Identification of cis- and trans- Melilotoside within an Artemisia annua Tea Infusion

Transcription

1 European Journal of Meicinal Plant 4(): 26, 204 SCIENCEDOMAIN international Ientification of ci an tran Melilotoie within an Artemiia annua Tea Infuion J. Mouton an F. Van er Kooy 2* Natural Prouct Laboratory,Intitute of Biology, Leien Univerity, PO Box 902, 2 CC Leien, Netherlan. 2 Centre for Complementary Meicine Reearch, Univerity of Wetern Syney, Locke Bag 797, Penrith, NSW 27, Autralia. Author contribution Author JM manage the analye an practical apect of the tuy. Author FVK eigne the tuy an wrote the manucript. All author rea an approve the final manucript. Short Reearch Article Receive th Augut 20 Accepte 6 th September 20 Publihe th October 20 ABSTRACT Aim: To ientify the major chemical component containe within the Artemiia annua tea infuion, which ha been traitionally ue to treat fever an chill. Stuy Deign: Currently, little chemical ata exit on the tea infuion a previou work only focue on LCMS analyi an although a valuable analytical tool it remain ifficult to ientify new compoun. Our tuy therefore employe LCMS an NMR analyi in orer to confirm the ientitie of the major compoun. Place an Duration of Stuy: Natural Prouct Laboratory, Intitute of Biology, Leien Univerity between Jan 202 an December 202. Methoology: A thorough chemical analyi of the tea infuion wa complete uing both LCMS an NMR analye. Tea infuion were prepare uing eionie water an were ubjecte to LCMS analyi followe by emipreparative fractionation an NMR analyi to confirm the ientitie of the major compoun. Reult: Eleven major compoun were ientifie incluing chlorogenic aci, feruloylquinic aci, flavonol, coumarin, of which two compoun, ci an tranmelilotoie, are new for Artemiia pp. Concluion: The melilotoie are known to be active againt iarrhoea cauing pathogen an therefore might explain the traitional ue of A. annua to treat iarrhoea. Future work will focu on the quantification of the melilotoie an the ientification of *Correponing author: f.vanerkooy@uw.eu.au;

2 European Journal of Meicinal Plant, 4(): 26, 204 other compoun (major an minor) in the tea infuion. Keywor: Artemiia annua; tea infuion; malaria; melilotoie; HIV; cancer.. INTRODUCTION Many of toay rug an rug lea are erive from traitional meicinal plant []. One uch example i Artemiia annua L. (Ateraceae) which ha been ue for centurie in China to treat fever. In the 970 Chinee cientit ientifie the active principle, artemiinin, uring a largecale creening for new antiplamoial lea compoun. Toay, artemiinin an it erivative are being ue a a firtline treatment for uncomplicate malaria an once again highlight the importance of cientific reearch into meicinal plant [2]. However, herbalit claim that uing A. annua in it traitional way mainly in the form of a tea infuion will have a comparable or even better efficacy againt Plamoium falciparum than the purifie artemiinin. The plant material, an the tea infuion thereof, contain many ifferent compoun incluing the active principle artemiinin. The coextracte compoun in the tea infuion can have a ynergitic effect on many ifferent level againt P. falciparum, although to prove ynergim will be very ifficult, ue to it inherent complexity an the fact that we preominantly ue in vitro bioaay. The ynergitic action between compoun within A. annua wa however recently emontrate by Onimu et al. [] who aminitere powerie A. annua leave, known to contain a very low amount of artemiinin (>0.%), to 2 patient. The treatment reulte in a 62% reuction in the number of paraite although the aily oe of artemiinin wa only 0. mg. They conclue that the aminitration of powerie A. annua leave wa apparently more effective than the tea infuion but ue to logitical reaon the tea infuion i till the preferre treatment option. It i however alo claime that the tea infuion i not only ueful to treat uncomplicate malaria but alo to treat HIV, iarrhoea an even cancer [47]. Thee aertion are irecte by Nongovernmental organiation that provie the plant material too many African an Aian communitie. A thee ieae are lifethreatening an where epecially chilren are vulnerable to malaria an iarrhoea it i extremely important to invetigate thee claim, a many thouan of people live are potentially affecte. Invetigating uch a complex iue coniting of many cientific quetion will be a time conuming eneavour. In orer to fin anwer to thi complex iue, we tarte with the invetigation of the extraction efficiency of artemiinin uring the tea making proce. We foun that by boiling the leaf material for 2 min almot all the artemiinin in the plant material wa effectively extracte, an the artemiinin content of the tea remaine table for at leat 24 h [8]. Thi wa followe by teting the tea infuion againt HIV (in vitro) where we foun it to be remarkably active an it appear that artemiinin play only a very minor role in the oberve activity [4]. We have alo tete artemiinin againt 44 breat cancer cell line an foun it to be active againt all cell line tete with electivity for the multirug reitant cell line [6]. The ynergitic effect of the A. annua tea infuion againt P. falciparum wa alo invetigate. Our reult inicate that the in vitro antiplamoial activity of the A. annua tea infuion coul only be acribe to artemiinin an therefore no ynergim wa preent in our bioaay ue [9]. Thi fining wa however contraictory to the tuie performe by Wright et al. [0] who foun an 68 fol enhance activity a compare to pure artemiinin.

3 European Journal of Meicinal Plant, 4(): 26, 204 With increaing report on the variou biological activitie acribe to the tea infuion an alo the lack of any toxicity reporte thu far, we fin it urpriing that very little reearch ha been conucte on the tea infuion itelf. At the heart of all of thee claime activitie lie the chemitry of the tea infuion, which ha been completely unertuie. Thi lack of information an the preence of a large amount of contraictory reult were reviewe by Van er Kooy an Sullivan [] an the poible reaon an implication of thi were reviewe by Van er Kooy []. Stuie conucte on the chemitry of the tea infuion are limite to Bilia et al. [2] who quantifie artemiinin an flavonoi but mot of thi work wa performe on aqueou methanol extract. Liu et al. [] employe NMR metabolomic to compare an aqueou methanol extract of A. annua with A. afra for quality control purpoe an alo tete the tea infuion againt P. falciparum. Polar extract (mainly methanol) of the leaf material were hown to contain a large amount of flavonoi an other phenolic [4]. Carbonara et al. [6] performe a LCMS analyi on a 24 h water extract of A. annua an reporte a large amount of phenolic compoun to be preent. However, no tuie were conucte uing both LCMS an NMR in orer to ientify poible new compoun in the tea infuion. In thi tuy only compoun that gave aequate MS an NMR ata were ientifie. Due to the low enitivity of NMR pectrocopy the tea infuion wa fractionate an concentrate in orer to confirm the ientitie of the main compoun. Thee two analytical ytem reveale both their trength an weaknee a the LCMS wa able to etect a large number of minor compoun which coul not be etecte with NMR. On the other han many metabolite coul be etecte with NMR (e.g. ugar) which were not etectable with both UV an MS pectrocopy. We mut alo keep in min that a tea infuion i prepare by aing boiling water to the plant material after which it i conume within a matter of minute. Thi i the main reaon why we ecie to prepare the tea a precribe an analyze it within a matter of minute an, importantly, without any ample treatment (except filtering). Therefore, the main objective of thi tuy wa to ientify the major chemical component in the tea infuion with MS an NMR analyi. 2. MATERIALS AND METHODS 2. Chemical an Reagent LCMS grae mobile phae were purchae from SigmaAlrich (Steinheim, Germany). Deionie water wa obtaine from a Millipore itille water unit (ph.7). The euterate olvent, D 2 O, CD OD an CDCl for NMR analyi were purchae from Anover (MA, USA). 2.2 Plant Material Artemiia annua (Aname A) plant material wa obtaine from the breeing program of ANAMED an ientifie by Dr HanMartin Hirt. The plant were grown in Ammerbuch (Germany), harvete in September 200 an conite of rie leaf material. 2. Sample Preparation Artemiia annua tea wa prepare a ecribe earlier [8]. In hort, tea infuion were prepare by aing 0 ml of eionie boiling water to 90 mg of A plant material. The 4

4 European Journal of Meicinal Plant, 4(): 26, 204 mixture wa allowe to boil for 2 min after which. ml wa filtere (0.4 µm PTFE yringe filter) into an HPLC vial for analyi. 2.4 LCMS Analyi The LCMS analyi wa performe on an Agilent 00 equippe with an autoampler, a binary pump ytem, a photoioe array an MS ingle quarupole etector. The mobile phae conite of water with 0.% formic aci (FA) (olvent A) an methanol with 0.% FA (olvent B). We ue the following elution graient: 0 min, 20% B; min, 20% B; 0 min, 00% B; min, 00% B; 6 min, 20% B, an 40 min 20% B. The flowrate wa.0 ml/min an UV etection wa performe at 20, 24 an 0 nm. The MS conition were a follow: APCI poitive an negative ioniation in the Scan moe (0900 m/z). The fragmentor wa et to 0V, rying ga flow 0 L/min, nebulizer preure 0 pig, rying ga temperature 0 C, vaporizer temperature 00 C, capillary voltage 4000 V an the corona current 7 μa for poitive an 2 µa for negative ioniation. For improve ioniation of peak 8, 9 an the fragmentor wa et to 0 an the elution graient wa change to: 0 min, 20% B; min, 20% B; 40 min, 00% B; 4 min, 00% B; 46 min, 20% B; 0 min, 20% B. 2. Semipurification of Compoun In orer to ai the ientification of the electe compoun, we fractionate the tea ample (A) by emipreparative HPLC. The column ue to eparate the compoun wa a Phenomenex Luna C 8 (2) (20x0.00 mm,.0 µm) column. The mobile phae wa the ame a for LCMS analyi but the flowrate wa increae to 2.0 ml/min an the elution graient wa a follow: 0 min, 40% B; 2 min, 40% B; min, 00% B; 40 min, 00% B; 4 min, 40% B; 4 min, 40% B. Fraction were collecte for. min tarting at 4 min after injection an ening at 40 min, yieling 24 fraction. Each fraction wa reinjecte into the LCMS in orer to obtain the UV an MS ata of the main compoun in each fraction an match thi ata with the original chemical profile of the tea infuion. 2.6 HNMR Analyi HNMR wa recore at 2ºC on a 00 MHz Bruker DMX00 pectrometer (Bruker, Karlruhe, Germany) operating at a proton NMR frequency of 00. MHz. Each HNMR pectrum conite of 28 can requiring 0 min an 26 ec acquiition time with the following parameter: 0.6 Hz/point, pule with = 0º (. µec), an relaxation ela y =. ec. A preaturation equence wa ue to uppre the reiual H 2 O ignal with low power elective irraiation at the H 2 O frequency uring the recycle elay. Free inuction ecay were Fourier tranforme with line broaening of 0. Hz. The reulting pectra were manually phae an baeline correcte, an calibrate to the internal tanar trimethylilyl propanoic aci at 0.0 ppm, uing XWIN NMR (verion., Bruker). All 24 fraction were rie uner vacuum an reiolve in D 2 O an bae on the preliminary compoun ientification all fraction were reanalyze in organic olvent (MeOD or CDCl ) in orer to compare the chemical hift with exiting literature.

5 European Journal of Meicinal Plant, 4(): 26, 204. RESULTS. Compoun Ientification The choice of which component to regar a the main contituent wa bae on the UV aborbance at 20, 24, 0 nm an eae of ioniation in the MS. Fig. how the chemical profile of a typical A. annua tea infuion at 24 nm an Table lit the ientitie of the main component ientifie in the tea bae on their MS an NMR pectral propertie. Due to artemiinin not containing a chromophore it oe not appear in the chromatogram. That artemiinin i preent in the tea infuion in appreciably quantitie ha been hown by a number of author a reviewe by Van er Kooy an Sullivan []. Fig.. Chemical profile of the A. annua tea infuion at 24 nm. : copolin; 2: ci 6: melilotoie; : chlorogenic aci; 4: feruloylquinic aci; : tranmelilotoie; copoletin; 7:,icaffeoylquinic aci; 8: rutin; 9: caffeoylferuloylquinic aci; 0: chryoplenol D; : chryoplenetin 6

6 European Journal of Meicinal Plant, 4(): 26, 204 Table. Ientification of the main phenolic compoun in the A. annua tea infuion bae on their MS (+ an ) an H NMR ata n RT (min) MS (+) MS () MW (D 2 O).79 ; 9 99; 9; 76 2 (MeOD).0 27; 6; 47 (MeOD).6 ; 9; 6 2; 6; ; H NMR Propoe compoun Reference δ (ppm) Integ. Pattern J (Hz) H ' t t m , ' 7' ' 8' copolin [7] cimelilotoie [8,9] chlorogenic aci (caffeoylquinic aci) [,6,20,2] 4 (MeOD) ; 209; 77; 67; m , ' ' 8' feruloylquinic aci [,6,20,2] 7

7 European Journal of Meicinal Plant, 4(): 26, 204 Table continue. (MeOD) ; 6; 47 2; 6; (MeOD) ; (MeOD) 8.7 7; 499; ; 7; 9; 6 8 (MeOD) 9.7 6; 49; ; 0 ; 497; 609; 49; t t m m , , , ' 4 8 7' 7'' ' ' ' '' 8' 8'' ' 8 6 '' ''' Me tranmelilotoie [8,9] copoletin [],icaffeoylquinic aci [,6,20,2] rutin [22,2] 8

8 European Journal of Meicinal Plant, 4(): 26, 204 Table continue.. 9 (MeOD) ; 499; ; 209; 77; 6 29; ; 67; (CDCl ) ; 0 9; (CDCl ) ; m m ,.9 8.0, , , ' 7'' ' ' ' '' 8' 8'' OH ' 8 OH ' 8,caffeoylferuloylquinic aci chryoplenol D [24] chryoplenetin [2] [,6,20,2] 9

9 European Journal of Meicinal Plant, 4(): 26, 204 Peak an 6 were ientifie a copolin an copoletin repectively, bae on their H NMR, MS an UV pectral ata a compare to literature [,7]. The HNMR ata of matche to Fliniaux et al. [7], for copolin but all ignal were hifte by ppm, ue to a ifference in the ph of D 2 O ue in the analyi. The ma pectrum of, reveale a quaimolecular ion of in poitive moe with the major fragment of 9 an 9 in poitive an negative moe repectively, inicating the lo of one glucoe molecule. The NMR ata of peak 6 reveale thi compoun to be a coumarin an the MS (quaimolecular ion of 9 an 9 in the poitive an negative moe repectively) an UV ata confirme it to be copoletin []. To a further evience for the ientity of thee compoun, a frehly prepare tea infuion wa hyrolye by the aition of HCl (0. ml N HCl ae to ml of tea an allowe to react at 80 ºC for hour). Upon hyrolyi the peak area of ecreae while the area of 6 increae inicating a correlation between an 6. Peak 2 an were ientifie a cimelilotoie an tranmelilotoie repectively (Fig. 2). Accoring to our knowlege thi i the firt report that thee compoun are ientifie in any Artemiia pp. Ientification wa bae on UV propertie an ma pectral ata a compare to literature [8]. For both compoun, a quaimolecular ion of 27 an 2 were oberve in the poitive an negative ioniation moe repectively. The fragment at m/z 6 an 6 oberve in poitive an negative moe, repectively, correpon to the coumaric aci part of the molecule. The area of thee peak ecreae upon hyrolyi, confirming the preence of a ugar moiety. HNMR ata of the emipurifie fraction of 2 gave the itinctive ring ignal at δ H 7.00 (H, t, J=7.8 Hz, H), 7.22 (H,, J=7.8 Hz, H ), 7.2 (H, t, J=7.8 Hz, H 4), 7.7 (H,, J=7.8 Hz, H 6) an the H of the glucoe moiety at δ H 4.97 ppm (H,, J=7.4 Hz, H ). The typical ci configuration ouble bon ignal coul clearly be oberve at δ H.99 (H,, J=2. Hz; H8), 7. (H,, J=2. Hz, H7). A imilar pattern wa oberve for : δ H 7.09 (H, t, J=7.9 Hz, H), 7.29 (H,, J=7.9 Hz, H), 7.4 (H, t, J=7.9 Hz, H4), 7.66 (H,, J=7.9 Hz, H6),.02 (H,, J=7.8 Hz, H ) but with the itinctive tran ouble bon configuration at δ H 6.6 (H,, J=6.2 Hz; H8), 8. (H,, J=6.2 Hz, H7) [8,9]. O OH H H OH HO HO O O H (a) OH HO HO O O H (b) OH O OH OH Fig. 2. a) ci an b) tran Melilotoie Peak, 4, 7 an 9 were ientifie a compoun belonging to the chlorogenic aci family, name repectively chlorogenic aci ( caffeoylquinic aci), feruloylquinic aci,, icaffeoylquinic aci, caffeoylferuloylquinic aci. Peak wa poitively ientifie bae on the NMR, MS an UV pectral ata an wa confirme by the analyi of a pure reference tanar of chlorogenic aci. The poition of the bon between caffeoyl or feruloyl moiety an quinic aci were efine bae on Ge et al. [20]. To confirm the ientity of thee compoun, UV, MS an HNMR pectral propertie were compare with literature [,6,2]. 60

10 European Journal of Meicinal Plant, 4(): 26, 204 Peak 8, 0 an belong to the flavonoi cla of compoun an were ientifie a rutin [22,2], chryoplenol D [24] an chryoplenetin repectively. For peak the reporte literature ata were not very clear. Inee, peak wa ientifie a chryoplenetin by Sy an Brown, [2] but the HNMR ata an UV propertie i not match thoe reporte by Horie et al. [26] an Ahme et al. [27]. Peak can therefore alo poibly be caticin or a chryoplenetin iomer a reporte by Horie et al. [27]; Aker et al. [28] an Meaik et al. [29]. 4. DISCUSSION AND CONCLUSIONS The main objective of thi tuy wa the ientification of the main compoun in the tea infuion. In orer to achieve thi we ecie to irectly inject the ample without any treatment, yieling a chemical profile of the tea infuion within 0 min of it preparation (Fig. 2). All major compoun that gave aequate pectral ata were ientifie. The main component conit of a wie variety of phenolic compoun incluing chlorogenic aci, feruloylquinic aci, flavonol, coumarin an two new ocoumaric aci erivative. Although only the main metabolite have been ientifie uring thi tuy, at leat 0 minor component coul be etecte. Some major compoun (e.g. the peak between compoun 7 an 8) i not give aequate pectral ata an alo remain unientifie. Thi i however the firt report on the ientification of ci an tran melilotoie in A. annua an any other Artemiia pp. No report on any activity againt P. falciparum, cancer or HIV coul be foun for thee compoun. Thee relatively rare compoun were hown to exhibit goo activity againt the iarrhoea cauing pathogen Entamoeba hitolytica an Giaria lamblia (IC0 = 2. an 6.8 μg/ml repectively) inicating that the traitional ue of A. annua for the treatment of iarrhoea might be effective [0]. It i however well known that mot phenolic compoun uffer from having a low in vivo bioavailability, but thi apect oe however not apply to the melilotoie an their activity againt E. hitolytica an G. lamblia. Thee paraite occur in the lumen of the igetive tract an they houl be inhibite here without the melilotoie having to be aborbe into the blootream. In concluion, we report here on the ientity of the major component in the A. annua tea infuion an by combining LC MS an NMR analyi we were able to ientify two new compoun for Artemiia pp. With thi tuy we have taken only the firt mall tep in unlocking the full meicinal potential of A. annua. Future work will focu on the quantification of the melilotoie in ifferent A. annua ample an the ientification of other compoun (major an minor) in the tea infuion. ACKNOWLEDGEMENTS We thank Aname for proviing the plant material an Ro Priet an Suzannah Bourchier for proof reaing the manucript. We alo thank CompleMe, Univerity of wetern Syney for proviing the facilitie to complete thi work an Shaun Sullivan for converting the manucript into the journal format.no funing wa receive for thi work. CONSENT Not applicable. ETHICAL APPROVAL Not applicable. 6

11 European Journal of Meicinal Plant, 4(): 26, 204 COMPETING INTERESTS Author have eclare that no competing interet exit. REFERENCES. Newman DJ, Cragg GM. Natural prouct a ource of new rug over the 0 year from 98 to 200. J Nat Pro. 202;7():. 2. De Rier S, Van er Kooy F, Verpoorte R. Artemiia annua a a elfreliant treatment for malaria in eveloping countrie. J Ethnopharmacol. 2008;20(): Onimu M, Carteron S, Lutgen P. The urpriing efficiency of Artemiia annua power capule. Me Aromat Plant. 20;2():2. 4. Lubbe A, Seibert I, Klimkait T, Van er Kooy F. Ethnopharmacology in overrive: The remarkable antihiv activity of Artemiia annua. J Ethnopharmacol. 202;4(): Van er Kooy F. Revere pharmacology an rug icovery: Artemiia annua an it antihiv activity. In: Artemiia annua Pharmacology an Biotechnology. t e. eitor. Aftab T, Ferreira JF, Khan MA an Naeem M, Heielberg: SpringerVerlag; 20(In pre). 6. Liu NQ, Pragervan er Smien W, Ozturk B, Smi M, Van er Kooy F, Foeken JA, et al. Molecular portrait of breat cancer cell line an repone to artemiinin. Eur J Cancer. 20;47():2. 7. ANAMED. Aktion Natürliche Meizin. Germany. Accee 6 December 202. Available: 8. Van er Kooy F, Verpoorte R. The content of artemiinin in the Artemiia annua tea infuion. Planta Me. 20;77(): Mouton J, Janen O, Fréérich M, Van er Kooy F. I artemiinin the only antiplamoial compoun in the Artemiia annua tea infuion? An in vitro tuy. Planta Me. 20;79(6): Wright CW, Linley PA, Brun R, Wittlin S, Hu E. Ancient Chinee metho are remarkably effective for the preparation of artemiininrich extract of Qing Hao with potent antimalarial activity. Molecule. 200;(2): Van er Kooy F, Sullivan SE. The complexity of meicinal plant: The traitional Artemiia annua formulation, current tatu an future perpective. J Ethnopharmacol. 20:(DOI: 0.06/j.jep ) 2. Bilia AR, Magalhae PM, Bergonzi MC, Vincieri FF. Simultaneou analyi of artemiinin an flavonoi of everal extract of Artemiia annua L. obtaine from a commercial ample an a electe cultivar. Phytomeicine. 2006;(V): Liu NQ, Cao M, Fréérich M, Choi YH, Verpoorte R, Van er Kooy F. Metabolomic invetigation of the ethnopharmacological ue of Artemiia afra with NMR pectrocopy an multivariate ata analyi. J Ethnopharmacol. 200;28(): Carvalho IS, Cavaco T, Broeliu M. Phenolic compoition an antioxiant capacity of ix Artemiia pecie. In Crop Pro. 20;(2): Han J, Ye M, Qiao X, Xu M, Wang B, Guo DA. Characterization of phenolic compoun in the Chinee herbal rug Artemiia annua by liqui chromatography couple to electropray ionization ma pectrometry. J Pharmaceut Biome. 2008;47():

12 European Journal of Meicinal Plant, 4(): 26, Carbonara T, Pacale R, Argentieri MP, Papaia P, Fanizzi FP, Villanova L, et al. Phytochemical analyi of a herbal tea from Artemiia annua L. J Pharmaceut Biome. 202;62: Fliniaux MA, GilletManceau F, Marty D, Macek T, Monti JP, JacquinDubreuil A. Evaluation of the relation between the enogenou copoletin an copolin level of ome olanaceou an papaver cell upenion an their ability to bioconvert copoletin to copolin. Plant Sci. 997;2(2): Yang L, Nakamura N, Hattori M, Wang Z, Bligh SW, Xu L. Highperformance liqui chromatographyioe array etection/electropray ionization ma pectrometry for the imultaneou analyi of ci, tran an ihyro2glucoyloxycinnamic aci erivative from Denrobium meicinal plant. Rapi Commun Ma Sp. 2007;2(2): Canuto KM, Silveira ER, Bezerra AME. Etuofitoquímico e epécimencultivao e cumaru ( Amburana ceareni AC Smith). Quím Nova. 2007;(): Portuguee. 20. Ge F, Ke C, Tang W, Yang X, Tang C, Qin G, et al. Iolation of chlorogenic aci an their erivative from Stemona japonica by preparative HPLC an evaluation of their antiaiv (HN) activity in vitro. Phytochem Analyi. 2007;8(): Pauli GF, Poetch F, Nahrtet A. Structure aignment of natural quinic aci erivative uing proton nuclear magnetic reonance technique. Phytochem Analyi. 998;9(4): Mabry TJ, Markham KR, Thoma MB. t e. The ytematic ientification of flavonoi. SpringerVerlag; Ibraheim ZZ, Ahme AS, Goua YG. Phytochemical an biological tuie of Aiantum capilluveneri L. Saui Pharma J. 2008;9(2): Krau G, Roy S. Direct ynthei of chryoplenol D. J Nat Pro. 2008;7(): Sy LK, Brown GD. Three equiterpene from Artemiia annua. Phytochem. 998;48(7): Horie T, Kawamura Y, Yamaa T. Revie tructure of a natural flavone from Artemiia lanata. Phytochem. 989;28(0): Ahme D, Chouhary MI, Turkoz S, Sener B. Chemical contituent of Buxu emperviren. Planta Me. 988;4(2): Aker E, Akin S, Hökelek T., Dihyroxy,6,7,4 tetra methoxy flavone. Acta Crytallogr E. 2006;62(9): Meaik MA, Azizuin, Mura S, Khan KM, Tareen RB, Ahme A, et al. Iolation an immunomoulatory propertie of a flavonoi, caticin from Vitex agnucatu. Phytother Re. 2009;2(): CalzaaF, Velazquez C, CeilloriveraR, Equivel B. Antiprotozoal Activity of the contituent of Teloxy graveolen. Phytother Re. 200;7(7): Mouton et al.; Thi i an Open Acce article itribute uner the term of the Creative Common Attribution Licene ( which permit unretricte ue, itribution, an reprouction in any meium, provie the original work i properly cite. Peerreview hitory: The peer review hitory for thi paper can be accee here: 6