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Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 93
components [166]. The combination of MS with GC is a suitable method for the structural determination of target components present in small quantities within complex
mixtures. When necessary, the derivatization of target compounds and the GC-MS
analysis of the resulting derivatives can provide valuable information about the parent compound. Another important consideration is that the removal of oxygencontaining functional groups prior to analysis may be beneficial [167].
In phytochemical analyses, the main applications of MS-MS spectra include the
partial determination of sugar sequences in glycosides, the detection of characteristic
losses, such as in prenylated compounds, the fragmentation of flavonoids to determine the positions of substituents on the A or B rings, and the differentiation of isomers. However, for the structural elucidation of completely unknown compounds,
MS-MS alone may be insufficient; therefore, it must be combined with other complementary methods [168].
High-resolution mass spectrometry (HRMS) can be used for both qualitative and
quantitative determination of metabolite profiles [169]. HRMS analysis is generally
characterized by high selectivity. Among mass spectrometers, HRMS has features
such as high mass precision, superior resolution, fast scanning capabilities, and excellent sensitivity [145]. Since HRMS-based analytical techniques offer fast and precise
solutions for the characterization of secondary metabolites found in highly complex
matrices and the detailed elucidation of their structures, they have become widely
preferred techniques today [170].
The presence of a wide variety of isomers and their derivatives in plant extracts
complicates the overall plant matrix, leading to the co-elution of isomeric species with
the same mass. This makes it difficult to accurately identify and quantify the targeted
components. Traditional precise techniques such as LC-MS and HPLC-MS also face
these challenges. Some of these limitations can be overcome by using multistage analyzers (MS/MS) or at least one high-resolution, specially equipped instrument. In MS/
MS or HRMS analyses, narrowing the mass range analyzed reduces background signal
noise from interfering ions detected by the detector, thus increasing sensitivity [171].
The UHPLC-HRMS technique combines the high sensitivity and excellent separation
capability of UHPLC with HRMS. UHPLC-HRMS is an advanced analytical technique
used for the separation and identification of components in complex mixtures [166].
2.3.5.3 Infrared spectroscopy (IR) and Fourier transform infrared
spectroscopy (FTIR)
Infrared (IR) spectroscopy is a technique that provides information about the presence of functional groups such as hydroxyl, primary and secondary amines, carbonyl
groups, alkenes, and arenes. The fingerprint region of the infrared spectrum is particularly useful for identifying and distinguishing substances due to its unique and
highly distinctive absorption patterns [172].
94 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
For the characterization of chemicals or chemical bonds (functional groups) contained in an unknown combination of plant extract, Fourier Transform Infrared Spectroscopy (FTIR) has proven to be a very helpful approach. Furthermore, FTIR spectra of
pure substances are typically sufficiently distinct that they resemble a chemical “fingerprint.” For the majority of common plant chemicals, comparing an unknown compound’s spectrum to a library of known compounds can help identify them [159]. For
FTIR analysis, both liquid and solid materials can be used. A drop of liquid sample can
be placed between the plates to form a thin film. Solid materials, on the other hand, can
be milled with potassium bromide and pressed into a thin pellet for analysis [173].
2.3.5.4 UV-visible spectroscopy
UV-visible spectroscopy is a fast and easy-to-apply analytical technique based on measuring the absorption or transmittance of light. The wavelength range in which UVvisible spectroscopy operates is between 200 nm and 800 nm. Ultraviolet-visible (UVVis) spectrophotometers primarily consist of a light source that passes light through a
sample and a detector on the opposite side that records the transmitted light. Covalently bonded unsaturated compounds capable of absorbing light at specific wavelengths known as chromophores, exhibit electronic transition energy differences that
match the energy of UV-visible light. Covalently bonded saturated groups that influence the absorption of chromophores but do not themselves absorb UV-Vis electromagnetic radiation are known as auxochromes. When UV-Vis radiation strikes chromophores, ground-state electrons are excited to higher energy states. Auxochromes
act as electron donors and, while they do not change color themselves, they influence
the color of chromophores. Since water and alcohols do not absorb light in the UV-Vis
range, they are transparent and therefore serve as suitable media for UV-visible spectroscopy. Chromophores have characteristic absorption bands; however, changes
such as the addition of another compound to the medium or an increase in temperature can alter energy levels and, consequently, absorption intensity [174].
UV-Vis spectroscopy is a versatile and powerful analytical technique. One of its key
advantages is its ability to simultaneously measure the electronic transitions of organic
molecules (primarily through n → π
✶
and π → π✶ transitions) and transition metal oxides or ions (via d-d and charge transfer transitions). However, absorption bands in the
UV-Vis range are often broad and may overlap, making the interpretation of results occasionally challenging. Combining UV-Vis spectroscopy with other analytical methods
provides more comprehensive insights. Examples of such complementary techniques
include X-ray absorption spectroscopy and diffraction, vibrational spectroscopy, and
magnetic resonance [175].
UV-visible spectroscopy is a widely used method for the qualitative and quantita-
tive analysis of phytochemicals, elucidation of the structure of plant components, and
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 95
examination of the potency, quality, and purity of compounds due to its suitability for
small-scale studies, simplicity, and low equipment cost [176].
2.3.6 Evaluation of therapeutic efficacy with bioassays
The physiological activities exhibited by natural products stem from their capacity to
engage with different cellular targets [177]. In the process of developing drugs from
plant-based sources, it is essential to thoroughly evaluate these interactions. Biological
activity studies focus on determining both the therapeutic effects and the potency of
these effects. To conduct an appropriate biological experiment, steps such as literature review, evaluation of the stability of the sample product or compound, performance testing in biosystems, and determination of 50% effective concentration (EC50)
or cytotoxic concentrations (IC50) must be carried out [178].
Testing biological activity after purification ensures more reliable results by eliminating the matrix surrounding the compound in the extract and removing unwanted
interactions [136]. Additionally, bioactivity assays can be conducted at multiple stages
of the drug development process. In the preliminary screening phase, plant samples,
extracts, various fractions of the extracts, or even libraries of pure compounds may
undergo biological analyses to detect bioactivity potential. Conducting these analyses
in a HTS format can increase efficiency while reducing costs and time. In later stages,
these tests can assist fractionation processes, such as purification or bioactivityguided approaches, in isolating and identifying bioactive compounds [179].
Conducting bioactivity assays in a stepwise manner is an appropriate approach.
The first phase emphasizes high capacity and low cost, and if a positive effect is detected in this screening, the process advances to the second phase of biological activity
testing, which is more precise and accurate. In the second phase, pure compounds
with potential as drug candidates should be investigated under additional models and
test conditions to select candidates for clinical trials. Biological experiments can be
classified based on the target used. These primarily include lower organisms (bacteria, fungi, insects, lower plants, etc.), live cells under tissue culture conditions (e.g.,
cancer cells), animal or human-derived tissues, animals, isolated organs of vertebrates, and isolated subcellular systems (e.g., enzymes, receptors, etc.) [180].
Biological analyses in the drug development process are generally classified into
in vitro and in vivo experiments conducted prior to preclinical and clinical research.
For drug discovery from medicinal and aromatic plants, it is essential to select appropriate biological assays to evaluate the activity and potency of bioactive compounds
(hits or leads) against the relevant disease. In vitro research focuses on primary activity, specificity, cellular toxicity, and physiologically significant activity of compounds
using various assays [181]. These analyses include:
– Comparative screening
– Interaction studies
96 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
– Bioactivity-guided fractionation
– Biological characterization
– Stability studies
– Investigation of mechanisms of action
While in vitro researches are indispensable for the primary evaluation of natural
products, they are limited by the absence of pharmacokinetic data, lack of direct compatibility with in vivo/clinical doses, and the narrow scope of physiological mechanisms represented in the assay systems [182].
In vivo bioassays aim to evaluate the complete effects of bioactive compounds in
disease models and assess toxicity and safety on a cellular or organismal basis. These
experiments involve the use of animal models, isolated systems (e.g., enzymes, receptors), or isolated organ preparations to investigate biological activity, toxicity, pharmacokinetics, and pharmacodynamics. However, ethical concerns regarding animal
use and the physiological and metabolic differences between animals and humans
can limit the applicability of the data obtained from these studies [183].
In vitro and in vivo tests can be used to conduct toxicity studies and perform toxicological evaluations of compounds. While in vitro studies are often sufficient to examine direct effects on cell proliferation and phenotype, in vivo studies provide more
detailed qualitative and quantitative assessments of toxicological effects. Since the effects and toxicity of many drugs vary by species, selecting appropriate animal models
for toxicity studies is crucial. Almost all in vivo studies evaluating pharmacological
and toxicological effects, including the mode of action, are conducted to generate fundamental data for the proposed use of the product in subsequent clinical trials [184].
2.3.7 Preclinical and clinical researches
The discovery of a bioactive compound is the first step toward its development as a
drug. The subsequent steps involve preclinical and clinical studies. A plant-derived
molecule discovered through in vitro and in silico experiments demonstrating bioactivity must be designed according to the characteristics of the target cell or mechanism in the body to exhibit the desired effect in vivo [36].
Drug discovery from medicinal and aromatic plants is essentially a stepwise optimization process of a pharmacologically active lead compound. For this, it is first necessary to investigate the bioactivity of the plant-derived molecule. However, this is
not sufficient for the molecule to be clinically applicable. Although the therapeutic effects of a medicinal plant or its components have been demonstrated through bioactivity tests, the molecule cannot be converted into a drug if there are deficiencies in
preclinical and clinical research. Especially in vivo studies are needed to determine
parameters such as effect profile, bioavailability, side effects, and toxicity. Numerous
studies are conducted daily on the pharmacological activities of plant extracts and iso-
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 97
lated plant components. However, most of these studies have not resulted in drug production due to the lack of clinical trials.
The purpose of preclinical studies in drug discovery is to present one or more
clinical candidate molecules that are effective, safe, and possess drug-like properties,
supported by sufficient data demonstrating biological activity at a disease-related target. These molecules are then selected for further clinical trials. Drug discovery programs generally aim to generate data through the collaboration of chemistry, biology,
toxicology, and pharmacology, evaluating compounds at different doses and across
multiple experiments. Theoretically, discovering a bioactive molecule is of no practical use if the molecule cannot be tolerated by the target or fails to produce a therapeutic effect when administered to humans. Compounds intended for drug development must have appropriate pharmacokinetic parameters, including dose, speed,
extent and duration of reaching the site of action, and binding to the relevant target.
Thus, comprehensive preclinical and clinical studies must be conducted for drug candidate compounds [185].
Clinical trials are conducted on either patients or healthy volunteers, depending on
the phase being carried out. Their primary goal is to provide data on the safety and efficacy of drugs. Clinical trials are conducted in accordance with a protocol designed by
the investigator or sponsor. The study begins once the conditions regarding the number
and selection criteria of participants, dosage information, study duration, parameters,
and data analysis procedures are established. The phases, general characteristics, and
analyses involved in preclinical and clinical studies are presented in Figure 2.17 [184].
For traditionally used medicinal plants and herbal products, an exception exists
under the European Medicines Agency (EMA) regulations regarding clinical trials. According to this regulation, herbal medicines with documented traditional use for at
least 15 years within the EU and 30 years in other countries do not require clinical trials
to demonstrate their safety and efficacy for their traditionally indicated uses [186].
2.3.8 Structural modifications and developing new analogues
Chemical modifications of natural products play a significant role in drug development, as altering the chemical structures of these compounds can lead to the discovery of new therapeutic options and improve existing treatments. Such small modifications in the synthesis of natural compounds can enhance their biological activity,
improve pharmacokinetic profiles, and result in more successful clinical applications [187].
Despite their intricate chemical structures, plant secondary metabolites demonstrate enhanced drug-likeness compared to synthetic molecules. However, some natural products, while demonstrating therapeutic efficacy, may have poor oral bioavailability, excessive side effects, or suboptimal activity. To address these limitations,
functional group modifications are often required to produce drugs that are more sol-
• First-in-human (FIH)
trials
• Cell or animal studies
• 10 to 15 volunteers
• Human micro dose
studies
• Pharmacokinetic
data
Preclinical
Trials-Phase 0
• A small number of
healthy volunteers
• 20 to 80 volunteers
• Pharmacodynemic
data
• Dosage,effectiveness,
safety, toxicity,
bioavailability
Phase 1
Clinical Trial
• Larger groups of
patient
• Optimal dose and
dosage ranges
• Therapeutic Efficacy
Phase 2
Clinical Trial
• 300 to 3,000
volunteers
• Long-term outcomes
and identification of
common side effects
• Efficacy and adverse
drug reactions
monitoring
Phase 3
Clinical Trial
• Post-FDA approval
• Post-Market Drug
Safety Monitoring
• Pharmacovigilance
Phase 4
Clinical Trial
Figure 2.17: Preclinical and clinical studies.
98 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 99
uble, better absorbed and distributed, more selective, and less toxic. Addressing these
challenges necessitates the chemical production of natural product analogs, which
serve as lead compounds for the creation of more potent drugs. Structural modifications are implemented through strategies such as the genomes to natural products
(GNP) platform to enhance activity [7, 13].
The extraction of natural compounds from their original sources is constrained
by the availability of source plants. Additionally, the extraction process is often complex and inefficient, and acquiring the large amounts of raw materials needed for
drug approval and distribution is typically expensive. Consequently, once plantderived drugs are discovered, synthetic production often becomes the preferred
method. Structural information is crucial for the synthesis of natural product analogs.
Understanding the configuration of stereogenic centers is essential for designing an
effective synthetic strategy [33]. Advances in NMR spectroscopy, HPLC, microfluidic
systems, and algorithmic developments have been effectively applied in medicinal
chemistry, enabling the synthesis of numerous natural compound analogs. Computational chemistry tools have further contributed to drug discovery by facilitating the
development of structural analogs from natural molecules [2, 151, 188].
An excellent example of clinical application through structural modifications is
the transformation of the natural product vinblastine into vinorelbine by adding
methyl groups and an oxygen atom. These changes improved the pharmacokinetic
properties of the compound and enhanced its efficacy in cancer treatment. Specifically, the addition of oxygen increased the compound’s bioavailability and facilitated
better targeting to cellular regions [189].
Prodrug and isomerism strategies are other prominent approaches in structural
modifications. The term “prodrug” was introduced by Adrien Albert in 1958. Prodrugs
are biologically inactive derivatives that can be converted into pharmacologically active drug molecules. These designs typically include functional groups such as esters,
amides, phosphates, carbonates, or carbamates, which can be enzymatically or chemically cleaved in the body. The prodrug strategy addresses physical barriers related to
solubility, bioavailability, chemical instability, and therapeutic effects at target sites,
aiming to optimize absorption, distribution, metabolism, excretion, and toxicity
(ADMET) processes to enhance therapeutic efficacy [190].
Isomerism has been one of the most groundbreaking results in clinical research
for improving the pharmacokinetics and efficacy of compounds used as drugs. Findings from studies on isomerism play a critical role in discovering new drugs and improving the bioavailability of existing ones. Most drugs currently in use have undergone chiral switching, transitioning from racemic mixtures to one of their isomers
[191]. By definition, isomers are molecules with the same atomic composition but different bonding arrangements or spatial orientations of atoms, meaning they are distinct substances sharing the same molecular formula. Isomerism leads to different
therapeutic applications; for instance, quinine exhibits antimalarial activity, while
quinidine has antiarrhythmic properties [192].
100 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
2.4 The use of omics technologies in drug discovery and development
Today, studies utilizing omics approaches are increasingly preferred, with the most
common omics technologies including genomics, transcriptomics, proteomics, and
metabolomics [193]. The application of omics studies to phytotherapy is referred to as
“phytomics” [194].
The therapeutic effects of plants are often not attributed to a single compound
but rather to the combinatorial effects of the components within the extract. Therefore, focusing solely on a single isolated compound in drug development studies from
plants may not always be the most accurate approach. Since many diseases are already treated with pharmacological combinations, a combination strategy should be
taken into consideration instead. The effects of these combinations on genes and proteins involved in various cellular processes should be thoroughly investigated using
available “-omics” platforms. Such an approach can capture an effect that operates
through synergistic mechanisms on multiple targets within a physiological system,
rather than searching for a specific molecule targeting a single objective. Integrating
technologies such as genomics, transcriptomics, proteomics, metabolomics/metabonomics, automation, and computational strategies into the research process will enable the development of a systems biology approach, paving the way for more efficient and innovative drug designs [188].
Recent technological advancements in genomics, proteomics, and metabolomics
have introduced significant innovations across various scientific research fields,
sparking great interest and excitement among scientists. Genomics aims to study genetic information, proteomics focuses on proteins, and metabolomics involves the
qualitative and quantitative analysis of all low-molecular-weight metabolites within a
cell or organism and their dynamics in biological systems [193]. These new platforms,
referred to as “-omics” technologies, are high-throughput systems capable of simultaneously detecting tens of thousands of genes and proteins. They enable detailed analysis and comprehensive characterization of biological systems. These technologies
have the potential to correlate complex mixtures with intricate effects in the form of
gene/protein expression profiles, providing evidence of the efficacy of phytochemical
components and defining their activity profiles [195].
2.4.1 Genomics
When the therapeutic properties of plants were first discovered, humanity was far
from the rigor of scientific evidence, the principles of philosophical and experimental
methodologies, and the ability to identify bioactive molecules through advanced genomic technologies that we possess today. Plant genomics has emerged as a transforma-
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 101
tive approach, enabling the comprehensive exploration and analysis of vast botanical
diversity and the intricate biochemical repertoire it encompasses. This progress has
been supported by rapid advancements and decreasing costs in genetic sequencing
technologies [196]. Recent developments in genomic techniques, such as DNA barcoding and other innovative methods, have established accurate identification criteria
for plants and other natural product sources. These techniques provide much faster
and more accurate identification compared to the commonly used morphological and
other traditional methods [197].
Genomics can serve various roles in the target identification process during drug
discovery from natural products. Specifically, genomic analysis can identify targets
such as cellular signaling pathways and enzymes metabolizing specific compounds.
Genome-based methods, including sequencing and transcriptomic studies, have enabled the evaluation of many systems for compound targeting [198].
Genome-wide association studies in humans have identified thousands of genetic
polymorphisms associated with diseases. The complex interactions of genes within
the human genome underpin conditions such as diabetes, autoimmune diseases, cancer, and neurological disorders. Functional genomics is an innovative field aimed at
elucidating the relationship between genotype and phenotype. Leveraging genetic editing tools and large datasets, it allows for deeper exploration of gene functions and
biological interactions. It also plays a vital role in revealing disorder mechanisms and
discovering new drug targets. Integrating functional genomic approaches into drug
development pipelines is expected to accelerate the creation of innovative and effective therapies [199].
2.4.2 Metabolomics
Metabolomics is a nonselective, universally applicable, comprehensive, and simultaneous analytical method used for the identification and quantification of metabolites
in biological samples. Metabolomics provides meaningful and useful data for largescale analysis of primary and secondary metabolites in plant extracts, holistic interpretation of results, and the monitoring and evaluation of cellular function or systems
biology. This research field aims to profile metabolites and detect differences between
them. In metabolomics, various analytical strategies are used to determine the phytochemical composition of a specific plant extract or matrix [200].
Advancements in instrumental analysis methods, such as chromatography and
spectroscopy, have accelerated progress in metabolomic technologies. In recent years,
omics technologies, including metabolomics, have become widely used in the research
of plant-based drugs. Through metabolomic methods, secondary metabolites found in
medicinal and aromatic plants can be accurately and comprehensively analyzed. This,
in turn, speeds up the processes of identification and characterization of these metabolites. In addition, metabolomics is a method that can be used to comprehend the
102 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
mechanisms of action of plant components at the molecular level [12]. Although metabolomics does not aid in the initial selection of plants, it provides a rapid analysis of
the active components in the selected plant extract. The concentrations and properties
of phytochemical components can be determined through metabolomics, and by utilizing various statistical analysis methods, these components can be studied in relation to their physiological activities. The aim of metabolomic studies is to evaluate
and identify bioactive components within a complex plant extract before isolating the
relevant compound. The strength of the metabolomic approach in plant-based drug
discovery lies in its ability to identify all components contributing to the plant’s therapeutic effects and, consequently, account for potential synergistic interactions [11].
Metabonomics primarily focuses on analyzing how living systems respond metabolically to biological stimuli or genetic changes on a global and dynamic scale. In recent years, the term has evolved beyond its traditional usage and is now often associated with a systems biology-driven approach. This perspective examines the
functional changes and disruptions within biological systems triggered by pharmacological effects, providing a thorough understanding of both the natural product and
its impact on the organism. Metabolomic profiling of natural products using technologies such as ultra-performance liquid chromatography–quadrupole TOF MS (UPLC–
MS) enables the identification of components responsible for therapeutic effects in
plants. Metabolomic and metabonomic profiling conducted using NMR, MS, and UPLC
also provides insights into the pharmacodynamic, pharmacokinetic, and toxicological
properties of natural products [188].
2.4.3 Proteomics
The proteome represents the cumulative composition of all proteins expressed in a
cell, tissue, or organism. Proteomics, on the other hand, is the scientific field that studies the flow of information through pathways and networks to understand the functional relationships of proteins. Proteomics involves the detailed analysis of proteins
in a sample, covering protein mapping and characterization, as well as the study of
their associated structures and functions. However, proteomics is highly complex due
to the vast scope of the analyzed domain (over 100,000 proteins) and the challenges in
detecting rare proteins. Nonetheless, the dynamic responsiveness of the proteome to
both genetic and environmental variations makes it a highly promising area for biomarker discovery. The potential of proteins to be widely affected in disease conditions
highlights their role in the diversity of disease biomarkers discovered to date, enabled
by proteomic technologies. Therefore, proteomic data analysis provides a multifaceted perspective for understanding disease mechanisms and developing new therapeutic strategies [201].
Advancements in MS-based proteomics have significantly contributed to unraveling biological systems, understanding disease mechanisms, and establishing links be-