INTRODUCTION:

The term ringworm or ringworms refers to fungal infections that are on the surface of the skin. The name is derived from the early belief was that the infection was due to a worm, which it is not. Ringworm is a fungal infection in the skin. Nevertheless, the name ringworm has stuck. Some of these fungi produce round spots on the skin but many do not. On the other hand, many round spots on the skin are not due to a fungal infection. A physical examination of the affected skin, evaluation of skin scrapings under the microscope, and culture tests can help doctors make the appropriate distinctions. A proper diagnosis is essential to successful treatment¹.

The medical term for ringworm is tinea. (Tinea is the Latin name for a growing worm.) Doctors add another word to indicate where the fungus is located. Tinea capitis, for instance, refers to scalp ringworm, tinea corporis to fungus of the body, tinea pedis to fungus of the feet, and so on.

Although the world is full of yeasts, molds, and fungi, only a few cause skin problems. These agents are called the dermatophytes, which means "skin fungi." An infection with these fungi is sometimes known as dermatophytosis. Skin fungi can only live on the dead layer of keratin protein on top of the skin. They rarely invade deeper into the body and cannot live on mucous membranes, such as those in the mouth or vagina. Scientific names for the most common of the dermatophyte fungi include Trichophyton rubrum, Trichophyton tonsurans, Trichophyton interdigitale, and/or Trichophyton mentagrophytes, Microsporum canis, and Epidermophyton floccosum. Some fungi live only on human skin, hair, or nails. Others live on animals and only sometimes are found on human skin. Still others live in the soil. It is often difficult or impossible to identify the source of a particular person's skin fungus. The fungi may spread from person to person (anthropophilic), from animal to person (zoophilic), or from the soil to a person (geophilic).

Heat and moisture help fungi grow and thrive, which makes them more commonly found in skin folds such as those in the groin or between the toes. This also accounts for their reputation as being caught from showers, locker rooms, and swimming pools. This reputation is exaggerated, though, since many people with "jock itch" or "athlete's foot" have not contracted the infection from locker rooms or athletic facilities

· Tinea barbae: Ringworm of the bearded area of the face and neck, with swelling and marked crusting, is often accompanied by itching, sometimes causing the hair to break off. In the days when men went to the barber daily for a shave, tinea barbae was called barber's itch.

· Tinea capitis: Ringworm of the scalp commonly affects children, mostly in late childhood or adolescence. This condition may spread in schools. Tinea capitis appears as scalp scaling that is associated with bald spots (in contrast to seborrhea or dandruff, for instance, which do not cause hair loss).

· Tinea corporis: When fungus affects the skin of the body, it often produces the round spots of classic ringworm. Sometimes, these spots have an "active" outer border as they slowly grow and advance. It is important to distinguish this rash from other even more common rashes, such as nummular eczema. This condition, and others, may appear similar to ringworm, but they are not due to a fungal infection and require different treatment.

· Tinea cruris: Tinea of the groin ("jock itch") tends to have a reddish-brown color and to extend from the folds of the groin down onto one or both thighs. Other conditions that can mimic tinea cruris include yeast infections, psoriasis, and intertrigo, a chafing rash which results from the skin rubbing against the skin.

· Tinea faciei (faciale): ringworm on the face except in the area of the beard. On the face, ringworm is rarely ring-shaped. Characteristically, it causes red, scaly patches with indistinct edges.

· Tinea manus: ringworm involving the hands, particularly the palms and the spaces between the fingers. It typically causes thickening (hyperkeratosis) of these areas, often on only one hand. Tinea manus is a common companion of tinea pedis (ringworm of the feet). It is also called tinea manuum.

· Tinea pedis: Athlete's foot may cause scaling and inflammation in the toe webs, especially the one between the fourth and fifth toes. Another common form of tinea pedis produces a thickening or scaling of the skin on the heels and soles. This is sometimes referred to as the "moccasin distribution." In still other cases, tinea causes blisters between the toes or on the sole. Aside from athlete's foot, tinea pedis is known as tinea of the foot or, more loosely, fungal infection of the feet. Tinea pedis is an extremely common skin disorder. It is the most common and perhaps the most persistent of the fungal (tinea) infections. It is rare before adolescence. It may occur in association with other fungal skin infections such as tinea cruris (jock itch).

· Tinea unguium: Finally, fungal infection can make the fingernails and, more often, the toenails yellow, thick, and crumbly. This is referred to as fungal nails or onychomycosis. Often, the diagnosis of ringworm is obvious from its location and appearance. Otherwise, skin scrapings for microscopic examination and a culture of the affected skin can establish the diagnosis of ringworm.

MATERIALS AND METHODS:

Plant material:

The plant of Aristolochia bracteolate Lam., was collected from Thirumalai samudram 7km away from Thanjavur (Tamil Nadu) in the month of December 2010. The plants was identified by local people of that village and authenticated by Dr. N. Ravichandran, Asst. Professor, Drug Testing Laboratory, CARISM, SASTRA University Thanjavur, and the Voucher specimen is preserved in laboratory for future reference.

Chemicals:

All the reagents used were of analytical grade obtained from S.D. fine chemicals, Ltd, and Hi Media, Mumbai.

Pharmacognostical Screening of Plants:

Macroscopic Characters, Microscopic characters and Physiochemical Parameters of Aristolochia bracteolate Leaf and leaf powder: The Macroscopic evaluation was carried out for shape, size, color, odor, taste and fracture of the drug. The Microscopic evaluation was performed the Transverse section of midrib and lamina region of the leaf. Different physio-chemical values such as Ash value, extractive values, loss on drying, foreign organic matter, Crude fiber content, were determined.

Fluorescence analysis study of Aristolochia bracteolate Lam., leaves powder:

Fluorescence analysis study of powdered drug material with different reagents was carried out to observe the color reactions.

Study of Plant cell inclusions:

Plant cell inclusions study of powdered drug material with different reagents was carried out to observe the colour reactions.²

General chemical and Micro chemical Tests:

General chemical and Micro chemical tests of powdered drug material with different reagents was carried out to observe the colour reactions to identify the compound.²

Preparation of Extract from Aristolochia bracteolate Lam., leaf powder:

The leaves were dried under shade, powdered and passed through 40meshes and stored in closed vessel for further use. The dried powder material (150g) was subjected to soxhelt extraction with Ethanol for continuous hot extraction for 24 hours. The extracts were concentrated under reduced pressure to obtain the extracts solid residues. The percentage value of extract was 28 (%w/w).

Phytochemical Evaluation of Ethanolic Leaf extract of Aristolochia bracteolate Lam.,^3,4

The Ethanolic Extract of Abutilon indicum (Linn) Sweet (Leaf) was subjected to preliminary Phytochemical tests followed by the methods of Harbone (1998), and Trease and Evans (1983) and the phytoconstituents reported in table.

Screening of Thin layer Chromatography:

TLC for Alkaloids

Stationary phase : Silicagel G

Mobile Phase :Butanol: Acetic acid: Water (4:5:1)

Detecting Reagent : Dragendorffs reagent

TLC for Terpenes:

Stationary Phase :Silica gel G

Mobile Phase :Hexane: acetone (9:1)

Detecting Reagent : Iodine Chamber

TLC for Saponins:

Stationary Phase :SilicagelG

Mobile Phase :Chloroform: Methanol: Water (7:4:1)

Detecting Reagent : Iodine Chamber

Enumeration (Counting) of bacteria by plate count or serial dilution agar plate technique:

Procedure:

1. Label the dilution blanks as 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 and 10^-7.

2. Prepare the initial dilution by adding 1 gm of the sample into a 9 ml dilution blank (Distilled water) labeled 10^-1 thus diluting the original sample 10 times (1/1+9 = 1/10 and is written 1:10 or 10^-1).

3. Mix the contents by rolling the tube back and forth between your hands to obtain uniform distribution of organisms (cells).

4. From the first dilution, transfer 1ml of the suspension while in motion, to the dilution blank 10^-2 with a sterile and fresh 1ml pipette diluting the original suspension to 100 times (1/10 x 1/10 = 1/100 or 10^-2).

5. From the 10^-2 suspension, transfer 1 ml of suspension to 10^-3 dilution blank with a fresh sterile pipette, thus diluting the original sample to1000 times (1:1000 or 10^-3).

6. Repeat this procedure till the original sample has been diluted 10,000,000 (10^-7) times using every time a fresh sterile pipette.

7. From the appropriate dilutions (10^-1 to 10^-7) transfer 1ml or 0.1 ml of suspension while in motion, with the respective pipettes, to sterile Petri dishes. Three Petri dishes are to be used for each dilution (if 0.1 ml is plated; the dilution is increased 10 times).

8. Add approximately 15 ml of the nutrient medium, melted and cooled to 45°C, to each plate containing the diluted sample. Mix the contents of each plate by rotating gently to distribute the cells throughout the medium.

9. Allow the plates to solidify. Incubate these plates in an inverted position for 24-48 hours at 37°

Test for Escherichia coli:

1. 1gm / 1ml of the sample or as specified at individual monograph is transferred to 100ml of fluid lactose medium for Escherichia coli.

2. Incubate the fluid lactose medium at 35 ± 2°C for 24 hours.

3. Observe the fluid lactose medium for growth and if growth is present, mix by gently shaking.

4. Streak a loop full of fluid lactose medium on the plates containing Mac-Conkey agar and inoculate the plate at 35 ± 2°C for 24 hours.

5. After incubation observe the growth and if none of the colonies are bright pink in colour, the sample complies the test for absence of Escherichia coli.

6. If colonies are bright pink in colour, transfer the suspect colonies to plates containing Levine Eosin methylene blue agar. Incubate the plate at 35 ± 2°C for 24 hours.

7. Similarly inoculate into a 5ml of peptone water and incubate at 35 ± 2°C for 24 hours.

8. Typical E. coli colonies on Levine Eosin methylene blue agar appear as dark colonies with metallic sheen.

9. After the incubation period, perform a test for indole production on peptone water.

10. A positive test will give a pink/red colour in the reagent layer and a negative test will give (retains) a yellow colour upon addition of 0.5ml of Kovac’s reagent.

11. Perform gram staining test for suspect colonies. Typical E. coli colonies are appeared as gram negative rods.

Test for Salmonella Species:

1. 1gm / 1ml of the sample or as specified at individual monograph is transferred to 100ml of fluid lactose medium for Salmonella.

2. Incubate the fluid lactose medium at 35 ± 2°C for 24 hours.

3. Observe the fluid lactose medium for growth and if growth is present, mix by gently shaking.

4. Transfer 1ml fluid lactose medium each to both tubes containing 10ml of Fluid selenite cysteine medium and Tetrathionate medium, mix and incubate the tubes at 35 ± 2°C for 24 hours.

5. Streak a portion of media from both Fluid selenite cysteine medium and Tetrathionate medium on the surface of Brilliant green agar and Bismuth sulphate agar contained in petriplates and incubate the plates at 35 ± 2°C for 24 hours.

6. After incubation observe the growth and if none of the colonies conforms to the following description, the sample complies of the test for absence of salmonella species.

a.) On Brilliant green agar medium – Small, transparent, Colourless, or opaque, pinkish or white colonies frequently surrounded by a pink or red zone.

b.) On Bismuth Sulphite agar – Black or green colonies

7. If colonies show characteristic colony morphology, transfer the suspect colonies to tubes containing triple sugar iron agar medium by first streaking the surface of the slant and then stabbing the inoculation wire well beneath the surface. Incubate the tubes at 35 ± 2°C for 24 hours.

8. Typical salmonella colonies on triple sugar iron agar produce alkaline (red) slant and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulphide production).

Determination of Minimum Inhibitory Concentration:

Plant extract which is active against the tested microorganisms was taken for the determination of minimum inhibitory concentration. Inoculate the two fungal strains and then inoculate plant extract of 3 different concentrations like (8mg, 4mg and 2mg) in sabouraud dextrose broth and incubate at 30°C for 24 hours. The concentrations are measured in spectrophotometer at the wavelength of 660 nm and readings are taken. The lowest reading was taken as MIC value.

Antifungal activity by Disc diffusion Method:

Sabouraud dextrose agar medium was prepared and sterilized at 121°C for 15 minutes. The medium was poured on the sterile petriplates and allowed to solidify. After solidification sterile cotton swab and dip it into culture containing two fungal strains (MTCC 3272 and MTCC 3270) separately. Inoculate the organisms first in horizontal and then vertical direction for even distribution, using the swab and dry for 15 minutes. A sterile filter paper disc was dipped in four different concentrations (100mg, 50mg, 25mg and 12.5mg) of Ethanolic Extract of Aristolochia bracteolata using sterile forceps. The disc was placed on the agar surface of the inoculated plate. The standard disc (Ketaconazole) as positive control was also placed on inoculated plate. The inoculated plate was incubated at 30°C for 3 days in an inverted position.

Results:

Macroscopic Characters of Aristolochia bracteolate Lam.,

Prostate herb and often the maximum height is 50cm. Leaves are cordiform or reniform, 4-7× 5-9cm, 5-nerved from base. Bracts are cordate-orbicular, leaf margin entire or wavy. Flowers dark purple, 5cm long the limb entire, 1-lipped; lip 3cm long, rolled back, emarginated. Fruits are Capsule 2.5×1.5cm in size and the seeds are numerous and the seeds cordate in shape.

Microscopical Character of Aristolochia bracteolate Lam., Leaf:

Fig : 1 T.S of Aristolochia bracteolate Lam., Leaf. Midrib region

Fig: 2 (T.S of Aristolochia bracteolate Lam., Leaf Lamina region) and Stomata

The T.S. of leaf midrib consists of epidermis, cortex and vascular bundle. The epidermis made up of single rows, short ovoid cells and the outer cell wall contains cuticle. The cortex consists of 4 rows of short, ovoid parenchymatous cells. The vascular bundles is single, globular shaped consists of xylem, cambium and phloem. The xylem cells are surrounded by 2 rows of phloem cells. The lamina consists of single rows of elongated cells and the outer cell was contains cuticle. The palisade cells are single rows, elongated with chloroplasts and arranged without intercellular space. The spongy parenchyma cells are 2-3 rows polygonal with intercellular space. The bundle sheath xylem cells are spiral thickening. The epidermal cells of the leaf having anomocytic type of stomata.

Table No: 1 Physiochemical Parameters of Aristolochia bracteolate Lam., Leaf Powder

S. No	Parameters	%W/W
1.	Hexane Soluble extractive	8%
2.	Pet ether Soluble extractive	2.4%
3.	Chloroform Soluble extractive	4.5%
4.	Acetone soluble extractive	4.8%
5.	Ethanol soluble extractive	14.4%
6.	Ethyl acetate soluble extractive	8%
7.	Methanol soluble extractive	13.6%
8.	Water soluble extractive	24%
9.	Foreign organic matter	2%
10.	Loss on drying	3%
11.	Crude fiber content	21%
12.	Total Ash	5%
13.	Acid insoluble ash	2%
14.	Sulphated ash	12%
15.	Water Soluble ash	1%

Table No: 2 Fluorescence analysis study of Aristolochia bracteolate Lam., leaves powder

S. No	Sample	Colour in Day light	Colour in UV
1.	Powder	Green	Green
2.	Powder + 0.1N Sodium Hydroxide	Dark green	Pale green
3.	Powder + Acetic anhydride	Dark green	Pale green
4.	Powder + 0.1N Hydrochloric acid	Pale green	Dark green
5.	Powder + water	Pale green	Dark green

Table No: 3 General Chemical and Micro chemical tests for Leaf powder of Aristolochia bracteolate

S. No	Test	Results
1.	Test with water /aqueous extract	+
2.	Test For Tannins	+
3.	Test for Anthra quinine	-
4.	Test for Mucilage	+
5.	Test for Carbohydrate	+
6.	Test for alkaloids	+

Table No: 4 Study of Plant cell inclusions

S. No	Test	Result	Colour
1.	Cellulose	+	Pale yellow
2.	Lignin	+	Deep blue
3.	Suberin	+	Deep yellow
4.	Chitin	+	Violet
5.	Starch	+	Blue
6.	Mucilage	+	Pink
7.	Proteins	+	Brick red
8.	Alkaloids	+	Reddish brown
9.	Tannins	+	Bluish black
10.	Calcium oxalate	+	Needle shaped crystals
11.	Calcium carbonate	+	Needle shaped crystals

Table No: 5 screening of Thin layer Chromatography

S. No	Colour of Spot	Rf Value
1.	TLC for Alkaloids - Brown	0.5
2.	TLC for Terpenes – Orange brown	0.6
3.	TLC for Saponins – Dark Brown	0.8

Table No:6 Preliminary phytochemical Analysis of Ethanolic Leaf Extract of Aristolochia bracteolate Lam.,

S. No	Phytoconstituents	Ethanolic Extract
1.	Alkaloids	+
2.	Aminoacids	+
3.	Anthraquinones	-
4.	Carbohydrates	+
5.	Flavonoids	+
6.	Phenolic groups	+
7.	Resins/gums	-
8.	Saponins	+
9.	Steroids	-
10.	Tannins	+

+ = Present - = Absent

Table No:7 Enumeration (Counting) of Total bacteria and Pathogens by plate count or serial dilution agar plate technique

S. No	Test organisms	Microbial count
1.	Total bacterial Count	3 x 10⁵ bacteria / gm of sample
2.	Escherichia coli	+
3.	Pseudomonas aeruginosa	-
4.	Salmonella	+

+ Present - Absent

Table No: 8 Determination of Minimum Inhibitory Concentration

Name of the strains	Minimum Inhibitory concentration (Tube method)
Name of the strains	8mg/ml	4mg/ml	2mg/ml
MTCC 3272	0.056	0.102	0.121
MTCC 3270	0.033	0.125	0.137

Table No: 9 Antifungal activity of Aristolochia bracteolata against two fungal strains

Name of the strains	Ketaconazole (Positive Control)	*Aristolochia bracteolate*
Name of the strains	Ketaconazole (Positive Control)	100mg	50mg	25 mg	12.5mg
Trichophyton rubrum MTCC 3272	34 ± 0.05	-	-	10 ± 0.02	14 ± 0.01
Microsporum canis MTCC 3270	38 ± 0.05	-	-	-	-

DISCUSSIONS:

Ringworm refers to a fungus infection that affects the scalp, feet, and nails. It is also known as Tinea. In ringworm a red ring appears on the infected person’s skin. It is contagious and may spread with direct or indirect contact. Ringworm is caused by several different fungus organisms that belong to a group called Dermatophytes, Derma means skin and Phytes means organisms. The duration of scalp ringworm is 10-14 days and that of skin ringworm is 4-10 days. The plant Aristolochia bracteolate Lam., authenticated by Dr. N.Ravichandran, Asst. Professor, Drug Testing Laboratory, CARISM, SASTRA University Thanjavur, and the Voucher specimen is preserved in laboratory for future reference. The Macroscopic evaluation was carried out for shape, size, color, odor, taste and fracture of the drug. The Microscopic evaluation was performed the Transverse section of midrib and lamina region of the leaf, Physiochemical parameters including Extractive vales determined by according to polarity of solvents, the extractive value was highest in water and was recorded to be 24%w/w, and ethanol soluble extractive value was about 14.4 %w/w .The different ash values (Total ash-5%, Acid insoluble ash 2%, Sulphated ash-12% and Water soluble ash – 1%) and the different physiochemical parameters were screened. Ethanolic extract of Aristolochia bracteolate Lam., was performed by using Soxhelt apparatus. The percentage value of extract was 28%w/w, Florescence analysis, General and microchemical analysis for crude powder and Plant cell inclusions. Qualitative and Total microbial load showed that they all within limit. Ethanolic extract effective against list out the fungal like Trichiophyton rubrum and Microsporum canis. The Ethanolic extract of Arisolochia bracteolate Lam., showed highest activity at minimum concentration. Thus from our findings, it was concluded that the bioactive principles present in the extracts may be responsible in the treatment of ringworm infection.

CONCLUSION:

Ringworm is a fungal skin infection. There are many types of fungus germs (fungi) and some can infect the skin, nails, and hair. This leaflet just deals with ringworm of the skin (sometimes called tinea corporis). Other leaflets deal with fungal infections of the scalp (scalp ringworm), foot (athlete's foot), groin (tinea cruris), and nails. A small area of infected skin tends to spread outwards. It typically develops into a circular, red, inflamed patch of skin. The outer edge is more inflamed and scaly than the paler centre. So, it often looks like a ring that becomes gradually larger - hence the name ringworm. Sometimes only one patch of infection occurs. Sometimes several patches occur over the body, particularly if you catch the infection from handling an infected animal. The rash may be mildly irritating, but sometimes it is very itchy and inflamed. The rash may vary depending on which type of fungus causes the infection. Sometimes fungal skin infections look similar to other skin rashes, such as psoriasis. Antibiotics and other antimicrobial agents are effective in the prevention and treatment of ring worm, but they also cause undesirable side effects such as ecologic disturbance of oral and gut flora. Therefore, search for the anti fungal herbs could offer an effective alternative to antibiotic strategies for fungal infection disease like ring worm. The plant Aristolochia bracteolate Lam., was screened for its Macroscopic, Microscopic, Physiochemical parameter, Florescence analysis, General and microchemical analysis for crude powder and Plant cell inclusions. Qualitative and Total microbial load showed that they all within limit. Extraction was carried out by using soxhlet apparatus. Ethanolic extract effective against list out the fungal like Trichiophyton rubrum and Microsporum canis. The Ethanolic extract of Arisolochia bracteolate Lam., showed highest activity at minimum concentration. Thus from our findings, it was concluded that the bioactive principles present in the extracts may be responsible in the treatment of ringworm infection. Developing countries like India having the percentage of poor people more, to meet with the demand of the poor public, the Aristolochia bracteolate Lam., may serve the purpose once the evaluation and detailed studies may over.

ACKNOWLEDGEMENT:

The authors would like to thank Dr N. Ravichandran, Asst. Professor, Drug Testing Laboratory, CARISM, SASTRA University Thanjavur, Tamil Nadu.

REFERENCE:

1. Rashid, Rashid M., and Andrew C. Miller. "Tinea." (2009) eMedicine.

2. Khandelwal K.R .Practical Pharmacognosy 19^th Edition (2002) P 24-25, 163-164

3. Trease GE, Evans WC. Pharmacognosy, London; Bailiere Tindal;(1983)

4. Harborne JB. A Guide to Modern techniques of plant Analysis. USA: Kluwer Academic Publishers; (1998)

Received on 07.04.2011 Modified on 14.04.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1123-1128