The Effect of Altitude Growth on The Profile of Typical Layer Chromatography (TLC)

Areca Seed Extract (Areca catechu L.) Using Densitometry Method

Subehan, Tayeb, R., dan Lestari, A.

E-mail:ayuulestarii28@gmail.com

Abstract. Areca seed (Areca catechu L.) contains various kinds of bioactive
compounds that it can be used as antibacterial, antiviral, anticancer, anti-
inflammatory, and anti-allergy. One factor that affect the content of compounds in
a plant is the environment, especially the altitude of the place to grow. The study
aims to determine the effect of the altitude of the growing place throught the thin
layer chromatography profile of Areca catechu L. extract with densitometry and
chemometric method using FT-IR data. The research was carried out by extracting
areca seed obtained from several growing places using methanol solvents. The
obtained extract was analyzed for its compound by using the TLC method, by
spraying several reagents on the TLC results, while the analysis by TLC-
Densitometry at a wavelength of 200-800 nm using ethyl acetate: acetone: formic
acid (2 : 5: 4 drops) as the mobile phase. The analysis results obtained by two
overlapping compounds on Rf 0.61-0.71 and Rf 0.87-0.91, whereas for the results
of FT-IR analysis are estimated to contain alkaloid and tannin compounds as well
as, the results of chemometric analysis using FT-IR data is possible that areca seed
extract A and B have characteristics similar compared to areca seed extract C.

Keywords: Areca seed, Altitude, TLC (Thin Layer Chromatography), Densitometry,

FT-IR (Fourier Transform Infrared), Chemometrics.

1. Introduction
Indonesia is a country that has a tropical climate and geographical conditions that
support the growth of various plants. One of the plants that thrives in Indonesia and can
be used as a medicinal plant is areca seed (Areca catechu L.). medicinal plants that are
used as simplicial or herbal medicinal raw material in the production of a preparation
must be standardized in advance in order to guarantee the quality of raw materials [1].
One of the quality parameters of the herbal medicine raw material is its chemical content.
The chemical content contained in plants is influenced by several factors, one of which is
the growing environment that can cause differences in chemical content both in quality
and quantity[2].
Areca seed is a part of plants that can be used as traditional medicine. The
compounds in areca seed (Areca catechu L.) include alkaloids, such as arekolin,
arekolidine, arekain, guvakolin, guvasine and isoguvasine, tannis, flavonoids, phenolic
compounds, catechins and acetylcholine [3]. Nonaka (1989) states that areca seeds
contain proantocyanidine, a condensed tannin belonging to the flavonoid group
proantocyanidin has effects as antibacterial, antiviral, anti-carcinogenic, anti-
infalmmatory and anti-allergy [4].
 The efficacy of medical plants is closely related to the content of active
compounds. Genetic and environmental factors affect active compounds in plants. One of
the environmental factors is the height of the place to grow. The difference in each height
causes the metabolic process in a plant to be different, so that the production of
secondary metabolites is different [5]. According to Karamoy (2009), the influence of the
height of the place of growth is related to the metabolic processesof plants, such as
biochemical processes and the synthesis of secondary metabolites, this will affect growth,
morphological forms, and the content of active compounds in a plant. The higher a place,
the lower the air temperature or the air gets colder and vice versa. Temperatures that are
too high will inhibit plant growth and can even lead to death for plant. Sugiyarto 92012),
states that the higher a place, the lower the flavanois content of a plant, this is caused by
environmental factors such as lack of sunlight intensity.
TLC (Thin Layer Chromatography) and HPTLC (Hight Performance Thin Layer
Chromatography) are methods used for the analysis of herbal grugs in determining the
characteristics of plant fingerprints and have been used in laboratories for analysis and
quality control [6]. The use of TLC densitometry is quite economical because it uses a
small mobile phase, a relatively short time and can be done on several samples
simultaneously by comparing all the chromatograms that exist with one another to
determine the compound profile of all chemical components of each simplicial [7].
2. Materials and Methods
2.1 Sampling
Areca catechu L. samples were obtained from several different altitude regions. The
samples obtained were taken from three locations, namely in Bababulo village, Lalampanua
village, Majene Regency with an altitude 0f 58 meters above sea level (location A), Makatta
village, Batulappa district, Majene Regency with an altitude 0f 554 meters above sea level
(location B), and Kassa village, Batulappa district, Pinrang Regency with an altitude of 1020
meters above sea level (location C).
2.2 Sample Preparation
Areca seed (Areca catechu L.) from each location A, B and C are separated first between
the skin on the fruit the seeds. Then areca seeds (Areca catechu L.) sre sorted wet with running
water until they are clean. Then drying using the simplicial oven, after drying. Dry sorting is
done. Then the samples were pollinated and sieved with a mesh size of 60 to obtain simplicial
powder.
2.3 Preparation of Methanol Extract
Areca seeds (Areca catechu L.) in each location A, B and C were extracted by
multilevel macerations method using two solvents. As much as 100 grams of areca seed are
weighed, then put into macerations containers and soaked in 1 liter of n-Heksan solvent, the
purpose of which is to draw the oil present in simplicial (1 part simplicial in parts n-
Hekssane). Stored for 3x24 hours while occasionally strring and then filtering. Re-extraction
is done using n-Heksane 5 times replication. The pulp obtained was dried and extracted
using ethanol solvent, the extract obtained was then combined and concentrated using a
Rotary Evaporator until a thick extract was obtained. The same is done for second and third
samples.
2.4 Partitions
As much as 1 gram of methanol extract of areca seed was partitioned with ethyl acetate
solvent until an extract that was solube in ethyl acetate and ethyl acetate insoluble. The same
is done to extract locations A, B and C.
2.5 Making Sample Test Solutions
Ethyl acetate insoluble extract from each location A, B and C each weighed 5 g dissolved
with methanol and sufficient volume up to 50 mL, the same was done to extract locations B and
C, so that the sample test solution was obtained by 10%.
2.6 Thin Layer Chromatography
To see the visible spot, extracts from three different locations were bottled on the TLC
plate with the same size of 3x10 cm using a capillary pipe with a distance of 1.5 cm from the
bottom of the plate, then left for a while until dry and put into the chamber (chromatography
vessel) already saturated with eluting fluid (mobile phase) Ethyl acetate : Acetone: Formic acid
(2mL : 5 mL: 4 drops). Elutions is carried out to a limit of of 0.5 cm from the top edge of the
plate. The plate is removed from from the vessel and aerated until the bleaching liquid
evaporated. The results were observed in UV 254 nm and 366 nm and sprayed H2SO4 10%.
Indentification of compound results of TLC was carried out using Cytroborate reagents for
identification of flavonoids, Dragendorf reagents for alkaloids, Liebermann-Bouchard reagents
for terpenoids and 1% FeCl3 reagents for tannins.
2.7 Measurement with Densitometry
To find out the thin layer areca seed chromatography profile (Areca catechu L.), then it is
tested with a densitometer. All extracts from locations A, B and C were bottled with a 4.0 μL
micropipette in one plate equal to 10x10 cm in size with three replications of each extract of
locations A, B and C obtained. The TLC plate results of the scanned test using TLC Scanner at
wavelength of 200-800 nm then the existing chromatogram profile areplotted and compared
with each other and the area under the curve (AUC) is calculated.
2.8 Measurement by Spectrophotometry Fourier-Transform Infrared (FT-IR)
Areca seed extract (Areca catechu L.) is crushed together with potassium bromide, then
pressed to form a pellet that resembles a thin disk. The pellet was inserted into the FT-IR
device, the a running process was carried out to measure the IR uptake of areca seed extract.
Then the functional groups was identified based on spectrum data recorded by the FT-IR
spectrophotometer detector.
2.9 Chemometric
Data obtained from the FT-IR results were then analyzed using MINITAB® software
using the PCA (Principal Componen Analysis) method.
2.10 A data analysis, discussion and conclusion
Data from the results of the TLC and FTIR test were analyzed, then discussed, then
concluded. For densitometry test the data analysis uses WinCATS® Software.
3. Results and Discussion
Areca palm is a type of palm that grows in the Pasific region, Asia and eastern Africa and
is included in the Arecaceae tribe with an upright stem shape, fiber roots, pinnate compound
leaves, compound flowers, and elongated egg-shaped fruit [8].
This research was conducted to analyze the compound content in areca seed taken from
three different places based on the height of the growth place, Bababulo village, Lalampanua
village, Majene regency with an altitude of 58 m asl (location A), Makatta village, Malunda
district, Majene regency with an altitude of 554 m asl (location B), and Kassa village,
Batulappa distric, Pinrang regency with an altitude og 1020 m asl. The selection of these
three places is based on the reason that the spread of areca plants in these three places is
extensive so that it is easly obtained. In addition, the community generally uses this plant as a
traditional medicinal plant. The soil conditions for each growing place form each cultivation
location, namely the soil with the composition mostly having larger sand grains.
The sample taken is the fruit that has seed yellow and has not fallen to the ground. The
sampling takes place in the morning, around 00:09-10:00 WITA. Furtheremore, the samples
from each location are separated first between the skin of the fruit with the seeds.
Then the areca seed is dried and processed into simplicial powder which pollination aims
to reduce the sample size so that the surface area is larger and maximize the extractions
process.
3.1 Extraction results
Areca seed (Areca catechu L.) was extracted from each location using two solvents,
namely n-Hexan to remove oil content in areca seed and methanol using maceration method,
the following results were obtained :
Table 1. Results of extraction of areca beans ( Areca catechu L.)

% Rendamen
Extract type Simplicia weight Amount of extract
extract
A 100 grams 12.88 grams 12.88%
B 100 grams 13.53 grams 13.53%
C 100 grams 14.09 grams 14.09%

Information :
A. Areca seed extract methanol location A (58 m asl)
B. Location areca nut methanol extract B (554 m asl)
C. Areca nut methanol extract location C (1020 m asl)

Based on table 1. it can be seen that% of renditions in each location are different. Location
A has the smallest% rendition value of 12.88%, location B is 13.53% and location C is
14.09%. Extract rendamen can be influenced by several factors, one of which is the extraction
method used. Maceration methods have the advantage of being simple equipment and
workmanship and easy to obtain [9].

3.2 Profile of TLC-Densitometry

The TLC-Densitometry Plate of Areca Catechu L. methanol extract of each location A,
B and C was bottled as follows:

A1 A2 A3 B1 B2 B3 C1 C2 C3 A1 A2 A3 B1 B2 B3 C1 C2 C3

Figure 2. Profile of TLC-densitometry of methanol extract of areca nut in UV

visualization of 254 nm (A) and 366 nm (B)
Information:
A1-A3 methanol extract of areca seed location A 58 mdpl (replication 1-3)
B1-B3 areca bean extract methanol B location 554 masl (replication 1-3)
C1-C3 methanol extract of areca seed location C 1020 masl (replication 1-3)
Silent phase: Silica gel GF 254
Motion phase: Ethyl acetate: Acetone: Formic acid (2: 5: 4 drops)

Analysis of the compound content in areca nut was carried out by qualitative and
quantitative methods, namely using TLC-densitometer and chemometrics using FT-IR
( Fourier Transform-Infrared ) data . The initial extract was bottled as much as 4μl on
silica g el GF 254 plate measuring 10x10 cm with each replication three times bottling. The
mobile phase used was ethyl acetate: acetone: formic acid (2 ml : 5 ml : 4 drops) with the
appearance of three spots on the same sample A, B, and C seen on 254 nm UV light and four
stain spots after spraying H 2 SO 4 10% solution . The difference in appearance of stain spots
on observations of UV light of 254 nm wavelength can be caused by chemical compounds
contained in the sample used. In addition, it is caused by the interaction between UV light
and the chromophore group bound by ausochrome which occurs on the stain (Wall, 2005 ).
The profile of TLC was seen in the spots observed under UV light 254 nm with
an Rf value almost equal to each track, so there is a possibility that there are similarities in
the types of compounds that appear. The results of the TLC profile were followed by
densitometry measurements using a densitometer (TLC Scanner ). The results obtained from
densitometers show compounds that have similar spectra, area, Rf value and peak height
( lamda max ). The KLT pacifier analyzed using a densitometer scanned at a wavelength of
200-800 nm, the data obtained is plotted, compared and calculated area (AUC) of each track.

9
8
67
45
1 23

Figure 3. Results of scanning KLT-Densitometer areca cat extract ( Areca catechu L.) at
254 nm wavelength with the mobile phase of ethyl acetate: acetone: formic acid (2: 5: 4
drops); tracks 1-3 are sample location A, tracks 4-6 are samples of location B, and tracks
7-9 are samples of location C

The TLC plate test results were analyzed using TLC-densitometry tools and scanned at
wavelengths of 200-800 nm. The scan shows several compounds in the measurement using 254
nm wavelength, but there are only two compounds that have the same Rf value , overlapping
area and spectrum, namely the 1st compound ( Rf 0.61-0.71) and compounds 2nd (Rf 0.87-
0.91). Based on the results of TLC-densitometry it can be seen that the compound content of
locations A, B and C is similar and the difference is only in the concentration of compounds
from each location.

Figure 4. The spectrum of the methanol extract 1 (compound 1) overlaps (254 nm)
 In the methanol extract there are two compounds, namely compound 1 with a range of
values of Rf 0.61-0.71 with the value of each wavelength in sequence from tracks 1-9 ie 217
nm, 217 nm, 237 nm, 217 nm, 217 nm , 216 nm, 214 nm, 216 nm, and 216 n m (Figure
4). Methanol extract 1 ( compound 1 )meets standardization requirements as compound markers
because it has an Rf value and the wavelength is almost the same and is found on all tracks.

Figure 5. The spectrum of the methanol extract 2 (compound 2) overlaps (254 nm)

Methanol extract 2 ( compound 2 ) with a range of values of Rf 0.87-0.91 and the value of
each wavelength sequentially from tracks 1-9 which are 261 nm, 259 nm, 258 nm, 200 nm, 255
nm, 257 nm, 258 nm, 261 nm and 259 n m (Figure 5). Methanol 2 ( compound 2 ) extracts meet
standardization requirements as compound markers because they have Rf values and
wavelengths are almost the same and are found on all tracks.

3.2 Profile of FT-IR Spectrophotometry

C- C-
N O
C-
H

O- C=C
H

Figure 6. Analysis of FT-IR spectrum of areca seed extract of location A (58

masl); Bababulo Village, Lalampanua Village, Majene Regency
 C-
N C-
C-
H O

O- C=C
H

Figure 7 . FT-IR spectrum analysis of areca seed anol extract extract location B (554
masl); Makatta Village, Malunda District , Majene Regency

C- C-
N O
C-
H
O- C=C
H

Figure 8. Analysis of the FT-IR spectrum of ol betel nut methane extract location C
(1020 m asl); Kassa Village, Batulappa District, Pinrang Regency

FTIR spectroscopy is a fast, simple , and non-destructive analysis technique with chemical
properties in the sample can be knownand appear p there is an FTIR spectrum. FTIR spectrum
profile of methanol extract of areca nut from location A, B and C used provide very identical
patterns with each other except the transmittance value of each spectrum which indicates that the
chemical compounds contained almost the same differ only in concentration .
Interpretation of the results of FT-IR instruments (Figures 6, 7, and 8) shows the presence
of wave absorption bands 3412 cm -1 , 3415 cm -1 , and 3415 cm -1 , respectively, locations A, B
and C showing the -OH group with a size strong and wide peak . This is indicated by the
presence of widening near 3550-3200 cm -1 .
Absorption bands at wave numbers in locations A, B and C 1612 cm -1 , 1610 cm -1 , and
1608 cm -1 respectively show absorption from C = C which usually appears at wave numbers
1650-1600 cm -1 with size peak medium.
The absorption bands at wave numbers 1444 cm -1 , 1444 cm -1 , and 1444 cm -1 in locations A, B
and C respectively show absorption from CH groups which usually appear at wave numbers near
1450 cm -1 with peak medium sizes.
The absorption band at wave number 1284 cm -1 , 1284 cm -1 , and 1284 cm -1 in locations
A, B and C respectively show the absorption band of the CN group which usually appears at
wave numbers 1342 - 1266 cm - 1 with a strong peak size .
Absorption bands at wave number 1107 cm -1, 1058 cm -1 (location A), 1109 cm -1, 1058
cm (location B) and 1107 cm - 1, 1056 cm -1 (site C) showed an absorption band group COs
-1

that overlap and usually appear at wave numbers 1124-1050 cm -1 with a strong peak size .
The presence of bullet s functions OH, C = C, CH, CN and CO indicates that the
compound is an alkaloid and tannin group. But there are still several other compounds that are
seen by the presence of other clusters of measurement results.
 Based on the results of spray reagents, TLC-Densitometry and FT-IR showed the same
results, namely compounds can be indicated as alkaloid and tannin compounds.

3.3 Kemometric Analysis of FT-IR

Score Plot of Peak, ..., Area
2
Sampel
C A
C B
C C C
C
1 BB
A
B
A
C
BA
A
B
CC BA C C
AC
CCA
A
BBABAB
C AB C
Second Component

C
AB C B
CA
CA
A
C B C C AB A
B
B A
A B
0 B

C
C
-1 AAB
CB
A
C
CB A C
BC

-2
A
BC

-3
-2 -1 0 1 2 3 4 5 6 7
First Component
Figure 9 . S core plots of areca seeds A, B and C

Figure 10 . Loading plots from areca seeds A, B and C

Distinguishing the three areca nuts from locations A, B and C is important to identify and
authenticate areca beans before being used in certain products. During this time the process of
identification and authentication generally uses microscopic and macroscopic analysis which
are sometimes not very accurate. At present, the combination of FTIR and chemometric
spectrum is an attractive choice. This combination has been widely used in the identification
and authentication of medicinal plants for the purpose of classification of geographical origin,
detection of counterfeiting materials and distinguishing plants that are closely related
(Purwakusumah, et. Al ., 2014). In this study the method of Principal component
analysis (PCA) was used for identification and authentication of areca seeds in locations A, B
and C.
From the results of FT-IR testing using the chemometric technique the eigenvalues are 9
which are related to each major component with a proportion of 100%, this is equal to the
number of variables used in PCA ( Principal Component Analysis ) analysis . Next are the
values that contain variable contributions for each component. The greater the coefficient value,
the greater the contribution of the variable to the value of the principal component .
PCA is a method that is often used in making models of plant discrimination with close
relatives. PCA is used to reduce data and provide information to find combinations of variables
or factors that can explain trends in a data set (Gad, et. Al ., 2012). The results of the analysis
with PCA (Figure 9 ) are expressed as plot scores or PC ( Principal component ) which can
identify plants and types of plant varieties. Score plots are also called latent variables because
the observed samples have adjacent score plot values , the sample with the score
plot valuehas the same physical properties. The grouping of areca beans using PCA is shown by
plots of PC values. Plots for two initial PC values are usually most useful in analysis because
these two PCs contain the most variations in data. The closer the sample is to another sample,
the greater the similarity between the samples.
To determine the correlation between samples, a loading plot is performed (Figure 10 )
which will show how strong each variable affects the principal components . In loading
plots can be seen each vector that shows how much the variable load has an effect on the PC.
Based on correlation data, it is likely that the most similar compounds with similar
characteristics are areca nut A and B with a positive correlation on the characteristics of the
chromatogram of peak, intensity and area while areca nut A and B against areca nut C
negatively correlate to the area and intensity only shows a low positive correlation on the peak
chromatogram observed (Appendix 5).
4. Conclusions
Based on the results of the research that has been done, it can be concluded that there is an
influence of the height of the growing place on the profile of a plant. The results of TLC-
Densitometry show that the compounds from locations A, B and C have similarities and the
differences are only found in different concentrations per altitude of growth, whereas based on
FT-IR results obtained groups –OH, CH, C = C, CN and CO, so that it is estimated to contain
alkaloid and tannin compounds. For chemometric results using FT-IR data, it was found that
areca bean extract A and B had almost the same characteristics of the compound compared to
C. areca seed extract.

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