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Determining method for paclitaxel in yew extract and homolog

A method of determination, the technology of paclitaxel, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high detection cost, long analysis time, cumbersome procedures, etc., and achieve the effect of economical sensitivity, high sensitivity, and good separation effect

Active Publication Date: 2006-12-13
GUANGDONG KELUN PHARMACEUTICAL CO LTD
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AI Technical Summary

Problems solved by technology

For the treatment of leukemia, cephalomannine and dexamethasone have a better effect on myeloid leukemia. However, cephalomannine and paclitaxel have very similar molecular structures and properties, and are the most difficult to separate from taxanes. The two substances of paclitaxel became a difficult point in the separation and purification process of paclitaxel
This method requires a second sample injection and step-by-step determination. The disadvantages are that the measurement time is long, the procedure is cumbersome, the workload is large, and the detection cost is relatively high.
And use Phenomenex IB-SIL Phenyl column and adopt another binary gradient ratio elution, mobile phase is acetonitrile and contain 0.015mmolKH 2 PO 4 The buffered aqueous solution of 10-deacetyl baccatin III, baccatin III, and paclitaxel were separated, and the retention time of paclitaxel was about 9.5 minutes, but the above two methods did not mention the effect of cephalomannine. determination
In addition, V.Bringi et al. (WO97 / 44476.1997) used a Diphenyl column, eluted with a binary gradient ratio, and the mobile phase was acetonitrile and 0.015mmolKH 2 PO 4 At this time, although the above four components and several other taxane compounds can be separated at the same time, the retention time of paclitaxel is 31 to 33 minutes, and it takes at least 40 minutes to complete an analysis. Therefore, this method is not suitable. Suitable for daily large-scale analysis
It should be pointed out that the above methods all use a buffer solution. Although in many liquid chromatography separations, adding a buffer solution to the mobile phase can increase the resolution of the chromatographic peaks, but it is very easy to produce crystalline salts in the liquid chromatography system. It will cause pipeline blockage, wear of pump head and shorten the life of chromatographic column, etc. Therefore, it is better not to use buffer solution as much as possible
[0007] In short, the above methods either have a long analysis time, or cannot achieve baseline separation of the cephalomannine peak and the paclitaxel peak, and the gradient ratio elution method uses a buffer solution, which has the problem of cumbersome operation

Method used

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  • Determining method for paclitaxel in yew extract and homolog
  • Determining method for paclitaxel in yew extract and homolog
  • Determining method for paclitaxel in yew extract and homolog

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of yew cell extract: Accurately weigh 0.2g of Chinese yew freeze-dried cell powder, add 20mL of methanol to soak for 24 hours, oscillate with ultrasonic wave for 15 minutes, filter the cells with filter paper, add 20mL of methanol and repeat the ultrasonication three times, combine the filtrate and depressurize Concentrate to dryness, add 10mL dichloromethane and 10mL distilled water for extraction, extract three times in total, collect the dichloromethane solution and concentrate to dryness, dissolve and elute with methanol and constant volume 10mL, filter with a 0.45μm filter membrane, and the filtrate is used for HPLC determination. Inject 5 μL each time.

[0036] Taxus cells adopt two kinds of methods to measure: 1, under above-mentioned chromatographic conditions, adopt binary gradient ratio elution of the present invention, see gradient table 1, make above-mentioned four component peaks realize baseline separation on HPLC chromatogram simultaneously , ...

Embodiment 2

[0045] Preparation of Taxus cell culture fluid extract: Taxus yunnanensis cells were filtered with a 200-mesh filter cloth, and the filtrate was Taxus cell culture fluid. Accurately measure 5 mL of the culture medium, directly add 10 mL of dichloromethane for extraction, extract three times in total, collect the dichloromethane solution and concentrate it to dryness, dissolve and elute with methanol and settle to a volume of 5 mL. Filter with a 0.45 μm filter membrane, and the filtrate is used for HPLC determination, and 5 μL is injected each time.

[0046] The cell culture solution was measured by the two methods described in Example 1, and the content determination results are compared in Table 2.

[0047] Table 2:

[0048]

[0049] In the table, D represents 10-deacetylbaccatin III, B represents baccatin III, C represents cephalomannine, and T represents paclitaxel.

Embodiment 3

[0051] Preparation of yew plant extract: accurately weigh 5 grams of dried powder of southern yew branches and leaves, add 30mL of acetone and 20mL of water, that is, the volume ratio of acetone and water is 6:4 for leaching, and stir and filter 4 times with a magnetic stirrer. Each time for 60 minutes, combine the filtrates to recover acetone with a rotary evaporator, add 50 mL of dichloromethane to the remaining water after recovering acetone and extract 3 times, collect the dichloromethane solution and concentrate it to dryness under reduced pressure, dissolve and elute with methanol and constant volume 100mL, shake well, filter with 0.45μm filter membrane, and the filtrate is used for HPLC determination, 5μL for each injection.

[0052] The branches and leaves of Taxus chinensis were determined by the two methods described in Example 1, and the content determination results are compared in Table 3.

[0053] table 3:

[0054]

[0055] In the table, D represents 10-deace...

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Abstract

The invention relates to a determining method for a kind of plant yew and the taxol in its cell culture and its homologen. Its characteristic lies in that the chromatographic column is Nova-Pak C18, the mobile phase composed of two chromatographic reagent which is acetonitrile and water, balancing the chromatography system with 30 % acetonitrile and 70 % water.and the cell extractor or the extractor of the cell culture solution of the yew and plant yew enter the system, gradient eluting, the proportion changes from 30:70 to 50:50 and the gradient changing curve is 0.5-0.7. Successively, continuing to elute at the proportion of 50:50 for three minutes; Last, balancing the system for 5 minutes with the proportion of 30:70, the preserval time of taxol is 11.12-11.8 minutes. The invention can determine the yew and its cell culture taxol,10-, simultaneously, easily and quickly. It has the characteristics of accuracy, high sensitivity and economy.

Description

technical field [0001] The invention relates to a method for determining paclitaxel and homologues in yew plants and their cell cultures by using high-performance liquid chromatography, in particular to a method for simultaneously determining paclitaxel and 10 in yew plants and their cell cultures by using high-performance liquid chromatography - Methods of desacetylbaccatin III, baccatin III and cephalomannine. Background technique [0002] Paclitaxel has been clinically proven to be an anticancer drug with obvious curative effect on ovarian cancer, breast cancer and lung cancer. Both yew plants and yew cell cultures contain paclitaxel and abundant taxane compounds. Among the taxane compounds, 10-deacetylbaccatin III and baccatin III are precursors for the synthesis of paclitaxel. The content of these two components in Taxus chinensis is more than that of Taxol, and the content of Baccatin III in Pacific Taxus is as high as 0.2%. The recovery rate of separation and extract...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/34G01N30/06G01N30/26G01N30/88
Inventor 黄巧明夏惠青侯嵩生王国亮李志良
Owner GUANGDONG KELUN PHARMACEUTICAL CO LTD
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