Antimicrobial activity of volatile constituents extracted by hydrodistillation and headspace solid-phase microextraction methods from different parts of Saudi Anethum graveolens L.

Anethum graveolens L. (AG) is one of the most important aromatic herb, used in folk medicine to cure many illinesses. The current work was carried to compare the chemical composition and the antimicrobial potency of Saudi AG essential oils obtained from different parts. The oils constituents were extracted by two techniques; the Headspace Solid-phase Microextraction (HS-SPME) and traditional hydrodistillation (HD), then the constituents of each extracted oil were quantitavly and qualitatively identied. The essential oil isolated from AG seeds exhibited the highest antimicrobial activity comparing to other essential oils isolated from other parts of the same plant followed by owers, leaves and stems, respectively. Interestingly, all tested AG essential oil samples showed stronger antifungal activity against Aspergillus parasiticus than the control antifungal itraconzole used in the study. Our nding suggest that AG seed essential oil may be considered as an alternative safe remedy derived from a natural source used to cure many uncomplicated infections. Up to our knowledge, this is the rst report on the chemical compostion of the Saudi AG oils isolated by different methods with a comparable testing against different pathogenic microorganisms. HD extraction method. Then, the GC/MS analyzed the volatile constituents. Fiber coatings divinylbenzene/carboxen on polydimethylsiloxane (DVB/CAR/PDMS) was used for SPME methods. The current study is the rst report involving rapid analysis of volatile components of seeds, owers, leaves and stems of AG essential oils growing in Jazan province, Saudi stems were subjected to hydrodistillation for 4 h using a modied Clevenger-type apparatus, with yields of 1.42%, 1.56%, 0.93%, and 0.83% (v/w), respectively. All oils showed a pale yellow, light green, intense yellow and yellow coloration with a strong fragrance. Also, oils of AG were obtained by HS-SPME by using PDMS/DVB/Carboxen ber. To our knowledge, this study is the rst report involving rapid analysis of volatile components of seeds, owers, leaves and stems of AG essential oils growing in Jazan province, Saudi Arabia by HS-SPME and HD methods. Myrcene, α-phellandrene, p-cymene, limonene, dill ether, carvone and dill apiole were found to be the common components of the AG plant seeds, owers, leaves and stems essential oils by both extraction methods. Dill ether is the most important constituent of AG essential oils, which found in sucient quantities in all parts; 46.9, 33.3, 16.0, 31.9% in owers, seeds,


Introduction
Anethum graveolens L. is an important aromatic annual herb. It is a member of the Apiaceae family which is distributed in Southwest Asia, Mediterranean and native to South-East Europe. It is commonly known as dill in English and "Shabat " in Arabic 1 . It is cultivated since ancient times and found closely related to the species of Indian dill (A. sowa) and European dill (A. graveolens). Mediterranean region countries in Eastern Europe, India and Russia are main producers of dill essential oil 2 .
The seeds of A. graveolens are used in folk medicine as appetizer, carminative, diuretic, stomachic, digestive, sedative and in hemorrhoids 3,4 . Chewing of the seeds improves bad smell of breath or halitosis. Anethum graveolens stimulates milk ow in lactating/nursing mothers, and is often given to livestock for this reason. It also cures mental disorders, urinary infections and piles 5 . This plant is one of the additive used in gripe water usually given for relief of colic pain and hiccups in babies/infants and atulence in young children 6 . Furthermore, it is used as a natural avor enhancer in many food items, particular in sauces, salads, soups, sea foods, fried meats and mainly in pickles. The essential oil is extracted from leaves, seeds and stems, used as a avoring agent in food and beverages due to its pleasant aroma. As well, the essential oil is used as perfume to aromatize soaps, detergents and cosmetics 7 .
Hydrodistillation (HD) is the most well-known extraction technique used to isolate the volatile constituents from the plant tissue, but it is time consuming and needs large quantity of sample as well as, losses of low-boiling-point volatile constituents during solvent expulsion. Headspace solid-phase microextraction (HS-SPME) is an attractive alternative method for the extraction and enrichment of volatile constituents from various plant materials. It utilizes a ne quartz ber with a polymeric coating to extract organic compounds from their matrix and directly transfer them into the injector of a gas chromatograph for thermal desorption and analysis. It is now considered a new extraction method. However, the extraction depends on the characteristics of the bers used and the volatile constituents' characteristic. Thusly, the volatile pro le may not precisely re ect the proportion of volatile components from the medicinal plant by HS-SPME sampling 8 .
The essential oils of the seeds, owers, leaves and stems of AG plant were separately extracted by using HS-SPME and HD extraction method. Then, the GC/MS analyzed the volatile constituents. Fiber coatings divinylbenzene/carboxen on polydimethylsiloxane (DVB/CAR/PDMS) was used for SPME methods. The current study is the rst report involving rapid analysis of volatile components of seeds, owers, leaves and stems of AG essential oils growing in Jazan province, Saudi Arabia by HS-SPME and HD methods. As well as, the antimicrobial activity for obtained essential oils that isolated from different part of AG were compared and assessed against various bacteria and fungus strains.

Results
The volatile compounds in AG essential oil samples (seeds, owers, leaves and stems) were extracted by HS-SPME, followed by desorption and analysis with GC-MS. The volatile compounds in AG oil samples were also extracted by using HD method.
The extracted compounds are tabulated in Table 1. Figs. S1-4 (a&b), showed the GC chromatograms of AG essential oils of seeds, owers, leaves and stems extracted by HS-SPME and HD methods. The typical total ion chromatograms (TIC) of the seeds, owers, leaves, and stems essential oils obtained by HS-SPME and HD were shown in Fig. 1 and indicated the differences in the volatiles composition between the two methods. The chemical analysis results obtained by HD and HS-SPME methods from AG seeds, owers, leaves and stems are presented in Table 1 (were present in amount more than 0.05%) in which compounds are listed in order of their elution on the column HP-5 MS. Retention indices (RI) calculated by GC capillary columns and obtained from literature were also listed in the tables.
The essential oils of AG samples (seeds, owers, leaves and stems) were tested for antimicrobial activity gram-positive like Staphylococcus aureus (CP011526.1), Bacillus licheniformis (KX785171.1), Listeria innocua (DSM 20649) and gram-negative like Enterobacter xiangfangensis (CP017183.1), Escherichia fergusonii (CU928158.2) and Pseudomonas aeruginosa (NR-117678.1) bacterial strains. In addition to, three pathogenic fungi Candida albicans (MF942350), Candida parapsilosis (MF942354) and Aspergillus parasiticus (CBS 100926), as shown in Table 2, were included to the assays. The oil from seeds at 20µL exhibited highest antimicrobial activity. The best activity was observed against Staphylococcus aureus strain with a diameter of inhibition equal to 25.0 ± 0.00, 17.0 ± 0.10, and 15.0 ± 1.10 mm for the essential oil isolated from seeds, owers and leaves, respectively. This strain had a lower sensitivity to the essential oil isolated from stems (11.0 ± 0.6 mm) comparing to the commercial antibiotic tetracycline. The halo of inhibition for Bacillus licheniformis (22.0 ± 1.60 and 18.0 ± 1.90 mm) of essential oils from seeds and owers, respectively are greater than those for the remaining samples. By contrast, in antifungal assay the inhibitory actions of all the tested samples against Aspergillus parasiticus were signi cantly higher than the action of the control antifungal drug itraconazole Table 2.

Discussions
The aerial parts of AG seeds, owers, leaves and stems were subjected to hydrodistillation for 4 h using a modi ed Clevengertype apparatus, with yields of 1.42%, 1.56%, 0.93%, and 0.83% (v/w), respectively. All oils showed a pale yellow, light green, intense yellow and yellow coloration with a strong fragrance. Also, oils of AG were obtained by HS-SPME by using PDMS/DVB/Carboxen ber. To our knowledge, this study is the rst report involving rapid analysis of volatile components of seeds, owers, leaves and stems of AG essential oils growing in Jazan province, Saudi Arabia by HS-SPME and HD methods.
Comparing the composition of AG essential oils extracted by two different methods indicated that HS-SPME and HD, respectively, had the strng different numbers of extracted components. It seems that HS-SPME can isolate signi cantly more volatiles at small quantities in contrast to the HD method.
Although HD is the most popular, common, widespread and worthwhile conventional method for extracting essential oils from plant parts, it has some frailty. This method is a long-standing and laborious process and requires a substantial amount of plants sample. Furthermore, in the presence of water and lengthy heating time, it can be concluded that the essential oil has a higher percentage of sesquiterpenes. Although, monoterpenes might be susceptible to a chemical process, and several highly volatile compounds, such as α/β-pinene and α-thujene evaporate during removal of the water/solvent by distillation. On the contrary, HS-SPME is a straightforward, expeditious method, environment friendless since it was solvent free, fast as well was better for more thermally sensitive volatile compounds and free from wastage of water. This method can be used for the qualitative analysis of volatile fractions from various plant samples simultaneously, requiring fewer samples. SPME is a method for the analysis of compounds, whereas HD can be used both for analysis and commercial production of essential oils. HD is a quantitative method for the extraction of volatiles from plant material, whereas SPME is not a quantitative technique. SPME is good if you want to get an overview of the composition of volatiles in a sample. It can be regarded as a semi-quantitative method at the most.
The oils or some of their constituents are very effective against many organisms, including bacteria and fungi. As typical lipophiles, they disrupt the structure of the cytoplasmic membrane and permeabilize them. In bacteria, the membranes' permeabilization is associated with loss of ions and reduction of membrane potential 9,10 . For that, the present work was carried to compare and investigate the activity of AG essential oils isolated from different parts against various strains of bacteria and fungi, the positive results will con rm the folkloric use of AG for curing many infectious diseases.
In vitro antimicrobial activity of the AG oil samples (seeds, owers, leaves and stems) Table 2 possessed great antimicrobial activity against all tested microorganisms. Data analysis showed that the essential oil isolated from AG seeds exhibited the highest antimicrobial activity comparing to other essential oils isolated from other parts of the same plant following by owers, leaves and stems, respectively. Previous ndings showed that essential oils rich in carvone and limonene had stronger antimicrobial activity 11,12 . Our study carvone and its precursor limonene are the major components constituents in the AG essential oil seeds sample that probably originate its high antimicrobial property. The former sample's highest activity was observed against Staphylococcus aureus strain with a diameter of inhibition equal to 25.0 ± 0.00 mm followed by Bacillus licheniformis with a diameter of inhibition 22.0 ± 1.60 mm comparing to the commercial anti-biotics gentamicin and tetracycline. Interestingly, all tested AG essential oil samples showed stronger antifungal activity against Aspergillus parasiticus than the control antifungal itraconzole used in the study

Essential oils preparation for analysis
Plant materials were placed in a 5 ml headspace vial and enriched for 1 h on a SPME ber (PDMS/DVB/Carboxen, SUPELCO part no. 57298-U) at a temperature of 80ºC in a metal block in such a way that the plant material was subjected to the elevated temperature while the SPME ber was kept cold (room temperature). The enriched ber was placed in the GC injector and the ber desorbed for 1 minute at 250ºC.

Essential oils extraction
According to the standard procedure described in the literature (European Pharmacopeia, the freshly cut of AG seeds, owers, leaves and stems (300g) were subjected to HD by using Clevenger Apparatus for 4 h, according to the standard procedure

Essential oils preparation for analysis
Isolated EOs were diluted in dichloromethane (1:20) and 1 µL of this solution was injected in the GC at a split ratio of 1:100.

GC-FID and GC-MS analysis
Gas chromatography-ame ionization detector (GC-FID) and GC-MS analyses were performed in one run using a MS-FIDsplitter consisting of a quartz Y-splitter, a short (ca. 20 cm) 0.1 mm id fused silica restrictor column as an inlet to the GC-MS interface, and a ca. 1 m ⋅ 0.25 mm deactivated fused silica column serving as a transfer line to the FID detector. The restrictor column was used to limit the MS vacuum ow and prevent the insertion of combustion gases from the FID, which operated at atmospheric pressure. Moreover, the analytical column ow had to be greater than the in ow to the MS detector, which was limited to about 1 mL/min by the restriction line. The GC column ow should also be constant, otherwise the FID/MS split ratio would change with temperature. This con guration yielded an FID and MS chromatogram with almost identical retention times (RT), thus facilitating the FID peaks' assignment to each substance. We used a Thermo Fisher Scienti c Trace GC Ultra with a split/split less injector heated at 250°C and connected to a 50 m ⋅ 0.25 mm ⋅ 1.0 µm SE-52 (95% polydimethylsiloxane, 5% polydiphenylsiloxane) capillary column (prepared and tested for deactivation and separation e ciency in our lab, Kurt,

Antibacterial assay
Agar diffusion methods were used in this study to assay the antibacterial activity of the essential oils against 9 different microbial strains including; Gram-positive; Staphylococcus aureus (CP011526.1), Bacillus licheniformis (KX785171.1) and Listeria innocua (DSM 20649) and the Gram-negative; Enterobacter xiangfangensis (CP017183.1), Escherichia fergusonii (CU928158.2) and Pseudomonas aeruginosa (NR-117678.1) bacterial strains. TSB tryptone soya broth media were used previously to grow the microorganisms as mentioned above for 24h. Nutrient agar plates were used to distribute 0.1 mL of the microbial suspensions. 10 and 20 µl of each essential oil were spotted on the inoculated plates. Under sterile condition and after 15 min, plates were then incubated at optimal growth condition culture of each strain. Free area without microbial growth was measured three times to detect the diameter of zone of inhibition and the mean were calculated. Gentamycin 10 µg and Tetracycline 10 µg were used as a positive control while ethanol used as a negative control.

Antifungal activity
All essential oils were tested for their antifungal activity, using well diffusion and broth microdilution method according to Each experiment was carried out in three independent replicates and the result is the average with standard deviation.
Itraconazole was used as the positive control and ethanol was used as the negative control.

Statistical analysis
Data analysis was expressed as mean ± standard deviation (SD) of three replicates. The data were subjected to one-way analysis of variance (ANOVA). Based on Microsoft Excel 2010 statistical package analyses, the signi cant differences were considered statistically signi cant values p < 0.05.

Conclusions
This study described and analyzed the chemical pro le of AG seeds, owers, leaves and stems essential oils by using HS-SPME and traditional HD extraction method. The results indicated that studies involving HS-SPME volatile oils are most effective and safe. Additionally, by using HS-SPME technique we can isolate many volatiles with small quantities which were not indicated by HD methods. Based on the current study, we recommend that the HS-SPME technique be suitable for the routine quality control analysis of medicinal plants at the analytical level. Finally, AG seeds oils' potent antimicrobial activity, perhaps attributed to the presence of carvone and its precursor limonene. Our data suggest that AG seed essential oil may be considered as an alternative method to cure uncomplicated super cial infections of staphylococcal etiology, such as folliculitis and impetigo.  Tables   Table 1 Composition of the flowers, seeds, leaves & stems of AG essential oils extracted using HS-SPME & HD methods.
No. Compounds Ret.time Flowers % Seeds % Leaves % Stems % HS-SPME HD HS-SPME HD HS-SPME HD HS-SPME HD   Structures of major constituents in essential oils of AG seeds, ower, leaves, and stems

Supplementary Files
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