Apr 26, 2026
Preparation of botanical extracts for in vitro and in planta evaluation
- Hemant Joshi1,
- Ashish Ghimire1
- 1School of Agriculture, Far Western University, Tikapur, Nepal
Protocol Citation: Hemant Joshi, Ashish Ghimire 2026. Preparation of botanical extracts for in vitro and in planta evaluation. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoezndl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 25, 2026
Last Modified: April 26, 2026
Protocol Integer ID: 315688
Keywords: reproducible preparation of standardized aqueous plant extract, standardized aqueous plant extract, preparation of botanical extract, planta evaluation this protocol, planta evaluation, botanical extract, planta experimental system, fresh biomass into dry weight equivalent, aqueous extraction, preparing plant, derived extract, extract, crude extract, extraction method, standardized concentration for application, experimental use in biological assay, fresh biomass, moisture content, reproducible preparation, plant material, standardized concentration, variability in moisture content, biological assay, membrane filtration under sterile condition, dry weight equivalent
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Abstract
This protocol was developed to ensure reproducible preparation of standardized aqueous plant extracts for experimental use in biological assays. The need for this protocol arises from variability in moisture content and extraction methods, which can lead to inconsistent concentrations and reduced comparability across studies. The method addresses this by standardizing all extracts on a dry weight basis.
Moisture content is first determined using oven drying to convert fresh biomass into dry weight equivalents for accurate concentration adjustment. Plant material is then homogenized and subjected to aqueous extraction under controlled conditions. The crude extract is clarified through sequential filtration, centrifugation, and membrane filtration under sterile conditions. Stock and working solutions are prepared based on dry weight equivalence and adjusted to a standardized concentration for application.
The protocol provides a consistent and reproducible approach for preparing plant-derived extracts suitable for in vitro and in planta experimental systems.
Guidelines
- Stock solutions are prepared based on the dry weight equivalent of plant material. About 1:1 w/v (fresh weight) for low moisture plant materials and 2:1 or 3:1 w/v for high moisture plant materials such as ginger rhizomes.
- Allow the mixture to stand for approximately 1 hour at room temperature to facilitate the extraction of bioactive compounds.
- Filter the mixture through four layers of muslin cloth to remove coarse plant debris and obtain a crude aqueous extract. Centrifuge the filtrate at an appropriate speed and duration (e.g., 6,000 rpm for 10–15 min) to further clarify the extract and remove fine particulate matter. Collect the supernatant as the clear extract and further pass through a 0.22 μm membrane filter to prevent contamination. Perform all extraction steps under semi-sterile conditions and use the extracts within 6 hours of preparation to minimize degradation of bioactive compounds.
- Prepare a final spray or working solution by diluting the stock solution to maintain a 5% concentration on a dry weight basis. Add a wetting agent (Phytostick) to improve the spray solution's adherence to leaf and fruit surfaces.
Materials
- Ginger rhizome (_Zingiber officinale_)
- Garlic cloves (_Allium sativum_)
- Mugwort leaves (_Artemisia vulgaris_)
- Cinnamon leaves (_Cinnamomum verum_)
- Distilled water
- Oven
- Desiccator
- Electric mixer grinder
- Muslin cloth
- Centrifuge
- 0.22 μm membrane filter
- Wetting agent (Phytostick)
Preparation of botanical extracts for in vitro and in planta evaluation
Collect fresh plant materials such as ginger rhizome (Zingiber officinale), garlic cloves (Allium sativum), mugwort leaves (Artemisia vulgaris), and cinnamon leaves (Cinnamomum verum), used to extract active ingredients.
Clean the materials with distilled water to remove dust and debris.
To express all extract concentrations on a dry weight basis, calculate the moisture content of each botanical sample using the oven-drying method. Weigh approximately 10–15 g of each fresh sample (fresh weight) in four replicates. Place the samples in a hot air oven at 105 °C and dry for 16–18 hours. After drying, cool the samples in a desiccator and record the dry weight. Repeat the drying, cooling, and weighing steps until a constant weight is obtained. Finally, calculate the dry matter content for each replicate and average the values to determine the conversion factor for each botanical.
Calculate the required fresh weight of plant material needed to obtain a dry weight equivalent (w/v) (e.g., 50 g dry matter per liter of water to get 5% solution) using the following formula:
C = desired concentration (%)
V = volume (mL)
D = dry matter percentage (%)
Use the calculated fresh weight to ensure consistent dosing across all botanical treatments regardless of moisture variability.
Clean the required quantity of fresh plant material and chop it into small pieces. Homogenize the material using an electric mixer grinder to obtain a fine paste.
Add the appropriate volume of distilled water and mix thoroughly to achieve the desired concentration. Prepare concentrated stock solutions using minimal distilled water.
Stock solutions are prepared based on the dry weight equivalent of plant material. About 1:1 w/v (fresh weight) for low moisture plant materials and 2:1 or 3:1 w/v for high moisture plant materials such as ginger rhizomes.
Allow the mixture to stand for approximately 1 hour at room temperature to facilitate the extraction of bioactive compounds.
Filter the mixture through four layers of muslin cloth to remove coarse plant debris and obtain a crude aqueous extract. Centrifuge the filtrate at an appropriate speed and duration (e.g., 6,000 rpm for 10–15 min) to further clarify the extract and remove fine particulate matter. Collect the supernatant as the clear extract and further pass through a 0.22 μm membrane filter to prevent contamination. Perform all extraction steps under sterile conditions and use the extracts within 6 hours of preparation to minimize degradation of bioactive compounds.
Prepare a final spray or working solution by diluting the stock solution to maintain a 5% concentration on a dry weight basis. Add a wetting agent (e.g., Phytostick) to improve the spray solution's adherence to leaf and fruit surfaces.