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PHYTOCHEMICAL Screening of crud drugs.pptx
- 1. PHYTOCHEMICAL SCREENINGOFCRUDE DRUGS By: Jyoti Singh Ph.D. Scholar Department of Rasa Shastra and Bhaishajya Kalpana, Faculty of Ayurveda, IMS, BHU
- 2. Phytochemicals are chemical compounds produced by plants, generally to help them resist fungi, bacteria and plant virus infections, and also consumption by insects and other animals.
- 3. Types of Metabolites Primary metabolites Secondary metabolite Primary Metabolites Required for growth and maintenance of cellular function. Same in every species. Ex: Carbohydrates, vitamins etc. Secondary Metabolites Derivatives of primary metabolites. Different in every species. Produced in small amounts. Eg: Phenolics, steroids, alkaloids, flavanoids etc.
- 4. Quality Evaluation of Herbal Drugs • Moisture content • Extractive value • Ash value • Fluorescence Analysis • Foreign matter • Qualitative • Quantitative • Chromatography • Heavy metal • Colour • Odour • Taste • Texture • Fracture Microbial contamination • Toxicological • Pharmacological • Other specific activities • Bacterial • Fungal Macroscopic Microscopic • Qualitative • Quantitative • Powder studies • Shape • Size • External • Marking
- 5. 5 Chemical evaluation This covers screening, isolation, identification and purification of the chemical components. Chemical analysis of the drug is done to assess the potency of material in terms of its active principles.
- 7. PRELIMINARYPHYTOCHEMICAL SCREENING Preliminary screening of phytochemicals is a valuable step, in the detection of the bioactive principles present in medicinal plants and subsequently may lead to drug discovery and development. 7 Qualitative phytochemical screening Quantitative phytochemical screening
- 9. FLUORESCENCE ANALYSIS The plant drug showed different colors under day light and UV light (at 254 nm and 365 nm) after treated with various chemical reagents. 9
- 10. Alkaloid Determination 5g of the sample were weighed into a 250ml beaker and 200ml of 20% acetic acid in ethanol was added and covered to stand for 4 hours. This was filtered and the extract was concentrated using a water-bath to one quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the preparation was complete. The whole solution was allowed to settle and the precipitate was collected by filtration and weighed . Phase 2 Month, Year Phenol Determination For the extraction of the phenolic component, the fat free sample was boiled with 50 ml of ether for 15 minutes. 5 ml of the extract was pipette into a 50 ml flask, and then 10 ml of distilled water was added, 2 ml of ammonium hydroxide solution and 5 ml of the extract was pipette into a 50 ml flask, and then 10 ml of distilled water was added, 2 ml of ammonium hydroxide solution and 5 ml of concentration amyl alcohol were also added. The samples were left to react for 30 minutes for color development. The absorbance of the solution was read using a spectrophotometer at 505 nm wavelengths. Flavonoid Determination 100g of the plant sample were extracted repeatedly with100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No. 42 (125mm). The filtrate was later transferred into a crucible and evaporated to dryness over a water bath and weighed. 10
- 11. Saponin Determination The samples were ground. 20g of each plant samples were dispersed in 200 ml of 20% ethanol. The suspension was heated over a hot water bath for 4 hours with continuous stirring at about 55°C. The mixture was filtered and the residue re-extracted with another 200 ml of 20% ethanol. The combined extracts were reduced to 40 ml over water bath at about 90°C. The concentrate was transferred into a 250 ml separator funnel and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n- butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to a constant weight. The saponins content was calculated in percentage. Tannin Determination 500 mg of the sample was weighed into 100 ml plastic bottle. 50 ml of distilled water was shaken for one hour in a mechanical shaker. This was filtered into a 50 ml volumetric. flask and made up to the mark. Then 5 ml of the filtrate was pipette out into a tube and mixed with 3 ml of 0.1M FeCl3 in0.1N HCl and 0.008M potassium ferrocyanide. The absorbance was measured in a spectrophotometer at 120 nm wavelengths, within 10 minutes. A blank sample was prepared and the colour also developed and read at the same wavelength. A standard was prepared using tannin acid to get100 ppm and measured. Cont. 11
- 12. CHROMATOGRAPHY Separation, identification, and purification of the components of a mixture The three components of the chromatography technique are: Stationary Phase: It is composed of solid or liquid component. Mobile Phase: It is always composed of liquid or gas component. Separated Molecules The individual components in the mixture are partitioned between the stationary and mobile phases. This process allows the separation of components in the mixture because different components in the mixture have different interactions with the stationary and mobile phases.
- 13. 13 To identify and analyze samples for the presence of trace elements or chemicals. Separation of compounds based on their molecular weight and element composition. Detects the unknown compounds and purity of mixture. In drug development. Applications of Chromatography
- 14. Adsorption chromatography Partition chromatography Size exclusion chromatography Affinity chromatography Ion exchange chromatography 14 Basedonthe mechanism of separation
- 15. Gas chromatography • Gas-solid chromatography (GSC) • Gas-liquid chromatography (GLC) Liquid chromatography • Liquid-solid chromatography Ex- TLC, Column, HPTLC, Ion exchange, UPLC • Liquid-liquid chromatography-Paper 15 Basedonthe nature ofthemobile phase
- 16. • Column Chromatography- Column, HPLC, Ion exchange, UPLC, Affinity, Size exclusion • Planar Chromatography -TLC, Paper chromatography 16 Based on the Geometry
- 17. ThinLayerchromatography TLC can be used to help determine the number of components in a mixture, the identity of compounds, and the purity of a compound. For Non-volatiles samples TLC consists of three steps - spotting, development, and visualization Rf ꓿distance traveled by the substance ∕the distance traveled by the solvent. Rf value is always between zero and one. 17 Spotting Development Visualization
- 18. Automated and sophisticated form of thin layer chromatography Advanced separation efficiency and detection 18
- 19. ColumnChromatography 19 The main principle involved in column chromatography is the adsorption. It is a solid-liquid technique in which the stationary phase is a solid & the mobile phase is a liquid.
- 20. ColumnEfficiency N= Number of theoretical plates H= Height of a theoretical plate L= Length of the column . 20 The higher the number of plates, the more efficient the column.
- 21. High-performance liquidchromatography (HPLC) 21 HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture. HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. Mass spectroscopy (MS)-coupled HPLC technique that is LC-MS (liquid chromatography-mass spectroscopy), in which the technique highly improves the sensitivity of detection.
- 22. GasChromatography It is applicable to thermal stable substances, which are volatilized under the temperatures employed. GC technique applied for separation, identification. Attached with mass spectrometry (GC-MS) for quantification of components . 22
- 23. Spectroscopy is the study of the absorption and emission of light and other radiation by matter. Spectrometry is the measurement of the interactions between light and matter, and measurements of radiation intensity and wavelength. SPECTROSCOPY 23 SPECTROMETRY
- 24. 1376 cm-1 (O-H bending; alcohol), 1085 cm-1 (C-O str; aliphatic ether), 779 cm-1 (C-H bending; 1,2,3 trisunstituted) 24 Infrared Spectroscopy Infrared Spectroscopy generally refers to the analysis of the interaction of a molecule with infrared light.
- 25. AAS is an analytical technique used to determine the concentration of metal atoms/ions in a sample. 25 Atomicabsorption spectroscopy During the atomic absorption spectroscopy process, these atoms will absorb electromagnetic radiation at a specific wavelength. This produces a measurable signal. By looking at these signals, it is then possible to determine the parts per million, or ppm, levels of specified metals in the material. How does Atomic Absorption Spectroscopy work?
- 26. 26 Mass spectrometry Mass spectrometry is an analytical tool useful for measuring the mass- to-charge ratio (m/z) of one or more molecules present in a sample. These measurements can often be used to calculate the exact molecular weight of the sample components.
- 27. GUIDELINES 27 WHO Guidelines for standardization of herbal drugs. Ayurvedic pharmacopoeia of india (A.P.I.), -Part-II, Vol. II CCRAS: Drug Development Of Ayurvedic Formulations
- 28. 28 IMPPAT is a manually curated database which has been constructed via digitalization of information from more than 100 books on traditional Indian medicine, 7000+ published research articles and other existing resources. IMPPAT captures three different types of associations: (a) Indian medicinal plant- Phytochemical (b) Indian medicinal plant- Therapeutic use (c) Indian medicinal plant - Traditional Indian medicinal formulation
- 29. • (TKDL) is a pioneering initiative of India to protect Indian traditional medicinal knowledge and prevent its misappropriation at International Patent Offices. • Available in English, Japanese, French, German and Spanish. • TKDL is available to 13 Patent Offices (European United State Patent & Trademark Office, Japan Patent Office, United Kingdom Patent Office, Canadian Intellectual Property Office, German Patent Office, Intellectual Property Australia, Indian Patent Office, Chile Patent Office, Intellectual Property Corporation of Malaysia, Rospatent- Intellectual Property Office of Russia, Peru Patent Office and Spanish Patent and Trademark Office) • As on date, more than 3.6 lakh formulations/ practices have been transcribed into the TKDL database. 29
- 30. Conclusion 30 Medicinal plants are widely recognized to be of great health importance. The crude drug that reaches the market and pharmaceutical industry must have some basic characteristics and quantity of active constituents for their claim therapeutic potential. The efficacy, safety and quality of medicinal plants are the main concerns, which are highly dependent on the comprehensive analysis of chemical components in the medicinal plants.
- 31. 31 Phytochemical screening not only helps to reveal the constituents of the plant extracts and the one that predominates over the others but also is helpful in searching for bioactive agents. Phytochemical analysis gives a good monitoring method of the seasonal changes of the active constituents and during cultivations and harvesting which assists in collecting the largest amounts of the active constituents.
- 33. ThankYou 33