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. 2019 Aug 3;8(8):265. doi: 10.3390/plants8080265

Antioxidant, Antidiabetic, and Anticholinesterase Activities and Phytochemical Profile of Azorella glabra Wedd

Immacolata Faraone 1,2,*, Dilip K Rai 3, Daniela Russo 1,2,*, Lucia Chiummiento 1, Eloy Fernandez 4, Alka Choudhary 3, Luigi Milella 1,2
PMCID: PMC6724412  PMID: 31382601

Abstract

Oxidative stress is involved in different diseases, such as diabetes and neurodegenerative diseases. The genus Azorella includes about 70 species of flowering plant species; most of them are commonly used as food and in particular as a tea infusion in the Andean region of South America in folk medicine to treat various chronic diseases. Azorella glabra Wedd. aerial parts were firstly analyzed for their in vitro antioxidant activity using different complementary assays. In particular, radical scavenging activity was tested against biological neutral radical DPPH; ferric reducing power and lipid peroxidation inhibitory capacity (FRAP and Beta-Carotene Bleaching tests) were also determined. The Relative Antioxidant Capacity Index (RACI) was used to compare data obtained by different assays. Then, the inhibitory ability of samples was investigated against α-amylase and α-glucosidase enzymes involved in diabetes and against acetylcholinesterase and butyrylcholinesterase enzymes considered as strategy for the treatment of Parkinson’s or Alzheimer’s diseases. Moreover, the phytochemical profile of the sample showing the highest RACI (1.35) and interesting enzymatic activities (IC50 of 163.54 ± 9.72 and 215.29 ± 17.10 μg/mL in α-glucosidase and acetylcholinesterase inhibition, respectively) was subjected to characterization and quantification of its phenolic composition using LC-MS/MS analysis. In fact, the ethyl acetate fraction derived from ethanol extract by liquid/liquid extraction showed 29 compounds, most of them are cinnamic acid derivatives, flavonoid derivatives, and a terpene. To the best of our knowledge, this is the first report about the evaluation of significant biological activities and phytochemical profile of A. glabra, an important source of health-promoting phytochemicals.

Keywords: Apiaceae, Azorella glabra, DPPH, Beta-Carotene Bleaching, RACI, phenolic characterization, UHPLC-MS/MS, polyphenols, flavonoids, health-promoting compounds

1. Introduction

Azorella glabra Wedd., also known as Azorella diapensioides or yareta, is an endemic Bolivian species belonging to the Apiaceae (Umbelliferae) family. In the Andean region of South America, the plants belonging the genus Azorella are commonly used to treat several chronic diseases in folk medicine [1]. The Azorella genus is rich in diterpenoids, with mulinane and azorellane skeletal, compounds with a variety of important biological activities [2] that could explain the traditional use of the native food plant species and health benefits of the infusions. Moreover, in a previous study, A. glabra was suggested as the “future in the past” [3] for its dose and time dependent anti-proliferative effect on the multiple myeloma cells (MM), but is not present in any South American pharmacopoeias. In particular, the chloroform and n-hexane fractions were the most active and their cytotoxic effect could be attributed to the greater content of terpenoids. The influence of polarity of the solvents on total polyphenolic, flavonoid, and terpenoid contents was also reported. In fact, the highest total polyphenolic and flavonoid contents were reported in fractions obtained with polar solvents. On contrary, the highest total terpenoid content was reported in non-polar fractions. Further research studies are needed to explore the biological activities of A. glabra regarding its phytochemical composition to support its potential effects on the human health.

In this study, the antioxidant activity of A. glabra samples were ascertained by using three different complementary assays, namely 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), Ferric Reducing Antioxidant Power (FRAP), and lipid peroxidation by Beta-Carotene Bleaching test (BCB) in addition to the previously radical scavenging activity against synthetic 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and physiological superoxide anion (SO) and nitric oxide (NO) radicals [3]. The oxidative stress has been implicated in different diseases such as ageing, neurodegenerative disorders (Alzheimer’s disease and Parkinson’s disease), and diabetes [4,5]. For these reasons, we focused our attention also on the investigation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes inhibition, the two enzymes involved in regulation of ACh levels in brain, therapeutic targets to cholinergic deficit [4]. Moreover, we evaluated the inhibition of α-amylase and α-glucosidase enzymes, an effective strategy to lower the levels of postprandial hyperglycemia typical in diabetic patients [6]. Then, the phytochemical profile was performed by LC-ESI-MS/MS analysis and the identification and quantification of polyphenols were achieved using commercially available standards [7]. To the best of our knowledge, this is the first report about the evaluation of biological activity, including antioxidant, antidiabetic, and anticholinesterase activities, and phytochemical profile of A. glabra.

2. Results and Discussion

The aerial parts of A. glabra were extracted by exhaustive dynamic maceration (four times for 3 h each) using 96% ethanol [7] with an extraction yield of 9.01%. To our knowledge, this is the first time that A. glabra was extracted by 96% ethanol, usually other species of the Azorella genus were extracted by petroleum ether [8,9]. Then, the present compounds in ethanol extract (Ag EtOH) were separated on the basis of the solvent affinity by liquid/liquid extraction using an increasing solvent polarity obtaining the extraction yields shown in Table 1, as reported previously [3].

Table 1.

Extraction yields of Azorella glabra ethanol extract and its fractions.

Samples Extraction Yield (%)
Ag EtOH 9.01
AgH 31.52
AgC 44.50
AgEA 2.23
AgB 5.66
AgW 16.10
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Samples are crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW). Data are expressed as percentage (%).

2.1. Antioxidant Activity

The measurement of antioxidant activity on foods and plants are performed by more than one in vitro method in order to establish the antioxidant ability of samples [7]. For this reason, the antioxidant activity of the ethanol extract of A. glabra and its fractions were analyzed using three different complementary in vitro antioxidant assays.

The neutral DPPH radical was used to evaluate the radical scavenging activity. The samples were able to reduce the radical DPPH to the yellow coloured diphenylpicrylhydrazine in a concentration dependent manner. The AgEA showed the highest radical scavenging activity (Table 2) with 240.33 ± 10.73 mg TE/g value, followed by AgB. Instead, the lowest activity was found in AgC, and AgH was not active. Highly significant results were reported by Lamorte et al. for the radical scavenging activity of A. glabra samples against cationic (ABTS) and physiological (superoxide anion) radicals [3].

Table 2.

Results of DPPH scavenging activity, Ferric Reducing Antioxidant Power (FRAP), and β-Carotene Bleaching assay (BCB) of A. glabra samples.

Samples DPPH (mgTE/g) FRAP (mgTE/g) BCB %AA
Ag EtOH 28.17 ± 2.32 a 73.58 ± 0.71 a 26.70 ± 0.61 a
AgH nc 18.79 ± 0.66 b 22.50 ± 0.65 b
AgC 5.94 ± 0.27 b 15.45 ± 0.44 b 22.18 ± 1.54 b
AgEA 240.33 ± 10.73 c 410.29 ± 5.69 c 34.93 ± 1.37 c
AgB 224.91 ± 4.84 d 318.57 ± 2.77 d 21.68 ± 0.57 b
AgW 43.12 ± 1.23 e 95.33 ± 3.86 e nc
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Samples are crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW). Data are expressed as means ± standard deviation from three experiments; mg TE/g = mg of Trolox Equivalents per gram of dried sample; % AA = percentage of Antioxidant Activity at initial sample concentration of 1 mg/mL; different superscripts in the same row indicate significant difference (p < 0.05); nc = not calculable.

The FRAP test was used to evaluate the ferric reducing antioxidant power of samples and AgEA presented the highest FRAP value (410.29 ± 5.69 mg TE/g) followed by AgB (318.57 ± 2.77 mg TE/g). AgC and AgH were again the least active (Table 2).

The inhibition of lipid peroxidation was evaluated by the β-Carotene Bleaching assay (BCB) and the most active sample was again the AgEA (34.93 ± 1.37% AA), whilst the other fractions had similar BCB values. As expected, there was no BCB activity for the polar AgW fraction (Table 2).

The Relative Antioxidant Capacity Index (RACI) was calculated to integrate the results of the antioxidant activity obtained by DPPH, FRAP, and BCB assays in order to compare the different phytocomplex antioxidant ability [3,7]. The results obtained so far agreed with RACI values (Figure 1). In particular, the RACI evidences the ethyl acetate fraction presented the highest value (1.35), followed by the butanol fraction (0.74). The AgW fraction presented the lowest index (−0.87) and, therefore, a relative lack of antioxidant activity.

Figure 1.

Figure 1

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Relative Antioxidant Capacity Index (RACI) of Azorella glabra samples. Samples are crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW).

These antioxidant data are in line with other complementary antioxidant assay, i.e., ABTS [3]. Except Lamorte et al., there are no other studies that reported the antioxidant activity of A. glabra. Although other species of Azorella, in particular the antioxidant activity of Azorella madreporica aerial parts, was evaluated [10]. The aerial parts of A. madreporica were extracted with petroleum ether followed by methanol. Then, the methanolic extract was subjected to Total Polyphenolic Content (TPC), Total Flavonoid Content (TFC), and DPPH assays. A. madreporica had shown potent antioxidant property on DPPH radical (IC50 of 96.57 ± 1.00 μg/mL) and showed higher inhibition than Ag EtOH extract obtained in this study (IC50 of 1109.04 ± 33.84 μg/mL), which may be due to isoflavonoids identified by HR-ESI-ToF-MS [10]. These results exemplify how different extraction solvents (ether and methanol for A. madreporica and ethanol for A. glabra, respectively) influence the various in vitro protocols used to determine the radical-scavenging activity.

The RACI values illustrated in Figure 1 may explain the high total polyphenol content of the ethyl acetate and butanol fractions and the correlation between TPC and antioxidant activity in the earlier study [3]. Moreover, the fractions obtained by polar solvents (ethyl acetate and n-butanol) reported the highest total polyphenol content, indicating that the majority of polyphenolic compounds in the aerial parts of A. glabra could be of polar nature [11].

2.2. Determination of Anticholinesterase Activity of A. glabra Samples

The A. glabra samples had a concentration-dependent activity on acetylcholinesterase and butyrylcholinesterase enzymes (Figure 2). In particular, the AgC and AgH fractions showed a moderate activity by in vitro AChE assay (IC50 of 30.75 ± 0.67 and 99.19 ± 6.18 μg/mL), compared with the positive control galantamine (IC50 of 4.68 ± 0.31 μg/mL) (Table 3). BChE enzyme was inhibited only by Ag EtOH extract, and AgC and AgB fractions that showed butyrylcholinesterase activity weaker than galantamine (IC50 16.07 ± 1.04 μg/mL). Again, the AgC fraction gave the lowest inhibition concentration (IC50 240.28 ± 8.91 μg/mL) (Table 3).

Figure 2.

Figure 2

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Acetylcholinesterase (AChE) (A) and butyrylcholinesterase (BChE) (B) inhibition activity of galantamine and Azorella glabra samples. Samples are galantamine, crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW). Data are mean ± standard deviation from three experiments performed in triplicate.

Table 3.

AChE and BChE inhibition by galantamine and Azorella glabra samples expressed as IC50 values in μg/mL.

Samples AChE Inhibition (IC50) BChE Inhibition (IC50)
Galantamine 4.68 ± 0.31 a 16.07 ± 1.04 a
Ag EtOH 193.81 ± 13.32 b 421.50 ± 39.38 b
AgH 99.19 ± 6.18 c nc
AgC 30.75 ± 0.67 d 240.28 ± 8.91 c
AgEA 215.29 ± 17.10 b nc
AgB 113.08 ± 5.18 c 362.06 ± 28.60 b
AgW nc nc
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Samples are galantamine, crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW). Enzymatic inhibition not calculable (nc). Data are mean ± standard deviation from three experiments performed in triplicate. The concentration of the sample required to inhibit the activity of the enzyme by 50% (IC50) in μg/mL was calculated by nonlinear regression analysis. In each test, the values with the same letter are not significantly different at the p < 0.05 level, 95% confidence limit, according to one-way analysis of variance (ANOVA).

This is the first report on anticholinesterase activity of A. glabra. In a previous study, the effect on the AChE enzyme of several terpenes (three lanostane-, two cycloartane-type triterpene, and two mulinane-type) isolated from Azorella trifurcata was reported. All compounds showed moderate inhibitory activity toward the enzyme, which may be due to the presence of acetate groups in these diterpenes [8].

2.3. Potential Antidiabetic Activity of A. glabra Samples

The inhibition of α-amylase and α-glucosidase enzymes is an important strategy in the treatment of obese and/or diabetic patients. Different concentrations of the A. glabra samples were subjected for inhibitory activities of both the enzymes. The samples were concentration-dependent and acarbose was used as positive control (Figure 3).

Figure 3.

Figure 3

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α-amylase (A) and α-glucosidase (B) inhibition activity of acarbose and Azorella glabra samples. Samples are acarbose, crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW). Data are mean ± standard deviation from three experiments performed in triplicate.

In α-amylase inhibition assay, only the Ag EtOH reached the IC50 (172.25 ± 7.25 μg/mL), which was higher than acarbose (IC50 of 22.78 ± 0.29 μg/mL, Table 4).

Table 4.

α-Amylase and α-glucosidase inhibition by acarbose and Azorella glabra samples expressed as IC50 values in μg/mL.

Samples α-Amylase Inhibition (IC50) α-Glucosidase Inhibition (IC50)
Acarbose 22.78 ± 0.29 a 401.15 ± 25.94 a
Ag EtOH 172.25 ± 7.25 b 207.70 ± 2.56 b
AgH nc 159.91 ± 5.59 b
AgC nc nc
AgEA nc 163.54 ± 9.72 b
AgB nc 373.77 ± 29.84 a
AgW nc nc
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Samples are acarbose, crude ethanol extract (Ag EtOH), n-hexane fraction (AgH), chloroform fraction (AgC), ethyl acetate fraction (AgEA), n-butanol fraction (AgB), and water fraction (AgW). Data are mean ± standard deviation from three experiments performed in triplicate. Enzymatic inhibition not calculable (nc). The concentration of the sample required to inhibit the activity of the enzyme by 50% (IC50) in μg/mL was calculated by nonlinear regression analysis. In each test, the values with the same letter are not significantly different at the p < 0.05 level, 95% confidence limit, according to one-way analysis of variance (ANOVA).

Whereas in the α-glucosidase assay, all samples except for AgC and AgW fractions showed inhibition activity. Moreover, Ag EtOH, AgH, AgEA, and AgB fractions showed IC50 values lower than acarbose (IC50 of 401.15 ± 25.94 μg/mL) and the best of all were the AgH and AgEA fractions (IC50 of 159.91 ± 5.59 and 163.54 ± 9.72 μg/mL, respectively) (Table 4).

This is the first report on antidiabetic activity of A. glabra. A previous study on Azorella compacta had also shown a potent antidiabetic property [12]. The Azorella species has been used as antidiabetic medicine by the Andean natives. Fuentes et al. in 2005 [12] suggested that the diterpenic compounds mulinolic acid, azorellanol, and mulin-11,13-dien-20-oic acid isolated from A. compacta are responsible for the antidiabetic activity in ethnomedicine. It is also possible that the anti-diabetic properties of A. compacta is due to its ability to increase insulin secretion as mentioned in a review by Prabhakar and Doble [13].

2.4. Identification and Quantification of Phytochemicals

In order to evaluate the compounds responsible for the various bioactivities examined above, the sample with the best antioxidant activity and the best enzyme inhibition was subjected to liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS). The A. glabra ethyl acetate fraction (AgEA) was therefore chosen for the LC-MS/MS (Figure 4) as it showed the highest antioxidant activity, the best RACI value, and antidiabetic activity. In other species of Azorella genus were identified compounds belonging mainly to the class of diterpenoids [8,12], but this is the first report of phytochemical profile of A. glabra. The profile of AgEA obtained by LC–MS analysis is shown in Figure 4.

Figure 4.

Figure 4

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Ethyl acetate fraction of Azorella glabra base peak intensity (BPI) chromatogram.

More than 29 compounds were detected and tentative identification of most of them was reached through accurate mass and fragmentation pattern and aided by the existing literature (Table 5). For the first time in A. glabra, 11 compounds were identified by comparing their retention times with those of the available commercial standards (Table S1). In particular, we performed calibration curves for both identified and used standards. The R2 values for all calibration curves were over 0.99.

Table 5.

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) of ethyl acetate fraction of Azorella glabra.

Peak No. RT (min) [M-H]− Observed m/z [M-H]−Calculated m/z Molecular Formula MS/MS Tentative Identity mg/g DW References
1 3.17 353.0888 353.0873 C16H18O9 191, 173, 135, 127, 93, 85 Chlorogenic acid 7.12 ± 0.83 [25]
2 6.48 427.1980 427.1968 C21H32O9 367, 327, 297, 285, 179, 161, 135, 101, 73, 61, 59 Methyl chlorogenate derivative nq [26]
3 6.64 463.0859 463.0877 C21H20O12 300, 271, 255, 179, 151 Quercetin-3-O-glucoside 0.07 ± 0.00 [26,27]
4 6.68 447.0918 447.0927 C21H20O11 357, 339, 327, 311, 299, 297, 285, 269, 253, 191, 175, 149, 133, 109 Iso-orientin 13.22 ± 1.43 [28]
5 6.84 447.0910 447.0927 C21H20O11 357, 339, 327, 311, 299, 297, 285, 269, 253, 191, 175, 149, 133, 109 Orientin 104.22 ± 4.01 [28]
6 6.92 367.1038 367.1029 C17H20O9 191, 179, 161, 107 Chlorogenic acid methyl ester 12.33 ± 0.04 [26]
7 7.14 515.1180 515.1190 C25H24O12 353, 179 Cynarin isomer 0.15 ± 0.03 [29]
8 7.41 447.0921 447.0927 C21H20O11 285, 151 Luteolin-7-O-glucoside 0.65 ± 0.10 [30]
9 7.67 515.1411 515.1401 C22H28O14 353, 191, 179, 173, 161, 135 Chlorogenic acid glucoside nq
10 7.83 515.1197 515.1190 C25H24O12 353, 335, 191, 179, 161, 135 3,5-di-O-caffeoyl quinic acid 44.70 ± 4.14 [26]
11 8.07 515.1209 515.1190 C25H24O12 353, 179, 173, 135, 93 3,4-di-O-caffeoyl quinic acid 23.12 ± 1.64 [26]
12, 13, 14, 16 8.49, 8.86, 9.12, 9.56 529.1365 529.1346 C26H26O12 367, 349, 191, 179, 161, 135 Feruloyl-caffeoylquinic acid isomers nq [23]
15 9.32 285.0380 285.0399 C15H10O6 151, 133 Luteolin 0.39 ± 0.01 [25]
17 9.98 325.1651 325.1651 C17H26O6 281, 263, 235, 219, 203, 191, 151, 111, 83, 59 Unknown nq
18 10.55 853.4720 853.4738853.4679 C48H70O13C55H66O8 584, 513, 191, 179, 161, 135, 119, 113, 101, 89, 85, 71, 59 Caffeoylquinic acid derivative nq
19 10.97 649.3929 649.3952 C36H58O10 407, 191, 129, 113, 85, 75 Unknown nq
20 11.37 691.4073 691.4057 C38H60O11 631, 191, 113, 85, 95 Unknown nq
21 11.87 867.4739 867.4742 C45H72O16 513, 408, 333, 285, 191, 179, 173, 153, 139, 89 Unknown nq
22 14.14 391.1744 391.1757 C21H28O7 391, 347, 305, 287, 259, 245, 217, 165 Unknown nq
23 15.17 677.3729 677.3748 C33H58O14 415, 397, 279, 179, 161, 119, 101 Unknown nq
24 15.46 504.3098 504.3087 C29H45O7 279, 242, 224, 168, 153, 79, 59 Unknown nq
25 15.76 426.9764 426.9785 C15H8O15 407, 387, 293, 283, 255, 217, 81 Unknown nq
26 16.22 480.3083 480.3087 C27H45O7 255, 242, 224, 168, 153, 79 Unknown nq
27 16.95 579.3354 579.3381 C28H52O12 269, 255, 89 Unknown nq
28 18.10 553.3193 553.3165 C33H46O7 523, 345, 97, 84, 73 Unknown nq
29 21.16 455.3539 455.3525 C30H48O3 407, 377 Oleanolic acid 0.23 ± 0.05 [31]
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Identification of compounds based on m/z, fragmentation pattern and retention time of standards. Quantities of the detected compounds were determined using commercial standards; nq = not quantified.

These compounds are cinnamic acid derivatives (chlorogenic acid (1), chlorogenic acid methyl ester (6), cynarin isomer (7), 3,5-di-O-caffeoyl quinic acid (10) and 3,4-di-O-caffeoyl quinic acid (11)), flavones (iso-orientin (4), orientin (5), luteolin-7-O-glucoside (8) and luteolin (15)), flavonol (quercetin-3-O-glucoside (3)) and a triterpene (oleanolic acid (29)).

The flavone orientin and the cinnamic acid derivative 3,5-di-O-caffeoyl quinic acid were the most abundant (104.22 ± 4.01 mg/g DW and 44.70 ± 4.14 mg/g DW, respectively). The identified phenolic compounds are known for their antioxidant properties [14,15,16,17,18,19,20] that could explain the good antioxidant activity of ethyl acetate fraction of A. glabra. In particular, flavonoids such as luteolin and luteolin-glycosides have been reported to have anti-inflammatory and antioxidant activities. Moreover, plant species containing these compounds are known for their anti-inflammatory properties. The antioxidant abilities of flavonoids are widely acknowledged. The two antioxidant structural features of flavonoids are the presence of a B-ring catechol group and of a C2-C3 double bond in conjugation with an oxo group at C4. Luteolin (15) and its glycosides iso-orientin (luteolin 6-C-glucoside, 4), orientin (luteolin 8-C-glucoside, 5), and cynaroside (luteolin 7-O-glucoside, 8) fulfil these two structural requirements and the antioxidant activity of these compounds has been related to their ability to scavenge reactive oxygen and nitrogen species. Moreover, luteolin could be developed as a cancer chemopreventive agent and be useful in cancer therapy to sensitize tumor cells to the cytotoxic effects of some chemotherapeutic drugs [21,22].

Four compounds were tentatively identified as feruloyl-caffeoyl quinic acid isomers (Table 5) with m/z 529.13 (12, 13, 14 and 16) and ion fragmentations at m/z 367, 349, 191, 179, 161, and 135 [23,24]. In addition, one compound was tentatively identified as chlorogenic acid glucoside (9) with m/z 515.14 and ion fragmentations at m/z 353, 191, 179, 173, 161, and 135.

3. Materials and Methods

3.1. Chemicals, Reagents, and Equipment

Solvents as ethanol, n-hexane, chloroform, ethyl acetate, n-butanol, hydrochloric acid, glacial acetic acid, methanol, and phosphoric acid were purchased from Carlo Erba (Milan, Italy). Acetonitrile and formic acid were purchased from Merck (Wicklow, Ireland).

2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), iron (III) chloride (FeCl3*6H2O), β-carotene, linoleic acid, Tween 20, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), acetylcholinesterase (AChE) from Electrophorus electricus (electric eel, type VI-s, lyophilized powder, CAS number: 9000-81-1), acetylthiocholine iodide (ATCI), butyrylcholinesterase (BChE) from equine serum (lyophilized powder, CAS number: 9001-08-5), s-butyrylthiocholine chloride (BTCC), trizma hydrochloride (Tris-HCl), bovine serum albumin (BSA), potassium phosphate monobasic, sodium chloride, sodium hydroxide, sodium phosphate, potassium sodium tartrate tetrahydrate, α-amylase from hog pancreas (CAS number: 9000-90-2), 3,5-dinitrosalicylic acid, starch, 4-p-nitrophenyl-α-d-glucopyranoside, and α-glucosidase from Saccharomyces cerevisiae (CAS number: 9001-42-7), were purchased from Sigma-Aldrich (Milan, Italy). Standards as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), butylhydroxytoluen (BHT), acarbose, galantamine, and Leucine-Enkephalin were purchased from Sigma-Aldrich (Milan, Italy) and Merck (Wicklow, Ireland), respectively. Standards for LC-MS/MS analysis (chlorogenic acid, chlorogenic acid methyl ester, cynarin isomer, 3,5-di-O-caffeoyl quinic acid, 3,4-di-O-caffeoyl quinic acid, iso-orientin, orientin, luteolin-7-O-glucoside, luteolin, quercetin-3-O-glucoside and oleanolic acid) were purchased from Extrasynthese (Genay, France). Water was deionized using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

All spectrophotometric measurements were done in 96-well microplates or cuvettes on a UV–VIS spectrophotometer (SPECTROstarNano BMG Labtech, Ortenberg, Germany). LC-MS/MS analyses were performed on a Q-Tof Premier mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA). Mass spectrometry quantification of the polyphenols was performed using Multiple Reaction Monitoring (MRM) experiments by Waters Acquity (Waters Corporation, Milford, MA, USA) ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS).

3.2. Plant Material and Samples Preparation

A voucher specimen of aerial parts A. glabra was stored at the University of La Paz after the collection in Bolivia near the Aymaya population/community (18.45° S to 66.46° W; 3750 msnm), Bustillo province, Potosí department, Bolivia. Samples of the species are found in the herbal medicinal plants of the National University Siglo XX, Llallagua, Potosí, Bolivia. A total of 140 g of aerial parts of A. glabra were subjected first to dynamic maceration with 96% ethanol and then to liquid/liquid extraction, as previously reported by Lamorte et al. in 2018 [3], obtained the initial ethanol extract (Ag EtOH) and the n-hexane, chloroform, ethyl acetate, n-butanol, and water fractions (AgH, AgC, AgEA, AgB and AgW, respectively). These six samples were analyzed for their biological activities.

3.3. Antioxidant Activity

3.3.1. Radical Scavenging Activity

The samples of A. glabra were tested for their radical scavenging capacity by in vitro DPPH assay.

The reaction between different sample concentrations (50 μL) able to reduce the radical DPPH (200 μL) to the yellow colored diphenylpicrylhydrazine was monitored by spectrophotometer at 515 nm after 30 min and quantified as milligrams of Trolox Equivalents per gram of dried sample (mg TE/g) [7,32,33].

3.3.2. Ferric Reducing Antioxidant Power Assay (FRAP)

The reduction of the Fe3+ complex of tripyridyltriazine [Fe(III)(TPTZ)2]3+ to Fe2+ complex [Fe(II)(TPTZ)2]2+ (225 μL) by antioxidants present in acidic medium of different sample concentrations (25 μL) was monitored at 593 nm after 40 min at 37 °C and Trolox was used as reference antioxidant standard (FRAP values were expressed as mg TE/g) [7].

3.3.3. β-Carotene Bleaching Assay (BCB)

This assay was used to evaluate the inhibition of lipid peroxidation. The β-carotene emulsion (950 μL) was mixed with sample at initial concentration of 1 mg/mL (50 μL) and BHT was used as positive control. The microplate with 250 μL of solution of each samples was placed at 50 °C for 3 h and the absorbance was measured at 470 nm at 0′, 30′, 60′, 90′, 120′, 150′, and 180′. The results were expressed as percentage of β-carotene bleaching inhibition (% Antioxidant Activity, % AA) [7].

3.4. Determination of Anticholinesterase Activity

The anticholinesterase inhibition of samples was investigated by Ellman’s reaction [4]. In the in vitro assays, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) catalyze the hydrolysis of acetyl- and butyryl-thiocholine, two alternative analogs of natural substrate acetylcholine. The quantification of free sulfhydryl groups was performed by 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) and the absorbance was measured at 405 nm for 2 min. The galantamine, a drug used to treat Parkinson’s and Alzheimer’s diseases, was used as positive control. The results were expressed on the basis of the concentration of the sample required to inhibit the activity of the enzyme by 50% (IC50) in μg/mL calculated by nonlinear regression analysis.

In particular, in AChE inhibition in vitro assay, different concentrations of sample (0.10–1000 μg/mL), buffer B (50.00 mM Tris-HCl, pH 8.00 containing 0.10% BSA), 3.00 mM DTNB, and 15.00 mM acetylthiocholine iodide were mixed and the reaction was started by adding 0.18 U/mL of AChE enzyme.

The BChE inhibition in vitro assay was performed in a similar way by using 15 mM butyrylthiocholine iodide as substrate and 0.10 U/mL of BChE enzyme [4].

3.5. Antidiabetic Activity

The enzymes involved in postprandial hyperglycemia are the α-amylase enzyme, which breaks down large and insoluble starch molecules into absorbable molecules and then in maltose, and the α-glucosidase enzyme that catalyzes the end step of digestion of starch and disaccharides. For this reason, the inhibitors of α-amylase and α-glucosidase enzymes were used in diabetic patients and the inhibitory ability of samples against both enzymes was screened.

3.5.1. α-Amylase Inhibition

Different concentrations of each sample (0.50–300 μg/mL) were mixed to α-amylase enzyme from hog pancreas and incubated for 10 min at 25 °C. After pre-incubation, a substrate solution of 1% starch solution was added to the reaction mixture that was again incubated for 10 min at 25 °C. Then, the reaction was stopped by the yellow-orange 3,5-dinitrosalicylic acid color reagent and the vials were incubated for 10 min at 100 °C. Then, distilled water was added to the reaction mixture and the absorbance was measured at 540 nm as described by Milella et al. [6,34]. The acarbose, a clinical antidiabetic drug, was used as positive control and the results were expressed as IC50 in μg/mL on the basis of the concentration of the sample required to inhibit the activity of the enzyme by 50% calculated by nonlinear regression analysis.

3.5.2. α-Glucosidase Inhibition

The ability of samples to inhibit the α-glucosidase enzyme was assessed using a previously reported procedure [6,34]. Different concentrations of sample (1–1600 μg/mL), phosphate buffer, 4-p-nitrophenyl-α-d-glucopyranoside, and α-glucosidase enzyme were mixed and incubated at 37 °C for 10 min. Then, the release of glucose and p-nitrophenol (yellow) by α-glucosidase enzyme was monitored spectrophotometrically at 405 nm. Again, acarbose was used as positive control and the results were expressed as IC50 in μg/mL.

3.6. Identification and Quantification by Liquid Chromatography Mass Spectrometry

The characterization of ethyl acetate of A. glabra was carried out on a Q-Tof Premier mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA) and the quantification of compounds was used a Waters Acquity (Waters Corporation, Milford, MA, USA) ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) as described previously [7]. Electrospray mass spectra data were acquired on a negative ionization mode for a mass range m/z 100 to m/z 1000. In particular, an Atlantis T3 C18 column was used (Waters Corporation, Milford, USA, 100.00 × 2.10 mm; 3.00 μm particle size) at 40 °C. The mobile phases were 0.10% aqueous formic acid (solvent A) and 0.10% formic acid in acetonitrile (solvent B). The stepwise gradient from 10% to 90% solvent B was applied at flow rate of 300 μL/min for 25 min. Cone voltage and capillary voltage were set at 30 V and 3 kV, respectively. Argon was used as collision gas and the collision induced fragmentation (CID) of the analytes was achieved using 12 to 30 eV energy. The quantification of present compounds was performed on a Waters Acquity HSS T3 C18 column (2.10 × 100.00 mm; 1.80 μm particle size) using water containing 0.10% formic acid (mobile phase A) and acetonitrile containing 0.10% formic acid (mobile phase B). The following gradient program was carried out: 0–2.50 min 2% B, 2.50–3.00 min 10% B, 3.00–7.50 min 15% B, 7.50–8.50 min 35% B, 8.50–9.50 min 98% B, and 9.50–10.00 min 2% B at a flow rate of 0.50 mL/min. The injection volume for all the samples and the standards was 3.00 μL. All the standards in the concentration ranging from 0.01 to 50.00 μg/mL were dissolved in 80% methanol. The Multiple Reaction Monitoring (MRM) quantitative method was developed for each of the standard compound using the Waters Intellistart software, and the quantifications of the data were carried out using the Waters TargetLynx™ Software (Waters Corporation, Milford, MA, USA) [35]. The ionization source conditions were as follows: capillary voltage 3 kV, cone voltage 35 V, source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow 200 L/h, cone gas flow 50 L/h, and collision gas flow 0.10 mL/min.

3.7. Statistical Analysis

The data were expressed as mean ± standard deviation (SD) at least three independent experiments performed in triplicate. The correlation among used assays was verified by the calculation of p values by one-way analysis of variance (ANOVA) using GraphPad Prism 5 Software (San Diego, CA, USA). Only p values of 0.05 or less were considered significant. The R2 values for all calibration curves were over 0.99.

4. Conclusions

The biological activities demonstrated for A. glabra samples in this study might partially justify its ethnobotanical uses in Bolivian populations. In particular, the phytochemical profile of ethyl acetate fraction of A. glabra revealed the presence of compounds with antioxidant, anti-inflammatory, anti-tumoral activities [21,22]. In addition, it was previously reported that aerial parts of A. glabra reduced the cell viability, induced the apoptosis, and arrested the cell cycle on multiple myeloma cells in G0/G1 phase [3].

In conclusion, the combination of A. glabra extract, a “health food,” with common drugs may offer a significant advantage for therapeutic efficacy in several treatments as diabetes, neurodegenerative diseases, and cancer and it may have economic implications in the health and pharmaceutical fields.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/8/8/265/s1, Table S1: LC-Q-Tof chromatograms showing the retention times of standard compounds mix used for the identification and quantification of various polyphenols and a terpene in the ethyl acetate fraction of Azorella glabra Wedd.

Click here for additional data file. (475.7KB, pdf)

Author Contributions

Conceptualization, L.C., D.K.R. and L.M.; Methodology, D.R., I.F., A.C., D.K.R., E.F., L.C. and L.M.; Software, D.R., I.F. and A.C.; Validation, L.C., D.K.R. and L.M.; Formal Analysis, D.R., I.F., A.C. and E.F.; Investigation, D.R., I.F., A.C., E.F. and D.K.R.; Resources, L.C. and L.M.; Data Curation, L.C., D.K.R. and L.M.; Writing—Original Draft Preparation, D.R., I.F., A.C. and D.K.R.; E.F. and L.M; Writing—Review and Editing, L.C. and L.M; Supervision, L.M.; Project Administration, L.M.; Funding Acquisition, L.C., D.K.R. and L.M.

Funding

This work was supported by the Regione Basilicata; the Fondazione Enrico Mattei DGR n. 1490 del 4/12/2014, vs. rep. n. 163 n8; and the Regional Project ALIMINTEGRA, GO NUTRIBAS financed on 16.1 PSR Basilicata founding ex D.G.R. No. 312/17 CUP: C31G18000210002.

Conflicts of Interest

The authors report no conflict of interests and they alone are responsible for the content and writing of the paper.

Data Availability

All dataset used for this study is available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Click here for additional data file. (475.7KB, pdf)

Data Availability Statement

All dataset used for this study is available on request.

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