Autecological and biochemical study Of

Leucas aspera Spreng

A WIDELY USED MEDICINAL PLANT”

A project report submitted to department of Botany , Moran College in

Partial fulfillment requirement for the award of Bachelor Degree in
Botany

Submitted by: Guided by:

Name: DIPU PAUL. Dr. Bhanu Prova Saikia

Roll No: 15120017 Associate Professor

Regd. No: S1223962 Dept. of Botany

Year: 2015 Moran College

Department of Botany

Moran College
 CERTIFICATE

It is certified that………………………………………..........................
bearing the Roll no………………………….............................and Registration No
………………………………………..of the year 2015 , a student of B. Sc.
Botany 6th Semester of Moran College have carried out the project entitled
“Autecological and Biochemical study of Leucas aspera Spreng, a widely used
Medicinal Plant” under my personal guidance supervision.

I wish him/her success in him/her future endeavour.

Mrs. Devajani Patgiri Dr. Bhanu Prova Saikia

Associate Professor (HOD) Associate Professor
Dept. of Botany, Dept. of Botany
Moran College, Moran College,

Moran, Sivasagar Moran, Sivasagar

 ACKNOWLEDGEMENT

It gives me immense pleasure to express my deepest sense of gratitude to

those who have been my source of inspirations and helped in carrying out and
preparation of my project.

It is a great privilege to express my heartfelt gratitude and profound

indebtness to my respected guide, Dr. Bhanu Prova Saikia, Lecturer, Department
of Botany, Moran College whose kind guidance and deepest supervisior has been
indispensable from inception till completion of the study.

I would like to offer my hearthy thanks to our respected Madam Mrs.

Devajani Patgiri (HOD), Madam Neelima Talukdar, Sir Rituraj Boruah for their
suggestions.

I would also like to express my sincere gratitude to Monisikha Deori, JRF,

IBT Hub (Funded by DBT, Govt. of India, New Delhi) Moran College, for her
great value of help in completion of the project and finding necessary information
about our project work.

Lab Bearer Krishna Bhumiz and Hemanta Dutta are also very thankful for
their help in carrying out the project.

Lastly, I would like to thank my co-workers for helping me to take every

steps very carefully during the time of project work.

Place: Moranhat

Dipu Paul

6th Semester,

Department of Botany,

Moran College,
 CONTENTS

Chapters :- Page no :-

I. Introduction

II. Review of Literature

III. Study Area

a) Map

b) Photo Plates

IV. Materials and Methods

V. Morphological and phytochemical study

a) Photo Plates

VI. Results and Discussion

a) Tables and Graphs

VII. Conclusion

VIII. Reference
 INTRODUCTION

. The branch of ecology which is concerned with the study of individuals or

the populations of a particular species with its environmental complex is called
autecology.

The study of autecology is important because it helps us to understand the

various factors that control the growth and yield of economically important plants
which are grown in pure stands as in agriculture or agronomy, floriculture,
horticulture, silviculture, etc. (Shukla 2014).

The morphology includes the study of all external features like height of the
plant, length of root, shoot, number of leaves, flowers, seeds, length of internode,
length and breadth of leaves, etc.

During the last few decades, there has been increasing interest in the study
of traditional plants and their medicinal values in different parts of the world. The
autecology and biochemical study of these plants are of paramount importance.

Plant products have been part of phytomedicines since time immemorial.

This can be obtained from barks, leaves, flowers, roots, fruits, seeds. Knowledge of
chemical constituents of plants is desirable because such information will be
valuable for synthesis of complex chemical substances. Medicinal plants contain
organic compounds which provide definite physiological action on the human
bodies and these bioactive substances includes the primary and secondary
metabolites. The primary metabolites are carbohydrates, proteins, fats, etc. The
secondary metabolites include alkaloids, flavonoids, steroids, volatile oils, etc.
These are biosynthetically derived from the primary metabolites. (Illango 2008)

Leucas aspera, commonly known as ‘Thumbai’ is a well-known medicinal

plant which is being used traditionally as antipyretic and insecticide. The plant is
an annual erect herb of the family lamiacea. It grows in both sunny and shady
places. It is strictly terrestrial plant growing along the roadside, open fields and
vegetable gardens. The whole plant is green in colour and the flower i.e, corolla are
white in colour and smooth in touch. The word Leucas is derived from the word
‘Leukos’ means white : alluding to the whiteness of the flowers.

. In the present investigation, an attempt has been made to study the

morphology, root – shoot ratio, qualititative and quantitative biochemical
constituents from extracts of plant parts of Leucas aspera collected from two
different sites of Moran - Moran Peoli Nagar and Moran College. The qualitative
study has been done on alkaloid and phenolic content on leaves. The quantitative
estimation of protein and carbohydrate has been studied.
 REVIEW OF LITERATURE

Prajapati, Purohit and Sharma, the authors of the book ‘A Handbook of

Medicinal Plants’ had discussed in this book about the medicinal plants found in
India. In this volume, the medicinal value of Leucas aspera has been discussed
(Page 45-47).

Shukla, Chaldel (2014) had discussed in the book ‘A text book of Plant
Ecology including Ethnobotany and Soil Science’, the definitions and concepts of
plant ecology along with autecology. The book seemed useful to know about the
concept of autecology.

Sunojkumar in (2008) studied ‘The botanic history and identity of the

species of Leucas appeared in clasasical theory’. The information has been
recorded from the field while collecting Leucas specimen from Indian territories.
He found out that Leucas aspera is known as ‘Dronapushpi’ in Ayurvedic
Nighantas and samhitas.

Saikia has studied “The morphological and Phenological characters of

Leucas aspera” in 2014. The plants were collected from Sunny and shady places
and studies were made on morphology such as root and shoot length, height of
plant, length of internode, leaf , number of flowers, seeds, etc. The phenological
studies were made on germination, vegetative growth, emergence of flowers, fruits
and seed formation. The morphology and phenological characters were compared
between plants collected from two sites (shady and sunny).

Lallianwalla et. al (2013) had carried out their researches on ‘Determination

of total phenolic content, total flavonoid content and antioxidant capacity of
Leucas aspera’, in 2013.
 Latha, et. al (2013) had worked on ‘Phytochemical studies of Leucas
aspera,. During their research work, alkaloid, carbohydrate, glycosides, protein
and phenol were found on the plant Leucas aspera. Phenolic compounds were to
be found in both edible and inedible parts of plants.

Ilango, et. al (2008) in their work ‘Antibacterial activity of Leucas aspera’

had made a phytochemical screening of extracts in Leucas aspera and found that
the phytochemical constituents – alkaloid, phenol, tannins were responsible for
antibacterial activity in the plant.

Hussain (2012) had worked on ‘Phytochemical screening and extraction of

polyphenol from stems and leaves of Leucas aspera.

Kardukmi (2009) had made an study on the Biomolecular and

Phytochemical analysis of three aquatic angiosperms. Test for estimation of
biomolecules – carbohydrate, proteins, and fats were done.

In the instruction manual UV-VIS spectrophotometer, the principle, working

principle, construction and uses of UV-VIS spectrophotometer had been discussed.

Jantra , Das (2010) in their book ‘College Botany Practical vol-1’ had
concentrated on the techniques commonly used in Biology, particularly Botany.
The techniques of qualitative and quantitative analysis of the biomolecules had
been discussed thoroughly.
 STUDY area

Moranhat is situated in NE of Sivasagar district of Assam. The area lies

between 94o8’ and 95o2’ E longitude and 270’ and 272’ N latitude. The east and
north sides of Moranhat are separated by the boundary of Dibrugarh district and
those of west and south sides are separated by the border area of Sivasagar, Nazira
and Sonari respectively. The study area is shown in the Map.

Moran covers an area of about 3.3 sq kms. It is the midpoint of Sivasagar

and Dibrugarh district. Sivasagar covers an area of about 1.6 sq. kms and
Dibrugarh District covers an area of 1.7 km area. The 37 No. National Highway
separates both the districts. The places situated in the four directions of Moran
Town are Bamunbari on the North, Rangoli on the east, Botamara on the south and
Teloi on the West.

The climate of Moran is very suitable for the growth of a variety of plants
which are of great economic importance. A highly humid atmosphere, abundant
rains and general coolness characterize the Moranhat. Moran exhibits a moderate
type of climate. The South west monsoon season is observed from June to October
and winters extended from November to February. The plants grow well in the
summer season due to abundant rainfall in this area.

The plants Leucas aspera was collected from two different sites of Moran –
Peoli Nagar (Dibrugarh District) and Moran College of (Sivasagar District) for
Study.
 MATERIALS AND Methods

Leucas aspera Spreng is an important medicinal plant available in the study

area, for this reason this plant is selected for the ecological study.

For the study of morphological characters, plants were collected from two
different localities and bagged and then brought to the laboratory. Roots were
collected carefully by excavating plot from the underground. From each sites, three
individuals were collected. The roots were washed carefully in running tap water
and the necessary measurement of the plants were recorded. The morphological
characters like height of plant, length of root, shoot, internode, leaf, breadth of leaf,
number of leaves, flowers, seeds per plant, etc. were observed and necessary
measurements were taken. The characters of flowers were also observed.

The root shoot ratio of each plant of both the sites has been taken. The roots
and shoots are at first separated and fresh weight are measured in weighing
balance. Then they are wrapped with paper and kept in Hot air oven for 24 hours at
110oC. After drying, the dry weight are measured and recorded.

Leucas aspera contains various phytochemical constituents like alkaloids,

flavonoids, terpenoids, steroids, volatile oils, etc. Besides these it also contains
carbohydrates, proteins, fats, etc. This shows that Leucas aspera is a source of
medicinally active compounds and is of great medicinal importance.

For the study of chemical studies, following tests are performed.

a) Detection of Alkaloid:- The plant material is dried in hot air oven and
finally powdered. The extraction is made with ethanol and the extract is
used for chemical tests
 Mayer’s test: To about 3 ml of extract, a few drops of Mayer’s reagent are
added. Yellow coloured precipitate indicate the presence of alkaloid.
 Wagner’s test: To about 3 ml of extract, a few drops of Wagner’s reagent
(Iodine in Potassium Iodide). Formation of brownish precipitate indicates
the presence of alkaloid.

a) Detection of Phenol: The plant sample is dried in an oven at a temperature

of 35o – 40o C for two days and crushed using a morter and pastle to
obtained a powdered form. The aqueous ethanol extract of Leucas aspera is
prepared by soaking 10g of powdered samples in 100ml of ethanol and
water. The extract is filtered.
 Ferric chloride test:- Extract are treated with 3 – 4 drops of Ferric chloride
solution. Formation of bluish black colour indicates the presence of Phenol..

b) Estimation of protein in various samples by spectrophotometric

method:
Extraction of protein from sample (plant leaf) is usually carried out with
buffers (CTAB) used for assay. 500 mg of the sample is weighed and
grinded well with a pastle and morter in 5 – 10 ml of buffer. After that,
centrifugation is done and supernatant is used for protein estimation.
 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standard (1ml of solution contain
200 mg of Protein) is pipetted out in a series of test tubes.
 0.1 and 0.2 ml of the samples extract is pipette out in two other test tubes.
 The volume is made up to 1 ml in all test tubes. A test tube with 1 ml of
water serves as a blank.
 5 ml of reagent C (Alkaline copper solution) is mixed well and allowed to
stand for 10 minutes.
 Then 0.5 ml of reagent D (Folin Ciocalteau reagent solution) is added,
mixed well and incubated at room temperature in the dark for 30 minutes.
 Reading is taken at 660 nm.
 A standard graph is drawn and the amount of protein in the sample is
calculated.
c) Estimation of Starch by Anthrone method:-

0.1 to 0.5 gm of the sample is homogenised in hot 80% ethanol to remove

sugars and then centrifuged to retain the residue. The residue are washed
repeatedly with hot 80% ethanol till the washing do not give colour with
anthrone reagent. The residue is dried well over a water bath. 5.0 ml of water
are added to the residue and 5.5 ml of 52% of perchloric acid. At 0o C, extracted
for 20 minutes and the centrifuged and the supernatant are saved. The extraction
is repeated using perchloric acid. Then it is centrifuged and the supernatant are
pooled and made upto 100 ml. 0.1 or 0.2 ml of the supernatant are pipetted out
and volume is made to 1 ml with water. The standard is prepared by taking 0.2,
0.4, 0.6, 0.8 and 1 ml in each tube. The samples in tube are heated for 1 minute
in a boiling water bath and then cooled rapidly. The intensity of green to dark
colour are read at 630 nm in spectrophotometer.
 Morphological and
phytochemical study

Leucas aspera commonly known as “ Doron” in Assam is a well known

medicinal plant which is being used traditionally as antipyretic and insecticide. The
plant is an annual erect herb of the family lamiaceae. Leaves are sub – sessile or
shortly petiolate, linear, acute. Flowers white, sessile small, in dense axillary
whorls. Calyx tubular, straight or recurved at the apex, corolla are white in colour
and smooth in touch. .

It is a very common weed in India. The plant has high medicinal values. It is
generally used in various diseases like headache, rheumatism, sinusitis, tonsils,
jaundice, chronic skin eruptions and respiratory diseases. It has various
pharmacological activities like antimicrobial, antifungal and antioxidant activities
(Lallianwalla 2013). Further study reveals the presence of various phytochemical
constituents – alkaloids, glucoside (Latha. Et al 2013). Leucas aspera is known as
Dronapushpi in Ayurvedic Nightants and Samhitas (Sunojkumar,2008)

During the project work morphological characters like height of plant,

length of root, shoot, internode, leaf, breadth of leaf, number of leaves, flowers,
seeds per plant etc. were studied. The morphological characters show variance
between the plants of two localities (Peoli Nagar and Moran College Campus).

Root – shoot ratio is the quotient of the dry weight of the roots divided by
the dry weight of the shoots produced during a given period. In the root – shoot
system of individual plant, there is some kind of balance. The root – shoot ratio is
one measure to access the overall health of plants.

Besides phytochemical constituents like alkaloids, flavonoids, terpenoids,

steroids, volatile oils, glucoside, etc. it is also a source of carbohydrates, proteins,
fats, etc. The chemical analysis of phenols, alkaloids, carbohydrates and proteins
are shown in this project.
 RESULTS AND DISCUSSION

During the study it is observed that plant collection from both the places
(Locality A and Locality B) have been found to vary in their growth due to
different habitat condition. The maximum average height was found to be 56.33
cm in locality – A and in locality – B the average height of the plant was 51.66 cm.
The root length of locality - B ( 14.33 cm) was more than root length of locality –
A (12.6 cm). The shoot length of locality A (43.66 cm) was more than locality B
(37.33 cm). The average length of internode was more in locality – B ( 5 cm) as
compared to locality A ( 4.8 cm). The length of leaf at third internode is more or
less same in both the localities. The breadth of leaf at third internode Is found to be
same (1.3 cm). The highest number of leaves (203), branches (12), inflorescence
per plant (16), flowers per inflorescence (34) and seeds (2782) were found in
locality A. Table I is represented below for morphological characters of Leucas
aspera

In the root – shoot ratio, the ratio of locality B (0.129) is higher than root -
shoot ratio of locality – A (0.120). An increase in root – shoot ratio is an indicator
of a healthier plant, provided the increase came from a greater root size and not
from decrease in shoot weight. It has been found that, in the plants of locality A,
the average root weight and the average shoot weight is higher than locality B.
Thus, the plant Leucas aspera in locality A is healthier than in locality B, although
the root – shoot ratio of locality B is greater than locality A. Table II is
represented for root – shoot ratio.

The tests of secondary metabolites shows the presence of Phenol and

Alkaloid in leaves and stems. These phytoconstituents may be responsible for
various pharmacological actions of these plant parts like antimicrobial, antiulcers,
antifungal and antioxidant activities.
 In the Table IV it is seen that in the sample 1 of Leucas aspera, the protein
content is 0.988 against concentration of 0.1 and in the sample 2 the protein
content is 1.022 against concentration 0.2. So, the average protein content is 1.022

In the Table V it is seen that the carbohydrate content in the Leucas aspera is
0.65 Thus, it is seen that the protein content in the leaves of Leucas aspera is more
than the carbohydrate content
 TABLE – I

Sl. CHARACTERISTICS LOCALITY – A LOCALITY – B

No. ( AV ± S.E ) (AV ± S.E )

1 Plant Height (cm) 56.33 ± 0.66 51.66 ± 3.35

2 Root length (cm) 12.66 ± 1.52 14.33 ± 1.5

3 Shoot length (cm) 43.66 ± 0.84 37.33 ± 1.98

4 Number of leaves/ plant 204 ± 9.02 112 ± 18.66
5 Length of internode 3rd/ 4th (cm) 4.8 ± 0.16 5 ± 0.38

6 Length of leaf at 3rd internode 5.5 ± 0.20 5.4 ± 1.51

(cm)
7 Breadth of leaf at 3rd internode 1.03 ± 0.05 1.33 ± 0.102
(cm)
8 No. of branches / plant 13 ± 1.03 4 ± 0.39
9 Length of longest branches (cm) 33.66 ± 2.75 18.4 ± 3.08

10 No. of inflorescence / plant 17 ± 1.58 5 ± 0.68

11 No. of flowers / inflorescence 34 ± 2.98 34 ± 2.4
12 No. of seeds / plant 2782 ±328.09 705 ± 116.12
 Table – II

Sl. Locality A Locality B

No. ( AV ± S.E) (AV±S.E)

1 Fresh weight of root ( gm) 2.14±0.26 1.15±0.29

2
Fresh weight of shoot ( gm) 22.43±2.6 10.9±2.54

3
Dry weight of root ( gm) 0.71±0.03 0.18±0.03

4
Dry weight of shoot ( gm) 5.88±1.24 1.46±0.35

5 Fresh 0.095 0.104

Average
Root-shoot ratio Dry 0.120 0.129
F .

*Locality – A : Peoli Nagar

*Locality – B : Moran college

Table- III

Active compounds Stems Leaves

Phenols +++ +++

Alkaloids +++ +++

 Table - IV

Sl. No. BSA (ml) Distilled Reagent Reagent Absorbance

water (ml) (C) (D)
1 Blank 1 5 0.5 0.046
2 0.2 0.8 5 0.5 0.87
3 0.4 0.6 5 0.5 0.91
4 0.6 0.4 5 0.5 0.78
5 0.8 0.2 5 0.5 1.30
6 1 0 5 0.5 1.364
7 0.1(sample) 0.9 5 0.5 0.988
8 0.2(sample) 0.8 5 0.5 1.022

BSA Curve
1.6
1.4 1.364
1.3
1.2
1 0.988 1.022
0.87 0.91
0.8 0.78 BSA Curve
0.6
0.4
0.2
0 0.046
0 0.2 0.4 0.6 0.8 1 0.1 0.2

Fig. 1 : - Graphical representation of quantitative estimation of protein

 Table : V

Sl. No. Standard (ml) Distilled Anthrone Absorbance

water (ml) reagent
1 0.1 0.9 4 0.001
2 0.2 0.8 4 0.77
3 0.4 0.6 4 0.6
4 0.6 0.4 4 0.8
5 0.8 0.2 4 0.83
6 1(sample) 0 4 0.65

Standard
0.9
0.8 0.83
0.8
0.77
0.7
0.65
0.6 0.6
0.5
0.4 Standard

0.3
0.2
0.1
0 0.001
0.1 0.2 0.4 0.6 0.8 1

Fig 2 : - Graphical representation of estimation of carbohydrates

 Conclusion

In conclusion, the morphological study demonstrates that Leucas aspera

collected from two different localities of Moran area shows slight variation in
morphological characters depending on varied environmental conditions. Besides
meterological factors, the soil content of the habitats also play very important role.
The climatic conditions play a vital role in the growth and development of the
species.

The qualitative biochemical analysis indicated the presence of alkaloid and

phenol which have high therapeutic and insecticidal value. So, Leucas aspera can
be recommended to the people for ethnomedico use such as for treatment of
headache, asthma, bronchitis, snakebites, fever, typhoid, skin diseases and also as
insect repellent. The parts of plants may also be recommended to the people to use
as food.

Further investigation on quantitative Estimation showed the presence of high

content of protein than carbohydrates.

The presence of protein and carbohydrate are very important for the
consumer as well as the plant itself.
 REFERENCE

Prajapati ND, Purohit SS : A handbook of Medicinal plants. Satyam

and Sharma A.K (2006) Publication Delhi: 45 – 47.

Shukla R.S, Chandel P.S : A textbook of Plant Ecology including

(2014) Ethnobotay and Science. S.Chand Publications

Sunojkumar P.(2008) : Medicinal Leucas R.Br (Lamiacea)–Botanic history

Identity of Species appeared in Classical literature

Indian. Journal. Botanical. Research 433 – 438

Saikia B.P ( 2014) : Study of Morphological and Phenological

characters of Leucas aspera . Spreng a widely
used medicinal plant.

Lallianwala S, et. al : Determination of total phenolic content, total

flavonoid content and total antioxidant capacity of
Leucas aspera spreng 3 (6) : 62 – 65
Ilangok, et. al (2008) : Antibacterial activity of Leucas aspera Spreng,

International Journal of Chemical Science : 6(2)

526- 530.

Latha B, et. al (2013) : Phtochemical studies of Leucas aspera

Jour. Chem. Pharm. 5(4) : 222- 228

Farhan Hussain (2012) : Phytochemical Screening and extraction of

polyphenol from stems and leaves of Leucas

aspera 3(1) : 149 – 156

Kandukmi et. al (2008) : Biomolecular and phytochemical analysis of

Three aquatic angiosperms. Af. J of MicRes Vol

3(8) : 418-421

R. Srinivasan, B. Ravali, : Leucas aspera- Medicinal Plant, International

Journal of Pharma and Bio Science 2(1) : 41- 43

Instruction manual UV - : Systronics ( An ISO 9001: 2008 Co. ) 2- 3

VIS Spectrophotometer.