Alkaloids are natural nitrogen-containing organic compounds, but do not include amino acids, proteins, nucleic acids, chlorophyll, choline, methylamine and other open-chain simple fatty amines. Its nitrogen atom is often on the ring, alkaloids have complex ring structure and strong physiological activity. Most of the alkaloids in plants have obvious physiological activities. For example, they are antibacterial, anti-inflammatory, analgesic, antitussive, anticancer, antitoxic shock, etc. They are often used as medicinal herbs. Determination of total alkaloids in plants can help to control the quality of Chinese herbal medicines, reflect the therapeutic efficacy of herbal medicines, and provide a good applied basic research for the development of health food and natural preservatives.
Principle
The basic principle of the determination of plant alkaloid content is that most of alkaloids are soluble in organic solvents, and only a few are soluble in water. In the plant body, alkaloids are often combined with acid to form salts, so when extracting, the plant material should be mixed with a small amount of alkali (e.g. 10% ammonia), so that the alkaloids can be converted into free state and then extracted with organic solvents. The main methods of determination are titration (direct titration and back titration), acid dye colorimetry and derivative spectrophotometry. The acid dye colorimetric method has been widely used in the literature and is considered to be the preferred method. The dyes used in the literature include olfactory cresol green, olfactory cresol blue, etc., and there are many kinds of buffers. In this experiment, the spectrophotometric method was used for the determination of total alkaloids in lotus leaves, using olfactory cresol green buffer as the color developer and hordenine as the control.
Operation method
Determination of plant alkaloid content
Principle
The basic principle of the determination of plant alkaloid content is that most of alkaloids are soluble in organic solvents, and only a few are soluble in water. In the plant body, alkaloids are often combined with acid to form salts, so when extracting, the plant material should be mixed with a small amount of alkali (e.g. 10% ammonia), so that the alkaloids can be converted into free state and then extracted with organic solvents. The main methods of determination are titration (direct titration and back titration), acid dye colorimetry and derivative spectrophotometry. The acid dye colorimetric method has been widely used in the literature and is considered to be the preferred method. The dyes used in the literature include olfactory cresol green, olfactory cresol blue, etc., and there are many kinds of buffers. In this experiment, the spectrophotometric method was used for the determination of total alkaloids in lotus leaves, using olfactory cresol green buffer as the color developer and hordenine as the control.
Materials and Instruments
Materials: Lotus leaves (commercially available) and other plant materials. Move The basic procedure for the determination of plant alkaloid content can be divided into the following steps:1 . Preparation of control solution: Weigh about 10 mg of Hollandine in a 25 mL volumetric flask, add trichloromethane to the graduated scale, shake well, suck 2.5 mL in a 25 mL volumetric flask, add trichloromethane to the graduated scale, shake well, and spare.2. Preparation of test solution: take the powder of lotus leaf crushed to a certain extent, extracted with aqueous hydrochloric acid pH 3 ~ 4 at constant temperature for 10 h, filtration, get reddish-brown slightly viscous filtrate, the first filtrate is concentrated to a certain volume in a vacuum, and then filtered, the second filtrate is extracted with chloroform for two times, in order to remove the fat cones and resinous impurities, the aqueous layer of the pH of 6 ~ 7 filtration in order to remove the blended substances and boot acid impurities, continue to add alkaline salt impurities, continue to add alkaline salt, and then add alkali. Filter the aqueous layer to pH 6~7 to remove blends and bootstrap acid salts, continue to add alkali to pH 9 or so to obtain the lotus leaf alkaloid extract, and then evaporate and concentrate it into 10 mL of concentrated solution by rotary evaporator, and then leave it for use.Or take lotus leaves, crushed into coarse powder, weigh about 4 g in Soxhlet extractor, add 80 mL of trichloromethane, heat reflux extraction for 4 h, cooled, filtered, evaporated, residue with trichloromethane dissolved in batches into a 100 mL volumetric flask, add trichloromethane to the scale, shaking well, precision aspiration of 1 mL in a 25 mL volumetric flask, add trichloromethane to the scale, shaking well, standby. It can also be extracted by ultrasonic wave, the better extraction conditions are 65% ethanol, material-liquid ratio 1:50 (m/V), pH 5, ultrasonic frequency 45 kHz, ultrasonic time 0.5 h, ultrasonic temperature 80°C. The extract can be extracted by ultrasonic wave.3. Preparation of Desiccated Cresol Green buffer: 125 mg of Desiccated Cresol Green (alias: Desiccated Cresol Blue) was weighed precisely and dissolved in 0.2 mol - L 1 NaOH solution 12.5 mL. Add potassium hydrogen phthalate 2.50 mg, add a small amount of water to dissolve, transfer to 250 mL volumetric flask, add water to the scale, shake well, spare. (Note: 0.05% desert muscovite blue buffer can be replaced)4. Standard curve production: respectively, 0, 0.5, 1.0, 1.5, 2.0, 2.5 mL of the control solution in a test tube (or 5 mL volumetric flask), add trichloromethane to 5 mL, shaking, transferred to the dispensing funnel, add 0.2 mol - L 1 NaOH solution and desert cresol green buffer each 1 mL, shaking, standing, take the clarified trichloromethane solution at 415 nm wavelength for determination. The concentration of trichloromethane was measured at 415 nm, and the regression equation was obtained by taking the concentration as the vertical coordinate and the absorption value as the horizontal coordinate, and then the regression equation was obtained by taking the concentration as the vertical coordinate and the absorption value as the horizontal coordinate.5. Determination of total alkaloids in lotus leaves: Take three batches of lotus leaves, use trichloromethane plus Cresol green buffer and 0.2 mol - L 1 NaOH solution as blank, according to the procedure of determining the content of 4 standards, substitute the colorimetric results into the regression equation to calculate the total alkaloids content of lotus leaves.6. Calculation of results:c X VAlkaloid content (卩 mol・g・) = - ___.Where: c i Alkaloid concentration from the standard curve, 卩 mol - mL'1;V a sample volume,mL;W - sample weight, go Caveat 1. Different plants have different alkaloid species and their extraction methods should be different. 2. If different color developing dyes are selected, the determination wavelength needs to be re-scanned. For more product details, please visit Aladdin Scientific website.
Equipments: Visible-ultraviolet spectrophotometer, electronic balance, R201 rotary evaporator.
Reagents:
①Holcimine Control
② Bun
③Trichloromethane
③Trichloromethane
⑤Potassium hydrogen phthalate
⑥ Sodium hydroxide (commercially available in analytical purity)