PREPARATION THEREOF

FIELD OF INVENTION

[0001] The present disclosure relates to nano-emulsions, and more particularly, the present invention relates to a nano-emulsion formulation of saffron and a method of preparation thereof.

BACKGROUND

[0002] Saffron is one of the most palatable spices, derived from the flower of Crocus sativus L, commonly known as the "saffron crocus." As a spice, saffron is used to add flavor, color, smell, and aroma to different types of foods, clothing dyes, and supplements. Besides the main use as species, saffron also has many health benefits and therapeutic properties. Saffron is known for ages having numerous usage and benefits. Many therapeutic actions of saffron have been scientifically evaluated, some of the actions include oxytocic, anti-carcinogenic, exhilarant, anti-depressant, and anti-asthmatic. Also, saffron is a medicine in many traditional systems of medicine. Saffron is known for use in rheumatism, alcohol addiction, cold, asthma, heart disease, tumor, and cancer. Saffron is a well-known antioxidant and has hypolipidemic effects.

[0003] Saffron is known to be originated mostly in Iran which is also the world's biggest producer of saffron owning 90% of global production. The species crocus sativus L. belongs to the family Iridaceae, crocus genus, and crocus species. It is a triploid plant with contractile roots, three stigmas, yellow anthers, and purple petals. The comparatively bitter taste and iodoform-like fragrancy of saffron come from the major phytochemicals picrocrocin and safranal. The golden-yellow hue of saffron is related to crocin, a carotenoid pigment. There are about 150 compounds in saffron among which picrocrocin and apocarotenoid compounds, such as crocetin, crocin, safranal, which are the bio-oxidative segmentation products of zeaxanthin, are considered as the most bioactive ingredients. The antibacterial and antifungal activities of Saffron and its byproducts have also brought many achievements for researchers and scientists. Safranal and picrocrocin inhibitory and bactericidal concentrations cause the antibacterial activities of Saffron which expand the horizon of both food and medicine science.

[0004] Considering the large market of saffron and its potential medical uses, serious competition over its bountiful markets has led many corporations to study and invest in other parts and byproducts of this Red Gold. Despite being of extraordinary therapeutic and commercial standing, no significant research is available that focuses on enhancing the properties of saffron or developing a stable formulation.

[0005] A need is always appreciated for innovative formulations that have improved stability and physical properties. A need is always appreciated for a formulation that has enhanced properties of saffron.

SUMMARY OF THE INVENTION

[0006] The principal object of the present invention is therefore directed to a formulation of saffron with improved physical properties. [0007] It is another object of the present invention that the formulation is stable.

[0008] It is still another object of the present invention that the formulation has a long shelf life.

[0009] In one aspect, disclosed is a method of preparing a water-in-oil nano-emulsion formulation of essential oils of saffron. The method includes the steps of dropwise adding an aqueous extract of saffron to an emulsified oil phase. Preferably the aqueous extract can be present in the composition at about 15 %. The emulsified oil phase having an oil phase i.e. n- octanol in 75 % concentration and 10 % of the emulsifier combination. The emulsifier combination includes equal proportions of Span 80 and Tween 80.

[0010] In one aspect, an emulsifier combination can be prepared by adding equal proportions of the Span 80 and Tween 80. The emulsifier combination can then be mixed to the oil phase i.e. n-octanol. To the emulsified oil-phase can then be added drop-wise saffron extract while stirring on a magnetic stirrer. The mixture can then be subjected to sonification while cooling the mixture.

[0011] For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there is illustrated preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying figures, which are incorporated herein, form part of the specification and illustrate embodiments of the present invention. Together with the description, the figures further explain the principles of the present invention and to enable a person skilled in the relevant arts to make and use the invention.

[0013] Fig. 1 shows the stability of nano-emulsion samples 4, 6, 7, 8, 9, and 10 over 15 days period.

[0014] Fig. 2 shows the spectrum differences for the samples with small and large sizes. [0015] Fig. 3 is a dynamic light scattering graph of sample 7.

[0016] Fig. 4 is a dynamic light scattering graph of sample 8.

[0017] Fig. 5 is a dynamic light scattering graph of sample 9.

DETAILED DESCRIPTION

[0018] Subject matter will now be described more fully hereinafter. Subject matter may, however, be embodied in a variety of different forms and, therefore, covered or claimed subject matter is intended to be construed as not being limited to any exemplary embodiments set forth herein; exemplary embodiments are provided merely to be illustrative. Likewise, the reasonably broad scope for claimed or covered subject matter is intended. Among other things, for example, the subject matter may be embodied as apparatus and methods of use thereof. The following detailed description is, therefore, not intended to be taken in a limiting sense.

[0019] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Likewise, the term embodiments of the present invention" does not require that all embodiments of the invention include the discussed feature, advantage, or mode of operation.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises", "comprising,", "includes" and/or "including", when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0021] The following detailed description includes the best currently contemplated mode or modes of carrying out exemplary embodiments of the invention. The description is not to be taken in a limiting sense but is made merely for the purpose of illustrating the general principles of the invention since the scope of the invention will be best defined by the allowed claims of any resulting patent.

[0022] Disclosed is a method of preparing a nano-emulsion from the essential oils of saffron. The nano-emulsion can be a water-in-oil emulsion wherein the aqueous phase can be the saffron extract. The aqueous saffron extract can be prepared by known methods. For the emulsification, a combination of span 80 and tween 80 can be used. Span 80 and Tween 80 in equal proportions can be combined and added to the oil phase. The mixture can be stirred to form a solution. To the solution can then be added saffron extract dropwise while stirring the mixture. The mixture can then be subjected to sonification for a predetermined duration. In a preferred embodiment, the mixture can be cooled while sonification to dissipate the heat generated by sonification.

EXPERIMENTAL SECTION

[0023] Materials: Sorbitan monooleate surfactant with commercial name of span80 (SMO- 80) and n-octane, both with 99% purity, purchased from Sigma Aldrich, polysorbate 80 (Tween80) and ethanol with 99% purity, acetone with 96% purity, purchased from Merck. Deionized water was used in all steps of the experimentation.

[0024] Example 1: The emulsification process was evaluated at different concentrations of Span 80 as the emulsifier and n-Octanol as the oil-phase. In the experiment, different sample nano-emulsions were prepared of different concentrations of the Span 80, n-Octanol, and saffron extract shown in Table 1. Span80 was mixed with n-octanol on a magnetic stirrer (Heidolph MR Hei-Standard, made in Germany) at 200rpm for 15 minutes. The Saffron extract was added to the solution dropwise while mixing at the rotational speed of 600rpm for 30 minutes. Subsequently, the beaker containing the mixture was placed in a cold-water bath for dissipating the heat generated by sonification which otherwise may have destructive effects on the compounds of Saffron extract. Probe ultrasonic device (HUP-400, built by the development of ultrasonic technology) was employed for nano-emulsion formulation. The ultrasonic waves were applied in the order of 7 seconds of sonification and 3 seconds of rest for 5 minutes at 150 nominal power of the device.

Table 1. Different nano-emulsion formulation based on Span80 emulsifier:

[0025] Samples 1, 2, S, and 5 were sedimented within a range of S to 10 hours and resulted in a lucid solution. Samples having a concentration of span80 to less than 2% showed higher stability. Samples 4, 6, and 7 were found to be more stable than the samples with concentrations of Span80 of more than 2%. The main factor of low stability of samples 1, 2, 3, and 5 was found to be the low hydrophilicity of span80 emulsifier.

[0026] Example 2: A combination of emulsifiers Span80 and Tween80 in equal proportions was prepared. Sample nano-emulsions using different concentrations of emulsifier combination, n-octanol, and saffron extract were prepared as shown in Table 2. Emulsifier combination was added to the organic phase i.e. n-octanol on a magnetic stirrer (Heidolph- MR Hei-Standard, Germany) at 300rpm for 15 minutes. Then, the saffron extract (5-15%) was added to the solution drop by drop and mixed at 600rpm for 30 minutes. Afterward, the beaker containing the mixture was placed in a cold-water bath to neutralize the generated heat through applying sonic waves and also reduce its probable destructive effects on saffron compounds. In the present experiment, the ultrasonic device (HUP-400, built by the development of ultrasonic technology) was utilized by the order of 7 seconds of application and 3 seconds of rest for 5 minutes at the nominal power of 150W.

Table 2. Different nano-emulsion formulation based on Span80 and Tween80 emulsifiers.

NANOEMULSION STABILITY TEST

[0027] After the preparation of nano-emulsions, 10ml of different samples were poured into screw cap vials and the caps were closed properly to prevent evaporation. The vials were placed at rest. This examination was done 15 days after the nano-emulsification and the stability percentage was obtained through the following equation by calculating the height of the separated layer at the top of the vial (HC) and the height of the primary nano-emulsion

(HE).

[0028] Fig. 1 exhibits the stability percentage of various samples. According to the figure, the stability of samples 4,6, and 7 declined after 15 days since the stability percentage of the three samples were 52%, 61%, and 88% respectively. While in samples 8,9, and 10 there were no conversion in the nano-emulsions stability after 15 days.

[0029] Samples containing only Span80 emulsifier, exposed stability reduction after 15 days, which was due to the emulsifier hydrophobicity and no complete dissolution of the emulsifier in water. The instability of samples was resolved by adding tween80- the hydrophilic emulsifier- to the samples 8,9, and 10 and samples with 100% stability were achieved.

DLS TEST

[0030] DLS analysis is a physical method for the determination of particle distribution in solutions and suspensions. In this method, illustrated in Fig. 2, after the interaction of laser light with the particle, the light scattering, and intensity changes, based on Brownian motion of the particles were evaluated, then, particle size distribution was determined. Explicitly, the Brownian motion of the particles in the DLS test was characterized by calculating the fluctuations in the intensity of light beams scattered by small and large particles. This specification was done through changes in pointwise patterns which is exposed in either light increasing or decreasing of dark and light points. The determination of the intensity of light scattering resulted in particle determination.

[0031] This examination was operated by dynamic light scattering (DLS- Cordon Tech, made in France), at the central laboratory of Amirkabir University. Figs. 3-5 and below table 3 shows the particle size distribution for synthesized samples. Sample No. 7 had an average particle size of 1021nm. But in samples 8 and 9, a particle size reduction to 203nm and 49nm was observed, respectively. In samples 8 and 9 the particle size was decreased from 10% to 20% by adding an emulsifier. Moreover, the relevance of particle size to sample stability could be evaluated. According to the aforementioned fact, sample 7 contained larger particles and the stability was reduced near to 12% during the mentioned time. While as for samples 8 and 9, due to the small nanometric size of particles, magnificent stability was observed, and after the same time, the stability was 100% constant.

Table 3: Analysis from DLS spectra shown on in Figs. 3, 4, 5.