Presented at the DLSU Research Congress 2015

De La Salle University, Manila, Philippines

March 2-4, 2015

Effect of Particle Size, Solvent and Extraction Time on Tannin Extract

from Spondias purpurea Bark Through Soxhlet Extraction
Hilary Y. Baldosano, Ma. Beatriz Micaela G. Castillo,
Chantal Danica H. Elloran, Florinda T. Bacani *
Chemical Engineering Department, De La Salle University, 2401 Taft Ave., 1004 Manila
*Corresponding author: florinda.bacani@dlsu.edu.ph

Abstract: In this study, the tannin content of Spondias purpurea or siniguelas bark
was explored due to the lack of information and studies on the species. This study
aimed to investigate the effects of particle size, solvent type and extraction time on
the extraction of tannin from its bark through Soxhlet extraction. The ratios of
solvent used were ethanol-water solutions at 0:1, 1:1, and 0.5:9.5. The extraction time
was also varied at 4, 6, and 8 hours. The extracts were then analyzed using a Perkin
Elmer High-Performance Liquid Chromatography (HPLC) with UV/VIS detector. The
HPLC was performed using a reversed phase C-18 column as the stationary phase,
and methanol (Solvent A) and 1:25 aqueous acetic acid solution (Solvent B) as the
mobile phase. It was confirmed that extraction parameters varied in this study
affected the yield of tannin in the extract. Finer particle sizes can produce a higher
yield of tannin as long as agglomeration in the extractor does not occur. Moreover, a
longer extraction time produces a higher yield. Solvents with higher amount of
ethanol gave better yields of tannin. Pure water, as a solvent, gave the lowest
amounts of tannin due to the solubility of proteins; the 95% ethanol was the most
effective. It was observed that 95% ethanol with the longest extraction time of 8
hours gave the highest percentage yield of 19.19% and 17.13% from 10 grams of bark.
The model generated that gave an insignificant lack of fit was quadratic. Using
ANOVA, the P-value obtained from the model was less than 0.0001, which implies
that it is significant.

Key Words: tannin; solvent extraction; soxhlet extraction; Spondias purpurea; High-
Performance Liquid Chromatography (HPLC);

1. INTRODUCTION are have a wide range of uses in industries such as

the leather tanning, textile dyeing, ink, wood
adhesive, and wine industries. Their ability to
Tannins are phytochemical polyphenols interact with and precipitate proteins, such as
found in most plants and trees, particularly in their proteins found in animal hides, are what make them
bark and wood (Steiner, 1989). They possess several important raw materials in industries.
beneficial characteristics such as being astringent, Since ancient times, tannins have been used
an antioxidant, and anti-bacterial (Sher, 2004). They in the traditional craft of textile dyeing as one of

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Proceedings of the DLSU Research Congress Vol. 3 2015
 Presented at the DLSU Research Congress 2015
De La Salle University, Manila, Philippines
March 2-4, 2015

these mordants. Other examples of natural mordants extraction of tannin from the plant's bark through
include tannic acid, tartaric acid, guava and banana Soxhlet extraction, which is an effective method in
leaves and bark (Mathur & Gupta, 2003). However, extracting phenolic compounds (Roberts et al., 2013).
as the industry expanded and more advanced In addition, the effect of the extraction parameters
technologies were developed, the use of natural particle size, solvent type, and extraction time on the
materials in the dyeing industry decreased. The yield of tannin was investigated.
advancement of technologies led to the development
of synthetic mordants such as alum, copper sulfate, 2. METHODOLOGY
and ferrous sulfate, which are relatively cheaper and
more reproducible (Bechtold et al., 2003). However,
many sources and experts believe that the use of
2.1 Collection and sample preparation
such synthetic materials cause several health risks The plant used for this experiment was
and environmental pollution (Forgacs et al., 2004). In obtained from Barangay Ulingan, Bagac, Bataan. A
modern times, the apparent need for safe and branch of the tree was brought to the National
environment-friendly materials have re- Museum of the Philippines for taxonomic
established the significance of natural mordants in identification. Phytochemical test was done by
the textile dyeing industry (Mathur & Gupta, 2003). Standards and Testing Division of the Department of
In the Philippines, dyes, whether synthetic Science and Technology to determine the present
or natural, have usually been imported into the constituents in the bark sample, as well as to confirm
country rather than manufactured (PTRI, 2005). The the presence of tannin. The bark was freeze dried to
Philippine Statistics Authority (PSA) has published determine its moisture content. The freeze-dried
in its August 2014 Preliminary External Trade bark was then ground into the desired particle sizes
Performance Report that the country's imports of using the laboratory mill. The weight of the bark
dyeing, tanning and coloring materials is a US sample used per run was 10 grams.
$29.01 million commodity. Also, the report showed After determining the moisture content,
that the annual growth rate of the commodity from Soxhlet extraction of the bark samples were
2013 to 2014 was 11.5%. To reduce the country's performed. The factors observed in the extraction
heavy reliance on synthetic dye imports and to were the type of solvent and the extraction time. The
establish local natural dyeing technologies, the crude extracts obtained were then analyzed in the
Department of Science and Technology's Philippine HPLC. After acquiring the results of the HPLC, the
Textile Research Industry (PTRI) conducted a study percentage yields of each were calculated and the
in 2005 that identified 26 indigenous plant species as data were analyzed using the Design Expert
promising sources of natural dyes and one of them Program.
was Spondias purpurea, known as sineguelas (PTRI,
2005).
Sineguelas or Spondias purpurea, from the 2.2 Soxhlet extraction
Anacardiaceae family, is a fruit-bearing tree mostly Each run used 10 grams of bark with a
found in the tropics such as South and Central particle size of -20+48 mesh, which was determined
America, Africa, and Asia (Bicas et al. 2011). It is in the preliminary experiment. The solvents used,
also commonly known as the spanish plum, jocote, were distilled water, 50% ethanol (v/v) or 95%
ovo, ciruela, or siriguela in other countries. The ethanol (v/v) with a constant volume of 300 ml. The
family where Spondias purpurea belongs to, solvent were heated up until it reached its boiling
Anacardiacea, is considered to be one of the tannin- point. The extraction process was conducted for 4
rich Dicotyledons (Mahlo & Chauke, 2012). The hours, 6 hours and 8.
recent discovery of the plant's natural dyeing The solvent used and the extraction time
abilities could be due to its tannin content. According were varied. The responses obtained were the
to a study on natural dyeing, the ability of plant to concentration of the sample and the percentage yield.
act as a natural dye or biomordant could be The number of runs was 9 with 2 trials, adding up to
attributed to its high tannin content (Prabhu et al., a total of 18 runs. The interpretation of the General
2011). Furthermore, a study on the tannin content of Factorial design is that for example for the first run
the leaves of the plant affirms that both plant parts the values that will be used for the solvent and
contain tannins (Asaad et al., 2006). Several sources extraction time are 50% ethanol and 6 hours. Also,
have also indicated the presence of tannin in its bark, the response of the design was the percentage yield.
which is known to be a primary source of tannin
(Steiner, 1989). Thus, this study focused on the

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Proceedings of the DLSU Research Congress Vol. 3 2015
 Presented at the DLSU Research Congress 2015
De La Salle University, Manila, Philippines
March 2-4, 2015

3. RESULTS AND DISCUSSION The results of the percentage yields are

summarized in Table 2; while, Figures 1 and 2 shows
the graph of the percentage yield as a function of the
3.1 Percentage Yield of Tannin type of solvent and extraction time.
The results of the first trial, as shown on
Figure 1, determined 95% ethanol as the solvent Table 2 Percentage Yield of the Samples
ratio that gave the highest percentage yields for the Solvent Time (hour) Percentage Yield (%)
extraction times of 4, 6, and 8 hours with 3.60%, 50% ethanol 6 4.27
8.26%, and 19.19%, respectively. On the other hand, 95% ethanol 8 19.19
extraction with water resulted to the lowest Water 6 2.45
percentage yields of 2.23%, 2.45%, and 3.96% for 4, 6, Water 4 2.23
and 8 hours. Using 50% ethanol gave percentage 95% ethanol 4 3.60
yields that were in between those of water and 95%
95% ethanol 6 8.26
ethanol.
Water 8 3.96
Water 6 2.51
50% ethanol 8 7.24
50% ethanol 4 3.16
Water 8 3.31
95% ethanol 4 2.76
50% ethanol 6 6.73
95% ethanol 6 6.49
50% ethanol 8 8.37
50% ethanol 4 4.14
95% ethanol 8 17.13
Water 4 2.16

Fig. 1 Percentage Yields of Samples (1 st Trial) As expected, a longer extraction time

generally led to a higher percentage yield of tannin.
The results of the second trial, shown on This could have been due to the longer amount of
Figure 2, also determined that a solvent ratio of 95% time the solute and solvent were in contact with each
ethanol with an extraction time of 8 hours gave the other. Longer contact time favored the system to
highest percentage yield of 17.13%. However, for the have more mass transfer. However, excessive
extraction times of 4 and 6 hours, the use of 95% extraction time would be unnecessary as the solvent
ethanol only gave percentage yields of 2.76% and and sample would be in final equilibrium after
6.49%. Rather, it was 50% ethanol that gave the certain duration. This is based on Ficks second law
highest percentage yields of 4.14% and 6.73% for of diffusion. By then, the rate of extraction of
extraction times of 4 and 6 hours. Water gave the
compounds would decelerate (Tan et al. 2013).
lowest percentage yields of 2.16%, 2.51%, and 3.31%
Moreover, a higher percentage yield was
for extraction times of 4, 6, and 8 hours.
also observed for solvents that had higher
percentages of ethanol. Although tannins are highly
soluble in water, water proved to be an ineffective
extraction solvent for tannins. This could be due to
the formation of tannin-protein complexes. The
effectiveness of ethanol as an extraction solvent for
tannin could be due to it being an organic polar
solvent. The polarity of ethanol makes it able to have
strong interactions with polar substances such as
tannin. Also, the results actually affirms the theory
that extracting at high temperatures could degrade
and lose some phenolic compounds ("Quantification
of Tannins in Tree Foliage," 2000). Since of all the
solvents used, 95% ethanol had the lowest boiling
point, it was able to extract the most tannin.
Fig. 2 Percentage Yields of Samples (2nd Trial) Moreover, the result was in accordance with other

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Proceedings of the DLSU Research Congress Vol. 3 2015
 Presented at the DLSU Research Congress 2015
De La Salle University, Manila, Philippines
March 2-4, 2015

previous studies stating that mixture of alcohols and studentized residuals versus predicted values. Since
water as the solvent could extract phenolic the points are in random and show no pattern, the
compounds better compared to mono-solvents like model is suitable to the data. It also satisfies the
pure water (Dent, 2012; Spigno et al. 2007; Tomsone independent normally distributed residuals that are
et al. 2012). usually assumed (Hoerl, 2008).
Also, based on the results of the preliminary
experiment, it was found that extracting with
smaller particle sizes could produce a higher yield.
Smaller particle sizes offer greater surface area for
mass transfer. Finer particles, however, are more
prone to agglomeration. Even if 0.297mm (-48 mesh)
was the smallest particle size used in the study, it
did not give the highest yield of tannin. The solvent
might have had a hard time passing through the
sample due to its compactness which resulted to low
extraction efficiency.

3.2 Analysis using Response Surface

Methodology
The Design Expert Program was used in
order to analyze the data acquired. A probability of
less than 0.05 shows that the lack of fit is significant. Fig. 3 Normal Plots of Residuals
An insignificant lack of fit means that there is no
need to look for a higher order type of model. In this
case, the only model with an insignificant lack of fit
is quadratic with a probability value of 0.0515.
Analysis of Variance (ANOVA) was done to
test the validity of the model as well as whether the
factors, solvent and time of leaching, observed have a
significant effect on the percentage yield of tannin.
The F-value of the model is 44.10 which implies that
it is significant. Also, the probability of it occurring
due to noise is only 0.01%. The values of the "Prob >
F" should be less than 0.05 for the model terms to be
considered as significant. Since the values are less
than 0.0001, the model terms (A, B, AB) are
significant.
From the ANOVA results, the final
equations of the model were generated: Equation (1) Fig. 4 Residuals versus Predicted Graph
is for the water as the solvent, Equation (2) is for
50% ethanol, and Equation (3) is for 95% ethanol. Figure 5 shows the graph of the interactions of the
factors, type of solvent and time of leaching, observed
%Yield=11.60306(3.5975*Time)+(0.32979*Time2 (1) in the study. It can be seen that at 4 hours, the
%Yield=10.41222(2.9187*Time)+(0.32979*Time 2 (2) percentage yield points are overlapping. There is no
%Yield=-1.90528(0.2125*Time)+(0.32979*Time2 (3) type of solvent that actually stands out for that
specific time. However, for the times 6 and 8 hours,
The normal plot of residuals can be seen in the 95% ethanol solvent performed well compared to
Figure 3. The points on the graph represent the the other solvents.
normal percentage probability of the percentage
yields with respect to the residuals. It can be seen
that the points are approximately linear. Since the
plot of residuals fit the expected pattern, it shows
that the residuals are distributed normally. On the
other hand, Figure 4 shows the plot of externally

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Proceedings of the DLSU Research Congress Vol. 3 2015
 Presented at the DLSU Research Congress 2015
De La Salle University, Manila, Philippines
March 2-4, 2015

Supplementary Feeding of Multinutrient

Blocks, Controlling Internal Parasites and
Enhancing Utilization of Alternate Feed
Resources, 239

Bechtold, T., Turcanu, A., Ganglberger, E., &

Geissler, S. (2003). Natural dyes in modern
textile dyehouseshow to combine
experiences of two centuries to meet the
demands of the future. Journal of Cleaner
Production, 11(5), 499-509.
Bicas, J., Molina, G., Dionsio, A., Barros, F. C.,
Fig. 5 Interactions of the Factors Wagner, R., Marstica, M., et al.
(2011).Volatile constituents of exotic fruits
from Brazil. Food Research International,
4. CONCLUSIONS 44(7), 18431855. Retrieved January 10,
2013,
Even though finer particle sizes offer greater from http://www.sciencedirect.com/science/ar
surface area for mass transfer, it does not guarantee ticle/pii/S096399691100024X
a higher yield of tannin. Finer particle sizes are more
prone to agglomeration which could hinder the Dent, M., Uzelac, V., Peni, M., Brni, M., Bosiljkov,
extraction process, but larger particle sizes are also T., & Levaj, B. (2012). The Effect of
ineffective. Thus, the most effective particle size for Extraction Solvents, Temperature and Time
extraction should be able to maximize the surface on the Composition and Mass Fraction of
area for mass transfer, yet prevent agglomeration. Polyphenols in Dalmatian Wild Sage (Salvia
It was confirmed that the factors, extraction officinalis L.) Extracts. Food Technology and
time and type of solvent, investigated in this study do Biotechnology. Retrieved March 21, 2013,
affect the yield of tannin in the extract. Statistical from http://www.ftb.com.hr/index.php/compo
analysis showed that there are significant nent/content/article/133-volume-51-issue-no-
interactions between the factors and the response. 1/1076-the-effect-of-extraction-solvents-
The result in this study is in agreement with the temperature-and-time-on-the-composition-
previous studies stating that as the extraction time and-mass-fraction-of-polyphenols-in-
increases, the extraction yield also increases in dalmatian-wild-sage-salvia-officinalis-l-
various solvents. A longer extraction time would give extracts
the bark and solvent better equilibrium and mass
transfer. Forgacs, E., Cserhati, T., & Oros, G. (2004). Removal
Comparing the three solvents used, water of synthetic dyes from wastewaters: a
was the least effective in extracting the tannin due to review. Environment international, 30(7),
the solubility of proteins in water. The solvent that 953-971
gave the highest percentage yield of tannin was 95%
ethanol. Hoerl, R. (2008). Statistics Roundtable: The Reality
The optimum conditions that afforded the of Residual Analysis. Retrieved November 7,
highest percentage yield of 19.19% (Tannic Acid 2014, from http://asq.org/quality-
Equivalent) were 95% ethanol solvent and 8 hours of progress/2008/06/statistics-roundtable/the-
extraction time. It was known that the bark of reality-of-residual-analysis.html
Spondias purpurea has a higher tannin content
compared to its other parts. Mahlo, S., & Chauke, H. (2012). The effectiveness of
polyethylene glycol (PEG) and polyvinyl
5. REFERENCES polypyrrolidone (PVPP) on removal of
tannins from leaf extracts of selected
Asaad, C. O., De Vera, A. C., Ignacio, L. M., & Tigno, medicinal plants in Limpopo Province.
P. B. (2006). Evaluation of lesser-known and African Journal of Biotechnology, 11(43),
lesser-utilized feed resources in the 10141-10145. Retrieved December 2, 2012,
Philippines. Improving Animal Productivity by from http://www.academicjournals.org/ajb/P

FNH-I-008 5
Proceedings of the DLSU Research Congress Vol. 3 2015
 Presented at the DLSU Research Congress 2015
De La Salle University, Manila, Philippines
March 2-4, 2015

DF/pdf2012/29May/Mahlo%20and%20Chauk March 2, 2013,

e.pdf from http://gjms.com.pk/ojs786/index.php/gj
ms/article/download/165/164
Mathur, J. P., & Gupta, N. P. (2003). Use of natural
mordant in dyeing of wool. Indian Journal of Spigno, G., & De Faveri, D. (2007). Antioxidants from
Fibre and Textile Research, 28, 90-93. grape stalks and marc: Influence of
extraction procedure on yield, purity and
Philippine Statistics Authority (PSA). (2014). antioxidant power of the extracts.Journal of
External Trade Performace: August 2014. Food Engineering, 78(3), 793801-793801.
Retrieved Novemer 13, 2014 Retrieved November 3, 2014,
from http://web0.psa.gov.ph/content/external from http://www.sciencedirect.com/science/ar
-trade-performance-august-2014
ticle/pii/S026087740500779X

Philippine Textile Research Institute (PTRI). (2005). Spigno, G., Tramelli, L., & De Faveri, D. M. (2007).
The Philippine Scene: 26 Dye-Yielding Trees Effects of extraction time, temperature and
Identified. Textile Developments, 20(2), solvent on concentration and antioxidant
0118-34X. Retrieved activity of grape marc phenolics. Journal of
from http://faculty.mu.edu.sa/public/uploads/ Food Engineering, 81(1), 200-208. Retrieved
1345909499.9763TD_Ap-Je05.pdf March 21, 201,
from http://www.sciencedirect.com/science/ar
Prabhu, K., & Bhute, A. (2012). Plant based natural ticle/pii/S0260877406006649
dyes and mordants: A Review.Journal of
Natural Product and Plant Resources, 2(6), Steiner, P. R. (1989). Tannins as specialty chemicals:
649-664 An overview. In Chemistry and Significance
of Condensed Tannins (pp. 517-523).
Prabhu, K., & Teli, M. (2011). Eco-dyeing using Springer US.
Tamarindus indica L. seed coat tannin as a
natural mordant for textiles with Tan, M., Tan, C., & Ho, C. (2013). Effects of
antibacterial activity. Journal of Saudi extraction solvent system, time and
Chemical Society. Retrieved April 5, 2014, temperature on total phenolic content of
from http://0- henna (Lawsonia inermis)
www.sciencedirect.com.lib1000.dlsu.edu.ph/s stems.International Food Research Journa,
cience/article/pii/S131961031100202X 20(6), 3117-3123. Retrieved November 12,
2014,
Prabhu, K., Teli, M., & Waghmare, N. (2011). Eco- from http://www.ifrj.upm.edu.my/20%20(06)
friendly dyeing using natural mordant %202013/21%20IFRJ%2020%20(06)%20201
3%20Ho%20446.pdf
extracted from Emblica officinalis G. Fruit
on cotton and silk fabrics with antibacterial
Tomsone, L., Kruma, Z., & Galoburda, R. (2012).
activity. Fibers and Polymers, 12(6), 753-759
Comparison of Different Solvents and
Extraction Methods for Isolation of Phenolic
Quantification of Tannins in Tree Foliage. (2000). Compounds from Horseradish Roots
Joint Fao/Iaea Division Of Nuclear (Armoracia rusticana). World Academy of
Techniques In Food And Agriculture. Science, Engineering and Technology, 6,
Retrieved November 9, 2014, 1155-1160. Retrieved November 11, 2014,
from http://www- from http://waset.org/publications/8584/comp
naweb.iaea.org/nafa/aph/public/pubd31022ma arison-of-different-solvents-and-extraction-
nual-tannin.pdf methods-for-isolation-of-phenolic-
compounds-from-horseradish-roots-
Roberts, T., Wilkes, W., & Khoddami, A. (2013). armoracia-rusticana-
Techniques for Analysis of Plant Phenolic
Compounds. Molecules, 18, 2328-2375

Sher, A. (2004).Antimicrobial activity of natural

products from medicinal plants. Gomal
Journal of Medical Sciences , 7(1). Retrieved

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