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Article Contents

Decoding of life: genome analyses and functional characterization, kiss and ride: molecular interactions, femto is no limit: the content of plant tissues, seeing is believing: microscopy as an indispensable technique for studying plants, rewrite ‘whatever’: gene editing, conclusions, acknowledgements, conflict of interest, methods in plant science.

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Martin Janda, Methods in plant science, Journal of Experimental Botany , Volume 75, Issue 17, 11 September 2024, Pages 5163–5168, https://doi.org/10.1093/jxb/erae328

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The development of new techniques and technical advances in existing methods are the driving force in any area of research, including experimental botany. Improving our knowledge about the mechanisms of how the ‘world works’ is a necessary first step in creating a new technology. On the other hand, very often, it needs an advance in methods in order to make a new scientific discovery. Research in plant biology is no exception to this, and it often paves the way for the development of new methods or approaches that can have applications across wider subject areas.

The idea to put together this special issue came via the support of the Journal of Experimental Botany for the conference ‘Methods in Plant Sciences 2023’, held in Srní, Czech Republic. The meeting brought together more than 200 plant biologists and focused exclusively on techniques applicable to plant sciences. Several participants of the conference accepted the offer to write reviews about methodology approaches within their fields of expertise, and they represent the core of this issue, whilst other contributors have provided original reseach papers and also additional reviews. The result is that the papers cover a wide range of methods and approaches, from molecular dynamics through -omics and microscopy to monitoring plant–herbivore interactions. The articles can broadly be divided into two categories, namely texts focusing on a single methodological approach, its potential applications, and associated pitfalls, and those focusing on complex biological phenomena that require multiple methodological approaches in order to be properly characterized and understood.

In early studies, the variety of available methods was limited, and often only one technique was used. Thus, the findings were burdened by assumptions and potential shortcomings in the method. For example, in 1671, Grew and Malpigni used a recently developed microscope to describe pollen grains ( Manten, 1969 ): nowadays, basic research in experimental plant biology is not satisfied just with mere description but is focused on gaining an in-depth understanding of the underlying mechanism. In such studies, using one approach is insufficient, and scientists need to integrate multiple available methods—and sometimes improving them—to push our knowledge further. I am convinced that the papers in this special issue provide insightful information about recent advances in a wide spectrum of plant methods and will be useful for a broad audience.

Knowledge of hereditary information is one of the fundamental building blocks in understanding how organisms work, and plant science has benefited greatly in recent years from advances in sequencing technologies. The first plant genome to be fully sequenced was published in 2000, the same year as the first human genome, and it was that of the flowering plant Arabidopsis thaliana ( The Arabidopsis Genome Initiative, 2000 ). The Arabidopsis genome project took approximately 10 years from its beginnings and cost around 100 million USD; nowadays, it costs under 1000 USD to obtain a high-quality Arabidopsis genome, and the results are available within a week ( Li and Harkess, 2018 ). Thus, it is not surprising that more than a thousand Arabidopsis ecotypes have been sequenced ( 1001 Genomes Consortium, 2016 ), and projects aiming at sequencing tens of thousands of plants have become realistic ( https://db.cngb.org/10kp/ ).

Among other uses, such large datasets form the basis of so-called genome-wide association studies (GWAS). GWAS are capable of providing information on the functional involvement of genes within the studied biological process ( Nordborg and Weigel, 2008 ). To perform GWAS effectively, in addition to the availability of a high-throughput screening method, such as seedling growth analysis, it is also necessary to use the best possible algorithm to search for mutations within the collection. In this issue, John et al . (2024) present technical improvements regarding the approach to analysing sequence data using a permutational GWAS method.

Hereditary information is not just stored in the plant cell nucleus, with DNA also being found in mitochondria and plastids. The development of techniques known collectively as next-generation sequencing has in particular made it possible to analyse DNA sequences from these organelles. In their review, Štorchová and Krüger (2024) focus on the analysis of mitochondrial genomes in plants, describing the bottlenecks that stand in the way of obtaining hereditary information from them while highlighting that different next-generation sequencing techniques do not all provide the same results, and that using a combination of them appears to be the most efficient way to obtain high-quality and reliable mitochondrial DNA sequences. Hereditary information and its expression in mitochondria is also the subject of a review in this issue by Kwasniak-Owczarek and Janska (2024) . The authors focus on the translation of genes encoded in semi-autonomous organelles, pointing out that translation is a fundamental regulatory element in the expression of these genes. They also describe techniques that have not yet been applied to plants.

The development of the polymerase chain reaction (PCR) method, for which Kary Mullis was awarded the Nobel Prize in 1993, enabled gene transcription analysis and was a milestone for studying the involvement influence of individual genes on events in living organisms. Normally, samples prepared for transcriptomic analysis consist of the genetic information of many plant cells and cell types. For example, researchers collect RNA from the whole or at least part of the leaf. However, in recent years, attention has been turned to being able to analyse transcription in single cells ( Tang et al. , 2009 ). This technological advance has led to the expansion of analyses at the single-cell level beyond transcriptomics, and techniques are now also being developed for other ‘-omics’ ( Bennett et al ., 2023 ). In this special issue, Tenorio Berrío and Dubois (2024) discuss the potential of single-cell transcriptomics in the context of plant stress responses. Data from single-cell studies, which can discern spatial patterns of cell responses within whole organs, are likely to provide an explanation for the events where homogenizing of the ‘whole leaf’ is too low-resolution, such as transcriptomic patterns within the leaf as a response to the pathogens.

Genome analysis does not end with the knowledge of the nucleotide sequence. The mixture of DNA and proteins that form the chromosomes is folded into a higher structure—chromatin—and its 3D structure has a very significant influence on the subsequent manifestation of hereditary information. Our current knowledge is summarized by Šimková et al. (2024) , and they consider the possibilities and limitations of different approaches to analyse genome-folding into a 3D architecture. They give particular focus on describing proximity ligation Hi-C methods. Detailed knowledge of the chromatin structure of individual chromosomes leads us to the goal of studying the functional wiring of individual chromosomes. In this context, sex chromosomes are an interesting research target, and this topic is reviewed by Hobza et al. (2024) .

Gene expression ends with the creation of the desired molecules, such as enzymes. Enzymes are essential components of metabolic processes that generate further compounds. All the molecules produced are transported to the site of their function, whether as enzymes, structural components, or energy sources. Molecules do not exist in a vacuum and are constantly interacting with other molecules. In this special issue, three reviews examine methods used to study the interactions of specific types of molecules: Cuadrado and van Damme (2024) focus on advances in protein–protein interactions, whilst Neubergerová and Pleskot (2024) and Škrabálková et al. (2024) focus on interactions between proteins and lipids, with each paper looking at the subject from a different perspective. The usage and power of molecular dynamics are described by Neubergerová and Pleskot, and biochemical, ‘wet’ molecular biology and microscopic approaches are presented by Škrabálková et al .

The molecules that influence most physiological phenotypes in plants are phytohormones, also referred to in the past as plant growth regulators. Perhaps surprisingly, phytohormones are also involved in the regulation of epigenetic events in plants: by their interactions with receptors or binding proteins, they are able to cause epigenetic changes. These interactions, their mechanisms of action, and the methods used to study the links between phytohormones and epigenetic regulation are discussed by Rudolf et al . (2024) .

Plants are ingenious chemical engineers. As such, they are able to synthesize a plethora of molecules, and they serve as a source of inspiration for organic chemists. In this special issue, Ćavar Zeljković et al. (2024) present an improved protocol for determining nitrogen-related metabolites, which include amino acids together with biogenic amines and their acetylated and methylated derivates. The method that they present enables the determination of the concentration of a total of 74 metabolites, some of the which have a limit of detection in the lower units of femtomoles per injection. The authors demonstrate the functionality of the protocol in the model dicot plant Arabidopsis thaliana , as well as in a variety of crop species: Solanum lycopersicum and Nicotiana tabacum (C 3 dicots), Zea mays (C 4 monocot) and Hordeum vulgare and Triticum aesetivum (C 3 monocots).

Petrik et al. (2024) focus on the analysis of phytohormones. A detailed knowledge of phytohormone concentrations in different parts of the plant and under different growing and stress conditions is needed to understand their effects. Phytohormones represent diverse types of molecules, and Petrik et al. refer to the art of their analysis as ‘hormonomics’, thereby placing it alongside traditional ‘-omics’ such as transcriptomics, proteomics, and metabolomics. They highlight the boom in phytohormonal analyses as evidenced by the large increase in publications focusing on their determination in recent years. Phytohormones can be analysed by targeted or non-targeted approaches and, in combination with microscopic techniques, it is now becoming possible to consider the analysis of their concentrations not only at organ-level resolution, but to approximate their concentrations at the cellular and even subcellular levels as well ( Petrik et al ., 2024 ), thus approaching that of single-cell transcriptomics as noted above. It is expected that using parallel reaction monitoring for high-resolution mass spectrometry, a sensitivity at 50 amol can be achieved, which makes the future use of this approach in ‘hormonics’ look promising.

Microscopy is a technique without which it is hard to imagine plant research. Its use in plants dates back to 1665 when Robert Hooke published the book Micrographia . From this early origin, the technique expanded massively, and today it is divided into many branches, such as bright-field, fluorescence, confocal, and electron microscopy. Each has its distinct limits in resolution and its own (dis)advantages. This special issue includes two original research articles that use microscopy as their main method. Daněk et al . (2024) improve the analysis and quantification of autophagosomes in a 3D perspective by critically evaluating the limitations of previously used techniques and comparing them with the modified approach that they present. Narasimhan et al. (2024) present a tool box for analysing the binding of peptides to their receptors, for which they have developed genetic tools to use at good spatiotemporal resolution in vivo . In addition, a review by Müller-Schüssele (2024) describes the creation of biosensors for the determination of redox dynamics that are analysed using microscopy..

A microscope is not always necessary to prove that ‘seeing is believing’. Sotta and Fujiwara (2024) describe the development of a new, inexpensive, and reliable high-throughput method for monitoring herbivore insect feeding in Arabidopsis, which ultilizes a scanner and custom-built macro software for the free ImageJ and R packages.

Good quality visualization contributes to a better understanding of the processes taking place in plants, and Jo and Kajala (2024) introduce a new open-source ggPlantmap programme for visualization of experimental data, in a similar fashion to that provided by ePlant ( Waese et al. , 2017 ).

Since the description of the revolutionary gene-editing technology using CRISPR/Cas9 ( Jinek et al ., 2012 ), researchers in nearly all fields of biology have harnessed its possibility of creating desired mutant organisms. In plants, the technology is being used and improved extensively. The database created by the EU-SAGE initiative, which aims to open the European Union to the use of new genomic techniques in agriculture, contains more than 800 studies that have already utilized CRISPR to create genome-edited crop plants ( https://www.eu-sage.eu/genome-search ).

In this special issue, Přibylová and Fischer (2024) have written a detailed ‘cookbook’ for planning and performing gene editing using CRISPR. The authors highlight details that are not easy (or are indeed impossible) to obtain from original research articles, such as the effect of the chromatin state on CRISPR/Cas9 activity. In another paper, Vats et al . (2024) discuss prime editing in plants, noting its advantages and, at the same time, the challenges that need to be overcome to use this technique effectively, such as the fact that prime editing works with very low efficiency in dicots.

CRISPR/Cas9 has opened up unexpected possibilities for transcribing genomes and made it possible to think about editing in virtually all plants. As a result, the current bottleneck in research is no longer editing itself but determining the most efficient and reliable transformation process for the plant chosen for gene editing.

From the very beginning, plant research has benefited from the most advanced methodological approaches that have been available and, in turn, it has often been part of their evolution, such as the birth of genetics ( Mendel, 1865 ) or gene silencing ( Baulcombe, 2002 ). It is rather like the ‘chicken-and-egg’ question: which came first, knowledge or method? One cannot be without the other. In the past, one available method (e.g. microscopy) and excellent observational talent were often enough to make significant scientific progress: such a situation is perhaps not imaginable today even in basic research. Now, significant advances in knowledge require a detailed description of the mechanisms of how the plant works, and this in turn requires a combination of the multiple, often state-of-the-art, methods that are available, many of which have only been developed just recently.

I am confident that the collection of articles assembled in this special issue will help a broad range of our colleagues obtain a good overview and better understanding of the techniques now available in plant biology. As an example, the fictional case study in Box 1 summarizes the possibilities of the methods and approaches presented in this issue to deal with a complex, long-term scientific goal.

This virtual case study examines the effects of herbivore-associated molecular patterns on plants, and serves to highlight the application of methods considered in this special issue. All the ‘results’ referred to and the associated discussion are fictitious.

(A) We have identified a novel peptide from larvae that can be used to treat plants and trigger their resistance. We monitored the damage caused by larvae to the plant using the methodology presented by Sotta and Fujiwara (2024) , [1] , which provides a very cheap and high-throughput approach. In addition to protection against larvae, we also observed a triggered immune response after the peptide treatment. This prompted us to ask what the molecular mechanism was, and we assumed that there must be a plant receptor recognizing the peptide. (B) Due to the availability of a collection of sequenced ecotypes of our plant, we were able to perform a genome-wide association study (GWAS) analysis and obtained a candidate gene for the receptor ( John et al ., 2024 ) [2] . The structure of the receptor revealed its localization on the plasma membrane. (C) We analysed the binding of the peptide to its receptor using the techniques described by Cuadrado and van Damme (2024) and Narasimhan et al . (2024) , [3,4] . We next characterized the receptor complex and the interaction between the two proteins. So far, we have not been able to perform prime editing on our plant to confirm the direct site of the peptide binding to the receptor ( Vats et al ., 2024 ) [5] . Using molecular dynamics, we have predicted the local composition of the plasma membrane around the receptor in the absence and presence of the peptide ( Neubergerová and Pleskot, 2024 ) [6] . A combination of molecular biology, biochemistry, and microscopy techniques confirmed the results obtained using molecular dynamics ( Škrabálková et al ., 2024 ) [7] . Using the approach described by Daňek et al. (2024) , [8] , we were able to demonstrate that the binding of the peptide to the receptor does not lead to autophagy. We performed a detailed single-cell transcriptomic analysis, as described by Tenorio Berrío and Dubois (2024) , [9] , and the results showed a clear similarity to typical biotic stress transcriptomic responses. Utilizing complex methods described by Rudolf et al . (2024) , [10] , we did not find any influence of the peptide treatment on the plant epigenetic landscape. One of the typical responses to biotic stress is changes in redox, which we analysed using the biosensors described by Müller-Schüssele (2024) , [11] . Another typical stress response is a change in the concentration of phytohormones, and we used the state-of-the-art methods described by Petrik et al . (2024) , [12] to examine these at a detailed level. Unsurprisingly, we found significant changes in the jasmonic and salicylic acid levels in distinct parts of the leaf. An intriguing observation from our transcriptomic analysis was that there might be changes in the abundance of nitrogen-related compounds. Using the techniques described by Ćavar Zeljković et al . (2024) , [13] , we were able to analyse these changes and identify altered concentrations in particular nitrogen-related compounds. We visualized the results obtained in (C) using the ggPlantap package ( Jo and Kajala, 2024 ) [14] . We have found that plants from other families, for example, sunflowers, do not respond well to the peptide. Focusing on sunflower, we determined that the homolog receptor protein contains a number of structural differences, effectively abolishing its interaction with the aphid peptide. (D) Therefore, we decided to edit the receptor gene in sunflower to make its sequence identical to that of our original model plant. For this, we utilized the CRISPR/Cas9 approach, as described in detail by Přibylová and Fischer (2024) [15] . As predicted, our genetic modification led to decreased damage caused by the larvae in sunflowers. Created with BioRender.com.

Many thanks to all the people who contributed to the success of the conference ‘Methods in Plant Sciences 2023’ in Srní, Czech Republic, and to all the authors who have contributed to this special issue, on whose work this editorial is based. I would like to thank Dr Štěpán Jeřábek of Columbia University (NY, USA) for help with editing the final draft of this text. Last but not least, I would like to thank the editorial office of the Journal of Experimental Botany for all their help and patience with the special issue.

The author declares that he has no conflict of interest in relation to this work.

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Cuadrado AF , Van Damme D. 2024 . Unlocking protein–protein interactions in plants: a comprehensive review of established and emerging techniques . Journal of Experimental Botany 75 , 5220 – 5236 . https://doi.org/10.1093/jxb/erae088

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SPECIALTY GRAND CHALLENGE article

Plant biology research: what is next.

• Department of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, United States

Plant biology is a key area of science that bears major weight in the mankind's ongoing and future efforts to combat the consequences of global warming, climate change, pollution, and population growth. An in-depth understanding of plant physiology is paramount to our ability to optimize current agricultural practices, to develop new crop varieties, or to implement biotechnological innovations in agriculture. The next-generation cultivars would have to withstand environmental contamination and a wider range of growth temperatures, soil nutrients and moisture levels and effectively deal with growing pathogen pressures to continue to yield well in even suboptimal conditions.

What are the next big questions in plant physiology, and plant biology in general, and what avenues of research should we be investigating and training students in for the next decade? As a plant scientist surrounded by like-minded individuals, I hear a lot of ideas that over time turn into buzz words, such as plant resilience, genotype-to-phenotype, data science, systems biology, biosensing, synthetic biology, neural networks, robustness, interdisciplinary training, new tool development, modeling, etc. What does it all mean and what are the main challenges that we should all be working on solving? Herein, I present my personal perspective on what the immediate questions and the biggest longer-term issues in plant science are. I suggest some themes and directions for future research in plant biology, some relatively obvious and some potentially unique, having been shaped by my own professional interests, experiences and the background in plant molecular genetics and physiology.

Integration, Packaging, Visualization and Interpretation of Existing OMICS and Genetic Data

For the past three decades, a lot of emphasis has been made on a small set of plant model organisms, primarily on Arabidopsis. There is no other plant on earth we know as much about as we do about this mustard weed. One clear need in the area of plant sciences is to make sense of the vast amount of descriptive phenotypic data that have been generated for this species and a handful of others—the transcriptome, metabolome, proteome, phenome, interactome, etc.—and the amazing genetic resources that have been built: mutants, transgenic lines and natural accession germplasm collections, tools and protocols, genomic sequences and other resources ( Koorneef and Meinke, 2010 ). Now, how do we organize these data into a series of integrated, comprehensive, user-friendly, cross-communicating databases that are easily accessible, searchable, trackable, and visual, with data that are downloadable and compatible with comparative analyses? How do we display the available data at a variety of scales, from the subcellular to the organismal and population level—think Google Earth but for an ecosystem or an agricultural field that allows you to zoom in and out to see the overview and the closeup—perhaps, by integrating and expanding existing initiative likes Plant Cell Atlas and ePlant ( Waese et al., 2017 ; Rhee et al., 2019 )? With the genome sequences of these select organisms in hand, often of multiple accessions of each, what can we learn about the genotype-to-phenotype relations? How can we use that knowledge to extrapolate the rules or patterns we discover in model organisms to species for which we have no experimental data beyond possibly a draft-quality genomic sequence and a few fragmentary phenotypic datasets? In other words, can the data obtained in reference organisms be leveraged to infer useful information relevant to a wide range of species of agricultural, ecological or, perhaps, ethnobotanical importance? Let's look into some examples of that.

Translational Research: Moving Foundational Discoveries From Models to Crops

It comes as no surprise that for the past 10–20 years the emphasis has been gradually shifting from Arabidopsis to non-model organisms, including crops and rare plant species. The key reason for that is the pressing need to move fast on crop improvement and plant conservation in light of the worlds' fast-growing population, climate change, pollution, habitat and agricultural land loss, and ever-increasing pathogen pressures. This shift of research focus is also steered by changing governmental policies and funders' priorities. To make the transition to studying crops and other non-models as smooth as possible, robust computational pipelines are needed that produce high-quality genome assemblies from combinations of short- and long-read sequences. In this regard, tackling the much more complex genomes of polyploid species presents an even greater challenge. With the genome sequences and high-quality assembles on hand, orthologous genes that have previously been studied only in reference organisms need to be tested for function in candidate processes in the non-model species of interest to determine what aspects of their function are conserved and what features are divergent. The key bottleneck in this process is, of course, the recalcitrance of many non-models to genetic transformation and plant regeneration ( Anjanappa and Gruissem, 2021 ). Thus, a major effort would need to be invested into new method development to improve the plant in vitro culturing, genetic transformation and regeneration pipelines, with the ectopic activation of morphogenesis genes like BABY BOOM, WUSCHEL, LEAFY COTYLEDON1 and 2 , and several others holding major promise for boosting the regeneration efficiency of otherwise recalcitrant plant species and cultivars ( Gordon-Kamm et al., 2019 ). Further optimization of genome editing technologies, including classical gene disruption through indels as well as more targeted gene edits via base- and prime-editing or homologous-recombination-based methods, should enable highly tailored manipulation of genes of interest. The foundational knowledge gained in both model and non-model organisms can then be leveraged by applied plant biologists and environmentalists in crop improvement and plant conservation.

Interpreting the Code

One aspect of experimental research we have become good at over the past 10 years is genome and transcriptome sequencing. The current challenge is to learn to infer what the sequence tells us about what a gene does and how it is regulated based on the code alone. Can we look at gene's genomic sequence and infer not only the gene function, but also the different levels of gene regulation, all from just the sequence without any additional experimentation? To elaborate on that distinction between function and regulation, we can already infer the likely function of an orthologous gene in a crop (previously studied in another species) based on the degree of conservation of its genomic sequence, and deduce, for instance, an enzymatic reaction a protein may catalyze, or a DNA element a transcription factor may bind, or a specific ion the channel may transport, or an array of ligands or other molecules a protein may interact with. What we cannot yet reliably do is to predict based on the gene sequence alone when and where the gene is transcribed and what environmental or developmental stimuli alter its expression, how stable its transcript is, what splicing patterns the transcript has in specific cell types or conditions, or what factors dictate these patterns, or how well the transcript is translated, how the protein folds, where in the cell the protein is targeted, what its half-life is, and so on. Can we someday look at the gene sequence and predict whether the gene is essential or what organ or tissues will be affected in the loss- or gain-of-function mutant, and what phenotype the mutant will show, all without having to run an experiment? Once we learn to do that for a diploid model plant, can the knowledge be translated to polyploids that may have a greater level of gene redundancy and potentially more cases of neofunctionalization? How do we gain that extraordinary power?

One of the critical components of the inferring-the-function or genotype-to-phenotype challenge will involve machine learning and neural network models, with the size and quality of the training datasets presenting as the likely bottleneck that would determine the accuracy of neural networks' predictions ( Ching et al., 2018 ). While the role of computational biologists in this endeavor would be to develop new algorithms or adapt existing pipelines and test the models, the irreplaceable function of experimental plant biologists in this effort will be to generate the most complete and robust datasets for model training. This inevitably brings us to the next big theme, data quality.

Data Quality: Standardization, Reliability, Robustness and Tracking

As experimental scientists, most if not all of us have had the negative experience of not being able to reproduce an important result (sometimes even our own) or confirm the identity of a material someone has shared with us (e.g., a strain, a plasmid, or a seed stock from a colleague or another lab). Issues with biological variation (e.g., differences in germination between seed batches), small sample size (due to prohibitive cost, time or material constraints, or other limitations), human error (suboptimal labeling nomenclature, poor tracking, inadequate record keeping, substandard experimental design, miscalculation, personnel changes, or outright sloppiness) or malfunctioning instrumentation (in many cases, due to the lack of funding or time to upkeep or upgrade the equipment) can all contribute to the limited reproducibility of experimental data or sample mix-up. Rarely is the wrongdoing intentional, but the consequences of these errors can be enormous. What can we do to minimize mistakes, standardize internal lab protocols and record keeping, and ultimately improve the reproducibility of published data? I would support a universal funder's mandate for detailed electronic note keeping (much like private companies require), automatic data backups and regular equipment upgrades, meticulous planning before an experiment is run (including developing a comprehensive sample labeling nomenclature, beyond the common 1, 2, 3), inclusion of universal controls (e.g., Arabidopsis Columbia accession included in every Arabidopsis experiment irrespective of what other germplasm is being tested), extensive sample replication, validation of the results at multiple steps in the process (like Sanger sequencing of construct intermediates), and other common-sense but often time-consuming practices (such as regrowing all genotypes side by side and using fresh seed stocks in an experiment to minimize seed batch effects, or resequencing every construct before donating it to the stock center or sharing it with others).

A different yet related constraint we often encounter in plant sciences is the inability to track and/or obtain the materials or datasets reported by other research groups or oftentimes even by prior members of one's own lab. To ensure the long-term availability and unrestricted access to published constructs, germplasm, omics datasets and other resources generated by the public sector, funding agencies should make it mandatory for all materials and data to be deposited in relevant stock centers, sequence repositories, etc. immediately upon publication. I often wonder whether this practice could be encouraged if one's scientific productivity and impact were to be evaluated not only by the number of papers published, but also by the number of stocks or datasets deposited and their usage by the community (e.g., the frequency of stock orders or data downloads). Publishers, on the other hand, should fully enforce the old rules that all submitted manuscripts must adhere to the established guidelines for proper scientific nomenclature (e.g., gene accession numbers, mutant names, or chemical structures) and include community access codes (e.g., gene identifiers, mutant stock numbers, Genbank accession codes, etc.) and detailed annotations for all materials and data utilized or generated in a study, with the compliance being a prerequisite for publication. These simple steps would reduce ambiguities, facilitate resource tracking, and make published materials and datasets universally available.

The extra effort invested into careful experiment planning, execution, record keeping, and making published materials and datasets trackable and accessible will undoubtedly lead to fewer but higher-quality research papers being published and ultimately save time and resources down the road. Of course, an external mandate for greater rigor and accountability would also mean the need for funding agencies to financially support the extra effort and develop ways to monitor the labs' adherence to the new stricter rigor and dissemination practices, but it is commonsense that in the long run it is cheaper to do the experiment right the first time around than waste years trying to reproduce or follow up on erroneous data or remaking the resource that has been generated previously.

Synthetic Biology

An exciting and highly promising area of sciences that plant biologists are starting to embrace more widely is synthetic biology. First, what is synthetic biology? To a plant biologist, it is a useful extension of classical molecular genetics that integrates basic engineering principles and aims to rebuild biology from the ground up. Traditionally, classically trained biologists approach learning about nature from top to bottom, much like a curious child trying to break a toy apart to see what it is made of. Synthetic biologists, vice versa, try to rebuild a functional system from its pieces to understand what its minimal required components are. In plant biology, we are still very far from being able to rebuild entire plants or plant cells from scratch, but we can reconstitute the pathways, e.g., those that we have previously studied in their native context, in a heterologous host cell, aka the chassis, or introduce simple gene regulatory circuits we have artificially built. Why would we want to do that? For one, to see if we can recreate the native behavior to ensure that we fully understand the pathway or the mechanism of regulation. In addition, this can be a useful endeavor from a practical perspective, as is the case in metabolic engineering, where a native or semi-synthetic biosynthetic pathway is expressed in a heterologous host (an intact plant or a cell suspension) to produce a valuable metabolite ( Lu et al., 2016 ; Birchfield and McIntosh, 2020 ), or in biosensing, where a synthetic genetic construct is introduced to turn the host into a bio-detector for a particular stimulus or ligand of interest, e.g., a metabolite ( Garagounis et al., 2021 ).

We do not fully comprehend what we cannot ourselves recreate. We may know, for example, that a gene is induced, for example, by heat stress, but that observation does not tell us anything about the developmental regulation of that gene, or what other biotic or abiotic factors control this gene's expression. An illustrative example of how limited our current knowledge is and how synthetic biology can help us to bypass the lack of comprehensive understanding is to try the following mental exercise. How would one go about conferring a desired pattern of expression to a gene of interest, so that the gene is transcribed, for example, only in a flower, in the anthers at a particular stage of flower development, and only in response to heat stress? If we are talking about a model organism, we can scavenge available transcriptomic data in hopes of finding a native gene with such a pattern, but chances are that most anther-enriched genes will be expressed elsewhere and/or will be regulated by stimuli other than the heat stress. With the vast amount of transcriptomic data and limited ChIP-seq, DAP-seq and chromatin availability data (ATAC-seq, DNase-seq, etc.), we still have no reliable ways to infer transcription patterns of a native gene across all tissues and conditions. A combination of bioinformatic analysis (to identify putative transcription factor binding sites based on sequence conservation) ( Zemlyanskaya et al., 2021 ), classical transgene promoter bashing (that involves building a series of transgenes with chunks of the promoter deleted or replaced in an effort to characterize the effect of these targeted DNA modifications on the expression of a reporter gene in a systematic manner) ( Andersson and Sandelin, 2020 ), and/or more recently, in planta promoter bashing via genome editing (i.e., generating targeted promoter modifications directly in the native genomic context) ( Pandiarajan and Grover, 2018 ) are often relied upon to identify regulatory cis -elements in the promoters of interest. However, these approaches will not be enough to identify the full array of the DNA cis -elements that dictate the spatiotemporal regulation of a gene of interest, but these strategies may be helpful at pinpointing some candidate cis -elements and experimentally validating which elements are required.

If a particular DNA element is experimentally shown to be necessary, let's say, for heat stress upregulation, the next step is to test if the element is sufficient. This could be done by building a tandem of these elements, making a synthetic proximal promoter and placing it upstream of a well-characterized core promoter like that of 35S to drive a reporter ( Ali and Kim, 2019 ). In the best-case scenario, if we are successful with finding an element that can confer heat-inducible expression to the reporter, we have no easy way of restricting this heat-activated expression to just the anthers, let alone at a specific stage of anther development. Even if we had another DNA element at hand that confers tissue-specific expression (in this example, in anthers), we have no straightforward way of implementing what computer scientists would view as the Boolean AND logic—to combine these DNA elements (e.g., in a single proximal promoter) in a manner that the transcription of the gene will now only be triggered specifically in anthers in response to heat, but not in any other conditions or tissues. Synthetic biology makes the implementation of that AND logic (and other types of Boolean logic gates) possible, e.g., through the use of heterodimeric transcription factors, with one monomer active in anthers (through the use of an anther-specific promoter) and another monomer expressed only in response to heat stress (through the use of a heat-regulated promoter) ( Figure 1 ). In this scenario, the full heterodimeric transcription factor would only be reconstituted in the anthers of heat-treated plants and will activate its target genes only in those flower tissues specifically under heat stress.

Figure 1 . An example of a hypothetical genetic Boolean logic AND gate. AB is a heterodimeric transcription factor. If subunit A is expressed in anthers and subunit B is inducible by heat, the full transcription factor is reconstituted only in heat-stressed anthers. The AND logic restricts the expression of the output gene of interest specifically to the tissues and conditions where/when both A and B are-co-expressed.

Thus, synthetic biology enables us to build genetic devices capable of controlling specific processes of interest despite the lack of the full mechanistic understanding of all the moving parts in those processes. In the near future, more and more plant biologists will adopt synthetic biology as a powerful way to bypass some of the technical bottlenecks in plant sciences. Who knows, someday futuristic concepts of a minimal plant genome and a minimal plant cell ( Yang et al., 2020 ) may even become a reality. How soon will we have a thorough enough understanding of plant molecular genetics and physiology, so that we can determine the minimal set of genes to make a functional plant that can stay alive in a single stable (optimal) environment? What would we need to add to the minimal system to make the plant now capable of responding to stress and thriving in less-than-optimal conditions? Although one would agree that we have a very long way before we can get there, it is not too early to start thinking about those more ambitious projects, while working on still very difficult but more achievable shorter-term goals where synthetic biology will play a central role, such as developing nitrogen-fixing cereal crops ( Bloch et al., 2020 ) or C4 rice ( Ermakova et al., 2020 ).

Other Directions and Concluding Remarks

Several other areas relevant to plant sciences will have paramount importance to our ability to propel plant biology research forward. Advanced automated high-throughput imaging and phenotyping will provide a more systematic, robust way to collect reliable morphometric data on a diversity of plant species in the lab, the greenhouse, and the field. New computational tool development and the implementation of novel experimental methods, along with the optimization and streamlining of existing tools and protocols, will remain the main driver of research progress, with single-cell omics approaches likely taking center stage for the next few years. Data science will play an even more predominant role given the vast amount of new data being generated and the need to handle and make sense of all that information. Systems-level approaches, mathematical modeling and machine learning will become a more integral part of plant biology research, enabling scientists to systematize and prioritize complex data and provide plant researchers with experimentally testable predictions.

If we want to see the breakthroughs we are making at the bench or in the field implemented in real-life products, we also need to work on shifting the public perception of biotechnologies. Critical steps toward rebuilding public trust in science include a greater understanding of the societal impacts of proposed innovations through collaboration with social scientists, the engagement of researchers with the science policy making process, and the active participation of all scientists (students, postdocs, technicians, faculty, industry professionals, etc.) in community outreach programs to make our work—and its implications—accessible to the general public. Lastly, one essential factor that would make the scientific advancements sustainable in the long run is a generous investment into the robust, trans-disciplinary training of the next generation of plant scientists. Our ability to create a welcoming environment for trainees from all backgrounds and paths of life would allow these students and postdocs to feel that their research team is their second family. Today's trainees are the ones who will be solving the world's pressing issues for years to come. Our ability to provide young scientists with the solid knowledge base and diverse skills would ensure that they are well equipped to take on the next big challenge.

Looking ahead, fundamental research on model organisms, applied work on crops, and conservation studies on rare plants will all continue to be of vital importance to modern plant biology. High-throughput inquiries and gene-specific projects done by mega-groups and small labs in state-of-the-art facilities or traditional field labs will all remain indispensable to the progress of plant sciences. In the end, addressing pressing societal issues like feeding the world's growing population and mitigating climate change ultimately rests on our ability as scientists to come together and harness the power of plants. Plant biology research is positioned to play a central role in this critical endeavor. It is an exciting and urgent time to be—or become—a plant scientist.

Author Contributions

The author confirms being the sole contributor of this work and has approved it for publication.

The work in the Stepanova lab is supported by the National Science Foundation grants NSF 1750006, NSF 1444561, NSF 1940829.

Conflict of Interest

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Keywords: plant biology, plant physiology, synthetic biology, translational research, data reproducibility

Citation: Stepanova AN (2021) Plant Biology Research: What Is Next? Front. Plant Sci. 12:749104. doi: 10.3389/fpls.2021.749104

Received: 05 August 2021; Accepted: 06 September 2021; Published: 30 September 2021.

Edited and reviewed by: Joshua L. Heazlewood , The University of Melbourne, Australia

Copyright © 2021 Stepanova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Anna N. Stepanova, atstepan@ncsu.edu

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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• J Pharm Bioallied Sci
• v.12(1); Jan-Mar 2020

Preparation of Medicinal Plants: Basic Extraction and Fractionation Procedures for Experimental Purposes

Abdullahi r. abubakar.

1 Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Bayero University, Kano, Nigeria

Mainul Haque

2 Unit of Pharmacology, Faculty of Medicine and Defence Health, National Defence University of Malaysia, Kuala Lumpur, Malaysia

Preparation of medicinal plants for experimental purposes is an initial step and key in achieving quality research outcome. It involves extraction and determination of quality and quantity of bioactive constituents before proceeding with the intended biological testing. The primary objective of this study was to evaluate various methods used in the preparation and screening of medicinal plants in our daily research. Although the extracts, bioactive fractions, or compounds obtained from medicinal plants are used for different purposes, the techniques involved in producing them are generally the same irrespective of the intended biological testing. The major stages included in acquiring quality bioactive molecule are the selection of an appropriate solvent, extraction methods, phytochemical screening procedures, fractionation methods, and identification techniques. The nitty-gritty of these methods and the exact road map followed solely depends on the research design. Solvents commonly used in extraction of medicinal plants are polar solvent (e.g., water, alcohols), intermediate polar (e.g., acetone, dichloromethane), and nonpolar (e.g., n-hexane, ether, chloroform). In general, extraction procedures include maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, superficial extraction, ultrasound-assisted, and microwave-assisted extractions. Fractionation and purification of phytochemical substances are achieved through application of various chromatographic techniques such as paper chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography. Finally, compounds obtained are characterized using diverse identification techniques such as mass spectroscopy, infrared spectroscopy, ultraviolet spectroscopy, and nuclear magnetic resonance spectroscopy. Subsequently, different methods described above can be grouped and discussed according to the intended biological testing to guide young researchers and make them more focused.

I NTRODUCTION

Medicinal plants are extracted and processed for direct consumption as herbal or traditional medicine or prepared for experimental purposes. The concept of preparation of medicinal plant for experimental purposes involves the proper and timely collection of the plant, authentication by an expert, adequate drying, and grinding. This is followed by extraction, fractionation, and isolation of the bioactive compound where applicable. In addition, it comprises determination of quantity and quality of bioactive compounds.[ 1 , 2 , 3 , 4 , 5 ] Recently, plant as a source of medicine is gaining international popularity because of its natural origin, availability in local communities, cheaper to purchase, ease of administration, and perhaps less troublesome. Also, herbal medicine may be useful alternative treatment in case of numerous side effects and drug resistance.[ 1 , 2 , 3 , 4 , 5 ] Extraction of medicinal plants is a process of separating active plant materials or secondary metabolites such as alkaloids, flavonoids, terpenes, saponins, steroids, and glycosides from inert or inactive material using an appropriate solvent and standard extraction procedure. Plant materials with high content of phenolic compounds and flavonoids were found to possess antioxidant properties, and hence are used to treat age-related diseases such as Alzheimer’s disease, Parkinsonism, anxiety, and depression.[ 2 , 5 ] Several methods were used in the extraction of medicinal plants such as maceration, infusion, decoction, percolation, digestion and Soxhlet extraction, superficial extraction, ultrasound-assisted, and microwave-assisted extraction. In addition, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), paper chromatography (PC), and gas chromatography (GC) were used in separation and purification of the secondary metabolites.[ 1 , 2 , 3 , 4 , 5 ] The choice of an appropriate extraction method depends on the nature of the plant material, solvent used, pH of the solvent, temperature, and solvent to sample ration. It also depends on the intended use of the final products.[ 1 , 2 , 3 , 4 , 5 ] This study aimed to assess various solvents of extractions, methods of extraction, fractionation, purification, phytochemical screening, and identification of bioactive compounds in medicinal plants.

Definition of terms

Medicinal plant . It refers to a plant comprising active ingredients or secondary metabolites that possess biological activity. A whole plant may be medicinally active or plant parts.[ 4 , 6 , 7 ] Herbal medicine . These are medicinal preparations comprising active ingredients obtained from the herbal plant. The product can be made from the whole plant or any part. Preparations from by-product herbal plants such as oils, gums, and other secretions are also considered as herbal medicine.[ 4 , 6 , 7 ] Menstruum . It is a liquid or a suitable solvent chosen for an effective extraction process.[ 2 , 3 ] Marc . It is an insoluble or inert drug material that is left behind at the end of the extraction process.[ 2 , 3 ] Micelle . It is the mixture of both the extracted drug material and the solvent of extraction.[ 2 , 3 ] Primary plant constituents . These are mainly nutritional components of plants such as common sugars, amino acid, proteins, and chlorophyll. These have little or no medicinal properties.[ 6 , 7 ] Secondary plant constituents . These are also known as secondary metabolites such as alkaloids, terpenoids, saponins, phenolic compounds, flavonoids, and tannins. These are responsible for many biological or pharmacological activities.[ 6 , 7 ] Bioassay-guided fractionation . It involves extraction of plant material followed by testing for biological activity. Once the extract tested is found to be biologically active, the next step is to proceed with fractionation. Subsequently, various fractions obtained are tested for biological activity. Also, the most productive portion is then taken for compound isolation. Finally, the compound isolated is identified and tested for biological activity.[ 1 , 5 , 8 ] Bioautography . It is a process that uses both TLC and antimicrobial testing to establish the identity of a compound extracted as well as its antimicrobial activity.[ 5 , 9 ] Finger printing in medicinal plants . It involves the use of chromatographic techniques, identification techniques, and chemical analysis to characterize a pharmacologically active compound from a medicinal plant.[ 4 , 5 ] Immunoassay . It is a process of identification of bioactive molecule as well as its biological activity via immune reaction, receptor binding, and enzyme-mediated reactions. The extract and low-molecular-weight secondary metabolites first interact with monoclonal antibody to detect drug–receptor binding. This is followed by application of enzyme-linked immunoassay (ELISA) to determine its enzymatic activities.[ 5 ]

S OLVENTS OF E XTRACTION

The solvent used for the extraction of medicinal plants is also known as the menstruum. The choice of solvent depends on the type of plant, part of plant to be extracted, nature of the bioactive compounds, and the availability of solvent. In general, polar solvents such as water, methanol, and ethanol are used in extraction of polar compound, whereas nonpolar solvents such as hexane and dichloromethane are used in extraction of nonpolar compounds.[ 3 , 5 , 10 ] During liquid–liquid extraction, the conventional way is to select two miscible solvents such as water–dichloromethane, water–ether, and water–hexane. In all the combinations, water is present because of its high polarity and miscibility with organic solvent. The compound to be extracted using liquid–liquid extraction should be soluble in organic solvent but not in water to ease separation.[ 11 ] Furthermore, solvent used in extraction is classified according to their polarity, from n -hexane which is the least polar to water the most polar.[ 3 , 5 , 10 ] The following are 11 various solvents of extractions arranged according to the order of increasing polarity[ 3 , 9 ]:

During fractionation, the selected solvent is added according to the order of increasing polarity, starting from n -hexane, the least polar to water with the highest polarity.[ 3 , 9 ] If a researcher wishes to select five solvents during fractionation, the usual practice is to choose two solvents with low polarity ( n -hexane, chloroform), two with medium polarity (dichloromethane, n -butanol), and one with the highest polarity (water).

P ROPERTIES OF S OLVENT OF E XTRACTIONS

• (i) Water . It is the most polar solvent and is used in the extraction of a wide range of polar compounds.[ 9 , 12 ] Advantages . It dissolves a wide range of substances; it is cheap, nontoxic, nonflammable, and highly polar.[ 9 , 12 ] Disadvantages . It promotes bacterial and mold growth; it may cause hydrolysis, and a large amount of heat is required to concentrate the extract.[ 9 , 12 ]
• (ii) Alcohol . It is also polar in nature, miscible with water, and could extract polar secondary metabolites.[ 9 , 12 ] Advantages . It is self-preservative at a concentration above 20%. It is nontoxic at low concentration, and as small amount of heat is required for concentrating the extract.[ 9 , 12 ] Disadvantages . It does not dissolve fats, gums, and wax; it is flammable and volatile.[ 9 , 12 ]
• (iii) Chloroform . It is a nonpolar solvent and is useful in the extraction of compounds such as terpenoids, flavonoids, fats, and oils.[ 3 , 12 , 13 ] Advantages . It is colorless, has a sweet smell, and is soluble in alcohols. It is also well absorbed and metabolized in the body.[ 3 , 12 , 13 ] Disadvantages . It has sedative and carcinogenic property.[ 3 , 12 , 13 ]
• (iv) Ether . It is a nonpolar solvent and is useful in the extraction of compounds such as alkaloids, terpenoids, coumarins, and fatty acids.[ 3 , 12 , 13 ] Advantages . It is miscible with water, has low boiling point, and is tasteless in nature. It is also a very stable compound and does not react with acids, bases, and metals.[ 3 , 12 , 13 ] Disadvantages . It is highly volatile and flammable in nature.[ 3 , 12 , 13 ]
• (v) Ionic liquid (green solvent) . This is a unique solvent of extraction and is highly polar and extremely heat stable. It can remain in a liquid state even at 3,000oC and usable where high temperature is applicable. It has extreme miscibility with water and other solvent and is very suitable in the extraction of polar compounds.[ 14 ] Advantages . It has excellent solvent that attracts and transmit microwave, and hence it is suitable for microwave-assisted extraction. It is nonflammable and is useful for liquid-liquid extraction and highly polar.[ 14 ] Disadvantage . It is not ideal for preparation of tinctures.[ 14 ]

Factors to be considered in selecting solvents of extraction

Various factors enumerated below should be taken into consideration when choosing a solvent of extraction.[ 3 , 9 , 15 ] (i) Selectivity . The ability of a chosen solvent to extract the active constituent and leave the inert material. (ii) Safety . Ideal solvent of extraction should be nontoxic and nonflammable. (iii) Cost . It should be as cheap as possible. (iv) Reactivity . Suitable solvent of extraction should not react with the extract. (v) Recovery . The solvent of extraction should be quickly recovered and separated from the extract. (vi) Viscosity . Should be of low viscosity to allow ease of penetration. (vii) Boiling temperature . Solvent boiling temperature should be as low as possible to prevent degradation by heat.[ 3 , 9 , 15 ]

M ETHODS USED IN E XTRACTION OF M EDICINAL P LANTS

Quite numbers of procedures were technically used in the extraction of medicinal plants. Some newer methods are still evolving, whereas the existing ones are undergoing modifications.[ 2 , 5 ] The choice of an appropriate way of extraction is very vital, which in some cases depends on the intended use of an extract.

Factors to be considered in choosing extraction method

(a) Stability to heat . Heat-stable plant material is extracted using Soxhlet extraction or microwave-assisted extraction, whereas plant materials that are not heat stable are extracted using maceration or percolation.[ 2 , 11 ] (b) Nature of solvent . If the solvent of extraction is water, maceration is a suitable method but for volatile solvent percolation and Soxhlet extraction are more appropriate.[ 2 , 11 ] (c) Cost of the drug . Cheap drugs are extracted using maceration, whereas costly drugs are preferably extracted using percolation.[ 2 , 11 ] (d) Duration of extraction . Maceration is suitable for plant material requiring long exposure to the menstruum, whereas techniques such as microwave- or ultrasound-assisted extraction are used for a shorter duration.[ 2 , 11 ] (e) Final volume required . Large volume products such as tinctures are prepared by maceration, whereas concentrated products are produced by percolation or Soxhlet extraction.[ 2 , 11 ] (f) Intended use . Extracts intended for consumption by human are usually prepared by maceration, whereas products intended for experimental testing are prepared using other methods in addition to maceration.[ 2 , 11 ]

Commonly used methods in the extraction of medicinal plants

• (i) Maceration . This is an extraction procedure in which coarsely powdered drug material, either leaves or stem bark or root bark, is placed inside a container; the menstruum is poured on top until completely covered the drug material. The container is then closed and kept for at least three days.[ 1 , 2 , 3 , 4 , 11 , 16 ] The content is stirred periodically, and if placed inside bottle it should be shaken time to time to ensure complete extraction. At the end of extraction, the micelle is separated from marc by filtration or decantation. Subsequently, the micelle is then separated from the menstruum by evaporation in an oven or on top of water bath.[ 1 , 2 , 3 , 4 , 11 , 16 ] This method is convenient and very suitable for thermolabile plant material.
• (ii) Infusion . This is an extraction process such as maceration. The drug material is grinded into fine powder, and then placed inside a clean container. The extraction solvent hot or cold is then poured on top of the drug material, soaked, and kept for a short period of time.[ 1 , 2 , 3 , 11 ] This method is suitable for extraction bioactive constituents that are readily soluble. In addition, it is an appropriate method for preparation of fresh extract before use. The solvent to sample ratio is usually 4:1 or 16:1 depending on the intended use.[ 1 , 2 , 3 , 11 ]
• (iii) Digestion . This is an extraction method that involves the use of moderate heat during extraction process. The solvent of extraction is poured into a clean container followed by powdered drug material. The mixture is placed over water bath or in an oven at a temperature about 50 o C.[ 1 , 3 , 11 ] Heat was applied throughout the extraction process to decrease the viscosity of extraction solvent and enhance the removal of secondary metabolites. This method is suitable for plant materials that are readily soluble.[ 1 , 3 , 11 ]
• (iv) Decoctiona . This is a process that involves continuous hot extraction using specified volume of water as a solvent. A dried, grinded, and powdered plant material is placed into a clean container. Water is then poured and stirred. Heat is then applied throughout the process to hasten the extraction.[ 1 , 2 , 3 , 11 ] The process is lasted for a short duration usually about 15min. The ratio of solvent to crude drug is usually 4:1 or 16:1. It is used for extraction of water soluble and heat stable plant material.[ 1 , 2 , 3 , 11 ]
• (v) Percolation . The apparatus used in this process is called percolator. It is a narrow-cone-shaped glass vessel with opening at both ends. A dried, grinded, and finely powdered plant material is moistened with the solvent of extraction in a clean container. More quantity of solvent is added, and the mixture is kept for a period of 4h. Subsequently, the content is then transferred into percolator with the lower end closed and allow to stand for a period of 24h.[ 2 , 3 , 11 ] The solvent of extraction is then poured from the top until the drug material is completely saturated. The lower part of the percolator is then opened, and the liquid allowed to drip slowly. Some quantity of solvent was added continuously, and the extraction taken place by gravitational force, pushing the solvent through the drug material downward.[ 2 , 3 , 11 ] The addition of solvent stopped when the volume of solvent added reached 75% of the intended quantity of the entire preparations. The extract is separated by filtration followed by decantation. The marc is then expressed and final amount of solvent added to get required volume.[ 2 , 3 , 11 ]
• (vi) Soxhlet extraction . This process is otherwise known as continuous hot extraction. The apparatus is called Soxhlet extractor made up of glass. It consists of a round bottom flask, extraction chamber, siphon tube, and condenser at the top. A dried, grinded, and finely powdered plant material is placed inside porous bag (thimble) made up of a clean cloth or strong filter paper and tightly closed.[ 1 , 2 , 3 , 4 , 11 , 17 , 18 ] The extraction solvent is poured into the bottom flask, followed by the thimble into the extraction chamber. The solvent is then heated from the bottom flask, evaporates, and passes through the condenser where it condenses and flow down to the extraction chamber and extracts the drug by coming in contact. Consequently, when the level of solvent in the extraction chamber reaches the top of the siphon, the solvent and the extracted plant material flow back to the flask.[ 1 , 2 , 3 , 4 , 11 , 17 , 18 ] The entire process continues repeatedly until the drug is completely extracted, a point when a solvent flowing from extraction chamber does not leave any residue behind. This method is suitable for plant material that is partially soluble in the chosen solvent and for plant materials with insoluble impurities. However, it is not a suitable method for thermolabile plant materials. Advantages . Large amount of drug can be extracted with smaller amount of solvent. It is also applicable to plant materials that are heat stable. No filtration is required, and high amount of heat could be applied.[ 1 , 2 , 3 , 4 , 11 , 17 , 18 ] Disadvantages . Regular shaking is not possible, and the method is not suitable for thermolabile materials.[ 1 , 2 , 3 , 4 , 11 , 17 , 18 ]
• (vii) Microwave-assisted extraction . This is one of the advanced extraction procedures in preparation of medicinal plants. The technique uses mechanism of dipole rotation and ionic transfer by displacement of charged ions present in the solvent and drug material. This method is suitable for extraction of flavonoids. It involves the application of electromagnetic radiation in frequencies between 300 MHz and 300 GHz and wavelength between 1cm and 1 m.[ 1 , 4 , 10 , 14 ] The microwaves applied at frequency of 2450 Hz yielded energy between 600 and 700 W. The technique uses microwave radiation to bombard an object, which can absorb electromagnetic energy and convert it into heat. Subsequently, the heat produced facilitates movement of solvent into the drug matrix.[ 1 , 4 , 10 , 14 ] When polar solvent is used, dipole rotation and migration of ions occur, increase solvent penetration, and assist extraction process. However, when nonpolar solvent is used, the microwave radiation released will produce only small heat; hence, this method does not favor use of nonpolar solvents.[ 1 , 4 , 10 , 14 ] Advantages . Microwave-assisted extraction has special advantages such as minimizing solvent and time of extraction as well as increase in the outcome.[ 1 , 4 , 10 , 14 ] Disadvantages . This method is suitable only for phenolic compounds and flavonoids. Compounds such as tannins and anthocyanins may be degraded because of high temperature involved.[ 1 , 4 , 10 , 14 ]
• (viii) Ultrasound-assisted extraction . This process involves application of sound energy at a very high frequency greater than 20 KHz to disrupt plant cell all and increase the drug surface area for solvent penetration. Consequently, secondary metabolites will be released. In this method, plant material should dry first, grinded into fine power, and sieved properly. The prepared sample is then mixed with and appropriate solvent of extraction and packed into the ultrasonic extractor.[ 2 , 3 , 10 ] The high sound energy applies hasten the extraction process by reducing the heat requirements. Advantages . Ultrasound-assisted extraction is applicable to small sample; it reduces the time of extraction and amount of solvent used, and maximizes the yield.[ 2 , 3 , 10 ] Disadvantages . This method is difficult to be reproduced; also, high amount of energy applied may degrade the phytochemical by producing free radical.[ 2 , 3 , 10 ]

P HYTOCHEMICAL S CREENING M wETHODS

Phytochemical screenings are preliminary tests conducted to detect the presence of both primary and secondary metabolites in an extract. Several qualitative analyses described below have been used to detect the presence of alkaloids, flavonoids, tannins, saponins, flavones, sterols, terpenes, cardiac glycosides, protein, carbohydrates, and fats.[ 3 , 19 , 20 , 21 ]

Test for alkaloids

(a) Dragendorff’s test . 1mL of extract was taken and placed into a test tube. Then 1mL of potassium bismuth iodide solution (Dragendorff’s reagent) was added and shaken. An orange red precipitate formed indicates the presence of alkaloids.[ 3 , 19 , 20 , 21 ] (b) Wagner’s test . 1mL of extract was taken and placed into a test tube. Then 1mL of potassium iodide (Wagner’s reagent) was added and shaken. Appearance of reddish brown precipitate signifies the existence of alkaloids.[ 3 , 19 , 20 , 21 , 22 ] (c) Mayer’s test . 1mL of extract was taken and placed into a test tube. Then 1mL of potassium mercuric iodide solution (Mayer’s reagent) was added and shaken. Emergence of whitish or cream precipitate implies the presence of alkaloids.[ 3 , 19 , 20 , 21 ] (d) Hager’s test . 1mL of solution of an extract was taken and placed into a test tube. Then 1mL of saturated ferric solution (Hager’s reagent) was added and shaken. Formation of yellow-colored precipitate indicates the existence of alkaloids.[ 3 , 19 , 20 , 21 ]

Test for glycosides

(a) Bontrager’s test (modified) . One gram of the crude extract was first weighed, placed into a test tube, and dissolved in 5mL of dilute hydrochloric acid. Then 5mL of ferric chloride (5%) solution was added. The mixture was shaken and placed over water bath. Then the mixture was allowed to boil for 10min, cooled, and filtered.[ 3 , 19 , 20 , 21 ] Afterward, the mixture was then extracted again with benzene. Finally, equal volume of ammonia solution was added to benzene layer. Appearance of pink color indicates the presence of anthraquinone glycosides.[ 3 , 19 , 20 , 21 ] (b) Legals test . 1mL of an extract was taken, and then an equal volume of sodium nitroprusside was added followed by few quantity of sodium hydroxide solution and shaken. Formation of pink-to-blood-red precipitate signifies the existence of cardiac glycoside.[ 3 , 19 , 20 , 21 ] (c) Keller–Killiani test . 2mL of the extract was taken and diluted with equal volume of water. Then 0.5mL of lead acetate was added, shaken, and filtered. Again, the mixture was extracted with equal volume of chloroform, evaporated, and dissolved the residue in glacial acetic acid. Then few drops of ferric chloride was added.[ 3 , 19 , 20 , 21 ] Again, the whole mixture was placed into a test tube containing 2mL of sulfuric acid. Emergence of reddish brown layer that turns bluish green implies the presence of digitoxose.[ 3 , 19 , 20 , 21 ]

Test for steroids and triterpenoids

(a) Libermann Burchard’s test . This method is utilized for an alcoholic extract. Extract need to dry out first through evaporation, then extracted again with chloroform.Add few drops of acetic anhydrites followed by sulfuric acid from the side of the test tube. Formation of violet to blue-colored ring at the junction of the two liquids indicated the presence of steroids.[ 3 , 19 , 20 , 21 , 22 ] (b) Salkowski’s test . 1mL solution of the extract was taken and 2mL of chloroform was added, shaken, and filtered. Few drops of concentrated sulfuric acid were added to filtrate, shaken, and allowed to stand. Development of golden-yellow precipitate indicates the presence of triterpenes.[ 3 , 19 , 20 , 21 , 22 ]

Test for tannins

(a) Gold Beater’s skin test . A Gold Beater’s Skin was obtained from Ox skin. The Gold Beater’s Skin was soaked in 2% hydrochloric acid and washed with distilled water. Then it was placed in a solution of an extract for 5min and washed with distilled water. Finally, it was placed in 1% ferrous sulfate solution. If the Gold Beater’s Skin changed to brown or black tannins is present.[ 3 , 19 , 20 , 21 ] (b) Gelatin’s test . 1mL of extract was taken and placed in a test tube. Then 1% gelatin solution containing sodium chloride added and shaken. Appearance of white precipitate indicates the presence of tannins.[ 3 , 19 , 20 , 21 , 22 ]

Test for flavonoids

(a) Shinoda’s test . 1mL of extract was taken and placed into a test tube. Then, few drops of concentrated hydrochloric acid was added followed by 0.5mg of mRimandoium turnings and shaken. Emergence of pink coloration indicates the presence of flavonoids.[ 3 , 19 , 20 , 21 ] (b) Lead acetate test . To detect the presence of flavonoids, 1mL of extract was taken and placed into a test tube. Then few drops of lead acetate added and shaken. Formation of yellow precipitate signifies the presence of flavonoids.[ 3 , 19 , 20 , 21 ] (c) Alkaline reagent test . 1mL of extract was taken and placed into a test tube. Then few drops of sodium hydroxide solution were added and shaken. Emergence of intense yellow color that turns to colorless after adding dilute acid implies the existence of flavonoids.[ 3 , 19 , 20 , 21 , 22 ]

Test for phenols

(a) Ferric chloride test . 1mL solution of an extract was taken and placed into a test tube. Then 1% gelatin solution containing sodium chloride was added and shaken. Formation of bluish-black color indicates the presence of phenols.[ 3 , 19 , 20 , 21 ] (b) Lead acetate test . 1mL solution of an extract was taken and placed into a test tube. Then 1mL of alcoholic solution was added, followed by dilution with 20% sulfuric acid. Finally, solution of sodium hydroxide was added. Formation of red-to-blue color signifies the occurrence of phenols.[ 3 , 19 , 20 , 21 ] (c) Gelatin test . A solution of plant extract was placed into test tube followed by 2mL of 1% gelatin solution and shaken. Appearance of white precipitate indicates the presence of phenols.[ 3 , 19 , 20 , 21 ] (d) Mayer’s reagent test (potassium mercuric iodide test). To a solution of plant extract, 1mL of Mayer’s reagent was added in an acidic solution. Manifestation of white precipitate shows the existence of phenolic compounds.[ 3 , 19 , 20 , 21 ]

Test for protein

(a) Biuret test . Some quantity of an extract was taken and 4% sodium hydroxide solution of the drug was produced. This is followed by the addition of 1 % copper sulfite. Appearance of violet color implies the existence of peptide linkage.[ 3 , 19 , 20 , 21 ] (b) Ninhydrin test . 1mL of an extract was taken and placed into a test tube. Then 0.25% of ninhydrin reagent was added and shaken. The mixture was then boiled for few minutes. Formation of blue color signifies the presence of protein. (c) Xanthoproteic test . 1mL of the extract was taken and placed it into a test tube. Then few drops of nitric acid were added and shaken. Emergence of yellow-color indicates presence of protein.[ 3 , 19 , 20 , 21 ]

F RACTIONATION AND P URIFICATION M ETHODS

Fractionation is a process of separation of plant extracts into various fractions. It further segregates the fractions into portions comprising a number of compounds. The process continues until pure compound is isolated.[ 4 , 8 , 23 ] When several solvents are required for the fractionation, they should be added according to the order of increasing polarity. Fractionation techniques are basically classified into physical or chemical method.

Chemical methods

This extraction method is based on the type of functional groups possessed by a compound in the given mixture. Separation or purification can be achieved by chemical reactions using appropriate reagents.[ 4 ]

Physical methods

Physical methods used in separation of compounds from mixtures include separation funnel method, chromatographic techniques, fractional distillation, fractional crystallization, fractional liberation, and sublimation.[ 4 ]

• (a) Separation funnel method . When four different solvents ( n -hexane, chloroform, acetone, and n -butanol) are selected, fractionation begins by moistening or complete dissolution of crude extract with 250mL of water. This is followed by transfer into a separating funnel, shaken, and allowed to settle. Furthermore, to 250mL of n -hexane, the least polar solvent was added and shaken. The content can settle, and the bottom of the separating funnel opened to remove the aqueous layer. The remaining content in the separating funnel was poured into a clean container to get n -hexane fraction.[ 1 , 5 , 8 , 24 ] Equal volume of n -hexane was added again, shaken, and separated. The addition continued until after adding n -hexane and shaken no reasonable quantity of extract appeared to move into the n -hexane portion.[ 1 , 5 , 8 , 24 ] Similar cycle was performed for chloroform, acetone, n -butanol to get chloroform, acetone, and n -butanol fractions. The remaining portion left after the fractionation is termed as residual aqueous fraction (RAF) as the crude extract was first dissolved in water.[ 1 , 5 , 8 , 24 ]
• (b) Fractional distillation . This is a process of separating or purifying compounds from a mixture. It is usually used in separation of hydrocarbons such as crude oil, citral, and eucalyptol. Purification is achieved based on the differences in their boiling points. Fractional distillation apparatus is constructed in such a manner that when heat is applied each compound will evaporate and separates at its boiling point. Consequently, each compound fractionated will condense and collected as a separate entity through several siphons attached to fractional distillation apparatus.[ 4 ]
• (c) Fractional crystallization . Large numbers of compounds that exist naturally in plant extracts are crystal in nature. Separation is achieved via formation of crystals during concentration of an extract using heat or refrigeration.[ 4 ]
• (d) Fractional liberation . This method is suitable for separating compounds that can easily form precipitate from the mixture. The precipitate is usually formed by changing the compounds into their salt form. Fractional liberation is commonly applicable in purification cinnamon alkaloids.[ 4 ]
• (e) Sublimation . This method involves changing from solid to gaseous state without passing through liquid state. Substances such as camphor and volatile oils when heated get separated and converted directly into gas.[ 4 ]
• (f) Chromatographic techniques . These are special techniques used in separation of compounds from mixtures based on their size, shape, and charge. The concept of chromatography involves the use of mobile phase, which is the solvent of extraction and the stationary phase such as silca gel and sephadex mixed with a calcium sulfate as a binder.[ 1 , 4 , 5 , 23 , 24 ] Silica gel is used for parting amino acids, sugars, fatty acids, lipids, and alkaloids. Sephadex is applicable in isolation of proteins and amino acids. Aluminum is useful in separation of vitamins, carotenes, phenols, steroids, and alkaloids. Cellulose powder is used in separation of amino acids, food dyes, and alkaloids. Celite is applicable in separation of organic cations and steroids.[ 1 , 4 , 8 , 23 ] Various mechanisms were involved in separation compounds using chromatographic techniques, namely, adsorption, partition, affinity, ion exchange, or size exclusion.[ 1 , 4 , 5 , 23 , 24 ] Chromatographic techniques include PC, TLC, column chromatography (CC), liquid chromatography (LC), GC, and HPLC.[ 1 , 4 , 5 , 8 , 23 , 24 ]

Mechanisms of separation in chromatography

• (i) Adsorption chromatography . Separation is performed based on the interaction between compounds to be separated and the stationary phase. In this case, the stationary phase will pull and remove compounds via hydrophobic, non-covalent Van der Waals forces of attraction. The compound that is loosely bound will first be eluted by the mobile phase.[ 1 , 5 , 24 ]
• (ii) Partition chromatography . Compounds are separated by addition of two or more immiscible solvents in to the mixture of an extract. Each compound will part away from the mixture by dissolving in the portion of solvent where it is soluble.[ 1 , 5 , 24 ] Subsequently, the immiscible liquids will be separated using separating funnel to obtain the individual compounds. The partition chromatography is otherwise known as liquid/liquid separation.[ 1 , 5 , 24 ]
• (iii) Affinity chromatography . The stationary phase is a ligand positioned in a separating column. The mobile phase applied washed down the compounds that have no affinity for the stationary phase. As such, compounds with high affinity for stationary phase get attracted and separated.[ 1 , 5 , 24 ]
• (iv) Ion exchange chromatography . The concept of ion exchange is useful in separation of polar compounds based on the type of charge they possessed. As such like charges attract, whereas unlike charges repelled. Like-charge substances attracted to each other and get separated the mixture or extract.[ 1 , 5 , 24 ]
• (v) Size exclusion chromatography . This method considers separating compounds based on their molecular size by application of mesh of different diameters. It is also known as gel filtration or molecular sieving.[ 1 , 5 , 24 ] A smaller size mesh was first applied followed by medium size, and finally larger pores size mesh.

Chromatographic techniques used in the separation of compounds from a mixture or extracts

• (1) PC . The mechanism of separation involved in adsorption chromatography. The apparatus comprises a glass chamber and a stationary phase, which is a filter paper made from cellulose. The filter paper is hanged from the top and suspended into the glass chamber.[ 1 , 5 , 24 ] The mixture to be separated is placed at the bottom of the filter paper. In addition, the solvent is then poured into the bottom of the container to serve as a mobile phase. The mobile phase immediately begins to ascend along with the filter paper; separation is carried out by the upward movement of the mobile phase via capillary action. The compounds that are soluble will move together with the solvent and stick to the filter paper based on their solubility.[ 1 , 5 , 24 ] The speed of separation depends on the type of filter paper used. Movement of liquid and the separation process is faster when thick filter paper is used, whereas porous filter paper slowed the whole process. Identification of each compound separated is done by calculating the retardation factor, which is the ratio of distance traveled by the compound to the distance traveled by the solvent.[ 1 , 5 , 24 ] The advantages of this technique include simplicity and cost-effectiveness, very sensitive to small quantity of material. The disadvantages include time-consuming and fragility of paper, which can be destroyed by chemicals.[ 1 , 5 , 24 ]
• (2) TLC . This technique also involves the use of adsorption mechanism to separate a compound from a mixture. Separation is based on the interaction between the compounds in a mixture and stationary phase. It is applicable in the separation of compounds with low molecular weight.[ 1 ] The stationary phase usually is 100g of silica gel dissolved in distilled water to make a slurry. Meanwhile, in some instances Sephadex is applicable. The solution of silica gel is then poured into a glass plate with dimension 20cm × 20cm to produce a thickness of 1.5mm. It is then kept for 1h at 105°C to solidify.[ 1 , 23 ] Afterward, 10mL of extract is injected into the lower part of the plate and allowed to spread. The plate is then carefully inserted into the separation chamber containing mobile phase and allowed to stand for 30min. The compounds contained in the mixture will ascend to various positions on the plate based on their solubility. Each compound separated is identified by calculating its retardation factor which is the ratio of distance traveled by the compound to the distance traveled by the solvent and compare it with that of a known compound).[ 1 , 5 , 23 , 24 ] The compounds spotted are scrapped at different position using spatula and finally re-extracted using various solvents.[ 1 , 23 ] Advantages include less time-consuming, producing clear spots, and stable to acid as solvent.
• (3) CC . It involves the use of several mechanisms such as adsorption chromatography, molecular sieve, and ion exchange to achieve the desired outcome.[ 1 ] The column is made up of a long glass tube (5–50mm in diameter, 5 cm–1 m long) with a tap and glass wool filter at the bottom. In addition, silica gel, alumina, cellulose, or Sephadex are used as stationary phase, whereas the mobile phase is liquid. The process begins by packing 30g of silica gel (70/35) into a transparent glass column (80cm long, 5cm diameter) without introducing air bubbles. Subsequently, the extract to be partitioned is added from the top. Least polar solvent ( n -hexane) was first added as a mobile phase and allowed to stand for 1h in a closed column. The bottom of the column opened and various fractions of n -hexane collected at an interval. In addition to that, other solvents such as chloroform, ethyl acetate, n -butanol, and methanol added. Fractions of these solvents were collected individually at different time intervals and finally characterized.[ 1 ]
• (4) GC . The mechanism implicated is partition. Two immiscible solvents are used: one in gaseous form (mobile phase) and the other is in liquid form adsorbed into the surface of inert solid to serve as the stationary phase.[ 1 , 4 , 23 ] Substances that are soluble in the gaseous phase will leave the liquid, move to the gaseous phase, and get separated. Similarly, compounds that are soluble only in liquid form will remain in the stationary phase.[ 1 , 4 , 23 ] Inert helium gas was used as mobile phase, at a constant flow rate. The crude extract to be analyzed was first diluted with methanol and injected into the system.[ 4 , 17 , 23 ] Advantages of this method include ability to separate plant material contaminated with volatile pesticides, also used in quality control testing.
• (5) HPLC . This technique uses the mechanism of adsorption to achieve effective separation. It is suitable for the partitioning of both organic and inorganic compounds. The mobile phase is a suitable solvent, whereas the stationary phase is solid particles tightly joined together. Separation is initiated via interaction of the compounds in the mixture with the solid particle of the stationary phase.[ 1 , 4 , 5 , 16 , 23 ] The apparatus consists of a solvent reservoir, sample injector, pressure pump, HPLC tube, and diode detector. The process begins by injecting the mixture to be separated at the bottom of HPLC. In addition, a suitable solvent is poured into the solvent reservoir. The tap is now opened to allow the movement of solvent downward, which is then pushed by a pressure pump to mix up with the injected sample. Finally, the mixture moved into the diode detector, which separated the compounds, removed the waste, and pumped the final content to processing units.[ 1 , 4 , 5 , 16 , 23 ]

I DENTIFICATION T ECHNIQUES

Several methods were used in the identification of compounds from medicinal plant extracts. It comprised detection of functional group, presence of multiple bonds and rings, hydrogen and carbon arrangement as well as full structural elucidation.[ 1 , 4 , 10 , 17 , 23 ] The techniques used include mass spectroscopy (MS), ultraviolet spectroscopy (UV), nuclear magnetic resonance spectroscopy (NMR), and infrared spectroscopy (IR).

• (i) MS . This method is useful in the identification of compounds based on chemical structure and molecular weight. The aim is to sequence and identify the unknown compound in a mixture. The substances usually identified include oligonucleotides and peptides.[ 1 , 4 , 10 , 17 , 23 , 25 ] The process begins by bombarding an organic molecule with an electron and converts it into very energetic charged ions. The signal was first detected using electron ionization energy of 70eV; also, the sample spectra are detected and recorded as percentage peak. Compounds are identified based on their relative molecular mass and molecular weight. This can be achieved by plotting mass of the fragmented ions against the charges of individual ion.[ 1 , 4 , 10 , 17 , 23 , 25 ] Notably, MS provides abundant information on organic molecules. Therefore, one of the standard procedures in processing medicinal plant is the combination of MS/HPLC.[ 4 , 17 , 23 , 25 ]
• (ii) UV . This method is suitable for qualitative and quantitative analysis of compounds present in the plant’s extract. Various secondary metabolites such as phenols, anthocyanins, tannins, and polymer dyes could be detected at certain frequencies. Total phenolic content and other secondary metabolites can be established using this technique. Specific frequencies were used to identify flavonoids (320nm), phenolic compounds (280nm), anthocyanins (520nm), and phenolic acids (360nm).[ 4 , 10 , 23 , 25 ]
• (iii) NMR . This technique pays more attention to the physical properties of the bioactive molecule such as number and array of the carbon atom, presence of isotopes of carbon, hydrogen atom, and protons. It also described how atoms are arranged in a molecule.[ 1 , 4 , 10 , 17 , 23 , 25 ]
• (iv) IR . This method tries to assess functional groups present in a compound. Knowledge of the functionalgroup helps in defining the physical and chemical properties of a given compound. Also, single, double, and multiple bonds are identified through this process.[ 1 , 4 , 5 , 10 , 23 , 25 ] The technique involves passing an organic compound through infrared radiation, which is absorbed in certain frequencies. Liquid samples are identified using sodium chloride plates, whereas solids samples are determined using potassium bromide milled together and compressed into a thin pellet. The result is recorded as a spectrum that is percentage transmittance. Lastly, the spectra are analyzed; the peaks obtained at certain wave number are compared with standard reference.[ 1 , 4 , 5 , 10 , 23 , 25 ]

C ONCLUSION

Several works have been done on medicinal plant either to investigate and prove a reported claim of biological activity or to mimic its traditional medicinal use based on ethnomedicinal survey. Large numbers of medicinal plants have been extracted, fractionated, and compounds isolated successfully. In addition, compounds obtained were tested for biological or pharmacological activity, and in most cases, they were found to be active. Nonetheless, the rate of success and the authenticity of these findings depends on the accuracy in selection of solvents, selection and proper execution of extraction methods, phytochemical screening, fractionation, and identification techniques. Lastly, proper understanding and implementation of these techniques are indispensable. Advancement and modification of these methods periodically will ease research processes and improve the outcome.

Financial support and sponsorship

Conflicts of interest.

There are no conflicts of interest.

Acknowledgement

Our special gratitude goes to the staffs of the Department of Pharmacology and Therapeutics and Faculty of Pharmaceutical Sciences, Bayero University, Kano, Nigeria.

R EFERENCES

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Collection  18 May 2018

Top 100 in Plant Science

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