Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876

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Alkaloids Profile of Clitoria ternatea Linn by High Performance Thin Layer

Chromatography (HPTLC)
Selvamaleeswaran Ponnusamy1, Wesely Ebenezer Gnanaraj2, Johnson Marimuthu Antonisamy3*
1
Research and Development centre, Bharathiar University, Coimbatore,
Tamil Nadu, India
2
Department of Botany, Arignar Anna Government Arts College, Namakkal,
Tamil Nadu, India
3
Department of Botany, St. Xavier’s College (Autonomous), Palayamkottai, India – 627 002
ARTICLE INFO ABSTRACT
Article history The present study was aimed to reveal the alkaloid profile of Clitoria ternatea
Received: 31/1/2013 seed, stem and leaves using HPTLC. Preliminary phytochemical screening was
Available online: carried out by Harborne method. HPTLC studies were followed by Harborne
3/3/2013 and Wagner et al. method. The ethyl acetate-methanol-water (100: 13.5: 10)
was employed as mobile phase for alkaloids. The methanolic extract of stem,
leaves and seeds of Clitoria ternatea showed the presence of 26 different types
of alkaloids with 21 different Rf values with range 0.02 to 0.93. In general
Keywords: more degree of alkaloids diversity has been observed in reproductive parts
HPTLC; Alkaloids; (seeds) when compared to the vegetive parts leaves and stem. Maximum
Clitoria ternatea; number (10) of alkaloids has been observed in seeds followed by leaves (9).
Phytochemistry Among the ten different alkaloids of seeds, seven (0.15, 0.23, 0.41, 0.52, 0.62,
0.67 and 0.79) are unique to the seeds only and they are not present in the
vegetaive parts of the plant. The proposed HPTLC profile can be used for the
identification of the medicinally important plants and distinguish from its
adulterant.

Corresponding author

Johnson Marimuthu Antonisamy, ptcjohnson@gmail.com

Please cite this article in press as Johnson Marimuthu Antonisamy et.al. Alkaloids Profile of Clitoria ternatea Linn by High
Performance Thin Layer Chromatography (HPTLC). Indo American Journal of Pharmaceutical Research, 201:3(3).
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Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Vol 3, Isuue 2, 2013. Johnson Marimuthu Antonisamy et. al. ISSN NO: 2231-6876

INTRODUCTION:

Phyto-constituents impart the specific characteristics and properties of plants. Therefore, it is obligatory
ettoal.resolve all of the phytochemical
ISSN NO: 2231-6876 present in the plants in order to ensure the consistency and
constituents
repeatability of pharmacological, antimicrobial and clinical research, to understand their bioactivities, identify
the active principles (components) and possible side effects of active compounds and to enhance product quality
control [1]. In pharmacognosy, the phytochemical assessment is one of the important and vital tool for quality
assessment, which includes preliminary phytochemical screening, chemoprofiling and marker compound
analysis using modern analytical techniques such as fluorescence, UV-VIS, FT-IR, HPLC, HPTLC and GC-
MS. The chromatographic technniques are used to differentiate the medicinal sources from its adulterants,
standardization of plant products and is accepted as a strategy for identification and evaluation of the quality of
plant medicines [2,3].
HPLC and HPTLC both emerged as efficient tool for the phytochemical evaluation and widely accepted
technique for its high accuracy, precision and reproducibility of results. Of which, HPTLC has many advantages
because of high sample throughput at low operating cost, easy sample preparation, short analysis time and
analytical assurance [4,5]. Chromatographic methods play an important role in the pharmaceutical field, hence,
need to validate the method when they are developed and intended to be for routine use. Clitoria ternatea L.
(butterfly pea in English) belongs to the family Fabaceae and subfamily Papilionaceae is an herbaceous
perennial legume valued for its forage and medicinal importance. The plant has been adopted in the traditional
Indian system of medicine (folk medicine) due to its multiple pharmaceutical applications. The active
constituents include lactones, aparajitin, taraxerol, phenol glycoside, alkaloid, phydroxycinnamic acid
polypeptide, hexacosanol, anthoxanthin, kaempferol, clitorin, stigmast-4-ene 3, 6-dionie, cyanine chloride,
palmitic, stearic, oleic, linolcic, linolenic acids, tannins, resins, finotin etc. It has been recommended as a
rejuvenating brain tonic having anxiolytic, anti-depressant, anticonvulsant, and anti-stress properties and is
believed to promote memory and intelligence and Anti-inflammatory, analges antipyretic activities of the plant
were attributed to its flavonoid content [6, 7].
The whole plants and seed extract are useful in stomaitis piles, sterility in female, hematemesis,
insomnia, epilepsy, psychosis, leucorrhea and polyurea. The seeds are purgative, cathartic, and useful in
visceralgia [8]. Clitoria ternatea is claimed to treat central nervous systems (CNS) problems; stress, anxiety,
depression and convulsions, etc. Kumar et al [9] have been developed a simple, precise, sensitive and selective
protocol for the determination of taraxerol in Clitoria ternatea L. But there is no report on the HPTLC profile of
Clitoria ternatea seed, stem and leaves. With this background the present study was aimed to reveal the alkaloid
profile of Clitoria ternatea seed, stem and leaves using HPTLC.
MATERIALS AND METHODS
Clitoria ternatea was collected from natural habitats, Coimbatore District, Tamil Nadu, India, and
authenticated by Dr. E.G. Wesely. The fresh materials of Clitoria ternatea stem, leaves and seeds were
separated shade dried and powdered using the electric homogenizer. The powdered samples were extracted with
150 ml of solvent methanol for 8 - 12 h by using the soxhlet apparatus. Preliminary phytochemical screening
was done by following the method of Harborne [10], HPTLC studies were carried out following Harborne [11]
and Wagner et al [12]. For the present study CAMAG HPTLC system equipped with Linomat V applicator,
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TLC scanner 3, Reprostar 3 with 12 bit CCD camera for photo documentation, controlled by WinCATS‐ 4
software were used. All the solvents used for HPTLC analysis was obtained from MERCK. The 100 mg extract
was dissolved in 5ml of Methanol and the solution was centrifuged at 3000 rpm for 5 min and used for HPTLC
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analysis as test solution. The samples (5µl) were spotted in the form of bands of width 5mm with a Camag

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Vol 3, Isuue 2, 2013. Johnson Marimuthu Antonisamy et. al. ISSN NO: 2231-6876

microlitre syringe on pre-coated silica gel glass plate 60F-254 (20cm × 10cm with 250μm thickness (E. Merck,
Darmstadt, Germany) using a Camag Linomat IV (Switzerland). The plates were pre-washed by methanol and
activated at 60°C for 5 min prior to chromatography. The sample loaded plate was kept in TLC twin trough
et al. ISSN NO: 2231-6876
developing chamber (after saturated with Solvent vapor) with respective mobile phase (alkaloids) and the plate
was developed in the respective mobile phase up to 90 mm. The Ethyl acetate-methanol-water (100: 13.5: 10)
was employed as mobile phase for alkaloids. Linear ascending development was carried out in 20 cm x 10cm
twin trough glass chamber (Camag, Mutenz, Switzerland) saturated with the mobile phase and the
chromatoplate development for two times with the same mobile phase to get good resolution of phytochemical
contents. The optimized chamber saturation time for mobile phase was 30 min at room temperature (25 ± 2°C).
The developed plate was dried by hot air to evaporate solvents from the plate.
The developed plate was sprayed with Dragendorff’s reagent followed by 10% Sodium nitrite reagent as
spray reagent and dried at 100 0C in hot air oven for 3 min. The plate was photo-documented at UV 366 nm and
daylight using Photo-documentation (CAMAG REPROSTAR 3) chamber. Finally, the plate was fixed in
scanner stage and scanning was done at 366 nm. The plate was kept in Photo-documentation chamber
(CAMAG REPROSTAR 3) and captured the images under White light, UV light at 254 and 366 nm.
Densitometric scanning was performed on Camag TLC scanner III and operated by CATS software (V 3.15,
Camag).

RESULTS:

The results of the preliminary phytochemical studies confirm the presence of alkaloids in the methanolic
extracts of Clitoria ternatea stem, leaves and seeds. Different compositions of the mobile phase for HPTLC
analysis were tested in order to obtain high resolution and reproducible peaks. The desired aim was achieved
using Ethyl acetate-methanol-water (100: 13.5: 10) as the mobile phase (Table - 1 to 4; Fig.1.A - J). The
methanolic extract of stem, leaves and seeds of Clitoria ternatea showed the presence of 26 different types of
alkaloids with 21 different Rf values with range 0.02 to 0.93 (Table – 1 to 4).
In general more degree of alkaloids diversity has been observed in reproductive parts (seeds) when
compared to the vegetive parts leaves and stem. Maximum number (10) of alkaloids has been observed in seeds
(Table – 1 & 2) followed by leaves (9). Among the sixteen different alkaloids, one alkaloid (0.93) was showed
its presence in all the tested parts of Clitoria ternatea; next to that three alkaloids with Rf values 0.06, 0.14 and
0.93 are shared by the of vegetivie parts (leaves and stem) of Clitoria ternatea, (Table - 1, 3, 4).
The alkaloid (0.10) was showed it jointly presence in the methanolic extracts of Clitoria ternatea seeds and
stem. The alkaloid with 0.3 Rf value was displayed its presence in the methanolic extracts of seed and leaves.
Ten different types of alkaloids have been observed in seeds of Clitoria ternatea. Among the ten different
alkaloids of seeds, seven (0.15, 0.23, 0.41, 0.52, 0.62, 0.67 and 0.79) are unique to the seeds only and they are
not present in the vegetaive parts of the plant. Three different alkaloids with Rf values 0.24, 0.29, and 0.74 are
showed their unique presence only in the stem. Similar to that the alkaloids with Rf values 0.02, 0.2, 0.54, 0.65
and 0.75 are present only in the leaves of Clitoria ternatea.
The alkaloids with the Rf value 0.14 is present commonly in the vegetative parts of the (stem and leaves) of
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the plant. The well resolved HPTLC profile of Clitoria ternatea methanolic extracts of stem leaves and seeds
were presented in Fig 1. A-J and Table -1. HPTLC chromatogram of the standard colchicines the alkaloid
profile of Clitoria ternatea are depicted in Fig 1.
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Vol 3, Isuue 2, 2013. Johnson Marimuthu Antonisamy et. al. ISSN NO: 2231-6876

et al. ISSN NO: 2231-6876

Fig. 1. HPTLC Profile and Chromatogram of Clitoria ternatea L.

A - HPTLC profile of the Clitoria ternatea under UV 366; B - HPTLC profile of the Clitoria ternatea under UV 254; C
- HPTLC profile of the Clitoria ternatea under Day Light - After Derivation; D - HPTLC Chromatogram of Petroleum
ether extracts of Albizia lebbeck - Baseline display - Scanned at 366 nm; E - HPTLC Chromatogram of standard
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Colchicine Peak densitogram display - Scanned at 366 nm; F - HPTLC Chromatogram of Standard Colchicine - Scanned at
366 nm; G - HPTLC Chromatogram of methanolic extracts of Clitoria ternatea seed - Peak densitogram display -
Scanned at 366 nm; H - HPTLC Chromatogram of methanolic extracts of Clitoria ternatea stem - Peak densitogram
display - Scanned at 366 nm; I - HPTLC Chromatogram of methanolic extracts of Clitoria ternatea leaves - Peak
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densitogram display - Scanned at 366 nm; J - 3D display of HPTLC Chromatogram of Clitoria ternatea – seed, stem and
leaves methanolic extracts.

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 Vol 3, Isuue 2, 2013. Johnson Marimuthu Antonisamy et. al. ISSN NO: 2231-6876

Table 1: HPTLC Alkaloid Profile of Clitoria ternatea methanolic extract

A B C Colchicine
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0.02 +

0.06 + +

0.10 + +

0.14 + +

0.15 +

0.20 +

0.23 +

0.24 +

0.29 +

0.30 + +

0.38 +

0.41 +

0.52 +

0.54 +

0.62 +

0.65 +

0.67 +

0.74 +

0.75 +

0.79 +

0.93 + + +
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10 7 9
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Note: A – HPTLC Alkaloid Profile of methanolic extract Clitoria ternatea Seed; B - HPTLC Alkaloid Profile of methanolic
extract Clitoria ternatea Stem; C- HPTLC Alkaloid Profile of methanolic extract Clitoria ternatea Leaves

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Vol 3, Isuue 2, 2013. Johnson Marimuthu Antonisamy et. al. ISSN NO: 2231-6876

Table 2: HPTLC Alkaloid Profile of methanolic extract Clitoria ternatea Seed

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Rf Height Area Assigned substance

0.38 606.3 15691.4 Colchicine standard

0.10 64.9 1219.3 Unknown
0.15 327.3 7583.5 Alkaloid 1
0.23 73.2 1765.4 Unknown
0.30 233.4 4862.0 Alkaloid 2
0.41 28.2 1019.3 Unknown
0.52 10.9 265.2 Unknown
0.62 12.3 324.0 Unknown
0.67 11.9 319.2 Unknown
0.79 44.2 1500.3 Unknown
0.93 50.6 2765.1 Unknown

Table 3: HPTLC Alkaloid Profile of methanolic extract Clitoria ternatea Stem

Rf Height Area Assigned substance

0.38 606.3 15691.4 Colchicine standard

0.06 37.2 623.2 Unknown

0.10 206.1 3946.7 Unknown

0.14 168.4 3699.0 Alkaloid 1

0.24 36.5 1264.6 Unknown

0.29 114.0 2349.7 Alkaloid 2

0.74 17.1 427.0 Unknown

0.93 157.9 8017.5 Unknown

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Vol 3, Isuue 2, 2013. Johnson Marimuthu Antonisamy et. al. ISSN NO: 2231-6876

Table 4: HPTLC Alkaloid Profile of methanolic extract Clitoria ternatea Leaves

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Rf Height Area Assigned substance
0.38 606.3 15691.4 Colchicine standard
0.02 26.0 266.4 Unknown
0.06 100.4 1623.5 Unknown
0.14 302.7 6859.1 Alkaloid 1
0.20 31.7 948.7 Unknown
0.30 447.0 12553.8 Alkaloid 2
0.54 71.7 2566.3 Unknown
0.65 14.4 319.9 Unknown
0.75 16.9 404.6 Unknown
0.93 300.2 15589.7 Unknown

DISCUSSION:
Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants, and animals especially
by higher plants – about 10 to 25% of those contain alkaloids and are part of the group of natural products (also
called secondary metabolites). They often have pharmacological effects and are used as medications,
as recreational drugs, or in entheogenic rituals [13]. Depending on the type of plants, the maximum
concentration is observed in the leaves (black henbane), fruits or seeds (Strychnine tree), root (Rauwolfia
serpentina) or bark (cinchona) [13]. Furthermore, different tissues of the same plants may contain different
alkaloids. Similar to the previous observations, in the present also we observed 26 different alkaloids in the
different parts of Clitoria ternatea. Many alkaloids are used in medicine, usually in the form of salts, including
the following: anti-arrhythmic, anti-cholinergic, anti-tumor, vasodilating, anti-hypertensive, cough medicine,
anesthetic, anti-protozoal agent [14]. The results of the present study confirms the folkloric usage and
pharmacological studies of the medicinally important plant Clitoria ternatea and suggest that some of the plant
extracts possess compounds with bioactivity properties that can be used as active principles or agents in new
drugs for the therapy of infectious diseases. The proposed HPTLC profile can be used for the identification of
the medicinally important plants and distinguish from its adulterant.
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REFERENCES:
1. Amani M, El-Mousallamy D. Leaf flavonoids of Albizia lebbeck. Phytochemistry 1998; 48(4): 759 -761.
et al.2. Anonymous. Quality control methods
ISSN NO: for medicinal plant material. WHO/PHARM, Geneva 1992.
2231-6876
3. Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo ZG. Medicinal plants in therapy. Bull. WHO
1985; 63: 965.
4. Di X, Chan KKC, Leung HW, Huie CW. Fingerprint profiling of acid hydrolyzates of polysaccharides
extracted from the fruiting bodies and spores of Lingzhi by high--performance thin-layer
chromatography. Journal of Chromatography A 2003; 1018: 85-89.
5. Larsen T, Axelsen J, Ravn HW. Simplified and rapid method for extraction of ergosterol from natural
samples and detection with quantitative and semi-quantitative methods using thin layer chromatography.
Journal of Chromatography A 2004; 1026: 301-304.
6. Parimaladevi B, Boominathan R, Mandal SC. Antiinflammatory, analgesic and antipyretic properties of
Clitoria ternatea root. Fitoterapia 2003; 74(4): 345-349.
7. Mukherjee PK, Kumar V, Kumar NS, Heinrich M. The ayurvedic medicine Clitoria ternatea from
traditional use to scientific assessment. J Ethnopharmacol 2008; 120: 291-301.
8. Mhaskar AV, Prakash K, Vishwakarma KS, Maheshwari VL. Callus induction and antimicrobial activity
of seed and callus extracts of Clitoria ternatea L. Current trends in Biotechnology and Pharmacy 2010;
3(4): 561-567
9. Kumar V, Mukherjee K, Kumar S, Mal M, Mukherjee PK. Validation of HPTLC method for the analysis
of taraxerol in Clitoria ternatea. Phytochem Anal 2008; 19: 244-250.
10. Harborne JB. Phytochemical Methods, London: Chapman and Hall, 1973;
11. Harborne JB. Phytochemcial methods. 3rd ed. London: Chapman and Hall; 1998;
12. Wagner H, Baldt S, Zgainski EM. Plant Drug Analaysis. Berlin: Springer; 1996;
13. David RF. Evolution of Plant Chemical Defense against Herbivores. In: Rosenthal, Gerald A, Janzen,
Daniel H. Herbivores: Their Interaction with Secondary Plant Metabolites. New York: Academic Press
1979; p. 41.
14. http://en.wikipedia.org/wiki/Alkaloid

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