EVALUATION OF THE ANTIFUNGAL ACTIVITY OF THE SUCCESSIVE

EXTRACTS FROM AERIAL PARTS OF Agrimonia Pilosa Ledeb.

A THESIS SUBMITTED TO ASSAM SCIENCE AND TECHNOLOGY UNIVERSITY,
GUWAHATI, ASSAM

IN THE PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

DEGREE OF BACHELOR OF PHARMACY

SUBMITTED BY-

Gopal Krishna Nath

ROLL NO.- 200510011032

REGN. NO.- 264505120

B. PHARM 8TH SEMESTER

UNDER THE GUIDANCE OF

Dr. Damiki Laloo (Professor), HOD Department of Pharmacogosy

GIRIJANANDA CHOUDHURY INSTITUTE OF PHARMACEUTICAL SCIENCE

(A Unit of Shrimanta Shankar Academy)

Approved by AICTE, PCI, affiliated to ASTU

N.H. 37, Hatkhowapara, Azara, Guwahati-781017

 GIRIJANANDA CHOWDHURY INSTITUTE OF PHARMACEUTICAL SCIENCE,
HATHKHOWAPARA, AZARA, GUWAHATI-781017, KAMRUP, ASSAM (INDIA)

GIRIJANANDA CHOWDHURY INSTITUTE OF PHARMACEUTICAL SCIENCE

(GIPS)

(A Unit of Shrimanta Shankar Academy)

Approved by AICTE & PCI & NBA Accredited, affiliated to Assam Science and
Technology University, N.H.37, Hathkhowapara, Azara, Guwahati- 781017

CERTIFICATE FROM THE PRINCIPAL

This is to certify that the topic incorporated in this dissertation “Evaluation of the antifungal
activity of the successive extracts from the aerial parts of Agrimonia pilosa Ledeb ” being
submitted by Gopal Krishna Nath, Roll no.-200510011032, Regn. No.-264505120, in the
partial fulfilment of the requirements for the award of Degree of Bachelor in Pharmacy of
Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati, affiliated to
ASSAM SCIENCE AND TECHNOOGY UNIVERSITY, GUWAHATI, ASSAM is a
bonafide assignment which has been carried out under the supervision of Dr. Damiki Laloo,
Professor,(HOD) Department of Pharmacognosy during the academic session 2023-2024.

Supervised by- Verified by-

Dr. Damiki Laloo Prof. ( Dr.) Bhanu P. Sahu

HOD of Pharmacognosy Principal , GIPS

Date- Date-
 DECLARATION

I hereby declare the thesis entitles “Evaluation of the antifungal activity of the successive
extracts from the aerial parts of Agrimonia pilosa Ledeb” is a bonafide assignment which has
been carried out under the supervision of Dr. Damiki Laloo, HOD, Department of
Pharmacognosy, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati,
affiliated to ASSAM SCIENCE AND TECHNOOGY UNIVERSITY, GUWAHATI,
ASSAM. The work embodied in the dissertation is original and has not been submitted in part or
full for the award of degree, diploma or fellowship of any other university or institute.

Gopal Krishna Nath

B. Pharm 8th semester

Roll no.- 200510011032

Regn. no.- 264505120

DATE:
 ACKNOWLEDGEMENT

First and foremost, from the core of my heart I would like to thank the society of Shrimanta
Shankar Academy (SSA), founder of this Institute of learning.

Any accomplishment requires the effort of many people and this work is no different. With
immense pleasure, I express my profound gratitude, heartfelt thanks and indebtness to my
teacher and supervisor Dr.Damiki Laloo, HOD, Department of Pharmacognosy, GIPS whose
patience and support was instrumental in accomplishing the project. I shall remain indebted to
her for her diligent efforts and professional competencies, which contributed towards the
completion of the project. I feel very much grateful and privileged to get an opportunity to word
under his.

I am extremely grateful to Prof. (Dr.)Bhanu P. Sahu, Principal, GIPS, for providing necessary
facilities for my work.

I convey my heartfelt thanks to all the teacher of GIPS, for their timely guidance, most valuable
suggestion and help throughout my course.

At last, I am greatly thankful to my B. Pharm friends for their constant inspiration. Words cannot
express my gratitude for my parents for making me capable of reaching this height of education.

Gopal Krishna Nath

8TH SEMESTER

BACHELOR OF PHARMACY

ROLL NO.- 200510011032

REGN. NO.- 264505120

DATE:
 CONTENTS

CHAPTERS PAGE NO

CHAPTER 1- INTRODUCTION -------------------------------------------- 1-4

CHAPTER 2- LITERATURE REVIEW------------------------------------ 5-10

CHAPTER 3- AIM & OBJECTIVES --------------------------------------- 11-13

CHAPTER 4- MATERIALS AND METHODOLOGY ----------------- 14-21

CHAPTER 5- RESULTS AND DISCUSSION ---------------------------- 22-32

CHAPTER 6- CONCLUSION ------------------------------------------------ 33-34

CHAPTER 7- REFERENCE -------------------------------------------------- 35-38

 LIST OF TABLES

List of tables Page no.

Table 1: Taxonomical classification and 7
synonyms.
Table 2: Percentage yield of successive 23
extracts.
Table 3: Solubility of Extracts. 23-24
Table 4: Preliminary Phytochemical 24
Evaluation for hexane extracts.
Table 5: Preliminary Phytochemical 25
Evaluation for chloroform extracts.
Table 6: Preliminary Phytochemical 26
Evaluation for ethyl acetate extracts.
Table 7: Preliminary Phytochemical 27
Evaluation for methanol extracts.
Table 8: Preliminary Phytochemical 28
Evaluation for aqueous extracts.
Table 9: Effect of different successive extracts 29
of Agrimonia pilosa on zone of inhibition (in
mm) against fungal strain.
 LIST OF FIGURES

 LIST OF FIGURE  PAGE NO

 Figure-1: Agrimonia pilosa  6

Ledeb.aerial parts.

 Figure-2: Agrimonia pilosa Ledeb.  6

Flowers and Fruits .

 Figure-3: Geographical distribution of  8

Agrimonia pilosa Ledeb.

 Figure-4: Diameter of zone of  30

inhibition (in mm) of Candida
albicans by different extracts
Methanol(MEAP), Hexane(HAP),Ethyl
acetate(EAAP).
 Figure-5: Diameter of zone of
inhibition (in mm) of Candida albicans  31
by different extracts Aqueous(AQAP)
and Chloroform(CAP).
ABSTRACT

Background: The plant Agrimonia pilosa is mainly distributed in China, Korea, Japan and
India's North Eastern regions particularly Meghalaya and Sikkim. The plant is employed in both
Ayurvedic formulations and a number of folk medicine. Malaria can be treated by the medicinal
paste of A. pilosa. Agrimonia pilosa is also used to treat dysentry by many tribes in Arunachal
Pradesh.

Aim: The aim of the present study was undertaken to perform the antifungal evaluation of the
sucessive extract from aerial parts of the Agrimonia pilosa .

Methods: Antifungal evaluation from the aerial parts of Agrimonia pilosa was evaluated based
on the standard method available in the literature that is disc diffusion method. The antifungal
activity of the aerial parts extract was examined against one fungal strain Candida albicans.

Results: When tested by the disc diffusion method and zone of inhibition, HAP (hexane extract),
CAP (chloroform extract), EAAP (ethyl acetate extract) and MAP (methanol extract), AQAP
(Water extract) of the aerial parts showed antifungal activity against tested microorganisms when
compared to Ketoconazole as standard. The successive extract showed antifungal activity
against tested microorganism Candida albicans.

Conclusion: The antifungal activity obtained in the present study will provide referential
information to researches to prevent microbial diseases and will maintain the pharmaceutical,
botanical and economical importance.
 CHAPTER-I

INTRODUCTION
1.INTRODUCTION:

Herbal medicines are the synthesis of therapeutic experiences of generations of practising

physicians of indigenous systems of medicine for over hundreds of years while nutraceuticals are
nutritionally or medicinally enhanced foods with health benefits of recent origin and marketed in
developed countries. The marketing of the former under the category of the latter is unethical.
Herbal medicines are also in great demand in the developed world for primary health care
because of their efficacy, safety and lesser side effects. They also offer therapeutics for age-
related disorders like memory loss, osteoporosis, immune disorders, etc. for which no modern
medicine is available. India despite its rich traditional knowledge, heritage of herbal medicines
and large biodiversity has a dismal share of the world market due to export of crude extracts and
drugs. WHO too has not systematically evaluated traditional medicines despite the fact that it is
used for primary health care by about 80% of the world population. However, in 1991 WHO
developed guidelines for the assessment of herbal medicine. Suggestions for herbal medicine
standardization are outlined. The scenario and perceptions of herbal medicine are discussed. i

Modulation of immune functions using medicinal plants and their products as a possible
therapeutic measure has become an accepted therapeutic approach. Plants and minerals have
been used since ancient times for the treatment of many ailments and diseases. It is now being
recognized that immunomodulation of immune response could provide an alternative to
conventional chemotherapy for a variety of disease conditions, especially when the host's
defense mechanism has to be activated under conditions of impaired immune responsiveness or
when a selective immunosuppressant has to be induced in situation like autoimmune disorders
and organ transplantation. Immunity is a homeostatic process, a series of delicately balanced
complex, multicellular and physiologic mechanisms that allow an individual to distinguish
foreign material from “self” and neutralize and/or eliminate the foreign matter. ii

Herbal medicine is still the mainstay of about 75 - 80% of the world population, mainly in the
developing countries, for primary health care (Kamboj, 2000). This is primarily because of the
general belief that herbal drugs are without any side effects besides being cheap and locally
available (Gupta and Raina, 1998). According to the World Health Organization (WHO), the use

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of herbal remedies throughout the world exceeds that of the conventional drugs by two to three
times (Evans, 1994). The use of plants for healing purposes predates human history and forms
the origin of much modern medicine. Many conventional drugs originated from plant sources: a
century ago, most of the few effective drugs were plant based. Examples include aspirin (willow
bark), digoxin (from foxglove), quinine (from cinchona bark), and morphine (from the opium
poppy) (Vickers and Zollman, 1999).iii

Antifungal agents are prescribed to treat and stop fungal infections. Due to the rising incidence
of immunocompromised people, extensive use of broad-spectrum antibiotics, and emergence of
drug-resistant fungal strains, fungus infections have become a serious public health concern. To
combat these infections and lower the rates of morbidity and mortality that go along with them, it
is imperative that efficient antifungal medications to be developed. Antifungal agents function by
concentrating on the fungal cells and preventing or eliminating their growth. Aspergillus niger,
Candida albicans, Aspergillus fumigatus are some of the example fungi.iv

Plant phytochemical with potent antimicrobial effect:v

Plants have long been known to produce a diverse range of phytochemicals, which possess
beneficial characteristics that have been harnessed by humans for thousands of years. The
discovery of potent and effective synthetic antimicrobial compounds revolutionized the treatment
and prevention of microbial infections. However, the rise of antibiotic-resistant bacterial strains,
along with concerns regarding the diminishing effectiveness and safety of synthetic
antimicrobials, has led to a renewed interest in phytochemicals as an alternative antimicrobial
solution. Researchers have conducted numerous studies, both in laboratory settings and in living
organisms, to investigate the antimicrobial properties of plant-derived phytochemicals.
Throughout history, various phytochemicals have been utilized in traditional folk medicines and
ethnomedicines. These phytochemicals can be classified into different major groups based on
their chemical structures, botanical origins, biosynthesis pathways, or biological properties. One
such group is phenolic compounds, which are characterized by the presence of an aromatic
benzene ring with at least one hydroxyl substituent, or a phenol structure.

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Phenolic compounds are commonly found throughout the plant kingdom and serve as a defense
mechanism against microbial diseases, UV radiation, and chemical stresses. The subclasses of
phenolic compounds include simple phenolic compounds (such as resorcinol and
phloroglucinol), phenolic acids and aldehydes, coumarins, flavonoids, chalcones, aurones,
benzophenones, xanthones, stilbenes, benzoquinones, naphthaquinones, anthraquinones,
betacyanins, lignans, and polyphenols (such as proanthocyanidin, galloyl, hexahydroxydiphenyl
ester, hydroxycinnamic acid, and phloroglucinol derivatives). Simple phenols and phenolic acids
have been shown to possess antibacterial, antiviral, and antifungal properties against various
microorganisms at different concentrations.

Coumarin, a chemical compound, can be categorized into five distinct groups based on its
structure. These groups include coumarins with substituents in the benzene ring, coumarins with
substituents in the pyrone ring, furocoumarins, pyranocoumarins, and coumarin dimmers.
Coumarins display a wide range of antimicrobial activity, showcasing their versatility in
combating various microorganisms.

Polyphenols found in plants consist of tannins, which are commonly categorized into two types:
hydrolysable tannins (HT) and condensed tannins (CT). Furthermore, these polyphenols exhibit a
range of antibacterial and antifungal properties.

Flavonoids are considered the most significant group of secondary metabolites present in
numerous plant species. Within the category of flavonoids, the main classes include flavones,
flavonols, flavanones, isoflavones, and anthocyanidins.

Alkaloids are a class of N-heterocyclic basic metabolites, as defined by their chemical structure.
The categorization of alkaloids can vary depending on the characteristics of their carbon
skeletons. Within plants, various alkaloids can be found, including pyridine (such as piperine),
piperidine, quinoline, indole, pyrrolidine, quinazoline, isoquinoline, glyoxaline, lupinane, tropan,
phenanthridine, imidazoline, and alkaloidal amines. These alkaloids possess strong antibacterial
properties, making them effective against both gram positive and gram negative bacteria.

Saponins are a group of high molecular weight glycosides that consist of a saccharide chain
moiety linked to a triterpene or steroid aglycone moiety. Various plant extracts, as well as pure

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saponins derived from different plants, have been found to possess antibacterial properties at
different concentrations.

Terpenoids, also known as essential oils, are composed of C5 isoprene units as their fundamental
structure. These compounds are classified into different categories based on the number of
isoprene units required for their synthesis. Monoterpenoids consist of 10 carbon atoms (C10),
sesquiterpenoids have 15 carbon atoms (C15), diterpenoids contain 20 carbon atoms (C20),
sesterterpenoids consist of 25 carbon atoms (C25), and triterpenoids are composed of even more
isoprene units.

Antimicrobial susceptibility testing (AST) is a crucial component of microbiology and clinical

practice as it aids in determining the appropriate antimicrobial drugs for the treatment of
bacterial infections. This testing involves evaluating the effectiveness of antimicrobial
medications against specific bacterial strains to determine their susceptibility or resistance. There
are several methods available to measure microbial growth or inhibition, such as viable counts,
direct microscopic count, turbidity measurement, bioluminescence, and fluorimetry. To assess
the impact of plant extracts or other antimicrobial agents on disease-causing pathogens, various
techniques like disc diffusion methods, broth dilution method, and cup plate method are
employed.vi

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 CHAPTER-II

LITERATURE REVIEW

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2.LITERATURE REVIEW:

2.1.Plant Profile:

Figure-1:Agrimonia pilosa Ledeb.aerial parts

Figure-2:Agrimonia pilosa Ledeb. Flower and Fruits

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 Table-1: Taxonomical classification and synonyms:vii

TAXONOMICAL CLASSIFICATION SYNONYMS

Kingdom-Plantae Agrimonia conopses Tschern ex CA Mey.
Phylum-Tracheophyta Agrimonia dahurics Schltdi patr ex Ledeb.
Class-Magnoliopsida Agrimonia eupatora vat japonica (Miq) Masarı
Order-Rosales Agrimonia glabrata CA Mey
Family-Rosaceae Agrimonia gadeliana Andrz
Genus-Agrimonia Agrimonia granulosa Juz
Species-Agrimonia pilosa Ledeb Agrimonia obtusifole Bar & Skv.

Vernacular name:viii ix x

1.Taniom(Arunachal)

2.Bherakuro(Nepali)

3.Hairy agrimony(English)

4. 짚신나물(Korean)

5.Gold mizuhiki(Japanese)

Agrimonia pilosa Ledeb., also known as hairy agrimony, is a flowering species in the family
Rosaceae and is found predominantly in the temperate zones of Eastern Europe and Eastern
Asia.xi xii
A. pilosa is a rich resource of bioactive constituents, essential oils, and homogenous
polysaccharides. Pharmacological publications reported that A. pilosa consisted of potentials in
anti-cancer, anti-bacteria, anti- oxidant, anti-inflammation, anti-virus, anti-diabetes, and so on. xiii
xiv

It has many lateral roots and a small tuberous rhizome. Its stem is yellowish-green or green
in color, with scanty pubescence. The leaf is oval and has similar-sized pointed teeth on the
edges. Length of 3-6 cm and width of 1.5-3.5 cm are the dimensions of the leaf. The leaf is also.

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hairy on both sides . The stem is associated with long and short glandular hairs without leaf
rosette. The petals reach is 2-4 mm, pale yellow, and oblong. The fruit is in the form of an
inverted cone, 5.5-6.5 x 3-4 mm. The bristles are 1-2.5 mm, inner and outer, converting to disc.
Flower blooms from June to August. The plant image is now available in several publications. xv
xvi

2.2.Geographical Distribution:

Figure-3: Geographical distribution of Agrimonia pilosa Ledeb.

Agrimonia pilosa Ldb. is a perennial herbaceous belonging to the Agrimonia genus in the
Rosaceae family, which is widely distributed in Northern Asia and Eastern Europe. xviiAgrimonia
pilosa, commonly known as hairy agrimony, is primarily distributed across East Asia, including
China, Japan, Korea, Russia (Far East), and Mongolia. It also occurs in parts of South Asia, such
as Nepal and Bhutan, and Southeast Asia, including northern Vietnam and Thailand. In Europe,
its presence extends to Eastern and Central regions like Russia (European part), Ukraine, Poland,
the Czech Republic, and Slovakia. This herbaceous perennial typically thrives in meadows,

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forest edges, grassy slopes, and roadsides, preferring well-drained soils and a variety of altitudes
from lowlands to mountainous areas. Agrimonia pilosa, commonly known as hairy agrimony, is
found in regions close to India, particularly in Nepal and Bhutan. It grows in the Himalayan
region, thriving in meadows, forest edges, and grassy slopes within these countries. Additionally,
it is also found in parts of northern Vietnam and northern Thailand, which are relatively close to
the northeastern borders of India.

Phytochemical profile of Agrimonia Pilosa:

Research on Agrimonia pilosa Ledeb (AP), a member of the Rosaceae family, renowned in
traditional Chinese medicine, has revealed significant pharmacological attributes. According to
studies, this plant exhibits anti-nociceptive, anti-inflammatory, antioxidant, anticancer, and α-
glucosidase inhibitory properties. The identified constituents of AP include 3-methoxy quercetin,
quercitrin (QU), quercetin (QC), tiliroside, ursolic acid, tormentic acid, and corosolic acid. Major
flavonoids found in AP consist of catechin (CT), luteolin (LT), quercetin (QC), isoquercetin
(IQC), hyperin (HP), apigenin (AG), vitexin (VT), kaempferol (KP), astragalin (AS), and afzelin
(AZ). These flavonoids play crucial roles in plant metabolism while possessing antioxidant,
antidiabetic, anticancer, and various inhibitory activities. The exploration of these bioactive
compounds in Agrimonia pilosa Ledeb sheds light on its potential therapeutic applications and
contributes to a deeper understanding of its medicinal properties.xviii

The investigation focused on the medicinal plant Agrimonia pilosa Ledeb, covering various
aspects such as its botanical description, traditional uses, phytochemistry, and pharmacological
properties. The research delved into comprehensive details concerning Agrimonia pilosa,
including its botanical characteristics, historical and traditional uses, chemical composition, and
the pharmacological effects associated with this plant. By studying these different facets,
researchers aimed to provide a thorough understanding of Agrimonia pilosa Ledeb and its
potential applications in medicine and healthcare.xix

The investigation into the constituents of Agrimonia pilosa Ledeb (AP) revealed the presence of
3-methoxy quercetin, quercitrin (QU), quercetin (QC), tiliroside, ursolic acid, tormentic acid, and
corosolic acid. Additionally, significant flavonoids identified in AP include catechin (CT),

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 9

luteolin (LT), QC, isoquercetin (IQC), hyperin (HP), apigenin (AG), vitexin (VT), kaempferol
(KP), astragalin (AS), and afzelin (AZ). These constituents are generally associated with
metabolic processes and possess antioxidant properties.xx

Research conducted on Agrimonia pilosa Ledeb., a member of the Agrimonia genus within the
Rosaceae family, has highlighted its significant medicinal properties, which have long been
recognized in traditional Chinese medicine. Historically, it has been utilized to address various
health issues such as tumors, trichomoniasis, vaginitis, diarrhea, and dysentery. Phytochemical
investigations have identified over 100 secondary metabolites in Agrimonia, which fall into six
categories: flavonoids, isocoumarins, triterpenes, phloroglucinol derivatives, tannins, and organic
acids. This research delves into the pharmacological aspects of these compounds, including their
potential as antitumor, anti-inflammatory, antioxidant, antibacterial, and antidiabetic agents.
Moreover, it examines the likelihood of parasites developing resistance to these substances. xxi

The investigation into Agrimonia pilosa Ledeb reveals its rich content of flavonoids, including
catechin, hyperoside, quercitrin, quercetin, and rutin. These compounds are identified as the
primary bioactive constituents with functions spanning anti-inflammatory, antitumor, antiviral,
antibacterial, and antioxidant properties. The antioxidant capabilities of the methanol extract
from Agrimonia pilosa Ledeb and its fractions were assessed using various radical scavenging
assays, including 2,2'-diphenyl-1-picrylhydrazyl(DPPH) and 2,2'-azinobis-(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays.xxii

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 CHAPTER-III

AIM AND OBJECTIVES

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3.AIM AND OBJECTIVES:

3.1 .Aim : Evaluation of the antimicrobial activity of the successive extracts from aerial parts of
Agrimonia pilosa Ledeb.

3.2 .Objectives:

 To perform the extraction of various soluble extracts by successive extraction methods

from the aerial parts of Agrimonia pilosa Ledeb.

 To perform the phytochemical evaluation of the various successive extracts of the aerial
parts of Agrimonia pilosa Ledeb.

 To evaluate the antifungal activity of various successive extracts from aerial parts of
Agrimonia pilosa Ledeb.

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3.3 . Plan of work:

Collection of the plant material and process it

Dring and grinding of the plant materials

Extraction of the plant material

Hexane Chloroform Ethyl acetate Methanol water

HAP CAP EAAP MEAP AQAP

Evaluation of Antifungal activity

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 CHAPTEFR-IV

MATERIALS AND METHODS

4.MATERIALS AND METHODS:

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4.1. Material for extraction:

Soxhlet apparatus, a round bottom flask, a condenser, petri-dish plates, a mechanical grinder
(GRYPHON), a desiccator (JSGW), a hot air oven, a digital balance (WENSAR), a heating
mantle, and various solvents such as hexane, chloroform, ethyl acetate, methanol, and water.

4.2. Material for antimicrobial activity:

Mueller Hinton agar, sterile petri dishes, sterile 6mm discs of Whatman filter paper no 1, sterile
forceps, distilled water, DMSO (Dimethylsulphoxide), chloroform, ethyl acetate, hexane,
methanol, ketoconazole and a micropipette.

4.3 Methodology:

4.3.1 Collection of plant materials and authentication:

The plant material was collected from the Jowai region (local market) of Jaintia hills in the state
of Meghalaya (India) between the months of April-May 2023.

4.3.2. Preparation of the crude plant material for extraction:

The plant material that was acquired was longitudinally divided into small fragments and
subsequently subjected to the tray drier for drying, with a consistent temperature of 35 to 37°C.
Afterward, a mechanical grinder was employed to crush the dried seeds into a coarse powder,
which was then sifted through an 80-mesh sieve.

4.3.3. Extraction process:

The extraction process of the dried coarse powdered drug (60gm) of A. pilosa was carried out
using a Soxhlet apparatus with 200ml solvents such as hexane, chloroform, ethyl acetate, and
methanol. After methanol extraction, the leftover marc was dried and macerated in water for 24
hours. Each extract was then stored in a desiccator before use. Specific codes were assigned to
each extract: HAP (Hexane extract), CAP (Chloroform extract), EAAP (Ethyl acetate extract),

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MeAP (Methanol extract), and AQAP (Aqueous extract). Calculations were performed to
determine the characteristics and percentage yield of each successive extract.

4.4. Preliminary phytochemical evaluation:

The preliminary phytochemical screening of all the successive extracts was performed for the
investigation of the different constituents. Procedures and the test for the specific chemicals were
done as per the standard methods available in the literature.xxiii

Tests for alkaloids:

50mg of solvent-free extract is taken and it is then stirred with a few ml of dilute hydrochloric
acid & filtered. The filtrate is tested carefully with various alkaloidal reagents as follows:

Mayer's test: To a few ml of filtrate add 1 or 2 drops of Mayer's reagent (potassiomercuric iodide
solution) by the side of the test tube. A white or creamy precipitate indicates the test is positive
for alkaloids.

Wagner’s Test: To a few ml of filtrate, a few drops of Wagner's reagent (solution of iodine in
potassium iodide) are added by the side of the test tube. A reddish-brown ppt indicates the test as
positive.

Hager’s Test: To a few ml of filtrate, 1 or 2ml of Hager's reagent (saturated solution of picric
acid) is added by the side of the test tube. A prominent yellow ppt indicates test as positive.

Dragendorff’s Test: To a few ml of filtrate, 1 or 2ml of Dragendorff's reagent (solution of

potassium bismuth iodide) is added by the side of the test tube. A prominent reddish brown ppt
indicates the test as positive.

Tests for carbohydrates:

100mg solvent free extract is dissolved in 5ml of water & filtered. The filtrate is subjected to the
following tests.

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Molish’s Test: To 2 ml of filtrate, 2 drops of alcoholic soln of alpha-naphthol are added, the
mixture is shaken well & Iml of conc. H2SO4 is added slowly along the side of the test tube &
allowed to stand. A violet ring indicates the presence of carbohydrates.

Fehling’s Test: 1ml of filtrate is boiled on water bath with 1ml each of Fehling solution A
(copper sulphate in distilled water) & Fehling solution B (alkaline sodium potassium tertarate
distilled water); a red ppt indicates the presence of sugar.

Benedict’s Test: To 0.5ml of filtrate, 1ml of Benedict's reagent (combination of copper sulphate,
sodium citrate and sodium carbonate) is added. The mixture is heated on a boiling water bath for
2 min. a characteristic colored ppt indicates the presence of sugar.

Barfoed’s Test: To 1 ml of filtrate, 1 ml of Barfoed's reagent (copper acetate and acetic acid) is
added & heated on a water bath for 2 min. Red ppt. indicates presence of sugar.

Tests for saponins:

50 mg extract is taken and diluted with distilled water and volume made up to 20ml. The
suspension is shaken for 15 min. A layer of 2 cm of foam indicates the presence of saponins.

Tests for phenolic compounds:

Ferric chloride test: 50mg of extract is dissolved in 5ml of distilled water. To these few drops of
natural 5% ferric chloride solution is added. A dark green color indicates the presence of
Phenolic compounds.

Lead Acetate test: 50mg of extract is dissolved in distilled water & to this; 3ml of 10% lead
acetate solution is added. A bulky white ppt. indicates the presence of Phenolic compounds.

Tests for glycosides:

Legal’s test: 50mg of extract is dissolved in pyridine, sodium nitroprusside solution is added &
made alkaline using 10% NaOH & presence of glycoside is indicated by pink color.

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Borntrager’s test: 50 mg of extract is hydrolyzed with concentrated hydrochloric acid for 2hr on
a water bath and then filtered it. To 2ml of filtered hydrolysate, 3ml of CHC13 is added &
shaken, CHC13 layer is separated & 10% NH3 solution is added to it; pink color indicates the
presence of glycosides.

Keller-Killiani’s test: To the extract add glacial acetic acid and to this few drops of ferric
chloride & conc. Sulfuric acids are added. A reddish-brown color is formed at the junction of
two layers & the upper layer turns bluish green.

Tests for tannins:

Gelatin test: 50mg of extract is dissolved in 5ml of distilled water & add 1% solution of gelatin
containing 10% sodium chloride solution is added. White ppt. indicates the presence of Tannins.

Goldbeater’s skin test: Soak a small piece of goldbeater's skin (a membrane prepared by the skin
of ox) in 2% hydrochloric acid rinse with distilled water and placed in the solution to be tested
for 5 minutes. Wash with distilled water and transferred it to 1 percent solution of ferrous
sulphate. A brown or black colour on the skin denotes the presence of tannins.

Phenazone test: 50mg of extract is dissolved in 5ml of distilled water & add 0.5gm of sodium
acid phosphate; warm, cool and filter. To the filtrate add 2% solution of phenazone. Bulky
coloured ppt. indicates the presence of tannins.

Tests for steroids and triterpenoids:

Libbermann-Burchard test: The extract is treated with few drops of acetic anhydride and boil
cool then concentrated H2SO4 is added from the side of the test tube, brown ring is formed at the
junction of two layers and the upper layer turns green which shows the presence of steroids and
formation of deep red colour indicates the presence of triterpenoids.

Tests for flavonoids:

Alkaline reagent test: An aqueous solution of the extract is treated with 10% NH4OH solution.
Yellow fluorescence indicates the presence of flavonoids.

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 18

Aqueous sodium hydroxide test: An aqueous solution of the extract is treated with sodium
hydroxide solution it gives blue to violet (anthocyanins), yellow (flavones), and yellow to orange
(flavonones).

Concentrated sulfuric test: An aqueous solution of the extract is treated with concentrate
sulphuric acid it gives yellowish orange (anthrocyanins), yellow to orange (flavones), orange to
crimson (flavonones).

Magnesium & hydrochloric acid reduction: 50mg of extract is dissolved in 5ml of alcohol & few
fragment of magnesium ribbon & conc. HCl acid (drop wise) is added. If any pink to crimson
color develops, presence of flavanol glycosides is inferred.

Tests for protein & amino acids:

100mg of extract is dissolved in 10ml of distilled water & filter through Whattmann filter paper
no.1& the filtrate is subjected to tests for proteins & amino acids.

Millon’s Test: To 2ml of filtrate, few drops of Millon's reagent (combination of mercury, fuming
nitric acid and distilled water) are added. A white ppt. indicates the presence of proteins.

Biuret Test: An aliquot of filtrate is treated with one drop of 2% copper sulfate solution. To this
Iml of ethanol (95%) is added, followed by an excess of potassium hydroxide palate. The pink
color in the ethanolic layer indicates the presence of proteins

Ninhydrin Test: Two drops of Ninhydrin solution (10mg of Ninhydrin in 200 ml of acetone) are
added to 2 ml of aqueous filtrate. A characteristic purple color indicates the presence of amino
acids.

Tests for fixed Oils & Fats:

Spot Test: Press a small quantity of extract separately between two filter papers. Oil stains on the
paper indicate the presence of fixed oil.

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Saponification test: Add a few drops of 0.5 N alcoholic KOH to a small quantity of extract along
with a drop of phenolphthalein. Heat the mixture on a water bath for 1-2 hr. Formation of soap or
partial neutralization of alkali indicates the presence of fixed oils & fats.

Tests for gums & mucilages:

100 mg of the extract is dissolved in 10 ml of distilled water & to this 25 ml of absolute alc. is
added with constant stirring. White or cloudy ppt. indicates the presence of gums & mucilage

4.5.Collection of microbial strains:

The strain Candida albicans (ATCC-10231) was sourced from the Department of Microbiology
at Girijananda Chowdhury Institute of Pharmaceutical Science, Azara.

4.6. Antifungal activity study:xxiv

The antifungal properties of Agrimonium pilosa was tested against one standard fungal strain
Candida albicans. The microbial cultures utilized in this research were sourced from the
Department of Microbiology at Girijananda Chowdhury Institute of Pharmaceutical Science in
Guwahati, Assam, India.

The antimicrobial activity was assessed using the disc diffusion method, with Muller Hinton agar
serving as the nutrient media. To prepare the agar, 6g of Muller Hinton agar was added to 150ml
of distilled water in a conical flask and heated over a flame. In order to ensure sterility, the Petri
plates, forceps, discs, cotton, and agar media were sterilized in an autoclave at a temperature of
121°C. The standards and extracts were prepared at a concentration of Img/ml. As a positive
control, ketoconazole was utilized. Specifically, 1mg of ketoconazole was mixed with 1ml of 1%
DMSO. Additionally, a blank was prepared using 0.9ml of methanol and 0.1ml of DMSO.

The fungal cultures were delicately inoculated onto the agar plates using sterile cotton.
Subsequently, 100 µg/ml concentration of standard and consecutive extracts were applied onto 6
mm discs of Whatman filter paper 1 using a micropipette. After drying, the discs were placed in
contact with the organism-containing surface, labeled, and then incubated for 24 hours at 37°C.

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 20

The experiments were conducted in replicates, and the outcomes were presented as the diameter
(mm) of the inhibition zones.

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 CHAPTER-V

RESULTS AND DISCUSSION

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5.RESULTS AND DISCUSSION:

5.1.Percentage yield of successive extracts:

Table-2: Percentage yield of successive extracts:

Solvents Colour of Nature of the Weight of Yeild of % Yeild

the extracts the plant extracts (in (% w/w)
extracts material gm)

HEXANE Green Sticky mass 100g 82.80g 82.80%

CHLOROFORM Green Dry 100g 10.80g 10.80%

ETHYL Brown Sticky mass 100g 28.50g 28.50%

ACETATE

METHANOL Brown Sticky mass 100g 47.83g 47.83%

AQUEOUS Reddish Dry 100g 25.68g 25.68%

Brown

5.2. Solubility of Extracts:

Table -3: Solubility of Extracts:

Successive Extract Solvent solubility

Hexane Chloroform Methanol

Hexane extract Soluble Soluble Soluble

Chloroform extract Soluble Soluble Soluble

Ethyl Acetate extract Partially soluble Insoluble Insoluble

Methanol extract Insoluble Soluble Soluble

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 23

 Aqueous extract Insoluble Insoluble Insoluble

5.3. Preliminary Phytochemical Evaluation:

The results for the preliminary phytochemical screening for various successive
extracts of the Agrimonia pilosa a tabular format

Table-4:For Hexane extract

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Table-5:For Chloroform Extract

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Table-6:For Ethyl acetate Extract

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Table-7:For Methanol Extract

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Table-8: For Aqueous Extract

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 28

5.4.Antifungal activity: The tested successive extracts of Agrimonia pilosa Ledeb showed
antifungal effects when tested with Candida albicans. The zone of inhibition (in mm) in MEAP
(Methanol extracts) ,EAAP (Ethyl acetate extracts) ,AQAP (Aqueous extracts) when tested with
Candida albicans fungal strain showed significant efficacy when compared to Ketoconazole
taken as the standard control(Table-9) and HAP (Hexane extracts) ,CAP (Chloroform extracts)
showed no noticeable antifungal effects over Candida albicans.

Table-9:Effect of different successive extracts of Agrimonia pilosa on zone of inhibition (in

mm) against fungal strain.

Extract Concentration Zone of inhibition

(in mm)
Candida albicans

Ketoconazole 100µg/ml 2.5

(standard control)

HAP(Hexane) 100µg/ml NIL

CAP(Chloroform 100µg/ml NIL

)

EAAP(Ethyl 100µg/ml 0.5

acetate)

MEAP(Methanol) 100µg/ml 0.5

AQAP(Water) 100µg/ml 0.3

Blank 100µg/ml 0.0

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 29

Figure-4: Diameter of zone of inhibition (in mm) of Candida albicans by different extracts
Methanol(MEAP), Hexane(HAP),Ethyl acetate(EAAP).

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 30

Figure-5: Diameter of zone of inhibition (in mm) of Candida albicans by different extracts
Aqueous(AQAP) and Chloroform(CAP).

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 31

DISCUSSION:

The antimicrobial activity of Agrimonia pilosa has been assessed. Phytochemical constituents
exhibit in plants to be specified alkaloids, flavonoid, tannins and terpenoids are delivering an
energizing open door for expansion of modern therapies against an extensive variety of
microorganisms. In the present study, fungal strain was chosen for screening antifungal impact
of the extract to check the antimicrobial spectrum. The extracts of the studied plants exhibited
varying zone of inhibition activity against tested fungal strain. The Hexane (HAP), Chloroform
(CAP), Ethyl acetate (EAAP), Methanol (MEAP) and Aqueous (AQAP) extracts are used and
the standards used in this study is Ketoconazole.

The antifungal activity against Candida albicans shown by the MEAP (Methanol extract),
EAAP ( Ethyl acetate extract), AQAP (Aqueous extract) and CAP (Chloroform extract),
HAP(Hexane extract) were not active against Candida albicans.

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 32

 CHAPTER-VI

CONCLUSION

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 33

CONCLUSION:

Plant has been a very vital source of drugs. It is imperative to use herbal products for the
biocontrol of diseases as a novel, developing alternative to antimicrobial therapies that results
nontoxic and more environmentally friendly controlling for microbial diseases.

It is now necessary to consider some alternative and effective therapies, such herb,due to rapid
development of resistance against chemotherapeutic medicines. The plant phytochemical
constituents for example tannins, alkaloids, flavonoids, phenolic compounds and several other
aromatic compounds are secondary metabolites of plant that fill in as barrier component against
predation by numerous microorganisms.

In this study, the antifungal activities of Agrimonia pilosa was evaluated by disc difussion
method. The microorganisms chosen to be studied was C. albicans . The study showed that it
has antifungal activity against tested microorganism. Since the inhibition was observed under
100 ug/ml, the aerial parts of Agrimonia pilosa was considered to have a decent antifungal
action.

Further research is necessary to shed information on the biological effects of Agrimonia pilosa
and its bioactive components against a variety of disorders.

Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 34

 CHAPTER-VII

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Girijananda Chowdhury institute of Pharmaceutical Science,Guwahati-17 Page 35

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