CHEMICAL CONSTITUENTS OF THE ROOT BARK OF TABERNAEMONTANA CATHARINENSIS (APOCYNACEAE) CONSTITUINTES QUÍMICOS DAS CASCAS DAS RAÍZES DE TABERNAEMONTANA CATHARINENSIS (APOCYNACEAE)

The Tabernaemontana genus, belonging to the Apocynaceae family, consists of approximately 110 species. Among these, the species under study, Tabernaemontana catharinensis is popularly known in Brazil as "leitero de vaca". This species is especially rich in indole alkaloids, which are compounds with p r o m i s i n g b i o l o g i c a l p o t e n a l , s u c h a s an cholinesterase and an oxidant ac vi es. The objec ve of the study was isolate, purify, and iden fy indole alkaloids from the root bark of T. catharinensis. The plant material was dried at room temperature, ground, and subjected to methanolic extrac on. The extract was par oned with dichloromethane/water. The phytochemical study was performed by column c h ro m ato g ra p hy a n d a n a l y ca l t h i n l aye r chromatography. The structures were established on the basis of spectroscopic methods, including 1D and 2D NMR. The phytochemical study of the T. catharinensis species led to iden fica on of four alkaloids, 12-methoxy-Nb-methylvoachalo ne (1), coronaridine (2), 5,6-dioxoibogamine (3), and 19 (S) heyneanine (4). Alkaloids (3) and (4) are being reported for the first me in the species.


INTRODUCTION
The Tabernaemontana genus contains  approximately 110 species, 27 of which are Brazilian  (Morales, 2009). The genus is especially rich in monoterpenic indole alkaloids, which are useful chemical markers of the genus and have great value for the classifica on of the species, in addi on to demonstra ng considerable variety of carbonic skeletons and diverse biological ac vi es.
In folk medicine, this species is used as an an dote for snake bites, to relieve toothache, and as a dewormer. Alkaloids and extracts containing alkaloids of this species showed several biological ac vi es: an -inflammatory (Camponogara et al. 2019), analgesic (Brum et al 2019), an tumor (Rosales et al., 2019), an oxidant (Pergher et al 2019), an microbial and an leishmanial (Pereira et al., 2005;Reis, 2018), and trypanocidal (Pereira et al., 1999).
In the present work, we report the inves ga on of the methanolic extract from the root bark of T. catharinensis, which allowed us to characterize four already known monoterpenic indole alkaloids, however, two of these are being reported here for the first me in the species.

OBJECTIVES
Isolate, using classical chromatographic methods, and iden fy, using spectrometric methods, indole alkaloids from the root bark of T. catharinensis, family Apocynaceae.

General experimental procedures
The plant material was powdered using a TECNAL hammer mill.
The extract was concentrated under reduced pressure on a rotary evaporator, FISATOM 802.
Analyses using the analy cal tool thin layer chromatography (TLC) were performed with silica gel 60 F254 MERCK. The compounds were visualized by irradia on with an ultraviolet lamp, with wavelengths of 254 nm and 365 nm and/or with chromogenic developers (Dragendorff reagent and 2% vanillin solu on in concentrated sulfuric acid), followed by hea ng.
The purity criteria adopted were the visualiza on of a single spot in TLC, using different eluent systems.
The solvent mixtures were expressed in % v/v. Nuclear Magne c Resonance (NMR) spectra were recorded on a BRUKER spectrometer, model DPX-500, opera ng at a frequency of 500 MHz for 1H and 125 MHz for 13C. The solvent used for all spectral analyses was CDCl3.

Plant Material
The vegetal material made up of the root bark from Tabernaemontana catharinensis was collected in the surroundings of the municipality of Bom Jesus do Itabapoana, RJ. The material was subsequently classified and iden fied by Professora Luiza S. Kinoshita from the State University of Campinas (UNICAMP). The exsicca on was deposited at the UNICAMP Herbarium with voucher specimen UEC117862.

Extrac on and isola on
The root bark was subjected to successive extrac ons at room temperature, using methanol as the solvent, giving rise to the crude extract (42.4g). T h i s w a s s u b s e q u e n t l y p a r o n e d i n dichloromethane/water, obtaining the organic phase (20.6g) and the aqueous phase (19.6g).
A por on of the organic phase (18.5g) was subjected to silica gel column chromatography, using dichloromethane/methanol as eluent in increasing polarity concentra ons. The frac ons were combined by analy cal thin layer chromatography, obtaining 14 frac ons in total, where frac on 13 gave rise to compound 1 (369.6mg).
Frac on 8 (1.8g) was rechromatographed on a s i l i c a g e l c o l u m n , u s i n g dichloromethane/methanol in increasing polarity concentra ons as eluent and, a er the frac ons were combined, provided 8 new frac ons in total, where frac on 8.2 resulted in compound 2 (7.6mg). T h e 8 . 3 f r a c o n ( 8 1 3 . 2 m g ) w a s a l s o rechromatographed on a silica gel column, using dichloromethane/ethyl acetate as an eluent in increasing polarity concentra ons, which, a er the frac ons were combined, provided 16 new frac ons, where the 16.6 frac on gave rise to compound 3 (4.50g) and frac on 16.9 gave rise to compound 4 (46.1mg).

RESULTS AND DISCUSSION
In the current research, we report the inves ga on of the methanolic extract of the root bark from T. catharinensis, which led to the characteriza on of the four known monoterpenic i n d o l e a l k a l o i d s : 1 2 -m e t h o x y -N bmethylvoachalo ne (1), coronaridine (2), 5,6dioxoibogamine, (3) and 19-(S)-heyneanine (4), which were iden fied with spectral data from the literature for 1H and 13C NMR spectra (Figueiredo et al, 2010;Gonçalves, 2011;Souza et al, 2010). It should be emphasized that only the spectral data of alkaloids 3 and 4 will be discussed, since they are being reported for the first me in this species. The structures of all iden fied compounds are shown in Figure 1.  (Azoug et al., 1995).
In alkaloid 3 there is the absence of a carbomethoxy group linked to C-16, common in the skeleton for monoterpenic indole alkaloids (Zenk, 1980). Analysis of the 13C NMR spectrum (Table 1, Figure 3-Annex) showed the presence of a signal at C 169.72, which is consistent with the chemical displacement of a carbonyl group from a lactam and also the presence of one more signal referring to a carbonyl carbon at C 184.62. The absence of the signals related to the coupling of the two methyleneic groups, 2H-5 to the nitrogen atom with 2H-6, suggests the proposal of an indole nucleus free of subs tuents, but with the presence of two carbonyl groups linked to the carbon atoms C-5 and C-6, respec vely.
The posi on of the carbonyl linked to the carbon atom C-5, forming a lactam with N-4, was confirmed by the long distance 3JCH correla on presented in the HMBC spectrum (Table 2, Figure 9  A hydroxyethyl group linked to CH-sp3 is recognized by changes in the signals referring to hydrogen atoms linked to carbon atoms CH3-18 (3H; H 1.13; d) and H 4.18;qui) in rela on to alkaloid 3 (Table 2, Figure 11 and Figure 12-Annex). The a ribu on of the chemical shi s of the remaining methyleneic and methyleneic hydrogen atoms in the alipha c chain is described in Table 2, together with the correla ons observed in the HSQC spectrum (Figure 15 -Annex).
The set of data above, added to those presented in the spectrum of 1H-1H-COSY (Figure 14 -Annex) allowed us to propose structure 4 for the iden fied alkaloid. The rela ve configura on of the carbon atom C-19 (19S) was established based on the analysis of the 1H NMR and 13C NMR spectra, and through comparisons with literature data for heyneanine alkaloids and their epimer 19-(R)heyneanine (A a-ur-Rahman et al., 1987;Matos et al., 1976;Lemos et al., 1996;Wenkert et al., 1976).
The hydrogen bond formed between the hydroxyl group, allocated on carbon atom C-19, and nitrogen N-4 forces the hydroxyethyl group to a more rigid conforma on (par al structures 4a and 4b, Figure 17), causing differences in chemical displacements of the hydrogen and carbon atoms of CH3-18 and CH-19 of alkaloid 4 and its epimer. The literature records values of H 1.11 (3H-18) and 4.12 (H-19) for 4 and H 1.27 (3H-18) and 3.92 (H-19) for its epimer (Matos et al., 1976) which corroborate the proposed structure 4 for the isolated alkaloid. In addi on, the chemical displacement values of CH2-15 (C 22.9) and CH-21 (C 59.7) carbon atoms (Table 2, Figure 7) recorded in the literature for 4 (A a-ur-Rahman et al., 1987) are different from those reported for the epimer: C 28.6 (CH2-15), 54.7 (CH-21) (Lemos et al., 1996;Wenkert et al., 1976). These differences are explained by the protec ve γ effect of the methyl group (CH3-18) on carbon CH2-15 in 4 (par al structure 4a) and on CH-21 carbon in the epimer (par al structure 4b - Figure 17).

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hydrogen integra on at H 0.98 with J = 7.3 Hz, rela ve to a methyl group in the alipha c part of the molecule, presented in the 1H NMR spectrum (Table 1, Figure 6-Annex), confirms the presence of an ethyl group a ached to the C-20 carbon atom.
The data set above allowed us to propose the structure of the 5,6-dioxoibogamine alkaloid, previously iden fied in T. hystrix (Souza et al., 2010). Alkaloid 4 tested posi ve with Dragendorff reagent. The analysis of the 13C NMR spectrum (Table 2, Figure 10 and Figure 11-Annex) enabled recogni on of the presence of twenty-one carbon atoms. The signals at C 174.95 and C 52.00 are consistent with the displacements for carbon atoms of a carbomethoxy group, and the signals at C 118.48 (C-9); 119.51 (C-10), 122.33 (C-11), and 110.48 (C-12) are related to an indole core with an unsubs tuted A ring.