ORGANIC SOL-GEL NANO-COMPOSITION FROM PANAX NOTOGINSENG, RUTIN, GINKGO BILOBA EFFECTIVE IN ENHANCING CEREBRAL AND CARDIOVASCULAR PROTECTION AND METHOD OF PRODUCING THEREOF

Abstract

An organic sol-gel nano-composition containing three solid lipid nano-components loaded with phytochemical active ingredients is proposed. Panax notoginseng saponins are extracted from Vietnamese Panax notoginseng. Rutin is extracted from Styphnolobium japonicum in Thai Binh Province, Vietnam, and Ginkgo biloba is extracted from Ginkgo biloba trees. The composition has shown to have low toxicity and great bioavailability. In particular, the composition is effective against local myocardial ischemias and arrhythmias, improves the blood rheology, platelet circulation and antiaggregation, and thrombosis, lowers the blood lipids to a certain limit, and has good effects against atherosclerosis. The composition has an antioxidant effect of preventing free radical damages, and has an effect of enhancing capillary resistance, strengthening the vessel walls that reduces hypertension risks, which accordingly protects the cerebral nerves, prevents strokes, and is also used to recover after strokes and other hemorrhagic diseases for the enhancement effects and reconstruction of the damaged blood vessels.

Claims

1. An organic sol-gel nano-composition containing Panax notoginseng saponins, rutin, and Ginkgo biloba effective in enhancing cerebral and cardiovascular protection, in which the composition comprise the following: (a) solid lipid nano Ginkgo biloba: 15-30%; (b) solid lipid nano rutin: 2.78%; (c) solid lipid nano Panax notoginseng saponins: 13.89%; (d) soy bean oil: 40-65%; (e) lecithin: 1-10%; (f) Tween 80: 0.1-5%.

2. A process for preparing the composition of claim 1, comprising the following steps: (i) preparing a carrier solution: soy bean oil is added to a reaction flask under heating at 70-100° C., preferably 80° C., and evenly stirred at 250-350 rpm, preferably 300 rpm; then lecithin is added to the reaction flask, boiled and stirred until complete dissolution; after about 30-50 minutes, preferably 45 minutes, the surfactant Tween 80 is further added to the flask, mildly boiled and evenly stirred for about 80-100 minutes, preferably 90 minutes until the carrier solution becomes transparent; (ii) preparing a conjugated lipid nano-presuspension of three active ingredients/carriers: solid lipid nano Panax notoginseng saponins are slowly added to reaction flask, maintained at 80-90° C. under fast stirring at 400-600 rpm, preferably 500 rpm, stirred and boiled until the solid lipid nano Panax notoginseng saponins are completely dissolved; solid lipid nano rutin is further added, maintained at the same temperature and stirring speed until the solid lipid nano rutin is completely dissolved, then, solid lipid nano Ginkgo biloba is added thereto while maintaining the same temperature and stirring speed until the solid lipid nano Ginkgo biloba is completely dissolved; (iii) preparing a conjugated pre-sol-gel lipid nano-composition of three active ingredients: the carrier mixture containing three solid lipid nano components is mixed in about 3-24 hours, wherein the reaction time depends on the content of each product batch: in lab for about 3 hours, semi-industrial production for about 7 hours, and industrial production for 18-24 hours; iv) preparing a conjugated sol-gel lipid nano-composition of three active ingredients: adjust the contents of the organic sol-gel components to achieve the standard technical parameters including the contents of Panax notoginseng saponins, rutin, and Ginkgo biloba; and (v) obtaining a finished composition: the gel formation at gel resolution temperature comprises gelation at normal temperature (room temperature) and storage temperature (below 24° C.), and gel resolution at 35-37° C. (human body temperature).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the chemical structures of the main components of Panax notoginseng saponins.

[0024] FIG. 2 shows the chemical structure of rutin.

[0025] FIG. 3 shows the chemical of Ginkgo biloba.

[0026] FIG. 4 illustrates a process of preparing a composition effective in enhancing cerebral and cardiovascular protection.

[0027] FIG. 5 represents Graph 1: Baseline pain threshold of the mouse batches over time.

[0028] FIG. 6 represents Graph 2: Pain threshold after treatment of the mouse batches over time.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The organic sol-gel nano-composition according to the invention comprises the following components: [0030] (a) Solid lipid nano Ginkgo biloba: 15-30%; [0031] (b) Solid lipid nano rutin: 5-25%; [0032] (c) Solid lipid nano Panax notoginseng saponins: 0.1-10%; [0033] (d) Soy bean oil: 40-65%; [0034] (e) Lecithin: 1-10% [0035] (f) Tween 80: 0.1-5%

[0036] Panax notoginseng saponins (PNS) is extracted from Vietnamese Radix notoginseng (Panax pseudo-ginseng) with ethyl alcohol solvent.

[0037] Panax Notoginseng Saponins (PNS) solid lipid nanoparticles purchased from Vietlife Nano Biological Manufactory have the following parameters: an average nanoparticle size of 9.94 nm, and the PNS content in nanoparticles is over 22%.

[0038] Rutin is extracted in ethyl alcohol solvent from Styphnolobium japonicum (Sophora japonica) from Thai Binh province.

[0039] Rutin solid lipid nanoparticles purchased from Vietlife Biological Nano Manufactory have the following parameters: an average nanoparticle size of 11.25 nm, and the rutin content in the nanoparticles is over 22%.

[0040] Ginkgo biloba is purchased from Shaan Meihe Biochemics Co. Ltd (China).

[0041] Solid lipid Ginkgo biloba (GBLB) nanoparticles purchased from Vietlife Biological Nano Manufactory have the following specifications: an average nanoparticle size of 9.943 nm, and the GBLB content in the nanoparticles is over 22%.

[0042] In addition, there are included excipient materials such as glyceril monostearate, stearic acid, magnesium stearate, Tween 80, lecithin, Gac oil, and soybean oil. All of these are raw materials for use according to Vietnamese standards as pharmaceutical formulative materials in accordance with the regulations.

[0043] Equipments used according to the invention include a heated gelating device, a temperature controller, a vacuum forming device, and a controllable mixer.

[0044] The process for preparing the composition according to the invention comprises the following steps: [0045] Step 1: Preparing a carrier solution: Add soy bean oil to a reaction flask. Heat the solution at 70-100° C., preferably 80° C. Evenly stir at 250-350 rpm, preferably 300 rpm. Next, add lecithin to the reaction flask. Boil and stir until complete dissolution. After about 30-50 minutes, preferably 45 minutes, further add the surfactant Tween 80 to the flask. Mildly boil and evenly stir in about 80-100 minutes, preferably 90 minutes, until the carrier solution becomes transparent. [0046] Step 2: Preparing a conjugated lipid nano-presuspension of three active ingredients/carriers: Slowly add solid lipid nano Panax notoginseng saponins to the reaction flask while maintaining the temperature at 80-90° C. under fast stirring at 400-600 rpm, preferably 500 rpm. The mixture is stirred and boiled until the solid lipid nano Panax notoginseng saponins are completely dissolved. Further add solid lipid nano rutin thereto while maintaining the same temperature and stirring speed until the solid lipid nano rutin is completely dissolved. Then, add solid lipid nano Ginkgo biloba thereto while maintaining the same temperature and stirring speed until the solid lipid nano Ginkgo biloba is completely dissolved. [0047] Step 3: Preparing a conjugated pre-sol-gel lipid nano-composition of three active ingredients: The carrier mixture containing three solid lipid nano components is mixed in about 3-24 hours. The reaction time depends on the content of each product batch: in lab for about 3 hours, semi-industrial production for about 7 hours, and industrial production for 18-24 hours. [0048] Step 4: Preparing a conjugated sol-gel organic lipid nano-composition of three active ingredients: Adjust the contents of the organic sol-gel components to achieve the standard technical parameters, including the contents of Panax notoginseng saponins, rutin, and Ginkgo biloba. [0049] Step 5: Obtaining a finished composition: The gel formation at gel resolution temperature comprises gelation at normal temperature (room temperature), storage temperature (below 24° C.), and gel resolution at 35-37° C. (human body temperature).

EXAMPLES

Example 1: Method of Preparing a Composition According to the Present Invention

[0050] One soft 900 mg capsule of the composition was produced using the following components: [0051] (a) Solid lipid nano Ginkgo biloba: 250 mg; [0052] (b) Solid lipid nano rutin: 25 mg; [0053] (c) Solid lipid nano Panax notoginseng saponins: 125 mg; [0054] (d) Soy bean oil: 450 mg; [0055] (e) Lecithin: 40 mg; [0056] (f) Tween 80: 10 mg.

[0057] 450 mg of soy bean oil was added to a reaction flask, heated at 80° C. while evenly stirring at 300 rpm. Then, 40 mg of lecithin was added to the flask under boiling ad stirring until complete dissolution. 10 mg of Tween 80 was further added to the flask, lowered to below 80° C. and evenly stirred in 90 minutes. 125 mg of solid lipid nano Panax notoginseng saponins was slowly added to the reaction flask, with the temperature maintained at 90° C. and the stirring speed raised to 500 rpm. The stirring and boiling continued until complete dissolution. A similar procedure was conducted with solid lipid nano rutin and solid lipid nano Ginkgo biloba. The mixing was continued for 3 hours. The sol-gel components were adjusted to reach the content standards.

Example 2: Evaluation of the Vasomotor Pain Relieving Effects of the Composition on Chronic Migraine

[0058] In the test, the white mice were randomly divided into 5 batches, each batch having 10 animals: Batch 1 (biological control) was subjected to oral administration of 0.2 ml/10 g water; batch 2 (model) injected 10 mg/kg with nitroglycerin (NTG) was subjected to oral administration of 0.2 mL/10 g water; batch 3 (positive control) injected with 10 mg/kg nitroglycerin was subjected to oral administration of 40 mg/kg propranolol; batch 4 injected with 10 mg/kg nitroglycerin was subjected to oral administration of the composition at dose of 0.96 pill/kg/day (equivalent to the clinical dose); batch 5 injected with 10 mg/kg nitroglycerin was subjected to oral administration of the composition at dose of 2.88 pill/kg/day (3 times as much as the clinical dose).

[0059] Test Results

[0060] Baseline Pain Threshold

TABLE-US-00001 TABLE 1 Effects of the composition on the pain-inducing force in the dolorimeter Pain-inducing force in the dolorimeter (g) Mouse batch nn Day 3 Day 5 Day 7 Day 9 Day 11 Batch 1: 110 7.6 ± 1.7 7.9 ± 0.9 7.5 ± 1.4 7.9 ± 1.2 7.6 ± 1.1 (biological control) Batch 2: 110 7.3 ± 1.6 6.2 ± 0.8 6.4 ± 0.7 6.2 ± 1.1 6.2 ± 0.5 (NTG 10 mg/kg) p in comparison >0.05 <0.001 <0.05 <0.01 <0.01 with the control Batch 3: 40 110 7.6 ± 1.5 6.5 ± 1.1 6.4 ± 1.0 7.1 ± 0.9 7.1 ± 1.0 mg/kg Propranolol p in comparison >0.05 <0.001 >0.05 >0.05 >0.05 with the control p in comparison >0.05 >0.05 >0.05 <0.05 <0.05 with the model Batch 4: 0.96 110 7.1 ± 1.3 7.1 ± 0.7 6.5 ± 1.1 6.3 ± 1.4 6.2 ± 0.9 pill/kg composition p in comparison >0.05 <0.05 >0.05 <0.05 <0.01 with the control p in comparison >0.05 <0.05 >0.05 >0.05 >0.05 with the model p in comparison >0.05 >0.05 >0.05 >0.05 <0.05 with propranolol Batch 5: 2.88 110 7.3 ± 1.1 6.2 ± 0.7 6.5 ± 0.8 6.2 ± 1.1 6.7 ± 0.6 pill/kg composition p in comparison >0.05 <0.001 >0.05 <0.01 <0.05 with the control p in comparison >0.05 >0.05 >0.05 >0.05 <0.05 with the model p in comparison >0.05 >0.05 >0.05 <0.05 >0.05 with propranolol p in comparison >0.05 <0.05 >0.05 >0.05 >0.05 with the low dose

[0061] Graph 1. Baseline Pain Threshold of the Mouse Batches over Time

[0062] Results from Table 1 and Graph showed that the baseline pain threshold of the mice in the model batch lowered at all time points of the study, most remarkably from day 5 to day 11 (p<0.05, p<0.01, and p<0.001).

[0063] The baseline pain threshold of the mouse batch subjected to oral administration of 40 mg/kg propranolol was greater than that of the model batch at almost all time points, most remarkably on day 9 and day 11 (p<0.05).

[0064] The baseline pain threshold of the mouse batch subjected to oral administration of the composition at dose of 0.96 pill/kg was greater than that of the model batch on day 5 (p<0.05), but was not different at other time points (p>0.05).

[0065] The baseline pain threshold of the mouse batch subjected to oral administration of the composition at dose of 2.88 pill/kg was greater than that of the model batch on day 11 (p<0.05), but was not different at other time points (p>0.05).

[0066] The effect of the composition at both doses was poorer in comparison with the 40 mg/kg propranolol.

[0067] Pain Threshold after Treatment

TABLE-US-00002 TABLE 2 Effects of the composition on the pain-inducing force in the dolorimeter Pain-inducing force on the dolorimeter (g) Mouse batch nn Day 3 Day 5 Day 7 Day 9 Day 11 Batch 1: 110 8.3 ± 1.2 7.6 ± 0.7 7.2 ± 1.1 7.8 ± 1.3 7.2 ± 0.7 (biological control) Batch 2: 110 6.1 ± 0.9 5.1 ± 1.1 6.2 ± 0.5 5.8 ± 0.6 5.8 ± 0.8 (NTG 10 mg/kg) p in com- <0.001 <0.001 <0.05 <0.001 <0.01 parison with the control Batch 3: 110 6.8 ± 1.2 7.0 ± 1.0 6.7 ± 0.5 6.7 ± 0.5 7.8 ± 0.7 40 mg/kg Propranolol p in com- <0.05 >0.05 >0.05 <0.05 <0.01 parison with the control p in com- >0.05 <0.01 <0.05 <0.01 <0.001 parison with the model Batch 4: 110 6.2 ± 1.2 6.4 ± 1.3 6.1 ± 1.1 6.6 ± 0.7 6.7 ± 1.0 0.96 pill/kg composition p in com- <0.001 <0.05 >0.05 <0.05 >0.05 parison with the control p in com- >0.05 <0.05 >0.05 <0.05 <0.05 parison with the model p in com- >0.05 >0.05 >0.05 >0.05 <0.05 parison with propranolol Batch 5: 110 6.0 ± 1.1 6.0 ± 0.8 6.8 ± 0.6 6.3 ± 0.3 7.4 ± 1.4 2.88 pill/kg composition p in com- <0.001 <0.01 >0.05 <0.01 >0.05 parison with the control p in com- >0.05 <0.05 <0.05 <0.05 <0.01 parison with the model p in com- >0.05 >0.05 >0.05 >0.05 >0.05 parison with propranolol p in com- >0.05 >0.05 >0.05 >0.05 >0.05 parison with the low dose

[0068] Graph 2. Pain Threshold after Treatment of Mouse Batches over Time

[0069] Results from Table 2 and Graph 2 showed that:

[0070] The pain threshold after NTG injection of the mice in the model batch was remarkably lower at all time points of the study (p<0.05, p<0.01, and p<0.001).

[0071] Pain threshold after the oral drug administration of the mice in the oral 40 mg/kg propranolol batch was greater than that of the model batch at all time points, which was most remarkable from day 5 to day 11 (p<0.05, p<0.01, and p<0.001).

[0072] The pain threshold after the oral drug administration of the mice in the oral 0.96 pill/kg composition batch was greater than that of the model batch at almost all time points, most remarkably on day 5, day 9, and day 11 (p<0.05). The effect was poor in comparison with the 40 mg/kg propranolol.

[0073] The pain threshold after the oral drug administration of the mice in the oral 2.88 pill/kg composition batch was greater than that of the model batch at almost all time points, most remarkably from day 5 to day 11 (p<0.05, and p<0.01). The effect was equivalent to the 40 mg/kg propranolol.

[0074] Conclusion

[0075] The composition at doses of 0.96 pill/kg and 2.88 pill/kg were effective in lowering the pain threshold after treatment in the chronic migraine model.

[0076] The composition at dose of 2.88 pill/kg was more effective than at dose of 0.96 pill/kg and was equivalent to the 40 mg/kg propranolol.

Example 3: Studies on the Sedative Effect of the Composition on the Advanced Plus Model

[0077] In the test, the white mice were randomly divided into 4 batches, each batch having 10 animals: Batch 1 (biological control) was subjected to oral administration of 20 mL/kg/day distilled water; batch 2 (positive control) was subjected to oral administration of 2.4 mg/kg/day diazepam; batch 3 was subjected to oral administration of the composition at dose of 0.96 pill/kg/day (equivalent to the clinical dose); batch 4 was subjected to oral administration of the composition at dose of 2.88 pill/kg/day (3 times as much as the clinical dose).

[0078] Results

TABLE-US-00003 TABLE 3 Effect of the composition on the number of times and time of mouse introduction into the the close branch Number of times of mouse Time of mouse introduction introduction into the close branch into the close branch (second) After 7 days After 7 days Before oral of oral Before oral of oral administration administration administration administration Mouse batch n of the drug of the drug of the drug of the drug Batch 1: 10 11.10 ± 10.50 ± 176.70 ± 182.10 ± Biological control 2.56 2.80 33.77 36.27 Batch 2: Diazepam 10 11.50 ± 6.40 ± 173.70 ± 121.20 ± at 2.4 mg/kg/day 2.32 2.07 36.50 27.18 p in comparison >0.05 <0.01 >0.05 <0.01 with batch 1 Batch 3: 10 10.60 ± 7.50 ± 187.10 ± 136.70 ± Composition 3.34 2.42 41.02 40.32 of 0.96 pill/kg/day p in comparison >0.05 <0.05 >0.05 <0.05 with batch 1 p in comparison >0.05 >0.05 >0.05 >0.05 with batch 2 Batch 4: 10 10.90 ± 6.10 ± 182.10 ± 124.90 ± Composition 2.38 2.02 39.03 26.48 of 2.88 pill/kg/day p in comparison >0.05 <0.01 >0.05 <0.01 with batch 1 p in comparison >0.05 >0.05 >0.05 >0.05 with batch 2

[0079] Results from Table 3 showed that after 7 days of oral administration of 2.4 mg/kg/day diazepam and the composition at doses of 0.96 pill/kg/day and 2.88 pill/kg/day, the number of times and time mouse introduction into the close end was lower in comparison with the drug pretest and in comparison with the biological control batch, wherein the difference was statistically significant (p<0.05). The effect of the composition at both doses was equivalent to the 2.4 mg/kg/day diazepam.

TABLE-US-00004 TABLE 4 Effect of the composition on the number of times and time of mouse introduction into the open branch Number of times of mouse Time of mouse introduction introduction into the open branch into the open branch (second) After 7 After 7 Before oral days of oral Before oral days of oral administration administration administration administration Mouse batch n of the drug of the drug of the drug of the drug Batch 1: Biological 10 3.90 ± 3.70 ± 84.50 ± 80.40 control 1.20 1.06 24.66 26.73 Batch 2: Diazepam 10 4.30 ± 6.90 ± 90.40 ± 150.10 ± at 2.4 mg/kg/day 1.42 2.33 29.51 26.36 p in comparison >0.05  <0.001 >0.05  <0.001 with batch 1 Batch 3: 10 3.70 ± 6.10 ± 81.70 ± 131.80 ± Composition 1.16 2.13 26.59 39.40 of 0.96 pill/kg/day p in comparison >0.05 <0.05 >0.05 <0.01 with batch 1 p in comparison >0.05 >0.05 >0.05 >0.05 with batch 2 Batch 4: 10 4.10 ± 6.50 ± 83.80 ± 148.40 ± Composition 1.10 2.12 27.58 33.08 of 2.88 pill/kg/day p in comparison >0.05 <0.01 >0.05  <0.001 with batch 1 p in comparison >0.05 >0.05 >0.05 >0.05 with batch 2

[0080] Results from Table 4 showed that after 7 days of oral administration of 2.4 mg/kg/day diazepam and the composition at doses of 0.96 pill/kg/day and 2.88 pill/kg/day, the number of times and time of mouse introduction into the intake end increased in comparison with the drug pretest and in comparison with the biological control batch, wherein the difference was statistically significant (p<0.05). The effect of the composition at both doses was equivalent to the 2.4 mg/kg/day diazepam.

TABLE-US-00005 TABLE 5 Effect of the composition on avoidance rate at the open branch of the mice Avoidance rate at the open branch After 7 days Before oral of oral administration administration Mouse batch n of the drug of the drug p.sub.before−after Batch 1: Biological 10 70.47 ± 7.55 71.37 ± 7.75 >0.05 control Batch 2: Diazepam 10 69.21 ± 8.88 46.37 ± 9.21 <0.0001 at 2.4 mg/kg/day p in comparison with >0.05 <0.0001 batch 1 Batch 3: Composition 10 71.44 ± 8.08 52.90 ± 7.84 <0.001 of 0.96 pill/kg/day p in comparison with >0.05 <0.0001 batch 1 p in comparison with >0.05 >0.05 batch 2 Batch 4: Composition 10 70.20 ± 9.23 47.15 ± 9.96 <0.0001 of 2.88 pill/kg/day p in comparison with >0.05 <0.0001 batch 1 p in comparison with >0.05 >0.05 batch 2

[0081] Results from Table 5 showed that after 7 days of oral administration of 2.4 mg/kg/day diazepam and the composition at doses of 0.96 pill/kg/day and 2.88 pill/kg/day, the avoidance rate at the intake end of the mice reduced in comparison with the drug pretest and in comparison with biological control batch, wherein the difference was statistically significant (p<0.001). The effect of the composition at both doses was equivalent to the 2.4 mg/kg/day diazepam.

Example 4: Anticonvulsant Effect Evaluation of the Composition

[0082] In the test, the white mice were randomly divided into 4 batches, each batch having 10 animals: Batch 1 (biological control) was subjected to oral administration of 20 mL/kg/day distilled water; batch 2 (positive control) was subjected to oral administration of 2.4 mg/kg/day diazepam; batch 3 was subjected to oral administration of the composition at dose of 0.96 pill/kg/day (equivalent to the clinical dose); batch 4 was subjected to oral administration of the composition at dose of 2.88 pill/kg/day (3 times as much as the clinical dose). After the mice were injected with nikethamide, the time of convulsion onset and the death time of the mice were taken.

TABLE-US-00006 TABLE 6 Effects of the composition on the time of convulsion onset and the death time of the mice after nikethamide injection Time of Death time of convulsion the mice after onset nikethamide injection Mouse batch n (minute) (minute) Batch 1: Mô hình 10 1.37 ± 0.36 8.31 ± 2.41 Batch 2: Diazepam 10 2.08 ± 0.55 14.91 ± 3.67  at 2.4 mg/kg/day p in comparison with <0.01 <0.001 batch 1 Batch 3: Composition 10 1.41 ± 0.44 8.83 ± 2.55 at dose of 0.96 pill/kg/day p in comparison with >0.05 >0.05 batch 1 p in comparison with <0.05 <0.05 batch 2 Batch 4: Composition 10 1.45 ± 0.48 8.92 ± 2.72 at dose of 2.88 pill/kg/day p in comparison with >0.05 >0.05 batch 1 p in comparison with <0.05 <0.05 batch 2

[0083] Results from Table 6 showed that 2.4 mg/kg/day diazepam took effects in prolonging the time of convulsion onset and the death time of the mice after nikethamide injection, which was statistically significant in comparison with the model batch (p<0.05).

[0084] The composition at doses of 0.96 pill/kg/day and 2.88 pill/kg/day had a tendency to prolong the time of convulsion onset and the death time of the mice after nikethamide injection in comparison with the model batch, but the difference was not statistically significant with p>0.05.