A sustainable process for the recovery of volatile constituents from Gracilaria lemaneiformis in agar production and evaluation of their antioxidant activities

Shengliang Yuan; Kefeng Wu; Zhihong Duan; Yanxia Huang; Yingnian Lu; Xiaoli Ma

doi:10.1186/s13065-019-0590-y

Research article
Open Access
Published: 31 May 2019

A sustainable process for the recovery of volatile constituents from Gracilaria lemaneiformis in agar production and evaluation of their antioxidant activities

Shengliang Yuan¹^na1,
Kefeng Wu¹^na1,
Zhihong Duan¹,
Yanxia Huang¹,
Yingnian Lu ORCID: orcid.org/0000-0003-1517-9851¹ &
Xiaoli Ma²

BMC Chemistry volume 13, Article number: 74 (2019) Cite this article

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Abstract

Background

Gracilaria lemaneiformis is a common red alga used as a raw material source for the agar industry. Its extract is rich in natural volatile constituents (VCs) having antioxidant activities. Herein, a sustainable method was used to recover VCs from the alga. The chemical composition of VCs present in the n-hexane fraction was analyzed by gas chromatography–mass spectroscopy (GC–MS) and the antioxidant potential was measured using a series of in vitro biochemical assays, including DPPH, hydroxyl, and superoxide radical scavenging assays.

Results

The recovery yield of the VCs was 0.823 wt% of the dry mass of G. lemaneiformis. A total of 25 VCs were successfully identified, comprising approximately 99.94% of the total volume. The major component was n-hexadecanoic acid (38.57%), followed by oleic acid (25.48%), arachidonic acid (12.84%), and tetradecanoic acid (2.52%). In addition, The VCs displayed strong free radical scavenging activity in the DPPH (IC₅₀ = 21.56 mg/L), hydroxyl (IC₅₀ = 18.34 mg/L), and superoxide (IC₅₀ = 391.12 mg/L) radical scavenging assays. The antioxidant activities of the VCs exhibited a dose-dependence at concentrations ranging from 5 to 200 mg/L.

Conclusion

The results indicated that the sustainable process improved the agar quality and that the extract contained many natural VCs with antioxidant activities, which have the potential to be used in functional food and cosmetics instead of as a discarded byproduct of the agar industry.

Introduction

Gracilaria lemaneiformis (G. lemaneiformis) a species of Gracilariales, is abundantly cultivated along the coast of China due to its high commercial value [1]. This seaweed is edible as a sea-flavor vegetable [2], and is also used as a traditional Chinese medicine because of its abundant ingredients beneficial for human health [3, 4]. More importantly, this seaweed is used in the agar industry as a raw material because of its high polysaccharide content [5, 6]. In current agar production, dried seaweed is used as a raw material to produce agar via several methods including alkali pretreatment or acid hydrolysis [7, 8], and the volatile constituents (VCs) of G. lemaneiformis are destroyed and discarded as waste. The VCs of G. lemaneiformis are reported to exhibit cytotoxic activity and tyrosine phosphatase 1B inhibitory activity [9, 10], suggesting that these VCs could be useful in the food, cosmetic, and pharmaceutical industries [11, 12]. Therefore, it is worthwhile to recover VCs to improve the economic value of G. lemaneiformis.

VCs are composed of significant amounts of lipids that exhibit biological activities such as antioxidant, antitumor, anti-Rhizopus activities [13,14,15]. For these applications, natural antioxidants have attracted significant research attention. Compared to synthetic antioxidants, safe and inexpensive natural antioxidants are more popular with consumers. Many studies have reported that antioxidant compounds are effective for protecting human health and food safety against the oxidizing reactions by reactive oxygen species (ROS). Excessive production of ROS causes oxidative stress which damages cell structure, leading to a number of chronic diseases including stroke, cancer, diabetes, atherosclerosis, and other degenerative diseases [16, 17]. Therefore, there is significant demand for effective natural antioxidant VCs that exhibit strong antioxidant activity to eliminate the ROS and other free radicals. However, few studies have investigated the VCs from the edible seaweed as important natural antioxidants and flavor agents in food industry.

In this study, a sustainable method was developed for the recovery of VCs from the red algae G. lemaneiformis. The chemical composition of VCs present in the n-hexane fraction was analyzed by GC–MS: gas chromatography–mass spectroscopy. In addition, the antioxidant activities of the extracted VCs were evaluated by a series of biochemical assays in vitro including the DPPH, hydroxyl, and superoxide radical scavenging assays. The sustainable extraction process of VCs improved agar quality and reduced the amount of effluent, providing a source of natural antioxidants rather than waste.

Methods

Biological material

Red seaweed, G. lemaneiformis, was harvested in June 2016 from the Zhanjiang Naozhou coastline of Guangdong province (N 21°12′; E 110°4′) in Southern China. The seaweed was washed thoroughly with tap water to remove salt and impurities and subsequently air-dried at 60 °C. The dried sample was cut and mixed with a blender and stored in a refrigerator at 4 °C until use. The sample was identified by the School of Marine Science, Guangdong Ocean University, China.

Chemicals and reagents

1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). The hydroxyl radical and superoxide anion detection kits were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Deionized water was supplied by a Milli-Q water purification system from Millipore (USA). All other reagents used in this study were of analytical grade.

Sustainable extraction for the recovery of VCs

The natural VCs were extracted as previously described [10] with some modifications. Briefly, the dry G. lemaneiformis (150 g) was mixed with 70% ethanol (3000 mL) and ultrasonically pretreated 10 min, and then the mixture was refluxing at 80 °C for 2 h. After twice extractions, the extracts were combined and concentrated using a rotary evaporator at 40 °C. The crude extract was dissolved in deionized water and fractioned with n-hexane. Subsequently, the n-hexane fraction was concentrated using a rotary evaporator to recovery the solvent and obtain the VCs.

Analysis of VCs by GC–MS

The chemical constituents of the VCs from G. lemaneiformis were determined via GC–MS using a standard procedure [18]. The system was equipped with an Agilent 6890 gas chromatograph/5973 N mass selective detector (Palo Alto, Calif.) and separated using an HP-FFAP (HP-free fatty acid phase) capillary column (30 m × 0.25 mm i.d.; film thickness = 0.25 μm). Helium was used as the carrier gas at a constant 1 mL min⁻¹ flow rate and the oven temperature was set at 50 °C for 5 min, raised to 230 °C at 4 °C min⁻¹, and held for 20 min. The detector and injector temperatures were maintained at 250 °C. The ionizing energy of the mass selective detector was set at 70 eV, with a scanning mass range of m/z 50–500. The VCs sample (1 µL of 100 times-diluted samples in methanol) was pass through a splitless injector in manual mode. The relative percentages of the constituents of the VCs were expressed as percentages calculated from the normalized peak areas. The various chemical constituents of VCs were identified by GC retention times on a DB-5 capillary column, similarity index, and mass spectra, which were consistent with the mass spectra in the computer library (Wiley 275L program). Chromatographic peaks were checked with the mass chromatograms of the characteristic fragment ion peaks for homogeneity. The chemical structures of the dominant compounds were drawn using ACD software.

Antioxidant activities

DPPH radical scavenging activity

The DPPH radical scavenging activity assay was used to determine the antioxidant activity of the isolated VCs. The assay was performed according to a method previously described with slight modifications [19]. Briefly, a sample aliquot (100 μL) was mixed with 100 μL of 0.2 mM DPPH ethanol solution and the intermixture was incubated at room temperature for 30 min. The sample absorbance was measured at 517 nm. All experiments were performed in triplicate and the percentage of scavenged DPPH was calculated using the following equation:

$${\text{DPPH scavenging activity }}\left( \% \right) \, = \, \left[ {\left( {{\text{A}}_{\text{C}} - {\text{A}}_{\text{S}} } \right)/{\text{A}}_{\text{C}} } \right] \, \times { 1}00$$

where A_c is the absorbance value of the control (mixture of 100 μL DPPH solution and 100 μL ethanol) and A_s is the absorbance value of the sample. Vitamin C was used as a positive reference.

Hydroxyl radical scavenging activity

Hydroxyl radical (HOR) scavenging activity of the VCs was determined according to a previously described method with some modification [19]. Briefly, 1.0 mL of a ferrous sulfate solution (6.0 mM), 1.0 mL hydrogen peroxide (6.0 mM), 0.5 mL salicylic acid (2.0 mM), and 1.0 mL sample solutions (at various concentrations) were thoroughly mixed and incubated at room temperature for 40 min. The mixture absorbance was then measured at 550 nm and vitamin C was used as a positive control. The hydroxyl radical scavenging activity was calculated using the following equation:

$${\text{HOR scavenging activity }}\% \, = \, \left[ {\left( {{\text{A}}_{ 1} - {\text{A}}_{ 2} } \right) \, / \, \left( {{\text{A}}_{ 1} - {\text{A}}_{0} } \right)} \right] \, \times { 1}00;$$

where A₀ is the absorbance value of the reagent blank, A₁ is the positive control absorbance, and A₂ is the absorbance value of the sample.

Superoxide radical scavenging activity

The superoxide anion radical (SOAR) scavenging activity of the VCs was measured using the procedure described previously with some modifications [20]. All solutions were prepared in 0.2 M phosphate buffer (pH 7.4). The reaction mixture consisted of 0.5 mL of the reaction buffer solution (pH 7.4), 100 μL PMS (15 μM), 100 μL NADH (73 μM), 100 μL NBT (50 μM), and 200 μL sample of various concentrations completely blended and incubated at room temperature for 30 min and the absorbance was measured at 550 nm. The control was a mixture without any sample and the assay was performed in triplicate. The superoxide anion radical scavenging activity (%) was analyzed using the following equation:

$${\text{SOAR scavenging activity }}\left( \% \right) \, = \, \left[ { 1- \left( {{\text{A}}_{\text{S}} /{\text{A}}_{\text{C}} } \right)} \right] \, \times 100,$$

Vitamin C was used as a reference.

Statistical analysis

The data were analyzed using a one-way analysis of variance (ANOVA) followed by the Duncan’s test (SPSS 12.0, SPSS Inc., USA). The data are expressed as mean ± standard deviation. All experiments were performed in triplicate and p < 0.05 was considered statistically significant.

Results and discussion

Recovery yield of VCs from G. lemaneiformis

To manufacture high quality agar from G. lemaneiformis, many redundant constituents such as fatty acids, essential oils and pigments should be removed. However, it may be beneficial to recovery the VCs from the raw material seaweed as natural antioxidants. In this study, using the reflux condensing extraction shown in Fig. 1, the recovery yield of the VCs was 0.823 wt% on a dry basis from G. lemaneiformis. The recovery of VCs was lower for seaweeds such as P. tenera (1.41%) obtained by the hydro-distillation [21], but higher than that of U. pinnatifida (0.260%) obtained by microwave-assisted hydro-distillation [22]. The differing results of the various seaweeds can be attributed to species-specific factors, environmental differences, as well as varied extraction methods and conditions such as solvents, times, and temperatures [23] used. Therefore, it was valuable to recover and further analyze the chemical constituents of the VCs obtained from red seaweed. In this extraction process, the VCs of G. lemaneiformis were obtained and ethanol was recovered, the seaweed was next used as raw material to produce agar. All solvents can be recovered and recycled, this result showed no harmful pollutant was release into the environment and it was a green sustainable process.

GC–MS analysis of VCs in the n-hexane fraction

The chemical constituents of VCs were identified via GC–MS and the results are shown in Table 1 and Fig. 2. A total of 25 volatile constituents were successfully identified comprising 99.94% of the total volume. The major constituents of the VCs obtained from G. lemaneiformis were n-hexadecanoic acid (38.57%), oleic acid (25.48%), arachidonic acid (12.84%), cholesterol (4.90%), tetradecanoic acid (2.52%). It was clear that n-hexadecanoic acid and tetradecanoic acid was non-reducing saturated fatty acid, in contrast, oleic acid, arachidonic acid and cholesterol was reductive.

Table 1 VCs identified by GC–MS in the n-hexane fraction of G. lemaneiformis

Full size table

The n-hexadecanoic acid content was the highest (38.57%), nearly twice as high as the maximum content of n-hexadecanoic acid (20.08%) in n-hexane extracted previously from the same type of seaweed [10]. It has been reported that n-hexadecanoic acid exhibits various biological activities including anti-inflammatory activities, anticancer effects, and antioxidant properties [24, 25].

Tetradecanoic acid is commonly called myristic acid and is found in seaweeds such as edible kelps [26]. The tetradecanoic acid content was significant different in different alga including G. lemaneiformis (2.52%) (shown in Table 1), L. japonica (51.75%) [18], and U. pinnatifida (31.32%) [22]. Tetradecanoic acid has been reported to possess potential antibacterial activity [27]; it is also applied as a flavoring agent in food industry, and a brain drug additive in medicine [18].

The total amounts (43.22%) of oleic acid, arachidonic acids and cholestrol suggested that significant amounts of unsaturated ethylenic bonds (C=C) were present, therefore the VCs were imparted reductive properties. The antioxidant activity of the VCs can be ascribed to the donation of electrons to reactive oxygen radical, reducing them to more stable and non-reactive species. These polyunsaturated fatty acids represent a valuable natural source of antioxidants from seaweed extract [28, 29].