Biochemical Evaluation of Selenium Cleome Droserifolia Nanoparticles on

Diabetes Mellitus

Omayma A. R. Abou Zaid; Abdel Maksoud H. A.; Omnia M.A and Alaa. E. A.Department of Biochemistry, Faculty of Veterinary Medicine, Benha University, Egypt.

Abstract

Diabetes Mellitus (DM) is a group of metabolic disorders have great challenge in its treatment due to its pathological complication. In recent decade, there is extensive use of applying nanotechnology to medicinal plants as a trend in diabetes treatment because of phytochemicals constituents. The present study aimed to evaluate the hypoglycemic effect of Selenium Cleome droserifolia nanoparticles (Se-CNPs) and/or Galvus met® treatment on streptozotocin induced diabetes mellitus in male rats. Fifty male rats were divided into 2 groups: group (A) consists of 10 rats and act as normal control. Group (B): consists of 40 rats. Rats were injected with single dose of STZ (50 mg/kg b.wt.) intraperitoneally (I/P) for induction of type-2 diabetes mellitus. And after 48 hrs rats divided into 5 subgroups. Subgroup (1) (diabetic control) diabetic rats not treated. Sub group (2) Diabetic rats treated daily with Se-CNPs serum at a dose (3.5mg/kg b.wt./orally/ 45 days). Subgroup (3) Diabetic group treated with Galvus met® at a dose (5 mg/100 mg/kg of rats orally/45 days). Subgroup (4) Diabetic rats treated with Se-CNPs and Galvus met® at doses described above for each one. Glucose and insulin levels, Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), Total Cholesterol (TC), Triacylglycerol (TG), High Density Lipoprotein (HDL-c), Very Low Density Lipoprotein cholesterol (VLDL-c), Low Density Lipoprotein cholesterol (LDL-c) and (NEFAs), urea and creatinine were evaluated. Moreover, histopathological changes in pancreatic tissue were also examined. The results showed significant elevation in serum glucose concentration, ALT and AST activities, TG, LDL-c, VLDL-c and Non Estratified Fatty Acids (NEFAs), urea and creatinine levels while a significant decrease in serum insulin and HDL-c concentration in diabetic rats when compared with control. On other hand daily administration of Se-CNPs and/or Galvus met® to diabetic rats showed significant amelioration of these parameters.

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Keywords: Diabetes Mellitus, Selenium Cleome droserifolia nanoparticles, Galvus met®, Antidiabetic, insulin, lipid profile1- Introduction

Diabetes mellitus (DM) is highly recognized as the most common metabolic

and endocrine disorder worldwide. It is linked to disturbances in carbohydrate,

protein, and fat metabolism (Moore et al., 2004). Just fewer than half a billion

people are living with diabetes worldwide and the number is projected to

increase by 25% in 2030 and 51% in 2045. The prevalence is higher in

urban than rural areas, and in high-income than low- income countries

(Saeedi et al., 2019).Diabetes requires continuous medical care to reduce risk of

hyperglycemia, preventing acute complications and reducing the risk of long-term

complications (American Diabetes Association, 2020).

In recent years, nanotechnology and smart nano structures have shown

considerable potential for a large number of biomedical applications like monitoring,

diagnosis, repair, and treatment of human biological systems. Nowadays, several

nanoparticles used as alternative therapy due to their multifunctional biological

activities, which treat diabetic complications and combat inflammation (Abdulmalek

and Balbaa, 2019). The management of diabetic conditions by insulin therapy has

several drawbacks like insulin resistance. Also, chronic treatment causes anorexia

nervosa, brain atrophy and fatty liver. Several research studies are currently on going

with the aid of nano size particles to overcome such limitations in diabetes

management (Rahiman and Tantry, 2012).

The chief advantages of nanoparticles are improved bioavailability by

enhancing aqueous solubility, increasing residence time in the body and targeting

drug to specific location in the body lead to concomitant reduction in the amount of

the drug required and dosage toxicity, thus allowing safe delivery of toxic therapeutic

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drugs and the protection of non- target tissues and cells from severe side effects

(Neuberger et al., 2005).

Green synthesis using medicinal plant extract has obtained specific importance

in the formation of nanoparticles especially, phytochemicals which are backbone of

plants and easily produce nanoparticles with less toxicity (Bashir et al., 2013). All of

the plants which used for nanoparticles synthesis are known to be rich in polyols and

antioxidant. Also, Hydroxyl and carboxylic groups present may act as reducing agent

and stabilizing agents in the synthesis of nanoparticles (Vilchis-Nestor et al., 2008).

The synthesis of metallic nanoparticles can be done by reducing metal ion

using some chemical molecules. Most of the plants contain an ample of free radical

scavenging molecules such as phenolic compounds, nitrogen compounds, vitamins,

reducing sugar, terpenoids and some other metabolites that are rich in antioxidant

activity (Salam et al., 2012).

Metallic nanoparticles are inert in the body, but are able to selectively deliver

their targeted cells when their surfaces are functionalized with antibodies specifically

recognizing membrane proteins of the diseased cell. In addition, with the proper

targeting moieties, metallic nanoparticles might even be able cross Blood Brain

Barrier (Abou Zaid- Omayma et al., 2015).

Selenium has a narrow therapeutic window and the toxicity margins are very

delicate whereas the Se nanoparticles (Se-NPs) possess remarkably reduced toxicity.

SeNPs have been explored in various oxidative stress and inflammation mediated

disorders like arthritis, cancer, diabetes and nephropathy with potential therapeutic

benefits. SeNPs constitute an attractive carrier platform to ferry various drugs to the

site of action (Khurana et al., 2019).

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Globally, there is renewed interest toward natural products (NPs) as a

starting point for drug discovery and development for different diseases (Ríos et

al., 2015). Medicinal plants are used as a medical resource in almost all cultures

because of ensuring the safety, quality and effectiveness in addition, herbal drugs

very recently became a key issue in industrialized and developing countries

(Jamshidi-Kia et al., 2018). Many substances extracted from plants are used to

treat diabetes, including flavonoids, terpenes, alkaloids, phenolic, and phenolic

coumarins compounds which are useful for the development of new drugs or the

treatment of diseases such as diabetes and its complications (Lamba et al.,

2000).

In Egypt, Medicinal plants have been a part of the country’s natural and

cultural heritage for thousands of years. However, many species are threatened due to

human impacts, loss of natural habitat or overexploitation (Abdel Wahab et al.,

2004; Ramadan et al., 2009; Moustafa et al., 2019).

Cleome droserifolia is a species of striking medicinal value. Considerable

research efforts confirmed its utility as a hypoglycemic herb (Shtaiwi et al.,

2013).Cleome droserifolia (Forssk) Del. (syn. Roridula droserifolia Forssk.), locally

known as “samwah”, is an aromatic flowering shrub of the genus Cleome with ~ 200

species, belonging to the family Cleomaceae (Kindt, 2020). It is distributed

regionally in Egypt, Libya, Syria, Jordan and Palestine. In Egypt it appears in South

Sinai, Red sea coast, the Oasis and Mediterranean coast. It is used by herbalists in

Egypt as a hypoglycemic agent, plus it’s widely used by the Bedouins of the southern

Sinai for treating diabetes (Mostafa et al., 2019).

Also, it exhibited different biological activities as anticancer, anti-schistomiasis,

antibacterial, antidiarrheal, analgesic, anti-inflammatory and antimalarial effect

(Abdullah et al., 2016).

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Phytochemical screening of Cleome droserifolia indicated the presence of

volatile oil, which consists of 3- butenylisothiocyanate, 2-methyl

butenylisothiocyanate, α, β, and γ-caryophyllene and 2-naphthyl-npropyl ether

(Boulos, 2000; Mirza et al., 2005). Essential oils and fatty acids isolated from C.

droserifolia were 3,7,11-trimethyldodeca-1,6,10-triene (11.8%), carotol (10.1%), δ-

cadinene (8.9%), β-eudesmol (7.0%) and benzyl isothiocyanate (5.9%) (Muhaidat et

al., 2015; Abdullah et al., 2016).

In addition, alkaloids, tannins, saponins, coumarins, ocosaniocacid, catechins,

amino acids, hydrocarbons, sterols such as (β-sitosterol and stigmasterol),

glucosinolates with sulfur aglycones such as glucocapparin, sesquiterpenes like

(carotol and dihydrodihydroxycarotol), methoxylated flavones, four flavone

aglycones (5-hydroxy3,6,7,3´,4´,5´- hexamethoxyflavone, chrysosplenetin, 5, 3´-

dihydroxy-3,6,7,4´,5´pentamethoxyflavone and penduletin) and four flavone

glycosides (kaempferitin, kaempferol-7-O-rhamnoside, kaempferol-3O-glucoside-7-

O-rhamnoside, and isorhamnetin-3- O-glucoside-7-Orhamnoside) (Diab, 1992;

Hussein et al., 1994; El-Askary, 2005).

The vildagliptin-metformin single-tablet combination is the first DPP-4

inhibitor-metformin combination to reach the market and is indicated for the

treatment of patients with type 2 diabetes who are unable to achieve sufficient

glycemic control at their maximally tolerated dose of oral metformin alone, or who

are already treated with the combination of vildagliptin and metformin as separate

tablets (Tahrani et al., 2009). This combination has demonstrated favorable effects

on pancreatic α- and β-cells. Whether, the effect on parameters of β-cell function will

translate in long term ß-cell preservation, which may modify the course of the

disease, remains to be shown by long-term clinical studies (Halimi et al., 2008).

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In this study we developed Selenium cleome droserifolia nanoparticles (Se-

CNPs) for oral administration is an attempt to potentiate selenium nanoparticles and

cleome droserifolia extract nanoparticles hypoglycemic effect and compare it with

Galvus met® an oral hypoglycemic drug. The oral hypoglycemic effect and

bioavailability of Se-CNPs were investigated in normal and diabetic STZ induced

rats.

2- Material and Methods1- Animals:

Fifty male albino rats, (6-8) weeks old, with average body weight 150-180 g

are used in the experimental investigation of this study, and purchased from "The

Laboratory Animals Research Center", Faculty of Veterinary Medicine, Benha

University. Rats were housed in separate wire mesh cages, exposed to good

ventilation, humidity and to a 12-hr light/dark cycle, and provided with a constant

supply of standard pellet diet and plenty of fresh, clean drinking water ad-

libitum.Animals were kept at constant environmental and nutritional conditions

throughout the period of the experiment and left for 15 days adaptation period prior

to the inception of experiment throughout the course of the experiment in the form of

concentrated diet.

2- Chemicals and Drugs:The chemicals and drugs in the present study were:

A- Streptozotocin:

Manufactured in Sigma Chemical Co. (St. Louis, Mo, USA) and purchased

from Schnelldorf, Germany through the Egyptian International Center for Import

Cairo, Egypt.

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Dosage: STZ was freshly dissolved in citrate buffer and injected to rats as a

single dose (50mg/kg b.wt I/P) for induction of type-2 diabetes mellitus (Ramanthan

et al., 1999).

B- Galvus Met®: (50/1000 mg Tablet) (Vildagliptin/ Metformin HCL) Manufactured by Novartis Singapore Pharmaceutical Manufacturing Pte. Ltd., Tuas Bay Lane, Singaphore for Novartis Pharma AG, Basal, Switzerland.

Dosage : Galvus Met was freshly dissolved in saline solution and administered

at a dose of (5mg/100mg/kg b.wt/ day/45 days) orally (Paget and Barnes, 1964).

3- Plant material:Plant (samwah) obtained dried from Haraz herbal store in Cairo. By following

data they obtained it from south Sinai, Egypt.

4- Preparation of plant extract and Synthesis of Selenium-Cleome Nanoparticles (Se-CNPs):

For preparation of the plant extract, dried plant (50 g) were grounded to

powder and soaked overnight with 200 ml ethanol. Then, the system was refluxed for

2 hrs. The obtained extract was filtered three times and then the solution was

lyophilized by vacuum freeze dryer for further use. For the synthesis of plant extract

mediated SeNPs, 0.01 M sodium oxide was mixed with 2 mg/ml plant extract under

magnetic stirring for 12 h and 40 °C. The obtained solution of SeNPs was dialyzed in

ultra-pure water for 48 hrs to remove the excess sodium selenite. The SeNPs were

characterized by using spectroscopic and microscopic methods.

5- Characterization of the Se-CNPs:Nanoparticles of Se-CNPs were characterized by Ultraviolet

spectrophotometer to confirm sample formation by showing the plasmon resonance,

Dynamic light scattering (DLS) to show size distribution by Zetasizer (ZS) (Malvern,

United Kingdom),Transmission Electron Microscopy (TEM)to analyze size, nature

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and morphology of obtained nanoparticles using a high resolution transmission

electron microscope (HR-TEM; JEOL, JEM2100, Electron Microscope, Japan) and

Fourier transforms infrared spectroscopy (FT-IR)analyzed for functional

groupsusing JASCO FTIR 6300 spectrophotometer, at resolution of 4 cm-1.

6- Determination of the Median Lethal Dose (LD50) of Se-CNPs :LD50 was calculated according to the method described by (Chinedu et al., 2013)

7- Experimental design:

Fifty male rats were divided into 2 groups: Group (A): consists of 10 rats and act

as normal control. Group (B): consists of 40 rats. Rats were injected with single

dose of STZ (50 mg/kg b.wt.) intraperitoneally (I/P) for induction of type 2

diabetes. And after 48 hrs rats divided into four sub groups as following:

Sub group (1): (Diabetic control) diabetic not treated (10 rats).

Sub group (2): Diabetic treated with Se-CNPs at a dose (3.5mg/kg b.wt. /orally/

45 days), given as 1 ml by intra-gastric tube daily (10 rats).

Sub group (3): Diabetic treated with Galvus Met at a dose of (5 mg/100 mg/kg of

rats orally/ 45 days), given as 1 ml by intra-gastric tube daily (10 rats).

Sub group (4): Diabetic treated with Se-CNPs + Galvus Met at a dose of (3.5

mg/kg b.wt. /orally/ 45 days) and (5 mg/100 mg/kg of rats orally/ 45 days),

respectively (10 rats).

During the experimental period, the dosage was adjusted every week according

to any change in body weight to maintain similar dose per kg body weight of rat as

(3.5 mg/kg b.wt.) for Se-CNPs and (5/100 mg/kg b.wt.) for Galvus met® over the

entire period of study for each group.

Sampling:

8.1. Blood Samples:

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Blood samples were collected after overnight fasting from retro-orbital plexus

of rats at the end of experiment (after 45 days). Blood samples were collected in dry,

clean plain test tubes and incubated for 1/2 hr at room temperature to allow clotting

for serum separation.Clear sera were separated by centrifugation at 3500 r.p.m. for 15

minutes, then serum collected in Eppendorf's tubes using automatic micropipettes

glucose and lipid profile were estimated immediately. The rest of serums were kept in

a deep freeze at -20 ºC until used for estimation of other biochemical parameters.

Glucose concentration (Trinder, 1969) , insulin concentration Frosig et al.,

(2007); Liver transaminases: ALT and AST activities (Murray, 1984a) ,Total

Cholesterol (TC) (Searcy, 1969), Triacylglycerols (Stein, 1987), High density

lipoprotein-cholesterol (HDL-c) (Burstein et al., 1970), Very low density

lipoprotein-cholesterol (VLDL-c) (Friedewald et al., 1972), Low density

lipoprotein-cholesterol (LDL-c) (Bauer, 1982), Non-Esterified Fatty Acids (NEFA)

(Bakker and Mücke, 2007); Urea (Kaplan, 1984) and Creatinine concentration

(Schirmeister et al., 1984).

8.2. Tissue Samples:

Pancreatic tissue for Histopathological examination: For histopathological

examination, pancreas specimens were collected, and preserved in 10% buffered

neutral formalin and subjected for microscopical examination according to the

technique described by Bancroft and Stevens (1996).

9. Statistical analysis:

The Statistical analysis was carried out using ANOVA with one way under

significance level of 0.05 for the whole results using SPSS (ver. 22). Data were

treated as complete randomization design according to (Steel et al., 1997). Multiple

comparisons were carried out applying LSD.

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3- Result 1. Characterization of Se-CNPS :Biologically synthesized Se-NPs

nanoparticles were characterized by:1.1UV–Visible absorption (UV/VIS) spectroscopy:

Se-CNPs sample was scanned using JASCO Spectrophotometer; model V-750,

Serial No. A009261799, and there was one peak at maximum absorbance 3.68276

obtained at wavelength 211.5 nm as shown in figure (1).

1.2 Transmission electron microscopy (TEM) analysis:

Transmission electron microscopy photos were used to investigate the surface

morphology, size and shape in Se-CNPs. As shown in this image, the observed

morphology of Se-CNPs showed almost spherical shape with different sizes in nano

scale (50-190 nm), Figure (2)

1.3. Dynamic Light Scattering (DLS):

DLS measurement of the hydrodynamic effective diameter of the

biosynthesized Se-CNPs showed that the zeta average diameter (d.nm) was 166.9

with distribution intensity of 17.3 for 50.73 nm, and 25.1 % for 55.78 nm, 22% for

68.06 nm, of 5.1 for 43.82 nm and 14.5% for 78.82 nm. The poly-dispersity index

(PDI) was 0.392 indicating the homogeneity and uniform dispersion of the

synthesized Se-CNPs Figure (3).

1. 4. Fourier Transforms Infrared Spectroscopy of Se-CNPs (FTIR):

The FT-IR signals recorded for Se-NPs biosynthesized by Cleome droserifolia

extract indicated the presence of the broad peaks at 3418.94 , 2122.98 , 1645.55 ,

1219.59cm-1 represents the presence of functional groups such as alcohols, phenols

(O–H stretch, H–bonded), carboxylic acids (O–H stretch), aromatics (C–C stretch

(in–ring), aliphatic amines (C–N stretch) and alcohols, carboxylic acids, esters,

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ethers(C–O stretch). These results confirmed the presence of bioactive molecules and

functional groups in Se-CNPs preparations (Figure 4).

Ultraviolet-Visible Spectrum

3.68276

0

0.5

1

1.5

2

2.5

3

3.5

4

0 100 200 300 400 500 600 700 800 900

Wave length (nm)

Ab

sorb

ance

Max. abs. = 3.68276

Figure (1): UV–Vis spectra of Se-CNPs aqueous solution.

Figure (2): Representative TEM images of biologically synthesized Se-BNPs

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Figure (3): DLS intensity-based size distribution histograms of Se- CNPs

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

2.0

5

10

15

20

25

30

35

40

45

50.0

cm-1

%T

3418.94

3005.53

2917.84

2122.98

1645.55

1436.31

1407.00

1316.81

1219.591121.27

1019.77

952.86

902.29

708.08

674.94

Figure (4): FTIR analysis for Se-CNPs

2. Serum Biochemical Parameters:The obtained data in table (1&2)revealed that injection of STZ to normal rats

exhibited a significant increase in serum glucose, total cholesterol, triacylglycerols,

LDL-c, VLDL-c ,NEFA, urea and creatinine concentrations in addition significant

increase in ALT and AST activities, while serum insulin and HDL-c concentrations

showed a significant decrease when compared with control.

Diabetic rats administrated Se-CNPs and/or treated with Galvus Met®

exhibited a significant decrease in serum glucose concentration, total cholesterol,

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triacylglycerols, LDL, VLDL, NEFA, urea and creatinine concentrations, in addition

to decrease in ALT and AST activities. On other hand, a significant increase in serum

insulin and HDL concentrations were observed when compared with diabetic control

group.

3. Histopathological findings:Normal histological structure is seen in the pancreatic islet of the control group

Figure (A). In untreated diabetic rats, there were sever degenerative changes in the

islets of Langerhans with mild mononuclear leukocytic infiltration particularly

mononuclear types. Moreover, complete cirrhosis of the islets of langerhans was

also detected in some examined rats in this group Figure (B). While, in Diabetic

group treated with Se-CNPs extract the pancreas of rats in this group showing mild

changes in the islets of Langerhans which represented by shrinkage in the size.

Moreover, mild changes in β-cells with few mononuclear leukocytic infiltrations

were also observed Figure (C).Diabetic group treated with Galvus met® and

diabetic group treated with mix between Galvus met® + Se-CNPs extract the

examination of the pancreas in those groups showing clear regeneration or nearly

normal histological structure of the islets of Langerhans which showed vesicular

nuclei and abundant cytoplasm. Moreover, mild mononuclear leukocytic

infiltrations were also seen Figure (D) and mild atrophy of the islets of

Langerhans was detected in some examined rats Figure (E).

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Figure (A):H&E X 400 Figure (B):H&E X 200

Figure (C): H&E X 200Figure (D): H&E X 200

Figure (E): H&E X 200

4- Discussion:Diabetes mellitus is becomes the ninth major cause of death all over the world

(Zheng et al., 2018).

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This study provides new insight into the use of integrative nanomedicine

containing phytomedicines (cleome droserifolia) and selenium for D.M treatment in

comparison with chemical drug Galvus met® which is an oral hypoglycemic agent.

Our results showed a significant increase in serum glucose concentration;

and significant decrease in serum insulin concentration in STZ- induced diabetic

rats when compared with control group. This result may be due to that STZ

generate reactive oxygen species (ROS), which contributed to DNA fragmentation

and evoked other deleterious changes within the pancreatic tissue as STZ is a

structural analogue of glucose (Lin et al., 2019).

Administration of Se-CNPs and/or Galvus Met® to STZ-induced diabetic rats

significantly adverse the previous results showed in diabetic group. The

hypoglycemic and hyperinsulinemic action of the studied Se-CNPs might be

attributed to presence of certain compounds as flavonoids which have insulin mimetic

functions, so it is reduced blood glucose in animals. In addition, Flavonoid extracts

can suppress a-glucosidase activity and may inhibit the non-Na +dependent smooth

diffusion of monosaccharides in epithelial cells of intestine (Andrade-Cetto et al.,

2008; Helal et al., 2015).

Treatment of diabetic rats with Galvus met® resulted in hypoglycemic and

hyperinsulinemic action. These may be due to that Galvus met is combination of

Vildagliptin with metformin; they caused improving in efficacy over time due to

GLP-1 induced increase in beta cell numbers and mass without weight gain and

hypoglycemia which are the common side effects with other antidiabetic drugs

(Gullapalli and Desai, 2018).

The present study showed a significant elevation in serum activities of AST

and ALT enzymes in diabetic rats when compared with the control group. This is

may be due to hyperglycemia-induced oxidative stress and subsequent disturbance in

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carbohydrate, protein and lipid metabolisms are the most important causes of liver

damage in diabetic patients (Mohamed et al., 2016). Thus, increased

gluconeogenesis and ketogenesis were observed (Maiti et al., 2004).

However, daily administration of Se-CNPs and/or Galvus Met to diabetic rats

exhibited a significant decrease in serum ALT and AST activities. This result may be

attributed to flavonoids content of Se-CNPs which have long been recognized to

possess antioxidant, hepatoprotective and anti-inflammatory activities. They can

exert their antioxidant activity by various mechanisms, e.g., by scavenging free

radicals, by chelating metal ions, or by inhibiting enzymatic systems responsible for

free radical generation. So, they can ameliorate the functions of the liver by inhibition

the pro-inflammatory mediators and protection of hepatocytes (Sharaf et al., 1997;

Yi et al., 2013).

However , Galvus met® exhibit hepatoprotective through DPP-4 inhibitors can

improve hepatic steatosis in mice and humans and ameliorate changes in liver

enzymes in patients with nonalcoholic steatohepatitis (Shirakawa et al., 2011; Itou

et al., 2012; Yilmaz et al., 2012). Together, these findings support the present results

of vildagliptin on liver enzymes. Metformin prevented liver damage due to functions

of liver may be affected by the changes in the levels of insulin, which provides rapid

uptake, storage as glycogen and usage of glucose, especially in liver (Yanardag et

al., 2005).

Moreover diabetic rats showed significant increase in serum total cholesterol,

triacylglycerols, LDL, VLDL, and NEFA concentrations; and significant decrease in

serum HDL in relative to the corresponding controls. Change in lipid metabolism

associated with diabetes mellitus is attributed to increased flux of free fatty acids into

the liver secondary to insulin deficiency/ resistance. This results lead to excess fatty

acid accumulation in the liver, which is converted to triglycerides. The impaired

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ability of insulin to inhibit free fatty-acid release leads to elevated hepatic VLDL-

cholesterol production. The increased VLDL-cholesterol and triglyceride levels

decrease the level of HDL-cholesterol and increase the concentration of small dense

LDL-cholesterol particles by activation of lipoprotein lipase and lecithin acyl-

cholesterol transferase (Nagy and Mohamed, 2014).

While, treatment of diabetic rats with Se-CNPs and/or Galvus Met

significantly improve this parameters. This may attributed to Flavonoids inSe-CNPs

inhibit the activity of cAMP - dependent protein phosphokinase; the consequence is

that the cAMP concentration increases and that phosphorylation of the Hydroxy

methyl glutaryl-CoA reductase, but endogenous cholesterol production is diminished.

In addition, the flavonoids can interact with the enzyme protein phosphatase, which

liberates the aliphatic phosphor esters from Hydroxy methyl glutaryl-CoA-CoA

HMG-CoA reductase, thus restoring the activity of this (Shabrova et al., 2011).

Galvus met improve lipid profile parameters due to its active constituents of

vildagliptin and metformin. vildagliptin can exert several possible mechanisms of

actions to improve lipid profile. Most simply, the influence of vildagliptin on

postprandial lipaemia could reflect incretin-mediated changes in pancreatic hormone

secretion, an improved metabolic state and reduced insulin resistance secondary to

improved islet function, incretin-mediated effects to slow gastric emptying, or direct

effects of GLP-1 and/or GIP on lipoprotein metabolism (Matikainen et al., 2006).

Moreover, metformin may decrease TG and TC levels through action of HDL-c that

could raise the efflux of TG and TC to liver tissue for catabolism (Jiang et al., 2015).

It reduces ectopic lipid depots (i.e. liver and skeletal muscle) through increased fat

oxidation and decreased lipid synthesis (Malin and Kashyap, 2014).

The current study revealed significant elevation in serum levels of urea and

creatinine concentrations along the period of the experiment in diabetic group when

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compared with normal control group. This may attributed to STZ injection in rats

causes inflammatory cell infiltration within the pancreas followed by the onset of

insulin deficiency, and activates protein kinase-C, poly (ADP-ribose) polymerase and

NAD(P)H oxidase, with consequent generation of ROS and advanced glycation end

products resulting in renal damage and nephropathy (Szabo, 2005; Arora et al.,

2009).

Moreover, administration of Se-CNPs and/or Galvus Met® to diabetic rats

exhibited a significant decrease in serum urea and creatinine concentrations when

compared with diabetic non-treated group. this improvement in kidney functions in

Se-CNPs treated diabetic group may be due to the presence of flavonoids, which

lowered creatinine, urea concentrations as they possess free radical scavenging

properties(Van Hoorn et al., 2006).While, Galvus Met®decrease serum urea and

creatinine in diabetic rats may be attributed to that, vildagliptin effectively inhibited

early pathologic changes of diabetic kidney disease, such as glomerular basement

membrane (GBM) thickening, glomerulosclerosis, and interstitial expansion (Liu et

al., 2012). Nephron protection that offered by DPP-4 inhibitors include reduction in

oxidative stress and inflammation and improvement of endothelial function (Haluzık

et al., 2013). In addition, metformin treatment ameliorated the majority of these

kidney dysfunctions, indicating the protective effects of metformin administration in

rats with T2DN. The underlying mechanism of renoprotective effects of metformin

against the development and progression of T2DN may be associated with glycemic

control, lipid metabolism, and anti-oxidative and anti-inflammatory functions (Zhang

et al., 2017). 

In this study our histopathological examination of pancreas confirmed the

biochemical results.

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5- Conclusion & RecommendationsThe findings of the present research demonstrated that Se-CNPs and/or Galvus

Met administration to STZ-induced diabetic rats provided an effective treatment

against hyperglycemia, hyperlipidemia, and hepato-renal dysfunction; since these

compounds were able to ameliorate the related serum biochemical parameters, as

they provide a novel mechanism of action for treating patients with type 2 diabetes

mellitus and may be useful either as monotherapy or in combination with other

antidiabetic agents. They are well tolerated, effectively improve glycemic control,

preserve β-cell function, and reduce lipotoxicity and insulin resistance.

So we recommend administration of Galvus Met and/or Se-CNPs for treatment

of type-II diabetes mellitus. Also, we strongly support the extensive use of nano-

technology in pharmacological industry for production of new drugs used as

therapeutics for treatment of diabetes mellitus.

6. References

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Abdullah, W., Elsayed, W. M., Abdelshafeek, K. A., Nazif, N. M. and Singab, A. N. B. (2016): Chemical constituents and biological activities of Cleome genus: A brief review. Int. J. Pharmacogn. Phytochem. Res, 8, 777-787.

Abdulmalek, S. A. and Balbaa, M. (2019): Synergistic effect of nano-selenium and metformin on type 2 diabetic rat model: Diabetic complications alleviation through insulin sensitivity, oxidative mediators and inflammatory markers. PloS one, 14(8).

Abou Zaid- Omayma. A. R., Abdel-maksoud, A.H., Elsenosy, Y.A. and Omnia, I .A. (2015): Biochemical study on the effect of cetyl trimethyl ammonium bromide surfactant coated spirulina zinc oxide nanocomposite as antimammary carcinogenesis in rats. Benha Veterinary Medical Journal, 29(1), 170-182.

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Table (1): Effect of Se-CNPs and/or Galvus Met administration on some blood parameters in diabetes mellitus induced experimentally in rats.

Parameters

Animal groups

Glucose(mg/dl)

Insulin(μIU/ml)

ALT(U/L)

AST(U/L)

Urea(mg/dl)

Creatinine(mg/dl)

Control normal 82.44 ± 3.91d 3.06 ± 0.12a 52.18 ± 3.47c 81.97 ± 3.03c 23.20 ± 1.37d 0.76 ± 0.02d

Diabetic control 401.66 ± 35.99a 0.81 ± 0.08d 96.77 ± 3.86a 155.84 ± 3.92a 45.45 ± 2.15a 1.47 ± 0.05a

Diabetic Se-CNPs-treated

198.27 ± 8.43b 1.35 ± 0.07c 73.09 ± 3.56b 126.62 ± 2.91b 35.98 ± 2.26b 1.17 ± 0.07b

Diabetic Galvus-treated

113.42 ± 5.31cd 2.42 ± 0.09ab 61.70 ±3.49bc 101.89 ± 2.48bc 26.68 ± 1.64c 0.86 ± 0.03c

Diabetic Se-CNPs + Galvus treated

154.80 ± 6.92bc 1.89 ± 0.06b 68.84 ± 3.72b 115.46 ± 3.72b 30.21 ± 1.97bc 0.99 ± 0.04bc

Data are presented as (Mean ± S.E). S.E = Standard error.Mean values with different superscript letters a, b, c &d in the same column are significantly different at (P>0.05).

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Table (2): Effect of Se-CNPs and/or Galvus Met administration on lipid profile in

diabetes mellitus induced experimentally in rats.Parameter

Animal groups

T. Ch.

(mg/dl)

TGs

(mg/dl)

HDL

(mg/dl)

VLDL

(mg/dl)

LDL

(mg/dl)

NEFA

(mg/dl)

Control normal 81.04 ± 2.64d 56.30 ± 2.79d 24.04 ± 0.84a 11.26 ± 0.56d 45.74 ± 2.39d 12.45 ± 0.67d

Diabetic control 165.75 ± 4.11a 159.80 ± 4.31a 11.87 ± 0.92c 31.96 ± 1.07a 121.92 ± 4.61a 40.80 ± 2.06a

Diabetic Se-CNPs-

treated143.97 ± 4.91b 121.37 ± 3.98b 15.95 ± 0.95bc 24.27 ± 0.94b 103.75 ± 4.27b 29.61 ± 1.80b

Diabetic Galvus-treated 87.86 ± 2.71d 68.41 ± 2.71d 21.98 ± 0.81a 13.68 ± 0.59d 52.20 ± 2.46d 18.39 ± 1.39c

Diabetic Se-CNPs +

Galvus treated117.88 ± 2.62c 102.82 ± 2.29c 18.13 ± 0.62b 20.56 ± 0.68c 79.19 ± 1.39c 23.06 ± 1.85bc

Data are presented as (Mean ± S.E). S.E = Standard error.Mean values with different superscript letters a, b, c &d in the same column are significantly different at (P>0.05).

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