Mulinum crassifolium Phil; Two New Mulinanes, Gastroprotective Activity and Metabolomic Analysis by UHPLC-Orbitrap Mass Spectrometry

Mulinum crassifolium Phil. (Apiaceae) is an endemic shrub from Chile commonly used as infusion in traditional medicine to treat diabetes, bronchial and intestinal disorders and stomach ailments, including ulcers. From the EtOAc extract of this plant, the new mulinane-type diterpenoids 3 and 5 were isolated along with three known diterpenoids. The gastroprotective effect of the infusion of the plant was assayed to support the traditional use and a fast HPLC analysis using high resolution techniques was performed to identify the bioactive constituents. The EtOAc extract and the edible infusion showed gastroprotective effect at 100 mg/kg in the HCl/EtOH induced gastric ulcer model in mice, reducing lesions by 33% and 74%, respectively. Finally, a metabolomic profiling based on UHPLC-ESI-MS/HRMS of the edible infusion was performed and thirty-five compounds were tentatively identified including quercetin, caffeic acid, apigenine glucoside, p-coumaric acid, chlorogenic acids, and caffeoylquinic acids, which have been associated previously with gastroprotective and antiulcer properties. This scientific evidence can support the contribution of polyphenols in the gastroprotective activity of the edible infusion of this plant, and can validate at least in part, its ethnopharmacological use.

Liquid chromatography coupled to mass spectrometry is considered a powerful method of analysis in environmental, food, and antidoping tests, very important in forensic laboratories as well as in applied research. Analytical challenges and developments in performance have been found not only in ion sources, especially those using atmospheric-to-vacuum interfaces such as atmospheric pressure chemical ionization (APCI), or the most common electrospray (ESI) but also on the increasing use of high-resolution mass spectrometry (HR-MS) instead of low-resolution mass spectra (ion trap MS or triple quadrupole MS). The first HR instruments started with time-of-flight (TOF) instruments and has continued with the most expensive ion cyclotron Fourier transform (ICFT) or Orbitrap mass spectrometers hybridized with quadrupoles. Indeed, Q-TOF and Q-Orbitrap mass analyzers are the most commonly used with HPLC or UHPLC systems. In general, TOF instruments show a mass resolving power of 10,000-60,000 FWHM (full width at half maximum) with a mass accuracy of 1-5 ppm, while the Q-Orbitrap instrument can reach up to 450,000 FWHM at m/z 200 with <3 ppm mass accuracy. The increased selectivity and accuracy provided by HR-MS is particularly important when studying non-targeted complex organic biological mixtures, such as plants and food extracts. Accurate detection and quantitation rely on the differences between the compounds of interest from background ions and other interferences. Furthermore, the simplification of workflows and fast and flexible scanning functions make LC-HR-MS, particularly Orbitrap MS, suitable for a myriad of applications such as food, drugs, pharmaceuticals, antidoping, environment, and forensic analysis [10]. Orbitrap is particularly useful for the fast analysis of phenolics, fatty acids and diterpenes [11,12].
As part of our studies on mulinane-type diterpenoids from Apiaceae, we describe in this paper the isolation and structural elucidation of two new diterpenoids from M. crassifolium. In addition, we discuss here the gastroprotective action of the infusion of this plant (edible form), including a metabolomic profiling of it based on UHPLC-ESI-MS/HRMS for the first time.

Isolation of Diterpenoids
The air-dried plant of M. crassifolium was extracted with EtOAc at room temperature for nine days. The crude extract obtained after evaporation of the organic solvent was fractionated in four fractions by flash column chromatography (SiO 2 ). Each fraction was subjected to repeated permeation with Sephadex LH-20 and chromatography over silica gel to yield two new mulinane-type diterpenoids (3 and 5) along with three known diterpenoids ( Figure 1).
Compound 3 (Figure 1, 9,13-epoxymulin-11-en-20-oic acid) showed a molecular formula of C 22 (12.6; H-12). The 13 C-NMR signals suggested the presence of a carboxyl group at δ C 178.7, two oxygenated carbon at δ C 70.1 and 91.8 (ether bridge), and an alkene at δ C 134.9 (C-11) and 135.1 (C-12). In the HMBC spectrum, the methyl groups of the isopropyl (H 3 -18 and H 3 -19) showed cross peaks with C-3 and C-4, which placed the isopropyl group at position C-3. The methine H-10 showed correlations to C-5 and C-20, which placed the carboxyl group at position C-20. The methyl group H 3 -16 displayed connectivities with a quaternary carbon (C-13) and with carbons C-12 and C-14. In turn, H 3 -17 showed correlations to C-7, C-8, C-9, and C-15. Therefore, the ether bridge was placed at position C-9 and C-13 supported on the cross peak between H-12 and C-9. Analysis of the NOESY spectrum of the 3 did not show dipolar interaction between the signal at δ H 1.36 for H 3 -16 and the H 3 -17 methyl at δ H 1.06 indicating that they were not on the same face of the molecule. No correlation was observed between H-10 and H 3 -17. These data indicated that H-10 and H 3 -17 have αand β-configuration, respectively.
Compound 5 (Figure 1, 14α-hydroxymulin-12-en-11-one-20-oic acid) was isolated as a yellow gum. HRESIMS and NMR data were consistent with formula molecular of C 20 H 30 O 4 (m/z obsd 333.2076; calcd for 333.2071 [M − H] − ). The 1 H-NMR spectrum indicated the presence of the typical mulinane isopropyl group at δ= H 0.82 d and 0.97 d (3H each, J = 4.9), two methyl groups at δ H 1.02 s and 1.96 s, and one trisubstituted olefine at δ H 5.74 s, which showed to be another mulinane-type diterpenoid. The 1 H-NMR data were similar to those of 11α-hydroxymulin-12-ene-14-one-20-oic and 14α,17-diacetoxymulin-12-ene-11-one-20-oic acid [9,11]. The 13 C-NMR data of 5 showed signals at δ C 178.3 s (C-20) and 205.8 s (C-11) corresponding to carboxylic acid and ketone groups, respectively. Two carbons at δ C 125.3 d and 155.1 s confirmed the presence of a trisubstituted double bond in the seven-membered ring, while the presence of a signal at δ C 69.3 was associated to a secondary alcohol. In the HMBC spectrum, the methyl signal at δ H 1.96 s (H 3 -16) showed cross peaks with the carbon signals at δ C 69. The experiments sel-pfg-1D NOESY supported these findings. In these experiments, irradiation of H-14β resulted in NOE enhancements of the H 3 -17β and H-15β signals at δ H 1.02 and 2.62, respectively. In turn, irradiation of the H 3 -17β signal resulted in enhancement of the H-14β, H-15β and H-9β signals. Based on these data, compound 5 was identified as 14α-hydroxymulin-12-en-11-one-20-oic acid and had the same relative stereochemistry of previously reported mulinane skeletons ( Figures S3 and S4; Supplementary Materials). these data, compound 3 was identified as 9,13-epoxymulin-11-en-20-oic acid (Figures S1 and S2; Supplementary Materials).

Metabolomic Profiling of the Infusion by Using UHPLC-ESI-MS/HRMS
The aqueous infusion of chopped M. crassifolium Phil. (3.0 g, tea bag) was performed by maceration in 200 mL of distilled water at 85 °C. The edible aqueous infusion was lyophilized to obtain 0.3 g of extract. Afterwards, 10 mg of the extract was re-dissolved in fresh water, filtered and injected in the UHPLC-MS instrument (10 μL) to obtain the chromatograms ( Figure 2). This edible extract was chosen for metabolomic profiling due to the high biological activity it showed in the HCl/EtOH-induced gastric lesions model in mice.

Metabolomic Profiling of the Infusion by Using UHPLC-ESI-MS/HRMS
The aqueous infusion of chopped M. crassifolium Phil. (3.0 g, tea bag) was performed by maceration in 200 mL of distilled water at 85 • C. The edible aqueous infusion was lyophilized to obtain 0.3 g of extract. Afterwards, 10 mg of the extract was re-dissolved in fresh water, filtered and injected in the UHPLC-MS instrument (10 µL) to obtain the chromatograms ( Figure 2). This edible extract was chosen for metabolomic profiling due to the high biological activity it showed in the HCl/EtOH-induced gastric lesions model in mice.

Simple Organic Acids
Peak 2 was identified as malic acid (C 4 H 5 O 5 − ) and peak 3 as citric acid (C 6 H 7 O 7 − ) [12]. Malic acid is founded naturally in fruits such as apples and pears as well as in vegetables. Citric acid occurs in citrus fruits and is used as an acidifier, flavoring and chelating agent. Both compounds are sold as nutritional supplements and their production is industrial [13].

Fatty Acids
Peak 31 showing an ion at m/z 271.22803 was tentatively identified as hydroxypalmitic acid (C16H31O7 -).

Gastroprotective Activity
The two new mulinane diterpenoids were not evaluated as gastroprotective agents since the amount isolated was not enough. However, the organic extract (EtOAc-E) and edible infusion (INF-E) showed gastroprotective effect at 100 mg/kg in HCl/EtOH induced gastric ulcer model in mice, reducing lesions by 33% and 74%, respectively (Table 1). This dose was taken in account based on traditional medicine and to validate their ethnopharmacological effectiveness of these crude extracts. Thus, the gastroprotective effect of the edible infusion was related to the presence of secondary

Gastroprotective Activity
The two new mulinane diterpenoids were not evaluated as gastroprotective agents since the amount isolated was not enough. However, the organic extract (EtOAc-E) and edible infusion (INF-E) showed gastroprotective effect at 100 mg/kg in HCl/EtOH induced gastric ulcer model in mice, reducing lesions by 33% and 74%, respectively (Table 1). This dose was taken in account based on traditional medicine and to validate their ethnopharmacological effectiveness of these crude extracts. Thus, the gastroprotective effect of the edible infusion was related to the presence of secondary metabolites, mainly phenolics and some diterpenes, which were detected using UHPLC-ESI-MS/HRMS. During the last century, many reports have been published in relation to new gastroprotective agents from natural sources. Some examples such as carbenoxolone from Glycyrrhiza glabra, solon from sophoradin and gefarnate from cabbage have been marketed [31]. Some mulinane-type diterpenoids from Azorella species, desertic shrubs that are closely related to M. crassifolium, and are generally called "Yaretas" by local people, have shown gastroprotective activity on the HCl/EtOH-induced gastric lesion model in mice. Among the compounds studied, azorellanol, 13β-hydroxyazorellane and mulin-11,13-dien-18-acetoxy-16,20-dioic acid showed gastroprotective effect, being as active as lansoprazole and reducing the gastric lesions by at least 69%. The same study demonstrated that the mulinane diterpenoids have increased the activity due to the presence of an additional carboxylic acid [32]. In another study, the hydro-carbonated compounds mulin-11,13-diene, mulin-11,13-dien-20-ol, and mulin-11,13-dien-20-oic acid were less active than lansoprazole reducing the gastric lesions by 18%, 26% and 39%, respectively [32,33]. Regarding the mode of gastroprotective action for mulin-11,13-dien-20-oic acid evaluated at 55 mg/kg, its effect was reduced by pre-treatment with indomethacin and N-ethylmaleimide, suggesting that prostaglandins and sulfhydryl compounds are positively involved in the gastroprotective activity of this mulinane diterpenoid [11]. Taking into consideration that many crude drugs have shown gastroprotective activity [29,[34][35][36], we decided to evaluate the edible extracts obtained from M. crassifolium since it is used in folk medicine to treat stomach sickness [1]. Metabolomic profiling of the edible infusion (74% gastroprotection) shows the presence of polyphenols and terpenoids ( Table 2) and it is well known that phenolics have a beneficial role in disease prevention as well as in gastroprotection [29,35]. Moreover, it has been suggested that phenolic compounds stimulate PGE 2 formation, decrease of histamine secretion, and inhibit H. pylori, which explain the gastroprotective effect [29,37,38]. The presence of those compounds in the infusion of M. crassifolium associated to the gastroprotective effect could be due to the synergistic effect produced by the compounds detected. In fact, quercetin (peak 27), caffeic acid (peak 17), apigenine glucoside (peak 20), p-coumaric acid (peak 21), chlorogenic acid (peak 8), and caffeoylquinic acids (peaks 11, 13, 15 and 23-25) have been associated with ulcer preventing capacity [39][40][41][42]. We thus demonstrated that the edible infusion at 100 mg/kg had gastroprotective effect, which could be linked to the presence of several phenolic compounds that could increase the activity of the diterpenoids, but further studies are required to isolate and evaluate the individual gastroprotection of these compounds. The results are expressed as mean ± sem * p < 0.01, significantly different compared with the control; ** p < 0.01, significantly different compared with lansoprazole (ANOVA followed by Dunnett's test). n = number of mice.

Plant Material
Mulinum crassifolium were collected in 2016 at "El Tatio" (Antofagasta, II Región, Chile) and identified by Prof. J. Bórquez. A voucher specimen (Nº 160316) is kept at the Natural Product Lab of the University of Antofagasta.

UHPLC-ESI-MS/HRMS Studies
For this study, 3.0 g of dried and chopped aerial parts were macerated with ethyl acetate (3 times, 50 mL at 25 • C), and an infusion prepared using 3.0 g of dried chopped aerial parts adding deionized water (200 mL) at 85 C, for 1 h. The solvents were concentrated in vacuo at 45 • C and the infusion lyophilized (Labconco, MA, USA) to obtain 125, and 300 mg of EtOAc and edible aqueous extracts, respectively.
A UHPLC Thermo Scientific Ultimate 3000, with all accessories including a quaternary pump, TCC-3000RS vial compartments, WPS-3000RS autosampler and PDA detector with Chromeleon 7.2 Software (Thermo Fisher Scientific, Germany) was connected to a Thermo high-resolution Q Exactive focus spectrometer (Thermo, Bremen, Germany) by means of an Electrospray Ionization Source II (HESI II). The nitrogen was produced by a Genius NM32LA nitrogen generator (purity > 99.999%, Peak Scientific, Billerica, MA, USA) and used as both damping and collision gas. Mass calibration for Orbitrap was performed every day, in both negative and positive modes, to ensure a mass accuracy lower than 5 ppm.
Chromatography was run using a C18 column (25 mm ID, Acclaim, 150 mm × 4.6 mm, Thermo Fisher Scientific, Bremen, Germany) at 25 • C. The detection used 354, 254, 280, and 330 nm, and PDA recorded from 200 to 800 nm. Mobile phases were formic aqueous formic acid (0.1% A) and acetonitrile formic acid 0.1% (B). The gradient program started at 5% B, maintaining for 5 min, then up to 15% B for 10 min, then to 30% B until 15 min, then 70% B until 25 min, then coming back to 5 min at 35 min., and waiting 12 min using 5% B for column equilibration before each injection, at a flow rate of 1.00 mL min −1 , using an injection volume of 10 µL. Standards and the extracts were kept in vials at 10 • C during storage.
The HESI parameters were: 75 units of sheath gas flow rate; 20 units of auxiliary gas unit flow rate; 400 • C of capillary temperature; 500 • C auxiliary gas heater temperature; 2500 V of spray voltage; S lens; and RF level 30. Full scan data were acquired at 70,000 FWHM (full width half maximum) at m/z 200. A scan range of m/z 100-1000 was chosen; the automatic gain control (AGC) was set at 3 × 10 6 and the injection time was set to 200 ms. The scan-rate was set at 2 scans s −1 . External calibration was performed using a calibration solution in positive and negative modes, with the Orbitrap spectrometer operating both in positive and negative modes at 17,500 FWHM (m/z 200). The AGC target was set to 2 × 10 5 , with injection time of 20 ms. The ultrahigh vacuum, fore vacuum and high vacuum and were maintained at 1010, 2, and 105 mbar, respectively. Collision energy in the HCD cell was operated at 30 kv. Detection operated on calculated exact mass and on retention time of compounds, as shown in Table 2. The tolerance window for mass detection was set to 5 ppm for the two modes.

Animals
Swiss albino mice were acquired at the Instituto de Salud Pública de Chile, Santiago, Chile. Mice weighing 30 ± 3 g were fasted for 24 h before the tests. The animals were fed on Champion certified diet with access to free water under 12 h dark-light period at standard conditions, 50% humidity and 22 • C of temperature. The protocols were approved by the Universidad de Chile (Animal Care Committee) that follows the recommendations of the Canadian Council on Animal Care and with the ethical guidelines for investigations in conscious animal.

HCl/EtOH-Induced Lesions in Mice
The gastroprotective activity of the extracts was assessed in the HCl/EtOH-induced lesion model, as already described [43]. Mice were randomly distributed into groups of seven animals each and fasted for 24 h with free access to water prior to the experiment. Fifty minutes after oral administration of the extracts (100 mg/kg), lansoprazole (30 mg/kg) or water (control), all groups were treated orally with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for the induction of gastric lesion. Approximately 1 h after the administration of HCl/EtOH, Animals were sacrificed and the stomachs were inflated and excised by injection of 1 mL of saline solution. A concentration of 5% formalin was used to fix the ulcerated stomachs for 30 min, which were opened along the greater curvature. Visible gastric damage was then observed in the gastric mucosa as elongated black-red lines, parallel to the long axis of the stomach similar to the HCl/EtOH-induced lesions in rats. The length in mm of each lesion was measured, and the sum of the length of all lesions was expressed as lesion index.

Statistical Analysis
Results were expressed as the mean ± S.D. In all experiments, statistical differences between treatments and their respective control were determined by one-way analysis of variance (ANOVA) followed by Dunnett's test. The level of significance was set at p < 0.01. All statistical analyses were performed using the software GraphPad Prism 4 for Windows.

Conclusions
In the present study, we isolated two new mulinane-type diterpenoids from the ethyl-acetate extract from M. crassifolium using a combination of chromatographic techniques and identification by NMR. In addition, we prepared an infusion (edible aqueous form) and both organic and water extracts were evaluated as gastroprotective agents using the HCl/EtOH-induced gastric lesion model in mice. Bioactivity assay results show that the infusion protected the gastric lesions by 74%, while the organic extract by 33%. Finally, to correlate the gastroprotective activity with the presence of bioactive compounds, some UHPLC-ESI-MS/HRMS analysis was performed. Thirty-five compounds were tentatively identified, being mostly polyphenolics the main compounds. Among the compounds identified, quercetin, caffeic acid, apigenine glucoside, p-coumaric acid, chlorogenic acid, and caffeoylquinic acids have been previously associated with gastroprotective and antiulcer properties. Gastric ulcers are becoming a serious public health problem due to stress and other factors and new therapeutic agents for the prevention of them are necessary. Our findings based on traditional medicine provide the basis for the utilization of M. crassifolium as a source of potential compounds for the prevention of gastric ulcers. Moreover, these extracts can be utilized as a natural alternative to synthetic gastroprotective agents for the use in the current therapy.