A Tricholoma matsutake Peptide with Angiotensin Converting Enzyme Inhibitory and Antioxidative Activities and Antihypertensive Effects in Spontaneously Hypertensive Rats

Hypertension is a major risk factor for cardiovascular disease. A crude water extract of the fruiting bodies of a highly prized mushroom Tricholoma matsutakei exerted an antihypertensive action on spontaneously hypertensive rats (SHRs) at a dosage of 400 mg/kg. An angiotensin converting enzyme (ACE) inhibitory peptide with an IC50 of 0.40 μM was purified from the extract and designated as TMP. Its amino acid sequence was elucidated to be WALKGYK through LC-MS/MS analysis. The Lineweaver-Burk plot suggested that TMP was a non-competitive inhibitor of ACE. A short-term assay of antihypertensive activity demonstrated that TMP at the dosage of 25 mg/kg could significantly lower the systolic blood pressure (SBP) of SHRs. TMP exhibited remarkable stability over a wide range of temperatures and pH values. It also demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The aforementioned activities of TMP were corroborated by utilizing the synthetic peptide. Hence T. matsutake can be used as a functional food to help prevent hypertension- associated diseases.

Purification and identification of ACE inhibitory peptide from T. matsutake. The purification protocol entailed extraction with distilled water, ultrafiltration through a membrane, followed by two ion-exchange chromatography steps on Q-Sepharose and Mono-Q, respectively, and finally fast protein liquid chromatography (FPLC) on a Superdex Peptide column. The procedure was efficacious for purifying T. matsutake ACE inhibitory peptide. The water extract was subjected to ultrafiltration through a 5-kDa molecular weight cut-off membrane. Filtrate with a molecular weight (M.W.) over 5 kDa elicited 27% inhibition of ACE activity while filtrate with a M.W. below 5 kDa showed 63% inhibition of ACE activity. Thus the active filtrate with a M.W. below 5 kDa was employed for isolation of ACE inhibitory peptide. After a series of purification procedures, three peaks were observed upon FPLC-gel filtration on a Superdex Peptide 10/300 GL column. Among these peaks, fraction P3 expressed the strongest ACE inhibitory activity (Fig. 1).
Five peptides were obtained in P3 by linear trap quadrupole (LTQ) LC-MS/MS analysis (Table 2). To confirm the ACE inhibitory activity of these peptides, they were chemically synthesized. The chemically synthesized peptide WALKGYK designated as TMP exhibited the highest ACE inhibitory activity (IC 50 = 0.40 μM) and was further studied.
Antioxidant property (free radical scavenging activity) of ACE inhibitory peptide from T. matsutake. Interestingly, it was found that the isolated ACE inhibitory peptide TMP demonstrated DPPH radical scavenging activity (Fig. 2). In this study, the DPPH radical scavenging activity of TMP increased in a concentration-dependent manner. At a higher concentration (10 mg/mL), 50% of the DPPH radicals were scavenged. TMP exhibited a dose-dependent scavenging action on DPPH radicals in the range of concentrations studied 23 . Effects of pH and temperature on ACE inhibitory activity of peptide from T. matsutake. As shown in Fig. 3, the isolated inhibitory peptide TMP retained ACE inhibitory activity after exposure to different pH values and different temperatures for 2 hours. TMP displayed good thermostability and pH stability.
Mode of inhibition of ACE inhibitory peptide from T. matsutake. The mode of inhibition of the isolated inhibitory peptide WALKGYK on ACE was ascertained from the Lineweaver-Burk plot. According to the plot presented in Fig. 4, which showed ACE activity in either the presence or absence of 0.325 mg/mL or 0.675 mg/mL TMP, the three straight lines intersected at the same point on the horizontal axis of the graph plotting 1/S, indicating that the same Km was attained regardless of inhibitor concentration, whereas the maximum velocity differed. The findings indicate that TMP was a non-competitive inhibitor of ACE 24 .
Antihypertensive action of the purified ACE inhibitory peptide from T. matsutake. The antihypertensive action of the isolated inhibitory peptide TMP was investigated by measuring the change of SBP in SHRs at 0.5, 2, 4, 6 and 8 h after oral administration of TMP as shown in Fig. 5a,b. The average blood pressure of the SHRs in the test groups was 190 mmHg just before the oral administration. Half an hour after the oral administration of captopril (100 mg/kg body weight [BW]), in the inhibitory peptide-treated groups (25 and 50 mg/kg BW) as well as the crude extract-treated group (400 mg/kg BW), the blood pressure had undergone a slight decrease. There were significant differences in SBP after treatment with the inhibitory peptide, the high dose of crude extract and the control 2 h after oral administration. The maximum decrease in SBP value caused by 100 mg/kg BW of captopril, 25 and 50 mg/kg BW of the inhibitory peptide and 400 mg/kg BW of the crude extract    was observed at 2 h, and the decrements in SBP produced were 38 mm Hg, 18 mm Hg, 36 mm Hg and 36 mm Hg, respectively. The blood pressures increased gradually 4 h after oral administration in all groups. The peptide group of 50 mg/kg BW exerted an antihypertensive effect equipotent to the positive group until 8 h following administration. There was no significant difference in SBP between rats receiving 200 mg/kg BW of the crude extract and the control rats. Furthermore, no allergic reactions or coughing was noted on the day of the experiment and on the following day.

Discussion
The present account represents the first report on the isolation of the peptide WALKGYK from the renowned edible mushroom T. matsutake which inhibited ACE with an IC 50 value of 0.4 μM and exhibited a clear antihypertensive effect on spontaneously hypertensive rats. A tripeptide GQP from T. giganteum belonging to the same genus manifested ACE inhibitory activity 13 .
There is a paucity of information about the constituents of mushrooms belonging to the genus Tricholoma. Hence the ACE inhibitory activities of eight mushrooms belonging to the genus of Tricholoma were examined in the present study (Table 1). There was a large disparity among the different Tricholoma species regarding ACE inhibitory activity, signifying that ACE inhibitory activity was not correlated with the genus. It was noted that the water extract of T. matsutake had potent ACE inhibitory activity.
At present, more and more studies are focused on bioactive peptides like ACE inhibitory peptides. The bioactive peptides from mushrooms documented for reducing blood pressure are listed in Table 3. The highest ACE inhibitory activity was shown by peptides from Pholiota adiposa 10 , Grifola frondosa 9 and T. giganteum 13 with the lowest IC 50 values which were 0.1, 0.12 and 0.13 μM, respectively. The peptide isolated from T. matsutake in the current study and peptide from Hypsizygus marmoreus 14 exhibited a lower IC 50 value, which were a little higher than the peptide from T. giganteum but substantially lower than other mushroom peptides, including peptides from Pleurotus cystidiosus 12 and Agaricus bisporus 15 .
Food-derived peptides with antihypertensive activity have been identified mostly based on their ability to inhibit ACE in vitro 2 . Potent ACE inhibitory peptides are usually composed of 2-12 amino acid residues 25 , which contain aromatic or hydrophobic residues at the C-terminus, positively charged amino acids in the intermediate domain, and hydrophobic amino acids residues such as alanine, proline, tyrosine, and valine residues at the N-terminus 26 . This is in accordance with the findings of Rohrbach et al. that, the most potent ACE inhibitors always contain hydrophobic amino acid residues at the three C-terminal positions 27 . Peptide TMP contained four hydrophobic amino acid residues (representing an abundance of 57%), and one of the tyrosines was present in the C-terminal tripeptide sequence. Moreover, in a previous study, the amino acids proline and lysine or aromatic  amino acid residues appeared in most naturally occurring ACE inhibitory peptides 28 . Peptide TMP contained two aromatic amino acid residues (tryptophan and tyrosine) and the amino acid residue lysine, in keeping with the characteristic feature of naturally occurring ACE inhibitory peptides.
It was disclosed by many researchers that peptides with ACE inhibitory activity usually possessed some antioxidant activity, such as DPPH radical scavenging activity at the same time. A native peptide RPSYT in commercial soybean infant formulas has both antioxidant activity and antihypertensive properties 29 . Antioxidant and ACE inhibitory peptides have been extracted and isolated from two different types of Thai traditional fermented shrimp pastes 30 . The peptide TMP isolated in the present study exhibited high ACE inhibitory activity and concurrently DPPH radical scavenging effect. On account of the relationship between hypertension and oxidant stress, these active peptides expressing both antihypertensive and antioxidant activities merit further investigations. They may have considerable potential as multifunctional ingredients in functional foods for controlling chronic diseases.   Table 3. ACE inhibitory peptides from mushrooms of different genera. "ND" means "Not Detected".
Scientific RepoRts | 6:24130 | DOI: 10.1038/srep24130 The isolated peptide TMP was a non-competitive ACE inhibitor. Non-competitive inhibitors have been located in Agaricus bisporus 15 , Hypsizygus marmoreus 14 and P. cornucopiae 11 as shown in Table 3. In recent years, some other food sources containing peptidic non-competitive ACE inhibitors have been isolated, for example, peptides from cuttlefish 31 and peptide from fertilized eggs 32 . Unfortunately, the relationship between the mode of inhibition and structure of these peptides has not been fully unravelled 24 .
ACE inhibitory activity in vitro is not always directly related to an antihypertensive effect in vivo 33 . Thus, demonstration of an in vivo antihypertensive effect is of paramount importance, and there have been many investigations on the antihypertensive effect of edible mushrooms and other foodstuff on SHRs. The peptide TMP identified from T. matsutake manifested a significant antihypertensive action on SHRs, which may be beneficial to hypertensive patients and advocates the peptide TMP as a candidate for development into an antihypertensive drug. The water extract of T. matsutake (400 mg/kg) engendered a 19% decline in blood pressure in SHRs from 190 mm Hg to 154 mm Hg while the water extract of H. marmoreus at the dosage of 800 mg/kg lowered the blood pressure by 14.4%, less potently than T. matsutake. The water extract of P. coruncopiae displayed a more pronounced effect on suppressing the blood pressure than T. matsutake and H. marmoreus. There are many bioactive substances for lowering the blood pressure encompassing various peptides, phenolic compounds, proteins, polysaccharides, saponins, sterols, pigments, fiber as well as minerals 34 , which may explain why the IC 50 values of ACE inhibitory activity of peptides from H. marmoreus and T. matsutake were almost identical while the crude water extract of T. matsutake was superior to H. marmoreus in reducing blood pressure 14 . The dosage of the crude extract of T. matsutake was 400 mg/kg, which corresponded to 14.5 g of fruiting bodies of T. matsutake. The peptide TMP from T. matsutake evinced good thermostability, which ensures preservation of the ACE inhibitory activity when T. matsutake is employed in tea or other food items. All this may furnish evidence for T. matsutake being a functional food to help prevent hypertension-associated diseases.
Twelve-week-old male spontaneously hypertensive rats (SHRs) were purchased from Beijing Vital River Experimental Animal Technical co., Ltd.
Assay of ACE inhibitory activity. ACE was prepared using fresh rabbit lungs according to the protocol reported by Hayakari et al. 35  The ACE inhibitory activity was assayed by using a spectrophotometric method following the protocol described by Cushman 36 with modifications. Briefly, an aliquot (20 μL) of the sample was mixed with 20 μL ACE and 150 μL 5 mM HHL in 0.1 M borate buffer (pH 8.3) containing 0.3 M NaCl. The reaction mixture was incubated at 37 °C for 30 min, and then the reaction was terminated by adding 500 μL 1 M HCl. The hippuric acid formed was extracted with 1.2 mL ethyl acetate and centrifuged at 4,000 rpm for 10 min. Then 800 μL of the upper organic phase was heat-evaporated at 95 °C for 35 min to remove ethyl acetate. The dried residue was dissolved in 800 μL distilled water and vortex mixed, and the absorbance was measured spectrophotometrically at 228 nm to estimate ACE inhibitory activity. ACE-inhibitory activity was calculated by using the equation 1: where A was the absorbance of the solution containing ACE and inhibitor, B was the absorbance of solution containing ACE and inhibitor but terminated in advance, C was the absorbance of the solution containing ACE without inhibitor, and D was the absorbance of the solution containing ACE without inhibitor but terminated in advance. The concentration of the inhibitor required to inhibit 50% of the ACE activity under the above assay conditions was defined as IC 50 .
Purification of ACE inhibitor. Fresh fruiting bodies of the mushroom T. matsutake (stored at − 20 °C) were soaked in distilled water (weight: volume = 1:2) at 4 °C for 1 hour, and then homogenized in a Waring blender. The homogenate was extracted overnight at 4 °C before centrifugation at 9,000 rpm for 20 min at 4 °C. The supernatant was centrifuged again at 9,000 rpm for 20 min after boiling for 10 min. Then the supernatant was subjected to ultrafiltration with a 5 kDa M.W. cut-off membrane. The filtrate and solution of the filter-cake were assayed for ACE inhibitory activity as described above. The active fraction was applied on an anion exchange Q-Sepharose column (2.5 cm × 20 cm) in Tris-HCl buffer (10 mM, pH 8.8) with a flow rate of 2 mL/min. After removal of the unadsorbed fraction Q1, adsorbed material was fractionated using a linear concentration gradient of 0-1 M NaCl in the same buffer. The fraction with ACE inhibitory activity was loaded on another strong anion exchange Mono-Q column (4.6 cm × 10 cm) in Tris-HCl buffer (10 mM, pH 8.0) with a flow rate of 1 mL/min. Following removal of the unadsorbed fraction, the column was further eluted with a linear gradient of 0-1 M NaCl in the same buffer. Fractions with ACE inhibitory activity were pooled, and final purification by fast protein liquid chromatography (FPLC) on a Superdex peptide gel filtration column at a flow rate of 0.8 mL/min using an AKTA Purifier was carried out. The most active fraction was lyophilized for further research.
Identification and synthesis of the purified peptide. After lyophilization, the most active fraction was dissolved in 0.1% formic acid and 2% acetonitrile for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a LTQ-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany). Samples were loaded onto a reversed-phase C18 column with a diameter of 0.1 mm × 10 cm and a particle size of 3 μm. Separation of the peptides was achieved by using a gradient of 5 to 35% acetonitrile in 0.1% formic acid over 120 min. The acquired MS/MS data were transformed to MGF by BIWORKS, and the data were searched against the NCBI database using MASCOAT software.
To investigate antihypertensive activity on SHRs, antioxidant capacity and mode of inhibition of ACE, the purified peptide was synthesized by Shanghai Bootech BioScience & Technology Co., Ltd. The purity of the synthetic peptide was 99% as evidenced by HPLC analysis. The IC 50 value of the peptide was investigated according to the method described above.
Antioxidant property (free radical scavenging activity). The DPPH radical scavenging activity of the inhibitor was determined according to the method described by Cheng et al. 23 with slight modifications. An aliquot of 200 μL of the inhibitor at different concentrations (1-10 mg/mL) was mixed with 600 μL 0.004% DPPH dissolved in methanol. The mixture was left at room temperature in the dark for 30 min. Scavenging capacity was measured spectrophotometrically by monitoring the decrease in absorbance at 517 nm. The DPPH radical scavenging activity was calculated by using the equation 2: where A control was the absorbance in the absence of sample, A sample was the absorbance in the presence of the sample.
Effects of pH and temperature on ACE inhibitory activity. The inhibitory peptide was dissolved in distilled water and subsequently incubated for 2 h at different pH values ranging from 2 to 11. The pH value was then adjusted to 7.0. ACE inhibitory activity was determined with the abovementioned method. The inhibitory peptide was incubated for 2 h at different temperatures covering a range from 40 °C to 90 °C. After acclimation to room temperature, the ACE inhibitory activity was assayed at 37 °C as mentioned above.

Determination of mode of inhibition of ACE.
The mode of inhibition of ACE by the peptide was determined spectrophotometrically using HHL as the substrate to distinguish between competitive, non-competitive and uncompetitive inhibition. Basically, a series of concentrations (0.63, 1.25, 2.5 and 5 mM) of HHL were incubated with ACE in the presence or absence of the peptide (0, 0.312 and 0.625 mg/mL). The ACE inhibitory activity was determined according to the aforementioned method. The kinetic parameters Vmax (maximum velocity) and Km (Michaelis constant) were investigated by constructing Lineweaver-Burk plots.
Antihypertensive action in spontaneously hypertensive rats. All experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China and also with the approval of the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong which had been procured prior to the animal experiments. SHRs with systolic blood pressure (SBP) higher than 180 mmHg were randomly divided into six groups after acclimation for 1 week. During the acclimatization period prior to the test, the blood pressures of the rats were measured four times weekly. Each group containing four rats. A blank control group receiving oral administration of 0.9% saline solution and a positive control group receiving oral administration of captopril (100 mg/kg body weight) were included. The ACE inhibitory peptide-treated group received the peptide at two different concentrations: 25 mg/kg body weight and 50 mg/kg body weight. The group treated with crude water extract of T. matsutake received the extract at two different concentrations: 200 mg/kg body weight and 400 mg/kg body weight. SBP was measured thrice at each of the following time points: 0, 0.5, 2, 4, 6 and 8 h after drug/sample administration, using a tail-cuff method with a programmable BP-100A electro-sphygmomanometer (Chengdu Taimeng Software Co., Ltd., Chengdu, China).