Quercetin Lab Report
Abstract A natural polyphenolic compound Quercetin was extracted from widely available and consumed plant Allium fistulosum which has shown promising chemo preventive and chemotherapeutic activities in cancer. In this study, quercetin is encapsulated and loaded onto PLGA (Poly lactic glycolic acid) microspheres using double emulsification methodology for improving its efficiency in cancer therapy. Quercetin is extracted and purified using chromatographic and nuclear magnetic resonance (NMR) techniques. PLGA-heparin (PLGAh) and PLGA-heparin-quercetin (PLGAh+Q) loaded microspheres were characterized for morphology using scanning electron microscopy (FESEM). The PLGAh and PLGAh-Q microspheres were tested for its cytotoxicity against MCF-7 lines.
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The PLGAh-Q showed excellent anti-cancer activities tested using Annexin-V/ Propidium iodide staining analysis.
Keywords: Apoptosis; Annexin-v, Electron microscopy; Nuclear magnetic resonance; Cancer. 1. Introduction
Biodegradable polymers are primarily used in targeted drug delivery system as vehicles and in tissue engineering as scaffolds for comfortable growth of specific tissues. Depending on upon the purpose, polymers are fabricated by considering specific chain compositions, mechanical strength and morphology [1]. Controlled release is an effective methodology and approach to optimize the dosage of the drug released to a particular site for particular duration [2]. Poly (lactic-co-glycolic acid (PLGA) is one such polymer prepared either as a scaffold or microsphere for medical resorbable sutures and delivery vehicles [3]. PLGA is a biocompatible, biodegradable polymer which has been used in medical resorbable sutures [4]. Many biomaterials used in tissue engineering cause immune reaction which reduces the efficiency for generation of tissue. PLGA was found to be an effective alternative as it physically interacts with hormones and facilitate the transport of drug [5]. Encapsulation of drug such as curcumin in PLGA nanoparticles increases the bioavailability [6] which is very crucial in an targeted drug delivery studies. Several techniques have been developed for loading of drug into PLGA including water-oil-water [7], phase separation [8], nano precipitation [9] and spray drying [10], [11]. Quercetin (3,3',4',5,7-pentahydroxyflavone) belongs to the family of poly phenolic flavonoid compound [12] and present in all edible fruits and vegetable [12]. It has wide range of biological activities such anti-cancer activity anti- oxidant activity, anti-inflammatory activity and it exist in form of glycosides [13]. Studies indicate that Quercetin at a low concentration induces mitochondria-mediated apoptosis in HeLa cells by activating the p53 gene [14] and reduces the cytotoxic side effects of doxorubicin in non-tumor cells [15]. Quercetin also down-regulate the phos¬phatidylinositol and phosphatidylinositol phosphate kinase activities in human carcinoma cells, leading to a significant reduction in IP3(inositol triphosphate) concentration and cell death [16] for treating carcinomas by down-regulated signal transduction capacity [17]. These examples clearly indicate the presence of potential anti-cancer properties in quercetin. The Annexin V-labeling method can also be improved by the Z staining with the vital dye 7-amino-actinomycin D (7AAD) [18]. Heparin is a significant biological polyelectrolyte consisting of chains of 2 to 23 units of 2 (2, 6-disulfoglucosamine- 2-sulfoiduronic acid)-(2, 6-disulfoglucosamine-glucuronic acid. The unique properties of heparin as a mixture of biological anionic polyelectrolytes are important in understanding its action as a drug. Heparin is an anticoagulant agent which prevents the coagulation of the blood stream allowing the free passage of blood in the arteries of the system. Allium fistulosum belongs to the family of Amaryllidaceae herbaceous perennial plant with flowers and generally addressed as Welsh onion. These are grown from solitary or clustered tunicate bulb and found to possess large quantity of quercetin which has potent antifungal property [19], anti-ischemic property [20], Anti-influenza A virus agents and anti-cancer property. In the present investigation, Quercetin loaded nanoparticles has been tested for its anti-cancer activity on michigan cancer cells (MCF-7) cell lines using Annexin V FITC staining studies.
3. Materials and Methods:
3.1 Chemicals required
Ethanol, methanol, hexane, toluene, ethyl acetate dichloromethane, 2% isopropanol were obtained from Merck, India. PLGA (50:50), PVA was obtained from Sigma Aldrich, USA Aldrich, Fluorescein isothiocyanate (FITC)-labeled Annexin V, propidium iodide (PI), kit were obtained from Novobiochem USA, Heparin was obtained from Fisher Scientific, Hank’s balanced salt solution (HBSS),were purchased from Invitrogen, USA. All other reagents used in reaction were of analytical grade and obtained from MERCK, India.
3.2 Isolation and purification of compound Fresh disease free young leaves samples of Allium fistulosum were collected from latitude 11° 00' N and longitude 78° 00' E Tiruvannamalai in Tamil Nadu. The leaves were washed using double distilled water and air dried in laboratory for 3 days. The dried leaves was homogenized and pulverized as fine powder. Dried and finely powder (500 G) soxhlet apparatus for 2 days..The crude residue of approximately 15 G was obtained after filtration and evaporation of solvent using rotary evaporator. 15 g of extract was treated with the hexane to remove fat and oil. The hexane extract was (6 Gm) was resolved in the by silica gel column chromatography (100 – 200 mesh, Merck, toluene, ethyl acetate, methanol gradients) and 30 fractions were collected. TLC was prepared using Silica gel 60 F 254 (Merck, Darmstadt, Germany) precoated plates (10 * 10 cm) were taken and the solvent system used was toluene: ethyl acetate: methanol in the ratio of 5:3:2(v/v/v).From 1000 µG/mL of around 10 µL and 20µL of the samples applied to the precoated TLC plates. The movement of the samples was found to be ascending and it moved up to 6cm. The developed plates were scanned under UV transilluminator at 264 nm.
3.3 Isolation and purification of Quercetin.
The confirmation of the bioactive compound was ascertained by C1 and H13 NMR using Bruker Ultrashield 600 WB PLUS.
3.4 Synthesis of the PLGA microsphere (Bare and Loaded)
PLGAh Microspheres were prepared by double emulsification method. Approximately, 500 mg PLGA in the ratio 50:50 was added to 8 mL of dichloromethane (CH2Cl2) and 1 mL of heparin. The mixed solution was further emulsified by vortexing for 40 sec. Proper care was taken to ensure the mixing of heparin. The primary solution was added drop wise using a 2G syringe into 100 mL of 1 % of PVA aqueous solution, which was mixed for 2 minutes at 9500 rpm with a turbine homogenizer resulting in a secondary emulsion. To this emulsion was further added 100 ml of 2 % isopropanol aqueous solution which was magnetically stirred at for 4 hours at 300 rpm. The beads were magnetically stirred for 4 hours and centrifuged 3 times at 2000 rpm in Eppendorf Centrifuge 5430 R machine. After the complete removal of solvent by decanting the supernatant the beads were freeze dried for 3 hours and stored for further use. The PLGAh-Q was also prepared in the above method by taking 20 mg/mL of Quercetin.
3.5In vitro anti-cancer activity
3.5.1 Cell line and culture medium
The Human monocytic cell lines and (THP-1) and Michigan cancer foundation cell lines (MCF-7) were obtained from National Center for cell science, Pune, India. The cells were cultured under standard conditions in DMEM medium, supplemented with 10% of FBS, 100U/mL of streptomycin and 100U/mL of penicillin in a humidified incubator set at 37 °C with 5% of CO2.The cells were subcultured after formation of the monolayer. The cells were detached by treating with trypsin (0.25% trypsin containing 0.01% EDTA) for 2 min and washed with PBS for further analysis.
3.5.2 Cytotoxicity assay using THP-1 and MCF-7 Human monocytic cell lines (THP-1) and Michigan cancer foundation (MCF-7) (5×103 cells/well) were incubated with various concentrations (0.6-10 µg/mL) of PLGAh. The culture media were removed and the cells were incubated with 5 mg/mL MTT in fresh medium at 37 °C for an additional 4 hrs. After this period, the supernatants were removed and 100 µL DMSO was added to each well to dissolve the formazan crystals. The plates were read on a micro plate reader at a test wavelength of 540 nm and a reference wavelength of 650 nm.
3.5.3 Annexin V / Propidium iodide double staining assay
Annexin V-FITC apoptosis detection kit (Calbiochem, CA, USA) was used to determine the number of apoptotic cells. According to the instruction of the manfacturer the treated cells (MCF-7) (1–2 × 105cells/well) were incubated at 37 °C with 5 % CO2 for 24 hours in the presence or absence of carrier PLGAh / PLGAh+Q (10 and 15 μM/L). Cells were trypsinized, washed twice with Hanks balanced salt solution (HBSS), suspended in 100 μL Annexin V binding buffer and then incubated for 20 min, in 5 μL of Annexin V Fluorescein isothiocyanate (FITC) / Propidium iodide. 1 μL of 1 mg/mL Propidium iodide solution was added to each cell suspension and were run immediately in flow cytometer (FACS Jazz Becton Dickinson, USA) and analyzed in Submit software
3.6 Characterization of microspheres
The surface morphology of the PLGAh and PLGAh-Q was determined using Field Emission Scanning Electron Microscope (FESEM-SUPRA 55) - Carl Zeiss, Germany.
4. Results
4.1 Column chromatography
The column chromatography was performed using different concentrations of elution solution and the column is eluted in increasing order of polarity of solvent from 100 % of extract, 25 % of ethyl acetate in hexane, 50 % of ethyl acetate in hexane, 75 % of ethyl acetate in hexane and 100 % of ethyl acetate. Then the polarity was increased using 2 % Methanol in Ethyl acetate to 50 % Methanol in ethyl acetate. 8 % of methanol and 92 % of ethyl acetate concentration was found to be the best concentration and after concentrating all fractions of these concentrations, approximately 7 to 8 mg of quercetin was obtained.
4.2 Thin layer chromatography
TLC profiles were carried out for the fractions, performed using the solvent mixture of toluene: ethyl acetate: methanol in the ratio of 5:3:2 (v/v/v) and this mobile phase enabled excellent separation of the bioactive compound. The results showed that there was good amount of correlation between the standard quercetin band and the band of bioactive sample plates when visualized under UV at 254 …show more content…
nm.
4.3 Morphology of microspheres
The FE-SEM micrograph provides an insight on the morphology of synthesized microspheres which showed PLGAh microspheres which are spherical in shape, PLGAh-Q showed high amount of porosity and the spheres were mono dispersed. The size of the spheres of PLGAh was found to be 36.20 µm and PLGAh-Q were 90.16 µm. This result showed that it is feasible to load the drug for optimum drug delivery to the system. The SEM images of Rifamycin / PLGA micro sphere which were also found to be spherical and mono dispersed and were prepared by the four fluid nozzle spray. Here the Rifamycin loaded microsphere was used for the anti-tuberculosis activity [21]. Another study revealed the SEM size of the periyl alcohol – bearing PLGA micro particles of were 768 ± 215 nm. These particles were prepared by oil-in-water-based emulsion solvent evaporation technique [22] Porous and non-porous PLGA microspheres prepared by oil/water emulsion technique, showed that the porous microspheres were bigger and size had high density of pores whereas the non-porogen microspheres were found to be smaller in size with less pores [23]. Studies on SEM images have also revealed that novel controlled release formulation for the anticancer drug paclitaxel in PLGA nanoparticles containing vitamin E showed the spherical shape with various degree of smoothness in the microsphere [24].
4.4 Cytotoxicity assay in THP-1 (MTT)
The cytotoxicity assay was performed in THP -1 (Human monocytic cell) lines for evaluating the toxicity of PLGAh carrier the normal cell lines and to evaluate the dosage beyond which the carrier is toxic to the cell lines. It was found that the 80 % cells are viable in the range of 0.625 µg / mL to 5 µg / mL. From these range the dosage of carrier was decided. This study shows the cytotoxicity assay done curcumin loaded PLGA nanoparticles prepared by single emulsion–solvent evaporation method showed destruction of amyloid aggregates and no cytotoxic effect was found in the formulation as 70 % of cells were found to be viable [25].
4.5 Cytotoxicity assay (MCF-7) MTT The PLGAh carrier and the PLGAh-Q carrier were tested for their anti- cancer activities against the Michigan cancer foundation (MCF-7) cancer lines. In the study it was found out that the PLGAh carrier has very less or negligible amount of anti-cancer activity but in comparison the PLGAh+Q carrier displayed excellent anti-cancer activity by decreasing the cell viability to 40 % in the range of concentration of 0-2 µg / mL. The inhibition concentration (IC 50) value was found in the concentration of 3.648 µg. So comparing the above, cytotoxicity of PLGAh carrier in normal cell lines and cytotoxicity of the PLGAh-Q carrier in the cancer cell lines the range of 1.5 µg and 3.0 µg concentrations was found to be optimum for anti-cancer activity. Studies revealed that cell viability assay in pancreatic cell lines treated in various concentrations of free curcumin and nanocurcumin. It was found that the nanocurcumin showed high cytotoxicity to pancreatic cell lines in higher concentration of 15 µM [26]. It is also known that the quercetin enhances anti-cancer activity of trichostatin A through up regulation of p53 protein as Chan and co-worker have studied both in in vivo and in vitro [27]. The PLGA- curcumin Nano formulation was targeted on to MCF-7 breast cancer cell lines by Verderio and group and they observed high toxicity to cancer cells showing potential anti-cancer activity [6]. Other studies have also indicated that cell proliferation has decreased significantly in PLGA-curcumin formulation when was subjected to human colon cancer cell lines 116 in comparison to native curcumin within 6-24 hours [28].
4.6 Annexin v / propidium iodide double staining assay staining
Apoptosis is the mechanism of programmed cell death which occurs in eukaryotic organisms [29] has promoted active research into new methods of detecting these subcellular lesions at the single-cell level in complex cell mixtures both in vivo and in vivo cultured cells [30], [31], [32].
The PLGAh and PLGAh+Q carrier was tested by these assay for rate of apoptosis in the in Michigan cancer foundation (MCF-7) cell lines. The control was the normal cancer cell lines without any PLGAh and PLGAh-Q carrier. The PLGAh carrier was tested in assay in two different concentrations which was found above 1.5 µg and 3.0 µg. The summation of early and late apoptosis was just 8 % in concentration of 1.5 µg and in concentration of the 3.0 µg it was found to be 15 %. But PLGAh-Q in the concentration of 1.5 µg showed approximately 45 % of apoptosis (early+ late). But when concentration was increased to the 3.0 µg the apoptosis was (early + late) found to be 60 %. So it is concluded that PLGAh carrier in various concentration (1.5 µg and 3.0 µg) does not show any significant apoptosis but the PLGAh-Q carrier showed increase in apoptotic activity by increasing the concentration of drug from 1.5 µg to 30 µg. Studies have indicate that the viability of cancer cell decreased when it was given silver nanoparticles in different concentration and the confirmation of different stages of apoptosis were observed by separating the live cell and dead cells by annexin V / PI staining which also
determined the % of apoptosis [33]. During early apoptosis membrane blebbing of cells occur so phosphatidyl serine comes out from the inner side of the plasma membrane to the outer membrane leaflet, where it becomes exposed [34] to the available of fluorochrome-labeled recombinant soluble annexin V which is an useful tool for detecting and quantifying early apoptotic cells by flow cytometry [31], [35], [34]. It is well known that quercetin induces apoptosis by activating caspase-3 and regulating B cell lymphoma-2 (Bcl-2) and cyclooxygenase-2 pathways as Niu and co-workers have studied its effects on human leukemia cell lines [36]. Studies have also been carried out on the effects of intracellular drug release from Curcumin-Loaded PLGA Nanoparticles and it was concluded that it Induces G2/M Block in Breast Cancer Cells and it was confirmed by 7-AAD/Annexin-V double staining and a prominent increase in death of cancer cell was observed [6]. It has also been studied that low concentration of curcumin PLGA formulation is far more effective than native curcumin and showed enhanced anti-cancer activity by initiating apoptosis of the human colon cancer lines [28].
5. Conclusion
In this study, the effect of PLGAh-Q microspheres on cancer cell lines has been evaluated. Allium fistulosum was used to extract quercetin and purified using chromatographic techniques. With the help of 1H and 13C NMR, the isolated compound was confirmed as quercetin. The electron micrographs of PLGAh and PLGAh-Q microspheres suggest that the particles are found to have spherical shaped structures for PLGAh microspheres and PLGAh-Q microsphere shows pores on the surface suitable for drug delivery studies. The PLGAh-Q showed excellent anti-cancer activities tested using Annexin–V FITC/PI staining. These results suggest that PLGAh-Q can be potent anticancer agent and tested further on other cell lines for development of novel anticancer agents.
The PLGAh-Q showed excellent anti-cancer activities tested using Annexin-V/ Propidium iodide staining analysis.
Keywords: Apoptosis; Annexin-v, Electron microscopy; Nuclear magnetic resonance; Cancer. 1. Introduction
Biodegradable polymers are primarily used in targeted drug delivery system as vehicles and in tissue engineering as scaffolds for comfortable growth of specific tissues. Depending on upon the purpose, polymers are fabricated by considering specific chain compositions, mechanical strength and morphology [1]. Controlled release is an effective methodology and approach to optimize the dosage of the drug released to a particular site for particular duration [2]. Poly (lactic-co-glycolic acid (PLGA) is one such polymer prepared either as a scaffold or microsphere for medical resorbable sutures and delivery vehicles [3]. PLGA is a biocompatible, biodegradable polymer which has been used in medical resorbable sutures [4]. Many biomaterials used in tissue engineering cause immune reaction which reduces the efficiency for generation of tissue. PLGA was found to be an effective alternative as it physically interacts with hormones and facilitate the transport of drug [5]. Encapsulation of drug such as curcumin in PLGA nanoparticles increases the bioavailability [6] which is very crucial in an targeted drug delivery studies. Several techniques have been developed for loading of drug into PLGA including water-oil-water [7], phase separation [8], nano precipitation [9] and spray drying [10], [11]. Quercetin (3,3',4',5,7-pentahydroxyflavone) belongs to the family of poly phenolic flavonoid compound [12] and present in all edible fruits and vegetable [12]. It has wide range of biological activities such anti-cancer activity anti- oxidant activity, anti-inflammatory activity and it exist in form of glycosides [13]. Studies indicate that Quercetin at a low concentration induces mitochondria-mediated apoptosis in HeLa cells by activating the p53 gene [14] and reduces the cytotoxic side effects of doxorubicin in non-tumor cells [15]. Quercetin also down-regulate the phos¬phatidylinositol and phosphatidylinositol phosphate kinase activities in human carcinoma cells, leading to a significant reduction in IP3(inositol triphosphate) concentration and cell death [16] for treating carcinomas by down-regulated signal transduction capacity [17]. These examples clearly indicate the presence of potential anti-cancer properties in quercetin. The Annexin V-labeling method can also be improved by the Z staining with the vital dye 7-amino-actinomycin D (7AAD) [18]. Heparin is a significant biological polyelectrolyte consisting of chains of 2 to 23 units of 2 (2, 6-disulfoglucosamine- 2-sulfoiduronic acid)-(2, 6-disulfoglucosamine-glucuronic acid. The unique properties of heparin as a mixture of biological anionic polyelectrolytes are important in understanding its action as a drug. Heparin is an anticoagulant agent which prevents the coagulation of the blood stream allowing the free passage of blood in the arteries of the system. Allium fistulosum belongs to the family of Amaryllidaceae herbaceous perennial plant with flowers and generally addressed as Welsh onion. These are grown from solitary or clustered tunicate bulb and found to possess large quantity of quercetin which has potent antifungal property [19], anti-ischemic property [20], Anti-influenza A virus agents and anti-cancer property. In the present investigation, Quercetin loaded nanoparticles has been tested for its anti-cancer activity on michigan cancer cells (MCF-7) cell lines using Annexin V FITC staining studies.
3. Materials and Methods:
3.1 Chemicals required
Ethanol, methanol, hexane, toluene, ethyl acetate dichloromethane, 2% isopropanol were obtained from Merck, India. PLGA (50:50), PVA was obtained from Sigma Aldrich, USA Aldrich, Fluorescein isothiocyanate (FITC)-labeled Annexin V, propidium iodide (PI), kit were obtained from Novobiochem USA, Heparin was obtained from Fisher Scientific, Hank’s balanced salt solution (HBSS),were purchased from Invitrogen, USA. All other reagents used in reaction were of analytical grade and obtained from MERCK, India.
3.2 Isolation and purification of compound Fresh disease free young leaves samples of Allium fistulosum were collected from latitude 11° 00' N and longitude 78° 00' E Tiruvannamalai in Tamil Nadu. The leaves were washed using double distilled water and air dried in laboratory for 3 days. The dried leaves was homogenized and pulverized as fine powder. Dried and finely powder (500 G) soxhlet apparatus for 2 days..The crude residue of approximately 15 G was obtained after filtration and evaporation of solvent using rotary evaporator. 15 g of extract was treated with the hexane to remove fat and oil. The hexane extract was (6 Gm) was resolved in the by silica gel column chromatography (100 – 200 mesh, Merck, toluene, ethyl acetate, methanol gradients) and 30 fractions were collected. TLC was prepared using Silica gel 60 F 254 (Merck, Darmstadt, Germany) precoated plates (10 * 10 cm) were taken and the solvent system used was toluene: ethyl acetate: methanol in the ratio of 5:3:2(v/v/v).From 1000 µG/mL of around 10 µL and 20µL of the samples applied to the precoated TLC plates. The movement of the samples was found to be ascending and it moved up to 6cm. The developed plates were scanned under UV transilluminator at 264 nm.
3.3 Isolation and purification of Quercetin.
The confirmation of the bioactive compound was ascertained by C1 and H13 NMR using Bruker Ultrashield 600 WB PLUS.
3.4 Synthesis of the PLGA microsphere (Bare and Loaded)
PLGAh Microspheres were prepared by double emulsification method. Approximately, 500 mg PLGA in the ratio 50:50 was added to 8 mL of dichloromethane (CH2Cl2) and 1 mL of heparin. The mixed solution was further emulsified by vortexing for 40 sec. Proper care was taken to ensure the mixing of heparin. The primary solution was added drop wise using a 2G syringe into 100 mL of 1 % of PVA aqueous solution, which was mixed for 2 minutes at 9500 rpm with a turbine homogenizer resulting in a secondary emulsion. To this emulsion was further added 100 ml of 2 % isopropanol aqueous solution which was magnetically stirred at for 4 hours at 300 rpm. The beads were magnetically stirred for 4 hours and centrifuged 3 times at 2000 rpm in Eppendorf Centrifuge 5430 R machine. After the complete removal of solvent by decanting the supernatant the beads were freeze dried for 3 hours and stored for further use. The PLGAh-Q was also prepared in the above method by taking 20 mg/mL of Quercetin.
3.5In vitro anti-cancer activity
3.5.1 Cell line and culture medium
The Human monocytic cell lines and (THP-1) and Michigan cancer foundation cell lines (MCF-7) were obtained from National Center for cell science, Pune, India. The cells were cultured under standard conditions in DMEM medium, supplemented with 10% of FBS, 100U/mL of streptomycin and 100U/mL of penicillin in a humidified incubator set at 37 °C with 5% of CO2.The cells were subcultured after formation of the monolayer. The cells were detached by treating with trypsin (0.25% trypsin containing 0.01% EDTA) for 2 min and washed with PBS for further analysis.
3.5.2 Cytotoxicity assay using THP-1 and MCF-7 Human monocytic cell lines (THP-1) and Michigan cancer foundation (MCF-7) (5×103 cells/well) were incubated with various concentrations (0.6-10 µg/mL) of PLGAh. The culture media were removed and the cells were incubated with 5 mg/mL MTT in fresh medium at 37 °C for an additional 4 hrs. After this period, the supernatants were removed and 100 µL DMSO was added to each well to dissolve the formazan crystals. The plates were read on a micro plate reader at a test wavelength of 540 nm and a reference wavelength of 650 nm.
3.5.3 Annexin V / Propidium iodide double staining assay
Annexin V-FITC apoptosis detection kit (Calbiochem, CA, USA) was used to determine the number of apoptotic cells. According to the instruction of the manfacturer the treated cells (MCF-7) (1–2 × 105cells/well) were incubated at 37 °C with 5 % CO2 for 24 hours in the presence or absence of carrier PLGAh / PLGAh+Q (10 and 15 μM/L). Cells were trypsinized, washed twice with Hanks balanced salt solution (HBSS), suspended in 100 μL Annexin V binding buffer and then incubated for 20 min, in 5 μL of Annexin V Fluorescein isothiocyanate (FITC) / Propidium iodide. 1 μL of 1 mg/mL Propidium iodide solution was added to each cell suspension and were run immediately in flow cytometer (FACS Jazz Becton Dickinson, USA) and analyzed in Submit software
3.6 Characterization of microspheres
The surface morphology of the PLGAh and PLGAh-Q was determined using Field Emission Scanning Electron Microscope (FESEM-SUPRA 55) - Carl Zeiss, Germany.
4. Results
4.1 Column chromatography
The column chromatography was performed using different concentrations of elution solution and the column is eluted in increasing order of polarity of solvent from 100 % of extract, 25 % of ethyl acetate in hexane, 50 % of ethyl acetate in hexane, 75 % of ethyl acetate in hexane and 100 % of ethyl acetate. Then the polarity was increased using 2 % Methanol in Ethyl acetate to 50 % Methanol in ethyl acetate. 8 % of methanol and 92 % of ethyl acetate concentration was found to be the best concentration and after concentrating all fractions of these concentrations, approximately 7 to 8 mg of quercetin was obtained.
4.2 Thin layer chromatography
TLC profiles were carried out for the fractions, performed using the solvent mixture of toluene: ethyl acetate: methanol in the ratio of 5:3:2 (v/v/v) and this mobile phase enabled excellent separation of the bioactive compound. The results showed that there was good amount of correlation between the standard quercetin band and the band of bioactive sample plates when visualized under UV at 254 …show more content…
nm.
4.3 Morphology of microspheres
The FE-SEM micrograph provides an insight on the morphology of synthesized microspheres which showed PLGAh microspheres which are spherical in shape, PLGAh-Q showed high amount of porosity and the spheres were mono dispersed. The size of the spheres of PLGAh was found to be 36.20 µm and PLGAh-Q were 90.16 µm. This result showed that it is feasible to load the drug for optimum drug delivery to the system. The SEM images of Rifamycin / PLGA micro sphere which were also found to be spherical and mono dispersed and were prepared by the four fluid nozzle spray. Here the Rifamycin loaded microsphere was used for the anti-tuberculosis activity [21]. Another study revealed the SEM size of the periyl alcohol – bearing PLGA micro particles of were 768 ± 215 nm. These particles were prepared by oil-in-water-based emulsion solvent evaporation technique [22] Porous and non-porous PLGA microspheres prepared by oil/water emulsion technique, showed that the porous microspheres were bigger and size had high density of pores whereas the non-porogen microspheres were found to be smaller in size with less pores [23]. Studies on SEM images have also revealed that novel controlled release formulation for the anticancer drug paclitaxel in PLGA nanoparticles containing vitamin E showed the spherical shape with various degree of smoothness in the microsphere [24].
4.4 Cytotoxicity assay in THP-1 (MTT)
The cytotoxicity assay was performed in THP -1 (Human monocytic cell) lines for evaluating the toxicity of PLGAh carrier the normal cell lines and to evaluate the dosage beyond which the carrier is toxic to the cell lines. It was found that the 80 % cells are viable in the range of 0.625 µg / mL to 5 µg / mL. From these range the dosage of carrier was decided. This study shows the cytotoxicity assay done curcumin loaded PLGA nanoparticles prepared by single emulsion–solvent evaporation method showed destruction of amyloid aggregates and no cytotoxic effect was found in the formulation as 70 % of cells were found to be viable [25].
4.5 Cytotoxicity assay (MCF-7) MTT The PLGAh carrier and the PLGAh-Q carrier were tested for their anti- cancer activities against the Michigan cancer foundation (MCF-7) cancer lines. In the study it was found out that the PLGAh carrier has very less or negligible amount of anti-cancer activity but in comparison the PLGAh+Q carrier displayed excellent anti-cancer activity by decreasing the cell viability to 40 % in the range of concentration of 0-2 µg / mL. The inhibition concentration (IC 50) value was found in the concentration of 3.648 µg. So comparing the above, cytotoxicity of PLGAh carrier in normal cell lines and cytotoxicity of the PLGAh-Q carrier in the cancer cell lines the range of 1.5 µg and 3.0 µg concentrations was found to be optimum for anti-cancer activity. Studies revealed that cell viability assay in pancreatic cell lines treated in various concentrations of free curcumin and nanocurcumin. It was found that the nanocurcumin showed high cytotoxicity to pancreatic cell lines in higher concentration of 15 µM [26]. It is also known that the quercetin enhances anti-cancer activity of trichostatin A through up regulation of p53 protein as Chan and co-worker have studied both in in vivo and in vitro [27]. The PLGA- curcumin Nano formulation was targeted on to MCF-7 breast cancer cell lines by Verderio and group and they observed high toxicity to cancer cells showing potential anti-cancer activity [6]. Other studies have also indicated that cell proliferation has decreased significantly in PLGA-curcumin formulation when was subjected to human colon cancer cell lines 116 in comparison to native curcumin within 6-24 hours [28].
4.6 Annexin v / propidium iodide double staining assay staining
Apoptosis is the mechanism of programmed cell death which occurs in eukaryotic organisms [29] has promoted active research into new methods of detecting these subcellular lesions at the single-cell level in complex cell mixtures both in vivo and in vivo cultured cells [30], [31], [32].
The PLGAh and PLGAh+Q carrier was tested by these assay for rate of apoptosis in the in Michigan cancer foundation (MCF-7) cell lines. The control was the normal cancer cell lines without any PLGAh and PLGAh-Q carrier. The PLGAh carrier was tested in assay in two different concentrations which was found above 1.5 µg and 3.0 µg. The summation of early and late apoptosis was just 8 % in concentration of 1.5 µg and in concentration of the 3.0 µg it was found to be 15 %. But PLGAh-Q in the concentration of 1.5 µg showed approximately 45 % of apoptosis (early+ late). But when concentration was increased to the 3.0 µg the apoptosis was (early + late) found to be 60 %. So it is concluded that PLGAh carrier in various concentration (1.5 µg and 3.0 µg) does not show any significant apoptosis but the PLGAh-Q carrier showed increase in apoptotic activity by increasing the concentration of drug from 1.5 µg to 30 µg. Studies have indicate that the viability of cancer cell decreased when it was given silver nanoparticles in different concentration and the confirmation of different stages of apoptosis were observed by separating the live cell and dead cells by annexin V / PI staining which also
determined the % of apoptosis [33]. During early apoptosis membrane blebbing of cells occur so phosphatidyl serine comes out from the inner side of the plasma membrane to the outer membrane leaflet, where it becomes exposed [34] to the available of fluorochrome-labeled recombinant soluble annexin V which is an useful tool for detecting and quantifying early apoptotic cells by flow cytometry [31], [35], [34]. It is well known that quercetin induces apoptosis by activating caspase-3 and regulating B cell lymphoma-2 (Bcl-2) and cyclooxygenase-2 pathways as Niu and co-workers have studied its effects on human leukemia cell lines [36]. Studies have also been carried out on the effects of intracellular drug release from Curcumin-Loaded PLGA Nanoparticles and it was concluded that it Induces G2/M Block in Breast Cancer Cells and it was confirmed by 7-AAD/Annexin-V double staining and a prominent increase in death of cancer cell was observed [6]. It has also been studied that low concentration of curcumin PLGA formulation is far more effective than native curcumin and showed enhanced anti-cancer activity by initiating apoptosis of the human colon cancer lines [28].
5. Conclusion
In this study, the effect of PLGAh-Q microspheres on cancer cell lines has been evaluated. Allium fistulosum was used to extract quercetin and purified using chromatographic techniques. With the help of 1H and 13C NMR, the isolated compound was confirmed as quercetin. The electron micrographs of PLGAh and PLGAh-Q microspheres suggest that the particles are found to have spherical shaped structures for PLGAh microspheres and PLGAh-Q microsphere shows pores on the surface suitable for drug delivery studies. The PLGAh-Q showed excellent anti-cancer activities tested using Annexin–V FITC/PI staining. These results suggest that PLGAh-Q can be potent anticancer agent and tested further on other cell lines for development of novel anticancer agents.