Phytochemical Extraction and identification methods.pptx

Phytochemical extraction methods are techniques used to isolate bioactive compounds from plant materials. These methods aim to separate and purify the desired phytochemicals, such as alkaloids, flavonoids, tannins, and terpenoids, which possess various therapeutic properties. The extraction process typically begins with the selection of a suitable solvent that can effectively dissolve the target compounds. Common methods include maceration, where the plant material is soaked in solvent; Soxhlet extraction, which involves continuous solvent reflux; and ultrasonic-assisted extraction, which uses ultrasonic waves to enhance extraction efficiency. More advanced techniques, such as supercritical fluid extraction and microwave-assisted extraction, offer higher efficiency and selectivity. The choice of extraction method depends on factors like the nature of the plant material, the stability of the compounds, and the intended application of the extract. These methods are crucial in pharmacognosy, nutraceuticals, and cosmetic industries for obtaining high-purity phytochemicals for further research and product development.Chromatography is a versatile and widely-used analytical technique for separating and analyzing complex mixtures of substances. It operates on the principle of partitioning components between a stationary phase and a mobile phase. In chromatography, the sample mixture is dissolved in the mobile phase, which carries it through the stationary phase. Different components in the mixture interact with the stationary phase to varying degrees, leading to their separation as they travel at different speeds. Various types of chromatography, such as High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Thin Layer Chromatography (TLC), are utilized based on the nature of the sample and the desired resolution. Chromatography is essential in many fields, including pharmaceuticals, biochemistry, environmental science, and food industry, for its ability to provide precise qualitative and quantitative analysis of complex mixtures.

1. Raman Yadav PhD scholar Department of Pharmacology SRMC & RI, SRIHER, Chennai-600116, Tamil Nadu, India “Extraction methods and identification of phytochemicals”

2. Source of drugs Extraction 3. Screening of phytochemicals 4. Chromatography Contents 2.1 Type of extraction 4.1 Types of chromatography

3. Drug: Any substance or product that is used or intended to be modify to explore physiological system or pathological state for the benefit of the patient. Plant source eg : atropine , vincristine, reserpine etc Animal source eg : heparin, gonadotropin , cod liver oil, urokinase Mineral source eg : iron, iodine, zinc oxide, gold Microbiological sources: penicillin, griseofulvin, streptomycin Synthetic sources:   phenytoin, chlorpromazine, ampicillin, morphine Recombinant DNA technology eg : insulin,  Hepatitis B vaccine Source of drug

4. Plant source of drug S.No Source Plant Chemical compounds Uses 1 Leaf Digitalis Digoxin CHF T ulsi Cineole R espiratory infection 2  Bark Cinchona Quinine, Quinidine Antimalarial, Antiarrhythmic Atropa belladonna Atropine Anticholinergic 3  flower Vinca rosea Vincristine, Vinblastine Anticancer Chamomile Coumarin, Apigenin Anti-inflammatory 4  Fruit Opium Morphine Analgesic Seena pod Anthracene Purgative 5  seed Isabgol Mucilage, Arabinose Laxative Nux vomica strychnine and brucine  CNS stimulants 6  Root Rauwolfia serpentina Reserpine Antihypertensive Ginseng Ginsenoside Strength immune

5. Extraction : An extraction is the process of removing the desired chemical components from a material to separate it from its initial source. Aim of p lant extraction is to extract components present in the plants. It is a solid/liquid separation operation: a solid object (the plant) is placed in contact with a fluid (the solvent). The plant components of interest are then solubilized and contained within the solvent. Marc:  The unwanted or insoluble substances left behind after the process of extraction. Menstruum: The solvent used in the process of extraction.

6. 1. Infusion 2. Decoction 3. Digestion 4. Maceration 5. Percolation 6. Soxhlet extraction Supercritical Fluid Extraction (SFE) M icrowave Assisted E xtractions (MAE) U ltrasound Assisted Extraction (UAE) Type of extraction Advance technique of extraction

7. I nfusions are prepared by macerating the crude drug for a short period of time with cold or boiling water. This used for readily soluble constituents of crude drugs. eg : infusion of Quassia, infusion of gentian. Infusion:

8. Decoction is the process in which the water soluble and heat stable constituents of hard and woody crude drugs are extracted out. The drug, cut in small pieces, is boiled with the menstruum for 10 to 15 minutes. After boiling, the liquid is cooled and filtered, more water is passed through the marc to produce the required volume. Eg ; tea, coffee Decoction:

9. In this process, the whole or coarsely powdered crude drug is placed in a stoppered container with the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then is strained, the marc is pressed, and the combined liquids are clarified by filtration or decantation after standing. Maceration:

10. Digestion   is a form of maceration with gentle heating extraction process. Temperature should not alter the active ingredients of plant material. It is done to increase the efficiency of menstruum. Eg ; extraction of morphine Types of maceration Simple maceration: E.g ; roots, stems, leaves etc. Maceration with adjustment: E.g ; oleo-resins and gum resins. Digestion:

11. A percolator is generally used for percolation extraction. The solid ingredients are moistened with an appropriate amount of the specified menstruum and allowed to stand for approximately 4 h in a well closed container, after which the mass is packed on the top of the percolator and it closed for 24 h. Percolation:

12. Use for preparation of tinctures and fluid extracts. The outlet of the percolator is opened and the liquid contained therein is allowed to drip slowly. Sufficient menstruum is added to produce the required volume, and the mixed liquid is clarified by filtration or by standing followed by decanting.

13. Soxhlet extraction Soxhlet extraction is ideal for extracting soluble analyte from a solid sample into an organic solvent. These technique allow for its continuous process of treatment of a sample with a solvent over a period of hours or days to extract compounds of interest.

14. The source material containing the compound to be extracted is placed inside the thimble. The thimble is loaded into the main chamber of the Soxhlet extractor. The extraction solvent to be used is placed in a distillation flask. The flask is placed on the heating element. The Soxhlet extractor is placed on the top of the distillation flask. Assembly 7. A reflux condenser is placed on top of the extractor.

15. The solvent is heated to reflux. The solvent vapor travels up a distillation arm, and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapor cools, and drips back down into the chamber housing the solid material. The chamber containing the solid material slowly fills with warm solvent. Working

16. The desired compound will then dissolve in the warm solvent. When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times, over hours or days. Soxhlet extraction has been used widely for extracting valuable bioactive compounds from various natural sources.  Biofuel extraction from coffee beans. Extraction of caffeine from beverage plants. Oil extraction from plants. In the food industry for analyzing food components. Applications:

17. The desired components must be soluble in the solvent at a high temperature. C annot extract from more than one sample at a time. There is no automation. It needs less solvent to yield concentrated extract. Continuous extraction and efficient. Advantages Disadvantages

18. Supercritical Fluid Extraction (SFE) Sample is stored in extraction cell and supercritical fluid (SCF) is pumped from a reservoir. Solvent Critical temperature (K) Critical pressure (bar) Carbon dioxide 304.1 73.8 Ethane 305.4 48.8 Propane 369.8 42.5

19. SCF is heated and pressurized to reach the supercritical conditions that enters the extraction chamber where contact with crude drug bed occurs and the more volatile substances are dissolved into the supercritical fluid. Solute and SCF leave extractor and extract is precipitated in separators, where SCF becomes gaseous is recycled by condensation before returning to liquid reservoir. SFE is used for extraction of essential oils and caffeine from the plant materials.

20. Microwaves are electromagnetic radiations with a frequency from 0.3 to 300 GHz. The plant material is immersed inside a microwave with the solvent, the heat of microwave radiation directly reaches to the solid without being absorbed by the solvent, resulting in continuous heating of the solid. Used for extraction of Azadiractin from Azadirachta indica, glycyrrhizic acid from Glycyrrhizia glabra roots Heating causes the moisture to evaporate and creates a high vapour pressure that breaks the cell wall of plant material and releases the content into solvent. M icrowave Assisted E xtractions (MAE)

21. Ultrasound-Assisted Extraction (UAE) The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz. The ultrasound waves causes disruption in the plant tissue through physical forces developed during acoustic cavitation and helps in release of extractable components in the solvent. It required in very less time by enhancing the mass transport. Use to extract oils from almond, apricot and rice bran

22. Choice of Solvent Selectivity: High selectivity of solvent is required. Boiling temperature: The boiling point of the solvent is as low as possible in order to facilitate removal of the solvent from the product. Reactivity: The solvent should not react chemically with the extract, nor should it readily decompose. Safety: The solvent should be non-flammable and non-corrosive, and should not present a toxic hazard; its disposal should not imperil the environment. Cost: The solvent should be readily available at low cost. Recovery: The solvent should separated easily from the extract to produce a solvent-free extract.

23. S.No Solvents Solvent polarity Index 1.         Cyclohexane 0.04 2.         Hexane 0.1 3.         Carbon tetrachloride 1.56 4.         Isopropyl ether 1.83 5.         Toluene 2.4 6.         Methyl-t-butyl ether 2.4 7.         Chloroform 2.7 8.         Diethyl ether 2.8 9.         Dichloromethane 3.1 10.     Isopropanol 3.92 11.     Tetrahydrofuran 4 12.     Ethanol 4.3 13.     Ethyl acetate 4.4 14.     Methanol 5.1 15.     Acetone 5.1 16.     Acetonitrile 5.8 17.     Water 10.2

24. S. No Solvent Phytochemicals 1 Hexane Waxes, Fats 2  Cyclohexane Waxes, Fats 3  Diethyl ether Aglycones, Alkaloids 4  Dichloromethane Terpenoids, Flavonoids, Aglycones 5  Chloroform Terpenoids, Flavonoids, Alkaloids, Aglycones 6  Ethyl acetate Aglycones, Alkaloids, Glycosides 7  Acetone Flavonols , Alkaloids, Aglycones. 8  Ethanol Tannins, Polyphenols, Flavanols, Terpenoids, Sterols, Alkaloids, Polyacetylenes, Propolis 9  Methanol Saponins, Tannins, Phenones , Flavones, sugars, Lectins, Terpenoids, Anthocyanins, Starches and Polypeptides, Aminoacids , Xanthoxyllines , Quassinoids , Lactones and Polyphenols, 10  Water Sugars, Aminoacids , Saponins, Tannins, Lectins, Terpenoids, Anthocyanins, Starches and Polypeptides.

25. Test for alkaloids Dragendorff ’s test: 1mL of extract + 1mL Dragendorff’s reagent was added and shaken. An orange red precipitate formed indicates the presence of alkaloids. Phytochemical screening of the plant extract Hager’s test: 1mL of solution of an extract + 1mL Hager’s reagent was added and shaken. Formation of yellow colored precipitate indicates the existence of alkaloids. Wagner’s test: 1mL of extract + 1mL Wagner’s reagent was added and shaken. Appearance of reddish brown precipitate signifies the existence of alkaloids. Mayer’s test: 1mL of extract + 1mL of Mayer’s reagent was added and shaken. Emergence of whitish or cream precipitate implies the presence of alkaloids.

26. Keller – kiliani test : Alcoholic extract of drug + equal volume of water + 0.5 ml of lead acetate solution → shake → filter→ filtrate extracted with same amount of CHCl3 → CHCl3 extract evaporated to dry → remainder dissolved in 3 ml of glacial acetic acid + few drops of FeCl3 solution + 2ml of conc. H2SO4→ reddish brown layer → turns to bluish green → indicate the presence of aglycon ( digitoxose ). Borntrager’s test : 1gm of drug sample + 5-10 ml of dilute HCl + 10 min. boil on water bath and filter + extract of filtrate with CCl4 or benzene + equal amount of ammonia solution to filtrate + shake → appearance of pink to red colour → indicate presence of anthraquinone moiety. Test for glycosides:

27. Test for flavonoids Shinoda test: Pieces of magnesium ribbon + Hcl concentrated mixed with extract after few minutes and pink color showed the presence of flavonoid. Lead acetate test: 1mL of extract + few drops of lead acetate added and shaken. Formation of yellow precipitate signifies the presence of flavonoids. Alkaline reagent test: 1mL of extract was + few drops of sodium hydroxide and shaken. Emergence of intense yellow color that turns to colorless after adding dilute acid implies the existence of flavonoids. Test for Terpenoids Salkowski’s test: 1mL of the extract + 2mL of chloroform, shaken and filtered. Few drops sulfuric acid and allowed to stand. Golden-yellow precipitate indicates the presence of triterpenes.

28. Libermann Burchard’s test : 2 ml of chloroform and concentrated H 2 SO 4  were added with the 5 ml crude extract. In the lower chloroform layer red color appeared that indicated the presence of steroids Test for steroids Test for tannins Gelatin’s test: 1mL of extract + 1% gelatin solution containing sodium chloride added and shaken. Appearance of white precipitate indicates the presence of tannins. Test for phenols Ferric chloride test: 1mL of a extract + 1% gelatin solution containing sodium chloride was added and shaken. Formation of bluish-black color indicates the presence of phenols

29. Chromatography Chromatography is a technique for the separating mixture into their components to analyze, identify, purify or quantify the mixture or components. Principle Chromatography technique is based on different interaction of compound with two phases i.e mobile phase and stationary phase as the compounds travels through the supporting medium. Mobile phase: A solvent that follows through the supporting medium. Stationary phase : A layer or coating on the supporting medium that interacts with the analyte.

30. Types of chromatography Thin layer chromatography Column chromatography Paper chromatography Gas chromatography High-performance liquid chromatography (HPLC)

31. Thin Layer Chromatography (TLC): TLC is a method separation of a mixture of component into individual components by using adsorbent (solid) spread over the glass plate and liquid as a mobile phase. Separation is based on the adsorption. Component which has more affinity towards stationary phase travels slowly and compound which has less affinity towards stationary phase travels faster. Principle TLC plates 2. TLC chamber 3. Mobile phase 4. F ilter paper   TLC system consists of:

32. Procedure Preparation of slurry Coating of plates Activation of TLC plate Application of sample Development of tank Detecting/ visualizing agent Steps in TLC

33. Thin mark on the bottom TLC plate’s with a pencil. Then, samples solutions are applied on the spots marked on the line in equal distances. The mobile phase is poured into the TLC chamber and filter paper placed on the inner wall of the chamber to maintain the humidity.  Finally prepared stationary phase plate is put inside the chamber.  At this point, the sample spots are kept above the mobile phase’s. Sufficient time is given for the development of spots. The plates are then removed and allowed to dry. The sample spots are then seen in a suitable UV light chamber, or using derivatizing agent.

34. UV light Iodine Staining: I n detecting carbohydrates since it turns black on contact with Iodine Ninhydrin Reagent: Used to detect amino acids and proteins Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics TLC plates do not have long stationary phases. Therefore, the length of separation is limited. Only soluble component of the mixture are possible. Applications Limitations Techniques for visualizing the results of a TLC plate R f = distance travel by the compounds/distance travel by solvent

35. High performance thin layer chromatography (HPTLC) HPTLC is the automated, sophisticated and improved form of TLC. Principle Principle is same as TLC, based on adsorption. Component which has more affinity towards stationary phase travels slowly and compound which has less affinity towards stationary phase travels faster. HPTLC is automated in some steps, to increase the resolution and to make more accurate quantitative measurements.

36. Steps involved in the HPTLC 1. Selection of chromatographic plate Precoated plates: silica gel 60F, Aluminium oxide, cellulose silica gel 2. Leyer of prewashing Purification step: to remove water vapour and volatile substance eg ; sorbent silica gel 60 F contain iron as impurities removed by the using methanol: water (9:1) 3. Activation of precoated plate Plates are kept in oven at 110 C - 120 C to remove water.

37. 4. Sample preparation and application Concentration: 0.1- 1 µL Automatic sample applicator 5. Selection of mobile phase Trial and error Literature based Coumarin : Diethyl Ether: Toluene (1:1) Alkaloids: Toluene: Ethyl Acetate: Diethyl Amine

38. 7. Chromatographic development and drying Plates are spotted with sample and air dried are placed in the development chamber. Plates are allow to run for optimum After development, plates are dry in vacuum desiccator 8. Detection and visualization Plate are scanned at white light illumination or at different selected UV regions (254 nm and 366 nm) 6. Pre-conditioning : Chamber is saturated with filter paper for 30 min

39. TLC visualization HPTLC visualization

40. Forensics HPTLC used to investigate drug and alcohol abuse . Eg : detecting cannabis; saturated ammonium metavanadate and p-anisidine reagent used to detect cannabis from urine samples. Pharmaceuticals HPTLC is used in the identification, purity testing and determination of the concentration of active ingredients, auxiliary substances and preservatives in drugs and drug preparations, HPTLC is broadly used in food industries for the analysis of vitamins and pesticides in vegetables and fruits and other food stuffs. S.No Chemical compounds (Herbal) Mobile phase  1 Alkaloid Toluene: Ethyl Acetate: Diethyl Amine (70:20:10)  2 Terpenes Chloroform: Methanol: Water (65:25:4)  3 Essential Oil Toluene: Toluene (93:7)  4 Flavonoids Ethyl Acetate: Formic Acid: Glacial Acetic Acid: Water (100:11:11:26)  5 Saponin Chloroform: Glacial Acetic Acid: Methanol: Water (64:32:12:8) Applications of HPTLC

41. Column chromatography Column chromatography is a technique in which the substances to be separated are introduced onto the top of a column packed with an adsorbent, passed through the column at different rates that depend on the affinity of each substance. The components that move rapidly are taken out first through the components that move slowly are eluted out last. 

42. The column mostly consists of a glass tube packed with a suitable stationary phase. Cotton wool or an asbestos pad is placed at the bottom of the column before packing the stationary phase. After packing, a paper disc kept on the top, so that the stationary layer is not disturbed during the introduction of sample or mobile phase. Steps in Column Chromatography Preparation of the Column The sample is dissolved in minimum quantity of the mobile phase. The sample mixture is introduced into the column from the top portion of the column. Introduction of the sample

43. Through this technique, the individual components are separated completely from the column. The process of elution can be carried out by employing two techniques: Isocratic elution technique Example: Use of chloroform alone Gradient elution technique – Example: Benzene → Chloroform → Ethyl acetate Elution technique

44. If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually If the compounds collected from column chromatography are colorless. In this case, small fractions of the eluent are collected sequentially in labelled tubes and the composition of each fraction is analyzed by TLC. To remove impurities or purification process. To isolate metabolites from biological fluids. To separate natural compounds like alkaloids, glycoside etc. Detection of Components Applications :

45. Paper chromatography  Stationary Phase Whatman filter papers of different grades like No.1, No.2, No.3, etc , Hydrophilic Papers, Hydrophobic papers. Principle of paper chromatography based on partition and sometimes adsorption chromatography. In paper chromatography, the sample mixture is applied to a piece of filter paper, the paper is immersed in a solvent, and the solvent moves up on the paper by capillary action. Instrumentation

46. Selection of Mobile Phase : Butanol: Acetic acid: Water (12:3:5) is a suitable solvent for separating amino acids. Saturation of Tank : The inner wall of the tank is wrapped with filter paper.

47. Detection Analytes detect by staining with reagents such as iodine vapor, ninhydrin, etc. Sample Preparation and Loading : Sample is dissolved in a suitable solvent. Sample (2-20ul) is added on the baseline as a spot using a micropipette or capillary tube. Development of the Chromatogram : Sample loaded filter paper is dipped carefully into the solvent and waited until the solvent front reaches near the edge of the paper. Drying   of Chromatogram After the development, the solvent front is marked and left to dry in a dry cabinet or oven.

48. To check the control of purity of pharmaceuticals. For detection of adulterants. Detect the contaminants in foods and drinks. Analysis of the reaction mixtures in biochemical labs. Applications of Paper Chromatography Large quantity of sample cannot be applied on paper chromatography. In quantitative analysis paper chromatography is not effective. Complex mixture cannot be separated by paper chromatography. Less Accurate compared to HPLC or HPTLC Limitations of Paper Chromatography  

49. Principle Separation based on the properties of the sample and the solvent, as well as the nature of the stationary phase. The compound which possess strongest interactions with the stationary phase exit the column the slowly and the compound which possess weak interaction with the stationary phase exit faster. Time taken analyte to exit the column time determines the retention time of analyte. High-Performance Liquid Chromatography (HPLC) HPLC is a form of column chromatography that pumps at high pressure a sample dissolved in a solvent (mobile phase) through a column with an immobilized chromatographic packing material (stationary phase).

50. Instrumentation of High-Performance Liquid Chromatography (HPLC) Solvent Reservoir: mixture of polar and non-polar liquid components Pump : 42000 KPa is the operating pressure of the pump. This operating pressure depends on column dimensions, particle size, flow rate and composition of mobile phase.

51. Sample Injector : syringe, autosampler ( 0.1 mL to 100 mL ) Columns: stainless steel around 50 mm and 300 mm long and an inward distance across of somewhere around 2 and 5 mm. They are generally loaded with a stationary phase with a molecule size of 3 μm to 10 μm . Recorder: The output is recorded as a series of peaks. Detector: The HPLC detector, situated toward the end of the column distinguishes the analytes as they elute from the chromatographic column. Regularly utilized detectors are UV-spectroscopy, mass spectrometric and Photoioniztion detector (PID) .

52. P harmaceutical Applications To control drug stability.  Pharmaceutical quality control. Applications of HPLC Applications in Forensics Quantification of drugs in biological samples. Identification of steroids in blood, urine, etc. Forensic analysis of textile dyes. Determination of cocaine and other drugs of abuse from blood, urine, etc.

53. Environmental Applications Detection of phenolic compounds in drinking water. Bio-monitoring of pollutants. Applications in Clinical Tests Antibiotics analysis in blood. Analysis of bilirubin, biliverdin in hepatic disorders. Detection of endogenous neuropeptides in the extracellular fluid of the brain etc. HPLC is much more costly. Complexity Volatile substances are much better to be separated by gas chromatography. Limitations

54. Gas chromatography (GC) Gas chromatography is an analytical technique used for separation, identification and quantitative estimation of sample components in gaseous and liquid mixture. In gas-solid chromatography, solid adsorbent is used as a stationary phase & separation takes place through adsorption process while in gas- liquid chromatography, the stationary phase consists of thin layer of non-volatile liquid bound to solid support & separation takes place through the process of partition. Principle

55. Instrumentation Sample injection system: Calibrated microsyringe (0.1 -0.2 ml) Carrier Gas : Helium , Nitrogen, Argon & Hydrogen Column: separation of sample components takes place in the column. Two types of column are used in the gas chromatography packed column Length : 0.5 – 10 m Inner diameter : 2- 4 mm Column material: stainless steel, glass, quartz Column completely filled with adsorbent

56. Length : 5 – 100 m Inner diameter: less than 1 mm Column material: glass, fused silica with outer protective coating of polyimide and fused silica coated stainless steel. Stationary phase only coated to inner column wall 2. Capillary column Column Oven : The thermostat oven are used to control the temperature of the column to conduct precise work. The oven can be operated in two manners: isothermal programming or temperature programming.

57. Detectors: Detector is present at the end of the column & gives the quantitative measurement of the components of the mixture as they elute in combination with the carrier gas. Most common types of detectors used in GC are: Mass Spectrometer , Flame ionization detector (FID), Electron capture detector (ECD), Thermal conductivity detector (TCD), Photoioniztion detector (PID) Recorder system: The recorder records the signals that come from the detector. Recorder amplify the signals and display in graphical format that represents several peaks of the constituents of the sample.

58. Working Sample is injected into the chromatographic column. Sample moves through the column with the flow of inert gas & results in the separation of the components of sample which are recorded as a sequence of peaks as they leave the column. The different components of the sample separated & eluted at different & particular time which is called retention time.

59. Applications Identification of volatile compounds from sample. Identification & quantification of pollutants of environment. Quantification of drugs & their metabolites in blood & urine for both pharmacological & forensic applications. Quantification of pollutants in drinking & waste water. Identification of hazardous compounds in waste damps. Analysis of pharmaceutical products for quality control.

60. only volatile and thermally stable compounds can be separated using gas chromatography. Only compounds with less than 1,000 Da, analyzed because it is difficult to vaporize larger compounds. Can not recover individual component Limitations

61. In-silico study Structure identification : 2D, 3D structure : Pubchem , drugbank Physiochemical properties: Pubchem , drugbank Molecular docking : Autodock , Autoduck Vina Drug likeness property: swiss ADME, PreADMET Pharmacokinetic properties: swiss ADME, PreADMET Toxicity: Protox II

62. Clinical evaluation Plant material Extraction of plant material Qualitative and quantitative analysis Identification of bioactive compounds In-silico study of compounds Isolation of compounds Preclinical evaluation In-vitro evaluation

63. References Abubakar AR, Haque M. Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. Journal of pharmacy & bioallied sciences. 2020 Jan;12(1):1. Roopashree KM, Naik D. Advanced method of secondary metabolite extraction and quality analysis. Journal of Pharmacognosy and Phytochemistry. 2019;8(3):1829-42 Shivatare RS, Nagore DH, Nipanikar SU. HPTLC’an important tool in standardization of herbal medical product: A review. J Sci Innov Res. 2013;2(6):1086-96. Moulishankar A, Ganesan P, Elumalai M, Lakshmanan K. Significance of TLC and HPTLC in Phytochemical Screening of Herbal Drugs. J. Glob. Pharma Technol. 2020;13:30-45. Coskun O. Separation techniques: chromatography. Northern clinics of Istanbul. 2016;3(2):156. Boswell HA, Dorman FL. Uncertainty of Blood Alcohol Concentration (BAC) results as Related to Instrumental Conditions: Optimization and Robustness of BAC analysis headspace parameters. Chromatography. 2015 Dec;2(4):691-708.

64. Thank you