Targeted Isolation of Anti-mycobacterial Labdane Terpenes from Grindelia stricta var. platyphylla

Abstract

This dissertation describes experimental work dealing with the targeted isolation and biological activity of grindelanes from Grindelia stricta var. platyphylla (Asteraceae), a plant variety found in areas of coastal California, USA. This species and variety has not been characterized before for its profile of labdane terpenes or for its activity against mycobacteria. In general, although Grindelia is a widely used and well-known medicinal plant genera , the more than 70 different species of Grindelia have not been much studied for the characterization of the diterpene compounds unique to each species. The last extensive study isolating diterpene compounds from a Grindelia sp. was conducted in the 1980-1990’s on Grindelia tarapacana native to Peru and Chile. The lack of studies may be due to the difficulty in separating these very similar low polarity compounds, combined with their lack of UV chromophore. Two innovations and five stated research aims from the approved research proposal defense were investigated in this dissertation work. The first stated innovation was to target and isolate diterpenes from the heretofore unstudied traditional medicinal plant variety Grindelia stricta var. platyphylla collected from the California coast. This was met by the isolation of six diterpene compounds, five known and one novel as well as a known flavonol. The second stated innovation was to the isolated grindelane compounds for antimicrobial activity not yet reported in the literature. This was met by testing for activity against mycobacteria. These innovations were met by completing the following five specific aims described and approved in the dissertation research proposal. Specific Aim 1: Identification, collection and extraction of Grindelia stricta platyphylla flower buds. Separation of mixed fractions from the ethyl acetate extract of Grindelia stricta platyphylla flower buds, by column chromatography. Specific Aim 2: Bioactivity testing of mixed fractions of Grindelia stricta platyphylla against the screening organism Mycobacterium phlei. Specific Aim 3: isolation and structural elucidation of terpenes from Grindelia stricta platyphylla. Specific Aim 4: Preliminary studies of the molecular mechanism of action by assay for anti-kinase activity of column fractions and isolated terpene compounds. Specific Aim 5: bioactivity testing of mixed fractions and single chemical terpene compounds of Grindelia stricta platyphylla against Mycobacterium tuberculosis (Mtb). In this dissertation study of Grindelia stricta var platyphylla, labdane diterpenes were targeted and seven compounds (six labdanes and one very similar-structured flavonol) were isolated by various chromatographic techniques for use in the investigation of specific biological activity (anti-mycobacterial). Of these, one is new (3) and five (1)(2)(4)(5)(6) are known labdane diterpenoids of the grindelane type. One known flavonol (7) kaempferol, was isolated and determined by x-ray crystallography. The new diterpenoid was carboxyl 3,4,4a,5,6,7,8,8a-octahydro-β-hydroxy-β,2,5,5,8a-pentamethyl-3-oxo-1-naphthalenepentanoate, (compound 3, ring opening). The chemical structure was established on the basis of spectral analysis including 1D and 2D NMR. Stereochemistry was not established however, stereochemistry of C-20 and H-5 of the structures can be predicted based on the biogenetic considerations of the grindelanes. The known grindelanes were labd-7-en-15-oic acid, 9,13-epoxy- (compound 1, grindelic acid), 2-(2',3,3,6a,7a-pentamethylspiro[1a,2,2a,4,5,6-hexahydronaphtho[2,3-b]oxirene-7,5'-oxolane]-2'-yl)acetic acid (compound 2, epoxide), spiro[furan-2(3H),1′(4′H)-naphthalene]-5-acetic acid, 4,4′a,5,5′,6′,7′,8′,8′a-octahydro-2′,5,5′,5′,8′a-pentamethyl-, ethyl ester,4′aα,8′aβ]]- (compound 4, ethyl ester), spiro[furan-2(5H),1'(4'H)-naphthalene]-5-acetic acid, 3,4,4'a,5',6',7',8',8'a-octahydro-2',5,5',5',8'a-pentamethyl- (compound 5, methyl ester), methyl 2-[4-(hydroxymethyl)-2',4,7,8a-tetramethylspiro[2,3,4a,5-tetrahydro-1H-naphthalene-8,5'-oxolane]-2'-yl]acetate (compound 6, methyl 18-hydroxy-grindelate). The known flavonol was the kaempferol, 5-Hydroxy-3,7,4'-trimethoxyflavone (compound 7, kaempferol 3,7,4'-trimethyl ether). Throughout the bioassay-guided separations, in various screening assays (disk, TLC overlay, broth) for anti-mycobacterial activity; an ethyl acetate extract of the flower resin, the majority of diterpene rich fractions and the isolated compounds showed activity. In addition, during preliminary screening, the ethyl acetate extract of the flower resin and the isolated compound grindelic acid, showed a kinase inhibitory activity and lack of cytotoxicity in a Streptomyces 85E kinase inhibition assay. This same assay simultaneously screens for possible anti-mycobacterial activity due to the eukaryotic-like kinase’s found in both Streptomyces 85E and mycobacteria. The correlation between compounds testing positive for Streptomyces 85E kinase inhibition and for anti-mycobacterial activity is described in the 2002 paper (“Identifying Protein Kinase Inhibitors Using an Assay Based on Inhibition of Aerial Hyphae Formation in Streptomyces”) by Julian Davies et al, the originator of the Streptomyces 85E kinase inhibition assay. In the final broth dilution assays, fast growing non-pathogenic models M. phlei (ATCC354) and M. smegmatis (ATCC14468) were used as screening organisms. The following MIC’s were determined for M. phlei and M. smegmatis respectively, ethyl acetate extract of the flower resin (25 and 125 µg/ml); compound 1, grindelic acid (16 and 78 µg/ml); compound 2, epoxide (63 and 250 µg/ml); compound 3 (not tested, too small amount); compound 4, ethyl ester (16 and 250 µg/ml); compound 5, methyl ester (63 and 500 µg/ml); compound 6, methyl 18-hydroxy-grindelate (250 and 125 µg/ml); and compound 7 (250 and 125 µg/ml); MIC’s of the anti-mycobacteria agent rifampin (0.16 and 1.3 µg/ml) and the antibiotic/β lactamase inhibitor combo drug, amoxicillin/clavulanate (10 and 313 µg/ml) were also determined. Of the plant compounds, grindelic acid and compound 4, the ethyl ester (MIC’s 16 µg/ml) showed the best activity against M. phlei and grindelic acid also showed favorable activity (78 µg/ml) against M. smegmatis. It was interesting to note that there was a magnitude of difference between the MIC’s determined using rifampin, against the two mycobacteria species. In a subsequent broth dilution study, the MIC of rifampin (0.13 and 1.25 µg/ml) was reduced when in combination with sub-inhibitory doses of amoxicillin/clavulanate, the ethyl acetate extract of the flower resin and some of the isolated compounds, in some cases. Results varied against the two different species of mycobacteria with no clear pattern. Grindelic acid, the most abundant isolated compound, showed the greatest effect, lowering the MIC’s of rifampin to (0.001 and 0.01 µg/ml) respectively. In an assay against M. tuberculosis (Mtb) strain: Mrb mc2 6206 (MtbAux) it was found that the crude ethyl acetate extract, the major compound (1) grindelic acid and the methyl ester compound(5) demonstrated an anti-mycobacterial MIC of 0.125mg/ml. Terpene compounds are difficult to isolate due to their low polarity, relatively low concentration in plants and the lack of a chromophore in the structure. This means that many interesting terpenes present in plants may go undetected into the waste stream when using a conventional UV detector at the end of a separating column. By taking advantage of the modern development of a universal detector, the evaporative light scattering detector (ELSD), it is now possible to more easily detect low concentration compounds that lack a UV chromophore. In the past, the ELSD was a problematic detector not widely used, as it was not robust and lacked the ability to operate at low temperature and concentrations. In this dissertation, using a modern Sedex LT-ELSD, a chromatography method was developed in which grindelanes may be detected at low concentrations and temperatures at the end of a separating column.