Hyaluronidase Inhibitory Activity of Polysaccharides Separated from a Fermented Beverage of Plant Extracts

Abstract

Super Ohtaka^®, a fermented beverage of plant extracts, is prepared from approximately 50 kinds of vegetables and fruits is a naturally fermented mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). In this study, we separated water-soluble polysaccharides from Super Ohtaka^® using dialysis and chromatography, yielding four polysaccharide fractions. The polysaccharide fraction designated as OEP3 exhibited hyaluronidase inhibitory activity. The half-maximal inhibitory concentration was 860 µg/mL. This polysaccharide not only stimulated macrophages but also inhibited hyaluronidase activity and showed weak 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.

Abbreviations

Ara, arabinose; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EPS, extracellular polysaccharide; Gal, galactose; Glc, glucose; IC₅₀, half-maximal inhibitory concentration; Man, mannose; p-DMAB, p-dimethyl-aminobenzaldehyde.

Super Ohtaka^® (a fermented beverage of plant extracts: Ohtakakohso Co., Ltd, Otaru, Japan) is produced by fermentation of an extract of 50 fruits and vegetables [1]. The extract is obtained using sucrose osmotic pressure in a cedar barrel for 7 days and fermented with lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.) for 180 days. After fermentation, this beverage turns brown (Fig. 1). This beverage has been reported to have scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and significantly reduces ethanol-induced damage to the gastric mucosa in rats [1]. We have previously examined a preparation of novel oligosaccharides obtained from Super Ohtaka^® containing several types of fructopyranose residues [2, 3, 4, 5]. To further investigate the functionality of this beverage, water-soluble polysaccharides from Super Ohtaka^® were separated using dialysis, ion-exchange chromatography, and gel filtration chromatography to obtain four polysaccharide fractions [6]. We have previously reported the ratios of constituent monosaccharides, molecular weights, and phosphorus contents of these polysaccharides. Among these polysaccharides, OEP2 (average molecular mass > 670 kDa; Glc:Gal:Man:Ara = 1.0:2.6:6.4:0.5) and OEP3 (average molecular mass = 320 kDa; Glc:Gal:Man = 1.0:0.2:0.4) stimulated macrophages and induced the production of various cytokines. Induction of these cytokines depended on the phosphorus content of the polysaccharides. The phosphorus contents of OEP2 and OEP3, which showed cytokine induction, were approximately 0.7 and 5.1 % [w/w], respectively [6].

Fig. 1. Fermented beverage of plant extracts (Super Ohtaka^®) and its dialyzed freeze-dried product.

Hyaluronidase (EC 3.2.1.35) is an enzyme that is involved in the hydrolysis of (1→4)-linkages between N-acetyl-β-D-glucosamine and D-glucuronate residues in hyaluronate, with inflammation caused by histamine released from mast cells [7, 8, 9]; accordingly, inhibitors of hyaluronidase have been established to be effective in suppressing allergies and inflammation [7, 8, 9]. Among these inhibitors is pectin isolated from immature fruit of Citrus reticulata BLANCO [10] and a coffee “Silverskin” hot-water extract [11]. The hyaluronidase inhibitory activity of sulfated polysaccharides, such as fucoidan, is well established, and the hyaluronidase inhibitory activities of different sulfated polysaccharides have been compared previously [12]. A phosphorylated polysaccharide [1.0 μmol phosphorylate residue per 1 mg of extracellular polysaccharide (EPS)] produced by the lactic acid bacteria Lactobacillus plantarum SN35N has been similarly shown to inhibit hyaluronidase activity [13], and numerous studies have previously investigated the hyaluronidase inhibitory activities of polysaccharides [14, 15].

It has also been reported that some plant polysaccharides [14, 15] and EPS produced by L. plantarum [16] and Bifidobacterium animalis RH [17] have the ability to scavenge DPPH radicals.

In the present study, we report that one of our previously separated polysaccharides exhibits hyaluronidase inhibitory and antioxidant activities.

Hyaluronidase inhibitory activity was determined as described by Kakegawa et al. [7]. The polysaccharide solution (20 µL) and 10 µL of an enzyme solution [Type VI-S from bovine testes, Sigma-Aldrich, Inc. (St. Louis, MO, USA) dissolved in 100 mM sodium acetate buffer (pH 4.0) to 1 mg/mL] were mixed, and the mixture was preincubated at 37 °C for 20 min. An activating reagent consisting of 0.5 mg/mL of compound 48/80 (Sigma-Aldrich, Inc.) and 3.75 mg/mL CaCl₂ (20 µL) was added, and the mixture was further incubated at 37 °C for 20 min. Next, a 0.8 mg/mL substrate solution [hyaluronic acid sodium salt obtained from the human umbilical cord (Sigma-Aldrich, Inc.)] prepared in 50 µL of the same buffer was added to this mixture, and the mixture was further incubated at 37 °C for 40 min. After incubation, 20 µL of 0.4 M sodium hydroxide solution and 20 µL of 0.4 M potassium hydroxide solution were added to terminate the reaction. After heating in a boiling water bath for 3 min and cooling on ice for 1 min, 80 µL of the reaction mixture was placed in a new test tube. This solution along with 400 µL of p-dimethyl-aminobenzaldehyde reagent (0.5 g of p-DMAB was dissolved in 600 µL of 10 M HCl and 4.4 mL of acetic acid to make a stock solution; this stock solution was diluted 10 times with acetic acid before use) was incubated at 37 °C for 20 min. Absorbance was measured at 585 nm. The percentage inhibition was calculated as follows:

Inhibition (%) = 100 × {1 − (A_s − A_sb) / (A_e − A_eb)}, where A_s and A_e are absorbance values in the presence and absence of the sample, respectively; A_sb and A_eb are absorbance values of the blank in the presence and absence of the sample, respectively. λ-Carrageenan (Nacalai Tesque, Inc., Kyoto, Japan), a commonly known hyaluronidase inhibitor, was used as a positive control. A part of the polysaccharides separated in a previous study [6] was used in this experiment. The polysaccharides were separated from Super Ohtaka^® using the following method. Super Ohtaka^® (1 L) was diafiltered using a Viva Flow 200 (membrane: 10,000 MWCO, Sartorius AG, Göttingen, Germany) and lyophilized. The lyophilizate was suspended in water and further dialyzed against distilled water using a dialysis tube (12,000-14,000 MWCO) for 5 days. The dialysate thus obtained was lyophilized to yield 0.3 g of a brown powder (Fig. 1). The freeze-dried powder was resuspended in distilled water and centrifuged [2,000 × g, 10 min, room temperature (approx. 25 °C)]. The supernatant thus obtained was filtered through a 0.45 μm filter and lyophilized to obtain 0.2 g of a water-soluble fraction (crude separated). This crude separated exhibited hyaluronidase inhibitory activity (Table 1). However, no inhibitory activity was observed when the crude separated was added at a concentration of 0.1 mg/mL, and at a concentration of 0.2 mg/mL, the inhibitory activity was only one-fourth that of λ-carrageenan added at a concentration of 0.1 mg/mL. The crude separated was further separated using DEAE-Sepharose Fast Flow (Sigma-Aldrich, Inc.) column chromatography. The crude separated (50 mg) was dissolved in 0.02 M Tris-HCl buffer (pH 8.6, 2 mL), loaded onto the column (1 × 15 cm), and fraction were eluted using a stepwise gradient with 50 mL of 0.02 M Tris-HCl buffer (pH 8.6) containing 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 1 M NaCl at a flow rate of 18 mL/h. The carbohydrate content of each fraction was monitored at 490 nm using the phenol-sulfuric acid method [18]. Fifty mg of the crude separated was subjected to ion-exchange chromatography four times; totally 0.2 g of the crude separated was fractionated. As previously reported, polysaccharide peaks were observed in the non-adsorbed fraction and in 0.1 M and 0.2 M NaCl elution fractions [6]. The non-adsorbed fraction was designated as OEP1. The 0.1 M and 0.2 M NaCl eluted fractions were further purified by gel filtration chromatography using a Toyopearl HW-65s column (Tosoh Corporation, Tokyo, Japan). Gel filtration chromatography was performed according to previously reported conditions [6]. OEP2 was obtained by gel filtration chromatography of 0.1 M NaCl elution fraction. Figure 2 shows the results of gel filtration chromatography of the 0.2 M NaCl elution fraction. It was divided into two fractions, a fraction that did not exhibit absorption at 280 nm (OEP3) and a fraction that showed strong absorption (OEP3-1). Table 1 shows results of the estimated hyaluronidase inhibitory activity of the four fractions. Inhibitory activity was observed with OEP3 and OEP3-1. OEP3 and OEP3-1 inhibited hyaluronidase activity in a dose-dependent manner (data not shown). Half-maximal inhibitory concentration (IC₅₀) for OEP3 and OEP3-1 were calculated to be 860 and 1,240 µg/mL, respectively (Table 1). In subsequent experiments, OEP3, which possessed relatively strong activity and almost no absorption at 280 nm, was used.

Table 1. Comparison of hyaluronidase inhibitory activity between crude separated and four separated fractions from Super Ohtaka^®, and λ-carrageenan.

Sample	Protein (%) (Lowry method)a)	Carbohydrate (%) (Phenol-sulfuric acid)b)	Yieldc) (mg)	Final concentration (mg/mL)	Inhibition ratiod) (%)	IC50 (µg/mL)
Crude separated			200	0.1	0
				0.2	21.1 ± 6.0
				0.4	37.8 ± 7.8
OEP1	2.5	71.2 ± 3.4	6.4e)	1.0	0	-
OEP2	8.4e)	86.4 ± 3.2e)	5.5e)	1.0	0	-
OEP3	7.4e)	68.5 ± 4.1e)	11.5e)	1.0	56.6 ± 6.8	860
OEP3-1	60.4	36.2 ± 1.9	2.3	1.0	43.6 ± 5.4	1,240
λ-Carrageenan			-	0.1	78.7 ± 9.4

^a) BSA standard.

^b) Glucose standard.

^c) Yield was the value obtained from 1 L of fermented liquid of plant extract.

^d) Mean ± SD, (n = 3).

^e) Okada et al. [6].

Fig. 2. Toyopearl HW-65s column chromatogram.

The fermented beverage was applied to a DEAE-Sepharose Fast Flow column, and the fraction eluted with 0.2 M NaCl was further fractionated via gel filtration chromatography using a Toyopearl HW-65s.

To confirm that the hyaluronidase inhibitory activity was not caused by pectin etc., the activity was measured after digestion with pectinase and sulfuric acid. λ-Carrageenan and pectin (citrus pectin; Kishida Chemical Co., Osaka, Japan) were used as a control. Treatment with pectinase (MP Biomedicals LLC, Irvine, CA, USA) was performed using the following method. Three µL of pectinase solution (0.02 units) dissolved in 100 mM sodium acetate buffer (pH 4.0) was added to 60 µL of each polysaccharide solution at a concentration of 5 mg/mL, and the mixture was incubated at 25 °C for 24 h. The mixture was then boiled for 5 min to terminate the reaction. Decomposition with sulfuric acid was performed using the following procedure. Distilled water (400 µL) and 0.1 M sulfuric acid (500 µL) were added to 100 µL of each sample solution (2.5 mg/mL) and the mixture was heated at 100 °C for 3 h. After cooling, a small amount of barium carbonate was added to neutralize the mixture, and the sample was filtered through a 0.45 µm filter to obtain a sulfuric acid decomposition sample. An undecomposed sample was prepared by adding distilled water instead of sulfuric acid. As shown in Table 2, the hyaluronidase inhibitory activity of OEP3 was not reduced by pectinase treatment. This suggests that pectin may not have remained behind as an impurity in the sample. Because the inhibitory activity almost disappeared when the sample was decomposed with sulfuric acid, this activity may possibly have been due to the separated polysaccharides.

Table 2. Hyaluronidase inhibitory activity of each sample after digestion with sulfuric acid and treatment with pectinase.

		Final concentration (mg/mL)	Inhibition ratio (%)
			Sulfuric acid	Pectinase
OEP3	treated	1.0	0	54.73
	untreated		54.21	54.21
λ-Carrageenan	treated	0.1	0	50.3
	untreated		54.6	50.3
Pectin	treated	1.0		17.71
	untreated			62.23

Having assessed the hyaluronidase inhibitory activity of OEP3, we subsequently investigated the DPPH radical scavenging activity of the separated OEP3. DPPH radical-scavenging assays were performed using the Blois method [19]. Samples (50 µL) were added to 150 µL of 62.5 µM DPPH, and after incubation at room temperature for 30 min, absorbance of the reaction mixture was measured at 492 nm using a Multiskan JX microplate reader (Thermo Electron LLC, Waltham, MA, USA). At a final concentration of 1.0 mg/mL, OEP3 showed a weak DPPH radical-scavenging activity of approximately 14.6 %, whereas OEP2 exhibited almost no DPPH radical-scavenging activity.

In the current study, we identified a polysaccharide with hyaluronidase inhibitory activity in fermented beverages (OEP3: IC₅₀ = 860 µg/mL; Table 1). Hyaluronidase inhibitory activity of polysaccharides with sulfate groups extracted from Caulerpa lentillifera (sea grape) and their IC₅₀ value (IC₅₀ = 163.3 µg/mL) has been reported [20]. IC₅₀ value of rhamnan sulfate present in Monostroma nitidum (Hitoegusa) is 145 µg/mL [21]. Moreover, IC₅₀ values of pectin isolated from the immature fruit of C. reticulata BLANCO (IC₅₀ = 91 µg/mL) and commercial orange pectin (IC₅₀ = 47 µg/mL) have been reported [10]. In our study, the separated polysaccharides exhibited weak inhibitory activities as compared to polysaccharides containing sulfate groups and orange pectin.

IC₅₀ values of hyaluronidase inhibitory activity of neutral polysaccharides and acidic polysaccharides produced by lactic acid bacteria of the L. paracasei IJH-SONE68 (IC₅₀ = neutral EPS: 550 µg/mL; acidic EPS: 1,240 µg/mL) [22] and Pediococcus pentoaceus LY45 (IC₅₀ = neutral EPS: 380 µg/mL; acidic EPS: 1,300 µg/mL) [23] have been previously reported. Phosphorylated polysaccharide (1.0 µmol phosphorylate residue per 1 mg of EPS) produced by lactic acid bacteria (L. plantarum SN35N) also inhibits hyaluronidase, and its IC₅₀ value is 240 µg/mL [13]. Although the polysaccharide in our study showed weak inhibitory activity compared to algal sulfated polysaccharides and pectin, it showed IC₅₀ values comparable to those of lactic acid bacteria EPS. Although the phosphate content of OEP3 [6] was 1.7 times higher than that of the EPS produced by L. plantarum SN35N, the IC₅₀ value for hyaluronidase inhibitory activity was approximately 3.6 times higher. OEP2 had a phosphorus content of approximately 0.7 % but showed no inhibitory activity at all. Also, no relationship was observed between the inhibitory activity and phosphorus content, suggesting that the phosphorus content of OEP3 is not the only factor involved in its inhibitory activity. It has been reported that the hyaluronidase inhibitory activity of rhamnan sulfate tends to increase with increasing sulfate group content [21]; however, it has also been reported that it is difficult to conclude that acidic residues present in the molecule are essential for hyaluronidase inhibition [22].

An EPS isolated from a culture of L. plantarum C88 and named LPC-1 has been reported to capture 52.23 % of DPPH radicals when added at a concentration of 4 mg/mL, and even at a concentration of 1 mg/mL, the capture rate exceeded 40 % [16]. By contrast, isolation of three types of polysaccharides has been reported from Prunella vulgaris [15], but even the one with the highest DPPH radical-scavenging ability showed an activity only of about 15 % at 1 mg/mL concentration. In our study, the activity of OEP3 was observed to be weaker than that of LPC-1 but was almost the same as that of P. vulgaris.

The results of this study suggest that the polysaccharide separated from the fermented liquid of plant extract, OEP3, not only stimulated macrophages but also exhibited hyaluronidase inhibitory activity. However, the mechanism of production and structure of this polysaccharide remain unknown. Therefore, further investigations are necessary to explore the relationship between the structure and function of this polysaccharide.

CONFLICTS OF INTEREST

Hideki Okada, Akira Yamamori, and Naoki Kawazoe are employees of Ohtakakohso Co., Ltd.

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