INTRODUCTION:

Cadmium is a well known industrial pollutant. It is industrially used in plating of steel, alloys, plastics, nickel-cadmium batteries and in electronic and nuclear engineering¹. Environmental cadmium exposure occurs due to industrial pollution and also in heavy smokers because tobacco smoke yields high cadmium concentrations². Exposure to cadmium leads to the production of reactive oxygen species (ROS) and it damages kidney as cadmium concentrates in the kidney. The biologic half life of cadmium is more than 30 years, so even the low level and long time exposure can accumulate in kidney. The plant Ixora coccinea (family Rubiaceae) has several names like ‘Flames of woods’ or ‘Jungle geranium’ or ‘burning love’ or chethi or kepala.

The use of almost all parts of the plant has been documented in the traditional Indian system of medicine and also in various folk system of medicine in India and as well as in Sri Lanka. The flowers, leaves, stem and roots of Ixia coccinea are used to treat various medical ailments³. The leaves are known to be of use in diarrhoea. The roots of the plant are generally used to treat hiccup, nausea, loss of appetite and externally for the treatment of sores, eczema and chronic ulcers⁴ and so far its protective effects on kidney have not been investigated. Traditional/folklore drug practitioners claim that Ixora coccinea removes heavy metals i.e lead, cadmium and mercury from contaminated water and the literature survey also indicates that it possesses several antioxidant principles⁵. Thus we hypothesise that this plant may also have nephroprotective properties. The aim of this study was to evaluate the protective efficacy of I. coccinea against CdCl₂ induced renal toxicity. The extent of the protective effect of I. coccinea against nephroprotective effects was determined by studying biochemical estimation and histopathological changes of wistar rats.

MATERIAL AND METHODS:

Plant material:

The flowers of Linn was purchased from the local market in Mangalore, Karnataka.

Preparation of extract of Ixora coccinea:

The aqueous extract of IACF was prepared by keeping 15g of dry flowers in 1000ml of water at 95°C for 15mins. The extract obtained was concentrated and evaporated under reduced pressure and controlled temperature and stored in desiccators until further use.

Preliminary phytochemical screening:

Preliminary phytochemical screening was carried out with aqueous extract of Ixora coccinea flowers (AICF) for the detection of various phytoconstituents using standard methods.

Animals:

Thirty male Wistar rats (150-200g) bred at Nitte University Center for Animal Research and Experimentation (NUCARE), Deralakatte, Mangalore were used in the study. The animals were randomly grouped, housed in cages and maintained under standard laboratory conditions (temperature 25 ± 3°) with dark and light cycle (12h/12h). Animal were allowed free access to standard dry pellet diet and water ad libitum.

The study protocol was approved by the Institutional Animal Ethics Committee (NGSMIPS/IAEC/MAY-2016/15). The animals were handled with care and experiments were carried out as per CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines⁶.

Study design:

In the present experiment rats were randomly divided into five groups each group comprised of six rats and were treated as follows^7-8:

Group I: Animals received vehicle (10 mg/kg body weight/day, p.o) for 10 days.

Group II: Animals received vehicle (10 mg/kg body weight/day) for 8 days followed by a single dose of cadmium chloride (20 mg/kg body weight, s.c) on 9^th day.

Group III: Animals received AICF (100 mg/kg body weight/day) for 8 days followed by a single dose of cadmium chloride (20 mg/kg body weight, s.c) on 9^th day.

Group IV: Animals received AICF (200 mg/kg body weight/day) for 8 days followed by a single dose of cadmium chloride (20 mg/kg body weight, s.c) on 9^th day.

Group V: Animals received AICF (400 mg/kg body weight/day) for 8 days followed by a single dose of cadmium chloride (20 mg/kg body weight, s.c) on 9^th day.

On 11^th day, blood samples were collected from retro-orbital venous plexus and serum was separated for the estimation biochemical parameters i.e blood urea, serum creatinine, total protein and uric acid. Animals were sacrificed and their kidneys were isolated, weighed and used for histopathological studies.

Statistical analysis:

The data was expressed as Mean ± SEM and analysed by using one way analysis of variance (ANOVA), followed by Post hoc Scheffe’s test using SPSS software version 16.0. A p value less than 0.05 was considered as statistically significant.

RESULTS:

Investigation of photochemicals:

Preliminary phytochemical investigation revealed the presence of various phytoconstituents i.e flavonoids, triterpinoides, proteins and sugars.

Body weight:

The initial and final body weights of animals of control and experimental groups were taken and percentage change in body weight was calculated. Weight of the animals was significantly reduced in CdCl₂ treated animals when compared with normal control. The decreased body weight was restored to near normal levels by (AICF) in extract treated animals (Table no.1). The animals of Group V (400 mg/kg B.W, p.o.) showed significant improvement in the body weight.

Relative kidney weight:

Kidney weight was significantly increased in CdCl₂ treated animals (Group II) when compared with normal control (Group I). Animal received AICF at dose of 400 mg/kg body weight (Group V) showed a significantly reduction in the increased kidney weight caused by the administration of CdCl₂when compared with toxic control (Table 1).

Table no 1: Effect of AICF in body weight and kidney weight in CdCl₂induced renal toxicity

Groups	Treatment	% Change in body weight	Kidney weight as % of body weight
I	Normal Control	7.67±4.47^b	0.40±0.02^b
II	CdCl₂treated (20 mg/kg)	-11.73±3.71^a	0.56±0.03^a
III	AICF 100 mg/kg +CdCl₂(20 mg/kg)	-5.38±0.55	0.52±0.01^a
IV	AICF 200 mg/kg + CdCl₂ (20 mg/kg)	1.31±1.30	0.49±0.03
V	AICF 400mg/kg + CdCl₂(20 mg/kg)	4.07±3.30^b	0.42±0.02^b

All the values are expressed as Mean ± SEM (n=6), a: p<0.05 when compared with Group I (Normal control), b: p<0.05 when compared with Group II (Toxic control)

Effect on blood urea:

The blood urea level of the normal control rats (Group I) was 29.5 ±1.98 mg/dl, while it was significantly elevated to 92.83±2.39 mg/dl in animals treated with CdCl₂ (Group II). The animals which received AICF at dose of 100, 200 and 400 mg/kg body weight (Group III, IV and V) significantly reduced the blood urea to 80.33±1.05 mg/dl, 62.17±1.70 mg/dl and 46.17± 3.86 mg/dl respectively when compared with toxic control.

Effect on serum creatinine:

The serum creatinine level of the normal control rats (Group I) was 0.43±0.02 mg/dl, while it was significantly elevated to 2.15±0.11 mg/dl in animals treated with CdCl₂ (Group II). The animals which received AICF at dose of 200 and 400 mg/kg body weight (Group IV and V) significantly reduced the serum creatinine level to 1.61±0.10 and 1.03±0.12 mg/dl respectively when compared with toxic control.

Effect on levels of uric acid:

The serum uric acid level of the normal control rats (Group I) was 1.36±0.09 mg/dl, while it was significantly elevated to 2.39±0.08 mg/dl in animals treated with CdCl₂ (Group II). The animals which received AICF at dose of 200 and 400 mg/kg B.W (Group IV and Group V) significantly reduced the serum uric acid level to 1.74±0.11 and 1.52±0.12 mg/dl respectively when compared with toxic control.

Effect on total proteins:

The Total protein level of the normal control rats (Group I) was 6.092±0.28 g/dl, while it was significantly reduced to 4.043±0.10 g/dl in animals treated with CdCl₂ (Group II). The animals which received AICF at dose of 100, 200 and 400 mg/kg B.W (Group III, IV and V) significantly elevated the uric acid level to 4.821±0.09, 5.027±0.11 and 5.753±0.09 g/dl respectively when compared with toxic control.

Table 2: Effect of AICF on Blood urea, Serum creatinine, Uric acid and Total protein content in CdCl₂ treated rats.

Treatment	Blood parameters
Treatment	Blood urea mg/dl	Serum creatinine mg/dl	Uric acid mg/dl	Total proteing/dl
Normal Control	29.5±1.98^b	0.43±0.02^b	1.36±0.09^b	6.09±0.28^b
CdCl₂treated (20 mg/kg, s.c.)	92.83±2.39^a	2.15±0.11^a	2.39±0.08^a	4.04±0.10^a
AICF 100 mg/kg +CdCl₂ (20 mg/kg, s.c.)	80.33±1.05^a	1.89±0.09^a	2.15±0.05^a	4.82±0.09^ab
AICF 200 mg/kg+ CdCl₂ (20 mg/kg, s.c.)	62.17±1.70^ab	1.61±0.10^ab	1.74±0.11^ab	5.03±0.11^ab
AICF 400 mg/kg +CdCl₂(20 mg/kg, s.c.)	46.17±3.86^ab	1.03±0.12^ab	1.52±0.12^ab	5.75±0.09^b

All the values are expressed as Mean ± SEM (n=6), a: p<0.05 when compared with Group I (Normal control), b: p<0.05 when compared with Group II (Toxic control).

Histopathological studies:

The histological observations basically support the results obtained from blood biochemical analysis. When CdCl₂ intoxicated rat kidney sections were compared to normal sections, showed noticeable congestion of the glomeruli with marked peritubular edema and congestion. The interstitium showed infiltration with inflammatory cells and congestion. Groups treated with AICF showed almost normalization of kidney section. However mild peritubular, glomerular congestion and inflammatory cells were observed.

Normal Kidney (Group I)

Kidney with CdCl₂toxicity (Group II)

Kidney with CdCl₂ + Extract 400mg/kg

DISCUSSION:

CdCl₂exposure show signs of a marked impairment of kidney functions, indicated by the decrease in the body weight, increase in kidney weight and increase of urea, serum creatinine and uric acid levels, decrease of total protein levels and histopathological variations^7-8. Urea and creatinine is the end product of protein and muscle metabolism respectively. Uric acid is which is formed by the natural breakdown of body’s cells and foods and these end products are constantly removed with the help of kidney. In case of kidney damage kidney fails to remove these markers and their concentration increases in the blood and because of proteinuria excess of protein excreted in the urine leads decreased levels of total protein in the blood^9-10.

The Ixora coccinea flowers contain several flavanoids like quercitin, rutin, kaempferitrin and procyanidin¹¹, it exhibits several”biological activities including antioxidant and free radical scavenging abilities and it is responsible for the nephroprotective activity. From the present study, it is understandable that injecting CdCl₂to the rats with AICF considerably protected the kidney function and structure as compared to the normal rats. These were confirmed by insignificant changes in urea, serum creatinine, total protein and uric acid levels and manifestation of normal structures of kidney, AICF may be beneficial in preventing progressive kidney diseases that are drastically accelerated by oxidative stress.“Hence the probable mechanism of nephroprotection by Ixora coccinea could be due to its antioxidant and free radical-scavenging property. This plant could play a promising role in the treatment of acute renal failure induced by CdCl₂.

CONCLUSION:

The Ixora coccinea is a well known traditional medicine in south India the chutney made from Ixora flower is popularly known in DK District and it is commonly added to well for water purification and people also believe this plant removes heavy metals such as lead, cadmium, and mercury from polluted water. It also reported to have antimicrobial, antioxidant, analgesic, chemo-protective, liver-protective, anti-mitotic and anti-inflammatory property. From the literature survey it was learnt that no substantial work related to its effect on CdCl₂induced nephrotoxicity was carried out. Hence an effort was made to evaluvate the nephroprotective potential of the aqueous extract of the flowers of Ixora coccinea against CdCl₂induced nephrotoxicity. Extract was orally administered to experimental animals in three different doses. The effect of plant extract was assessed in terms of serum urea, serum creatinine, total protein and serum uric acid. Histopathological studies were also supported the results. Extract treated groups of animals showed significant improvement in serum markers and the kidney damage caused by CdCl₂. Based on the results obtained, it can be concluded that aqueous extract possess significant nephroprotective activity against CdCl₂ induced kidney damage. However the present study did not include the test for establishing the exact mechanism of action. Further study is required using more specific models and isolated compounds which are responsible for its nephroprotective activity.

ACKNOWLEDGEMENT:

The authors are thankful to the Nitte University, Mangalore for providing support to this study.

REFERENCES:

1. Adams RG, Harrison JF, Scott P. The development of cadmium induced proteinuria, impaired renal function and osteomalacia in alkaline battery workers. Q J Med. 38;1969:425-43.

2. Maheswari C, Venkatnarayanan R. Protective effect of Orthosiphon stamineus leaves against lead acetate and cadmium chloride induced renal dysfunction in rats. Int Res J Pharm. 4(4);2013:232-235.

3. Sunitha D, Hemalatha K, Bhagavanraju M. Phytochemical and pharmacological profile of Ixora: A review. Int J Pharmaceut Sci Res. 6(2);2015:567-584.

4. Vadivu R, Jayashree N, Kasthuri C, Rubhini K, Rukmankathan G. Pharmacognostical standardization of leaves of Ixora coccinea Linn. J Pharmaceut Sci Res.1(4);2009:127-134.

5. Joshi H, Sayati D, Gururaj MP, Arun BJ, Chandrashaker KS and Dinesh NA review on a potential herb: Ixora coccinea Linn. J Pharmacog. 2(1);2014:1-12.

6. Joshi H. An Alternative Approach to Experimental Pharmacology. Himdeep Publication, 2015.

7. Uma C, Poornima K, Surya S, Ravikumar G, Gopalakrishnan VK. Nephroprotective Effect of ethanolic extract of Tabernaemontana Coronaria in Mercuric Chloride induced renal damage in Wistar Albino rats. Int J Chem Eng App. 3(4);2012:269-273.

8. Mohammad A, Shiekh TA, Rameshver D, Kalim J, Sadiq U, Asia A, Sana N, Jalaluddin B, Arjumand W, Mohammad S, Surender S. Nephroprotective action of Peucedanum grande against cadmium chloride induced renal toxicity in wistar rats. EXCLI Journal. 11;2012:444-452.

9. Shanmugam V, Kulanthaivel L, Subbaraj GK, Rajendran R, Maruthaiveeran PB. Nephro-protective significance of kaempferol on mercuric chloride induced toxicity in Wistar albino rats. Biomed Aging Path. 3; 2013: 119–124.

10. Madani WA, Siddiqi NJ, Alhomida AS. Renal toxicity of mercuric chloride at different time intervals in rats. Biochem. Insights. 2; 2009:37-45.

11. Cooke TCIE. The Flora of Presidency of Bombay. Ixora coccinea Linn. India: Botanical survey of India. 9(49);1902:75-75.

Received on 25.05.2018 Modified on 11.06.2018

Research J. Pharm. and Tech 2018; 11(11): 4949-4952.

DOI: 10.5958/0974-360X.2018.00901.0