molecules Article Mulinane and Azorellane Diterpenoid Biomarkers by GC-MS from a Representative Apiaceae (Umbelliferae) Species of the Andes Bernd R.T. Simoneit 1,*, Daniel R. Oros 2, Rudolf Jaffé 3 , Alexandra Didyk-Peña 4,*, Carlos Areche 5, Beatriz Sepúlveda 6 and Borys M. Didyk 7 1 Department of Chemistry, College of Science, Oregon State University, Corvallis, OR 97331, USA 2 Consultant, 72 Marina Lakes Drive, Richmond, CA 94804, USA; [email protected] 3 Southeast Environmental Research Center and Department of Chemistry and Biochemistry, Florida International University, 3000 NE 151st Street, North Miami, FL 33181, USA; jaffer@fiu.edu 4 Escuela de Química y Farmacia, Facultad de Medicina, Universidad Andres Bello, Viña del Mar 2520000, Chile 5 Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago 8320000, Chile; [email protected] 6 Departamento de Ciencias Químicas, Universidad Andres Bello, Viña del Mar 2520000, Chile; [email protected] 7 Consultant, Casilla 942, Viña del Mar 2520000, Chile; [email protected] * Correspondence: [email protected] (B.R.T.S.); [email protected] (A.D.-P.); Tel.: +1-541-737-2081 (B.R.T.S.) Academic Editor: Motoo Tori Received: 16 January 2019; Accepted: 13 February 2019; Published: 14 February 2019 Abstract: Extracts of bled resin from Azorella compacta, of the Azorelloideae family from the Andes (>4000 m), were analyzed by gas chromatography-mass spectrometry. The mass spectra of the dominant compounds of the resin and its hydrogenation products were documented. The most abundant compounds were oxygenated diterpenoids, namely mulinadien-20-oic (D11,13 and D11,14) acids, azorell-13-en-20-oic acid, 13α,14β-dihydroxymulin-11-en-20-oic acid, and azorellanol, with a group of azorellenes and mulinadienes. The mass spectra of the novel diterpenoid hydrocarbons with the azorellane and mulinane skeletons were also presented. This study documents the molecular diversity of these diterpenoid classes, and could be of great utility for future organic geochemical, environmental, archeological, pharmaceutical, and forensic chemistry studies. Keywords: Azorella compacta; diterpenoids; GC-MS; standards 1. Introduction Natural products or their derivatives in the ambient environment or geological record are used by organic geochemists as tracers for sources, transport and alteration processes of organic matter, e.g., [1–7]. Remote locales, for example, the high Andes region in South America, are ideal sites as pristine references for background studies of such processes. An early report on the lipids and biomarkers in sediment from Lake Lejia in a caldera in Antofagasta, Chile, found traces of diterpenoids typical of resinous vegetation [8]. The region is barren of trees, especially gymnosperms that produce resin, and potential sources such as the Araucariaceae grow far to the south [9]. Subsequently, we reported on the lipid and resin composition of a dried sample of Laretia compacta (common name Llareta) from San Pedro de Atacama [10]. The dried material had a strong resinous odor and thus was implied as a possible source of diterpenoids in the Altiplano environment. The extract yield of the whole plant based on dry weight comprised 4.2 mg/g (0.2%) lipids and 2.5 mg/g (0.12%) Molecules 2019, 24, 684; doi:10.3390/molecules24040684 www.mdpi.com/journal/molecules Molecules 2019, 24, x 684 FOR PEER REVIEW 22 of of 15 extract yield of the whole plant based on dry weight comprised 4.2 mg/g (0.2%) lipids and 2.5 mg/g (0.12%)terpenoids terpenoids [10]. The [10]. terpenoid The terpenoid distribution distribution consisted consisted of mono-, of sesqui- mono‐ and, sesqui diterpenoids,‐ and diterpenoids, and the latter and theare latter of interest are of here interest because here tri- because and tetracyclic tri‐ and tetracyclic diterpenoids diterpenoids were the were dominant the dominant compounds. compounds. Laretia compacta is now called called AzorellaAzorella compacta compacta ofof the the Apiaceae:Azorelloideae Apiaceae:Azorelloideae family, family, also known as Umbelliferae [11]. [11]. These plants grow slowly at high altitudes (>4000 m) in in the northern Andes of Chile and Peru as dense dense pillow pillow-like‐like structures structures close close to to the the ground ground with with subterranean subterranean stems stems and and roots. roots. The Azorelloideae family evolved from thethe firstfirst UmbelliferaeUmbelliferae duringduring thethe Cretaceous,Cretaceous e.g., [12,13]. [12,13]. Azorella sp. have historically been used on the Altiplano as firewood firewood and cooking fuel by local cultures, andand laterlater in in early early industrial industrial and and mining mining activities activities as as fuel, fuel, which which contributed contributed to its to scarcity its scarcity and andcurrent current protection. protection. The speciesThe species is now is now more more common, common, and represents and represents the dominant the dominant biomass biomass in these in Andeanthese Andean environments. environments. The plants The andplants resin and exudates resin exudates have been have used been by Andeanused by cultures Andean for cultures medicinal for medicinalpurposes where, purposes for example,where, for hot example, water infusions hot water serve infusions as herbal serve remedies as herbal for various remedies ailments for various [14,15]. ailmentsConsequently, [14,15]. natural Consequently, product characterizationnatural product andcharacterization pharmacological and pharmacological studies have been studies carried have out beenon A. carried compacta outand on related A. compacta species. and Numerous related species. investigations Numerous reported investigations structure determinations reported structure and determinationspharmacological and potential pharmacological studies of potential the resin studies compounds, of the thatresin were compounds, isolated that and were purified isolated from and the purifiedmajor Andean from the Apiaceae major Andean species, Apiaceae namely Mulinum species, namelyand Azorella Mulinum[16– and35]. Azorella This has [16–35]. led to a This description has led toof a mainly description two novel of mainly diterpenoid two novel skeletons diterpenoid among skeletons the natural among products, the natural specifically products, the specifically mulinanes theand mulinanes azorellanes and (Figure azorellanes1), with the(Figure assignment 1), with of theirthe assignment skeletal numbering of their conventionsskeletal numbering [ 16,21]. conventionsThe biosynthetic [16,21]. pathway The biosynthetic for the mulinane pathway and for azorellane the mulinane diterpenoids and azorellane has been diterpenoids proposed as has starting been proposedfrom a phytatetraene as starting from [24]. a Laboratory phytatetraene syntheses [24]. Laboratory of polar mulinane syntheses diterpenoids of polar mulinane have diterpenoids also recently havebeen reportedalso recently [36, 37been]. reported [36,37]. Skeleton numbering convention 16 16 16 16 12 12 12 12 13 13 13 13 11 11 14 14 11 11 17 17 17 14 17 14 1 9 1 9 9 1 9 10 10 1 10 15 15 10 15 15 8 8 8 2 2 8 5 5 2 2 3 3 5 5 7 7 3 7 3 7 6 6 4 4 4 6 4 6 18 20 18 20 18 20 18 20 19 19 19 19 13(H)-Mulinane 13(H)-Mulinane 13(H)-Azorellane 13(H)-Azorellane Figure 1. CarbonCarbon numbering numbering convention for the mulinane and azorellane skeletons. Here, we characterize the molecular composition of Azorella compacta resin based on mass spectrometric interpretation, interpretation, and and correlation correlation and and comparison comparison with with known known standards. standards. We Wereport report the massthe mass spectra spectra and and gas gas chromatography chromatography-mass‐mass spectrometry spectrometry (GC (GC-MS)‐MS) characteristics characteristics of of the the dominant dominant compounds of of the the resin resin and and of ofthe the hydrogenated hydrogenated resin resin as the as thefree freeand andderivatized derivatized products. products. This informationThis information is unique, is unique, because because prior prior reports reports in inthe the natural natural product product literature literature only only provided provided mass spectrometric data on some compounds. Our data further documents the molecular diversity of this novel diterpenoidditerpenoid class class (mulinane (mulinane and and azorellane), azorellane), information information which which could could be of be great of great utility utility for future for futureorganic organic geochemical, geochemical, environmental, environmental, archeological, archeological, pharmaceutical, pharmaceutical, and forensic and chemistryforensic chemistry studies. studies. 2. Results and Discussion 2. ResultsThe relative and Discussion composition of the fresh resin was 1% sesquiterpenoid and 99% diterpenoids, excludingThe relative a low amountcomposition of bornyl of the acetate fresh resin (<0.5%). was No1% lipidsesquiterpenoid components and from 99% plant diterpenoids, wax were excludingdetected. Ita islow of amount interest toof notebornyl the acetate breakdown (<0.5%). of theNo diterpenoidslipid components into 19% from hydrocarbons plant wax were and detected.80% polar It oxygenated is of interest compounds, to note the breakdown and the same of the proportions diterpenoids were into retained 19% hydrocarbons upon hydrogenation. and 80% polarThis observationoxygenated wascompounds, unexpected and becausethe same most proportions bled conifer were resinsretained are upon characterized hydrogenation.