CHEMICAL COMPOSITION OF OILS CONTAINED IN THE SEEDS OF LOCAL Punica granatum VARIETIES

ABSTRACT

Pomegranate (Punica granatum L.) is a valuable fruit crop widely cultivated in arid and semi-arid regions. The seeds of pomegranate contain bioactive oils rich in essential fatty acids, which play a crucial role in health and industrial applications. This study analyzes the chemical composition of oils extracted from the seeds of local Punica granatum L. varieties. The lipid content was determined using Soxhlet extraction with Petroleum ether (b.p. 72–80°C), while the fatty acid profile was assessed through gas chromatography (GC). The results indicate a high concentration of punicic acid (C18:3), a conjugated linolenic acid with significant antioxidant and anti-inflammatory properties. Additionally, other major fatty acids, such as linoleic acid (C18:2), oleic acid (C18:1), and palmitic acid (C16:0), were identified in varying proportions among the studied varieties. The findings highlight the potential of pomegranate seed oil as a valuable source of functional lipids with nutraceutical and pharmaceutical applications. Understanding the variation in oil composition among local varieties contributes to optimizing its utilization in food, cosmetics, and medicinal industries.

АННОТАЦИЯ

Гранат (Punica granatum L.) является ценным плодовым растением, широко культивируемым в аридных и семиаридных регионах. Семена граната содержат биологически активные масла, богатые незаменимыми жирными кислотами, которые играют важную роль в здравоохранении и промышленности. В данном исследовании проведен анализ химического состава масел, выделенных из семян местных сортов Punica granatum L. Липидный состав определяли методом экстракции по Сокслету с использованием экстракционным бензином (т. кип. 72-80⁰С), а профиль жирных кислот исследовали методом газовой хроматографии (ГХ). Результаты показали высокую концентрацию пуниковой кислоты (C18:3) – конъюгированной линоленовой кислоты с выраженными антиоксидантными и противовоспалительными свойствами. Кроме того, были идентифицированы другие основные жирные кислоты, такие как линолевая (C18:2), олеиновая (C18:1) и пальмитиновая (C16:0), содержание которых варьировалось в зависимости от изучаемых сортов. Полученные данные подчеркивают потенциал масла семян граната как ценного источника функциональных липидов, обладающих нутрицевтическими и фармацевтическими свойствами. Понимание вариаций в составе масла среди местных сортов способствует его оптимальному использованию в пищевой, косметической и медицинской промышленности.

Keywords: Punica granatum, pomegranate seed oil, fatty acid composition, punicic acid, gas chromatography.

Ключевые слова: Punica granatum, масло семян граната, жирнокислотный состав, пуниковая кислота, газовая хроматография.

Introduction. In recent years, pomegranate seed oil (Punica granatum L.) has emerged as a subject of extensive scientific research due to its exceptional nutraceutical and pharmacological potential. This oil is particularly rich in conjugated linolenic acids, with punicic acid (C18:3) being the most abundant component, accounting for up to 80% of the total fatty acid composition. This unique profile endows pomegranate seed oil with significant antioxidant, anti-inflammatory, and cardioprotective properties [1,2].Numerous studies have demonstrated the bioactive potential of pomegranate seed oil in various health applications. It has been found to modulate inflammatory pathways, protect against oxidative stress, and regulate lipid metabolism [3,4]. Additionally, it has shown promising effects in cancer prevention, particularly in reducing tumor progression by inducing apoptosis in malignant cells [5,6]. The high concentration of polyphenols and flavonoids further enhances its free-radical scavenging ability, making it a valuable ingredient in functional foods and dietary supplements [7]. The impact of pomegranate seed oil on metabolic disorders, such as type 2 diabetes and dyslipidemia, has also been studied extensively. Research suggests that it can activate PPAR-γ receptors, which play a crucial role in glucose homeostasis and lipid metabolism, thereby exerting a hypoglycemic effect and improving insulin sensitivity [8]. These findings align with investigations into omega-5 fatty acids, where punicic acid has been shown to mimic the beneficial effects of conjugated linoleic acid (CLA) in reducing adiposity and improving metabolic health [9]. Beyond its health benefits, pomegranate seed oil has industrial applications, particularly in cosmetics and pharmaceuticals. Its high oxidative stability and unique fatty acid composition make it a sought-after ingredient in anti-aging formulations, moisturizing products, and wound-healing treatments [10,11]. Furthermore, it is widely incorporated into nutraceutical supplements, offering an alternative to synthetic antioxidants due to its natural ability to protect cellular membranes from oxidative damage [12]. Extraction methods significantly influence the quality and yield of pomegranate seed oil. While Soxhlet extraction remains a standard technique, supercritical CO₂ extraction has gained attention for its ability to preserve delicate bioactive compounds without residual solvents [13,14]. Studies comparing different extraction methods highlight the importance of optimizing processing conditions to maintain the oils functional integrity and enhance its bioavailability [15].

Research Location and Methods. This study focuses on analyzing the chemical composition of oils extracted from the seeds of local Punica granatum L. varieties. The sample collection was conducted in (specific location and geographical coordinates can be added), a region characterized by arid and semi-arid climatic conditions, where pomegranate cultivation is widely practiced. The selected samples were obtained from local farms growing different pomegranate varieties.

• The seeds were dried, ground, and then subjected to extraction using a Soxhlet apparatus with petroleum ether (b.p. 72–80°C).
• The obtained extract was purified using a rotary evaporator to remove the solvent, and the oil content was determined gravimetrically [16]. The extracted oil samples were transesterified into methyl esters and analyzed using gas chromatography (GC).
• The GC analysis was performed on an Agilent 6890 N gas chromatograph equipped with a capillary column (30 m × 0.32 mm, HP-5) and a flame ionization detector (FID).
• The temperature program started at 150°C and gradually increased to 270°C.
• The results were compared with standard methyl esters and identified using the NIST library.
• Each sample was analyzed at least three times, and the mean values were calculated.
• Statistical analysis was performed using the ANOVA method, and differences were considered significant at a confidence level of p < 0.05.

These methods allow for the qualitative and quantitative assessment of pomegranate seed oil composition and provide valuable insights into its potential applications in the food, pharmaceutical, and cosmetic industries.

Result and discussion. Analysis Results of Four Punica granatum L. Seed Samples

• Sample №1 – "Qozoqi"
• Sample №2 – "Achchiq dona"
• Sample №3 – "Qora don"
• Sample №4 – "Qayum"

Neutral lipids were extracted from air-dried, ground seeds using petroleum ether (b.p. 72–80°C) in a Soxhlet apparatus for 24 hours [17]. The obtained extract was concentrated using a rotary evaporator. The residual petroleum ether was removed by drying the oil in an oven at 100–105°C until a constant weight was achieved, after which it was stored in a desiccator. The oil content was determined as a percentage of the extracted sample weight, considering its moisture content.

Table 1.

Indicators and Content (%)

The analysis of Punica granatum seed samples demonstrated variations in moisture content and lipid composition among different varieties. Moisture and volatile substances ranged from 6,52% to 7,01%, indicating relatively stable water content across samples. The neutral lipid content at actual moisture levels varied between 11,13% (Sample №3) and 15,78% (Sample №1), while on a dry matter basis, it ranged from 11,97% to 16,90%, confirming that Sample №1 ("Qozoqi") had the highest oil yield, making it the most promising variety for oil extraction. Conversely, Sample №3 ("Qora don") exhibited the lowest oil content, which may affect its industrial applications.

Extraction of Total Fatty Acids from the Sample and Preparation for Gas Chromatographic Analysis

To determine the fatty acid composition, the thoroughly mixed oil sample was placed in a 50 mL round-bottom flask, and 20 mL of 2N methanolic KOH solution was added. The flask was then heated in a water bath, and lipid saponification was carried out by boiling the mixture for 1 hour [18].

A 50% aqueous sulfuric acid (H₂SO₄) solution was added to the resulting soap solution to decompose the soap and release free fatty acids (FFA). The acid was added until the solution turned pink when tested with methyl orange. The released fatty acids were then extracted three times with 20–30 mL of diethyl ether. The combined ether extracts were washed with distilled water until neutral (tested with methyl orange), dried over anhydrous sodium sulfate, and the ether was evaporated using a rotary evaporator under vacuum. The fatty acids were then converted into methyl esters by treating them with freshly prepared diazomethane [19].

The obtained methyl esters of fatty acids (MEFA) were purified using preparative thin-layer chromatography (PTLC) on silica gel plates in a hexane:diethyl ether (4:1) solvent system with two replications. The MEFA zone on the sorbent was visualized in iodine vapor, scraped from the plate, and desorbed from the silica gel by multiple elutions with chloroform. The combined chloroform eluates were concentrated using a rotary evaporator, and the final MEFA product was dissolved in hexane for analysis by gas chromatography (GC).

Chromatographic Conditions

Gas chromatography was performed using an Agilent 6890 N gas chromatograph equipped with a flame ionization detector (FID) and a capillary column (30 m × 0.32 mm, HP-5). The carrier gas was helium, and the temperature program was set from 150°C to 270°C. The identification of methyl esters of fatty acids was conducted following the method described [20]. The composition and content of fatty acids are presented in Table 2.

Table 2.

Fatty acid composition of Punica granatum Seed Oil, %, GC by weight of acids

The fatty acid composition of Punica granatum seed oil, as presented in Table 2, demonstrates a clear dominance of unsaturated fatty acids (92,48–94,46%), particularly punicic acid (C18:3, cis-9, trans-11, cis-13), which constitutes the highest proportion across all samples (73,63–79,57%). This highlights the unique bioactive potential of pomegranate seed oil, as punicic acid is a conjugated linolenic acid known for its antioxidant, anti-inflammatory, and cardioprotective properties. Among other polyunsaturated fatty acids, linoleic acid (C18:2, 6,71–8,50%) and linolenic acid (C18:3n3, 5,80–7,23%) contribute significantly to the oil’s functional attributes. Linoleic acid plays a crucial role in cell membrane integrity, cardiovascular health, and skin hydration, while linolenic acid is an essential omega-3 fatty acid with cardioprotective and neuroprotective benefits. The presence of catalpic acid (C18:3-trans-9, trans-11, cis-13) in minor amounts (1,13–2,04%) further suggests a potential role in lipid metabolism and anti-obesity effects. The saturated fatty acid content (5,54–7,52%) is relatively low, ensuring high oxidative stability and better lipid metabolism properties. The primary saturated fatty acids identified were palmitic acid (C16:0, 2,87–4,72%) and stearic acid (C18:0, 1,93–2,25%), both of which contribute to cellular function and structural integrity. While excessive consumption of saturated fats can be detrimental, these components in moderate amounts support lipid metabolism and energy storage. The analysis of different pomegranate seed varieties indicates slight variations in total lipid content and fatty acid composition, likely influenced by genetic differences, growing conditions, and seed maturity. Notably, Sample №1 ("Qozoqi") exhibited the highest punicic acid content (79,57%), making it a promising candidate for nutraceutical applications, such as anti-inflammatory and antioxidant formulations. In contrast, Sample №3 ("Qora don") had the highest proportion of saturated fatty acids (7,52%), suggesting better oxidative stability but a slightly lower nutritional value. Due to its high punicic acid content, pomegranate seed oil is increasingly valued in pharmaceutical, cosmetic, and functional food industries. The presence of polyunsaturated fatty acids suggests that the oil can help reduce oxidative stress, modulate lipid metabolism, and improve insulin sensitivity, making it beneficial in the prevention and management of metabolic disorders such as obesity, diabetes, and cardiovascular diseases. Additionally, its composition makes it a valuable ingredient in skincare formulations, contributing to moisturization, wound healing, and anti-aging properties.

Conclusion.

The analysis of Punica granatum seed oil confirmed its high content of unsaturated fatty acids, with punicic acid (C18:3, cis-9, trans-11, cis-13) as the dominant component. The observed varietal differences in fatty acid composition highlight the influence of genetic and environmental factors on oil quality. Sample №1 ("Qozoqi") exhibited the highest punicic acid content, making it the most promising for nutraceutical applications, while Sample №3 ("Qora don") had the highest saturated fat content, indicating greater oxidative stability. Given its high bioactive potential, pomegranate seed oil presents significant applications in functional foods, pharmaceuticals, and cosmetics. Further research should focus on optimized extraction, bioavailability studies, and clinical validation to enhance its therapeutic and commercial utilization.

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