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Chapter 2 Review of literature
Department of Botany, Jamia Hamdard (Hamdard University) 9
2. Review of literature
“Shankhapushpi” is an important drug of indigenous system of medicine. According to
Ayurveda, Shankhpushpi is bitter, pungent, alexiteric, alternative, tonic, anti-helmintic,
brightens intellect, improves complex, increase appetite, useful in bronchitis,
biliousness, epilepsy, leucoderma and teething troubles of infants etc. It contributes
considerably to the improvement of the memory power and intellect (Charaka, 1941;
Nadkarni, 1954). The whole plant is medicinal. It is astringent, hot, aphrodisiac and
rejuvenating (Sharma et al., 1965; Chaturvedi et al., 1997; Mudgal et al., 1975; Singh and
Mehta, 1977; Shukla, 1981). Investigators have reported barbiturate hypnosis potentiation
effect in this drug. It improves strength, digestive power, and complexion. The herb is
reported to be useful in fever and bronchitis (Kirtikar and Basu, 1918). The drug also
supposedly cures diseases due to evil spirits (Chuneker et al., 1969), the morbidity of
tridosis and is useful in epilepsy insanity, insomnia, heart diseases and haemetemesis
(Chuneker, 1982). Many formulations containing Shankhpushpi as a single drug or in
combination with other drugs are available in Indian market. Shankhpushpi is routinely
advertised for memory enhancement. The important Ayurvedic formulations using the
drug are “Abhrak bhasma”, “Brahmi ghrita”, “Brahmi vati”, “Brahm rasayan”,
“Manasmrita gutika”, Manjisthadi Kasayam, “Mukta vati”, Memorex tablets, Stress guard
capsules, “Medhya kashaya”, and “Shankhapushpi panaka”, Dimagheen (Dawakhana
Tibiya College, Aligarh), Shankhpushpi syrup (Unjha), Shankhavali Churna (Narnaryan
Pharmacy), BR-16A (Himalaya Drug. Co. Ltd.) etc.
Except for the term Shankhpushpi, which indicates the resemblance of its flower to a
conch shell, the various Sanskrit synonyms given to this drug do not give any clue in
identifying the source plant of the drug. There seems to be a lot of confusion in equating
the Sanskrit terms Vishnukranta, Shankhpushpi, Aparijata, Girikarni etc. to their respective
botanical source. Therefore, the botanical identity of Shankhapushpi is highly
controversial. While some authors equate it with Convolvulus pluricaulis Choisy of
Convolvulaceae as the source plant (Singh and Chuneker, 1972; Anonymous, 1978; Dey,
1980; Chuneker, 1982), others consider Evolvulus alsinoides (L.) L. as the source plant of
Shankhapushpi (Vaidya, 1982). The former is extensively used as Shankhapushpi in North
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Department of Botany, Jamia Hamdard (Hamdard University) 10
India. However, Kerala physicians do not discriminate between Aparajita and
Shankhapushpi and use Clitoria ternatea L. of Fabaceae. Moose (1976) opines that the
purgative action of Shankhapushpi, combined with its property of being a good nerve tonic
in mental disorders is clearly evinced in Clitoria ternatea.
In addition to these three widely used plant species, further literature survey revealed that
some other plant species like Canscora decussata Schult, of Gentianaceae (Vaidya 1936;
Kapoor and Mitra, 1979), Lavandula bipinnata Kuntz (Lamiaceae), Goniogyne hirta
(Willd.) Ali (Fabaceae), Tephrosia purpurea Pers. (Fabaceae) (Singh and Viswanathan,
2000) and Cheilanthes farinosa, a fern (Daniel, 2004) have also been detected in the
market samples of Shankhpushpi.
The Ayurvedic Pharmacopoeia (Anonymous, 2001) mentions three plant species namely:
Convolvulus pluricaulis, Evolvulus alsinoides and Clitoria ternatea as the source of the
drug Shankhpushpi. Regardless of the source, the drug is used for its therapeutic effects on
Central Nervous System disorders like insanity, epilepsy, nervous debility and memory
enhancement (Gupta et al., 2007).
2.1. Plant species used as the source of “Shankhpushpi”
2.1.1. Clitoria ternatea L. (Papilionaceae)
2.1.1.1. Distribution
C. ternatea is commonly found as an escape in hedges and thickets throughout India, up to
an altitude of 1, 500 m and in the Andaman Islands. It is also cultivated in herbal gardens
and nurseries.
2.1.1.2. Botany
A climbing shrub with slender terete branches, up to 2-3 m height; leaves alternate,
stipulate, imparipinnate, 6-13 cm long; leaflets 5-7, ovate or oblong, 5.5 x 3.5 cms,
glabrous; flowers large, conch-shell shaped, white or blue, solitary axillary; bracts small,
persistent; bracteoles large, foliaceous, roundish, persistent; calyx gamosepalous, tubular,
5-cleft, lobe lanceolate, the upper two subconnate; corolla paplionaceous; stamens
diadelphous; ovary monocarpellary, many ovuled, style elongated, incurved; pod linear-
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Department of Botany, Jamia Hamdard (Hamdard University) 11
oblong, flat 10 x 1cm, sharpely beaked, appressed hairy; seeds 6-10, compressed to
subglobose.
2.1.1.3. Chemical constituents
Chemical constituents present in C. terneata are: -sitosterol, kaempferol-3-
monoglucoside, kaempferol-3-O-rhamnosyl-(1→6)-galactoside, kaempferol-3-O-
rhamnosyl-(1→6)-galucoside, kaempferol-3-O-rutinoside, kaempferol-3-neohesperidoside
and clitorin, which was characterized as kaempferol-3-O-rhamnosyl(1→2)-O-
[rhamnosyl(1→6) glucoside-(1→6)]- glucosidealvidin-3--glucoside. The leaves also
contain an essential oil, coloring-matter and mucilage. Kaempferol have also been reported
in flower (Mukherjee et al., 2008).
2.1.1.4. Traditional uses
C. ternatia is used as brain tonic to promote memory and intelligence (Gomez and
Kolamani, 2003). The plant extract is used in a rejuvenating recipe to treat neurological
disorders and is considered to be wholesome for the intellect (Rai et al., 2002). Tribes use
the root to induce abortion and to reduce abdominal swellings, sour throat and mucous
disorders (Parimaladevi et al., 2003). The juice of the root is mixed with cold milk and is
drunk to remove phlegm and to cure chronic bronchitis (Perimaladevi et al., 2004).
The roots are bitter, laxative, refrigerant, diuretic, anti-helmintic and tonic and are useful in
dementia, hemicrania, burning sensation, leprosy, inflammation, pulmonary tuberculosis,
ascites and fever. The leaves are cathartic and useful in otalgia and hepatopathy (Jain et al.,
2003).
2.1.1.5. Biological activities
2.1.1.5.1. Memory enhancement activity studies
The oral treatment of C. ternatea roots extract significantly increased memory in rats
(Sethiya et al., 2009; Rai et al., 2000). The alcoholic extracts of aerial parts and roots of C.
ternatea were reported to attenuate electroshock induced amnesia. The acetylcholine
(AcH) content of the whole brain and acetyl cholinesterase activity at different regions of
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Department of Botany, Jamia Hamdard (Hamdard University) 12
the rat brain viz- cerebral cortex, mid-brain, medulla oblongata and cerebellum was
evaluated by Taranalli and Cheeramkuzhy (2000). It was suggested that an increase in
(AcH) in rat hippocampus may be the neurochemical basis for improved learning and
memory (Rai et al., 2002; Mukherjee et al., 2007). Rai et al. (2001) by using passive
avoidance test and spatial learning T-maze have also shown that the aqueous root extract of
C. ternatea enhances memory in rats. In another reported study, the effect of aqueous root
extract on the dendritic cytoarchitecture of neurons of the amygdale was studied (Rai et al.,
2000). This improved dendritic arborisation of amygdaloidal neurons, correlates with the
increase passive avoidance learning and memory in the C. ternatea treated rats (Rai et al.,
2005).
2.1.1.5.2. Anti-epileptic activity studies
Methanol extract from the aerial parts of C. ternatea was screened by using
pentylenetetrazol (PTZ) and maximum electroshock (MES) – induced seizures in mice at
the dose of 100 mg/kg p.o. C. ternatea significantly delayed the onset of convulsions and
also delayed the duration of tonic hind limb extension in MES-induced convulsions
(Sethiya et al., 2009).
2.1.1.5.3. Anti-inflammatory, analgesic and anti-pyretic activity studies
The anti-inflammatory activity of the methanolic extract from the roots of C. ternatea has
been studied in rat models (Parimaladevi et al., 2003). In the same study the ethanolic
extract was also evaluated for analgesic activity in mice with the acetic acid-induced
writhing response and mechanical stimulus by tail clip method. In another study, the
methanol extract of C. ternatea was evaluated for its anti-pyretic potential in albino rats
and the anti-pyretic effect of the extract was comparable to that of paracetamol (PCM)
(150 mg/kg b.w. p.o) a standard anti-pyretic agent (Parimaladevi et al., 2004).
2.1.1.5.4. Anti-oxidative studies
It has been established that oxidative stress is among the major causative factors of many
chronic and degenerative diseases (Vadlapudi and Naidu, 2010). C. ternatea petals have
been recognised to possess anti-oxidant activity (Kankonen et al., 1999; Hinneburg et al.,
2006). Extracts of C. ternatea flowers are used in Thailand as a component of cosmetics
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Department of Botany, Jamia Hamdard (Hamdard University) 13
and the chemical composition of the flowers suggest that they may have anti-oxidant
activity. Aqueous extracts were shown to have stronger anti-oxidant activity than ethanol
extracts (Kamkaen and Wilkinson, 2009). The antioxidant potential of aqueous leaf
extracts of C. ternatea were evaluated by determining the levels of enzymatic and non-
enzymatic antioxidants. In vitro antioxidant capacity was also determined using different
assays such as Ferric reducing power assay (FRAP), Reducing activity assay,
diphenypicrylhydrazyl (DPPH) assay and Hydroxyl radical scavenging activity and the
results were comparable with standard antioxidants such as butylated hydroxyl toluene
(BHT), ascorbic acid and rutin (Rao et al., 2009).
2.1.1.5.5. Blood platelet aggregation inhibition studies
An anthocyanin ternatin D1 isolated from petals of C. ternatea was evaluated for in vitro
platelet aggregation inhibitory activity in rabbits and the results of various reported studies
showed significant inhibition of collagen and adenosine diphosphate (ADP) induced
aggregation of platelets (Mukherjee et al., 2008; Honda et al., 1991).
2.1.1.5.6. Anti-diabetic studies
Oral administration of aqueous extract of C. ternatea leaves (400mg/kg body weight) and
flowers (400mg/kg body weight) for 84 days showed significantly reduced serum glucose,
glycosylated hemoglobin, total cholesterol, triglycerides, urea, creatinine and the activity
of gluconeogenic enzyme glucose-6-phosphatase, but increased serum insulin, HDL-
cholesterol, protein, liver and skeletal muscle glycogen content and the activity of
glycolytic enzyme glucokinase. For all the above biochemical parameters investigated, C.
ternatea leaves treated rat showed a little better activity than C. ternatea flowers treated
diabetic rats (Terahara and Rajathi, 1996; Daisy et al., 2009).
2.1.1.5.7. Other activities
The root juice of white-flowered variety is blown up the nostrils as a remedy for
hemicrania. A decoction or powder of the root is given in rheumatism and ear diseases.
The roots are also demulcent and given in chronic bronchitis and fevers; they cause gripe
and tenesmus (Banerji and Chakravarti, 1963; Moose, 1976).
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Department of Botany, Jamia Hamdard (Hamdard University) 14
The roots and leaves are diuretic, laxative, emetic and antiperiodic and are used in the
treatment of a number of ailments including, infections, urinogenital disorders, and as
anthelmentic, antidote to animal stings. Of the two varieties, the white flowered is found to
be more active and hence preferred. The blue-flowered variety is generally used as the
substitute for the white flowered one (Fantz, 1991).
2.1.2. Convolvulus pluricaulis Choisy (Convolvulaceae)
2.1.2.1. Distribution
C. pluricaulis is known from margins and within the Sahara and Sind deserts. In India it is
widely distributed in and grows on the waste land in the plains of Punjab, U. P, Haryana,
Rajasthan, Bihar and Chhotanagpur.
2.1.2.2. Botany
It is a prostrate spreading wild herb. The tap root is about 20cm long, tortuous or straight
and is without lateral branches. The stem measures about 6-8cm long. Leaves are simple,
entire, alternate, tapering below, almost sessile, oblong, nerves 3-4. Flowers are small in
size, white or light pinkish, axillary, bracteolate. Fruit is capsule; Seeds are black, Plano-
convex faintly ridged on the convex surface, dehiscing terminally.
2.1.2.3. Chemical constituents
C. pluricaulis shows the presence of 6-Methyl-7-hydroxy coumarin, maltose, -sitosterol,
kaempferol, kaempferol 3-glucoside, 3, 4 dihydroxy Cinnamic acid along with glucose
rhamnose, sucrose and starch, n-octacosanol, n-dotria contanol, - and £-sitosterols.
2.1.2.4. Traditional uses
Traditionally it is recommended as a brain tonic to promote intellect and memory,
eliminate nervous disorders and to treat hypertension (Bala and Manyam, 1999). It is
described as anti-helmintic, good in dysentery, brain and hair tonic, cures skin ailments and
reduces high blood pressure (Rai, 1987). In Gonda Uttar Pradesh, India, the leaves are
recommended for depression and mental disturbance (Singh et al,. 1997). C. pluricaulis
has been widely used in Ayurvedic medicine to treat nervous disorders, similar to the use
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Department of Botany, Jamia Hamdard (Hamdard University) 15
of kava kava (Piper methysticum) and valerian (Valeriana officinalis) prescribed by
American herbalists (Husain et al., 2007).
2.1.2.5. Biological activities
2.1.2.5.1. Effect of C. pluricaulis on learning and memory
The ethanolic extract of C. pluricaulis and its ethyl acetate and aqueous fractions were
evaluated for their memory enhancing properties. Two doses (100 and 200 mg/kg/p.o.) of
ethyl acetate and aqueous fractions of the ethanolic extract were administered in separate
groups of animals. Both the doses of all the extracts of C. pluricaulis significantly
improved learning and memory in rats (Nahata et al., 2008).
2.1.2.5.2. Anxiolytic, antidepressant, antistress, neurodegenerative and
antiamnesic activity
An alcoholic extract of C. pluricaulis was found to cause an antagonist effect against
amphetamines and tremorine, a potentiation of the acetylcholine effect of pentobarbitone
induced hypnosis and morphine analgesia, without having own sedative properties. A
protective action on muscle against electroshocks has been shown (Sharma et al., 1965;
Barar and Sharma, 1966; Mudgal, 1975). The chloroform fraction of the total ethanolic
extract of C. pluricaulis elicited a significant antidepressant like effect in mice by
interaction with the adrenergic, dopaminergic, and serotonergic systems (Dhingra and
Velecha, 2007a; 2007b). A methanolic extract of the whole plant produced alterations in
the general behaviour pattern, reduction in spontaneous motor activity, hypothermia,
potentiation of pentobarbitone sleeping time, reduction in exploratory behavioural pattern
and suppression of aggressive behaviour (Pawar et al., 2001). Ethyl acetate and aqueous
fractions of the ethanolic extract showed an anxiolytic effect as evidenced by an increase in
the time spent in open arms and the number of open arm entries compared with the control
group. The ethyl acetate fractions at doses of 200 mg/kg p.o. significantly reduced the
neuromuscular coordination indicative of the muscle relaxant activity at a higher dose
(Nahata et al., 2009). A nitrogen containing active principle of the drug produced marked
reduction in I-131 uptake, PBI and acetylcholine suggesting its effect on various glands
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Department of Botany, Jamia Hamdard (Hamdard University) 16
through neurohumors particularly acetylcholine (Prasad et al., 1974). Upadhyay (1986)
studied the therapeutic role of Ayurvedic herbs in mental disorders and classified C.
pluricaulis as a brain tonic. C. pluricaulis in a dose of 100 mg/100 g body weight has a
barbiturate potentiation effect in albino rats; this effect was weaker than that of diazepam,
but stronger than that of Centella asiatica L. and urban Hydrocotyle asiatca L. This plant
has also been reviewed and reported for its potent anxiolytic, neurodegenerative and
antistress activity by various researchers (Singh and Mehta, 1977; Shukla, 1981; Sinha et
al., 1986; Dandiya, 1990; Dubey et al., 1994; Sharma et al., 2009).
2.1.2.5.3. Anticonvulsant activity
It was observed that the animals treated with the methanolic extracts of stem callus, leaf
callus and whole plant (200 mg/kg oral) of C. pluricaulis showed significant protection
against tonic convulsion induced by transcorneal electroshock, which was also comparable
with that of the standard drug phenytoin (Ahmad et al., 2007). C. pluricaulis has also been
shown to possess a potent anticonvulsant activity (Shukla, 1981).
2.1.2.5.4. Antioxidant activity
An ethanolic extract of C. pluricaulis possesses significant antioxidant activity when tested
in vitro (Nahata et al., 2009).
2.1.2.5.5. Hypolipidemic
An ethanolic extract of the whole plant when administered to cholesterol fed gerbils,
reduced serum cholesterol, LDL cholesterol, triglycerides and phospholipids significantly
after 90 days (Chaturvedi et al., 1997).
2.1.2.5.6. Analgesic activity
The C. pluricaulis extract caused a reduction in the fighting behaviour in mice but was
devoid of analgesic activity although it potentiated morphine analgesia (Sharma et al.,
1965).
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Department of Botany, Jamia Hamdard (Hamdard University) 17
2.1.2.5.7. Antiulcer and anticatatonic activity
The antiulcerogenic effect of C. pluricaulis was found to be due to augmentation of
mucosal defensive factors such as mucin secretion, lifespan of mucosal cells and
glycoprotein rather than on the offensive factors such as acid pepsin (Sairam et al., 2001).
2.1.2.5.8. Other activities
C. pluricaulis was found to be an effective remedy for treatment of diabetes (Alam et al.,
1990). It possesses neuropharmacologocal actions such as nootropic (Nahata et al., 2008),
antistress, anxiolytic, antidepressant (Dhingra and Valecha, 2007a; 2007b), anticonvulsant
(Sharma et al., 1965) and sedative activities which justify its use in CNS diseases in the
Ayurvedic System of Medicine. It has antibacterial, antipyretic, anti-inflammatory,
analgesic, diuretic, anti-diabetic and insecticidal properties.
2.1.3. Evolvulus alsinoides (L) L. (Convolvulaceae)
2.1.3.1. Distribution
It is found throughout India in grassy, sandy localities, river beds, forest edges, forest
clearings, scrub jungles and Sal forests.
2.1.3.2. Botany
E. alsinoides L. (dwarf morning glory) belonging to the family Convolvulaceae is a
perennial herb with a small woody and branched rootstock. Its branches are annual,
numerous, more than 30 cm long, often prostrate, slender and wiry with long hairs. Leaves
are small, entire, elliptic to oblong, obtuse, apiculate, base acute and densely hairy. Petiole
is minute or nearly absent. Bracts are linear and persistent. Flowers mostly solitary in
upper axils. Corolla blue rotate and broad funnel shaped, Calyx 4 is lobed, lanceolate and
the tip acute. Peduncle is long and axillary. Capsule is globose and 4 valved. Seeds are 4
and glabrous.
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Department of Botany, Jamia Hamdard (Hamdard University) 18
2.1.3.3. Chemical constituents
Some of the predominantly reported chemical constiuents in Evolvulous alsinoides are
kaempferol-7-O--glucopyranoside, -3-O--lucopyranoside, quecetine-3-O--
glucopyranoside, 2, 3, 4-trihydroxy-3-methylbutyl 3-[3-hydroxy-4-(2, 3, 4-trihydoxy-2-
methylbutoxy)-phenyl]-2 propenoate, 1, 3-di-O-caffeoyl quinic acid methyl ester, caffeic
acid, 6-methoxy-7-O--glucopyranoside coumarin, 2-C-methyl erythritol, triacontane,
pentatriacontane, -sitositisterol and two alkaloids betain and shankpushpi. Besides, four
unidentified alkaloids A, B, C and evolvine have also been identified (Gupta et al., 2007).
2.1.3.4. Traditional uses
E. alsinoides L. is used mainly in traditional medicine of East Asia. The plant is used in
Ayurveda as a brain tonic in the treatment of neurodegenerative diseases, asthma and
amnesia. In Sri Lanka, roots and stem extract of the plant are used to treat dysentery and
depression. Leaves are recommended for asthma and mental disturbances (Rajaqkaruna et
al., 2002). Decoction of roots, thrice a day, is consumed in Eastern Ghats of Andhra
Pradesh, India for three days for curing cough and cold (Rajaqkaruna et al., 2002).
According to an ethno botanical survey conducted among Kani/Kanikaran ethnic groups in
Southern Western Ghats of India, whole plant of E. alsinoides is used for the treatment of
venereal diseases (Ayyanar and Ignacimuthu, 2005). In Uttara Kannada district of
Karnataka, E. alsinoides is used as spermopiotic ((Hedge et al. 2006). The Valaiyan
communities of Piranmalai hills, Tamilnadu consumes leaf juice of E. alsinoides internally
for three days for fever (Sandhya et al., 2006).
2.1.3.5. Biological activities
2.1.3.5.1. Antistress activity
Phenolics and flavonoides isolated from nBuOH soluble fraction from the ethanolic extract
of E. alsinoides were screened for anti stress activity in acute stress induced Sprague-
Dawley rats. The extract displayed most promising anti-stress effect by normalising
hyperglycemia, plasma corticosterone, creatine kinase and adrenal hypertrophy (Gupta et
al., 2007).
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Department of Botany, Jamia Hamdard (Hamdard University) 19
2.1.3.5.2. Anti ulcer and anticatatonic activity
The in vivo evaluation of the alcoholic extract of E. alsinoides revealed it’s marked anti
ulcer and anticatatonic activity (Purohit et al., 1996).
2.1.3.5.3. Immunomodulator activity
The crude extract of Emblica officinalis and E. alsinoides were evaluated for
immunomodulator activity in adjuvant induced arthritic model. The anti-inflammatory
response of both the extracts was determined by lymphocyte proliferation activity and
histopathological severity of synovial hyperplasia. Both the extracts showed a marked
reduction in inflammation and edema. The suppression occurred during the early phase of
the disease. There was a mild synovial hyperplasia and infiltration of few mononuclear
cells in Emblica officinalis and E. alsinoides treated animals. The induction of nitric oxide
synthase was significantly decreased in treated animals as compared to control (Ganju et
al., 2003).
2.1.3.5.4. Other activities
The whole herb is used medicinally in the form of decoction with cumin and milk in fever,
nervous debility, loss of memory and syphilis. Decoction of the drug, with Ocimum
sanctum is administered in fevers accompanied by indigestion or diarrhoea. Decoction was
given in cases of malarial fever. The root is used by the santals, for intermittent childhood
fever. Leaves are recommended for asthma and mental disturbances (Rajaqkaruna et al.,
2002). Decoction of roots, thrice a day, is consumed in Eastern Ghats of Andhra Pradesh,
India for three days for curing cough and cold (Rajaqkaruna et al., 2002).
2.2. Molecular markers
A molecular marker or genetic marker is a gene or DNA sequence with a known location on a
chromosome that can be used to identify cells, individuals or species. Molecular markers are used
to identify a particular sequence of DNA. As the DNA sequences are very highly specific, they can
be identified with the help of the known molecular markers which can find out a particular
bequence of DNA from a group of unknown.
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Department of Botany, Jamia Hamdard (Hamdard University) 20
2.2.1. Types of molecular markers
The range of molecular markers available has expanded rapidly. The commonest are restricted
fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),
random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSRs), also
known as microsatellites. Single-nucleotide polymorphisms (SNPs) are the newest type. No
absolute hierarchy exists between the marker types-all having advantages and disadvantages
(Table 1). The choice of a type of marker therefore will depend on the user’s objectives and
thus be determined case by case. From the historical viewpoint, RFLPs were the first to be
used as markers. The techniques involve digesting DNA with a restriction enzyme. The
fragments are separated by gel electrophoresis and transferred to a filter for hybridization with
radioactively labeled probes. Given that single-copy probes are selected, RFLP markers are
locus specific and co-dominant. Their polymorphism is due to variations in the restriction sites.
The positions on the genetic map of the most commonly used probes are known for species of
interest. RFLP markers are technologically demanding, requiring large quantities of good
quality DNA. They have therefore been progressively replaced by markers based on the
polymerase chain reaction (PCR-based markers).
Table 1. Main characteristics of major types of molecular markers
Characteristic RFLP SSR AFLP RAPD SNP
Type of
visualization
Single
locus
Single locus Multi-loci Multi-loci Single
locus
Allelism Co-
dominant
Co-
dominant
Dominant Dominant Co-
dominant
Type of
polymorphism
Sequence No. of
repeats
Sequence Sequence Sequence
Level of
polymorphism
Good Excellent Excellent Good Excellent
Polymorphism
at the locus
2 to 5
alleles
Multiple
alleles
Presence/absence Presence/absence 2 alleles
Quantity of
DNA needed
Large Small Small Small Small
Quality of
DNA needed
Good No
restrictions
Good Good Good
Reproducibility Good Good Good Low Good
Time Long Fast, once
markers are
developed
Fast Fast Fast, once
markers are
developed
Cost Expensive Average Average Average Expensive
Technical
difficulty
High Low Medium Medium High
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Department of Botany, Jamia Hamdard (Hamdard University) 21
2.2.1.1. PCR based markers
PCR is a molecular biology technique for enzymatically replicating (amplifying) small
quantities of DNA without using a living organism. It is used to amplify a short (usually up
to 10 kb), well defined part of a DNA strand from a single gene or just a part of a gene.
This technique has enabled the development of various types of PCR-based techniques:
2.2.1.2. Random amplification of polymorphic DNA (RAPD)
The RAPD (Williams et al., 1990) technology utilizes short synthetic oligonucleotides (10-
12 base pairs) as primers to amplify nanogram amounts of genomic DNA under low
annealing temperatures by PCR. Amplification products are generally separated on agarose
gels and stained with ethidium bromide. At an appropriate annealing temperature
oligonucleotide primers bind several priming sites in the template DNA and produce
discrete DNA products. The profile of amplified DNA depends on nucleotide sequence
homology between the template DNA and the primer at the end of each amplified product.
Although the RAPD primers are arbitrarily chosen, two basic criteria must be meant: a
minimum of 40% GC content and the absence of palindrome sequence; the GC bond
consists of three hydrogen bridges and the A-T bond of only two, a primer-DNA hybrid
with less than 40% GC will probably not with stand the 72ºC temperature at which DNA
elongation takes place.
Most RAPD fragments result from the amplification of one locus resulting into two types
of polymorphisms i.e. the band may be present or absent. The band intensity differences
may result from copy number or relative sequence abundance (Devos and Gale, 1992) and
may serve to distinguish homozygote dominant from heterozygote as more bright bands
are expected for the former. However, some authors (Thormann et al., 1994) found no
correlation between copy number and band intensity. The fact that fainter bands are less
robust in RAPD experiments (Ellsworth et al., 1993; Heun and Helentjaris, 1993) suggest
that varying degrees of primer mismatch may account for many band intensity differences.
The major disadvantage of RAPD reaction is the low reproducibility. However, the
concern of reproducibility can be overcome through the choice of an appropriate DNA
extraction protocol to remove any contaminants (Micheli et al., 1994), by optimising the
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Department of Botany, Jamia Hamdard (Hamdard University) 22
parameters used (Ellsworth et al., 1993; Skroch and Nienhuis, 1995), by testing several
oligonucleotide primers and by scoring only reproducible DNA fragments (Kresovich et
al., 1992; Yang and Quiros, 1993).
2.2.1.3. Amplified fragment length polymorphism (AFLP)
This technique is based on the detection of restriction fragments by PCR amplification and
can be used for DNA of any origin or complexity (Martin et al., 1991). The fingerprints are
produced, without any prior knowledge of sequence, using a limited set of primers. Both
good quality and partially degraded DNA can be used for digestion, but the DNA should
be free from restriction enzyme and PCR inhibitors.
The first step in AFLP analysis involves restriction digestion of genomic DNA. Double
stranded oligonucleotide adapters are then designed in such a way that the initial restriction
site is not restored after ligation. Such adaptors are ligated to both ends of the fragments to
provide known sequence for PCR amplification. PCR amplification will only occur where
the primers are able to anneal to fragments which have the adaptor sequence plus the
complementary base pairs to the additional nucleotides called selective nucleotides. The
mixture is then subjected to two subsequent PCR amplifications under highly stringent
conditions with primers complementary to the adaptors, and possessing selective
nucleotides of 1-3 bases. The pre amplification is performed with primer combinations
containing a single bp extension while final amplification is performed using primer pairs
with up to 3-bp extension. Because of the high selectivity, primers differing by only a
single base in the AFLP extension amplify a different subset of fragments.
AFLP fragments are visualized either on agarose gel or on denaturing polyacrylamide gels
with autoradiography, AgNO3 staining or automatic DNA sequences. Polyacrylamide gel
electrophoresis provides maximum resolution of AFLP banding patterns to the level of
single-nucleotide length differences, where as fragment length differences of less than ten
nucleotides are difficult to score on agarose gels.
2.2.1.4. Inter-simple sequence repeats (ISSR)
ISSR involves amplification of DNA segment present at an amplifiable distance in
between two identical microsatellite repeat regions oriented in opposite direction. This
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Department of Botany, Jamia Hamdard (Hamdard University) 23
technique uses microsatellite as primers targeting multiple genomic loci to amplify mainly
inter simple sequence repeats of different sizes. The microsatellite used as primers for
ISSRs dinucleotide-penta nucleotide. The primers used can be either unanchored (Meyer et
al., 1993; Gupta et al., 1994; Wu et al., 1994) or more usually anchored at 3`-5` end with 1-
4 degenerate bases extended in to the flanking sequences (Zietkiewicz et al., 1994). The
ISSRs use longer primers (15-30 mers) as compared to RAPD primers (10 mers), which
permit the subsequent use of high annealing temperature leading to higher stringency. The
amplified products are usually 200-2000 bp long and amenable to detection by both
agarose and polyacrylamide gel electrophoresis.
ISSR markers usually show high polymorphism (Kojima et al., 1998) although the level of
polymorphism has been show to vary with the detection method used. Fang and Roose
(1997) reported a reproducibility level of more than 99% after performing repeatedly tests
for ISSR markers by using DNA samples of the same cultivar grown in different locations,
DNA extracted from different aged leaves of the same individual, and by performing
separate PCR runs. In other cases, the reproducibility of ISSRs amplification products
ranged from 86-94%, with the maximum being when polyacrylamide gel electrophoresis
and AgNO3 staining were used and faint bands excluded from scoring (Moreno et al.,
1998).
2.2.2. Applications of molecular markers
2.2.2.1. Molecular markers and Genetic diversity
The ability of a species to adapt to environmental changes depends greatly on the genetic
diversity in the species (Neel and Ellstrand, 2003; Anand et al,. 2004). Narrowing of gene
pool and reduced genetic diversity pose challenges in the selection pressure brought in by
environmental changes (Caro and Laurenson, 1994). Existence of low genetic diversity
within species has been mostly attributed to self pollination, unless other environmental
pressures are influencing genetic diversity (Archak et al., 2002). Small populations with
low gene diversity will be affected much by lack of gene flow, limited gene drift and
reduced mutation rate. Similarly high genetic diversity could result from higher cross
pollination and higher effective population size.
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Department of Botany, Jamia Hamdard (Hamdard University) 24
The assessment of genetic diversity within and between populations is routinely performed
at the molecular level using various laboratory-based techniques such as allozyme or DNA
analysis, which measure levels of variation directly. Genetic diversity may be also gauged
using morphological, and biochemical characterization and evaluation. Morphological
characterization does not require expensive technology but large tracts of land are often
required for these experiments, making it possibly more expensive than molecular
assessment. These traits are often susceptible to phenotypic plasticity; conversely, this
allows assessment of diversity in the presence of environmental variation. Biochemical
analysis is based on the separation of proteins into specific banding patterns. It is a fast
method which requires only small amounts of biological material. However, only a limited
number of enzymes are available and thus, the resolution of diversity is limited. Molecular
analyses comprise a large variety of DNA molecular markers, which can be employed for
analysis of variation. Different markers have different genetic qualities (they can be
dominant or co-dominant, can amplify anonymous or characterized loci, can contain
expressed or non-expressed sequences, etc.).
Random Amplified Polymorphic DNA (RAPD) is one of the most efficient molecular
methods in terms of ability to produce abundant polymorphic markers within a short time
and limited budget. The basis of RAPD based genetic diversity is high mutation, inversion,
deletion and near neutrality of alleles. RAPD polymorphism should be governed by
mutation drift balance. In this method, a single short primer is used in a polymerase chain
reaction (PCR) to amplify random DNA sequences. Since its introduction just over two
decade ago (Williams et al., 1990), RAPD has become widely used in various areas of
plant research and it has proved to be a valuable tool in studying inter and intra-specific
genetic variations, patterns of gene expression, and identification of specific genes using
nearly isogenic variants (Kuddus et al., 2002). Sequence-based analyses sometimes fail to
distinguish between species because of the significant similarity between their DNA
sequences in the amplified region. RAPD primers are able to distinguish taxa below the
species level (Choo et al., 2009), because RAPD analysis reflects both coding and non-
coding regions of the genome (Vanijajiva et al., 2005). The RAPD markers have been used
for detecting genomic variations within and between varieties of sweet potato. A total of
160 primers were tested and eight showed consistent amplified band patterns among the
plants with variations within and between varieties (Lin et al., 2009) of sweet potato.
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Department of Botany, Jamia Hamdard (Hamdard University) 25
Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a
total of 42 accessions of Barberton daisy (Gerbera jamesonii) employing a set of 12 primer
pairs (da-Mata et al., 2009). Germplasm accession of 80 Plantago spp. was studied by
using RAPD with the help of 20 random primers (Singh et al., 2009). Recently, RAPD has
been used for estimation of genetic diversity in various endangered plant species (Wang et
al., 2005; Lu et al., 2006; Liu et al., 2007; Zheng et al., 2008). The other examples in
which this technique has been use for the analysis of genetic diversity studies include the
studies of (Tassanakajon et al., 1997; Selbach and Molina, 2000; Fu et al., 2003; Viccini et
al., 2004; Ghafoor et al., 2007; Sharma et al., 2008; Verma et al., 2009; Arif et al., 2009;
Jan et al., 2011; Lu et al., 2012). RAPD-based molecular markers have been found to be
useful in differentiating different accessions of neem (Farooqui et al., 1998), Codonopsis
pilosula (Zhang et al., 1999), Allium schoenoprasum L. (Friensen et al., 1999),
Andrographis paniculata (Padmesh et al. 1999), Juniperus communis L. (Adams et al.,
2002) Taxus wallichiana (Mohapatra et al., 2009), collected from different geographical
regions.
AFLP technique has also been widely used for genetic diversity in many plants some of
which include Azadirachta indica (Singh et al., 2002), Brassica nigra (Negi et al., 2004),
Brassica rapa (Zhao et al., 2005), Zea mays (Menkir et al., 2005), Tribulus terrestris
(Sarwat et al., 2008), Origanum vulgare (Azizi et al., 2009), Jatropha curcus (Zang et al.,
2011).
ISSR markers have widely been used for the assessment of clonal fidelity of
micropropagated plants. For example Fang and Roose (1997) in trifoliate orange,
Ratnaparkhe et al. (1998) in chickpea, Blair et al. (1999) in rice, Lakshmanan et al. (2007)
in banana and Joshi and Dhawan (2007) in Swertia chirayita. The ISSR technique finds its
application in analysing genetic diversity of plants. For example these markers were used
to analyze genetic diversity of Swertia chiratiya genotypes collected from temperate
Himalayas of India. Fourteen ISSR markers were screened for detecting the genetic
diversity in wild populations of Glycrrhiza uralensis Finch (Yao et al., 2008); the
comparison of genetic diversity in Humulus lupulus was done using RAPD, STS, ISSR and
AFLP molecular techniques (Patzak, 2001). Genetic diversity and geographical
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Department of Botany, Jamia Hamdard (Hamdard University) 26
distribution of disjunct Psychotria ipecacuanha was done by using ISSR markers (Rossi et
al., 2009).
2.2.2.2. Molecular markers and medicinal plant authentication
Besides macro- and microscopic studies and chemical profiling, DNA- based markers are
now becoming a popular means for the identification and authentication of medicinal
plants (Yip et al., 2007). Molecular markers have the advantage over chemical markers as
the genetic composition is unique for each individual and is least affected by age (Kumble,
2003), environmental factors and physiological conditions (Macbeath, 2000), harvest,
storage and processing of the samples (Scheitzer et al., 2003). DNA extracted from leaves,
stems or roots of an herb all carry the same genetic information. In general, extracted DNA
is stable and can be stored at -20ºC for a long period of time (about 3-5 years), hence
eliminating the time constraint in performing the analysis. A small amount of sample is
sufficient for analysis and the physical form of the sample does not restrict the detection.
Molecular markers are not tissue specific and thus can be detected at any stage of plant
development. This is particularly true for similar looking herbal materials that can often
vary greatly in their medicinal properties and market value. DNA- based technology can
provide an efficient, accurate and cheaper means of testing the authenticity of hundreds of
samples simultaneously while conventional chemical methodologies usually take several
days for verification. DNA based authentication of medicinal plants can be useful as a tool
for quality control and safety monitoring of herbal pharmaceuticals and neutraceuticals and
will significantly add to the medical potential and commercial profitability of herbal
products.
DNA based molecular markers have proved their utility in the fields like taxonomy,
physiology, embryology, genetics and recently in pharmacognostic characterization of
herbal medicine (Joshi, 2004; Wang et al., 2005; Passinho-Soars et al., 2006; Yip et al.,
2007). There are successful cases of using DNA methods to identify a number of medicinal
plants such as Panax, Dendrobium, Codonopsis, Fritllaria etc. (Joshi, 2004; Lum et al.,
2005; Shim et al., 2005; Zang et al., 2005; Yip et al., 2007). DNA methods can also be
used to identify components in medicine preparations in which the components have been
grounded, boiled, filtrated, concentrated, dried and blended.
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Department of Botany, Jamia Hamdard (Hamdard University) 27
DNA based technology is especially useful in case of those drugs that are frequently
substituted or adulterated with other plant species that are morphologically and
phytochemically indistinguishable. This application of DNA profiling will not only benefit
the herbal drug industry but can also facilitate the identification of herbal products by
regularity authorities.
DNA-based techniques have been widely used for authentication of plant species of
medicinal importance. Dried fruit samples of Lycium barbarum were differentiated from
its related species using RAPD markers (Zhang et al., 2001). RAPD and PCR–RFLP
analysis have been used for authentication of P. ginseng among ginseng populations
(Chang et al., 1988; Zhang et al., 2001). RAPD technique was adopted to identify eight
types of dried Coptis rhizomes and one type of Picrorrhiza rhizome, a substitute for the
former in the Chinese herbal market (Um et al., 2001). DNA fingerprinting of Taraxacum
mongolicum (herba taraxaci) and its adulterants was demonstrated using AP–PCR and
RAPD. The other examples where RAPD technique finds immense use in authentication of
medicinal plants that are morphologically indistinguishable include Panax species (Shaw
and But, 1995), Coptis species (Cheng et al., 1997; Cao et al., 1997), Echinacea species
(Wolf et al., 1999), Scutellaria species (Hosokawa et al., 2000), Atractylodes species
(Chen et al., 2001 ) bulb of Fritillaria cirrhosa ( Li et al., 2003), Aloe arborescens (Shioda
et al., 2003), Mimosa tenuiflor (Arce et al., 2007), Glycirrhiza glabra (Khan et al., 2009)
and Piper nigrum (Khan et al., 2010).
AFLP markers have also been used for authentication and interpretation of medicinal plant
phylogeny at different taxonomic levels. For example AFLP markers have been used to
discriminate species of Erythroxylum (Johnson et al., 2005), Mentha (Shasany et al., 2005),
Safed Musli (Misra et al., 2007), Plectranthus (Passinho-Soares et al., 2006), and Zingiber
(Ghosh et al., 2011). Applications for AFLP in plant mapping include establishing linkage
groups in crosses, saturating regions with markers for gene landing efforts (Yin et al.,
1999) and assessing the degree of relatedness or variability among cultivars (Mian et al.,
2002).
ISSR markers have been used to authenticate various medicinally important plants. Micro
satellite markers were developed to fingerprinting of Eucalyptus. The primers developed
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Department of Botany, Jamia Hamdard (Hamdard University) 28
were able to amplify the corresponding microsatellite loci from five different species of
Eucalyptus namely E. grandis, E. nitens, E. globulus, E. camaldulensis, and E. urophylla
(Van Der Nest et al., 2000). DNA profiling of disputed chilli (Capsicum annumm) was
done using ISSR and FISSR-PCR markers (Lekha et al., 2001). ISSR markers are proving
very useful for correct botanical identification. They can clearly distinguish intra and inter
species variation e.g. identification of Mediterranean Diplodus spp. and Dentex dentex was
done by means of ISSR markers (Casu et al., 2009). RAPD, ISSR and SRAP were
employed for identification and genetic diversity of 35 elite late bolting radish cultivars
(Wang et al., 2008).
2.2.2.3. Molecular markers and population genetics
The area of research that has shown the most growth with respect to the use of RAPD
technology is that of population genetics (Hedrick, 1992). RAPD markers have been used
to create DNA fingerprints for the study of individual identity and taxonomic relationship
in both eukaryotic and prokaryotic organisms (Caetano-Anollés et al., 1991; Hu and
Quiros, 1991; Welsh et al., 1991; Wostemeyer et al., 1991; Hadrys et al., 1992; Kresovich
et al., 1992; Lark et al., 1992; Wilde et al., 1992; Stiles et al., 1993). RAPD bands of equal
mol. wt. that are shared between individuals are homologous characters (characters
inherited from a common ancestor) or homoplastic characters (characters that arise
independently within a population). It seems likely that closely related individuals would
co-inherit a shared character state from a common ancestor and unlikely that they would
acquire the same character independently. Williams et al. (1993) demonstrated this to be
the case by using single RAPD bands as hybridization probes to detect homologous
characters on a DNA blot of RAPD products. Within the limits of the resolution of an
agarose gel, RAPD bands were amplified from different species of the genus Glycine
(Mienie et al., 1995) and scored as homologous by relative mobility, were also shown to be
homologous by hybridization.
The potential of the RAPD method to identify diagnostic markers for different OTUs is
that RAPD can be applied to analyse fusion of genotypes at different taxonomic levels. At
the level of the individual, RAPD markers may be applied to parentage analysis; at the
population or species level RAPD may be used to detect hybrid populations or species. The
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Department of Botany, Jamia Hamdard (Hamdard University) 29
detection of genome hybrids relies upon the identification of diagnostic RAPD markers for
the parental genotypes under investigation. Several groups have reported on the utility of
RAPD markers as a source of phylogenetic information. Arnold et al. (1991) were
successful in using RAPD markers to test for interspecific nuclear gene flow between lris
fulva and 1. hexagona, and to study the presumed hybrid origin of I. nelsonii. Using the
AP-PCR modification of RAPD, Welsh et al. (1991) identified F1 hybrids from different
inbred maize lines. Other groups have begun to use RAPD for analyses of hybridization
events where allozyme studies have not proven to be sensitive enough for hybrid
genotypes, for example, in hybridization along vertical zonations in natural populations of
Dnphnia or in plant breeding programmes (Crowhurst et al., 1991; Hu and Quiros, 1991;
Martin et al., 1991; Quiros et al., 1991). Hilfiker et al. (2004) used RAPD markers to study
the effect of genetic drift in small populations of Taxus baccata. It was revealed that there
is reduced genetic variation and an increased female biased sex ratio.
2.2.2.4. Limitation of DNA methods
Molecular authentication methods have several advantages which make them suitable for
the identification of herbs used in traditional medicine, as compared to macroscopic,
microscopic, and phytochemical analyses. The DNA-based techniques are not affected by
environmental factors, independent from the physical form of the plant material, and only a
low amount of material is required.
Although DNA analysis is currently considered to be cutting-edge technology, it has
certain limitations.
The applicability of a DNA-based method depends generally on the quality and
quantity of the DNA, which might be a problem for dried or processed materials.
Important drug-processing conditions, for example, temperature and pH, may lead
to degradation (fragmentation) of the DNA, rendering PCR analysis impossible.
However, depending on the degree of degradation of DNA some methods can still
be used in processed materials. For these, it is necessary to develop very short
amplicons to have a certain probability of successful application.
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Department of Botany, Jamia Hamdard (Hamdard University) 30
High concentrations of secondary plant compounds (polysaccharides, tannins,
essential oils, phenolics, alkaloids, etc.) may influence DNA extraction or PCR
reaction. In tissues of medicinal plants, secondary compounds generally get
accumulated and the problem becomes severe as the material gets older.
Polysaccharide contaminations are particularly problematic as they can inhibit the
activity of many commonly used enzymes, such as polymerases, ligases, and
restriction endonucleases. Polyphenol contamination of DNA makes it resistant to
restriction enzymes and interacts irreversibly with proteins and nucleic acids.
Choosing the most suitable DNA extraction procedure may help to eliminate the
PCR inhibitors.
Sometimes plant materials are contaminated with endophytic fungi, which might
influence DNA sequencing and can be eliminated with a plant-specific primer
design.
DNA related methods can generally not be applied when the herb is processed to an
extract.
DNA markers, such as the internal transcribed spacer (ITS) region of the 18S, 5.8S,
and 26S nuclear ribosomal cistron, sometimes show intraspecific sequence
variation due to nonfunctional paralogous sequences (pseudogenes). For DNA
barcoding as a practical molecular method to identify species, only orthologous
DNA sequences can be used. Consequently cloning of PCR products is sometimes
inevitable.
In order to establish a marker for identification of a particular species, DNA
analysis of closely related species and/or varieties and common botanical
contaminants and adulterants is necessary, which is a costly and time-consuming
process.