VARIABILITY in the CHEMICAL COMPOSITION of WILD Rosmarinus Officinalis L

VARIABILITY IN THE CHEMICAL COMPOSITION OF WILD Rosmarinus officinalis L.

Varela, F.1; Navarrete, P.1, Cristobal, R.2 Fanlo, M. 2; Melero, R.2; Sotomayor, J.A.3; Jordán, M.J. 3; Cabot, P.4; Sánchez de Ron, D.5; Calvo, R.6; Cases, A.1

1 MA-INIA. Apdo. 8111. 28040 Madrid 2 CTFC. Pujada al Seminari s/n. 25280 Solsona (Lleida) 3 IMIDA. C/ Mayor s/n. 30150 La Alberca (Murcia) 4 IRTA. Ctra. de Cabrils, s/n. 08348 Cabrils (Barcelona) 5 INIA-CIFOR. Apdo. 8111. 28040 Madrid 6 INIA-Servicio de Biometría. Apdo. 8111. 28040 Madrid

Introduction Rosemary (Rosmarinus officinalis L.) is a spontaneous shrub growing in all Mediterranean countries. Different species of rosemary exist all over the world: R. officinalis, R. erriocaly, R. laxiflorus and R. lavandulaceus. R. officinalis is the only one that grows naturally in the Mediterranean region. Manuta et al. reported that the differences between plants and oils composition were correlated to differences in the substrate. Elamrani et al. studied the chemotaxonomy of Moroccan rosemary essential oils, finding three chemotypes: α-pinene, camphor and 1,8-cineole. Moreover, rosemary collected in Portugal is rich in myrcene (25%) 1.8-cineole, and camphor, and is similar to Argentinean and Brazilian oils.

Materials and method Collection methodology. Representative samples of flowering branches of Rosmarinus officinalis L plants were collected from wild flora of 87 populations from different regions of Spain: Andalucia, Baleares, Castilla La-Mancha, Cataluña, Madrid and Murcia. Essential oils. The essential oils were obtained by hydridistillation of the dried ground material in a Clevenger-like apparatus for 2h at atmospheric pressure on about 100g of sample. Time was measured from the falling of the first drop of distillate. For each sample two replications of each extraction were done. The essential oil yield was evaluated gravimetrically. Gas chromatography: The GC analysis was carried out with a Hewlett Packard 6890 Series instrument equipped with an FID and HP-5 capillary columns (30m x 0.25mm, 0.25μm film thickness), working with the following temperature program: 70ºC for ten minutes and then ramp of 3ºC/min to 220ºC; injector and detector temperature, 250ºC; carrier gas nitrogen was adjusted to a flow of 2ml/min. The samples were injected using the split mode (split ratio 1:30) and an injection volume of 0.2μL. Identification of the components was performed by comparison of their retention times with those of pure authentic samples and by means of their linear retention indices (LRI) relative to the series of n-hydrocarbons.

Results The 87 samples collected were grouped into four eco-regions (Fig. 1), following the biogeoclimatic classification of Peninsular Spain and the Balearic Islands used by Elena (1996). This divides the whole territory into seven eco-regions by grouping territories as defined by their geographic, physiographical, climatic and lithological characteristics. It should be pointed out that some transition areas with unclear ecological limits may be assigned to more than one eco-region. In this work, we decided that the grouping of samples should be concentrated amongst the smallest possible number of eco-regions because of the limited number of populations sampled in such a large territory in which there are extensive areas from which no material was collected.

Figure 1. Eco-regions of Peninsular Spain and the Balearic Islands The following are the results of the distillation of the samples collected and of the chromatographic analysis of their essential oil.

Table 1. Essential oil yield

% Essential oil yield sms No. A No. B No. C No. D 441A 1.94 312A 2.48 310A 2.35 101 1.90 630B 1.83 313A 1.92 321 1.48 102 1.70 728A 1.95 313B 1.71 401 1.53 104 1.64 728B 1.83 313C 2.00 427A 1.97 104A 1.74 731A 1.63 314 2.76 428 2.11 105 1.94 731B 1.81 316 2.49 432A 1.14 105A 1.70 807A 2.02 317 1.92 433 1.64 106 1.56 824A 1.70 318 2.28 435 2.16 112 2.10 824B 1.04 318A 1.74 436 1.50 113 2.03 824C 1.63 318B 2.28 442 1.98 114 2.02 824D 1.78 319 2.52 501 1.54 116 1.30 825 1.98 320 2.08 501A 2.18 120 1.91 826 1.66 320A 1.61 502 2.12 123 1.32 832A 1.88 322A 1.89 503 2.30 124 2.29 838 1.74 322B 1.87 506 2.24 125 1.76 323 1.73 507 0.82 130 1.17 326 2.03 508 1.38 136 2.02 328 1.92 509 1.45 140A 2.10 428A 1.70 510 2.07 412 1.58 429 2.23 511 2.21 728C 1.82 516 2.02 731C 2.33 517 2.25 731D 1.65 522 2.26 1001A 2.83 524 1.85 1001B 2.99 630A 1.95 630C 1.87 630D 1.97 630G 2.25 M 1.76 2.11 1.88 1.78 MAX 2.02 2.99 2.35 2.29 MIN 1.04 1.61 0.82 1.17

In A, with an average essential oil yield of 1.76%, only the population 807A from Biosca (LE) exceeded 2%, with an essential oil concentration of 2.02%. The populations from B are the richest in essential oil, with an average RT of 2.11%. Within this group there are two populations from Formentera (Balearic Islands) with very high essential oil RT of 2.99% in sample 1001B and 2.83% in sample 1001A. In this group, 50% of the populations analysed had a content of more than 2% of essential oil. In C, with an average of 1.88%, 46% of the populations also exceeded 2%, with a maximum of 2.35% in sample 310A from Maria (AL). In D, with an average yield of 1.78%, only 6 populations exceeded 2% and the maximum of 2.29% was in sample 124 from Ardales (MA).

In the chromatographic analysis of the essential oils, 54 components were identified as having a concentration in excess of 0.1%. Of these, the following were chosen for this work: α-pinene, 1-8 cineole, camphor and verbenone, these being the most characteristic components of this specie.

The essential oil composition amongst the group A, Table 2, samples was seen to be fairly uniform, with not very large differences between the maximum and minimum concentrations. However, samples 731A from Mas de Barberans (TA) and 824B from Naves (LE) had a high content of 1.8-cineole, 28.44% and 26.36% respectively. Sample 824C from Oden (LE) was rich in camphor with 38.42%. With regard to verbenone, sample 838 from Algerri (LE) had 4.93%, much more than the other samples, and was followed by 728A from La Pobla de Benifassa (CS) with 3.29%. The other samples had over 1.0%, and six of them had over 2.0%.

Table 2. Essential oil components group A

α-pinene 1,8-cineol camphor verbenone 441A 11.39 21.74 29.58 1.36 630B 12.49 27.96 27.18 1.60 728A 15.88 20.46 23.80 3.29 728B 11.62 20.12 29.89 2.99 731A 10.92 28.44 22.60 0.01 731B 12.93 25.64 23.18 2.75 807A 12.12 20.84 26.38 2.77 824A 14.75 20.16 28.18 1.94 824B 12.86 26.36 27.30 1.75 824C 8.98 20.22 38.42 2.40 824D 14.83 19.91 31.21 2.37 825 14.91 19.18 32.03 1.82 826 17.41 20.14 22.10 2.02 832A 12.49 19.05 31.10 1.86 838 16.75 23.55 17.38 4.93 M 13.16 22.40 27.36 2.26 MAX 17.41 28.44 38.42 4.93 MIN 8.98 19.18 17.38 0.01

Table 3. Essential oil components group B

α-pinene 1,8-cineol camphor verbenone 312A 17,68 34,59 13,40 2,48 313A 16,83 22,42 16,72 3,65 313B 21,75 26,36 16,61 0,01 313C 20,07 27,42 17,57 2,00 314 22,18 21,69 15,76 1,40 316 18,66 31,19 19,26 3,18 317 16,81 34,99 16,81 2,20 318 19,80 30,18 13,10 3,36 318A 27,54 33,09 12,31 0,01 318B 32,56 37,03 9,09 0,80 319 22,22 27,84 15,61 2,94 320 17,08 27,12 20,91 2,73 320A 23,14 25,14 14,22 3,53 322A 20,03 22,99 21,01 1,34 322B 19,66 30,24 17,52 2,37 323 16,60 28,47 18,45 2,98 326 14,30 19,04 31,84 1,06 328 17,81 29,81 14,00 3,36 428A 16,15 28,04 17,88 3,20 429 21,07 29,03 13,29 2,11 728C 13,05 20,94 31,35 2,28 731C 13,45 21,73 24,70 2,36 731D 12,39 24,34 20,75 3,65 1001A 16,53 28,85 19,58 0,01 1001B 14,39 28,24 23,46 4,59 M 18.87 27.63 18.21 2.30 MAX 32.56 37.03 31.84 4.59 MIN 12.39 19.04 9.09 0.01 The group B samples, showed a high degree of variability in their composition, with the concentrations of the four main components varying over very large intervals as shown in Table 3. Camphor is the component with the largest variability, between a maximum value of 31.84% in sample 326 from Santomera (MU), and a minimum of 9.09% in sample 318B from Cartagena (MU), which had the highest value for α-pinene of 32.56% and 1.8-cineole of 37.03%. With regard to verbenone content we find the maximum value of 4.59% in the sample 1001B Formentera (IB). Of the other samples from B, seven had in excess of 3%, ten in excess of 2% and seven under 2%.

In this group C, Table 4, the concentrations also varied greatly and the composition was not uniform amongst the different samples. There was nothing of note, except for sample 501 from Villar del Olmo (MD) with 34.23% of camphor. Regarding verbenone content, three samples had in excess of 2%, fifteen more than 1% and ten less than 1%.

Table 4. Essential oil components group C

α-pinene 1,8-cineol camphor verbenone 310A 18.13 26.58 17.50 1.44 321 18.53 22.98 20.01 2.41 401 13.41 26.60 21.94 1.37 427A 13.46 19.03 20.95 0.16 428 17.09 22.93 22.20 1.47 432A 9.39 20.00 23.86 2.25 433 11.65 24.68 23.46 1.73 435 13.17 27.46 23.91 1.80 436 15.05 23.57 22.00 1.81 442 11.55 26.51 27.18 1.50 501 7.74 21.42 34.23 1.22 501A 11.53 29.45 24.52 1.36 502 11.51 26.76 24.37 1.03 503 11.04 28.39 26.72 0.97 506 12.35 16.76 19.97 1.03 507 9.66 15.64 18.46 0.71 508 8.93 20.44 21.15 0.92 509 11.80 19.13 20.33 0.60 510 13.70 20.29 20.81 0.69 511 7.10 15.00 26.46 0.69 516 11.58 17.99 15.65 0.89 517 10.75 17.79 23.82 0.94 522 12.21 23.75 29.73 2.17 524 11.50 25.05 28.75 0.89 630A 12.47 28.03 23.04 1.38 630C 14.19 22.96 27.44 1.43 630D 11.60 27.30 22.52 1.17 630G 13.48 22.12 29.31 1.57 M 12.31 22.81 23.58 1.27 MAX 18.53 29.45 34.23 2.41 MIN 7.10 15.00 15.65 0.16

In the composition of the group D samples, Table 5, again there was a high degree of variability, the largest interval being in camphor with a maximum of 35.19% in sample 113 from Bornos (CA), which also had the minimum concentration of α-pinene with 5.34%, and a minimum of camphor with 6.22% in sample 130 from Competa (MA). The maximum verbenone content was found in samples 120 of 5.49% from Ojen (MA), 104A of 5.37% from Fuente Obejuna (CO) and 136 of 4.41% from Motril (GR). Another two samples in this group had more than 3% of verbenone, eight had more than 1% and the remainder had less than 1%. Table 5. Essential oil components group D

α-pinene 1,8-cineol camphor verbenone 101 12,35 20,83 21,30 1,04 102 12,39 22,91 23,39 1,74 104 11,18 19,86 21,56 0,97 104A 18,63 20,77 12,60 5,37 105 10,00 20,01 18,89 0,85 105A 8,30 18,11 23,40 0,67 106 10,02 17,17 22,35 0,45 112 10,23 28,64 25,46 1,00 113 5,34 24,91 35,19 1,59 114 10,20 23,78 17,45 1,82 116 9,37 24,08 21,55 1,06 120 19,03 21,23 16,97 5,49 123 12,92 21,88 21,45 2,47 124 19,08 23,97 11,36 3,60 125 17,00 26,04 11,57 3,97 130 29,94 12,23 6,22 0,01 136 24,84 22,86 14,27 4,41 140A 16,74 19,97 26,65 1,64 412 16,30 19,97 14,87 0,38 M 14,41 21,54 19,29 2,03 MAX 29,94 28,64 35,19 5,49 MIN 5,34 12,23 6,22 0,01

Table 6 shows and compares the average values for the majority components in the four collection areas:  The samples from B have the highest essential oil content and are the richest in α-pinene, 1.8- cineole and verbenone.  The samples from A are the richest in camphor and have high verbenone content.  In line with Elamrani et al., the camphor chemotype was found in A whereas in B we found the α- pinene and 1.8-cineole chemotypes. Like this author, we found in C the 1.8-cineole and camphor chemotypes and in D the predominant chemotype was 1.8-cineole.  The populations of Rosmarinus officinalis collected in this study tally with the chemical characteristics of Moroccan rosemary.

Table 6. Average values

A B C D Essential oil yield 1.76 2.11 1.88 1.78 α-pinene 13.16 18.87 12.31 14.41 1,8-cineol 22.40 27.63 22.81 21.54 camphor 27.36 18.21 23.58 19.29 verbenone 2.26 2.30 1.27 2.03

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