Magnesium Enhances the Hepatorenal Protective Effects of Lippia multiflora Aqueous Leaves Extract in Streptozotocin-Diabetic Rats

Purpose: This study was conducted in order to evaluate the antidiabetic effects of the aqueous extract of Lippia multiflora supplemented with magnesium on some biochemical markers of the kidneys and liver in type 2 diabetic rats. Method: 7 groups of 4 STZ-diabetic rats received separately orally Glu-cophage ® (Glu 10 mg/kg), the plant extract (LiMAE 200 - 600 mg/kg) and the plant extract supplemented with magnesium (LiMAE-Mg 200 - 600 mg/kg). After a daily treatment of 21 days, serum biochemical parameters were assayed in 16 hr-fasted rats. Results: Diabetes caused a significant (p < 0.0001) elevation of urea, creatinine, ALT and AST. Treatment of diabetic rats with plant extracts (LiMAE and LiMAE-Mg) and Glucophage significantly restored levels of these biochemical markers at p < 0.0001. However, the restoration induced by LiMAE-Mg was greater than those of LiMAE and Glucoco-phage, a reference antidiabetic. Conclusion: The addition of magnesium to the extract of Lippia multiflora caused a greater reduction in the levels of urea, creatinine, ALT and AST increased in STZ-diabetic rats. Magnesium would therefore enhance the nephroprotective and hepatoprotective effects of Lippia multiflora in diabetic rats.


Introduction
Diabetes is a chronic metabolic disease caused by an inability of the body to produce enough insulin (type 1) or to use it effectively (type 2).It is a non-communicable disease characterized either by chronic hyperglycemia or by fasting blood glucose above 1.26 g/l.Diabetes, because of its manifestation and its treatment, constitutes a real public health problem.Its expensive and lifelong treatment leads poor populations in developing countries to use medicinal plants.In view of the controversies on the efficacy and safety of the use of medicinal plants, studies have been undertaken on Lippia multiflora, a plant reputed to be antidiabetic [1].
Several studies have been carried out on the effects of Lippia multiflora in diabetic rats.They revealed on the one hand the non-toxicity of Lippia multiflora aqueous leaves extract [2] and on the other hand its antidiabetic properties.Lippia multiflora aqueous extract (LiMAE) corrected several disturbances caused by diabetes in Wistar rats.This antidiabetic activity of LiMAE would, moreover, be reinforced by magnesium.Indeed, the aqueous extract of Lippia multiflora supplemented with magnesium (LiMAE-Mg) compared to LiMAE resulted in a greater restoration of the levels of glycemia, insulinemia, glycosylated hemoglobin, lipid profile and cardiovascular parameters in STZ-diabetic rats [3].Magnesium would also potentiate the antioxidant activity of LiMAE [4].These results are in agreement with those of previous work which demonstrated that magnesium would positively increase the effects of certain antidiabetic substances [5] [6] [7].
The results of this work, although interesting, are not sufficient to fully demonstrate the antidiabetic action of Lippia multiflora aqueous leaves extract supplemented with magnesium.Indeed, diabetes is associated with many complications including not only endocrine and cardiovascular disorders but also dysfunctions of several vital organs.Persistent hyperglycemia over time can affect almost any organ, especially the kidneys and liver [8].
And it is in this sense that the present research was carried out in order to evaluate the effects of Lippia multiflora aqueous leaves extract supplemented with magnesium on some biochemical markers of kidney and liver functionalities in streptozotocin-diabetic rats.
The plant aqueous extract was obtained according to the method used by Allo et al. [3].The fresh leaves were washed and dried at room temperature (28˚C ± 2˚C).Dried leaves were pulverized to powder with the use of a laboratory blender-About 100 g of powder were macerated during 24 hrs in distilled water (1 L), thereafter filtered.An oven at a temperature of 50˚C was used to concentrate the Journal of Biosciences and Medicines filtrate.And the concentrated extracts obtained (Lippia multiflora aqueous leaves extract: LiMAE) was stored at 4˚C until experiments.LiMAE was supplemented with magnesium (1 g per 9 g LiMAE) to give the supplemented Lippia multiflora aqueous extract (LiMAE-Mg).

Animals and Ethics
Wistar rats (Rattus norvegicus) weighing 200 -250 g and 8 -12 weeks old were used.These animals were provided by the Pasteur Institute of Côte d'Ivoire (IPCI).They were acclimatized for 2 weeks before being used for experiments.
Animals were housed and maintained under standard laboratory conditions (temperature 25˚C ± 2˚C) with dark and light cycle (12/12h).They were allowed free access to standard dry pellet diet and water ad libitum.Rats were treated according to good laboratory practices [9].The experimental protocols were conducted in accordance with the protocols for the protection of experimental animals of the European Council on Legislation 2012/707 [10].

Experimental Induction of Diabetes
The method used to induce type 2 diabetes in rats is similar to those of previous works [3] [4] [11] [12].Fifteen minutes after receiving a single dose of streptozotocin (STZ, 65 mg/kg), 16 hr-fasted rats were treated with nicotinamide (230 mg/kg).STZ which was freshly prepared in citrate buffer solution (0.1 M, pH = 4.5) and nicotinamide were administered intraperitoneally.From D14 to D21, augmented levels of blood glucose (2 -3 g/L), urea, creatinine, AST and ALT in 16 hr-fasted rats and body weight decrease confirmed the development of the diabetes.

Experimental Design
The experimental protocol is that described by Allo et al. (2022) [3] and Konan et al. (2023) [4].Four healthy rats (Group 1) and 32 STZ-diabetic rats divided into 8 groups of 4 animals (Groups 2-9) were used.Group 1 (HeR: healthy rats) and Group 2 (uDR: untreated-diabetic rats) received distilled water.Group 3 received Glucophage (Glu 10 mg/kg).Groups 4, 5, 6 were treated with LiMAE at 200, 400 and 600 mg/kg respectively while Groups 7, 8 and 9 received LiMAE-Mg at 200, 400 and 600 mg/kg respectively.Drugs were administered orally for 21 days.And all animals had free access to water and normal diet during the expe-rimentation.Rats were weighed at the start and end of the phase.Intermediate weighings at regular intervals of 2 days were also made.After 21 days administration of drugs studied to the experimental animals, they were starved overnight, anaesthetized with Isoflurane (Forene ® ) and sacrificed.A thoracotomy was performed.Blood samples were collected from the animals through cardiac puncture.
Blood samples collected in non-heparinized tubes were allowed to clot for about 15 min and centrifuged at 3000 rpm for 5 min.Serum was separated from the clot with pasteur pipette and dispensed into clean tube for the measurement of the biochemical indices.Analysis of the selected serum biochemical indices were carried out on each sample.Parameters were measured using Chemistry Analyzer (HITACHI 704R ® auto-analyser).The serum biomarkers measured were urea and creatinine for the kidney and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) for the liver.Indeed, these parameters have been measured by several authors to study the functions of the kidney and liver [13] [14] [15].

Data Analysis
All the data were expressed as Mean ± Standard Error of Means (SEM).Statistical analyses were performed by one way analysis of variance (ANOVA) and differences between means were determined by Turkey's Multiple Comparison test using Graph Pad Prism 7.0 program (Microsoft, San Diego California, USA).
A value of p < 0.05 was considered significant.

Effects of Studied Drugs on Biochemical Markers of Kidney Functionality
A significant elevation (p < 0.05) of the urea level at 1.00 ± 0.02 g/L was measured in untreated-diabetic rats (uDR) against 0.49 ± 0.02 g/L for healthy rats (HeR), an increase of 51% (Figure 1).At a dose of 200 mg/kg, the extracts (Li-MAE and LiMAE-Mg) reduced the urea level to 0.87 ± 0.05 g/L and to 0.77 ± 0.02 g/L giving respective reductions of 13% and 23% compared to that of uDR.
LiMAE and LiMAE-Mg at a dose of 400 mg/kg caused a respective decrease of urea levels to 0.72 ± 0.03 g/L and 0.66 ± 0.02 g/L corresponding to respective reductions of 28% and 34% compared to that of uDR.At a dose of 600 mg/kg, LiMAE and LiMAE-Mg caused a decrease in urea levels in diabetic rats with respective values of 0.59 ± 0.02 g/L and 0.57 ± 0.05 mg/L.These values correspond respectively to a reduction of 41% and 43% compared to that of uDR.With Gluco 10 mg/kg, a urea value of 0.73 ± 0.02 g/L was measured.Treatment with Gluco 10 mg/kg therefore resulted in a 27% decrease in urea in treated diabetic rats compared to uDR.
Induction of diabetes also caused a significant elevation in creatinine level (Figure 2).The rate was 60.89 ± 2.25 mg/L in uDR versus 13.02 ± 1.56 mg/L for Journal of Biosciences and Medicines

Effects of Studied Drugs on Biochemical Markers of Liver Functionality
Diabetes increased AST transaminase activity significantly at p < 0.05 in uDR (186.36 ± 2.17 IU/L) compared to that in HeR (154.81 ± 3.59 IU/L).This in-crease was estimated at 20.67% (Figure 3).

Discussion
The induction of diabetes caused an increase in serum levels of AST, ALT, urea and creatinine in animals as revealed by numerous works [3] [4] [11] [16].In general, an increase in transaminases (AST and ALT) in the blood implies liver  It therefore emerges from these observations that magnesium interacts positively with the secondary metabolites contained in Lippia multiflora aqueous leaves extract.These could include flavoids, alkaloids, tannins highlighted by various studies [2] [38] and whose hepatoprotective and nephroprotective actions have been demonstrated [16] [33] [39].

Conclusion
Lippia multiflora aqueous leaves extract supplemented with magnesium caused a greater reduction in the levels of kidney and liver biomarkers that were elevated in diabetic rats.Magnesium would therefore enhance the hepatoprotective and nephroprotective effects of Lippia multiflora aqueous extract.This provides scientific support for the use of medicinal plants such as Lippia multflora in the treatment of liver and kidney disorders caused by diabetes.Treatment of the diabetic rats with Lippia multiflora leaves extract associated with magnesium is more effective in the amelioration of diabetes and hepato-renal injuries in STZ induced diabetic male rat.And therefore, magnesium supplementation of Lippia multiflora aqueous extract could be an interesting alternative for the develop- ment of new products to treat diabetes.
Administration of LiMAE and Li-MAE-Mg caused a reduction in AST activity in diabetic rats.Thus, 181.66 ± 0.86 IU/L, 175.52 ± 1.72 IU/L and 164.24 ± 3.09 IU/L were measured with LIMAE at 200, 400, 600 mg/kg respectively.Compared to uDR, these are respective de- son to uDR.LiMAE-Mg showed statistically greater reductions in ALT activity in diabetic rats than LiMAE.In diabetic rats treated with LiMAE-Mg, recorded ALT activity decreased from 83.54 ± 2.31 IU/L (200 mg/kg) to 66.38 ± 3.57 IU/L (600 mg/kg).Thus, LiMAE-Mg (200 -600 mg/kg) reduced decreases in ALT activity from 8.47% to 27.27%.As for Glucophage at 10 mg/kg, it caused a reduction in ALT activity of 15.83% in diabetic rats.