RESEARCH ON PHLOROTANNIN COLLECTION FROM BROWN ALGAE (SARGASSUM SERRATUM) IN NHA TRANG AND ASSAY APPLY TO ANTIOXIDANT BEVERAGE

NHA TRANG UNIVERSITY --- Dang Xuan Cuong RESEARCH ON PHLOROTANNIN COLLECTION FROM BROWN ALGAE SARGASSUM SERRATUM IN NHA TRANG AND ASSAY APPLY TO... Therefore, a study of “Research on

NHA TRANG UNIVERSITY -

Dang Xuan Cuong

RESEARCH ON PHLOROTANNIN COLLECTION

FROM BROWN ALGAE (SARGASSUM SERRATUM) IN

NHA TRANG AND ASSAY APPLY TO

Thesis was completed in:

Nha Trang university and Insititute of Technology Application and Research

Science instructor:

1 Dr Vu Ngoc Boi

Nha Trang university

2 Ass Pro Dr Tran Thi Thanh Van

Insititute of Technology Application and Research

Reviewer 1: Prof Sci Dr Nguyen Trong Can

Reviewer 2: Assoc Dr Phung Thi Thanh Tu

Reviewer 3: Assoc Dr Nguyen Anh Tuan

Thesis will be defened at the doctor thesis assessment committee of Nha Trang university ………

At …………hours ……… Date …… ……… ………

The thesis can be found in:

The library of Nha Trang university or National library

INTRODUCTION

1 Necessity of thesis

Brown algae (Phaeophyta) is bioactive-rich algae such as lamilaran,

fucoidan, alginate, phlorotannin, … The substances have many bioactives, inside, antioxidant, antibacterial, antifungal,… is being interested Brown

algae phylum (Phaeophyta) has many families as: Alariaceae, Fucaceae and Sargassaceae

In Vietnam, brown algae genus (Sargassum) belong to brown algae

family (Sargassaceae) The genus has large output and high value on

medicine because they consists of many bioactive substances as fucoidan, laminaran, alginate, phlorotannin,… Inside, a noteworthy biocompound is phlorotannin – a compound of secondary metabolism belong to phenolic group Phlorotannin has different bioactives such as: antioxidant, antibacterial, antifungal, antitumor, anticancer,… Antioxidant activity of phlorotannin is one of the most extensively studied activities and phlorotannin was known as non-toxic and safe antioxidants for human healthy Thus, phlorotannin can be exploited and used to support free radical removal and health improvement for human body Thence, phlorotannin of brown algae was interested by researchers

Nha Trang sea has abundant and diverse resource of brown algae

Sargassum in numerous and category Alternatively, brown algae in Nha

Trang sea contains a high content of bioactive substances as fucoidan,

phlorotannin, alginate,… Therefore, a study of “Research on

phlorotannin collection from brown algae (Sargassum serratum) in Nhatrang and assay apply to antioxidant beverage” is necessary

2 The target of the thesis:

- Research on antioxidant phlorotannin collection from brown algae

(Sargassum serratum) collected in Nhatrang sea, Khanhhoa

- Assay apply phlorotannin into the antioxidant beverage

3 The object and scope of research

3.1 The object of research

Brown algae (Sargassum serratum) growed in Nhatrang sea was

collected, cleaned by using salt water, dried until the humidity of 19%, storaged in the room temperature and used for the research of phlorotannin

collection

3.2 The scope of research: contain 5 parts

1) Extraction and optimization study on phlorotannin collection from brown algae (Sargassum serratum) grown in Nhatrang sea, Khanhhoa 2) Initial fraction and purification of phlorotannin collected from brown algae (Sargassum serratum)

3) Study on a spray drying of brown algae S Serratum extract to receive phlorotannin powder

4) Evaluation of a toxic of phlorotannin obtained from brown algae S serratum

5) Assay apply phlorotannin to antioxidant beverage

4 The method of research

The standard methods of the world and Vietnam used for the study of

phlorotannin obtainment from brown algae (Sargassum serratum) were

utilized in the thesis The mathematic methods were also exploited to discovery new rules, natures, and a relation between the quantity and hyppothesis verification in the thesis

5 The new contribution to the science

- This is the first time the comprehensive research on phlorotannin of

brown algae (Sargassum serratum) harvested at Nhatrang sea, Khanhhoa

was done The research was from a optimization of the extraction step to purification, assay of the toxic on mice, spray drying to create high antioxidant phlorotannin powder and test a production of antioxidant phlorotannin beverage Thence, the research results of thesis have high significance in science These results contribute to affirm the value of brown algae resource of Vietnam Alternatively, the result of thesis will be the reference to serve the study and teach-in the field of active element of

marine seaweed

- The thesis results will be the basis for development and commercialization of some products originated from brown algae

(Sargassum serratum) of Nhatrang sea at company of food production

6 The structure of thesis

The thesis contains 171 pages, inside, a review of 34 pagas, a study method of 22 pages, a result of 89 pages, a conclusion of 2 pages, a references of 24 pages and a appendix

CHAPTER I OVERVIEW

1.1 INTRODUCTION OF BROWN ALGAE

Brown algae is a bioactive abundant and bioactive element – rich resource, as antioxidant, antifungal, antibacterial, anticoagulant and anti-

radiation, healing effect and cell regeneration (Kang et al., 2003) In fact, Khanhhoa province has 21 brown algae species, inside, Sargassum serratum

species is large distribution, great reserves and new species, they simultaneously contains many bioactive compounds such as fucoidan, phlorotannin,… but they have never been fully studied

1.2 INTRODUCTION OF PHLOROTANNIN

Phlorotannin is one of strong antioxidant compounds originated from

brown algae (Ahn et al (2007); Kang et al (2003); LIM (2002); Kuda

(2007) and Shibata (2008)) Phlorotannin contains basis units of phloroglucinol (1,3,5-trihydroxybenzene), and their molecular size oscillate between 126 Da and 650 kDa (Singh et al (2006)), and characteristic linkages of phlorotannin structure are the linkage of ether, phenyl, ether and phenyl, dibenzodioxin (La Barre et al (2010)) It is very interesting, a content, structure and antioxidant mechanism of polyphenol/ phlorotannin

in brown algae closely related to antioxidant activity Phlorotannins is a phenolic compound and their nature is polymer of phloroglucinol They

exist in many families of brown algae phylum, such as: Alariaceae, Fucaceae and Sargassaceae Thus, phlorotannin is worth bioactive agent to

study

1.3 SITUATION OF PHLOROTANNIN STUDY IN THE WORLD AND VIETNAM

* Situation of phlorotannin study in the world

Many studies showed that, phenolic compound in brown algae is

phlorotannin (Jormalainen et al (2004); Koivikko et al (2007); Swanson et

al (2002)) Siti A Budhiyanti et al (2012) published free radical scavenging (DPPH) oscillated from 14.61 to 48.71% and 0.17 to 44.05%, respectively, when extract of cell membrane and cytoplasm has a concentration of 0.45 mg/ml; reducing power activity vacillated conforming 2.05 to 12.51% and 26.77 to 68.80%, when extract of cell membrane and

cytoplasm of some Sargassum species harvested in Indonesia sea has the

concentration of 0.45 mg/ml, all extract consist of phlorotannin EC50 oxidant index performed DPPH free radical scavenging activity of species

S ramifolium and S pteropleuron was from 6.64 to 7.14 mg/ml (Mayalen Zubia et al., 2007) Free radical scavenging activity of species S thunbergii and S fulvellum was 97.41% and 84.66%, respectively, was simultaneously

showed by Soo Jin Heo et al (2005) Strong antioxidant activity of purify phlorotannin originated from some brown algae species has closely relation

to skeleton molecular (Ahn et al 2007) A activity and vitality of

phlorotannin is diverse in different species of Sargassum in the worldwide,

therefore All over the study and a announcement of Barry Halliwell (2001) performed, phlorotannin has a minimizing power of cell damage under a impact of free radicals such as: superoxide, hydroxyl, peroxyl, alkoxyl, hydroperoxyl, nitric oxide and nitrogen dioxide The thing was also evidenced by Naja et al., 2012, (.OH) and (O2-) radical scavenging ability of

S ringgoldianum species got 78% and 88.4%, respectively, and free radical scavenging activity (DPPH) of S vulgare species was 0.2% ÷ 20.3% Total antioxidant activity of S pallidum species also oscillated from 0.04 ± 0.02

to 52.08 ± 0.02µmol FeSO4/mg (Hong Ye et al., 2009)

* Situation of phlorotannin study in Vietnam

Early studies of antioxidant phlorotannin extracted from Vienamese brown algae were announced in the years 2009 However, first publications

about antioxidant activity of algae species grown in Nhatrang sea - Khanhhoa, Vietnam were announced in the years 2010 and the publications were noticed by the group of author

Over analysis shows that it have not any publication of extraction, optimization, spray drying of powder preparation, application, determination of phlorotannin structure or screening and evaluation of phlorotannin content and reserve according to a crop and growing time of each parts of algae or storage time of brown algae Sargassum serratum grown in Nhatrang sea, Khanhhoa, Vietnam Therefore, the thesis focus on over researchs

CHAPTER II MATERIAL AND METHOD OF RESEARCH 2.1 Material

Brown algae (Sargassum serratum) belong to: phylum (Phaeophyta), class (Phaeophyceae), order (Fucales), family (Sargassaceae), genus (Sargassum)

Brown algae (Sargassum serratum) grown in Nhatrang sea - Khanhhoa

was collected, cleaned by using a salt water, dried until the humidity of 19% and storaged in a room temperature to use in the research

2.2 The method of research

2.2.1 The analysis method

2.2.1.1 The quantification of phlorotannin: determined according to Swanson et al 2002 Phloroglucinol was used as a standard

2.2.1.2 Determination of antioxidant activity

+ Determination of total antioxidant activity (TA): quantified in

accordance with Prieto et al (1999) Acid ascorbic was used as the standard

+ Reducing power activity (RP): evaluated by the method of Zhu et al

(2002) The standard of FeSO4

+ (DPPH) free radical scavenging activity: measured on Blois M S (1958)

Absorbant value was measured by using UV-Vis Spectrophotometer JenWay 6400/6405 machine

2.2.1.3 Determine of colour degree of beverage: determined by the

method of UF treatment of Neslihan et al (2005)

2.2.1.4 Quantification of other compositions

- Carbohydrate content: quantified by the method of AOAC (1990)

- Pb content: measured on the Vietnamese standard of 7602:2007-Food

- Citric acid content: determined according to AOAC (1932)

- Sucrose sugar: explained by AOAC (2000)

2.2.2 The method of purification

Each fraction is analysed by thin layer chromatography and the solvent system of elution is chloroform: methanol: acid formic (90: 9: 1) Phlorotannin have been detected with 5% FeCl3 reagent (5% FeCl3 in 96% ethanol) The purification of sample on thin layer chromatography was tested by LC/MS and phlorotannin analysis according to the method of Swanson et al The most purificatory fraction of phlorotannin was loaded onto the Sephadex LH 20 column and the solvent system of chloroform : methanol : acid formic was used to elute, the ratio of 90 : 9: 1, respectively The fraction of purificatory phlorotannin got from LH 20 column was analysed by 1H and 13C NMR spectroscopy (mobile phase contains: A phase of H2O and 0.1% formic acid, B phase of acetonitril and 0.1% formic acid) The analysis method of HPLC-MS, recognition of element by using a m/z score

- A gradient programme run LC/MS as follow:

Table 2.1 Gradient programme on LC/MS

2.2.3 The method of sensory evaluation: theo phương pháp cho điểm

theo tiêu chuẩn Việt Nam 3215-79 với thang điểm 20 để đánh giá chất

lượng cảm quan của sản phẩm

2.2.4 The method of bacterial quantification

- Determine of total aerobic bacteria (KL/ ml) according to the Vietnam standars of 4884:2005

- Deterimine of E coli according to the Vietnamese standars of

2.3 Diagram of general experiment

Layout of general experiment of studying process was shown in fig 2.1

Fig 2.1 Diagram of general experiment layout

+ Degree of colour (for beverage)

+ Microorganism (for storage of

beverage)

Dialysis

Purification

Spray drying phun

Phlorotannin powder

Storage

Based on diagram of general experiment layout, suitable factors of each studying step were established

2.3 CHEMICAL AND ACCESSORIES

2.3.1 Chemical

- Chemicals were used for the study: HCl, Na2CO3, H2SO4, sodium phosphate, ammonium molybdate, đệm phosphate, K3[Fe(CN)6], CCl3COOH, FeCl3, the standard of FeSO4 and ascorbic acid, phloroglucinol,…, DPPH, ethanol, n-hexan, ethyl acetate, chloroform, All chemicals were pure and provided by Merck – Germany and Sigma - American 96% ethanol of Vietnamese

- Addition of food: citric acid, ascorbic acid, carrageenan, sacharose, were pure additions and used for the medicine and food All additions were provided by Merck – Germany

2.3.2 Accessories

Accessories of an analysis chemical and technology development laboratary of Nhatrang Institute of Technology Development and Research and a lab of biotechnology – a centre of practise analysis – Nhatrang university were used to obtain: thermostat equipment - Memment (Germany), machine of pH HANA (American), equipment of vacuum concentration (Germany), the machine of spray drying Bucchi - Thụy Sỹ, máy so mầu UV- VIS - American,…

2.4 DATA ANALYSIS

Data analysis and estimation of surface by using a software of Design Expert 8 trial, Nemrodw and Excell Each experiments was repeatted 3 times Unusual value was removed by using Dulcan method

CHAPTER III RESEARCH RESULTS AND DISCUSSION

3.1 PHLOROTANNIN EXTRACTION FROM BROWN ALGAE

S SERRATUM

3.1.1 Determine of extraction solvent of phlorotannin

The results showed the effect of extracting solvent on phlorotannin content (Phc) and antioxidant of extract is very strong In 2 extracting methods (reflux and maceration), Phc and antioxidant activity of different extract decrease in the following order: ethanol > acetone > ethyl acetate > chloroform > n-hexan The results showed the effect of maceration method

is better than that of reflux method This thing exhibite Phc and antioxidant activity is extracted more when the maceration method was used In the method of reflux, phlorotannin content, the activity of total antioxidant (TA) and reducing power (RP) of extract only got 98.19%, 91.12% and 93.89%, respectively, compared to the method of maceration

Thus, ethanol was choiced as the extracting solvent and the maceration method was used to extract phlorotannin from brown algae

3.1.2 Determine of the concentration of ethanol

The results noticed 96% ethanol was collected to extract phlorotannin

from brown algae S serratum, Phc, TA, and RP of collected extract is the

highest ANOVA and regression analysis showed the significant difference between the target functions (Phc, TA, and RP) in various conditions is exhibited and their close correlation is also detected (R2 > 0.8) In addition,

a changes of these target functions is exhibited according to the non-linear

function of level 2 Thus, 96% ethanol was used to extract phlorotannin from brown algae S serratum

3.1.3 Determine of the ratio of solvent-to-material

Phc, TA and RP of extract got the highest value and 4.102 ± 0.005mg phloroglucinol equivalent /g dry weight (DW), 4.279 ± 0.001 mg ascorbic

acid equivalent /g DW and 24.228 ± 0.014 mg FeSO4 equivalent /g DW, respectively, when phlorotannin was extract by 96% ethanol and the

DM:NL ratio of 40:1 (v/w) from brown algae S serratum If the ratio of

solvent-to-material of 10:1 (v/w) was used to extract, Phc, TA, and RP of extract only got 57.80%, 73.63%, and 86.50%, respectively, compared to the solvent-to-material ratio of 40:1 (v/w) If the solvent-to-material score

of 30:1 (v/w), Phc, TA, and RP of extract only got 86.57%, 96.79%, and 86.89%, respectively, in comparation to the ratio of solvent-tot-material of 40:1 (v/w) When the solvent-to-material score of 50/1 (v/w) was used, Phc,

TA, and RP was 96,32%, 96,61%, 96,65%, respectively, compared to the ratio of solvent-to-material of 40:1 (v/w) Consequently, the ratio of solvent-to-material of 40:1 (v/w) was choiced to study the phlorotannin

extraction from brown algae S serratum

3.1.4 Determine of the temperature of extraction

The analysing results of ANOVA and regression showed phlorotannin content and antioxidant activity of the extract get the highest value when

phlorotannin was extracted from brown algae (S serratum) at the

temperature of 500C Therefore, the temperature of 50 0 C was choiced to extract antioxidant phlorotannin from brown algae

3.1.5 Determine of the time of extraction

The analysing results noticed a change of Phc on the extracting time exhibited in non-linear function of level 2, the results is also similar in the publication of Dang Xuan Cuong, Tran Thi Thanh Van, Vu Ngoc Boi, Bui

Minh Ly (2014) about the phlorotannin extraction from brown algae S mcclurei [10] RP of extract on various extracting time is a statistical

signification and a good correlation between the extracting time and TA is happened Moreover, a close relation between Phc, TA and RP is found, a relation of Phc and TA is stronger than other relations The relation is

exhibited through the changes of phlorotannin on the extracting time When the extracting time is enough, diffusion of phlorotannin from brown algae outgoing is the highest When the extracting time is too long, phlorotannin

be destroyed and phlorotannin content was decreased The results showed Phc, TA and RP of extract get the highest value in the extracting time of 32 hours Hence, the extracting time of 32 hours was choiced to extract phlorotannin and antioxidant from brown algae S serratum

3.1.6 Determine of pH of extracting solution

The analysing results of ANOVA and regression showed a positive correlation between pH of extracting solution and antioxidant activity of extract is exhibited (R > 0.9) In addition, a strong correlation between Phc and TA was detected (R2 = 0.95) and the correlation is changed according

to the non-linear function of level 2 in the strong effect of solvent pH A normal correlation between Phc and RP is also found (R2 = 0.65) The results is similar in the publication of Dang Xuan Cuong, Tran Thi Thanh Van, Vu Ngoc Boi, Bui Minh Ly (2014) about the phlorotannin extraction

from brown algae S mcclurei

The results of over analysis showed that solvent of pH 7 is suitable to

the extracting process of phlorotannin from brown algae S serratum Thus, solvent of pH 7 was selected to extract phlorotannin from brown algae S serratum

From over studies announced some suitable factors of antioxidant phlorotannin extraction from brown algaae Sargassum serratum were choiced as the basis condition of processing optimization of phlorotannin The condition is as follow: the temperature of 50 0 C, the time of 32 hours, 96% ethanol solvent, the ratio of solvent-to-material of 40:1 (v/w)

3.2 OPTIMIZATION OF EXTRACTING PROCESS OF PHLOROTANNIN USING BOX-BEHNKEN MODEL

After some conditions of phlorotannin and antioxidant activity

extraction from brown algae S serratum were determined The experiments

of step optimization of phlorotannin from brown algae S serratum were

designed and inlet factors such as: the temperature (U1: 24 - 600C), the time (U2: 16 - 42 giờ) and the ratio of solvent-to-material (v/w) (U3: 10 - 50) Target functions were phlorotannin content (mg phloroglucinol euquivalent

/g), Y2: total antioxidant activity (mg ascorbic acid equivalent /g), Y3: reducing power activity (mg FeSO4 equivalent /g) The opitimal result was performed in table 3.1 and 3.2, fig 3.1

Table 3.1 The optimal results of extracting step of phlorotannin from

brown algae S serratum using Box-behnken model

43 33 27:1 34.00 58.60 71.50 80.32 82.11

43 33 27:1 33.70 57.40 69.90 79.70 82.59

43 33 27:1 36.00 59.80 70.23 81.40 81.30

Note: DPPHtb: DPPH free radical scaevenging activity

The optimal result is suitable for the data of experiment tested in the

extracting step of antioxidant phlorotannin from brown algae Sargassum serratum From the result, the optimal condition of phlorotannin collection from brown algae S serratum was as follow: the solvent of 96% ethanol, the temperature of 43 0 C, the ratio of solvent-to-material (DM:NL) of 27:1 (v/w) and the time of 33 hours

* Determine of extracting times

The results showed total Phc of 3 extracting times increase trivial, compared

to Phc of 1st extracting times when 96% ethanol was used to extract phlorotannin

from brown algae S serratum In addition, if 3 extracting times was realized,

total volumn of used ethanol is very much Moreover, the economical effect do not has, because Phc was collected very few in 2nd extracting times and 3rdextracting times Thus, phlorotannin was only extracted 1 times to ensure a economical and efficient extraction

3.3 STUDY OF CONCENTRATION OF PHLOROTANNIN

SERRATUM

After the filtered extract collected from brown algae S serratum was

inspissated at various temperatures of 400C, 500C, and 600C, respectively, the results noticed the condensing temperature increase, Phc and antioxidant activity of jelly were decreased and changed according to a linear function and the condensing temperature of 400C impacted less into

phlorotannin content and antioxidant activity Thus, the temperature of

40 0 C, the presure of 3mbar and the condition of cooled reflux was choiced

to concentrate antioxidant phlorotannin extract from brown algae

3.4 STUDY OF PHLOROTANNIN PURIFICATION USING THE TECHNIQUE OF FRACTION AND GEL SEPHADEX LH 20 GEL CHROMATOGRAPHY

3.4.1 Purification by using gel chromatography of Sephadex LH 20

Phlorotannin-rich concentrated extract was fracted by using various solvent and exhibited the following order: n-hexan, chloroform, ethyl acetate, n-butanol Most pure fraction was loaded onto the column of sephadex LH 20, 2x25cm column, 10ml were collected from each fractions The results were shown in fig 3.2 and 3.3

Fig 3.2 Phlorotannin content and antioxidant activity of diferent

fractions

0 5 10 15 20 25 30 35 40