A Flavonoid compound of Turbinaria decurrens Bory with The Potential Antioxidant and Anticancer Activity

 

Fitriyanti Jumaetri Sami1,2*, Nunuk Hariani Soekamto1, Tatsufumi Okino3, Firdaus1, Jalifah latip4

1Department of Chemistry, Hasanuddin University, Indonesia.

2Sekolah Tinggi Ilmu Farmasi Makassar, Indonesia.

3Faculty of Environmental Earth Science, Hokkaido University, Japan.

4School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, Selangor.

*Corresponding Author E-mail: nunukhariani@unhas.ac.id

 

ABSTRACT:

Isolation and characterization of quercetin flavonoid compound from Dutungan Island, South Sulawesi Province, Indonesia has been successfully done from ethyl acetate extract. Extraction method used maceration, isolation used chromatography, anticancer activity with MTT method and antioxidant test used DPPH radical. Structure was discussed with the FT-IR, NMR spectrophotometer and compared with the literature. Total flavonoids from ethyl acetate extract were 4.8 mgEQ/g, IC50 value of antioxidant activity was 4.23 μg/ml using the DPPH method (2,2-diphenyl-1-picrylhydrazyl), and anticancer activity of H460 cells IC50 value was 10.95 μg/ml. The quercetin compound is potential as an anticancer and was first report in the T. decurrens Bory species.

 

KEYWORDS: Turbinaria decurrens Bory, Antioxidant, Quercetin, Anticancer, Total flavonoids.

 

 


INTRODUCTION:

Turbinaria is a species of seaweed that is included in the class phaeophyceae or brown algae. Some of Indonesian waters have various type of seaweed, including species T. ornata, T. conoides, and T. decurrens Bory. Dutungan Island is a coral island in South Sulawesi which is found of brown algae, one of which is T. decurrens Bory. Phytochemical screening of this species contains phenolics, flavonoids, terpenoids and steroids1, however the research for chemical compounds is still very limited for this species.

 

Information on the chemical content of the genus Turbinaria that have been reported are (22E)-3β-hydroxycholesta-5,22-dien-24-on, 24-ketocholesterol, Saringosterol and fucoxanthin in T. ornate2. Sterol also found in T. conoides including 3,6,17-trihydroxy-stigmasta-4,7,24-triene, 14,15,18,20-diepoxiturbinarine, and fucosterol3. Turbinaric acid was found in T. conoides4, as for the phenol group, myricetin was found in T. ornata5. Besides other phenolic compounds that are also found in brown algae, namely tannins, phlorotannins are found in Turbinaria ornata6.

 

Ethyl acetate extract was previously reported to has higher antioxidant activity than other extracts to inhibiting DPPH radicals, this extract was also toxic to shrimp larvae Artemia salina.7 The anticancer test begins with a toxicity test against Artemia salina Leach as a preliminary test. Flavonoids are the main ingredients and are found in almost all medicinal plants and have a very good pharmacological effect.8 Flavonoid compounds can be efficacious as an antioxidant or anticancer. Antioxidants are compounds that are able to inactivate the development of oxidation reactions by inhibiting the formation of free radicals9,10. Therefore, in our study were interested to isolating antioxidant compounds from ethyl acetate extract. In this paper we also report structure elucidation, total flavonoids, antioxidant activity and anticancer activity on H460 cells from isolate. The structure was determined using FT IR, NMR spectroscopy and compare the reported data.

 

MATERIAL AND METHODS:

Chemicals and Reagents:

H460 cell ATCC HTB-177, Roswell Park Memorial Institute (RPMI 1640), MTT (3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide), Phosphate Buffer Saline, Fetal Bovine Serum, DMSO Trypsin EDTA, cisplatin, thin layer chromatography, silica gel 60 Merck F245. 2,2-diphenyl-l-picrylhydrazyl (DPPH), H460 cell line, aluminum chloride, and all solvents were analytical reagent grade.

 

Instrumentation:

FT IR Shimadzu, 1H, 13C, HMBC, HSQC, and COSY (Bruker 500 MHz) spectrophotometers, ultra violet lamps radiation (254 and 365 nm), ELISA reader, and UV VIS spectrophotometers.

 

Collection and Preparation of seaweed:

Brown seaweeds, T. decurrens Bory was collected freshly from Dutungan Island, South Sulawesi Province, Indonesia. Algae had been taken by pulling from the substrate where the brown algae are attached, then put in a bag. After collected, the sample was sorted to remove of impurities. After sorting and cleaning, then washed with water until clean and put in a cool box that has been labeled. The next sample was dried in the oven simplisia, before the extraction process the dried sample was mashed with a blender. Furthermore, the sample powder was stored in a dry condition, for the next process extraction.

 

Extraction and Isolation:

Dried powder 1 kg was extracted by maceration method with step gradient polarity solvent (n-hexane, ethyl acetate, and methanol). The combined extracts were evaporated to produce crude extracts of n-hexane (3g), ethyl acetate (10g), and methanol (8g).

 

The dried ethyl acetate extract (10g) was separated by liquid vacuum chromatography (LVC) using silica gel as the stationary phase, and eluted with n-hexane: ethyl acetate: methanol gradient method as mobile phase. LVC results obtained 5 combined fractions (T1-T5), T4 fraction was separated by flash column chromatography eluent n-hexane: ethyl acetate (8:2 - 2:8) and obtained 12 sub-fractions (T1-T12). T10 sub-fraction was re-purified by flash column chromatography and obtained 8 sub-fractions. T8 sub-fraction was recrystallized with ethyl acetate and a white solid of 15mg was obtained. This solid was tested for antioxidant and anticancer activity against H460 cells.

 

Determination of Total Flavonoid Content11

Total flavonoid content in ethyl acetate extract was carried out by colorimetric method and use quercetin as standard solution.12 Preparation of quercetin series by pipetted from 100μg/mL stock solution of 0.1mL, 0.2 mL, 0.3mL, 0.4mL, and 0.5mL. Each inserted into a 5 mL volumetric flask, from each concentration added 0.2 mL AlCl3, and 0.1mL sodium acetate, then added volume up to 5ml with 70% alcohol to obtain a concentration of 2, 4, 6, 8, and 10μg/mL, shake until homogeneous. Incubated for 30 minutes at room temperature, the quercetin concentration was measured at the wavelength of 420nm. Furthermore, the relationship curve between absorption and concentration was made.

 

Flavonoid content analysis of 10mg ethyl acetate extract dissolved with 10mL ethanol with 1000μg/mL solution then pipette 1mL with 100μg/mL stock solution. Pipette as much as 0.2mL into a 5 mL volumetric flask, added 0.2mL AlCl3, 0.1mL sodium acetate, then sufficient volume up to 5mL with 70% alcohol. Homogenized and incubated for 30 minutes at room temperature, measured sample absorption at a wavelength of 420 nm.

 

(a x v) X1000

Total Flavonoids = --------------------------- X Df

G

Description:

a = Quercetin concentration in the test sample (mg/L)

V =Total volume of test solution (mL)

G = Weight of extract used (g)

Df = Dilution factor

1000= conversion factor for total solution volume

 

Measurement of Antioxidant Activity:

The antioxidant activity was carried out by Garg and Mittal (2018), with slight modification. The isolate was made with serial concentration of 2, 4, 6, 8, and 10 μg/mL. Then each concentration was pipette as much as 1 mL and added with 1 mL of 0,4 mM DPPH reagent solution, the mixtures were sufficient to 5 mL with methanol p.a. Homogenize the mixture and incubated for 30 minutes, absorption at a wavelength of 515 nm was measured with UV-VIS spectrophotometer 13.

 

Antioxidant activity was calculated using the formula:

 

Abs Blanko Abs sample

Antioxidant = --------------------------------------- x 100%

activity Abs blanko

 

Cytotoxicity Test on Cancer Cells by MTT assay:

H460 cells in RPMI-1640 media with 10% FBS, were put 90L into 96-well plates with a density of 1.0 104 cells per well, incubated 24 hours at 37C with 5% CO2. Pipette 10L of sample solution containing different concentrations of 10, 20, 30, 40, and 50g/mL and Cisplatin as a positive control, incubated for 72 hours under the same conditions. Discard the media and replace with 100L MTT in RPMI-1640 with 10% FBS (0.5mg/mL), and the cells are incubated for 3 hours at 37C with 5% CO2. The MTT solution was discarded, and formazan crystals were dissolved in DMSO. After 15 minutes of incubation, absorbance was measured with Elisa reader at 570nm14.

 

RESULTS AND DISCUSSION

This research is a follow-up study from previous research, where in this study the results were obtained that the ethyl acetate extract of T. decurrens Bory has antioxidant activity with an IC50 value of 180.54 g/mL. Ethyl acetate extract in our study also determined its total flavonoid by colorimetric method and the results of total flavonoid was obtained of 4.8 mgEQ/g (mg Equivalent quercetin/g) extract. The existence of this information, so that this research continues the process of isolation and purification in order to obtain compounds that have bioactivity. The isolation process several fractions were obtained and then purification was carried out. This process obtained a pure isolate named T-8 with a weight of 15 mg. The T-8 isolates obtained were then tested for bioactivity as antioxidants and anticancer using H460 cells.

 

Determination of antioxidant activity in our study was carried out using the DPPH method (2,2-diphenyl-1-picrylhydrazyl). Free radical reduction activity that was approved with an IC50 value15. The smaller IC50 value mean higher antioxidant activity where the T-8 isolate from T.decurrens Bory has antioxidant activity obtained IC50 value of 4.23 μg/ml with a very strong activity category16. This study was also complemented with an in vitro anticancer against H460 cell and compared with cisplatin as positive control. Table 1 shows that the anticancer activity of T-8 isolates. Isolate which had an IC50 value of 10.95 μg/ml and cisplatin as a positive control of 5.64 μg/ml.

 

Table 1. Anticancer activity of isolate T-8 against H460 by MTT Assay

Sample

Concentration g/ml

Cytotoxicity

(%)

IC50

μg/ml

T-8

10

33.673.01

10.95

20

78.381.13

30

85.050.09

40

96.40.21

50

100.00.09

Cisplatin

5

41.820.21

5.64

10

66.450.04

15

75.400.14

20

83.190.05

25

91.210.39

The data were express as meanSD with triplicate.

 

Identification of isolate is substance form yellow solids with FT IR spectroscopy shows the wave number at 3412.06 cm-1 is -OH, 3288.63 cm-1 is -CH unsaturated, 2850.79 cm-1 characteristic for aliphatic, 1688.43 cm-1 is carbonyl (C = O), 1517.98 cm-1 is C=C aromatic, and 1166.93 cm-1 is C-O (ether). ESI-TOFMS analysis was obtained [M + H]+ C15H10O7 m/z 303.0495. This isolate structure was determined by 1H and 13C NMR spectroscopy by comparing the data in Table 1. The existence of ABX proton aromatic system at δH 7,729 (1H, d, J = 2.0 Hz, H-2'), δH 7,621 (1H, dd, J = 7.0 Hz, H-6′), and δH 6,878 (1H, d, J = 7.0 Hz, H-5′) because 3′, 4′ is mated to ring B and the typical meta-coupled pattern for H-6 and H-8 protons (δH 6,181 and 6,385, d, J = 2.0 Hz). The NMR 13C spectrum shows the presence of 15 aromatic carbon, based on NMR data and compared with previous data17.

 

Table 2. 1H-NMR and 13C-NMR (500 MHz), compound 1a (CDOD3, in ppm) and Quercetinb (in CDOD3)

Position C

1H NMRa

13C NMRa

1H NMRb

13C NMRb

2

 

148.92

 

147.7

3

 

137.36

 

135.7

4

 

177.46

 

176.8

5

 

162.64

 

160.7

6

6.181 1H d, J= 2.0 Hz

99.42

6.20 1 H d, J= 2.0 Hz

98.2

7

 

165.71

 

163.9

8

6.385 1H d, J=2.0 Hz

94.59

6.40 1H d, J= 2.0 Hz

94.5

9

 

158.41

 

156.1

10

 

104.69

 

103

1'

 

121.86

 

121.9

2'

7.729 1 H, d, J= 2.0 Hz

116.19

7.65 1H, d, J=2.1 Hz

115

3'

 

146.37

 

145

4'

 

148.2

 

145.8

5'

6.878 1H d, J= 7 Hz

116.4

6.85 1H, d, J=8.4 Hz

115.6

6'

7.621 1H dd, J= 7.0, 2.0 Hz

124.32

7.50 1H dd, J=8.4, 2.1 Hz

124.5

 

Figure 1 Correlation of HMBC, HSQC and COSY from isolate

 

To confirm the position of the functional group in isolate, the COSY, HSQC, and HMBC experiments were carried out and the results can be seen in Figure 1. Two aromatic protons at δH 6.878 and 7.621 are interlocked with each other and correlated with C-4' (δC 148.2) and C1' (δC 121.86), while the aromatic protons at δH 7.729 correlate with C-4' (δC 148.2) and C-1' (δC 121.86), from the HMBC spectrum. The aromatic proton δH 6.181 correlates with C-5 (δC 162.64) and C-7 (δC 165.71), while the aromatic proton at δH 6.385 correlates with C-7 (δC 165.71) and C-9 (δC 158.41). The HSQC spectrum shows the aromatic proton δH 6.181 correlates with C-6 (δC 99.42), δH 6.385 with C-8 (δC 94.59) shows that the hydroxyl group is located at C-5 and C-7 on ring A. The aromatic proton δH 7.729 correlates with C-2' (δC 116.19), δH 6.878 correlates with C-5' (δC 116.4), suggesting that the two hydroxyl groups at C-3' and C-4' on ring B of the flavonols skeleton. COSY data shows that δH 6,878 correlates with δH 7.621 shows a very high conformity, so the isolate is identified as quercetin18. Myricetin a flavonoid group has also been found in T. ornata from Mandapan coast 18, quercetin for the first time reported from the T. decurrens Bory species. Base on bioactivity data indicate Quercetin has potential as an anticancer, also got quercetin inhibits HepG2 liver cancer cell19.

 

CONCLUSION:

In this study, the total flavonoids produced from ethyl acetate extract were 4.8 mgEQ/g. One flavonoid compound, quercetin was isolated from the ethyl acetate extract of T. decurrens Bory, this compound is the first report of T. decurrens Bory. Quercetin showed very strong antioxidant activity with a value of IC50 4.23 μg/ml. This compound also has the potential to inhibit H460 lung cancer cells with IC50 values < 20 μg/ml, but when compared with positive controls (cisplatin), quercetin activity was still lower.

 

ACKNOWLEDGEMENTS:

The author thankful to the Ministry of Research, Technology and Higher Education, Directorate General of Science and Technology Resources and Higher Education Indonesia for scholarships enhancing international publication program (sandwich like 2019) at Hokkaido University.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 09.07.2020 Modified on 18.01.2021

Accepted on 10.04.2021 RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6207-6210.

DOI: 10.52711/0974-360X.2021.01074