Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa

Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa Optimization of microwave, ultra sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from curcuma longa

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Separation and Purification Technology

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / s e p p u r

Optimization of microwave, ultra-sonic and supercritical carbon dioxide assisted extraction techniques for curcumin from Curcuma longa

P.S Wakte∗, B.S Sachin, A.A Patil, D.M Mohato, T.H Band, D.B Shinde

Pharmaceutical Technology division, Department of Chemical Technology, Dr Babasaheb Ambedkar Marathwada University, Aurangabad 431004, India

a r t i c l e i n f o

Article history:

Received 30 August 2010

Received in revised form 27 October 2010

Accepted 19 March 2011

Keywords:

Curcumin

Microwave assisted extraction

Soxhlet assisted extraction

Supercritical carbon dioxide assisted

extraction

Ultra-sonic assisted extraction

a b s t r a c t

A bio-active phytochemical, curcumin, was isolated from dried rhizomes of Curcuma longa using Soxhlet, microwave, ultra-sonic and supercritical carbon dioxide assisted extraction techniques The quantifica-tion of curcumin in resultant extracts was performed using pre-validated HPLC methodology The critical parameters viz effect of pre-irradiation and soaking solvent on the curcumin yield were studied The extraction efficiency of all the above described techniques was established in terms of percent curcumin yields and extraction rate constants Prior to extraction, microwave and ultra-sonic irradiation of dry curcuma powder resulted in 68.57 and 40.00% curcumin yield, respectively, whereas water soaked irradi-ated curcuma powder yielded 90.47 and 71.42% curcumin recovery respectively, during a total extraction period of five minutes The maximum extraction rate constant of 47.49× 10−2min−1was observed when using microwave assisted acetone extract of water soaked curcuma rhizomes The comparison of Soxhlet, microwave, ultra-sonic and supercritical carbon dioxide assisted extraction in terms of percent yield and required extraction period showed that microwave assisted extraction technique was more efficient for the curcumin extraction from powdered C longa rhizomes

© 2011 Elsevier B.V All rights reserved

1 Introduction

Phytochemicals are, naturally occurring plant-based substances

are considered as potential therapeutic and prophylactic agents

Among several examples, Curcumin, 1,7-bis(4-hydroxy 3-methoxy

phenyl)-1,6-heptadione-3,5-dione (Fig 1) is a dietary

phytochem-ical obtained from the dried rhizomes of the turmeric plant

(Curcuma longa)

Turmeric has been used traditionally in “ayurvedic medicine” as

an antiseptic, wound healing, and anti-inflammatory compounds

The yellow colour, which is characteristic of the turmeric rhizome,

is due to presence of 3–5% of curcuminoids The curcuminoids

includes curcumin, demethoxycurcumin, bisdemethoxycurcumin

[1]and cyclocurcumin[2]of which curcumin is the major bio-active

constituent

Traditionally curcumin has been used as a colouring, as a

flavouring substance and as a food preservative[3] Because of

its wide spectrum of biological activity, an extensive number of

studies have been focused on curcumin Recently, curcumin has

also been shown to display anti-oxidant [4], anti-cancer [5–7],

Abbreviations: MAE, microwave assisted extraction; SAE, Soxhlet assisted

extraction; SC CO 2 E, supercritical carbon dioxide extraction; UAE, ultra-sonic

assisted extraction.

∗ Corresponding author Tel.: +91 240 2403307; fax: +91 240 240013.

E-mail address: pravinwakte@gmail.com (P.S Wakte).

anti-viral, anti-infectious [8] and anti-amyloidogenic properties [9]

For isolating curcumin as well as other curcuminoids from C longa rhizomes, numerous methods are reported: conventional solvent extraction [10,11], hot and cold percolation[12], use of alkaline solution[13]and insoluble salt[14]

Moreover, the extraction of curcuminoids using supercritical carbon dioxide extraction (SC CO2E)[15–19], microwave assisted extraction (MAE) [20]and ultra-sonic assisted extraction (UAE) techniques[21]have also been reported Despite the fact that SC CO2 extraction of curcumin has previously been reported, some ambiguity in the use of modifier percentage remains[16] More-over, no single report specifically delineates the curcumin yields Finally, the length of the MAE and UAE curcuminoids extraction cycle was quite long[20,21] Thus, the objectives of this work were

to establish cost effective and efficient extraction procedures for curcumin by SC CO2E, MAE and UAE, to compare the obtained yields and to describe the processes using mass transfer and extraction rate concepts

2 Experimental procedures

2.1 General The authenticated dried rhizomes of C longa were ground to

a powder using a pulveriser (K.C Engineers, Ambala, HR, India)

To select uniform particle size, rhizomes powder was sifted in a 1383-5866/$ – see front matter © 2011 Elsevier B.V All rights reserved.

cumin (purity 98% by HPLC) was purchased from Sigma–Aldrich

(St-Louis, MO, USA) All solvents used for the extraction and the

chromatographic purpose were of analytical grade (Finar

Chemi-cals Ltd., Ahmedabad, GJ, India) and HPLC grade (Qualigens Fine

Chemicals, Mumbai, MH, India), respectively

A commercial microwave oven with extraction assembly

(Model: MicroSYNTH, Max power: 1000 W, M/s Milestone, Shelton,

CT, USA), ultrasonic bath (Model: UCB 40, power: 150 W, M/s

Spec-tralab, Thane, MH, India) and a bench top SC CO2E unit (Model:

SFE 2000 series, Jasco International Co Ltd., Hachioji, Tokyo, Japan)

were used for the extraction purposes The extracts were prepared

freshly and stored temporarily in desiccators (Riviera Glass Pvt Ltd.,

Mumbai, MH, India) under vacuum until the analysis

2.2 HPLC method for curcumin analysis

The HPLC analysis of curcumin was essentially performed as

described earlier with the following modifications[22] The Jasco

HPLC system was equipped with a PU 2089 quaternary gradient

pump, a UV-2075 plus detector, a LC-NetII/ADC communication

module and a HiQ Sil C-18 W column (4.6 mm× 250 mm, 5 ␮m

particle size, KYA Tech corporation, Tokyo, Japan) was used for

chromatographic purpose Data analysis was carried out using a

ChromPass version 1.8.6.1 software

The mobile phase consisted of 40% THF and 60% water

contain-ing 1% citric acid (v/v), adjusted to pH 3.0, with concentrated KOH

solution It was filtered under vacuum through a 0.45␮m

mem-brane filter before use The eluent flowed isocratically at a flow

rate of 1 ml/min Sample detection was acquired at 420 nm; and

the injection volumes were 10␮l

The analytical method was validated to meet the acceptance

criteria as per International Conference on Harmonization of

Tech-nical Requirements for registration of Pharmaceuticals for Human

Use (ICH) guidelines

Stock I (1 mg/ml) of curcumin was prepared by weighing

5 mg curcumin using a pre-calibrated weighing balance (Model:

CPA225D, Sartorius, Goettingen, Lower Saxony, Germany),

trans-ferring to 5 ml volumetric flask (Riviera Glass Pvt Ltd., Mumbai,

MH, India), dissolving by ultra-sonication and diluting with mobile

phase

Stock I was diluted serially with the mobile phase to obtain 7

calibration standards (10, 20, 40, 80, 160, 320 and 500 ng/ml), 3

quality control standards (15, 200 and 450 ng/ml) and 1 system

suitability standard (300 ng/ml)

The system suitability test was performed using 9 replicate

injections of system suitability standard before analysis of samples

The acceptance parameters were less than 0.5 and 1.5% relative

standard deviation (RSD) for retention time and peak area,

respec-tively, along with more than 3500 theoretical plates The linearity

and range was established using 7 calibration standards of

cur-cumin The peak area vs concentration plots were subjected to

linear least square regression analysis Intra- and inter-day

accu-tion with acceptable accuracy and precision was reported as limit

of quantification (LOQ) for curcumin

System suitability test showed that retention time (% RSD < 0.5), peak area (% RSD < 1.5) and number of theoretical plates (<3500) for curcumin met the acceptance criteria on all the experimental days Analysis of the calibration standards showed good correlation between concentration and resulting peak area (r2> 0.999) for cur-cumin The intra- and inter-day accuracy (Diff %) for curcumin was

in the range of−2.81 to +2.53 and −3.11 to +3.61, respectively The intra- and inter-day precision (% RSD) for curcumin was in the range

of 1.11 to 2.09 and 2.34 to 3.14, respectively The LOQ of the method was determined to be 9 ng/ml The typical HPLC chromatogram of standard curcumin, curcumin after Soxhlet, MAE, UAE and SC CO2E

is shown inFig 2A–E

2.3 Soxhlet assisted extraction (SAE) SAE was used for the maximum recovery of curcumin from the C longa rhizomes Twenty grams of powdered rhizomes were placed

in thimble (Borosil, Mumbai, MH, India), which was inserted into a Soxhlet apparatus and extracted with 100 ml acetone The extrac-tion was performed for 8 h After the extracextrac-tion period, the sample was collected, filtered and analyzed for curcumin content by HPLC

as described earlier The SAE of C longa rhizomes was performed

in five replicates

2.4 Microwave assisted extraction (MAE) The MAE was carried out using two different experimental pro-cedures The first condition consisted of placing 20 g of dry C longa powder in a glass dish (thickness approximately 0.3 cm) and irradi-ated for a pre-defined time period (1, 3, 5 or 7 min) The irradiation was performed at 140 W microwave power

After irradiation, the powder was suspended in ethanol or acetone (mass:solvent ratio 1:3 w/v) and poured into an extrac-tion assembly, which consisted of a mono-block rotor and nine cylindrical vessels (height 8.5 cm× internal diameter 3.5 cm, SK-10 digestion rotor, M/s Milestone, Shelton, CT, USA) The stirring speed

of 400 rpm was kept constant throughout the extraction period The extraction was performed at 90 W (extraction solvent: ethanol) and

60 W (extraction solvent: acetone) microwave power

After the pre-defined extraction period of 5 min, the samples were collected from the extraction assembly, filtered and analyzed for its curcumin content by HPLC

The second experimental condition consisted of placing 20 g of the dry powder of C longa in water or ethanol (mass:solvent ratio 1:2 w/v) for a period of 24 h at 20◦C Before irradiation, the excess solvent content was removed and the wet powder was irradiated for a pre-defined time period as described in the first experimen-tal condition The water soaked powder was irradiated at 270 W whereas ethanol soaked powder was irradiated at 50 W microwave power

Fig 2 Chromatographic profiles of curcumin: curcumin standard (A) and curcumin obtained after Soxhlet assisted extraction (B), microwave assisted extraction (C),

ultra-sonic assisted extraction (D) and supercritical carbon dioxide assisted extraction (E).

The microwave-irradiated powder was suspended in solvents,

extracted and collected samples were analyzed as described in first

experimental condition

2.5 Ultra-sonic assisted extraction (UAE)

The UAE was performed at the fixed power of 150 W using

iden-tical experimental conditions as described for MAE Under the first

condition, dry powder (20 g) was placed in a 500 ml glass beaker

and exposed to ultrasonic waves for pre-defined time periods (1,

3, 5 or 7 min) The irradiated powder was suspended in ethanol

or acetone (mass:solvent ratio 1:3 w/v) and sonicated for 5 min

at 21± 2◦C The samples were collected, filtered and analyzed for

curcumin content by HPLC

In the second experimental condition, the dry powder of C longa

(20 g) was soaked in water or ethanol (mass:solvent ratio 1:2 w/v)

for a period of 24 h at 20◦C Before irradiation, the excess solvent

was removed and the wet powder was exposed to ultrasonic waves,

suspended in solvents and after extraction, samples were analyzed

as described in the first experimental condition

2.6 Supercritical carbon dioxide extraction (SC CO2E)

A flow diagram of SC CO2E system is presented inFig 3 The

extractor column was densely packed with 10 g of C longa powder

The column was carefully fixed in a column oven The CO2from

the cylinder was passed through chiller unit (∼277 K) via a siphon tube, delivered and compressed to the desired working pressure

by CO2 delivery pump (PU 2080-CO2Plus, Jasco International Co Ltd., Hachioji, Tokyo, Japan) mounted with a pressure regulator (BP-2080 Plus, Jasco International Co Ltd., Hachioji, Tokyo, Japan), respectively The organic modifier (10% ethanol) was introduced into system by solvent pump (PU 2080 Plus, Jasco International Co Ltd., Hachioji, Tokyo, Japan) The temperature and pressure of CO2

Fig 3 Flow diagram of SC COE system.

Fig 4 Effect of microwave irradiation on dry rhizomes () % Extraction of curcumin

using ethanol and ( ) % extraction of curcumin using acetone.

was manipulated with a pressure regulator The SC CO2was passed

through an extraction column (150 mm length× 15 mm i.d., 10 g

capacity) which was placed in a thermostatically controlled oven

(CO-2060 Plus, Jasco International Co Ltd., Hachioji, Tokyo, Japan)

After the pressure and the fluid flow rate reached the desired values,

the six-port valve was turned on so that SC CO2was passed through

the extractor; this was counted as the start of the extraction cycle

In the first operating mode, sufficient SC CO2was introduced for a

60 min static conditioning so that sufficient contact with C longa

powder was established The second operating mode was followed

by a steady flow of SC CO2 under the dynamic extraction

condi-tion for 300 min The exit fluid from the extractor was expanded to

ambient pressure by a pressure regulator The extract was collected

in a glass vial and analyzed for curcumin content by HPLC

2.7 Extraction rate constant

At constant volume, assuming that the curcumin mass transfer

process from solid to liquid boundary could be formally treated

as an irreversible first-order reaction, the equation of curcumin

extraction rate constant is:

k =1

t



lnS0

where k is the extraction rate constant (min−1), t the extraction time

(min), S0the total yield of curcumin (w/w) and Stis the remained

extractible curcumin after extraction time t (w/w)[23]

2.8 Statistical analysis

Each experiment was performed in five replicates and the data

was subjected to calculations of mean± S.E The mean values were

used for drawing the graphs

3 Results and discussion

3.1 Soxhlet assisted extraction (SAE)

The conventional SAE of powdered curcuma rhizomes was

car-ried out to recover the maximum extractable amount of curcumin

After SAE, 2.1± 0.1% curcumin was obtained

3.2 Microwave assisted extraction (MAE)

3.2.1 Effect of microwave irradiation

Fig 4depicts the effect of microwave irradiation on the

extrac-tion of curcumin The dry powder of curcuma rhizomes was

exposed to microwave irradiation and further extracted with

Fig 5 Effect of microwave irradiation on pre-soaked rhizomes () % Extraction of curcumin using ethanol from ethanol soaked curcuma powder, ( ) % extraction of curcumin using ethanol from water soaked curcuma powder and ( ) % extraction

of curcumin using acetone from water soaked curcuma powder.

ethanol or acetone The extraction solvents were selected on the basis of pilot experiments [Data not shown] and prior reports of curcumin extraction[20]

The curcumin percentage in the extract increased proportion-ally with longer microwave irradiation exposure periods The 1,

3 and 5 min exposures of microwave irradiation resulted in 9.71, 29.14 and 48.57% curcumin recovery, respectively, while using ethanol as the extraction solvent When acetone was used as the extraction solvent, the curcumin concentration in the extract were 13.71, 41.14 and 68.57% for microwave irradiation periods of 1,

3 and 5 min, respectively Further increase in microwave irradia-tion exposure period did not show any increment in % curcumin extraction

3.2.2 Effect of soaking solvent Fig 5represents the effect of different soaking solvents on cur-cumin extraction On the basis of dipole moment and sensitivity towards microwave irradiation, water and ethanol were selected

as soaking solvents The rhizome powder was soaked in ethanol or water for 24 h at 25◦C The ethanol soaked material was extracted with ethanol whereas water soaked material was extracted with ethanol or acetone

While using ethanol as a soaking and an extraction solvent, the obtained curcumin content was greater by 15.54–57.14% when compared to the microwave irradiation treatment with exposure period of 1–5 min The water soaked powder, when extracted with ethanol, showed curcumin content up to 67.01% after 5 min microwave irradiation The acetone extract of water soaked pow-der demonstrated an increase in curcumin content with increased microwave irradiation exposure period The net curcumin content

in resulted extract was enhanced from 37.49 to 90.69% which was found to be constant even after 7 min of microwave irradiation exposure

Earlier Dandekar and Gaikar have reported the effect of water soaking of C longa on the extraction of curcuminoids The study was performed using MAE technique wherein the soaked powder was extracted without any pre-treatment like microwave irradiation The maximum yield of curcuminoids was obtained after 24 h of soaking[20]

The preliminary experiments of the present work showed the prominent effect of microwave irradiation on the curcumin extraction so the effect of microwave irradiation was studied in combination with different soaking solvents The soaking solvents viz water and ethanol were used as modifier to bring about effective extraction of curcumin from C longa

Fig 6 Effect of ultra-sonic irradiation on dry rhizomes () % Extraction of curcumin

using ethanol and ( ) % extraction of curcumin using acetone.

3.3 Ultra-sonic assisted extraction (UAE)

3.3.1 Effect of ultra-sonic irradiation

Fig 6shows the effect of ultra-sonic irradiation on curcumin

extraction from C longa rhizome powder The dry powdered

mate-rial was extracted with ethanol or acetone after exposure to

ultra-sonic irradiation The ethanol extract showed 6.57–32.85%

curcumin content after the ultra-sonic irradiation exposure period

of 1–5 min Further increases in the exposure period did not show

any increase in the curcumin content of the extract The acetone

extract showed an increase in curcumin concentration, from 8 to

40%, after the ultra-sonic irradiation period of 1 to 5 min

3.3.2 Effect of soaking solvent

The effect of soaking solvents on curcumin extraction is depicted

in Fig 7 When ethanol was used as the soaking and

extrac-tion solvent, 1–5 min ultra-sonic irradiaextrac-tion exposure resulted in

17.55–61.90% curcumin extraction, which was found to be constant

even after 7 min irradiation period

After ultra-sonic irradiation over the period of 1–5 min,

water soaked powder was extracted with ethanol and

ace-tone The ethanol and acetone extract showed 19.70–66.66% and

22.15–71.42% curcumin content, respectively

Mandal et al has reported the UAE of curcumin wherein the

maximum curcumin extraction was achieved in 70 min[21] The

present work demonstrated the maximum curcumin yield by UAE

within 5 min extraction period

Fig 7 Effect of ultra-sonic irradiation on pre-soaked rhizomes () % Extraction of

curcumin using ethanol from ethanol soaked curcuma powder, ( ) % extraction of

curcumin using ethanol from water soaked curcuma powder and ( ) % extraction

Fig 8 Curcumin extraction by supercritical CO2 () % Extraction of curcumin from curcuma powder using supercritical CO 2 .

3.4 Supercritical carbon dioxide extraction (SC CO2E) The SC CO2E of curcumin from C longa powder was achieved using following optimal extraction conditions: pressure – 30 Mpa, temperature – 50◦C, CO2 flow rate – 5 ml/min, modifier – 10% ethanol, static time – 60 min and dynamic time – 300 min.Fig 8 presents the extraction period vs the curcumin concentration of the extract After the extraction period of 240 min, the result-ing extract showed 69.37% curcumin concentration which was observed to be constant, even after 300 min of extraction The HPLC chromatogram of SC CO2extract showed presence of only one peak representing curcumin It has been demonstrated

by Leal et al that slight modification in pressure and CO2 flow rate in SC CO2E changed the composition of C longa extract[19] Boumann et al showed that 10% ethanol when used as SC CO2 mod-ifier, yielded 100% curcumin from two commercial finely ground dry C longa samples[15] This is in good agreement with obtained results of SC CO2E

Although SC CO2E of curcumin from C longa powder has pre-viously been reported by Chassagnez-Mendez et al., the curcumin recovery was around only 1% over the extraction period of 100 min using ethanol as modifier wherein the ethanol content in gaseous phase was not mentioned[16] Moreover, the time vs wt % cur-cuminoids extraction profile showed the presence of curcumin, demethoxy curcumin, bis-demethoxy curcumin and total curcum-inoids

3.5 Extraction rate constant The extraction rate constant was calculated on the basis of total curcumin yield that was obtained In this study S0 (0.021 g cur-cumin/g C longa) was obtained with a Soxhlet extraction for 8 h with acetone The extraction rate constant (k) for each irradiation exposure point (1, 3, 5 and 7 min) was calculated and the values are presented inTables 1 and 2

As depicted inTable 1, the k value was increased with increased microwave and ultra-sonic irradiation exposure period The treat-ment of water and ethanol as soaking solvent further increased the

k value In the case of microwave and ultra-sonic assisted extrac-tions, the water soaked powder after irradiation when extracted with acetone resulted in a higher extraction rate constant When the SC CO2E technique was used for the extraction of curcumin, the k value was increased from 0.09× 10−2min−1 to 0.48× 10−2min−1when the extraction period was increased from

60 to 300 min (Table 2)

b The % yield of curcumin is calculated after total extraction time of 5 min.

Table 2

Supercritical carbon dioxide assisted extraction technique based curcumin extraction rate constants.

Extraction technique Extraction solvent Extraction rate constant a (min −1 ) % Yield b

a Extraction rate constant is expressed in min −1 at 60, 120, 180, 240 and 30 min extraction period.

b The % yield of curcumin is calculated after total extraction time of 24 min.

3.6 Comparison of SAE, MAE, UAE and SC CO2E on the basis of

curcumin yield and extraction time

The conventional Soxhlet assisted acetone extract of C longa

powder resulted in a 2.1% curcumin yield after 8 h of extraction

Using the Soxhlet extraction as the basis, the curcumin yields of

MAE, UAE and SC CO2E techniques were calculated The MAE of C

longa for 5 min recovered a maximum of 90.47% curcumin, whereas

UAE resulted in 71.42% of curcumin with acetone as the extraction

solvent (Table 1) The SC CO2E showed a 69.36% curcumin recovery

after an extraction period of 240 min (Table 2) The comparison of

yield and the time required for the extraction of curcumin

demon-strated that MAE technique is more efficient than SAE, UAE and SC

CO2E techniques

4 Conclusion

Efficient MAE, UAE and SC CO2E methods for curcumin from

C longa has been developed Compared among each other, high

extraction recovery at minimum extraction time was obtained with

MAE Water soaking effectively contributed in curcumin extraction

Extraction rate constant of curcumin has been calculated based on

the experimental results The value of curcumin extraction rate

constant k increased with use of water as soaking solvent and

ace-tone as extraction solvent

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