WO2016043349A1

WO2016043349A1 - Solution of bio gold nanoparticles produced by extracts of plants

Info

Publication number: WO2016043349A1
Application number: PCT/KP2015/000036
Authority: WO
Inventors: Song Sik Kim
Original assignee: Ri Kyong Min
Current assignee: Ri Kyong Min
Priority date: 2014-09-18
Filing date: 2015-01-24
Publication date: 2016-03-24
Anticipated expiration: 2017-03-18

Abstract

Disclosed is the process producing solution of bio gold nanoparticles using natural physiological activators of phylogenesis such as several medicinals and spices, and etc as reductant and dispersant. Particularly, this proposal presents the process producing solution of bio gold nanoparticles by reducing chloroauric acid by adding aqueous aolution of chloroauric acid to plant extract including polyphenolic compound, and solution of bio gold nanoparticles gained through this process. The plant using in this proposal can be chosen at will according to the using field and the purpose. A bio gold nanoparticle of this proposal has the surface Plasmon price of 520-540mm, the average particle size of 15-70mm, and concentration of 50-300ppm.

Description

Solution of Bio Gold Nanoparticles produced by extracts of plants

Specification Field of Invention

This invention relates to the process producing bio gold nanoparticles and solution of bio gold nanoparticles gained through the mentioned process.

Particularly, it relates to the process producing bio gold nanoparticles by the bio reduction method using extract of several medicinal and spice plants as reductant and stabilizer, and solution of bio gold nanoparticles gained through this process.

Background and purpose of invention

Gold nanoparticles(GNPs) is being widely studied for application of optoelectronics, chemical and biological receptor of super sensitivity, and catalyzer and ect at present.

Gold nanoparticles is one of materials most widely studied. These can be easily compounded, and produces strong surface Plasmon resonant effect. And. its heat stability is surely stabilized chemically.

In the realm of biomedicine. gold nanoparticles is used for several purposes of treating leukemia, immobilizing bioparticles and projecting biosensing. Usage of gold nanoparticles as an anti-blood rebirth factor, an anti-malaria factor and an anti-rheumatic factor has already been reported.

Nano-gold in a living body strengthens the system of immunity and helps the absorption of nutrient. It also keeps the balance of mineral matter and strengthens the circulatory system. Particularly, it activates the transmission of signal between cells by raising the function of transmitting information between nervous synapsis over 10000 so that it strengthens the activitv of whole bodv like enceohalon nervous activitv.

Nanoparticles are produced by the method of top down method (physical method) like cracking, diffusion, radiation esposure and arc discharge, and bottom-up method (chemical and biological method) like seed growing method, polyol method, electro-chemical method. chemically reducing method and biological method.

In top down method, solid matter is comminuted into nano sized matter by the machinery comminution and corrosion technology. Constrstingly with this, in bottom-up method, atoms or molecules are compounded in molecule structure within the scope of nano metre. Here. l chemical and biological synthesis method is often used. Generally, the methods used in synthesis of nano particles require high temperature and high pressure, and use sodium borohydride, sodium citrate, thiosodium cyanide, and hydrazine and etc as a certain chemical reductant and protective.

In the patent document CN101450386, a method producing nano-silver or nano-gold particles by using inorganic phosphate as a protective is described. In the patent document CN1736637A, a method producing monodisperse nano gold particles for immunochromato analysis. Here, sodium citrate is used as a reductant and a protective, and particules gained by reducing chloroauric acid is 20~40 run. According to some research data, sodium borohydride, sodium citrate and nitromethane has been used to produce gold nanoparticles, but not a few problems are being raised in its usage as the mentioned matters are unsuitable for a living body.

Although sodium borohydride is the good reductant, even existence of its trace quantity is unsuitable for biomedicinal usage of gold nanoparticles. As its reducing ability is very strong, it reduces functional group of biology in peptide skeleton so that it removes or changes the characteristics of biomolecule.

Particularly, in case of gold nanoparticles produced by sodium citrate, as residual air of citric acid laied on particles is existent so it toxic to blood vessels or endothelial cells originated by brain to obstruct the increasing, the fact that the higher concentration will be, the bigger obstacle for existence of cell has been revealed.

As the thiol compounds used in stabilizing gold nanoparticles have very strong interaction of thiol gold nanoparticles, they make gold nanoparticles very stable. Therefore, once stabilized as such gold nanoparticles, they cannot be combined with useful medicines like peptide, protein, and several biochemical vectors so that they function that gold nanoparticles for diagnosis and treatment make not to adhere in tumors and several focal parts to a living body. This means that thiol-stabilized gold nanoparticles are limited in using bio molecule of gold nanoparticles mark. Other methods are not intimate in environment and ha e many faults to disturb the effective usage of gold nanoparticles in biomedical usage.

In WO 2013/008061, a method to gain nano gold is presented as handling red heated gold tips by using extracts of several plants like CITRUS and FERULA. This method raises complicated problems in process as it includes several process like red heating, communiting and ashing the gold tips. This invention is originated to solve the problems like the above so the process producing bio gold nanoparticles with good bio synthesis and high dispersion stability of particles, and solution of bio gold nanoparticles gained through this process are provided.

The purpose of this invention is to provide bio gold nanoparticles with high dispersion stability of particles in vitro, and good synthesis produced by the method of bio reduction using extracts of medicinal plants and spice plants such as Panax ginseng C.A.Meyer and Aloe which are known as physiological active material of human body, as reductant and stabilizer. Brief description of Invention

This invention relates to bio gold nanoparticles which is avirulence with good bio synthesis and high stability of dispersion, and produced by using natural bio physiological active material of phytegenesis like several medicinal and spices which are rich in our country as a reductant and dispersant, and the process producing it.

This invention suggests a process producing bio gold nanoparticles rapidly under the calm condition by bio reducing method without the aid of a chemical reductant like sodium citrate or sodium borohydride.

Bio gold nanoparticles are produced by heating and agitating the plant extract including total flavonoid quantity of 0.01~0.10% and simultaneously adding aqueous solution of chloroaurid acid of 0.01-0.06% to it, and finally reacting them for about 3min-2h. After production, it is used by keeping in 4^*C after cooling it in the room temperature and germ-tight filting.

This invention also suggests bio gold nanoparticles produced according to the mentioned process. The value of surface plasmon resonance: SPR of bio gold nanoparticles produced according to the process presented in this invention was 520~540nm, and the average particle size was 15~70nm, and its concentration was 50~300ppm, pH5~9.

As the dispersed stability of bio gold nanoparticles is very high, adding 25% of 5M NaCl does not change the value of SPR and it shows that bio gold nanoparticles is covered sufficiently by biomass.

This invention also shows that hydroxyl and carbonyl and so on of phenolic compounds with high anti-oxidation activity like flavonoid and terpenoid of plant extracted solution is participated in formation of gold nanoparticles and dispersion stability of particles by measuring infrared absorption spectrum of produced bio gold nanoparticles. As bio gold nanoparticles have good bio synthesis, there' re no other changes in adding 30% to serum of men.

Brief description of figures

Figure 1 is an absorption spectrum of ultraviolet rays of bio gold nanoparticles.

Figure 2 is a FTIR absorption spectrum of bio gold nanoparticles.

Figure 3 is a graph showing the characteristics of light absorption of bio gold nanoparticles according to ultrapure added quantity.

Figure 4 is a graph showing the changes of absorption spetrum of gold nanoparticles by adding salt solution of 5M.

Figure 5 is a graph showing changes of light absorption characteristics of gold nanoparticles according to added quantity of serum.

Figure 6 is a graph shwoing distribution according to size of bio gold nanoparticles.

Figure 7 is distribution according to size of bio gold nanoparticles of 59ppm measured by a nano laser particle size analyser.

Figure 8 is distribution according to size of bio gold nanoparticles of 300 ppm measured by a nano laser particle size analyser.

Detailed explanation of invention

As this invention relates to bio gold nanoparticles which is avirulence with good bio synthesis and high stability of dispersion, and produced by using natural bio physiological active material of phytegenesis like several medicinal and spices which are rich in our country as a reductant and dispersant, and the process producing it, it is described in detail through the following illustrations.

[Illustration 1 ] Producing plants extract

Leaves of plants which are dried in the shade after gathering them in plants at Pyongyang district from April to October and are powdered to pass through a sieve with a diameter of 0.64mm are used. It is extracted in a thermotank by inserting the sample of lOg into beaker of 250mL and pouring distilled water (other extracted solvent) of lOOmL. Total flavonoid quantity has been detennined by a method of AICI3.

[Illustration 2] Production of bio gold nanoparticles by using plants extracts

It is produced by a method of reducing into black red with addition into plants extracts of lOOmL by heating lOmL, 10% of chloroauric acid solution to a certain temperature. (1 ) Effect of reaction temperature

The reacting result by changing temperature to choose a suitable temperature in producing bio gold nanoparticles is same as table 1.

Table 1. Characteristic changes of gold nanoparticles solution according to reaction temperature

As table 1 shows, keeping the reaction temperature at 40— 95 ^"C is rational. (2) Effect of chloroauric acid concentration

The result measuring absorbance in λ=520 hm which is the value of SPR peculiar to bio gold nanoparticles produced by using tetrachloroauric acid solution of different concentration is same as table 2.

Table 2. Absorbance Changes of gold nanoparticles solution according to chloroauric acid concentration

N=3. * : absorbance of a diluted solution into 4 times

As table 2 shows, the absorbance is increasing according to the beginning concentration of tetrachloroauric acid solution and from over 0.03% it becomes dull. So the beginning concentration is determined on 0.02% from the viewpoint of dispersion stability of gold nanoparticles.

3) Effect of adding quantity of reductant

The result measuring the value of SPR of gold nanoparticles solution produced using the plants extracts of fixed quantity by converting into total quantity of fiavonoid is same as table 3. Table 3. Absorbance changes of gold nanoparticles solution according to total adding quantity of flavonoid

Reaction Temperature 70 "C As table 3 shows, the valuesof SPR decrease according to adding quantities, but as they are all close to standard value from over 0.01%, the adding quantity of extract is fixed on 0.01 ~0.1 % by converting into total quantity of flavonoid.

[Illustration 3] SPR Characteristics of bio gold nanoparticles

The absorption spectrum at band of ultraviolet-visible rays of bio gold nanoparticles produced by the plants extracts of different kinds is like as figure 1.

As figure 1 show, the maximum absorption wave of gold nanoparticles is within the scope of 510~540nm so according to kinds of used plants, the values by effect of SPR peculiar to gold nanoparticles are appeared.

Synthesizing the characteristics of light absorbability of bio gold nanoparticles produced by using extracts of several plants are like as tables 4 and 5.

Table 4. SPR characteristics of bio gold nanoparticles produced by using woody plants

Table 5. SPR characteristics of bio gold nanoparticles produced by using herbaceous plants

Leaf of Pinus densiflora

6 526±7 0.93±0.03 red

Sieb

7 Fruit of shizandra 535±6 0.94±0.03 pink violet

8 Flower of Vinca rosea L 530±15 0.71±0.02 pink

Leaf of Rhododendron

9 530±10 0.42d_0.02 pink

mucronulatum

Flower -bud of Eugenia

10 540±7 0. 75±0.06 pink violet

caryophyllata Thunb

As tables 4 and 5 shows, bio gold nanoparticles with different characteristics according to kinds of plants are gained, but they all indicates the value of SPR as 510-550nm.

This shows that formed particles are all included within the scope of nano-size.

[Illustration 4] Absorption spectrum characteristics of Fourier transformation infrared rays of bio gold nanoparticles

The result measuring infrared absorption spectrum of gold nanoparticles by the method of measuring the transmittance in the section between 500-4000 cm^*1 at resolution of 4cm^-1 using Fourier transmission intrared spectrograph (Nicolet 6700) are like figure 2.

As the curve 1 of figure 2 shows, the absorption of 3400 erf appeared in the extracted solution of persimmon leaves is concerned to stretching vibration of hydroxyl in phenol and alcohol compound, and absorption band of 2879.5 erf is concerned to stretching vibration of methyl in aromatic nucleus.

Absorption of 1702.23 cm^'1 is concerned to stretching vibration of carbonyl(C=0) in aldehyde, and absorption ban of 1633.94 erf and shoulder absorption band at 1560.93 erf are concerned to C=C stretching vibration of phenol. The existence of several functional group like O-H, C-H, C=0, C=C, C-O-C can be said that it is caused by water soluble heterocyclic compound of phenol same as flavonoid and terpenoid which are rich in extracted solution of persimmon leaves.

As shown in curve 2 of figure 2, in bio gold nanoparticles, most absorption bands appeared in extract of leaves are transitted as high wave numbers so that the absorption bands of 1735.35, 1649.73, 1557.90-1508.86, 1456.89, 1418.52, 1373.86-1244.92, 1052.48crf are newly appeared. This shows that flavonoid and phenol compounds added to the reduction of auric acid functions as a stabilizer of covering. Such characteristics can be found in Panax ginseng C.A.Meyer, Aloe and ect. (figure 2-5)

[Illustration 5] Dilution stability of bio gold nanoparticles and salt stability

In order to evaluate the dilution stability of gold nanoparticles, the result measured the value of SPR by adding 1.8mL of gold nanoparticles solution and 0.2mL of ultrapure water is like as figure 3.

As figure 3 shows, the value of SPR of bio gold nanoparticles solution is not changed as 520±2nm according to increasing the adding quantity of ultrapure water, and only the value of absorbance is decreased linearly. This shows that there're no changes in size or dispersion of bio gold nanoparticles.

Next, in order to evaluate the salt stability of bio gold nanoparticles, the values of SPR over 30min after adding 0.5mL of 5M salt solution to 1.5mL of bio gold nanoparticles solution are measured. Gold nanoparticles which are produced by the method of sodium citrate and the method of sodium borohydride are contrasted, (figure 4)

As figure 4 shows, the value of absorbance is low, but the value of SPR is conformable perfectly with the begining value as it is 520±2 nm. In the meantime, gold nanoparticles produced by the method of sodium citrate and the method of sodium borohydride. Sodium which is used as a contrast forms the sludge within lOmin by changing into blue upon adding salt solution and the absorption characteristics peculiar to nanoparticles are fully disappeared.

[Illustration 6] Stability of bio gold nanoparticles in serum

In order to valuate the bio synthesis of bio gold nanoparticles, the result measured the value of SPR over 30min after adding serum at different ratio is like figure 5.

As figure 5 shows, the valuesof SPR are not changed by adding serum, but only absorbance are decreased linearly. This shows that bio gold nanoparticles are existed very stably even in serum.

These results shows that bio synthesis is good as bio gold nanoparticles are very stable.

[Illustration 7] Particles distribution characteristics of bio gold nanoparticles

The result measured the distribution according to the size of gold nanoparticles in dispersion solution of bio gold nanoparticles by using the PCS (naho laser particle size analyser) is like as figures 7 and 8. In case that gold concentration is 50ppm, as figure 7 shows, we can know thatbio gold nanoparticles are very unified because their D50 is 18.4nm and size distribution is 14~22.3nm. In case that concentration is 300ppm, as D50 is 69.5nm, we can know in figure 8 that nanoparticles are fully formed.

[Illustration 8] Structual characteristics of bio gold nanoparticles

The result observating the size and the shape of bio gold nanoparticles produced as mentioned in illustrations 1 and 2 using transmission electron microscope is like as Photo 1.

As photo 1 shows, we can know that shapes of most gold nanoparticles are not only rectangle, but also their size is 15~20nm so they are comparatively unified and their dispersiveness are very good.

The result observed by the Atomic Force Microscope (AFM) of the mentioned bio gold nanoparticle is like as photo 2. As photo 2 shows, nanoparticles is rectangular and 2nm so it is conformable fundamentally with the value measured by the nano laser particle size analyser.

Claims

1. Solution of bio gold nanoparticles characterized as gaining by adding solution of chloroauric acid to plant extract including polyphenolic compounds.

2. Solution of bio gold nanoparticles of claim 1. wherein polyphenolic compounds are mixture of fiavonoid, terpenoid, tannin and ect.

3. Solution of bio gold nanoparticles of claim 1, wherein the plants used are chosen in the group consisted of EBENACEAE, FAGACEA, EUCOMMIACEAE, CUPRESSACEAE, ARONIA, PINACEAE, APOCYNACEAE, ERICACEAE, SYRINGA, ARALIACEAE, LILIACEAE, BERBERIDACEAE, RABITAE, FABACEAE, CUCURBITACEAE, ALLIACEAE, and PLANTAGINACEAE.

4. Plants group of claim 3, wherein plants are chosen in group consisted of Diospyros kaki, Quercus acutissima, Eucommia ulmoides Oliv, Biota orientalis, Aronia melanocarpa, Pinus densiflora Sieb, Vinca rosea L, Rhododendron mucronulatum, Eugenia caryophyllata Thunb, Panax ginseng C.AMeyer, Aloe arborescens Mill, Epimedium koreanum Nakai, Salvia miltiorrhiza Bunge, Phaseolus radiatus, Luffa cylindrical(L)M.Roem, Allium cepa, Allium fistulosum, Allium sativum, Allium thunbergii, Plantago asiatica

5. Solution of bio gold nanoparticles of claim 1, wherein adding amound of plants extracts is 0.01~0.1%, and 0.04-0.08% which is good, and 0.05% which is better by converting into total fiavonoid.

6. Solution of bio gold nanoparticles of claim 1, wherein concentration of chloroauric acid solution is 0.01—0.06%, and 0.02-0.05% which is good, and 0.03% which is better.

7. Solution of bio gold nanoparticles of claim 1, wherein the producing temperature is 30-90Γ, 50-70 which is good, 60 ^"C which is better.

8. Solution of bio gold nanoparticles of claim 1, wherein time of reaction is 3min-2h, 4min-lh which is good, lOmin which is better.

9. Solution of bio gold nanoparticles of claim 1, wherein size of bio gold nano particle is 15~~70nm and concentration of nanoparticles solution is 50~300pp.