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990Namrata K Satam. et al. / International J ournal of Biological & Pharmaceutical Research. 2012; 3(8): 990-995.

e- ISSN 0976 - 3651

Print ISSN 2229 - 7480

International Journal of Biological

&Pharmaceutical ResearchJournal homepage:www.ijbpr.com

SUPERCRITICAL FLUID EXTRACTION OF FLAVONOIDS FROM

SOLANUM MELONGENA Linn. FRUIT AND ITS EVALUATION OFFREE RADICAL SCAVENGING ACTIVITY

Namrata K. Satam1, Lavu S. Parab

1, Ashok M. Bhagwat

2, Suvarna I. Bhoir

2

1School of Science, SVKMs NMIMS, Vile Parle (West), Mumbai400 056, Maharashtra, India.

2Shri C.B Patel Research Centre, 3 rd floor, Bhaidas Sabhagriha Bldg, Vile Parle (West),

Mumbai400 056, Maharashtra, India.

ABSTRACT

Supercritical fluid extraction (SFE) represents an efficient and environmentally friendly technique for isolation of

phytoconstituents from different plant sources. The present study is focused on extraction of flavonoids from Solanum

melongena Linnfruit using Supercritical fluid (SCF-CO2) technology. The fruit extract containing flavonoids has been reported

to have hypolipidemic activity. The objective of this work is to evaluate the SCF extraction of flavonoid from S. melongena L.

fruit at different operational conditions and discuss the temperature, pressure and flow rate of CO 2 dependence in the extract

composition profile. Since Carbon dioxide is a non-polar solvent, ethanol is used as co-solvent to increase the polarity of the

fluid. HPLC-PDA determination of flavonoids is done for each condition and the extraction conditions are optimized.

Chromatographic conditions employed for determination of flavonoids component in fruit extract for PDA analysis are mobilephase (Methanol: Acetonitrile: Orthophosphoric acid: Acetic acid: Water, 200:100:10:10:200 v/v), at a flow rate of 1mL/min.

The optimized SC-CO2 extraction conditions under which maximum yields of flavonoids are obtained are temperature 450 C,

Pressure 19.61MPa, co-solvent at 11.5% with a CO2 flow rate 3.0mL/min. Further, in vitro free radical scavenging activity of

SFE extract was evaluated by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging method. Ascorbic acid was used as

the reference. SFE extract exhibited promising antiradical effects in a concentration dependent manner. Linear regression

analysis was used to calculate IC50 value. Results showed that, the extract exhibited significant DPPH radical scavenging

activity with IC50 value of 66.0630.15 g/mL.

Key Words: Solanum melongena, Flavonoids, Supercritical fluid extraction, Antioxidant.

INTRODUCTIONOne of the most important areas of research in

food technology is the isolation of natural compounds with

functional properties from natural sources (Ramirez P et

al.,2005).Therefore plant sources are being re-evaluated

for presence and extraction of pharmacologically active

natural compounds. These phytochemicals are often

Corresponding Author

Namrata K.SatamEmail: [email protected]

present in low concentration in the plants and are

chemically sensitive. One class of natural products,

ubiquitous in vascular plants, is the flavonoids, which are

made up of over 8000 compounds comprising 12

subclasses including flavones, flavanones and flavanols

(Merken HM et al., 2000; Piett, 2005). The medicinal and

pharmacological activities of flavonoids against

inflammation, allergies, viruses, cancer and other ailments

are well documented (Merken HM et al., 2000; Harborne

JB et al., 2000).

Eggplant, Solanum melongena, is a common and

popular vegetable crop grown in the subtropics and tropics

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(Sarker RH et al., 2006). The eggplant is

commonly known as brinjal and aubergine in India and in

Europe, respectively. There are several research

publications describing the health benefits of the phenolic

compounds extracted from eggplant. Studies have shown

that eggplant extracts suppress the development of blood

vessels required for tumor growth and metastasis(Matsubara K et al., 2005) and inhibit inflammation that

can lead to atherosclerosis (Han SW et al., 2003). Research

on hypolipidemic beneficial effects of phenolic

phytochemicals from Solanum melongena in normal and

cholesterol fed rats has been reported. (Sudheesh S et al.,

1999; Sadilova E et al., 2006).

Extraction is the main step for the recovery of

these bioactive phytochemicals and the ultimate goal is the

preparation of a sample extract uniformly enriched in all

components of interest and free from interfering matrix

components (Pyrzynska K and Biesaga M, 2009). An

alternative method to recover the phytochemicals from raw

materials is Supercritical fluid extraction (SFE). SFE is a

rapidly developing method to produce bioactivecompounds by pure technology, under mild conditions

(Simandi B et al., 2002). In recent years, several

researchers studied the extraction of natural compounds

from plant matrix by using supercritical carbon dioxide

(SC-CO2) (Povh NP et al., 2001; Ebrahimzadeh H et al.,

2003; Sonsuzer S et al., 2004). Carbon dioxide (CO2) is

the most widely used solvent in SFE, since it is

physiologically harmless, environmentally safe, non-

explosive, and readily available and it can be easily

removed from products (Cavero S et al., 2006).

Supercritical fluid extraction has previously been

used to extract flavonoids from various plants. The use of

supercritical fluid extraction (SFE) for the extraction ofpolyphenols and optimization of the experimental

conditions are focused on the evaluation of the effect of the

variables that control the whole process. The main targets

are to provide maximum yields, preserving highest quality

with antioxidant activity, making the final product suitable

for use in food, cosmetic or pharmaceutical industries

(Spigno G et al.,2006).To date, there are no publications

found on flavonoid extraction from S. melongena L fruitby

supercritical fluid extraction. In this study supercritical

carbon dioxide was employed to extract flavonoid

bioactive compounds from S. melongena Linn. The

objective of this work were to (i) to investigate the

influence of parameters such as Temperature, Pressure,CO2flow rate and co-solvent flow rate on extraction of

flavonoids.(ii) to evaluate antioxidant activity of SFE

extract.

MATERIALS AND METHODS

Plant materialFruits ofSolanum melongena Linn were collected

from local market. They were thoroughly washed with tap

water and rinsed with distilled water. Identification of the

species was confirmed at Agharkar Research Institute,

Pune.

Fruits were cut in small pieces and were dried in

ventilated drying oven at 40C. Dried fruits were stored in

dark place at room temperature. To avoid degradation, the

dried plant material was ground just before extraction to

form powder in order to increase surface area of sample.The dried plant leaves were grinded in a blender for 10 sec

to produce powder and sieved by 0.85mm mesh screen to

maintain constant particle size throughout the study.

Chemicals and ReagentsCarbon dioxide (purity 99.99%) was purchased

from Rakhangiz gases, India. Ethanol and Orthophosphoric

acid of AR grade and Methanol, Glacial acetic acid of

HPLC grade were purchased from E-Merck, India.

Quercetin (purity 99.99%), DPPH (2, 2-diphenyl-1-

picrylhydrazyl) and Ascorbic acid were purchased from

Sigma- Aldrich.

Supercritical F luid Extraction [SFE] ApparatusThe supercritical carbon dioxide extraction system

and components were acquired from JASCO (Japan

Spectroscopic Co.) 900 series Supercritical fluid

extractor/Chromatograph, included the following: 100 ml

extraction vessel, temperature control unit (JASCO C0-

965), high-pressure pump (JASCO-PU-980), automated

back pressure regulator (JASCO 880-81). The refrigerating

coolant circulator was manufactured by Scinics Co. Ltd.

L.R. Grade methanol was used as a coolant and circulated

at -5C for cooling the SC-CO2 extraction apparatus.

Absolute ethanol (95% EtOH) acted as the co-solvent. The

independent variables were Pressure (7.84MPa to

29.41MPa), Temperature (35C to 70C), CO2 Flow rate(1.8mL/min to 3.5mL/min) and Co-solvent percentage

(6.97% to 13.04%).Before the liquid CO2 was passed into

the extraction vessel, it was pressurized to the desired

pressure and heated to the specified temperature to reach

the supercritical state. The powdered fruit (10g) was well

mixed with 2.0mm diameter glass beads, placed in the

extractor vessel. The introduction of some rigid materials

such as glass beads with the ground sample, contributed to

maintaining a proper flow rate of CO2 in the extractor

vessel as well as in maintaining the desired permissibility

of the particle during extraction process (Chemat S et al.,

2004; Wang CL and Weller T, 2006). The supercritical

CO2 flow rate was maintained at 2 mL/min and thedynamic extraction time was fixed to 60 min.

During the dynamic extraction time, CO2 carrying the

crude extract flowed out of the extraction vessel unit and

into a collection vessel. The ethanolic SFE extracts for

each parameter were stored in amber coloured tubes in a

refrigerator (4 C) until further analysis.

Sample preparationAll collected SFE extracts were processed to


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remove chlorophyll interference. Chlorophyll was

separated from the SFE extract by adding chloroform and

water to the ethanolic extracts in the ratio of 4/3/5 (v/v/v),

respectively. The mixture was mixed vigorously and the

phases were allowed to separate. Upper layer consisted of

flavonoids which was been separated and used for further

analysis (Choinski JS and Johnson JM, 1993).

Determination of flavonoid components in extract byHPLC analysisHPLC Instrumentation and chromatographic conditions

HPLC analysis was performed using equipment

from JASCO HPLC-PDA system (Japan) consisting of

pump (JASCO PU-2080), injector (JASCO AS-155510),

JASCO MD-2010(PDA detector) and CHROMNAV

offline processing software. SFE extracts were analyzed

using a Kromasil RP-C18 column (2504.6mm i.d, 5m)

and mobile phase consisted of methanol-acetonitrile-acetic

acid - phosphoric acid-H2O (200:100:10:10:200, v/v)

(Chen X and Xiao J,2005). All the solutions were

degassed by suction-filtration through a nylon membrane.The detecting wavelength was set at 254 nm. The flow rate

was 1.0 ml/min and the loading volume of injecting sample

was 80.0l. The HPLC system was operated at ambient

temperature (252C).Quercetin was used as standard and

calibration curve was studied in range (8-60g/mL).

Determination of extraction yieldEthanolic extract under optimized SFE conditions

was collected and the residue of the co-solvent from the

extract was removed by a rotary evaporator under vacuum

at 40C. The residue was then completely dried in the oven

at 40C and the final constant weight was recorded. All the

steps were performed with the exclusion of light. Theresult of the extraction yield was calculated as follows:

Yextract (mg/g) = mextract / mherb X 100

Where Yextract is the % extraction yield, mextract is the crude

extract mass (g) and mherb is the extracted herb mass (g)

Free Radical Scavenging Activity evaluated by 1, 1-Diphenyl-2-picrylhydrazyl

Free radical scavenging activity of the SFE

eggplant extract was determined by using a stable 2, 2-

diphenyl-1-picrylhydrazyl radical (DPPH) with slight

modifications (Patwekar F et al., 2010). Different

methanolic dilutions of extract were prepared (25-

125g/ml). Briefly, 2.0 mL SFE extract was added to 2mLDPPH solution (90M in methanol) as the free radical

source. The mixtures were shaken vigorously and allowed

to stand at room temperature for 1hr.The decrease of

solution absorbance due to proton donating activity of

components of each extract was determined at 517nm.

Lower absorbance of the reaction mixture indicates higher

free radical scavenging activity. Vitamin C (Ascorbic acid)

was used as the positive control. The DPPH with

corresponding solvents (without plant material) serves as

control. The DPPH radical scavenging activity was

calculated using the following formula:

DPPH Radical Scavenging Activity (%) = [(A0 A1 /A0)

100], where A0 is the absorbance of the control, and A1

is the absorbance of extract or standard sample.

RESULTS AND DISCUSSIONEffect of Temperature of flavonoid extractionFig. 2 represents the effect of temperature on

flavonoid content of S. melongena fruit in SC-CO2 at

different temperature levels. In this study, results showed

that the flavonoid content increased with increase in

temperature from 35C to 45C. This can be ascribed to the

fact that with rise in temperature there is enhancement of

vapor pressure of analytes thereby increasing extraction of

compounds (Reverchon E et al., 1999). However increase

in temperature also results in reduction of density of CO 2and therefore there was decrease in flavonoid content from

45 to 70C.

Effect of Pressure on flavonoid extractionFig. 3 shows the effect of pressure on the

flavonoid content of S. melongena fruit in SC-CO2 at

different pressure levels. According to the results, as

pressure increases from 7.84MPa to 19.61 MPa, the

flavonoid content increased. At a constant temperature,

there is increase in density of SC-CO2 with increase in

pressure. As the density increases, the distance between the

molecules decrease and therefore the interaction between

the analytes and CO2 increases, leading to greater solubility

of the analytes in SC-CO2 (De Castro et al., 1999). In this

study, the flavonoid content increased with increasing

pressure to a certain value. However, an increase of

pressure also results in an increase in the fluid density,which alters solute solubility and thereby decreasing

extraction (Gomes PB et al., 2001). Above this range of

pressure, decreasing flavonoid content with increasing

pressure is obtained and therefore flavonoid content

reduced after 19.61MPa.

Effect of CO2 Flow rate on flavonoid extractionFig. 4 shows the effect of fluid flow rate (SC-

CO2) on flavonoid content ofS. melongena fruitin SC-CO2at different flow rate. It shows that the flavonoid content

increases with increase in SC-CO2 flow rate, reaches a

maximum value, and then decreases with a further increase

in the flow rate. The results obtained here can be explainedas a trade-off between a mass transfer process and a

thermodynamic equilibrium state (Elkanzi EM and Singh

H, 2001). At low flow rates of the solvent, the mass

transfer resistance limits the amount of solute transported

into the bulk of the solvent and the SC-CO2 leaves the

extractor unsaturated. As the flow rate is increased, mass

transfer resistance continues to decrease until the exiting

solvent is saturated; and allows equilibrium to be achieved

enabling a maximum yield to be attained. Further, increase


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of the flow rate reduces the residence time causing the

system to deviate from equilibrium and the solvent leaves

the extractor unsaturated despite the high mass transfer rate

(Mira B et al., 1999).

Effect of Co-solvent (Ethanol) percentage on flavonoid

extractionThe results of co-solvent percentage on theextraction of the flavonoids are shown in Fig.5. Different

percentage of ethanol (co-solvent) used exhibited different

effect in changing the fluid polarity and thus resulted in

change in the solubility enhancement of the flavonoids.

Co-solvent basically interacts with analyte complex

causing rapid desorption in SC-CO2 and enhances

solubility of fluid (Castro L et al., 1996). In the present

study, the results indicated that the optimal 95% ethanol

concentration for extraction of flavonoids ofS. melongena

was 11.5%.

Determination of final extraction yield under optimizedSFE conditions

The best conditions obtained for the extraction of

flavonoids from S.melongena fruit extract were pressure at

19.61MPa, temperature at 45C, CO2 flow rate at 3mL/min

and Co-solvent at 11.5%.The extract obtained at optimum

SFE conditions was dried to obtain final extraction yield.

The final extraction yield obtained under optimized SFE

conditions is 76.5%.

DPPH radical-scavenging activityThe SFE extract of Solanum melongena was

found to be effective free radical inhibitor or scavenger, as

well as a primary antioxidant that reacts with free radicals,

which may limit free radicals. The results are depicted in

Table 1. Free radical scavenging activity increased with

increasing concentrations of the extract in the range of (25-

125) g/ml. Based on the results of this study, it is clear

that the test plant extract have powerful in vitro free radical

scavenging properties against DPPH model in a

concentration dependent manner.

Table 1. Percentage free radical scavenging activity of SFE extract by DPPH method

All values in this table represents the mean + SEM (n=3).

IC50 values, from the data, were calculated by regression analysis.

Fig.1 Schematic design of supercritical fluid extraction

(SFE) unit

Fig.2 Effect of Temperature on flavonoid content

Concentration (g/ml) Percentage free radical scavenging activity

25 20.36+ 0.35

55 41.67+ 0.32

65 48.88+ 0.12

75 57.86+ 0.07

90 68.86+ 0.01

125 88.12+ 0.05

IC 50 66.063+ 0.15


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Fig.3 Effect of Pressure on flavonoid content Fig.4 Effect of CO2 Flow rate on flavonoid content

Fig.5 Effect of Co-solvent percentage on flavonoid

content

Fig 6. Effect of extract of S. melongena on DPPH radicalscavengingactivity

CONCLUSIONThe results presented in this work indicated that

Supercritical fluid Extraction was feasible for extraction of

flavonoids from Solanum melongena L. fruit which is

reported to have multiple biological activities. The results

show that SFE may be a valuable alternative technique for

the extraction of the flavonoids from S. melongena L. The

optimum conditions of SC-CO2 for flavonoid compounds

are pressure at 19.61MPa, temperature at 45C, CO 2 flow

rate at 3.0mL/min and co-solvent at 11.5%. DPPH study

revealed the in vitro antioxidant activity of SFE extract of

S. melongena fruit. The presence of flavonoids and related

polyphenols may be responsible for the activity. Further

investigations are required to find active component of the

extract and to confirm the mechanism of action.

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