{"id":57880,"date":"2024-06-25T11:18:39","date_gmt":"2024-06-25T11:18:39","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=57880"},"modified":"2024-07-03T17:10:23","modified_gmt":"2024-07-03T17:10:23","slug":"effects-of-methanolic-leaf-extract-of-costus-pictus-d-don-on-swiss-albino-mice-with-hyperglycemia-associated-renal-complications","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/effects-of-methanolic-leaf-extract-of-costus-pictus-d-don-on-swiss-albino-mice-with-hyperglycemia-associated-renal-complications\/","title":{"rendered":"Effects of Methanolic Leaf-extract of Costus pictus D. Don on Swiss Albino Mice with Hyperglycemia Associated Renal Complications"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Diabetes mellitus (DM) has gained international attention as a remarkable health threat. Unusually prolonged hyperglycemia, a major hallmark of diabetic individuals, results in several pathophysiological problems1<\/sup> often affecting vital organs like the eye, kidney, heart and nerves2<\/sup>. There are two major forms of DM, one is type-I and other is type-II, which are also known as Insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Type-I diabetes is an autoimmune disorder that causes a localized inflammatory response, in and around the islets of Langerhans, followed by the selective death of \u03b2-cells that secrete insulin. Type-II diabetes is characterized by reduced insulin secretion and peripheral insulin resistance3,4<\/sup>. Diabetes prevalence is predicted to increase from 2.8% in 2000 to 4.4% in 2030 across all age groups and according to projections, there will be 366 million people with diabetes worldwide by 2030, up from 171 million in 20005<\/sup>. Diabetic nephropathy (DN) is the most prevalent and serious consequence of prolonged diabetes and has been identified as the leading cause of kidney damage and eventual renal failure, which ultimately necessitates regular dialysis or kidney transplantation6,7<\/sup>. According to reports, DN or end-stage renal failure can develop in 30\u201340% of people worldwide sooner than later8,9<\/sup>. The hallmarks of DN include thickening of the basement membrane, growth of mesangial cells and podocyte cell death, which result in morphological and functional abnormalities in the filtration barrier10,11<\/sup>. In such nephropathic individuals, the impaired renal basement membrane causes an increase in the excretion of urine albumin, urea and creatinine which lowers the glomerular filtration rate (GFR)12<\/sup>.<\/p>\n\n\n\n

One of the often used naturally occurring diabetes-inducing substances used on laboratory animals is streptozotocin (STZ), derived from Streptomyces achromogenes,<\/em> which is chemically known as N (Methylnitrosocarbamoyl)-\u00b1-<\/em>D-glucosamine<\/em>13<\/sup>. According to studies, oral or intravenous administration of STZ to animals causes pancreatic beta cells to die, which causes the animals to develop diabetes14<\/sup>. STZ enters the \u03b2 cell via the GLUT-2 transporter and causes alkylation of DNA, thereby inducing the activation of polyADP ribosylation, leading to depletion of cellular NAD+ and ATP, which without medical intervention can lead to the condition of DN15<\/sup>. STZ was intraperitoneally employed in our investigation to establish the DN mouse model.<\/p>\n\n\n\n

In addition to conventional diabetic\ntreatments, including insulin therapy and oral hypoglycemic medications, many\ndiabetic patients adopt supplementary and alternative approaches, as it is\nbelieved to have fewer side effects and are cost-effective as well16<\/sup>.\nHence, adopting a plant-based therapeutic approach in context with the\nmanagement of diabetes and associated complications is an essential\nstrategy.  It has also been reported that\na related plant of C. pictus<\/em> known as C. afer, <\/em>originated in\nAfrica having anti-nephrotoxicity effects17<\/sup>. Moreover, the mixture\nof C. pictus<\/em> with metformin and enalapril showed beneficial effects in the\nmanagement of DN18<\/sup>. So, the current study investigated the effects\nof methanolic extract of C. pictus<\/em> for attenuation of DN. A wide range\nof phytochemical substances, including alkaloids, glycosides, tannins, phenols,\nsteroids, terpenoids, and flavonoids, were found in C. pictus<\/em>. In particular,\nquercetin, a flavonoid available in C. pictus<\/em> leaf extract has a strong\nantioxidant effect, which helps scavenge superoxide radicals and inhibit\nxanthine oxidase19<\/sup>. Among all the parts of C. pictus<\/em>,\nmethanolic leaf extract demonstrated the best antioxidant activity20<\/sup>.\nIt has also been revealed that the leaf extract of C. pictus<\/em> has anti-inflammatory\nproperties. These extracts reduced the levels of pro-inflammatory cytokines\nsuch as tumor necrosis factor-\u03b1 and C-reactive protein by inhibiting the\nmolecules involved in the stress-sensitive signaling cascade. Additionally, it\nsuppressed the expression of monocyte chemotactic protein and interleukin 6\n(IL-6), the inflammatory cytokines often linked to insulin resistance, obesity,\ndiabetes and metabolic syndrome21<\/sup>. The potent anti-inflammatory of\nthe plant qualities could be attributed to the rich phenolic compounds present in\nC.<\/em> pictus<\/em> leaves22<\/sup>. The biochemical pathways\ninfluenced by the bioactive components present in the methanolic extract of C.\npictus <\/em>leaves,leading\nto beneficial effects on diabetic kidney injury advocate for further molecular\ninvestigations. <\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Collection\nand Identification of C. pictus<\/em><\/strong><\/p>\n\n\n\n

Naturally, growing C.\npictus <\/em>D. Don, also known as insulin plant or Sugar\nplant (Family: Costaceae), were\ncollected in and around the area of Siliguri (26.7271\u00b0N, 88.3953\u00b0E), West\nBengal, India during January-February in the year of 2020. The Taxonomy of\nAngiosperm and Biosystematics Laboratory, Department of Botany of the\nUniversity of North Bengal authenticated the collected plant specimens. The\nvoucher specimen was deposited in the herbarium of the Department of Botany,\nUniversity of North Bengal, with the depository accession number (Accession no:\n11783). <\/p>\n\n\n\n

Preparation of methanolic extract of C. pictus <\/em>leaves<\/strong><\/p>\n\n\n\n

Collected\nC. pictus <\/em>leaves were shed and dried at room temperature for two\nweeks. Then the leaves were ground into fine powder and then stored in airtight\ncontainers at room temperature. For extraction, the powder was suspended in\nabsolute methanol in a 5 g: 250 ml (w\/v) ratio for 72 hours in the Soxhlet apparatus.\nThe extract was concentrated using a Buchi-style rotary evaporator (Cole Parmer\nRV1010D596, India) at a lower pressure and temperature (45\u00b0C). Then, the\nconcentrated semi-liquid component was taken into a Petri dish and dried at\nroom temperature. Then, the final yield efficiency was calculated. The dried\nsample was collected into a glass vial and kept at -20o<\/sup> C for future\nuse. <\/p>\n\n\n\n

Induction\nof renal injury<\/strong><\/p>\n\n\n\n

For\nthe induction of the renal injury associated with hyperglycemia, a batch of\nanimals (n=6) were given intraperitoneal injections of STZ, dissolved in\nfreshly prepared 0.5 mol citrate buffer (<\/strong>pH 8) at the fixed dosage of\n30mg\/kg body weight (bw) for five days with a gap of one day between 2 dosages.\nAnimals that had fasting blood glucose levels above 215 mg\/dL were selected for\nfurther study on the 10th<\/sup> day of the experiment23,24<\/sup>.\nAnimals were monitored for another seven days for any changes in blood glucose\nlevels to ensure no reversal of the hyperglycemic state. On the 17th<\/sup>\nday of the experiment, animals were grouped for further experimentation.  <\/p>\n\n\n\n

Animal Experimental Design<\/strong><\/p>\n\n\n\n

The animals were divided into five groups: CMC (vehicle control group; animals that were given carboxy methyl cellulose along with food and water ad libitum<\/em> for 28 days duration), DC (Disease control group; renal injury-induced disease group that were subjected to only food and water ad libitum<\/em> without any specific treatment for 28 days duration), GLD (positive control group; renal injury induced group that were treated with glibenclamide dissolved in carboxy methyl cellulose along with food and water ad libitum<\/em> for 28 days duration), LD (Treatment groups with low dose; renal injury induced group that were treated with low dose of  CPLE at 250 mg\/kg bw dissolved in carboxy methyl cellulose along with food and water ad libitum<\/em> for 28 days duration) and HD (Treatment groups with high dose; renal injury induced group that were treated with a high dose of  CPLE at 500 mg\/kg bw dissolved in carboxy methyl cellulose along with food and water ad libitum<\/em> for 28 days duration)<\/p>\n\n\n\n

Determination\nof OGTT-based Area Under Curve of Plasma Glucose (AUCglucose<\/sub>)<\/strong><\/p>\n\n\n\n

OGTT-based\nAUCglucose<\/sub> was performed at the end of the experimental duration.\nThe levels of blood glucose were measured by pricking the tail vein of 16\nhours-fasted animals, aseptically with a sterile needle. For this study, the\nmice were administered a single oral dose of glucose (2 g\/kg bw) and the\nreading was taken just before the glucose administration (0 min) and after 30,60,\n90, and 120-minute time intervals using a handheld glucometer (Dr. Morepen\nBG03, Morpen Laboratories Ltd., India)<\/strong>. Then, using the values of the respective\nglucose levels at different time intervals, AUCglucose<\/sub> was measured25<\/sup>.<\/p>\n\n\n\n

Kidney\nIndex<\/strong><\/p>\n\n\n\n

Before\nsacrificing, each mouse was weighed individually and after sacrifice, both\nkidneys were weighed24<\/sup>. Finally, the kidney index was calculated by\ndividing the kidney weight by the bw and multiplying by 100.<\/p>\n\n\n\n

Biological samples collection<\/strong><\/p>\n\n\n\n

Urine, blood and tissue samples were collected aseptically by\nfollowing standard protocols. Specimens except blood were stored at -20o<\/sup>C\nuntil further evaluations. For the blood sample, serum was separated by\ncentrifuging the clotted blood samples at 3000 rpm for 5 min and stored at\n-20\u00b0C until analysis.<\/p>\n\n\n\n

Serological\nLipid Profiling<\/strong><\/p>\n\n\n\n

Standard\ncolorimetric kits were used (Coral Clinical System, India) to evaluate the\nlipid profile, which included serum total cholesterol (TC, Cat No. 1102040275),\nhigh-density lipoprotein-C (HDL-C, Cat No. 1102150040) and Triglyceride (TG,\nCat No. 1102220075). The manufacturer’s instructions were followed for\nperforming serum lipid profile testing, and LDL-C was calculated, following the\nMartin-Hopkin calculation26<\/sup>.<\/p>\n\n\n\n

Serological\nand urological profiling of renal injury parameters<\/strong><\/p>\n\n\n\n

Standard\ncolorimetric kits from Coral Clinical System, India were used following the\nmanufacturer’s instructions to estimate Albumin (Cat No. 1101021150),\nCreatinine (Cat No. 1101070275) and Urea (Cat No. 1102240075) parameters of\nserum and urine as renal injury markers.<\/p>\n\n\n\n

In-vivo<\/em><\/strong>\nantioxidant study<\/strong><\/p>\n\n\n\n

Preparation of kidney homogenate<\/strong><\/p>\n\n\n\n

After sacrifice, the kidneys\nwere dissected and washed in phosphate buffer saline (PBS). The kidney tissue\nwas homogenized to make 10% homogenate with phosphate buffer (PB) in a 50 mM\nconcentration at 7.4 pH. The homogenate was centrifuged at 1000 g for 15\nminutes at 4\u00b0C27<\/sup>.<\/p>\n\n\n\n

Estimation of lipid peroxidation (MDA)<\/strong><\/p>\n\n\n\n

Lipid peroxidation was\nestimated by thiobarbituric acid (TBA) method with slight modification28<\/sup>. In short, a reaction mixture was prepared with 1 ml\nof TBA (0.5%) and 1 ml of trichloroacetic acid (20%). In this mixture, 50 \u03bcl of\ntissue homogenate was mixed. The whole mixture was kept in a water bath at 95\u00b0C\nfor 30 minutes. After that, to stop the reaction, it was ice-cooled for 5\nminutes, followed by centrifugation at 2000 g for 12 minutes. Absorbance was\ntaken at 532 and 600 nm, respectively.<\/p>\n\n\n\n

Estimation of superoxide dismutase (SOD)<\/strong><\/p>\n\n\n\n

SOD was estimated by the\nnitroblue tetrazolium (NBT) method with slight modification29<\/sup>. A reaction mixture of 1 ml sodium carbonate (50 mM),\n400 \u03bcl of NBT (25 mM) and 200 \u03bcl of hydroxylamine hydrochloride (0.1 mM) was\nmade. On that mixture, 100 \u03bcl of tissue homogenate was mixed and absorbance was\ntaken at 560 nm.<\/p>\n\n\n\n

Estimation of reduced glutathione (GSH)<\/strong><\/p>\n\n\n\n

GSH was estimated by the Elman\nreagent method with slight modification30<\/sup>. At first, Elman reagent was prepared by mixing 19.8\nmg of DTMB and it was dissolved in 100 ml of sodium nitrate (0.1%). On that\nreagent, 1 ml of tissue homogenate and 3 ml of phosphate buffer were added and absorbance\nwas taken at 412 nm.<\/p>\n\n\n\n

Estimation of catalase activity<\/strong><\/p>\n\n\n\n

Catalase activity was\nestimated by the hydrogen peroxide (H2<\/sub>O2<\/sub>) method with\nslight modification31<\/sup>. A reaction mixture of 500 \u03bcl of H2<\/sub>O2\n<\/sub>(0.34 nM) and 2.5 ml distilled water was made. In that reaction mixture,\n40 \u03bcl of tissue homogenate was added. After that, absorbance was taken at 240\nnm. <\/p>\n\n\n\n

Histopathological\nEvaluation<\/strong><\/p>\n\n\n\n

By\nfollowing standard protocol, histological evaluations were carried out from the\nanimal tissue. Surgically, the kidneys were removed and washed with chilled PBS\nand placed in Bouins fixative for fixation. After that, serial ethanol\ndilutions were used to dehydrate the tissues before embedding them in paraffin\nwax. Haematoxylin and eosin were used for staining and six \u03bcm thick sections were\ncut from a paraffin-embedded block32<\/sup>. Following that, using a light\nmicroscope, microscopic inspections were performed (Nikon Eclipse E200, Nikon,\nTokyo, Japan) with a 40X magnification.<\/p>\n\n\n\n

Statistical\nanalysis<\/strong><\/p>\n\n\n\n

With the aid of a statistical package program (KyPlot 6.0), Dunnett’s test was done to compare the groups statistically, and p<\/em> \u2264 0.05 was considered significant for all statistical analyses in this study. <\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

AUCglucose<\/sub> <\/strong><\/p>\n\n\n\n

Plasma Glucose determined by OGTT-based Area Under Curve (AUCglucose<\/sub>) It has been found that the values of AUCglucose<\/sub> were significantly higher (p<\/em>\u22640.05) in the DC group (469.28 \u00b1 87.93 mg h\/dL;) when compared with that of the CMC group (246.13 \u00b1 7.30 mg h\/dL); whereas, the values of GLD (252.76 \u00b1 8.88 mg h\/dL), LD (277.75 \u00b1 24.26 mg h\/dL) and HD (275.77 \u00b1 11.19 mg h\/dL) group were statistically non-significant with CMC group (Fig.1). <\/p>\n\n\n\n\n\n
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Figure 1:<\/strong> AUCglucose <\/sub>values of the different experimental groups, where, *** p<\/em> \u22640.001<\/strong><\/p>\n

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Kidney Index<\/strong><\/p>\n\n\n\n

Statistical\nanalysis showed that the kidney index of the DC (1.24 \u00b1 0.47 gm;) group\nsignificantly (p<\/em>\u22640.05) elevated compared to the CMC (0.64 \u00b1 0.25 gm)\ngroup. Whereas, LD (0.84 \u00b1 0.25 gm), HD (0.84 \u00b1 0.20 gm) and GLD (0.62\u00b1 0.27\ngm) group shows insignificant results compared to the CMC group (Fig. 2).<\/p>\n\n\n\n\n\n
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Figure 2:<\/strong> Kidney index value of different experimental groups, where, *p<\/em> \u2264 0.05<\/strong><\/p>\n

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Lipid\nProfile Analysis<\/strong><\/p>\n\n\n\n

Colorimetric\nkits were used to evaluate the lipid profile (serum TC, HDL-C and TG) of the\nexperimental animals on the last day of the experiment. The HDL-C levels were\nsignificantly (p<\/em>\u22640.05) lower in the disease group (26.68\u00b131 mg\/dl)\ncompared to the CMC (34.44\u00b17.47 mg\/dl), and GLD, LD, and HD group shows values\nare close to the CMC group. Whereas, serum total cholesterol was significantly\nhigher in the disease group (156.27\u00b14.50 mg\/dl)<\/em> compared to the CMC (136.66\u00b112.20\nmg\/dl) group, the GLD, LD, and HD parameters showed non-significant variation.\nThe TG values in the disease group (141.37\u00b12.57 mg\/dl) and CMC group (122.55\u00b17.04\nmg\/dl) also significantly (p<\/em>\u22640.05) differed, but comparison with LD, HD\nwith CMC showed non-significant differences. In the case of bad cholesterol\nlike LDL-C, the value in disease control (101.67\u00b15.31mg\/dl) is considerably\nhigh compared to the CMC (77.66\u00b113.51 mg\/dl) group; the GLD, LD, and HD groups\nshow non-significant variations (Fig. 3). <\/p>\n\n\n\n\n\n
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Figure 3:<\/strong> Serum lipid profile among experimental groups: A. Concentration of serum cholesterol B. Concentration of serum triglycerides<\/strong><\/p>\n

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Serum\nparameters of Albumin, Creatinine and Urea<\/strong><\/p>\n\n\n\n

At\nthe end of the experimental duration, it was found that the levels of serum\nalbumin were significantly lower in the DC group (2.90 \u00b1 0.18 g\/dL) when\ncompared with the CMC group (4.12 \u00b1 0.59 g\/dL). Whereas, the values of the same\ntests observed in the treated groups (HD and LD) and GLD were close to that of\nthe CMC group. For the serum creatinine, the DC group showed significantly\nelevated (p<\/em>\u22640.05) levels (0.98 \u00b1 0.25 mg\/dL) when compared with the CMC\ngroup (0.77 \u00b1 0.03 mg\/dL). Other experimental groups LD, HD and GLD showed\nslight elevations in the serum creatinine levels (0.87 \u00b1 0.03 mg\/dL, 0.85 \u00b1\n0.03 and 0.82 \u00b1 0.07 mg\/dL respectively), but these are not significant (p<\/em>\u22640.05)\nwhen compared with the CMC group. Serum urea parameter was also significantly (p<\/em>\u22640.05)\nhigher (42\u00b11.67 mg\/dL) in the DC group compared to the CMC group (29.66 \u00b1 2.94\nmg\/dL), but the treatment groups (33.66 \u00b1 2.87 mg\/dL and 32.5 \u00b1 3.61 mg\/dL\nvalue of LD and HD respectively) and GLD (31.83 \u00b1 3.06 mg\/dL) group shows\ninsignificant result compared with CMC group (Fig. 4). <\/p>\n\n\n\n\n\n
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Figure 4: <\/strong>Concentration of serum urinary parameters among different groups; A. Concentration of albumin B. Concentration of urea<\/strong><\/p>\n

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Urine\nparameters of Albumin, Creatinine, and urea<\/strong><\/p>\n\n\n\n

At\nthe end of the experimental duration, it was found that the levels of urine\nalbumin were significantly higher (p<\/em>\u22640.05) in the DC group (3.83 \u00b1 0.25\nmg\/dL) when compared to the CMC group (2.87 \u00b1 0.22 mg\/dL). Whereas, the levels\nof the same in the groups LD, HD (3.29 \u00b1 0.28 mg\/dl, 3.14 \u00b1 0.43 mg\/dL\nrespectively) is a bit higher than in the CMC group but not significantly (p<\/em>\u22640.05)\ndiffer. With regards to the urine creatinine, the DC group showed significantly\nelevated (p<\/em>\u22640.05) levels (1.85 \u00b1 0.49 g\/L) when compared with the CMC\ngroup (1.26 \u00b1 0.30 g\/L). The groups LD, HD and GLD showed slight elevations in\nthe levels (1.41 \u00b1 0.35 g\/L, 1.34 \u00b1 0.32 and 1.32 \u00b1 0.31 g\/L respectively), but\nthese are not significant (p<\/em>\u22640.05) when compared with the CMC group. For\nthe Urine urea parameter, it was also significantly higher levels (5.53\u00b1 0.53\nmg\/dL) in the DC group compared with the CMC group (3.22 \u00b1 0.44 mg\/dL), but the\nLD (3.95 \u00b1 0.44 mg\/dL), HD (3.55 \u00b1 0.57) and GLD (3.08 \u00b1 0.50 mg\/dL) group\nshows insignificant result compared with CMC group. (Fig. 5).<\/p>\n\n\n\n\n\n
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Figure 5: <\/strong>Concentration of urinary parameters among different groups; A. Concentration of urine albumin B. Concentration of urine urea.<\/strong><\/p>\n

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In-vivo<\/em><\/strong>\nantioxidant study<\/strong><\/p>\n\n\n\n

The MDA level in the case of DC (0.08\u00b10.01nM\/mg tissue)\nwas significantly higher (p<\/em>\u22640.05)\nthan other groups (0.048\u00b10.015; 0.066\u00b10.014; 0.066\u00b10.011; 0.058\u00b10.10 nM\/mg\ntissue values of CMC, LD, HD and GLD respectively). The SOD level in the case\nof DC (0.079\u00b10.023 Unit\/mg tissue) was significantly lower (p <\/em>\u2264 0.05), but the treatment group was\nable to increase the level of SOD (LD: 0.145\u00b10.053; HD: 0.154\u00b10.050; GLD: 0.151\u00b10.11)\nwhich was close to CMC group (CMC: 0.149\u00b10.058). The relative GSH concentration\nlevel in the case of DC (33.66\u00b19.52) significantly (p <\/em>< 0.05) reduced than the CMC group (CMC: 87.5\u00b14.13). The\ntreatment group namely HD (0.77.83\u00b19.70) and GLD (86.5\u00b12.07) showed an increase\nin the GSH level toward normalcy. However, the LD (76.33\u00b17.50) group GSH\nrelative concentration value shows a bit significant difference compared to the\nCMC group. In terms of catalase levels, the group treated with DC (634.16\u00b147.55\nUnit\/g tissue) displayed significantly (p\n<\/em>\u2264 0.05) lower levels of GSH compared to the CMC group (912.66\u00b1223.14 Unit\/g\ntissue). All the HD (856.16\u00b1143.06 Unit\/g tissue), LD (813.16\u00b1262.08 Unit\/g\ntissue) and GLD (901.33\u00b1131.42 Unit\/g tissue) groups demonstrated a trend\ntowards normalization (Fig. 6).<\/p>\n\n\n\n\n\n
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Figure 6:<\/strong> Kidney tissue antioxidant profile among experimental groups; A. Concentration of SOD B. Concentration of catalase.<\/strong><\/p>\n

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Histopathological\nevaluation:<\/strong><\/p>\n\n\n\n

In\nthe histological study, we have found the normal architecture of the kidney and\nglomerular size in the CMC group. However, in the case of STZ-induced mice, it\nshowed moderate to severe vascular degeneration of the glomerular tubule,\nincreased glomerular and finally, atrophy in the glomerulus. At the same time,\nit is observed that among the treatment groups, the HD and the GLD groups\nshowed a tendency toward normalcy of glomerulus architecture. However, LD also\nshows effects on restoration glomerulus architecture but is not as promising as\nHD (Fig. 7). <\/p>\n\n\n\n\n\n
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Figure 7:<\/strong> Kidney tissue histology at 40X magnification (25\u00b5m scale bar) of different experimental groups shows the effects of CPLE on glomerulus.<\/strong><\/p>\n

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Discussion<\/strong><\/p>\n\n\n\n

Nephropathy resulting from diabetes poses a substantial danger to human health due to its high morbidity and mortality rates. DN affects about 15\u201325% of type 1 diabetes patients and 30\u201340% of type 2 diabetes patients33<\/sup>. Despite the availability of therapeutics that slow the development of DN, there is increased interest in using herbal remedies to stop the development of this condition. When mice with STZ-induced hyperglycemia were administered ethanolic and methanolic extracts from C. pictus<\/em> leaves, the hyperglycemic condition was significantly reduced34,35<\/sup>. C. pictus<\/em> methanolic leaf extract is also reported to restore renal function, lower cholesterol levels and bring blood sugar levels back to normal in STZ-induced hyperglycemic mice36<\/sup>.In the initial and crucial stage of performing a study on phytomedicine, the choice of solvent holds immense importance. Opting for polar solvents such as methanol yields the greatest concentration of natural compounds37<\/sup>. Additionally, the methanolic extract boasts the highest levels of bioactive substances, further enhancing its extraction efficiency. Therefore, methanol is widely regarded as the optimal solvent for achieving elevated levels of phytochemical components38<\/sup> and in our study, the yield efficiency of the methanolic leaf extract was 9.80%.  However, our investigation observed that in the DC group, the glucose levels were elevated throughout the entire experimental duration (0 to 120 mins). However, the LD, HD, and GLD groups restored the glycemic profile just like the CMC group. These findings indicated that the CPLE helps to restore metabolic stress along with glycemic parameters positively. Because of the hyperglycemia and hypoinsulinemia caused by STZ-induced diabetes, there is a considerable loss in bw39<\/sup> and increased protein loss from tissues as well as muscle atrophy40<\/sup>. The kidney index parameter of our study reveals that the bw of STZ-diabetic mice gradually decreased, while treatment with CPLE considerably increased the bw, indicating a reduction in the risk of hyperglycemia-induced damage to muscle tissue; it was also observed that in STZ-induced hyperglycemic mice, kidney weight (hypertrophy) increases proportionally to bw. Hypercholesterolemia and hypertriglyceridemia are brought on by diabetes mellitus41,42<\/sup>. After receiving CPLE, these elevated levels returned to normal, suggesting the potential of CPLE to enhance lipid metabolism. Albumin is by far the most abundant protein in nephrotic urine43<\/sup>. The relationship between albuminuria and declining kidney function was shown by the fact that the serum albumin concentration was significantly lower, and urine albumin levels were higher in STZ-diabetic mice. Treatment with CPLE normalized these levels, demonstrating the drug’s protective effects against microalbuminuria. Reduced excretion of creatinine in the urine and elevated urea and serum creatinine levels are signs that DN is developing22<\/sup>. These effects were reversed in STZ-diabetic Swiss albino mice treated with CPLE.Hyperglycemia also leads to an increase in reactive oxygen species (ROS), which disrupts normal tissue and cellular function, contributing to various diabetic complications, including DN44<\/sup>. These elevated ROS levels impair cellular defences by interfering with the normal functioning of lipids, DNA and proteins. Glutathione (GSH) plays a crucial role in preserving the plasma’s normal state by scavenging free radicals. Superoxide dismutase (SOD) captures superoxide molecules, converting their activity into less reactive ROS. When catalase combines with hydrogen peroxide (H2<\/sub>O2<\/sub>), it neutralizes ROS45<\/sup>. Malondialdehyde (MDA) is a byproduct of lipid oxidation and serves as an indicator of ROS44<\/sup>. In our experiment, we observed that both high and low doses of CPLE effectively reduced MDA levels. Furthermore, GLD also demonstrated the ability to decrease MDA levels. However, we observed low levels of SOD, GSH and catalase, indicating a diminished antioxidant status in STZ-induced DN mice. Notably, the HD CPLE group exhibited a more significant tendency toward restoring normal GSH levels than the LD group. Additionally, both HD and LD groups displayed the capacity to normalize SOD and catalase levels. In conclusion, our findings suggest that CPLE can help maintain the balance between oxidants and antioxidants by mitigating the effects of ROS. Kidney sections from STZ-diabetic mice underwent histopathological evaluation and the results revealed significant tubular vascular degeneration, increased glomerular space, thickening of the basement membrane and atrophy of the glomerulus in the DC group. Comparing the treatment groups and positive control group to the disease control group, the number of completely and partially malformed glomeruli is likewise deficient, which shows the restoration power of CPLE extract. Though the HD group’s restoration of glomerulus architecture proved to be more intense than that of the LD group, the LD group still shows the ability to restore glomerulus architecture compared to the DC group. It can be concluded, that treatment with CPLE significantly reduced the alterations of the glomerulus and restored other important parameters, like oxidative stress management. While compared with LD and HD, all the data shows promising results for managing renal complications except the value of GSH of the LD group, which showed a significant difference (\u2264 0.05) compared with the CMC group. Thus, our experiments demonstrate a protective role of CPLE in controlling the nephrological complications and glycaemic parameters.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In the current study, STZ-induced diabetic Swiss albino mice that received CPLE treatment were found to have normalized blood sugar levels, cholesterol and triglyceride levels, improved serum and urine parameters of albumin, creatinine, urea and maintained the antioxidant level of the tissue. Histopathological investigations suggest that CPLE treatment helps repair the renal basement membrane, possibly through its anti-inflammatory and antioxidant activities. Although the mechanism of the nephroprotective action of the prepared leaf extract is yet to be established, the preliminary data suggest that CPLE possesses potent antioxidant, antihyperglycemic and antihyperlipidemic activity, thus exhibiting a protective action in STZ-induced DN. Further investigation may elucidate the detailed mechanism of action of CPLE at the cellular and molecular levels.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

The authors thank the University of North Bengal, Siliguri College, Council of Scientific and Industrial Research (CSIR), Govt. of India, and University Grants Commission.<\/p>\n\n\n\n

Conflicts of\ninterest <\/strong><\/p>\n\n\n\n

The authors\ndeclare no conflict of interest.<\/p>\n\n\n\n

Funding source<\/strong><\/p>\n\n\n\n

The authors thank the University of North Bengal for partial financial support (Ref. No. 2274\/R-2021 Dated 24.06.2021) to carry out the works. Support from Siliguri College is hereby acknowledged for SS. Fellowship support from the Council of Scientific and Industrial Research (CSIR), Govt. of India for AJG (Ref. No. 09\/285(0089)\/2019-EMR-I Dated 07.10.2019) and from University Grants Commission (UGC), Govt. of India for RI (UGC- No. F.16-6 (DEC-2018)\/2019(NET\/CSIR UGC Ref. No. 711\/ (CSIR-UGC NET DEC.2018) and SG (NTA Ref. No.201610130713 Dated 01.04.2021) is also acknowledged.<\/p>\n\n\n\n

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