KR20240033978A - Quercetin-mediated silver nanoparticles and manufacturing method thereof - Google Patents
Quercetin-mediated silver nanoparticles and manufacturing method thereof Download PDFInfo
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- KR20240033978A KR20240033978A KR1020220112961A KR20220112961A KR20240033978A KR 20240033978 A KR20240033978 A KR 20240033978A KR 1020220112961 A KR1020220112961 A KR 1020220112961A KR 20220112961 A KR20220112961 A KR 20220112961A KR 20240033978 A KR20240033978 A KR 20240033978A
- Authority
- KR
- South Korea
- Prior art keywords
- quercetin
- agnps
- mixed solution
- sodium hydroxide
- silver nanoparticles
- Prior art date
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 title claims abstract description 316
- 235000005875 quercetin Nutrition 0.000 title claims abstract description 155
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 154
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 title claims abstract description 154
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 154
- 229960001285 quercetin Drugs 0.000 title claims abstract description 154
- 230000001404 mediated effect Effects 0.000 title claims abstract description 67
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 177
- 239000011259 mixed solution Substances 0.000 claims abstract description 107
- 239000000243 solution Substances 0.000 claims abstract description 79
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims abstract description 48
- 229910001961 silver nitrate Inorganic materials 0.000 claims abstract description 24
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims description 57
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000008213 purified water Substances 0.000 claims description 23
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 20
- 229910052709 silver Inorganic materials 0.000 claims description 20
- 239000004332 silver Substances 0.000 claims description 20
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 18
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G5/00—Compounds of silver
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/07—Metallic powder characterised by particles having a nanoscale microstructure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F9/00—Making metallic powder or suspensions thereof
- B22F9/16—Making metallic powder or suspensions thereof using chemical processes
- B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
- B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F2301/00—Metallic composition of the powder or its coating
- B22F2301/25—Noble metals, i.e. Ag Au, Ir, Os, Pd, Pt, Rh, Ru
- B22F2301/255—Silver or gold
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- General Health & Medical Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Dentistry (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
본 발명은 케르세틴 용액과 질산은 용액을 혼합한 혼합용액에, 수산화나트륨을 가하여 형성된 수산화나트륨 첨가 혼합용액을, 원심분리하여 수집한 것으로, 항산화 효과, 항균 효과가 우수하며 및 생체에 적합한 케르세틴 매개 은나노 입자 및 그 제조방법에 관한 것이다. The present invention is a mixture of a quercetin solution and a silver nitrate solution, formed by adding sodium hydroxide to a mixed solution of sodium hydroxide, collected by centrifugation, and quercetin-mediated silver nanoparticles that have excellent antioxidant and antibacterial effects and are biocompatible. and its manufacturing method.
Description
본 발명은 나노 입자에 관한 것으로, 더 자세하게는 케르세틴 용액과 질산은 용액을 혼합한 혼합용액에, 수산화나트륨을 가하여 형성된 수산화나트륨 첨가 혼합용액을, 원심분리하여 수집한 것으로, 항산화 효과, 항균 효과가 우수하며 및 생체에 적합한 케르세틴 매개 은나노 입자 및 그 제조방법에 관한 것이다. The present invention relates to nanoparticles, and more specifically, the sodium hydroxide added mixed solution formed by adding sodium hydroxide to a mixed solution of quercetin solution and silver nitrate solution, collected by centrifugation, has excellent antioxidant and antibacterial effects. and biocompatible quercetin-mediated silver nanoparticles and their manufacturing method.
나노 기술은 전자, 재료 과학 및 의학 분야에서 광범위하게 응용되고 있다. 의학 분야에서 나노물질은 정형외과, 심장병 치료, 암 치료, 치과 치료 등에 사용된다. Nanotechnology has widespread applications in electronics, materials science, and medicine. In the medical field, nanomaterials are used in orthopedics, heart disease treatment, cancer treatment, and dental treatment.
나노입자란 100nm 이하의 입자를 말하며, 나노입자를 개발하기 위해서는 물리화학적 특성을 근본적으로 제어할 수 있는 합성이 가능해야 한다. Nanoparticles refer to particles of 100 nm or less, and in order to develop nanoparticles, synthesis that can fundamentally control their physical and chemical properties must be possible.
나노입자는 물리적, 화학적, 열적, 기계적, 촉매적, 전기적, 광학적 등 다양한 특성을 갖고 있어 활용 면적이 크다. 이 중 금, 은, 구리, 아연, 백금 등의 금속 나노입자는 고유한 특성으로 인해 큰 장점을 가지고 있으며 나노의학, 바이오약리학, 생명공학 등 다양한 분야에 적용되고 있다. 나노 크기의 금속 입자의 물리적 및 생물학적 특성(생체적합성 및 항균성)은 합성 과정에서 제어할 수 있다.Nanoparticles have a variety of properties such as physical, chemical, thermal, mechanical, catalytic, electrical, and optical, and have a wide range of applications. Among these, metal nanoparticles such as gold, silver, copper, zinc, and platinum have great advantages due to their unique characteristics and are applied in various fields such as nanomedicine, biopharmacology, and biotechnology. The physical and biological properties (biocompatibility and antibacterial properties) of nanosized metal particles can be controlled during the synthesis process.
일반적으로 금속 나노입자를 제조하는 방법으로는 다양한 화학적, 물리적 기술이 사용된다. 그러나 이러한 방법론은 매우 비싸고 경제적이지 않으며 잠재적인 환경 위험을 초래할 수 있는 화학 물질이 때때로 사용된다. In general, various chemical and physical techniques are used to manufacture metal nanoparticles. However, these methodologies are very expensive and uneconomical, and chemicals are sometimes used that may pose potential environmental hazards.
따라서 최근에는 식물추출물을 기반으로 금속나노입자를 합성하는 "생화학적 방법"이 중요해지고 있다. 또한, 식물성 금속 나노입자 합성법은 또 다른 친환경 합성법인 '미생물법'에 비해 정교한 미생물 배양 공정을 생략할 수 있어 주목받고 있다. Therefore, recently, “biochemical methods” for synthesizing metal nanoparticles based on plant extracts have become important. In addition, the plant-based metal nanoparticle synthesis method is attracting attention because it can omit the elaborate microbial culture process compared to the 'microbiological method', another eco-friendly synthesis method.
그러나 나노입자 합성을 위한 벌크 환원제/캡핑제의 사용은 생물학적 시스템에서 제한되지 않는 특성(크기, 모양, 안정성, 독성 등)과 작용 방식을 초래한다.However, the use of bulk reducing/capping agents for nanoparticle synthesis results in unrestricted properties (size, shape, stability, toxicity, etc.) and mode of action in biological systems.
비특이적 독성 및 특정 메커니즘(항균, 항산화, 항암 등)을 피하기 위해 AgNPs는 환원제 및 캡핑제 역할을 하는 특정 분자를 사용하여 합성되었다. To avoid non-specific toxicity and specific mechanisms (antibacterial, antioxidant, anticancer, etc.), AgNPs were synthesized using specific molecules that act as reducing and capping agents.
한편, 케르세틴(Qn)은 다양한 과일, 채소 및 종자에서 발견되는 가장 항산화제 플라보노이드 중 하나이며 양파에서 가장 흔하다. 강력한 항산화력 외에도 Qn은 신경, 심장 및 혈관 기능을 보호하고 항염 및 항알레르기 특성을 향상시키며 보고된 노화 관련 대사 질환을 약화시킨다. 그러나, 아직까지는 케르세틴(Qn)의 사용처가 나노입자 분야로 발전해 있지는 않다.Meanwhile, quercetin (Qn) is one of the most antioxidant flavonoids found in various fruits, vegetables and seeds, and is most common in onions. In addition to its powerful antioxidant properties, Qn protects nervous, cardiac and vascular functions, improves anti-inflammatory and anti-allergic properties, and attenuates reported age-related metabolic diseases. However, the use of quercetin (Qn) has not yet developed into the field of nanoparticles.
한편, 은 나노입자(AgNPs)는 다양한 항균 및 상처 치유 응용을 위한 AgNP 및 AgNP 기반 나노물질을 생산하기 위해 연구원을 끌어들이는 다양한 금속 중에서 독특한 항균 특성을 가지고 있다. AgNPs의 합성에 다양한 식물이 사용되었다. 식물 추출물에 존재하는 폴리페놀, 알칼로이드, 플라보노이드, 지방산 및 단백질과 같은 식물 성분은 환원제 및 캡핑제로 작용하여 금속 나노 입자 합성에 관여한다. 그러나, 아직까지는 은 나노입자(AgNPs)의 생체적합성에 대한 의구심을 품는 의견들이 존재한다. Meanwhile, silver nanoparticles (AgNPs) have unique antibacterial properties among various metals, which attracts researchers to produce AgNPs and AgNP-based nanomaterials for various antibacterial and wound healing applications. Various plants have been used for the synthesis of AgNPs. Plant components such as polyphenols, alkaloids, flavonoids, fatty acids and proteins present in plant extracts are involved in the synthesis of metal nanoparticles by acting as reducing and capping agents. However, there are still doubts about the biocompatibility of silver nanoparticles (AgNPs).
이에, 케르세틴(Qn)의 나노입자 분야로써의 사용 가능성을 늘리며, 은 나노입자(AgNPs)의 생체적합성을 높일 수 있음은 물론, 항산화 효과, 항균 효과 등 다양한 효과를 갖는 나노입자 및 그 제조방법에 대한 필요성이 커지고 있다. Accordingly, the possibility of using quercetin (Qn) in the field of nanoparticles is increased, and the biocompatibility of silver nanoparticles (AgNPs) can be increased, as well as nanoparticles with various effects such as antioxidant and antibacterial effects, and methods for producing the same. The need for this is growing.
상기와 같은 문제점을 해결하기 위하여 본 발명은 케르세틴 용액과 질산은 용액을 혼합한 혼합용액에, 수산화나트륨을 가하여 형성된 수산화나트륨 첨가 혼합용액을, 원심분리하여 수집한 것으로, 항산화 효과, 항균 효과가 우수하며 및 생체에 적합한 케르세틴 매개 은나노 입자 및 그 제조방법을 제공하는데 목적이 있다. In order to solve the above problems, the present invention collects by centrifuging a mixed solution of sodium hydroxide, which is formed by adding sodium hydroxide to a mixed solution of quercetin solution and silver nitrate solution, and has excellent antioxidant and antibacterial effects. And the purpose is to provide biocompatible quercetin-mediated silver nanoparticles and a method for producing the same.
상기와 같은 목적을 달성하기 위하여 본 발명은 케르세틴 용액;과 질산은 용액;을 혼합한 혼합용액;에, 수산화나트륨을 가하여 형성된 수산화나트륨 첨가 혼합용액;을 원심분리하여 수집한 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)를 제공한다. In order to achieve the above object, the present invention provides a quercetin-mediated silver nano, which is collected by centrifugation; a mixed solution of a quercetin solution and a silver nitrate solution; and a mixed solution of sodium hydroxide formed by adding sodium hydroxide. Particles (Qn-AgNPs) are provided.
또한, 상기 케르세틴 용액은, 케르세틴을 정제수에 2 ~ 4mM의 농도가 되도록 용해하여 형성된 것을 특징으로 한다.In addition, the quercetin solution is characterized in that it is formed by dissolving quercetin in purified water to a concentration of 2 to 4mM.
또한, 상기 혼합용액은, 상기 케르세틴 용액과, 8 ~ 12mM의 질산은 용액을 95 : 5 ~ 85 : 15의 부피비로 혼합하여 형성된 것을 특징으로 한다.In addition, the mixed solution is characterized in that it is formed by mixing the quercetin solution and an 8 to 12mM silver nitrate solution at a volume ratio of 95:5 to 85:15.
또한, 상기 수산화나트륨 첨가 혼합용액은, 상기 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 형성되며, 35 ~ 39℃에서 8 ~ 16시간 동안 방치된 것을 특징으로 한다.In addition, the sodium hydroxide added mixed solution is formed by adding 8 to 12mM of sodium hydroxide to the mixed solution, and is left at 35 to 39°C for 8 to 16 hours.
또한, 상기 케르세틴 매개 은나노 입자(Qn-AgNPs)는 수산화나트륨 첨가 혼합용액을 10000 ~ 14000rpm의 속도로 10 ~ 20분간 원심분리하여 수집된 것을 특징으로 한다.In addition, the quercetin-mediated silver nanoparticles (Qn-AgNPs) were collected by centrifuging a mixed solution containing sodium hydroxide at a speed of 10,000 to 14,000 rpm for 10 to 20 minutes.
또한, 상기 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하여 -50℃에서 24 ~ 72시간 동안 동결건조한 것을 특징으로 한다.In addition, the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) were washed with purified water and freeze-dried at -50°C for 24 to 72 hours.
또한, 본 발명은 케르세틴을 정제수에 용해하여 케르세틴 용액을 제조하는 케르세틴 용액 제조단계(S10); 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 질산은 용액을 혼합하여 혼합용액을 제조하는 혼합용액 제조단계(S20); 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 수산화나트륨을 가해 수산화나트륨 첨가 혼합용액을 제조하는 수산화나트륨 첨가 혼합용액 제조단계(S30); 및, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 원심분리하여, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 수집하는 케르세틴 매개 은나노 입자 수집단계(S40);를 포함하는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법을 제공한다.In addition, the present invention includes a quercetin solution preparation step (S10) of dissolving quercetin in purified water to prepare a quercetin solution; A mixed solution preparation step (S20) of preparing a mixed solution by mixing the quercetin solution prepared in the quercetin solution preparation step (S10) and a silver nitrate solution; A sodium hydroxide addition mixed solution preparation step (S30) of preparing a sodium hydroxide addition mixed solution by adding sodium hydroxide to the mixed solution prepared in the mixed solution preparation step (S20); And, a quercetin-mediated silver nanoparticle collection step (S40) of collecting quercetin-mediated silver nanoparticles (Qn-AgNPs) by centrifuging the sodium hydroxide-added mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30). A method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs) is provided.
또한, 상기 케르세틴 용액 제조단계(S10)에서의 케르세틴 용액은, 케르세틴을 정제수에 2 ~ 4mM의 농도가 되도록 용해하여 형성되는 것을 특징으로 한다.In addition, the quercetin solution in the quercetin solution preparation step (S10) is characterized in that it is formed by dissolving quercetin in purified water to a concentration of 2 to 4mM.
또한, 상기 혼합용액 제조단계(S20)에서의 혼합용액은, 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 8 ~ 12mM의 질산은 용액을 95 : 5 ~ 85 : 15의 부피비로 혼합하여 형성되는 것을 특징으로 한다.In addition, the mixed solution in the mixed solution preparation step (S20) is a mixture of the quercetin solution prepared in the quercetin solution preparation step (S10) and an 8 to 12mM silver nitrate solution at a volume ratio of 95:5 to 85:15. It is characterized by being formed.
또한, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30에서의 수산화나트륨 첨가 혼합용액은, 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 제조되며, 35 ~ 39℃에서 8 ~ 16시간 동안 방치되는 것을 특징으로 한다.In addition, the sodium hydroxide addition mixed solution in the sodium hydroxide addition mixed solution preparation step (S30) is prepared by adding 8 to 12mM of sodium hydroxide to the mixed solution prepared in the mixed solution preparation step (S20), at 35 to 39°C. It is characterized by being left for 8 to 16 hours.
또한, 상기 케르세틴 매개 은나노 입자 수집단계(S40)에서의 케르세틴 매개 은나노 입자(Qn-AgNPs)는, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 10000 ~ 14000rpm의 속도로 10 ~ 20분간 원심분리하여 수집하는 것을 특징으로 한다.In addition, the quercetin-mediated silver nanoparticles (Qn-AgNPs) in the quercetin-mediated silver nanoparticle collection step (S40) are obtained by adding the sodium hydroxide mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30) at a speed of 10,000 to 14,000 rpm. It is characterized by collection by centrifugation for 10 to 20 minutes.
또한, 상기 케르세틴 매개 은나노 입자 수집단계(S40)를 수행한 다음, 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하고 -50℃에서 24 ~ 72시간 동안 동결건조하는 동결건조 단계(S50)를 더 포함하는 것을 특징으로 한다.In addition, after performing the quercetin-mediated silver nanoparticle collection step (S40), the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) are washed with purified water and freeze-dried at -50°C for 24 to 72 hours (S50). ) is characterized in that it further includes.
본 발명의 제조방법에 따라 제조된 케르세틴 매개 은나노 입자는 케르세틴 용액과 질산은 용액을 혼합한 혼합용액에, 수산화나트륨을 가하여 형성된 수산화나트륨 첨가 혼합용액을, 원심분리하여 수집한 것으로, 항산화 효과, 항균 효과가 우수하며 및 생체적합성이 우수하다. Quercetin-mediated silver nanoparticles prepared according to the production method of the present invention are collected by centrifuging a mixed solution of sodium hydroxide, which is formed by adding sodium hydroxide to a mixed solution of quercetin solution and silver nitrate solution, and have antioxidant and antibacterial effects. It has excellent biocompatibility.
도 1은 질산은 용액(AgNO3), 케르세틴 용액(Qn) 및, 케르세틴 매개 은나노 입자(Qn-AgNPs)에 대한 UV-visible spectrum 그래프.
도 2는 Qn-AgNPs의 50nm 및 10nm 배율의 투과 전자 현미경 이미지(A, B), Qn-AgNPs의 입자 크기 분포 그래프(C) 및, Qn-AgNPs의 에너지 분산 X선 분광계(EDS) 매핑 이미지(D).
도 3은 Zeta size analysis에 따른 An-AgNPs의 유체역학적 입자 크기 그래프(A) 및, An-AgNPs의 제타 전위 그래프(B)
도 4는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 XRD 스펙트럼 이미지(A) 및 Qn과 Qn-AgNPs의 FTIR 스펙트럼 이미지(B).
도 5는 Qn-AgNPs의 DPPH 및 ABTS 소거 활성 실험에 관한 그래프.
도 6은 Qn-AgNPs의 Cytotoxicity assay 결과를 나타낸 그래프.
도 7은 Qn-AgNPs의 용혈 분석 실험에 관한 그래프(A) 및 Qn-AgNPs의 Chorioallantoic membrane(CAM) in-ovo 분석 실험에 관한 이미지(B, ((-)는 Qn-AgNPs 처리전, (+)는 Qn-AgNPs 처리 후).
도 8은 Qn-AgNPs에 대한 TEM(A) 및 EDX mapping(B)이미지. Figure 1 is a UV-visible spectrum graph for silver nitrate solution (AgNO 3 ), quercetin solution (Qn), and quercetin-mediated silver nanoparticles (Qn-AgNPs).
Figure 2 shows transmission electron microscopy images (A, B) at 50 nm and 10 nm magnification of Qn-AgNPs, particle size distribution graph of Qn-AgNPs (C), and energy dispersive X-ray spectrometry (EDS) mapping image of Qn-AgNPs ( D).
Figure 3 shows a hydrodynamic particle size graph of An-AgNPs (A) and a zeta potential graph of An-AgNPs (B) according to Zeta size analysis.
Figure 4 shows an XRD spectrum image of quercetin-mediated silver nanoparticles (Qn-AgNPs) (A) and an FTIR spectrum image of Qn and Qn-AgNPs (B).
Figure 5 is a graph of DPPH and ABTS scavenging activity experiments of Qn-AgNPs.
Figure 6 is a graph showing the results of Cytotoxicity assay of Qn-AgNPs.
Figure 7 shows a graph of the hemolysis analysis experiment of Qn-AgNPs (A) and an image of the chorioallantoic membrane (CAM) in-ovo analysis experiment of Qn-AgNPs (B, ((-) before Qn-AgNPs treatment, (+) ) is after Qn-AgNPs treatment).
Figure 8 shows TEM (A) and EDX mapping (B) images of Qn-AgNPs.
이하의 본 발명에 관한 상세한 설명들은 본 발명이 실시될 수 있는 실시 예이고 해당 실시 예의 예시로써 도시된 첨부 도면을 참조한다. 이들 실시 예는 당 업자가 본 발명의 실시에 충분하도록 상세히 설명된다. 본 발명의 다양한 실시 예는 서로 다르지만 상호 배타적일 필요는 없음이 이해되어야 한다. 예를 들어, 여기에 기재되어 있는 특정 형상, 구조 및 특성은 일 실시 예에 관련하여 본 발명의 사상 및 범위를 벗어나지 않으면서 다른 실시 예로 구현될 수 있다. 또한, 각각의 기재된 실시 예 내의 개별 구성요소의 위치 또는 배치는 본 발명의 사상 및 범위를 벗어나지 않으면서 변경될 수 있음이 이해되어야 한다.The following detailed description of the present invention is an embodiment in which the present invention can be practiced and refers to the accompanying drawings, which are shown as examples of the corresponding embodiments. These embodiments are described in sufficient detail to enable any person skilled in the art to practice the invention. It should be understood that the various embodiments of the invention are different from one another but are not necessarily mutually exclusive. For example, specific shapes, structures and characteristics described herein may be implemented in one embodiment without departing from the spirit and scope of the invention. Additionally, it should be understood that the location or arrangement of individual components within each described embodiment may be changed without departing from the spirit and scope of the invention.
따라서 후술되는 상세한 설명은 한정적인 의미로서 취하려는 것이 아니며, 본 발명의 범위는 적절하게 설명된다면 그 청구항들이 주장하는 것과 균등한 모든 범위와 더불어 첨부된 청구항에 의해서만 한정된다. 도면에서 유사한 참조부호는 여러 측면에 걸쳐서 동일하거나 유사한 기능을 지칭한다.Accordingly, the detailed description set forth below is not intended to be taken in a limiting sense, and the scope of the invention is limited only by the appended claims together with all equivalents to what those claims would assert if properly described. Similar reference numbers in the drawings refer to identical or similar functions across various aspects.
본 발명에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.The terms used in the present invention are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person working in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.
본 발명에서 어떤 부분이 어떤 구성요소를 “포함”한다고 할 때, 이는 특별히 반대되는 기재가 없는 한, 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있음을 의미한다.In the present invention, when a part “includes” a certain component, this means that, unless specifically stated to the contrary, it does not exclude other components but may further include other components.
이하, 본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)에 대하여 자세히 설명한다. Hereinafter, the quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention will be described in detail.
본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)은 케르세틴 용액;과 질산은 용액;을 혼합한 혼합용액;에, 수산화나트륨을 가하여 형성된 수산화나트륨 첨가 혼합용액;을 원심분리하여 수집한 것을 특징으로 한다.Quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention are characterized in that they are collected by centrifuging a mixed solution of quercetin solution and silver nitrate solution, formed by adding sodium hydroxide to the mixed solution. .
상기 케르세틴 용액은, 케르세틴을 정제수에 2 ~ 4mM의 농도가 되도록 용해하여 형성된 것을 특징으로 한다.The quercetin solution is characterized in that it is formed by dissolving quercetin in purified water to a concentration of 2 to 4mM.
보다 구체적으로, 상기 케르세틴 용액은 케르세틴 이수화물(Quercetin dihydrate)을 정제수에 3mM의 농도가 되도록 용해하여 형성된 것이 가장 바람직하다. More specifically, the quercetin solution is most preferably formed by dissolving quercetin dihydrate in purified water to a concentration of 3mM.
상기 혼합용액은, 상기 케르세틴 용액과, 8 ~ 12mM의 질산은 용액을 95 : 5 ~ 85 : 15의 부피비로 혼합하여 형성된 것을 특징으로 한다.The mixed solution is characterized in that it is formed by mixing the quercetin solution and an 8 to 12mM silver nitrate solution at a volume ratio of 95:5 to 85:15.
보다 구체적으로, 상기 혼합용액은, 상기 케르세틴 용액과, 10mM의 질산은 용액을 90 : 10의 부피비로 혼합하여 형성된 것이 가장 바람직하다. More specifically, the mixed solution is most preferably formed by mixing the quercetin solution and a 10mM silver nitrate solution at a volume ratio of 90:10.
상기 수산화나트륨 첨가 혼합용액은, 상기 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 형성되며, 35 ~ 39℃에서 8 ~ 16시간 동안 방치된 것을 특징으로 한다.The sodium hydroxide added mixed solution is formed by adding 8 to 12mM of sodium hydroxide to the mixed solution, and is left at 35 to 39°C for 8 to 16 hours.
이때, 상기 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 형성된 수산화나트륨 첨가 혼합용액은 35 ~ 39℃에서 8 ~ 16시간 동안 방치됨에 따라 케르세틴으로 캡핑된 은나노 입자가 서서히 형성된다. At this time, as the sodium hydroxide-added mixed solution formed by adding 8 to 12mM of sodium hydroxide to the mixed solution is left at 35 to 39°C for 8 to 16 hours, silver nanoparticles capped with quercetin are gradually formed.
보다 구체적으로, 상기 수산화나트륨 첨가 혼합용액은, 상기 혼합용액에 10mM의 수산화나트륨을 가해 형성되며, 37℃에서 12시간 동안 방치된 것이 가장 바람직하다. More specifically, the sodium hydroxide added mixed solution is formed by adding 10mM sodium hydroxide to the mixed solution, and is most preferably left at 37°C for 12 hours.
상기 케르세틴 매개 은나노 입자(Qn-AgNPs)는 수산화나트륨 첨가 혼합용액을 10000 ~ 14000rpm의 속도로 10 ~ 20분간 원심분리하여 수집된 것을 특징으로 한다.The quercetin-mediated silver nanoparticles (Qn-AgNPs) were collected by centrifuging a mixed solution containing sodium hydroxide at a speed of 10,000 to 14,000 rpm for 10 to 20 minutes.
보다 구체적으로, 상기 케르세틴 매개 은나노 입자(Qn-AgNPs)는 수산화나트륨 첨가 혼합용액을 12000rpm의 속도로 15분간 원심분리하여 수집된 것이 가장 바람직하다. More specifically, the quercetin-mediated silver nanoparticles (Qn-AgNPs) are most preferably collected by centrifuging a mixed solution with sodium hydroxide added at a speed of 12000 rpm for 15 minutes.
한편, 본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)는, 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하여 -50℃에서 24 ~ 72시간 동안 동결건조한 것일 수 있다.Meanwhile, the quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention may be obtained by washing the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) with purified water and freeze-drying them at -50°C for 24 to 72 hours.
보다 구체적으로, 본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)는, 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하여 -50℃에서 48시간 동안 동결건조한 것이 가장 바람직하다. More specifically, the quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention are most preferably obtained by washing the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) with purified water and freeze-drying them at -50°C for 48 hours.
이하 본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법에 대하여 자세히 설명한다. Hereinafter, the method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention will be described in detail.
본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법은 케르세틴을 정제수에 용해하여 케르세틴 용액을 제조하는 케르세틴 용액 제조단계(S10); 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 질산은 용액을 혼합하여 혼합용액을 제조하는 혼합용액 제조단계(S20); 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 수산화나트륨을 가해 수산화나트륨 첨가 혼합용액을 제조하는 수산화나트륨 첨가 혼합용액 제조단계(S30); 및, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 원심분리하여, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 수집하는 케르세틴 매개 은나노 입자 수집단계(S40);를 포함한다.The method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention includes a quercetin solution preparation step (S10) of dissolving quercetin in purified water to prepare a quercetin solution; A mixed solution preparation step (S20) of preparing a mixed solution by mixing the quercetin solution prepared in the quercetin solution preparation step (S10) and a silver nitrate solution; A sodium hydroxide addition mixed solution preparation step (S30) of preparing a sodium hydroxide addition mixed solution by adding sodium hydroxide to the mixed solution prepared in the mixed solution preparation step (S20); And, a quercetin-mediated silver nanoparticle collection step (S40) of collecting quercetin-mediated silver nanoparticles (Qn-AgNPs) by centrifuging the sodium hydroxide-added mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30). do.
상기 케르세틴 용액 제조단계(S10)에서는 케르세틴을 정제수에 용해하여 케르세틴 용액을 제조한다.In the quercetin solution preparation step (S10), quercetin is dissolved in purified water to prepare a quercetin solution.
상기 케르세틴 용액 제조단계(S10)에서의 케르세틴 용액은, 케르세틴을 정제수에 2 ~ 4mM의 농도가 되도록 용해하여 형성되는 것이 적절하다. It is appropriate that the quercetin solution in the quercetin solution preparation step (S10) is formed by dissolving quercetin in purified water to a concentration of 2 to 4 mM.
보다 구체적으로, 상기 케르세틴 용액 제조단계(S10)에서는 케르세틴을 정제수에 3mM의 농도가 되도록 용해하여 케르세틴 용액을 제조하는 것이 가장 바람직하다. More specifically, in the quercetin solution preparation step (S10), it is most preferable to prepare a quercetin solution by dissolving quercetin in purified water to a concentration of 3mM.
상기 혼합용액 제조단계(S20)에서는 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 질산은 용액을 혼합하여 혼합용액을 제조한다.In the mixed solution preparation step (S20), the quercetin solution prepared in the quercetin solution preparation step (S10) is mixed with the silver nitrate solution to prepare a mixed solution.
상기 혼합용액 제조단계(S20)에서의 혼합용액은, 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 8 ~ 12mM의 질산은 용액을 95 : 5 ~ 85 : 15의 부피비로 혼합하여 형성되는 것이 적절하다. The mixed solution in the mixed solution preparation step (S20) is formed by mixing the quercetin solution prepared in the quercetin solution preparation step (S10) and an 8 to 12mM silver nitrate solution at a volume ratio of 95:5 to 85:15. It is appropriate.
보다 구체적으로, 상기 혼합용액 제조단계(S20)에서는 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 10mM의 질산은 용액을 90 : 10의 부피비로 혼합하여 혼합용액을 제조하는 것이 가장 바람직하다. More specifically, in the mixed solution preparation step (S20), it is most preferable to prepare a mixed solution by mixing the quercetin solution prepared in the quercetin solution preparation step (S10) with a 10mM silver nitrate solution at a volume ratio of 90:10. .
상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서는 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 수산화나트륨을 가해 수산화나트륨 첨가 혼합용액을 제조한다.In the sodium hydroxide addition mixed solution preparation step (S30), sodium hydroxide is added to the mixed solution prepared in the mixed solution preparation step (S20) to prepare a sodium hydroxide addition mixed solution.
상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서의 수산화나트륨 첨가 혼합용액은, 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 제조되며, 35 ~ 39℃에서 8 ~ 16시간 동안 방치되는 것이 적절하다. The sodium hydroxide addition mixed solution in the sodium hydroxide addition mixed solution preparation step (S30) is prepared by adding 8 to 12mM of sodium hydroxide to the mixed solution prepared in the mixed solution preparation step (S20), at 35 to 39°C. It is appropriate to leave it for 8 to 16 hours.
상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 혼합용액에 10mM의 수산화나트륨을 가해 35 ~ 39℃에서 8 ~ 16시간 동안 방치하는 과정에서, 케르세틴으로 캡핑된 은나노 입자가 서서히 형성된다. In the sodium hydroxide addition mixed solution preparation step (S30), 10mM sodium hydroxide is added to the mixed solution and left at 35 to 39°C for 8 to 16 hours, and silver nanoparticles capped with quercetin are gradually formed.
보다 구체적으로, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서는 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 10mM의 수산화나트륨을 가해 제조되며, 37℃에서 12시간 동안 방치하여, 수산화나트륨 첨가 혼합용액을 제조하는 것이 가장 바람직하다. More specifically, in the sodium hydroxide addition mixed solution preparation step (S30), 10mM sodium hydroxide is added to the mixed solution prepared in the mixed solution preparation step (S20), and left at 37° C. for 12 hours to produce sodium hydroxide. It is most desirable to prepare an additive mixed solution.
상기 케르세틴 매개 은나노 입자 수집단계(S40)에서는 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 원심분리하여, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 수집한다. In the quercetin-mediated silver nanoparticle collection step (S40), the sodium hydroxide-added mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30) is centrifuged to collect quercetin-mediated silver nanoparticles (Qn-AgNPs).
상기 케르세틴 매개 은나노 입자 수집단계(S40)에서의 케르세틴 매개 은나노 입자(Qn-AgNPs)는, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 10000 ~ 14000rpm의 속도로 10 ~ 20분간 원심분리하여 수집하는 것이 적절하다. The quercetin-mediated silver nanoparticles (Qn-AgNPs) in the quercetin-mediated silver nanoparticle collection step (S40) are obtained by adding the sodium hydroxide mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30) at a speed of 10,000 to 14,000 rpm for 10 minutes. It is appropriate to collect by centrifuging for ~20 minutes.
보다 구체적으로, 상기 케르세틴 매개 은나노 입자 수집단계(S40)에서는 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 12000rpm의 속도로 15분간 원심분리하여, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 수집하는 것이 가장 바람직하다. More specifically, in the quercetin-mediated silver nanoparticle collection step (S40), the sodium hydroxide-added mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30) was centrifuged at a speed of 12000 rpm for 15 minutes to produce quercetin-mediated silver nanoparticles ( It is most desirable to collect Qn-AgNPs).
한편, 상기 케르세틴 매개 은나노 입자 수집단계(S40)를 수행한 다음, 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하고 -50℃에서 24 ~ 72시간 동안 동결건조하는 동결건조 단계(S50)를 더 포함할 수 있다. Meanwhile, after performing the quercetin-mediated silver nanoparticle collection step (S40), the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) are washed with purified water and freeze-dried at -50°C for 24 to 72 hours (S50). ) may further be included.
보다 구체적으로, 상기 동결건조 단계(S50)에서는 상기 케르세틴 매개 은나노 입자 수집단계(S40)에서 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하고 -50℃에서 48시간 동안 동결건조하는 것이 가장 바람직하다. More specifically, in the freeze-drying step (S50), the quercetin-mediated silver nanoparticles (Qn-AgNPs) collected in the quercetin-mediated silver nanoparticle collection step (S40) are washed with purified water and freeze-dried at -50°C for 48 hours. Most desirable.
이하, 하기 실시예, 비교예 및 실험예를 통하여 본 발명에 따른 케르세틴 매개 은나노 입자(Qn-AgNPs)가 갖는 효과에 대하여 자세히 설명한다. Hereinafter, the effects of the quercetin-mediated silver nanoparticles (Qn-AgNPs) according to the present invention will be described in detail through the following examples, comparative examples, and experimental examples.
1. 재료 및 방법1. Materials and Methods
1.1. 재료1.1. ingredient
케르세틴 이수화물(HPLC 등급), 에리트로마이신, 트리톤 X-100, 수산화나트륨(NaOH) 2,2'-아지노-비스-3-에틸벤즈티아졸린-6-설폰산(ABTS) 및 디페닐-2-피크릴-하이드라질(DPPH) Sigma-Aldrich(St. Louis, MO, USA)에서 구입했다. 질산은 및 한천 분말은 대한민국 시흥시에 위치한 대중화학금속(주)에서 구입하였다. Sheep Blood는 Carlina(한국)에서 구입했다. 영양액(NB)과 Muller-Hinton 한천(MHA)은 대한민국 Thermo Fisher Scientific의 BD DifcoTM에서 구입했다. 세포 생존력 분석 키트는 Cellomax™MediFab에서 구입했다. 테트라사이클린-염산염은 베링거 만하임(독일 만하임)에서 구입했다.Quercetin dihydrate (HPLC grade), erythromycin, Triton Picril-hydrazyl (DPPH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Silver nitrate and agar powder were purchased from Daeung Chemical Metal Co., Ltd., Siheung-si, Korea. Sheep Blood was purchased from Carlina (Korea). Nutrient broth (NB) and Muller-Hinton agar (MHA) were purchased from BD DifcoTM, Thermo Fisher Scientific, Korea. Cell viability assay kit was purchased from Cellomax™ MediFab. Tetracycline-hydrochloride was purchased from Boehringer Mannheim (Mannheim, Germany).
1.2. 1.2. 실시예Example 1. 케르세틴 매개 1. Quercetin mediation 은나노silver nano 입자( particle( QnQn -- AgNPsAgNPs )의 제조)Manufacture of
하기 제조방법에 따라, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 제조하였다. Quercetin-mediated silver nanoparticles (Qn-AgNPs) were prepared according to the following manufacturing method.
케르세틴 용액 제조단계(S10): 케르세틴(케르세틴 이수화물, Quercetin dihydrate)을 정제수에 3mM의 농도가 되도록 용해하여 케르세틴 용액 90mL를 제조하였다. Quercetin solution preparation step (S10): Quercetin (Quercetin dihydrate) was dissolved in purified water to a concentration of 3mM to prepare 90mL of quercetin solution.
혼합용액 제조단계(S20): 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액 90mL과, 10mM의 질산은 용액 10mL를 혼합하여, 총 100mL의 혼합용액을 제조하였다. Mixed solution preparation step (S20): 90 mL of the quercetin solution prepared in the quercetin solution preparation step (S10) was mixed with 10 mL of a 10 mM silver nitrate solution to prepare a total of 100 mL of mixed solution.
수산화나트륨 첨가 혼합용액 제조단계(S30): 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액 100mL에, 10mM의 수산화나트륨을 100μL 가하고, 37℃에서 12시간 동안 방치하여, 수산화나트륨 첨가 혼합용액을 제조하였다. Sodium hydroxide addition mixed solution preparation step (S30): 100 μL of 10mM sodium hydroxide was added to 100 mL of the mixed solution prepared in the mixed solution preparation step (S20), and left at 37° C. for 12 hours to prepare the sodium hydroxide addition mixed solution. Manufactured.
케르세틴 매개 은나노 입자 수집단계(S40): 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 12000rpm의 속도로 15분간 원심분리하여, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 수집하였다. Quercetin-mediated silver nanoparticle collection step (S40): The sodium hydroxide-added mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30) was centrifuged at a speed of 12000 rpm for 15 minutes to produce quercetin-mediated silver nanoparticles (Qn-AgNPs). collected.
동결건조 단계(S50): 상기 케르세틴 매개 은나노 입자 수집단계(S40)에서 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하고 -50℃에서 48시간 동안 동결건조하였다. Freeze-drying step (S50): The quercetin-mediated silver nanoparticles (Qn-AgNPs) collected in the quercetin-mediated silver nanoparticle collection step (S40) were washed with purified water and freeze-dried at -50°C for 48 hours.
1.3. 1.3. 비교예Comparative example 1. 케르세틴 용액( 1. Quercetin solution ( QnQn )의 준비) preparation of
케르세틴(케르세틴 이수화물, Quercetin dihydrate)을 정제수에 3mM의 농도가 되도록 용해하여, 비교예 1의 케르세틴 용액을 준비하였다. Quercetin (Quercetin dihydrate) was dissolved in purified water to a concentration of 3mM to prepare the quercetin solution of Comparative Example 1.
1.4. 1.4. 비교예Comparative example 2. 질산은 용액( 2. Silver nitrate solution ( AgNOAgNO 33 )의 준비) preparation of
10mM의 질산은 용액을 준비하여, 비교예 2로 하였다. A 10mM silver nitrate solution was prepared and used as Comparative Example 2.
2. 실험 방법2. Experimental method
2.1. 은 나노 입자의 특성 확인2.1. Characterization of silver nanoparticles
Qn-AgNPs의 합성은 처음에 UV-Visible 분광 광도계(SpectraMax®Plus 384 Microplate Reader, Molecular Devices)에서 확인되었다. 그 다음, Qn 및 Qn-AgNPs의 기능기를 FTIR 분광 광도계(PerkinElmer Paragon 500(USA))에서 측정했다. Qn-AgNPs 측정 특성은 X선 분말 회절(PANalytical, X'pert-pro MPD, 네덜란드)에서 측정되었다. 10-80°의 2θ스캔 범위로 분석 또한 EDS 분석과 결합된 투과 전자 현미경(TEM, JEM-2100F, JEOL, Japan)을 사용하여 Qn-AgNPs의 크기와 모양을 관찰했다. Qn-AgNPs의 제타 전위는 제타 전위 입자 크기 분석기(Malvern PANalytical Netherland)를 사용하여 측정되었다.The synthesis of Qn-AgNPs was initially confirmed on a UV-Visible spectrophotometer (SpectraMax®Plus 384 Microplate Reader, Molecular Devices). Then, the functional groups of Qn and Qn-AgNPs were measured in an FTIR spectrophotometer (PerkinElmer Paragon 500 (USA)). The measured properties of Qn-AgNPs were determined by X-ray powder diffraction (PANalytical, X'pert-pro MPD, Netherlands). The size and shape of Qn-AgNPs were observed using a transmission electron microscope (TEM, JEM-2100F, JEOL, Japan) coupled with EDS analysis and analysis with a 2θ scanning range of 10–80°. The zeta potential of Qn-AgNPs was measured using a zeta potential particle size analyzer (Malvern PANalytical Netherland).
2.2. 생물학적 응용2.2. biological applications
2.2.1. 시험관 내 세포독성2.2.1. In vitro cytotoxicity
HEK293 세포는 공지된 방법에 따라 Qn-AgNPs 세포 독성을 평가하는 데 사용되었다. 간단히, HEK293 세포는 초기에 10%의 태아 소 혈청 및 1% 항생제를 함유하는 DMEM 배지에서 배양되었고 37℃에서 CO2(5%) 인큐베이터에서 유지되었다. Qn-AgNPs의 세포 독성 효과는 WST-1(수용성 테트라졸륨염) 분석 키트로 테스트되었다. 세포 생존력 분석을 위해 HEK293 세포를 96-웰 플레이트에서 배양하고 위에서 언급한 조건에서 배양하고, 다른 농도로 연속적으로 희석된 Qn-AgNPs를 처리하고 세포 컨플루언스의 70%에 도달한 후 24시간 동안 배양했다. 그런 다음 10 μL의 WST-1을 각 웰에 첨가하고 인큐베이터에서 2시간 동안 반응시켰다. 그런 다음 마이크로플레이트 리더를 통해 450 nm에서 흡광도를 확인하였으며 세포 독성을 계산하였다.HEK293 cells were used to evaluate Qn-AgNPs cytotoxicity according to known methods. Briefly, HEK293 cells were initially cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics and maintained in a CO2 (5%) incubator at 37°C. The cytotoxic effect of Qn-AgNPs was tested with WST-1 (water-soluble tetrazolium salt) assay kit. For cell viability analysis, HEK293 cells were cultured in 96-well plates and cultured under the above-mentioned conditions, treated with serially diluted Qn-AgNPs at different concentrations for 24 h after reaching 70% of cell confluence. cultured. Then, 10 μL of WST-1 was added to each well and incubated in an incubator for 2 hours. Then, the absorbance was checked at 450 nm using a microplate reader, and cytotoxicity was calculated.
2.2.2. 항균 분석2.2.2. Antibacterial analysis
2.2.2.1. 2.2.2.1. WellWell diffusiondiffusion methodmethod
Qn-AgNPs의 항균 효과는 well 확산 방법을 사용하여 분석되었다. 간단히, 서로 다른 박테리아 병원체(Escherichia coli, Salmonella enterica, Bacillus cereus 및 Staphylococcus aureus)가 초기에 영양액에서 배양되었다. 그런 다음 무균 환경에서 무균 코르크 천공기를 사용하여 웰을 만든 후 무균 MHA 플레이트에서 배양했다. 그 후, 50 μL의 상이한 농도의 Qn-AgNPs(62.5, 125, 250, 500 μg/mL)를 각 웰에 첨가했다. 동시에 테트라사이클린-염산염 20μL(1000μg/mL)을 양성 대조군으로 사용했다. 이어서 플레이트를 37℃에서 밤새 인큐베이션하였다. Incubation 후 Induction zone의 직경을 측정하여 비교하였다.The antibacterial effect of Qn-AgNPs was analyzed using the well diffusion method. Briefly, different bacterial pathogens (Escherichia coli, Salmonella enterica, Bacillus cereus and Staphylococcus aureus) were initially cultured in nutrient broth. Wells were then created using a sterile cork borer in a sterile environment and then cultured on sterile MHA plates. Afterwards, 50 μL of different concentrations of Qn-AgNPs (62.5, 125, 250, and 500 μg/mL) were added to each well. At the same time, 20 μL (1000 μg/mL) of tetracycline-hydrochloride was used as a positive control. The plates were then incubated overnight at 37°C. After incubation, the diameter of the induction zone was measured and compared.
2.2.2.2. 최소 억제 농도(2.2.2.2. Minimum inhibitory concentration ( MICMIC ) 테스트) test
최소 억제 농도(MIC) 분석을 사용하여 미생물의 검출 가능한 성장을 억제하는 항균제의 최저 농도를 측정했다. Qn-AgNPs의 MIC는 미세희석법으로 수행하였다. 간단히, Qn-AgNPs를 증류수에 6가지 농도(3.9, 7.8, 15.62, 31.25, 62.5μg/mL)로 희석하고 멸균된 신선한 MHB(150μL)가 포함된 플레이트에 농도당 20μL을 추가했다. 그런 다음 E. coli 및 S. aureus에 각각 10μL를 접종하고 미리 측정된 시간 간격(0, 1, 3, 6, 12, 24 및 48시간) 동안 37℃에서 배양했다. 음성대조군으로 증류수(20μL)를 사용하였고, 600nm에서 흡광도를 확인하였다.Minimum inhibitory concentration (MIC) analysis was used to determine the lowest concentration of an antimicrobial agent that inhibits detectable growth of microorganisms. The MIC of Qn-AgNPs was performed by microdilution method. Briefly, Qn-AgNPs were diluted to six concentrations (3.9, 7.8, 15.62, 31.25, and 62.5 μg/mL) in distilled water, and 20 μL per concentration was added to a plate containing sterilized fresh MHB (150 μL). E. coli and S. aureus were then inoculated with 10 μL each and incubated at 37°C for predetermined time intervals (0, 1, 3, 6, 12, 24, and 48 h). Distilled water (20 μL) was used as a negative control, and absorbance was checked at 600 nm.
2.2.3. 항산화 분석2.2.3. Antioxidant analysis
DPPH, ABTS 및 기타 자유 라디칼의 제거는 일반적인 항산화 특성을 측정하는 기초다. DPPH 및 ABTS 자유 라디칼의 솔루션은 공지된 방법에 따라 준비되었다. 100μL 연속 희석된 상이한 농도의 Qn-AgNP(0.9, 1.9, 3.9, 7.8, 15.6, 31.25 μg/mL)를 96개의 플레이트에 첨가하고 100μL의 DPPH 및 ABTS를 각각 첨가했다. Ascorbic acid와 phosphate buffer(pH 7.4)를 각각 positive control과 negative control로 사용하였다. 어두운 조건에서 10분간 배양한 후 UV-Visible 분광광도계를 사용하여 DPPH와 ABTS의 흡광도를 각각 517 nm와 734 nm에서 측정했다.Scavenging of DPPH, ABTS and other free radicals is the basis for measuring general antioxidant properties. Solutions of DPPH and ABTS free radicals were prepared according to known methods. 100 μL serially diluted different concentrations of Qn-AgNPs (0.9, 1.9, 3.9, 7.8, 15.6, 31.25 μg/mL) were added to 96 plates, followed by 100 μL of DPPH and ABTS, respectively. Ascorbic acid and phosphate buffer (pH 7.4) were used as positive and negative controls, respectively. After incubation in dark conditions for 10 minutes, the absorbance of DPPH and ABTS was measured at 517 nm and 734 nm, respectively, using a UV-Visible spectrophotometer.
2.2.4. 용혈 분석2.2.4. Hemolysis assay
Qn-AgNPs 혈액 독성을 확인하기 위해 이전 방법에 따라 체외 용혈 분석을 수행했다. 간단히 말해서, 1mL의 Sheep Blood(Carlina, Korea)를 10mL PBS(pH 7.2)에 용해시켜 4%의 적혈구(RBC)를 제조하였다. 그런 다음, 4℃에서 2000rpm으로 10분 동안 원심분리하여 적혈구를 수집하고 PBS를 사용하여 철저히 세척하고 PBS(10 mL)에 분산시켰다. 분석을 위해 200μL의 서로 다른 농도의 AgNP를 200μL의 RBC와 함께 37℃에서 1시간 동안 인큐베이션했다. Triton X-100(1%) 및 PBS는 각각 양성 및 음성 대조군으로 사용되었다. 원심분리 후 상층액은 UV-vis 분광광도계(SpectraMax®Plus 384 Microplate Reader, Molecular Devices)를 사용하여 545nm에서 관찰하고 용혈의 백분율을 계산하였다.To confirm the hematological toxicity of Qn-AgNPs, an in vitro hemolysis assay was performed according to a previous method. Briefly, 4% red blood cells (RBCs) were prepared by dissolving 1 mL of Sheep Blood (Carlina, Korea) in 10 mL of PBS (pH 7.2). Then, red blood cells were collected by centrifugation at 2000 rpm for 10 min at 4°C, washed thoroughly using PBS, and dispersed in PBS (10 mL). For analysis, 200 μL of different concentrations of AgNPs were incubated with 200 μL of RBCs at 37°C for 1 hour. Triton X-100 (1%) and PBS were used as positive and negative controls, respectively. After centrifugation, the supernatant was observed at 545 nm using a UV-vis spectrophotometer (SpectraMax®Plus 384 Microplate Reader, Molecular Devices), and the percentage of hemolysis was calculated.
2.2.4. 2.2.4. CAMCAM 분석 analyze
병아리 배아 융모막요막(CAM) 분석은 여러 암(결장, 전립선 및 뇌)에서 혈관신생 및 종양 침습의 메커니즘을 이해하기 위해 널리 수행된다. 수정란은 대한민국 춘천에 있는 양계장에서 구입했다. 난자의 표면을 살균한 후 기포 공간의 외벽을 구형으로 잘라 조심스럽게 떼어냈다. 그런 상이한 농도의 Qn-AgNPs(62.5, 125, 250μg/mL)와 1N NaOH 및 PBS 200μL를 각각 주입하고 실온에서 5분 동안 방치합니다. Qn-AgNPs가 계란에 미치는 영향을 처리 전과 처리 후로 각각 비교했다.Chick embryo chorioallantoic membrane (CAM) analysis is widely performed to understand the mechanisms of angiogenesis and tumor invasion in several cancers (colon, prostate and brain). Fertilized eggs were purchased from a poultry farm in Chuncheon, South Korea. After sterilizing the surface of the egg, the outer wall of the bubble space was cut into a sphere and carefully removed. Then, different concentrations of Qn-AgNPs (62.5, 125, and 250 μg/mL) and 200 μL of 1N NaOH and PBS were respectively injected and left for 5 min at room temperature. The effect of Qn-AgNPs on eggs was compared before and after treatment.
2.3. 통계 분석2.3. statistical analysis
수집된 실험 데이터는 일원 분산 분석을 사용하여 분석되었으며 데이터는 평균 ± 표준 오차로 표시되었다. 0.05 미만의 P-값은 통계적으로 유의한 것으로 간주된다.The collected experimental data were analyzed using one-way analysis of variance and data were expressed as mean ± standard error. A P-value of less than 0.05 is considered statistically significant.
3. 결과 및 논의3. Results and discussion
3.1. 3.1. AgNPs의AgNPs 합성 및 특성확인 Synthesis and characterization
약 10mM의 무색 AgNO3를 1mM의 밝은 노란색 Qn과 혼합했다. 옅은 노란색에서 짙은 갈색으로의 형성은 처음에 AgNPs의 합성을 입증했다. 광학 이미지와 UV-가시광선 스펙트럼은 AgNP의 합성을 확인했다(도 1). 케르시틴(Qn)은 cinnamoyl 시스템의 B와 C 고리로 인해 300 - 450 nm에서 광대역을 보여주었다. Qn-AgNPs의 광학 스펙트럼은 은의 플라즈몬 공명 효과로 인해 420 nm에서 최대 흡수를 보였다. Approximately 10mM of colorless AgNO 3 was mixed with 1mM of bright yellow Qn. The formation of light yellow to dark brown color initially demonstrated the synthesis of AgNPs. Optical images and UV-visible spectra confirmed the synthesis of AgNPs (Figure 1). Quercitin (Qn) showed a broad band at 300 - 450 nm due to the B and C rings of the cinnamoyl system. The optical spectrum of Qn-AgNPs showed maximum absorption at 420 nm due to the plasmon resonance effect of silver.
TEM 및 EDS 분석은 합성된 Qn-AgNPs의 크기, 모양 및 원소 존재를 측정했다(도 2). 그 결과 Qn-AgNPs가 100nm 미만의 크기로 단분산되고 모양이 구형임을 확인하였다(도 2A 및 2B). 또한, Qn-AgNP는 응집되지 않아 입자가 더 안정적임을 나타냈다. 또한, ImageJ 소프트웨어를 사용하여 입자 크기를 측정했으며 분포 데이터는 7.92 ± 2.79(d.nm)로, Qn-AgNP의 평균 크기를 나타낸다(도 2C). 종래 연구에서는 생리활성 분자를 사용하여 합성된 AgNP가 높은 안정성을 갖는 독특한 물리적 구조를 갖는다는 것을 입증한 바 있다. 케르세틴을 사용하여 AgNPs를 합성했지만 나노입자는 농도, 비율, 조건을 포함하는 합성 방법으로 인해 더 작은 크기와 균일한 모양을 가지지 못했다. Qn-AgNPs의 원소 매핑은 Ag의 존재를 확인했다(도 8). 또한, Qn-AgNPs의 원소 분석은 더 높은 비율로 Ag의 존재를 보여주었다(도 2D). TEM and EDS analyzes determined the size, shape, and elemental presence of the synthesized Qn-AgNPs (Figure 2). As a result, it was confirmed that Qn-AgNPs were monodisperse with a size of less than 100 nm and were spherical in shape (Figures 2A and 2B). Additionally, Qn-AgNPs did not aggregate, indicating that the particles were more stable. Additionally, the particle size was measured using ImageJ software, and the distribution data was 7.92 ± 2.79 (d.nm), which represents the average size of Qn-AgNPs (Figure 2C). Previous studies have demonstrated that AgNPs synthesized using bioactive molecules have a unique physical structure with high stability. Although AgNPs were synthesized using quercetin, the nanoparticles did not have a smaller size and uniform shape due to the synthesis method including concentration, ratio, and conditions. Elemental mapping of Qn-AgNPs confirmed the presence of Ag (Figure 8). Additionally, elemental analysis of Qn-AgNPs showed the presence of Ag in a higher proportion (Figure 2D).
또한, 제타 크기 분석을 이용하여 Qn-AgNPs의 유체역학적 입자 크기, 다분산 지수(polydispersity index, PDI) 및 ζ전위를 확인하였다(도 3). 그 결과 Qn-AgNPs의 PDI는 0.27이었고, 평균 유체역학적 크기는 92.91(d.nm)이었다(도 3A). 또한, 도 3B는 Qn-AgNPs의 제타 전위가 -31.36(mV)으로 입자의 안정성이 양호함을 나타낸다. 해당 결과는 Qn-AgNP가 단분산이고 크기가 작아 입자의 안정성을 보장한다는 것을 보여준다.Additionally, the hydrodynamic particle size, polydispersity index (PDI), and ζ-potential of Qn-AgNPs were confirmed using zeta size analysis (Figure 3). As a result, the PDI of Qn-AgNPs was 0.27, and the average hydrodynamic size was 92.91 (d.nm) (Figure 3A). Additionally, Figure 3B shows that the zeta potential of Qn-AgNPs is -31.36 (mV), indicating good particle stability. The results show that Qn-AgNPs are monodisperse and small in size, ensuring the stability of the particles.
또한, XRD 분석을 통해 Qn-AgNPs의 결정질 특성을 확인하였다(도 4A). 그 결과 (111), (200), (220), (311)의 결정면에 해당하는 38.17°, 44.23°, 64.64°, 77.6°에서 회절 피크가 나타나는 것으로 나타났다. 38.17°(111)에서 주요 회절 피크는 금속 Ag 입자의 형성을 나타낸다. Qn-AgNPs의 측정 패턴은 표준 은(Ag)과 밀접하게 일치했다. Additionally, the crystalline properties of Qn-AgNPs were confirmed through XRD analysis (Figure 4A). As a result, diffraction peaks appeared at 38.17°, 44.23°, 64.64°, and 77.6°, corresponding to the crystal planes of (111), (200), (220), and (311). The main diffraction peak at 38.17° (111) indicates the formation of metallic Ag particles. The measurement pattern of Qn-AgNPs closely matched that of standard silver (Ag).
또한, Qn 및 Qn-AgNP의 기능적 특성은 FTIR 분석에서 측정되었다(도 4B). 스펙트럼은 Qn 및 Qn-AgNPs가 3251cm-1 및 3231cm-1에서 유사하게 넓은 피크를 나타내는 것으로 나타났는데 이는 Qn의 히드록시기의 O-H 스트레칭에 기인한다. 또한, Qn은 C=O stretching(1661 cm-1), C-H bending(1447 cm-1, 1378 cm-1, 864 cm-1) 및 C-O stretching(1257 cm- 1)에 해당하는 몇 가지 특징적인 밴드를 보였다. 또한, Qn-AgNPs의 투과율은 일반적으로 quercetin만큼 강하지 않았고, C=C stretch(1653 cm-1, 1599 cm-1, 815 cm-1), N-O stretch(1517 cm-1)에서 특징적인 피크가 관찰되었다. 1), Ag와의 배위 때문에 AgNPs의 표면에 존재하는 Qn과 관련된 C-O 스트레칭(1163 cm-1). 전반적으로, Quercetin으로 합성된 Qn-AgNPs는 AgNPs가 Qn에 의해 성공적으로 캡핑되었음을 나타내는 유사한 피크를 갖는 것으로 밝혀졌다.Additionally, the functional properties of Qn and Qn-AgNPs were measured in FTIR analysis (Figure 4B). The spectra showed that Qn and Qn-AgNPs exhibited similarly broad peaks at 3251 cm -1 and 3231 cm -1 , which were attributed to OH stretching of the hydroxy group of Qn. Additionally, Qn has several characteristic bands corresponding to C=O stretching (1661 cm -1 ), CH bending (1447 cm -1 , 1378 cm -1 , 864 cm -1 ) and CO stretching (1257 cm -1 ) . showed. In addition, the transmittance of Qn-AgNPs was generally not as strong as that of quercetin, and characteristic peaks were observed at C=C stretch (1653 cm -1 , 1599 cm -1 , 815 cm -1 ) and NO stretch (1517 cm -1 ). It has been done. 1), CO stretching (1163 cm-1) associated with Qn present on the surface of AgNPs due to coordination with Ag. Overall, Qn-AgNPs synthesized with Quercetin were found to have similar peaks, indicating that the AgNPs were successfully capped by Qn.
3.2. 생물학적 분석3.2. biological analysis
3.2.1. 항산화 분석3.2.1. Antioxidant analysis
항산화제는 다양한 질병의 예방제로 사용되는 생리활성 분자로 인식되고 있지만 대부분의 항산화 분자(커큐민, 케르세틴 등)는 투과성이나 흡수 및 용해도가 낮다는 한계가 있다. 또한, 항산화 분자를 표적 부위에 효율적으로 전달하기 위해 여러 나노 약물 전달 시스템이 개발되었다. 따라서 본 발명에서는 케르세틴(Qn)을 이용하여 AgNPs를 합성하고 DPPH와 ABTS 라디칼 소거법을 통해 항산화능을 평가하였다(도 5). Qn-AgNPs는 농도에 따라 DPPH와 ABTS free radical 모두에서 더 높은 라디칼 억제 활성을 보였다. IC50 농도 Qn-AgNPs는 DPPH 및 ABTS에 대해 각각 3.42μg/mL 및 3.66μg/mL였다. 더 낮은 IC50 농도는 나노입자의 넓은 표면적과 표면의 Qn 캡핑으로 인해 더 높은 자유 라디칼 소거 활성을 나타낸다. 본 발명은 케르세틴(Qn)에 의해 생합성된 AgNPs가, 케르세틴(Qn)의 캡핑을 통해, 페놀과 플라보노이드의 농도 의존적으로 강력한 항산화 능력을 가지고 있음을 뒷받침했다.Antioxidants are recognized as bioactive molecules used as preventive agents for various diseases, but most antioxidant molecules (curcumin, quercetin, etc.) have limitations such as low permeability, absorption, and solubility. Additionally, several nano-drug delivery systems have been developed to efficiently deliver antioxidant molecules to target sites. Therefore, in the present invention, AgNPs were synthesized using quercetin (Qn) and their antioxidant activity was evaluated through DPPH and ABTS radical scavenging methods (Figure 5). Qn-AgNPs showed higher radical inhibition activity on both DPPH and ABTS free radicals depending on concentration. The IC 50 concentration of Qn-AgNPs was 3.42 μg/mL and 3.66 μg/mL for DPPH and ABTS, respectively. Lower IC 50 concentrations indicate higher free radical scavenging activity due to the large surface area of the nanoparticles and Qn capping of the surface. The present invention supported that AgNPs biosynthesized by quercetin (Qn) have strong antioxidant ability in a concentration-dependent manner of phenol and flavonoid through capping of quercetin (Qn).
3.2.2. 항균 분석3.2.2. Antibacterial analysis
Qn-AgNPs의 항균 특성은 좋은 확산 방법을 통해 확인되었다. 실험 결과 Qn-AgNPs는 그람 양성균(B. cereus, S. aureus, L. monocytogenes)과 그람음성균(E. coli, S. enterica)에 대해 상당한 항균 활성을 보였다(표 1). 하기 표 1은 세균성 병원체에 대한 다양한 농도의 Qn-AgNPs의 항균 활성을 나타낸 것으로, TCH(테트라사이클린 염산염)은 대조군으로 사용되었다. The antibacterial properties of Qn-AgNPs were confirmed through a good diffusion method. As a result of the experiment, Qn-AgNPs showed significant antibacterial activity against Gram-positive bacteria (B. cereus, S. aureus, L. monocytogenes) and Gram-negative bacteria (E. coli, S. enterica) (Table 1). Table 1 below shows the antibacterial activity of Qn-AgNPs at various concentrations against bacterial pathogens, and TCH (tetracycline hydrochloride) was used as a control.
(μg/mL)Qn-AgNPs
(μg/mL)
Qn-Ag 나노입자의 항균 활성은 농도(62.5 - 500 μ/mL)의존적으로 상이하게 나타났다. Qn-AgNPs의 최대 농도(500 μg/mL)에서, B. cereus, S aureus, E. coli, S. entrica 및 L. monocytogenes의 경우, 각각, 12 ± 1.2 mm, 13 ± 1 mm, 13 ± 0.8 mm, 12 ± 1.4 mm, 13 ± 1.6 mm에서 억제 영역을 나타냈다(표 1). The antibacterial activity of Qn-Ag nanoparticles varied depending on concentration (62.5 - 500 μ/mL). At the maximum concentration of Qn-AgNPs (500 μg/mL), 12 ± 1.2 mm, 13 ± 1 mm, and 13 ± 0.8 for B. cereus, S aureus, E. coli, S. entrica, and L. monocytogenes, respectively. mm, showing zones of inhibition at 12 ± 1.4 mm and 13 ± 1.6 mm (Table 1).
그러나 균주에 특이적인 항균 활성에는 어떠한 차이도 발견되지 않았다. AgNPs의 가설적인 항균 기작은 박테리아 세포벽, 단백질 및 핵산 합성을 억제하는 것으로 보고된 바 있다. 본 실험의 결과는 Qn-AgNPs가 그람 양성균과 그람 음성균 모두에 효과적인 항균제가 될 수 있음을 확인시켜 주었다. However, no differences were found in strain-specific antibacterial activity. The hypothetical antibacterial mechanism of AgNPs has been reported to inhibit bacterial cell wall, protein, and nucleic acid synthesis. The results of this experiment confirmed that Qn-AgNPs can be an effective antibacterial agent against both Gram-positive and Gram-negative bacteria.
최소 억제 농도(MIC)는 12시간 배양 후 박테리아의 가시적인 성장을 완전히 억제하는 최저 농도로 정의된다. Qn-AgNPs(<50 nm)의 MIC는 B. cereus, S. aureus, E. coli, S. enterica 및 L. monocytogenes에 대해 3.9 μg/mL, 3.9 μg/mL, 7.8 μg/mL, 3.9 μg/mL 및 3.9 μg/mL로 나타났다(표 1). 이러한 결과는 Qn-AgNP가 낮은 농도로도 병원체 박테리아의 성장을 효과적으로 억제한다는 것을 나타낸다.Minimum inhibitory concentration (MIC) is defined as the lowest concentration that completely inhibits visible growth of bacteria after 12 hours of incubation. The MICs of Qn-AgNPs (<50 nm) were 3.9 μg/mL, 3.9 μg/mL, 7.8 μg/mL, and 3.9 μg/mL against B. cereus, S. aureus, E. coli, S. enterica, and L. monocytogenes. mL and 3.9 μg/mL (Table 1). These results indicate that Qn-AgNPs effectively inhibit the growth of pathogenic bacteria even at low concentrations.
3.2.4. 3.2.4. 시험관내in vitro 세포독성 cytotoxicity
HEK-293 세포에서 24시간 동안 Qn-AgNPs의 세포독성을 측정하여 도 6에 나타내었다. 그 결과 Qn-AgNPs의 농도에 따라 세포독성이 증가하였으나 유의하지는 않았다. HEK-293 세포는 62.5μg/mL 농도에서 95%의 높은 세포 생존율을 보였고, 500μg/mL에서는 76.84%의 세포 생존율이 현저히 감소했다. 그러나 1000 μg/mL의 Qn-AgNPs에서도 유의한 독성은 관찰되지 않았고 세포 생존율은 76%였다. 유사하게, AgNPs의 농도에 의존하는 DNA 손상 및 mRNA 발현 측면에서 HEK-293 세포에서 독성이 관찰되었다. 종래의 연구에서는 독성이 나노입자의 크기에 따라 달라지며 "트로이 목마" 메커니즘으로 인해 더 작은 크기의 AgNPs(10nm)가 더 높은 독성을 유발한다고 지적한 바 있다. 일반적인 AgNPs는 더 낮은 농도에서도 HEK-293 세포에서 독성을 유의하게 유발한다. 이러한 결과는 나노입자의 크기와 환원제가 물성을 측정하는데 중요한 역할을 한다는 것을 보여준다. The cytotoxicity of Qn-AgNPs was measured in HEK-293 cells for 24 hours and is shown in Figure 6. As a result, cytotoxicity increased depending on the concentration of Qn-AgNPs, but it was not significant. HEK-293 cells showed a high cell viability of 95% at a concentration of 62.5 μg/mL, and the cell viability was significantly reduced to 76.84% at 500 μg/mL. However, no significant toxicity was observed even at 1000 μg/mL of Qn-AgNPs, and the cell survival rate was 76%. Similarly, toxicity was observed in HEK-293 cells in terms of DNA damage and mRNA expression depending on the concentration of AgNPs. Previous studies have indicated that toxicity depends on the size of nanoparticles, with smaller size AgNPs (10 nm) causing higher toxicity due to the “Trojan horse” mechanism. Common AgNPs cause significant toxicity in HEK-293 cells even at lower concentrations. These results show that nanoparticle size and reducing agent play an important role in measuring physical properties.
3.2.5. 용혈 분석3.2.5. Hemolysis assay
나노 입자는 독특한 물리적 특성으로 인해 일반 저분자에 비해 세포 및 혈액에 독성을 유발할 수 있다. 따라서 다양한 농도의 Qn-AgNPs로 용혈분석을 수행하여 혈액독성을 확인하였다(도 7A). 분석 결과 Qn-AgNPs의 농도가 31.2㎍/mL까지 농도가 6% 미만으로 독성이 매우 낮은 것을 확인할 수 있었다. Qn-AgNPs의 높은 농도(>31.2)가 증가하고 적혈구에 부착된 입자의 수가 많아지면 혈구의 분해로 이어질 수 있기 때문에 용혈 활성이 점차 증가한다. 종래의 연구 결과에 따르면 AgNPs(20 nm)가 40 μg/mL의 농도에서 ~19%의 용혈을 보였고, 용혈은 나노입자 농도에 크게 의존한다는 것을 발견한 바 있다. 게다가, 종래의 연구에 따르면 작은 크기의 AgNP(20 nm)는 큰 크기(≥50 nm)에 비해 더 많은 용혈을 유발한다고 지적한 바 있다. 따라서 본 발명에 따른 케르세틴 매개 은나노입자는 케르세틴의 캡핑으로 인한 유체역학적 크기의 최적화를 통해, 낮은 용혈 활성을 가진다. 이와 같은 연구는 효율적인 나노 입자 및 나노 물질 기반 약물 전달 시스템 개발에 중요한 포인트라 할 수 있다. Nanoparticles can cause toxicity to cells and blood compared to ordinary small molecules due to their unique physical properties. Therefore, hemolysis analysis was performed with various concentrations of Qn-AgNPs to confirm hematological toxicity (Figure 7A). As a result of the analysis, it was confirmed that the concentration of Qn-AgNPs was less than 6% up to 31.2㎍/mL, which was very low in toxicity. As the high concentration (>31.2) of Qn-AgNPs increases and the number of particles attached to red blood cells increases, the hemolytic activity gradually increases, which may lead to the decomposition of blood cells. According to previous research results, AgNPs (20 nm) showed ~19% hemolysis at a concentration of 40 μg/mL, and hemolysis was found to be highly dependent on nanoparticle concentration. Moreover, previous studies have indicated that small-sized AgNPs (20 nm) cause more hemolysis compared to large-sized AgNPs (≥50 nm). Therefore, the quercetin-mediated silver nanoparticles according to the present invention have low hemolytic activity through optimization of hydrodynamic size due to capping of quercetin. Such research can be considered an important point in the development of efficient nanoparticle and nanomaterial-based drug delivery systems.
3.2.6. 3.2.6. CAMCAM 분석 analyze
CAM 분석은 세포 기반 실험과 동물 기반 실험 사이의 중간 모델로 사용된다. 따라서 CAM 독성을 확인하기 위해 Qn-AgNPs의 다른 농도를 테스트했다(도 7B). 실험 결과 Qn-AgNPs(7.8 - 62.5 μg/mL)는 양성대조군으로 사용된 NaOH보다 독성이 낮은 것으로 나타났다. CAM에 대한 나노입자의 독성은 혈관 손상 및 혈액 응고 측면에서 측정되었다. 일반적인 AgNPs는 독성을 가지는 것으로 알려져 있으나, 본 발명에서는 케르세틴(Qn)을 사용하여 은나노 입자를 캡핑함에 따라, 혈관 조직 손상과 같은 심각한 독성을 유발을 보호하는 결과를 보여주었다. CAM assays are used as an intermediate model between cell-based and animal-based experiments. Therefore, different concentrations of Qn-AgNPs were tested to determine CAM toxicity ( Figure 7B ). The experimental results showed that Qn-AgNPs (7.8 - 62.5 μg/mL) had lower toxicity than NaOH used as a positive control. The toxicity of nanoparticles to CAM was measured in terms of vascular damage and blood coagulation. General AgNPs are known to be toxic, but in the present invention, by capping silver nanoparticles using quercetin (Qn), it was shown to protect against serious toxicity such as vascular tissue damage.
4. 결론4. Conclusion
AgNO3는 녹색 합성 방법을 통해 환원제 및 캡핑제로 식물성 플라보노이드 케르세틴(Qn)을 사용하여 AgNPs로 성공적으로 환원되었다. Qn-AgNPs의 기능적 특성과 안정성은 UV-vis, FTIR, XRD, Zeta size 분석을 통해 확인하였다. 작은 크기(< 50 nm) 및 단분산된 Qn-AgNP는 TEM 분석에 의해 측정되었다. Qn-AgNPs는 최소 농도(≤7.8 μg/mL)에서 병원성 박테리아 성장을 상당히 억제했다. 환원제로 사용된 케르세틴(Qn)으로 인해 Qn-AgNPs는 더 나은 DPPH 및 ABTS 자유 라디칼 소거 특성을 나타냈다. 또한, 낮은 농도의 Qn-AgNPs(≤31.2μg/mL)는 일반적인 AgNPs와 달리 유의한 세포독성, 용혈을 나타내지 않았으며, 이는 본 발명의 egg CAM 손상 실험을 통해 입증되었다. 따라서 본 발명은 Qn-AgNPs가 강력한 항산화제 및 항균제임은 물론, 독성이 감소된 나노입자이며, 생물의학 응용에 사용될 수 있다고 결론지었다. 이에 본 발명은 케르세틴을 매개로 합성되어, 케르세틴의 유효성분으로 캡핑된 은나노 입자로써, 케르세틴 매개 은나노 입자 및 그 제조방법을 개발하였음을 명시한다. AgNO 3 was successfully reduced to AgNPs using the plant flavonoid quercetin (Qn) as a reducing and capping agent through a green synthesis method. The functional properties and stability of Qn-AgNPs were confirmed through UV-vis, FTIR, XRD, and Zeta size analysis. Small size (<50 nm) and monodisperse Qn-AgNPs were measured by TEM analysis. Qn-AgNPs significantly inhibited pathogenic bacterial growth at the minimum concentration (≤7.8 μg/mL). Due to quercetin (Qn) used as a reducing agent, Qn-AgNPs exhibited better DPPH and ABTS free radical scavenging properties. In addition, low concentrations of Qn-AgNPs (≤31.2 μg/mL), unlike general AgNPs, did not show significant cytotoxicity or hemolysis, which was proven through the egg CAM damage experiment of the present invention. Therefore, the present invention concluded that Qn-AgNPs are not only powerful antioxidants and antibacterial agents, but also nanoparticles with reduced toxicity, and can be used in biomedical applications. Accordingly, the present invention specifies that silver nanoparticles synthesized using quercetin and capped with the active ingredient of quercetin have been developed, including quercetin-mediated silver nanoparticles and a method for producing the same.
본 발명을 첨부된 도면과 함께 설명하였으나, 이는 본 발명의 요지를 포함하는 다양한 실시 형태 중의 하나의 실시 예에 불과하며, 당 업계에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 하는 데에 그 목적이 있는 것으로, 본 발명은 상기 설명된 실시예에만 국한되는 것이 아님은 명확하다. 따라서, 본 발명의 보호범위는 하기의 청구범위에 의해 해석되어야 하며, 본 발명의 요지를 벗어나지 않는 범위 내에서 변경, 치환, 대체 등에 의해 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리에 포함될 것이다. 또한, 도면의 일부 구성은 구성을 보다 명확하게 설명하기 위한 것으로 실제보다 과장되거나 축소되어 제공되는 것임을 명확히 한다.Although the present invention has been described with the accompanying drawings, this is only one example among various embodiments including the gist of the present invention, and is intended to enable those skilled in the art to easily implement the present invention. For this purpose, it is clear that the present invention is not limited to the embodiments described above. Accordingly, the scope of protection of the present invention should be interpreted in accordance with the following claims, and all technical ideas within the equivalent scope by change, substitution, substitution, etc. without departing from the gist of the present invention are subject to the rights of the present invention. will be included In addition, it is clarified that some of the configurations in the drawings are provided in an exaggerated or reduced form compared to the actual figure for the purpose of explaining the configuration more clearly.
(S10): 케르세틴 용액 제조단계
(S20): 혼합용액 제조단계
(S30): 수산화나트륨 첨가 혼합용액 제조단계
(S40): 케르세틴 매개 은나노 입자 수집단계(S10): Quercetin solution manufacturing step
(S20): Mixed solution manufacturing step
(S30): Sodium hydroxide addition mixed solution manufacturing step
(S40): Quercetin-mediated silver nanoparticle collection step
Claims (12)
Quercetin-mediated silver nanoparticles (Qn-AgNPs), which are collected by centrifuging a mixed solution of a quercetin solution and a silver nitrate solution, and a mixed solution of sodium hydroxide formed by adding sodium hydroxide.
상기 케르세틴 용액은, 케르세틴을 정제수에 2 ~ 4mM의 농도가 되도록 용해하여 형성된 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs).
According to paragraph 1,
The quercetin solution is quercetin-mediated silver nanoparticles (Qn-AgNPs), which are formed by dissolving quercetin in purified water to a concentration of 2 to 4mM.
상기 혼합용액은, 상기 케르세틴 용액과, 8 ~ 12mM의 질산은 용액을 95 : 5 ~ 85 : 15의 부피비로 혼합하여 형성된 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs).
According to paragraph 1,
The mixed solution is quercetin-mediated silver nanoparticles (Qn-AgNPs), which are formed by mixing the quercetin solution and an 8 to 12mM silver nitrate solution at a volume ratio of 95:5 to 85:15.
상기 수산화나트륨 첨가 혼합용액은, 상기 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 형성되며, 35 ~ 39℃에서 8 ~ 16시간 동안 방치된 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs).
According to paragraph 1,
The sodium hydroxide-added mixed solution is formed by adding 8 to 12mM of sodium hydroxide to the mixed solution, and is left at 35 to 39°C for 8 to 16 hours. Quercetin-mediated silver nanoparticles (Qn-AgNPs).
상기 수산화나트륨 첨가 혼합용액을 10000 ~ 14000rpm의 속도로 10 ~ 20분간 원심분리하여 수집된 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs).
According to paragraph 1,
Quercetin-mediated silver nanoparticles (Qn-AgNPs), which are collected by centrifuging the sodium hydroxide-added mixed solution for 10 to 20 minutes at a speed of 10,000 to 14,000 rpm.
상기 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하여 -50℃에서 24 ~ 72시간 동안 동결건조한 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs).
According to paragraph 1,
Quercetin-mediated silver nanoparticles (Qn-AgNPs), characterized in that the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) were washed with purified water and freeze-dried at -50°C for 24 to 72 hours.
상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 질산은 용액을 혼합하여 혼합용액을 제조하는 혼합용액 제조단계(S20);
상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 수산화나트륨을 가해 수산화나트륨 첨가 혼합용액을 제조하는 수산화나트륨 첨가 혼합용액 제조단계(S30); 및,
상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 원심분리하여, 케르세틴 매개 은나노 입자(Qn-AgNPs)를 수집하는 케르세틴 매개 은나노 입자 수집단계(S40);를 포함하는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법.
A quercetin solution preparation step (S10) of dissolving quercetin in purified water to prepare a quercetin solution;
A mixed solution preparation step (S20) of preparing a mixed solution by mixing the quercetin solution prepared in the quercetin solution preparation step (S10) and a silver nitrate solution;
A sodium hydroxide addition mixed solution preparation step (S30) of preparing a sodium hydroxide addition mixed solution by adding sodium hydroxide to the mixed solution prepared in the mixed solution preparation step (S20); and,
A quercetin-mediated silver nanoparticle collection step (S40) of collecting quercetin-mediated silver nanoparticles (Qn-AgNPs) by centrifuging the sodium hydroxide-added mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30). Characterized by a method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs).
상기 케르세틴 용액 제조단계(S10)에서의 케르세틴 용액은, 케르세틴을 정제수에 2 ~ 4mM의 농도가 되도록 용해하여 형성되는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법.
In clause 7,
A method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs), wherein the quercetin solution in the quercetin solution preparation step (S10) is formed by dissolving quercetin in purified water to a concentration of 2 to 4mM.
상기 혼합용액 제조단계(S20)에서의 혼합용액은, 상기 케르세틴 용액 제조단계(S10)에서 제조된 케르세틴 용액과, 8 ~ 12mM의 질산은 용액을 95 : 5 ~ 85 : 15의 부피비로 혼합하여 형성되는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법.
In clause 7,
The mixed solution in the mixed solution preparation step (S20) is formed by mixing the quercetin solution prepared in the quercetin solution preparation step (S10) and an 8 to 12mM silver nitrate solution at a volume ratio of 95:5 to 85:15. A method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs).
상기 수산화나트륨 첨가 혼합용액 제조단계(S30에서의 수산화나트륨 첨가 혼합용액은, 상기 혼합용액 제조단계(S20)에서 제조된 혼합용액에 8 ~ 12mM의 수산화나트륨을 가해 제조되며, 35 ~ 39℃에서 8 ~ 16시간 동안 방치되는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법.
In clause 7,
The sodium hydroxide addition mixed solution in the sodium hydroxide addition mixed solution preparation step (S30) is prepared by adding 8 to 12mM of sodium hydroxide to the mixed solution prepared in the mixed solution preparation step (S20), at 35 to 39°C. A method of producing quercetin-mediated silver nanoparticles (Qn-AgNPs), characterized in that they are left for ~16 hours.
상기 케르세틴 매개 은나노 입자 수집단계(S40)에서의 케르세틴 매개 은나노 입자(Qn-AgNPs)는, 상기 수산화나트륨 첨가 혼합용액 제조단계(S30)에서 제조된 수산화나트륨 첨가 혼합용액을 10000 ~ 14000rpm의 속도로 10 ~ 20분간 원심분리하여 수집하는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법.
In clause 7,
The quercetin-mediated silver nanoparticles (Qn-AgNPs) in the quercetin-mediated silver nanoparticle collection step (S40) are obtained by adding the sodium hydroxide mixed solution prepared in the sodium hydroxide-added mixed solution preparation step (S30) at a speed of 10,000 to 14,000 rpm for 10 minutes. A method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs), characterized by collection by centrifugation for ~ 20 minutes.
상기 케르세틴 매개 은나노 입자 수집단계(S40)를 수행한 다음, 수집된 케르세틴 매개 은나노 입자(Qn-AgNPs)를 정제수로 세척하고 -50℃에서 24 ~ 72시간 동안 동결건조하는 동결건조 단계(S50)를 더 포함하는 것을 특징으로 하는 케르세틴 매개 은나노 입자(Qn-AgNPs)의 제조방법.
In clause 7,
After performing the quercetin-mediated silver nanoparticle collection step (S40), the collected quercetin-mediated silver nanoparticles (Qn-AgNPs) are washed with purified water and freeze-dried at -50°C for 24 to 72 hours (S50). A method for producing quercetin-mediated silver nanoparticles (Qn-AgNPs), further comprising:
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