Extraction of the active compounds of Chenopodium quinoa Willd. was performed using a percolation method with 95% ethanol. Both plant samples were treated separately. The plant matrix was suspended in the solvent for 24 h, and then the extracts were drained at a flow rate of 60 drops/min. Finally, the ethanol was evaporated with a rotary evaporator system (BÜCHI).
Rotary evaporator system
Manufactured by Büchi
Sourced in Switzerland
The Rotary Evaporator System is a laboratory equipment designed to efficiently remove solvents from liquid samples through the process of evaporation. It consists of a rotating flask that is immersed in a heated water bath, which facilitates the evaporation of the solvent. The system also includes a condenser that collects and recovers the evaporated solvent, allowing for its reuse or proper disposal.
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6 protocols using rotary evaporator system
1
Extraction of Chenopodium quinoa Compounds
2
Extraction and Preparation of Herbal Formulation
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Dried forms of Mori Fructus [Sangsimja in Traditional Korean Medicine (TKM)], Atractylodis Rhizoma (Changchul in TKM), and Lycii Radicis Cortex (Jigolpi in TKM) were purchased from the Department of Medicine, Dongguk University International Hospital (Goyang, Republic of Korea). Extraction and preparation of the herbal formulation were performed according to our laboratory-optimized procedure with modifications. Briefly, these three herbs were ground separately into powder form which was then combined together (3:1:1, w/w, respectively). The resultant mixture was subjected to reflux extraction with 10 vol. of 30% ethanol for 1 h, followed by evaporation at 95 °C using a rotary evaporator system (Buchi, Flawil, Switzerland) The extract was filtered through a 0.2 μm syringe filter (Merck Millipore, Billerica, MA, USA) and concentrated by vacuum evaporation at 60 °C. The final product was stored at −20 °C prior to experimental use.
3
Extraction and Characterization of B. racemosa Plant Parts
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The plant materials of B. racemosa were collected from Nasuha Herbal Farm, Johor, Malaysia. The determination of botanical term for plant parts used was made by referring to glossary of botanical terms [16 ] and Prance [17 ]. Four plant parts were studied which were infloresence axis, leaf [Figure 1 ], endosperm, and pericarp part of fruits [Figure 2 ]. The samples were air dried under the shade at ambient temperature ranging from 28°C to 32°C. The dried plant samples were further crushed into coarse powder using a domestic electric grinder (Philips, Netherlands). The extracts were prepared by soaking 5 g of samples in 200 ml of 70% methanol (Merck, Germany) diluted with 30% deionized water (Arium® pro Sartorius, Germany). The macerated samples were filtered using Whatman filter paper Number 1 (70 mm) (GE Healthcare, UK) placed on Buchner funnel whereby the filtration was assisted by a vacuum pump (GAST, USA). The resulting filtrates were then concentrated under vacuum using rotary evaporator system (Buchi, Switzerland) to yield concentrated extracts of samples. The resulting extracts were kept in glass vials and refrigerated at 4°C until further use.
4
Extraction and Characterization of Medicinal Plant Extracts
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Coltsfoot leaves, common butterbur roots teas (produced by Stef Mar, Ltd., Ramnicu Valcea, Romania) and comfrey roots tea (produced by Fares, Orastie, Romania) were purchased from retail stores. Senecio vernalis aerial part was harvested in May 2013, from Craiova Botanical Garden (Craiova, Romania), naturally dried and conserved in laboratory conditions. The morphological characters of the vegetal material were compared with the ones quoted by literature. A voucher specimen is available in the collection of the Department of Botany and Cell Biology, ‘Carol Davila’ University of Medicine and Pharmacy (Bucharest, Romania), and at ‘Dimitrie Brandza’ Botanical Garden (Bucharest, Romania; no. 405789).
The dry extracts were obtained as previously described (24 ). Briefly, the dried plants were ground (Tyler mesh 48), and 20 g of each plant material were refluxed twice for 2 h with 1,000 ml of 50% methanol acidified with citric acid to pH 2.0–3.0. The combined extracts were evaporated, under reduced pressure with a rotary evaporator system (Buchi, Flawil, Switzerland) to about 300 ml and atomized with a Mini Spray Dryer B-290 (Buchi). The extracts were coded as follows: SEN (Senecio vernalis), SYM (Symphytum officinale), PET (Petasites hybridus) and TUSS (Tussilago farfara).
The dry extracts were obtained as previously described (24 ). Briefly, the dried plants were ground (Tyler mesh 48), and 20 g of each plant material were refluxed twice for 2 h with 1,000 ml of 50% methanol acidified with citric acid to pH 2.0–3.0. The combined extracts were evaporated, under reduced pressure with a rotary evaporator system (Buchi, Flawil, Switzerland) to about 300 ml and atomized with a Mini Spray Dryer B-290 (Buchi). The extracts were coded as follows: SEN (Senecio vernalis), SYM (Symphytum officinale), PET (Petasites hybridus) and TUSS (Tussilago farfara).
5
Liposome Preparation and Extrusion
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DSPC, DSPG, and cholesterol
were added into a 50 mL round-bottom flask at a 1:1:1 mol/mol/mol
ratio. Dried thin films were created by heating the round-bottom at
50 °C, spinning, and pumping the pressure down to <25 mTorr
for at least 1 h using a rotary evaporator system (Buchi). For NPs
used in confocal microscopy, 0.4 mol % DSPE-Cy5 lipid was added to
replace DSPC, resulting in a 32.9:33.3:33.4:0.4 DSPC/DSPG/Chol/DSPE-Cy5
molar ratio.
After the evaporation of all organic solvents,
the round-bottomed flask was partially submerged in a sonicator bath
(Branson 1800 Ultrasonic Bath) filled with 65 °C water, and the
thin film was resuspended at 1 mg/mL using fresh 65 °C milli-Q
water while sonicating. The resulting colloidal suspension was extruded
at 65 °C by using an Averin LipsosFast LF-50 liposome extruder.
Liposomes were first passed three times through a 200 nm filter, then
three times through a 100 nm filter, and finally at least once through
a 50 nm filter.
were added into a 50 mL round-bottom flask at a 1:1:1 mol/mol/mol
ratio. Dried thin films were created by heating the round-bottom at
50 °C, spinning, and pumping the pressure down to <25 mTorr
for at least 1 h using a rotary evaporator system (Buchi). For NPs
used in confocal microscopy, 0.4 mol % DSPE-Cy5 lipid was added to
replace DSPC, resulting in a 32.9:33.3:33.4:0.4 DSPC/DSPG/Chol/DSPE-Cy5
molar ratio.
After the evaporation of all organic solvents,
the round-bottomed flask was partially submerged in a sonicator bath
(Branson 1800 Ultrasonic Bath) filled with 65 °C water, and the
thin film was resuspended at 1 mg/mL using fresh 65 °C milli-Q
water while sonicating. The resulting colloidal suspension was extruded
at 65 °C by using an Averin LipsosFast LF-50 liposome extruder.
Liposomes were first passed three times through a 200 nm filter, then
three times through a 100 nm filter, and finally at least once through
a 50 nm filter.
6
Liposome Entrapment Efficiency Comparison
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In order to compare the entrapment efficiency of our DAC-produced liposomes with a more well-known method, liposomes of the same composition as for the ones produced by DAC were prepared by probesonication. The lipid-drug films were prepared using a rotary evaporator system (Büchi Labortechnik AG, Flawil, Switzerland) with a vacuum pump (20 minutes at 150 mbar, followed by 1 hour at 50 mbar) and a water bath (45 ± 1 °C) to remove the organic solvents. Dried films were then hydrated into more dilute liposome dispersions (10 mg/ml PC) with PBS pH 7.4 and 2 mL samples were sonicated (40 % amplitude) for 2 x 2 minutes on ice bath using a GEX500 high intensity ultrasonic processor (Sonics & Materials Inc., Newtown, USA) with a 19 mm probe.
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