TLC of selected sesquiterpenoids of the Asteraceae family

Visual chromatography has been employed for a preliminary identification of natural compound of a group of sesquiterpene lactones – Centaurea bella Trautv., C. crocodylium L., C. lusitanica Boiss. et Reuter, Helenium hoopesii = hoopesii (A. Gray) Rydb., Stizolophus balsamita (Lam.) Cass., baldschuanica – Santolina pinnata subsp. neapolitana (Jord. et Fourr.) Guinea = S. neapolitana Jord. et Fourr. A dependence of the colour of the spots, induced anisaldehyde reagent, on the presence of several substituents in the germacranolide ring – derivatives of partnenolide and salonitenolide – has been identified. The structures of the skeleton in the ring of sesquiterpene lactones in Helenium hoopesii can also be established by thin layer chromatography (TLC). The analysis of the chromatograms of extracts from dry and fresh Centaurea crocodylium herb has shown significant differences as for the chemical composition. Two sorts of germacranes of Santolina pinnata subsp. neapolitana display a characteristic colour of the spots.


Introduction
Among natural compounds in the species of family Asteraceae, sesquiterpenoids play an important role.They constitute a valuable chemotaxonomic material and might be also decisive for the medicinal value of the raw material, as in Chamomillae anthodium, Cnici benedicti herba, Arnicae anthodium, Millefolii herba, Chrysanthemi partheni herba, Cichorii radix et herba.A recent study has revealed antibacterial and cytotoxic activities of sesquiterpene lactones [1].These compounds can be detected through visual chromatography and isolated by simple column chromatography, with silica gel as the adsorbent.
The possibility of identification of several substituents at guaianolides by TLC with concentrated sulphuric acid as the developer had been mentioned earlier [2,3].Now, visual chromatography of compounds of four other types of sesquiterpenoids has been presented: germacranolides, germacranes, seco-pseudoguaianolides and pseudoguaianolides.
To develop the spots, the sole anisaldehyde reagent, which proved selective enough to rate the colours of the spots of the compounds in question, was used.

Plant material
The plant materials used for the studies employed aerial parts of Centaurea bella, C. crocodylium, C. lusitanica, Helenium hoopesii, Santolina pinnata subsp.neapolitana, Stizolophus balsamita, Zoegea baldschuanica, plants cultivated and identified in the garden of Department of Medicinal and Cosmetic Natural Products, University of Medical Sciences in Poznań (Poland), where their voucher specimens are deposited.

Isolation and identification of compounds
The herbs, collected right before blooming (about 450 g each) were extracted with methanol three times.The methanol extracts were inundated with distilled water (ca.600 cm 3 ), after the evaporation of the solvent.The water phase was extracted with chloroform.The chloroform extracts, in turn, having been dried with anhydrous sodium sulphate, were used for thin layer chromatography and isolation of compounds.Furthermore, some methanol/water/chloroform extracts were prepared from Centaurea crocodylium herb, which had been collected at the same time and from the same plot as the herbs designed to be dried.
The extracts were separated by column chromatography on silica gel (Merck art.7733).The fractions were subjected to repeated column chromatography on silica gel (Merck art.7729) and eluted by right mobile phases.The structures of the isolated compounds (Fig. 1) were identified on the basis of 1H NMR, IR and EI mass spectroscopy and by comparing the obtained data with those of the reference compounds or reported data [4][5][6][7][8].

TLC analysis
Thin-layer chromatrography was performed at room temperature on aluminium-backed silica gel plates DC Alufolien Kieselgel 60 (Merck art.5553).15-20 µg of each isolated compounds were applied per plate.Developed and dried chromatograms were sprayed by anisaldehyde reagent and heated at 100 o C by 3 minutes.Spots of isolated compounds exhibited, mauve, violet, red-brown, black-blue, orange, yellow, cherryred and gray colours when examined 3-15 minutes from the time of spraying (Tab.1).

Biosynthesis of cnicin during the drying of Centaurea crocodylium herb
While an attempt was being made to obtain compounds of C. crocodylium by column chromatography [silica gel, CH 2 Cl 2 -CO(CH 3 ) 2 8:1], from the extract from the fresh herb on large quantity (665 mg), salonitenolide (Fig. 1e) was isolated.Another compound, also in large quantity (845 mg), was obtained [silica gel, CH 2 Cl 2 -CO(CH 3 ) 2 4:1] from the dry herb of the plant.To our astonishment, rather than salonitenolide (Fig. 1e), it turned out to be its ester derivative: cnicin (Fig. 1f).It can be stated, thus, that at the time of drying, the ester had been connected to the hydroxyl group at C8 of e on Fig. 1.It is also worth noting that with the methods used so far in this work, the attempts to isolate either cnicin (Fig. 1f) from the fresh herb, or salonitenolide (Fig. 1e) from the dried herb have been unsuccessful.

Germacranes
Two out of the five germacranes of Santolina neapolitana [7], with the 4,5 epoxide and a substituent at C2 (Fig. 1i) exhibit dark-blue colour of their spots on the chromatograms (Fig. 3a,c).The spots of compounds j, k, l on Fig. 1 have a different, namely mauve, colour (Fig. 3b,d,e).They lack of the 4,5-epoxide ring and are characterised by the presence of the OH group at C5 and the =CH 2 substituent at C4.The 1,10 epoxide of l on Fig. 1 has no influence on the colour of the spot.

Seco-pseudoguaianolides
Another type of sesquiterpene lactones occurs in the herb of Helenium hoopesii [8], with seco-pseudoguaianolides (Fig. 1m-p) as the dominant feature.Hymenovin -mixture of C3 and/or C4 diastereoisomers (Fig. 1m) and hymenoratin B (Fig. 1n) with pyran skeleton with at least one OH group at C2 or C3 and C4 exhibited orange colour on the chromatograms.Hymenoratin B 2-O-β-D-(6'-O-acetyl)-glucopyranoside (Fig. 1o), on the other hand, with a pyran ring and blocked hydroxyl  groups at C2 and C4, changes its colour to yellow on the chromatograms, while floribundin (Fig. 1p), without a substituent at these places, is invisible in the anisaldehyde reagent (but fast crystallizes as white, spectrally clear needles).This compound was presented in this paper for the first time as one of compounds isolated from H. hoopesii.

Guaianolides and pseudoguaianolides
The other compounds of this plant: guaianolides (Fig. 1r,s) and pseudoguaianolides (Fig. 1t,u) with a five-part ring exhibited cherry-red colour.The fading of the cherry-red colour can be observed in acetylhymenograndin (Fig. 1v).Three acetyl groups in this compound are responsible for the grey colour of the spot.

Conclusions
Sesquiterpenoids constitute an important group of natural compounds occurring in species of family Asteraceae.By means of the simple TLC method, it is possible to estimate several details of the structure within guaianolides, germacranolides, pseudoguaianolides, seco-pseudoguaianolides and germacranes.
The simple thin layer chromatography may surely rank among methods of comparative analysis of natural compounds.It is often easier to differentiate and classify species based on TLC than through the traditional botanical analysis.One might assume that with high probability.Zoegea species will biosynthesize "violet" germacranolides with substituent at C9, and that Stizolophus species will biosynthesize "mauve" germacranolides with substituents at C8, and some Centaurea species will biosynthesize "brown-red" ones, with the characteristic CH 2 OH group at C4.