Intestinal anti-inflammatory activity of Ulva ohnoi oil in DSS-induced experimental mouse model

This study was conducted to examine the physiological activity of Ulva ohnoi, some of which may be used for food or natural products but could disturbing coastal ecosystems due to large scale green-tide, to check values of U. ohnoi oil through experimental results. U. ohnoi oil was extracted from bulk of Ulva biomass to confirm its antioxidant and antibacterial activity, and the efficacy of U. ohnoi oil in the state of inflammation was confirmed through animal experiments. To confirm the anti-inflammatory effect, a mouse model induced with DSS was used. As a result of measuring NO using plasma after induction of inflammation, the amount of NO produced in the U. ohnoi oil group was decreased compared to the control group. Expression of inflammatory cytokines TNF-α, IL-6, and IL-1β was decreased compared to the control group. As a result of observing H&E staining, lower crypt loss and inflammatory cell infiltration were found in the U. ohnoi oil group compared to the control group. Consequently, U. ohnoi oil appears to have great anti-inflammatory properties.


Scientific Reports
| (2021) 11:15087 | https://doi.org/10.1038/s41598-021-94475-z www.nature.com/scientificreports/ The Antarctic krill (Euphausia superba) is a zooplankton found in the Antarctic water that feeds directly on minute phytoplankton. This species is receiving the spotlight as a food resource in the future due to its excellent nutritional value 20 , as it contains large amounts of unsaturated fatty acids and antioxidants such as EPA and DHA 21 . However, krill contains a large amount of fluoride, which is harmful to the human body 22 . There are also reports that global warming is accelerating due to the decline in krill populations. Therefore, it is urgent to find a replacement because overfishing of krill threatens the survival of other marine life such as whales, penguins and seals 23 .
Seaweeds are known to accumulate many elements on from the surrounded environments. In particular, seaweeds absorb these elements over their entire surface area and are a treasure trove of minerals up to tens of thousands of times compared to pelagic organisms 24 . Although seaweeds have low nutritional value due to their low protein and fat content, they have antioxidant effects such as scavenging free radicals within the tissue 25,26 due to their high content of various inorganic salts, such as potassium, iodine, and calcium, and vitamins A and C 27 . Recently, as the interest in finding substances with pharmacological action from natural plants has increased, efforts to extract effective active ingredients and use them as food additives or pharmaceuticals are growing alongside research on the physiological activity of sea weeds 28,29 .
Ulva ohnoi is a green alga in the family Ulvaceae (Chlorphyta) widely distributed around the North America, South Korea, Japan, and Mediterranean sea, usually attached to hard bottom on the rocky shore. The rise of sea surface temperature and nutritive salts due to global warming causes mass reproduction of U. ohnoi 30 , causing large scare green-tide in some contaminated costal ecosystem, such as Japan 31 , and South Korea 30 . U. ohnoi has a fast growth rate, so no problem of material depletion is raised, and it is thought that it is valuable as a new marine natural product while solving environmental problems. About 700 tons of U. ohnoi were collected in blooming area in Japan, and some algal biomass were used as food ingredients 31 . In addition, morphologically similar green alga, U. lactuca has been used for food in Europe for a long time, and multiple studies have been performed on its various food and physiological activities. It contains a large amount of polysaccharides containing sulfuric acid, and this acid polysaccharide has been reported to have anti-viral, immunity-boosting, and anti-cancer effects 32 . Some studies in South Korea reported anti-cancer effects and immune activities by extracting glycoproteins from single-cell layered green algal, Monostroma nitidum 32 .
The purpose of this study is to extract oil from U. ohnoi using an organic solvent extraction method to find out the utility of U. ohnoi oil for its physiological activity. There have been various efficacy studies using U. ohnoi, but studies using U. ohnoi oil are insufficient. Conventionally, oily ingredients such as krill oil and omega-3 are exerting the function of maintaining human health. We would like to consider the aspect of recycling discarded U. ohnoi through the use as a new material by proving the functionality of U. ohnoi, the subject of our research.

Materials and methods
Instruments and reagents. In this experiment, DPPH (1,1-diphenyl-2-picrylhydrazyl) (Sigma-Aldrich, USA), folin&ciocalteu's reagent (sigma-Aldrich, USA), bacto agar (BD DIFCO, USA), bacto tryptic soy broth (BD DIFCO, USA), NO (nitric oxide) kit (Cell Biolabs. INC., USA), ELISA (enzyme-linked immunosorbent assay) kit (Abfrontier, KOR), TRIzol reagent (Sigma-Aldrich, USA), chloroform (Daejung, KOR), isopropyl alcohol (Sigma-Aldrich, USA), ethanol (Daejung, KOR), DEPC (diethyl pyrocarbonate) water (Thermo Scientific, USA), agarose (Bioneer, KOR), RT-PCR premix kit (Bioneer, KOR), Hematoxylin (Sigma-Aldrich, USA), Eosin Y (Sigma-Aldrich, USA ), centrifuge (Labogene, KOR), enzyme-linked immunosorbent assay (ELISA) reader (Thermo Scientific, USA), fluoro box (Neo Science, KOR), and Permount (Fisher, USA) were used. Experimental material. The U. ohnoi used in this study was collected from the north-east part of Jeju Island (A : Gujwa-eup, 33°33′33.0"N, 126°45′33.7", B : Jochon-eup, 33°32′57.9"N, 126°38′58.9"E, and C : Sinyang sand beach, 33°26′06.0"N, 126°55′23.4"E) in 2019 (voucher specimen number ELMV 03; deposited in the herbarium of ELMV laboratory in Kunsan National University; collector: JuHyoung Kim). We have transported small amount of Ulva biomass collected from the blooming area to the laboratory, thus sample collection was not threatened with endangered species and have not destroyed natural seaweed habitats. In addition, sampling was carried out after permission of National Research Foundation (NRF) of South Korea, thus government is permitting the collection of Ulva because it is working on a research project to manage Ulva biomass. Sample were washed with tap water and distilled water several times, and dried in an oven at 60℃. Organic solvent extraction was performed to extract U. ohnoi oil. The weight of the dried sample in the beaker was 29.3 g, 33.5 g, and 30.7 g of A, B, and C, respectively, and 200 ml of hexane was added per 10 g. Thereafter, extraction was performed at room temperature for 48 h and the organic solvent was removed using speed vacuum. The yields are 0.19%, 0.23%, and 0.21% for A, B, and C, respectively. All the experiments involving plants adhered to relevant ethical guidelines on plant usage. Krill oil is manufactured by Neptune (Neptune Technologies & Bioressources INC., Canada), and omega-3 is manufactured by Now Foods (Now Foods, USA). The composition of krill oil, omega-3, and U. ohnoi oil used in the experiment is shown in Table S1 of Supplementary Information. DPPH radical scavenging activity measurement. DPPH radical scavenging activity was used to eliminate absorption characteristics when the compound was stabilized by receiving electronic or hydrogen radicals and measured in accordance with using 1,1-diphenyl-2-picryhydrazyl (DPPH) 33 . DPPH was dissolved in ethanol at a final concentration of about 0.2 mM. U. ohnoi oil (100 µl) by concentration was added to 0.1 ml of ethanol solution of DPPH. After blocking the light and reacting for 20 min, the absorbance was measured at 517 nm. DPPH scavenging effect was calculated from [1-(Abs sample -Abs blank )/Abs control ] × 100, where Abs sample is the absorbance of the sample, and Abs blank is the absorbance of the color control, and Abs control is the absorbance www.nature.com/scientificreports/ of the negative control. As a control group, ethanol was added instead of the sample. L-ascorbic acid, which is known to have high antioxidant capacity, was used as a control to compare antioxidant activity.
Total polyphenol content assay. The total polyphenol content assay (TPA) was determined using the Folin-Denis reagent 34 . 200 µl of the U. ohnoi oil was added to 1 ml of 0.2 N folin-ciocalteu reagent and 800 µl of 7.5% Na 2 CO 3 was added. After blocking the light and reacting at room temperature for 2 h, the absorbance was measured at 620 nm using a spectrophotometer. Gallic acid was used as a standard reagent, and polyphenol content was calculated using a standard curve. The TPA was expressed as gallic acid equivalents (GAE) in mg/1 g material.
URDA (ultrasensitive radical diffusion assay). The antibacterial activity measurement using U. ohnoi oil was performed by URDA (ultrasensitive radical diffusion assay) method 35 . The sample was prepared at a concentration of 10 mg/ml using 0.01% acetic acid (HAc). The strains used were gram-positive bacteria Bacillus subtilis KCTC1021, gram-negative bacteria Escherichia coli D31, and fungus Candidia albicans KCTC76965. Each strain was inoculated with 1 platinum loop in tryptic soy broth (TSB) medium and pre-culture was performed at 37℃ for 24 h. The activated bacteria measured absorbance at 630 nm to make the O.D value of the bacteria 0.01 (≒ 0.5 McF). 0.03% TSB, sabouraud's dextrose broth (SDB), 1% agarose, and 10 mM phosphate buffer (pH 6.5) were added to the underlay gel at 9.5 ml concentrations and then poured into the plate to solidify. The plate was pierced with a diameter of 2.5 mm and 5 µl of sample was injected into each well. All samples were tested with 0.01% acetic acid 5 µl, which was not affected by solvent. After the first culture at 37℃ for 3 h, 10 ml overlay gel containing 6% TSB, SDB, 1% agarose, and 20 mM phosphate buffer (pH 6.5) was poured into the cell, and then solidified at room temperature, and then the clear zone was confirmed after the second culture at 37℃ for 24 h.

Characterization of antibacterial activators using enzymatic treatment.
In order to confirm the properties of the antibacterial activity in the U. ohnoi oil, various enzymes were treated to determine whether there was any change in antibacterial activity. The enzymes used in the experiment are trypsin, chymotrypsin, lipase, α-amylase, pronase, and proteinase. 5 µl of U. ohnoi oil at a concentration 10 mg/ml was mixed with 1 µl of each enzyme and incubated for 3 h at 37 ℃, followed by secondary culture at 37 ℃ for 24 h. The cultured extracts were tested for URDA against B. subtilis, E. coli, and C. albicans.

Experimental animals and inflammatory intestine disease induction.
Male ICR mouse aged 4 weeks were obtained from Orient bio (Orient bio, Gwangju). They were housed in a controlled environment (25 ± 2 ℃, 50 ± 10% humidity) with a 12 h light/dark cycle. The animals allowed to free access to food and filtered water. They were quarantined and acclimatized for 1 week before use. The animals were kept in groups of 6 per cage. After 1 week habituation period, the experimental animals were classified into five groups as follows: one is normal, another is control and the others is DSS + krill oil (KO), DSS + omega-3 (ω-3), and DSS + U. ohnoi oil (UO). Krill oil, ω-3, and U. ohnoi oil are prepared using distilled water at concentrations of 100 mg/kg, 100 mg/ kg, and 25 mg/kg and administered orally at 100 µl each for 10 days (Table S2). U. ohnoi oil used for mouse administration was prepared by mixing a certain amount of A, B, and C. To induce inflammatory bowel disease, the Con, KO, ω-3, and UO groups freely ingested 5% dextran sulfate sodium (DSS) dissolved in distilled water for 7 days after 4 days of sample administration. The body weight of the experimental animals was measured at the same time every day and is shown in Figure S1 of Supplementary Information. The animal studies were approved by Kunsan National University animal experiment ethics committee (Approval No: 2020-02). This study was carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All the experiments involving animals adhered to relevant ethical guidelines on animal usage.
Measurement of NO production. The amount of NO production was measured using plasma of 6 mice per group, and Griess reagent was used 36 . Blood was collected to obtain plasma and centrifuged at 7000 rpm for 10 min at 4 ℃. NO concentration was then determined by the addition of 50 µl each Griess reagents 1 and 2. After incubation for 10 min at room temperature, the absorbance was read at 540 nm.

Measurement of inflammatory cytokine in colon tissue.
In this study, colon tissue from 6 mice per group was removed and used, 1 ml of 1 × phosphate-buffered saline (PBS) was added, crushed, and centrifuged at 7000 rpm for 10 min, and then the supernatant was taken. The amount of inflammatory cytokine TNF-α, IL-6, and IL-1β was measured using an enzyme-linked immunosorbent assay (ELISA) kit. After adding 100 µl of antigen to the antibody-coated plate, incubated at 37℃ for 2 h, and washed 3 times using a washing buffer. After the addition of 100 µl of the secondary antibody, it was incubated at 37℃ for 1 h and washed 3 times. After adding 100 µl of the TMB solution, incubate at 37℃ for 30 min, and reacted at room temperature for 10 min after adding 100 µl of stop solution, absorbance was measured at 450 nm.
RT-PCR. RNA used in the experiment was extracted from the colon tissue of the mouse using TRIzol reagent, and it was dissolved in 50 µl diethyl pyrocarbonate (DEPC) water. Reverse transcription-polymerase chain reaction (RT-PCR) is performed using and RT-PCR premix kit, and 300 ng of total RNA, 20 pmol of each of forward and reverse primers are added to use a total of 20 µl of the reaction solution. The cDNA was synthesized by reacting at 50℃ for 30 min and 94℃ for 5 min, followed by 35 cycles of denaturation at 94℃ for 40 s, annealing at 58℃ for 40 s, and extension at 72℃ for 90 s. The PCR product was confirmed by electrophoresis on 1.2% agarose gel, and GAPDH was used as control. The primer sequence used in the experiment is as follows  37 . H&E stains are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis 38 . Each specimen was sectioned into a thickness of 7 µm and histochemical staining was performed using hematoxylin and eosin. For deparaffinization, xylene was added and repeated three times for 5 min, and xylene was removed in the order of 100%, 95%, and 70% ethanol. For nuclear staining, it was stained with hematoxylin solution for 5 min, and then washed with flowing water. Hematoxylin was removed using 1% HCl solution and 1% ammonia solution, followed by immersion in eosin solution for 2 min to stain the cytoplasm. Then, it was dehydrated in the order of 70%, 95%, and 100% ethanol. To remove alcohol, it was put in xylene and repeated three times for 3 min, and then the cover glass was and sealed.

Results and discussion
Antioxidant activity evaluation. The antioxidant activity of U. ohnoi oil was measured using DPPH and Folin-Denis reagent. The radical scavenging ability was measured by concentrations of 0.1, 0.3, 0.5, 0.7, and 1 mg/ml, and ascorbic acid was used as a positive control (Fig. 1A). It was confirmed that the DPPH radical scavenging activity of A and B U. ohnoi oil increased to 0.7 mg/ml, but decreased at the concentration of 1 mg/ml. At the concentration of 0.7 mg/ml, B U. ohnoi oil showed the most similar value to ascorbic acid at 86.71 ± 8.36%. Ascorbic acid showed 88% scavenging activity independent of concentration. The total phenolic compound content of the sample was curve using gallic acid as the standard and converted to gallic acid equivalents (GAE) (Fig. 1B). The total polyphenol content was 107.45 ± 0 mg GAE/g in B and 69.44 ± 3.46 mg GAE/g in C, which was about 1.5 times higher than that of B U. ohnoi oil. Krill oil showed a lower polyphenol content than B U. ohnoi oil at 70.6 ± 1.99 mg GAE/g. Antibacterial activity of U. ohnoi oil by strain. The antibacterial activity of U. ohnoi oil was measured against B. subtilis, E. coli, and C. albicans strains using the URDA method. In general, B. subtilis and E. coli showed antibacterial activity, but not against the fungus C. albicans. The size of the clear zone was 10.36 mm in B and 7.35 mm in A in the B. subtilis strain, indicating that the clear zone size was 1.41 time higher than that of B U. ohnoi oil. As a result of measuring the activity against E. coli strains, all three types of U. ohnoi oil showed high antibacterial activity of 10 mm or more (Table 2A).

Changes in antibacterial activity by enzyme treatment.
To confirm the characteristics of substances exhibiting antibacterial activity in U. ohnoi oil, six enzymes such as protease and lipolytic enzyme were treated, respectively, and then it was confirmed whether there was any change in antibacterial activity. As enzymes, trypsin, chymotrypsin, lipase, α-amylase, pronase, and proteinase K were used, and the size of the clear zone was measured to show antibacterial activity according to the enzyme treatment (Table 2B). B. subtilis, E. coli, and C. albicans strains were tested for activity changes, but the antibacterial activity of U. ohnoi oil was not significantly affected by the enzymes used. Therefore, it was confirmed that the substance exhibiting antibacterial activity in U. ohnoi oil is considered to an organic compound rather than a fat or proteinaceous substance that is cleaved by enzyme treatment and has stability in enzyme reaction. NO production measurement. NO (nitric oxide) is known as a mediator of the inflammatory response secreted when cells stimulated by inflammatory substances such as LPS or TNF-α induce inflammation 39 . In this study, the amount of NO production was measured using plasma of mice ( Fig. 2A). The NO production of the control group was 31.26 ± 5.04 µM, which was about 1.3 times higher than that of the normal group, and it was found that there is an effect of inducing inflammation by DSS. The production amount of the UO group was 26.26 ± 4.08 µM, which was about 84.0% lower than that of the control group, and it was confirmed that the production amount of NO was like that of the normal group. www.nature.com/scientificreports/ Inflammatory cytokine production. The amount of TNF-α, IL-6, and IL-1β production were measured by ELISA method. All three cytokines were increased in the control group. In the case of TNF-α and IL-6, the amount of production of 43.9 ± 20.2 pg/ml and 17.41 ± 2.68 pg/ml, respectively, in the UO group and significantly decreased compared to the control group (Fig. 2B,C). The amount of IL-1β produced in the UO group was 54.68 ± 14.14 pg/ml, which was like that of the normal group, but there was no statistical significance (Fig. 2D).
Confirmation of inflammatory cytokine mRNA expression. RT-PCR was performed using the colon tissue of experimental animals induced with 5% DSS to determine whether inflammatory cytokine affects mRNA expression. The results are as follows (Fig. 3). When compared with the normal group, it was confirmed that the amount of inflammatory factor expression in the control group was increased. It was confirmed that the expression amount of the U. ohnoi oil administration group decreased compared to the control group, but there was no significant difference in the case of TNF-α.
Histochemical observation. The histological examination of the intestine was evaluated for morphological changes through hematoxylin and eosin (H&E) staining, and observed using an optical microscope. The normal group represents typical colon tissue, but the control group lost crypt and destroyed the nucleus (Fig. 4).
On the other hand, in the U. ohnoi oil-administered group, submucosal infiltration was observed compared to the normal group, but the degree of crypt and tissue damage was lower than that of the control group.

Discussion
This study recognized the superiority of U. ohnoi, a species of green algae known for its immune activity, based on the results of prior research using U. ohnoi extracts to investigate its effects on physiological activity. This study conducted experiments to evaluate the antioxidant, antibacterial activities of the U. ohnoi oil, and measured its anti-inflammatory activity by using a DSS-induced inflammatory bowel disease mouse model. As a result of measuring DPPH radical scavenging activity to evaluate the antioxidant activity of U. ohnoi oil, the radical scavenging activity of A and B U. ohnoi oil was 77.85 ± 5.39% and 86.71 ± 8.36%, respectively, at 0.7 mg/ml, showing the highest activity. In particular, B U. ohnoi oil showed similar activity to the control substance, ascorbic acid. When the total polyphenol was measured, it was confirmed that among the three types of U. ohnoi oil, U. ohnoi oil of B contained the highest polyphenol as 107.45 ± 0 mg GAE/g. This confirms that U. ohnoi oil exhibits a function that can replace krill oil, an animal oil. In addition, the efficacy of antioxidants was repeatedly confirmed through additional experiments. Since the existing oils with excellent antioxidant efficacy are being used in abundance in the food and cosmetics industry, it is possible to industrially use U. ohnoi oil, which has antioxidant properties. Meanwhile, as a result of evaluating the antibacterial activity of U. ohnoi oil using URDA method, B. subtilis and E. coli strains showed strong antibacterial activity, but no antibacterial activity was observed for C. albicans. In the B. subtilis strain, the U. ohnoi oil from B showed higher activity than the U. ohnoi oils from other regions, and there was no significant difference in the C. albicans strain. The change in antibacterial activity was measured by treating six enzymes to investigate the properties of the antibacterial active ingredients of U. ohnoi oil, but there was no significant change compared with before treating enzymes. Ulva ohnoi oil showed some antibacterial activity, and this is a result that can prove the wide utility of U. ohnoi oil. In addition, it can be suggested as one of the many causes of reduced inflammation in the research results related to colitis.
After inducing inflammatory bowel disease with DSS, this study examined the anti-inflammatory effect by administering krill oil, ω-3, and U. ohnoi oil. Compared with the normal group, weight loss and diarrhea were observed in all groups administered with DSS. In terms of proof, the U. ohnoi oil administered group and the normal group produced a similar amount of NO (an inflammation mediator) compared to the control group, indicating the effect of U. ohnoi oil to inhibit NO production. The inhibition of production of TNF-α IL-6, and IL-1β of U. ohnoi oil was measured through ELISA. The production of cytokines increased significantly the control group compared to the normal group, and the production was inhibited in the U. ohnoi group. The exact www.nature.com/scientificreports/ mechanism for the effectiveness of Ulva ohnoi oil to inhibit inflammatory cytokines will need to be investigated through future studies. In case of TNF-α and IL-6, the U. ohnoi oil group showed a significant decrease compared to the control group. In case of the mRNA expression of inflammatory factors (TNF-α, IL-6, IL-1β, and COX-2) in the colon tissue, the U. ohnoi oil group showed similar expression levels to the krill oil administered group. These results support the results of measuring inflammatory cytokines in colon tissue.
As a result of analyzing the inflammatory response of the colon tissue using H&E staining, the group that induced bowel disease with DSS showed crypt loss and inflammatory cell infiltration, but this was mitigated in the mice treated with DSS U. ohnoi. These results show that U. ohnoi oil has a higher anti-inflammatory effect than krill oil in mice with DSS-induced inflammatory bowel disease, and that it was effective in inhibiting inflammatory expression.
Krill oil, which has previously been helping health, needs an alternative material due to the decrease in krill shrimp. It is similar to the efficacy of krill oil, which is the U. ohnoi oil used in this study, and the one-minute efficacy is more beneficial. The U. ohnoi is being multiplied due to the warming of the climate, and the part that can be used in the industry is extremely weak. As the result of this study, it is expected that if the usefulness of U. ohnoi oil is utilized in industry, there will be a synergistic effect of reducing environmental pollution and utilizing biomass. Statistical analysis. Data were analyzed using the predictive analytics software (PASW 18 for Windows) and presented as mean ± S.D. A one-way analysis of variance (ANOVA) test was performed to assess the NO production in mouse plasma and the generation of inflammatory cytokines in the colon. The significance level was set at * p < 0.05.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.