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Coptis accumulation of active ingredients_715

By Rodney Wallace,2014-10-30 14:15
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Coptis accumulation of active ingredients_715

Coptis accumulation of active ingredients

     Author: Ma Kai-sum, Shi-Jiang Chen De-Li Zhang

    Zhong-Yue

     Abstract Objective To study the Coptis (flavored with) the dynamics of accumulation of the active ingredient, in

    order to determine the optimal harvest time provide the basis Coptis. Fixed-point method on a regular basis collected 2 to 5 year-old Coptis samples, analysis of berberine hydrochloride Coptis rhizome and total alkaloid content, respectively, by high performance liquid chromatography (HPLC) method and UV method. Results Coptis rhizomes of dynamic accumulation of the active ingredients have certain laws, berberine content in April each year (flowering and fruiting period) is almost the whole year were the lowest, 4 ~ 5 years old to 10 November

    Coptis Coptis rhizomes of berberine content in at their annual maximum value, while a 5-year-Coptis of berberine content was

    the highest; the total alkaloid content of an upward trend in each of the 10 levels at their annual maximum in November,

    while a 5-year-total alkaloid content of berberine maximum. Conclusion According to the accumulation of Coptis dynamics of the active ingredient, berberine best harvesting period of 10 to 11 months of 5-year-Coptis rhizomes.

     Key words berberine dynamic accumulation of active ingredients

     Abstract: ObjectiveTo study the dynamic accumulation regulation of the effective components of Coptis chinensis, so to provide the experimental data of optimal harvest time of Coptis chinensis. MethodsThe samples of two to five years were gathered from the same glebe and time. Berberine and the total alkaloids were analyzed by HPLC and UV methods. ResultsThe

    dynamic accumulation of effective components in the rootstalk of the Coptis chinensis had regularity in the certain extend. The contents of berberine in samples were almost the lowest in April in every year, which were mostly the highest in October and November from the samples grown 4 ~ 5 years, but the content of the component was mostly the highest in 5-year-old

    Coptis chinensis in the series samples. The content of the total alkaloids increased year by year in the rootstalk of the Coptis chinensis, which was mostly the highest in October and November in every year, but the content of the component was

    mostly the highest in 5-year-old Coptis chinensis in the

    samples. ConclusionAccording to the accumulation pattern of the effective components in the Coptis chinensis, the optimal harvest time is from September to November.

     Key words: Coptis chinensis; Effective components; Dynamic accumulation

     Coptis Coptis chinensis Franch cultivation history in China planted earlier and export of Chinese herbal medicines commonly used medicinal herbs, there are more than 600 years of cultivation history [1], produced in Chongqing, the main pillars of its national production output accounts for 40% ~ 60%, quality. To this end, Shizhu have "Coptis town" reputation. Coptis rhizomes of berberine and total alkaloid berberine is the main active ingredients, is to examine the

    quality of key indicators Coptis this end, we have studied the Coptis rhizomes of berberine and total alkaloid content of the law of accumulation, in order to determine the optimum harvest period of Coptis to develop standard operating procedures

    provide a scientific basis Coptis.

     1 Materials and methods

     2002-04 onwards, regular, fixed collection Shizhu yellow water Coptis farm house Cong Tan (elevation 1 300 m) the same plot, the same growth and age, the size of basically the same

    2 ~ 5 year-old plant samples Coptis rhizome dry thermostat back.

     Coptis rhizome 1.1 Determination of berberine

    hydrochloride in

     1.1.1 Instrument and test drugs instruments: high-

    performance liquid chromatography: waters the company's

    Alliance2690 pump, 996 diode array detector; Millennium32 chromatography workstation, ultrasonic cleaner (KQ-50B type);

    four-hole separate constant temperature water bath pot .

     Test drugs: Berberine hydrochloride reference substance

    by the Chinese pharmaceutical and biological products to offer batch 0713-200107 (for HPLC use) chromatography pure

    acetonitrile, methanol and other reagents were of analytical grade, water-deionized water .

     1.1.2 Column Methods: Dima's Dimonsil C18 (4.6 mm × 250

    mm, 5 μm); pure mobile phase, acetonitrile -0.1 mol potassium

    dihydrogen phosphate (35:65) detection wavelength of 345 nm, column Greenhouse temperature, flow rate 0.7 ml * min-1.

     1.1.3 linear relationship between the study said

    precision reference substances 5.45 mg of berberine hydrochloride to take home 25 ml Liang Ping, add the amount of dissolved methanol, together with methanol was diluted to scale, blending, attainable standards of reserve liquid. Precision drawing this standard reserve liquid

    0.2,0.5,1.0,2.0,4.0,5.0 ml, set 10 ml Liang Ping, add methanol

    was diluted to scale, get six different concentrations of contrast solution, respectively, precise drawing 20 μl, into

    the liquid chromatography in the determination of record peak area, and to the peak area (Y) of the sample volume (X) regression, have standard curve equation:

     Y =- 421 5 1 660 625X (r = 0.999 8)

     To peak area (Y) - sample volume (X) is graphed, it was a straight line, experimental results show that: The 0.087 2 ~ 2.18 μg range berberine peak area and sample volume has a

good linear relationship between . The results in Table 1.

     Linear relationship between the experimental results in Table 1 (omitted)

     1.1.4 draw on the control precision of experimental precision solution of 20 μl (concentration of 0.087 2 mg /

    ml) repeated injection five times, the determination of berberine peak area recorded the results in Table 2.

     Precision of experimental results in Table 2 (omitted)

     Experiments show that good accuracy.

     1.1.5 stability test to take samples for test solution, 0,4,8,12,16 h, respectively, were injection 20 μl, the

    determination of berberine hydrochloride in the peak area recorded the results in Table 3.

     Table 3 Stability test results (omitted)

     Experiments show that the sample for the test solution was stable in the 16 h period.

     1.1.6 Preparation for the test materials will be berberine solution samples at 60 ? for drying, crushing, over on the 3rd screen, the powder obtained is about 0.1 g, precision that set, and placing 100 ml Liang Ping, add hydrochloric acid - methanol (1:100 ) 95 ml, set the 60 ?

    constant temperature water bath heated 15 min, removed with ultrasonic cleaning machine ultrasonic 30 min, removed, placed filter, using hydrochloric acid - methanol (1:100) constant

    volume, mixing with a 0.2 μm micro - pore membrane

    filtration, take samples of liquid added to the filtrate.

     1.1.7 Sample measurement precision drawing reference substance, respectively for the test solution and the product

    solution 20 μl, according to the above chromatographic conditions were determined, record the peak area using external standard method content. Reposted elsewhere in the paper for free download http://

     Coptis rhizome 1.2 Determination of total alkaloid

     1.2.1 Instrumentation and reagents

     Instrument: UV-Vis spectrophotometer (Shimadzu UV-1604

    type).

     Test drugs: ethanol, methanol, were analytically pure alumina.

     1.2.2 Methods for the preparation and determination of

    test solution: Coptis samples of low-temperature drying,

    grinding, over on the 3rd screen, the sample powder dryer in the home stand. Precision that is about to take Coptis Powder 0.8 g, set in Soxhlet extractor, using hydrochloric acid methanol (1:100) amount of heat reflux extraction, to extract colorless, will extract the appropriate concentration and transferred to 50 ml Liang Ping, the use hydrochloric acid -

    methanol (1:100) a small amount of washing containers, washing liquid into the extraction solution, and add to the scale,

    according to column chromatography [2] ( "Chinese Pharmacopoeia" 2000 edition of the Department of Appendix ?

    32 tests), precision volume take 5 ml, plus which has been handled well in alumina column (glass column, inner diameter

    of about 9 mm, neutral alumina 5 g, wet-mounted column, pre-

    washed with 30 ml of ethanol), the use of ethanol 25 ml sub-

    sub-elution collect eluate, set 50 ml Liang Ping, add ethanol was diluted to scale, precision drawing 2 ml, set 50 ml Liang Ping, add sulfuric acid solution (0.05 mol / L) was diluted to scale, shake, according to spectrophotometric France [2] (Appendix 26), was measured at 345 nm wavelength absorbance, according to C20H17NO4 * HCl absorption coefficient for the 728, that is to have: the total alkaloid content of berberine in berberine terms, the formula is as follows:

     Percentage of total alkaloid content (%) = A × 50 ml ×

    50 ml × 50 ml728 × 100 × 2 ml × 5 ml × w × 100%

     2 Results

     2.1 Coptis rhizomes of berberine hydrochloride content

    change of the results shown in Table 4 and Figure 1 ~ 2.

     From Table 4 and Figure 1 shows that the 2-year-Coptis

    rhizomes of berberine hydrochloride by the end of August and September in the middle of the year to reach a peak by the end

    of December its roots in the berberine content of berberine content in the year The first two peak values, in the flowering and fruiting period in April, the berberine content in Coptis rhizome is almost the lowest throughout the year,

    this period of flowering and fruiting and consume a large amount of nutrient substances.

     From Table 4 and Figure 2 shows three-year-Coptis

    rhizomes of the active ingredients of berberine hydrochloride content change of trend reached its peak by the end of June,

    in the period of flowering and fruiting in April, the berberine content in Coptis rhizomes almost the whole year minimum, to gradually rise in mid-May, the remaining content

    of berberine hydrochloride on basically the same.

     Table 4 Coptis Rhizome medicinal active ingredient at different times of variation of berberine hydrochloride (abbreviated)

     Figure 1 year Health berberine berberine at different times of the content (abbreviated)

     Figure 23-year-Berberine in Coptis different time

    (omitted)

     Can be seen from Table 4, 4-year-Picrorhiza and 5-year-

    Coptis rhizomes content of berberine hydrochloride in each year measured the highest concentrations of this two years in particular, the content of berberine hydrochloride from 9 October to the highest, the use of statistical regression analysis, 4-year-roots levels of berberine hydrochloride in the period and the harvesting quadratic regression

relationship between the fitted equation as follows:

     Y = 5.64 0.591X-0.029X2 r = 0.86

     5-year-Coptis rhizome content of berberine hydrochloride in harvesting date is quadratic regression relationship between the fitted equation is:

     Y = 6.22 0.38X-0.017X2 r = 0.74

     2.2 Coptis rhizome alkaloid content of the total dynamic results shown in Figure 3 and Table 5.

     Figure 3 Coptis rhizome alkaloid content in different years, the total change (slightly)

     From Table 5 and Figure 3 shows Coptis rhizomes in

    different years in the total alkaloid content was an upward trend year by year, the highest content of 10 in November, especially in the highest 5-year-berberine. 2 ~ 5 year-old

    Coptis rhizomes to 10 November Berberine in Coptis rhizomes in

    the highest value, particularly among the 5-year-Coptis the

    highest levels of berberine. This is also the dry matter accumulation of berberine in October reached a peak of about approximation.

     3 Discussion

     Berberine and total alkaloid berberine physical activity are secondary metabolites [3], our results show that berberine in the later growth stage of its dry matter and content of secondary metabolites increased significantly. Coptis the physiological activities of late fertility may contribute to

    the formation of secondary metabolites Coptis.

     Table 5 Coptis rhizomes of total alkaloid content of dynamic (abbreviated)

     From the point of view to determine the quality of harvest was based primarily on Coptis Coptis rhizomes of

    berberine and total alkaloid accumulation rule. The results show that: The best harvesting period Coptis 9 to 11 months for 5 years, the highest, it is recommended as the best harvest period, which is the traditional practice of harvesting period coincides. Note the formation of centuries Shizhu producing Coptis Coptis Coptis collection period can guarantee the quality, with a scientific basis.

     Coptis rhizome about the content of berberine hydrochloride in larger studies [3,4], individual studies have

    some differences, which may be related to sampling and the different eco-geographical environment. On the total alkaloid content of berberine dynamics rarely reported, we believe that the active ingredient of Coptis rhizomes of the Accumulation

    of research should be combined with the total alkaloid berberine herbs as the basis for quality indicators.

     References

     [1] Kong Wenyan, yellow square. Practical Coptis cultivation techniques [M]. Chongqing: Chongqing Publishing House, 1984:3.

     [2] National Pharmacopoeia Committee. Chinese Pharmacopoeia [S]. Beijing: Chemical Industry Press, 2000:

    Appendix 32, Appendix 26.

     [3] Peng Qiang, Liu Khan, Lan, Zhou David. Berberine berberine content changes in different growing period [J]. Chinese herbs, 1989,2 (3): 7.

     [4] WU Chun, Xie Ying. RP-HPLC Determination of Berberine

    in Coptis study [J]. West Medicine, 2006,21 (3): 467. Reposted elsewhere in the paper for free download http://www.hi138 . com

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