Research Note Production of astragaloside and flavones from adventitious root cultures of Astragalus membranaceus var. mongholicus

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1 POJ 5(5): (212) ISSN: Reserh Note Proution of strglosie n flvones from ventitious root ultures of Astrglus memrneus vr. mongholius Aye Aye Thwe 1, Nguyen Thi Thnh Mi 1, Xiohu Li 1, Yeji Kim 1, Yeon Bok Kim 1, M. Romij Uin 1, Young Seon Kim 2, Hnhong Be 3, Heng Hoon Kim 4, Mi Young Lee 2,* n Sng Un Prk 1,* 1 Deprtment of Crop Siene, College of Agriulture & Life Sienes, Chungnm Ntionl University, 79 Dehngno, Yuseong-gu, Dejeon, , Kore 2 TKM-Bse Herl Drug Reserh Group Reserher, Kore Institute of Orientl Meiine, 1672 Yuseongero, Yuseong-gu, Dejeon, , Kore 3 Shool of Biotehnology, Yeungnm University, Gyeongsn , Kore 4 Deprtment of Well-eing Resoures, Sunhon Ntionl University, 413 Jungngno, Sunheon, Jeollnm-o, , Kore * Corresponing uthors: Mi Young Lee (mylee@kiom.re.kr), Sng Un Prk (suprk@nu..kr) Astrt Mny plnts ontin vrious seonry metolites tht hve meiinl vlue. Their ontent vries ross plnt prts. We rrie out in vitro ventitious root inution from lef explnts of Hung-qi (Astrglus memrneus) using ifferent nutrient mei supplemente with vrious plnt hormones. The level of strglosie n flvones were nlyze from the ventitious roots of A. memrneus grown uner these ifferent mei onitions. Among the ifferent mei n plnt hormones, Murshige n Skoog meium supplemente with 1. mg/l 1-nphthleneeti i resulte in the gretest egree of ventitious root inution. The highest onentrtion of lyosin (11.7 µg/g ry weight), lyosin-7-glu (13.3 µg/g ry weight), n gluorphnin (241.9 µg/g ry weight) were oserve in roots grown in hlf-strength Shenk & Hilernt minerl solution, hlf-strength Gmorg s B5 meium, n full-strength Gmorg s B5 mei, respetively. Keywors: ventitious root, strglosie, Astrglus memrneus, flvones. Arevitions: IAA- inole-3-eti i; IBA- inole-3-utyri i; B5- Gmorg s B5; HPLC- high-performne liqui hromtogrphy; MS- Murshige n Skoog; NAA- 1-nphthleneeti i; SH- Shenk & Hilernt. Introution Astrglus memrneus (Fish.) Bge. or Astrglus mongholius Bge. (Fee) re perennil flowering plnts ntive to the northern, north-estern, n north-western prts of Chin, (M et l., 2). Astrglus memrneus is lso known s Hung-qi or yellow leer, n the root hs een use in tritionl Chinese meiine for thousns of yers to tret vrious iseses. The root is onsiere toni to enhne metolism n igestion, to strengthen the immune system, n promote the heling of wouns n injuries. It is si to prevent ner, nemi, ietes, heptitis, n liver n hert iseses. It is often use s n ntiperspirnt, immunostimulnt, iureti, n s supplementry meiine uring ner therpy (Wgner et l., 1997; Zheng, 25). A. memrneus lso hs ntiteril, ntivirl, n nti-inflmmtory properties. Moreover, it ontins ntioxints tht prevent ell mge use y free rils. Extrts of A. memrneus ontin mny vlule plnt onstituents, suh s triterpenoi sponins (etylstrglosies, strglosies, n strgenol), mino is, flvonois, isoflvonois, n polyshries. Among these, strglosie is generlly onsiere to e the primry tive ingreient in A. memrneus extrt n is well known for its nti-ging properties. Astrglosie IV (Fig. 1-A) is the mjor tive omponent extrte from A. memrneus n is use in the tretment of mny isorers, inluing riovsulr iseses. Astrglosie IV hs een shown to hve protetive effets ginst ishemi injury in the myorium n entrl nervous system (Zhng et l., 26; Luo et l., 24; Zhou et l., 2; Qu et l., 29). Furthermore, it hs shown promise s nturl prout with oth heling n nti-srring effets for woun tretment. These results provie support for the pplition of strglosie IV in the tretment of injuries (Chen et l., 212). Moreover, lyosin (Fig. 1-B) n lyosin-7-o-β-d-gluosie (lyosin-7-glu; Fig. 1-C) re 2 mjor isoflvones relte to the iotivity of the her (To n Shirtki, 1998; Wu et l., 2). Clyosin hs een shown to protet enothelil ells from hypoxi-inue rrier impirment (Fn et l., 23). Clyosin-7- β-d-gluosie ppers to e potentil nturl nti-inflmmtory n nti-osteorthriti gent n might e use effetively to protet ginst proteoglyn egrtion (Choi et l., 25) n s hyluronise inhiitory omponent (Lee et l., 25). It hs een propose tht these two ompouns oul e use s mrker ompouns for the hemil evlution or prout stnriztion of A. memrneus (Nkmur et l., 1999). The roots of numerous plnt fmilies re the site for iosynthesis or umultion of mjor seonry metolites, inluing lklois, polyetylene, sesquiterpenes, n npthoquinones. In vitro root ulture is n lterntive metho for the proution of vlule 466

2 seonry metolites on ommeril sle. Aventitious roots, inue y in vitro methos, show high rte of prolifertion n tive seonry metolism (Hhn et l., 23; Yu et l., 25). They grow vigorously in phytohormone-supplemente meium n hve shown tremenous potentil for umultion of vlule seonry metolites (Murthy et l., 28). The evelopment of n effiient metho for inution n estlishment of ventitious A. memrneus root ultures, s well s ientifition of onitions for optiml flvonol proution in this plnt, is wrrnte for the effiient utiliztion of this meiinl plnt. The ojetives of this stuy were to fin out suitle meium with pproprite uxin onentrtion n nlysis of strglosie n flvones from ventitious root ultures of A. memrneus. Fig 1. Chemil struture of strglosie IV (A), lyosin (B), n lyosin-7-o-β--gluosie (C). Results Aventitious root inution Lef explnts were ulture on MS meium supplemente with vrious uxins, suh s IAA, IBA, n NAA. After 6 weeks, the ventitious roots, pproximtely 2%, were proue only on MS meium supplement with 1. mg/l NAA (Fig. 2-A). No ventitious roots were oserve with other mei n hormone omintions (t not shown). Emerging roots were then further ulture in Erlenmeyer flsks ontining MS liqui meium supplemente with 1. mg/l of NAA (Fig. 2-B). Effet of ifferent mei on ventitious root growth of A. memrneus Roots were seprte from the soli meium n inoulte into NAA-supplemente MS liqui meium. Culture flsks were mintine uner rk onitions n serve s soures of inoulums for further experiments. Aventitious roots were ulture y growing the root inoulums in vrious onentrtions of MS, B5, n SH liqui mei, fortifie with 1. mg/l NAA n 3% (w/v) surose (Fig. 3). Among the mei teste, MS meium sustine etter root growth (455.7 mg), followe y ½ MS, ½ SH, ½ B5, B5, n SH mei. These results inite tht MS meium ws the est for ventitious root inution n tht this metho n e useful for the lrge-sle proution of A. memrneus. Chemil nlysis for flvones n strglosie ompoun in A. memrneus Among the ifferent flvonois in the roots of A. memrneus, we ssesse the ontent of lyosin n lyosin-7-glu in sl meium (Fig. 4). The lyosin ontent ws foun to e the highest in ½ SH meium (11.7 µg/g ry weight), followe y tht in ½ B5, ½ MS, SH, n B5 mei, while MS meium h the lowest ontent (3.8 µg/g ry weight). Levels of lyosin-7-glu were the highest in roots grown in ½ B5 meium (13.3 µg/g ry weight), while those of roots grown in SH meium were the lowest (7. µg/g ry weight). However, one of the strglosie ompouns, gluorphnin, ws more onentrte in roots grown in B5 meium (241.9 µg/g ry weight), followe y those grown in MS, ½ B5, ½ MS, n SH mei, n the lowest (183.6 µg/g ry weight) in roots grown in ½ SH meium (Fig. 5). Fig 2. Aventitious root inution n ulture of Astrglus memrneus vr. mongholius. A: Aventitious rootinution from lef explnts ulture on MS meium ontining 1 mg/l NAA fter 6-week ulture. B: Aventitious root ulture in MS meium ontining 1 mg/l NAA fter 3-week ulture. Dry weight (mg) Bsl meium Fig 3. Effet of mei on growth of ventitious roots of Astrglus memrneus vr. mongholius fter 3-week ulture. Vlues represent LSD (p=.5). Disussion Lef explnts were ulture on MS sl meium with ifferent onentrtions n omintions of uxins. MS sl meium supplemente with 1. mg/l NAA ws foun to e the est for ventitious root inution. This result ws onurre with the finings of Kitto n Young (1981), whih inite tht mong ommon uxins, NAA is the most effetive uxin for inuing root regenertion. Therefter, the prolifertion of ventitious roots ws teste using 6 ifferent onentrtions of 3 ifferent sl mei supplemente with 1. mg/l of NAA. 467

3 Agin, MS meium supplemente with 1 mg/l NAA ws oserve to e the est for ventitious root growth mong the ifferent mei teste. This onfirme tht MS meium is the most suitle sl meium for ventitious root inution of A. memrneus. The sme result ws lso reporte y Khlfll et l., (29) in in vitro fst-growing norml root ulture of Vernoni myglin. The stimultory effet of sl meium on ventitious root inution n root qulity hs lrey een reporte (Bskrn n Jyln, 25). Similrly, in Bupleurum fltum ventitious root ultures, full strength MS meium ws foun to e suffiient for oth root evelopment n sikosponin proution (Kuskri et l., 2). Amzllg et l. (1992) reporte tht the promoter effet of minerl onentrtion of the ulture meium on rooting oul e ttriute to the prtiiption of inorgni ions in proesses regulting hormonl lne. Sometimes, high levels of uxin re eleterious to seonry metolite proution in plnts (Dornenurg n Knorr, 1995; Chn et l., 25). Wu et l. (26) emonstrte tht inresing the NAA onentrtion h negtive effet on iomss, phenol n flvonoi ontents in ventitious roots of Ehine ngustifoli. However, the response of ventitious roots to ifferent uxins epens on the plnt speies. For exmple, tretment with IBA is more effetive thn NAA in promoting iomss proution from root ultures of Pnx ginseng (Kim et l., 23). In ontrst, NAA is etter t inuing the elongtion of tomto lterl roots (Tylor n vn Sten, 1998). In the present stuy, roots grown on MS meium h low flvone ontent, even though this meium proue the gretest growth of ventitious roots when supplemente with 1. mg/l of NAA (Fig. 3). Moreover, the flvone n strglosie ontents vrie wiely, epening on the meium use. In this stuy, ½ SH, ½ B5, n B5 meium resulte in the highest onentrtion of lyosin, lyosin-7- glu, n strglosie, respetively. Aoring to this fining, Astrglus memreus requires high nutrient onentrtions whih re ritil eterminnt in ontrolling the growth of ventitious roots. Therefore, in this stuy, the ifferene in rooting ility etween sl mei might e ue to their sl slt formultion n the low numer of roots otine on explnt ulture on B5 n SH mei is proly ue to their low mmonium ontent ompre to MS meium. Kim et l. (29) emonstrte tht lyosin from A. memrneus root reues melnin proution y regulting the tyrosinse enzyme. Furthermore, it hs een suggeste lyosin mye potentil skin-whitening gent. Mterils n methos See steriliztion n germintion Sees of A. memrneus were surfe sterilize with 7% (v/v) ethnol for 3 s n 2% soium hypohlorite solution for 1 min. Sees were then rinse thoroughly with sterilize istille wter n inute on 25 ml of hormone-free Murshige n Skoog (MS) sl meium (Murshige n Skoog, 1962) in Petri ishes uner pproprite light onitions. The sl meium onsiste of minerl slts-n-vitmins supplement together with 3 g/l of surose n 8 g/l of Phytgr s the soliifying gent. The ph of the meium ws juste to 5.8 efore ing the Phytgr; mei were sterilize y utolving t 121 C for 2 min. The germinte sees were trnsferre to Mgent ox ontining 5 ml MS sl meium n mintine uner ontrolle environmentl onitions until use. Flvone (µg/g ry wt.) Bsl meium Clyosin Clyosin-7-glu Fig 4. Effet of mei on proution of flvones in ultures of ventitious roots of Astrglus memrneus vr. mongholius fter 3-week ulture. Vlues represent LSD (p=.5). Astrglosie (µg/g ry wt.) Bsl meium Fig 5. Effet of mei on strglosie proution in ventitious root ulture of Astrglus memrneus vr. mongholius fter 3-week ulture. Vlues represent LSD (p=.5). Aventitious root inution Leves (Four weeks ol) from in vitro-grown plnts were exise n ut into smll segments (.5 1. m). The exise lef segments were ulture on MS meium (Murshige n Skoog, 1962) supplemente with inole-3-eti i (IAA), inole-3-utyri i (IBA) n 1-nphthleneeti i (NAA) t.1,.5, n 1. mg/l, respetively. The ultures were kept uner rk onitions for ventitious root evelopment. After 6 weeks, the emerging roots were trnsferre to 1-mL Erlenmeyer flsks ontining 3 ml MS liqui meium with 1. mg/l of NAA for root multiplition. Then, ventitious root ulture of A. memrneus ws initite y trnsferring 2 g of the inoulum from the Erlenmeyer flsks into hlf-strength (½) MS, full-strength MS, ½ Gmorg s B5 (Gmorg et l., 1968), full-strength B5, ½ Shenk & Hilernt (SH) (Shenk n Hilernt, 1972), n full-strength SH liqui mei, eh fortifie with 1. mg/l NAA n 3% (w/v) surose. These flsks were ple on rotry shker t 1 rpm, in rk onitions, for 3 weeks. Approximtely 2 leves were use per experiment, n the experiment ws repete thrie in the sme environment. The growth rte ws mesure y tking into ount the fresh n ry weight of the roots fter 3 weeks of ulture. Fresh weight ws etermine fter ompletely removing the meium y lotting with tissue pper; roots were then ple onto pre-weighe luminum foil, weighe, n then kept t -8 C for few hours to freeze-ry the smple for hemil nlysis. 468

4 Chemil nlysis of flvones n strglosie ompouns Chemil nlysis ws rrie out y high performne liqui hromtogrphy (HPLC) nlysis. Freeze-rie smples of A. memrneus ventitious roots were groun into fine power using mortr n pestle. Two hunre milligrms of powere smples were extrte with 5 ml of 8% (v/v) ethnol t room temperture for 3 minutes. The smples were extrte for three times for the quntifition of flvones n strglosie ompouns. Then, the solvent ws evporte n 1 ml of 8 % methnol. Therefter, the extrts were entrifuge n the superntnt ws filtere with.45-μm Arois syringe filter (Pll Corp.; Port Wshington, NY), for HPLC nlysis. HPLC nlysis ws performe with C18 olumn (25 4.6mm, 5 μm; RSteh; Dejeon, Kore). The moile phse ws grient prepre from mixtures of etonitrile n.3 % formi i n the olumn temperture ws mintine t 3 C. The flow rte ws mintine t 1. ml/min. Injetion volume of 2 µl n 28 nm wvelengths were use for etetion of flvones n ELSD (Evportive Light Sttering Detetor) ws use for strglosie. The onentrtions of flvones n strglosie ompouns were etermine y using stnr urve. All smples were nlyze in triplite. Sttistil nlysis All t re given s the men n stnr evition of triplite experiments. The t were nlyze y using the omputer softwre Sttistil Anlysis System (SAS version 9.2). Tretment men omprisons were rrie out using the Lest Signifint Differene (LSD). Conlusion Aventitious root ulturing is n effiient metho for prouing useful phytomoleules. Here, we evelope the most suitle sl meium n uxin onentrtion for ventitious root inution in A. memrneus. In ition, we oul nlyze the onstitutient of flvone n strglosie from the ventitious root of A. memrneus. Although previous investigtions hve ientifie flvone n strglosie ontents from ifferent plnt prts n hiry root ultures of A. memrneus, to te, no other stuy hs yet investigte this in the ontext of in vitro ventitious root ulture using ifferent nutrient mei. Therefore, our finings provie useful sis for inresing the proution of seonry metolites in A. memrneus. Aknowlegements This work (K1111) ws supporte y the Kore Institute of Orientl Meiine (KIOM) grnt fune y the Kore government. Referenes Amzllg G, Lerner N, Poljkoff-Myer HRM (1992) Intertion etween minerl nutrients, ytokinin n gierilli i uring growth of sorghum t high NCl slinity. J Exp Bot 43: Bskrn P, Jyln N (25) Role of sl mei, ron soures n growth regultors in miropropgtion of Elipt l vlule meiinl her. KMITL Si J 5 (2): Chn LK, Dewi PR, Boey PL (25) Effet of plnt growth regultors on regenertion of plntlets from u ultures of Cymopogon nrus L. n the etetion of essentil oils from the in vitro plntlets. J Plnt Biol 48: Chen X, Peng LH, Li N, Li QM, Li P, Fung, KP, Leung PC, Go JQ (212) The heling n nti-sr effets of strglosie IV on the woun repir in vitro n in vivo. J Ethnophrmol 139: Choi S, Prk SR, Heo TR (25) Inhiitory effet of strgli rix on mtrix egrtion in humn rtiulr rtilge. J Miroiol. Biotehnol 15: Dornenurg H, Knorr D (1995) Strtegies for the improvement of seonry metolite proution in plnt ell ultures. Enzyme Miroil Tehnol 17: Fn Y, Wu DZ, Gong YQ, Zhou RY, Hu ZB (23) Effets of lyosin on the impirment of rrier funtion inue y hypoxi in humn umilil vein enothelil ells. Eur J Phrmol 481: Gmorg OL, Miller RA, Ojim K (1968) Nutrient requirement of suspension ultures of soyen root ells. Exp Cell Res 5: Hhn EJ, Kim YS, Yu KW, Jeong CS, Pek KY (23) Aventitious root ultures of Pnx ginseng, Meyer CA n ginsenosie proution through lrge sle ioretor systems. J Plnt Biotehnol 5: 1-6. Khlfll MM, Dffll HM, El-Shemy HA, Aelltef E (29) Estlishment of in vitro fst-growing norml root ulture of Vernoni myglin - potent Afrin meiinl plnt. Afrin J Biotehnol 8 (21): Kim JH, Kim MR, Lee ES, Lee CH (29) Effets of Clyosin Isolte from the Root of Astrglus memrneus on Melnin Biosynthesis. Biol Phrmeut Bull 32(2): Kim YS, Hhn EJ, Yeung EC, Pek KY (23) Lterl root evelopment n sponin umultion s ffete y IBA or NAA in ventitious root ultures of Pnx ginseng CA Meyer. In Vitro Cell Dev Biol Plnt 39: Kitto SL,Young MJ (1981) In vitro propgtion of Crrizo itrnge. Hort Siene 16: Kuskri K, Yokoym M, Inomt S (2) Enhne Proution of sikosponin in ulture roots of Bupleurum fltum L. using two-step ontrol of sugr onentrtion Plnt Cell Rep. 19: Lee YM, Choi SI, Lee JW, Jung SM, Prk SM, Heo TR (25) Isoltion of hyluronise inhiitory omponent from the roots of Astrglus memrneus Bunge Astrgli rix. Foo Si Biotehnol 14: Luo Y, Qin Z, Hong Z, Zhng XM, Ding D, Fu JH, Zhng WD, Chen J (24) Astrglosie IV protets ginst ishemi rin injury in murine moel of trnsient fol ishemi. Neurosi Lett 363: M XQ, Dun JA, Zhu DY, Dong TTX, Tsim KWK (2) Speies ientifition of Rix Astrgli (Hung-qi) y DNA sequene of its 5S-rRNA sper omin. Phytohem 54: Murshige T, Skoog F (1962) A revise meium for rpi growth n iossys with too tissue ultures. Physiol Plnt 15: Murthy HN, Hhn EJ, Pek KY (28) Aventitious roots n seonry metolism. Chinese J Biotehnol 24: Nkmur T, Hshimoto A, Nishi H, Kokuseny Y (1999) Investigtion on the mrker sustnes of rue rugs in formultions. I. Mrker sustnes for the ientifition of Astrgli Rix in kmpo n rinkle preprtions. J Phrmeut So Jp 119:

5 Qu YZ, Li M, Zho YL,Zho ZW, Wei XY, Liu JP, Go L, Go GD (29) Astrglosie IV ttenutes ererl ishemi reperfusion-inue inrese in permeility of the loo-rin rrier in rts. Eur J Phrmol 66: Shenk RU, Hilernt AC (1972) Meium n tehniques for inution n growth of monootyleons n iotyleonous plnt ell ultures. Cn J Bot 5: Tylor JLS, vn Sten J (1998) Plnt-erive smoke solutions stimulte the growth of Lyopersion esulentum roots in vitro. Plnt Growth Reg 26: To S, Shirtki Y (1998) Inhiitory effets of isoflvones in roots of Astrglus memrneus Bunge (Astrgli Rix) on lipi peroxition y retive oxygen speies. Phytother Res 12: Wgner H, Buer R, Xio PG, Chen JM, Mihler G (1997) Chinese Drug Monogrphs n Anlysis: Rix Astrgli (Hungqi); Verlg Wl Germny pp Wu CH, Dewir YH, Hnh EJ, Pek KY (26) Optimiztion of ulturing onitions for the proution of iomss n phenolis from ventitious roots of Ehine ngustifoli. J Plnt Biol 49(3): Wu DZ, Fn Y, Hen ZF, Song CQ, Hu ZB (2) Effet of flvonois isolte from Astrglus memrneus on the hnges of enothelil permeility inue y low osmolrity. Phrmol Clin Chin Mter Me 16: Yu KW, Hhn EJ, Pek KY (25) Proution of ventitious roots using ioreters. Koren J Plnt Tissue Cult 27: Zhng WD, Chen H, Zhng C, Li HL, Liu RH, Chen HZ (26) Astrglosie IV from Astrglus memrneus shows rioprotetion uring myoril ishemi in vivo n in vitro. Plnt Me 72: 4-8. Zheng XY (25) Phrmopoei of the Peoples Repuli of Chin, Vol. 1, Chinese en. Beijing: Chemil Inustry Press Zhou JY, Fn Y, Kong JL, Wu DZ, Hu ZB (2) Effets of omponents isolte from Astrglus memrneus Bunge on ri funtion injure y myoril ishemi reperfusion in rts. Chin J Chin Mter Me 25:

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