Received 2024-02-16

Revised 2024-03-25

Accepted 2024-05-15

GMJ Copyright© 2024, Galen Medical Journal. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/) Email:gmj@salviapub.com

 Correspondence to: Hamid Zaferani Arani; Department of Surgery, Shiraz University of Medical Sciences, Shiraz, Iran. Telephone Number: +987132305410 Email Address: Hamid_zaferani73@yahoo.com

GMJ.2024;13:e3317

www.salviapub.com

Dahmardnezhad M, et al.

Novel Persian Herbal Syrups and Urolithiasis

2	GMJ.2024;13:e3317 www.salviapub.com

Introduction

Urolithiasis is the third common chronic disorder of the urinary system and its incidence is estimated from 2% to 20 % in the Middle East countries [1]. Near 80% of kidney stones are composite of calcium, oxalate, and calcium phosphate [2]. Currently, extracorporeal shock wave lithotripsy, local calculus disruption using laser, and medication therapy are widely used against urolithiasis [3]. However, these procedures are highly costly and may lead to serious complications such as hemorrhage, decrease in renal function, and hypertension [4, 5]. Therefore, the use of herbal plants can be replaced with conventional treatments to reduce these side effects [6, 7]. Increasing interest in the use of medicinal plants around the world, as well as research and scientific fields for their beneficial effects, have also been performed [7].

Achillea millefolium L., (named Bumadaran in the Persian language) is a genus of the Asteraceae family that is found in Europe, Asia, and North America [8], and in Iran, it grows in the northern areas in some provinces, including Azerbaijan, Lorestan, and Isfahan [9]. A. millefolium has long been used in traditional Persian medicine. A. millefolium L. was first mentioned in the Makhzan-ol-Advieh book written by Aghili-Shirazi in the 18th century [10]. Previous evidence reported anti-hemorrhoids, anti-inflammatory, and wound healing effects for A. millefolium [9, 10]. In addition, other activities of A. millefolium were mentioned in traditional medicine books, including anti-bacterial, astringent, anti-oxidative, hepatoprotective, anti-spasmodic, anti-dysenteric, antipyretic, diuretic, anti-urolithiasis, urinary anti-septic by native peoples [11, 12]. Also, its anti-nociceptive and calcium-antagonist activities are beneficial in the prevention of urolithiasis [13, 14]. Previously, in the pilot study, we reported the beneficial effect of hydroalcoholic extract of A. millefolium on kidney stone formation [15]. However, the solubility of the active substance varies in the aqueous and ethanolic phases; hence, the aim of this study was to evaluate two novel A. millefolium syrups with different formulations for preventive and curative effects on urolithiasis animal models.

Materials and Methods

Animals

This manuscript has been done in two steps, in the first step, 16 rats were used to perform acute toxicity, and in the second step, 36 rats were used to perform animal trials. Thirty-six male Wistar rats (200-250 g) were purchased from the Karaj branch of Pasteur Institute (Iran) and kept in standard cages under 25±2 ◦C with 12/12 hours of light/dark cycles for a week. and fed with a standard pelleted diet and water.

Plant Collection and Identification

The aerial parts of A. millefolium were obtained from northwest Iran and identified by the Pharmacognosy Department of Tehran University recognized it with herbarium number: 83001. The plants were then dried and powdered in a standard manner.

Preparation of Plant Extracts

The dried A. millefolium was pulverized and extracted with 95% ethanol for a total of seven hours, followed by a 1.5 hour extraction with distilled water. The resulting ethanol and water extracts were filtered, concentrated, and dried in an oven at 50◦C. The ethanolic extract yielded 9.2% and the aqueous extract yielded 13.41%. The extracts were refrigerated at four degrees Celsius and diluted with distilled water before being tested [16].

Preparation of Suspension-I Formulation

Six g of the dried, powdered ethanolic extract of A. millefolium was suspended in water using 6g of Arabic gum (Sigma-Aldrich, Germany) as a suspending agent. Then 0.2 g and 0.06 of methyl paraben and propyl paraben (Sigma-Aldrich, Germany) were added as additive agents, respectively.

Preparation of Suspension-II Formulation

Six g of the dried, powdered aqueous extract of A. millefolium was triturated by using a mortar and pestle and it was added 1g sodium carboxymethyl cellulose (Sigma-Aldrich, Germany) as a suspending agent then made volume by sugar syrup. Also, 0.05 g, 0.06 g, and 0.2 g of Amaranth (Sigma-Aldrich, Germany), methyl paraben, and propyl paraben were added as coloring and additive agents, respectively.

Acute Toxicity

The acute toxicity study followed guidelines from the Organization for Economic Cooperation and Development (OECD) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) [12], using healthy Wistar rats of both sexes weighing 200-250 g. The rats were divided into two groups of 8, with each group receiving different doses. The rats were deprived of food overnight but had access to water. They were then given two different doses of oral suspensions, 1500 and 3000 mg/kg of body weight, and observed for 24 hours with no negative effects or fatalities. According to OECD guidelines 420, the median lethal dose (LD50) was determined to be greater than 3000 mg/kg, so one-tenth of that dose was used for the anti-urolithiasis treatment [12].

Experimental Groups

Rats were grouped randomly (n=6 per group) as follows:

Group A that considered as control rats and received only regular food and drinking water ad libitum. Groups B to F were fed ethylene glycol (EG) 1% (Merck, Germany) by the use of drinking water to induce nephrolithiasis [15] in rats from the first day till the 28th day. Group B was considered as sham group and received no any treatments. Groups C and D (curative groups) received 300 mg/kg b.wt Suspension-I and -II from days 15 to 28, respectively. Groups E and F (preventive groups) received Suspension-I (300 mg/kg b.wt) and suspension-II (300 mg/kg b.wt) from the first to 28th day, respectively [15]. Rats were fed with extracts once daily via oral route.

Urine Analysis

Urine-24-hour samples were collected using standard metabolic polypropylene cages at the end of the study. The urine volume of each rat was calculated for each group and a drop of hydrochloric acid was added to the collected urine before being stored at 4◦C. Urinary parameters (such as oxalate, calcium, phosphorus, and citrate) were measured by commercial kits (Darman Kaw, Tehran, Iran) with an auto-analyzer (SB 501 Plus, Sinduri Biotec, India).

Blood Sample Collection

At the conclusion of the experiment, blood was obtained through retro-orbital puncture while the animal was under ether anesthesia. The serum was then separated by centrifugation at 2500 rpm for 10 minutes [15].

Serum Analysis

The levels of serum creatinine, blood urea nitrogen, and uric acid were measured using kits and a spectrophotometer from MAN, Tehran, Iran, following the manufacturer’s instructions. The concentrations were calculated using the formula (mg/dl)=[(Ablank- Asample)/Ablank] where Ablank represents the absorbance of the control reaction (containing all reagents except the test compound) and Asample represents the absorbance of the test compound. The results were reported in mg/dl.

Histopathology Examination

On the 28th day’s conclusion, anesthesia was administered to all the rats, followed by decapitation using a guillotine. Subsequently, both kidneys were extracted and preserved in formalin (10%) for histological investigations. From each kidney, three 5µm sections were meticulously prepared, and the resulting slides were subjected to staining with hematoxylin-eosin (H&E). These stained slides were then examined under a light microscope. The number of calcium oxalate (CaOx) deposits in the renal tubules was quantified by counting them in 10 microscopic fields.

Ethics Statement

This study and all protocols were performed based on the Principles of Laboratory Animal Care (NIH publication, 9th edition). Also, the Ethics Committee of Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran approved the current study via number: 12/4/3109.

Statistical Analysis

The data was expressed as the mean±SD and analyzed using SPSS version 14 (SPSS Inc, Chicago, IL, USA) with one-way ANOVA and Tukey post hoc test for multiple comparisons. A significant difference was determined at P<0.05.

Results

A. millefolium Administration Could Improved Urine Parameters Against Urolithiasis

There was a marked increase in oxalate, calcium, and phosphate excretions in the sham group (group B) in comparison to control rats (P<0.05, Table-1). However, administration of A. millefolium significantly prevented these changes in urinary oxalate, calcium, and phosphate excretion in groups C to F compared to group B (P<0.05). As shown in Table-1, there was a significant difference between A. millefolium-treated groups in terms of oxalate (P<0.001).

The mean urinary citrate level in group A (control rats) was 19.23±0.03 mg/dl, while citrate excretion was significantly decreased by urolithiasis to 7.27±0.02 mg/dl (Table-1, P=0.011). However, administration of A. millefolium significantly prevented these changes (P<0.001). Also, citrate changes between A. millefolium-treated groups were significant (Table-1, P<0.01). Based on the results of urine analysis, among A. millefolium-treated groups, rats in group F had significantly lower oxalate and higher citrate in comparison to group B (P=0.018 and P=0.003, respectively).

Serum Cr, BUN, and Uric Acid Concentrations

Regarding Table-2, the mean Cr concentration in groups A and B was 0.49±0.03 and 2.57±0.06, respectively. Indeed, serum Cr was increased significantly following urolithiasis; however, A. millefolium administration significantly reduced the serum Cr in group C to F in comparison to the sham group (Table-2, P<0.001).

Also, the mean BUN and uric acid concentrations of the rats in the sham group were significantly higher than those of all other groups (Table-2, P<0.05). However, those concentrations in the curative and preventive groups were significantly decreased in contrast to the sham group (Table-2, P<0.01).

CaOx Deposits

Based on histopathological examination, no CaOx deposits were seen in the nephron segment of rats in the control group. Indeed, many CaOx deposits were observed in the rats of groups B to F (Figure-1). Regarding Table-3 , the mean CaOx deposits in groups C and D were 18.17±0.69 and 15.5±0.76, respectively, which was significantly lower than that in group B (P=0.03 and P=0.002, respectively). In addition, in the preventive groups (E and F) the CaOx deposits were significantly lower than those in group B (Table-3).

Discussion

In the current study, the effects of two formulations of A. millefolium were evaluated in prevention as well as treatments of urolithiasis in rat models. Urinary parameters, including oxalate, Cr, and phosphate were significantly increased after EG-induced urolithiasis, while citrate level was reduced. A. millefolium administrations could significantly show both preventive and treatment effects. Also, serum parameters changes in rats that received A. millefolium syrups markedly improved against control and sham groups. In addition to urine and serum biochemical parameters, the histopathological study revealed the beneficial effects of A. millefolium in CaOx deposit prevention.

In traditional medicine, A. millefolium was administered usually through the oral route [10]. Hence, in our study, the same route was taken for evaluating the anti-urolithiasis effect of the A. millefolium in the rat model. Previous evidence indicates that the rate of kidney stone formation in male rats, as in humans, was higher than the females. Hence, male rats were used to induce urolithiasis in the present study [15, 17].

Previous studies have provided evidence suggesting that the administration of EG to young rats for a duration of two weeks led to the formation of renal calculi primarily composed of CaOx [18, 19]. The development of nephrolithiasis in EG-fed animals can be attributed to hyperoxaluria, resulting in the increased retention and excretion of oxalate in the kidneys. [17, 20, 21]. Therefore, this model was used to evaluate the protective effect of A. millefolium syrups against urolithiasis.

In the current study, urinary oxalate and calcium excretions were increased in EG-induced urolithiasis rats. Also, a reduction in oxalate and calcium was observed in A. millefolium treated groups. The plant extract may have contributed to the decrease in oxalate excretion by inhibiting oxalate formation. Rats with calculi, specifically in group B without A. millefolium syrups displayed a notable reduction in urinary citrate levels.

Some evidence has shown that the reabsorption of citrate in tubules plays a regulating role by forming a complex with calcium thereby reducing the concentration of CaOx [22-24]. In our study, it was found that A. millefolium treatment brought the urinary citrate excretion level near to its level among control rats to decline the risk of stone formation.

In rats with EG-induced urolithiasis, there was an increase in the amount of phosphorus excreted in their urine. This, along with oxalate, has been linked to the formation of stones through the formation of calcium phosphate crystals which induces CaOx deposition [17, 22, 25]. However, treatment with A. millefolium reduced phosphorus excretion and the risk of stone formation, while the administration of an aqueous extract-based suspension decreased oxalate and increased citrate compared to other treatment groups.

The accumulation of waste products, particularly nitrogenous substances such as Cr, BUN, and uric acid, in the blood is a consequence of the decreased glomerular filtration rate (GFR) observed in urolithiasis [26-28]. The current study reveals that nephrotoxicities induced by EG are characterized by a notable rise in serum Cr, BUN, and uric acid levels. The administration of both suspensions significantly mitigated nephrotoxicity, as evidenced by an improvement in GFR and a subsequent reduction in the presence of nitrogenous waste products. While there was no significant difference between the two suspensions.

Microscopic evaluation of renal sections obtained from rats with calculi revealed the existence of polymorphic and irregular crystal deposits inside the tubules. These deposits resulted in the enlargement of the proximal tubules and the development of interstitial inflammation, which could potentially attributed to the presence of oxalate. Nonetheless, the administration of A. millefolium suspensions exhibited a significant reduction in both the quantity and dimensions of CaOx deposits in various regions of the renal tubules. So, results showed the protective effect of A. millefolium syrups with ethanolic and aqueous bases in the EG-induced urolithiasis model.

The previous study [15] reported high antioxidant and anti-inflammatory effects of A. millefolium. It can be speculated that the anti-urolithiasis formation activity of the kidney may be through of oxidant activity and free radicals. In other words, the administration of ethanolic and aqueous extracts of A. millefolium reduces and prevents the growth of urinary stones. However, better protective effects were observed following the administration of A. millefolium aqueous extract from the first day of the study.

This study has some limitations. The mechanism underlying the anti-urolithiasis effect of A. millefolium was not exactly evaluated, but it could related to diuresis and the lowering of urinary concentrations of stone-forming constituents. Also, in this study we used the whole of A. millefolium extracts; however, the main bioactive components were not evaluated. Hence, further studies are still recommended on the various aspects and side effects of this herbal agent.

Conclusion

Our findings indicate that the use of both A. millefolium formulations showed preventive and curative effects on EG-induced urolithiasis, providing further support for the traditional use of A. millefolium in treating urolithiasis.

Conflict of Interest

The authors report no conflicts of interest.

Novel Persian Herbal Syrups and Urolithiasis

Dahmardnezhad M, et al.

GMJ.2024;13:e3317 www.salviapub.com

3

Dahmardnezhad M, et al.

Novel Persian Herbal Syrups and Urolithiasis

4	GMJ.2024;13:e3317 www.salviapub.com

Table 1. Effect Of Plant Extracts On Urinary Biochemical Parameters On The 28th Day

Groups	Oxalate (mg/dl)	Calcium (mg/dl)	Citrate (mg/dl)	Phosphate (mg/dl)
A	0.79±0.04	1.73±0.03	19.23±0.03	3.19±0.05
B	4.11±0.06**	3.5±0.15**	7.27±0.02**	7.2±0.01**
C	2.61±0.01*	2.42±0.15*	10.16±0.07*	5.25±0.03*
D	2.04±0.09*	2.7±0.03*	11.21±0.02*	5.59±0.1*
E	2.5±0.01*	2.55±0.03*	10.25±0.01*	5.26±0.03*
F	1.79±0.02*	2.57±0.02*	11.34±0.07*	5.51±0.01*

* P<0.05, Achillea groups vs. groups A and B

** P<0.05, group A vs. group B

Novel Persian Herbal Syrups and Urolithiasis

Dahmardnezhad M, et al.

GMJ.2024;13:e3317 www.salviapub.com

5

Table 2. Effect Of Plant Extracts On Serum Biochemical Parameters On The 28th Day

Groups	Uric acid (mg/dl)	Creatinine (mg/dl)	BUN (mg/dl)
A	1.62±0.03	0.49±0.03	23.17±1.34
B	4.24±0.03**	2.57±0.06**	33.67±1.11**
C	2.89±0.01*	1.77±0.01*	16.5±0.96*
D	2.64±0.01*	1.43±0.01*	15.5±1.26*
E	2.75±0.04*	1.66±0.03*	21.5±0.96*
F	2.38±0.02*	1.12±0.03*	18.5±0.96*

* P<0.05, Achillea groups vs. groups A and B

** P<0.05, group A vs. group B

Dahmardnezhad M, et al.

Novel Persian Herbal Syrups and Urolithiasis

6	GMJ.2024;13:e3317 www.salviapub.com

Novel Persian Herbal Syrups and Urolithiasis

Dahmardnezhad M, et al.

GMJ.2024;13:e3317 www.salviapub.com

7

Table 3. Effect of Plant Extracts On Calcium Oxalate Deposition In The Kidneys Of Rats On The 28th Day

Parameter	Groups
	A	B	C	D	E	F
CaOx deposition	-	33.67±1.11**	18.17±0.69*	15.5±0.76*	8.83±0.89*	5.83±0.69*

* P<0.05, Achillea groups vs. groups A and B,

** P<0.05, group A vs. group B

References

1. Lang J, Narendrula A, El-Zawahry A, Sindhwani P, Ekwenna O. Global trends in incidence and burden of urolithiasis from 1990 to 2019: an analysis of global burden of disease study data. European urology open science. 2022 Jan 1;35:37-46.
2. Samantha C, Avani S, Kumar E, Prasobh G. A review on urinary calculi-types, causes, its mechanism, diagnosis, prevention and medical expulsion therapy of calculi. World Journal of Pharmaceutical Research. 2021 May 27;9(10):473-86.
3. Lv G, Qi W, Gao H, Zhou Y, Zhong M, Wang K, Liu Y, Zhang Q, Zhou C, Li Y, Zhang L. Safety and efficacy of extracorporeal shock wave lithotripsy vs flexible ureteroscopy in the treatment of urinary calculi: A systematic review and meta-analysis. Frontiers in Surgery. 2022 Nov 7;9:925481.
4. Malhi M, Hanumanthrao P, Kumari PR. A Update On Current Diagnostic Modalities And Treatment Of UrolithiasisReview. Journal of Survey in Fisheries Sciences. 2023 Jul 12:710-7.
5. Riedler I, Trummer H, Hebel P, Hubmer G. Outcome and safety of extracorporeal shock wave lithotripsy as first-line therapy of lower pole nephrolithiasis. Urol Int. 2003;71(4):350-4.
6. Bawari S, Sah AN, Tewari D. Anticalcifying effect of Daucus carota in experimental urolithiasis in Wistar rats. J Ayurveda Integr Med. 2020 Jul 1;11(3):308-15.
7. Vikas M, Anju D, Chhavi S. An Update on Urolithiatic Plant Drugs as Alternative Treatment Option for Mitigation of Kidney Stones. Annals of the Romanian Society for Cell Biology. 2020:507-37.
8. Barda C, Grafakou ME, Tomou EM, Skaltsa H. Phytochemistry and evidence-based traditional uses of the genus Achillea L.: an update (2011–2021). Scientia Pharmaceutica. 2021 Nov 22;89(4):50.
9. Mehrnia M, Akaberi M, Amiri MS, Nadaf M, Emami SA. Ethnopharmacological studies of medicinal plants in central Zagros, Lorestan Province, Iran. Journal of Ethnopharmacology. 2021 Nov 15;280:114080.
10. Benedek B, Kopp B. Achillea millefolium L. sl revisited: recent findings confirm the traditional use. Wien Med Wochenschr 2007;157:312-4.
11. Jangjoo M, Joshaghani A, Tahernejadgatabi F. The role of Achillea millefolium in traditional medicine: A review of its use in different cultures. Journal of Multidisciplinary Care. 2023 Sep 29;12(3):152-6.
12. Bashir S, Noor A, Zargar MI, Siddiqui NA. Ethnopharmacology, phytochemistry, and biological activities of achillea millefolium: a comprehensive review. Edible plants in health and diseases: volume II: phytochemical and pharmacological properties. 2022 Mar 15:457-81.
13. Pires JM, Mendes FR, Negri G, Duarte-Almeida JM, Carlini EA. Antinociceptive peripheral effect of Achilliea millefolium L. and Artemisia vulgaris L.: both plants known popularly by brand names of analgesic drugs. Phytother Res 2009;23:212–9.
14. Candan F, Unlu M, Tepe B, Daferera D, Polissiou M, Atalay Sökmen A, Akpulat HA. Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp Millefolium Afan (Asteraceae). J Ethnopharmacol. 2003;87:215–20.
15. Bafrani HH, Parsa Y, Yadollah-Damavandi S, Jangholi E, Ashkani-Esfahani S, Gharehbeglou M. Biochemical and pathological study of hydroalcoholic extract of Achillea millefolium L. on ethylene glycol-induced nephrolithiasis in laboratory rats. North Am J Med Sci 2014;6:638-42.
16. Zhang L, Hu J-J, Lin J-W, Fang W-S, Du G-H. Anti-inflammatory and analgesic effects of ethanol and aqueous extracts of Pterocephalus hookeri (CB Clarke) Höeck. J Ethnopharmacol. 2009;123(3):510-4.
17. Karadi RV, Gadge N, Alagawadi KR, Savadi RV. Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol 2006;105:306–11.
18. Huang HS, Ma MC, Chen J, Chen CF. Changes in the oxidant- antioxidant balance in the kidney of rats with nephrolithiasis induced by ethylene glycol. J Urol. 2002;167:2584-93.
19. Atmani F, Slimani Y, Mimouni M, Hacht B. Prophylaxis of calcium oxalate stones by Herniaria hirsute on experimentally induced nephrolithiasis in rats. BJU Int. 2003;92:137-40.
20. Liu J, Huang J, Gong B, Cheng S, Liu Y, Chen Y, Feng Q, Li J, Qiu M, Yu G, Liao Y. Polydatin protects against calcium oxalate crystal-induced renal injury through the cytoplasmic/mitochondrial reactive oxygen species-NLRP3 inflammasome pathway. Biomedicine & Pharmacotherapy. 2023 Nov 1;167:115621.
21. Alenzi M, Rahiman S, Tantry BA. Antiurolithic effect of olive oil in a mouse model of ethylene glycol-induced urolithiasis. Investigative and clinical urology. 2017 May;58(3):210.
22. Soundararajan P, Mahesh R, Ramesh T, Begum VH. Effect of Aerva lanata on calcium oxalate urolithiasis in rats. Indian J Exp Biol. 2006;44:981–6.
23. Divakar K, Pawar AT, Chandrasekhar SB, Dighe SB, Divakar G. Protective effect of the hydro-alcoholic extract of Rubia cordifolia roots against ethylene glycol induced urolithiasis in rats. Food Chem Toxicol. 2010;48:1013–18.
24. Chauhan CK, Joshi MJ. Growth inhibition of Struvite crystals in the presence of juice of Citrus medica Linn. Urol Res. 2008;36(5):265-73.
25. Patel VB, Acharya N. Effect of Macrotyloma uniflorum in ethylene glycol induced urolithiasis in rats. Heliyon. 2020;6(6):e04253.
26. Grover PK, Resnick MI. Evidence for the presence of abnormal proteins in the urine of recurrent stone formers. J Urol. 1995;153:1716-21.
27. Vaidya AD. Reverse pharmacological correlates of ayurvedic drug actions. Indian J Pharmacol. 2006;38(5):311.
28. Joshi VS, Parekh BB, Joshi MJ, Vaidya AD. Inhibition of the growth of urinary calcium hydrogen phosphate dihydrate crystals with aqueous extracts of Tribulus terrestris and Bergenia ligulata. Urol Res. 2005;33(2):80-6.

Dahmardnezhad M, et al.

Novel Persian Herbal Syrups and Urolithiasis

8	GMJ.2024;13:e3317 www.salviapub.com