# Patent

## Title
Biomimetic synthesis of antihyperglycemic silver nanoparticles

## Abstract

A biomimetic synthesis of antihyperglycemic nanoparticles using silver nitrate and Lavatera cretica is a method for the green synthesis of silver nanoparticles. These nanoparticles may be produced by extraction of fresh L. cretica leaves and mixing and incubation of the resulting L. cretica extract with silver nitrate to produce a nanoparticle composition including the silver nanoparticles. The nanoparticle composition may protect against hyperglycemia.

## Background
BACKGROUND
1. Field
The disclosure of the present patent application relates to nanotechnology, and particularly to a biomimetic synthesis of nanoparticles using silver nitrate and Lavatera cretica: 
2. Description of the Related Art
Recently, metal nanoparticles have demonstrated important uses in a variety of fields. In particular, silver nitrate derived nanoparticles have been of interest to researchers, due to their wide range of applications including electronics, biosensing, plasmonics, optics, and medicine.
Synthesis of silver nanoparticles (AgNPs) have been achieved by a variety of methods, including physicochemical, thermal decomposition, electrochemical, microwave assisted, sonochemical, solvothermal, photosynthesis, photochemical reduction, chemical reduction and continuous-flow methods. These methods are often costly or produce byproducts that pose increased risks to human health and the environment.
Thus, an affordable method of biologically synthesizing silver nanoparticles solving the aforementioned problems is desired.
SUMMARY
The biomimetic synthesis of antihyperglycemic AgNPs using silver nitrate and Lavatera cretica includes providing a solution including silver nitrate; providing an extract of the Lavatera cretica plant or plant part; mixing the silver nitrate solution and the extract solution to form an aqueous mixture; and resting the aqueous mixture for a period of time to form L. cretica AgNPs. The AgNPs may be used for their protective effect against hyperglycemia. The synthesis method is inexpensive and environmentally friendly.
These and other features of the present disclosure will become readily apparent upon further review of the following specification and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the UV-Visible spectrum of L. cretica AgNPs after about 24 hours and about 48 hours of incubation.
FIG. 2 is a Transmission Electron Microscopy scan of the surface morphology of the L. cretica AgNPs.
FIG. 3 is an Energy Dispersive X-Ray Diffraction spectrum of L. cretica AgNPs, illustrating the additional organic components present.
FIG. 4 is a Fourier Transform Infrared Spectroscopy spectrum of the surface functional groups of the L. cretica AgNPs.
FIG. 5 is an X-Ray Diffraction pattern of the L. cretica AgNPs.
FIG. 6 is a graph of glucose tolerance testing in normal and diabetic control mice.
FIG. 7 is a graph of blood glucose over time in diabetic control and diabetic mice treated with different concentrations of L. cretica AgNPs.
FIG. 8 is a graph of plasma glucose and plasma insulin in control and diabetic mice either treated or untreated with L. cretica AgNPs over a period of fifteen days.



Similar reference characters denote corresponding features consistently throughout the attached drawings.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Biomimetic synthesis of antihyperglycemic nanoparticles can include mixing silver nitrate with an extract of Lavatera cretica (L. cretica) to provide silver nanoparticles (AgNPs). The extract can be an aqueous leaf extract of L. cretica. The nanoparticles can be spherical in shape with sizes ranging from about 5 nm to about 24 nm (average size about 11 nm).
The extract can be prepared by collecting one or more plant parts of the L. cretica plant for use as the extraction substrate. Suitable plant parts can include, for example, the leaves, flowers, stems, and/or roots, of L. cretica. Prior to extraction, the L. cretica plant or plant part may be washed thoroughly one or more times with tap water and/or distilled water, e.g., triple distilled water. The washed L cretica may then be dried, e.g., shade dried, at room temperature to provide dried L cretica. The shade drying may proceed for about two days. The dried L. cretica may then be powdered, by grinding, blending, or any other conventional means. Powdered L. cretica may then be suspended in water, e.g., triple distilled water to produce the L. cretica extract. For example, about 5 grams of the powdered L. cretica may be suspended in about 500 mL of triple distilled water for about 24 hours. The L. cretica extract may be filtered using filter paper, a muslin cloth, or any other conventional means, producing filtered L. cretica extract.
The biomimetic synthesis of the AgNPs may be achieved by mixing the L. cretica extract with silver nitrate (AgNO3). For example, about 10 mL of the filtered L. cretica extract can be mixed with about 250 mL of the AgNO3. The mixture may be incubated at room temperature in a darkened location until its color changes from colorless to brown, indicating the formation of AgNPs. The mixture may then be centrifuged, e.g., at about 15,000 rpm for about 20 minutes, and re-suspended in distilled water. The centrifugation and resuspension steps may then be repeated multiple times in order to remove impurities. The final resuspension may then be dried using conventional means such as an oven, producing L. cretica AgNPs or a nanoparticle composition including AgNPs and L. cretica extract components.
The nanoparticle composition including AgNPs and L. cretica extract components can be administered to a patient in need thereof. For example, a therapeutically effective amount of the composition can be administered to a patient suffering from Diabetes mellitus (diabetes) and/or hyperglycemia. The therapeutically effective amount can be about 1 mg/kg.
The nanoparticle composition can be admixed with a suitable pharmaceutical carrier, including, but not limited to water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like. The composition can be administered by any conventional route of administration, including, but not limited to, intravenous, oral, subcutaneous, intramuscular, intradermal and parenteral. Depending on the route of administration, the composition can be constituted into any form. For example, forms suitable for oral administration include as pills, gelcaps, tablets, caplets, capsules (each including immediate release, timed release and sustained release formulations), granules, powders, and syrups.
Example 1
Extraction of L. cretica Leaves
Leaves were collected from fresh L. cretica from Riyadh, Saudi Arabia. An aqueous extraction was performed using the leaves to produce L cretica leaf extract. The leaves were washed first with tap water and then with triple distilled water. The washed L. cretica leaves were shade dried for about two days. The shade dried L. cretica leaves were then powdered using a blender. 5 grams of powered L. cretica leaves were suspended in 500 mL of triple distilled water for 24 hours, producing L. cretica leaf extract. The L. cretica leaf extract was filtered using a muslin cloth, producing filtered L. cretica leaf extract.
Example 2
Biomimetic Synthesis of AgNPs
About 10 mL of filtered L. cretica leaf extract was mixed with about 250 mL of 1 mM AgNO3 and kept at room temperature in a dark place. Color of the reaction mixture changed from colorless to brown, indicating the formation of AgNPs. The mixture containing AgNPs was centrifuged at 15,000 rpm for 20 mM and the supernatant was discarded and the pellet was dispersed in distilled water. This process was repeated three times for removal of impurities and the pellet was dried in an oven at 40° C. The resulting dried L. cretica AgNPs were used for further studies.
Example 3
Characterization of AgNPs
Bio-reduction of Ag+ ions to colloidal nanoparticles was visually observed by a color change from colorless (AgNO3 solution) to brown (AgNPs).
UV-Vis spectra were used for further confirmation of the AgNPs synthesis reaction. The absorption of light by the nanoparticles at different wavelengths provides an indication of particle size, while the breadth of the peaks signifies the particle size distribution. FIGS. 1(A) and 1(B) represent the UV spectra recorded at 24 and 48 hours, respectively. The maximum absorbance appears at 440 nm, confirming nanoparticles formation (AgNPs).
Formation of nanoparticles was further confirmed by Transmission Electron Microscopy (TEM). FIG. 2 shows that the nanoparticles are spherical in shape with size ranging from 5 nm to 24 nm (average size 11 nm).
The presence of additional organic components (L. cretica extract components) in the synthesized AgNPs is demonstrated by FIG. 3. The Energy Dispersive X-Ray Diffraction spectra demonstrates intense signal at about 3 keV, confirming the presence of Ag as the major element; however, the spectra also indicates the presence of other elements, including C, O, and Cl. This indicated biomolecules overlapping the silver nanoparticles.
Fourier Transform Infrared Spectroscopy (FTIR) measurements were performed to identify the biomolecules that bound specifically on the AgNP's surface and the local molecular environment of the capping agent. FIG. 4 presents the FTIR spectra of synthesized AgNPs.
The band at about 3293 cm−1 is assigned to the N—H stretch. The band at about 2922 cm−1 is assigned to the C—H stretch. The absorption bands observed at about 1647 cm1 are assigned to the amide groups of proteins or to the C═O stretching vibration group. The band at about 1377 cm−1 is assigned to the O—H stretch. The bands appearing at about 1038, 1063, 1158 and 1235 cm−1 are assigned to the C—O group. The prominent bands at about 750, 698 and 629 cm+1 are assigned to the aromatic class. This observed spectrum demonstrates the presence of active biomolecules in the L. cretica AgNps.
The X-Ray Diffraction (XRD) pattern of the L. cretica AgNPs is shown in FIG. 5. The crystalline nature of the synthesized AgNPs was confirmed by the XRD analysis. The lattice planes (111), (200), (220) and (311) were indexed for face-centered cubic (fcc) silver.
Example 4
L. cretica Leaf AgNPs Antihyperglycemic Effect
Male C57BL/6J mice of 3 weeks of age were maintained under standard conditions with a 12 hour light/dark cycle. The animals received a standard pellet diet and water ad libitum. After an acclimatization period of 1 week, mice were used for experiments. The normal group was provided with a standard pellet diet, which had a fat composition of 4.2%. The high fat diet (HFD) experimental group was provided with beef tallow-based HFD containing 17.7 g protein, 35.8 g fat, 34.5 g carbohydrate, 3.4 g fiber, 6.8 g minerals and 1.8 g vitamins. On the 8th week, animals with blood glucose more than 180 mg/dL were considered to be diabetic and were used for the experiment.
The mice were divided into 5 groups, containing 5 mice per group. Group 1 included control mice, and Groups 2, 3, 4, and 5 included diabetic mice. After 6 hours of fasting, 2 g/kg body weight of glucose was loaded orally to all groups. Three doses of L. cretica AgNPs (about 500 μg/kg, about 1 mg/kg, and about 2 mg/kg body weight) were given to diabetic mice orally. The OGTT were recorded at 0 mins, 30 mins, 1 hr and 2 hr. Only the active dose of L. cretica AgNPs was continued for 15 days' evaluation study.
Analysis of glucose tolerance tests of the different experimental groups is shown in FIG. 6. The results show that in the healthy control or normal control mice there is an increase in blood glucose after the glucose load at the first minute, which then decreases to the baseline level (FIG. 6). In contrast, in diabetic rats with the glucose load, blood glucose levels increased significantly at the first minute and did not return to the baseline level. In diabetic mice treated with L. cretica AgNPs, there was no increase in blood glucose levels after the glucose load (FIG. 7). Among the three different doses, the 1 mg/kg dose of L. cretica AgNPs showed the maximum prevention of increase in blood glucose levels (FIG. 7). Diabetic rats that were treated with L. crectica AgNPs attenuated their increase in blood glucose level in the glucose tolerance test, suggesting that the L. crectica AgNPs exert a protective effect (FIG. 7).
Of the three different doses, 1 mg/kg was observed to provide maximum prevention of increase in blood glucose levels. Hence, the 1 mg/kg dose was used for a further 15 days' study. FIG. 8 represents the result of 15 days' treatment of L. cretica AgNPs on plasma glucose and insulin in high fat diet (HFD) induced C57BL/6J diabetic mice. As demonstrated in FIG. 8, L. cretica AgNP treated HFD-fed diabetic mice demonstrated reduced levels of plasma glucose and insulin when compared to the diabetic controls.
It is to be understood that the biomimetic synthesis of nanoparticles using silver nitrate and Lavatera cretica is not limited to the specific embodiments described above, but encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter.

## Claims

1. A method of biomimetic synthesis of silver nanoparticles, comprising:
providing an aqueous extract of Lavatera cretica prepared by suspending about 5 g of powdered Lavatera cretica in about 500 ml of water for about 24 hours, and
mixing about 10 ml of the aqueous extract of Lavatera cretica with about 250 ml of 1 mM silver nitrate at room temperature to provide silver nanoparticles.

2. The method of biomimetic synthesis of silver nanoparticles of claim 1, wherein the powdered Lavatera cretica comprises one or more plant parts of the Lavatera cretica.

3. The method of biomimetic synthesis of silver nanoparticles of claim 2, wherein the Lavatera cretica plant parts include Lavatera cretica leaves.

4. The method of biomimetic synthesis of silver nanoparticles of claim 1, further comprising purifying the silver nanoparticles.

5. The method of biomimetic synthesis of silver nanoparticles of claim 1, wherein the silver nanoparticles have a size ranging from about 5 nm to about 24 nm.

6. The method of biomimetic synthesis of silver nanoparticles of claim 1, wherein the silver nanoparticles have an average size of about 11 nm.

7. The method of biomimetic synthesis of silver nanoparticles of claim 1, further comprising the step of purifying the silver nanoparticles by centrifugation at about 15,000 rpm for about 20 minutes.

8. The method of biomimetic synthesis of silver nanoparticles of claim 7, further comprising the steps of repeating the purifying step at least three times and drying the purified silver nanoparticles in an oven at about 40 degrees Celsius.

9. A nanoparticle composition, comprising:
silver nanoparticles prepared according to the method of claim 1 and having an average size ranging from about 5 nm to about 24 nm,
wherein the composition includes silver nanoparticles and Lavatera cretica extract components.

10. A method of treating hyperglycemia, comprising administering to a patient in need thereof a therapeutically effective amount of the nanoparticle composition of claim 9.

11. The method of treating hyperglycemia of claim 10, wherein the nanoparticle composition is orally administered to the patient.

12. The method of treating hyperglycemia of claim 10, wherein the therapeutically effective amount of the nanoparticle composition comprises about 1 mg/kg.