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Original article
13 (
4
); 5096-5106
doi:
10.1016/j.arabjc.2020.02.009

Green synthesis of gold nanoparticles from Fritillaria cirrhosa and its anti-diabetic activity on Streptozotocin induced rats

, , ,
a Department of Nephrology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China
b Department of Neurology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China

⁎Corresponding author at: Department of Nephrology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China. jdyinmin01@sina.com (Min Yin)

Received: , Accepted: ,
© The Author(s) 2020
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

To evaluate the anti-diabetic effect of Fritillaria cirrhosa gold nanoparticles on Streptozotocin (STZ) stimulated diabetic preclinical models. The albino rats of either sex were equally distributed to five different groups. Group-I represented as Control; Group-II represented as diabetic control (STZ alone); Group-III represented as 10 mg/kg body weight of Fritillaria cirrhosa gold nanoparticles + diabetes; Group-IV represented as 20 mg/kg body weight of Fritillaria cirrhosa gold nanoparticles + diabetes; Group-V represented as 0.1 mg/kg body weight of glibenclamide + diabetes. The animals were killed after the experimental period. The blood and organs samples were gathered and stored for the additional investigations. The gold nanoparticles were inspected via the UV spectrophotometer, HR-TEM, XRD and FT-IR techniques. The bodyweight, kidney and liver weight were tabulated. The food and water consumption were monitored in all the experimental rats. The serum markers, hepatic markers and renal markers were quantified in both normal and investigational rats. The lipid peroxidation and antioxidant status were quantified in the control and experimental rats. The histopathological alterations were also studied in all the experimental animals. The standard drug glibenclamide was used to compare the synthesized AuNPs. The study revealed that the AuNPs treatment restores the serum, hepatic and renal marker in the STZ-challenged diabetic rats. The AuNPs treatment modulates the antioxidants level and decreased the lipid peroxidation by its antioxidant properties. The pathology results revealed that the AuNPs treatment induces the regeneration of islets cells of pancreas in the experimental rats. The research study proved that the Fritillaria cirrhosa AuNPs exerts anti-diabetic properties.

Keywords

Diabetes
Fritillaria cirrhosa
Gold nanoparticles
Streptozotocin
Glibenclamide
1

1 Introduction

Diabetes Mellitus (DM) is an impaired condition due to defect in glucose homeostasis, malfunction of pancreatic β-cells and worsens the insulin secretion in the peripheral tissues mainly in skeletal muscle and adipose tissue. Metabolic disturbance in diabetes directs to structural and functional alteration of tissues and can seriously impact the quality of life (Samadder et al., 2013; Vijayan et al., 2017). The alarming rate of global occurrence of Diabetes Mellitus accounts a global health issue. In 2016, The International Diabetes Federation (IDF) recorded that 415 million individuals were diagnosed with diabetes mellitus and are projected at 642 million by 2040 with an average increasing case of 3 people every second (Rahelic, 2016). More than 150 million Chinese people are diagnosed with this multifaceted illness currently and instigating severe socioeconomic crisis (Cheng, 2005). The prevalence of the disease in China is multi-factorial which includes genetic disorders, environmental factors such as obesity associated with sedentary lifestyle (Sarah et al., 2004).

There are several oral anti-hyperglycemic drugs are in the market to mollify diabetes. But, it is still a challenge for the scientific community to alleviate it with slight or null side effects. Plants offer strong efficacy with least or none side effects especially against metabolic disorders as their effectiveness are from a blend of active molecules eliciting at the same time. Attention towards traditional practices has been rekindled due to research in alternative medicine and natural therapies. A necessity has risen to quest for more anti-diabetic drug candidates without adverse effects (Ahmed et al., 2010). The Fritillariae cirrhosae (FC) was also named in Chinese as “Chuan-Bei-Mu”, related to the Lilliaceae family. It was extensively utilized as a conventional herb to treat common cold, cough and asthma for 2000 years in Chinese medicine (Wang et al., 2011). Various studies showed that the Fritillariae cirrhosae plant extract contains alkaloids as a major bioactive compound (Wang et al., 2014). The scientific study confirms the Fritillariae cirrhosae exerts antiasthmatic, antitussive, antibacterial and anticancer properties (Li et al., 2013; Wang and Wang, 2014).

Nanotechnology is a technique which used to bind the nanoparticles (1–100 nm) with desired therapeutic compounds to increases its efficacy (Shu and Tang, 2020; Liu et al., 2016; Shi et al., 2017; Cai et al., 2018; Shi et al., 2017). The noble metallic nanoparticles were gained immense awareness because of their elevated catalytic, electronic, fluorescence, nanoprobes, and biological actions (Huang et al., 2018; Huang et al., 2017; Zeng et al., 2018). The nanoparticles and nanostructured materials illustrates a dynamic area among the researchers and technology-economical zone (Huang et al., 2017; Zhang et al., 2017; Huang et al., 2018). They attained a distinctive place in technological progressions that owing to their individual physic-chemical features like conductivity, wettability, melting point, light absorption, catalytic, biomedical, and pharmacological fields (Huang et al., 2020; Gan et al., 2020). Gold nanoparticles are widely used in traditional medicine against various chronic diseases due to its medicinal properties. The green synthesis method was effective, inexpensive and reliable to develop nanomedicines (Cai et al., 2018). The toxic compound streptozotocin was widely used by the researchers to induce diabetes in rodent models (Ibrahim and Abd-El-Maksoud, 2015). However, there were none reports on the metallic nanoparticles from Fritillariae cirrhosae towards their biological activities. Hence in this current exploration, we investigated the anti-diabetic activity of Fritillaria cirrhosa gold nanoparticles on streptozotocin induced diabetes in in vivo models. The physical properties of synthesized particles were also studied. In this research exploration, we utilized Glibenclamide as a standard drug to compare with the synthesized Fritillaria cirrhosa gold nanoparticles.

2

2 Materials and methods

2.1

2.1 Sample collection, preparation of extract and synthesis of AuNPs from Fritillaria cirrhosa

The whole plant Fritillaria cirrhosa was identified and collected in the province of china. The species of the plant sample was confirmed and approved by the botanist. The whole plant materials were washed with tap water to eliminate the foreign particles. Then the samples were washed thrice thoroughly with the double distilled water. The plant should be air dried at the sun shaded place for 15 days. The dried plant material was made into a fine course powder using electric mixer. 25 g of fine powdered sample was mixed with 100 ml of Millipore water in a rounded bottom conical flask. The sample was boiled at 60 °C with magnetic stirrer in the electric oven for the 30mins. The samples were cooled and it was centrifuged at 5000 rpm for 15mins. Filter the samples with the Whatmann filter paper to obtain the crude extract of Fritillaria cirrhosa. 10 ml of crude extract was mixed with the 100 ml of 10 mM auric chloride solution for the bio-reduction AuNOs. The phytochemicals present in the F. cirrhosa was work as a stabilizing, capping, and reducing agent for the formation of Au3+ ions to Au0. It should be placed in orbital shaker at 30 °C. The pale color sample which tends to develop deep red color solution confirms the synthesis of AuNPs. The AuNPs were collected from the extract by centrifuged thrice at 10,000 rpm for 20 mins. The supernatant was subsequently discarded. The collected AuNPs were lyophilized and it was used to treat the diabetic induced in vivo models.

2.2

2.2 Characterization of gold nanoparticles

The peak values of prepared AuNPs were measured using Agilent Cary UV/Vis System at the range of 300–700 nm. The Fritillaria cirrhosa extract was pale in color. The deep color was formed after adding the auric chloride solution. This confirms the generation of gold nanoparticles. The peak value of AuNPs was observed periodically (24 h, 48 h, 5th day, 15th day and 30th day). The AuNPs size, shape and electron diffraction pattern was captured with the HR-TEM. The morphological structure and functional group of AuNPs were studied by XRD and FT-IR.

2.3

2.3 Cytotoxic assay

The cytotoxic effects of AuNPs on 3T3 cells were appraised by MTT analysis. 3T3cells were cultured onto 24 well plates (1 × 104 cells/well) for 24 h. After 24 h, different dosages of AuNPs (5, 10, 25 and 50 µg) were supplemented and incubated for 24 h. Afterward, supernatant was collected, added MTT (200 µl) solution to all well for 4 h and measuring the absorbance at 570 nm through ELISA reader.

2.4

2.4 Experimental protocol

The adult wistar rats were randomly parted into five groups. Every group possessed with six rats. This investigation was permitted by the committee members and the protocol was followed according to the guidelines. The streptozotocin (STZ) at the dose of 55 mg/kg in citrate buffer was injected through intraperitoneal region to induced the diabetes in the experimental rats expect the control groups. The control group (G-I) received the standard diet and water. The STZ alone represented as G-II. The AuNPs at the dose of 10 mg/kg body weight represented as G-III. The AuNPs (20 mg/kg b.wt) supplementation represented as G-IV. The FDA approved drug Glibenclamide at the dose of 0.1 mg/kg body weight represented as G-V. The AuNPs and the Glibenclamide were orally given to the experimental rats for 28 days. The final bodyweight, food and water consumption was noted in the end of research study. After the termination of investigational regimen, the rats were killed via cervical displacement. The blood samples were collected in the vials through cardiac puncture. The organs (Liver, Kidney and Pancreas) were collected and instantly rinsed with ice chilled phosphate buffer solution. The tissues were weighed and stored for the further investigations.

2.5

2.5 Quantification of biochemical parameters

The blood samples were placed in a disposable strip of the glucometer. The glycemic levels were quantified in experimental rats. The blood samples were centrifuged at 2000 rpm for 5mins to attain the serum samples. The AST and ALT were quantified in the serum of investigational rats (Reitman and Frankel, 1957). The Serum Alkaline Phosphatase was determined (Seligman et al., 1951). The serum protein levels were quantified (Zhang et al., 2012). The serum creatinine levels were quantified by modified jaffe assay kit method. The remaining blood sample was stored in heparinized vial to obtain the plasma. The plasma was used to quantify the levels of insulin in the experimental rats by ELISA method (Cat. No. abx050131, Abbexa Ltd, United Kingdom). The glycosylated haemoglobin (HbA1c) was measured by using commercially available kit (BioSystem SA, Spain).

2.6

2.6 Determination of hepatic enzymes in liver

The liver tissue was weighed and minced with ice cooled buffer to obtain the supernatant. The supernatant was used to determine the activity of hepatic enzymes hexokinase (Racker, 1947), glucose-6-phosphatase (Zak et al., 1977) and fructose-1, 6-bisphosphatase (Gancedo and Gancedo, 1971). The enzymes were quantified by using the values obtain from the UV spectrophotometer.

2.7

2.7 Determination of oxidative stress and antioxidants parameters in pancreas

The pancreas was excised and minced with ice cooled buffer to obtain the supernatant to perform the lipid peroxidation and antioxidant assay. The levels of TBARS were quantified (Ohkawa et al., 1979). The lipid hydroperoxide content was evaluated (Buege and Aust, 1978). The SOD (Kakkar et al., 1984), catalase (Sinha, 1972), Glutathione peroxidase (Rotruck et al., 1973) and GSH (Moron et al., 1979). The UV spectrophotometer was used to analysis the content of oxidative stress and antioxidant parameters.

2.8

2.8 Histopathology and histomorphometric analysis

The part of pancreas was expunged and stored in 10% formalin. Then tissues were cut to 5 µm thickness and place on the slide. The haematoxylin and eosin stain were used to evaluate the pathological alterations in the experimental animals at 40X magnification. On the other hand, the morphometric investigation was executed by inspecting the total number of islets in the pancreas of every group, area of the pancreatic islets, volume, and the diameter of the pancreatic islets for every section. The total number of islets regeneration was investigated via the preceding method described by Adeyemi et al. (2010).

2.9

2.9 Statistical study

Data were analysed through the SPSS version 22 software. The one-way ANOVA (Duncan test) were utilized to find the significant values between the groups. The obtained values were expressed as mean ± SD. The p ≤ 0.05 regarded as a statistically relevant.

3

3 Results

3.1

3.1 Assessment of Fritillaria cirrhosa AuNPs by UV method and SAED pattern

The Fritillaria cirrhosa extract initially exhibited pale color then it forms a reaction with auric chloride, then the extract becomes intense red, which indicate the bio-reduction of gold nanoparticles. Fig. 1A depicts the gold nanoparticles revealed the maximum peak at 544 nm. The peak was maintained at 544 nm at varying reaction time (24 h, 48 h, 5th day, 15th day and 30th day). This confirms the nature of AuNPs constancy and accuracy.

Assessment of Fritillaria cirrhosa AuNPs by UV/Vis-spectrophometer method and SAED analysis. [A] The peak value of 544 nm was constantly maintained at the varying time period. This proves the good stability of synthesized AuNPs. [B] The shape, size and diffraction pattern (SAED) of synthesized AuNPs was analysed. The gold nanoparticles crystalline structure from diffraction pattern confirms the faced centered cubic structure.
Fig. 1 Assessment of Fritillaria cirrhosa AuNPs by UV/Vis-spectrophometer method and SAED analysis. [A] The peak value of 544 nm was constantly maintained at the varying time period. This proves the good stability of synthesized AuNPs. [B] The shape, size and diffraction pattern (SAED) of synthesized AuNPs was analysed. The gold nanoparticles crystalline structure from diffraction pattern confirms the faced centered cubic structure.

The Selected area electron diffraction (SAED) of synthesized AuNPs was crystalline in nature. The observed SAED ring pattern of AuNPs showed different planes (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) indicates face centered cubic structure (Fig. 1B).

3.2

3.2 Analysis of Fritillaria cirrhosa AuNPs using HR-TEM and XRD

The size, pattern and disperses of AuNPs were inspected through HR-TEM (Fig. 2A). The microscopic photographs of HR-TEM evidenced that the formulated nanoparticles possessing the magnitude at the range of 40–45 nm and also it was revealed the spherical in shape.

Analysis of Fritillaria cirrhosa AuNPs using HR-TEM and XRD analysis. [A] The synthesized AuNPs size was about 40–45 nm. [B] The intensity was measured in the synthesized gold nanoparticles. The synthesized AuNPs showed the peak value at 2θ angles 40.79(1 1 1) and 45.11(2 0 0) confirms the face cubic centered structure. This confirms the high purity of synthesized gold nanoparticles.
Fig. 2 Analysis of Fritillaria cirrhosa AuNPs using HR-TEM and XRD analysis. [A] The synthesized AuNPs size was about 40–45 nm. [B] The intensity was measured in the synthesized gold nanoparticles. The synthesized AuNPs showed the peak value at 2θ angles 40.79(1 1 1) and 45.11(2 0 0) confirms the face cubic centered structure. This confirms the high purity of synthesized gold nanoparticles.

The XRD investigation was executed to validate the SAED-TEM outcome. The formulated AuNPs diffraction peaks located at 2θ angles: 40.79, 45.11 corresponded to (1 1 1), (2 0 0) indicates face centered cubic structure (Fig. 2B). The intense peaks were evidenced the highest purity of AuNPs. This outcome of Full Width at Half Maximum (FWHM), the size of the fabricated AuNPs was determined as 40–45 nm.

3.3

3.3 Analysis of Fritillaria cirrhosa AuNPs using FT-IR

The FT-IR technique was executed to recognize the functional groups occur in the surface of AuNPs (Fig. 3). The spectrum at 3331.36 and 1634.38 cm−1 denotes the existence of essential metabolites that exists on the formulated AuNPs. The peak at 3331.36 reveals the occurrence of O—H stretch of polyphenols and the peak at 1634.38 denotes the existence of —C⚌C— stretch of alkynes. The binding ability of functional groups with AuNPs denotes the existence of alkaloids.

Analysis of Fritillaria cirrhosa AuNPs using FT-IR. The gold particles bounded to hydroxyl and alkynes functional groups present in the Fritillaria cirrhosa extract. This confirms the availability and abundance of flavanoids.
Fig. 3 Analysis of Fritillaria cirrhosa AuNPs using FT-IR. The gold particles bounded to hydroxyl and alkynes functional groups present in the Fritillaria cirrhosa extract. This confirms the availability and abundance of flavanoids.

3.4

3.4 Effect of Fritillaria cirrhosa AuNPs in 3T3 cells

3T3 were added with AuNPs at dosages of 5, 10, 25, and 50 µg and measured cell viability by the MTT technique. As shown in Fig. 4, there is no toxic effect were found in the different concentration of Fritillaria cirrhosa AuNPs in 3T3 cells which revelas the nontoxic nature in the normal cells.

The cytotoxicity effect of AuNPs on normal mouse fibroblast 3T3 cells measured by MTT assay. The 3T3 cells were treated with different concentrations of AuNPs (5–50 μg/ml). Values are represented mean ± S.D. of three experiments.
Fig. 4 The cytotoxicity effect of AuNPs on normal mouse fibroblast 3T3 cells measured by MTT assay. The 3T3 cells were treated with different concentrations of AuNPs (5–50 μg/ml). Values are represented mean ± S.D. of three experiments.

3.5

3.5 Assessment of bodyweight, kidney and liver weight

Fig. 5A represents the bodyweight, kidney and liver weight of the experimental and control rats. The bodyweight was decreased in STZ-challenged diabetic rats (G-II) due to the muscle wasting and insulin deficiency. The 10 mg/kg AuNPs treated rats (G-III) moderately gaining the body weight. The 20 mg/kg AuNPs treated rats (G-IV) gradually attain the healthy body weight. The 0.1 mg/kg glibenclamide treated rats (G-V) drastically gaining the body weight. The 20 mg/kg AuNPs treatment showed more significant changes in bodyweight than the (G-II) rats. Likewise, the kidney and liver weight was noticeably (p ≤ 0.05) found to be diminished in STZ-provoked diabetic rats (G-II) than the non-diabetic rats (G-I, G-III - G-V). The AuNPs treatment prevents the muscle loss by maintaining the insulin levels in normal.

Assessment of Body weight, Kidney and liver weight and Evaluation of food and water intake of control and experimental rats. Values are demonstrated as Mean ± SD by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. [A] Units = grams; [B] Units = Food intake = grams/rat/day and water intake = millilitre/rat/day.
Fig. 5 Assessment of Body weight, Kidney and liver weight and Evaluation of food and water intake of control and experimental rats. Values are demonstrated as Mean ± SD by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. [A] Units = grams; [B] Units = Food intake = grams/rat/day and water intake = millilitre/rat/day.

3.6

3.6 Evaluation of food and water intake

Fig. 5B represents food and water consumption of the experimental and normal rats. The consumption of food and water was increased in STZ-challenged diabetic rats (G-II) owing to exaggerated appetite (glucose loss via urine) and increased diuresis. The AuNPs (G-III and G-IV) and Glibenclaminde (G-V) treatment prevent the food intake and water consumption in experimental rats. The Control rats (G-I) food and water intake was not found to be significant when compared with (G-III – G-V). The 20 mg/kg AuNPs treatment showed more potential by preventing food and water intake.

3.7

3.7 Anti-diabetic effect of AuNPs on AST, ALT and ALP markers in serum

Fig. 6 shows the anti-diabetic effect of Fritillaria cirrhosa AuNPs on serum AST, ALT and ALP in experimental and control rats. The serum markers were increased in the STZ-provoked diabetic rats (G-II) when correlated to normal (G-I). The 10 mg/kg AuNPs treated rats (G-III) AST, ALT and ALP were noticeably declined to the normal range when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) AST, ALT and ALP were appreciably declined to the normal range when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) AST, ALT and ALP were significantly declined to the normal range when correlated to diabetic rats. The AuNPs treatment prevents the hepatic damage on STZ-challenged experimental diabetic rats.

The anti-diabetic effect of Fritillaria cirrhosa AuNPs on serum AST, ALT and ALP in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. Units = IU/L.
Fig. 6 The anti-diabetic effect of Fritillaria cirrhosa AuNPs on serum AST, ALT and ALP in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. Units = IU/L.

3.8

3.8 Anti-diabetic effect of AuNPs on protein level in serum

Fig. 7 depicts the anti-diabetic potential of Fritillaria cirrhosa AuNPs on protein level in experimental and control rats. The serum protein content was diminished in the STZ-provoked diabetic rats (G-II) when correlated to normal (G-I). The 10 mg/kg AuNPs treated rats (G-III) serum protein content was inclined when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) serum protein content was inclined when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) serum protein content was inclined when correlated to diabetic rats. The AuNPs treatment prevents the renal damage on STZ-challenged experimental diabetic rats.

The anti-diabetic effect of Fritillaria cirrhosa AuNPs on serum protein and serum creatinine in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method).). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. Units of protein = g/L, Units of creatinine = mg/dl.
Fig. 7 The anti-diabetic effect of Fritillaria cirrhosa AuNPs on serum protein and serum creatinine in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method).). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. Units of protein = g/L, Units of creatinine = mg/dl.

3.9

3.9 Anti-diabetic effect of AuNPs on creatinine level in serum

Fig. 7 represents the anti-diabetic effect of Fritillaria cirrhosa AuNPs on serum creatinine in experimental and control rats. The serum creatinine level was high in the STZ-stimulated diabetic rats (G-II) when correlated to normal (G-I). The 10 mg/kg AuNPs treated rats (G-III) serum creatinine level was significantly lowered when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) serum creatinine level was significantly lowered when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) serum creatinine level was significantly lowered when correlated to diabetic rats. The AuNPs treatment prevents the renal damage on STZ-stimulated experimental diabetic rats.

3.10

3.10 Anti-diabetic effect of AuNPs on blood glucose levels in experimental rats

Fig. 8A shows the anti-diabetic potential of Fritillaria cirrhosa AuNPs on blood glucose level in experimental and control rats. The blood glucose status was peaked in the STZ-challenged diabetic rats (G-II) when correlated to control (G-I). The 10 mg/kg AuNPs treated rats (G-III) blood glucose content was markedly lowered when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) blood glucose content was appreciably lowered when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) blood glucose content was significantly lowered when correlated to diabetic rats. The AuNPs treatment prevents the hyperglycemia on STZ-provoked experimental diabetic rats.

The anti-diabetic effect of Fritillaria cirrhosa AuNPs on blood glucose and plasma insulin level in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. [A] Units = mg/dl, [B] Units=µU/ml.
Fig. 8 The anti-diabetic effect of Fritillaria cirrhosa AuNPs on blood glucose and plasma insulin level in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. [A] Units = mg/dl, [B] Units=µU/ml.

3.11

3.11 Anti-diabetic effect of AuNPs on plasma insulin in experimental rats

Fig. 8B represents the anti-diabetic effect of Fritillaria cirrhosa AuNPs on plasma insulin in experimental and normal rats. The plasma insulin status was diminished in the STZ induced diabetic rats (G-II) when correlated to Control (G-I). The 10 mg/kg AuNPs treated rats (G-III) insulin content was moderately increased when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) insulin content was moderately increased when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) insulin content was moderately increased when correlated to diabetic rats. The AuNPs treatment prevents the pancreatic damage on STZ-stimulated experimental diabetic rats.

3.12

3.12 Anti-diabetic effect of AuNPs on glycosylated haemoglobin (HbA1c) in experimental rats

Fig. 9A reveals the anti-diabetic capacity of Fritillaria cirrhosa AuNPs on glycosylated haemoglobin in experimental and control rats. The glycosylated hemoglobin was high in the STZ-stimulated diabetic rats (G-II) when correlated to normal (G-I). The 10 mg/kg AuNPs treated rats (G-III) glycosylated haemoglobin was lowered when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) glycosylated haemoglobin was lowered when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) glycosylated haemoglobin was lowered when correlated to diabetic rats. The AuNPs treatment prevents the storage of glucose in hemoglobin on STZ-challenged experimental diabetic rats.

The anti-diabetic effect of Fritillaria cirrhosa AuNPs on glycosylated haemoglobin and hepatic enzymes in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. [A] Units expressed as %, [B] Hexokinase (Units) = µmol glucose phosphorylated/min/g protein). Glucose-6-phosphatase (Units) = µmol Pi liberated/min/mg protein. Fructose-1,6-bisphosphatase (Units) = µmol Pi liberated/min/mg protein).
Fig. 9 The anti-diabetic effect of Fritillaria cirrhosa AuNPs on glycosylated haemoglobin and hepatic enzymes in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and ** p ≤ 0.01 are statistically significant. [A] Units expressed as %, [B] Hexokinase (Units) = µmol glucose phosphorylated/min/g protein). Glucose-6-phosphatase (Units) = µmol Pi liberated/min/mg protein. Fructose-1,6-bisphosphatase (Units) = µmol Pi liberated/min/mg protein).

3.13

3.13 Anti-diabetic effect of AuNPs on hepatic enzymes in experimental rats

Fig. 9B exhibits the anti-diabetic potential of Fritillaria cirrhosa AuNPs on hepatic enzymes in experimental and control rats. The glucose-6-phosphatase, fructose 1,6-bisphosphatase activity was high and the hexokinase activity was lowered in the STZ induced diabetic rats (G-II) when correlated to Control (G-I). The 10 mg/kg AuNPs treated rats (G-III) glucose-6-phosphatase, fructose 1,6-bisphosphatase function was lowered and the hexokinase function was gradually increased when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) glucose-6-phosphatase, fructose 1,6-bisphosphatase function was lowered and the hexokinase function was gradually increased when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) glucose-6-phosphatase, fructose 1,6-bisphosphatase function was lowered and the hexokinase function was gradually increased when correlated to diabetic rats. The AuNPs treatment promotes glycolysis by the activation of hexokinase on STZ-provoked experimental diabetic rats.

3.14

3.14 Anti-diabetic effect of AuNPs on pancreatic TBARS and lipid hydroperoxide (HPX) in experimental rats

Fig. 10A represents the anti-diabetic effect of Fritillaria cirrhosa AuNPs on pancreatic TBARS and Lipid hydroperoxide in experimental and control rats. The TBARS and HPX were drastically raised in the STZ-stimulated diabetic rats (G-II) when correlated to normal (G-I). The 10 mg/kg AuNPs treated rats (G-III) TBARS and HPX were lowered when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) TBARS and HPX were lowered when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) TBARS and HPX were lowered when correlated to diabetic rats. The AuNPs treatment prevents lipid peroxidation on STZ-stimulated experimental diabetic rats.

The anti-diabetic effect of Fritillaria cirrhosaAuNPs on pancreatic TBARS and antioxidants in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and **p ≤ 0.01 are statistically significant. [A]TBARS (Units) = mmol/100 g tissue. Lipid hydroperoxides (Units) = mmol/100 g tissue. [B] SOD (Units) = µmole/min/mg protein. Catalase (Units) = mmole of H2O2 consumed/min/mg protein. GPx (Units) = mg of GSH consumed/min/mg protein. GSH (Units) = mg/100 mg tissue.
Fig. 10 The anti-diabetic effect of Fritillaria cirrhosaAuNPs on pancreatic TBARS and antioxidants in experimental and control rats. Values are demonstrated as Mean ± S.D by One way ANOVA (Duncan method). *p ≤ 0.05 and **p ≤ 0.01 are statistically significant. [A]TBARS (Units) = mmol/100 g tissue. Lipid hydroperoxides (Units) = mmol/100 g tissue. [B] SOD (Units) = µmole/min/mg protein. Catalase (Units) = mmole of H2O2 consumed/min/mg protein. GPx (Units) = mg of GSH consumed/min/mg protein. GSH (Units) = mg/100 mg tissue.

3.15

3.15 Anti-diabetic effect of AuNPs on pancreatic antioxidants level in experimental rats

The anti-diabetic potential of Fritillaria cirrhosa AuNPs on pancreatic antioxidants level in experimental and control rats were shown in Fig. 10B. The enzymatic functions of SOD, Catalase, GPx and GSH were lowered in the STZ-challenged diabetic rats (G-II) when correlated to normal (G-I). The 10 mg/kg AuNPs treated rats (G-III) antioxidants were upraised when correlated to diabetic rats. The 20 mg/kg AuNPs treated rats (G-IV) antioxidants were upraised when correlated to diabetic rats. The 0.1 mg/kg glibenclamide treated rats (G-V) antioxidants were upraised when correlated to diabetic rats. The AuNPs treatment prevents free radicals by modulating the antioxidants on STZ induced experimental diabetic rats.

3.16

3.16 Anti-diabetic effect of AuNPs on pathological alterations in pancreas of experimental rats

Fig. 11 depicts the anti-diabetic effect of Fritillaria cirrhosa AuNPs on pathological alterations in pancreas of experimental and control rats. Fig. 10(A) the pathology of pancreas was normal in control rats (G-1). Fig. 10(B) the islets cells of pancreas were severely damaged and the secondary granules were formed in β-cells of STZ-provoked diabetic rats (G-2). Fig. 10(C) the 10 mg/kg AuNPs treated rats (G-3) islets cells of pancreas were partially prevented from the degeneration. It also prevents the necrosis of β-cells in experimental rats. Fig. 10(D) the 20 mg/kg AuNPs treated rats (G-4) islets cells of pancreas was moderately prevented from the degeneration. It also prevents the necrosis of β-cells in experimental rats. Fig. 10(E) the 0.1 mg/kg glibenclamide treated rats (G-5) islets cells of pancreas were prevented from the degeneration. It also prevents the necrosis of β-cells in experimental rats. The AuNPs treatment prevents the pancreatic damage on STZ induced experimental diabetic rats.

The anti-diabetic effect of Fritillaria cirrhosa AuNPs on pathological alterations in pancreas of experimental and control rats. The AuNPs and Glibenclamide treatment activates islets cells of pancreas for insulin stimulation to normalize the glucose level in the blood.
Fig. 11 The anti-diabetic effect of Fritillaria cirrhosa AuNPs on pathological alterations in pancreas of experimental and control rats. The AuNPs and Glibenclamide treatment activates islets cells of pancreas for insulin stimulation to normalize the glucose level in the blood.

4

4 Discussion

Diabetes is rapidly securing the highest place among the public health issues in China with nearly 150 million peoples living with diabetes presently. A metabolic compromise in the insulin secretion and tissue insensitivity to insulin secreted in the body leads to the impaired regulation of glucose homeostasis. World Health Organization (WHO) has also mentioned exploring the medicinal plants that could facilitate the way for the development of new chemical entities (Patel et al., 2012). Preceding finding highlighted the existence of numerous active compounds in the Fritillaria cirrhosa like imperialine; verticinone, peimisine, verticine, etc., which may work as a perfect agent for the capping and reducing of metallic nanoparticles (Wang et al., 2016). The utilization of gold nanoparticles was gained much awareness in immense field especially in bio-medical area. They have reached the extensive usages in diagnostics, photo-thermal therapy, biolabeling, and drug carrier (Abadeer and Murphy, 2016; Pei et al., 2013). The present study showed the anti-diabetic potential effect of Fritillaria cirrhosa AuNPs on STZ-challenged diabetic rats. The I.P intervention of streptozotocin causes severe damage and degeneration to the islets cells and β-cells of pancreas. This directs to the development of diabetes in the rodents (Fu et al., 2010). In our study, all the experimental rats expect control were induced with STZ to develop the diabetic models. The bodyweight was deduced in STZ-stimulated diabetic rats owing to the muscle wasting and protein deterioration (Rajkumar and Govindarajulu, 1991). Oral administration of Fritillaria cirrhosa (10 and 20 mg/kg b.wt) and glibenclamide eliminates excess blood glucose in the experimental rats. AuNPs and glibenclamide treatment prevents the muscle wasting due to diabetes and improves the body weight of the animals. The higher intake of water and food was noted in STZ-provoked experimental diabetic animals (Sunmonu and Afolayan, 2013).

The supplementation of fabricated AuNPs and glibenclamide manage the water and food intake in the experimental animals. The organs like kidney and liver weight was diminished in the STZ-stimulated experimental diabetic rats. The AuNPs and glibenclamide treatment prevents the weight loss of liver and kidney in the investigational animals. The activity of alkaline phosphatase was augmented due to hepatic tissue injury in STZ-provoked experimental diabetic rats (Rai et al., 2010). The AST and ALT were increased in the blood stream due to hepatic injury in the STZ-stimulated diabetic rats (Maruthupandian et al., 2010). The AuNPs and glibenclamide treatment prevent the hepatic damage in the STZ-challenged experimental rats. So our outcomes concluded that the hepatic markers were found to be normal in the serum. The creatinine was elevated and protein level was diminished in the serum of STZ-challenged experimental diabetic rats. Usually creatinine level was upraised in poor renal function (Perez et al., 2006). The AuNPs and glibenclamide treatment prevent creatinine excretion in serum of STZ induced diabetic animals.

The glycosylated haemoglobin was drastically augmented in the diabetes rats owing to the presence of enormous glucose which reacts with haemoglobin in the blood (Jayaprasad et al., 2015; Sundaram et al., 2014). Our study proved that the glycosylated haemoglobin and blood glucose was upraised in the STZ-stimulated diabetic rats. The Fritillaria cirrhosa AuNPs and glibenclamide treatment decreased the glycosylated haemoglobin by lessening the blood glucose in the experimental rats. The insulin secreting β-cells was destroyed by the streptozotocin resulted diabetes in the experimental animals. The AuNPs and glibenclamide treatment stimulates the insulin production by regenerating the islets and β-cells of pancreas in the experimental animals. The histology study also confirms that the AuNPs and glibenclamide treatment regenerates the islets cells and β-cells of pancreas (Nurdiana et al., 2017). Hexokinase, an enzyme promotes glycolysis in the cell. The hexokinase activity was decreased in the diabetes owing to the deficiency of insulin excretion (Ghosh, 1984). Glucose-6-phosphatase and fructose-1, 6-bisphosphatase regulates gluconeogenesis pathway. These enzymes were found be increased in the diabetes (Baquer et al., 1998; Raju et al., 2001).

The hexokinase activity was decreased and the Glucose-6-phosphatase and fructose-1, 6-bisphosphatase was upraised in the STZ-stimulated diabetic rats. In our investigation, we proved that the AuNPs and Glibenclamide treatment increase insulin secretion which restores the hexokinase, Glucose-6-phosphatase and fructose-1, 6-bisphosphatase functions in the experimental animals. Previous data proved that the increased TBARS and lipidhydroperoxides were observed in the STZ-provoked experimental diabetic rats (Latha and Pari, 2003; Ananthan et al., 2004).

The AuNPs and glibenclamide treatment reduce the activity of TBARS and hydroperoxides by its antioxidant properties. The enzymatic functions of SOD, Catalase, GPx and GSH were diminished in the diabetic induced rats owing to raised oxidative stress. The antioxidants were deduced in the islets cells of pancreas due to the damage in the islets cells (Saravanan and Pari, 2005; Lei and Vatamaniuk, 2011; Gupta et al., 2012; Karunakaran and Park, 2013). The AuNPs and glibenclamide treatment restores the antioxidant enzymes by regenerating the islets cells of pancreas.

5

5 Conclusion

Oral administration of Fritillaria cirrohosa AuNPs possessed anti-diabetic capacity in STZ-challenged diabetic rats. The outcomes also proved the favourable benefits of this plant in promoting the insulin secretion during diabetes. It also prevents the hepatic, renal and pancreatic tissue damage in STZ-challenged rats. The serum, hepatic and renal markers were appreciably restored to normal by the Fritillaria cirrohosa AuNPs treatment. So we concluded that the AuNPs from F. cirrohosa has anti-diabetic effect in preclinical models. However, the extensive and molecular studies were needed in future to explore the precise therapeutic action of the AuNPs against the diabetes.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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