The effect of Lime Leaf Ethanol extract on Diabetic Nephropathy in Male White Rats
College of Pharmaceutical Sciences Pelita Mas Palu, Central Sulawesi 94111, Indonesia.
*Corresponding Author E-mail: jonitandi757@yahoo.com, viani.anggi@gmail.com, dinasafitraumran.pasau@gmail.com, edbbatara@gmail.com, vionazalsabila@gmail.com
ABSTRACT:
Background: Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia caused by defects in insulin secretion, insulin action, or both. Hyperglycemia in chronic DM can trigger various complications, both macrovascular and microvascular, one of which is diabetic nephropathy. Diabetic nephropathy or diabetic kidney disease (PGD) is a complication that often occurs in diabetics. Objective: This study aims to determine the content of secondary metabolites in lime leaf extract on reducing blood glucose levels, pancreatic histopathology and diabetic nephropathy in male white rats (Rattusnorvegicus) induced by streptozotocin. Methods: This study used 30 male white rats which were divided into 6 groups, namely group I as normal control, group II as positive control (glibenclamide), group III as negative control (Na-CMC 0.5%), group IV, V and VI as control. the test group was given ethanol extract of lime leaves with doses of 150mg/kgBW, 250mg/kgBB, 350mg/kgBW, respectively. Data were analyzed using a one way ANOVA test at a 95% confidence level, followed by a post hoc LSD test and using a nonparametric test. Result: Showed that the ethanol extract of lime leaves (citrus aurantifolia (christm.) swingle) contained 1.419% w/w alkaloids, 0.008% w/w tannins, 0.687% w/w flavonoids, and 15.805% w/w saponins. b and can reduce glucose levels at a dose of 350mg/kg BW and can regenerate pancreatic tissue at a dose of 350mg/kg BW damage 0.8 and repair renal tubular injury at a dose of 250mg/kg BW damage 1.2.Conclusion:this study contains secondary metabolites and can reduce blood glucose levels in rats and can regenerate pancreatic tissue and kidney tubular injury.
KEYWORDS: Streptozotocin, Lime Leaf, Diabetes, Pancreas, Diabetic Nephropathy.
INTRODUCTION:
Secondary metabolites are compounds that are not essential for the growth of organisms and are found in unique forms or differ from one species to another. Secondary metabolites in the form of small molecules, are specific (not all organisms contain similar compounds), have a varied structure, each compound has a different function or role12. In general, secondary metabolites function to defend themselves or to maintain their existence in the environment where they are located. Secondary metabolites are biomolecules that can be used as lead compounds in the discovery and development of new drugs.
Secondary metabolites commonly found in plants are alkaloids, flavonoids, steroids, saponins, terpenoids and tannins.Indonesia is a country enriched with medicinal plants (herbal medicine). The slogan is back to nature to minimize the side effects of chemical drugs and increase the use of medicinal plants. Regarding this aspect, one of the potential medicinal plants is red betel (Piper crocatum). Ret betel is considered to have efficacy as a drug based on Evidence-Based Medicine. The widely reported medicinal properties of red betel the anti-hyperglycemic, anti-proliferative on cancer cells, antibacterial activity gram-positive and gram-negative against, antioxidant activity, and other. The chemical contents of red betel leave 96% ethanol extract are tannins, saponins, triterpenoids, and flavonoids. Flavonoids in red betel leave formanthocyanidins flavonon, Auron, isoflavones, catechins, and chalcones7.
Diabetes Mellitus is a metabolic disease characterized by high blood glucose levels (hyperglycemia) because the body cannot produce insulin normally or occurs due to increased insulin resistance3.
The pancreas is an important glandular organ in the body consisting of exocrine and endocrine tissues. The pancreas functions to produce pancreatic juice which contains the enzymes trypsinogen, amylase and lipase. These enzymes mix with food in the duodenum and carry out digestive functions in the intestine, the exocrine part consists of acinar cells that secrete enzymes through the duct to the duodenum5.
The condition of hyperglycemia in chronic DM can trigger many complications, both macrovascular and microvascular, one of which is diabetic nephropathy. Diabetic nephropathy or diabetic kidney disease (PGD) is a complication that often occurs in diabetics. In this disease there is damage to the kidney filter, known as the glomerulus. Due to glomerular damage, a number of blood proteins are excreted in the urine abnormally11.
Previous research stated that the ethanol extract of lime leaves (Citrus aurantifolia (Christm.) Swingle) with a dose of 250mg/kg BW was an effective dose in lowering glucose levels in diabetic rats2.
Based on this research, the researchers were interested in conducting research on the potency of lime leaf (Citrus aurantifolia (Christm.) Swingle) in male white rats (Rattusnorvegicus) induced by streptozotocin at a dose of 150mg/kg BW, 250mg/kg BW and 350mg/kg. mg/kg body weight.
MATERIALS AND METHODS:
Tool:
40 mesh sieve, stirring rod, maceration vessel, blender (kirin), rat drinking bottle, porcelain cup, glass funnel, 100 ml beaker (pyrex ®), 25ml, 50ml, 100ml (pyres ®) measuring cups glucometer (Accu-Chek ®), glukotest test strip (Accu-Chek®), animal cage, 50ml, 100ml volumetric flask (pyrex ®), Olympus Cx-21 microscope, mortar and stamper, water bath, dropper, tube rack, Rotavapor (Heidolph), surgical kit (Renz), 1ml injection syringe, 3ml (One Med Health Care), 3ml oral syringe (One Med Health Care), steroform, EDTA tube, test tube (pyrex®), analytical balance (Ohaus), gram balance and Water Bath.
Alcohol 70%, alcohol 90%, alcohol 95%, alcohol 100%, distilled aqua (aqua), hydrochloric acid (Merck), Citrate– buffered saline, lime leaf (Citrus aurantifolia (Christm.) Swingle) Dragendrof LP, ethanol 96% (Merck), ether, FeCL3, 10% formalin, glibenclamide, handskun (sensi), cotton, label paper, filter paper, Mayer Hematoxylin-Eosin solution, mask (sensi), 10% Na CMC, 0.5% Na CMC , magnesium powder (Merck), and streptozotocin (Bioworld USA).
Phytochemical screening:
Alkaloids:
The lime leaf extract was weighed as much as 0.5grams and then added 5ml of 2N hydrochloric acid and heated on a water bath for 2 minutes then added 3 drops of Dragendrof reagent. If the results give a yellow orange to brick red precipitate, the sample contains alkaloids6.
Flavonoid:
The lime leaf extract was weighed as much as 0.5grams of extract mixed with 5ml of ethanol (95%) then shaken and heated and after that it was shaken again and then filtered, then 0.2grams of mg powder and 3 drops of HCl were added to each filtrate. Flavonoids are said to be positive if a red-orange to red-purple color change is formed indicating the presence of flavonoids and if there is a yellow color indicates the presence of flavones, chalcones and aurons4.
Saponin:
The lime leaf extract was weighed as much as 0.5grams was put into a test tube, then added 10ml of hot water, cooled and then shaken vigorously for 10 seconds. If a foam is formed which persists for not less than 1 minute as high as 10 cm or when the addition of 1 drop of 2N hydrochloric acid does not disappear, it indicates the presence of saponins4.
Tannin:
The ethanol extract of lime leaves was weighed as much as 0.5grams was put into a cup and then added with 20 ml of hot water and 3 drops of 10% NaCl solution. Then a solution of FeCl3, if a blue-black color is formed, indicates the presence of tannins6.
Making Red Castor Ethanol Extract:
Making ethanol extract of lime leaves is done by maceration method. 1200grams of lime leaf simplicia powder was put into 3 maceration vessels, each containing 400grams. The powder was then dissolved using 96% ethanol solvent as much as 2 liters per vessel until the entire simplicia was submerged (± 2.5cm from the upper limit of the simplicia). Maceration was carried out for 3 days in a room protected from sunlight and occasionally stirred to prevent saturation. The filtrate obtained was filtered using filter paper, then concentrated using a rotary evaporator (temperature 40oC - 60oC) and evaporated in a water bath to obtain a thick extract of lime leaves, then the yield was calculated.
As much as 0.5grams of sodium carboxymethyl cellulose (Na CMC) was weighed and sprinkled in a mortar containing 10ml of heated distilled water, allowed to stand for 15 minutes until a transparent mass was obtained, then mixed until homogeneous. The NaCMC suspension was transferred to a 100 ml volumetric flask. The volume is made up with distilled water up to 100ml.
Glibenclamide Suspension Manufacturing:
Streptozotocin was weighed as much as 0.32grams and then dissolved using citrate-buffered saline with a pH of 4.5 to 100ml, then induced in rats via intraperitoneal (ip). The dose of streptozotocin is 40mg/kg BW.
Test Animal Experiment:
The experimental animal protocol was approved by the Medical and Health Research Ethics Committee, Faculty of Medicine, Tadulako University with number 7662/UN 28.1.30/KL/2020. Male wistar rats weighing 200-250g were obtained from VailaWistar and acclimatized in local animal cages for 2 weeks.
The sample data obtained in the study were in the form of blood glucose levels of male white rats (Rattusnorvegicus). Then, a statistical test was carried out which was preceded by a normality test in order to find out the sample data was normally distributed and then the Shapiro-Wilk test was carried out at a 95% confidence, level to determine the distribution of data for each group. The results of the analysis in the form of a normal data distribution were then analyzed with the F test at a 95% confidence level to determine the homogeneity of the group data. If the data is homogeneous and normally distributed, then it is continued using the one way ANOVA test analysis to determine the significant difference between the treatment groups and continued with the post hoc least significant difference (LSD) further test and the Kruskal-Wallis test was carried out and the Man-Whitney test, the data were analyzed using the program spss 25.
DISCUSSION:
Table 1: Phytochemical screening and amount of phytochemichals
S. No |
Bioactive |
Reagent |
Result |
|
|
Ethanol leaf extract of lime (Citrus aurantifolia (Christm.) Swingle). |
Screening test |
Amount Phytochemichals |
|||
1 |
Alkaloid |
Dragendorff |
Formation of red-orange color |
+ |
1.419 mg/g |
2 |
Flavonoid |
Magnesium danHCl |
Brick red color |
+ |
0.687 mg/g |
3 |
Saponin |
Foam test |
Fixed foam |
+ |
15.805 mg/g |
4 |
Tanin |
FeCl3 1% |
Formation of dark blue color |
+ |
0.008 mg/g |
+ = Positive
Table 2: Average Blood Glucose Level
Day |
A |
B |
C |
D |
E |
F |
0 |
91.0 ± 9.51 |
82.2 ± 8.7 |
45.3 ± 4.2 |
86.4 ± 12.5 |
79.4 ± 6.2 |
86.4 ± 7.7 |
7 |
115.6 ± 11.4 |
469.8 ± 174.2 |
430.4 ± 171.8 |
395.0 ± 169.7 |
501.4 ± 108.0 |
349.2 ± 61.9 |
14 |
92.0 ± 5.4 |
374.2 ± 56.7 |
157.6 ± 56.3 |
218.6 ± 107.4 |
250.0 ± 64.8 |
198.4 ± 44.8 |
21 |
88.8 ± 4.4 |
360.8 ± 76.9 |
115.6 ± 20.3 |
152.2 ± 53.3 |
130.8 ± 16.2 |
117.2 ± 11.1 |
28 |
88.0 ± 3.8 |
350.6 ± 75.4 |
99.2 ± 13.7 |
117.2 ± 16.7 |
115.6 ± 15.0 |
99.6 ± 7.8 |
A. Normal control group, B. Negative control group, C. Positive control group, D. Lime leaf ethanol extract therapy group at a dose of 150mg/kgBW, E. Lime leaf ethanol extract therapy group at a dose of 250 mg/kgBW, F. Therapeutic group of lime leaf ethanol extract dose of 350mg/kgBB
Figure 1: Blood Glucose Levels of Male White Rats
Figure 2: Cell Histopathological Appearance Male White Mouse Pancreas 400x Magnification with H&E Staining.
A. Score 0 : Normal
B. Score 1 : Degenerative Cells 25%
C. Score 2: 25%-50% Necrotic Cells and Nearly 25%-50% Lysis
D. Score 3 : Necrotic Cells >50% and Lysis 50%
Table 3 Scoring of the Histopathological Damage Level of the Male White Rat Pancreas
Treatment Group |
Animal Pancreatic Damage Score |
Average±SD |
||||
1 |
2 |
3 |
4 |
5 |
||
A |
0 |
0 |
0 |
0 |
0 |
0±0a |
B |
2 |
2 |
3 |
2 |
2 |
2.2±0.44b |
C |
1 |
0 |
1 |
1 |
0 |
0.6±0.54cd |
D |
2 |
1 |
1 |
1 |
2 |
1.4±0.54d |
E |
2 |
1 |
2 |
1 |
1 |
1.4±0.54d |
F |
0 |
1 |
1 |
1 |
1 |
0.8±0.44d |
A. Normal control group, B. Negative control group, C. Positive control group, D. Lime leaf ethanol extract therapy group at a dose of 150mg/kgBW, E. Lime leaf ethanol extract therapy group at a dose of 250 mg/kgBW, F. Therapeutic group of lime leaf ethanol extract dose of 350mg/kgBB
Figure 3. Image of Histopathological Assessment with a scoring system on Kidney Tubular Injury with HE staining of DM rat model, magnification 400 times and scale 100 m.
Figure 3: A. Score 0= Normal
B. Score 1= Tubular injury < 25% field of view
C. Score 2= Tubular injury involves 25–50% of visual field
D. Score 3= Tubular injury involves more than 51–75% of the visual field
E. Score 4= Tubular injury involves more than 75% of the visual field
Table 4 Scoring of Kidney Tubular Injury Rates in Diabetic Rat Models
Treatment Group |
Average damage value scoring of kidney |
A |
0.16±0.16a |
B |
3.08±0.30b |
C |
0.6±0.28c |
D |
1.3±0.66d |
E |
1.2±0.54dc |
F |
1.52±0.10d |
A. Normal control group, B. Negative control group, C. Positive control group, D. Lime leaf ethanol extract therapy group at a dose of 150 mg/kgBW, E. Lime leaf ethanol extract therapy group at a dose of 250 mg/kgBW, F. Therapeutic group of lime leaf ethanol extract dose of 350 mg/kgBB
Lime leaves were extracted using the maceration method using 96% ethanol as a solvent. The viscous extract obtained after separating the solvent using a rotary vacuum evaporator (rotavapor) and obtained a thick extract of 60 grams with a yield percentage of 3.037%. The maceration method was chosen because to prevent damage to the compounds contained in the ethanol extract of lime leaves. Then a phytochemical screening test was carried out to determine the content of compounds contained in the ethanol extract of lime leaves. The results of the phytochemical screening test showed that the ethanolic extract of lime leaves contained alkaloids, flavonoids, saponins and tannins. The results of the phytochemical screening.1 test can be seen in table 1.This study was used to examine the effect of lime leaf ethanol extract on reducing blood glucose levels, regenerating pancreatic histopathology and preventing kidney tubular injury in diabetic rats. by measuring glucose levels, on the 7th, 14th and 21st days. The rats were sacrificed and their kidneys removed for examination of the kidneys of male white rats2.This study used 30 male white rats (Rattusnorvegicus). The reason for using male white rats as test animals is because they have a fast metabolism and can provide more stable research results11. It is useful in experimental research related to body metabolism. Then the test animals were adapted in the Anatomy Laboratory of StifaPelita Mas Palu for 2 weeks to adapt to the surrounding environment. Before treatment, the test animals were fasted for ± 16 hours while still being given water. The purpose of the test animals being fasted before treatment is to normalize the metabolism in the test animal's body and reduce food interaction factors and accelerate gastric emptying time10. After that, it was continued with the administration of streptozotocin via intraperitonial in the thigh muscle area. The purpose of giving streptozotocin is to increase blood glucose levels (hyperglycemia) in male white rats. Then the test animals were treated by measuring blood glucose levels on the 7th day, 14th day and 21st day. Where negative control was given 0.5% NaCMC suspension, positive control was given glibenclamide suspension and 3 treatment groups were given lime leaf extract at a dose of 150 mg/kg body weight, 250 mg/kg body weight, and 350 mg/kg body weight. on day 14 and day 21 by testing the results of the One Way ANOVA test showed that lime extract can reduce blood glucose levels. The effect of lowering glucose levels is caused by the content of secondary metabolites such as alkaloids, flavonoids, saponins and tannins. This can be seen in table 2.Based on the results of histopathological preparations of rat pancreas and statistics, it can be seen that the ethanol extract of kaffir lime leaves at a dose of 150 mg/kg BW and 250 mg/kg BW had an average score of 1.4. This is because doses of 150 mg/kg BW and 250 mg/kg BW have the same effect in the regeneration of pancreatic cells, seen where the pancreas is moderately damaged, Langerhans cells and exocrine cells undergo degenerative and necrotic lysis by 25-50%. In this case the doses of 150 mg/kg BW and 250 mg/kg BW had a higher level of damage than normal controls and positive controls had an average damage score of 0.6 (mild damage) while compared to negative controls who had an average damage. an average damage score of 2.2 (moderate damage) the level of damage is still low. At a dose of 350 mg/kg BW had an average damage score of 0.8 (mild damage). This is because at a dose of 350 mg/kg BW contains more secondary metabolites which are seen where the pancreas is lightly damaged, Langerhans cells and exocrine cells are degenerative. Based on the scoring data in table 4.2, it can be seen that doses of 150 mg/kg BW, 250 mg/kg BW, and 350 mg/kg BW have therapeutic effects in the regeneration of pancreatic cells. this can be seen in table 3.
Based on the results of rat kidney histopathology preparations and the statistics carried out, it can be seen that the ethanol extract of kaffir lime leaves at a dose of 150 mg/kg BW caused mild damage with an average score of 1.3 mice, the level of damage was higher than normal controls, while compared to the negative control group. . lower damage rate. In the group given the ethanolic extract of lime leaves at a dose of 250 mg/kg BW suffered minor damage with an average score of 1.2 rats, where the level of damage was higher than the normal control, while compared to the negative control group the level of damage was lower. In the group giving lime leaf ethanol extract at a dose of 350 mg/kg BW there was mild damage with an average score of 1.52, where at this dose kidney damage was still higher than the normal control group, compared to the control group. the negative control group the damage was still higher. low. Based on the scoring data in table 4.3, it can be seen that doses of 150, 250 and 350 mg/kg BW had a therapeutic effect in regenerating cells in the kidneys and a dose of 250 mg/kg BW had a better effect than the dose. 150 and 350 mg/kg BW. This is because at a dose of 250 mg/kg BW has a better effect in preventing damage to kidney tubule injury in diabetic rats. These three therapeutic doses did not show a statistically significant difference but showed a clinically significant difference at a dose of 250 mg/kh BW. this can be seen in table 4. In diabetes mellitus, type, strategies with special emphasis on advancement in treatment of diabetes mellitus13.
CONCLUSION:
The ethanol extract of lime leaves (Citrus aurantifolia (Christm.) Swingle) contains secondary metabolites, namely: alkaloids, flavonoids, saponins and tannins. The ethanolic extract of lime leaf (Citrus aurantifolia (Christm.) Swingle) has an effect on reducing blood sugar levels of male white rats (RattusNovergicus) induced by streptozotocin.
ABBREVIATIONS:
Na CMC: Natrium carboxyl cellulosa, Mg/Kg: miligram/kilogram, BW: Body Weight, DL: Deciliter, NaCl: Natrriumclorida
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Received on 06.04.2022 Modified on 10.08.2022
Accepted on 09.11.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(4):1601-1606.
DOI: 10.52711/0974-360X.2023.00262