Rhodiola Injection Extraction and Refining Technology
Authors: Yang Ting-yuan, Li San-ming Liu Hongzhuo
Hui-Min Yao Li Hongju
【Key Words】 Rhodiola rosea extract injection refining Salidroside tyrosol
Abstract: ObjectiveTo study the extraction and
purification process of Hongjingtian Injection. MethodsAn orthogonal method was adopted to optimize the extraction and purification process with the transference rate of Salidroside and p-Tyrosol as criterion. ResultsThe optimized process of
extraction was that eight times water as extracted solvent, the comminuted medical material was extracted three times, each for an hour. Alcohol precipitation method and gel flocculating method were both used to optimize purification
process. Alcohol precipitated concentration was 70%. The optimized process of gel flocculating method was that the extracted solution was condensed to 0.5g * ml-1 (equivalent to
raw materia1) and its pH was adjusted to 5.0. The 5.0% gelatin solution was added into the condensed solution with agitation rate of 60 r * min-1 at 60 ? until there was no more
precipitation. ConclusionThe results of impurity test meet the standard for injection. The established methods can be used for extraction and purification of Hongjingtian injection.
Key words: Hongjingtian Injection; Extraction; Purification; Salidroside; P-Tyrosol
Sedum Rhodiola plants for the dry root and rhizome of Rhodiola, cold, taste slightly bitter, astringent, clinical medicine often simmered them in water with heat-clearing and
detoxifying, promoting blood circulation Huayu of the effect. Modern research shows that Rhodiola rosea on the cardio-
cerebral vascular diseases have a strong therapeutic effect, and no obvious side effects, has an excellent prospect.
Transfusion medicine, compared with the small injection in the insoluble particulate control and related substances to enhance effective component analysis to detect aspects, has a higher sensitivity and accuracy, while avoiding the
compatibility of the small injection may cause adverse reactions, although the much more difficult preparations, but the effectiveness and safety of clinical use of the controllability and other products all had played a "qualitative" change in [1]. Rhodiola glycosides and tyrosol [2,3] is known Rhodiola two active ingredients, this paper salidroside and tyrosol as an indicator of the overall transfer rate of extraction and injection Rhodiola Refining research.
An instrument and reagent
LC-10ATVP high-performance liquid chromatograph, Shimadzu Zhushikuaishe; RE-52A rotary evaporator, the Yahya Rong
Biochemical Instrument Factory; micro-mill plant samples,
Tianjin Taisite Instrument Co., Ltd.; DF-101S Collectors
constant temperature heating magnetic stirrer, Gongyi City, England valley to the Chinese Instrument Factory.
Rhodiola, origin Tibet; salidroside reference substance, purchased from the laboratory of Pharmaceutical and Biological Products; tyrosol reference substance, were purchased from
Japan; acetonitrile, methanol were HPLC pure; water twice distilled water; other reagents were AR.
2 Methods and Results
2.1 salidroside and tyrosol analytical conditions of HPLC column: ODS (Kromasil C18, 250 mm × 4.6 mm, 5 μm, Tianjin
Science and Technology Instrument Co., Ltd.); mobile phase of methanol - acetonitrile -0.06% phosphoric acid ( 10:10:80, v / v / v), flow rate 1.0 ml * min-1, detection wavelength 275 nm,
injection volume 20 μl.
2.2 Extraction Process
2.2.1 examines the choice of extraction solvent of water and different concentrations of ethanol as an extraction solvent, extraction, and results showed no significant differences, it was defined as the water as extraction solvent.
2.2.2 Optimization of extraction obtained after crushing herbs Rhodiola 9 copies to solvent (A), extraction time (B) and extraction times (C) the three process parameters for the study factors, each select three horizontal (see Table 1). Accordance with the L9 (34) orthogonal experimental table experiments to detect tyrosol in Rhodiola glycosides and the transfer rate of the overall evaluation index, filter optimum process conditions. The results in Table 2.
Table 1 Extraction of factors and levels (omitted)
According to very poor analysis, the results showed that the number of solvent and extraction of Rhodiola glycosides and tyrosol on the total transfer rate has a significant influence, while the extraction time had no significant effect on the results. Taken together, determine the optimal extraction process for the A2B1C3, herbs Rhodiola be shattered, with eight times the amount of water, heat reflux extraction three times, extracted 1 h / time.
2.3 Refining traditional Chinese medicine study and
optimization of injection affect the stability and security of impurities are mainly protein (Protein), oxalate (0xalate), potassium (K), tannin (Tannin) and resin (Resin) followed by use of sulfonamide Base salicylic acid precipitation, calcium
chloride precipitation, sodium tetraphenylborate
precipitation, egg white precipitation method, hydrochloric acid precipitation method to be checked. Reposted elsewhere in the paper for free download http://
Table 2 Orthogonal experimental results (omitted)
2.3.1 Alcohol precipitation of water to take concentrated extract, plus 95% ethanol, adjusted to 70% alcohol concentration, refrigerated 24 h, filtration, recovery of ethanol and the filtrate was concentrated, determined salidroside and tyrosol content to calculate the total transfer rate, and alcohol precipitation of impurities in the samples after the inspection. The results in Table 3.
Table 3 impurity examination results (omitted)
"(-)" That the results showed that the results showed
that the positive-negative ;"(+)"
Alcohol Shen's pre-transfer rate of 73.52%, and alcohol precipitation of total post-transfer rate of 68.52%, the
results showed that the alcohol precipitation of Rhodiola glycosides and less impact on the content of tyrosol; alcohol precipitation after the extract is not containing protein, oxalate, potassium ion and resin, but the tannins are still there.
2.3.2 remove the liquid gelatin law is currently the method of Tannins in alkaline alcohol precipitation, heat
treatment and cold storage method, gelatin and chitosan flocculation method, etc. [4], this method uses gelatin as the addition to tannin method, and its influence factors orthogonal experimental study to determine the optimum
process.
After taking alcohol Shen extract 9 copies, in order to concentrate ratio (A), control the temperature (B), pH value (C) and stirring speed (D) This four process parameters In order to investigate factors, each of three levels of choice .
Table 4, according to L9 (34) orthogonal experimental table experiments, each with 5% gelatin solution to precipitate until they no longer produce, refrigerated 24 h, filtration, concentration, adjusted with 95% ethanol alcohol concentration to 85 %, refrigerated 24 h, filtration, concentration and determination of salidroside and tyrosol content, and conduct
tannin, protein examination. The results in table 5.
Table 4 gelatin method factors and the level of (omitted)
Tannins in each group inspection and examination of
protein were negative, indicating tannin had been removed, excess gelatin have also been removed. According to very poor analysis, results showed that the concentration ratio and pH value on the overall transfer rate significantly affect the
temperature and stirring speed on the overall transfer rate did not affect only when the temperature is high, speed is larger, the sedimentation rate of settlement faster, therefore, to determine the optimum purification process for the A2B3C3D3, soon after the alcohol precipitation liquid ratio of 1:2, that is, 0.5 g * ml-1 (equivalent to the native
drug), adjusting pH value to 5.0, at 60 ?, speed of 60 r *
min-1 under the conditions of 5% gelatin solution to precipitate until they are no longer generated.
2.4 The extraction and purification of the best programs and the results obtained after crushing herbs Rhodiola, plus 8
times the amount of water, heat reflux extraction three times, extracted 1 h / times, liquid filtration, vacuum concentration, plus 95% ethanol, adjusted alcohol concentration to 70%, stir well, refrigerated 24 h, filtration, vacuum concentrated, adding water to a
concentration of 0.5 g * ml-1 (equivalent to the native drug)
of the solution, adjusting pH to 5.0 at 60 ?, speed of 60 r *
min-1 under the conditions of 5% gelatin solution to precipitate until they no longer produce, refrigerated 24 h,
filtration, concentration, plus 95% ethanol, adjusted to 85% alcohol concentration, refrigerated 24 h, filtration the filtrate concentrated to the relative density of 1.20 to 1.30 of the extract, that is, too. Of the extract to TLC, HPLC and impurities inspection. The results in Table 6 ~ 7.
Table 5 gelatin France orthogonal experimental results (omitted)
Table 6 extract assay results (omitted)
Table 7 impurity examination results (omitted)
Thin-layer chromatography using extracts of Rhodiola
Rhodiola glycosides and tyrosol are identified, we can see extracts with the reference substance salidroside and tyrosol in the same location show the same color spots. The results show that using the above-mentioned extraction purification
method, retaining the active ingredient in Rhodiola Rhodiola glycosides and tyrosol at the same time, which can effectively remove the protein, oxalate, potassium, tannin and resin and other impurities, and the Rhodiola glycosides and tyrosol higher overall metastatic rate, the system obtained Rhodiola extract, Rhodiola rosea injection can be used to prepare the next step.
3 Discussion
The active ingredients of Rhodiola Rhodiola glycosides and alcohols such as casein, are alcohol-water double soluble
components, this article examines water and different concentrations of ethanol as an extraction solvent, extraction, and results showed no significant differences, taking into account industry safety factors of production and economic factors, so using water as extraction solvent.
From the experimental results can be seen, alcohol precipitation is still feasible in traditional Chinese medicine preparation purification method used more often. Affect the clarity of traditional Chinese medicine injection
the main factor is due to the presence of tannins, this paper gelatin Removal of tannin, gelatin method is to make tannin in acidic aqueous solution into the tannic acid and protein binding protein, the formation of precipitation, filtration to remove tanning quality, and then add alcohol to the filtrate concentrated solution of alcohol concentration, excess gelatin solution at high concentration ethanol precipitation, put it aside, filtration to remove excess gelatin.
From the TLC, HPLC and impurity inspection results show that the extract used in this paper refined methods, retains the active ingredient in Rhodiola Rhodiola glycosides and
tyrosol, it is also effectively removed the impact of
traditional Chinese medicine injection stability and security of impurities, and so salidroside and tyrosol has a high transfer rate, the method is simple, reliable, and adapted to industrial production.
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