A Synopsis on

Development and Evaluation of Polyherbal Antifungal Formulation

Submitted in the partial fulfillment of the requirement for a Degree of

MASTER OF PHARMACY

IN

PHARMACOGNOSY & PHYTOCHEMISTRY

In the faculty of Pharmaceutical Sciences (Science and Technology)

Sant Gadge Baba Amravati University, Amravati

Submitted By

RAJASHRI R. DHONNAR

(B. Pharm)

Research Guide

MRS. MONA Y. AGARWAL

(M. Pharm)

Assistant Professor

GOVERNMENT COLLEGE OF PHARMACY, AMRAVATI

KATHORA NAKA- 444604(M.S)

2022-2023
 INDEX

SR NO. CONTENT PAGE NO.

1. Introduction 1-5

2. Literature Review 6-13

3. Aim and Objective 14

4. Plant profile 15-20

5. Plan of work 21

6. Reference 22-24
 DEVELOPMENT AND EVALUATION OF POLYHERBAL ANTIFUNGAL FORMULATION

INTRODUCTION:
The human skin, among its other functions, serves as an effective shield against an invasion
of the body by microorganisms, including fungi. This protective function fails when the surface
integrity is changed by environmental factors such as moisture, humidity and heat, friction,
trauma, and other immunosuppressive factors. There is wide variation in the prevalence and
aetiological agents of superficial fungal infections reported in different parts of the world. Skin
diseases are a public health problem in developing countries. Superficial fungal infections are
known to occur in areas characterized by poor hygiene and low socioeconomic status.1
Infections caused by pathogenic fungi and limited to the human hair, nails, epidermis, and
mucosa are referred to as superficial fungal infections. Despite the fact that these infections
rarely are dangerous or life-threatening, they are important because of their worldwide
distribution, frequency, person-to-person transmission, and morbidity. Dermatophytosis (tinea
or ringworm), pityriasis Versicolor (formerly tinea versicolor), and candidiasis (moniliasis) are
the three most common types of superficial fungal infections.
A. The dermatophytes are a large group of related fungi that can infect human skin, hair,
and nails; they are found in soil (geophilic organisms), on animals (zoophilic), and on
humans (anthropophilic). Dermatophytosis is named by the involved area of the body
(e.g., tinea corporis, tinea capitis¸ tinea pedis, tinea unguium).
B. Pityriasis Versicolor is caused by the yeast form of a dimorphic fungus that is

considered part of normal human skin.

C. Candidiasis is caused by a yeast-like fungus of the genus Candida (most commonly C.

Albicans) that is part of the microflora in the human gastrointestinal tract (including the
mouth) and the vagina.2
Skin Fungal Infection Symptoms:

• Irritation
• Scaly skin
• Redness
• Itching
• Swelling
• Blisters

Causes: common causes of skin fungal infection include poor hygiene, dermatophytes, and
environmental factor like moisture, humidity, heat, etc.

Treatment of fungal infection depends on the type of infection. Medicated creams can treat most
skin infections. Treatment usually involves antifungal medications that you put on your skin. You
might use an over-the-counter cream such as:

• Clotrimazole (Lotrimin, Mycelex)

• Miconazole (Micatin, Monistat-Derma)
• Terbinafine (Lamisil)

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If you use too much allopathic or any over-the-counter (OTC) cream, spray, or solution or use
it more often than you need to, it may make your skin red or irritated. Other side effects of
OTC drugs include Burning, stinging, swelling, irritation, redness, pimple-like bumps,
tenderness, or flaking of the treated skin may occur.3

Herbal medicines in systems have been used for a long time and are documented with their
special theories and concepts, and accepted by the countries. Plants and natural products were
used by humankind over the years as food and medicines to cure and prevent diseases.4
The people of India are rich in ethnomedical tradition and use many herbal preparations to cure
skin infections and other diseases instead of conventional synthetic drugs. The tribals and the
local people of this region use various plants either in crude form or as decoctions, poultices,
etc. against a variety of dermatological disorders. The use of medicinal plants against various
bodily ailments is a common practice here mainly among those residing in the hilly and rural
areas, having easy access to these natural products. The plant parts used include leaves, bark,
seeds, shoots, latex, roots, and in many cases the whole aerial parts together. In particular cases,
two to three plants are mixed together in equal proportion in the preparations and given to the
patients. The commonest methods of preparing skin ethnotherapeautics in this region were
direct topical applications of the leaf juice, latex, or macerated parts of the medicinal plants
confined to the affected part of the body.5
Some medicinal plants which having antifungal properties
Allium sativum
Zingiber Officinalis
Glycyrrhiza Glabra
Curcuma longa
Adhatoda vasica
Ocimum sanctum
Azadiracta Indica
Vitex negundo
Murraya koenigii
Withania somnifera
Piper betel6
The present investigation deals with the use of herbs and prepare topical semisolid formulation
in the treatment of fungal infection.
Topical semi-solid formulations are used to treat various skin and systemic diseases. They help
avoid hepatic first-pass metabolism and serum absorption is minimal; furthermore, high drug
concentration in the skin is achieved by topical administration. This is important for long-term

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therapy, for example, therapy of fungal diseases, which can be classified into superficial,
subcutaneous, and systemic mycoses. The most common fungal infections are
dermatophytosis. We hypothesized that the incorporation of antifungal active substances in a
semisolid formulation could ensure suitable contact of the drug and the skin and its sufficient
release. semi-solid preparation comprises small amounts of a solid dispersed in a relatively
large amount of a liquid. it might provide faster drug release compared to solid dosage form,
regardless of the water solubility of the substance.7
Neem consists of all aerial parts of the plant known as Azadirachta indica, family: Meliaceae.
It contains diterpenes, nimbiol(bark), and triterpenes: beta-sitosterol, stigmasterol(leaf),
limonoids: maliantriol, nimbidinine, nimbebdiol, and azadiractin, flavonol glycoside: nimaton,
quercetin, myricetin, kaempferol. Ethanolic extract of the leaves is fungicidal and antibacterial.
Nirgundi consists of dried leaves and flowers of the vitex negundo family: Verbenaceae. It
contains vitexin and isovitexin, the leaves contain iridoid glycosides, isomeric flavanones, and
flavonoids, besides casticin and the glucosides, luteolin-7-glucoside and alpha D-glucoside of
a tetrahydroxy monomethoxy flavone. Dried powder of roots contains hentriacontane, beta-
sitosterol, and its aetat and stigmasterol. Leaf used (also the root) in excessive vaginal
discharges, oedema, skin diseases, pruritus, helminthiasis, rheumatism, and puerperal fever.
Curry leaves consist of leaves and aerial part of the plant known as Murraya koenigii family:
Rutaceae. All parts of the plant, especially the leaves, are rich in carbazole alkaloids (several
carbazole bases have been isolated). The leaves also gave a coumarin glucoside, scopolin. the
steam distillate of the leaves is reported to exhibit antifungal and insecticidal activities. The
ethanolic extract of the stem bark showed an anti-inflammatory effect.8

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MARKETED FORMULATION:
1) Brand name: Bioayrveda
Product name: Antifungal healing cream
Type: Cream
Content: Turmeric
Garlic
Coconut oil
Lemongrass oil

2) Brand name: Vidhmaan Ayurvedic

Product name: Itch coat antifungal Malam
Type: Cream
Content: Sudha gandhak
Neem
Karanj
Vetas
Turmeric
Camphore

3) Brand name: Al Arsh

Product name: antifungal cream
Type: Cream
Content: Vaseline
Coconut oil
Khaskari
Panwad

4) Brand name: ABHINAV

Product name: Fungon antifungal cream
Type: Cream
Content: Sesam oil
Yashtimadhu
Jati
Kumari
Sonthi
Neem oil
Chakaramarda oil
Shuda gandhak

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5) Brand name: Inducare Pharma

Product name: Fungiwin cream
Type: Cream
Content: Shuddha gandhak
Shuddha hingu
Karan tail
Shatavar

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LITERATURE SURVEY:
Ethnopharmacological survey
Venugopalan Santhosh Kumar et al. (2013)
Neem (Azadirachta indica) is principally cultivated in the Indian subcontinent.
According to historical records, neem has been used extensively by humankind to treat various
health issues. World health organization stated that all of the population living in developing
countries depends on traditional medicine. On the Indian subcontinent, the Azadirachta indica
tree has been used for more than 4500 years. The earliest documentation of neem mentioned
the fruit, seeds, oil, leaves, roots, and bark for their advantageous medicinal properties. For
hundreds of years, millions have cleaned their teeth with neem twigs, smeared skin disorders
with neem leaf juice, taken neem tea as a tonic, and placed neem leaves in their beds, books,
grain bins, cupboards, and closets to keep away troublesome bugs. The tree has relieved so
many different pains, fevers, infections, and other complaints that it has been called “the village
pharmacy9.”

Rakesh Kumar Singh (2019)

Neem (Azadirachta indica) has been a significant constituent formula of traditional
healers and homeopathic, ayurvedic in the faculty of the Indian medicine system. The neem
tree is also found in places such as Australia, Africa, Asia, and America, and some small-scale
plantations are also reportedly successful in Europe and the United States of America. The
neem is a very well-known plant in India its uses are also mentioned in Ayurveda. The whole
plant of Azadirachta indica shows its medicinal property.
Flowers: These neem flowers can be used fresh, dried, or in powder form. These are used in
south India for cooking and garnishing dishes. Neem flowers can be used to treat anorexia,
nausea, intestinal worms, and bleaching.
Pulp: The pulp of fruits is useful for medicines and for the production of methane gas.
Wood: Wood is used to make furniture, it is very long-lasting.
Bark: The bark of the neem used for healthy hair detoxifies the blood and balances blood sugar
level
Seeds: The seeds are used to treat leprosy, and kill intestinal worms. It is also used for birth
control.
Seeds oil: used to treat the symptoms of eczema, leprosy, and other skin diseases10.

Balraj Singh Gill et al. (2018)

Vitex negundo is an ethnobotanically important plant with enormous medicinal
properties. The Vitex is used as a folk medicine in Bangladesh, India, China, Indo-China,
Indonesia, Nepal, Pakistan, Philippines, and Sri Lanka. Traditionally leaves are used for eye
diseases, inflammatory disorders, skin infections, and arthritis. Dry nirgundi leaves are placed
with stored woolen garments, as the leaves prevent wool-destroying insects and worms. There
are different species of Vitex such as Vitex agnus-castus, V. rotundifolia, V. negundo, and
others, which have medicinal importance11.

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Nurkhalida Kamal et al (2022)

The Vitex negundo tree, including its leaves and fruits, has been traditionally used for
herbal remedies in the form of pastes, decoctions, and dried fruits since ancient times.
Traditional Medicinal practices including Ayurveda, Unani medicine, Chinese traditional
medicine, Malay traditional medicine, European medicine, and ancient Greek medicine noted
and recognize the medicinal value of the herb nirgundi in their health practices. They
mentioned the use of Vitex for a wide range of conditions, such as treating women with
reproductive disorders, improving body health after childbirth, suppressing libido, treating skin
problems, cure symptoms of gastrointestinal afflictions, reducing fever, healing rheumatism,
etc12.

Vandana Jain et al (2012)

In the Indian system of medicine like Ayurveda, Unani, Siddha Murraya koenigii
mention as a medicinally important herb and have lots of therapeutic properties. especially leaf,
stem, bark, and oil show therapeutic action. M. koenigii is widely used in Indian cookery for
hundreds of years and has a versatile role to play in traditional medicine. The plant is credited
with tonic and stomachic properties. Bark and roots are used as stimulants and externally to
cure eruptions and bites of poisonous animals. Green leaves are eaten raw to cure dysentery
and diarrhoea and for checking to vomit. Leaves and roots are also used traditionally as a bitter,
anthelmintic, analgesic, curing piles, inflammation, and itching, and are useful in leukoderma
and blood disorders13.

Dheeraj K. Gahlawat et al (2015)

Murraya koenigii is a native of India, Sri Lanka, and other south Asian countries.
Murraya koenigii is distributed throughout India and is abundantly found from Sikkim to
Garhwal, Bengal, Assam, Western Ghats, and Kerala. It reached Malaysia, South Africa, and
Reunion Island from India along with South Indian immigrants. Murraya koenigii is an
aromatic leaf commonly known as curry leaves. Leaves and aerial parts of plants have some
phytoconstituents that show ethnopharmacological activity.
For example- Stem: Used as Datum for cleaning, strengthening gums and teeth
Leaves: Used as Stomachic Purgative Febrifuge Anti-Anemic Anti-Helminthic Analgesic
Anti-ulcer Antinociceptive Antiamnesic Anti Inflammation, cooling and itching Hair tonic
Stimulant of hair growth14.

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Phytochemical survey:

Santosh Kumar Srivastava et al. (2020)

Azadirachta indica contains the phytochemicals which are active against different
pathogens, nimbolinin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, salannin, and
quercetin. Leaves contain ingredients such as nimbin, nimbanene, 6-desacetylnimbinene,
nimbandiol, nimbolide, ascorbic acid, n-hexacosanol and aminoacid,7-desacetyl-7-
benzoylazadiradione,7-desacetyl-7-benzoylgedunin,17hydroxyazadiradione, and nimbiol”.
“Fresh leave extract of Neem gives quercetin and -sitosterol, polyphenolic flavonoids, they
have antibacterial and antifungal properties, and the neem seeds contains constituents including
gedunin and azadirachtin in it”. Antifungal activity has been found in aqueous extracts of Neem
oil, leaf extract, bark extract and the other parts of Neem extracts15.

M. Mukunda Vani et al (2016)

Azadirachta indica is a tree which having number compounds that can be used for
medicinal purposes. Extraction process of Azadirachta indica compounds has been done using
solvents namely ethanol, methanol, benzene, ethyl acetate, toluene which are widely used in
industries as solvents. All the compounds present in Azadirachta indica leaves are supposed to
be soluble in solvents. The semi quantitative analysis has been done with the help of thin layer
chromatography (TLC) which shows the efficiency of every individual solvent to extract
compounds from Azadirachta indica. Thin layer chromatography can physically separate the
compounds from pure extracts hence this method is preferred for analysis purpose. The
standard RF values of Nimbin, Ascorbic acid, Amino acid, Azadirachtin are 0.09, 0.74, 0.62,
0.70 respectively which are obtained from literature. data we can say that alcoholic solvents
are most effective throughout the study since they are able to extract maximum number of
compounds present in Azadirachta indica leaves. The capacity of particular solvent to extract
the compounds present in Azadirachta indica leaves is as follows, Benzene < Toluene < Ethyl
acetate < Alcohol16.

Imam Hashmat et al (2012)

Neem contain Biologically active principles isolated from different parts of the plant
include: Azadirachtin, meliacin, gedunin, nimbidin, nimbolides, salanin, nimbin, valassin,
meliacin forms the bitter principles of Neem oil, the seed also contain tignic acid responsible
for the distinctive odour of the oil. Neem kernels contain 30-50 % of oil mainly used by the
soap, pesticide and pharmaceutical industries and contain many active ingredients which are
together called triterpene or limnoids. The four best limnoids compounds are: Azadirachtin,
Salannin, Meliantriol, and Nimbin. Limonoids contain insecticidal and pesticidal activity17.

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Benjamin Gosse et al (2005)

The seed of Azadirachta indica (Meliaceae) was analyzed by various chromatographic
methods. The hexane extract yielded six fatty acids and six unsaponified compounds resolved
by high-speed counter current chromatography (HSCCC) and confirmed by GC. These
compounds are the main constituents of the solid substance of the hexane extract of the seed
used in traditional medicine since ancient times. GC analysis of the fatty acids showed that it
contained nine fatty acids. The most abundant are 43.1% of D1 -oleic acid, 19.4% of palmitic
acid, 17.6% of D2 -linoleic acid, 16.4% of stearic acid, 1.3% of arachidic acid; GC analysis of
the unsaponifiable fraction showed that it contained sixteen steroid alcohols. The most
abundant are 62.92% of b-sitosterol, 19.17% D7 -stigmasterol, 15.65% D7 -avenasterol, 1.18%
4-methylcholest-7-enol, and the minor compounds are 0.80% D5 - campesterol and 0.20%
cholesterol. A series of simple phenols were found, such as hydroxy-tyrosol, tyrosol, vanillic
acid, and p-counaric acid. Vanillin 4-(acetoxyethyl)-1,2- dihydroxybenzene were also found18.

Sunil pawar, Vanita kamble (2017)

Phytochemical screening of leaves extracts of vitex negundo revealed the presence of
carbohydrates, proteins, amino acids, steroids, glycosides (cardiac, anthraquinone and
saponin), flavonoids, tannins and phenolic compounds.
The ICP-MS analysis study revealed the presence of major elements like Ca, Mg, Na, K and P
have been found in maximum concentrations which are the quantity elements. Micro/trace
elements such as Cr, Mn, Fe, Co, Ni, Cu, Zn, Se and Mo were quantified in sufficient
concentrations.
The FTIR analysis study has confirmed the presence of vital functional groups which may be
important in the synthesis of pharmaceutically active phytoconstituents. Detection of hydroxyl
groups is an indication of the presence of flavonoids, and alcoholic and phenolic compounds
[28]. Vitex negundo is an aromatic plant that is confirmed in present research by the detection
of aromatic functional groups and oxidized nitrogen functional aromatic groups. The
occurrence of alkaloids in Vitex negundo leaves extracts is confirmed by the presence of
alkanes, amines, primary and secondary amines, and aromatic compounds. Some lipids and
proteins have asymmetric streching due to C-H vibration and saturated fatty acids, which were
detected in the present research. Many organic acids contain carboxylic acids and their
derivatives and which are responsible for several medicinal properties19.

Suganthi N. and Sonal Dubey (2015)

Vitex negundo Linn which is distributed throughout India. It is a large aromatic shrub
belonging to the family Verbenaceae growing in moist or along water courses. Phytochemical
investigation shows the presence of flavonoids, essential oils, flavonoid glycosides, terpenes,
lignans, stilbene derivatives, and irridoid glycosides and possesses enormous pharmacological
activities like anti-inflammatory, antioxidant, analgesic, antipyretic, antibacterial, antitumor,
anti-arthritic, anti amnesic, anxiolytic, larvicidal activity, nephroprotective activity, anti-HIV
activity, anti-eosinophilic and anti-snake venom activity. Here in we present a review of all
such data related to Vitex negundo Linn20.

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Bansod M.S.*, Harle U.N (2009)

Phytochemical investigation of this plant (vitex negundo) indicated the presence of
monoterpenesagnuside, flavonoids- casticin, chryso-splenol and vitexin, flavonoids
(vitexicarpin)also, 5,3’-dihydroxy-3,6,7,4’-tetramethoxyllavone and hydroxy-3,6,7,3’,4’-
pentamethoxy flavone from the leaves. Seeds of Vitex negundo L agorded a new lignan
characterized as 6-hydroxy-4-4-hydroxy-3-methoxyphenyl), 3-hydroxymethyl-7-methoxy-
3,4-dihydro-2-naphthaldehyde by spectroscopic methods and triterpenoids (betulinic acid and
ursolic acid), lignans (negundins, vitedonin),alkaloid (vitrafalal)and diterpene (vitedoin)
investigated. Isolation of the acetoaceta fraction yielded two major lignans:6-hydroxy-4-
(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2napthededyde and
andvitedoamine A. Chromatography of an ethanolic extract of Vitex negundo L. resulted in
the isolation of another new iridoid glucoside which was characterized as 6’-p-
hydroxybenzoyl mussaenosidic acid with derivatives 2-p hydroxyl benzyl mussaenosidic acid,
6-p-hydroxybenzoylmussaenosidic acid , p-hydroxybenzoyl ester of mussaenosidic acid, p-
hydroxybenzoyl methyl ester of mussaenosidic acid. Sequiterpenes flavone glycosides, iridoid
glycosides, eurotoside aucubin, stilbenes have been isolated from roots of Vitex negundo
L.Chasteberry is thought to contain a progesterone-like compound21.

API (2004)
TLC of alcoholic extract of vitex negundo on silica gel G using chloroform: methanol
(8:2) shows in visible light two spot at Rf 0.14 and 0.95(both yellow). Under UV (366nm) six
fluorescent zones are visible at Rf 0.14(dirty yellow), 0.40(blue), 0.66(blue), 0.82(light blue),
0.90(blue,) and 0.95(bluish green). On exposure to iodine vapour five spots appear at Rf 0.14,
0.40, 0.66,0.82 and 0.95 (all yellow). On spraying with dragendroff reagent followed by 5% of
methanolic sulfuric acid reagent two spots appear at Rf 0.03 and 0.95(both orange)
Constituent – alkaloid (nishindine)22.

CE Igara et al (2016)
The phytochemical compositions of the Murraya koenigii leaf that flavonoids content
was the highest while glycoside was detected to be the least. Flavonoids have been shown to
possess anti-fungal and anti-bacterial activity. Flavonoids have strong anti-oxidant and anti-
inflammatory properties. They have ability to scavenge hydroxyl radicals, superoxide anions
and lipid peroxy radicals . These free radicals have been implicated in causing some age-long
diseases such as diabetes, cardiovascular, Parkinson and Alzheimer’s diseases. The presence
of flavonoids in the curry leaf may account for its use in treatment of these diseases. Saponins
was detected in an appreciable amount. Saponins are known as immune booster. Plants rich in
saponnins have been shown to demonstrate anti-inflammatory, cholesterol-lowering and ant-
cancer properties. Alkaloid was found in curry leaf. Alkaloids have anti-microbial properties
owing to their ability to intercalate with DNA of the micro-organisms. Phenol was found.
phenols and phenolics have anti-tumour and anti-oxidant effects. They also have anti-
inflammatory and anti-carcinogenic properties and the plant leaves contain a substantial
amount of carbohydrate and moisture. This shows that the leaf is a good source of carbohydrate
while the moisture content is high. With its content of these anti-oxidants flavonoids, phenols,
vitamins E and C, curry leaf possess anti-cancer and cardio-protective agents23.

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Ram S. Vermaa et al (2013)

Chemical (GC/FID and GC/MS) and statistical analyses of the volatile-oil constituents
of M. koenigii, populations collected from different regions of foot and mid hills of
Northwestern Himalaya of India, indicated significant phytochemical diversity. In conclusion,
the yields and compositions of the essential oils isolated from M. koenigii varied considerably,
depending on the origin and chemotype of the plant material. Essential oil yield and
composition could not be correlated with altitude and growing site. It seems that M. koenigii
chemotypes are not growing in a geographical region- or altitude-specific manner. Sabinene-
rich populations, explored for the first time for M. koenigii, may be utilized as a potential
natural resource for high-value aromatic oil. This study has contributed to the primary
knowledge of M. koenigii populations from an under-explored area. The genetic resources of
this plant growing wild in this region can be utilized for genetic improvement and, hence, for
the production of the high-value fragrance chemicals, viz., sabinene, a-pinene, (E)-
caryophyllene, and b-pinene24.

Norhakimy Abdul Majid, Aiza Harun (2019)

This study aimed to identify antioxidative constituents from Murraya koeginii leaves
using the thin layer chromatographic (TLC) technique. The screening of phytochemicals was
conducted using methanol extracts as well as the developed TLC of methanolic extracts.
Isolation of antioxidative constituents was held using preparative TLC. Secondary metabolites
that had been screened from methanol extract were recognized as tannins, alkaloids, flavonoids,
and terpenoids. Screening for phytochemicals using developed TLC of extracts revealed the
presence of antioxidative constituents from dichloromethane extract after spraying with DPPH
reagent. The antioxidative constituents are proposed as (A) Cyclic Terpene ester, (B) 4-
benzyloxy-4- [2,2,-dimethyl-4-dioxolanyl] butyraldehyde, (C) Oleic acid, (D) Piperidione
(alkaloid derivatives) and (E) 9-oxo-methyl ester after analyzing with GCMS aided with Wiley
Database matching individually. Based on its potent antioxidant properties, the use of M.
koenigii leaves in the development of natural medicine for the treatment of various diseases
cannot be neglected25.

Harish K Handral et al (2012)

Curry Leaves are aromatic and contain proteins, carbohydrates, fiber, minerals, carotene,
nicotinic acid and vitamin C. The leaves contain high amount of oxalic acid, leaves also
contains crystalline glycosides, carbazole alkaloids, koenigin and resin. Fresh leaves contain
yellow-colored volatile oil conversely also rich in vitamin A and calcium. It also contains
girinimbin, iso-mahanimbin, koenine, koenigine, koenidine and koenimbine. Mahanimbicine,
bicyclomahanimbicine, phebalosin, coumarine as Murrayone imperatoxin etc are isolated from
leaves. Triterpenoid alkaloids like cyclomahanimbine and tetrahydromahanmbine are also
present in the leaves26.

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Pharmacological survey:
Daniel Iván ospina salazar et al (2015)
The antifungal activity of methanolic extracts from neem tree (Azadirachta indica),
several bioassays were conducted following M38-A2 broth microdilution method on 14
isolates of the dermatophytes Trichophyton mentagrophytes, Trichophyton rubrum,
Microsporum canis, and Epidermophyton floccosum. Neem extracts were obtained through
methanol-hexane partitioning of mature green leaves and seed oil. Furthermore, high-
performance liquid chromatography (HPLC) analyses were carried out to relate the chemical
profile with their content of terpenoids, of widely known antifungal activity. The antimycotic
Terbinafine served as a positive control. Results showed that there was total growth inhibition
of the dermatophytes isolates at minimal inhibitory concentrations (MIC) between 50 μg/mL
and 200 μg/mL for leaves extract, and between 625 μg/mL and 2500 μg/mL for seed oil extract.
The MIC of positive control (Terbinafine) ranged between 0.0019 μg/mL and 0.0313 μg/mL.
Both neem leaves and seed oil methanol extracts exhibited different chromatographic profiles
by HPLC, which could explain the differences observed in their antifungal activity. This
analysis revealed the possible presence of terpenoids in both extracts, which are known to have
biological activity. The results of this research are a new report on the therapeutic potential of
neem for the control of dermatophytosis27.

Imam Hashmat et al (2012)

Antibacterial Activity: The petroleum ether, methanol, and aqueous extracts of the
leaves of Azadirachta indica (Meliaceae), bulbs of Allium cepa (Liliaceae), and methanol
extract of gel of Aloe vera (Liliaceae) were screened for their anti-microbial activity using the
cup plate agar diffusion method. They were tested against six bacteria; two Gram-positive
bacteria (Bacillus subtilis and Staphylococcus aureus) and four Gram-negative bacteria
(Escherichia coli, Proteus Vulgaris, Pseudomonas aeruginosa, and Salmonella typhi).
Antioxidant: Extracts from young flowers and leaves have strong antioxidant potential. An
indicator of oxidative stress, malondialdehyde (MDA), was reduced by 46.0% and 50.6% for
flower- and leaf-based extracts, respectively, prompting the recommendation to use neem as a
vegetable bitter tonic to promote good health
Skin Disorders: Neem can treat many skin disorders, including scabies and lice; in a paste
combination with Curcuma longa (turmeric), neem was used to treat scabies in 814 people—
97% of them were cured within 3 to 15 days of application, and no adverse reactions were
observed17.

Anbalagan S, Sankareswaran M et al (2017)

the Vitex negundo ethanol and methanol leaf extracts showed a broad spectrum of activity
against fungal strains. It showed maximum activity against Candida albicans, Aspergillus
niger, Cryptococcus neoformans, and Penicillium sp. The knowledge of the extent and mode
of inhibition of specific bioactive compounds which are present in plant extracts may contribute
to the successful application of such natural compounds for the treatment of infection caused
by fungal and bacterial diseases28.

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Zahoor Ullah et al (2012)

Antioxidant activity: Preliminary studies showed that Vitex negundo leaf exhibited
antioxidant properties and contain natural antioxidants. Thus, the objective of this study
was to analyse the antioxidant activity of methanol and hexane extract and essential oil
from V. negundo leaf using different in vitro antioxidant assays. In addition, total phenolic
contents, flavonoids, tocopherol and carotenoids content of leaf of V. negundo were also
Quantified using high performance liquid chromatography (HPLC).
The plant is traditionally reported for its use for the treatment of cough, asthma, fever,
eye disease, inflammation, intestinal worms, skin diseases, nervous disorders, leprosy and
rheumatism. Roots are tonic, anodyne, febrifuge, bechic, expectorant and diuretic. This
review is short review of last two years reporting the natural products isolated and
biological potential of Vitex negundo Linn29.

Shashank Tiwari, Shreya Talreja (2020)

Used as a Antibacterial agent Murraya koenigii plant extract possess anti-bacterial
activity against some bacteria includes Bacillus cereus, Escherichia coli (E.coli),
Staphylococcus aureus, Salmonella typhi (S.typhi). The ethanol, methanol and acetone extract
of plant shown the significant effect against these bacteria strain. The acetone extract of plant
shown maximum effect against Bacillus cereus and the methanol extract shown high inhibition
efficacy compare to ethanol extract. Moreover, methanol extract shown less inhibition efficacy
against E.coli, while acetone extract shown higher inhibitory effect compare to methanol
extract.
Useful for Wound Healing The Ethanol leaves extract of plant possess wound healing
property. The researchers study has found in albino rat, male albino rat is sacrificed and applied
the extract in wounds after some day the study showed decrease the wound affected
inflammation in day by day. Hence, the study proved that the plant leaves extract exhibit wound
healing activity
Murraya koenigii useful for Haematological property The whole plant is useful for the
haematological study. According to researchers study that found the normal human intake dose
administered in rat and after administration the study not shown any adverse effect and no
adverse effect on counts of RBC,WBC, haemoglobin, total serum protein, Fibrin level, blood
urea and no any histopathological (disease on tissue/ cell) changes in rat liver. Hence, the study
proved that the plant possess no any adverse effect.
Useful for Skin pigmentation The leaves oil of this plant possess sun protection factor (SPF)
property (useful for protect skin against sun burn), cream of the murraya koenigii plant useful
against skin pigmentation, erythema (skin redness). And cosmetic industries also use of this
plant for making formulations such as cream, lotion, soap and add some other additives for
enhance/increase product efficacy30.

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AIM AND OBJECTIVE:

A literature survey revealed that medicinal plants play a vital role in the treatment of fungal
diseases. Neem, nergundi, and curry leaves are used in traditional medicine for their
antifungal property.
The marketed survey revealed that there are many formulations available for treating fungal
infection but no formulation is available for these plants in combination. Thus, it was thought
worthwhile to develop and evaluate semisolid preparation containing these three potent
antifungal plants.

Objective:
• Develop antifungal polyherbal formulation.
• Evaluate antifungal polyherbal formulation.
• Comparative study of laboratory preparation with marketed formulation.

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PLANT PROFILE:

Neem (Azadirachta indica)

Biological source: Neem consists of all aerial parts of the plant known as Azadirachta indica.
Family: Meliaceae.
Common name: Nimba, Nimbaka, Arishta, Arishtaphala, Pichumarda, Pichumanda,
Pichumandaka, Tiktaka, Sutiktak, Paaribhadra.

Taxonomical classification:

Kingdom Plantae

Order Rutales

Suborder Rutinae

Family Meliaceae
Subfamily Melioideae
Tribe Melieae
Genus Azadirachta

Species Indica

Distribution: A native to east India and Burma, it grows in much of south East Asia and West
Africa, and more recently Caribbean and south and Central America. In India it occurs naturally
in Siwalik Hills, dry forests of Andhra Pradesh, Tamil Nadu, and Karnataka. It is also grown
and often naturalized in Peninsular Malaysia, Singapore, Philippines, Australia, Saudi Arabia,
Tropical Africa, the Caribbean, Central, and South America17.
Description: It is a tree 40-50 feet or higher, with a straight trunk and long spreading branches
forming a broad round crown; it has rough dark brown bark with wide longitudinal fissures

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separated by flat ridges. The leaves are compound, and imparipinnate, each comprising 5-15
leaflets. The compound leaves are themselves alternating with one another. It bears many-
flowered panicles, mostly in the leaf axils. The selel are ovate and about one cm long with
sweet scented white oblanciolate petals. It produces yellow drupes that are ellipsoid and
glabrous, 12-20 mm long. Fruits are green, turning yellow on ripening, aromatic with a garlic-
like odor. Fresh leaves and flowers come in March-April. Fruits mature between April and
August depending upon locality.
Cultivation: It generally performs well in areas with annual rainfall varying from 400 - 1200
mm. It thrives under the hottest conditions where the maximum day temperature reaches50
degree Celsius. But it cannot withstand freezing or extended cold. Neem grows on almost all
kinds of soils including clayey, saline, and alkaline soils but does well on black cotton soils.
The nursery could be either a temporary or permanent one. The site, in either case, should have
a perennial water source and locate on flat ground with well-drained soil. Neem starts bearing
fruits after 5 years and comes to full bearing at the age of 10-12 years. Fruit yield is 5-20 kg
per tree per year in the initial years. A mature tree produces 35-50 kg of fruit/per year. Oil yield
varies from 40-43% of seed on a dry weight basis. Neem can be easily raised through direct
sowing, entire/poly pot seedlings, or root-shoot cuttings. For degraded areas, direct sowing is
more successful. Direct sowing is done either by dibbling in bushes, broadcast sowing, line
sowing, sowing on mounds or ridges, sowing in trenches in sunken beds in circular saucers, or
by aerial sowing. The choice varies with the edaphic, climatic, biotic, and economic conditions
of the site. Planting in pits is carried out by using 20 to 45 cm tall seedlings. Taller ones promise
better survival. Planting stumps prepared from year-old seedlings in crowbar holes also give
good results.
Chemical Composition: Azadirachtin, meliacin, gedunin, nimbidin, nimbolides, salanin,
nimbin, valassin, meliacin. the seed also contains tignic acid responsible for the distinctive
odour of the oil. The four best limonoids compounds are Azadirachtin, Salannin, Meliantriol,
and Nimbin. Limonoids contain insecticidal and pesticidal activity.
Uses: Neem extracts are used as insecticides, pesticides and fungicides. Neem oil has
antibacterial, and antiviral properties and is used in skin and dental problems. Neem products
are being used for malaria, fever, pain and also as contraceptives. Neem is also being used in
cosmetics, lubricants and fertilizers. Neem bark is used in villages for rope making. Neem oil
is used in soap manufacturing31.

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Nirgundi (Vitex negundo)

Biological source: it consists of dried leaves and flowers of the Vitex negundo.
family: Verbenaceae.
Common name: Five-leaved Chaste tree, Shveta nirgundi, Sugandhikaa.

Taxonomical classification:

Kingdom Plantae
Sub Kingdom Tracheobionta
Super Division Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Sub Class Asteridea
Order Lamilales
Family Verbenaceae

Genus Vitex linn

Species Vitex negundo Linn.
(Chaste tree)

Distribution: The plant is found throughout India, Ceylon- Afghanistan, tropical Africa,
Madagascar, China and the Philippines.5 The plant occurs in Bengal, Southern India, and
Burma also.6 It is common in waste places around villages, river banks, moist localities and in
deciduous forests.7 It is common throughout India from the coastal belt to subtropical Western
Himalayas and Andaman Islands, abundant in drier zones. It is particularly found in Karnataka
and Tamilnadu32.

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 DEVELOPMENT AND EVALUATION OF POLYHERBAL ANTIFUNGAL FORMULATION

Botanical description: Vitex negundo is a woody, aromatic deciduous shrub growing to a

small tree. Vitex negundo is also known as the five-leafed chaste tree or monk’s pepper. Its
most striking feature centers on a cluster of five pointed leaves resembling a palm. It is an
erect, 2-5 m in height, slender tree with quadrangular branchlets. The leaves have five leaflets
palmately arranged, which are lanceolate, acute, glabarous, 4-10 cm long, hairy beneath and
pointed at both ends. The terminal leaflet has a long petiole whereas, the lateral ones have short
petiole. Flowers are bluish purple in colour borne on axillary or terminal panicle up to 30 cm
long. The fruit is succulent globose, and black when ripe with four seeds, rounded and about 4
mm in diameter33.

Cultivation: Vitex negundo is a much-branched shrub up to 5 m tall or sometimes a small,

slender tree with thin, gray bark. Leaves are palmately compound, 3–5 foliate; leaflet are
lanceolate; margins are entire or crenate; terminal leaflet are 5–10 cm × 1–3 cm; lateral leaflet
are smaller; all nearly glabrous above, whitish tomentose beneath and aromatic when crushed.
Flowers are bluish-purple, small, in peduncled cymes, forming large, terminal, often
compound, pyramidal panicles. In central India, flowering occurs between June and December
and fruiting from September to February. The fruit is a succulent drupe, black when ripe, 5–6
mm in diameter. Seeds are 5–6 mm in diameter. The mature seeds sown in nursery beds
normally germinate within 2–3 weeks. Four- to six-month-old seedlings are used for
transplanting in the field. It can be reproduced readily from shoot cuttings. Vitex negundo roots
are strong and deep and suckers profusely. It produces root suckers which can also be utilized
as planting material. Vishvavallabha points out that V. negundo can be grown both from seeds
and stalks.

Chemical constituents: It contains vitexin and isovitexin, the leaves contain iridoid
glycosides, isomeric flavanones, and flavonoids, besides casticin and the glucosides, luteolin-
7-glucoside and alpha D-glucoside of a tetrahydroxy monomethoxy flavone. Dried powder of
roots contains hentriacontane, beta-sitosterol, and its aetat and stigmasterol.
Uses: leaf (also the root) in excessive vaginal discharges, oedema, skin diseases, pruritus,
helminthiasis, rheumatism, and puerperal fever. Flowers—astringent, febrifuge, antidiarrhoeic;
prescribed in liver complaint. Oil— applied to sinus, scrofulous sores33.

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 DEVELOPMENT AND EVALUATION OF POLYHERBAL ANTIFUNGAL FORMULATION

Curry leaves (Murraya koenigii):

Biological source: consists of leaves and aerial part of the plant known as Murraya koenigii.
Family: Rutaceae.
Common name: Curry-Leaf tree, Surabhini-Nimba, Unani Karipattaa.

Taxonomical classification:

Kingdom Plantae

Sub-kingdom Tracheobionta

Superdivision Spermatophyta

Division Magnoliophyta

Subclass Rosidae

Order Sapindales

Family Rutaceae

Genus Murraya J. Koenig ex L

Species Murraya Koenigii L. Spreng

Distribution: curry-leaf tree, is a native of India, Sri Lanka and other south Asian countries. It
is found almost everywhere in the Indian subcontinent, it shares aromatic nature, more or less
deciduous shrub or tree up to 6 m in height and 15-40 cm in diameter with short trunk, thin
smooth grey or brown bark and dense shady crown 5. Most part of plant is covered with fine
down and has a strong peculiar smell. M. koenigii is genus of tree, native to tropical Asia from
Himalaya foothills of India to Srilanka eastward through Myanmar, Indonesia, Southern China
and Hainan30.

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Plant description: A small spreading shrub, about 2.5 metres high; the main stem, dark green
to brownish, with numerous dots on it; its bark can be peeled off longitudinally, exposing the
white wood underneath; the girth of the main stem is 16 cm. Leaves, exstipulate, bipinnately
compound, 30 cm long, each bearing 24 leaflets, having reticulate venation; leaflets, lanceolate,
4.9 cm long, 1.8 cm broad, having 0.5-cm-long petiole. Flowers, bisexual, white, funnel-
shaped, sweetly scented, stalked, complete, ebracteate, regular, actinomorphic, pentamerous,
hypogynous, the average diameter of a fully opened flower being 1.12 cm; inflorescence, a
terminal cyme, each bearing 60 to 90 flowers; calyx, 5-lobed, persistent, inferior, green;
corolla, white, polypetalous, inferior, with 5 petals, lanceolate; length, 5 mm; androecium,
polyandrous, inferior, with 10 stamens, dorsifixed, arranged into circles of five each; smaller
stamens, 4 mm. long whereas the longer ones, 5 to 6 mm; gynoecium, 5 to 6 mm long; stigma,
bright, sticky; style, short; ovary, superior. Fruits, round to oblong, 1.4 to 1.6 cm long, 1 to 1.2
cm in diameter; weight, 880 mg; volume, 895 microlitres; fully ripe fruits, black with a very
shining surface. Seed, one in each fruit, 11 mm long, 8 mm in diameter, colour spinach green.
Cultivation: Flowering starts in the middle of April and ends in the middle of May. The peak
flowering season under the Sanwara conditions was observed to be the last week of April. The
fruiting season was observed to continue from the middle of July to the end of August. The
peak fruiting season, however, was found to continue from the last week of July to the 1st week
of August. Curry leaf is Native to India. Large shrub to small tree. Pinnate leaves are used in
many South Indian curries. Full sun or light shade. Fertilize with palm or citrus fertilizer to
promote leaf production. Grows well in containers. Use a well-drained potting mix. Can be
grown outdoors in Southern California, South Texas and South Florida. Protect from freezing.
Seeds are fragile so handle with care. Seeds are shipped in moist peatmoss/coir mix and should
be planted immediately.

Chemical constituents: All parts of the plant, especially the leaves, are rich in carbazole
alkaloids (several carbazole bases have been isolated). The leaves also gave a coumarin
glucoside, scopolin, beta carotene.
are- α - caryophyllene, δ - elemene, β- elemene, α & β- phellandrene due to the presence of
these compounds the plant ability to prevent food spoilage. Some other compounds include
cis-β-ocimene, cis-Piperitol, Linalool, γ-Terpinene, terpinyl acetate, m-Cymene, α-Terpinene,
β-Myrcene, Camphene, α-Thujene, Eucalyptol, Caryophyllene oxide. The plant also in rich
source of girinimbiol, girinimbine (carbazole alkaloids), koenine, koenigine, koenidine,
calcium, phosphorus, iron, thiamine (vitamin B1), riboavin (vitamin B2), niacin (nicotinic
acid), sitosterol, carotene, 1,4 methanoazulen-9-ol, Caryophyllene oxide, Phytol, pinene,
phenolic compounds, etc.

Uses: steam distillate of the leaves is reported to exhibit antifungal and insecticidal activities.
The ethanolic extract of the stem bark showed an anti-inflammatory effect. They are also used
externally to cure eruptions and the bites of poisonous animals. The green leaves are stated to
be eaten raw for curing dysentery, and the infusion of the washed leaves stops vomiting. Curry
leaves are also used in calcium deficiency34.

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 DEVELOPMENT AND EVALUATION OF POLYHERBAL ANTIFUNGAL FORMULATION

PLANT OF WORK:
• Collection and authentication of plant material
• Preparation of plant extract
• Phytochemical extraction
• Estimation of phytoconstituents
• Preparation of herbal cream
• Evaluation of cream
• In-vitro antifungal activity of cream
• Comparative study of marketed and our formulation

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REFERANCE:
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14] Dheeraj K.Gahlawat, Savita Jakhar and Pushpa Dahiya. Murraya koenigii (L.) Spreng: an
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phytochemical and nutritional profile of Murraya Koenigii (Linn) Spreng leaf. Journal of
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26] harish k handral1, anup pandith, and shruthi sd. a review on murraya koenigii:
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OROZCO SÁNCHEZ, Myrtha ARANGO ARTEAGA, Luisa Fernanda GÓMEZ LONDOÑO.
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28] Anbalagan S, Sankareswaran M, Moorthy M, Elakkia B and Fahamitha E.Phytochemical
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December 2017, pp. 119-125.
29] Zahoor Ullah*1, Riaz Ullah 1, 2, Azhar-ul-Haq Ali Shah 2, Ijaz Ahmad 2 and Sajjad
Haider. phytochemical and biological evaluation of vitex negundo linn: a review. ijpsr, 2012;
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Rajashri R. Dhonnar Mrs. Mona Y. Agarwal

(Student) (Research Guide)

Date:
Place:

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