Patent Application
20240342108
The disclosure of the present patent application relates to a green method of synthesizing circle shaped silver nanoparticles using ethyl acetate extract of Urtica dioica leaves, and particularly to the use of the such-produced silver nanoparticles to assist in bone regeneration.
Urtica dioica, also known as the common nettle (Stinging nettle), is a perennial flowering plant in the family Urticaceae. Urtica dioica plants are spread out all over the world and have been a staple in herbal medicine since ancient times. The Urtica dioica plants contain several biological active compounds, which are capable of reducing inflammation, reducing blood sugars, and used to maintain sugar level in static conditions. While fresh nettle leaves may cause irritation, cooked, dried, or freeze-dried leaves are safe to consume. The medicinal values of nettle plant crude extract have been tested with several treatments, and have been reported to reduce capacity of bleeding, promote wound healing, serve as a natural diuretic and contain burn healing properties with animal models. Further, neck sores, infertility, lactation, abdominal pain, and internal injury have been generally known to be treated with nettle leaves, as disclosed by Pande, P. C. et al., “Ethnoveterinary plants of Uttaranchal—A review”, Ind. J. Trad. Know. (2007) 6, 444-458.
Another study showed the capacity to regenerate bone in calvarial defects of mice using a combination of Chitosan/dicarboxylic acid, which compounds were identified in the nettle leaves by Sukpaita T et al., “In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells”, Int J Mol Sci, 2019 October 1; 20(19). Neophytadiene has demonstrated analgesic, antipyretic, anti-inflammatory, antimicrobial, and antioxidant properties, per Swamy MK1 et al., “GC-MS Based Metabolite Profiling, Antioxidant and Antimicrobial Properties of Different Solvent Extracts of Malaysian Plectranthus amboinicus Leaves”, Evid-Based Complement Alternat Med.
Thus, the synthesis of a composition including Urtica dioica leaves extract solving the aforementioned problems is desired.
The current subject matter relates to the synthesis of silver nanoparticles using an acetyl extract of Urtica dioica leaves and their biological benefits. The nanoparticles were confirmed by different characterization measurements. At the same time, biologically active compounds were identified by using GC- MS analysis. Three very active compounds in the extract have been identified. The innovative nanoparticles were tested with a variety of treatments.
In this regard, in one embodiment, the present subject matter relates to Urtica diocia silver nanoparticles comprising nanoparticles synthesized from Urtica diocia extract and silver. Also contemplated herein are pharmaceutical compositions comprising the Urtica diocia silver nanoparticles and a pharmaceutically acceptable carrier, as well as methods of providing a therapeutic effect to a subject, such as antimicrobial, analgesic, antipyretic, anti-inflammatory, antioxidant, wound healing, or homeostasis effects. Further included herein are methods for treating bone loss, or promoting bone regeneration, in a subject.
In another embodiment, the present subject matter relates to a method for obtaining an ethyl acetate Urtica diocia extract comprising: blinding cleaned and dried leaves of Urtica diocia to a fine powder; combining the fine powder with an ethyl acetate solvent to obtain a mixture; and obtaining the ethyl acetate Urtica diocia extract using a Soxlet extractor.
In an embodiment, the present subject matter relates to a silver nanoparticle composition including silver nanoparticles having a silver nanoparticle core and an organic layer coating over the core, the organic layer coating comprising Urtica diocia extract.
In a further embodiment, the present subject matter relates to a method of synthesizing Urtica diocia silver nanoparticles, comprising: dissolving silver nitrate in water to provide a silver solution; and adding the ethyl acetate Urtica diocia extract to the silver solution to provide a mixture including Urtica diocia silver nanoparticles. Urtica diocia silver nanoparticles prepared by this method are further contemplated herein.
These and other features of the present subject matter will become readily apparent upon further review of the following specification.
Similar reference characters denote corresponding features consistently throughout the attached drawings.
The following definitions are provided for the purpose of understanding the present subject matter and for construing the appended patent claims.
Throughout the application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps.
It is noted that, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein.
The use of the terms “include,” “includes”, “including,” “have,” “has,” or “having” should be generally understood as open-ended and non-limiting unless specifically stated otherwise.
The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term “about” is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.
The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.
Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter.
Throughout the application, descriptions of various embodiments use “comprising” language. However, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of”.
For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
The current subject matter relates to the synthesis of silver nanoparticles using an acetyl extract of Urtica dioica leaves and their biological benefits. The nanoparticles were confirmed by different characterization measurements. At the same time, biologically active compounds were identified by using GC-MS analysis.
In this regard, in one embodiment, the present subject matter relates to Urtica diocia silver nanoparticles comprising nanoparticles synthesized from Urtica diocia extract and silver. In an embodiment in this regard, the Urtica diocia extract is an ethyl acetate extract. In other embodiments, the nanoparticles have an average particle diameter of about 8 nm to about 86 nm, an average particle diameter of about 25 nm to about 30 nm, or an average particle diameter of less than about 30 nm. In certain embodiments, a silver nanoparticle composition can include a Ag nanoparticle core coated with an organic layer comprising the Urtica diocia extract.
In certain embodiments, the Uritica diocia extract comprises one or more components selected from the group consisting of 2,3,6-TRIMETHYL-1,5-HEPTADIENE, 2-(2-BUTOXYETHOXY)-ETHANOL, FARNESOL, 3,7,11,15-TETRAMETHYL-2-HEXADECENE, 2,6,10-TRIMETHYL-NEOPHYTADIENE, 3,7,11,15-TETRAMETHYL-2-HEXADECANE, 1,2-BENZENEDICARBOXYLIC ACID, PHYTOL ACETATE, (E,E)-FARNESYLACETONE, 2,3,5,8-TETRAMETHYL-1,5,9-DECATRIENE, HEXADECANOIC ACID, 3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL, [[(3.BETA.)-GORGOST-5-OL, LINOLENIC ACID, 1,2-BENZENEDICARBOXYLIC ACID, PALUSTROL, and a mixture or combination thereof. In this regard, the Urtica diocia extract, in some embodiments, comprises one or more components selected from the group consisting of 2,6,10-TRIMETHYL-NEOPHYTADIENE, HEXADECANOIC ACID, 1,2-BENZENEDICARBOXYLIC ACID, and a mixture or combination thereof, which are each effective for treating bone loss.
Also contemplated herein are pharmaceutical compositions comprising the Urtica diocia silver nanoparticles and a pharmaceutically acceptable carrier, as well as methods of providing a therapeutic effect to a subject, such as antimicrobial, analgesic, antipyretic, anti-inflammatory, antioxidant, wound healing, or homeostasis effects. Further included herein are methods for treating bone loss, or promoting bone regeneration, in a subject. In this regard, the present methods are effective for promoting bone regeneration in a subject.
In this regard, some of the present methods for treating bone loss in a subject can comprise administering to a subject in need thereof a therapeutically effective amount of the Urtica diocia silver nanoparticles (Urtica diocia-AgNPs) described herein for reducing bone loss, wherein the Urtica diocia silver nanoparticles can comprise one or more of HEXADECANOIC ACID, TRIMETHYL-NEOPHYTADIENE, and BENZENEDICARBOXYLIC ACID from the Urtica diocia extract, which are each effective for treating bone loss. Similarly, the present Urtica diocia silver nanoparticles can, in some embodiments, be useful to promote hemastosis, wound healing, bone production, and bone remodeling.
In another embodiment, the present subject matter relates to a method for obtaining an ethyl acetate Urtica diocia extract comprising: blinding cleaned and dried leaves of Urtica diocia as a fine powder; combining the fine powder with an ethyl acetate solvent to obtain a mixture; and obtaining the ethyl acetate Urtica diocia extract using a Soxlet extractor. In this regard, the mixture containing the blinded fine powder can be heating to its boiling point, for example, 77.1° C., by using the Soxlet extractor.
In a further embodiment, the present subject matter relates to a method of synthesizing Urtica diocia silver nanoparticles, comprising: dissolving silver nitrate (AgNO3) in water to provide a silver solution; and adding the ethyl acetate Urtica diocia extract to the silver solution to provide a mixture including Urtica diocia silver nanoparticles. In certain embodiments in this regard, after the ethyl acetate is added to the Urtica diocia extract, an oxidation reduction reaction occurs for about 24 hours to obtain the Urtica diocia silver nanoparticles. In this regard, once the initiated oxidation and reduction process starts, the color of the mixture will typically change from clear to light brown, after about 8 hours or more, and it will typically completely turn dark brown after about 24 hours or more. Urtica diocia silver nanoparticles prepared by these methods are further contemplated herein. In this regard, the thus produced Urtica diocia silver nanoparticles can have a spherical shape when taken individually.
Synthesis of Ethyl Acetate Extract of Urtica dioica
Urtica dioica (stinging nettle), a perennial plant belonging to the family of Urticaceae, genus Urtica, was collected from two different countries (Saudi Arabia, Riyadh Region and Tamil Nadu, Tanjore District, India). The fresh leaves were harvested in the field. The harvested leaves were cleaned and dried under laboratory temperature conditions. The dried leaves were collected and cleaned with running double distilled water and dried again under the same conditions. Finally, the completely cleaned and dried leaves were blinded as a fine powder.
The blinded fine powder (20 gm) was flowed rapidly in 250 ml of ethyl acetate solvent, followed by heating to a boiling point of 77.1° C. by using a Soxlet extractor. The final product was collected and filtered with Whatman No. 1 filter paper. The purity purpose of the chemical constituents from the ethyl acetate extract of Urtica dioica was maintained, the dried compounds again washed with ethyl acetate, and the ethyl acetate was evaporated off. The final pure dried extract was collected, and the active constitutions from the ethyl acetate extract were identified by Gas chromatography-mass spectrometry (GC-MS). The GC-MS results found the chemical constitutions having the therapeutic properties of bone regeneration components are TRIMETHYL-NEOPHYTADIENE, (RT:20.86; area:19.590), ENZENEDICARBOXYLIC ACID, (RT: 27.40; area: 28.110), and HEXADECANOIC ACID (RT:23.04; area: 3.290), as shown in
1 mM silver nitrate was dissolved in 250 ml of double distilled water and 10 ml of ethyl acetate plant extract was added in the 1 mM silver nitrate solution and monitored for completion of an oxidation reduction process. The interaction of silver nitrate and plant extract was deeply monitored. The initiated oxidation and reduction process started (indicated by color changes) 8 h onwards and completely turned into dark brown within 24 h.
Characterization of Silver Nanoparticles Confirmation from Urtica dioica
The UV-Visible spectra demonstrate the formation of silver nanoparticles from ethyl acetate extract of nettle plant leaves exhibit a characteristic absorption peak at 420 nm due to surface Plasmonic resonance (SPR), as seen in
FTIR spectral measurements were carried out to identify the potential biomolecules in the ethyl acetate extract of the Urtica dioica leaf, which is used herein for reducing and capping the bioreduced AgNPs, as shown in
The surface morphology and size of the silver nanoparticles was analyzed by Scanning Electron Microscope (SEN) with different magnifications, 5 μm and 50 μm, in
The energy dispersive spectrum (EDX) of the synthesized silver nanoparticles from Urtica dioica leaves extract is shown in
Transmission electron microscope (TEM) analysis with different magnifications of 100 nm and 200 nm are shown in
The nanoparticles disbursement details, including minimum, maximum, and average size are provided in Table 2 below.
The synthesis of silver nanoparticles achieved a minimal size of particles that are less than 20 nm and a maximum size of particles that are less than 100 nm. The average particle size was achieved at less than 30 nm, which was uniformly distributed without significant agglomeration.
The Zeta potential image of Urtica dioica AgNPs is shown in
Zeta potential size distribution analysis was used to measure the dispersion of AgNPs from ethyl acetate leaves extract of Urtica dioica. The synthesized nanoparticles were shown to have the particle size distribution in
Table 3, below, gives the information of the nanoparticle distribution with a less than 100 nm (35.94) size being achieved. The nano range of the AgNPs was given more effective fast recovery in wound healing.
The total number of male rats (1) was selected for an initial bone remodelling experiment. Before the experiments, the health of the rats was examined. The selected rat was healthy and very active with a body weight at 525 g and an age of 14 weeks. Then, a local aesthetic injection of Ketamine was injected to control the pain and movement during the removal of hair from the left thigh of the selected rat. Afterwards, the skin was cleaned with a gauze sponge containing 10% povidone-iodine solution to avoid any infections from microorganisms.
The initial protocol was completed and then the rat was anesthetized with Tekam 1 ml containing 50 mg ketamine, followed by a sevoflurane vaporizer with a single route channel for 20 minutes. The surgical site was approached through a 5 mm skin cut using a sterilized scalpel blade. The bleeding was swapped with a surgical sponge. After exposing the left thigh, the femur was cut with two holes of 3 mm in diameter.
All animal care and experimental procedures followed the guidance of the Experimental Surgery and Animal Laboratory and all animals were kept in individual cages with free access to food and water. The study was approved by the Institutional Animal Care and Use Committee of King Saud University (Approval No: SE-19-100), Riyadh, Saudi Arabia.
To measure the biological effects, cessation of bleeding, reduction of inflammation, and bone remodeling by the nanomaterials produced using ethyl acetate Urtica dioica leaves extract were monitored. 10 mg of the AgNPs was filled into the two femur bone defects and the opened skin was then stitched closed.
The rat survived the surgery without any complications, such as soft tissue damage at the defect site or stiffness of the adjacent knee and ankle joints. The rat was visually observed to be very active with healthy movement. There was no wound or bleeding seen at the defect site. The rat was deeply monitored every day, but it exhibited faster recovery and healthy movements. This provided evidence the present nanoparticles are capable of exhibiting wound healing properties, likely due to the phytoconstituents 3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL; 2,6,10-TRIMETHYL-NEOPHYTADIENE; and 1,2-BENZENEDICARBOXYLIC ACID in the Urtica dioica leaves extract.
The rat was sacrificed exactly after the 7th week post-surgery and monitored for any inflammation in the upper part of the femur bone cut area, bone production (any soft tissue formation), and bone regeneration. Inflammation was found in the upper region of the femur bone, which was attached with the surgery bone. The inflammation was detached from the bone.
Another interesting effect was observed in the surgery bone. to which the new nanoparticles were applied. The nanoparticles interacted with blood flow from the cut bone, and they covered the entire cut bone and produced the soft tissue upper region of the bone. The soft cartilage tissue turned harder and mature or stronger bone occurred with bone remodeling in the rat.
Accordingly, the Urtica dioica AgNPs with active components adequately provided wound healing to enhance the blood circulation in the rat. The nanoparticles helped to enhance support for the recovery process, including hemastosis, wound healing, bone production, and bone remodeling.
The study was conducted over an at least 12 week period.
It is to be understood that the silver nanoparticles prepared using ethyl acetate extract of Urtica dioica leaves are not limited to the specific embodiments described above, but encompass 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.
