Research Paper

Preventive treatment of calcium oxalate crystal depositionwith immortal flowers

Nilüfer Orhan a,n, Metin Onaran b, İlker Şen b, İpek Işık Gönül c, Mustafa Aslan a

a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkeyb Department of Urology, Faculty of Medicine, Gazi University, 06560 Ankara, Turkeyc Department of Pathology, Faculty of Medicine, Gazi University, 06560 Ankara, Turkey

a r t i c l e i n f o

Article history:Received 29 August 2014Received in revised form24 November 2014Accepted 7 January 2015Available online 21 January 2015

Keywords:Calcium oxalate crystallizationHelichrysumAsteraceaeImmortal flowersKidney stoneUrolithiasis

a b s t r a c t

Ethnopharmacological relevance: A number of medicinal plants are used for their diuretic, urolithiatic andanti-inflammatory effects on urinary system problems in Turkey and the most common traditionalremedy for kidney stones is the tea of immortal flowers. The aim of this study is to evaluate thepreventive effect of infusions prepared from capitulums of Helichrysum graveolens (M.Bieb.) Sweet (HG)and Helichrysum stoechas ssp. barellieri (Ten.) Nyman (HS) on formation of kidney stones.Materials and method: Sodium oxalate (Ox-70 mg/kg intraperitoneally) was used to induce kidney stoneson Wistar albino rats. At the same time, two different doses of the plant extracts (HG: 62.5 and 125 mg/kg; HS: 78 and 156 mg/kg) were dissolved in the drinking water and administered to animals for 5 days.Potassium citrate was used as positive control in the experiments. During the experiment, water intake,urine volume and body weights of the animals were recorded. At the end of the experiments, liver,kidney and body weights of the animals were determined; biochemical analysis were conducted onurine, blood and plasma samples. Histopathological changes in kidney tissues were examined andstatistical analysis were evaluated.Results: HS extract showed the highest preventive effect at 156 mg/kg dose (stone formation score: 1.16),whereas a number of kidney stones were maximum in sodium oxalate group (stone formation score:2.66). Helichrysum extracts decreased urine oxalate and uric acid levels and increased citrate levelssignificantly. In addition, Helichrysum extracts regulated the negative changes in biochemical andhematological parameters occurred after Ox injection.Conclusions: We conclude that Helichrysum extracts could reduce the formation and growth of kidneystones in Ox-induced urolithiasis and can be beneficial for patients with recurrent stones. In addition,this is the first study on the preventive effect of immortal flowers.

& 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Urolithiasis is a growing health problem in industrializedcountries and often correlated with habits such as hypertension,high purine intakes, diabetes, obesity, and metabolic syndrome(Rosa et al., 2013). Unexpectedly, 12% of the world's population isaffected by urinary system stone disease (Pak, 1998; Basavarajet al., 2007). Urolithiasis is formation of kidney stones in urine-collecting spaces of the kidneys. Under certain conditions, sub-stances normally dissolved in the urine can separate out as crystalsand accumulate to form a solid mass called kidney stone. Stones canmigrate into the ureters, the bladder and finally be evacuated in the

urine. Even surgical procedures, drug treatment, extracorporealshock wave lithotripsy and endoscopic techniques are commonlyused to treat stones; none of these options inhibit formation of newkidney stones. It is established than the recurrence rate of calciumoxalate stones is 10.5% in the first year; but it reaches 50% at thetenth year (Menon et al., 1998). Thus, after treatment, there is agreat demand for nonsurgical methods to prevent the recurrence ofurolithiasis.

In recent years, beside drug treatment, herbal medicines and theircomponents have been widely investigated (Rosa et al., 2013). Datafrom in-vitro, in-vivo and clinical trials revealed that phytotherapeuticagents could be usefull as either an alternative or a complementarytherapy in the management of urolithiasis (Butterweck and Khan,2009). Therewithal, various plants and herbal preparations have beenused for treatment of kidney stones since ancient times. Amongthese, infusions prepared from capitulums of Helichrysum species are

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2015.01.0090378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

n Corresponding author. Tel.: þ90 312 2023173; fax: þ90 312 2235018.E-mail address: nsendogdu@gazi.edu.tr (N. Orhan).

Journal of Ethnopharmacology 163 (2015) 60–67

used all around Turkey (Baytop, 1999). Many ethnobotanical studiesrevealed that different species of Helichrysum genus such asHelichrysum arenarium (Sargın et al., 2013), armenium (Özüdoğru etal., 2011), compactum (Kargıoğlu et al., 2010), orientale (Gürdal andKültür, 2013), pallasi (Altundağ and Öztürk, 2011), plicatum (Honda etal., 1996), and stoechas (Tuzlacı and Sadıkoğlu, 2007) have been usedto pass kidney stones (Tabata et al., 1993). Helichrysum stoechas andHelichrysum graveolens, most common and widely grown species inTurkey, are sold for their medicinal purposes including diuretic andanti-urolithiatic effects in herbal shops called aktar.

Nineteen Helichrysum species (Asteraceae) grow in Turkey(Sümbül et al., 2003), and many of them have been widely usedfor stomachache, as anti-asthmatic, anti-diabetic, diuretic, lithagogue,to pass kidney stones, and as herbal tea in Turkish folk medicine(Sezik et al., 1991; Sezik et al., 2001). According to traditional usage,number of research works have been worked on different biologicalactivities of Helichrysum species (Süzgeç et al., 2005; Aslan et al.,2007a, 2007b; Sezik et al., 2010; Orhan et al., 2014).

In vitro models of experimental urolithiasis relate to only oneevent and aspect of the kidney stone disease. Thus, animal modelsare widely preferred in order to understand all aspects of thepathogenesis, including the anatomic and pathological role ofkidneys. The objective of this study is to evaluate the preventiveand beneficial effects of Helichrysum species on experimentalcalcium oxalate crystallization in rats.

To determine the preventive effect of the extracts on renalcalculi formation, Helichrysum graveolens and Helichrysum stoechasssp. barellieri capitulum extracts were administered to rats withsodium oxalate (Ox-70 mg/kg i.p.) simultaneously for 5 days. Meta-bolic cages were used to measure water intake and urine volumesfor 24 h. Plasma and urine biochemical parameters were measuredand histopathological studies were waged in the experiments.

2. Material and methods

2.1. Plant materials

Helichrysum graveolens (M.Bieb.) Sweet (HG)was collected fromIlgaz Mountain, Kastamonu/Turkey (July 2010) and Helichrysumstoechas ssp. barellieri (Ten.) Nyman (HS) was collected from theopen field near Saint Pierre Church, Antakya/Turkey (May 2011).Plant species were identified by Prof. Dr. Mustafa Aslan andherbarium specimens are deposited in the Herbarium of GaziUniversity Faculty of Pharmacy (GUE 2490, 2491).

2.2. Preparation of the extracts

Infusions [3% (w/v)] of dried capitulums of Helichrysum specieswas prepared in water on a water bath by mixing for 30 min. Theextract was filtered using filter paper and concentrated undervacuum using rotary evaporator at 45 1C and extracts were dried ina freeze dryer. The yields of the extracts were: HG: 18.0%, andHS: 22.5%.

The doses of the plant extracts were calculated according tofolkloric knowledge and yield of the extracts. In Turkey, a cup ofinfusion or decoction (3%) of the Helichrysum capitulums is drunkthree times a day for 10 days to pass kidney stones (Baytop, 1999).Before the experiments, daily water intake of the rats in metaboliccages were monitored. The quantity of the dried extract ofHelichrysum capitulums to be weighed was calculated pursuantto the doses and dissolved in drinking water according to themean of daily water consumption.

2.3. Animals

Wistar Albino male rats (200–250 g) were used in this studywith the approval of Animal Experiments Local Ethical Committee(GUET-09.079). Experiments were performed according to theinternational rules considering the animal experiments and bio-diversity rights. Animals were purchased from the animal breed-ing laboratories of Experimental Animal Research Center of GaziUniversity (GUDAM) (Ankara, Turkey) and the experiments werecarried on at the same research center. The animals were main-tained on standard pellet diet and water ad libitum throughout theexperiment.

2.4. Experimental procedures

Animals were divided into seven groups of six rats each.Sodium oxalate (70 mg/kg) dissolved in physiological saline wasadministered intraperitoneally (i.p.) for 5 days to induce urolithia-sis (Bouanani et al., 2010). To evaluate the effect of Helichrysumextracts on stone formation, extracts were given to animals bymixing to drinking water during Ox administration. Only daily dietand water was given to control group. Potassium citrate wasadministered to positive control (4 meq/daily per rat) by addingto drinking water. HG (62.5 and 125 mg/kg) and HS extracts (78and 156 mg/kg) were given to rats at two different doses by addingto drinking water for 5 days.

On the 5th day, body weights of the animals were recorded thenrats were anesthetized with ketamine/xylazine and the bloodsamples were withdrawn by cardiac puncture. The liver and kidneyswere removed from the abdominal cavity quickly, cleaned with icecold physiological saline and weighted. In order to compare thechanges in the organ weights, relative organ weights were calcu-lated after the experiment according to the formula

Relative Organ Weight¼[Organ Weight/Body Weight (at thelast day)]�100

Kidneys were used to examine histopathological changes andcalcium oxalate crystals in tissues.

2.5. Hematological and biochemical analysis

Blood samples were collected from the heart into two tubes forbiochemical and hematological analysis (a heparinized tube and atube with EDTA). In order to determine biochemical parameters,blood samples collected in heparinized tubes were centrifuged at3000g for 10 min to obtain plasma. For the hepatic functions, totalbilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT),and aspartate aminotransferase (AST) were determined from plasmasamples. In addition, some lipit parameter such as cholesterol,triglyceride, high density lipoprotein (HDL), very low density protein(VLDL) and low density lipoprotein (LDL), blood urine nitrogen(BUN), creatinine, uric acid, total protein, albumin and levels of someelements (Ca2þ , Mg2þ , Naþ , Kþ , and Cl- ions) were evaluated. Alsohematological analysis were carried out by a Beckman Coulter LH780blood autoanalyzer.

Urine samples of each group were collected from metaboliccages after 24 h. Urine citrate, oxalate, uric acid, BUN, creatinine,total protein, albumin, and levels of some elements (Ca2þ , Naþ ,Kþ , and Cl- ions) were measured from the urine samples of the lastday. All biochemical parameters were determined by BeckmanCoulter AU2700, oxalate and citrate levels were measured by aHitachi U2700 spectrophotometer.

2.6. Histopathological analysis

For the histopathological studies, the kidney tissue samples fromboth kidneys of the animal were fixed in 10% formalin for a period

N. Orhan et al. / Journal of Ethnopharmacology 163 (2015) 60–67 61

of at least 24 h. The paraffin sections were then prepared and cutinto 5-mm thick sections in a rotary microtome. The sections werestained with Haematoxylin-eosin dye and mounted with Canadabalsam. The histopathological examination of slides were per-formed under plain and polarized light microscope and photo-graphed by an Olympus Digital Camera. Histopathologic changes,aggregation of calcium oxalate crystals and stones in the kidneytissues were observed. All animals were scored according to thedensity of the crystals observed in the unit area and average groupscores were calculated pursuant to scores of all animals in thesame group.

2.7. Statistical analysis

Values were presented as means7standard deviation (S.D.).Statistical differences between the treatments and the controlswere tested by one-way analysis of variance (ANOVA) followed bythe Student-Newman-Keuls test using the “GraphPad Instat 3”statistic computer program. A difference in the mean values ofpo0.05 was considered to be statistically significant.

3. Results

In this study, the preventive and beneficial effects of Helichrysumextracts on stone formation were investigated. After examining allkidney sections, the stone formation rate of the kidney tissues wasscored from 0–3 and average group scores were calculated. Fig. 1shows photomicrography of the kidney sections that demonstratedifferent group scores having different density of kidney stones. Nostructural abnormalities, bleeding, inflammation, calcium oxalateand calculus accumulation were observed in kidney tissues of thecontrol group in the histopathological examinations. However, largeamounts of calcium oxalate crystals, accumulated in large aggre-gates were examined in the kidney sections of negative control (Ox)group animals. As shown in Table 1, the stone formation scores ofall groups were considered significant compared to control group.Stone formation and accumulation in Ox group was too much asexpected and its stone formation score was found to be the highest(2.66). The preventive effect of Helichrysum graveolens extracts onstone formation was promising at both doses. The stone formationscore of HG at 62.5 mg/kg dose (1.83) was lower than the score ofpositive control group (2.16). HS extracts were found to havesignificant and dose dependent preventive effect on stone forma-tion (stone formation score is 2.0 at 78 mg/kg dose and 1.16 at156 mg/kg).

It was observed that urine volumes and water consumptions of allanimals were similar at the beginning of the experiment. But in thelast day of the experiment, urinary volume/water consumption ratioof positive control (potassium citrate) group was decreased to 0.35. Aparallel reductionwas observed in HG 62.5 mg/kg (0.48), HS 78 mg/kg(0.48) and HS 156mg/kg (0.43) dose groups. According to the analysisof the urines, high doses of the Helichrysum extracts have increasedurine citrate levels (HG: 9.6 and HS: 12.2 mg/dl). In addition, HS at78 mg/kg and HG at 125 mg/kg dose have decreased urine oxalatelevels significantly compared to control (Table 1). As known thatincreased amount of uric acid in the urine, lead to the formation ofuric acid stones. Helichrysum extracts have decreased uric acid levelsat all tested doses in this experiment significantly (0.40–0.73 mg/dl).This finding is an outstanding result of our experiment.

Body weights of animals were recorded in the beginning, 3rd and5th day of the experiment. As demonstrated in Table 2, all the groupstreated with Ox lost weight during the experiment. The highestdecrease was observed in the HS 156 mg/kg dose group. On thecontrary, relative organ (liver and kidney) weights of the positivecontrol and extract groups were not changed significantly (Table 2).

Effect of Helichrysum extracts on blood liver enzyme levels ofWistar rats was given in Table 3. It was concluded that Ox injectionincreased all measured enzyme (AST, ALT, ALP) levels significantly,whereas HG and HS reverse that effect significantly withoutchanging total bilirubin levels. Also, potassium citrate decreasedthese enzyme levels, but its effect was not as much as Helichrysumextracts. On the other hand, elevations in lipid levels (cholesterol,LDL and HDL) by Ox injection were reduced by both doses of HGand HS. No significant change was observed in triglyceride andVLDL levels.

Clinical biochemistry and hematology results of blood and urinesamples are shown in Tables 4–6. The blood analysis reported inTable 4 shows that BUN and creatinine levels were significantlyincreased in Ox group. HG and HS decreased BUN and creatininelevels at both tested doses and also lowered uric acid, total protein,and albumine levels significantly. Magnesium levels of all groupswere increased compared to control but no significant differencewas observed in the other ion concentrations of blood samples.

The hematological results given in Table 5 reveal significantdecreases in RBC, hemoglobin, hematocrit, lymphocytes and sig-nificant increases in WBC levels in the groups treated with HG andHS. On the other hand, all the changes in hematological parametersof all groups were in the normal ranges (Teixeira et al., 2000). Inaddition, administration of HG and HS lowered urine levels of BUN,creatinine and calcium, sodium, potassium ions at all tested dosessignificantly (Table 6).

4. Discussion

Ethnobotanical studies in Turkey revealed that a large number ofplants were used as diuretics in urological problems such as kidneydiseases, calculi in the kidney and ureter, dysuria, nocturia, cystitis,inflammation, for prostatitis, gynecological disorders, fertility inwoman or as an aphrodisiac (Sezik et al., 2001). Among these,Helichrysum infusions and decoctions are the most common andwell known remedies to pass and restrain urinary stones (Baytop,1999). Although many plants are popular in traditional medicineand used in urolithiasis, there are only a few studies on their exactrole, efficacy and side effects (Günocak and Küpeli, 2006).

Calcium, struvite, uric acid, cystine stones and others are the fivemain types of kidney stones. In fact, nearly 80 percent of all kidneystones are composed of calcium and oxalate. On the literature survey,it was concluded that sodium oxalate injection is one of the best wayto induce experimental oxalate stones in animals. Thus, in this study,effects of Helichrysum extracts on Ox induced urolithiasis werestudied in detail. According to histopathological examinations, itwas determined that HG and HS inhibited formation and accumula-tion of oxalate crystals without causing cell degeneration, bleedingand inflammation significantly. HS extracts were found to havepotent dose dependent preventive effect on stone formation.

It is known that hypocitraturia, a low amount of citrate in theurine, is an important risk factor for kidney stone formation. Inaddition, increased concentrations of oxalate in the urine, can leadto the deposition of calcium oxalate in the kidney tissue or urinarytract. Moreover, increased amount of uric acid in the urine leads tothe formation of uric acid stones. In our study, Helichrysum extractsincreased urine citrate levels and also decreased oxalate and uricacid levels significantly. These data supported the histopathologicalfindings shown in Table 1 and Fig. 1.

Liver function panel test that is commonly used to evaluate theliver for injury, infection, or inflammation, measures the bloodlevels of total protein, albumin, bilirubin, and liver enzymes (AST,ALT, and ALP). Elevated liver enzymes and high bilirubin levelsare important indicators of liver and red blood cell damage. Inour study, it was observed that Helichrysum extracts decreased all

N. Orhan et al. / Journal of Ethnopharmacology 163 (2015) 60–6762

measured liver enzyme (AST, ALT, ALP) levels significantly by Oxinjection without changing total bilirubin levels. Clinical biochem-istry and hematology results of blood and urine samples showedthat HG and HS regulated the negative effects of Ox injection andimproved many biochemical parameters significantly.

Serum creatinine and BUN levels are often monitored to evaluaterenal function. While serum creatinine increases only with nephrondamage, the BUN is affected by hydration, hepatic metabolism ofprotein and reduced glomerular filtation rate. Also significant increa-seas in protein, urea, creatinine, uric acid levels in blood and urineare the evidences of nephrotoxicity (Grover and Resnick, 1995). In ourstudy, creatinine, BUN and uric acid levels in blood and urea isdecreased significantly thus nephrotoxicity caused by Ox injection isabated by HG and HS. Our results suggest that both Helichrysumspecies has protective effect on kidney and liver tissue and pre-ventive effect on crystal deposition in urolithiasis model of rats.

There is only one study on the effect of Helichrysum species onkidney stones. In this study, Bayır et al. (2011) have investigated thepreventive effect of Helichrysum plicatum ssp. plicatum (HP) at 125,250 and 500 mg/kg doses in 1% ethylene glycol and 1% ammoniumchloride-induced urolithiasis model in rats. 21 days administration ofHP extracts have shown that increased plasma and urine biochemicalparameters after induction of urolithiasis have been significantlydecreased compared to healthy rats. Urine calcium oxalate levelshave been also decreased by HP extracts. It is suggested that HP hasboth protective effect on tubular structures and preventive effect onintratubular crystal deposition in urolithiasis model of rats. Even if adifferent method has been used to induce urolithiasis, the results arecompatible with the data obtained from our study.

The pathogenesis of renal calculi formation is not a simpleprocess. Although several theories exist, the exact pathway is stillunclear. Various herbs have been used in urolithiasis therapy since

Fig. 1. Photomicrography of the kidney sections that demonstrate different group scores having different density of stones and calcium oxalate aggregation, Score 0: Nostones and aggregation, Score 1: 1–5 stones, þaggregation of calcium oxalate crystals, Score 2: 5–10 stones, þþ aggregation of calcium oxalate crystals, Score 3: More than10 stones, þþþ aggregation of calcium oxalate crystals.

N. Orhan et al. / Journal of Ethnopharmacology 163 (2015) 60–67 63

ancient times (Rosa et al., 2013). Increased excretion of urinarycitrate, decreased excretion of urinary calcium and oxalate, diure-tic and antiseptic features are some of the known mechanisms ofthese herbal extracts (Günocak and Küpeli, 2006).

As mentioned above, diuretic action is an important factor inkidney stone treatment, as an increase in the volume of fluid flowingthrough the kidney will help to dissolve the stones, assist their passingto avoid further retention, and flush out the deposits. Among the

Table 1Effect of Helichrysum extracts on daily water consumption, urine volume, urine citrate, oxalate, uric acid levels and stone formation score of Wistar rats c.

Group Dose (mg/kg) Urine volume/water consumption ratio Citrate7S.D. Oxalate7S.D. Uric acid7S.D. Stone formation score7S.D.

Control – 20/40¼0.50 7.671.50 2.670.11 1.4470.21 0.0070.00Sodium oxalate 70 20/40¼0.50 7.672.28 2.670.43 1.2070.43 2.6670.52annn

Potassium citrate 4 # 15/42¼0.35 7.070.81 4.271.76ann,bnnn 1.0470.52 2.1670.41 annn

HG 62.5 20/41.5¼0.48 7.071.37 4.270.58a,bnn 0.7370.21ann,bn 1.8371.17annn

125 14.5/24¼0.60 9.672.36 1.070.23a,bnn 0.4870.08annn,bnn 2.3370.52annn

HS 78 12/25¼0.48 6.871.51 0.970.10a,bnn 0.4070.13annn,bnn 2.0071.10annn

156 18/42¼0.43 12.273.40a,bnnn 2.170.31 0.6670.42ann,bn 1.1670.75a,bn

HG: Helichrysum graveolens, HS: Helichrysum stoechas ssp. barellieri.#: 5 meq/daily, n¼6 animals/group.Urine citrate, oxalate and uric acid levels were expressed as mg/dl.

a Compared with control group.b Compared with negative control group (sodium oxalate).n po0.05.nn po0.01.nnn po0.001.c All measurements were conducted on the last (5th) day of the experiment.

Table 2Effect of Helichrysum extracts on the body and relative organ weights of Wistar rats.

Group Dose (mg/kg) Body weight (g) Relative organ weights

Initial7S.D. 3rd Day7S.D. (Change %) 5th Day7S.D. (Change %) Liver Kidney

Control – 237.00716.57 238.33716.57 (þ 0.56) 234.50716.08 (�1.06) 3.7770.56 0.8870.14Sodium oxalate 70 233.60725.66 222.00722.57 (�4.96) 217.00723.05 (�7.10) 3.9370.29 1.1270.08Potassium citrate 4 # 190.83715.27 176.00714.17 (�7.77) 175.83712.17 (�7.86) 4.0670.47 1.3271.10HG 62.5 218.20725.96 206.40726.12 (�5.40) 208.00724.69 (�4.67) 3.9370.30 1.1670.13

125 189.83713.89 180.50713.32 (�4.91) 181.17712.04 (�4.56) 4.2270.41 1.2270.15HS 78 213.00721.26 198.60715.63 (�6.76) 201.00714.21 (�5.63) 4.2170.43 1.6070.28

156 224.17728.88 196.83712.75 (�12.12) 200.33719.83 (�10.63) 3.9470.38 1.3170.23

HG: Helichrysum graveolens, HS: Helichrysum stoechas ssp. barellieri.n¼6 animals/group. Change % is calculated according to initial body weight of each group.Relative organ weights were measured on the last (5th) day of the experiment.

# meq/daily.

Table 3Effect of Helichrysum extracts on blood lipid parameters and liver enzyme levels of Wistar rats #.

Parameter Units Controlgroup

Sodium oxalategroup

Potassium citrategroup

HG groups HS groups

62.5 mg/kg 125 mg/kg 78 mg/kg 156 mg/kg

Total Bil.7S.D. mg/dl 0.2470.03 0.1770.08 0.1370.07 an 0.1670.08 0.1470.06 0.1670.03 0.1870.04AST7S.D. U/l 110.67711.56 161.00731.94ann 129.40733.87 158.67714.38 ann 131.67714.62 114.6077.09 bnn 120.50714.72 bn

ALT7S.D. U/l 45.5076.89 57.5076.27ann 24.8074.32a,bnnn 34.67710.02 ann,bnnn 24.5076.02 a,bnnn 24.8074.60 a,bnnn 24.0074.20 a,bnnn

ALP7S.D. U/l 130.1777.51 148.7579.08 annn 122.8076.73 an,bnnn 107.0074.44 a,bnnn 90.1773.84 a,bnnn 106.4071.24 a,bnnn 104.0074.04 a,bnnn

Cholesterol7S.D.

mg/dl 48.8377.86 65.7577.90 an 44.4079.07 bnn 57.33710.41 51.83713.24 46.6078.47 bn 51.33710.37

HDL7S.D. mg/dl 31.00710.00 38.5071.70 an 25.8074.97 bnn 29.3373.06 bn 28.6777.63 bn 25.8074.82 bnn 26.1772.48 bnn

LDL7S.D. mg/dl 9.2076.30 22.1576.52 an 10.3676.03 bn 20.0776.06 an 15.8376.50 8.6875.43 bnn 16.1076.08VLDL7S.D. mg/dl 8.6372.25 7.6072.07 8.2471.88 7.9372.21 7.0470.71 12.1276.12 9.0773.73Triglyceride

7S.D.mg/dl 43.17711.27 38.0075.36 41.2079.42 39.67711.06 36.6774.80 60.6073.62 ann, bnnn 45.3378.65

HG: Helichrysum graveolens, HS: Helichrysum stoechas ssp. barellieri, Bil.: Bilirubin, AST: Aspartate Aminotransferase, ALT: Alanine Aminotransferase, ALP: AlkalinePhosphatase, HDL: High Density Lipoprotein, VLDL: Very Low Density Lipoprotein, LDL: Low Density Lipoprotein, S.D.: Standard Deviation, n¼6 animals/group.

a Compared with control group.b Compared with negative control group (sodium oxalate).n po0.05.nn po0.01.nnn po0.001.# All measurements were conducted on the last (5th) day of the experiment.

N. Orhan et al. / Journal of Ethnopharmacology 163 (2015) 60–6764

diverse roles of adenosine A1 receptor (AA1R) antagonists in renalprotection, many studies have shown that they can induce diuresisand sodium excretion. In this respect, a number of flavonoids and theirderivatives have been examined for their ability to compete for AA1Rradioligand binding and found to be strong AA1R antagonists (Kartonet al., 1996; Alexander, 2006; Yuliana et al., 2009).

Many study support the notion that renal tubular injury resultingfrom free radical production by oxalate is closely related to thepathogenesis of urolithiasis. Hence, it is shown that flavonoidspossess powerful antioxidant properties which prevented the devel-opment of papillary and intratubular calcification of the kidney,consequently preventing the development of papillary calculi in ratswith ethylene glycol induced lithiasis (Grases et al., 2009). Also, Parket al. (2008) found that quercetin has potent antioxidant effects andit decreases the lipid peroxidation production induced by oxalate inMDCK cells and inhibits renal crystal deposition in the rats.

Helichrysum species contain many secondary metabolites suchas flavonoids, acetophenones, phloroglucinols, pyrones, terpenes andfatty acids (Facino et al., 1990, Eroglu et al., 2009). Capitulums of theplant contain many flavonoids, including apigenin, luteolin, galangin-3-methyl ether, 3,5-dihydroxy-6,7,8-trimethoxy flavone, kaempferol,

quercetin, naringenin, apigenin-7-glucoside, apigenin-40-glucoside, lute-olin-7-glucoside, luteolin-40-glucoside, kaempferol-3-glucoside, heli-chrysin A, B, naringenin-4-glucoside, and isosalipurposide (Çubukçuand Meriçli, 1977, 1979; Çubukçu et al., 1981). Due to the rich flavonoidcontent of Helichrysum capitulums, these components having highantioxidant activity (Aslan et al., 2007a, 2007b), may be suggested asthe main active principles having preventive effect in urolithiatic rats.

For many years, it is known that metabolic syndrome and type2 diabetes is associated with an increased risk of nephrolithiasis.Diabetic patients, having low urine pH, produce uric acid stones.Insulin resistance is also involved in the pathogenesis of primaryuric acid nephrolithiasis observed in overweight subjects with themetabolic syndrome (Daudon and Jungers, 2007). In connectionwith this hypothesis, it can be concluded that antidiabetic effect ofHG extract (Aslan et al., 2007b) might have contributed to thepreventive effect of the plant on kidney stone formation.

In conclusion, the folkloric utilization of Helichrysum specieswas enlightened by the present study. Both extracts were found tohave significant inhibitory effect on stone formation. Nature isthe best combinatorial chemistry and has possible answers to alldiseases for humanity. Our future aim is completing scientific and

Table 4Effect of Helichrysum extracts on blood biochemical parameters of Wistar rats #.

Parameter Units Controlgroup

Sodium oxalategroup

Potassiumcitrate group

HG groups HS groups

62.5 mg/kg 125 mg/kg 78 mg/kg 156 mg/kg

BUN7S.D. mg/dl 15.6273.80 28.9875.70 an 33.24711.32 ann 24.7076.40 24.8877.10 27.7473.58 an 30.2775.87 ann

Creatinine7S.D. mg/dl 0.2970.02 0.6170.14 annn 0.4970.12 ann 0.4370.09 an,bn 0.4770.04 ann 0.5770.09 annn 0.5570.06 annn

Uric acid7S.D. mg/dl 70.5270.51 65.1972.04 annn 69.1870.35bnnn 47.6270.52 a,bnnn 58.4672.56 a,bnnn 51.0370.07 a,bnnn 53.0472.09 a,bnnn

Total Prt. 7S.D. g/dl 6.6770.24 5.8070.73 an 5.2371.09 ann 5.6170.23 an 5.7370.29 an 5.6370.32 an 5.8670.38 an

Albumine7S.D. g/dl 3.3170.19 2.6170.30 annn 2.7570.21 annn 2.5270.18 annn 2.6870.17 annn 2.7270.24 annn 2.6670.15 annn

Calcium7S.D. mg/dl 9.8970.32 9.5770.86 9.6670.16 9.4770.11 10.0170.71 9.3870.39 9.8070.48Magnesium7S.D. meq/l 1.7570.04 2.4370.52 an 2.1870.34 2.0770.20 2.5870.58 ann 2.2170.25 2.4870.32 an

Sodium7S.D. mmol/l 139.1772.14 139.7574.79 143.0071.22 140.0072.65 142.3371.21 144.0072.00 139.0077.13Potassium7S.D. mmol/l 3.7970.20 4.5171.05 4.0570.44 4.3270.65 5.0271.77 3.5170.23 4.0170.56Chlorine7S.D. mmol/l 107.5071.52 103.5074.04 103.0074.06 107.6773.06 105.5072.17 108.2071.92 103.0074.82

HG: Helichrysum graveolens, HS: Helichrysum stoechas ssp. barellieri, BUN: Blood Urea Nitrogen, S.D.: Standard Deviation, n¼6 animals/group.a Compared with control group.b Compared with negative control group (sodium oxalate).n po0.05.nn po0.01.nnn po0.001.# All measurements were conducted on the last (5th) day of the experiment.

Table 5Effect of Helichrysum extracts on the hematological parameters of Wistar rats #.

Parameter Units Control group Sodium oxalategroup

Potassium citrategroup

HG groups HS groups

62.5 mg/kg 125 mg/kg 78 mg/kg 156 mg/kg

WBC7S.D. �10�3/ml 2.1471.75 3.9070.79 5.4172.10 an 7.5470.59 annn,bnn 8.3073.47 ann 4.2670.06 5.9070.30 ann

RBC7S.D. �10-6/ml 7.0070.46 6.9170.46 6.8670.29 6.0770.42 an 6.6270.54 6.4270.76 6.1370.35 an

Hemoglobin7S.D. g/dl 13.3670.91 13.6470.47 12.8370.27 11.7770.72 an,bnn 12.6470.81 12.1571.43 bn 11.7870.52 an,bnn

Hematocrit7S.D. % 41.2272.71 41.9971.54 39.3671.11 34.8972.15 ann,bnnn 39.2873.22 37.0574.05 an,bn 36.0071.95 an,bnn

MCV7S.D. fl 58.9271.11 60.8673.06 57.3872.22 63.4675.04 an 59.3672.76 57.7971.21 58.6970.39MCH7S.D. pg 19.0970.27 19.6270.93 18.7270.81 19.4070.91 19.1370.78 18.9470.53 19.2370.32MCHC7S.D. g/dl 32.4570.41 32.4770.14 32.6170.34 33.7571.21 a,bnnn 32.2170.57 32.7670.38 32.7670.43Platelet count

7S.D.�10-3/ml 850.80723.51 916.33713.53 annn 748.50726.77 a,bnnn 855.93723.12 bnnn 878.35722.48 bnn 836.18717.17 bnnn 747.42721.89 a,bnnn

Lymphocytes7S.D. % 76.2572.94 68.0672.71 an 56.7175.91 annn, bnn 45.0674.27 a,bnnn 44.7675.74 a,bnnn 64.5777.33 ann 41.5078.44 a,bnnn

HG: Helichrysum graveolens, HS: Helichrysum stoechas ssp. barellieri, WBC: White Blood Cell, RBC: Red Blood Cell, MCV: Mean Corpuscular Volume, MCH: Mean CorpuscularHemoglobin, MCHC: Mean Corpuscular Hemoglobin Concentration, S.D.: Standard Deviation. n¼6 animal/group.

a Compared with control group.b Compared with negative control group (sodium oxalate).n po0.05.nn po0.01.nnn po0.001.# All measurements were conducted on the last (5th) day of the experiment.

N. Orhan et al. / Journal of Ethnopharmacology 163 (2015) 60–67 65

clinical studies about the long term efficacy, safety and mechanismof Helichrysum extracts on treatment of urolithiasis.

Acknowledgments

This study was financially supported by the Scientific ResearchFund of Gazi University (GUBAP-02/2010-06).

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Table 6Effect of Helichrysum extracts on urine biochemical parameters of Wistar rats #.

Parameter Units Controlgroup

Sodium oxalategroup

Potassium citrategroup

HG groups HS groups

62.5 mg/kg 125 mg/kg 78 mg/kg 156 mg/kg

BUN7S.D. g/l 0.2970.03 0.4070.04 an 0.2170.06 bnnn 0.2170.12 bnnn 0.1970.03 bnnn 0.2170.07 bnnn 0.2070.10 bnnn

Creatinine7S.D. mg/dl 9.0370.13 10.9270.23 annn 9.7470.93 bnn 7.3970.27 a,bnnn 6.2370.53 a,bnnn 7.2270.75 a,bnnn 9.1870.89 bnnn

Calcium7S.D. mg/dl 1.6470.18 3.8570.32 annn 1.1470.10 a,bnnn 1.0170.31 a,bnnn 1.2970.08 ann,bnnn 1.4070.06an,bnnn 1.1570.20 a,bnnn

Sodium7S.D. mmol/dl 1.1070.05 0.8670.01 annn 0.9370.03 0.3570.01 a,bnnn 0.8570.02 annn 0.5770.01 a,bnnn 0.4070.01 a,bnnn

Potassium7S.D. mmol/dl 3.7370.01 5.4270.03 annn 5.5470.02 a,bnnn 2.7670.01 a,bnnn 2.8570.04 a,bnnn 2.5670.01 a,bnnn 2.3570.02 a,bnnn

Chlorine7S.D. mmol/dl 1.0270.10 1.1070.02 0.5470.09 a,bnnn 0.3370.04 a,bnnn 0.8770.12 a,bnnn 0.4670.01 a,bnnn 0.3370.01 a,bnnn

Protein7S.D. mg/dl 14.2670.21 16.6270.17 annn 17.4870.25 a,bnnn 12.0370.14 a,bnnn 21.6670.31 a,bnnn 11.2070.17 a,bnnn 12.5770.23 a,bnnn

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