Study of the Antifungal Activity of Shikonin and Alcoholic – Oily Extracts of Iranian Arnebia euchroma L

Introduction: Today regarding drug resistance of fungi and bacteria, many researches have focused on herbal-based medication. As these herbal-based medications can show better adaptivity, the minimum advantage of them compared to synthetic drugs is that they are harmless. This article aimed to study the antifungal effect of alcoholic extract and essence of Arnebia euchroma L (Abukhalsa) roots on saprophytic and dermatophytic fungi. Methods: In this research, the roots were collected from Zagros heights in spring. Then they were dried and 300 mL ethanol was added to each 100 g dried powder. The alcoholic extraction was performed by maceration and the extract was concentrated by distillation in vacuum. The clevenger apparatus was used to extract the essence; then it was extracted by boiling water at vacuum for 4–6 hours. Shikonin was provided in commercial form. The antifungal activities of alcoholic extract, essence and Shikonin were studied and recorded using cylinder test based on the diameter of inhibition zone in Sabouraud-Dextrose agar. Minimum inhibitory concentration (MIC) and Minimum fungicidal concentration (MFC) were measured by broths macrodilution tests. Results: The results from cylinder, MIC and MFC tests showed that 30% of shikonin was more effective than root on fungi. Our data demonstrated that alcoholic extract was better than oily extract. Conclusion: The alcoholic extract had better characteristics than the essence. To confirm the final findings, further researches are required.


Introduction
One of the most common herbal drugs that are used in traditional medication is Abukhals (Arnebia euchroma) from the family of Boraginaceae.This plant is herbaceous, with sharp silver pubes and the flower is cluster shaped with stretched and alternate leaves.One of the most common habitats of this plant is Iran, especially Kerman. 1,2The root of this plant was used as an ointment for wounds and burnings.It was used in reducing the swellings and had anticancer activity.It caused mild constipation, was used in nourishing the liver, kidneys and spleen, and had vulnerary effect. 37][8] Shikonin and alkanin are red and lipophilic pigments seen in most species.The other species of Arnebia such as nobilis, hispidissima, densiflora, and decumbens are found all over the world. 9nother substance that is found in Arnebia's root is naphthazarin (5,8-dihydroxy-1,4-naphthoquinone).The other substances such as cycloshikonin, acylshikonin, acetylshikonin, beta and beta dimethyl acrylate, isovalerate, beta acetoxy isovalerate, and arnebin 5,6 were also extracted. 10,11n this research, we tried to use Iranian native fungi species to provide logical and significant results.The article aimed to study the antifungal effect of alcoholic extract and essence of Arnebia euchroma L (Abukhalsa) roots on saprophytic and dermatophytic fungi.

Extracts and Essence
The roots were collected from Zagros heights in spring and delivered to Kosha Faravar Giti Institute.After confirmation, the samples were dried in a dark and dry place and then the roots were separated. 3After drying, 300 mL of ethanol was added to 100 g of dried powder.In order to complete the extraction process, the mixture was placed on shaker for 72 hours. 11The alcoholic extraction was performed by maceration and the extract was concentrated by distillation in vacuum. 12The clevenger apparatus was used to extract the essence, then it was extracted by boiling water at vacuum for 4-6 hours.The essence was separated by N-hexane from the water, and anhydride sodium sulfate was used to complete the separation process.The essence was collected in a dark dish, then stored in 4°C. 13Shikonin was supplied from market in commercial form.

Dry Weight of Extracts
In order to evaluate the antifungal activity of extracts, 5 mL of alcoholic concentrated extracts were added to 3 pre-weighted test tubes and dried after incubation for 24 hours.All tubes were remeasured and the dry weighs were calculated. 12ngal Strains For this research, the dermatophytic fungal strains including Trichophyton mentagrophytes (PTCC5054), Trichophyton rubrum (PTCC5143), Microsporum canis (PTCC5069), and Candida albicans (PTCC5027) and saprophytic strains such as Aspergillus fumigatus (PTCC5009) and Penicillium chrysogenum (PTCC5076) were provided from Microbial Collection of Iran, Industrial Research Organization.

Preparation of Fungal Suspension
All the fungi were cultured in Dextrose agar.Fresh colonies were diluted by physiological saline and agitated steadily by vortex.The concentration of the suspension was 1.5 × 10 8 cfu/mL equal to the standard of half McFarland. 14aluating the Antifungal Sensitivity to Cylinder Test For this test, prepared fungal suspension was cultured on Sabouraud-Dextrose agar using swap.The sterile cylinders were applied in determined intervals inside the plate (15 mm from plate's wall and 24 mm from center of 2 cylinders).Then, 0.2% of alcoholic extracts and oily extracts as well as 30% of shikonin were added to each cylinder.In one cylinder, ethanol and in the other DMSO were used as negative blanks, and fluconazole was used as positive blank.All the plates were incubated in 25°C for 72 hours and the results were recorded and compared based on the diameter of colonies.All the experiments were repeated 3 times. 14,15termination of MIC and MFC The Broth microdilution was applied to determine the MIC.In a 96-well plate, 22 wells contained Sabouraud-Dextrose broth.The concentration series of 0.01 to 0.9 mL were prepared for each well and 1.5 × 10 6 CFU/ mL fungi were added to each well.Three wells were selected as blanks for fungal growth, lack of contamination of culture, and contamination of extracts.Finally, all test tubes were incubated in 25°C for 72 hours.The last dilution that did not show any growth was MIC value.All the wells that did not show any growth, were cultured again with Sabouraud-Dextrose agar and incubated in 25°C for 48 hours.The extract dilutions that did not show any fungal growth, were selected as MFC values. 14

Statistical Analysis
The data was analyzed by SPSS software (20th edition), using independent t test and one-way analysis of variance (ANOVA) with P value < 0.05.

Results
The results from cylinder, MIC and MFC tests showed that the effect of 30% shikonin on fungi are more efficient than root.Alcoholic extract was better than oily extract (Figure 1 and Table 1).It is necessary to mention that there is a significant difference among T. mentagrophytes, T. rubrum, M. canis, C. albicans, A. fumigatus, and P. chrysogenum.

Results of MIC-MFC tests
The results from MIC and MFC tests using broth microdilution are shown in Tables 2 and 3.

Discussion
Herbal drugs have been used since ancient times.Recently regarding the drug resistance of fungi and bacteria, herbal drugs as natural reservoirs have received much attention from researchers.Many of these plants were seen in human and animal food and it was proven that they have no side effects.Type and amount of the metabolites in different parts of plants vary based on ecological conditions. 1,15European and developed countries are in a period of transition to production of herbal drugs.It has been proven that high dose of these drugs has no adverse effect on human.This view leads to efforts to provide herbal products and derivatives.Today, production of herbal compounds for treating some microorganism-based illnesses are followed by large pharmaceutical companies.As chemical drugs are synthetic or semi-synthetic, they might be harmful and cause side effects on humans.Iran and other ancient countries such as China, Greece, and Italy have used herbal products to treat the diseases. 16,17aranek et al showed that shikonin had positive effects on inhibition of cells. 18Haghbeen et al prepared 2 different cultures for A. euchroma which had high amounts of pigments.However, antimicrobial experiments showed that they had no effects on gram-negative bacteria and fungi, but optimal effects were observed on gram-positive bacteria. 2Doulah et al showed that the extracts of other species of Arnebia had good antimicrobial effects on gram-negative and gram-positive bacteria. 7Ashkani Esfahani et al illustrated that extracts of A. euchroma compared to sulfadiazine silver had better effects on second-degree burns. 19irbalouti et al determined the antimicrobial activities of the extracts of eight plant species endemic in Iran.The antimicrobial activities of these extracts of 8 Iranian traditional plants were investigated against Escherichia coli O157:H7 and Bacillus cereus.Most of the extracts showed a relatively high antimicrobial activity against all the tested bacteria. 20asiri et al showed that A. euchroma ointment was an effective treatment for healing burn wounds in comparison with SSD and could be regarded as an alternative topical treatment for burn wounds. 21n cylinder test, we studied the antifungal effects of shikonin, alcoholic and oily extracts on saprophytic, dermatophytic and yeast Candida.We found that shikonin had better effects compared to alcoholic and oily extracts, as.In our study, MIC-MFC tests provided different results with previous works.These tests were used in mentioned fungi and good results were obtained.According to these tests, comparing to alcoholic and oily extracts, shikonin had better effects on fungi as shown in figures and tables.

Conclusion
Our results indicated that 30% shikonin extract had better antifungal effects than alcoholic extracts and essence.The alcoholic extract had better characteristics than the essence.To confirm the final findings, further researches are recommended.

Competing Interests
Authors declare that they have no competing interests.

Fig. 1 :
Fig.1: Mean Values of Cylinder Test Between Tested Fungi and Extracts Based on milliliter

Figure 1 .
Figure 1.Mean Values of Cylinder Test Results Regarding Different Fungi and Extracts.

Table 1 .
Comparison of Cylinder Test Between Alcoholic Extract and 30% Shikonin (Compared

Table 1 .
Comparison of Cylinder Test Results Between Alcoholic Extract and 30% Shikonin (Compared Means for 2 Independent Populations)

Table 2 .
MIC-MFC of Fungi Corresponding to Shikonin and Alcoholic and Oily Extracts

Table 3 .
Comparison of MIC-MFC Tests Between Alcoholic Extract and 30% Shikonin (Compared Means for 3 Independent Populations)