Composition of Guayule (Parthenium argentatum Gray) resin

Guayule (Parthenium argentatum Gray) is a semi-arid shrub, native from the Chihuahan desert. This plant produces polyisoprene and resin. Polyisoprene is the main focal point of many researches, from structure to properties. Today, some processes are used to extract polyisoprene under its dry form, using solvent extraction, to produce rubber (used in truck or airplane tires) or as an emulsion, to make latex products by dipping (used in medical gloves, condoms, etc.). This article focuses on guayule resin which has some interesting applications in adhesives, coatings, pharmaceuticals, etc. In order to better know the resin composition and to be able to perform comparisons between varieties or seasons, liquid and gas chromatographic analysis methods have been described, for the groups of molecules composing the resin (polyphenols, guayulins, free fatty acids, di and triacylglycerols, argentatins, alkanes, alkanals, sugars, organic acids). Unlike other articles, this study aims to analyze all components of the same resin; the average composition of a guayule resin is given.

. Guayulins were detected in positive mode. Guayulin A and C were quantified by injection of cinnamic acid standard and guayulins B and D, by p-anisic acid standard. Free fatty acids, Di and Triacylglycerols. The same system UPLC-DAD-ESI-MSn was used to identify and quantify the neutral lipids in the extract with the same column. The temperature of the column was 40 °C and the total flow rate 0.4 mL/min. The initial mobile phase consisted of 100% solvent A (acetonitrile:water 80:20 10 mM ammonium formate) and 0% solvent B (isopropanol), which was run during 6 min. The percentage of solvent B was increased to reach 40% at 15 min, 90% at 17 min. The column was then equilibrated with the initial mobile phase composition prior to the next run. Identification was made by negative mode for all the fatty acids (M-H) − and positive mode for the acylglycerols, by detecting the adduct ion [M + NH 4 ] + . Quantification was made with trilinolein equivalent for the di and triacylglycerols and with all the fatty acids standards.
Argentatins were determined using a UPLC-MS system Waters with a C18 CSH Waters (100 m × 2.1 mm × 1.7 µm) column. The temperature of the column was 45 °C and the total flow rate 0.4 mL/ min. The initial mobile phase consisted of 95% solvent A (acetonitrile:water 80:20 and 0.1% formic acid) and 5% solvent B (acetonitrile: isopropanol 50:50 and 0.1% formic acid), which was run during 1.5 min. The percentage of solvent B was increased to reach 99% at 16.5 min, then hold up to 18.75 min and decreased to go back to the initial condition at 19 min. Argentatins A standard was used to quantify all argentatins. Data were acquired in positive ionisation mode in high resolution mode from 50 to 1200 m/z with a scan time of 0.08 s/scan using MSe (low energy 4 eV and high energy ramp from 25 to 50 eV). Source temperature was set up at 120 °C with a capillary voltage of 0.8 kV and a cone voltage of 40 V with a gas flow of 50 L/h. Desolvation temperature was 450 °C with a gas flow of 650 L/h. A solution of 1 ng/mL of Leu-Enk was infused at 10 µL/min during each injection in order to correct for accurate mass acquisition. Argentatin A was used to quantify Argentatin A, Incanilin, Argentatin C, Isoargentatin C and one unknown compound and Argentatin B was used to quantify all Argentatin B and Isoargentatins B isomers.
Alkanals and alkanes were determined using a GC-MS system (Shimadzu QP2010) with a ZB-5MS Phenomenex (30 m × 0.25 mm × 0.25 µm). Extracts in acetone were diluted with dichloromethane. A 2 µl sample was injected in split mode (ratio 10). The injection temperature was maintained at 300 °C, and Helium was the carrier gas at a constant flow rate of 1.3 mL.min −1 . The column temperature program consisted initial temperature 60 °C, increases at 20 °C/min to 200 °C, then 5 °C/min to 280 °C, then 3 °C/min to 320 °C, followed by an isothermal hold at 320 °C for 5 min. Mass spectra were recorded in Electronic Ionisation at 70 eV. The scan range was set from 40 to 600 m/z at 0.3 scan.s −1 . Alkanes were quantified by injection of standard mixture C10-C40 (Merck). When the alkane did not exist in the standard mixture, the closest alkane was used for the quantification. Docosanal was used to quantify all the alkanals.
Sugars, glycerol and organic acids were determined using a GC-MS system (Shimadzu QP2010, Kyoto, Japan) with a ZB-5MS Phenomenex (30 m × 0.25 mm × 0.25 µm). 50 µL of 10 mg/mL ASE resin in acetone solution were dried under nitrogen. 50 µL of 20 mg/mL methoxyamine in pyridine were added and heated at 80 °C during 30 min. Then of 80 µL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added and the mix was heated at 80 °C during 30 min.
A 2 µl sample was injected with split mode (ratio 10). The injection temperature was maintained at 300 °C and Helium was the carrier gas at a constant flow rate of 1.3 mL.min −1 . The column temperature program consisted of injection at 60 °C, temperature increases of 20 °C/min to 200 °C, then 5 °C/min to 280 °C, then 3 °C/min to 320 °C, followed by an isothermal hold at 320 °C for 5 min. Mass spectra were recorded in Electronic Ionisation

Results and discussion
20 samples of ASE resins from four-year-old plants of two different varieties: CL1 (corresponding to USDA 11591) and CL3 (corresponding to USDA N 593) and from the same field in Lansargues (south of France) were analyzed. The plant samples were collected and extracted from February to December. No trends were observed that could differentiate the two varieties and the results given are an average and biological range between shrubs (same age, two varieties).

Polyphenols and guayulins.
An analytical method to quantify polyphenols and guayulins (sesquiterpene esters) in the same run has been developed. In relation to polyphenolic compounds, six compounds that belong to the family of chlorogenic acids were found:  Table 1) Guayulins A and C consist of the chromophore of cinnamic acid and anisic acid respectively, bound to the patheniol form. Due to the absence of the hydroxyl group from the spatulenol form, their signal in positive mass detection is weak 4 . Guayulins B and C contain the presence of this hydroxyl group, but concentrations in samples are low.
A study of 13 cultivars of 4-year-old plants in India 12 found that Guayulin A content accounts for 0.7 to 5.9% of the ASE resin coming from the entire plant (and not only from stems) and Guayulin B content, between 0.1% and 1.8%. Guayulins are present at higher proportions in guayule stems 14 , so these results are consistent with those obtained here. As Rozalén describes 4 in an article, "guayulin similars" were also found in some samples but in too little amount to be quantified. This same team worked on neighbor joining trees clustering the content of guayulins A, B, C and D and total guayulins. They placed CL-1 (corresponding to CL1 here) and 593 (corresponding to CL3 here) on related branches. Model groups including CL1 and CL3 varieties, of two-year-old plants contain guayulin contents between 8.18 and 9.3% of the ASE resin. The results obtained are comparable with those given by previous authors even if quantification is different. They both used guayulins extracted in the laboratory as analytical standards.
As for polyphenols, less than 1% in total (0.01 to 0.9%) were found. To the authors' knowledge, the quantification of polyphenols in the ASE resin had not yet been carried out.     Neutral lipids comprise between 14.1% and 26.3%; 20.0 ± 5.1% of the ASE resin in average. Free fatty acids represent 34.5% of lipids (6.9 ± 1.8% of the resin) in average (ranging from 4.9 to 9.0% of the resin), diacylglycerols 5.0% (1.0 ± 0.2% of the resin; between 0.8 and 1.2% of the resin) and triacylglycerols 56% (12.2 ± 4.8% of the resin; between 4.3 and 18.6% of the resin). Linoleic, linolenic and palmitic acids are the major free fatty acids; diacylglycerol 18:2/18:2 is the mean diacylglycerol; triacylglycerols 18:3/18:3/18:2, 18:3/18:2/18:2 and 18:2/18:2/18:2 are the major triacylglycerols. For the first time, details of the composition of neutral lipids are given. This work completes Schloman team's identification of triacylglycerols and free fatty acids 15 : resin was obtained by percolation with acetone (after a first extraction with boiling water), from three and four-year-old 593 (corresponding to CL3 variety here) shrubs. A percentage of 13% of triglycerides in the resin was determined by high-efficiency gel permeation chromatography, with trilinolein as the external standard. Despite the slight difference which may be due to the extraction technique as well as the cultivation conditions, fatty acids found after saponification correspond to those of the triglycerides and free fatty acids: linoleic, linolenic, palmitic, oleic and stearic. Those fatty acids, in particular linolenic and linoleic, are also found in guayule rubber particles 30,31 under the triacylglycerols form, mainly linear and including mostly unsaturated but also in hydroxy-functional structures. The neutral lipids found in the resin might be derived from the rubber particles.

Argentatins.
A recent paper gives details of 12 cycloartane-and lanostane-type triterpenoids 21 . All these molecules are defined here as belonging to the argentatin family. A simple UPLC-HRMS method was developed to quantify the argentatin family. Low energy ionization was used so the fragments found differ from those given in previous publications.    21 (group D on Fig. 8, retention time: 9.3 min). (Table 3) Resins contain between 40.8% and 67.7% of argentatins/isoargentatins, 56.4 ± 7.6% in average. The major ones are argentatins/isoargentatins A and B, accounting for 29.9 ± 7.2% and 24.3 ± 6.9% in average respectively (Fig. 9) . Schloman 15 and his team found only 27% of total triterpenoids in a resin extracted with cold acetone (after previous boiling water extraction) from three and four-year-old 593 guayule plants (corresponding to CL3 variety here). This difference can be due to the extraction method. Indeed, Komoroski 19 writes that it is usually about 55% of argentins that is found in guayule resin from southern Texas and northern Mexico.
Alkanes and alkanals. Waxes were analyzed by gas chromatography in order to determine their composition. Alkanes and alkanals were recognizable with their spectra patterns: 57, 71, 85, 99 (alkanes), and 82, 96 and pseudomolecular ion (M − H 2 O) (alkanals). To quantify them, an alkane standard mixture and a dososanal standard were used. (Fig. 10 and Table 4). Between 2.3% and 8.3% of alkanals were found, 4.9 ± 1.4% in average. The major ones were tetracosanal and hexacosanal. Alkanes were present as traces (> 0.01%). Banigan 23 described 3-4% of "waxlike substance" in the leaves so our results seems to match with previous analysis. However Marwah 7 fractionated guayule wax and obtained 3% of alkanes ranging from C19 to C36, 92% of esters and a high polar part.
Sugars, glycerol and organic acids. Glucose and fructose have already been found in the bagasse 5 but not in the resin. Glycerol and organic acids have already been described in the resin. Sucrose was added to the identification list and sugars, organic acids and glycerol were quantified. ASE resin was derivatized in two steps: methoxamine hydrochloride (aldehyde and cetone function) and BSTFA (alcool and acid function): MS spectra were identified thanks to NIST database. (Fig. 11 and Table 5).
The percentage of each sugar is less than 1% but the average sum over all samples is 0.9 ± 0.4% (between 0.3 and 1.5%). The major ones are fructose and sucrose. Caffeic acid content is really low (> 0.2%). Cinnamic acid accounts for 4.1 ± 1.5% in average (between 1.6 and 6.5%). Glycerol content is 1.0 ± 0.8% (between 0.3 and 3.3%).

Monoterpenes and phytosterols.
As the plants were dried at 50 °C and stored at room temperature for a few days, the analysis of monoterpenes is not performed in this study. The volatile part of the ASE resin (mainly α and β-pinene) usually accounts for between 2 and 4% 25 . Analysis of monoterpenes and phytosterols (β-sitosterol and stigmasterol are present in guayule 15,32 ) can be carried out using GC.

Conclusions
This article provides chromatographic methods for the characterization of guayule resin, a complex mixture of organic chemicals produced by acetone extraction of the guayule plant. The work is of interest to researchers working with the crop, to those working in natural product characterization methods, and for general interest in plant secondary metabolites.
To the author's knowledge, it is the first time that the majority of compounds are analyzed on the same resin. Analytical methods used are principally MS methods, customized to allow detection of multiple metabolites simultaneously. Guayule resin contains hundreds to thousands of chemicals and with this work, supplemental informations are given (new polyphenols, identification of alkanes, alkanals, sugars, di and triacylglycerols) ( Table 6).   www.nature.com/scientificreports/ Samples of various varieties and differing plant age have been analyzed in order to ensure not to miss any important compounds. To give an idea of possible contents, average percentages are given for each family of molecules (Fig. 12). These percentages can help other scientists in their experiments on guayule and given methods allows them to analyze their own resin.
The stated objective (identifying and quantifying all components of the same resin) is mostly achieved: less than 5% of compounds remain unqualified and quantified. Knowing the composition of guayule resin and the quantity of each molecule is of great interest as many of them have a high added value: guayulins have shown repellent and anti-feedant activities 33 and argentatins could be used in cancer treatments 6 . The aim is now to extract and fractionate this resin to valorise it.

Data avilabilty
The datasets analyzed during the current study available from the corresponding author on reasonable request.