Gastroprotective Activity of Parastrephia quadrangularis (Meyen), Cabrera from the Atacama Desert

Forty-three metabolites including several methoxylated flavonoids, tremetones, and ent-clerodane diterpenes were accurately identified for the first time in the ethanolic extract of P. quadrangularis by means of hyphenated UHPLC-quadrupole Orbitrap mass spectrometry, and seven isolated compounds were tested regarding gastroprotective activity using the HCl/EtOH-induced lesion model in mice. A new tremetone (compound 6) is reported based on spectroscopic evidence. The isolated clerodanes and tremetones showed gastroprotective activity in a mouse model, evidenced by compound 7 (p-coumaroyloxytremetone), which showed the highest gastroprotective activity (76%), which was higher than the control drug lansoprazole (72%). Our findings revealed that several constituents of this plant have gastroprotective activity, and particularly, p-coumaroyloxytremetone could be considered as a lead molecule to explore new gastroprotective agents. This plant is a rich source of biologically active tremetones and terpenoids which can support the ethnobotanical use of the plant.


Introduction
Parastrephia quadrangularis (Meyen) Cabrera, (Figure 1) commonly known as Tola-Tola (Alpachtola or Burru suputula, Aymara names) since pre-Hispanic times, is a resinous shrub that grows up to 2 m high, typically found in dry semi-arid places in the Cordillera of the Central Andes in the Puna habitats, at altitudes of 3500 to 5000 m above the sea level.This plant is medicinal and used for gastrointestinal ailments, plus the treatment of urinary and respiratory diseases, fever, altitude sickness, and to treat bone dislocations and bruises [1][2][3][4], besides cattle feeding in the Atacama Desert [5].Furthermore, Parastrephia (tola) is an important highland genus of South American perennial plants in the aster (sunflower) family (Asteraceae) growing in the Altiplano of Chile, Bolivia, Argentina, and Peru.Many interesting bioactivities were reported for plants within this genus.For example, the plant Parastrephia lucida (Meyen) Cabrera is used in traditional medicine as an antiseptic and anti-inflammatory [6].The plant resin is also used for the healing of wounds and showed inhibition of arachidonic acid metabolism [7].The related plant P. lepidophylla showed antifungal activity on some phytopatogenic fungi of lemon [8] and inhibition of cell proliferation using Caco-2 cells [9].In addition, the infusions of P. lepidophylla and P. lucida showed a protective effect agaist oxidative damage on human erythrocytes [10].From these genera, antioxidant and analgesic tremetone (5-acetyl-2,3-dihydro-2-isopropenyl-benzofuran) derivatives were isolated [11,12].Moreover, bioactive constituents of snakeroot (Eupatorium rugosum) and several rayless goldenrods (especially Haplopappus heterophyllus) and other species are reported to be tremetones, causing milk sickness in humans and trembles in livestock [13,14].
Chile, Bolivia, Argentina, and Peru.Many interesting bioactivities were reported for plants within this genus.For example, the plant Parastrephia lucida (Meyen) Cabrera is used in traditional medicine as an antiseptic and anti-inflammatory [6].The plant resin is also used for the healing of wounds and showed inhibition of arachidonic acid metabolism [7].The related plant P. lepidophylla showed antifungal activity on some phytopatogenic fungi of lemon [8] and inhibition of cell proliferation using Caco-2 cells [9].In addition, the infusions of P. lepidophylla and P. lucida showed a protective effect agaist oxidative damage on human erythrocytes [10].From these genera, antioxidant and analgesic tremetone (5-acetyl-2,3-dihydro-2-isopropenyl-benzofuran) derivatives were isolated [11,12].Moreover, bioactive constituents of snakeroot (Eupatorium rugosum) and several rayless goldenrods (especially Haplopappus heterophyllus) and other species are reported to be tremetones, causing milk sickness in humans and trembles in livestock [13,14].Regarding the phytochemical components of P. quadrangularis, a poly-methylated flavonoid: 5,7dihydroxy-3,8,3′,4′-tetramethoxyflavone, some common coumarins and tremetones were reported [12,15].Recently, other tentative molecules (coumaroyloxytremetone-O-hexoside and coumaroyloxytremetone C-hexoside) found in a sample from Argentina were suggested using only low-resolution mass spectrometry [16].Some extracts of this species showed antifungal properties against Fusarium verticilloides which were attributed to the presence of tremetones in the active fractions of the plant [16].On the other hand, medicinal tinctures are extracts of the active metabolites of the most usable part of a medicinal plant; For this, an extraction method is used in which the plants are submerged or macerated for days in mixtures of edible ethanol water or edible pure ethanol [17,18].Moreover, one out of five persons suffer from ulcers associated to diet, stress, and certain drugs, due to an imbalance among aggressive factors (bile and hydrochloride acids, pepsins, hypoxia, drugs, and alcohol) and defensive factors (mucose blood flow, nitric oxide, sulfhydryl, growth factors bicarbonate, prostaglandins and mucus) in the stomach.Medicines used in the treatment of gastric ulcers are mainly H2-receptor antagonists, anti-acids, and proton-pump inhibitors and when the gastric ulcer is produced by Helicobacter pylori, antibiotics are included in the treatment.In this regard, numerous pharmacological agents with known anti-ulcer activity produce severe collateral effects, showing the need for new agents, including natural products which can be valuable as antiulcer agents [19][20][21].
Following our program to analyze and isolate interesting bioactive compounds from the Atacama Desert flora [22][23][24], we report in this study the gastroprotective activity of several compounds isolated from this plant; furthermore, the high resolution UHPLC fingerprinting analysis of the ethanolic extract (medicinal tincture) of this important Aymara plant is reported for the first time.Regarding the phytochemical components of P. quadrangularis, a poly-methylated flavonoid: 5,7-dihydroxy-3,8,3 ,4 -tetramethoxyflavone, some common coumarins and tremetones were reported [12,15].Recently, other tentative molecules (coumaroyloxytremetone-O-hexoside and coumaroyloxytremetone C-hexoside) found in a sample from Argentina were suggested using only low-resolution mass spectrometry [16].Some extracts of this species showed antifungal properties against Fusarium verticilloides which were attributed to the presence of tremetones in the active fractions of the plant [16].On the other hand, medicinal tinctures are extracts of the active metabolites of the most usable part of a medicinal plant; For this, an extraction method is used in which the plants are submerged or macerated for days in mixtures of edible ethanol water or edible pure ethanol [17,18].Moreover, one out of five persons suffer from ulcers associated to diet, stress, and certain drugs, due to an imbalance among aggressive factors (bile and hydrochloride acids, pepsins, hypoxia, drugs, and alcohol) and defensive factors (mucose blood flow, nitric oxide, sulfhydryl, growth factors bicarbonate, prostaglandins and mucus) in the stomach.Medicines used in the treatment of gastric ulcers are mainly H2-receptor antagonists, anti-acids, and proton-pump inhibitors and when the gastric ulcer is produced by Helicobacter pylori, antibiotics are included in the treatment.In this regard, numerous pharmacological agents with known anti-ulcer activity produce severe collateral effects, showing the need for new agents, including natural products which can be valuable as antiulcer agents [19][20][21].

Isolation and Identification of the Compounds in Parastrephia Quadrangularis Extract
Following our program to analyze and isolate interesting bioactive compounds from the Atacama Desert flora [22][23][24], we report in this study the gastroprotective activity of several compounds isolated from this plant; furthermore, the high resolution UHPLC fingerprinting analysis of the ethanolic extract (medicinal tincture) of this important Aymara plant is reported for the first time.
p-Cinnamoyloxytremetone (compound 6, Figure 3) IR (CCl4, neat) 1650 cm −1 (CO), 1685 (PhCO), 1610, 1590 (Ph).).HR-ESI-MS spectra: See Table 1.After data comparison with other tremetones [12], and examination of the NMR spectra we realized that compound 6 (Figure 2) showed similar NMR data to that reported for compound 7 [26], particularly the missing of a doublet of doublets in the proton spectra corresponding to the aromatic ring of the coumaroyl moiety (Table S1 and Figure S2, Supplementary Material) plus similar IR bands, but differences in the substitution pattern of only the aromatic protons of the cynnamoyl attached to the tremetone, in the 1 H-and 13 C-NMR spectra evidenced by 1-D and 2-D NMR analyses (HMBC, HMQC, Tables S1 and S2, Supplementary Material), which led to the identification of 6 as a p-cinnamoyloxyltremetone, a newly reported compound.

Coumarins
Peaks 42 and 43 were identified as umbelliferone and scopoletin by spiking experiments with authentic standards.Peak 2 with a pseudomolecular ion at m/z 353.02919 was identified as the dicoumarin euphorbetin (Figure S1b) [29].

Coumarins
Peaks 42 and 43 were identified as umbelliferone and scopoletin by spiking experiments with authentic standards.Peak 2 with a pseudomolecular ion at m/z 353.02919 was identified as the dicoumarin euphorbetin (Figure S1b) [ S1k).

Tremetones
Peak 27 with a [M -H] − ion at m/z: 363.12385 was identified as p-coumaroyloxyltremetone, identity confirmed by spiking experiments with a standard sample, and peak 37 with a [M -H] − ion at m/z: 347.12855 as a derivative of the latter, which was isolated and used as standard for spiking experiments (see experimental).

Gastroprotective Capacities of Isolated Compounds (1-7) from Parastrephia Quadrangularis
The results of the gastroprotective effects of compounds 1-7 in the HCl/EtOH-induced gastric lesion model are presented in Table 2.All compounds tested showed gastroprotective activity at a dose of 20 mg/kg (p.o) except compound 2 and 4. The best effect was shown by compound 7 (76%) which was close to that observed with lansoprazole (72%).In addition, the protection displayed by compound 6 (41%) was nearly half of that showed by the positive control, showing that an addition of an OH group in the cinnamoyl moiety for compound 7 is key for the bioactivity.Among the most lipophilics compounds (1-5), the lowest gastroprotective activity was evidenced by compound 5 (bacchalineol 18-O-acetate, 19%), compound 1 (bacchalineol, 12%), and compound 3 (bacchalineol methyl malonate, 11%).Compound 2 (bacchalineol malonic acid) and 4 (bacchalineol 18-O-malonate ethyl ester) did not show any significant difference with the control group.
The gastroprotective activity of several terpenoids have been reported in the literature.Among them, plaunotol, ferruginol and their derivatives, mulinane diterpenoids, dehydroabietic acid derivatives, carnosol and carnosic acid derivatives, labdane diterpenoids, poligodial sesquiterpenoids, oleanolic acid and their derivatives, suaveolol diterpenoid, ent-beyerene derivatives, and lupeol [35][36][37][38][39][40].The gastroprotective effects of these terpenoids reported in those studies were comparable with our results at the same oral dose.A high amount of compound 7 could explain in part for some of the putative medicinal properties assigned to this species.Further studies are necessary to explain the mechanism of action of compound 7.

Plant Material
Parastrephia quadrangularis (Meyen), Cabrera aerial parts were collected in El Tatio, Atacama Desert, in November 2015 at 4000 m.a.s.l. and were identified by the botanist Alicia Marticorena from the University of Concepción, Chile.Voucher herbarium specimens are kept at the Natural Products Laboratory of the Universidad de Antofagasta under reference number: PQ20151115.

Extraction
Dried and chopped aerial parts of P. quadrangularis (500 g) collected in Northern Chile were extracted with absolute ethanol (1 L, per 3 times in the dark, 24 h each time) to obtain a medicinal tincture, at room temperature.The tincture was then concentrated in vacuum below 40 • C to yield 36 g of a dark gummy extract.

UHPLC-PDA-MS Instrument
A Thermo Scientific Dionex Ultimate 3000 UHPLC system hyphenated with a Thermo Q exactive focus machine was used [24].For the analysis, 5 mg of the extract were dissolved in 2 mL of methanol, filtered (thorough PTFE filter) and 10 µL were injected in the instrument, with all specifications set as previously reported [24].

Animals
Animals were acquired from the Chilean Institute of Health, Chile, Santiago.Swiss albino mice weighing 30 ± 3 g were fasted for 24 h before the experiments.The animals were fed on certified Champion diet with free access to water under standard conditions of 12 h dark-light period, 50% relative humidity and room temperature (22 • C).The protocols were approved by the Animal Use and Care Committee of the Universidad de Chile (07022010) following the recommendations of the Canadian Council on Animal Care as stated previously [40].

Gastroprotective Effects
The gastroprotective activity of the compounds 1-7 was performed in the HCl/EtOH-induced lesion model as described previously [19].Briefly, mice were distributed into groups of seven animals each and fasted for 24 h with free access to water prior to the experiments.Fifty min after oral administration of the compounds (20 mg/kg), lansoprazole (20 mg/kg) or 12% Tween 80 (10 mL/kg), all groups were orally treated with 0.2 mL of a solution containing 0.3 M HCl/60% ethanol (HCl/EtOH) for gastric lesion induction.Animals were sacrificed 1 h after the administration of HCl/EtOH, and the stomachs were excised and inflated by injection of saline (1 mL).The ulcerated stomachs were fixed in 5% formalin for 30 min and opened along the greater curvature.Gastric damage visible to the naked eye was observed in the gastric mucosa as elongated black-red lines, parallel to the long axis of the stomach.The length (mm) of each lesion was measured, and the lesion index was expressed as the sum of the length of all lesions.

Statistical Analysis
The statistical analysis was carried out using the originPro 9.1 software packages (Originlab Corporation, Northampton, MA, USA).The determination was repeated at least three times for each sample solution.Analysis of variance was performed using ANOVA.Significant differences between means were determined by Dunnet comparison test (p values < 0.05 were regarded as significant).

Conclusions
The ethanol extract of an endemic species from the Atacama Desert showed several metabolites which were isolated using chromatography and detected using a hybrid UHPLC-PDA-OT-MS instrument.The isolated tremetones and clerodanes showed gastroprotective activity in a mouse model, evidenced by compound 7, which showed better gastroprotective capacity than the control drug lansoprazole (76%).The hyphenated machine equipped with orbitrap-PDA detectors and high-resolution collision cell is an outstanding tool for accurate and fast metabolomics analysis of the Atacama Desert flora, and allowed for the first time the identification of several ent-clerodane and kaurene diterpenes.P. Quadrangularis is rich in phenolic compounds and terpenoids and thus can be useful for the preparation of nutritional supplements.This study might support in part the putative medicinal properties of the plant as a gastroprotective agent.

Figure 1 .
Figure 1.Photographs of aerial parts of Parastrephia quadrangularis collected in El tatio, Atacama Desert, in November 2015.

Figure 1 .
Figure 1.Photographs of aerial parts of Parastrephia quadrangularis collected in El tatio, Atacama Desert, in November 2015.

Table 1 .
UHPLC PDA and HR-MS analysis of P. quadrangularis ethanol extract.
* Identification made using authentic standards.

Table 2 .
Gastroprotective effect of compounds isolated from P. quadrangularis at 20 mg/kg on HCl/EtOH-induced gastric lesions in mice.