Antioxidative Activities and Angiotensin I-Converting Enzyme Inhibition of Extracts Prepared from Chum Salmon (Oncorhynchus Keta) Cartilage and Skin

Abstract

The extracts from cartilage and skin of chum salmon were prepared using a pressure cooker. As a result, protein and collagen contents in extracts from cartilage and skin was higher than those only in cartilage. The inhibition activity of linoleic acid oxidation was high in extract from cartilage. The scavenging activity of cartilage extract was higher than those of cartilage and skin against all reactive oxygen species, such as superoxide anion, hydroxyl, and DPPH radicals. On the other hand, angiotensin I-converting enzyme inhibitory activity of cartilage extract was about seven times as high as that of cartilage and skin extract. The present studies indicate that the extracts, particularly from cartilage, have angiotensin I-converting enzyme inhibitory activity that functions to depress hypertension, and antioxidant activity, which acts to prevent of life-style related diseases such as cancer, cardiovascular diseases, and diabetes. The data should be useful for developing a novel type of functional seasoning.

Keywords:

• Chum salmon
• Extract
• Cartilage
• Skin
• Antioxidant activity
• Angiotensin I-converting enzyme inhibitory activity

INTRODUCTION

Salmon species such as chum salmon Oncorhynchus keta, coho salmon Oncorhynchus kisutch, blueback Oncorhynchus nerka, and chinook salmon Oncorhynchus tshawytscha are an important fish and shellfish having animal protein source. The art of cooking of salmon are as follows: broiled fish, meuniere, deep-fried food, mousse, steeped in sake, a hot-pot, a dish of raw fish and vegetables, mehun (salted fish guts using kidney), sujiko, and ikura (salty food). There are also many processed foods such as salted salmon, canned salmon, and smoked salmon. Typically salmon muscle comprises water (about 70%), proteins (about 22%), and lipids (about 4%), carbohydrates (about 0.1%), and ash (about 1.2%). Moreover, it was richer in retinol, vitamins B₁₂, D, and E, folic acid, and pantothenic acid than mammalian meats. Salmon muscle is now being recognized as a healthy food because its muscle is rich in astaxanthin that shows about 1000 times higher antioxidant activity than representative antioxidant, α-tocopherol. For these reasons, salmon species attract a great deal of attention among consumers that eat sea food, although an edible portion in salmon species is about 60–70% after the removal of skin, bones, head portion, and the internal organs.

The Japanese consume a wide range of fish species everyday. So great quantities of wastes such as skin, bones, and fins are also produced at home, in fish shops, and fish processing and refrigerating factories. Dumping of these wastes could cause pollution that would emit an offensive odor. Although there is an effort to decrease the food waste, the quantity of fish waste produced is increasing with time. Consequently, there has been interest in investigating possible means of making more effective use of underutilized resources and industrial wastes. In fact, these resources may be wasted except for that used in fishmeal manufacture, although the nutritional value of these discards may be fairly high. In this study, we prepared the extracts from the cartilage and skin of chum salmon as an underutilized resources and tested the antioxidant activities of the extracts by measuring their ability to inhibit the autoxidation of linoleic acid and scavenging the oxygen free radicals superoxide and hydroxyl, and the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) using a spectrophotometer. This article also deals with angiotensin I-converting enzyme inhibitory activities of these extracts. The data developted in this study could be useful for developing a novel type of functional seasoning.

MATERIALS AND METHODS

Sample

The cartilage and skin of chum salmon Oncorhynchus keta were obtained from Abe Atsushi Co. Ltd., Miyagi, Japan. Batch A of cartilage and skin were boiled for 1 hour with distilled water (cartilage and skin : distilled water = 1 kg : 0.4 kg) using a pressure cooker. The extracts were filtered by cheesecloth. The filtrate was centrifuged at 20°C for 1 hour, and the suparnatants were pooled. Batch B of cartilage was boiled for 1 hour (cartilage : distilled water = 0.5 kg : 0.3 kg) with distilled water using a pressure cooker, and then the extracts were filtered by cheesecloth. The extracts were centrifuged at the same condition, and then the supernatants were collected. Each extract (0.1, 1.0, 10, and 100 % solutions) was used in this study.

Chemicals

Linoleic acid, α-tocopherol, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), nitroblue tetrazolium salt (NBT), xanthine, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2-deoxy-d-ribose, ACE (from bovine lung; 1U), substrate peptide (hippuryl-l-histidyl-l-leucine), and ethyl acetate for spectrochemical analysis grade were purchased from Wako Chemicals Co. Ltd. (Osaka, Japan). Xanthine oxidase from butter milk (XOD; 0.33 U/mg powder) was obtained from Oriental yeast Co., Ltd. (Tokyo, Japan). Other chemicals used were of regent grade.

Measurements of Protein Content and Collagen Content

The protein content was determined as the method of Lowry et al.[1] using bovine serum albumin as standard. The collagen content was measured by method of Bergman and Loxley.[2]

Inhibition of Linoleic Acid Oxidation

The antioxidant activity was evaluated in a linoleic acid oxidation system. A 0.0833 ml of sample solution and 0.2083 ml of 0.2 M sodium phosphate buffer (pH 7.0) were mixed with 0.2083 ml of 2.5% (w/v) linoleic acid in ethanol. The preoxidation was initiated by the addition of 20.800 μl of 0.1 M AAPH and carried out at 37°C for 200 minutes in the dark. The degree of oxidation was measured according to the thiocyanate method[3] for measuring peroxides by reading the absorbance at 500 nm after coloring with FeCl₂ and ammonium thiocyanate. A control was performed with linoleic acid but without sample solution. Ascorbic acid (1 and 5 mM) and α-tocopherol (1 mM) were used as positive control.

Superoxide Anion Radical Scavenging Ability

The effect of superoxide anion radical was evaluated by the method of Nagai et al.[4] This system contained 0.48 ml of 0.05 M sodium carbonate buffer (pH 10.5), 0.02 ml of 3 mM xanthine, 0.02 ml of 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA), 0.02 ml of 0.15% bovine serum albumin, 0.02 ml of 0.75 mM NBT, and 0.02 ml of sample solution. After preincubation at 25°C for 10 minutes, the reaction was started by adding 6 mU XOD and carried out at 25°C for 20 minutes. After 20 minutes, the reaction was stopped by adding 0.02 ml of 6 mM CuCl. The absorbance of the reaction mixture was measured at 560 nm, and the inhibition rate was calculated by measuring the amount of the formazan that was reduced from NBT by superoxide. Ascorbic acid (1 and 5 mM) and α-tocopherol (1 mM) were used as positive control.

Hydroxyl Radical Scavenging Ability

The effect of hydroxyl radical was assayed by using the method of deoxyribose. The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7.0), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO₄-EDTA, 0.15 ml of 10 mM H₂O₂, 0.525 ml of H₂O, and 0.075 ml of sample solution in Eppendorf tube. The reaction was started by the addition of H₂O₂. After incubation at 37°C for 4 hours, the reaction was stopped by adding 0.75 ml of 2.8% trichloroacetic acid and 0.75 ml of 1.0% of TBA in 50 mM NaOH; the solution was boiled for 10 minutes, and then cooled in water. The absorbance of the solution was measured at 520 nm. Hydroxyl radical scavenging ability was evaluated as the inhibition rate of 2-deoxyribose oxidation by hydroxyl radical.[4] Ascorbic acid (1 and 5 mM) and α-tocopherol (1 mM) were used as positive control.

DPPH Radical Scavenging Ability

The effect of DPPH radical was evaluated by the method of Okada and Okada[5] with a slight modification. The assay mixture contained 0.3 ml of 1.0 mM DPPH radical solution, 2.4 ml of 99% ethanol, and 0.3 ml of sample solution. The solution was rapidly mixed, and this scavenging capacity was measured spectrophotometrically at 517 nm after incubation for 30 minutes. Ascorbic acid (0.1 and 1.0 mM) and α-tocopherol (1 mM) were used as positive control.

Angiotensin I-converting Enzyme Inhibitory Activity

The ACE inhibitory activity assay was performed using a modified version of the method of Cushman and Cheung.[6] Twenty five microliters of sample solution and 75 μl of 0.1 M sodium borate (pH 8.3) containing 5.83 mM hippuryl-l-histidyl-l-leucine and 1.0 M NaCl were preincubated at 37°C for 5 minutes, and then incubated with 25 μl of 0.1 M sodium borate buffer (pH 8.3) containing 1 mU ACE and 1.0 M NaCl at 37°C for 60 minutes. The reaction was stopped by the addition of 125 μl of 1.0 M HCl. The resulting hippuric acid was extracted with 750 μl of ethyl acetate by mixing for 15 seconds. After centrifugation at 6000 rpm for 3 minutes, 500 μl of the upper layer was transported into the tube and evaporated at 40°C for 2 hours. The hippuric acid was dissolved in 500 μl of distilled water, and the absorbance was measured at 228 nm using a PerkinElmer model Lambda 11 (PerkinElmer, Tokyo, Japan) UV/VIS spectrometer. The IC₅₀ value was defined as the concentration of inhibitor required to inhibit 50% of the ACE inhibitory activity.

Statistical Analysis

Results were statistically analyzed by analysis of variance (ANOVA) followed by Fisher's PLSD test at p < 0.05.

RESULTS

Protein and Collagen Contents

The extracts of cartilage and skin from chum salmon were prepared using a pressure cooker. As a result, the protein content of extract of cartilage and skin (24.1 mg/ml of extract) was higher than that of cartilage (16.4 mg/ml of extract) (Table 1). Moreover, collagen contents of these extracts were as follows: 2.06 mg/ml (cartilage and skin) and 0.90 mg/ml (cartilage). This suggested that extract of cartilage and skin was higher than that of cartilage both in protein content and collagen content because of the extraction of a large amount of type I collagen from skin.

Table 1 The contents of collagen and protein in extracts from chum salmon cartilage and skin.

Sample	Collagen (mg/ml)	Protein (mg/ml)
Extract from cartilage and skin	2.06 ± 0.12	24.1 ± 0.98
Extract from cartilage	0.90 ± 0.09	16.4 ± 0.87
Significant difference (p < 0.05).

Inhibition of Linoleic Acid Oxidation

To evaluate the inhibition effects at the initiation stage of lipid peroxidation, the antioxidant activity was investigated in vitro. The extract of cartilage and skin (0.1 and 1.0% solutions) showed low activities, but the extract (10% solution) was showed the same activity as 1 mM ascorbic acid (Table 2). Moreover, the activity on an undiluted extract was similar to that (10% solution). On the other hand, the activities of the extract from cartilage (0.1 and 1.0% solutions) against crude extract were low. The extract of cartilage (10% solution) exhibited the same activity as 1 mM ascorbic acid. Among these sample species, an undiluted extract of cartilage was higher activity than 1 mM ascorbic acid and lower than 5 mM ascorbic acid and 1 mM α-tocopherol.

Table 2 Antioxidant abilities of extracts from chum salmon cartilage and skin.

Absorbance at 500 nm
	Sample
Time (min)	A	B	C	D	E	F	G	H	I	J	K	L
0	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
50	0.099 ± 0.005	0.064 ± 0.003	0.072 ± 0.003	0.111 ± 0.012	0.108 ± 0.007	0.076 ± 0.005	0.068 ± 0.002	0.035 ± 0.008	0.022 ± 0.001	0.016 ± 0.001	0.006	0.379 ± 0.008
100	0.333 ± 0.021	0.262 ± 0.015	0.141 ± 0.010	0.203 ± 0.013	0.329 ± 0.024	0.278 ± 0.011	0.170 ± 0.010	0.081 ± 0.009	0.135 ± 0.002	0.032 ± 0.002	0.025 ± 0.002	0.715 ± 0.025
200	1.462 ± 0.040	1.178 ± 0.031	0.372 ± 0.021	0.343 ± 0.017	1.258 ± 0.038	1.231 ± 0.029	0.590 ± 0.020	0.175 ± 0.009	0.469 ± 0.029	0.090 ± 0.008	0.028 ± 0.002	1.406 ± 0.041
(A) × 1/1000 cartilage and skin extract; (B) × 1/100 cartilage and skin extract; (C) × 1/10 cartilage and skin extract; (D) × 1 cartilage and skin extract; (E) × 1/1000 cartilage extract; (F) × 1/100 cartilage extract; (G) × 1/10 cartilage extract; (H) × 1 cartilage extract; (I) 1 mM ascorbic acid; (J) 5 mM ascorbic acid; (K) 1 mM α-tocopherol; (L) control. Significant differences between the results were determined by ANOVA methodology followed by Fisher's PLSD test. Differences were considered significant for p < 0.05.

Superoxide Anion Radical Scavenging Activities

Superoxide anion radical scavenging activities of extracts from chum salmon were measured and the results were indicated as the superoxide productivity. The extracts (0.1–10% solutions) did not showed the scavenging activity (Table 3). Even the undiluted extract of cartilage and skin showed the scavenging activity about only 13% and the activity was similar to that of 1 mM ascorbic acid. On the other hand, the undiluted extract of cartilage exhibited extremely high scavenging activity about 88%, and the activity was similar to that of 5 mM ascorbic acid and was higher than that of 1 mM α-tocopherol.

Table 3 Superoxide anion radical, hydroxyl radical, and DPPH radical scavenging abilities of extracts from chum salmon cartilage and skin.

	Scavenging activity (%)
Sample	Superoxide anion radical	Hydroxyl radical	DPPH radical
A	0.0	10.1 ± 0.08	0.0
B	0.0	15.4 ± 0.12	0.0
C	0.0	15.4 ± 0.11	2.5 ± 0.02
D	13.2 ± 0.10	67.7 ± 5.02	2.5 ± 0.03
E	0.0	20.0 ± 0.24	0.0
F	0.0	24.1 ± 0.45	0.3
G	0.0	32.4 ± 2.78	5.3 ± 0.09
H	87.9 ± 4.98	89.6 ± 6.04	26.8 ± 1.44
I	13.3 ± 0.14	13.2 ± 0.21	3.1 ± 0.04∗
J	90.0 ± 5.43	16.1 ± 0.64	34.1 ± 2.49∗∗
K	52.5 ± 4.16	67.3 ± 4.33	87.5 ± 6.77
See sample nomenclature in Table 2. ∗0.1 mM ascorbic acid; ∗∗1.0 mM ascorbic acid. Significant differences between the results were determined by ANOVA methodology followed by Fisher's PLSD test. Differences were considered significant for p < 0.05.

Hydroxyl Radical Scavenging Activities

Hydroxyl radical scavenging activities of extracts from chum salmon were measured using Fenton reaction system, and the results were indicated as the inhibition rate. Each extract showed hydroxyl radical scavenging activity and the activity increased with increasing the concentration of sample (Table 3). The extract of cartilage and skin (0.1–10% solutions) showed the same activities as those of ascorbic acid (1 and 5 mM). The activity on an undiluted extract from cartilage and skin was similar to that of 1 mM α-tocopherol. On the contrary, the activities of cartilage (0.1 and 1.0% solutions) were low and were similar to those of ascorbic acid (1 and 5 mM). The extract (10% solution) showed moderate activity between 5 mM ascorbic acid and 1 mM α-tocopherol. The undiluted extract of cartilage showed highest scavenging activity among these extracts and scavenged about 90% against hydroxyl radical.

DPPH Radical Scavenging Abilities

DPPH radical scavenging activity of extracts from chum salmon was measured, and the results were indicated as relative activity. Most of these extracts of cartilage and skin and cartilage hardly exhibited DPPH radical scavenging activities. The activities of extracts (10% solution) were similar to that of 0.1 mM ascorbic acid (Table 3). Among these extracts an undiluted extract of cartilage showed the activity about 40% that was similar to 1 mM ascorbic acid.

Angiotensin I-converting Enzyme Inhibitory Activities

Angiotensin I-converting enzyme inhibitory activities of extracts from chum salmon were measured and the results were indicated in Table 4. The activities of extract from chum salmon were as follows: 62.6 mg protein (cartilage and skin) and 8.87 mg protein (cartilage), respectively.

Table 4 ACE inhibitory abilities of extracts from chum salmom cartilage and skin.

Sample species	(mg protein) IC50
Extract of cartilage and skin	62.6 ± 1.03
Extract of cartilage	8.87 ± 0.52
Significant difference (p < 0.05).

DISCUSSION

The functional food market is growing at a rate of 15–20% per year, and the industry is clamed to be worth $33 billion.[7] Functional foods, also called as nutraceuticals, designer foods, medicinal foods, therapeutic foods, superfoods, foodiceuticals, and medifoods, are defined as “foods that contain some health-promoting compounds beyond traditional nutrients.”[8] Foods can be modified by addition of phytochemicals, bioactive peptides, polyunsaturated fatty acids, and probiotics, and/or prebiotics to become functional.[8] As a part of a study looking at the important physiological functions of foods, the antioxidant properties of foods were reported: honey species,[9–12] beverages produced by sea algae,[13] plant,[4,14] and lee produced during sake making using fugu muscle and fin[15] in the previous papers. All of sample species tested showed high antioxidative activity on a linoleic acid oxidation and scavenging activities against reactive oxygen species, such as superoxide anion and hydroxyl radicals. On the contrary, there are many reports of angiotensin I-converting enzyme inhibitory activities in peptides derived from casein,[16–18] fish muscle,[19,20] and other food proteins.[21–23] Although the inhibitory activities of these food-derived peptides are weaker than those of drugs such as captopril and enarapril, some of them have been shown to be effective in lowering blood pressure of spontaneously hypertensive rats (SHR) after oral administration.[24] In the present study, it was found that the antioxidant activity and scavenging abilities against superoxide anion, hydroxyl, and DPPH radicals of extract from cartilage were fairly higher than those of cartilage and skin, although the protein content and collagen content of extract from cartilage were lower than those from cartilage and skin. In addition, the extract from cartilage exhibited high angiotensin I-converting enzyme inhibitory activity than that from cartilage and skin. Thus, it was suggested that the component, except for collagen in extract of cartilage from chum salmon, for example chondroitin sulfate, may contributed to these functional properties. In fact, Takeda et al.[25] reported that they prepared chondroitin sulfate and its oligosaccharide mixture from the nasal cartilage of the salmon Onchoryncus keta, and investigated the effects of these samples on intestinal absorption of glucose. As a result, the samples inhibited glucose uptake in brush border membrane vesicles prepared from rat jejurum. Moreover, they investigated the effects of chondroitin sulfate from the nose cartilage of salmon on the digestion and absorption of dietary triacylglycerol, and the antiobesity effect of salmon chondroitin sulfate in mice with obesity induced by a high-fat diet.[26] The result indicated that salmon chondroitin sulfate inhibited the hydrolysis of dietary triacylglycerol by pancreatic lipase and also the absorption of fatty acids, in brush border membrane vesicles prepared from rat jejunum. At the end of feeding, body weights, parametrial adipose tissue weight, liver weight and lipid concentration, and serum lipid concentration were measured. These values were significantly lower in the groups fed on salmon chondroitin sulfate. Thus, salmon chondroitin sulfate suppresses obesity, fatty liver and hyperlipidemia by inhibiting the hydrolysis of dietary triacylglycerol, and the intestinal absorption of fatty acids. At present, the problem of Bovine Spongiform Encephalopathy (BSE) infection in land animals such as bovines is not resolved, the production of industrial collagen is limited or is discontinued. From this point, aquatic materials, particularly the cartilage in chum salmon, as an alternative source of collagen are attract much attention in cosmetic and medical fields. Recently, Ohba et al.[27] reported that the physiological functions, angiotensin I-converting enzyme inhibitory activity, of enzymatic hydrolysate of collagen in scales and bone from yellowtail. These hydrolysates possessed strong angiotensin I-converting enzyme inhibitory activities (IC₅₀ = 2.0–2.9 mg/ml). It is known that many peptides contained proline showed angiotensin I-converting enzyme inhibitory activity. From the reason, it suggests that the hydrolysate of collagen exhibited angiotensin I-converting enzyme inhibitory activity in order to contain large amounts of proline and hydroxyproline as a constituent amino acid in collagen. Moreover, they also reported that the hydrolysates of collagen from scales and bone showed DPPH radical scavenging activities.[27] Further characterization of the inhibitor from the extract of cartilage and skin from chum salmon is the subject of ongoing investigation.

The extracts, particulally from cartilage, have angiotensin I-converting enzyme inhibitory activity, which functions to depress hypertension, although the activity was weak because of crude extract in comparison with the other food-derived proteins.[10–13,15–18] It was also found that these extracts possessed antioxidant activity, which acts to prevent of life-style related diseases such as cancer, cardiovascular diseases, and diabetes. As these extracts are rich in collagen, it is expected various functions in the constituents that is contained in chum salmon.

CONCLUSION

The extract from cartilage and skin of chum salmon Oncorhynchus keta has antioxidative activities against superoxide anion and hydroxyl radicals and angiotensin I-converting enzyme inhibitory. The extract could be used for developing a novel type of functional seasoning.