Total Phenolic, Antioxidant and Cytotoxic Activities of Saurauia

vulcani (Korth.) Leaves towards RAW 264.7 Cells

Rosidah

Yuandani

, Sri Suryani

, Novycha Auliafendri

and Denny Satria

Faculty of Pharmacy, Department of Pharmacology, Universitas Sumatera Utara Jl. Tri Dharma ,Medan, Indonesia

Faculty of Medicine, Department of Biochemistry, Universitas Sumatera Utara Jl. Tri Dharma ,Medan, Indonesia

Faculty of Pharmacy, Doctoral Programme, Universitas Sumatera Utara Jl. Tri Dharma ,Medan, Indonesia

Keywords: Total phenolic, Antioxidant, Cytotoxicity, Saurauia vulcani Korth. Leaves

Abstrak : This study aims to evaluate total phenolic content (TPC), antioxidant and cytotoxic activities of Saurauia

vulcani Korth leaves towards RAW 264.7 cells. Saurauia vulcani Korth. leaves powder was extractioned by

maceration method, TPC were determined by Folin-Ciocalteu and antioxidant capacity was assesed by DPPH

assay and cytotoxicity resolved by MTT assay. n-hexane extract of Saurauia vulcani Korth leaves (NESL)

was found to contain hight levels of phenolic (88.16±0.71 mg GAE/g), ethylacetate extract of Saurauia

vulcani Korth leaves (EEASL) (153.22 ±0.71 mg GAE/g); and ethanol extract of Saurauia vulcani Korth

leaves (EESL) (242.21±1.11 mg GAE/g),antioxidant capacity NESL; EEASL; EESL from DPPH assay was

measured as inhibitory concentration (938.33±41.08 ppm; 94.66±0.63 ppm; 48.88±0.18 ppm). Viability of

RAW 264.7 cell toward extracts of Saurauia vulcani Korth leaves showed no toxicity with best concentrations

at 12.5 and 25 μg/mL. The results reveal that NESL, EEASL, EESL has hight levels of phenolic and strong

antioxidant capacity. Cell viability testing showed that extract of Saurauia vulcani Korth leaves didn’t induce

cytotoxicity to RAW 264.7 cells.

1 INTRODUCTION

In this modern era human population is very

susceptible to various diseases, especially inveterate

diseases such as cardiovascular, cancer, infectious

diseases, diabetes, heart disease, Alzheimer's, aging

and others. It is caused by an uncontrollable

composition of oxygen free radicals and unequal

process of anti-oxidant care outcome causes of much

illness so it is necessary immunomodulator that can

improve the human body's immune system that can

prevent various diseases (Satria, 2017) (WHO, 2015)

(Kusmardi, 2007). One of the potential plants is

Pirdot (Saurauia vulcani Korth.). Saurauia vulcani

Korth. Is one of the plant which used as antidiabetic

traditionally in Tapanuli Utara, North Sumatera,

Indonesia. Saurauia vulcani Korth. is efficacious as

wound healing, hypoglycemic, antihyperlipidemic

(Sitorus, 2015) (Sitorus, 2018) (Hutahaean, 2018).

The results of phytochemical screening Saurauia

vulcani Korth. contains flavonoids, steroide/

triterpenoide, glycosides, saponin, tannins

(Marpaung, 2016) (Saragih, 2016). Steroide/

triterpenoide and flavonoide which is also believed to

be efficacious as an immunomodulator (Durga,

2014).

Therefore, the function of this research was to

determine of total phenol value, activity of

antioxidant and cytotoxicity of Saurauia vulcani

Korth leaves extract toward RAW 264.7 cells.

2 MATERIALS AND METHODS

2.1 Plants and Chemicals Reagent

Fresh leaf of S. vulcani Korth. were obtained from

Dolog Huluan Village, Raya District, Simalungun

Regency, Sumatera Utara province, Indonesia.

Chemicals used were AlCl

.6H

O (Merck), distilled

water, DPPH (Sigma), Folin-Ciocalteu (Sigma),

Quercetin (Sigma), gallic acid (Sigma), sodium

acetate (Merck), and sodium bicarbonate (Merck),

phosphate buffer saline (PBS), dimethylsulfoxide

(DMSO), Dexamethasone (Harsen). RAW 264.7

cells used are from the Department of Parasitology

Medical Faculty, University of Gadjah Mada

Rosidah, ., Yuandani, ., Suryani, S., Auliafendri, N. and Satria, D.

Total Phenolic, Antioxidant and Cytotoxic Activities of Saurauia vulcani (Korth.) Leaves towards RAW 264.7 Cells.

DOI: 10.5220/0010091608090813

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

809-813

ISBN: 978-989-758-449-7

809

Yogyakarta. Penicillin/streptomycin (100×) and

Phosphate buffer saline (PBS).

2.2 Preparation of n-hexane Extract of

Saurauia vulcani Korth Leaves

(NESL), ethylacetate Extract of

Saurauia vulcani Korth Leaves

(EEASL) and Ethanol Extract of

Saurauia vulcani Korth. Leaves

(EESL)

The air-dried and powdered of S. vulcani Korth.

leaves (1 kg) were repeatedly extractioned by

maceration with n-hexane (3×3 day, 8 L), the dust

was dried in the air and extractioned with ethylacetate

(3×3 day, 8 L), the dust was dried in the air and

extractioned with ethanol (3×3 day, 8 L) at 25-30°C

with periodical intrusion. The liquid was

congregated, and then vaporized to get an viscid

extract and then freeze dried to dry (Satria, 2015)

(Satria, 2017).

2.3 Determination of Total Phenol

Value (TPV)

The TPV of sample was resoluted used Folin reagent.

Shortly, 100 μL of NESL; EEASL; EESL (500

μg/mL) was mingled with 7.9 mL of filtered water

and 0.5 mL of Folin-Ciocalteu’s reagent (1:10 v/v)

and mixed using vortex for 1 minute. Total phenolic

value was calculated as previously described (Satria,

2017).

2.4 Activity of Free Radical Scavenging

The DPPH testing was carried out according to the

previous study with some modifications. 0.2 mM

solution of DPPH in methanol was available, and 100

μl of this solution was attached to various

concentrations of NESL; EEASL; EESL. Inhibitor

concentration was calculated as previously described

(Satria, 2017) (Jamuna, 2012).

2.5 Cell Culture

RAW 264.7 cells, cell line of a mouse macrophage,

was achieved from the Department of Parasitology

Medical Faculty, University of Gadjah Mada

Yogyakarta. RAW 264.7 cells were preserved in

DMEM enhanced with 100 units/mL of penicillin,

100 μg/mL of streptomycin, and 10% FBS at 37

C in

a humidified environment containing 5% CO

Phosphate buffer saline (PBS) containing EDTA

0.02% and trypsin 0.25% was used to detach RAW

264.7 cells (Susanto, 2018) (Yuandani, 2017).

2.6 Viable Cell Assay

An MTT cell proliferation test was used to appraised

the cytotoxicity of n-hexane extract (NESL),

ethylacetate extract (EEASL) and ethanol extract

(EESL) of Saurauia vulcani Korth. leaves on RAW

264.7 cells. Briefly, RAW 264.7 cells (3×10

cells/well) were planted into a 96-well plate (Iwaki)

and incubated for 24 h. The cells were then medicated

with NESL, EEASL and EESL of Saurauia vulcani

Korth. leaves at series (12.5; 25; 50; 100 and 200

μg/mL) concentrations. After 24 h stimulation, MTT

was attached to each well to a final concentration of

12.5 μg/mL. The plates were further incubated for 3-

6 hrs, and then the produced formazan crystals were

soluble in DMSO. The absorbance at 595 nm was

quantified in a microplate reader Benchmark.

(Yuandani, 2017) (Nugroho, 2013).

2.7 Analysis of Statistical

Data were presented as mean ± standard deviation,

which were analyzed using the SPSS 22 edition.

3 RESULTS AND DISCUSSION

3.1 Total Phenolic Value (TPV)

TPV was determined by the Folin-Ciocalteau method.

The n-hexane extracts (NESL), ethylacetate extracts

(EEASL) and ethanol extracts (EESL) of Saurauia

vulcani Korth. leaves was found to include hight rate

of phenolic value (88.16 ± 0.71 mg GAE/g;

153.22±0.71 mg GAE/g; 242.21±1.11 mg GAE/g).

Phenolic composite are known as an antioxidant, and

they are very necessary plant constituents because of

their free radical scavenging capaability due to their

hydroxyl groups (Satria, 2017) (Jamuna, 2017)

(Nugroho, 2013) (Sitorus, 2017). Total phenol

content for extract and fractions in DPPH assay were

shown on Figure 1.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

810

Figure 1. Total phenol value of n-hexane extract

(NESL), ethylacetate extract (EEASL) and ethanol extract

(EESL) of Saurauia vulcani Korth. leaves

3.2 Free Radical Scavenging Activity

Antiradical ability of the plant samples was measured

in term of hydrogen donating capability using DPPH

which is a constant, nitrogen centered free radical and

produces deep purple color in methanol solution (Pan,

2008). The capacity of reducing composite could

serve as an indicator of potential antioxidant property

(Meir, 1995) (Dalimunthe, 2016) (Shah, 2015);

Satria, 2017). DPPH assay, which is based on the

ability of DPPH, a steady free radical, to decolourize

in the existence of antioxidants, is a direct and

dependble method for determining radical

scavenging behavior (Hasan, 2006) and has been

mainly used as a fast, credible and reproduciblec at in

vitro antioxidant activity assay (Koleva, 2002).

Inhibitory concentration (IC

) for NESL; EEASL;

EESL and Quercetin in DPPH assay was

(938.33±41.08 ppm; 94.66±0.63 ppm; 48.88±0.18

ppm and 4.94±0.05 μg/mL, respectively. NESL is a

weak antioxidant, EEASL strong antioxidant, EESL

as a powerful antioxidant. IC

for extract and

fractions in DPPH assay were shown on Figure2.

Figure 2. Activity of antioxidant, IC

of n-hexane extract

(NESL), ethylacetate extract (EEASL) and ethanol extract

(EESL) of Saurauia vulcani Korth. leaves and Quercetin as

positive control.

3.3 Cells Viability

The results of cell viability test on extract of n-

hexane, extract of ethylacetate and extract ethanol of

Saurauia vulcani Korth. leaves and dexamethasone as

positive control. The best results were shown on

extract n-hexane of Saurauia vulcani Korth. leaves

with concentration at 12.5 and 25 μg/mL which

resulted in the highest viability percentage. The

higher result of viability percentage then the greater

the viability of the cell. Percent of viable cells of

RAW 264.7 cells for extract and fractions in DPPH

assay were shown on Figure 3.

Figure 3. Percentage of viable cells of RAW 264.7 cells

treated by n-hexane extract (NESL), ethylacetate extract

(EEASL) and ethanol extract (EESL) of Saurauia vulcani

Korth. leaves and dexamethasone as a positive control

100

150

200

250

300

Total Phenol

NESL

EEASL

EESL

0,000

20,000

40,000

60,000

80,000

100,000

120,000

12,5

100

200

Viable Cells

Concentration (μg/mL)

Dexamethasone

NESL

EEASL

EESL

200

400

600

800

1000

1200

IC50

NESL

EEASL

EESL

Quercetin

Total Phenolic, Antioxidant and Cytotoxic Activities of Saurauia vulcani (Korth.) Leaves towards RAW 264.7 Cells

811

The effects of extract of Saurauia vulcani Korth.

leaves on survival of RAW 264.7 cells.

Cell viability testing was performed to

determine the cytotoxicity of that Saurauia vulcani

Korth leaves extract toward RAW 264.7 cells. RAW

264.7 cells were medicated with culture media

containing that extract of Saurauia vulcani Korth

leaves with various concentrations. After 24 hours of

incubation, culture medium was aspirated and cell

viability was measured using an MTT solution. As

shown in Figure 3, cell viability testing showed that

extract of Saurauia vulcani Korth leaves didn’t

induce toxicity toward

RAW 264.7 cells even at the

maximum concentration (Susanto, 2018) (Yuandani,

2017).

4 CONCLUSION

The results reveal that NESL, EEASL, EESL has

hight levels of phenolic and weak, strong and

powerful

antioxidant capacity. Cell viability testing

showed that extract of Saurauia vulcani Korth leaves

didn’t induce toxicity toward RAW 264.7 cells.

ACKNOWLEDGEMENTS

This research was funding by PDUPT 2018 ministry

of research technology and higher education.

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