Introduction

In the past few decades, a worldwide increase in the incidence of fungal infections has been observed & antifungal drugs have led to the emergence of resistant strains. The challenge has been to develop effective strategies for the treatment of candidiasis and other fungal diseases considering the increase in opportunistic fungal infections in humans.1 Statistically fungal diseases have caused over 1.6 million deaths annually and over one billion people suffer from severe fungal diseases.2

In present conditions effective, complete and timely management of these microbes is a must. Though the available antifungal drugs have been tried in most of the conditions, their reoccurrence is common, thus making it necessitate the need of an alternative and effective therapy. The drug Nimbooka (Citrus limon Linn.), which is easily available everywhere and is known to have both krimighna & kandughna activity.3

Dravyaguna shastra, a part and partial of Ayurveda, describes in detail the pharmacological properties, administration and modes of actions of drugs. Among the various actions explained, krimighna is one. The word ‘krimi’ has multifaceted meanings referring to macro and micro organisms responsible for causing diseases and includes all microbes irrespective of whether bacteria, fungi or virus. Krimighna drugs act as antimicrobials as well as antimacrobials.4

Nimbooka (Citrus limon Linn.) belongs to Rutaceae family. In Nighantus, Nimbooka has been explained under Amradiphala varga of Bhavaprakasha Nighantu5, Aushadhi varga of Kaiyadeva Nighantu6, Phala varga of Shaligrama Nighantu.7 It is described as ‘Jantumari’ in Raja Nighantu,8 as ‘krimi samuha nashanam’ in Bhavaprakasha Nighantu. This drug is widely used in many krimihara yogas. Hence, the trial drugs selected for the study viz. Nimbooka Patra and Phala twak are known to have krimighna activity.

The two different strains of fungi namely Aspergillus niger and Candida albicans9 selected for the study as representative microorganisms, owing to greater susceptance of Candida albicans, which is a opportunistic pathogen in the oral cavity & vagina and Aspergillus niger, an opportunistic pathogen of the uro-genital tract and of the blood stream. In the present study Agar well diffusion/Cup plate method has been followed to detect the Antifungal property of the drugs in different dosage forms and concentrations by serial tube dilution method.

Objective of the study:

• To evaluate krimighna activity of Nimbooka phala twak Swarasa by Serial Tube Dilution method.
• To evaluate krimighna activity of Nimbooka patra Swarasa by Serial Tube Dilution method.
• To compare the results of krimighna activity of Swarasa of Nimbooka phala twak and patra in selected fungal strain.

Material and Methods:

The drug was identified and authenticated by the botanist and HOD of Dravyaguna, S.J.G.A.M.C- Koppal. Phytochemical studies and In Vitro Antifungal activity were carried out at ‘Drug Testing lab’, BLDEA’s College of Pharmacy, BLDE campus, Vijayapur. Preparation of different dosage forms were done at ‘Drug Testing lab’, BLDEA’s College of Pharmacy, Vijayapur.  Microscopic studies of Nimbooka Patra and Phala twak were carried out at Laboratory, Department of Botany, KCP Science College- Vijayapur.

 

A. Source of the drugs selected for the study:

Bright yellow, botanically identified fresh Nimbooka (Citrus limon Linn.) phala was collected of average size and with thick rind. The leaves collected were kept for drying under shade for 10 days and later made in to coarse powder.

B. Preparation of the dosage forms10:

a. Preparation of Swarasa of Nimbooka patra and Nimbooka phala twak: 100 gm of each trial drug were kept in clean plastic jar and 400 ml of water was added and kept for 48 hours and macerated then filtered. Filtrate was evaporated on Magnetic stirrer evaporator; yield obtained was 9.59 gm of extract from Nimbooka phala twak and 11.750 gm of extract from Nimbooka patra. Due to its hygroscopic nature it was stored in the desiccators.

 b. Preparation of methanol extract of Nimbooka patra and phala twak: Extraction was carried out by Maceration method. 100 gm of Nimbooka Patra powder yielded 7.52 gm and Nimbooka phala twak yielded 11.65 gm of the extract which was stored in an air tight container and then stored in desiccators.

Source of microorganisms: Microorganisms were procured Department of microbiology, BLDEA’s College of Pharmacy, Vijayapur. 

Preparation of the media: Sabouraud Dextrose Agar Media (HIMEDIA –M096)

Preservation of the cultures: Cultures from Department of microbiology, BLDEA’s College of pharmacy, Vijayapur were supplied on Agar media in cotton plugged test tubes.

Agar Diffusion/Cup Plate method: To determine the antimicrobial activities of the different samples of Nimbooka Patra and Phala twak.

Preparation of the samples11: Required amount of 100 mg of the extract was diluted in 10 ml sterile distilled water to get concentration of 10 mg/ml. 1ml of concentration of 10 mg/ml was taken in separate test tube and to that 1 ml of sterile distilled water was added to achieve the concentration of 5 mg/ml. 1 ml of concentration of 10 mg/ml was taken in separate test tube and to that 4 ml of sterile distilled water was added to achieve the concentration of 2 mg/ml. 1 ml of concentration of 10 mg/ml was taken in separate test tube and to that 9 ml of sterile distilled water was added to achieve the concentration of 1 mg/ml. Control – distilled water was taken as negative control. Standard drug – 100 mg of Fluconazole tablet was dissolved in 100 ml of Phosphate buffer saline of pH 7.4. The Phosphate buffer saline is prepared by adding Disodium hydrogen phosphate= 10.9 gm, Sodium di-hydrogen phosphate=3-2 gm, Sodium chloride= 90 gm in 1000 ml water.

Addition of the samples:

A sterile cork borer of 6 mm diameter was used to make bores in the medium which was already solidified. 4 bores were made in the plate in order to accommodate 2 different concentrations of 1 sample – for eg. 1 mg/ml and 2 mg/ml concentration of aqueous extract of Nimbooka patra, Standard-Fluconazole and negative control-distilled water. Same procedure is done for different concentrations of other extracts i.e., aqueous extract of Nimbooka phala twak and alcohol extract of Nimbooka patra and phala twak. Sterile 1ml pipette used to fill 0.2 ml of samples, Standard and Negative control into the boxes. Plates were covered with sterile lid immediately. Proper markings were done to identify the respective bores pertaining to different samples. All the plates were allowed for the diffusion of the solution at room temperature for an hour. After an hour of pre-incubation, the plates were transferred to the incubator. It was incubated at 370 C for 24 hours. After 24 hrs of incubation, the zone of inhibition produced by different dosage forms of Nimbooka Patra and Phala twak, Standard drug were measured in mm and recorded.

Observations during pilot study:

• Solution of Water Extracts:  Water extracts of Nimbooka patra was Blackish Green and that of Nimbooka phala twak was Brownish yellow in color.   
• Solution of Methanol Extracts:

Methanol Extracts of Nimbooka patra and Nimbooka phala twak dissolve in water easily and was Blackish Green and Brownish yellow in color respectively.

During the pilot study of the antimicrobial assay it was found that

• Drugs exhibited the zone of inhibition by 24 hrs.
• Zone of inhibition was maintained till 48 hrs.
• However by 72 hrs there was complete fungal growth.

Antifungal Activity:

Group I: Aspergillus niger

• All the 6 test samples showed antifungal activity against Aspergillus niger
• Nimbooka peel Water extract at a concentration of 10mg/ml showed highest Zone of inhibition of 52 mm against Aspergillus niger.
• Nimbooka peel Methanol extract at a concentration of 5 mg/ml and 10 mg/ml showed highest Zone of inhibition of 49 mm against Aspergillus niger.
• Nimbooka leaf Water extract at a concentration of 10mg/ml showed highest Zone of inhibition of 43 mm against Aspergillus niger.
• Standard drug Fluconazole showed highest zone of inhibition of 40mm against Aspergillus niger. 
• Nimbooka leaf Methanol extract at a concentration of 10 mg/ml showed highest Zone of inhibition of 39.5 mm against Aspergillus niger.

Note: Nimbooka peel water extract was found to be very effective in Aspergillus niger when compared to other test dosage forms.

Group II: Candida albicans

• All the 6 test samples showed antifungal activity against Candida albicans
• Nimbooka leaf water extract at a concentration of 10 mg/ml showed a zone of inhibition of 40mm against Candida albicans
• Nimbooka leaf methanol extract at a concentration of 5 mg/ml showed a zone of inhibition of 30 mm against Candida albicans
• Nimbooka peel water extract at a concentration of 10 mg/ml showed a zone of inhibition of 30mm against Candida albicans
• Nimbooka peel methanol extract at a concentration of 5 mg/ml showed a zone of inhibition of 33 mm against Candida albicans
• Standard drug Fluconazole at concentration 100 mcg/ml showed zone of inhibition of 21 mm against Candida albicans.  

• Note: Nimbooka leaf water extract was found to be very effective in Candida albicans when compared to other test dosage forms.

Comparison between dosage forms of each drug (the mean value of zone of inhibition against the 2 organisms) – Anti fungal Activity

Nimbooka patra

• Among the different dosage forms viz. Methanol extract and water extract, the mean difference was insignificant at the significance level at 5% between the organisms Aspergillus niger and Candida albicans

Nimbooka phala twak

• Among the different dosage forms viz. Methanol extract and water extract, the mean difference was insignificant at the significance level at 5% between the organisms Aspergillus niger and Candida albicans.

Comparison of antifungal activity of different drug delivery forms against each test organism.

1. Standard – Fluconazole :

• Aspergillus niger was sensitive to Fluconazole with a zone of inhibition of 25.5 ± 8.43 mm
• Candida albicans was sensitive to Fluconazole with a zone of inhibition of 19. ± 1.78 mm

1. Nimbooka patra methanol extract :

• Candida albicans was sensitive to Nimbooka patra methanol extract with a zone of inhibition 24 ± 3.4mm.
• Aspergillus niger was sensitive to Nimbooka patra methanol extract with a zone of inhibition 29.5 ± 7.95mm

1. Nimbooka patra water extract :

• Aspergillus niger was the most sensitive to Nimbooka patra water extract with a zone of inhibition 27.3 ± 10.69 mm.
• Candida albicans  was  less sensitive to Nimbooka patra water  extract with a zone of inhibition 22 ± 11.28 mm

1. Nimbooka phala twak methanol extract :

• Candida albicans was the less sensitive to Nimbooka phala twak methanol extract with a zone of inhibition 21.5 ± 8.46 mm
• Aspergillus niger was  most sensitive to Nimbooka phala twak methanol extract with a zone of inhibition 32.34 ± 11.8 mm

1. Nimbooka phala twak water extract :

• Aspergillus niger  was the most sensitive to Nimbooka phala twak water  extract with a zone of inhibition 30 ± 14.6 mm
• Candida albicans  was  sensitive to Nimbooka phala twak water  extract with a zone of inhibition 24 ± 4.9 mm

Note:  Aspergillus niger was found to be more sensitive when compared to Candida albicans.

Discussion

Pilot study:

• The methanol extracts of Nimbooka Patra and Phala twak dissolved in water completely, wherein both dissolved easily and uniform solution was obtained.
• Agar cup plate method- was preferred than other methods because of the requirement of the minimum sample size; several samples can be tested on one micro organism.
• Pilot study was carried out to determine the effective concentration at which the trial drugs showed the anti fungal activity.
• It was observed that during the pilot study both fungal strains showed encouraging growth in sabouraud dextrose agar media. Hence it was taken up for the study proper.
• Standard drug Fluconazole showed good activity at 100mcg/ml. Hence this concentration was selected for the study proper.

• Distilled water was selected as the negative control group, to rule out the role of methanol in antifungal activity.

Study proper:  

• The parameters selected for the antifungal activity were zone of inhibition in milligram in agar cup plate method.
• The study proper was carried out in 2 dosage forms viz. methanol and water extract, because in vitro studies allow extracts as dosage because different extracts reveal the different phyto-constituents present in it.
• In agar well diffusion/cup plate method, Nimbooka Phala twak water extract showed good activity against Aspergillus niger whereas Nimbooka Patra water extract showed good activity against Candida albicans. It showed antifungal activity at 10 mg/ml.

Test group I: Aspergillus niger 

Nimbooka phala twak water extract was found to be very effective in Aspergillus niger when compared to other test dosage forms. This may be due to presence of  Hesperidin, Quercetin, Volatile oil, Pectin, Calcium oxalate, Bitter substances, α-terpineol, Linailye acetate, Gerange acetate, Luteolin-7-rutinoside, Quercetin-3, 5-diglucoside.

Test group II: Candida albicans

Nimbooka leaf water extract was found to be very effective in Candida albicans when compared to other test dosage forms. This may be due to presence of Rectin, Hesperidin, Neral, Geraniol, Limonene, Citral.

Comparison between the groups:

Nimbooka Phala twak in different dosage forms was more active and sensitive against Aspergillus niger when compared with Candida albicans. This shows that Nimbooka Phala twak water extract is more sensitive for Aspergillus niger when compared with that of Candida albicans.

            Nimbooka phala twak water extract has high significant activity with 52 mm when compared to that of methanol extract (49 mm).

            Though all the forms of Nimbooka Phala twak are sensitive against both fungal strains, the sensitivity of Nimbooka Phala twak may be due to the presence of Amla, Katu rasa, ushna veerya and Tiksna guna. Whereas in patra, presence of Amla, katu rasa, ushna veerya and laghu guna. 

            The well known standard drug Fluconazole showed better antifungal activity against Aspergillus niger and Candida albicans.        

Krimighna activity of Nimbooka Patra and Phala twak (Ayurvedic Perspective):

• For a krimi to manifest it requires drava with kledamsha and this is very true in fungal infection also. Thus the drug due to the kaphahara and kleda achushana ability has a direct action on the krimi.

• Nimbooka Patra as well as Phala twak possesses Amla & Katu rasa, laghu guna and ushna veerya12, 13, 14. The Amla & katu rasa is considered to be Dhatu hrasaka (causes the depletion of the dhatus)
• Nimbooka has its action like Agnivardhana, Rochana, Pachana, Deepana, Varnya, Balakara, Shleshma and Vatahara due to Tikshana, Ushna guna and hence does the Prakruti Vighata by reducing the lakshanas of krimi like Chardi, Vibandha, Mandanala, Arochaka, etc12, 13, 14, 15, 16.
• Hence it can be inferred that Nimbooka due to its above properties succeeds in checking the growth of the microbe. This can be very much compared to the fungistatic activity or prakruthi vighatha where in an unfavorable environment for the growth of fungus is created.

The activities of krimighna dravyas in In-vitro studies is limited to the antimicrobial activity alone but in-vivo they may have wide spectrum of activities such as Anti inflammatory,  phagocytosis, neutralizing the circulating endotoxins produced by the micro organisms etc. thus proving that the krimighna dravyas have a holistic

Conclusion

Nimbooka Phala twak has a higher yield in methanol extract when compared to Nimbooka Patra. Nimbooka Patra has a higher yield in water extract when compared to Nimbooka Phala twak.  Agar diffusion/cup plate method is the most convenient method to screen the herbal extracts.  Among both the drugs, Nimbooka Patra has shown better activity than Nimbooka Phala twak against Candida albicans where as Nimbooka Phala twak has shown better activity than Nimbooka Patra against Aspergillus niger. Among the two extracts of Nimbooka, water extracts showed better activity than methanol extracts. It is observed from the in-vitro study that the drug Nimbooka Phala twak and patra has shown comparative antifungal activity with that of standard drug Fluconazole. Thus it can be concluded that at a lower dose of Nimbooka Patra and phala twak is having statistically significant antifungal activity when compared with standard drug.