THE ANTIFUNGAL PROPERTY OF RADISH

EXTRACT (Raphanus sativus)

A Thesis
Presented to
The Faculty of the Graduate School
University of Perpetual Help Laguna

In Partial Fulfillment of the

Requirements for the Degree of
Master of Science in Microbiology
 By
 
Antonina L. Hipolito
Lab-tech
UPH- DJGT Med. Univ.

January 20ll

 
THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT
(Raphanus sativus)
Introduction:

• Herbal medicines
• Alternate medicine
• Radish root extract ( Raphanus sativus)
• Plant defensin
• Antifungal solution
• Alkanoids, saponins, tannins and glycosides
• Antifungal ointment
• Skin diseases
Operational Framework:
• INDEPENDENT VARIABLE :
Effects of:
- Radish root crude extract (Raphanus sativus)
• DEPENDENT VARIABLE
Antifungal property against Trichohyton mentagrophytes
- Halozone of inhition
• Treat skin diseases through the application of two solutions:
Radish extract and antifungal ointment (Canesten).
• Antifungal solution: synthetically made or solutions that
come from medicinal plants.
• Antifungal property
Procedure to prove the antifungal property of Radish
crude extract:

• Collection of fresh plant sample: Radish (Raphanus sativus

Linne) (air dry for 2 days)
• Maceration (80% ethyl alcohol)
• Continuous Extraction using: Rotavap apparatus with 80 %
Ethyl Alcohol
• Filtration
• Preliminary Test for Glycosides
• Partial-purification of Glycosides
• Physical and Chemical Test
• Microbiological Test
Statement of the Problem :
This study will deal with the investigation for the
presence of antifungal property of Radish root extract in
treating fungal skin diseases caused by Trichophyton
Mentagrophytes in vitro.

Specific Problems:
1. What is the effect of Radish root crude extract on the
growth of Trichophyton mentagrophytes?
2. What method of extraction is best use in radish root
crude extract?
3. Is there a significant difference in the antifungal
property of Radish root extract and antifungal
ointment in vitro?
Statement of Hypothesis :

There is no significant difference in antifungal

property of Radish root crude extract and antifungal
ointment against Trichophyton mentagrophytes.
Scope and Delimitation :
The study will focus on the antifungal property of
Radish root extract against T. mentagrophytes in vitro.
It also takes into consideration the physical test,
chemical test and microbiological test, wherein the
radish root crude extract will be subjected to rigorous
experimentations in order to obtain the exact results,
testing its ability to inhibit the growth of Trichophyton
mentagrophytes that cause fungal skin infection.
Significance of the Study:
• Beneficial to manufacturers
• Filipino masses
• Medical students especially the Medical Technologist
Definition of Terms:
• Antifungal
• Fungi
• Extract
• Extraction
• Maceration
• Herbal medicine
• Radish
• Plant defensin
• Tannins
• Raphanus sativus Linne. (fam. Brasicaceae)
• Every part of the radish plant is useful leaves and roots maybe
eaten raw or cooked as vegetable
• Each plant part has been used as medication for hyperacidity,
vomiting, intestinal obstruction, loss of voice and speech,
dysentery, nosebleeding, hemoptysis, melena, diphtheria,
constipation in children, furuncles and other fungal skin diseases
• Radishes are a great source of vitamin C and are rich in minerals
like sulphur, iron, and iodine.
• Radishes can be added to vegetable juice to spice up the flavor a
little they can help clear your sinus cavities and soothe your sore
throat.
• seeds, leaves and roots eaten raw or cooked as vegetable
• Juice of the fresh leaves is used as diuretic and laxative
• Great source of vitamin C and are rich in minerals like sulphur,
iron, and iodine. Leaves with Vitamin C
• It can be added to vegetable juice to spice up the flavor a little
they can help clear your sinus cavities and soothe your sore
throat.
• Seeds contain fatty oil 30 percent ash, ash 3.5 percent, volatile
oil, sulfuric acid, erudic acid
REVIEW OF RELATED LITERATURES AND STUDIES
• Phytoactive substance of Radish seeds contain an anti bacterial
principle , raphanin, stable components glycosinolates, enzymes
trace elements, acid, aldehydes, anthocyanin, pectin,
arabinogalactan protein
• Seeds are used as a diuretic, laxative, and lithoriptic
• Seed are believed to have also emmenangogue properties
• Chinese radish and White Icicle , daikon( Oriental specialty
markets )
• Daikon is even better, a source of vitamin C, potassium,
magnesium, and folate as well as sulphur, iron, and iodine.
• Dressing or poultice to burns, scalds, fetid feet, and ecchymoses
• Radish root is used as stomachic, anthelmintic, and nervine tonic
• Useful in diseases of the heart, amenorrhea, hiccups, leprosy and,
cholera
• flowers are becnic and cholagogue.
• Useful also in gonorrhea and employed in cancer stomach.
• leaves and roots are used as diuretic and laxative
• Root is also considered as carminative and corrective
REVIEW OF RELATED LITERATURES AND STUDIES

• Raphanus sativus Linne. (fam. Brasicaceae)

• Roots are fleshy, pungent and variable in size and form. It is
a coarse annual popular common and cheap vegetable which
are eaten raw or cooked
• Roots contain raphanol, rettichol, volatile oil, sinapin,
methylmercaptan, vitamins B1, C2, C10H­1­ 1NS, and oxidase.
• Radish oil doesn’t dry up like ordinary oil used in soap
making the meat after juice extraction is used as fertilizer
• Roots are regarded as stimulant and or a reputed medicine
for piles and gastrodynamic pain. The juice of the fresh root
is considered powerfully as antiscorbutic
• Storage for tuberous roots of Radish (Raphanus sativus)
for the production of spice (mustard) and oil which has
glycosinate compounds known as mustard oil that produce
the pungent odor
REVIEW OF RELATED LITERATURES AND STUDIES

• Roots are regarded as carminative and corrective. They are

crushed and applied locally as addressing or poultice to
burns, scalds, fetid feet, and ecchymoses, used as a
stomachic, antihelmithic, and nervic tonic, and is useful in
diseases of the heart, amenorrhea, hiccups, leprosy and
cholera. Seeds are also reported to be employed in cancer of
the stomach.
• Studies report its antibacterial effect against growth of S.
aureus, E. coli, S. aeruginosa, S. typhi and S. subtilis in
carminative and digestive
• Saponins that helps cleanse and soften the skin and Sulfur
that can heal and disinfect.
• It has interesting properties such as cleansing and
antibacterial effect
• Fungal ointment is used to treat some skin diseases but
indeed quite expensive
 REVIEW OF RELATED LITERATURES AND STUDIES

• antiscorbutic because of quantity of nitrous juice

• An excellent food remedy for stone, gravel and scorbutic
conditions, its juice has been used in the treatment of
cholelithiasis as an aid in preventing the formation of
biliary calculi.

Related Studies :

• Plant antifungal protein I (Rs-AFP I) contains 51 amino

acid residue plant defensin which is isolated from radish
seeds.
• Radish extract can be possibly used as topical ointment for
fungal infection, according to Nakamura(2000)
REVIEW OF RELATED LITERATURES AND STUDIES

• Trichophyton mentagrophytes : can cause superficial

infections of the skin, hair and nails.
• Trichophyton mentagrophytes var. interdigitale: frequent
causative agents of chronic infection of the feet, the nails,
and the groin.
• T. mentagrophytes var. mentagrophytes: inflammatory
lesions of the scalp, the glabrous skin, the nails, and the
bear region. 
• Keratinophylic fungus belonging to a homogeneous group of
fungi called the dermatophytes.
• Organism has been recovered from a variety of sources such
as soil, floor of swimming pools, hairs of wild boar, cats and
dogs, farm animals, foot wears, shower stalls and from
human toewebs without clinical lesions.
RESEARCH DESIGN AD METHODOLOGY

Laboratory Procedure:
1. Collection and. Preparation of the Sample
Fresh radish were bought in Biñan, Laguna Public Market.
The radish were washed with running water and drained immediately
after collection and then air dry for two days then cut into small
pieces as possible and reserved it for the next procedure.
1.1 Moisture Determination
A clean and dry crucible was weighed and placed with ____ g of Radish
and weighed again. The porcelain crucible with radish sample was
heated for about thirty minutes. After heating, the crucible with the
radish sample was allowed to cool in the desiccators for l5 minutes.
After cooling the crucible with the radish sample was weighed again
and then the moisture content was computed.
1.2 Ash Determination
The accurately weigh quantity of the ground drug
representing from ___ g of dried radish sample in tarred plain
crucible and incinerate at low temperature, not exceed very dull
redness, until free from carbon. Cool and determine the weight of
the ash. If the carbon free ash cannot be obtained in this way, the
charged mass was exhausted with hot water, and then the insoluble
residue was collected on the ashless filter paper. The residue was
incinerated and filtered until the ash is nearly so, then the filtrate
was added and evaporated to dryness and was heated a whole to a
dull redness. If again the carbon free ash cannot be obtained by this
manner the crucible was cooled. Fifteen milliliters of alcohol was
added to break up the ash with a glass rod, burned of the alcohol,
and again was heated with the whole to a dull redness. The ash was
cooled, weighed and calculated the percentage of the total ash
from the weight of the plant sample taken.
2. Method of Extraction
The Researcher use the Maceration method using 80% ethyl
alcohol and continuous extraction using the rotavap apparatus. From
those extractions, the researcher computed the
glycoside extract.

3. Preliminary Screening for Glycosides

Five grams of the weighed plant extract is added with three
milliliter (3 ml) of lead acetate T.S., and was filtered to remove the
precipitate. The first procedure was repeated until no precipitate is
formed. Five drops (5 gtts.) of basic lead acetate was added to the
final filtrate. Formation of white precipitates indicates the presence
of glycosides.
4. Partial Purification of Glycosides
The alcoholic extract obtained from the continuous extraction
was placed in a tarred evaporating dish and was heated in a water
bath until dried. The dried extractive was treated with hot water,
stirred and then transferred in a beaker. More hot water was added
into a beaker to completely dissolve the extractive and then filtered.
The filtrate was treated again with lead acetate solution to remove
any unwanted constituents and filtered. This process was repeated
several times until it no longer gives precipitate when treated with
one to two drops (1-2 gtts.) of ferric chloride T.S. and the filtrate
obtained was placed in a tarred evaporating dish and heated in a
water bath until dried to obtain the glycoside extractive.
4.1Percentage Yield of the Semi-purified Glycoside Extract.

A portion was computed for the percentage yield. The weight

of the evaporating dish with the residues was subtracted to the
weight of the evaporating dish alone to get the weight of the
residues alone. The weight of the residues was then divided to the
weight of the plant sample used then multiplied by 100 for the
purpose of determining the percentage yield of the plant extract.
 4.2 Physical test
4.2.1 Organoleptic Test.
After obtaining the glycosides, color, odor and the
appearance or texture was observed and noted.
4.2.2 Solubility Test.
A small amount of the semi-purified glycosides extracts were
divided into four test tubes labeled each as “A, B, C, D”. Each test
tube contains five milliliters (5ml) of water, petroleum ether,
ethanol and chloroform. The degree of solubility of glycosides was
observed and recorded
4.3 Chemical Test.
4.3.1Screening of Anthraquinone Glycosides
A)Borntrager Test
B)Modified Borntrager Test
4.3.2 Screening for Flavonoids
A)Bate-Smith and Metcalf Test for Leucoanthocyanins
B)Wilstatter “Cyanidin” Test.
5. Microbiological Test for determining the Antifungal Property of
Radish extract
The following are the culture media, microorganism,
standards and apparatus used in the microbiological screening:
Saboraud Dextrose Agar (SDA) is the culture media; the fungi
Trichophyton mentagrophtes; the standard antibiotic Canesten and
the apparatus used are the Petri dishes, pipette cork borer and
vernier caliper.

5.1Test Organism:
Fungi (yeast): Candida albicans and Trichophyton
mentagrophytes
5.2 Preparation of inoculums - Preparatory to the assay, the
surface of agar slants contained in the test tubes are inoculated
from a recently grown slant. After incubation at room
temperature for eighteen to twenty-four hours, a stock
suspension is prepared by collecting the surface growth in
about 10 ml of sterile distilled water.
 A portion of this stock suspension is diluted with the
volume of sterile distilled water and the inoculum density of
this trial dilution is compared by the addition of Antifungal
standard. This suspension corresponds to an approximate
bacterial density of 300 ml million per milliliter.
5.3 Preparation of assay plates. Melted SDA is poured into each
sterile petri dish.The agar is distributed evenly in the plate
and allowed to solidify. After the agar solidified the inoculums
was transferred to the petri dish by a cotton swab. To spread
the organism evenly in the petri dish multiple method of
streaking was used.
5.4 Preparation of filter paper disc. Several pieces of ashless
paper was cut into disc with the aid of a puncher. Then wrap
with clean sheet of bond paper in lots of four. They are then
sterilized in an autoclave together with the media for 15
minutes and dried in oven.
5.5 Microbiological Assay Method. Dip forceps in methanol,
drain excess solvent, flame and allow to cool. Pick the
sterile filter paper discs from the solution of alcohol
(control), the extract and/ or the final product and the
standard antifungal. Sterilize the forceps every time you
pick the discs from each solution. Drain off excess solution
by letting the disc touch the lip of the container. Gently
press down the discs with the tip of the flamed forceps to
ensure contact with the agar. Follow the pattern on the trace
paper. Indicate the starting point on the plate. Allow three
readings for alcohol, radish root extract and final product
and standard antifungal. Incubate the plates at 37ºC for 24
hours. After incubation measure the zone of inhibition in mm.
using vernier caliper. Get the average of the three
measurements and record the results.
5.6 Ash determination - The ___ g of dried Radish roots were
placed in tared porcelain casserole. Then it was heated
until it was free of carbon and placed in a desiccators and
the weight of the ash and% total ash was computed
5.7 Percentage Yield Determination
The extract obtained from 80% ethyl alcohol was weighed
and measured for the determination of the percentage yield
6. Physical and Chemical Methods of Analysis:
6.1 Physical Test
6.1.1 Organoleptic Test.
By observation the odor, color, appearance, taste and
the physical state of the tannin was determined.
6.1.2 Solubility test
About ___ g of the tannin was dissolved in 3ml of
alcohol, water, ether, chloroform, benzene and glycerin to
determine its solubility.
6.2 Chemical Test
About ____ g of the tannin extract was dissolved in l2
ml of water and divided in four test tubes and treated with 1
ml of Ferric chloride T.S., Lead acetate T.S. ,Gelatin T.S.,
and Copper sulfate T.S.
 RESEARCH DESIGN AD METHODOLOGY

Laboratory Procedure :
1. Collection and. Preparation of the Sample
Fresh radish were bought in Biñan, Laguna Public
Market. The radish were washed with running water and
drained immediately after collection and then air dry for
two days then cut into small pieces as possible and
reserved it for the next procedure.

1.1 Moisture Determination

A clean and dry crucible was weighed and placed with
____ g of Radish and weighed again. The porcelain crucible
with radish sample was heated for about thirty minutes.
After heating, the crucible with the radish sample was
allowed to cool in the desiccator for l5 minutes. After cooling
the crucible with the radish sample was weighed again and
then the moisture content was computed.
 4. Partial Purification

The alcohol filtrate is then treated with 1 ml of Sodium hydroxide

T.S., and 1 ml of Calcium chloride T.S. The researcher repeated
filtration and again, obtained filtrate and residue. The filtrate is now
treated with Lead acetate T.S. and proceeded to filtration, this time,
the filtrate was discarded and the residue was first added with hot
water and then Hydrogen sulfide. Again, filtration was done until it
turned to crystals. The crystals obtained are now the tannins.
5. Quantitative Test
• 5.1 Moisture Content Determination Using the oven in
moisture determination, prepare the plant sample by cutting the
material so that the parts are about 3 mm in thickness. The sample
should be reduced to smaller pieces if its seed or fruit. Accurately
weigh __ g of the drug as prepared in evaporating dish. Dry at 105º C
for 5 hours; cool and weigh. Continue heating, drying, and weighing at
1 hour intervals until the loss in not more than 0.25% in one drying.
Determine the moisture content from the weight of the plant sample
taken using this formula:
1.2 Ash Determination.
The accurately weigh quantity of the ground drug
representing from ___ g of dried radish sample in tarred
plain crucible and incinerate at low temperature, not exceed
very dull redness, until free from carbon. Cool and determine
the weight of the ash. If the carbon free ash cannot be
obtained in this way, the charged mass was exhausted with
hot water, and then the insoluble residue was collected on
the ashless filter paper. The residue was incinerated and
filtered until the ash is nearly so, then the filtrate was added
and evaporated to dryness and was heated a whole to a dull
redness. If again the carbon free ash cannot be obtained by
this manner the crucible was cooled. Fifteen milliliters of
alcohol was added to break up the ash with a glass rod,
burned of the alcohol, and again was heated with the whole
to a dull redness. The ash was cooled, weighed and
calculated the percentage of the total ash from the weight
of the plant sample taken.
2. Method of Extraction
The Researcher use the Maceration method using 80%
ethyl alcohol and continuous extraction using the rotavap
apparatus. From those extractions, the researcher computed
the glycoside extract.
3. Preliminary Screening for Glycosides
Five grams of the weighed plant extract is added with
three milliliter (3 ml) of lead acetate T.S., and was filtered to
remove the precipitate. The first procedure was repeated
until no precipitate is formed. Five drops (5 gtts.) of basic
lead acetate was added to the final filtrate. Formation of
white precipitates indicates the presence of glycosides
4. Partial Purification of Glycosides.
The alcoholic extract obtained from the continuous
extraction was placed in a tarred evaporating dish and was
heated in a water bath until dried. The dried extractive was
treated with hot water, stirred and then transferred in a
beaker. More hot water was added into a beaker to
completely dissolve the extractive and then filtered. The
filtrate was treated again with lead acetate solution to
remove any unwanted constituents and
filtered. This process was repeated several times until it no
longer gives precipitate when treated with one to two drops
(1-2 gtts.) of ferric chloride T.S. and the filtrate obtained was
placed in a tarred evaporating dish and heated in a water
bath until dried to obtain the glycoside extractive.
 4.1 Percentage Yield of the Semi-purified Glycoside Extract.
A portion was computed for the percentage yield. The
weight of the evaporating dish with the residues was subtracted to
the weight of the evaporating dish alone to get the weight of the
residues alone. The weight of the residues was then divided
to the weight of the plant sample used then multiplied by 100 for the
purpose of determining the percentage yield of the plant extract.
4.2 Physical test
4.2.1 Organoleptic Test.
After obtaining the glycosides, color, odor and the
appearance or texture was observed and noted.
  4.2.2 Solubility Test.
A small amount of the semi-purified glycosides extracts
were divided into four test tubes labeled each as “A, B, C,
D”. Each test tube contains five milliliters (5ml) of water,
petroleum ether, ethanol and chloroform. The degree of
solubility of glycosides was observed and recorded.
 4.3 Chemical Test.
4.3.1 Screening of Anthraquinone Glycosides
A) Borntrager Test
B) Modified Borntrager Test
4.3.2 Screening for Flavonoids
A) Bate-Smith and Metcalf Test for Leucoanthocyanins
B) Wilstatter “Cyanidin” Test.

5. Microbiological Test for determining the Antifungal Property of

Radish extract. The following are the culture media,microorganism,
standards and apparatus used in the microbiological screening:
Saboraud Dextrose Agar (SDA) is the culture media; the fungi
Trichophyton mentagrophtes; the standard antibiotic Canesten and
the apparatus used are the Petri dishes, pipette cork borer and
vernier caliper.
5.1 Test Organism:
Fungi (yeast): Candida albicans and Trichophyton
mentagrophytes
5.2 Preparation of inoculums - Preparatory to the assay, the surface of
agar slants contained in the test tubes are inoculated from a
recently grown slant. After incubation at room temperature for
eighteen to twenty-four hours, a stock suspension is prepared by
collecting the surface growth in about 10 ml of sterile distilled water
A portion of this stock suspension is diluted with the volume of
sterile distilled water and the inoculum density of this trial dilution is
compared by the addition of Antifungal standard. This suspension
corresponds to an approximate bacterial density of 300 ml million
per milliliter.
5.3 Preparation of assay plates. Melted SDA is poured into each sterile
petri dish.The agar is distributed evenly in the plate and allowed to
solidify. After the agar solidified the inoculums was transferred to
the petri dish by a cotton swab. To spread the organism evenly in the
petri dish multiple method of streaking was used.
5.4 Preparation of filter paper disc. Several pieces of ashless paper
was cut into disc with the aid of a puncher. Then wrap with clean
sheet of bond paper in lots of four. They are then sterilized in an
autoclave together with the media for 15 minutes and dried in oven.
5.5 Microbiological Assay Method. Dip forceps in methanol, drain
excess solvent, flame and allow to cool. Pick the sterile filter paper
discs from the solution of alcohol (control), the extract and/ or the
final product and the standard antifungal. Sterilize the forceps
every time you pick the discs from each solution. Drain off excess
solution by letting the disc touch the lip of the container. Gently
press down the discs with the tip of the flamed forceps to ensure
contact with the agar. Follow the pattern on the trace paper.
Indicate the starting point on the plate. Allow three readings for
alcohol, radish root extract and final product and standard
antifungal. Incubate the plates at 37ºC for 24 hours. After incubation
measure the zone of inhibition in mm. using vernier caliper. Get the
average of the three measurements and record the results.
5.6 Ash determination - The ___ g of dried Radish roots were placed in
tared porcelain casserole. Then it was heated until it was free of
carbon and placed in a desiccators and the weight of the ash and%
total ash was computed.
5.7 Percentage Yield Determination The extract obtained from 80%
ethyl alcohol was weighed and measured for the determination of
the percentage yield.
6. Physical and Chemical Methods of Analysis:
6.1 Physical Test
6.1.1 Organoleptic Test.
By observation the odor, color, appearance, taste and
the physical state of the tannin was determined.
6.1.2 Solubility test
About ___ g of the tannin was dissolved in 3ml of alcohol,
water, ether, chloroform, benzene and glycerin to determine its
solubility.

6.2 Chemical Test

About ____ g of the tannin extract was dissolved in l2 ml of
water and divided in four test tubes and treated with 1 ml of
Ferric chloride T.S., Lead acetate T.S. ,Gelatin T.S., and
Copper
sulfate T.S.