INTRODUCTION:

Antioxidants are commonly used as food additives in food preservation ¹. Antioxidants also play a crucial role in preventing many diseases related to lifestyle and aging. Recently, volatile oils have become popular as antioxidants, despite no clear scientific evidence proving the effectiveness of these oils, but their use has become very popular due to fear of the toxicity of industrial additives ².

Potential antioxidant activity has been evaluated in multiple plant species, among these volatile oils are Anise and Dill fruit oils; herbaceous plants belonging to the Apiaceae family and native to the Mediterranean origin ^3,4.

In folk medicine, Anise fruits are traditionally used to relieve migraine pain, carminate, galactagogue, stimulate menstruation, and secrete sweat ⁵.

Dill fruits were widely used as a gas repellent and for treating gastroenteritis, liver disease, and kidney stones ⁶.

The extracted volatile oils from these fruits are used as antispasmodics and carminatives; but Anise oil is not recommended for infants, because it can be quite dangerous to infants under six months old, so in this case, Dill fruit oil is used.

The goal of this study was to determine the yield of the volatile oils extracted from Anise and Dill's fruits grown in Al-Ghab Plain in Syria using stem distillation, in addition to studying the chemical composition of these volatile oils using Gas Chromatography - Mass Spectrometry, and to study the antioxidants activity of them using DPPH method.

MATERIALS AND METHODS:

Chemicals:

2,2-diphenyl-1-picrylhydrazyl DPPH (from TCI), distilled water, absolute Ethanol (from Merck), ascorbic acid (from Panreac Quimica SLU), Anhydrous sodium sulfate.

Apparatuses:

Gas chromatography- spectrometry (GC–MS) apparatus.

sensitive scale.

Electric heater.

Spectrophotometry apparatus.

Hydro distillation apparatus (Clevenger-type).

Plant materials:

Anis and Dill fruits were obtained from Al-Ghab Plain in Hama, Syria in November 2019. Where the temperature was 23 ° C and the air humidity was 58%.

Extraction of essential oils:

The essential oils were extracted from fruits of Anise and Dill by hydro distillation, where 100 g of Anise fruits (and 200 g of Dill fruits) were placed after grinding in the flask of the distillation apparatus, and placed on an electric heater at a temperature of 100 ° C, for 4 hours using a Clevenger-type apparatus. The extracted essential oils were exhausted from water using anhydrous sodium sulfate.

The volatile oils were placed in airtight glass tubes, covered with aluminum sheets, and kept in the refrigerator until their components were examined.

GC-MS Analysis of Essential Oils:

The volatile oils of Anise and Dill were analyzed by gas chromatography and mass spectrometry (GC/MS) at the faculty of science (Damascus University), using a non-polar column (HP-5MS 5% Phenyl Methyl Silox) (30 m × 0.25 mm i.d., 0.25 μ film thickness). A selective electronic impact (EI) detector set at 70 eV.

The volatile oils were expanded by 1/1000 with pure hexane and 2 μL of the dilution injected in GC using the following:

· The injector's temperature was at 250°C.

· The column's initial temperature is 50°C, holding for two minutes, and then raised to 220°C at a rate of 4.3°C/min, then raised to 250°C at a rate of 15°C/min, and the temperature remains constant at 250°C for two minutes.

· Helium carrier gas flows 3 ml/min.

· Draw speed 50:1

· The injection sample volume is 2 μl.

By comparing the mass spectra and retention times of volatile oils with those of the pure chemicals in the data bank (NIST MS Search 2.0), the components of volatile oils were determined using the chemical data of the library (Agilent Chemstation).

DPPH Radical Scavenging Assay:

(DPPH) assay was used to quantify the free radicals scavenging activity. 3ml of DPPH solution was added to 100 µL of samples at the concentrations (1, 2.5, 5, 10, mg/ml) for Anise fruits and (0.25, 0.5, 1.2 mg/ml) for Dill fruits. The mixtures were shaken and left in a dark area for thirty minutes. The absorbance of the solutions was determined by using a spectrophotometer at 517 nm. The following equation was used to calculate the percentage (I%) of DPPH:

Acontrol – Asample

AA% = ---------------------------- x100

Acntrol

A_control is the absorbance of the control and A_sample is the absorbance of the test compound. Vitamin C was used as a standard.

Statistical Analysis:

Data from three experimental determinations are shown as means ± standard deviations. The average absorbance of the volatile oil samples of Anise and Dill fruits was calculated along with the standard deviation and P-value using the t-test method. The statistical analysis was carried out using SPSS (version 13.0).

RESULTS:

Extraction of essential oils:

It is noteworthy that the Al-Ghab Plain Anise sample produced good yields of volatile oil (2.9%). This yield agreed with the range of 2-6% reported in the flora Syria, Palestine, and Sinai reference ⁷, and the percentage mentioned in Reference Treas and Evans 2-3% ⁸. This percentage of volatile oil was higher than the percentages of volatile oil extracted from Anise fruits grown in Turkey (Isparta) (1.45 ml) ⁹.

Whereas Al-Ghab Plain Dill sample produced 1.6% of volatile oil. This percentage was in the range of 2–4% ⁽⁷⁾ reported in the Flora Syria, Palestine, and Sinai reference, and 2–3% ⁸ in the Treas and Evans reference, but it was lower than the percentage reported in Turkey (Isparta) 3.23 ml ⁹.

Chemical composition analyses by GC-MS:

Anise fruits:

The chromatograms of the tested oils are shown in (Figs 1, 2).

Figure 1. GC-MS chromatogram of Al-Ghab Plain Anise.

Table 1. The chemical components identified in Al-Ghab Plain Anise oil

Compound Name	Peak (Pk)	Retention time (RT)	Percentage %	Chemical structure ¹⁰	Nature of compound
Methyl chavicol	1	15.6615	0.6205		Phenyl propane
(Estragole)	1	15.6615	0.6205		Phenyl propane
Trans- Anethole	2	18.6485	96.2829		Phenyl propane
Alpha -Fenchene	3	19.9995	0.1068		Monoterpene
Gamma - himachalane	4	24.2356	1.1315		Sesquiterpene
Zingiberene	5	24.67	0.4307		Sesquiterpene
Beta -Bisabolene	6	25.0434	0.1226		Sesquiterpene
Iso -Eugenol	7	33.6446	1.1945		Phenyl propane
Butanoic acid, 2-methyl-, 4-methoxy-2-(3-methyloxiranyl) phenyl ester	8	34.9209	0.1104		Sesquiterpene

Dill fruits:

Figure 2. GC-MS chromatogram of Al-Ghab Plain.

Table 2. The chemical components identified in Al-Ghab Plain Dill oil.

Compound Name	Peak (Pk)	Retention time (RT)	Percentage %	Chemical structure ¹⁰	Nature of compound
Chloroacetic acid	1	4.175	0.3967		Monohalogenated acetic acid
Alpha-Pinene	2	6.9788	0.0572		Monoterpene
Myrcene	3	8.7642	0.0929		Monoterpene
l-Phellandrene	4	9.1783	0.9679		Monoterpene
l-Phellandrene	4	9.1783	0.9679		Monoterpene
p-Cymene	5	9.8572	0.2531		Monoterpene
dl-Limonene	6	10.0201	28.3177		Monoterpene
Styrene, p. alpha -dimethyl (p- cymene)	7	12.0024	0.1833		Monoterpene
Dill ether	8	15.2881	0.1274
Cis- Dihydrocarvone	9	15.6411	1.0437		Monoterpene
Trans- Dihydrocarvone	10	15.8923	2.4977		Monoterpene
l-Carvone	11	17.3111	49.9549		Monoterpene
Trans- anethole	12	18.4719	0.5004		Phenyl propane
Myristicin	13	25.471	0.6889
Dillapiole	14	28.3358	14.7595		Phenyl propane

DPPH free radical scavenging assay of volatile oils:

DPPH free radical scavenging was used to determine the antioxidant activity of volatile oils extracted from Anise fruits, Dill fruits, and the control Vit C.

We note that the antioxidant activity increased with increasing volatile oil concentrations.

Figure 3. DPPH radical scavenging activity of Anise oil sample.

Figure 4. DPPH radical scavenging activity of Dill oil sample.

Figure 5. IC50 values for standard and oil samples.

In Fig 5, Anise and Dill volatile oils exhibited good antioxidant activity (IC₅₀ = 0.98 mg/ml, 0.8 mg/ml) respectively, but lower than that of the standard antioxidant VIT C (IC₅₀ = 0.06 mg/ml).

DISCUSSION:

Chemical composition analyses by GC-MS

Anise fruits:

The number of compounds found in Anise oil reached 8, representing 99.97% of the total oil (Tab 1).

The major constituents of the volatile oil were Anethole (96.28%), Iso-eugenol (1.19%), ɣ- himachalane (1.13%), Methyl chavicol (Estragole) (0.62%), Zingiberene (0.43%) and a-Fenchene (0.1%).

It is notable that the volatile oil extracted from Anise fruits included 1.6% mono and sesquiterpene derivatives, while derivatives of phenylpropane formed 98.2% of these compounds.

Anethole formed the largest percentage 96.28%, this percentage exceeds the percentage of Anethole found in the volatile oil extracted from Anise fruits grown in Serbia and Iraq, (87.4%, 87.9%) respectively ^{(11) (12)}. In contrast, this percentage is close to the percentage of Anethole found in the volatile oil extracted from Anise fruits grown in Turkey and Algeria, (92.4% and 94.8%) respectively^13,14.

Although Estragole was present in a concentration of 0.62% in our study, we observe that this chemical is significant in the oil of Anise fruits grown in Serbia, Iraq and Turkey, (5.3%, 3% and 3.3%) respectively ^11,12,13.

Zingiberene and Iso -Eugenol were present in concentration of 0.43% and 1.19, We notice the absence of these compounds in most studies ^{11, 12, 14}.

Overall, these studies support the idea that Anethole is the most abundant compound in anise seed essential oil, and the composition may vary slightly depending on the location of cultivation.

Dill fruits:

The number of compounds found in Dill oil reached 14, representing 99.8% of the total oil (Tab 2).

The major constituents of the volatile oil were l-Carvone (49.95%), dl-Limonene (28.31%), Dillapiole (14.75%), l-Phellandrene (0.96%), Myristicin (0.68%) and Trans-Anethole (0.5%).

It is notable that the volatile oil extracted from Dill fruits included 82.5% monoterpene derivatives, while derivatives of phenylpropane formed 15.7% of these compounds.

Carvone compound formed the highest percentage of Dill oil components 49.95%. This percentage is agreed with the percentage of Carvone found in the oil of Dill fruits grown in Bulgaria (50.1%)¹⁵. On the other hand, the percentage of this compound was high compared to the percentage found in the oil of Dill fruits grown in Turkey, Serbia and India, (45.22%, 42.4% and 30%) respectively ^{13, 11, 16}.

The second significant chemical in Dill seed oil was Limonene, and its percentage was 28.31%. We observe that the percentage of this compound in Dill fruit oil grown in Turkey and Serbia was higher than the percentage of Limonene found in our sample (35.9% and 29%) respectively^13,11. Whereas the percentage of Dill seed oil grown in India is small, reaching 9.6% ¹⁵.

Dillapiole, which was found at a percentage of 14.75%, was absent in many studies; however, it was found in a study on the oil extracted from Dill fruits grown in India, 15.7% ¹⁶.

The only compound shared by dill fruit oil and anise, Trans-Anethole, was detected at a percentage of 0.5%.

These findings support the idea that the composition of essential oils is influenced by a range of environmental factors, including temperature, humidity, and soil type.

Table 3. Comparison of the chemical composition of the volatile oil of anise and dill fruits with reference studies

Compound	Anise (Syria)	Dill Syria	Anise Serbia	Anise Turkey	Anise Algeria	Anise Iraq	Dill Turkey	Dill India	Dill Serbia
Trans- Anethole	96.2	0.5	87.4	92.4	94.8	87.9	3.7	-	0.1
Estragole	0.6	-	5.3	3.3	1.6	3	0.1	-	-
Gamma - himachalane	1.13	-	1.3	1.2	-	-	-	-	-
Zingiberene	0.43	-	-	0.1	-	-	-	-	-
Iso -Eugenol	1.19	-	-	-	-	-	-	-	-
Carvone	-	53	-	-	-	-	45.22	30	42.4
Limonene	-	28.3	-	-	-	-	35.9	9.6	29
Alpha-Pinene	-	0.05	-	-	-	-	0.04	-	-
Phellandrene	-	0.96	-	-	-	-	0.3	0.6	13.1
Dill ether	-	0.12	-	-	-	-	0.1	-	6.5
Dillapiole	-	14.7	-	-	-	-	1.6	15.7	-
Myristicin	-	0.68	-	-	-	-	0.9	11.7	-

DPPH radical scavenging assay of volatile oils:

Although both plants are members of the same family (Apiaceae), their chemical compositions of volatile oils are varied, as we have shown in the previous study.

Due to this difference, we chose to compare the volatile oils' capacity to scavenge free radicals. Since these oils typically have similar therapeutic effects, their potent scavenging capacity suggests using them as natural antioxidants.

The oil extracted from Dill fruits has lower IC₅₀ (0.8 mg/ml) than the oil extracted from Anise fruits (0.98 mg/ml), respectively. As a result, Dill oil has a greater capacity to scavenge free radicals than Anise oil grown in Al-Ghab Plain.

By comparing the antioxidant activity of volatile oil extracted from Dill fruits with reference studies, the Syrian dill fruits showed stronger antioxidant activity than Dill fruits grown in Tunisia (IC₅₀ = 3 mg/ml ¹⁷ and Bulgaria (IC₅₀ = 2.6 mg/ml) ¹⁸.

Also, we found that Syrian Anise, oil has better antioxidant activity than the oil of anise fruits grown in Thailand (IC₅₀= 86.88 mg/ml) ¹⁹, but our results agreed with a study conducted on the oil of Anise fruits grown in Iran (IC₅₀= 0.8 mg/ml) ²⁰.

Table 4. IC₅₀ values and the chemical composition of Anise and Dill oils.

	Dill oil	Anise oil
IC₅₀	0.8 mg/ml	0.98 mg/ml
Monoterpenes compounds %	82.5 % (carvone 50% + Limonene 28%)	1.6 %
Phenylpropane compounds %	15.7 %	98.2% (Anethole 96.2%)

From table 4, we note the high phenolic content in the volatile oil extracted from anise fruits (98%) compared to the high monoterpene content in dill oil (82.5%).

It has been reported that polyphenols contribute significantly to the total antioxidant potentials ¹².

Phenols (Anethole 96.2%) are mainly responsible for the antioxidant capacity of anise oil and are identified as chain-breaking antioxidants. Phenols are capable of donating an H-atom from the phenolic hydroxyl group to peroxyl radicals. This process can lead to a lower rate of peroxidation of unsaturated lipids ²¹.

But from the previous results we noticed that dill oil which contains less phenols showed stronger antioxidant activity than anise oil.

The primary compound in dill oil is Carvone (50%), a ketone monoterpene with strong antioxidant properties. Literature studies have shown that oxygenated monoterpenes, particularly ketone ones, have strong antioxidant properties and thus contribute most to the volatile oil's antioxidant activity ²².

Additionally, the monoterpene Limonene (28.5%), is a highly effective antioxidant due to double bonds in its structure that trap free radicals within the terpene molecule, enhancing the volatile oils containing limonene's ability to scavenge free radicals ²³.

So, antioxidant activity is not only related to the high phenolic content of the volatile oil but also depends on the presence of a good number of unsaturated terpenes, and it should be considered that the antioxidant activity can be attributed to the synergistic effect of all compounds that make up the extracted oil.

CONCLUSION:

Antioxidant activity of volatile oils is not limited to phenolic compounds. It may also come from the presence of other antioxidant secondary metabolites like monoterpenes.

Anise and dill volatile oils are good natural antioxidants, which can extend shelf life and prevent the spoilage of seasoned food. Therefore, they are widely applied in the food industry ²¹.

Furthermore, dill essential oil can act as a cheap, safe, environment friendly and effective substitute for synthetic chemicals.

CONFLICT OF INTEREST:

There is no conflict of interest in the research.

ACKNOWLEDGMENTS:

We would like to thank the faculty of Science at Damascus University and D. Khaldoun Tiba at the Agriculture Research Center.

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Received on 17.12.2024 Revised on 07.04.2025

Accepted on 10.06.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5207-5215.

DOI: 10.52711/0974-360X.2025.00751

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.