Int J Pharm Pharm Sci, Vol 7, Issue 9, 505-506Short Communication


HISTOMORPHOMETRIC ANALYSIS IN KIDNEY TISSUE OF MICE TREATED WITH INDIGOFERA SUFFRUTICOSA MILL

MARLLON ALEX NASCIMENTO SANTANA1, IVANISE BRITO DA SILVA1, JÉSSICA ANDRÉIA PEREIRA BARBOSA2, IZABELA RANGEL LIMA1, JEYMESSON RAPHAEL CARDOSO VIEIRA1, ROBERTA MARIA PEREIRA LEITE LIMA1, SÔNIA PEREIRA LEITE1.

1Department of Embryology and Histology/Universidade Federal de Pernambuco, Recife-PE, Brazil, 2Department of Antibiotics/Universidade Federal de Pernambuco, Recife-PE, Brazil
Email: marllon_gb@hotmail.com  

 Received: 22 May 2015 Revised and Accepted: 30 Jul 2015

ABSTRACT

Objective: To investigate by histomorphometry the effect of aqueous extract of Indigofera suffruticosa leaves on proximal convoluted tubules and renal corpuscles of mice with sarcoma 180.

Methods: Male swiss albino mice (Mus musculus) with 60 days of age were divided into 3 groups of 6 animals: Group 1-sarcoma 180 treated with aqueous extract (50 mg/kg, intraperitoneally (i. p.)), Group 2-sarcoma 180 treated with saline solution (15 ml/kg, i. p.) and Group 3–without sarcoma 180 treated with saline (15 ml/kg, i. p.). After 48 hours of tumor implantation, the treatment was initiated during 7 days. Histomorphometric analysis of kidney tissue was performed using IMAGEJ software.

Results: Antitumoral treatment with aqueous extract of I. suffruticosa leaves at a dose of 50 mg/kg has not caused mice renal tissue histological changes.

Conclusion: After treatment with aqueous extract of leaves of I. suffruticosa, renal architecture was preserved, suggesting its safety use as an antitumor agent.

Keywords: Histomorphometry, Indigofera suffruticosa, Kidney, Sarcoma 180.

According to the World Health Organization (WHO), about 80% of the world’s population makes use of medicinal plants as the main treatment alternative in primary care [1]. Throughout human history, natural products have been used as a resource to cure diseases. Knowledge about plant popular use and the effectiveness significantly contributes to therapeutic properties spread, although chemical constituents sometimes are not known [2].

Indigofera suffruticosa Mill (Fabaceae) occurs in abundance in Northeastern Brazil and has intense popular use in treating infections, inflammation and other processes, with no reports of harmful side effects to humans. This species has compounds in its chemical constitution such as flavonoids derived from quercetin glycosides and alkaloids [3].

Pharmacological studies have shown that aqueous extract of leaves of I. suffruticosa have the following properties: anti-inflammatory [4], antiparasitic [5], embryotoxic [6], antimicrobial [7], anti-tumor [8], cytotoxic [9], embryotoxic on Aedes aegypti eggs and larvae and repellent to Aedes aegypti mosquitoes [10].

Many pharmaceutical products are lipophilic, which permits the drug to go through cellular membranes or to the target tissue. The same chemical property which enhances the drugs bioavailability can also hinder their renal excretion, since kidney debugging requires these drugs to become more hydrophilic. Thus, biotransformation reactions often increase compounds hydrophilicity, in order to make them more susceptible to renal excretion [11].

Computer new methods are being developed to evaluate tissue micro-architecture [12], such as histomorphometry, which is a technique that allows assessing tissue structure by quantitative analysis [13]. It is considered a well-established and valuable research tool for metabolic diseases study [14]. Its measurement and quantification, through statistical parameters, gives information that enables operation and organization understanding of the tissue or organ.

This study analyses the effect of aqueous extract of I. suffruticosa on renal tissue of mice, and it shows the importance of investigating pharmacological effects of natural compounds in metabolizing organ like kidney. The lack of toxicological studies relating the effect of I. suffruticosa on morphological changes mainly using quantitative parameters as histomorphometry analysis highlights the importance of this proposal, suggesting its safety use as an antitumor agent.

Indigofera suffruticosa species was collected in Nova Cruz city, state of Pernambuco, in July 2013. The sample is cataloged under the number 43694 in Federal University of Pernambuco (UFPE) Herbarium. Leaves were reduced to small fragments (300g) and macerated for extract preparation. After 24 hours, the plant material was subjected to aqueous extraction, and the extract was concentrated through a lyophilizer. After total water extraction, dry weight and yield were determined. Swiss albino mice (Mus musculus) male young adults with 60 days of age and weighing on average 30 to 40g were used. Mice were kept in polypropylene cages in central air conditioning, with 12 hours light/dark cycle. Experiments were conducted with the approval of the ethics committee on animal experimentation of the Federal University of Pernambuco (UFPE), process No. 500/12, October 2012.

Sarcoma 180 ascitic form was induced in the experimental group (G1 and G2) by injection into animals’ right axillary area containing 0.3 ml (approximately 3x106 cancer cells). After 48 hours of tumor implantation, treatment was initiated. Indigofera suffruticosa aqueous extract (50 mg/kg) was administered intraperitoneally (i. p.) for 7 consecutive days in group G1, while groups G2 and G3 received saline 15 ml/kg i. p. for the same period.

At the end of treatment, animals were sacrificed and renal tissues fragments were fixed in 70% alcohol and stained by Hematoxylin and Eosin (H. E). Photomicrographs of histological preparations were taken through an optical microscope with a camera attached. Images were projected and enlarged, having a final increase of 790 μm. Histomorphometric analysis of proximal convoluted tubules perimeters and renal corpuscle diameter was performed using the IMAGEJ software. For statistical analysis, the Kruskal-Wallis test (p<0.05) was used and data were expressed as mean and standard deviation.

Fig. 1 (A, B and C) shows the histomorphology and fig. 2 and 3 show histomorphometric analysis of proximal tubules and the renal corpuscles of mice. Sarcoma 180/aqueous extract of I. suffruticosa (50 mg/kg i. p) (A); Sarcoma 180/saline (15 mg/kg i. p) (B); without Sarcoma 180/saline (15 mg/kg i. p) (C).

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Fig. 1: Histomorphology of renal tissue. G1 (A) Sarcoma 180/aqueous extract (50 mg/kg) i. p.; G2 (B) Sarcoma 180/saline i. p.; G3 (C) without sarcoma 180/saline. Proximal tubules–PT; RC-Renal Corpuscle. Magnification of 400X

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Fig. 2: Histomorphometry of renal proximal convoluted tubules of mice (n=150). Data are expressed as mean and standard deviation and analyzed through Kruskal-Wallis test (p<0.05)

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Fig. 3: Histomorphometry of renal corpuscles of mice (n=150). Data are expressed as mean and standard deviation and analyzed through Kruskal-Wallis test (p<0.05)

The histomorphological analysis of renal tissue shows the maintenance of cellular architecture in group treated with aqueous extract and the group without sarcoma/saline; however, sarcoma 180/saline group presents cellular disarray with hypertrophied nuclei. The histomorphometric analysis of the perimeters of proximal tubules and renal corpuscles did not show statistical difference between the groups. Histological studies are important tools to detect histophysiological changes in gastrointestinal tract functions. Studies on histology and renal morphology are common in literature, revealing important role especially when it is wanted to assess new substances with nephrotoxic potential. However, no published articles with renal tissue histomorphological assessment of mice treated with Indigofera suffruticosa extracts were found. Similar studies using the same species of Indigofera demonstrated that the plants contain pharmacological substances with hepatoprotective properties [17, 18]. The kidney is a very important organ and plays an important role in blood pressure regulation and body fluid volume in acid-base balance, and in certain hormones formation and release. [15, 16].

In this study, no histological alterations were identified in the renal tissue of sarcoma 180-bearing mice treated with daily doses of aqueous extract of leaves of I. suffruticosa (50 mg/kg i. p.) when compared to the control. There were no changes in the proximal convoluted tubules perimeter and renal corpuscles diameter.

This study showed that the aqueous extract obtained by maceration, when used in mice with sarcoma 180 treatment, did not induce any histomorphometric changes in the proximal convoluted tubules and renal corpuscles, suggesting a low nephrotoxic effect order.

AKCNOWLEDGEMENT

The authors thank CNPq for financial support.

COMPETING INTERESTS

The authors declare no competing interests.

REFERENCES

  1. Elizabetsky E. Resenha: fitoterapia com base científica. Ciência Hoj, 2002;31:78-9.
  2. López, CAA. Considerações gerais sobre plantas medicinais. Ambiente: Gestão e Desenvolvimento 2006;1:19-27.
  3. Calvo TR, Cardoso CR, Silva MAC, Santos LC, Colus IM, Vilega W, et al. Mutagenic activity of Indigofera truxillensis and I. suffruticosa aerial parts. J Evidence-Based Complementary Altern Med 2009;20:1-8.
  4. Leite SP, Silva LLS, Catanho MTJA, Lima EO, Lima VLM. Anti-inflammatory activity of Indigofera suffruticosa extract. Revbrasa 2003;7:47–52.
  5. Leite SP, Silva EC, Cassilhas APP, Torquanto GN, Silva LLS, Medeiros PL, et al. Atividade antiparasitária de frações aquosas de Lonchocarpus araripenses (Papilionaceae) e Indigofera suffruticosa (Fabaceae). 55ª Reunião Anual da SBPC–Resumo 4694 C 13.4. Recife; 2003.
  6. Leite SP, Medeiros PL, Silva EC, Maia MBS, Lima VLM, Saul DE. Embryotoxicity in vitro with extract of Indigofera suffruticosa leaves. Reprod Toxicol 2004;18:701-5.
  7. Leite SP, Vieira JRC, Leite RMP. Antimicrobial activity of Indigofera suffruticosa. J Evidence Based Complementary Altern Med 2006;3:261-5.
  8. Vieira JRC, Souza IA, Nascimento SC, Leite SP. Antitumoractivity of indigofera suffruticosa. An Fac Med Univ Recife 2007;52:112-5.
  9. Vieira JRC, Souza IA, Nascimento SC, Leite SP. Indigofera suffruticosa:an alternative anticancer therapy. J Evidence Based Complementary Altern Med 2007;4:355-9.
  10. Vieira JRC. Investigação farmacognóstica e biológica de folhas de Indigofera suffruticosa Mill sobre Aedes aegypti. Tese (Doutorado em Ciências Farmacêuticas)–Departamento de Ciências Farmacêuticas. Recife: Universidade Federal de Pernambuco; 2011.
  11. Golan DE, Tashjian AH, Armstrong EJ, Armstrong AW. Princípios de farmacologia-a base fisiopatológica da farmacoterapia. 3nd ed. Guanabara;  2014. p. 972.
  12. Laib A, Newitt DC, Lu Y, Majundar S. New model-independent measures of trabecular bone structure applied to in vivo high-resolution MR images. Osteoporosis Int 2002;13:130-6.
  13. Freemont T, Malluche HH. Utilization of bone histomorphometry in renal osteodystrophy: demonstration of a new approach using data from a prospective study of lanthanum carbonate. Clin Nephrol 2005;63:138-45.
  14. Weinsten R. Clinical use of bone biopsy. In: Marcus R, Feldman D, Nelson D, Rosen C J. editors. Osteoporosis. 3nd ed. 2008;448-65.
  15. Gartner LP, Hiatt JL. Atlas Colorido de Histologia. 4 nd ed. Guanabara Koogan, Rio de Janeiro; 2007. p. 431.
  16. Kuppuswamy R, Govindara A, Velusamy G, Balasubramanian R, Balasundarm J, Sellamuthu M. Effect of dried fruits of solanum nigrum LINN against CCl4-induced hepatic damage in rats. Biol Pharm Bull 2003;26:1618-9.
  17. Lima IR, Vieira JRC, Silva IB, Leite RMP, Maia MB, Leite SP. Indican from Añil (Indigofera suffruticosa Miller) as an herbal protective agent to the liver. Anal Quant Cytol Histol 2014;36:41-5.
  18. Silva IB,  Lima IR, Santana MAN, Leite RMP, Leite, SP. Indigofera suffruticosa Mill (Fabaceae): hepatic responses in mice bearing sarcoma 180. Int J Morphol2014;32:1228-33.