Comparative Evaluation/Optimization of Soxhlet, Ultrasonic and Microwave Assisted Extraction of Ficus religiosa Linn.
Panchawat S.* and Sisodia S.S.
Bhupal Noble’s Girls’ PG College of Pharmacy,
Udaipur (Rajasthan), India.
ABSTRACT:
Plant derived Phytochemicals
have been the focus of recent research due to their health promoting effects.
Previous studies to estimate the leaves of these bioactive compounds made use
of traditional extraction procedures. Recently modern
techniques (such as ultrasonication and microwave)
has been shown to be an efficient non thermal extraction method. The
purpose of this study was to compare three methods of extraction (soxhlet, ultrasonication and
microwave assisted extraction) for Ficus religiosa Linn. (Stem bark) phenolic
contents and to determine optimal conditions (solvent, time, temperature and
power) for their extraction. The yields obtained by ultrasonication
and microwave extraction were found to be higher than soxhlet
extraction. The total phenolic compounds were found
to be higher in soxhlet extraction compared to modern
techniques. Due to considerable saving of time and high extraction efficiency,
Microwave assisted extraction and ultrasonication
under specific conditions can be used successfully for extraction of phytoconstituents.
KEYWORDS: Extraction,
Microwave, Ultrasonication, Phenolic,
Ficus religiosa Linn.
INTRODUCTION:
Extraction is one of the most crucial points in the
analytical chain in the effort of achieving a complete recovery of targeted
compounds1. Plant derived phytochemicals
have been the focus of recent research due to their health promoting effects2.
Previous studies on the estimation of the amounts of bioactive compounds from
plant sources made use of traditional solvent extraction methods such as
leaching, homogenization and soxhlet methods3-6.
Other classical methods that have emerged in recent
years and are in use include percolation, supercritical fluid extraction,
microwave assisted solvent extraction (MAE) and ultrasonication7-9.
Among the techniques used for exhaustive extraction in
a soxhlet apparatus is one of that has been
frequently employed for more than a century. The advantages of this method are
its continuous character and matrix independence. There are significant
drawbacks associated with soxhlet extraction are long
time, large amount of solvent and thermal degradation of thermolabile
analytes may occur10.
Microwave assisted extraction methods
required shorter time, less solvents, higher extraction rate and better
products with lower costs11. Microwave extraction follows the same
principal as maceration or percolation, but the speed of breaking up plant
cells and plant tissues is much higher. This reduced processing time is not
only an economic advantage, but also there is less risk of decomposition or
disintegration and oxidation of the valuable plant constituents.
The
penetration of microwaves into the plant tissues depends very much on dielectrical properties of the plant material. The energy
requirement can be controlled much well than with conventional thermal energy12.
Numerous biologically active compounds have been extracted with application of
microwave assisted extraction such as extraction of Taxus bravifolia needle13, azadirachtine related limonoids
from Azadirachta indica seeds
Kernel14 and phenolic compounds from grape
seeds15.
Ultrasonication involves the use of shear force created by the implosion of cavitation bubbles of ultrasonic waves (sound waves in the
KHZ range) to alter material properties thereby further disrupting
plant tissues and facilitating extraction16, 17. This makes it a
more efficient non thermal extraction method. More recently, ultrasonication techniques have found applications in the
food and pharmaceutical industries and its application in the phytochemistry is currently under study18-20.
Ficus
religiosa
Linn. (Moraceae)
commonly known as peepal is a large perennial tree
grows throughout India. It is widely cultivated in south East Asia. In Ayurveda, Ficus religiosa Linn. belongs to a class of drugs called Rasayana.
Rasayana are rejuvenators, antioxidants and relieve
stress in the body21,22. Bark is useful in
healing ulcers, various skin diseases and scabies and in treatment of diabetes,
the root bark is stated to be aphrodisiac. Fruit is laxative and digestive. All
plant parts are acrid, sweetish, cooling and are useful in diseases of blood,
vagina, uterus, leucorrhoea, burning sensation and ulcers23-26. Although it is used extensively in traditional medicine as an
anti-inflammatory and analgesic agent27.
Comparative
studies of soxhlet, microwave assisted extraction and
ultrasonication extraction method for Ficus religiosa
Linn. (stem bark) have not been attempted previously.
The purpose of present study is to examine different extraction methods for Ficus religiosa
Linn. (stem bark) and compare them relative merits.
MATERIAL AND
METHODS:
Plant Material- The stem bark of F.
religiosa was collected from Bhupal
Nobles institute campus, Udaipur (Raj.) India, in the month
of Sep-Oct 2008. It was dried under shade. Drug sample was identified by
Dr. S.S. Katewa (Department of Botany), College of
Science, MLSU, Udaipur (Raj.) India.
Apparatus and Chemicals- Microwave oven (IFB Company, 30SC2),
Ultrasonic bath (power 250 w), UV-1800, Shimadzu was used for extraction
analysis. All the reagents and solvents used in the experimental work were of
analytical grade chemicals.
Soxhlet Extraction-
Fifteen grams of dried and ground stem bark of F. religiosa were placed in a soxhlet apparatus and 200ml of an appropriate solvent for
16, 24, 32 and 48 hrs at 400C, 500C and 600C
with ethanol (90%), ethanol (60%) and acetone. After
extraction solvent was removed from the extract in a vacuum evaporator at 500C.
All extractions were performed in duplicate.
Table 1-
Percentage yields of different extracts of Ficus religiosa Linn. by
soxhlet (%w/w)
|
Time |
Temperature |
Ethanol
(90%) |
Ethanol
(60%) |
Acetone |
|
16 hrs |
400C |
4.0% |
5.0% |
3.9% |
|
500C |
4.5% |
5.5% |
4.5% |
|
|
600C |
5.0% |
6.0% |
4.5% |
|
|
24 hrs |
400C |
6.0% |
7.0% |
5.0% |
|
500C |
6.5% |
7.5% |
5.55% |
|
|
600C |
7.0% |
8.0% |
6.66% |
|
|
32 hrs |
400C |
8.6% |
9.0% |
7.5% |
|
500C |
8.9% |
9.6% |
8.99% |
|
|
600C |
9.0% |
9.99% |
9.6% |
|
|
48 hrs |
400C |
9.0% |
9.99% |
8.99% |
|
500C |
9.0% |
10.1% |
9.20% |
|
|
600C |
9.99% |
10.5% |
10.1% |
Microwave-assisted
extraction- A household microwave oven
(IFB, 30SC2) was used for extraction. Stem bark powder of F. religiosa
L. was mixed with an appropriate solvent. The suspensions were irradiated with
microwave (at 480 htz) for 30 sec., 60sec., 90 sec.,
120 sec., 150 sec. and 180 sec. with ethanol (90%), ethanol (60%) and acetone.
Super boiling of the solution did not occur. After extraction, the vessels were
left for 30 min. to cool down. Each extract was filtered through filter paper
and the filter was washed with 5 ml of an appropriate solvent. The extract was
concentrated in a vacuum evaporator at 500C. All extractions were
performed in duplicate.
Table 2-
Percentage yields of different extracts of Ficus religiosa Linn. by
ultrasonication (%w/w)
|
Time |
Temperature |
Ethanol
(90%) |
Ethanol
(60%) |
Acetone |
|
50 min |
300C |
7.9% |
8.0% |
8.5% |
|
400C |
8.0% |
8.5% |
8.9% |
|
|
500C |
8.5% |
8.99% |
9.0% |
|
|
70 min |
300C |
8.9% |
8.99% |
9.0% |
|
400C |
9.0% |
9.2% |
9.5% |
|
|
500C |
9.5% |
9.70% |
9.80% |
|
|
90 min |
300C |
9.5% |
9.70% |
9.80% |
|
400C |
9.6% |
9.70% |
9.99% |
|
|
500C |
10.0% |
10.5% |
10.99% |
Ultrasonication Extraction-
Five gram each of dried powder of stem bark of of F. religiosa
L. were suspended in 100 ml of appropriate solvents i.e. ethanol (90%), ethanol
(60%) and acetone in mason jars. The jars were placed in a sonication
tank filled with water, the suspensions were sonicated at fixed frequency. The temperature of the sonication could be controlled and extractions were
performed for 30 min., 50 min, 70 min and 90 min. Each suspensions
were filtered and then evaporated to dryness in rotary evaporator at 380C.
All extractions were performed in duplicate.
Total Phenol
content estimation- The total phenolic content of all the soxhlet,
microwave and ultrasonication extracts which shows
higher percentage yield were determined by using standard Folin-Ciocalteu
method28. Phenol react with phosphomolybdic acid in Foilin-Ciocalteu
(FC) reagent in alkaline medium and produce blue color complex (molybdenum
blue). 10 mg of extract dissolved in 10 ml of distilled water. 0.2 ml of
extract solution, 1.0 ml of FC reagent
and 0.8 ml of Na2CO3 (7.5%) were mixed in a 20 ml vial and the
mixture was allowed to stand for 2 h with intermittent shaking at room
temperature. The absorbance of the blue color that developed was read at 760
nm. The concentration of total phenols was expressed as mg/gm of dry extract.
The concentration of total phenolic compounds in the
extract was determined as mg of gallic acid
equivalent using an equation obtained from the standard gallic
acid graph.
Table 3-
Percentage yields of different extracts of Ficus religiosa Linn. by
microwave (%w/w)
|
Power |
Time |
Ethanol (90%) |
Ethanol (60%) |
Acetone |
|
50
Pha |
30
sec |
8.9% |
9.2% |
9.4% |
|
60
sec |
9.0% |
9.4% |
9.5% |
|
|
90
sec |
9.2% |
9.5% |
9.7% |
|
|
120
sec |
9.3% |
9.7% |
9.75% |
|
|
150
sec |
9.5% |
9.75% |
9.8% |
|
|
180
sec |
9.0% |
9.8% |
9.99% |
|
|
70
Pha |
30
sec |
9.2% |
9.4% |
9.5% |
|
60
sec |
9.2% |
9.6% |
9.7% |
|
|
90
sec |
9.4% |
9.7% |
9.8% |
|
|
120
sec |
9.5% |
9.8% |
9.99% |
|
|
150
sec |
9.7% |
9.9% |
10.0% |
|
|
180
sec |
9.75% |
10.0% |
10.1% |
|
|
100
Pha |
30
sec |
9.7% |
9.9% |
10.0% |
|
60
sec |
9.8% |
9.95% |
10.5% |
|
|
90
sec |
9.99% |
9.99% |
10.7% |
|
|
120
sec |
10.0% |
10.0% |
10.85% |
|
|
150
sec |
10.1% |
10.2% |
10.99% |
|
|
180
sec |
10.2% |
10.5% |
11.0% |
Table 4- Phenolic
contents of different extracts of Ficus religiosa Linn.
|
S. No. |
Solvents |
Extraction Method Used |
Total Phenolic Content
(mg/gm) |
|
1. |
Acetone |
Microwave |
347.5 |
|
2. |
Ethanol (60%) |
Microwave |
322.5 |
|
3. |
Ethanol (90%) |
Microwave |
302.5 |
|
4. |
Acetone |
Ultrasonication |
327.5 |
|
5. |
Ethanol (60%) |
Ultrasonication |
307.5 |
|
6. |
Ethanol (90%) |
Ultrasonication |
297.5 |
|
7. |
Acetone |
Soxhlet |
367.5 |
|
8. |
Ethanol (60%) |
Soxhlet |
372.5 |
|
9. |
Ethanol (90%) |
Soxhlet |
352.5 |
RESULT AND
DISCUSSION:
Optimization
of extract solvent- The phenolic contents (mg/gm) and percentage yield (w/w) of
extracts of stem bark of F. religiosa L. prepared by soxhlet
extraction, microwave assisted extraction and ultrasonication
with ethanol (90%) are 352.5 mg, 302.5 mg, 297.5 mg and 9.99% w/w, 10.2% w/w,
10.0% w/w, with ethanol (60%) are 372.5 mg, 322.5 mg, 307.5 mg and 10.5% w/w,
10.5% w/w, 10.5% w/w and with acetone are 367.5 mg, 347.5 mg, 327.5 mg and
10.1% w/w, 11.0% w/w, 10.99% w/w respectively, after accurate determination.
The evaluation indexes of extracting efficiencies of different extracting
methods were used here.
Graph 1-
Standard curve of gallic acid.
Optimization
of extraction time- Results obtained
showed that the percentage yield of extracts in all the three methods used i.e.
soxhlet microwave assisted extraction and ultrasonication were significantly increased with
increasing time. The highest percentage yield of extract of F. religiosa
L. (stem bark) was obtained for soxhlet method after
48 hrs, for microwave assisted extraction highest yield was obtained after 3
min. and for ultrasonication it was obtained after 90
min.
Optimization
of extraction temperature- The results
showed that the percentage extraction of F.
religiosa L. (stem bark) extract increased with
the increase of temperature. Optimum temperature for soxhlet
is 600C, for microwave extraction 600C (480 htz), for ultrasonication 500C.
Above these temperature percentage yield of extract
decreased due to decomposition29.
Comparison of
soxhlet, microwave assisted extraction and ultrasonication extraction techniques- The result shows that almost similar percentage
extraction was reached after 48 hrs., 90 min and 3 min., when soxhlet, microwave assisted extraction and ultrasonication extraction techniques were used. Microwave
and ultrasonication extraction techniques can
obviously reduce the extraction time. The result showed that MAE and ultrasonication method gave similar or higher results in
comparison to soxhlet extraction method (more time
consuming). Less amount of solvents were used in microwave and ultrasonication to soxhlet
extraction technique.
Total Phenolic Content-
The concentration of total phenolic compounds in all
extracts was expressed as mgs of gallic acid
equivalents (GAE) per gm dry weight of plant. Total phenolic
contents of extracts were studied using Folin-Ciocalteu
method. Absorbances of samples were measured at 760
mm and the amount of total phenolics in mg/gm extract
were then analysed. By manipulating the regression
equation of gallic acid
calibration curve (y = 0.004x - 0.059, R2 = 0.995), the total phenolic content of each extract was calculated. As results
shown that, the hydroalcoholic (ethanol 60%) extract
prepared by soxhlet method had the highest contents
of phenolic compounds (372.5 mg/gm) followed by
acetone extract (367.5 mg/gm) and ethanolic extract
(352.5 mg/gm).
CONCLUSION:
Phenolic
compound decomposed easily when they were exposed at higher temperature for a
longer period of time. The efficiencies F.
religiosa L. (stem bark) extract in to various
solvents by three alernative extraction techniques
were compared. The effect of extraction solvent, time, temperature and the
recoveries were studied. Microwave assisted extraction was a fast heating
method in a very short time. The percentage yield of extracts were almost
similar or higher prepared by soxhlet, microwave and ultrasonication but preliminary phytochemical
analysis and phenolic content shows that soxhlet extraction is best method for extraction of F. religiosa
L. (stem bark).
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Received
on 02.05.2010
Accepted on 13.05.2010
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
2(4): July-Aug. 2010, 309-312