Isolation and Characterization of Compounds from Cinnamon Oil (Cinnamomum burmanii) Distillation Residu

This study aimed to isolate and characterize compounds from the distillation residue of cinnamon oil from Loksado, South Kalimantan. Cinnamon (Cinnamomum burmanii) distillation residue was extracted with methanol as solvent. The methanol extract was fractionated by liquid vacuum chromatography to obtain fractions A, B, C, and D. The crystals contained in fraction C were washed with cold n-hexane to obtain 5.4 mg of yellow isolate (FC1). FC1 isolates were characterized by UV-Vis, IR, H-NMR, and C-NMR spectrophotometers. UV spectra showed a maximum wavelength at 307, 316, and 321 nm indicating the presence of a conjugated or aromatic system. The infrared spectra showed -C=O, -OH, C-O, C-H, C-N, and C=N groups. The H-NMR spectra showed the presence of aromatic protons at 6.38 ppm (1H, d, J=9.5 Hz), 7.67 ppm (1H, d, J=9.5 Hz), 7.29 ppm (1H, d, J=8 Hz), 7.44 ppm (1H, d, J=8 Hz), and 7.49 ppm (1H, t) and there was a methyl proton (acetyl group) at H 2.13 ppm (3H,s). The C-NMR spectra showed the presence of a C=O ketone group at 207.26 ppm and there were 9 C-sp at 116.9; 119.0;124.6; 128.1;132.0;143.7; 154.3; 161.0 ppm, which δC 161.0 ppm was C-oxyaryl. Based on UV, IR, H and CNMR spectra data, FC1 isolate was suggested as an isoquinoline alkaloid substituted by OH and acetyl groups.


INTRODUCTION
Cinnamon (Cinnamomum burmanii) belongs to the Lauraceae family and is one of the leading commodities of South Kalimantan, particularly the South Hulu Sungai area. The productivity of cinnamon from the Loksado sub-district was 1,184.43 tons, and the Haruyan sub-district was 0.9 tons. Another cinnamon producer area was the Kotabaru district, with 24 tons (BPS South Kalimantan, 2014).
These various bioactivities and the distinctive aroma of cinnamon are caused by the various compounds, such as essential oils, consisting of various compounds from the monoterpenoid, sesquiterpenoid, or phenylpropanoid groups. In addition to essential oils, cinnamon contains other compounds such as flavonoids, saponins, tannins, alkaloids, steroids, and lignans (Safratilofa, 2016;Yuan et al., 2017).
Cinnamon essential oil (cinnamon oil) can be obtained by distillation. The distillation process produces dregs or residues that most likely still contain nonvolatile chemical compounds such as flavonoids, saponins, tannins, or alkaloids. These compounds have various bioactivities. Flavonoids have antioxidant, anti-inflammatory, antimutagenic, and anticancer activities (Panche et al., 2016). Alkaloids act as an antibacterial, antiinflammatory, antifungal, analgesic, and antiviral (Bribi, 2018). Saponins have anticancer and anticholesterol activity (Thakur et al., 2011). Tannins are used as lung anticancer therapy (Rajasekar et al., 2021). Based on the description above, it can be seen that flavonoid compounds, alkaloids, saponins, and tannins have various bioactivities, and it is suspected that these compounds are still present in the cinnamon distillation residue. Therefore, it is necessary to isolate and characterize the chemical compounds from the cinnamon distillation residue from Loksado.

Extraction
One kilogram of chopped cinnamon bark distillation residue was air-dried and then ground into a coarse powder. Cinnamon residue was macerated with methanol for 24 hours and filtered. Maceration was repeated three times. The filtrate was concentrated with a rotary evaporator and continued by heating on a waterbath until 39.79 g of methanol extract was obtained.

Isolation and purity analysis.
Fractionation of cinnamon methanol extract was carried out by Vacuum Liquid Chromatography (KVC) with 60G silica gel as stationary phase (t=5 cm and column d=7 cm). The mobile phase used a gradient elution system, namely n-hexane with increased polarity with chloroform, then continued with ethylacetate single eluent. A total of 10 g of methanol extract was fractionated by KVC, obtained 4 combined fractions, namely fractions A (vials 1), B (vials 2-3), C (vials 5-10), and D (vials 11-12). There were yellow crystals in the evaporator flask at fraction C when concentrated with a rotary evaporator. Crystals were collected and washed with cold n-hexane. The washed crystal was called FC1. The rotary evaporator flask containing fraction C was rinsed with chloroform and then concentrated with a rotary evaporator, called fraction FC2. Furthermore, the FC2 fraction (60 mg) was fractionated by gravity column chromatography with n-hexane:chloroform (1:1) as eluent to produce 2 fractions, namely FC2a and FC2b fractions. FC1 and FC2b fractions were spotted on the same TLC plate with n-hexane:chloroform (1:1) eluent and showed the same Rf value. The FC1 fraction purity test was carried out by TLC in various eluents, namely nhexane:chloroform (1:1); nhexane:ethylacetate (1:1) and chloroform:ethylacetate (1:1), and 2dimensional TLC with eluent 1, namely nhexane:ethylacetate (1:1).and eluent 2, namely n-hexane:chloroform (1:1).

Structure characterization
The structural characterization of FC1 isolates was carried out using spectrophotometer UV, infrared, 1 H-NMR, and 13 C-NMR.
Isolation and Characterization of Compounds from Cinnamon Oil...

Isolation of FC1 Compound
The yield of methanol extract of cinnamon distillation residue was 3.98%. The eluent selection was carried out before fractionating the methanol extract. TLC chromatograms of methanol extracts in various eluent compositions are presented in Fig. 1. Fig. 1f reveals that n-hexane:chloroform (1:1) TLC eluent produces better separation than n-hexane:ethyl acetate (1:1) (Fig. 1e) so that n-hexane:chloroform eluent was used as eluent for fractionation of methanol extract using KVC. Fig. 1a, 1b, 1c, and 1d show that the methanol extract was not separated properly.
Fractionation of methanol extract by KVC method employed a gradient elution system. The elution process started with a single eluent of n-hexane, followed by nhexane:chloroform (9:1), (8:2), (7:3), and ended with ethyl acetate. Fig. 2 shows the chromatogram of the methanol extract fractionated by KVC. Based on the TLC chromatogram pattern, the resulting fractions were grouped into 4 combined fractions, namely Fraction A, Fraction B, Fraction C, and D. There were crystals on the walls of the flask at the concentration of fraction C with a rotary evaporator, then separated and washed with cold n-hexane, called FC1. A total of 5.4 mg of FC1 isolate in the form of a yellow crystalline solid was obtained. The remaining fraction C in the evaporator flask was dissolved with chloroform, dried, and 60 mg FC2 was obtained.
In Fig. 3, it can be seen that FC2(b) has one stain, so to determine whether FC2(b) and FC1 are the same compounds, the two isolates were spotted on the same TLC plate. Fig. 4 shows that FC1 and FC2(b) have the same Rf, so it can be said that FC1 and FC2(b) are the same compounds.
The purity test was carried out on FC1 isolates in different eluents and 2dimensional TLC. All TLC chromatograms ( Fig. 5 and 6) showed one spot, so it can be stated that the FC1 isolate was pure enough to proceed with structural characterization   The UV spectra of the FC1 isolate ( Fig.  7) showed maximum wavelengths at 321, 316, and 307 nm. The presence of absorption at wavelengths in the UV region indicates the presence of a conjugated system chromophore or an aromatic ring, according to the simple isoquinoline framework as reported by Saidi et al. (2011). Isoquinoline alkaloid compounds, namely papralline, isolated from the Cryptocarya rugulosa plant (Lauraceae), showed maximum wavelengths at 325, 317, 312 nm (Saidi et al., 2011). Sulaiman et al. (2011 reported that the UV spectra of the Litsea lancifolia (Lauraceae) plant had a maximum wavelength at 307 nm and were a compound of N-alyllaurolitsine, an alkaloid with a benzylisoquinoline framework.
The infrared spectra show the presence of some characteristic functional group vibrations. The absorption at 3415.45 cm -1 is caused by the vibration of the hydroxy functional group (-OH), which is strengthened by the presence of C-O vibrations at 1174.37 cm -1 . Absorption at wave number 1697.76 cm -1 indicates the presence of a carbonyl group (C=O). A characteristic vibration of C=C indicates the aromatic ring at 1618.95; 1560.62; 1486.74 cm -1 , which is also supported by the =C-H stretching vibration at a wavenumber of 3057.10 cm -1 and =C-H bending at 686.87 to 994.33 cm -1 . The aliphatic -C-H vibration appears at wave number 2954.41 cm -1 . Absorption at wave number 1397.20 cm -1 indicates the presence of C-N vibrations. The imine vibration (C=N) was found at a wavenumber of 1599.94 cm -1 , in accordance with the imine vibration (1599 cm -1 ) typical of isoquinoline alkaloids as in the study of Sulaiman et al. (2011). The presence of aromatic C=C, =C-H functional groups, C-N, and C=N vibrations support the isoquinoline alkaloid framework in FC1.
Based on the UV, infrared, 1 H-NMR, and 13 C-NMR spectra data, the structure of FC1 isolate is estimated to be an alkaloid compound with an isoquinoline skeleton substituted by an OH group and an acetyl group, namely 1-(5-hydroxyisoquinoline-1yl)ethanol, as shown in Fig. 8.  Alkaloid compounds with an isoquinoline framework are commonly found in the Lauraceae family. The structure of several alkaloid compounds that have been isolated