In Vitro Studies on Synergistic Effects of Limonia Acidissima And Apple Cider Vinegar on AntiUrolithiatic Activity

Abstract

Abstract
Traditionally Wood apple (Limonia acidissima) and Apple cider vinegar was found to be used to treat and prevent kidney stones. The present study is dedicated towards proving the same and providing scientific proof to these traditional ideas. Minerals like calcium, oxala tes, phosphates, uric acid etc. form insoluble aggregates through formation of monohydrates and di -hydrates. This process is initiated by nucleation and then aggregation of insoluble masses. Phytoconstituents like flavonoids, terpenoids, ascorbic acid, acetic acid, potassium, magnesium etc. contain inhibitory properties and hence are urolithiatic in nature. In vitro tests for nucleation and aggregation has been conducted for Wood apple, Apple cider vinegar and their (1:1) synergistic sample and absorbance at 620 nm was recorded to obtain a turbidity slope. The tests were conducted in presence of sodium acetate/ NaCl buffer of pH 5.7 to mimic the biological conditions. 1:10 dilution of commercially obtained Apple cider vinegar sample showed better % inhibition of nucleation and aggregation when compared to the inhibition shown by standard drug cystone. 10 mg/ mL methanolic extract of dried pulp of wood apple showed higher % inhibition when compared to cystone and Apple cider vinegar. Synergistic sample containing both wood apple and apple cider vinegar in equal proportion showed the highest % inhibition for nucleation and aggregation of calcium oxalate crystals. This proved the synergistic activity of wood apple and apple cider vinegar for urolithiasis. Further studies are being conducted check for aggregation and degradation activities of the mentioned test extracts.

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International Journal of Pharmacy and Biological Sciences
ISSN: 2321-3272 (Print), ISSN: 2230-7605 (Online)
IJPBS | Volume 9 | Special Issue 1- NCBTIL | 2019 | 14-20
National Conference on Biochemistry –Transcending and Integrating Life Sciences (NCBTIL) -2019
Held @ Kristu Jayanti College, K Narayanapura, Kothanur PO, Bengaluru-560077, January 17th & 18th
| Conference Proceedings | Research Article | Biological Sciences |Open Access |MCI Approved|
|UGC Approved Journal|
International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
www.ijpbs.com or www.ijpbsonline.com
14
In Vitro Studies on Synergistic Effects of
Limonia Acidissima And Apple Cider
Vinegar on AntiUrolithiatic Activity
Kavita. G. Singh*1, Devyani.Purohit1 and G. L. Sai Ramya1
1Department of Biochemistry, Mount Carmel College Autonomous, Bengaluru, India
Received: 10 Dec 2018
/
Accepted: 30 Dec 2018
/
Published online: 10 Jan 2019
Corresponding Author Email: kavi182@yahoo.co.in
Abstract
Traditionally Wood apple (Limonia acidissima) and Apple cider vinegar was found to be used to
treat and prevent kidney stones. The present study is dedicated towards proving the same and
providing scientific proof to these traditional ideas. Minerals like calcium, oxalates, phosphates,
uric acid etc. form insoluble aggregates through formation of monohydrates and di -hydrates. This
process is initiated by nucleation and then aggregation of insoluble masses. Phytoconstituents like
flavonoids, terpenoids, ascorbic acid, acetic acid, potassium, magnesium etc. contain inhibitory
properties and hence are urolithiatic in nature. In vitro tests for nucleation and aggregation has
been conducted for Wood apple, Apple cider vinegar and their (1:1) synergistic sample and
absorbance at 620 nm was recorded to obtain a turbidity slope. The tests were conducted in
presence of sodium acetate/ NaCl buffer of pH 5.7 to mimic the biological conditions. 1:10 dilution
of commercially obtained Apple cider vinegar sample showed better % inhibition of nucleation and
aggregation when compared to the inhibition shown by standard drug cystone. 10 mg/ mL
methanolic extract of dried pulp of wood apple showed higher % inhibition when compared to
cystone and Apple cider vinegar. Synergistic sample containing both wood apple and apple cider
vinegar in equal proportion showed the highest % inhibition for nucleation and aggregation of
calcium oxalate crystals. This proved the synergistic activity of wood apple and apple cider vinegar
for urolithiasis. Further studies are being conducted check for aggregation and degradation
activities of the mentioned test extracts.
Keywords
Anti-urolithiatic activity, Apple Cider Vinegar, Wood Apple, Synergistic effect, Nucleation and
Aggregation.
*****
1. INTRODUCTION:
Kidney stones are hard masses of crystals formed due
to aggregation of dissolved minerals in the kidneys or
urinary tract or the bladder. These are also known as
renal calculus. Commonly the minerals responsible for
the formation of theses masses are calcium, oxalates,

International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
www.ijpbs.com or www.ijpbsonline.com
ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.
15
phosphates, uric acid, cystine and other similar
minerals. Medically kidney stones are categorised into
four different types of which the most prevalent form
is calcium oxalate monohydrate/dihydrate stones
followed by struvite stones, uric acid stones and the
least common kind is cystine stones. Kidney stones can
be treated through invasive keyhole surgeries or
noninvasive methods like oral medications or laser
treatment. The urinary calculi (= urinary stones, renal
stones or kidney stones) may arise due to chemical
activities within the body [1]. The condition favorable
for the formation of the stones includes high
concentrations of calcium salts (Ca-oxalate and Ca-
phosphate), infections of urinary tracts, pH, and a
decrease in the body’s natural ability to inhibit the
formation of the crystals [ 1].
Limonia acidissima commonly known as wood apple is
very rich in its phytochemicals like flavonoids,
terpenoids, minerals like potassium, magnesium etc.
Ascorbic acid is one of the major components present
in it which plays a key role in inhibiting the growth of
kidney stones. This fruit has various activities such as
antioxidant, wound healing, anti-diabetic, anti-
hyperlipidemic, anti-cancer, diuretic, hepato-
protective activity [ 2].
Apple cider vinegar is used in many home remedies to
cure various small-scale ailments such as treatment for
kidney stones. This might be due to the presence of
various minerals like potassium, magnesium, many
nutrients and high amount of acetic acid. It has been
found to contain various activities like antioxidant
activity, antimicrobial activity [ 3], anti-diabetic activity,
anti-obesity activity[4].
The present study is dedicated towards identifying the
potential of anti-urolithiatic activity of the fruit pulp of
Limonia acidissima (wood apple) and Apple Cider
vinegar (ACV). Along with this we are checking for the
synergistic/antagonistic effect of the combined
samples on urolithiasis.
2. MATERIALS AND METHODS:
Wood apple was procured from the local market in
Bengaluru and was ripened. The extracted pulp was
sun dried along with the seeds, powdered and stored
in airtight container. The Apple Cider Vinegar was
bought at a local organic store and used in various
dilutions. The chemicals and solvents required were
provided by the laboratory, Department of
Biochemistry, Mount Carmel College, Bangalore, India.
2.1. Phytochemical screening: Phytochemical
evaluation of dried extracts of wood apple
was conducted with methanol and water to
check the solubility and activity. 10% extract
of methanol and water was prepared by
placing the mixtures on magnetic stirrer for 45
mins and centrifuged at 8000 rpm, 15 mins,
24°C. The obtained clear supernatants was
subjected to various phytochemical tests. ACV
was diluted with distilled water and filtered.
The different dilutions tested were 1:5, 1:10
and 1:20. These dilutions were also subjected
to various phytochemical tests like
carbohydrates, proteins, alkaloids, glycosides,
tannins/phenolics, flavonoids and terpenoids
[5,6,7,8].
2.2. Quantitative estimation of Flavonoids: 1g of
dried sample was macerated with 10 mL of
(8:2) methanol: water and filtered with
whatman filter paper. The filtrate was
partially dried and 6 drops of 2M H2SO4 was
added. Chloroform was added to acidified
filtrate in 3:1 ratio in separating funnel. The
mixture was shaken well for 10 mins and
chloroform layer was extracted from the
separating funnel after 30 mins which was
dried for 12 hours. The dried extract was
dissolved in minimal amount of methanol for
further tests [9].
0.2 to 1.0 mL (10𝛍g/mL) aliquots of standard quercetin
solution was pipetted out into different test tubes
which was made upto 2.0 mL with methanol. 2.0 mL of
methanol was taken as blank and 1.0 mL of extracts
were taken separately for flavonoids estimation. 0.1
mL of 10% AlCl3 and 0.1 mL of 1M potassium acetate
solutions were added to all the test tubes which were
incubated for 30 minutes at room temperature. The
absorbance was checked at 415 nm for all the test
tubes and standard graph was plotted to estimate the
flavonoid content of extracts as quercetin equivalents.
The test was conducted in triplicates [10].
2.3. Quantitative estimation of Ascorbic acid in
Wood apple: 10% methanolic extract of dried
pulp of wood apple was used for Ascorbic acid
estimation. Standard solutions of 2,6 -
dichlorophenol indophenol and 1% ascorbic

International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
www.ijpbs.com or www.ijpbsonline.com
ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.
16
acid was prepared with oxalic acid-acetic acid
activation mixture. The DCPIP solution was
standardized by titrating with 1 mL of
standard ascorbic acid solution mixed with 10
mL of activation mixture. This standardized
DCPIP solution was used to titrate with 1 mL
of 10% methanolic wood apple extract mixed
with 10 mL of activation mixture. The end
point was pale pink. All the tests were
conducted in triplicates [11].
2.4. Quantitative estimation of Acetic acid in
Apple Cider Vinegar: 1:1 dilution of ACV with
distilled water was prepared and filtered and
used for acetic acid estimation. Standard
solutions of sodium hydroxide and oxalic acid
were prepared with distilled water. NaOH was
standardized with 10 mL of 0.05M of oxalic
acid. 0.5% phenolphthalein was used as
indicator which turned the solution to pale
pink in presence of base which was
considered as the end point. 25 mL of extract
was titrated against standardized NaOH in
presence of indicator until end point was
obtained. All the tests were conducted in
triplicates [ 12].
2.5. Parameters to test for Urolithiasis
2.5.1. Nucleation and Aggregation assay:
Nucleation and Aggregation assay were
performed as per method previously
described by Hess et al. [2000] with
minor modifications [13]. Stock solutions
of 10 mM CaCl2 and 1 mM of Sodium
oxalate solutions were prepared with
buffer solution containing 200 mM
NaCl and 10 mM Sodium acetate (pH
5.7). All the solutions were prepared
with filtered Millipore water and again
filtered after preparations. 15 mL of
CaCl2 solution was added into a clean
beaker and maintained on magnetic
stirrer with continuous stirring at 37°C.
1.5 mL of Control (Millipore water)/
Standard (10 mg/ mL cystone)/ 10 mg/
mL methanolic extract of wood apple/
1:10 diluted ACV/ 1:1 mixture of wood
apple and ACV was added under
continuous stirring. Incubation time
was started as soon as 15 mL of sodium
oxalate solution was added.
Absorbance was noted every minute at
620 nm for 30 minutes [14]. The blank
was set with millipore water. All the
crystallization experiments were
conducted in triplicates. Percentage
inhibition of the standard and extracts
were calculated as [1-(Tsi/Tsc)] ⨯100
where Tsc indicated the turbidity slope
of the control and Tsi indicates the
turbidity slope in presence of inhibitor
like cystone and extracts [ 15,16].
3. OBSERVATIONS AND RESULTS:
3.1. Preliminary phytochemical screening:
Wood Apple - Dried Pulp 10% w/v Extract
Apple Cider Vinegar - Dilutions with Water
Test
Water
Methanol
1:5
1:10
1:20
Molish
++
+++
+++
++
++
Fehlings
+++
+++
+++
++
++
Proteins
-
+
+
-
-
Alkaloids
+
+
-
-
-
Glycosides
+
++
++
+
-
Tannins/Phenolics
-
+
-
-
-
Flavonoids
+++
+++
+++
++
+
Terpenoids
++
+++
+++
++
+

International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
www.ijpbs.com or www.ijpbsonline.com
ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.
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From the above table it was found that methanolic
extract of wood apple gives better results when
compared to the aqueous extracts. Hence methanolic
extract was taken for further analysis. In case of ACV,
better results were found in 1:10 dilution and hence
this dilution was considered for further analysis. It was
found that wood apple and ACV were rich in flavonoids
and terpenoids and they were subjected to
quantitative estimations.
3.2. Quantitative estimation of Flavonoids:
Wood apple was found to contain 6.9 ± 0.15 𝛍g QE/g sample dry weight of flavonoids. Similarly, ACV was found
to contain 11.7 ± 0.3 𝛍g QE/ mL sample of flavonoids.
3.3. Quantitative estimation of Acetic acid and Ascorbic acid
Samples
Estimation compound
Trial 1
Trial 2
Trial 3
Concentration
Wood apple
Ascorbic acid (w/w)
0.2
0.25
0.2
6.5% ± 0.5%
Apple Cider Vinegar
Acetic acid (v/v)
16.87
17
17.1
8.9% ± 1.2%
Through titrimetric analysis it was found that wood apple contained 6.5% ± 0.5% (w/w) of total Ascorbic acid in
the dried pulp and ACV was found to contain 8.9% ± 1.2% (v/v) of acetic acid in it.

International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.
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3.5. Nucleation and Aggregation Assay
samples
slope
% inhibition
standard (cystone)
10mg/ml
0.008678629032
4.418687526
apple cider vinegar
1:10
0.008274193548
8.872902115
wood apple
10mg/ml
0.006822580645
24.86011233
synergistic sample
1:1 mixture
0.005703225806
37.18802736

International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
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From the above data, it is well understood that the
nucleation and aggregation of the kidney stone is
maximally inhibited by the synergistic sample
(37.18802736%) i.e.,combination of wood apple and
ACV followed by wood apple (24.86011233%), ACV
(8.872902115%) and Standard drug (cystone -
4.418687526%).
4.DISCUSSION:
Hyperoxaluria is a major risk factor for CaOx
nephrolithiasis, which in turn is associated with renal
injury. High level of oxalate causes a variety of changes
in the renal epithelial cells, such as an increase in free
radical production and a decrease in antioxidant
status, followed by cell injury and cell death [15]. These
changes are significant predisposing factors for the
facilitation of crystal adherence and retention [ 17]
.Oxalate induced toxicity and free radical production
are attenuated in vivo[18] and in vitro[19] by
antioxidants. Phytotherapy for urolithiasis treatments
is an ancient method but it has garnered the attention
of researchers for its huge unexplored domain as it is
an easier, cost effective alternative compared to other
complicated treatment strategies.
Wood apple possess a high amount of Ascorbic acid,
flavonoids, terpenoids and other minerals which
contribute towards inhibiting the growth of renal
calculi. Ascorbic acid content was estimated through
titrimetric method based on its reducing nature and
the obtained results expressed in percentage weight
followed by estimation of flavonoids through
colorimetric method which was expressed in
Quercetin Equivalents. Similarly, total flavonoid
content was estimated in ACV followed by estimation
of acetic acid by titrimetric method. The obtained
results were compared with the % inhibition of
nucleation and aggregation shown by the respective
samples. Kidney stone formation is initiated by
forming small nuclear molecules of calcium oxalates
which aggregate to form insoluble crystals/masses. In
vitro studies of 10mg/mL methanolic extract of Wood
apple showed promising results in inhibition of
nucleation and aggregation of Calcium oxalate crystals.
The herb extracts may contain substances that inhibit
the growth of CaOx crystals. This property of plants
may be important in preventing the growth of kidney
stone. Aggregation may be an important factor in the
genesis of stones [20,21]. 1:10 diluted ACV showed better
% inhibition than the standard drug cystone and finally
the synergistic sample of 1:10 diluted ACV and 10 mg/
mL methanolic extract of Wood apple (mixed in a ratio
of 1:1) showed the highest % inhibition in comparison
to all the other samples.
5.SCOPE OF STUDY:
This study demonstrated the possession of inhibitory
properties of Wood apple and ACV towards Renal
calculi nucleation and aggregation in the in vitro
conditions. Also, when the two test extracts were
mixed and tested for inhibitory properties, the %
inhibition was shown to be the highest. This concludes
that there was no antagonism in between active
components of WA and ACV but in turn the samples
showed better inhibition in presence of each other.
Thus, proving their synergistic activity in urolithiasis.
The present work is underway and many more related
activities are yet to be tested such as - Calcium oxalate
crystal growth assay which checks the % inhibition of

International Journal of Pharmacy and Biological Sciences Kavita. G. Singh* et al
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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print)
Int J Pharm Biol Sci.
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aggregation of minerals around formed stones, Single
gel diffusion Growth assay in which the ability of the
sample to degrade synthetically grown stones is
checked and Kidney stones degradation assay in which
degradation of biologically formed (surgically
removed) kidney stones in presence of test samples is
tested. Further studies can be conducted to isolate the
active compounds and conducting invivo studies.
6. ABBREVIATIONS:
ACV - Apple Cider Vinegar
DCPIP - 2,6 -dichlorophenol indophenol
WA - Wood apple
NaCl - Sodium Chloride
QE - Quercetin equivalents
CaCl2 - Calcium Chloride
Tsc - Turbidity slope of control
AlCl3 - Aluminium Chloride
Tsi - Turbidity slope of indicator
NaOH - Sodium Hydroxide
CaOx - Calcium oxalate
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