Introduction
Life on earth mainly depends on plants and it is very important for the survival of human
beings. Biodiversity is the vast array of all flora and fauna inhabiting the earth either in aquatic
or the terrestrial habitats. It provides enormous direct and indirect essential services through
natural ecosystem function and stability to humankind. Plants and plant products have been the
primary sources upon which the modern civilization has been built. In the tropics alone it has
been estimated that 25,000 - 30,000 plants are in use (Shanley and Luz, 2003). Recent studies
have reported that out of the total 4, 20, 000 flowering plants in 440 families of plants from the
world more than 50,000 are used for medicinal purposes (Schippmann et al., 2002). India is one
of the twelve mega-biodiversity centres of the world having rich vegetation with a wide variety
of plants with medicinal value (Patrick, 2002). India is also the largest producer of medicinal
herbs and is appropriately called the botanical garden of the world. In India, more than 43% of
the total flowering plants are reported to be of medicinal importance (Pushpangdan, 1995). The
exploitation of plants for medicinal purposes in India has been documented long back in ancient
literature (Charak and Drdhbala, 1996). In rural India, 70 percent of the population is dependent
on traditional systems of medicine. The officially documented plants with medicinal potential are
3000 but traditional practitioners use more than 6000 plants (Alam, 2008).
Economical, social, ecological, cultural and aesthetic cases have been made for
identification, quantification and understanding the distribution and relationship of biological
diversity (Kunin and Lawton, 1996). Biological diversity may be assesed at three diferent levels:
the community, the species and the gene (Frankel et al., 1995). Efficient utilization,
improvement and conservation of taxa must be based on a sound understanding of phylogeny,
1
the amount and distribution of genetic variation. Genetic markers that are observable traits are
classified into five broad groups viz., morphological, cytological, chemical, protein and DNA
(Szmidt and Wang, 2000).
Nature has myriads of life forms on this planet among which variations are of ubiquitous
occurrence. Variations between individuals of the species have occured the millennia and are
considered as real “hotspot” of evolution. Down the years, variations among plants have always
fascinated the inquistive mind and helped an evolutionary biologist or a breeder to select a
desirable variant or breed a new form of greater agronomic value. In the wild, plants grow in
extreme situations along longitudinal, latitudinal and temperature gradients. Therefore variations
within and between populations of a species are not uncommon (Lei et al., 2006). Much of the
variations in phenotype observed in natural populations of a species were earlier attributed to
environmental influences (Briggs and Walters, 1984). Many botanists reasoned that distinct
intra-specific variations of plants were merely due to habitat modifications and adaptation to
environment was by phenotypic plastic responses. Phenotype was accepted as a resultant product
of interaction between genotype, environment and different phenotypes. In certain plants
developmental variations were observed as evident from differential morphological
characterstics of the juvenile and adult forms (Smila et al., 2007; Johnson, 2007). The problem
of variations are further compounded in medicinal plants which apart from displaying visible
interactions, synthesize and accumulate an array of plant specific chemicals. These compounds
are biosynthetically derived from primary metabolism and accumulated by certain plants or
group of plants in trace quantities. A study of variation in the active principles is often an
important element in the investigation of variation in such plants.
2
A wide spectrum of simple and overlapping variations is now documented in plants
(Connely et al., 1993; James and Ashburner, 1997; Sen et al., 2009). All observed variations are
broadly grouped into two categories: epigenetic and genetic. Genetic variations in plants are
strictly heritable. They occur invariably due to alterations in the genetic material and may affect
both phenotypic and chemical characteristics of a medicinal plant. In general the variations in
plants are classified as inter-specific and intra-specific variations. Inter-specific variation means
the variations which are found between the populations, i.e. between the different species of a
single genus or among different genera themselves. Intra-specific variation is the variations
within populations which are found within a single species of a particular genus. Intra-specific
and inter-specific variations are the building blocks for trait/character improvement through
breeding or genetic selection and hence has become very important in botanical breeding, plant
science research and gene bank activities related to preservation of integrity in viable seed
materials (Mercandante and Pfander, 1998). Chemo-profiling and morphological evaluation is
routinely used for the identification of genotypic variation. In addition to variation in chemical
production resulting from environmental and temporal conditions, many studies have looked at
the variation of chemical groups within genetically related taxa as well. For example,
polyacetylenes and related compounds have been shown to segregate in various intra-generic
groups within Artemisia (Greger et al., 1982; Albasini et al., 1983). Variation in chemical
production by non-specific individuals has also been documented in wild Artemisia dracunculus.
Both French and wild tarragon have been analyzed to determine if there are differences in their
phytochemical compositions (Balza et al., 1985; Deans et al., 1988).
Secondary metabolites can be derived from any part of the plant like bark, leaves,
flowers, seeds, etc (Cragg and Newman, 2001). It is necessary to check the quality of the
3
material by estimating the phytomarkers available in the plant parts. Knowledge of the chemical
constituents of plants is desirable because such information will be of value for the production of
complex chemical constituents. Secondary metabolite screening in various plants was reported
by many workers (Siddiqui et al., 2009; Chitravadivu et al., 2009; Ashok Kumar et al., 2010).
Chemical complexity and lack of therapeutic markers are some of the limitations associated with
the identification of genotype. Chemical profiling establishes a characteristic chemical pattern
for a plant material. Identification of DNA markers that can co-relate DNA fingerprinting data
with quantity of selected phytochemical markers associated with that of particular plant would
have extensive applications in quality control of raw materials (Lee et al., 1990; Sikorska et al.,
2001; Joshi et al., 2004).
In pharmacognosy, chemoprofiling and marker compound analysis was carried out using
modern analytical techniques such as fluorescence, UV-Vis, FTIR, TLC, HPLC, HPTLC and
GC-MS. These have become firmly established as a key technological podium for secondary
metabolite profiling in both plant and other species (Fernie et al., 2004; Biren Shah, 2009). Gas
Chromatography is a very good method for separating components of a mixture and mass
spectrometry is a powerful technique for identifying components in a mixture. The combination
of these two techniques may be considered as a natural marriage (Merlin et al., 2009).
With the advancement in the separation and isolation of natural product chemistry
scientists have shown that phytochemical constituents can be used to distinguish, illustrate and
classify species into taxa. Correlations between traditional morphological and chemical
classifications can be traced as early as 1699 (Fairbrothers, 1968). However, a real interest in the
understanding of possible relationships between plant constituents and systematics is more
recent. Interest in this aspect of systematics has increased with the development of rapid and
4
precise analytical techniques, and there is a consensus that data from as many sources as possible
should be employed in plant classification (Stace, 1980; Datta, 1988). Evidence from chemical
constituents has already led to the reconsideration of many plant taxa. For example a number of
taxonomically difficult families have been successfully grouped on the basis of their secondary
metabolite profiles. At lower taxonomic levels, several metabolites have proved useful in
establishing taxonomic relationships (Waterman, 1998).
Molecular markers have provided a powerful new tool for breeders to search for new
sources of variation and to investigate genetic factors controlling quantitatively inherited traits.
The molecular approach for the identification of plant varieties/genotypes seems to be more
effective than traditional morphological markers because it allows direct access to the hereditary
material and makes it possible to understand the relationships between individuals (Paterson et
al., 1991; Sangwan et al., 2001). References for genetic variation among medicinal plants are
scanty although analysis of such variation holds great promise owing to the location specific
attributes of the herbs and the attendant diversity of plant specific compounds. It is also now
understood that the genetic variation within a given species is usually much more serious and
occurs much earlier than the total extinction of the species itself (He and Sheng, 1997). For
efficient conservation and management of the selected medicinal plants diversity, the genetic and
phytochemical composition of species needs to be assessed. Based on morphology, it is very
difficult to identify the medicinal plants in the form of crude drug and juvenile stage.
Genetic diversity among inter and intra-specific populations can be determined using
morphological, phytochemical, cytological and molecular markers. Phenotypic characters have
limitations since they are influenced by environmental factors and the developmental stage of the
plant. Measurement of morphological traits alone may not serve as a useful criterion for
5
assessing genetic diversity of plant germplasm. The environmental influence on these traits may
sometimes render this measure relatively insensitive particularly where differences are very
small. However, several molecular and biochemical analyses make it possible to establish
differences at various taxonomic levels which in turn helps the researchers to assess genetic
diversity in the investigated germplasm (Vaughan, 1983; Rabbani et al., 2010; Pervaiz et al.,
2010). Electrophoretic separation methods are increasingly playing an important role in genetic
diversity analysis and conservation of plant genetic resources. These methods are being used as
complementary strategies to traditional approaches for assessment of genetic diversity. This can
be performed at any growth stage using any plant part and it requires only small amount of
materials. SDS-PAGE is a practical and reliable method for species identification because seed
storage proteins are largely independent of environmental fluctuation (Gepts, 1989). Molecular
markers, based on DNA sequence polymorphisms, are independent of environmental conditions
and show higher levels of polymorphism (William et al., 1990). The basic premise is that
variation in the nucleotide sequence of DNA can be exploited to produce characteristic
fingerprints (Mace et al., 1999). The barcode of life is a short DNA sequence from a uniform
locality on the genome, which is used for identifying species (Schori and Showalter, 2011). DNA
barcoding has rapidly achieved recognition as an important tool with the power to aid many
basic research and applied endeavors in taxonomy and species identification (Savolainen et al.,
2005; Hajibabaei et al., 2007).
In the present study three medicinally important Plumbago species were selected to reveal
the inter and intra-specific variation. The roots of Plumbago zeylanica extract has antiplasmodial
(Simonsen et al., 2001), antimicrobial (Ahmad et al., 2000), antifungal (Mehmood et al., 1999),
anti inflammatory and anticancer (Oyedapo, 1996), antihyperglycemic (Olagunju et al., 1999),
6
hypolipidaemic and anti atherosclerotic activities (Sharma et al., 1991). The aerial parts or roots
of Plumbago auriculata is taken to treat black water fever (Dorni et al., 2006). Various parts
of P. auriculata contain the naphthoquinone plumbagin (2-methyl juglone), which blisters the
skin. P. auriculata has insecticidal properties as an antifeedant and as a moulting inhibitor (Paiva
et al., 2003). Plumbagin is also a yellow pigment, occurring in a colourless combined form in the
plant and is liberated by acid treatment. P. auriculata contains an antifungal protein that inhibits
spore germination in Macrophomina phaseolina (Gangopadhyay et al., 2011). Plumbago rosea
also contains the napthoquinone plumbagin. Plumbagin possess several pharmacological
activities i.e. antimicrobial, anticancer, cardiotonic, anti rheumatism and anti fertility actions (Lal
et al., 1993). It is also a powerful irritant. In small doses, the compound is asudorific and it
stimulates the central nervous system; large doses may cause death from respiratory failure and
paralysis, anti implantation and abortive activity. The ethanolic leaf extract of P. rosea is active
against herpus simplex virus type I. The root of P. rosea was used to treat digestive problems,
dyspepsia, colic cough and bronchitis (Dinda et al., 1997).
In addition, previous studies on the Plumbago species were focused on the roots only, very
few reports are available on the preliminary phytochemical analysis on P. zeylanica and aerial
parts of the selected Plumbago species. To supplement the previous observations, an attempt has
been made to reveal the phytochemical properties of the aerial parts of three Plumbago species
and find out the inter-specific and intra-specific variation among the selected three species of
Plumbago using preliminary phytochemical screening, spectroscopic, chromatographic and
electrophoretic (protein and DNA) analysis. These phytochemical and molecular analyses of
intra and inter-specific variation in particular may find application in resolving disputes of
taxonomic identities, relations and authentication of the species in the pharmaceutical industries.
7
With this knowledge the present investigation aims to reveal the phytoconstituents present in
the aerial parts of medicinally important Plumbago species viz., P. zeylanica L. P. auriculata
Lam. and P. rosea L. In addition it also aimes to assess the variation at genus and species level
(inter and intra-specific) among the selected three Plumbago species (P. zeylanica, P. auriculata
and P. rosea) using phytochemical (UV-Vis qualitative analysis, FTIR analysis, TLC, HPLC,
HPTLC and GC-MS analysis. The biochemical and molecular variation among the selected three
species was studied using SDS-PAGE, MALDI-TOF MS and DNA barcoding.
8