Identification of Chemical Compounds in Ziziphus mauritiana Fruit Juice by GC-MS and LC-MS/MS Analysis

| ABSTRACT Ziziphus mauritiana is an edible fruiting plant commonly found in Asia. In Indonesia, this plant grows wild and thrives on the islands of Bali, Lombok, and Sumbawa. Although it is edible and has good potential as food and medicinal ingredient, in Indonesia, the fruits of Z. mauritiana are underutilized and have almost no economic value. Information about the bioactivity and chemical content of the fruit is limited. To evaluate its possibility of being developed as functional food, a GC-MS and LC-MS/MS analysis was carried out to identify the phytochemical content of the fruit juice. From the GC-MS chromatogram, four compounds were identified with a quality match of 85% and above. The compounds were 5-( hydroxymethyl)-2 – furancarboxaldehyde (43.45%), 5, 5'-(oxybis(methylene)) bis-2- furancarbox-aldehyde (25.99%), 2,3 – dihydro-3,5 – dihydroxy – 6 – methyl-4H – pyran – 4-one (6.05%), and hexadecanoic acid (2.16%). The result of the LC-MS/MS analysis showed 42 peaks of different chemical compounds and included several groups of compounds such as flavonoids, alkaloids, phenols, terpenoids, and organic acids. From these results, it can be concluded that Ziziphus mauritiana fruit juice contains various chemical compounds that are likely to have medicinal activity and therefore has good potential to be used and developed as a functional food.


Introduction
Plants have an essential role in maintaining human health and well-being; this is based on the idea that plants contain natural substances that can help human health as a preventive and curing disease (Jangde, 2015). Therefore, plants that have the potential as drugs have curative properties due to various phytochemicals, such as flavonoids, tannins, alkaloids, saponins, essential oils, and glycosides (Shrivastava & Dwivedi, 2015). Phytochemicals are biologically active chemical compounds that occur naturally in plants, so they have a role as a natural defense system for host plants and provide aroma, taste, and color to these plants (Arts & Hollman, 2005).
One of the plants with the potential as a medicine is Ziziphus Mauritiana from the Rhamnaceae family. The Rhamnaceae family is a multipurpose plant used for traditional medicine, food, and environmental protection (Sheng Guo et al., 2017). Z. Mauritiana is an edible fruit plant widely found in tropical and subtropical regions and is widespread in Asia and America, as well as in the Mediterranean area (Plastina et al., 2012;Prakash et al., 2021). In Indonesia, this plant grows wild and thrives on the islands of Bali, Lombok

Plant Materials
Ripe and fresh Ziziphus mauritiana fruit was collected from Bugis Village, Medang Island, West Nusa Tenggara, Indonesia, in September 2021. The plant and its fruit were identified and authenticated at the Bogoriense herbarium, Center for Biological Research, Badan Riset dan Inovasi Nasional (BRIN), West Java, Indonesia, with a voucher number B-205/V/DI.05.07/10/2021 Z. mauritiana fruit is washed with water, then processed with a slow juicer. The juices are collected and immediately frozen and stored in the freezer. The frozen fruit juices were then freeze-dried at 50˚C and stored in the refrigerator until used for further analysis.

Sample Preparation 3.2.1 GC-MS Analysis
A total of 5 g of dried Ziziphus mauritiana fruit juice was weighed, then extracted in ± 25 mL of methanol for 24 hours. The extract was filtered through Whatman filter paper no. 41 (110 mm). Then 10 mL of the extract was pipetted into a tube and then dried at 60°C for 1 hour and, after drying, dissolved again with the remaining extract as much as 200 L. Afterward, the sample was injected into the GC/MSD (Gas Chromatography/Mass Selective Detector).

LC-MS/MS QTOF Analysis
A total of 0.5 g of freeze-dried fruit juice was dissolved in methanol in a 10 mL volumetric flask, followed by sonication for 30 minutes. Then it was diluted further with methanol, homogenized to the right concentration, passed through a syringe filter with 0.22 m GHP/PTFE membrane, and finally injected into the UPLC system.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was performed in Agilent Technologies 7890 Gas Chromatography equipped with an Auto Sampler and a 5975 Mass Selective Detector (MSD). Capillary column (HP Ultra 2) measuring 30 m × 0.20 mm with a film thickness of 0.11 m and helium as carrier gas at a 1.2 ml/min flow rate and constant flow in column mode. The initial temperature of the oven is 80°C for 0 minutes. The temperature increased at a rate of 3°C/min to 150°C, stabilized for 1 min, and finally rose by 20 °C/min to 280 °C, which lasted 26 min. The injector temperature, ion temperature, interface temperature, and quadrupole temperature in scan mode are set at 250 °C, 230 °C, 280 °C, and 140 °C, respectively. The electron impact ionization mode is set at 70eV. The structure of the reported compounds was assessed by comparing the fragmentation patterns obtained by 1:8 separation. The chromatograms were analyzed, and the relative percentages of the compounds were calculated. Compounds were identified by comparing the mass spectra with the reference mass spectra in the Willey 275 database. The relative content of the compounds was calculated based on the total peak area of the integrated ion chromatogram (TIC) for the co-eluting peaks, and the results were expressed as total abundances.

LC-MS/MS QTOF Analysis
The LC-MS/MS QTOF analysis was carried out in the C18 column of the Waters Acquity UPLC system (2.1 mm × 100 mm, 1.7 mm) equipped with an auto-sampler, column manager, and adjustable MS detector. The mobile phase was 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in aquabides (solvent B). For gradient elution, the flow rate of the mobile phase was kept at 0.6 mL/min. The total chromatography run time was 2.0 min. The temperatures for the column and auto-sampler were maintained at 40°C and 15°C. The injection volume was 10 L. MS analysis was performed with an electrospray ionization source (ESI) in positive and negative ion modes. MS data is obtained in the m/z range of 50-1200 and MSE Tof mode operation. The compounds were identified using a UNIFI data processor with a mass spectrum library of natural active substances from the Waters Traditional Medical Scientific Library database based on UPLC/QTof MSE Data Acquisition, which is integrated with an automatic identification process.

LC-MS/MS QTOF Analysis of Ziziphus mauritiana fruit juice
In Z. mauritiana fruit juice, 43 compounds were identified using LC-MS/MS QTOF analysis. The basic peak ion chromatogram (BPI) is shown in Figure 2. The list of compounds is given in Table 2   While the compounds that were confirmed in the negative mode consisted of 32 compounds, including sanjoinine A, Quinic acid, and Kukoamine A, these three compounds also have biological activity that can contribute medicinally, namely the compound sanjoinine A, which has potential as an antioxidant (Foyet et

Conclusion
From these results, it can be concluded that Ziziphus mauritiana fruit juice contains various chemical compounds that have medicinal potential. Four compounds were identified by GC-MS analysis with a quality match of 85% and above, and 42 compounds were identified by LC-MS/MS analysis.
Funding: This research was funded by The International Research Training Center for Botanicals and Chronic Disease (CBCD).