Analysis of Total Phenolics and Flavonoids from the

oot Bark of

Flacourtia rukam

Muharni

, Elfita, Julinar, and Heni Yohandini

Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sriwijaya, Jl. Raya Palembang-

Prabumulih Km 32, Indralaya, Ogan Ilir, South Sumatera, 30662 Indonesia

Keywords: Phe

nolic, Flavonoid, Flacourtia rukam

Abstract:

Flacourtia rukam have been used in the to folk medicine. The roots was used by women after giving birth

as antiseptic and the stem bark was used to antihypertensive in the Regency of Musi Banyu Asin South

Sumatera Indonesia. Total phenolic and flavonoid content were determined for methanol, ethyl acetate and

n-hexane extracts from the root bark of F. rukam. Each extracts were determined antioxidant activity using

1,1-diphenyl-2-picryl-hydrazyl (DPPH) method. The results of the study showed that phenolic higher in

methanol extract (71.8868 mg GAE/g) compared with ethyl acetate extract (56.6094 mg GAE/g), while n-

hexane extract no detection. For flavonoids content showed, methanol extract (2.3116 mg QE/g) and ethyl

acetate extract (2.3304 mg QE/g) where the flavonoid content is not significantly different, while n-hexane

extract only contained flavonoid 0.8129 mg QE/g. The antioxidant activity the methanol, and ethyl acetate

extracts showed IC

52.91µg/mL and 86.92 µg/mL and in categorized as active antioxidant, while n-hexane

extract obtained IC

values 268.02 µg/ml in category weak antioxidant.

1 INTRODUCTION

Phenolic compounds is antioxidant properties and

antiradical activities that are beneficial to health.

Phenolic compound also showed activity as anti-

inflammatory, enzyme inhibition and induction,

detoxification and process of skin rejuvenation or

the premature aging process through stimulate

collagen production (Fuad et al., 2016). Antioxidant

activity of phenolic pompounds in small

concentrations can protect, prevent or induction the

level of oxidative damage to biomolecules. One of

the plants used as traditional medicine is Flacourtia

rukam.

This plant in the south of Sumatra the stem bark

is used as an antihypertensive drug. Besides that the

fruit is used to treat diarrhea and dysentery, the

leaves are used to treat swollen eyelids, and the roots

are used by women after giving birth (Yustian et al.,

2012. The biological activity of a plant extract is

closely related to the secondary metabolites it

contains, including flavonoid, phenol, terpenoid,

steroid and alkaloid. Secondary metabolites can be

utilized in the field of pharmacology (Mustarichie et

al., 2013), including as an antioxidant, antibiotic,

anticancer, blood anticoagulant, inhibiting

carcinogenic effects. Besides that secondary

metabolites can also be used as environmentally

friendly pest controllers (Samsudin and Khoiruddin,

2008).

Some information on the chemical content and

biological activity of F. rukam has been reported.

Saree et al., (1998) reported that the Flacourtia

rukam contained stigmastan-3,6-dione and friedelin.

Besides that, Ikram et al., (2009) reported that the

fruit contained phenolic and showed antioxidant

activity with a value of % inhibition of 70.9%. From

the fruit section 5 compounds have been reported,

namely monogalactosyl diacylglycerol, β-sitosterol-

3β-glucopiranoside-6β-fatty acid esters, β-sitosterol,

triacylglycerol, and chlorophyll a (Ragasa et al.,

2016). Monogalaktosildiasilgliserol compound is

active as anti-inflammatory (Imbs et al., 2013).

Meanwhile for the parts of the stem bark of

Flacourtia rukam was reported to contain friedelin,

poliothrysoside, and β-sitosteryl-3β-glucopyranoside

(Muharni et al., 2018). The phytochemical test of the

Flacourtia rukam plant has been shown contain

triterpenoid, steroids, flavonoids and phenolic

compounds (Muharni et al., 2016). In this paper we

will report the analysis of total phenolic and

Muharni, ., Elﬁta, ., Julinar, . and Yohandini, H.

Analysis of Total Phenolics and Flavonoid from the Root Bark of Flacourtia rukam.

DOI: 10.5220/0008839200300035

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 30-35

ISBN: 978-989-758-415-2

flavonoids from the root extracts of Flacourtia

rukam

2 MATERIALS AND METHODS

2.1 Chemicals

1,1-diphenyl-2-picryl-hydrazyl (DPPH), quercetin,

gallic acid, Folin-Ciocalteu, dimethylsulfoxide,

sodium carbonat, aluminum chloride, potassium

acetate (reagent were obtained from Sigma Aldrich,

USA), methanol, ethyl acetate, n-hexane, and

aquadest. The solvents is analytical grade and were

destilation before used.

2.2 Preparation of Sampel and

Identification

The root bark of plant of F. rukam was collected at

month of january 2018 2018 from Musi Banyuasin,

South Sumatera, Indonesia. The taxonomic identity

of the plant was confirmed by Dr. Laila Hanum

(number specimen VIC 2702) at of the Botanical

laboratory Departement of Biology University of

Sriwijaya. The plant materials (1Kg) was cleaned

from the pollutants and the materail plants were

cut into smaller sizes and then to air dried at

room temperature until the weight constant. The

dried sample was powdered busing a grinding

machine and obtained (300 g) and used for further

studies.

2.3 Extraction

Powdered of root bark F. rukam (300 g) was place

in bottle and extracted with organic solvents with

step gradien polarity: using n-hexane, ethyl acetate,

and methanol and placed at room temperature.

Maceration was done for 24 h and then were

filtered using paper filter.

The extraction was repeated

three times for each solvent, and extraction has been

complete. The maserate obtained was concentrated

by rotary vacuum evaporator, at temperature 55 ºC

and then air dried so that we obtained eachs crude

extract.

2.3.1 Yield Precentage of Extracts

The extraction yield precentage is the amount of

extract to obtained compared with the amount of

root plant used. Yield precentage (%) was

determined for each solvent with equations:

%Yield= Wa/ Wb  100 %

(1)

Wa = weight of crude extract

Ws = weight of sample

2.4 Analysis of Total Phenolic

Determination of the total phenolic content of each

extracts of obtained used spectrometrically

analysis to the Folin-Ciocalteu method. (Santoso et

al., 2012). The Standard gallic acid solution of

1000 µg/mL was prepared by dissolving 5 mg gallic

acid in 2 mL ethanol and the volume made up to a

volume of 5 mL with aquadest. The standard gallic

acid with a concentration series of 5, 10 , 15, and 20

µg/mL were preparated with multilevel dilution

from a standard solution of 1000 µg/mL . 100 μL of

each standard gallic acid add 1 mL of 5% Na

solution. The mixture incubated for 5 min, then

added 1 mL Folin-Ciocalteu reagent 50%,

incubation to continued for 1 hour at room

temperature, then measured absorbance at λ

maxs

765

nm. The calibration curve is made through the

relationship between the concentration of gallic acid

(µg/mL) and absorbance.

The total phenolic in the sample was determined

by means of 5 mg of the each extracts were

dissolved in 2 mL ethanol and the volume made up

to a volume 5 mL with distilled water. Then each

sample 1 ml were added 1 mL sodium carbonat

solution (5%) 0.5 mL, and incubated for 5 minutes,

then add folin ciocalteu’s reagent 50%) and

incubated for 1 hour. Absorbance was recorded at

maxs

765 nm used spectrofotometer UV-Vis. The

total phenolics were expressed as gallic acid

equivalents (GAE). Base on the calibration curve

total phenolic calculations used the following

formula:

Total phenolic = x Fp / 

(2)

note :

c = equivalent levels of gallic acid (mg GAE / L)

v = volume of extract solution (mL)

m = mass of extract (g)

Fp = Dilution factor

2.5 Determining of Flavonoid Contents

The Determining of flavonoid contents eachs

extracts were according to the method described

(Nugroho et al., 2013). The standard concentration

of quercetin 1000 µg/mL 25 mL was prepared by

means of 0.025 g quercetin dissolved in aquadest in

a 25 mL flask. The quercetin standard was prepared

with a concentration of 50, 100, 150 and 200 µg/mL

by piping 0.5 mL; 1 mL; 1.5 mL; and 2 mL of a

Analysis of Total Phenolics and Flavonoid from the Root Bark of Flacourtia rukam

standard solution of quercetin 1000 µg/mL into a

100 ml volumetric flask and added distilled water.

500 µL each standard solution were added 1.5 mL

of methanol, 0.1 mL of AlCl

10%, 1 mL of NaNO

0.5 M and 2.8 mL of aquadest, then homogenized

the mixture by means of the vortex, and incubation

to continued until 30 min and followed the

addition of 1 mL of NaOH (1 M). After that

absorbance was measured at at the λ

maxs

415 nm.

The flavonoid content in the extract is

determined by means of the sample 1000 µg/mL

was prepared by means of 5 mg dissolved in

aquadest in a 5 mL flask. Sample solution (500 µL)

of different extract wereadded 1.5 ml of methanol,

0.1 mL of AlCl

10%, 1 mL of NaNO

1M and 2.8

mL of aquadest, incubating for 30 min at room

temperature and then to continued with addition of

1 mL of NaOH (1 M). As the blank using test tube

containing 2.5 mL of aquadest. The sample

containing of flavonoid was show by pink color to

the read with UV-spectrophotometrically at λ

maxs

415 nm. For the determining quantification of

flavonoid content of sampel used Quercetin

compound as the standard. This experiment with

with triplicates and the results were determined in

quercetin equivalents (QE). Flavonoid content

calculations use the following formula

Flavonoid content = x Fp / 

(3)

note :

c = equivalent levels of quercetin (mg QE / L)

v = volume of extract solution (mL)

m = mass of extract (g)

Fp = Dilution factor

2.6 In vitro Antioxidant with DPPH

Method

The radical DPPH methd was used to measure

antioxidant activity ( Nataraj et al., 2013). 200 µL

the samples at various concentrations (62.5, 125,

250, 500, and 1000 µg/mL of extracts with

respective organic solvents) were taken and added

3.8 mL of a 0.05 mM. As a negative control used .

200 µL of methanol in 3.8 ml of DPPH solution.

The reaction mixtures were incubated for 30 min at

room temperature. Antioxidant activity determined

as inhibition of DPPH by the samples with reduced

content of DPPH radical characterized by decreased

absorbance. against the blank (methanol) and was

measured at λ

maxs

517 nm.

3 RESULT AND DISCUSISON

3.1 Yield Percentage

The dry powder of plant roots F. rukam (300 g) were

extracted successively using solvent with increased

polarity of n-hexane, ethyl acetate and methanol and after

concentrating the extracts of each extract were obtained.

he choice of maceration method has many

advantages compared to other methods. such as

procedures and tools that are used simply, do not use

heating so that the compounds contained therein will

not be damaged or decomposed, the solvents used

are also less than the other cold methods of

percolation.

The yield percentage showed in Table 1.

Each extracts after concentrating to obtained n-hexane

extract (1.101 g), ethyl acetate fraction (3.221 g) and

methanol fraction (17.701 g).

Extract yield percentage

of methanol (5.90%)

higher compared ethyl acetate

(1.07%) and n-hexane extract (0.36%).

Among the

different solvent extractions, methanol solvent found to

have higher ecovery over other.

Table 1: Yield percentage of extracts

Solvent

weight of crude

extract (g)

yield

percentage (%)

n-Hexane 1.101 0.36

Ethyl acetate 3.221 1.07

Methanol 17.701 5.90

3.2 Determining of Total Phenolic, and

Flavonoid Contents

The total phenolic contents of different extracts were

analyzed with used curva standard gallic acid. The

gallic acid standard curve with 4 series of standard

quercetin solutions in ethanol with folin ciocalteu

and sodium carbonate solution. The results are

shown in Table 2.

Table 2: Effect of concentration on the absorbance value

of gallic acid

Concentration ( µg/mL) Absorbance

0 0

5 0.226

10 0.445

15 0.739

20 1.003

The coefficient correlation of the gallic acid

standard curve based on the regression equation y

= 0,050x - 0,021 R² = 0.996, is greater than 0.98,

this shows that the curve linearity obtained is very

good. The total phenol content of each extract was

determined with standard gallic acid calibration

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

curve, were stated in mg gallic acid in each g of

extract (Mg QE/g) and presented in Table 3. In this

study, methanol extract (71.8868 mg/g) shows

higher total phenolic content compared ethyl acetate

extract (56.6094) and n-hexane extract phenolic

compound was no detection. The methanol solvent

was more effective in extracting phenolic

constituents.

Table 3: Total phenolic contents of the root of F. rukam

Extract Total phenolics (mg GAE/g

n-hexane -

Ethyl acetate 56,6094

Methanol 71,8868

Analysis of flavonoid content was determined

based on the quercetin standard curve. Standard

curve was done by reacting 4 series of standard

quercetin solutions in ethanol and the results are

shown in Table 4.

Table 4: Effect of concentration on the absorbance

valueof quercetin

Concentration ( µg/mL) Absorbance

0 0

0.5 0,032

1 0.063

2 0.138

5 0.346

Based on the regression equation, y = 0,069x -

0,002, R² = 0,999 the correlation coefficient value is

approaching 1. This shows that the curve has a good

linearity value. The flavonoid content of each

extract was determined based on the quercetin

standard curve regression equation. Flavonoid

content is expressed in mg qercetin in each gram of

extract (Mg QE/g) as shown in Table 5. This data

show, methanol extract (2.3116 mg QE/g) and

ethyl acetate extract ( 2.3304 mg QE/g) shows The

flavonoid content is not significantly different,

Meanwhile n-hexane extract shows lower

flavonoid content than other extracts. Base on this

data methanol and ethyl acetate solvent having the

same effectiveness were used to isolate flavonoid

constituents.

Phenolic and flavonoids compounds have been

know to have multiple biological effects oxidative

stress related disorders such as antioxidant,

anticancer, antidiabetic, anticholesterol, hypertence,

and anti-inflammatory properties (Amarowicz,

2007). Phenolic and flavonoid constituents of root

bark of F. rukam allegedly plays a role in the

used of this plant traditionally for the treatment of

diseases related to oxidative stress related disorders.

Table 5: Flavonoid contents of the root bark of F. rukam

Extract Flavonoid content (mg QE/g)

n-hexane 0.8129

Ethyl acetate 2.3304

Methanol 2.3116

3.3 In vitro Antioxidant Activity by

DPPH Method

The DPPH method, has been widely used for

determining the antioxidant activity of plant extracts.

The results of all extract show decrease Inhibition

percentage with decrease concentration test (Table

6) .

Table 6: The effect of concentration on % inhibition

values each extracts

concentration

(µg/ml)

Percentage Inhibition extrcts

n-hexane Ethyl

acetate

Metanol

1000 71.96 98.20 93.53

500 68.94 78.71 72.86

250 53.30 69.06 68.10

125 44.42 59.08 57.57

62.5 25.76 39.28 39.33

To determine the IC

value of each extracts, base

on a linear regression equation between the %

inhibition value eachs extracts and the concentration

variation. Based on the regression equation

obtained IC

values for n-hexane, ethyl acetate, and

methanol extract 268.02, 86.92, and 52.91 µg/mL

respectively. The IC

value which is getting lower,

the extract is stated to be more active. Based on this

data, the methanol extract showed higher antioxidant

activity compared to other extracts (Table 7).

Table 7.: IC

value eachs extracts

Extracts

(µg/ml)

n-hexane 268.02

Ethyl acetate 86.92

Methanol 52.91

According to Molyneux (2004) extract is

categorized as active as an antioxidant if it has an

of less than 200 µg/mL. If the IC

value is

found at around 200 to 1000 µg/mL in category

weak antioxidant and if above 1000 µg/mL is

categorized as in active. Base on data we conclusion

that methanol and ethyl acetate extract in category

active antioxidant and n-hexane extract category

weak antioxidant. Based on this study, the search

for antioxidant compounds from root bark of F.

Analysis of Total Phenolics and Flavonoid from the Root Bark of Flacourtia rukam

rukam plants can be carried out on ethyl acetate and

methanol extracts. This data also proves that the

used of F. rukam as traditional medicine for the

treatment of diseases related to degenerative

diseases can be explained by the presence contained

of antioxidant compounds found in F. rukam.

Antioxidant compounds are generally phenolic

groups which are soluble in semi-polar solvents or

polar solvents. However, the discovery of the

antioxidant properties of non-polar n-hexane fraction

is thought to be due to the presence of flavonoid

compounds extracted into n-hexane solvents.

according to the results of previous studies where in

the n-hexane extract was found to contain flavonoid

compounds. Fidrianny et al., (2014) also found

flavonoid compounds as antioxidant from n-hexane

extract Tamarindus indica L. Atioxidant activity

was also reported from the n-hexane fraction

Euphorbia tirucalli (Mawadah et al., 2016).

Antioxidant activity is proportional to the total

phenolic content and flavonoids of the extract

(Esmaeilli et al., 2015). At the tested extracts found

the significant correlation between the antioxidant

activity of extracts with value total phenolic and

flavonoid content. According to the findings, the

extract of methanol possesses a comparable

antioxidant activity to the total phenolic and

flavonoid content.

4 CONCLUSIONS

The result of the study showed that phenolic higher

in methanol extract compared with ethyl acetate

extract while n-hexane extract no detection. For

flavonoids content showed, methanol extract and

ethyl acetate extract, the flavonoid content was not

significantly different, while n-hexane extract only

contained flavonoid very less. The antioxidant

activity the methanol, and ethyl acetate extractin

category active antioxidant, while n-hexane extract

in category in weak antioxidant.

ACKNOWLEDGMENTS

The authors are thankful to ministry Ristekdikti

Republic of Indonesia, who have funded this

research through grant Basic Research superior

university periode of 2019. Thank you also to the

department of chemistry University of Sriwijaya

and Basic Laboratory of University on providing

facilities for the research work

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Analysis of Total Phenolics and Flavonoid from the Root Bark of Flacourtia rukam