Identification of Naturally Occurring Carbazole Alkaloids Isolated from Murraya koenigii and Glycosmis pentaphylla by the Preparation of HPLC Fingerprint

2021 Abstract The plants Murraya koenigii and Glycosmis pentaphylla are rich sources of different carbazole alkaloids. A number of monomeric carbazole alkaloids with C 13 , C 18, and C 23 carbon frames and a good number of dimeric carbazole alkaloids were isolated from these two plants. Scientists are still working on these two plants in search of more and more novel compounds. Many of these alkaloids have potential biological activities. Scientists have determined the structures of these compounds by detailed analysis of spectral data like UV, IR, Mass, 1 H NMR, and 13 C NMR (1D and 2D). These procedures require expertise in spectral data analysis and huge time, and also these instruments are very costly. In this paper, I report the preparation of the HPLC fingerprint of some known carbazole alkaloids. These HPLC data will be helpful in quick and unambiguous identification of the natural


Introduction
Desoky [1] has published his work on isolating several phytosterols from Murraya exotica using column chromatography accompanied by HPLC. Similarly, several flavonoids from fruits of Murraya paniculata were purified by Ferracin et al. [2] using reverse-phase -HPLC. The use of HPLC is limited to the separation and purification of organic molecules. However, the HPLC profile can also be used for the identification and authenticity check of the compounds. In 2021, Chang and the group reported screening of anti-lipase components of Artemisia Argui leaves based on spectrum-effect relationship and HPLC-MS/MS [3].
Murraya koenigii, commonly known as curry leaf tree, and Glycosmis pentaphylla, commonly known as 'toothbrush plant,' belongs to the family Rutaceae and are rich sources of carbazole alkaloids [4][5][6]. The monomeric and dimeric carbazole alkaloids isolated from these plants have important biological activities [7][8][9]. Many scientists working in this field have also prepared several derivatives of these naturally occurring carbazole alkaloids [10][11][12][13]. In our present work, five carbazole alkaloids viz. murrayanine, girinimbine, koenidine, mahanimbine, and o-methylmurrayamine-A were isolated from the leaves and bark of Murraya koenigii. Two more carbazole alkaloids glycozoline and glycozolidine were obtained from the root bark of Glycosmis pentaphylla. Structures of all the isolated compounds were determined by a detailed investigation of 1D and 2D NMR spectral data analysis. HPLC fingerprint of each compound was prepared using a Water 515 HPLC pump. In this paper, the details of sample preparation for HPLC, selection of appropriate solvent system, selection of the optimum concentration of the compounds, and their retention time will be discussed. These data will be helpful in the quick and unambiguous identification of natural products.

Drugs and chemicals
HPLC grade solvents such as methanol, petroleum ether, chloroform, and other necessary chemicals and reagents were collected from Merck, Germany.

General experimental procedure
HPLC fingerprints were prepared using a Waters 515 HPLC pump equipped with model 680 automated gradient controller and Waters 2487 dual λ absorbance detector. Solvents (HPLC grade, Merck) were filtered by using a Millipore system, and analyses were performed on a Waters RP C18 (7 μm) column (300 mm × 7.8 mm, ID) in isocratic mode. For injection into the HPLC system, solutions of the pure compounds were prepared in methanol. Injection volume was 50 L for all the cases. All the sample solutions were detected at a UV wavelength of 254 nm, using methanol as a mobile phase in each case. 1 H NMR spectra were recorded using a BRUKER AVANCE 600 MHz NMR with TLCcryoprobe using TMS as an internal standard.

Plant material
The leaves and stem bark of Murraya koenigii were collected from Shantiniketan, West Bengal, India. The root bark of Glycosmis pentaphylla was collected from Jhargram, West Bengal, India.

Extraction and separation
The stem bark of Murraya koenigii (1 kg) was dried in air and cut into small pieces. Then, at that point, it was extracted with MeOH at 25 o C and concentrated utilizing a vacuum evaporator at 40 ºC. Then, at that point, the MeOH extract (8 g) was fractionated into three sections: petroleum ether, ethyl acetate, and water. The ethyl acetate portion was kept undisturbed for 24 h. A solid was precipitated from it, which was insoluble in chloroform. The solid was filtered, washed with cold chloroform, and dried. The compound was described to be girinimbine 1 by TLC (Benzene) and examination of the spectroscopic information with reference. The EtOAc extract was chromatographed on a column of silica gel (size 60-120). At first, elution was completed with petrol ether (bp 60-80 ºC) trailed by various combinations of petroleum ether and benzene (

Results and Discussion
The stem bark of Murraya koenigi was extracted with MeOH. The MeOH extract was fractionated into three parts: petroleum ether, ethyl acetate, and aqueous. The EtOAc extract was chromatographed on a silica gel column yielding girinimbine 1 [14] and murrayanine 2 [15]. The air-dried powdered leaves of Murraya koenigii were extracted with MeOH. The MeOH extract was chromatographed on a silica gel column, yielding three more known pyrano carbazoles, viz. koenidine 3 [16], mahanimbine 4 [17], and Omethylmurrayamine-A 5 [18] (Fig. 1). Structures of these compounds were determined by the detailed NMR spectral data analysis (Fig. 4) and then by comparing these data with that of the reported compounds. Petroleum ether extract of the root bark of Glycosmis pentaphylla on chromatographic resolution over neutral alumina yielded glycozoline 6 [19] and glycozolidine 7 [20] (Fig. 2). Structures of these two compounds were also determined by NMR spectral data analysis (Fig. 4) and then by comparing these data with that of the reported compounds.

Conclusion
Carbazole alkaloids are a very important group of natural products which possess various therapeutic activities. They are found in various medicinal plants belonging to the family Rutaceae. Scientists from different countries have isolated an innumerable number of carbazole alkaloids, and their structures have been determined by detailed spectral data analysis. However, it is not possible to go through the detailed spectral data analysis in some cases, as it is costly, time-consuming, and needs a high level of expertise. The HPLC fingerprint region data discussed in this paper will help the researchers in quick and unambiguous identification of some known carbazole alkaloids without taking the hazards of spectral data analysis. These HPLC data can also be taken as a confirmatory measure for the identification of these alkaloids.