Non-Destructive Raman Analyses – Polyacetylenes in Plants

Spectrochimica Acta Part A 61 (2005) 1395–1401

Non-destructive Raman analyses – polyacetylenes in plants

Bernhard Schrader a, ∗, Hartwig Schulz b, Malgorzata Baranska b, c, George N. Andreev d, Caroline Lehner e, Juergen Sawatzki e

a Institut f¨ur Physikalische und Theoretische Chemie, Universit¨at Duisburg-Essen, Soniusweg 20, D-45259 Essen, Germany b Federal Centre for Breeding Research on Cultivated Plants, Institute for Plant Analysis, Neuer Weg 22-23, D-06484 Quedlinburg, Germany c Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland d Department of Chemistry, University of Plovdiv, Bulgaria e Bruker Optik GmbH, Rudolf-Planck-Strasse 27, D-76275 Ettlingen, Germany Received 22 September 2004; received in revised form 15 October 2004; accepted 15 October 2004

Dedicated to Professor James Durig thanking for more than 30 years of friendship, personal and scientiﬁc contacts.

Abstract

Ferdinand Bohlmann has described the isolation, the identification and the structure elucidation of acetylene compounds in many plants, and confirmed it by its synthesis. We have recorded the Raman spectra of most of these plants non-destructively by FT–Raman spectroscopy using radiation at 1064 nm. We could not observe any interfering fluorescence. We found acetylene compounds in some plants, even distinct compounds with different concentration in various parts of it. The distribution of the different compounds over the plant can be observed and their changes during the ontogenesis can be followed by a FT–Raman mapping technique. Of special help is a library of Raman and IR spectra and the structure of the compounds, synthesized by Bohlmann. Thus, the Raman technique allows analyses in a very short time replacing the usual time-consuming separation procedures and avoiding artefacts during clean-up procedures. © 2004 Elsevier B.V. All rights reserved.

Keywords: Non-destructive analyses by NIR–FT–Raman spectroscopy; Distribution of natural compounds in plants; Library of IR and Raman spectra of natural compounds of plants; Application in breeding; Cultivation and quality control

1. Introduction of various terpenes in plants had been recorded already [3,4]. We were especially surprised to see strong Raman bands at By exploring the most promising applications of about 2230 cm−1 in the Raman spectra of several plants. Of FT–Raman spectroscopy with excitation by a Nd:YAG course, these bands give a proof for acetylenic bonds. laser at 1064 nm we found that this technique allows non- Raman spectra of acetylene and di-acetylene have been destructive analyses of works of art, of animal and plant tis- published already in 1935 [5,6]. Acetylene shows a Raman sues [1,2] perfectly. Particularly, the disturbing ﬂuorescence band at 1973 cm−1, diacetylene (having a center of sym- of the enzymes and coenzymes of all cells, especially the metry) produces a Raman band at 2183 and an IR band at −1 photosynthetic machinery in plants, produced mostly by all 2085 cm of the (C≡C)2-in-phase- and out-of-phase vibra- other Raman techniques using excitation with visible light tions, respectively. The ﬁrst Raman analysis of an acetylene and even at 785 and 830 nm, is avoided. The Raman spectra compound in a plant, the carlinaoxide, has been published already in 1935 [7]. Unsubstituted polyacetylenes are extremely unsta- ∗ Corresponding author. Tel.: +49 201 460638; fax: +49 201 466650. ble and are known to explode violently. Nevertheless, E-mail addresses: [email protected] (B. Schrader), Else Kloster–Jensen succeeded in preparing triacetylene, [email protected] (H. Schulz), [email protected] (M. Baranska), tetraacetylene and even pentaacetylene and in recording their [email protected] (G.N. Andreev), [email protected] (C. Lehner). Infrared, UV and NMR spectra [8]. Their UV spectra and

Fig. 1. FT–Raman spectrum of below: the bottom of the flower heads of Erigeron neclectus; above: compound I [16] lachnophyllum lactone. The drawing copied into the figure is taken from [23]. their photoelectron spectra were discussed in detail [9]. The 1064 nm and a Germanium detector, cooled with liquid ni- Raman spectrum of triacetylene allowed the calculation of its trogen. Most spectra have been recorded using a sample ar- force field [10]. rangement shown in Fig. 1candfof[1] with a resolution of Baranovic and coworkers discussed the spectra of 4cm−1 using a laser power of 150 mW supplied by an unfo- oligo(phenyldiacetylenes) [11]. Furthermore, Raman and in- cused laser beam and with a recording time of 15–30 min. frared spectra of various acetylenes have been discussed in Two-dimensional mappings were performed using an xy [12] and are published in the Raman/IR Atlas [13]. stage, a mirror objective, a prism slide and a suitable software We were especially attracted by the work of Ferdinand to control the xy stage and the three-dimensional data process- Bohlmann. He has found polyacetylenes in many plants, es- ing. Raman mapping of the inflorescence of Bidens ferulifo- pecially in species of Umbelliferae and Compositae fam- lia was performed from above over an area of 9 mm × 9mm ilies already since about 1955. Bohlmann has been by a with a spatial resolution of 250 ␮m and from the side over an wide margin the most productive of all chemists dealing area of 7.2 mm × 10 mm with a spatial resolution of 200 ␮m with natural substances in higher plants. Winterfeldt re- (Fig. 8). The sample was irradiated with a focused laser beam viewed Bohlmann’s life’s work and gives the list of his 1453 of 100 mW with a diameter of about 0.1 mm. With a spec- publications [14], published during the years 1948–1992. tral resolution of 4 cm−1, eight or six scans respectively were Bohlmann’s early work concerning acetylenes in plants un- collected at each measured point. til 1963 have been summed up by Bohlmann and Sucrow in the paper: ‘Naturlich¨ vorkommende Acetylenverbindungen’ [15]. We sent a list of 57 botanical names given in this paper 3. Results and discussion to several Botanical Gardens in Germany, asking for samples of living plants with this name. We investigated the Raman The concentration of polyacetylenes in plants is in the or- spectra of many of them, especially of the flowers, leaves and der of 0.01–1%. Bohlmann studied the natural compounds in roots separately and found weak or strong bands in the range −1 plants usually by chopping the whole plants, extracting them of about 2000 cm in several samples. The most important with a petrolether/diethylether mixture and sending the solu- of these measurement results are described in the present pa- tion via a small chromatographic column through a sample per. cell of an UV spectrometer. Polyacetylenes are shown up by strong bands, due to the system of triple and double bonds in the molecule [14]. This allowed him to optimise the extrac- 2. Experimental tion procedure and to prepare pure products. Bohlmann eluci- dated their structure by interpreting these bands and combin- The Raman spectra presented in this paper were ing this with the results given by the other instrumental tech- mainly recorded using an instrument equivalent to the niques of that time. Usually he confirmed it by preparation of NIR–FT–Raman Spectrometer BRUKER RFS 100 with a the corresponding synthetic substances. Polyacetylenes are diode-pumped Nd:YAG laser, emitting maximal 350 mW at very sensitive, especially against heat and light, they some- B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401 1397 times explode during distillation or by determination of the diacetylene groups have a very strong band at 2230 cm−1, melting point. They are best stored in the dark and under especially if the adjoining group(s) of the triple bond(s) are nitrogen. aliphatic. Some of the molecules show a weak satellite band The function of polyacetylenes in plants is not yet clear. at about 2190 cm−1, which may be due to the 13C isotopic Some of them are very poisonous, and phototoxic. Poly- substitution of the triple bonds [17]. Satellite bands at about acetylenes in fungi may show antibiotic properties [15]. 2290 cm−1 were attributed to Fermi resonance [12]. Only in In the Bohlmann/Sucrow paper [15] the compounds are the group of the six triine-molecules, the strongest bands are ordered by the structural features, especially the number of at lower frequencies: 2190–2118 cm−1. It is interesting that C≡C and C C bonds and their substituents. The formulae also the compounds with octadehydrodibenzo[12]annulene are arranged together with the names of the compounds, the and dodekadehydrotribenzo[18]annulene [12] showed bands names of the plants from which they were extracted and the at 2191 and 2198 cm−1, respectively. citation of the publication, describing the isolation, the anal- The Raman spectrum of the unsubstituted triacetylene yses and its syntheses. showed two very strong bands, at 2212 and 2019 cm−1, due We investigated many plants described in the publication to the in-phase stretching vibration of all three triple bonds [15] by FT–Raman spectroscopy and found the bands of poly- and the out-of-phase vibration of the same bonds [10]. acetylenes in some of them. We noticed that they are not uni- In the bottom of the flower of Erigeron neclectus we found formly distributed over the whole plant, but concentrated on a strong band at 2198 cm−1 (Fig. 1). According to Bohlmann specific parts of the plant—blossoms, leaves, roots, or seeds. it can be attributed to the lachnophyllumlactone, I in [15]. We even found distinctive compounds in different parts of We could not find a spectrum of exactly this compound in the same plant. the library, however only one with a further double bond in Fortunately, a library of the infrared and Raman spectra the side chain. This explains its lower frequency (2178 cm−1) obtained from 1505 samples of the Bohlmann collection of compared to that in the plant (2198 cm−1). natural compounds, which have been stable enough to survive Coreopsis grandiflora (Fig. 2) shows in the roots a band at the storage, is now available [16]. These spectra are combined 2194 cm−1. According to Bohlmann [14] the isolated com- with the molecular structures, given by the individual mol- pound V is a monoacetylene with a substitution by a thio- files. They include spectra of 43 mono-, di- and triacetylenes, phene ring. We did not find this compound in [16],how- further of alkaloids, coumarins and many other rarely known ever an example with one thiophene ring substituted by secondary metabolites. monoacetylenes has a similar spectrum. Of the 43 Raman spectra with acetylene groups from the Measurements of the flowers of the ox-eye daisy library [16] we related the observed bands in the region about (Chrysanthemum leucanthemum) show the FT–Raman spec- − 2200 cm 1 to the molecular structure. In the library, there are trum presented in Fig. 3; it can be seen there that the isolated 14 molecules with one C≡C bond, 23 with (C≡C)2 and 6 with trans-dehydromatricariaester (LII in [15]), of which the spec- (C≡C)3 groups. All acetylene groups are disubstituted. It is trum is shown above exactly coincides with the spectrum of surprising that most spectra of the molecules with mono- and this flower.

Fig. 2. FT–Raman spectrum of below: the roots of Coreopsis grandiﬂora; above: reference spectrum of a thiophene substituted monoacetylene (compound V [16]). 1398 B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401

Fig. 3. FT–Raman spectrum of below: the blossoms of Chrysanthemum leucanthemum; above: compound LII [16] trans dehydromatricariaester. The drawing copied into the ﬁgure is taken from [23].

The blossoms of cornflowers (Centaurea cyanus) show bitter taste of carrots [18]. Although these substances were the spectrum of Fig. 4 which is in good accordance with already identified in various Apiaceae species before, their the spectrum of LIV in [15]. The intensity of both ν(C≡C) sensoric properties were yet not known [19]. Fig. 6 shows a bands are reverse which may point out that another acetylene spectrum of a substance of the library, which shows a simi- compound or other isomer is also present in the blossoms. lar spectrum as the natural diacetylenes occurring in carrots. The flower heads of Centaurea ruthenica and the leaves of The intensity of the bands, due to the carotene at 1520 and Carthamus lanatus show polyacetylene bands of which the 1156 cm−1 are the strongest in the spectrum. position (2166.7 and 481.4) is nearly identical with that of Fig. 7 shows the spectrum of the leaf and the flower of LXII in [15] (Fig. 5). apache beggarticks (Bidens ferulifolia, family Compositae). When exposed to abiotic stress during harvesting, trans- This species, native to Guatemala and Mexico, is exclusively portation, storage and processing, carrots are able to produce used as horticultural plant for hanging baskets and window a bitter off-flavour. Recently, it has been found, that several di- boxes. Other species of the genus Bidens are widely used in acetylenes such as falcarinol and falcarindiol contribute to the Chinese medicine, such as Bidens pilosa and Bidens campy-

Fig. 4. FT–Raman spectrum of below: the heads of Centaurea cyanus (cornﬂower); above: compound LIV [16]. The drawing copied into the ﬁgure is taken from [23]. B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401 1399

Fig. 5. FT–Raman spectrum of line a: bottom of the flowers of Centaurea ruthenica; line b: leaves of Carthamus lanatus; above: compound LXII [16]. The drawing copied into the figure is taken from [23]. lotheca. Previous examinations of these species resulted in necessary to allow a more precise interpretation of these spec- the identification of several lipophilic polyacetylenes and tra. polyacetylene glucosides [20,21] which seem to be mainly The Raman map of the inflorescence of B. ferulifolia gives responsible for the described medicinal properties. Chang et another unexpected information: The upper row in Fig. 8 al. [22] described one of the acetylene compounds, occurring shows Raman maps and the picture from above—the left map in B. pilosa, as a phenyl substituted triine. We found that the shows the distribution of carotenes, which occur obviously in reference spectrum of this substance is similar to that ob- the petals, whereas the polyacetylenes (middle maps) occur tained from the flowers of B. ferulifolia (Fig. 7). In addition mainly in the stamen, this is also shown in the middle map of both carotene bands at 1527 and 1158 cm−1 are very intense. the lower row in Fig. 8 showing a greater enlargement of the Contrary to that, the Raman spectrum of the leaves show flower from aside. The presented data correspond very well a polyacetylene signal at lower wavenumbers (2135 cm−1). with previously published results reporting the polyacetylene More detailed work, including HPLC-MS identification of distribution in the chamomile inflorescence based on the Ra- the individual unknown polyacetylenes in B. ferulifolia is man mapping technique [4].

Fig. 6. FT–Raman spectrum of below: carrot root containing higher amounts of polyacetylenes; above: reference spectrum of a C18 diine. 1400 B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401

Fig. 7. FT–Raman spectrum of line a: the ﬂower; line b: the leaf of Bidens ferulifolia; line c: reference spectrum of a phenyl-substituted triine (compound L [16]. The drawing copied into the ﬁgure is taken from [23].

Fig. 8. Upper row: maps and photo of the ﬂower of Bidens ferulifolia from above, left map: distribution of carotenes, middle map: distribution of polyacetylenes; lower row: middle and right: map of the polyacetylenes and photo of the ﬂower from aside. The colours of the map describe the integral Raman intensity. B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401 1401

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