AU2021101319A4 - A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity - Google Patents
A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity Download PDFInfo
- Publication number
- AU2021101319A4 AU2021101319A4 AU2021101319A AU2021101319A AU2021101319A4 AU 2021101319 A4 AU2021101319 A4 AU 2021101319A4 AU 2021101319 A AU2021101319 A AU 2021101319A AU 2021101319 A AU2021101319 A AU 2021101319A AU 2021101319 A4 AU2021101319 A4 AU 2021101319A4
- Authority
- AU
- Australia
- Prior art keywords
- cells
- silver nanoparticles
- piper nigrum
- nps
- concoction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 238000000034 method Methods 0.000 title claims abstract description 60
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 244000203593 Piper nigrum Species 0.000 title claims abstract description 52
- 235000008184 Piper nigrum Nutrition 0.000 title claims abstract description 51
- 230000008569 process Effects 0.000 title claims abstract description 32
- 230000001093 anti-cancer Effects 0.000 title claims abstract description 18
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 10
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 21
- 239000000203 mixture Substances 0.000 claims abstract description 20
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 claims abstract description 16
- 235000020744 piper nigrum extract Nutrition 0.000 claims abstract description 14
- 239000000843 powder Substances 0.000 claims abstract description 13
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 11
- 238000004458 analytical method Methods 0.000 claims abstract description 11
- 238000012512 characterization method Methods 0.000 claims abstract description 11
- 238000002173 high-resolution transmission electron microscopy Methods 0.000 claims abstract description 11
- 239000013580 millipore water Substances 0.000 claims abstract description 11
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 11
- 201000011510 cancer Diseases 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 8
- 238000003917 TEM image Methods 0.000 claims abstract description 7
- 230000009467 reduction Effects 0.000 claims abstract description 7
- 238000004611 spectroscopical analysis Methods 0.000 claims abstract description 7
- 238000009835 boiling Methods 0.000 claims abstract description 5
- 239000000084 colloidal system Substances 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 238000000227 grinding Methods 0.000 claims abstract description 5
- 239000004570 mortar (masonry) Substances 0.000 claims abstract description 5
- 238000003756 stirring Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 32
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 claims description 23
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims description 23
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 22
- 229960005542 ethidium bromide Drugs 0.000 claims description 22
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 16
- 229910052709 silver Inorganic materials 0.000 claims description 11
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims description 10
- 239000004332 silver Substances 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 8
- -1 Ag+ ions Chemical class 0.000 claims description 7
- 238000010186 staining Methods 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 229910052802 copper Inorganic materials 0.000 claims description 5
- 239000010949 copper Substances 0.000 claims description 5
- 238000000116 DAPI staining Methods 0.000 claims description 4
- 206010023825 Laryngeal cancer Diseases 0.000 claims description 4
- 230000003833 cell viability Effects 0.000 claims description 4
- 231100000433 cytotoxic Toxicity 0.000 claims description 4
- 235000013399 edible fruits Nutrition 0.000 claims description 4
- 201000005264 laryngeal carcinoma Diseases 0.000 claims description 4
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 3
- 101000868789 Drosophila melanogaster Carboxypeptidase D Proteins 0.000 claims description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- 239000006145 Eagle's minimal essential medium Substances 0.000 claims description 3
- 241000408529 Libra Species 0.000 claims description 3
- 238000003222 MTT reduction assay Methods 0.000 claims description 3
- 229930182555 Penicillin Natural products 0.000 claims description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 239000000356 contaminant Substances 0.000 claims description 3
- 230000001472 cytotoxic effect Effects 0.000 claims description 3
- 239000012470 diluted sample Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 230000002538 fungal effect Effects 0.000 claims description 3
- 230000003993 interaction Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 229940049954 penicillin Drugs 0.000 claims description 3
- 230000021419 recognition of apoptotic cell Effects 0.000 claims description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 claims description 3
- 239000003381 stabilizer Substances 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 abstract description 13
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 9
- 230000003013 cytotoxicity Effects 0.000 abstract description 9
- 239000002245 particle Substances 0.000 abstract description 8
- 238000000338 in vitro Methods 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 abstract description 2
- 241000722363 Piper Species 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 73
- 230000001640 apoptogenic effect Effects 0.000 description 12
- 230000008901 benefit Effects 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 7
- 238000002149 energy-dispersive X-ray emission spectroscopy Methods 0.000 description 6
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 5
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 3
- 238000010170 biological method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 238000000024 high-resolution transmission electron micrograph Methods 0.000 description 3
- 150000002576 ketones Chemical class 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000001338 necrotic effect Effects 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 241001483078 Phyto Species 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 238000005452 bending Methods 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 238000009388 chemical precipitation Methods 0.000 description 2
- 238000005229 chemical vapour deposition Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000001027 hydrothermal synthesis Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 239000011824 nuclear material Substances 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000004098 selected area electron diffraction Methods 0.000 description 2
- 238000003980 solgel method Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000000305 Fourier transform infrared microscopy Methods 0.000 description 1
- 208000009565 Pharyngeal Neoplasms Diseases 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 235000013614 black pepper Nutrition 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000010428 chromatin condensation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 201000008006 pharynx cancer Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004347 surface barrier Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/38—Silver; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- B22F1/05—Metallic powder characterised by the size or surface area of the particles
- B22F1/054—Nanosized particles
-
- 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
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
- G01N2021/3155—Measuring in two spectral ranges, e.g. UV and visible
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N2021/3595—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using FTIR
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nanotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Optics & Photonics (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Crystallography & Structural Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Alternative & Traditional Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- General Physics & Mathematics (AREA)
- Manufacturing & Machinery (AREA)
- Zoology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The present disclosure relates to a process for synthesizing silver nanoparticles using Piper
nigrum concoction for anticancer activity against. The process reports emphasis on silver
nanoparticles are synthesized using Piper nigrum extract for in vitro cytotoxicity efficacy
against MCF-7 and HEP-2 cells. The silver nanoparticles (Ag-NPs) are formed within 20
minutes and preliminarily confirmed by UV-Visible spectroscopy. Further, it is characterized
by FT-IR and HR-TEM. The strong SPR broad peak observed at ~441 nm which confirms
the formation of silver nanoparticles and further, the same sample are taken into the
additional supporting characterization analysis of FT-IR, HRTEM-EDX, cytotoxicity cancer
cell lines at room temperature. TEM images of biosynthesized Ag-NPs predominantly
spherical shape with particle size in the range 5 nm to 40 nm. At various concentrations,
biosynthesized silver nanoparticles showed a significant anticancer activity against both
MCF-7 and Hep-2 cells compared to Piper nigrum extract.
18
100
grinding Pipernigrum(fuit) into fine powder using mortar and pestleandthereby dissolving 2ozoffiper
nigrumpowderin,100 nil ofMillipore waterto formand mixture
boiling the mixture at 80 degree Celsius for 10 min and thereafterfiltering the mixturethrough a syringe filter of
0.A5 micronto prepare Piper nigrum concoction
a dding the Piper nigrumconcoction(10 ml) to 90 nl of 1 mMilver nitratesolution for the reduction ofAg* 10
ions and maintaining various temperatures such as RT, 40, 60, and 8O
0
C using water bath to optimize the
synthesis
stirring solutionat1000rpm for10 minutesandobservingcolor modulationatvarioustemperaturestoensure 108
theformationofsilvernanoparticles
removingexcesssilver ionsbycentnfuging thesilver colloids at10,000rpmfor 15 minutesandthereafter 110
washing three times with Millipore water
obtaining dried powder ofsilver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further 112
characterization
Figure1
Figure2
Description
grinding Pipernigrum(fuit)into fine powder using mortar and pestleandthereby dissolving 2ozoffiper nigrumpowderin,100 nil ofMillipore waterto formand mixture
boiling the mixture at 80 degree Celsius for 10 min and thereafterfiltering the mixturethrough a syringe filter of 0.A5 micronto prepare Pipernigrumconcoction
a dding the Pipernigrumconcoction(10ml) to 90 nl of 1 mMilver nitratesolution for the reduction 0 ofAg* 10 ions and maintaining various temperatures such as RT, 40, 60, and 8O C using water bath to optimize the synthesis
stirring solutionat1000rpm for10 minutesandobservingcolor modulationatvarioustemperaturestoensure 108 theformationofsilvernanoparticles
removingexcesssilver ionsbycentnfuging thesilver colloids at10,000rpmfor 15 minutesandthereafter 110 washing three times with Millipore water
obtaining dried powder ofsilver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further 112 characterization
Figure1
Figure2
The present disclosure relates to a process for concoction of Piper nigrum to synthesize the silver nanoparticles characterized by Fourier transform infrared spectroscopy (FTIR), UV-Visible spectroscopy, High resolution transmission electron microscopy (HR TEM) carried out for the first time for anticancer activity against MCF-7 and Hep-2 cell lines.
Recently, metals nanoparticles have received considerable attention of researchers due to their wide unique properties as compared to bulk and possess immense applications in the fields of diagnostics, cell labeling, antimicrobial agents, drug delivery and cancer therapy. Silver nanoparticles have established substantial consideration for different reasons such as valuable antimicrobial agent, reveals low toxicity etc. Plant mediated synthesis is gaining cost effective, economic, eco-friendly and aiding to scale up synthesis of nanoparticles. Biosynthetic processes are an effective way to prepare silver nanoparticles using plants or their extract in a restricted approach due to their dispersion, size and shape. The plant-based nanoparticles has enhanced the opportunity of using beneficial nanoparticles in the diagnosis and treatment of human cancers. Noble metal silver (Ag), is a potential application in medicine traditionally due to its unique properties. There are various syntheses methods for Ag-NPs preparation, for example; sol-gel process, chemical precipitation, reverse micelle method, hydrothermal method, microwave, chemical vapor deposition and biological methods, etc. However; biological methods are preferred for being eco-friendly, cost effective, and don't involve the use of toxic chemicals.
Piper nigrum (black pepper) is a spicy plant in which the whole plant is medicinally high in nutritional and therapeutic compounds. It possesses many medicinal properties such as antipyretic, anti-inflammatory, analgesic, and antimicrobial properties.
However, there are various syntheses methods for Ag-NPs preparation, for example; sol-gel process, chemical precipitation, reverse micelle method, hydrothermal method, microwave, chemical vapor deposition and biological methods, etc. In order to overcome the aforementioned drawbacks, there exists a need to develop a process for synthesizing silver nanoparticles using Pipernigrum concoction for anticancer activity.
The present disclosure seeks to provide a process for synthesizing silver nanoparticles using Pipernigrum concoction for anticancer activity against MCF-7 and Hep-2 Cell Lines.
In an embodiment, a process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against. The process comprises:
grinding Piper nigrum (fruit) into fine powder using mortar and pestle and thereby dissolving 20g of Pipernigrum powder in 100 ml of Millipore water to form and mixture; boiling the mixture at 80degree Celsius for 10 min and thereafter filtering the mixture through a syringe filter of 0.45 micron to prepare Piper nigrum concoction; adding the Piper nigrum concoction (10 ml) to 90 ml of1 mM silver nitrate solution for the reduction of Ag+ ions and maintaining various temperatures such as RT, 40, 60, and °C using water bath to optimize the synthesis; stirring solution at 1000 rpm for 10 minutes and observing color modulation at various temperatures to ensure the formation of silver nanoparticles; removing excess silver ions by centrifuging the silver colloids at 10,000 rpm for 15 minutes and thereafter washing three times with Millipore water; obtaining dried powder of silver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further characterization;
In an embodiment, the Piper nigrum seed concoction employed as a reducing and stabilizing agent for 1mM of silver nitrate. In an embodiment, the preliminary characterization of silver nanoparticles is carried out using UV-visible spectroscopy and UV Vis spectroscopy analysis is done using nanodrop 2000r in a scanning range of 200 nm to 800 nm.
In an embodiment, Millipore water is used as a blank, wherein the interactions between protein-silver nanoparticles are analyzed by Fourier transform infrared spectroscopy
(FTIR) in the range of 4000 to 400 cm-1 and transmission electron microscope (TEM) images of biosynthesized Ag NPs are obtained for size and shape determination using libra 200 HR TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200kV.
In an embodiment, the Ag-NPs sonicated for 5 minutes and a drop of diluted sample placed onto the carbon-coated copper grid and liquid fraction is allowed to evaporate at room temperature.
In an embodiment, the breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer (Hep-2) cell lines are cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, wherein the cell lines are maintained at 5% CO 2 in CO 2 incubator and cultures are examined using an inverted microscope to evaluate the quality of confluency and confirming the absence of bacterial and fungal contaminants.
In an embodiment, a method for determining cytotoxic effect of silver nanoparticles and Piper nigrum extract and performing cell viability study with the MTT reduction assay comprises: seeding MCF-7 and Hep-2 cells in a 96-well plate at the density of 5x103 cells /well; allowing cells to attach and grow in 96-well plate for 24 h, in 200 g of EMEM with % FBS; removing the media and replacing with the suspension of various concentrations of silver nanoparticles 10 to100 mg/ml (minimum 4 wells are seeded with each concentration); incubating the cells for 48 h inside an incubator and thereafter adding 10 ml, 5 mg/ml MTT; incubating the cells are at 37 0 Celsius for another 4 h and adding 200 pl of DMSO to each well upon removing the incubator; and evaluating optical density (OD) of the formazan product at 620 nm using multi well spectrophotometer.
In an embodiment, a method for straining Acridine Orange/ Ethidium Bromide (AO/EtBr) for detection of apoptotic cells comprises: mixing acridine orange (AO) and ethidium bromide (EtBr) staining approximately 5 gl of dye mixture (100 mg.ml-1 acridine orange (AO) and 100 mg.ml-1 ethidium bromide
(EtBr)) with 9 ml of cell suspension (1 x 105 cells.ml-1 ) on a clean microscopic cover slip; and incubating the mixture for 2-3 min and visualizing the cells under fluorescence microscope at 40x magnification with excitation filter at 510-590 nm.
In an embodiment, a method for DAPI staining comprises: treating HEP-2 cells with the above methods for 48 h, and then fixed with methanol: acetic acid (3:1, v/v) prior to washing with PBS; staining the washed cells with 1 1 mg.ml- DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) for 20 min in the dark; and recording the stained images with fluorescent microscope with appropriate excitation filter.
In an embodiment, the grouped data are statistically evaluated using GraphPad Prism 6 and Origin8.5 user interface.
An objective of the present disclosure is to characterize silver nanoparticles extracted from Piper nigrum by Fourier transform infrared spectroscopy (FTIR), UV-Visible spectroscopy, High resolution transmission electron microscopy (HR-TEM).
Another object of the present disclosure is to evaluate anticancer activity against MCF-7 and Hep-2 Cell Lines.
Yet another object of the present invention is to deliver an expeditious and cost effective process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against.
To further clarify advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Figure 1 illustrates a flow chart of a process for synthesizing silver nanoparticles using Piper nigrum concoction for anticancer activity against in accordance with an embodiment of the present disclosure; Figures 2A and 2B illustrate visual observation of silver nitrate solution and biosynthesized silver nanoparticles in accordance with an embodiment of the present disclosure; Figure 3 illustrates an UV-visible spectrum of synthesized Ag-NPs from Piper nigrum extract at different temperature conditions in accordance with an embodiment of the present disclosure; Figures 4A and 4B illustrate FTIR spectrum of Piper nigrum Ag NPs biosynthesized and as synthesized Pipernigrum in accordance with an embodiment of the present disclosure; Figures 5A and 5B illustrate HRTEM image of Ag-NPs and magnified portion of Ag-NPs in accordance with an embodiment of the present disclosure; Figure 6 illustrates an elemental compositional analysis of Ag NPs by TEM image with EDAX in accordance with an embodiment of the present disclosure; Figure 7 illustrates a cytotoxicity of the green synthesized silver nanoparticles against the MCF-7 and the Hep-2 cell line in accordance with an embodiment of the present disclosure; Figures 8A and 8B illustrate efficacy of Piper nigrum and biosynthesized Ag-NPs against MCF-7 cells at different concentration and efficacy of Piper nigrum and biosynthesized Ag-NPs against Hep-2 cells at different concentration in accordance with an embodiment of the present disclosure; and Figures 9A, 9B, 9C and 9D illustrate apoptotic effects of fluorescence microscopy study of AO/EtBr stain respective, control cells appear in live cells in green colour, orange colour apoptotic cells and necrotic cells appearing in red colour, DAPI nuclear stain of control cells, and DAPI stain of Ag-NPs treated cells exhibited condensed form of nuclear materials in apoptotic cells in accordance with an embodiment of the present disclosure.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to "an aspect", "another aspect" or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.
Referring to Figure 1, a flow chart of a process for synthesizing silver nanoparticles using Pipernigrum concoction for anticancer activity against is illustrated in accordance with an embodiment of the present disclosure. At step 102, the process 100 includes grinding Piper nigrum (fruit) into fine powder using mortar and pestle and thereby dissolving 20g of Piper nigrum powder in 100 ml of Millipore water to form and mixture. The Piper nigrum (fruit) are collected from the local market and authenticated. The color modulation was observed at various temperatures to ensure the formation of silver nanoparticles.
At step 104, the process 100 includes boiling the mixture at 80degree Celsius for 10 min and thereafter filtering the mixture through a syringe filter of 0.45 micron to prepare Pipernigrum concoction.
At step 106, the process 100 includes adding the Pipernigrum concoction (10 ml) to ml of1 mM silver nitrate solution for the reduction of Ag+ ions and maintaining various temperatures such as RT, 40, 60, and 80degree Celsius using water bath to optimize the synthesis. Silver nitrate (AgNO 3) and MTT are purchased from Hi Media Laboratories Pvt. Ltd. India. The MCF-7 and Hep-2 cancer cell line is purchased from King Institute of Preventive Medicine and Research, Chennai-600025, India.
At step 108, the process 100 includes stirring solution at 1000 rpm for 10 minutes and observing color modulation at various temperatures to ensure the formation of silver nanoparticles.
At step 110, the process 100 includes removing excess silver ions by centrifuging the silver colloids at 10,000 rpm for 15 minutes and thereafter washing three times with Millipore water.
At step 112, the process 100 includes obtaining dried powder of silver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further characterization;
In an embodiment, the Piper nigrum seed concoction employed as a reducing and stabilizing agent for 1mM of silver nitrate. In an embodiment, the preliminary characterization of silver nanoparticles is carried out using UV-visible spectroscopy and UV Vis spectroscopy analysis is done using nanodrop 2000r in a scanning range of 200 nm to 800 nm.
In an embodiment, Millipore water is used as a blank, wherein the interactions between protein-silver nanoparticles are analyzed by Fourier transform infrared spectroscopy (FTIR) in the range of 4000 to 400 cm-1 and TEM images of biosynthesized Ag NPs are obtained for size and shape determination using libra 200 HR-TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200 kV.
In an embodiment, the Ag-NPs sonicated for 5 minutes and a drop of diluted sample placed onto the carbon-coated copper grid and liquid fraction is allowed to evaporate at room temperature.
In an embodiment, the breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer (Hep-2) cell lines are cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, wherein the cell lines are maintained at 5% CO 2 in CO 2 incubator and cultures are examined using an inverted microscope to evaluate the quality of confluency and confirming the absence of bacterial and fungal contaminants. Breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer
(Hep-2) cell lines are purchased from King Institute of Preventive Medicine and Research, ICMR, Chennai, India.
In an embodiment, a method for determining cytotoxic effect of silver nanoparticles and Piper nigrum extract and performing cell viability study with the MTT reduction assay includes seeding MCF-7 and Hep-2 cells in a 96-well plate at the density of 5x103 cells /well. The method includes allowing cells to attach and grow in 96-well plate for 24 h, in 200 gl of EMEM with 10% FBS. The method includes removing the media and replacing with the suspension of various concentrations of silver nanoparticles 10 tol00 mg/ml (minimum 4 wells are seeded with each concentration). The method includes incubating the cells for 48 h inside an incubator and thereafter adding 10 ml, 5 mg/ml MTT. The method includes incubating the cells are at 37degree Celsius for another 4 h and adding 200 pl of DMSO to each well upon removing the incubator. The method further includes evaluating optical density (OD) of the formazan product at 620 nm using multi well spectrophotometer. The results are given as mean of four independent experiments. The OD value is subjected to sort out percentage of viability by using the following formula,
% Cell Viability = OD Sample x 100% OD Control
In an embodiment, a method for straining Acridine Orange/ Ethidium Bromide (AO/EtBr) for detection of apoptotic cells comprises mixing acridine orange (AO) and ethidium bromide (EtBr) staining approximately 5 pl of dye mixture (100 mg.ml 1 acridine orange (AO) and 100 mg.ml-1 ethidium bromide (EtBr)) with 9 ml of cell suspension (1 x 105 cells.ml- 1) on a clean microscopic cover slip. The method further comprises incubating the mixture for 2-3 min and visualizing the cells under fluorescence microscope at 40x magnification with excitation filter at 510-590 nm.
In an embodiment, a method for DAPI staining comprises treating HEP-2 cells with the above methods for 48 h, and then fixed with methanol: acetic acid (3:1, v/v) prior to washing with PBS. The method comprises staining the washed cells with 1 mg.ml-1 DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) for 20 min in the dark. The method further comprises recording the stained images with fluorescent microscope with appropriate excitation filter.
In an embodiment, the grouped data are statistically evaluated using GraphPad Prism 6 and Origin8.5 user interface. Values are presented as the mean SD of the four replicates of each experiment.
Figures 2A and 2B illustrate visual observation of silver nitrate solution and biosynthesized silver nanoparticles in accordance with an embodiment of the present disclosure. Silver nitrate solution (Figure 2A) is colourless and after adding Piper nigrum plant extract to silver nitrate solution, the colourless silver nitrate solution became dark red in colour (Figure 2B). This confirms that the silver nitrate is reduced and transformed into silver nanoparticles.
Figure 3 illustrates an UV-visible spectrum of synthesized Ag-NPs from Piger nigrum extract at different temperature conditions in accordance with an embodiment of the present disclosure. The UV-Vis spectra of synthesized Ag-NPs from Piper nigrum extract at different temperature conditions are as shown in the Figure 3. The colour change depicts the existence of the formation of silver nanoparticles in the Piper nigrum extract. It shows that the UV-Vis spectra of silver nanoparticle formation at different temperature using Piger nigrum nanoparticle extract (i) Room temperature (RT), (ii) 40 °C, (iii) 60 °C, (iv) 80 °C in aqueous medium. The surface Plasmon resonance (SPR) bands of colloidal silver for different temperatures are observed in the range 420 to 446 nm. At higher temperature (80 °C), intense SPR band observed compared other samples. The strong SPR broad peak observed at -441 nm which confirms the formation of silver nanoparticles and further, the same sample are taken into the additional supporting characterization analysis of FT-IR, HRTEM-EDX, cytotoxicity cancer cell lines at room temperature.
Figures 4A and 4B illustrate FTIR spectrum of Piper nigrum Ag NPs biosynthesized and as synthesized Pipernigrum in accordance with an embodiment of the present disclosure. FTIR spectrum of Ag-NPs Piper nigrum is as shown in the Figure 4A. Addition of silver nitrate, still, have the band at 3421 cm-1 which corresponds to "polymeric" OH stretching mode. An intense peak observed at 1645 cm-1 attributed to the C=O stretching mode of ketone (Figure 4B). The peaks at 1230 cm-1 and 1026 cm-1 which are corresponding to the amine C-N stretching, and the C-C stretching and the aliphatic fluoro compounds C-F stretching respectively present in the Ag-NPs and the peaks suppressed in comparison with Piper nigrum. The stretching vibration of nitro group of leaf (1388 cm-1), iodo compounds
OH group out of plane bending (521 cm-1, 565 cm-1) are completely suppressed due to the addition of silver nitrate, which means the complete reduction and stabilization of the silver nanoparticles. Here, it is confirmed that the modulated transmittance percentage of ketone, fluoro compounds and amine groups play a vital role for the bio reduction of silver nitrate to silver nanoparticles.
FTIR finger print spectrum of as synthesized Piper nigrum is depicted in Figure 4B inset. The extracted materials of vibrational bands assignments of Piper nigrum observed at 3421 cm- 1, 2933 cm- 1, 2388 cm- 1, 1632 cm- 1, 1388 cm- 1, 1221 cm-1, 1021 cm-1 565 cm-1 and 521 cm-1. The molecular vibrational bands at 3421 cm-1 which is corresponds to normal "polymeric" OH stretching mode. The peak at 2933 cm-1 associated to the methylene C-H asymmetric and symmetric stretching mode. The peak at 2388 cm-1 indicates the symmetric stretching of alkanes and the peak at 1632 cm-1 attributed to the C=O stretching mode of ketones. The peak at 1388 cm-1 corresponds to the N=O stretching of nitro groups of leaf extract. The 1021 cm-1 peak corresponds to the C-C stretch and the aliphatic fluoro compounds C- F stretch. The peaks at 521 cm-1 and 565 cm-1 which are corresponds the aliphatic iodo compounds, C-I stretch, alcohol and OH out-of-plane bending.
Figures 5A and 5B illustrate HRTEM image of Ag-NPs and magnified portion of Ag-NPs in accordance with an embodiment of the present disclosure. The surface morphology of green synthesized Ag NPs investigated by HRTEM and their surface crystalline, particle shape/size and compositional properties at the atomic-scale level. HRTEM image of Ag-NPs is shown the Figure 5A observed with particle size ~ 5 nm Ag nanoparticles shows uniform with well-crystallized particles. The overall particle size of Ag NPs range of 5 to 40 nm. The nanoparticles are embedded in a dense matrix which may be the organic stabilizing components of Pipernigrum extract. The shape of silver nanoparticles is spherical with core-shell structure indicated in the round red-circle of the Figure 5B.
Figure 6 illustrates an elemental compositional analysis of Ag NPs by TEM image with EDAX in accordance with an embodiment of the present disclosure. The elemental composition observer by silicon surface barrier detector determined using the selected area electron diffraction (SAED) (IH-300X) analysis shows (Figure 6) is performed at several points in the HRTEM system respectively. The identified elements are indexed in the EDAX spectrum as follows C, Cu, Ag and their Ka, Kp X-ray lines. The preeminent peaks of silver (Ag) concentrations from the extracted P. nigrum, C and Cu from the sample gird.
Figure 7 illustrates a cytotoxicity of the green synthesized silver nanoparticles against the MCF-7 and the Hep-2 cell line in accordance with an embodiment of the present disclosure. This study shows that the minimum dose shows enhanced anticancer activity. Respectively the Inhibitory concentration (IC 5 )0 value of the Phyto mediated Ag-NPs are observed at 43tg.ml- 1 against Hep-2 cells. The bar diagram indicates the efficacy of Piper nigrum and biosynthesized Ag-NPs against MCF-7 cells at different concentrations Figures 7(a-b). The bar diagram shows the efficacy of Piper nigrum and biosynthesized Ag-NPs against Hep-2 cells at different concentrations are shown in the Figures 7(c-d). In fact, silver nanoparticles may stimulate reactive oxygen species and effect in damage cellular components which lead to cell death.
Figures 8A and 8B illustrate efficacy of Piper nigrum and biosynthesized Ag-NPs against MCF-7 cells at different concentration and efficacy of Piper nigrum and biosynthesized Ag-NPs against Hep-2 cells at different concentration in accordance with an embodiment of the present disclosure. The cytotoxicity of the silver nanoparticle (Ag NPs) and Pipernigrum extract is studied against the MCF-7 (Figures 6a-b) and Hep-2 cell line by MTT assay (Figures 6c-d). The cytotoxicity effect of cancer cell is studied at different concentration (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 pg.ml-1). The inhibitory concentration (IC 5 0) value of the Phyto mediated Ag-NPs are observed at the concentration of 52 pg.ml-lagainst MCF-7 cells and Piper nigrum plant extract are observed as 74tg.ml' show in the bar diagramFigure 8.
Figures 9A, 9B, 9C and 9D illustrate apoptotic effects of fluorescence microscopy study of AO/EtBr stain respective, control cells appear in live cells in green colour, orange colour apoptotic cells and necrotic cells appearing in red colour, DAPI nuclear stain of control cells, and DAPI stain of Ag-NPs treated cells exhibited condensed form of nuclear materials in apoptotic cells in accordance with an embodiment of the present disclosure. The induction of apoptosis, after the treatment with IC5 0 concentration of Ag-NPs is assessed by fluorescence microscopy after staining with acridine orange/ethidium bromide (AO/EtBr). Because AO can penetrate the normal cell membrane, the cells are observed as green fluorescence, while in apoptotic cells and apoptotic bodies are formed as a result of nuclear shrinkage, blebbing and are observed as orange-coloured bodies whereas, necrotic cells are observed as red colour fluorescence due to their loss of membrane integrity when viewed under fluorescence microscope Figures 9(a-b).
DAPI is a popular nuclear counter stain and the Ag-NPs induced nuclear fragmentation is observed by DAPI staining. The untreated cells showed normal nuclei (smooth nuclear) whereas after treatment of MCF-7 cells with Ag-NPs, the apoptotic nuclei (condensed or fragmented chromatin) are observed as shown in Figures 9 (c-d). Nuclear morphology analysis showed characteristic apoptotic changes, such as chromatin condensation, fragmentation of the nucleus, and formation of apoptotic bodies in the MCF-7 cells. Interestingly, some studies have reported that Ag-NPs can also induce DNA damage and apoptosis in cancer cells. With an increase the concentration of Ag-NPs, number of apoptotic cells are increased, which suggested that Ag-NPs could induce cell apoptosis. Respectively, UV-visible spectrum higher wavelength shift and TEM surface morphology particle crystalline size confirmation expose the similar observation.
In an embodiment, a simple, facile, inexpensive, eco-friendly and green synthesis of silver nanoparticles from the Piper nigrum in aqueous medium without employing manmade chemicals is reported. The UV-Vis spectroscopy and FT-IR analysis is confirmed the preliminary confirmation of the formation of silver nanoparticles. TEM image showed spherical shape with an average particle size of 20-40 nm. The biosynthesized silver nanoparticles and Piper nigrum extract showed promising anticancer activity against breast cancer cells (MCF-7) and human pharynx cancer cell line (Hep-2). From the study, it can be concluded that the silver nanoparticles synthesized using plant possess high anticancer activity against cell lines which further suggested the potential therapeutic use of these nanoparticles.
In the present innovative report emphasis on silver nanoparticles are synthesized using Piper nigrum extract for in vitro cytotoxicity efficacy against MCF-7 and HEP-2 cells. The silver nanoparticles (Ag-NPs) are formed within 20 minutes and preliminarily confirmed by UV-Visible spectroscopy. Further, it is characterized by FT-IR and HR-TEM. The strong SPR broad peak observed at ~441 nm which confirms the formation of silver nanoparticles and further, the same sample are taken into the additional supporting characterization analysis of FT-IR, HRTEM-EDX, cytotoxicity cancer cell lines at room temperature. TEM images of biosynthesized Ag-NPs predominantly spherical shape with particle size in the range 5 nm to nm. The compositional analysis are observed by EDAX. MTT assays for cytotoxicity are carried out using various concentrations of biosynthesized silver nanoparticles and Piper nigrum extract ranging from 10 to 100 pg are performed. At various concentrations, biosynthesized silver nanoparticles showed a significant anticancer activity against both MCF-7 and Hep-2 cells compared to Piper nigrum extract. Therefore, the results reveal excellent applications of green synthesis of silver nanoparticles using Piper nigrum. From the study, it can be concluded that the silver nanoparticles synthesized using plant possess high anticancer activity against cell lines which further suggested the potential therapeutic use of these nanoparticles.
The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.
Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.
Claims (10)
1. A process for synthesizing silver nanoparticles (Ag-NPs) using Piper nigrum concoction for anticancer activity against, the process comprises:
grinding Piper nigrum (fruit) into fine powder using mortar and pestle and thereby dissolving 20 g of Piper nigrum powder in 100 ml of Millipore water to form and mixture; boiling the mixture at 80degree Celsius for 10 min and thereafter filtering the mixture through a syringe filter of 0.45 micron to prepare Pipernigrum concoction; adding the Piper nigrum concoction (10 ml) to 90 ml of 1 mM silver nitrate solution for the reduction of Ag+ ions and maintaining various temperatures such as RT, 40, 60, and 80 °C using water bath to optimize the synthesis; stirring solution at 1000 rpm for 10 minutes and observing color modulation at various temperatures to ensure the formation of silver nanoparticles; removing excess silver ions by centrifuging the silver colloids at 10,000 rpm for 15 minutes and thereafter washing three times with Millipore water; obtaining dried powder of silver nanoparticles by freeze-drying in Alpha Christ 2.0 lyophiliser for further characterization;
2. The process as claimed in claim 1, wherein the Piper nigrum seed concoction employed as a reducing and stabilizing agent for 1mM of silver nitrate.
3. The process as claimed in claim 1, wherein the preliminary characterization of silver nanoparticles is carried out using UV-visible spectroscopy and UV-Vis spectroscopy analysis is done using nanodrop 2000r in a scanning range of 200 nm to 800 nm.
4. The process as claimed in claim 3, wherein Millipore water is used as a blank, wherein the interactions between protein-silver nanoparticles are analysed by Fourier transform infrared spectroscopy (FTIR) in the range of 4000 to 400 cm-1 and TEM images of biosynthesized Ag NPs are obtained for size and shape determination using libra 200 HR-TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200 kV.
5. The process as claimed in claim 1, wherein the Ag-NPs sonicated for 5 minutes and a drop of diluted sample placed onto the carbon-coated copper grid and liquid fraction is allowed to evaporate at room temperature.
6. The process as claimed in claim 1, wherein the breast cancer cells (MCF-7), and Human Larynx Carcinoma cancer (Hep-2) cell lines are cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, wherein the cell lines are maintained at 5% CO2 in CO 2 incubator and cultures are examined using an inverted microscope to evaluate the quality of confluency and confirming the absence of bacterial and fungal contaminants.
7. The process as claimed in claim 1, wherein a method for determining cytotoxic effect of silver nanoparticles and Piper nigrum extract and performing cell viability study with the MTT reduction assay comprises:
seeding MCF-7 and Hep-2 cells in a 96-well plate at the density of 5x103 cells /well; allowing cells to attach and grow in 96-well plate for 24 h, in 200 gl of EMEM with 10% FBS; removing the media and replacing with the suspension of various concentrations of silver nanoparticles 10 tol00 mg/ml (minimum 4 wells are seeded with each concentration); incubating the cells for 48 h inside an incubator and thereafter adding 10 ml, 5 mg/ml MTT; incubating the cells are at 37degree Celsius for another 4 h and adding 200 gl of DMSO to each well upon removing the incubator; and evaluating optical density (OD) of the formazan product at 620 nm using multi well spectrophotometer.
8. The process as claimed in claim 1, wherein a method for straining Acridine Orange/ Ethidium Bromide (AO/EtBr) for detection of apoptotic cells comprises:
mixing acridine orange (AO) and ethidium bromide (EtBr) staining approximately 5 pl of dye mixture (100 mg.ml-1 acridine orange (AO) and 100 mg.ml-1 ethidium bromide (EtBr)) with 9 ml of cell suspension (1 x 105 cells.ml- 1) on a clean microscopic cover slip; and incubating the mixture for 2-3 min and visualizing the cells under fluorescence microscope at 40x magnification with excitation filter at 510-590 nm.
9. The process as claimed in claim 1, wherein a method for DAPI staining comprises:
treating HEP-2 cells with the above methods for 48 h, and then fixed with methanol: acetic acid (3:1, v/v) prior to washing with PBS; staining the washed cells with 1 mg.ml-1 DAPI (4',6-diamidino-2 phenylindole, dihydrochloride) for 20 min in the dark; and recording the stained images with fluorescent microscope with appropriate excitation filter.
10. The process as claimed in claim 1, wherein the grouped data are statistically evaluated using GraphPad Prism 6 and Origin 8.5 user interface.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021101319A AU2021101319A4 (en) | 2021-03-14 | 2021-03-14 | A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021101319A AU2021101319A4 (en) | 2021-03-14 | 2021-03-14 | A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2021101319A4 true AU2021101319A4 (en) | 2021-06-17 |
Family
ID=76327650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021101319A Ceased AU2021101319A4 (en) | 2021-03-14 | 2021-03-14 | A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2021101319A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114226712A (en) * | 2021-12-14 | 2022-03-25 | 英特派铂业股份有限公司 | Efficient fusion coating process for silver powder surface |
-
2021
- 2021-03-14 AU AU2021101319A patent/AU2021101319A4/en not_active Ceased
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114226712A (en) * | 2021-12-14 | 2022-03-25 | 英特派铂业股份有限公司 | Efficient fusion coating process for silver powder surface |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Urnukhsaikhan et al. | Antibacterial activity and characteristics of silver nanoparticles biosynthesized from Carduus crispus | |
| Venugopal et al. | Synthesis of silver nanoparticles (Ag NPs) for anticancer activities (MCF 7 breast and A549 lung cell lines) of the crude extract of Syzygium aromaticum | |
| Varadavenkatesan et al. | Dye degradation and antibacterial activity of green synthesized silver nanoparticles using Ipomoea digitata Linn. flower extract | |
| Ahmed et al. | Eco-friendly green synthesis of silver nanoparticles and their potential applications as antioxidant and anticancer agents | |
| Sankar et al. | Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles | |
| Krishnan et al. | Green synthesis of silver nanoparticles using Piper nigrum concoction and its anticancer activity against MCF-7 and Hep-2 cell lines | |
| Raja et al. | Antibacterial and anticoagulant activity of silver nanoparticles synthesised from a novel source–pods of Peltophorum pterocarpum | |
| Mani et al. | Environmental and biomedical applications of AgNPs synthesized using the aqueous extract of Solanum surattense leaf | |
| Peddi et al. | Green synthesis, characterization, antioxidant, antibacterial, and photocatalytic activity of Suaeda maritima (L.) Dumort aqueous extract-mediated copper oxide nanoparticles | |
| Shnawa et al. | Scolicidal activity of biosynthesized zinc oxide nanoparticles by Mentha longifolia L. leaves against Echinococcus granulosus protoscolices | |
| Ayinde et al. | Biosynthesis of Ultrasonically Modified Ag‐MgO Nanocomposite and Its Potential for Antimicrobial Activity | |
| Wongyai et al. | Exploration of the antimicrobial and catalytic properties of gold nanoparticles greenly synthesized by Cryptolepis buchanani Roem. and Schult extract | |
| Singh et al. | Biosynthesis of silver nanoparticles by marine invertebrate (polychaete) and assessment of its efficacy against human pathogens | |
| Khalili et al. | Anticancer properties of phyto-synthesized silver nanoparticles from medicinal plant Artemisia tschernieviana Besser aerial parts extract toward HT29 human colon adenocarcinoma cells | |
| KR102091166B1 (en) | Manufacturing method of silver nanoparticles using gardenia seed extract | |
| AU2021101319A4 (en) | A process for synthesizing silver nanoparticles using piper nigrum concoction for anticancer activity | |
| Mohanta et al. | Phyto‐assisted synthesis of bio‐functionalised silver nanoparticles and their potential anti‐oxidant, anti‐microbial and wound healing activities | |
| Khan et al. | Antioxidant, antibacterial, and catalytic performance of biosynthesized silver nanoparticles of Rhus javanica, Rumex hastatus, and Callistemon viminalis | |
| Almessiere et al. | Green synthesis, characterization and anti-cancer capability of Co0. 5Ni0. 5Nd0. 02Fe1. 98O4 nanocomposites | |
| Akwu et al. | Two temperatures biogenic synthesis of silver nanoparticles from Grewia lasiocarpa E. Mey. ex Harv. leaf and stem bark extracts: characterization and applications | |
| Kumar et al. | A Green approach for the synthesis of iron oxide nanoparticles by using roots of A. racemosus and its deg-radation of dye methyl orange. | |
| Ullah et al. | Green synthesis, in vivo and in vitro pharmacological studies of Tamarindus indica based gold nanoparticles | |
| Chandrika et al. | Adsorptive removal of acridine orange dye by green tea/copper-activated carbon nanoparticles (Gt/Cu-AC np) | |
| Alahmdi et al. | Green nanoarchitectonics of ZnO nanoparticles from Clitoria ternatea flower extract for in vitro anticancer and antibacterial activity: inhibits MCF-7 cell proliferation via intrinsic apoptotic pathway | |
| Deshi et al. | Antimicrobial efficacy of biosynthesized silver nanoparticles from different solvent extracts of Waltheria americana root |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |