A Pickering Emulsion Comprising Nano Crystals of Silybin and A Method of Preparation Thereof CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims benefit of Chinese application having Serial No. 201410064969.5 filed on 25 Feb 2014, which is hereby incorporated by reference herein in its entirety. FIELD OF INVENTION [0002] This invention relates to a pharmaceutical formulation. In particular, this invention relates to a Pickering emulsion and a method of preparation thereof BACKGROUND OF INVENTION [0003] Silybin is a flavonolignans compound extracted from Silybum marianum (L) Genrtn. The structure of silybin is. N0 H OH [0004] Silybin is a hepatocellular membrane stabilizer, which can effectively protect and enhance the hepatocellular mucous membrane, protect the enzyme system of hepatic cells and scavenge reactive oxygen free radicals in hepatic cells. Silybin can also reduce the concentration of blood cholesterol and further scavenge the fat deposit in liver tissue. It has been mainly used in clinical treatment of hepatitis and cholecystitis. Silybin has been recently demonstrated to have anti-tumor effect, especially to prostatic cancer. Therefore, silybin has generated great interest since its discovery. However, its low water solubility 1 severely affects the absorption and distribution thereof in vivo; its low oral bioavailability severely weakens the efficacy thereof [0005] Silybin is commonly administered to patients as tablets and capsules. It has been demonstrated that such formulations are of low bioavailability, i.e. around 5%, which severely limit the efficacy thereof To date, new formulations of silybin mainly include emulsion, microemulsion, self-microemulsion, microspheres and so on. However, the preparation processes of these formulations are complex, presenting challenges in quality control. In addition, organic solvents, represented by surfactant, are used in large quantity. Surfactant may lead to side effects, such as, allergy, hemolysis, intestinal cytoxicity, etc., and thus surfactant is not suitable for long-term use. SUMMARY OF INVENTION [0006] In the light of the foregoing background, it is an object of the present invention, in view of the low water solubility of silybin and disadvantages of current oral formulation, to provide a new type of oral formulation, i.e. a Pickering emulsion comprising nano crystals of silybin and a method of preparation thereof This preparation process is simple. The composition of the formulation is simple, and does not include organic solvents, such as surfactant. [0007] Accordingly, the present invention, in one aspect, provides a pharmaceutical formulation comprising, [0008] a) a carrier solution comprising emulsion of water phase and oil phase; and [0009] b) nano crystals of silybin which stabilize said emulsion; [0010] wherein the ratio of mass of said silybin to volume of said oil phase is in the range of 60:1-200:1 (m/v, mgml- 1 ), wherein said formulation is free of additional surfactant. [0011] In an exemplary embodiment, the oil phase is selected from the group consisting of Capmul MCM C8 (glyceryl mono caprylate), Capmul MCM C1O (glyceryl mono 2 caprate), Capmul MCM (medium chain mono-and diglyceride), CAPTEX 355 (glyceryl tricaprylate/caprate/laurate), MAISINE 35-1 (Glyceryl monolinoleate) and soybean oil. [0012] In an exemplary embodiment, the ratio of mass of the silybin to volume of the oil phase is in the range of 60:1-160:1. [0013] In a further aspect of the present invention, a method of preparing a silybin Pickering emulsion is provided, comprising [0014] a) mixing silybin with water; after vortex, shearing for 1.5-2.5 min at the speed of 12000-14000rpm.min- 1 by high speed shearing machine, to obtain primary suspension; and [0015] b) mixing said primary suspension with oil phase, and homogenizing the mixture for 8-20 cycles under the pressure of 600-1200bar to obtain said emulsion. [0016] In an exemplary embodiment, the primary suspension is processed into suspension of nano crystals by homogenizing said primary suspension for 8-20 cycles under the pressure of 600-1200bar, before mixing with oil phase. [0017] In an exemplary embodiment, the oil phase is selected from the group consisting of Capmul MCM C8, Capmul MCM C1O, Capmul MCM, CAPTEX 355, MAISINE 35-1 and soybean oil. [0018] In a further exemplary embodiment, the homogenizing pressure of the high pressure homogenizer in steps (a) and (b) is 900-1000bar; the homogenization goes through 8-12 cycles. [0019] In an exemplary embodiment, the size of nano crystal of silybin wrapping on the surface of oil phase in the Pickering emulsion is 100nm-400nm. The size of emulsion droplet in the Pickering emulsion is 10pim- 1 00ptm. 3 [0020] In a further exemplary embodiment, the size of nano crystal of silybin wrapping on the surface of oil phase in the Pickering emulsion is 310nm-370nm. The size of emulsion droplet in the Pickering emulsion is 25ptm-70ptm. [0021] The advantages and beneficial effects of the Pickering emulsion and the preparation method thereof in the present invention include the followings: [0022] (1) The preparation method of a Pickering emulsion disclosed in the present invention uses high-pressure homogenization method, through a large number of experiments and researches by the inventor, to obtain the primary suspension, nano crystals, and the optimal condition to prepare a Pickering emulsion by nano crystals and preferred oil phase. Nano crystals of silybin in the resulting Pickering emulsion are adsorbed to the surface of oil droplets to make the oil droplets wrapped by silybin. Silybin is partially dissolved in the oil phase, and partially in the form of nano crystals adsorbed to the surface of oil droplets, which keeps oil droplets stably dispersed in water. The size of the emulsion droplet formed by oil droplet and nano crystal is 10 im-100 im, which makes the emulsion droplets stably dispersed in water to form stable emulsion. Silybin in the emulsion is active ingredient and also plays a role in stabilizing the emulsion. [0023] (2) The Pickering emulsion comprising nano crystals of silybin in the present invention does not contain any surfactant or organic solvents. It has good stability, rapid release rate and sound bioavailability. BRIEF DESCRIPTION OF FIGURES [0024] Figure 1 shows images of the Pickering emulsion comprising nano crystals of silybin captured by optical microscopy; the one on the right is a partially enlarged view. [0025] Figure 2 shows images of the Pickering emulsion comprising nano crystals of silybin captured by scanning electron microscope. Figure 2B is a partially enlarged view of Figure 2A while Figure 2C is a partially enlarged view of Figure 2B. 4 [0026] Figure 3 shows images of the Pickering emulsion comprising nano crystals of silybin captured by laser confocal microscopy. [0027] Figure 4 shows the in vitro release curves of Pickering emulsion comprising nano crystals of silybin (m), nano crystals of silybin (A) and crude ingredients (e) (Figure 4A shows the in vitro release curve in simulated gastric fluid while Figure 4B shows the in vitro release curve in simulated intestinal fluid). [0028] Figure 5 shows the in vivo pharmacokinetics curves of Pickering emulsion comprising nano crystals of silybin (A), nano crystals of silybin (o) and crude ingredients (0) in rats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0029] As used herein and in the claims, "comprising" means including the following elements but not excluding others. [0030] The water in the present invention is ultrapure water. The oil phase and silybin are both pharmaceutical grade. [0031] In this invention, the inclusion of oil phase with nano crystals of silybin was characterized by the combination of optical microscopy and laser confocal microscopy. The mechanism of inclusion of oil phase with nano crystals of silybin was studied. The oil phase wrapped with nano crystals of silybin and the external morphological characteristics thereof were observed by scanning electron microscope. The short-term stability and long-term stability of the Pickering emulsion comprising nano crystals of silybin as prepared in the present invention were investigated. The Pickering emulsion was demonstrated to have a more rapid release rate compared with the crude ingredients by the in vitro release test. The oral bioavailability of the Pickering emulsion compared with the conventional formulation was investigated through animal experiments. [0032] The present invention will be further illustrated by the following examples. Example 1 Method of preparing Pickering emulsion 5 [0033] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0034] (1) Weighing 300mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 2 min at the speed of 13000rpm-min- 1 by high speed shearing machine, to obtain primary suspension A; [0035] (2) Homogenizing the primary suspension A for 10 cycles under the pressure of 1 000bar to obtain suspension B of nano crystals; [0036] (3) Mixing suspension B with 5ml Capmul MCM C8, and homogenizing the mixture for 10 cycles under the pressure of 1 000bar to obtain the Pickering emulsion. [0037] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 35.8±4.2ptm, wherein the average size of the nano crystal is 336.3+20.7nm. Example 2 Method of preparing Pickering emulsion [0038] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0039] (1) Weighing 800mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 2 min at the speed of 13000rpm-min- 1 by high speed shearing machine, to obtain primary suspension A; [0040] (2) Homogenizing the primary suspension A for 10 cycles under the pressure of 1 000bar to obtain suspension B of nano crystals; [0041] (3) Mixing suspension B with 5ml Capmul MCM C8, and homogenizing the mixture for 10 cycles under the pressure of 1 000bar to obtain the Pickering emulsion. [0042] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 32.4±2.7ptm, wherein the average size of the nano crystal is 350.4+4.8nm. 6 Example 3 Method of preparing Pickering emulsion [0043] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0044] (1) Weighing 800mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 2 min at the speed of 13000rpm-min- 1 by high speed shearing machine, to obtain primary suspension A; [0045] (2) Homogenizing the primary suspension A for 10 cycles under the pressure of 1 000bar to obtain suspension B of nano crystals; [0046] (3) Mixing suspension B with 5ml Capmul MCM C1O, and homogenizing the mixture for 10 cycles under the pressure of 1 000bar to obtain the Pickering emulsion. [0047] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 30.5±2.8jim, wherein the average size of the nano crystal is 338.4±14.3nm. Example 4 Method of preparing Pickering emulsion [0048] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0049] (1) Weighing 800mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 2 min at the speed of 13000rpm min- 1 by high speed shearing machine, to obtain primary suspension A; [0050] (2) Mixing the primary suspension A with 5ml Capmul MCM C8, and homogenizing the mixture for 10 cycles under the pressure of 1000bar to obtain the emulsion. [0051] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 28.6+3.1pjm, wherein the average size of the nano crystal is 326.4±5.2nm. 7 Example 5 Method of preparing Pickering emulsion [0052] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0053] (1) Weighing 800mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 2 min at the speed of 13000rpm-min-1 by high speed shearing machine, to obtain primary suspension A; [0054] (2) Mixing the primary suspension A with 5ml Capmul MCM C1O, and homogenizing the mixture for 10 cycles under the pressure of 1000bar to obtain the formulation. [0055] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 30.5+2.8gm, wherein the average size of the nano crystal is 344.5±21.2 nm. Example 6 Method of preparing the Pickering emulsion [0056] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0057] (1) Weighing 1000mg crude silybin and adding to 80ml ultrapure water, after vortex for 2 min, shearing for 2.5 min at the speed of 14000rpmmin- 1 by high speed shearing machine, to obtain primary suspension A; [0058] (2) Homogenizing the primary suspension A for 20 cycles under the pressure of 1200bar to obtain suspension B of nano crystals; [0059] (3) Mixing suspension B with 5ml Capmul MCM, and homogenizing the mixture for 20 cycles under the pressure of 1200bar to obtain the Pickering emulsion. [0060] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 11.7+1.4pm, wherein the average size of the nano crystal is 116.9+13.6nm. 8 Example 7 Method of preparing the Pickering emulsion [0061] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0062] (1) Weighing 600mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 2.5 min at the speed of 14000rpmmin- 1 by high speed shearing machine, to obtain primary suspension A; [0063] (2) Mixing the primary suspension A with 5ml CAPTEX 355, and homogenizing the mixture for 20 cycles under the pressure of 1200bar to obtain the emulsion. [0064] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 15.8+2.2pm, wherein the average size of the nano crystal is 226.4+3.8nm. Example 8 Method of preparing the Pickering emulsion [0065] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0066] (1) Weighing 300mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 1.5 min at the speed of 12000rpmmin- 1 by high speed shearing machine, to obtain primary suspension A; [0067] (2) Homogenizing the primary suspension A for 8 cycles under the pressure of 600bar to obtain suspension B of nano crystals; [0068] (3) Mixing suspension B with 5ml MAISINE 35-1, and homogenizing the mixture for 8 cycles under the pressure of 600bar to obtain the Pickering emulsion. [0069] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 90.5+5.8gm, wherein the average size of the nano crystal is 383.6+15.9nm. 9 Example 9 Method of preparing the Pickering emulsion [0070] A method of preparing a Pickering emulsion comprising nano crystals of silybin comprises the following steps: [0071] (1) Weighing 400mg crude silybin and adding to 80ml ultrapure water; after vortex for 2 min, shearing for 1.5 min at the speed of 12000rpmmin- 1 by high speed shearing machine, to obtain primary suspension A; [0072] (2) Mixing the primary suspension A with 5ml soybean oil, and homogenizing the mixture for 10 cycles under the pressure of 800bar to obtain the emulsion. [0073] The particle size was measured by photon correlation spectroscopy (PCS). The average size of the obtained emulsion is 88.6+10.1gm, wherein the average size of the nano crystal is 379.4+19.8nm. Example 10 Study on the inclusion of oil phase with nano crystals of silybin [0074] 1. Objective [0075] Particle size distribution and the inclusion of oil droplets with nano crystals of silybin in the Pickering emulsion as prepared in Example 2 were observed and studied by optical microscope and laser confocal microscope. [0076] 2. Methods [0077] A. Study on the particle size distribution in Pickering emulsion [0078] The Pickering emulsion comprising nano crystals of silybin as prepared in Example 2 was placed under the optical microscope for image acquisition. The size of particles was analyzed statistically by Image Pro Plus 6.0. [0079] B. Study on the inclusion of oil droplets with nano crystals of silybin [0080] A small amount of the Pickering emulsion comprising nano crystals of silybin as prepared in Example 2 was placed on the surface of silicone membrane. After natural 10 evaporation, the surface was treated by gold sputtering process, and then observed by scanning electron microscope. [0081] In addition, when preparing the Pickering emulsion comprising nano crystals of silybin in Example 2, a small amount of Nile red dye was added to oil phase for fluorescence labeling. The resulting product was captured through fluorescent location by laser confocal microscope. [0082] 3. Results [0083] A. The particle size distribution in Pickering emulsion [0084] The result on the particle size distribution in Pickering emulsion is shown in Fig. 1 (the right one is a partially enlarged view). From Fig. 1, the result observed under optical microscope shows that the size of emulsion droplet in the Pickering emulsion comprising nano crystals of silybin is in the range of 64.3±3.5p.m-27.4±2.8p.m. The particle size was determined through image analysis method by optical microscope in this Example, thus the determined size would be different from the average size determined by photon correlation spectroscopy (PCS). If the size determined by the latter method falls within the scope of the size determined by the former one, the results obtained by these two methods will be considered as substantially consistent. [0085] B. The inclusion of oil droplets with nano crystals of silybin [0086] The images of the Pickering emulsion comprising nano crystals of silybin under scanning electron microscope are shown in Fig.2 (Fig.2B is a partially enlarged drawing of Fig.2A; Fig.2C is a partially enlarged drawing of Fig. 2B). Fig. 2 further illustrates Fig. 1 as captured by optical microscope and the distribution of particle size, and proves that nano crystals of silybin are completely adsorbed on the surface of oil droplets and completely wrap oil droplets. [0087] The fluorescence-labeled image of the Pickering emulsion comprising nano crystals of silybin under scanning electron microscope is shown in Fig.3. The two images 11 on the right side of Fig. 3 are fluorescence distribution images of the emulsion, and the two images on the left side are images of the emulsion. It can be seen from Fig.3 that fluorescence distribution coincides with the images of the emulsion, which shows that nano crystals of silybin completely wrap oil phase. Example 11 Release test in vitro [0088] 1. Objective [0089] The release rates of the Pickering emulsion comprising nano crystals of silybin, nano crystals of silybin, and crude silybin were studied by release test in vitro. [0090] 2. Methods [0091] The method of release test in vitro refers to the stirring paddle method used in drug release test in Chinese pharmacopoeia, with the speed at 100rpm and the temperature at 37'C C [0092] Each of crude silybin and the Pickering emulsion comprising nano crystals of silybin as prepared in Example 1 was added to 900ml of simulated intestinal fluid and 900ml simulated gastric fluid, respectively. 2ml of sample was taken from simulated gastric fluid at the time point of 5min, 10min, 15min, 30min, 1h and 2h, respectively. 2ml of sample was taken from simulated intestinal fluid at the time point of 5min, 10min, 15min, 30min, lh, 2h, 4h and 6h, respectively. The samples were filtered by 0.22p.m PVDF millipore filter. Primary filtrate was removed. Samples were analyzed by High Performance Liquid Chromatography (IHPLC). [0093] 3. Results [0094] The results are shown in Fig.4. It can be seen from Figs.4A and 4B that the release rates of the Pickering emulsion comprising nano crystals of silybin in both simulated gastric fluid and simulated intestinal fluid are higher than that of crude silybin. The Pickering emulsion comprising nano crystals of silybin in simulated gastric fluid and simulated intestinal fluid at the time point of 2h releases 69.9% and 80.2%, respectively, 12 while at this time point crude ingredients only release 22% and 33%, respectively. This shows that the release rate of the emulsion is significantly higher than that of crude ingredients. It also can be seen from Figs.4A and 4B that the release rates of the Pickering emulsion comprising nano crystals of silybin in both simulated gastric fluid and simulated intestinal fluid are higher than that of nano crystals of silybin. Higher release rate is beneficial for the absorption of drug and the increase of oral bioavailability. Example 12 Study on the pharmacokinetics of rats in vivo [0095] 1. Objective [0096] The bioavailability of the Pickering emulsion comprising nano crystals of silybin was studied by comparison experiments. [0097] 2. Methods [0098] Rats (each has a weight of about 200g) were divided into 3 groups in a random manner. 10 rats in parallel were used at each time point. Samples were administrated intragastrically to rats. Rats fasted for 12 hours before administration with access to drinking water. The first group was a suspension of crude silybin (10mg crude ingredients were dispersed into 1.5ml aqueous solution containing 0.25% hypromellose ). The second group was a suspension of nano crystals of silybin as prepared according to the method in Example 2. The third group was the Pickering emulsion comprising nano crystals of silybin as prepared according to the method in Example 2. The dose at 50mg/kg (50mg silybin per 1 kg of actual weight of rat) for each group was administrated. [0099] 500ptl orbital venous blood was collected at 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12h after administration, respectively, placed into centrifuge tube containing heparin sodium, and then centrifuged for 15min (3500rpm). 200 pl supernatant was taken, and added with 50ptl 1-naphthol solution (9.95 ptg/ml, internal standard) and 200 ptl potassium dihydrogen phosphate solution (0. 1mol/L). After vortex for mixture for 2min, 4ml ether was added and mixed by vortex for 5min. Organic layer was isolated by centrifuge for 10min at 5000rpm, dried by nitrogen, added with 200ptl mobile phase, mixed by vortex, 13 and then centrifuged at 5000rpm for 10min. Supernatant was isolated and the content of silybin was determined by HPLC. [00100] 3. Results [00101] The results on AUC (Area Under the Curve), Cmax (peak concentration) and MRT (Mean Residence Time) for three kinds of samples are shown in Fig. 5 and Table 1. It can be seen from the variance analysis of these results that the parameters AUC, Cmax and MRT for the Pickering emulsion comprising nano crystals of silybin have highly significant difference compared with those of crude ingredients (p<0.01). The AUC result of the Pickering emulsion comprising nano crystals of silybin has highly significant difference compared with that of nano crystals of silybin (p<0.01), however, there is no significant difference on Cmax and MRT. The relative bioavailability of the Pickering emulsion comprising nano crystals of silybin are increased 1.60-fold and 4.02-fold, compared with that of nano crytals of silybin and crude ingredients, respectively. MRT of rats in vivo for the Pickering emulsion comprising nano crystals of silybin was extended 2.03-fold compared with crude ingredients. It shows that the oral bioavailability of the Pickering emulsion comprising nano crystals of silybin increases significantly compared with that of crude ingredients or nano crystals. [00102] Table 1 The parameters of pharmacokinetics in rats in vivo for the Pickering emulsion comprising nano crystals of silybin, nano crystals and crude ingredients thereof AUC(0-t) AUC(0-o) Tmax Cmax T/2 MRT (ng/ml*h) (ng/ml*h) (h) (ng/ml) (h) (h) Suspensionof crude 202.3±18.7 235.8±12.3 3 81.4±10.1 1.3±0.5 3.3±0.2 ingredients Nano crystals 508.1±47.1 628.9±15.1 6 73.8±4.5 3.6±0.5 6.4±0.4 Pickering emulsion 812.7±25.1 898.2±25.1 5 184.9±8.2 2.5±0.4 6.7±0.3 [00103] The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific 14 details. Hence this invention should not be construed as limited to the embodiments set forth herein. [00104] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and disclose specific information for which the reference was cited in connection with. [00105] The practice of the invention is exemplified in the above non-limiting examples. The scope of the invention is defined solely by the appended claims, which are in no way limited by the content or scope of the examples. <DocRef#00123529-CX > 15