In vitro study to determine antimicrobial activity of selected Ayurvedic preparations against bacteria and fungi causing superficial skin infections

The increasing antimicrobial resistance exhibited by microorganisms causing superficial skin infections has led to extensive research on the therapeutic potential of Ayurvedic preparations. Medicinal plants contain many types of naturally occurring and side effectsfree anti microbial compounds that can be effectively used against microbial infections. We tested the antimicrobial activity of twenty-eight Ayurvedic preparations used to treat superficial infections in a local Ayurvedic healthcare institution. They were tested against Trichophyton rubrum, Microsporum gypseum, Candida albicans, Malassezia furfur, Staphylococcus aureus and Streptococcus pyogenes. Twelve preparations showed significant antimicrobial activity and gave inhibition zones >10 mm. Two Ayurvedic preparations (Mixture containing Terminella chebula, Terminella bellerica and Emblica officinalis and one of Terminella chebula only ) showed antimicrobial activity against all the microorganisms tested. We suggest that these two decoctions could be effectively used against all the above tested strains.


Introduction
Superficial skin infections are very common in general practice and are caused by bacteria, fungi and viruses.Bacteria such as Staphylococcus aureus and Streptococcus pyogenes are responsible for the majority of superficial infections. 1 Fungal infections of the skin, nails and hair are commonly caused by dermatophytes, Candida albicans and Malassezia furfur. 2 Treatment of superficial skin infections is mainly based on the use of topical antibacterial and antifungal agents.However, the indiscriminate use of antimicrobial agents has led to antimicrobial resistance.[5][6] The increasing antimicrobial resistance exhibited by microorganisms causing superficial skin infections has led to extensive research on the therapeutic potential of Ayurvedic preparations.Medicinal plants contain many types of naturally occurring anti microbial compounds that can be effectively used against microbial infections. 7Alkaloids, flavones (flavonoids, flavonols, Quinones), essential oils, lectins, polypeptides, phenolics, polyphenols, tannins and terpenoids are the major groups of naturally occurring antimicrobial agents that can be extracted from medicinal plants. 8rrently there are about 29 Ayurvedic preparations used to treat superficial skin infections.In vitro testing has been sparsely carried out to evaluate the effectiveness of these preparations against standard strains.A comprehensive in vitro study will provide valuable scientific information towards the development of more effective topical Ayurvedic applications for superficial infections.Therefore, in order to determine the antimicrobial activity, we investigated 28 Ayurvedic preparations against six common microorganisms causing superficial skin infections including Trichophyton rubrum, Microsporum gypsum, Candida albicans, Malassezia furfur, Staphylococcus aureus and Streptococcus pyogenes.

Ayurvedic preparations
Twenty-eight Ayurvedic preparations used to treat superficial skin infections were kindly provided by a local Ayurvedic healthcare institution.The medicinal plants used for the preparations are listed in table 1.

Antimicrobial activity against bacteria
The antibacterial activity of each Ayurvedic preparation was tested against Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes using the agar well method. 9,10The diameter of a well was 7mm and had a height of 4mm.For each strain, a suspension was prepared in sterile normal saline and turbidity adjusted to 0.5 McFarland standard.The suspension of organisms was used to inoculate blood agar plates to obtain a confluent growth.Wells were cut in the agar surface with the help of a cork borer.Each well was filled with 100µl of the Ayurvedic preparation.At the same time, cefoxitin 30 µg and penicillin 10 µg discs were tested against Staphylococcus aureus and Streptococcus pyogenes respectively.All the plates were incubated 24 hours at 37°C before measuring the inhibition zone sizes.Any zone of inhibition around the decoction containing wells was considered as sensitive.

Antimicrobial activity against dermatophytes and Candida
Using the same method, the Ayurvedic preparations and ketoconazole 10µg disc were tested against standard strains of Trichophyton rubrum, Microsporum gypseum and Candida albicans on Sabouraud agar. 11,12For dermatophytes, the plates were incubated at 30°C for 7 days.Zones for Candida albicans were interpreted after incubating the plates for 24 hours at 37°C.

Antimicrobial activity against Malassezia furfur
Similarly the activity of the Ayurvedic preparations and ketoconazole 10µg disc against a standard strain of Malassezia furfur was tested on Sabouraud agar supplemented with 3% tween 20.The results were interpreted after an incubation period of 48 hours at 37°C.

The effect of freezing on the antimicrobial activity
An aliquot of each Ayurvedic preparation was frozen for 7 days at -80°C and tested simultaneously for all the microorganisms along with the normal solution.The inhibition zones were compared in order to determine any effect of freezing on the antimicrobial activity of the preparations.

The potency of the Ayurvedic preparations
For each strain, a suspension was prepared in sterile normal saline and the turbidity was adjusted to 0.5 McFarland standard.The suspension (100 µl) was mixed separately with 900 µl of each decoction that showed antimicrobial activity.The mixture was incubated at 37°C (30 °C for dermatophytes) and plated on appropriate media at 30 minutes and 60 minutes intervals.The plates were incubated overnight at 37°C (30 °C for dermatophytes) and observed for complete inhibition of the microorganism.A mixture with 100 µl of suspension and 900 µl of sterile normal saline was used as the control.Five ten-fold dilutions were prepared from each decoction that inhibited growth after overnight incubation at 37°C (30 °C for dermatophytes) and observed for complete inhibition of the microorganism.The above experiment was performed for each dilution.
All experiments were performed in triplicates to ensure reproducibility of results.

Results
Out of the 28 Ayurvedic preparations, 12 showed antimicrobial activity against at least one microorganism tested.The antimicrobial activity of these 12 preparations is summarized in Table 2.All preparations that showed antimicrobial activity gave inhibition zones >10 mm.Two Ayurvedic preparations [the mixture of T chebula, T belerica and E officinalis and T chebula ] showed antimicrobial activity against all the microorganisms tested.Emblica officinalis also showed activity against all the test organisms except Candida albicans.Nardostachys jatamansi was only effective against Streptococcus pyogenes and Syzygium cumini showed activity against Staphylococcus aureus only.Apart from the two preparations that showed antimicrobial activity against all the microorganisms tested, decoctions made from A.indica, G glabra, E. officinalis, R cordifolia, C. fistula and M indica were also effective against both dermatophytes tested.
Only the decoction prepared from T chebula, T belerica, E officinalis mixture and T chebula were active against C albicans and M furfur.C. fenestratum, A. indica, E. officinalis, and C. fistula are the medicinal plants used for other decoctions that were effective against both S aureus and S pyogenes.We did not notice any effect of freezing (for 7 days at -80°C) on the microbial activity of the medicinal plant preparations.
The results show (Table 2) that even the 10 -5 dilution of most Ayurvedic preparations could inhibit the growth of tested microorganisms in exposure intervals as low as 30 minutes.For instance, an exposure of 30 minutes to 10 -5 dilution of T chebula, T belerica, E. officinalis mixture inhibited growth of all the tested microorganisms except S aureus and C albicans.When T chebula alone was used such an exposure was effective only against S pyogenes and M gypsum.An overnight exposure to non-diluted preparation was necessary for a broad-spectrum activity.

Discussion
The results show that a high percentage of the tested herbal plants have potent antimicrobial activity.Even the 10 -5 dilution of most Ayurvedic preparations showed efficient inhibitory action.To determine whether the preparations can be effectively used in superficial skin infections, a case control study on humans should be done.Most of the plants used in the preparations are widely distributed and used in ethnomedical preparations in Sri Lanka.We selected these 28 preparations since they are already used to treat superficial skin infections at the local Ayurvedic healthcare institution.S aureus and S pyogenes were selected as the main bacterial strains since they are responsible for the majority of superficial infections. 1 T rubrum, M gypseum C albicans and M furfur are the commonest fungal species responsible for skin infections. 2 Decoctions made from T. chebula, T. belerica, E. officinalis mixture and T. chebula showed the widest antimicrobial activity in our study.4][15][16] Broad-spectrum antibacterial activity of E. officinalis and T. belerica has also been shown previously. 17,18 ccording to our results (Table 2) several other preparations were also effective against specific groups of microorganisms when used un-diluted for longer durations.For instance, C. fenestratum, A. indica, and C. fistula decoctions showed inhibition zones against both S aureus and S pyogenes.Inhibition zones >10 mm indicate strong antimicrobial activity 19 .
The activity of medicinal plant extracts has been intensely studied during recent times.However, varying results have also been observed in different studies.For instance, our results showed that E. officinalis extract is not effective against Candida albicans, which contradicts finding of several other researchers. 17,18 he effectiveness of the preparation might be influenced by the extraction method.Antimicrobial activity of medicinal plants has been demonstrated to be higher in organic extracts than aqueous extracts 20,21 .
In conclusion, Ayurvedic preparations show promising antimicrobial activity against microorganisms causing superficial skin infections.We suggest the use of decoctions prepared from T. chebula, T. belerica, E. officinalis mixture and T. chebula as a universal treatment against microorganisms causing superficial skin infections including T rubrum, M gypsum, C albicans, M furfur, S aureus and S pyogenes.