Column chromatography - Determination of total flavonoids
Author: Yu Danni, Desheng, FENG Yi, Wei Hong-
【Abstract】 Objective To establish Motherwort Determination of Flavonoids in medicinal herbs, and medicinal resources, a
preliminary investigation. Methods rutin as reference substance, samples purified by polyamide column, three aluminum chloride color reagent, the use of ultraviolet spectrophotometry at 412 nm wavelength determination of total
flavonoids. The results of rutin in the 4.8 ~ 29.0 μg / ml
and the absorbance within the framework of a good linear relationship, r = 0.999 6, the average recovery was 102%, RSD = 3%. Conclusion The method is simple, accurate, and can be used as the quality of medicinal herbs Leonurus analysis.
Key words Motherwort; total flavonoids; column chromatography - spectrophotometry
Abstract: ObjectiveTo establish the method for the quantitative determination of total flavonoids in Herba Leonuri, make the prime examination of the
resources.MethodsThe sample was treated with a polyamide column for separation and purification, and was colorated with aluminium trichloride, and then, the content of flavone in the sample was determined at the wavelength of 412 nm by
ultraviolet spectrophotometer with rutin as control.ResultsIt showed a good linear relationship in the range of 4.8 ~ 29.0 μg / ml, r = 0.9996 the average recovery of the added sample was 102 % and RSD was 3%. ConclusionThe method is simple and
accurate. It can be used for the quality control.
Key words: Herba Leonuri; Total flavonoids; Column chromatogram UV spectrophotometry
Labiatae Leonurus Motherwort plants for Leonurous heterophyllus Sweet dry ground parts of bitter, Sim, slightly
cold, owned by the liver, pericardium, with blood circulation Huayu, the efficacy of diuretic swelling . Mainly contains alkaloids, diterpenes, iridoid glycosides, flavonoids, organic acids and other ingredients . "Chinese Pharmacopoeia" to alkaloid determination as indicators of Leonurus herbs. In recent years, flavonoid constituents of experts and scholars began to receive attention. Motherwort contains a variety of flavonoids such as rutin, quercetin, kaempferol, and glycosides, apigenin, genkwanin and glycosides, etc. ,
studies have shown that flavonoids in cardiovascular Motherwort significant pharmacological effects . Motherwort medicinal herbs and preparations in the determination of total flavonoids reported that small, mostly herbs extracted
directly after a preliminary determination, половдмид
р the first time the introduction of column chromatography, medicinal herbs and preparations Motherwort content analysis . At present, the determination of total flavonoids Motherwort there are still large enough, such as extract,
purify mode selection confusion caused interference components measured are more obvious method of color is no basis for selection and so on, while herbal medicine Motherwort no reports of total flavonoids of resources. This experiment
simulated by thin-layer chromatography conditions for column chromatography to determine the impurity of the herbs way right Motherwort Determination of Flavonoids in medicine conducted a systematic study, and measured six areas of Flavonoids in herbs Leonurus.
An instrument and reagent
Aglient8453 UV-visible spectrophotometric analyzer ,752-
N-type spectrophotometer (Shanghai Analysis Instrument Factory), BP211D sartorius electronic balance.
Motherwort (6 origin herbs were purchased from Shanghai
Traditional Chinese Medicine Cambridge Pieces Co., Ltd. Chinese Herbal Pieces Qiqihar Lily Processing Co., Ltd., Henan
Guangshan County medicine company, the Guangzhou Health and Herbal Pieces Co., Ltd., Anhui Bozhou medicine market,
Guangdong City, medicinal herbs the market, via Shanghai Traditional Chinese Medicine Pharmaceutical Students Teaching and Zhou Jia teacher appraisal are origanum Motherwort Leonurus heterophyllus Sweet).
Rutin, quercetin, kaempferol reference substances (all
purchased from Shanghai Standardization Center); polyamide film, polyamide column chromatography, 80 ~ 100 mesh (Zhejiang Taizhou Bridge 4 Green Biochemical Material Factory Product ); methanol, ethanol, aluminum chloride, sodium hydroxide,
aluminum nitrate, nitrite were AR.
2 Methods and Results
2.1 Choice of color, sodium nitrite - aluminum nitrate -
sodium hydroxide and aluminum chloride for the determination of total flavonoids most commonly used color reagent [6,7]. The former measured after color around 510 nm wavelength, which is in the vicinity of 410 nm or 270 nm. Leonurus herbs at 500 nm due to more interference with absorption of office, and the sample solution in the presence of quercetin, kaempferol flavonoids and other ingredients, in order to
nitrite - aluminum nitrate - sodium hydroxide color absorption
at 500 nm after the weak or non-absorbing  Therefore, the
use of sodium nitrite - aluminum nitrate - sodium hydroxide
reagent is not ideal to do. So, the selection of aluminum
chloride as chromogenic agent, rutin as reference substance, for determination of total flavonoids.
2.2 Impurity take Motherwort medicine to determine the conditions appropriate, Chi Soxhlet extractor, add chloroform to extract reflux extraction colorless chloroform solution evaporated and the residue add 1 ml of methanol for the test allows the dissolved for product solution A. Dregs to shake off chloroform, add methanol to extract to the colorless, extracts evaporated and the residue add 1 ml of methanol for
the test items for the solution allows the dissolved B. The above samples and the corresponding reference substance solutions were point-like on the polyamide film, the first
water start, water film will start to dry out, and then with
different concentrations of ethanol to commence after the commencement of the polyamide thin-film spray with
trichloroacetic Aluminum test solution at 365 nm UV lamp observation of fluorescence. Thin-layer chromatography shown
in Figure 1 ~ 3.
Thin-layer indicates that note, polyamide adsorption can remove chlorophyll (for the test solution A section). Water elution and 20% ethanol elution can be removed with color interfering with water-soluble components. 70% ethanol elution of total flavonoids can be enriched.
2.3 medicinal flavonoids extraction methods herbs were collected from fine powder 3 g, by adding methanol, 50 ml, in order to return (45 min), Soxhlet extraction (to colorless), ultrasound (200 w, 59 kHz, 45 min) 3 kinds of methods of
extraction, measured absorbance to calculate the yield of flavonoids. Choosing to return the results of extraction methods extract.
2.4 Motherwort Determination of flavonoids in medicinal herbs
2.4.1 Preparation of reference substance solution
precision Weigh 105 ? drying to constant weight of rutin
standard 0.002 5 g buy 25 ml flask, add 60% ethanol, dissolved into a fixed volume 0.1 mg / ml solution, that is, too.
2.4.2 Preparation for the test product solution to take medicine powder 3 g, precision that set. Purchase Round-bottom
flask, the precision by adding methanol, 60 ml, weighing, heating reflux 45 min, let cool, put it aside, and then said that given the weight reduction weight loss supplement with methanol, shaken, filtration, sophisticated take the amount of filtrate added to 30 ml evaporating dish in the home, bath evaporated. Residue plus a small amount of 50% ethanol dissolved in water bath to shake off 1 / 3 the volume, add distilled water to the original volume, Suspension, the
suspension over polyamide column (80 ~ 100 mesh, 1 g, diameter 1 cm, wet loaded column). To 20 ml of water rinse, abandoned to the water liquid, and then to 20% ethanol 15 ml rinse, and to the alcohol solution. Finally, 70% ethanol elution, a
yellow ribbon to view the flavonoid movement, when the ribbon reaches the lower part of collected eluate, the eluate collected into 25 ml flask, the collection to nearly scale, plus 50% ethanol fixed Yung, shaking, as a solution for the
2.4.3 Determination of the wavelength selection for the trial to take reference substance and the amount of product solution, by adding 0.1 mol / L AlCl3 solution, 1 ml color 30 min after the 200 ~ 800 nm for wavelength scanning, scan the results shown in Figure 4.
The results showed that rutin reference standards and samples at both 273 nm maximum absorption peak at 412 nm Department, rutin maximum absorption, the sample does not appear as strong absorption maximum absorption. Experiment found that, after the polyamide impurity at 273 nm
interference is still significant. Therefore chosen for the determination of wavelength 412 nm.
2.4.4 Precision linear relationship study lessons 0,0.5,1,1.5,2,2.5,3 ml solution of the above-mentioned
reference substance were placed in 10 ml Liang Ping, add 0.1 mol / L AlCl3 solution 1 ml, with 60% ethanol constant volume to the scale, place 25 min, in order to sample No. 1, blank, 412 nm absorbance was measured. With the concentration of abscissa, absorbance standard curve drawn for the vertical axis. Calculated regression equation was Y = 25.089X 0.0153, r = 0.999 6, shows that reference substance in the concentration range of 4.8 ~ 29.0 μg / ml and the absorbance have a good
2.4.5 precision experiment to take a certain
concentration of test items for the solution of the absorbance measurement, continuous measurement 5 times, the RSD was 0.26%, good precision instruments.
2.4.6 stability test for the test materials obtained
solution were placed 5,10,15,20,25,30,35,45,55,65,75,85 min at room temperature measured absorbance for the solution of test items to place 25 min After the color completely, and 1 h to stabilize, RSD is 0.15%. Reposted elsewhere in the paper for free download http://
2.4.7 Experimental repeatability precision obtained with the batches of samples, said five copies, according to assay method of determination of total flavonoids average 3.25 mg. RSD was 1.4%, reproducibility was good.
2.4.8 The average recovery obtained experimental
precision that is known medicinal powder Motherwort content of five copies, respectively, by adding precise amount of rutin reference substance, according to determination under the method of measurement, the average recovery was 102%, RSD 3 %.
2.4.9 Determination of content of the sample taken six different areas of medicine powder Motherwort appropriate, in accordance with the standard curve method under the absorbance was measured at 412 nm. Flavonoids in medicinal herbs in the
calculation. The results in Table 1. Table 16 regions Motherwort sample size determination results (omitted)
The results showed that total flavonoids in Heilongjiang Motherwort the highest, 3.27 mg / g; Guangxi total flavonoids
production Motherwort the lowest, 0.7 mg / g. Tip Motherwort the effective content and geographic location, species, harvesting season and other factors are closely linked.
3.1 Polyamide thin-layer pre-test and the results agree
medicinal polyamide column separation, experimental study through the thin layer of 10%, 20%, 30% ethanol impurity effect, with 20% ethanol for the best selection of 60% eluted , 70%, 80% ethanol compared to 70% less eluting impurities, and eluting a smaller dosage, therefore, to
determine with 70% ethanol eluted flavonoids and elution with water and alcohol consumption are eluting thin-layer analysis.
3.2 With regard to medicinal herbs and extracts the content of total flavonoids determination to reports, has been
very chaotic, with various different types of herbs containing flavonoids, in the reference substance, impurity removal methods, testing methods should have a choice [9,10], the experiments, polyamide refined, except for a large number of interfering substances, so that more reasonable determination of the results, this method can be used Motherwort extract of herbs and total determination.
3.3 The experiment had a different determination methods are compared to sodium nitrite - aluminum nitrate - sodium
hydroxide color aluminum chloride than by three high color
determination results. Impurity of different methods also have an impact on the determination results, as compared with the chloroform impurity, impurity by polyamide column lower post-
test results obtained. Therefore, the impurity removal methods and determination of the choice of methods in the spectrophotometric determination of total flavonoids in the process is essential.
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