| After entering WTO, western medicine is in a period of low tide in our country due to the ownership of the intellectual property right. Traditional Chinese Medicine is regarded as our country's rarity, but as traditional medicinal herbs, its exporting amount just accounts for 4% in international traditional medicine market. Because of its effective composition and the mechanism which being failed to understand, and the obsolete processing technology, so the directly result is that our traditional medicine's dominant position has been challenged in the competition with exotic natural medicine. Now, our traditional medicinal industry is facing huge difficulties. One of key reasons is that we lack "heavy bomb" innovative medicine with intellectual property right. Especially, medicalmarket recently has gradually accepted natural compound medicine in western developed countries and this gives us a rare chance to modernize our traditional medicine, which is rich in resource, and has special cure effect as well as little side effect. If we want to make our traditional medicine famous on in international medical circle and attract attention from different countries in the world, we are forced to study with advanced science and technology and break through greatly traditional exploring ideas and methods, shorten the period for screening out the effective composition from traditional medicine, speed up the development of its pharmacological study, and promote the exploration and development of innovative Traditional Chinese Medicine.1. Thinking and methodsThis present study takes advantage of multicomponent Litsea cubeba (Lour) Per's essential oil as compound model of traditional Chinese medicine, made citral the major antibacterial component of the essential oil as "target part", and let single cell of Aspergillus flavus secreting aflatoxin causing cancer as the object of drug. In process of our study, We adopted advanced technologies from medical image area, and the strategies that many subjects were combined. To investigate how the imitated target part of prescription influenced on cell, multi-dimensional microscopy, and Rayleigh light scattering and biochemical analysis technology were used to succeed in it at three levels of cell, sub-cell and molecule. It is found that citral damaged the morphological structure of cell membrane of A. flavus and changed its physical property after it invaded the cell. Citral not only damaged organelles but also changed the status of intracellular macromolecular crowding. These changes resulted in the directional local concentration of organelles, the conformation change of macromolecules, the intensification of the irreversible aggregation reaction of macromolecules with the highest content in protoplasm (i.e. proteins) and the metabolic disorder caused by the lost of regionality of biochemical reactions. So, cells, organelles and macromolecules lost their normal structures, andfunctions. Consequently, the spores of Aspergillus flavus lost their germination ability; and its mycelia lost their ability to produce conidia. When the study about citral against the growth of A. flavus is under proceeding, it is important to put forward its pharmacological study to break through "bottleneck" and speed up the explorment and development of new traditional medicine. And we should have an idea to shine our old race's rarity, and make a new research in exploring thinking and methods.2 Results and analysis2.1 Single living cell is the ideal object of exploring drug mechanismCell is the basical structure and functional unit of living beings that is connected with the state of movement of the whole cell. But heterogeneity makes cell different in the composition of chemistry, and physiology responding to time, etc. It has great significance to analyze and determine the structure and function of single special cell from these kinds of heterogeneous cells. In that case, we can understand the responding between signal cell and tissue, and the relationship between different tissues, even considers it as the important base for us to take an imitated biological study.Different from traditional tissue slice, optical microscope and electronic microscope, this study used natural antibacterial Litsea cubeba (Lour) Per's essential oil and let its major antifungal component as "target part" and a single active A. flavus spore damaged by citral as the target to exploit the drug effects on the whole process of spore from germination to growth and propagate. The spores were placed into the culture chamber adding a drug medium with a series of concentrations, and were carried on the non-invasive, in situ, real time measurement. After computer storaged, processed, and analyzed on information from phase contrast microscope, the changes of structure, function and state of the same kind of cells could be investigated in one week. For example, the morphological parameters including cell area, perimeter, major axe, and minor axe,irreversibly decreased with the increasing concentration of citral; The functional parameters of membrane showed the dosage relationships; The nucleus DNA were damaged by comet assay and the correlative parameters were tested; The absorption spectrum curved at regions of a single living cell moved on ultraviolet region; the aggregation reaction of macromolecules with the highest content in protoplasm (i.e. proteins) were irreversibly intensified; and so on. These results are good answers to some questions of biomedicine, such as changing convergent character of homogeneous cell in stress condition of structure (i.e. function, chemical constitute, variation and communication). It is more correct and fast than the test by flowing cytometer at present.2.2 Measuring the mechanics characteristic variation of membrane is an effective way to identify the effect of drug fastly.Membrane is the cell barrier where the exchange of substance and energy performs. It's on the membrane that action of drug to cell can be realized at first. Drug affects the cell membrane in two ways. One is physical effect, which changes the mechanics parameters of membrane by influencing its fluidity directly. The other is chemical effect, that is to say medical agent reacts with some macromolecules on the membrane, making them lose activity or change conformation and destroy the channels in membrane which makes the substance transport can't go on the rails, so that membrane loses selective permeability. The variations of mechanics parameters and chemical status of cell membrane both reflect the changes on vital movement of cell, like metabolism, respiration, photosynthesis, signal transduction, transportation of substances and so on. If a kind of drug has no vividly effect on physics parameter of membrane, that means it certainly loses the meaning of researching on the drug entrance into cell and its antibacterial.Experiment was based on the fact that Aspergillus flavus spores (AFS) have the flicker phenomenon similar to the red blood cell. According to some related theoretic models, the frequency spectrum and light intensity of the flicker were analyzed by using digital imaging microscopy and micro-image analysis technique. Comparing with theconditions before citral acts, the bending modulus Kc, shear modulus fx and e of AFS respectively increased by 3, 5,1.6 times. And as the concentration of citral increased, these three parameters took on irreversibly increasing, that is to say the membrane of A.flavus increases its mechanical intensity, decreases its elasticity, and deformability, also it is more prone to crash, which indicates citral injured the AFS' membrane. At the same time, it got that, comparing with control group, the electric conductivity was increased by 52.8%, [Na+, k+] —ATPase activity decreased by 21.9% (taking the amount of decomposing the inorganic phosphorus), and the utilization rate to protein and sugar decreased by 61.5% and 44.3% respectively. These results obtained from physiologic and biochemical analyses on A.flavus were identical with those from physical parameters analysis on the membrane of AFS. All of these suggest that citral can influence the selective permeability and the utilization rate on nutrition of A. flavus' membrane by changing its physical property.2.3 Obtaining the information of intracellular macromolecular crowding in living cells with the effects of drugs is a way to explore its mechanism.Cytoplasm is a place where organelle and macromolecules perform their physiological functions. The information that drug works on cell, on top of getting from the tissue slice, isotopic tracing and change of cell surface, was also obtained from the living cell inner, so that the function of the "target part" from drug can be commented objectively. There is a high-order macromolecular crowding in a cell. The status of macromolecules crowding not only determines of the conformation of all macromolecules but also affects their association and disassociation. So, the status plays an important role in all biological functions relying on non-covalent union and or conformational change.Comparing with other pharmacological methods, in order to know more about the mechanism of drug, the intracellular photo absorption, the diameter of particles and its distribution were determined via non-invasive, in situ real time measurement. According to the result that whether intracellular macromolecules took place the conformational and aggregation changes, then it was judged whether the intracellular macromolecule crowdingsystem was interrupted by citral or not. The present study determined the variation of photoabsorption in single living cell, which it is in citral and it isn't in, in 350nm-800nm of the whole wavelengths, via the fast multi-channel micro-spectrophotometer. According to the theory of Rayleigh light scattering, in this way, quasi-elastic laser light scatting technique was used to determine the diameter of intracellular particles and its distribution.It was found that all absorbencies increased at 350nm - 700nm in AFS damaged by citral, especially at 350nm - 410nm, and also the absorbency integral value was positively related to the concentration of citral. It seems that citral increased the amount of substance that can absorb the light of a specific wavelength in the tested micro-region, namely, the same class substance gathered rapidly, or that citral changed the molecular conformation of such substance, which increased the number of functional groups that can absorb the light of a specific wavelength in the tested micro-region. Because monomer A and B of citral absorb the light of 230nm and 330nm, respectively, there was no possibility that citral itself could absorb the light of wavelengths 350nm-750nm. The results of the experiment of quasi-elastic laser light scatting suggested that citral could neither cause a new macromolecular association reaction because the kind of particle between the control group and tasted group without changing, nor cause to change the distribution of particle diameters, because control group and test one appeared the same that particles with higher contents were distributed in the small-diameter region and particles with lower content in the large-diameter increased. In the small-diameter region, all particle diameters increased. In the large-diameter region, the relative content of particles increased quickly, which showed, from the two aspects, that citral sped up the association of macromolecules. The diameter of particles with highest content increased as the concentration of citral increased, this furthermore suggested that citral firstly interfered with the highest content in protoplasm (i.e. proteins). So however whether the change of photoabsorption or the acceleration of non-functional aggregation of macromolecules in AFS is just a response to the changes of normal macromolecular crowding status. That suggested after citral entered into cell, it caused the conformational variation and the irreversible aggregation ofmacromolecules, through perturbing the normal status of macromolecular crowding finally, and lead to block of pathways of biosynthesis and biodegradation.2.4 Researching the influence of drug to important organelle in cell and the system of oxidation-reduction needs more connected methods.Life is a nonlinear system with a structure of interconnected rank order. Therefore, biological phenomenon of cell, even the whole life, has the property of connecting a whole, interconnected rank and statistically fluctuating, as well as the property of randomicity of internal and external, inequality, opening and historic evolvement. The nonlinear interaction and support of different kinds of physical and chemical factors propel the life forward. However, different biological phenomomena, not only has its independence, but also has its unity of overlapping and coordination. Being not in the normal statement, the changes on function and relevant structure couldn't be clarified their orders by the unique subject. So it needs more subjects and probing in different ranks and fields.According to the chemical property of citral, the methods of using maloualdehyde, electron microscope and comet electrophoresis were adopted. It was examined the effects of radical from the lipid peroxidation of membrane on organelle, such as membrane, mitochondrion and nuclear DNA, etc. It was also measured the respiratory rate of oxidation-reduction system, using suuinate, pyruiate and a-ketoglutarate as substrates. Then, it was investigated the status about cell metabolism though the activity of malate dehydrogenate (MDH) and succinicate dehydrogenate (SDH). Thus, we expounded that the order of macromolecular crowding was disturbed, as correspond that some of metabolism and function are weaken. We found that citral could induce the lipid peroxidation and it contained maloualdehyde 1.23n mol/ml. Comparing with control group, it was increased over 50%. This may be the reason why organelle got different lesions. According to the result of comet electrophoresis, the nuclear DNA was damaged by citral. Besides, according to the results observed under electron microscope, irregular proliferation, abnormal forms and disordered structure of mitochondrion in injured AFM occurred.Therefore, it suggested that citral damaged these kinds of genes, which control Mt replication and its form. With the biochemical method, we also measured that the electric conductivity of AFM was increased by 52.8%, the activity of [Na+, K+]—ATPase was decreased by 15.8%, the utilization rate of AFM to reduced sugar and protein in culture medium was decreased by 23.3% and 30.6%, the respiratory rate of Mt, using suuinate, pyruiate and a-ketoglutarate as substrates, was decreased by 24.1%, 14.3%, 36.1% and the activity of MDH and SDH was decreased by 31.8% and 19.6% (the activity of MDH in cytoplasm was decreased by 27.5%). These results suggested that the damaged structure had the unity with the weak function of cell, organelle and macromolecule. And the mechanism of the citral affecting germination and growth of A. flavus was investigated from different points of view.3 Innovations during the investigationIt is concluded from the above discussion that the strategy of multi-subject cross research was adopted in the investigation of the elucidating the inhibitory effects of citral refined from Litsea cubeba oil on A. flavus. With the professional advantage of biochemistry and the absorption of new theory and technique in physics, many analytical techniques were combined organically. At three levels of cell, sub-cell and molecule, the interaction processes of citral on single active AFS were studied and the non-invasive, in situ and real time measurements were performed. Furthermore, the changes of structure and function that occurs on cell, organelle and molecule were investigated after citral acted on cell, and the mechanism of effective component screened from the medicine was elucidated. The research time was shortened greatly. The concrete innovation can be concluded as follows:(1) The research workbench was founded which was organically made up of many analytical technique, as multi-dimensional microscopy, multi-channel microspectro-photometer, dynamic image analyzing technique, single cell gel electro-phoresis analysis,Rayleigh light scattering, the image storage and analysis of computer, electron microscopy, biochemical assay and so on. It also established a new model, which contains the screening out effective component from traditional Chinese medicine and the studying of its mechanism.(2) It begins with that the effective component of traditional Chinese medicine enters into cell and changes its normal status of macromolecular crowding. Then it is investigated that the drug affects macromolecular conformations and aggregation reaction of macromolecules. All above upgrades the level of mechanism research from tissue-cell to subcell-macromolecule.(3) With the fast multi-channel micro-spectrophotometer, it is realized that the non-invasive, in situ, real time observation and quantitative measurement on single living cell replaces the static watching and qualitative testing on tissue section after the effective component of drug acts on cell, which greatly increases the accuracy of investigating "target part". Using dynamic image analyzing technique, it is realized that the analysis on drug efficiency substitutes the quantitative analysis on the mechanical parameters of single living cell membrane for the qualitative observation and statistical test on tissue section or its extract, which greatly enriches the fast and directive appraisal of drug effect, breaks the "bottleneck" in researching of traditional Chinese medicine, and extends the thinking of study on the "target part".(4) With the investigating of the influence of effective component of traditional Chinese medicine on the structure and function of multipotential cell, the law of dosage effect is discovered at many aspects via the research workbench, which reduces space-time errors of subjective estimation and shortens the process of cell metabolism and pharmacology investigation. It also provides scientific theory basis for natural drug and its effective monomer to screening, exploiting and entering market.
|
PDF Full Text Request