J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research.

(2016)

Chemical Engineering Research

Determination of the Effect of Hydro-alcoholic Blumea balsamifera Extract

on the Nucleation, Aggregation and Morphology of Calcium Oxalate
Crystals in Aritificial Urine Using Turbidimetric and Microscopic Analyses
Jirah Emmanuel Nolasco*, John Irvin Bautista, Ramon Paolo Quintero
Department of Chemical Engineering, University of the Philippines Diliman, Quezon City, Philippines
*Corresponding Author. Tel.: +63905 604 8325; Email address: jtnolasco@gmail.com

A R T I C L E

I N F O

Article History:
Date Received 04 June 2016
Date Accepted 04 June 2016
Adviser:
Asst. Prof. Charlimagne M. Montealegre
Department of Chemical Engineering
University of the Philippines Diliman
Keywords:
Sambong
Crystallization
Calcium oxalate
Artificial urine
Turbidimetry

1.

A B S T R A C T
Blumea balsamifera, or sambong is a known natural cure for nephrolithiasis or
kidney stone formation; however, there are limited scientific studies which quantify
and explain how the extract inhibits kidney stone formation. The study aims to
describe and quantify the effect of the extract on the nucleation, aggregation, and
the resulting morphology of the formed calcium oxalate crystals (major component
of kidney stones) via turbidimetric and microscopic analyses.
Results showed a 50-70% reduction in the sizes of the crystals. However, no
significant difference was found in crystal sizes as extract dosage varies. Images
showed that the calcium oxalate dihydrate (COD) form dominated over calcium
oxalate monohyrate (COM) for solutions with extract. Also, it was found out that the
extract promoted nucleation via a decrease in surface free energy, from 1.58 mJ/m 2
to1.55 mJ/m2, 1.52 mJ/m2 and 1.33 mJ/m2 at 1.0, 2.0 and 5.0 mg/mL of extract. The
extract inhibited aggregation with the consistent declining trend of the turbidity
slopes of the samples with respect to the control. Furthermore, the extract has been
found out to have an IC 50 of 3.38 mg/mL, which is comparable to conventional antiurolithiatic drugs. These results suggest that sambong can help in avoiding the
formation of significantly large kidney stones.

Introduction

Nephrolithiasis, commonly known as kidney stone

formation, is a globally prevalent disease. It has an
approximate incidence rate of 1 out of 272 people in the world
and 1 out of 86 people in the United States. Meanwhile, in the
Philippines, it was determined to be the seventh leading cause
of death in the Philippines in 2014 according to the
Department of Health (DOH).
Nephrolithiasis is caused by urine supersaturation, a
condition where solute crystallizes out of the solution. The
formed crystals undergo crystal growth and aggregation to
form larger crystals, which upon retention, are harmful and
can impair kidney functions. Crystals are composed of various
minerals such as struvite, uric acid, cysteine and calcium
oxalate. Among those minerals, the major constituent of most
kidney stones is calcium oxalate. Calcium oxalate was
accounted as the main component of kidney stones in roughly
70% of the total number of clinically recorded cases of
nephrolithiasis. Calcium oxalate has various crystalline
manifestations, but the two most common are calcium oxalate
monohydrate (COM) and calcium oxalate dihdyrate (COD).
The former is picket fence-shaped while the latter is
octahedral in shape. Due to this difference in crystal
geometry, COM crystals are more likely to adhere to kidney
tissue walls, while COD crystals are more easily flushed out

during urination. COM crystals are the major component of

most kidney stones and will therefore be the crystal type
which will be addressed by this study.
Several medications and treatment methods currently
exist to treat the said disease, the most notable of which are
shockwave lithotripsy and nephrolithotomy. The former
involves sending shockwaves of certain frequency down the
renal area to break down the formed kidney stones to smaller
pieces, while the latter involves the insertion of a hollow tube
into the kidneys which removes the stones through suction. In
addition, various dissolution drugs are available in the market.
However, the performance of such drugs are not very
satisfactory; also, the two mentioned treatment methods are
not a hundred percent effective, not to mention its high cost.
The effectiveness of both lithotripsy and nephrolithotomy is
lowered by the possibility of leaving small fragments of stones
which become new sites for stone formation.
This turns the attention of experts in the field of medical
science to exploring cheaper, yet effective alternatives, such
as naturopathic medicines with phytochemicals from different
plants and herbs as active ingredients. Examples of these are
medications from B. balsamifera, locally known in the
Philippines as sambong.

J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research. (2016)

Medicines derived from the sambong plant are already

well-known alternatives for the treatment of nephrolithiasis,
and are already being commercially mass-produced and sold
in the market. They are also more readily available, cheaper,
and have fewer side effects, as compared to other over-thecounter (OTC) drugs. There is, however, a very limited
number of scientific studies which provide explanations with
concrete proof, as well as quantifications, for its effectiveness.
Thus far, no research has been able to pinpoint the exact
compound, or group of compounds, which is responsible for
the plants anti-nephrolithiatic effect. Furthermore, there is
also a lack of quantification of its inhibitory effects, leaving
medical practitioners and patients alike with no clear idea on
proper subscription of the said medication.
By conducting this study, some of the knowledge gaps in
the anti-nephrolithiatic effects of sambong may be filled. The
effect of a hydro-alcoholic, i.e. extracted using an alcoholwater solution, B. balsamifera extract on the crystallization of
renal stones in artificial urine will be explored and quantified.
This will be able to provide concrete scientific grounds, on
which the efficacy of sambong-derived medications may be
based on.

2.

Experimental Procedure

2.1. Extract Preparation

Commercially-available sambong leaves were cut into
small bits with petioles removed. Petioles were removed
because they contain only a small amount of extractable
material and they make leaf grinding harder. Cut leaves were
placed in an oven at 40C for six days to remove all moisture
content, as well as volatile components present in the leaves
which are unwanted. The leaves are then retrieved and
crushed into its powder form. Powdered sambong leaves are
then stored until used for extraction.
Extraction was conducted through the use of a Soxhlet
apparatus, with ethanol as the extracting solvent. The use of
the said apparatus is advantageous, as compared to multiple
batch extraction, which is the commonly used extraction
method in literature, because for the given time, the Sohxlet
apparatus continuously extracts the compounds present in
the sambong leaves. This translates to less effort required, as
well as less material loss. The apparatus was heated at a
temperature of 80C and a magnetic stirrer was deployed
which was set to spin at a rate of 400 revolutions per minute.
Ethanol was used due to safety issues with other solvents and
it was also found out that ethanol is the solvent which is able
to extract the greatest number of organic compounds from
sambong leaves. Most of the compounds in sambong leaves
are also polyphenolic compounds which have a high affinity
with alcohols, making ethanol the most suited solvent. A total
of 7g of crushed sambong leaves and 700mL of 99.5% ethanol
were used for every run. Extraction was run for a total of 6
hours.
After extraction, the ethanol solvent was then boiled-off to
obtain the crude extract, which is placed in a water bath at
80C to evaporate the ethanol.
After the solvent has been boiled-off, deionized water is
added to the crude extract and then filtered using a
Whatman-40 filter paper to remove any impurity in the
extract such as leaf particulates. Deionized water is further
added depending on the desired extract dosage. Extract
dosages used in the study are 0.5mg/mL, 1.0mg/mL,
2.0mg/mL, and 5.0mg/mL. The unit of the dosage, mg/mL,
refers to the milligrams of crude extract per milliliter of the
resulting solution.

2.2. Artificial urine preparation

Chutipongtanate et al. (2010) conducted a systematic
comparative study on various formulations of artificial urine
for in vitro studies. Most of the formulations present in the
study were recommended for biological applications, such as
bacterial culture and dermatology. A formulation by Burns and
Finlayson (1980) was recommended for studies involving
crystallization of calcium oxalate. Furthermore, this
formulation was used by most related studies, thus, increasing
its reliability.
Artificial urine was prepared using a formulation by Burns
and Finlayson (1980) and buffered to pH = 6.0, using a
phosphate buffer. The components listed in the formulation
are dissolved using deionized water in a 50-mL volumetric
flask. A fresh batch of artificial urine is prepared for every day
of turbidimetric analysis. Fresh urine is required as the
components start to precipitate out of the solution within 24
hours. This changes the concentration of the different
components in the solution and makes it ineffective.
Furthermore,
precipitated
components
interfere
with
absorbance
readings
during
spectrophotometry.
The
formulation is summarized in Table 1.
Table 1
Artificial urine formulation by Burns and Finlayson (1980).
Compound
Sodium Chloride
Sodium Phosphate
Tri-Sodium Citrate
Magnesium Sulfate
Sodium Sulfate
Potassium Chloride
Calcium Chloride
Sodium Oxalate
Ammonium Hydroxide
Ammonium Chloride

Amount (mmol/L)
105.5
32.3
3.21
3.85
16.95
63.7
3.5
0.32
17.9
0.0028

2.3. Pythochemical Assay

To gain a deeper understanding and more insights
regarding the active compounds and probable mechanism by
which these substances may inhibit calcium oxalate
crystallization, a phytochemical assay was performed by the
Standards and Testing Division of the DOST-ITDI after giving
them 50 g of cut sambong leaves.
2.4. Turbidimetry
Turbidimetry involves collecting time vs. absorbance data
for a solution undergoing crystallization. From the
turbidimetric data, the induction time (time required for
crystals to become optically detectable) and the turbidity
slope (rate of increase in absorbance) obtained from the data
are used in the nucleation and aggregation studies,
respectively.
A full-factorial design was implemented for the
experiment. Turbidimetry was performed at various levels of
supersaturation ratios (17.5, 20.0, 22.5, 25.0) and extract
dosages (0.0mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL,
5.0mg/mL). Three replicates were made for every
combination.
Analysis of each sample was facilitated through the use of
a Perkin Elmer Lambda 850 UV-Vis spectrophotometer. For
every 1-mL cuvette replicate, volumes of artificial urine,
extract, calcium chloride solution, and sodium oxalate solution
were added in the order they were mentioned. Water replaces
the extract in the control replicates (those with extract dosage
of 0.0mg/mL). The spectrophotometer was set to record
absorbance readings vs. time at a wavelength of 620nm.
Every sample ran for 300s. The data obtained from the

J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research. (2016)

spectrophotometer were then analyzed to give values for the

induction time and turbidity slopes.

2.5. Microscopic Analysis

Microscopic analysis aims to determine the effect of the
extract on the morphology of the crystals. The contents of the
cuvette use in the turbidimetric analysis were left undisturbed
for three days to facilitate crystallization for microscopic
analysis. Each sample was placed under light microscopy with
the aid of a hemocytometer which contains grids that can
help estimate crystal sizes. Pictures were taken from each
sample at magnifications of 100x and 400x under the light
microscope. The pictures were then analyzed via the software
ImageJ and crystal sizes were estimated. Particle sizes of the
crystals were reported as Area Mean Feret Diameter.

growth rate dominates nucleation rate. Otherwise, a dominant

nucleation rate results in smaller crystal sizes as
supersaturation increases.
This means that when no extract is added, the crystals
growth rate is faster than the nucleation rate at the said
supersaturation ratios. A trend discontinuity between
supersaturation ratio of 17.5 and 20 may mean that the site in
the hemocytometer for imaging does not represent well the
crystal sizes formed in that specific sample.
In order to determine how the addition of Sambong extract
affected the sizes of calcium oxalate crystals, the area mean
Feret diameters were obtained for every supersaturation ratio
and extract dosage. The resulting plot is shown in Fig. 2.

A Scanning Electron Microscope (SEM) of model S-3400N

was also used to see detailed images of the crystals at higher
magnifications of 500x, 2000x, and 4000x. The samples used
for SEM analysis came from bulk crystallization wherein
500mL of artificial urine was prepared along with fresh
extract, calcium chloride and sodium oxalate solutions.
Samples
used
for
bulk
crystallization
followed
combinations of extract dosages of 0.0, 1.0, and 5.0 with
supersaturation ratios of 17.5 and 25.0. The samples were
placed inside centrifuge tubes with a volume of 50mL and
were left undisturbed for three days to facilitate
crystallization. Afterwards, the samples were centrifuged at a
rate of 2000rpm for 30 minutes. The centrifuge tubes were
decanted and the remaining solids were placed on filter
papers which were left overnight inside an oven to dry. The
dried precipitates were then sputtered coated with a thin
platinum layer for better SEM imaging and subjected under
SEM analysis.

3.

Results and Discussion

3.1. Morphology Study

Fig. 1 shows the arithmetic and area mean Feret diameters of
calcium oxalate crystals per supersaturation ratio for samples
with no extract.

Fig. 2. Area mean Feret diameters of calcium oxalate crystals as a

function of supersaturation ratio and extract dosage.

From the plot, it can be seen that the crystal sizes for
samples with extract does not differ significantly as
supersaturation ratio and extract dosage varies. This is
supported by the use of two-way ANOVA, with supersaturation
ratio and extract dosage having p-values of 0.73 and 0.32,
respectively, at = 0.05.
The behavior of the crystal sizes as supersaturation varies
when extract is added is different when there is none. As seen
from Figure 8, adding the sambong extract removes the effect
of changing supersaturation ratio to the size of the crystals.
This means that sambong extract either decreases the crystal
growth rate, increases the nucleation rate, or both, so that
crystal growth rate does not dominate nucleation rate.
The insignificant effect of varying extract dosage on the
crystal sizes, on the other hand, is different from the results of
the related works reviewed for this study. In those works,
increasing the extract dosages of herbal extracts decreases
the crystal sizes. This could be attributed to the selected
dosages of the extract, which might be too large in order to
see a trend. If the chosen dosages are too large, this means
that their effect is already at maximum, and varying the
dosage would have no effect.

Fig. 1. Mean Feret diameters vs. super saturation ratios for control
samples.

It can be seen here that from supersaturation ratios of 20

to 25, the mean Feret diameters are increasing. Increasing
crystal sizes as supersaturation increases means that crystal

Regardless of the lack of effect of increasing extract

dosage on the crystal sizes, the extract still significantly
reduces the crystal sizes as shown in Fig. 3. The plot shows
that the percent reduction in crystal sizes when extract is
added is around 50-70%, with the highest percent reduction
occurring at the supersaturation of 25.
Furthermore, it was found out that although increasing
extract dosage does not affect crystal sizes, it affected
aggregation as shown in Fig. 4. Higher extract dosage resulted
in the loss of crystal aggregation, depicted as clustered dark
spots in the images.

J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research. (2016)

COD crystals were already detected when no extract is

added, contrary to the results of other related works. This is
due to the use of the artificial urine formulation, with crystals
already expected to form because of the presence of
numerous ions in the solution. This presence of COD even
though no extract is added is supported by SEM images
obtained for the supersaturation ratios of 17.5 and 25 at
extract dosages of 0, 1, and 5 as shown in Fig. 6.

Fig. 3. Percent Reduction of crystal sizes for samples with extract

at different supersaturation levels.

Fig. 4. Sample microscopic images for extract dosages 1 mg/mL

(left) and 5 mg/mL (right).

Related works results show that herbal extracts effect on

the crystal morphology is the change from COM to COD. This
result is also the one obtained in this study as shown in Fig. 5.
COM fractions were determined by manually counting the
COM crystals, based from its shape as described in
references, and dividing it by the total number of crystal
found in that specific supersaturation ratio and extract
dosage.

Fig. 6. SEM images for supersaturation ratios 17.5 (left column)

and 25.0 (right column) at different extract dosages: control (top),
1 mg/mL (middle), and 5 mg/mL (bottom),

The presence of COD in all of the SEM images makes it

inconclusive in supporting the large conversion of COM
crystals to COD if extract is added. However, the presence of
smaller crystals as supported by microscopy is evident
particularly in 5 mg/mL dosage for both supersaturation ratio
of 17 and 25.
3.2. Nucleation Study
The induction time and the turbidity slope were
determined from the absorbance plots obtained through the
use of UV-Vis spectrophotometer. Fig. 7 and 8 show two
sample absorbance plots.

Fig. 5. COM fractions at various supersaturation ratios and extract

dosages.

The above plot shows that there is a large conversion of

COM to COD as extract is added. Higher extract dosages tend
to convert COM crystals more to COD, although their
differences with other extract dosage is small only.

The induction time is the point in which the crystals

become detectable. In the absorbance plots, it was the time
elapsed when the slope of the absorbance plots become
positive. It is characterized by a well at the start of an
absorbance plot. However, not all absorbance plots exhibited
time induction, particularly those that are at the lower extract
dosage. This is due to the equipments slow detection of the
crystals at higher extract dosages; however, the reason for
this is still inconclusive at this point.

J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research. (2016)

that it cannot be detected readily by the equipment. On the

other hand, the increase in induction times because of
increasing supersaturation ratio is linear, particularly for the
control and extract dosage of 1 mg/mL.
The equations of the best fit line can be used to calculate
the overall Gibbs free energy, in accordance with the study of
Abdel-all et al (2009). The calculated Gibbs free energies are
plotted with respect to extract dosages as shown in Fig. 10.

Fig. 7. Absorbance plots for supersaturation ratio 17.5 at various

extract dosages

Fig. 10. Calculated Gibbs free surface energy per extract dosage.

Fig. 8. Absorbance plots for supersaturation ratio 25.0 at various

extract dosages.

In cases where the induction time is not obtainable by

visual inspection, the first derivative plot of the absorbance
curves was generated. The time corresponding to the
maximum, corresponds to the induction time. As such, there is
a need to calculate some of the thermodynamic variables
related to the crystallization. One of which is the overall Gibbs
free energy, where the calculations need Fig. 9 as a precursor.

Fig. 9. Plot of natural logarithm of induction time versus inverse

square of the natural logarithm of supersaturation.

The above plot shows that the induction time increases as

supersaturation ratio and extraction dosage is increased.
Increasing extract dosage hinders the formation of crystal, so

Fig. 10 shows that the Gibbs surface free energy decreases

when extract is added. Gibbs free energy is the energy
needed in starting crystallization. This means that increasing
extract dosages resulted in an easier start of crystallization. In
order to understand this further and how it does exactly affect
the crystallization mechanism, the Gibbs free energy is
plotted against nucleus size per supersaturation ratio as
shown in Fig. 11.
The maxima of the curves found in Fig. 11 correspond to the
critical radii, the size of the crystal needed in order for crystal
growth to proceed. From the plot, it can be seen that there is
a consistent decreasing trend in the critical Gibbs free energy
and the critical radii with an increase in supersaturation ratio.
This supports the expected effect of supersaturation ratio in
the nucleation of calcium oxalate crystals: higher saturation
promotes nucleation, i.e. formation of crystals is more evident
with high values of supersaturation ratio.

Fig. 11. Overall Gibbs Free energy as a function of nuclei radius at

various supersaturation ratios.

By varying the extract dosage at a constant level of

supersaturation ratio, the effect of extract dosage on the
nucleation of calcium oxalate crystals may be determined. As
shown in Fig. 12, it can be seen that the plots exhibited the
same behaviour as Fig. 11: the critical Gibbs free energy and

J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research. (2016)

the critical radii consistently decrease with increasing values

of extract dosages. Using the same argument made earlier, it
can be theorized that the compounds found in the extract are
actually promoting the nucleation of the crystals. It is worth
noting that the critical Gibbs energy is lowest at the highest
extract dosage.

Fig. 12. Overall Gibbs free energy as a function of nuclei radius at

various extract dosages (S = 17.5).

The effect of both supersaturation ratio and extract dosage

on the critical radius is seen clearly on Fig. 13. Both
parameters decrease the critical radius, and consequently,
promote nucleation. Fastest nucleation would theoretically
occur at S = 25 and the 5 mg/mL dosage.
Both parameters have significant effects on the critical
radius, and were verified by a two-way ANOVA at a confidence
level of 95%. F is equal to 188.344 and 385.885 for
supersaturation ratio and extract dosage, respectively, both of
which are much greater than the critical F (3.862).

aggregation of crystals. From the absorbance plots, the

turbidity slopes were determined for each supersaturation
ratio and extract dosages as shown in Fig. 14.

Fig. 14. Turbidity slopes at various supersaturation ratios and

extract dosages

Similar with the results of microscopy, it was found out

that aggregation is inhibited as extract dosage is increased.
This is evident from the consistent decline of turbidity slope as
extract dosage becomes higher as shown in Fig. 14. This can
be attributed to the compounds present in sambong extract,
which interacts with the crystals and/or ions and ultimately
inhibits attaching to other particles.
In order to assess the efficacy of the extract in inhibiting
calcium oxalate aggregation, the percent inhibition for each
extract dosage was plotted, as shown in Fig. 15.

Fig. 15. Determination of the IC50 of the extract.

Fig. 13. Effect of supersaturation ratio and extract dosage on

calcium oxalate critical radius.

3.3. Aggregation Study

Due to the complex nature of aggregation, only empirical
means of quantification is possible. In order to quantify the
inhibitory effect of the extract, the turbidity slope of each
absorbance plot is determined. The turbidity slope is defined
as the linear rate of change in property due to the

The slope of the lines for each curves were determined

and the IC50 of sambong was calculated from it. IC 50 is the
potency of an extract. It measures how much of an extract is
needed in order to have a 50% inhibition of aggregation. From
the calculations, it was found that the average IC 50 of
sambong is 3.38 mg/mL. This is comparable to conventional
anti-urolithiasis drugs such as Cystone, with IC50s of 3.07
8.01 mg/mL (Saha et al, 2013). For more accurate
comparisons, the IC50 of the conventional drug must be
calculated from experiment, and not just compared with what
is written in literature.
3.4. Pythochemical Assay

J.E. Nolasco, J.I. Bautista, R.P. Quintero / Chem. Eng. Research. (2016)

In order to understand further the reasons behind the

results of the study, sambong samples were sent to DOST-ITDI
Standards and Testing Division for a phytochemical assay. The
following table shows the results of this test:
Table 2
Phytochemical Assay Results
Compound
Sterols
Triterpenes
Flavonoids
Alkaloids
Saponins
Glycosides
Tannins

Relative Amount
+++
+
++
++
+
-

Based from the assay results, it can be seen that sterols

are the most abundant compound group in the extract,
followed by alkaloids and saponins. Flavonoids have trace
amounts in the extract, while tannins and triterpenes are
absent.
The polyphenolic compounds have structural similarities
between the three compound groups. Therefore, it can be
theorized that something in those compounds structure
makes them good inhibitors of crystallization.
Other authors have taken notice of this similarity. Saha &
Verna (2013) attributed the inhibitory effects of the extract on
polyphenolics, such as saponins, alkaloids, flavonoids and
terpenoids, but the mechanism by which these compounds
inhibit crystallization is still unknown.
Patent WO2015025294 A1, filed by different French
institutions, namely, National Institute of Health and the
Medical Research, Pierre and Marie Curie University Paris
and the National Institute of Scientific Research, details the
use of green tea for renal lithiasis treatment. They attributed
the inhibitory effects of green tea to catechins found in the
drink.
A striking similarity in structure can be seen between
catechins and the compounds present in the extract.
According to the patent, the inventors have observed that the
green tea extract, rich with catechins, specifically
epigaliocatechin (EGC), are able to chelate calcium, or to
solubilize, or to favour solubilisation of, calcium crystals or
kidney stones made of calcium crystals. Thus, two possible
mechanisms of inhibition may be attributed to catechins: (1)
chelation of calcium ions, inhibiting the formation of oxalate
crystals, and; (2) solubilisation of the crystals. Because of the
compounds structural similarity with catechin, it can be
theorized that the polyphenolic compounds present in the
extract have chelating and solubilizing effect on calcium
crystals make them useful as crystallization inhibitors.
4.

Conclusions and Recommendations

The study aims to characterize and quantify the effect of

the active compounds of sambong leaves on the nucleation,
aggregation and morphology of calcium oxalate crystals in
artificial urine by the use of turbidimetric and microscopic
analyses.
Microscopic analysis showed that sambong extract
generally changes COM to COD and aggregation decreases as
extract dosage increases. However, no significant effect on
the size of the COD has been found as extract dosage
changes. Nevertheless, the percent reduction of crystal sizes
when extract is added is still significantly high.
Based from the nucleation study, the extract promoted
nucleation and increased the nucleation rate, as seen by the

decrease in the overall Gibbs free energy and critical radius

upon the addition of extract. On the other hand, aggregation
study showed that the extract inhibited aggregation of
crystals, as seen by the consistent decrease of turbidity slope
for increasing extract dosages. As a combined effect of the
extracts promotion of nucleation and inhibition of
aggregation, the crystals formed are larger in number but
smaller in size. This conclusion is consistent to that of the
microscopic analysis.
The extracts potency has been found out to be
comparable with conventional medicine, with an IC50 of 3.38
mg/mL, which is comparable to that of conventional antiurolithiatic drugs, such as Cystone.
Results of the phytochemical analysis show that the
extract is abundant in various polyphenolics, such as
saponins, flavonoids and alkaloids. Based from a patent on
catechins as anti-nephrolithatic compounds, it was found out
that catechins have inhibitory, as well as dissolution
properties, due to their chelating and solubilising action on
calcium crystals. Due to the striking similarity between the
chemical structure of catechins and the compounds present in
the extract, it is theorized that the compounds in the extract
manifest the same actions, which gives the sambong extract
its inhibitory properties.
Based from the nucleation study, the extract promoted
nucleation and increased the nucleation rate, as seen by the
decrease in the overall Gibbs free energy and critical radius
upon the addition of extract. On the other hand, aggregation
study showed that the extract inhibited aggregation of
crystals, as seen by the consistent decrease of turbidity slope
for increasing extract dosages. As a combined effect of the
extracts promotion of nucleation and inhibition of
aggregation, the crystals formed are larger in number but
smaller in size. This conclusion is consistent to that of the
microscopic analysis.
The extracts potency has been found out to be
comparable with conventional medicine, with an IC50 of 3.38
mg/mL, which is comparable to that of conventional antiurolithiatic drugs, such as Cystone.
The results of the study concretizes claim that sambong
extract can indeed help in treatment of kidney stones. With
the dominance of COD form, smaller crystal sizes, and little
aggregation of calcium oxalate crystals, the retention in the
kidney of calcium oxalate stones is expected to be low with
the presence of sambong extract.
This research could be extended to the assessment of
dissolution properties of the extract and its efficacy,
optimization of the extraction process, exploration of other
popular methods of analysis such as the double gel growth
technique. Furthermore, more sophisticated techniques in
microscopic and particle analysis could be used to better
describe the changes in the morphology of crystals due to the
addition of the extract. It is also recommended to extend the
study of the effect of sambong extract on real kidney stones
and in a system that simulates the kidney more than this
study.
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