Antifungal Potential of Aqueous Extract of Boswellia carteri

To assess the antifungal activity of the crude aqueous extract of B. carteri. Three independent concentrations (1%, 2.5%, and 5%) of the crude aqueous extract of B. carteri were tested for their in vitro activity against selected fungal strains. The treated (5% concentration) and untreated A. alternata samples were analyzed for morphological changes using scanning electron microscopy (SEM). FourierTransform Infrared (FTIR) spectrometry of the extract was done to identify the phytoconstituents responsible for the antifungal activity. Results showed that the crude aqueous extract of B. carteri inhibited the growth of all the selected fungal species. The percentage of mycelial growth of the tested fungi decreased as the concentration of the aqueous extract increased from 1% to 5%. SEM-based studies of A. alternata treated with 5% showed significant morphological changes including shrunken hyphae, membrane disintegration, and distorted conidial structures compared to the untreated fungal cells. The crude aqueous extract of B. carteri has the potential to be used as a natural and effective fungicidal agent for controlling the growth of pathogenic fungi.


INTRODUCTION
Boswellia carteri (B. carteri; also known as Birdwood or Birdw) is a moderate-to-large sized endemic tree mostly found in the dry mountainous regions of Somalia, Saudi Arabia, Sudan, and Yemen 1 . The extracts from Boswellia species have been widely plethora of conditions such as asthma, arthritis, rhinitis, analgesic effect, gastric & hepatic disorders, skin diseases, cancer as well as inflammatory associated diseases in human [2][3][4][5][6] .
Although the antifungal activity of Boswellia species has been well documented [11][12][13][14] , the antifungal activity of the aqueous extract of B. carteri against A. alternata, H. rostratum, and F. solani remains elusive. Therefore, the present study was designed to assess the antifungal properties of crude aqueous extracts of B. carteri against three plant pathogenic fungi (A. alternata, H. rostratum, and F. solani). Furthermore, scanning electron microscopic (SEM) and Fouriertransform infrared (FTIR) spectroscopic studies were undertaken to characterize the antifungal attributes of B. carteri.

MATERIALS AND METHODS Sample Collection
The oleo-gum resin of B. carteri was procured from authorized suppliers (Bin mingash store, Riyadh, Saudi Arabia), or collected from the plant (B. carteri) grown in the region of Riyadh, Saudi Arabia. The sourced plant material was stored overnight at -80°C. The collected gum resins were powdered using mortar, pestle, blender, and electric sieve. The powdered resins were stored at -20°C until further use.

Preparation of Crude Aqueous Extract
Briefly, 30 g of the crushed material was soaked for 24 hours in 300 mL distilled water (10% w/v) at room temperature (37°C). The soaked material was macerated with 50 mL distilled water (10% w/v) in a conical flask and kept in an orbital shaker (250 rpm at 45°C for 24 hours). The extracts were then concentrated and dried under reduced pressure and 40°C using a rotary evaporator (Rotavapor® R-215, BUCHI). All the filtered extracts were preserved aseptically in glass bottles at 4°C until further use.
Three concentrations of the aqueous extract (1, 2.5, and 5 mg/mL) were prepared using sterile aqueous extracts and with distilled water (10% w/v). The reconstituted aqueous extracts were passed through 0.45µM bacterial filter papers (Millipore Inc., Riyadh, Saudi Arabia) prior to using them for in vitro studies.

Fungal specimens
Fungal strains of A. alternata, H. rostratum, and F. solani were obtained from the Department of Plant Protection, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia. Fungal isolates were maintained on potato dextrose agar, stored at 4°C and subcultured once a month.

Antifungal screening
Antifungal activity was determined using the poisoned food technique 18 . One milliliter (mL) of the aqueous extract (1%, 2.5%, and 5%) was aseptically poured into sterile petri dishes (9 cm in diameter) followed by the addition of 19 mL of molten potato dextrose agar. After solidification of the medium, mycelial plugs (6 mm in diameter) of A. alternata, H. rostratum, and F. solani from the periphery of 9 days old culture were aseptically inoculated into the center of the petri dishes and incubated at 25±2°C for 7 days. Plates without extract served as controls. The experiments were carried out in triplicates. Percent reduction of mycelial growth was measured using the formula: % inhibition= (AC -AT)/AC ‫׳‬ 100 Where AC = mean diameter of the mycelial growth in the control plate; AT = mean diameter of the mycelial growth in the treatment plate 19 .

Scanning Electron Microscopic Analysis
The morphological changes induced by the extract was assessed using SEM. In brief, small agar pieces (6 mm) were aseptically cut from the inhibition zone and fixed in 2.5% (v/v) glutaraldehyde buffered with 0.1 M sodium phosphate buffer (pH 7.4). The suspension was centrifuged after 48 hours, rinsed thrice with phosphate-buffered saline, and was dehydrated through sequential ethanol washes (60 % -100%). The dehydrated specimens were freeze-dried and mounted onto stubs using double-sided carbon tape, and then coated with a thin layer of gold. The processed specimens were finally examined under a scanning electron microscope (JSM 6060LV JEOL, Japan LTD).

Structural Characterization by Fourier-Transform Infrared Spectrometry
The dried powder of the aqueous extract of B. carteri was used for Fourier-Transform Infrared (FTIR) analysis. In brief, 10 mg of the dried extract was encapsulated in 100 mg of KBr pellet. The resulting pellet was loaded in the FTIR spectrophotometer (Nicolet 6700, Thermo Scientific, USA) equipped with a beam splitter, a detector (DTGS) and OMNIC software to generate the FTIR spectra in the mid-region of 500 -4000 cm 1 .

Statistical Analysis
The data are expressed as mean and standard deviation (SD). Significant differences were analyzed using one-way analysis of variance (ANOVA). p-value <0.05 was considered statistically significant. As the overall mean difference between the three groups was not significant, post-hoc test was not required.

Antifungal Activity of the Aqueous Extract of B. carteri
The crude aqueous extract of B. carteri showed mild inhibition at 1% concentration against all three fungal strains (A. alternata, H. rostratum, and F. solani), with a percentage inhibition ranging    Fig. 1a to 1c).

Scanning Electron Microscopy
The microphotographs of the untreated mycelia (controls) of A. alternata showed normal tubular structure with intact mycelia (Fig. 2a). The treated A. alternata mycelia appeared distended, flaccid and showed condensed hyphal branches with a rough and wrinkled surface (Fig. 2b). Similarly, distorted structures were noted for H. rostratum and F. solani treated with the aqueous extract of B. carteri (results not shown).

DISCUSSION
This study showed that the aqueous extract of B. carteri exhibited potent in vitro activity against A. alternata, H. rostratum, and F. solani. SEM confirmed the presence of  ultrastructural changes in the mycelia treated with the extract compared with the untreated mycelia. FTIR spectroscopy of the extract showed the presence of various aliphatic and aromatic compounds, which could be responsible for the antifungal activity. Chemical pesticides have widely been used in contemporary agriculture to control various plant diseases including those caused by pathogenic fungi 20 . However, the lethal effect exerted by the synthetic fungicides on the soil microbiota, plants, and water coupled with the development of fungicide resistance highlights the need for the development of natural and effective antifungals for combating fungal infections. Plant-based antifungals offer an eco-friendly and effective alternative to conventional synthetic fungicides 21 .
The arid and semi-arid regions of the Middle East is the home for many medicinal plants 22 . B. carteri is one of the valuable medicinal plants distributed in the dry mountain areas of the Middle East 1 . Metabolites derived from oleogum resin have been used in the management of myriads of plant diseases 23 . However, there is a paucity of studies exploring the antifungal activity of B. carteri 11,13 . In the current study, three different concentrations (1%, 2.5%, and 5%) of the crude aqueous extract of B. carteri were screened against three phytopathogenic fungal species (A. alternata, H. rostratum, and F. solani). With subtle exceptions, the extract of B. carteri showed mild activity at lower concentrations (1% and 2.5%) and moderate activity at 5% concentration against the three fungal species tested. The variations in the antifungal activity of the crude aqueous extract may be due to the differences in concentrations of the bioactive compounds (aliphatic and aromatic compounds) in the crude extract. Other factors ascribed for the differential activity include the extraction method, pH, solubility, volatility, diffusion characteristics in the growth medium, and the tested fungal species 1,24 .
The SEM photographs of the fungal mycelia treated with the crude aqueous extract of B. carteri showed structural alterations in the mycelial and conidial structures compared with the untreated mycelia, thereby endorsing the antifungal activity of the crude aqueous extract. In general, crude aqueous extracts are a rich source of several bioactive compounds. Exposure to such extracts may result in cellular deformation by inducing loss of water, electrolytes and other vital intracellular components essential for the survival of the fungal cells. The phytochemical screening of crude extract using FTIR spectroscopy revealed the presence of diverse bioactive compounds (aliphatic and aromatic) which could play a vital FTIR, Fourier-transform infrared. The antifungal activity of Boswellia species is well established [25][26][27] 26 Limited studies have assessed the antifungal activity of B. carteri 11,13 . A Serbian study investigating the antifungal potential of B. carteri essential oils showed variable inhibition of the tested fungal species (A. niger, Stachybotrys chartarum [S. chartarum], and Trichotecium roseum [T. roseum]). While S. chartarum and T. roseum were the most sensitive fungal isolates, A. niger was found to be least susceptible 11 . In another study, essential oil from B. carteri inhibited the growth of A. niger, C. albicans, and C. neoformans 13 . The antifungal activity of the essential oil of B. carteri was due to the presence of limonene (22.4%), b-caryophyllene (22.2%), p-cymene (10.0%), d-cadinene (9.4%), and a-copaene (4.8%) 13 . In line with these findings, FTIR analysis of the crude aqueous extract of B. carteri in the present study showed the presence of several bioactive aliphatic and aromatic compounds (alcohol, carboxylic acid, ester, alkene, alkane and alkyl amine, alkyl halides, halogen and cycloalkane), which may account for the antifungal activity against A. alternata, H. rostratum, and F. solani.

CONCLUSION
The crude aqueous extract of B. carteri has great potential as a natural antifungal in the treatment of plant phytopathogenic fungi. However, further studies are required to understand the molecular mechanism by which B. carteri extract inhibits the growth of the fungal mycelia.