Surface-sterilized seeds were germinated in sterile petri dishes lined with moistened Whatman filter paper number 2, as described by Khan et al. (2016). After seed germination for 1 week, equally sized seedlings (5 per Petri dish) were transferred to new sterilized Petri dishes containing a sterile Whatman filter paper moistened with sterile distilled water. The RSC-14-inoculated treatment received 5 mL of an RSC-14 suspension with an optical density at 600 nm (OD600) of 0.5. The seedlings were allowed to grow in a growth chamber with a 14-h photoperiod (350 μmol m−2 s−1) at 25°C and 60% relative humidity for 15 days, and the root/shoot lengths were measured on the final day of harvesting.
Whatman number 2 filter paper
Manufactured by Cytiva
Sourced in United Kingdom
Whatman number 2 filter paper is a general-purpose filter paper designed for a variety of filtration applications. It is made from high-quality cellulose fibers and is suitable for the filtration of both aqueous and organic solutions. The paper has a medium-fast flow rate and a nominal retention rating of 8 microns, making it suitable for the removal of fine particulates.
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23 protocols using whatman number 2 filter paper
1
Seed Germination and Growth Assay
2
Extraction and Analysis of Basil and Rosemary
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Two grams (2.0 ± 0.05 g) of powdered samples were extracted with 20 mL of nano-pure water. The suspension was heated to the boiling point, and results were observed at 5, 10, and 15 min. The boiling time was selected based on the results of pre-trials. The pre-trials were conducted for different boiling times and data were obtained for TPC and the reducing power. The boiling times which were suitable for both the basil and the rosemary were selected for this study. Once the boiling was finished for their respective time, the mixture was cooled at room temperature. The obtained mixture was then centrifuged at room temperature for 10 min at 3000× g (HERMLE Labortechnik GmbH. Siemensstr. 25 D-78564 Wehingen, Germany) and filtered using Whatman filter paper number 2. The obtained extracts were stored at 4 °C and used for analyses.
3
Quantification of Starch and Sugars
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Starch and sugar were extracted from 0.02 g of the pulverized sample using 80% hot ethanol. The mixture was then centrifuged at 2000 rpm for 10 min after which the supernatant was decanted and used for free sugar analysis, while the residue was used for starch analysis [17 ]. For sugar analysis, 0.2 mL of the diluted supernatant was mixed with 0.5 mL of phenol solution (5%) and 2.5 mL of H2SO4 (absolute). The mixture was allowed to cool to room temperature before reading the absorbance at 490 nm. The residue was hydrolyzed with 7.5 mL of perchloric acid for 1 hr, diluted to 25 mL with distilled water, and filtered through Whatman filter paper (number 2). Then 0.05 mL of the filtrate was mixed with 0.5 mL of phenol solution (5%) and 2.5 mL H2SO4 (absolute). The mixture was allowed to cool to room temperature and the absorbance was read at 490 nm. Starch and total free sugar contents of the sample were calculated from a glucose standard curve prepared along with the sample.
4
Extraction of Leaves from C. tricuspidata
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C. tricuspidata leaves were obtained from traditional Korean medical center (Goseong, Gangwon-do, Korea), which were pulverized using a blender. The pulverized leaves were subjected to reflux extraction twice using 20 times its volume of distilled water at 100°C for 8 h. The extracts were filtered using Whatman filter paper number 2 (pore size; 8 μm), concentrated by using a rotary vacuum evaporator (EYELA, Tokyo, Japan), and lyophilized using a freezer dryer (PVTF20R, Ilshinbiobase, Dongduchun, Korea). CTe extraction and processing were performed in a good manufacturing practice (GMP) facility according to industry standards. The CTe yield was 28.7%.
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C. tricuspidata leaves were obtained from traditional Korean medical center (Goseong, Gangwon-do, Korea), which were pulverized using a blender. The pulverized leaves were subjected to reflux extraction twice using 20 times its volume of distilled water at 100°C for 8 h. The extracts were filtered using Whatman filter paper number 2 (pore size; 8 μm), concentrated by using a rotary vacuum evaporator (EYELA, Tokyo, Japan), and lyophilized using a freezer dryer (PVTF20R, Ilshinbiobase, Dongduchun, Korea). CTe extraction and processing were performed in a good manufacturing practice (GMP) facility according to industry standards. The CTe yield was 28.7%.
5
Extraction of Soluble Compounds from Desalinated Cherry Blossom Petals
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Salted cherry blossom petals were washed, and distilled water (20×) was added. After soaking at 25 °C for 30 min, the distilled water was replaced, and the procedure was repeated four times. Then, these desalinated cherry blossom petals were mixed with a cellulose softening agent (2×). Soluble components were extracted at 80 °C for 90 min. The extract was filtered using Whatman filter paper number 2 (Whatman, Maidstone, UK) and then freeze-dried (Figure 1 ).
6
Microencapsulation Efficiency of Phenolic Compounds
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The TPC and SPC of the extract and powder was determined by the Folin-Ciocalteu method [4] (link) with some modifications. The absorbance of the solution was measured at 760 nm using a spectrophotometer (1205 Vis Spectrophotometer UNICO). The results were expressed as µg of equivalent gallic acid (GAE) per gram of microcapsules (powder). All analyzes were done in triplicate. For the determination of the surface phenolic content (SPC), 24 mg of microcapsules were dissolved in 4.5 mL of methanol and stirred using a vortex for 1 min and then filtered through a Whatman filter paper number 2. The surface phenolic content was measured according to the same method described for TPC determination.
The percentage of efficiency of TPC microencapsulation was calculated using the following equation: Percentage Efficiency (%) = [(TPC) -(SPC)/TPC] × 100.
The percentage of efficiency of TPC microencapsulation was calculated using the following equation: Percentage Efficiency (%) = [(TPC) -(SPC)/TPC] × 100.
7
Evaluating Alfalfa Polyphenols and Antioxidants
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Based on the nutritive values, the autumn sample was selected for further determination of total polyphenol, total flavonoid, diphenylpicryl hydrazine (DPPH) scavenging, and reduction power. For extraction, 1 g dried alfalfa powder was extracted with 20 mL of either absolute ethanol or distilled water using a shaker (200 rpm) at 25 °C for 1 h. Then the mixture was centrifuged at 6000 rpm for 10 min at room temperature. The supernatant was filtered using a Whatman filter paper number 2 and the obtained extract was used for further analyses.
The total polyphenol content (TPC) was determined according to the method of Hayat (2020) . In summary, 25 μL extract were mixed with 1500 μL water and then 125 μL reagent (undiluted Folin Ciocalteau) was added to the mixture. After 1 min incubation, 375 μL 20% sodium carbonate were added and the final volume of the mixture was made to 2500 μL by adding 475 μL water. Spectrophotometer (Jenway 6705 UV/Vis., Cole-Parmer, Staffordshire, UK) was used to determine absorbance at 760 nm after 30 min incubation time at room temperature. Gallic acid (gallic acid equivalents, GAE) was used as standard to express TPC per gram dry weight of the sample (mg GAE g -1 DW).
The total polyphenol content (TPC) was determined according to the method of Hayat (2020) . In summary, 25 μL extract were mixed with 1500 μL water and then 125 μL reagent (undiluted Folin Ciocalteau) was added to the mixture. After 1 min incubation, 375 μL 20% sodium carbonate were added and the final volume of the mixture was made to 2500 μL by adding 475 μL water. Spectrophotometer (Jenway 6705 UV/Vis., Cole-Parmer, Staffordshire, UK) was used to determine absorbance at 760 nm after 30 min incubation time at room temperature. Gallic acid (gallic acid equivalents, GAE) was used as standard to express TPC per gram dry weight of the sample (mg GAE g -1 DW).
8
Enhancing Seed Germination with Nano-Silver and Biofertilizer
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The surfaces of the seeds were sterilized by using 0.5% (v/v) sodium hypochlorite for 20 min, and then they were rinsed several times with deionized water (Habib, Kausar & Saud, 2016 (link)). As a control (T1), seeds were soaked in distilled water for 48 h. Seeds in sample T2 were soaked in 40 mg l−1 AgNPs (based on the MIC results) for 48 h at 31 °C. For sample T3, seeds were soaked in 40 mg l−1 AgNPs for 48 h at 31 °C, and then kept in bio fertilizer containing bacteria UPMR bio1 and UPMR bio2 for 20 min. For sample T4, seeds were soaked in 40 mg l−1 nano-silver for 48 h at 31 °C, and then placed in a culture of R. solanacearum for 20 min. For sample T5, seeds were soaked in 40 mg l−1 nano-silver for 48 h at 31 °C, followed by soaking in a culture of R. solanacearum for 20 min, and then kept in bio fertilizer for 20 min. Finally, two moist sterilized sheets of Whatman number 2 filter paper (Whatman International Ltd, Maidstone, England) were placed in each petri dish (9 mm diameter). After the seeds had been treated they were placed in these petri dishes. Four seeds were placed in each petri dish and after 7 days, the percentage of seed germination was calculated (Table 1 ).
9
Extraction of Polyphenol-Rich V. amygdalina Leaves
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The procedure for obtaining polyphenol-rich extract of leaves of V. amygdalina (PEVA) was carried out using standard protocol and as described by Mutiu et al. [15 ] and Comfort et al. [16 ]; V. amygdalina leaves were air-dried and pulverized with an electric pulverizer (DIK-2910, Daiki Rika Kogyo Co. Ltd, Tokyo-Japan). The pulverized leaves were weighed, and the value was recorded. This was further crushed in 80% acetone (1:2 w/v) using a Waring blender (Waring Commercial, Torrington, CT). The sample was homogenized in a Polytron Homogenizer (Glen Mills Inc., Clifton, NJ) for 3 min, and the homogenates were filtered under vacuum using Buchner funnel and Whatman number 2 filter paper (Whatman PLC, Middlesex, UK). The filtrate was concentrated under vacuum using a rotary evaporator (HahnShin Scientific, HS-2005-N) and freeze-dried in a Lyophilizer (Ilshin Lab. Co. Ltd, Seoul, Republic of Korea). The powdered yield that was obtained (PEVA) was weighed and kept in a desiccator until when needed. The percentage (%) yield of PEVA was calculated as shown below;
% yield of PEVA = yield of PEVA ÷ weight of pulverized leaves × 100% [17 ]
The extraction process was repeated for three different samples and the final % yield of PEVA was expressed as mean ± standard error of the mean (SEM) (n = 3).
% yield of PEVA = yield of PEVA ÷ weight of pulverized leaves × 100% [17 ]
The extraction process was repeated for three different samples and the final % yield of PEVA was expressed as mean ± standard error of the mean (SEM) (n = 3).
10
Preparation and Characterization of QRQS Herbal Extract
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The preparation of the QRQS extract followed the standardized process outlined in a previous study [8 (link)]. Briefly, four herbs (listed in Table 1 ) were immersed in water at a 1:10 w/v ratio and subjected to boiling twice, each for a duration of 1 h. Following the addition of an adequate amount of distilled water, the extract was filtered using Whatman number 2 filter paper (Maidstone, UK). The final sample was a clear, brown liquid. Tests conducted for bacteria and heat sources yielded negative results. The extract was initially concentrated at 2.7 g/mL and then diluted with ultrapure water before its application in experiments.Table 1
The composition of QRQS.
| Herb name | Latin name | English name | Use part | Amount (g) |
|---|---|---|---|---|
| Ku shen | Sophora flavescens Aiton | Lightyellow Sophora | Root | 10 |
| Bai hua she she cao | Hedyotisdiffusa Willd. | Hedyoti scorymbosa | Whole herb with root | 10 |
| Pu gong ying | Taraxacum mongolicum Hand.-Mazz. | Mongolian Dandelion | Aerial parts | 10 |
| Cang er cao | Xanthium sibiricum | Cocklebur grass | Stem-leaf | 2.5 |
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