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20 Phytopharmaceutical Biotechnology: Integration of Botany, Pharmacology and…
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Micropropagaon of herbs for phytopharmaceucal producon
Phytochemical screening of mother plant to
determine secondary metabolite profile
Explant selecon and preparaon for micropropagaon process
Explant sterilisaon methods standardizaon
Establishmentof different types of dedifferenated
callus cellslike friable or non-friable callus
Opmisaon of processes for biomass accumulaon
Mass producon of callus, Connuous Screening for
Idenficaon and quanficaon of precursors, elicitors,
type of stress that induce phytochemical producon
Establishment of sterile homogenous suspension
Phytocompounds
Scale-up of process : Preparatory phase for producon of secondary metabolites
Phytochemical Producon Phase
cultures with standardized addives
Determinaon of ideal explant for micropropagaon
Growth substrate composion and growth
parameters standardizaon
Culture inaon
Establishment of organ culture like shoot, root,
petal, vesicles, trichomes, hairy roots etc
Mass producon of organs, Connuous Screening for
Idenficaon of permeability, immobilisaon
(Bioreactors)
Phytocompounds
techniques
Mass organ culture in liquid medium
453
Screening for phytocompound content and
Harvesng of dedifferenaon of callus
(HPLC/ HPTLC / GC-MS / LC-MS / NMR / UV Visble / AAS)
(SCE, Liquid Nitrogen assisted extracon, and other extracon methods)
Downstream processing to obtain crude extract
Phytochemical analysis
Screening for phytocompound content and
Extracon Phase
Harvesng of organs
Fraconaon, characterisaon , isolaon and purificaon
Phytopharmaceucal 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 etal. 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 signicantly 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 specic 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 andBiomarkers
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 specic 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 efcacies 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 efcacy 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 efcacy of complex herb mixtures. For instance, a polyherbal treatment for healing of non-healing wounds should reduce inammation,
arrest infection, remove bad odour and improve granulation to achieve even minimal healing. Here, reduced inammation, 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 etal. 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-inammatory, 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 etal. 2020).
• Andrographolide: The chief bioactive component of Andrographis paniculata, a
herb used in ethnomedicine for respiratory infections, inammation 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 etal. 2023).
• Curcumin: The principal phytochemical curcumin, displaying marked anti-
inammatory, 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 etal. 2008; Gopumadhavan etal. 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 efcacy 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 proles of plant-derived compounds. Pharmacological studies help determine
the efcacy of herbal extracts and compounds in treating specic diseases or conditions. This can involve testing for activities such as anti- inammatory, antimicrobial,
antioxidant, neuroprotective, immunomodulatory or analgesic effects.
20.4.6 Formulation Development
Extraction
Phytopharmaceutical biotechnology also focuses on developing efcient 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 etal. 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 1–22.1MPa 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
etal. 2020; Di Domenico Ziero etal. 2020).
Further purication and isolation of phytochemicals are accomplished through
preparative column chromatography and fermentation through biotechnological
techniques using microbial strains such as Bidobacterium bidum, Lactobacillus
acidophilus and others to achieve greater efcacy 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 etal. 2013). SFCE-treated cells
showed signicant neuroprotection at 500μg/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 etal. 2022). These results highlight the role
biotechnological processes play in bringing out valuable synergistic efciencies 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 efcacies 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 100nm in size and are visible to the naked eye (Alsaiari etal. 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 efcacies as a result of synergies (Letchumanan etal. 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 etal. 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
etal. 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 efcient 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 nanobers, polymeric nanomicelles and polymeric nanoliposomes. For example, green
extract nanoparticles may be dispersed in a semi-solid cream to deliver antioxidants
of high efcacy. 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 specic 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 identication of critical process parameters in a given process and the
generation of data that aligns with the pre-set specications.

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20.4.8 Pre-clinical Studies
The safety and efcacy of phytopharmaceutical drugs are evaluated during preclinical
studies before they are tested on humans. These studies include invitro and invivo
experiments to determine the pharmacological properties of the drug, its toxicity prole 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), invitro (cell culture or partial
biological systems) and in vivo (animal studies or complete nonhuman biological
systems). Preclinical research can be classied into two distinct categories depending
on the aim and purpose of the study, such as “hypothesis- generating” (exploration)
and “hypothesis-testing” (conrmation) 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
etal. 2015). Knowledge of pathophysiology is vital in target validation, biomarker
identication, patient grouping or stratication 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 efcacy forecasting, drug-dose optimisation, biomarker identication 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. Qualied 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 identication 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 andRegulatory Compliance
The third phase of preclinical trials collates the potential outcomes of administering
a new therapy or drug targeted to alleviate a specic 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 efcacy. Regulatory compliance ensures that
phytopharmaceutical products meet the requirements of health authorities and are
safe for consumers. Compliance with specic 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 specic chemical markers with one
biomarker. This new classication 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 efcacious 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
invitro 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 efcacy 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 sufcient quantities to be commercially viable.
Optimising the biotechnological processes to enhance efcacy 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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