ultrasound-assisted extraction of bioactive...

Research ArticleUltrasound-Assisted Extraction of Bioactive Compounds fromAnnatto Seeds Evaluation of Their Antimicrobial andAntioxidant Activity and Identification of Main Compounds byLCESI-MS Analysis

Julian Quintero Quiroz 1 Ana Maria Naranjo Duran1 Mariluz Silva Garcia2

Gelmy Luz Ciro Gomez1 and John Jairo Rojas Camargo1

1College of Pharmaceutical and Food Sciences University of Antioquia Street 67 No 53-108 Medellin Colombia2Institute of Food Science and Technology (INTAL) Carrera 50g No 12S-91 Itagui Colombia

Correspondence should be addressed to Julian Quintero Quiroz julianquinteroudeaeduco

Received 18 January 2019 Accepted 15 June 2019 Published 16 July 2019

Academic Editor Mitsuru Yoshida

Copyright copy 2019 Julian Quintero Quiroz et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study evaluated the antimicrobial activity (ie against Bacillus cereus and Staphylococcus aureus) and the antioxidant activity(ie ABTS FRAP and DPPH) of annatto seeds extract obtained by ultrasound-assisted extraction A response surface design withthree levels such as pH (2-11) solvent concentration (50-96 ) seed-to-solvent ratio (12ndash110) and treatment time (0-30min) wasemployed to determine the optimal experimental conditions Thus a pH of 70 seed-to-solvent ratio of 17 and treatment time of20 min were selected as optimal rendering an extract having a 062 of bixin 381 mg gallic acidmg equivalent of polyphenolcompounds (ABTS 10357 FRAP 4247 and DPPH 11615 120583M troloxL) and a minimal inhibitory concentration against Bacilluscereus and Staphylococcus aureus of 32 and 16 mgL respectively Further the main bioactive compounds identified by LCESI-MSwere bixin and catechin chlorogenic acid chrysin butein hypolaetin licochalcone A and xanthohumol

1 Introduction

The seeds of annatto (Bixa orellana L) are widely employedas a dye for foods such as butter cheese soft drinks andconfectionery The pigment obtained from this plant isrich in carotenoids (ie bixin) and polyphenol compoundssuch as hypolatin and caffeic acid [1] The antioxidantability of the extract is attributed to the high conjugationof these compounds and their ability to quench singletoxygen deactivation of the excited triplet state of sensitizersusually associated with photosensitization sweeping freeradicals and denaturation of proteins from cell membranesFurther the extract exhibits anti-inflammatory antioxidantand antimicrobial activities [2] However the extractionmethod affects the functional properties of the extractTraditionally different extractionmethods for bioactive com-pounds include percolation solvent extraction or leaching

enzyme-assisted extraction and liquid-liquid microextrac-tion [3] However conventional methods such as solventextraction or leaching require a long processing time expos-ing the bioactive compounds to degradation factors leadingto a low efficiency [4]

To date ultrasound and microwaves-assisted extractionshave been reported for the extraction of several com-ponents Particularly mechanical methods spouted bedultrasound-assisted extraction microwave-assisted extrac-tion and supercritical fluids (CO2) have been employedfor annatto seed extraction [3 5ndash7] Nonetheless thesestudies have been focused exclusively on the extraction ofthe pigment leaving aside the evaluation of the effect of theextraction treatment on activities such as antimicrobial andantioxidant The increase of the extraction yield of the bioac-tive compounds obtained by the ultrasound and microwave-assisted techniques is attributed to the acceleration of mass

HindawiInternational Journal of Food ScienceVolume 2019 Article ID 3721828 9 pageshttpsdoiorg10115520193721828

2 International Journal of Food Science

transfer from the solid-phase to the liquid-phase [3] Theultrasound-assisted extraction (UAE) creates cavitation ofsmall bubbles in the solvent due to the passage of ultrasoundwaves allowing for a greater penetration of the solventwithin the material increasing the surface area On the otherhandmicrowave-assisted extraction implies the formation ofhigh-energy electromagnetic waves changing the molecularrotation and ionicmobility of themediumwithout destroyingthe sample resulting in heating and possibly denaturationof bioactive compounds The last two methods force themigration of all active compounds rapidly from the solid-phase to the solvent-phase However the UAE shows a betterprotection of those compounds due to the lower temperaturegenerated in the medium [8 9]

Further not only the evaluation of the functional activityof the extracts but also the identification of the compoundsresponsible for that activity is crucial In general bixinphenolic acids lignans and flavonoids are the main bioactivecompounds However the isolation and identification ofthese compounds require techniques having a high separa-tion efficiency since coeluting compounds could be missed[10] Currently HPLC coupled with mass spectrometry atatmospheric pressure chemical ionization (APCI) or elec-trospray ionization (ESI) represents the most selective ana-lytical technique for the identification and quantificationof compounds isolated from plants and food sources Theidentification of carotenoids and polyphenol compounds byLCndashESI-MS using a LTQ Orbitrap XL renders an extremelyhigh mass accuracy (lt5 ppm of resolution) This techniqueis capable of multiple levels of fragmentation to reveal themolecular weight (MW) of unknown compounds Hence byusing this technique unknown compounds are identified onthe basis of nominal MW and MSMS fragmentation [10]

The goal of this study was to enhance the extraction ofbioactive compounds from the seeds of annatto and optimizethe extraction conditions (ie solvent concentration pHtreatment time and seed-to-solvent ratio) as compared toa conventional method Further it is expected that theantimicrobial and antioxidant activity is due to the high yieldof extracted compounds as identified by LCESI-MS

2 Materials and Methods

21 Materials Annatto seeds were purchased from a farmersmarket of Medellın (Colombia) which were sun-dried untilreaching amoisture content of 1058plusmn 098 All the reagentsused were synthesis grade

22 Experimental Design The UAE conditions were opti-mized using a response surface plot experimental design(Box-Behnken (BBD)) using the Design Expert Software(Version 806 Stat-EaseUSA)Thedependent variables suchas treatment time (min) (X1 0-30) pH (X2 4-11) solventconcentration () (X3 50-96) and seed-to-solvent ratio (X42-10) were studied The solvent used was absolute ethanol(lot k4958171742 Merck Darmstadt Germany) With theindependent variables studied the statistical program estab-lished 30 experimental units which are presented in Table 1with their respective randomization The two independent

variables studied were the bixin content (Bix) and the totalpolyphenol content found by the Folin-Ciocalteu methodusing a uvvis spectrophotometer (UV-1700 Shimadzu) [11]

The operational conditions of the equipment used for theUAE were a frequency of 37 KHz and a power of 320 W(Elmasonic E30h) Adjustment of the pHwas donewith 01Macetic acid (lot k47109963539 Merck Darmstadt Germany)and 01 M sodium hydroxide solution (lot b0895598319MerckDarmstadt Germany)Themethod ofmultiple regres-sions of least squareswas used in order to optimize the extrac-tion conditions and investigate the effect of the independentvariables The experimental data was adjusted by the second-order polynomial equation (see (1)) by comparison of thedetermination coefficient (r2 ) and the adjusted determinationcoefficient (r2-adj) The quadratic model follows the expres-sion

Y = 1205730 + 1205731X1 + 1205732X2 + 1205733X3 + 1205734X4 + 12057311X11

+ 12057322X2

2+ 12057333X

2

3+ 12057344X

2

4+ 12057312X1X2 + 12057313X1X3

+ 12057314X1X4 + 12057323X2X3 + 12057324X2X4 + 12057334X3X4

(1)

where Y represents the predicted response 1205730 is the interceptof the model 1205731 1205732 1205733 1205734 12057311 12057322 12057333 12057344 y 12057312 12057313 1205731412057323 12057324 and 12057334 are the linear and interaction coefficientsrespectively and X1 X2 X3 and X4 correspond to the inde-pendent variables Analysis of variance (ANOVA)was used toanalyze the significant effects (with a confidence level of 95)of the independent variables over the dependent variablesOnce the significant mathematical models were obtained theextraction process was optimized seeking to maximize theextraction of polyphenols and bixin (weight of 1 for both) andminimizing the seed-to-solvent ratio The calculation of therelative and absolute errors was accomplished between theresponses predicted by the model versus the ones obtainedexperimentally under optimal conditions

23 Characterization of Optimized Annatto Seed ExtractsThe antioxidant and antimicrobial activities of the optimizedextracts obtained by the UAE were compared with (i) anextract obtained by conventional extraction (with ethanolseeds-to-solvent ratio of 12 and continuous agitation for 48h) and (ii) previous results from an optimized microwave-assisted extraction [12] The results are presented as meansand standard deviation (SD) The analysis was performedusing the Statgraphics Centurion XVI software

24 Quantification of Total Polyphenol The total phenolicconcentration in the annatto seed extract was measured bythe Folin-Ciocalteu method [13] Twenty microliter of samplewas mixed with in 158 120583L of distilled water 100 120583L of Folin-Ciocalteu phenol reagent (lot hc43368401Merck DarmstadtGermany) and 300 120583L of 20 sodium carbonate (lota0594092339 Merck Darmstadt Germany) The absorbancewas read at 725 nm in a UVVIS spectrophotometer (UV-1700 Shimadzu) after 1h of storage under darkness Theexperiments were performed in triplicate and the results wereexpressed as mg of gallic acid (GA) (lot SLBM8746V MerckDarmstadt Germany) per gram of seeds (mgGAg seed)

International Journal of Food Science 3

Table 1 The experimental matrix for the UAE of bioactive compounds from annatto seeds

Treatmentnumber

UAETreatment

time(min)

pHSolvent

Concentration()

Seed-tosolvent ratio

(1X4)

UAEPolyphenol content(mg AGg seed)

n=3

Bixin content ()n=3

1 000 750 7300 1000 157 plusmn 0094 017 plusmn 00532 1500 400 5000 600 150 plusmn 0014 015 plusmn 00383 1500 750 5000 1000 192 plusmn 0017 032 plusmn 00304 1500 400 9600 600 215 plusmn 0135 054 plusmn 00495 1500 750 7300 600 206 plusmn 0068 031 plusmn 00296 000 1100 7300 600 190 plusmn 0155 038 plusmn 00357 3000 1100 7300 600 180 plusmn 0023 067 plusmn 00668 1500 1100 9600 600 217 plusmn 0032 057 plusmn 00689 000 750 5000 600 159 plusmn 0119 017 plusmn 001310 1500 750 5000 200 111 plusmn 0137 012 plusmn 008611 3000 400 7300 600 203 plusmn 0056 039 plusmn 001812 1500 400 7300 200 111 plusmn 0029 039 plusmn 001913 1500 750 7300 600 186 plusmn 0073 031 plusmn 001914 3000 750 9600 600 205 plusmn 0157 050 plusmn 003015 1500 1100 7300 1000 249 plusmn 0154 020 plusmn 002516 1500 1100 5000 600 157 plusmn 0065 019 plusmn 000417 000 750 9600 600 176 plusmn 0134 022 plusmn 001618 1500 750 9600 200 153 plusmn 0099 054 plusmn 000819 1500 750 7300 600 211 plusmn 0143 033 plusmn 001220 1500 750 9600 1000 285 plusmn 0066 064 plusmn 003121 3000 750 7300 200 150 plusmn 0132 039 plusmn 002922 1500 750 7300 600 185 plusmn 0246 027 plusmn 005823 1500 1100 7300 200 157 plusmn 0180 047 plusmn 002224 3000 750 7300 1000 272 plusmn 0140 038 plusmn 002525 000 400 7300 600 142 plusmn 0113 024 plusmn 003726 1500 750 7300 600 216 plusmn 0121 033 plusmn 001727 3000 750 5000 600 133 plusmn 0098 027 plusmn 001428 000 750 7300 200 147 plusmn 0064 038 plusmn 004129 1500 400 7300 1000 233 plusmn 0094 028 plusmn 000730 1500 750 7300 600 203 plusmn 0014 035 plusmn 0047Values are expressed as mean plusmn standard deviation (n = 3) UAE ultrasound-assisted extraction AG gallic acid

241 Quantification of Bixin 100 120583L of sample with 2 mLof tetrahydrofuran (lot dg643 Merck Darmstadt Germany)and 10 mL of acetone (lot k43912814243 Merck DarmstadtGermany) were mixed to obtain an absorbance of less than015 at 487 nm taken in a UVVIS spectrophotometer (UV-1700 Shimadzu) The concentration of bixin in the samplewas determined using the following equation [14]

119861119894119909119894119899 (119898119892119898119871) =119860 lowast 100 lowast 119881

11986011119888119898lowast 100

(2)

where11986011119888119898

= 3090 (1g100 mL)minus1lowast1cm minus1 (specific absorptivitycoefficient of bixin in acetone) [14]

A = absorbance value of the sampleV = dilution volume of the sample (mL)

242 Antioxidant Activity ABTS+ Method The ABTS assaywas performed following the method described by Contreraset al 2011 [15] One hundred microliter of sample (dilutedappropriately with water) was mixed with 1 mL of ABTS+solution which was prepared with ABTS standard (221015840-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammo-nium salt lot slbp9592v sigma-Aldrich St Lois USA) dilutedin ethanol The change in coloration was read after 30 minat 730 nm in a spectrophotometer (UV-1700 Shimadzu)Theresults were expressed as trolox equivalents (TE) (6-hydroxy-2578-tetramethylchromane-2-carboxylic acid to 97 lotstbb6668 sigma-Aldrich St Lois USA) or TE 120583mol L

243 Antioxidant Activity Ferric ReducingAntioxidant Power(FRAP) Method The FRAP was measured according to


Benzie and Strain (1996) with modifications [16] Briefly 30120583L of sample 90 120583L of deionized water (diluted appropriatelywith water) and 900 120583L of the FRAP reagent (prewarmed at37∘C) were mixed The sample was incubated for 30 minutesat 37∘C Then the change in coloration was read at 593 nmin a spectrophotometer (UV-1700 Shimadzu) A Trolox (6-hydroxy-2578-tetramethylchromane-2-carboxylic acid to97 lot stbb6668 sigma-Aldrich St Lois USA) calibrationcurve was obtained for quantification purposes and theresults were expressed as TE 120583mol L

244 Antioxidant Activity DPPH Method The DPPH assaywas performed as previously described [17] Briefly 2 mLof 05 mM 22-diphenyl-1-picrylhydrazyl or DPPH (067klot 1154 sigma-Aldrich St Lois USA) reagent was mixedwith 2 mL of methanol (lot l823009611 Merck DarmstadtGermany) and 02 mL of sample (diluted appropriatelywith water) The change in coloration was read after 30min of incubation in darkness at 517 nm in a spectropho-tometer (UV-1700 Shimadzu) A Trolox (6-hydroxy-2578-tetramethylchromane-2-carboxylic acid to 97 lot stbb6668sigma-Aldrich St Lois USA) calibration curve was obtainedfor quantification purposes and the results were expressed asTE 120583molL

245 Minimal Inhibitory Concentration of Annatto SeedExtracts The antibacterial activity was evaluated againstBacillus cereus (ATCC 11778) and Staphylococcus aureus(ATCC 6538) Bacterial strains were incubated for 24 h at37∘C in a Mueller-Hinton broth (lot b1223098542 MerckDarmstadt Germany) and adjusted to a McFarland scale 05(106 CFUmL) Samples were dissolved in dimethylsulfoxide(DMSO) (lot 190260 Merck Darmstadt Germany) to reachconcentrations ranging from 4 to 4096 mgL Ninety-six wellmicroplates were prepared by adding 20 120583L of sample 120120583L of Mueller-Hinton broth and 10 120583L of inoculum Thepositive and negatives controls were prepared according toprevious reports [12] After incubation for 5 h at 37∘C 25120583L of 3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazoliumbromide (MTT) (lot p31b064 Alfa Aesar Tewksbury USA)was added to each well and incubated for 1 h The minimuminhibitory concentration (MIC) was considered as the con-centration of the first well that did not suffer from any colorchange (from yellow to purple) The procedure was repeatedthree times for each microorganism and pH (4 7 and 11) [2]

25 Identification of Bioactive Compounds (Polyphenols andBixin) by LCESI-MS Analysis The extract obtained at theoptimal operational conditions by UAE was analyzed forthe identification of the bioactive compounds LCESI-MSanalyses were performed using an UHPLC (Thermo FisherScientificUltimate 3000) coupledwith an orbitrapQ-exactivemass spectrometer (Thermo Fisher Scientific Waltham MAUSA) Chromatographic separation was done by revers-phase elution equippedwith a SiliaChromPlusC18 (SILICY-CLE) HPLC column (46 x 150 mm 5 120583m 100 A)Themobilephase consisted of a combination of A (01 formic acid(lot 798381 PanReac AppliChem Barcelona Spain) in water)and B (01 formic acid in acetonitrile (lot 830491 PanReac

Table 2 ANOVA table for the response variables for the UAE ofbioactive compounds from annatto seeds

Variable

UAEPolyphenol

(mg AGg seed)Bixin ()

p-value p-valueModel lt 00001 lt 00001X1-Treatment time (min) gt0050 0006X2-pH gt0050 0065X3- Solvent concentration () 00001 lt 00001X4- Seed-to-solvent ratio (1X4) lt 00001 0003X3X4 gt0050 lt 00001X4X1 0036 0023Lack of Fit 0058 0057r2 0768 0897r2-adj 0731 0872AG gallic acid

AppliChem Barcelona Spain)) at a flow rate of 02 mLminminus1(injection volume of 90 120583L)The gradient was linear from 0to 40 B for 60 min followed by 100 B from 65 to 70 minand held with 100 B from 70 to 75 min The composition ofthe eluent was then restored to 100A for 2min and then thesystem was reequilibrated for 14 min The mass spectrometerwas equipped with an electrospray interface (ESI) whichwas operated in positive-ion mode and controlled by theXcalibur software (version 20) The parameters of the ESIsource were as follows positive-ion mode spray voltage of 40kV capillary voltage of 49 V capillary temperature of 275∘Cand tube lens of 120 V The identification of the polyphenolcompounds was based on their molecular weight using thePhenol-Explorer database containing 502 polyphenols Bixinwas identified according to itsmolecularweight (3945 gmol)[10 18]

3 Results and Discussion

31 Experimental Design for Optimization of UAE Theexper-imental matrix was composed of 30 runs The experimentswere performed in triplicate andTable 1 lists the experimentalconditions

The ANOVA was used to evaluate the significance of thequadratic polynomial models For each term in the modelsa large F-value and a small p-value would imply a moresignificant effect on the respective response variable [19]TheANOVA table for the UAE (Table 2) for both dependentvariables shows that most of the factors studied were statis-tically significant (p lt005) in their linear terms p-value forbixin () of X2-pH is 0065 which is also gt0050 p-value forpolyphenol of X1-treatment tine and X3X4 is gt0050 too

The statistical significance of the independent variables isattributed to the ionization states of the aliphatic moleculesupon the modification of the pH and physical processesof mass transfer attributed to the effect of ultrasounds andsolvent concentration Bixin is a carotenoid having a highly


300

250

200

150

100

1000800

600400

200 000600

12001800

24003000Po

lyph

enol

ic (m

g AG

g se

ed)

Seed to solvent ratio (1X4) Ultrasound time (min)

(a)

000

600

1200

1800

2400

3000

Ultrasound time (min)

070062

054

045

037

029

021

012

Bixi

n (

)

9600

86807760

68405920

5000

Concentration of the solvent ()

(b)

Figure 1 Response surface plots for the significant effects of seed-to-solvent ratio vs treatment time and concentration of the solvent vstreatment time for (a) polyphenol compounds and (b) bixin

Table 3 Predicted local maximum in the optimization of the UAE applying a Box-Behnken experimental design

Parameter pHSolvent

concentration ofthe ()

Seed-to-solventratio (1X4) Treatment time (min)

Polyphenolcontent

(mg AGg seed)

Bixin()

Predicted 75 9598 782 3000 271 043Experimental 70 9600 700 2000 381 062

Relative error -110 -019Absolute error () 4074 4442

Values are expressed as mean plusmn standard deviation (n = 3) AG gallic acid

conjugated structure and presents a carboxyl group It is alsohighly soluble at basic pH On the other hand polyphenolcompounds are slightly acidic molecules and their solubilityis not significantly influenced by the medium pH Thereforethe concentration of the extraction solvent (ie ethanol)was the variable of greater statistical significance for allcompounds The statistical significance of seed-to-solventratio was attributed to the saturation of the extractionmedium Further the significance of the treatment time inthe extraction process was related to the residence time of thecavitation bubbles in contact with the seeds easing the releaseof the metabolites During the first 15 minutes of ultrasoundtreatment mass transfer occurred mainly by convection andcorresponded to the solubility of the extracted solute thendiffusion phenomena took place involving the internal partof the seed particles where the extraction of the compoundsis maximized [20 21] The response surfaces plots obtainedfrom the polynomial models are shown in Figure 1 Allthe models were subjected to an optimization process Thepolynomial equations for the response variables are describedas follows

ln (bixin) = minus561 + 003 lowast 1198832 + 007 lowast 1198833

+ 0461198834 minus 001 lowast 1198831 minus 001

lowast 1198833 lowast 1198834 + 001 lowast 1198834 lowast 1198831

(3)

ln (polyphenols) = minus017 + 001 lowast 1198833 + 003 lowast 1198834

minus 001 lowast 1198831 + 001 lowast 1198834 lowast 1198831(4)

The plots describe how the application of ultrasounds accel-erates the extraction process of both type of bioactive com-pounds Once both treatments were optimized the estimatedconditions for the UAE along with their relative error arelisted in Table 3 The absolute bias of the UAE process washigh and thus the experimental results obtained were greaterthan those predicted by the statistical analysis

Results from the UAE were compared with a previouslyreported microwave-assisted extraction (MAE) conduced onthe same seeds [12] The MAE is based on the exposure ofthe extract to high-energy electromagnetic waves generatingheating ofmedium and in turn increasing the energy transferrateThe reported extraction yield of polyphenol compounds(mg AGg seed) and bixin () by MAE corresponds to 308plusmn 001 and 058 plusmn 002 respectively These yields are closeto those obtained reported in this study for UAE (Table 3)A detailed analysis of the MAE showed a treatment time (5min) solvent concentration (ethanol) (96) and a solvent-to-seed ratio (5951) as optimal This means that a shortertreatment time is needed for MAE as compared to the UAEto get about the same yield [12] Emine and collaboratorsobtained similar results when compared both extractiontechniques for polyphenol compounds from nettle In this


Table 4 Minimum inhibitory concentration (MIC) at different pHs against B cereus and S aureus and antioxidant activity of the extractsobtained by UAE and leaching

Assayextract UAE Leaching MAE

Bacillus cereuspH 11 (mg L) 37 a 48 b 16 c

pH 7 (mg L) 37 a 48 b 16 c

pH 4 (mg L) 37 a 48 b 16 c

Staphylococcus aureuspH 11 (mg L) 9 a 48 b 8 a

pH 7 (mg L) 9 a 48 b 8 a

pH 4 (mg L) 9 a 48 b 8a

Bixin () 0621a plusmn 0031 0165b plusmn 0002 0576 a plusmn 0015

Polyphenols mg AGg seed 3814a plusmn 0201 0343b plusmn 0003 3078 a plusmn 0012

ABTS 120583M TroloxL extract 1035652a plusmn 189517 174782b plusmn 8700 57768 c plusmn 5

FRAP 120583M TroloxL extract 424700a plusmn 7000 127033b plusmn 2517 31637 c plusmn 10

DPPH 120583M TroloxL extract 1161524a plusmn 28938 811048b plusmn 5774 104390 c plusmn 50

Values are expressed as mean plusmn standard deviation (n = 3) Different letters indicate statistically significant differences (p lt 005)

case they used a treatment of 10 min at 407 W retrievinggallic acid (1125 mgg db) caffeic acid (1223 mgg db)chlorogenic acid (4798 mgg db) p-coumaric acid (1157mgg db) naringenin (5582 mgg db) and naringin (0665mgg db) These compounds were statistically comparableto those obtained by UAE conducted at 80 power and30 min of treatment time (1209 1289 4453 1100 5735and 0784 mgg db respectively) In annatto seeds MAEshows the same extraction efficiency in a shorter period oftime [22] The major difference between both techniquesis focused on the diffusion resistance for the mass andheating transfer of bioactive compounds Thus the UAEgenerates microdomains and cavitations of the bubbles caus-ing a greater temperature ramp whereas the MAE changesthe molecular rotation and ionic mobility of the mediumgenerating heating in the whole system and not in form ofmicrodomains

32 Effect of the UAE on the Antimicrobial and AntioxidantActivities of the Extract The antioxidant and antimicrobialproperties of the extracts obtained at the optimal operationalconditions for the UAE were compared with an extractobtained by conventional extraction and a previous studyconducted byMAE Table 4 shows a larger antimicrobial andantioxidant activities for the UAE and MAE than thatobserved for the extract obtained by convectional extractionThis is explained by the higher content of polyphenol andbixin compounds present in both emerging extraction meth-ods

The extract of the seeds of annatto contains mainly bixinand polyphenol compounds which are responsible for theantioxidant and antimicrobial activities [2] Particularlybixin is a highly conjugated molecule and polyphenol com-pounds present hydroxyl groups in its structure having theability to capture electrons extinguishing the singlet oxygendeactivating the excited triplet state of sensitizers whichare usually associated with photosensitization and sweepingfree radicals during their transition states As a result thesecompounds showed a large antioxidant activity On the otherhand the antimicrobial activity is attributed mainly to the

content of polyphenol compounds in the extract These com-pounds have the ability to denature the proteins of microbialcell membranes and thus generate their inactivation or deathwithout being affected by the medium pH [2] Consequentlythe antimicrobial and antioxidant activities increased in theextracts obtained by UAE and MAE due to the greateramount of bioactive compounds in comparison to the extractobtained by leaching Similar results were obtained by Zhanget al (2010) and Yolmeh et al (2014) when UAE was used forthe extraction of astaxanthin and bixin respectively showingan increased antioxidant capacity [3 23]

33 Identification of Bixin and Polyphenol Compounds ofthe Extract Obtained by UAE under Optimal ConditionsThe extract obtained by UAE was subjected to an LCESI-MS using an orbitrap as an MS analyzer for the identifica-tion bioactive compounds [2] The monoisotopic mass wassearched in a database of plant phenolic compounds and theresults were listed in Figure 2 Seven polyphenol compoundswere identified including catechin (flavonoids-flavanols)chlorogenic acid or 5-caffeoylquinic acid (phenolic acids-hydroxycinnamic acids) chrysin (flavonoids-flavanols) bu-tein (flavonoids-chalcone) hypolaetin (flavonoids-flavanols)licochalcone A (flavonoids-chalcone) xanthohumol (flavon-oids-chalcone) Furthermore compounds such as hypolaetinand chlorogenic acid or 5-Caffeoylquinic acid were previ-ously identified by Campos et al (2011) who designed achromatographic method for the quantification of bixin andpolyphenol compounds from the extract of annatto seedsThey reported exclusively bixin as the main carotenoid com-pound accounting for the 80 in the annatto seed extractsFurther they also reported hypolaetin and caffeic acid deriva-tives as the main polyphenol compounds However they didnot identify any other type of polyphenols in the extract[1 10]

Despite the verified antioxidant activity of the com-pounds extracted from annatto seeds and identified bythe orbitrap some of them particularly catechin chloro-genic acid chrysin and licochalcone A present antimi-crobial activity against gram (+) gram (-) bacteria and


Compound MF MF (adduct H) MW MW (adduct H)

Bixin 39422169 39522169 733

Catechin 29007903 29108631 159

Chlorogenic acid 35410236 35510236 736

Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772

MF molecular formula MW molecular weight TR retention time

25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO

bixin (6-Methyl hydrogen (9Z)-66-diapocarotene-6)

(b)

Peak spectrum for bixin

Catechin Chlorogenic acid Chrysin Butein

Hypolaetin Licochalcone Xanthoangelol

OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)

Figure 2 (a) Bioactive compounds identified in annatto seeds extract by LCESI-MS analysis (b) Experimental MS2 spectrum of the ion atmz 39422169 with the predicted structures of the ion at mz 39522070 arising from bixin (6-Methyl hydrogen (9Z)-661015840-diapocarotene-6)(c) Structures of polyphenol compounds identified from the annatto seeds extract

fungi [24ndash27] Further Dzotam and collaborators in 2017reported the antimicrobial activity of six Cameroonians foodplants (Psidium guajava Persea americana Citrus sinensisCoula edulis Mangifera indica and Camellia sinensis) against

enteric multiresistant bacteria (Providencia stuartii Kleb-siella pneumoniae and Escherichia coli) They declared thatthe antimicrobial activity is partly attributed to polyphenolcompounds such as catechin This compound either affects


the membrane fluidity in the hydrophilic and hydrophobicregions of lipid bilayers of the microorganism or inhibitsthe action of DNA polymerases [24] Further chlorogenicacid chrysin and licochalcone A have also been reported aspolyphenol compounds with antimicrobial activity against Ecoli M Luteus and S aureus The latter is more susceptibledue to differences of the proteins of the cell wall Forinstance the cell wall of E coli contains of a thin layerof peptidoglycans and an external layer of lipoproteinslipopolysaccharides and phospholipids whereas the cell wallof S aureus includes a peptidoglycan layer with many poresThe porous structure of the cell wall of S aureus makes it easyfor interaction with chlorogenic acid or other antimicrobialcompounds inhibiting the synthesis of nucleic acids thefunction of the cytoplasmic membrane or the energeticmetabolism [25ndash27]

4 Conclusions

The UAE technique accelerated the extraction processreduced energy expenditure and increased the yield of thebioactive compounds present in the annatto seeds The UAEpreserved the metabolites with a greater efficiency and thusfavored their functional activities In addition the presenceof bixin and seven polyphenol compounds such as catechinchlorogenic acid chrysin butein hypolaetin licochalcone Aand xanthohumol found in the extract was responsible fortheir antimicrobial and antioxidant activities

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was sponsored by Colciencias through the grant727 of 2015 for the formation of Colombian PhDs and 715 forresearch and development projects in engineering-2015 Theauthors also thank the sustainability strategy 2018-2019 of theUniversity of Antioquia

References

[1] R C Chiste F Yamashita F C Gozzo and A Z MercadanteldquoSimultaneous extraction and analysis by high performanceliquid chromatography coupled to diode array and mass spec-trometric detectors of bixin and phenolic compounds fromannatto seedsrdquo Journal of Chromatography A vol 1218 no 1 pp57ndash63 2011

[2] M Viuda-Martos G L Ciro-Gomez Y Ruiz-Navajas et al ldquoInvitro antioxidant and antibacterial activities of extracts fromannatto (Bixa orellana L) leaves and seedsrdquo Journal of FoodSafety vol 32 no 4 pp 399ndash406 2012

[3] M Yolmeh M B Habibi Najafi and R Farhoosh ldquoOptimisa-tion of ultrasound-assisted extraction of natural pigment fromannatto seeds by response surface methodology (RSM)rdquo FoodChemistry vol 155 pp 319ndash324 2014

[4] Z Lianfu and L Zelong ldquoOptimization and comparison ofultrasoundmicrowave assisted extraction (UMAE) and ultra-sonic assisted extraction (UAE) of lycopene from tomatoesrdquoUltrasonics Sonochemistry vol 15 no 5 pp 731ndash737 2008

[5] S C Alcazar-Alay J F Osorio-Tobon T Forster-Carneiro andM A AMeireles ldquoObtaining bixin from semi-defatted annattoseeds by a mechanical method and solvent extraction processintegration and economic evaluationrdquo Food Research Interna-tional vol 99 pp 393ndash402 2017

[6] T TahamDO Silva andMA Barrozo ldquoImprovement of bixinextraction from annatto seeds using a screen-topped spoutedbedrdquo Separation and Purification Technology vol 158 pp 313ndash321 2016

[7] L M Rodrigues S C Alcazar-Alay A J Petenate and MA Meireles ldquoBixin extraction from defatted annatto seedsrdquoComptes Rendus Chimie vol 17 no 3 pp 268ndash283 2014

[8] K Ghafoor Y H Choi J Y Jeon and I H Jo ldquoOptimization ofultrasound-assisted extraction of phenolic compounds antiox-idants and anthocyanins from grape (Vitis vinifera) seedsrdquoJournal of Agricultural and Food Chemistry vol 57 no 11 pp4988ndash4994 2009

[9] T Zou Q Jia H Li C Wang and H Wu ldquoResponse surfacemethodology for ultrasound-assisted extraction of astaxanthinfrom haematococcus pluvialisrdquoMarine Drugs vol 11 no 5 pp1644ndash1655 2013

[10] G Aldini L Regazzoni A Pedretti et al ldquoAn integrated highresolutionmass spectrometric and informatics approach for therapid identification of phenolics in plant extractrdquo Journal ofChromatography A vol 1218 no 20 pp 2856ndash2864 2011

[11] M Barbosa C Borsarelli and A Mercadante ldquoLight stabilityof spray-dried bixin encapsulatedwith different edible polysac-charide preparationsrdquo Food Research International vol 38 no8-9 pp 989ndash994 2005

[12] J Q Quiroz A C Torres L M Ramirez M S Garcia G CGomez and J Rojas ldquoOptimization of the microwave-assistedextraction process of bioactive compounds from annatto seeds(Bixa orellana L)rdquo Antioxidants vol 8 no 2 article 37 2019

[13] T Swain andW E Hillis ldquoThe phenolic constituents of Prunusdomestica Imdashthe quantitative analysis of phenolic con-stituentsrdquo Journal of the Science of Food and Agriculture vol 10no 1 pp 63ndash68 1959

[14] FAOWHO Rome 1982[15] J Contreras-Calderon L Calderon-Jaimes E Guerra-Her-

nandez and B Garcıa-Villanova ldquoAntioxidant capacity pheno-lic content and vitamin C in pulp peel and seed from 24 exoticfruits from Colombiardquo Food Research International vol 44 no7 pp 2047ndash2053 2011

[16] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of rsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[17] HHNurhudaM YMaskat SMamot J Afiq andAAminahldquoEffect of blanching on enzyme and antioxidant activities oframbutan (nephelium lappaceum) peelrdquo International FoodResearch Journal vol 20 no 4 pp 1725ndash1730 2013

[18] V Neveu J Perez-Jimenez F Vos et al ldquoPhenol-explorer anonline comprehensive database on polyphenol contents infoodsrdquo Database vol 2010 pp bap024ndashbap024 2010


[19] L Quanhong and F Caili ldquoApplication of response surfacemethodology for extraction optimization of germinant pump-kin seeds proteinrdquo Food Chemistry vol 92 no 4 pp 701ndash7062005

[20] C Da Porto E Porretto and D Decorti ldquoComparison ofultrasound-assisted extraction with conventional extractionmethods of oil and polyphenols from grape (Vitis vinifera L)seedsrdquo Ultrasonics Sonochemistry vol 20 no 4 pp 1076ndash10802013

[21] F Rubio-Senent J Fernandez-Bolanos A Garcıa-Borrego ALama-Munoz and G Rodrıguez-Gutierrez ldquoInfluence of pHon the antioxidant phenols solubilised from hydrothermallytreatedolive oil by-product (alperujo)rdquoFoodChemistry vol 219pp 339ndash345 2017

[22] A E Ince S Sahin and G Sumnu ldquoComparison of microwaveand ultrasound-assisted extraction techniques for leaching ofphenolic compounds from nettlerdquo Journal of Food Science andTechnology vol 51 no 10 pp 2776ndash2782 2014

[23] L-L Zhang M Xu Y-M Wang D-M Wu and J-H ChenldquoOptimizing ultrasonic ellagic acid extraction conditions frominfructescence of platycarya strobilacea using response surfacemethodologyrdquoMolecules vol 15 no 11 pp 7923ndash7932 2010

[24] J K Dzotam and V Kuete ldquoAntibacterial and antibiotic-modifying activity of methanol extracts from six cameroo-nian food plants against multidrug-resistant enteric bacteriardquoBioMed Research International vol 2017 Article ID 1583510 19pages 2017

[25] M Zhao H Wang B Yang and H Tao ldquoIdentification ofcyclodextrin inclusion complex of chlorogenic acid and itsantimicrobial activityrdquo Food Chemistry vol 120 no 4 pp 1138ndash1142 2010

[26] A Uzel K Sorkun O Oncag D Cogulu O Gencay andB Salih ldquoChemical compositions and antimicrobial activitiesof four different Anatolian propolis samplesrdquo MicrobiologicalResearch vol 160 no 2 pp 189ndash195 2005

[27] T P Cushnie and A J Lamb ldquoAntimicrobial activity offlavonoidsrdquo International Journal of Antimicrobial Agents vol26 no 5 pp 343ndash356 2005

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


transfer from the solid-phase to the liquid-phase [3] Theultrasound-assisted extraction (UAE) creates cavitation ofsmall bubbles in the solvent due to the passage of ultrasoundwaves allowing for a greater penetration of the solventwithin the material increasing the surface area On the otherhandmicrowave-assisted extraction implies the formation ofhigh-energy electromagnetic waves changing the molecularrotation and ionicmobility of themediumwithout destroyingthe sample resulting in heating and possibly denaturationof bioactive compounds The last two methods force themigration of all active compounds rapidly from the solid-phase to the solvent-phase However the UAE shows a betterprotection of those compounds due to the lower temperaturegenerated in the medium [8 9]

Further not only the evaluation of the functional activityof the extracts but also the identification of the compoundsresponsible for that activity is crucial In general bixinphenolic acids lignans and flavonoids are the main bioactivecompounds However the isolation and identification ofthese compounds require techniques having a high separa-tion efficiency since coeluting compounds could be missed[10] Currently HPLC coupled with mass spectrometry atatmospheric pressure chemical ionization (APCI) or elec-trospray ionization (ESI) represents the most selective ana-lytical technique for the identification and quantificationof compounds isolated from plants and food sources Theidentification of carotenoids and polyphenol compounds byLCndashESI-MS using a LTQ Orbitrap XL renders an extremelyhigh mass accuracy (lt5 ppm of resolution) This techniqueis capable of multiple levels of fragmentation to reveal themolecular weight (MW) of unknown compounds Hence byusing this technique unknown compounds are identified onthe basis of nominal MW and MSMS fragmentation [10]

The goal of this study was to enhance the extraction ofbioactive compounds from the seeds of annatto and optimizethe extraction conditions (ie solvent concentration pHtreatment time and seed-to-solvent ratio) as compared toa conventional method Further it is expected that theantimicrobial and antioxidant activity is due to the high yieldof extracted compounds as identified by LCESI-MS

2 Materials and Methods

21 Materials Annatto seeds were purchased from a farmersmarket of Medellın (Colombia) which were sun-dried untilreaching amoisture content of 1058plusmn 098 All the reagentsused were synthesis grade

22 Experimental Design The UAE conditions were opti-mized using a response surface plot experimental design(Box-Behnken (BBD)) using the Design Expert Software(Version 806 Stat-EaseUSA)Thedependent variables suchas treatment time (min) (X1 0-30) pH (X2 4-11) solventconcentration () (X3 50-96) and seed-to-solvent ratio (X42-10) were studied The solvent used was absolute ethanol(lot k4958171742 Merck Darmstadt Germany) With theindependent variables studied the statistical program estab-lished 30 experimental units which are presented in Table 1with their respective randomization The two independent

variables studied were the bixin content (Bix) and the totalpolyphenol content found by the Folin-Ciocalteu methodusing a uvvis spectrophotometer (UV-1700 Shimadzu) [11]

The operational conditions of the equipment used for theUAE were a frequency of 37 KHz and a power of 320 W(Elmasonic E30h) Adjustment of the pHwas donewith 01Macetic acid (lot k47109963539 Merck Darmstadt Germany)and 01 M sodium hydroxide solution (lot b0895598319MerckDarmstadt Germany)Themethod ofmultiple regres-sions of least squareswas used in order to optimize the extrac-tion conditions and investigate the effect of the independentvariables The experimental data was adjusted by the second-order polynomial equation (see (1)) by comparison of thedetermination coefficient (r2 ) and the adjusted determinationcoefficient (r2-adj) The quadratic model follows the expres-sion

Y = 1205730 + 1205731X1 + 1205732X2 + 1205733X3 + 1205734X4 + 12057311X11

+ 12057322X2

2+ 12057333X

2

3+ 12057344X

2

4+ 12057312X1X2 + 12057313X1X3

+ 12057314X1X4 + 12057323X2X3 + 12057324X2X4 + 12057334X3X4

(1)

where Y represents the predicted response 1205730 is the interceptof the model 1205731 1205732 1205733 1205734 12057311 12057322 12057333 12057344 y 12057312 12057313 1205731412057323 12057324 and 12057334 are the linear and interaction coefficientsrespectively and X1 X2 X3 and X4 correspond to the inde-pendent variables Analysis of variance (ANOVA)was used toanalyze the significant effects (with a confidence level of 95)of the independent variables over the dependent variablesOnce the significant mathematical models were obtained theextraction process was optimized seeking to maximize theextraction of polyphenols and bixin (weight of 1 for both) andminimizing the seed-to-solvent ratio The calculation of therelative and absolute errors was accomplished between theresponses predicted by the model versus the ones obtainedexperimentally under optimal conditions

23 Characterization of Optimized Annatto Seed ExtractsThe antioxidant and antimicrobial activities of the optimizedextracts obtained by the UAE were compared with (i) anextract obtained by conventional extraction (with ethanolseeds-to-solvent ratio of 12 and continuous agitation for 48h) and (ii) previous results from an optimized microwave-assisted extraction [12] The results are presented as meansand standard deviation (SD) The analysis was performedusing the Statgraphics Centurion XVI software

24 Quantification of Total Polyphenol The total phenolicconcentration in the annatto seed extract was measured bythe Folin-Ciocalteu method [13] Twenty microliter of samplewas mixed with in 158 120583L of distilled water 100 120583L of Folin-Ciocalteu phenol reagent (lot hc43368401Merck DarmstadtGermany) and 300 120583L of 20 sodium carbonate (lota0594092339 Merck Darmstadt Germany) The absorbancewas read at 725 nm in a UVVIS spectrophotometer (UV-1700 Shimadzu) after 1h of storage under darkness Theexperiments were performed in triplicate and the results wereexpressed as mg of gallic acid (GA) (lot SLBM8746V MerckDarmstadt Germany) per gram of seeds (mgGAg seed)



Treatmentnumber

UAETreatment

time(min)

pHSolvent

Concentration()


(1X4)


n=3

Bixin content ()n=3



119861119894119909119894119899 (119898119892119898119871) =119860 lowast 100 lowast 119881

11986011119888119898lowast 100

(2)

where11986011119888119898











Variable

UAEPolyphenol









300

250

200

150

100

1000800

600400

200 000600

12001800

24003000Po

lyph

enol

ic (m

g AG

g se

ed)


(a)

000

600

1200

1800

2400

3000


070062

054

045

037

029

021

012

Bixi

n (

)

9600

86807760

68405920

5000


(b)



Parameter pHSolvent



Polyphenolcontent

(mg AGg seed)

Bixin()








(3)









pH 7 (mg L) 37 a 48 b 16 c

pH 4 (mg L) 37 a 48 b 16 c


pH 7 (mg L) 9 a 48 b 8 a

pH 4 (mg L) 9 a 48 b 8a















Bixin 39422169 39522169 733

Catechin 29007903 29108631 159


Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772


25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO


(b)




OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)






4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Treatmentnumber

UAETreatment

time(min)

pHSolvent

Concentration()


(1X4)


n=3

Bixin content ()n=3



119861119894119909119894119899 (119898119892119898119871) =119860 lowast 100 lowast 119881

11986011119888119898lowast 100

(2)

where11986011119888119898











Variable

UAEPolyphenol









300

250

200

150

100

1000800

600400

200 000600

12001800

24003000Po

lyph

enol

ic (m

g AG

g se

ed)


(a)

000

600

1200

1800

2400

3000


070062

054

045

037

029

021

012

Bixi

n (

)

9600

86807760

68405920

5000


(b)



Parameter pHSolvent



Polyphenolcontent

(mg AGg seed)

Bixin()








(3)









pH 7 (mg L) 37 a 48 b 16 c

pH 4 (mg L) 37 a 48 b 16 c


pH 7 (mg L) 9 a 48 b 8 a

pH 4 (mg L) 9 a 48 b 8a















Bixin 39422169 39522169 733

Catechin 29007903 29108631 159


Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772


25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO


(b)




OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)






4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








Variable

UAEPolyphenol









300

250

200

150

100

1000800

600400

200 000600

12001800

24003000Po

lyph

enol

ic (m

g AG

g se

ed)


(a)

000

600

1200

1800

2400

3000


070062

054

045

037

029

021

012

Bixi

n (

)

9600

86807760

68405920

5000


(b)



Parameter pHSolvent



Polyphenolcontent

(mg AGg seed)

Bixin()








(3)









pH 7 (mg L) 37 a 48 b 16 c

pH 4 (mg L) 37 a 48 b 16 c


pH 7 (mg L) 9 a 48 b 8 a

pH 4 (mg L) 9 a 48 b 8a















Bixin 39422169 39522169 733

Catechin 29007903 29108631 159


Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772


25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO


(b)




OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)






4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



300

250

200

150

100

1000800

600400

200 000600

12001800

24003000Po

lyph

enol

ic (m

g AG

g se

ed)


(a)

000

600

1200

1800

2400

3000


070062

054

045

037

029

021

012

Bixi

n (

)

9600

86807760

68405920

5000


(b)



Parameter pHSolvent



Polyphenolcontent

(mg AGg seed)

Bixin()








(3)









pH 7 (mg L) 37 a 48 b 16 c

pH 4 (mg L) 37 a 48 b 16 c


pH 7 (mg L) 9 a 48 b 8 a

pH 4 (mg L) 9 a 48 b 8a















Bixin 39422169 39522169 733

Catechin 29007903 29108631 159


Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772


25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO


(b)




OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)






4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






pH 7 (mg L) 37 a 48 b 16 c

pH 4 (mg L) 37 a 48 b 16 c


pH 7 (mg L) 9 a 48 b 8 a

pH 4 (mg L) 9 a 48 b 8a















Bixin 39422169 39522169 733

Catechin 29007903 29108631 159


Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772


25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO


(b)




OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)






4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Bixin 39422169 39522169 733

Catechin 29007903 29108631 159


Chrysin 25406519 25506519 702

Butein 27207575 27307575 726

Hipolaetin 30204993 30304993 772

Licochalcone A 33815909 33915909 654

Xanthoangelol 39220604 39320604 772


25（314

15（156

16（199

15（114

15（135

15（117

21（234

25（295

25（304

15（146

16（189

15（104

15（125

15（107

21（224

25（284

４２ (min)

(a)

393 394 395 396 397 398 399mz

0

10

20

30

40

50

60

70

80

90

100 39421622

39522070

3962252939322160

39725324 39826068 39928878

O

O

OH

Rela

tive A

bund

ance

（3CO


(b)




OH

OH

OH

OHOHOHOH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

HOHO

HOHO

HO

HO

HOHO

OO

OO

O

O

O

O

OO

OH

（3CO

（2C

（2CC（3

C（3

C（3OC（3

C2H

(c)






4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




4 Conclusions


Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018














Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


ultrasound-assisted extraction of bioactive...

Documents