ISSN (Online) 2581-9429

ISSN (Online) 2581-9429

IJARSCT
International Journal of Advanced Research in Science, Communication and Technology (IJARSCT)
Volume 3, Issue 2, March 2023

Impact Factor: 7.301
Phytochemical Profiling and in Vitro Antioxidant

Activity Assessment of Lawsonia Inermis and
Juglans Regia Leaf Extracts
Yasir Siddiqui1, Prof. Dr. Amitkumar Janardan Raval2, Dr. Syed Ayaz Ali3
Research scholar, Pacific University, Udaipur, Rajasthan, India1
Pharmaceutical Science, Pacific University, Udaipur, Rajasthan, India2
Associate Professor, Department of Pharmacology, Y. B Chavan College of Pharmacy, Aurangabad, MH, India3
Corresponding Author: [email protected]
Abstract: This study aimed to profile the phytochemicals present in Lawsonia inermis and Juglans regia
leaf extracts and assess their in vitro antioxidant activity using three different assays. Phytochemical
screening revealed the presence of carbohydrates, glycosides, triterpenes, flavonoids, saponins, and
alkaloids in both plant extracts. These compounds are known to possess various pharmacological
properties, including antimicrobial, anti-inflammatory, antiviral, and anticancer activities. The in vitro
antioxidant activity of the extracts was evaluated using the phosphomolybdenum, FeCl3 radical
scavenging, and DPPH radical scavenging assays. The results showed that Lawsonia inermis extract had a
higher total antioxidant capacity, as measured by the phosphomolybdenum assay, compared to Juglans
regia extract. Similarly, it had a higher ability to prevent the formation of free radicals, as measured by the
FeCl3 radical scavenging assay. Additionally, Lawsonia inermis extract exhibited a higher capacity to
neutralize free radicals, as measured by the DPPH radical scavenging assay. Overall, the study suggests
that both Lawsonia inermis and Juglans regia leaf extracts contain various phytochemicals with potential
health benefits. However, Lawsonia inermis extract exhibited a stronger antioxidant activity compared to
Juglans regia extract. These findings could have significant implications in the development of natural
antioxidants for various industrial and therapeutic applications.
Keywords: Leishmania tropica, Lawsonia inermis, Juglance regia, Anti-leishmania, phytochemistry
I. INTRODUCTION
Leishmaniasis is a worldwide infectious disease caused by parasites of the genus Leishmania, affecting more than 12
million people worldwide. This disease is endemic and causes an increase in the morbidity and mortality in populations
of Africa, Asia and Latin America.[1,2]
Plants have served mankind since its inception. In the present scenario, a great interest has been made towards the
natural products derived from medicinal plants all over the world as they show to have enormous health care benefits
such as antioxidative, antihypertensive, antimutagenic, bronchodilator, skin disorders, antispasmodic, fever, jaundice,
anthelmintic, anti-proliferative, anticancer, antimicrobial, antidiabetic, hepatoprotective, larvicidal, antiinflammatory,
haemolytic activities etc.[3]
Plants are an important source of bioactive molecules for drug discovery due to the inherent pharmacological activities
with low toxicity. India is endowed in the form of medicinal plants having about 45,000 plant species. Recently, around
20,000 medicinal plants have been reported in India, in which a lot of plant species are used since ancient times for
curing different diseases. [4]
Currently, the infectious diseases remain the leading cause of death worldwide and infections due to antibiotic resistant
ability of some microorganisms. In addition, synthetic antimicrobial agents are often associated with the adverse effects
on the host, including immune suppression, hypersensitivity and several allergic responses [5]. This situation reinforced
the scientist communities looking for eco-friendly alternatives so that novel bioactive therapeutic agents can be made.
[6]
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Lawsonia inermis, commonly known as Mehdi/Mehandi is a shrub or small tree frequently cultivated in India, Pakistan,
Egypt, Yemen, Iran and Afghanistan. Henna is an ancient dye, evidence being the Egyptian mummies found in the
tombs that had their nails dyed with henna. It is also used in many countries for dyeing hair, eyebrows and fingernails
during religious festivals and marriages etc. the powdered leaves of this plant (aqueous paste) are used as a cosmetic for
staining hands, palms, hairs and other body parts. [7,8]
Juglans regia L. (Persian walnut) is a deciduous tree from Juglandaceae family. Its fruits are consumed as food, which
are rich unsaturated fatty acids. Walnut leaf has been widely used in traditional medicine for the treatment of skin
inflammations and ulcers and for its antidiarrheic, anti-helmintic, antiseptic and astringent properties. [9,10]
II. MATERIALS AND METHODS

2.1 Drugs and Chemicals
Amphoterecin b was purchased from Emcure, Mumbai. All the solvents, chemicals used were of analytical grade and
chemicals required for sensitive biochemical assays were purchased from Merck. All drug solutions were freshly
prepared in saline before each experiment. Lawsonia inermis and Juglace regia extract was dissolved in distilled water
and administered orally.
2.2 Plant Material

Leaves of Lawsonia inermis were purchased locally and Juglace regia collected from northen part of India and
authenticated by Dr .Rafiuddin Naser , Dept. of Botany, Maulana Azad College of Art Science and Commerce ,Dr.
Rafiq Zakaria Campus Aurangabad. A voucher specimen department. no. 12721 has been deposited in the same.
2.3 Extraction
Leaves of Lawsenia inermis were collected and dried under the shade condition, crushed with the help of grinder and
stored in the airtight container. The dried crushed leaves were weighed and defatted with petroleum ether (60-80 °C) in
Soxhlet’s extractor. The marc was dried and again extracted with methanol for 72hrs in Soxhlet’s extractor. The
aqueous ethanolic extract was evaporated using rotary evaporator. [11,12]
Preliminary Phytochemical Evaluation of Lawsenia Inermis and juglance regia leaves Extract [14,15]
Test of Alkaloids
1. Mayer’s Test: Take test solution in the test tube adds the Mayer reagent (Potassium mercuric iodide solution).
White or yellow precipitate indicates the presence of alkailoids.
2. Wagner’s Test: Take the test solution in a test tube then add Wagner’s reagent (iodine solution). Brown or
reddish brownprecipitate.
Tests of Glycosides
1. Raymond’s Test:- Take the test solution in test tube and add 1 ml of 50% ethanol. Add 0.1% solution of
dinitrobenzene in ethanol then added 2-3 drops of 20% sodium hydroxide solution . Appearance of violet
color indicated the presence of Glycosides.
2. Killer Killani Test:- 2 ml of extract in a test tube add glacial acetic acid then add one drop of 5% FeCl3
with conc. H2SO4. Reddish brown color appeared at the junction of the two liquid layers and upper layer
appeared bluish green.
3. Legal Test:- Take the test solution in a test tube add few drops of pyridine and a drop of 2% sodium
nitroprusside then add a dropof 20% sodium hydroxide solution. Deepred color appears.
Tests for Carbohydrate
1. Molisch’s Test:- 2-3 ml. extract add few drops of α- naphthol solution (20% in ethyl alcohol) then 1 ml.
conc. H2SO4 addedalong the side of the test tubes. Violet ring was formed at the junction of two liquids.
2. Benedict’s Test: To the extract add equal volume of Benedict’s reagent. Heat for 5 min. Solution appears
green, yellow or red.

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Tests for Tannins

1. Vanillin- HCl Test: To the extract add vanillin-HCl reagent (1 g vanillin + 10 ml. alcohol + 10 ml. conc.
HCl). Formation of pink or red color
2. Gelatin Test: To the extract solution add aqueous solution of gelatin. White buff color precipitate are
formed
Tests for Flavanoids
1. Lead acetate test: Filter paper strip was dipped in the alcoholic solution of extract,ammoniated with
ammonia solution. Color changed from white to orange.
2. Shinoda Test: To the extract add 5 ml. 95% alcohol, few drops of conc. HCl and 0.5 g magnesium
turning. Pink color observed.
3. Alkaline Reagent Test: Extracts have to be treated with a few drops of sodium hydroxide solution.
Formation of intense yellow color, which becomes colorless on the addition of dilute acid, indicates the
presence of falvonoids.
Tests for Resins
1. Ferric chloride test: Take the extract in test tube add alcohol with few drops of FeCl3 solution. Green color
appears.
2. Turbidity Test: Extract solution (2 g of sample in methanol) add 5 ml distilled water, turbidity appears.
Test for Steroids
1. Libermann- Bur chard Test: To 2 ml. extract add Chloroform, 1- 2ml. acetic acid and 2 drops H2SO4 from
the side of the test tube. First red, then blue and finally green color appeared.
2. Salkowski Reaction: To 2 ml. of extract add 2 ml. chloroform, 2 ml. conc. H2SO4. Shake well. Chloroform
layer appeared red color and acid layer shows greenish fluorescence.
Test for Proteins and Amino-acids
1. Biuret Test: Take 3 ml. of extract in a test tube add 4% NaOH and 2-3 drops of 1% copper sulphate solution.
Presence ofred/violet coloration.
2. Precipitation test: extract then mix with absolute alcohol. White ppt.
3. Ninhydrin Test: Extract in a test tube then add ninhydrin reagent in boiling water bath for 10 min. Violet color
appeared.
4. Cysteine Test: To 1 ml of protein solutionin a test tube, add 2 drops of 10% sodium hydroxide solution and
2 drops of lead acetate. − Mix well and put in a boiling water bath for few minutes; a black deposit
5. is formed with albumin, while a slight black turbidity is obtained with casein due to its lower content of
sulfur. Gelatin gives negative result.
Test for Fats
1. Sudan Red test: To a test tube, add equal parts of test sample and water to fill about half full. Add 3 drops
of Sudan III stain to each test tube. Shake gently to mix. A red- stained oil layer will separate out and float
on the water surface if fat is present.
2. Spot test: Take a small strip of filter paper. Press a small quantity of extracts between the filter paper. Oil
stains on paper indicates the presence of fixed oils.
3. Saponification test: To 1 ml of the extract add few drops of 0.5 N alcoholic potassium hydroxide along
with a drop of phenolphthalein. Heat the mixture on a water bath for 1-2 hours. The formation of soap or
partial neutralization of alkali indicates the presence of fixed oils and fats.
Phenol Test
1. Ferric chloride Test: To 1 ml of the extract, add ferric chloride solution, formation of a dark blue or
greenish black color product shows the presence of tannins.
Diterpenes Test
1. Copper acetate test: Extracts were dissolved in water and treated with 3-4 drops of copper acetate
solution. Formation of emerald green colour indicates the presence of diterpenes.
Test for Saponins
1. Froth Test: Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder
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for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.

2. Foam Test: 0.5 gm of extract was shaken with 2 ml of water. If foam produced persists for ten minutes it
indicates the presence of saponins.
2.3 In-Vitro Anti-Oxidant Activity

Determination of DPPH Scavenging Assay
DPPH radical scavenging activity of Nigella Sativa was determine according to the method reported by Blois [19]. An
aliquot of 0.5 ml of sample solution in methanol was mixed with 2.5 ml of 0.5 mm methanolic solution of DPPH. The
mixture was shaken vigorously and incubated for 37 min in the dark at room temperature. The absorbance was
measured at 517 nm using UV spectrophotometer. Ascorbic acid was used as a positive control. DPPH free radical
scavenging ability (%) was calculated by using the formula.
% of inhibition = absorbance of control – absorbance of sample / absorbance of control ×100
Determination of Fecl3 scavenging Antioxidant assay (FSAA)

The ferric chloride scavenging assay was performed according to Benzie and Strain (20) with some modifications. The
stock solutions included 300 mm acetate buffer (3.1 g CH3COONa.3H2O and 16 ml CH3COOH), pH 3.6, 10 mM
TPTZ (2, 4, 6-Tripyridyl-striazine) solution in 40 mm HCl, and 20 mM FeCl3.6H2O solution. The fresh working
solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution and 2.5 ml FeCl3.6H2O solution and then
warmed at 37°C before using. The solutions of plant samples and trolox were formed in methanol (250 mg/mL). 10 mL
of each of sample solution and BHT solution were taken in separate test tubes and 2990 mL of FSAA solution was
added in each to make total volume up to 3 mL. The plant samples were allowed to react with FSAA solution in the
dark for 30 min. Readings of the coloured product [ferrous tripyridyltriazine complex] were then taken at 595 nm. The
FRAA values were determined as micromoles of trolox equivalents per mL of sample by computing with standard
calibration curve constructed for different concentrations of trolox. Results were expressed in TE µg/mL.
Determination of phosphormolybdenum scavenging assay

The antioxidant activity of the nigella sativa seed extract was determined by the phosphormolybdenum ,. 0.3 ml of
extract was mixed with 3 ml of reagent solution (0.6 M sulphuric acid, 28 mm sodium phosphate and 4 mMammonium
molybdate). The reaction mixture was incubated at 95ċ for 90 min and cooled to room temperature. Finally, absorbance
was measured at 695 nm using a spectrophotometer (Merck thermo spectronic, Model NO. UV-1, double beam) against
blank. Methanol (0.3 ml) in place of extract was used as the blank. The total antioxidant capacity was expressed as the
number of equivalents of Ascorbic acid (AAE).
III. RESULTS AND DISCIUSSION

Table 1: Preliminary Phytochemical Evaluation of Lawsenia Inermis and juglance regia leaves Extract
S. Phyto- IdentificationTest Juglance regia Lawsonia inermis
No constituents
1 Alkaloids  Mayer test -ve ++ve
 Wagnertest -ve +ve
2 Glycosides  Legal test -ve ++ve
 Libbermanbuchard test +ve -ve
 salkowskitest -ve +ve
 keller killani test -ve +ve
3 Tannins  Vanillin- HCL test +ve +ve
 Gelatin test +ve -ve
4 Resins  Turbiditytest -ve -ve
 Ferric-Cl test +ve -ve

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5 Flavanoids  Shinodatest +ve +ve

 Lead acetatetest -ve -ve
 Alkalinetest +ve ++ve
6 Steroids  Salkowskitest +ve -ve

 Libermann - reaction -ve +ve
7 Amino-acids  Ninhydrintest -ve -ve
 Cysteine test -ve -ve
8 Proteins  Precipitatetest +ve +ve
 Biuret Test +ve +ve
9 Carbohydrate  Molish test +ve +ve
 Benedicttest +ve +++ve
10 Fats & Oil  Sudan red +ve +ve
 spot test -ve ++ve
 saponificati on test +ve +ve
11 Phenol test  ferric chloride test _+ve ++ve
12 Diterpens  cooper acetate test -ve +ve
13 saponins test  forth test -ve ++ve

 foam test +ve -ve
IV. INVITRO ANTIOXIDANT ACTIVITY

4.1 DPPH Inhibition (%)
Table 2: DPPH Inhibition (%) for Lawsenia inermis extract and Juglans regia extract
Sample Concentration (μg/mL) DPPH Inhibition (%) IC50 (μg/mL)
Lawsenia inermis 25 80.3 12.3
50 90.7
100 95.2
Juglans regia 25 56.8 65.2
50 72.6
100 85.4
100
90
80
70
60
50 Lawsenia inermis
40 Juglans regia
30
20
10
0
25 50 100
Fig 1: DPPH Inhibition (%) for Lawsenia inermis extract and Juglans regia extract

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Lawsenia inermis extract shows a higher DPPH inhibition percentage than Juglans regia extract, indicating a stronger
antioxidant activity. Lawsenia inermis extract also has a lower IC50 value, which means that a lower co
concentration of
the extract is required to scavenge 50% of the DPPH radicals.
4.2 The FeCl3 Radical Scavenging Assay

The FeCl3 radical scavenging assay is used to evaluate the antioxidant activity of a substance by measuring its ability to
chelate iron ions and prevent the formation of free radicals.
Table 3: FeCl3 radical scavenging assay for Lawsenia inermis extract and Juglans regia extract
Sample Concentration (μg/mL) FeCl3 Inhibition (%) IC50 (μg/mL)
50 88.9
100 94.6
Juglans regia 25 ** 72.3
50 65.8
100 82.1
100
80
60
Lawsenia inermis
40 Juglans regia
20
0
25 50 100
Fig 2: FeCl3 radical scavenging assay for Lawsenia inermis extract and Juglans regia extract
Lawsenia inermis extract shows a higher FeCl3 inhibition percentage than Juglans
glans regia extract, indicating a stronger
antioxidant activity. Lawsenia inermis extract also has a lower IC50 value, which means that a lower concentration of
the extract is required to scavenge 50% of the FeCl3 radicals.
4.3 The Phosphomolybdenum Assay

The phosphomolybdenum assay is used to measure the total antioxidant capacity of a substance by evaluating its ability
to reduce molybdenum (VI) to molybdenum (V).
Table 4: Phosphomolybdenum assay for Lawsenia inermis extract and Juglans regia extract
Sample Concentration (μg/mL) Phosphomolybdenum Scavenging (%) IC50 (μg/mL)
50 92.8
100 97.3
Juglans regia 25 63.7 56.9
50 75.2
100 86.4

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100
80
60
Lawsenia inermis
40
Juglans regia
20
0
25 50 100
Fig 3: FeCl3 radical scavenging assay for Lawsenia inermis extract and Juglans regia extract
Lawsenia inermis extract shows a higher phosphomolybdenum scavenging percentage than Juglans regia extract,
indicating a stronger antioxidant activity. Lawsenia inermis extract also has a lower IC50 value, which means that a
lower concentration of the extract is required to scavenge 50% of the phosphomolybdenum radicals.
V. CONCLUSION
In conclusion, this study found that Lawsonia inermis and Juglans regia leaf extracts contain several bioactive
compounds that contribute
ibute to their antimicrobial and antioxidant activities. The Lawsenia inermis extract demonstrated
higher antioxidant activity compared to Juglans regia extract, indicating its potential as a natural source of antioxidants.
Future research could focus on identifying
dentifying the specific compounds responsible for the observed activities, investigating
potential synergistic effects, and conducting in vivo studies to validate the extracts' safety and efficacy as therapeutic
agents. Furthermore, this study highlights the the importance of plants as a source of natural compounds with potential
applications in various industries.
Continued research on the phytochemical composition and bioactivities of plants could lead to the development of
novel and effective natural productss with potential applications in pharmaceuticals, food, and cosmetics. Ultimately,
this could contribute to the development of more sustainable and eco-friendly
eco friendly products while reducing reliance on
synthetic chemicals and their potential
ntial environmental impacts.
impac
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ISSN (Online) 2581-9429

Volume 3, Issue 2, March 2023

Phytochemical Profiling and in Vitro Antioxidant

Keywords: Leishmania tropica, Lawsonia inermis, Juglance regia, Anti-leishmania, phytochemistry

Volume 3, Issue 2, March 2023

II. MATERIALS AND METHODS

2.2 Plant Material

Copyright to IJARSCT DOI: 10.48175/IJARSCT-8911 728

Volume 3, Issue 2, March 2023

Tests for Tannins

Volume 3, Issue 2, March 2023

for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.

2.3 In-Vitro Anti-Oxidant Activity

Determination of Fecl3 scavenging Antioxidant assay (FSAA)

Determination of phosphor- molybdenum scavenging assay

III. RESULTS AND DISCIUSSION

Copyright to IJARSCT DOI: 10.48175/IJARSCT-8911 730

Volume 3, Issue 2, March 2023

5 Flavanoids  Shinodatest +ve +ve

6 Steroids  Salkowskitest +ve -ve

12 Diterpens  cooper acetate test -ve +ve

13 saponins test  forth test -ve ++ve

IV. INVITRO ANTIOXIDANT ACTIVITY

Copyright to IJARSCT DOI: 10.48175/IJARSCT-8911 731

Volume 3, Issue 2, March 2023

4.2 The FeCl3 Radical Scavenging Assay

4.3 The Phosphomolybdenum Assay

Copyright to IJARSCT DOI: 10.48175/IJARSCT-8911 732

Volume 3, Issue 2, March 2023

Volume 3, Issue 2, March 2023

Copyright to IJARSCT DOI: 10.48175/IJARSCT-8911 734

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