Analysis on the formation principle and present situation of nano phase state of multi-component self-assembly of traditional Chinese compound medicine decoction

GUAN Qing-xia, ZHOU Xiao-ying, Lü Shao-wa, YANG Fang-fang, NIE Ze-hui, LIN Ze-yu,WANG Yan-hong

College of pharmacy Heilongjiang University of Traditional Chinese Medicine,Harbin 150000,China

Keywords:

ABSTRACT Traditional Chinese medicine decoction is a complex polydispersed phase system containing real solution, colloid solution, emulsion and suspension.The compound decoction of traditional Chinese medicine has complex components, including saponins, alkaloids,polysaccharides, flavonoids, amino acids and so on, which can be self-assembled to form gels, fibers, micelles, vesicles and so on.The self-assembled nano-phase not only neutralizes the single drug and reduces the toxicity and side effects, but also has its own pharmacological effects, which complement each other to achieve synergistic effect, so as to achieve the role of drug supplement, which is of research significance.The formation principle, solubilization and synergism principle and characterization method of multi-component self-assembly of traditional Chinese medicine compound decoction are discussed in this paper.

1.Introduction

The compound decoction of traditional Chinese medicine is the treasure of traditional Chinese medicine.taking “monarch, minister,assistant and envoy” as the principle of prescription and the law of action of “mutual necessity, mutual affiliation and mutual evil”,it not only solves the problem that it is difficult to use drugs in complex conditions, but also overcomes the taste bias of single traditional Chinese medicine.it is the main method of traditional Chinese medicine to reduce adverse reactions and improve clinical curative effect.It is also the concentrated embodiment of the overall concept of traditional Chinese medicine and syndrome differentiation and treatment[1,2].In modern medicine, in order to solve the problems of inconvenient use, difficult to carry and low utilization rate of medicinal materials, traditional decoction is prepared into modern new dosage forms, such as granules, extracts,prepared slices and so on.However, in the process of preparation,the efficacy of compound decoction of traditional Chinese medicine is narrowed and weakened in varying degrees because of the guidance of “only component theory”.As a result, the efficacy of compound decoction of traditional Chinese medicine has been narrowed and weakened in varying degrees.Traditional Chinese medicine contains insoluble and fat-soluble active ingredients such as puerarin, baicalin, tanshinone Ⅱ A, emodin and so on, which are very low in water solubility, so that the effective components of traditional Chinese medicine are difficult to be used effectively.Modern science uses liposomes, solid dispersions, microcapsules and other techniques to increase its solubility[3], but there are also many hidden dangers, such as hemolytic phospholipids in lecithin,residual hydrogen peroxide in Tween-80, and gastrointestinal irritation or toxicity caused by synthetic surfactants or high concentrations of surfactants[4-5].

Through the compatibility of multiple medicines, the compound prescription of traditional Chinese medicine can not only neutralize the problem of single taste, but also achieve synergism, without the need to add excipients or with the help of special technology and equipment, so as to reduce the safety risks caused by the use of excipients.After the co-decoction of traditional Chinese medicine, there are complex physical and chemical changes, and the components in the decoction exist not only in the form of single components, but also in the aggregates of metabolites such as lipids,proteins, polysaccharides and carbohydrates, and the aggregates between monomolecules and metabolites form gels, fibers, micelles,vesicles and so on in aqueous solution, which has an external effect on the solubility[6].

2.Research status of self-assembled Nanophase State of traditional Chinese Medicine compound decoction

Traditional Chinese medicine decoction is a complex polydispersed phase system containing real solution, colloid solution, emulsion and suspension.Some researchers have proposed that the self-assembly process of traditional Chinese medicine compound decoction is different from chemical synthesis, it means that molecules or some molecules spontaneously form ordered polymers without human intervention through non-covalent bonds such as hydrogen bond, van der Waals force, π-π stacking, cation-π adsorption and so on.the drugs of insoluble or fat-soluble components are wrapped in gels,fibers, micelles and vesicles, thus playing the role of solubilization and solubilization[7,8].It is also possible that a variety of natural components, such as protein, saponins, gum, mucus, sterols, etc.,can be self-assembled to form micelles in the preparation process to play their solubilization role; it may also be caused by other factors,such as pH, solvents and other physical and chemical environment changes[9].

The components of traditional Chinese medicine are complex,including saponins, alkaloids, polysaccharides, flavonoids and so on.Among them, amino acids, sugars, nucleoside bases, steroids,triterpenes and coumarins can form hydrogen bonds, π-π interactions, van der Waals forces and other non-covalent bonds between molecules, thus promoting the orderly arrangement of molecules to form aggregates[10].For example, the base molecule itself has many hydrogen bond donor and receptor sites, which can form hydrogen bond and π-π interaction, which is beneficial to the formation of self-assembly; steroids, mainly cholesterol and cholesterol acid, have hydrophobic skeleton and hydrophobic alkyl side chain, and have π-π interaction provided by aromatic ring,which can cooperate with hydrogen bond provided by connecting arm and van der Waals force provided by steroids to realize selfassembly.Coumarin has a nuclear structure of α-benzopyranone,which has a strong π-π stacking effect, and the coumarin group is also a strong fluorescent group, so it is often used in the assembly system; multiple hydroxyl groups on the ascorbic acid skeleton are easy to form hydrogen bonds between molecules and promote the orderly arrangement of molecules to form assemblies.Amino acids have easily modified amino and carboxyl functional groups,which are easily connected by amide bonds to obtain polypeptide structures of different sequences.the hydrogen bonds formed between molecules can promote the ordered arrangement of amino acid units to form assembly.and in the process of self-assembly,the chirality of amino acid molecules is usually magnified to form a nano-phase[10].Gum, cellulose, starch, mucus, inulin, tannin and protein are also widely found in traditional Chinese medicine, which are not easily soluble in water and have high molecular weight,which are often removed because they are considered invalid in the process of preparation.In the process of boiling, these components will form hydrophilic colloidal solution, and polysaccharides can be hydrolyzed to form monosaccharides or oligosaccharides, and when the aggregates of protein decomposition products are dispersed in the medium, they can form polymer solutions or multiple inhomogeneous dispersion systems.thereby increasing the stability and solubility of the solution[11].However, the compatibility of the prescriptions is different, the properties of the micelles formed are different, and their solubilization is also different.

3.Self-assembly Nano-phase formation principle of traditional Chinese Medicine compound decoction

Molecular self-assembly is very common, peptide chains are assembled into proteins and nucleic acids, and the relationship between ligands and receptors is in the form of self-assembly, and their molecular structure determines the properties of the selfassembly units.they are usually in equilibrium or metastable state[12].The self-assembly process has the following five remarkable characteristics: (1) the aggregation of fragments of a group of identical or different molecules or interacting macromolecules transforms the molecules from a more disordered state (solution,disordered polymerization) to a more organized final state (crystals,composite macromolecules); (2) the self-assembly of molecules needs to balance the interaction between molecules through the attraction and repulsion of non-covalent bonds.(3) the polymer must be reversible, that is, the tendency force of intermolecular polymerization and separation should be equal, which will be significantly affected by thermal energy.thus, the components in the polymerization are allowed to adjust their position, the intensity of the interaction, etc.(4) the self-assembly process usually occurs in the solution or at the interface, and its components have a strong interaction with the surrounding media, and this effect is from beginning to end.(5) the molecule must be movable, and the solution causes the molecule to enter the self-assembly aggregate in an appropriate direction during hot stirring[6].The molecular selfassembly process means that the entropy of the system increases.When a force is large enough to overcome entropy, spontaneous self-assembly may occur in the system.In addition, biologically,amphiphilic molecules are the key to the formation of self-assembly.The self-assembly of amphiphilic molecules in aqueous solution is driven by hydrophobic effect[6].Saponins, polysaccharides, proteins and other components in traditional Chinese medicine compound decoction all play a certain role in the process of self-assembly,which may occur in the process of self-assembly as shown in figure 1.

Fig 1 Possible self-assembly process of traditional Chinese medicine compound decoction in the process of decoction

4.Principle of multi-component self-assembly nanophase formation of traditional Chinese medicine compound decoction

4.1 the principle of nano-phase formation by self-assembly of saponins

Saponins are amphiphilic compounds composed of hydrophobic regions containing triterpenes or spirogenins and one or more hydrophilic sugar chains, called glycoside ligands, which not only show good surface activity and emulsification by Maillard reaction.It can also reduce the possibility of systemic toxicity[13].Pentacyclic triterpenes have rigid and chiral central nano-skeletons.Most pentacyclic triterpenes have molecular lengths of 1.2-1.3 nm.Natural components of both acyclic and alicyclic triterpenes are suitable for nano-phase self-assembly because of their flexible side chains that can be folded into many different conformations.The selfassembly of pentacyclic triterpenes is mainly due to the hydrogen bonds of carboxyl and hydroxyl groups between molecules and the dispersion of nonpolar triterpene skeletons, while the differences in the spatial direction of triterpenoid skeletons and functional groups may have a significant impact on the self-assembly process[15].It has also been found that saponins self-assemble to form micelles under certain conditions, aglycones have different degrees of lipophilicity, sugar chains have strong hydrophilicity, which can significantly increase the dissolution of insoluble substances, while the size, shape and structure of micelles depend on their plant origin,temperature, pH, electrolyte concentration, etc.[16-17].The surface activity of saponins can wrap hydrophobic drugs in hydrophobic core and play a solubilizing role as single or composite carrier materials of drugs.Bag et al.[15] studied the self-assembly ability of oleanolic acid in aqueous and non-aqueous medium.it was found that oleanolic acid can encapsulate fluorophore or adriamycin in aqueous medium to form nano-gels, including adriamycin and other molecules, and that this self-assembly is considered to be driven by intermolecular forces, such as hydrogen bonds involving carboxyl and hydroxyl groups.Because of the diversity of glycosides and sugar chains, saponins are different as surfactants, so the selfassembly mechanism of saponins is also different.the structural characteristics of saponins in traditional Chinese medicine and the principle of nano-phase formed by self-assembly are shown in Table 1.Saponins can be divided into triterpenoid saponins and steroidal saponins according to the structure of saponins.According to the number of sugar chains, saponins can be divided into monosaccharides, disaccharides and trisaccharide saponins, which usually contain one or more monosaccharides[18].The sugar chain of triterpenoid saponins is mainly connected to Cmur3 and Cmur28 of aglycone, and the sugar chain of steroidal saponins is usually connected to Cmur3 and Cmur6 of spirostane saponins and furan sterane saponins, in which disaccharide saponins form a hydrophilichydrophobic-hydrophilic triblock structure, which is easy to selfassemble to form micelles and vesicles[18].For example, licorice saponins have internal hydrophobic groups (triterpenoid glycosides)and external hydrophilic groups (two kinds of glucuronic acid), the internal hydrophobic groups link the insoluble components, and the hydrophilic groups make the nano-phase dissolve in the aqueous solution, so that it can form spherical micelles in the aqueous solution and improve the insoluble solubility[19].

4.2 Formation principle of polysaccharide self-assembly nanophase

Most natural polysaccharides have hydrophilic groups such as hydroxyl and carboxyl groups, and amphiphilic copolymers are synthesized when hydrophilic polymer chains are grafted with hydrophobic segments[24].When exposed to water, amphiphilic polymers spontaneously form micellar or micellar aggregates by intramolecular or intermolecular associations between hydrophobic groups.Because there are many groups can be derived in the molecular chain, polysaccharide can easily for chemical and biological chemical modification, produced a variety of polysaccharide derivatives, in order to minimize the interfacial free energy, amphiphilic molecules through self-assembly at the interfaces and components form the spherical micelles, and the internal restructuring into a cylindrical or spherical micelles formed film, the final form vesicles.Iitsuka et al.[25] detected nanoparticles in a variety of soups and found that they were mainly composed of carbon (93.49%), followed by oxygen (3.95%).By comparing the similarity between nanoparticles and Raman spectral waveforms in the database, they predicted that nanoparticles were composed of arabinogalactan, cellulose and other polysaccharides.Hydrophilic groups can form non-covalent bonds with biological tissues to form biological adhesion.Nanoparticle carriers with bioadhesive polysaccharides as carriers can prolong their residence time, so that the residence time loads the drug absorption rate[24,26].Wang[27]through the study found that white and polysaccharides such as main structure by 1, 2 - or 1, 4 - chain of mannose residues, mannose C -2 and hexose residues on the branched chain, the presence of large amounts of hydroxyl, easy happening after the water crosslinking,long chain molecules from stretches the state gradually tangles form intermolecular hydrogen bond hampering the free movement of water molecules, which has a viscous, When pH 5.0, the viscosity increases significantly, and insoluble components can be encapsulated, which can be used as excipients for controlled release systems and biological adhesion targeting systems.Many kinds of Traditional Chinese medicines are rich in polysaccharides because of their unique structural characteristics.

Tab 1 Structural characteristics of saponins and the principle of nanophase formation by self-assembly

4.3 Principles of nanophase formation of self-assembly of protein components

Natural protein is a complex biological macromolecule with spherical shape or elliptic shape.The inner part of the molecule is hydrophobic amino acid residue, and the outer part is hydrophilic amino acid residue.The protein has three domains, namely the amphiphilic N-terminal region of 50-70 residues, the central hydrophobic domain of 72 residues and the amphiphilic C-terminal region of variable length[36], which enable most protein molecules to be adsorbed on oil-water and air-water interfaces and can be effectively used as stabilizer for emulsion and foam.In water medium structure, it is usually combined with polysaccharide to form gel microstructure, so as to achieve the balance of proteinprotein and protein-water interaction[37].The intramolecular or intermolecular interactions such as hydrogen bonding, hydrophobic interaction, electrostatic and van der Waals forces make it have specific recognition ability and binding effect, which makes it easy to self-assemble micelles with other components.The decocting process of Traditional Chinese medicine decoction is not a simple process of hot water extraction, but a possible chemical reaction (Maillard reaction) and physical process (supramolecular self-assembly).Nonenzymatic Browning reaction, also known as Maillard reaction,can occur between reducing sugars of traditional Chinese medicine plants and animals and compounds containing free amino acids,polypeptides and proteins[36].Heating is the driving force of protein structure change.Protein molecules contain hydrophobic groups inside.When the protein structure changes, hydrophobic groups are exposed, and non-polar molecules or non-polar groups are accumulated close to each other in aqueous solution Hydrophobicity of proteins[38].The process of heating and boiling causes structural changes in proteins, namely partial expansion of tertiary structures and conformational changes of secondary structures, and then some specific regions, such as hydrophobic points or free SH groups,are exposed, and proteins form aggregates to form micelles through hydrophobic interactions[39].Amino acids composed of proteins mostly contain either positively or negatively charged dissociable side chains.Dissimilar charges attract each other, like charges repel each other, and charge interactions generate static electricity[40].If the protein contains sulfhydryl groups, the molecule can also be heated to form disulfide bonds, which are crosslinked and self-assembled to form gel structures[41].Glycyrrhiza glycyrrhiza protein can achieve self-assembly of insoluble components into nanophase states due to electrostatic interaction between its special amide groups and quaternary ammonium ions in aqueous solution[42].Protein self-assembly is a process of intermolecular and intramolecular interaction and balance.Therefore, different types of protein will lead to different self-assembly and formation processes.The structural characteristics of proteins in Traditional Chinese medicine and the principle of nano-phase self-assembly are shown in Table 3.

4.4 other

On this basis, the formation and the researchers found that traditional Chinese medicine medicinal broth self-assembly nanometer phase,reach the role of solubilization efficiency not only limited to the above ingredients, researchers have found that traditional Chinese medicine (TCM) in the natural molecules such as japonica rice starch, the inorganic calcium sulfate in the gypsum, lignin in plants,flavonoids, alkaloids, organic acids, trace elements such as[46-49]can self-assemble to form nano phase.The structural characteristics of other components in Traditional Chinese medicine and the principle of self-assembly forming nanophase state are shown in Table 4.Natural rhubaric acid can be self-assembled by π-πstacking, hydrogen bonding and other non-covalent interactions without any structural modification or transport vector [50].Natural small molecules berberine and cinnamic acid can also self-assemble into natural nano-phase states with modified antibacterial activity,which are mainly controlled by hydrogen bond and superposition interaction, and their inhibition is far better than some first-line antibiotics, such as norfloxacin, amoxicillin and tetracycline [47].Using natural ingredients in Traditional Chinese medicine to form nano-phase state after decocting and self-assembling not only solves a series of problems brought by additives, but also effectively inherits the property of traditional Chinese medicine compound which is lost in the research of modern preparations.It is likely to be a very promising direction of modern preparations research.

Tab 2 Structural characteristics of polysaccharides and the principle of nanometer phase state self-assembly

Tab 3 Structural characteristics of proteins and the principle of nanophase formation by self-assembly

Tab 4 Structural characteristics of other kinds of traditional Chinese medicine components and the principle of nano-phase formation by self-assembly

5.Synergistic mechanism of self-assembly of TCM compound decoction in nano-phase state

Licorice in the calendar with priorities, mediate the effect of the medicine, modern medicine of liquorice and a thorough study of the effect of the medicine licorice with biological activity in the secondary metabolites of triterpenoid saponins, flavonoids,coumarins, polysaccharide and other phenolic compounds, including triterpenoid saponins (glycyrrhizic acid, etc.) and flavonoids(licorice glycosides, licorice glycosides, etc.) and composition of glycyrrhiza polysaccharide are amphiphilic, Triterpenoid saponins in glycyrrhiza glycyrrhiza contain non-polar saponins and watersoluble side chains, which can significantly increase the solubility of insoluble components and easily form nanoparticles during water decoctions[21].The insoluble components, such as saponin-A,saponin-B1 and baicalin, are the main active components of Radix Bupleurum and Scutellaria baicalensis, but their bioavailability is often difficult to improve because of solubility problems.Some researchers have found that glycyrrhiza saponin and its derivatives in compound decoction can significantly solubilize Bupleurin A and bupleurin B1[54].Wang Shuqi[55] verified the solubilization effect of glycyrrhizin by studying the oil-water distribution coefficient and index component content in Taohe Chengqi Decoction, and the solubilization was realized by self-assembly of glycyrrhizin acid to form micels.Zhou[56] et al successfully isolated colloidal particles from Maxing Shigan Decoction and found that the active components ephedrine (99.7%) and pseudoephedrine (95.5%) selfassembled into nanomicelles through glycyrrhizin, and the active components could be dissolved in different areas of micelles, such as hydrophobic core, fence layer or outer surface of micelles, thus effectively improving their bioavailability.

In addition to licorice, there are many ingredients in Traditional Chinese medicine can play the role of surfactant, emulsifier or solvent.Li Chang et al.[31] found that icariin polysaccharide formed a new micellar complex with icariin and Baicariin I, thus achieving the solubizing effect of ICariin polysaccharide on icariin and Baicariin I, insoluble flavonoid components.Meng et al.[57]respectively studied the supernatant and natural precipitate of Huanglian Jiudu Decoction and found that baicalin and berberine could self-assemble to form nanoparticles.Li et al.[58] further study found that the synergistic effect of natural nanoparticles formed by berberine and baicalin on irritable bowel syndrome was realized through self-assembly rather than simple mixing.Liu et al.[59]systematically studied the effects of ASTRagalus polysaccharides on 15 flavonoids in water Solution on the solubility and stability, the effect of astragalus polysaccharides found on the solubilization of flavonoids is greater than the solubilization of flavonoid glycosides and astragalus polysaccharides and flavonoids can form 1:1 complex,this complex wrap low solubility of flavonoids in nano phase state,to realize the solubilization to improve bioavailability.Vinegar fry bupleurum polysaccharide[27] is a kind of acidic polysaccharides, the uronic acid content was 61.16%, its unique branched structure and hyaluronic acid residue can enhance within the molecular hydrogen bonding interaction, in addition, hyaluronic acid residues also can strengthen the electronegativity of polysaccharide molecules, thereby strengthening the intermolecular hydrogen bond interaction, make its can be converged in aqueous solution to form nano phase, The solubility of baicalin and rhein was increased by 15 times and 49.6 times, respectively, by wrapping baicalin and rhein.

Most of the components of Traditional Chinese medicine form nano-phase through self-assembly process in vitro or in vivo to improve the efficacy.In vivo, the active ingredient can be selfassembled by a sequence of enzyme reactions, such as the interaction with a protein-peptide pair in Escherichia coli to form a nano-phase state, isolated by drug effluence pump and lysosome, exocytosis,and targeted delivery of the active ingredient.Nanoparticles can also naturally absorb foreign particles with reticuloendothelial cells in liver or spleen or circulating monocytes to achieve internal cell accumulation[51].In vitro, it forms nanometer phase and can affect drug level, antigenicity, location and duration of action.The hydrophilic group of the nanoparticles is outward, which can directionally bind the hyaluronic acid receptor on the surface of the cancer cells and then enter the cell, while the hydrophobic group is inward, which can wrap the hydrophobic drug.After the drug carrier interacts with the reducing agent, the disulfide bond is broken, leading to the rupture of the spherical micelles, and the drug molecule is released.Controllable reduction response micelles can enable the delivery and release of hydrophobic drug molecules in targeted cells and effectively prevent excessive accumulation of drugs in cells.

6 Characterization method of self-assembled nano-phase state of Chinese herbal compound decoction

The self-assembled nanophase of TCM compound decoction can be comprehensively analyzed by means of electrospray mass spectrometry, capillary electrophoresis, nuclear magnetic resonance,UV-vis spectrophotometer, Fourier transform infrared and fluorescence spectroscopy, etc.[58-59].To determine the nanophase state, parameters such as its shape, size and crystal structure need to be determined.Scanning electron microscopy (SEM),confocal laser scanning microscopy (LSCM), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scatterometer (DLS), differential scanning calorimetry (DSC), etc.,are also required for preliminary characterization and analysis of the microstructure of the nanophase[54].SEM and TEM techniques are widely used in the characterization of nanophase states[61].Chen et al.[54] characterized the morphology of baicalin and berberine complex by SEM and TEM, and found that the self-assembled nanophase was uniform spherical nanoparticles, which were detected by DLS and showed that the average particle size was 174 nm.Ping et al.[46] conducted TEM analysis of Baihu Decoction and showed that its nano-phase particle size was 100 nm, and the particle edge was smooth and clear.

7.Discussion and outlook

Decoction is one of the oldest and most widely used traditional classical dosage forms in China.For thousands of years, decoction has been respected by doctors and trusted by patients for its advantages of multi-effect, comprehensive effect and fast absorption.However, in order to improve its portability and lose its potency in the modern preparation process, modern researchers have made a series of suggestions on how to inherit its potency completely,such as “component medicine”, “Design theory of Multiple drug release of Traditional Chinese medicine Compound”, and “material rough set Theory”[1,2].In the process of decocting, the substance base of TCM compound is complex, including saponins, alkaloids,polysaccharides and flavonoids.