antiurolithiatic effects of hydroalcoholic extract of lawsonia inermis l ...

International Journal of Universal Pharmacy and Life Sciences 1(2): September-October 2011INTERNATIONAL JOURNAL OF UNIVERSALPHARMACY AND LIFE SCIENCESPharmaceutical SciencesResearch Article……!!!Received; acceptedANTIUROLITHIATIC EFFECTS OF HYDROALCOHOLIC EXTRACT OFLAWSONIA INERMIS L LEAVESK.J. Kore*, R.V. Shete., P.J. Jadhav, M.P.KabraDepartment of Pharmacology, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor, Dist. Pune,Maharashtra, Pin – 412 206.Keywords:Lawsonia inermis L,kidney stone,Antiurolithiatic activity,EG induced urolithiasisFor Correspondence:K.J. KoreDepartment ofPharmacology, RajgadDnyanpeeth’s College ofPharmacy, Bhor, Dist.Pune, Maharashtra, Pin –412 206.E-mail:k_kore_2000@yahoo.comABSTRACTIn the traditional system of medicine in India, the plantLawsonia inermis L is claimed to be useful for the treatmentof kidney disorder. The major purpose of this study is toinvestigate the potential of Lawsonia inermis L in thetreatment of renal calculi. The leaf of Lawsonia inermis Lwas extracted with ethanol and evaluated for antiurolithiaticpotential. The antiurolithiatic activity was evaluated byperforming ethylene glycol with ammonium chloride model.The parameters monitored in the present study are serumurea, BUN, uric acid, creatinine, kidney weight, urinevolume, urine pH,urinary excretion of total protein, calcium,phosphorus and magnesium. The hydroethanolic extract ofLawsonia inermis L leaves (HELI) showed significantantiurolithiatic activity against calcium oxalate-type stones.The results obtained in this study provide evidence for theefficacy of HELI as antiurolithiatic agent.81 Full Text Available On www.ijupls.com (Free)

1. Introduction:Urolithiasis is the third most common disorder of the urinary tract, the othersbeing frequently occurring urinary tract infection and being prostatic hyperplasia. Theworldwide incidence of urolithiasis is quite high and in spite of tremendous advances inthe field of medicine, there is no truly satisfactory drug for treatment of renal calculi(Mitra et al., 1998). Presently medical management of urolithiasis mainly involves thesurgical removal of stones. Techniques such as extracorporeal shock wave lithotripsy andpercutaneous nephrostolithotomy do not assure the prevention of recurrence of the stone.They cause side effect such as haemorrhage, hypertension, tubular necrosis, andsubsequent fibrosis of the kidney. Hence the search of new herbal medicines is still goingon. Diuretics have been introduced and medically been used as prophylactic agents forurolithiasis, due to their key role in regulating kidney function and alleviating the urinaryrisk factors for stone formation (Arafat et al., 2008). Plant medicine was commonly usedfor traditional treatment of some significant diuretic activity. Many investigators havedemonstrated that studies of herbal plan used in traditional medicine as diuretic haveincreased recent years (Maghrani et al., 2005) and might be a useful tool in the treatmentof urolithiasis. A large number of Indian medicinal plants have been used in the treatmentof urolithiasis and they have been reported to be effective with fewer side effects (Selvamet al., 2001). Even today, plants provide a cheap source of drugs for majority of world’spopulation. Several pharmacological in vitro and in vivo investigations on the medicinalplants used in traditional antiurolithiatic therapy revealed their therapeutic potential(Bashir and Gilani, 2009).Lawsonia inermis Linn. (Lythraceae) which is known as Henna, Mehandi inHindi and Mendhi, Mendi in Marathi (Kirtikar and Basu, 2005). Henna, a traditionalproduct with religious associations, has been widely used over the centuries for medicaland cosmetic purposes in Africa, Asia, the Middle East and many other parts of the world(Jallad et al., 2008) It is much branched, deciduous, glabrous, sometime spinescent shrubor small tree with greyish brown bark, attaining a height of 2.4-5 m (Kirtikar and Basu,2005.).The compounds obtained from the Leaves of this plant are Flavonoids such asluteolins, apigenin, and their glycosides; Coumarins such as esculetin, fraxetin,82 Full Text Available On www.ijupls.com (Free)

scopletin;Steroids such as β-sitosterol (Dev, 2006). The leaves of Lawsonia inermis alsoreported to contain soluble matters like tannin acid, gallic acid, glucose, mannitol, fat,resin and mucilage (Nayak et al., 2007).This plant has been described in Charaka Samhita for the treatment of epilepsyand jaundice, and for dyeing grey hair. In Sushruta Samhita it has been recommended asa remedy for malignant ulcers (Dev, 2006). The Ayurvedic Pharmacopoeia of Indiaindicated the use of leaves in dysuria, bleeding disorder, prurigo and other obstinate skindiseases. (Khare, 2007). The leaves have a bitter bad taste; vulnerary, diuretic, useful inheadache, hemicranias, lumbago, bronchitis, boils, ophthalmia, syphilitis, sores,amenorrhoea, scabies, diseases of the spleen; favour the growth of the hair (Kirtikar andBasu, 2005).Besides its traditional claim as for kidney disorder, its antiurolithiatic potentialremained unclear. Hence the present study was undertaken to explore antiurolithiaticproperties of Hydroalcoholic extract of Lawsonia inermis (HELI).2. Materials and Methods:2.1. Plant materials and extraction:The leaves of Lawsonia inermis Linn. was collected from Ambajogai area ofMaharashtra in the month of October and were authenticated by the Agharkar ResearchInstitute, Pune. Authentication No. is: 09-101.The 200 gm of coarsely powdered form of dried leaves of Lawsonia inermis,Linn. was subjected to exhaustive extraction in percolater apparatus using 70% aqueousethyl alcohol. Then obtained extract were evaporated at 45˚c, the semisolid mass obtainedwas 46 gm (% yield- 23%). The extract was stored in air tight container in refrigerator forfurther use.The extract was converted into a suspension and used for experimentalpurpose. Suspension was prepared using carboxymethyl cellulose powder in distilledwater.2.2. Preliminary phytochemical evaluation of HELI:The qualitative analysis of HELI was carried out for the evaluation of thepresence of various phytoconstituents by using the standard phytochemical tests83 Full Text Available On www.ijupls.com (Free)

(Khandelwal, 2006), which revealed the presence of carbohydrates, steroids,triterpenoids, tannins, and flavonoids in HELI.2.3 Experimental animals:Male Wistar rats (150-100 g) and female Swiss albino mice (20-30 gm) procuredfrom the Yash Farm and National Toxicological Centre, Pune were used for the study.Animals were housed in groups of 5-6 in standard polypropylene cages with wire meshtop at standard environmental condition of temperature 25±2ºC and relative humidity of45-55% under 12: 12 h light/ dark cycle in the institutional animal house. Animals hadfree access to standard pellet rodent diet (Lipton India Ltd., Mumbai, India) and waterwas provided ad libitum. All experiments were carried out between 09:00 to 17:00. Theexperimental protocol was approved by the Institutional Animal Ethics Committee ofRajgad Dnyanpeeth’s College of Pharmacy, Bhor, India constituted as per the rules andguidelines provided by the Committee for the Purpose of Control and Supervision ofExperiments on Animals, Chennai, India (RDCOP/IAEC/10/3).2.4 Acute toxicity (AOT) studies:Healthy adult female Swiss albino mice (20-30 gm) were subjected to AOTstudies as per Organization for Economic Co-operation and Development (OECD)guidelines 2001 (AOT-423). Animals were observed individually after dosing at leastonce during the first 30 min, periodically during the first 24 h, with special attentiongiven during the first 4 h, and daily thereafter, for a total of 14 days.The changes in skin,fur, eyes, mucous membranes, respiratory, circulatory, autonomic, central nervoussystem, somatomotor activity and behaviour pattern were noted.The animals treated with HELI did not show any sign of toxicity and mortalityduring the 14 days of observational period after the administration of a limit dose of 2000mg/kg, p.o. Hence for the further pharmacological studies doses of HELI selected were100, 200, and 400 mg/kg, p.o.2.5 Evaluation of antiurolithiatic activity in ethylene glycol induced urolithiasismodel (Touhami et al., 2007):The rats were randomly divided into six groups (n=6) and treated for 10 days asNormal control: 0.5 % CMC in distilled water (5 ml/kg, p.o.); urolithiasis control: 0.5 %84 Full Text Available On www.ijupls.com (Free)

CMC in distilled water (5 ml/kg, p.o.); Cystone 750: Cystone (750 mg/kg, p.o.); HELI100, 200, and 400: HELI (100, 200, and 400 mg/kg, p.o.). Additionally rats from allgroups, except normal control, were had free access to regular food and drinking watercontaining 0.75% (v/v) ethylene glycol (EG) and 2% (w/v) ammonium chloride (AC) adlibitum to induce urolithiasis. The rats were kept individually in metabolic cages andurine samples of 24 h were collected on 10 th day. A drop of concentrated hydrochloricacid was added to the urine, stored at 4ºC and urine volume, urine pH, andCalcium,Phosphorus,Magnesium and protein content of urine using commerciallyavailable kits were estimated. On 10 th day, blood samples from rats from each treatmentgroup were collected by retro orbital puncture under light ether anesthesia.2.5.2 Estimation of biomarkersSerum was separated by centrifugation of the collected blood at 4000 xg for 10min and subjected to the estimation of creatinine, urea, blood urea nitrogen (BUN), anduric acid using commercially available kits.2.6 Statistical analysis:The data was expressed as mean±SEM for each experimental group. The resultswere analyzed for statistical significance using Student’s t- test, one-way analysis ofvariance (ANOVA) followed by Dunnett’s test. p < 0.05, p < 0.01were considered asstatistically significant.3. Results:3.1 Evaluation of HELI in ethylene glycol induced urolithiasis in rats:Urine volume and urinary pH:Administration of 0.75 % ethylene glycol with 2 % ammonium chloride indrinking water to rats for 10 days showed significant (p

Electrolyte content of urine:Administration of 0.75 % ethylene glycol with 2 % ammonium chloride indrinking water to rats for 10 days showed significant (p

(p

Table 3: Effect of HELI on urinary level of total protein in ethylene glycol inducedurolithiasis in rats.TreatmentsTotal Protein (mg/dl)Normal control 2.00± 0.19Control8.84±0.64 bCystone 750 3.72±0.29**HELI 100 8.49±0.59HELI 200 6.13±0.44*HELI 400 4.00±0.27**Data was expressed as mean ± S.E.M. (n=6); b p

acid by metabolism in vivo. CaOx crystalluria could be induced by ethylene glycol andammonium chloride in rats without severe renal damage and this animal model is alwaysused to mimic the etiology of urinary stone formation in humans (Zhao et al., 2007). Thebiochemical mechanism for this process is related to an increase in the urinaryconcentration and renal retention of oxalate (hyperoxaluria) (Divakar et al., 2010).Oxalate metabolism is considered to be almost identical between rats and humans(Doddola et al., 2007). Ethylene glycol disturbs oxalate metabolizes by ways of increasethe substrate availability that increases the activity of oxalate synthesizing enzymes likeglycolic acid oxidase and lactate dehydrogenase in rats. These enzymes catalyses thecoupling of oxidation and reduction of glycoxilate results in the formation glycolate andoxalate (Soundararajan et al., 2006). This oxalate precipitates as CaOx crystals in thekidneys (Tsai et al., 2008). Ammonium chloride causes urinary acidificationconsequently decreases the urinary citrate excretion. This in turn increased the depositionof CaOx crystal in the kidney and accelerated the lithiaisis (Fan et al., 1999). Evidence inprevious studies indicated that 6 h after injection of sodium oxalate in rats, crystals ofCaOx were present mainly as large aggregates near the papillary tip with increasedtubular necrosis. Loss of tubular epithelium was more pronounced and there were morecellular fragments in the tubular lumens. The biochemical mechanisms for this processare related to an increase in the urinary concentration of oxalate. Similar results havebeen obtained when rats were treated with ethylene glycol and with ethylene glycol andammonium oxalate (Bouanani et al., 2010). Thus, a rat model of CaOx urolithiasis can beused to investigate the mechanisms involved in human kidney stone formation and also toscreen new agents with antiurolithiatic activity (Doddola et al., 2007).The present study was undertaken to investigate the effect of AAP in ethyleneglycol and sodium oxalate induced urolithiaisis paradigms using cystone ® , antiurolithiaticherbal formulation, as reference standard (Dandia et al., 1975; Mitra et al., 1998). In thepresent study HELI significantly increased urine volume in both the paradigms.It is accepted that hypercalciuria is one of the risk factor in the pathogenesis ofrenal stone. An increased urinary calcium concentration is a factor favoring nucleationand precipitation of CaOx or apatite (calcium phosphate) from urine and subsequent89 Full Text Available On www.ijupls.com (Free)

crystal growth (Selvam et al., 2001; Karadi et al., 2006). This fact, combined with theincreased urinary calcium leads to their supersaturation in urine and finally stoneformation. One mechanism currently proposed as an important to prevent the formationof urinary stone is the presence of substance in urine that prevents calcium saltcrystallization (Tostes et al., 2004). In present study the significant hypercalciuria wasobserved in control groups from both the paradigms studied. Treatment with HELI (200and 400 mg/kg) showed significant reduction in calcium in urine in both the paradigmsstudied. By inhibiting calcium excretion the drug decreases the supersaturation of theurine with respect to CaOx and thereby decreased the risk of stone formation. Apart fromurinary calcium excretion, decrease in serum calcium was evident in urolithiatic rats(Touhami et al., 2007; Tsai et al., 2008). In present study the significant decrease inserum calcium level was observed in control groups from both the paradigms studied.Treatment with HELI (200 and 400 mg/kg) significantly increased serum calcium level inboth the paradigms studied, thereby prevented stone formation.Normal urine contains many organic and inorganic inhibitors of crystallization;magnesium is one such well known inhibitor (Selvam et al., 2001). Magnesiumcomplexes with oxalate, thus reducing CaOx supersaturation in urine (Vidya andWaralakshmi, 2000; Soundararajan et al., 2006) as a consequence decrease the growthand nucleation rates of calcium crystals (Grases et al., 1989; Selvam et al., 2001). Inpresent study the significant decrease in urinary magnesium was observed in controlgroups from both the paradigms studied. Treatment with HELI (200 and 400 mg/kg) wassignificantly increased urinary magnesium in both the paradigms studied.Increased urinary phosphorus was observed in calculi induced rats. Increasedurinary phosphorus excretion along with oxalate stress seems to provide an environmentappropriate for stone formation by forming calcium phosphate crystals, which epitaxiallyinduces CaOx deposition (Karadi et al., 2006).Treatment with HELI (200 and 400 mg/kg) restored phosphorus levelssignificantly, thus reduced the risk of stone formationIn urolithiasis, the glomerular filtration rate decreases due to the obstruction to theflow of urine by stones in urinary system (Divakar et al., 2010). This causes impairment90 Full Text Available On www.ijupls.com (Free)

of renal function resulting decreased excretion of waste products, particularly nitrogenoussubstances such as urea, creatinine, and uric acid with concurrent accumulation in blood(Bahuguna et al., 2009; Divakar et al., 2010; Touhami et al., 2007).Rats treated with ethylene glycol with ammonium chloride showed marked renaldamage as evident from increase in serum creatinine, serum urea, BUN, urinary urea andBUN and decrease in urinary creatinine. However preventive treatment with HELI (200and 400 mg/kg) prevents impairment of renal function which was evidenced fromdecrease in serum creatinine serum urea, BUN, urinary urea and BUN and normalizingurinary creatinine.Uric acid interferes with CaOx solubility and it binds and reduces the inhibitoryactivity of glycosaminoglycans. The predominance of uric acid crystals in calciumoxalate stones and the observation that uric acid binding proteins are capable of bindingto calcium oxalate and modulate its crystallization also suggests its primary role in stoneformation (Selvam et al., 2001). Apart from increase in urinary uric acid concentration,some studies suggested increase in the serum uric acid concentration in urolitiasis (Karadiet al., 2006; Bahuguna et al., 2009).In the current study, higher concentration of serum uric acid was observed insodium oxalate and ethylene glycol with ammonium chloride induced urolithiatic rats.Treatment with HELI (200 and 400 mg/kg) restored the uric acid level to normal levelthus reducing the risk of stone formation.Renal injury whether a result of renal calculi decreases nephron population. Theeffect of this reduction in renal mass is to place an increased workload on survivingnephron. Through a series of microcirculary adaptations, glomerular blood flow andcapillary hydrostatic pressure are increased, and the filtration rate in each glomerulus isaugmented. Gomeruli undergoing this hyperfiltration process are subjected tohemodynamic stress; which damages capillary integrity and predisposes to leakage ofprotein into the urine (Clive, 1991b).Protein excretion was increased in hyperoxaluric rats (Selvam et al., 2001;Soundararajan et al., 2006; Varalakshmi and Vidya, 2000). Supersaturation of urinarycolloids results in precipitation as a crystal initiation particle, which when trapped, act as91 Full Text Available On www.ijupls.com (Free)

a nidus and leading to subsequent crystal growth (Selvam et al., 2001). Proteinureareflects proximal tubular dysfunction (Varalakshmi and Vidya, 2000; Soundararajan etal., 2006).Favoring this discussion, protein excretion was increased in sodium oxalate andethylene glycol with ammonium chloride induced lithogenic rats. Administrations ofHELI (200 and 400 mg/kg) had profound effect on reducing the protein excretion andthus prevented the nidus formation for nucleation and thereby minimizing the extent oftubular dysfunction.Significant increase in kidney weight in urolithiatic rat is associated withcalculogenesis which is considered as a result of urinary supersaturation with respect tostone forming constituents (Mitra et al., 1998). The significant increase in kidney weightof lithogenic rats was reported in many studies (Tsai et al., 2008; Bashir and Gilani et al.,2009; Mitra et al., 1998).This effect was significantly prevented with treatment of HELI (200 and 400mg/kg). There are several phytochemicals which could be responsible for antiurolithiaticeffect are flavonoids and triterpenes (Arafat et al., 2008; Soundararajan et al., 2005)saponins and tannins (Doddola et al., 2008).The preliminary phytochemical studies have confirmed the presence ofcarbohydrates, saponnins, tannins and flavonoids in HELI. Hence the antiurolithiaticeffects of aqueous extract of Lawsonia inermis L. leaves may be due to presence ofsaponins, tannins and flavonoids which may act individually or in combination.The result of present study revealed that aqueous extract of Lawsonia inermis L.leaves (200 and 400 mg/kg) has antiurolithiatic activity equipotent to that of Cystone ® , athigher dose tested. The antiurolithiatic activity may be due to increased diuresis;increased excretion of urinary salt constituting stone; inhibition of oxalate induced toxicmanifestation; disintegration of mucoproteins by saponins; formation of soluble complexof tannins with calcium.However, further studies are necessary to isolate and characterize thephytoconstituents responsible for antiurolithiatic action and to explore the exactunderlying mechanism of action as well as clinical utility of Lawsonia inermis L.92 Full Text Available On www.ijupls.com (Free)

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