CHEN Yu, LIN Hong, LI Zhenxing,, and MOU Quangui

1)Food Safety Laboratory,Ocean University of China,Qingdao266003,P. R.China

2)National R&D Branch Center for Kelp Processing of China,Shandong Haizhibao Ocean Science and Technology,Co. Ltd.,Yantai264300,P. R.China

The Anti-allergic Activity of Polyphenol Extracted from Five Marine Algae

CHEN Yu1), LIN Hong1), LI Zhenxing1),*, and MOU Quangui2)

1)Food Safety Laboratory,Ocean University of China,Qingdao266003,P. R.China

2)National R&D Branch Center for Kelp Processing of China,Shandong Haizhibao Ocean Science and Technology,Co. Ltd.,Yantai264300,P. R.China

Natural polyphenol has been widely believed to be effective in allergy remission. Currently, most of the natural polyphenol products come from terrestrial sources such as tea, grape seeds among others, and few polyphenols have been developed from algae for their anti-allergic activity. The aim of the study was to screen some commercial seaweed for natural extracts with anti-allergic activity. Five algae includingLaminaria japonica,Porphyrasp.,Spirulina platensis,Chlorella pyrenoidosaandScytosiphonsp. were extracted with ethanol, and the extracts were evaluated for total polyphenol contents and anti-allergic activity with the hyaluronidase inhibition assay. Results showed that the total polyphenol contents in the ethanol extracts ranged from 1.67% to 8.47%, while the highest was found in the extract fromScytosiphonsp. Hyaluronidase inhibition assay showed that the extracts fromScytosiphonsp. had the lowest IC50, 0.67 mg mL-1, whileChlorella pyrenoidosaextract had the highest IC50, 15.07 mg mL-1. The anti-allergic activity ofScytosiphonsp. extract was even higher than the typical anti-allergic drug Disodium Cromoglycate (DSCG) (IC50= 1.13 mg mL-1), and was similar with natural polyphenol fromEpigallocatechin gallate(EGCG) (IC50= 0.56 mg mL-1). These results indicated that the ethanol extract ofScytosiphonsp. contains a high concentration of polyphenol with high anti-allergic activity. PotentiallyScytosiphonsp. can be developed to a natural anti-allergic compound for allergy remission.

alga; polyphenol; hyaluronidase; anti-allergic activity

1 Introduction

With advancements in food processing technology and the internationalization of the distribution and consumption of food products, food allergy is becoming an increasing concern and therefore gaining more attention of the scientific community (Sicherer, 2011). Allergic or hypersensitivity reactions can be divided into four general categories based on the mechanism of immunological response. Food allergy is considered as type I allergy, with a clinical syndrome resulted from the release of allergenic media by mast cells and basophils (Breiteneder and Ebner, 2000). Allergic reactions can cause erythrism, asthma, anaphylactic shock and a number of other ailments (Helm and Burks, 2000).

Hyaluronidase (HAase, EC 3.2.1.35), a hyaluronic acidsplitting enzyme, is a principal compound involved in allergic reaction (Shibataet al., 2002). It is reported that anti-allergic agent such as DSCG (disodium chromoglycate) has a strong inhibitory effect on the activation of hyaluronidase. Several natural extracts from plants such aspolyphenols (Kojimaet al., 2000), polysaccharides (Tianet al., 2012), flavonoids (Shimosakiet al., 2011)etc. are known to possess anti-allergic activity in addition to having low side effects and high safety to human health. Recently, marine source has attracted more and more attentions for the development of active compounds. Currently, some algal extracts such as ethanol extract or hydro extract are reported to possess immune-related (Katayamaet al., 2011), anti-inflammatory (Boonchumet al., 2011), anti-tumor (Jiaoet al., 2009), and bacteriostatic activities (Kajiwaraet al., 2006). However, few studies have been reported on the anti-allergic activity from marine sources (Sameeet al., 2009).

A large number of algae species are abundantly distributed along the coast of China. Additionally, China produced 9900 thousand tons of algae and other aquatic plants through aquaculture in 2008 alone (FAO, 2008). Definitive information on the anti-allergic activities of algae from China is lacking, and little is known about the variations among different species of algae. In the present study, we selected five algae belonging to four different groups with the objectives of (a) measuring their antiallergic properties in terms of inhibition of hyaluronidase, (b) selecting algae with high anti-allergic activity, and (c) providing a sound basis for the development of anti-al-lergic compounds and functional foods from algae.

2 Materials and Methods

2.1 Algae

Laminaria japonica(Phaeophyta) (LJ) andPorphyrasp. (Rhodophyta) (PP) were collected from the coastal area of Qingdao, China, in March 2011.Spirulina platensis(Cyanophyta) (SP), andChlorella pyrenoidosa(Chlorophyta) (CP) were purchased from Lvqi Biological Engineering Corporation of Shandong, China in March 2011.Scytosiphonsp. (Phaeophyta) (SS) was collected from the coastal area of Tuoji Island, China, in April 2011. Collected algae were washed three times with water to remove epiphytes, sandand salt. Washing was followed by air-drying in a shady area to avoid direct sunlight. Dried algae were crushed and finely ground into a powder and passed through a 40-mesh sieve and stored at room temperature in air-tight containers.

2.2 Chemicals

Folin-Ciocalteu’s phenol reagent (2 mol L-1), hyaluronidase from bovine testes (500 U mL-1), disodium cromoglycate (DSCG) (95%, purity), epigallocatechin gallate (EGCG) from green tea and polyphenol from green tea (TP) were purchased from Sigma-Aldrich, USA. Hyaluronic acid sodium salt fromStreptococcus equiwas purchased from Fluka, Germany. All other chemicals were at AR grade. Milli-Q purified water was used to prepare the reagents.

2.3 Extraction of Anti-allergic Compounds

Crude extracts ofLJ,PP,SP,CP, andSSwere prepared using the method standardized in our laboratory (Sameeet al., 2009). Approximately, 10 g of dried seaweed samples were immersed in 100 mL 85% ethanol in a conical flask and stirred for 24 h at 25℃. The extracts were centrifuged and the supernatant was obtained. The residues were subjected to extractions for two times. The supernatant were pooled, filtered and then concentrated to about 50 mL under vacuum by a rotary evaporator at 40℃for each variety separately. Concentrated samples were washed five times with one volume of chloroform to remove fats and pigments by precipitation. The upper layer was mixed with 50 mL ethyl acetate thrice and the ethyl acetate fraction was removed using a rotary evaporator at 40℃. Resulting residues were weighed and dissolved in distilled water with a ratio 1：3.

2.4 Total Polyphenol Content

Total polyphenol content of the ethanol extracts were determined using Folin-Ciocalteu’s method (Singleton, 1999) with some modifications. Briefly, the samples were diluted considering the measurable range of the spectrophotometer. About 0.1 mL of the diluted sample was mixed in a test tube with 0.5 mL of 2 mol L-1Folin-Ciocalteu’s reagent and 0.5 mL of water, followed by addition of 2.0 mL of 20% Na2CO3after 3 min. Samples were incubated in dark at room temperature for 45 min followed by centrifugation at 9000 r min-1for 10 min. Absorbance of the supernatant were measured at 730 nm using spectrophotometer (TU-1810, China). Phloroglucinol was used as the standard. Total polyphenol content was calculated using the standard curve.

2.5 Anti-hyaluronidase Activity Assay

Anti hyaluronidase activity was assayed using modified Morgan-Elson method (Muckenschnabel,et al., 1998). Initially, hyaluronidase was dissolved in a buffer solution (0.2 mol L-1sodium formate, 0.1 mol L-1NaCl, and 0.2 mg mL-1bovine serum albumin (BSA) and adjusted to the pH 3.0-4.0 with formic acid) to get a final concentration of 500 U mL-1. A solution of 5 mg mL-1hyaluronic acid was prepared in triple distilled water and stored at 4℃. About 17.3 g H3BO3and 7.8 g KOH was dissolved in 100 mL water to get borate solution to which 0.8 g mL-1K2CO3solution was added at ratio of 10：1 before use. The streak reagent consisted of 20 gp-dimethylaminobenzaldehyde, 25 mL of concentrated hydrochloric acid and 75 mL of glacial acetic acid. The solution was diluted for four times with glacial acetic acid before use.

The anti-allergic activity assay was performed as described below. Ahead of assay, 50 μL of the hyaluronic acid stock solution, 100 μL of buffer and 250 μL of water were mixed and equilibrated at 37℃ for 10 min. Initially, 50 μL of hyaluronidase and 100 μL of sample each were mixed and incubated at 37℃ for 20 min. The enzymatic reaction was terminated by adding 110 μL of borate solution and holding for 4.5 min in a boiling water bath, followed by cooling the test tubes in ice-cold water for 20 min and adding 1.5 mL of streak reagent. The tubes were then incubated at 37℃ for 20 min to maximize the color. Finally, absorbance was measured on a TU-1810 spectrophotometer at 585 nm. Percentage inhibition was calculated by the formula：

whereAis the control absorbance, test sample of A was replaced by the buffer solution;Bis the control blank absorbance, test samples and the hyaluronidase solution were replaced by the buffer solution;Cis the sample absorbance; andDis the absorbance of the blank sample in which the hyaluronidase solution was replaced by the buffer solution. IC50was calculated using the mean of three observations from each of the five concentrations for all the samples including the positive controls. All reactions were performed in triplicate including those for the three positive controls.

2.6 Statistical Analysis

In the same experiments, the treatments were performed at least in triplicate on the same turbot muscle samples. Data were analyzed using SPSS 19.0 softwareValues are represented as mean ± SD. Analysis of variance was done to determine the significance of the changes during different treatments.

3 Results

The ethanol extracts of five commercial seaweed samples were tested and the activity of anti-hyaluronidase was compared. The typical anti-allergic drug disodium cromoglycate (DSCG), epigallocatechin gallate (EGCG) and tea polyphenol (TP) were used as positive control. Following our previous extract procedure, the ethanol extracts obtained with 85% ethanol concentration gave maximum yield of crude polyphenol. The total polyphenol content of the five seaweed samples are listed in Table 1. Total polyphenol content ofScytosiphonsp. was the highest at 8.47% as compared to other four algae, while inChlorella pyrenoidosait was 1.98%, inLaminaria japonicait was 3.00%, inPorphyrasp. it was 6.84%, inSpirulina platensisit was 1.67%. The total polyphenol content was calculated according to the standard curve using phloroglucinol as the standard (R2＞ 0.99) (Fig.1).

Table 1 Total polyphenol contents of the crude extracts

Fig.1 Standard curve of phloroglucinol absorbance at 730 nm.

Meanwhile, the inhibitory effects of the five seaweed ethanol extracts on hyaluronidase were evaluated. As shown in Table 2, IC50values were 15.07 mg mL-1for CP, 5.17 mg mL-1forLJ, 6.86 mg mL-1forPP, 0.67 mg mL-1forSS, 3.10 mg mL-1forSP, respectively. For the positive control, the IC50values for DSGC, EGCG and TP were 1.13, 0.54 and 0.41 mg mL-1, respectively. Among the algal tests, SS has the lowest IC50value which was lower than that of DSGC and closely similar to those of EGCG and TP. These results indicated that ethanol extract of SS was the most effective as comparison with ethanol extracts from other algae and has potential to be used as an inhibitor against hyaluronidase.

Table 2 Anti-hyaluronidase activity of the crude seaweed extracts and the positive controls

Fig.2 Anti-hyaluronidase activity of algae and DSCG, EGCG and TP. DSCG, disodium chromoglycate; EGCG, (-)-Epigallocatechin gallate; TP, tea polyphenol;CP,Chlorella pyrenoidosa;LJ,Laminaria japonica;PP,Porphyra;SS,Scytosiphon;SP,Spirulinaplatensis. All values of inhibition (%) are the means ± SD.

4 Discussion

Five algae belonging to four different groups were collected from the coast of Qingdao, Shandong Peninsula, and analyzed for their anti-allergic or anti-hyaluronidase activity. Based on preliminary trials conducted in our laboratory, 85% ethanol solvent extraction method was used with chloroform precipitation to remove fat and pigments. Phloroglucinol was used as the standard to determine total polyphenol contents. A common anti-allergic drug, DSCG, was used as the positive control. It has been reported that natural polyphenol from land-based materials such as EGCG (Maeda and Tachibana, 2012) and TP (Liet al., 2008) have high anti-allergic activity. Here, we extracted polyphenol with hyaluronidase inhibition activity from seaweeds, a marine source.

The inhibitory effects of different concentrations of the seaweed extracts were determined. The inhibitory effect was deemed stronger if the activity could be achieved with a lower concentration. The total polyphenol contents of SS, SP, LJ, PP and CP extracts were 8.47%, 1.67%, 3.00%, 6.84%, 1.98% (Table 1) and their corresponding IC50values were 0.67, 3.10, 5.17, 6.86, 15.07 mg mL-1(Table 2), respectively. Results showed thatSShas remarkable inhibitory effect on hyaluronidase, which may correlate with its higher total polyphenol content among five algae tested (Fig.2). However, no clear relation wasevident between the total polyphenol contents and the inhibitory effects of the algae tested. Other studies have shown that the inhibition activity varied with not only the total content of polyphenol, but also the polyphenol species (Yamamotoet al., 2004; Sameeet al., 2009). Overall, the total polyphenol yield by ethanol extraction was very low. It is possible that some other active ingredients may also be contributing toward the inhibitory activity against hyaluronidase.

The inhibitory effects were not obvious for other four algae probably due to their low total polyphenol contents. It is also possible that the structures of polyphenol might be different among the seaweed products. Additionally, the inhibitory effects could be influenced or inhibited by the presence of a variety of bioactive ingredients such as algal polysaccharides, phycocyanin among others (Sugiuraet al., 2009). For example, algal polysaccharides are known to enhance organismic immune response (Kanget al., 2008), and phycocyanin can improve the function of immune system (Romayet al., 2003).

In conclusion, we demonstrated that ethanol extract ofScytosiphonsp. is a natural inhibitor of hyaluronidase. The inhibitory effect ofScytosiphonsp. is higher than that of the anti-hyaluronidase drug DSGC. Further research is required to identify and analyze the anti-allergic components fromScytosiphonsp. The results will provide scientific basis for producing a natural inhibitor of food allergy by utilizing the rich natural seaweed resource.

Acknowledgements

This work was financially supported by Shandong Province Young and Middle-Aged Scientists Research Awards Fund (BS2011HZ020), the National Natural Science Foundation of China (No. 31371730) and Program for Changjiang Scholars and Innovative Research Team in University.

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(Edited by Qiu Yantao)

(Received February 20, 2014; revised May 9, 2014; accepted June 6, 2015)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-532-82032389 E-mail： lizhenxing@ouc.edu.cn