Essential and Recovery Oils from Matricaria chamomilla Flowers as Environmentally Friendly Fungicides Against Four Fungi Isolated from Cultural Heritage Objects

Recovery oils, obtained from the hydro-distillation of the fresh flowers of Matricaria chamomilla, as well as essential oils, were studied for their environmental purposes in cultural heritage. These oils were assayed for their antifungal activity against the growth of four molds isolated from archaeological manuscripts (Aspergillus niger), museum gypsum board Antique (A. flavus), museum archaeological tissue (A. terreus), and museum organic materials (Fusarium culmorum) of cultural heritage objects. Oils were applied to inhibit the growth of fungi at amounts of 25, 50, 75 and, 100 μL/mL, and compared with negative controls (0 μL/mL) or positive controls (Sertaconazol 3g/L). Using GC/MS analysis, the main chemical compounds identified in the essential oil were (Z)-β-farnesene (27%), D-limonene (15.25%), and α-bisabolol oxide A (14.9%), while the compounds identified in the recovery oil were α-bisabolol oxide A (18.6%), d-limonene (8.82%), and α-bisabolol oxide B (7.13%). A low amount of chamazulene was observed in both essential and recovery oils, with amounts of 0.73% and 3.50%, respectively. Recovery oil, at a concentration of 75 and 100 μL/mL, showed fungal mycelial inhibition (FMI) percentage for the growth of A. niger, with values of 78% and 85%, respectively. At a concentration of 100 μL/mL, both oils showed 100% FMI of A. terreus. Oils showed weak activity against the growth of A. flavus. Essential oils at 100 μL/mL had good activity against the growth of F. culmorum, with FMI of 86.6%. The results suggest the potential use of essential and recovery oils from M. chamomilla fresh flowers as environmentally friendly bio-fungicides.


Introduction
In domestic environmental conditions, fungi are known as a major biodeteriogens of cultural heritage. Fungi are able to colonize and degrade materials (wood, paper, textiles, leather, plastic, stones, metal, and clay) that have been used for the construction of cultural heritage sites, such as monuments and artifacts, causing stains in their surfaces or changing their morphological characterizations [1][2][3][4][5][6][7][8][9][10]. Therefore, the chemical treatment applications used in cultural heritage conservation must be non-toxic and non-destructive [2,11].

Tested Fungal Isolates
All fungi used in this study were isolated from different organic and inorganic substrata of cultural heritage objects in Egypt (Table 1). For DNA extraction, each isolate was grown in potato dextrose broth for three to four days. The mycelia of each isolate were harvested and processed for genomic DNA extraction, using a protocol published by Saitoh [36]. Analyses of DNA sequences of partial ITS gene were performed according to our previous published article [37].

GC-MS Analysis of Essential Oil and the n-Hexane Recovered Oil
The essential oil and n-hexane recovered oil from flowers of Matricaria chamomilla were analyzed for their chemical constitutes, using Focus GC-DSQ Mass Spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness) apparatus, at Atomic and Molecular Physics Unit, Experimental Nuclear Physics Department, Nuclear Research Centre, Egyptian Atomic Energy Authority, Inshas, Cairo, Egypt. The column oven temperature programs were initially held at 45 • C, and then increased by 5 • C/min to 200 • C hold for 5 min, and then increased to 300 • C, with 30 increments of 5 • C/min [15].
The compounds were identified through a comparison of their retention times and mass spectra with those of the WILEY 09 and NIST 11 mass spectral database. Further confirmation of chemical compounds was reported by measuring the Standard Index and Reverse Standard Index with Xcalibur 3.0 data system of GC/MS, where the value ≥ 650 is acceptable to confirm the compounds [14,15,38].

Antifungal Activity of Essential and Recovery Oils
The antifungal activity of oils were measured against the growth of A. niger, A. terreus, A. flavus, and F. culmorum. Oils of M. chamomilla were dissolved in a mixture of dimethyl sulfoxide (DMSO) 10%, and Tween 40 and distilled water (1:0.5:1) were added to a warm potato dextrose agar (PDA) medium (40 • C to 45 • C), at a concentration of 25 µL/mL, 50 µL/mL, 75 µL/mL, and 100 µL/mL, before immediately being poured into 9 cm Petri dishes. Using a sterile pipette, each Petri dish was given exactly 20 mm of a treated PDA medium. Sertaconazol 3 g/L (standard antibiotic) was used as a positive control. The negative control treatment contained DMSO 10%, Tween 40, and distilled water (1:0.5:1). The mixture of dilution was used as a negative control. Each treatment was tested in triplicate. A mycelial disc, with a 9 mm diameter of the pathogenic fungi from a seven-day-old colony, was transferred to the center of the treated PDA dishes and controls.
14 days from the incubation period, at 26 ± 1 • C, the inhibition percentage of mycelial growth was calculated using the following equation [39]: where A c and A t represent the average diameters of the fungal colony of control and treatment, respectively.

Statistical Analysis
Results of the inhibition percentage of the diameter growth for each fungus were statistically analyzed based on two factors (oil type and oil amount) using analysis of variance, SAS system [40]. The differences between the mean of each treatment were recorded using LSD 0.05 .

Statistical Analysis
Results of the inhibition percentage of the diameter growth for each fungus were statistically analyzed based on two factors (oil type and oil amount) using analysis of variance, SAS system [40]. The differences between the mean of each treatment were recorded using LSD0.05.          Table 2 presents the antifungal activity of essential and recovery oils from the fresh flowers of M. chamomilla. Recovery oils, at amounts of 75 and 100 µL/mL, showed a fungal mycelial inhibition (FMI) percentage for the growth of A. niger, with values of 78% and 85%, respectively. The same amount of essential oil showed FMI values of 73% and 84%, respectively. However, these values are lower that the FMI reported by the positive control (87%).

In vitro Antifungal Activity of Essential and Recovery Oils
Both oils, applied at 100 µL/mL, showed 100% FMI of A. terreus, higher than the value observed in the positive control (89.66%). Essential and recovery oils at 75 µL/mL showed FMI of 65.66% and 58.33%, respectively. Other concentrations showed limited impact on the growth of A. terreus. Oils showed limited impact on the growth of A. flavus, where all the studied concentrations from both oils presented a much lower FMI than the positive control (88.66%). Essential and recovery oils at 100 µL/mL showed FMI values of 52.33% and 47.33%, respectively, against A. flavus. Essential oil at 100 µL/mL had good activity against the growth of F. culmorum, with an FMI of 86.66%, and, at 75, µL/mL with a value of 65.33%, compared to 91% of positive control (Sertaconazol 3 g/L). Values are means ± SD, **: Highly significant at 0.01 level of probability.  Table 2 presents the antifungal activity of essential and recovery oils from the fresh flowers of M. chamomilla. Recovery oils, at amounts of 75 and 100 µL/mL, showed a fungal mycelial inhibition (FMI) percentage for the growth of A. niger, with values of 78% and 85%, respectively. The same amount of essential oil showed FMI values of 73% and 84%, respectively. However, these values are lower that the FMI reported by the positive control (87%). Both oils, applied at 100 µL/mL, showed 100% FMI of A. terreus, higher than the value observed in the positive control (89.66%). Essential and recovery oils at 75 µL/mL showed FMI of 65.66% and 58.33%, respectively. Other concentrations showed limited impact on the growth of A. terreus. Oils showed limited impact on the growth of A. flavus, where all the studied concentrations from both oils presented a much lower FMI than the positive control (88.66%). Essential and recovery oils at 100 µL/mL showed FMI values of 52.33% and 47.33%, respectively, against A. flavus. Essential oil at 100 µL/mL had good activity against the growth of F. culmorum, with an FMI of 86.66%, and, at 75, µL/mL with a value of 65.33%, compared to 91% of positive control (Sertaconazol 3 g/L). Table 3 presents the chemical composition of the essential oil from M. chamomilla (fresh flowers) as analyzed by GC/MS apparatus. The main compounds in the essential oil were (Z)-β-farnesene (27.00%), d-limonene (15.25%), α-bisabolol oxide A (14.90%), palmitic acid (6.44%), (E)-germacrene d (3.71%), γ-terpinene (3.54%), and citronellal (3.02%), while low amounts of chamazulene were observed (0.73%).  Table 4 presents the chemical compounds identified in the recovery oil from the hydrodistillation of M. chamomilla fresh flowers. The main compounds were α-bisabolol oxide A (18.60%), d-limonene (8.82%), α-bisabolol oxide B (7.13%), dodecane (5.92%), α-farnesene (5.16%), undecane (5.03%), oleic acid (4.51%), citronellal (4.24%), bisabolone oxide (3.81%), α-terpineol (3.73%), and chamazulene (3.50%).

Discussion
In the present study, essential and recovery oils from M. chamomilla fresh flowers were reported to have potential antifungal activity against the growth of fungi associated with the biodeterioration of cultural heritage (Aspergillus niger, A. terreus, A. flavus, and Fusarium culmorum).
These activities could be significantly related to the main identified compounds in both oils, such as (Z)-β-farnesene, d-limonene, α-bisabolol oxide A, α-bisabolol oxide B, and even chamazulene, which was present in low amounts.
It was reported that the two most prominent compounds found in oils of M. chamomilla, farnesol and α-bisabolol, have potential antifungal activity [52]. β-farnesene, α-farnesene, and α-bisabolol and its oxide were reported as its main compounds [53,54]. Essential oils with these were observed to have good antifungal activity against A. niger, Aspergillus sp. and Candida albicans [55]. An oil rich in α-bisabolol oxide A, extracted from M. chamomilla flowers from Neyshabur, Iran, showed potential activity against B. cereus, S. aureus, and Proteus vulgaris [56].
There are several reports regarding the use of natural products in the field of cultural heritage conservation [2]. EOs of Pimpinella anisum and Allium sativum showed the best antifungal activity against fungal strains isolated from Cuban and Argentine Documentary Heritage, including A. niger, A. clavatus, Penicillium sp. and Fusarium sp. [57].
Finally, it could be concluded that from the above data and from the literature, the essential oil from M. chamomilla flowers has a potential antifungal activity and, notably, that the recovered oil also showed potent antifungal activity.

Conclusions
This study highlighted the importance of using essential and recovery oils from fresh flowers of Matricaria chamomilla. Both oils presented potential bioactive molecules in their chemical compositions, demonstrating activity against the growth of four fungi isolated from cultural heritage objects. Interestingly, our results identified novel and strong antifungal agents against four deteriorating fungi by applying the recovery oil, which could be considered as an alternative source for the production of commercial antifungal agents. Further studies are required to develop new methods to apply the oils in the field of cultural heritage preservation.