TABLE OF CONTENTS

Title page i

Acknowledgement ii

Table of contents iii

Chapter 1: Introduction 1-2

Chapter 2: Method and methodology 3-6

Chapter 3: Result and discussion 7-8

Chapter 4: Conculsion 9

Chapter 5: Reference 10
TO STUDY ON PHYTOCHEMICAL PRESENT IN BACOPA MANNIERI
PLANT PRESENT IN NEPAL

A PRODECT WORK SUBMITTED FOR THE PARTIAL FULFILMENT OF THE

REQUIREMENT IN CHEMISTRY OF CLASS 11

Submitted By

Yuvraj Timilsena

Class 11

Class Roll Number: 33

Submitted to Mr.Prem Dhungena

SHREE MOUNT EVEREST SECONDARY SCHOOL

BRENDRANAGAR-1

SURKHET, NEPAL

DATE: 08/2079
ACKNOWLEDGEMENT
I would like to thank my chemistry teacher," Mr.Prem Dungena" for guiding me
throughout this project work. I should thank our lab assistant who helped me to
line up the project and helped me with practical work.

A special acknowledgement goes to our principal,"Mr.Hem Prakhash Dhungena"

who gave me the golden opportunity of this wonderful project, which also helped
me in doing a lot of research and I come to know about so many new things.

I wish to thank my parents as well for their support and encouragement without
which I could not have completed this project in the limited time frame.

In the end, I want to thank my friends who displayed appreciation for my work
and motivated me to continue my work.
 1 INTRODUCTION

1.1 Phytochemical
Phytochemicals are chemical compounds produced by plants, generally to help them
resist fungi, bacteria and plant virus infections, and also consumption by insects and other
animals. The name comes from Greek 'plant'. Some
phytochemicals have been used as poisons and others as
traditional medicine. Worldwide, between 50,000 and
80,000 flowering plants are used medicinally. Catalogues
have recorded 1515 to 2331 useful medicinal in Nepal.
At least 48 Important Plant Areas (IPAs) for medicinal
and aromatic plants (MAPs) are located in the Karnali
Province. Some uses of medicinal plant are that its help
to increase our health conduction. It also helps us to maintain good metal health. Medicinal
plants are considered as rich resources of ingredients which can be used in drug development
either pharmacopoeial and non-pharmacopoeial or synthetic drugs. A part from that, these
plants play a critical role in the development of human cultures around the whole world.

Phytochemical investigation of Brahmi revealed the presence of various important

secondary metabolites such as carbohydrates, proteins, amino acids, steroids, glycosides,
flavonoids, alkaloids and tannins in Methanolic, ethanolic and aqueous extracts. No activity was
observed against bacterial strains like staphylococus aureus, Bacillus subtilis, Escherichia coli
and Pseudononas aeruginosa when subjected to aqueous and Methanolic extract of Bacopa
monnieri. Methanolic extract showed significant anti-fungal activity against Candida albicans
and Aspergillus Niger at 2.5mg/ml and 1.25mg/ml concentrations.

Brahmi, Scientific name of Brahmi is Bacopa mannieri, belongs to the family

scrophulariacea. The species of Brahmi plant found in the world are 146. Brahmi is mainly
found in marshy areas throughout Asia, especially India, Sri Lanka, Pakistan, Nepal, Vietnam, as
well as in South America. Additionally, it grows in Hawaii and Florida. It is a small, succlent
(stems adapted to store water), glabrous (smooth), creeping or prostrater (branches above the
ground) annual herb. It is also known as herpestis monniera, water hyssop (small bushy) and is
locally known as Jalanimaba or Brahmi in India, Nepal. The term Brahmi is derived from the
Brahma, which refers to the mythical god of creation in Hindu mythology. Brahmi is primarily
has been used for nearly 3000 years by Ayurvedic medical professionals. As the brain is
considered the centre of creative activity, any substance the might boost the brain's health is
known as Brahmi. It also means 'bringing knowledge of supreme reality. Brahmi may be
recognized as a popular memory booster among locals. Brahmi plant is very healthy to health of
human being. It helps to increase capacity of brain. It helps to decrease anxiety, fear and
stress. It helps to improves liver function.

Some side effects of Brahmi

Commonly reported side effects of Brahmi include:

a. Nausea
b. Increased stomach motility (food moving from mouth to the throat).
c. Stomach upset

However, if you experience any adverse reactions to Brahmi, immediately contact a

doctor or your Ayurvedic physician who has prescribed it to you. They will be able
to guide you appropriately for you symptoms

Scientific studies
a. Memory enhancing activity:
EE improved the process of motor learning in rats (Prakash and Sirsi, 1962). At
40mg/kg orally for 3 or more days showed better acquisition, improved retention
and delayed extinction of treated rats (J.Ethnopharmacol. 1982, 5,250).
b. Phsychotropic activity:
EE (10-200mg/100g b.w.) had a sedative effect on frogs and dogs (Malhotra and
das, 1959). EE of whole plant (50mg/kg i.p.) exhibited transquilizing effect on
albino rats and dogs (Aithal and Sirsi, 1961). Hersaponin showed a sedative effect
in mine (Malhotra et al. 1960, 1961 and 1962). Plant extract showed significant
barbiturate hypnosis potentiation in rats (J. res. Ind. Med. Yoga and Homoeo.
1979, 14, 1).
c. Anti-cancer activity:
The EE of whole plant was found to have anti-cancer activity against walker
carcinosarcoma 256 (i.m.) in rat (Bhakuni et al. 1969).

 Objective of research
Each research study has its own purpose and objective. In this project, my
objective in this research is that to find about brahmi plant and its advantage and
phytochemical present in Brahmi plant.
METHOD AND METHODOLOGY OF PHYTOCHEMICAL OF BRAHMI

1) Methodology
I. Research design (Experimental Research design)
Experimental research design used to study phytochemical of Bacopa mannieri
II. Research area
This creeping and succulent plant commonly grows in marshy area. The places
where we can found Brami plant in Nepal are koshi Tappu in sunsari,
Ghodagahodi Lake and Beeshazari in Chitwan

III. Collection of Brahmi plant

We can collect brahmi from different part of terai region of Nepal. We should
collect this plant with very care. The upper portions of the stem should be cut up
to 4.5 cm of stem.

IV. Drying process of Brahmi plant

The plant should dried by spreading it clean area or sheets in sun for four to five
days, followed by shade drying for next seven to ten days. The dried material
should be stored in clean containers. Therefore, long storage should be avoided.

V. Powder process of Brahmi

You could dry the brahmi leaves and stem and make a powder by grinding the
dried leaves and stem beforehand. Otherwise, you can grind the raw leaves to
make a paste out of it.
VI. Method of extraction of Brahmi
We can extract brahmi from different method. Some of them are given below:
a. Plant tissue homogenization
DNA and RNA is isolation from plant tissues requires the homogenization
of sample. This often achieved by mortar and pestle tissue
homogenization or glass bead homogenization. The first of these
methods grinding the brahmi and other plants into a powder with a
mortar and pestle following the addition of liquid nitrogen.

b. Serial exhaustive extraction

The serial exhaustive extraction method using a solvent of increasing
polarity from hexane to water was designed to prepare crude extracts,
liquid partition was used to fractionate of active extracts.

c. Soxhlet extraction
Soxhlet extraction is an exhaustive extraction technique widely applied to
analysts that are sufficiently thermally stable. The extraction solvent is
continuously cycled through the matrix, by boiling and condensation,
with the brahmi being collected in the hot solvent.

d. Decoction
Decoction is a method of extraction by boiling brahmi which may include
stems, roots and bark and rhizomes to dissolve the chemicals of material.

e. Infusion
Infusion is the process of extracting chemical compounds or flavors from
plant material in a solvent such as water, oil or alcohol by allowing the
material to remain suspended in the solvent over time.

f. Percolation extraction
Percolation extraction is a conventional extraction method used in
processing of traditional medicines. After brahmi powder is placed in a
percolation tank, the extraction solvent is continuously added, and
percolation extract is collected simultaneously.
VII. Qualitative and Quantative analysis of phytochemical

A. Test for alkaloids

To 2ml of plant extract, 2ml of concentrated hydrochloric acid was
added. Then few drops of Mayer’s reagent were added. Then few drops
of Mayer's reagent were added. Presence of green color or white
precipitate indicates the presence of alkaloids.

B. Test for carbohydrates

To 2ml of the plant extract, 1ml of Molisch’s reagent and few drops of
concentrated sulphuric acid were added. Presence of purple or reddish
color indicates the presence of carbohydrates.

C. Test for flavonoids

To 2ml of plant extract, 1ml of 2N sodium hydroxide was added. Presence
of yellow color indicates the presence of flavonoids.

D. Test for Terpenoids

To 0.5ml of extract, 2ml of chloroform was added and concentrated sulphuric
acid was added carefully. Formation of red brown color at the interface
indicates presence of terpenoids.

E. Test for Phlobatannins

To 1ml of plant extract few drops of 2% HCL was added appearance of red color
precipitate indicates the presence of Phlobatannins.

F. Test for cardiac glycosides

To 0.5ml of extract, 2ml of glacial acetic acid and few drops of 5% ferric chloride
were added. This was under layered with 1 ml of concentrated sulphuric acid.
Formation of brown ring at the interface indicates presence of cardiac
glycosides.

G. Test for saponins

To 2ml of plant extract, 2ml of distilled water was added and shaken in a
graduated cylinder for 15minutes lengthwise. Formation of 1cm layer of foam
indicates the presence of saponins.
 H. Test for tannins
To 1ml of plant extract, 2ml of 5% ferric chloride was added. Formation of dark
blue or greenish black indicates the presence of tannins.

I. Test for phenols

To 1ml of the extract, a few drops of Phenol Ciocalteau reagent was added
followed by few drops of 15% Sodium carbonate solution. Formation of blue or
green color indicates presence of phenol.

J. Test for quinones

To 1ml of extract, 1ml of concentrated Sulphuric acid was added. Formation of
red color indicates presence of quinones.

VIII. Method of Detection

1 Chromatography method
1.1 High performance liquid chromatography(HPLC)
HPLC Analysis The separation was performed using a Shimadzu
HPLC system equipped with a SPD-M10AVP (PDA), an LC-10ATVP
pump and a Rheodyne injector with 20 µL loop. A Luna RP-18
column was used together with a Phenomenex RP-18 guard
column. The mobile phase consisted of 0.2% phosphoric acid and
acetonitrile. The pH of the mobile phase was adjusted to 3.0 with
5 M NaOH. The flow rate and total run time were 1.0 mL/min and
20 min, respectively. All peaks were integrated at the wavelength
of 205 nm. They were initially assigned by comparing retention
times with standards, and confirmed with characteristic spectra
obtained from the PDA.

1.2 High Performance Thin Layer Liquid

Chromatography(HPTLC)
In HPTLC, Bacoside A showed single peak at 0.43 Rf, which was
observed in the chromatogram of each samples. Calibration curves
were prepared by plotting concentrations versus average area of the
peak. The linear regression analysis of calibration plots of Bacoside A
exhibited linear relationship in the range of 100e600 ng with the
correlation coefficient (r2).
3. RESULT AND DISCUSSION

1. RESULT

i. Identification of phytocompounds from BmAE and BmME

Qualitative phytochemical analysis of aqueous and methanolic extracts of Brahmi plant

revealed the presence of phenolics and non-phenolics phytocompounds such as total phenols,
tannins and flavonoids, alkaloids, sterol, resins, terpenoids, xanthoproteins, quinones,
glycosides and saponins while the steroids and carboxylic acids were absent in both type of
extracts. Other compounds were found in fairly detectable quantity.

Table 1: Phytochemical composition of methanolic and aqueous extracts of Brahmi

Phytochemical constituents BmAE BmME

Alkaloids + +
Flavonoids + +
Steroids - -
Saponins + +
Phenols + +
Resins + +
Tannins + +
Terpenoids + +
Xanthoproteins + +
Quinones + +
Glycosides + +
Carboxylic acid - -

Evaluation of antioxidant potential of BmAE and BmME

 2. DISCUSSION

Brahmi has been traditionally used in Ayurvedic medicine to treat conditions such as fever,
inflammation, pain, asthma, epilepsy, and memory decline. In this study, the phytochemical
screening of aqueous and methanolic extract of whole plant of Brahmi revealed that among the
phytocompounds investigated, presence of phenolic (total phenols, tannins and flavonoids),
and non-phenolics (alkaloids, sterol, resins, terpenoids, xanthoproteins, quinones, glycosides
and saponins) compounds were detected while steroids and carboxylic acid were absent. The
presence of some of these secondary metabolites suggests that the plant might be of medicinal
importance. The methanolic extract of Brahmi gave more intense result as compared to the
aqueous extract. The phytochemical constituents present in Brahmi may be responsible for its
therapeutic role and other pharmacological properties, the constituents of its which are
reported here and detected during the experiments might be use for medicinal purpose. The
presence of phenolic compounds (total phenols, tannins and flavonoids) provides
pharmacological activities like anti-cancer, anti-oxidant, antimicrobial, and anti-inflammatory
that may suggest an association to the species here investigated.
4. CONCULSION

CONCLUSION

Brahmi is a very important and potent herbal plant use for various medicinal purposes. Its
antioxidant nature reflects its capability and helpful to prevent oxidative damage, hence
promoting health benefits. Animal experiments as well as in human studies, Brahmi have
shown pharmacological activities in brain improvement and neuroprotection effect. From the
present study it can be concluded that, the extractive value is useful to find the effective
solvent for extraction process. It gives idea about the nature of phytochemical constituents
present in the plant material. In the present study methanol has been found to be preeminent
solvent used for extraction. The ash value helps determine purity of a crude plant material
and foreign inorganic matter present as an impurity. For further analytical study of Brahmi
ash value is useful as it removes all traces of organic matter which may interfere further
experimentation. Loss on drying test is effective to measure the amount of moisture content
and volatile matters in a sample. Higher water content may prone to have chemical and
microbial decomposition of crude drug. The phytochemical screening is helpful for confirmation
of bioactive phytochemical constituents in Bacopa monnieri. The loss on drying has been found
less in Bacopa monnieri.
5 REFERENCES

I. http://ijrar.com/upload_issue/ijrar_issue_20542977.pdf- for gathering

information of Brahmi.
II. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778428/- for gathering
information of Brahmi.
III. https://en.wikipedia.org/wiki/Bacopa_monnieri-for gathering of
information of Brahmi.
IV. https://d-nb.info/1191207277/34- for gathering information of Brahmi.
V. https://www.researchgate.net/publication/
306038691_Study_of_Phytochemical_Screening_Physicochemical_Analysis
_and_Antimicrobial_Activity_of_Bacopa_monnieri_L_Extracts- for
gathering information of Brahmi.
VI. https://www.istockphoto.com/photos/brahmi-plant for photos of brahmi.
VII. https://chandigarh.gov.in/brahmi#:~:text=Introduction%3A%20This
%20plant%20is%20known,botanically%20identified%20as%20Centella
%20asiatica for gathering information of Brahmi.
VIII. https://www.ijpsonline.com/articles/qualitative-and-quantitative-
assessment-of-four-marketed-formulations-of-brahmi.html?view=mobile
for gathering information on uv spectroscopy to detect brahmi
IX. https://www.healthline.com/nutrition/bacopa-monnieri-benefits-for
gathering information on Brahmi.
X. https://innovareacademics.in/journals/index.php/ijpps/article/view/14739-
for gathering information on Brahmi.
XI. /8261#:~:text=The%20phytochemicals%20such%20as%20betulinic,The
%20Indian%20herb%20B- for gathering information on Brahmi.
XII.https://pharmeasy.in/blog/health-benefits-of-brahmi/ -for gathering
advantages of Brahmi.
XIII. https://www.keralaayurveda.biz/blog/brahmi-benefits-uses- for
gathering information on Brahmi.