impact of different extraction solvents on bioactive...

Research ArticleImpact of Different Extraction Solvents on BioactiveCompounds and Antioxidant Capacity from the Root ofSalacia chinensis L

Thanh Van Ngo12 Christopher James Scarlett1 Michael Christian Bowyer1

Phuong Duc Ngo3 and Quan Van Vuong1

1School of Environmental and Life Sciences The University of Newcastle PO Box 127 Ourimbah NSW 2258 Australia2Vietnam National University of Forestry Xuan Mai Chuong My Hanoi Vietnam3Investment and Development of Southern Herb JSC Quynh Luu District Nghe An Province Vietnam

Correspondence should be addressed toThanh Van Ngo thanhngovanuoneduauand Quan Van Vuong vanquanvuongnewcastleeduau

Received 29 July 2016 Revised 15 September 2016 Accepted 4 October 2016 Published 11 January 2017

Academic Editor Eduardo Puertolas

Copyright copy 2017 Thanh Van Ngo et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study aimed to study the impact of selected common organic solvents on extractable solids phytochemical compositionand antioxidant capacity of S chinensis The results showed that the tested solvents played an important role in extraction oftotal solid and phytochemical composition as well as antioxidant capacity of S chinensis Acetone (50 vv) was found to be theoptimal extraction solvent for extractable solids (122) phenolic compounds (60mg GAEg DW) flavonoids (100mg CEg DW)proanthocyanidins (474mg CEg DW) and saponins (754mg EEg DW) as well as antioxidant capacity (ABTS 334mMTEg DWDPPH 470mM TEg DW FRAP 347mM TEg DW and CUPRAC 310mM TEg DW) The extract prepared from 50 acetonehad high levels of bioactive compounds (TPC 555mg GAEg CRE flavonoids 819mg CEg CRE proanthocyanidins 392mg CEgCRE and saponins 1880mg EEg CRE) as well as antioxidant capacity (ABTS 414mM TEg DPPH 407mM TEg FRAP 320mgTEg and CUPRAC 623mM TEg) thus further confirming that 50 acetone is the solvent of choice Therefore 50 acetone isrecommended for extraction of phenolic compounds their secondary metabolites saponins and antioxidant capacity from theroot of S chinensis for further isolation and utilisation

1 Introduction

Salacia chinensis L (S chinensis) belongs to the genus Salaciaof the family Celastraceae The S chinensis tree can grow upto 3ndash10m in height and 16 cm in diameter in the tropicalforests of Africa China India and South East Asia includingLaos Cambodia and Vietnam S chinensis has been widelyused as a traditional medicine to treat various ailmentssuch as arthritis leucorrhoea inflammation fever as anastringent and amenorrhea [1] In Vietnam the root of Schinensis has been used for the treatment of rheumatismback-pain and debility [2] S chinensis has been linked withantimicrobial antidiabetic antioxidant antimutagenic andanticancer properties due to the material being found tocontain high levels of phenolic and flavonoid compounds andpossessing strong antioxidant capacity [1]

One of themost important factors affecting the extractionefficiency of bioactive compounds from plant materials andtheir consequent health benefits is the extraction solventTraditionally S chinensis has been brewed or decocted inwater for use as a traditional medicine in some Asian coun-tries such as India Sri Lanka and Vietnam [2 3] Previousstudies have used methanol petroleum ether chloroformethanol acetone and water as the solvents for extractingbioactive compounds from S chinensis for further analysis[4ndash6] Although extraction solvents have been extensivelystudied in other plant materials such as macadamia skinwaste [7] S chinensis fruit pulp [8] and basil leaf [9] noneof the previous studies have compared the impact of differentcommon solvents on the extraction efficiency of phenoliccompounds from the S chinensis root Therefore this studyaimed to determine the impact of different common solvents

HindawiJournal of Food QualityVolume 2017 Article ID 9305047 8 pageshttpsdoiorg10115520179305047

2 Journal of Food Quality

(water absolute methanol ethanol acetone 50 methanol50 ethanol and 50 acetone) on the extraction efficiency ofbioactive compounds aswell as antioxidant capacity from theroot of S chinensis in order to identify the most appropriatesolvent for further extraction and isolation of bioactivecompounds and antioxidant capacity from S chinensis

2 Materials and Methods

21 Materials The root of S chinensis L was collected fromNghe An Province (Vietnam) in May 2015 After collectionthe root was sun-dried which is the traditional preparationmethod to obtain the dried sample The dried root was thenground into small particles using a commercial blender (JohnMorris Scientific Chatswood NSW Australia) and thensieved using a steel mesh sieve (14mm EFL 2000 EndecottsLtd London England) The ground root was kept at minus20∘Cfor further analysis

22 Methods for Characterisation of the Root of S chinensis221 Extraction Process Seven common solvents were usedfor the extraction of bioactive compounds from the groundroot of S chinensis L including water absolute methanolethanol acetone (the polarity indexes are 102 51 43 and51 resp) 50 methanol 50 ethanol and 50 acetoneThe sample was extracted in these solvents by firstly adding1 g of sample into 100mL of solvent The mixture wasthen put in an ultrasonic bath (Soniclean 220V 50Hz and250W Soniclean Pty Ltd Thebarton Australia) with presetconditions temperature of 35∘C time of 30min and powerof 150W (the mixture was vortexed thoroughly once everyfive minutes) Next the extract was immediately cooled onice to room temperature and then filtered using filter paper(Whatman 11 120583m pore size) Subsequently the extract wasstored in the dark at minus18∘C for further determination ofthe extractable solids total phenols content (TPC) totalflavonoids content (TFC) total proanthocyanidins content(TPrC) total saponins content (TSC) and antioxidant capac-ity (DPPH FRAP CUPRAC and ABTS assays)

222 Preparation of Saponin and Phenolic Enriched Extractfrom S chinensis Firstly 100 g of sample was added into 2 Lof 50 acetone The mixture was then put in an ultrasonicbath (Soniclean 220V 50Hz and 250W Soniclean Pty LtdThebarton Australia) with preset condition temperature of35∘C time of 30min and power of 150W (the mixturewas vortexed thoroughly once every five minutes) Next theextract was immediately cooled on ice to room temperatureand then filtered using filter paper (Whatman 11 120583m poresize) Subsequently the extract was condensed to the volumeof 150mL using a rotary evaporator (Buchi Rotavapor B-480Buchi Australia Noble Park Victoria Australia) and thenfreeze-dried to yield the crude extract This crude extractwas stored in dark containers at minus20∘C for further analysisincluding TPC TFC TPrC TSC and antioxidant capacities(DPPH FRAP CUPRAC and ABTS assays)

223 Determination of Extractable Solids Extractable solidswere determined according to amethod described previously

with minor modification [10] 3mL of the extract was put ina pottery tray and then placed in an oven set at 120∘C fordrying during 5 h to remove all moisture Extractable solids(ES) were calculated by the following formula

ES () = 119882 times 1003

(1)

(119882 is weight of 3mL of the extraction after drying in grams)

224 Determination of Chemical Properties

Total Phenolic Content (TPC) TPC of Salacia chinensis rootwas determined as previously described by [11] 25mL of10 (vv) Folin-Ciocalteu reagent was mixed with 05mL ofdiluted sample The solution was then added to 2mL of 75(wv) Na2CO3 followed by thorough mixing and incubatingin the dark at room temperature for 1 h The absorbance at760 nm was taken using a UV spectrophotometer (VarianAustralia Pty Ltd Victoria Australia) a reagent blank wasset at base level (zero) Gallic acid was used as the standardfor a calibration curve and the results were expressed asmg ofgallic acid equivalents per g of sample dry weight (mg GAEgDW)

Total Flavonoids Content (TFC)TFC of Salacia chinensis rootwas determined as previously described by Dailey and Vuong[12] 2mL of deionized water was mixed with 015mL of 5(wv) NaNO2 and 05mL of diluted sample The solutionwas mixed thoroughly and then left at room temperaturefor 6min Subsequently 015mL of 10 (wv) AlCl3 wasadded and the solution was mixed well and allowed to standfor 6min Finally 2mL of 4 (wv) NaOH and 07mL ofdeionized water were added to get the final volume of 55mLThe solutionwas thenmixed thoroughly and allowed to standfor 15min at room temperature The absorbance at 510 nmwas taken using a UV spectrophotometer (Varian AustraliaPty Ltd Victoria Australia) a reagent blank was set atbase level (zero) Catechin was used as the standard for acalibration curve and the results were expressed as mg ofcatechin equivalents per gramof sample dry weight (mgCEgDW)

Total Proanthocyanidins Content (TPrC) TPrC of Salaciachinensis root was measured as previously described byDailey and Vuong [12] 05mL of diluted sample was mixedwith 3mL of 4 vanillin and followed by adding 15mL HCl37The solution was mixed and allowed to stand for 15minThe absorbance was measured using a spectrophotometer at500 nm a reagent blank was set at base level (zero) Catechinwas used as the standard for a calibration curve and the resultswere expressed as mg of catechin equivalents per gram ofsample dry weight (mg CEg DW)

Total Saponin Content (TSC) TSC of Salacia chinensis rootwas determined as previously described by Vuong et al[11] 05mL of diluted sample was mixed with 05mL of 8vanillin followed by adding 5mLH2SO4 (72)The solutionwas mixed thoroughly and placed on ice to coolThemixturewas then incubated in a water bath at 60∘C for 15min The

Journal of Food Quality 3

mixture was then cooled on ice for approximately 10min andthe absorbance was thenmeasured at 560 nm a reagent blankwas set at base level (zero) Escin was used as the standard fora calibration curve and the results were expressed as mg ofescin equivalents per gram of sample dry weight (mg EEgDW)

225 HPLC Analysis of Bioactive Components in the CrudeExtract of S chinensis Root The solution made of 002 gof crude extract of S chinensis root diluted in 2mL of50 acetone was filtered using a 045 120583m Phenex Syringefilter (Phenomenex) The bioactive components were thenmeasured using a Shimadzu HPLC system (Shimadzu Aus-tralia Rydalmere NSW Australia) using UV detection at254 nm on a 250mm times 46mm Prodigy 5 120583m ODS3ndash100Areversed-phase column (Phenomenex Australia Pty LtdLane Cove NSW Australia) which was maintained at 35∘CThe mobile phases consisted of solvent systems A and B sol-vent A was deionized water acetonitrile orthophosphoricacid 968 3 02 (vvv) solvent B was 100 acetonitrile

A gradient elution schedule was used as follows 100 Afrom 0 to 10min a linear gradient from 100 A to 90 Afrom 10 to 15min and remaining at 90 A to 25min from90 A to 85 A from 25 to 40min from 85 A to 10 Afrom 40min to 42min 10 A to 0 A from 42 to 52minand remaining at 0 A to 57min and then back to 100 Aat 60min with a postrun reequilibration time of 15min with100 A before the next injection The injection volume was50 120583L of the crude extract solution onto the HPLC and theflow rate was 1mLmin

226 Determination of Antioxidant Properties To obtain agreater understanding on the antioxidant properties of theSalacia chinensis root four antioxidant assays were employedincluding theABTS (221015840- azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) assay the DPPH (22-diphenyl-1-picrylhy-drazyl) assay the CUPRAC (cupric reducing antioxidantcapacity) assay and the FRAP (ferric reducing antioxidantpower) assay

ABTS Assay ABTS assay described by Thaipong et al [13]was applied with some modifications A stock solution wasprepared by adding 10mL of 74mMABTS solution to 10mLof 26mM K2S2O8 and left at room temperature in thedark for 15 h and then stored at minus20∘C until required Theworking solution was freshly prepared by diluting 1mL ofstock solution with approximately 60mL of methanol toobtain an absorbance value of 11 plusmn 002 at 734 nm at theday of analysis 285mL of the working solution was addedto 015mL of diluted sample and left in the dark at roomtemperature for 2 h before its absorbance was read at 734 nmusing a UV spectrophotometer (Varian Australia Pty LtdVictoria Australia) Trolox was used as a standard and theresults were expressed as mg trolox equivalents per gram ofdry weight (mg TEg dw)

DPPH Assay DPPH assay introduced by Thaipong et al [13]was applied with some modifications A stock solution wasprepared by dissolving 24mg DPPH in 100mLmethanol and

then stored at minus20∘C until required The working solutionwas then prepared fresh by mixing 10mL stock solution withapproximately 45mL methanol to obtain an absorbance of11 plusmn 002 at 515 nm 285mL of working solution was addedto 015mL of diluted sample and then left under darkness atroom temperature for 3 h before measuring the absorbanceat 515 nm using a UV spectrophotometer (Varian AustraliaPty Ltd Victoria Australia) Trolox was used as standard fora calibration curve and the results were expressed as mg oftrolox equivalents per g of dry weight (mg TEg dw)

CUPRAC Assay CUPRAC assay described by Apak et al [14]was employed with some modifications 1mL of CuCl2 wasmixed with 1mL of neocuproine and 1mL of NH4Ac and11mL of diluted sample The sample was mixed well andincubated at room temperature for 15 h before measuringthe absorbance at 450 nm using a UV spectrophotometer(Varian Australia Pty Ltd Victoria Australia) Trolox wasused as standard for a calibration curve and the results wereexpressed as mg of trolox equivalents per g of sample (mgTEg dw)

FRAPAssay FRAP assay described byThaipong et al [13] wasemployed with some modifications A working FRAP solu-tion was prepared by mixing 300mM Acetate buffer 10mMtripyridyl-s-triazine (TPTZ) in 40mM HCl and 20mMFeCl3 in the ratio of 10 1 1 and mixed at 37∘C in a water bath(Ratek Instruments Pty Ltd Victoria Australia) before use285mL of the working FRAP solution was added to 015mLof diluted sample and incubated at room temperature in thedark for 30min before its absorbance was read at 593 nmusing a UV spectrophotometer (Varian Australia Pty LtdVictoria Australia) Trolox was used as a standard and theresults were expressed as mg trolox equivalents per gram ofdry weight (mg TEg dw)

23 Statistical Analysis The one-way analysis of variance(ANOVA) and the Least Significance Difference (LSD) wereconducted using the IBM SPSS statistical software version23 Data were reported as averages plusmn standard deviationsDifferences between themean levels of the components in thedifferent experiments were taken to be statistically significantat 119901 lt 005 The Pearson correlation test was employedto determine the correlation coefficients among bioactivecompounds and different antioxidant assays

3 Results and Discussion

31 Impact of Extraction Solvents on Extractable SolidsThe result of this study showed that different solvents hadsignificant effects on the extractable solids yield of S chi-nensis root (Figure 1) Absolute methanol had the highestextractable solids (156) followed by 50 ethanol 50methanol and 50 acetone (143 123 and 122 resp)Water extracted half of extractable solids in comparison withabsolute methanol whereas absolute ethanol and absoluteacetone only extracted sim25 of extractable solids extractedby absolute methanol These findings indicated that recoveryyields of crude powder extract prepared from S chinensis


02468

1012141618

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

Extr

acta

ble s

olid

()

AAB

B B

C

D D

Figure 1 Effect of solvents on extractable solids from the root of Schinensis The values are the mean average of three replications foreach solvent plusmn standard deviation Columns not sharing the samesuperscript letter are significantly different 119901 lt 005 (DI waterdeionized water)

root could be significantly affected by the extraction solventsThese findings were in agreement with previous studies onLimnophila aromatica [15] and Phoenix dactylifera L [16]whereby the variation can be explained by the difference insolubility of different compounds in the sample In generalthese findings suggested that absolute methanol or mixtureof 50 (vv) water with methanol ethanol or acetone wasthe solvents of choice for yielding high levels of extractablesolids

32 Impact of Extraction Solvents on Total Phenolic Content(TPC) Our data showed that extraction solvents had asignificant impact on the extraction yields of TPC from theroot of S chinensis (Figure 2) The mixture of 50 (vv)water with methanol ethanol and acetone had the highestextraction yields of TPC followed by absolute methanoland absolute ethanol which accounted for approximately66 and 43 of the TPC extracted by 50 acetone Waterextracted approximately 30 of TPC in comparison to 50acetone whereas absolute acetone only extracted about 12of the TPC extracted by 50 acetone These findings furtherconfirmed that extraction solvents play an important role inthe extraction of phenolic compounds from the sample andthe mixture of 50 (vv) water with methanol ethanol andacetone is the best solvents for maximum extraction of TPC

These findings were supported by previous studies whichalso found that different extraction solvents significantlyaffected the extraction yields of TPC [6 7 15 17] Howeverthe extraction yields of TPC were different depending onthe types of solvent used For example Chavan et al [6]reported that methanol was the best extraction solvent forTPC from the fresh fruit pulp of S chinensis while Daileyand Vuong [7] reported that 50 acetone with water wasthe best solvent for the extraction of TPC from macadamiaskin Furthermore Do et al [15] found that absolute ethanoland acetone were the best extraction solvents for TPC fromLimnophila aromatic The differences can be explained by thevariation in polarities of the solvents which selectively extract

different hydrophobic or hydrophilic phenolic compounds inthe sample thus highlighting the importance of investigatingand identifying the optimal extraction solvent for eachsample type

33 Impact of Extraction Solvents on Total Flavonoid Content(TFC) Our study showed that the extraction solvents had asignificant effect on the extraction of flavonoids (119901 lt 005)(Figure 2) The mixture of 50 (vv) water with acetoneand ethanol had the highest extraction yields of flavonoids(100 and 89mg CEg DW resp) This was followed by50 methanol (85mg CEg DW) while absolute methanolabsolute ethanol and water could only extract 50 30 and20 of flavonoids respectively in comparison with those of50 acetone and 50 methanol Absolute acetone extractedthe lowest flavonoid levels from S chinensis Our findingswere supported by previous studies on S chinensis fruitpulp Limnophila aromatica andMacadamia tetraphylla skinwaste which reported that extraction solvents significantlyaffected flavonoids [6 7 15] The variation can be alsoexplained by the different polarities of compounds whichwere selectively more soluble in different solvents

34 Impact of Extraction Solvents on Total ProanthocyanidinContent The current study found that absolute acetonehad the highest extraction of proanthocyanidins (61mgCEg DW) followed by 50 acetone (474mg CEg DW)(Figure 2) 50 methanol or ethanol only extracted 50of proanthocyanidins in comparison to that extracted byabsolute acetone Water was found to extract the lowestcontent of proanthocyanidins These findings indicated thatextraction solvents play an important role in the extrac-tion efficiency of proanthocyanidins Water has the highestpolarity index whereas acetone has the lowest polarity indexamong the tested solvents thusmost proanthocyanidins fromS chinensis are more hydrophilic and thus acetone is the bestsolvent for extraction of these phenolic compounds

35 Impact of Extraction Solvents on Total Saponin ContentThe results of this study showed that the best solvent forextraction of saponins was 50 acetone (754mg EEg DW)followed by absolute methanol ethanol and 50 (vv) ofthese solvents with water The results also revealed that waterand absolute acetone had the lowest content of saponins (Fig-ure 2) Previous studies also reported that different extractionsolvents significantly affected the extraction efficiency ofsaponins [18 19] In comparison with some Chinese herbalmedicines reported by Chen et al [20] the total content ofsaponins in S chinensis is higher than those of various speciesof herbs such as Artemisia capillaries Codonopsis pilosulaEuryale ferox and Coix lacryma-jobi

36 Impact of Extraction Solvents on Antioxidant PropertiesFour antioxidant assays were used for determining the effectof extraction solvents on the antioxidant capacity of Schinensis extracts Figure 3 shows that the extraction solventsignificantly affected the antioxidant capacity of S chinensisAll four antioxidant assays revealed that antioxidant capacityis in decreasing order with the corresponding solvents used


0

10

20

30

40

50

60

70

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

TPrC

(mg

CEg

DW

)

A

BC

B

BCDCD

DEE

0100200300400500600700800900

TSC

(mg

EEg

DW

)

A

BBBB

C CD

I wat

er

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

0

10

20

30

40

50

60

70TP

C (m

g G

AE

g D

W)

AAA

B

C

De E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one 0

20

40

60

80

100

120

TFC

(mg

CEg

DW

)

AABB

D

EF

G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

Figure 2 Effect of solvents on recovery of total phenolics (TPC) flavonoids (TFC) proanthocyanidins (TPrC) and saponins (TSC) fromthe root of S chinensis The values are the mean of three replications for each solvent plusmn standard deviation Columns not sharing the samesuperscript letter are significantly different 119901 lt 005 (DI water deionized water GAE gallic acid equivalent CE catechin equivalent EEescin equivalent and DW dry weight)

50 acetone gt 50 ethanol gt 50 ethanol gt absolutemethanol gt absolute ethanol gt water gt absolute acetonePrevious studies found that extraction solvents significantlyaffected antioxidant capacities of Saptarangi (S chinensisL) fruit pulp [6] and macadamia skin [7] However theimpact of individual solvents on the antioxidant capacityof different solvents was different For example Chavan etal [6] reported that absolute methanol gave the highestantioxidant capacity followed by ethanol acetone and waterthat had the lowest antioxidant capacity from Saptarangi(S chinensis L) fruit pulp whereas Dailey and Vuong [7]revealed that the highest antioxidant capacities were seenin the combination of organic solvents (methanol ethanolacetonitrile and acetone) with water in the ratio of 1 1 (vv)

The differences in impact of solvents on antioxidantcapacity of S chinensis in the current study can be explainedby the variation of bioactive groups extracted by the differentsolvents Each bioactive group contributed with a differentantioxidant power as these groups were found to have differ-ing correlation with antioxidant capacity (Table 2) Table 2shows that phenolic compounds had a strong correlationwith the four antioxidant properties (119903 gt 095) followed by

flavonoids (119903 gt 067) and saponins (119903 gt 06) Proantho-cyanidins were found to have a weak correlation with antiox-idant capacity of S chinensis These findings revealed thatantioxidant capacity of S chinensis was mainly contributedby phenolic compounds flavonoids and saponins Thesefindings were supported by studies on fruit pulp of Salaciachinensis lilly pilly and bitter melon which reported thatphenolic compounds flavonoids and saponins were mainlyresponsible for antioxidant activity for these tested materials[6 21 22]

37 Phytochemical and Antioxidant Properties and HPLCAnalysis of the Enriched Extract As 50 acetone was foundto be the best extraction solvent for both phenolic andsaponin compounds from S chinensis root a crude extractwas prepared using 50 acetone and the results are shown inTable 1 The results showed that the crude extract had highlevel of phenolics and saponins as well as potent antioxidantcapacity Compared to the result of previous studies theTPC TFC and TPrC of enriched extract of S chinensis root(555mg GAE 819mg CE and 392mg CE per g crude extractresp) are much higher than those of Davidsonia pruriens F


050

100150200250300350400

ABT

S (m

M T

Eg

DW

)

C

A

B

DE

F G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(a)

0

100

200

300

400

500

600

DPP

H (m

M T

Eg

DW

) ABB

C

DE

F

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(b)

050

100150200250300350400

FRA

P (m

M T

Eg

DW

)

AB

B

C

DD

E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(c)

0

50

100

150

200

250

300

350

CUPR

AC (m

g TE

gr D

W)

ABC

EF

gG

D

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(d)

Figure 3 Effect of solvents on antioxidant properties from the root of S chinensis using various antioxidant assays including ABTS DPPHFRAP and CUPRAC The values are the mean of three replications for each solvent plusmn standard deviation Columns not sharing the samesuperscript letter are significantly different 119901 lt 005 (DI water deionized water)

Table 1 Physicochemical and antioxidant properties of the enrichedextract prepared by optimal solvent

Properties ValuesTotal phenolic compounds (mgGAEgCRE) 55522 plusmn 1122

Flavonoids (mgCEg) 81947 plusmn 2706

Proanthocyanidins (mgCEg) 39209 plusmn 238

Saponins (mg EEg) 188083 plusmn 24668DPPH (mMTEg) 40747 plusmn 1140

ABTS (mMTEg) 41438 plusmn 1821

CUPRAC (mMTEg) 62382 plusmn 977

FRAP (mMTEg) 32024 plusmn 1570

Muell (45mg GAE 22mg CE and 32mg CE per g crudeextract resp) [23] Similarly the saponin content of the Schinensis root enriched extract (1880mg EEg crude extract)is many times higher than that of Carica papaya leaf (32mgEEg) [24] Therefore these findings further confirmed that50 acetone is the solvent of choice for extraction of bioactive

2500

2000

1500

1000

500

0

(mV

)

0 10 20 30 40 50 60 70

(min)

Figure 4 HPLC chromatogram detected at 254 nm for the crudeextract of S chinensis root

compounds from S chinensis In addition these findings alsoindicated that the crude extract prepared from 50 acetonehas the potential for further testing biological activities suchas antimicrobial and anticancer properties

From the HPLC analysis (Figure 4) there are severalpeaks in the extract which can be further isolated foridentification as well as testing their properties


Table 2 Correlation between bioactive compounds and antioxidant properties of the root of S chinensis

TPC TFC TPrC TSC119903 119901 value 119903 119901 value 119903 119901 value 119903 119901 value

ABTS 0957 lowastlowastlowastlowast 0730 lowastlowastlowastlowast 0322 ns 0664 lowastlowastlowast

FRAP 0981 lowastlowastlowastlowast 0727 lowastlowastlowastlowast 0202 ns 0606 lowastlowast

CUPRAC 0989 lowastlowastlowastlowast 0739 lowastlowastlowastlowast 0184 ns 0613 lowastlowast

DPPH 0977 lowastlowastlowastlowast 0672 lowastlowastlowast 0248 ns 0602 lowastlowast

Note lowastlowastlowastlowast extremely significant (119901 value lt 00001) lowastlowastlowast extremely significant (00001 lt 119901 value lt 0001) lowastlowast very significant (0001 lt 119901 value lt 001) andns not significant (119901 value ge 005)

4 Conclusion

This study demonstrated that the extraction solvents playan important role in the extraction of important bioactivegroups from S chinensis Absolute organic solvents or waterwas not effective whereas 50 ethanol and 50 acetone weresolvents of choice for yielding high content of extractablesolids phenolic compounds and flavonoids Among thesetwo solvents 50 acetone was found to have the highestlevels of saponins as well as high antioxidant capacity Thisstudy also prepared phenolic and saponin enriched extractsusing 50 acetone and further confirmed that 50 acetonewas the solvent of choice for yielding high content ofphenolics saponins and antioxidant properties Therefore50 of acetone is recommended for extraction of phenoliccompounds their secondary metabolites and saponins fromthe root of S chinensis for further isolation and utilisation

Additional Points

Practical Applications The medicinal properties of herbalplants are mostly determined by the contents of bioactivecompounds such as phenolic compounds flavonoids andsaponins and the antioxidant capacities of the plants Salaciachinensis has been used widely for prevention and treatmentof various diseases such as arthritis diabetes and obesityand therefore it is a potential material for further researchIn this study we have optimised the conditions for extractionof bioactive compounds and determined the antioxidantproperties in S chinensis rootThe result showed that optimalconditions for extraction of bioactive compounds from S chi-nensis root can be applied for further isolation and utilisationin the food and pharmaceutical industries

Competing Interests

The authors declare no conflict of interests

Acknowledgments

The authors also kindly thank to Faculty of Science andIT University of Newcastle the Vietnamese Governmentthrough the Vietnam International Education Development(VIED) theMinistry of Education andTraining theMinistryof Agriculture and Rural Development and the University ofNewcastle for awarding a VIED-TUIT scholarship to ThanhVan Ngo

References

[1] J J Chavan D M Ghadage A S Bhoite and S D UmdaleldquoMicropropagation molecular profiling and RP-HPLC deter-mination of mangiferin across various regeneration stages ofSaptarangi (Salacia chinensis L)rdquo Industrial Crops and Productsvol 76 pp 1123ndash1132 2015

[2] V C Vo Dictionary of Vietnamese Medicinal Plants MedicinalPublishing House 1997

[3] M H S Jayawardena N M W De Alwis V Hettigodaand D J S Fernando ldquoA double blind randomised placebocontrolled cross over study of a herbal preparation containingSalacia reticulata in the treatment of type 2 diabetesrdquo Journal ofEthnopharmacology vol 97 no 2 pp 215ndash218 2005

[4] S S Periyar P M Balu S Kamalraj V Raja and S MurugesanldquoFree radical scavenging activity of Salacia chinensis rootextract in streptozotocin-induced diabetic ratsrdquo BioresearchBulletin vol 4 no 1 2014

[5] M S Sikarwar and M B Patil ldquoAntihyperlipidemic activ-ity of Salacia chinensis root extracts in triton-induced andatherogenic diet-induced hyperlipidemic ratsrdquo Indian Journal ofPharmacology vol 44 no 1 pp 88ndash92 2012

[6] J J Chavan U B Jagtap N B Gaikwad G B Dixit and VA Bapat ldquoTotal phenolics flavonoids and antioxidant activityof Saptarangi (Salacia chinensis L) fruit pulprdquo Journal of PlantBiochemistry andBiotechnology vol 22 no 4 pp 409ndash413 2013

[7] A Dailey and Q V Vuong ldquoEffect of extraction solvents onrecovery of bioactive compounds and antioxidant propertiesfrom macadamia (Macadamia tetraphylla) skin wasterdquo CogentFood amp Agriculture vol 1 no 1 Article ID 1115646 2015

[8] J J Chavan D M Ghadage P R Kshirsagar and S SKudale ldquoOptimization of extraction techniques and RP-HPLCanalysis of antidiabetic and anticancer drug Mangiferin fromroots of lsquoSaptarangirsquo (Salacia chinensis L)rdquo Journal of LiquidChromatography and Related Technologies vol 38 no 9 pp963ndash969 2015

[9] U Złotek S Mikulska M Nagajek and M Swieca ldquoThe effectof different solvents and number of extraction steps on thepolyphenol content and antioxidant capacity of basil leaves(Ocimum basilicum L) extractsrdquo Saudi Journal of BiologicalSciences vol 23 no 5 pp 628ndash633 2016

[10] Q V Vuong J B Golding M H Nguyen and P D RoachldquoProduction of caffeinated and decaffeinated green tea catechinpowders from underutilised old tea leavesrdquo Journal of FoodEngineering vol 110 no 1 pp 1ndash8 2012

[11] Q V Vuong S Hirun P D Roach M C Bowyer P APhillips and C J Scarlett ldquoEffect of extraction conditions ontotal phenolic compounds and antioxidant activities of Caricapapaya leaf aqueous extractsrdquo Journal of Herbal Medicine vol3 no 3 pp 104ndash111 2013


[12] A Dailey and Q Vuong ldquoOptimum conditions for microwaveassisted extraction for recovery of phenolic compounds andantioxidant capacity fromMacadamia (Macadamia tetraphylla)skin waste using waterrdquo Processes vol 4 no 1 article 2 2016

[13] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D Hawkins Byrne ldquoComparison of ABTS DPPH FRAPandORACassays for estimating antioxidant activity fromguavafruit extractsrdquo Journal of Food Composition and Analysis vol 19no 6-7 pp 669ndash675 2006

[14] R Apak K Guclu M Ozyurek and S E Karademir ldquoNoveltotal antioxidant capacity index for dietary polyphenols andvitamins C and E using their cupric ion reducing capabilityin the presence of neocuproine CUPRAC methodrdquo Journal ofAgricultural and Food Chemistry vol 52 no 26 pp 7970ndash79812004

[15] Q D Do A E Angkawijaya P L Tran-Nguyen et al ldquoEffectof extraction solvent on total phenol content total flavonoidcontent and antioxidant activity of Limnophila aromaticardquoJournal of Food and Drug Analysis vol 22 no 3 pp 296ndash3022014

[16] W Kchaou F Abbes C Blecker H Attia and S Besbes ldquoEffectsof extraction solvents on phenolic contents and antioxidantactivities of Tunisian date varieties (Phoenix dactylifera L)rdquoIndustrial Crops and Products vol 45 pp 262ndash269 2013

[17] C T Sulaiman K V Thushar S George and I BalachandranldquoPhenolic characterisation of selected Salacia species using LC-ESI-MSMS analysisrdquo Natural Product Research vol 28 no 13pp 1021ndash1024 2014

[18] O A Wintola and A J Afolayan ldquoPhytochemical constituentsand antioxidant activities of the whole leaf extract of Aloe feroxMillrdquoPharmacognosyMagazine vol 7 no 28 pp 325ndash333 2011

[19] T Pasaribu D A Astuti E Wina and A SumiatisetiyonoldquoSaponin content of Sapindus rarak pericarp affected by particlesize and type of solvent its biological activity on Eimeria tenellaoocystsrdquo International Journal of Poultry Science vol 13 no 6pp 347ndash352 2014

[20] Y-F Chen H-Y Roan C-K Lii Y-C Huang and T-S WangldquoRelationship between antioxidant and antiglycation ability ofsaponins polyphenols and polysaccharides in Chinese herbalmedicines used to treat diabetesrdquo Journal of Medicinal PlantsResearch vol 5 no 11 pp 2322ndash2331 2011

[21] S Tan C Stathopoulos S Parks and P Roach ldquoAn optimisedaqueous extract of phenolic compounds from bitter melon withhigh antioxidant capacityrdquo Antioxidants vol 3 no 4 pp 814ndash829 2014

[22] Q V Vuong S Hirun T L K Chuen et al ldquoPhysicochemicalcomposition antioxidant and anti-proliferative capacity of alilly pilly (Syzygium paniculatum) extractrdquo Journal of HerbalMedicine vol 4 no 3 pp 134ndash140 2014

[23] T L K Chuen Q V Vuong S Hirun M C Bowyer C DGoldsmith and C J Scarlett ldquoOptimum aqueous extractionconditions for preparation of a phenolic-enriched Davidsonrsquosplum (Davidsonia pruriens F Muell) extractrdquo InternationalJournal of Food Science and Technology vol 50 no 11 pp 2475ndash2482 2015

[24] Q V Vuong S Hirun T L K Chuen et al ldquoAntioxidantand anticancer capacity of saponin-enriched Carica papaya leafextractsrdquo International Journal of Food Science and Technologyvol 50 no 1 pp 169ndash177 2015

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


(water absolute methanol ethanol acetone 50 methanol50 ethanol and 50 acetone) on the extraction efficiency ofbioactive compounds aswell as antioxidant capacity from theroot of S chinensis in order to identify the most appropriatesolvent for further extraction and isolation of bioactivecompounds and antioxidant capacity from S chinensis

2 Materials and Methods

21 Materials The root of S chinensis L was collected fromNghe An Province (Vietnam) in May 2015 After collectionthe root was sun-dried which is the traditional preparationmethod to obtain the dried sample The dried root was thenground into small particles using a commercial blender (JohnMorris Scientific Chatswood NSW Australia) and thensieved using a steel mesh sieve (14mm EFL 2000 EndecottsLtd London England) The ground root was kept at minus20∘Cfor further analysis

22 Methods for Characterisation of the Root of S chinensis221 Extraction Process Seven common solvents were usedfor the extraction of bioactive compounds from the groundroot of S chinensis L including water absolute methanolethanol acetone (the polarity indexes are 102 51 43 and51 resp) 50 methanol 50 ethanol and 50 acetoneThe sample was extracted in these solvents by firstly adding1 g of sample into 100mL of solvent The mixture wasthen put in an ultrasonic bath (Soniclean 220V 50Hz and250W Soniclean Pty Ltd Thebarton Australia) with presetconditions temperature of 35∘C time of 30min and powerof 150W (the mixture was vortexed thoroughly once everyfive minutes) Next the extract was immediately cooled onice to room temperature and then filtered using filter paper(Whatman 11 120583m pore size) Subsequently the extract wasstored in the dark at minus18∘C for further determination ofthe extractable solids total phenols content (TPC) totalflavonoids content (TFC) total proanthocyanidins content(TPrC) total saponins content (TSC) and antioxidant capac-ity (DPPH FRAP CUPRAC and ABTS assays)

222 Preparation of Saponin and Phenolic Enriched Extractfrom S chinensis Firstly 100 g of sample was added into 2 Lof 50 acetone The mixture was then put in an ultrasonicbath (Soniclean 220V 50Hz and 250W Soniclean Pty LtdThebarton Australia) with preset condition temperature of35∘C time of 30min and power of 150W (the mixturewas vortexed thoroughly once every five minutes) Next theextract was immediately cooled on ice to room temperatureand then filtered using filter paper (Whatman 11 120583m poresize) Subsequently the extract was condensed to the volumeof 150mL using a rotary evaporator (Buchi Rotavapor B-480Buchi Australia Noble Park Victoria Australia) and thenfreeze-dried to yield the crude extract This crude extractwas stored in dark containers at minus20∘C for further analysisincluding TPC TFC TPrC TSC and antioxidant capacities(DPPH FRAP CUPRAC and ABTS assays)

223 Determination of Extractable Solids Extractable solidswere determined according to amethod described previously

with minor modification [10] 3mL of the extract was put ina pottery tray and then placed in an oven set at 120∘C fordrying during 5 h to remove all moisture Extractable solids(ES) were calculated by the following formula

ES () = 119882 times 1003

(1)

(119882 is weight of 3mL of the extraction after drying in grams)

224 Determination of Chemical Properties

Total Phenolic Content (TPC) TPC of Salacia chinensis rootwas determined as previously described by [11] 25mL of10 (vv) Folin-Ciocalteu reagent was mixed with 05mL ofdiluted sample The solution was then added to 2mL of 75(wv) Na2CO3 followed by thorough mixing and incubatingin the dark at room temperature for 1 h The absorbance at760 nm was taken using a UV spectrophotometer (VarianAustralia Pty Ltd Victoria Australia) a reagent blank wasset at base level (zero) Gallic acid was used as the standardfor a calibration curve and the results were expressed asmg ofgallic acid equivalents per g of sample dry weight (mg GAEgDW)

Total Flavonoids Content (TFC)TFC of Salacia chinensis rootwas determined as previously described by Dailey and Vuong[12] 2mL of deionized water was mixed with 015mL of 5(wv) NaNO2 and 05mL of diluted sample The solutionwas mixed thoroughly and then left at room temperaturefor 6min Subsequently 015mL of 10 (wv) AlCl3 wasadded and the solution was mixed well and allowed to standfor 6min Finally 2mL of 4 (wv) NaOH and 07mL ofdeionized water were added to get the final volume of 55mLThe solutionwas thenmixed thoroughly and allowed to standfor 15min at room temperature The absorbance at 510 nmwas taken using a UV spectrophotometer (Varian AustraliaPty Ltd Victoria Australia) a reagent blank was set atbase level (zero) Catechin was used as the standard for acalibration curve and the results were expressed as mg ofcatechin equivalents per gramof sample dry weight (mgCEgDW)

Total Proanthocyanidins Content (TPrC) TPrC of Salaciachinensis root was measured as previously described byDailey and Vuong [12] 05mL of diluted sample was mixedwith 3mL of 4 vanillin and followed by adding 15mL HCl37The solution was mixed and allowed to stand for 15minThe absorbance was measured using a spectrophotometer at500 nm a reagent blank was set at base level (zero) Catechinwas used as the standard for a calibration curve and the resultswere expressed as mg of catechin equivalents per gram ofsample dry weight (mg CEg DW)

Total Saponin Content (TSC) TSC of Salacia chinensis rootwas determined as previously described by Vuong et al[11] 05mL of diluted sample was mixed with 05mL of 8vanillin followed by adding 5mLH2SO4 (72)The solutionwas mixed thoroughly and placed on ice to coolThemixturewas then incubated in a water bath at 60∘C for 15min The















02468

1012141618

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

Extr

acta

ble s

olid

()

AAB

B B

C

D D











0

10

20

30

40

50

60

70

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

TPrC

(mg

CEg

DW

)

A

BC

B

BCDCD

DEE

0100200300400500600700800900

TSC

(mg

EEg

DW

)

A

BBBB

C CD

I wat

er

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

0

10

20

30

40

50

60

70TP

C (m

g G

AE

g D

W)

AAA

B

C

De E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one 0

20

40

60

80

100

120

TFC

(mg

CEg

DW

)

AABB

D

EF

G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one







050

100150200250300350400

ABT

S (m

M T

Eg

DW

)

C

A

B

DE

F G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(a)

0

100

200

300

400

500

600

DPP

H (m

M T

Eg

DW

) ABB

C

DE

F

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(b)

050

100150200250300350400

FRA

P (m

M T

Eg

DW

)

AB

B

C

DD

E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(c)

0

50

100

150

200

250

300

350

CUPR

AC (m

g TE

gr D

W)

ABC

EF

gG

D

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(d)











2500

2000

1500

1000

500

0

(mV

)

0 10 20 30 40 50 60 70

(min)












4 Conclusion


Additional Points


Competing Interests


Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















02468

1012141618

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

Extr

acta

ble s

olid

()

AAB

B B

C

D D











0

10

20

30

40

50

60

70

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

TPrC

(mg

CEg

DW

)

A

BC

B

BCDCD

DEE

0100200300400500600700800900

TSC

(mg

EEg

DW

)

A

BBBB

C CD

I wat

er

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

0

10

20

30

40

50

60

70TP

C (m

g G

AE

g D

W)

AAA

B

C

De E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one 0

20

40

60

80

100

120

TFC

(mg

CEg

DW

)

AABB

D

EF

G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one







050

100150200250300350400

ABT

S (m

M T

Eg

DW

)

C

A

B

DE

F G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(a)

0

100

200

300

400

500

600

DPP

H (m

M T

Eg

DW

) ABB

C

DE

F

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(b)

050

100150200250300350400

FRA

P (m

M T

Eg

DW

)

AB

B

C

DD

E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(c)

0

50

100

150

200

250

300

350

CUPR

AC (m

g TE

gr D

W)

ABC

EF

gG

D

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(d)











2500

2000

1500

1000

500

0

(mV

)

0 10 20 30 40 50 60 70

(min)












4 Conclusion


Additional Points


Competing Interests


Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40

50

60

70

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

TPrC

(mg

CEg

DW

)

A

BC

B

BCDCD

DEE

0100200300400500600700800900

TSC

(mg

EEg

DW

)

A

BBBB

C CD

I wat

er

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

0

10

20

30

40

50

60

70TP

C (m

g G

AE

g D

W)

AAA

B

C

De E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one 0

20

40

60

80

100

120

TFC

(mg

CEg

DW

)

AABB

D

EF

G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one







050

100150200250300350400

ABT

S (m

M T

Eg

DW

)

C

A

B

DE

F G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(a)

0

100

200

300

400

500

600

DPP

H (m

M T

Eg

DW

) ABB

C

DE

F

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(b)

050

100150200250300350400

FRA

P (m

M T

Eg

DW

)

AB

B

C

DD

E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(c)

0

50

100

150

200

250

300

350

CUPR

AC (m

g TE

gr D

W)

ABC

EF

gG

D

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(d)











2500

2000

1500

1000

500

0

(mV

)

0 10 20 30 40 50 60 70

(min)












4 Conclusion


Additional Points


Competing Interests


Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


050

100150200250300350400

ABT

S (m

M T

Eg

DW

)

C

A

B

DE

F G

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(a)

0

100

200

300

400

500

600

DPP

H (m

M T

Eg

DW

) ABB

C

DE

F

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(b)

050

100150200250300350400

FRA

P (m

M T

Eg

DW

)

AB

B

C

DD

E

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(c)

0

50

100

150

200

250

300

350

CUPR

AC (m

g TE

gr D

W)

ABC

EF

gG

D

DI w

ater

50

met

hano

l

Met

hano

l

50

etha

nol

Etha

nol

50

acet

one

Acet

one

(d)











2500

2000

1500

1000

500

0

(mV

)

0 10 20 30 40 50 60 70

(min)












4 Conclusion


Additional Points


Competing Interests


Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 Conclusion


Additional Points


Competing Interests


Acknowledgments


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

impact of different extraction solvents on bioactive...

Documents