GC-MS Analysis of Seeds from Different Pomegranate Varieties

Yun LI, Jianmei LU, Weiyu Wang, Qingying Wang, Luyang LU

College of Pharmacy, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives] This study aimed to study and analyze the main components of the oil from seeds of 8 different pomegranate varieties. [Methods] The oil in sour and sweet pomegranate seeds was extracted by ultrasonic-assisted method, and the composition of the soil after methyl esterification was identified by GC-MS. [Results] The oil yield of the four sweet pomegranate varieties and the four sour pomegranate varieties ranged from 9.43%-16.97%. A total of 6 compounds were identified in the methyl esterified pomegranate seed oil by GC-MS. [Conclusions] There were no significant differences in oil yield between sweet and sour pomegranate varieties (P>0.05). The content of methyl punicate in the four sweet pomegranate varieties was 52.18%-78.06%, and that in the four sour pomegranate varieties was 78.25%-84.55%. The content of methyl punicate in sour pomegranate was slightly higher than that in sweet pomegranate.

Key words GC-MS, Pomegranate seed oil, Ultrasonic-assisted extraction

1 Introduction

Pomegranate (PunicagranatumL.) (Punicaceae), is also known as Anshiliu, Ruoliu, Shanliye, Danruo, Tulin, Tianjiang, and so on. Dried seeds of pomegranate, as a common Tibetan medicinal material, are collected inDrugStandardoftheMinistryofPublicHealthofthePeople’sRepublicofChina:TibetanMedicine(Volume 1), and are commonly used to treat Bacon’s disease, stomach cold syndrome and all stomach diseases. In Tibetan medicine, they are called as "Saizhu". There are detailed records in the well-known classic Tibetan medicine booksTheFourMedicalTantrasandJingZhuMateriaMedica. Pomegranate seeds are the most commonly used medicinal material in Tibetan medicine (pomegranate peel for Chinese medicine) to dispel cold and enhance stomach fire. Pomegranate is rich in nutrients and has high medicinal value. It has a good therapeutic effect on anorexia, cold stomach, bloating,etc. "Pomegranate seeds are the king to eliminate all cold diseases, and most cold syndromes are caused by the weakness of stomach fire. Pomegranate seeds have the best effect on improving stomach fire, and other medicines cannot compare with it, so they are called ‘The Prescription of Monarch’. However, the long-term experience of Tibetan medicine in using pomegranate seeds to treat stomach diseases believes that the effect of sour pomegranate seeds is better than that of sweet pomegranate seeds. The difference in composition between the two types of pomegranate seeds with different taste is worthy of in-depth study. The weight of pomegranate seeds is as high as 40% of the total weight of pomegranate. They are an important medicine for treating stomach diseases in Tibetan medicine. However, they are usually abandoned in the development of pomegranate products, resulting in a great waste of resources. Punicic acid is mainly found in pomegranate seed oil. According to reports, the content of punicic acid in pomegranate seed oil is 64%-83%[1-2]. Foreign research results show that punicic acid has significant antioxidant and anti-tumor activity, and has the functions of preventing and treating hyperlipidemia and cardiovascular diseases. Research has shown that punicic acid can be effectively absorbed by the human body into plasma and red blood cell membrane, and is metabolized to cis-9, trans-11 conjugated linoleic acid, which can be effectively absorbed and utilized by the human body[3]. This experiment analyzes and evaluates the seeds of different varieties of pomegranate, in order to provide a basis for better development and utilization of pomegranate.

2 Materials and methods

2.1MaterialsThe instruments and equipment used included Agilent 6890N-5973N GC-MS equipped with DB-Wax (60 m×0.25 mm×0.25 μm) and high-purity helium (purity≥99.999%), KQ-250DE CNC ultrasonic cleaner, UPT ultrapure water device, high-speed multifunctional powdering machine, BCD-216ZDJ Haier refrigerator and DHG-9240A electric heating constant-temperature blast drying oven.

Pomegranate seeds were dried at 50 ℃, crushed by a high-speed multi-functional powdering machine, passed through a No.4 sieve, sealed and stored in dark for later use. KOH, petroleum ether (60°-90°), isooctane, ethanol and phosphoric acid were all analytically pure.

2.2Methods

2.2.1Extraction of pomegranate seed oil. A certain amount (20 g) of each of the 8 batches of sour and sweet pomegranate seeds (Table 1) was weighed, poured into a round-bottom flask, added with 100 mL of petroleum ether with a boiling range of 60-90 ℃, sonicated for 30 min, re-sonicated[4]for 10 min, and filtered, and the residue obtained was added 100 mL of petroleum ether, sonicated for 10 min, and filtered. The filtrate of the two times was blended, and rotary-evaporated at 55 ℃, to get pomegranate seed oil sample[5]. After letting standard at 35 ℃ for 10 h to let the residual solvent volatilize, the yield of the oil was calculated. Then, the oil sample was sealed and stored at -20 ℃ for later use.

Table 1 Sources of pomegranate seed samples

2.2.2Methyl esterification treatment of sample[6]. As pomegranate seed oil contains a lot of fatty acids, in order to facilitate the detection of punicic acid and other compounds in the sample during GC detection, the sample can be subjected to methyl esterification treatment to transform carboxylic acids into esters and reduce their boiling point, facilitating gasification and detection. Research has shown that the alkali method is more suitable for methyl esterification of pomegranate seed oil than other methyl esterification methods. It can prevent part of the conjugated double bonds of punicic acid from undergoing cis-trans isomerization, and avoid reducing the content of punicic acid, thus getting the correct content. A certain amount (0.1 g) of each sample was weighed, placed in a 10 mL test tube with a stopper, added with 2 mL of isooctane and 0.2 mL of K-OH-MeOH solution, let standard for 5 min, added with 2 mL of isooctane and 3 mL of distilled water, shaken well and let stand in success. Since the sample would emulsify during the shaking process, a saturated NaCl solution could be added to de-emulsify, and after standing still, the upper solution was collected for gas chromatography analysis.

2.2.3Gas chromatographic conditions. Column: DB-Wax, 60 m×0.25 mm×0.25 μm; carrier gas: high-purity helium (purity≥99.999%); inlet temperature: 230 ℃; injection mode: split injection; split ratio: 20∶1; carrier gas flow rate (constant flow): 1.0 mL/min; heating program: 80 ℃ for 2 min, rise to 180 ℃ at 15 ℃/min, rise to 230 ℃ at 1.5 ℃/min, for 10 min; GC-MS interface temperature: 230 ℃, ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; ionization method: EI; electron energy: 70 eV; scan mode: scan; scan range:m/z(35-350) amu; solvent delay time: 10 min.

3 Results and analysis

3.1YieldandcompositionofoilfrompomegranateseedsThe chemical composition of the oil from seeds of 8 different varieties of pomegranate was analyzed by GC-MS after methyl esterification. The gas chromatograms of the seed oil in sour pomegranate from Mengzi, Yunnan are shown in Fig.1 and Fig.2. A total of 12 chromatographic peaks were separated by gas chromatography. Deducting the peak area of the solvent, and taking the sum of the peak areas of the remaining peaks as 1, the relative content of each compound was calculated using the peak area normalization method. Compounds with a relative content greater than 1.00% were identified. Referring to literature[7]and SDBS organic compound spectral database, a total of 6 compounds in methyl esterified pomegranate seed oil were identified, and they were methyl palmitate, methyl linoleate, methyl oleate, methyl stearate, methyl punicate and methyl 6,9,11-octatrienoate. Due to the low content of methyl 6,9,11-octatrienoate in individual pomegranate seed oil samples, its content is not listed in Table 2.

Fig.1 Total ion current chromatogram of methyl esterified pomegranate seed oil

Fig.2 Mass spectrum of methyl punicate

As shown in Table 2, the oil yield of the four sweet pomegranate varieties and the four sour pomegranate varieties was 9.43%-16.97%, and there were no significant differences in seed oil yield between the sweet pomegranate varieties and the sour pomegranate varieties (P>0.05). The contents of methyl palmitate, methyl linoleate, methyl oleate and methyl stearate in methyl esterified pomegranate seed oil were 1.71%-2.64%, 3.21%-7.61%, 2.47%-7.32%, 1.06%-3.72%, respectively, and the differences between various varieties were also small. The content of methyl punicate in seed oil of the four sweet pomegranate varieties was 52.18%-78.06%, and that in the seed oil of the four sour pomegranate varieties was 78.25%-84.55%. The content of methyl punicate in sour pomegranate seed oil was significantly higher than that in sweet pomegranate seed oil.

Table 2 Yield and composition of oil from seeds of 8 pomegranate varieties %

3.2DataanalysisFig.3 shows that there was no significant difference in seed oil yield between the sweet pomegranate varieties and the sour pomegranate varieties (P>0.05). The oil yield ranged from 9.43% to 16.97%. The seed oil yield of all the pomegranate varieties except Maya Sweet from Zaozhuang, Shandong was greater than 10%. As shown in Fig.4, in the oil of pomegranate seeds, the content of punicic acid was the highest (52.18%-83.13%), and it was slightly higher in the sour pomegranate seed oil than the sweet pomegranate seed oil. The contents of palmitic acid, linoleic acid, oleic acid and stearic acid were relatively low, all less than 10%, and there was no significant difference between sour pomegranate and sweet pomegranate (P>0.05).

Fig.3 Yield of oil from seeds of pomegranate

Note: 1. Methyl palmitate; 2. Methyl linoleate; 3. Methyl oleate; 4. Methyl stearate; 5. Methyl punicate.

4 Discussion

Punicic acid is the main component of pomegranate seed oil, and its content is as high as 52.18%-83.13%. Due to the existence of conjugated triene structure, the chemical properties of punicic acid are active. Under the determination conditions, the double bond will be transformed[8]. Therefore, before the gas chromatography analysis, pomegranate seed oil should be subjected to alkaline methyl esterification first to ensure accurate identification and content analysis of punicic acid. This experiment found that the content of punicic acid in seed oil of the four sweet pomegranate varieties (52.18%-78.06%) was lower than that of the four sour pomegranate varieties (78.25%-84.55%), consistent with Tibetan medicine’s belief that sour pomegranate seeds are better than sweet pomegranate seeds when they are used to treat stomach diseases. Modern research found that punicic acid is the component with the highest content in pomegranate seed oil, and it has a significant antioxidant effect on the cardiovascular system. Therefore, the higher the content of punicic acid, the stronger the anti-oxidation of pomegranate seed oil. The results of this experiment show that the contents of punicic acid in the seed oil of sour pomegranate from Zaozhuang, Shandong, the sour pomegranate from Mengzi, Yunnan, the sour pomegranate from Lintong, Shaanxi, and Maya Sour from Zaozhuang, Shandong were higher than other varieties, and the seed oil of these pomegranate varieties is suitable for further development and application.