Extraction of phytocompounds from the medicinal plant Clinacanthus nutans Lindau by microwave-assisted extraction and supercritical carbon dioxide Extraction

Indus ial C ops and P oduc s 74 (2015) 83–94 Con en s lis s a ailable a ScienceDi ec Indus ial C ops and P oduc s jou nal homepage: www.else ie .com/loca e/indc op Ex ac ion o phy ocompounds om he medicinal plan Clinacan hus nu ans Lindau by mic owa e-assis ed ex ac ion and supe c i ical ca bon dioxide ex ac ion Ana N. Mus apaa,b, Ángel Ma ina,∗, Ra ael B. Ma oa, Ma ía José Coce oa aUni e si y o Valladolid, Indus ial Enginee ing School, Depa men o Chemical Enginee ing and En i onmen al Technology, High P essu e P ocesses G oup, C/D . Me gelina, s/n, 47011 Valladolid, Spain bFacul y o Chemical Enginee ing, Uni e si i Teknologi MARA, 40450 Shah Alam Selango , Malaysia a icle in o A icle his o y: Recei ed 15 Decembe 2014 Recei ed in e ised o m 24 Ma ch 2015 Accep ed 17 Ap il 2015 A ailable online 23 May 2015 Keywo ds: Mic owa e-assis ed ex ac ion (MAE) Supe c i ical fluid ex ac ion Soxhle Fla onoids polyphenols Snake g ass abs ac The composi ion and bioac i i y o na u al plan ex ac s s ongly depends on he ex ac ion echnique employed. Clinacan hus nu ans Lindau (C. nu ans) is a well-known medicinal plan in Sou h-Eas Asia ha has been adi ionally used o ea men o hepa i is, skin- ashes and snake enom poisoning, and ecen ly has a ac ed a en ion o i s applica ions o ea men and p e en ion o cance diseases. In p e ious s udies, he ex ac ion o bioac i e compounds om C. nu ans by con en ional Soxhle sol en ex ac ion has been desc ibed, bu his me hod shows limi a ions in e ms o selec i i y, ex ac ion yield and oxici y o he sol en s employed. In his s udy, phy ochemical compounds we e ex ac ed om lea es and s ems o C. nu ans by mic owa e-assis ed ex ac ion (MAE), p essu ized mic owa e-assis ed ex ac ion (PMAE), supe c i ical ca bon dioxide ex ac ion (SFE) and Soxhle me hod o in es iga e he bes echnique in e ms o yield, ex ac ion ime and eco e y o bioac i e compounds: phenols, fla onoids, phy os e ols and ␤-si os e ol. The ex ac ed phy ocompounds and phenolics we e cha ac e - ized by gas ch oma og aphy mass spec ome y (GC/MS) and ul a pe o mance liquid ch oma og aphy (UPLC). The esul s showed ha MAE was he bes echnique o achie e a high yield and a maximal o al polyphenol con en (11.30±0.39mg GAE/g DM) and fla onoids con en (and 4.66±0.20mg GAE/g DM), whe eas SFE was he bes me hod o phy os e ols and ␤-Si os e ol ex ac ion. P-MAE me ely enhanced he polyphenol and fla onoids yield o 14.56±0.77mgGAE/g DM and 5.29±0.30mg QE/g DM espec i ely, wi hou significan a ia ions on he ype o compounds ob ained. MAE appea s as he mos e ficien echnique o he ex ac ion o phy ochemical compounds om C. nu ans in a sho ime wi h a easonable yield and a good selec i i y owa d bioac i e nu aceu ical compounds, wi h high concen a ions o an ioxidan s, an i-inflamma o y and an imic obial compounds. © 2015 Else ie B.V. All igh s ese ed. 1. In oduc ion Clinacan hus nu ans Lindau (C. nu ans), commonly known as snake g ass, is a medicinal he b belonging o he amily o Acan- haceae widely g own in he opical egion, mainly in Sou heas Asia. I has been adi ionally used as he bal medicine o ea - men o he pes in ec ion, insec and snake bi es and alle gic esponses (Sakda a e al., 2009; Tun iwachwu ikul e al., 2004; Wanikia e al., 2008). I s uses in adi ional medicine ha e been scien ifically suppo ed by nume ous s udies ha demons a e ha C. nu ans ex ac s show an i-inflamma o y, an imic obial ∗Co esponding au ho . Tel.: +34 983423174. E-mail add ess: [email p o ec ed] (Á. Ma in). and an i- i al ac i i y agains he pes simplex i us (HSV) and a icella-zos e i us (VZV) lesions (Cha uwichi a ana e al., 1996; Janwi ayanuchi e al., 2003; Sakda a e al., 2009; Yoosook e al., 1999). In Thailand, he plan has been accep ed as an essen ial medicinal he b o p ima y heal hca e by he Minis y o Public Heal h o he coun y a e ex ensi e esea ch on he C. nu ans medicinal p ope ies (Wanikia e al., 2008). Mo eo e , in ecen yea s, C. nu ans has a ac ed conside able esea ch in e es due o i s alleged p ope ies o cance ea men (Pu wa ana e al., 2009; Yong e al., 2013; Yuann e al., 2012). Va ious ac o s mus be examined in o de o de e mine he e ec i eness o medicinal he b ex ac s. Among hem, he ex ac- ion echnique employed o ob ain he bioac i e compounds om he plan is a key ac o . This is because, he e ficiency o ex ac- ion o di e en bioac i e compounds om plan ma e ials is h p://dx.doi.o g/10.1016/j.indc op.2015.04.035 0926-6690/© 2015 Else ie B.V. All igh s ese ed. 84 A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 influenced by se e al ac o s, such as sol en pola i y and con- cen a ion, sol en - o- eed a io, ex ac ion ime and he mal deg ada ion. In addi ion, a easonable sample p epa a ion is also e y impo an o p e en he de e io a ion o he plan ex ac . Fo example, he use o ac i a ed cha coal o emo e in e e - ences, such as chlo ophyll in plan ex ac s, epo ed in some p e ious wo ks, should be a oided. We ha e ound in p elimina y expe imen s o his wo k ha ea men wi h ac i a ed cha coal elimina es many phy ocompounds om he medicinal plan , hus, educing he po en ial biological ac i i y o he ex ac . In p e ious s udies, C.nu ans was ea ed by Soxhle ech- nique using me hanol, chlo o o m, e hanol and hexane as sol en s. Depending on he sol en employed, he bioac i e cons i uen s o C. nu ans ex ac s we e disco e ed o comp ise s igmas e ol, ␤- si os e ol, lupeol (Dampawan e al., 1977), be ulin (Lin e al., 1983), six known C-glycosyl fla ones, i exin, iso i exin, sha oside, iso- mollupen in, 7-O-␤-glucopy anoside, o ien in, isoo ien in (Teshim e al., 1997), sul u con aining glucosides (Teshima e al., 1998), glycoglyce olipids,a mix u eo ninece eb osides, monoacylmono- galac osylglyce ol (Tun iwachwu ikul e al., 2004). F om hese p e ious s udies, i can be concluded ha o da e, only simple mace a ion and Soxhle ex ac ion echniques ha e been epo ed o ex ac ion o bioac i e compounds omC. nu ans medicinal he bs. In his wo k, i is hypo hesized ha he com- posi ion and p ope ies o he ex ac a e highly dependen on ex ac ion me hod as well as on he sol en employed. The appli- ca ion o enhanced ex ac ion echniques wi h non- oxic sol en s (supe c i ical ca bon dioxide ex ac ion and mic owa e-assis ed ex ac ion MAE wi h e hanol–wa e sol en mix u es) on C. nu ans is epo ed o he fi s ime and compa ed wi h he con en ional Soxhle ex ac ion me hod. Supe c i ical fluid ex ac ion is a well- known me hod o p oducing high quali y plan ex ac s in a sa e and clean way. On he o he hand, MAE ex ac ion has been de el- oped as a simple, as e and less sol en consump ion me hod o he p oduc ion o high aluable ex ac s om plan ma e ials. In addi ion, we in oduced he use o p essu e in mic owa e-assis ed ex ac ion (P-MAE) o u he enhance he ex ac ion o phy o- compounds om he medicinal plan . Theo e ically, inc eases o p essu e and he co esponding inc emen s in ex ac ion empe - a u e could inc ease he solubili y o he bioac i e compounds in he ex ac ing sol en . The aim o his s udy is o de e mine he bes echnique o he ex ac ion o phy ocompounds om C. nu ans, conside ing he ex ac ion yield and he con en on significan phy ocompounds: phenols,fla onoids,phy os e olsand␤-si os e ol.Chlo ophyllcon- en in he C. nu ans was also de e mined as complemen o he phy ocompounds cha ac e iza ion, ega dless he e ec o ex ac ion echnique. In addi ion, he phenolic compounds in C. nu ans ex ac s we e cha ac e ized by ul a-pe o mance liquid ch oma og aphy (UPLC) coupled o elec osp ay ioniza ion and quad upole- ime o fligh -mass spec ome y (ESI-QTOF/MS). This s udy is he fi s wo k epo ing on he e ec o mode n ex ac- ion echniques on he phy ocompounds and he cha ac e iza ion o phenolic compounds p esen in C.nu ans ex ac based on he UPLC–ESI-QTOF/MS analysis. 2. Expe imen al 2.1. Ma e ials ␤-si os e ol (analy ical ch oma og aphy g ade), gallic acid, que ce in (analy ical g ade) and e hanol (96%) we e pu chased om Sigma–Ald ich Co. (Spain). Wa e was pu ified by a Milli- Q wa e pu ifie sys em om Millipo e (Mil o d, MA, USA). The Folin–Ciocal eu eagen was ob ained om Me ck (Da ms ad , Ge many). D ied C. nu ans (Bu m. .) Lindau samples we e pu - chased om M . Lee Hua Lye Kuala Lumpu , Malaysia. 2.2. Sample p epa a ion The o al mois u e con en o lea es and s ems was de e mined g a ime ically by ai d ying in an o en se a 105◦C o 24h. P io o ex ac ion, samples we e d ied in he o en a 60◦C o 2h, in o de o educe he mois u e con en , g ound and sie ed o a pa - icle size in he ange o 500–100␮m. The p epa ed samples we e hen s o ed in ai igh bags, swep wi h ni ogen gas and kep in a e ige a o (−8◦C) un il used in ex ac ion expe imen s. 2.3. Mic owa e-assis ed ex ac ion (MAE) and p essu ised-MAE (P-MAE) MAEandP-MAE we eca iedou inalabo a o y CEMDisco e ® mic owa e o en (300W maximum powe ) ope a ing a a e- quency o 2.45GHz, using e hanol–wa e sol en mix u es. The mic owa e o en was equipped wi h an op ical fibe p obe o mea- su e he eal empe a u e p ofile du ing he ex ac ion p ocess. MAE was ope a ed as an open sys em whe eas P-MAE was pe - o med in closed sys em. Mic owa e i adia ion in P-MAE was e mina ed when 2.7ba o p essu e build-up in he sys em was achie ed. The pa ame e s s udied we e he concen a ion o he e hanol–wa e solu ion (44–86 ol%) wi h a sol en - o- eed a io o 14g/g, a a cons an powe o 300W. The ex ac ion ime (5, 10, 15, and 20s) was de e mined in p elimina y expe imen s o find an app op ia e ela ion o empe a u e- ime a a fixed i adia ion powe and o iden i y a su ficien ime o achie e a empe a u e close o he boiling poin o he e hanol–sample sys em a a con- s an i adia ionpowe o 300W.F om hep elimina ys udy,i was ound ha 15s was su ficien o achie e e hanol’s boiling poin a 300W based on sol en - o- eed a io o 14. In ex ac ion expe imen s, abou 1.0000±0.0002g o C. nu ans sample was mixed wi h 14mL o e hanol–wa e solu- ion (44–86 ol%) in a 100mL ound-bo om flask. The mixed e hanol–sample ma e ial sys em was kep unde magne ic s i ing o 3min o allow he samples o be soaked by he sol en . This p omo es he di usion o he sol en in o he sample ma ix and imp o es he mass ans e o ac i e compounds in o he sol en . Du ing he mic owa e i adia ion, he e hanol-sample was con- s an ly s i ed using a magne ic s i e o a oid he o ma ion o ho spo s wi hin he sample, hus, homogenizing he empe a u e o he mix u e du ing he ex ac ion p ocess. A e i adia ed by MAE, he sample was apidly cooled down o 40◦C in an ice ba h, 6mL o cold esh e hanol was added o he sample and he expe - imen p oceeded wi h a con en ional ex ac ion in wa m wa e (40◦C) o 80min. P io o he sample collec ion, ni ogen gas was flowed in o he ambe ials used o s o e he sample o emo e ai . Ex ac s we e subjec ed o fil a ion using 0.20␮m PTFE o emo e solid esidues be o e s o age. 2.4. Supe c i ical fluid ex ac ion (SFE) The supe c i ical fluid ex ac ion o C. nu ans was ca ied ou o e 80g o C. nu ans (7–8w % o mois u e) a 350ba and 60◦C in a cus om-made SFE se up (Fig. 1). The sys em consis ed o an ex ac ion essel madeo s ainlesss eel wi ha olumeo 4L, heigh o 49.5cm and inne diame e o 9.7cm. P essu e in he essel was egula edbymeanso a back-p essu e GO al eins alledin he line be ween he ex ac ion essel and he sepa a o . The educ ion o p essu e wi h his al e causes he ansi ion om a supe c i ical o a gaseous CO2s a e, and he e o e, he sepa a ion o he com- pounds ex ac ed om CO2by condensa ion and p ecipi a ion. The sepa a o was a essel o 2.5L, designed o gene a e a cyclonic flow A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 85 Fig. 1. Supe c i ical fluid ex ac ion plan . E-0: CO2 ank, E-1: e ige a o , E-2: pump, E-3: ex ac o , E-4: sepa a o , F-1: gas flowme e . pa e nandequippedwi hahea ing/coolingjacke ha canbeused o adjus he empe a u e desi ed o an op imum sepa a ion o he ex ac edcompounds omCO2.CO2wasp essu ized by means o a diaph agm pump. The sys em ope a ed in a closed CO2ci cui , and gaseous CO2lea ing he sepa a o was condensed in a coole ope - a inga −25◦C and ecomp essedwi h hepumpin o heex ac o . Tempe a u e and p essu e condi ions in he ex ac ion appa a us we e moni o ed h ough p obes connec ed o a Picolog da a acqui- si ion so wa e, and he amoun o CO2consumed was measu ed by a Co iolis gas flowme e . In a SFE expe imen , he ex ac ion essel was filled wi h he d ied sample o C. nu ans, co e ed a op and bo om wi h a mesh fil e . Be o e he ex ac ion s a ed, he appa a us was flushed wi h CO2 o emo e ai om he sys em, and a e wa ds he ex ac ion essel was filled wi h CO2up o he desi ed ope a ing p essu e and empe a u e (350ba and 60◦C). A e eaching hese condi ions, he back p essu e al e be ween ex ac o and sepa a o was eg- ula ed in o de o main ain he condi ions a he sepa a o a 50ba and40◦C oachie eane ficien ex ac -CO2sepa a ion. Theex ac- ion p oceeded o a o al ime o 120min wi h a CO2flow a e o 9kg/h. Finally, he sys em was dep essu ized and a sample o he ex ac was aken om he sepa a o . The sample was collec ed in an ambe ial o p o ec i om ligh , weighed and s o ed in a e ige a o (−8◦C) un il analysis. 2.5. Soxhle ex ac ion Sol en ex ac ion was ca ied ou in a Soxhle appa a us o 8h using 80mL o absolu e e hanol o e abou 3.0g o d y plan . A e wa ds, ex ac s we e subjec ed o o a y acuum e apo a ion (Heidolph) a 40◦C o emo e he sol en om he oil. The ex ac s we eweighedands o edinanambe ialsandkep ina e ige a o (−8◦C) un il analysis. 2.6. Gas ch oma og aphy mass spec ome y (GC/MS) analysis The phy ochemical composi ion o C. nu ans ex ac was analyzed on an Agilen gas ch oma og aph model 6890 (Agi- len Technologies, J&W Scien ific P oduc s, Palo Al o, CA, USA) equipped wi h an Agilen 122-7032 capilla y column, DB-WAX (30m×0.25mm×0.25␮m) and coupled o a mass selec i e de ec- o (MSD5973) wo king a 70eV o ioniza ion ol age. Helium was used as a ca ie gas a 1.0mL/min wi h injec ion in spli less mode. The o en empe a u e was p og ammed as ollows: 110◦C held du ing 3min, hen inc eased o 200◦C a a e o 5◦C/min, hen inc eased o 250◦C a a e o 10◦C/min and finally main ained a 250◦C o 10min. Componen s iden ifica ion was made based on compa ison o hei mass spec a wi h hose in Wiley Regis y o Mass Spec al Da a, 7 h edi ion (Agilen Technologies, Inc.) and Na ional Ins i u e o S anda ds and Technology 05 MS (NIST) mass spec al lib a y da a. 2.7. High pe o mance liquid ch oma og aphy (HPLC) analysis A e e sed-phase and isoc a ic HPLC me hod was se up o analyze he ␤-si os e ol con en in ex ac s. The HPLC appa- a us consis ed o pump (515HPLC pump wa e s), wa e s 717 plus au osample , wa e s 432 conduc i i y de ec o , wa e s 2487 dual ␭abso bance de ec o and column (symme y C18 5␮m, 4.6×150mm). The mobile phase was me hanol/2-p opanol (4:1 / ) a a flow a e o 0.7mL/min and he injec ion olume was 20 ␮L in each analysis. ␤-si os e ol was de ec ed by an e apo a ing ligh sca e ing de ec o (ELSD) and he ch oma og am was moni o ed a a UV wa eleng h o 210nm. In o de o quan i y he amoun o ␤-si os e ol in he sample, a linea calib a ion cu e was de eloped based on s anda d solu ions o ␤-si os e ol a six di e en concen- a ions, in he ange 50–500mg/L. Abou 0.005g o oil ex ac s om SFE expe imen s was dilu ed wi h me hanol (4mL). Fo MAE sample, he ex ac s we e subjec ed o saponifica ion echnique p io o he HPLC analysis. 2.8. Chlo ophyll analysis C. nu ans has been epo ed o con ain chlo ophyll a and b (Sakda a e al., 2009). To de e mine he o al chlo ophyll con en in C. nu ans, e hanol (abs) and ace one (abs) sol en s we e used in he MAE a 300W o 15s. The sample ex ac s we e fil e ed and subjec ed o UV– is spec opho ome e de ec ion. To al con- cen a ions o chlo ophyll a and b we e de e mined by measu ing he abso bance using a UV– is spec opho ome e . The equa ions p oposed by Win e mans and de Mo s (1965) o e hanol and by Wellbu n (1994) o ace one we e used o e alua e he chlo ophyll concen a ion. 86 A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 Fig. 2. Compa isono he ex ac ion yield, sol en consump ion and ex ac ion ime co esponding o he di e en ex ac ion echniques employed. 2.9. To al phenol con en (TPC) analysis The o al phenolic con en in he SSG ex ac s was de e mined by he Folin–Ciocal eu colo ime ic me hod and exp essed as gal- lic acid equi alen s (GAE) pe g am o d y ma e ial (mgGAE/g d ied ma e ial, DM) (Sólyom e al., 2014). Fo his, 40␮L o sam- ple ex ac s we e mixed wi h 3mL o dis illed wa e ollowed by addi ion o 200␮L o Folin–Ciocal eu eagen unde gen le s i ing. A e 5min,600␮Lo sa u a edNa2CO3solu ionwasadded,shaken gen ly and incuba ed in a wa m wa e ba h (40◦C) o 30min. Abso bance was measu ed a 765nm (UV-2550 Shidmadzu UV– is spec ome e ). S anda d solu ions o known concen a ion o gallic acid (50–900ppm) we e used o calib a ion. 2.10. To al fla onoids con en P io o hefla onoidsde e mina ion,i isnecessa y oelimina e chlo ophyll om e hanolic ex ac s as i causes in e e ence. Some au ho s sugges o use ac i a ed cha coal o elimina e he chlo o- phyll colo . Howe e , om ou p elimina y ials we ound ha he use o cha coal o mo e han 2h o emo e any in e e ence aces including chlo ophyll in he C. nu ans ex ac significan ly educed he phenols con en om 5.54 o 1.51mgGAE/g d ied ma e ial (DM). Thus, use o cha coal may cause deficiency on he he apeu- ic s eng h as some bioac i e compounds om medicinal plan ex ac could be abso bed. In ac , i has been es ablished ha cha - coal has been used as an ido e o subs ance poisoning including alkaloid (Ka ona e al., 1987). Thus, o emo e chlo ophyll in his wo k, he e hanolic ex ac s we e subjec ed o liquid–liquid ex ac ion by adding hexane in he a io o 5:6 ( / ) o ex ac :hexane. The mix u e was shaken igo ously and he phases we e allowed o sepa a e. The bo om laye ha con ained fla onoids was eco e ed o p oceed wi h he fla onoids analysis. A me hod pe o med by Sólyom e al. (2014) was e e ed whe e 1mL o C. nu ans ex ac was aken in o a es ube and mixed wi h 300␮L o 5% sodium ni i e solu ion (NaNO2). A e 5min, 500␮L o 2% aluminum chlo ide solu ion (AlCl3) was added in o he es ube ollowed by he addi ion o sodium hyd oxide (NaOH) a e 6min o equilib a ion. The mix- u e was ho oughly mixed du ing 10min and dilu ed wi h 10mL wi h Milli-Q wa e . The abso bance o he eac ion mix u e was hen measu ed a 510nm by UV– is spec opho ome e agains a blank which was p epa ed wi h he same p ocedu e as sam- ples. The o al fla onoids con en was calcula ed om a s anda d calib a ion cu e (100–500mg/L) and he esul s we e exp essed as que ce in equi alen , mg que ce in (QE)/g d ied ma e ial (DM). 2.11. To al phy os e ols con en The o al phy os e ols con en in C. nu ans ex ac s was de e - mined acco ding o he me hod desc ibed by A aújo e al. (2013) wi h sligh modifica ions. P io o he analysis, he ex ac s we e subjec ed o alkaline saponifica ion o clea e he ace al bond be ween he phy os e ol and he ca bohyd a e moie y (Liu e al., 2007). In his wo k, a di ec saponifica ion was pe o med as done by Xiao e al. (2013) wi h sligh modifica ion, unde mic owa e i adia ion o 20mL o 1.5mol/L e hanolic o KOH a 300W wi hin 15s. Unsaponifiable ma e was emo ed by wo consecu i e liquid–liquid ex ac ions wi h hexane (5mL) and washed wi h 0.5M KOH (2×5mL) and 2×5mL o Milli-Q wa e . Hexane was apo ized and he ex ac esidue was dissol ed in 5mL o chlo o- o m p io o he analysis. Fo SFE ex ac , he oil ex ac s (3mL) we e saponified wi h 3mL o 2mol/L KOH e hanolic solu ion a 80◦C o 1 h. A e he saponifica ion, he solu ion was cooled down o oom empe a u e and 2mL o Milli-Q wa e we e added o homogenize he solu ion. The unsaponifiable ma e was sepa- a ed om he ex ac by liquid–liquid ex ac ion as p e iously desc ibed and dissol ed in 5mL o chlo o o m. P io o he phy- os e ols de ec ion, abou 2mL o he Liebe mann–Bu cha d (LB) eagen was added o 5mL o chlo o o m sample ex ac s and olume was inc eased wi h chlo o o m o 10mL. A e 5min o eac ion ime, he abso bance o he samples was measu ed by UV– is spec opho ome e a 625nm agains a blank sample. The Liebe mann–Bu cha d (LB) eagen was p epa ed by adding abou 50mL o ace ic anhyd ide in o an ambe glass ial and kep in an ice ba h. A e 30min, 5mL o sulphu ic acid was added o he ace ic anhyd ide. Resul s o he o al phy os e ols measu emen s we e de e mined by e e ing o a s anda d calib a ion cu e o ␤- si os e ol (20–100mg/L) and exp essed as mg o ␤-si os e ol (BS)/g d ied ma e ial (DM). 2.12. Ul a-pe o mance liquid ch oma og aphy (UPLC) analysis Ul a-pe o mance liquid ch oma og aphy (UPLC) analyzes we e ca ied ou using a wa e s Acqui y UPLC sys em (Wa e s Co - po a ion, Mil o d, MA, USA). Samples we e sepa a ed on e e se phase in Acqui y UPLC BEH C18 (1.7␮m×2.1×50mm) column wi h mobile phases o 0 0.1% o mic acid in Milli-Q wa e and 0.1% o mic acid in ace oni ile in a g adien elu ion mode. The addi ion o o mic acid significan ly enhances he e ficiency o ioniza ion (No áko áe al.,2010). Theinjec ion olumewas 1␮Land heo en empe a u e was main ained a 25◦C. Fo he ESI/QTOF/MS, UPLC was coupled wi h o a quad upole- ime-o -fligh mic o-TOF-Q (B uke Dal onik GmbH, B emen, Ge many), an o hogonal accel- e a ed QTOF mass spec ome e , equipped wi h an ESI. A nega i e ion mode was se wi h spec a acqui ed o e a mass ange om m/z 50 o 1300. The op imum pa ame e s o he ESI/MS we e ound o be: capilla y ol age, +2.5kV; d ying gas empe a u e, 200◦C; d y- ing gas flow, 8.0L/min; nebulising gas p essu e, 2.0; collision RF, 300Vpp; ans e ime 120.0␮s; and p e-pulse s o age, 8.0␮s. An au oma ion o MS was se a collision ene gy as −10eV. The mass da a was p ocessed by Da a Analysis 4.1 so wa e (B uke Dal on- ics, Be men, Ge many), ha p o ided a lis o possible elemen al o mulae using he gene a e molecula o mula edi o . 2.13. S a is ical analysis All analyzes we e done in iplica e. Resul s a e epo ed as mean aluesand s anda d de ia ions.One-wayanalysis o a iance (ANOVA) es s we e pe o med in Mic oso Excel 2012 o analyze A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 87 Table 1 Phy ocomponen s iden ified by GC/MS in C. nu ans ex ac s ob ained by mic owa e assis ed ex ac ion (MAE), supe c i ical fluid ex ac ion (SFE) and Soxhle ex ac ion. Compounds Peak a ea (%) Na u e o compound Biological ac i i y MAE (86% / ) SFE (350 ba , 60◦C) Soxhle (abs e hanol) Neophy adiene 13.44 2.73 – Di e pene An i-mic obial, an i-flamma o y I on 3.00 – – Me al ions Haemoglobin o ma ion agen , main ain immune unc ion 7,9-Dodecadien-1-ol 4.25 – – Alcohol NA My is ic acid – 2.12 – Fa y acid Fla ou ing, skin ca e Palmi ic acid acid, me hyl es e 3.09 – 5.91 Fa y acid An ibac e ial, an i ungal Palmi ic acid 29.23 43.49 12.13 Fa y acid An ibac e ial, an i ungal Benzene hanol 3.74 – – Alcohol NA Phy ol 34.99 11.34 75.42 Di e pene An i adical, an imic obial, an i-cance Squalene – 5.58 – T i e pene An icance , an imic obial, an ioxidan , chemo p e en i e, pes icide, an i- umou , sunsc een S ea ic acid, me hyl es e 4.53 17.43 – Fa y acid An ibac e ial, an i ungal Ma ga ic acid, e hyl es e – 0.50 6.54 Fa y acid NA Lupeol 3.73 – – T i e pene An ip o ozoal, an imic obial, an i-inflamma o y, an i umo and chemop e en i e p ope ies Linoleic acid, e hyl es e – 15.77 – Fa y acid An ibac e ial, an i ungal Linolenic acid, me hyl es e – 1.04 – Fa y acid An i-mic obial, an i-inflamma o y hee ec o sol en concen a ion,de e mining hecaseswhe e he di e ence be ween indi idual means was s a is ically significan , wi h p<0.05. 3. Resul s and discussion Theex ac ionyieldsob ainedbyMAE,SFEandSoxhle me hods we e compa ed o each o he by conside ing sol en consump- ion and ex ac ion ime as p esen ed in Fig. 2. In his figu e, he ex ac ion yield is exp essed as g/g d y ma e ial while he mois- u e con en was 11.77w % in d y weigh basis. As i can be seen in Fig. 2, he lowes ex ac ion yield was ob ained by SFE me hod, wi h3.19w %ond ybasisa e 120mino ex ac iona 350ba and 60◦C, while he highes yield o 21.28w % was ob ained by Soxhle ex ac ion, and an in e media e yield o 17.39w % was achie ed by MAE me hod. The low yield ob ained by SFE is p obably due o he non-pola na u e o he ca bon dioxide sol en ha is un a o able o ex ac ing he abundan pola compounds p esen in C. nu ans oil, such as chlo ophyll and polyphenols (see Sec ion 3.3). Besides, a slowe ex ac ion a e in he cou se o ex ac ion (shown in Fig. 5 in Sec ion 3.6) indica ed ha he oil ex ac ion was con olled by mass ans e esis ance as he solu es we e s ongly bound wi h he plan ma ix. As consequences, he fluid-phase concen a ion became much lowe han he solubili y (So o á, 2005) and p o- longed he ex ac ion ime needed o achie e asymp o ic yield. The solubili y o C. nu ans oil was as low as 0.242mgoil/g CO2.A ela i ely high amoun o ca bon dioxide o 315gsol en /g eed comp essed o 120min needed in o de o achie e an ex ac ion yield o only 3.19w %, indica es ha he supe c i ical ex ac ion o he C. nu ans was an un a o able me hod conside ing he economic easibili y o he p ocess. On he o he hand, he highes yield achie ed by Soxhle ex ac- ion can be explained by applica ion o he high empe a u e (82◦C) du ing a longe pe iod (480min) compa ed o MAE which he empe a u e was 70◦C a 300W du ing 15s and comple ed wi h con en ional ex ac ion a 40◦C in 80min. The ele a ed empe a u e con ibu ed o he p ocess e ficiency as he sa u- a ion concen a ion was inc eased imp o ing he compounds ex ac abili y (Aspé and Fe nández, 2011). Besides, he di e en concen a ion o he e hanol solu ions used o he ex ac ion by Soxhle (100% e hanol) and MAE (86% / ) may also con ibu e o he lesse yield achie ed by MAE. Howe e , conside ing he ac o o sol en consump ion whe e MAE equi es only 13gsol en /g eed ins ead o 21gsol en /g eed in Soxhle he ex ac ion ime which is significan ly sho e o MAE (15s) han o Soxhle (480min) and he ene gy consump ion, mic owa e ea men appea s as a a o able me hod o he C. nu ans ex ac ion. 3.1. Phy ocompounds iden ifica ion GC/MS analyzes we e pe o med on he e hanolic ex ac s om MAE and Soxhle as well as on he SFE ex ac . The a i- ous phy ochemical compounds iden ified ha can con ibu e o he bioac i i y o C. nu ans a e p esen ed in Table 1. The analysis on he ex ac s e ealed ha C. nu ans ex ac comp ises di e - pene, i e pene, a y acids and o he bioac i e compounds. Mos o hecompoundsiden ifiedcanexhibi se e albiologicalac i i ies, such as an ibac e ial, an i-inflamma o y and an ioxidan s, as o example phy ol, squalene and lupel. Phy ol was ound as he majo compound in he MAE and Soxhle whils palmi ic acid was he majo componen in he SFE ex ac . These compounds ha e been claimed o ha e an i adical, an i-mic obial, an i-inflamma o y, an i umo and chemop e en i e p ope ies (Pejin e al., 2014; Saleem, 2009). O he bioac i e compounds, such as neophy adi- ene ha e been iden ified as s ong bac e icidal and an i ungal e penoids compounds (Ca e e o e al., 2008; Mendiola e al., Table 2 Chlo ophyll con en in C. nu ans ex ac s. Sol en Chlo ophyll a (g/g sample) Chlo ophyll b (g/g sample) To al chlo ophyll(g/g DM) E hanol 0.38 0.77 1.30 Ace one 0.48 0.45 1.06 88 A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 Fig. 3. Colou o ex ac s a di e en concen a ion o e hanol. (A) 44% / , (B) 50% / (C) 65% and (D) 86% / . 2008). Mo eo e , a y acids, such as palmi ic acid, linoleic acid and linolenic acid ha e also been iden ified o ac as an ibac e ial and an i ungalagen sagains Esche ichia coli,S aphylococcus au eus and Bacillus sub ilis (Ago amoo hy e al., 2007). In addi ion, SFE ex ac included in e es ing compounds based on he i e pene g oup, as squalene and se e al long a y acids ha we e no ound in MAE o Soxhle ex ac s. Squalene has been epo ed o exhibi an i-cance , an ioxidan , chemop e en i e, an i- umo (Ezhilan and Neelamegam, 2012), an i-cance ogenic and educing se um choles e ol le els (Rao e al., 1998). These esul s show ha he ype o compounds p esen in ex ac s is highly dependen on he na u e o sol en and on he echnique o ex ac ion employed. Due o he cha ac e is ics o sol en s employed, MAE ex ac ion enabled ob aining se e al mino i y pola compounds, while non-pola a y compounds mainly we e ob ained by SFE. In con as , Soxhle ex ac s did no include any o hese mino i y compounds. This can be due o a pa ial deg ada- ion o hese compounds du ing he long p ocessing imes a high empe a u e equi ed o Soxhle ex ac ion. 3.2. Chlo ophyll con en Sakda a e al. (2009) iden ified h ee chlo ophyl de i a- i es ela ed o he s uc u es o chlo ophyll a and b, namely 13(2)-hyd oxy-(13(2)-R)-phaeophy in b, 13(2)-hyd oxy-(13(2)- S)-phaeophy in a and 13(2)-hyd oxy-(13(2)-R)-phaeophy in a. These compounds we e in es iga ed by hese au ho s and exhib- i ed an i-he pes simplex ac i i y. I has been demons a ed by se e al esea che s ha he chlo ophyll pigmen s show a di e se ange o biological p ope ies, such as an ioxidan , an imu a- genicac i i ies, chemop e en i eac ionandinduc iono apop osis ac i i y in cance cells (Dashwood, 1997; Fe uzzi and Blakeslee, 2007). The e o e, i is conside ed ha he he p esence o chlo o- phyll in he ex ac also con ibu e o he medicinal p ope ies o C. nu ans. To alamoun so chlo ophyllaandbde e minedin hiswo ka e shown in Table 2. Resul s showed a high concen a ion o chlo o- phyll in he C. nu ans lea es and s ems. On he o he hand, as i can be obse ed om Table 2, e hanol ex ac ions a ained highe chlo ophyllcon en compa ed o ace one ex ac ions. E en hough, ace one has been used o many decades o chlo ophyll de e mi- na ionasi could gi ea e y sha p chlo ophyllabso p ionpeak, i is a poo ex ac ing sol en o chlo ophyll om many ascula plan s (Ri chie, 2008). In addi ion, e y low chlo ophyll con en s we e ob ained by ex ac ion wi h a 50% / e hanol/wa e mix u e, and he highes amoun s o chlo ophyll we e ob ained by ex ac ion wi h a 86% / e hanol–acidified wa e mix u e. I is in e es ing o no e ha di e en colo o ex ac s was ob ained a di e en e hanol concen a ion (see Fig. 3). I was obse ed ha he highe e hanol concen a ion he g eene he colo o ex ac . This is possibly due o he highe solubili y o chlo ophyll a high e hanol concen a ion. 3.3. To al phenols con en (TPC) The o al phenols con en o sample ex ac s a e p esen ed in Table 3. In gene al, a highe TPC was ob ained by e hanol ex ac- ion assis ed by mic owa e i idia ion (MAE) han by supe c i ical fluid ex ac ion (SFE). This phenomenon can be explained by he na u e o he sol en used in he ex ac ion, as he non-pola na u e o supe c i ical ca bon dioxide makes i less sui able han e hanol–wa e mix u es o he ex ac ion o pola polyphenol compounds (B ahmi e al., 2012; Le Floch e al., 1998). Phenolic compounds comp ise one o mo e hyd oxyl g oups (pola ag- men s) and phenyl g oups o a oma ic ings (non-pola agmen s). These compounds a e o en ound in plan s as es e s o glycosides a he han as ee molecules (Queimada e al., 2009). Wi h his s uc u e, hey can o m a hyd ogen bond wi h wa e molecules as well as wi h an elec onega i e oxygen o e hanol and ice e sa (Galanakis e al., 2013), making hem soluble in pola sol en s. Table 3 To al phenols con en (TPC) and o al fla onoids (TF) ob ained wi h di e en me hods o ex ac ion. Me hod E hanol concen a ion (% / ) TPC (mg GAE/gDM) TF (mg que ce in/gDM) MAE 44 11.09 ±0.28 4.24 ±0.12 50 11.30 ±0.39 4.66 ±0.20 65 9.31 ±0.18 4.54 ±0.20 86 5.74 ±0.29 3.41 ±0.76 aP-MAE 44 13.23 ±0.40 5.23 ±0.40 50 14.56 ±0.77 5.29 ±0.30 65 12.89 ±0.90 5.07 ±0.56 86 8.88 ±0.85 2.71 ±0.47 Con ol (MAE) 44 8.89 ±0.46 4.27 ±0.22 Soxhle Absolu e 7.95 ±0.21 3.04 ±0.02 SFE No e hanol CO2350ba , 60◦C 7.01 ±0.15 5.88 ±0.22 Values a e mean±S.D o iplica e expe imen s. aA p essu e=2.7ba . A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 89 Fig. 4. GC/MS Ch oma og ams o ex ac s ob ained by mic owa e assis ed ex ac ion (MAE) and p essu ized mic owa e assis ed ex ac ion (PMAE) wi h a 50% / e hanol/wa e sol en . In ou me hod, an addi ional amoun o sol en (6mL) was added a e he MAE i ida ion o inc ease he compounds solu- bili y in o he sol en and minimize he mass ans e limi a ion o compounds. The sol en amoun was de e mined by he sol en - o- eed- a io and he desi ed final olume o sol en . Indeed, om p elimina y expe imen s, i was ound ha by adding he sol en a e he MAE enhanced he o al phenols con en om 9.19 o 11.09mg GAE/g DM a 44% / e hanol (no shown in his wo k). Hence, his me hod imp o ed he ex ac abili y o polyphenols om sample ma ix in o ex ac ing sol en . The p opo ion be ween sol en s in wa e –e hanol mix u es mus be se depending on he specific compounds a ge ed. A low concen a iono e hanol(i.e.,50% / ),a highe amoun o polyphe- nols can be ex ac ed, whe eas less polyphenols can ob ained Table 4 GC/MS phy ochemical compounds iden ified in ex ac s ob ained by MAE and P-MAE wi h 50% / e hanol–wa e mix u es. No. Compound RT (min) Peak a ea (%) Na u e o compound Ac i i y/uses MAE50 P-MAE50 1 2-Bu anol 6.85 1.97 1.98 Ca boxylic acid An imic obial 2 Bu anamide 7.02 3.63 3.66 Amide An imic obial 3 2-cyclopen en-1-one, 2-hyd oxy 8.43 27.64 28.00 Cyclic ke one F ag ance 4 Glycine 9.33 22.05 22.24 Amino acid NA 5 Pen anal 9.99 3.18 3.21 Aldehyde An imic obial 6 Iso e aldehyde 10.16 2.15 2.11 Aldehyde Food fla ou ing 7 Dime hyl isulfide 10.68 1.05 1.09 O ganosul u An imic obial 8 Thiophene 10.87 2.94 2.82 He e ocyclic An imic obial 9 Succinic acid 11.21 1.17 1.25 O ganic acid An imic obial 10 Glycolic acid 11.54 1.30 1.44 ␣-Hyd oxy acid Food p ese a i e, skin ca e agen 11 Oxazolidine 11.80 3.87 3.99 Ni ogen compound An imic obial 12 Thiophene 12.09 5.56 5.74 He e ocyclic An imic obial 13 9-Azabicyclo (6.1.0) non-4-4en-9-amine 12.71 3.51 2.77 Azo compound NA 14 4-Vinyl-2-me hoxy-phenol 12.92 2.03 2.20 Phenol An ioxidan 15 Phenol,2,6-dime hoxy 13.70 1.20 1.31 Phenol An ioxidan 16 4H-Py an-4-one,2,3-dihyd o-3,5-dihyd oxy-6-me hyl 13.83 2.33 2.51 Fla onoid ac ion An imic obial, an i-inflamma o y 17 Glyce ine 14.30 6.46 6.95 Alcohol An imic obial, p ese a i e 18 4-Vinylphenol 15.14 6.36 6.73 Phenol An ioxidan 90 A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 Fig. 5. Oil (line) and ␤-si os e ol (columns) yields ob ained by Supe c i ical Fluid Ex ac ion as a unc ion o he sol en - o- eed a io. a high e hanol concen a ion due o he solubili y compe i ion be ween phenolics compounds and chlo ophyll ha is go e ned by pola i y ac o and also migh be a he modynamic p ope y i.e., ac i i y coe ficien . Galanakis e al. (2013) explained ha a edency o phenols o be ans e ed, solubilized and di used was depen- den on ac i i y coe ficien , as wi h smalle ac i i y coe ficien s he solubili y was highe and ice e sa. On he o he hand, he maximum con en s o polyphenols we e ob ained wi h e hanol/wa e mix u es in a 50% / p opo ion, wi h lowe con en s (wi h a s a is ically significan di e ence wi h p<0.05) when he p opo ion o e hanol was ei he inc eased o dec eased. This esul ag ees wi h he findings by Spigno e al. (2007) who ound ha he phenols concen a ion dec eased when he p opo ion o e hanol in he mix u e was inc eased beyond 50% / . This beha io is due o he molecula s uc u e o phenols which comp ises bo h pola and non-pola agmen s, limi ing i s solubili y in e hanol–wa e mix u e depending on he p opo ions be ween he wo sol en s. Mo eo e , he lowe phenols con en obse ed a 44% / o e hanol can also be due o he co-ex ac ion o o he compounds which dec ease he phenols con en in he ex ac . Spigno e al. (2007) men ioned ha he addi ion o wa e o e hanol imp o ed he ex ac ion a e, bu excessi e wa e in sol en mix u e could educe he phenols con en as o he con- aminan s we e also ex ac ed. As p e iously indica ed, he low TPC con en in SFE ex ac s can be expec ed due o he non-pola na u e o supe c i ical ca - bon dioxide. This esul is in ag eemen wi h he obse a ions o Rombau e al. (2014) who pe o med an ex ac ion o polyphenols om g ape seed by supe c i ical cabon dioxide wi hou he aid o any co-sol en also ound a e y low ex acion yield o polyphenols i.e., be ween 0.061 and 0.067gex ac /g d y ma e ial a e 120min a 53.8MPa and 104◦C. A highe TPC con en is expec ed i a co- sol en , such as e hanol is employed in he SFE p ocess due o he inc easing solubili ies o high molecula weigh phenolics (Sanjaya e al., 2014), as epo ed by se e al esea che s (Kazan e al., 2014). Howe e , he use o o ganic co-sol en s in SFE p ocesses consid- e aby complica es he pu ifica ion and ecycling o ca bon dioxide, inc easing cos s, and i is a ely used in comme cial SFE plan s. Fu he mo e, he TPC o Soxhle ex ac was also lowe com- pa ed o he con en s in MAE and P-MAE ex ac s, e en hough he o al ex ac ion yield ob ained by Soxhle was highe . This is p obably due o he absence o wa e in he e hanol sol en used in Soxhle , leading o a lowe solubili y o he polyphenols in he sol en . 3.4. Fla onoids con en Fla onoids a e seconda y me aboli es o plan s wi h polyphe- nolic s uc u e and ha e se e al pha mocological ac i i ies, such as an i-cance , an ioxidan , an i- i al and an i-inflamma o y. In his wo k, he end o o al fla onoids con en in ex ac s was simila as he end o polyophenol con en desc ibed in he p e ious sec ion. The o al fla onoids con en ob ained by MAE showed a maximum alue o . 4.66 ±0.20mg que ce in/g DM a 50% / when he concen a ion o e hanol was inc eased om 44% / o 50% / . Howe e , a u he inc ease in he e hanol con- cen a ion o 86% / insignifican ly (p>0.05) educed he o al fla onoids con en o 3.41±0.76mg que ce in/g DM. The lowes fla anoids con en ob ained a 86% / o e hanol was p obably due o he ex ac ion o non-pola aglycone fla onoids by hex- ane du ing he p e- ea men o liquid–liquid ex ac ion (LLE) o emo e chlo ophyll he eby lea es me ely pola fla onoids (glyco- sides fla onoids) in he ex ac s (Mab y e al., 1970), hus, educed he o al amoun o fla onoids con en . Fu he mo e, i also migh be due o he ex ac ion compe i ion be ween chlo ophyll and fla onoids om he plan lea es as a unc ion o e hanol con- cen a ion. P e iously, fla onoids con en in C.nu ans has been epo ed by se e al eseache s, o example Ho e al. (2013) ound 0.04±0.001mgQE/g o o al fla onoids con en a e ex ac ed C. nu ans using me hanol mace a ion o 3 days whils and Tiew e al. (2014) disco e ed 0.21±0.005mgQE/g d y ex ac by using he same ex ac ion p ocedu e. Di e ences in alues o he fla onoids migh be due o di e en ex ac ion me hod and sol en employed. In addi ion, a mic owa e p e- ea men could signfican ly enhance heex ac abili yo hepolyphenols om heC. nu ans plan . Mo e- o e , hedeg ada ion o fla onoidsdu ing he ex ac ionp ocedu e can be an impo an ac o , as fla onoids a e labile compounds ha can easily unde go deg ada ion eac ions in aqueous media. Wi h espec o his, i is ema kable he high fla onoid con en achie ed by SFE (Table 3), e en hough fla onoids a e pola compounds ha a e no easily ex ac able by SC–CO2. This esul is p obably a con- sequence o a lowe deg ada ion o he compounds du ing SFE, as wi h his p ocedu e a d y ex ac is ob ained along he cou se o he ex ac ion hus a oiding possible deg ada ion eac ions in aqueous media. 3.5. MAE s p essu ised-MAE (P-MAE) In his wo k, an ex ac ion assiss ed by mic owa e unde p es- su e (P-MAE) a 2.7ba was also ca ied ou wi h same pa ame e s as MAE expe imens. The ex ac s we e analyzed o de e mine he TPC and fla onoids, phy os e ols con en as well as polyphenols cha ac e iza ion by UPLC/MS. The esul s a e shown in Tables 3–5, espec i ely. In gene al, he TPC and fla onoids con en in he P- MAE ex ac s we e simila o he alues ob ained by MAE. The TPC alue dec eased om 13.23 o 8.88mg GAE/g DM signifi- can ly (p<0.05) when e hanol concen a ion was educed om 44 o 86% / . P essu ised-MAE (P-MAE) enhanced he ex ac abili y o he polyphenols abou 2–4mg GAE/g DM o he TPC in MAE ex ac s a di e en e hanol concen a ions. The inc easing em- pe a u e p oduced by a highe p essu e could ha e caused damage o he cell walls o he plan , s imula ing compounds eleasing and enhancing i s dissolu ion in o he sol en . The high empe - a u e achie ed in P-MAE ha is up o 108◦C also con ibu ed o he high ex ac abili y o he compounds om plan sample. This ac o has u he a ec ed he fla onoids ex ac ion when he i s con en was ound o be much highe compa ed o he fla onoids con en ob ained by con en ional MAE. The maximum alue o fla onoids, i.e., 5.29±0.33mg que ce in/g DM, was obse ed a 50% / and he e ec o a a ia ion in e hanol concen a ion om 44 o 86% / on his amoun was significan . Fu he mo e, as he A.N. Mus apa e al. / Indus ial C ops and P oduc s 74 (2015) 83–94 91 Fig. 6. UPLCQTOF ESI/MS ch oma og am o ex ac s ob ained by MAE and P-MAE a 86% / and 50% / o e hanol. e hanolconcen a ion wasinc eased o86% / , hefla onoidscon- en dec eased o he lowes amoun o 2.71±0.50mg que ce in/g DM. I is impo an o no e ha he applica ion o P-MAE did no imp o e any majo di e ence in he a ie y o compounds ex ac edcompa ed oMAE.Asi canbeseen omFig.4andTable4, bo h ex ac s o MAE50 and P-MAE50 comp ised simila phy o- chemical compounds. Howe e , he ex ac ion yields ob ained by P-MAE we e sligh ly highe han he yields ob ained by MAE. This esul sugges ed ha MAE ea men is su ficien o imp o e he ex ac abili y o compounds om he plan ma e ial. Mo e o e , he P-MAE solely enhanced he ex ac ion yield a he han he a ie y o compounds ex ac ed. In MAE and P-MAE p ocess, 50% / o e hanol was ound as he op imum concen a ion o he C. nu ans ex ac ion in p oduc- ing he highes TPC and TF. E en hough he fla onoids con en was inc eased wi h e hanol, small di e ences we e ound. Mo e- o e , e hanol wi h mo e han 50% / can caused local mucosal lesions h ough dehyd a ion and albumin p ecipi a ion (Eu ope, 1994) meanwhile e hanol concen a ion in excess o 60% / could be oxic o sho - e m and p olonged use o e hanol-con aining he bal medicinal p oduc s (Eu opean Medicines Agency, 2010). The e is also ano he s udy by Chung e al. (2002) conce ning he e hanol concen a ion o human consump ion, who s udied he an ioxidan ac i i y and hesa e y o 50% e hanolicex ac om ed bean e men ed. They claimed ha he 50% e hanolic ex ac was ound obe sa ein geno oxici yasno mu agenici yo oxici y e ec was ound on he es e s ains. Geno oxici y is a phenomenon desc ibing he p ope y o chemical sol en can cause a mu a ion o cells which lead o cance . Thus, om he economic, sa e y and polyphenols ex ac abili y, poin o iew, i is sugges ed o use 50% / o e hanol as an op imum concen a ion o ex ac o al phenols compounds om C. nu ans sample. 3.6. To al phy os e ols and ˇ-si os e ol Phy os e ols a e plan s e ols membe s o he ‘ e pene’ amily o na u al p oduc s and classified as low pola i y o non-pola compounds. Thei nu aceu ical ac i i ies include an i-inflamma o y, an i-bac e ial, an i-ulce a i e and an i- umo p ope ies (Be e idge e al., 2002; Lla e ias e al., 2013). The mos abundan phy os e ol componen s ound in plan oil a e ␤-si os e ol, s igmas e ols and campes e ol. In his wo k, ana- lyzes o o al phy os e ol (TP) and ␤-si os e ol by UV– is Table 5 To al phy os e ol (TP) con en and ␤-si os e ol (BS) yield ob ained wi h me hods o ex ac ion. Me hod E hanol concen a ion (% / ) TP (mg BS/g DM) BS (mg/g DM) MAE 50 0.19±0.13 – 65 0.41±0.20 0.16 ±0.22 86 0.70±0.10 0.52 ±0.10 aP-MAE 50 0.35±0.12 – 65 1.04±0.15 0.45 ±0.20 86 1.19±0.22 0.65 ±0.14 Con ol 44 – – Soxhle Absolu e 0.47±0.20 0.23 ±0.18 SFE 350ba , 60◦C 1.35±0.12 0.83 ±0.10 MAE–KOH 1.5mol/L e hanolic KOH 2.36±0.15 0.64 ±0.13 Con ol-e hanolic KOH 0.88±0.14 0.40 ±0.10 aA p essu e=2.7ba