Metabolite profile and in vitro activities of Phagnalon saxatile (L.) Cass. relevant to treatment of Alzheimer’s disease

Abstract

The present study describes for the first time the in vitro properties (inhibition of NO production and anticholinesterase) of Phagnalon saxatile (L.) Cass. (Asteraceae). The methanolic extract showed antioxidant activity that was measured by DPPH assay and β-carotene bleaching test. The same extract inhibited NO production in the murine monocytic macrophage cell line RAW 264.7. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition was assessed by modifications of Ellman’s method. Purification of the MeOH extract of P. saxatile allowed the isolation of phenolic compounds. Among them, the compounds that most effectively inhibited lipopolysaccharide-induced NO production were caffeic acid and methylchlorogenic acid, with IC₅₀ values of 7 μg/mL and 12 μg/mL, respectively. Luteolin and 3,5-dicaffeoylquinic acid exhibited the most promising activity against AChE with an IC₅₀ of 25.2 and 54.5 μg/mL, respectively, while caffeic acid and luteolin exhibited higher activity against BChE with an IC₅₀ of 32.2 and 37.2 μg/mL, respectively.

Keywords::

• Phagnalon saxatile
• Asteraceae
• phenolic compounds
• inhibition of NO production
• Alzheimer’s disease

Introduction

Oxidative damage, caused by the action of free radicals, may initiate and promote the progression of a number of chronic diseases, including cancer, cardiovascular diseases, inflammation, diabetes, and Alzheimer’s disease¹. Nitric oxide (NO^•) is a diatomic free radical produced from l-arginine by constitutive and inducible nitric oxide synthase (cNOS and iNOS) in numerous mammalian cells and tissues. Nitric oxide (NO), superoxide (O²⁻), and their reaction product peroxynitrite (ONOO⁻) may be generated in excess during the host response against viral and antibacterial infections and contribute to some pathogenesis by promoting oxidative stress, tissue injury, and even cancer^2,3. Recently, the effects of selected polyphenolics on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells have been reported. These studies have shown that some polyphenolics or methanol extracts of plants inhibit NO production^4–7. Most clinically important medicines belong to steroidal or non-steroidal anti-inflammatory chemical therapeutics for treatment of inflammation-related diseases. Though these have potent activity, long-term administration is required for the treatment of chronic disease. Furthermore, these drugs have various and severe adverse effects.

In recent years, oxidative stress has been described in the pathological changes that occur in Alzheimer’s disease (AD)^8,9. Acetylcholine (ACh) is found in the synapse of the cerebral cortex, and a deficiency in the cerebral cortex is one of the major features seen in sufferers of AD¹⁰. Acetylcholinesterase (AChE) inhibitors such as tacrine, donepezil, and the natural products rivastigmine and galantamine are currently the only effective treatment for AD. These approved drugs are limited in use due to their adverse side-effects, such as gastrointestinal disturbance, and bioavailability problems^11–13. The continuing search for novel anticholinesterases from plants as therapeutic agents for dementia and other central nervous system disorders is motivated by the current need for new substances of natural origin targeted to the brain areas affected, with effectiveness in the described fields of application and low degrees of toxicity and side-effects for man.

The genus Phagnalon (Asteraceae) is represented by about 30 species distributed worldwide, six of which are typical of the Mediterranean region¹⁴. Different Phagnalon sp. are used in popular medicine, and a variety of extracts have been examined^15–21. Among the Bedouins of the Negev desert, the bark of P. rupestre (L.) DC. is widely used to induce deliberate burns for the healing of various ailments¹⁵, while in the Palestinian area the whole plant is used to treat asthma and headache, and as an analgesic for toothache. The aqueous and ethanolic extracts of this plant showed antimicrobial activity against both positive and negative bacteria and C. albicans¹⁶. Phytochemical investigations on this species are mainly devoted to the study of P. rupestre, and report the presence of terpenoids²², flavonoids²³, hydroquinone glycosides, and caffeoylquinic acid derivatives²⁴. Pharmacological studies have been led only on P. rupestre: prenylhydroquinone glucosides and caffeoylquinic esters from this plant have been studied for a variety of biological activities.

Phagnalon saxatile (L.) Cass. is one of five suffruticose chamaephyte species growing wild in Italy, mostly in southern Italy²⁵. A recent ethnobotanical study classifies it as a nutritional plant utilized for medicinal purposes²⁶. A survey of the literature shows the presence in the aerial parts of the plant of an essential oil constituted mainly of sesquiterpenes, fatty acids, and waxes²⁷, 3,3-dimethylallyl-p-benzoquinone²⁸, and flavonoids such as apigenin, apigenin-7-glucoside, and luteolin²², but phytochemical data are incomplete and pharmacological information on the plant and its metabolites is lacking. The objectives of the present study were (1) to investigate the phytochemical characteristics of P. saxatile flowering aerial parts, which exhibit significant antioxidant, inhibition of NO production, and anticholinesterase activities, and (2) to relate these activities to pure isolated compounds.

Materials and methods

General experimental procedures

Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DRX-600 (Billerica, MA, USA) (¹H at 599.19 MHz, ¹³C at 150.86 MHz) spectrometer: δ (ppm), J in Hz, spectra referred to CHD₂OD as internal standard. Mass spectra were recorded with an API 2000 (Applied Biosystems, Foster City, CA, USA). Ultraviolet (UV) spectra were measured on a UV/VIS Jasco V530 spectrophotometer. Reverse-phase high performance liquid chromatography (HPLC) was performed with a TSP SpectraSeries P 100 equipped with a rheodyne injector and a refractive index detector, using a column C₁₈ β-Bondapack (Waters, Milford, MA, USA; p.s. 10 μm, 300 × 7.8 mm, flow rate 2.5 mL/min). Thin layer chromatography (TLC) was performed on plates coated with silica gel 60 F₂₅₄ (Merck, Whitehouse Station, NJ, USA), 0.25 mm. Elemental analysis was carried out with a PerkinElmer 240 apparatus. All solvents (analytical and deuterated grade) were purchased from Carlo Erba Reagenti, Milan, Italy.

Plant material

The flowering aerial parts of Phagnalon saxatile (L.) Cass. were collected in June 2005, in Cefalù, capo Playa (Sicily, Southern Italy). A voucher specimen of the plant (SN 49) is kept in the Herbarium Neapolitanum (NAP), Faculty of Pharmacy, University of Naples “Federico II”.

Extraction procedures and isolation of compounds

Plant material (452.5 g) was chopped and then sequentially extracted by cold maceration with 3 × 2.5 L of petroleum ether, CHCl₃, and CH₃OH. The methanolic solution was concentrated under reduced pressure obtaining a residue (15.8 g) that was chromatographed in 2 g lots on a Sephadex LH-20 (Pharmacia) column, eluting with CH₃OH. Fractions of 20 mL were collected and analyzed by TLC using BuOH/CH₃COOH/H₂O (60:15:25, v/v/v) as eluent and Ce(SO₄)₂ in H₂SO₄ as spray reagent. Fractions 12–14 (2.20 g), 15, 16 (1.14 g), 17 (0.178 g), 18 (0.263 g), 19–22 (1.09 g), and 23–27 (0.525 g) were gathered according to TLC analysis and further purified by reverse HPLC: fraction 12–14 (CH₃OH/H₂O, 40:60, as eluent) yielded compound (1) (6.6 mg; Rt = 9 min) and compound (2) (1.4 mg; Rt = 10.5 min); fraction 15, 16 (CH₃OH/H₂O, 40:60, as eluent) yielded compounds (3) (55.2 mg; Rt = 6.5 min), (4) (15.0 mg; Rt = 9 min), and (5) (13.4 mg; Rt = 13.5 min); fraction 17 (CH₃OH/H₂O, 55:45, as eluent) yielded compound (6) (10.6 mg; Rt = 10.5 min); fraction 18 (CH₃OH/H₂O, 60:40, as eluent) yielded compound (7) (0.8 mg; Rt = 6.5 min); fraction 19–22 (CH₃OH/H₂O, 60:40, as eluent) yielded compounds (8) (130.5 mg; Rt = 4.5 min) and (9) (22.4 mg; Rt = 12.5 min); fraction 23–27 (CH₃OH/H₂O 70:30 as eluent) yielded compounds (10) (4.7 mg; Rt = 6.5 min) and (11) (4.3 mg; Rt = 7.5 min). The structures of the compounds were determined by comparison of their NMR and mass spectrometry (MS) data with literature values²⁴.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay

This experimental procedure was adapted from Wang et al.²⁹, modified by our workgroup as reported previously³⁰. The absorbance was measured using a PerkinElmer Lambda 40 UV/VIS spectrophotometer at 517 nm against a blank, which was without DPPH. All tests were run in triplicate. Ascorbic acid was used as a positive control. A decrease of DPPH solution absorbance indicates an increase of DPPH radical scavenging activity. This activity is given as % DPPH radical-scavenging, calculated by the equation:

β-Carotene bleaching test

Antioxidant activity was determined using a modified β-carotene bleaching test³¹. Briefly, 1 mL of β-carotene solution (0.2 mg/mL in chloroform) was added to 0.02 mL of linoleic acid and 0.2 mL of 100% Tween 20. After evaporation of chloroform and dilution with water, 5 mL of the emulsion was transferred into different test tubes containing 0.2 mL of samples in 70% ethanol at different concentrations. Standard (propyl gallate) at the same concentration as samples was used for comparison. The tubes were then gently shaken and placed at 45°C in a water bath for 60 min. The absorbance of the samples, standard, and control was measured at 470 nm using a PerkinElmer Lambda 40 UV/VIS spectrophotometer against a blank, consisting of an emulsion without β-carotene. The measurement was carried out at initial time (t = 0) and successively at 30 and 60 min. All samples were assayed in triplicate and results averaged. The antioxidant activity (AA) was measured in terms of successful bleaching of β-carotene by using the following equation:

where A₀ and A°₀ are the absorbance values measured at the initial incubation time for samples/standard and control, respectively, while A_t and A°_t are the absorbance values measure in the samples/standard and control, respectively, at t = 30 min and t = 60 min.

Cell culture

The murine monocytic macrophage cell line RAW 264.7 (European Collection of Cell Cultures, London, UK) was grown in a plastic culture flask in Dulbecco’s modified Eagle’s medium (DMEM) with l-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimitotic solution (penicillin/streptomycin) under 5% CO₂ at 37°C. After 4–5 days cells were removed from the culture flask by scraping and were centrifuged for 10 min at 1500 rpm. The medium was then removed and the cells were resuspended with fresh DMEM. Cell counts and viability were assessed using a standard trypan blue cell counting technique. The cell concentration was adjusted to 1 × 10⁶ cells/mL in the same medium, and 100 μL of the above concentration was cultured in a 96-well plate for 1 day to become nearly confluent. Concentrations ranging from 10 to 100 μg/mL of the samples were prepared from stock solutions by serial dilution in DMEM to give a volume of 100 μL in each well of a microtiter plate (96-well). Then cells were cultured with vehicle, P. saxatile MeOH extract, and isolated compounds in the presence of 1 μg/mL LPS for 24 h.

Assay for cytotoxic activity

Cytotoxicity was determined using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay reported by Tubaro et al.³², with some modification. The assay for each concentration of samples was performed in triplicate and the culture plates were kept at 37°C with 5% (v/v) CO₂ for 1 day. After 24 h of incubation, 100 μL of medium was removed from each well. Subsequently, 100 μl of 0.5% (w/v) MTT (Sigma, Italy), dissolved in phosphate buffered saline, was added to each well and allowed to incubate for a further 4 h. After that, 100 μL of dimethylsulfoxide (DMSO) was added to each well to dissolve the formazan crystals. Absorbance values at 550 nm were measured with a microplate reader (GDV DV 990 B/V; Roma, Italy). Cytotoxicity was expressed as CD₅₀, which is the concentration to reduce the absorbance of treated cells by 50% compared with the control (untreated cells).

Inhibition of nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells

The presence of nitrite, a stable oxidized product of NO, was determined in cell culture media using Griess reagent (1% sulfanamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H₃PO₄)³³; 100 μL of cell culture supernatant was removed and combined with 100 μL of Griess reagent in a 96-well plate, followed by spectrophotometric measurement at 550 nm using a microplate reader (GDV DV 990 B/V). The nitrite concentration in the supernatant was determined by comparison with a sodium nitrite standard curve.

Microtiter cholinesterase inhibition assay

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition was assessed by modification of Ellman’s method³⁴, which is based on the reaction of released acetyl- or butyrylthiocholine to give a colored product with a chromogenic reagent. AChE or BChE 20 μL (0.20 U/mL in buffer pH 8) and P. saxatile isolated compounds (25 μL) were added to 50 μL of buffer, pH 8, and pre-incubated in an ice bath at 4°C for 30 min. Duplicate wells were also treated this way with 20 μL of physostigmine (0.1 mM) to allow interference of the test substances in the assay to be assessed, and to control for any hydrolysis of acetylcholine not due to enzyme activity. The reaction was started by adding dithionitrobenzene (DTNB) solution (125 μL of 0.05 mM in buffer, pH 7) and acetylthiocholine (ATCI) or butyrylthiocholine BTCI (25 μl, 0.018 mM in buffer, pH 7), and tubes were incubated in a water bath for 20 min at 37°C. The reaction was stopped by placing the assay solution tubes in an ice bath and adding physostigmine (20 μL, 0.018 mM in buffer, pH 7). Blanks were used of reagents without compounds, and a positive control was set up which was the same as the blank except that physostigmine (20 μL, 0.018 mM in buffer, pH 7) was added. The production of yellow anion was immediately recorded on a spectrophotometer at 405 nm. The inhibition rate (%) was calculated by the equation:

Results and discussion

Compound characteristics in P. saxatile MeOH extract

The major constituents in the MeOH extract were isolated using several repeated procedures of RP-18 silica gel HPLC. The structures of the known compounds (Figure 1), 1-O-β-glucopyranosyl-2(3′,3′-dimethylallyl) hydroquinone (1), 1-O-β-glucopyranosyl-2(3′-hydroxymethyl-3′-methylallyl) hydroquinone (2), chlorogenic acid (3), caffeic acid (4), methylchlorogenic acid (5), apigenin 7-O-β-glucopyranoside (6), 3,5-di-O-caffeoylquinic acid methyl ester (7), 3,5-di-O-caffeoylquinic acid (8), luteolin-4′-O-glucopyranoside (9), luteolin (10), and apigenin (11), were determined by comparison of their NMR and MS data with literature values²². All these compounds have been previously investigated for their antioxidant activity. In fact, Chiang et al.³⁵ have accredited to chlorogenic acid, caffeic acid, and 3,5-di-O-caffeoylquinic acid a very high antioxidant activity. The antioxidant activity of flavonoids was confirmed by Bae et al.³⁶. The compounds present in a sufficient amount (3–6, 8–11) were investigated for their anti-inflammatory and anticholinesterase activities in order to relate biological activities of the extract to pure isolated compounds.

Figure 1.  Structures of isolated compounds from P. saxatile.

Free radical scavenging and antioxidant activity

The DPPH radical-scavenging activity of all samples was measured. The effect of antioxidants on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was thought to be due to their hydrogen-donating ability. The scavenging effects of the methanolic extract and fractions on DPPH were examined at different concentrations (10, 25, 50, 100, 250, 500, and 1000 μg/mL). As shown in Table 1, the methanolic extract showed significant activity (IC₅₀ 25 μg/mL). Antioxidant activity, which was demonstrated by the ability of the samples to inhibit the bleaching of β-carotene, was measured and compared to that of the control, which contained no antioxidant component. The methanolic extract of P. saxatile inhibited oxidation of linoleic acid. As shown in Table 1, the methanolic extract showed antioxidative activity after 30 min of incubation as well as after 60 min of incubation (IC₅₀ 14 μg/mL and 41 μg/mL, respectively), indicating that its activity was not correlated with time of heating.

Table 1.  Free radical scavenging and lipid peroxidation inhibition of methanolic extract from P. saxatile.

	% Inhibition	IC50 (μg/mL)^a
		DPPH	β-Carotene bleaching test
			30 min	60 min
DPPH assay (μg/mL)		25 ± 0.18
1000	92.5
500	93.9
250	93.6
100	92.0
50	86.9
25	50.8
10	21.8
β-Carotene bleaching test^b			14 ± 0.08	41 ± 0.21
100	78.1/76.0
50	59.6/59.7
25	50.8/43.7
10	36.7/38.8
5	13.1/22.0
1	11.0/18.5
Propyl gallate^c			1 ± 0.01	1 ± 0.01
Ascorbic acid^c		2 ± 0.01

^aValues are given ± SEM (n = 3).

^bInhibition values are 30 min/60 min.

^cPropyl gallate and ascorbic acid were used as positive controls.

Inhibition of NO production

The anti-inflammatory activity of P. saxatile MeOH extract and isolated compounds was studied in vitro, analyzing their inhibitory effects on chemical mediators released from macrophages. Once activated by inflammatory stimulation, macrophages produce a large number of cytotoxic molecules. Treatment of RAW 264.7 macrophages with LPS (1 μg/mL) for 24 h induces NO production, as assessed by measuring the accumulation of nitrite, a stable metabolite of NO, in the medium, based on the Griess reaction. NO, a macrophage-derived mediator, is considered to play a key role in the inflammatory response, based on its occurrence at inflammatory sites and its ability to induce many of the hallmarks of the inflammatory response. The beneficial effect of P. saxatile extract on inhibition of the production of inflammatory mediators in macrophages can be mediated through oxidative degradation of the products of phagocytes, such as O²⁻ and HOCl. As shown in Table 2, incubation of RAW 264.7 cells with the methanol extract and isolated compounds of P. saxatile induced a significant inhibitory effect on LPS-induced nitrite production. The MeOH extract of P. saxatile showed significant inhibition of LPS-induced NO production in RAW 264.7 cells in a dose-dependent manner, with an IC₅₀ value of 82 μg/mL. The pure compounds that most effectively inhibited LPS-induced NO production were caffeic acid (4) and methylchlorogenic acid (5), with IC₅₀ values of 7 μg/mL and 12 μg/mL, respectively (Table 2). Although the anti-inflammatory activity of caffeic acid has already been studied³⁷, methylchlorogenic acid was investigated for the first time. In contrast with Wang and Mazza³⁸, who showed that chlorogenic acid has no inhibitory effect, but on the contrary may induce the production of NO, we found that both chlorogenic acid and its methyl ester showed anti-inflammatory activity. As regards flavonoids, several articles^39,40 have discussed their anti-inflammatory activity, particularly of apigenin and luteolin. No data are available for the glucopyranosides of these compounds, which in our study showed significant activity on NO production. In fact luteolin-4′-glucopyranoside (9) showed an IC₅₀ value of 30 μg/mL, while apigenin-7-glucopyranoside (6) showed an IC₅₀ value of 25 μg/mL. Phenolic compounds are one of the most important groups of secondary metabolites in edible plants. Epidemiological studies have indicated that naturally occurring phenolic compounds may play an important role in the maintenance of human health and prevention of several diseases⁴¹. This is also believed to be correlated to the anti-oxidative properties of phenolics. In particular, flavonoids have a broad spectrum of biological properties, e.g. antinflammatory. It has been previously reported that the presence of free hydroxyls on a flavonoid nucleus enhances the inhibition of inflammation, whereas substitution by methoxy groups or sugars diminishes the activity⁴². Based on structure and activity relationship analysis, we observed that substitution of the C4′ hydroxyl group with glucose, as in the case of luteolin-4′-O-glucopyranoside (9), and substitution of the C7 in the case of apigenin 7-O-β-glucopyranoside (6), resulted in an approximately two-fold increase in IC₅₀ values for inhibition of NO production relative to that of luteolin (10) or apigenin (11), containing free hydroxyl groups in the C4′ and C7 position, respectively.

Table 2.  Inhibition of NO production in LPS-induced RAW 264.7 macrophages by methanolic extract and compounds isolated from P. saxatile.

Compound	Concentration (μg/mL)	% Inhibition	IC50
MeOH extract	100	65 ± 1.4**	82.09 ± 1.6**
	50	30 ± 0.8**
	25	27 ± 0.7**
	10	25 ± 0.5**
Chlorogenic acid (3)	100	95 ± 1.9**	17.59 ± 0.75**
	50	50 ± 1.2**
	25	33 ± 0.7**
	10	22 ± 0.5**
Caffeic acid (4)	100	97 ± 1.9**	7.21 ± 0.63**
	50	65 ± 1.5**
	25	29 ± 0.6**
	10	40 ± 0.7**
Methylchlorogenic acid (5)	100	99 ± 2.2**	12.86 ± 0.71**
	50	57 ± 1.3**
	25	48 ± 0.9**
	10	29 ± 0.7**
Apigenin 7-O-β-glucopyranoside (6)	100	99 ± 2.1**	25.35 ± 0.87**
	50	43 ± 0.9**
	25	26 ± 0.7**
	10	15 ± 0.4**
3,5-Dicaffeoylquinic acid (8)	100	10 ± 0.5**	—
Luteolin-4′-O-glucopyranoside (9)	100	90 ± 1.9**	30.55 ± 0.91**
	50	38 ± 0.8**
	25	26 ± 0.6**
	10	10 ± 0.3**
Luteolin (10)	100	91 ± 1.8**	20.76 ± 0.82**
	50	38 ± 0.8**
	25	36 ± 0.7**
	10	15 ± 0.4**
Apigenin (11)	100	90 ± 2.0**	16.75 ± 0.72**
	50	37 ± 1.0**
	25	22 ± 0.6**
	10	18 ± 0.5**
Indomethacin	200	95 ± 2.1**	52.8 ± 1.2**
	100	66 ± 1.3**
	50	46 ± 1.1**
	25	5 ± 0.3**
	12.5	2.5 ± 0.2**

Note. Values are mean ± SD (n = 3); multicomparison Dunnett’s test: ** p < 0. 01. Indomethacin was used as positive control

Cytotoxicity

The cytotoxic effect of all samples (P. saxatile MeOH extract and isolated compounds) in the presence of LPS (10 μg/mL) was evaluated. The P. saxatile MeOH extract and isolated compounds did not show any cytotoxicity at concentrations of 10–100 μg/mL.

Anticholinesterase activity

This article reports for the first time the cholinesterase inhibitory properties of P. saxatile and derived compounds (Table 3). Results evidenced that the MeOH extract was able to inhibit BChE (IC₅₀ 523.75 μg/mL) but was unable to affect AChE activity (Table 3). As regards the isolated compounds, aglycons were more potent than glycosides in inhibition of the enzyme; in fact IC₅₀ against AChE was 25.2 μg/mL for luteolin and 66.1 μg/mL for luteolin-4′-O-glucopyranoside, and 27.5 and 102.8 μg/mL for apigenin and apigenin-4-glycoside, respectively. Luteolin exhibited the same pattern also against BChE (IC₅₀ of 37.2 and 99.5 μg/mL for aglycon and glycoside, respectively). Previously, our research group reported the ability of linariin and isolinariin A and B, three flavones isolated from Linaria reflexa, to inhibit AChE in a dose-dependent manner⁴³. Moreover, recent studies have examined the permeability of flavonoids and their known circulating metabolites across the blood–brain barrier (BBB). Flavonoids are hydrolyzed in the gut and then glucuronized, so it is quite possible that a glycosidal form reaches the central nervous system (CNS)⁴⁴. In particular, Youdim et al.⁴⁵ reported that flavonoids from particular families are able to permeate the BBB, whereas the entry of others is limited by the actions of efflux transporters expressed at the endothelium surface. However, it should be noted that penetration of the BBB does not necessarily equate to entry into neurons, where flavonoids are believed to elicit their neuroprotective effects. The inhibitory activity of caffeic acid against AChE and BChE is also reported (IC₅₀ of 32.4 and 32.2 μg/mL, respectively). Previously, Borrelli et al.⁴⁶ showed the ability of caffeic acid phenethyl ester (CAPE) to inhibit AChE. Luteolin and 3,5-dicaffeoylquinic acid exhibited the most promising activity against AChE with IC₅₀ of 25.2 and 54.5 μg/mL, respectively.

Table 3.  Cholinesterase inhibitory activity of methanolic extract and compounds isolated from P. saxatile.

	Concentration (μg/mL)	% InhibitionAChE	IC50AChE	% InhibitionBChE	IC50BChE	SI(IC50 BChE/AChE)
MeOH extract	1000	—	> 1000	100	523.75 ± 2.6**	—
	750			55.1
	500			49.2
	250			21.9
	100			0
Chlorogenic acid (3)	—		—		—	1.48
Caffeic acid (4)	100	100	32.4 ± 1.7**	100	32.2 ± 1.5**	0.99
	75	88.7		88.6
	50	52.4		52.3
	25	46.9		47.0
	10	23.8		24.1
Methylchlorogenic acid (5)	—		—		—	—
Apigenin 7-O-β-glucopyranoside (6)	200	100	102.8 ± 2.5**	100	85.7 ± 1.4**	0.83
	100	50.3		57.6
	50	25.9		41.3
	25	10.6		36.4
	10	0		9.6
3,5-Dicaffeoylquinic acid (8)	100	100	54.5 ± 1.6**	100	73.2 ± 1.4**	1.3
	75	67.4		51.7
	50	46.9		42.4
	25	31.2		20.8
	10	23.5		0
Luteolin-4′-O-glucopyranoside (9)	100	100	66.1 ± 2.3**	50.3	99.5 ± 2.3**	1.50
	75	56.4		34.2
	50	44.3		10.9
	25	23.4		0
	10	5.5		0
Apigenin (11)	100	100	27.5 ± 1.3**	100	42.7 ± 1.6**	1.55
	75	76.4		79.2
	50	52.9		52.8
	25	46.6		40.8
	10	10.8		12.6
Physostigmine			0.07 ± 0.02		0.17 ± 0.04	2.42

Note. Values are mean ± SD (n = 3); multicomparison Dunnett’s test: ** p < 0. 01.

Conclusions

Oxidative damage may initiate and promote the progression of a number of chronic diseases, including inflammation and Alzheimer’s disease. The present work showed for the first time the in vitro activity of P. saxatile methanolic extract in the treatment of inflammation and Alzheimer’s disease and its metabolite profile. The beneficial effects may be attributable to the ability of P. saxatile extract to act in different ways. In this study we have demonstrated that the MeOH extract of P. saxatile exhibited significant antioxidant activity and an inhibitory effect on NO production (an inflammatory mediator) in macrophages. Furthermore, anticholinesterase assays on the methanolic extract and isolated phenolic compounds showed that luteolin was the most potent inhibitor of both AChE and BChE. We also observed that the extract of P. saxatile could be a good source of phenolic compounds, which are one of the most bioactive groups of secondary metabolites in plants. The metabolism and bioavailability of these phenolic compounds in vivo are suggested to correlate well to their observed antioxidant properties in vitro.

Therefore, we propose here the potential benefits of P. saxatile extract on the basis of the phytochemical characteristics and the observed bioactive properties. Naturally occurring phytocompounds possess antioxidative, anti-inflammatory, and anticholinesterase properties, which appear to contribute to their chemopreventive or chemoprotective activity. Further studies of the plant extracts and/or the identified compounds from P. saxatile on the pharmacokinetics or mode of action on mechanisms of chemopreventive properties are warranted.

Acknowledgement

The authors thank the CSIAS of University of Naples “Federico II” for technical assistance.

Declaration of interest: The authors report no conflicts of interest.