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20 Phytopharmaceutical Biotechnology: Integration of Botany, Pharmacology and…
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drug (https://www.cdsco.gov.in/opencms/opencms/en/Acts- and- rules/New-
Drugs/ NDCTR_G.S.R. 175(E) dt_09.03.2023_ Amendment in First Schedule
regarding use of induced Pluripotent Stem Cells additional testing methods).
20.3.2 Efcacy
Phytopharmaceutical compounds are complex mixtures of various lead compounds, their intermediates, by-products and precursors. The choice of a drug
substance as a lead compound will depend on its therapeutic value at lower
concentrations, safety at higher limits and interactions with other drug components in a given formula. The effectivity and harmlessness of any pharmaceutical product are determined by its constituents and the response time taken to
achieve the desired outcome and extent of recovery. In a clinical trial, efcacy
is determined as primary and secondary outcomes. During a phase 2, multicentred, randomised controlled open-label, clinical trial evaluating potency and
lack of toxicity of antiviral phytopharmaceutical drug AQCH (abbreviation for
aqueous extract of Cocculus hirsutus) obtained from Cocculus hirsutus, treat-
ment for COVID-19, the measurable outcomes were recorded as follows
(Table20.2) (Joglekar etal. 2022).
However, this kind of in-depth study to ascertain efcacy and safety is usually
lacking in traditional herbal medicines.
Table 20.2 Evaluation of safety and efcacy of a phytopharmaceutical drug
List of criteria for evaluation of safety and efcacy of a phytopharmaceutical drug derived from
Cocculus hirsutus for treatment of COVID-19
Primary outcomes Secondary outcomes Safety outcomes
Clinical improvement by 14th day Number of patients showing clinical
Reduction in disease severity Time to clinical improvement Serious adverse
betterment by 7th and 28th days
Time to normalisation of fever without
the use of antipyretics in the past 24h
Time to alleviate cough
Time to viral clearance (rst negative
respiratory tract SARS-Cov-2 RT-PCR
test)
Duration of supplemental oxygen
therapy
Proportion of patients showing
deterioration of clinical condition
Duration of hospitalisation
Number of deaths
Treatmentemergent adverse
events
events
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S. Krishnaswamy
20.3.3 Consistency
The quality of phytopharmaceuticals depends on multiple factors such as accuracy
in identication of plant, time and season of harvesting, improper harvesting (mixture of immature plant parts or plant parts that do not contain active ingredient
resulting in dilution of bioactive concentration), presence of foreign matter such as
soil, pebbles, seeds of weeds and sticks resulting in dilution and adulteration of the
herb, improper drying and processing methods, pests, diseases and contaminants
such as fungus and insect infestation. The consistent yield of biomass and phytopharmaceutical content in the biomass may be achieved only by the cultivation of
specic herbs and standardising the agronomy protocols. For instance, the total
alkaloid content in Holarrhena antidysenterica is highest (3.2–3.9%) only during
the two owering seasons: March–May and September–October. Adhatoda vasica ,
grown in different agroclimatic zones during different seasons, still yields the highest content of total alkaloids at 2.5% only between July and October. Thus, unseasonal harvesting may result in poor-quality raw materials with low or no efcacy.
Cataloguing the different quality parameters for each herb may be valuable for
establishing consistent herb quality leading to the quality of phytopharmaceuticals
(Agrawal 2012).
20.3.4 Toxicity
Toxicity pharmacology refers to the preliminary tests for the evaluation of adverse
reactions to a new phytopharmaceutical drug. Toxicity may be due to some factors
that irritate the skin in the case of dermal products, due to the presence of heavy
metals, drug interactions and unstable formulations leading to degradation of original drug and so on. The presence of alarming levels of heavy metals in herbs indicates the extent of pollution in soils, air and water. Heavy metal stress may possibly
stimulate different, variable or enhanced bioactive production in some herbs.
Growing specic herbs on heavy metal-contaminated soils may prove benecial
provided they are tolerant to high concentrations of soil heavy metals and growth
and secondary metabolite production are optimised (Maleki etal. 2017). Two of the
traditionally used neuromodulators, Centella asiatica and Bacopa monnieri, are
known to accumulate heavy metals such as lead, nickel, cadmium or arsenic from
the soil (Biswas etal. 2020). This process, known as bioremediation, involves the
uptake of metal ions by the plant’s roots and their translocation to different parts. It
was found that both Centella asiatica and Bacopa monnieri store cadmium and lead
in the roots with very little translocation to the aerial parts (Chandra etal. 2010). If
the whole plant of either Centella or Bacopa is taken for processing as is the norm
in the market, it is very likely that the resultant extract may have higher than accepted
levels of cadmium or lead. Heavy metal contamination is a concern for both the
phytopharmaceutical industry and consumers alike. This risk can be mitigated only
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with proper cultivation and harvesting practices, the adoption of heavy metal trapping strategies and strict quality control measures during production. Plants that are
known hyperaccumulators may be cultivated through soil-less cultivation methods
such as hydroponics and aquaponics that use inert growth substrates such as cocopeat and light expanded clay aggregate (LECA) balls, to reduce the risk of heavy
metal contamination in herbs.
Toxicity could be due to drug–drug interactions between phytochemicals that are
part of a polyherbal formulation. The physical parameters and solvents used to
extract the bioactives may also impact the toxicity potential of a phytopharmaceutical drug or ingredient.
Detailed toxicity studies of an investigational product, while evaluating the
safety prole, also provide important information about the absorption, distribution,
metabolism and excretion (ADME) of the compound in the body (Govindaraghavan
and Sucher 2015). The preclinical toxicity testing on various biological systems
reveals the species-, organ- and dose-specic toxic effects of the candidate compound being tested. The toxicity of substances can be observed by a. studying the
accidental exposures to a substance, b. cells/cell line-based invitro study and c.
invivo testing on experimental animals. The pre-clinical toxicity testing helps to
derive the important “No Observed Adverse Effect Level” without which further
clinical evaluation of investigational product cannot continue.
Though traditional herbal medicine has a long history of providing therapeutic
benets, it is an undeniable fact that the risk potential for underdosing or overdosing
has been quite high.
Plant-based medicines have indeed been traditionally derived based on approximations or ratios that were often rooted in empirical knowledge and observations
rather than precise scientic measurements. These approximations were used to
determine the intended dosage of the active compounds or drugs within the plant
material. Here are a few reasons why this approach was common:
• Speculative Knowledge: Traditional herbal medicine systems, such as ethno-
medicine, Ayurveda, Siddha, traditional Chinese medicine (TCM) and other
indigenous healing practices, have evolved through centuries based on trial and
error. Practitioners relied on their observations of the effects of various plant-
based remedies on patients to determine dosages. These observations formed the
basis for approximate dosing guidelines. Classical Ayurveda has clear-cut bound-
aries for harvesting, processing and mixing drugs in specic methods docu-
mented in the Samhitas, Nighantus and other treatises, which are considered best
practices. Ethical manufacturers usually conform to these recommendations with
no or few deviations.
• Variability in Plant Material: Plants are complex biological entities, and the con-
centration of active compounds can vary signicantly based on numerous factors
such as the plant’s age, growing conditions, seasons, agroclimatic differences
and even the time of harvest. This variability poses a great challenge to establish
unequivocal standards.
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• Lack of Analytical Tools: In the past, there were limited analytical tools and
techniques available to quantify the exact content of active compounds within
plant materials. Without these tools, it was difcult to determine precise dosages.
• Cultural and Historical Practices: Traditional medicine often incorporated cultural
and historical practices that were passed down verbally through generations. There
were few instances of documentation on palm leaves, but largely, oral transfer of
formulation details was the norm. Any deviations or misrepresentation of the origi-
nal formula or practices may have been lost through time. These practices included
specic preparation methods, combinations of herbs and dosing regimens that
were considered effective based on tradition or individual experiences.
In recent years, however, there has been growing interest to standardise herbal medicine by scientic validation of plant bioactives through exhaustive analysis, pre-clinical
study and clinical trials, stringent quality control to standardise various parameters leading to the consistency of potency, dosage and safety and governed by strict regulatory
guidelines demanding accountability from the grower/collector, researcher, manufacturer and medical practitioners alike. It was against this backdrop that a need for an
integrated approach to standardisation of herbal therapeutics was envisaged. The Indian
Pharmacopoeia, which is the ofcial book of standards for drugs, has also included
phytopharmaceuticals and species quality standards of identication, purity and
potency of the drugs imported, manufactured for sale, stocked or exhibited for sale or
distributed in India (Phytopharmaceuticals—Indian Pharmacopoeia Commission (ipc.
gov.in)). The specic herbal monograph should state the title and synonym, if any, de-
nition, limits of active ingredient/marker compounds, description, category, identication, relevant chemical tests, design of assay of the marker constituents, adulterants and
contaminants, specic tests, stability and storage conditions, etc.
S. Krishnaswamy
20.4 Phytopharmaceutical Biotechnology
Phytopharmaceutical biotechnology, an interdisciplinary eld, integrates the botanical knowledge of plants, their efcacies and the use of plant biotechnology methods to devise novel strategies to deliver the best therapeutic potential of herbs and
other plant-derived compounds, best practices for the authentication, cultivation,
extraction and utilisation of medicinal plants. Phytopharmaceutical biotechnology
forms a vital bridge between traditional herbal medicine and modern science. It
enables the development of evidence-based herbal remedies that can be integrated
into mainstream healthcare systems, providing patients with safe and effective treatment options. Additionally, it contributes to the conservation and sustainable use of
natural biodiversity. When built over the solid foundation of time-tested and proven
tenets of ethnomedicine, phytopharmaceutical biotechnology can help develop
innovative strategies for the optimisation of the processes that lead to evidencebased, safe, effective sets of novel bioactive compounds. The main aspects of the
critical study of phytopharmaceutical biotechnology include:
20 Phytopharmaceutical Biotechnology: Integration of Botany, Pharmacology and…
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1. Botany
2. Standardisation
3. Plant biotechnology
4. Phytochemistry
5. Pharmacology
6. Formulation development
7. Validation and proof of concept
8. Preclinical Studies
9. Clinical trials
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20.4.1 Botany
A deep study and analysis of various features of an herb/medicinal plants such as
morphology, anatomy, histochemistry, ecological preferences and associations and
unique adaptations help identify morphological markers unique to each plant species. These markers serve as differentiators or identiers, aiding varietal authentication and raw material validation. For instance, in an exhaustive botanical investigation
of Andrographis serpyllifolia (Rottl. Ex. Vahl) Wight, an Acanthaceae member,
presence of peltate and glandular trichomes on the leaves, presence of cystoliths
(calcium carbonate crystals) and presence of numerous white hairs at the base of
anthers were identied as markers unique to this species as all three features were
preserved in crude drug powder as well (Figs.20.2 and 20.3) (Krishnaswamy and
Kushalappa 2017).
Pharmacobotanical evaluation, a term used in the eld of pharmacognosy,
explores the quality of herbs, their derivatives and their potential therapeutic properties. Pharmacobotanical evaluation involves the comprehensive assessment and
analysis of botanical materials, such as plants, plant parts (leaves, roots, stems, etc.)
and plant extracts, to determine their pharmacological and medicinal potential.
Further, pharmacobotanical analysis aids in highlighting the plant part most likely
to be therapeutic by virtue of its structure and the presence of organs/organelles
meant for producing and storing bioactives. These organs or organelles may be cultured through plant biotechnology processes to scale up targeted production of bioactive. This evaluation typically includes several key aspects:
• Botanical Identication: Accurate botanical identication of the live plant and
the features of dry material is essential. Taxonomical studies, chemotaxonomy
and DNA ngerprinting are conducted to ensure that the correct plant species or
variety is being used for medicinal purposes.
• Morphological Characterisation: The physical characteristics of the plant, includ-
ing its appearance, size, shape, colour and any distinctive features, are
documented.
• Microscopic Evaluation: Microscopic analysis involves examining the plant
material under a microscope to study its anatomical features and cellular struc-
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condions
2.Standardisaon
andadulterants,storage
Raw material authencaon,
cleaning methods contaminants
3. Plant Biotechnology
Callus and suspensioncultures,
vesicle culture, co-culture
techniquesfor biopharming
specialised culture likeTrichome,
S. Krishnaswamy
4.Phytochemistry
procedures, Purificaon,
Phytochemicals,Screening,
Bioacvesprofile, Extracon
5.Pharmacology
isolaon of marker compounds
phrmacokinecstudies
Bioequivalence, dermato-
drug molecule, Bioavailability,
Knowledgeof efficacy of herbal
1.Botany
Knowledge of plantecology,
morphology,plant-plant interacons,
10.MARKET
edaphicfactorinteracons
plant-predaon interacons, plant-
mullocaon, randomised /
studiesregulatory compliances
placebo basedpaent volunteer
9. Clinical Trials Mulcentric,
Development
6.Formulaon
Biotechnology
Phytopharmaceucal
Drug interacons, dose,dosage
Invitro, in vivo
8. Preclinical Tesng
safety,efficacy
tesng,toxicityBioassays,
formulaon properes,
analysis,mechanism of acon
Phytochemical, molecular and
7. Validaon Phytochemical
biomarkers,microbial analysis,
delivery methods, stability
form, green nano parcles,
microemulsions, liposomes, drug
heavy metals, stability
Fig. 20.2 Stages in the development of phytopharmaceutical drug/formulation
20 Phytopharmaceutical Biotechnology: Integration of Botany, Pharmacology and…
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Fig. 20.3 Biomarkers (persistent hairs on anther in fresh and dried ower) in dried herbage of
Andrographis serpyllifolia (Reused from Krishnaswamy and Kushalappa, 2017, distributed under
Creative Commons Attribution 4.0 International Licence, which permits unrestricted use, distribution and reproduction in any medium. http://creativecommons.org/licenses/by/4.0) (Krishnaswamy
and Kushalappa 2017)
449
ture, which can reveal important information about the plant’s identity, cellular
contents and quality.
• Toxicological Assessment: Safety is an important aspect of pharmacobotanical
evaluation. Toxicological studies help determine whether the plant material or its
extracts have any harmful effects on humans or animals.
20.4.2 Standardisation
Establishing quality control parameters and standards for a safe, effective and stable
phytopharmaceutical ingredient or formulation is necessary at multiple levels, starting from planting material to the conduct of clinical trials and post-market surveillance. Standardisation helps to set non-negotiable guidelines ensuring high levels of
compliance to maintain standards, processes and documentation, which are absolutely necessary for achieving batch-to-batch consistency, efcacy and stability.
The preliminary standards of cultivation or collection of herbs need to be set at
the eld level, with good cultivation practices or good collection practices at the
source. The eld staff responsible for cultivation or collection need to be trained to
be able to distinguish salient features of clones of selected varieties and their substitutes or adulterants. Authentication of the botanical specimen and periodical botanical analysis through DNA ngerprinting to ensure genetic purity are necessary. If
the herb is cultivated using the conventional method, adopting soil test crop response
(STCR) along with integrated pest management techniques that avoid the use of
harmful chemical pesticides may be preferred. STCR is a nutrient management
method of determining the optimal fertiliser dose for an herb based on the soil
analysis values and the target yield. STCR is used to improve productivity, soil
health, nutrient availability and nutrient use efciency. The use of hydroponics for
the production of herbal biomass may be expensive as compared to the conventional
cultivation model but is valuable in producing heavy metal-free herbage. However,
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the adoption of plant biotechnology techniques can ensure the production of pure
plant material provided the tissue culture protocols are managed well under aseptic
conditions that avoid microbial contamination and resultant endotoxins in the plant
extract.
S. Krishnaswamy
20.4.3 Plant Biotechnology
Plant biotechnology takes advantage of modern techniques to improve the production,
quality and consistency of herbs and their bioactive principles. This can include
genetic modication, tissue culture, biopharming and other advanced methods. Plant
biotechnology helps in developing high-yield and disease-resistant plant varieties,
enhancing the production of bioactive compounds and ensuring product uniformity.
Plant tissue culture has the potential to develop alternate phytochemical pathways
generating de novo synthesis of new molecules that may be potentially valuable as
phytopharmaceuticals. Thus, plant tissue culture may aid herbal drug discovery by
creating a vast pool of bioactive intermediates. Plant stem cell extracts are derived
from callus cultures of specic plants, and their pathways may be modulated to overproduce certain phytochemicals. Specic plant organ culture is a specialised technique where minute plant parts storing a minuscule quantity of phytochemicals can be
mass propagated to provide a valuable source of phytomedicine. Some plants have
specialised hair-like structures called glandular trichomes on their leaves, owers or
other aerial parts. These trichomes bear secretory glands that can produce and store
phytochemicals, such as essential oils or secondary metabolites, which serve various
ecological roles, including defence against herbivores and pathogens.
Phytopharmaceutical Biotechnology
Phytopharmaceutical biotechnology is an interdisciplinary eld that combines elements of botany, pharmacology and plant biotechnology to utilise the therapeutic
potential of herbs and other plant-derived compounds. This approach seeks to
develop innovative strategies for the propagation, cultivation, extraction and utilisation of medicinal plants to deliver safe and effective herbal medicines and natural
remedies. The combination of phytopharmaceuticals and biotechnology has aided
in the development of new drugs, dosage forms and treatments. By using contemporary methods of biotechnology, plants with unique chemical compositions are propagated and genetically upgraded for the mining of pharmaceuticals (Singh etal.
2022). Some key aspects of phytopharmaceutical biotechnology are to modify sec-
ondary product metabolic pathways by genetic modication of plants and the design
of robust invitro plant regeneration systems. Continued advancements in genetic
tools and techniques, such as gene cloning, advancement in the identication of
secondary plant metabolites and high-throughput monitoring technologies, have
made it easier to implement strategies for manipulating a plant’s secondary products.
20 Phytopharmaceutical Biotechnology: Integration of Botany, Pharmacology and…
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Plant Tissue Culture
Plant stem cells are propagated by adopting the technique of plant tissue culture,
resulting in aseptic cultures of a whole new plant, tissue or specic types of single
cells from which plant metabolites may be harvested. Tissue material obtained for
culture from a selected elite plant is called an explant. Regeneration of new cells
occurs along the cut surfaces and edges of the explant. This is similar to a woundhealing reaction. Normal plant cells undergo dedifferentiation to develop into a
colourless/coloured cell mass called callus or plant stem cells. Totipotent meristematic tissues are the source of plant stem cells. The callus cells mimic the intact
mother plants’ meristematic cells in terms of cell division rates, physiology and cell
contents. Plant stem cells may be grown as individual cells or small cell clusters in
a liquid culture for higher yield. In certain plant cells that accumulate compounds
such as anthocyanins and carotenoids, the callus cells may be coloured, blue, purple, red, pink, black or orange depending on the pH, light or dark conditions provided during the culture regime.
Researchers globally are focused on the optimised development of high-value
stem cell extracts with promising potential in the cosmetic, biomedical and therapeutic domains (Aggarwal et al. 2020). Stem cells derived from plants such as
grapes (Vitis vinifera), lilacs (Syringa vulgaris) and others possess strong antioxi-
dant and anti-inammatory properties and are invaluable in cosmetic applications
(Barthel and Aberdam 2005). Plant stem cells, their extracts and formulations have
a wide range of cosmetic applications including skin whitening, de-tanning, moisturising and cleansing. Despite all the promising developments, plant stem cell technology remains hugely unexplored due to the staggering number of varieties of
plant species and the exhaustive array of their phytochemical components.
In an experiment to enhance the production of metabolite β-pinene in callus culture developed in light conditions, using petals of Rosa damascene (Olgunsoy etal.
2017), it was found that deep pink-coloured callus cells were formed, accumulating
about 236 times more metabolite than was found in intact petals. The inuence of
light on anthocyanin production is visible in the rapidly proliferating callus where
the rose petal stem cells grown in the dark appear creamish yellow, whereas those
grown in light developed pigment-bearing stem cells. This exponentially increased
bioactive production was achieved using phenylalanine as a precursor in light
conditions.
The stem cell of a rare, bitter apple variety, Malus Domestica (“Uttwiler
Spätlauber”), with excellent keeping quality over extended periods, perhaps due to
the presence of certain unique metabolites, was derived through plant cell culture
technology (Trehan etal. 2017). Large-scale cultivation of apple callus (stem) cells
was undertaken to overproduce the metabolites. It was found that human young
epidermal cells treated with this extract had the potential to grow into multi-layers
resulting in skin renewal. Untreated older epidermal cells could not develop into
layers under normal laboratory conditions but resumed epithelial growth on treatment with 2% emulsion of apple stem cell extract consisting of phytocomponents
such as carotenoids and avonoids (Prhal etal. 2014).
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S. Krishnaswamy
Another product was developed by using biotechnological production methods
in cloudberry (Rubus chamaemorus) cells (Martinussen etal. 2004). Bioreactors
were used to develop entrenched callus and suspension cultures of Rubus
chamaemorus. Optimal growth of callus was achieved on Murashige and Skoog
medium, supplemented with kinetin and α-naphthalene acetic acid as plant growth
hormones at specic concentrations. This process was standardised and proved to
be a sustainable technique for consistent and continuous sources of fresh cells/
extracts from cell fractionation for isolating biologically potent compounds, freezedried cell products, aromatics or colouring agents. Stem cells cultured from tomato
(Lycopersicon esculentum) cells possess tremendous potential to protect skin from
adverse effects due to heavy metal toxicity (Tito etal. 2011).
A cosmetically active hydrophilic ingredient, extracted from liquid cultures of
L. esculentum, consisted of comparatively higher concentrations of avonoids and
phenolic acids such as quercetin, rutin, coumaric, protocatechuic and chlorogenic
acids. The tomato stem cell extract also contained higher phytochelatins, an antioxidant and chelating agent responsible for capturing metal ions and protecting cellular
materials and organelles from potential toxicity. Extracts obtained by this method
were very effective at enhancing healthy skin growth and complexion, contributing
a valuable phytopharmaceutical in the area of skincare cosmetics.
The short-wave (UVC 200–280nm), mid-wave (UVB 280–315nm) and longwave (UVA 315–400nm) UV rays can cause cancer, erythema or photoageing on
constant exposure. Ginger extract with skin-preserving bioactives (Zingiber ofci-
nale) is derived through biotechnology-based cell multiplication of improved cosmetic cell lines. Reduced pores and mattifying effect, contributing to almost 50%
enhancement of skin quality, were achieved in female subjects in a clinical study
(http://www.naolys.com/media/rene_ginger_en.pdf. Accessed 15 Jul 2018). This
effect was further augmented by a consequent reduction in skin sebum and oily
shininess. Increased synthesis of elastin bres in the skin consequently reduced the
rate of sebum production. The protective and potent anti-collagenase and hyaluronidase activity of an anti-ageing component obtained from stem cell extracts of edelweiss (Leontopodium alpinum) was reported to be rich in leontopodic acids A and
B, which are responsible for exhibiting a strong and potent antioxidant effect on the
skin (Cho etal. 2020).
Specialised organ culture is another phytopharmaceutical biotechnology-driven
route to elicit the overproduction of pure bioactives from plant cell cultures, with
fewer intermediates, which may otherwise appear as impurities. The juice-bearing
vesicles from the fruits of citrus species have been cultured using tissue culture
techniques to harvest vitamin C, naringin and hesperidin (Einset 1978). Citrus vesicle stem cells may also accumulate high concentrations of kinetin, a cytokine that
strongly inhibits ageing processes in human cells. The innovations and technological advancement in the eld of plant biotechnology enable novel product designs
such as nourishing and hydrating, easily absorbed skin nutrients. Such highly efcacious plant stem cell-based cosmetics are in great demand.
The process ow for the production of phytopharmaceuticals using biotechnology methods is given in Fig.20.4.
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