Name: Evaluation of the Antioxidant and Cytotoxic Effects of Gracilaria gracilis Extracts
Uploaded: 2023-11-21T14:20:00
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evaluation of the antioxidant and cytotoxic effects of gracilaria gracilis extracts

Gracilaria gracilisの利用:持続可能なイノベーションのための生物活性の可能性

&#160;Seaweeds can be regarded as an interesting natural raw material to be profited by the pharmaceutical, food, feed, and environmental sectors. They biosynthesize a panoply of molecules, many not found in terrestrial organisms, with relevant biological properties1,2. However, seaweed-optimized cultivation protocols need to be implemented to ensure a large biomass stock.
Cultivation methods must always consider the nature of the seaweed thalli and overall morphology. Gracilaria gracilis is a clonal taxon, meaning the attachment organ produces multiple vegetative axes. Propagation by fragmentation (vegetative reproduction) is thus achieved, as each of these axes is fully able to adopt an independent life from the main thallus3. Clonal taxa can be successfully integrated with simple and fast one-step cultivation methodologies, as large amounts of biomass are obtained by splitting the thallus into small fragments that quickly regenerate and grow into new, genetically identical individuals. Both haplontic and diplontic thalli may be used in this process. Although the genus exhibits a complex haplo-diplontic isomorphic triphasic life cycle, sporulation is rarely necessary except when genetic renewal of the stocks is required to achieve improved crops. In this case, both tetraspores (haplontic spores formed by meiosis) and carpospores (diplontic spores formed by mitosis) give rise to macroscopic thalli that can then be grown and propagated by vegetative reproduction4. Growth cycles are dictated by environmental conditions and the physiological state of the individuals, among other biological factors such as the emergence of epiphytes and the adhesion of other organisms. Therefore, optimizing growing conditions is crucial to ensure high productivity and produce good quality biomass5.
The extraction of bioactive compounds from seaweed, including G. gracilis, can be achieved through various methods6,7. The choice of the extraction method depends on the specific compounds of interest, the target application, and the characteristics of the seaweed. In this study, we focused on solvent extraction, which involves using green solvents, such as water or ethanol, to dissolve and extract bioactive compounds from the seaweed biomass. The extraction can be carried out through maceration in a versatile and effective way and can be used for a wide range of compounds. It is a simple and widely used method involving soaking biomass in a solvent for an extended period, typically at room or slightly elevated temperatures. The solvent is stirred to enhance the extraction process. After the desired extraction time, the solvent is separated from the solid material by filtration or centrifugation.
Water is a commonly used solvent in food applications due to its safety, availability, and compatibility with a wide range of food products. Water extraction is suitable for polar compounds such as polysaccharides, peptides, and certain phenolics. However, it may not effectively extract non-polar compounds. Ethanol is also a widely used solvent in food applications and can be effective for extracting a variety of bioactive molecules, including phenolic compounds, flavonoids, and certain pigments. Ethanol is generally recognized as safe for use in food and can be easily evaporated, leaving behind the extracted compounds. It is worth noting that the choice of extraction method should consider factors such as efficiency, selectivity, cost-effectiveness, and environmental impact. The optimization of extraction parameters, such as solvent concentration, extraction time, temperature, and pressure, is crucial to achieving optimal yields of bioactive compounds from G. gracilis or other seaweeds.
Seaweeds have been found to exhibit antimicrobial activity against a wide range of microorganisms, including bacteria, fungi, and viruses8. This activity is attributed to bioactive components, including phenolics, polysaccharides, peptides, and fatty acids. Several studies have demonstrated their efficacy against pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella sp., and Pseudomonas aeruginosa, among others9. The antimicrobial activity of seaweeds is attributed to the presence of bioactive compounds that can interfere with microbial cell walls, membranes, enzymes, and signaling pathways10. These compounds may disrupt microbial growth, inhibit biofilm formation, and modulate immune responses.
Red seaweeds, also known as rhodophytes, are a group of algae that can exhibit antimicrobial activity against a variety of microorganisms. Within this group, G. gracilis contains various bioactive compounds that may contribute to its reported antimicrobial activity. While the specific molecules can vary, the common classes that have been reported in G. gracilis and may possess antimicrobial properties are polysaccharides, phenolics, terpenoids, and pigments11. However, it is important to note that the presence and amounts of these components can vary depending on factors such as the location of seaweed collection, seasonality, physiological condition of the thalli, and environmental conditions. Therefore, the specific class and concentration of antimicrobial compounds in G. gracilis may vary accordingly.
G. gracilis has also been found to hold antioxidant properties, containing various phenolic compounds, which have been shown to scavenge free radicals and reduce oxidative stress12. Antioxidants help to protect cells from damage caused by reactive oxygen species and have potential health benefits. Antioxidant capacity can be evaluated directly through different methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and, indirectly, through the quantification of total polyphenolic content (TPC)13.
Even though an ingredient is reported to have a prominent bioactivity, its cytotoxicity assessment is indispensable in evaluating natural and synthetic substances to be used in contact with living cells or tissues. There are several methods for measuring cytotoxicity, each one with advantages and limitations. Overall, they offer a range of options to evaluate the harmful effects of many substances on cells and, at the same time, to investigate the mechanisms of cell damage and death14.
In this work, we use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a colorimetric method introduced by Mosmann (1983)15. This method measures the reduction of tetrazolium salts to a purple formazan product by metabolically active cells. The higher the amount of formazan crystals, the higher the number of viable cells, thus providing an indirect measure of cytotoxicity14. Since in this work, G. gracilis water and ethanol extracts are intended to be incorporated into dermo-cosmetic formulations, the in vitro cytotoxicity evaluation is performed in a keratinocyte (HaCaT) cell line.
Concerning the food application, seaweeds are generally low in calories and nutritionally rich in dietary fibers, essential elements and amino acids, polysaccharides, polyunsaturated fatty acids, polyphenols, and vitamins2,16. G. gracilis is no exception, having an interesting nutritional value. Freitas et al. (2021)4 found that cultivated G. gracilis had higher levels of protein and vitamin C and maintained the level of total lipids compared to wild seaweed. This may represent an economic and environmental advantage, as nutritionally speaking, production is preferable to the exploitation of wild resources. In addition, consumers are increasingly concerned about the type of food they eat, so it is important to introduce new ingredients for food enrichment and use new resources to obtain extracts that can add value to a product and claim a &#34;clean label.&#34; Besides, the current market is very competitive, requiring the development of new products and innovative strategies to differentiate manufacturers from their competitors17.
The enrichment of products with poor nutritional value, such as pasta, with marine resources, including seaweed, is a strategy to introduce this resource as a new food and a market differentiation strategy through a product with distinct nutritional value. On the other hand, G. gracilis is a source of natural red pigments such as phycobiliproteins18, having high potential for applications in the food industry. This seaweed has shown high interest in several areas, and its application can be made using the whole seaweed, extracts and/or the remaining biomass. In this work, we demonstrate some examples of such applications.

著者スポットライト:産業における持続可能なイノベーションのための海藻の生物活性化合物の探索 

バイオリファイナリーアプローチによる紅海藻 Gracilaria gracilis の価値化

Our research is focused on the prospection of new bioactive compounds from seaweed that can be useful in different sectors such as food, cosmetic, and pharmaceutical industries. There is a growing awareness in society about the potential of marine organisms to deliver bioactive compounds. These compounds can be used to produce sustainable and safer functional cosmetics to improve the quality of food products and serve as source of new chemical structures for the development of new pharmaceutical drugs.One of the challenges of using seaweed in the development of new food products is to avoid using high amounts that could lead to unpleasant sensory properties in food. Using seaweed extracts is a way to get around this problem. With this protocol, we were able to develop pasta, wheat, add nutritional value by incorporating whole algae.Usually, the addition of these ingredients provides less common sensory properties. However, with the developed formulation, a consumer acceptance was still achieved.

Watch this Scientific Journal Video about Evaluation of the Antioxidant and Cytotoxic Effects of Gracilaria gracilis Extracts at JoVE.com

Evaluation of the Antioxidant and Cytotoxic Effects of Gracilaria gracilis Extracts  

Gracilaria gracilis抽出物の抗酸化作用および細胞毒性作用の評価

まず、乾燥したGracilaria gracilisから得られた水性およびエタノール抽出物を取ります。96ウェルマイクロプレートに、各サンプル2マイクロリットルと無水エタノールに溶解した198マイクロリットルのDPPHを分注します。次に、マイクロプレート分光光度計を用いて、517ナノメートルの吸光度を測定する。96ウェルプレートで増殖させたヒトケラチノサイトを一晩採取します。2マイクロリットルのDMSO溶解抽出物を、マイクロプレートウェル中の培地198マイクロリットルに加えます。培地を取り出します。100マイクロリットルのMTTを細胞に加え、暗所で30分間インキュベートします。細胞内ホルマザン結晶を可溶化するには、100マイクロリットルのDMSOを添加します。マイクロプレートリーダーを使用して、570ナノメートルの吸光度を測定します。DPPH試験では、室温または70°Cで得られた抽出物と比較して、摂氏40度の温度で酸化活性の阻害値が高いことが示されました。ヒトケラチノサイトに対する細胞毒性作用は観察されず、最大アッセイ濃度では、水性抽出物とエタノール抽出物の両方が皮膚での使用に安全であることが示唆されています。

Research

JoVE Journal

Biology

Evaluation of the Antioxidant and Cytotoxic Effects of Gracilaria gracilis Extracts

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we ...

生物学

Extraction of the Bioactive Ingredients from Gracilaria gracilis

Extraction of the Bioactive Ingredients from Gracilaria gracilis  

Gracilaria gracilisからの生理活性成分の抽出

Pasta Production Using Gracilaria gracilis, Nutritional Evaluation, and Sensory Evaluation

Pasta Production Using Gracilaria gracilis, Nutritional Evaluation, and Sensory Evaluation  

Gracilaria gracilisを用いたパスタ製造、栄養評価、官能評価

Incorporation of Seaweed Extract in Yogurt and Color Stability Evaluation 

ヨーグルトへの海藻エキスの配合と色安定性評価 