proessed, or semi proessed forms.. Due to the derth of informtion on these vegetles vis-à-vis their therpeuti uses, It is therefore hypothesised tht the rrys of nutrient nd non-nutrient phytohemils in these lefy vegetles ould e hrnessed in the mngement of type 2 dietes nd hypertension whih neessitted this study. EXPERIMENTAL SECTION Smple olletion Fresh green lefy vegetles [Struhium sprgnophor (Wter itterlef), Amrnthus ruentus (Amrnth), Telfiri oidentlis (Fluted pumpkin), Oimum grtissimum (Wild sil), Tlinum tringulre(wter lef), Cnidosolous onitifolius (Chy) nd Vernoni mygdlin (Bitter lef) nd Corhorus olitorius (Jute) leves] were olleted in vrious frm settlements in South-western Nigeri. They were uthentited in the Deprtment of Crop, Soil & Pest Mngement of Federl University of Tehnology Akure, Nigeri. Susequently the edile portions of the vegetles were seprted from the inedile portion, then the edile portion ws hopped into smll piees efore sundrying nd milling. The milled vegetles were kept in the refrigertor for susequent nlysis. Extrtion of free solule phenols The free solule phenolis of vegetles were extrted out using the method reported y [9]. Briefly, 5g of the vegetles ws homogenized with 8% etone (1:2 w/v) in hilled lender for 5 minutes. Then the smples were homogenized further for 3 minutes to otin thoroughly homogenized smple. The homogentes were filtered (filter pper Whtmn no. 2) under vuum. The filtrte ws then evported using rotry evportor under vuum t 45 C until out 9% of the filtrte hd een evported. The phenoli extrts were frozen t -4 C until further nlysis. Determintion of totl phenol ontent The totl phenol ontent ws determined ording to the method of [12]. Briefly, pproprite dilutions of the extrts were oxidized with 2.5ml 1% Folin-Ciolteu s regent (v/v) nd neutrlized y 2.ml of 7.5% sodium ronte. The retion mixture ws inuted for 4minutes t 45 o C nd the sorne ws mesured t 765nm in the spetrophotometer. The totl phenol ontent ws susequently lulted s glli id equivlent. Determintion of totl flvonoid ontent The totl flvonoid ontent ws determined using slightly modified method reported y [13], riefly.5ml of ppropritely diluted smple ws mixed with.5ml methnol, 5µl 1% AlCl 3, 5µl 1M Potssium ette nd 1.4ml wter, nd llowed to inute t room temperture for 3min. The sorne of the retion mixture ws 149

3 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): susequently mesured t 415 nm; the totl flvonoid ontent ws susequently lulted. The non-flvonoid polyphenols were tken s the differene etween the totl phenol nd totl flvonoid ontent. In vitro Antioxidnt Studies 2,2'-zino-is(3-ethylenzthizoline-6-sulphoni id) (ABTS) Rdil Svenging Aility The ABTS* svenging ility of the extrts were determined ording to the method desried y [14]. The ABTS* ws generted y reting n (7 mmol/l) ABTS queous solution with K 2 S 2 O 8 (2.45 mmol/l, finl onentrtion) in the drk for 16 h nd djusting the As734nm to.7 with ethnol..2ml of pproprite dilution of the extrt ws dded to 2.ml ABTS* solution nd the sorne were mesured t 734nm fter 15mins. The trolox equivlent ntioxidnt pity ws susequently lulted. Determintion of reduing property The reduing property of the extrts ws determined y ssessing the ility of the extrt to redue FeCl 3 solution s desried y [15]. 2.5ml liquot ws mixed with 2.5 ml 2 mm sodium phosphte uffer (ph 6.6) nd 2.5 ml 1% potssium ferriynide. The mixture ws inuted t 5 o C for 2 min. nd then 2.5 ml 1 % trihloroeti id ws dded. This mixture ws entrifuged t 65 rpm for 1 min. 5 ml of the superntnt ws mixed with n equl volume of wter nd 1 ml.1% ferri hloride. The sorne ws mesured t 7 nm. The ferri reduing ntioxidnt property ws susequently lulted. Lipid peroxidtion ssy Preprtion of Tissue Homogentes The rts were depitted under mild diethyl ether nesthesi nd the pnres ws rpidly isolted nd pled on ie nd weighed. This tissue ws susequently homogenized in old sline (1/1 w/v) with out 1-up-nd down strokes t pproximtely 12 rev/min in Teflon glss homogenizer. The homogente ws entrifuged for 1 min t 3xg to yield pellet tht ws disrded, nd low-speed superntnt (S1) ws kept for lipid peroxidtion ssy [16]. Lipid Peroxidtion nd Thioriutri Aid Retions The lipid peroxidtion ssy ws rried out using the modified method of [17], riefly 1µl S1 frtion ws mixed with retion mixture ontining 3µl of.1m ph 7.4 Tris-HCl uffer, extrt ( - 1µl) nd 3µl of 25µM freshly prepred FeSO 4. The volume ws mde up to 3µl y wter efore inution t 37 o C for 1h. The olour retion ws developed y dding 3µl 8.1% SDS (Sodium doudeyl sulphte) to the retion mixture ontining S1, this ws susequently followed y the ddition of 6µl of eti id/hcl (ph 3.4) mixture nd 6µl.8% TBA (Thiorituri id). This mixture ws inuted t 1 o C for 1h. TBARS (Thiorituri id retive speies) produed were mesured t 532nm nd the sorne ws ompred with tht of stndrd urve using MDA (Mlondildehyde). Enzyme Inhiition Assys α-amylse inhiition ssy Approprite dilution of the phenoli extrts (5µl) nd 5 µl of.2m sodium phosphte uffer (ph 6.9 with.6 M NCl) ontining Hog pnreti α-mylse (EC ) (.5mg/ml) were inuted t 25 C for 1 minutes. Then, 5µl of 1% strh solution in.2 M sodium phosphte uffer (ph 6.9 with.6 M NCl) ws dded to eh tue. The retion mixtures ws inuted t 25 C for 1 minutes nd stopped with 1. ml of dinitrosliyli id olour regent. Therefter, the mixture ws inuted in oiling wter th for 5 minutes, nd ooled to room temperture. The retion mixture ws then diluted y dding 1 ml of distilled wter, nd sorne mesured t 54 nm [18]. α-gluosidse inhiition ssy Approprite dilution of the phenoli extrts (5µl) nd 1 µl of α-gluosidse solution (1. U/ml) in.1 M phosphte uffer (ph 6.9) ws inuted t 25 C for 1 min. Then, 5 µl of 5 mm p-nitrophenyl-α-dgluopyrnoside solution in.1 M phosphte uffer (ph 6.9) ws dded. The mixtures were inuted t 25 C for 5 min, efore reding the sorne t 45 nm in the spetrophotometer [19]. The α-gluosidse inhiitory tivity ws expressed s perentge inhiition. 15

4 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): Angiotensin I onverting enzyme (ACE) inhiition ssy The Angiotensin I- onverting enzyme (ACE) inhiitory tivity ws mesured ording to the priniples of [2]. ACE solution (5µl, 4 mu) ws preinuted with eh phenoli extrt (5µl) for 15 min t 37 o C. The enzymti retion ws initited y dding 15µl of 8.33 mm of the sustrte Bz Gly His Leu in 125 mm Tris HCl uffer (ph 8.3) to the mixture. After inution for 3 mins t 37 o C, the retion ws rrested y dding 25µl of 1M HCl. The Gly His ond ws leved nd the Bz Gly produed y the retion ws extrted with 1.5 ml ethyl ette. Therefter the mixture ws entrifuged to seprte the ethyl ette lyer; then 1 ml of the ethyl ette lyer trnsferred to len test tue nd evported. The residue ws redissolved in distilled wter nd its sorne mesured t 228 nm ginst series of stndrds. The ACE inhiitory tivities will expressed s perentge inhiition. Dt Anlysis The results of the three replites were pooled nd expressed s men ± stndrd error (S.E.). Student t-test, onewy nlysis of vrine (ANOVA) nd the lest signifine differene (LSD) were rried out[21]. Signifine ws epted t p.5. RESULTS AND DISCUSSION Studies on the reltionship of phenoli ontents, ntioxidnt properties nd enzyme inhiitory tivities of plnts foods, in order to survey the nutreutil signifine of plnt foods re numerous [22, 23, 24]. Therefore, the phenoli ontent ws determined. The totl phenoli ontent of the vegetles ws shown in figure 1. The totl phenoli ontent vries in the vegetles. The result showed tht OG nd CO hs the highest totl phenoli ontents when ompred to others. However, TT hs the lowest totl phenoli ontent. Totl phe noli ontent(mg/1g) d d Vegetles Figure 1: Totl phenol ontents of free phenols of some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis,ca=celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre Flvonoids re the most undnt polyphenols reported to possess ntioxidnt tivity in plnt foods [25]. The totl flvonoid ontent ws shown in figure 2. There ws signifint vrition in the flvonoid ontent of the vegetles.co lso hs the highest totl flvonoid ontent while SS hs the lowest. 151

5 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): The totl ntioxidnt pity of the extrts, funtion of the rdil svenging ility of the extrts, ws studied using modertely stle nitrogen-entered rdil speies, 4 Totl flvonoid ontent(mg/1g) d e Vegetles Figure 2: The totl flvonoid ontent of some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis,ca=celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre 1 ABTS svenging ility(mmolteac/g) d d d Vegetles Fig.3: ABTS* svenging ility of some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis,ca=celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre 152

6 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): ABTS+[14].The result in figure 3 reveled tht the free phenoli extrts of the vegetles exhiited svenging ility on ABTS rdil. However, TT showed the lowest svenging ility.this grees with the phenoli ontents of these vegetles. Ferri reduing ntioxidnt property(mgaae/g) Vegetles Fig.4:Ferri reduing ntioxidnt properties of some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis, CA=Celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre. % MDA produed f d e de de I B Vegetles Fig.5:Inhiition of lipid peroxidtion y some green lefy vegetles Brs with different letters re signifintly different from the others t P<.5. I=Indued,B=Bsl, TO=Telfri oidentlis,ca=celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre. 153

7 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): % α-mylse inhiition Vegetles. Fig.6:Inhiition of α mylse tivity y some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis,ca=celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre. %α-gluosidse inhiition Vegetles Fig.7: Inhiition of α-gluosidse tivity y some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis, CA=Celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre. Reduing power is potent ntioxidnt defense mehnism whih is sed on either eletron trnsfer or/ nd hydrogen tom trnsfer y the ntioxidnt moleule[26]. 154

8 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): Ferri reduing ntioxidnt properties of some green lefy vegetles ws shown in figure 4. The result reveled tht only SS hs etter ferri reduing ntioxidnt property while others showed less effet. Inthe pnres, Fe 2+ umultes in inr ells nd in the islets of Lngerhns, therey resulting in the destrution of et-ells ssoited with DM[27]. The ility of the phenoli extrts to inhiit Fe2+-indued lipid peroxidtion in rt s pnres ws investigted. The results lerly showed tht inution of the rt pnres in the presene of Fe 2+ used signifint inrese (P<.5) in the MDA ontents of the rt pnres (131%) when ompred with the sl pnres homogente (1.%). The inresed lipid peroxidtion in the presene of Fe 2+ ould e ttriuted to the ft tht Fe 2+ n tlyze one-eletron trnsfer retions tht generte retive oxygen speies. The inution of rt pnres in presene of Fe2+used signifint inrese (P<.5) in MDA ontent; however, ll the extrts exhiited inhiitory effets on pnreti MDA produed. This inhiition ould e ttriuted to the phenoli phytohemils present in the extrts; whih ould hve formed omplexes with the Fe(II), therey preventing them from tlyzing the initition of lipid peroxidtion. Figure 5 showed the inhiition of lipid peroxidtion y some green lefy vegetles.there ws mild redution of mlondindehyde produed however, OG seems to show the highest redution of MDA. Inhiition of α mylse tivity y some green lefy vegetles s shown in figure 7 depited tht CO hs the highest lph mylse inhiition. % Angiotensin-1-onverting enzyme inhiition Vegetles Fig.8: Inhiition of Angiotensin-1-onverting enzyme tivity y some green lefy vegetles. Brs with different letters re signifintly different from the others t P<.5. TO=Telfri oidentlis,ca=celoi rgenti, OG=Oimum grtiimum, SS=Strutium sprejnophor,, CO=Corhorus olitorius nd TT=Tlinum tringulre. Fig.8 shows the inhiition of α-gluosidse tivity y some green lefy vegetles.ca shows the highest inhiitory tivity while TT shows the lowest inhiitory tivity on α-gluosidse. These results(figure 6 nd figure7) show tht there is higher inhiition of lph gluosidse thn their respetive lph-mylse inhiitory tivities. This result grees 155

9 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): with previous works tht plnt phytohemils re mild inhiitors of lph-mylse nd strong inhiitors of lphgluosidse tivities[22,23,28]. Therefore, stronger inhiition of lph-gluosidse tivity nd mild inhiition of lph-mylse tivity exhiited y the extrts my e of gret nutreutil importne, in minimizing some of the side effet (suh s dominl distention, fltulene,meteorism nd possily dirrhe) ssoited with the drugs (Arose nd Vogliose), presently used for the mngement of dietes. It n therefore e suggested tht the inhiition of lph-mylse nd lph-gluosidse tivities ould e prt of the possile mehnisms involved in the use of green lefy vegetles in therpeuti/dietry mngement of dietes, y retrdtion of strh hydrolysis in the gstrointestinl trt sine polyphenols suh s flvonoid hve potent lph-gluosidse inhiitory tivities[24]. As shown in figure 8, ll the vegetle extrts demonstrted strong inhiition ginst ngiotensin-1- onverting enzyme. Angiotensin-I- onverting enzyme (ACE) leves ngiotensin I to produe ngiotensin II, powerful vsoonstritor tht hs een identified s mjor ftor in hypertension [29]. As result, ACE inhiitors hve een widely developed to prevent ngiotensin II prodution in rdiovsulr diseses, nd these hve een utilized in linil pplitions sine the disovery of ACE inhiitors in snke venom[3]. Hene, this finding revels possile ntihypertensive potentils of these polyphenoli rih-extrts of these vegetles in folk mediine. Tken together ll these ntioxidnt potentils nd inhiitions of key enzymes linked to dietes nd hypertension y the free polyphenol-rih extrts of these vegetles, our findings hve shown tht these vegetles ould serve s soures of heper nd sfer lterntives to the syntheti drugs tht re in use now for the mngement of oxidtive stress indued diseses espeilly in type 2 dietes nd hypertension. REFERENCES [1] JE Shw; RA Siree; PZ Zimmet. Dietes Res. Clin. Pr,, 21, 87(1),4-14. [2] MI Hrris; P Zimmer. Interntionl Textook of Dietes Mellitus,1 st Edition, John Wiley, London, 1992; [3] T Nishikw, D Edelstein ; XL Du, S Ymgishi; T Mtsumur ;Y Kned, MA Yorek;D Beee;PJ Otes;HP Hmmes; I Girdino;M Brownlee. Nt.., 2,44(), [4] S Horii; K Fuksse; T Mtsu; K Kmed,; N Asno; Y Msui.. J. Med. Chem..,1987, 29(), [5] YI Kwon;HD Jng; K Shetty. Asi P. J. Clin. Nutr., 26,15, [6] LW Oerley. Free Rdi. Biol., 1988,.5, [7] SP Wolff..Br Med Bull., 1993, 49, [8] JW Bynes., Dietes, 1991,4: [9] Y Chu; J Sun; X Wu; RH Liu..J Agri Food Chem., 22, 5(23), [1] G Ooh; JBT Roh. Antioxidnt in foods: A new hllenge for food proessors: Leding edge ntioxidnts reserh. New York: Nov Siene Pulishers In.; 27; [11] G Ooh; H Rddtz; T Henle Antioxidnt properties of polr nd non-polr extrts of some tropil green lefy vegetles. J. Si. Food Agri. 28, 88(14), [12] VL Singleton;R Orthofer;RM Lmuel-Rventos. Met. Enzym. 1999, 299: [13] A Med;CE Lmien;M Romito;J Millogo;OG Noulm. Food Chem. 25, 91, [14] R Re ; N Pellegrini; A Proteggente;A Pnnl;M Yng ;C Rie-Evns. Free Rd Biol Med. 1999, 26(9-1), [15] M Oyizu. Jpn J Nutr. 1986, 44, [16] NAV Belle ;GD Dlmolin; G Fonini; MA Ruim; JBT Roh. Brin Res.,24,18(2), [17] H Ohkw; N Ohishi; K Ygi. Anl Biohem., 1979, 95(2): [18] V Worthington. Alph mylse. In: Worthington Enzyme Mnul.Worthington Biohemil Corp,New Jesey Freehold; 1993, [19] E Apostolidis;YI Kwon,K Shetty. Innov. Food Si. Emerg., 27,8, [2] DW Cushmn;HS Cheung. Biohem. Phrm., 1971, 2, [21] JH Zr. Biosttistil nlysis. Upper Sddle River: Prentie-Hll, In.; 1984; 62. [22] YI Kwon;E Apostolidis; YC Kim;K Shetty. J Med Food 27, 1(2): [23] G Ooh; AJ Akinyemi; AO Ademiluyi; SA Adefegh. J Food Nutr Res. 21, 49(1), [24] G Ooh;AO Ademosun.Riv Itl Sostnze Gr.211,88(1), [25] T Rong.Nutr., 21, 2(12), [26] A Villr;M Py;MC Terenio;. Fitot.,1986,57,

10 J. A. Sliu et l J. Chem. Phrm. Res., 213, 5(2): [27] R Pulido;L Brvo; F Sur-Clixto. J Agri Food Chem., 2; 48(8), [28] SV Shh; VA Fonse. Dietes Cre 211,34(7), [29] LG Rnill;YI Kwon ; E Apostolidis;K. Shetty.Bioresour Tehnol 21, 11(12), [3] I Ahnfelt-Ronne Enzyme inhiitors s drugs. In A Textook of Drug Design nd Development (P.Krogsgrd-Lrsen nd H. Bundgrd, eds.), Hrvood Ademi Pulishers, Chur, Switzerlnd. 1991,