Chemical constituents of essential oil of endemic Rhanterium suaveolens Desf. growing in Algerian Sahara with antibiofilm, antioxidant and anticholinesterase activities

Abstract Twenty compounds were detected in the essential oil of Rhanterium suaveolens representing 98.01% of the total oil content. Perillaldehyde (45.79%), caryophyllene oxide (24.82%) and β-cadinol (5.61%) were identified as the main constituents. In β-carotene–linoleic acid assay, both the oil and the methanol extract exhibited good lipid peroxidation inhibition activity, with IC50 values of 17.97 ± 5.40 and 11.55 ± 3.39 μg/mL, respectively. In DPPH and CUPRAC assays, however, the methanol extract exhibited a good antioxidant activity. The highest antibiofilm activity has been found 50.30% against Staphylococcus epidermidis (MU 30) at 20 μg/mL for essential oil and 58.34% against Micrococcus luteus (NRRL B-4375) at 25 mg/mL concentration for methanol extract. The in vitro anticholinesterase activity of methanol extract showed a moderate acetylcholinesterase inhibitory (IC50 = 168.76 ± 0.62 μg/mL) and good butyrylcholinesterase inhibitory (IC50 = 54.79 ± 1.89 μg/mL) activities. The essential oil was inactive against both enzymes. Graphical abstract


Introduction
The genus Rhanterium is distributed over western North Africa, the Arabian Peninsula, Iraq and Iran. Three species; namely, R. adpressum Coss. & Durieu, R. epapposum Oliver and R. suaveolens Desf. of this genus have been reported in the literature. R. intermedium Coss. & Durieu ex Pomel is another species, but it is a hybrid between R. suaveolens and R. adpressum. R. suaveolens Desf., a member of the Asteraceae (Compositae), is locally known as 'Arfadja' . Quézel and Santa reported that it is an endemic desert plant growing in North Africa (Quézel & Santa 1963;Wiklund 1986). Some of the Rhanterium species are used in folk medicine as an antidiuretic (Hamia et al. 2013).
The essential oil composition of the R. suaveolens and biological activities have not been reported earlier, except the antiradical activity using ABTS and DPPH assays (Bouaziz et al. 2009).
This paper is the first study of the chemical composition of essential oil of R. suaveolens. Antibiofilm and anticholinesterase activity of essential oil and methanol extract of R. suaveolens against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the chief enzymes of Alzheimer's disease, were also reported for the first time. Hence, the main objectives of this work were: determine the chemical composition of hydrodistillated oil of the aerial parts of R. suaveolens using gas chromatography and gas chromatography/mass spectrometry (GC/MS). The antibiofilm effect of sub-MICs of essential oil and methanol extract was assessed by the microplate biofilm assay. The antioxidant capacity of the essential oil and methanol extract was evaluated using three complementary assays; namely, β-carotene-linoleic acid, DPPH • scavenging and CUPRAC assays.

Anticholinesterase activity
The anticholinesterase activity of the EO and MeOH extract of R. suaveolens, against AChE and BChE enzymes was given in Table S2. Galantamine was the standard drug used for comparison. The MeOH extract exhibited moderate to good inhibitory activity against AChE and BChE enzymes. The IC 50 values were 168.76 ± 0.62 and 54.79 ± 1.89 μg/mL, respectively. On the contrary, the EO was inactive against both AChE and BChE.

Total phenolic and total flavonoid contents and antioxidant activity
The total phenolic and total flavonoid contents were performed spectrophotometrically using pyrocatechol and quercetin as standard compounds, respectively. The phenolic content of MeOH extract of R. suaveolens was 35.58 ± 0.04 μg pyrocatechol equivalents/g extract and the flavonoid content was 5.5 ± 0.02 μg quercetin equivalents/g extract. It can be said that the R. suaveolens is poor in flavonoids. Supportively, in a previous study (Bouaziz et al. 2009), 625 ± 75 mg pyrogallol/100 g and trace milligrams of quercetin/100 g were reported for the same plant collected in November in Douz area in Tunisia. The season and area of collection of the plant may lead to the different results. Table S3 shows the DPPH • scavenging activity and lipid peroxidation inhibitory activity by β-carotene/linoleic acid assay of the EO and the MeOH extract of R. suaveolens. Antioxidant activity of the extract and oil increased dose dependently in both assays. In DPPH assay, the MeOH extract demonstrated good DPPH • scavenging activity (IC 50 = 0.017 ± 0.004 mg/mL), while the EO exhibited weak activity. In the previous study, the DPPH assay was performed only for MeOH extract and the IC 50 value was calculated as 1.09 ± 0.19 μg/mL (Bouaziz et al. 2009). These differences may be due to the collection time and locality of the plant.
In lipid peroxidation inhibition assay, the MeOH extract exhibited higher inhibition (IC 50 = 11.55 ± 3.39 μg/mL) against lipid peroxidation, while the EO exhibited (IC 50 = 17.97 ± 5.40 μg/mL). The EO composition was given in Table S1. As seen, there are no phenolic compounds to scavenge the DPPH radicals. However, compounds as well as the conjugated mono and sesquiterpenoids are responsible for the lipid peroxidation activity. These compounds can scavenge the singlet oxygen and therefore protect the β-carotene colour against bleaching, indirectly. Figure S1 shows the results of the CUPRAC (cupric reducing antioxidant capacity) of both the MeOH extract and the EO of R. suaveolens. α-Tocopherol and BHT were used as positive controls. As shown in (Figure S1), the activity of the MeOH extract of the R. suaveolens was compatible with α-tocopherol at all concentrations. As expected the EO indicated a less reducing activity.

Determination of MIC's and antibiofilm activity
The MIC and antibiofilm activity results of the EO and methanol extract against six bacteria species and Candida albicans are given in Table S4. The oil inhibited the growth of all micro-organisms between 10 and 80 μg/mL concentrations. EO at the MIC's inhibited biofilm formations of all microorganisms tested in various percentages. The oil exhibited the highest antimicrobial activity against S. epidermidis MU 30 at 20 μg/mL (MIC/1) and at 10 μg/mL (MIC/2) concentrations with 50.3% and 32.96%, respectively.
According to the results, B. subtilis was found to be the most susceptible strain against MeOH extract of R. suaveolens. The extract has low activity on the growth of M. luteus NRRL B-4375 and S. epidermidis MU 30 which were only inhibited at high concentration (25 mg/ mL). In the presence of 25 mg/mL extract (MIC), the mean biofilm formation values were equal to 58.34% for M. luteus NRRL B-4375 and 49.06% for S. epidermidis MU 30.
In the current investigation, the EO exhibited antimicrobial activity, particularly against S. epidermidis MU 30, M. luteus NRRL B-4375 and B. subtilis ATCC 6633. Perillaldehyde, the major compound of the oil is an antimicrobial agent. It demonstrated antimicrobial activity against B. cereus, E. coli and S. aureus (Friedman et al. 2006). Concerning the antimicrobial activity against B. subtilis, the oil supported the previous results. However, the oil that showed a weak activity against against S. aureus. This may be due to its percentage (45.79%) in the oil which did not reach to the extent necessary to inhibit the growth of those micro-organisms.

Conclusion
The anticholinesterase activity against both enzymes was performed for the first time in this study. According to the results the EO indicated no activity. However, the methanol extract exhibited good activity against BChE. Thus, the plant can be used as an anticholinesterase agent, particularly against BChE. However, further studies are necessary to evaluate the origin of the activity. Also, the methanolic extract and the oil proved to be effective antioxidants and antimicrobials in different in vitro assays and can be suggested as a natural additive in food and pharmaceutical industries. In the case of antioxidant activities, results obtained from β-carotene/linoleic acid bleaching test were found to be stronger than those obtained from DPPH and CUPRAC systems.