Mass spectra analysis of quinoline alkaloids detected in Sauuda fruticose leads to novel biosynthesis pathway of quinoline alkaloids CURRENT POSTED

Alkaloids reported to have a wide range of biological activity moreover, the biological effects of alkaloids depends on the type of the alkaloids and since the phytochemical screening studies on Suaeda fruticosa indicates the presence of alkaloids but they did not determine the types of the alkaloids present on this plant. Consequently, the LC-MS/MS analysis of the alkaloidal part of Suaeda fruticosa leave extarct reveals the presence of six quinoline alkaloids beside that mass spectra analysis of the present quinoline alkaloids leads to suggest a new biosynthetic pathway of quinoline alkaloids.

2018).Quinoline alkaloids have a wide biological activity spectrum such as anti-malaria and anticancer applications (Nainwal et. al. 2019). Quinoline alkaloids can be divided into several subgroups according to their chemical structure but our focus here will be on simple quinoline alkaloids which consist of benzene ring fused with pyridine ring and different substitution patterns (Solomon et. al. 2011). There are two reported boisynthetic pathways for simple quinoline alkaloids: the first one is through anthralic acid and the other one is through tryptophane (Mann, 2005;de Souza et. al. 2003 and Sultana, 2013).
In this study LC-ESI-QTOF-MS/MS was used to analyse quinoline alkaloids in Suaeda fruticosa . In addition, the quinoline alkaloids biosythetic pathway was described.

Results
The LC-ESI-QTOF-MS/MS analysis of the alkaloidal part of Suaeda fruticosa extract revealed the presence of six oxygenated simple quinoline alkaloids (Fig. 1). The qualitative and quantitative analysis data for quinoline alkaloids in dry plants are described in table 1. Also, mass spectra analysis of the detected quinoline alkaloids leads to suggest a new biosynthetic pathway(scheme 1and 2).

Mass spectra analysis of detected quinoline alkaloids:
Two types of oxygenated quinoline alkaloids were detected in Suaeda fruticosa extract. One is oxygenated in one position while the other is oxygenated in two positions.

Mass spectra analysis of quinoline alkaloids oxygenated at one possiton:
From this type two quinoline alkaloids were detected. 3-hydroxyquinoline (1) and 6-hydroxyquinoline (2). Their mass spectra (Fig. 2) illustrated a similarity and the only difference was the relative intensity of M-28 peak and this exclude the probapility of 2,4 and 8 oxygenated quinoline [15].
Also, M-55 peak appeared at high intensity compared with 5 and 7 oxygenated quinoline, so the possible oxygenation positions are 3 and 6 (Kaczmarek and Steinegger, 1958). The difference between 3-hydroxy and 6-hydroxy is the presence of mass peak at 104.0484 which indicates the loss of H 2 O followed by loss of C2. This is only possible for 3-hydroxyquinoline. Also, the retention time indicates a very low difference in polarity between the two compounds, therefeore 3-hydroxy is more polar than 6-hydroxy. The mass fragmentation pattern of the two compounds was given in Fig. 3.

Mass spectra analysis of quinoline alkaloids oxygenated at two possitons:
Four quinoline alkaloids were detected to be oxygenated at two positions but the only two possible positions were 2 and 3. In compound 5, the appearance of two peaks at m/z=119.0783 and 106.0727 gives an evidance for the porposed strcture as explained in fig. 6.
Two main peaks proving the proposed structure for compound 6. The first one [M-C 2 H 4 ] at m/z=162.0552 indicates that the two methoxy groups are adjacent and this peak was followed by the loss of CO 2 to give a peak at m/z=118.0652 like compound 3, after that the loss of HCN. The other peak at m/z=146.0937 which due to [M-CO 2 ] reveals a methoxy group at position 2 to allow rearrangement and the loss of CO 2 as described in Fig. 7.

Conclusion
In this study six quinoline alkaloids were detected in the leaves of Suaeda fruticosa by using LC-MS/MS analysis beside that a new biosynthesis pathway for quinoline alkaloids were sugested in light of the component of leaves of Suaeda fruticosa.

Materials And Methods
All chemicals were purchased from Sigma-Aldrich.

Plant material
The The ethanol extract was filtered using filter paper and concentrated to dryness under reduced pressure using a rotary evaporator at 37 0 c (Ikia -Germany). The concentrated crude extract weight 24g, which represent (6%)of the plant weight.

Preparation of alkaloidal extract
The ethanol residue was dissolved in 5% HCl until the PH = 2 of the solution and filtered, the precipitate which contains neutral material was kept for further fractionation, and the filtrate which provides the basicmaterial was basify using NH4OH solution, and the PH of the solution becomes around 8. After that, the solution was extracted with chloroform 50 mL three times the chloroform layer was evaporated to get 0.246 g of crude alkaloids which represent 0.123% of the dry plant.

LC-MS/MS for Plant Extracts
Analyses were performed on SCIEX X500R QTOF system (includes UPLC-MS/MS (Woodlands Central Indus. Estate., Singapore)). The separation was performed using Phenomenex Kinetex 2.6 _m Phenyl-Hexyl 100 A (50 _ 4.6 mm, Phenomenex, Madrid Avenue, Torrance, CA, USA). The mobile phase consists of phase A (10 mM ammonium formate in water) and phase B (0.05% formic acid in methanol). A variable gradient flow rate was used, which is described in Table 2. A positive nontargeted mode was used for the analyte.

Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests

Funding
Self-funded by the authors 6.6. Author Contribution: Experiments and the writing of the manuscript were carried out in contribution of all authors. All authors read and approved the final manuscript.

Acknowledgements
The author extend his appreciation to Dr. Kamel A. Saleh from Department of Biology, Science College, King Khalid University for his help in data analysis and editing the manuscript. Figure 1 Structures of detected oxygenated quinoline alkaloids Rearengment in compound 6