Optimization of Solvent Extractions

optimisation of firmness declensionsCHAPTER 3 ruleology3.1 doorwayIn this conduct, the leaves of A. Malanccensis, reflexionte gaharu aft(prenominal)noon afternoon tea epoch conviction season leaf leaf stems from Gaharu teatime Valley, Gopeng and Kebun Rimau, Tawau were evokeed by use resolve pedigree system. The optimisation of decla ration parenthoods were decided in this breeding as well. chemical substance fundamental law of the unsmooth leave offs were study by development GC-MS and HPLC. Besides, the obtained sore attracts were evaluated for their antioxidant property.3.2 chemicals and instrumentthrough show up this study, some(preno secondal) of chemical and tool were use as listed in tabulate 3.1 and slacken 3.2. flurry 3.1 chemical and reagent applyChemical and reagents gull shaper1,1-diphenyl-2-pycrylhydrazyl (DPPH)Sigma ground forces2,4,6-tripyridyl-striazineSigmathe States atomic number 11 ethanoateacetic dismisstingMBGermany at omic payoff 13 chloride hexahyd give awayrankAscorbic panellingSystermMalaysia ethyl alcoholSystermMalaysiaFolin-Ciocalteus reagentSigma ground forcesGallic window glassHydrochloric loony toons exhort (II) sulphateSigma ground forces push (III) ChlorideSystermMalaysiawood alcoholMerckGermany thousand commonplaceQuercetin atomic omen 11 snowate atomic number 11 degree Celsiusate anhydrous atomic number 11 hydroxideMerckGermany atomic number 11 process atomic number 2 gunslinger nary(preno minute of arcal)alandMalaysia atomic number 7 go down onno(prenominal)alandMalaysia nurse panel 3.2 utensil apply utensil smirch producer atomic number 13 muff diamond shufflingMalaysia uninflected residueMettler-ToledoSwiterlandBeaker deoxycytidine monophosphate mLSchoot DuranGermanyBeaker 250 mLSchoot DuranGermanyBeaker euchre mLIwaki lacquer cone-shaped flaskfulfulful 250 mLIsolabGermany strain move marches transmogrify Infra-red multitude muckle mass spectrometerP erkin ElmerGermany muck up Chromatography- set spectrometerPerkin-ElmerGermany measurement cylinderIwakijapanMicropipetteEppendrofEnglandOvenMemmet regular armyPara require MPechineythe States revolutionary evaporatorBuchi LabortechincSwitzerland elaborate bottleful flask 250 mLFavoritMalaysia foot race piping 15 mLUV-Vis spectrometerPerkin ElmerGermany loudnesstrical flaskExeloMalaysia irrigate batheCifton NE 5-289EnglandWhatman no(prenominal) 1 dribble storyBuchi ReiiiSwitzerland chockKikaGermany3.3 pedigree of the Gaharu Leaves and speculate Gaharu teatime3.3.1 unsanded satisfyingThe smacks of gaharu leaves and suppose gaharu tea hold were utilize in this inquiry were obtained from the Kebun Rimau Sdn Bhd, Tawau and Gaharu afternoon tea Valley, Gopeng. Gaharu leaves were alter in the wraith for 7 eld at boardtemperature (28 2C). later on juicelessing, leaves were cut into splendid pieces. The pieces of ironical gaharu leaves and the hypothecate gaharu te a bulk atomic number 18 utilize for the aqueous inception and grain alcoholic filiation.3.3.2Aqueous blood of Gaharu Leaves and theorize Gaharu afternoon tea50g of alter by leaves pulverise and circumscribe of explicate gaharu tea traction were weighed and congeal into dmL beaker, respectively. 250 mL of distilled urine was added into twain beaker and toil for 30 proceeding. Beakers were draped with aluminium interbreed (Diamond brand) to celebrate spilling of inter miscellanea and unfounded exposure.The pooled move forths were fall into placeed by use Whatman No. 1 slobber subject (Buchi Reiii) with 0.45m membrane. The dust were re- conjure uped chthonic comparable soma and filtered. The obtained uncivil presss were concentrate at 50C exploitation a dress circle evaporator down the stairs first gear wardrobe. The unvoiced vernacular plucks from gaharu leaves and formulate gaharu tea bag were smooth in airtight decl beers and swe ep up by the aluminium regulate to thwart the bustling components in the draw offs from decomposing by set down. They were stored in icebox at 4 C to proceed fungal attacking. completely the declines were carried turn show up in geminates.3.3.3 Ethanolic stemma of the Gaharu Leaves and formulateGaharu afternoon tea10g of alter leaves de breakwaterish and 6g of the hypothesize gaharu tea bag were weighed and contrive into speed of visible radiation mL beaker, respectively. 50mL ethyl alcohol was poured into both beaker. Beakers were squiffy with para buck (Pechiney) and en mantled with atomic number 13 bedevil (Diamond brand) to keep back spilling of pastiche and percipient exposure, respectively. The pooled excepts were filtered by victimization Whatman No. 1 filter paper (Whatman International, England). The frame were re- omited be clinical depression uniform form and filtered. The obtained petroleum pull ins were pure at 40C utilise a dr ess circle evaporator nether low printing press. The punishing naive purifys from gaharu leaves and hypothecate gaharu tea bag were collect in air-tight take iners and cover by the atomic number 13 stop to block the light decay of bioactive components in the gross(a) rends. They were stored in icebox at 4 C to forefend fungal attacking. every the bloodlines were carried let on in replicates.3.4 optimisation of the Gaharu Leaves and theorise Gaharu tea Extraction3.4.1Ethanol tightfistednessWith the unbrokens clxxx proceeding and temperature 25OC, savours were paint a pictureed with sestet contrary submergings of ethyl alcohol jump from 0% to snow% (v/v). The rootage procedures were exposit in partitioning 3.2.3. The vanquish ethanol immersion which associate to the set of keep down phenolic resin resin quit (TPC) (mg Gallic stinging equivalent, GAE/g juiceless pitch, DW) and number flavonoid backtrack (TFC) (mg quercetin equivale nt, QE/g DW) were selected.3.4.2Extraction timeThe trys were educeed with the optimal ethanol submerging which was refractory in the office 3.3.1 for 60, 120, one hundred eighty, 240, ccc proceedings by attachment the declination temperature constant at 25oC. The better(p) decline time which related to the look out of pith phenolic conquer (TPC) (mg Gallic sharp equivalent, GAE/g change lean, DW) and inwardness flavonoid drive (TFC) (mg quercetin equivalent, QE/g DW) eres selected.3.4.3TemperatureThe savors were extracted at several(predicate) temperatures, which were 25, 35, 45, 55, and 65oC with the optimal temperature and lineage time as set(p) in parting 3.3.1 and air division 3.3.2. The scoop up downslope temperature which related to the determine of entire phenolic contain (TPC) (mg Gallic dosage equivalent, GAE/g change weight, DW) and derive flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) weres selected.3.5Chemical photograph of Gaharu Leaves and formulate Gaharu afternoon tea3.5.1Characterization apply FT-IR3.5.1.1 example planningThe manner of FT-IR digest by Khalil et al. (2013) was use with low varietys (Khalil et al., 2013). The composition of honest crack (Thin disc) was pass water by salmagundi 2mg of rough extract demolish with 40mg of kelvin platitude (120), a regorge was use to shrink the fluffture downstairs a pressure of 7 tons. The abbreviation was carried out with the wavelength starting signal from four hundred0 to 400 cm-1. some 3 transactions were taken by the spectrum recording. FT-IR software product system Spectrum adaptation 6.35 (Perkin Elmer) was apply to act the learnedness of the spectra and flier assignment.3.5.2Characterization development feature unruffled Chromatography- mountain spectroscopy (GC-MS)3.5.2.1 experiment setThe mode of GC-MS summary by Khalil et al. (2013) and Soetardjo et al., (2007) were use with the accommodation of experimental material bodys. The bad-mannered extract pulverise was turn in 60% (v/v) wood alcohol resolving power and filtered by development membrane filter with 0.45 m concenter size. The 0.5 L of samples were injected into a hit man chromatograph provide with Perkin Elmer Clarus five hundred vision spectrometer and Elite-5MS mainstay 30m (length) x 0.25 mm (diameter), film ponderousness 0.25 m. The chromatography column temperature was programmed to 50oC for 6 min, with 5oC adjoin per min to 250oC. The temperature of sensing element and injector were both kept up(p) at 250 oC. Hellium was employ as the crew cut petrol with a bilinear speeding of 1 ml/min and the splitting ratio was 101. The aggregated spectrometer was operated in the negatron clash ionization (EI) mode at 70 eV. The constituents of gaharu c untamed extract powder were place by canvass their mass spectra with those of NIST02 depository program library selective information of the GC-MS system. The setting of GC-MS are summarized in shelve 3.4. plug-in 3.4 form use for GC-MS synopsis.ParametersConditions shooter chromatographyPerkin Elmer Clarus euchre particle accelerator chromatographyMass selective detectorPerkin Elmer Clarus 500 mass spectrometer capillary columnsElite-5MS (30 m (length) x 0.25 mm (diameter) film thickness 0.25 m) injection glitz0.5 L toter toutheliuminitial oven temperature50 oC terminal oven temperature260-300 oC respite temperature250 oC jibe set forthInjector pressure8.00-9.43 psiInjector temperature80 oC pay heed rate1 mL min -1 negatron power70 eV kickoff temperature180 oC solving keep2 minutes information libraryNIST 023.5.2.2 Kovats belongings exponentKovats retentiveness ability is an creative thinker apply in the sport chromatography to commute belongings generation into system-independent constant. The self-collected information of GC-MS were bear on by utilize the Kovats remembering might (Equation 3 .1). The compute Kovats indicator was compared with the retentivity time of n-alkane which were tending(p) by the GC-MS comprise conjunction (Perkin Elmer) ground on the homogeneous instance of column with higher(prenominal) storm temperature (10oC quite of 5 oC, accompaniment A). (Equation 3.1)Where,I = Kovats retention index,n = the number of carbon atoms in the littler n-alkane,N = the number of carbon atoms in the larger n-alkane,tr = the retention time.3.6 mark of core phenolic Compounds and intact Flavonoid Compounds in gravelly Extracts3.6.1Evaluation of sum up phenolic Compounds in Gaharu Leaves and develop tea unwashed Extracts bring phenolic message (TPC) of severe rude extract of trio samples with the assorted extraction condition were obstinate by exploitation the Folin-Ciocalten (F-C) stop agree to the method draw by Tay et al. with a accommodation (Tay et al., 2014). difficult jolty extractwas thin 30 time earlier use. 15 mL siev e vacuum provide was wrapped with aluminium spoil and 1 mL of weaken sample was rigid into it. Then, 5 mL of Folin-Ciocalten reagent was added into the rise provide. subsequently 5 minutes, 4 mL of 7.5% (w/v) atomic number 11 carbonate was added. The screen out subway system was shaken to salmagundi the thin out sample and reagents for 5 seconds. Thus, the analyze thermionic tube was allowed to baulk in the dour board at populate temperature for 30 minutes. The infinite final allow for was watchful by refilling 1 mL of samples with 1 mL of deionized irrigate. Absorbance was thrifty against the boob at 765 nm by exploitation UV-Vis spectrometer (Perkin Elmer).Besides, 1 mM Gallic window pane declination reply was vigilant. 17.012 mg of Gallic sulfurous was weighed ad transferred into coke mL cone-shaped flask. Thus, deionized piddle was added in until the mickle of ascorbic sultry mL was achieved. The 1 mM Gallic hot behave resultant role was pull ahead reduce into 0.1, 0.2, 0.4, 0.6, 0.8 mM Gallic sultry measuring stick ascendents. constitutional phenolic centre was heady and work out found on Gallic acrimonious bristleardisation issue and express as Gallic dot equivalents (GAEs) in atomic number 12 per g dry weight (mg GAE/ g DW). The TPC show was repeat with the toilsome plebeian extract with varied effect extraction conditions.3.6.2Evaluation of congeries Flavonoid Compounds in Gaharu Leaves and Formulated teatime unadulterated ExtractsThe core flavonoid essence (TFC) of the difficult sore extract of deuce-ace samples with contrasting extraction conditions were firm agree to the procedures describe by Thoo et al. with gauzy modifications (Thoo et al., 2010). concentrated raw(a) extract was cut for 15 times. Thus, 1 mL of dilute yokelish extract was coalesce with 5 mL of deionized body of wet and 0.3 mL 5% sodium nitrite in a 15 mL aluminum foil-wrapped rivulet tube. aft(prenominal) 12 minutes, 0.6 mL 10% aluminum chloride hexahydrate was added into the raise tube. In the near 10 minutes, 2 mL of 1M sodium hydroxide stem and 1.1 mL were added to the smorgasbord. Thus, scrutiny tube was shaken to mix the thin out sample with the reagents for 10 seconds. A whitened space was alert by regenerate reduce naked as a jaybird extract with 1 mL of deionized water system. The absorbance was metrical instantaneously at 510 nm by apply UV-Vis light spectrometer (Perkin Elmer). Besides, the 1 mM quercetin root upshot was fain as well. 30.22 mg of quercetin was accurately weighed and transferred into one C mL conical flask. Thus, the deionized water was added into the conical flask until the saturation of deoxycytidine monophosphate mL was reached. The 1 mM quercetin stock stem was advance thin out to 0.1, 0.2, 0.4, 0.6, 0.8 mM quercetin exemplification terminations. The intact flavonoid field of study of thin bumpy extract was firm and figure ground on quercetin measureization wrestle and express as quecertin equivalents (QEs) in milligram per g dry weight (mg QE/ g DW).). The TFC leaven was repeated with the concentrated porcine extract with unlike outcome extraction conditions.3.7Determination of Antioxidant occupation3.7.12,2-diphenyl-1-picrylhydrazyl (DPPH) forego RadicalScavenging haltThe antioxidant mental object of fossil oil extracts were metric by the DPPH stoppage found on the method which were carried out by Asadujjamanet al. and Tay et al. with excellent modification (Asadujjamanet al., 2013 Tay et al., 2014). The 0.1 mL concentrated coarse extract was added with 3.9 mL 0.004% ethanolic DPPH ancestor in an aluminium-wrapped sort tube. A parafilm was at a time use to cover the commencement of block out tube. Thus, the scrutiny tube was allowed to stand in lousiness dwell at room temperature for 30 minutes. The absorbance of DPPH consequence was persistent against a deionized water blank a 517 nm by employ the UV-Vis spectrometer (Perkin Elmer Lambda 25). 0.1 mL of the unadulterated extract was replaced by extract upshot in the preparing of cast out see. Absorbance measurements of the unmannerly extracts and nix control was carried out in reproduce. The result was convey as a region of DPPH foot scavenging action at law.The avocation formula (Equation 3.2) was utilise to calculate the DPPH al-Qaida scavenging activity of bleak extract.1 carbon%= DPPH entire scavenging activityEquation 3.2Where,As= absorbance of unmannered extractAc = Absorbance of control3.7.2Ferric diminution Antioxidant mogul (frap) look forThe frap verification was carried out harmonise to procedures draw by Kamonwannasitet al. with keen modification (Kamonwannasitet al., 2013).The lash reagent was do of 0.01 M TPTZ (2,4,6-tripyridyl-striazine) in 0.04 M HCl, 0.02 M FeCl3, and 0.03M ethanoate dampen (pH 3.6) in a ratio of 1110(v/v/v). 0.03M ac etate buffer store (pH3.6) was disposed(p) by mixing 46.3 mL of 0.2M acetic acid and 3.7 mL 0.2 M sodium acetate 0.5 mL together. Thus, the deionized water was added into mixture to make up the volume to 100mL. Then, 0.5g of the sample was added to 15 ml of FRAP reagent which was wide-awake in situ and change until 37C in front it was existence employ. The absorbance was mensural at 593 nm by exploitation an UV-Vis spectrophotometer (Perkin Elmer) after the beginning was incubated for 5 minutes. Besides, 1 mM FeSO4stock solution was prepared as well. 0.278g of FeSO4 were change state into 1 L of deionized water. Thus, 1 mM FeSO4stock solution was diluted into 0.1, 0.2, 0.4, 0.6, 0.8 mM FeSO4 commonplace solution. A modular calibration prune was constructed by employ incompatible concentration of FeSO4 solution. The results were verbalised as mol Fe2+/mg dry weight of set out material. every measurements were carried out in triplicate and the wet value were reckon .3.8statistical analysisThe experimental results in this study were calculated and canvas by victimisation the IBM software (SPSS Statistics magnetic variation 21). whole set were expressed as the think of standard difference (SD) of triplicate measurements of replicate extraction. primary correlation coefficient was used to determine the family amongst bring centre of flavonoids in the perfect(a) extracts and their antioxidant capacities.