UHPLC-MS Phenolic Fingerprinting, Aorta Endothelium Relaxation Effect, Antioxidant, and Enzyme Inhibition Activities of Azara dentata Ruiz & Pav Berries

Azara dentata Ruiz & Pav. is a small Chilean native plant from Patagonia, a producer of small white reddish berries. For the first time, the proximal analysis of the fruits, phenolic fingerprinting, the antioxidant activity, and the enzymatic inhibition and relaxation effects in rat aorta induced by the ethanolic extract of these fruits were investigated. The proximal composition and the mineral (Ca: 2434 ± 40 mg/kg; Mg: 702 ± 13 mg/kg; Fe: 117.1 ± 1.6 mg/kg; Zn: 16.1 ± 0.4 mg/kg) and heavy metal (As: 121 ± 11 µg/kg; Cd: 152 ± 5 µg/kg; Hg: 7.7 ± 1.3 µg/kg; Pb 294 ± 4 µg/kg) contents were analyzed. Anthocyanins, flavonoids, phenolic acids, and coumarins were identified using UHPLC-PDA-QTOF-MS. The ethanolic extracts showed a total phenolic content of 23.50 ± 0.93 mg GAE/g extract. In addition, the antioxidant activity was assessed using both DPPH and TEAC (28.64 ± 1.87 and 34.72 ± 2.33 mg Trolox/g of dry fruit, respectively), FRAP (25.32 ± 0.23 mg Trolox equivalent/g dry fruit), and ORAC (64.95 ± 1.23 mg Trolox equivalents/g dry fruit). The inhibition of enzymatic activities (acetylcholinesterase IC50: 2.87 + 0.23 µg extract/mL, butyrylcholinesterase IC50: 6.73 + 0.07 µg extract/mL, amylase IC50: 5.6 ± 0.0 µg extract/mL, lipase IC50: 30.8 ± 0.0 µg extract/mL, and tyrosinase IC50: 9.25 ± 0.15 µg extract/mL) was also assessed. The extract showed 50–60% relaxation in rat aorta (intact), mediated thorough the release of endothelial nitric oxide. Our results suggest that A. dentata is a good source of compounds with the capacity to inhibit important enzymes, can be hypotensive, and can thus have good potentiality as supplements in the amelioration of neurodegenerative diseases and could also have potential to be used to develop new functional foods. The study highlights the benefits of these neglected small fruits and could boost their consumption.


Introduction
In the last few years, the search for plant extracts rich in polyphenolic compounds from local plants in special ecosystems such as the Valdivian Forest or the Atacama gerprinting and antioxidant activity of crude ethanolic extracts of A. dentata fruits; to evaluate the enzymatic inhibitory potential against several enzymes, (cholinesterases, tyrosinase, lipase, amylase, and glucosidase), which are implicated in several NCDs such as neurodegenerative diseases (Alzheimer's and Parkinson's disease) and metabolic syndrome; and finally, to evaluate the hypotensive potential of the A. dentata fruits' ethanolic extract in rat aorta obtained from a normotensive animal model. The fruit extracts were studied using ultra-high-resolution chromatography (Ultra HPLC) coupled with mass spectrometry (Q-TOF-M-S), which is a rapid technique which provides precise information regarding the chemical profile of several phenolic compounds in food sources [13,14]. Using this technique, our group has explored the chemistry of Chilean plants, shrubs, and fruits combined with their antioxidant and enzyme inhibitory capacities [15,16].The aims of this study are: (i) to perform a proximal analysis and determine the mineral content and the content of heavy metals of A. dentata fruits; (ii) analyze the phenolic fingerprinting and the antioxidant activity of the crude ethanolic extracts of A. dentata fruits; (iii) evaluate the enzymatic inhibitory potential against several enzymes, such as cholinesterases, tyrosinase, lipase, amylase and glucosidase, which are implicated in several NCDs such as neurodegenerative diseases (Alzheimer's and Parkinson's disease) and metabolic syndrome; iv) evaluate the hypotensive potential of the A. dentata fruits' ethanolic extract in rat aorta obtained from a normotensive animal model. For the first time, the previously mentioned bioactivities for A. dentata fruits are reported.

Chemicals
Purified water (TOC<5 µg/L) was obtained from a reverse osmosis system (Arium 126 61316-RO and Arium 611 UV unit (Sartorius, Goettingen, Germany)). Methanol (HPLC grade) and formic acid (for mass spectrometry, puriss. P.a.) were obtained from J. T. Baker (Phillipsburg, NJ, USA). Folin-Ciocalteu (FC) reagent, 2,2-diphenyl-1picrylhydrazyl (DPPH), ferric chloride hexahydrate, 2,4,6-tris(2-pyridyl)-s-triazine, Trolox, quercetin, gallic acid, Amberlite ® resin (XAD4), dimethyl sulfoxide (DMSO), acetylcholinesterase (AchE), butyrylcholinesterase (BchE), phosphate buffer, L-DOPA, tyrosinase, kojic acid, and trichloroacetic acid were obtained (Merck, Darmstadt, Germany). HPLC standards (quercetin and gallic acid purified to 95% and up via HPLC) were purchased from Sigma-Aldrich Chem. Co. (St Louis, MO, USA) and Extrasynthese The fruit extracts were studied using ultra-high-resolution chromatography (Ultra HPLC) coupled with mass spectrometry (Q-TOF-M-S), which is a rapid technique which provides precise information regarding the chemical profile of several phenolic compounds in food sources [13,14]. Using this technique, our group has explored the chemistry of Chilean plants, shrubs, and fruits combined with their antioxidant and enzyme inhibitory capacities [15,16].The aims of this study are: (i) to perform a proximal analysis and determine the mineral content and the content of heavy metals of A. dentata fruits; (ii) analyze the phenolic fingerprinting and the antioxidant activity of the crude ethanolic extracts of A. dentata fruits; (iii) evaluate the enzymatic inhibitory potential against several enzymes, such as cholinesterases, tyrosinase, lipase, amylase and glucosidase, which are implicated in several NCDs such as neurodegenerative diseases (Alzheimer's and Parkinson's disease) and metabolic syndrome; (iv) evaluate the hypotensive potential of the A. dentata fruits' ethanolic extract in rat aorta obtained from a normotensive animal model. For the first time, the previously mentioned bioactivities for A. dentata fruits are reported.

Plant Material
A. dentata fruits were collected by hand in the Calera de Tango (−33, Chile. The voucher specimen AS-012052022 was coded and maintained in the Natural Products lab of the Universidad Austral de Chile, in Valdivia, Chile.

Extraction Procedure
One hundred grams of sample was lyophilized (Labconco Freeze Dry Systems, Model 7670541 2.5 Liter Palo Alto, CA, USA), and then, the dehydrated fruit material was milled using a Grindomix blade mill (GM 200, Retsch, Haan, Germany); the fine powder was sieved trough a stainless steel sieve No. 100 (150 µm) and wrapped in plastic bags and stored at −20 • C. Afterward, 5 g of powder was disposed in a flask, and then, 20 mL of ethanol with 1% formic acid was added as an extraction solvent and protected from the light; to obtain the extract, the flask was placed in an ultrasonic water bath (UC-60A Biobase medical equipment, Shangai, China) at room temperature for 30 min. Three extractions were performed with the same material. The extraction was filtered, and the solvent was removed with a rotary evaporator at 36 • C to obtain the crude extract (125 mg).

Determination of Proximal Composition
AOAC procedures were employed for the physicochemical determinations [17,18]. The moisture content was obtained using the oven method, and the crude protein was obtained with the Kjeldahl (N × 6.25) method using a Kjelmaster k375 (Büchi AG, Flawil, Switzerland) apparatus. The fiber content was determined via the gravimetric method followed by the hydrolysis of the samples, and the total lipid content was obtained via Soxhlet methodology (Sigma-Aldrich, St. Louis, MO, USA) by performing extractions with petroleum ether. The ash content was analyzed by employing a muffle furnace for incineration at 550 ± 15 • C. Total carbohydrates were obtained by difference using the equation: 100 − (g fat +g water +g fiber + g protein + g ash). The results are indicated in grams per 100 g of fresh weight (g/100 g fw).

Determination of Heavy Metals and Multi-Element Determination via ICP-MS
The minerals and heavy metals were analyzed according to the methodology reported previously [19,20]. For the digestion of the fruit samples, approximately 10 mg of the samples was digested with 0.5-1 mL of HNO 3 69% in a microwave oven, at a temperature of 180 • C, and taken to a final volume of 5 mL with purified water and then sent to the multielement determination. The ICP-MS equipment used was an Agilent 7900, equipped with a Micromist-type concentric nebulizer, a Scott-type spray chamber, platinum interface cones, an off-axis double lens system, a hyperbolic quadrupole as a mass filter and collision cell, and an octopolar reaction that uses He and H 2 gases, with a 15-place rotor. The standards were 20 µg/g Ge, Rh, and Ir from ISC Science and Ca, Mg, Fe, Zn, As, Cd, Hg, and Pb certified to 10,000 µg/mL by High-Purity Standards.

Determination of Total Phenolic and Flavonoid Content
The total phenolic content (TPC) was determined using the colorimetric method with Folin-Ciocalteu' reagent, and the results are expressed as mg of gallic acid equivalents (GAE) per g of dry fruit (mg GAE/ g). The total flavonoids content (TFC) was determined using the aluminum chloride method, and the results are expressed as mg of quercetin equivalents (QE) per g of dry fruit (mg QE/ g) [21]. Both assays were carried out in a microplate and measured in triplicate using a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA). The values are shown as the mean ± standard deviation (SD).

DPPH Scavenging Activity
The potential of A. dentata extract dissolved in methanol as a free radical scavenger was determined using the DPPH assay in a microplate following the procedure previously reported [22]. This analysis was performed at 517 nm in triplicate using a FC Microplate Multiskan spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the results are reported as the mean ± SD expressed in mg Trolox equivalent per gram of dry fruit.

Ferric-Reducing Antioxidant Power Assay (FRAP)
A FRAP assay of A. dentata extract was performed as previously described [23] and by considering details from other reports [24]. For the preparation of the FRAP solution, 10 mL of acetate buffer (300 mM, pH 3.6) was mixed with 1 mL of FeCl 3 (20 mM) (dissolved in water) and 1 mL of 2,4,6-tris(2-pyridyl)-s-triazine (10 mM) dissolved in HCl 40 mM. Then, 10 µL of sample was mixed with 190 µL of FRAP solution. The results were obtained via linear regression from a calibration graph obtained with Trolox (0-1 mM). The assay was performed in triplicate, and the results are reported as the mean ± SD expressed as mg of Trolox equivalent (TE) per gram of dry fruit.

Trolox Equivalent Antioxidant Activity (TEAC) Assay
The TEAC assay was performed according to a previous study [25] with some modifications. First, 10 µL of the sample was mixed with 200 µL of ABTS •+ . The absorbance was measured at 734 nm at 30 • C. The results were interpolated in a calibration curve of Trolox (0-1 mM). The results are expressed as mg of Trolox equivalents (TE) per gram of dry fruit; all the assays were performed in triplicate, and the values are reported as the average ± SD.

Assay of Oxygen Radical Absorbance Capacity (ORAC)
The ORAC capacity was determined as previously described [26]. The reaction was prepared with 50 µL of fluorescein, 50 µL of sample/Trolox/blank and 25 µL of AAPH (2,2 -Azobis(2-amidinopropane) dihydrochloride). Values were obtained using a regression equation between the sample concentration of Trolox (100 µM) and the area under the decay curve. Data are depicted as mg of Trolox equivalents (TE) per gram of dry fruit (mg of TE/g).

Lipid Peroxidation in Human Erythrocytes
The lipid peroxidation in human erythrocytes (LP) assay was performed as described in a previous study [22] with some modifications. Human blood cells (red) were used. After washing, the cells were suspended in phosphate-buffered saline (hemoglobin density of 1 mM). The final cell suspension with different concentrations of the test compounds was incubated and dissolved in DMSO and PBS for 10 min at 37 • C. After incubation, the cells were added to tert-butylhydroperoxide (1 mM) for 15 min at 37 • C. After this, the lipid peroxidation was obtained via the TBARs' formation. The evaluation was performed in quadruplicate, and the results are shown as the inhibition percentage (IP) of lipid peroxidation in human erythrocytes (IPLP) contrasted to the control of catechin at 100 µg/mL and reported as the average ± SD. Healthy volunteer donors gave their informed consent for inclusion before participating in the study. The protocol assay was conducted in accordance with the Declaration of Helsinki and is included in the approved project by CICIT-CA-UNSJ-Argentina (Project code 80020190100277SJ, N 0591-20-R-UNSJ), referred to as an ethical and environmental safeguard and to preserve the hygiene and safety conditions in the activities to be carried out in laboratories.

Determination of Enzymatic Activity
The inhibition of AChE was obtained according to the Ellman method, as previously reported [27]. A solution of 2 mg of extract per milliliter was prepared. The assay was performed by mixing the sample or standard with the enzyme AChE or BChE dissolved in Tris-HCl buffer (50 mM, pH 8.0) and 5-dithiol-bis (2-nitrobenzoic acid) at a pH of 8.0; to start the reaction, the substrate acetyl-thiocholine iodide or butyryl-thiocholine chloride was added. The absorbance was read at 405 nm for 30 min at 37 • C. The experiments were carried out in triplicate, and the results are reported as the average ± SD. The results are expressed as IC 50 (µg of extract per mL). Galantamine was used as the positive control.
Tyrosinase activity was assessed using the dopachrome method [28]; 20 µL of A. dentata extract dissolved in ethanol was mixed with 30 µL of pH 6.8 PBS, (67 mM), 40 µL of tyrosinase (100 U/mL), and 40 µL of the substrate L-DOPA (2.5 mM). The absorbance was measured at 492 nm. The results are expressed as IC 50 (µg of extract per mL. The experiments were run in triplicate, and the results are reported as the average ± SD. Kojic acid was the positive control compound.
The amylase inhibition was implemented by agreeing to the method previously described by H. Ali, P.J. Houghton and A. Soumyanath [29]. The glucosidase inhibition assays were evaluated using the method described by L. Liu, M. Deseo, C. Morris et al. [30]. Finally, the lipase inhibition evaluation was performed according to D. Lewis and D.J. Liu [31].

Isolation of Rat Aorta-Vascular Reactivity Assays
This study included the use of male Sprague Dawley rats (6-8 weeks old; n = 3) weighing between 170 and 200 g. The experimental procedure was approved (CEIC #366/2022) by the local ethics research committee of Universidad de Antofagasta, and the research was performed in agreement with this. The rats were randomized and maintained at room temperature and a humidity of 45-51% with full access to tap water and food ad libitum.
After the euthanasia of animals via cervical dislocation, the aortas were taken, and aortic rings we placed in an organ bath with Krebs-Ringer bicarbonate solution. The integrity of the vascular endothelium was evaluated with 10 −5 M acetylcholine (ACh); then, the assays were performed. For the evaluation of the relaxation capacity produced by the A. dentata ethanolic extract, the tissue was pre-contracted with 10 −6 M phenylephrine (PE); then, increasing concentrations of the extract were added to the organ bath on the vascular plateau response, as reported previously [32,33]. In addition, other similar protocols were performed to evaluate the role of the endothelium in the relaxation effect produced by A. dentata ethanolic extract; for these, the removal of the endothelium of the aortic rings and experiments with the protocol which includes the use of N ω -Nitro-L-arginine methyl ester (L-NAME, 10 −4 M) was performed.

Statistical Analysis
Statistical analysis of the data was carried out using one-way or two-way analysis of variance (ANOVA) followed by the post hoc Dunnett test where applicable. In addition, the sensitivity determination (EC 50 or IC 50 ) was performed using nonlinear regression (sigmoidal) via Graph Pad Prism version 5.0, software package. (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined at p = 0.05. Table 1 shows the proximal composition of A. dentata fruits such as the crude fiber, ashes, humidity, proteins, fatty matter, carbohydrates, and the mineral content of the fruits (Ca, Mg, Fe, and Zn). The results of proximal composition showed that A. dentata fruits are rich in carbohydrates (72.1%), have good protein content (4.3%)-which is similar to that in guava fruits [34] and more than that in kiwis (1.1%) [35]-and the mineral content showed that A. dentata fruits are rich in calcium. This mineral is most frequently associated with fruit firmness, is essential, and plays crucial roles in the metabolism, since it is used in muscle contraction, bone formation, blood clotting, and nerve function, among others, so it is very important to assess the amount of calcium intake in the diet [36,37]. Magnesium is very important for bone formation, cardiovascular and muscular function, and the synthesis of proteins and nucleic acids. The content of magnesium, iron, and zinc are connected at both the cellular and molecular levels and are important, because deficiencies can cause psychological and cognitive symptoms, as well as manifestations of fatigue and lack of energy [38]. Fruits with high magnesium and calcium contents include avocados, dried figs, bananas, guavas, kiwi fruit, blackberries, papayas, cantaloupes, raspberries, and grapefruit [39,40]. The daily value (DV) for magnesium is 420 mg per day, so 500 g of A. dentata fruit can provide the daily intake and can be helpful in hypertension. To consider food safety, we also report the heavy metal contents of A. dentata fruits.  [43]. Regarding the content of heavy metals, the levels of arsenic, a harmful metal and one of the principal factors for public health risks [44], in A. dentata fruits were 121 ± 11 µg per kilogram of fruit. On the other hand, cadmium values were 152 ± 5 µg per kilogram of fruit. Cadmium is an industrial and environmental pollutant that has harmful effects on human health, being a carcinogenic agent [45]. The mercury content in A. dentata fruits was 7.7 ± 1.3 µg/kg, while the values of lead, a harmful pollutant that has high toxic effects on many body organs [44], in A. dentata fruits were 294 ± 4 µg per kilogram of fruit. The values for arsenic, mercury, and lead were below or equal to the maximum limits set by the EFSA [42] and Codex Alimentarius [43]: 0.1mg/kg (in fruits), 0.5 mg/kg (in fishery products), and 0.20 mg/kg (in berries), respectively. Cadmium was the only case in which the concentration found in A. dentata fruits was higher than the maximum limit (0.050 mg/kg). However, further analysis must be carried out to estimate the daily intake and carcinogenic risk due to the consumption of this berry.

UHPLC-MS Analysis of Extracts
The fingerprint analysis via UHPLC MS (Figure 2) of the crude extracts of corcolen (A. dentata) was investigated through high-resolution mass spectrometric analysis. A negative mode of detection was used. Some metabolites have been pointed out for the first time in this species. Twenty-nine compounds were detected and twenty-seven were tentatively identified based on UV absorption and HR-MS n patterns (Table 2), and the major compounds of this plant are presented in Figure 3. Eleven detected compounds were assigned to phenolic acids, (peak 1-3, 6-8, 12, 15, 17, 18, and 24), seven to flavonoids (peaks 4, 11, 14, 18, 19, and 20), one flavanol (peak 13), two coumarins (peaks 5 and 27), five cyanidins (peaks 21-26), and one quinone (peak 9). The detailed fingerprinting analysis is explained below.

Total Phenolic and Flavonoid Contents and Antioxidant Activity
The results are depicted in Table 3; the phenolics contents (23.50 ± 0.93 mg gallic acid equivalent/g of dry fruit) and flavonoids 10.75 ± 0.62 mg of quercetin/ g dry fruit) support the antioxidant potential of the ethanol extracts which was measured using several methods, namely DPPH (28.64 ± 1.87 mg TE/g dry fruit) methods, FRAP (25.32 ± 0.23 mg Trolox equivalents (TE)/g dry fruit),TEAC (34.72 ± 2.33 mg TE/g dry fruit), and ORAC (64.95 ± 1.23mg TE/g dry fruit). Additionally, A. dentata extract exhibited a strong inhibition of lipoperoxidation in human erythrocytes with an IPLP of 98% at 50 µg of A. dentata extract/mL compared to catechin, which showed 70% of IPLP at 100 µg/mL. These results are in the range of superfruit berries from Chile such as maqui berries (DPPH 28.18 ± 0.37 mg TE/g, TEAC 18.66 ± 0.26 mg TE/g, and FRAP 25.22 ± 0.38 mg TE/g), [55] as well as other Chilean berries: for instance, the reported TFC of murta, highbush blueberries, arrayán, calafate, chequén, and meli were in the range of 2.57-45.72 mg quercetin equivalent/g [46], and the TEAC from the popular goji berries was reported as 24.86 ± 2.15 mg Trolox/g, and its FRAP was 16.91 ± 1.95 mg Trolox/g (Licium barbarum cv. Erma) [56].

Enzymatic Inhibitory Activity
The enzymatic inhibitory properties (cholinesterases, amylase, lipase, glucosidase, and tyrosinase inhibitory potential) of A. dentata extracts were evaluated for the first time; it is important to mention that no previous research exists related to the anti-enzymatic potential of this species reported. The enzymes that were included in this study are impor-tant in the treatment of noncommunicable diseases such as neurodegenerative diseases and metabolic syndrome. The results are outlined in Table 4 and are depicted as IC 50 values (µg/mL). The employment of vegetables and shrubs has been important through the years to prevent neurodegenerative diseases due to the high amount of phenolics [57]. Neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, are the NCDs linked to prolonged disability in the older adult population. For the treatment of Alzheimer's Disease, usually, anticholinergic agents are employed; however, it is known that this treatment can produce many adverse effects that reduce the quality of life of the patient, despite collaborating with the delay of the progression of the disease. Parkinson's disease (PD) is the second most common neurodegenerative disorder after AD and remains essentially untreatable. Experimental evidence suggests that it is a significant contributor to dopaminergic neuronal loss and that targeting tyrosinase is a frequent strategy in the prevention of neuronal degeneration in this NCD [58]. In the AChE assay, A. dentata extract showed an IC 50 of 4.58 ± 0.04 µg/mL, while in the BChE assays, the IC 50 was 23.44 ± 0.03 µg/mL (Table 4), and in the tyrosinase assays the inhibition was IC 50 9.25 ± 0.15 µg/ml. Regarding the metabolites identified in the extract of A. dentata (Table 2), we can highlight the importance of some biologically active components such as anthocyanin flavonoids, coumarins, and phenolic compounds in this species, which could be worthy for use in the prevention of chronic noncommunicable or neurodegenerative diseases. On the other hand, A. dentata ethanolic extract showed good inhibition capacities against amylase (IC 50 : 5.6 + 0.0 µg/mL) and lipase (IC 50 : 30.8 ± 0.0 µg/mL), enzymes, which are relevant in the study of NCDs because they are related to the treatment of metabolic syndrome (Table 4). They describe the association between many chronic pathologies such as obesity, diabetes mellitus type 2, and CVDs, among others. Indeed, natural products can be inhibitors of the enzymes α-glucosidase and α-amylase and can be used to control post-prandial hyperglycemia [8]. Some fruits from Chañar from northern Chile-which were studied by some of us-containing flavonoids such as glycosides of kaempferol, isorhamnetin plus procyanidin dimers, etc., were able to inhibit those enzymes [8]. Some other berry extracts with phenolic compounds have been reported to inhibit α-amylase in vitro, but the most potent were from rowanberry and raspberry (IC 50 values of 4.5 and 21.0 µg/mL, respectively) [59], the latter was similar to our corcolen berries and was also similar to the standard compound, acarbose. On the other hand, corcolen berries did not show good anti-glucosidase activity (360.9 µg/mL) as compared to other berries, for instance, rowanberry and blackcurrant, which inhibited α-glucosidase with IC 50 values of 20 and 30 µg GAE/mL and were as effective as the current inhibitor, acarbose [60]. Meanwhile, some berries were able to inhibit pancreatic lipase activity: blueberry showed slight inhibition, whilst arctic bramble, strawberry, lingonberry, cloudberry, and raspberry were considerably more potent (the most active EC 50 below 20 µg/mL) [61]. However, the enzymatic inhibitory activity of A. dentata ethanolic extract has demonstrated its capacity to inhibit enzymes that are linked to NCDs. The ability of phenolic compounds to modulate the activity of various enzymes, and therefore to interfere with cell signaling mechanisms, can be attributed in part to their chemical structures, while the antioxidant properties of the compounds may explain some of their beneficial effects [32]. Regarding the bioactive compounds, it was proposed that anthocyanins, such as peaks 21-26, may attenuate the symptoms of metabolic syndrome via improving impaired glucose tolerance, insulin resistance, dyslipidemia, blood glucose, hypertension and cholesterol levels; protecting β cells; and preventing ROS production [62]. Catechins such as peak 13 (catechin 4-ol-3-O-glucoside) plus other phenolic compounds and flavonoids were indicated as beneficial in the amelioration of chronic diseases, such as cancer and metabolic syndrome [63]. Rutin, a main flavonoid detected in this study, has been shown to cause the significant inhibition of α-amylase (p < 0.001; 53.66%) and α-glucosidase (p < 0.001; 52.56%) [64]. On the other hand, it was found that chlorogenic acid has a great inhibitory effect on lipase (IC 50 : 0.647 mg/mL) [65] and amylase (IC 50 0.498 ± 0.013 mg/mL) [66]. It also inhibited cholinesterases (IC 50 : 8.01 ± 0.01 and 6.30 ± 0.02 for AChE and BChE, respectively [67]. All values are expressed as means ± SD (n = 3).

Effect of A. dentata on Vascular Relaxation: Role of the Endothelium
The search for new vasodilatory functions of natural products is a main topic, since only 40 percent of the hypotensive or vasoactive plants from traditional medicine have been investigated so far [68]. Traditional medicines used for vascular relaxation, such as Vernonia amygdalina Del., Tridax procuinbens L., Mikania glomerata Spreng., and Kaempferia galanga L., have significant vasodilatory bioactivities, found via tests in vitro, through acting on eNOS and Ca 2+ channels, while others, such as Gaultheria procumbens L., H. cere, Bidens pilosa Linn., etc. have vasodilatory activity which were investigated in vivo; they also showed their activity based upon other mechanisms, including sGC-cGMP, potassium channels, cyclooxygenase pathways, muscarinic receptors, prostaglandin I2, etc. [68]. In this study, the role of the endothelium in vascular relaxation shown by A. dentata fruits was investigated. The endothelium denudation in aortic rings produced a drastic decrease in relaxation for the A. dentata extract. Thus, the relaxation was approximately 20-50% lower in endothelium-rubbed rat aorta (rubbed); 32 ± 8% intact aorta (control) versus 6 ± 2% (rubbed) at 2 log µg/mL or 100 µg/mL (p < 0.001); and 78 ± 1% intact aorta (control) versus 27 ± 3% (rubbed) at 3 log µg/mL or 1000 µg/mL (p < 0.001) (Figure 4). The negative logarithm of the half-maximal effective concentration (pEC 50 ) was significantly (p < 0.05) lower in rubbed aortic rings in the presence of the extract (Table 5).
ds 2023, 12, x FOR PEER REVIEW [68]. In this study, the role of the endothelium in vascular relaxation s fruits was investigated. The endothelium denudation in aortic rings decrease in relaxation for the A. dentata extract. Thus, the relaxation 20-50% lower in endothelium-rubbed rat aorta (rubbed); 32 ± 8% intac sus 6 ± 2% (rubbed) at 2 log μg/mL or 100 µg/mL (p < 0.001); and 7 (control) versus 27 ± 3% (rubbed) at 3 log μg/mL or 1000 µg/mL (p < 0 negative logarithm of the half-maximal effective concentration (pEC (p < 0.05) lower in rubbed aortic rings in the presence of the extract (T  Table 5. Relaxation effect of A. dentata in pre-contracted aortic rings with PE i thelium (rubbed), presence (control), and pre-incubated with 10 −4 M L-NAME  Table 5. Relaxation effect of A. dentata in pre-contracted aortic rings with PE in the absence of endothelium (rubbed), presence (control), and pre-incubated with 10 −4 M L-NAME.
The aortic ring tissue was pre-incubated with L-NAME, an endothelial nitric oxide synthase inhibitor, to evaluate whether endothelial factors were released. The results show that the use of 10 −4 M L-NAME for pre-incubation with the aortic rings significantly decreased the vascular relaxation to 100 µg/mL A. dentata extract concentration ( Figure 5A,B). The values for 2 log µg extract/mL (100 µg/mL) were 44 ± 4% control versus 5 ± 3% L-NAME, p < 0.001. In addition, the values for 3 log µg/mL (1000 µg/mL) extract were 77 ± 7% control vs. 14 ± 3% L-NAME, p < 0.001 ( Figure 5C). The pIC 50 of L-NAME vs. control was not significantly different (Table 5). The denudation of the endothelium in aortic rings reduced the vascular relaxation induced by A. dentata in the vascular tissue pre-contracted with PE. These results suggest that the extract produces relaxation in intact rat aorta mediated by the release of factors, such as NO, from the vascular endothelium and vascular smooth muscle [69]. Certainly, the pre-incubation of the rat aorta with an inhibitor of endothelial nitric oxide synthase (L-NAME) reduced the relaxation induced by A. dentata, strongly suggesting that the Previously, vascular endothelium was tested with 10 −5 M acetylcholine in pre-contracted aortic rings with 10 −6 M phenylephrine (PE). Effect of the extract on the aortic ring in absence of L-NAME (black line) (A) versus aortic ring pre-incubated with 10 −4 M L-NAME for 20 min (red line) (B). Aortic rings pre-incubated with 10 −4 M L-NAME or absence (Control) (C). Rat aorta was precontracted with 10 −6 M phenylephrine (PE) for 10 min, and then, rising concentrations of A. dentata (0.1 to 1000 µg/mL) were added in organ bath at 7 min intervals. Data are presented as the average standard error of the mean (SEM) of 5-7 independent experiments. * p < 0.05, *** p < 0.001.
The denudation of the endothelium in aortic rings reduced the vascular relaxation induced by A. dentata in the vascular tissue pre-contracted with PE. These results suggest that the extract produces relaxation in intact rat aorta mediated by the release of factors, such as NO, from the vascular endothelium and vascular smooth muscle [69]. Certainly, the pre-incubation of the rat aorta with an inhibitor of endothelial nitric oxide synthase (L-NAME) reduced the relaxation induced by A. dentata, strongly suggesting that the nitric oxide (NO) pathway produced the response in some way. Therefore, these findings suggest that A. dentata shows a potential hypotensive effect.

Conclusions
In this study, the proximal analysis, the metal content, plus the chemical profile of the native berry Azara dentata is reported. The phenolic fingerprinting was investigated using UHPLC-PDA-Q-TOF-MS, and twenty-seven metabolites were detected, and of those, eleven detected compounds were assigned to phenolic acids, (peak 1-3, 6-8, 12, 15, 17, 18, and 24), seven to flavonoids (peaks 4, 11, 14, 16, 18, 19, and 20), one to a flavanol (peak 13), two to coumarins (peaks 5 and 27), five to cyanidins (peaks [21][22][23][24][25][26], and one to quinone (peak 9) for the first time. In addition, the content of phenolic and flavonoid compounds plus the antioxidant activity using different methods, including a strong inhibition of lipoperoxidation in human erythrocytes with an IPLP of 98% at 50 µg of A. dentata extract/mL compared to catechin, and the inhibition of different enzymes related to noncommunicable diseases such as metabolic syndrome and Alzheimer disease (AChE, BChE, tyrosinase, amylase, glucosidase, and lipase) are reported for the first time. The amylase inhibition was IC 50 5.6 ± 0.0, more potent than standard acarbose, while for tyrosinase, it was found to be a good inhibitor, with IC 50 9.25 ± 0.15, as well as for cholinesterases (IC 50 2.87± 0.23 and IC 50 6.73 ± 0.07 for AChE and BChE, respectively). Furthermore, the A. dentata extract showed 50-60% relaxation in rat aorta mediated thorough the release of endothelial nitric oxide, so it has potential for use in the treatment of hypertension as a dietary supplement. These findings are important to position these neglected fruits as interesting sources of phenolic compounds and as fruits with nutritional and health-promoting properties. The study highlights the nutritional benefits of these fruits and could boost their consumption.