Tian Hongxing, Yin Shaowen
Guiyang College of Traditional Chinese Medicine, Guiyang 550002, Guizhou
Abstract: The rate of pollen wall breaking is often considered as one of the important indicators for evaluating the method of pollen wall breaking and the quality of pollen wall breaking, but there is no unified standard method for detecting the rate of pollen wall breaking. Therefore, different detection methods of pollen wall breaking rate were discussed, the advantages and disadvantages of various methods were analyzed, and the existing shortcomings and development trend were put forward, in order to lay a foundation for further research on detection methods of pollen wall breaking rate.
[Key words] Pollen wall breaking rate; Detection method; Research progress
[CLC No.] R914 [Document Code] A [Article No.] 1007 － 8517 (2017) 13 － 0052 － 04
Pollen is the male gametophyte of plants, which exists in the male pollen sac. It is a complex mixture of powdery granular cells on male stamens and special secretions of bees [1]. Studies have proved that pollen is not only the source of life of plants, but also known as the “micro nutrient treasure house” (containing 25%~30% protein, more than 20 free amino acids that are easily absorbed by the human body, more than 10 vitamins, trace elements, natural active enzymes and flavonoids), with high nutritional value and medical and health care functions [2-6].
The hard outer wall of the pollen is mainly composed of dense substances such as sporopollen, cellulose, cutin and pollen element. The chemical properties are very stable, and it has the characteristics of acid resistance and alkali resistance. As a result, the extraction of effective substances in the pollen wall is greatly hindered, making its effective substances not fully utilized by the human body [7]. In order to improve the utilization value of pollen, people have done a lot of exploration on pollen wall, among which “pollen wall breaking technology” is one of the main research directions. The results showed that the utilization rate of pollen could be improved by wall breaking treatment. At present, pollen wall breaking methods mainly include compound wall breaking method [8-9], physical wall breaking method [10-11], biochemical wall breaking method [12-13], etc. However, no matter which wall breaking method and process conditions are used, there should be a wall breaking rate detection method to test the degree of pollen wall breaking. In view of the fact that there is no unified standard detection method for pollen wall breaking rate at present, this paper discusses different detection methods for pollen wall breaking rate, analyzes the advantages and disadvantages of various methods, and puts forward the shortcomings and development trend, which is intended to lay a foundation for in-depth research on the detection method for pollen wall breaking rate.
- Micrometry
1. 1 The slide direct counting method and the improved group Pap blood cell counting method Wang Huakai et al
Method and the modified group were used to count Pap’s blood cells. The results show that,
The slide direct counting method is simple and easy to operate, but because the cover glass has the effect of extrusion, the distribution of pollen cells is not easy to be uniform, counting according to the field of vision, with Overlap or omission and poor reproducibility. The direct slide counting method is to use a micro syringe (or hemoglobin pipette) to suck the diluted solution of broken and unbroken wall pollen plasma10 prepared into an aqueous solution of equal concentration μ L on ordinary glass slide, add 20mm × Place the 20mm cover glass under the microscope, move the propeller, and count the field of view one by one with a high-power mirror from left to right and from top to bottom at the center of the cover glass. Measured pollen wall breaking rate（ η) The formula is as follows:
Wall breaking rate (%) × (Number of pollen cells in the control sample? Number of intact pollen cells retained in the test sample)/Number of pollen cells in the control sample × 100% ; In the improved group, the counting method of Pap’s blood cell counter is more reasonable than the direct reading method of slide. Because the counting room has a depth of 0.1 mm, the pollen cells are not squeezed, and the four grids are evenly distributed with good reproducibility. In addition, if there is no micro syringe, a small drop of sample can also be put into the counting room with an eye drop stick, but the filling should be moderate. The operation method is made of a thick glass slide, and the whole counting room is divided into nine grids. The area of each large grid is 1mm2, and its depth is 0.1mm. Four square cells, each cell is divided into 16 middle squares, and the wall broken pollen slurry and the wall unbroken pollen slurry diluent 10 that have been prepared into an aqueous solution of equal concentration are absorbed respectively μ 50. Fill the counting chamber along the edge of the cover glass, wait for 3~5min, Count under high power microscope and divide the total number of cells in the four corners by 4 Number of pollen cells per large cell. Compared with the number of cells per mm3 and volume, Measured pollen wall breaking rate（ η) The formula is as follows:
Wall breaking rate (%)=100% – {100% × ( A ÷ 4 × 10) / ( B ÷ 4 × 10) }
During test: total number of four bars of test sample A;
B The total number of four cells of the control sample.
In addition, more than 50 batches of pollen wall breaking rate were tested by the improved group of Pap’s blood cell counter counting method. Compared with the slide direct counting method, this method is more rapid, simple, reproducible, and reliable. It can be used as one of the evaluation indicators for detecting the degree of pollen wall breaking in the future.
1. 2 Staining smear method This method is mainly based on the direct counting method of glass slides. It uses the principle that the protoplast of pollen after dyeing is light red and the wall is purple red, forming a large contrast between the two, and the pollen is easy to count to measure the pollen wall breaking rate. Xue Miaonan [15] dyed the broken and unbroken Chinese nectar source pollen with a quantitative alkaline fuchsin dye solution for 1~1.5 hours, then diluted the dyed pollen solution by 5 times, stirred evenly, took the quantitative pollen solution with a pipette, placed it on the carrier, smeared it on a certain area, dried it in a 40 ℃ oven, transparent it with xylene for 2~3 minutes, took it out, sealed it with Canadian gum, and then observed and counted. Under the light microscope, with an eyepiece micrometer, use the 5-point sampling method to calculate the number of broken and unbroken pollen within 0.5 cm2 of each point, and calculate the wall breaking rate of pollen according to the following formula:
Pollen wall breaking rate (%)=100% × (Number of complete pollen before wall breaking – number of complete pollen after wall breaking)/Number of complete pollen before wall breaking; It can be seen from the above that this method is more reasonable than the slide direct counting method. The contrast in color makes it easy to count the pollen, which further provides the reliability of microscopic observation results. However, there are also uneven distribution, overlapping or omission of pollen in the slide direct counting method. Therefore, this method is worth further discussion.
1.3 Gravimetric method combined with microscopic quantitative method This method is based on the above slide direct counting method, referring to the microscopic quantitative method of traditional Chinese medicine, taking complete pollen grains as the reference material, and using gravimetric method combined with microscopic quantitative method to determine the pollen wall breaking rate [16]. Measured pollen wall breaking rate（ η) The formula is as follows:
Pollen wall breaking rate (%)={1 – (number of sample pollens/number of control pollens) × (Weight of control pollen/number of sample pollen)} × 100%.
The number of sample pollen is the total average value of 9 visual fields observed by the second sample of sample pollen;
The number of control pollen is the total average of 9 visual fields of the second sample of control pollen;
The weight of the control pollen is the actual weight (mg) of the control pollen when preparing the suspension;
The weight of sample pollen is the actual weight (mg) of the sample weighed when preparing the suspension.
This method is simple and easy to operate. The chloral hydrate test solution is used for loading, and the average value of pollen grains in 9 visual fields is observed in the same sample suspension twice, and the relative deviation is less than 5%. At the same time, the gravimetric analysis principle is added to improve the accuracy and reliability of the measurement results. However, the raw material must be a single pollen and must not be mixed with other pollen and impurities, otherwise the measurement results will be affected easily.
1.4 Determination of standard curve The principle of standard curve method is a quantitative analysis method that uses points of different concentrations to explore the relationship between independent variables and dependent variables. Li Ying et al. [17] used this principle to explore the relationship between the broken wall rate of pine pollen and the number of unbroken pollen cells, and optimized various factors affecting the determination by using orthogonal experimental design, and obtained the best experimental conditions. The obtained method curve has good linear relationship, high accuracy and is easy to operate. The first step of the experiment was to weigh 0g, 1.00g, 5.00g, 10.00g, 15.00g and 20.00g of the control sample of unbroken pine pollen respectively, Dilute 100% wall broken pine pollen to 20.00 g and mix well to prepare standard samples with wall broken rates of 100%, 95%, 75%, 50%, 25% and 0% respectively. Weigh 0.50g of each standard sample, dissolve it with water and fix the volume to 25.00mL, make a suspension of 20.00mg/mL, then take 0.25mL of the suspension after shaking respectively on the glass slide, develop it into an oval sheet with an area of about 5cm2 × 15. Objective lens × 10) Observe 5 visual fields, record the number of unbroken pollen cells in each visual field, operate each standard sample 3 times in parallel, and calculate the arithmetic mean. The standard curve is drawn with the wall breaking rate as the ordinate and the arithmetic mean of the number of unbroken pollen cells detected in each field as the abscissa. According to the number of unbroken pollen cells detected in the sample, calculate the pollen wall breaking rate according to the following formula:
Y = a + bX
Where: wall breaking rate of Y pine pollen (%);
X Arithmetic mean value of unbroken pine pollen (piece);
a. B Constant.
This method is applicable to the determination of the wall breaking rate of pine pollen, and it is also applicable to the determination of wall breaking rate of many kinds of pollen. The method has the characteristics of high precision, high accuracy, simplicity and wide linear range.
2. Laser particle size analysis
In recent years, with the development of the research on the wall breaking rate of traditional Chinese medicine, more and more researchers are no longer limited to the traditional microscopic measurement of wall breaking rate, but constantly seeking new methods to determine the wall breaking rate. Among them, the laser particle size analysis method is a more forward-looking analysis method, which has great development advantages. It can not only overcome the characteristics of large subjective influence factors of microscopic determination method, but also measure the rate of cell wall breaking of traditional Chinese medicine with insignificant microscopic characteristics of traditional Chinese medicine. In addition, it has the advantages of simple operation, reliable results, and high accuracy [18]. The principle is mainly based on Mie scattering theory. The energy distribution of scattered light is directly related to the distribution of particle diameter. The particle size distribution characteristics can be obtained by receiving and measuring the energy distribution of scattered light [19-20]. According to the characteristics of the tested medicinal materials, wet method and dry method can be selected. Wet method measurement is to mix the sample with a liquid to prepare a suspension, and disperse it by means of stirring, ultrasonic and other physical conditions, so that the aggregated particles are dispersed into the original particles, and the original particles are kept in a good dispersion state in the dispersion solution; Dry method measurement Solid samples used to determine water solubility or without suitable dispersion medium, impact and disperse the agglomerated particles through the high-speed air flow provided by the air compressor, and then collect them by the connected vacuum cleaner after measurement in the closed air tank [21]. He Yu et al. [22] discussed for the first time the relationship between the average cell diameter of traditional Chinese medicine, the particle size after ultramicro pulverization, and the cell wall breaking rate. As a result, an ideal calculation method of breaking model and a formula of wall breaking rate were obtained（ η) :
η 1 = ［1 － ( 1 － 1 / n) 3］ × 100% ( n ＞ 1) ;
η 2 = 100% ( n≤1) ;
η = η1 + η2;
Where n is the ratio of particle size to cell diameter.
On this basis, Yang Ying et al. In addition, Sun Huili et al; The results of wall breaking rate measured by the two methods were basically the same by comparing the changes in the microscopic and particle size distribution of Typhus pumila before and after wall breaking. It can be seen that the laser particle size method is feasible to replace the traditional Chinese medicine microscopic quantitative method to determine the wall breaking rate of pollen, and at the same time, it lays a solid foundation for the research of other pollen wall breaking rate laser particle size analysis. The method is simple, fast and objective.
3. Chemical composition method
Relevant literature research shows that there is a certain relationship between wall breaking rate and chemical composition [18, 24-26]. On this basis, Liu Yingmei et al. [27] discussed the relationship between different wall breaking rate and proline content in pollen with different wall breaking rate, and proposed a new method to determine wall breaking rate of black pine pollen Law. After measuring the absorbance of proline content at 520nm in samples with different wall breaking rates by spectrophotometry, calculate the wall breaking rate of pine pollen according to the following formula:
Y ( % ) = ( a + bX) × 100%
Where: Y: wall breaking rate of pollen (%);
10: The absorbance of the sample with proline content at 520nm;
a. B: is a constant.
The results showed that the relationship equation between pollen wall breaking rate and its corresponding proline absorbance was y=11.886x-0.0637, R2=0.9897. It can be seen that the linear relationship is good. As long as the proline absorbance of a kind of pollen after wall breaking is measured and substituted into the equation, the pollen wall breaking rate can be obtained, which is simple and accurate, and can avoid the tedious operation process caused by microscopic measurement of wall breaking rate. However, there are some defects. Different Pinus thunbergii pollens have different internal available proline content, and the measured wall breaking rate is different. Therefore, the reliability of the results is affected.
4.Summary of advantages and disadvantages of various detection methods
Looking at the current detection methods of pollen wall breaking rate, although each method has its own advantages, there are still some shortcomings as shown in belows:
Direct counting method using glass slides
Advantages: Simple and easy to operate.
Disadvantages: Cover glass has a squeezing effect, uneven distribution of pollen cells, easy overlap or omission, and poor reproducibility.
Modified group Pasteur blood cell counting method
Advantages: Compared to the direct fragment counting method, this method is faster, simpler, more reliable, and has good reproducibility.
Disadvantage: The filling of the counting chamber should be moderate, otherwise it may cause inaccurate readings of pollen cells.
Staining method, smear method
Advantages: This method is more reasonable than the direct counting method using glass slides, and the contrast in color makes pollen easy to count, further providing reliability for microscopic observation results.
Disadvantages: Uneven pollen distribution with overlapping or missing phenomena.
Gravimetric analysis combined with microscopic quantitative method
Advantages: Simple and easy to operate, with the addition of the principle of gravimetric analysis, making the data more objective and further improving its measurement accuracy.
Disadvantage: It can only be a single pollen and should not be mixed with other pollen and impurities, otherwise it may affect the measurement results.
Standard curve measurement method
Advantages: It has the characteristics of high precision, accuracy, simplicity, and wide linear range.
Disadvantage: Compared with laser particle size method and chemical composition, it has a certain degree of subjectivity.
Laser Particle size analysis
Advantages: The method is simple, fast, and has the characteristic of objectivity.
Disadvantages: It is easily affected by dispersants, the properties of the medicinal material itself, the water content of the medicinal material, particle size, and over time. In addition, the correlation between particle size and wall breaking rate needs further research.
Chemical composition method
Advantages: Simple and accurate, further establishing and exploring the correlation between pollen wall breaking rate and composition.
Disadvantages: Different pollen contents may vary, and there are many influencing factors, such as collection time, variety, batch, etc. Therefore, the reliability of measurement results is not very strong, and there are certain drawbacks.
5.Summary and Prospect
At present, it is generally believed in the academic circles that the effective components of natural pollen can not be fully released without shell breaking treatment. Therefore, the rate of pollen wall breaking has become one of the important indicators to evaluate the method of pollen wall breaking and the quality of pollen wall breaking powder. According to the current literature research, the detection methods of pollen wall breaking rate mainly include microscopic determination, laser particle size determination and chemical composition determination, but which detection method is the more reasonable one.
There is no unified conclusion before. Through the summary of advantages and disadvantages of various detection methods in Table 1, it can be seen that each detection method has certain advantages and disadvantages. Therefore, which detection method is reasonable needs further research in the future. However, as far as the author is concerned, the laser particle size analysis method may be a hot spot in future research on pollen wall breaking rate, and is expected to become a reasonable standard detection method [24].
In addition, when analyzing or studying which detection method of pollen wall breaking rate is more reasonable, we should first determine whether the detection method itself is scientific, and whether there is a certain detection process.
Rationality, whether the factors causing error sources can be reduced to the minimum, so that the experimental results are more reliable. For example, Lu Jingci et al. [28] provided a good example for the analysis of the uncertainty of the analysis method of the wall breaking rate of Ganoderma lucidum spores by evaluating the measurement uncertainty and improving and optimizing the method. Therefore, when discussing the detection methods of pollen wall breaking rate, we should compare and verify various detection methods according to the specificity of each pollen, so as to develop a reasonable standard method for detection of wall breaking rate.
Based on the above discussions, the author puts forward the following suggestions in order to provide reference for formulating reasonable detection standards in the future: strengthen the research on the uncertainty of wall breaking rate detection method itself to reduce the error source factors and improve the reliability of experimental results; Strengthen the research on the correlation between particle size and pollen wall breaking rate and the discussion on the influencing factors, so as to realize the measurement of pollen wall breaking rate by laser particle size analysis, make the detection more simple and easy to operate, and overcome the shortcomings of traditional microscopy; Strengthen the research on the determination of pollen wall breaking rate by component analysis. The wall breaking rate is in direct proportion to the chemical components in pollen to some extent, but the research on their correlation is still lack of in-depth research; When measuring the wall breaking rate of a certain kind of pollen, it is necessary to carry out systematic research on multiple detection methods or joint analysis of multiple methods to make the experimental results more reasonable and reliable.
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