Biological activities of derived pigments and polyphenols from the newly recorded alga Phyllymenia gibbesii

The newly recorded Phyllymenia gibesii in the Mediterranean Sea at Alexandria coast of Egypt is regarded as a significant source of bioactive substances and is applied as an antioxidant, anti-inflammatory, and antimicrobial agent. According to the HPLC chromatograms, the acetone extract of P. gibesii comprised ten photosynthetic pigments (chlorophyll-a, chlorophyll-d, α-carotene, β-carotene, phycocyanin, allophycocyanin, antheraxanthin, β-cryptoxanthin, lutein, and violaxanthin). Total carotenoids were the dominant class in the pigments’ profile, achieving a concentration of 257 g/g dry weight. The P. gibbesii extract had a total content of phenols (146.67 mg/g) and a total content of flavonoids (104.40 mg/g). The capacity of all the investigated biological activities augmented with the concentration of the algal extract. The maximal DPPH scavenging capacity was 81.44%, with an inhibitory concentration (IC50) of 9.88 μg/mL. Additionally, the highest ABTS scavenging capacity was 89.62%, recording an IC50 of 21.77 μg/mL. The hemolytic activity of P. gibbesii attained a maximum capacity of 49.88% with an IC50 of 100.25 μg/mL. Data also showed the maximum anti-inflammatory effectiveness at 81.25%, with an IC50 of 99.75 μg/mL. Furthermore, the extract exhibited antimicrobial capacity against all reference strains, particularly at high concentrations (0.1 mg/mL), with the greatest effect on C. albicans and E. coli.

www.nature.com/scientificreports/food, paper, and textile industries, water technology, and agricultural practices 4 .Interestingly, pigments exhibit advantageous biological activity as antioxidants and anticancer agents, as well as several key features that make them suitable in these various industrial contexts 4 .Therefore, they have a significant potential to meet current market needs, which increasingly focus on the health and biotechnology sectors in search of natural substances having positive effects on human health.However, there are still issues with optimizing and then scaling up extraction and purification processes, particularly when taking into account the needs for both quantity and quality from an industrial and commercial perspective 4 .
In particular, the red alga Grateloupia gibbesii was newly introduced to the Egyptian coast of the Mediterranean Sea 7 .Currently, this species has been identified as a synonym of Phyllymenia gibbesii 8 .Many studies are focused on the alien species in the Mediterranean Sea, which cause many impacts on the introducing area, such as changes in biodiversity, competition between these alien species and native ones 9 .In addition, some of them have the potential to be detrimental to the ecosystem and to other macroalgal species.Thus, it would be of interest to find out valuable substances in the domain of health and nutrition from the biomass of alien species in order to compensate the expense of eradication and as a safe and eco-friendly solution to the problem of introduction.
The basic target of the current study is to investigate several biological activities of different pigments detected in the newly recorded alga Phyllymenia gibbesii and to assess the concentration of these pigments.

Results and discussion
Macroalgae are a significant basis for therapeutically valuable compounds.They are regarded as a source of unique types of biologically active substances used in several domains due to their diversity and chemical differences.Herein, the antioxidant, anti-inflammatory, hemolytic, and antibacterial properties of P. gibbesii acetone extract were evaluated.Its pigments were also examined to further clarify the chemical constituents.
Because of a substance known as sodium-copper-chlorophyllin, chlorophyll has anti-cancer and antioxidant properties.Supplementation with chlorophyllin before meals has been proven to diminish the harm caused by aflatoxin, a highly carcinogenic mold that grows on some foods 10 .In high-risk populations, the use of chlorophyllin may aid in the prevention of liver cancer caused by aflatoxin.Moreover, chlorophyll metabolites, like phytanic acid, can be used to treat and prevent diabetes 11 .Phytanic acid also has an effect on the expression of genes related to glucose metabolism via different natural peroxisome proliferator-activated receptor isoforms 11 .
According to Dasgupta 12 , chlorophyll-a and chlorophyll-d are two of the pigments of red algae.In contrast, Kato and his co-workers 13 mentioned that red seaweeds have only chlorophyll-a.However, the detection of chlorophyll-d is dependent on the choice of solvent for the extraction process 14 .In this trend, Osório and his co-workers 15 investigated the red alga Porphyra sp. for pigment content by using a variety of solvents.Their findings indicated that acetone was the most suitable solvent for the detection of chlorophyll-d, resulting in a maximum of 16.25 μg/g, while it was absent using the N-dimethyl formamide solvent.In the present study, using acetone as solvent gave the opportunity to determine chlorophyll-d in P. gibbesii (25.95 μg/g) and chlorophyll-a (41.65 μg/g).In contrast to our study, Paransa and his co-workers 16 (2020) found that chlorophyll-d was higher (16.36-21.42μg/g) than chlorophyll-a (6.14-18.32μg/g) in Kappaphycus alvarezii.This may be attributed to that chlorophyll-d can support the role of chlorophyll-a in red algae 17 .
Phycobiliproteins (PBP) have many health advantages as antioxidants and free-radical scavengers 4 besides their potent neuroprotective, anti-bacterial 18 , anti-inflammatory 19 , anti-allergic 19 , antitumoral 19 , anti-ageing 19 , anti-Alzheimer 18 , immunomodulatory 18 , and hypocholesterolemic agents 18 .Basically, phycobiliproteins (PBP) are classified into four pigment families according to their absorption properties.Red algae comprise phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC), while phycoerythrocyanin (PEC) is absent in Fig. 6.HPLC analysis of β-cryptoxanthin in P. gibbesii acetone extract.this group 4 .Actually, PBP have been found to be vital in proliferating and adapting the red algae by maximizing the capabilities of algal light-harvesting in the deep layers of the water column 4 .In the present study, PE was not detected by the HPLC.This may be attributed to the methodologies of extraction of the PE.In the present study, the extraction process was based on adding acetone as a solvent to the dry powder alga, followed by HPLC for separation and isolation of pigments, while in many previous different studies, methodologies were used to determine PE based mainly on using different buffer solutions such as phosphate buffer 19 , acetic-acid sodium acetate buffer 20 , and EDTA 21 .The extraction was followed by purification, using many efficient methods like precipitation with ammonium sulfate 22 , gel filtration and ion-exchange chromatography 23 , expanded bed absorption and ion-exchange chromatography 24 .
Considering the other two pigments, allophycocyanin content was higher (41.95 μg/g) than that of phycocyanin (20.60 μg/g) in P. gibbesii.Francavilla and his co-workers 20 reported that PC was the less abundant phycobiliprotein in Gracilaria gracilis, which is consistent with our results.Pina and his co-workers 25 showed that the concentration of PC in Chondrus crispus was 149 μg/gdw, while Sukwong and his co-workers 26 isolated from Gelidium amansii 56 μg/g dw of PC.These differences in PC and APC contents compared with the previous studies may be due to many factors, such as the aforementioned methodologies for extraction, the species, the time of the year when harvesting, or environmental conditions such as light intensity 27 , nutrient cycles, and photoperiod 27 .
Carotenoids in red algae are divided into carotenes (β-carotene and α carotenes) and the major xanthophylls (antheraxanthin, β-cryptoxanthin, lutein, violaxanthin, and zeaxanthin) 28 .Schöbel 29 reported that rhodophytes lack distinctive carotenoid profiles due to either the existence or absence of specific carotenoids, which are constituents in the xanthophyll group.On the other hand, Mimuro & Akimoto 30 noted that the existence of particular biosynthetic routes or the distribution of carotenoids with different molecular structures can be considered an index for algae classification.In this trend, Shubert and his co-workers 6 estimated the carotenoid content of 65 subtropical red algal species from many taxonomic orders and families.The findings demonstrated that there were three main carotenoid profiles identified, with the exception of the Ceramiales and Corallinales.Shubert and his co-workers 6 reported that most of the species had lutein as the major xanthophylls (first profile), while zeaxanthin was the major one in some other species (second profile).The third profile (Xanthophyll Cycle -related carotenoids) showed that violaxanthin or antheraxanthin were also detected in some species.In addition to these major xanthophylls, there are minor ones that depend on the interconversion of the major pigments 31 .
The variation in these trace pigments may be the reason for the unclear relationship between the carotenoid content of red algae and their phylogenetics 32 .The findings of Takaichi 33 synergized with the previous study of Czeczuga 32 , where he reported in five red algal species belonging to different families different profiles of carotenoids than that of Shubert and his co-workers 6 .Czeczuga 32 explained the different pigment profiles on the basis of the presence of additional minor xanthophylls such as γ-carotene, α-cryptoxanthin, lutein epoxide, mutatoxanthin, fucoxanthin, neoxanthin, and others.Our results are in agreement with those of Czeczuga 32 and Takaichi 33 and disagree with those of Shubert and his co-workers 6 .It is also noteworthy to mention that the taxonomic position of the Grateloupia, Phyllymenia, and Prionitis genera remains controversial, and many species Table 1.Pigments content of P. gibbesii acetone extract (μg/g) analyzed by HPLC.

Pigments
Pigments concentration (μg/g) Chlorophylls: Chlorophyll-a 41.65 Chlorophyll are still re-identified or transferred from one family to another.Currently, the species G. gibbesii is considered a synonym of P. gibbesii 8 , which was transferred to the genus Phyllymenia based on the female reproductive structures and other attributes 8 .Thus, it is necessary to screen again the pigment profiles of the members of order Halymeniales, which was previously included in order Cryptomeniales and has recently undergone many taxonomic changes and may show different pigment profiles.
In the present study, the profile of P. gibbesii showed that β-cryptoxanthin was the principle xanthophyll component (42.50 μg/g) forming 42.37%, followed by lutein (31.30μg/g) forming 31.21% of total carotenoids, with antheraxanthin and violaxanthin forming collectively 26.42% and with the absence of zeaxanthin.On the other hand, β-carotene and α carotenes were comparable in P. gibbesii forming (80.6 μg/g) and (76.1 μg/g), contributing 31.36% and 29.61% of total carotenoids, respectively.Thus, the percentage of total carotenes (60.97%) was higher than that of xanthophylls (39.03%).However, the content of the two carotenes is dependent on the lycopene pathway, whether it is mainly cyclized into either β-carotene, or α-carotene or both 33 .Also, the differences between algal species and their geographical distribution, seasonality, environmental conditions, and light regime are controlling factors of carotenoids content 34 .In the present study, total carotenoids content was higher (257.00μg/g) than chlorophyll-a (41.65 μg/g).This is consistent with the findings of Barsanti & Gualtieri 35 , who reported that algal species exposed to high light intensity typically exhibit a relatively high amount of carotenoid pigments in comparison with chlorophyll-a.

Total phenolics and flavonoids contents (TFC)
The acetone extract of P. gibbesii had a total content of phenolics of 146.67 ± 0.61 mg/g and a total content of flavonoid of 104.40 ± 0.10 mg/g (Table 2).
Numerous biological actions, like antimicrobial, anti-diabetic, antioxidant, and anticancer capabilities, are provided by phenolic compounds, which include one or more phenolic rings that may be halogenated 36 .The main classes of phenolics in the red algae are bromophenols, terpenoids, phenyl propanoid derivatives, polymerized hydroxycinnamyl alcohols, and mycosporine-like amino acids 37 .In the current study, phenolic content in P. gibbesii was much higher (146.67 ± 0.61mgGAE/g) than that of Shabaka & Moawad 38 in the same alga during the same season (May, 2019) at the same site (Eastern Harbor) utilizing different solvents (chloroform, ethyl acetate, and distilled water), resulting in 4.745, 6.629 and 3.211 mgGAE/g, respectively.The great difference between the phenolic content of the two algal samples can be attributed to the site of algal collection and its environmental conditions, algae growth stage, sample preparation, and the method used for determination of the phenolic content, in addition to the choice of the extracting solvent, where the acetone in our study showed very high efficiency compared to the other solvents.
The significance of total flavonoid content is mostly due to its redox properties, that could explain its antioxidant, anti-inflammatory, and antibacterial properties towards many microbes 39 .In addition, the antioxidant property of the flavonoid content is attributed to its hydroxyl group structuring, which may be responsible for capturing and stabilizing much more free radicals 39 .However, in the current study, flavonoid content in P. gibbesii was much higher (104.40 ± 0.10 mg CAT/g) than that of Khristi and his co-workers 40 in G. indica (0.0241QUE mg/g) using methanol as extracting solvent, while Ruiz-Medina and his co-workers 5 found that flavonoid content was 1.44 mg CAE/g in G. imbricata using 80% methanol.This may be attributed to using different methodologies and different solvents and other limitations like species of algae, harvesting season, and environmental conditions 37 .

Biological activities of P.gibbesii extract
The results of the biological activities of the newly recorded alga P. gibbesii are shown in (Tables 3-5).www.nature.com/scientificreports/

Antioxidant activity
The DPPH was a significant metric for assessing the antioxidant activity of an extract.Also, the ABTS assay has been extensively used to test the ability of different extracts, fractions, and/or pure substances to scavenge free radicals.

Activity of DPPH radical scavenging
The results indicated that increasing the extract concentration boosted the DPPH' s capacity (Table 3).Knowingly, the better the antioxidant properties, the smaller the value of IC 50 .Indeed, it was 9.88 ± 0.01 g/mL for the P. gibbesii, while it was 3.44 ± 0.01 g/mL for the positive control (L-ascorbic acid) (Table4).

ABTS radical scavenging assay
The results demonstrated that, like the DPPH, increasing the concentration of the acetone extract of P. gibbesii enhanced the scavenging activity scored by the ABTS assay (Table 3).Moreover, the P. gibbesii acetone extract showed ABTS activity with an IC 50 value of 21.77 ± 0.01 μg/mL, corresponding to7.73 ± 0.01 μg/mL for L-ascorbic acid (Table 4).Antioxidants are efficacious in safeguarding cells against ROS-mediated oxidative damage, particularly under unfavorable conditions 41 .Several synthetic antioxidants are commercially available, but due to safety concerns, there has been a lot of interest in switching to natural plant-based antioxidants 41 .The current data showed high DPPH radical scavenging activity in P. gibbesii, with a maximum of 81.44%, which was higher than that of G. indica, showing a maximum of 69.90% 40 .Similarly, the ABTS assay showed high activity, with a maximum of 89.62%.Noticeably, the free radical scavenging capacity increased with increasing the concentration of the algal extract.This scavenging capacity may be due to the hydrogen-donating ability of the antioxidant properties of P. gibbesii 42 .This might be interpreted by the view that there is a positive correlation between pigment concentration and the antioxidant activity of the algae species.Increasing pigment production is induced by increasing light intensity, which in turn boosts antioxidant activity 28 .Furthermore, according to Zhang and his co-workers 43 , the antioxidant activity of seaweeds may be influenced by the presence of flavonoids and polyphenols components.However, their concentrations affected the activity potential.
Hemolysins are lipid and protein substances that cause the erythrocyte to burst and release hemoglobin, harming a number of vital organs, including the heart, liver, and kidney 44 .Moreover, hemolysis may result in anemia or jaundice.As a result, while testing the biological activities of algal extracts and derivatives for pharmaceutical purposes, it is necessary to investigate the hemolytic activity 45 .

Invitro anti-inflammatory activity
The anti-inflammatory activity of P. gibbesii extract at different concentrations (10, 20, 40, 80, and 100 μg/mL) is represented in (Table 3).This extract exhibited a lower IC 50 value of 99.75 ± 0.05 μg/mL compared with that of the commercial Diclofenac (331.24 ± 0.04 μg/mL) (Table 4).The development of many severe diseases has been linked to complicated biochemical events that distinguish the inflammation process 2 .The anti-inflammatory activity of the extracts obtained from different red algae species has been confirmed by several studies (e.g., Chen and his co-workers 47 ).Within the current study, theIC 50 value of the algal extract (99.75 ± 0.05 μg/mL) was much lower than that of the commercial diclofenac (331.24 ± 0.04 μg/mL).This showed that P. gibbesii is of great potential to develop natural sources of antioxidants that may help in combating inflammatory diseases.www.nature.com/scientificreports/

Antimicrobial activity
All of the examined microorganisms were susceptible to the effects of P. gibbesii acetone extract, particularly at the highest concentration (0.1 mg/mL) (Table 5).The extract had the greatest impact on the Gramme-negative bacterium E. coli and the yeast C. albicans.Nowadays, the bioactive compounds derived from seaweed metabolites have been documented as antiviral, antibacterial, antifungal, and antiprotozoal agents 48 .Abu-Ghannam & Rajauria 49 explained the antimicrobial mechanism in view of carotenoids role, which causes the accumulation of the immunological enzyme lysozyme that breaks down bacterial cell walls.The antimicrobial activity of P. gibbesii acetone extract was promising, as all the tested microorganisms were inhibited to varying degrees.This was confirmed by the minimum inhibitory concentration (MIC) (0.025 mg/L), which showed the low resistance and the high sensitivity of these microorganisms to the algal extract, particularly E. coli and C. albicans.Moreover, the efficiency of the algal extract augmented with the high concentration.In this trend, Abdel-Latif and his co-workers 50 carried out a study on Grateloupia doryphora, which exhibited a wide range of antimicrobial action towards the investigated microbial species, namely Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, and C. albicans.Obviously, P. aeruginosa was the most affected microbe, with higher inhibition zones (30, 28, and 27 mm) for methanolic, ethanolic extracts and ethyl acetate, respectively.In addition, Negm and his coworkers 51 prepared silver nanoparticles from Grateloupia sp., showing activity versus E. coli, S. faecalis, S. aureus, P. aeruginosa, and V. damsela.Moreover, Hajri and his co-workers 52 studied the antibacterial, among other biological activities, of the aqueous extract of Grateloupia sparsa in the formula of cobalt oxide nanoparticles (Co 3 O 4 NPs).Amazingly, their data showed that Co 3 O 4 NPs had stronger antibacterial activity than Ciprofloxacin, a common antibiotic.

Conclusion
Recently, the red alga P. gibesii was recorded in the Mediterranean Sea of the Egyptian coast.Herein, it was investigated for phytochemical content and pigments' profiles.The P. gibesii chromatograms showed ten individual photosynthetic pigments: chlorophyll-a, chlorophyll-d, α-carotene, β-carotene, phycocyanin, allophycocyanin, antheraxanthin, β-cryptoxanthin, lutein, and violaxanthin.The results of P. gibbesii acetone extract showed many biological activities, including antioxidant, anti-inflammatory, and antimicrobial activity, which confirmed their potential biochemical components.As a result, the alga might be seen as a potential source of bioactive substances that can be applied in several industries, especially pharmaceutical, biotechnological, and nutraceutical.This gives the opportunity to recycle this alien species in a safe and eco-friendly manner.

Area of the study
The algal samples of the newly recorded P. gibbesii were collected from the Eastern Harbor, Alexandria, Egypt, during spring (May, 2019).The algal samples were collected at a depth of 0.5-1 m at the Scout Club, which is situated in the Eastern Harbor at 31°13.3`N latitude and 29°53.10`Elongitude.

Sample collection
The algal samples were manually collected, then rinsed in seawater at the site of collection to get rid of the adherent sediments and other impurities before being placed in clean plastic bags.The samples were kept in an ice box at 4 °C.Upon arrival at the lab, algal samples were promptly rinsed with tap water to remove any remaining epiphytes and contaminants.Rodrguez-Prieto and his coworkers 7 collected the species from the Eastern Harbor during the same period of the current study (May-June, 2019) and identified the newly introduced alga as Grateloupia gibbesii Harvey by using the rbcL gene and provide a detailed description of the morphology, anatomy and in details the pre-and post-fertilization stages of the female reproductive structures.The phylogeny based on the chloroplast-encoded rbcL gene of Grateloupia sensu lato clusters the Grateloupia samples from Alexandria, Egypt, with a single sequence of G. gibbesii Harvey from Charleston, South Carolina.The species belongs to the Class Florideophyceae, Order Halymeniales, Family Grateloupiaceae, and is currently considered to be the synonym of Phyllymenia gibbesii (Harvey) 8 .
About 250 g of the alga was air dried to a consistent weight at ambient temperature, and then the algal sample was chopped into tiny chunks and ground to a fine powder for the next procedures.www.nature.com/scientificreports/

Preparation of algal extract
A weight of 100 g of algal powder was macerated in 400 mL of acetone for 48 h.In a rotating evaporator (Stuart RE300), the acetone was filtered and concentrated at 40 °C under pressure.The acetone extract was then stored at −20 °C for further analysis 53 .
Phycobiliproteins (phycoerythrin and phycocyanin) were analyzed by HPLC by using a UV detector set at detection wavelengths: 260 nm under isocratic conditions, which included the following: mobile phase-acetonitrile/methanol in the ratio of 80:20, respectively.The flow rate was 1 mL/min at a temperature of 25 °C.
Allophycocyanin and lutein were analyzed using an HPLC system with an Echromatech Mod.C1620, column 18, by using a detector set at 620 nm.The isocratic conditions were a Na-phosphate buffer (pH 3.0) as the solvent (A) and NaCl and methanol as the solvents (B) in the ratio of 45:55, respectively.The flow rate of the process was 0.5 mL/min at a temperature of 25 °C.
Carotenoids (α-carotene, β-carotene, and zeaxanthin) were assessed using an HPLC with a diode array detector and data collected by a Chemstation.Sample (20 mL) was loaded on an ODS-1 reversed phase column (250′4.6 mm i.d., 5 mm particle size).Acetonitrile, 2-propanol, and water were eluted isocratically in the following proportions: 39:57:4.The detection wavelength was 450 nm, while the detection flow rate was 1 mL/min at a temperature of 25 °C.
Antheraxanthin analysis was carried out by HPLC analysis using the system echromatechMod.C1620, column C18, with a detector set at detection wavelengths of 490 nm, and under isocratic conditions, with the mobile phase made up of methanol, water, acetonitrile, and dichloromethane in the ratio of 70:4:13:13.The temperature was 25 °C, and the flow rate was 1 mL/min.The conditions were the same for β-cryptoxanthin, except a detector set at 450 nm was used, and the mobile phase was methanol/acetone in the ratio of 70: 30.The process detected violaxanthin using a 450 nm detector under isocratic conditions with mobile phase (A) acetonitrile/water in a ratio of 90:10 and mobile phase (B) ethyl acetate.The flow rate was 1 mL/min, and the temperature was 25 °C.
Different standards for each pigment were utilized to prepare its calibration curve for quantification.All standards were bought from Sigma and were 95% pure (HPLC grade).The retention times and spectral data were compared to those of the authentic standards in order to identify the HPLC peaks.

Determination of total phenolic content (TPC)
According to Dewanto et al. 54 , the Folin-Ciocalteu reagent was used to determine the total phenolic content.200 μL of the dissolved sample (one mg of extract after being dissolved in one mL of deionized water) was added to 600 μL of distilled water and 100 μL of Folin-Ciocalteu reagent.Before 2% Na 2 CO 3 was added, the mixture was agitated and allowed to stand for 6 min.A final volume of 3 mL was adjusted with distilled water, which was then well mixed.The absorbance at 650 nm was measured in comparison to the blank produced after 30 min of incubation in the dark.A standard curve was produced utilizing various gallic acid concentrations (standard, 0-100 μg /mL).Based on the calibration curve, the following linear equation was used to compute the total phenol content (TPC), which was expressed as gallic acid equivalent (GAE)/mg of dry weight: y = 0.0095x-0.0409& R 2 = 0.9953.Where x is the concentration (GAE mg/g extract), y is absorbance, and R 2 is the correlation coefficient.

Determination of total flavonoid content (TFC)
Total flavonoids of the algal extract were estimated using a modified colorimetric method published by Sakanaka et al. 55 Catechol was used as a standard at concentrations of 20-200 μg /mL.Precisely, 250 μL of extract or standard solutions were added to 1.25 mL of distilled water, 75 μL of 5% sodium nitrite solution (NaNO 2 ), and 150 μL of 10% aluminium chloride solution (AlCl 3 ) five minutes later.0.6 mL of distilled water and 0.5 mL of 1 M sodium hydroxide (NaOH) were added after 6 min.Afterward, the mixture was shaken well, and the absorbance was determined at 510 nm.Based on the calibration curve, the following linear equation was used to assess total flavonoid content (CE): Y = 0.0079 x−0.109 & R 2 = 0.986.Where R 2 is the correlation coefficient, X is the concentration (CE mg/g extract), and y is absorbance.

DPPH radical scavenging activity
The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique, developed by Brand-Williams et al. 56 , was modified to test the algal extract's capacity to scavenge free radicals.One milliliter of the radical solution (0.2 Mm DPPH in methanol, 0.0078 g/100 mL) was added to 1 mL of the sample, or standard solution, at various concentrations (1:1, v/v).The mixture was incubated at room temperature in the dark for 30 min, and the absorbance was detected at 517 nm.To create a calibration curve, the ascorbic acid solution was employed as the standard with a concentration range of 5-200 μg/mL.However, DPPH radical scavenging activity was qualified as mg ascorbic acid equivalent (AAE)/g dried sample.Using the following equation, the percentage of DPPH radical-scavenging activity was determined: www.nature.com/scientificreports/All reagents were added to the control sample, with the exception of the algal extract.All measurements were made in triplicate, and the average result was tabulated.

ABTS radical scavenging assay
The assay of 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid; ABTS) was used to determine the ABTS radical scavenging activity 57 .The extract's ability to scavenge ABTS was compared with that of dibutyl hydroxytoluene (BHT), and inhibition% was calculated as follows: Where, Abs.sample is the absorbance of ABTS radical + sample extract/standard, whereas Abs.control is the absorbance of ABTS radical + methanol.

Hemolytic activity assay
The experiment was carried out in 2 mL microtubes via a modified method of Farias et al. 58 .First, a two-fold serial dilution of each extract with 0.9% NaCl between 1000 and 1.9 μg/mL was made and set aside.Afterward, 900 μL of each extract dilution were placed in a new microtube, and 100 μL of a 1% red blood cell suspension (A, B, and O human blood types or rabbit blood) were added.The microtube was then incubated at 37 °C for an hour.All tubes were centrifuged for 5 min at 3,000 × g.A 96-well plate containing the supernatant (200 μL) was connected to a microplate reader to measure different absorbances at 540 nm.The cell suspensions of each human blood type (100 μL) were mixed with distilled water or 0.9% NaCl (900 μL).The following is how the percentage of hemolysis was determined: Hemolysis% = Abs test /Abs pc × 100.
Where Abs pc = Abs 540 of the 1% cell suspension treated with distilled water and Abs test = Abs 540 of the 1% cell suspension treated with a sample test.

Anti-inflammatory activity assay
Using a few minor adjustments, the human red blood corpuscles (HRBCs) membrane stabilizing method 59 was used to evaluate the extracts' anti-inflammatory activity in vitro.Blood was drawn from a healthy volunteer without using any inflammatory medications for the 14 days prior to such an experiment.The blood was then transferred to heparinized centrifuge tubes and spun at a speed of 3000 rpm.After being cleaned with isosaline, the packed cells were converted into a 10% suspension in regular saline.As a reference, diclofenac potassium (50 μg/mL) was employed.About 5 mL of the reaction mixture includes 1 mL of 0.15 M phosphate buffer (pH 7.4), 1 mL of test solution (1 mg/mL) in normal saline, and 0.5 mL of 10% HRBC in normal saline, together with 2 mL of hypotonic saline (0.25% NaCl).One molesters of isotonic saline was adjusted as the control instead of the test solution.The mixtures were incubated for 30 min at 56 ºC, cooled under running water, and then centrifuged for 20 min at 3000 rpm.Using a visible spectrophotometer, the supernatant's absorbance at 560 nm was measured.The experiment was carried out three times.100% of lyses are represented by the control.The following calculation was used to compute the percentage of membrane stabilization: Protection% = 100−(OD of test/OD of control) × 100.
By graphing the percentage of inhibition with respect to the control versus the treatment concentration, the extract/drug concentration for 50% inhibition (IC 50 ) was revealed.

Antimicrobial activity assay
Reference microbial species, including Klebsiella pneumonia ATCC700603 and Escherichia coli ATCC25922, were used as Gram negative bacteria.In addition, Staphylococcus aureus ATCC25923 and Streptococcus pyogenes EMCC1772 were used as Gram-positive bacteria.Moreover, one yeast strain (Candida albicans EMCC105) was used.For these pathogens, the antimicrobial effect of P. gibbesii extract was evaluated.
The yeast strain was cultivated in potato dextrose broth at 28 °C for 24 h, while the bacterial strains were grown in nutritional broth in g/L at 37 °C.A sterile 100 mm diameter Petri plate was then filled with 100 μL of the inoculums (1 × 10 8 cfu/mL) and inculcated on nutrient agar medium in g/L.The well-cut diffusion method was applied to detect the antibacterial activity 60 .The wells were prepared in the plates by utilizing a cork-borer (0.5 cm) in nutrient agar plates inoculated with the reference microbes.The algal extract was added with a volume of 100 μL to each well.All tested bacteria were incubated for 24 h at 37 °C, while C. albicans EMCC 105 was incubated at 28 °C for 3-5 days.To ascertain the minimum inhibitory concentration (MIC), P. gibbesii extract was used at three concentrations (0.025, 0.05, and 0.1 mg/mL).The diameter of the inhibition zone surrounding the well (mm) was measured in three triplicates, and the average values were estimated.

Table 2 .
Total phenolics and total flavonoids in P. gibbesii.

Table 5 .
Antimicrobial activity of acetone extract of P. gibbesii.MIC is abbreviated to minimum inhibition concentration (mg/mL).ND not detected.**Diameter includes 5 mm well diameter.