Protective Effects of Chives Leaves (Allium Schoenoprasum, L.) Infusion Against Ethylene Glycol and Ammonium Chloride Induced Nephrolithiasis in rats

Abstract

The present day medical management of calculi is either costly or not without side effects. Hence, the search of anti-calculi drugs from natural sources has been assumed greater importance. Chives (Allium schoenoprasum, L.) is a long-lived plant which is very easy to grow. It contained a variety of phytochemical compounds and various minerals. This aim of this research was to evaluate exposed of anti-calculi activity of fresh and dried chives leaves infuse on male albino Wistar rats using 0.75 % ethylene glycol and 2 % ammonium chloride induction method. Animals in treatment group are given 10 % of fresh and dried chives leaves infuse. Anticalculi activity in the chives leaves infuse examined by observing the kidney weight ratio, calcium level in kidney, urinary calcium level, urinary oxalate level, and creatinine serum level. From the data obtained, there were significant differences in treated group from negative control group. The biggest effect of dissolving the calcium oxalate crystal in kidney was the dried chives leaves infuse. Based on the above results, it could be concluded that chives leaves could be an alternative to calculi disease treatment.

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Journal of Applied Pharmaceutical Science Vol. 7 (08), pp. 222-225, August, 2017
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2017.70830
ISSN 2231-3354
Protective Effects of Chives Leaves (Allium Schoenoprasum, L.)
Infusion Against Ethylene Glycol and Ammonium Chloride Induced
Nephrolithiasis in rats
Ginda Haro1, Siti Morin Sinaga1, I. Iksen1*, N. Nerdy1, Suthinee Theerachetmongkol2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sumatera Utara, Medan, Indonesia.
2Department of Internal Medicine, Surin Hospital, Surin, Thailand.
ARTICLE INFO
ABSTRACT
Article history:
Received on: 06/05/2017
Accepted on: 21/06/2017
Available online: 30/08/2017
The present day medical management of calculi is either costly or not without side effects. Hence, the search of
anti-calculi drugs from natural sources has been assumed greater importance. Chives (Allium schoenoprasum,
L.) is a long-lived plant which is very easy to grow. It contained a variety of phytochemical compounds and
various minerals. This aim of this research was to evaluate exposed of anti-calculi activity of fresh and dried
chives leaves infuse on male albino Wistar rats using 0.75 % ethylene glycol and 2 % ammonium chloride
induction method. Animals in treatment group are given 10 % of fresh and dried chives leaves infuse.
Anticalculi activity in the chives leaves infuse examined by observing the kidney weight ratio, calcium level in
kidney, urinary calcium level, urinary oxalate level, and creatinine serum level. From the data obtained, there
were significant differences in treated group from negative control group. The biggest effect of dissolving the
calcium oxalate crystal in kidney was the dried chives leaves infuse. Based on the above results, it could be
concluded that chives leaves could be an alternative to calculi disease treatment.
Key words:
Anticalculi, Chives, Infuse,
Ethylene Glycol, Calcium
Oxalate, Atomic Absorption
Spectrophotometry.
INTRODUCTION
Kidney stone or calculi disease has become a rising
problem and the third relevant disorder affecting the urinary tract
with high recurrence. The majority (up to 80%) of all stone is
mainly composed of Calcium oxalate. The present day medical
management of calculi is either costly or not without side effects.
Hence, the search of anti-calculi drugs from natural sources has
assumed greater importance (Amin, et al., 2015). Herbal drugs
are the oldest sources for the preventing, medication, treatment
and even diagnostic tools know to mankind.
* Corresponding Author
Iksen, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Sumatera Utara, Medan, Indonesia.
Mailing address: Jl. A.R.Hakim No. 179, Medan 20216, Indonesia
Phone: +6283189352214, Email: ikseniksen08 @ gmail.com
Most of the drugs commonly used today are herbal
origin. Herbal drugs are inexpensive and effective with fewer side
effects (Manimala, et al., 2014). Chives (Allium schoenoprasum
L.) is a long-lived plant which is very easy to grow (Iksen, 2017).
Chives can be used to treat many diseases such as vaginal
discharge, constipation and infectious germs in the intestines,
speed up the blood flow, prevent blood clotting, antioxidants,
breast cancer, antiinflammatory and antihypertensive (Andarwulan
and Faradilla, 2012; Amalia, et al., 2008; Jalkumar and Jasmine,
2016; Parvu, et al., 2014; Sachinm, et al., 2010) .
Chives leaves contain a variety of phytochemical
compounds included alkaloids, flavonoids, glycosides, steroids,
tannins and various minerals such as potassium, magnesium and
sodium, which contains high potassium is believed to dissolve
calcium oxalate in kidney stones, where one of the main factors
affecting solubility calcium is potassium (Al-Snafi, 2014; Iksen, et
al., 2017).

Haro et al. / Journal of Applied Pharmaceutical Science 7 (08); 2017: 222-225 223
Infusion technique has several advantages when
compared with the manufacture of the extract that is cheaper,
faster and a tool used quite simple (Alvin, 2015).In our previous
study, we used fresh and dried chives leaves infuse to dissolve
calcium oxalate and found that chives leaves infuse can dissolve it
(Iksen, et al., 2017).
Therefore, the aim of the present study was to evaluate
the effect of the anticalculi activity of fresh and dried chives leaves
infuse on male albino wistar rats using 0.75 % ethylene glycol and
2 % ammonium chloride induction method.
MATERIALS AND METHODS
Materials
Ammonium chloride, Batugin Elixir®, demineralized
water, ethylene glycol, fresh and dried chive leaves, nitric acid,
and the standard solution of calcium 1000 mg/mL
Methods
Preparation of fresh and dried chive leaves infuse solution
Chives leaves infusion solution was made with
two kinds of which are fresh and dried chives leaves infuse
solution. The dose used was 10% (both 10 g of fresh and dried
chives leaves voiled in 100 mL 90oC water) w/ v in accordance
with the infuse criteria in the Indonesian Pharmacopoeia Edition
IV.
Selection of Animals for In Vivo Studies
For the purpose of anticalculi studies, adult male Wistar
albino rats weighing around 170 to 180 g were selected. The
animals were acclimatized to standard laboratory conditions and
maintained for 12 hours light and dark cycle. They were provided
with regular rat chow and drinking water ad libitum. Our
Institutional Animal Research Ethics Committees (AREC)
Approval No: 697/KEPH-FMIPA/2016.
Animal Groupings
In this study, animals were divided into five groups,
each with five rats. The group I act as normal group. Group II, act
as calculi negative control where animals received 0.75% ethylene
glycol with 2% ammonium chloride in drinking water for 20 days.
Group III (positive control) animals received 0.75% ethylene
glycol with 2% ammonium chloride in drinking water along with
Batugin Elixir® drug from the first day till the tenth day.
Rats in group IV and V were treated respectively with fresh
and dried chives leaves infuse solution from day eleven till last
day.
Parameters Assayed for Anticalculi Studies
The biochemical parameter for the anticalculi studies
were the kidney weight ratio, calcium level in kidney, urine
volume, calcium and oxalate level in urine and serum creatinine
level. All parameters was analysed by standard procedures.
Preparation of Sample
Preparation of Calcium Calibration Curve
A total of 5 mL of 1000 ppm calcium (the mother liquor)
was added to a 100 mL columetric flask and then added aquabidest
right to mark boundaries, the obtained raw calcium was 50 ug/mL.
Each of 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL pipetted calcium
standard solution 50 ug/mL in a 50 mL volumetric flask to obtain
successive concentration of 2 ppm; 4 ppm; 6 ppm; 8 ppm and 10
ppm and measured by atomic absorption spectrophotometry at a
wavelength of 422.7 nm. Then obtained a calibration curve of
calcium
Destruction of Samples
Each kidney obtained from the rats will be dried in oven
(1000C) for 24 hours and then heated in 5400C for eight hours and
then diluted with nitric acid and demineralized water
Determination of Calcium Levels in Kidney
The sample solution that has been prepared with the
absorbance measured using flame atomic absorption spectro-
photometry at a wavelength of 422.7 nm for calcium (Iksen, et al.,
2017). Absorbance values obtained should be within the range of
the calibration curve of calcium standard solution. The calcium
level will be calculated based on the regression equation of the
calibration curve.
Collection and Analysis of Urine for Biochemical Assays.
All animals were kept in individual metabolic
cages and 24 hours urine samples were collected on 20th day of
calculi induction treatment. The volume of urine, urine calcium
and oxalate were measured at the respective time points. Animals
had free access to drinking water during the urine collection
period. A drop of concentrated hydrochloric acid was added to
urine before being stored at -20oC until analyzed.
Serum Analysis
Blood was collected from rats using standard procedure.
Separated serum was analysed for creatinine.
Statistical Data Analysis
Results were given as Mean ± SEM from five animals in
each groups. Comparisons were made by ANOVA method with
SPSS 21. P (probability value) < 0.05 was considered significant.
RESULTS AND DISCUSSION
Selection of Animals
Adult male abino Wistar rats were selected to induce
nephrolithiasis (calculi) because of the urinary system of male rats
resembles that of humans. Previous studies have shown that the
amount of calcium oxalate stone deposit in female or young rats

224
Haro et al. / Journal of Applied Pharmaceutical Science 7 (08); 2017: 222-225
was significantly less. That is why in this research we used the
adult male albino Wistar rats.
Calibration Curve of Calcium
The calibration curve of calcium has the range for the
concentration which were measured on concentration of 2 ppm,4
ppm, 6 ppm, 8 ppm and 10 ppm. The correlation coefficient
obtained from this metal can be accepted as the appropriate
requirements for the correlation coefficient which should not
smaller than 0,995. Coefficient above suggested a linear
relationship between the concentration of the metal and
absorbance (Iksen, et al., 2017). The calibration curve of calcium
is shown in Figure 1.
Fig. 1: The calibration curve of calcium standard solution.
The kidney body weight ratio and calcium level in kidney
Metabolites of ethylene glycol such as glycolaldehyde,
glycolate and oxalate can induce tissue damage, hyperoxaluria and
the calcium oxalate calculi (Hadjzadeh, et al., 2008).In these
studies, chronic administration of 0.75% ethylene glycol and 2 %
ammonium chloride to male albino Wistar rats increased the
kidney weight ratio and the calcium level in kidney (Group II/
Control Negative). The calcium level was significantly increased
when compared to normal animals are shown on Table 1 and
Figure 2.
The calcium levels were decreased while treated with
fresh and dried chives infuse solution. The treatment with chives
leaves infuse solution significantly decreased the calcium levels.
These results indicated that chives leaves infuse treatment shows
improvement in renal function compared to the group II/ control
negative. Potassium in chives leaves infuse was believed can
dissolve the calcium oxalate precipitate caused by the location of
potassium in the Volta’s row to the left side from calcium, so it
will get rid of potassium calcium oxalate compound to join, and
will dissolve calcium compounds. The more to the left of an
element in Volta row, the reductant power will getting stronger.
This means that the element will be able to reduce ions of the
elements from its left side (Anggraini, 2015; Iksen, 2017). The
other factor to dissolve calcium oxalate is flavonoids in chives
leaves infuse. The other research investigation also said that the
effect of inhibiting calculi is from the antioxidant effect of the
flavonoids. It seems that flavonoids in chives could play an
antioxidant role against oxidative stress that is induced by the
ethylene glycol (Shekha, et al., 2015).
Table 1: The biochemical parameter results in kidney.
Group
Kidney weight
ratio
(mg/100 g)
Calcium level in
kidney
(mg/ g)
Group I (Normal)
0.66± 0.006
2.587 ± 0.221
Group II (Negative Control)
0.95± 0.03*a
4.377 ± 0.170*a
Group III (Positive Control)
0.735± 0.025*b
2.815 ± 0.137*b
Group IV (Fresh chives leaves infuse)
0.75± 0.025*b
2.838 ± 0.091*b
Group V (Dried chives leaves infuse)
0.75± 0.011*b
2.726 ± 0.08*b
Values are expressed as Mean ± SEM, n=5; Comparison: a (Group I vs Group
II), b (Group II vs Group III, IV, and V); * P<0.05.
Fig. 2: Calcium Level in Kidney
Effect on the Urinary and Serum Parameters
The super-saturation of urine is the main reason
responsible for the calculi stone forming. The results ethylene
glycol and ammonium chloride induction administration increased
the level of calcium, oxalate and creatinine level in urine.
Ammonium chloride can accelerating stone formation through
metabolic acidosis (Iksen, 2017).
The 24 hour excretion of calcium was increased
significantly on 20th day in negative control group rather than other
treatment group. Similarly increased in urinary oxalate was
increased significantly on 20th day in negative control group rather
than other treatment group. Simultaneous treatment with fresh and
dried chives leaves infuse solution reduced the increase in urinary
calcium and oxalate. The level of urinary oxalate and urine volume
are shown on Table 2.
In Figure 3, we can see that the creatinine level was
increased significantly on 20th day in negative control group
compared with other treatment group. This creatinine level was
reduced in both positive control and chives leaves infuse solution.
0
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
4,500
5,000
Group I
Group II
Group III
Group IV
Group V
Calcium Level in Kidney (mg/g)

Haro et al. / Journal of Applied Pharmaceutical Science 7 (08); 2017: 222-225 225
CONCLUSION
From the anticalculi in vivo test result, it can be
concluded that fresh and dried chives leaves infuse solution have
the potential as anticalculi. It can be seen from the decreased
kidney weight ratio, calcium level in the kidney, and the
biochemical parameters in urine such as the calcium and oxalate
level.
ACKNOWLEDGMENTS
Administrative Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Universitas Sumatera Utara and Surin
Hospital, Thailand.
Financial support and sponsorship: Nil.
Conflict of Interests: There are no conflicts of interest.
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How to cite this article:
Haro G, Sinaga SM, Iksen I, Nerdy N, Theerachetmongkol S.
Protective Effects of Chives Leaves (Allium Schoenoprasum, L.)
Infusion Against Ethylene Glycol and Ammonium Chloride
Induced Nephrolithiasis in rats. J App Pharm Sci, 2017; 7 (08):
222-225.
Table 2: The biochemical parameter results in urine.
Group
Calcium Level (mg/24 h)
Oxalate Level (mg/ 24 h)
Volume of Urine (mL)
Group I (Normal)
2.27 ± 0.58
2.98 ± 0.18
4.4 ± 0.40
Group II (Negative Control)
5.65 ± 0.61*a
6.70 ± 0.34*a
2.48 ± 0.26*a
Group III (Positive Control)
2.72 ± 0.58*b
3.83 ± 0.21*b
5.94 ± 0.32*b
Group IV (Fresh chives leaves infuse)
2.87 ± 0.17*b
4.11 ± 0.61*b
4.8 ± 0.76*b
Group V (Dried chives leaves infuse)
2.46 ± 0.32*b
3.43 ± 0.19*b
5.43 ± 0.26*b
Values are expressed as Mean ± SEM, n=5; Comparison: a (Group I vs Group II), b (Group II vs Group III, IV, and V); * P<0.05.
Fig. 3: The creatinine level in blood serum.
0
0.5
1
1.5
2
2.5
Group I
Group II
Group III
Group IV
Group V
Creatinine Level
Creatinine Level