Plant-derived Pharmaceuticals Research Articles

Inula viscosa (L.) Aiton Ethanolic Extract Inhibits the Growth of Human AGS and A549 Cancer Cell Lines.

The present study shows the chemical profile and cytotoxic properties of the ethanolic extracts of Inula viscosa from Northeast Algeria. The extract was obtained by maceration using ethanol. Its phenolic profile was determined using ultra-high-performance liquid chromatography coupled with a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI/MS), which allowed the identification and quantification of 17 compounds, 1,5-O-caffeoylquinic acid being the most abundant. The cytotoxic activity was assessed against human gastric cancer (AGS) and human non-small-cell lung cancer (A549) cell lines, whereas ethanolic extract elicited nearly 60 % and 40 % viability loss toward AGS and A549 cancer cells, respectively. Results also showed that cell death is caspase-independent and confirmed the involvement of RIPK1 and the necroptosis pathway in the toxicity induced by the I. viscosa extract. In addition, the ethanolic extract would not provoke morphological traits in the cancer cells. These findings suggest that I. viscosa can be a source of new antiproliferative drugs or used in preparation plant-derived pharmaceuticals.

Emerging mechanistic insights into the regulation of specialized metabolism in plants.

Plants biosynthesize a broad range of natural products through specialized and species-specific metabolic pathways that are fuelled by core metabolism, together forming a metabolic network. Specialized metabolites have important roles in development and adaptation to external cues, and they also have invaluable pharmacological properties. A growing body of evidence has highlighted the impact of translational, transcriptional, epigenetic and chromatin-based regulation and evolution of specialized metabolism genes and metabolic networks. Here we review the forefront of this research field and extrapolate to medicinal plants that synthetize rare molecules. We also discuss how this new knowledge could help in improving strategies to produce useful plant-derived pharmaceuticals.

An integrated computational approach towards the screening of active plant metabolites as potential inhibitors of SARS-CoV-2: an overview.

COVID-19 and its causative organism SARS-CoV-2 paralyzed the world and was designated a pandemic by the World Health Organization in March 2020. The worldwide health system is trying to discover an effective therapeutic measure since no clinically authorized medications are present. Screening of plant-derived pharmaceuticals may be a viable technique to fight COVID-19 in this vital situation. This review discusses the potential application of in silico approaches in developing new therapeutic molecules related to preventing SARS-CoV-2 infection. Also, it describes the binding affinity of various phytoconstituents with distinct SARS-CoV-2 target sites. In this perspective, an extensive literature survey was carried out to find the potential phytoconstituents to develop new therapeutic entities to treat COVID-19 in different online academic databases and books. Data retrieved from databases were analyzed and interpreted to conclude that many phytochemicals will bind with the 3-chymotrypsin-like (3CLpro) and papain-like proteases (PLpro), spike glycoprotein, ACE-2, NSP15-endoribonuclease, and E protein targets of SARS-CoV-2 main protease using in silico molecular docking approach. The present investigations reveal that phytoconstituents such as curcumin, apigenin, chrysophanol, and gingerol are significantly binding with spike glycoprotein; laurolistine, acetoside, etc. are bound with Mpro for anti-SARS-CoV-2 therapies. Using virtual applications of in silico studies, the current study constitutes a progressive data analysis on the mechanism of binding efficiency of distinct classes of plant metabolites against the active sites of SARS-CoV-2. Furthermore, the current review also demonstrates the fundamental necessity of the alternative and complementary medicine for future therapeutic uses of phytoconstituents by phytochemists in the fight against COVID-19.

In vitro evaluation of antimicrobial and anticancer potential of Artemisia absinthium growing in Kashmir Himalayas

Herbal medicines are an important and growing part of International pharmacopeia. Research and testing enhance our understanding of their medical properties, making them a safer alternative or preferable option to allopathic medication. Plant-derived pharmaceuticals are gaining popularity due to the belief that "green medicine" is safer and more trustworthy than expensive synthetic drugs. This study aimed to evaluate the antimicrobial and anticancer potential of the methanolic leaf extract of Artemisia absinthium against human lung cancer A549 cell line by well diffusion method and MTT assay, respectively. The A. absinthium leaf extract showed the highest activity against Enterococcus faecalis (20 ± 0.7 mm), and Escherichia coli (18 ± 0.8 mm), followed by Pseudomonas aeruginosa (16 ± 0.6 mm), Candida albicans (14 ± 0.9 mm) and Staphylococcus aureus (13 ± 0.8 mm), with MIC values 128, 128, 128, 256 and 256 µg/mL respectively. The methanolic extract of A. absinthium showed significant (p ≤ 0.05) cytotoxicity against the A549 cancer cell line with an IC50 value of 36.8 µg/mL. The present study's findings give strong evidence for using the methanolic leaf extract of A. absinthium as an effective ethnomedicinal agent and a possible candidate for treating various human diseases and a potent bioactive agent in anticancer medications.

Comparative Content, Biological and Anticancer Activities of Heracleum humile Extracts Obtained by Ultrasound-Assisted Extraction Method.

As the safety and effectiveness of synthetic drugs remain in doubt, researchers are trying to develop natural medicines from medicinal plants. Herein, ethyl acetate, methanol and water extracts from the Heracleum humile plant were obtained by an ultrasonic-assisted extraction process and the aim was to evaluate some biological effects of the extracts due to the limited data on the pharmacological properties of Heracleum humile in the literature. Weak antibacterial activity was observed on tested bacterial species. The minimum inhibitory concentration and the minimum bactericidal concentration values ranged from 250 to 500 μg/mL. In addition, cytotoxic activity was determined using the MTT test. The strongest findings were determined for ethyl acetate extract on the MDA-MB-231 cell lines at the 48th hour (IC50 :97.94 μg/mL), followed by the MCF-7 cell lines at the 24th hour (IC50 :103.9 μg/mL). All extracts of Heracleum humile contained mainly flavonoids, phenolic acids and their derivatives, i. e., well-known compounds that possess numerous biological activities such as antioxidant, anti-inflammatory, anticancer, antimicrobial etc. The study results could provide important information that Heracleum humile could be a potential candidate as a natural enzyme inhibitor. It can be concluded that these extracts could be useful in the elementary step of improving novel plant-derived multifunctional pharmaceuticals.

Chromosome-scale genomes throw light on plant drug biosynthesis

Recent chromosome-scale genomes from prominent medicinal plants have provided unprecedented insight into the architecture and evolution of some prominent metabolic pathways. These new technologies also facilitate the identification of plant drug biosynthetic genes and would likely accelerate the development of new bioengineering procedures to secure the supply of important plant-derived pharmaceuticals.

The methods behind transgenic plant production: a review

The ability to insert a gene into a plant’s nuclear or chloroplast genome enables the transformation of higher plants (e.g. tobacco, Arabidopsis thaliana, potato, tomato, and banana) into Bioreactors for the production of plant-derived pharmaceuticals. Biopharmaceuticals are generally produced on a commercial basis by scale fermentation in bacteria, yeast, or animal cells. Several plant-derived pharmaceuticals have undergone clinical trials and are close to market authorization, with antibodies and vaccines being the front runners. Plant-derived vaccines have been produced using recombinant plant viruses as transgenic expression vectors and Agrobacterium tumefaciens transformation systems. During the last decade, several efficient plant-based expression systems have been examined, and more than 100 recombinant proteins, including plant-derived vaccine antigens. Besides, regulatory protocols are slowing down production. Industry requirements and public acceptance of the technology are important aspects in establishing successful products. This paper reviews the current status of development in the area of biopharmaceuticals and vaccines produced from transgenic plants.

Biotechnology of Viscum Album L. and Cancer Treatment: A Review

Background: This review aims to highlight biotechnological techniques applied for the identification, isolation and propagation of mistletoe-derived bioactive compounds and the preclinical evaluation of their anticancer properties. Methods: We undertook a structured search of bibliographic databases to identify and evaluate the literature, regarding biotechnology of mistletoe and malignancy treatment. The selected articles were classified into five modules according to the study design. Results: Firstly, we have provided an overview of the role of biotechnology in plant-derived pharmaceuticals, by reviewing twelve articles. Then, V. album L. botany and a retrospection of the plant pharmaceutical applications are presented from a set of ten articles. Subsequently, mistletoe derived compounds and their anticancer properties are highlighted, throughout sixty-two articles, which emphasized the mechanisms of antitumor action. The next module is referred to as the biosynthesis of mistletoe bioactive compounds, covering nine cases of ‘in vitro’ propagation and genetic manipulation. Finally, fourteen assays regarding mistletoe preparations and preclinical testing for chemosensitivity are presented. Conclusion: Biotechnology provides essential tools for the identification, production and evaluation of anticancer compounds derived from Viscum album L. The preclinical studies indicate the potential use of V. album preparations in chemotherapy but further research is essential for standardizing the therapeutic benefits of mistletoe compounds in the treatment of malignancies.

Back to the plant: overcoming roadblocks to the microbial production of pharmaceutically important plant natural products.

Microbial fermentation platforms offer a cost-effective and sustainable alternative to plant cultivation and chemical synthesis for the production of many plant-derived pharmaceuticals. Plant alkaloids, particularly benzylisoquinoline alkaloids and monoterpene indole alkaloids, and recently cannabinoids have become attractive targets for microbial biosynthesis owing to their medicinal importance. Recent advances in the discovery of pathway components, together with the application of synthetic biology tools, have facilitated the assembly of plant alkaloid and cannabinoid pathways in the microbial hosts Escherichia coli and Saccharomyces cerevisiae. This review highlights key aspects of these pathways in the framework of overcoming bottlenecks in microbial production to further improve end-product titers. We discuss the opportunities that emerge from a better understanding of the pathway components by further study of the plant, and strategies for generation of new and advanced medicinal compounds.

Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum

Anthraquinones (AQs) are secondary metabolites widely distributed in nature. Interesting applications of plant extracts containing AQs include the treatment of Hepatitis C and cancer. Plant in vitro culture is an alternative for producing plant-derived pharmaceuticals in controlled conditions and with low environmental impact. Moreover, it allows the application of different strategies for enhancing secondary metabolite production. Hairy root cultures, obtained after Agrobacterium rhizogenes infection, are able to produce high amounts of secondary metabolites at high growth rates. In this work, growth kinetics of hairy root cultures of Rubia tinctorum and AQ production were evaluated in two different culture media, Gamborg B5 with half of the saline strength (B51/2) and Lloyd & Mc Cown’s Woody Plant Medium (WPM). Although WPM allowed higher biomass production (58.6% higher) than B51/2, specific AQ production was higher in B51/2 (between 1.2 and 2.1 fold increases from day 21 to the end of the experiment). Moreover, AQ release to the culture medium was observed in B51/2 (∼10% of total AQs). The different performance of hairy roots in these culture media may be due to a limiting nutrient (other than carbon source) in B51/2. Elicitation in B51/2 with methyl jasmonate (100μM) resulted in a massive accumulation of intracellular (between 1.5 and 2.4-fold increases) and also extracellular AQs (up to 8.1 fold-increase compared with control at 4days post-elicitation), which could ease AQ purification. These results prove the usefulness of combining different approaches to enhance secondary metabolite accumulation in plant in vitro cultures, in order to develop an optimized productive process.

Natural Deep Eutectic Solvents (NADES) as a Tool for Bioavailability Improvement: Pharmacokinetics of Rutin Dissolved in Proline/Glycine after Oral Administration in Rats: Possible Application in Nutraceuticals.

There is a need for innovation in plant-derived pharmaceuticals, food supplements and nutraceutical products regarding the use of more eco-sustainable solvents for their extraction. Furthermore, the poor oral bioavailability of several phytochemicals with health promoting effects stimulates the research in the field of pharmaceutical formulations. Natural Deep Eutectic Solvents (NADES) are formed by natural compounds, and can be considered as future solvents being especially useful for the preparation of nutraceuticals and food-grade extracts. In this paper various NADES were prepared using sugars, aminoacids and organic acids. Rutin (quercetin-3-O-α-l-rhamnopyranosyl-(1→6))-β-d-glucopyranose) was used as a model compound to study NADES. Moreover, the effect of various eutectic mixtures on rutin’s water solubility was studied. Proline/glutamic acid (2:1) and proline/choline chloride (1:1) mixtures have a solubility comparable to ethanol. The proline/glutamic acid (2:1) eutectic containing rutin was used in a pharmacokinetic study in Balb/c mice while bioavailability was compared to oral dosing of water suspension. Plasmatic levels of rutin were measured by HPLC-MS/MS showing increased levels and longer period of rutin permanence in plasma of NADES treated animals. This paper reports the possible use of non-toxic NADES for pharmaceutical and nutraceutical preparations.

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

Process Development Strategies in Plant Molecular Farming.

Protein-based biopharmaceuticals are often produced in mammalian cell cultures, which are more expensive than microbial systems but capable of authentic post-translational modifications. The costs are lower if plants are used as an alternative platform to produce complex proteins such as monoclonal antibodies, vaccines and enzymes. This review highlights recent advances that have been achieved in plant-based biopharmaceutical production platforms in terms of expression strategies, product yields and process development. The first generation of plant-derived pharmaceuticals now entering the market is also discussed. Finally, the review considers the downstream processing of plant-derived pharmaceuticals which can account for up to 80% of the production costs. In this context, recent improvements in clarification and integrated process methods will have a strong impact on the economic feasibility of production, especially if supported by and combined with process analytical technology as part of the quality-by-design initiative.

Biological studies concerning the antioxidant and antimicrobial activities of Pelargonium species cultivated in Egypt (Family-Geraniaceae)

Plant-derived pharmaceuticals have become prominent in the market place, making it a favored healthcare choice. In this study, air dried samples of aerial parts of Pelargonium X fragrans Willd. and Pelargonium peltatum L’Hérit. were separately extracted using successive extraction with a soxhlet apparatus. Each extract was tested for its antimicrobial activity using two Gram-negative bacterial strains (Pseudomonas aeruginosa and Escherichia coli), two Gram-positive bacterial strains (Bacillus subtillus and Staphyllococcus aureus), and clinical fungal isolates (Aspergillus niger and Candida albicans). Also, their antioxidant activity was tested using a DPPH free radical assay. The ethyl acetate, n-Butanol and the total extracts showed moderate activity against the tested microorganisms with significant high activity against E. coli. The free radical scavenging property was found to be in a concentration dependent manner in all the tested fractions. The most effective antioxidant fractions in both spp. was the n-Butanol fraction (85% and 85.2%) at the concentration of 0.375µg/ml followed by the total ethanolic extracts (78.1% and 84.62%), respectively, with the same concentration compared to the standard reference ascorbic acid which showed a significant radicals scavenging potential (79.1%) in the concentration of 1µg/ml.DOI: http://dx.doi.org/10.3329/icpj.v4i2.21481 International Current Pharmaceutical Journal, January 2015, 4(2): 340-342

Transcriptome analysis of Catharanthus roseus for gene discovery and expression profiling.

The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals.

Molecular farming of human cytokines and blood products from plants: Challenges in biosynthesis and detection of plant‐produced recombinant proteins

Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms.

Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.

Albumin from rice: Why and wherefore?

The work by He et al. (1) expressed concern that human albumin derived from plasma fractionation is associated with “an increasing public health concern with plasma-derived HAS with its potential risk for transmission of infectious pathogens such as hepatitis and HIV” (1). The work by He et al. (1) offered two references to support this statement, one of which included the unequivocal statement that “[b]oth HSA and PPF are manufactured with pasteurization procedures that have led to an excellent viral safety record based on 50 years of clinical use” (2). The other reference does not mention albumin but describes emerging pathogens in the context of blood safety. We note that none of the agents noted, in addition to mainstream transfusion-transmitted agents, have ever been transmitted by albumin. This aspect of albumin's safety derives from its robust purification and pathogen clearance processes, which have proven reliable against the threat of known and unknown pathogens. Albumin's safety in other aspects is also high (3). Much of this safety is because of the establishment of good manufacturing practices overseen by regulatory agencies. The required features of such practices for plant-derived pharmaceuticals have been reviewed (4), and we suggest that the introduction and enforcement of such measures is crucial for all manufacturing processes, including the measures described in the work by He et al. (1). We note the interest of He et al. (1) in cost-effectiveness and their assertion that this cost-effectiveness can be deduced for their process from the level of protein expression achieved; however, it seems that the manifold safety measures needed to achieve environmental protection as described in addition to factors not mentioned have not been factored into this nonquantified estimation. Consideration of the measures described in the work by Howard and Hood (5) may contribute to a more concrete assessment of the facility by which the process in the work by He et al. (1) may be developed into a therapeutic product.

Greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression

The production of plant-derived pharmaceuticals essentially requires stable concentrations of plant constituents, especially recombinant proteins; nonetheless, soil and seasonal variations might drastically interfere with this stability. In addition, variability might depend on the plant organ used for production. Therefore, we investigated the variability in plant constituents and antigen expression in potato plants under greenhouse and field growth conditions and in leaves compared to tubers. Using potatoes expressing VP60, the only structural capsid protein of the rabbit haemorrhagic disease virus (RHDV), CTB, the non-toxic B subunit (CTB) of the cholera toxin (CTA-CTB(5)) and the marker protein NPTII (neomycinphosphotransferase) as a model, we compare greenhouse and field production of potato-derived antigens. The influence of the production organ turned out to be transgene specific. In general, yield, plant quality and transgene expression levels in the field were higher than or similar to those observed in the greenhouse. The variation (CV) of major plant constituents and the amount of transgene-encoded protein was not influenced by the higher variation of soil properties observed in the field. Amazingly, for specific events, the variability in the model protein concentrations was often lower under field than under greenhouse conditions. The changes in gene expression under environmental stress conditions in the field observed in another event do not reduce the positive influence on variability since events like these should excluded from production. Hence, it can be concluded that for specific applications, field production of transgenic plants producing pharmaceuticals is superior to greenhouse production, even concerning the stability of transgene expression over different years. On the basis of our results, we expect equal or even higher expression levels with lower variability of recombinant pharmaceuticals in the field compared to greenhouse production combined with approximately 10times higher tuber yield in the field.

Expression of the Viral Antigen VP60 in Transgenic Potatoes and its Effect on the Nutritional Composition of Tubers

Recombinant plant-derived pharmaceuticals have been investigated for the last two decades and some products will soon be brought to market. Since veterinary pharmaceuticals seem to be the front-runners of plant-derived vaccines, we selected one model subunit vaccine, the structural capsid protein VP60 against rabbit haemorrhagic disease, and analyzed the expression of three different sequences representing the VP60 open reading frame in potato plants. The generation of antigenic VP60 molecules in the leaf and tuber tissue of potato was tremendously enhanced by replacing virus-derived sequences with plant-optimized codons. In order to identify potentially undesirable alterations in the composition of these genetically modified food components, we studied their nutrient composition and nutritional value in comparison to two parental conventional breeding varieties (Albatros and Desiree). The largest differences in nutrient composition were found between the two conventional breeds and between conventional Desiree and its near-isogenic genetically modified potato plant, indicating that genetic modification as well as conventional breeding can influence nutrient composition. Nevertheless, most parameters of nutritional value seemed to be more affected by conventional breeding than by genetic modification.