Phytochemical analysis of eight medicinal plants of Lamiaceae

Keywords:
Phytochemical analysis, Qualitative phytochemical screening, TLC, Lamiaceae.
ABSTRACT:


The study has been designed with the objective to examine the methanol
extracts of eight medicinal plants belonging to the family Lamiaceae. Fresh matured
leaves were collected; shade dried and aqueous-extracted to screen the active
chemical constituents. Qualitative phytochemical analysis of these plants confirm the
presence of various phytochemicals like alkaloids, flavonoids, tannins, terpenoids,
saponins, steroids and phenolic compounds in their aqueous leaf extracts.
Quantitative analysis was carried out using TLC scanning method, a combination of
Petroleum ether, Methanol and Benzene in the ratio of 8:1:1.5 respectively was
employed as the mobile phase. The TLC chromatograms constituted different
coloured phytochemical compounds with different R
f
values. It can be conveniently
used to evaluate the quality of different area samples. Present paper deals with the
significance of these phytochemicals with respect to the role of these plants in
traditional medicinal system.

001-006 | JRPS | 2011 | Vol 1 | No 1
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www.plantsciences.info
Authors:
John De Britto A,
Steena Roshan Sebastian
and Mary Sujin R.




Institution:
Plant Molecular Biology
Research Unit,
St.Xavier's College,
(Autonomous),
Palayamkottai. Tamilnadu,
India.



Corresponding author:
John De Britto A



Email:
bjohndesxc@gmail.com









Web Address:
http://plantsciences.info/
documents/PS0001.pdf.


Dates:
Received: 30 Aug 2011 /Accepted: 08 Sep 2011 /Published: 12 Sep 2011
Article Citation:
John De Britto A, Steena Roshan Sebastian and Mary Sujin R.
Phytochemical analysis of eight medicinal plants of Lamiaceae.
Journal of Research in Plant Sciences (2011) 1: 001-006
Original Research Paper
Journal of Research in Plant Sciences
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An International Scientific Research Journal
Journal of Research in
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An International Scientific
Research Journal


INTRODUCTION
Plants have an almost limitless ability to
synthesize aromatic substances, most of which are
phenols or their oxygen-substituted derivatives
(Geissman, 1963). Most are secondary metabolites,
of which at least 12,000 have been isolated, a
number estimated to be less than 10% of the total
are used appropriately (Schultes, 1978). In many
cases, these substances serve as the molecules of
plant defense against predation by microorganisms,
insects and herbivores. Furthermore, some of which
may involve in plant odour (terpenoids),
pigmentation (tannins and quinines) and flavor
(capsacin). However, several of these molecules
possess medicinal properties (Mallikharjuna et al.,
2007).
In recent times, focus on plant research has
increased all over the world, and a large body of
evidence has been collected to show the immense
potential of medicinal plants used in traditional
systems (Nazoora et al., 2010). Plant medicines are
ideal tools to restore health and treat disease
precisely because they consist of a multiplicity of
chemical components. It is now clear that, the
medicinal value of these plants lie in the bioactive
phytochemical constituents that produce definite
physiological effects on human body. These natural
compounds formed the base of modern drugs as we
use today (Edeoga et al., 2005; Akinmo et al.,
2007; Rout et al., 2009). Various medicinal plants
have been identified and studied using modern
scientific approaches (Dipak et al., 2010).
Lamiaceae species are important for its
medicinal properties among plants. This family is
represented by 45 genera and 574 species with 256
endemic species (Erik and Tarikahya, 2004).
Naturally growing Lamiaceae members have been
used as tea, spice and for various medicinal
purposes by the public for centuries. The plants are
used as diuretic, sedative, digestive, antiparasitic,
carminative, appetizer, anticonvelsant, anti-
inflammatory and stimulant. It is also used to treat
fever, cough, headaches, stomachaches, wound
healing, heart diseases, and dysmenorrhea (Nurdan
and Aysel, 2007). Numbers of chromatographic
methods were established for the analysis of herbal
medicine. Since application of medicinal plants
have been growing steadily, development of a
suitable quality control method for it is urgently
required. Thin layer chromatography (TLC) is an
option that has distinct advantages over several
other chromatographic methods such as its
simplicity, low cost, high sample capacity and rapid
availability of results. It is unique due to the fact
that it presents the result as an easy-to-interpret
image or chemical fingerprint (Marston, 2007).
In the present study, we have concentrated on the
preliminary screening and qualitative separation of
secondary metabolites from leaves of eight
Lamiaceae species through TLC.

MATERIALS AND METHODS
1. Materials
The leaves of Mentha spicata L. (L
1
),
Anisomeles malabarica (L.) R.Br. ex Sims (L
2
),
Hyptis suaveolens (L.) Poit. (L
3
), Ocimum
gratissimum L. (L
4
), Ocimum sanctum L. (L
5
),
Leucas aspera (Willd.) (L
6
), Coleus aromaticus
Benth. (L
7
) and Ocimum bascilicum L. (L
8
) were
identified and collected from different areas of the
Tirunelveli district, Tamilnadu. The specimens
were compared and checked with the Herbarium
(XCH), St. Xaviers College, Palayamkottai.
2. Preliminary screening
The shade dried leaf materials were
powdered using mortor and pestle, it was then
continuously extracted with 100 ml of methanol for
18 hrs at 60C using Soxhlet apparatus. The
condensed extracts were used for preliminary
screening of phytochemicals such as alkaloids,
st er oi ds, r educi ng sugar s, cat echi ns,
anthroquinones, flavonoids, terpenoids, sugars,
phenols, saponins, tannins and aminoacids. The
presence of phytochemicals from methanol extract
of all the samples were qualitatively determined by
adopting standard method of Brinda et al., (1981).
TLC
10 ml methanol extract of each sample were
taken and evaporated, the paste of the evaporated
extracts were used for TLC analysis. A combination
of Petroleum ether, Methanol and Benzene in the
ratio 8:1:1.5 was used as solvent mixture. The
precoated silica aluminum plates were used for
TLC studies of secondary metabolites. The extracts
to be analysed were spotted on the plate. The plates
were placed in TLC chamber and the chromatogram
was developed with the solvent mixture. The TLC
plates were taken out and visualized in visible light,
UV light (254 nm & 366 nm) and iodine chamber
and spots were marked. The migration pattern was
recorded.
3. Data analysis
R
f
value of each spot was calculated using
the formula, R
f
value=Distance traveled by the
solute/Distance traveled by the solvent and the
values were tabulated.
Britto et al.,2011
002 Journal of Research in Plant Sciences (2011) 1: 001-006
RESULTS
The present investigation was carried out on
eight medicinal plants of Lamiaceae to study the
presence of medicinally active phytochemicals in
the leaves. Alkaloids, reducing sugars, tannins,
phenolic compounds and aminoacids showed high
scores in the extract while triterpenoids and
saponins indicated low scores. Steroids, flavonoids
and catechins showed moderate scores. Sugars and
anthroquinones were not detected in any of the
plant extract. Alkaloids, Phenolic compounds,
amino acids, Reducing sugars and tannins were
present in all plants investigated, while catechins
was present only in M. spicata, A. malabarica, H.
suaveolens, O. gratissimum, O. sanctum and L.
aspera. Similarly flavonoids were present in five
plants M. spicata, A. malabarica, H. suaveolens, O.
sanctum and L. aspera. Steroids were present in all
the seven plants studied except C. aromaticus.
Triterpenoids was present only in C. aromaticus.
Saponins were present only in two plants A.
malabarica and L. aspera (Table 1).
Thin layer chromatographic technique is a
useful analytical tool for the isolation and
identification of organic compounds. The data of
quantitative separation of secondary metabolites
from leaves of Lamiaceae by thin layer
chromatography is tabulated. R
f
value obtained by
thin layer chromatography patterns are useful to
establish their identity and purity of the herbs. The
plates were first exposed to visible light, then
viewed through UV (254 & 366 nm), and kept in
iodine chamber to observe the variously coloured
bands.
TLC chromatogram under visible light
revealed 4 bands for L
1
, L
5
, L
6
and L
7
, 5 bands for
L
2
and L
4
, 2 bands for L
8
and 7 bands for L
3
,

which
are given in Table 2(a). The highest R
f
value was
0.97 with dark yellow coloured band in L
1
and L
3
and yellowish green coloured band in L
2
and L
6
. L
4

had least R
f
value of 0.52 with a brown coloured
band. Under UV light (254 nm) the chromatogram
revealed 4 bands in L
1
, L
2
and L
6
, 5 bands in L
3
and
L
8
, 6 bands in L
4
and L
5
and 2 bands in L
7
species
Britto et al.,2011
Journal of Research in Plant Sciences (2011) 1: 001-006 003
S.No Phytochemicals L
1
L
2
L
3
L
4
L
5
L
6
L
7
L
8

1. Steroids + + + + + - + +
2. Triterpenoids - - - - - - + -
3. Reducing sugar + + + + + + + +
4. Sugars - - - - - - - -
5. Alkaloids + + + + + + + +
6. Phenolic compounds + + + + + + + +
7. Flavonoids + + + - + + - -
8. Catechin + + + + + + - -
9. Saponins - + - - - + - -
10. Tannins + + + + + + + +
11. Anthroquinones - - - - - - - -
12. Amino acids + + + + + + + +
Table 1. Qualitative analysis of the phytochemicals of eight different taxa of Lamiaceae
+ = Present; - = absent
S. No. Colour of the bands L
1
L
2
L
3
L
4
L
5
L
6
L
7
L
8

1. Dark Yellow 0.97 - 0.97 0.95 - - 0.96 0.96
2. Yellowish green - 0.97 - - - 0.97 - -
3. Light yellow - - 0.95 - - - 0.93 -
4. Ash green - - 0.93 0.92 - - - -
5. Light geen - 0.94 0.86 0.86 0.97 - - -
6. Green 0.94 0.91 - - 0.95 0.89 0.87 0.81
7. Yellow 0.86 0.86 - - 0.90 0.79 - -
8. Dark green 0.81 0.75 0.81 - 0.87 0.66 - -
9. Brown - - - - - - 0.78 -
10. Pale green - - 0.68 0.77 - - - -
11. Yellowish brown - - 0.59 - - - - -
12. Brownish green - - - 0.52 - - - -
Table 2(a). TLC fingerprint profile of eight different taxa of Lamiaceae under visible light


which is given in the table 2(b). In all the species
the highest R
f
value was 0.97 with brown coloured
bands in L
5
and L
6
and Orange coloured band in L
1
,
the least R
f
value was 0.47 with fluorescent green
coloured band in L
4.

Under UV light (366 nm), the chromatogram
revealed 4 bands in L
1
, L
2
and L
4
, 3 bands in L
5,
L
7

and L
8,
6 bands in L
3
and 2 bands in L
6
species
which is tabulated in table 2(c). The highest R
f
value was 0.97 with dark green coloured band in L
1
,
L
5
and L
6
and fluorescent green coloured band in
L
2
, The least R
f
value was 0.52 with pale green
coloured band in L
4
species. When the
chromatograms were exposed to iodine vapour, it
revealed 4 bands in L
1
, L
5
, L
7
and L
8,
7 bands in L
3
,
3 bands in L
2
,

5 bands in L
4
and 2 bands in L
1

species which is given in the table 2(d). The highest
R
f
value was 0.97 with brown coloured band in L
1

and L
6
, ash green coloured band in L
4
and dark
yellow coloured band in L
3.
The least R
f
value was
0.50 with brown coloured band in L
3
. A
chromatogram under various lights are shown in
Fig1.

DISCUSSION
The curative properties of medicinal plants
are perhaps due to the presence of various
secondary metabolites such as alkaloids, flavonoids,
glycosides, phenols, saponins, sterols etc. The
above results indicates that, the leaves of the plants
investigated are rich in alkaloids, flavonoids,
reducing sugars, phenols and also showed presence
of aminoacids. They are known to show medicinal
potential and physiological activities (Sofowara,
1993). Thus the plants under investigation showed
their medicinal potential and can be a source of
useful drugs.
Dipak et al. (2010) did the preliminary
phytochemical analysis in eight ethnomedicinal
plants, Ocimum sanctum L., Hyptis suaveolens (L)
Poit., Croton tiglium L., Physalis minima L.,
Tephrosia villosa (L) Pers., Malachra capitata L.,
Cleome viscosa L., and Galphimia glauca Cav. The
qualitative phytochemical analysis of these plants
confirms the presence of various phytochemicals
like alkaloids, flavonoids, tannins, phlobatannin,
terpenoid, saponin, steroid and cardiac glycosides
in their aqueous leaf extracts. Anyanwu and Dawet
Britto et al.,2011
004 Journal of Research in Plant Sciences (2011) 1: 001-006
S.No. Colour of the bands L
1
L
2
L
3
L
4
L
5
L
6
L
7
L
8

1. Brown - - - - 0.97 0.97 - -
2. Orange 0.97 - 0.95 0.95 0.90 0.87 0.96 0.96
3. Light green 0.94 0.94 - - - - - -
4. Fluorescent green 0.86 0.97 - - - - - -
6. Brown - 0.91 - 0.92 - - - -
7. Dark brown 0.78 0.77 0.86 0.80 - - 0.90 0.93
8. Brown - - - 0.71 - - - 0.87
9. Black - - - - 0.87 0.76 - 0.81
10. Yellow - - - - 0.82 - - -
11. Light brown - - 0.72 - 0.70 - - -
11. Yellowish orange - - 0.68 0.66 - - - -
12. Flourescent green - - 0.59 0.47 0.50 0.64 - 0.75
Table 2(b) TLC fingerprint profile of eight different taxa of Lamiaceae under UVlight (254 nm)
S.No. Colour of the bands L
1
L
2
L
3
L
4
L
5
L
6
L
7
L
8

1. Dark green 0.97 - - - 0.97 0.97 - 0.96
2. Fluorescent green - 0.97 - - - - - -
3. Yellowish green 0.94 - - 0.95 - - 0.96 -
4. Light green 0.86 0.94 0.97 - 0.92 0.94 - -
5. Ash green - - 0.93 0.80 - - - -
6. Fluorescent green 0.81 0.86 - - - - - -
7. Brown - - 0.81 - - - - -
8. Green - 0.75 0.77 - - - 0.84 0.87
9. Light brown - - - - 0.80 - - -
10. Light green - - 0.72 0.71 - - 0.72 0.75
11. Pale green - - 0.59 0.52 - - - -
Table 2(c). TLC fingerprint profile of eight different taxa of Lamiaceae under UV light (366 nm)
(2005) stated that these constituents found in plants
are known to have anti bacterial activities.
Flavonoids especially, are of a potential benefit to
human health (Jouad et al., 2001).
A simple, robust and reproducible TLC
method for the separation of phytochemicals were
reported in Radix Polygoni by Gao et al. (2007) and
in Mucuna pruriens by Misra and Wagner (2007)
and in Vitex trifolia by Alfi et al. (2010). In the
work carried out, all the species had a highest R
f

value of 0.97, but each species had different
coloured bands. A new brown coloured band was
found only in L
7
with R
f
value 0.78 and ash green
coloured band was found only in L
3
with R
f
value
0.77. Gabriela (2009) suggested that the colours of
the separated spots in TLC and their position
relative to standard substances are important
characteristics for the plant extract identification.


CONCLUSION
Therefore, the wide use of these medicinal
plants is attributable to the presence of these
bioactive compounds, which may explain its
traditional uses against various ailments. However,
further studies are required to isolate the active
principal compound from the crude plant extract for
proper drug development. These results are needed
to further carry out the advanced hyphenated
spectroscopic studies in order to elucidate the
structure of these compounds. Furthermore, this
data may be handy in probing the structure of the
biochemical compound of this plant in the future.

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Britto et al.,2011
Journal of Research in Plant Sciences (2011) 1: 001-006 005
Fig 1. TLC chromatogram of Ocimum gratissimum
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L
2
L
3
L
4
L
5
L
6
L
7
L
8

1. Brown 0.97 - - - - 0.97 - -
2. Ash green - - - 0.97 - - - -
3. Dark yellow - - 0.97 - - - - -
4. Light yellow - - 0.95 - - - - -
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006 Journal of Research in Plant Sciences (2011) 1: 001-006
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