Study On The Production Process Optimization And The Quality Standard Of Fructus Citri Sarcodactylis Steaming Process

ObjectiveThrough the use of modern analytical chemistry methods, measured the main chemical components in fructus citri sarcodactylis samples in different steamed times and fructus citri sarcodactylis samples in different drying ways. With a reference to the difference resistance of oxidation, to explore the relationship between the length of steamed time and the content of the chemical components in the statistics method, and to analyze the mechanism of the steaming process to find a better scale of steamed time and a better way of drying. By modern analytical chemistry methods and the TCM fingerprint. Establishment of a comprehensive and feasible system of quality control method of bergamot and fingerprint, provide methodology reference for comprehensive exploitation and utilization of bergamot.Methods1a study on the steaming processBy ultravidlet-visible spectrophotometry, the hesperidin as a control article, to determinate the content of total flavonoids in fructus citri sarcodactylis samples in different steamed times. By HPLC method, the hesperidin and the5,7-Dimethoxycoumarin as control articles, to determinate and analyze the flavonoids in fructus citri sarcodactylis samples in different steamed times.2a preliminary study on the mechanism of the steaming processTo determinate the A420nm value in fructus citri sarcodactylis samples in different steamed times by ultravidlet-visible spectrophotometry method. By Ninhydrin Color Reaction, the glutamic acid as a control article, to determinate the content of the total amino acid in fructus citri sarcodactylis samples in different steamed time. By the3,5-dinitrosalicylic acid assay and a phenol-sulfuric acid colorimetric method, the glucose as a control article, to determinate the content of reducing sugar and sugar in fructus citri sarcodactylis samples in different steamed time. By HPLC method, the5-hydroxymethylfurfural as a control article, to determinate and analyse the flavonoids in fructus citri sarcodactylis samples in different steamed times.3a study on the capacity of oxidation resistanceDetermination the antioxidant activity in fructus citri sarcodactylis samples in different steamed times, by the positive reaction of ascorbic acid as a control article, DPPH free radical scavenging capacity as indicators in vitro. Study the relationship between the scale of steamed time and the content of different components by SPSS17.0SPSS Statistics.4a study of drying processTo find out a better method for fructus citri sarcodactylis drying process by, determination the content of components, determination and analysis the rehydration rate and the out looking of fructus citri sarcodactylis samples dryed in different methods of dryer model RTI-1, constant temperature drying and sun-dried.5a study on quality standard To establish characteristic spectrum of the TLC by, a preliminary study to fructus citri sarcodactylis samples from different collecting times with reference to "Chinese pharmacopoeia"2010edition, the physical and chemical identification, determination of moisture, ash content and extract content, a TLC analysis to ethanol extract from Fructus Citri Sarcodactylis by TLC method, observation the TLC change in two expansion systems of different polarity.To determinate the content of hesperidin,5,7-Dimethoxycoumarin and5-hydroxymethylfurfural in Fructus Citri Sarcodactylis samples from different collecting times by HPLC method.By HPLC-PDA method for full wavelength scanning to methanol extract citron slices to determine the baseline stability and provide detection wavelength of information, with the hesperidin as a control article to establish fingerprint and obtain the Common Peaks in the Chromatographic Fingerprints of Fructus Citri Sarcodactylis from different collecting times, then using "traditional Chinese medicine fingerprint atlas of similarity evaluation system2004A version" to analyze the Common Peaks similarity.Results1a study in the steaming processThe change of the content of total flavonoids in samples is increased at the beginning and then decreased. After a steamed time of2.5hours, the content of total flavonoids is MAX, of50.682mg/g. After a steamed time of4hours, the content of total flavonoids is MIN, of1.038mg/g. Compared to the content of29.355mg/g before steaming, the total flavonoids now is doubled.The content values of hesperidin in samples in different steamed time are different. After a steamed time of2.5hours, the content of total flavonoids is MAX, of1.038mg/g.The content values of the5,7-Dimethoxycoumarin in samples in different steamed time are different, it is2.195mg/g, MAX before steaming and it is1.628mg/g after a steamed time of2.5hours, at the second stage.2a preliminary study on the mechanism of the steaming processThere is color difference in different steamed times. With the color changing from light to dark, the A420nm value is increasing. The MAX is0.671(sample#9) after a steamed time of4hours. In sample#9, the A420nm value before steaming is0.090.After steaming process, the content of the total amino acid in samples is decreased, as steaming time extended. The content of the reducing sugar and total sugar is increased and then stand still. The content is33.18%(sample#1) before steaming, after a steamed time of2hours and afterwards, the content keeps in56%～60%more or less.There is quite big difference in content of5-hydroxymethylfurfural in samples before and after steaming process. It is0.0106mg/g (sample#1) before and it is increased after steaming. After a steamed time of4hours, it is0.520mg/g (sample#9), increased by50times compared.3a study on the capacity of oxidation resistanceThe inhibiting effect to DPPH free radical scavenging capacity is different by water extract and alcohol extract. The alcohol extract has a worse effect. As time extended, the IC50value is decreased at the beginning and then increased, also, referred as the capacity of oxidation resistance is increased at the beginning and then decreased. After a steaming time of2.5hours, the IC50value is MIN of0.20mg/ml (sample#6), while the capacity of oxidation resistance is MAX, of2.39mg/ml (sample#6). When the IC50value is MIN and the capacity of oxidation resistance is MAX, the water extract is0.72mg/ml (sample#1) before steaming. After steaming time of4hours, the alcohol extract is5.47mg/ml (sample#9), while it is5.35mg/ml (sample#1) before steaming, which are not bit different.The relationships between different steamed times and the IC50value, the A420nm value, the content of the total amino acid, the content of the total sugar, the content of the5-hydroxymethylfurfural of the water extract in samples, are significant (P<0.05), their values are-0.915、0.983、-0.885、0.671、0.921. The relationships between the IC50value of the water extract and the A420nm value, the content of the total amino acid, the content of the total sugar, the content of the5-hydroxymethylfurfural, are significant(P<0.05), their values are-0.880、0.847、-0.710、-0.717. The relationships between the IC50value of the alcohol extract and the content of the total flavonoids, the hesperidin, are significant, their values are-0.891and-0.702. The content of the total flavonoids, the hesperidin are negatively interrelated with the IC50value of the alcohol extract, the content value is higher, the lower the IC50value (the higher the capacity of oxidation resistance). The relationship between the A420nm value and the steaming time, the content of the total amino acid, the content of the total5-hydroxymethylfurfural, are significant (P<0.05), their values are0.983、-0.820and0.950.4a study of drying processTime used in drying by dryer RTI-1is the shortest one, compared with the other methods, as well as the out looking, the smell, the content of the hesperidin, the rehydration rate.5a study on quality standardThe out looking of Fructus Citri Sarcodactylis steamed is oval or orbicular-ovate shape, shrunk and curled, in different sizes. Top ruptures in several finger-shape strips, bottom in round-shape, base slightly narrow, some visible fruit stems,5～10cm in length and3～5 cm in width. Hard and brittle, turn soft after damped, fragrance smell and brown or dark brown color.To establish TLC analysis method by1.0g powder of Fructus Citri Sarcodactylis steamed,10ml alcohol added, sonic20min provided, strained, drying, and then the rest of powder,0.5ml alcohol added, to make it become the sample. Drops on a Gypsum plate, in an expansion system by1.5:1cyclohexane and ethyl acetate, the spot on the TLC is identical and clear.The percentage of water is9.19%～12.84%, the percentage of total ash is3.14%～9.71%, water soluble extraction by cold dip is42.48～60.65%, water soluble extraction by hot dip is54.23～74.33%, ethanol soluble extraction by cold dip with ethyl alcohol liquid is4.07～20.29%, ethanol soluble extraction by hot dip with ethyl alcohol liquid is22.90～50.30%.The content of the hesperidin, the5,7-Dimethoxycoumarin, the5-hydroxymethylfurfural in Fructus Citri Sarcodactylis samples from14different collecting times, are different. The content of the hesperidin is0.142～0.788mg/g, with an average of0.142～0.788mg/g. The content of the5,7-Dimethoxycoumarin is0.642～1.747mg/g, with an average of1.038mg/g. The content of the5-hydroxymethylfurfural is0.028～0.901mg/g, with an average of0.238mg/g.There are12Common Peaks in HPLC for Fructus Citri Sarcodactylis samples from14different collecting times. The similarity between control article and sample is0.044～0.930, to give a result that the chemical components of fructus citri sarcodactylis samples from14different collecting times, is different.Conclusion1a study in the steaming process The steaming production process can increase the content of the total glutamic acid and the hesperidin. The MAX amount is obtained after a steaming time of2.5hours.The steaming production process can decrease the content of the5,7-Dimethoxycoumarin. The MAX amount is obtained after a steaming time of2.5hours.To sum up, the steaming time of2.5hours is the best time scale of Fructus Citri Sarcodactylis steaming production process.2a preliminary study on the mechanism of the steaming processAfter steaming production process, the content of the total flavonoids and the hesperidin is increased, while the content of the5,7-Dimethoxycoumarin is decreased. Meantime, maillard reaction happens to these Fructus Citri Sarcodactylis samples, generating melanoidin and inducing polymerization then forming complicated macromolecular pigment. The A420mm value increases, so the color of the Fructus Citri Sarcodactylis sample becomes darker. The amino acid degrades and generates the5-hydroxymethylfurfural.3a study on the capacity of oxidation resistanceAfter steaming production process, the capacity of oxidation resistance has a quite big change. It increased. The MAX is after a steaming time of2.5hours. Thus, a steaming time of2.5hours is thought to be the best.The scale of steaming time has a big effect to the IC50value, the A420mm value, the content of the total flavonoids, the total sugar, the5-hydroxymethylfurfural. And the content of the total flavonoids, the total sugar, the5-hydroxymethylfurfural directly effects the capacity of oxidation resistance of the water extract. The content of the total flavonoids and the hesperidin is higher, the better the capacity of oxidation resistance of the alcohol extract. As steaming extended, the amino acid complete hydrolysis, the5-hydroxymethylfurfural increases, as well as the A420mm value, the color becomes darker. Thus, maillard reaction happens to these Fructus Citri Sarcodactylis samples.4a study of drying processQuality of Fructus Citri Sarcodactylis samples dried by dryer RTI-1is the best one. Thus, this method can be widely used.5Study on quality standardThe content of water for Fructus Citri Sarcodactylis steamed is not more than13.0%, according to the Committee of National Pharmacopoeia requirements for initial formulation.The content of total ash for Fructus Citri Sarcodactylis steamed is not more than10.0%, according to the Committee of National Pharmacopoeia requirements for initial formulation.The water soluble extraction by hot dip is not less than45.0%, the ethanol soluble extraction by hot dip with ethyl alcohol liquid is not less than15.0%, according to the Committee of National Pharmacopoeia requirements for initial formulation.The content of (C28H34O15) is not less than0.030%, by dry matter basis, according to the Committee of National Pharmacopoeia requirements for initial formulation.The content of (C28H34O15) is not less than0.50%, by dry matter basis, according to the Committee of National Pharmacopoeia requirements for initial formulation.