The Moringa oleifera nanoparticles may be synthesized by harvesting Moringa leaves, drying the Moringa leaves, powdering the dried Moringa leaves, suspending the powdered Moringa leaves in a solution, and spraying the solution into boiling water under ultrasonic conditions to obtain Moringa nanoparticles. The Moringa nanoparticles may be encapsulated by dissolving the Moringa nanoparticles and gum olibanum in ethanol to produce a mixture, injecting the inert organic phase of the mixture into an aqueous solution containing PVA, and homogenizing the aqueous solution. The Moringa nanoparticles may be useful in preventing the growth of cancer cells and in treating diabetes by inhibiting α-glucosidase and/or α-amylase activity.

BACKGROUND

The disclosure of the present patent application relates to nanotechnology, and particularly toMoringa oleiferananoparticles, methods of synthesizingMoringa oleiferananoparticles, and to methods of administeringMoringa oleiferananoparticles to inhibit the growth of cancer cells.

2. Description of the Related Art

Recently, nanoparticles have demonstrated important uses in a variety of fields. Nanoparticles have been used in a vast array of applications, including electronics, sensing, optics, and medicine.

Synthesis of nanoparticles has 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. Encapsulation of nanoparticles with synthetic or bio polymers ensures more stability and controlled delivery, hence improving the bioavailable status of some nano-formulations. These methods are often costly or produce by-products that pose increased risks to human health and the environment.

In recent years, green or environmentally friendly chemical methods have been developed to prepare nanoparticles using plant extracts. Green chemistry has the advantage of being safer, faster, environmentally friendly, and economical. However, the rise of green methods of preparing nanoparticles has also demonstrated that the activities and characteristics of the nanoparticles vary significantly, depending upon the detailed method of synthesis and specific plant extract used.

Moringa oleiferaLam. is commonly known as the drumstick tree of the Moringaceae family. It is a native of India and currently grown widely in many tropical and sub-tropical countries (Mbikay, 2012). Traditionally,Moringa oleiferais considered to be one of the most therapeutically useful trees, as almost all the parts (pods, leaves, flowers, roots and bark) have been reported to have a gamut of medicinal and nutritional properties (Singh et al., 2014).Moringaleaves are a good source of natural antioxidant due to the presence of various types of antioxidant compounds such as ascorbic acid, flavonoids, phenolics and carotenoids (Anwar et al., 2005; Makkar and Becker, 1996; Mbikay, 2012). These bioactive compounds are thought to be found inMoringaleaves and have been widely used in various studies as wound healing, anti-tumor, anti-fertility, hypotensive, antipyretic, antihepatotoxic, antiepileptic, anti-inflammatory, antiulcer, diuretic hypocholesterolemic, antifungal, antibacterial and anti-cardiovascular and anti-diabetic agents (Hussain et al., 2014; Jung, 2014).

Due to the pharmacological properties and rich nutritional value of theMoringaleaves, there has been a growing interest in promoting it as a food supplement or nutraceutical in the human diet.

Thus,Moringa oleiferananoparticles solving the aforementioned problems are desired.

SUMMARY

TheMoringa oleiferananoparticles may be synthesized by harvestingMoringaleaves, drying theMoringaleaves, powdering the driedMoringaleaves, suspending the powderedMoringaleaves in a solution, and spraying the solution into boiling water under ultrasonic conditions to obtainMoringananoparticles. In an embodiment, theMoringa oleiferaleaves may be harvested fromMoringa oleiferatrees grown in Riyadh, Saudi Arabia. In an embodiment the powderedMoringaleaves may be suspended in an appropriate solvent, including but not limited to methanol.

In an embodiment, encapsulatedMoringa oleiferananoparticles may be synthesized by harvestingMoringaleaves, drying theMoringaleaves, powdering the driedMoringaleaves, suspending the powderedMoringaleaves in a solution, spraying the solution into boiling water under ultrasonic conditions to obtainMoringananoparticles, dissolving theMoringananoparticles and gum olibanum in ethanol to produce a mixture, injecting the inert organic phase of the mixture into an aqueous solution containing polyvinyl alcohol (PVA), and homogenizing the aqueous solution. In an embodiment, theMoringa oleiferaleaves may be harvested fromMoringa oleiferatrees grown in Riyadh, Saudi Arabia. In an embodiment the powderedMoringaleaves may be suspended in an appropriate solvent, including but not limited to methanol. In an embodiment, the ratio ofMoringananoparticles:gum olibanum:PVA may be 1:5:7 (w/w/w).

An embodiment of the present subject matter is directed to a pharmaceutical composition including theMoringananoparticles and a pharmaceutically acceptable carrier.

An embodiment of the present subject matter is directed to a method of making a pharmaceutical composition including mixing theMoringananoparticles under sterile conditions with a pharmaceutically acceptable carrier and preservatives, buffers, or propellants to create the pharmaceutical composition; and providing the pharmaceutical composition in a form suitable for daily, weekly, or monthly administration.

An embodiment of the present subject matter is directed to a method of inhibiting cancer cell growth, including administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the present subject matter.

An embodiment of the present subject matter is directed to a method of treating diabetes, including administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the present subject matter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

TheMoringa oleiferananoparticles may be synthesized by harvestingMoringaleaves, drying theMoringaleaves, powdering the driedMoringaleaves, suspending the powderedMoringaleaves in a solution, and spraying the solution into boiling water under ultrasonic conditions to obtainMoringananoparticles. In an embodiment, theMoringa oleiferaleaves may be harvested fromMoringa oleiferatrees grown in Riyadh, Saudi Arabia. In an embodiment the powderedMoringaleaves may be suspended in an appropriate solvent, including but not limited to methanol.

In an embodiment, encapsulatedMoringa oleiferananoparticles may be synthesized by harvestingMoringaleaves, drying theMoringaleaves, powdering the driedMoringaleaves, suspending the powderedMoringaleaves in a solution, spraying the solution into boiling water under ultrasonic conditions to obtainMoringananoparticles, dissolving theMoringananoparticles and gum olibanum in ethanol to produce a mixture, injecting the inert organic phase of the mixture into an aqueous solution containing PVA, and homogenizing the aqueous solution. In an embodiment, theMoringa oleiferaleaves may be harvested fromMoringa oleiferatrees grown in Riyadh, Saudi Arabia. In an embodiment the powderedMoringaleaves may be suspended in an appropriate solvent, including but not limited to methanol. In an embodiment, the ratio ofMoringananoparticles:gum olibanum:PVA may be 1:5:7 (w/w/w).

In an embodiment, the synthesis ofMoringananoparticles may include mixing about 400 mg of powderedMoringa oleiferaleaves with about 20 ml of methanol and spraying the resulting solution into about 50 ml of boiling water dropwise at a flow rate of about 0.2 ml/min for about 5 minutes under ultrasonic conditions (ultrasonic power of 750 W and frequency of 20 kHz). The resulting solution may then be stirred at about 200-800 rpm at room temperature for about 20 minutes to produce theMoringananoparticles.

TheMoringa oleiferaleaves may be powdered by any available means of rendering a dried leaf into a powder, including but not limited to grinding, blending, and other similar techniques known in the art.

In an embodiment, the synthesis of encapsulatedMoringananoparticles may include mixing about 150 mgMoringananoparticles with about 750 mg of gum olibanum and about 30 ml of ethanol to form a mixture, injecting the inert organic phase of the mixture into about 150 ml of an aqueous solution containing about 1,050 mg PVA, and homogenizing the resulting solution at about 22,000 rpm for about 25 minutes.

In an embodiment, theMoringananoparticles may have an average particle diameter of about 141.6 nm with a polydispersity of about 0.32.

In an embodiment, the encapsulatedMoringananoparticles may have an average particle diameter of about 222 nm with a polydispersity of about 0.2.

As used herein, the term “about,” when used to modify a numerical value, means within ten percent of that numerical value.

An embodiment of the present subject matter is directed to a pharmaceutical composition comprising theMoringananoparticles and a pharmaceutically acceptable carrier.

An embodiment of the present subject matter is directed to a method of making a pharmaceutical composition including mixing theMoringananoparticles with a pharmaceutically acceptable carrier. For example, the method of making a pharmaceutical composition can include mixing theMoringananoparticles under sterile conditions with a pharmaceutically acceptable carrier with preservatives, buffers, and/or propellants to create the pharmaceutical composition.

An embodiment of the present subject matter is directed to a pharmaceutical composition including theMoringananoparticles. To prepare the pharmaceutical composition, theMoringananoparticles, as the active ingredient, are intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques. Carriers are inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorings, sweeteners, preservatives, dyes, and coatings. In preparing compositions in oral dosage form, any of the pharmaceutical carriers known in the art may be employed. For example, for liquid oral preparations, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like. Further, for solid oral preparations, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.

The present compositions can be in unit dosage forms such as tablets, pills, capsules, powders, granules, ointments, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampules, auto-injector devices or suppositories, for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. TheMoringananoparticles can be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if required, any needed preservatives, buffers, or propellants. The composition can be presented in a form suitable for daily, weekly, or monthly administration. The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful, suppository and the like, an amount of the active ingredient necessary to deliver an effective dose. A therapeutically effective amount of theMoringananoparticles or an amount effective to treat a disease, such as a bacterial infection, may be determined initially from the Examples described herein and adjusted for specific targeted diseases using routine methods.

TheMoringananoparticles can be administered to a subject in need thereof. In an embodiment, theMoringananoparticles can be administered to a subject in need thereof to inhibit cancer cell growth and/or prevent or treat a cancer. In a further non-limiting embodiment, the cancer can be a breast cancer or a liver cancer. In a further embodiment, theMoringananoparticles can be administered to a subject in need thereof to treat the effects of diabetes. In a further non-limiting embodiment, theMoringananoparticles can be administered to a subject in need thereof in order to inhibit the activity of at least one of α-glucosidase, α-amylase, and a combination thereof.

An embodiment of the present subject matter is directed to a method of preventing cancer cell growth, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to the present subject matter.

An embodiment of the present subject matter is directed to a method of inhibiting α-amylase activity, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to the present subject matter.

An embodiment of the present subject matter is directed to a method of inhibiting α-glucosidase activity, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to the present subject matter.

TheMoringananoparticles or pharmaceutical compositions thereof can be administered to a subject by any suitable route. For example, the compositions can be administered orally (including bucally and sublingually), nasally, rectally, intracisternally, intra vaginally, intraperitoneally, topically, transdermally (as by powders, ointments, or drops), and/or parenterally. As used herein, “parenteral” administration refers to modes of administration other than through the gastrointestinal tract, which include intravenous, intramuscular, intraperitoneal, intrasternal, intramammary, intraocular, retrobulbar, intrapulmonary, intrathecal, subcutaneous and intraarticular injection and infusion. Surgical implantation may also be contemplated, including, for example, embedding a composition of the disclosure in the body such as, for example, in a tissue, in the abdominal cavity, under the splenic capsule, brain, or in the cornea.

The following examples illustrate the present subject matter.

FreshMoringaleaves were collected fromMoringa oleiferatrees grown in Riyadh, Saudi Arabia. The leaves were air dried and powdered to form powderedMoringa oleiferaleaves. 400 mg of the powderedMoringa oleiferaleaves was added to 20 ml of methanol, and the resulting solution was sprayed into 50 ml of boiling water dropwise at a flow rate of 0.2 ml/min for 5 minutes under ultrasonic conditions (ultrasonic power of 750 W and frequency of 20 kHz). The contents were then stirred at 200-800 rpm at room temperature for about 20 minutes.

EncapsulatedMoringa oleiferananoparticles were synthesized using a ratio ofMoringananoparticles:gum olibanum:PVA of 1:5:7 (w/w/w) using the nano precipitation technique as previously described (Bilati et al., 2005; Zili et al., 2005). Briefly, 150 mg ofMoringa oleiferananoparticles prepared according to Example 1 and an appropriate amount of olibanum were dissolved in 30 ml of ethanol. The internal organic phase of the resulting solution was then injected into 150 ml of an external aqueous solution containing the appropriate amount of PVA, and the solutions were homogenized at 22,000 rpm for 25 minutes.

Dynamic light scattering (DLS) analysis on a Zetasizer (ZEN 3600, Malvern, United Kingdom) was used to observe physical characteristics of theMoringananoparticles and the encapsulatedMoringananoparticles. As shown inFIG.1and Table 1, theMoringananoparticles had an average diameter of 141.6 nm with a polydispersity of 0.32 and diverse sizes. In comparison, the encapsulatedMoringananoparticles were more monodisperse, with an average diameter of 222 nm, a polydispersity of 0.2, and diverse sizes (SeeFIG.2and Table 2).

Transmission electron microscopy (TEM) was performed using a JEM-1011 (JEOL, Japan), to study the surface morphology, shape, and size of theMoringananoparticles. As shown inFIGS.3and4, theMoringananoparticles are spherical in shape with diverse sizes. As shown inFIGS.5and6, the encapsulatedMoringananoparticles are encapsulated and covered by a polymer layer.

Evaluation of the Cytotoxic Effects ofMoringaNanoparticles Against Cancer Cells

Mammalian MCF-7 cells (a human breast cancer cell line) and HepG-2 cells (a human liver cancer cell line) were obtained from the VACSERA Tissue Culture Unit. Dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA). Fetal Bovine Serum (FBS), Dulbecco's modified Eagle's medium (DMEM), RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza. Crystal violet stain (1%) was prepared by mixing 0.5% (w/v) crystal violet and 50% methanol, brought up to volume with ddH2O and filtered through Whatman No. 1 filter paper.

MCF-7 and HepG-2 cell lines were propagated in DMEM supplemented with 10% heat-inactivated FBS, 1% L-glutamine, HEPES buffer and 50 μg/ml gentamycin. All cells were maintained at 37° C. in a humidified atmosphere with 5% CO2and were sub-cultured two times a week.

Cytotoxicity assays were performed by seeding cells from each cell line into 96-well flat-bottomed microtiter plates (Falcon, N.J., USA) at a concentration of 1×104cells per well in 100 μl of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 hours of incubation. Specifically, serial two-fold dilutions of each sample to be tested were added to the confluent cell monolayers using a multichannel pipette. The microtiter plates were then incubated at 37° C. in a humidified incubator with 5% CO2for a period of 48 hours. Three wells were used for each concentration of the tested samples. Control cells were incubated without adding any of the tested samples, and with or without the addition of DMSO. The percentage of DMSO present in the wells (a maximum of 0.1%) was found not to affect the experiment. After the incubation period, the respective concentrations of the sample used for each well were added to the wells once again, and the incubation was continued for a further 24 hours. Viable cells were then counted by a colorimetric method.

Briefly, media was aspirated from each well and crystal violet solution (1%) was added for at least 30 minutes. The stain was then removed and the plates were rinsed with tap water to remove all excess stain. Glacial acetic acid (30%) was then added to the wells and mixed thoroughly and the absorbance was measured after gentle shaking using a Microplate reader (Tecan, Inc.) at a wavelength of 490 nm. The results were corrected for background absorbance detected in control wells without any added stain. Treated samples were then compared with stained control wells. All experiments were carried out in triplicate. The cytotoxic effect of each tested composition was calculated. Briefly, the optical density of each well was measured with a microplate reader (SunRise, Tecan, Inc., USA) to determine the number of viable cells and the percentage of viability was calculated according to Equation 1:

In Equation 1, ODtis the mean optical density of wells treated with a particular test sample and ODcis the mean optical density of untreated (control) cells. The relation between surviving cells and test sample concentration is plotted to reveal the survival curve of each tumor cell line after treatment with a particular composition. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was then estimated from the graphic plots of the dose response curve using Graphpad Prism software (San Diego, Calif., USA).

As illustrated inFIGS.7-12, a significant decrease in cell survival was observed in both MCF-7 and HepG-2 cancer cell lines when treated withMoringaleaf powder,Moringananoparticles, and encapsulatedMoringananoparticles. For MCF-7, an IC50of 168±27.04 μg/ml was observed forMoringaleaf powder (Mo). (See Table 4). The inhibitory activity ofMoringananoparticles (MoN) was significantly enhanced when compared toMoringaleaf powder alone, with an IC50of 65.6±13.97 μg/ml. (See Table 3). This inhibitory activity was further enhanced for the encapsulatedMoringananoparticles (Mo CapN), with an IC50of 22.5±0.63 μg/100 μl. (See Table 5) For HepG-2, an IC50of 113.5±6.59 μg/ml was observed forMoringaleaf powder (Mo). The inhibitory activity ofMoringananoparticles (MoN) was significantly enhanced when compared toMoringaleaf powder alone, with an IC50of 53.4±1.93 μg/ml. (See Tables 6 and 7). This inhibitory activity was further enhanced for the encapsulatedMoringananoparticles (Mo CapN), with an IC50of 15.2±2.37 μg/100 μl. (See Table 8). Thus, encapsulating theMoringananoparticles resulted in a significantly enhanced inhibitory activity against both MCF-7 and HepG-2 cancer cell lines.

Evaluation of the Antidiabetic Activity ofMoringaNanoparticles

Current treatments of diabetes mainly focus on reducing fluctuations in blood sugar and subsequent complications. α-amylase and α-glucosidase inhibitors are currently used for diabetic treatment as oral hypoglycemic agents. Acarbose is a commercially available enzyme inhibitor for type II diabetes. In the present study,Moringaleaves (Mo) andMoringanano-formulations, includingMoringananoparticles (MoN) and encapsulatedMoringananoparticles (MoCapN) were evaluated for their inhibitory effect on α-amylase and α-glucosidase enzymes using in-vitro methods and using Acarbose as a reference anti-diabetic drug.

The α-glucosidase and α-amylase inhibitory activity ofMoringaleaf powder andMoringananoparticles was tested, using Acarbose as a reference drug. Briefly, α-glucosidase inhibitory activity was determined according to standard methods with minor modifications (Shai, L. J. et al., “Inhibitory effects of five medicinal plants on rat alpha-glucosidase: Comparison with their effects on yeast alpha-glucosidase,” J. Med. Plant Res. 5: pp. 2863-2867 (2011)). In a 96-well plate, a reaction mixture containing 50 μl phosphate buffer (100 mM, pH=6.8), 10 μl alpha-glucosidase (1 U/ml), and 20 μl of varying concentrations of each tested composition (1000 to 7.81 μg/ml) was preincubated at 37° C. for 15 minutes. Then, 20 μl P-NPG (5 mM) was added as a substrate and incubated for a further 20 minutes at 37° C. The reaction was stopped by adding 50 μl Na2CO3(0.1 M). The absorbance of the released p-nitrophenol was measured at 405 nm using a Multiplate Reader. Acarbose at various concentrations (1000 to 7.81 μg/ml) was included as a standard. Control wells were maintained without adding any of the tested compositions. Each experiment was performed in triplicate. The results are expressed as percent inhibition, which was calculated using Formula 2:

Wherein Asis the absorbance of the tested composition and Acis the absorbance of the control samples. Results are expressed in terms of mean standard deviation and IC50values were calculated using GraphPad.

The α-amylase inhibitory activity of theMoringaleaf powder and theMoringananoparticles was also tested as previously described. Briefly, a volume of 150 μl of each composition to be tested or of the standard drug (acarbose) at concentrations varying from 2000-7.81 μg/ml was incubated with 50 μl of porcine pancreatic amylase (2 U/ml) in phosphate buffer (100 mM, pH 6.8) at 37° C. for 20 minutes in a 96-well plate. Then 100 μl of 1% starch dissolved in 100 mM phosphate buffer (pH 6.8) was further added to the reaction mixture and incubated at 37° C. for 1 hour. Dinitrosalicylate colour reagent (1 ml) was then added and boiled for 10 minutes. The absorbance of the resulting mixture was read at 540 nm and the inhibitory activity was again calculated according to Formula 2 above. All measurements were performed in triplicate and results are expressed in terms of mean±standard deviation and IC50values were calculated using GraphPad.

The inhibitory potency ofMoringaleaves against α-amylase activity resulted in an IC50value of 719.92±4.41 μg. (See Table 9 andFIG.17). In comparison, the nano-formulations were significantly more efficacious as is evident from the IC50values of 15.9±1.11 μg (MoN) and 94.02±1.50 μg (MoCapN). (See Tables 10 and 11 andFIGS.16and18).

Further, the inhibitory potency ofMoringaleaves against α-glucosidase activity demonstrated an IC50value of 231.08±3.73 μg/m. (See Table 12 andFIG.14). In comparison, the nano-formulations were significantly more efficacious as is evident from their IC50values of 59.38±1.42 (MoN) and 30.13±0.83 μg (MoCapN). (See Tables 13 and 14 andFIGS.13and15).

It can be concluded that,Moringaleaves andMoringanano-formulations exhibited potent anti-cancer and anti-diabetic activities, with theMoringanano-formulations being more effective anti-cancer agents and anti-diabetic agents, and with the encapsulatedMoringananoparticles being even more effective than theMoringananoparticles alone. This could be attributed to an enhanced bioavailability of the phytochemical constituents ofMoringaleaves in the nano-formulations, allowing for synergistic effects of these phytochemical constituents.