Moukette et al. Biological Research (2015) 48:15 DOI /s

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1 Moukette et l. Biologil Reserh (2015) 48:15 DOI /s RESEARCH ARTICLE Open Aess In vitro ntioxidnt properties, free rdils svenging tivities of extrts nd polyphenol omposition of non-timber forest produt used s spie: Monodor myristi Bruno Moukette Moukette 1, Constnt Antole Pieme 1*, Jques Romin Njimou 2,4, Cbrl Prosper Ny Bip 3, Brvi Mro 4 nd Jenne Yonkeu Ngogng 1 Abstrt Bkground: Exessive prodution of free rdils uses diret dmge to biologil moleules suh s DNA, proteins, lipids, rbohydrtes leding to tumor development nd progression. Nturl ntioxidnt moleules from phytohemils of plnt origin my diretly inhibit either their prodution or limit their propgtion or destroy them to protet the system. In the present study, Monodor myristi non-timber forest produt onsumed in Cmeroon s spie ws sreened for its free rdil svenging properties, ntioxidnt nd enzymes protetive tivities. Its phenoli ompound profile ws lso relized by HPLC. Results: This study demonstrted tht M. myristi hs svenging properties ginst DPPH,OH,NO, nd ABTS rdils whih vry in dose depending mnner. It lso showed n ntioxidnt potentil tht ws omprble with tht of Butylted Hydroxytoluene (BHT) nd vitmin C used s stndrd. The queous ethnol extrt of M. myristi brks (); showed signifintly higher ontent in polyphenoli ompounds (21.44 ± 0.24 mg ffei id/g dried extrt) nd flvonoid (5.69 ± 0.07 queretin equivlent mg/g of dried weight) s ompred to the other studied extrts. The HPLC nlysis of the brks nd leves reveled the presene of severl polyphenols. The ids (3,4-OH-benzoi, ffei, glli, O- nd P- oumri, syringi, vnilli), lohols (tyrosol nd OH-tyrosol), theobromine, queretin, rutin, tehine nd pigenin were the identified nd quntified polyphenols. All the tested extrts demonstrted high protetive potentil on the superoxide dismutse (SOD), tlse nd peroxidse tivities. Conlusion: Finlly, the different extrts from M. myristi nd speifilly the queous ethnol extrt revel severl properties suh s higher free rdil svenging properties, signifint ntioxidnt pities nd protetive potentil effets on liver enzymes. Keywords: Antioxidnt, Rdil svenging, HPLC, Monodor myristi, Non-timber forest produt Bkground A growing humn popultion, globl limte hnge nd the hnge of terrestril food resoures for energy needs in reent times hve rised serious globl food seurity onerns [1]. Further, the globliztion of mrkets hs lso brought growing globliztion of foods, wekening the boundries of humn res nd geogrphil regions * Correspondene: pieme@yhoo.fr 1 Lbortory of Biohemistry, Deprtment of Biohemistry nd Physiologil Sienes; Fulty of Mediine nd Biomedil Sienes, University of Youndé I, PO Box 1364, Younde, Cmeroon Full list of uthor informtion is vilble t the end of the rtile of the ountries throughout the world [1]. Also, there hs been quest to explore nd use foods from diverse soures to enhne nd supplement the nutritionl qulity of humn foods [1]. Non timber forest produts used in diet hve reently gotten n inresing interest s they onstitute developing soure of food. Moreover, herbs nd spies derived from plnts re widely used for their romti properties s ondiments or sesonings ross Afrin ountries inluding Cmeroon nd hve high impt on the food qulity in this re [2,3]. Nturl ompounds hve been reported to possess ntioxidnt 2015 Moukette et l.; liensee BioMed Centrl. This is n Open Aess rtile distributed under the terms of the Cretive Commons Attribution Liense ( whih permits unrestrited use, distribution, nd reprodution in ny medium, provided the originl work is properly redited. The Cretive Commons Publi Domin Dedition wiver ( pplies to the dt mde vilble in this rtile, unless otherwise stted.

2 Moukette et l. Biologil Reserh (2015) 48:15 Pge 2 of 17 properties, biotivities nd pplitions of preprtions isolted from plnt speies, whih most frequently inlude berries, fruits, vegetbles, mediinl, romti plnts, spies nd other botnils hve been well doumented [4-7]. Polyphenols re biotive ompounds brodly spred in plnts nd they re lso signifint onstituents of the humn diet [2]. Plnts re onsidered s the min soures of ntioxidnts, whih onstitute rih diversity of ompounds suh s flvonoids (nthoynins, flvonols, flvones) nd severl lsses of nonflvonoids (phenoli ids, lignins, stilbenes, terpenoids, et.). These ompounds differ in struture, the number of phenoli hydroxyl groups nd their lotion, leding to vrition in their ntioxidtive nd biologil potentil [8]. Some ulinry herbs nd spies hve been proved to be more effetive ntioxidnts thn ommon food dditives (butylted hydroxynisole, butylted hydroxytoluene nd propyl gllte) nd vitmins (sorbi id, α-toopherol nd β-rotene) [9]. Therefore herbs nd spies rih in ntioxidnts nd other phyto-ompounds re ble to prevent oxidtive stress nd its relted disorders suh s hroni diseses [10,11]. Oxidtive stress origintes from n imblned redox sttus between the prodution of retive oxygen speies (ROS) nd the biologil system ble to remove them [12]. ROS, whih inlude superoxide (O 2 ), hydroxyl rdil ( OH) nd hydrogen peroxide (H 2 O 2 ), re ontinully produed in erobi orgnisms. The endogenous soures of ROS re oxidtive phosphoryltion, P450 metbolism, peroxisomes nd inflmmtory ell tivtion [12,13]. Typilly, ROS viewed s host defending moleules re relesed by the neutrophils for bolishing exogenous pthogens suh s bteri. They n be lso formed s result of exposure to ionizing rdition (IR), hemotherpeuti drugs nd environmentl ontt to trnsition metls nd hemil oxidnts [14]. Cellulr ntioxidnt defense enzymes, ginst oxidtive stress, ontin the superoxide dismutse (SOD), glutthione peroxidse (GPx) nd tlse [13]. SOD nd GPx present in the ytosol nd mitohondri redue the superoxide nion to hydrogen peroxide nd wter, nd remove lso the mjority of hydrogen peroxide respetively [12,15]. Menwhile, tlse, found in the peroxisomes, lso elimintes high levels of hydrogen peroxide. Nonenzymti ntioxidnts suh s vitmin E, vitmin C, β-rotene, glutthione, nd oenzyme Q funtion to quenh ROS [16]. ROS n rbitrrily ret with lipids, proteins nd nulei ids using oxidtive stress nd dmge in these mromoleules leding to pthogenesis of ge-relted nd hroni diseses suh s rdiovsulr disese, dibetes, hroni inflmmtion, neurodegenertive disorders nd ners [15,17-19]. Ative oxygen my be involved in rinogenesis through two possible mehnisms: the indution of gene muttions tht result from ell injury nd the effets on signl trnsdution nd trnsription ftors [20]. Monodor myristi (M. myristi) grows nturlly in evergreen forests from Liberi to Nigeri, Cmeroon, Angol nd Ugnd nd west Keny [21]. The fruits nd seedsredriedndsoldinwholeorgroundtobeusedin stews, soups, kes nd desserts. They re used s stimulnts, stomhi, ginst hedhes, sores nd lso s inset repellent. In the folk mediine, the brk is used in tretments of stomh-hes, febrile pins, eye diseses nd hemorrhoids [21]. The explortion, hrteriztion nd pplition of nturl ntioxidnts re the fous of severl reserh tems in the Sub-Shrn Afri [4,22]. The present study imed to (i) investigte the ntioxidnt properties of M. myristi through different in vitro ssys, (ii) nlyze the phenoli ontent of its leves nd brk using HPLC; nd (iii) determine the protetive effet of these extrts on liver enzymes. Results nd disussion Free rdil svenging potentil nd ntioxidnt properties The svenging bility of DPPH free rdil is widely used to nlyze the ntioxidnt potentil of nturlly derived foods nd plnts. Ethnol nd wter-ethnol mixture were used in this study s solvents for the extrtion of the low moleulr weight nd moderte polr substnes [23]. The DPPH rdil svenging potentil of the different extrts is represented in Tble 1. From this Tble, ll the extrts showed n inhibitory potentil ginst DPPH free rdil. The inhibitory perentges vry from ± 0.55% for the queous ethnol extrt from the brks of M. myristi () to 95.25% ± 0.23% for the vitmin C. The ethnoli extrt from the brks of M. myristi () hs the highest nd signifint (p < 0.05) inhibitory potentil mong the extrts smples tested t the different onentrtions ompred to the other extrts. Plnt ts s eletron donors beuse of their ontent in phenoli ompounds [24]. This my justify the DPPH rdil svenging power noted in the extrts tested. This result orrobortes previous study whih demonstrted tht DPPH svenging properties of plnt extrts inrese with the onentrtion of extrts [25-27]. Severl omplementry methods hve been proposed to ssess the ntioxidnt tivity of plnt extrts nd pure ompounds [28]. In vitro ssys for the free rdil svenging pity re usully bsed on the intivtion of rdils, suh s hydroxyl (OH) nd nitri oxide (NO) rdils. The Tble 2 presents the results of the OH rdil svenging tivity of the extrts. At the lowest onentrtion of extrt (25 μg/ml), the svenging properties of (25.03 ± 0.57%) nd (25.24 ± 0.91%) re higher (p < 0.05) thn those of (17.45 ± 0.81%) nd (19.33 ± 1.47%). As the onentrtion rises, n inrese of

3 Moukette et l. Biologil Reserh (2015) 48:15 Pge 3 of 17 Tble 1 DPPH svenging potentil of the different plnt extrt DPPH inhibition (%) Conentrtions smples (μg/ml) ± ± ± ± ± ± 0.82 b ± 0.41 b ± 2.51 b ± 2.54 b ± 0.63 b ± 041 b ± 0.40 b ± 0.63 b ± 0.54 b ± ± ± ± ± ± 0.25 d Vit C ± 1.69 d ± 0.83 d ± 0.63 d ± 0.63 d ± 0.23 e Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter ( - e) re signifintly different t p < : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) queous ethnolextrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) queous ethnolextrt; VIT C = Vitmin C. the perentge of inhibition is observed. At the highest onentrtion (300 μg/ml), (79.79 ± 0.56%) nd (77.28 ± 0.44%) showed the highest inhibition. Vitmin C used s positive ontrol hs the best svenging tivity with perentge of inhibition of ± 0.28%. The NO rdil svenging property of the extrt is represented in Tble 3. These results demonstrted tht (28.02 ± 1.36%) nd (29.71 ± 0.18%) showed the higher (p < 0.05) svenging potentil t the onentrtion of 25 μg/ml. The inhibitory potentil of the extrts tested rise with the ugmenttion of onentrtion. At 300 μg/ ml, showed the highest (p < 0.05) potentil ± 0.00% followed by (76.37 ± 0.26%). Vitmin C used s ontrol showed the overll highest inhibitory potentil. The mehnism involved in the svenging tivity of the smples my be ttributed to the phenoli ompounds found in the plnt extrts s desribed in previous studies [15,29]. Furthermore, the rdil-svenging tivity of polyphenols is relied to the moleulr struture, the substitution pttern of the hydroxyl groups, the vilbility of phenoli hydrogen nd the possibility of stbiliztion of the resulting HO nd NO rdils vi hydrogen dontion or through expnsion eletron deloliztion [8]. The ABTS method is known to be rpid method for the determintion of the ntioxidnt tivity nd ould be useful tool to sreen smples nd ultivrs in order to obtin high ontent of nturl ntioxidnts in foods [30]. The ABTS + svenging tivity is presented in the Tble 4. This results show tht t ll the onentrtion, the showed higher inhibitory potentil ompred to other smples with mximum of the perentge of inhibition of ± 0.05% t 300 μg/ml. Previous study suggests tht the flvonoid found in the plnts exerts the ntioxidnt tion by donting of hydrogen tom to brek the free rdil hin [31]. Our results demonstrted the presene of higher totl phenol ontent (Tble 5) in the nd ompred to the ethnoli extrt of the leves from M. myristi () nd hydro-ethnoli extrt of the leves from M. myristi (). The Figure 1 represents the redutive potentil of ll the tested smples. The results showed tht ll the extrts hve redutive tivity, whih inrese proportionlly with the onentrtion. The smples nd demonstrted the highest (p < 0.05) redutive potentil ompred to nd but this vlue remin lower thn vitmin C. Phenoli ids re one of the min phenoli lsses within the Plnt Kingdom nd our in the form of esters, glyosides or mides, but rrely in free form [32]. Within this group, flvonoids re some of the most ommon phenolis, widely distributed in plnt tissues, nd often responsible longside the rotenoids nd hlorophylls for their blue, purple, yellow, ornge nd red olors [32]. The totl phenol, flvonoid nd Tble 2 Hydroxyl (OH) rdil svenging potentil of the different plnt extrts OH inhibition (%) Conentrtions smples (μg/ml) ± ± ± ± ± ± 0.81 b ± 0.93 b ± ± 1.79 b ± 0.56 b ± ± ± 0.08 b ± ± ± ± ± ± ± 2.02 Vit C ± 0.28 d ± 1.48 d ± 0.56 d ± 1.46 d ± 0.28 d Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter ( - e) re signifintly different t p < : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) hydroethnoli extrt; VIT C = Vitmin C.

4 Moukette et l. Biologil Reserh (2015) 48:15 Pge 4 of 17 Tble 3 Nitri oxide (NO) rdil svenging potentil of the different plnt extrts NO inhibition (%) Conentrtions smples (μg/ml) ± ± ± ± ± ± 0.55 b ± 0.12 b ± 1.12 b ± 0.34 b ± 0.51 b ± ± ± ± ± ± 0.27 d ± ± 1.70 b ± 0.96 d ± 2.31 b Vit C ± 0.22 e ± 0.64 d ± 0.18 d ± ± 0.01 d Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter ( - e) re signifintly different t p < : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) hydroethnoli extrt; VIT C = Vitmin C. flvonol ontent of the different plnts extrt re represented in the Tble 5. Results in this tble showed tht the smple hs the higher totl phenol ontent (21.44 ± 0.24 mg CA/g DE) followed by, nd. Conerning the flvonoid nd flvonol ontents, the sme extrt () lso showed the higher (p < 0.05) ontent followed by, nd. The phenoli ompounds from plnts re tive ntioxidnts owing to their redox ssets nd hemil struture. They lso hve signifint role in reduing the effets of free rdils, helting trnsitionl metls nd quenhing singlet nd triplet oxygen, by deloliztion or deomposing peroxides [15]. The potentil ntioxidnt tivity of these phenoli ompounds is demonstrted in this study through their svenging effets on vrious rdils. The Tble 6 presents the different vlues of fifty perent inhibitory onentrtion (IC 50 ) of the tested extrts. The results demonstrted tht displyed the lowest IC 50 ginst the DPPH, HO, NO rdils while showed the lowest inhibitory potentil for the ABTS rdil. This results orrelte the sttement tht the IC 50 of the plnt smples is inversely proportionl to its ntioxidnt power thus to its phenoli ontent. The Person orreltion nlysis showed positive orreltion between the polyphenol ontent nd the DPPH svenging potentil (Tble 7). Therefore our study supports tht rdil svenging power of the plnt extrt relies with their phenoli ontent [24]. The use of biohemil ssys to ssess the ntioxidnt power of plnts hs emerged nd beome the best relible nd redily vilble methods. Beuse of vrible response engendered by speifi ntioxidnt in vrious testing systems, it is importnt to utilize diverse ntioxidnt ssys to ppreite the mehnism of tion of the biotive priniple involved [33]. The FRAP nd the phosphomolybdenum ssys re good inditors to hieve suh work. The FRAP ntioxidnt tivities of the different tested smples re displyed in Figure 2. This result demonstrted tht mong these extrts, exhibited the highest (p < 0.05) tivity (35.57 ± 0.14 mg equivlent vitmin C/g of dried extrt (mg eq VtiC/g DE)) ompred to the other extrts. The Figure 3 represents the phosphomolybdenum inhibition potentil of the different plnt extrts. This results show tht ( ± 2.50 mg of sorbi id equivlents/g of dried extrt (mg eq AS/g DE)) nd ( ± 0.73 mg eq AS/g DE) exhibited the higher (p < 0.05) ntioxidnt potentil ompred to ( ± 0.81 mg eq AS/g DE) nd ( ± 0.24 mg eq AS/ g DE). However these vlues remin lower thn tht of BHT ( ± 1.32 mg eq AS/g DE) used s ontrol. Aording to this result, we n suggest tht the different phenoli ompound present in our smples t s ntioxidnt by ting s reduing gents whih onvert free rdils into stble ompounds [34]. These results re similr to previous studies whih demonstrted the ntioxidnt potentil of plnt phytohemils [29,35]. Tble 4 ABTS rdil svenging potentil of the different plnt extrts ABTS inhibition (%) Conentrtions smples (μg/ml) ± ± ± ± ± ± 1.25 b ± 0.26 b ± 0.62 b ± ± ± ± ± ± 2.18 b ± 0.37 b ± 0.26 b ± ± ± ± 0.38 Vit C ± 0.23 d ± 1.36 d ± 1.23 d ± 1.50 d ± 1.73 d Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter ( - e) re signifintly different t p < : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) hydroethnoli extrt; VIT C = Vitmin C.

5 Moukette et l. Biologil Reserh (2015) 48:15 Pge 5 of 17 Tble 5 Totl phenol, flvonoid nd flvonol ontents of the different plnt extrts Smples Phytohemils Polyphenol(CAE/g dried extrt) Flvonoid(QE/g dried extrt) Flvonol(CAE/g dried extrt) ± ± ± ± ± 0.07 b 3.63 ± 0.00 b ± ± ± ± ± ± 0.07 b Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter ( - e) re signifintly different t p < : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) hydroethnoli extrt. The inhibitory effet of the extrts from M. myristi on the oxidtive ell dmge used by H 2 O 2 nd HO through Fenton medited retion ws investigted by lipid peroxidtion ssy. In the biologil system number of end produts of lipid peroxidtion suh s MDA onstitute signifint soure of ell membrne oblitertion nd ell injuries [36]. The results of protetive potentil of extrts ginst lipid peroxidtion re represented in the Figure 4. This result showed tht the oxidnt (positive) ontrol hs higher onentrtion of MDA ( ± 0.88 μmol/l) ompred to the norml (negtive) ontrol (65.13 ± 0.58 μmol/ L). The vitmin C used s ontrol hs signifintly (p < 0.05) inhibited the lipid peroxidtion s showed the level of MDA (70.80 ± 1.46 μmol/l) nd demonstrted the higher inhibitory potentil (42.80 ± 0.75%) ompred to the extrt smples (Figure 5). Among the extrt, the smple, showed the lowest MDA onentrtion (75.82 ± 0.53 μmol/l) ompred to the other smples (Figure 5). The inhibition of lipid peroxidtion by ntioxidnt ompounds is ruil property by whih they n diminish the indution nd/or propgtion of oxidtive stress [29]. Thus, we n onlude tht Mmyristiextrts hve potentil propriety s protetive ompound ginst oxidtive stress [36]. Enzymti nd non-enzymti systems re used by the living orgnism to fight ginst free rdil produe during oxidtive stress [26,29]. The Figure 6 represents the protetive effet of the plnt extrt on the SOD ginst oxidnt. We n observe on this Figure tht the oxidnt (positive) ontrol group exhibited the lowest SOD tivity (2.22 ± 0.27 Unit/min/mg of protein (UI/mg Prot.)) ompre to the other extrts inluding the norml group (negtive) ontrol (7.92 ± 0.13 UI/mg Prot.). Among the extrt tested, (5.51 ± 0.72 UI/mg Prot.) nd AEF (4.05 ± 0.27 UI/mg Prot.) showed the highest protetive tivities. Polyphenols re well-known to possess effetive svenging tivity of free rdils [2]. Therefore, phenoli ompounds of Mmyristipresent in the medium during the inubtion time most likely quenh the rdils formed in the queous phse before reting with enzymes when it is lter dded. Our results orroborte with other studies whih demonstrted tht polyphenols re ble to protet mromoleules from oxidtive stress or inrese their resistne to dmge used by oxidnts [4,37]. The protetive effet 1,95 Redutive tivity (nm) 1,90 1,85 1,80 Vit C 1,75 1, Smple onentrtions (µg/ml) Figure 1 Redutive tivities of the different plnt extrts. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) hydroethnoli extrt; VIT C = Vitmin C.

6 Moukette et l. Biologil Reserh (2015) 48:15 Pge 6 of 17 Tble 6 Different vlues of IC 50 of the plnt extrts on the different rdils tested Smples tests IC 50 (μg/ml) DPPH OH NO ABTS 65,10 ± 0,02 157,57 ± 0,98 148,28 ± 0,13 155,16 ± 0,66 65,80 ± 0,27 99,74 ± 0,11 b 171,38 ± 0,22 b 134,17 ± 0,74 b 14,66 ± 0,31 b 28,32 ± 0,23 80,07 ± 0,50 190,06 ± 0,25 150,66 ± 1,56 154,77 ± 0,44 d 180,18 ± 0,01 d 228,43 ± 0,18 d Vit C 2,55 ± 0,46 d 27,75 ± 0,51 e 41,43 ± 0,58 e 93,05 ± 0,07 e Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter ( - e) re signifintly different t p < : M. myristi (Leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (Leves) hydroethnoli extrt; VIT C = Vitmin C. of the different extrts of M. myristi on the tlse tivity is represented in the Figure 7. These results showed the tlse tivity is signifintly deresed (p < 0.05) in the oxidnt (positive) ontrol (32.30 ± 3.95 UI/mg Prot.) ompred to the other groups. Among the extrt, showed the highest tivity (74.44 ± 1.36 UI/mg Prot.) ompred to (67.37 ± 0.65 UI/mg Prot.), (60.34 ± 0.88 UI/mg Prot.) nd (43.20 ± 0.91 UI/ mg Prot.). Iron ontins unpired eletrons tht enble them to prtiipte in one-eletron trnsfer retions. Hene, they re powerful tlysts of utoxidtion retions. Thus from this results, the endogenous ntioxidnts of M. myristi my be benefiil by voiding the deleterious effets of ion medited oxidtive stress nd ndidtes for the prevention of oxidtive dmge used by ROS [38]. The protetive effets of the different smples on the peroxidse tivity re represented in the Figure 8. From this Figure, we n observe tht the oxidnt (positive) ontrol exhibited the lowest tivity (5.15 ±.63 UI/mg Prot.) ompred (p < 0.05) to the other groups inluding the norml (negtive) ontrol (12.30 ± 0.12 UI/mg Prot.). Among the extrt smples, by (7.90 ± 0.28 UI/mg Prot.) showed the highest tivity followed by (7.14 ± 0.31 UI/mg Prot.). The mehnism of ntioxidnts to remove free rdils implies the trnsfer of hydrogen to free rdil nd hene its redution to n unretive speies through removing the odd eletron feture whih is responsible for rdil retivity [38]. The orreltions between the free rdil svenging properties, ntioxidnt tivity ssys were lso studied using the Person s orreltion nlysis nd the results re presented in the Figure 9 nd Tble 7. All the M. myristi extrts demonstrted positive nd signifint orreltion between ABTS, OH rdils nd totl polyphenol with oeffiient respetively of 0.984; (Tble 7). In the sme wy other positive signifint (p < 0.05) orreltions were found between phosphomolybdenum; FRAP nd flvonoids or between totl phenol ontent. These results were supported by the results of the orreltion of the different extrt effets on liver enzymes in vitro (Figure 10 nd Tble 8). From these results it n be notied positive signifint (p < 0.05) (r 2 = 0.969) orreltion between the totl phenol ontent nd the lipid peroxidtion inhibition. Also these results showed positive orreltion between the totl phenoli ontent nd the tivities of SOD (r 2 = 0.858), tlse (r 2 = 0.758), peroxidse ssys (r 2 = 0.489) supporting the hypothesis tht phenoli ompound svenge these free Tble 7 Results of the Person orreltion of the different in vitro ntioxidnt ssys TESTS OH NO ABTS DPPH RED ACT FLAVONOLS POLYPHENOL FRAP MOLYBDAT FLAVONOIDS OH 1 NO 0,683 1 ABTS 0,965* 0,815 1 DPPH 0,880* 0,761 0,838 1 RED ACT 0,811 0,715 0,766 0,983* 1 FLAVONOLS 0,831 0,265 0,673 0,703 0,612 1 POLYPHENOL 0,984* 0,572 0,906* 0,861 0,786 0,917* 1 FRAP 0,633 0,143 0,433 0,667 0,604 0,920* 0,752 1 MOLYBDAT 0,816 0,456 0,689 0,812 0,718 0,941* 0,888* 0,935* 1 FLAVONOIDS 0,802 0,261 0,633 0,724 0,640 0,994* 0,895* 0,957* 0,962* 1 *:signifint vlues p = 0,050 (bilterl test). MOLYBDAT: Phosphomolybdenum test; FLvonols: Flvonol ssy; Polyphenol: Polyphenol ssy; Flvonoids: Flvonoid ssy; NO: NO rdil svenging test; ABTS: ABTS rdil svenging test; DPPH: DPPH rdil svenging test; OH: OH rdil svenging test; RED ACT: redutive tivity test.

7 Moukette et l. Biologil Reserh (2015) 48:15 Pge 7 of 17 FRAP (mg eq Vitmin C/ g of extrt 38 d 36 b BHT Extrt smples Figure 2 FRAP ntioxidnt tivities of the different plnt extrts. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt; BHT:Butylted hydroxyl Toluene. rdils by proteting therefore these mromoleules from oxidtive dmges. Furthermore positive orreltion hs been found between the FRAP ssy, Phosphomolybdenum test nd SOD, tlse, peroxidse tivity ssys (Tble 8) demonstrting tht the inrese of the ntioxidnt potentil is proportionlly relted to the inrese in the tivity of ntioxidnt enzymes in vitro nd thus derese in the free rdils ontent in the medium. To overwhelm misunderstndings onerning the hoie on the most effetive ntioxidtive extrt in vitro nd in inhibiting lipid peroxidtion, nd lso to help reporting the most relible ntioxidnt tivity order of M. Miristi extrts bsed on sttistil pproh, prinipl omponent nlysis (PCA) ws pplied to the ntioxidnt ssys dt. The totl phenoli flvonoid nd flvonol ontent ssys were not onduted on the pure moleules. Thus, ftor nlysis ws performed on the dt obtined only for plnt extrts. A ftor rottion using the Vrimx method ws performed for two ftor lodings to see the orreltions between ssys tht Antioxidnt power (mg eq Vitmin C/ g of extrt) 180 d 160 b BHT b 40 Extrt smples Figure 3 Phosphomolybdenum ntioxidnt tivities of the different plnt extrts. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt; BHT:Butylted hydroxyl Toluene.

8 Moukette et l. Biologil Reserh (2015) 48:15 Pge 8 of f Vit C Neg ontrol Pos ontrol MDA (µmol/l) 80 b d e Extrt smples Figure 4 Protetive properties of plnt extrts ginst lipid. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt; Vit C: Vitmin C. Pos Control: oxidnt (positive) ontrol. Neg Control: Norml (negtive) ontrol. ounted for the totl ovrine of the plnt extrts [8]. In Figure 9, the vrines used by F1 nd F2 were found 78.87% nd 15.65% respetively. As n be found from the PCA grph, the results from OH, DPPH, redutive tivity, ABTS svenging, Phosphomolybdenum test, flvonoid, flvonol nd totl phenol ontent ssys re respetively losely loded to F1. In ontrry, the NO svenging nd FRAP ssy results pper to be loded highly lose to F2. Therefore, it derived tht the overll ntioxidnt tivity of different solvent extrts of M. Miristi inreses in the following order < < < bsed on the ABTS svenging, Phosphomolybdenum test, flvonoid, flvonol nd totl phenol ontent ssys vlues, hving muh higher ontributions to F1 thn NO ssy. Similr observtionsremdeinfigure10wherethephosphomolybdenum test, flvonoid, flvonol nd totl phenol ontent ssy results re losely burdened to the F1 xis whih hs vrineof82.07%. Inhibition of peroxidtion (%) b d Vit C 25 Extrt smples Figure 5 Lipo-peroxidtive Inhibitory potentil of plnt extrts. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt; Vit C: Vitmin C. Pos Control: oxidnt (positive) ontrol. Neg Control: Norml (negtive) ontrol.

9 Moukette et l. Biologil Reserh (2015) 48:15 Pge 9 of SOD tivity (UI/mg Prot.) b b d f Vit C Pos Control Neg Control 0 Extrt smples Figure 6 Protetive properties of plnt extrts: SOD tivity. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt; Vit C: Vitmin C. Pos Control: oxidnt (positive) ontrol. Neg Control: Norml (negtive) ontrol. HPLC phenoli profile of M. myristi extrts Plnt derived ntioxidnts suh s polyphenols inluding phenoli ids, phenoli diterpenes, flvonoids, tehins, oumri nd rutin re beoming progressively more importnt s dietry ftors. Vrious plnts hve been investigted to identify the presene of phenoli ompouds using HPLC methods. The determintion of phenoli ompounds in the plnt extrts help for their hrteriztion nd their effiient uses s importnt plnt resoures [23]. The identifition of phenoli ompounds in the extrts of M. myristi ws rried out in this study. The results of the phenoli profile of the leves nd brk extrts were presented in the Figures 11 nd 12 while the levels of identified phenols ontent re found in the Tble 9. This result showed tht similr phenoli ompounds were presented both in the leves nd the brk of M. myristi, butin 180 Ctlse tivity (UI/mg Prot.) b d e f g Vit C Pos Control Neg Control 20 0 Extrt smples Figure 7 Protetive properties of plnt extrts: tlse tivity. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt; Vit C: Vitmin C. Pos Control: oxidnt (positive) ontrol. Neg Control: Norml (negtive) ontrol.

10 Moukette et l. Biologil Reserh (2015) 48:15 Pge 10 of Peroxidse tivity (UI/mg Prot.) b bd d f Vit C Pos Control Neg Control 2 0 Extrt smples Figure 8 Protetive properties of plnt extrts: peroxidse tivity. Vlues re expressed s men ± SD of three replites. In the sme olon the vlues ffeted with different letter re signifintly different t p<0.05. Vit C: Vitmin C; : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt. different onentrtions. In generl, the levels of these phenoli moleules were higher in the brk thn the leves (Tble 9). The presene of the moleules n be lssified in three groups bsed on the onentrtion. In the brk extrt of M. myristi, the first group is mde of polyphenol whih onentrtion is higher thn 100 mg/g of dry mteril (Queretin, Eugenol, OH-tyrosol, Rutin), the seond group (with the onentrtion higher thn 35 mg/g of dry mteril) inlude O-oumri id, Tyrosol, Ctehin, Cffei id, Vnilli id nd the lst group with lower onentrtion of phenols (Tble 9). The leves extrt of M. myristi showed moderte or lower onentrtion of phenols ontent. In two extrts three lsses of polyphenols n lso be identified. These inlude phenoli ids (Cffei id, 3,4-OH benzoi id, Syringi id), flvonoids (Queretin, Ctehin) nd other phenoli ompounds. These results orroborte with those presented in the Tble 5. Totl phenol vlue is known to reflet the overll phenolis ontent of plnt or food smple. Beuse of unidentified peks tht were not quntified in the hromtogrms A Individus (xes F1 et F2 : 93,73 %) B Biplot (xes F1 et F2 : 93,73 %) xe F2 (15,65 %) VIT C xe F2 (15,65 %) NO REDACT ABTS DPPH OH 0 POLYPHEN MOLYBDAT FLAVNOLS FLAVNOIDS FRAP VIT C xe F1 (78,07 %) xe F1 (78,07 %) Figure 9 Correltion between ntioxidnt pity nd free rdil svenging properties of the extrts. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydro-ethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. Miristi (leves) hydro-ethnoli extrt; MOLYBDAT: Phosphomolybdenum test; Flvonol: Flvonol ssy; Polyphenol: Polyphenol ssy; Flvonoid: Flvonoid ssy; NO: NO rdil svenging test; ABTS: ABTS rdil svenging test; DPPH: DPPH rdil svenging test; OH: OH rdil svenging test; RED ACT: redutive tivity test; A: distribution of the smples round the F1 nd F2 xis; B: projetion of the smples nd tests round the F1 nd F2 xis.

11 Moukette et l. Biologil Reserh (2015) 48:15 Pge 11 of 17 A Individus (xes F1 et F2 : 93,81 %) B Vribles (xes F1 et F2 : 94,52 %) 6 1 PEROXIDASE xe F2 (6,80 %) VIT C xe F2 (16,10 %) FLAVNOLS FLAVNOIDS FRAP MOLYBDAT 0 POLYPHEN INHIB MDA CATALASE 4 SOD xe F1 (87,02 %) xe F1 (78,42 %) Figure 10 Correltion between enzymes involved in oxidtive stress nd phenol ontents of the extrts. : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. Miristi (leves) hydroethnoli extrt; SOD: SOD tivity test; Ctlse: Ctlse tivity test; Peroxidse: Peroxidse tivity test; FLvonols: Flvonol ssy; Polyphen: Polyphenol ssy; Flvonoids: Flvonoid ssy; FRAP: FRAP ntioxidnt test; MDA: MDA ssy; INHIB MDA: MDA inhibition perentge; A: distribution of the smples round the F1 nd F2 xis; B: projetion of the smples nd tests round the F1 nd F2 xis. (Figures 11 nd 12), the determintion of the totl phenol ontent seems to be inomplete. However the results of HPLC orrelted nd onfirmed the previous results. Also this result onfirmed tht the lssifition of nd extrts s the most powerful ntioxidnt mong tested the extrt. Phenolis re group of non-essentil dietry omponents nd their hydrogen donting property is responsible for the inhibition of free rdil indued LPO [39]. The orreltion index shows tht phenolis re minly responsible for produing lipo-protetive tivity in the extrts. In ddition other non-phenoli phytohemils present in extrts might lso be involved in imprting some degree of enzyme protetion. Thus phenoli ontents present in M. myristi extrts ould be ountble for their ntioxidnt nd protetive tivities. However, other studies need to be rried out to isolte individul tive priniples nd determiner other phrmologil properties of these extrts. Conlusion From the present study, we n onlude tht M. myristi extrts exhibited higher free rdil svenging properties s well s the protetive potentil ginst few mrkers of liver homogente involved on oxidtive stress nd this ould be linked to their ntioxidnt potentil pity. This informtion is supported by the results of HPLC of phenoli profile of the extrts. However, further investigtions need to be done either to isolte the ntioxidnt ompounds or to determine the in vivo biologil tivity of these extrts. Tble 8 Results of Person orreltion for in vitro ntioxidnt ssys with liver enzymes nd extrts Assys SOD CATALASE PEROXIDASE INHIB MDA FLAVNOLS POLYPHEN FRAP MOLYBDAT FLAVNOIDS SOD 1 CATALASE 0,926* 1 PEROXIDASE 0,064 0,197 1 INHIB MDA 0,897* 0,849* 0,315 1 FLAVNOLS 0,793 0,565 0,631 0,872* 1 POLYPHEN 0,858* 0,758 0,489 0,969* 0,917* 1 FRAP 0,817 0,556 0,405 0,749 0,920* 0,752 1 MOLYBDAT 0,906* 0,690 0,432 0,843* 0,941* 0,888* 0,935* 1 FLAVNOIDS 0,829 0,594 0,562 0,862* 0,994* 0,895* 0,957* 0,962* 1 *: signifint vlues p = 0,050 (bilterl test). SOD: SOD tivity test; Ctlse: Ctlse tivity test; Peroxidse: Peroxidse tivity test; FLvonols: Flvonol ssy; Polyphen: Polyphenol ssy; Flvonoids: Flvonoid ssy; FRAP: FRAP ntioxidnt test; MDA: MDA ssy; INHIB MDA: MDA inhibition perentge.

12 Moukette et l. Biologil Reserh (2015) 48:15 Pge 12 of 17 Figure 11 HPLC hromtogrms of phenoli extrts from the leves of M. myristi reorded t 280 nm (TR: 19.10: 3,4-OH benzoi id; 33.49:pigenin; 25.67: ffei id; 23.48: tehine; 29.43: eugenol; 14.38; glli id; 25.11: O-oumri; 21.91:OH-tyrosol; 30.52: P-oumri id : queretin; 29.45: rutin; 25.55: syringi id; 17.35: theobromine; 21.77: tyrosol nd 25.27: vnilli id.). Figure 12 HPLC hromtogrms of phenoli extrts from the brks of M. myristi reorded t 280nm (TR: 19.10: 3,4-OH benzoi id; 33.49:pigenin; 25.67: ffei id; 23.48: tehine; 29.43: eugenol; 14.38; glli id; 25.11: O-oumri; 21.91:OH-tyrosol; 30.52: P-oumri id : queretin; 29.45: rutin; 25.55: syringi id; 17.35: theobromine; 21.77: tyrosol nd 25.27: vnilli id.).

13 Moukette et l. Biologil Reserh (2015) 48:15 Pge 13 of 17 Tble 9 Representtion of the mounts of phenoli ompounds in the different plnt prts Phenoli stndrds Stndrd retention time M. myristi (brks) M. myristi (leves) hrteristis T.R (min) A(mUA) Con (mg/g DW) A(mUA) Con (mg/g DW) 3,4-OH benzoi id 19,10 ± ± ± ± ± 0.00 Apigenin 33,49 ± ± ± ± ± 0.00 Cffei id 25,67 ± ± ± ± ± 0.00 Ctehine 23,48 ± ± ± ± ± 0.00 Eugenol 29,43 ± ± ± ± ± 0.00 Glli id 14,38 ± ± ± ± ± 0.00 O-oumri id 25,11 ± ± ± ± ± 0.00 OH-tyrosol 21,91 ± ± ± ± ± 0.00 P-oumri id 30,52 ± ± ± ± ± 0.00 Queretin 42,19 ± ± ± ± ± 0.00 Rutin 29,45 ± ± ± ± ± 0.00 Syringi id 25,55 ± ± ± ± ± 0.00 Theobromine 17,35 ± ± ± ± ± 0.00 Tyrosol 21,77 ± ± ± ± ± 0.00 Vnilli id 25,27 ± ± ± ± ± 0.00 Con: onentrtion; DW: Dried Weight; T.R: retention time; A: re. Methods Plnt mteril The leves nd brks of M. myristi were olleted t the Kl Mountin in the Center region of Cmeroon. They were uthentited by M. NANA, botnist of the Ntionl Herbrium of Cmeroon, in omprison to the vouher speimens (27690/SFR/CAM). Preprtion of plnt extrts The olleted leves nd brks were dried t mbient temperture nd rushed. The powders were then merted in the rtio of 1:10 (w/v) for 48 h in ethnol for the ethnoli extrt nd in mixture of wter + ethnol (30/ 70); ph = 3 for the hydro-ethnoli extrt. The mixtures were then filtered using Buhner funnel nd Whtmn No 1 filter pper. This proess ws repeted one on the residue. The filtrte ws onentrted using rotvpor nd the solution ws dried in the oven t 55 C for two dys. Eh rude extrt obtined ws lbelled using the following odes: : M. myristi (leves) ethnoli extrt; : M. myristi (Brks) hydroethnoli extrt; : M. myristi (Brks) ethnoli extrt; : M. myristi (leves) hydroethnoli extrt. The different smples were then kept t 4 C. Prior to the experimenttion, the solutions of the four plnt extrts were dissolved using ethnol different dilutions (25, 50, 75, 150, 300 μg/ml) of eh. Animls Mle lbino Wistr rts weighing g were used in this study. The rts were mintined t room temperture under the lb onditions nd were fed with stndrd diet nd wter d libitum. Livers were olleted fter depittion of the rts under mild ether nesthesi from overnight fsted rts. This study ws rried out with pprovl from the niml Ethis Committee of university of Youndé I. Determintion of the free rdil svenging potentil of the smples Svenging tivity of DPPH rdil The DPPH ssy mesures the free rdil svenging pity of the extrts s desribed previously [40]. Three milliliters of eh of the diluted extrts were put in the test tube nd 1 ml of methnol solution of DPPH (0.1 mm) ws dded. The mixture ws kept in the drk t room temperture for 30 min nd bsorbne ws mesured t 517 nm ginst blnk. The sme proedure ws used for the vitmin C used s stndrd. The following eqution ws used to determine the perentge of the rdil svenging tivity of eh extrt. Svenging effet ð% Þ ¼ 100 ða 0 A S Þ=A 0 where Ao is the bsorbne of the blnk nd As the bsorbne of the smple. Svenging effet of the ABTS+ rdil TheABTSssywsbsedonpreviouslydesribed method [41] with slight modifitions. ABTS rdil tion (ABTS+) ws produed by the retion of 7 mm ABTS solution with potssium persulphte

14 Moukette et l. Biologil Reserh (2015) 48:15 Pge 14 of 17 (2.45 mm). The ABTS + solution ws diluted with ethnol to n bsorbne of 0.70 ± 0.05 t 734 nm. The mixture ws stored in the drk t room temperture for 12 h before used. After ddition of 25 μl of extrt smple or vitmin Cusedsstndrdto2mLofdilutedABTS + solution, bsorbne ws mesured t 734 nm fter extly 6 min. The derese in bsorption ws used for lulting svenging effet vlues. The following eqution ws used to determine the perentge of the rdil svenging tivity of eh extrt. Svenging effet ð% Þ ¼ 100 ða 0 A S Þ=A 0 where Ao is the bsorbne of the blnk; As is the bsorbne of the smple. Nitri oxide svenging tivity Nitri oxide svenging tivity ws determined ording previous uthors [42]. The retion mixture ontined 2 ml of sodium nitroprusside (10 mm) in 0.5 ml phosphte buffer (0.5 M; ph 7.4). Vrious onentrtions (25, 50, 75, 150, 300 μg/ml) of the extrts (0.5 ml) were dded in finl volume of 3 ml. After inubtion for 60 min t 37 C, Griess regent [α-npthyl-ethylenedimine (0.1%) nd sulphnili id (1%) in H 3 PO 4 (5%)] ws dded. The pink hromophore generted during dizotiztion of nitrite ions with sulphnilmide nd subsequent oupling with α-npthylethylenedimine ws mesured spetrophotometrilly t 540 nm. Asorbi id ws used s positive ontrol. The svenging bility (%) of the nitri oxide ws lulted using the formul: Svenging effet ð% Þ ¼ 100 ða 0 A S Þ=A 0 where Ao is the bsorbne of the blnk nd As the bsorbne of the smple. Hydroxyl rdil svenging tivity The svenging tivity of the extrts on hydroxyl rdil ws mesured ording to previously desribed method [43]. To 1.5 ml of eh diluted extrt, 60 μl of FeCl 3 (1 mm), 90 μl of 1,10-phennthroline (1 mm), 2.4 ml of phosphte buffer (0.2 M; ph 7.8) nd 150 μl of H 2 O 2 (0.17 M) were dded respetively. The mixture ws then homogenized using vortex nd inubted t room temperture for 5 min. The bsorbne ws red t 560 nm ginst the blnk. The perentge of the rdil svenging tivity of eh extrt ws lulted from the eqution below: Svenging effet ð% Þ ¼ 100 ða 0 A S Þ=A 0 where Ao is the bsorbne of the blnk nd As the bsorbne of the smple. Determintion of the totl ntioxidnt potentil of the different smples Totl ntioxidnt tivity by Ferri Reduing Antioxidnt Power ssy (FRAP) The FRAP ws determined using previously desribed method [44] with slight modifitions. The fresh FRAP regent onsisted of 500 ml of ette buffer (300 mm; ph 3.6), 50 ml of 2,4,6- Tri (2-pyridyl)-s-trizin (TPTZ) (10 mm), nd 50 ml of FeCl 3 6H 2 O (50 mm). The olorimetri mesurement ws performed t 593 nm nd the mesurement ws monitored up to 12 min on 75 μl of eh extrt nd 2 ml of FRAP regent. The vitmin C ws used to drw stndrd urve nd the Butylted Hydroxy Toluene (BHT) ws used for the omprison. The results were expressed s mg equivlent vitmin C/ g of dried extrt (mg eq VitC/g DE). Phosphomolybdenum Antioxidtive Power (PAP) The totl ntioxidnt tivity of the extrts ws evluted by green phosphomolybdenum omplex [45]. An liquot of 10 μl of the extrt solution ws mixed with 1 ml of regent solution (0.6 M sulphuri id, 28 mm sodium phosphte nd 4 mm mmonium molybdte) in miro entrifuge tube. The tubes were inubted in dry therml bth t 95 C for 90 min. After ooling, the bsorbne of the mixture ws mesured t 695 nm ginst blnk. The vitmin C ws used s referene to drw the stndrd urve nd BHT ws used for the omprison. The reduing pities of the nlysed extrts were expressed s mg of sorbi id equivlents/g of dried extrt (mg eq AS/g DE). Reduing power ssy The reduing power of the extrts ws determined by method desribed by Oyizu [46]. Different onentrtions of extrts in 1 ml of distilled wter were mixed with 2.5 ml of phosphte buffer (0.2 M, ph 6.6) nd 2.5 ml of potssium ferroynide (1%). The mixtures were inubted t 50 C for 20 min. Aliquots 2.5 ml of trihloroeti id (10%) were dded to the mixtures nd entrifuged t 3000 rpm for 10 min. The superntnt of the solution (2.5 ml) ws mixed with 2.5 ml of distilled wter nd 0.5 ml of FeCl 3 (0.1%). The bsorbne ws mesured t 700 nm. Determintion of the phenoli ontent of the extrts Totl phenol determintion The totl phenol ontent ws determined by the Folin Ciolteu method [47]. The retion mixture ontined 200 μl of extrt, 800 μl of freshly prepred diluted Folin Ciolteu regent nd 2 ml of sodium rbonte (7.5%). The finl mixture ws diluted to 7 ml with deionized wter nd kept in the drk t mbient onditions for 2 h to omplete the retion. The bsorbne ws

15 Moukette et l. Biologil Reserh (2015) 48:15 Pge 15 of 17 mesured t 765 nm. Cffei id ws used s stndrd nd the results were expressed s mg ffei id/g dried extrt (mg CA/g DE). Determintion of totl flvonoid ontent Totl flvonoid ontent ws determined using luminium hloride (AlCl 3 ) ording to known method [48]using queretin s stndrd. A volume of 0.1 ml of spie extrt ws dded to 0.3 ml distilled wter followed by 0.03 ml of NNO 2 (5%).After5mint25 C,0.03mLof AlCl 3 (10%) ws dded. After further 5 min, the retion mixture ws mixed with 0.2 ml of 1 mm NOH. Finlly, the retion mixture ws diluted to 1 ml with wter nd the bsorbne ws mesured t 510 nm. The results were expressed s queretin equivlent mg/g of dried extrt (QE/g dried ext). Determintion of totl flvonols Totl flvonols in the plnt extrts were estimted using known method [49] with modifitions. To 2.0 ml of smple, 2.0 ml of 2% of ethnoli solution of AlCl3 nd 3.0 ml (50 g/l) sodium ette solutions were dded. After 2.5 h of inubtion t 20 C, the bsorbne ws red t 440 nm. The results were expressed s queretin equivlent (mg/g) dried extrt (QE/g dried ext). Quntifition of phenoli ompounds by HPLC High Performne Liquid Chromtogrphy (HPLC) with UV detetion is frequently used for the seprtion nd detetion of phenoli ompounds in extrts. Smples were dissolved in pure wter ording to the rtio (0.3 g/10 ml) nd entrifuged t 4706 rpm for 10 min. The superntnt ws filtered through ellulose ette membrne filter (0.20 μm or 0.45 μm, Shleiher & Shuell) nd used for nlysis. The nlysis ws performed on n Agilent Tehnologies 1200 HPLC system fitted with SUPELCOSIL LC-18 olumn (length 250 mm, dimeter 4.6 mm, pkging size 5 mm). The olumn temperture ws set t 20 C. The mobile phse onsisted of mixture of n queous solution of eti id (0.5%) by volume ( A ) nd eti nitrile ( B ). Elution ws performed by using 100% of A for the first 2 min of the run, 40% of A nd 60% of B from 2 to 60 min. The flow rte ws set equl to 1 ml/min nd the injetion volume ws 25 mirolitres. Polyphenols were deteted by UV detetor (280 nm). The retention times of the identified polyphenoli ompounds of interest were mesured by single stndrd solution t onentrtion of 100 mg/l. Protetive properties of the plnt ginst oxidtive dmge Preprtion of liver homogente The liver ws isolted from 3 norml lbino Wistr rts. The orgns were weighed nd 10% (w/v) homogente ws prepred in phosphte buffer (0.1 M, ph 7.4 hving 0.15 M KCl) using the homogenizer t 4 C [29]. The homogente ws entrifuged t 3000 rpm for 15 min nd the ler ellfree superntnt obtined ws used for the study. Preprtion of the pro-oxidtive solution The oxidnt solution ws prepred, diretly before its utiliztion by dding solution of ferri hloride 100 mm to H 2 O % prepred in phosphte buffer (0.1 M, ph 7.4). This solution ws used for the investigtion of the protetive ssys on liver enzymes. Totl protein ontent The totl protein ontent of the mixture of liver ws mesured ording to the protein kit supplier method (Humn Kit-Hu102536, Boehringer Ingelheim, Germny). This result ws used to express the tivities of the different enzymes per grm of orgns. In vitro lipid peroxidtion ssy Lipid peroxidtion ssywsperformedbyformerlydesribedprotool[50]. Phosphte buffer 0.58 ml (0.1 M; PH 7.4), 200 μl smple, 200 μl liver homogente nd 20 μl ferri hloride (100 mm) were ombined to form mixture whih ws pled in shking wter bth for 1 h t 37 C. The retion wstermintedbydding1mltca(10%),tba1ml (0.67%) to ll the tubes whih were pled in boiling wter bth for 20 min. Then test tubes were shifted to rushed ie bth nd were entrifuged t 3000 trs/rpm for 10 min. Absorbne of the superntnt ws heked t 535 nm nd ws lulted s nm of MDA tissue using molr extintion oeffiient of /M.m. Determintion of peroxidse tivity Peroxidse tivity ws determined by the peroxidse kit (CAS Number , Sigm Aldrih) supplier with modifitions. A solution ontining the mixture of 1 ml of the oxidnt solution (FeCl 3, 100 mm) nd extrt or vitmin C (stndrd) for finl onentrtion of 100 μg/ml ws inubted for 1 h in wter bth t 37 C. An liquot of PBS (0.1 ml), hydrogen peroxide (50 μl), nd pyrogllol solution (110 μl) were dded to distilled wter (625 μl) tht ws erlier dispensed into n Eppendorf tube. The plnt extrt (75 μl) from the mixture ws therefter dded. For the blnk, the ontrol oxidnt solution nd the vitmin C s stndrd, the sme regents were used, exept the extrt whih ws repled by distilled wter (75 μl). The retion ws mixed nd inubted for t lest 10 min. The solution ontining 100 mm, ph 6.0 PBS (40 μl) nd 0.002% (v/v) diluted liver homogente (40 μl) were dded to the blnk nd test mixtures respetively. These were mixed, nd the inrese in bsorbne t 420 nm ws mesured t every 10 s for 3 min using spetrophotometer (BioMte 3S UV-Visible, Thermo Sientifi Mnufturer, Wohlen,