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Eucalyptus leaves youmanii Determination of total flavonoids and quality of Identification of_355

By Jacob Rose,2014-10-30 17:23
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Eucalyptus leaves youmanii Determination of total flavonoids and quality of Identification of_355

    Eucalyptus leaves youmanii Determination of total flavonoids and quality of Identification of

     Abstract Objective To study the youmanii eucalyptus leaves Determination of total flavonoids and quality of identification methods. Method with UV spectrophotometry,

    colorimetry aluminum chloride, aluminum nitrate colorimetric Sichuan youmanii Introduced Eucalyptus leaves flavonoids were determined and high performance liquid chromatography and the results were compared. Results Introduced youmanii methanol

    extract of leaves of Eucalyptus existence of A259/A364 ? 1.71

    and A259/A364 ? 2.34 of the two kinds of quality of different plants by liquid chromatography analysis of their content and chemical composition of rutin significantly different.

    A259/A364 ? 1.71 of the four kinds of plants were measured result does not differ greatly, while the A259/A364 ? 2.34

    aluminum nitrate plants were higher than the results of colorimetric determination of high-performance liquid

    chromatography and aluminum chloride, France 46.4%, 39.9%. Conclusion A259/A364 ? 2.34 plants may contain aluminum

    nitrate assay component interference. 3 aluminum chloride colorimetric method youmanii eucalyptus leaves flavonoids than UV and aluminum nitrate method; using hot water extraction,

    ultraviolet spectrophotometry and high performance liquid chromatography can easily be two types of plants leaf identification.

     Key words youmanii eucalyptus leaves; flavonoids; determination method; the quality of identification

     Study on Determination Methods of Total Flavonoids and Quality Identification about Eucalyptus youmanii Leaves

     Abstract: ObjectiveTo study the determination methods of

    total flavonoids and quality identification about Eucalyptus youmanii leaves.MethodsThe content of total flavonoids in leaves of Eucalyptus youmanii which was induced to Sichuan was detected by UV spectrophotometric method, AlCl3 colorimetric method, Al (NO3) 3 colorimetric method and HPLC. Comparisons of the four methods were also discussed. ResultsThere were two

    types with different quality and chemical components in which the A259/A364 of extracting liquid from leaves by methanol was 1.71 and the other one was 2.34. The determination results among the four methods in leaves (A259/A364 ? 1.71) had no

    obvious difference but the results by Al (NO3) 3 colorimetric method in leaves (A259/A364 ? 2.34) was preponderate 46.4%

    and 39.9% over HPLC and AlCl3 colorimetric method. ConclusionThe trees (A259/A364 ? 2.34) are likely to hold

    some components disturbing Al (NO3) 3 colorimtric method. AlCl3 colorimetric method is superior to UV spectrophotometric method and Al (NO3) 3 colorimetric method. The two types are easy distinguished by method of extraction with hot water, UV spectrophotometric method and HPLC.

     Key words: Eucalyptus Youmanii leaves; Total flavonoids; Determination method; Quality identification

     Eucalyptus Eucalyptus youmanii youmanii are Myrtaceae Eucalyptus Gaia is a single capsule, renal medicine group of trees, Sichuan Academy of Forestry in 1997, the success of introduction from Australia. Its leaves are rich in rutin, in addition to elements such as eucalyptus flavonoids [1], there is a greater development of medicinal and food value. This paper studies the introduction of Eucalyptus leaves youmanii Determination of total flavonoids and quality of

    identification methods, designed to provide a reference for their development and utilization.

     1 Materials and methods

     1.1 Instrument and reagents Dalian high-performance

    liquid chromatography according to Bartlett, EC2000 chromatography workstation; UNICO UV-2100 spectrophotometer,

    Soxhlet extraction devices, vacuum pump, oven, water bath temperature control pot. Anhydrous ether, methanol, aluminum

    chloride, aluminum nitrate, sodium nitrite, sodium hydroxide,

    the above reagents were analytical pure. Rutin standard were purchased from Bio-Technology Co., Ltd., Tianjin peak (content 98%).

     1.2 Preparation of experimental materials

     1.2.1 Preparation of leaf extract of leaves collected from Yaan Mingshan youmanii 1998 youmanii Eucalyptus from Australia and the introduction of breeding forest planting the seeds.

     Site conditions and the crop is growing in line to take youmanii eucalyptus trees (large leaf) 4 (?, ?, ?, ?),

    select the location and bloom per plant the same branches, the removal of the lower part (1), Central (2), the upper (3) start near the upper leaves and brown leaves (4) and branch tip leaves (5) a number of, washed after 60 ? dried 5 h,

    after grinding than 60 mesh sieve.

     1.2.2 Preparation of sample extract, said to take an accurate sample preparation of a good dry 0.500 0 g, using filter paper wrap and put it into Soxhlet extraction device, the extraction with anhydrous ether to colorless after. Change to the ether until the ether Hui dry samples by adding 90 ml of methanol at 90 ? for extraction of 5 ~ 6 h to the

    colorless until extract into 100 ml volumetric flask using methanol fixed volume sample solution obtained.

     1.3 UV spectrophotometry, colorimetry aluminum chloride, aluminum nitrate colorimetric method and high performance liquid chromatography youmanii eucalyptus leaves flavonoids [2 ~ 5]

     1.3.1 Preparation of standard solution of rutin will

    rutin standard 2 h baking at 120 ? to remove crystal water

    contained rutin, rutin standard Weigh accurately 29.7 mg dissolved in 70 ml micro-thermal methanol, transferred to 100 ml volumetric flask at room temperature (18 ?) under constant

    volume obtained with the methanol concentration of 0.297 mg / ml of rutin standard solution.

     1.3.2 High Performance Liquid Chromatography Conditions

    Hyper ODS2 C18 column, mobile phase was glacial acetic acid: methanol: water = 3:49:48, detection wavelength UV355 nm,

    column temperature 30 ?, flow 0.8 ml / min.

     1.3.3 four kinds of methods according to the regression equation [2 ~ 5] methods. According to measurement results, the regression equation was:

     Y364 = 0.830 76X +0.005 33 r = 0.999 8 (UV

    spectrophotometry)

     Y432 = 0.611 59X 0.000 65 r = 0.999 9 (3 aluminum chloride method)

     Y500 = 2.086 50X 0.005 25 r = 0.999 8 (aluminum nitrate method)

     Y = 0.000 594 6X 0.107 r = 0.999 9 (liquid

    chromatography)

     As can be seen from the standard curve, UV-

    spectrophotometry in 0.1 ~ 1.0 mg / ml concentration range, at 259 nm and 364 nm Department of rutin concentration and absorbance have a good linear relationship. 3 aluminum chloride method in 0.1 ~ 1.0 mg / ml concentration range, the

    432 nm absorbance at rutin concentration and have a good linear relationship. Aluminum nitrate method in 0.1 ~ 2.0 mg / ml concentration range, at 500 nm absorbance at rutin concentration and have a good linear relationship. High

    Performance Liquid Chromatography in 1.49 ~ 7.42 ng / μl

    within the chromatographic peak area of rutin volume and have a good linear relationship.

     Eucalyptus youmanii 1.3.4 Determination of Flavonoids in

the sample by UV spectrophotometry, aluminum chloride, France,

    France to take samples of liquid aluminum nitrate amount, add 25 ml volumetric flask by standard curve method of colorimetry, calculated on total rutin Total flavonoid content.

     High-performance liquid chromatography injection volume

    of 10 μl, detection wavelength of 355 nm in order to record and flavonoid rutin peak area of chromatographic peak area to calculate the sum of the content of rutin youmanii eucalyptus leaves and in terms flavonoid rutin content. Reposted elsewhere in the paper for free download http://

     2 Results

     2.1 Determination of four kinds of methods of total flavonoids ? Comparison of high-performance liquid

    chromatography flavonoid specificity strong, free from the interference of impurities present, many scholars as a

    reference method to determine the accuracy of other chemical methods. High-performance liquid chromatography determination of total flavonoid content, a sample is the main flavonoids relatively clear, with samples of the main flavonoids as

    standard sample, you can more accurately determine the total flavonoid content. One is not entirely clear the sample or the lack of flavonoids in the case of the standard sample, according to flavonoids in the 225 ~ 280 nm and 300 ~ 400 nm are more obvious in these two regions to identify and determine the absorption of flavonoids of chromatographic peak in order to rutin or quercetin as a standard sample in order to flavonoids, and to calculate the peak area of the total flavonoids. In this study, comparing the samples tested at 255,280,355 nm 3 wavelength chromatogram confirmed that, in Figure 2, the detection wavelength of 355 nm chromatogram of 1,2 peak for the flavonoid peak, according to their size and to calculate the content of flavonoids, from which you can see

    youmanii total flavonoids rutin eucalyptus leaves 85% ~ 95%; ? UV spectrophotometry, also known as standard direct determination method, by comparing the four ether extraction plant After the mention of methanol extract of the A259/A364

    ratio found in the existence of the average ratios were 1.71 and 2.34 of the two types of plant, the same plant at

    different growth stages, with a total flavonoid content of different leaf samples A259/A364 ratio of the basic remain unchanged. UV spectrophotometry of total flavonoids and two kinds of plant performance liquid chromatography determination of the results of the average error of 15.4% and 10.8%, an

    average of 13.1%, its calibration coefficients were 0.846 and 0.892, averaging 0.869 ; ? Determination of aluminum chloride results and high performance liquid chromatography results are very close, less than the UV spectrophotometry. Aluminum

    chloride method, two kinds of errors per plant were 8.7% and 4.6%, with an average of 6.6%, the correction coefficient of 0.913 and 0.954 for the other, with an average of 0.934. Therefore, I believe that the determination of aluminum chloride method after correction for the results to be able to compare accurately reflect Eucalyptus youmanii flavonoids (rutin) levels, it is more appropriate ways; ? the most

    commonly used method of two kinds of aluminum nitrate plant in measurement error of difference is very obvious, the A259/A364 ? 1.71 in plant error is only 6.1%, while the A259/A364 ?

    2.34 in plant error of up to 46.4%, from the liquid chromatogram of rutin, inter alia in terms found in other high content of flavonoids and may therefore interfere with such a

    plant containing aluminum nitrate determination of non-

    flavonoids [6 ~ 8]; ? In addition to aluminum nitrate method A259/A364 ? 2.34 of the plant, the four measurement methods have a strong correlation between the results of, they reflect

    the total flavonoids in Eucalyptus youmanii change in the trend is completely consistent, can be selected according to the experimental conditions, the appropriate method.

     Table 1 Flavonoids Eucalyptus youmanii Determination of four kinds of results and the correction coefficient

    (abbreviated)

     2.2 youmanii identify the quality of Eucalyptus leaves can also be seen from Table 1, youmanii eucalyptus leaf rutin content in very different, there are differences in average levels of plant. Randomly selected 15 selected 4 branches per plant removal of all leaves measured average levels of 12 A259/A364 ? 1.71 in plant average content of rutin in 9% and 11.5%, and while three of the A259/A364 ? 2.34 The average

    content per plant at 4% ~ 6.5%, and from the perspective of

    rutin extracted significant difference in quality. As the current youmanii Eucalyptus breeding also depends on seed

    multiplication, so the two kinds of plant to the quality of identification is necessary.

     2.2.1 Introduced plants of the original form of

    identification of Sichuan, a total of four kinds of Eucalyptus youmanii sources, of which one kinds of source leaves small and slender, the other three blades is relatively wider, larger leaves. Lobules of the A259/A364 ? 1.71, rutin content

    of about 10%, while the big-leaf there A259/A36 ? 1.71 and

    A259/A364 ? 2.34 two kinds of plant. No significant

    difference between their biological properties, but A259/A364 ? 2.34 per plant in Nenshao color slightly darker, solely from the character speaking it is difficult to identify them.

     2.2.2 Identification of the two kinds of hot water extract of fresh leaves per plant (10 g) or dry leaves (3 g), after grinding by adding 100 ml distilled water, boil 20 min filtration put it aside, after cooled to 10 ~ 15 ?, 2 h

    A259/A364 ? 1.71, after the plant has a large number of yellow and rutin precipitates, the solution was light yellow; and A259/A364 ? 2.34 in plant no or very small amount of rutin precipitates, the solution was brown. Put it aside for

    more than 10 h after the solution of the color difference is obvious. The results shown in Figure 1.

     2.2.3 Identification of ultraviolet spectrophotometry to take two kinds of dry leaf powder of 0.01 g per plant in the flask, accurate by adding 50 ml of methanol in the ultrasonic extractor extracted 30 min, filtered after cooling to room temperature control in methanol 259,364 nm UV

    spectrophotometer measured absorbance to calculate the ratio of A259/A364 to be identified.

     From left to right 1-3 for A259/A364 ? 1.71 per plant,

    4-5 A259/A364 ? 2.34 in plant

     Figure 1, two kinds of plant extract of hot water (put it aside for 4 h) identification (abbreviated)

     2.2.4 High Performance Liquid Chromatography according to 3.3.1 extract preparation, chromatographic conditions: glacial acetic acid / methanol / water = 3/49/48, detection wavelength: UV256 nm, flow rate 0.8 ml / min, injection volume of 10 μl. In addition to rutin peak height differences are

significant, the No. 2 peak for the A259/A364 ? 1.71 in plant

    characteristic peak of 1,2,4,5-peak, the plant for the

    A259/A364 ? 2.34 characteristic peaks of the two single -

    strain extract the detection wavelength was 355 nm chromatogram of the time there is no significant difference.

    The results shown in Figure 2.

     Detection wavelength: 256 nm detection wavelength: 355 nm

     Figure 2 two kinds of plant leaves extract by high performance liquid chromatography diagram (omitted)

     3 Discussion

     Determination of total flavonoids are more, but more commonly is used in this study, four kinds of ways. Especially in the chemical composition of the sample is unclear circumstances, in particular the use of a maximum of aluminum nitrate method, but its specificity is not strong. Number of

    non-flavonoids, such as protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, phenolic hydroxyl group with o-2

    structure after the reaction to a substance at 500 nm has absorption [6 ~ 8], A259/A364 ? 2.34 in plant may also

    contain such ingredients. I Determination of aluminum nitrate in several other Eucalyptus leaves flavonoids, also found that measured values was significantly higher than aluminum chloride method, and therefore discussion of eucalyptus leaves flavonoids content of aluminum nitrate, when laws may not apply, of course, trichloride Method in the Determination of Aluminum other eucalyptus leaves flavonoids also need to verify the applicability of the time. For many eucalyptus leaves flavonoids clearer and more accurate high performance

    liquid chromatography.

     According to extract chromatograms, two kinds of plant chemical composition significantly different, A259/A364 ?

    2.34 for a single plant may be a subspecies of Eucalyptus youmanii, or other eucalyptus species and hybrid species of Eucalyptus youmanii and may even be for other eucalyptus species, the full identification of their remains to be

    further studied. Although this plant's flavonoid content is relatively high, but I found that the leaf extract on

    Staphylococcus aureus (Staphy lococcus aureus), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichiu coli), and Fusarium oxysporum (Fusari - um oxysporum

    f.sp.cucumerinum) of the antibacterial activity was significantly higher than the A259/A364 ? 1.71 of the plant,

    also higher than lemon eucalyptus, and antibacterial activity of Dayean rather, its medicinal value is worth in-depth study.

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