Post on 26-Jan-2022
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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.
kmtmanjula@gmail.com
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Manjula K T et al. World Journal of Pharmaceutical Research
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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