CHEMICAL SCREENING OF LICHENS
Natural products from higher plants, marine organisms or even toxic venoms have shown a growing interest for their anti-biologic, neutraceutic or cosmetic potential applications. The present study concerns the development of a method to screen raw plant extracts in regard to characterize some molecules of interest. This survey on lichens will start by a description of the vegetal material and the chemistry analyses chosen to then present the main results. Finally, spectroscopic and chromatographic techniques will discuss in the validation of this chemical screening to efficiently characterize secondary metabolites.
I. Material and Methods
A. Plant collections
Lichens are symbiotic organisms resulting from the association of a microscopic green alga with a filamentous fungus. These plants are a really source of bioactive compounds and they have been described for the diversity of their secondary metabolites (J. Barnes et al. 2000). A variable set of these metabolites, ranging from terpenes, xanthones or depsides, are synthetised in lichens. Such compounds are easily detected by chemical techniques and can then be purified to allow further studies.
B. Species identifications
Tweenty-three samples of fresh materials were collected at four different locations on Tasmanias East Coast. Each lichen sample was divided into two parts: a representative specimen kept for morphological identification while the rest of the collection was for extraction purposes. The TLC technique is a reliable and easy way to characterize lichen compounds and thus identify different species. The different extracts were spotted onto glass plates coated with silica gel. Plates were then put in a tank containing a specific solvent which is elueting the lichen compounds at different distances on the silica plate. This allows the different substances present in the extract to be separated by the solvent according to their polarity. Lichen specimens (0.5 cm3) were placed in test tubes and extracted with 1 mL of acetone at room temperature for 24 hours. Three different solvent systems that are still widely used for chemotaxonomic characterization as described by A. Orange et al. (2001) and Culthberson et al. (1972) was used. In that experiment, the different acetone extracts were run in solvent A, C and E (Table 1).
Table 1. Solvent composition in mL used for lichen chemotaxonomic identification (A. Orange et al., 2001).
|Highly Polar compounds|
|Acetic acid: 5|
|Acetic acid: 30|
|Non polar compounds|
|Ethyl acetate : 25|
After development, spots were viewed under a 245 nm UV light. The TLC visualisation was made by applying 10% H2SO4 directly onto the plates and then heating for 10 min at 120? C. Rf of these spots are characteristics of a particular compound in a particular solvent. The final identification of the species was done in relation to lichen morphology, the colour and the Rf of the TLC spots in comparison with a control of known chemistry.
C. Secondary Metabolite Extractions
1. Chemical isolation
The air-dried and ground lichen thalli were treated by stirring at room temperature with 400 ml of acetone over night. The raw extracts were concentrated by rotary evaporation at reduced pressure and spotted on TLC plates in order to determine the number of secondary metabolites to isolate. The optimal resolution for flash chromatography was defined by TLC screening of crude extracts. A Rf within the range of 0.25 to 0.35 was used for each particular compound. Each extract was purified and fractionated by flash chromatography with silica gel of 320-630 m under isocratic elution gradient.
2. Structure elucidation of extracted compounds
Each pure fraction was then analyzed by 1H-NMR and 13C-NMR supplemented by MS when necessary to characterize their structure. Chemical shifts are given in ppm with TMS as reference point. 1H-NMR signals are described as singlets (s), doublets (d), doublet of doublets (dd) triplets (t) or multiplets (m). Each value of the spectra is given for a particular carbon of the molecule. Analyses were realized in deutereted chlochloroform with the addition of dimethylsulfoxide when needed to improve the dissolution. Mass spectrometry was required when no chemical shifts could be compared to the one found.
A. Species identifications and selections
Fifteen lichen species were identified by their morphological and chemical characteristics (G. Kantvilas et al. 2002) thanks to the expertise of the botanist Dr. Gintaras Kantvilas. Two species were then selected because of the TLC screening: Flavoparmelia rutidota to extract usnic acid a well known secondary metabolites (M. Cochietto et al. 2002) and Xanthoparmelia scabrosa. This last species showed two main secondary metabolites by TLC screening.
B. Secondary metabolite extractions
The acetone crude extract of F. rutidota was fractionated on a silica gel column using 80% hexane /20% ethyl acetate. One compound  was identified by spectroscopy as usnic acid.
The acetone crude extract was fractionated using 30% EA/hexane and then 85% toluene/EA in flash chromatography. Two fractions were then collected and  was identified as loxodin while  was identified as norlobaridone (S.Huneck et al.1996).
C. Compound elucidations
1. Usnic acid
1H-NMR (CDCl3): 1.75 (3H, s), 2.09 (3H, s), 2.66 (3H, d), 5.98 (1H, s), 11.03 (1H, s)
13C-NMR (CDCl3): 7.6, 27.9, 29.7, 32.1, 59.5, 98.4, 101.5, 104, 105.2, 109.3, 155.2, 157.5, 163.9, 179.4, 191.7, 198.0, 200.4, 201.8
1H-NMR (CDCl3+ DMSO-d6 ):0.94 (6H, t), 1.39 (2H, m), 1.45 (2H, m), 1.51 (2H, m) 1.73 (4H, t), 2.74 (2H,t), 3.12 (2H, t), 3.96 (3H, s), 6.74 (1H,d), 6.75 (1H,d), 6.77 (1H,s), 11.03 (1H, s).
13C-NMR (CDCl3 + DMSO-d6 ): 13.9, 13.9, 21.6, 22.0, 25.5, 27.5, 31.0, 31.5, 41.2, 52.5, 105.9, 106.1, 111.2, 111.6, 119.8, 134.2, 140.7, 144.5, 148.7, 152.8, 160.1, 160.4, 162.9, 166.5, 203.0.
1H-NMR (CDCl3 ):0.83 (6H, t), 1.291 (2H, m), 1.45 (2H, m), 1.54 (2H, m),1.73 (4H,t), 2.74 (2H,t), 3.18 (2H, t), 6.41( 1H,d), 6.50 (1H,d), 6.61 (1H, s, H-1′), 6.64 (1H,s), 11.03 (1H, s).
13C-NMR (CDCl3): 13.7, 13.9, 22.0, 22.4, 25.9, 29.8, 30.0, 31.5, 42.3, 105.6, 108.3, 110.3, 111.8, 113.6, 136.5, 141.4, 144.1, 148.9, 153.8, 162.9, 163.6, 164.4, 206.9.
A. Lichen distribution and known secondary metabolites
Flavoparmelia rutidota is a wide spread species of the Parmeliaceae family and is common in Tasmania, temperate and subtropical Australia as well as southern USA. It is known to contain usnic acid, protocetraric acid and caperatic acid (G. Kantvilas et al., 2002). The Parmeliaceae Xanthoparmlia scabrosa is distributed in Tasmania, Australia, New-Zealand, New-Guinea, Fidji, southern south America, south Africa, and Japan. Usnic acid, loxodin, norlobaridone, and related compounds as well as some trace of scabrosin ester have been recorded in this species (G. Kantvilas et al., 2002).
B. Chromatography techniques
As many secondary metabolites have the same range of Rf and the same color on the plates, this technique requires a good knowledge of the TLC secondary metabolite characteristics and the solvent system to allow the identification of these compounds.
TLC was a reliable, cheap and quick way to assess the number of secondary metabolites present in the crude lichen fractions. It was a very powerful tool to isolate different secondary metabolites present in the twenty-three lichen acetone extracts and provided indications on the polarity of these compounds. Crude extracts were screened with solvent E to allow the separation of compounds showing the same range of Rf At the same time, TLC was very effective to assess the best solvent to use in Flash Chromatography. Solvents allowing an optimal Rf of 0.3, were primary worked out on the plates and then made for the use Flash Chromatography.
2. Flash chromatography
Flash chromatography on silica gel was used to isolate and purify crude lichen extracts was quite efficient in this study. However, finding the appropriate solvent giving an optimal Rf could be time consuming and two or three columns had often to be run to obtain a pure compound.
During the present survey, polar compounds with a Rf x100 ranging from 4 to 15 in A solvent such as protocetraric acid and caperatic acid commonly found in Flavoparmelia rutidota were not extracted. While these polar compounds were easy to spot by TLC in the crude extracts, their low Rf made the elaboration of a flash chromatography solvent delicate. The purification of the crude fractions was made to the detriment of these polar compounds and favored non polar secondary metabolites such as usnic acid which shows a high Rf
In addition, flash chromatography was not powerful to separate compounds with a similar range of Rf .In the case of the lichen Xanthoparmelia scabrosa, fractions containing loxodin and norlobaridone were hard to purify by this technique and minor related compounds such as loxodinol and norlobaridol were suspected to be present in these fractions. Separation of atranorin and usnic acid was performed using solvent E which was quite appropriate for the purification of relatively non polar compounds.
C. Spectroscopic technique
13C and 1H NMR were used in a first time to assess the purity of the compounds extracted and allow confirmation of structure. Purification of loxodin and norlobaridone fractions was achieved by differential solubility using DMSO to extract loxodin while norlobaridone stayed in the chloroform phase. Identification of the extracted secondary metabolites, usnic acid, loxodin and norlobaridone, was allowed by comparison of their chemical shifts to the one describes in the literature. This spectrometric technique was a very accurate and reliable tool in the identification of the structures.
IThis study has firmly established that the method developed, the 2 chromatography techniques associated to NMR, was an effective way to extract and characterise major secondary metabolites of crude lichen extracts. Thus, 15 lichens were chemically screened for their secondary metabolites and 3 of these compounds were eventually extracted from 2 lichen species.
Extraction and purification of further minor secondary metabolites could be realised by using HPLC, another technique commonly used in the isolation and extraction of lichen metabolites. Secondary metabolites are identified by their retention time and fractionations are more quantitative for the extraction of minor secondary metabolites (G. Feige et al. 1993). NMR is the most powerful and accurate technique to characterize chemical structures even though fraction purities remain critical for an optimal elucidation.
Finally purified, it would be interesting to test the bioactive potential of individual compounds through the bacterial or fungal screening to assess their anti-biological potential.
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