Ethnopharmacological relevance: A perennial medicinal herb, Curcuma caesia Roxb. Zingiberaceae known as black turmeric in Ayurveda is used in ethnomedicine to treat ailments such as inflammation, wounds and kidney disorders.
Aim of the study: To evaluate the protective effect of methanolic extract of Curcuma caesia (CCE) against gentamicin (GM) induced renal toxicity.
Materials and methods: Nephrotoxicity was induced in rats with GM (100 mg/kg b.w.; i.p. for 8 days) and were treated with CCE (250 mg/kg and 500 mg/kg b.w.; p.o. for 8 days) or 0.9% sodium chloride (vehicle). Plasma (urea, creatinine) and urine (urea, creatinine); renal enzymatic and non-enzymatic antioxidants along with lipid peroxidation were evaluated in various experimental groups.
Results: GM treatment induced significant elevation (p<0.01) in plasma and urine urea, creatinine, renal lipid peroxidation along with significant decrement (p<0.001) in renal enzymatic and non-enzymatic antioxidants. CCE treatment to GM treated rats (GM+CCE) recorded significant decrement (p<0.001) in plasma and urine urea and creatinine, renal lipid peroxidation along with significant increment (p<0.01) in renal enzymatic and non-enzymatic antioxidants.
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Conclusion: CCE rhizome extract ameliorates GM induced nephrotoxicity and renal dysfunction and thus validates its ethnomedicinal use.
Key words: Curcuma caesia Roxb. (CCE), Gentamicin (GM), Nephrotoxicity, Reduced glutathione (GSH), Oxidised glutathione (GSSG), Malonaldehyde (MDA), Catalase (CAT).
The Zingiberaceae family comprises a variety of medicinal plants used in the treatment of various human ailments. A perennial medicinal herb, Curcuma caesia Roxb. (Zingiberaceae) known as black turmeric in Ayurveda is widely cultivated and found in Southeast Asian countries like India, Indonesia, Thailand & Malaysia for traditional purposes (Paliwal et al., 2011). It is a wonder herb with highest contents of curcumin, a chemical substance with many curative properties. It is perhaps one of the few medicinal plants that are used in black magic as well as herbal treatment. However, scientific studies suggests that the rhizome possess characteristic properties like antioxidative, anti-inflammatory, anti-diarrheal, diuretic, anti-emetic, wound healing, hypoglycemia, anticoagulant, and antimicrobial activities (Charles Santhanaraju V et al., 2013).
Recent research showed that the volatile oils from rhizomes of Curcuma caesia contains 30 major components such as; camphor, ar-turmerone, (Z)-ocimene, ar-curcumene, 1,8-cineole, borneol bornyl acetate etc., which have been responsible for major pharmacological activities (Satyavama D AsemÂ et al., 2012).
The present study was designed to evaluate the protective effect of methanolic extract of C. caesia against gentamicin induced renal toxicity. However the antioxidant and free radical scavenging effect of phenolic compounds, flavonoids and volatile oils remarkably showed nephroprotective action (Pandey Ashok K et al., 2003). Thus, the biological screening of this plant was carried out to find nephroprotective potential, which is remarkably a new herbal treatment in kidney disorders. The extent of protective effect was determined by studying serum kidney toxicity markers and biochemical estimation of antioxidant enzymes.
Materials and methods
The rhizomes of Curcuma caesia Roxb. was collected in the month of September from foothills of Himalayan region, Pithoragarh (Uttarakhand, India). The plant was identified by Mr. Gaurav Upadhyay, Assistant Professor, Siddhartha Institute of Pharmacy, Dehradun, India.
Preparation of extract
About 450g fine powder of dried rhizomes of Curcuma Caesia was subjected to successive extraction in a Soxhlet apparatus with 750 ml methanol by at a temperature of 55Â±5Â°C for a period of 16 hrs. The obtained crude extract was filtered and evaporated to dryness at vacuum in a Buchi evaporator. The residue was weighed (30g) and stored at a temperature 4°C until use (Karmakar Indrajit et al., 2011).
Preliminary phytochemical screening
A preliminary phytochemical screening were carried out to assess presence/absence of various groups of phytochemicals (Trease and Evans, 1987).
Healthy adult male wistar rats (180-250g) were obtained from Siddhartha Institute of Pharmacy, Dehradun, India. They were housed in groups of four under standard laboratory conditions of temperature (25 Â± 2Â°C) and 12/12 h light/dark cycle. Animals had free access to standard pellet diet and water ad libitum. The distribution of animals in the groups, the sequence of trials and the treatment allotted to each group were randomized, throughout the experiment. Laboratory animal handling and experimental procedures were performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and experimental protocol was approved by Institutional Animal Ethical Committee Siddhartha Institute of Pharmacy, Dehradun, India (Approval No. 1891/PO/A/05/CPCSEA).
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Gentamicin induced nephrotoxicity in rats
Animals were divided into four groups consist of 6 animals in each group. Group I (CON) received 10ml/kg/day oral dose of 0.9% of NaCl, Group II (GM) received 100 mg/kg/day i.p. of gentamicin and Groups III (GM + CCE-250) & IV (GM + CCE-500) were received CCE at a dose of 250mg/kg/day and 500mg/kg/day, respectively with 100 mg/kg/day i.p. of gentamicin for eight days. The body weights of the animals were also recorded daily. After 8 days, Animals were anaesthetized with ether for collection of blood serum (using retro orbital plexus) for estimation of various haematological and biochemical analysis. The rats were sacrificed then by cervical dislocation and kidneys were isolated for estimation of tissue parameters and histopathological study.
Plasma and urine markers of renal damage
Animals were individually housed in metabolic cages as per divided in separate groups for 24 h and urine was collected on the 8th day of the treatment. Blood samples were collected from these overnight fasted animals through retro-orbital sinus puncture in ethylene diamine tetra acetic acid (EDTA) coated vials and plasma was separated by cold centrifugation of vial (REMI Centrifuge R23) at 3000rpm for 10 min. Urea and creatinine were assayed in plasma and urine using commercially available kits (DIATEK Healthcare Pvt. Ltd. Kolkata, India) and the hematological variables viz. RBC, WBC and Hb were recorded by using haematological auto-analyzer using Lal pathology lab dehradun, India.
Preparation of homogenate of renal tissue
A small portion of the kidney tissue (300-600 mg) were excised and homogenized in 5-8 mL of 0.02M EDTA and then 4mL of cold distil water was added to it. 2mL of suspension medium was taken from the supernatants of the 10% (w/v) tissue homogenate in 1.15% KCl and centrifuged at 10,000 rpm for 20 min (0 â-‹C) in a high speed cooling centrifuge and supernatant and sediment were used for further biochemical estimations.
Measurement of renal lipid peroxidation
Measurement of malonaldehyde (MDA) as an index for lipid peroxidation was done using thiobarbituric acid assay as per Buege and Aust (1978).
Measurement of renal antioxidants
Superoxide dismutase (SOD) was assayed in tissue supernatant by the method of Kakkar et al. (1984), based on the inhibition of the formation of nicotinamide adenine dinucleotide-phenazine methosulfate-nitro blue tetrazolium formazan. Catalase (CAT) activity in tissue supernatant was measured spectrophotometrically at 240nm by calculating the rate of degradation of hydrogen peroxide, the substrate of the enzyme (Sinha, 1972). Reduced glutathione (GSH) content in tissue supernatant was measured spectrophotometrically by using Ellman's reagent (Dinitrothiobenzoic acid) as a colouring reagent, following the method described by Beutler et al. (1963). Whereas, oxidised glutathione (GSSG) was measured according to the method described by Aseni et al. (1999) based on the principle of glutathione reductase enzyme reducing GSSG to GSH with the concomitant oxidation of NADPH to NADP+.
Acute oral toxicity study
The toxicity of extract was studied as per organization for economic co-operation and development (OECD) guideline number 425. Male wistar rats weighing 180-250g were used in the toxicity study. Six rats were serially administered a dose of 2000 mg/kg bodyweight of CCE prepared in water as recommended in the guideline. After dose administration, each animal was observed after every hour for signs of toxicity and abnormality in behaviour up to the 48th hour. After this, daily observations for toxicity and mortality were made up to the 14th day. The body weights of the animals were recorded every third day. On the 14th day after dosing, all the mice were sacrificed and processed for gross necropsy (OECD, Adopted in December 2001).
Preparation of extract dose
A dose of 250mg/kg and 500mg/kg (based on toxicity study) methanolic extract Curcuma caesia (CCE) were suspended in distilled water using 1% tween 80 and administered orally to experimental animals. Suspension of extract was prepared freshly. The extracts were administered at a constant volume of 10 ml/kg for each animal (Arulmozhi et al., 2006).
Kidney tissues of rat was removed and washed with normal saline. Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely employed universal fixative particularly for routine paraffin embedded sections. The cleared tissue was fixed in 10% natural buffered formalin solution (pH 7.0-7.2). All the sections of the tissues were examined under microscope for the analyzing the altered architecture due to the Kidney tissue due to Gentamicin challenge and improved Kidney architecture due to pretreatment with test extracts and standard drug. These were examined under the microscope for histopathological changes such as glomerular congestion, degeneration of epithelial cells of tubular cells.
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Numerical results are expressed as Mean Â± SEM. Data was analyzed using one way analysis of variance (ANOVA) test. Calculations were performed using commercial software (GraphPad Software, San Diego, CA). ANOVA followed by Bonferroni post hoc test. p values < 0.05 were considered as statistically significant. *P<0.05; **<0.01 and ***<0.001 significant when compared with normal group as applicable.
Preliminary phytochemical screening of methanolic extract of Curcuma caesia showed that the plant contains phenolic compounds, flavonoids, carbohydrates, proteins, tannins, alkaloids, glycosides, saponins and volatile oils which may possessing antioxidant and free radical scavenging activity. There were no any abnormal behaviour and physiological changes recorded in CCE fed rats up to a dose of 2000mg/kg bodyweight.
Measurement of Curcuma caesia Roxb extract (CCE) on bodyweight, kidney weight and kidney volume in gentamycin (GM) induced nephrotoxicity
The graded dose (250mg/kg and 500mg/kg) of methanolic extract of CCE was given as pre-treatment dose by oral route for both the treatment group with gentamicin (100mg/kg i.p.) induced nephrotoxicity in rats (n=6). The measured parameters are mentioned in Table 1 which summarised that, the GM treated group has been scored significant (P<0.001) decrease in body weight, while significant (P<0.05 and <0.01) increase in kidney weight and kidney volume as compared to CON group. Whereas, these sets of changes were remains same in graded dose of CCE treatment groups as compared to GM treated animals.
Effect of CCE on RBC, WBC count and Haemoglobin in GM induced nephrotoxicity
As per above mentioned treatment sequence, the haematological parameters are expressed in Table 2. In which, the GM treated group showed significant (p<0.01) increase in WBC and RBC count as compared to CON group, while Hb showed significant (p<0.01) decrease in parameter when compared to the same group. The pre-treated group of CCE at graded dose of 250mg/kg and 500mg/kg showed significant (p<0.01 and p<0.001) improvements in WBC, WBC count and Hb parameter as compared to GM treated group.
Effect of CCE on plasma (urea & creatinine) and urine (urea & creatinine) level in GM induced nephrotoxicity
Table 3 shows that, the GM treated animals registered significant (P<0.01 and P<0.001) increase in urea and creatinine levels in plasma and urine compared to CON rats whereas, these set of changes were assumed base line gradually in CCE treated group of animals.
Effect of CCE on renal activity levels of superoxide dismutase (SOD), catalase (CAT), Malonaldehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSH) level in GM induced nephrotoxicity
As summarized in Table 4, GM treated animals registered significant (p < 0.01) decrement in the enzymatic and non-enzymatic antioxidants along with significant increment (p < 0.05) in renal MDA as compared to CON rats whereas, GM+CCE (250mg/kg and 500mg/kg) groups significantly (P<0.05, <0.01 & <0.001) prevented these set of changes and achieved base line of GM+CCE-500 treatment group.
GM lead to major histopathological changes including glomerular congestion and degeneration of the tubular epithelial cells and necrosis of cells in compared to control group. The renal tissue of rats treated with CCE at a graded dose of 250mg/kg and 500 mg/kg bodyweight per day for 8 days, showed the excellent recovery with little epithelial cell degeneration and few blood vessels congestion (Figure 1).
Discussion and conclusions
In the present study, we investigated the nephroprotective activity of Curcuma caesia rhizomes extract in animal model of nephrotoxicity, various biochemical parameters such as blood urea, serum creatinine and antioxidant parameters such as lipid peroxidation, glutathione and oxidized glutathione in tissue and serum MDA and serum GSH were chosen to assess the antioxidant activity of Curcuma caesia extract on animal model. These parameters were assessed after treatment with extract in two different doses (250 & 500 mg/kg. orally) and compared them with toxicant agent gentamicin (100 mg/kg, i.p.).
In case of serum marker, a significant reduction in elevated levels as compared to gentamicin treated group were observed and significant decrease in kidney weight after treatment with Curcuma caesia extract compared to gentamicin group of rats conferred the nephroprotective effect. More ever significant decrease in renal blood urea (p<0.05), serum creatinine (p<0.05) and lipid peroxides (p<0.05) and significant increase in glutathione (p<0.05) when compared with Gentamicin treated group further confirm the protective effect of Curcuma caesia extract. Based on improvement in serum marker levels and antioxidant it is conferred that Curcuma caesia extract possess nephroprotective activity and thus supports the traditional application of the same under the light of modern science.
Curcuma caesia extract has shown the ability to maintain the normal functional status of the kidneys and shown the nephroprotective effect which confirmed by the following parameters: