PHYTOCHEMICAL ANALYSIS OF Lepidium didymum

Abstract

Phytochemical screening is a chief phase, which results in the isolation of novel and new compounds. In the current study, Lepidium didymum roots, stems and leaves extracts in N-hexane, ethanol and water were examined for phytochemicals in order to investigate various groups of phytochemicals. Secondary metabolites like cardiac glycosides, phlobatannins, tannins, steroids, flavonoids, saponins, terpenoids, proteins and carbohydrates in Lepidium didymum were investigated. N-hexane, ethanolic and Aqueous extracts of stems revealed the presence of flavonoids, carbohydrates, anthra-glycosides and saponins. Likewise, the N-hexane ethanolic, and aqueous extracts of roots revealed the existance of carbohydrates, terpenoids, anthra-glycosides, cardiac glycosides and saponins. Phlobatannins and Hager were not present from the given extracts in the plant. Hence, it was concluded that Lepidium didymum may be employed in medicinal purposes as it is rich in secondary metabolites.

Full text

Pak. J. Weed Sci. Res., 23(2): 155-164, 2017
PHYTOCHEMICAL ANALYSIS OF Lepidium didymum
Wajidullah
1
, Naveed Akhtar*, 1, Syed Shaukat Ali 2, Sajjad Ahmed1,
Samin Jan1, Barkatullah1, Muhammad Azim Khan3 and Muhammad
Saleem Khan1
ABSTRACT
Phytochemical screening is a chief phase, which results in the
isolation of novel and new compounds. In the current study, Lepidium
didymum roots, stems and leaves extracts in N-hexane, ethanol and
water were examined for phytochemicals in order to investigate
various groups of phytochemicals. Secondary metabolites like cardiac
glycosides, phlobatannins, tannins, steroids, flavonoids, saponins,
terpenoids, proteins and carbohydrates in Lepidium didymum were
investigated. N-hexane, ethanolic and Aqueous extracts of stems
revealed the presence of flavonoids, carbohydrates, anthra-glycosides
and saponins. Likewise, the N-hexane ethanolic, and aqueous extracts
of roots revealed the existance of carbohydrates, terpenoids, anthra-
glycosides, cardiac glycosides and saponins. Phlobatannins and Hager
were not present from the given extracts in the plant. Hence, it was
concluded that Lepidium didymum may be employed in medicinal
purposes as it is rich in secondary metabolites.
Key words: Carbohydrates, cardiac glycosides, Lepidium, saponins,
terpenoids.
Citation: Wajidullah, N. Akhtar, S.S. Ali, S. Ahmed, S. Jan,
Barkatullah, M.A. Khan and M.S. Khan. 2017. Phytochemical analysis
of Lepidium didymum. Pak. J. Weed Sci. Res. 22(2): 155-164.
INTRODUCTION
Medicinal plants have a great role in improvement of health of
community and individuals. Since ancient times, the world has relied
upon the use of herbal drugs for the treatment of different type of
diseases across (Prajapati and Prajapati, 2002; Shinwari et al., 2006).
Over 80% of the population in the developing world depends upon the
traditional medicinal plants extracts for the provision of health
coverage (Fransworth and Soejarto, 1998; Latif et al., 2003). Study of
1
Dept. of Botany, Islamia College, Peshawar, Pakistan
2Dept. of Chemistry, Islamia College, Peshawar, Pakistan
3Dept. of Weed Science, The University of Agriculture, Peshawar,
Pakistan
*Corresponding author’s email: n.akhtar@icp.edu.pk

Wajidullah et al., Phytochemical analysis of Lepidium ...
2
medicinal plants and the invention of synthetic drugs is much needed,
considering the indication of AIDS (acquired immune deficiency
syndrome) like fatal diseases and the threats of newly arising diseases
like bird flu and SARS (Severe acute respiratory syndrome) etc.
Fractional plants and plant extracts have been a good source of natural
phytochemicals and herbal medicines over the years (Robert and
Lewis, 1998; Brodie, 2010). Data obtained from various studies
showed that an estimated thirty five thousand to seventy thousand
species of plants are used in folk medicines worldwide (Lewington,
1990). Lepidium L. is a large genus of about 250 species, cosmopolitan
in distribution on all continents excepting Antarctica. Lepidium belongs
to Brassicaceae, the Mustard family. One of the representative of the
genus is L. didymum (common name lesser swine-cress), which is a
herbaceous annual to biennial plant. The continent of South America is
the site from where the origination and geographic distribution of L.
didymum occurred. Lepidium plant is known very well for the
production of a variety of natural products that are biologically active.
These products include flavonoid, coumarins, phlobatannins and
terpenoids etc (Kashani et al., 2012)
Species of genus Lepidium are well reputed medicinally and
work as folk medicinal remedies for healing of various diseases
(Abebe, 2013). In India, traditional medicine has valued the plant as a
source for the treatment of wounds and allergies. The use of its seeds
improves lung function in asthmatics thereby alleviating the asthma
symptoms. In Mauritius, the application of Lepidium sativum leaf
poultice externally on the head and the consumption of the decoction
of the whole plant orally has been used for the treatment of headache.
Oral consumption of the decoction also helps in relieving of fever
(Archana and Anita, 2006). Research data have revealed the efficacy
of L. didymum leaves for the treatment of other skin disease and
diabetes. The present study was undertaken to screen different parts
of L. didymum for qualitative chemical analysis so that these
phytochemicals can be further quantified.
MATERIALS AND METHODS
Chemicals
N-hexane, Distilled water, chloroform, ethyl acetate, olive oil,
ferric chloride, ammonia, hydrochloric acid, sulphuric acid, lead,
benzene, ethanol, acetate, Hager reagent, glacial acetic acid, nephtol
solution, ferric chloride, million reagent and iodine solution.
Glassware
Funnel, bottle, graduated cylinder, beakers, tripod stand, test
tubes, beakers, pipette, spirit lamp, test tubes tray, stirrer and
aluminum foil.
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Pak. J. Weed Sci. Res., 23(2): 155-164, 2017
3
Plant collection
Leaves, stems and roots of Lepidium didymum were collected
during October, 2015, from Pakha-ghullam area (ring road) in district
Peshawar. Dr. Naveed Akhtar, Plant taxonomist, (Department of
Botany, Islamia Collage, Peshawar, Pakistan) identified and
characterized the plant to be Lepidium didymum L.
Extraction
The plant material was dried for one month in shade at room
temperature. The dried plant material (roots, stems and leaves) of
Lepidium didymum were crushed into a fine powder. The powdered
material was soaked for 3 days in n-hexane, water and ethanol and
subjected to extraction till plant material exhaustion.
Phytochemical investigation
Different tests were performed on the water, n- hexane and
ethanol extracts of roots, stems and leaves of Lepidium didymum
following the methods of Ali et al. (2014).
1. Reducing sugar (Benedict test)
Benedict reagents (2.5ml) and test solution (5ml) were heated
for 5 minutes in water bath. Depending on amount of reducing sugar
in the solution, the colour of the test solution changed to red, yellow or
green (Ali et al., 2014).
2. Flavonoid test
In this test, 3 ml of test solutions was taken and 5ml of NH3
solution was added to it, followed by the addition of conc. HCl. The
appearance of a yellowish color showed the flavonoids to be present.
3. Tannins test (lead acetate test)
3ml of lead acetate were mixed in a test tube with 3ml extract,
formation of the precipitate marked the presence of tannins.
4. Terpenoids test
The appearance of reddish brown color, after the mixing of
about 5 ml of the test solution, 2 ml of chloroform and 3 ml of
concentrated H2SO4, showed the terpenoids to be present.
5. Phlobatannins test
Test solution (3ml) was mixed with 1% HCl, formation of red
colored precipitate indicated the phlobatannins presence in the test
solution.
6. Steroids test (Salkowski’s reaction)
Test solution (2ml) was mixed with 2ml conc. H2SO4 and 2ml
chloroform and shacked well. The acid layer showed yellow greenish
florescence while chloroform layer appeared red indicating the
presence of steroids.
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Wajidullah et al., Phytochemical analysis of Lepidium ...
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7. Saponins test
Test solution (10 ml) was added to distilled water (5ml) and
vigorously shaken. Thereafter, 3 drops of olive oil were added to it and
shaken again. Formation of emulsion showed the existence of
saponins.
8. Cardiac glycoside test
Here 2ml of glacial acetic acid containing 1 drop of ferric
chloride solution was added to about 5 ml of test solution. Under layer
of 1ml of concentrated H2SO4 formed violet or brown color which
indicated a deoxy-sugar, characteristic of cardinolides sugars.
9. Anthraquinone glycoside (modified Borndragers test)
A few drops of ferric chloride and 5ml of dilute hydrochloric acid
was added to 5 ml of test solution and heated for one minute in water
bath then cooled. After cooling 2 ml of benzene were added to it and
shaken for the separation of organic layer. An equal amount of NH3
was then added to the solution. Presence of anthraquinone glycoside
was indicated by red coloration.
10. Test for alkaloid (Hager’s test)
Few drop of Hager mixture was added to 3ml of test solution
and mixed. Areddish brown coloration showed the presence of alkaloid.
11. Carbohydrate test (Molish test)
About 3ml of alpha nephtol solution were added into 3 ml of
test solution. After vigorous shaking, conc. sulphuric acid was added
from the side of test tube. Presence of carbohydrates was shown by
the appearance of violet coloration.
12. Starch test (Non-reducing sugar)
Three (3) ml of test solution were added to a few drops of
iodine solution. A blue color appeared and vanished on steaming which
showed the presence of starch.
13. Proteins test (Millions test)
About 5 ml of million reagents was added to 3ml of test solution
and mixed, a white precipitate, which turned brick red on heating, was
formed. This indicated the presence of proteins.
14. Anthocyanin test
Here test solution (2ml) was mixed to 2ml of ammonia and
hydrochloric acid. The pink red color of the mixture turned into blue
violet color which indicated the existence of anthocyanin.
15. Coumarins test ( zohra et al. 2012)
About 3 ml of ten percent ammonium hydroxide was mixed
with 2 ml of test solution which resulted in the formation of yellow
color; this indicated the presence of coumarins.
16. Emodins test (Zohra et al. 2012)
3 ml of test solution was mixed with 2 ml of NH4OH and 3 ml of
benzene. Appearance of red color indicated that emodins are present.
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Pak. J. Weed Sci. Res., 23(2): 155-164, 2017
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Table-1: Qualitative phytochemical results of Lepidium didymum
roots, stem and leaves in N-hexane, ethanol and water extracts
Component
s
Roots
Stem
Leaves
N-
Hexa
ne
Ethan
ol
Wat
er
N-
Hexa
ne
Ethan
ol
Wat
er
N-
Hexa
ne
Ethan
ol
Wat
er
Anthocyani
n
-
-
-
-
-
-
-
+++
++
Anthra-
glycosides
+
++
+
+
++
++
+++
+
+
Benedict
-
++
++
-
+
-
-
++
++
Carbohydra
tes
+++
++
++
+++
++
++
+++
++
+++
Cardiac
glycosides
+++
++
+
+
+++
-
+
+
-
Coumarins
-
+++
+
-
+++
++
+
+
++
Emodins
-
-
-
-
-
-
-
+
+
Flavonoids
-
++
+
++
+++
+
+
++
+++
Hager
-
-
-
-
-
-
-
-
-
Phlobatanni
ns
-
-
-
-
-
-
-
-
-
Proteins
-
-
+++
-
-
++
-
-
+++
Saponins
+++
++
++
++
++
++
+++
-
++
Starch
-
-
-
-
-
-
-
-
-
Steroids
-
-
-
-
+
-
-
++
-
Tannins
-
+++
-
-
+
+
+
+
++
Terpenoids
+
++
+
++
++
-
-
-
-
- = absence of phytochemicals; += presence of phytochemicals;
++ = moderate concentration; +++ = high concentration
RESULTS AND DISCUSSION
Table-1 represents the qualitative phytochemical results of
Lepidium didymum roots, stem and leaves extracts in the N-hexane,
ethanol and water. These results showed different compounds in L.
didymum that are medicinally active. Anthraquinone glycosides and
Carbohydrates were present in all parts of the plant (Table-1). Hager,
phlobatannins and starch were, however, absent at the given extracts
in the plant. In N-hexane extract Benedict test was negative for the
whole plant while positive in ethanolic and aqueous extracts of roots
and leaves.
Tests for proteins were positive in the roots, stems and leaves
only in the aqueous extracts. Tests for steroids were positive for stem
and leaves in the ethanolic extract only. Emodins and anthocyanin
were absent at all extracts in roots and stems. Except the ethanolic
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Wajidullah et al., Phytochemical analysis of Lepidium ...
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extract from leaves, saponins were present at all extracts in all the
plant parts. Tannins tests were positive in all leaves extracts however,
it was negative in the N-hexane extract in roots and stems as well as
water extract in roots. The table showed that except Hager,
phlobatannins and starch, various plant organs showed positive results
for many phytochemicals that are medicinally active.
Figure 1. Phytochemicals of roots extracts in ethanol, N-hexane and
water. Each bar represents the conc. of coloration for the
phytochemicals in these extracts.
Figure 2. Phytochemicals of stems extracts in ethanol, N-hexane and
water. Each bar represents the conc. of coloration for the
phytochemicals in these extracts.
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Pak. J. Weed Sci. Res., 23(2): 155-164, 2017
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Figure 3. Phytochemicals of leaves extracts in ethanol, N-hexane and
water. Each bar represents the conc. of coloration for the
phytochemicals in these extracts.
Phytochemicals exploration of the plant extracts has shown the
presence of medicinally as well as physiologically active compounds
(Sofowora, 1993). Qualitative phytochemical screening test of crude
extracts in methanol/chloroform has shown the presence of
phytochemicals like carbohydrates, cholesterol, terpenoids, flavonoid,
glycosides, steroids, phytosterols, tannins, phenols, proteins, saponins
and alkaloids in different plant species (Berehe and Boru, 2014).
Brassica genus is known to contain different phytochemicals like
carbohydrates, alkaloids, glycosides, flavonoids, proteins and tannins
(Talreja and Moon, 2014).
Examination of L. didymum extracts showed the presence of
various phytochemicals like flavonoids, tannins, phenols, glycosides,
saponins, terpenoids and steroids. One of the major groups of plant
metabolites are phenolic compounds (Singh et al., 2007). According to
Han et al. (2007), phenolic compounds have biological assets such as
antiaging, anti-apoptosis, anti atherosclerosis, anti-inflammation, anti-
carcinogenic, cardiovascular protection and enhancement in
endothelial functions, as well as angiogenesis events and inhibition of
cell proliferation. Medicinal plants contain phenolic compounds which
have antioxidant properties (Krings and Berger, 2001; Brown & Rice-
Evans, 1998). Natural antioxidant major source is from medicinal
plants, which are present as phenolic compounds like flavonoids,
tocopherols and phenolic acids, etc. (Ali et al., 2008). Tannins are
known to bind to proteins that are rich in proline thereby affecting
protein synthesis. Plants in reaction to microbial infection produce
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Wajidullah et al., Phytochemical analysis of Lepidium ...
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hydroxylated phenolic substance called flavonoids, which when used in
vitro, show antimicrobial properties against wide array of
microorganisms. Flavonoids shows their antimicrobial properties by
forming complexes with bacterial cell wall as well as extracellular and
soluble proteins (Marjorie, 1996). They are antioxidant and display
strong anticancer activities (Okwu, 2004; Benavente-García et al.,
1997; Salah et al., 1995).
Saponins, which are renowned for their inhibitory effect against
inflammation, were also reported from the plant extracts (Just et al.,
1998). Saponins are also shown to be coagulating and precipitating
red blood cells. Other characteristics shown by some saponins iclude
creation of foams in aqueous solutions, hemolytic activity, cholesterol
binding properties and bitterness (Sodipo et al., 2000). Steroids
compounds are also of significance because of the ability to complex
with sex hormones (Okwu, 2001), and antibacterial activity (Raquel,
2007). Cardiac glycosides, reported in plant extracts have been
employed for centuries as stimulants in the cases of cardiac failures
(Trease and Evans, 2001). Terpenoids were also found in L. didymum
especially in stems and roots. These are known for having
antimicrobial potential (Habtemariam, 1993). Hence presence of
terpenoids in the roots provides protection against bacterial infections.
CONCLUSION
Data obtained from the present study reveals that L. didymum
is a rich source of phytochemical compounds. The presence of such a
large array of bioactive constituents enables plant for its significant
medicinal value. Our present facts and figures are in accordance with
the literature, which confirms that those phytochemicals have strong
effects on physiology and hence used in cure of different diseases. The
practice of customary medication is strongly suggested for L.
didymum. More work is recommended for the isolation, purification
and characterization of those medicinally active constituents which are
medicinally active.
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