PYOCHEMICAL SCREENING, ANALYSIS AND ANTIMICROBIAL ACTIVITY OF

SOLVENT EXTRACTS OF WRIGHTIA TINCTORIA

VIGNESH S S , REDHANYA V K AND DR DEVAKUMAR J
UG and Research Department of microbiology
Dr. N. G.P. P Arts and Science College, Coimbatore -641048.

ABSTRACT

A plant with a long history of use in medicine, Wrightia tinctoria, continues to attract
scientific interest because of its wide range of secondary metabolites. The plant's medicinal
potential is enhanced by these bioactive substances, which include terpenoids, glycosides,
alkaloids, and flavonoids. To maximise yield and bio efficacy, this study investigates the
generation of secondary metabolites using a variety of extraction and cultivation techniques.
Significant antioxidant, anti-inflammatory, and antibacterial properties are revealed by
thorough phytochemical screening and active assays, underscoring W. tinctoria's
pharmacological worth. This study highlights the plant's potential as a sustainable supply of
natural medicinal compounds by fusing traditional knowledge with contemporary analytical
methods. Gas chromatography-mass spectroscopy (GC-MS) is a combined analytical
technique used to determine and identify compounds and phytochemical analysis Using
conventional qualitative techniques, this study was done to identify the secondary
metabolitesgroup identify phytochemical composition.position.

INTRODUCTION
Ivory tree, Sweet indrajao (English) is a small decidious tree which belong to the family
Apocynaceae.the botanical name of Ivory tree is Wrightia tinctoria.In India, it is locally
recognized by its different vernacular names, the most commonly used ones are Indrajava,
Svetkutaja, Krsnkutaja (Sanskrit), Kalakuada (Marathi) and Mitha indrajau (Hindi). The
whole plant or its specific parts (bark, leaf, seed and root) are known to have medicinal
properties and have a long history of use by indigenous communities in India. The medicinal
value of this plant for the treatment of many human ailments is mentioned in Ayurveda,
Siddha. It is commonly used for skin diseases, jaundice etc., In folk medicine it is used for
some breast related disorders. Most of the herbal drugs are having good immune response
and to prevent the diseases. Wrightia tinctoria is very effective jaundice plant in Indian
indigenous system of medicine. The juice of the tender leaves is used efficaciously in
jaundice and crushed fresh leaves when filled in the cavity of decayed tooth relieve
toothache. The juice from fresh unripe fruits is used for coagulating milk. The seeds are said
to be aphrodisiac and anthelminthic. The leaves are used to relieve toothache when chewed
with salt. In Nepal, the milky juice is used to stop bleeding. Also, the leaves and roots are
pounded in water for treatment of fever. The seeds yield deep red, semi-drying oil, which has
medicinal value. The presence of lipid, saponin, tannin, alkaloid, phenol, steroid, flavonoid,
and some other chemical constituents are observed.

MATERIALS AND METHODS

Source of the Plant material Fresh healthy leaves of Wrightia tinctoria, were collected
from the locality near the Coimbatore, Tamil Nadu, India.

INSTRUMENTS AND CHEMICAL

FTIR analysis of Wrightia tinctoria extract has been used to identify functional groups
present in its phytochemical composition, The GC-MS[Gas chromatography-mass
spectroscopy] analysis of ethanolic extract of Wrighti tinctoria flowers gives a fingerprint for
the identification and quantification of phytochemical present.petroeum ether,n-
butanol,carbinol,Acetone, Alkaloids.

1.Sample Preparation for Phytochemical analysis

Healthy leaf samples were washed thoroughly 2-3 times with running tap water and
followed by sterile water, shade-dried and ground into uniform powder, extracted in distilled
water and organic solvents like Petroleum ether,N-butanol,Acetone and carbinol for 24 hr.
The extract was filtered using Whatman No. 1 filter paper and used for the screening of
phytochemicals. Qualitative tests were carried out using solvent extract and powdered sample
according to standard procedures for identification of major secondary metabolites such as
alkaloids, Tannis, saponins, flavonoids and phenolic compounds.

1.1Preparation for water and solvent extractions

Fifty grams of shade dried, leaf powder of W. tinctoria was extracted in 200 ml of each
petroleum ether, N-butanol, acetone, carbinol and water using a Soxhlet extractor for 48
hours. The collected extracts were concentrated and stored in refrigerator at 4° C for further
use.

2.Plant Pathogens

Pure isolates of bacteria such as E.coli,S.aureus,Pseudomonas,Klebsiella and

Enterobacter were used for the antibacterial assay.

3.Antibacterial activity assay

Antibacterial activity of solvent extracts was determined by well diffusion

method.Muller Hinton Agar were prepared and poured in a sterile petri dish. After
solidification, the bacterial cultures were inoculated by cotton swab method. Wells were
punched through well puncher and for each organisms the extracted solvents were added at
different concentrations such as 50 µl,20 µl, positive and negative control. Streptomycin
antibiotic is used as positive control and distilled water is used as a negative control. Samples
were added and incubated at 37°c for 24 hours. After incubation the plates were observed for
zone of incubation.

3.1Antifungal activity assay

Antifungal activity of solvent extracts were determined by well diffusion

method.Sabouraud Dextrose Agar were prepared and poured in a sterile test tubes and fungal
sample such as Fusarium were added and the solvent extracts were added in the well at
different concentrations such as 25 µl,50 µl,75 µl,125 µl and 150 µl.The plates were incubated
at room temperature for 3-5 days.

4. ANALYSIS OF PHYTOCHEMICALS

4.1Analysis of Qualitative Phytochemical Components

Preliminary qualitative phytochemical analysis was done to determine if secondary

metabolites were present or absent in solvent extracts.

4.2 Test for Alkaloids: Wagner’s Test

Each extract was put into a test tube with a volume of around 2 mL. Wagner’s reagent was
applied, followed by 2 mL of diluted 1% HCL, and incubated for 15 min. Alkaloids are present when a
reddish-brown precipitate forms. Iodine (1.3 g) and potassium iodide (2.0 g) were combined to form
a stock solution of Wagner’s reagent in 100 m (about 328.08 ft) L of distilled water. The mixture was
placed in an amber container for storage.

4.3 Test for Flavonoids

A test tube containing 5 mL of each extract was Test for Flavonoids: Alkaline Reagent
Test Two milliliters (2 mL) of weak NaOH was added to 3 mL of each extract in test tubes.
Flavonoids are present when a strong yellow coloration develops. 4.25 g of NaOH was
dissolved in 50 mL of distilled water to obtain the diluted NaOH.

4.4 Test for Phenolics

Three drops of this combination were added to 3 mL of each extract, along with a
mixture of 10% ferric chloride and 10% ferrocyanide. The precipitate’s orange-brown color
indicates the presence of phenolics. 10 mg of ferric chloride and 10 mg of ferrocyanide were
dissolved in 100 mL of distilled water to create a working solution, and a ferric chloride test
was performed.

4.5 Test for saponins: froth test

A test tube containing 1 ml (about 0.03 oz) of extract were taken and mixed with 1%
lead acetate. Saponin is present when the extract forms white precipitate.1% lead acetate is
prepared by dissolving 1g of lead acetate in 100 ml (about 3.38 oz) of water

4.6Test for Tannis

A test tube containing 5 ml (about 0.17 oz) of solvent extracts were taken and mixed
with 10% lead acetate. Tannis is present when the extract forms yellow precipitate.10%
lead acetate is prepared by dissolving 10g of lead acetate in 100 ml (about 3.38 oz) of
distilled water.

RESULTS

Phytochemical Screening

The preliminary phytochemical screening indicated ethyl acetate, methanol and water
leaf extracts of Wrightia tinctoria showed the presence and absence of bioactive constituents
such as alkaloids, Tannis, saponins,flavanoids, saponin and phenolic compounds (Table 1).
These substances are said to be responsible for the antimicrobial activity of this plant.
TABLE 1: QUALITATIVE PHYTOCHEMICAL ANALYSIS OF WRIGHTIA
TINCTORIA
Phytochemical Petroleum N-butanol Carbinol Acetone Water
compound ether

Alkaloids - +++ +++ ++ +

Tannis - - - - ++

Saponins + +++ - - -

Flavonoids - + +++ - -

Phenolic - - - - -

5.ANTIBACTERIAL ACTIVITY OF LEAF EXTRACT OF WRIGHTIA TINCTORIA

The result of the antibacterial screening crude leaf extracts is shown in the Table 2. The maximum
inhibitory activity was observed in acetone extract followed by N-butanol extract and petroleum ether
extract against the growth of tested pathogenic bacteria such as Escherichia coli, Staphylococcus
aureus, Pseudomonas sp, Klebsiella sp and Enterobacter sp. Carbinol extracts shows low zone of
incubation which indicates the low inhibition against the microorganisms. The result of the
antibacterial screening crude leaf extracts is shown in the Table 2. The maximum inhibitory
activity was observed in acetone extract followed by N-butanol extract and petroleum ether
extract against the growth of tested pathogenic bacteria such as Escherichia coli,
Staphylococcus aureus, Pseudomonas sp, Klebsiella sp and Enterobacter sp. Carbinol extracts
shows low zone of incubation which indicates the low inhibition against the microorganisms.
Figures 1,2 and 3 demonstrates the Antibacterial activity of Wrightia tinctoria flower extract.

TABLE 2: ANTIBACTERIAL ACTIVITY OF WRIGHTIA TINCTORIA SOLVENT

EXTRACTS

ORGANIS C PETROLEU N- ACETON CARBINO

M CONCENTRATION M ETHER BUTANO E L
S OF THE L
SOLVENT
EXTRACTS

Escherichia 50 µl No zone No zone No zone No zone

coli
20 µl No zone 1.2 mm No zone No zone

Positive 20 µl 2 mm 2 mm 2 mm 2 mm
 Negative 20 µl No zone No zone No zone No zone

Staphylococc 50 µl 0.5 mm 0.6 mm 1 mm 0.1 mm

us aureus
20 µl No zone No zone 0.5 mm No zone

Positive 20 µl 2 mm 2 mm 2 mm 2 mm

Negative 20 µl No zone No zone No zone No zone

Pseudomonas 50 µl No zone 0.1 mm 0.5 mm 0.1 mm

20 µl No zone No zone No zone No zone

Positive 20 µl 2 mm 2 mm 2 mm 2 mm

Negative 20 µl No zone No zone No zone No zone

Klebsiella 50 µl 0.3 mm 0.5 mm No zone 1.5 mm

20 µl No zone No zone No zone No zone

Positive 20 µl 2 mm 2 mm 2 mm 2 mm

Negative 20 µl No zone No zone No zone No zone

Enterobacter 50 µl 0.1 mm No zone No zone 0.5 mm

20 µl No zone No zone No zone No zone

Positive 20 µl 2 mm 2 mm 2 mm 2 mm

Negative 20 µl No zone No zone No zone No zone

ANTIBACTERIAL ACTIVITY IMAGES

Fig 1 n-butanol Fig 3carbinol

 Fig 2 petroleum

5.1 ANTIFUNGAL ACTIVITY ASSAY

The result for the antifungal activity of solvent extracts is shown in table 3. Fungal
sample Fusarium were used to check the inhibition of Fusarium against the solvent plant
extracts. The inhibition is high in highest concentration and the inhibition is low in the lowest
concentration. This indicates that in highest concentration of the plant extracts the inhibition
of the growth of Fusarium is high is high and Fungal sample Aspergillus were used to check
the inhibition of Aspergillus the solvent plant extracts. The inhibition is high in highest
concentration and the inhibition is low in the lowest concentration. This indicates that in
highest concentration of the plant extracts the inhibition of the growth of Aspergillus is
high Figures 4 and 5 demonstrates the Antifungal activity of Wrightia tinctoria flower extract.

TABLE 3: ANTIFUNGAL ACTIVITY OF WRIGHTIA TINCTORIA

MICROORGNISMS CONCENTRATION IN N -BUTANOL
µl
25 µl
2mm
50 µl
ASPERGILLUS NO GROWTH
75 µl
NO GROWTH
100 µl NO GROWTH
125 µl 1.5mm

150 µl NO GROWTH
 25 µl 1.6mm

50 µl 1.9mm

75 µl 1.5mm
FUSARIUM
100 µl NO GROWTH

125 µl 1.9mm
150 µl 0.9mm

Secondary metabolites are resistant to Aspergillus.S, and no growth occurs in the

agar medium. Some concentration[50 µl,75 µl,100 µl,150 µl] has partial resistant to the
secondary metabolites and Secondary metabolites are resistant to Fusarium, and no growth
occurs in the agar medium. Some concentration [100 µl ]has partial resistant to the secondary
metabolites.

Fig 4 ASPERGILLUS
Fig 5 FUSARIUM
6.FT-IR ANALYSIS OF A WRIGHTIA TINCTORI
An FT-IR (Fourier Transform Infrared) test was conducted to analyze the functional
groups present in a sample by measuring its infrared absorption spectra. FTIR analysis of
Wrightia tinctori extract has been used to identify functional groups present in its
phytochemical composition. Studies indicate that FTIR spectra confirm the presence of
phenols, alkanes, aliphatic amines, Aliphatic hydrocarbons. The spectrum of betacyanins FT-
IR results from wrightia tinctoria flower extract can be seen in Figure 6.

Fig 6: FT-IR ANALYSIS OF A WRIGHTIA TINCTORI

TABE 4: FUNCTIONAL GROUPS OF WRIGHTIA TINCTORI FLOWER
OBSERVED BY FT-IR
PEAK POSITION [cm] FUNCTIONAL GROUP FUNCTIONAL GROUP
NAMES
3848.26 O-H Alcohols
3680.48 O-H Alcohols
2851.24 C-H Aliphatic hydrocarbons
2357.55 C=C Alkyne
1541.81 N-H Bending
1255.43 C-O Carboxylic acid
1034.62 C-O Alcohols

7.GC-MS ANALYSIS OF WRIGHTIA TINCTORIA FLOWER EXTRACT

The GC-MS [Gas Chromatography-Mass Spectrometry] analysis of Wrightia tinctoria
flower extract provided critical insights into its chemical profile, revealing the presence of
various bioactive compounds. The separation and identification of individual compohehts
through retention times and mass spectral data underscore the extract’ s complex
phytochemical composition. Chromatographic fingerprint analysis of Indian variety dried
flowers of n-Butanoic extract of Wright tinctoria using GC-MS has shown 28 peaks
indicating the presence of 28 phytochemical compounds. The peak observed at GC-MS
Analysis presented in Fig 7. The identification of the phytochemical compounds was based
on the peak are, Retention time and molecular formula [MW] and concentration% as shown
in table 5 The molecular activity of some of the identified compounds in table 6 and
structures of some of the identified compound’s table 7.
Fig 7 THE PEAKS OBSERVED GC-MS CHROMATOGRAM OF WRIGHTI TINCTORIA

S.N RETENTION COMPOUND NAME MOLECULAR MOLECULAR AREA MOLECULAR

O TIME FORMULA WEIGHT [g\
%
mol]
1 5.1419 Butanoic acid C4H8O2 88.11 308695 96.1
0
2 5.8738 1,3-Propanediol, C9H22OS 190.356 810987 81.9
TBDMS derivative i
3 6.2890 Propanoic acid, 2- C8H16O2 144.21 254763 95.9
 methyl-, butyl ester
4 6.3145 Butanoic acid, 2- C8H16O2 144.21 250842 97.5
methylpropyl ester 5
5 6.4565 Butanoic acid, butyl C8H16O2 144.2114 630832 97.0
ester 2
6 6.5694 n-Butyl methacrylate C8H14O2 142.2 731471 88.8
0
7 6.8352 2-Hexenal, 2-ethyl C8H14O 126.192 375425 97.2
8 7.1120 1-Hexanol, 2-ethyl C8H18O 130.23 606369 98.0
9 7.2795 2-Butenoic acid, C8H14O2 142.192 117819 98.2
butyl ester 6
10 8.2627 tert-Butyl glycidyl C7H14O2 130.18 249999 83.8
ether 8
11 8.8198 1,1-Diisobutoxy- C12H26O 202.33 882776 97.4
isobutane 2 2
12 9.4571 Butane, 1,1-dibutoxy C12H26O 170.328 495262 89.1
2 2
13 15.3672 3-O-Methyl-d- C7H14O6 194.182 643234 81.9
glucose 1
14 17.0022 Neophytadiene C20H38 278.52 694301 91.3
15 18.2658 n-Hexadecenoic acid C16H32O 256.43 243147 97.5
2 0
16 19.6933 Phytol C20H40O 296.53 318963 97.8
9
17 19.9628 9,12,15- C18H30O 278.42 147042 94.3
Octadecatrienoic 2 7
acid, (Z, Z,Z)-
18 20.3233 Hexadecanoic acid, C20H40O 312.538 220873 97.7
butyl ester 2 6
19 21.7981 Butyl 9,12- C22H40O 336.54 140853 92.8
octadecadienoate 2 6
20 21.8345 Cis-9-Octadecenoic C21H40O 282.46 132908 86.4
acid, propyl ester 2 5
21 21.8673 Butyl 9,12,15- C22H38O 334.524 386763 85.6
octadecatrienoate 2 3
22 22.0311 Octadecanoic acid, C22H44O 340.59 691433 93.7
butyl ester 2

23 23.0653 Hexadecenoic acid, C19H38O 330.50 526023 96.5

2-hydroxy-1- 4 2
(hydroxymethyl)ethy
l ester
24 23.2014 Campesterol C28H48O 400.68 336429 95.4
3
25 24.6239 Octadecanoic acid, C21H42O 358.56 269613 96.3
2,3-dihydroxypropyl 4 9
ester
26 24.6239 Stigmasterol C29H48O 412.69 528286 97.5
 6
27 25.4068 Supraene C30H5O 410.7180 199389 91.6
3
28 25.7309 . gamma-Sitosterol C29H50O 414.71 103365 97.4
55
TABLE 5 PHYTOCHEMICAL PROFILING AND GC -MS ANALYSIS OF WRIGHTIA TINCTORIA

THE MOLECULAR ACTIVITY OF SOME OF THE INDENTIFIED

TABLE 6
COMPOUNDS

S .NO NAME OF THE CLASS MOLECULAR AACTIVITY

COMPOUND
1 Butanoic acid Fatty acid Butanoic acid is used as a food
additive, disinfectionts and
pharmaceutical
2 1,3-Propanediol, TBDMS Alcohols -
derivative
3 Propanoic acid, 2-methyl-, Propionic acid Used in food and beverage Flavors
butyl ester
4 Butanoic acid, 2- Carboxylic acid esters, Antimicrobial, Pheromone component
methylpropyl ester fatty acid esters, organic
oxides
5 Butanoic acid, butyl esters Carboxylic acid, Fatty Antimicrobial, antioxidant, Anti-
acid esters, organic inflammatory
oxides
6 n-Butyl methacrylate Methacrylic acid -
7 2-Hexenal, 2-ethyl Alkenal Antifungal,antimcrobial
8 1-Hexanol, 2-ethyl Alcohol
9 2-Butenoic acid, butyl Cratonic acid -
ester
10 tert-Butyl glycidyl ether - -
11 1,1-Diisobutoxy-isobutane - -
12 Butane, 1,1-dibutoxy - -
13 3-O-Methyl-d-glucose+ Fattyalcohol,D- Anti-inflammatory, Anti-cancer
aldohexoses
14 Neophytadiene Diterpenoid, terpenoid Antimicrobial, antioxidant, antioxidant
15 n-Hexadecenoic acid Iong-chain fatty acids Antimicrobial, antioxidant, Anti-
inflammatory, cancer
prevention,hypocholesterolemic
16 Phytol Acyclic diterpenoids Antimicrobial, antioxidant, Anti-
inflammatory, Anti-cancer
17 9,12,15-Octadecatrienoic Polyunsaturated fatty Anti-inflammatory, Anti-
acid, (Z,Z,Z)- acid arthritic,hypocholesterolemic
18 Hexadecenoic acid, butyl Fatty acid ester Plant metabolite, animal metabolite
ester
19 Butyl9,12- Chemical compound Antileishmanial activity
octadecadienoate
20 cis-9-Octadecenoic acid, Oleic acid Antimicrobial,antioxidant,inflammatory
propyl ester properties
 21 Butyl 9,12,15- Chemical compound Anticancer,antioxidant,antileishmanial
octadecatrienoate
22 Octadecanoic acid, butyl Fatty acid ester Emollient,solvent,lubricant
ester
23 Hexadecenoic acid, 2- monoglyceride Antioxidant,antibacterial,pesticidal
hydroxy-1- properties
(hydroxymethyl)ethyl
ester
24 Campesterol phytosterols Anti-inflammatory, Anti-cancer
25 Octadecanoic acid, 2,3- Monoacylglycerols Antimicrobial and antioxidant
dihydroxypropyl ester
26 Stigmasterol stigmastanes Antimicrobial, antioxidant, Anti-
inflammatory, Anti-cancer,anti-allergy,
neuroprotective
27 Supraene General anethetic, -
Inhalation
28 gamma-Sitosterol Stigmastanes,derivatives Antihyperlycemic

TABLE 7

STRUCTURES OF SOME OF THE INDENTIFIED COMPOUNDS

S.NO COMPOUND NAME MOLECULAR IMAGES

1 Butanoic acid
2 1,3-Propanediol, TBDMS
derivative

3 Propanoic acid, 2-methyl-,

butyl ester

4 Butanoic acid, 2-
methylpropyl ester

5 Butanoic acid, butyl ester

6 n-Butyl methacrylate

7 2-Hexenal, 2-ethyl

8 1-Hexanol, 2-ethyl
9 2-Butenoic acid, butyl
ester

10 tert-Butyl glycidyl ether

11 1,1-Diisobutoxy-isobutane

12 Butane, 1,1-dibutoxy
13 3-O-Methyl-d-glucose

14 Neophytadiene

15 n-Hexadecanoic acid

16 Phytol

17 9,12,15-Octadecatrienoic
acid, (Z, Z, Z)-
181 Hexadecenoic acid, butyl
ester

19 Butyl 9,12-
octadecadienoate

20 cis-9-Octadecenoic acid,
propyl ester

21 Butyl 9,12,15-
octadecatrienoate

22 Octadecanoic acid, butyl

ester
23

Hexadecenoic acid, 2-
hydroxy-1-
(hydroxymethyl)ethyl
ester

24 Campesterol

25 Octadecanoic acid, 2,3-

dihydroxypropyl ester

26 Stigmasterol

27 Supraene
28 .gamma.-Sitosterol

DISCUSSION
Preliminary qualitative phytochemical analysis was done to determine if secondary
metabolites were present or absent in solvent extracts. The Wagner’s test is used to test the
alkaloids. If Alkaloids is present reddish-brown precipitate forms. In N-butanol, carbinol,
acetone, water, a reddish-brown precipitate occurred which indicate the presence of alkaloid
whereas in petroleum ether is does not form reddish brown precipitate which means a
absence of alkoids.Test for Flavonoids, if Flavonoids is present, a strong yellow coloration
develops. In N- butanol, carbinol a strong yellow color develops indicate the presence of
Flavonoids whereas in petroleum ether, acetone, water it doesn't form strong yellow color
means the absence of flavoinds.Test for Phenolics, if orange-brown color precipitate occur it
indicates the presence of phenolics. In petroleum -ether, acetone, water, N-Butanol, carbinol
they do not form orange, brown precipitate which indicate an absence of phenolics. Froth test
is used to test the saponins, if Saponin is present the extract forms white precipitate. In
petroleum ether, N-Butanol a white precipitate is formed which indicate the presence of
saponin. In carbinol, acetone, water a white precipitate doesn't occur which indicate an
absence of saponin. Test for Tannis: If Tannis is present the extract forms yellow
precipitate. In water, a yellow precipitate occur which indicate the presence of tannins. In
petroleum ether, N-Butanol, carbinol, acetone, white precipitate doesn't occur which indicate
an absence of tannins.

ACKNOWLEDGEMENT
The author thanks DBT Star Status Scheme, DBT, Govt. of India and Dr N.G.P Arts and
Science College, Coimbatore, Tamil Nadu, for providing necessary support for this review
and chapter work.

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