Preparation Technology And Quality Control Of Compound Jinlian Injection

Purified extracts for injection are direct raw materials of an injection of Traditional Chinese Medicine. Therefore, orthogonal experiment design and single factor tests were used in optimizing the extraction and purification technology for two of the medicinal materials （Flos Trollii and Flos Lonicerae） of Compound Jinlian Injection by taking the content of the total flavonoids and chlorogenic acid as the indices, respectively. The process for preparation of purified extracts from Flos Trollii for injection was as follows: dried flower of Flos Trollii was refluxed with water for 1h, and the extraction was repeated three times. The combined extracts were filtered, concentrated under reduced pressure and then deposited with ethanol aqueous solution. The filtration passed through a macroporous resin column after the ethanol was removed. The fraction of ethanol eluate from the column was collected to yield purified extract from Flos Trollii for injection. The content of the total flavonoids in total solid was 86.9%. The process for purified extracts from Flos Lonicerae for injection was as follows: Dried flower of Flos Lonieerae was refluxed with 80% ethanol for 1 h, and the extraction was repeated twice. The combined extracts were filtered and the ethanol was removed under reduced pressure. The extracts were adjusted to pH 2～3 with 0.2 mol/L HCl and then passed through a macroporous resin column. The fraction of ethanol eluate was collected to yield purified extract from Flos Lonicerae for injection. The content of chlorogenic acid in total solid was 27.5%.The prescription and technological process were optimized by the criterion of appearance, moisture and resolubility. Good products were produced with Mannitol （150 mg/ampule） as filler and the optimum freeze-drying process: pre-freezed for 5 h at -40℃, dried for 12 h under vacuum at -20℃and dried for another 3 h at 20℃under vacuum.Four flavonoids were isolated from Trollius ledibouri Reichb by using several techniques based on column chromatography. On the basis of their chemical properties and spectra, the compounds were fully characterized as orientin, vitexin, 2″-O-β-L-galactopyranosylorientin and 2″-O-β-arabinopyranosylorientin. Among them, 2″-O-β-arabinopyranosylorientin was isolated from the genus Trollius for the first time.A high-performance liquid chromatography （HPLC） method was developed for simultaneous quantitative determination of four flavonoids （2″-O-β-L-galactopyranosylorientin, 2″-O-β-arabinopyranosylorientin, orientin and vitexin） in Flos Trollii. The analysis was performed on a Thermo Hypersil BDS C₁₈ column （4.6 mm×250 mm, 5μm） using isocratic elution with the mobile phase of acetonitrile-water-glacial acetic acid （14:86:0.05） at 340 nm. The flow rate was 0.6 mL min^-1. The method was applied to the determination of four flavonoids in different species and different harvesting periods of Flos Trollii from multi sources. A significant variance in the contents of the flavonoids was found among the tested forty samples. The fully validated HPLC method for quantitative analyzing four flavonoids is applicable to the quality evaluation of Flos Trollii.The chemical variables as a whole were obtained by using HPLC fingerprint （HPLC-FPS） analysis. HPLC-FPS was developed on a C₁₈ column using gradient elution with a mobile phase of acetonitrile and 0.5% acetic acid with ultraviolet detection at 280 nm. Analyzed by Computer Aided Similarity Evaluation System, good similarities with cosine coefficient above 0.90 were obtained in fingerprints of 10 batches Flos Trollii. The HPLC-FPS similarities of 30 samples of different species and different harvesting periods of Flos Trollii from multi sources were evaluated by Similarity Evaluation System.A rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric （UPLC-ESI-MS/MS） method was developed for the qualitative and quantitative determination of the constituents of the flower of T. ledibouri Reichb. The analysis was performed on an AcQuity UPLCTM BEH C₁₈ column using gradient elution with a mobile phase of 0.1% acetic acid and acetonitrile over 20 min. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring （MRM） was used for the qualitative and quantitative analysis of the constituents, respectively. The molecular weights of the 33 constituents were concluded on the basis of their positive and negative ion electrospray mass spectra, which showed precursor ions ([M+H]⁺ and [M--H]^-. The possible structures of 15 constituents were deduced on-line by careful studies on their MS and MS/MS spectra and four of them, 2″-O-β-L-galactopyranosylorientin, 2″-O-β-arabinopyranosylorientin, orientin and vitexin, were quantified.A UPLC-ESI-MS/MS method was developed for the qualitative identification of constituents in the fower buds of seven Lonicera species. The optimal separation and detection were achieved within 17 min. Among the 33 compounds detected, 22 constituents including 6 organic acids, 8 flavonoids and 8 iridoid glycosides were characterized based on their fragmentation patterns in collision-induced dissociation experiments and/or by comparison with standard compounds. In addition, to statistically establish the correlation and discrimination of the Lonicera species, principle component analysis （PCA） was applied in this study. Lonicera samples were divided into well-defined groups directly related to their species based on PCA in terms of the log transformed relative contents of the major organic acids as the variables. All results indicated that the method presented here is able to classify the sample species and to reveal characteristic details of the chemical constituents of different Lonicera species.The major active constituents （chlorogenic acid, orientin, vitexin and indexⅣ） in Compound Jinlian Injection were analyzed quantitatively by HPLC method. Under the same chromatographic conditions, the HPLC-FPS of Compound Jinlian Injection was analyzed and evaluated. Ten common peaks were achieved after analyzing 10 batches of injection samples and HPLC-FPS common pattern of the injection was developed. Analyzed by Similarity Evaluation System for Chromatographic Fingerprint of TCM of the Chinese Pharmacopoeia Committee, a similarity threshold of 0.95 was set up for Compound Jinlian Injection. Furthermore, good correaltions were found by comparing the fingerprints of Compound Jinlian Injection and purified extracts for injection. The developed method offered guarantee for the stability of the preparation technology and final products of Compound Jinlian Injection and it was beneficial to improving its quality and the clinical safety could be ensured.An HPLC method was developed and validated for the determination of orientin in rabbit plasma using ultraviolet （UV） detection. Protein precipitation was used as the sample preparation technique. A Diamonsil C₁₈ column （150mm×4.6mm, 5μm） was equilibrated with a mobile phase composed of 0.1% acetic acid/methanol/acetonitrile （80/5/15, v/v）. This validated method was successfully applied to a pharmacokinetic study in rabbits after the intravenous administrations of orientin, purified extract （TRO PE） from Flos Trollii and Compound Jinlian Injection. Orientin showed nonlinear pharmacokinetics in rabbits in the studied dose range. Significant increase in AUC was found from TROPE and Compound Jinlian Injection dosing compared with orientin dosing at the same dosage levels in the comparative pharmacokinetic study. This result indicated that the pharmacokinetic characterisitic of orientin was affected by other constituents in TROPE. The pharmacokinetic results are useful for further study of the clinical applications of Compound Jinlian Injection.