Analytica Chimica Acta 447 (2001) 125134

Computer-aided method for identication of components in essential oils by 13C NMR spectroscopy
Marcelo J.P. Ferreira a , Mara B. Costantin a , Patr cia Sartorelli a , b c Gilberto V. Rodrigues , Renata Limberger , Amlia T. Henriques c , Massuo J. Kato a , Vicente P. Emerenciano a,
b a Instituto de Qu mica, Universidade de So Paulo, Caixa Postal 26077, 05513-970 So Paulo, Brazil Departamento de Qu mica, ICEx, Universidade Federal de Minas Gerais, 30161-000 Belo Horizonte, Brazil c Faculdade de Farmcia, Universidade Federal do Rio Grande do Sul, 90610-000 Porto Alegre, Brazil

Received 20 December 2000; received in revised form 6 June 2001; accepted 13 June 2001

Abstract The aim of this paper is to present a procedure based on analyses of 13 C NMR data for identication of known and new chemical constituents in essential oils. A novel program developed to analyze complex mixtures of terpenoid compound was evaluated for the identication of components in the essential oils from leaves of Piper cernuum and Piper regnellii. 2001 Elsevier Science B.V. All rights reserved.
Keywords: 13 C NMR; Essential oils; Computer-aided method; Expert system; Piper sp.; Terpenoid identication

1. Introduction Countless specialist systems have been developed in the last decades seeking the structural determination of novel organic substances which are not recorded in databases yet [110]. Among these systems, the SISTEMAT system [1122] was developed by our research group to assist the processes of structural determination of natural products. In this system and in others, the developed programs were proved to be very efcient to accomplish the analysis and identication for new isolated compounds. However, such systems do not allow the analysis of mixtures of substances, such as in the essential oils, without previous purication, due to the large number of signals. Fig. 1 shows
Corresponding author. Fax: +55-11-38155579. E-mail address: vdpemere@quim.iq.usp.br (V.P. Emerenciano).

an example of 13 C NMR spectra of the essential oil where it is easy to note the difculties faced by the method due to the great number of signals and overlapping of these. For such cases, we have developed a methodology specically addressed to analyze 13 C NMR data of constituents present in mixtures. This methodology was applied in the identication of a mixture of triterpenes obtained from the roots of Vernonia cognata (Asteraceae) [23]. From this mixture six triterpenes were recognized by the SISTEMAT system. However, the analysis of volatile oils was not possible due to large number of compounds found in this type of mixture and also because, at that time, SISTEMAT did not have databases of 13 C NMR data of monoterpenes and sesquiterpenes as well. The 13 C NMR spectroscopy has been recently introduced to analyze several classes of compounds

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 2 0 4 - 1

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in mixtures by the use of various specialist systems [2432]. The aim of this paper is to introduce a new methodology to the identication of chemical constituents present in essential oils through analyses of 13 C NMR data. The developed method differs from others [3133] based on signal intensity.

The sequence of information to be supplied to the SISCONST program is the following:

13 C chemical shifts and their multiplicities obtained

2. Methodology The identication of constituents present in essential oils was based on the analysis of their 13 C NMR spectra and comparison with these of monoterpenes and sesquiterpenes available in the literature. These data were encoded and stored in the SISTEMAT system [19,21]. After we have prepared the 13 C NMR databases that contain around 1300 mono- and 2500 sesquiterpenes, the SISCONST program [14] was developed, in order to analyze mixtures of these compounds. 2.1. The SISCONST program The SISCONST program [14] was developed to aid the process of structural determination of natural products by means of analysis of chemical shifts and multiplicities obtained from the 13 C NMR DEPT spectra. The program may predict the most probable skeleton type for a compound under analysis and suggest several substructures with the assigned signals of 13 C NMR. The program matchs the signals of the spectrum obtained with all spectra stored in the database. If a spectrum signal and the its respective multiplicity are present in a determined carbon atom, the signals of the interlinked carbons are matched with the spectrum in question. This searching process is repeated so that the largest fragments of the substructure bearing compatible chemical shifts with the 13 C NMR data from the spectrum are obtained. Except for the reduction process of associative groups [13], the system basically follows the Munks algorithm [2]. Earlier works using a similarity searching method are reported in the literature concerning the identication of known and new substances through infrared spectra, mass spectrometry, 1 H NMR and 13 C NMR spectroscopic databases [3446].

experimentally from the DEPT spectra; the minimum number of carbon atoms for the substructure search, i.e. this number is demanded by the user, and generally it is the half of the total carbon number of the chemical class studied; the error range admitted by the program, being generally 1.0 ; the gradient, that allows the automatic increase of the error range; specify the possible classes of compounds to be searched. The search process for a probable skeleton is limited to compounds having a substructure containing at least half of the total carbons number. So, from each chosen compound and for its skeleton, the program associates a statistical weight (Ws ) calculated by: W s = (N C TNC)2 (ER/ERM), where NC is

Fig. 2. Performance ow chart of the SISCONST program.

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the number of carbons found in the substructure, TNC the total number of carbons, ERM the absolute value of the largest difference between the substructure signals and the corresponding signal of the spectrum in question, and ER is the error range. Thus, the skeleton probability of the substance in question is computed.
Table 1 Chemical constituents of the P. cernuum volatile oil with their respective Kovats indexes and relative concentrations analyzed by GCMS KI-DB5a 924 961 966 978 1006 1013 1017 1034 1045 1074 1357 1366 1371 1374 1404 1421 1437 1444 1467 1472 1487 1489 1495 1509 1548 1551 1562 1566 1569 1576 1585 1610 1621 1626 1630 1636 1640 Chemical constituents -Pinene Sabinene -Pinene Myrcene -Terpinene p-Cymene Limonene (Z)--ocimene (E)--ocimene Terpinolene -Copaene -Bourbonene -Cubebene -Elemene -Caryophyllene Aromadendrene -Humulene Alo-aromadendrene Germacrene-D Viridiorene Bicyclogermacrene -Muurolene Germacrene-A -Cadinene (E)-nerolidol Ledol Spathulenol Caryophyllene oxide Globulol Epi-globulol Eudesmol (isomer not identied) 1-10-Di-epi-cubenol Iso-spathulenol -Cadinol + -muurolol Cubenol Not identied -Cadinol Total Non-identied Identied
a

Concentration (%) 7.16 0.27 6.15 0.50 0.97 0.61 0.52 0.14 1.88 0.68 1.48 0.20 0.45 3.02 20.69 0.36 1.74 0.23 6.68 0.62 21.88 0.31 4.15 1.54 1.28 0.67 2.26 0.74 3.08 2.21 0.82 0.20 0.26 1.72 0.60 0.49 2.84 99.40 0.49 98.91

For this search process an error range of the 13 C NMR chemical shift is xed at 1.0 for the substructure and 5.0 for skeleton. If this error range is too narrow and does not allow a successful matching, the ranges are automatically increased with a gradient of 0.5 and 1.0 for substructures and skeletons, respectively. If the error range of the search reaches the upper limit of 3.0 for substructures and 10.0 for skeletons, the program automatically will stop the search, and no substructures and/or skeletons will be presented. Since the system is supposed to create constraints in structural determination, the assignments of the carbon belonging to the substructures are important data as well as their respective chemical shifts. Due to this fact, we introduced the Kalchausers algorithm [47] which we modied slightly, because the only data

Table 2 Chemical constituents of the P. regnellii essential oil analyzed by GCMS KI-DB5a 917 924 962 966 984 996 1007 1014 1018 1045 1088 1161 1174 1358 1402 1422 1437 1477 1483 1509 1542 1548 1562 1626 1630 Chemical constituents Tricyclene -Pinene Sabinene -Pinene Myrcene -Phellandrene D3-carene p-Cymene Limonene+-phellandrene -Terpinene Linalool Terpinen-4-ol -Terpineol -Copaene -Caryophyllene Aromadendrene -Humulene Germacrene-D Bicyclogermacrene -Cadinene Germacrene-B (E)-nerolidol Spathulenol -Muurolol -Muurolol Total Non-identied Identied
a

Concentration (%) 0.30 0.50 1.30 0.20 52.6 0.20 0.20 1.50 4.10 0.20 15.9 0.70 1.20 0.30 8.50 0.40 0.40 0.30 2.90 0.50 0.50 4.20 0.90 0.30 0.70 100.0 1.30 98.70

Kovats index on DB-5 column [54].

Kovats index on DB-5 column [55].

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134 Table 3 13 C NMR DEPT spectral data obtained for crude essential oil from leaves of P. cernuum s 154.9 153.7 152.4 151.1 149.1 147.9 144.7 142.6 139.6 135.3 135.1 134.1 133.2 131.5 127.7 126.5 116.4 81.1 75.4 73.6 72.5 54.2 40.8 36.5 36.4 28.1 26.0 24.9 20.5 19.4 d 150.5 145.4 140.3 135.8 133.7 132.0 129.2 128.2 126.7 126.1 125.2 124.8 124.6 123.0 121.2 119.8 116.7 116.3 77.5 58.5 57.3 54.6 54.5 53.9 53.7 53.2 53.0 52.1 50.3 49.6 48.7 47.3 47.1 45.7 44.6 41.4 41.0 40.7 40.1 39.7 38.7 37.2 36.6 34.8 33.3 32.5 31.6 30.2 28.6 27.8 27.2 27.0 26.2 22.6 t 115.9 113.2 112.3 111.9 111.1 110.1 109.3 108.5 107.6 106.2 44.9 43.7 42.5 42.3 42.0 41.0 40.6 40.5 40.0 39.8 38.2 37.5 36.5 34.9 34.8 33.2 32.6 31.7 31.5 31.2 31.1 30.7 30.0 29.6 29.5 29.2 28.2 27.2 27.1 27.0 26.8 26.4 26.3 25.1 23.8 23.2 22.9 22.2 21.5 20.4 q 32.4 30.3 30.1 29.5 28.9 28.1 27.2 26.6 26.4 25.0 24.1 23.8 23.2 22.9 22.1 21.8 21.5 21.4

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21.1 21.0 21.0 20.5 20.0 19.2 18.2 16.9 16.8 16.5 16.3 16.1 15.9 15.7 15.4

which are necessary are the signal values and their multiplicities. Therefore, from this structure search process, the chemical shift values are attributed to the respective carbons during the similarity searching process. Therefore, the reliability of each assignment of the experimental spectrum is achieved by the program with the stored data obtained from the literature. However, so far, this program had only been previously tested for the individual identication, i.e. previously separated and puried compounds. In order to allow the analysis of mixtures, we have increased the data storage capacity of the program to a largest number of chemical shifts and multiplicities into the system during the analysis. The analyses with a minimum of nine coincident signals could be successfully carried out in the analysis of mono- and sesquiterpenes, once this value embodies structures of both compounds. The SISCONST program is able to recognize structures for which the 13 C NMR data are present in the database and structures whose 13 C NMR data are absent in the database. In the last case, the program searches for substructures, parts of a structure compatible with the experimental data that, in many cases, when overlapped, they furnish the complete structure. Various examples of new substances which were tested by the program are shown with excellent results [11,14,16,19,22]. The self-training is the main charac-

theristic of this program, since it identies known and new substances present in the sample. Fig. 2 shows the performance ow chart of the SISCONST program. 3. Results The identication capacity of the SISCONST program was evaluated in the analysis of terpene mixtures
Table 4 13 C NMR DEPT spectral data obtained for crude essential oil from leaves of P. regnellii s 154.7 146.1 134.2 131.9 129.6 129.0 126.3 110.0 108.4 73.5 40.0 19.7 d 146.2 145.1 139.0 131.8 126.3 124.4 124.3 124.2 53.6 48.5 40.4 30.1 26.8 t 115.7 113.0 111.7 42.1 40.0 39.7 34.8 31.5 31.4 29.4 28.4 27.9 26.0 24.1 22.6 q 30.2 27.5 26.8 26.7 25.7 22.8 20.8 19.5 17.7 16.3 16.1

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Fig. 3. Structures of compounds identied by the SISCONST program.

in volatile oil samples obtained from leaves of Piper cernuum and Piper regnellii (Piperaceae family). The system efciency was based on the comparison of results obtained in the identication of compounds by analysis of 13 C NMR spectra with those obtained by GCMS (gas cromatographymass spectrometry) and by GCKI (gas chromatographyKovats indexes). The data obtained through GCMS and GCKI for P. cer-

nuum and P. regnellii are listed in Tables 1 and 2, respectively. Both samples were also submitted to the aquisition of 13 C NMR data in which 167 chemical shifts and their respective multiplicities were obtained by 45, 90 and 135 DEPT for P. cernuum (Table 3) and 49 chemical shifts for P. regnellii (Table 4). All chemical shifts obtained from the list of the peaks in the 13 C NMR

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132 Table 6 Experimental

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13 C

NMR data from crude essential oil of P. regnellii assigned by SISCONST program Myrcene Lit. [19] 25.2 130.3 124.5 22.6 41.2 72.6 145.0 111.3 17.5 27.2 Exp. 26.8 126.3 131.8 27.9 31.5 146.1 139.0 115.7 16.3 113.0 Lit. [19] 27.7 124.4 131.5 27.0 31.7 146.3 139.1 115.5 17.7 112.9 -Caryophyllene Exp. 53.6 40.0 29.4 48.5 154.7 40.0 29.4 124.3 134.2 34.8 28.4 30.2 22.8 111.7 16.1 Lit. [50] 53.5 40.0 30.1 48.2 154.3 40.4 29.3 124.5 135.0 34.7 28.3 30.1 22.6 111.8 16.2 Bicyclogermacrene Exp. 124.2 26.0 42.1 129.0 126.3 26.8 30.1 26.0 39.7 19.7 30.2 16.1 16.3 20.8 Lit. [50] 124.8 26.0 41.0 128.0 126.5 26.8 30.0 26.7 37.2 140.6 19.7 29.2 15.3 16.5 20.7 Nerolidol Exp. 25.7 131.9 124.2 24.1 39.7 134.2 124.4 24.1 42.1 73.5 145.1 111.7 17.7 16.1 25.7 Lit. [57] 25.1 130.5 124.2 24.3 39.2 134.3 124.7 24.2 42.0 72.2 145.3 110.8 17.2 15.4 25.0

Linalool Exp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 25.7 129.6 124.4 22.6 42.1 73.5 145.1 111.7 17.7 27.5

spectra were entered in the program for analysis. The additional parameters of the obtained spectra are described in the Section 4.1. The data of P. cernuum were loaded into the SISCONST program which, at the rst stage of run, identied the sesquiterpene germacrene-D. The assignment of its 13 C NMR signal was in full agreement with the data previously published in [48]. Then, in the second step, the program continued the analysis and identied additionally three sesquiterpenes: -caryophyllene, germacrene-A and bicyclogermacrene. Thus, successively, the program identied the following compounds: -pinene, spathulenol, -elemene, -pinene and globulol (Fig. 3). The experimental 13 C NMR data for identied compounds was compared to those available in the literature, and they are listed in Table 5. The analysis of the components in the essential oil of P. regnellii was also carried out (Table 6) and the SISCONST program was able to identify sequentially linalool, myrcene, nerolidol, -caryophyllene and bicyclogermacrene. The reliability in each assignment of the compound was obtained in both samples by GCMS, GCKI and 13 C NMR data previously published in [4853]. In summary, the SISCONST program was able to identify two monoterpenes (- and -pinene) and eight sesquiterpenes (germacrene-D, germacrene-A,

-elemene, -caryophyllene, spathulenol, globulol, bicyclogermacrene and -cadinol) in the essential oil from P. cernuum. In P. regnelliis essential oil, two monoterpenes (myrcene and linalool) and three sesquiterpenes (-caryophyllene, nerolidol and bicyclogermacrene) were identied.

4. Discussion and conclusions The SISCONST program was capable to identifying efciently all the constituents present in the volatile oils at the same or superior concentration of 2.26%. The non-identication of minor compounds (<2.26%) is caused by interrelated factors: the small concentration of the constituent in our sample of the volatile oil which did not allow the achievement of the chemical shifts of the compound. This limitation is inherent to the own analysis type. However, if a larger amount of oil were obtained, starting, for example, from a larger biomass, and we had haven more time of accumulation to run the spectrum, we probably could have identied, through 13 C NMR, the other constituents present in smaller amounts. In summary, the method here described allows the correct identication of major compounds present in volatile oils. The method can be suggested as a complementary tool for analysis of essential oils based on

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GCMS data. One of the advantage of this method is the correct identication of isomers of a certain compound present in the sample or to detect the presence of new compounds in essential oils. 4.1. Experimental Plant material and extraction: leaves of Piper cernuum Vell. and Piper regnellii (Miq.) C.DC. were collected at the Campus of So Paulo University in So Paulo city, Brazil, in June 2000. Voucher specimens (Kato-0137 and E. Guimares-1961, respectively) were deposited at Herbarium of Instituto de Botnica, So Paulo, Brazil. The essential oils were obtained from steam distillation of 570 and 400 g of fresh leaves of P. cernuum and P. regnellii, respectively, in agreement with the literature [54], yielding 102 mg of the crude oil for P. cernuum and 400 mg for P. regnellii. 4.1.1. 13 C NMR analysis The 13 C NMR spectra were obtained through a Varian 300 MHz, using CDCl3 as solvent and TMS as internal standard. The 13 C NMR spectra were measured using 45 pulse (13.4 ms) and 3600 pulses; repetition time, 2 s; threshold, 15; signal to noise, 80:1; spectral width, 18.1 kHz; data set, 65.5 kilo-words zero-lled; temperature, 25 C. 4.1.2. GC and GCMS analysis Gas chromatography analysis was performed in a chromatograph (Shimadzu GC-17A) equipped with a Shimadzu GC 10 software, using a fused silica capillary column, DB-5 (30 m 0.25 mm 0.25 m), and a ame ionization detector. Injector and detector temperatures were set at 220 C. The oven temperature was programmed from 60 to 300 C at 3 C/min and helium was employed as carrier gas (1 ml/min) for both analyses. The percentage compositions were obtained from electronic integration measurements, using ame ionization detection without taking into account relative response factors. Gas chromatographymass spectrometry: the sample was analyzed by GCMS, using a Shimadzu capillary GC-quadrupole MS system (QP 5000) operating at 70 eV at the same conditions as described above. The identication of the compounds was performed by comparison of retention indexes (determined relatively to the retention times of a

series of n-alkanes) and mass spectra with that available in the system [55,56].

Acknowledgements This work was supported by grants from the Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Cient co e Tecnolgico (CNPq) and by the Fundao Coordenao de Aperfeioamento de Pessoal de N vel Superior (CAPES). The authors thank Antnio J.C. Brant for helpful discussion during the preparation of the manuscript. References
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