the phytochemical screening and profiling of …

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Manjula K T et al. World Journal of Pharmaceutical Research

THE PHYTOCHEMICAL SCREENING AND PROFILING OF

BIOCHEMICAL PARAMETERS IN DIFFERENT PARTS OF

PANDANUS UNIPAPILLATUS DENNST. IN SOUTH INDIA

*Manjula K.T1, J M Sasikumar1, V K Gopalakrishnan2

1Department of Biotechnology, Karpagam University,Coimbatore,Tamilnadu,India-652 101 2Department of Biochemistry and Bioinformatics, Karpagam University, Coimbatore,

Tamilnadu, India

ABSTRACT

Pandanus unipapillatus Dennst. is a shrub or tree belonging to the

family pandanaceae. It is widely distributed in Penisular India. In the

present study of preliminary screening, it was found that among the

different parts of methanolic extracts of Pandanus unipapillatus (root,

leaf, and stem) showing various phytochemicals. The total phenolics

and total flavanoids were qntitatively estimated in each extracts. Stem

extract showing more phenolic and flavanoids content than leaf and

root of the plant. The F/P ratio measures, the flavanoids among the

whole Phenolics. F/P ratio is very high in Root (0.80%) compare to the

other parts (Stem (0.68%) and Leaf (0.63%). In addition, some group

of Phenolic compounds shown as blue fluorescent band as in phenolic

specific thin layer chromatographic profile of different parts of the

plant. The generated data has provided the basis for its therapeutic value and used as a

therapeutant.

Keywords: Phytochemicals, phenolics, flavanoids, Thin layer chromatography.

INTRODUCTION

Tropical herbs and plants produce a large variety of phytochemicals or secondary

metabolites, which are important for synthesizing pharmaceuticals and botanical medicines

(MAO, 1999). The use of plants as medicines has involved the isolation of secondary

metabolites and bioactive compounds. The active principle is usually found in specific part of

World Journal of Pharmaceutical research

Volume 2, Issue 4, 878-889. Research Article ISSN 2277 – 7105

Article Received on 16 April 2013, Revised on 07 May 2013,

Accepted on 29 June 2013

*Correspondence for Author: Manjula K T

Department of Biotechnology,

Karpagam University,

Coimbatore, Tamilnadu,

India.

[email protected]

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plant such as aerial parts , leaves, flowers, fruits, seed, root, rhizome and bark and the crude

plant extract is a complex mixture of phytochemical constituents (peters&Whitehorse, 1999).

Pandanus unipapillatus (syn.Pandanus canaranus Warb.) is used in traditional medicine and

it also famous for its fragrance. It is endemic to peninsular India, common along the banks of

streams, canals, rice fields and bunds. The local names of the plant is “Thazha

kaitha”,”Anakaitha”.shrubs or small tree.,8-10m tall, stem more or stout erect branching near

top,dichotomous and few grayish brown,ringed,distinct brownscars.,prop root stout (Nicolson

et al.interprt.Hort.Malab.306.1988). Pandanus unipapillatus Dennst is the one of the major

plant source of the Ayurvedic drug known as “Kethaki” (Joshi et al., 2011).

Hence the present studies explained the comparative preliminary phytochemical screening,

Total phenolics and Total flavanoid content, F/P ratio and Phenolic specific TLC chemical

profiling of different parts of Pandanus unipapillatus.

This study is helpful to determine each parts (Root, Leaf, Stem) medicinal properties, class of

chemical constituents and their (Phenolic and Flavonoids) amount present in the plant.

Nowadays, phytochemicals and antioxidants in plants are raising interest in consumers for

their roles in the maintenance of human health. Phenolics and Flavonoids are known for their

health-promoting properties due to protective effects against cardiovascular disease, cancers

and other disease. The study supports the view that the amount of Flavonoids, phenols is

depend on antioxidant activity of the plant.To the best of our knowledge, the phytochemical

screening and quantitative test of phenolics and Flavonoids of the plant have not been

reported so far.

MATERIALS AND METHODS

Phytochemical screening

Phytochemical screening of the extract and fractions were carried out to identify the

constituents, using standard phytochemical methods as described by Sofowora (1993).

Collection of plant Material

The whole plant of P. unipapillatus Dennst. was collected from Village area of Calicut,

kerala, India on the month of December , 2012. The plant was shade dried for some days and

ground into powder with the help of an grinder and later it was stored in air tight bottles for

further use. It was taxonomically authenticated at the herbarium of the Department of

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Taxonomy, Calicut University, Kerala, India. A Voucher specimen (CU001 (Root), CU002

(stem), CU003 (leaf)) was deposited there for future reference.

Extract preparation

Leaves, stems and Roots were Shade dried to constant weights prior to being used in the

extraction. For Preliminary screening and quantitative analysis, the roots, leaves, and stems,

were powdered and 1 g of the powder was extracted continuously with polar protic solvent:

methanol, CH3 OH, 80%, The solution was then swirled for 1 h at room temperature using an

orbital shaker. Extracts were then filtered under suction and stored at -20°C for further use.

Chemicals and reagents

Deionized water, Folin-Ciocalteu phenol reagent , gallic acid (SD Fine

chemicals),Aluminium chloride,Hcl, Dragendroff reagent,Potassium acetate ,methanol,

hexane, chloroform, quercetin, Ferric chloride, Magnesium ribbon(Sigma Aldrich,Mumbai),

Merk Silica gel 60 F254 precoated aluminium sheet.

Qualitative screening

Test of Carbohydrate:

Molisch test: Filtrates of extracts of were treated with 2 drops of alcoholic alpha-naphthol

solution in a test tube. Formation of the violet ring at the junction indicates the presence of

carbohydrates.

Fehlings test: Filtrates were hydrolysed with dil.Hcl, neutralized with alkali and heated with

Fehling’s A&B solutions.Formation of red precipitate indicates the presence of reducing

suger.

Tannins: To 2 ml of aqueous extract 2 ml of 5% FeCl₃ was added. Formation of yellow

brown precipitate indicates that tannins are present (Jigna et al., 2007).

Alkaloids: To the 2 ml methanolic filtrate, 1.5 ml of 1% HCl was added. After heating the

solution in water bath,6 drops of Mayors reagents/Wagner’s reagent/Dragendroff reagent was

added. Formation of Orange precipitate indicates the presence of alkaloids (Oguyemi, 1979).

Test of Glycosides: Extract were hydrolysed with dil.Hcl and then subjected to test for

glycosides. .(Tiwari et al,2011)

Brontragers test: Extracts were treated with Ferric chloride solution and immersed in

boiling water for about 5 minutes.The mixture was cooled and extracted with equal volume

of benzene.The benzene layer separated and treated with ammonium solution.Formation of

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rose pink colour in the ammonial layer indicates the presence of anthranoid glycosides.

(Tiwari et al,2011)

Legal’s test: Extracts were treated with sodium nitroprusside in pyridine and sodium

hydroxide.Formation of pink to blood red colour indicates the presence of glycosides. (Tiwari

et al, 2011)

Saponins: Aqueous extract of 2 g powder was made and subjected to frothing test. Frothing

persistence indicated presence of saponins. Latter the froth was mixed with few drops of

olive oil. Formation of emulsion indicates presence of saponins (Sofowora, 1993).

Flavonoids: 2 g plant material was extracted in 10 ml alcohol or water. To 2 ml filtrate few

drops of concentrated HCl followed by 0.5 g of zinc or magnesium turnings was added. After

3 minutes magenta red or pink color indicated the presence of flavonoids (Jigna et al., 2007).

Phenolics: To 2 ml of alcoholic or aqueous extract, 1 ml of 1% ferric chloride solution was

added. Blue or green color indicates phenols (Martinez, 2003).

Test of sterols: Extracts were treated with chloroform and filtered.The filtrates were treated

with few drops of conc.Sulphuric acid, shaken and allow to stand.Appearence of golden

yellow colour indicates the presence of triterpenes. (Tiwari et al, 2011)

Fats & oils test: 1.ml of the extract was add to a filter paper. These extract was allow it for

evaporation on filter paper and the appearance of transparence on filterpaper indicates the

presence of fats &oils. (Tiwari et al, 2011)

Determination of Total phenolic content

The total phenolic content was determined using Folin–Ciocalteu reagents with analytical

grade gallic acid as the standard. 1 mL of extract or standard solution (0 to 500 mg/L) was

added to deionized water (10 mL) and Folin–Ciocalteu phenol reagents (1.0 mL). After 5

min, 20% sodium carbonate (2.0 mL) was added to the mixture. After being kept in total

darkness for 1 h.Ablue colour was developed in each tube, because the phenols undergo a

complex redox rection with phosphomolybdic acid in Folin-Ciocalteu reagent in alkaline

medium which resulted in molybdenum blue. A blue colored the absorbance of the reaction

mixture was measured at 650 nm using a spectrophotometer . Amounts of TP were calculated

using gallic acid calibration curve. The results were expressed as gallic acid mg /g of dry

plant matter (Kim et al., 2003).

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Determination of Total Flavonoids

The TF were measured by taken from the methodology of chang et al (2002).Methanolic

extract(3g)of each extracts were mixed with 3 ml of methanol,0.2 ml of 10% AlCl3,0.2 ml of

1M potassium acetate and 5.6 ml of distilled water. After being kept in room temperature in

30 minutes.The absorbence was measured at 415nm.For each sample reading were taken to

get the average results. The results were expressed in mg quercetin/g dry weight by

comparison with the quercetin standard curve, which was made under the same condition.

Phenolic specific- Thin Layer Chromatography

Mashed plant parts(leaf, stem) were extracted by soaking in MEOH for 24 hours by using

Soxhlet apparatus.The extract was spoted on Merk Silica gel 60 F254 precoated aluminium

sheet for thin layer chromatography analysis.The thin layer chromatography sheet was

developed in an ascending manner using different solvent system.After running of sovents

completed,the plate dried by using Drier .Then visualized under UV(365nm). (Plant drug

Analysis by Wagner. 1989).

RESULT AND DISCUSSION

The medicinal plants having various secondary metabolites such as alkaloids, flavanoids,

glycosides, phenols, tannins, saponins, sterols, fats and oils etc. The preliminary screening

tests may be usefull in the detection of the bioactive principles and subsequently may lead to

the drug discovery and development.The results of the phytochemical screening are shown in

the table.1.

The preliminary screening of the plant material reveals that the presence of Flavanoides,

Carbohydrate,saponins,Phenolics seems to be positive in leaves, but here

Tannins,alkaloids,fats and oils, Glycosides were absent.In roots, has been shown positive as

Flavanoides, Alkaloides, Tannins, Carbohydrate, phenolics.But Sterols,Saponins,Fats & oils

were absent.In Stem Part, showed positive results of Flavanoides Phenolics,

carbohydrate,saponin,Tannins.Here Sterols,Fats & oils,alkaloids,glycosides were absent.

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Table: 1 Preliminary phytochemical screening of different parts of Pandanus

unipapillatus

Sl no. Class of

compounds Test performed Root Stem Leaf

1 Carbohydrate Molisch’s test +++ +++ ++

Fehling’s test +++ +++ +++

2 Phenols Phosphomolybdic acid test +++ +++ +++

3 Flavanoids Lead acetate test +++ +++ +++

4 Tannins Braemer’s test + ++ --

5 Alkaloids Draggendorf’s test +++ -- ---

Wagner’s test +++ -- ---

6 Glycosides Legals test --- --- ---

Brontranger’s test --- --- ---

7 Saponin Foam test --- ++ +++

8 Sterols Salkowski’s test --- --- ---

9 Fats and Oils Paper test --- --- ---

+ = moderate - - - = absent +++ = strong - = less

1.1 Determination of Total Phenolics (TP) Content

Phenols or Polyphenols are secondary plant metabolites that are tremendously present in

plants and plant products. Many of the phenolics have been shown to contain high level of

antioxidant activities(Razali et al.,2008).Phenolic compounds contribute the overall

antioxidant activities of mainly due to their redox properties.Generally the mechanism of

phenolic compound preventing decomposition of hydroxyperoxide in to

freeradicals(Javanmardex.,2003:Li et al.,2009).

The result obtained from the preliminary analysis of TP is shown in Table 2. The total

phenolic content of the P.unipapillatus extract in terms of gallic acid equivalent (GAE mg g-1

drymass) which is a common reference compound. The phenolic constituents can react with

active oxygen radicals such as hydroxyl radical (Hussein et al., 1987), superoxide anion

radical (Afanaslev et al., 1989) and lipid peroxy radical (Torel et al., 1986). Literature reports

shown that there is high correlation between antioxidant activity and phenolic compounds

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(Odabasoglu et al., 2004).The result indicates that the MEOH extract of stem (52O.48 GAE

mg g-1) contain very high amount of polyphenolic compounds as compared to the other parts

of the plant(Root 140.96 GAE mg g-1 ,Leaf491.56 GAE mg g-1 ). Here, the root has shown

comparatively low phenolic content. The calibration curve of Gallic acid to determine

phenolic content was shown in fig. 1 and 2.

The F-C assay for total phenolics content is a simple method.F-C method is based on

oxidation of phenols by a molybdotungstate in F-C reagent to yield a coloured product λmax

745-750nm (Prior et al., 2005). F-c assay gives a crude estimated the total phenolic

compounds present in an extract. It is not specific to polyphenols, but many interfering

compound may react with the reagent giving elevated, apparent polyphenolic

compounds(Prior et al., 2005).

1.2 Determination of Total Flavanoid (TF) content

Flavanoides are most common and widely distributed group of plant phenolic compounds. It

is mostly occurred in plants, vegetables, Fruits. TF can be determined in the sample extracts

by reaction of Potassium acetate,followed by the development of coloured flavanoid-

aluminium complex formation using Aluminium chloride in alkaline condition which can be

monitored spectrophotometrically at maximum wave length of 415nm (Chang et al.,2002).

The total flavonoid content was expressed as quercetin equivalents(QE) in milligram per

gram dry materials of extracts. The calibration curve of quercetin to determine flavonoid

content was shown in fig. 3 and 4. Total flavonoid content of MEOH extract was compiled in

Table 2. The stem had the highest flavanoid content,compared to that of other

extracts(leaf,root).However flavonoids content of the three parts of plants(Root,leaf,stem)

was 113.92 QE mg g-1,311.39 QE mg g-1 ,356.96 QE mg g-1 respectively. The MEOH extract

of leaf shown average and root having comparatively low content of flavonoides.

Flavonoids and phenolics are most important groups of secondary metabolites and bioactive

compounds in plants [Kim et al., 2003]. Flavonoids have important roles in human life and

health. Their function in human health is supported by the ability of flavonoids to induce

human protective enzyme systems, and by number of epidemiological studies suggesting

protective effects against cardiovascular diseases, cancers and other related diseases [Cook et

al.,1996]. We examined The Total flavonoid /total phenolics ratio(F/P ratio was highest in

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root(0.80%) part of the plant as compared to leaf(0.63%) and stem(0.68%).The F/P ratio

measures the flavanoides among the whole phenolics.

Table:2 Total phenolics and flavanoides in the studied different parts of Pandanus

unipapillatus

Different parts of

Pandanus

unipapillatus

Total phenolics(mg

GAE/1 mg DW)

Total flavanoids(mg

QE/1mg DW)

F/P ratio

Stem 520.48 GAE mg g-1) 356.96 QE mg g-1 0.68%

Root 140.96 GAE mg g-1) 113.92 QE mg g-1 0.80%

Leaf 491.56 GAE mg g-1) 311.39 QE mg g-1 0.63%

0

0.5

1

1.5

2

20 40 60 80

Abs

orba

nce

at

650

NM

Different concentrations of extract and standard(GALLIC ACID)…

Fig:2 Total Phenolics of Methanolic extract of root of Pandanus unipapillatus DENNST.

STANDARD

EXTRACT

0

0.5

1

1.5

2

20 40 60 80Absorbance at 750nm

Different Concentrations of extract and standard(GALLIC ACID)…

Fig:1 Total Phenolics of Methanolic extract of stem of Pandanus unipapillatus DENNST.

STANDARD

EXTRACT

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0

0.5

1

1.5

2

20 40 60 80Abso

rban

ce a

t 650

NM

Different concentrations of extracts and standard(QUERCETIN)

Fig:3 Total Flavanoides of Methanolic Extract of root of Pandanus unipapillatus

DENNST.

STANDARD

EXTRACT

0

0.5

1

1.5

2

20 40 60 80

Abs

orba

nce

at 6

50 N

M

Different concentrations of extract and standard(Quercetin)

Fig:4 Total Flavanoids of Methanolic Extract of stem of Pandanus unipapillatus DENNST.

STANDARD

EXTRACT

2.1 Thin Layer Chromatography

For development of phenolic specific- TLC profiles for the parts of the plant, The solvent

system used is Toluene: Ethyl acetate (8:1) .The band will be vishualized under UV 365 NM.

The chromatographic plate (Fig: 1) shows that the phytochemical profile of the extracts

obtained by different parts of pandanus unipapilltus at stem(s) extract appears the blue

fluorescent single band of the plate bottom. At the moment compare to the stem extract(S) to

the Leaf extract (L) shown low intensity blue fluorescent band situated at the same hight of

gallic acid (standard) were detected, indicates the presence of the Phenolic compound in the

extracts. The phenolic compounds very high in the stem parts. But in visible spectrum no

band will be appeared. It will prove some class of phenolic compounds is present in the stem

part of P.unipappillatus.

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The phenolic compounds contribute largely to the colour and sensory characteristics of fruits

and vegetables. In addition, phenols participate in growth and reproduction processes, and

provide protection against pathogens and predators.

Std S L

Fig: 1 TLC of Methanolic extracts of different parts of Pandanus unipapillatus

obtained by standard (Std.) (Gallic acid),stem(S) extract, Leaf extract(L).

Solvent system: Toluene: Ethyl acetate (8:1)

Detection: U.V 365 nm

CONCLUSION

From the present investigations described that among the Methanolic extracts of different

parts(stem, root,leaf) of Pandanus unipapillatus showed the presence of a various

phytochemicals. So the preliminary study confirms that the MEOH extracts of each part may

have active high Phenolic and Flavonoid compounds. To the determination of total phenolics

and total flavanoids content of various parts of plant confirms the MEOH extract of stem part

showed high Phenolic and flavanoid content. The result will be proved that the stem is the

more potent part of the plant. Further studies are carried out to assess the invitoantioxidant

and antimicrobial property of the MEOH stem extract of Pandanus unipapillatus.

This work contribute an insight to understanding some Class of compounds basis of

therapeutic properties of Pandanus unipapillatus in traditional medicine. Furthermore,

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Revealed studies on the isolation and characterization of the plant extract as well as in vivo

and invitro assays will be necessary in discovering new biological compounds.

ACKNOWLEDGMENT

The authors are grateful the management, Karpagam University, Coimbatore, TamilNadu,

India for providing research facilities and Department of Pharmacology, MISAT (Manian

Institute of Science and Technology) Coimbatore, TamilNadu-India.

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