Nephroprotective Activity of Methanolic Extract of Lantana camara and Squash ( Cucurbita pepo ) on Cisplatin-Induced Nephrotoxicity in Rats and Identification of Certain Chemical Constituents of Lantana camara by HPLC-ESI-MS

Introduction: Cisplatin is a highly effective chemotherapeutic agent; its clinical use is severely limited by serious side effects as nephrotoxicity. The aim of this study is to evaluate the nephroprotective activity of defatted methanolic extract of two Egyptian plants: Lantana camara and Cucurbita pepo and certain fractions derived from the defatted methanolic extract of L. camara on cisplatin-induced nephrotoxicity in rats. Also, identification of certain chemical constituents of L. camara by HPLC-ESIMS. Methods: Nephrotoxicity was induced in rats by single dose of cisplatin. The effect of plants extract at doses 100-400 mg/kg.b.wt comparing with standard; ascorbic acid; was determined using serum urea, creatinine and some ions. Furthermore, the effect of these extracts on some renal antioxidant enzymes and histopathological examination of kidneys were examined. Results: The defatted methanolic extract and ethyl acetate fraction of L. camara showed the highest improvement of renal parameters. Also, HPLC-ESI-MS analysis of L. camara extracts exhibited bioactive phenolic compounds including phenyl ethanoid, flavonoids and phenolic acids. Conclusion: The phytochemical constituents of L. camara are responsible for their nephroprotective activity.

agents that provide nephroprotection against the renal impairment caused by drugs like cisplatin for which allopathy offers no remedial measures.Thus, it is obligated to turn towards alternative systems of medicine as natural products.So, plants which containing antioxidant properties are recommended as nephroprotective agents. 3,4,5ntana camara L. (Family Verbenaceae) is distributed throughout many countries.It is an important medicinal plant, which exhibits anti-inflammatory, antihyperglycemic, antioxidant activity and hepatoprotective activities. 6,7,8On the other hand, Several Squash plants (Cucurbita pepo) (Family Cucurbitaceae) have health benefits such as anti-diabetic, antifungal, antibacterial and anti-inflammation. 9,10herefore, the objective of this study is evaluating the nephroprotective effect of the defatted methanolic extract of the leaves of two plants grown in Egypt; Lantana camara and Cucurbita pepo (squash).Also, Ascorbic acid and all the extracts of above mentioned dose were administrated for 10 days before cisplatin.Whereas, the rats were recovered with free access to food and water ad libitum for five days after cisplatin injections (all experiment time 15 days). 14,15

Blood collection
The animals were sacrificed by decapitation after 15 days and blood samples were collected from the tested animals in clean centrifuge tubes and centrifuged at 3000 rpm for 15 min.Serum parameters including creatinine, urea, magnesium, potassium and sodium were estimated.The biochemical estimations were done using a Biochemical-semi-auto analyser by standard procedures.

Calculate relative kidney weight
The kidneys and livers were isolated, weighed and relative weight of them was calculated from the ratio of organ weight to body weight. 16lative weight = (The organ wt./Body wt.) × 100

Preparation of tissue sample
One kidney was homogenised in 5-10 ml/g tissue of cold phosphate buffer saline solution (PH 7.4) using an electronic homogenizer to prepare homogenate.The homogenate was centrifuged at 4000 rpm for 15 min at 4 º C then the supernatant was used for the estimation of lipid peroxidation as malondialdehyde (MDA), 17 superoxide dismutase (SOD), 18 catalase (CAT) 19 and Glutathione peroxidase (GPx). 20

Histopathology studies
The other kidney was weighed and preserved in 10% neutral buffered formalin.A 5μ thickness of tissue sections was stained withhematoxylin and eosin stain (H and E stain) for routine histopathological examination.

HPLC-ESI-MS conditions
The samples (5 mg/mL) of L. camara extracts were injected to HPLC hyphenated with mass spectrometer.LC-ESI-MS analysis system consists of HPLC (Waters Alliance 2695) and mass spectrometry (Waters 3100).The mobile phases were prepared daily by filtering through 0.45 mm membrane disc filter and degassed by sonication before use.The mobile phase for gradient elution consists of two solvents: solvent A (0.1% formic acid (FA) in H 2 O) and solvent B (0.1% FA in CH 3 CN/MeOH (1: 1; v/v).The linear gradient profile was as follows: 95% A (5 min), 95-90% A (10 min), 90-50% A (55 min), 50-95% A (65 min), and 95% A (70 min).The injection volume was 10 µL.The flow rate (0.6 mL/min) was split 1: 1 before the MS interface.The negative ion mode parameters were as follows: source temperature 150°C, desolvation temperature 350 °C, cone gas flow 50 L/h, cone voltage 50 eV, capillary voltage 3 kV, and desolvation gas flow 600 L/h.Spectra were recorded in the ESI negative mode between m/m 50-1000.The peaks and spectra were processed using the Masslynx 4.1 software.The compounds were tentatively identified by comparing their retention time (tR) and mass spectrum with literature.

Statistical analysis
The results are reported as mean ± SD.Data were analyzed by using oneway analysis of variance (ANOVA) by SPSS software package.P values less than 0.05 were considered significant.

Acute Toxicity
The purpose of preliminary acute toxicity studies is to determine the LD 50 values to help us in evaluating the safe dose range at which the drug can be used without any harmful or lethal effect on the experimental the identification of chemical constituents of Lantana camara by HPLC-ESI-MS was carried out.

Plant materials
L. camara leaves were collected from Garden of Theodor Bilharz Research Institute, Giza, Egypt whereas, Cucurbit pepo leaves (Squash) were collected from local markets in Giza, Egypt.Both plants were identified by Asst.Prof.Rim Samir Hamdy Professor of Plant Taxonomy, Faculty of Science, CairoUniversity. 11The voucher specimen of each plant was stored in Medicinal Chemistry Department, Theodor Bilharz Research Institute.

Chemicals
Cisplatin and Ascorbic acid were purchased from Sigma -Aldrich (USA), Lipid Peroxide (Malondialdehyde), Superoxide dismutase, Catalase and Glutathione peroxidase were purchased from Biodiagnostics, Egypt.

Extraction and fractionation
Leaves of each plant under investigation were dried and powdered by grinder.500 g dry powders of each plant was separately extracted with 85% methanol (MeOH) then evaporated under vacuum using rotatory evaporator till dryness.The dried methanolic extract was defatted with petroleum ether.The aqueous defatted methanolic extract of the leaves of L. camara was fractionated using dichloromethane, ethyl acetate (EtOAc) and n-butanol (N-BuOH).Each fraction was concentrated and dried using rotatory evaporator then kept away from moisture.

Experimental animals
Male Wister albino rats, weighing 120-200 g were purchased from animal house of National Research Centre, Giza, Egypt.Animals were housed in polypropylene cages for one week under standard laboratory conditions (25±2˚C, 55±5% humidity and 12h light/ dark cycle).They were fed standard diet and water ad libitum.The experiment followed the ethical standards for care and use of laboratory animals.

Acute oral toxicity studies
Acute toxicity studies of the defatted methanolic extract of the leaves of each plant were performed according to. 12The detailed experiments and results were reported in our previous study. 13
animals.In our study, results of each defatted methanolic extract of L. camara and C. pepo leaves revealed that there is no death or any clinical signs of toxicity in the tested rats.Therefore, the LD 50 value of the defatted methanolic extract of each plant extract was found to be higher than 5000 mg/kg. 13

Nephroprotective activity
Unfortunately, kidney diseases may be silent for a long time.Early detection is the key to preventing kidney diseases.The kidneys provide the final common pathway for the excretion of many drugs and their metabolites.Therefore, they are frequently subjected to high concentrations of potentially toxic substances. 21he present study is an effort to the evaluation of the potency of a famous natural antioxidant vitamin C. 22,23 The body, relative kidney and liver weights The results in (Table 1) exhibited that the initial body weights of all rats were almost the same.In the normal (control group), there is an obvious change in the body weights of rats between the initial and final body.The rat group treated with cisplatin (Toxic group) showed a significant decrease in their body weights as comparing with control group whereas, the relative weights of liver and kidney increased.These results suggested that the loss of weights of cisplatin-induced experimental animals may be due to the gastrointestinal toxicity of these animals and due to reducing of food ingestion by the animals. 24he administration of one dose of vitamin C (standard group) along with injection this group with of one dose of cisplatin on day 10 led to significant increase in the final body weights of the rats whereas decreased the relative kidney and liver compared with the toxic group.These results are in agreement with the previous results on other plant extracts. 25he groups treated with each dose 100, 200 and 400 mg /kg b.wt./day of the defatted methanolic extract of L. camara increased the final body weights of the rats whereas the relative kidney and liver were decreased comparing with the toxic group.The increasing of the final body rat weights is higher with a concentration of L. camara extract 400 mg / kg b.wt./day than concentration of 100 and 200 mg /kg b.wt./day.Also, the decreasing of the relative kidney and liver is associated with the high concentration of the plant extract 400 mg /kg b.wt./day as showed in (Table 1).In the groups treated with the defatted methanolic extract of C. pepo, it was obvious that the increasing of the final body rat weights and the decreasing of the relative kidney and liver of rats are lesser than as in the defatted methanolic extract of L. camara as showed in (Table 1).So, the defatted methanolic extract of L. camara had higher effect than the extract of C. pepo.Also the results appeared that the extract of each plant has the ability in protective of the rats against cisplatin-induced nephrotoxicity and improvement of the relative liver and kidney of the experimental rats.These results agree with previous studies on other plant extracts. 26,27wing to the high nephroprotective activity of the defatted methanolic extract of L. camara, this extract was submitted to fractionation process using EtOAc and n-BuOH and the two fractions were evaluated as protective agents against cisplatin-induced nephrotoxicity for the experimental animals (Rats).Also, results in (Table 1) indicated the two fractions (EtOAc and n-BuOH) increase the final weight of rats and improve the relative kidneys of the rats as comparing with the toxic group.Also, EtOAc fraction showed higher effect than an n-BuOH fraction.Therefore this result means that the two fractions have nephroprotective effect against cisplatin-induced nephrotoxicity.

Kidney parameters
In the present study, the effect of oral doses 100, 200 and 400mg/kg b. wt./day of each defatted methanolic extract of L. camara and Squash (C.Pepo) on serum urea, creatinine, and certain ions in cisplatin-induced nephrotoxic rats were performed.The results in (Table 2) revealed that: In a toxic group, it appears that there is the significant elevation of serum urea and creatinine values compared to the normal group.Also, the levels of sodium and potassium ions in the toxic group were significantly decreased compared to control group whereas, the magnesium ion was significantly increased.These results indicate that injury of the kidney of the rats occurred. 28dministration of the standard substance vitamin C (standard group) to rats which injected with one dose of cisplatin on day 10 led to significant reduction to the elevated levels of serum urea and creatinine as well as Mg ions whereas Na and K ions increased significantly comparing to the toxic group.The groups treated with different concentrations 100, 200 and 400 mg/kg/b.wt./day of each defatted methanolic extract of L. camara and C. pepo led to significant reduction of the levels of serum urea and creatinine as well as Mg ions values whereas Na and K significant increased compared to the toxic group.400 mg/kg.b.wt./day of each plant extract gave good results.Also L. camara extract gave higher effect than C. pepo extract.The results mean that the two plant extracts have nephroprotective effect against cisplatin-induced nephrotoxicity and improve the kidney functions.These results are in agreement with a study on other plant extracts. 25esults in (Table 2) indicated the two fractions (EtOAc and n-BuOH) reduce the levels of serum urea and creatinine as well as Mg ions values whereas Na and K significant increased compared to the toxic group.400 mg/kg b.wt./day of two fractions gave good results than 100 mg/kg b. wt./ day.Also, EtOAc fraction showed higher effect than an n-BuOH fraction.Therefore this result means that the two fractions have nephroprotective effect against cisplatin-induced nephrotoxicity.

Enzymatic antioxidants
It was reported that cisplatin increases the production of free oxygen radicals and decreases the antioxidants; this led to the disturbance of the oxidant/antioxidant balance. 29,30The reactive oxygen radicals accumulated in tissues resulted in the production of hydroxyl radicals, superoxide anions, nitric oxide and hydrogen peroxide. 31Thus the reactive oxygen species effect on the antioxidant defensemechanism through enhances lipid peroxidation process and led to the elevation of MDA levels 32 as well as reduce CAT, GPx and SOD levels. 33he results in a (Table 3) exhibited that: In (Toxic group), the antioxidant enzymes such as CAT, GPx and SOD significant decrease but MDA levels significant increase in the cisplatin group compared to the control.These observations reflected that the rat kidney is injured and this support that the nephrotoxicity induced by cisplatin in rats is occurred and partially related to the depletion of the renal antioxidant system.DeWoskin and Riviere 34 reported that this decreasing may either be due to loss of the certain mineral as copper and zinc, which are essential for the activity of the enzyme or due to ROS-induced inactivation of the enzyme.Moreover, the decreasing of CAT and GPx activities in the cisplatin group led to increasing the H 2 O 2 concentration and enhanced the lipid peroxidation. 35In the same time, the concentration of MDA as a result of lipid peroxidation increased in the toxic group.Administration of vitamin C or the defatted methanolic extract of each plant L. camara and C. pepo prevents the lipid peroxidation by enhancing the renal SOD, CAT, and GPx activities.So, vitamin C or the defatted methanolic extract of each plant L. camara and C. pepo could significantly protect the cisplatin-induced renal damage.However, the nephro- protective activity at (400 mg/kg.b. wt./ day) dose of each plant extract was higher than that at (100 mg/kg.b. wt./ day).Results in (Table 3) revealed that both two fractions of L. camara (EtOAc and n-BuOH) showed a significant elevation in SOD, CAT, GPx enzymes while significantly decreased MDA levels.Although, the high dose (400 mg/kg.b.wt.) of the two fractions has a significant effect, but EtOAc fraction showed higher significant effect than an n-BuOH fraction.Preliminary phytochemical screening of L. camara and C. pepo showed the presence of major antioxidant polyphenolic compounds in a chemical constituent of the two plants.These compounds protect the cisplatininduced renal oxidative damage in rat and led to increasing of the renal antioxidant status, such as SOD, CAT, GPx activities by attack the free radicals (scavenges the free radicals) which generated during cisplatininduced oxidative damage of renal parenchyma and improve the kidney functions. 24,36,37,38isplatin represents a class of antineoplastic drugs containing a heavy metal, platinum.It has been shown to cause nephrotoxicity in patients as well as in a variety of animal species. 26A higher dose of cisplatin (10 mg/kg.b. wt.) was sufficient to induce nephrotoxicity in rats corresponds to that currently being used in clinical practice. 15,26Although, the exact mechanism of cisplatin-induced nephrotoxicity is still not fully understood, however, cisplatin causes free radical production and lipid peroxidation in tubular cells.So, it has been suggested that these free radicals are responsible for the oxidative renal damage. 39,40Therefore, the antioxidants especially natural antioxidants are a good nephroprotective agent against cisplatin-induced renal injury. 23,28,40,41

Histopathological examination
Histopathological examination of the control group, toxic group, standard group and treated groups of kidney tissue of the experimental animals (Table 4) showed that: the control group exhibit normal renal cells as shown in (Figure 1).Cisplatin group (Toxic group) showed smaller glomeruli, interstitial oedema in tubular cells with mild hyaline casts in their lumens as in (Figure 2) comparing to control group.The standard group exhibits congested glomeruli as well as remarkable degeneration of the renal tubules with the presence of luminal hyaline casts and mild stromal interstitial mononuclear cellular infiltration as showed (Figure 3) compared to the toxic group.These results agreed with previous reports. 42,43he groups treated with concentrations 100 and 200 mg/kg.b. wt. of the defatted methanolic extract of L. camara showed average sized glomerulus with mild congestion as well as mild tubular degeneration and few luminal casts as shown in (Figure 4) compared to the toxic group.The group treated with concentration 400 mg/kg.b. wt. of the defatted methanolic extract of L. camara showed lesser glomerular congestion and milder degree of tubular degeneration as shown in (Figure 9) compared to the toxic group.The group treated with concentration 100 and 200 mg/kg.b. wt. of ethyl acetate fraction derived from the defatted methanolic extract of L. camara showed average sized glomeruli and mild tubular degeneration as shown in (Figure 5) compared to the toxic group.The group treated with concentration 400 mg/kg.b. wt. of ethyl acetate fraction derived from the defatted methanolic extract of L. camara showed normalisation of the histopathological features (Figure 7) Compared to the toxic group.The groups treated with concentrations 100 and 200 mg/kg.b. wt. of the butanolicfraction derived from the defatted methanolic extract of L. camara showed histopathological changes comparable to the cisplatin-treated group, except for the absence of stromal inflammation and mild tubular degeneration as showed in (Figure 6) compared to the toxic group (Figure 2).The group treated with concentration 400 mg/kg.b. wt. of the butanolicfraction derived from the defatted methanolic extract of L. camara has    not showed glomerular congestion but mild interstitial infiltration by mononuclear inflammatory cells as showed in (Figure 7) Compared to the toxic group (Figure 2).These results indicated the nephroprotective activity of L. camara as reported in other previous studies. 44,45he groups treated with concentrations 100, 200 and 400 mg/kg.b. wt. of the defatted methanolic extract of C. pepo exhibited marked tubular degeneration and frequent luminal hyaline casts associated with glomerular congestion and stromal inflammation comparable to the group treated with cisplatin (Figure 8).However, the 400 mg/kg.b. wt.treated group showed milder stromal inflammation as shown in (Figure 9) compared to the toxic group (Figure 2).The above results of histopathological examination of the rats exhibited that: The groups treated with the defatted methanolic extract of the two plants L. camara and C. pepo had a protective effect against degenerative kidney injury caused by cisplatin.The defatted methanolic extract L. camara gave better protective property against the injury of kidney induced with by cisplatin in rats than C. Pepo.Also, it was obvious that the higher concentration of the ethyl acetate fraction derived from the defatted methanolic extract of L. camara has high protective effect against kidney injury in rats induced bycisplatin than the defatted methanolic extract and the butanolic fraction of L. camara.The histopathological study of kidney against cisplatin-induced nephrotoxicity are in agreement with measurements of all parameters such as the changes in body weight, average kidney weights of rats and certain serum parameters as urea and creatinine as well as certain enzymatic antioxidants and ions of certain metals for toxic and treated groups.

HPLC-ESI-MS analysis of the defatted methanolic extract of L. camara as well as ethyl acetate and butanolic fractions
Owing to the high nephroprotective activity of the defatted methanolic extract of the leaves of L. camara as well as the ethyl acetate and butanolic fractions ,these extracts were submitted to HPLC ESI-MS analysis followed by identify of the major components of these extracts.ESI-MS full scan mode analyses were performed, in order to identify the deprotonated molecular ions [M-H] -, followed by ESI-MS/MS product ion experiments in negative ionization mode using the deprotonated molecular ion as a precursor to studying the fragmentation of the compounds.The identification of each compound based on its main molecular ions and different fragments as well as on the reported data of standards fragmentation especially sugar moieties as 162 = hexose; 146 = deoxyhexose; 176 = glucuronic or galacturonic acid; 86 = malonyl residue.The components of the defatted MeOH extract, EtOAc and BuOH fractions shown in (Figure 10) and (Tables 5, 6 and 7) respectively revealed that major components of these extracts are polyphenol groups such as phenolic acid derivatives, phenylethanoid glycosides, flavonoids and iridoids.

Figure 1 :
Figure 1: Section in kidney tissue of normal control rat, showing average sized glomeruli and proximal tubules with unremarkable pathological changes (Hematoxylin and eosin stain, X 200).

Figure 2 :
Figure 2: Section in kidney tissue of cisplatin-treated rat (toxic control), showing: (A) smaller glomeruli and degenerated proximal tubules.(B)degenerated distal tubules with many hyaline casts in their lumens (Hematoxylin and eosin stain, X 200).

Figure 4 :
Figure 4: Section in kidney tissue of cisplatin -treated rat preceded by MeOH extract of L. camara(C) (100 mg/kgb.wt.) and (D) (200 mg/ kg b. wt./ day), showing average sized glomerulus with mild congestion as well as mild tubular degeneration and few luminal casts compared to the toxic group (Hematoxylin and eosin stain, X 400)

Figure 9 :
Figure 9: Section in kidney tissue of cisplatin -treated rat preceded by (M) MeOH extract of L. camara (400 mg/ kg b. wt./ day), showing milder degenerative changes of glomeruli and proximal tubules compared to the toxic group, (N) MeOH extract of C. Pepo(400 mg/ kg b. wt./ day), showing mild collapse of glomeruli and degeneration of proximal tubules (Hematoxylin and eosin stain, X 400).

Table 6 : Tentative identification of polyphenol compounds in ethyl acetate fraction derived from the defatted methanolic extract of L. camara by HPLC-ESI -MS.
Pharmacognosy Journal, Vol 10, Issue 1, Jan-Feb, 2018