Molecular Pharmacognosy: Applying a New Techniques to Natural Product Science

Abstract

Molecular pharmacognosy is the branch of science that involves the application of molecular biology, biochemistry, and computational techniques in conjunction with conventional pharmacognosy for the analysis of natural products and their activities. Some of the important technologies include DNA fingerprinting for the identification of plants, DNA microarrays for gene expression, genome sequencing for lead identification and bioactive compound discovery, and DNA barcoding for species differentiation. Proteomics, genomics, metabolomics, and transcriptomics help in the understanding of the biological actions of natural products while reverse pharmacology involves the use of traditional knowledge to identify the target molecules. The idea and application of these methods in the detection, improvement, and characterization of medications produced from plants is the main topic of this review.

Keywords

Introduction

Molecular pharmacognosy is a subfield that studies the synthesis of effective components and categorization of crude pharmaceuticals. It is based on theories and techniques of pharmacognosy and molecular biology. Molecular pharmacology studies the origins of basic medicines found in plants and animals. The biological spectrum, which includes gene, cell, organ, organism, population, and community, is divided into six biological levels. The discovery of each level in the biological spectrum has progressed over time ^[1]. Independent branches of life science have emerged due to research on different scientific issues at different levels. Pharmacognosy focuses on tissue, organ, organism, and population levels, developing theories and techniques like pharmacognosy histology and morphology. Molecular pharmacognosy deals with crude pharmaceuticals at the genetic level. Genetic markers, like genes, indicate individual genetic variations across organisms. Three categories of molecular markers exist: morphological indicators, biochemical markers, and DNA markers.^[2]

Benefits of employing molecular markers:

Recent biotechnology advancements have enabled the discovery of numerous genetic polymorphisms at the DNA level, making molecular markers more relevant and accessible for genetic research. Their applications include DNA tracing, marker-aided selection, twin zygosity, sexing preimplantation embryos, identifying disease carriers, and determining genetic distance.^[3]

DNA indicators:

Genetic indicators, such as nucleotide sequences, are used to study population polymorphisms. Ideal DNA markers, such as hybridized, PCR-based, and transposable element markers, are dominant, uniformly distributed, and capable of detecting larger polymorphism degree.^[4]

Six methods for conducting research: From genes and molecular markers to MAS:

Plant breeders can use molecular markers linked to QTLs or candidate genes to expedite breeding programs and improve genotype selection, using six MAS development methodologies to illustrate their similarities and differences.

Method 1: SSR or comparable biological indicators

Method 2: The relationship between variants and SNPs and associated markers

Method 3: During evolution, plants react to the stress in a variety of ways.

Method 4: Gene expression validation and verification "since DNA markers towards MAS." Method 5: Technology using RNA-seq microarrays

Method 6: Genetic Reversal goes from discovered alleles towards traits, genetic markers, and finally MAS^[5].

Current Advances in Molecular Pharmacognosy:

1. DNA Fingerprinting:

Fingerprinting techniques offer nonselective information on food to characterize or authenticate a dish. These methods, including metabolic profiling and infrared energy profiling, help detect, categorize, and track dietery components and goods more effectively.^[6] Dietery profiling ensures traceability, authenticity, and constancy of foods, ensuring the food industry's sustainability and transparency to customers. Various stakeholders, including food producers, regulators, technology suppliers, and researchers, work together to develop new applications. Traditional Chinese Medicine (TCM) fingerprinting captures the chemical properties of TCM medicines using analytical techniques, supporting consistency, authenticity verification, and quality control. Creating a single, standardized TCM fingerprint database is essential for enhancing quality assurance, assisting research, and advancing TCM's globalization. Fingerprints are recognized as an efficient way to assess the quality of TCM due to their systematic, integral, and proprietary nature. Despite numerous methods and tools available, no comprehensive fingerprint retrieval system has been implemented for TCM fingerprint database technology^[7].

2. DNA Microarray:

The exponential growth of information in life sciences has led to the development of novel techniques for high-throughput gene analysis. DNA array technology was created to allow simultaneous assessment of thousands of genes, including expression monitoring, polymorphism analysis, and sequencing. Microarrays consist of three main parts: a solid substrate, a coating that connects organic biological molecules to inorganic glass, and a variety of biomolecular probes for hybridization. Glass is the preferred substrate due to its chemical inertness, low fluorescence, great flatness, and low price. There are two types of DNA-probe hybridization technologies for microarrays: high-density and low-density. High-density microarrays can test for hundreds of possible diseases at once, while low-density microarrays are CE-marked techniques that allow in vitro identification of viral diseases in humans. These assays have the potential to detect and diagnose various prevalent and recently discovered viral infections in humans. cDNA microarrays, which employ mostly unknown patterns or variable show PCR, have proven effective for searching for undiscovered genes. GeneChip, one of the oligonucleotide microarrays, can measure over 45,000 transcripts simultaneously, indicating that we have a technology that can measure almost the entire "transcription tome."Microarray research has gained recognition as a technique that can simultaneously quantify gene expression on a massive scale. As the genome is fully sequenced, it will soon be used in the development of pharmaceuticals. Understanding every functional component of the immune system is increasingly essential, and microarrays have been employed as a high-throughput test technique up to this point.[^8,9,10,11]

3. DNA Barcoding:

In recent decades, DNA barcoding has become a popular method for identifying herbs, allowing for creativity and security in the herbal medicine industry. In order to offer suggestions for the future advancement and use of this technology, we provide an overview of recent developments in DNA bar coding for herbal medicine in this article. Most significantly, there are two ways in which the conventional DNA barcode has been expanded. First , Plasmid genome-based super-barcodes have emerged quickly and demonstrated benefits in identification of species at low taxonomy categories, whereas traditional DNA barcode have been extensively marketed for their adaptability in identifying materials that are either fresh or stored. Second, miniature barcodes work with degraded DNA from herbal materials, which makes them appealing. Furthermore, some molecular methods, like isothermal amplification and high-throughput sequencing, are used in conjunction with DNA barcodes to identify species. This has led to the post-DNA-barcoding age and increased the uses of DNA barcoding for herb identification. Additionally, reference sequences for species identification are provided by genomic identifier reference libraries that are both standard and have significant species coverage, which improves the precision and legitimacy of employing DNA barcodes to distinguish between species. In conclusion, DNA barcoding need to be a major component of both the global herb commerce and the quality assurance of traditional herbal therapy^[12,13].

Arguments against DNA barcoding :

Traditional  taxonomists  first  criticized  the  idea  of  DNA  barcoding. Low resolutions for recently evolved species, species complexes, and hybrids are among the drawbacks of DNA barcoding. Some researchers draw attention to the existence of mitochondrial introgression and pseudogenes. In maritime environments, reproductive isolation—a crucial component of the biological species concept is challenging to study. Divergent barcode clusters were found to correlate with reproductively isolated groups in a study involving cosmopolitan marine bryozoans, suggesting a connection between DNA barcoding and the idea of biological species. The process of finding and identifying species will be enhanced by the combination of DNA barcode data with morphological, ecological, and physiological data. Several flaws in the integration of DNA barcode data were discovered. Collins and Cruickshank evaluated seven shortcomings and listed possible fixes for each. These personnel addressed the following seven flaws in the experimental design:

1. Not testing well-defined hypotheses
2. Insufficient specimen identification beforehand
3. The phrase "species identification" is used.
4. Using neighbor-joining trees inappropriately.
5. Using bootstrap resampling incorrectly.
6. Using fixed distance thresholds inappropriately.
7. Reading the barcoding gap incorrectly.

These problems must be carefully addressed during DNA barcoding. According to many studies, it is more difficult than initially believed to find an overall genome for all living species.^[14].

DNA barcoding's advantages:

For taxonomy users, DNA barcoding is quite helpful. Compared to traditional taxonomic labor, it offers faster advancement. DNA barcoding allows taxonomists to rapidly classify specimens by highlighting dissimilar species that may represent new species. Taxonomists have the chance to significantly increase and finally finish a worldwide inventory of life's diversity thanks to DNA barcoding. DNA barcoding proponents assert that it has revitalized biological collecting and expedited inventories and identification of species; opponents counter that it would obliterate traditional systematics and turn it into a lucrative service sector. When DNA barcoding is fully established, it could fundamentally alter our understanding of the variety of living things and how we interact with the natural world. DNA bar coding will speed up the discovery of thousands of unnamed species and enable many people to rapidly and affordably identify recognized species and access information about them. Bar coding may offer a crucial new tool for maintaining and valuing the vast variety of the planet. The necessity of establishing tissue banks has been acknowledged, and methods for connecting DNA samples to taxonomic vouchers for a variety of organisms are now being developed. The reported effectiveness of employing the barcoding area to find obscure species and differentiate species from a variety of taxa is impressive, despite several disadvantages of DNA barcoding. However, it is well recognized that using a s. DNA barcoding allows taxonomists to rapidly classify specimens by highlighting dissimilar species that may represent new species. ingle DNA sequence to identify a species would almost always result in some inaccurate findings. Consequently, in order to complement the already utilized barcoding area, measures should be made to develop nuclear barcodes.Given the benefits and drawbacks of barcoding, it is obvious that taxonomic methods that combine morphology, DNA sequencing, and ecological research will be the most effective in identifying  species.  DNA  barcoding  is  used  for  the  following  primary  reasons: It does the following: (a) it operates using segments; (b) it operates across the entirety of life stages; (c) it shows species with similar lookalikes; (d) it removes contradiction; and (e) it develops competence to recognize new species quickly and to speed up the identification of recognized creatures. (f) DNA barcoding makes access more accessible; that is, a standardized database of barcodes will allow more people to recognize and call adjacent species by title, regardless of their physical dimension (220 M). (g) New leaves are sprouted on the tree of life by DNA barcoding^[15].

1. Genome sequencing:

Pharmacokinetics is (different drug metabolic function), pharmacodynamics (differential attaching to receptors impacting beneficial accessibility), unique responses (immune- mediated sensitivity responses), and illness reaction to therapies (having or without targets for specific therapies) are all influenced by genetic factors, as studies have long recognized accounting for some of the variability in individual responses to pharmaceutical agents. The enzymes that carry out these tasks are extremely diverse, frequently exhibiting notable variations among ethnic groups. These genetic variations that affect how well medications work can be evaluated specifically, but they are also part of WGS. Controlled by hundreds of enzymes,  xenobiotic  metabolism  developed  to  deal  with  molecules substances that were not naturally present in the body, as those present in other plants that were eaten. Variations in these biogolical enzymes are characterized by their impact on drug activity rather than being categorized as pathogenic or nonpathogenic. Lipophilic molecules are easily dissolve in the water by the The cytochrome P450 gene family of 58 human CYP genes and further further stage II enzyme processes complete the transformation to create excretable, innocuous chemicals. There are also an increasing number of reports of clinically significant genetic variations in non cytochromics enzymes that are prevalent in the general population. "More than 97% of Americans have at least twelve pharmacogenes having at least one recognized, treatable, hereditary variation, one high-risk diplotype.are taken into accountWhole-genome sequencing of drug-gene pairings in healthy individuals. However, there are many such barriers like kack of pharmacogenomic education, clinician opposition to change, and the infrastructure needed to manage genetic data in health records and provide support for clinical decisions at the point of treatment in the fragmented US healthcare system; and issues with reimbursement and cost, which are exacerbated by the absence of proof that this kind of genetic examination is ultimately economical.^[16]. One of the most fundamental techniques for genome sequence investigations is genome alignment. These freshly completed genomes pose difficulties for instruments of aligning to handle the heightened intricacy and scale due to the quick advancements in sequencing and assembly methods. Plant genome alignment is technically challenging due to the high nucleotide diversity, extensive structural variation, chromosomal rearrangements and fractionation, strong transposable element (TE) activity that results in large proportions of repetitive elements, and frequent whole-genome duplications . We highlight methods that are commonly employed or being created by the community of plant researchers, and we provide an overview of traditional Multi chromosomal alignment and pairwise methods. We also list the remaining difficulties in accurately aligning the plant genome and interpreting the alignment results^[17].

Difficulties Complex sequencing and assembly:

High frequency of recurring patterns : A significant and ubiquitous component of genomes' dark matter consists of recurring patterns that are identical or comparable to to other sequences inside the genome. Numerous satellites, rDNA, short and DNA transposons, extended terminal repetition retrotransposons, and long interleaved nuclear elements and other repetitive sequences are found throughout many plant genomes . In the genomes of maize ,wheat, and tea plants, for example, the total repetitive sequences make up 85% of the genome. These many repeating sequence types range from one to two bases (repeats of mono- and dinucleotides) to millions of bases, with copies ranging from two to millions. Numerous significant genetic functional areas, including multicopy genes, center points, the ends of tel and extremely heterochromatic, non-recombining chromosomes such as the Y & W sex chromosomes are typically included in these repeat-rich regions. The requirement to correctly assemble these areas has become a challenge in intrinsic genome research since they are crucial to the genome's function and evolution

1. Elevated Heterozygosity: Small-scale differences exist between the two haplotypes in species with low heterozygosity. Consensus sequences are produced during assembly thanks to these minute changes, which allow precise alignment (Figure 2A). Nevertheless, a lot of large-scale structural differences between the two haplotypes are present in genomes with high heterozygosity, which results in assembly "bubbles" that are redundant allelic sequences.Due to self-incompatibility and remote hybridization, many plants have substantial genome heterozygosity. As a result, the assembled size of these extremely heterozygous genomes is typically greater than the haploid genome's projected size.
2. Polyploidy: Plant polyploidizations can result via either whole-genome doubling of one species (autopolyploidy) or interrelated hybridization followed by chromosomal doubling (allopolyploidy).Allopolyploid genomes are easier to assemble, and allopolyploid crops like peanuts (A. hypogaea), cotton (G. hirsutum), and rape (B. napus) were the primary participants in the initial wave of genome assembly for polyploidy. Subgenomes from several ancestral species can be distinguished from one another with reasonable ease because they have preserved a significant percentage of genetic variants over the course of their lengthy evolutionary history. However, because homologous chromosomes are so identical, autopolyploid species with more than two homologous sets of chromosomes present serious difficulties with genome assembly and haplotype phasing^[18].

RNA Sequencing:

The RNA sequencing (RNA sequencing) technology was developed just before the second- generation scanning technique was introduced in 2008. RNA-seq has transformed biological research since its inception. For further information, see a prior review . It has made it easier to examine mutations/SNPs, gene fusion, alternative splicing transcripts, RNA alterations, and variations in gene expression across time or among various populations or therapies.. Conventional RNA-seq gives a snapshot of the transcriptome, or collection of RNA molecules in the sample, primarily mature RNAs. Stated differently, mRNA-seq quantifies the outcomes of metabolisms (degradation) and RNA synthesis (transcription and processing)^[19]. Single-cell RNA sequencing (scRNA-seq) technology.Animals and plants are both sophisticated entities made up of several cell types that share almost the same genetic makeup. However, different cells have varied functions as a result of stochastic developmental events that alter gene expression.When complete tissues are homogenized for sequencing using bulk RNA-seq approaches, all cells are treated equally, which causes RNAs to mix during library formation and obstruct detection. Additionally, expression of genes and transmissible polymorphisms in particular cells are obscured by the average levels of gene expression in each of the tissue's cells. In order to get over these restrictions, The gene transcriptomes of every cell may be examined using scRNA-seq technology. This allows for the recognition of rare cells as well as in-depth understanding of molecular mechanisms and transcriptomic heterogeneity at the the level of a single cell^[20]

2. Proteomics:

The methodical investigation of a wide range of proteins' identities, variable abundances, distributions, modifications, interactions, structures, and functions, as well as their role in disease, is known as proteomics .Because proteins have a direct role in every aspect of physiology, proteomics makes it possible to dynamically monitor changes in protein expression in order to shed light on the underlying mechanisms of disease and further discover particular biomarkers and possible treatment targets.In the proteomics analysis process, gathering and preparing samples is a wonderful place to start when trying to get reliable data . Generally speaking, there are two types of samples utilized in proteomics analysis: tissues (such as the brain, kidneys, lungs, or stomach) various bodily fluids, such as serum, urine, tears, plasma, cerebrospinal fluid, and saliva. The more widely used sampling techniques. The fresh-frozen method and the formalin-fixed paraffin-embedded method were used for tissue collection and preservation . Protocols and tutorials on best practices for collecting bodily fluid samples were also available Nonetheless, the sample collection procedure still faces numerous difficulties. For instance, variations in freezing temperatures, centrifugation techniques, and blood collection timing can all affect the outcome when it comes to plasma . Enough consideration should be given to the sample collection process's complexity.The ability to identify and analyze proteins as well as protein separation techniques are somewhat necessary for the development of proteomics . The most widely utilized proteomics methods at the moment are mass spectrometry (MS), 2D electrophoresis (2-DE), arrays of antibodies and antigens^[21]. A interdisciplinary method called proteome-based systems biologyintegrates systems biology with proteomics to clarify the intricate relationships and dynamics of biological events at the protein level. In proteomics-based systems biology, the combination of various datasets and computer modeling is essential. Based on experimental data, cellular behavior is simulated and predicted using using systems biology models and mathematical simulations. By combining proteomic data with other omics information and employing computational modeling, proteome-based systems biology offers a comprehensive approach to clarifying the complicated nature of biological systems.^[22].

1. Metabolomics:

There are two types of metabolomic analyses: targeted and untargeted. Untargeted studies focus on the entire metabolic profile of a sample (a "fingerprint") in order to measure and recognize metabolites that are differently expressed, such as those associated with certain illnesses, metabolic pathways, or prescribed medications.. Targeted analyses, on the other hand, concentrate on particular metabolites present in a sample. The most popular approaches for characterizing metabolites are spectroscopy and spectrometry techniques like NMR and MS. Spectrometry examines how light and matter interact, but spectroscopy quantifies how much light and other radiation is absorbed and emitted by matter. Although these terms have different meanings, they are frequently used interchangeably. Both techniques are employed to ascertain the molecular structures of both big and tiny compounds, such as medications and metabolites^[23]. It is anticipated that the use of Metabolomics in mycology of food will continue to advance, with a number of developments anticipated. The metabolomics' sensibility, resolution, and throughput of metabolomics analysis will be further improved by ongoing advancements in analytical equipment, such as MS and imaging techniques. More work needs to be done to enhance fungal metabolomics data bases, such as extensive ungal metabolite libraries and their spectrum that go along with them, such as spectrum from tandem mass spectroscopy (tandem MS).Data analysis with artificial intelligence will make it possible to mine massive The study of metabolism data sets for food mycology pattern detection, biomarker identification, and predictive modeling. This will improve Risk evaluation and quality assurance procedures by making it easier to identify important metabolites linked to to food product contamination, deterioration, and fungal growth ^[24].

Transcriptomics:

Numerous factors, including cell type, developmental stage, environmental conditions, and disease status, can affect the transcriptome's composition, which is temporally dynamic .Because the genome is static , it is impossible to collect information on how gene expression is regulated under biological factors like development, disease progression, and environmental stress . Consequently, one of the most researched biological databases is the human transcriptome. Numerous studies on the human transcriptome have facilitated quick developments in personalized medical strategies, cancer detection , and the comprehension of complex diseases. Prior to the introduction of next- generation sequencing (NGS), a highly effective and massively parallel sequencing technology, the production of sequencing data constituted a bottleneck in transcriptomics. The effectiveness of sequencing an entire transcriptome or genome has increased over time. Using Illumina's NGS techniques, the predicted cost of sequencing a human genome for cancer research by 2020 was USD 4000, which is often within the means of the majority of research organizations^[25]. Because it allows for the investigation of individual cell transcriptomes and reveals cell-to- cell diversity, scRNA-seq, or single-cell RNA sequencing, has become a powerful technique for researching plant biology. High-throughput, parallel analysis is made possible by droplet- based scRNA-seq, which uses microfluidic technology to encapsulate individual cells with barcoded beads. By employing unique molecular identifiers (UMIs) for every transcript, it gets beyond the biases associated with large PCR amplification. While less customizable, commercial alternatives to early systems are easier to use. Research on plants has used this technique to look into things like root primordia, leaf vasculature, and stomatal development. In order to overcome the drawbacks of single-cell RNA sequencing (scRNA-seq) in plant research, spatial transcriptomics techniques are essential. Problems with traditional scRNA- seq include skewed cell type representation, loss of spatial information, and changes in gene expression       brought           on        by protoplasting. Fluorescence of single molecules in situ hybridization and the more modern Stereo-seq are examples of spatial transcriptomics that enable spatially resolved transcriptome analysis to address these problems. Barcoded oligo(dT) arrays are used in spatial transcriptomics (e.g., 10x Genomics Visium) to extract mRNA from tissue cryosections. The technique overcomes some of the drawbacks of scRNA-seq by enabling high-resolution spatial mapping of gene expression without the need for protoplast isolation. Numerous plant species have benefited from its successful application, which has shed light on processes like maize grain filling, vascular tissue development, and flower organogenesis. Through the use of DNA nanoball sequencing, stereo-seq provides higher resolution spatial transcriptomics, making it possible to capture spatiotemporal transcriptomes at the single-cell level. By mapping mRNA and cell identity using high-resolution chips, it tackles issues like plant cell wall diffusion. However, for in-depth tissue analysis, border recognition and chip resolution still require work. These cutting-edge methods offer comprehensive insights into tissue-specific gene activity and cellular interactions, marking a substantial advancement in spatially resolved gene expression investigations in plants ^[26].

Reverse Pharmacology:

The Reverse Pharmacology (RP) strategy differs from the conventional classical pharmacology approach for developing and discovering drugs, which proceeds from molecule to mice to man, by starting from man to mice to molecule. RP seeks to advance the therapeutic benefits of conventional wisdom. new biological science technology, new medical treatment techniques, and conventional healthcare knowledge bases can all interact through RP, An route for the development of new medication actions an route for the development of new medication actions. Accessing experiential hits, conducting exploratory research to find leads, and finally carrying out meticulously planned trials to uncover medication candidates are all part of the RP strategy. Safe, efficient, cost-effective, and innovative pharmacotherapeutics for improved global health are the anticipated results of RP. Numerous treatment leads have been found using this method in a variety of domains, including neurological diseases, musculoskeletal and arthritic disorders, Cervicovaginal infections, cancer and precancer diseases, liver and hepatitis problems, insulin resistance and metabolic abnormalities, and other ailments ^[27].

CONCLUSION:

Molecular pharmacognosy is a modern approach to the study of natural commodities that helps understand the chemical, biological, and therapeutic properties of plants and other natural sources using state-of-the-art molecular methods. The shift from traditional herbal medicine research methods to molecular-based approaches, which allow for more precise bioactive chemical synthesis, characterization, and identification, has altered the field. The application of DNA fingerprinting or genetic identification and verification of plant species has helped to boost the credibility of herbal medicines. New technologies based on DNA microarrays and the analysis of whole plant genomes have enhanced their our mechanisms and knowledge of action. molecular Reverse profile pharmacology of which plants is and the hence study lead of to the the effects identification of of herbal new products bioactive on compounds the and body at a molecular level is one of the most important strategies used in drug discovery. The knowledge of proteomics and transcriptomics assist in the identification of proteins and compounds genes and that the are identification affected of by pathways bioactive and mechanisms of action. In addition, metabolomics gives a systematic profile of the metabolites, which facilitates the identification of biomarkers and the analysis of plant metabolism in a therapeutic context. Altogether, these advanced techniques help to gain a better perspective of natural products, which facilitates the creation of more efficient and less hazardous medications. Molecular pharmacognosy is expected to have a significant part in the advancement of the technology in the area of herbal medicine research, leading to a better appreciation of the interactions between plants, their constituents, and human health.

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