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20 Phytopharmaceutical Biotechnology: Integration of Botany, Pharmacology and…
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Micropropagaon of herbs for phytopharmaceucal producon
Phytochemical screening of mother plant to
determine secondary metabolite profile
Explant selecon and preparaon for micropropagaon process
Explant sterilisaon methods standardizaon
Establishmentof different types of dedifferenated
callus cellslike friable or non-friable callus
Opmisaon of processes for biomass accumulaon
Mass producon of callus, Connuous Screening for
Idenficaon and quanficaon of precursors, elicitors,
type of stress that induce phytochemical producon
Establishment of sterile homogenous suspension
Phytocompounds
Scale-up of process : Preparatory phase for producon of secondary metabolites
Phytochemical Producon Phase
cultures with standardized addives
Determinaon of ideal explant for micropropagaon
Growth substrate composion and growth
parameters standardizaon
Culture inaon
Establishment of organ culture like shoot, root,
petal, vesicles, trichomes, hairy roots etc
Mass producon of organs, Connuous Screening for
Idenficaon of permeability, immobilisaon
(Bioreactors)
Phytocompounds
techniques
Mass organ culture in liquid medium
453
Screening for phytocompound content and
Harvesng of dedifferenaon of callus
(HPLC/ HPTLC / GC-MS / LC-MS / NMR / UV Visble / AAS)
(SCE, Liquid Nitrogen assisted extracon, and other extracon methods)
Downstream processing to obtain crude extract
Phytochemical analysis
Screening for phytocompound content and
Extracon Phase
Harvesng of organs
Fraconaon, characterisaon , isolaon and purificaon
Phytopharmaceucal drug / substance
Minimum of four phytocompounds + one biomarker
Fig. 20.4 Flowchart depicting the production of phytopharmaceutical or secondary metabolites through plant biotechnology methods
Another under-explored but highly promising aspect is the culture of glandular trichomes (Atito etal. 2018), which stores volatile compounds such as terpenes, essential oils, avours and fragrances for targeted production. Glandular trichomes are specialised hair-like structures on the surfaces of various plants, including some species of owering plants, herbs and even certain trees. These trichomes are involved in the production and secretion of various chemicals, often playing
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important roles in the plant’s interactions with its environment, including defence mechanisms and adaptation to environmental conditions. The botanical compounds sequestered in these glandular heads are of huge therapeutic value. Cleome droseri- folia plant has been overexploited to be used in folk medicine for diabetes. Glandular trichomes of Artemisia annua, the annual wormwood, contain artemisinin, a sesqui­terpene lactone effective against most species of the pathogenic protozoan, Plasmodium spp., that causes malaria (Duke and Paul 1993). Glandular trichome proliferation through tissue culture techniques may enable the isolation of purer volatile oils that get stored only in the trichome and not in the leaves. This way, there may be less interference with other bioactive compounds produced and stored in the whole leaf or organ.
S. Krishnaswamy
20.4.4 Phytochemistry
Higher plants display a great deal of phytochemical diversity in response to varied environmental, physiological and agroclimatic stimuli and species within the same genus can produce a plethora of related or dissimilar biologically active chemicals. Plant-based bioactives are described as primary or secondary metabolites based on their role in plant metabolism. Primary metabolites, responsible for the structure of the plant body and their basic vital functions, include common sugars, amino acids, proteins, lipids, purines and pyrimidines of nucleic acids, which constitute the DNA and RNA, chlorophylls, etc. Secondary metabolites include all other plant chemi­cals such as alkaloids, terpenes, avonoids, lignans, plant steroids, curcumins, sapo­nins, phenolics and glucosides, which may be produced as by-products of primary metabolism. Both primary and secondary metabolites can signicantly impact human health and well-being. Traditional ways of using plant extracts may not be consistent with their phytochemical content, as some phytochemicals may be pres­ent in minute quantities and may remain undetected through normal testing methods followed by traditional practitioners, but in the realm of phytopharmaceuticals, the chemical constitution of the plant material is analysed through a range of highly sensitive chemical and analytical tests to detect, identify and quantify the even trace quantities of bioactive compounds or secondary metabolites, which are responsible for the plant’s medicinal properties. Sophisticated laboratories adopt high­throughput, rapid and precise screening methods to isolate phytochemicals for spe­cic targeted pharmacological activity. A thorough evaluation of the plant material/ crude drug quality, including factors such as moisture content, ash content and other quality parameters, is crucial to ensure consistency and effectiveness in medicinal preparations.
According to regulatory stipulations, a phytopharmaceutical formulation manda­torily comprises a minimum of four biologically active phytochemical compounds in addition to a biomarker.
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Phytopharmaceuticals andBiomarkers
It is important to distinguish the difference between phytochemical compounds and biomarkers. Phytopharmaceuticals and biomarkers are distinct but associated with each other in the elds of biology, pharma and medicine.
Phytopharmaceuticals are also known as plant-derived biologically active sub­stances with curative or therapeutic properties. The bioactives or phytochemicals are usually secondary metabolites or by-products of primary metabolic processes taking place in a plant species and may be found stored in any or all regions of the plants such as leaves, stem, owers and roots and are extracted from the plant parts using specic methods and conditions.
The usual phytochemicals used for therapy can be categorised as phenolics, tan­nins, saponins, alkaloids, polysaccharides, terpenes, gums, etc.
Biomarkers, however, are characteristics or a means of evaluating efcacies or outcomes of the use of phytopharmaceuticals, biological processes or conditions response to treatment. Biomarkers could be of different types:
1. Molecular or DNA-Based: genetic markers
2. Chemical Moieties: unique to a plant species, genus or family
3. Biochemical: proteins, enzymes
4. Response-Based: reduced blood sugar levels in the treatment of diabetes with a
polyherbal formulation, reduction in itching and scaling in the treatment of pso­riasis using a polyherbal skin cream
Biomarkers are necessary to assess the efcacy and safety of numerous interact­ing phytochemical molecules found in polyherbal formulations. In this context, a biomarker can help determine whether the formulation has the desired therapeutic effects and whether it can be safely used. Multiple biomarkers may also be neces­sary in ensuring safety and efcacy of complex herb mixtures. For instance, a poly­herbal treatment for healing of non-healing wounds should reduce inammation, arrest infection, remove bad odour and improve granulation to achieve even mini­mal healing. Here, reduced inammation, lack of bad odour and clear red zones, indicating restored blood circulation, may be considered biomarkers.
In certain cases, phytopharmaceutical compounds themselves may be considered biomarkers. For instance, polysaccharides are complex carbohydrates that consist of many monosaccharide units linked together by glycosidic bonds. Polysaccharides have various biological functions, such as energy storage, structural support and cell recognition. Polysaccharides can also be considered biomarkers in a polyherbal phytopharmaceutical formulation, which is a medicine derived from natural sources and containing multiple herbs. Polysaccharides can reveal the source and authentic­ity of the herbs, determine the optimal dosage and extraction method and aid in the evaluation of the stability and shelf-life and the pharmacological and therapeutic effects of the formulation (Mohammed etal. 2021). Some examples of polysaccha­rides that have been used as biomarkers in polyherbal formulations are:
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• Mangiferin: A xanthone glycoside is found in various plants, especially in
mango. Mangiferin has anti-inammatory, antioxidant, antidiabetic, anticancer
and neuroprotective properties. Mangiferin can be used as a biomarker to stan-
dardise Mahasudarshan Churna, an Ayurvedic polyherbal medicine that is used
for fever, cold, malaria and other conditions (Kaur etal. 2020).
• Andrographolide: The chief bioactive component of Andrographis paniculata, a
herb used in ethnomedicine for respiratory infections, inammation and liver
disorders, is a diterpenoid lactone, andrographolide. It exerts immunomodula-
tory, antiviral, antibacterial, antimalarial and hepatoprotective effects.
Andrographolide can be used as a biomarker to standardise PartySmart capsule,
a polyherbal formulation that is used for preventing hangovers by preventing
acetaldehyde build-up in the liver and protecting the liver (Khanvilkar etal. 2023).
• Curcumin: The principal phytochemical curcumin, displaying marked anti-
inammatory, antioxidant, anticancer, antidiabetic and neuroprotective proper-
ties in turmeric, is a polyphenol. Curcumin can be designated as a biomarker to
indicate standardisation in the PartySmart capsule along with andrographolide
(Venkataranganna etal. 2008; Gopumadhavan etal. 2008).
Thus, the choice of biomarker needs to necessarily align with the intended thera­peutic effect or expected outcome desired by the treatment regime or medicine.
S. Krishnaswamy
20.4.5 Pharmacology
Pharmacological effect or efcacy is the study of how drugs and compounds interact with the body and produce therapeutic effects. In phytopharmaceutical biotechnology, pharmacological investigation focuses on unravelling the mechanisms of action and safety proles of plant-derived compounds. Pharmacological studies help determine the efcacy of herbal extracts and compounds in treating specic diseases or condi­tions. This can involve testing for activities such as anti- inammatory, antimicrobial, antioxidant, neuroprotective, immunomodulatory or analgesic effects.
20.4.6 Formulation Development
Extraction
Phytopharmaceutical biotechnology also focuses on developing efcient methods for extracting and formulating plant-derived compounds. This includes optimising processes and extraction techniques to maximise compound yield, recovery and stability. Quality control standardisation is a crucial step, needing strict vigilance and enforcement. Novel methods of extraction of wholesome extracts and fraction­ated molecules have been developed. Some examples of these innovative extraction technologies are counter-current extraction, oil-as-solvent extraction (using
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bio-renewable, bio-based solvents such as citrus peel and rapeseed oil), ultra-soni­cation-assisted solvent extraction (Azmir etal. 2013).
Subcritical water hydrolysis (SWH), also known as subcritical water extraction (SWE), is a novel modern extraction process that exploits the unique properties of water below critical points of 100–374°C and 1–22.1MPa temperature and pres­sure, respectively, where the distinction between liquid and gaseous phases is oblit­erated and only one phase with strong catalytic character prevails. At subcritical ranges of temperature and pressure, low dielectric levels and varying densities cause a marked shift in the behaviour and properties of water. Under these conditions, non-polar substances such as proteins and amino acids can be recovered from bio­mass with tough cell walls (e.g. leftover biomass from coffee and tea processing), without breakdown of their structural integrity. This highly compressed water pro­vides a clean, green alternative for extraction of biological/bioactive compounds through “valorisation” of biological raw material to valuable nutrients (Maheshwari etal. 2020; Di Domenico Ziero etal. 2020).
Further purication and isolation of phytochemicals are accomplished through preparative column chromatography and fermentation through biotechnological techniques using microbial strains such as Bidobacterium bidum, Lactobacillus
acidophilus and others to achieve greater efcacy of the phytopharmaceuticals. Codonopsis lanceolata (C. lanceolata), Campanulaceae, consists of many phyto-
compounds such as saponins, alkaloids, tannins and polysaccharides (Ushijima et al. 2008). Using steam and fermentation in sequential processes, the extract (SFCE—steam fermented Codonopsis extract) was derived and used for the treat­ment of scopolamine-treated memory-impaired mice. Steamed C. lanceolata fol­lowed by fermentation recovered more phenolic acid including gallic acid and vanillic acid than original C. lanceolate (Weon etal. 2013). SFCE-treated cells showed signicant neuroprotection at 500μg/ml. The combination of steam and fermentation to obtain C. lanceolata extract enhanced cognitive activity related to memory processes and neuroprotective effects as compared to the conventionally derived C. lanceolata extract (Shinde etal. 2022). These results highlight the role biotechnological processes play in bringing out valuable synergistic efciencies in phytopharmaceutical formulations.
The plant stem cell extracts are then used as single drugs or combined with mul­tiple other extracts to derive a polyherbal formulation. The preferred solvents for extraction are water and ethyl alcohol as they are considered generally accepted as safe (GRAS). The phytopharmaceutical drug may be dispensed as such or run through other biotechnological techniques that augment the efcacies multifold.
Drug Delivery Systems
Biologically active materials such as plant parts, fungi, algae, bacteria or their extracts are incorporated into metal ions to develop green microscopic nanoparticles (NP) in a process known as “green synthesis”. Advances in the eld of nanotechnol­ogy enable the synthesis of a wide class of microscopic materials in the nanoscale
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range (ranging from 1 to 100 nanometres) as opposed to bulk materials, which range more than 100nm in size and are visible to the naked eye (Alsaiari etal. 2023). The green synthesis process, while being environmentally friendly and degradable, ensures the delivery of optimally loaded doses of the drug onto metallic nanoparti­cles. These drug-impregnated nanoparticles have better penetrability, faster action and greater efcacies as a result of synergies (Letchumanan etal. 2021). Developed using metal/metal oxides of gold (Au), silver (Ag) and copper (Cu), nanoparticle­mediated delivery methods possess various advantages, such as long-term persis­tence, preservation of enzyme structure, precision targeting of cells and upregulation of the immune system (Mohammed etal. 2018). Copper being relatively cheaper is most preferred for green synthesis. Furthermore, it is a natural trace component in the human body and is necessary for important enzymes such as tyrosinase, super­oxide dismutase and cytochrome oxidase, which mediate vital metabolic processes. On oxidation, Cu NPs form inorganic NPs of copper oxides (CuOs). Both Cu and CuO NPs possess anticancer, antimicrobial and antioxidant properties (Huang etal. 2020).
Formulation studies involve creating herbal medicines in various forms, such as capsules, tablets, creams and tinctures, to enhance their bioavailability and ease of use. Plant biotechnology methods support efcient impregnation of plant extracts in novel drug delivery systems such as liposomes, phytosomes, green synthesised nanoparticles, polymeric nanoparticles (PNPs), nanocapsules and nanospheres, polymeric nanogels and nanosuspensions, carbon nanotubes (CNTS) and nano­bers, polymeric nanomicelles and polymeric nanoliposomes. For example, green extract nanoparticles may be dispersed in a semi-solid cream to deliver antioxidants of high efcacy. Among the recently developed technology-driven drug delivery carriers, polymeric nanoparticles (PNPs) with high drug loading capacity, long half­life and high capacity to prevent drug deterioration are very effective. The PNPs offer easy surface penetration possibilities for ligands to pass through the blood­brain barrier (BBB) in Alzheimer’s disease, dementia, Parkinson’s disease and other CNS-related maladies.
S. Krishnaswamy
20.4.7 Validation
Phytopharmaceuticals are manufactured through various processes of pharmacol­ogy and biotechnology resulting in specic dosage forms with different drug deliv­ery mechanisms. Validation is vital across the entire gamut of the phytopharmaceutical value chain, encompassing research, manufacturing, testing and distribution activi­ties from analytical methods used for quality control, checking infrastructure/equip­ment facilities and processes adopted for manufacturing. Validation protocols that set testing guidelines are developed for the kind of tests or assays to be carried out, frequency of testing and expected and realised outcomes. Process validation includes the identication of critical process parameters in a given process and the generation of data that aligns with the pre-set specications.
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20.4.8 Pre-clinical Studies
The safety and efcacy of phytopharmaceutical drugs are evaluated during preclinical studies before they are tested on humans. These studies include invitro and invivo experiments to determine the pharmacological properties of the drug, its toxicity pro­le and its mechanism of action. The preclinical studies also help to identify potential drug interactions and prediction of adverse effects that may occur when the drug is administered to humans. Preclinical research generally falls into one of three catego­ries: in silico (computer modelling or simulations), invitro (cell culture or partial biological systems) and in vivo (animal studies or complete nonhuman biological systems). Preclinical research can be classied into two distinct categories depending on the aim and purpose of the study, such as “hypothesis- generating” (exploration) and “hypothesis-testing” (conrmation) research (Venkatraman and Mehta 2023).
The pharmaceutical industry relies heavily on preclinical PK/PD modelling to select, predict and optimise effective human doses and/or dose regimens (Schuck etal. 2015). Knowledge of pathophysiology is vital in target validation, biomarker identication, patient grouping or stratication and impacts preclinical/clinical study success rates. Quantitative systems pharmacology (QSP) is another tool that pharmaceutical companies are adopting for evaluating all stages of drug discovery/ development. Though preclinical QSP modelling is still in nascent stages, it may be designed to optimise processes of lead generation, proof-of-concept validation, clinical efcacy forecasting, drug-dose optimisation, biomarker identication and elucidation and compound selection (van der Graaf and Benson 2011).
Laboratory experimentation is followed by the rst phase of pre-clinical study, which aims to establish proof of safety of the new drug and is conducted on a limited number of individuals. The procedures to determine the effectiveness of the drug in the rst phase are similar except for the increased number of individuals deployed in the third phase. Qualied doctors administer the new drug to a small group of 10–20 recruited patients, in small doses initially. The gradual increase in the dosage known as dose escalation enables the identication of the dosage that produces optimal results without causing harsh side effects. Drug metabolism and absorption in the human body are studied. Vital data collected on the dose, dosage timing, method of drug administration and the suitability of the treatment set the stage for the next phase. Usually, side effects are not observed in the rst administration of the drug to a patient. The rst phase of preclinical trials usually goes on for about a year.
20.4.9 Clinical Trials andRegulatory Compliance
The third phase of preclinical trials collates the potential outcomes of administering a new therapy or drug targeted to alleviate a specic ailment, to a small number of patients and compares them with the responses to the standard of care for similar conditions. Once the pre-clinical raw data are obtained and analysed, the phytophar­maceutical product moves on to phase 1–4 clinical trials. Phytopharmaceutical
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biotechnology leans heavily on the three pillars namely, quality, safety and consis­tency of herbal products. This includes implementing good manufacturing practices (GMP) to establish quality control standards, conducting quality assessments and validation of the therapeutic potential of herbal medicines. Human clinical trials are conducted to assess their safety and efcacy. Regulatory compliance ensures that phytopharmaceutical products meet the requirements of health authorities and are safe for consumers. Compliance with specic regulations and guidelines related to herbal medicines and dietary supplements is necessary, depending on the region and the intended use of the plant material.
S. Krishnaswamy
20.5 Conclusion
Plant-based natural products including extracts, enriched fractions, essential oils, phytomolecules, avours and fragrances are popular among the pharmaceutical, nutraceutical and cosmetic industries. Phytopharmaceuticals include enriched frac­tions of plant drugs consisting of at least four specic chemical markers with one biomarker. This new classication provides the way forward for the plant-based enriched fractions to be used as a drug, which do not belong to any part of Ayurvedic, Siddha or Unani literature. It has been very important to know the chemical compo­sition and precise proportion of pharmacologically active ingredients in the formu­lation. The provisions stipulated for synthetic drugs are not appropriate or relevant for botanical-based products. Regulatory guidelines have been framed to bridge science-based conventional medicine and traditional medicine with anecdotal prom­ise through a new class of evidence-based, safe and efcacious phytopharmaceuti­cal drugs. This class of drugs in a new avatar may encourage a deeper investigation into the vast potential of plant-based therapeutics to provide quick-acting healthcare solutions to unmet medical needs under the standard of care.
The current trend is the domestication of medicinal plants through various inno­vative methods of cultivation, production and biotechnological interventions for invitro over-production through plant tissue culture and genetic improvement of herbs. Discouraging the use of wild-crafted plants will force the industry to resort to the cultivation of herbs, taking away the huge stress on rapidly depleting biodiver­sity and natural populations. It will then be possible to obtain uniform and high­quality raw materials, which are fundamental to the efcacy and safety of herbal drugs. Furthermore, the development of skincare products based on plant stem cell extracts is an emerging trend at present due to the vast potential of plant stem cells that may be developed from different plant species. Presently, various forms of plant stem cells and the products derived from their extracts are commercially accessible to the cosmetic industry.
However, plant stem cells as a sustainable source of curative phytopharmaceuti­cals will be highly valuable in treating cases of antimicrobial resistance (AMR) and orphan diseases apart from NCDs. Plant stem cells have been found to amass plant constituents and therapeutically relevant plant products such as phytohormones
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(kinetin) and other bioactives, in sufcient quantities to be commercially viable. Optimising the biotechnological processes to enhance efcacy and yield resulting in augmented production of phytopharmaceuticals may make them economically affordable. The rich natural biodiversity offers an excellent source of plant stem cells that may be utilised by the phytopharmaceutical industry. Destructive harvest­ing should be penalised heavily, and the biodiversity should only serve as the gene pool for initiating experiments in phytopharmaceutical biotechnology and not as the nal product of commerce. Earth’s biodiversity, comprising millions of unique bio­logical specimens, has evolved over nearly 3.5 billion years. Unfortunately, man, armed with technology, needs only a few decades to decimate them. After all, every living organism in the earth’s ecosystem has a right to exist, irrespective of their contribution towards the betterment of the human race. Responsible utilisation of natural resources is not only necessary but is critical for the sustainable develop­ment of the phytopharmaceutical industry.
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