Page 1 of 8
European Journal of Applied Sciences – Vol. 10, No. 1
Publication Date: February 25, 2022
DOI:10.14738/aivp.101.11798. Maryanti, K., Ginting, C. N., Chiuman, L., & Ginting, S. F. (2022). The Effect of Elixir of the Ethanol Extract of Rosa damascena on the
Solubility of Urine Struvite Crystal in Wistar Rats. European Journal of Applied Sciences, 10(1). 409-416.
Services for Science and Education – United Kingdom
The Effect of Elixir of the Ethanol Extract of Rosa damascena on
the Solubility of Urine Struvite Crystal in Wistar Rats
Kistia Maryanti
Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia
Chrismis Novalinda Ginting
Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia
Linda Chiuman
Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia
Sahna Ferdinand Ginting
Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia
ABSTRACT
Kidney stones are hard and solid masses formed by crystals in the urinary tract.
Hence it should be treated appropriately. The use of Rosa damascena might be an
option to help the recovery of the kidney and urinary tract. This study aims to show
the potential of the elixir of the ethanol extract of Rosa damascena on the solubility
of urine struvite crystal in Wistar rats. The experimental rats were divided into six
groups: the control group (K0); magnesium oxide as an inducer (K-); inducer and
Batugin elixir (K+); inducer and the elixir of the ethanol extract of Rosa damascena
250 mg/kg body weight (P1); 500 mg/kg body weight (P2) and 1000 mg/kg body
weight (P3). The treatment was carried out for 28 days by examining urine crystals;
urine pH was carried out on days 14 and 28, rat body weight measurements were
carried out on days 14 and 28. At the end of the treatment period, the rats were
necropsied using a combination of ketamine xylazine, and their kidneys were taken.
Observations were made on the ratio of kidney of rats (w/w) and measurements of
blood serum creatinine and urea levels using a spectrophotometer. The elixir of
Rosa Damascena’s ethanolic extract demonstrated the potential of extract
administration by raising weight gain, diminishing kidney weight ratio, reducing
creatinine and blood urea concentrations, and lowering the number of kidney
crystal deposits. The elixir of the ethanol extract of Rosa damascena has shown its
potential to dissolve struvite crystals with the best dose of 250 mg/kg BW based on
the number of urine crystals.
Keywords: Struvite crystal; R. Damascena; kidney crystal deposit; ethanol extract.
INTRODUCTION
Struvite stones are also known as infection stones or magnesium, ammonium, phosphate
stones. These stones are associated with urea-splitting bacterial infections (Proteus mirabilis,
Klebsiella pneumonia, Pseudomonas, Staphylococcus aureus, and Staphylococcus epidermidis)
[1]. Urea-breaking bacteria can make urine alkaline by hydrolyzing urea into ammonia, thus
Page 2 of 8
410
European Journal of Applied Sciences (EJAS) Vol. 10, Issue 1, February-2022
Services for Science and Education – United Kingdom
facilitating the formation of magnesium ammonium phosphate (MAP) stones and carbonate
apatite in an alkaline environment [2].
Modern management of kidney stones can be done using Extracorporeal Shockwave
Lithotripsy (ESWL), Percutaneous Nephro Litholapaxy (PCNL), open surgery, and conservative
therapy or expulsive medical therapy (TEM) [3]. However, modern medicine requires relatively
more expensive costs, so research is needed from natural ingredients for traditional medicine
at a more affordable price. Research on the treatment of kidney stones has been carried out by
several previous researchers with conventional plants that have a nephroprotective effect on
the ethanolic extract of balakka fruit [4] anticalculi activity of cucumber juice [5]. From the
studies that have been done, it was found that these plants have effectiveness in treating kidney
stones.
Roses are ornamental plants that have many benefits and uses in the form of extracts and oils
[6]. Roses contain geraniol and citronellol. Citral, linalool, nerol, eugenol, phenyl ethyl alcohol,
farnesol, nonialdehyde [7]. Rose flowers contain terpenoids, glycosides, flavonoids, and
anthocyanins [8]. The essential oils in roses are damascenone, damascone, and ionone, which
are obtained from the degradation of carotenoids. Roses also contain phosphorus, potassium,
calcium, magnesium, sodium, iron, copper, manganese, zinc, and boron [9]. Some of the
pharmacological activities of the rose plant are as an antioxidant and hepatoprotective [10],
analgesic, hypnotic, anti-spasmodic, anti-inflammatory [8], antidiabetic [11], anti-microbial,
anti-cancer [12]. Some reports on the nephroprotective effects of Rosa Damascena, such as rose
petals [13] and mill flowers [14], have been reported. To the best of our knowledge, the
potential use of elixir of ethanolic extract of Rosa damascena to fight against kidney stones is
still rarely found.
In this study, we will evaluate the elixir effect of the ethanolic extract of Rosa damascena on
urinary struvite crystal solubility and kidney histopathology in Wistar rats induced with
magnesium oxide. This study might be helpful in the development of new herbal medicine to
cure kidney stones recently.
MATERIALS AND METHODS
pH indicator, oral probe, rat cage, rat scale, analytical balance, light microscope, dropper
pipette, test tube, funnel, filter paper, blender, measuring cup, beaker glass, a set of surgical
instruments, rotary evaporator, syringe, microhematocrit, Eppendorf tube, micropipette,
centrifuge, microplate reader, spectrophotometer were among the materials used in this study.
The secondary chemical metabolites, including alkaloids, flavonoids, glycosides, tannins,
saponins, terpenoids, and steroids, were examined by phytochemical screening on Rosa
damascena extract [15], [16].
Experimental rats were randomly divided into six groups; each group had four rats. The group
divisions are:
a. Group K0 = consisting of 4 male Wistar rats who were given drinking plain water.
b. Group K- = 4 male Wistar rats induced with magnesium oxide 0.4%
c. Group K+ = 4 male Wistar rats induced magnesium oxide 0.4% and given Batugin elixir orally.
d. Group P1 = 4 male Wistar rats induced with magnesium oxide 0.4% and given the elixir of
the ethanol extract of Rosa damascena orally at a dose of 250 mg/kg body weight.
Page 3 of 8
411
Maryanti, K., Ginting, C. N., Chiuman, L., & Ginting, S. F. (2022). The Effect of Elixir of the Ethanol Extract of Rosa damascena on the Solubility of
Urine Struvite Crystal in Wistar Rats. European Journal of Applied Sciences, 10(1). 409-416.
URL: http://dx.doi.org/10.14738/aivp.101.11798
e. Group P2 = 4 male Wistar rats induced with magnesium oxide 0.4% and given the elixir of
the ethanol extract of Rosa damascena orally at a dose of 500 mg/kg body weight.
f. Group P3 = 4 male Wistar rats induced with magnesium oxide 0.4% and given the elixir of the
ethanol extract of Rosa damascena orally at a dose of 1000 mg/kg body weight.
The treatment was carried out for 28 days by examining urine crystals; urine pH was carried
out on days 14 and 28, rat body weight measurements were carried out on days 14 and 28. At
the end of the treatment period, the rats were necropsied using a combination of ketamine
xylazine, and their kidneys were taken. Observations were made on the ratio of kidney weight
to body weight of rats and measurements of blood serum creatinine and urea levels using a
spectrophotometer.
Quantitative data in the form of urine pH, the number of struvite crystals, body weight, the ratio
of the kidney (w/w) of rats, blood serum creatinine, and urea levels. Qualitative data in the form
of the chemical content of Rosa damascena extract and kidney histopathology. Statistical tests
were carried out with SPSS on quantitative data; if the data were homogeneous, the Anova test
was carried out. The Kruskal Wallis test is also carried out [17].
RESULTS AND DISCUSSION
Phytochemical content and pH analysis
The content in Rosa damascena extract consists of alkaloids, flavonoids, glycosides, saponins,
tannins, and triterpenes/steroids. The pH analysis on days 14 and 28 are shown in Table 1.
Table 1. The urine pH examination on days 14 and 28
Group
s Day-14 Day-28
K0 7.2 + 0.75 8.8 + 0.41
K- 6.5 + 0.55 8.2 + 0.75
K+ 6.8 + 0.75 9.2 + 0.41
P1 6.4 + 0.55 7.8 + 1.10
P2 6.3 + 0.82 8.2 + 1.17
P3 6.7 + 0.82 9.0 + 0.89
The results of the examination of the pH of the urine of rats on day 14 in all groups showed a
pH < 7 except for the K0 group, which was 7.2 + 0.75. On day 28, all groups showed an alkaline
pH outside the normal urine pH value of rats, namely 7.3-8, except for the P1 group with a pH
value of 7.8 + 1.10. A pH value > 7 facilitates the formation of struvite crystals [18]. Acid-base
regulation in the kidney determines the pH value [19].
Rat body weight analysis
The rat’s body weight was measured on days 0, 14, and 28, as shown in Table 2.
Page 4 of 8
412
European Journal of Applied Sciences (EJAS) Vol. 10, Issue 1, February-2022
Services for Science and Education – United Kingdom
Table 2. Bodyweight of rats on days 0, 14th and 28th
Groups Day-0 Day-14th Day-28th
K0 241.83 + 32.69
241.50 +
20.89 237.50 + 24.59
K- 229.67 + 31.74
240.00 +
30.49 222.83 + 25.14
K+ 240.17 + 23.84
255.83 +
37.62 238.33 + 16.32
P1 243.33 + 27.66
229.67 +
22.55 237.33 + 31.58
P2 220.67 + 24.13
220.67 +
21.00 215.20 + 19.01
P3 246.17 + 16.87
251.50 +
41.05 250.00 + 30.88
Weighing was conducted to see how each experimental animal was doing overall health. Day 0
refers to the day after the mice had adapted for a week before receiving treatment. Weight
increase was observed in the K-, K+, and P3 groups on day 14, but weight loss was observed in
the P1 group. Except for group P1, which gained weight after the treatment period (day 28), all
treatment groups (K0, K-, K+, P2, P3) lost weight at the end of the treatment period (day 28).
The status of a healthy and stable animal can be described by its body weight being generally
constant or gradually increasing. A drop or change in body weight, on the other hand, might
represent the status of an experimental animal suffering from health difficulties. The
restoration of physical condition is assumed to be attributed to a decrease in the concentration
of crystal components in the urine and antioxidant and anti-free radical action [20].
Urine crystal analysis
A light microscope was used to observe urine crystals at magnifications of 100 and 400 times.
Table 3 and Figure 1 show each animal group's different kinds of crystals.
Table 3. Type of Urine Crystal on day 14th and 28th
Groups Day-14th Day-28th
K0
Struvite
crystals
Struvite
crystals
K- Struvite
crystals
Struvite
crystals
K+
Struvite
crystals
Struvite
crystals
P1
Struvite
crystals
Struvite
crystals
P2
Struvite
crystals
Struvite
crystals
P3
Struvite
crystals
Struvite
crystals
Page 5 of 8
413
Maryanti, K., Ginting, C. N., Chiuman, L., & Ginting, S. F. (2022). The Effect of Elixir of the Ethanol Extract of Rosa damascena on the Solubility of
Urine Struvite Crystal in Wistar Rats. European Journal of Applied Sciences, 10(1). 409-416.
URL: http://dx.doi.org/10.14738/aivp.101.11798
Figure 1. The formation of urine crystals under a microscope
Number of urinary struvite crystals
The amount of the struvite crystal is shown in Table 4.
Table 4. Examination number of urinary struvite crystals
Groups
Day-14 Day-28
Number of
crystals Scoring
Number of
crystals Scoring
K0 7.50 + 2.35 ++ 6.83 + 2.71 ++
K- 31.50 + 14.47 +++ 31.50 + 16.08 +++
K+ 14.17 + 5.49 ++ 7.33 + 4.23 ++
P1 17.67 + 3.50 ++ 7.17 + 2.71 ++
P2 19.40 + 8.08 ++ 8.60 + 2.88 ++
P3 22.50 + 10.96 ++ 12.33 + 9.20 ++
The same type of crystal was found in all treatment groups, namely struvite crystals. Struvite
stones are formed by supersaturation of urine, where they grow into larger particles and turn
into crystals. If deposited in the urinary tract, larger crystals can form into struvite stones. In
humans, the enzyme urease converts urea into ammonia, and carbon dioxide is reduced to
carbonate, increasing urine pH and forming crystals [21], [22]. Struvite is usually prepared by
crystallizing three components: magnesium, ammonium, and phosphate [23].
On examination of the severity of crystals, namely struvite crystals, it was seen that all groups
showed the same range of crystal numbers, namely 6-20 crystals (++) on both the 14th and
28th days of observation. However, on the 28th day of observation, the number of struvite
crystals found in the K-group increased from the 14th-day observation, namely > 20 crystals
(+++). The number of struvite crystals in the K+, P1, P2, and P3 groups was less than the K- group on the 14th and 28th-day observations. On the 28th day of observation, the amount of
Page 6 of 8
414
European Journal of Applied Sciences (EJAS) Vol. 10, Issue 1, February-2022
Services for Science and Education – United Kingdom
calcium oxalate dihydrate in the P1 group was less than the K+, P1, and P3 groups. This
indicated that the P1 group could dissolve crystals and that there was an inhibitory activity of
struvite crystal formation for 28 days based on the number of crystals found. The P2 and P3
groups can also dissolve struvite crystals, although the more soluble crystals in the P1 group.
The ethanolic extract of roses contains flavonoid compounds; flavonoids have a role in
dissolving struvite crystals and have a urolithiasis effect [24].
Kidney to rat weight ratio
The ratio of kidney weight to rat weight can be seen in Table 5.
Table 5. Kidney to Weight Ratio of Rats
Groups Kidney weight
Kidney weight ratio
(g/100g)
K0 0.75 + 0,10 0.32 + 0,04
K- 0.73 + 0.09 0.33 + 0,02
K+ 0.82 + 0,08 0.34 + 0,02
P1 0.81 + 0,12 0.34 + 0,02
P2 0.70 + 0,10 0.33 + 0,05
P3 0.85 + 0,10 0.34 + 0,02
All groups had a kidney weight ratio value more significant than the control group (K0). Kidney
stones can cause an increase in kidney weight; this is due to high mineral levels in the kidneys
and swelling due to an inflammatory reaction. Rats will experience an increase in kidney weight
and the ratio of kidney weight to body weight due to the induction of magnesium oxide, which
forms struvite crystals [25].
Creatinine and urea levels
The results of measuring blood serum creatinine and urea levels can be seen in Table 6.
Table 6. Blood serum creatinine level measurement results
Groups
Creatinine level
(mg/dL)
Ureum level
(mg/dL)
K0 0.19 + 0.05 14.38 + 5.90
K- 0.21 + 0.04 13.85 + 2.99
K+ 0.18 + 0.03 11.20 + 2.15
P1 0.21 + 0.04 12.74 + 3.14
P2 0.44 + 0.08 14.45 + 7.00
P3 0.40 + 0.07 11.43 + 1.77
One of the screening tests for the renal function is creatinine analysis. Changes in serum
creatinine concentrations can be used to assess the health of nephrons in the urinary system.
Creatinine is a metabolite of creatine that has stable amounts in the blood and urine. This will
alter if the tubule is injured due to kidney stones [26]. Compared to the K+, P1, and P3 groups,
the P2 group had lower creatinine levels than the control group (K0) and the K- group. All of
Page 7 of 8
415
Maryanti, K., Ginting, C. N., Chiuman, L., & Ginting, S. F. (2022). The Effect of Elixir of the Ethanol Extract of Rosa damascena on the Solubility of
Urine Struvite Crystal in Wistar Rats. European Journal of Applied Sciences, 10(1). 409-416.
URL: http://dx.doi.org/10.14738/aivp.101.11798
the groups had creatinine levels that were within the normal range. Normal male white rats
had blood creatinine values of 0.20-0.89 mg/dL [27].
Because the kidneys generally eliminate urea and other nitrogen-rich waste products from the
blood vessels, a rise in urea levels can suggest renal failure [28]. Urea levels were lower in the
K+, P1, P2, and P3 groups than control (K0) and K- groups. All of the groups' urea levels were
within the usual range. Normal male white rats had blood urea values of 15.00-21.00 mg/dL
[29]. In normotensive rats, an elixir made from an ethanol extract of Rosa damascena, which
includes flavonoids, stimulates urine and electrolyte expenditure. Following flavonoid delivery,
the glomerular filtration rate (GFR) increased significantly. Because a high GFR causes the
kidneys to eliminate waste materials from the body quickly, small changes in glomerular
filtration or tubular reabsorption can result in quite substantial changes in urine excretion [30].
CONCLUSION
The elixir of Rosa damascena ethanol extract has the effect of dissolving struvite crystals in
wistar rats induced by 0.4 percent magnesium oxide. This study revealed that R. Damascena
extract might help the kidney and urinary tract healing. Further studies on the
pharmacokinetics and safety assessment of the new developed herbal drugs using Rosa
Damascena.
References
Rasyid N, Kusuma DGW & Atmoko W. Panduan penatalaksanaan klinis batu saluran kemih. Edisi Pertama. Ikatan
Ahli Urologi Indonesia (IAUI). 2018.
Halim P, Girsang E, Nasution AN, Lister INE & Lie S. Nephroprotective effect of ethanolic extract of balakka
(Phyllanthus Emblica L.) on rats induced ethylene glycol and ammonium chloride. Indonesian Journal of
Pharmaceutical and Clinical Research (IDJPCR). 2019; 2(2): 36-42.
D.V. Kishore, Fouzia Moosavi, DR. R. K. Varma. Effect of ethanolic of Portulaca oleracea Linn. on the ethylene
glycol and ammonium chloride-induced urolithiasis. International Journal of Pharmacy and Pharmaceutical
Sciences. 2013; 5(2): 134-140.
Ula DQ, Azizah N, Suryanto A. Pembungaan kembali tanaman mawar (Rosa Sp.) sebagai tanaman taman melalui
pemangkasan dan pemberian pupuk. Plantropica Journal of Agricultural Science. 2019; 4(1): 1-10.
Hariana, HA. Tumbuhan obat dan khasiatnya seri 3. Cetakan 3. Penebar Swadaya. Jakarta. 2007.
Boskabady MH, Shafei MN, Saberi, Amini S. Pharmacological effects of Rosa damascena. Iranian Journal of Basic
Medical Sciences. 2011; 14(4): 295-307.
Farnsworth NR Biological and phytochemical screening of plants. Journal of Pharmaceutical Sciences.1966;
55(3): 225-276.
Boskabady, Mohammad H, Mohammad NS, Zahra S, and Somayeh A. 2011. “Pharmacological Effects of Rosa
Damascena.” Iranian Journal of Basic Medical Sciences 14(4):295–307.
Akram, Muhammad, Muhammad R, Naveed M, Naheed A, Sadia Z, Farhat J, Mohammad AS, Naheed A Zarfishan R,
Syed HA, Muhammad D, Rabia Z, and Fahad SK. 2019. “Chemical Constituents, Experimental and Clinical
Pharmacology of Rosa Damascena: A Literature Review.” Journal of Pharmacy and Pharmacology 72(2):161–74.
Achuthan, C. R., B. H. Babu, and Jose Padikkala. 2003. “Antioxidant and Hepatoprotective Effects of Rosa
Damascena.” Pharmaceutical Biology 41(5):357–61.
Gholamhoseinian, A., H. Fallah, and F. Sharifi far. 2009. “Inhibitory Effect of Methanol Extract of Rosa Damascena
Mill. Flowers on α-Glucosidase Activity and Postprandial Hyperglycemia in Normal and Diabetic Rats.”
Phytomedicine 16(10):935–41.
Page 8 of 8
416
European Journal of Applied Sciences (EJAS) Vol. 10, Issue 1, February-2022
Services for Science and Education – United Kingdom
Mahboubi, Mohaddese. 2016. “Rosa Damascena as Holy Ancient Herb with Novel Applications.” Journal of
Traditional and Complementary Medicine 6(1):10–16.
Suherman, Chrismis NG, Linda C, Ali NN. 2021. " Nephroprotective Effect from Rose Petals (Rosa damascena)
Extract Against Cadmium Toxicity." 2021 IEEE International Conference on Health, Instrumentation &
Measurement, and Natural Sciences (InHeNce).
Khaliq T, F Mumtaz, ZU Rahman, I Javed and A Iftikhar, 2015. The nephroprotective potential of Rosa damascena
mill flowers, Cichorium intybus Linn roots, and their mixtures on gentamicin-induced toxicity in albino rabbits.
Pak Vet J, 35(1): 43-47.
Januar R, Wientarsih I, Widhyari SD. Uji kelarutan kristal kalsium oksalat urin tikus Spraque dawley setelah
pemberian eliksir ekstrak etanol daun pelawan. Jurnal Veteriner. 2020; 21(1): 106-114.
Verdes S, Fogazzi GB, Messa GPP, Daudon M. Crystalluria: prevalence, different types of crystals and the role of
infrared spectroscopy. Clin Chem Lab Med. 2011; 49(3): 515-520.
Steven L. Stockham, Michael A. Scott. 2008. Fundamentals of Veterinary Clinical Pathology. Blackwell Publishing.
The USA.
Narendra Nalwaya, E. Edwin Jerald, Suhail Asghar & Showkat Ahmad. 2009. Diuretic activity of a herbal product
UNEX. International Journal of Green Pharmacy. 225-226.
S. Bouanani, C. Henchiri, E. Migianu-Griffoni, N. Aouf, M. Leucovey. 2010. Pharmacological and toxicological
effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats. Journal of
Ethnopharmacology. 129: 38-45.
Nancy A. Brunzel. 2013. Fundamentals of urine and body fluid analysis. Third edition. Elsevier Saunders.
Missouri (US).
Jie Fan, Michael A. Glass & Paramgit S. Chandhoke. 1999. Impact of ammonium chloride administration on a rat
ethylene glycol urolithiasis model. Scanning Microscopy. 13(2-3) : 299-306.
G.B. Fogazzi. 1996. Crystalluria: a neglected aspect of urinary sediment analysis. Nephrol Dial Transplant. 379-
387.
Touhami M, Laroubi A, Elhabazi K, Loubna F, Zrara I, Eljahiri Y, Oussama A, Grasses F, Chait A. 2007. Lemon juice
has protective activity in a rat urolithiasis model. BMC Urology. 2007; 7:18.
Ghelani H, Chapala M, Jadav P. Diuretic and antiurolithiatic activities of an ethanolic extract of Acorus calamus L.
rhizome in experimental animal models. Journal of Traditional and Complementary Medicine. 2016; (30): 1-6.
Cruzan G, Corley RA, Hard GC, Mertens JJWM, McMartin KE, Snellings WM, Gingell R, Deyo JA. Subchronic toxicity
of ethylene glycol in Wistar and F-344 rats related to metabolism and clearance of metabolites. Toxicological
Sciences. 2004; 81:502-551.
Wientarsih I, Madyastuti R, Prasetyo BF, Aldobrata A. Anti lithiasis activity of avocado (Persea Americana Mill)
leaves extract in white male rats. Journal of Biosciences. 2012; 19(1): 49-52.
Dalton RN. Serum creatinine and glomerular filtration rate: perception and reality. Clinical Chemistry. 2010;
56(5): 687-689.
Laksmi NLGMC, Dada IKA, Damriyasa, IM. The bioactivity of the tapakdara leaf extract (Catharanthus roseus) on
creatinine and blood urea of rats (Rattus novergicus). Bulletin Veteriner Udayana 2014; 6(2): 147-152.
Suryawan DGA, Arjani IAMS, Sudarmanto IG. Gambaran kadar ureum dan kreatinin serum pada pasien gagal
ginjal kronis yang menjalani terapi hemodialisis di RSUD Sanjiwani Gianyar. Meditory. 2016; 4(2): 145-153.
Jouad H, Lacaille-Dubois MA, Lyoussi B, Eddouks M. Effecct of the flavonoid extracted from Spergularia purpurea
Pers. On the arterial blood pressure and renal function in normal and hypertensive rats. Journal of
Ethnopharmacology. 2001; 76: 159-163.