Research

HYPERICUM TRIQUETRIFOLIUM CALLUS CULTURES A POTENTIAL SOURCE OF PHENOLICS AND FLAVONOIDS

Description
HYPERICUM TRIQUETRIFOLIUM CALLUS CULTURES A POTENTIAL SOURCE OF PHENOLICS AND FLAVONOIDS
Categories
Published
of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Share
Transcript
     HYPERICUM TRIQUETRIFOLIUM   CALLUS CULTURES A POTENTIAL SOURCE OF PHENOLICS AND FLAVONOIDS Hoshyar A. AZEEZ   * ,  Kadhim . I!RAHI *** Biology Dept., Faculty of Science and Science Education, School of Science, Sulaimani University, Sulaimania, IRA, e!mail" hoshyar#$mm%yahoo.com ** Biotechnology Dept., &ollege of Sciences, Al!'ahrain University, Baghdad, IRA,  e!mail"  (adhimm)**+%yahoo.co.u(  A"s#ra$# A% &'(&rim&%# )as $o%d$#&d #o i%d$& a%d i%$r&as& #h& (rod$#io% o+ som& s&$o%dary m&#a"oi#&s i% #iss& $#r&s o+  Hypericum triquetrifolium  Trra si%- som& "io#&$h%oo-i$a a((roa$h&s.   Cas )as i%i#ia#&d o% &a+ dis$s $#r&d o% rashi&-& a%d Soo- m&dim /S0 s((&m&%#&d )i#h Thidia1ro% /TDZ0 a# #h& $o%$&%#ra#io%s 2.3, 2.45, 2.5, 4.3, or 4.5 m-   L 62  a%d i%do&676a$&#i$ a$id /IAA0 a# 3.5 m-L 62 8 $as )as i%i#ia#&d o% s#&m a%d roo# &'(a%#s o% S m&dim s((&m&%#&d )i#h 2.45 m-L 62  96"&%1y6ami%o(ri%& /!AP0 a%d 3.5 m-   L 62  o+ i%do a$&#i$ a$id /IAA0. R&s#s sho)&d #ha# #h& $om"i%a#io% o+ TDZ a# 4 m-   L 62  )i#h 3.5 m-   L 62  IAA )as #h& mos# &++&$#i:& i% i%d$i%- $as +orma#io% o% &a+ &'(a%#s. Th& sam& $om"i%a#io% )as s&d +or $as mai%#&%a%$&. HPLC )as s&d #o d&#&rmi%& #h& #y(& a%d ;a%#i#y o+ s&$o%dary m&#a"oi#&s i% $om(ariso% )i#h s#a%dards. Th& di++&r&%$&s i% (hy#o$h&mi$a $om(osi#io% amo%- &a+ /L0, s#&m /S0, a%d roo# /R0 )&r& i%:&s#i-a#&d. Th& hi-h&s# &:&s o+ (h&%oi$ $om(o%ds a%d +a:o%oids )&r& (r&s&%# i% L $om(ar&d #o S a%d R &'#ra$#s, &'$&(# #h& (rod$#io% o+ r#i% a%d hy(&rsoid i% S )hi$h )as mor& #ha% L a%d R. R a$$ma#&d ar-&r amo%#s o+ $hor-&%i$ a%d $a#&$hi% #ha% L a%d S, )hi& #h& $a++&i$ a$id a%d #a%%i$ a$id (rod$#io% i% $& ss(&%sio% $#r&s d&ri:&d +rom &a+ /LCs0 i%$r&as&d si-%i+i$a%#y ( #o 5.9 a%d <.4 +ods r&s(&$#i:&y $om(ar&d #o L a%d $as d&ri:&d +rom &a+ /LC0. P6OH6"&%1oi$ a$id, $a++&i$ a$id, ;&r$i#i% a%d #a%%i$ a$id )&r& a"s&%# i% S, )h&r&as a o+ #h&s& $om(o%ds )&r& (r&s&%# i% $as d&ri:&d +rom s#&m /SC0 a%d $& ss(&%sio% $#r&s d&ri:&d +rom s#&m /SCs0, "# i% roo#s o%y (6OH6"&%1oi$ a$id )as %o# (rod$&d. K&y )ords=  Hypericum triquetrifolium Trra, Cas, ss(&%sio% $#r&s, Ph&%oi$s, Fa:o%oids.   1  I. I%#rod$#io%  : he genus  Hypericum  encompasses appro-imately $* different species of annuals, perennials, shru/s and small trees, 0idespread all over the 0orld, /ut only si-teen species are found in Ira12 the most a/undant her/s are  Hypericum perforatum  3. and  H. triquetrifolium  urra 456. In this genus, some species have /een used since ancient times as fol( remedies and credited 0ith a long list of medicinal uses, including antiviral, antimicro/ial, antifungal, antitumor, analgesic, sedative and for the treatment of neurological disordered and depression 4)6. 7revious  phytochemical studies on  H. perforatum  and other related species have led to the isolation and identification of several groups of  phytochemicals including phenolic compounds 4chlorgenic acid, tannic acid and caffeic acid6, flavonoids 4rutin, hypersoid, iso1uercitrin, 1uercitrin, 1uercitin, and catechin6, naphtodianthrones 4hypericin and  psudohypericin6, and the phloroglucinols 4hyperforin and adhypeforin6 and essential oil 4+and $6.   he developing field of plant /iotechnology may provide a /etter alternative for the large scale production of secondary meta/olites2 the use of /iotechnological approaches, specifically plant cell and tissue cultures for the large!scale production of secondary meta/olites has so far achieved only limited success due to the lo0 and unrelia/le yields of the secondary products 486. Although significant improvements in product yields have /een achieved through conventional  /iochemical approaches and the manipulation of the culture and process factors, most applications of plant cell cultures in  /iotechnology are aimed at the production of  /ioactive secondary meta/olites. hus, in this e-periment, an attempt 0as carried out in order to increase some secondary meta/olites in tissue cultures of this vital plant using  /iotechnological means. II.a#&rias a%d &#hodsA. Pa%# ma#&ria= Seeds of  H. triquetrifolium  urra 0ere collected from intact plant during Decem/er )*5* to 9anuary )*55 from 0ild population in the locality of aslu:a, in dry roc(y soil of Sulaimania city, altitude 5*** m.a.s.l. 7lant samples 0ere dried at room temperature 4)8!); <&6 for ; days. Dried plant material 0as then pac(ed in paper /ags and (ept in a dar(, dry and cool place.   !. S&&d s#&rii1a#io% a%d S&&d -&rmi%a#io%=   hey 0ere   surface sterili=ed /y immersing in a ;*> 4v?v6 ethanol for +* sec, and then in a )*> 4v?v6 commercial /leach 48> 'a@&l6 plus *.5 0een )* 4polyo-ythylene sor/itan monolaurete6 for )8 min. su/se1uently, they 0ere 0ashed $ times 0ith sterili=ed distilled 0ater 46. Seeds 0ere germinated /y t0o methods either /y pre!soa(ing treatments 0ith different A + ,  ) S@ $ 2  doses, and D.C. /efore placing in 7etri dishes. hey 0ere soa(ed in 8*, 5**, and 58* mg3 !5 A + , 5.8>  ) S@ $  and D.C. for +* min. reated seeds 0ere placed individually in sterili=ed 7etri dishes containing moisture!retaining paper liners. 7aper liners in the 7etri dishes 0ere (ept moist throughout the germination period2 seedling 0as inoculated to test tu/es 48 cm.8 cm6 containing )8 ml of the 5?) strength S medium solidified 0ith *.G 0?v agar. &ultures 0ere su/:ected to a  photoperiod of 5?G hrs 4light?dar(6 in a gro0th cham/er. emperature 0as set at )8<&. ermination 0as measured after )* days, seeds sho0ed radical emergence and 0ere recorded as germinated 4;62 or sterili=ed seeds 0ere cultured on *.G> 40Hv6 0ater!agar medium, after     pre!soa(ing treatments. @ne seed placed in one test tu/e 48 cm  .8 cm6 containing )8 ml of 0ater!agar. &ultures 0ere maintained at )8<& under a photoperiod of 5?G hrs 4light?dar(6, and monitored every day for a period of )* days 4G6.  C. Cas i%d$#io%= Seedlings o/tained from aseptically germinated seeds 45$!)* days old6 0ere cut into 5 cm long e-plants 4leaf, stem and root6 using surgical /lade and forceps under aseptic conditions. hey 0ere inoculated into S medium 45* ml? test tu/e6 that supplemented 0ith the au-in IAA 4*.* or *.86 mg3 !5  and the cyto(inin D 4*.*, 5.*, 5.)8, 5.8, ).* or ).86 mg3 !5  for leaf e-plants. For stem and root e-plants the au-in IAA 4*.* or *.86 mg3 !5  and the cyto(inin BA7 4*.* or 5.)86 mg3 !5  0ere added to the culture medium. hose 0ere used to investigate the response of callus induction on dissected e-plants. he cultures 0ere incu/ated at )8 o & for 5?G hrs 4light?dar(6  photoperiod at light intensity of 5*** lu-. &allus fresh and dry 0eights 0ere measured after +* days 46. D. C& ss(&%sio% $#r&s=  H. triquetrifolum  cell suspension cultures 0ere initiated from )5!)G days callus cultures, appro-imately 8!5* g of callus fresh 0eight 0ere cultured in )8* ml S li1uid medium supplemented 0ith the same components as in callus maintenance medium 0ithout agar. Flas(s 0ere placed on a rotary sha(er at 5**!5)* rpm, );<& under 5? G hrs 4light?dar(6 0ith a light intensity of 5*** lu- in a gro0th room for 5$ days 46. E. S&(ara#io% a%d ;a%#i+i$a#io% o+ s&$o%dary m&#a"oi#&s=   Secondary meta/olites compounds 0ere separated from e-tracted samples for the three plant parts 4leaf, stem and root6 of  H. triquetrifolium urrra using a method descri/ed /y 4$6. A Shimadu= li1uid chromatograph 4Shimadu= corp, Jyoto, 9apan6 consisting of a 3&!)*A 1uaternary pump, a DU!)*A+ degasser, an S7D!)*Adiode array detector and a manual rheodyne in:ector 0ith a )*Kl loop 0ere used. 7ea(s 0ere identified /y comparison of their retention times 0ith those of the reference standard and UL spectra in the range of )**!G**nm. he standard curves 0ere o/tained /y plotting the  pea( areas of standard concentrations for 3   phenolic acids 4*.*8, *.5, *.), *.+, or *.8 mg ml !5 6, for flavonoids 4*.**), *.*5)8, *.*)8, *.*8, or *.5 mg ml !5 6. Standard solutions 0ere (ept in dar( at !)*<& to prevent o-idation 45*6. 73& conditions, 3& time program" +8 min., otal of mo/ile phase" 5ml?min, In:ection volume" )*Kl, emperature of column" room temp. 7ressure on pumps" 4A, B, &6 ;. M .* milpas(al 4pa6. o/ile phase" A" 0ater > ! phosphoric acid *.+>, B" Acetonitrile 5**>, &" ethanol 5**> F. E'(&rim&%#a d&si-% a%d s#a#is#i$a a%aysis= Statistical analysis 0as conducted using a completely randomi=ed design 4one!0ay6 0ith fifteen replicates for tissue culture e-periments 0ere used. he same design 0as used for sample analysis /ut 0ith three replicates /y DuncanNs multiple range test for mean comparison at 47 O *.*86 using the Statistical 7ac(age for the Social Sciences 4S7SS, version 5;6 4556. III. R&s#s a%d Dis$ssio% he standard curves and values for the studied compounds are sho0n in Fig. 5 A, B and a/le 5 0hich 0ere compared 0ith those separated from different plant parts. &omposition of secondary meta/olites that are found in the samples is reported in a/le ). a:or     phenolic compounds 4p!@!/en=oic acid, chlorgenic acid, caffeic acid, and tannic acid6 and flavonoids 4catechin, rutin, hypersoid, and 1uercitin6, 0ere identified in methanol leaf e-tracts, stems and roots 43, S, and R6 in seedlings gro0n in vitro  on P S!semi solid medium 0ithout plant gro0th regulators, in callus and cell suspension cultures 0hich derived from these parts 43&, S&, and R&6, 43&s, S&s, and R&s6 of  H. triqutrifolium  urra respectively. Differences in chemical composition among 3, S, and R 0ere found in the present study. he highest levels of phenolic compounds and flavonoids 0ere present in 3 compared to S and R e-tracts, e-cept the production of rutin and hypersoid in S 0hich 0as higher than those in 3 and R, /ut R accumulated greater amounts of cathechin and chlorgenic compounds compared 0ith the 3 and S. 3eaves produced all ma:or phenolic and flavonoid compounds that 0ere mentioned a/ove, 0hereas the p!@! /en=oic acid, caffeic acid, 1uercitin and tannic acid did not produce in S, 0hereas all of these compounds 0ere present in S& and S&s. In roots, only p!@!/en=oic acid 0as not  produced. Results indicated that 3 gro0n in vitro , dissected from seedlings had the highest level of p!@!/en=oic acid, chlorgenic acid, caffeic, rutin, hypersoid, 1uercitin and tannic acid compared 0ith 3& and 3&s. Data also e-hi/ited a significant increase in catechin in 3& 4*.GG; mg.g !5  d0t.6 compared 0ith the 3 and 3&s recording *.G;+ and *.)8* mg g !5 d0t. respectively. &affeic acid and tannic acid  production in 3&s increased significantly up to 8. and $.) folds compared 0ith those 4  0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 m in- 2 502 55 07 51 0 01 2 51 5 01 7 52 0 02 2 52 5 02 7 5m A U2 5 4 n m , 4 n m ( 1 . 0 0 )        3 .       4       7       3       /       1       0       7       5       3       3 .       5       6       9       /       1       5       8       5       0       3 .       8       6       7       /       7       9       7       2       1       7 .       7       5       0       /       8       2       7       7       7       1       9 .       6       1       1       /       6       0       3       8       4       1       9 .       8       9       6       /       1       4       6       1       2       2       0 .       1       7       1       /       7       3       6       0       2       0 .       5       6       7       /       1       0       7       6       9       4       4       2       0 .       8       0       8       /       1       4       0       3       2       1 .       2       6       3       /       1       2       1 .       8       3       1       /       1       8       2       2 .       2       7       0       /       4       2       0       9       9       2       2 .       6       1       2       /       2       1       2       3 .       2       7       9       /       3       2       2       4 .       0       9       3       /       4       9       8       3       5       2       4 .       4       1       6       /       5       0       7       2       2       4 .       8       4       1       /       1       0       5       3       6       2       5 .       0       6       7       /       1       7       3       6       2       5 .       6       9       8       /       4       9       4       6       1       2       6 .       0       0       2       /       5       5       7       2       2       6 .       3       2       3       /       4       2       9       7       2       6 .       6       2       4       /       1       0       8       2       2       6 .       8       2       7       /       1       0       0       7       2       2       6 .       9       5       0       /       7       4       0       8       2       7 .       1       5       7       /       7       2       4       3       2       7 .       5       8       1       /       3       4       2       5       8       2       8 .       6       0       9       /       9       9       6       8       2       9 .       6       3       4       /       5       6       6       6       2       9 .       9       5       1       /       3       2       2       0       3       3 .       8       5       6       /       1       1       0       9       3       4 .       3       7       7       /       2       1       6       4       8  produced /y 3 and 3&. Stem e-plants did not  produce p!@!/en=oic acid, caffeic acid, 1uercitin and tannic acid, 0hereas all of these compounds 0ere present in S& and S&s. Qield of chlorgenic acid and catechin in S& increased significantly up to 5.) and 5. folds compared to S 4*.558; and *.GG;+ mg.g !5  d0t.6 respectively. 7!@!/en=oic acid 0as not found in R, /ut it 0as produced only in R&, 0hile R&s produced greater 1uantities of caffeic acid and rutin among other compounds and increased significantly to )8 and ) folds than R 0hich produced *.*)* and *.*)5; mg.g !5  d0t respectively. Based on the results of the present study, the accumulation of some active compounds in cultured cells 4callus and cell suspension6 at higher levels than those in 0ild type plants through optimi=ation of cultural conditions may /e due to the presence of plant gro0th regulators 47Rs6 supplemented to the medium for callus initiation. he effects of different 7Rs on accumulation of secondary meta/olites have previously /een studied and confirmed in various plants and their tissue cultures 45) and 5+6.   5   0 . 0 5 . 0 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 m in- 2 502 55 07 51 0 01 2 51 5 01 7 52 0 02 2 52 5 0m A U2 5 4 n m , 4 n m ( 1 . 0 0 )        3 .       2       8       7       /       7       6       3       5       3 .       5       3       6       /       3       3       6       6       1       3 .       8       4       4       /       1       1       3       5       5       1       0 .       6       2       8       /       1       6       2       7       2       2       1       3 .       9       2       3       /       2       0       3       9       1       5 .       4       2       8       /       2       4       5       5       1       6 .       9       7       6       /       1       0       0       3       9       1       7 .       9       8       3       /       1       0       4       9       3       7       0       1       9 .       4       3       7       /       1       9 .       8       9       2       /       1       0       0       3       2       0 .       5       2       3       /       1       9       8       4       1       2       0 .       7       3       9       /       9       3       7       3       0       2       0 .       9       9       1       /       8       1       3       2       2       1 .       2       1       8       /       1       2       1 .       6       5       9       /       1       6       2       2 .       0       0       3       /       5       6       6       6       2       2 .       2       6       9       /       1       4       7       4       1       2       2 .       6       8       7       /       5       2       1       6       0       2       3 .       3       4       1       /       3       0       3       8       9       2       3 .       8       0       8       /       1       4       5       3       2       4 .       0       5       2       /       2       9       9       0       7       2       4 .       3       9       0       /       4       3       5       2       2       4 .       8       2       8       /       5       6       3       6       2       5 .       0       4       9       /       1       9       9       4       2       5 .       6       8       7       /       5       8       6       5       9       2       6 .       3       0       3       /       6       4       9       7       2       6 .       7       9       4       /       1       1       4       3       7       2       6 .       9       2       8       /       7       4       7       1       2       7 .       1       4       6       /       8       4       8       6       2       7 .       5       7       0       /       3       9       9       9       3       2       8 .       6       1       1       /       1       0       3       4       3       2       9 .       6       3       5       /       5       3       7       2       2       9 .       9       7       5       /       2       8       8       8       3       4 .       4       3       7       /       1       0       3       6       1       4   -A-   -B-   Figure 456" @riginal chromatogram of standard compound A" Flavonoids mi-ture conc. *.*5)8 mg ml !5  B" i-ture of phenolic acids at a concentration of *.*8 mg ml !5
Search
Similar documents
View more...
Tags
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x