| ObjectiveExocarpium Citri Grandis (ECG)-Citrus grandis’tomentosa’is one of the genuine medicinal materials from our province. One of its characteristics is its rich content of flavonoid compositions. The purpose of this research is to extract its effective part, to research systematically on the development of Exocarpium citri Gandis antioxidant health foods, and to lay down a solid research foundation as well as the scientific basis for the development and utilization of the precious and rare medicinal material resource.MethodsThe reasonable prescription composition and proportion was determined, and formula optimization design was carried out by the application of the Peroxyl Radical Scavenging Capacity (PSC) Method for the antioxidation evaluation on different formulae, and the application of the Caco-2cell model for the examination on the transport mechanism of both naringenin and the formula.The quality specifications for the crude drug was standardized by the application of the UPLC method for the simultaneous determination of the content of active components from the ECG as naringin, rhoifolin, naringenin and apigenin etc.; the application of the three methods of health food method, pharmacopeia method and SBC method for the comparison study on the determination of total flavonoids, and for the fixing of the most optimized method for the determination of total flavonoids content; the application of HPLC method for the setting up of the chromatograph fingerprint database for the different cultivars of ECG.The most optimized process parameters were fixed and the technology roadmap was formulated by the application of the Central Composite Design plus response surface methodology in the total flavonoids extraction process research, selecting the three factors of ethanol concentration, ethanol dosage and pH value, taking the extraction rate of the total flavonoids and the flavonoid clearance rate as the responses, having the best extraction process for the total flavonoids from ECG thus determined; and by the application of the Box-Behnken experimental design plus response surface methodology for the optimization of the naringinase enzymolysis technology, selecting the four factors of the enzyme quantity, pH value, temperature and time, taking the debittering power as the response; and by the application of the rotary-type tablet granulating machine for tabletting, the application of the Box-Behnken experimental design principles, selecting the four factors of acid alkaline proportion, the mannitol consumption quantity, PEG6000consumption quantity and PVP-K30concentration, taking the hardness and disintegration time as the responses, having the effervescent tablet preparation process thus optimized.The quality standard for ECG-Vit C effervescent tablet was established and tested by the application of UPLC method for the rapid determination of naringenin and apigenin content within the ECG-Vit C effervescent tablets, by the application of TLC method for the identification of naringenin and apigenin within the ECG-Vit C effervescent tablets, by the application of the total flavonoids determination method regulated by the Inspection and Assessment Standard for Health Food issued by the Country for the determination of the total flavonoids content in the ECG-Vit C effervescent tablets and, by the application the determination method and requirements on disintegration, weight and variation from the Pharmacopoeia of China2010Revision.The antioxidant function evaluation was researched according to the requirements from the Inspection and Assessment Standard for Health Food2003Revision. The antioxidant activity of the product under the research was initially assessed by the application of the bromobenzene liver lipid peroxidation mice model, and the application of kit assay method for the determination of the content of lipid peroxide degradation products MDA&LPO, and antioxidant enzyme indexes as SOD&GSH-Px etc. within the blood and the liver.Results1Formula designThe total flavonoids of ECG, such as naringin, rhoifolin and naringenin were selected as the effective components for the realization of the antioxidant function. Vitamin C and myo-inositol-the unique component from ECG were selected as the source of nutrition for vitamin supplement. The unique and advanced biological enzyme technology was applied for the debittering of ECG extracts. Aspartame and mannitol were aselected as the composite sweetener. The pH value was controlled by regulating the ratio of citric acid and sodium bicarbonate.Use the ECG naphtha beta entrapped cyclodextrin complex as the fragrance-imparting substance. Keep the claybank color which is inherent in ECG flavonoids. Take citric acid-tartaric acid, sodium bicarbonate polyethylene glycol clathrate compound as the effervescing agent. Take the sodium carboxymethyl starch as the disintegrating agent for the effervescent tablet. With organic integration according to the scientific theories, through the synergy effect from naringenin, a better antioxidant function is reached with the matching of a small amount of Vitamin C with naringenin.The formula proportioning is optimized with the advanced PSC method and concluded that the best matching is debittered flavonoids vs. Vitamin C=2:1when the antioxidant activity was equal to65.219umol Trolox PSC value. The approval from Caco-2cell model reveals that the combination with vitamin C enhances the naringenin in absorption and reduces its exocytosis action.2Quality specifications of the crude drug2.1The position of sampling has a certain impact on the content of active components, among which the impact on naringin shoed more regularity, e.g. there is always a relatively higher content of naringin in samples from the sampling positions at the top. There are different level of flavonoids from different ECG cultivars, among which Jia Xiyang has higher naringin, and Huang Rongguo has higher rhoifolin, naringenin and apigenin.2.2By comparison on different methods for the determination of total flavonoids it is shown that the output value from the Pharmacopoia method is remarkably higher than the values from Health food method and SBC method. SBC method is an advanced internationally recognized determination method for total flavonoids. However, as it has relatively more steps of reaction, the stability of it still needs to be further determined. Health food method is a kind of direct measurement without coloration. It’s accurate in output, simple in operation. So it is a reasonable determination method. Through synthetic consideration, we select the Health food method for the determination of the total flavonoids content in ECG2.3The HPLC fingerprint database for50batches of ECG was established. When the data is processed with the A edition of the Similarity Evaluation System, the similarity of HPLC fingerprint of ECG from the different cultivars are all higher than0.93. Similarity calculation was carried out between the reference fingerprint for each of the cultivars and the fingerprint for the50batches under the "TCM Chromatographic Fingerprint Similarity Evaluation System2004B Revision", and the ones with the highest similarity were selected as the decision outcome of the cultivar. The results show that, except the erroneous judgement on No.12,32,33,&34, the rest of46batches ECG can all be judged on their kinds of cultivar through the similarity. The success rate of the judgement reached92%.3Technologies for effervescent tablet preparation3.1Extraction process for total flavonoids from ECG:Further optimization was carried out to the ECG total flavonoids extraction process with the response surface methodology. The most optimized process is:Get ECG coarse particles, Add25times of water, Immerse0.5hours, Boil2hours, Filtration, Add20times water to the residue, Boil1.5hours, Filtration, Combine the filtrate. Evaporate the filtrate under negative pressure to lg/mL. Add slowly95%ethanol to the residue with stirring, reaching78.5%ethanol. Keep adding ethanol to reach the8times quantity of raw material. Adjust the pH value to7.0by40%NaOH. Stir for15minutes. Filtrate. Wash the residue with ethanol. Combine with the filtrate. Recover the ethanol. Evaporate under negative pressure to1g/ml. Add water to0.6g/ml. Adjust the pH by5mol/L hydrochloride to1.3. Freeze for72hours. Filtrate. Wash the residue by water. Dried at60" C. Shatter. Sieve. The powder of total flavonoids thus obtained.3.2Debittering technology of flavonoids from ECG:According to the results from response surface optimization tests, the optimized ECG total flavonoids debittering conditions are:naringinase consumption1.6U/mL, enzymolysis temperature54℃, and enzymolysis time132minutes. The debittering rate reached98.88%.3.3Process for preparation of effervescent tabletsThe result of optimization is:Take ECG extracts. Add mannitol to form the medicinal softwood. Take citric acid14units, tartaric acid7units, sodium carboxymethyl starch2.6units, vitamine C3.4units, aspartame0.3units and mannitol6units and make the acid system softwood. Pass it through24mesh sieve. Take sodium bicarbonate17.5units, polyethylene glycol (PEG-6000)2units and make the alkali system softwood. Pass it through24mesh sieve. Have the three kinds of softwood granulated, dried, mixed respectively. Then have them tableted by the rotary-type tablet granulating machine under the conditions of25℃temperature and30%humidity.4. Quality standard for effervescent tabletThin-layer differentiation shows that when the ECG Vit C effervescent tablet is inspected under the UV lamp (365nm) the same color of flueresent spots are both shown at the same Rf value positions respectively with the reference samples of naringenin&apigenin. The tablet weight difference of the effervescent tablets is between-3.91%-3.89%which is in conformity with the±5%limit by the Pharmacopoeia. The tablet weight difference inspection passed. The disintegration time for the4batches of effervescent tablets are all in conformity with the5minute of complete disintegration requirement from the Pharmacopoeia. The tablet passed its disintegration time limit inspection. The naringenin content of the4batches of effervescent tablet determined with UPLC method should be5.4±0.5mg/tablet. The total flavonoids content within the effervescent tablet shall be determined with the total foavonoids determination method regulated by the Health Food Inspection Guide issued by the country, and the average total flavonoids content is14.13%.5Evaluations on Antioxidant FunctionThe result from DPPH method in vitro evaluation of antioxidant activity showed that the ability in scavenging free radicals i.e. the antioxidant activity of ECG extracts from the optimized process (inhibition rate up to26.24%) is significantly higher than the ECG extracts from the process before optimization (inhibition rate up to16.13%).After the intragastric administration of ECG vitamin C effervescent tablets for45days, the results of the superoxide dismutase (SOD) and glutathione peroxidase activity (GSH-Px) in mice blood indicated that, in high dose group and positive control group, the SOD activity had significant difference (P<0.05) with normal control group, and GSH-Px activity had significant difference (P<0.01) with normal control group; in the medium and low dose group, the GSH-Px activity had significant difference (P<0.05) with normal control group.After intragastric bromobenzene oil modeling, MDA and LPO content in livers of mice increased by0.73±0.22nmol/ml and0.68±0.31μmol/gprot respectively compared with normal control group, which had very significant difference (P<0.01). Compared with model group,,the high dose group was effective in suppressing bromobenzene liver lipid peroxidation, reducing MDA&LPO content within the liver, which had very significant difference (P<0.01). The medium and low dose group can reduce LPO content, which also had very significant difference (P<0.01); Meanwhile, the SOD activity of medium dose group had significant difference (P<0.05) with model group, the GSH-Px activity of high and medium dose group had significant difference (P<0.01) with model group. The above results indicated that taking reasonable dosages of ECG-Vit C effervescent tablet will enhance the ability of scavenging free radicals and the ability of antioxidant in mice.Conclusions1The health food formula after the optimization is scientific and reasonable. The basis the project sets up on is in conformity with the modern medical theory and the traditional healthcare theory in Chinese Medicine. The expectation on the healthcare functions to be reached is scientific.2The quality control level of the newly developed product under this research project shall be guaranteed effectively by the application of multi-dimensional quality control from the crude drugs to the extracts and, to the effervescent tablet preparation as well as, by the comprehensive application of quality control indexes as the appearance character, thin-layer chromatography, disintegration time and the content determination of active components.3The enzyme method is the first time used in the debittering technological research on ECG extracts. The ECG extract thereof obtained has got dramatic improvements from the bitter taste of the traditional ECG extracts and at the same time, the antioxidant activeness and effectiveness are enhanced. This technology can be applied to the preparation of ECG foods and health foods. The selected form of effervescent tablet has the characteristics of cool tasting, radpid disintegrating, conveniency in usage, easy keeping and carrying. The newly developed product by the research under this project is an ECG health food with confirmed effectiveness and rich characteristics.4The quality specification of ECG-Vit C effervescent tablet was established with specific data. The standard can control effectively the quality of the effervescent tablet.5The ECG-Vit C effervescent tablet is effective in suppressing bromobenzene caused liver lipid peroxidation, in rising up remarkably the SOD&GSH-Px activity within the blood and the liver of mice, in reducing remarkably the MDA&LPO content within the liver. These indicate that taking a reasonable dosage of ECG-Vit C effervescent tablet may enhance the ability of scavenging free radicals and the ability of antioxidant. |
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