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Extraction methods for preparation of bioactive plant extracts: A comparative study
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Acknowledgement
x All authors thank Nirma University

x Vijay Kothari is thankful to his parents, sisters and his wife
Harsha for their continuous support
x Ankit and Madhu are thankful to all their family members
1

2

INDEX
Page No.
1. Prelude 6
1.1 Introduction
1.2 Extraction technology
1.3 Extraction methods
1.4 Antioxidant activity
1.5 Phenols
1.6 Flavonoids
1.7 Antibacterial activity
1.8 Experimental material
1.9 Objectives
2. Materials and Methods 34
2.1 Materials
2.2 Methods
2.3 Statistical analysis
3

3. Results and Discussion 45
3.1 Optimization
3.2 Extraction efficiency
3.3 Total flavonoids
3.4 Total phenols
3.5 Total antioxidant activity
3.6 Total antibacterial activity
3.6.A Staphylococcus epidermidis
3.6.B Pseudomonas oleovorans
4. Final comments 122
4.1 Soxhlet
4.2 MAE and MAEC
4.3 ERT
4.4 UAE
5. Appendices 137
6. References 144
4

List of abbreviations
DMSO ± Dimethyl sulfoxide
ERT ± Extraction at room temperature
GAE ± Gallic acid equivalent
MAE ± Microwave assisted extraction
MAEC± Microwave assisted extraction continuous heating
MH ± Mueller Hinton
QE ± Quercetin equivalent
UAE ± Ultrasonic assisted extraction
5

1. PRELUDE
6

1.1 INTRODUCTION
For thousands of years mankind is using plant source to alleviate or

cure illnesses. Plants constitute a source of novel chemical
compounds which are of potential use in medicine and other
applications. Plants contain many active compounds such as
alkaloids, steroids, tannins, glycosides, volatile oils, fixed oils, resins,
phenols, and flavonoids which are deposited in their specific parts
such as leaves, flowers, bark, seeds, fruits, root, etc. The beneficial
medicinal effects of plant materials typically result from the
combination of these secondary products (Balandrian et al., 1985). In
1985 Farnsworth et al. identified 119 secondary plant metabolites
which were used as drugs. Out of 255 drugs which are considered as
basic and essential by the World Health Organization (WHO), 11%
are obtained from plants and a number of synthetic drugs are also
obtained from natural precursors. Phytochemicals are known to
possess antioxidant (Wong et al., 2009), antibacterial (Nair et al.,
2005), antifungal (Khan and Wassilew, 1987), antidiabetic (Singh and
Gupta, 2007; Kumar et al., 2008), anti-inflammatory (Kumar et al.,
2008), antiarthritic (Kumar et al., 2008), and radio-protective activity
(Jagetia et al., 2005), and due to these properties they are largely used
for medicinal purpose. The development of drug resistance and the
undesirable side effects of certain antibiotics have led to the search for
new antimicrobial agents, mainly among plant kingdom, in order to
find leads with unique chemical structures which may exert a hitherto
unexploited mode of action (Evans, 2002).
The phytochemical investigation of a plant may involve following

steps: authentication and extraction of the plant material, separation
and isolation of the constituents of interest, characterization of the
7

isolated compounds, and quantitative evaluation (Evans, 2002). For
the development of new plant derived medicines, proper extraction of
active components is required and it can be achieved by an
appropriate extraction method. A considerable effort has been made
by researchers to find efficient extraction methods in order to get high
efficiency and efficacy (Appendix - 1).
For isolation of biological components, extraction from plant is one of

the more sustainable approaches (Jadhav et al., 2009). Obtaining
better quality and high efficiency of extraction from herbs being
significant, one has to optimize the extraction methods for better
extraction efficiency. Efficacy of plant extracts may in some cases be
dependent on extraction efficiency. A strong positive linear
correlation (r = 0.96) between extraction efficiency and total
antibacterial activity was found during investigation on antibacterial
activity of plant seed extracts (Kothari, 2010). The analytical
procedures have several steps like sampling, sample preparation,
quantification, statistical evaluations, etc. Each of these steps is
critical for obtaining correct sample preparation (Pawliszyn, 1997).
The need for selection of most appropriate extraction methodology

is evident from the fact that when different methods are applied on
same plant material with same solvent, extraction efficiency can vary
significantly (Table 1). In addition, the method selected as the most
appropriate one also needs to be standardized so as to achieve
acceptable degree of reproducibility.
8

Table 1. Effect of extraction methodology on extraction efficiency
Plant Activity Extraction
Method Solvent Ref.
material reported efficiency (%)
Methanol Antibacterial 16.4 Kothari, 2010
MAE 50% Ethanol Antibacterial 29.0
Anti-
inflammatory 10.36 Kumar et al., 2008
Anti-diabetic
Methanol Kumar et al., 2008
Syzygium Cold Anti-arthritic 10.38
cumini percolation
(seed) CNS Activity Kumar et al., 2007;
Ethyl 1.81
Radio protective Jagetia et al., 2005
Acetate
50% Ethanol Anti-diabetic 20.0
Singh and Gupta,
100%
Soxhlet - 0.9 2007
Ethanol
Table 2. Type of phytochemicals extracted in various solvents

(Houghton and Raman, 1998; Cowan, 1999)
Polarity Solvent Chemical class extracted
Chloroform Terpenoids, Flavonoids, Alkaloids, Aglycones

Low Cyclohexane Waxes, Fats
Hexane Waxes, Fats
Dichloromethane Terpenoids, Alkaloids, Aglycones
Diethylether Alkaloids, Aglycones,
Ethylacetate Alkaloids, Aglycones, Glycosides
Acetone Flavonols, Alkaloids, Aglycones
Medium Ethanol Tannins, Polyphenols, Flavonol, Terpenoids, Sterols,

Alkaloids, Polyacetylenes, Propolis.
Methanol Saponins, Tannins, Phenones, Flavones, Sugars,
Aminoacids, Anthocyanins, Terpenoids, Xanthoxyllines,
Totarol, Quassinoids, Lactones, Polyphenols
High Water Sugar, Aminoacids, Saponins, Tannins, Lectins,
Terpenoids, Anthocyanins, Starches, Polypeptides
It should be noted that choice of appropriate solvent is of essential

importance along with application of a compatible extraction method.
9

For selection of solvents µOLNH GLVVROYHV OLNH¶ principle is applicable.
Thus polar solvents will extract out polar substances and non-polar
material will be extracted out by non-polar solvents. Solvent
extraction is the most popular method of extraction. Table 2 lists
some solvents suitable for extraction of particular classes of plant
compounds. Sample preparation is the crucial first step in analysis of
herbs, because it is necessary to extract the desired chemical
components from the herbal material for further separation and
characterization (Huie, 2002). For the extraction of therapeutically
desired active constituents various solvents such as water, ethanol,

chloroform, ethyl acetate, methanol, etc. are commonly used
(Appendix - 3). Sometimes mixtures of solvents are also used to get
better extraction efficiency (Table 3).
Table 3. Binary mixtures of solvents of differing polarity used for

extraction
Method Solvent mixture Reference

Kothari et al., 2009;
MAE Chloroform: Methanol
Kothari et al., 2010
PMAE Hexane: Acetone
Soxhlet Hexane: Acetone Camel, 2001
UAE Dichloromethane: Acetone
The development of modern sample preparation techniques has

significant advantages over conventional methods (Box 1) in terms of
reduction in organic solvent consumption and in minimizing sample
degradation. They also result in the elimination of undesirable and
insoluble components from the extract. The modern methods include
microwave assisted extraction (MAE), ultrasonication assisted
10

extraction (UAE), supercritical fluid extraction (SFE), solid phase
micro extraction (SPME), Soxhwave, etc. Latter is a combination of
Soxhlet with microwaves.
Classical methods are fairly simple, standard and continue to have

widespread use, but these methods can also be insufficient and slow,
consume large quantities of organic solvents, and cause degradation
of heat-labile constituents. While using conventional methods, quality
related problems viz. lack of consistency, safety, and efficacy are also
the issues. Furthermore, elimination of additional sample clean-up
and concentration steps before chromatographic analysis,
improvement in extraction efficiency, selectivity are also the benefits
of modern processes (Kothari et al., 2010).
The purposes of standardizing extraction procedures for production of

crude drugs are to obtain the therapeutically desired portion and to
eliminate the inert material by treatment with selective solvents and
methods. With the increasing demand for herbal medicinal products,
and natural products for health care all over the world, herbal
manufacturers aim at using the most appropriate extraction
technologies to produce extracts of defined quality with least batch to
batch variation, which can also help in scale-up of extraction.
Standardization of extraction procedures contributes significantly to
the final quality of the herbal drug. To have a complete idea of the
bioactivity of crude extracts, it becomes necessary to optimize the
extraction methodology to achieve the broadest possible range of
phytochemicals. The selection of method to isolate active components
with best yield and highest purity from natural sources is mainly
dependent on the nature of compounds and raw material which is
going to be processed (Kothari et al., 2009).
11

Box 1. Conventional methods used to recover natural products
(Handa et al., 2008)
x Soxhlet extraction x Pressurized liquid extraction

x Thermal desorption x Steam distillation
x Maceration x Percolation
x Phytonic desorption x Membrane process
x Infusion x Decoction
x Extraction leaching x Sample disruption method
x Surfactant mediated x Counter current extraction
extraction
x Accelarated solvent
x Enfleurage
extraction
1.2 EXTRACTION TECHNOLOGY
Fresh plant materials once collected from a particular region in a

particular season, taxonomic identity (ethno botanical details) are
determined by a botanist. Overall process may contain following
steps. (Handa et al., 2008):
1. Collection of plant material & drying 2. Size reduction

3. Extraction 4. Filtration
5. Concentration 6. Drying & reconstitution
Quality of an extract is influenced by several factors such as, plant

parts used as starting material, solvent used for extraction, extraction
procedure, and plant material: solvent ratio, etc. From laboratory scale
to pilot scale all the parameters are optimized and controlled during
extraction. Extraction techniques separate the soluble plant
metabolites through selective use of solvents.
12

Table4. Comparative account of various extraction methods (28, 29)
Method Sample Solvent Operating Suitability for Suitability Time Possibility Automation
size volume temp thermolabile forvolatile (h) of solvent level
(g) (ml) (°C) components components recovery
Traditional Soxhlet 10-20 200-500 40-100 No No 12-24 Yes None
Modern Soxhlet 10-20 50-100 40-100 No No 1-4 Yes Mostly
Sonication 20-50 100-300 Ambient-40 Yes Yes 0.5-1.0 No None
SFE 5-10 10-20 50-150 Yes Yes 0.5-1.0 No Fully
Pressurized fluid extraction 1-30 10-45 50-200 Yes Yes 0.2-0.3 No Fully
Closed ±vessel MAE 2-5 30 100-200 Yes Yes 0.1-0.2 No Mostly
Open-vessel MAE 2-10 20-30 Ambient Mostly No 0.1-0.2 No Mostly
13

1.3 EXTRACTION METHODS
A brief description of methods employed during this work follows.

Besides a comparative account of various extraction methods are
presented in the table 4.
1.3.1 Soxhlet extraction:
Soxhlet extraction was named after Baron Von Soxhlet, who

introduced this method in the mid-nineteenth century. It has been the
most widely used method until other extraction techniques were
developed in the 1980s.
A schematic diagram of a typical soxhlet apparatus is shown in figure

1. There are three components in the system: (1) The top part is a
solvent vapor reflux condenser, (2) In middle are a thimble holder
with a siphon device and a side tube, (3) The thimble holder connects
to a round bottom flask at the bottom. Because the sample is extracted
with cooled, condensed solvents, soxhlet is slow and can take 6 to 48
h. Soxhlet is rugged, well established technique that is often used as
the benchmark for comparing other methods. Few parameters like
long extraction time and relatively large solvent consumption are the
main drawbacks of this method. (Mitra, 2003).
The higher concentration of nerolidol from Piper gaudichaudianum
kunth was achieved in the soxhlet extraction with petroleum ether
(Peres et al., 2006).The percentage extraction of all tanshinones from
Salvia miltiorrhiza bunge increased with the increase of Soxhlet
extraction time from 30 to 90 min (Pan et al., 2002). The extracts of
Ficus bengalensis Linn. (hanging roots), Eugenia jambolana Lam.
(bark), Ficus racemosa Linn. (bark) and Leucas lavandulaefolia Rees
DHULDO SDUWV VKRZHG VLJQL¿FDQW LQKLELWRU\ DFWLYLW\ against castor oil
14

induced diarrhoea and the extracts was prepared by Soxhlet method
(Mukherjee et al., 1998). Extraction of phenolic compounds from
fruits of bitter melon (Momordica charantia) was done with Soxhlet
extraction (Budrat and Shotipruk, 2008). Soxhlet extraction technique
is employed for the extraction and separation of chemical constituents
in the medicinal plant, Herba Leonuri (Ahmad et al., 2010). Soxhlet
extractions were carried out in order to determine the best solvent was
methanol, used for Andrographis paniculata diterpenoid lactones
extraction (Kumoroa et al., 2009).
Figure 1. Soxhlet apparatus (adapted from Mitra, 2003)
1.3.2 Extraction at room temperature (ERT):
Extraction at room temperature is generally done at ambient

temperature (25-30°C) for particular time duration with continuous
shaking. This simple, low-cost method uses agitation in a shake-ÀDVN
SODFHG RQWR D URWDU\ VKDNHU ZLWK VXEPHUVHG LQWR WKH ÀDVN GLUHFWO\
Although it is an easy handling method with minimal glassware and
15

smaller volumes of extraction solvent, this method has not been as
widely used as the Soxhlet and sonication due to the lower extraction
eƥciency and unsatisfactory quantitative results. Comparable results
were only attainable with long shaking times to extend the contact
time with solvent (Lau et al., 2010). ERT was practiced for extraction
of phytochemicals from root of Saliva miltiorrhiza bunge with an
appropriate solvent (Pan et al., 2002).
1.3.3 Microwave assisted extraction (MAE):
Upon absorption by a material, electromagnetic energy of

microwaves is converted to heat energy. 2450 MHz (2.5 GHz) is the
most commonly used frequency for commercial microwave
instruments (in order to avoid interference with radio
communications), which has an energy output of 600-700 W (Jain et
al., 2009). MAE is a simple, environment friendly and economical
technique for the extraction of biologically active compounds from
different plant materials (Hemwimon et al., 2007). Samra et. al. had
first time used microwave domestic ovens for the treatment of
biological samples for metal analysis in 1975 (Letellier and
Budzinski, 1999). The application of MAE for plant materials was
first reported by Ganzler and co-workers in 1986 (Kaufmann and
Christen, 2002).
MicroZDYHV SRVVHVV HOHFWULF DQG PDJQHWLF ¿HOGV ZKLFK DUH

SHUSHQGLFXODUWRHDFKRWKHU7KHHOHFWULF¿HOGFDXVHVKHDWLQJYLDWZR
simultaneous mechanisms, namely, dipolar rotation and ionic
conduction. Dipolar rotation is due to the alignment on the electric
¿HOGof the molecules possessing a dipole moment in both the solvent
and the solid sample. This oscillation produces collisions with
16

surrounding molecules leading to liberation of thermal energy into the
medium. With a frequency of 2.45 GHz, this phenomenon occurs
4.9×109 times faster and thus the resulting heating is very fast. Indeed,
larger the dielectric constant of the solvent (Appendix - 2), more rapid
the heating is. Consequently, unlike classical conductive heating
methods, microwaves heat the whole sample simultaneously. In the
case of extraction, the advantage of microwave heating is the
disruption of weak hydrogen bonds promoted by the dipole rotation of
the molecules (Kaufmann and Christen, 2002).
When plant material is immersed inside a microwave transparent

solvent, the heat of microwave radiation directly reaches to the solid
without being absorbed by the solvent, resulting in instantaneous
heating of the residual moisture in the solid. Heating causes the
moisture to evaporate and creates a high vapour pressure, which
breaks the cell wall of substrate and releases the content into solvent.
Solvents employed for most MAE operations are those with a high
dielectric constant and capacity to strongly absorb microwave energy,
however, the extraction selectivity, and the ability of the medium to
interact with microwaves can be modulated by using mixtures of
solvents (Camel, 2001). The solvent which has been chosen for MAE
should absorb the microwave energy without leading to overheating
so as to avoid sample degradation. It is not uncommon to use binary
mixture of solvents (Table 3), with only one solvent capable of
absorbing microwave (Camel, 2001). Though polar solvents are
usually believed to be better than non-polar ones (Jagetia et al., 2005;
Proestos and Komaitis, 2007; Kothari et alµ%URNHQFHOO-wall
WKHRU\¶ UDWHV PLFURZDYH WUDQVSDUHQW VROYHQWV DERYH WKH PLFURZDYH
absorbing ones (Kothari et al., 2009). Addition of water to the solvent
17

may lead to increased yields. Microwave transparent solvents like
acetone proved to be best for extraction of phenolic compounds
(Proestos and Komaitis, 2007). In case of methanol:chloroform
mixture, former provides better overall heating efficiency because of
its high dissipation factor, and Because of low polarity chloroform
remains transparent (Proestos and Komaitis, 2007; Kothari et al.,
2009). Microwave transparent solvents (e.g., hexane) are particularly
suitable for extraction of thermolabile components (Mandal et al.,
2008).
MAE can be practiced in two different modes- one is closed vessel

operation, which is under controlled (elevated) pressure and
temperature, another is open vessel operation performed at
atmospheric pressure. These technologies are named as pressurized
microwave assisted extraction (PMAE) and focused microwave
assisted extraction (FMAE), respectively (Chemat and Esveld, 2001).
In closed vessel system the solvent may be heated much above their
atmospheric boiling point.
Both extraction speed and efficiency are enhanced in MAE

(Kaufmann and Christen, 2002). In closed vessels the temperature
may be elevated by simply applying the correct pressure. The closed
vessel system is most suitable for volatile compounds. In open vessel
system the maximum temperature is determined by the boiling point
of the solvent used (Camel, 2001). Compared to closed vessel
extractions, open cells offer increased safety in sample handling and,
furthermore, they allow larger amounts to be extracted (Kaufmann
and Christen, 2002). Open cells can accommodate multiple extraction
vessels at a time. Advantage of improved mass transfer due to
18

agitation is available in both modes of MAE (Mitra, 2003; Sarker et
al., 2006).
Though superheating has been indicated to occur during microwave

processes (Chemat and Esveld, 2001), MAE is not likely to suffer
from thermal degradation of phytoconstituents by superheating
because superheating is reported to occur in homogenous systems,
and not in heterogeneous ones -in which MAE falls. In microwave
assisted reactions degree of rate enhancement is associated with the
condition of the glass surface (whether smooth or not) and the wetting
properties of the solvent. Surface of the wall is generally not much
heated during microwave operations, and most energy is dissipated
inside the bulk liquid (Chemat and Esveld, 2001).
The ability of microwave radiation to heat solid material effectively

can be used for obtaining essential oils. This yields essential oils
consisting of relatively low volatile fractions as compared to hydro
distillation (Handa et al., 2008). MAE is highly effective for
obtaining extracts under mild conditions. MAE has shorter extraction
time, lesser solvent requirement, improved purity of the extract, low
cost, and better extraction yield in comparison to Soxhlet extraction.
Therefore it has been considered as a potential alternative to
conventional methods (Chemat and Esveld, 2001; Ahuja and Diehl,
2006). Microwaves have been reported to cause little or no quality
deterioration when applied to substances of plant origin such as
ascorbic acid, where as moist heat application resulted in quality
deterioration (Sasaki et al., 1998). MAE has been shown to be faster
than the reflux method for extraction of phenolic compounds.
19

MAE allows extraction of curcumin in lesser time with better
precision than conventional methods (Mandal et al., 2007). MAE is
an alternative technique for extraction of tanshinones from root of
Saliva miltiorrhiza Bunge, it provides higher extraction efficiency in
shorter time and is less labour intensive (Pan et al., 2002). A kinetic
study of the effect of solvent composition, solvent volume, extraction
temperature, and matrix characteristics on the MAE of leaves of
rosemary and peppermint revealed that for a sample matrix which
contains water as a component, the use of pure, microwave
transparent solvents such as hexane could result in the rapid
extraction of essential oil components. This was because of direct
interaction of microwaves with the free water molecules present in the
cell, which caused rupture of the cell and release of essential oil into
organic solvent used (hexane). More effective microwave heating for
leaves of rosemary and peppermint could be achieved by optimizing
the plant material:solvent ratio (Huie, 2002).
Extracts prepared by MAE method from seeds of Manilkara zapota,

Annona squamosa, Tamarindus indica, Phoenix sylvestris, Citrus
limon, Carica papaya, and Tricosanthes dioica have shown
significant antibacterial and/or antioxidant activity, with extraction
efficiency in the range 2-15 % (Kothari et al., 2010; Kothari and
Seshadri, 2010; Kothari, 2011). For A. squamosa seeds extraction
efficiency of 17% (in chloroform-methanol mixture) was reached
with a total microwave exposure of just 50 sec (Kothari et al., 2009).
MAE was used to extract paclitaxel in methanol-water mixture from
Iranian yew trees (Ahuja and Diehl, 2006).
20

1.3.4 Ultrasonication assisted extraction (UAE):
UAE involves application of high-intensity, high-frequency sound
waves and their interaction with materials. UAE is a potentially useful
technology as it does not require complex instruments (Fig. 2) and is
relatively low-cost. It can be used both on small and large scale (Dai
and Mumper, 2010). UAE involves ultrasonic effects of acoustic
cavitations. Under ultrasonic action solid and liquid particles are
vibrated and accelerated and, because of that solute quickly diffuses
out from solid phase to solvent (Cares et al., 2009). Several probable
mechanisms for ultrasonic enhancement of extraction, such as cell
disruption, improved penetration, and enhanced swelling, capillary
effect, and hydration process have been proposed (Huaneng et al.,
2007). If the intensity of ultrasound is increased in a liquid, then it
reaches at a point at which the intramolecular forces are not able to
hold the molecular structure intact, so it breaks down and bubbles are
created, this process is called cavitation (Baig et al., 2010). Collapse
of bubbles can produce physical, chemical and mechanical effects
which result in the disruption of biological membranes to facilitate the
release of extractable compounds and enhance penetration of solvent
into cellular materials and improve mass transfer (Cares et al., 2009;
Metherel et al., 2009). The beneficial effects of sound waves on
extraction are attributed to the formation and asymmetrical collapse
of microcavities in the vicinity of cell walls leading to the generation
of microjets rupturing the cells. The pulsation of bubbles is thought to
cause acoustic streaming which improves mass transfer rate by
preventing the solvent layer surrounding the plant tissue from getting
saturated and hence enhancement of convection (Kadkhodaee and
Kakhki). Skin of external glands of plant cell wall is very thin and can
be easily destroyed by sonication, and this facilitates release of
21

essential oil contents into the extraction solvent. Ultrasonication can
also facilitate the swelling and hydration of plant cell wall and this
will improve mass transfer, and occasional breakage of the cell wall,
thus resulting in reduced extraction time and increased extraction
efficiency (Huie, 2002). Application of heat along with ultrasound
may enhance mass transfer.
Extraction of the tea solids from dried leaves with water using
ultrasound gave 20% improvement in extraction yield. UAE also
proved better for extraction of carnosic acid by using different
solvents viz. ethanol, ethyl acetate, and butanone, and also reduced
the extraction time (Baig et al., 2010).
Figure 2. Schematic representation of UAE setup

(adapted from Jadhav et al., 2009)
In general, solvent type has a significant effect on both extraction rate
and the final yield of total isoflavones but in one study when UAE
was used for the extraction of isoflavones from the stem of Pueraria
lobata (Willd.), the increased extraction rate and yield was obtained
for all types of solvents. The higher the electrical power input in the
22

range of 0-650 W, the higher the extraction yield was observed
(Huaneng et al., 2007). UAE enhance extraction efficiency of vanillin
in much shorter time period in many solvents. Ultrasonic treatment on
commercial scale could be reliable and simple by applying ultrasound
to the pre-leached mixture for short time period (Jadhav et al., 2009).
UAE of resveratrol from grapes was considered to be very effective.
The degradation of resveratrol from grapes may be negligible within a
certain extraction time period with the use of UAE (Cho et al., 2006).
UAE has retaining effect on extraction of protein and pectin, which
improve the sensory quality of tea. UAE was found appropriate for
the extraction of aroma compounds and glycosidic aroma precursors
(Xia et al., 2006). UAE was also used for extraction of oil from
soyabean (Li et al., 2004), rapeseed (Ibiari et al., 2010), and
Monopterus albus (Abdullah et al., 2010). Studies concerning effect
of different solvents and their mixture, effect of solvent volume,
sonication power, and sonication time indicated that UAE has the
potential to improve extraction efficiency and reduce processing time
and, during processing the oil composition was also not affected by
the use of ultrasound. UAE gives the highest extraction yield of some
flavonoids such as- tectoridin, iristectorin B, iristectorin A,
tectorigenin, iris-WHFWRULJHQLQ $ DQG WRWDO LVRÀDYRQHV LQ OHVVHU WLPH
in comparison to maceration and Soxhlet extraction (Sun et al., 2011).
Important functional components from grape seeds were extracted by
UAE. Extraction variables, particularly extraction time and
temperature, strongly influence the UAE of total phenolics,
antioxidants, and anthocyanins from grape seeds (Ghafoor et al.,
2009). UAE was reported as a simpler and more effective alternative
to conventional extraction methods for the isolation of ginsenosides
(saponins) from various types of ginseng. UAE of ginseng saponins
23

was about three times faster than the traditional extraction method.
The ultrasonic extraction was not only more efficient but also
convenient for the recovery and purification of the active ingredients.
UAE can be carried out at lower temperatures which are favorable
for the thermally unstable compounds (Wu et al., 2001).
Sonication proved to be the most powerful tool for extraction of

certain phytochemicals- hypericin, pseudohypericin, hyperoside,
rutin, quercitrin, and hyperforin- from Hypericum perforatum L.,
when extraction efficiency of UAE was compared with that of
conventional maceration, indirect sonication, Soxhlet extraction, and
accelerated solvent extraction (ASE) (Smelcerovic et al., 2006). UAE
provides better extraction of vanillin in shorter time period with
different solvents as compared to the Soxhlet method (Jadhav et al.,
2009). Application of UAE significantly accelerated the analyte
(nicotine from pharmaceutical and plant formulations) extraction.
Each extraction step takes up to 24 h in conventional ERT technique,
whereas UAE took less than 20 min to achieve the same extraction
efficiency, and the consumption of environmentally harmful organic
solvent was also lowered (Ishtiaq et al., 2009).
UAE has the advantages of shorter reaction preparation time, usage of

small amounts of material, efficient and minimum expenditure on
solvents, and the increase in sample throughput. It is very useful for
the isolation and purification of bioactive principles (Ishtiaq et al.,
2009). One disadvantage of the procedure is the occasional but known
deleterious effect of ultrasound energy (>20 kHz) on the active
constituents of medicinal plants through formation of free radicals
and consequently undesirable changes in the drug molecules (Handa
et al., 2008).
24

1.4 ANTIOXIDANT ACTIVITY
Natural antioxidants occur in all higher plants, and in all parts of

plant. As plants produce a lot of antioxidants to control the oxidative
stress caused by sunbeams and oxygen, they can represent a source of
new compounds with antioxidant activity (Scartezzini and Speroni,
2000). Free radical species and antioxidants both playrole in
maintaining redox equilibrium in the body. Apart from their role in
the diseased conditions in the body, reactive oxygen species (ROS)
are also known to have a role in the spoilage of food by autoxidation
of lipids, enzymatic oxidation, during storage and processing in fats,
oils, and fat-containing foods (Kothari et al., 2010).
Recently there has been an upsurge of interest in the therapeutic

potentials of plants, as antioxidants in reducing free radical induced
tissue injury. Although several synthetic antioxidants, such as
butylated hydroxyanisole (BHA) and butylated hydroxytoluene
(BHT), are commercially available, but are quite unsafe and their
toxicity is a problem of concern. Hence, strong restrictions have been
placed on their application and there is a trend to substitute them with
naturally occurring antioxidants.
Natural antioxidants especially phenolics and flavonoids from tea,

wine, fruits, vegetables and spices are already exploited
commercially either as antioxidant additives or as nutritional
supplements. Also many other plant species have been investigated in
the search for novel antioxidants, but generally there is still a demand
to find more information concerning the antioxidant potential of
plant species as they are safe and also bioactive. Therefore, in recent
25

years, considerable attention has been directed towards the
identification of plants with antioxidant ability (Patel et al., 2010).
1.5 PHENOLS
Phenols, sometimes called phenolics, are a class of chemical

compounds consisting of a hydroxyl group (-OH) bonded directly to
an aeromatic hydrocarbon (Bones and Rossite, 1996). Phenolic
substances tend to be water-soluble since they most frequently occur
combined with sugar as glycosides and they are usually located in the
cell vacuole. Phenolic compounds are commonly found in both edible
and nonedible plants, and they have been reported to have multiple
biological effects, including antioxidant activity. In, addition, gallic
acid and related phenols in red wine have been suggested to be
UHVSRQVLEOHIRUWKH³)UHQFKSDUDGR[´WKDWLVWKHIDFWWKDWUHVLGHQWVRI
France have lower rates of cardiovesicular disease than those of other
countries, despite consuming a diet high in fats. Gallic acid
derivatives are often powerful antioxidants (Larson, 1997). Phenols
can be categorized into sub categories such as phenylpropenoids,
flavonoid pigments, anthocynin, flavones and flavonols, tannins and
quinine pigments, etc. (Harborne, 1988). The antioxidant activity of
phenolics is mainly due to their redox properties, which allow them to
act as reducing agents, hydrogen donators, and singlet oxygen
quenchers (Kahkonen et al., 1999). Phenolic compounds play an
important role in plant growth, development, reproduction and
defense. Based on their biological functions, phenolic compounds can
be classified as flower pigments, fruit pigments, fungicides,
phytoalexins, and pesticides (Kothari et al., 2010). Phenols get
oxidized in to brown colored compound upon exposure to air (Evans,
2002). The mechanism by which phenolics can be toxic to
26

microorganisms is enzyme inhibition by the oxidized compounds,
possibly through the reaction with sulfhydryl groups or through more
non specific interactions with proteins (Cowan, 1999).
1.6 FLAVONOIDS
Flavonoids, a broad class of polyphenolic compounds widely

distributed among photosynthesizing cells, possess an impressive
array of pharmacological activity (Schubert et al., 1999). Flavonoids
are structurally derived from the parent substance flavones (Harborne,
1988), which are 2-phenyl-Ȗ-benzopyrone, comprising two benzene
rings (Felix and Mello, 1997). Flvonoids are mainly water-soluble
compounds, and they can be extracted with 70% ethanol. Main
classes of flavonoid are: anthocynin, proanthocyanidins, flavonols,
flavones, glycoflavones, biflavonyls, flavanones, isoflavones.
Flavonoids show intense absorption bands in the UV and visible
regions of the spectrum (Harborne, 1988). Flavonoids are also
hydroxylated phenolic substances but occur as a C6-C3 unit links to an
aromatic ring. As anti-LQÀDPPDWRU\ DJHQWV ÀDYRQRLGV PD\ EH
effective against parodentitis and local pain, without the gastric
irritating effects of aspirin and other non-steroidal anti-LQÀDPPDWRU\
drugs. Flavonoids have also been suggested as cancer- protective
agents, if not therapeutic ones and the consumption of dietary
ÀDYRQRLGV ZDV LQYHUVHO\ FRUUHODWHG ZLWK FRURQDU\ KHDUW GLVHDVH LQ D
population of elderly men (Schubert et al., 1999). Since they are
known to be synthesized by plants in response to microbial infection,
it should not be surprising that they have been found in vitro to be
effective antimicrobial substances against a wide array of
microorganisms (Cowan, 1999).
27

1.7 ANTIBACTERIAL ACTIVITY
Plants are rich source of drugs either of direct remedies or production

template for synthetic drugs. Due to emergence of new drug resistant
strains of pathogenic organisms it has become necessary to
investigate plants as source of novel antimicrobials as they can inhibit
the drug resistant bacteria by a mechanism different than that of
conventional microbe-derived antibiotics (Kothari, 2011). Various
seed extracts have been reported by several researchers for their
antibacterial activity (Kothari and Seshadri, 2010).
Some of the common features of these organisms are described as

follows:
1.7.1 Staphylococcus epidermidis:
S. epidermidis is a gram-positive cocci, that occurs singly, in pairs,

tetrads, short chains, and irregular grape like clusters. They are non-
motile, non spore forming and usually have limited capsule
formation. S. epidermidis forms a confluent biofilm, in which the
cells are embedded in an amorphous material, referred to DV³VOLPH´
S. epidermidis is the most prevalent and persistant Staphylococcus
spcies on human skin. It is found over the much of the body surface
and produce the largest population where moisture content and
nutrition are high. The bacterium is responsible for a growing number
of infections among the among hospital patients whose immune
system are weakened. Such infections often start at skin wounds
caused by catheters. This species have been implicated in bacteremia,
native and prosthetic valve endocarditis, osteomylatis during
continuous ambulatory dialysis, vasicular grafts, cerebrospinal fluid
shunts, urethritis and pylonephritis. Little is known about mechanisms
28

of pathogenesis of S. epidermidis infections. S. epidermidis shows
resistance to various antibiotics like cephalosporin, penicillin,
methicillin, quinolone, and vancomycin (Dworkin et al., 2006).
1.7.2 Pseudomonas oleovorans:
Pseudomonas are gram-negative, motile by one or several polar

flagella; rarely non-motile, straight or slightly curved rods but not
helical. Common physiological properties of this organism are
chemoorganotrophic metabolism, absence of fermentation, absence of
photosynthesis, and capacity for growth of the expense of a large
variety of organic substrates. Pseudomonas species have a wealth of
habitats available to it, ranging from soil and water environments to
plant and animal tissue. P. oleovorans is a methylotrophic bacterium
that is a source of rubredoxin (part of hydroxylation-epioxidation
system). It was first isolated in water-oil emulsions used as lubricants
and cooling agents for cutting metals. Pili have been observed in P.
oleovorans which is one of the component of virulence factor
(Dworkin et al., 2006).
1.8 EXPERIMENTAL MATERIALS
1.8.1 Tamarindus indica L.:

Family: Caesalpiniaceae
Indian names: Tamarind, Indian date (English); Cinca,
Cincini (Sanskrit); Tamruhindi, Teter (Hindi): Amli, Huli
(Kannada); Puli, Amailam (Tamil); Puli, Madhurappuli,
Valanpuli (Malayalam); Chinta, Chinta ±pandu (Telugu)
(Ahmed, 2010).
29

(A) (B)
Plate 1. Tamarindus indica (A) Fruit (B) Seeds

Uses: The bark is used as to relives, sores, ulcers, boils and
rashes. It may also be administered as a decoction against
asthma and amenorrhea and as a febrifuge. Leaf extracts shows
antioxidant activity in the liver, and is a common ingredient in
cardiac and blood sugar reducing medicines. Leaves may be
used in fomentation for rheumatism, and administered as a
poultice for inflammation of joints to reduce swelling and
relieve pain (Orwa et al., 2009).
1.8.2 Annona squamosa L.:

Family: Annonaceae
Indian names: Custard apple, sugar apple, sweet-sop
(English); Gandagaatra, Sitaaphala (Ayurvedic); Sharifaa
(Unani); Sitaaphalam, Atta (Tamil).
(A) (
B)
Plate 2. Annona squamosa (A) Fruit (B) Seeds
30

Uses: leaves are used as insecticides. A combination of seed
power and leaf juice is used for removing lice from scalp.
Seeds are abortifacient.
Roots are used in blood dysentery. Fruits are sedative to heart,

antibilous, antiemetic, expectorant. Unripe fruits in form of
dried powered used for treating ulcers. Leaves, bark, unripe
fruits are strongly astringent; used for diarrhea and dysentery.
Fractions of total alkaloid from root of A. squamosa have
antihypertensive, antispasmodic, antihistaminic properties.
Leaves of A. squamosa contain a cardiotonic alkaloid,
quinoline. Squamone and bullatacinone were selectively
cytotoxic to human breast carcinoma (Khare, 2007).
1.8.3 Syzygium cumini L.:

Family: Myrtaceae
Indian names: Jambolan, Java plum (English); Jambava
(Sanskrit); Jaman, Jambhal, Jamun (Hindi); Jambuva, Nerale
(Kannada); Kottainaval, Naval (Tamil); Njaval, Njara
(Malayalam); Neredu, Jambu (Telugu) (Ahmed, 2010).
Plate 3. Syzygium cumini Fruit

Uses: The bark of the plant is astringent, sweet, refrigerant,
carminative, diuretic, digestive, antihelminthic, febrifuge,
31

constipating, stomachic and antibacterial. The fruits and seeds
are used to treat diabetes, pharyngitis, spleenopathy,
urethrorrhea and ringworm infection. The leaves are
antibacterial and used to strengthen the teeth and gums. The
leaves have been extensively used to treat diabetes,
constipation, leucorrhoea, stomachalgia, fever, gastropathy,
strangury, dermopathy and to inhibit blood discharges in the
faeces (Jagetia and Baliga, 2002).
1.8.4 Phoneix sylvestris Roxb.:

Family: Palmae; Arecaceae.
Indian names: Wild Date Palm (English); Kharjuuri
(Ayurvedic); Periyaitcham, Icham (Tamil); Sulemaani
Khajuur, Desi Khajuur
(A) (B)
Plate 4. Phoneix sylvestris (A) Fruit (B) Seeds

Uses: Fruits are restorative. Juice is cooling, gastric stimulant.
Seeds are used in ague. Roots are used for nervous debility.
Fresh, unfermented sap (Niraa) is a good source of ascorbic
acid, nicotinic and isonicotinic acids, riboflavin, thiamine,
sugars; cystine, leucine, isoleucine, phenylalanine, tyrosine.(
(Khare, 2007).
32

1.8.5 Manilkara zapota L.:
Family: Sapotaceae
Local names: Chickle gum, Sapodilla, Naseberry (English);
Chiku (Hindi); Ciku, Chiku (Malay); Lamut, Lamut-farang
(Thai).
Plate 5. Manilkara zapota Fruit

Uses: A leaf decoction is taken for fever, haemorrhage, wound,
and ulcers. Seeds are antipyretic and when ground with water
they act as diuretic. This plant is a source of sapotin, a
glucoside used in medicine as febrifuge (Orwa et al., 2009).
1.9 OBJECTIVES
x To study the variation in extraction efficiency with change in

extraction methodology.
x To check the correlation of extraction efficiency with
1. Total flavonoid content
2. Total phenol content
3. Total antioxidant capacity
4. Antibacterial activity
x To determine relative suitability of various methods for above
four parameters.
33

2. MATERIALS
AND
METHODS
34

2.1 MATERIALS
Solvents (Merck, Mumbai): Methanol, ethanol (Ureca consumers

Co.op stores Ltd.), dimethyl sulfoxide.
Total flavonoid content: Ammonium chloride (Merck,), potassium
acetate (Merck), quercetin (S-d fine chemicals, Mumbai).
Total phenolic content: Sodium carbonate (S-d fine chemicals,
Mumbai), Folin-Ciocalteu reagent (SRL), gallic acid (SRL).
Molybdate assay: Ammonium molybdate (S-d fine chemicals),
H2SO4 (Merck), gallic acid (SRL).
Antibacterial activity: Mueller Hinton (MH) broth (HiMedia,
Mumbai).
Apparatus: Mixer grinder (Maharaja whiteline bonus mixer grinder),
weighing balance (Shimadzu AUX 220, Uniblock), Microwave oven
(Electrolux EM30EC90SS), Ultrasonicator (Syclon JY92-11DN),
Microplate reader reader (BIO-RAD 680).
2.2 METHODS
x Collection of plant materials: Seeds of five plant materials
Tamarindus indica L., Annona squamosa L., Syzygium cumini
L., Phoenix sylvestris Roxb., and Manilkara zapota L., were
procured from the fruits purchased from local market in the city
of Ahmedabad and stored in air tight container. They were
authenticated for their unambiguous identity by Prof. Y. T.
Jasrai, Head of Botany Dept., Gujarat University, Ahmedabad.
x Grinding: Dried seeds were ground by mixer grinder at normal
speed. The grinding cycle was kept constant for all the seeds
that were 30-15-15 sec, but for P. sylvestris the grinding cycles
were changed to 30-30-30-30-30-30 sec, total time 3 min. Final
35

powdery form of all the seeds were kept constant and checked
by visual observation.
2.2.1 Extraction:
Sample to solvent ratio was kept constant for all the methods: 1 g
seed powder in 50 ml solvent.
2.2.1.1 Soxhlet extraction:
Soxhlet extraction was performed using classical Soxhlet apparatus

for 3 h for all the solvents. 250 ml round bottom flask (Borosil) was
used. The total amount of solvent in the system was 100 ml and seed
powder was 2 g, so that final seed to solvenW UDWLR ZDV UHPDLQ
FRQVWDQW 2SWLPXP ERLOLQJ WHPSHUDWXUH ZDV NHSW IRU UHVSHFWLYH
VROYHQW)RUZDWHUDQGHWKDQROWHPSHUDWXUHZDVNHSWࡈ&IRU
PHWKDQROࡈC.
2.2.1.2 Extraction at room temperature (ERT):
ERT was carried out in 250 ml (Borosil) flask on rotary shaker for 24
h at room temperature. Flask was covered with cotton plug and
aluminum foil in case of volatile solvents during extraction.
2.2.1.3 Microwave assisted extraction (MAE):
MAE was carried out in microwave domestic oven at 720 W with

intermittent cooling in 250 ml brown (Borosil) bottle. Cap of the
bottle was kept loose during extraction. Heating and cooling cycles
were different for all solvents as shown in table 5 (Kothari et al.,
2009).
36

Table 5. Heating and cooling cycles for different solvents
First heating Cooling Reheating Total heating

Solvent cycle cycle time time
(sec) (sec) (sec) (sec)
Methanol 10 40 40 90
Ethanol (50%) 40 40 10 70
Water 60 40 30 90
2.2.1.4 Microwave assisted extraction continuous (MAEC):
MAEC was carried out in the same microwave domestic oven at 720
W, without any intermittent cooling in 250 ml brown (Borosil) bottle.
Total heating time was 75 sec for all the solvents. Cap of the bottle
was closed more tightly as compared to that during MAE, however
evaporation was allowed during extraction.
Total heat exposure is lesser in MAEC but the highest temperature

attained may be higher than MAE.
2.2.1.5 Ultrasonic assisted extraction (UAE):
UAE was carried out in ultrasonicator at the power of 720 W in 100

ml (Borosil) beaker. 6 mm probe was used for all the plant material.
Working frequency was set at 20-25 kHz with the total time
(including several 5 sec cycles of working on and working off time)
of 2 min for all the solvents at room temperature. During working
with methanol and ethanol beaker was covered with aluminium foil.
2.2.2 Filtration and reconstitution:
After extraction, extract was centrifuged at 10,000 rpm for 15 min in

35 ml polypropylene tubes in centrifuge (Remi, BZCI-8729) at room
37

tempHUDWXUH $IWHU ZKLFK H[WUDFWV ZHUH ILOWHUHG LQ GDUN XVLQJ
:KDWPDQILOWHUSDSHU:KDWPDQ,QWHUQDWLRQDO/WG(QJODQGDQG
ZHUH DOORZHG WR HYDSRUDWH LQ SUH-ZHLJKHG PP SHWULSODWHV
%RURVLODWࡈ C for 18-24 h. After drying plates were weighed for
constant weight. Then dried extracts were reconstituted with the
respective solvent with the help of gel rocker (GeneI, Bangalore) at
20 rpm for 15 min. For flavonoid assay all the extracts were
reconstituted in 99% methanol, for phenol and antioxidant assay
reconstitution was done in 95% methanol. For antibacterial assay all
extracts were reconstituted in DMSO. The extracts were stored in
autoclaved flat bottom capped glass vials (15 ml, Merck). The vials
were protected from light to prevent the oxidation oI FRPSRXQGV
SUHVHQWLQH[WUDFWDQGUHIULJHUDWHGDWࡈC for further use. Inner side of
the cap of vial was covered with aluminum foil to prevent the
leaching of compounds from the plastic into the extract.
2.2.3 Estimation of flavonoid content:
Aluminum chloride colorimetric method was used for flavonoids

determination (Chang et. al., 2002). 0.5ml of each plant extract was
separately mixed with 1.5ml of methanol, 0.1ml of 10 percent
aluminum chloride, 0.1ml of 1M potassium acetate and 2.8ml of
distilled water. The reaction mixture was allowed to stand at room
temperature for 30min and the absorbance of the reaction mixture was
measured at 415 nm. The calibration curve was prepared by using
quercetin at concentrations of 12.5 to 100 mg/ml in methanol.
38

Growth control: 150 µl broth + 50 µl inoculum.
Positive control: 148 µl broth + 2 µl antibiotic (gentamycin)
+ 50 µl inoculum. The final concentration of the antibiotic
in the well was 2 mg/ml.
Negative control: 148 µl broth + 2 µl DMSO +50 µl
inoculum.
Experimental well: 148 µl broth + 2 µl extract (10 mg/ml) +
50 µl inoculum.
Turbidity control: 148 µl broth +2 µl extract + 50 µl
distilled water.
After that microplate was incubated aseptically at 35 ºc for 16-20 h.
then the readings were taken in microplate reader at 655 nm. The
plates were subjected to shaking for 5 sec before readings were taken.
% inhibition was calculated considering growth in negative control as
100%.
2.3 STATISTICAL ANALYSIS
2.3.1 ANOVA:
Single factor ANOVA was done for all data sets by MS®-Excel 2003
(Keller, 2001; Gurumani, 2005).
The null hypothesis was set up as follows.
H0 : M1 = M2 = M3 = M4 = M5
In ANOVA we would be failing to reject the null hypothesis (H 0)

only if there is no significant difference between any pairs of the
means. On other hand, we would be rejecting the H0 even if there is a
significant difference between one pair of means. Therefore, it
becomes necessary to identify which of the pairs differ significantly
42

2.3.3 t-test:
By using MS®-Excel 2003: Two tailed test was done for testing
variance between two methods. According to p value the data was
analyzed for all the methods. The minimum p value for significance
was kept 0.05.
44

3. RESULTS
AND
DISCUSSION
45

3.1 OPTIMIZATION
3.1.1 Optimization of MAEC:
Optimization of MAEC was done with the seeds of T. indica with water as a solvent.
MAEC was also done in brown bottles (Borosil).
Table 6. Extraction efficiencies at different time during optimization of MAEC
Time Extraction efficiency (%)

(sec) 1 2 3 Mean±SD
75 16.60 14.50 15.20 15.43±1.06
90 12.40 12.70 14.50 13.20±1.13
120 15.10 14.80 15.50 15.10±0.35
Microwaves treatment was provided for different time period (75, 90, 120 sec.)
without any cooling cycle and then observed that at 75 sec treatment MAEC was
giving extraction efficiency approximately same as 120 sec treatment so
overexposure of microwave was avoided and all the experiments was done for 75 sec
for all the seed materials.
3.1.2 Optimization of UAE:
Optimization was done with seeds of T. indica with water as solvent. UAE was done at
different times and power with different probes. UAE at 2 min was giving superior
extraction efficiency then all other experiments so we have performed all the
experiments for 2 min, at 720 W power and working time. So we have used 2 min total
time and above cycle for UAE.
Table 7. Extraction efficiencies at different time during optimization of UAE
Time Probe size Power Working time Working off Extraction
(min) (mm) (W) (sec) time Efficiency (%)
(sec)
20 3 680 5 5 6.1
20 3 720 5 5 5.6
20 6 720 5 5 7.7
15 3 680 5 5 6.7
15 6 720 5 5 8.3
10 6 720 5 5 7.4
5 6 720 5 5 7.5
3 6 720 5 5 7.5
2 6 720 5 5 7.6
1 6 720 5 5 6.3
46

3.2 EXTRACTION EFFICIENCY
3.2.1 T. Indica:
3.2.1.1 Water extract of T. indica seed:
Table 8. Extraction efficiency of T. indica water extract prepared by

different methods
Extraction efficiency (%) Significant
Method 1 2 3 Mean±SD Difference
Soxhlet 45.20 40.01 37.02 40.74±4.13
ERT 14.60 15.30 14.10 14.66±0.60
MAE 17.30 18.70 17.30 17.76±0.80 05.34
MAEC 12.60 13.60 11.20 12.46±1.20
UAE 7.60 6.90 6.90 7.13±0.40
(Each mean ± SD is derived from three independent experiments.)
Table 9. Comparison of different methods for T. indica water extract

Methods being compared Difference (%) Whether Significant
Soxhlet-ERT 26.08 Yes
Soxhlet-MAE 22.98 Yes
Soxhlet-MAEC 28.28 Yes
Soxhlet-UAE 33.61 Yes
MAE-ERT 3.16 No
ERT-MAEC 2.20 No
ERT-UAE 7.53 Yes
MAE-MAEC 5.30 ࡱYes
MAE-UAE 10.63 Yes
MAEC-UAE 5.33 ࡱYes
Null hypothesis for this extract was rejected because F calculated was
higher than Fcritical .Soxhlet was found to be most effective followed by
MAE and ERT for extraction of this plant material, but it requires a
longer time (3 h) so if we want a faster alternative MAE can be
47

considered, though with somewhat reduced efficiency. MAE and
0$(& DUH VLPLODU WR (57 EHFDXVH WKHVH DOO GRQ¶W KDYH DQ\
significant difference so we can use any of the methods, but ERT is
time consuming so preferably MAE will be chosen (for avoiding heat
degradation). MAE was providing high extraction efficiency than
MAEC so we can say intermittent cooling should be preferred. UAE
proved to be least effective with respect to extraction efficiency for
this extract.
3.2.1.2 Methanol extract of T. indica seed:

Table 10. Extraction efficiency of T. indica methanol extract prepared by
different methods
Method Extraction efficiency (%) Significant
1 2 3 Mean±SD Difference
Soxhlet 6.90 7.00 6.95 6.95±0.05
ERT 5.70 5.00 4.90 5.20±0.43
MAE 3.70 3.70 3.40 3.60±0.17 00.66
MAEC 3.90 3.70 3.40 3.66±0.25
UAE 2.50 2.60 2.80 2.63±0.15
Table 11. Comparison of different methods for T. indica methanol extract

ERT-MAE 1.60 Yes
ERT-MAEC 1.54 Yes
ERT-UAE 2.57 Yes
MAEC-MAE 0.06 No
MAE-UAE 0.97 Yes
MAEC-UAE 1.03 Yes
48

The null hypothesis for this extract was rejected because Fcalculated was
higher than Fcritical. Soxhlet was found to be most suitable method for
extraction of this plant material in methanol, and this was having
significant difference with all the other methods.
UAE proved to be inferior, as for water extract of same seed. MAE

DQG0$(&GRQ¶WKDYHDQ\VLJQLILFDQWGLIIHUHQFHVRZHFDQVD\WKDW
LQWHUPLWWHQWFRROLQJRUFRQWLQXRXVKHDWLQJGRQ¶WKDYHDQ\PDMRUHIIHFW
on extraction as far as extraction efficiency is considered for this plant
material.
For preparation of T. indica extract water was proven to be better than

methanol and soxhlet was found to be most suitable extraction
method. UAE provides lowest extraction efficiency with both the
solvents. Polarity of water and methanol, though vary much,
extraction efficiency them does, for T. indica seed.
3.2.2 Annona squamosa:
3.2.2.1 Water extract of A. squamosa seed:
Table 12. Extraction efficiency of A. squamosa water extract prepared by

different methods
Soxhlet 11.25 12.45 13.05 12.25±0.91
ERT 6.50 5.70 6.60 6.26±0.49
MAE 9.80 8.30 9.30 9.13±0.76 01.91
MAEC 10.60 10.60 10.70 10.63±0.05
UAE 11.70 13.40 11.90 12.33±0.92
49

Table 13. Comparison of different methods for A. squamosa water extract

Soxhlet-MAEC 1.62 No
UAE-Soxhlet 0.08 No
MAE-ERT 2.87 Yes
MAEC-ERT 4.37 Yes
UAE-ERT 6.07 Yes
MAEC-MAE 1.50 No
UAE-MAE 3.20 Yes
UAE-MAEC 1.70 No
The null hypothesis for this extract was rejected because Fcalculated was
higher than Fcritical. UAE was proven to be better from all the above
methods for extraction of A. squamosa LQ ZDWHU EXW LW GRHVQ¶W KDYH
any significant difference with MAEC and Soxhlet but Soxhlet is time
consuming and employs heat for a longer duration, so it should be
avoided. MAEC and UAE can be used as an alternative for Soxhlet.
ERT was an inferior method for extraction of this plant material.
3.2.2.2 Methanol extract of A. squamosa:

Table 14. Extraction efficiency of A. squamosa methanol extract prepared by
different methods

Soxhlet 12.75 12.20 11.75 12.23±0.50
ERT 11.70 11.60 11.70 11.66±0.05
MAE 7.80 7.10 7.80 7.56±0.40 01.53
MAEC 8.80 8.30 7.40 8.16±0.70
UAE 8.50 10.00 9.90 9.46±0.83
50

Table 15. Comparison of different methods for A. squamosa methanol
extract

Soxhlet-ERT 0.57 No
ERT-MAE 4.10 Yes
ERT-MAEC 3.50 Yes
ERT-UAE 2.20 Yes
MAEC-MAE 0.60 No
UAE-MAE 1.90 Yes
UAE-MAEC 1.30 No
Null hypothesis was rejected for this particular extract. Soxhlet was
VXSHULRU WR DOO WKH PHWKRGV ZKLFK ZHUH DSSOLHG DQG GRHVQ¶W KDYH
significant difference with ERT, so ERT can be used as an alternative
of Soxhlet if we want to extract out heat labile components.
Soxhlet and ERT are most effective methods but time is a limitation
so we can use UAE as a somewhat inferior but still acceptable
alternative. UAE and MAE can be used as an equal alternative of
MAEC so we can say inWHUPLWWHQW FRROLQJ GRHVQ¶W KDYH DQ\ QRWDEOH
effect during extraction with microwaves.
MAE was found to be inferior method for extraction of this particular

plant material.
Water is better solvent than methanol for the extraction of A.

squamosa seed and Soxhlet is superior with both solvent water and
methanol.
51

3.2.3 Syzygium cumini:
3.2.3.1 Methanol extract of S. cumini seed:

Table 16. Extraction efficiency of S. cumini methanol extract prepared by
different methods

Soxhlet 32.50 31.50 27.80 30.60±2.47

MAE 16.00 16.60 16.10 16.23±0.32 03.64
MAEC 21.00 20.70 21.30 21.00±0.30
UAE 17.30 16.10 18.50 17.30±1.2
Table 17. Comparison of different methods for S. cumini

methanol extract

MAEC-MAE 4.77 Yes
UAE-MAE 1.07 No
MAEC-UAE 3.70 Yes
We have rejected the null hypothesis for this extract because Fcalculated
was higher than Fcritical .Soxhlet was found to be superior followed by
MAEC and MAE was found to be inferior to all the methods for this
SDUWLFXODUH[WUDFW8$(DQG0$(GRQ¶WKDYHVLJQLILFDQWGLIIHUHQFHVR
both can be used for extraction of heat labile components. MAE and
MAEC are different from each other so we can say continuous
treatment by microwaves is beneficial for extraction of particular
seed. UAE and MAE are not similar so we can use UAE for
extraction of heat labile components.
52

3.2.3.2 Ethanolic extract of S. cumuni seed:
Table 18. Extraction efficiency of S. cumini ethanol extract prepared by

different methods
Method Extraction efficiency Significant

Soxhlet 40.35 42.45 40.10 40.96±1.29
MAE 27.70 30.10 30.20 29.33±1.41 03.36
MAEC 30.10 31.00 31.70 30.93±0.80
UAE 27.50 27.10 29.90 28.16±1.51
Table 19. Comparison of different methods for S. cumini ethanol extract

MAEC-MAE 1.60 No
MAE-UAE 1.17 No
MAEC-UAE 2.77 No
We have rejected the null hypothesis for this extract because Fcalculated
was higher than Fcritical .Soxhlet is superior followed by MAEC and
MAE and has significant difference from all the methods. But MAE
was found to be similar to UAE and MAEC; hence it can be used as
an alternative.
UAE proved to be inferior with respect to extraction efficiency for

WKLV SODQW PDWHULDO ,QWHUPLWWHQW FRROLQJ GRHVQ¶W have any significant
effect on extraction efficiency for this plant material. Soxhlet was
found to be better method and 50% ethanol is superior from methanol
for the extraction of S. cumuni.
53

3.2.4 P. sylvestris:
3.2.4.1 Methanolic extract of P. sylvestris:
Table 20. Extraction efficiency of P. sylvestris methanol extract prepared by
different methods

Soxhlet 13.10 15.90 13.70 14.23±1.47
ERT 12.50 15.20 13.70 13.80±1.35
MAE 12.50 10.60 11.40 11.50±0.95 03.58
MAEC 13.40 9.80 11.30 11.50±1.80
UAE 9.80 11.26 11.20 10.75±0.82
Table 21. Comparison of different methods for P. sylvestris methanol extract

Soxhlet-ERT 0.43 No
Soxhlet-MAE 2.73 No
ERT-MAE 2.30 No
ERT-MAEC 2.30 No
ERT-UAE 3.05 No
MAE-MAEC 0.00 No
MAE-UAE 0.75 No
MAEC-UAE 0.75 No
Null hypothesis is rejected for this particular extract. Though mean

extraction eIILFLHQF\ LV KLJKHVW IRU VR[KOHW EXW LW GRHVQ¶W KDYH
significant difference with MAE and MAEC, so both methods can
serve as faster alternative for extraction of this plant extract because
of higher time consumption in Soxhlet. ERT is similar to MAE, along
with MAEC and UAE with respect to extraction efficiency. MAE and
MAEC both provide same extraction efficiencies so we can say
neither continuous heating nor intermittent cooling causes any
54

difference in processing but then we can choose MAEC because it
requires shorter time and power in comparison to MAE. UAE was
found to be inferior amongst all the methods used for the extraction.
3.2.4.2 Ethanolic extract of P. sylvestris:

Table 22. Extraction efficiency of P. sylvestris ethanol extract prepared by
different methods
Soxhlet 11.45 13.30 12.00 12.25±0.95
ERT 10.00 10.50 10.10 10.20±0.26
MAE 6.90 7.20 7.90 7.33±0.51 02.69
MAEC 7.50 7.90 7.10 7.50±0.40
UAE 9.80 7.20 10.90 9.30±1.90
Table 23. Comparison of different methods for P. sylvestris ethanol extract

Soxhlet-ERT 2.05 No
ERT-MAE 2.87 Yes
ERT-MAEC 2.70 Yes
ERT-UAE 0.90 No
MAEC-MAE 0.17 No
UAE-MAE 1.97 No
UAE-MAEC 1.80 No
most effective as compare to all the methods but it was not having
any significant difference with ERT, then also we cannot use ERT
because it has disadvantage of time limitation. For extraction of heat
labile components ERT can be used for extraction. ERT, MAE and
55

MAEC can be used as an alternative of UAE. MAE and MAEC both
provides same extraction efficiencies so we can say neither
contentious heating nor intermittent cooling causes any difference in
processing but then we can choose MAEC because it requires shorter
time and power in comparison to MAE. Soxhlet is effective than all
the methods tasted and methanol is better than ethanol for extraction
of P. sylvestris seeds. MAE and MAEC give similar results with both
solvents fort his plant material.
3.2.5 M. Zapota:
3.2.5.1 Water extract of M. zapota seed:
Table 24. Extraction efficiency of M. zapota water extract prepared by
different methods

Soxhlet 11.35 10.70 12.80 11.61±1.07
ERT 6.30 6.50 6.10 6.30±0.2
MAE 10.00 8.10 8.60 8.90±0.98 02.10
MAEC 9.70 9.90 9.60 9.73±0.15
UAE 9.70 11.00 9.20 9.96±0.92
Table 25. Comparison of different methods for M. zapota water extract

Soxhlet-UAE 1.65 No
MAE-ERT 2.60 Yes
MAEC-ERT 3.43 Yes
UAE-ERT 3.66 Yes
MAEC-MAE 0.83 No
UAE-MAE 1.06 No
UAE-MAEC 0.23 No
56

Null hypothesis was rejected for this particular extract. Soxhlet was found
to be a superior method followed by UAE for this plant material and it
GRHVQ¶WKDYHDQ\VLJQLILFDQWGLIIHUHQFHZLWK0$(DQG(57KRZHYHU8$(
and MAEC can be used as an alternative for this extraction. Soxhlet needs
longer time for extraction so we can use UAE or MAEC for fast extraction.
If extraction of heat labile components is required then one should go for
UAE. MAE is similar to MAEC and UAE so we can say intermittent
FRROLQJ GRQ¶W KDYH VLJQLILFDQW UROH GXULQJ H[WUDFWLRQ (57 ZDV LQIHULRU
method for this plant material.
3.2.5.2 Methanolic extract of M. zapota:

Table 26. Extraction efficiency of M. zapota methanol extract prepared by
different methods

Soxhlet 11.80 11.70 12.35 11.95±0.35
ERT 11.20 10.30 11.30 10.93±0.55
MAE 7.50 7.60 6.40 7.16±0.66 01.19
MAEC 7.50 7.40 7.30 7.40±0.10
UAE 5.60 6.10 6.20 5.96±0.32
Table 27. Comparison of different methods for M. zapota methanol extract

Soxhlet-ERT 1.02 No
ERT-MAE 3.77 Yes
ERT-MAEC 3.53 Yes
ERT-UAE 4.97 Yes
MAEC-MAE 0.24 No
MAE-UAE 1.20 Yes
MAEC-UAE 1.44 Yes
57

Null hypothesis was rejected for this particular extract. Soxhlet is
superior to other methods EXW LW GRHVQ¶W KDYH VLJQLILFDQW GLIIHUHQFH
with ERT. ERT can serve as one the alternative methods for
extraction, but it also has time limitation. MAEC will be best
DOWHUQDWLYHIRUIDVWH[WUDFWLRQ0$(DQG0$(&GRQ¶WKDYHVRPXFK
difference in extraction efficiency so we can say intermittent cooling
GRHVQ¶W KDYH VLJQLILFDQW UROH GXULQJ H[WUDFWLRQ8$( LV PRVW LQIHULRU
method for this plant material.
3.2.5.3 Ethanolic extract of M. zapota seed:

Table 28. Extraction efficiency of M. zapota ethanol extract prepared by
different methods

Soxhlet 7.90 8.75 8.65 8.43±0.46
ERT 7.50 6.70 7.40 7.20±0.43
MAE 7.50 5.60 6.40 6.50±0.95 01.61
MAEC 7.30 7.00 7.50 7.26±0.25
UAE 8.10 8.60 7.30 8.00±0.65
Table 29. Comparison of different methods for M. zapota ethanol extract

Soxhlet-ERT 1.23 No
Soxhlet-UAE 0.43 No
ERT-MAE 0.70 No
MAEC-ERT 0.06 No
UAE-ERT 0.80 No
MAEC-MAE 0.76 No
UAE-MAE 1.50 No
UAE-MAEC 0.74 No
58

VXSHULRU WR RWKHU PHWKRGV ZH XVHG DQG LW GRHVQ¶W KDYH VLJQLILFDQW
difference with ERT, MAEC, and UAE. UAE is the best method for
fast extraction and for extraction of heat labile components.
Continuous heating is useful during microwave operation.
MAEC and ERT both gives similar extraction efficiency for this
plant material but ERT cannot be used due to time limitation. For this
particular extract only Soxhlet and MAE have significant difference
other methods have similar strength in term of extraction efficiency.
MAE was inferior method for extraction of this plant material.
Soxhlet is most effective method and methanol is better solvent than

water and methanol for M. zapota seeds if we want to get high
extraction efficiency.
3.3 TOTAL FLAVONOID
3.3.1 T. indica:
3.3.3.1 Water extract of T. indica:
Table 30. Flavonoid content of T. indica water extract prepared by different

methods
Total flavonoid Significant

Method
(mg/ml QE/g of dry extract) difference
Soxhlet 1.76±0.11
ERT Not detectable
MAE 2.49±0.00 0.23
UAE 4.72±0.00
(Each mean ± SD is derived from two independent experiments.)
59

Table 31. Comparison of flavonoid content in T. indica water extract
prepared by different methods
Methods being Difference Whether

compared (mg/ml QE/g of dry extract) Significant
MAE-Soxhlet 0.73 Yes
UAE-Soxhlet 3.03 Yes
MAE- ERT 2.49 Yes
UAE-ERT 4.72 Yes
UAE-MAE 2.23 Yes
Null hypothesis was rejected for this particular extract. UAE was
superior for total flavonoid extraction from plant material and ERT
was inferior.
ERT was unable to extract out any detectable flavonoid content from
T. indica seeds.
3.3.1.2 Methanol extract of T. indica:
Table 32. Flavonoid content of T. indica methanol extract prepared by

different methods

Method
Soxhlet 13.85±2.92
ERT 36.86±14.99
MAE 16.52±3.69 28.42
MAEC 5.02±0.37
UAE 11.75±2.00
60

Table 33. Comparison of flavonoid content in T. indica methanol extract

ERT-Soxhlet 23.01 No
MAE-Soxhlet 02.67 No
Soxhlet-UAE 02.10 No
ERT-MAE 20.34 No
ERT-MAEC 31.84 Yes
ERT-UAE 25.11 No
MAE-MAEC 11.50 No
MAE-UAE 04.77 No
UAE-MAEC 06.73 No
Null hypothesis is also rejected for this extract. ERT was superior for
extraction of total flavonoid for this plant material and MAEC was
found to be inferior.
We can say continuous heating is not suitable during microwave

application. As mentioned above heat degradation may be a reason
for low extraction of flavonoid from Soxhlet and MAEC.
(57GRHVQ¶WKDYHDQ\VLJQLILFDQWGLIIHUHQFHZLWK6R[KOHW0$(DQG
UAE, but it requires a longer time.
When fast extraction is required then MAE and UAE should be

preferred over soxhlet and ERT.
Methanol is better than water for extraction of flavonoids from T. indica seeds.
ERT was superior with methanol, but inferior with water.
61

3.3.2 Annona squamosa:
3.3.2.1 Water extract of A. squamosa:
Table 34. Flavonoid content of A. squamosa water extract prepared by

different methods

Method
Soxhlet Not detectable
ERT 2.73±0.22
MAE 3.87±1.56 3.35
MAEC 2.55±0.25
UAE 13.10±0.97
Table 35. Comparison of flavonoid content in A. squamosa water extract


ERT-Soxhlet 2.73 No
MAEC-Soxhlet 2.55 No
MAE-ERT 1.14 No
ERT-MAEC 0.18 No
UAE-ERT 10.37 Yes
MAE-MAEC 1.32 No
MAE-UAE 9.23 Yes
UAE-MAEC 10.55 Yes
Null hypothesis is rejected for this particular extract. UAE was

superior method for extract out total flavonoid from A. squamosa
seeds and Soxhlet was found inferior. Soxhlet was unable to extract
out any detectable flavonoid from this plant material. UAE has
significant difference with all the methods. UAE is best for the
62

flavonoid extraction in very short time period. MAEC and MAE has
some difference so we can say intermittent cooling has effect in
flavonoid extraction during microwave application.
Table 36. Flavonoid content of A. squamosa methanol extract prepared by

different methods

Method
Soxhlet 02.83±00.00
ERT 16.62±02.94
MAE 45.84±05.53 13.11
MAEC 43.04±03.76
UAE Not detectable
Table 37. Comparison of flavonoid content in A. squamosa methanol extract

ERT-Soxhlet 13.79 Yes
MAEC-Soxhlet 40.21 Yes
Soxhlet-UAE 2.83 No
MAE-ERT 29.22 Yes
MAEC-ERT 26.42 Yes
ERT-UAE 16.62 Yes
MAE-MAEC 2.8 No
MAE-UAE 54.84 Yes
MAEC-UAE 43.04 Yes
Null hypothesis was rejected for this particular extract. MAE was best
method for extraction of total flavonoid from this particular seed and
LWGRHVQ¶WKDYHDQ\VLJQLILFDQWGLIIHUHQFHZLWK0$(&VRZHFDQVD\
that intermittent cooling has no effect on the flavonoid extraction for
63

this plant material. Both MAE and MAEC are good for flavonoid
extraction from particular plant material; it means microwave has
some special mechanism for this seed. UAE is inferior method for
extraction of flavonoid from this plant material.
Methanol is better than water and MAE found to be effective then

other methods for extraction of total flavonoid from A. squamosa
seeds. UAE is giving a good extraction with water but unable to
extract flavonoid with methanol so we can say solvent also
significantly influence extraction by different methods.
3.3.3 S. cumini:
3.3.3.1 Methanolic extract of S. cumini:
Table 38. Flavonoid content of S. cumini methanol extract prepared by

different methods
Total flavonoid Significant difference
Method
(mg/ml QE/g of dry extract)
MAE 23.54±3.69
MAEC 13.28 ± 1.70 10.65
UAE 14.72 ± 3.28
Table 39. Comparison of flavonoid content in S. cumini methanol extract


MAE-MAEC 10.26 No
MAE-UAE 8.82 No
UAE-MAEC 1.44 No
64

Null hypothesis was rejected for this particular extract. MAE was best
method for extraction of total flavonoid from this plant material,
whereas Soxhlet proved to be an inferior. Soxhlet was again not able
to extract out any detectable flavonoid for particular material.
However, as MAEC and UAE did not show any significant
difference, the can be used instead of MAE. UAE can be preferred
ZKHQKHDWODELOHFRPSRQHQWVDUHGHDOWZLWK0$(DQG0$(&GRQ¶W
have significant difference according to this data but we can say
intermittent cooling has some advantage during microwave
application.
3.3.3.2 Ethanolic extract of S. cumini:
Table 40. Flavonoid content of S. cumini ethanol extract prepared by

different methods

Method
Soxhlet 15.46 ± 1.98
MAE 19.13 ± 3.01
MAEC 17.46 ± 0.85 15.91
UAE 21.08 ± 6.88
Table 41. Comparison of flavonoid content in S. cumini ethanol extract


MAE-Soxhlet 3.67 No
UAE-Soxhlet 5.62 No
MAE-MAEC 1.67 No
UAE-MAE 1.95 No
UAE-MAEC 3.62 No
65

Null hypothesis is rejected for this extract. The t-test was performed
for whole data set and we have got p value >0.05, so we have to
mention that all the methods are equivalent for extraction of flavonoid
for this particular extract. Methanol and MAE proved to be best for
extraction of flavonoid from S. cumini seeds. Soxhlet was proved to
be inferior with both the solvents.
3.3.4.1 Methanol extract of P. sylvestris:

Table 42. Flavonoid content of P. sylvestris methanol extract prepared by
different methods
Method
Soxhlet 3.63 ± 1.02
ERT Not detectable
MAE 1.26 ± 0.00 5.66
MAEC 10.10 ± 2.99
UAE Not detectable
Table 43. Comparison of flavonoid content in P. sylvestris methanol

extract prepared by different methods
Soxhlet-ERT 3.63 No
Soxhlet-MAE 2.37 No
Soxhlet-UAE 3.63 No
MAE-ERT 1.26 No
MAEC-ERT 10.10 Yes
ERT-UAE 0.00 No
MAEC-MAE 8.84 Yes
MAE-UAE 1.26 No
MAEC-UAE 10.10 Yes
66

Null hypothesis was rejected for this particular extract. MAEC was
found to be the best method for the extraction of flavonoids for this
plant material, whereas ERT and UAE were found to be unable to
extract out any detectable amount of flavonoid. MAE has a higher
significant difference with MAEC so we can say either intermittent
cooling or continuous heating have some significant role during
microwave application.
3.3.4.2 Ethanol extract of P. sylvestris:

Table 44. Flavonoid content of P. sylvestris ethanol extract prepared by
different methods
Method
ERT 6.01 ± 0.45
MAE Not detectable 5.94
MAEC 5.72 ± 0.21
UAE 4.49 ± 3.27
Table 45. Comparison of flavonoid content in P. sylvestris ethanol extract

Soxhlet-MAE 0.00 No
UAE-Soxhlet 4.49 No
ERT-MAE 6.01 Yes
ERT-MAEC 0.29 No
ERT-UAE 1.52 No
MAEC-MAE 5.72 No
UAE-MAE 4.49 No
MAEC-UAE 1.23 No
67

Null hypothesis was rejected for this particular extract. ERT was
found to be the best method for the extraction of flavonoids from this
plant material, whereas Soxhlet and MAE are unable to extract any
amount of flavonoid from particular material. MAEC and UAE can
be used as alternatives instead of ERT due to time limitation during
ERT. MAEC is proved to be more effective so we can say continuous
heating is good for flavonoid extraction. Methanol is better solvent
than ethanol for extraction and MAEC is better with both solvents for
flavonoid extraction from P. sylvestris seeds.
3.3.5 M. zapota:
3.3.5.1 Water extract of M. zapota:

Table 46. Flavonoid content of M. zapota water extract prepared by different
methods
Method
ERT 6.03 ± 1.15
MAE Not detectable 2.10
MAEC 1.26 ± 0.00
UAE 3.15 ± 0.19
Table 47. Comparison of flavonoid content in M. zapota water extract

Soxhlet-MAE 0.00 No
ERT-MAE 6.03 Yes
ERT-MAEC 4.77 Yes
ERT-UAE 2.88 Yes
MAEC-MAE 1.26 No
UAE-MAE 3.15 Yes
UAE-MAEC 1.89 No
68

Null hypothesis was rejected for this particular extract. ERT was
found to be the best method for the extraction of flavonoids for this
plant material, whereas Soxhlet and MAE were unable to extract
detectable amount of flavonoid. ERT requires a long time for
extraction so we can use UAE for faster extraction. UAE and MAEC
can be used as alternative methods as no significant difference is
observed between them. MAEC is again proved to be better then
MAE so we can say contentious heating us beneficial during
3.3.5.2 Methanol extract of M. zapota:

Table 48. Flavonoid content of M. zapota methanol extract prepared by
different methods
Method
Soxhlet 19.32 ± 0.39
ERT 6.07 ± 0.00
MAE 2.71 ± 0.00 5.57
MAEC 4.28 ± 3.08
UAE Not detectable
Table 49. Comparison of flavonoid content in M. zapota methanol extract

ERT-MAE 3.36 No
ERT-MAEC 1.79 No
ERT-UAE 6.07 Yes
MAEC-MAE 1.57 No
MAE-UAE 2.71 No
MAEC-UAE 4.28 No
69

Null hypothesis was rejected for this particular extract. For this
particular plant material, Soxhlet is the most effective method for the
extraction of flavonoids, whereas UAE was unable to extract out any
detectable amount of flavonoid. MAEC and MAE can also be used
alternatives instead of ERT when faster extraction is required. MAE
and MAEC are having some difference in extracting flavonoid so we
can say contentious heating is better during microwave treatment.
3.3.5.3 Ethanol extract of M. zapota:
Table 50. Flavonoid content of M. zapota ethanol extract prepared by

different methods
Method
Soxhlet 1.56±0.098
ERT 7.58±0.00
MAE 2.17±0.74 1.45
MAEC 9.30±0.28
UAE 2.23±0.09
Table 51. Comparison of flavonoid content in M. zapota ethanol extract

MAE-Soxhlet 0.61 No
UAE-Soxhlet 0.67 No
ERT-MAE 5.41 Yes
MAEC-ERT 1.72 Yes
UAE-ERT 5.35 Yes
MAEC-MAE 7.13 Yes
UAE-MAE 0.06 No
MAEC-UAE 7.07 Yes
70

Null hypothesis was rejected for this particular extract. MAEC is the
best method for the extraction of flavonoid for this plant material.
Soxhlet proved to be inferior in this case. MAEC is better than MAE so
we can say contentious heating is better during microwave treatment.
UAE and Soxhlet can be used as alternatives instead of MAE.Methanol
is better solvent than water and ethanol for extraction of flavonoid from
M. zapota seeds. In all cases MAEC proved to be better then MAE.
UAE was not found to be good for extraction of flavonoid from M.
zapota seed.
3.4 PHENOLS
3.4.1 T. indica:
Table 52. Phenol content of T. indica water extract prepared by different
methods
Total phenol Significant
Method
(mM GAE/g of dry extract) difference
Soxhlet 1139.39±147.26
ERT 999.99±188.56 385.19
MAE 1219.1±139.79
UAE 498.82±8.30
Table 53. comparison of phenpl content in T. indica water extract prepared

by different methods
compared (mM GAE/g of dry extract) Significant
Soxhlet-ERT 139.40 No
MAE-ERT 219.11 No
ERT-UAE 501.17 Yes
MAE-UAE 720.28 Yes
71

Null hypothesis was rejected for this extract. MAE was found to be
superior and UAE was inferior for extraction of phenols from this
particular plant material. ERT and MAE can be also used as an
alternative of Soxhlet. MAE and ERT can be used as alternative of each
other but MAE will be preferred because it will allow fast extraction.
This data shows that heat is better for extraction of phenols from T.
indica seeds because both ERT and UAE are inferior with respect to
phenol extraction. MAEC was not used for phenol extraction because
reconstitution of extract was not possible for this seed.

Table 54. Phenol content of T. indica methanol extract prepared by different
methods
Method
Soxhlet 10037.64±567.81
ERT 1429.33±2.53
MAE 6327.16±122.57 2087.96
MAEC 16328.24±1673.30
UAE 8581.04±391.11
Table 55. Comparison of phenol content in T. indica methanol extract prepared


Soxhlet-UAE 1456.60 No*
MAE-ERT 4897.83 Yes
MAEC-ERT 14898.91 Yes
UAE-ERT 7151.71 Yes
MAEC-MAE 10001.08 Yes
UAE-MAE 2253.88 Yes
MAEC-UAE 7747.20 Yes
*
p value <0.05 (from t-test)
72

Null hypothesis is also rejected for this extract. The t-test was
performed for Soxhlet-UAE, but the p value was >0.05, so both are
not similar. MAEC was found to be superior and ERT was inferior for
extraction of total phenols for this plant material.
MAEC is also having significant difference with all other methods.

Soxhlet and UAE can be used as an alternative for each other so UAE
should be used for fast extraction.
Soxhlet and MAEC were found better than all other methods so we
can conclude that heat is better for extraction of phenols from T.
indica seeds. MAEC is better than MAE so we can say continuous
heating is better than intermittent cooling during microwave
application.
Methanol is more efficient than water for phenol extraction.

Application of heat seems to be effective for extraction of phenols.
3.4.2 A. squamosa:
Table 56. Phenol content of A. squamosa water extract prepared by different

methods

Method
Soxhlet 102.08±8.83
ERT 2317.51±25.80
MAE 368.85±32.60 236.74
MAEC 1225.67±192.59
UAE 1113.39±58.32
73

Table 57. Comparison of phenol content in A. squamosa water extract
ERT-MAE 1948.66 Yes
ERT-MAEC 1091.84 Yes
ERT-UAE 1204.12 Yes
MAEC-MAE 856.85 Yes
UAE-MAE 744.54 Yes
MAEC-UAE 112.28 No
Null hypothesis is rejected for this particular extract. ERT was found
to be superior and Soxhlet was inferior method for extraction of total
phenol for this plant material.
In the case of Soxhlet, it can be said that direct application may be a

reason of degradation of phenols.
(57 GRHVQ¶W KDYH DQ\ VLmilarity with respect to extraction of total

phenols.
MAEC and UAE can be used as alternative methods for each other.
MAEC is again better than MAE so we can say continuous heating is

better than intermittent cooling during microwave application.
74

Table 58. Phenol content of A. squamosa methanol extract prepared by
different methods
Method
Soxhlet 2529.52±129.30
ERT 999.01±156.98
MAE 2390.81±57.31 285.06
MAEC 2206.51±107.60
UAE 1162.1±71.46
Table 59. Comparison of phenol content in A. squamosa methanol extract


Soxhlet-MAE 138.71 No
MAE-ERT 1391.80 Yes
UAE-ERT 163.09 No
MAE-MAEC 184.30 No
MAE-UAE 1228.71 Yes
Null hypothesis was rejected for this extract. Soxhlet was found to be
superior and ERT was inferior method for the extraction of phenols
IRUWKLVSDUWLFXODUSODQWPDWHULDO6R[KOHWGRHVQ¶WKDYHDQ\VLJQLILFDQW
difference with MAE so later can be used as an alternative for fast
extraction. MAE is also used as alternative method for MAEC. These
results show that heat application is better for the extraction of
phenols for this particular extract because cold methods such as UAE
75

and ERT are unable to extract a high amount of phenols.Methanol is
better than water for the extraction of phenols from A. squamosa
seeds. The choice of method will depend on solvent, because superior
method for one solvent is inferior with another solvent.
3.4.3 S. cumini:
3. 4.3.1 Methanol extract of S. cumini:

Table 60. Phenol content of S. cumini methanol extract prepared by different
methods
Significant
Total phenol
Method difference
(mM GAE/g of dry extract)
Soxhlet 1308.67±36.28
MAE 4679.44±472.70 905.05
MAEC 4437.56±93.67
UAE 4614.69±436.74
Table 61. Comparison of phenol content in S. cumini methanol extract

MAE-MAEC 241.88 No
MAE-UAE 64.75 No
UAE-MAEC 177.13 No
Null hypothesis is rejected for this particular extract. MAE is superior

and Soxhlet is inferior for the extraction of total phenols for this
extract. MAE is different from all the methods. MAE was found to be
similar to UAE and MAEC so both of these can be used as an
DOWHUQDWLYH 0$( GRHVQ¶W KDYH PXFK GLIIHUHQFH WKDQ 0$(& VR ZH
76

FDQ VD\ WKDW LQWHUPLWWHQW FRROLQJ GRHVQ¶W KDYH VR PXFK DGYDQWDJH
during microwave application.
3.4.3.2 Ethanol extract of S.cumini:

Table 62. Phenol content of S. cumini ethanol extract prepared by different
methods

Method difference
Soxhlet 1540.61±77.81
MAE 3431.24±179.72 1074.68
MAEC 3447.57±471.83
UAE 3960.69±580.73
Table 63. Comparison of phenol content in S. cumini ethanol extract

MAEC-MAE 16.33 No
UAE-MAE 529.45 No
UAE-MAEC 513.12 No
Null hypothesis was rejected for this particular extract. UAE is

superior and Soxhlet was found to be inferior for the total phenol
H[WUDFWLRQ IRU WKLV SODQW PDWHULDO 8$( GRHVQ¶W KDYH DQ\ VLJQLILFDQW
difference with MAE and MAEC so these can be used as an
alternative for UAE. Both MAE and MAEC can be used as an
alternative of each other for extraction of phenols so we can say
neither intermittent nor continuous heating affects extraction of
phenols.
77

Methanol is better than ethanol for extraction of phenols from S. cumuni seeds.
Soxhlet is not suitable for extraction of phenols for this plant material. MAE and
UAE proved no inferior to any other method for same purpose.

Table 64. Phenol content of P. sylvestris methanol extract prepared by
different methods
Method
Soxhlet 3476.92±114.33
ERT 6338.05±218.61
MAE 10929.14±2516.09 2960.87
MAEC 6938.93±375.86
UAE 12486.57±289.48
Table 65. Comparison of phenol content in P. sylvestris methanol extract

Methods being compared Difference Whether
(mM GAE/g of dry extract) Significant
MAE-ERT 4591.09 Yes
MAEC-ERT 600.88 No
UAE-ERT 6148.52 Yes
MAE-MAEC 3990.21 Yes
UAE-MAE 1558.43 No
UAE-MAEC 5547.64 Yes
Null hypothesis is rejected for this particular extract. UAE was found
to be superior and Soxhlet was inferior for the extraction of phenols
IRU WKLV SODQW PDWHULDO 8$( GRHVQ¶W KDYH DQ\ Vignificant difference
78

with MAE so it can serve as an alternative for UAE. Degradation of
phenols due to direct application of heat can be a reason of lower
performance of Soxhlet and MAEC. MAE is better than MAEC so we
can say intermittent cooling is useful during microwave application
for phenol extraction.
Table 66. Phenol content of P. sylvestris ethanol extract prepared by different

methods
Total phenol Significant difference
Method
Soxhlet 3466.96±397.54
ERT 4349.97±0.00
MAE 4173.07±281.03 6165.08
MAEC 20977.36±3903.48
UAE 9995.30±3634.84
Table 67. Comparison of phenol content in P. sylvestris ethanol extract prepared


ERT-MAE 176.90 No
UAE-ERT 5645.33 No
UAE-MAE 5822.23 No
Null hypothesis is rejected for this extract. MAEC is superior and

Soxhlet is inferior for extraction of total phenols for this plant
material. MAEC has significant difference with all other methods so
79

it GRHVQ¶W KDYH DQ\ EHWWHU DOWHUQDWLYH 8$( LV VLPLODU WR (57 DQG
MAE. MAE is similar to Soxhlet, CE and UAE. MAEC proved better
than MAE so we can say continuous heating increases phenol
extraction.
Ethanol is a better than methanol for extraction of phenols from P.
sylvestris seeds. With both solvents methanol and ethanol UAE gives
good extraction of phenols from this plant material. Soxhlet was
found to be inferior with both the solvents.
3.4.5 M. zapota:
Table 68. Phenol content of M. zapota water extract prepared by different
methods
Method
Soxhlet 754.71±97.02
ERT 355.98±11.44
MAE 478.02±0.00 124.39
MAEC 337.5±46.18
UAE 290.32±0.00
Table 69. Comparison of phenol content in M. zapota water extract prepared

MAE-ERT 122.04 No
MAEC-ERT 18.48 No
ERT-UAE 65.66 No
MAE-MAEC 140.54 Yes
MAE-UAE 187.70 Yes
MAEC-UAE 47.18 No
80

Null hypothesis is rejected for this particular extract. Soxhlet was
found to be superior and UAE was inferior for phenol extraction for
WKLVSODQWPDWHULDO6R[KOHWGRHVQ¶WKDYHDQ\DOWHUQDWLYHDPRQJWKRVH
tasted. ERT is similar to MAE, UAE and MAEC. ERT and UAE are
giving inferior results. MAE is proved to be better than MAEC so we
can say intermittent cooling during microwave helps to get good
extraction.
Table 70. Phenol content of M. zapota methanol extract prepared by

different methods
Method
Soxhlet 820.88±104.76
ERT 227.99±16.97
MAE 812.73±6.72 224.36
MAEC 769.46±148.57
UAE 432.09±67.89
Table 71. Comparison of phenol content in M. zapota methanol extract


Soxhlet-MAE 8.15 No
MAE-ERT 584.74 Yes
MAEC-ERT 541.47 Yes
UAE-ERT 204.10 No
MAE-MAEC 43.27 No
MAE-UAE 380.64 Yes
MAEC-UAE 337.37 Yes
81

superior and ERT was inferior for extraction of phenol for this plant
PDWHULDO 6R[KOHW GRHVQ¶W KDYH DQ\ VLJQLILFDQW GLIIHUHQFH ZLWK 0$(
and MAEC so both of these can be used as quicker alternative for
Soxhlet. UAE and CE proved to be worst for extraction of phenols so
we can say that heat is required for extraction of phenols for this
extract. UAE is similar to ERT so using UAE will provide a better
and faster alternative. MAE and MAEC are similar for extracting
phenols so we can say neither intermittent cooling nor continuous
heating have any effect during microwave treatment.

Table 72. Phenol content of M. zapota ethanol extract prepared by different
methods

Method
Soxhlet 3975.38±374.79
ERT 96.52±5.456
MAE 618.9±20.67 506.77
MAEC 2274.36±229.74
UAE 316.35±10.91
Table 73. Comparison of phenol content in M. zapota ethanol extract


MAE-ERT 522.38 Yes
UAE-ERT 219.83 No
MAE-UAE 302.55 No
82

superior and ERT was inferior for the extraction of phenols for this
plant material. Soxhlet has significant difference with all the methods
VRZHGRQ¶WKDYHDQ\DOWHUQDWLYHPHWKRGIRUSKHQROH[WUDFWLRQ8$(
is not significant to ERT and MAE so both can serve as alternative
method of UAE. UAE and ERT proved to be worst for extraction of
phenols so we can say that heat is required for extraction of phenols
for this extract. MAEC is better than MAE so we can say continuous
heating is useful for extracting phenols during microwave treatment.
Ethanol is best solvent for the extraction of phenols from M. zapota

seeds. Soxhlet was superior and ERT and UAE proved lesser efficient
then other methods so we can conclude that heat application is
required for phenol extraction form this seed. MAE is better than
MAEC with water and methanol.
3.5 TOTAL ANTIOXIDANTS
3.5.1 T. indica:
Table 74. Antioxidant content of T. indica water extract prepared by

different methods
Total antioxidant Significant
Method
Soxhlet 1597.22±128.81
ERT 433.32±47.142
MAE 476.43±162.63 437.31
UAE 249.99±29.11
83

Table 75. Comparison of antioxidant content in T. indica water extract

MAE-ERT 43.11 No
ERT-UAE 183.33 No
MAE-UAE 226.44 No
Null hypothesis is rejected. Soxhlet was superior and it was having

significant difference with all other methods. UAE was inferior
method. ERT was equivalent to MAE and UAE, so MAE can be
preferred over UAE and ERT. MAE will also give faster extraction of
antioxidant than ERT. Data shows that ERT and UAE give a lower
extraction of antioxidants then MAE and Soxhlet so we can conclude
that heat application positively affects the extraction of total
antioxidants.
Table 76. Antioxidant content of T. indica methanol extract prepared by

different methods

Method
Soxhlet 9006.27±1494.26
ERT 3108.10±1974.80
MAE 3260.82±0.00 4806.04
MAEC 2503.78±259.09
UAE 2807.00±992.43
84

Table 77. Comparison of phenol content in T. indica methanol extract prepared

MAE-ERT 152.72 No
ERT-MAEC 604.32 No
ERT-UAE 301.10 No
MAE-MAEC 757.04 No
MAE-UAE 453.82 No
UAE-MAEC 303.22 No
Null hypothesis was rejected. Soxhlet was superior and MAEC was
inferior method. Soxhlet was having significant difference with every
method so no method can serve as alternative.
Heat exposure for a long time seems helpful for extraction of total
antioxidants for this plant material. MAEC was inferior method.
MAE was better than MAEC so we can say intermittent cooling was
helpful in extraction of total antioxidants from plant material.
(57GRHVQ¶WKDYHDQ\VLJQLILFDQWGLIIHUHQFHZLWK0$(0$(&DQG
UAE so they can be used as an alternative. UAE and MAEC can be
used as alternative for MAE. MAE was better than MAEC so we can
use it as an alternative for faster extraction.
Methanol is better than water for antioxidants extraction from T.

indica seeds. Heat exposure increases antioxidant extraction from T.
indica seeds it can be the reason for superiority of Soxhlet.
85

3.5.2 A. squamosa:

Table 78. Antioxidant content of A. squamosa water extract prepared by
different methods
Method
Soxhlet 804.15±253.37
ERT 3666.65±261.89
MAE 1123.06±152.29 740.28
MAEC 305.18±82.65
UAE 1134.00±87.47
Table 79. Comparison of phenol content in A. squamosa water extract

UAE-Soxhlet 329.85 No
ERT-MAE 2543.59 Yes
ERT-MAEC 3361.47 Yes
ERT-UAE 2532.65 Yes
MAE-MAEC 817.26 No
UAE-MAE 10.94 No
UAE-MAEC 828.82 Yes
Null hypothesis was rejected. ERT is superior and MAEC is inferior

method. ERT has significant difference with all the methods. MAE,
MAEC and UAE can be used at the place of Soxhlet. UAE, Soxhlet
and MAEC can be used as an alternative of MAE. ERT and UAE are
superior then Soxhlet and MAEC so we can say over heating
negatively affect extraction of antioxidants. Intermittent cooling may
be the reason of superiority of MAE over MAEC.
86

Table 80. Antioxidant content of A. squamosa methanol extract prepared by

different methods
Method
Soxhlet 1790.41±377.10
ERT 787.03±327.36
MAE 939.07±107.68 2632.42
MAEC 2832.72±1376.41
UAE 2025.31±0.00
Table 81. Comparison of phenol content in A. squamosa methanol extract


MAE-ERT 152.04 No
MAEC-ERT 2045.69 No
UAE-ERT 1238.28 No
MAEC-MAE 1893.65 No*
UAE-MAE 1086.24 No
MAEC-UAE 807.41 No
*
p value <0.05
Null hypothesis was not rejected. For MAE and MAEC the p value
was <0.05 so both of these were having significant difference with
each other, so by this result we can say continuous heating increases
the antioxidant extraction from this plant material.
87

Water was better solvent for the extraction of antioxidants from A.
squamosa. UAE was found average for antioxidant extraction with
both solvents. Selection of suitable method will vary with solvent to
solvent.
3.5.3 S. cumini:
3.5.3.1 Methanol extract of S. cumini:
Table 82. Antioxidant content of S. cumini methanol extract prepared by
different methods
Method
Soxhlet 800.82±9.51
MAE 2104.01±185.96 447.26
MAEC 1771.86±0.00
UAE 2552.78±116.45
Table 83. Comparison of phenol content in S. cumini methanol extract

MAE-MAEC 332.15 No
UAE-MAE 448.77 Yes
UAE-MAEC 780.92 Yes
Null hypothesis is rejected. UAE is superior and Soxhlet is inferior

from all other methods. UAE and MAE has advantage over Soxhlet
and MAEC. Low efficiency of Soxhlet and MAEC shows negative
effect of heat application on extraction of antioxidants. MAE and
0$(&FDQEHXVHGDVDOWHUQDWLYHEHFDXVHWKH\GRQ¶WKDYHVLJQLILFDQW
difference so intermittent cooling can be said useless and time and
power consuming.
88

3.5.3.2 Ethanol extract of S. cumini:
Table 84. Antioxidant content of S. cumini ethanol extract prepared by
different methods

Method difference
Soxhlet 844.26±10.20
MAE 1541.56±106.05 216.98
MAEC 1283.39±0.00
UAE 1685±0.00
Table 85. Comparison of phenol content in S. cumini ethanol extract

MAE-MAEC 258.10 Yes
UAE-MAE 143.44 Yes
UAE-MAEC 401.61 Yes
Null hypothesis was rejected. UAE is superior and Soxhlet is inferior.

All methods are having significant difference with other methods so
every method is different from other. MAE was found to be better
than MAEC so we can say intermittent cooling is helpful in
antioxidant extraction for this plant material. MAE and UAE are
superior then Soxhlet and MAEC. Overexposure of heat causes
degradation of antioxidants and lower antioxidant extraction.
UAE is superior for both the solvents and Methanol is better than
ethanol for extraction of antioxidants from S. cumini seeds. Heat
exposure is not suitable for antioxidant extraction that may be the
reason of inferiority of Soxhlet with both solvents for this plant
material.
89


Table 86. Antioxidant content of P. sylvestris methanol extract prepared by
different methods
Method
Soxhlet 2111.30±75.01
ERT 2883.16±401.99
MAE 2933.28±0.00 1644.29
MAEC 3181.09±311.79
UAE 3650.99±759.31
Table 87. Comparison of phenol content in P. sylvestris methanol extract


ERT-Soxhlet 771.86 No*
MAE-ERT 50.12 No
MAEC-ERT 297.93 No
UAE-ERT 797.83 No
MAEC-MAE 247.81 No
UAE-MAE 757.71 No
UAE-MAEC 469.90 No
*
p value <0.05
Null hypothesis was not rejected. All methods seem same for
antioxidant extraction. The t-test was carried out for assessing the
significance of all methods, all methods were found to have
significant value > 0.05, but only ERT and Soxhlet were found to
90

have p value < 0.05 so we can say these methods differs from each
other and ERT is better than Soxhlet.
Table 88. Antioxidant content of P. sylvestiris ethanol extract prepared by

different methods
Method
Soxhlet 1290.24±86.88
ERT 1736.22±155.40
MAE 615.37±0.00 734.35
MAEC 3221.65±255.95
UAE 2967.11±265.57
Table 89. Comparison of phenol content in P. sylvestris ethanol extract


ERT-MAE 1120.85 Yes
UAE-ERT 1230.89 Yes
UAE-MAE 2351.74 Yes
MAEC-UAE 254.54 No
Null hypothesis was rejected. MAEC is superior and MAE is inferior.

Intermittent cooling cycles are not favorable for extraction. UAE is an
alternative to MAEC, MAE and ERT was found to be alternative of
91

Soxhlet for fast and better extraction of antioxidants. ERT and UAE
provide average extraction.
Methanol is better solvent then ethanol for antioxidants extraction
from P. sylvestris seeds. Both UAE and MAEC provide good
extraction for this plant material. MAEC was superior than MAE with
both the solvents.
3.5.5 M. zapota:
Table 90. Antioxidant content of M. zapota water extract prepared by
different methods
Method
Soxhlet 1802.55±0.00
ERT 493.91±17.17
MAE 980.69±10.91 1223.57
MAEC 280.63±3.85
UAE 2411.56±682.09
Table 91. Comparison of phenol content in M. zapota water extract prepared


MAE-ERT 486.78 No
ERT-MAEC 213.28 No
UAE-ERT 1917.65 Yes
MAE-MAEC 700.06 No
UAE-MAE 1430.87 Yes

UAE-MAEC 2130.93 Yes
92

Null hypothesis was rejected. UAE is superior and MAEC is inferior.
Soxhlet also provides good extraction so we can comment that heat
degradation of antioxidants is not occurring here. Intermittent cooling
KHOSV WR SURYLGH JRRG H[WUDFWLRQ E\ 0$( WKDW¶V ZK\ 0$( LV
providing better extraction than MAEC. UAE and Soxhlet can be
used as alternative of each other. MAE and MAEC can be used as
alternative methods at the place of ERT.

Table 92. Antioxidant content of M. zapota methanol extract prepared by
different methods
Method
Soxhlet 1181.94±1143.68
ERT 677.31±82.96
MAE 1209.52±40.40 2699.56
MAEC 976.18±303.04
UAE 1206.88±926.54
Table 93. Comparison of phenol content in M. zapota methanol extract

Methods being Difference Whether Significant
compared (mM GAE/g of dry extract)
MAE-ERT 553.21 No
MAEC-ERT 298.87 No*
UAE-ERT 529.57 No
MAE-MAEC 233.34 No
MAE-UAE 2.64 No
MAEC-UAE 230.70 No
*
p value <0.05
93

Null hypothesis was not rejected so all the methods can be said as
same. The t-test was carried out for significance testing and for the
whole data set p values were found > 0.05, but for MAEC and ERT p
values were < 0.05 so we can say MAEC was an alternative for faster
extraction of antioxidants.

Table 94. Antioxidant content of M. zapota ethanol extract prepared by different
methods
Total antioxidant Significant difference

Method
Soxhlet 7345.75±4222.41
ERT 571.54±120.15
MAE 751.20±151.76 7583.65
MAEC 990.92±19.19
UAE 789.4±150.40
Table 95. Comparison of phenol content in M. zapota ethanol extract prepared


compared (mM GAE/g of dry extract)
MAE-ERT 179.66 No
MAEC-ERT 419.38 No*
UAE-ERT 217.86 No*
MAEC-MAE 239.72 No*
UAE-MAE 38.20 No
MAEC-UAE 201.52 No
*
p value <0.05
94

Null hypothesis was not rejected so all the methods can be said as
same. But based on results of t-test Soxhlet can be used as the method
of choice and t-test has given p values > 0.05 but for MAEC-ERT,
UAE-ERT and MAEC-MAE the p values were < 0.05 so we can say
MAEC differs from MAE and ERT, and UAE differs from ERT for
antioxidant extraction. MAEC was better than MAE, so we can say
continuous heating was beneficial for antioxidant extraction from M.
Zapota seeds.
Ethanol was better than water and methanol, and Soxhlet was
providing average extraction of antioxidants from M. zapota seeds.
With all following solvents ERT was giving lower extraction of
antioxidant from this plant material.
3.6 ANTIBACTERIAL ACTIVITY
3.6.A S. epidermidis:
3.6.A.1 T. indica:
3.6.A.1.1 Water extract of T. indica:
Table 96. Antibacterial activity of T. indica water extract prepared by

different methods
Antibacterial activity Significant

Method
(% inhibition at 100 ȝg/ml) difference
Soxhlet 23.33±2.35
ERT 34.89±19.72
MAE 32.61±1.02 37.43
MAEC 38.39±1.26
UAE 33.3±6.20
(Each mean ± SD is derived from two independent experiment.)
95

Table 97. Comparison of antibacterial activity in T. indica water extract
compared (%) Significant
MAE-ERT 22.19 Yes
MAEC-ERT 4.35 No
ERT-UAE 13.64 No
MAE-MAEC 17.84 Yes
MAE-UAE 35.83 Yes
MAEC-UAE 17.99 Yes
*
p value <0.05
Null hypothesis was not rejected. So t-test was carried out for whole
data set and it was found that the p value was > 0.05, but for particular
methods MAE-Soxhlet, MAEC-Soxhlet, UAE-Soxhlet and MAEC-
MAE, p values were < 0.05, so we can say Soxhlet is different from
MAE, MAEC and UAE. MAEC and MAE were also having difference
so we can say continuous heating is helpful during microwave treatment
for better extraction of antioxidant compounds from T. indica seeds.
3.6.A.1.2 Methanol extract of T. indica:

Table 98. Antioxidant content of T. indica methanol extract prepared by
different methods
Method
Soxhlet 9.85±1.06
ERT 39.4±4.28
MAE 61.59±1.02 17.53
MAEC 43.75±1.26
UAE 25.76±8.57
96

Table 99. Comparison of antibacterial activity in T. indica methanol extract
MAE-Soxhlet 9.28 No*
MAEC-Soxhlet 15.06 No*
UAE-Soxhlet 9.97 No*
ERT-MAE 2.28 No
MAEC-ERT 3.50 No
ERT-UAE 1.59 No
MAEC-MAE 5.78 No*
UAE-MAE 0.69 No
MAEC-UAE 5.09 No
Null hypothesis was rejected. MAE is superior and Soxhlet is inferior

PHWKRG0$(GRHVQ¶WKDYHDQ\DOWHUQDWLYHPHWKRGIRUIDVWH[WUaction.
Intermittent cooling can be said useful for extraction of antibacterial
components by using microwaves because MAE is better than
MAEC. Soxhlet is inferior it means direct heat exposure is
responsible for degradation of compounds which might be responsible
for antibacterial activity. UAE and MAEC are alternative of ERT, so
MAEC and UAE can serve as a faster alternative to ERT for
extraction of antistaphylococcal compounds from T. indica seeds.
MAE is better than MAEC for extraction of antibacterial compounds
so we can say continuous heating may have caused degradation of
antibacterial compounds. Extract prepared by MAE is showing
antibacterial activity against test organism. It has IC50 Value below
100 µg/ml of the extract.
Methanol is better solvent than water and microwave application is

useful for extracting out antibacterial components from T. indica
97

seeds. Soxhlet is inferior for extracting antistaphylococcal compounds
from T. indica seeds with both the solvents.
3.6.A.2 A. squamosa:
3.6.A.2.1 Water extract of A. squamosa:
Table 100. Antibacterial activity of A. squamosa water extract prepared by
different methods
Method
Soxhlet 0.00±0.00
ERT 48.21±2.52
MAE 75.23±3.53 11.13
MAEC 12.76±1.68
UAE 55.08±4.10
Table 101. Comparison of antibacterial activity in A. squamosa water extract

compared (%)
MAE-ERT 27.02 Yes
ERT-MAEC 35.45 Yes
UAE-ERT 6.87 No
MAE-MAEC 62.47 Yes
MAE-UAE 20.15 Yes
UAE-MAEC 42.32 Yes
Null hypothesis was rejected for this extract. MAE is superior and
Soxhlet is inferior method. There is no alternative method of MAE.
Extract prepared by soxhlet is not giving any antibacterial activity it
means direct heat exposure is responsible for degradation of
98

compounds which might be responsible for antibacterial activity.
UAE can be used as faster alternative for ERT. Extracts prepared by
Soxhlet and MAEC both are giving lowest antibacterial activity so we
can say heat application is not suitable foUH[WUDFWLRQRIDQWLEDFWHULDO
FRPSRXQGV([WUDFWSUHSDUHGE\0$(LVVKRZLQJࡱ 0,&, prepared by
UAE is showing IC50DQGSUHSDUHGE\(57LVVKRZLQJࡱ ,&50 against
test organism.
3.6.A.2.2 Methanol extract of A. squamosa:

Table 102. Antibacterial activity of A. squamosa methanol extract prepared
by different method
Method
Soxhlet 48.49±0.00
ERT 43.18±16.07
MAE 44.70±20.35 48.22
MAEC 38.38±4.93
UAE 44.20±5.12
Table 103. Comparison of antibacterial activity in A. squamosa methanol

Soxhlet-ERT 5.31 No
Soxhlet-MAE 3.79 No
Soxhlet-UAE 4.29 No
MAE-ERT 1.52 No
ERT-MAEC 4.80 No
UAE-ERT 1.02 No
MAE-MAEC 6.32 No
MAE-UAE 0.50 No
UAE-MAEC 5.32 No
99

Null hypothesis was not rejected. The t-test was carried out for the
whole data set and obtained p values were > 0.05, so on the basis of
Q-test and t-test we can say all methods used here were similar for the
extraction of antibacterial compounds from A. squamosa seeds. We
can prefer MAE and UAE for particular extraction because both of
these can provide fast extraction. Extract prepared by Soxhlet, ERT,
UAE and MAE alODUHVKRZLQJࡱ ,&50 against test organism. Water is
better than methanol for extraction of antibacterial compounds. MAE
and UAE were found to be superior then other methods for both the
solvents. MAE is superior to MAE with both solvents.
3.6.A.3 S. cumini:
3.6.A.3.1 Methanol extract of S. cumini:
Table 104. Antibacterial activity of S. cumini methanol extract prepared by
different methods
Antibacterial activity
Method
(% inhibition at 100 ȝg/ml) Significant difference
Soxhlet 33.33±4.71
MAE 69.17±3.53 13.54
MAEC 60.00±2.35
UAE 70.83±1.18
Table 105. Comparison of antibacterial activity in S. cumini methanol

compared (%)
MAE-MAEC 9.17 No*
UAE-MAE 1.66 No*
UAE-MAEC 10.83 No*
*
p value <0.05
100

Null hypothesis was rejected for this extract. UAE was superior and
Soxhlet was inferior method. On the basis of results obtained by Q-
test MAE and MAEC can serve as an alternative for UAE, in another
case MAE was similar to MAEC. But the t-test was performed for
whole data set and it was showing p value < 0.05 so we have to reject
and the results which were obtained by Q-test. The result of t-test
indicates that all the methods use for extraction of this seed have
significant difference. So according to results of t-test MAE is better
than MAEC so we can say intermittent cooling has positive effect on
extraction of antibacterial compounds so use of MAE will be
preferred then MAE.
Extract prepared by soxhlet is showing lowest antibacterial activity it

UAE, MAE and MAEC are showing inhibition in between range of

IC50 and MIC at 100 µg/ml concentration so they seem to be potent
against test organism. Extract prepared by MAE again showing
average inhibition.
3.6.A.3.2 Ethanol extract of S. cumini:
Table 106. Antibacterial activity of S. cumini ethanol extract prepared by

different methods

Method
Soxhlet 63.33±4.71
MAE 60.83±1.18 20.07
MAEC 44.17±8.24
UAE 53.33±2.35
101

Table 107. Comparison of antibacterial activity in S. cumini ethanol extract
Soxhlet-MAE 2.50 No
MAE-MAEC 16.66 No
MAE-UAE 7.50 No
UAE-MAEC 9.16 No
Null hypothesis was not rejected so all methods are equivalent.

Results of t-test was showing the p value > 0.05 so we can say all the
methods are similar for extraction of antibacterial compounds from
particular plant material. Extract prepared by Soxhlet and MAE are
showing antibacterial activity higher then IC50, and prepared by UAE
and are showing IC50DQGࡱ ,&50 respectively. By above results using
MAE will be better.
Methanol is superior then ethanol and MAE proved better with both
the solvents for extraction of antibacterial compounds from S. cumini
seeds. Extract prepared with all methods and solvents by S. cumini
seeds was showing IC50 or ~IC50.
3.6.A.4 P. sylvesteris:
3.6.A.4.1 Methanol extract of P. sylvestris:
Table 108. Antibacterial activity of P. sylvestris methanol extract prepared
Method
Soxhlet 6.98±0.00
ERT 31.4±8.21
MAE 53.49±6.57 22.64
MAEC 43.03±4.93
UAE 43.03±4.93
102

Table 109. Comparison of antibacterial activity in P. sylvestris methanol

MAE-ERT 22.09 No
MAEC-ER 11.63 No
UAE-ERT 11.63 No
MAE-MAEC 10.43 No
MAE-UAE 10.43 No
MAEC-UAE 0.00 No
Null hypothesis was rejected for this extract.
MAE is superior and Soxhlet is inferior method. ERT, UAE and

MAEC can serve as an alternative for MAE. MAEC, UAE and MAE
can serves as faster alternative for ERT.
Extract prepared by soxhlet was not showing good antibacterial

activity it means direct heat exposure is responsible for degradation of
MAEC and UAE are similar in extraction of compounds which are

responsible for antibacterial activity.
103

3.6.A.4.2 Ethanol extract of P. sylvestris:
Table 110. Antibacterial activity of P. sylvestris ethanol extract prepared by

different methods
Antibacterial activity Significant difference
Method
(% inhibition at 100 ȝg/ml)
Soxhlet 56.98±1.64
ERT 62.79±6.58
MAE 40.70±1.64 15.32
MAEC 41.87±0.00
UAE 29.07±4.92
Table 111. Comparison of antibacterial activity in P. sylvestris ethanol

compared (%)
ERT-Soxhlet 5.81 No
ERT-MAE 22.09 Yes
ERT-MAEC 20.92 Yes
ERT-UAE 33.72 Yes
MAEC-MAE 1.17 No
MAE-UAE 11.63 No
MAEC-UAE 12.80 No
Null hypothesis was rejected for this extract. ERT was superior and
UAE was inferior. Both MAE and MAEC are also similar for the
extraction of antibacterial compounds. MAEC and ERT can be used
as an alternative for Soxhlet. Extract prepared by Soxhlet and ERT
are showing antibacterial activity over IC50. Ethanol is better than
methanol and MAE is fast method and average for extracting
antibacterial compounds from P. sylvestris seeds.
104

3.6.A.5 M. zapota:
3.6.A.5.1 Water extract of M. zapota:
Table 112. Antibacterial activity of M. zapota water extract prepared by

different methods

Method
Soxhlet 19.57 ± 5.11
ERT 50.76 ± 13.93
MAE 34.79 ± 2.05 27.08
MAEC 36.05 ± 1.64
UAE 48.55 ± 1.02
Table 113. Comparison of antibacterial activity in M. zapota water extract

Methods being compared Difference Whether Significant

(%)
ERT-MAE 15.97 No
ERT-MAEC 14.71 No
ERT-UAE 2.21 No
MAEC-MAE 1.26 No
UAE-MAE 13.76 No
UAE-MAEC 12.50 No
Null hypothesis was rejected. ERT is superior and Soxhlet is inferior

method. MAE, MAEC and UAE can be used as an alternative of
ERT. Extract prepared by soxhlet is showing lowest antibacterial
activity it means direct heat exposure is responsible for degradation of
105

,QWHUPLWWHQW FRROLQJ GRHVQ¶W FDXVH VR PXFK FKDQJH LQ DQWLEDFWHULDO
activity during MAE operation. Extract prepared by ERT is showing
IC50DQGSUHSDUHGE\8$(VKRZVࡱ,&50.
3.6.A.5.2 Methanol extract of M. zapota:
Table 114. Antibacterial activity of M. zapota methanol extract prepared by

different methods

Method
Soxhlet 32.5 ± 3.53
ERT 34.09 ± 1.06
MAE 34.85 ± 8.57 20.79
MAEC 30.24 ± 6.57
UAE 43.48 ± 2.05
Table 115. . Comparison of antibacterial activity in M. zapota methanol

ERT-Soxhlet 1.59 No
MAE-Soxhlet 2.35 No
UAE-Soxhlet 10.98 No*
MAE-ERT 0.76 No
ERT-MAEC 3.83 No
UAE-ERT 9.39 No
MAE-MAEC 4.61 No
UAE-MAE 8.39 No
UAE-MAEC 13.24 No*
*p value <0.05
106

Null hypothesis was not rejected. So t-test was performed for
significance testing. UAE-Soxhlet, UAE-ERT and UAE-MAEC were
having significant difference < 0.05, so we can say UAE will be
superior over all the methods and MAEC will be inferior. UAE can be
used as an alternative of ERT and Soxhlet for fast extraction. Not any
extract was able to inhibit the test organism near the IC50.
3.6.A.5.3 Ethanol extract of M. Zapota:
Table 116. Antibacterial activity of M. zapota ethanol extract prepared by

different methods

Method
Soxhlet 40.58±2.04
ERT 34.09±5.35
MAE 48.48±2.14 13.19
MAEC 0.00±0.00
UAE 39.13±4.09
Table 117. Comparison of antibacterial activity in M. zapota ethanol extract

Soxhlet-ERT 6.49 No
MAE-Soxhlet 7.90 No
Soxhlet-UAE 1.45 No
MAE-ERT 14.39 Yes
ERT-MAEC 34.09 Yes
UAE-ERT 5.04 No
MAE-MAEC 48.48 Yes
MAE-UAE 9.35 No
UAE-MAEC 39.13 Yes
107

Null hypothesis was rejected for this extract. MAE is superior
and MAEC is inferior. So we can say intermittent cooling have
significant role during microwave treatment for extracting
antibacterial compounds.
UAE and MAE can be used as an alternative of Soxhlet for fast

extraction. UAE will be similar to MAE and ERT. Extract
prepared by MAE shows IC50 against test organism.
Water is better solvent than methanol and ethanol for

antibacterial compounds extraction from M. zapota seeds and
MAE is an average method for all the solvents we have used for
extraction. Particular one method for all solvents may not be
best. MAE is better than MAEC for this seed.
3.6.B P. oleovorans:
3.6.B.1 T. indica:
3.6.B.1.1 Water extract of T. indica:

Table 118. Antibacterial activity of T. indica water extract

Method
Soxhlet 0.00±0.00
ERT 33.34±0.00
MAE 30.77±4.34 8.78
MAEC 27.48±0.63
UAE 10.77±2.17
108

Table 119. Comparison of antibacterial activity in T. indica water extract

(%)
ERT-MAE 2.57 No
ERT-MAEC 5.86 No
ERT-UAE 22.57 Yes
MAE-MAEC 3.29 No
MAE-UAE 20.0 Yes
MAEC-UAE 13.71 Yes
Null hypothesis was rejected. ERT was superior and Soxhlet was
inferior method. MAE and MAEC can be used at the place of ERT for
fast extraction when we have time limitation.
Extract prepared by soxhlet is showing no antibacterial activity so we

can say that heat application for long time is not suitable if we want
antibacterial action in extract.
MAE and MAEC both are similar so intermittent cooling effect can
be ignored so we can say MAEC use will be more suitable because it
requires low energy for short time.
Extract prepared by above methods was unable to show any

significant inhibition in IC50 range.
109

3.6.B.1.2 Methanol extract of T. indica:
Table 120. Antibacterial activity of T. indica methanol extract prepared by
different methods
Method
Soxhlet 0.00±0.00
ERT 27.15±0.00
MAE 27.11±0.83 5.64
MAEC 31.54±0.00
UAE 36.43±3.03
Table 121. Comparison of antibacterial activity in T. indica methanol extract


ERT-MAE 0.04 No
MAEC-ERT 4.39 No
UAE-ERT 9.28 Yes
MAEC-MAE 4.43 No
UAE-MAE 9.32 Yes
UAE-MAEC 4.89 No
Null hypothesis was rejected. UAE is superior and Soxhlet is inferior

method. MAEC can be used as an alternative of UAE. MAE and
MAEC can be used as an alternative of ERT because of time
limitation. Extract prepared by soxhlet is showing no antibacterial
activity so we can say that heat application for long time is not
suitable if we want antibacterial action in extract. Methanol proved to
be superior solvent than water.
110

Use of Soxhlet is not suitable for T. indica seed extraction preparation
if antibacterial activity is required. MAE was average method with
both solvents for extraction of antibacterial compounds.
3.6.B.2 A. squamosa:
3.6.B.2.1 Water extract of A. squamosa:

Table 122. Antibacterial activity of A. squamosa water extract prepared by
different methods
Method
Soxhlet 19.82±1.27
ERT 33.57±11.10
MAE 29.29±3.02 24.96
MAEC 18.02±1.27
UAE 22.31±7.61
Table 123. Comparison of antibacterial activity in A. squamosa water extract


compared (%)
ERT- Soxhlet 13.75 No*
MAE- Soxhlet 9.47 No*
UAE- Soxhlet 2.49 No
ERT-MAE 4.28 No
ERT-MAEC 15.55 No
ERT-UAE 11.26 No
MAE-MAEC 11.27 No
MAE-UAE 6.98 No
UAE- MAEC 4.29 No
*
p value<0.05
Null hypothesis was rejected for this extract, so on the basis of Q-test
all the methods are equal for extracting antipseudomonas compounds
111

from A. squamosa seeds. For further checking t-test was performed
for the whole data set and according to t-test ERT-Soxhlet and MAE-
Soxhlet were showing p value <0.05, so they are different for the
extraction purpose. ERT was found to be better then both MAE and
Soxhlet, heat application may be the reason of inferiority of MAE and
Soxhlet.
3.6.B.2.2 Methanol extract of A. squamosa:
Table 124. Antibacterial activity of A. squamosa methanol extract prepared

Method
Soxhlet 0.00±0.00
ERT 30.72±3.02
MAE 37.86±3.02 9.46
MAEC 24.78±1.90
UAE 24.80±2.43
Table 125. Comparison of antibacterial activity in A. squamosa methanol


(%) Significant
MAE-ERT 7.14 No
ERT-MAEC 5.94 No
ERT-UAE 5.92 No
MAE-MAEC 13.08 Yes
MAE-UAE 13.06 Yes
UAE-MAEC 0.02 No
112

Null hypothesis was rejected for this extract. MAE is superior and
Soxhlet is inferior method. MAE, UAE and MAEC can serve as an
alternative for ERT.
Extract prepared by soxhlet is not showing any antibacterial activity it

Extracts prepared by Soxhlet and MAEC both are giving lowest
antibacterial activity so we can say heat application is not suitable.
MAE was found to be better than MAEC so we can say intermittent

cooling is useful for extraction of antibacterial compounds from this
plant material.
Water is better solvent then methanol for preparation of the extract by

P. sylvestris seeds. Direct heat exposure or use of Soxhlet is not
suitable. ERT was found to be better with both the solvents for
extraction of antibacterial compounds.
3.6.B.3 S. cumini:
3.6.B.3.1 Methanol extract of S. cumini:
Table 126. Antibacterial activity of S. cumini methanol extract prepared by

different methods

Method
Soxhlet 0.00 ± 0.00
MAE 0.00 ± 0.00
MAEC 0.00 ± 0.00 0.00
UAE 0.00 ± 0.00
113

Table 127. Comparison of antibacterial activity in S. cumini methanol

(%)
Soxhlet-MAE 0.00 No
Soxhlet-UAE 0.00 No
MAE-MAEC 0.00 No
MAE-UAE 0.00 No
MAEC-UAE 0.00 No
P. oleovorans was not inhibited at all by methanolic extract of S.

cumini prepared by any method, thus methanol failed to extract any
antibacterial compound from this plant material. This is in striking
contrast with the notable antistaphyloccal activity exerted by the same
extract prepared through different methods (Table 104).This extract
has earlier been reported to be bacteriostatic against gram-negative
bacteria including P. oleovorans (Kothari, 2011), albeit at
concentrations higher than used here.
3.6.B.3.2 Ethanol extract of S. cumini:
Table 128. Antibacterial activity of S. cumini ethanol extract prepared by

different methods

Method
Soxhlet 0.00 ± 0.00
MAE 5.38 ± 7.61 15.66
MAEC 0.00 ± 0.00
UAE 0.77 ± 1.08
114

Table 129. Comparison of antibacterial activity in S. cumini ethanol extract
MAE-Soxhlet 5.38 No
UAE-Soxhlet 0.77 No
MAE-MAEC 5.38 No
MAE-UAE 4.61 No
UAE-MAEC 0.77 No
Null hypothesis was not rejected. For this whole data set t-test was
also performed but significant difference was not found because the p
value was > 0.05. MAE seems superior and Soxhlet and MAEC are
inferior. So we can say intermittent cooling during microwave
application increases the extraction of antibacterial compounds.
Extract prepared by soxhlet is showing no antibacterial activity so we
can say that heat application for long time is not suitable if we want
antibacterial action in extract. Methanol seems superior then water. Use
of Soxhlet is not suitable for S. cumini seed extraction preparation if
antibacterial activity is required.
3.6.B.4 P. sylvesteris:
3.6.B.4.1 Methanol extract of P. sylvestris:
Table 130. Antibacterial activity of P. sylvestris methanol extract prepared

Method
Soxhlet 33.34 ± 1.27
ERT 27.03 ± 1.27
MAE 25.23 ± 1.27 6.94
MAEC 26.58 ± 1.90
UAE 17.12 ± 2.54
115

Table 131. Comparison of antibacterial activity in P. sylvestris methanol

Soxhlet-ERT 6.31 No
ERT-MAE 1.80 No
ERT-MAEC 0.45 No
ERT-UAE 9.91 Yes
MAEC-MAE 1.35 No
MAE-UAE 8.11 Yes
MAEC-UAE 9.46 Yes
Null hypothesis was rejected. Soxhlet was superior and UAE was
inferior.
MAEC is better than MAE. Soxhlet and MAEC are superior then all
other methods so for this case we can say heat application for
preparation of extract is increasing the extraction of antibacterial
compounds.
Continuous heating can be a reason of superiority of MAEC over

MAE. MAE and MAEC can be used as an alternative of ERT for fast
extraction.
ERT and UAE can be used as an alternative for Soxhlet but use of
UAE will be better because it is a time saving operation. Use of MAE
can be preferred over ERT.
116

3.6.B.4.2 Ethanol extract of P. sylvestris:
Table 132. Antibacterial activity of P. sylvestris ethanol extract prepared by
different methods

Method
Soxhlet 25.23 ± 10.19
ERT 19.37 ± 0.63
MAE 25.23 ± 5.09 29.31
MAEC 18.47 ± 10.82
UAE 13.97 ± 4.45
Table 133. Comparison of antibacterial activity in P. sylvestris ethanol


(%) Significant
Soxhlet-ERT 5.86 No
Soxhlet-MAE 0.00 No
ERT-MAE 5.86 No*
ERT-MAEC 0.90 No
ERT-UAE 5.40 No
MAE-MAEC 6.76 No
MAE-UAE 11.26 No
MAEC-UAE 4.5 No
*
p value<0.05
Null hypothesis was rejected for this extract, so on the basis of Q-test
all the methods are equal for extracting antipseudomonas compounds
from P. sylvestris seeds when ethanol was used as a solvent. MAE
was found to be better than all methods we have used.
117

Both Soxhlet and MAE are better than other methods for extraction of
antibacterial compound from P. sylvestris. UAE was inferior with
both the solvents. Methanol was better than ethanol for extraction of
antipseudomonas compounds from P. sylvestris seeds. Both MAE and
MAEC were also found to be similar with both the solvents.
3.6.B.3 M. zapota:
3.6.B.5.1 Water:
Table 134. . Antibacterial activity of M. zapota water extract prepared by

different methods
Method
Soxhlet 33.85±2.17
ERT 54.27±2.04
MAE 44.29±14.14 25.98
MAEC 25.23±0.00
UAE 36.16±1.08
Table 135. Comparison of antibacterial activity in M. zapota water extract

ERT- Soxhlet 20.42 No
MAE- Soxhlet 10.44 No
ERT-MAE 9.98 No
ERT-MAEC 29.00 Yes
ERT-UAE 18.11 No
MAE-MAEC 19.06 No
MAE-UAE 8.13 No
UAE-MAEC 10.83 No
118

Null hypothesis was rejected. ERT is superior and MAEC is inferior. UAE,
MAE and Soxhlet can be used as an alternative of ERT if time limitation is
there MAE will be better option for extract preparation. ERT, MAE and
UAE are better than Soxhlet and MAEC. MAEC and Soxhlet are inferior to
all the methods so we can say heat application is not suitable for extraction
of this extract if we want antibacterial activity. Extract prepared by ERT is
giving IC50. ExtUDFW SUHSDUHG E\ 0$( LV VKRZLQJࡱ ,&50 against P.
oleovorans. Intermittent cooling helps in increase to extract the
components which are responsible for antibacterial activity during
3.6.B.5.2 Methanol extract of M. zapota:
Table 136. Antibacterial activity of M. zapota methanol extract prepared by
different methods
Antibacterial activity Significant difference
Method
(% inhibition at 100 ȝg/ml)
Soxhlet 3.08±4.35
ERT 22.86±0.00
MAE 0.00±0.00 16.46
MAEC 24.33±0.00
UAE 35.71±8.08
Table 137. Comparison of antibacterial activity in M. zapota methanol

(%)
Soxhlet-MAE 3.08 No
MAEC- Soxhlet 21.25 Yes
UAE- Soxhlet 32.63 Yes
ERT-MAE 22.86 Yes
MAEC-ERT 1.47 No
UAE-ERT 12.83 No
MAEC-MAE 24.33 Yes
UAE-MAE 35.71 Yes
UAE-MAEC 11.38 No
119

Null hypothesis was rejected. UAE is superior and MAE is inferior
method for extraction of compounds which are responsible for
antibacterial activity. UAE and MAEC can be used as an alternative
for ERT and alternative for each other too, so using UAE will be
more beneficial because it gives better extraction of antibacterial
compounds than MAEC. Soxhlet is again proved as inferior method
as compared to UAE CE and MAEC so we can conclude that heat
application causes degradation of antibacterial compounds.
3.6.B.5.3 Ethanol extract of M. zapota:

Table 138. Antibacterial activity of M. zapota ethanol extract prepared by
different methods
Method
Soxhlet 39.23±3.25
ERT 47.15±2.02
MAE 45.00±3.03 25.97
MAEC 10.63±7.89
UAE 57.86±11.11
Table 139. Comparison of antibacterial activity in M. zapota ethanol extract


ERT- Soxhlet 7.92 No
MAE- Soxhlet 5.77 No
ERT-MAE 2.15 No
ERT-MAEC 36.52 Yes
UAE-ERT 10.71 No
MAE-MAEC 34.37 Yes
UAE-MAE 12.86 No
UAE-MAEC 47.23 Yes
120

Null hypothesis was rejected. UAE is superior and MAEC is inferior
method for extraction of compounds which are responsible for
antibacterial activity. Soxhlet and MAEC are inferior than other
methods so heat application during extraction can be said as harmful
for antibacterial activity. MAE can be used as an alternative of ERT
and Soxhlet.
Intermittent cooling proved to be good during microwave application

that is why MAE was providing higher antibacterial compound
extraction than MAEC. Extract prepared by UAE is giving IC50
([WUDFW SUHSDUHG E\ 0$( DQG (57 DUH VKRZLQJࡱ,&50 against P.
oleovorans.
Ethanol is superior solvent than water and methanol. UAE is superior

method then all other methods we have used for extraction of M.
zapota seed extract if anyone wants to work with P. oleovorans.
Water and ethanol extracts shows IC50 against test organism.
121

4. FINAL
COMMENTS
122

A summary of all the assays carried out with all the extracts prepared by all
the methods is presented through table 140-144. Similarly statistical
significance among results of different methods is indicated through table
(145-149)#. Based on differences or similarities in extracts prepared by
different methods, scoresheet was prepared, one (Table 150) indicating
suitability of each method for a particular purpose (e.g., phenol extraction,
antibacterial activity, etc.); and another (Table151) indicating suitability of
each solvent used for a particular purpose. Each method was given a score
of 1, every time it registered maximum value for a particular parameter.
Scores for various methods ranged from 0-11, 11 being the maximum
possible score. On the same line a scoresheet for comparison of only two
microwave based methods (MAE and MAEC) was prepared (Table 152).
Following generalization can be drawn from the results of present study:
x ERT, MAE, MAEC and UAE were not as good as Soxhlet with respect
to extraction efficiency but these methods were better for providing
other activities.
x Extracts prepared by ERT, MAE and UAE were showing better
antibacterial activity against both tested organisms and were also better
than MAEC and Soxhlet. Extracts prepared by ERT, MAE and UAE
were showing equally good antibacterial activity against P. oleovorans,
thus direct heat application may be a reason for degradation of
compounds responsible for antipseudomonas activity.
x MAE was unable to provide good extraction efficiency but extract
prepared by MAE and UAE were showing better flavonoid extraction.
x The extracts prepared in methanol were showing flavonoids, phenols
and antioxidant activity better than those in water and ethanol, so we
can say methanol should be preferred for extraction of antioxidant
metabolites from plant seeds.
#
In table (145-µ\HV¶RUµQR¶LQGLFDWHVZKHWKHUVWDWLVWLFDOVLJQLILFDQFHLVHxistent between two
methods in question.
123

Table 140. Values of all the parameters for T. indica seed extract
#
Antioxidant Antibacterial activity
Extraction Total flavonoid Total phenol
Seed and capacity (% inhibition at 100 ȝg/ml)
Method efficiency (mg/ml QE/g of (mM GAE/g of
solvent (mM GAE/g of dry
(%) dry extract) dry extract) S. epidermidis P. oleovorans
extract)
Soxhlet 40.74±4.13 13.85±2.92 1139.39±147.26 1597.22±128.81 23.33±2.35 0.00±0.00
ERT 14.66±0.60 Not detectable 999.99±188.56 433.32±47.142 34.89±19.72 33.34±0.00
T. indica
MAE 17.76±0.80 2.49±0.00 1219.1±139.79 476.43±162.63 32.61±1.02 30.77±4.34
[water]
MAEC 12.46±1.20 Not done Not done Not done 38.39±1.26 27.48±0.63
UAE 7.13±0.40 4.72±0.00 498.82±8.30 249.99±29.11 33.3±6.20 10.77±2.17
Soxhlet 6.95±0.05 13.85±2.92 10037.64±567.81 9006.27±1494.26 9.85±1.06 0.00±0.00
ERT 5.20±0.43 36.86±14.99 1429.33±2.53 3108.10±1974.80 39.4±4.28 27.15±0.00
T. indica MAE 3.60±0.17 16.52±3.69 6327.16±122.57 3260.82±0.00 61.59±1.02 27.11±0.83
[methanol] 16328.24±1673.3
MAEC 3.66±0.25 5.02±0.37 2503.78±259.09 43.75±1.26 31.54±0.00
0
UAE 2.63±0.15 11.75±2.00 8581.04±391.11 2807.00±992.43 25.76±8.57 36.43±3.03
#
Ascorbic acid (2mM) registered a value of 30.80 mM GAE
124

Table 141. Values of all the parameters for A. squamosa seed extract
#
Extraction Total flavonoid Total phenol Antioxidant capacity Antibacterial activity
Seed and
Method efficiency (mg/ml QE/g of (mM GAE/g of (mM GAE/g of dry (% inhibition at 100 ȝg/ml)
solvent
(%) dry extract) dry extract) extract) S. epidermidis P. oleovorans
Soxhlet 12.25±0.91 Not detectable 102.08±8.83 804.15±253.37 0.00±0.00 19.82±1.27
ERT 6.26±0.49 2.73±0.22 2317.51±25.80 3666.65±261.89 48.21±2.52 33.57±11.10
A. squamosa
MAE 9.13±0.76 3.87±1.56 368.85±32.60 1123.06±152.29 75.23±3.53 29.29±3.02
(water)
MAEC 10.63±0.05 2.55±0.25 1225.67±192.59 305.18±82.65 12.76±1.68 18.02±1.27
UAE 12.33±0.92 13.10±0.97 1113.39±58.32 1134.00±87.47 55.08±4.10 22.31±7.61
Soxhlet 12.23±0.50 02.83±00.00 2529.52±129.30 1790.41±377.10 48.49±0.00 0.00±0.00
ERT 11.66±0.05 16.62±02.94 999.01±156.98 787.03±327.36 43.18±16.07 30.72±3.02
A. squamosa
MAE 7.56±0.40 45.84±05.53 2390.81±57.31 939.07±107.68 44.70±20.35 37.86±3.02
(methanol)
MAEC 8.16±0.70 43.04±03.76 2206.51±107.60 2832.72±1376.41 38.38±4.93 24.78±1.90
UAE 9.46±0.83 Not detectable 1162.1±71.46 2025.31±0.00 44.20±5.12 24.80±2.43
125

Table 142. Values of all the parameters for S. cumini seed extract
#
Seed and
solvent
Soxhlet 30.60±2.47 15.46 ± 1.98 1308.67±36.28 800.82±9.51 33.33±4.71 0.00 ± 0.00
S. cumini MAE 16.23±0.32 19.13 ± 3.01 4679.44±472.70 2104.01±185.96 69.17±3.53 0.00 ± 0.00
(methanol) MAEC 21.00±0.30 17.46 ± 0.85 4437.56±93.67 1771.86±0.00 60.00±2.35 0.00 ± 0.00
UAE 17.30±1.2 21.08 ± 6.88 4614.69±436.74 2552.78±116.45 70.83±1.18 0.00 ± 0.00
Soxhlet 40.96±1.29 Not detectable 1540.61±77.81 844.26±10.20 63.33±4.71 0.00 ± 0.00
S. cumini
MAE 29.33±1.41 23.54±3.69 3431.24±179.72 1541.56±106.05 60.83±1.18 5.38 ± 7.61
(ethanol)
MAEC 30.93±0.80 13.28 ± 1.70 3447.57±471.83 1283.39±0.00 44.17±8.24 0.00 ± 0.00
UAE 28.16±1.51 14.72 ± 3.28 3960.69±580.73 1685±0.00 53.33±2.35 0.77 ± 1.08
126

Table 143. Values of all the parameters for P. sylvestris seed extract
#
Seed and
solvent
Soxhlet 14.23±1.47 3.63 ± 1.02 3476.92±114.33 2111.30±75.01 6.98±0.00 33.34 ± 1.27
ERT 13.80±1.35 Not detectable 6338.05±218.61 2883.16±401.99 31.4±8.21 27.03 ± 1.27
P. sylvestris
MAE 11.50±0.95 1.26 ± 0.00 10929.14±2516.09 2933.28±0.00 53.49±6.57 25.23 ± 1.27
[methanol]
MAEC 11.50±1.80 10.10 ± 2.99 6938.93±375.86 3181.09±311.79 43.03±4.93 26.58 ± 1.90
UAE 10.75±0.82 Not detectable 12486.57±289.48 3650.99±759.31 43.03±4.93 17.12 ± 2.54
Soxhlet 12.25±0.95 Not detectable 3466.96±397.54 1290.24±86.88 56.98±1.64 25.23 ± 10.19
ERT 10.20±0.26 6.01 ± 0.45 4349.97±0.00 1736.22±155.40 62.79±6.58 19.37 ± 0.63
P. sylvestris
MAE 7.33±0.51 Not detectable 4173.07±281.03 615.37±0.00 40.70±1.64 25.23 ± 5.09
[ethanol]
MAEC 7.50±0.40 5.72 ± 0.21 20977.36±3903.48 3221.65±255.95 41.87±0.00 18.47 ± 10.82
UAE 9.30±1.90 4.49 ± 3.27 9995.30±3634.84 2967.11±265.57 29.07±4.92 13.97 ± 4.45
127

Table 144. Values of all the parameters for M. zapota seed extract
#
Seed and
Method efficiency (mg/ml QE/g (mM GAE/g of dry (mM GAE/g of dry (% inhibition at 100 ȝg/ml)
solvent
(%) of dry extract) extract) extract) S. epidermidis P. oleovorans
Soxhlet 11.61±1.07 Not detectable 754.71±97.02 1802.55±0.00 19.57 ± 5.11 33.85±2.17
ERT 6.30±0.2 6.03 ± 1.15 355.98±11.44 493.91±17.17 50.76 ± 13.93 54.27±2.04
M. zapota MAE 8.90±0.98 Not detectable 478.02±0.00 980.69±10.91 34.79 ± 2.05 44.29±14.14
[water] MAEC 9.73±0.15 1.26 ± 0.00 337.5±46.18 280.63±3.85 36.05 ± 1.64 25.23±0.00
UAE 9.96±0.92 3.15 ± 0.19 290.32±0.00 2411.56±682.09 48.55 ± 1.02 36.16±1.08
Soxhlet 11.95±0.35 19.32 ± 0.39 820.88±104.76 1181.94±1143.68 32.5 ± 3.53 3.08±4.35
ERT 10.93±0.55 6.07 ± 0.00 227.99±16.97 677.31±82.96 34.09 ± 1.06 22.86±0.00
M. zapota
MAE 7.16±0.66 2.71 ± 0.00 812.73±6.72 1209.52±40.40 34.85 ± 8.57 0.00±0.00
[methanol]
MAEC 7.40±0.10 4.28 ± 3.08 769.46±148.57 976.18±303.04 30.24 ± 6.57 24.33±0.00
UAE 5.96±0.32 Not detectable 432.09±67.89 1206.88±926.54 43.48 ± 2.05 35.71±8.08
Soxhlet 8.43±0.46 1.56±0.098 3975.38±374.79 7345.75±4222.41 40.58±2.04 39.23±3.25
ERT 7.20±0.43 7.58±0.00 96.52±5.456 571.54±120.15 34.09±5.35 47.15±2.02
M. zapota
MAE 6.50±0.95 2.17±0.74 618.9±20.67 751.20±151.76 48.48±2.14 45.00±3.03
[ethanol]
MAEC 7.26±0.25 9.30±0.28 2274.36±229.74 990.92±19.19 0.00±0.00 10.63±7.89
UAE 8.00±0.65 2.23±0.09 316.35±10.91 789.4±150.40 39.13±4.09 57.86±11.11
128

Table 145. Statistical significance between different methods for T. indica seed extracts
Water extract of T. indica seed Methanol extract of T. indica seed

Methods being Extraction Flavonoid Phenol Antioxidant Antibacterial activity Extraction Flavonoid Phenol Antioxidant Antibacterial activity
compared efficiency Content content activity S. epidermidis P. oleovorans efficiency content Content activity S. epidermidis P. oleovorans
Soxhlet-ERT Yes Yes No Yes No Yes Yes No Yes Yes Yes Yes
Soxhlet-MAE Yes Yes No Yes No* Yes Yes No Yes Yes Yes Yes
Soxhlet-MAEC Yes - - - No* Yes Yes No Yes Yes Yes Yes
Soxhlet-UAE Yes Yes Yes Yes No* Yes Yes No No Yes No Yes
ERT-MAE No Yes No No No No Yes No Yes No Yes No
ERT-MAEC No - - - No No Yes Yes Yes No No No
ERT-UAE Yes Yes Yes No No Yes Yes No Yes No No Yes
MAE-MAEC ~Yes - - - No* No No No Yes No Yes No
MAE-UAE Yes Yes Yes No No Yes Yes No Yes No Yes Yes
MAEC-UAE ~Yes - - - No Yes Yes No Yes No Yes No
Table 146. Statistical significance between different methods for A. squamosa seed extracts
Water extract of A. squamosa seed Methanol extract of A. squamosa seed
compared efficiency Content content activity S. epidermidis P. oleovorans efficiency content content activity S. epidermidis P. oleovorans
Soxhlet-ERT Yes No Yes Yes Yes No* No Yes Yes No No Yes
Soxhlet-MAE Yes Yes Yes No Yes No* Yes Yes No No No Yes
Soxhlet-MAEC No No Yes No Yes No Yes Yes Yes No No Yes
Soxhlet-UAE No Yes Yes No Yes No Yes No Yes No No Yes
ERT-MAE Yes No Yes Yes Yes No Yes Yes Yes No No No
ERT-MAEC Yes No Yes Yes Yes No Yes Yes Yes No No No
ERT-UAE Yes Yes Yes Yes No No Yes Yes No No No No
MAE-MAEC No No Yes No Yes No No No No No* No Yes
MAE-UAE Yes Yes Yes No Yes No Yes Yes Yes No No Yes
MAEC-UAE No Yes No Yes Yes No No Yes Yes No No No
129

Table 147. Statistical significance between different methods for S. cumini seed extracts
Methanol extract of S. cumini seed Ethanol extract of S. cumini seed
compared efficiency content Content activity S. epidermidis P. oleovorans efficiency content content activity S. epidermidis P. oleovorans
Soxhlet-ERT - - - - - - - - - - - -
Soxhlet-MAE Yes Yes Yes Yes Yes No Yes No Yes Yes No No
Soxhlet-MAEC Yes Yes Yes Yes Yes No Yes No Yes Yes No No
Soxhlet-UAE Yes Yes Yes Yes Yes No Yes No Yes Yes No No
ERT-MAE - - - - - - - - - - - -
ERT-MAEC - - - - - - - - - - - -
ERT-UAE - - - - - - - - - - - -
MAE-MAEC Yes No No No No* No No No No Yes No No
MAE-UAE No No No Yes No* No No No No Yes No No
MAEC-UAE Yes No No Yes No* No No No No Yes No No
Table 148. Statistical significance between different methods for P. sylvestris seed extracts
Methanol extract of P. sylvestris seed Ethanol extract of P. sylvestris seed

compared efficiency content Content activity S. epidermidis P. oleovorans efficiency content content activity S. epidermidis P. oleovorans
Soxhlet-ERT No No Yes No* Yes No No Yes No No No No
Soxhlet-MAE No No Yes No Yes Yes Yes No No No Yes No
Soxhlet-MAEC No Yes Yes No Yes No Yes No Yes Yes No No
Soxhlet-UAE Yes No Yes No Yes Yes Yes No Yes Yes Yes No
ERT-MAE No No Yes No No No Yes Yes No Yes Yes No*
ERT-MAEC No Yes No No No No Yes No Yes Yes Yes No
ERT-UAE No No Yes No No Yes No No No Yes Yes No
MAE-MAEC No Yes Yes No No No No No Yes Yes No No
MAE-UAE No No No No No Yes No No No Yes No No
130

Table 149. Statistical significance between different methods for M. zapota seed extracts
Water extract of M. Zapota Methanol extract of M. zapota
compared efficiency content content activity S. epidermidis P. oleovorans efficiency content content activity S. epidermidis P. oleovorans
Soxhlet-ERT Yes Yes Yes Yes Yes No No Yes Yes No No Yes
Soxhlet-MAE Yes No Yes No No No Yes Yes No No No No
Soxhlet-MAEC No No Yes Yes No No Yes Yes No No No Yes
Soxhlet-UAE No Yes Yes No Yes No Yes Yes Yes No No* Yes
ERT-MAE Yes Yes No No No No Yes No Yes No* No Yes
ERT-MAEC Yes Yes No No No Yes Yes No Yes No No No
ERT-UAE Yes Yes No Yes No No Yes Yes No No No* No
MAE-MAEC No No Yes No No No No No No No No Yes
MAE-UAE No Yes Yes Yes No No Yes No Yes No No Yes
MAEC-UAE No No No Yes No No Yes No Yes No No* No
131

Table 150. Score sheet for all methods with respect to different parameters
Parameter Soxhlet ERT MAE MAEC UAE
Extraction efficiency 11 0 0 0 0
Antioxidant capacity 3 1 1 2 5
Total flavonoid 1 2 3 2 3
Total phenol 4 2 0 3 2
Antibacterial activity against P. oleovorans 1 3 3 0 3
Antibacterial activity against S. epidermidis 2 3 2 1 2
Table 151. Score sheet for all solvents with respect to different parameters
Parameters Solvents
Water Methanol Ethanol (50%)
Extraction efficiency 2 2 1
Total flavonoids 0 5 0
Total phenols 0 3 2
Total antioxidants 1 3 1
Antibacterial activity against P. oleovorans 0 3 2
Antibacterial activity against S. epidermidis 2 2 1
Table 152. Scoresheet for comparison of MAE & MAEC

Method Extraction efficiency Total flavonoids Total phenols Total antioxidants Antibacterial activity
S. epidermidis P. oleovorans
MAE 1 4 5 6 8 7
MAEC 10 4 5 4 3 3
132

4.1 SOXHLET:
x For getting high extraction efficiency and high phenol content
(and antioxidant activity thereof) Soxhlet can be used as the
better option. Better extraction efficiency with Soxhlet method
for Chamomile flowers was reported by Scalia et al. (1999).
Soxhlet method was employed for extraction of phenolic
antioxidants from T. indica seeds and pericarp by Sudjaroen et
al. (2005).
x Soxhlet proved better for extraction of antioxidant compounds
on more number of occasions than ERT and microwave
assisted methods. A small degree of superiority of Soxhlet over
microwave extraction with respect to antioxidant activity of
anthraquinones from roots of Morinda citrifolia was reported
by Hemwimon et al. (2007).
x Soxhlet was found to be superior for getting high extraction
efficiency from all the plant seeds we have used, but for the
extraction of flavonoids and antibacterial compounds it was
found to be inferior.
4.2 MAE and MAEC:

x MAEC was better than MAE with respect to extraction
efficiency for plant seeds.
x Extract prepared by MAE exerted both antistaphylococcal and
antipseudomonas activities on more number of occasions than
those prepared by MAEC. It may be due to possible
degradation of antibacterial components in extracts due to
continuous heat application during MAEC, as continuous
heating can take temperature to a level higher than that
achieved with intermittent cooling in MAE.
133

x MAE and MAEC both proved suitable on equal number of
occasions for extraction of phenols and flavonoids (Table 152).
Suitability of microwave based method for flavonoid extraction
from dried cell cultures of S. medusa was reported by Gao et al.
(2006). However MAE was found suitable more number of
times than MAEC in context of total antioxidant capacity,
which again seems to be an indication of the positive role of
intermittent cooling during microwave extraction. Microwave
extraction with intermittent cooling was proposed for fast
extraction of plant phenolic compounds (Proestos and
Komaitis, 2007). Better flavonoid extraction by microwaves
than Soxhlet from Herba Epimedii was reported by Chen et al.
(2007).
x Though MAEC yields better extraction efficiency than MAE,
the latter is no inferior to former on any parameter.
Additionally more number of extracts prepared through MAE
contained antibacterial compounds than those through MAEC.
Differences in the bioactivity of extracts prepared by these two
methods may be attributed to the fact that though heat enhances
mass transfer during extraction (and makes the process faster),
maximum temperature attained during any heat-employing
method should not surpass a certain limit. Although MAEC
heats the plant material for lesser duration, the maximum
temperature reached during it is likely to be higher than that in
MAE.
x Microwave extraction is the one of the most advanced
extraction method, which has the potential to play a major role
in extraction and analytical quantification. Advantage of
134

microwave assisted extraction process over other processes has
been reviewed by Routray and Orsat (2011).
x MAE when applied with methanol as the extraction solvent
seems to be one of the better ways for extraction of
antibacterial compounds from plant seeds.
4.3 ERT:
x ERT proved to be a poor method with respect to extraction
efficiency and antioxidant capacity. Extracts prepared through
ERT showed good antibacterial activity. This again indicates
that heat may prove detrimental to antibacterial plant
metabolites, as may have been the case during MAEC. On the
same line Soxhlet (a heat employing method) proved no
attractive option for extraction of antibacterial compounds from
plant seeds.
4.4 UAE:
x UAE though not yielding high extraction efficiency on most
occasions was found to be superior on most number of
occasions with respect to total antioxidant capacity. Use of
ultrasonication for extraction of antioxidants from grape seeds
was reported by Ghafoor et al. (2009).
x UAE prepared extracts exerted good antibacterial activity on
more number of occasions that those prepared by Soxhlet or
MEAC. This again confirms better suitability of those methods
which either do not employ heat or employ it for lesser time
towards retention of antibacterial activity in given plant extract.
4.5 SOLVENTS:
x Methanol proved most inferior with respect to no parameter
evaluated. It proved better- for extraction of phenols,
135

flavonoids, antioxidant metabolites, and antibacterial
phytochemicals- than water and ethanol (50%). Better
suitability of methanol (as compared to ethanol and water) for
screening and isolation of antimicrobial compounds has earlier
been reported by Eloff (1998), and Parekh et al. (2005).
Methanolic extracts of plant leaves with antibacterial activity
better than that in aqueous extracts were reported by Nair and
Chanda (2008).
High phenol content in methanolic extracts was reported by
Kaneria et al., (2009).
x Water was found to be least effective for extraction of phenols
and flavonoids. Extracts prepared in water also exerted no
notable activity against P. oleovorans.
x Hydroalcoholic extracts prepared in 50% ethanol failed to
extract high quantity of flavonoids from different plant seeds.
From these results we can conclude that for evaluating specific type
of activity from plant extract selection of a particular extraction
method is required because all the methods and solvents differ in
mechanism of extraction from each other. Hence any one method
cannot be said as universally applicable for extraction of all types of
bioactive metabolites.
Extraction efficiency was not found to have any meaningful linear

correlation (Appendix 6) with any of the parameters assayed (i.e.,
phenol/flavonoid content, antioxidant capacity, or antibacterial
activity). This indicates that methods which are good with respect to
extraction efficiency may not be equally good in terms of efficacy.
Hence, selection of method for preparation of a particular plant
extract should be made in context of activity desired.
136

5. APPENDICES
137

Appendix - 1
Glossary
x Dissipation factor (WDQ į): A measure of the ability of the

solvent to absorb the microwave energy.
x Efficacy: The capacity to produce an effect. It has different
specific meanings in different fields. In pharmacology, efficacy
refers to the maximum response achievable from a drug.
x Extraction: Extraction is defined as the technique used for the
separation of soluble bioactive components and elimination of
undesirable insoluble constituents or, separation of medicinally
active portions of plant tissues using selective solvents through
standard procedures from the different herbs.
x Microwave: Electromagnetic waves within frequency range of
300 MHz to 300 GHz.
x Secondary metabolites: A wide range of compounds that can
be highly inducible in response to stress, these compounds are
not essential for cell structure and maintenance of life but are
often involved in plant protection against biotic and abiotic
stress.
x Superheating: In microwave reactor, the average temperature
of solvent can be reached at higher temperature than its
atmospheric boiling point. This is because microwave power is
dissipated over the volume of solvent, where nucleation points
are absent, which is necessary for boiling. This is known as
super boiling.
x Ultrasonication: The sound with frequency higher than what
human ear can detect (generally above 20 kH.
138

Appendix - 2
Properties of solvents used during present study (Mendham, et al., 2005;

Leonelli, and Mason, 2010)
Solvent B.P. Polarity index Dielectric Dissipation factor

(°C) constant WDQį
Ethanol 78.5 4.3 24.6 0.941
Methanol 64.7 5.1 32.6 0.856
Water 100 10.2 80.4 9.889
Appendix ± 3
Structure of (A) Ethyl alcohol (B) Methanol
(A) (B)
Appendix - 4
Preparation of 0.5 McFarland turbidity Standard:
McFarland turbidity standards are used to standardize the approximate number
of bacteria in a liquid suspension by visually comparing the turbidity of a test
suspension with the turbidity of a McFarland standard. McFarland standards are
prepared by adding barium chloride to sulfuric acid to obtain a barium sulfate
precipitate. The most commonly used standard in laboratories is 0.5,
representing 1.5x108 bacteria / ml.
139

Appendix - 5
Example of statistical analysis

ANOVA:
eg. T. indica water extract for extraction efficiency
Average.
soxhlet 45.2 40.01 37.02 40.74333
Cold 14.6 15.3 14.1 14.66667
MAE 17.3 18.7 17.3 17.76667
MAE
continuous 12.6 13.6 11.2 12.46667
UAE 7.6 6.9 6.9 7.133333
Anova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Row 1 3 122.23 40.74333 17.13143
Row 2 3 44 14.66667 0.363333
Row 3 3 53.3 17.76667 0.653333
Row 4 3 37.4 12.46667 1.453333
Row 5 3 21.4 7.133333 0.163333
ANOVA
Source of
Variation SS Df MS F P-value
Between 1.51E-
Groups 2026.755 4 506.6888 128.1798 08
140

Within
Groups 39.52953 10 3.952953 F crit
3.47805
Total 2066.285 14
Q ± Test:
significant
Q MS N
difference
4.66 3.952953 3 5.349167
t ± Test:
Following example shows t±test in between Soxhlet and ERT.
t-Test: Two-Sample Assuming Equal

Variances
Variable Variable
1 2
Mean 40.74333 14.66667
Variance 17.13143 0.363333
Observations 3 3
Pooled Variance 8.747383
Hypothesized Mean
Difference 26.08
Df 4
t Stat -0.00138
P(T<=t) one-tail 0.499482
t Critical one-tail 2.131847
P(T<=t) two-tail 0.998965
t Critical two-tail 2.776445
141

Appendix - 6
Correlation between different parameters
Parameters Correlation
coefficient
(r)
1. Extraction efficiency- Total flavonoid 0.0641
2. Extraction efficiency-Total Antioxidant -0.185
3. Extraction efficiency-Total Phenol -0.149
4. Extraction efficiency- Activity against S. epidermidis 0.157
5. Extraction efficiency- Activity against P. oleovorans -0.579
6. Total Phenol-Total Flavonoid -0.009
7. Total Phenol-Total Antioxidant activity 0.528
8. Total Phenol- Activity against S. epidermidis 0.064
9. Total Phenol- Activity against P. oleovorans -0.142
10.Total Flavonoid-Total Antioxidant activity 0.104
11.Flavonoid- Activity against S. epidermidis 0.216
12.Total Flavonoid- Activity against P. oleovorans -0.180
13.Total Antioxidant activity-Activity against S. epidermidis -0.049
14.Total Antioxidant activity- Activity against P. oleovorans -0.096
142

Appendix ± 7
Effect of vessel type and closure type during MAE
We have used brown screw capped bottle (250 ml) for extraction in MAE
because it was giving a better extraction efficiency than flask (250 ml)
covered with cotton plug, and flask with loose glass lid. And another
advantage was there were lesser chances of evaporation of solvent from
capped bottles. Brown bottles also protected inside plant material from
light.
Table 3. Optimization of vessel size and vessel type for MAE
Extraction
Seed and solvent Vessel size and type efficiency
(%) ± SD
100 ml flask with cotton plug,
T. indica (water) 13.93 ± 0.73
without oven preheating
100 ml flask with cotton plug, 3.33 ± 0.05
T. indica (methanol)
without oven preheating
250 ml flask with glass lead,
with preheating
250 ml flask with glass lead,
T. indica (methanol) 3.53 ± 0.25
with preheating
250 ml brown bottle, with
preheating
250 ml brown bottle, with
T. indica (methanol) 4.73 ±0.68
preheating
__________________________________________________________________________________
_____
NOTE: When T. indica water extraction was done with MAEC there was
a problem in reconstitution, as after drying the extract formed an
undissoluble shiny film in petridish. We were not able to reconstitute the
extract in 99% and 95% methanol. So for this particular extract
antioxidant, phenol, and flavonoid assay could not be done.
143

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Extraction methods for preparation of bioactive plant extracts: A comparative study

Book · August 2012

Vijay Kothari Ankit Gupta

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x All authors thank Nirma University

1.2 Extraction technology

1.3 Extraction methods

1.4 Antioxidant activity

1.7 Antibacterial activity

1.8 Experimental material

2. Materials and Methods 34

2.3 Statistical analysis

3. Results and Discussion 45

3.2 Extraction efficiency

3.3 Total flavonoids

3.4 Total phenols

3.5 Total antioxidant activity

3.6 Total antibacterial activity

3.6.A Staphylococcus epidermidis

3.6.B Pseudomonas oleovorans

4. Final comments 122

4.2 MAE and MAEC

DMSO ± Dimethyl sulfoxide

ERT ± Extraction at room temperature

GAE ± Gallic acid equivalent

MAE ± Microwave assisted extraction

MAEC± Microwave assisted extraction continuous heating

UAE ± Ultrasonic assisted extraction

For thousands of years mankind is using plant source to alleviate or

The phytochemical investigation of a plant may involve following

For isolation of biological components, extraction from plant is one of

The need for selection of most appropriate extraction methodology

Table 2. Type of phytochemicals extracted in various solvents

Polarity Solvent Chemical class extracted

Chloroform Terpenoids, Flavonoids, Alkaloids, Aglycones

Hexane Waxes, Fats

Dichloromethane Terpenoids, Alkaloids, Aglycones

Diethylether Alkaloids, Aglycones,

Ethylacetate Alkaloids, Aglycones, Glycosides

Acetone Flavonols, Alkaloids, Aglycones

Medium Ethanol Tannins, Polyphenols, Flavonol, Terpenoids, Sterols,

It should be noted that choice of appropriate solvent is of essential

desired active constituents various solvents such as water, ethanol,

Table 3. Binary mixtures of solvents of differing polarity used for

Method Solvent mixture Reference

The development of modern sample preparation techniques has

Classical methods are fairly simple, standard and continue to have

The purposes of standardizing extraction procedures for production of

x Soxhlet extraction x Pressurized liquid extraction

1.2 EXTRACTION TECHNOLOGY

Fresh plant materials once collected from a particular region in a

1. Collection of plant material & drying 2. Size reduction

Quality of an extract is influenced by several factors such as, plant

Table4. Comparative account of various extraction methods (28, 29)

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