Development of Fast Reliable Methods of Extraction and High-Throughoutput
Screening (HTS) of Crude Plant Extracts: New Challenges
Medicinal plants have formed the basis of health care throughout the world since
the earliest days of humanity and are still widely used and have considerable im-
portance in international trade. Recognition of their clinical, pharmaceutical, and
economic value is still growing, although this varies widely between countries.
Plants are important for pharmacological research and drug development, not on-
1.2 Development of Fast Reliable Methods of Extraction and High-Throughoutput Screening 3
ly when bioactive phytocompounds are used directly as therapeutic agents, but al-
so as starting materials for the synthesis of drugs or as models for pharmacologi-
cally active compounds. Regulation of their exploitation and exportation is there-
fore essential to ensure their availability for the future [1].
Plant preparations have a very special characteristic that distinguishs them from
chemical drugs: a single plant may contain a great number of bioactive phytocom-
pounds and a combination of plants even more. This complexity is one of the most
important challenges to phytoscientists attempting to identify a single bioactive
phytocompound or chemical group in the enormous universe that comprises a sin-
gle crude extract.
Biotechnology in the 1970s and 1980s made tremendous strides and ushered in
a new era for the pharmaceutical industry. Many enzymes and receptor proteins of
therapeutic interest were made available in large quantities by recombinant expres-
sion, while signal transduction pathways could be interrogated by reporter gene
carrying cellular constructs. Such mechanism-based in vitro assays are amenable to
large scales of operation, and the concept of high-throughput screening rapidly be-
came the paradigm for lead discovery [2].
High-throughput screening, often abbreviated as HTS, is a method of scientific
experimentation especially relevant to the fields of biology and chemistry. Through
a combination of modern robotics and other specialized laboratory hardware, it al-
lows a researcher to effectively conduct hundreds of scientific experiments at once.
In essence, HTS uses a brute-force approach to collect a large amount of experi-
mental data, usually observations about how some biological entity reacts to expo-
sure to various chemical compounds in a relatively short time. A screen, in this
context, is the larger experiment, with a single goal to which all this data may sub-
sequently be applied [3].
A necessary precondition for the success of the HTS approach is a large and di-
verse compound collection. In the early days, this largely comprised in-house ar-
chives and natural product extracts. The former represented the efforts of chemists
internally over the years, supplemented by purchase from external sources. Nei-
ther the total number of compounds, nor their chemical diversity, was appropriate
to feed HTS. These deficiencies created the science of combinatorial chemistry in
the late 1980s and early 1990s and an unanticipated repercussion of high-through-
put chemical synthesis was a steady waning of interest in natural product screen-
ing, leading to its complete abandonment by many companies [4].
Just like drugs of synthetic origin, bioactive phytocompounds range from simple
to complex structures. Either way, the evaluation of a bioactive phytocompound or
a natural product leads to benefits from modern HTS for the generation of analogs
[5]. Thus, paradoxically, the same combinatorial chemistry that initially caused the
decline in natural product screening now promises to be an essential tool in reju-
venating it. Academic groups in particular are used to allocating significant re-
sources of time and staff towards the total synthesis of bioactive phytocompounds.
The ability to adapt such routes for the preparation of analogs is an obvious strate-
gy for leveraging the initial expenditure, and is now increasingly evident in the lit-
erature. Because of the stricter timelines, large-scale combinatorial programs
4 1 Bioactive Phytocompounds: New Approaches in the Phytosciences
based on natural products are less common in industry, but are still practiced in
the absence of more tractable synthetic leads [6].
Combinatorial chemistry has come a long way in the past two decades. Industri-
ally, it competed with natural product extracts and purified bioactive phytocom-
pounds for HTS resources and emerged as the preferred option. Unfortunately
this technique has not produced a wealth of high-quality drug candidates. Instead,
the integration of combinatorial chemistry with other mechanisms for lead gener-
ation is now rightly considered the correct strategy. A natural product lead is a le-
gitimate starting point for combinatorial chemistry, and this process can often dis-
cover novel analogs [7]. In some cases, such compounds are more potent than the
natural product or can possess superior drug-like properties. In others, the synthet-
ic analogs display new biological activities not seen with the original molecule [4].
The ability to rapidly identify undesirable or desirable compounds in natural
product extract libraries is a critical step in an efficiently run natural products dis-
covery program. This process, commonly called dereplication [8], is important to
prevent the unnecessary use of resources for the isolation of compounds of little or
no value for development from extracts used in the screening process. Resources
can then be focussed on samples containing the most promising leads. The recent
application of HTS technologies to assay natural products extracts for biological ac-
tivity has intensified the need for efficient dereplication strategies [9].
Dereplication of the bioactive phytocompounds in crude natural product extracts
requires some form of feedback from the bioassay, which was initially used to de-
tect the biological activity. This is necessary regardless of the separation technique
and analytical method used. A common strategy has been to collect fractions from
the high-performance liquid chromatography (HPLC) separation in deep-dish mi-
crotiter plates or tubes and then resubmit the individual fractions to the original as-
say. This approach requires desiccation of fractions to remove the HPLC solvents,
which are usually incompatible with the bioassay, resuspending the fractions in a
compatible solvent (water, DMSO, or Tween), and then individual assaying of each
fraction. This process is not cost effective, being both time and labor intensive.
Consequently, as a result of the increasing emphasis on the generation of new lead
compounds, faster cycle times, and high efficiency, many pharmaceutical compa-
nies have moved away from the natural products area.
Currently, almost every large pharmaceutical company has established HTS in-
frastructures and possesses large combinatorial compound libraries, which cover a
wide range of chemical diversity. However, the ability to detect the desired biolog-
ical activity directly in the HPLC effluent stream and to chemically characterize the
bioactive phytocompound on-line, would eliminate much of the time and labor tak-
en in the fraction collection strategy. This way, cycle times, expenses, and the iso-
lation of known or undesirable compounds would be reduced dramatically, allow-
ing natural products to be screened in an efficient and cost effective manner [10].
Recently, such an on-line HPLC biochemical detection (BCD) system, in the fol-
lowing referred to as high-resolution screening (HRS) system, has been described
for a range of pharmacologically relevant targets, such as the human estrogen re-
ceptor, cytokines, leukotrienes, and the urokinase receptor [11]. In contrast to con-
1.2 Development of Fast Reliable Methods of Extraction and High-Throughoutput Screening 5
ventional microtiter-type bioassays, the interactions of the extracts and the bio-
chemical reagents proceed at high speed in a closed continuous flow reaction de-
tection system. When sufficient chromatographic separation is achieved, the indi-
vidual contribution of the bioactive phytocompounds to the total bioactivity is ob-
tained within a single run. Moreover, by combining on-line biochemical detection
with complementary chemical analysis techniques, such as mass spectrometry
(HRS-MS), chemical information that is crucial for the characterization and iden-
tification of bioactive phytocompounds is obtained in real time. Biochemical re-
sponses are rapidly correlated to the recorded MS and MS/MS data, thus providing
chemical information such as molecular weight and MS/MS fingerprints [12].
Compared with traditional screening approaches of complex mixtures, which are
often characterized by a repeating cycle of HPLC fractionation and biological
screening, HRS-MS analysis speeds up the dereplication process dramatically.
Moreover, the technology enables drug discovery programs to access the enor-
mous chemical diversity offered by complex mixtures as a source of novel drug-like
molecules [13]. The use of chromatographical assays is discussed in the next sec-
tion of this chapter.
Screening (HTS) of Crude Plant Extracts: New Challenges
Medicinal plants have formed the basis of health care throughout the world since
the earliest days of humanity and are still widely used and have considerable im-
portance in international trade. Recognition of their clinical, pharmaceutical, and
economic value is still growing, although this varies widely between countries.
Plants are important for pharmacological research and drug development, not on-
1.2 Development of Fast Reliable Methods of Extraction and High-Throughoutput Screening 3
ly when bioactive phytocompounds are used directly as therapeutic agents, but al-
so as starting materials for the synthesis of drugs or as models for pharmacologi-
cally active compounds. Regulation of their exploitation and exportation is there-
fore essential to ensure their availability for the future [1].
Plant preparations have a very special characteristic that distinguishs them from
chemical drugs: a single plant may contain a great number of bioactive phytocom-
pounds and a combination of plants even more. This complexity is one of the most
important challenges to phytoscientists attempting to identify a single bioactive
phytocompound or chemical group in the enormous universe that comprises a sin-
gle crude extract.
Biotechnology in the 1970s and 1980s made tremendous strides and ushered in
a new era for the pharmaceutical industry. Many enzymes and receptor proteins of
therapeutic interest were made available in large quantities by recombinant expres-
sion, while signal transduction pathways could be interrogated by reporter gene
carrying cellular constructs. Such mechanism-based in vitro assays are amenable to
large scales of operation, and the concept of high-throughput screening rapidly be-
came the paradigm for lead discovery [2].
High-throughput screening, often abbreviated as HTS, is a method of scientific
experimentation especially relevant to the fields of biology and chemistry. Through
a combination of modern robotics and other specialized laboratory hardware, it al-
lows a researcher to effectively conduct hundreds of scientific experiments at once.
In essence, HTS uses a brute-force approach to collect a large amount of experi-
mental data, usually observations about how some biological entity reacts to expo-
sure to various chemical compounds in a relatively short time. A screen, in this
context, is the larger experiment, with a single goal to which all this data may sub-
sequently be applied [3].
A necessary precondition for the success of the HTS approach is a large and di-
verse compound collection. In the early days, this largely comprised in-house ar-
chives and natural product extracts. The former represented the efforts of chemists
internally over the years, supplemented by purchase from external sources. Nei-
ther the total number of compounds, nor their chemical diversity, was appropriate
to feed HTS. These deficiencies created the science of combinatorial chemistry in
the late 1980s and early 1990s and an unanticipated repercussion of high-through-
put chemical synthesis was a steady waning of interest in natural product screen-
ing, leading to its complete abandonment by many companies [4].
Just like drugs of synthetic origin, bioactive phytocompounds range from simple
to complex structures. Either way, the evaluation of a bioactive phytocompound or
a natural product leads to benefits from modern HTS for the generation of analogs
[5]. Thus, paradoxically, the same combinatorial chemistry that initially caused the
decline in natural product screening now promises to be an essential tool in reju-
venating it. Academic groups in particular are used to allocating significant re-
sources of time and staff towards the total synthesis of bioactive phytocompounds.
The ability to adapt such routes for the preparation of analogs is an obvious strate-
gy for leveraging the initial expenditure, and is now increasingly evident in the lit-
erature. Because of the stricter timelines, large-scale combinatorial programs
4 1 Bioactive Phytocompounds: New Approaches in the Phytosciences
based on natural products are less common in industry, but are still practiced in
the absence of more tractable synthetic leads [6].
Combinatorial chemistry has come a long way in the past two decades. Industri-
ally, it competed with natural product extracts and purified bioactive phytocom-
pounds for HTS resources and emerged as the preferred option. Unfortunately
this technique has not produced a wealth of high-quality drug candidates. Instead,
the integration of combinatorial chemistry with other mechanisms for lead gener-
ation is now rightly considered the correct strategy. A natural product lead is a le-
gitimate starting point for combinatorial chemistry, and this process can often dis-
cover novel analogs [7]. In some cases, such compounds are more potent than the
natural product or can possess superior drug-like properties. In others, the synthet-
ic analogs display new biological activities not seen with the original molecule [4].
The ability to rapidly identify undesirable or desirable compounds in natural
product extract libraries is a critical step in an efficiently run natural products dis-
covery program. This process, commonly called dereplication [8], is important to
prevent the unnecessary use of resources for the isolation of compounds of little or
no value for development from extracts used in the screening process. Resources
can then be focussed on samples containing the most promising leads. The recent
application of HTS technologies to assay natural products extracts for biological ac-
tivity has intensified the need for efficient dereplication strategies [9].
Dereplication of the bioactive phytocompounds in crude natural product extracts
requires some form of feedback from the bioassay, which was initially used to de-
tect the biological activity. This is necessary regardless of the separation technique
and analytical method used. A common strategy has been to collect fractions from
the high-performance liquid chromatography (HPLC) separation in deep-dish mi-
crotiter plates or tubes and then resubmit the individual fractions to the original as-
say. This approach requires desiccation of fractions to remove the HPLC solvents,
which are usually incompatible with the bioassay, resuspending the fractions in a
compatible solvent (water, DMSO, or Tween), and then individual assaying of each
fraction. This process is not cost effective, being both time and labor intensive.
Consequently, as a result of the increasing emphasis on the generation of new lead
compounds, faster cycle times, and high efficiency, many pharmaceutical compa-
nies have moved away from the natural products area.
Currently, almost every large pharmaceutical company has established HTS in-
frastructures and possesses large combinatorial compound libraries, which cover a
wide range of chemical diversity. However, the ability to detect the desired biolog-
ical activity directly in the HPLC effluent stream and to chemically characterize the
bioactive phytocompound on-line, would eliminate much of the time and labor tak-
en in the fraction collection strategy. This way, cycle times, expenses, and the iso-
lation of known or undesirable compounds would be reduced dramatically, allow-
ing natural products to be screened in an efficient and cost effective manner [10].
Recently, such an on-line HPLC biochemical detection (BCD) system, in the fol-
lowing referred to as high-resolution screening (HRS) system, has been described
for a range of pharmacologically relevant targets, such as the human estrogen re-
ceptor, cytokines, leukotrienes, and the urokinase receptor [11]. In contrast to con-
1.2 Development of Fast Reliable Methods of Extraction and High-Throughoutput Screening 5
ventional microtiter-type bioassays, the interactions of the extracts and the bio-
chemical reagents proceed at high speed in a closed continuous flow reaction de-
tection system. When sufficient chromatographic separation is achieved, the indi-
vidual contribution of the bioactive phytocompounds to the total bioactivity is ob-
tained within a single run. Moreover, by combining on-line biochemical detection
with complementary chemical analysis techniques, such as mass spectrometry
(HRS-MS), chemical information that is crucial for the characterization and iden-
tification of bioactive phytocompounds is obtained in real time. Biochemical re-
sponses are rapidly correlated to the recorded MS and MS/MS data, thus providing
chemical information such as molecular weight and MS/MS fingerprints [12].
Compared with traditional screening approaches of complex mixtures, which are
often characterized by a repeating cycle of HPLC fractionation and biological
screening, HRS-MS analysis speeds up the dereplication process dramatically.
Moreover, the technology enables drug discovery programs to access the enor-
mous chemical diversity offered by complex mixtures as a source of novel drug-like
molecules [13]. The use of chromatographical assays is discussed in the next sec-
tion of this chapter.
PCR Machine
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