Selective monoamine oxidase B inhibition by an Aphanizomenon flos-aquae extract and by its constitutive active principles phycocyanin and mycosporine-like amino acids.
Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga that has been traditionally used for over 25 years for its health-enhancing properties. Recent studies have shown the ability of a proprietary AFA extract (Klamin[R]) to improve mood, counteract anxiety, and enhance attention and learning. Aim of this study was to test the monoamine oxidase (MAO) inhibition activity of the same AFA extract and of its constituents phycocyanin (AFA-PC) and mycosporine-like aminoacids (AFA-MAAs). All compounds showed a dose-dependent selective inhibition of MAO-B activity as compared to MAO-A. The [IC.sub.50] values of the AFA extract (concentration 10 mg/ml), AFA-PC and AFA-MAAs were 6.4 [micro]l/ml, 1.33 [micro]M and 1.98 [micro]M, respectively, evidencing a mixed-type of inhibition for the AFA extract ([K.sub.i] 0.99 [micro]l/ml), a non-competitive inhibition for AFA-PC ([K.sub.i] 1.06 [micro]M) and a competitive inhibition for AFA- MAAs ([K.sub.i] 0.585 [micro]M). These results are important to explain the neuromodulating properties of the AFA extract Klamin[R], which is rich in phenylethylamine, a general neuromodulator, that would nevertheless rapidly destroyed by MAO-B enzymes without the inhibitory activity of the synergic active principles AFA-PC and AFA-MAAs. The present investigation thus proposes the extract as potentially relevant in clinical areas such as mood disorders and neurodegenerative diseases.
AFA-mycosporine-like amino acids
Klamath algae extract
The cyanophyta Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga that is consumed as a nutrient-dense food source and for its health-enhancing properties (Benedetti et al., 2004; Jensen et al., 2000, 2001; Pugh and Pasco, 2001; Pugh et al., 2001). Recent studies have shown that a proprietary extract from Klamath algae is able to improve mood alterations, including depression and anxiety, as well as to enhance learning (Genazzani et al., 2010; Scoglio et al., 2009; Sedriep et al., 2011).
As a possible explanation of these reported effects, it has been found that Klamath Lake's AFA contains varying but significant quantities of phenylethylamine (PEA), ranging from 1 to 3 mg/gr, which are brought to a concentration of 15 mg/gr in the Klamin[R] extract. PEA is considered responsible for affective behavior (Sabelli and Javaid, 1995); indeed, the most important action of PEA is to promote the neurotransmission of catecholamines, not only by stimulating their release but also by acting as a fast and physiological reuptake-inhibitor of dopamine (Barroso and Rodriguez, 1996). At the same time, PEA can also modulate the transmission of serotonin (Sabelli and Javaid, 1995) and depress neurotransmission if needed (Federici et al., 2005). However, PEA is subjected to a rapid deamination by peripheral monoamine oxidase B (MAO-B) which, like monoamine oxidase A (MAO-A), catalyzes the degradation of neuroactive and vasoactive amines in the central nervous system and in peripheral tissues. As a consequence, once ingested, PEA needs to be protected by some MAO-B inhibiting substances to be effective (Sabelli et al., 1996). In this study, we hypothesized that, besides the content of PEA in Klamath Lake's AFA, there might be in the algae one or more molecules endowed with the ability to inhibit MAOs.
Most of the various natural plant and food-derived molecules endowed with a MAO inhibition ability are antioxidant and/or anti-inflammatory substances, whole or purified, including some which have long been used for the treatment of some mental diseases and as anti-aging factors in traditional folk medicine (Kong et al., 2000, 2001; Lin et al., 2003; Mazzio et al. 1998; Pan et al., 2000; Zhou et al., 2001). Research on these natural substances manifests a need to find new and safe MAO inhibitors, and among such substances devoid of any serious side effects we can list Rhodiola rosea L. (Van Diermen et al., 2009), coffee (Herraiz and Chaparro, 2005), constituents of common benign foods and herbs such as green tea catechins, curcumin, sylimarin, sesame oil (Mazzio et al., 1998), as well as licorice (Hatano et al., 1991).
Klamath Lake's AFA contains a significant amount of a specific AFA-phycocyanin (AFA-PC) and a complex of mycosporine-like amino acids (MAAs), principally porphyra-334 (International patent WO 2008/000431 A2). C-Phycocyanin (C-PC) has been described as a strong antioxidant (Bhat and Madyastha, 2000,2001; Romay and Gonzalez, 2000), anti-inflammatory (Reddy et al., 2000; Romay et al., 1998) and neuroprotective (Rimbau et al., 1999) natural compound. The specific AFA-PC, characterized by a peculiar structure which includes both C-PC and phycoerythrocyanin, has also been found to be a strong antioxidant than ordinary C-PC (International patent WO 2008/000431 A2). In accordance, a previous study on the Oxygen Radical Absorbance Capacity (ORAC) of AFA-PC evidenced that it was the most effective antioxidant among some purified molecules (i.e. trolox, ascorbic acid and glutathione), showing a ORAC value 3 times higher than catechine (Benedetti et al., 2010).
On the other hand, MAAs are water-soluble nitrogenous compounds which have UV-absorbing properties and antioxidant activities (Cockell and Knowland, 1999; de la Coba et al., 2009; Sinha et al., 1998). Accordingly, it has been demonstrated that porphyra-334 from AFA algae presents UVA protective capacity (Torres et al., 2006).
This is the first time that the inhibition properties on MAO-A and MAO-B activity of the novel AFA extract Klamin[R] and of its constituents AFA-PC and AFA-MAAs have been investigated.
Materials and methods
The AFA extract Klamin[R] was kindly provided by Nutratec (Urbino, Italy) and stored in the dark at 4[degrees]C. MAO-A, MAO-B, R-(-) deprenyl hydrochloride, clorgyline, kynuramine dihydrobromide and benzylamine hydrochloride were purchased from Sigma (Milan, Italy).
Preparation of the hydrosoluble AFA extract
The dried extract was first dissolved in phosphate saline buffer (PBS) pH 7.4 (concentration 10 mg/ml) and centrifuged at 2500 x g at 4[degrees]C for 10min to remove any insoluble material. The spectrophotometric analysis of the blue supernatant revealed the characteristic peaks of PC at 620 nm (Benedetti et al., 2006) and of MAAs (i.e. porphyra-334) at 334 nm (Torres et al., 2006) (Fig. 1). Taking into consideration that porphyra-334 has a molecular weight of approximately 300 Da and a molar extinction coefficient ([epsilon]) of 42,300 [M.sup.-1] [cm.sup.-1], we obtained that 1 gr of Klamin[R] extract contained 14mg of MAAs (1.4% DW). Similarly, taking into consideration that PC has a specific extinction coefficient ([E.sup.1%]) of 701[g.sup.-1] [cm.sup.-1], we obtained that 1 gr of Klamin[R] extract contained 86 mg of PC (8.6% DW). The hydrosoluble extract was stored at 4[degrees]C until assessed.
Purification of AFA-PC
AFA-PC was purified from the dried AFA extract by a single step chromatographic run using a hydroxyapatite column (Bio-Rad Laboratories, CA, USA) as previously described (Benedetti et al., 2006; Rinalducci et al., 2009). Pure AFA-PC (ratio [A.sub.620]/[A.sub.280] of 4.78) was stored at -20[degrees]C; before use, the concentration of the protein was evaluated spectrophotometrically at 620 nm ([OD.sub.620] 770,000 [M.sup.-1] [cm.sup.-1]).
Extraction of AFA-MAAs
AFA-MAAs were extracted as previously reported (Torres et al., 2006). Briefly, 20 mg of AFA powder were extracted in 2 ml of 20% (v/v) aqueous methanol (HPLC grade) by incubating in a water bath at 45 [degrees]C for 2.5 h. After centrifugation (5000 x g; GS-15R Centrifuge, Beckman, Palo Alto, USA), the supernatant was evaporated to dryness and redissolved in 2 ml 100% methanol, vortexed for 2-3 min and centrifuged at 10,000 x g for 10 min. The supernatant was evaporated and the extract redissolved in the same volume of 0.2% acetic acid. Before use, AFA-MAA concentration (principally porphyra-334) was evaluated spectrophotometrically at 334 nm ([OD.sub.334] 42,300 [M.sup.-1] [cm.sup.-1]).
MAO-A and MAO-B assay
MAO-A and MAO-B activity were assessed by using the respective substrates kynuramine (0.05 mM) and benzylamine (0.4 mM) (Yoshida et al., 2004). The test was performed spectrophotometrically at 316 nm (MAO-A) or 250 nm (MAO-B) by incubating at 30 [degrees]C MAO-A or MAO-B (2 [micro]g/ml) with different concentrations of the hydrosoluble AFA extract (2.5-40 [micro]l/ml), and diverse concentrations of AFA-PC and MAAs (0.5-4 [micro]M). The compounds clorgyline (a selective MAO-A inhibitor) and deprenyl (a selective MAO-B inhibitor) were used as positive controls.
The mechanism of MAO inhibition was assessed by analyzing the corresponding double reciprocal Lineweaver-Burk plots. The apparent inhibition constants ([K.sub.i])) for the AFA extract, AFA-PC and MAAs were calculated using Lineweaver-Burk plots, estimated from the secondary plot (slope versus concentration of inhibitor). The 50% inhibitory concentration ([IC.sub.50]) values were calculated by adjusting the experimental data (% inhibition versus concentration of inhibitor) to non-linear regression curves. The selectivity for MAO-B was calculated using the [IC.sub.50] (MAO-A)/(MAO-B) ratio.
Statistics and data processing
Results are expressed as means [+ or -] standard deviation (SD). Values are at least from duplicates experiments. Statistics and graphs were obtained using the software Microcal Origin 6.0 (Microcal Software, Inc., Northampton, MA, USA).
Inhibition of the AFA extract on MAO-A and MAO-B activity
The spectrophotometric analysis of MAO-A and MAO-B activity evidenced that the hydrosoluble AFA extract was a selective inhibitor of MAO-B (Fig. 2). Indeed, at the concentrations tested (2.5-40 [micro]l/ml), it inhibited in a dose-dependent manner MAO-B but not MAO-A activity. Specifically, [IC.sub.50] values were equal to 6.4 [micro]l/ml for MAO-B (corresponding to 0.064 mg/ml of AFA extract) and >40 [micro]l/ml for MAO-A (Table 1).
The Lineweaver-Burk plots, determined using various concentrations of benzylamine (0.1-1.25 mM), indicated that the AFA extract acted as a mixed-type inhibitor (Fig. 3) and had a [K.sub.i] value of 0.99 [micro]l/ml (corresponding to 0.01 mg/ml of AFA extract) (Fig. 3 insert). Selectivity was more than 6.25 times higher for MAO-B than for MAO-A, as calculated using the MAO-A/MAO-B [IC.sub.50] ratio (Table 1).
Inhibition of the purified AFA-PC on MAO-A and MAO-B activity
MAO-A and MAO-B inhibition by AFA-PC was reported in Figs. 4 and 5, respectively. We found that AFA-PC inhibited MAOB activity in a dose-dependent manner with a [IC.sub.50] value equal to 1.33 [micro]M, the [IC.sub.50] value of the positive control deprenyl was 0.28 [micro]M.
The purified AFA-PC was selective for MAO-B; indeed, by comparing the respective MAO-A and MAO-B [IC.sub.50] values (MAO-A/MAO-B ratio), selectivity was more than 3.76 times in favor of MAO-B (Table 1). The Lineweaver-Burk plots revealed that AFA-PC inhibited MAO-B activity in a non-competitive way (Fig. 6), with a [K.sub.i] value equal to 1.06 [micro]M (Fig. 6 insert).
Inhibition of AFA-MAAs on MAO-A and MAO-B activity
MAO-A and MAO-B inhibition by AFA-MAAs was reported in Figs. 4 and 5, respectively. AFA-MAAs inhibited MAO-B activity in a dose-dependent manner, with a [IC.sub.50] value equal to 1.98 [micro]M. The selectivity of MAAs for MAO-B was more than 2.02 times higher than for MAO-A, as established by comparing the respective MAOA and MAO-B [IC.sub.50] values (MAO-A/MAO-B ratio) (Table 1). The Lineweaver-Burk plots revealed that AFA-MAAs inhibited MAOB activity in a competitive way (Fig. 7), with a [K.sub.i] value equal to 0.59 [micro]M (Fig. 7 insert).
The present study investigated for the first time the potential effect of a proprietary extract of A.flos-aquae (AFA), and of its constitutive active principles PC and MAAs on MAO-A and MAO-B activity in vitro. Results show that the tested substances are powerful selective MAO-B inhibitors, yet with the reversible type of inhibition typical of natural substances and plausibly devoid of any significant side effects. Indeed, we found that AFA-PC and AFA-MAAs have [IC.sub.50] and [K.sub.i] values much lower than those of other natural substances such as catechine and piperine, and were very close to that of the synthetic selective MAO-B inhibitor deprenyl (selegiline) (Table 2).
The first generation of MAO inhibitors (non-selective and irreversible) was characterized by a lack of selectivity in their MAO inhibition, inhibiting both MAO-A and MAO-B with serious side-effects (Cooper, 1989). The new generation of MAO inhibitors has been developed with a high degree of selectivity in the inhibition of either MAO-A or MAO-B. Deprenyl (a MAO-B selective inhibitor), even though has drastically reduced the side effects of the first generation of MAO inhibitors, especially when used in combination with levodopa can cause anorexia/nausea, dry mouth, dyskinesia, and orthostatic hypotension in patients with Parkinson's disease, the latter being most problematic (Volz and Gleiter, 1998). Although the main role of AFA-PC and AFA-MAAs in the extract Klamin[R] appears to be that of protecting PEA naturally contained in the extract in its journey through the gastro-intestinal system and to the brain, the extract itself, as well as the two specific molecules, can provide a relevant alternative for those who want new and natural MAO inhibitors devoid of adverse effects, mainly due to their reversible type of inhibition.
As regards the type of MAO-B inhibition, the AFA extract shows a mixed-type mechanism, certainly due to the complex nature of the extract itself, which is likely to contain other molecules beside PC and MAAs endowed with a MAO-B inhibition ability, some of which competitive and others non-competitive. This warrants the need for further research to identify such molecules.
Interestingly, we found that AFA-MAAs, which are soluble low molecular weight compounds (approximately 300 Da), show a competitive type of inhibition toward MAO-B activity, indicating that, thanks to their chemical structure, MAAs compete with the substrate for the link to the active site of the enzyme. This is the first time that MAAs from any species or organism are identified as capable to significantly inhibit MAO-B activity; above all, their low molecular weight and the consequent possibility to easily cross the blood-brain barrier make MAAs very interesting compounds for their actions in vivo.
On the other hand, PC is a phycobiliprotein of large dimensions (121,000 Da in its trimeric form, and approximately 40,000 Da in its monomeric form). This could support the hypothesis that the in vitro MAO-B inhibition may be simply due to its steric volume. However, AFA-PC selectivity is more than 3.75 times higher for MAO-B than for MAO-A, indicating a functional and not simply volumetric selectivity toward MAO-B. The value of this first in vitro evaluation is supported by previous studies showing that the oral administration of PC in rats reduces microglial and astroglial activation induced by kainic acid (Rimbau et al., 1999), thus suggesting that some active metabolites of the protein actually cross the blood brain barrier. Further studies are needed to determine which portion of PC is able to cross the barrier and inhibit the MAO-B enzyme at the neuronal level.
A recent study confirmed the ability of the Klamin[R] extract to cross the blood brain barrier, not only to stimulate through its PEA and MAO-B inhibitor content the brain dopaminergic activity and cascade, enhancing learning ability and attention in mice, but also to reduce, thanks to its AFA-PC and other antioxidants, the level of lipoperoxidation in mice brain (Sedriep et al., 2011).
A further area of research, as indicated above, is the synergy between AFA-PC and AFA-MAA physiological MAO-B inhibitory activity and PEA naturally contained in the AFA extract. PEA is a powerful dopaminergic neuromodulator, and previous works show that PEA, when administered together with a selective MAO-B inhibitor, can significantly relieve depression without side effects (Sabelli et al., 1989,1996). Therefore, we can expect that the synergy between PEA, AFA-PC and MAAs, all molecules present on the whole extract, may significantly improve dopamine synaptic level and activity, and therefore the many physiological cascades in which dopamine is involved.
Finally, AFA-PC has proven to possess a strong antioxidant activity (Benedetti et al., 2010). Since PC from other microalgae have shown significant neuroprotective activity (Rimbau et al., 1999), it is plausible to expect a similar or higher neuroprotective activity from AFA-PC, and at least one study has already shown that in vivo (Sedriep et al., 2011). Oxidation of dopamine and PEA by MAO-B can lead to the production of neurotoxic compounds such as hydrogen peroxide, semiquinones (Klegeris et al., 1995), hydroxyl radicals (Smith et al., 1994) and superoxide anions (Youdim and Lavie, 1994). The formation of these radicals can induce subsequent lipid peroxidation and neurodegeneration (Chiueh et al., 1994). Elevated levels of lipid hydroperoxides have been found in the central nervous system tissue taken from patient with Parkinson's disease (Dexter et al., 1994). Thus, the potential neuroprotective activity of AFA-PC may add a further significant angle to the synergic activity of the whole AFA extract.
In conclusion, the present investigation provides for the first time evidence that the AFA extract Klamin[R] and its constituents PC and MAAs are MAO-B selective inhibitors, and that a fruitful synergy of different molecules and functions can be expected from specific extracts from AFA algae. This finding may be of importance to obtain a better understanding of the working mechanism and the potential use of the AFA extract Klamin[R].
Received 31 July 2013
Received in revised form 9January 2014
Accepted 2 March 2014
Abbreviations: AFA, Aphanizomenon flos-aquae; MAAs, mycosporine like aminoacids; MAO-A, monoamine oxidase A; MAO-B, monoamine oxidase B; PC, phycocyanin; PEA, phenylethylamine.
Conflict of interest statement
The authors declare that they (except Francesca Benvenuti) are the inventors of the patent relative to the Klamin[R] extract; and that Stefano Scoglio is the R8&D Director of the company which owns the patent.
We gratefully acknowledge Sonia Francogli for technical assistance.
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Stefano Scoglio (a),*, Yanina Benedetti (a-b), Francesca Benvenuti (b), Serafina Battistelli (b), Franco Canestrari (b), Serena Benedetti (b)
(a) Centro di Ricerche Nutriterapiche, via I Maggetti 14, 61029 Urbino, Italy
(b) Dipartimento di Scienze Biomolecolari, Sezione di Biochimica Clinica e Biologia Cellulare, Universita di Urbino "Carlo Bo" via Ubaldini 7, 61029 Urbino, Italy
* Corresponding author at: Centro di Ricerche Nutriterapiche, Via I Maggetti 14 61029 Urbino, PU, Italy. Tel.: +39 0722 351483; fax: +39 0722 327453.
E-mail addresses: firstname.lastname@example.org, email@example.com (S. Scoglio).
Table 1 Kinetic analysis on MAO inhibition by the tested compounds. Compound [MAO-AIC.sub.50] [MAO-BIC.sub.50] AFA extract ([micro]l/ml) >40 6.40 AFA-PC ([micro]M) >5 1.33 AFA-MAAs ([micro]M) >4 1.98 MAO-B vs. MAO- Compound A selectivity Inhibition type AFA extract ([micro]l/ml) >6.25 Reversible mixed AFA-PC ([micro]M) >3.76 Reversible non competitive AFA-MAAs ([micro]M) >2.02 Reversible competitive Table 2 Kinetic parameters ([IC.sub.50] and K, values) on MAO-B inhibition by AFA-PC and AFA-MAAs in comparison with known synthetic and natural inhibitors. Inhibitors [IC.sub.50] [K.sub.i] Deprenyl 0.28 [micro]M 0.02 [micro]M AFA-PC 1.33 [micro]M 1.06 [micro]M AFA-MAAs 1.98 [micro]M 0.59 [micro]M Non-Harman 6.47 [micro]M 1.12 [micro]M Alcaloids (b) Emodin (c) 35.4 [micro]M 15.1 [micro]M Paeonol (c) 42.5 [micro]M 38.2 [micro]M Epicatechine (d) 58.9 [micro]M 21 [micro]M Catechine (d) 88.6 [micro]M 74 [micro]M Piperine (c) 91.3 [micro]M 79.9 [micro]M Inhibitors Inhibition type Deprenyl Irreversible11 AFA-PC Reversible non competitive AFA-MAAs Reversible competitive Non-Harman Reversible mixed Alcaloids (b) Emodin (c) Reversible mixed Paeonol (c) Reversible competitive Epicatechine (d) Reversible mixed Catechine (d) Reversible mixed Piperine (c) Reversible competitive (a) Yan et ai. (2004). (b) Herraiz and Chaparro (2005). (c) Kong etal. (2004). (d) Hou et al. (2005).