Extraction of bioactive

Extraction of bioactive

Antimicrobial Bioactive Phytocompounds from Extraction to Identification:
Process Standardization
Different approaches to drug discovery using higher plants can be distinguished:
random selection followed by chemical screening; random selection followed by
one or more biological assays; biological activity reports and ethnomedical use of
plants [14]. The latter approach includes plants used in traditional medical sys-
tems; herbalism, folklore, and shamanism; and the use of databases. The objective
is the targeted isolation of new bioactive phytocompounds. When an active extract
has been identified, the first task to be taken is the identification of the bioactive
phytocompounds, and this can mean either a full identification of a bioactive phy-
tocompound after purification or partial identification to the level of a family of
known compounds [15].
In Fig. 1.2 an extraction-to-identification flowchart is proposed in order to opti-
mize bioactive phytocompound identification. For screening selection, plants are
collected either randomly or by following leads supplied by local healers in geo-
graphical areas where the plants are found. Initial screening of plants for possible
antimicrobial activities typically begins by using crude aqueous or alcohol extrac-
tions followed by various organic extraction methods [16]. Plant material can be
used fresh or dried. The aspects of plant collection and identification will be dis-
cussed further in this chapter. Other relevant plant materials related to antimicro-
bial activity are the essential oils. Essential oils are complex natural mixtures of vol-
atile secondary metabolites, isolated from plants by hydro or steam distillation and
by expression (citrus peel oils). The main constituents of essential oils (mono and
sesquiterpenes), along with carbohydrates, alcohols, ethers, aldehydes, and ke-
6 1 Bioactive Phytocompounds: New Approaches in the Phytosciences
tones, are responsible for the fragrant and biological properties of aromatic and
medicinal plants. Due to these properties, since ancient times spices and herbs
have been added to food, not only as flavoring agents but also as preservatives. For
centuries essential oils have been isolated from different parts of plants and are al-
so used for similar purposes.
The activities of essential oils cover a broad spectrum. Various essential oils pro-
duce pharmacological effects, demonstrating anti-inflammatory, antioxidant, and
anticancerogenic properties [17–19]. Others are biocides against a broad range of
organisms such as bacteria, fungi, protozoa, insects, plants, and viruses [20–22].
The dispersion of the hydrophobic components of essential oils in the growth
medium is the main problem in testing the activity of essential oils. Different or-
ganic solvents must be used as solubilizing agents, which may interfere with the
results of antimicrobial assays. The solution to this problem is the use of nonionic
emulsifiers, such as Tween 20 and Tween 80. These molecules are relatively inac-
tive and are widely applied as emulsifying agents. Control tests must guarantee
that these emulsifying agents do not interfere in the experiments.
Plants can be dried in a number of ways: in the open air (shaded from direct sun-
light); placed in thin layers on drying frames, wire-screened rooms, or in buildings;
by direct sunlight, if appropriate; in drying ovens/rooms and solar dryers; by indi-
rect fire; baking; lyophilization; microwave; or infrared devices. Where possible,
temperature and humidity should be controlled to avoid damage to the active
chemical constituents. The method and temperature used for drying may have a
considerable impact on the quality of the resulting medicinal plant materials. For
example, shade drying is preferred to maintain or minimize loss of color of leaves
and flowers; and lower temperatures should be employed in the case of medicinal
plant materials containing volatile substances [23]. The drying conditions should
be recorded. In the case of natural drying in the open air, medicinal plant materi-
als should be spread out in thin layers on drying frames and stirred or turned fre-
quently. In order to secure adequate air circulation, the drying frames should be lo-
cated at a sufficient height above the ground. Efforts should be made to achieve
uniform drying of medicinal plant materials to avoid mold formation [24].
Drying medicinal plant material directly on bare ground should be avoided. If a
concrete or cement surface is used, the plant materials should be laid on a tarpau-
lin or other appropriate cloth or sheeting. Insects, rodents, birds and other pests,
and livestock and domestic animals should be kept away from drying sites. For in-
door drying, the duration of drying, drying temperature, humidity and other con-
ditions should be determined on the basis of the plant part concerned (root, leaf,
stem, bark, flower, etc.) and any volatile natural constituents, such as essential oils.
If possible, the source of heat for directs drying (fire) should be limited to butane,
propane or natural gas, and temperatures should be kept below 60 °C [25]. If other
sources of fire are used, contact between those materials, smoke, and the medici-
nal plant material should be avoided.
Since researches are trying to identify bioactive phytocompounds in medicinal
plant extracts generally used by local population to treat diseases and based on em-
piric knowledge that they have the searched bioactivity, the solvent chosen must be
1.3 Antimicrobial Bioactive Phytocompounds from Extraction to Identification 7
8 1 Bioactive Phytocompounds: New Approaches in the Phytosciences
1.3 Antimicrobial Bioactive Phytocompounds from Extraction to Identification 9
Fig. 1.2 Standardization flowchart: from
extraction to identification of bioactive
phytocompounds. (1) Plants can be chosen
either randomly, based on the literature or
following consulation with local healers. After
choosing the right material, plant collection
must be followed by botanical identification
and a voucher specimen must be placed in
the local herbarium. All data about the
collection must be observed and
documented, such as climate conditions,
season, geographical localization,
environmental conditions, etc. in order to
elucidate future differences in bioactivity
compared with other results found. Any plant
part can be used but consultation of the
literature or with local healers is very useful to
reduce research time. (2) Collected plant
material can be used fresh or dried. Several
studies have started extractions with both
fresh and dried material in order to compare
the chemical composition of the extracts.
They must be ground to optimize the solvent
contact during the extraction process. Weight
standardization must be used (i.e. 300 g of
plant material to 1000 mL of solvent). More
than 90% of the studies for antimicrobial
activity in the literature start extraction with
methanol, ethanol or water because it is
proved that ethanol extraction is more
effective in isolating the bioactive phytocompound.
The primary extractions methods
are very variable but the idea is to research
activity cited in popular use, and to choose
the same extraction method. This is especially
useful to corroborate the in vivo activity found
in popular use. (3) After extraction the
volume must be concentrated by lyophilization
or another concentration technique
before screening. Usually, after the lyophilization
process ground powder is obtained. This
must be resuspended in water at a higher
concentration (i.e. 1 g mL–1) for initial drop
test screening. The high concentration of the
extract guarantees the identification of the
bioactivity, if present. Using low concentrations
in drop tests may lead to false negative
results. (4) Due to the complex composition
of the extract primary separation may be used
to facilitate the identification process.
Micromolecules can be separated from
macromolecules (proteins and carbohydrates)
by very simple techniques such as
ethanol precipitation (30% v/v), centrifugation
(10 000g for 10 min) and filtration
systems such as Centricon and Amicon
(Millipore). Supernatant and precipitate
phases are obtained and can be separated in
drop tests. As discussed previously, antimicrobial
activity is commonly present in
micromolecules (supernatant) phase. (5) The
antimicrobial screening by drop test (formerly
disk diffusion agar assay) is the most efficient
and inexpensive assay to identify antimicrobial
activity. The extract is dropped (i.e.
15 μL) onto an agar surface previously
inoculated with the desired microorganism.
Note that is very important to count by
McFarland scale or Newbauer chamber (i.e.
105 UFC mL–1 for bacteria; 106 cells mL–1 for
fungi) the microorganism inoculums; this
permits the antimicrobial activity to be
compared within antibiotic controls and
between different microorganism groups. (6)
When antimicrobial activity is detected the
minimum inhibitory concentration (MIC)
must be determined to continue other
antimicrobial assays of interest. The MIC is
usually established by the broth dilution
method. The use of 96-microwell plates to
minimize costs is very effective, reducing the
culture media quantities drastically and
enhancing the test capacity (in one plate up
to eight different extracts can be tested in 10
different concentrations plus 1 negative and 1
positive controls, also see Fig. 1.3). (7) Bioguided
chromatography techniques such as
bioautography preceded by solvent separation
is essential to initiate the bioactive
phytocompound identification process;
fraction collection with HPLC or FPLC assays,
preparative TLC are also valid techniques.
Bio-guided fraction and purification confirms
previous results leading to isolation of a
bioactive phytocompound. (8) By TLC assays,
Rf values can be determined and polarity or
even chemical groups (use of specific dyes)
elucidated (Fig. 1.3). (9) NMR, HPLC/MS,
and GC/MS are used to identify a bioactive
phytocompound as discussed in this chapter.
the same as that used in popular treatment. As we know, water and ethanol are by
far the most commonly used, and for this reason most studies begin with water or
ethanol as solvents.
Water is almost universally the solvent used to extract activity. At home, dried
plants can be ingested as teas (plants steeped in hot water) or, rarely, tinctures
(plants in alcoholic solutions) or inhaled via steam from boiling suspensions of the
parts. Dried plant parts can be added to oils or petroleum jelly and applied external-
ly. Poultices can also be made from concentrated teas or tinctures.
Since nearly all of the identified components from plants active against microor-
ganisms are aromatic or saturated organic compounds, they are most often ob-
tained initially through ethanol and water extraction [26]. Some water-soluble com-
pounds, such as polysaccharides like starch and polypeptides, including fabatin
[27] and various lectins, are commonly more effective as inhibitors of virus adsorp-
tion and would not be identified in the screening techniques commonly used [28].
Occasionally tannins and terpenoids may be found in the aqueous phase, but they
are more often obtained by treatment with less polar solvents (Fig. 1.2).
Another concern during the extraction phase is that any part of the plant may
contain active components. For instance, the roots of ginseng plants contain the
active saponins and essential oils, while eucalyptus leaves are harvested for their
essential oils and tannins. Some trees, such as the balsam poplar, yield useful sub-
stances in their bark, leaves, and shoots [29]. The choice of which part to use must
be based on ethnopharmacological studies and review of the literature.
For alcoholic extractions, plant parts are dried, ground to a fine texture, and then
soaked in methanol or ethanol for extended periods. The slurry is then filtered and
washed, after which it may be dried under reduced pressure and redissolved in the
alcohol to a determined concentration. When water is used for extractions, plants
are generally soaked in distilled water, blotted dry, made into slurry through blend-
ing, and then strained or filtered. The filtrate can be centrifuged (approximately
10000g for 10 min) multiple times for clarification [30]. Crude products can then
be directly used in the drop test and broth dilution microwell assays (Fig. 1.2) to
test for antifungal and antibacterial properties and in a variety of assays to screen
bioactivity (Fig. 1.3).
In order to reduce or minimize the use of organic solvents and improve the ex-
traction process, newer sample preparation methods, such as microwave-assisted
extraction (MAE), supercritical fluid extraction (SFE) and accelerated solvent ex-
traction (ASE) or pressurized liquid extraction (PLE) have been introduced for the
extraction of analytes present in plant materials. Using MAE, the microwave ener-
gy is used for solution heating and results in significant reduction of extraction
time (usually in less than 30 min). Other than having the advantage of high extrac-
tion speed, MAE also enables a significant reduction in the consumption of organ-
ic solvents. Other methods, such as the use of SFE that used carbon dioxide and
some form of modifiers, have been used in the extraction of compounds present in
medicinal plants [31].
To identify the bioactive phytocompounds, liquid chromatography with an iso-
cratic/gradient elution remains the method of choice in the pharmacopeia, and re-
10 1 Bioactive Phytocompounds: New Approaches in the Phytosciences
versed octadecyl silica (C-18) and ultraviolet detection mode is the most common-
ly used method. Gradient elution HPLC with reversed phase columns has also
been applied for the analysis of bioactive phytocompounds present in medicinal
plants extracts [32].
The advantages of liquid chromatography include its high reproducibility, good
linear range, ease of automation, and its ability to analyze the number of constitu-
ents in botanicals and herbal preparation. However, for the analysis of multiple bi-
oactive phytocompounds in herbal preparations with two or more medicinal
plants, coeluting peaks were often observed in the chromatograms obtained due to
1.3 Antimicrobial Bioactive Phytocompounds from Extraction to Identification 11
Fig. 1.3 Current assays to identify bioactivity
and start molecule identification. (A/B)
Bioauthography technique: (A) Thin-layer
chromatography (TLC) of aqueous extracts of
(1) Ocimun gratissimum, (2) Anadenanthera
macrocarpa, (3) Croton cajucara Benth. (4)
Cymbopogon citrates, and (5) Juglans regia
performed in silica gel G60 F254 aluminum
plates (5 _ 8). Plates were developed with nbutanol:
acetic acid:water (8:1:1, v/v) and were
visualized under ultraviolet light or after
staining with cerric sulfate plate. (B)
Alternatively, plates were placed inside Petri
dishes and covered with over solid media
(10 mL BHI with 1% phenol red). After
overnight incubation for diffusion of the
separated components, the plate was
inoculated with Candida albicans (ATCC
51501) 106 cells per plate and incubated for
48 h at 37 °C. Growth inhibition can be seen
in (1, 2, and 3) after spraying with
methylthiazollyltetrazolium chloride (MTT) at
5 mg mL–1. (C) Drop test at same concentrations
(200 μg mL–1) of (1) aqueous extract
from Punica granatum and commercially
available antifungal agents, (2) fluconazole,
(3) flucytosine, and (4) anphotericin.
(D) MIC microwell dilution test of (L1)
Punica granatum, (L2) fluconazole, and (L3)
flucytosine against Candida albicans (ATCC
51501). (C+) positive control, (C–) negative
control, (1) 200 μg mL–1, (2) 100 μg mL–1, (3)
50 μg mL–1, (4) 25 μg mL–1, (5) 12.5 μg mL–1,
(6) 6.75 μg mL–1, (7) 3.4 μg mL–1, (8)
1.7 μg mL–1, and (9) 0.8 μg mL–1. (+) means
fungi growth.
the complexity of the matrix. The complexity of matrix may be reduced with addi-
tional sample preparation steps, such as liquid–liquid partitioning, solid-phase ex-
traction, preparative LC and thin-layer chromatography (TLC) fractionation.
Capillary electrophoresis (CE) proved to be a powerful alternative to HPLC in the
analysis of polar and thermally labile compounds. Reviews on the analysis of natu-
ral medicines or natural products in complex matrix by CE are well reported. Many
publications showed that all variants of CE, such as capillary zone electrophoresis
(CZE), micellar electrokinetic capillary chromatography (MEKC), and capillary iso-
electric focusing (cIEF), have been used for the separation of natural products. The
separation in CZE is based on the differences in the electrophoretic mobilities re-
sulting in different velocities of migration of ionic species in the electrophoretic
buffer in the capillary. For MEKC, the main separation mechanism is based on so-
lute partitioning between the micellar phase and the solution phase. Factors that
are known to affect separation in CZE and MEKC include the pH of the running
buffer, ionic strength, applied voltage, and concentration and type of micelle add-
ed. From the review articles, CE has been used to determine the amount of cate-
chin and others in tea composition, phenolic acids in coffee samples and flavo-
noids and alkaloids in plant materials.
Chromatographic separation with mass spectrometry for the chemical character-
ization and composition analysis of botanicals has been growing rapidly in popu-
larity in recent years. Reviews on the use of mass spectrometry and high-perfor-
mance liquid chromatography mass spectrometry (HPLC/MS) on botanicals have
been reported. The use of hyphenated techniques, such as high-resolution gas
chromatography mass spectrometry (HRGC/MS), high performance liquid
chromatography/mass spectrometry (HPLC/MS), liquid chromatography tandem
mass spectrometry (HPLC/MS/MS) and tandem mass spectrometry (MS/MS) to
perform on-line composition and structural analyses provide rich information that
is unsurpassed by other techniques.
HRGC/MS remains the method of choice for the analysis of volatile and semi-vol-
atile components, such as essential oils and others in botanicals and herbal prepar-
ations, along with high-resolution separation with capillary column coupling with
mass spectrometry using electron impact ionization (EI).
In analyzing bioactive phytocompounds, HPLC/MS has played an increasingly
significant role as the technique is capable of characterizing compounds that are
thermally labile, ranging from small polar molecules to macromolecules, such as
peptides/proteins, carbohydrates, and nucleic acids. The most common mode of
ionization in HPLC/MS includes electrospray ionization (ESI) and atmospheric
pressure chemical ionization (APCI). Mass analyzers, such as single quadruple,
triple quadruple, ion-trap, time-of-flight, quadruple time-of-flight (Q-TOF) and
others, are also used. With tandem mass spectrometry, additional structural infor-
mation can be obtained about the target compounds. However, methods using
HPLC/MS are still limited to conditions that are suitable for MS operations. There
are restrictions on pH, solvent choice, solvent additives and flow rate for LC in or-
der to achieve optimal sensitivity.
12 1 Bioactive Phytocompounds: New Approaches in the Phytosciences