CN113215077A - Method for obtaining honeysuckle glandular hair tissue - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for obtaining honeysuckle glandular hair tissues, which comprises the steps of firstly cutting off the upper parts of honeysuckle buds to avoid pollen interference; secondly, the epidermis glandular hair tissues are efficiently and quickly exfoliated by a method of combining liquid nitrogen low-temperature cooling with vortex mixer oscillation friction; and finally, successfully separating the honeysuckle high-quality glandular hair tissue by filtering through a 100-micron cell filter screen and filtering through a 70-micron cell filter screen. The invention firstly uses the liquid nitrogen low-temperature cooling combined vortex mixer vibration friction method in honeysuckle glandular hairs collection, establishes the purification technology of honeysuckle glandular hairs tissues by filtering through a cell filter screen, optimizes the traditional freezing and brushing method for collecting honeysuckle glandular hairs, avoids the interference of pollen grains, reduces the consumption of manpower and material resources, improves the efficiency and obtains the high-purity glandular hairs tissues. The honeysuckle glandular hair tissue obtained by the method has high purity, and lays a foundation for further researching the content and molecular biology of the glandular hair tissue.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for obtaining honeysuckle glandular hair tissues.
Background
Glandular hairs are an important epidermal tissue of plants, and can be divided into non-secretory glandular hairs and secretory glandular hairs according to different secretory behaviors. The tip of the non-secretory glandular hair lacks synthesis and storage cells and has no capacity of synthesizing and accumulating secondary metabolites; secretory glandular hairs can synthesize, store or secrete a large number of secondary metabolites (Konarska, 2020), and the glandular hairs of the type contain quite complex secondary metabolites including various chemical components such as terpenes, flavonoids, alkaloids, lignins, polysaccharides, glycosides, fatty acids, proteins, alkaloids and the like (Balcke, 2017). The active ingredients of the traditional Chinese medicinal materials are mostly plant secondary metabolites, so that the glandular hairs of the plants are used as the entry points to explore the quality of the traditional Chinese medicinal materials, and the traditional Chinese medicinal materials have great research value.
Among the current methods for isolating and purifying glandular hairs, the most commonly used are direct extraction with chemical reagents and mechanical separation. The methylene chloride extraction method (Asahando et al, 2021) is commonly used as a chemical reagent direct extraction method, and although this method is simple and rapid and does not require isolation and purification of glandular hairs, the accuracy of the experimental results is questioned because the obtained glandular hair extract is not an organic solvent extract of plant tissues. The conventional method for mechanical separation comprises freezing the leaf containing glandular hair tissue in liquid nitrogen for 2min, brushing with a brush with moderate hardness to obtain glandular hair (Jianzhen et al, 2016) and obtaining a large amount of glandular hair in a short time. However, when the method of brushing the leaf gland with a freezing brush is used to brush the leaf gland with a thin thickness, such as the leaf gland of plants like peppermint, wrinkled giant hyssop, etc., the frozen leaf gland is extremely broken, and the obtained gland contains a large amount of plant tissue fragments. Although the method realizes glandular hair separation to some extent, the size, shape and variety of plant glandular hair are various, and the purification method has applicability obstacle among different species. The isolation and purification of glandular hair tissues are the core and difficulty of glandular hair research.
Honeysuckle is a dry flower bud or a flower with an initial bloom of Lonicera japonica Thunb, a honeysuckle plant of Caprifoliaceae, is a common Chinese medicinal material in China, is sweet in nature and cold in nature, has the effects of clearing heat, removing toxicity and dispelling wind-heat, is clinically used for treating wind-heat cold, wind-warm cold, sore and carbuncle pyogenic infections, and can also be used for treating various pyrexia (Liangfu, 2016; Huangyong, etc., 2012; Marling, 1997). The honeysuckle glandular hairs play an important role in regulating the normal growth and physiological activities of plants and occupy an important position in the quality evaluation, variety breeding and local research of traditional Chinese medicinal materials (Lijianju et al, 2013; Wufeiyan et al, 2014; Zhang shan et al, 2015; Wang Linna et al, 2016). Therefore, the method has important significance for the quality control of the honeysuckle medicinal materials, the variety breeding and other works through the precise analysis of the variety, the content and the distribution of the content in the glandular hairs of the honeysuckle. However, how to obtain high-purity glandular hair tissue in plants with high efficiency is still a technical problem.
For a long time, students adopt a freezing and brushing method to obtain plant glandular hair tissues (Jiangzhe, etc., 2016), however, the efficiency of brushing honeysuckle glandular hair with a brush after freezing by liquid nitrogen is relatively low, and honeysuckle buds are easy to break, so that pollen grains are mixed in the brushing and taking products. Therefore, the traditional freezing and brushing method is not suitable for separating the glandular hair tissues of the honeysuckle and has long time consumption and relatively low efficiency.
Disclosure of Invention
In view of the above, the present invention aims to overcome the drawbacks and disadvantages of the prior art, and provides a method for obtaining honeysuckle glandular hair tissue.
The method for obtaining the glandular hair tissue of the honeysuckle is characterized in that honeysuckle buds are sequentially subjected to low-temperature freezing, oscillation friction and two-stage cell filter screen filtering separation to obtain the high-purity glandular hair tissue.
The method comprises the following specific steps:
(1) preprocessing the collected honeysuckle buds;
(2) placing the centrifugal tube in an ice block for precooling, adding the pretreated honeysuckle flower buds into the precooled centrifugal tube, and slightly shaking to ensure that the flower buds are tightly arranged in the centrifugal tube;
(3) quickly pouring liquid nitrogen into the centrifuge tube until buds in the centrifuge tube are just hidden, quickly covering the centrifuge tube cover, and not screwing; quickly placing the centrifugal tube on a vortex mixer, and carrying out vortex for 1.5-2.0min at the rotating speed of 2500-;
(4) after the vortex is finished, adding distilled water into the centrifugal tube until the buds in the tube are just hidden, after slight oscillation, filtering by using a cell filter screen of 100 mu m, adding distilled water into the centrifugal tube again, slightly oscillating and washing, passing through the cell filter screen of 100 mu m, and combining the two filtrates;
(5) and (4) filtering the filtrate obtained in the step (4) through a cell filter screen with the particle size of 70 microns, slightly washing the filter screen with distilled water, placing the cell filter screen with the particle size of 70 microns in a cool and ventilated place for air drying, and slightly scraping residues on the filter screen after air drying, namely the honeysuckle epidermal glandular hairs.
The pretreatment in the step (1) is to cut off one third of the upper part of the honeysuckle buds, so that pollen grain interference can be removed.
And (3) placing the centrifugal tube in an ice block for precooling in the step (2), wherein the purpose of precooling is to prevent the liquid nitrogen from volatilizing rapidly when the liquid nitrogen is added. The addition of honeysuckle is not more than one half of the volume of the centrifugal tube, the addition of honeysuckle is too much, the oscillation friction amplitude is too small, the quantity of glandular hairs obtained by vortex friction separation is small, and the experimental efficiency is reduced.
The operation process is rapid after the liquid nitrogen is poured in the step (3), otherwise, the honeysuckle is softened too fast, and the quantity obtained by frictional separation of glandular hairs is little. The rotation speed is set to be 2500-3000rpm during vortex, and the time is 1.5-2.0min, so that a large amount of glandular hairs are obtained under the condition of less broken tissues, and the rotation speed is preferably 2500rpm, and the time is preferably 1.5 min.
Repeating the steps for many times, and collecting a large amount of honeysuckle epidermal glandular hairs.
In the invention, the flower bud can be poured out for observation after the vortex is finished, and if the surface of the flower bud is smooth and almost has no epidermal plush, and a large amount of macroscopic tiny hairy objects are attached to the tube wall, the vortex result is good.
The working principle and the beneficial effects of the invention are as follows: the working principle of the invention is that the honeysuckle glandular hair texture is changed from flexible to hard by using liquid nitrogen low-temperature freezing, and the honeysuckle glandular hair texture is easy to fall off. Then, the vortex mixer is used for oscillating and rubbing, so that the efficient and rapid exfoliation of the glandular hairs of the epidermis is realized. The honeysuckle glandular hairs are relatively large in size and high in density, do not float on the water surface, and the supernatant does not contain glandular hair tissues, so that the high-purity glandular hair tissues are obtained by utilizing a cell filter screen for filtration and separation. Because the traditional freezing and brushing method is not suitable for separating the honeysuckle glandular hair tissues, the method improves the traditional freezing and brushing method, and the improved method is used for successfully obtaining the honeysuckle glandular hair tissues with high purity.
The concrete expression is as follows: the honeysuckle flower buds are pretreated, the upper parts of the flower buds are cut off, and the interference of pollen grains is avoided. The vortex mixer vibrates and rubs, and the efficiency of separating the epidermal glandular hairs is improved. The rotation speed is set at 2500-. Filtering with 100 μm cell sieve to remove other large plant tissues; and filtering again by using a 70-micron cell filter screen to filter other small plant tissues into filtrate, wherein the glandular hair tissues of the honeysuckle left on the filter screen are more and complete, and the purity of the collected glandular hair tissues is higher.
Compared with the traditional freezing and brushing method, the method has the advantages that the improved method is suitable for separating the glandular hair tissue of the honeysuckle, the labor is greatly saved, the efficiency is improved, and the requirement on operators is not high. Traditionally, glandular hair tissues are concentrated in supernatant by a hydro-sedimentation method and then further screened by a cell filter screen, and the glandular hair tissues exist in filtrate. However, the experimental study finds that the honeysuckle glandular hair tissue and the volume density are greater than those of the glandular hair of the common plant leaf, the supernate almost does not contain the honeysuckle glandular hair tissue, and the honeysuckle glandular hair tissue cannot be enriched by a wet separation sedimentation method. Therefore, the experiment adopts a vortex oscillation friction method, the efficiency of obtaining honeysuckle glandular hair tissues is improved, and other broken plant tissues are less than that of glandular hairs brushed by a traditional freezing and brushing method. Then filtering through two cell filter screens with different specifications, filtering a large amount of other tissues into filtrate, and collecting high-purity honeysuckle glandular hair tissues on a 70-micron cell filter screen.
In conclusion, the method firstly cuts the upper parts of the honeysuckle buds to avoid pollen interference; secondly, the epidermis glandular hair tissues are efficiently and quickly exfoliated by a method of combining liquid nitrogen low-temperature cooling with vortex mixer oscillation friction; and finally, successfully separating the honeysuckle high-quality glandular hair tissue by filtering through a 100-micron cell filter screen and filtering through a 70-micron cell filter screen. The invention firstly uses the liquid nitrogen low-temperature cooling combined vortex mixer vibration friction method in honeysuckle glandular hairs collection, establishes the purification technology of honeysuckle glandular hairs tissues by filtering through a cell filter screen, optimizes the traditional freezing and brushing method for collecting honeysuckle glandular hairs, avoids the interference of pollen grains, reduces the consumption of manpower and material resources, improves the efficiency and obtains the high-purity glandular hairs tissues. The honeysuckle glandular hair tissue obtained by the method has high purity, and lays a foundation for further researching the content and molecular biology of the glandular hair tissue.
Drawings
FIG. 1 is a 70 μm cell strainer residue (4X 40) obtained in example 1 observed under an optical microscope;
FIG. 2 shows the pellet (4X 40) of the centrifugal tube obtained in example 1 observed under an optical microscope;
FIG. 3 shows the supernatant (4X 40) obtained in example 2 observed under an optical microscope;
FIG. 4 shows the residue of the 70 μm cell strainer obtained in example 2 (4X 40) observed under an optical microscope;
FIG. 5 shows the pellet (10X 40) of the centrifugal tube obtained in example 2 observed under an optical microscope.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments.
Material reagent
Fresh honeysuckle flower buds are collected from Shandong Chinese medicine university nursery garden in 2021, 5 middle of the month.
Instrumentation and equipment
100 μm and 70 μm cell screens (Beijing Lanjike technologies, Inc.), Sickleike vortex mixers.
Example 1: a method for obtaining honeysuckle glandular hair tissues comprises the following three steps:
step one, honeysuckle glandular hair tissue is separated
Cutting off one third of the collected flower buds of the honeysuckle flower VI, weighing about 3 g, putting into a 50ml centrifugal tube after precooling for 10min in an ice block, and slightly shaking to ensure that the flower buds are tightly arranged in the centrifugal tube, wherein the adding amount of the flower buds of the honeysuckle flower is not more than one half of the volume of the centrifugal tube.
Liquid nitrogen is poured into the centrifuge tube quickly until the flower buds in the centrifuge tube are just submerged, and the centrifuge tube cover is covered quickly, so that the centrifuge tube cover is not screwed tightly. The tube was placed rapidly on a vortexer and vortexed at 2500rpm for 1.5 min.
Step two, purifying the glandular hair tissue of the honeysuckle
After the vortex is finished, 10-15ml of distilled water is added into the centrifuge tube until the flower buds in the tube are just submerged, after the gentle shaking, the mixture is filtered by a cell filter screen with the size of 100 mu m, 10-15ml of distilled water is added into the centrifuge tube again, the mixture is washed by gentle shaking, the mixture passes through the cell filter screen with the size of 100 mu m, and the two filtrates are combined.
The resulting total filtrate was passed through a cell strainer of 70 μm and the strainer was gently rinsed 2-3 times with distilled water. After centrifugation of the filtrate at 1000r/min for 1min, the clear liquid was allowed to absorb light, leaving a precipitate containing a small amount of water. The cell strainer of 70 μm and the centrifuge tube containing the pellet were placed in a cool and ventilated place to air dry the water.
Step three, collecting glandular hair tissues of honeysuckle
After the 70 μm cell sieve and the centrifuge tube containing the precipitate were air-dried, a small amount of residue was scraped off, and the results were collected by observation under an optical microscope.
Experimental control
Example 2: a method for obtaining honeysuckle glandular hair tissues comprises the following three steps:
step one, honeysuckle glandular hair tissue is separated
Freezing the collected flower buds of the honeysuckle flower of Huajin VI in liquid nitrogen for 2min, taking out the flower buds with forceps, and immediately and lightly brushing the glandular hairs with a brush with moderate hardness. In this step, the flower buds should be brushed immediately after being taken out, otherwise, the flower buds will turn soft and black. This was repeated several times until a large number of glandular hairs were collected.
Step two, purifying the glandular hair tissue of the honeysuckle
And (3) putting the brushed plant tissues into a centrifugal tube, adding a small amount of distilled water, and slightly shaking and uniformly mixing. Standing for 10min, sucking a small amount of supernatant, flaking, and observing under a microscope. The supernatant was observed to contain pollen grains indicating that pollen grains were mixed in the brush; the honeysuckle glandular hair tissue is hardly contained, which indicates that the honeysuckle glandular hair tissue density is higher than that of water. Therefore, the wet separation and sedimentation method is not suitable for separating and purifying the glandular hair tissue of the honeysuckle.
Distilled water containing the brush extract was passed through a cell strainer of 100 μm and 70 μm, and the strainer was gently rinsed 2-3 times with distilled water. After centrifugation of the filtrate at 1000r/min for 1min, the clear liquid was allowed to absorb light, leaving a precipitate containing a small amount of water. The cell strainer of 70 μm and the centrifuge tube containing the pellet were placed in a cool and ventilated place to air dry the water.
Step three, collecting glandular hair tissues of honeysuckle
After the 70 μm cell sieve and the centrifuge tube containing the precipitate were air-dried, a small amount of residue was scraped off, and the results were collected by observation under an optical microscope.
According to the observation results under the optical microscope of examples 1 and 2, it can be seen that the residue on the 70 μm cell strainer of example 2 contains pollen grains and other plant tissues which are relatively obvious although it contains glandular hair tissues, whereas the 70 μm cell strainer of example 1 contains a large amount of glandular hair tissues, contains no pollen grains and contains a small amount of plant tissue fragments; the precipitates of the centrifuge tubes of the two types of the plants contain more other plant tissues.
In combination with the above analysis, the residue of the 70 μm cell strainer contained the most glandular hair tissue and had the highest purity glandular hair tissue under the operating conditions of example 1.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (5)
1. A method for obtaining glandular hair tissue of honeysuckle is characterized in that honeysuckle flower buds are sequentially subjected to low-temperature freezing, vibration friction and two-stage cell filter screen filtering separation to obtain high-purity glandular hair tissue.
2. The method for obtaining glandular hair tissue of honeysuckle as claimed in claim 1, comprising the following steps:
(1) preprocessing the collected honeysuckle buds;
(2) placing the centrifugal tube in an ice block for precooling, adding the pretreated honeysuckle flower buds into the precooled centrifugal tube, and slightly shaking to ensure that the flower buds are tightly arranged in the centrifugal tube;
(3) quickly pouring liquid nitrogen into the centrifuge tube until buds in the centrifuge tube are just hidden, quickly covering the centrifuge tube cover, and not screwing; quickly placing the centrifugal tube on a vortex mixer, and carrying out vortex for 1.5-2.0min at the rotating speed of 2500-;
(4) after the vortex is finished, adding distilled water into the centrifugal tube until the buds in the tube are just hidden, after slight oscillation, filtering by using a cell filter screen of 100 mu m, adding distilled water into the centrifugal tube again, slightly oscillating and washing, passing through the cell filter screen of 100 mu m, and combining the two filtrates;
(5) and (4) filtering the filtrate obtained in the step (4) through a cell filter screen with the particle size of 70 microns, slightly washing the filter screen with distilled water, placing the cell filter screen with the particle size of 70 microns in a cool and ventilated place for air drying, and slightly scraping residues on the filter screen after air drying, namely the honeysuckle epidermal glandular hairs.
3. The method for obtaining glandular hair tissue of honeysuckle as claimed in claim 2, wherein: the pretreatment in the step (1) is to cut off one third of the upper part of honeysuckle buds.
4. The method for obtaining glandular hair tissue of honeysuckle as claimed in claim 2, wherein: and (3) adding the honeysuckle in the step (2) in an amount not more than one half of the volume of the centrifugal tube.
5. The method for obtaining glandular hair tissue of honeysuckle as claimed in claim 1, wherein: and (4) setting the rotating speed to be 2500rpm during the vortex in the step (3), and setting the time to be 1.5 min.
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| NL2028631B1 (en) * | 2021-07-06 | 2022-07-06 | Univ Shandong Traditional Chinese Medicine | Method for Acquiring Honeysuckle Glandular Hair Tissue |
| CN115807010A (en) * | 2023-01-10 | 2023-03-17 | 青岛农业大学 | Honeysuckle leaf glandular hair development gene and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| NL2028631B1 (en) * | 2021-07-06 | 2022-07-06 | Univ Shandong Traditional Chinese Medicine | Method for Acquiring Honeysuckle Glandular Hair Tissue |
| CN114136738A (en) * | 2021-12-08 | 2022-03-04 | 河南中医药大学 | Microscopic flaking method for observing lower epidermis characteristics of folium artemisiae argyi |
| CN114136738B (en) * | 2021-12-08 | 2024-05-24 | 河南中医药大学 | Microscopic tabletting method for observing lower epidermis characteristics of mugwort leaves |
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| CN115807010B (en) * | 2023-01-10 | 2024-04-05 | 青岛农业大学 | Honeysuckle leaf glandular hair-growing gene and application thereof |
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