Ginkgo biloba polysaccharide extraction, purification and the determination of
【Abstract】 Objective To study the extraction and
purification of polysaccharides of Ginkgo biloba and determination. Methods of extraction of different frequency,
solid-liquid ratio and extraction time of experiment, and the study of purified polysaccharide to establish conditions for various factors, using phenol - sulfuric acid method
polysaccharide content determined. The results of various factors, conditions of extraction temperature 80 ?,
extraction time 3 h, solid-liquid ratio 1:30, extracted two
times, purified with activated carbon. Ginkgo biloba may result in Polysaccharide yield was 12.2%, purified polysaccharides yield of 9.8%. Determination of polysaccharide
extracted crude polysaccharide content of 35.7%, purified polysaccharide content of 43.3%, polysaccharide determination of the relative standard deviation less than 3.0%. Conclusion This method is a good purification effect measured with good
reproducibility obtained in the polysaccharide content of Ginkgo biloba 4.3%.
【Key Words】 Ginkgo biloba polysaccharide determination of extraction and purification of polysaccharides
Abstract: ObjectiveThe extraction and purification methods for polysaccharide in Gingko leaves were studied and the content of polysaccharide in it was determined.MethodsThe
condition of each factor for extraction and pruification from the leaves of Gingko was established through the experiments of extraction times, different ratios of material to extraction solvent and extraction time.The method of phenol-
sulfuric acid was applied to determination of the content of the polysaccharide.ResultsThe conditions of the factors were
extraction temperature 80 ?, extraction time 3h, material to
extraction solvent 1:30, extraction 2 times and activated carbon for purification.Raw polysaccharide in gingko leaves
was 12.2%, the polysaccharide after prurified was 9.8%, the content of polysaccharide in the raw polysaccharide was 35.67%, and in the pure one was 43.32%, ConclusionThe purification method is efficient and stable. The content of
polysaccharide in gingko leaves is 4.3%.
Key words: Ginkgo leaves; Polysaccharide; Extraction; Purification; Determination of polysaccharide
Polysaccharide also known as polysaccharides
(polysaccharide), the four major life activities constitute
one of the basic material, not only the body primarily for energetic materials, but also has a wide range of biological functions in life activities involved in the cells activities. Currently known active polysaccharides have anti-tumor, anti-
virus, enhance immunity, anti-aging, blood fat, lowering blood
sugar, detoxification, anti-radiation effects [1 ~ 5], its
role increasingly attention.
Ginkgo (Ginkgo) is the period of survival in glacial relict plant on the planet, known as a "living fossil," said.
China is the birthplace of Ginkgo biloba and its resources accounted for about 70% of the world total [6,7]. At present, the Ginkgo biloba extract from the medicinal pure gradually applied to the food and beverage, health care products and
other industries, including Ginkgo biloba extract hair care, hair growth, skin care cosmetics such as the development has made some progress. Ginkgo leaves contain an important role in physiological and pharmacological function of the active ingredient, but on the polysaccharides in Ginkgo biloba is currently very little coverage because people are concentrating their efforts are concentrated in the leaves of the Ginkgo Flavonoids research on [7 ~ 12], the neglect of polysaccharides. The experiments of extracting polysaccharide
from Ginkgo biloba in the influencing factors, purification carried out a series of studies to determine the conditions for extraction and purification. At the same time on the extraction and purification of the polysaccharide content were
measured to determine the polysaccharide content in the leaves of the Ginkgo biloba. This is a comprehensive development and utilization of Ginkgo biloba leaves of great significance.
1 Materials and methods
1.1 Instrument and reagents constant temperature water
bath, centrifuge, constant temperature drying oven, vacuum drying, UV - visible spectrophotometer.
95% ethanol, anhydrous ethanol, chloroform were of analytical grade; glucose as biochemical reagents.
1.2 Sample source and pretreatment of the samples of Ginkgo biloba, in 2005 11 taken from the Dalian
Nationalities University campus. Will be mined by the Ginkgo biloba washed with tap water, then washing with distilled water, splashing water, after dry, with 70 ? constant
temperature drying oven.
1.3 extraction and purification methods of Ginkgo biloba polysaccharide extraction, purification steps are as follows:
a) Ginkgo biloba treatment: before the experiment with the 70 ? constant temperature drying oven to bake another 1
of Ginkgo biloba, and then use high-speed grinder grinding,
over 80 mesh sieve, placed in brown bottle to save.
b) Extraction temperature: according to a certain solid-
liquid ratio by adding water, extracted in constant temperature water bath temperature.
c) centrifugation: centrifuge extracted later to 5 000 r / min centrifugation 20 min, collect supernatant, residue re-
extracted one time with the former, merging two supernatant.
d) purification: the supernatant obtained after
centrifugation, purified with activated carbon.
e) Concentration: vacuum drying of the purified liquid for centrifuge enrichment.
f) alcohol precipitation: Add 5 times the volume of 95% ethanol, placed in the refrigerator put it aside for 24 h.
g) centrifugation: The alcohol precipitation of a white floc sedimentation centrifuge polysaccharide.
h) washing: With the polysaccharide with anhydrous ethanol and chloroform followed by washing and then placed a small beaker, evaporated in the water bath after the pot placed in 80 ? oven drying.
2 Results and Conclusion
2.1 Experimental Factors Affecting Polysaccharide extraction
2.1.1 solid-liquid ratio on the yield of crude
polysaccharide obtained five copies of 5 g of Ginkgo biloba
samples were placed in 250 ml flask with plug in, according to solid-liquid ratio 1:10,1:20,1:30,1 : 40,1:50 were added 50,100,150,200,250 ml of distilled water, pH 7, at 80 ? water
bath extraction 3 h, then centrifuged, concentrated with
ethanol precipitation, in the refrigerator put it aside for 24 h, re-centrifugation, crude polysaccharide yield shown in Figure 1.
Can be seen from Figure 1, solid-liquid ratio the bigger
the better, but to carry out two times extraction, therefore,
solid-liquid ratio not too big. As can be seen from Figure 1, solid-liquid ratio of 1:30 after the crude polysaccharide yield increases slowly. Solid-liquid ratio of 1:30, the crude
polysaccharide yield was 10.4%, solid-liquid ratio of 1:40,
when crude polysaccharide yield was 10.5%, only an increase of 0.1%; solid-liquid ratio of 1:45, the coarse polysaccharide yield was 10.7% over the solid-liquid ratio of 1:40 increased
by 0.2% over the solid-liquid ratio of 1:30 to increase 0.3%. The solid-liquid ratio of 1:20, the crude polysaccharide yield was 9.4%, solid-liquid ratio of 1:30 compared to 1:20 and the yield of crude polysaccharide increased by 1.0%. So, use the solid-liquid ratio 1:30.
2.1.2 extraction time on the yield of crude
polysaccharide take six copies of 5 g of Ginkgo biloba samples were placed in 250 ml flask with plug in, according to solid-
liquid ratio 1:30 by adding distilled water 150 ml, pH 7, at 80 ? in a water bath were extracted 0.5,1,2,3,4,5 h, then centrifuged, concentrated, ethanol precipitation, in the refrigerator put it aside for 24 h and then centrifuged, and crude polysaccharide yield shown in Figure 2.
Can be seen from Figure 2, distilled water and extracted 3 h later to extend the extraction time on the extraction rate
of increase is not significant. When the extraction time was 3 h, the crude polysaccharide yield was 9.3%, extraction time of 4 h, the crude polysaccharide yield was 9.6%, the yield increased by only 0.3%; when the extraction time was 5 h, the
coarse multi - sugar yield of 9.8%, the yield increased by 0.2% over 4 h compared with 3 h increased 0.5%. The extraction time was 3 h compared with 2 h the yield increased 2.1%, so 3 h extraction time should be better.
2.1.3 Extraction of the number of pairs of crude
polysaccharides obtained a copy of the rate of 5 g of Ginkgo biloba powder put in a 250 ml flask in Cyprus, according to the solid-liquid ratio 1:30 by adding distilled water, pH 7, at 80 ? water bath extraction 3 h, centrifuge. Extraction of
the residue collected after centrifugation, according to the above conditions and then extracted two times. 3 Extraction of extracts obtained from three separate flask placed in concentration, ethanol precipitation, in the refrigerator put
it aside for 24 h, centrifugation, crude polysaccharide yield shown in Figure 3.
Can be seen from Figure 3, the first one Extraction of crude polysaccharides was the highest rate was 9.0%; the first 2 times the second, 2.8%; the first three times only 0.4%, so
this experiment extraction times selected two times.
2.1.4 of temperature on the yield of crude polysaccharide were obtained five samples of 5 g of Ginkgo biloba be put in a 250 ml flask in Cyprus, according to solid-liquid ratio 1:30
by adding distilled water pH 7, respectively, in 70,75, 80,85,90 ? water bath extraction 3h, then centrifuged, concentrated, ethanol precipitation, in the refrigerator put it aside for 24 h and then centrifuged, and crude polysaccharide yield Figure 4.
Can be seen from Figure 4, with the extraction
temperature increased, the polysaccharides yield increased gradually, peaked at 80 ?, polysaccharide yield of 9.2%. But
to reach 85 ? after the yield of crude polysaccharide has been slowly decreasing. 85 ? when the yield was 9.0%, 80 ?
lower than 0.2%; 90 ? when the yield was 7.6%, 1.4% lower
than 85 ?, 80 ?, lower than 1.6%. This is because the
temperature is too high, polysaccharide degradation occurred, indicating at this time conditions are not conducive to the
extraction of polysaccharides, so the present study, the best extraction temperature 80 ?. Reposted elsewhere in the paper
for free download http://
Extracted according to the above conditions, the leaves of the Ginkgo biloba crude polysaccharide yield was 12.2%,
showing brown yellow.
2.2 Purification of Ginkgo biloba polysaccharide study obtained crude polysaccharide extract of Ginkgo biloba refined, first thing to consider is to remove impurities and polysaccharide precipitation, such as flavonoids and other
substances. As Ginkgo biloba contains a large number of flavonoids, in the extraction process of polysaccharide will inevitably occur with the polysaccharide precipitation. Flavonoids dissolved in ethanol, so first consider the use of
ethanol to refining, but also refined the method using activated carbon.
2.2.1 unrefined crude polysaccharide obtained 0.100 g of dry extract crude polysaccharide dissolved in distilled water and transferred to 50 ml flask in distilled water diluted to
scale, shake. By UV spectrophotometer at 190 ~ 320 nm within the scan, obtained UV spectrum shown in Figure 5.
Be seen from Figure 5, in the crude polysaccharide extract without any treatment, the extracted crude polysaccharide absorbance up to 5.0.
2.2.2 ethanol purification methods of a crude
polysaccharide extracted 24 h after ethanol precipitation, centrifugation before washing with 50 ml anhydrous ethanol, then centrifuged. Take the above-mentioned methods were dry
polysaccharide 0.100 g, dissolved in distilled water and transferred to 50 ml flask in distilled water diluted to scale, shake. By UV spectrophotometer at 190 ~ 320 nm within the scan, obtained UV spectrum shown in Figure 6.
As can be seen from Figure 6, crude polysaccharide by
ethanol washing, some impurities have been removed, so spectrum 6 compared with the spectrum of 5 in the vicinity of
the absorbance at 320nm decreased slightly.
2.2.3 ethanol purification methods of the two crude polysaccharide extracted after centrifugation and drying the former with 50 ml anhydrous ethanol twice washing, centrifugation. Take the above-mentioned methods were dry
polysaccharide 0.100 g, dissolved in distilled water and transferred to 50 ml flask in distilled water diluted to
scale, shake. By UV spectrophotometer at 190 ~ 320 nm within the scan, obtained UV spectrum shown in Figure 7.
As can be seen from Figure 7 using this method of washing of ethanol than the ethanol purification methods of an effect of better, in the wavelength range 255 ~ 320 nm, the
absorbance decreased significantly, indicating this method is superior to ethanol, a refined method.
2.2.4 Method 3 will be refined ethanol extracted crude polysaccharide in the centrifuge, dried and dissolved with 60
ml distilled water, and then ethanol precipitation, centrifugation, dry. Take the above-mentioned methods were
polysaccharides 0.100 g, dissolved in distilled water and transferred to 50 ml flask in distilled water diluted to scale, shake. By UV spectrophotometer at 190 ~ 320 nm within the scan, obtained UV spectrum shown in Figure 8.
As can be seen from Figure 8, this approach results in two ways is better than the previous number, in the wavelength range 230 ~ 320 nm, the absorbance decreased, indicating this
method is superior to ethanol purification method 2, but the purification effect is not very ideal.
2.2.5 refined to take 5.0 g of activated carbon have been shattered by the Ginkgo biloba extract has been established in the best conditions in 80 ? water bath extraction two times,
obtained extract with activated carbon purification, and then the filtrate was concentrated, add ethanol precipitation, are polysaccharides . Weigh the extracted polysaccharide 0.1 g, dissolved in distilled water and transferred to 50 ml flask in distilled water diluted to scale, shake. By UV
spectrophotometer at 190 ~ 320 nm within the scan, obtained UV spectrum shown in Figure 9.
As can be seen from Figure 9 using activated carbon
adsorption method of flavonoids than the method of ethanol to effect much better. In the wavelength range 190 ~ 320 nm, the absorbance decreased significantly. Thus determine the activated carbon adsorption method to purify crude polysaccharide extract of Ginkgo biloba.
Refined using activated carbon, the obtained
polysaccharides yield 9.8%, was white.
2.3 Ginkgo biloba polysaccharide content in testing the present study, phenol - sulfuric acid colorimetric method of Ginkgo biloba polysaccharide were determined.
2.3.1 standard sample solution, blank solution and determination from a prepared 10.0,20.0,30.0,40.0,50.0 mg / L of glucose standard solution were removed 2.0 ml in 10ml test
tubes with plug, and then adding 6% phenol, respectively solution 1.0 ml, slowly adding concentrated sulfuric acid 5.0 ml, shake. Put it aside for 30 min, at 490 nm wavelength under the colorimetric analysis. Taking 2.0 ml of distilled water in 10 ml test tube with plug in, instead of glucose standard solution, that is, a blank solution, measured with the standard samples were determined. Standard curve correlation coefficient of 0.999 7.
2.3.2 Determination of polysaccharide samples were collected from four copies of refined polysaccharide and polysaccharide samples, dissolved in distilled water were transferred to a 50 ml flask with distilled water and diluted to scale, shake. Were removed from 2.0 ml to 10 ml test tube with plug, determined in accordance with the standard samples were measured the same way. The results in Table 1. Polysaccharide determination results in Table 1 (omitted)
2.3.3 precision experiments crude polysaccharide extracted and refined polysaccharide measured 8 times
respectively. The results in Table 2. Precision of experimental results in Table 2 (omitted)
Table 2 shows, 8 refined polysaccharide and
polysaccharide determination of the relative standard deviations were less than 3.0%, refined polysaccharide
polysaccharide content of the sample was 43.1%, crude polysaccharide samples in the polysaccharide content of 35.1%.
2.3.4 Ginkgo biloba in the polysaccharide content was informed by the determination of the above-mentioned
polysaccharides, ginkgo leaf extract crude polysaccharide
yield was 12.2%, the measured content of 35.67% of which polysaccharide, thus calculate the Ginkgo biloba leaf polysaccharide content of 4.35%; empathy with the refined calculation of Ginkgo biloba polysaccharide polysaccharide
content of 4.25%, the two extraction methods were calculated polysaccharides in Ginkgo biloba polysaccharide content of error was 2.33%. Note on the extraction using activated carbon purification process of crude polysaccharides basically no loss of polysaccharide.
Ginkgo biloba polysaccharide extraction conditions of 80 mesh ginkgo leaf samples in 80 ?, the solid-liquid ratio of
1:30 in pH 7.0 distilled water in the extraction 3 h, extracted two times, and then by centrifugation, concentrated,
and alcohol precipitation steps such as ginkgo biloba be polysaccharide. According to the above conditions of the Ginkgo biloba crude polysaccharide yield was 12.2%.
Ginkgo leaves Polysaccharide purified using activated carbon results much more desirable. Refined polysaccharides from Ginkgo biloba leaves yield of 9.8%.
Using phenol - sulfuric acid colorimetric method of
Ginkgo biloba extract in the polysaccharide were determined, the standard sample solution of the correlation coefficient of
0.999 7, 8 Determination of polysaccharides relative standard deviation less than 3.0%. Measured in crude polysaccharide polysaccharide content 35.67%, purified polysaccharide polysaccharide content of 43.32%. Last fall acquisition of the
floor to calculate the yellow ginkgo leaves polysaccharide content of 4.3%.
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