research article green extraction: enhanced extraction

Hindawi Publishing CorporationJournal of Applied ChemistryVolume 2013 Article ID 460168 7 pageshttpdxdoiorg1011552013460168

Research ArticleGreen Extraction Enhanced Extraction Yield of Asiatic Acidfrom Centella asiatica (L) Nanopowders

M Z Borhan123 R Ahmad3 M Rusop12 and S Abdullah2

1 NANO-SciTech Centre (NST) Institute of Science Universiti Teknologi MARA (UiTM) Selangor 40450 Shah Alam Malaysia2 NANO-Innovation Centre (Nano-IC) Faculty of Applied Sciences Universiti Teknologi MARA (UiTM) Selangor40450 Shah Alam Malaysia

3 Faculty of Applied Sciences Universiti Teknologi MARA (UiTM) Selangor 40450 Shah Alam Malaysia

Correspondence should be addressed to M Z Borhan mzborhanyahoocom

Received 29 May 2013 Revised 4 September 2013 Accepted 24 October 2013

Academic Editor Rassoul Dinarvand

Copyright copy 2013 M Z Borhan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nanopowders of Centella asiatica (L) were produced using planetary ball mill in order to investigate the differences of waterextraction yield of asiatic acid as compared tomicropowders ofCentella asiatica Effect of extraction time (20ndash60min) on extractionyield of asiatic acid from Centella asiaticawas examined Results showed that water extraction of asiatic acid usingCentella asiaticananopowders exhibits was almost 50 higher extraction yield with 709mgg as compared to themicropowders ofCentella asiaticaIt was concluded that nanopowders contributed in enhancing water extraction yield of asiatic acid as compared to micropowdersThereby utilizing nanopowders in water extraction could improve the extraction of asiatic acid via clean eco-friendly and lessexpensive process

1 Introduction

Centella asiatica (L) is one of the medicinal plants that hasbeen declared to have a variety of medicinal effects Its bioac-tive compounds include asiatic acid asiaticoside madecassicacid and madecassoside Asiaticoside (Figure 1(a)) is theprincipal bioactive ingredient among them since asiaticosidegave a better effect on antibacterial and fungicidal activityagainst bacteria and fungi [1] Asiatic acid (Figure 1(b)) alsodisplays good biological effects such as promoting collagensynthesis stimulating extracellular matrix accumulation andpromoting fibroblast proliferation in a rat wound model [2]

Asiatic acid is an aglycone form of asiaticoside andeasily formed by hydrolyzing the sugar moiety of asiaticosidestructure in acid condition Therefore some data suggestthat the therapeutic effect of asiaticoside may come fromasiatic acid [3] Some studies show the significance of sugarmoiety in the structure of asiaticosidewhichmay enhance thebioavailability of asiatic acid compared to the only aglyconestructure alone [4] But once again enriching asiatic acid inthe extract is important since all the synthesizing alkyl groups

require hydrolyzing of asiaticoside first prior modificationof asiatic acid functional groups [4] By modification theasiatic acid may provide us with data in relationship betweenstructural and hepatoprotetive properties in order to developnew novel drugs for treating disease [5 6]

Extraction of active chemical compounds from naturalsources is a most important research area in pharmaceuticaland chemical industry Bioactive compound extraction aimsto get a better extraction yield and less time consumingwithout altering the structure of compounds extractedLarge organic solvents consumption in extraction processis the main problem in conventional extraction processIn conventional extraction the usage of organic solvent ismandatory as a result large amount of chemical waste wasdumped and affected the environmental Clean extractiontechniques have been encouraged by government healthand environmental regulations due to environment con-cern Recently clean extraction methods such as pressur-ized hot water extraction (PHWE) supercriticalsubcriticalwater extraction (SFE) and ultrasonic-assisted extraction(UAE) have been introduced to overcome toxicity problem

2 Journal of Applied Chemistry

OHO

HO

OH

H

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

HCH3

(a)

HO

HO

OH

OH

HO

(b)

Figure 1 Chemical structure of (a) asiaticoside and (b) asiatic acid

encountered in the extraction industry [7ndash9] However theextraction efficiency of these methods is greatly dependenton the particles size of plant material Among the methodsultrasonic-assisted extraction (UAE) has been known for itssimplicity such as short processing time and low solventconsumption [10]

Herbal nanopowders are the herbal powders rangingfrom 10 to 1000 nm in size This nanopowder has uniqueproperties such as larger surface area thus improvingdissolve-out properties of bioactive components in the cellu-lar matrix Several studies have shown increase of extractionratio of bioactive components from 17 to 50 increasescompared to the normal powders [11 12] Beside a bet-ter extraction yield the extraction of nanopowders doesnot require longer extraction time Thus it will save timeconsumed for extraction and more than that it just onlyrequires small portion of powderThus it is cost effective andconcerned with the limited supply of raw material of herbalplant itself [13]

In this study we investigated the effectiveness of nano-powders compared to themicropowders for the water extrac-tion of asiatic acid from Centella asiatica assisted by theultrasonic probe The influence of extraction time also hasbeen investigated in order to define the optimal condition forextraction

2 Methodology

21 Chemicals and Materials Asiaticoside was purchasedfrom Chengdu Biopurify China asiatic acid standard waspurchased from the Sigma Aldrich acetonitrile (LC grade)from Fisher Chemicals and methanol (HPLC grade) fromFriedemann Schmidt Distilled water was purified using aMilli-Q water purification system (Millipore Bedford MAUSA)

22 Sample Preparation Plant material was bought fromlocal market in Tanjung Malim Perak Malaysia duringMarch 2010 Centella asiatica was washed with runningtap water and rinsed by distilled water to remove any dirtand contaminant on the plant material Then cleaned plantmaterial was stored in the 50∘C controlled oven and left todry for 3 days The dried plant material was ground using

conventional rotor The ground powder was sieved through250120583m sieve and stored in the 4∘C prior ball milling process

23 Production of Centella asiatica Nanopowders The driedand powdered plant material (03 g) first was mixed with25mL of 01 (wv) Pluronic F127 and milled at 12 wvof mass concentration and 550 rpm milling speed at 4 hoursof milling time in the 50 cm3 stainless steel jar containing 25gram of 2mm diameter size of zirconia bead These param-eters were chosen because that the early preliminary resultsshow at these conditions the content of asiatic acid is highestamong nanopowders produced at difference conditions ofgrinding (mass concentration milling time and amount ofbead) The resulting milling product was in the form ofnanosuspension This nanosuspension was further used forextraction

24 Particles Size Analysis andMorphology Prior to themea-surement nanosuspensions of each sample were taken outand adjusted to 001 (wv) concentration and introducedinto a disposable cuvette to be measured in triplicate Z-Average and polydispersity (PdI) were recorded FESEM(Carl Zeiss SMT SUPRA 40VP) imaging was carried out byplacing 10 120583L nanosuspension onto a glass slide and stored inan electronic desiccators (temperature 20∘C humidity 18RH) for drying purpose The dried samples were then coatedwith gold (sim10 nm thick) and were placed onto an adhesivetape on the FESEM stub

25 HPLC Analysis

251 Extraction A 06 g micropowder was weighed andmixed with 50mL water Then this mixture was sonicatedfor 20 40 and 60 minutes by ultrasonic processor (UP400SUltrasonic Processor Hielscher) at frequencies wattage out-put and 125W Then mixture was centrifuged to separatethe particles and filtered by gravitational filtration Filtratewas collected and water was removed by freeze dryer andweights of crude extracts were recorded The extracts weredissolved in methanol to 1mgmL and filtered through022120583mfilter paper before being subjected to HPLC analysisThe nanopowders were also extracted in the same manner asabove

Journal of Applied Chemistry 3

16

14

12

10

8

6

4

2

0

1 10 100 1000 10000

Inte

nsity

()

Size (dnm)

100

90

80

70

60

50

40

30

20

10

0

Und

ersiz

e

(a)

10

09

08

07

06

05

04

03

02

01

00

01 10 1000 100000 10000000 1000000000

Time (120583s)

Cor

relat

ion

coeffi

cien

t

(b)

Figure 2 Particles size analysis of nanopowder (a) size distribution (b) correlogram

(a)

(a)

(b)

(b)

Figure 3 FESEMmicrographs of Centella asiatica (a) raw powder (b) nanopowders

15

10

5

001 1 10 100 1000 10000

Size (dnm)

Inte

nsity

()

Figure 4 Size distribution of nanopowders (red curvemdashnanopow-ders in suspension green curvemdashfreeze-dried nanopowders)

252 HPLC Condition Individualrsquos content in the extractwas analysed using HPLC analysis The separation wasperformed on an Agilent 1200 HPLC (Agilent USA) with aZorbax Eclipse XDB-C18 (46mm times 150mm 5 120583m) Solvent

A (methanol) and solvent B (acetonitrile) were selected asthe mobile phases Gradient elution was used as follows0min 80 20 30min 45 55The injection volumewas 40120583Lthe flow rate was 10mLmin and the column temperaturewas maintained at 25∘C Sample was introduced into theHPLC using an Agilent 1200 G1367B autosampler The signalwas monitored at 206 nm using the diode array detector(Agilent USA) The peaks were characterized by compar-ing the retention time with the standards Retention timesfor asiaticoside and asiatic acid were approximately 8807and 23809mins respectively Asiatic acid and asiaticosidestandard stock solution were prepared by dissolving approx-imately 10mg each of accurately weighed pure compound in10mL methanol-water (9 1) respectively Standard workingsolutions used for calibration were prepared by dilutingthe above standard solutions with methanol to the desiredconcentrations (10ndash100120583gmL) All standard solutions were


120

100

80

60

40

20

00 5 10 15 20 25

8807

23809

Area 10238Asiaticoside

Asiatic acid

(mAU

)(min)

DAD1 A Sig = 206 16 Ref = 360 100 (ZUHAIRI 2012-07-02 10-25-19004-0401D)

(a)

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

1301

1915

2231

2525

2867

3903

4580

4856

5236

6442

7097

8368

18232

23016

Asiaticoside Asiatic acid


(b)

1330

1881

2057

2616

3184

38854570

5242

5657

5861

6374

13953

18755

19011 23523

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

Asiatic acid

DAD1 A Sig = 206 16 Ref = 360 100 (DEF_ LC2012-07-26 10-46-28001-0101D)

(c)

Figure 5 HPLC chromatogram (a) mixture standards (b) micropowders extract (c) nanopowders extract

analyzed in triplicate and the average peak areas were mea-sured UV peak areas of the external standards (119910) wereplotted against the corresponding concentrations (119909 120583gmL)to generate standard curves 119910 = 1222119909 minus 0296 was forasiatic acid (1199032 = 0994) and 119910 = 6191119909 minus 2125 was forasiaticoside (1199032 = 0994) The content of each compound(120583gmL) in the extracts was then calculated from the exper-imental peak areas by interpolation to standard calibrationcurves

26 Extraction Yields Determination The extraction yieldsof asiatic acid and asiaticoside were calculated using thefollowing

Yield (mgg) =

weight of asiatic acid (mg)weight of dried sample (g)

(1)

The percentage total asiatic acid and asiaticoside extractionyield in nanopowders over micropowders were calculated asfollows

Extraction yield

= ((Total asiatic acid + asiaticoside)119873

minus (Total asiatic acid + asiaticoside)119872)

times ((Total asiatic acid + asiaticoside)119872)minus1

times 100

(2)

where119873 is nanopowders and119872 is micropowders

3 Results and Discussions

31 Particles Size Analysis and Surface Morphology Theaverage size of the nanoparticles was determined as 285 nm


as shown in Figure 2(a) The ground nanoparticle sizesvaried from 50 nm to 800 nm with the 342 nm being thehighest size amount with 14 of intensity The correlationcoefficient shown in Figure 2(b) shows a good correlationof analysis with interception of y= axis around 09 and thebaseline of curve is almost flat It represents the mixtureof smaller and larger particles present in the sample [14]This can be understood by referring to the polydispersityindex (PdI) value of 0469 indicating that distribution size ofnanoparticles is in themedium rangeThismediumPdI valuealso indicates the presence of larger particles after millingsince the plant material is hard to grind below 100 nm [11 1215] due to the fibrous structure of plant material and limitsthe size reduction of particle [16]

The surface morphology between nanoparticles andmicroparticles ground powders was shown in Figure 3 Thenanopowders showd a significant difference in size compareto the micropowder which indicates that the size reduc-tion of particles occurred through high impact forces asa consequence of the collision of high accelerated beadsand wall of grinding jar Redispersibility of agglomeratedparticles to individual particles is another important featurein nanotechnology and extraction process specifically Theparticles that readily exist in the individual form will have agreater surface area compared to the agglomerated particlesand will affect extraction efficiency by increasing the contactratio between solvent and particles Thus the determinationof the degree of redispersibility of freeze-dried nanopowderswas carried out The measurement was carried out by dis-persing nanopowders in one-minute ultrasonic time and thesuspension was measured as stated earlier Figure 4 showsthe size distribution comparison between nanopowders inthe suspension and nanopowders after freeze-drying processAs can be seen in Figure 4 the majority sizes of freeze-dried nanopowders located at microsize range (gt1000 nm)compared to the size of nanopowders before freeze-dryingThis indicates that after solvent removal the particles start toagglomerate During freezing step phase separation occursbetween the aqueous phase and nanoparticles This sepa-ration between water and nanoparticles will enhance theinteraction between them leading to their aggregation orfusion [17]

32 HPLC Analysis of Asiaticoside and Asiatic Acid HPLCchromatograms were shown in Figure 5 As can be seenfrom chromatogram asiaticoside and asiatic acid were wellresolved with this method and the retention time is 8807and 23809mins respectively Both extracts showmany polarcompounds appearing in the first 10 minutes since water isvery polar solvent thus it is easy to extract polar compoundThis explained why asiaticoside is more preferably extractedin a water extraction compared to the asiatic acid This lowwater extraction yield of asiatic acid was in line with whatwas early reported in [8 18ndash20] Micropowders extract showsthe existing of asiaticoside (119905

119877= 8324mins) and asiatic

acid (119905119877= 22951mins) while nanopowders extract shows

the disappearance of asiaticoside peaks and only asiatic acid(119905119877= 21884mins) peaks exist in the analysis The hydrolysis

8

7

6

5

4

3

2

1

0

M20 M40 M60 N20 N40 N60

Sample

Extr

actio

n yi

eld

(mg

g)

AsiaticosideAsiatic acid

Figure 6 The effect of ultrasonic extraction time on the yield ofasiaticoside and asiatic acid usingmicropowders (119872) and nanopow-ders (119873)

of asiaticoside merely occured in the presence of acid or baseas early reported In this study we also try to figure outhow hydrolysis of asiaticoside occurs in the milling processthis possible mechanism will be explained later in the nextsection

To investigate the influence of extraction time on yieldof asiaticoside and asiatic acid sample was extracted at theconditions of 25∘C 125W and 50mL water at different times(20 40 and 60min) Figure 6 showed that the extractionyield of the asiaticoside and asiatic acid decreased with theincrease of extraction time The extraction yields of activecompound for micropowders and nanopowders at 20minwere 475 and 709mgg respectively Increasing extractiontime from 20 to 60min the extraction yields of asiaticosideand asiatic acid in the micropowders and nanopowders werereduced to 307 and 349mgg respectivelyThis ismaybe dueto degradation of bioactive compound under a long periodof exposure to ultrasound activity Ultrasound extractionthrough high-energy sound wave induces cavitation anddestroys plant cells promoting the penetration solvent todissolve of target constituent of plant [21] As extraction timeincreases the number of ruptured plant cell increases andresults in increasing extraction yieldNevertheless increasingnumber of ruptured plant cell will also increase insolublecompound such as insoluble substances and cytosol suspendin the extraction liquid which will affect the permeabilityof the solvent [22] Beside that readsorption of interestedcompound on the flake of plant particles will also contributeto the low extraction yield at longer time of extraction time[23] Therefore it is not necessary to do overtime extractionwhen the maximum extraction yield has been achieved andthus 20minutes of extraction time was selected for extractiontime


HO

HO

OH

H

H

HH

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

H

OH

H

OHOH

OHOH

OHOH

OHO

OO

O

H

H

O

OH

H

OHOH

OHOHO

OH

OHO

HO

OH

OHH

O

CH3

+

H2O H3O+

Asiaticoside

Asiatic acid

+2 sugars + alpha L-rhamnose

HO

HO

HO

HO

O

O

Figure 7 Possible mechanism of hydrolysis of asiaticoside

As can be seen in Figure 6 extraction of asiatic acid innanopowders is higher than asiatic acid in micropowders atdifferent ultrasonic times Some asiatic acid in nanopowdersmight come from hydrolysis of asiaticoside thus to be fairthe total of asiaticoside and asiatic acid extracted was used asa comparison to expose the useful application of nanopow-ders on the extraction yield Figure 6 also revealed a totalasiaticoside and asiatic acid extracted in micropowders wasmuch lower with 475mgg compared to the nanopowderswith 709mgg By using nanopowders the water extractionyield of bioactive compounds from Centella asiatica couldbe improved by 4926 and almost 50 improvement Sincenanopowders have higher surface area due to the smallerparticles size then it will help to increase the contact ratiobetween cell and solvent With the help of ultrasonic energythe disruption of cell wall becomes easy [10 24] This actionresults in surface peeling erosion and particle breakdown[25] thus nanopowders used in this extraction help speedup the rupture of cell wall and increase the mass transfer ofbioactive compounds from cell cytoplasm to the surroundingsolvent

33 Possible Hydrolysis Mechanism in Milling Process Pro-duction of asiatic acid from asiaticoside can be easily pro-duced by hydrolyzing asiaticoside in acidic conditions Theexplanation of hydrolysis of asiaticoside was shown in earlyresearch paper [6] Hydrolyzation of asiaticoside throughmilling process is still not explained yet Thus we comeout with a possible mechanism that could happen duringmilling process The conversion asiaticoside to asiatic acidwas converted by the hydronium ion (H

3O+) generated from

the reaction zirconium ligand and water The formation ofzirconium ligand due to the interaction between grindingball and grinding jar consequently allows the zirconium

ion (Zr4+) to form complex ion as illustrated in chemicalequation (1) Therefore this allows the hydronium ions to beattacked by the oxygen in the asiaticoside structures duringthe milling process The sugar structure was hydrolyzed partby part until producing an aglycone (asiatic acid) as shownin Figure 7 This hydrolysis process will produce aglyconeand 2 sugar molecules and one rhamnose [26 27] which ofthese compounds are a part of sugarmoiety in the asiaticosidestructure [28]

ZrO2+H2O 997888rarr Zr(H

2O)3+6

Zr(H2O)4+6

+H2Olarrrarr Zr(H

2O)5(OH)3+ +H

3O+

(3)

The hydrolysis of glycoside also has been explained in[29] Several articles have been reported onmetal oxides suchas titanium dioxide (TiO

2) silicon oxide (SiO

2) zeolite and

ZrO2were capable to act as the solid acid catalyst and has

been demonstrated as a catalyst in the reaction of convertingcomplex sugar to simple sugar [30 31] In this method byusing the residue of ZrO

2to generate acidic condition in the

milling jar will it be possible to break the sugar moiety fromasiaticoside to produce asiatic acid The possible mechanismwas illustrated below

4 Conclusions

Enhancement of water extraction of asiatic acid yield usingnanopowders was investigated in this study assisted byultrasound energy at different extraction times Extractionof asiatic acid using nanopowders was compared to microp-owders and the results show that the extraction of asiaticacid using nanopowders is 50 higher compared to themicropowder with 709mgg extraction yield Extractiontime was 20 minutes due to the maximum yield was obtained


at this extraction period In this research we also found aninteresting finding that is ball milling process using zirconiabead also has a great potential in hydrolyzing glycosides totheir aglycone with fast efficient and clean green approachItmay revolutionize the preparation of herbalmedicine in thefuture

Acknowledgment

Financial support from Universiti Teknologi MARA (UiTM)is gratefully acknowledged

References

[1] B M Hausen ldquoCentella asiatica (Indian pennywort) an effec-tive therapeutic but a weak sensitizerrdquo Contact Dermatitis vol29 no 4 pp 175ndash179 1993

[2] F XMaquart G Bellon P Gillery YWegrowski and J P BorelldquoStimulation of collagen synthesis in fibroblast cultures by atriterpene extracted from Centella asiaticardquo Connective TissueResearch vol 24 no 2 pp 107ndash120 1990

[3] W R Rush G R Murray and D J M Graham ldquoThe com-parative steady-state bioavailability of the active ingredientsof Madecassolrdquo European Journal of Drug Metabolism andPharmacokinetics vol 18 no 4 pp 323ndash326 1993

[4] P-J Shim J-H Park M-S Chang et al ldquoAsiaticoside mimeticsas wound healing agentrdquo Bioorganic and Medicinal ChemistryLetters vol 6 no 24 pp 2937ndash2940 1996

[5] B-S Jeong C K Young and E-S Lee ldquoModification ofC232328 functional groups on asiatic acid and evaluationof hepatoprotective effectsrdquo Bulletin of the Korean ChemicalSociety vol 28 no 6 pp 977ndash982 2007

[6] B-S Jeong K L Mi C K Young and E-S Lee ldquoModificationof C2 functional group on asiatic acid and the evaluation ofhepatoprotective effectsrdquo Archives of Pharmacal Research vol30 no 3 pp 282ndash289 2007

[7] E S Ong J S H Cheong and D Goh ldquoPressurized hot waterextraction of bioactive or marker compounds in botanicals andmedicinal plant materialsrdquo Journal of Chromatography A vol1112 no 1-2 pp 92ndash102 2006

[8] W-J Kim J Kim B Veriansyah et al ldquoExtraction of bioactivecomponents from Centella asiatica using subcritical waterrdquoJournal of Supercritical Fluids vol 48 no 3 pp 211ndash216 2009

[9] R Konwarh S Pramanik D Kalita C L Mahanta and NKarak ldquoUltrasonicationmdasha complementary ldquogreen chemistryrdquotool to biocatalysis a laboratory-scale study of lycopene extrac-tionrdquoUltrasonics Sonochemistry vol 19 no 2 pp 292ndash299 2012

[10] S R Shirsath S H Sonawane and P R Gogate ldquoIntensificationof extraction of natural products using ultrasonic irradiationsmdasha review of current statusrdquoChemical Engineering and Processingvol 53 pp 10ndash23 2012

[11] P Y Ma Z Y Fu Y L Su et al ldquoModification of physicochemi-cal and medicinal characterization of Liuwei Dihuang particlesby ultrafine grindingrdquo Powder Technology vol 191 no 1-2 pp194ndash199 2009

[12] Y L Su Z Y Fu C J Quan and W M Wang ldquoFabrication ofnano Rhizama Chuanxiong particles and determination oftetramethylpyrazinerdquo Transactions of Nonferrous Metals Societyof China vol 16 pp S393ndashS397 2006

[13] S Huang and W H Chang ldquoAdvantages of nanotechnology-basedChinese herb drugs on biological activitiesrdquoCurrentDrugMetabolism vol 10 no 8 pp 905ndash913 2009

[14] Dynamic Light Scattering An Introduction in 30 MinutesMalvern Instruments Malvern Wis USA 2005

[15] P Ma Z Fu Y Su and J Ma ldquoNano pulverization of traditionalChinese medicine Liuwei Dihuangrdquo Journal Wuhan Universityof Technology vol 21 no 2 pp 105ndash108 2006

[16] R Rajkhowa L Wang J Kanwar and X Wang ldquoFabrication ofultrafine powder from eri silk through attritor and jet millingrdquoPowder Technology vol 191 no 1-2 pp 155ndash163 2009

[17] W Abdelwahed G Degobert S Stainmesse and H FessildquoFreeze-drying of nanoparticles formulation process and stor-age considerationsrdquo Advanced Drug Delivery Reviews vol 58no 15 pp 1688ndash1713 2006

[18] N R Barbosa F Pittella and W F Gattaz ldquoCentella asiaticawater extract inhibits iPLA2 and cPLA2 activities in rat cere-bellumrdquo Phytomedicine vol 15 no 10 pp 896ndash900 2008

[19] M C Kwon W Y Choi Y C Seo et al ldquoEnhancement of theskin-protective activities ofCentella asiatica L urban by a nano-encapsulation processrdquo Journal of Biotechnology vol 157 no 1pp 100ndash106 2012

[20] A Soumyanath Y-P Zhong E Henson et al ldquoCentella asiaticaextract improves behavioral deficits in a mouse model ofAlzheimerrsquos disease investigation of a possible mechanism ofactionrdquo International Journal of Alzheimerrsquos Disease vol 2012Article ID 381974 9 pages 2012

[21] T J Mason L Paniwnyk and J P Lorimer ldquoThe uses of ultra-sound in food technologyrdquoUltrasonics Sonochemistry vol 3 no3 pp S253ndashS260 1996

[22] S ZhaoK-CKwok andH Liang ldquoInvestigation onultrasoundassisted extraction of saikosaponins from Radix BupleurirdquoSeparation and Purification Technology vol 55 no 3 pp 307ndash312 2007

[23] J Dong Y Liu Z Liang andWWang ldquoInvestigation on ultra-sound-assisted extraction of salvianolic acid B from Salviamiltiorrhiza rootrdquo Ultrasonics Sonochemistry vol 17 no 1 pp61ndash65 2010

[24] M Vinatoru ldquoAn overview of the ultrasonically assisted extrac-tion of bioactive principles from herbsrdquo Ultrasonics Sonochem-istry vol 8 no 3 pp 303ndash313 2001

[25] L Paniwnyk H Cai S Albu T J Mason and R Cole ldquoTheenhancement and scale up of the extraction of anti-oxidantsfrom Rosmarinus officinalis using ultrasoundrdquo UltrasonicsSonochemistry vol 16 no 2 pp 287ndash292 2009

[26] B Burlando L Verotta L Cornara and E Bottini-MassaHerbal Principles in Cosmetics Properties and Mechanisms ofAction Taylor amp Francis Group 2010

[27] C K Kokate A P Purohit and S B Gokhale PharmacognosyNIRALI PRAKASHAN 2009

[28] D Monti A Candido M M Cruz Silva V Kren S Rivaand B Danieli ldquoBiocatalyzed generation of molecular diversityselective modification of the saponin asiaticosiderdquo AdvancedSynthesis and Catalysis vol 347 no 7-8 pp 1168ndash1174 2005

[29] T W G Solomons and C B Fryhle Organic Chemistry JohnWiley amp Sons 9th edition 2008

[30] H Wang C Zhang H He and L Wang ldquoGlucose productionfrom hydrolysis of cellulose over a novel silica catalyst underhydrothermal conditionsrdquo Journal of Environmental Sciencesvol 24 no 3 pp 473ndash478 2012

[31] K D O Vigier and F Jerome Heterogeneously-CatalyzedConversion of Carbohydrates vol 295 2010

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Applied ChemistryJournal of



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Analytical ChemistryInternational Journal of


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Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


OHO

HO

OH

H

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

HCH3

(a)

HO

HO

OH

OH

HO

(b)

Figure 1 Chemical structure of (a) asiaticoside and (b) asiatic acid

encountered in the extraction industry [7ndash9] However theextraction efficiency of these methods is greatly dependenton the particles size of plant material Among the methodsultrasonic-assisted extraction (UAE) has been known for itssimplicity such as short processing time and low solventconsumption [10]

Herbal nanopowders are the herbal powders rangingfrom 10 to 1000 nm in size This nanopowder has uniqueproperties such as larger surface area thus improvingdissolve-out properties of bioactive components in the cellu-lar matrix Several studies have shown increase of extractionratio of bioactive components from 17 to 50 increasescompared to the normal powders [11 12] Beside a bet-ter extraction yield the extraction of nanopowders doesnot require longer extraction time Thus it will save timeconsumed for extraction and more than that it just onlyrequires small portion of powderThus it is cost effective andconcerned with the limited supply of raw material of herbalplant itself [13]

In this study we investigated the effectiveness of nano-powders compared to themicropowders for the water extrac-tion of asiatic acid from Centella asiatica assisted by theultrasonic probe The influence of extraction time also hasbeen investigated in order to define the optimal condition forextraction

2 Methodology

21 Chemicals and Materials Asiaticoside was purchasedfrom Chengdu Biopurify China asiatic acid standard waspurchased from the Sigma Aldrich acetonitrile (LC grade)from Fisher Chemicals and methanol (HPLC grade) fromFriedemann Schmidt Distilled water was purified using aMilli-Q water purification system (Millipore Bedford MAUSA)

22 Sample Preparation Plant material was bought fromlocal market in Tanjung Malim Perak Malaysia duringMarch 2010 Centella asiatica was washed with runningtap water and rinsed by distilled water to remove any dirtand contaminant on the plant material Then cleaned plantmaterial was stored in the 50∘C controlled oven and left todry for 3 days The dried plant material was ground using

conventional rotor The ground powder was sieved through250120583m sieve and stored in the 4∘C prior ball milling process

23 Production of Centella asiatica Nanopowders The driedand powdered plant material (03 g) first was mixed with25mL of 01 (wv) Pluronic F127 and milled at 12 wvof mass concentration and 550 rpm milling speed at 4 hoursof milling time in the 50 cm3 stainless steel jar containing 25gram of 2mm diameter size of zirconia bead These param-eters were chosen because that the early preliminary resultsshow at these conditions the content of asiatic acid is highestamong nanopowders produced at difference conditions ofgrinding (mass concentration milling time and amount ofbead) The resulting milling product was in the form ofnanosuspension This nanosuspension was further used forextraction

24 Particles Size Analysis andMorphology Prior to themea-surement nanosuspensions of each sample were taken outand adjusted to 001 (wv) concentration and introducedinto a disposable cuvette to be measured in triplicate Z-Average and polydispersity (PdI) were recorded FESEM(Carl Zeiss SMT SUPRA 40VP) imaging was carried out byplacing 10 120583L nanosuspension onto a glass slide and stored inan electronic desiccators (temperature 20∘C humidity 18RH) for drying purpose The dried samples were then coatedwith gold (sim10 nm thick) and were placed onto an adhesivetape on the FESEM stub

25 HPLC Analysis

251 Extraction A 06 g micropowder was weighed andmixed with 50mL water Then this mixture was sonicatedfor 20 40 and 60 minutes by ultrasonic processor (UP400SUltrasonic Processor Hielscher) at frequencies wattage out-put and 125W Then mixture was centrifuged to separatethe particles and filtered by gravitational filtration Filtratewas collected and water was removed by freeze dryer andweights of crude extracts were recorded The extracts weredissolved in methanol to 1mgmL and filtered through022120583mfilter paper before being subjected to HPLC analysisThe nanopowders were also extracted in the same manner asabove


16

14

12

10

8

6

4

2

0

1 10 100 1000 10000

Inte

nsity

()

Size (dnm)

100

90

80

70

60

50

40

30

20

10

0

Und

ersiz

e

(a)

10

09

08

07

06

05

04

03

02

01

00

01 10 1000 100000 10000000 1000000000

Time (120583s)

Cor

relat

ion

coeffi

cien

t

(b)


(a)

(a)

(b)

(b)


15

10

5

001 1 10 100 1000 10000

Size (dnm)

Inte

nsity

()





120

100

80

60

40

20

00 5 10 15 20 25

8807

23809


Asiatic acid

(mAU

)(min)


(a)

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

1301

1915

2231

2525

2867

3903

4580

4856

5236

6442

7097

8368

18232

23016



(b)

1330

1881

2057

2616

3184

38854570

5242

5657

5861

6374

13953

18755

19011 23523

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

Asiatic acid


(c)




Yield (mgg) =


(1)


Extraction yield




times 100

(2)











8

7

6

5

4

3

2

1

0

M20 M40 M60 N20 N40 N60

Sample

Extr

actio

n yi

eld

(mg

g)






HO

HO

OH

H

H

HH

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

H

OH

H

OHOH

OHOH

OHOH

OHO

OO

O

H

H

O

OH

H

OHOH

OHOHO

OH

OHO

HO

OH

OHH

O

CH3

+

H2O H3O+

Asiaticoside

Asiatic acid


HO

HO

HO

HO

O

O




3O+) generated from




2O)3+6

Zr(H2O)4+6

+H2Olarrrarr Zr(H

2O)5(OH)3+ +H

3O+

(3)



2) zeolite and





4 Conclusions




Acknowledgment


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


16

14

12

10

8

6

4

2

0

1 10 100 1000 10000

Inte

nsity

()

Size (dnm)

100

90

80

70

60

50

40

30

20

10

0

Und

ersiz

e

(a)

10

09

08

07

06

05

04

03

02

01

00

01 10 1000 100000 10000000 1000000000

Time (120583s)

Cor

relat

ion

coeffi

cien

t

(b)


(a)

(a)

(b)

(b)


15

10

5

001 1 10 100 1000 10000

Size (dnm)

Inte

nsity

()





120

100

80

60

40

20

00 5 10 15 20 25

8807

23809


Asiatic acid

(mAU

)(min)


(a)

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

1301

1915

2231

2525

2867

3903

4580

4856

5236

6442

7097

8368

18232

23016



(b)

1330

1881

2057

2616

3184

38854570

5242

5657

5861

6374

13953

18755

19011 23523

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

Asiatic acid


(c)




Yield (mgg) =


(1)


Extraction yield




times 100

(2)











8

7

6

5

4

3

2

1

0

M20 M40 M60 N20 N40 N60

Sample

Extr

actio

n yi

eld

(mg

g)






HO

HO

OH

H

H

HH

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

H

OH

H

OHOH

OHOH

OHOH

OHO

OO

O

H

H

O

OH

H

OHOH

OHOHO

OH

OHO

HO

OH

OHH

O

CH3

+

H2O H3O+

Asiaticoside

Asiatic acid


HO

HO

HO

HO

O

O




3O+) generated from




2O)3+6

Zr(H2O)4+6

+H2Olarrrarr Zr(H

2O)5(OH)3+ +H

3O+

(3)



2) zeolite and





4 Conclusions




Acknowledgment


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


120

100

80

60

40

20

00 5 10 15 20 25

8807

23809


Asiatic acid

(mAU

)(min)


(a)

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

1301

1915

2231

2525

2867

3903

4580

4856

5236

6442

7097

8368

18232

23016



(b)

1330

1881

2057

2616

3184

38854570

5242

5657

5861

6374

13953

18755

19011 23523

40

30

20

10

0

minus100 5 10 15 20 25

(mAU

)

(min)

Asiatic acid


(c)




Yield (mgg) =


(1)


Extraction yield




times 100

(2)











8

7

6

5

4

3

2

1

0

M20 M40 M60 N20 N40 N60

Sample

Extr

actio

n yi

eld

(mg

g)






HO

HO

OH

H

H

HH

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

H

OH

H

OHOH

OHOH

OHOH

OHO

OO

O

H

H

O

OH

H

OHOH

OHOHO

OH

OHO

HO

OH

OHH

O

CH3

+

H2O H3O+

Asiaticoside

Asiatic acid


HO

HO

HO

HO

O

O




3O+) generated from




2O)3+6

Zr(H2O)4+6

+H2Olarrrarr Zr(H

2O)5(OH)3+ +H

3O+

(3)



2) zeolite and





4 Conclusions




Acknowledgment


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








8

7

6

5

4

3

2

1

0

M20 M40 M60 N20 N40 N60

Sample

Extr

actio

n yi

eld

(mg

g)






HO

HO

OH

H

H

HH

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

H

OH

H

OHOH

OHOH

OHOH

OHO

OO

O

H

H

O

OH

H

OHOH

OHOHO

OH

OHO

HO

OH

OHH

O

CH3

+

H2O H3O+

Asiaticoside

Asiatic acid


HO

HO

HO

HO

O

O




3O+) generated from




2O)3+6

Zr(H2O)4+6

+H2Olarrrarr Zr(H

2O)5(OH)3+ +H

3O+

(3)



2) zeolite and





4 Conclusions




Acknowledgment


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


HO

HO

OH

H

H

HH

OHOH

OHOH

OHOH

OH

OH

OH

OO

O

OO

O

H

H

H

OH

H

OHOH

OHOH

OHOH

OHO

OO

O

H

H

O

OH

H

OHOH

OHOHO

OH

OHO

HO

OH

OHH

O

CH3

+

H2O H3O+

Asiaticoside

Asiatic acid


HO

HO

HO

HO

O

O




3O+) generated from




2O)3+6

Zr(H2O)4+6

+H2Olarrrarr Zr(H

2O)5(OH)3+ +H

3O+

(3)



2) zeolite and





4 Conclusions




Acknowledgment


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Acknowledgment


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article green extraction: enhanced extraction

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