Assessment of Antidiabetic Activity by Using Silvernanoparticles of Arctium minus Leaves in Wistar Albino Rats

Received: 10-Sep-2024, Manuscript No. DPC-24-147702; Editor assigned: 14-Sep-2024, Pre QC No. DPC-24-147702 (PQ); Reviewed: 28-Sep-2024, QC No. DPC-24-147702; Revised: 01-Oct-2024, Manuscript No. DPC-24-147702 (R); Published: 29-Oct-2024, DOI: 10.4172/0975-413X.16.5.488-494

Introduction

The diabetes mellitus is persistent metabolic sickness. It’s far a fast-developing international problem with huge social, fitness and monetary consequences. Hyperglycemia or high blood sugar is a commonplace effect of uncontrolled diabetes and over time leads to serious damage to a number of the body’s structures, especially the nerves, kidney and blood vessels [1].

Herbal drug treatments do now not have any aspect outcomes, as they're loose from chemical compounds. They’re additionally milder than allopathic drugs. The herbal detoxing method of the frame is efficaciously enhanced by way of natural medicines. They may be used to cleanse the colon, enhance digestion and food absorption. natural drugs are also superb in boosting the immune system [2].

Nanoparticles (NP) are particles discovered in natural, accidental or synthetic substances, 50% or extra of which fall inside this length range of one- 100 nm. Inside the gift take a look at green synthesis is seemed as a crucial tool to reduce the damaging outcomes associated with metabolic sickness.

The fundamental techniques and mechanisms of “inexperienced” synthesis techniques are particularly for steel and metallic oxide (e.g., gold (Au), silver (Ag), Copper Oxide (CuO) and Zinc Oxide (ZnO)) nanoparticles the usage of natural extracts. Plant-based Silver Nanoparticles (AgNPs) are the various simplest to put together and to lessen and stabilize Ag ions the usage of an aggregate of biomolecules, along with polysaccharides, vitamins, amino acids, proteins, phenolics, saponins, alkaloids. Where aqueous plant extract acts as a reducing in addition to the stabilizing agent of silver nanoparticles [3-6].

Plant profile

Arctium minus

Herbalism or "Herbal medicines” have a long history to cure several kinds of human diseases from the various parts of the plants such as leaf, stem, bark, root, etc. Arctium minus is a biennial plant, normally found along roadsides. The leaves are basal and alternative, it is a tall, purple brush like flower heads which appears July to September. The common names are Burdock, cuckoo button, button burs, beggar’s button. The World Health Organization (WHO) estimates that 80 percent of the population of some Asian and African countries presently uses herbal medicines for some aspect of primary health care. It was realized from the various reports that Arctium minus, belongs to the family Rubiaceae, have displayed plethora’s of potential biological activities such as anticancer, hepatoprotective, anti-inflammatory, anti-fertility, anti-amoebic, antinociptive etc. from its various parts (Figure 1).

Figure 1. Whole plant of Arctium minus.

Materials and Methods

Preparation of extraction J

The leaves were shade dried at room temperature for 10 days. Then these were milled into powder by mechanical grinder. This powder was sequentially extracted to their increasing polarity with petroleum ether, ethyl acetate, ethanol respectively. About 500 gm of powdered leaves was uniformly packed into a thimble in a soxhlet apparatus and extracted with 1000ml Petroleum ether, Ethyl acetate and Ethanol, respectively. Finally, ethanol extract was collected and filtered.

Synthesis of silver nanoparticles

Synthesis of Silver Nanoparticles (AgNPs) using leaves of Arctium minus was synthesized by a green method, through the reduction of silver nitrate using 90ml at a concentration of 1 mM mixed with 10 ml of Arctium minus extract. The reaction was carried out at 85°C for 2 hrs.

Characterization of synthesized AML-AgNPs

The synthesized AML-AgNPs were subjected to the following spectral study for characterization UV-Vis spectroscopy, Energy Dispersive X-ray (EDX) and attenuated total reflectance fourier transform infrared spectroscopy are among the analytical techniques used in the characterization (ATR-FTIR), Scanning Electron Microscopy (SEM) and High-resolution Transmission Electron Microscopy (HR-TEM).

UV-spectrophotometry analysis of AML-AgNPs

UV-Vis spectroscopy the nanoparticle solution was analyzed using a UV-Visible spectrophotometer in the wavelength range of 300 nm-800 nm with a resolution of 1 nm to monitor the bio reduction of silver (Ag and AgO). Because of the colloidal suspension’s high Optical Density (OD), a 1 ml aliquot of the solution was diluted with 3 ml of distilled water. For 24 hours the absorbance spectra of silver nanoparticles were examined on a regular basis. As a control Milli Q water was employed.

Attenuated Total Reflectance Fourier Transform Inflectance (ATR-FTIR) analysis of AML-AgNPs

FTIR were employed to characterize the composition, structure, structure and morphology of the silver nanoparticle. The nanoparticle solution was centrifuged for 30 mts at 6000 rpm and the supernatant was discared. To eliminate any non-reacting molecules in the colloidal matrix the pellets in a hot air oven for 2 hrs a powder sample was obtained. The Bruker Platinum-ATR spectrophotometer was used to conduct FTIR analyses on powder AM-AgNPs and crude extracts FTIR mesurements were performed at an average of 32 scans per sample in the wavenumber range 4000^-l-400 cm^-l with a resolution of 4 cm^-1.

High Resolution Transmission Electron Microscopy (HR-TEM) analysis of AML-AgNPs

HR-TEM is a useful, extensively used and vital method for determining particle and grain size, size distribution and morphology. The distance between the objective lens and specimen as well as the distance between the objective lens its image plane, define the magnification of TEM particles were sonicated in 100% ethanol for 30 minutes to an hour. Allowing a drop of the suspension to dry on a carbon-coated formvar grid specimens were analyzed using a 200 KV VEGA3 TESCAN SOC MK-University transmission electron microscope. Digital micrograph software was used to capture micrographs with a Gatan bottom mount camera.

Scanning Electron Microscopy (SEM) analysis of AML-AgNPs:

Scanning Electron Microscopy (SEM) was carried out to determine the structure, size and forms of nanoparticles. Samples were placed on 12 mm aluminum specimen stubs using double-sided carbon tape, coated with gold palladium and analyzed with a 10KV VEGA3 TESCAN SOC-MK University SEM.

X-ray Energy Dispersive (EDX) analysis of AML-AgNPs:

Energy Dispersive X-ray (EDX) studies revealed the spherical nature of particles synthesized from silver metal. It reveals strong signal in the silver region and confirms the formation of AgNPs. Metallic silver nanocrystals generally show typical peak approximately at 3KeV due to surface plasmon resonance.

Pharmacological activity

Acute toxicity study: In the research study, when a drug is administered to a biological system there will be some interactions may happen. In most case these are desired and useful, but many effects are not advantageous. Acute, sub-acute and chronic toxicity studies are performed by the manufacturers in the investigation of a new drug. Acute toxicity is involved in estimation of LD50 (It is the lethal dose (causing death) to 50% of tested group animals).

LD₅₀ (median lethal oral dose)

LD₅₀ (median lethal oral dose) is a statistically derived oral dose of a substance that can be expected to cause death in percent of animals when administered by the oral route. The LD₅₀ value is expressed in terms of weight of test substance per unit weight of animal (mg/kg). In this study acute toxicity study was carried out in wistar albino rats. The procedure was followed by using OECD 423 (acute toxic class method). The rats are fasted overnight, prior to dosing. The three dose levels are administered by the help of oral feeding needle over the prior of 24 hours. After the extract has been administered, food may be withheld for a further 3-4 hours in rats. The purpose of sighting study is to allow selection of the appropriate starting dose for main study. The test substance is administered to a single animal in a sequential manner following from the fixed dose levels of 5, 50, 300 and 2000 mg/kg. The interval between dosing of each level is determined by the mortality/onset, duration and severity of toxic signs over the period of 24 hours, special attention given during the first 4 hours. Four hours after the drug administration, provide the food and water for 14 days and daily observed some parameters such as food intake, water intake, mortality, onset, duration and severity of toxic signs. The animal weight is recorded on weekly once. On the day fourteen all the animals are sacrificed, to isolate the organs and observe the histopathological changes. Based on the mortality result of sighting is decided and carried out with five animals per dose level (5 or 50 or 300 or 2000 mg/kg). Based on the mortality result on 14th day of observation, the doses for in vivo study are selected.

In vivo anti-diabetic activity of nanoparticles Arctium minus leaf in streptozotocin induced diabetic wistar albino rats

Wistar albino rats (150 grams-200 grams) of both sexes were procured from TANWAS laboratories, Chennai-TN, India. Prior to the experiment the rats were housed in a clean polypropylene cages (6 rats/cages) for a period of 7 days under standard temperature (25°C-30°C), relative humidity (45%-55%), dark/light cycle (12/12 hrs). The studies were performed with the approval of CPCSEA (887/Po/Re/S/2005) IAEC/M.PHARM/07/2018. The animals were put in overnight fasting were deprived of food for 16 hrs but allowed free access of water.

Hypoglycemic test: Groupings were done as follows: Group I served as control-Carboxy Methyl Cellulose (CMC) 0.5% (0.3 ml/100 g rat), group II served as positive control-Glibenclamide (2 mg/kg), group III served as aqueous nanoparticles of Arctium minus (200 mg/kg), group IV served as aqueous nanoparticles of Arctium minus (400 mg/kg). Blood samples were collected by the tail nipping method and glucose level checked by glucometer. After drug Administration blood samples have been collected different time intervals at 30, 60 and 120.

Oral glucose tolerance test: Groupings were done as follows: Group I served as control-Carboxy Methyl Cellulose (CMC) 0.5% (0.3 ml/100 g rat), group II served as positive control-Glibenclamide (2 mg /kg), group III served as aqueous nanoparticles of Arctium Minus (200 mg/kg), group IV served as aqueous nanoparticles of Arctium minus-(400 mg/kg). All the groups of animals were fasted for 24 h and blood samples were collected before drug or solvent treatment. The drug, extract and solvent, have been administered to different groups and 30 mins later all the groups of rats were treated with glucose orally at dose 10 gm/kg body weight by using oral feeding needle. Blood samples were collected by the tail nipping method and glucose level checked by glucometer. After drug Administration blood samples have been collected different time intervals at 30, 60 and 120.

Induction of diabetes to animals: A single dose (100 mg/kg, i.p.) of streptozotocin monohydrate dissolved in sodium citrate buffer was used for the induction of diabetes in rats after overnight fasting. After 1 hr of streptozotocin monohydrate administration, the animals were given feed and libitum and 5% dextrose solution was also given in feeding bottle for a day to overcome early hypo-glycaemic phase. The animals were stabilized for a week and animals showing blood glucose level more than 200 mg/dl were selected for the study.

Experimental design

Group treatment: AML-AgNPs, standard drug and normal saline were administered with the help of oral feeding needle. Group I serve as normal control which received normal saline for 14 days. Group II to group V were diabetic control rats. Group IV and group V (which previously received streptozotocin 100 mg/kg) were given fixed doses of AML-AgNPs (200 mg/kg, P.O, 400 mg/kg, P.O) and group III received standard drug Glibenclamide (2 mg/kg,P.O) for 14 consecutive days.( AML-AgNPs ) (Table 1).

Table 1: Five groups of rats six in each groups received the following treatment schedule for 14 days.

Collection of blood samples: Fasting blood samples were drawn from retro orbital puncture of rats at weekly intervals till the end of the study 1, 7 and 14 days.

Estimation of biochemical parameters serum blood glucose: On 1, 7 and 14 days fasting blood samples were collected and analyzed the blood glucose.

Blood glucose level: The blood glucose level test measures the amount of glucose in the blood sample obtained from the animals. The test is usually performed to check for elevated blood glucose levels which can be an indication of diabetes or insulin inhibition.

Statistical analysis: Statistical analysis was done by using Graph Pad Prism 5.0. All the values of Biochemical parameters and body weight were expressed as Mean ± Standard Error Mean (SEM). The values were analyzed for statistical significance using one-way Analysis of Variance (ANOVA), comparison was done by using Dunnett’s t test. P values <0.05 were considered as significant, P values <0.01 were considered as very significant, P values < 0.001 were considered as highly significant and ns were considered as not significant.

Results and Discussion

The glucose levels were analyzed by using glucometer and each value is the mean ± standard error (n=each group consist of 6 animals) (p<0.05)^*, (p<0.001)^** and (p<0.0001)^*** as compared to control and positive control group evaluated by one way, ANOVA followed by Dunnet ‘t’ test (Table 2).

Table 2: Hypoglycemic test.

The hypoglycemic test results have shown Table 2, which indicated aqueous extract of Arctium minus treated animals 200 and 400, significantly decreased in blood glucose level (0.84 ± 1093↓ and 18.83 ± 3.879↓) (P<0.05)^*, (P<0.001)^** and (P<0.0001)^*** when compared to control and positive control (Figures 2 and 3).

Figure 2. Hypoglycemic Test at 30 minutes.

Figure 3. Hypoglycemic test at 1 hour.

In vivo antidiabetic study

The experimental results have indicated on Table 3. The negative control group glucose levels were significantly increased when compared to each other groups. All the groups of animals were affected in diabetes, which indicated blood glucose levels were slight changes in the blood glucose level (4.13 ± 1.207↓ and 1.0 ± 0.93↑) for normal control group at 7^th and 14^th days. On day 7^th glucose levels were significantly decreased glibenclamide 2 mg/kg treated group (120.2 ± 1.414↓ and 23 ± 1↓) (P<0.05)^*, (P<0.001)^** and (P<0.0001)^*** when compared with control group at 7^th and 14^th days. The AML-AgNPs treated groups 200 and 400 mg/kg were dose dependent manner decreased (P<0.001)^** and (P<0.0001)^*** (10 ± 0.362↓ and 90±1.67↓) when compared with control group but positive control have more anti diabetic activity at 7^th day. The aqueous AML-AgNPs at the dose level 400 mg/kg have potent activity (90 ± 1.67↓ and 120.2 ± 1.414↓when compared with positive control at 7^th day. The nanoparticles of Arctium minus leaf 200 and 400 mg/kg have been expressed dose dependent antidiabetic action (P<0.001)^** and (P<0.0001)^*** when compared to control and positive control. On day 14^th, nanoparticles of Arctium minus leaf treated animals 200 and 400 mg/kg significantly decreased and maintain the blood glucose level (5.1 ± 0.07↓ and 11 ± 1.08↓), (P<0.001)^** and (P<0.0001)^*** when compared to control and positive control (Table 4 and Figure 5).

Table 3: Results of the effects of AML-AgNPs on blood glucose levels.

Table 4: Oral glucose tolerance test.

Figure 4. Oral glucose tolerance test at 2 hours.

Oral Glucose Tolerance Test (OGTT) results have been expressed on Table 4: Half hour after the glucose treatment, all the groups of animal blood glucose levels were significantly increased (74.5 ± 3.863↑, 35.2 ± 6.6905↑, 59.5 ± 3.764↑ and 65.58 ± 3.762↑). The blood glucose levels were significantly decreased for, aqueous nanoparticle of Arctium minus 200 and 400 mg/kg (59.5 ± 3.764↓ and 65.58 ± 3.762↓ ) ↓), (P<0.001)^** and (P<0.0001)^** when compared to control and positive control at 1hour and each and every ½ hour blood glucose levels(200 mg/kg: 8.8 ± 3.26,10 ± 0, 6.66 ± 1.164 and 3 ± 0.696, 400 mg/kg:17.9 ± 1.422, 9.97 ± 0,14.7 ± 1.093 and 5 ± 0.696) (P<0.05)^*, (P<0.001)^** and (P<0.0001)^*** were changes in the dose dependent manner extract treated group of animals compared to control and positive control but 400 mg/kg produce the potent activity [7- 10].

Sequential extraction was done according to increasing polarity order (petroleum ether, ethyl acetate and ethanol). In this present study acute toxicity study was carried out in rats. The procedure was followed by using OECD 423 (Acute Toxic Class Method). The acute toxic class method is a step wise procedure with three animals of a single sex per step. The method used defined doses (2000, 1000, 500, 50, 5 mg/kg body weight, Upand- Down Procedure). The starting dose level of EEKAGA was 2000 mg/kg body weight p.o as most of the crude extracts possessed LD₅₀ value more than 2000 mg/kg p.o and also found to be the maximum safe dose. They were observed for signs of toxicity for 14 days. The onset of toxicity and signs of toxicity also noted [11-15].

Hence, 1/10^th (200 mg/kg) and 1/5^th (400 mg/kg) of this dose were selected for further study. Streptozotocin, a beta cytotoxic, induces diabetes in a wide variety of animal species by damaging the insulin secreting cells of the pancreas. Literature sources indicate that the Streptozotocin induced rats are hyperglycaemic. The treatment of lower doses of streptozotocin (100 mg/kg b.w.) produced partial destruction of pancreatic β-cells even though the animals become permanently diabetic. Thus, these animals have surviving β-cells and regeneration is possible. It is well known that the sulfonylurea’s act by directly stimulating the β-cells of the Islets of Langerhans, to release more insulin and these compounds are active in mild streptozotocin-induced diabetes. In vivo anti-diabetic screening was performed for the confirmation of above mechanism of action, of nanoparticles of Arctium minus in biological system. At the end nanoparticles of Arctium minus (200 mg/kg p.o, 400 mg/kg p.o.) showed statistically significant decrease in blood glucose levels. Hence, the silver nanoparticles of Arctium minus showed antidiabetic activity. This work may serve as a preliminary work and may be used by researchers to evaluate experimental anti-diabetic models and its related diseases, to develop new entity, for the treatment of diabetes mellitus [15-20].

Conclusion

Extraction of leaf was done by sequential extraction method. The leaves of Arctium minus was extracted using the solvent with increasing polarity order (petroleum ether, ethyl acetate and ethanol). Then Successfully synthesized AgNPs. The AgNPs exhibited potent carbohydrate degrading enzymes inhibitor for further animal studies and clinical methodology. Based on this result, it is suggested the AgNPs should be used as a nano antidiabetic drugs. Finally, the in vivo anti-diabetic activity of silver nanoparticles of Arctium minus leaf was tested using streptozotocin-induced diabetic rats. Acute toxicity study was carried out in rats using OECD 423. 1/10^th (200 mg/kg) and 1/5^th (400 mg/kg) of the maximum safe dose (2000 mg/kg) was selected for further study. Fasting blood sample were drawn from retino-orbital puncture of rats at weekly intervals till the end of the study (1, 7 and 14 days). On these days, fasting blood glucose were collected and analyzed for glucose. At the end of the study, (14^th day) the silver nanoparticles of Arctium minus leaf (200 mg/kg p.o and 400 mg/kg p.o) treated diabetic groups showed statistically significant decrease in blood glucose levels similar to the standard drug glibenclamide (2 mg/kg). The synthesized silver nanoparticles showed higher level of antidiabetic activity by inhibiting the carbohydrate metabolizing enzymes. In addition, it also exhibited good antioxidant activity which will scavenge the free radicals produced due to hyperglycemia.

References

1. Agiostratidou G, Anhalt H, Ball D, et al. Diabetes Care. 2017; 40(12): p. 1622-1630.

   [Crossref] [Google Scholar] [PubMed]

2. Mathers CD, Loncar D. PLoS medicine. 2006; 3(11): p. 442.

   [Crossref] [Google Scholar] [PubMed]

3. Wang TJ, Larson MG, Vasan RS, et al. Nat Med. 2011; 17(4): p. 448-453.

   [Crossref] [Google Scholar] [PubMed]

4. Young KA, Snell-Bergeon JK, Naik RG, et al. Diabetes Care. 2011; 34(2): p. 454-458.

   [Crossref] [Google Scholar] [PubMed]

5. Joergensen C, Hovind P, Schmedes A, et al. Diabetes Care. 201l; 34(5): p. 1081-1085.

   [Crossref] [Google Scholar] [PubMed]

6. Kohei KA. JMAJ. 2010; 53(1): p. 41-46.

   [Google Scholar]

7. Sigal RJ, Kenny GP, Wasserman DH, et al. Diabetes Care. 2006; 29(6): p. 1433-1438.

   [Crossref] [Google Scholar] [PubMed]

8. Yki-Järvinen H. Diabetes Care. 2001; 24(4): p. 758-767.

   [Crossref] [Google Scholar] [PubMed]

9. Rasool Hassan BA. Pharmaceut Anal Acta. 2012; 3(10): p. 2153-2435.

   [Google Scholar]

10. Tag H, Kalita P, Dwivedi P, et al. J Ethnopharmacol. 2012; 141(3): p. 786-795.

    [Crossref] [Google Scholar] [PubMed]

11. Tuso PJ. Perm J. 2002; 6(4): p. 22-26.

    [Google Scholar]

12. Balan K, Qing W, Wang Y, et al. Rsc Adv. 2016; 6(46): p. 40162-40168.

    [Google Scholar]

13. Awwad AM, Salem NM, Abdeen AO. Int J Ind Chem. 2013; 4: p. 1-6.

    [Crossref] [Google Scholar]

14. Sharker SM, Hossain MK, Haque MR. Asian Pac J Trop Biomed. 2012; 2(3): p. 1317-1322.

    [Crossref] [Google Scholar]

15. Gad MZ, El-Sawalhi MM, Ismail MF, et al. Mol Cell Biochem. 2006; 281: p. 173-183.

    [Crossref] [Google Scholar] [PubMed]

16. Dzhafarova RE, GSh G, Dzhafarkulieva ZS. Georgian Med News. 2009; 1(170): p. 110-114.

    [Google Scholar] [PubMed]

17. Kuroda M, Mimaki Y, Nishiyama T, et al. Biol Pharm Bull. 2005; 28(5): p. 937-939.

    [Crossref] [Google Scholar] [PubMed]

18. Mondal SK, Chakraborty G, Bhaumik UK, et al. Adv Tradit Med. 2008; 8(3): p. 236-242.

    [Google Scholar]

19. Gupta R, Gupta RS. DiabetolCroatica. 2011; 40(4):107-113.

    [Google Scholar]

20. Yoshino K, Miyauchi Y, Kanetaka T, et al. Biosci, Biotech Biochem. 2009; 73(5): p. 1096-1104.

    [Crossref] [Google Scholar] [PubMed]