Exudate Compounds of Origanum Species  ^†

Abstract

Origanum species are valuable medicinal and culinary herbs whose biocidal properties are very important for organic farming. The first substances involved in allelopathic interactions in nature were the exudate (surface) compounds. In the present study, acetone exudates of ten samples of the Origanum species were comparatively analyzed by GC/MS and TLC. Plant material of Origanum dictamnus L., Origanum vulgare L. and Origanum vulgare subsp. hirtum (Link) Ietsw. as the latter taxa represented by eight patterns with different origins were studied. Flavonoid aglycones, terpenes, fatty acids and alcohols, triterpene acids and phenolic derivatives were identified. Methylated derivatives of flavones and non-methylated flavanones (naringenin and eriodictyol) were identified as the most common flavonoid aglycones. The most complex flavonoid profile was detected for O. vulgare ssp. hirtum samples. A few differences in the flavonoid profiles of O. vulgare ssp. hirtum from different origins were found. Carvacrol was determined to be a main component of O. vulgare subsp. hirtum samples, whereas in O. vulgaris exudate long-chain fatty alcohol was found to be an abundant compound. The data obtained complement the knowledge of the distribution and role of exudate compounds.

1. Introduction

Exudate flavonoids and their distribution among the plant kingdom have been comprehensively studied by Prof. Eckhard Wollenweber and the followers of this approach [1,2,3,4,5,6,7]. In addition to being taxonomically important, these compounds have important ecological functions due to their location on the surface on the plant. Exudate flavonoids are often defined as surface, external and lipophilic exudate flavonoids [3,5]. Besides flavonoid aglycones, exudates also contain terpenes, fatty alcohols and acids, alkanes and phenolic compounds.

Origanum species are valuable medicinal and culinary herbs, and their biocidal activity has been very important for organic farming in recent decades [8,9,10]. The results of the research on allelopathic interactions reveal a scientific basis for the selection of plant products with biocidal properties [11,12]. The first substances involved in these interactions in nature were the exudate compounds. The variability of production of metabolities in Origanum species has been reported not only during vegetation season [12] but also depends on the environmental conditions [13,14].

In the present study, the acetone exudates of ten samples of the Origanum species were comparatively analyzed by GC/MS and HPTLC. Plant material from Origanum dictamnus, Origanum vulgare and Origanum vulgare subsp. hirtum (Greek, oregano) as the latter taxa represented by eight patterns with different origins were examined.

2. Experiments

2.1. Plant Material

Aerial parts of studied samples were collected from plant collections of experimental fields at the Institute of Roses, Essential and Medicinal Cultures Kazanluk and the Institute of Biodiversity and Ecosystem Research (IBER) as well as from natural populations. The detailed information is presented at

Table 1. Description of studied plant material.

No	Taxon	Description of Origin
Od	O. dictamnus	Plant collection Kazanlak, source material (seeds) purchased from seed plot https://zelena-prolet.com/
Ov	O. vulgare	Natural population, Trigrad, Bulgaria CO1408
Oh1	O. vulgare subsp. hirtum	Natural population, at the Struma valley BulgariaC01409
Oh2	O. vulgare subsp. hirtum	Plant collection IBER, source material (seeds) from natural population http://www.iber.bas.bg/sites/default/files/projects/plantscollection
Oh3	O. vulgare subsp. hirtum	Plant collection Kazanlak, source material (seeds) purchased from Germany company https://www.pharmasaat.de
Oh4	O. vulgare subsp. hirtum	Plant collection Kazanlak, source material from natural population, northern Greek
Oh5	O. vulgare subsp. hirtum	Plant collection Kazanlak, Hebros variety
Oh6	O. vulgare subsp. hirtum	Plant collection Kazanlak, candidate variety
Oh7	O. vulgare subsp. hirtum	Plant collection Kazanlak, hybrid 1, seed progeny of O. vulgare subsp. hirtum obtained by free pollination of O. vulgare subsp. hirtum and O. vulgare
Oh8	O. vulgare subsp. hirtum	Plant collection Kazanlak, hybrid 2, seed progeny of O. vulgare subsp. hirtum obtained by free pollination of O. vulgare subsp. hirtum and O. vulgare

. Voucher specimens for the samples collected from natural populations are deposited at the Herbarium, Institute of Biodiversity and Ecosystem Research (SOM), Bulgaria.

2.2. Preparation of Extracts

Plant exudates were prepared from 2 g of air-dried, whole aerial parts by rinsing them with 20 mL of acetone for several minutes to dissolve compounds accumulated on the surface of plant tissue. The obtained extracts were concentrated for further GC/MS and TLC analysis.

2.3. TLC Analysis

Three TLC sorbents and mobile phases were used for the analysis of the flavonoid exudates. Toluene/dioxane/acetic acid (95:25:4, v/v/v) was used for the development of exudates on silica gel plates Kieselgel 60 F₂₅₄. Toluene/methylethylketon/methanol (60:25:15, v/v/v) was used for Polyamide 11 F₂₅₄ plates. Chromatograms were viewed under UV light at 336 nm before and after spraying them with “Naturstoffreagenz A”, 1% solution of diphenylboric acid ethanolamine complex in methanol.

2.4. GC/MS Analysis

For GC/MS analysis 300 µL of each exudate was transferred to a vial and evaporated to dryness, and then silylated with 50 µL of N, O-Bis-(trimethylsilyl)trifluoroacetamide (BSTFA) in 50 µL of pyridine for 2 h at 50 °C. The spectra were recorded on a Thermo Scientific Focus GC combined with a Thermo Scientific DSQ mass detector as described previously [15].

3. Results

The ten exudates of Origanum samples listed in Table 1 were comparatively analyzed for the determination of their main constituents. Flavonoid aglycones, fatty acids and alcohols, terpenes and phenolic derivatives were identified. The flavonoid profiles of the studied samples were determined by TLC. The results are presented in

Table 2. Identified flavonoid aglycones in the studied samples by TLC. Od O. dictamnus; Ov O. vulgare; Oh1–Oh8 O. vulgare subsp. hirtum (details in Table 1) Me—methyl ether.

Compounds	Od	Ov	Oh1	Oh2	Oh3	Oh4	Oh5	Oh6	Oh7	Oh8
Apigenin	●	●	●	●	●	●	●	●	●	●
Scutellarein 6,7-diMe	●			●			●		●
Scutellarein 6,7,4ˈ-triMe					●
Scutellarein 6,7,8-triMe (Xantomicrol)	●						●	●		●
Luteolin	O	●	●	●	●	●	●	●	●	●
Naringenin			●	●	●	●	●	●	●	●
Eriodictyol		o	●	●	●	●	●	●	●	●

.

Methylated derivatives of the flavones apigenin and luteolin and the non-methylated flavanones naringenin and eriodictyol were identified as the most common flavonoid aglycones. The flavonoid profile of O. vulgare was found to be the simplest. Only simple flavonoids—apigenin and luteolin as well as eriodictyol in trace amounts—were established. The most complex flavonoid profile was detected for the O. vulgare ssp. hirtum samples. A few differences in the flavonoid profiles of O. vulgare ssp. hirtum from different origins were found. The exudates of Hebros variety, candidate variety and hybrid 2 contain a xanthomicrol compound that was abundant in O. dictamnus exudate. Additionally, scutellarein 6,7-dimethyl ether, which was abundant in the O. dictamnus profile, was found in a few patterns of O. vulgaris ssp. hirtum—Hebros variety, hybrid 1 and plant material of the IBER ex situ collection (Oh2).

The exudates of the studied samples were analysed for the identification of other components by GC/MS analysis. The results are presented in Table 2 and

Table 3. Identified compounds of studied exudates of Origanum species; Od O. dictamnus; Ov O. vulgare; Oh1–Oh8 O. vulgare subsp. hirtum (details in Table 1). The quantities are expressed in relative percentages (area %).

Compounds	Od	Ov	Oh1	Oh2	Oh3	Oh4	Oh5	Oh6	Oh7	Oh8
Monoterpenes
Carvacrol	13.6	0.9	49.2	14.7	30.8	5.1	29.1	31.6	39.1	14.5
Sesquiterpenes
Copaene	4.7
Caryophyllene	1.4	0.8	3.3		0.8			1.3
Caryophyllene oxide	2.7	0.4	0.4		0.8				0.3	0.1
Fatty alcohols
Tetradecanol							1.9
Octadecanol						0.4		0.2		0.1
Hexadecanol			1.1		0.2
Tetracosanol						1.7		0.6		1.2
Hexacosanol	13.4	61.1	2.3	36.5	31.2	46.5	8.9	12.6	5.7	33.4
Fatty acids
Hexadecanoic acid		1.1		0.8	0.4	0.3	0.6	0.1		0.1
Octadecatrienoic acid				0.7						0.1
Fatty acid	1.9
Polyunsaturated fatty acid					8.5	10.8	1.9
Unsaturated fatty acid	1.8		1.2	1.1	8.5		22.5		0.6	9.8
Antifugal agents
Polyene					1.5	2.4	1.2
Phenolics
Hydroquinone derivative	3.4		19.6	0.5	0.4	0.4	5.6	3.2	7.6	1.6
Triterpenes
ß-Sitosterol						1.4		0.2		0.6
Triterpene	2.1	0.5	4.6	1.1	0.3	2.9	2.2	1.2	1.1	0.6
Oleanolic acid		2.9
Ursolic acid	8.2	6.9	10.4		0.2	0.8	1.3	0.2

. Monoterpene: carvacrol and long-chain alcohol: 1-hexacosanol were identified as the main components in the exudates. Differences among species levels were found. The O. vulgaris exudate contained about 60% of 1-hexacosanol, but carvacrol was found in a low amount, whereas in O. vulgare ssp. Hirtum, carvacrol was found to be dominant component. Carvacrol content varied between different patterns of the last taxon in the range 5—49%. The sample of the natural locality from Bulgaria (Oh1) contained carvacrol in the largest amount. Exudates of hybrid 1, of the Hebros variety, contained a significant amount of carvacrol as well. Hexacosanol, except in O. vulgare profile, was found in significant amounts, as well as in the sample from the natural Greek population (Oh4) and ex situ collection of IBER (Oh2). Triterpene acids and derivatives were also identified as the most common metabolite for the studied samples. Ursolic acid was found in a larger amount in exudates of O. dictamnus, O. vulgare and O. vulgare ssp. hirtum from the natural population (Oh1). Oleanolic acid was found only in the O. vulgare exudate.

4. Discussion

The first study on exudate flavonoids of Origanum species was conducted by Tomás-Barberán et al. [16], but a detailed summary, supplemented with new research, has been provided by Skoula et al. [17]. The data obtained in the present study are consistent with the results obtained by Skoula et al., [17]. In addition to the flavonoids, data on the other components in the exudates was reported for the first time in the present study. Carvacrol and hexacosanol were established as the main components of the studied samples. Strong antifungal, antibacterial and phytotoxic properties have been proven for carvacrol [18]. Hexacosanol’s inhibitory effect on acetylcholinesterase and larvicidal activity has been demonstrated [19]. This fatty alcohol has been reported for Origanum vulgare subsp. virens [20]. Methylated flavonoids such as xanthomicrol have been found to display acetylcholinesterase activity [21], which implies a manifestation of insecticidal action [22]. The presence of methylated flavonoids in certain patterns of O. vulgare ssp. hirtum (Oh2, Oh7, Oh8) can be interpreted as an effect of being synthesized as a result of hybridization or as a protective response to stressors, because these compounds have established insecticidal and antimicrobial properties. Detected ursolic acid of the exudates possesses significant biological activity including antibacterial properties [23]. The presence of bioactive compounds on the surface of plants suggests their protective role against abiotic and biotic stress factors.

5. Conclusions

In the present study, metabolite profiles of the exudates of ten samples on three Origanum taxa were determined. Monoterpene phenol (carvacrol), long-chain primary fatty alcohol (hexacosanol), ursolic acid, methylated flavones and non-methylated flavanones were determined to be main bioactive compounds. These are substances with proven strong biocidal activity that suggests their protective role in plants.