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Malliga et al, 2015/ Preliminary phytochemical screening and HPTLC fingerprinting profile

International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2 (2) 32- 40 32

ORIGINAL ARTICLE

International Research Journal of Pharmaceutical and Biosciences

Pri -ISSN: 2394 - 5826 http://www.irjpbs.com e-ISSN: 2394 - 5834

Preliminary Phytochemical Screening and HPTLC

Fingerprinting Profile of Leaf Extracts of Moringa oleifera

and Phyllanthus emblica. N. Malliga Elangovan1*, M.S. Dhanarajan 2, I. Elangovan3

1 Research Scholar, Department of Biochemistry, Manonmaniam Sundaranar University, Tirunelveli. 2 Principal, Jaya College of Arts and Science, Thirunindravur, Chennai. 3Biochemist, Meenakshi Mission Hospital, Madurai, Tamil nadu.

Article info Abstract

Article history:

Received 10 March 2015

Accepted 18 March 2015

*Corresponding author:

[email protected]

Copyright 2015 irjpbs

Objective: To establish the physical constants and fingerprint profile of Phyllanthus emblica and Moringa oleifera using high performance thin layer chromatography (HPTLC) technique. Methods: Preliminary phytochemical screening was done, physical constants were evaluated and HPTLC studies were carried out. CAMAG make HPTLC system equipped with Linomat 5 applicator, TLC scanner 3, Reprostar 3 and WIN CATS-1.4.3 software were used. Results: Preliminary phytochemical screening of the extract showed the presence of alkaloids, flavonoids, glycosides, saponins, tannins, terpenoids, phenolic compounds, proteins, reducing sugars, fats and oils. HPTLC finger printing of petroleum ether extract of Moringa oleifera leaf powder under white light reveal presence of eight components 0.24, 0.29, 0.34, 0.54, 0.76, 0.83, 0.93, 0.99. Component number 8 at Rf 0.99 showed maximum concentration. Petroleum ether extracts of Phyllanthus emblica leaf powder showed seven peaks with Rf values in the range with their Rf value - 0.26 0.34 0.55, 0.77 0.80, 0.95. Component number 6 at 0.95 Rf value showed maximum concentration. Conclusions: It can be concluded that HPTLC fingerprint analysis of Petroleum ether extract of leaf powder extracts of Phyllanthus emblica (PELPE) and Moringa oleifera (PELMO) can be used as a diagnostic tool for the correct identification of the plant and it is useful as a phytochemical marker and also a good estimator of genetic variability in plant populations.

Keywords: Phytochemicals, HPTLC, Petroleum ether extracts, Phyllanthus emblica and Moringa oleifera.

mailto:[email protected]



INTRODUCTION

The history of plants being used for medicinal purpose is probably as old as the history of mankind. Herbal

medicine is an achievement of popular therapeutic diversity since they may possess hundreds of medicinal

materials and produce their curative effects. Awareness of the herbal plant’s chemical constituent is

helpful in the discovery of effective therapeutic agents. Extraction and characterization of several active

phytocompounds from the medicinal plants is the foundation for the formation of some high activity

profile drugs [1]. Chromatographic and spectral fingerprints play a vital role in the complex herbal

medicines in support of the enhancement of the quality control [2]. Since they represent the chemical

integrities of the herbal medicines and its products and therefore used for authentication and identification

of herbal plant [3].

Moringa oleifera Lam. Also called as drumstick tree or horseradish tree (Moringaceae) is an indigenous

tree from northwestern India, often cultivated in hedges and home backyards. Many recent reports on

disease prevention by M. oleifera have been reported. Malliga et al (2014) reported that the enzymatic and

non-enzymatic activities such as SOD, Catalase, Peroxidase, Vitamin C, Carotenoids, and reduced

Glutathione were high in petroleum ether extracts of Moringa oleifera and thus the leaves can be a good

source of natural antioxidants. The leaf extract is competent in decreasing hyperglycemia and dyslipidemia

[4] with effective antiproliferative activity and apoptosis inducing ability on tumor (KB) cell line [5].

Phyllanthus emblica (L.) or Emblica officinalis Gaertn. commonly known as “amla” (family-Euphorbiaceae) is

indigenous to tropical South-East Asia. It is extremely nutritious and might be a chief dietary source of

vitamin C, amino acids, and minerals. Plant parts proved to be with antidiabetic, hypolipidemic,

antibacterial, antioxidant, antiulcerogenic, gastroprotective, hepatoprotective, and chemopreventive

properties [6]. Phytochemicals in the plant extract offer protection against cellular damage due to their

ability of inhibiting lipo-oxygenases [7].

HPTLC is more efficient, faster with more reliable and reproducible results. HPTLC in combination with

digital scanning profile, also provides accurate and precise retention factor (Rf) values and quantitative

analysis of sample by in situ scanning densitometry aided by formation of easily detected derivatives by

post-chromatographic chemical reactions as needed, as well as a record of the separation in the form of a

chromatogram with fractions represented as peaks with defined parameters including absorbance

(intensity), Rf value, height and area [8]. HPTLC fingerprints could be used in proper identification of

medicinal plants, as a valuable analytical tool in the routine quality control and standardiza-tion of herbal

drugs [9] and as a chemotaxonomical tool in the plant systematic [10], for determination of bioactive

components of the herbal medicine [11]. The present study sought to investigate HPTLC profile of

petroleum ether leaf extracts of Phyllanthus emblica and Moringa oleifera for analyzing marker chemical

constituents.

MATERIALS AND METHODS

Plant Collection

The fresh mature healthy leaves of Phyllanthus emblica and Moringa oleifera plant were collected from

Algar kovil hills, Madurai, Tamil nadu, India. The selected plants were authenticated by Dr. P. Jayaraman,

Institute of Herbal Botany, Plant Anatomy Research Centre, Chennai-05. Leaves were shade-dried at room

temperature to avoid loss of essential oil and milled with the aid of grinding machine.



Preparation of Plant extract

The extraction process was carried out using soxhlet extraction method. About 200gm of dry powdered

plant material was extracted in Soxhlet apparatus with 250ml of petroleum ether for 8 hours. After

extraction, the solvent was removed using rotary vacuum evaporator to give a concentrated extract at 60˚C

in a water bath. It was then dried aseptically with the help of drier and subjected to spectroscopic analysis.

Phytochemical Screening

The different qualitative phytochemical tests were for establishing the phyto-constituents present in the

petroleum ether extracts of Phyllanthus emblica and Moringa oleifera leaves for the identification of the

various active chemical constituents such as alkaloids, phenols, flavonoids, tannins, saponins, terpenoids,

glycosides, reducing sugars, fats and oils [12]. The positive tests were noted as present (+) and absent (-).

HPTLC Fingerprinting Equipments.

Sample preparation: The plant extracts residue was redissolved in 1ml of chromatographic grade

methanol, which was used for sample application on HPTLC plates pre-coated silica gel 60F 254 aluminium

sheets.

Developing solvent system: A number of solvent systems were tried, for extract, but the satisfactory

resolution was obtained in the solvent Toluene: Ethyl acetate: Methanol (7:2:1).

Sample application: Application of bands of each extract was carried out (8mm band in length and 2ul in

concentration) using spray technique. Sample were applied in duplicate on pre-coated silica gel 60F 254

aluminium sheets (5 x 10 cm) with the help of Linomat 5 applicator attached to CAMAG make HPTLC

system, which was programmed through WIN CATS software (1.4.3).

Development of chromatogram: After the application of sample, the chromatogram was developed in

Twin trough glass chamber 10x 10 cm saturated with the solvent Toluene: Ethyl acetate: Methanol (7:2:1)

for 15 min.

Detection of spots: The air-dried plates were viewed in ultraviolet radiation to mid day light. The

chromatograms were scanned by densitometer at 520nm, 254nm & 366nm in Densito-metry TLC Scanner

3.The Rf values and finger print data were recorded by WIN CATS software.

RESULT

The preliminary qualitative phytochemical screening of the crude powder of Phyllanthus emblica and

Moringa oleifera was done to assess the presence of bioactive components. The presence of various

phytoconstituents like alkaloid, carbohydrate, glycoside, steroid, protein, tannin, terpenoid, flavonoid and

phenol was determined (Table 1).



Table 1: Preliminary Qualitative phytochemical screening on petroleum ether leaf extracts of Phyllanthus

emblica and Moringa oleifera

S. No Phytochemicals Test Name Observation Phyllanthus

emblica

Moringa

oleifera

1. Alkaloids a. Mayer’s White precipitate + +

b. Wagner’s Reddish brown

colour

+ +

2. Carbohydrates a. Molisch’s Purple ring at the

junction

+ +

b. Fehling’s Brick red colour + +

c. Benedict’s Red precipitate + +

3. Glycosides Keller- Killiani’s Brown ring

interphase

+ +

4. Steroids a. Libermann-

Burchard

Bluish-green + +

b. Salkowski Reddish Brown

interphase

+ +

5. Flavonoids With NaOH Yellow colour + +

Conc. H2SO4 Yellow colour + +

10% NaCl Yellow colour + -

6. Saponins Frothing test Layers of form + -

7. Fixed Oil & Fats Spot test Oil strain on paper + +

8.

Tannins &

Phenolic

compounds

a. Lead acetate

solution

White precipitate + +

b. With FeCl3 Greenish black

colour

+ +

9. Proteins and

Amino acids

a. Biuret test Purple colour + +

b. Ninhydrin test

Blue colour

+ +

High Performance Thin Layer Chromatography (HPTLC) technique is most simple and fastest separation

technique available today which gives better precision and accuracy with extreme flexibility for various

steps. The results showing number of peaks, the Rf values are presented in Table 2 and Table-3. HPTLC

spectral analysis of both the plant extracts were done in three different wavelengths- under UV at 254 and

366 nm, and white light (Figure 1 and Figure-3). The Petroleum ether leaf extract of Phyllanthus emblica

and Moringa oleifera were subjected to HPTLC fingerprinting (Figure 2 and Figure-4).

Table 2-: Rf values of petroleum ether leaf extract of Phyllanthus emblica

Visualization / Detection (Rf values)

Sample Under White light Under UV 254 nm Under UV 366 nm

2l of petroleum ether

leaf extract

0.26, 0.34, 0.55, 0.77,

0.80, 0.95

0.26, 0.34, 0.39, 0.41,

0.55, 0.66, 0.68, 0.77,

0.80, 0.92

0.24, 0.30, 0.34, 0.39,

0.48, 0.51, 0.60, 0.75,

0.80, 0.90, 0.99



Table 3-: Rf values of petroleum ether leaf extract of Moringa oleifera

Visualization / Detection (Rf values)

Sample Under White light Under UV 254 nm Under UV 366 nm

2l of petroleum ether

leaf extract

0.24, 0.29, 0.34, 0.54,

0.76, 0.83, 0.93, 0.99

0.24, 0.29, 0.34, 0.43,

0.49, 0.55, 0.64, 0.77,

0.95

0.24, 0.29, 0.34, 0.38,

0.46, 0.50, 0.59, 0.69,

0.75, 0.84, 0.93

White light @ 254nm @366nm

Figure 1: HPTLC profile of PELMO

White light @ 254nm @ 366nm

Figure 2: Fingerprinting of PELMO



White light @ 254nm @366nm

Figure 3 : HPTLC profile of PELPE


Figure 4: Fingerprinting of PELPE

The results showed 10 peaks at 254 nm, 11 peaks at 366 nm and 6 peaks under white wavelengths (Figure-

5) for Phyllanthus emblica extract. Peak number 6 covered maximum % of area at white light with 0.95 Rf

value. Peak number 10 covered maximum % of area at 254 nm with 0.92 Rf value. Peak number 10 covered

maximum % of area at 366 nm with 0.90 Rf value. Moringa oleifera extract showed11 peaks at 254 nm, 8

peaks at 366 nm and 9 peaks under white wavelengths (Figure-6). Peak number 11, 8 and 9 covered

maximum % of area with 0.93, 0.99 and 0.95 Rf values respectively.




Figure 5.HPTLC chromatograms of Phyllanthus emblica petroleum ether leaf extract showing different

peaks of phytoconstituents


Figure 6.HPTLC chromatograms of Moringa oleifera petroleum ether leaf extract showing different peaks

of phytoconstituents



DISCUSSION

Authentication of medicinal plants at chemical and genetic level is a crucial step for both research purposes

and commercial preparations. Recently anatomical, biochemical, cytological, and molecular markers are

also being used to classify the organisms in addition to morphological markers. Mostly, the entire

chromatographic profiles are used to identify or to evaluate the quality of the herbs investigated.

A thorough understanding of their chemical composition is essential for conducting a safety risk

assessment. Chemical constituents of Moringa plants were performed by many researches but they are not

always exactly similar due to their geographic location. The chemical constituents and their amounts in

herb can be different, due to growing conditions, such as climate, soil fertility, the harvest season, age of

the leaves, the drying process, etc.

HPTLC studies have shown that it is more resourceful than ordinary TLC methods, as the spots are well

resolved. It is an invaluable quality assessment tool for the assessment of botanical materials, and it allows

for the analysis of a broad number of compounds both efficiently and cost effectively. It is helpful as a

phytochemical marker and also a good estimator of genetic variability in plant populations. The exclusive

characteristic of the picture like image of HPTLC coupled with digital scanning profile is progressively

attractive to herbal analysis to construct the herbal chromatographic fingerprint.

To our knowledge HPTLC profile of Phyllanthus emblica and Moringa oleifera were generated for the first

time in petroleum ether solvent system in order to establish the total number of bioactive compounds. The

HPTLC images shown in Figure 2 indicate that both the sample constituents were obviously separated

without any shadowing and diffuseness. The chromatographic finger prints obtained can be stored as

electronic images without any errors and change for further usage.

CONCLUSION

The results of phytochemical analysis showed that the petroleum ether extract of both the selected

medicinal plants showed highest content than water and chloroform extracts. Thus, the plants studied here

can be seen as a potential source of new useful drugs. The phytochemical characterization of the extracts,

the identification of responsible bioactive compounds and quality standards are necessary for future study.

HPTLC profile is a rapid, precise and powerful procedure to place the plant in taxonomic categories by

analysing the presence or absence of chemical constituent. The main goal of pharmacognosy is to assess

the value of raw materials and to ensure that the final product is of the required standard. Strict

standardization procedures and pharmacognostical studies of medicinal plants would reduce drastically

much of the accidents in wrong prescriptions of traditional herbal medicines [13]. The enzymatic and

non_enzymatic antioxidant activities are significantly high in petroleum ether extracts of Moringa oleifera leaves,

must be due to the presence of bioactive phytochemicals in it [14].

HPTLC and preliminary phytochemical study of Phyllanthus emblica and Moringa oleifera could be useful

for the grouping of a suitable monograph for its proper identification and also in designing the method of

isolation, purification and characterization of marker chemical compounds. In the present study, both the

plant extracts possesses several phytoconstitutents with biological activities. The isolation and

identification of these bioactive compounds can be used to formulate new drugs to treat a variety of



diseases and disarray. The chromatographic finger prints obtained in this study can be stored as electronic

images without any errors and change for further usage.

ACKNOWLEDGMENT

We would like to thank N. Rama Varier Ayurveda Foundation, Research & Development, Madurai, Tamil

nadu, India for providing laboratory facilities and instruments for smooth conduction of research work.

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