Phytochemical composition of Denhamia obscura (A. Rich.) Meisn. Ex Walp. root bark, seeds and leaves

An investigation of the chemical composition of the root bark, seeds and leaves from the Australian plant, Denhamia obscura was carried out. Despite the traditional medicinal use of this plant by Indigenous communities in Australia and the comprehensive studies of other Denhamia species, the phytochemical profile of this plant has not been reported previously. Twelve known pentacyclic triterpenes, as well as seven abietane compounds were characterised. Two of the abietane compounds are new natural products, previously reported as synthetic compounds, obscurol and 13-methoxy-sempervir-6-ene. Friedelin was isolated from the root bark and leaves


Introduction
The plant genus Denhamia (family Celastraceae) is endemic to Australia and the Pacific Islands region. 1 The genus contains 15 recognised species (to date) including eight species previously included in the genus Maytenus. 1,2Only three studies have endeavoured to characterise the composition of two plant species from this genus, Denhamia celastroides and Denhamia pittosporoides.4][5] The first study into the composition of D. celastroides leaves was performed in 2015 by Levrier and Davis et al. who identified eight compounds named denhaminols A-H. 4 These compounds are esters of polyhydroxy sesquiterpenes, all belonging to the dihydro--agarofuran compound family (Figure 1).While the compounds all shared a similar core structure, they were functionalised with acetate, benzoate, tigliate and/or cinnamate esters on various hydroxylated positions.These compounds inhibit growth of the human prostate carcinoma cell line LNCaP with denhaminol A and G the most effective.
Figure 1.Structure of two dihydro--agarofurans isolated from D. celastroides. 3 2018, Davis' group published a second paper 3 on the composition of D. celastroides.This study reported the isolation of new denhaminols K-N in addition to those identified in the initial work.These were minor compounds that evaded detection during the previous studies.In this study, Davis et al. also saponified a portion of the denhaminol A extracted to generate the core sesquiterpene lacking ester moieties, to assess the effect of the esters on uptake and cell growth inhibitory activity.Leucine transport in the LNCaP cells was inhibited by all denhaminols A-N, with denhaminol K being the most effective while the sesquiterpene aglycone exhibited no biological effect.
The third study investigated D. pittosporoides. 5Following the same experimental protocol 4 denhaminol I and J were isolated alongside four other structurally related compounds corresponding to a total of 0.3% of dry weight.
A distinctive characteristic of Denhamia obscura, investigated here, is the presence of a bright yellow layer in the root bark which is highly flammable and changes to a yellow-orange color after exposure to air.The function of such a pigmented layer in an underground organ raises some ecological questions.For example, does the layer provide some protection of the root from herbivores or microbial pathogens?An understanding of the chemistry of this layer will assist in understanding its function.The species also has a bright red fleshy aril surrounding the seed.Fleshy arils normally function as an attractant to seed dispersers by providing a food reward.The presentation of the seed in the open fruit capsule while still hanging on the tree suggests that birds are the targeted dispersal agent for the seeds.It is of interest to know if the aril pigmentation is based on the same chemistry as the root bark pigmentation given the very different functions proposed for the two materials, i.e., herbivore/pathogen deterrent and seed disperser attractant.
Denhamia obscura is a species endemic to Australia, which has been used by Aboriginal and Torres Strait Islander communities for centuries as a traditional medicine.Pharmacopoeias report that chewing the bright yellow-orange root bark alleviates toothaches, 6 and the aqueous extract from boiled leaves is used in the treatment of respiratory ailments. 7Hyland and colleagues report aphrodisiac effects resulted from the consumption of the root bark with limited details provided. 8The Tiwi people of the Northern Territory also attribute the yellow coloration as a "good luck" charm in various endeavours such as travel or significant life events. 7The characterisation of the natural products present in the root bark and leaves of D. obscura is the focus of this study, since these are parts of the plant used in traditional medicine.Alongside the phytochemical characterisation of the root bark and leaves, assays were conducted on the crude extracts to evaluate the antimicrobial potential of the root bark extracts.On the other hand, compounds isolated from the leaves shed light on the biosynthetic pathway by which this plant produces triterpene natural products.

Root bark
Denhamia obscura was collected from three locations in the Top End of the Northern Territory, Australia.Samples were dried, milled and transported to the University of Queensland, where they were suspended in methanol and sonicated to increase the efficiency of the extraction process.The methanolic extract was then partitioned with hexane.Water was added to the methanol partition, which was further partitioned with chloroform.These partitioning steps yielded three extracts for analysis: hexane; chloroform; and aqueous methanol.The hexane and chloroform extracts were subjected to normal phase high performance liquid chromatography (NP HPLC) using a mixture of hexane and ethyl acetate as eluting solvents.The aqueous methanol partition was fractionated using reverse phase (RP) HPLC using acetonitrile/water mixtures as the eluting solvent.Fractionation by NP HPLC of the chloroform partition yielded abietanes 1-7 and triterpenes 8-14 (Figure 2), with known natural products characterised as ferruginol (1), maytenoquinone (2), dispermol (4), 12-hydroxytotarol (5), sugiol (6), pristimerin (8), celastrol (9), tingenone (10), iguesterin (11), friedelin (12), multifluorenol (13), glutinol (14).Abietanes 3 and 7 were identified by a combination of NMR spectroscopy and mass spectrometry from simple mixtures.Fractionation of the methanolic partition only yielded compounds 2, 3, 8, 12 and 14 already identified in the other extracts.0][11] The chemistry therefore appears to support this taxonomic realignment.Compounds 1, 2, 5, 6, 8-14 have been previously isolated as natural products with some also being the focus of synthetic studies, [12][13][14][15] providing sufficient characterisation data for comparison.The synthesis of 3 has been reported 10 but there has been no previous reports of its occurrence as a natural product.
The abietane skeleton gives rise to characteristic 1 H NMR signals including five methyl signals (δC 21.0-33.0):three singlets corresponding to the methyl groups on ring A at C18, C19 and C20; and two doublets corresponding to the methyl groups of the isopropyl group attached to the aromatic C ring, positions 16 and 17.Both of these signals in the COSY spectrum exhibit correlation to the septet signal at approximately 3.00 ppm (δH 3.11 for ferruginol 1).
The NMR spectra of compound 3 exhibited peaks analogous to the characteristic ones described above: three methyl singlets in the 1 H NMR at 1.13, 1.18 and 1.21 ppm corresponding to H-18, 19 and 20, respectively; and two methyl doublets for H-16 and 17 at 1.36, 1.33 ppm with a vicinal coupling constant of 7.0 Hz, assigned to the isopropyl methyl groups.The latter proton signals exhibited coupling to the septet signal at 3.11 ppm (H-15), further supporting the presence of an isopropyl group.The methyl signals at 1.36 and 1.33 ppm show three bond correlation to a signal at 161.6 ppm (C-14), and four bond correlations to a signal at 135.8 ppm (C-8) and 126.2 ppm (C-13) in the HMBC spectrum.Another characteristic feature of unsaturated abietanes is the appearance of low field proton signals at 6.88 and 6.27 ppm corresponding to H-7 and 11 of the extended conjugated quinone methide system present in 3. The correlation between H-11 and C-7 that was observed in the HMBC supported the presence of this moiety.The hydroxyl moiety attached to C-13 was assigned to a singlet at 6.95 ppm.An interesting feature of compound 3 was the appearance of a broad doublet at 4.69 ppm, which exhibited a 10 Hz coupling to the signal at 1.89 ppm, assigned to the bridgehead H-5.These data suggested that in 3, C6 (δC 69.9) is an sp 3 hybridised carbon bearing an hydroxyl group, H-6 appears at 4.69 ppm and H-5 and H-6 are trans-diaxial.This is consistent with the trans A,B-ring junction observed in the other abietanes isolated and with the hydroxyl group at C-6 ring equatorial.Thus, compound 3 was characterised as 6,13-dihydroxytotar-6,8 (11),13-triene.This compound has not previously been isolated as a natural product, but has been reported synthetically; 12 the NMR data observed here matched that reported, and compound 3 is hereafter referred to as obscurol.Compound 7 was identified as a minor (~11%) component in a mixture with maytenoquinone (2).The compound was characterised initially through its diagnostic signals and then structural elucidation was completed using 2D NMR techniques to arrive at a proposed structure.The overall distribution of signals in the 1 H spectra suggested the abietane-like nature of the core, with three singlets at 1.01, 1.04, 0.94 ppm corresponding to H-18, 19 and 20, respectively, and two downfield singlets at 6.71 and 6.61 ppm assigned to H-11 and H-14.These assignments were supported by HSQC correlation of these signals to carbons with methyl (δC 20.8, 21.6, 33.1) and aromatic character (δC 114.0, 131.0), respectively.The COSY spectrum provided evidence for the isopropyl moiety due to the correlations between the doublets at 1.34, 1.32 ppm (H-16, H-17) and the septet at 3.06 ppm (H-15).These proton signals were then assigned to their respective carbons via HSQC (27.2, 21.3, 21.4 ppm, C-15, 16, 17, respectively).HMBC correlation between the methoxy 1 H NMR signal (3.75 ppm) and an aromatic carbon signal tentatively assigned as C-13 (147.3 ppm) supported the presence of a methyl ether in ring C. Other signals assigned based on the established abietane core were the signals associated with ring A (C-1-3).These assignments were possible by combining the HSQC data, which associated the methylene signals with carbon signals, and HMBC correlations between methyl signals for C-18-20 and these sp 3 hybridised carbons.Finally, the signals at 5.88 and 6.84 ppm each observed as a doublet of doublets were assigned to H-6 and H-7 of ring B. Each showed a vicinal coupling of 10 Hz, as expected for a cis coupling.COSY correlations suggested that both H-6 and H-7 coupled to H-5.The magnitude of the coupling constant (2.9 Hz) between H-5 and H6 is consistent with a dihedral angle of approximately 92° (Chem 3D Ultra 20.0.0.41,PerkinElmer, MM2 minimised energy), congruent with H-5 occupying the axial position which is the stereochemistry observed in other abietane compounds isolated from this plant.The smaller coupling ( 4 J = 3 Hz) exhibited by H-7 is likely a 'w'-coupling to H-5 due to the allylic nature of this 'H-C-C-C-H' segment of the structure.Since very few HMBC correlations were observed, with none connecting rings B and C, the structure could have been 6,7-dehydroferruginol methyl ether, reported by Cordova-Guerrero and colleagues, 16 or structure 7 in Figure 5.The NMR data from the previously reported natural product did not align with the compound indicated here.The structure of 7 was published as part of a synthetic protocol, 17 but no NMR or other characterisation data was given.Therefore, this is the first publication of the NMR data as well as the first report of it as a natural product, and it is named 13-methoxy-sempervir-6-ene (7), based on the similarity of the structures to sempervirol, the target natural product in the synthetic paper which reported these compounds.The phytochemical characterization discussed above was conducted on a sample collected from Thorak Road (see experimental for coordinates).Root bark samples were also collected from Buffalo Creek and Deleye Outstation (see experimental for coordinates), during different seasons.For comparison, a methanolic extract (21.1 mg) of samples from each collection site, was suspended in aqueous methanol (50%) and screened with the same method and conditions on LC-PDA-MS (10 µL), GCMS (2 µL) and 1 H NMR (CD3OD, 32 scans).Compounds were identified by matching retention time, UV-vis and ESIMS data.Once those compounds were identified, it was possible to compare the relative abundance of compounds qualitatively, based on UV-detected chromatogram intensities and total ion count.Compounds 4, 5 and 7 were observed inconsistently: 7 was not observed in the Deleye outstation sample, 4 and 5 were not observed in the Buffalo creek sample, while 1-3, 6, 8-11 were observed across all three samples.These samples were collected during different seasons, which may impact the phytochemical composition.Alternatively, there may be some geographical variation in the phytochemical composition of the bark.
With the phytochemical characterisation complete, it was possible to determine which compound contributed to the color of the bark.Maytenoquinone (2), λmax 345 nm, the most abundant abietane and found in all samples provides the yellow tones of the root bark.Pristimerin (8) (λmax 438 nm), celastrol (9) (λmax 438 nm) and other triterpenes provided a deep red hue, which combine to enrich the vivid yellow coloration of the root bark.
Members of the abietane family of compounds, e.g., 1-7, and pentacyclic triterpenes with extensive conjugation, e.g., 8-16, have been identified in various studies as exhibiting anti-inflammatory, anti-cancer, and some cytotoxic activity. 18The root bark crude methanolic extract from the Thorak Road site was screened for antimicrobial activity against a panel of microbes including Gram negative bacteria [Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25668)], gram positive bacteria [Staphylococcus aureus (ATCC 25923) and Streptococcus pyogenes (ATCC 19615)] and the yeast Candida albicans (ATCC 90029).Antimicrobial activity was examined at a single concentration (20 mg/mL) in a broth microtiter method based on Clinical Laboratory Standards Institute (CLSI) standards for Antimicrobial Susceptibility testing. 19,20Antimicrobial activity was defined as a reduction of 80% or greater in growth as compared to the control.The crude extract derived from the Thorak Road sample demonstrated antimicrobial activity against S. aureus.This data is in agreement with the previously reported effects of abietane and triterpenes against S. aureus and Propionibacterium acnes. 21The demonstration of the antimicrobial activity of the root bark compounds supports the hypothesis that these compounds may provide some protection against microbial pathogens in the soil.

Seeds
An analogous process of extraction and fractionation was carried out on D. obscura whole seeds, which contained compounds 1-4, 6, 8-10, 13 and 14, some of which are responsible for the red coloration of the fleshy aril covering the seeds.The difference in coloration between the root bark and the aril material stems from the variation in concentration of compounds, with a higher abundance of compounds 8 and 9 compared to compound 2 in the aril material.The identification of these compounds was carried out via LC-PDA-MS by comparison of retention time, UV-vis absorbance and ESIMS data to authentic compounds isolated from the root bark.
Characterization of the remaining components in the seed extract was achieved by GCMS.This led to the tentative characterization of eleven compounds by comparison to mass spectrometric libraries.Tentative identification was assigned to compounds presenting a mass spectrum similarity over 93% in at least two of three libraries (NIST17, FFNSC and WIST29).These compounds were; heptanal, E-2-decenal, 2,4-decadienal (E,E) and (E,Z), methyl of eicosanoate, palmitic acid, methyl 9,12-octadecadienoate, methyl 9-octadecanoate, methyl stearate, linoelaidic acid and stearic acid.
Friedelin (12) was easily isolated due to its abundance and proclivity to crystallise.This compound was the most prevalent in the extract of D. obscura leaves.In 1955, the absolute configuration of friedelin was determined by Corey and Ursprung through a lengthy series of degradation studies. 22To date, all friedelin isolated from natural sources has exhibited the same negative optical rotation, with slight fluctuations in the reported values due to differences in concentration and temperature.The crystallised sample of friedelin in this work exhibited an [a] = -12° (c = 0.4 in chloroform, at 24 °C and 589 nm).All naturally occurring friedelin samples have therefore always been assigned the absolute configuration consistent with the original 1955 determination.][25] 1D and 2D NMR analysis of the crystallised friedelin allowed for the complete assignment of both the 1 H and 13 C NMR spectra of the compound.Complete assignment of the 13 C NMR spectrum of friedelin has been published, 26 but no such data has been published for the 1 H NMR spectrum (Table 1).
Table 1.Assignment of 1 H (500 MHz) and 13 C NMR (125 MHz) data of friedelin (12) in chloroform-d and literature 13 C NMR (100 MHz) data 1 H NMR (J = Hz) 13 C NMR 13   Other triterpenes were obtained as mixtures but were still able to be confidently identified by high field NMR and GCMS analysis.Lupenone (15) was obtained either as a mixture with friedelin (12) or with germanicone ( 16) and -amyrone (17), while lupeol (18) was only isolated as a mixture with glutinol (14).The characterisation of these compounds was possible due to key resonances associated with the olefinic and methyl group.The olefinic CH2 group characteristic of lupenone (15) and lupeol ( 18) is observed as signals at 4.63 (br s, H-29b) and 4.50 (br s, H-29a) ppm in the 1 H spectrum of 15 and at 4.67 and 4.57 ppm in the case of 18. Compounds 15 and 18 were identified in different fractions, characterised by their distinct 1 H NMR chemical shifts and molecular weight as determined by GCMS.Germanicone (16) and -amyrone (17) have endocyclic olefins at C-18 and C-12, respectively.The olefinic protons of these trisubstituted double bonds are observed characteristically as broad singlets at 4.88 (H-19) ppm for 16 and at 5.20 (br s, H-12) ppm for 17.][29][30] A mixture of lupenone (15) and germanicone (16), at a 1:1.5 ratio respectively, had a measured specific optical rotation in chloroform [a] +38 (c = 0.2 at 24 °C and 589 nm).The comparable literature values (chloroform, at 24 °C and 589 nm) are [a] +37 for 16 31 and [a] +64 for 15. 32 The fact that the sign of the specific rotation for this mixture is consistent with these literature values suggests that the configuration of both 1 and 2 is that previously reported in the literature as 3aR and 4aR, respectively.
Compounds 12 and 15-17 have not previously been reported from the same plant but have been postulated to belong to the same biosynthetic pathway.The previously postulated biosynthetic pathway by Thimmappa and colleagues 33 is shown (Figure 7) and supported here by isolation of 12 and 15-17 from the same plant species and indeed the same plant part.

Figure 7.
Biosynthetic pathway as proposed by Thimmappa et al. 33 with isolated compounds identified here shown in red.
Silva's investigation into Maytenus gonoclada identified lupeol (18), -amyrin (22) and friedelin (12) within this Brazilian relative of D. obscura, with no report of the presence of germanicone (16). 34A biochemical study into Maytenus ilicifolia, 35 characterised an oxidosqualene cyclase, dubbed friedelin synthase which produced 12 only; a mutant of this enzyme yielded -amyrin and friedelin (12).Since germanicone (16) and -amyrin (22) differ only in the position of their double bond and friedelin 12 and glutinol 14 differ in the position of a methyl group and an unsaturation we postulate that all of these compounds may either arise from a nonspecific triterpene cyclase or possibly different but related synthase enzymes.

Conclusions
Abietanes 1-7 and pentacyclic triterpenes 8-14 were isolated and elucidated from the methanolic extract of D. obscura root bark, with compounds 1-4, 6, 8-10, 13, and 14 also isolated from the extract of the seeds.Compound 3 was characterised and compound 7 was tentatively characterised as new natural products and named obscurol and 13-methoxy-sempervir-6-ene, respectively.With the traditional use, 36 the antimicrobial results presented here and previously studied activity of maytenoquinone (2), sugiol (6), pristimerin (8) and celastrol (9), 11 suggest the logical progression to this research is biological testing to ascertain other medicinal uses of this plant.
Qualitative comparison of the phytochemical composition across three different collection sites, provided data on some diagnostic compounds associated with D. obscura and highlighted some differences.For a more conclusive evaluation of phytochemical diversity, samples would need to be collected across multiple seasons and locations for statistically significant conclusions to be drawn.
It is suggested that the bright yellow of the root bark is the result of the particular composition of compounds that provide the best defence against soil borne herbivores/pathogens.The red coloration in the aril on the other hand would seem to be positively selected for because it is a signal to seed dispersers that a food source is available.Red is typically seen as a color indicating ripeness and hence palatability of fruit or seeds.
Compounds 12 and 14-21 were isolated from the leaves of D. obscura and characterised including support of the 1955 absolute configuration assignment for friedelin (12) by X-ray crystallography.As the absolute configuration of all compounds isolated appear to belong to the same enantiomeric series, the suite of compounds isolated provides strong support for the previously proposed biosynthetic pathway of this suite of compounds. 33

Biological testing
The antibacterial and antifungal activities were evaluated in a microtiter broth assay based on Clinical Laboratory Standards Institute (CLSI) Standard Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically 19 and Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 20he extracts were screened for activity against bacterial reference strains Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 25668), Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes (ATCC 19615) and yeast reference strain Candida albicans (ATCC 90029).The crude extracts were assessed in 96 well microtiter plates in a final volume of 150 µL at a final concentration of 20 mg/mL in 2% DMSO.Growth controls included microbe in growth media, microbe in growth media with 2% DMSO solution.Growth inhibition control included microbe in growth media with 0.1% povidone-iodine (w/v, Betadine, Sanofi).Escherichia coli and P. aeruginosa were inoculated in cation adjusted Mueller Hinton broth, S. aureus and S. pyogenes in Todd Hewitt Broth with 2% Yeast Extract at a final concentration of 5 × 10 5 CFU/mL.Candida albicans was inoculated in Sabouraud broth at a final concentration of 1.25 × 10 5 CFU/mL.Microbe growth assessed by optical density at 595 nm using a plate reader (Victor X2, Perkin Elmer).The increase in OD at 20 h for bacteria, and 24 h for yeast compared to the initial OD reading was used to determine the percentage growth inhibition compared to the growth control.Each extract and control was tested in triplicate in at least three independent experiments. 19,20ewitt family are acknowledged for their contribution in supporting the project on their country.The authors also acknowledge the financial contribution to this research from the Cooperative Research Centre for Developing Northern Australia through the project "Enabling a Traditional Medicinal Plants Agribusiness" (HT.2.1718007).

Figure 3 .
Figure 3. Phylogenetic distribution of Denhamia genus based on works by Mckenna 1 and Simmons.2
57"E) vouchered by specimen Church 57 and Deleye Outstation, NT (1345'21.72"S,12958'47.52"E,)vouchered by specimen Church 15.Seeds were collected from Henry Wrigley Dr, Eaton, NT (1224'01.0"S,13052'38.0"E)by Dr Gregory Leach and vouchered by specimen Leach 4780.Leaves were collected at Thorak Rd, Berrimah, NT (1225'53"S, 13057'0"E) vouchered by specimen Leach 4800.All collections were authenticated by Dr Gregory Leach.All voucher specimens are deposited at the Northern Territory Herbarium in Darwin, NT.Samples were collected under NT Parks & Wildlife permit 60819 and Northern Land Council research permit where required.This work was conducted as part of a project reviewed and approved by Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (HREC reference number2018-3263).