Screening of Litsea salicifolia (Dighloti) as a mosquito repellent

Namrata Devi and Marmish Debbarma

Guided by Dr. Sthiti Porna Dutta
THE ASSAM ROYAL GLOBAL UNIVERSITY
GUWAHATI, ASSAM (781035) INDIA

Keywords: Mosquito repellent, photochemical analysis, mosquito

borne disease, traditional medicine.
 Introduction

Litsea salicifolia is one of the most diverse species of evergreen

trees or shrubs belonging to the family Lauraceae of genus Litsea. It
is distributed abundantly across tropical and subtropical Asia, and
North and South America. In India, the genus is represented by 46
species, one of which is Litsea salicifolia and 27 of which are endemic
to the country (Mohanan and Kumar, 2003).
It has been used globally in traditional medicine for thousands of
years. In China and other countries such as Japan, Korea, India,
Thailand, Myanmar, Bangladesh, Malaysia, Indonesia, Philippines,
Mexico, and Guatemala, some Litsea species are known for their
antipyretic, analgesic, and antidiarrheal activities, finding use in
traditional medicine to treat influenza, stomach aches, inflammatory
diseases, bruises, insect bites, and other ailments. Several classical
books including monographs, as well as modern texts, have
described the botanical, chemical, and pharmacological properties
and traditional uses of Litsea species. Every part of Litsea species,
including the fruit, leaf, stem, and roots, is used in the preparation of
mixtures and decoctions to treat various ailments. The
genus Litsea exhibit a wide spectrum of biological and
pharmacological activities, including antitumor, antimicrobial, anti-
inflammatory, antioxidant, hypothermic, and insecticidal effects;
they are used to treat diarrhea, vomiting, bone pain, colic in children,
and disorders of the central nervous system (Guzmán-Gutiérrez et
al., 2012, Jiménez-Pérez et al., 2011, Institute of Kunming Botany,
Chinese Academy of Sciences, 1984).
In the north eastern region of India towards Assam Litsea salicifolia is
known as Dighloti and is traditionally attached to the people of
Assam. Dighloti is a relevant plant associated with Rongali Bihu. This
medium-sized plant is found naturally in different parts of Assam.
The leafy branches of Dighloti are used in some rituals played on the
day of Goru Bihu while washing the cow’s body. It is believed that
the process is made to protect cows from various insects. The life
span of this plant is long. The stem and branches are somewhat
weak. The leaves are slightly elongated. The average height of this
plant is 10 to 12 feet. This plant is generally found in the forest areas
of Assam. Nowadays, people have started planting this tree in the
free spaces around their houses for traditional and other purposes.

Scientific Classification of Dighloti

 Scientific name: Litsea salicifolia

 Kingdom: Plantae
 Phylum: Angiospermae
 Sub-phylum: Magnolids
 Order: Laurales
 Family: Lauraceae
 Genus: Litsea
 Species: salicifolia

In Assam, Dighloti is used for the following reasons – 

1. In the time of Bohag Bihu.

2. As a mosquito repellent. 
3. Used as food for the worms while the production of Muga
silk. 
4. Dighloti is also used for its medicinal importance towards
human health.
Practical Aspects of Dighloti

Apart from the use of Dighloti in Bohag Bihu, the plant is used as
mosquito repellent. The plant has a history of rare use as a medicinal
remedy in households. But nowadays, studies are continuing on the
medicinal properties of this plant and still a long way to go to find
out its practical aspects.
OBJECTIVE

 Screening of Litsea salicifolia as a mosquito repellent

 In silico studies to analyse their binding affinity with specific
mosquito protein
 Histology/ultrastructural studies: to check for occurrence of
cellular change in larva upon treatment with plant extract

METHODS AND MATERIALS

COLLECTION AND IDENTIFICATION

Fresh parts of Litsea salicifolia leaves were collected from Dibrughar
(Assam) and was identified by the Department of Botany, The Assam
Royal Global University, Guwahati, Assam

PREPARATION OF SAMPLE
The leaves were plucked and washed multiple time with tap water to
remove dust. The leaves were allowed to shade dry for a few days at
room temperature until the water molecule evaporated and became
well dried for grinding. After drying, the leaves were grinded into fine
powder using grinder and transfer into airtight container with proper
labelling.

PREPARATION OF SAMPLE EXTRACT

50ml of 80% methanol was poured to a beaker containing 5gm of
sample powder. For four hours, the mixture was stirred with a
magnetic stirrer. After being transferred to a mechanical shaker, the
mixture was shaken for a week. The mixture was filtered after a
week using Whatman No. 1 filter paper, and the extracted was
collected and stored in Falcon tubes with the appropriate labelling.

QUALITATIVE PHYTOCHEMICAL ANALYSIS

Standard techniques were used for phytochemical screening on the
methanol extract to determine the components.
1. Test for Flavonoids
Alkaloid reagent test
A test tube containing 2 ml of the extract was used to treat with a
few drops of 2N sodium hydroxide solution. Flavonoids are present
when a yellow colour develops.
2. Test for Alkaloid
Mayer’s test
In a test tube, 2 ml of the extract was added with 2 ml of
concentrated hydrochloric acid. The Mayer's reagent was then added
in a few drops and mixed. A greenish colour or white precipitation
indicate the presence of alkaloids.
3. Test for Tannin
Ferric chloride test
In a test tube, 1 ml of the extract was added with 2 ml of the 5%
ferric chloride solution. Formation of dark blue colour and greenish
black colour indicate the presence of tannin.
4. Test for Saponin
2ml of extract was taken in a test tube and diluted with 2ml of
distilled water. Mixed well and shaken in a graduated cylinder for 15
minutes. Formation of 1cm layer of foam indicates the presence of
saponins.
5. Test for Phenol
A test tube was filled with 1 ml of the extract, 2 ml of distilled water,
and a few drops of 10% ferric chloride. Phenols are present when
blue or green colour develops.

6. Test for Glycosides

2 ml of extract was taken in a test tube and 3 ml of Chloroform and
10% Ammonia solution was added. Formation of Pink color shows
the presence of Glycosides.

QUANTITATIVE PHYTOCHEMICAL ANALYSIS

1. Total Alkaloids content
Harborne Method
2.5gm of sample was weighed and transferred into a beaker.10%
acetic acid in 100 ml of ethanol were added and the beaker was
covered and allowed to stand for 4 hours. The above mix was filtered
and the extract was concentrated on a water bath to 1/4 th of the
original volume. Concentrated ammonia hydroxide was added drop
wise to the mix until the precipitation was complete. The whole
solution was allowed to settle and the precipitation was collected
and wash with dilute ammonium hydroxide and then filtered. The
residue left was dried and weighed.
2. Total Saponins content
20gm of sample was taken into a beaker and 100 ml of 20% ethanol
was added. The mixture was heated over the water bath for 4 hours
at 55◦C with continuous stirring. The mix was then filtered and the
residue was re-extracted with another 200 ml of 20% ethyl alcohol.
The combined extract was reduced to 40 ml over water bath at 90◦C.
The concentration was then added with 20 ml of diethyl ether and
transferred into a 250 ml separating funnel. The aqueous layer was
recovered and the diethyl layer was discarded. The purification was
repeated once more. The aqueous layer was collected and 60 ml of
n- butanol was added. The combined extract was then washed with
10 ml of 5% Nacl solution. The remaining solution was then heated
over the water bath and after evaporation the samples were dried
and weighed.
3. Total Tannins content
Peri and Pompei method
0.1 ml of sample extract was added into a test tube and 0.9 ml of
distilled water was added to make 1ml. Add 0.5 ml of FC reagent. 1
ml of 35% sodium carbonate was prepared and diluted to 10 ml with
distilled water. The mixture was then shaken and kept at room
temperature for 30 minutes. A set of standard solution of tannic acid
was prepared (20,40,60,80 and 100 µg/ml). Absorbance for the test
and standard solution were measured against the blank at 700nm.
4. Total Phenolic content
Folin-Ciocalteu method
1 ml of extract was added with 1 ml of Folin-ciocalteu reagent in a
test tube and incubate for 3 minutes. 1 ml of saturated sodium
carbonate was added and mixed well. Make a final volume of 10 ml
with distilled water and incubated in dark room for 90 minutes. A set
of standard solution of gallic acid was prepared. Dissolve 10 mg of
gallic acid in 10 ml of methanol (concentration 1 mg /ml). From this
various standard concentration was prepared (20,40,60,80,100
µg/ml). Absorbance for the test and standard solution were
measured against the blank at 650 nm.
5. Estimation of flavonoid content
0.5 ml of plant extract was taken and 0.5 ml of distilled water and 0.3
ml of 5% NaNo2 was taken and incubated for 5 minutes at 25◦C. After
that 0.3 ml of 10% AlCl3 was added. This was followed by addition of
2ml of 1M NaOH.
Preparation of Rutin standard: 1 mg of Rutin was dissolved in 10 ml
of alcohol. From this various Concentration (20,40,60,80 and 100
µg/ml) were prepared. To each concentration 0.3 ml of 5% NaNO 2
and 0.3 ml of 10% AlCl3 and 2 ml of 1M NaOH was added. The
absorbance was taken at 510 nm against blank.

Method of larvicidal activity

The mosquito species used for the test was Anopheles gambiae,
larvae of A. gambiae were used to screen the larvicidal activity of
methanolic extracts obtained from Litsea salicifolia. The powdered
was macerated with 80% methanol for week and filtered.
10 larvae were taken into a beaker along with 9ml of water, 1ml of
extract was added and mix well. Larva maintain in distilled water
were used as control. Observed for maximum 48 hours and the
mortality was recorded, larva was considered dead if they stopped
moving for a prolonged period even after gentle probing with a small
spatula.

GCMS ANALYSIS
Gas chromatography-mass spectrometry (GC–MS) analysis
To identify the compound present in the methanolic extract of the
plant. we took 0.1/0.2 g and dissolved it in 1 ml of methanol. To carry
out the GC-MS analysis the sample were sent to Guwahati
Biotechnology Park. Upon obtaining the GC peaks of the separated
compounds, we choose the peaks with the highest area percentage
and proceeded for spectral detection of the compounds in those
peaks. The identity of the compounds was then established by
comparing their retention time, peak area, peak height and mass
spectral patterns with the compound library stored in the National
Institute of Standard and Technology.
 RESULTS

PHYTOCHEMICAL TEST
1. Qualitative results
Methanolic Alkaloid Flavonoids Saponin Glycoside Tannin Phenol
extract
Conc. + + + _ + +
0.1g/ml

Present: +
Absent: -

2. Quantitative result
Methanoli Alkaloi Flavonoi Saponin Tannin Phenol
c extract d d
Conc. 0.7g/ml 1.5mg/ml 43.6mg/ 0.0001mg/ 0.148mg/
0.1g/ml ml ml ml
 RESULT OF LARVACIDAL ACTIVITY

1st batch (conc. 0.1g/ml)

No. of larva Exposure Time Observation Inference
treated
10 48 hours Maximum 90%
effectiveness effectiveness

2nd batch (conc. 0.05g/ml)

No. of larva Exposure Time Observation Inference

treated

10 48 hours Medium 30%

effectiveness effectiveness
Result of GC-MS analysis
COMPOUND NAME COMPOUND NO.
BENZOIC ACID, 1-PHENYLETHYL ESTER Compound-1

BENZENE, (1-BROMOETHYL)- Compound-2

BENZENE, (1-NITROETHYL)- Compound-3

2-PHENETHYL PHENYL ETHER Compound-4

BENZENE, (2-IODOETHYL)- Compound-5

BENZALDEHYDE, 4-[(3- Compound-6

METHYLPHENYL)METHOXY]-

ETHER, P-METHYLBENZYL VINYL Compound-7

BENZENE, 1-(BROMOMETHYL)-2-METHYL- Compound-8

BENZENE, 1,1'-(OXYDIETHYLIDENE)BIS- Compound-9

BENZENE, (1-AZIDOETHYL)- Compound-10

BENZENE, 1-(BROMOMETHYL)-4-METHYL- Compound-11

2-METHYLBENZYL ISOTHIOCYANATE Compound-12

BENZENE, 1-(BROMOMETHYL)-2-METHYL- Compound-13

BENZENE, 1,1'-(1,2-DIMETHYL-1,2- Compound-14

ETHANEDIYL)BIS-, (R*,R*)-(.+/-.)-

BENZENE, 1,1'-(1,2-DIMETHYL-1,2- Compound-15

ETHANEDIYL)BIS-

D-MANNITOL, 1-DECYLSULFONYL- Compound-16

BETA.-D-MANNOFURANOSIDE, 1-O-(10- Compound-17

UNDECENYL)-

CYCLOTETRADECANONE OXIME Compound-18

1,3-DIOXOLANE, 4-PENTYL-5-PROPYL-2,2- Compound-19

BIS(TRIFLUOROMETHYL)-, CIS-

ARACHIDONIC AMIDE, N-[5-HYDROXY-N- Compound-20

PENTYL]-

4-NITRO-5-HYDROXY-1,2-DIMETHYLINDOLE Compound-21
BENZYL BENZOATE Compound-22

9-.BETA.-[TRI-O-BENZYL-D- Compound-23
ARABINOFURANOSYL]ADENINE N-
BENZOATE

PHENOL, P-(BENZYLOXY)-, BENZOATE Compound-24

2-(METHYLSULFONYL)ACETOPHENONE Compound-25

1,3,5-TRI-O-BENZOYL-2-O- Compound-26
METHANESULFONYL-.BETA.-D-RIBOSE

4H-OXAZOL-5-ONE, 2-PHENYL-4-(P- Compound-27

TOLYLHYDRAZONO)-

BENZENEMETHANOL, 2-NITRO- Compound-28

2-(METHYLSULFONYL)ACETOPHENONE Compound-29

4-(BENZYLIMINO)-1,4-DIHYDRO-1-(2- Compound-30
METHYLBENZYL)PYRIDINE

N-BENZYLBENZAMIDE Compound-31

N-BENZOYL-3,5,6-O- Compound-32
TRIBENZYLGLUCOSAMINE

2-PHENETHYL PHENYL ETHER Compound-33

BENZENE, (1-METHYLPROPYL)- Compound-34

2-TRIFLUOROMETHYLPHENYL-.BETA.- Compound-35
PHENYLPROPIONATE

TRIFLUOROMETHYL T-BUTYL DISULFIDE Compound-36

2,4,4-TRIMETHYL-1-PENTANOL Compound-37

D-FRUCTOSE, 1,3,6-TRIDEOXY-3,6-EPITHIO- Compound-38

1,4,2,5 CYCLOHEXANETETROL Compound-39

2,4,4-TRIMETHYL-1-PENTANOL Compound-40

BETA.-D-LYXOFURANOSIDE, O-NONYL- Compound-41

4H-PYRAZOLE, 3-TERT-BUTYLSULFANYL-4,4- Compound-42

BISTRIFLUOROMETHYL-

7-OCTEN-4-ONE, 2,6-DIMETHYL- Compound-43

AZIRIDINONE, 1,3-BIS(1,1-DIMETHYLETHYL)- Compound-44

2-PENTANOL, 1-T-BUTYLTHIO- Compound-45

1,2,3,4-CYCLOHEXANETETROL Compound-46

1,5-ANHYDRO-L-RHAMNITOL Compound-47

3-HEPTANONE, 5-ETHYL-4-METHYL- Compound-48

ETHER, TERT-BUTYL 3,3-DIMETHYLBUTYL Compound-49

ETHYL 3,3,3-TRIFLUORO-2- Compound-50

PROPIONAMIDOLACTATE

N-METHYLVALERAMIDE Compound-51

1-PROPENE, 3,3-DIETHOXY- Compound-52

1-BROMO-2-METHYL-2-BUTANOL Compound-53

SUCCINIC ACID, DI(BUT-2-EN-1-YL) ESTER Compound-54

FORMIC ACID, 2-METHYLHEX-3-YL ESTER Compound-55

FORMIC ACID, 2,4-DIMETHYLPENT-3-YL Compound-56

ESTER

[1,4]DIOXINO[2,3-B]-1,4-DIOXIN, Compound-57
HEXAHYDRO-2,3,6,7-TETRAMETHYL-

1,2,4,5-TETRAZINE, 1,4- Compound-58

DIETHYLHEXAHYDRO-

2,4-DIHYDROXY-2,5-DIMETHYL-3(2H)- Compound-59
FURAN-3-ONE

4-NONANOL Compound-60

5-METHYL-4-OCTANOL Compound-61

3-{4-[2- Compound-62
(CYCLOPROPYLMETHOXY)ETHYL]PHENOXY
}-N-(1-METHYLETHYL)-2-[(TRIM

2,4-DIHYDROXY-2,5-DIMETHYL-3(2H)- Compound-63
FURAN-3-ONE

N-(1-METHOXYCARBONYL-1- Compound-64
METHYLETHYL)-4-METHYL-2-AZA-1,3-
DIOXANE

1,1-DIMETHYL-1-SILACYCLOBUTANE Compound-65

2-PROPYL-TETRAHYDROPYRAN-3-OL Compound-66
4-HEPTANOL, 3-ETHYL- Compound-67

NITRO-TERT-BUTYL-ACETATE Compound-68

SUCCINIC ACID, DODECYL 2-ETHOXYETHYL Compound-69

ESTER

2,2,5,5,8-PENTAMETHYL-3,6-DIOXA-8-NONEN- Compound-70
1-OL

SUCCINIC ACID, 8-CHLOROOCTYL 2- Compound-71

ETHOXYETHYL ESTER

SUCCINIC ACID, 2-ETHOXYETHYL UNDECYL Compound-72

ESTER

1,4-BUTANEDIONE, 1,4-DIPHENYL- Compound-73

BENZOIC ACID, 1,3-DIOXAN-5-YL ESTER Compound-74

3-BENZOYL-5-(2,4- Compound-75
DICHLOROBENZYLIDENE)RHODANINE

1,3,4-DIOXAZINE, PERHYDRO-4-BENZOYL-6- Compound-76

METHYL-2-(3-NITROPHENYL)-

D-RIBOPYRANOSE 1-O-BENZOATE Compound-77

GLUCITOL, 1,6-DIBENZOATE, D- Compound-78

2-PHENYL-[1,3]DIOXOLANE-4,5- Compound-79
DICARBOXYLIC ACID DIAMIDE

2-ACETYL-3,5-DIBENZOYLRIBOSE Compound-80

2,3-DI-O-BENZOYL-D-RIBOPYRANOSE Compound-81

HIPPURIC ACID, TBDMS DERIVATIVE Compound-82

CARBAMODITHIOIC ACID, Compound-83

[(BENZOYLAMINO)CARBONYL]-, METHYL
ESTER

HIPPURIC ACID, TMS DERIVATIVE Compound-84

BENZAMIDE, N-(3-ETHYL-5- Compound-85

METHYLAMINOCARBONYLMETHYL-4-OXO-
2-THIOXO-1-I

2-BUTENOIC ACID, 4-OXO-3,4-DIPHENYL-, Compound-86

METHYL ESTER

2-BUTENE-1,4-DIONE, 1,4-DIPHENYL- Compound-87

1-BENZOYL-3-[2-HYDROXYPROPYL]-2- Compound-88
THIOUREA

3-BENZOYLPROPIONIC ACID, TMS Compound-89

DERIVATIVE

1-(2,5-DIHYDROPYRROL-1-YL)-3- Compound-90
PHENYLPROPANE-1,3-DIONE

DINOCAP Compound-91

DIBENZOYLMETHANE Compound-92

2-BUTEN-1-OL, 3-METHYL-, BENZOATE Compound-93

BUTANE, 2,3-DICHLORO-2-METHYL- Compound-94

DIBENZAL SEDOHEPTULOSAN Compound-95

BUTANE, 1,3-DICHLORO-2-METHYL- Compound-96

2,2,2-TRIFLUORO-N-(2-OXO-5-PHENYL-2,3- Compound-97
DIHYDROFURAN-3-YL)ACETAMIDE

BUTANE, 1,3-DICHLORO-3-METHYL- Compound-98

3-BUTEN-1-OL, 3-METHYL-, BENZOATE Compound-99

GERANYL BENZOATE Compound-100

HYDRATROPIC ACID, OCT-3-EN-2-YL ESTER Compound-101

3-PHENYLPROPIONIC ACID, OCT-3-EN-2-YL Compound-102

ESTER

1,5-PENTANEDIOL DIBENZOATE Compound-103

1-HYDROXY-1-PHENYL-1,2,5,7A- Compound-104
TETRAHYDRO-PYRROLIZIN-3-ONE

2-ACRYLAMIDOTHIAZOLE Compound-105

3,7,11-TRIMETHYL-3-HYDROXY-6,10- Compound-106
DODECADIEN-1-YL ACETATE

(2E,6E,10E)-3,7,11,15- Compound-107
TETRAMETHYLHEXADECA-2,6,10,14-
TETRAEN-1-YL FORMAT

TRANS-GERANYLGERANIOL Compound-108

(2E,6E)-3,7,11-TRIMETHYLDODECA-2,6,10- Compound-109
TRIENYL PROPIONATE
3,7,11-TRIDECATRIENOIC ACID, 4,8,12- Compound-110
TRIMETHYL-, METHYL ESTER, (Z,E)-

1,6,10,14,18,22-TETRACOSAHEXAEN-3-OL, Compound-111
2,6,10,15,19,23-HEXAMETHYL-, (ALL-E)-(.

2,6,10-DODECATRIEN-1-OL, 3,7,11- Compound-112

TRIMETHYL-

1-(3,5-DINITROPHENOXY)-3,7,11-TRIMETHYL- Compound-113
DODECA-2,6,10-TRIENE

SUCCINIC ACID, TRIDEC-2-YN-1-YL 3- Compound-114

METHYLBUT-3-EN-1-YL ESTER

(E,E)-7,11,15-TRIMETHYL-3-METHYLENE- Compound-115
HEXADECA-1,6,10,14-TETRAENE

1,6,10,14-HEXADECATETRAEN-3-OL, 3,7,11,15- Compound-116

TETRAMETHYL-, (E,E)-

2,6,10,14-HEXADECATETRAEN-1-OL, 3,7,11,15- Compound-117

TETRAMETHYL-, ACETATE, (E,E,E)-

3,7,11-TRIDECATRIENENITRILE, 4,8,12- Compound-118

TRIMETHYL-

UNDEC-10-YNOIC ACID, 3-METHYLBUT-2-EN- Compound-119

1-YL ESTER

FARNESYL BUTANOATE Compound-120

SQUALENE Compound-121

FARNESOL (E), METHYL ETHER Compound-122

(2-ISOPROPENYL-5-METHYL- Compound-123
CYCLOPENTYL)-ACETONITRILE

2,6,10,14-HEXADECATETRAENOIC ACID, Compound-124

3,7,11,15-TETRAMETHYL-, METHYL ESTER

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