Trends in bioprospecting of biodiversity in new drug design

Abstract

Medicinal plants constitute main resource base of almost all the traditional healthcare systems. Most of the herbal drugs produced currently in majority of the developing countries lack proper quality specification and standards. The technological advancement of 20 th century saw the collaboration of engineering science and biological science, thus explore the genomic basis and molecular structure of the active principles. In the 21 st century with the availability of sophisticated instruments led to the detailed structural and functional dynamics of the organism/ genes become so handy. This led to the newer opportunities in creating different pharmaceuticals with lesser side effects, and food with more nutritional value and emergence of novel field of research. The understanding of the genomics, proteomics, transcriptomics and metabolomics has contributed a great deal in pharamaceutical and nutraceutical science. The giant strides made by analytical and synthetic chemistry, electronics and science in general, have immensely contributed to the development of the science or biomedicine that has achieved miracles in medical practice. A new thinking centered on the concept of 'knowledge engineering' for building up future 'knowledge societies' and 'knowledge industries' is now gaining attention and acceptance both nationally and internationally.

Full text

[Received 21.10.2015; Revised 08.12.2015; Accepted 20.12.2015; Published 31.12.2015]
Trends in bioprospecting of biodiversity in new drug design
P. Pushpangadan1,3, T.P. Ijinu1, V.M. Dan2and V. George1
1Amity Institute for Herbal and Biotech Products Development, 3 Ravi Nagar, Peroorkada Post,
Thiruvananthapuram 695 005, Kerala, India
2Rajiv Gandhi Centre for Biotechnology, Thycaud Post, Thiruvananthapuram 695 014, Kerala, India
3Corresponding author, E-mail: palpuprakulam@yahoo.co.in
Abstract
Medicinal plants constitute main resource base of almost all the traditional healthcare systems.
Most of the herbal drugs produced currently in majority of the developing countries lack proper
quality specification and standards. The technological advancement of 20th century saw the
collaboration of engineering science and biological science, thus explore the genomic basis and
molecular structure of the active principles. In the 21st century with the availability of
sophisticated instruments led to the detailed structural and functional dynamics of the organism/
genes become so handy. This led to the newer opportunities in creating different pharmaceuticals
with lesser side effects, and food with more nutritional value and emergence of novel field of
research. The understanding of the genomics, proteomics, transcriptomics and metabolomics
has contributed a great deal in pharamaceutical and nutraceutical science. The giant strides made
by analytical and synthetic chemistry, electronics and science in general, have immensely
contributed to the development of the science or biomedicine that has achieved miracles in
medical practice. A new thinking centered on the concept of ‘knowledge engineering’ for building
up future ‘knowledge societies’ and ‘knowledge industries’ is now gaining attention and
acceptance both nationally and internationally.
Key words: Biodiversity, bioprospecting, metabolomics, knowledge engineering, high-throughput
screening
INTRODUCTION
Biodiversity and traditional knowledge together formed the cradle for bio-prospecting and
the technological advancements in the modern world provided the essential nourishment to
the mammoth development of this new age field. The prospects of exploring biodiversity for
new medicines, foods, crops, insecticides, pesticides and other commercially valuable genetic
and biological products and processes are booming, thanks to the rapid development in
biotechnology - particularly genomics, proteomics, transcriptomics, enzymatic and transgenic
technologies, herbal technology and information technology. This exploration of biodiversity
for commercially valuable genetic and biochemical resources is termed as “bio-prospecting”
- a concept pioneered by Thomas T Eisner as “chemical prospecting” (Eisner 1989; Reid et
al. 1993). The advancements in biotechnologies have further redefined the overall scope
and utility of bio-prospecting to encompass all relevant activities related to systematic search
for genes, natural compounds, designs and whole organisms in wildlife with a potential for
product development by biological observations and biophysical, biochemical and genetic
methods without disruption to nature (Mateo et al. 2001). In short, bio-prospecting involves
investigation of genetic resources or bio-chemicals for new commercial leads (Laird & ten
Pleione 9(2): 267- 282. 2015. ISSN: 0973-9467
© East Himalayan Society for Spermatophyte Taxonomy

Kate 2002) and includes three major areas such as chemical prospecting, gene prospecting
and bionic prospecting (Maeto et al. 2001).
Chemical prospecting
Modern high-throughput chemical screening and automated bioassay programs for identifying,
isolating, characterizing novel bioactive compounds from wild plants, fungi, animals (insects
and invertebrates especially) and microbes have opened up new vistas in natural product
research in general and drug and pharmaceutical discoveries in particular. Chemical
prospecting of wild plant resources is becoming increasingly applicable in agro-chemistry
(biopesticides and insecticides), drugs and pharmaceuticals, cosmetics, proteins, enzymes,
food additives and other industrially valuable chemical products (Eisner 1997).
Gene prospecting
Modern molecular technologies like DNA recombinant techniques and transgenic technologies
made it possible to identify, isolate and introduce desirable gene(s) from one organism to
another, transcending the biological/taxonomic barrier. Transgenic technologies are making
significant headways by facilitating transfer of the desirable agronomic traits or chemicals
from one organism (e.g., plants; animals, including humans) to bacteria and converting the
resultant transgenic bacteria to potential chemical factories producing desired products such
as enzymes, drugs, pharmaceuticals and other valuable products. Similarly, genetically modified
fungi are now proved to be potential sources for mass production of enzymes, proteins and
other biomolecules. New and improved crop varieties are developed through genetic
engineering techniques with desired genes that confer resistance/tolerance to pest, disease
and climatic/environmental stresses. Isolation of Bt gene (isolated from the bacterium,
Bacillus thuringiensis) having insect resistance and introducing this gene into crop plants
such as potato, cotton, corn, wheat, rice, tomato, etc., through transgenic techniques is a
classic example to demonstrate the prospects of gene prospecting for crop improvement and
sustainable agriculture. Proteomics is an interesting field of gene prospecting. This deals
with the identification and patterns of expression of gene(s) that encodes for the synthesis of
a specific protein of interest. Bioinformatics provide the key source for mining DNA / nucleotide
sequences data, based on which new genes responsible for the synthesis, expression and
function of a particular protein or enzyme can be made. Proteomics offer new promises to
gene therapy and enzymatic technologies.
Bionic prospecting
Bionic prospecting is a new area by which new designs, patterns, models and techniques are
evolved based on natural biodiversity. New sensor technologies, architecture, bioengineering
and bio- modeling are some of the interesting fields in bionic prospecting.
Major sectors of bioprospecting
Bio-prospecting covers wide range of commercial activities, including the pharmaceutical,
biotechnology, seed, crop protection, horticulture, botanical medicine, cosmetic and personal
care, and food and beverage sectors (ten Kate & Laird 1999; Laird & ten Kate 2002). The
major players of bio-prospecting include multinational companies (in private and public sectors),
R&D institutions, universities, botanic gardens, etc. Genetic resources and associated traditional
knowledge provide the key resources and biotechnologies offer the key tools relevant for
these bio-prospecting sectors. The ways in which they use genetic resources and
biotechnologies would vary among and between these sectors depending upon the ultimate
268 Trends in bioprospecting of biodiversity

aim and targets of each bio-prospecting activity. The quantum of genetic resources or their
derivatives used, the leads from associated traditional knowledge accessed or utilized, and
the methodological framework of various techniques and tools employed would differ
significantly in each bio-prospecting activity. These are guided by a number of requisite
factors such as the capability of the bio-prospecting companies or institutions in terms of
infrastructure, human resources and technological capabilities, as well as the existing national
and international policy and legal frameworks that facilitate free and regulated access to
genetic resources, or their derivatives and/or the associated traditional knowledge, and more
importantly the ultimate objectives of the bio-prospecting mission envisaged. For example,
among the above-mentioned major players in bio-prospecting programs, the pharmaceutical
and agro-technology industries are the prominent ones and have a major stake in the global
bio-industrial regimes. They use genetic resources in significantly different ways. There is
diversity of genetic resources use and biotechnological interventions within and between the
bio-prospecting sectors, which is influenced greatly by the following factors such as (1) size
of industries and markets for the products, (2) role of natural products in these markets and
percentage of sales contributed by genetic resources, (3) relationship between commercial
products and the genetic resources from which they are developed (Laird & ten Kate 2002).
The global estimates show a 900 billion in the annual sale values of pharmaceuticals,
crop protection, agricultural seed, horticulture, botanical medicines, cosmetics and personal
care. Within each sector, the share of sale value derived from genetic resources varies. For
example, in the pharmaceutical industry, natural products contributes about 25 – 50 percent
of medicines, whereas ornamental horticultural products and sales of agricultural seeds are
100 percent natural products and make up less than 10 percent of global sales. Biotechnology
is yet another largest sector wherein genetic resources are found increasingly used for the
development and manufacture of a plethora of products, from enzymes and metabolites to
process such as bioremediation systems.
The relationship between commercial products and genetic resources also varies within
each bio-prospecting sector. In the pharmaceutical and crop protection industries, the final
commercial product might be: (i) chemically identical to the pure natural product, and (ii)
start with a natural synthesized to a design based on a natural template. Similarly, natural
personal care and cosmetics products might be derived directly from natural genetic resources
through high-throughput chemical screening, standardized chemical extracts (with set amounts
of chemical markers), or extracts of whole plants containing all the constituents found in a
given plant (Laird & ten Kate 2002).
Bioprospecting methods and strategies
The modern bio-prospecting sectors employ all the latest tools and technologies in
biotechnologies along with appropriate designs and models of high-throughput screening,
combinatorial chemistry, computational biology, bioinformatics, biosynthesis, and production
and marketing strategies. While the inventive steps and technical inputs involved in development
of a new product or process in each of the above sector would vary significantly, the general
framework and overall strategies required by each sector contain certain essential common
elements. For example, all bio-prospecting enterprises would require collection and acquisition
of the essential raw or value-added or derivatives materials including naturally occurring
genetic resources and/or associated traditional knowledge, which would be followed by
random or selective screening for targeting a desired candidate/precursor or new leads for
developing the targeted product or process. Once the leads are obtained, these need to be
characterized and evaluated using appropriate techniques so as to pin point the most ideal
P. Pushpangadan et al. 269

and desirable hit for further evaluation, standardization, quality tests and trials (clinical trials
in case of biopharmaceuticals), synthesis, production, and marketing.
The success of any bio-prospecting programme depends on a number of factors such
as (i) demand for and constraints on access to biological resources and associated traditional
knowledge by potential users (ii) capability of providers of genetic resources to supply a
good service and constraints on capacity to supply (iii) legislation, policy and a political climate
suitable for bio-prospecting (iv) economic contribution that facilitates bio-prospecting and
(v) ability to guarantee enforcement of agreements (ten Kate 1995).
There are also a number of other factors that contribute to better choice of bio-
prospecting venues and partners. These, according to ten Kate & Laird (1999) include: (i)
“biological diversity of the region; (ii) confidence that partner collects and supplies specimens
in strict compliance with international and national law; (iii) ability of partner to obtain relevant
permits and prior informed consents; (iv) clear, simple, and unbureaucratic procedure for
securing permits and access agreements; (v) capacity of partner to ensure quality and quantity
of supply and resupply; (vi) caliber of scientific staff in partner institute; (vii) political stability
of the country and region in which collection takes place and where partners collaborate in
value – adding activities, law and policy on protection of IPRs, taxation, and foreign ownership
conducive to foreign investments.
Development in chemistry
Unfortunately, most of this modern therapeutics are so expensive that they are beyond the
reach of the vast majority of the world’s population. Also there are many ailments like
cancer, liver disorders and arthritis etc, which has no satisfactory cure in modern medicine
but traditional medicines like Ayurveda and Siddha claim to have satisfactory cure and
management of such dreadful diseases. Modern medicine generally serves only a minority
(about 30 to 35 per cent) of the total population in the developing countries (Naranjo 1981,
1995). The rest of the population attends to its health needs through the traditional medicine,
which is essentially based on the use of easily accessible low-cost medicinal plants. Several
considerations make the use of medicinal plants desirable. Among them are: a) their low
cost, while the new synthetic drugs are becoming increasingly inaccessible to the vast majority
of people; b) often they are the only recourse available; c) research has confirmed the
presence of therapeutically active compounds such as alkaloids, glycosides and others,
justifying a good many practices of folk medicine; and c) they have few, if at all, harmful side
effects and hence their direct administration in traditional medicine offers little risk of causing
iatrogenic (drug induced) disorders, unlike the modern synthetic drugs (Pushpangadan &
Govindarajan 2005).
The capacity of chemists to modify a molecular structure is almost unlimited, but the
capacity to create new structures with therapeutic properties has been found to be limited
(Naranjo 1995). Plants and animals offer thousands of new molecules (Evans et al. 1982;
Gottlieb 1982). An intensive and extensive study of the naturally occurring molecules identified
as ‘therapeutically active’ is desired urgently to come out with new therapeutic entities. The
very large number of alkaloids and several other classes of chemical compounds discovered
during the 1970s and 1980s found to be pharmacologically active, serve as models for new
synthetic compounds (Barz & Ellis 1981).
A number of plant-based drugs, such as vincristine, taxol, digoxin, quinine, reserpine,
ergotine, opioids, ephedrine, colchicine, rutin, coumarins, anthraquinones, etc., are still a part
of standard therapy. Most of these do not have any synthetic substitutes. Several other plant
products are used in formulations that are sold over the counter (OTC) in several countries.
270 Trends in bioprospecting of biodiversity

The role of plants in standard therapy will certainly be enhanced several fold in future,
provided we make the move in the right direction.
Chemo-profiling using HPLC, HPTLC, GC-MS, LC-MS, UV-Vis, FT-IR, LC-NMR-
MS, LC-ToF etc. have a great role in quality control of medicinal plants (raw drugs) and in
the finished herbal drugs. European Pharmacopoeia gives assay of quinine type alkaloids
and cinchonine type alkaloids.Cinchona bark using UV spectroscopy and US Pharmacopoeia
include an UV absorption test for the absence of foreign oils in oil of lemon and orange. UV
spectroscopic analysis has been used for the quantification and qualitative detection of marker
compounds from the herbal material. Infrared spectroscopy, NMR and mass spectroscopy
have been used for the structure elucidation of marker or active components of medicinal
plants. As mentioned earlier the active principles in most medicinal plants are highly variable.
This include intrinsic factors such as the genetic variations particularly in cross pollinated
plants and intrinsic factors such as agroclimatic, edaphic conditions, stage of growth and
developmental stage of the plant etc. to ensure a reasonable consistency in quality and
efficacy one needs to identify the right genotype and correct growing, collection and post
harvest handling practises. Withania somnifera an important medicinal plant of Ayurveda is
reported to have three chemotypes depending upon the presence of a class of closely related
steroidal lactones like withanolides, withaferin A etc. The content of withanolides and withaferin
A and other biological active compounds may vary depending upon the genotype, micro and
macro environment and developmental stage of the plant. All such finer details of the medicinal
plant species when compiled together, it is referred as the passport data of the plant.
DNA based molecular markers are proving to be a versatile tool in the plant genome
analysis and in differentiating different genotypes. Various techniques like Random Amplified
Polymorphic DNA (RAPD), Fragment Length Polymorphic DNA (AFLP), Restricted Length
Polymorphic DNA (RFLP) and Inter Simple Sequence repeat (ISSR) etc are now being
successfully used for such genetic analysis of medicinal plants and also for the characterization
of semi-processed and even fully processed herbal products. DNA markers are highly stable
and specific. It has immense applications in the standardization of medicinal plants and its
products.
Minimum requirements for medicinal plants and its products for global acceptance
should have:
Demonstrated safety
Mapped efficacy (Pharmacologically credible formulation)
Consistency in batch to batch quality
 For polyherbals, contribution of each herb to be proved along with synergims. It
should be synergistic rather than additive
Avoidance of endangered species
Easy availability and accessibility of the plant
New technologies are constantly being developed to isolate and identify the components
responsible for the activity of these plants. But these technologies should consider and possibly
use the fact that the biological activity of plant extracts often results from additive or synergistic
effects of its components. Another possibility is the qualitative and quantitative variations in
the content of bioactive phytochemicals, which are currently considered major detriments in
their use as medicine. Different stresses, locations, climates, microenvironments and physical
and chemical stimuli, often called elicitors, qualitatively and quantitatively alter the content of
P. Pushpangadan et al. 271

bioactive secondary metabolites. Enzymatic pathways leading to the synthesis of these
phytochemicals are highly inducible (Ebel & Costa 1994). This is particularly true for
phytochemicals that are well documented for their pharmacological activity, such as alkaloids
(Facchini 2001), phenylpropanoids (Dixon & Paiva 1995) and terpenoids (Trapp & Croteau
2001) whose levels often increase by two to three orders of magnitude following stress or
elicitation. Thus, elicitation-induced, reproducible increases in bioactive molecules, which
might otherwise be undetected in screens, should significantly improve reliability and efficiency
of plant extracts in drug discovery while at the same time preserving wild species and their
habitats. Standardization, optimization and full control of growing conditions should guarantee
a cost-effective and quality-controlled production of many plant-derived compounds. This
kind of standardization and quality control of the plant-based drugs will improve safety of
these drugs and promote their usage.
Alarming levels of antibiotic resistance in many human pathogens is likely to provoke
an increase in pharmaceutical bioprospecting, which remains a vital source of lead drug
discovery. Malaria, one of the world’s most deadly diseases, has been treated historically
with drugs derived from natural products - quinine, chloroquine, meoquine, and doxycycline
and today the artemisinins derived from the Chinese herb Qinghao (Artemesia annua)
are at the forefront of the battle against this parasite. Ethnomedical knowledge of some
plants led to the development of drugs like Aspirin. It was rst isolated from Filipendula
ulmaria because it had long been used in folk medicine of Europe to treat pain and fevers.
When the Bayer company developed a synthetic derivative of salicylic acid called
acetylsalicylic acid, they named it Aspirin - ‘‘a’’ for ‘‘acetyl’’ and ‘‘spirin’’ for Spiraea,
the former Latin name for the genus. Another European folk cure that became a drug was
derived from Digitalis purpurea, the leaves of which were rst used to treat congestive
heart failure. The active ingredients, digitoxin and diyoxin, remain an important treatment
for heart ailments.
The potential of plant metabolomics in drug development
Plant Metabolomics provides one of the pillars for studying the relation between the composition
of complex and variable mixtures of plant derived remedies and their - also complex - biological
effects. Plant metabolomics starts with the analysis of as many as possible detectable individual
components that are present in the material. Extracts made from individual herbs/ plants,
total mixtures or combinations of individual herbs/plants and extraction/mixing/preparation
methods as used in phyto medicine can be analyzed by means of different techniques (LC-
MS, GC-MS, NMR, LC-NMR etc.) resulting in total metabolite profiles.
Next, extracts (individual, total, combinations) are investigated for bioactivity (effects
in cell lines, animal models, human volunteers) studying their effects at various biological
levels using the body fluid studies. The databases from plant metabolic profiles, bioactivity
and animal studies can be linked and analyzed by means of Multivariate Data Analysis
(MVDA). MVDA is a powerful technique for the analysis of data sets with a large number
of variables. It enables, for example the visualization and interpretations of patterns in NMR
data that correlate with a target variable such as bioactivity. In this way, a plant metabolic
data base can be constructed which will be extended with single compounds and metabolic
profiles of all types of commercial extracts available from vegetables/crops/herbs that have
grown, harvested and stored under different conditions. In the past a major tool in identifying
new activity was testing unknown compounds or extracts on whole animals and making a
whole set of observations at regular time intervals (Hippocratic screening). By validating
this method with various known compounds, it was very useful for the identification of novel
activities. However, for determining which were the active compounds this approach had
the disadvantage that it was elaborate and quiet large samples were needed, something
272 Trends in bioprospecting of biodiversity

which is difficult to realize with bio-assay guided fractionation. By administering plant extracts
of different composition and using MVDA, it will be possible to calculate which compounds
or groups of compounds are associated with the highest bioactivity.
Reverse pharmacology
Contrary to what has been said earlier the first step in Reverse Pharmacology (RP) is to
select a remedy for development, through a retrospective treatment – outcome study. The
second step is a dose escalating clinical trial that shows a dose – response phenomenon
which helps in selecting the safest and most efficacious dose. The third step is a randomized
controlled trial to compare the phytomedicine to the standard first-line treatment. The final
step is to identify active compounds which can be used as marker for standardization and
quality control. Thus RP approach can help in development of phytomedicines more
economically and cheaply. The advantage of RP method in development of herbal
phytomedicine can be appreciated when we consider that the development of conventional
drugs is a slow and expensive processes taking up to 15 years and about $ 800 million
(Nwaka et al. 2009; DiMasi et al. 2003). The various steps involved in dose optimization in
RP studies are depicted in Fig. 1 and 2.
Vaidyaet al. (2003) proposed a new discipline called Ayurvedic pharmacoepidemiology.
Pharmacoepidemiology is a new field developed by synergy of fields of clinical pharmacology
and epidemiology taking roots in the Western countries. He felt that with the ever-growing
global interest in the Ayurvedic system of medicine, Ayurvedic Pharmacoepidemiology could
emerge in India. This was indeed the result of a novel highly competitive research programme
launched under NMITLI to develop globally acceptable herbal drugs from the Ayurvedic
therapeutic heritage. There projects have been already initiated in – diabetes mellitus,
osteoarthiritis and hepatitis.
Modern molecular technologies like DNA recombinant techniques and transgenic
technologies make it possible to identify, isolate and introduce desirable gene(s) from one
organism to another, transcending the biological/taxonomic barrier. Proteomics and
metabolomics are interesting field of gene and drug prospecting. This deals with the
identification and patterns of expression of gene(s) that encodes for the synthesis of a specific
protein of interest. Bioinformatics provide the key source for mining DNA / nucleotide
sequences data, based on which new genes responsible for the synthesis, expression and
function of a particular protein or enzyme can be made. Proteomics offers new promises to
gene therapy and enzymatic technologies.
Council of Scientific and Industrial Research (CSIR) has initiated a coordinated program
on drug discovery with a network of 19 CSIR laboratories and other R&D institutions working
in the area of traditional systems of medicines and universities. The program which was
initiated in 1996 aims at discovering new bioactive molecules from plants, fungi, microbes,
insects, etc. using new techniques of both synthesis (combinatorial chemistry) and bio-
evaluation (medium and high through put screening). The program also includes discovery of
molecules based on mechanism of the disease, functional genomics, anti-sense agents, etc.
Currently bio-evaluation of the following eleven major diseases is in progress: 1. Bacterial
infections 2. Malaria 3. Tuberculosis 4. Filaria 5. Hepatitis 6. Hypertension 7. Memory 8.
Leishmania 9. Inflammation and Arthritis 10. Diabetes 11. Cancer.
The Planning Commission, Govt. of India and CSIR has embarked on a few bio
prospecting programs with some specific targets/goals – such as the inter laboratory
collaborative programs on biomolecule/drugs, drug prospecting. The Planning Commission
P. Pushpangadan et al. 273

Range of traditionally recommended doses:
Start with lowest dose
Decrease dose
Increase dose
Clinical results
Good effectiveness Insufficient effectiveness
Safe and well tolerated Safe and well tolerated
No Yes No Yes
Optimal dose Stop the trial
(failure)
Fig. 1. Dose optimization[Adapted from Willcoxet al. 2011; George & Ijinu 2011]
sponsored the NMITLI (New Millennium Indian Technology Leadership Initiative), which is
one of the most innovative bioprospecting programs. NMITLI has major herbal drug
development program for developing effective herbal remedies for hepatic disorders, arthritis
and diabetes, which has shown highly encouraging results.
Systems biology and traditional medicine
It is now seen that systems biology can provide an important bridge function between TM
and Western Medicine, because it can reveal the effects of simple perturbations such as
single drug and/or complex perturbations such as TM or food. The use of systems biology to
study the effect of TM on humans is very promising but also one of the most complex
challenges in life science research. Moreover, within TM there is the challenge of the quality
control of the production.
274 Trends in bioprospecting of biodiversity

Stage 1:
Selection of a remedy
Retrospective treatment outcome study
Literature review (selected remedy)
Stage 2:
Dose escalating clinical trial
Increase dose sequentially
Observe clinical effects
Assay safety
Choose optimal dose
Stage 3:
Randomized controlled trial
Pragmatic inclusion criteria and outcomes
Compare to standard first line drug
Test effectiveness in the field
Stage 4:
Isolation of active compounds
In vitro tests of purified fractions and isolated
compounds
Standardization and quality control of
phytomedicine
For agronomic selection
For pharmaceutical development
Fig. 2. Summary of the methodology used to develop phytomedicine by Reverse
Pharmacology [Adapted from Willcox et al. 2011; George & Ijinu 2011]
The optimal bioactive fingerprint of the many components in a TM preparation is not
known. There is no direct control mechanism on the production and processing of plants and
therefore it may cause a varying success factor in the evaluation process. In fact differences
in harvesting conditions that occur even during intervals as short as less than one day may
already generate differences between batches. A more basic consideration in the evaluation
of TM comes from the fact that it is prescribed as a personalized preparation. Consequently
it cannot be evaluated in the more conventional way by applying clinical trials and generic
treatments.
P. Pushpangadan et al. 275

All the complicating factors mentioned above can be addressed in well designed studies
if technologies are used based on finger printing of not only the system but also the complex
perturbation mixtures and their linking both together using non-linear multivariate approaches.
The technology platform used in system biology comprises the elements as given in
Fig.3: transcriptomics, proteomics and metabolomics at different levels: cell, tissue, organ,
organism and system level. In addition, special attention is paid to profiling body fluids being
an important source of information regarding the systems control functions or its biochemical
“body” language. The extensive mapping of body fluids brings a new dimension into human
physiology and is especially suited for the evaluation of the effects of treatments. Typical
fluids are urine, blood, cerebrospinal fluid (CSF), saliva, lymph, synovial fluid etc. The ethical
consideration favors urine and blood, which opens up especially the elements of proteomics
and metabolomics measurements. In selected cases transcriptomics can be used in monitoring
studies when blood cells are harvested or tissue biopsies are available. As for TM, the
medical preparation itself is also of a complex and changing character. This introduces a
second set of variables. To make it even more challenging, not only the relative composition
of a remedy is subjected to variation, but the quality of the starting materials may also differ.
Genomics
Transcriptomics
Proteomics
Functional Proteomics
Metabolomics
Cell level
Tissue level
Organ level
Organism level
DNA
mRNA
Protein
Protein complex
Metabolites
F
L
U
I
D
S
S
Y
S
T
E
M
S
B
I
O
L
O
G
Y
Fig. 3. The different levels of measurement in a systems approach [Adapted from Wang et
al. 2005]
In TM most of the constituents of the preparations are derived from plants. Plants
constantly interact with their changing and often harsh environment during the different
phases of their life cycle. Plant secondary metabolites provide chemical protection against
invading pathogens and predators to attract, for e.g., pollinators and physical stress but may
also act to give a typical smell or color. Plants can make several thousands of these secondary
metabolites. This has resulted in a natural treasure house with highly diverse and often very
276 Trends in bioprospecting of biodiversity

potent compounds with a wide diversity of application in human health. A complicating factor
when using plants is the variability of the material. This can be caused by differences occurring
during growth, but also after harvesting the plant, due to decomposition during post harvest
processing, extraction and preparation. Quality control and standardization are therefore
highly relevant to assure proper preparation and standardization of a medication. In India the
Indian Council of Medical Research has developed quality standards for a large number of
Indian medicinal plants and are published in a series of volumes. However, standardization is
still a matter of debate. It has been demonstrated that the secondary metabolite concentration
in plants like Ginkgo biloba leaves vary considerably between the leaves harvested in the
morning and in the evening from the same tree. It is noted that the ginkgolide and bilobalide
concentration in the leaves were much higher when they are harvested in the evening. Also
it has been noticed that there is cultivar dependent variation on metabolites in plants such as
Cannabis sativa. In fact, in many studies on the activity of medicinal plants, and in particular
in clinical studies, the plant material was not properly defined, making the results very doubtful.
At the same time with all the possible variables it is almost impossible to measure each
separately to determine which one is more active. Again by using the multivariate analysis of
the data from either the treated animals or patients and of the metabolomes of the different
preparations tested, the optimal composition can probably be calculated.
More recently novel techniques such as Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry (FT-MS) represent a quantum leap forward in the capabilities of mass
spectrometers for metabolite analysis. Due to the exceptionally high resolution of these
instruments metabolites with mass differences of less than 2ppm can be separated on a
chromatographic scale. The accurate masses obtained give elemental compositions, which
enable unequivocal metabolite identification.
Nutrigenomics, nutrigenetics and personalized nutrition
With the revolutionary advancement now making in genomics and proteomics a new type of
food and nutrition under the head nutrigenomics are expected to emerge. With genome
analysis the genetic predisposition of an individual can be made and thereby it is possible to
know what kind of proteins or nutrients will suit to his/her constitutional type. Based on such
genetic/constitutional analysis it is possible to recommend specific food items or even can
breed or develop genetically modified food(s) that suit his/her genetic predisposition.
Alternatively it is also possible to introduce raw or modified functional gene(s) or administer
desired protein(s) / enzymes to human so that he/she may be able to assimilate/avoid the
allergy or certain proteins in the food. Thus one look forward for a custom-made or
personalized food and medicine that is genetically modified can be very soon expected to
flood in the market. In fact the concept of personalized/ person specific food/nutrition and
medicine is not a new one ancient Ayurvedic / Siddha masters have given top priority to this
concept.
The Human Genome Project (HGP) is the largest ever international collaboration in
biology. The result has been that the sequence of three billion chemical coding units in human
DNA is now known. The next challenge is to identify each of the sequences of codes that
are responsible for a specific activity or outcome. Genes are turned on and off according to
metabolic signals that the nucleus receives from internal factors (e.g., hormones), and external
factors (e.g., nutrients), which are among the most influential of environmental stimuli (Harland
2005). Unbalanced diets alter nutrient gene interactions, thereby increasing the risk of
developing chronic diseases. Numerous dietary components can alter genetic events, and
thereby influence health. In addition to the essential nutrients, such as carbohydrates, amino
acids, fatty acids, calcium, zinc, selenium, folate, and vitamins A, C and E, there is a variety
P. Pushpangadan et al. 277

of non-essential bioactive components that seem to significantly influence health (Corthésy-
Theulaz et al. 2005; Trujillo et al. 2006).
Nutritional genomics or nutrigenomics, is the study of how food and genes interact
and aims to understand the effects of diet on an individual’s genes and health. It attempts to
study the genome wide influences of nutrition and identify the genes that influence the risk of
diet related diseses on a genome wide scale, and to understand the mechanisms that underlie
these genetic predispositions (Muller & Kersten 2003). More practically, nutrigenomics
describes the use of functional genomic tools to probe a biological system following a nutritional
stimulus that will permit an increased understanding of how nutritional molecules affect
metabolic pathways and homeostatic control. Nutrigenetics, on the other hand, aims to
understand how the genetic makeup of an individual coordinates their response to diet, and
thus considers underlying genetic polymorphisms. It embodies the science of identifying and
characterizing gene variants associated with differential responses to nutrients, and relating
this variation to disease states. Therefore, both disciplines aim to unravel diet/genome
interactions; however, their approaches and immediate goals are distinct. Nutrigenomics will
unravel the optimal diet from within a series of nutritional alternatives, whereas nutrigenetics
will yield critically important information that will assist clinicians in identifying the optimal
diet for a given individual, i.e., personalized nutrition (Mutch et al. 2005).
Ayurgenomics
Many rare diseases like hemophilia, beta-thallasemia etc. are monogenic, caused due to
mutations in single genes. Most of the common diseases such as diabetes, asthma,
cardiovascular disease etc. are multigenic complex disorders involving many genes. It is
generally observed that common diseases are a consequence of cumulative effect of a large
number of variations in the genome which independently have small effects that are not
sufficient to cause the disease. However, it is now being increasingly realized that even
those diseases that were considered to be monogenic sometimes exhibit differences in
manifestation of disease in different individuals in spite of carrying the same mutations. This
is thought to be due to presence of variations in other genes that could modify the effect of
the primary mutation. Further, there is a complex interplay of gene and environment involved
in the majority of the diseases. Most of these diseases require long term drug administration
and there is a high variability in individual response to drug dosage and adverse effects due
to mainly variations in the genes responsible for drug transport and drug metabolism within
the individual’s system. Therefore, design of optimum dosage with least side-effects is difficult
to establish.
Tridoshas are not only genetically determined (Shukra Shonita) but also influenced by
the environment during development, especially maternal diet and lifestyle. Prakriti is fixed
at the time of birth and remains invariant throughout the individual’s lifespan. Ethnicity
(Jatiprasakta), familial characteristics (Kulanupatini), and geoclimatic regions (Deshanupatini)
are also implicated in influencing phenotypic variability through their effect on Tridoshas and
Prakriti. Thus, most of the factors such as ethnicity, geography, and environment that contribute
to inter-individual variability at the genetic or epigenetic levels are embedded in Ayurveda’s
concept of Prakriti. In an individual, the Tridoshas work in conjunction and maintain
homeostasis throughout the lifetime of the individual (Sethi et al. 2011).
There is no modern methods available to look at inter-individual differences within
ethnically matched healthy populations and no studies at the genome-wide scale have, however,
been attempted before. Mukherji and her team at the Institute of Genomics and Integrative
Biology, have been exploring the concept whether Ayurveda, can fill this gap and help in
278 Trends in bioprospecting of biodiversity

identification of predictive markers for some of these complex diseases (Mukherji & Prasher
2011).
In this regard, recently the Council of Scientific and Industrial Research (CSIR) in
association with Indian Centre for Social Transformation (Indian CST) has started major
program called TRISUTRA (Translational Research and Innovative Science Through
Ayurgenomics) at the Institute of Genomics and Integrative Biology (IGIB), New Delhi in
March, 2009. The European Institute of Systems Biology and Medicine (EISBM) and the
Institute of Genomics and Integrative Biology (IGIB) are already planning a research exchange
programme in Ayurgenomics.
Time and risk factors in high-tech bioprospecting
The underlying premise of any meaning bio-prospecting ventures should be conservation of
biological and sustainable human development. Acknowledging this fact, the partners need
to appreciate the strength and constraints on both parties and certainly have to make benign
compromises on acceptable realities and risks involved in long – term bio-prospecting
programmes. For example, drug prospecting is a multi – billion-dollar industry. The cost of
developing a single modern drug is to the tune of US $ 500 – 575 millions. It is well accepted
that the possibility of finding a potential bioactive compound is 1 in 10,000 samples and that
of discovering a marketable drug is a 1 in 4 bioactive compound. Moreover, drug-screening
programmes take a long term of say 15 to 18 years. The market opportunities prevailing at
the time of a drug discovery is also vital factor that prompts the drug or pharmaceutical
prospectors to compete for marketing their products. Under such circumstances, the drug
prospecting companies would neither be sure about the royalty to be fixed for its future
product nor would they like to forfeit their investments by taking sheer risks. It is therefore
recommended and followed in many bio-prospecting partnerships, like the In Bio – Merck,
that an ‘up – front’ payment may be made by the prospectors to the source countries to
support research and development programs, infrastructure development and human resource
development through training and capacity building in biotechnology and bio-prospecting.
Likewise, the biotechnology–rich countries should make certain commitments and concessions
to the developing countries to access the relevant biotechnologies required for sustainable
use of genetic resources.
Bioprospecting and biodiversity conservation
It may, however be recalled that the pros and cons of bio-prospecting need to be evaluated
against the backdrop of the increasing incidence of bio-piracy and more seriously against the
current crisis of bio-depletion and the likely impacts of predicted mass extinction spasm
impending in the tropical biomes (Myers 1987; Pimm et al. 1995). About 5 % of the earth’s
land surface is in protected area networks, and if human activities continue in the rest of the
95 % of the unprotected wild land habitats, about 50 % of the species would go extinct
(Pimm & Lawton 1998). This is a ground reality and any bio-prospecting program should,
therefore, be carried out with the end in view that apart from direct economic benefits, such
activities would contribute directly or indirectly to fund conservation, inventorying and
monitoring of biodiversity, both in- situ and ex- situ.
The practices and strategies for bio-prospecting should, therefore, focus on the above
mentioned components of (1) assessments (2) national policy and legislation (3) capacity
building (4) equitable benefit sharing mechanisms (5) participatory management involving all
stake holders in bio-prospecting, including local and indigenous communities (6) mobilizing
financial resources for bio-prospecting and other sustainable uses of biodiversity (ten Kate
& Laird 1999).
P. Pushpangadan et al. 279

Relationship between bioprospecting and access and benefit sharing
The recent trends in bio-prospecting necessitate an ever-increasing demand for access to
genetic resources and traditional knowledge that are available in in situ and ex situ sources.
This has also triggered conflicting interests and common concerns among all stakeholder states,
organizations, institutions, individuals, and communities involved in collection, characterization,
conservation, sustainable utilization, and documentation of genetic resources and traditional
knowledge at local, national, regional and global levels. The United Nations Convention on
Biological Diversity (CBD 2001) is the first international legal instrument that brought out a
radical change from the then prevailing common perception on genetic resources as “common
heritage of mankind” to a legally binding regime that confers “sovereign rights” to the states
over their own biological resources (including genetic resources and traditional knowledge).
CONCLUSION
With the emerging area of biotechnological intervention in medicinal plants for pharmaceutical
and nutraceutical discoveries include: “systematic search for genes, natural compounds and
designs with a potential for product development of biochemical and genetic methods without
disruption to nature”. Modern high-throughput chemical screening and automated bioassay
programs for identifying, isolating, characterizing novel bioactive compounds from wild
medicinal plants, fungi, animals (insects and invertebrates especially) and microbes have
opened up new vistas in natural product research in general and drug and pharmaceuticals
research in particular. The complexity of ingredients and the aspects of synergistic bioactivities
of poly-herbal medicines could now be well explained by system biology approach that enables
linking of the complex metabolic profile of herb with biological effects.
Acknowledgments
The authors express their sincere thanks to Dr. Ashok K. Chauhan, Founder President,
Ritnand Balved Education Foundation (RBEF) and Amity Group of Institutions for
constant support an d encouragement and to Dr. Atul Chauhan, President, RBEF and
Chancellor, AUUP, Noida for facilitating this work.
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