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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5217_Библиотеки_им_академика_М_И_Перельмана.pdf
Drug
Discovery
from Plants
Pharmacology
Pharmacognosy
Molecular
biology
Microbiology
Toxicology
Biochemistry
Botany
Chemistry
Figure 2.5: Interdisciplinary collaboration in drug discovery from plants.
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 53
The discovery of new therapeutic compounds from plants is made possible
through the collaboration of various fields: botany, taxonomy, ethnobotany, and plant
ecology in the selection, collection, and species identification of plants; pharmacognosy and chemistry in the extraction, analysis, and isolation of raw materials; and
molecular biology, biochemistry, microbiology, pharmacology, and toxicology in the
evaluation of therapeutic efficacy (Figure 2.5).
2.3.1 Plant selection
The first step in discovering new drugs from plant sources is identifying and collecting plant species with potential therapeutic effects. The successful discovery and development of drugs heavily rely on the effective and efficient selection of plants that
align with the research objectives [11].
Plants can be selected based on knowledge or randomly without prior information and subjected to screening for potential therapeutic effects. However, whether
selected based on knowledge or randomly, they remain extremely valuable resources
for the pharmaceutical industry [35].
54 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
Selecting plant species for drug development based on a well-designed, knowledgebased strategy increases the success rate compared to random plant selection [7]. In
knowledge-based plant selection, records of traditional uses and natural compound libraries should be thoroughly investigated [6]. For any natural product to be used as a
drug, it must first be identified as having a potential therapeutic effect on a symptom
or disease. Following this, it must undergo preclinical and clinical trials to establish its
efficacy and safety. This process is long and challenging. Proceeding without an appropriate and detailed screening and well-founded hypothesis may result in wasted time
and inefficient outcomes in bioactivity tests [36].
Knowledge-based plant selection refers to the process of choosing plants for drug
discovery or other applications based on existing information, such as traditional uses,
ethnopharmacological data, chemical composition, or biological activity. In some studies, plants are also selected using an ecological approach, which considers their biodiversity and chemical diversity, morphological characteristics, phylogenetic traits, and
chemical defense mechanisms, in addition to the ethnopharmacological approach [11].
Two primary approaches can be identified for knowledge-based plant selection in
drug discovery. The first approach is based on ethnopharmacological research, where
the starting point is the plant itself. In this approach, a potential plant is identified
first, and then, through various analyses, the compound responsible for the activity is
obtained. The second approach involves identifying potential active compounds using
modern screening technologies, where the starting point is the natural compound.
The plant or plants containing the desired active compound are then determined [35].
Over years of research, libraries composed of compounds derived from natural
sources have been developed. Studies aimed at examining the bioactivities of these natural compounds seek to link findings either to the original plant source or to another
potential plant source. These studies represent an approach where plant selection is
based on specific molecules. Through this method, it is possible to discover new biological activities from natural compounds with known structures and origins [11].
In recent years, HTS technologies that accelerate the selection process of plants
promising therapeutic effects have been developed. Since these technologies allow
the testing of very small sample quantities, it facilitates the screening of natural products that are difficult to isolate, purify, and synthesize. With the integration of AI into
the drug discovery process from plants, the library of natural compounds has expanded, and the number of potential target molecules with therapeutic effects has increased [13]. The application of genomics, transcriptomics, proteomics, and metabolomics in the evaluation of natural molecules with drug potential has enabled the
discovery of new therapeutic candidates through more effective and advanced screening methods. Various contemporary techniques, such as database mining, virtual
screening, natural product libraries, and molecular modeling, are also applied in the
transformation of natural compounds into pharmaceutical agents [7, 9]. The main
methods used in plant selection are shown in Figure 2.6.
Plant Selection
Random
selection
Etnopharmacological
approach
High-Througput
Screening
Virtual Screening
Figure 2.6: Selecting plants for new drug discovery and development.
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 55
2.3.1.1 Random plant selection
Random selection of medicinal and aromatic plants is a method of source plant research where ethnobotanical or ethnopharmacological knowledge plays almost no
role. In this approach, plants are typically screened regionally for activity or sometimes for specific target secondary metabolites (flavonoids, alkaloids, terpenoids, etc.)
[7]. The plant-based screening and research efforts by the United States National Cancer Institute (NCI) for cancer therapeutic agents serve as an example of this approach.
The NCI and the Central Drug Research Institute (CDRI) screened approximately
35,000 plant species for anticancer activity between 1960 and 1980 [37]. Paclitaxel and
camptothecin are notable results of this screening process and are now used in chemotherapy [38].
The large number and complex chemical composition of plants limit the approach
of randomly collecting plants in the drug discovery process. After random selection,
the processes become more complex and time-consuming, making it more challenging
to identify plant-based bioactive compounds and understand their mechanisms of action. Additionally, conducting trials with numerous plants to identify bioactive compounds that exhibit the desired efficacy can be a highly costly endeavor [11, 35].
2.3.1.2 Plant selection based on ethnopharmacology and traditional uses
The knowledge accumulated from centuries of plant use by humans has provided
valuable guidance for numerous scientific studies. The ways in which plants can be
used for various diseases, and the specific applications, have been passed down from
generation to generation, first through oral traditions and later through written documents and records. The scientific investigation of the effects of plants with traditional
uses, as well as the identification of their phytochemical contents and compounds potentially responsible for these effects, is extremely important for their preparation
and application as medicinal products.
56 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
The decision to investigate a specific plant species for drug discovery is often based
on its traditional use as a medicine, insect repellent, or for a cultural purpose. The analysis of plants or plant extracts is guided by their traditional uses [11]. The traditional
use of plants in medicine can provide insights into their efficacy and safety. Collaborating with local communities to leverage their knowledge and experience with plants is
essential for researching medicinal plants suitable for treatment and for advancing
new drug discovery. Ethnopharmacological field studies conducted prior to the plant
selection stage will serve as a guide for this purpose. As a result of these field studies,
the creation of regional ethnopharmacological databases will provide an invaluable resource for future research [5]. However, traditional medicinal plants cannot proceed to
the clinical usage phase unless the knowledge obtained from traditional uses is validated through a long and labor-intensive series of analyses. Only 1 in 10,000 of the
tested compounds from plants with traditional uses has successfully made it to the path
of becoming a drug over an approximate 10-year timeframe [35].
The investigation of bioactive compounds in traditional herbal products is the
focus of ethnopharmacology. The fundamental principle of ethnopharmacology is to
investigate traditional medicines and their uses regionally, by combining them with
field observations. Most of the natural products that have been developed into drugs
currently have ethnomedical uses [18]. Drug discovery studies based on a plant’s
known activity according to traditional uses are still frequently applied by scientists
today. Drugs developed from plants such as Rauwolfia serpentina and Digitalis pur-
purea, as well as morphine derived from Papaver somniferum, and berberine derived
from Berberis aristata, are examples of drug discovery through ethnopharmacological
approaches. Many important drug compounds, such as artemisinin, quinine, atropine,
taxol, and aspirin, demonstrate the successful use of knowledge-based strategies with
natural products on the path to drug development [6, 13].
Advancements in isolation and characterization techniques, the development of
specific chemoinformatics methods, the rise in bioassay techniques, and HTS technologies have established systematic methodologies that bridge traditional ethnopharmacology and modern drug discovery. Ethnopharmacological studies remain a vital
starting point for drug discovery, just as they were historically. However, in the past,
these studies would first identify a plant of interest, followed by the isolation of its
active components. In contrast, modern approaches typically identify the active compound initially, and then use existing ethnopharmacological data to pinpoint plants
that contain these active ingredients. In plant-based drug development using ethnopharmacological data, the first step is to determine whether extracts obtained from
plants exhibit efficacy against a specific disease, independent of their structureactivity relationship [35]. There are numerous scientific publications reporting positive activity in various tests for plant extracts selected using ethnopharmacological
criteria. Once the activity is confirmed, the next step involves isolating and identifying
the active compound. The isolated active compound becomes a verified potential drug
candidate or can be developed into a new drug through further studies. However,
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 57
combining this approach with other technologies can significantly increase the chances of success [11].
2.3.1.3 Plant selection by HTS technologies
For many years, natural products with potential as drug candidates have been
screened. HTS is a widely used technological method for this purpose [39].
Over years of research, libraries consisting of compounds derived from natural
sources have been created. Studies that aim to investigate the bioactivities of these
natural compounds and link the findings either to the original plant source or to another potential plant source focus on the selection of plants based on specific molecules. Through this method, it is possible to discover new biological activities from
natural compounds with known structures and sources [11].
The emergence of databases for natural compounds, the increase in computational power, and the application of new technologies such as AI have enabled the
development and application of computational methods for identifying new drug-like
compounds and their derivatives. Advancements in analytical and fractionation techniques used for the identification, isolation, and purification of natural compounds
have made the screening of natural compounds using HTS more compatible and efficient. This facilitates the screening of thousands of compounds for their therapeutic
effectiveness and safety in a short time and at significantly lower costs [40].
In the past, creating natural product libraries was a challenging, complex, and
slow process. However, with the emergence of new technologies, this process has become faster and more efficient, allowing natural compounds to be seamlessly integrated into modern screening technologies [34].
HTS is an advanced technology that plays a critical role in the drug discovery process. Essentially, it is a method supported by robots, detectors, and software that enables the rapid and efficient screening and testing of chemical compounds. The primary function of HTS is to accelerate drug development by identifying the potential
interaction of chemical compounds with biological targets. By allowing the simultaneous screening of large volumes of compounds, this method enables the analysis of
up to 100,000 compounds per day in modern applications [41].
HTS technologies are also crucial in screening plant-derived compounds. Plants
are a rich source of compounds with therapeutic potential. However, traditional
methods for studying plants often yield results over extended periods. To address this
issue, high-throughput pharmacological screening (HTPS) can be applied to crude
plant extracts. Using the “differential smart screening” method, the biological activities of compounds within crude extracts are measured, allowing plants that exhibit
the desired activity to be prioritized for further investigation [42].
Since the 2000s, automated HTS has become a central focus in drug discovery.
This innovation positioned combinatorial chemistry as a preferred method for devel-
58 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
oping drug candidates suitable for HTS leading numerous drug manufacturers to
move away from extract libraries developed through traditional screening. This shift
was driven by the perception that extract-based screening often led to the rediscovery
of known compounds. Additionally, the structural complexity of natural products was
seen as a significant challenge, requiring costly and time-intensive processes like total
synthesis and derivatization. Consequently, natural product-based drug discovery
was considered impractical, especially when compounded by supply chain issues and
long development timelines. In contrast, HTS technologies employ combinatorial
chemistry to rapidly generate large compound libraries, accelerating the identification of potential drug candidates. Over the past two decades, traditional natural product chemistry has been mostly supplanted by drug discovery that targets specific molecular pathways, which focuses on efficiently obtaining “hits” from these extensive
combinatorial libraries. Combinatorial chemistry has significantly transformed the
discovery of new chemical entities with biological activity, enabling the efficient development of structural analogues [43].
The success of the HTS process depends on the diversity of compound libraries
and the quality of screening assays. A successful HTS operation requires careful selection of biological activity tests. Through accurate assay methods, undesirable biological activities can be filtered out, leading to better outcomes [44]. As a result of numerous studies conducted on plants to date, libraries containing hundreds or even
thousands of natural compounds have been established [35]. This has accelerated the
discovery of drug candidate compounds by screening the natural compound chemical
space using advanced methods, including HTS technologies.
The isolation of the first natural protein tyrosine phosphatase 1B (PTP1B) inhibitor from Broussonetia papyrifera has been a successful example of the application of
HTS technology in drug discovery [13].
2.3.1.4 Plant selection through virtual screening
Virtual screening is a method that can be applied to both combinatorial chemistrygenerated molecular libraries and natural compound libraries. The primary goal of this
approach is to select potential drug candidate molecules from large libraries in a more
specific and reduced manner. Virtual screening focuses on two main approaches: ligandbased and structure-based screening. Ligand-based virtual screening selects potential
compounds for further testing based on the structural and activity data of known bioactive compounds. In contrast, structure-based virtual screening utilizes the threedimensional structure of a compound and techniques such as molecular docking to determine the compound’s optimal position and orientation within a binding site, thereby
predicting its potential bioactivity. When sufficient structural and activity data for a molecule are available, both techniques can be used together to achieve more successful results. Virtual screening has been shown to outperform HTS in certain cases [11].
PHYTOCHEMICAL
DATABASES
Chemical
Structures
Molecular
formulas
Three-dimensional
structures
Stereochemistry of
compounds
Pharmacological
Properties
Biological
activity profiles
Target
molecules
Mechanisms
of action
Physicochemical
Properties
Molecular
weight
Polarity
Solubility
Source
Information
Plant species
Geographic
distribution
Taxonomy
Figure 2.7: Basic information included in phytochemical databases.
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 59
2.3.1.5 Phytochemical databases
Bioactive phytochemicals possess the ability to bind to molecular targets or receptors
associated with specific diseases or physiological conditions. This characteristic of
plant-derived compounds makes them suitable for use in drug design through virtual
screening methods. Due to the high drug development potential of phytochemicals in
the field of computational drug design, database management systems are essential specifically for these compounds. Phytochemical databases are digital resources that systematically store chemical, biological, and pharmacological information about compounds derived from plant sources. A comprehensive database containing information
about medicinal plants and their components serves as a valuable resource for researchers working on drug development from medicinal plants. Such databases should
include detailed information about the chemical structures, pharmacological properties, and physicochemical characteristics of plant-derived compounds (Figure 2.7)
Examples of phytochemical natural product databases include CVDHD, KNAPSACK, Nutrichem, Phytochemica, TCMID, TCM@Taiwan, TCM-Mesh, MAPS, and Phytochemdb.
While all these databases generally provide basic information, some are also suitable
for virtual screening. However, there is a need to expand these resources to encompass
both phytochemical and pharmacological data comprehensively. By doing so, phytochemical databases can become even more effective tools for in silico drug design, playing a larger role in the discovery of drugs from medicinal plants [45].
60 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
2.3.2 Collection and identification of selected plants
and pretreatment of plant materials
The step following the selection of candidate plants for drug discovery and development is the identification of the plant species and the collection of the necessary plant
materials. The accurate identification of the plant species is of vital importance in the
subsequent processes. For species identification of the collected plant materials, various analyses, including macroscopic, microscopic, and instrumental techniques, are
conducted in collaboration with the fields of botany and taxonomy. Advanced methods such as chemometrics, immunoassays, and DNA fingerprinting can also be used
for this purpose [7]. The morphological and anatomical characteristics of the plant
material should be determined by experts, knowledgeable and experienced individuals in the field, particularly taxonomists [33].
The first step in correctly identifying selected medicinal and aromatic plants is to
determine the plant’s botanical origin and identify its species name in binomial nomenclature. At this stage, organoleptic properties such as color, smell, taste, shape,
size, fracture characteristics, surface, and textural features should first be examined
macroscopically. Subsequently, specific structural and anatomical characteristics at
the tissue and cellular levels should be evaluated microscopically. In addition to these
methods, DNA barcoding is a reliable technique that provides secure information,
identifying plant species and quality assessment of medicinal and aromatic plants. Botanical species can be performed using DNA barcoding, where a short region of the
plant’s DNA sequence is used as a genetic marker [7].
For the extraction, isolation, and characterization of a bioactive compound from
plants, it is essential to have sufficient biological material. This requires the collection
of an adequate amount of material in the correct manner [12]. Legal and ethical regulations must be followed when collecting plants and plant materials. A sample of the
species-verified reference material must be recorded and preserved in a herbarium
with a designated accession number [33].
The chemical makeup of medicinal plants is extremely complicated and can be
influenced by a variety of factors, including soil composition, growth and storage conditions, genetic makeup, harvest timing, processing techniques, and more [46]. Preserving the biomolecules in medicinal plants is crucial for all processes involving
these plants. Therefore, after plant materials are collected, they undergo certain pretreatment processes before the extraction stage. Sample pretreatment is an important
component of the sample preparation process in modern analytical methods. It is also
the most error-prone stage during analysis, and the pretreatment procedures applied
to plant materials significantly affect the phytochemicals in the final extract. The primary goal of pretreatment is to isolate target metabolites from the matrix and to enhance the selectivity, accuracy, reliability, reproducibility, and determinability of the
analysis. The proper preparation of plant material is one of the most important factors that enhance extraction efficiency [47, 48]. An effective plant material prepara-
Chapter 2 Methods of obtaining drugs from medicinal and aromatic plants 61
tion method should allow for the efficient and comprehensive isolation of both volatile and nonvolatile, alongside polar and nonpolar substances, irrespective of the compound’s location within the matrix, its classification, or the existence of other factors.
The method should also be durable and sensitive, for example, resistant to high temperatures. A well-chosen material pretreatment method enhances the accessibility of
phytoactive compounds and simultaneously facilitates their extraction [49].
Plant material to be processed is typically subjected to pretreatment steps such as
drying, lyophilization, crushing, grinding, homogenization, or steam distillation.
These pre-extraction processes enhance the active surface area, significantly improving extraction kinetics and, consequently, the yield of targeted metabolites [50, 51].
Drying
It is possible to extract plant samples from either fresh or dried plant material, including leaves, bark, roots, fruits, and flowers [48]. However, the analysis of plants and
herbal products is typically conducted on dried materials. This approach allows the
determination of component ratios on a dry mass basis [50].
Another reason for preferring dried materials in experimental studies is that
fresh materials tend to deteriorate more quickly over time, whereas dried materials
are more stable [48]. To prevent microbial and/or enzymatic degradation of the material, water activity must be eliminated through drying, freezing, or lyophilization [51].
The primary goal of the drying process is to prevent metabolic activities that could
lead to alterations in the chemical composition of the plant. This is achieved by reducing the water content in the plant material, which is essential for the proper functioning of plant enzymes. Thus, the drying process helps eliminate issues related to the
high water content in the material. The absence of water, coupled with high drying
temperatures, helps inhibit enzymes that might degrade the active compounds. Additionally, effective drying reduces the microbial load in the end product. It also substantially decreases the mass and volume of the material, leading to decreased expenditures on packaging, transportation, and warehousing [49].
The drying process can be carried out under natural or artificial conditions and
in various ways. The drying technique and temperature depend on the type and features of the components contained in the plant. The drying of natural products is typically performed in hot air or nitrogen-flow ventilated ovens. In the presence of volatile components, low-temperature drying is preferred. Drying with high heat can lead
to the depletion of these components and may also trigger the degradation of compounds in essential oils [52]. The drying process can lead to unpredictable degradation
of the phytochemical content of the plant, depending on the method used and the
characteristics of the plant components. Therefore, the appropriate drying method
should be selected after evaluating all necessary parameters [51]. The most commonly
used drying methods for plant materials are shown in Figure 2.8 [53].
One of the oldest drying methods used for plant-based raw materials is open-air
drying. While it can be carried out under sunlight or in the shade, drying under direct
Heat
Air Drying
Convection Drying
Microwave/Microwave-Vacuum
Drying (VM)
Freeze-Drying (Lyophilization)
-pump-assisted drying, Infrared
drying, Fluidized bed drying,
• Sun drying
• Shade drying
• Solar assisted drying
• Oven drying
• Heat transfer with convection
•
Hot air drying
••Heating with microwave
Vacuum pressure
Figure 2.8: Drying methods for plant materials.
62 İrem Yıldız Özbaş, Severina Pacifico, and Emre Özbaş
sunlight often leads to issues such as the degradation of the material’s aroma and
color. In the shade-drying method, the material is left to dry in an open or semi-open
space without direct exposure to sunlight. Drying is carried out under natural conditions, benefiting from air circulation. Shade drying allows for better preservation of
volatile compounds as well as the aroma and color of the material. However, all openair drying methods expose the material to environmental contaminants. Additionally,
due to the long drying times and the inability to control parameters such as temperature and pressure, this method has lost its importance in modern applications. Due to
the long drying times associated with sun and shade drying methods, a commonly
used alternative is hot air drying, also known as oven drying. Under artificial conditions, both temperature and pressure can be regulated as needed, depending on the
characteristics of the material. For this purpose, ventilated chambers heated using
various methods are preferred. This technique utilizes convection for heat transfer
and allows precise control over key parameters such as temperature, air circulation
speed, and drying duration. These adjustable features make it an efficient option for
drying plants and herbs while ensuring consistency and quality [53].
Due to the various limitations of traditional drying techniques, new and modern
methods have been developed for the drying of plant materials. Compared to traditional methods, these techniques offer improved preservation of bioactive compounds
as well as enhancements in the physical and chemical characteristics and organoleptic features of the dried products. Advanced drying techniques such as freeze-drying,
microwave drying, infrared drying, spray drying, and supercritical drying are particularly noteworthy. The most suitable drying techniques and conditions should be selected by considering the differences in the properties of the materials to be dried
and/or the target plant components of interest. Each drying method should be evaluated for its advantages and disadvantages in terms of drying kinetics and the quality
of the final dried products [54].