Analysis of Eugenol Content in Ethanolic Extract of Galangal

Rhizome (Alpinia galanga L. Willd) Ointment Using UV-VIS

Spectrophotometry Method

Adi Yugatama, Sholichah Rohmani and Rizky Apriliani

Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Indonesia

Keywords: eugenol, ointment, galangal rhizome, UV-Vis spectrophotometry

Abstract: Galangal (Alpinia galanga L.) is a plant that commonly used by people as traditional medicines. Galangal

rhizome contains various compounds such as galangin, methyl cinnamate, cineole, camphor, δ-pinene, and

eugenol. Eugenol has an analgesic, antioxidant, and antibacterial activity that is usually used in topical

preparations. In manufactured drugs, it is necessary to examine active compound which is one of the

requirements to ensure its quality. This study aims to find out eugenol levels in the ointment of ethanolic

extract from galangal rhizome. The method used to extract secondary metabolite from galangal rhizome is

digestion using ethanol 70%. Eugenol separation from ointment of ethanolic extract of galangal rhizome

was done by liquid-liquid extraction using chloroform as a solvent. The eugenol was analyzed using a UV-

Vis Spectrophotometer because eugenol has chromophore, a benzene ring, so able to absorb ultraviolet

light. Parameters of validation method used in this study are linearity, accuracy, precision, Limit of

Detection (LOD) and Limit of Quantification (LOQ). The result showed that analysis method of eugenol in

the ointment of ethanolic extract of galangal rhizome has good validation, with linearity, accuracy and

precision occupied the requirement, LOD level is 2.388 μg/mL and LOQ level 7.235 μg/mL. Determination

of eugenol level in the ointment of ethanolic extract from galangal rhizome obtained the result of 5.187

mg/gr sample.

1 INTRODUCTION

Galangal is a plant that has been used traditionally as

a medicinal plant. The galangal rhizome is easily

obtained and often used as food spices. Galangal

rhizome contains various compounds, such as

flavonoids, terpenoids, saponins, phenolic acids and

essential oils (Tang et al., 2018). In galangal

essential oils, eugenol has various activities as an

analgesic, antifungal, antitermitic, antibacterial, anti-

inflammatory, and antioxidant (Magalhães et al.,

2018; Park et al., 2011; Xie et al., 2015; Zhang et

al., 2017).

The utilization of ethanol extract of galangal

rhizome as topical medicines when used directly on

the skin is not optimal and is less comfortable, so it

is necessary to create an ointment preparation form.

This form of ointment is preferred, because it is

easier to use, practical, site-specific application of

drug on affected area, convenient for unconscious

patients having difficulty in oral administration,

chemically more stable, and avoid first-pass

metabolism of drug (Shelke and Mahajan, 2015). In

manufactured drugs, it is necessary to examine

active compounds which is one of the requirements

to ensure its quality. A good quality of drug

preparation will greatly support the achievement of

the expected therapeutic effect.

Eugenol has chromophore which is a group in

organic compounds that is capable of absorbing

ultraviolet light and visible light. The use of UV-Vis

spectrophotometry for analyzing eugenol levels in

drug preparation has great benefits in the industrial

world. The method is simple, fast, economical, able

to measure a solution with very small

concentrations, and can produce results accurately.

Until now, there has been no research that performed

the analysis of eugenol in ointment of ethanol

extract of galangal rhizome using UV-Vis

spectrophotometry. Based on the reasons mentioned

above, it is necessary to examine an analytical

method to determine the level of eugenol in the

Yugatama, A., Rohmani, S. and Apriliani, R.

Analysis of Eugenol Content in Ethanolic Extract of Galangal Rhizome (Alpinia galanga L. Willd) Ointment Using UV-VIS Spectrophotometry Method.

DOI: 10.5220/0008239900900095

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 90-95

ISBN: 978-989-758-349-0

ointment of ethanol extract of galangal rhizome

using UV-Vis spectrophotometry method.

2 MATERIALS AND METHOD

2.1 Materials

The tools used in this study are rotary evaporator

(RVO 400SD Boeco Germany), magnetic stirrer

(IKA C-MAG HS 7), stirrer bar, analytical scale

(KERN ALJ), UV-Vis Spectrophotometer (Thermo

Scientific Genesys 10S), waterbath. Galangal

rhizome obtained from Wonogiri regency, vaseline

album (Bratacco), cera alba (Bratacco), stearyl

alcohol (Bratacco), standard eugenol (Merck),

ethanol 70%, ethanol pa (Merck), KOH (Merck),

H2SO4 (Merck), n-hexane, ethyl acetate pa (Merck),

chloroform pa (Merck), filter paper, and aquadest.

2.2 Methods

2.2.1 Preparation of Ethanolic Extract

Galangal Rhizome

The galangal rhizomes were washed, finely

chopped, dried at 50ºC and then powdered. Galangal

powder was extracted using 70% ethanol with 1:10

ratio. The extraction was heated in waterbath at 40-

50ºC and stirred in 3000 rpm for 1 hour. The liquid

extract obtained was separated, then thickened using

rotary evaporator at 50ºC until viscous extract was

obtained.

2.2.2 Qualitative Test for Ethanolic Extract

of Galangal Rhizome

Extract and standard eugenol was spotted on TLC

plate (silica gel GF254) and eluted with mobile

phase (n-hexane:ethyl acetate, 4:1 v/v). Spots were

observed at UV 254 nm. The separation was done

and Rf value of extract was calculated and then

compared to Rf value of standard eugenol.

2.2.3 Formula Ointment of Ethanolic Extract

of Galangal Rhizome

Vaseline album, cera alba, stearyl alcohol were

heated in waterbath until they melted. Vaseline

album and cera alba were put into a warm mortar,

mixed and stirred until cool. Subsequently, stearyl

alcohol was added, stirred until homogeneous and

formed an ointment base. Then ethanol extract of

galangal rhizome was added and stirred until

homogeneous. The ointment obtained was tested by

organoleptic. The formula of the ointment is

described in Table 1.

2.2.4 Extraction Eugenol from Ointment

The separation process of eugenol from the ointment

was carried out by liquid-liquid extraction method

(LLE) using separating funnel. The sample used was

the ointment of ethanolic extract of galangal

rhizome. 2.5 grams of ointment was weighed and 20

mL of KOH solution 0,8 N was added to break the

ointment matrix, then stirred at 2000 rpm for 30

minutes at 25ºC. The base was then separated and

water-soluble phase was taken. The water phase was

added with chloroform and shaken for 10 minutes to

allow 2 layers to form. The layers were separated

and the aqueous phase taken, added with H

until pH 4 then added with 10 mL of chloroform and

extracted 3 times. The chloroform phase was

evaporated, then the residue was dissolved in 5 mL

of ethanol, filtered and inserted to the flakon.

2.2.5 Determination of Lambda Maximal of

Eugenol

Stock solution was prepared by dissolving 10 mg

standard eugenol in ethanol of up to 100 ml to obtain

1000 ppm of eugenol stock solution. From this stock

solution, 100 µL was dissolved in 10 mL of ethanol

(10 ppm), then the determination of λ max was

carried out.

2.2.6 Preparation of Standard Curve of

Eugenol

From eugenol stock solution, 3, 5, 15, 25, 30, 35

µg/mL were prepared and scanned in

spectrophotometry UV. The corresponding

absorbances were noted and then a calibration curve

was plotted.

2.2.7 Validation Method

Validation of the analysis method included linearity,

precision, accuracy, LOD and LOQ. Linearity was

Table 1. Ointment formula of ethanolic extract of

galangal rhizome

Ingredients

Quantity

(%)

Ethanolic extract galangal rhizome 10

Vaseline album 78

Cera alba 9

Stearyl alcohol 3

Analysis of Eugenol Content in Ethanolic Extract of Galangal Rhizome (Alpinia galanga L. Willd) Ointment Using UV-VIS

Spectrophotometry Method

performed by adding standard concentrations of 3, 5,

15, 25, 30, and 35 μg/mL to the samples prior to

extraction process. The absorbance was measured

and calibration curve was determined by linear

regression equation and correlation coefficient was

calculated. Precision was tested as repeatability

(intraday) and intermediate precision (interday).

Repeatability: analyzing the samples that have been

added with the series of eugenol standard solutions

5, 15, 25, 30, 35 μg/mL, measured three times in one

day. Percentage of RSD was calculated from the

obtained absorbance to determine variations within a

day. Intermediate precision: analyzing the samples

that have been added with the series of eugenol

standard solutions 5, 15, 25, 30, 35 μg/mL,

measured on three different days. The percentage of

RSD was calculated from the obtained absorbance to

determine the variation between days. Accuracy test

was performed by adding solution series of eugenol

concentrations of 5, 15, 25, 30, 35 μg/mL and

replicated 4 times. Accuracy was indicated as %

recovery. The analysis was repeated three times.

LOD and LOQ was performed by calibration curve

method using linear regression line. The standard

deviation was calculated using calibration curve then

calculated to find the value of LOD and LOQ.

2.2.8 Determination of Eugenol Level in

Ointment of Ethanolic Extract of

Galangal Rhizome

Eugenol levels were determined by liquid-liquid

extraction method (LLE) using separating funnel

and then measured using a UV-Vis

spectrophotometer. The method used is standard

additions. The absorbance of the sample without the

addition of standard solution was then inserted into

the calibration curve previously obtained, and the

regression equation was generated.

3 RESULT AND DISCUSSION

Ethanol was chosen as a solvent because it is a

universal solvent that can attract polar and non-polar

compounds. Ethanol is also selected because

eugenol has good solubility in alcohols (O’Neil and

Budavari, 2001). Digestion method was selected

because heating and stirring can reduce the

viscosity, therefore increasing the dissolution of

insolvent chemical content. The viscous extract

obtained was thick, blackish brown, had distinctive

smell and weighing 137 gr with a yield percentage

of 16.407%.

The advantages of TLC were easy to use,

relatively fast, and simple. The selection of mobile

phase for TLC was based on the polarity of the

compound. The mobile phase that was used is a

mixture of some organic solvents because the

elusions can be adjusted so that separation can be

optimal. In this study, eugenol is a nonpolar

tendency compound so that the mobile phase used

was a mixture of n-hexane and ethyl acetate (4:1

v/v) (Yugatama et al., 2017). Based on TLC result, it

can be seen that Rf value of standard eugenol was

0.662 (spot B) and Rf value of sample was 0.650

(spot A). The result of the quantification test can be

seen in Figure 1.

Ointment formulation was selected because it

allows longer contact with skin than other topical

preparation so that the release of the active

compound will be maximal. In addition, the ethanol

extract of galangal rhizome can be dissolved in the

suitable ointment base. The base of ointment used in

this study is hydrocarbon type. The base of this

ointment has properties that are difficult to be

washed by water so that contact with the skin will

last longer and does not allow evaporation into the

air. The fat base is an occlusive cover so it can

hydrate the skin (Khar et al., 2013). The ointment

was prepared by melting method; some of the

ingredients were mixed by way of melting and

cooled by constant stirring until they solidified

(Allen, 2014). The obtained ointment has a rather

dense, brownish shape and has distinctive odor.

Figure 1: Qualitative test of eugenol in ethanol extract

galangal rhizome (A) extract (B) eugenol standard.

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

Measurements of the maximum wavelength are

performed because it has maximum sensitivity. The

result of spectra obtained shows there are 3 peaks.

The existence of several peaks is due to ethanol

solvent effect with 205 nm UV cut-off 205 nm, so

that the absorption peak of maximum wavelength

eugenol that is at 282 nm.

The separation process of the analyte from the

sample (ointment base) can be broken down by

adding a chemical compound. This process is

intended for analysis of the analyte so it is not

disturbed by the existence of the sample matrix. In

this study, we used a strong base KOH 0.8 N to

break down the sample matrix, which would change

the eugenol into its salt form that can dissolve in

water. Eugenol has phenolic properties that are

highly influential in color change because phenols

are reactive to air and bases. The reaction will occur

when the phenol is in contact with air, strong bases

and heat, are oxidation reactions where phenol binds

the oxygen causing the change of color.

The process of breaking down the sample matrix

needs to be assisted by heating and stirring which

aims to accelerate and maximize the process (Putri

et al., 2014). The addition of KOH to the sample

will form two parts: K-eugenolate (water layer) and

other organic compounds. The KOH (strong base)

will react with phenol so that it will form its salt (K-

eugenolate). The soluble phenol (water layer) was

added with chloroform to attract any impurities that

may exist at the time of separation. It then formed

two layers (water layer and chloroform layer) that

was then separated and the water layer was taken.

To eliminate the phenol from the salt, H

5 N

was added to neutralize the remaining base. It was

intended to regain the analytes in the whole

molecule (eugenol) (Daryono, 2015; Fitri and

Kawira, 2006). Then, Liquid-liquid extraction (LLE)

was performed by partition using separating funnel

with chloroform as non-mixing solvent. The

selection of chloroform is based on the principle of

‘like dissolve like’ where the nonpolar eugenol tend

to be soluble in chloroform. The selection of the

separation funnel method was due to the ease of

separating the compound between the two non-

interfering solvents. Small droplets of the solvent

will create a larger surface area so that it will

accelerate the equilibrium of the solute between the

two solvents. The solvent obtained from the

extraction (chlorophorm phase) is evaporated. The

resulting eugenol was subsequently dissolved in

ethanol.

Linearity test is necessary because in this area we

will get the correct validation method of an analyte.

The curve between absorbance and concentrations is

linear because there is an increase in concentration

value followed by an increase in absorbance value.

At 282 nm wavelengths a linear range of

concentrations of 3-35 ppm yields a linear equation

y = 0.0093x + 0.2483 with R2 = 0.9976. According

to ICH, the requirement of linearity is when the

coefficient of determination (r

) ≥ 0.997 (Chan et al.,

2004). Calibration curve of eugenol can be seen in

Figure 2.

Precision is a measure that indicates the

suitability of individual test results as measured by

the average outcome distribution if the procedure is

repeatedly defined. In this study, the measurements

at concentrations of 5, 15, 25, 30 and 35 ppm in each

replication were then calculated to achieve the

standard deviation and relative standard deviation.

The variations that appear in precision results can be

caused by various factors that are difficult to control

such as disturbance and different conditions of each

measurement. The precision result of the analytical

method is described in Table 2.

In this accuracy test, standard addition method

was used where a number of known standard

solutions of concentration are added to the sample

and then analyzed. The selection of standard

solution series concentrations used in accuracy test

is based on the results of calibration curve that has

met the range and linearity. Given the addition of

standard eugenol series to sample, at 282 nm there

will be a significant increase in absorbance value.

The result obtained from the calculation of %

recovery is 98.195%.

Figure 2: Calibration curve of eugenol.

Analysis of Eugenol Content in Ethanolic Extract of Galangal Rhizome (Alpinia galanga L. Willd) Ointment Using UV-VIS

Spectrophotometry Method

The method that we used in this study to

determine LOD and LOQ is calculation. From the

result of linear equation y = 0.0093x + 0.2483, we

can calculate LOD value and LOQ value based on

standard deviation and slope of a standard curve

obtained. From the statistical calculation using the

standard curve equation, the LOD value that was

obtained was 2.388 μg/mL and LOQ value 7.235

μg/mL.

The determination of eugenol content in the

sample uses standard method addition. This method

is done by adding the standard eugenol to the

sample. The sample absorbance of 0 ppm (without

the standard addition) is plotted to the calibration

curve (Fig. 3). The results obtained from the

calculation of eugenol content in the ointment of

ethanol extracted from galangal rhizome with

accuracy 98.195% is 5.093 mg/g, so that the actual

eugenol content in the ointment extract of galangal

rhizome ethanol is 5.187 mg/g.

4 CONCLUSIONS

The study shows that the UV-Vis

spectrophotometric method is a good method for

analyzing eugenol in the ointment of ethanolic

extract of galangal rhizome with validation

parameters occupied the requirements. From the

result, we know that the content of eugenol in

ointment ethanolic extract of galangal rhizome is

5.187 mg/g.

ACKNOWLEDGEMENTS

We are thankful to Sebelas Maret University for the

financial support.

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Table 2: Repeatability and intermediate precision of UV-Vis spectrophotometry method.

(µg/mL)

Repeatability (n=4) Intermediate precision (n=4)

Mean Absorbance±SD %RSD Mean Absorbance±SD %RSD

5 0,295±0,0019 0,639 0,297±0,0026 0,864

15 0,395±0,0026 0,646 0,399±0,0051 1,282

25 0,478±0,0054 1,130 0,481±0,0085 1,763

30 0,524±0,0024 0,466 0,525±0,0034 0,649

35 0,571±0,0033 0,586 0,573±0,0041 0,715

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

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Analysis of Eugenol Content in Ethanolic Extract of Galangal Rhizome (Alpinia galanga L. Willd) Ointment Using UV-VIS

Spectrophotometry Method