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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

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Page 1: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT (Raphanus sativus)

A ThesisPresented to

The Faculty of the Graduate SchoolUniversity of Perpetual Help Laguna

In Partial Fulfillment of theRequirements for the Degree of

Master of Science in Microbiology By

 Antonina L. Hipolito

Lab-techUPH- DJGT Med. Univ.

January 20ll

 

Page 2: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT (Raphanus sativus)

Page 3: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

Introduction:

• Herbal medicines • Alternate medicine• Radish root extract ( Raphanus sativus)• Plant defensin• Antifungal solution• Alkanoids, saponins, tannins and glycosides• Antifungal ointment• Skin diseases

Page 4: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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

Page 5: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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

Page 6: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

Statement of the Problem :

This study will deal with the investigation for the presence of antifungal property of Radish root extract intreating fungal skin diseases caused by TrichophytonMentagrophytes 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?

Page 7: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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 rigorousexperimentations in order to obtain the exact results, testing its ability to inhibit the growth of Trichophytonmentagrophytes that cause fungal skin infection.

Page 8: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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

Page 9: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

• 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

Page 10: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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

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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, C10H 11NS, 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

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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

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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)

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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.

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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.

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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.

Page 17: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 18: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

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4.2 Physical test4.2.1 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 Test4.3.2 Screening for Flavonoids A)Bate-Smith and Metcalf Test for Leucoanthocyanins

B)Wilstatter “Cyanidin” Test.

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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 mentagrophytes5.2 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.

Page 21: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 22: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 23: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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 yield6. 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.

Page 24: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 25: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

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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:

Page 27: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 28: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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

Page 29: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 30: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 31: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

4.3 Chemical Test. 4.3.1 Screening of Anthraquinone Glycosides

A) Borntrager Test B) Modified Borntrager Test4.3.2 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.

Page 32: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

5.1 Test Organism: Fungi (yeast): Candida albicans and Trichophyton mentagrophytes5.2 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.

Page 33: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.

Page 34: THE ANTIFUNGAL PROPERTY OF RADISH EXTRACT   (Raphanus

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.  

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