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Extraction and phytochemical analysis of medicinal plants

This study investigated the phytochemical constituents and total phenolic content of several medicinal plants. Plant materials were extracted using different solvents and subjected to qualitative and quantitative analysis. Qualitative testing identified the presence of compounds like phenols, tannins, flavonoids, saponins, alkaloids, etc. Total phenolic and flavonoid content was determined using spectrophotometric methods. The results showed that the plants contained valuable phytochemicals and could be a potential source of drugs. The methanol extracts generally had the highest concentration of phytochemicals.

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2 Seminar on Extraction and Phytochemical Analysis of Medicinal Plants by Shameembanu A. ByadgiShameembanu A. Byadgi
Medicinal plants  Medicinal plants constitute an effective source of both traditional and modern medicines  Herbal medicine has been shown to have genuine utility  About 80% of rural population depends on it as primary health care. [WHO, (2005)]
Medicinal plants are the richest bio-resource  drugs of traditional systems of medicine  modern medicines  nutraceuticals  food supplements  folk medicines  pharmaceutical intermediates  chemical entities for synthetic drugs
 Natural bioactive compounds found in different parts of plant (fruit, flower, stem, leaf, root)  Provide definite physiological action on the human body  Bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids  Widely used in the human therapy, veterinary, agriculture, scientific research and countless other areas  Have inhibitory effects on all types of microorganisms in vitro Phytochemicals
Extraction ……… is the separation of medicinally active portions of plant tissues using selective solvents through standard procedures The basic parameters influencing the quality of an extract •Plant part used as starting material •Solvent used for extraction •Extraction procedure
Choice of solvents Successful determination of biologically active compounds depends on the type of solvent used in the extraction procedure Property of a good solvent in plant extraction •Low toxicity •Ease of evaporation at low heat •Promotion of rapid physiologic absorption of the extract •Preservative action
The factors affecting the choice of solvent •Quantity of phytochemicals to be extracted •Rate of extraction •Diversity of different inhibitory compounds extracted •Ease of subsequent handling of the extracts •Toxicity of the solvent in the bioassay process •Potential health hazard of the extractants
Solvents used for active component extraction Water Ethanol Methanol Chloroform Ether Acetone Anthocyanins Starches Tannins Saponins Terpenoids Polypeptides Lectins Tannins Polyphenols Polyacetylenes Flavonols Terpenoids Sterols Alkaloids Anthocyanins Terpenoids Saponins Tannins Xanthoxyllines Totarol Quassinoids Lactones Flavones Phenones Polyphenols Terpenoids Flavonoids Alkaloids Terpenoids Coumarins Fatty acids Phenol Flavonols
General techniques of medicinal plant extraction  Plant tissue homogenization  Maceration  Infusion  Percolation  Digestion  Decoction  Soxhlet extraction (Hot continuous extraction)  Sonication (Ultrasound extraction)
Plant tissue homogenization
Maceration Whole / coarsely powdered crude drug is placed in a stoppered container with the solvent Whole / coarsely powdered crude drug is placed in a stoppered container with the solvent Allow to stand @ room temperature for a period of at least 3 days with frequent agitation until the soluble matter gets dissolved Allow to stand @ room temperature for a period of at least 3 days with frequent agitation until the soluble matter gets dissolved The mixture then is strained, the marc (the damp solid material) is pressed The mixture then is strained, the marc (the damp solid material) is pressed The combined liquids are clarified by filtration or decantation after standing The combined liquids are clarified by filtration or decantation after standing
Infusion
Digestion • A form of maceration in which gentle heat is used during the process of extraction • Used when moderately elevated temperature is not objectionable • The solvent efficiency of the menstruum is thereby increased Microwave digestion system
Decoction  Suitable for extracting  water-soluble, heat-stable constituents  Typically used in preparation of Ayurvedic extracts
Percolation • Used most frequently to extract active ingredients in the preparation of fluid extracts • The solid ingredients are moistened with an appropriate amount of the specified menstruum • Allowed to stand for approximately 4 hours in a well closed container, After stand time, the mass is packed & the top of the percolator is closed • The mixture is allowed to macerate in the closed percolator for 24 h
, • Additional menstruum is added as required, until the percolate measures about three-quarters of the required volume of the finished product • The marc is then pressed and the expressed liquid is added to the percolate • Sufficient menstruum is added to produce the required volume • The mixed liquid is clarified by filtration or by standing followed by decanting
Soxhlet Extraction (Hot Continuous Extraction)
Sonication (Ultrasound Extraction) • Involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz • Increases the permeability of cell walls & produces cavitation Disadvantage Deleterious effect of ultrasound energy (>20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules
Effect of extracted plant phytochemicals depends on • The nature & origin of the plant material • Degree of processing • Moisture content • Particle size
Variation in extraction methods • Length of the extraction period • Solvent used • pH of the solvent • Temperature • Particle size of the plant tissues • Solvent-to-sample ratio
Phytochemical screening methodsPhytochemical screening methods Phytochemicals Tests Reagents Positive results Alkaloids Dragendorff test Dragendorff’s reagent Prominent yellow ppt Wagner test Wagner’s reagent Reddish brown ppt Mayer test 1% HCl, Mayer’s reagent Turbid extract is obtained Flavonoids Ammonia test 1% NH3 Yellow colour Sodium hydroxide test 20% NaOH, HCl Yellow colour turns to colourless Tannins Ferric chloride test 5% FeCl3 Blue-black or blue- green colouration Phenolic compounds Gelatine test 1% gelatine solution containing 10% NaCl White ppt Lead acetate test 10% lead acetate Bulky white ppt Saponins Foam test 20 mL distilled water (mixed vigorously for 15 minutes) Presence of froth Qualitative Analysis
Phytochemicals Tests Reagents Positive results Terpenoids Salkowski test 0.5 mL chloroform, 1 mL conc. H2SO4 Reddish brown colouration at the interface Carbohydrates Molish test Conc. HCl Violet ring Fehling test Conc. HCl & Mg turnings Yellow & brick red ppt Proteins Biuret test 4% NaOH, 1% CuSO4 Violet or pink colour Glycosides Legal’s test Pyridine, sodium nitroprusside Pink to red colour Kellar killani test Glacial acetic acid, 5% FeCl3 Reddish brown & bluish green colour Contd....
Quantitative Analysis Total Phenolic Content Determined by Folin-Ciocalteau assay method (Singleton and Rossi, 1965) Instrument: UV-Vis Spectrophotometer, absorbance measured at 765 nm Expressed as Gallic acid equivalent (GAE) in milligrams per gram of fresh leaf Total Flavonoid Content Determined by Colourimetric method (Yun et al., 2009) Instrument: UV-Vis Spectrophotomer, absorbance measured at λ415 nm Expressed as mg rutin equivalent (mg RE) per gram of fresh leaf
Yadav and Agarwala, 2011 Assam, India To carry out qualitative and quantitative phytochemical analysis of selected medicinal plants Phytochemical Analysis of Some Medicinal Plants Objective
MethodologyMethodology Plant sources Bryophyllum pinnatum (Leaves) Ipomea aquatica (Leaves) Oldenlandia corymbosa (Whole plant) Ricinus communis (roots) Terminalia bellerica (Leaves) Tinospora cordifolia (Leaves/Stem) Xanthium strumarium (Leaves)
Hot water extraction 5gm of dried finely powdered plant material mixed with 200ml of distilled water Heated on a hot plate with continuous stirring at 30º-40ºC for 20 minutes filtered through filter paper Solvent extraction 20gm powdered plant material packed into a thimble and extracted with 250ml of solvents Extraction continues for 24 hours or till the solvent in siphon tube of an extractor become colourless kept on hot plate and heated at 30-40ºC Preparation of extracts
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content FolinCiocalteu reagent expressed as gallic acid equivalent, mg/g of extracted compound  Total phenolic content FolinCiocalteu reagent expressed as gallic acid equivalent, mg/g of extracted compound  Total flavonoid content Aluminium chloride colourimetric method expressed as quercetin equivalent, mg/g of extracted compound  Total flavonoid content Aluminium chloride colourimetric method expressed as quercetin equivalent, mg/g of extracted compound  Proteins (Millon’s & Ninhydrin test)  Carbohydrates (Fehling’s, Benedict’s, Molisch’s & Iodine test)  Phenols & tannins (Ferric chloride test)  Flavonoids (Shinoda & alkaline reagent test)  Saponins (Foam test)  Glycosides (Liebermann’s, Salkowski’s & Keller-kilani test)  Steroids  Terpenoids  Alkaloids (Mayer’s & Wagner’s test)  Proteins (Millon’s & Ninhydrin test)  Carbohydrates (Fehling’s, Benedict’s, Molisch’s & Iodine test)  Phenols & tannins (Ferric chloride test)  Flavonoids (Shinoda & alkaline reagent test)  Saponins (Foam test)  Glycosides (Liebermann’s, Salkowski’s & Keller-kilani test)  Steroids  Terpenoids  Alkaloids (Mayer’s & Wagner’s test)
Table 1. Phytochemical constituents of medicinal plants Results and discussion L = leaves; S = stem
Fig 1. Total phenolic content Fig 2. Total flavonoid content
Conclusion  Results revealed that extracts from these plants can be used as a good source for drugs  Further work should be carried out to isolate, purify and characterize the active constituents responsible for the activity of these plants
Badugu, 2012 To screen the presence of phytochemicals and estimate the amount of total phenols and flavonoids in Cyamopsis tetragonoloba Phytochemical Screening, Quantitative Estimation Total Phenolics and Total Flavonoids, Antimicrobial Evaluation of Cyamopsis tetragonoloba Objective
MethodologyMethodology Seeds of Cyamopsis tetragonoloba Washed, shade dried & powdered mechanically Preparation of extracts Powdered material was weighed Soxhlet extraction using methanol, acetone, chloroform & hexane Solvent was recovered using Rotary Vacuum Evaporator Phytochemical screening Subjected to 48 hrs
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content Folin-Ciocalteu reagent expressed in terms of milligrams of catechol per gram of dry weight  Total phenolic content Folin-Ciocalteu reagent expressed in terms of milligrams of catechol per gram of dry weight  Total flavonoid content Aluminium chloride colourimetric method expressed as catechin equivalent, mg catechin/g dried extract  Total flavonoid content Aluminium chloride colourimetric method expressed as catechin equivalent, mg catechin/g dried extract  Carbohydrates, reducing sugars, monosaccharides, alkaloids, saponins & tannins (Evans, 1996)  Flavonoids (Shinoda’s test)  Terpenes/ steroids (Liebermann- Burchard’s test)  Anthraquinones (Borntrager’s test)  Cardiac glucosides (Sodium nitro proside method)  Proteins (Copper sulphate & Folin- Ciocalteau solution)  Amino acids (Ninhydrin test)  Carbohydrates, reducing sugars, monosaccharides, alkaloids, saponins & tannins (Evans, 1996)  Flavonoids (Shinoda’s test)  Terpenes/ steroids (Liebermann- Burchard’s test)  Anthraquinones (Borntrager’s test)  Cardiac glucosides (Sodium nitro proside method)  Proteins (Copper sulphate & Folin- Ciocalteau solution)  Amino acids (Ninhydrin test)
Table 2. Phytochemical screening of Cyamopsis tetragonoloba Sl. No. Secondary metabolites Tests Solvents Methanol Acetone Chloroform Hexane 1 Carbohydrates Molisch’s test +ve +ve +ve +ve 2 Reducing sugars Fehling test +ve +ve -ve -ve 3 Monosaccharide Barfoed’s test -ve -ve -ve -ve 4 Tannins Ferric chloride test Lead sub acetate test -ve -ve -ve +ve 5 Saponins Frothing test +ve -ve -ve -ve 6 Flavonoids Shinoda’s test +ve +ve -ve -ve 7 Terpenes/ steroids Liebermann Burchard’s test +ve -ve +ve -ve 8 Alkaloids Mayer’s test Wagner’s test +ve +ve -ve -ve 9 Cardiac glucosides Sodium nitroproside +ve +ve -ve +ve 10 Proteins Copper sulphate Folin Ciocalteau solution -ve -ve -ve -ve 11 Amino acids Ninhydrin -ve +ve -ve +ve 12 Anthraquinones Borntrager’s test -ve -ve -ve -ve Results and discussion
Table 3. Total phenolic and flavonoids content of different extracts of Cyamopsis tetragonoloba Extract Total phenolic content mg of Catechin equivalents/ 200mg dried extract Total flavonoids content mg of Catechol equivalents/ 200mg dried extract Methanol 14.51 17.34 Acetone 9.17 8.75 Chloroform 11.32 14.05 Hexane 5.53 6.41
Conclusion  The study indicated that methanol is used as best solvent for extraction of phytochemicals  Other solvents like chloroform, acetone and hexane are less commonly used for the extraction of phytochemicals  The herbal extract can be used for curing diseases without any side effects
Hasanuzzaman et. al., 2013 Bangladesh To investigate total phenolic content of methanolic extract of Averrhoa bilimbi fruits Evaluation of Total Phenolic Content, Free Radical Scavenging Activity and Phytochemical Screening of Different Extracts of Averrhoa bilimbi Objective
MethodologyMethodology Fruits of Averrhoa bilimbi Washed, sliced & dried under sun Preparation of extracts Powdered fruits (500g) 2500 ml methanol for 15 days at room temperature Extract filtered using filter cloth & Whatman’s filter paper Filtrate evaporated under ceiling fan & in a water bath below 40°C until dried Soaked in Cold maceration technique
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content Folin-Ciocalteu reagent (oxidizing agent) & gallic acid (standard) expressed as mg of GAE (gallic acid equivalent) / gm of the extractive  Total phenolic content Folin-Ciocalteu reagent (oxidizing agent) & gallic acid (standard) expressed as mg of GAE (gallic acid equivalent) / gm of the extractive  Alkaloids (Mayer’s & Dragendoff’s test)  Tannins (FeCl3 test)  Saponins (Foam test)  Flavonoids (chip of magnesium & HCl)  Glycosides (NaCl & Fehling’s solutions A & B)  Steroids & triterpenes (Ethylic, H2SO4 & anhydride acetic)  Phenols (FeCl3 & K3Fe(CN6))  Cardiac glycosides (Acetic, FeCl3 & conc. H2SO4)  Carbohydrates (Alcoholic α-naphthol, Benedict’s reagent)  Alkaloids (Mayer’s & Dragendoff’s test)  Tannins (FeCl3 test)  Saponins (Foam test)  Flavonoids (chip of magnesium & HCl)  Glycosides (NaCl & Fehling’s solutions A & B)  Steroids & triterpenes (Ethylic, H2SO4 & anhydride acetic)  Phenols (FeCl3 & K3Fe(CN6))  Cardiac glycosides (Acetic, FeCl3 & conc. H2SO4)  Carbohydrates (Alcoholic α-naphthol, Benedict’s reagent)
Table 4. Phytochemical screening of different extractives of Averrhoa bilimbi fruits Tests Extracts MEF PTSF CTSF CSF AQSF Alkaloids + + - + + Tannins + + + - - Saponins + + - - + Flavonoids + + + + + Cardiac glycosides + + - + + Glycosides + + + + + Phytosterols - - - - - Triterpenes + + - + - Phenols + + + + + Carbohydrates + - + + + MEF = methanolic extract; PTSF = pet-ether soluble fraction; CTSF = carbon tetrachloride soluble fraction; CSF = chloroform soluble fraction; AQSF = aqueous soluble fraction Results and discussion
Table 5. Total phenolic content of different extractives of Averrhoa bilimbi fruits Extractives Total phenol content (mg of GAE/g of extractive) Methanolic extract 65.16 ± 0.52 Pet-ether soluble fraction 55.31 ± 1.01 Carbon tetrachloride soluble fraction 52.00 ± 0.90 Chloroform soluble fraction 68.67 ± 0.94 Aqueous soluble fraction 50.23 ± 0.56 GAE = Gallic acid equivalent
Conclusion  It may be concluded that Averrhoa bilimbi fruits is a good source of phytochemicals  Different extractives showed significant total phenolic content
Pranoothi et. al., 2014 Andhra Pradesh, India To carry out qualitative and quantitative phytochemical analysis of aerial parts of Leucas indica (L) Studies on Qualitative, Phytochemical Analysis and Screening of In Vitro Biological Activities of Leucas indica (L) Objective
MethodologyMethodology Aerial parts of Leucas indica Cleaned, shade dried, mechanically grinded & coarsely powdered Preparation of extracts Powdered material Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening Subjected to
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content FolinCiocalteu reagent expressed as µg of gallic acid equivalents per gram dry mass of extract (µg GAE/gDM)  Total phenolic content FolinCiocalteu reagent expressed as µg of gallic acid equivalents per gram dry mass of extract (µg GAE/gDM)  Total flavonoid content Aluminium chloride colourimetric assay expressed with the rutin equivalents per g of dried fraction)  Total flavonoid content Aluminium chloride colourimetric assay expressed with the rutin equivalents per g of dried fraction)  Alkaloids  Steroidal compounds  Phenolic compounds  Flavonoids  Saponins  Tannins  Coumarins  Cardiac glycosides  Alkaloids  Steroidal compounds  Phenolic compounds  Flavonoids  Saponins  Tannins  Coumarins  Cardiac glycosides
Table 6. Physico-chemical evaluation Solvent Initial weight of the powder (g) Final weight of the powder (g) Weight of the crude extract (g) Crude extract (%) Colour of the extract Hexane 50 44.563 5.437 10.874 Dark brown Acetone 50 40.415 9.585 19.17 Dark green Methanol 50 35.552 14.448 28.896 Dark green Water 50 38.621 11.379 22.758 Dark red Figure 3. Yield of extracts Results and discussion
Table 7. Phytochemical analysis of whole aerial part extracts of Leucas indica (L) Sl. No. Tests Extracts Hexane Acetone Methanol Water 1 Alkaloids Mayers - + + + Dragon - + - + Wagners - + + + Hagers - + - + 2 Phenolics FeCl2 test - + + + 3 Flavonoids Lead acetate test - + + + NaOH test - + + - Ethyl acetate test - - - - 4 Anthraquinones Borntrager’s test - - - - 5 Steroids Salkowski’s test + + + +
6 Tannins FeCl2 test - + + - Lead acetate test - + + - Potassium dichromate test - + + - 7 Saponins Vigorous shaking test + + + + 8 Anthocyanins Ammonia-HCL test - - - - 9 Leuco-Anthocyanin Iso amyl alcohol test - - - - 10 Coumarins NaOH test - - - - 11 Reducing sugars Keller-Kiliani test + + + + Sl. No. Tests Extracts Hexane Acetone Methanol Water Contd...
Table 8. Total phenol content % of phenol content µg GAE/µg Concentration (µg/ml) Hexane extract Acetone extract Methanol extract Water extract 100 15.69 18.5 25.6 22.8 200 22.54 26.9 45.86 41.8 300 30.41 38.9 72.8 68.4 400 42.89 54.78 88.96 76.5 500 54.58 65.8 105.68 95.8 Figure 4. Total phenol content
Table 9. Total flavonoid content % of phenol content µg GAE/µg Concentration (µg/ml) Hexane extract Acetone extract Methanol extract Water extract 100 00 10.28 17.25 00 200 00 15.56 26.35 00 300 00 32.47 29.12 13.20 400 00 41.58 43.52 18.25 500 00 50.24 62.34 24.69 Figure 5. Total flavonoid content
Conclusion  The presence of most general phytochemicals in Leucas indica might be responsible for their therapeutic effects  It further reflects a hope for the development of many more novel chemotherapeutic agents from plants which in future may serve for the production of synthetically improved therapeutic agents
Rajesh et. al., 2014 Tamil Nadu, India To evaluate the phytochemicals from aqueous, ethanolic and petroleum ether extracts of four fern species Qualitative and Quantitative Phytochemical Analysis in Four Pteridophytes Objective
MethodologyMethodology Washed & cut into small pieces Shade dried & then hot air oven drying at 55-60ºC Dried leaves, stems & roots were pulverized using mixer Strained through 25 mm mesh diameter sieve to obtain fine dust Phytochemical screening Actinopteris radiata Pityrogramma calomelanos Drynaria quercifolia Dryopteris cochleata
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total tannins Folin-Ciocalteu reagent expressed as mg tannic acid equivalent per 100g of the sample)  Total tannins Folin-Ciocalteu reagent expressed as mg tannic acid equivalent per 100g of the sample)  Total phenols Folin-Ciocalteu reagent method expressed in mg of gallic acid equivalent (GAE) per g of dry weight of plant powdres  Total phenols Folin-Ciocalteu reagent method expressed in mg of gallic acid equivalent (GAE) per g of dry weight of plant powdres  Alkaloids (Mayer’s test)  Anthocyanin & betacyanin (NaOH test)  Cardio glycosides  Coumarins  Flavonoids (NaOH test)  Glycosides  Phenols  Quinones  Saponins (Foam test)  Steroids  Tannins  Terpenoids  Alkaloids (Mayer’s test)  Anthocyanin & betacyanin (NaOH test)  Cardio glycosides  Coumarins  Flavonoids (NaOH test)  Glycosides  Phenols  Quinones  Saponins (Foam test)  Steroids  Tannins  Terpenoids
Table 10. Qualitative analysis of phytochemicals in four fern species Table 11. Quantitative analysis of phytochemicals (mg/g) in four fern species Phytochemicals A.radiata D.quercifolia D.cochieata P.calamelanos Total tannin 12.189 ± 0.258 6.332 ± 0.187 9.405 ± 0.299 17.181 ± 0.441 Total phenol 10.962 ± 0.327 7.131 ± 0.184 8.912 ± 0.310 13.581 ± 0.481 Results and discussion
Conclusion  The results revealed that ethanolic solvent performed well exhibiting the presence of major phytochemicals compared to aqueous and petroleum ether extracts  Quantitative analysis showed highest content of tannins and phenols in Pityrogramma calomelanos fern extract followed with least content of tannins and phenols in Actinopteris radiata, Dryopteris cochleata and Drynaria quercifolia
Vastrad et. al., 2014 Karnataka, India To screen the presence of phytochemicals in leaf extracts of C. fistula, P. pinnata, T. grandis & J. curcas and assess total phenolic & total flavonoid content Characterization of Phytoconstituents in Leaf Extracts of Forest Species for Textile Applications Objective
MethodologyMethodology Cleaned, shade dried, chopped & ground in laboratory mortar & pestle Ground leaf was mixed with the solvent & incubated (200 strokes) in incubator cum shaker for 24 hours at 25ºC The extract was centrifuged (5000 rpm) at room temperature & supernatant separated Residue was re-extracted with the respective solvent The extract was filtered using Whatman filter paper & yield was measured Cassia fistula Pongamia pinnata Tectona grandis Jatropha curcas
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent in milligrams per gram of fresh leaf)  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent in milligrams per gram of fresh leaf)  Total flavonoid content Colourimetric method expressed as milligrams of rutin equivalent (mg RE) per gram of sample)  Total flavonoid content Colourimetric method expressed as milligrams of rutin equivalent (mg RE) per gram of sample)  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins
Table 12. Qualitative phytochemical screening of forest species Figure 6. Total phenolic content (mg per gram of fresh leaf) Results and discussion
Table 13. Total flavonoid content (TFC) of extracts in forest species Forest species TFC (mg/g of sample) Ethyl alcohol Methanol Aqueous Cassia fistula 106.4 126.21 9.49 Pongamia pinnata 121.53 148.33 8.96 Tectona grandis 153.52 179.1 16.71 Jatropha curcas 149.70 138.7 -
Conclusion  The results revealed that phytochemicals viz., alkaloids, flavonoids, tannins and terpenoids were found in appreciable amount in the selected forest species  Methanolic extract of C. fistula, P. pinnata and T. grandis exhibited maximum TPC and TFC content while J. curcas depicted higher amount of TPC and TFC content in ethanolic extract  Therefore it can be concluded that extracts from forest species such as C. fistula, P. pinnata and T. grandis and J. curcas can be used for applying eco friendly and healthy finishes to textile substrates
Geetha and Geetha, 2014 Tamil Nadu, India To carry out qualitative and quantitative phytochemical analysis of lemongrass leaves Phytochemical Screening, Quantitative Analysis of Primary and Secondary Metabolites of Cymbopogan citratus (DC) Leaves Objective
MethodologyMethodology Leaves of Cymbopogan citratus Washed & air dried under shade Preparation of extracts Powdered leaf Fresh leaf Extracted with chloroform, methanol & acetone using soxhlet apparatus Ground using distilled water & filtered; used as an aqueous extract Concentrated using rotary vacuum evaporator & dried
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative  Alkaloids (Wagner’s test)  Flavonoids (Shinoda & lead acetate test)  Phenols & tannins (Lead acetate, FeCl3 & NaOH test)  Steroids & sterols (Salkowski’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Saponins (Honey comb & foam test)  Glycosides (Glycoside test)  Protein & amino acids (Biuret & Ninhydrin test)  Anthraquinones (Borntragers test)  Alkaloids (Wagner’s test)  Flavonoids (Shinoda & lead acetate test)  Phenols & tannins (Lead acetate, FeCl3 & NaOH test)  Steroids & sterols (Salkowski’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Saponins (Honey comb & foam test)  Glycosides (Glycoside test)  Protein & amino acids (Biuret & Ninhydrin test)  Anthraquinones (Borntragers test)
Quantitative analysisQuantitative analysis  Total phenolics Expressed as tannic acid equivalents  Total phenolics Expressed as tannic acid equivalents  Total flavonoid content Colourimetric method expressed as rutin equivalent  Total flavonoid content Colourimetric method expressed as rutin equivalent  Carbohydrates  Total chlorophyll content  Proteins  Total lipid content  Carbohydrates  Total chlorophyll content  Proteins  Total lipid content  Total tannins Tannin (%) = Total phenolics (%) – Non tannin phenolics (%)  Total tannins Tannin (%) = Total phenolics (%) – Non tannin phenolics (%) Primary metabolites Secondary metabolites
Plant constituent Extracts TestsCholoroform Methanol Aqueous Acetone Alkaloids - - - - Wagners test Flavonoids - + - + Shimoda Lead acetate test Phenolics & tannins - + + - Lead acetate test Ferric chloride test Steroids & sterols + + - + Salkowski test Carbohydrates - + - - Fehlings test Benedicts test Saponins - - + + Honeycomb test Foam test Glycosides - + - - Glycoside test Protein & amino acids + + + - Biuret test Ninhydrin test Anthraquinone test + + + - Borntragers test Table 14. Preliminary phytochemical screening of lemongrass leaves Results and discussion
Table 15. Quantification of primary metabolites of lemongrass leaves Sl. No. Primary metabolites Weight (mg/g dw) 1 Carbohydrates 150.63 ± 26.83 2 Chlorophyll 2.03 ± 0.02 3 Protein 105.4 ± 2.78 4 Lipids 0.03 ± 0.001 Figure 7. Quantification of secondary metabolites
Conclusion The results indicates that lemon grass leaves can be used as a source of useful drugs because of presence of various phytochemical components
Pandey et. al., 2014 Uttarakhand, India To screen the phytochemicals present in Cinnamon zeylanicum aqueous bark extract Phytochemical Screening of Selected Medicinal Plant Cinnamon zeylanicum Bark Extract Objective
MethodologyMethodology Barks of Cinnamon zeylanicum Washed, shade dried and powdered Preparation of extracts Powdered material mixed with 150ml distilled water for 1 hour in rotary shaker Extract was filtered using muslin cloth & Whatman filter paper Concentrated by evaporation on water bath The extract was dried & used as powder Maceration technique
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative  Alkaloids (Dragendorff’s test)  Steroids (Salkowski test)  Tannins & polyphenols (Ferric chloride test)  Flavonoids (Shinoda test)  Saponins (Froth test)  Glycosides (Legal’s test)  Cardenoloids (Kellar Killani test)  Alkaloids (Dragendorff’s test)  Steroids (Salkowski test)  Tannins & polyphenols (Ferric chloride test)  Flavonoids (Shinoda test)  Saponins (Froth test)  Glycosides (Legal’s test)  Cardenoloids (Kellar Killani test)
Table 16. Phytochemical screening of secondary metabolites of plant extracts Phytoconstituents Extracts Cold water (15ºC) Hot water (70ºC) Warm water (45ºC) Ethanol Methanol Acetone Carbohydrates + + + - - - Steroids + + + + + + Proteins - - - - - - Glycosides - - - - - - Alkaloids + + + + + + Flavonoids + - - + - + Saponins + + + + + + Tannins & phenol - - - + + + Results and discussion
Conclusion Characterization and isolation of the active chemical components possessed by traditional plants may lead to the development of a potential drug that may treat various kinds of infections and may lead to full utilization by the local community
Singh and Bag, 2014 Manipur, India To identify and compare the bioactive constituents present in Hedychium species and determine total phenolic content Phytochemical Analysis and Determination of Total Phenolics Content in Water Extracts of Three Species of Hedychium Objective
MethodologyMethodology Hedychium rubrum Cleaned, shade dried, mechanically grinded & coarsely powdered Preparation of extracts Powdered material Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening Subjected to Hedychium spicatum Hedychium coronarium
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content Folin-Ciocalteu reagent expressed as gallic acid equivalent (mg of gallic acid equivalent / g of sample)  Total phenolic content Folin-Ciocalteu reagent expressed as gallic acid equivalent (mg of gallic acid equivalent / g of sample)  Alkaloids (Hager’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Proteins (Xanthoproteic test)  Flavonoids (Alkaline reagent test)  Saponins (Foam test)  Phenolic compounds (Lead acetate test)  Tannins (Lead acetate & FeCl3 test)  Steroids & terpenoids (Salkowski’s test)  Saponins (Froth test)  Cardiac glycosides (Keller Killiani test)  Oil  Phlobatannin  Alkaloids (Hager’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Proteins (Xanthoproteic test)  Flavonoids (Alkaline reagent test)  Saponins (Foam test)  Phenolic compounds (Lead acetate test)  Tannins (Lead acetate & FeCl3 test)  Steroids & terpenoids (Salkowski’s test)  Saponins (Froth test)  Cardiac glycosides (Keller Killiani test)  Oil  Phlobatannin T = (C x V) M T = TPC (mg/g plant extract) C = concentration of gallic acid (µg/ml) V = volume of extract (ml) M = weight of plant extract (g)
Table 17. Comparative analysis of phytochemical constituents of three different species of Genus Hedychium Phytochemical constituents Chemical tests Water extract H. spicatum H. coronarium H. rubrum Alkaloids Hager’s test - - - Carbohydrates (reducing sugar) Benedict’s test Fehling’s test - + - + + + Proteins Xanthoproteic test + + + Flavonoids Alkaline reagent test + + + Phenolic compounds Lead acetate test + + + Tannins Lead acetate test Ferric chloride test + - + + + + Steroids & terpenoids Salkowski’s test + + + Saponins Froth test + + + Cardiac glycosides Keller-killiani test + + + Oil + + + Results and discussion
Table 18. Total phenolic content in the water extracts of H. Spicatum, H. Coronarium and H. rubrum Water extracts Concentration (mg/ml) mg of gallic acid/g of extract (Mean ± Standard Deviation) H. Spicatum 1 29.39 ± 0.01 H. Coronarium 1 34.93 ± 0.01 H. rubrum 1 66.48 ± 0.01 Figure 8. Callibaration curve of gallic acid
Conclusion Results revealed that the water extracts of three different species of Hedychium contain a good quantity of phenolic compounds These plants can be studied further to know their biological effects which could be a beneficial in the treatment and controlling of various diseases
Vastrad et. al., 2015 Karnataka, India To screen various bio-active compounds present in the leaf extracts of A. vera, O. tenuiflorum and T. cordifolia and evaluate total phenolic content & total flavonoid content Identification of Bio-active Components in Leaf Extracts of Aloe vera, Ocimum tenuiflorum (Tulasi) and Tinospora cordifolia (Amrutballi) Objective
MethodologyMethodology Tinospora cordifolia Cleaned, shade dried, mechanically grinded & coarsely powdered Preparation of extracts Powdered material Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening Subjected to Aloe vera Ocimum tenuiflorum
Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative QuantitativeQuantitative  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent (GAE) in milligrams per gram of fresh leaf  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent (GAE) in milligrams per gram of fresh leaf  Total flavonoid content Aluminium chloride colourimetric method expressed as mg rutin equivalent (mg RE) per gram of fresh leaf  Total flavonoid content Aluminium chloride colourimetric method expressed as mg rutin equivalent (mg RE) per gram of fresh leaf  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins  Terpenoids  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins  Terpenoids
Figure 9. Yield of extracts Results and discussion
Table 20. Total phenolic content (TPC) of the plant leaf extracts Extraction solvent Total phenolic content (GAE* mg/g) A. vera O. tenuiflorum T. cordifolia Aqueous 94.42 ± 4.92 80.82 ± 8.63 465.82 ± 23.04 Ethanol 138.13 ± 6.63 113.07 ± 9.81 264.06 ± 18.41 Methanol 95.20 ± 3.23 114.34 ± 11.86 301.42 ± 29.69 GAE = Gallic acid equivalent Fig 10. Total phenolic content (TPC): Calibration curve
Table 21. Total flavonoid content (TFC) of the plant leaf extracts RE = Rutin equivalent Figure 11. Total flavonoid content (TFC): Calibration curve Extraction solvent Total flavonoid content (RE* mg/g) A. vera O. tenuiflorum T. cordifolia Aqueous 72.28 ± 8.70 61.84 ± 7.25 178.43 ± 6.61 Ethanol 76.50 ± 8.57 95.46 ± 4.12 208.36 ± 2.86 Methanol 88.59 ± 8.38 96.34 ± 5.85 132.59 ± 7.59
Conclusion  The results revealed that alkaloids were found to be present in all the extracts of A. vera, O. tenuiflorum and T. cordifolia.  Flavonoids were present in ethanol, methanol and aqueous extracts of A. vera and T. cordifolia  TPC was high in ethanol extract of Aloe vera, methanol extract of O. tenuiflorum and aqueous extract of T. cordifolia  TFC was high in methanol extract of A. vera, methanol and ethanol extracts of O. tenuiflorum and ethanol extract of T. cordifolia

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Extraction and phytochemical analysis of medicinal plants

  • 2.
    2 Seminar on Extraction andPhytochemical Analysis of Medicinal Plants by Shameembanu A. ByadgiShameembanu A. Byadgi
  • 3.
    Medicinal plants  Medicinalplants constitute an effective source of both traditional and modern medicines  Herbal medicine has been shown to have genuine utility  About 80% of rural population depends on it as primary health care. [WHO, (2005)]
  • 4.
    Medicinal plants arethe richest bio-resource  drugs of traditional systems of medicine  modern medicines  nutraceuticals  food supplements  folk medicines  pharmaceutical intermediates  chemical entities for synthetic drugs
  • 5.
     Natural bioactivecompounds found in different parts of plant (fruit, flower, stem, leaf, root)  Provide definite physiological action on the human body  Bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids  Widely used in the human therapy, veterinary, agriculture, scientific research and countless other areas  Have inhibitory effects on all types of microorganisms in vitro Phytochemicals
  • 6.
    Extraction ……… is theseparation of medicinally active portions of plant tissues using selective solvents through standard procedures The basic parameters influencing the quality of an extract •Plant part used as starting material •Solvent used for extraction •Extraction procedure
  • 7.
    Choice of solvents Successfuldetermination of biologically active compounds depends on the type of solvent used in the extraction procedure Property of a good solvent in plant extraction •Low toxicity •Ease of evaporation at low heat •Promotion of rapid physiologic absorption of the extract •Preservative action
  • 8.
    The factors affectingthe choice of solvent •Quantity of phytochemicals to be extracted •Rate of extraction •Diversity of different inhibitory compounds extracted •Ease of subsequent handling of the extracts •Toxicity of the solvent in the bioassay process •Potential health hazard of the extractants
  • 9.
    Solvents used foractive component extraction Water Ethanol Methanol Chloroform Ether Acetone Anthocyanins Starches Tannins Saponins Terpenoids Polypeptides Lectins Tannins Polyphenols Polyacetylenes Flavonols Terpenoids Sterols Alkaloids Anthocyanins Terpenoids Saponins Tannins Xanthoxyllines Totarol Quassinoids Lactones Flavones Phenones Polyphenols Terpenoids Flavonoids Alkaloids Terpenoids Coumarins Fatty acids Phenol Flavonols
  • 10.
    General techniques ofmedicinal plant extraction  Plant tissue homogenization  Maceration  Infusion  Percolation  Digestion  Decoction  Soxhlet extraction (Hot continuous extraction)  Sonication (Ultrasound extraction)
  • 11.
  • 12.
    Maceration Whole / coarselypowdered crude drug is placed in a stoppered container with the solvent Whole / coarsely powdered crude drug is placed in a stoppered container with the solvent Allow to stand @ room temperature for a period of at least 3 days with frequent agitation until the soluble matter gets dissolved Allow to stand @ room temperature for a period of at least 3 days with frequent agitation until the soluble matter gets dissolved The mixture then is strained, the marc (the damp solid material) is pressed The mixture then is strained, the marc (the damp solid material) is pressed The combined liquids are clarified by filtration or decantation after standing The combined liquids are clarified by filtration or decantation after standing
  • 13.
  • 14.
    Digestion • A formof maceration in which gentle heat is used during the process of extraction • Used when moderately elevated temperature is not objectionable • The solvent efficiency of the menstruum is thereby increased Microwave digestion system
  • 15.
    Decoction  Suitable forextracting  water-soluble, heat-stable constituents  Typically used in preparation of Ayurvedic extracts
  • 16.
    Percolation • Used mostfrequently to extract active ingredients in the preparation of fluid extracts • The solid ingredients are moistened with an appropriate amount of the specified menstruum • Allowed to stand for approximately 4 hours in a well closed container, After stand time, the mass is packed & the top of the percolator is closed • The mixture is allowed to macerate in the closed percolator for 24 h
  • 17.
    , • Additional menstruumis added as required, until the percolate measures about three-quarters of the required volume of the finished product • The marc is then pressed and the expressed liquid is added to the percolate • Sufficient menstruum is added to produce the required volume • The mixed liquid is clarified by filtration or by standing followed by decanting
  • 18.
  • 20.
    Sonication (Ultrasound Extraction) • Involvesthe use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz • Increases the permeability of cell walls & produces cavitation Disadvantage Deleterious effect of ultrasound energy (>20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules
  • 21.
    Effect of extractedplant phytochemicals depends on • The nature & origin of the plant material • Degree of processing • Moisture content • Particle size
  • 22.
    Variation in extractionmethods • Length of the extraction period • Solvent used • pH of the solvent • Temperature • Particle size of the plant tissues • Solvent-to-sample ratio
  • 23.
    Phytochemical screening methodsPhytochemicalscreening methods Phytochemicals Tests Reagents Positive results Alkaloids Dragendorff test Dragendorff’s reagent Prominent yellow ppt Wagner test Wagner’s reagent Reddish brown ppt Mayer test 1% HCl, Mayer’s reagent Turbid extract is obtained Flavonoids Ammonia test 1% NH3 Yellow colour Sodium hydroxide test 20% NaOH, HCl Yellow colour turns to colourless Tannins Ferric chloride test 5% FeCl3 Blue-black or blue- green colouration Phenolic compounds Gelatine test 1% gelatine solution containing 10% NaCl White ppt Lead acetate test 10% lead acetate Bulky white ppt Saponins Foam test 20 mL distilled water (mixed vigorously for 15 minutes) Presence of froth Qualitative Analysis
  • 24.
    Phytochemicals Tests ReagentsPositive results Terpenoids Salkowski test 0.5 mL chloroform, 1 mL conc. H2SO4 Reddish brown colouration at the interface Carbohydrates Molish test Conc. HCl Violet ring Fehling test Conc. HCl & Mg turnings Yellow & brick red ppt Proteins Biuret test 4% NaOH, 1% CuSO4 Violet or pink colour Glycosides Legal’s test Pyridine, sodium nitroprusside Pink to red colour Kellar killani test Glacial acetic acid, 5% FeCl3 Reddish brown & bluish green colour Contd....
  • 25.
    Quantitative Analysis Total PhenolicContent Determined by Folin-Ciocalteau assay method (Singleton and Rossi, 1965) Instrument: UV-Vis Spectrophotometer, absorbance measured at 765 nm Expressed as Gallic acid equivalent (GAE) in milligrams per gram of fresh leaf Total Flavonoid Content Determined by Colourimetric method (Yun et al., 2009) Instrument: UV-Vis Spectrophotomer, absorbance measured at λ415 nm Expressed as mg rutin equivalent (mg RE) per gram of fresh leaf
  • 26.
    Yadav and Agarwala,2011 Assam, India To carry out qualitative and quantitative phytochemical analysis of selected medicinal plants Phytochemical Analysis of Some Medicinal Plants Objective
  • 27.
    MethodologyMethodology Plant sources Bryophyllum pinnatum(Leaves) Ipomea aquatica (Leaves) Oldenlandia corymbosa (Whole plant) Ricinus communis (roots) Terminalia bellerica (Leaves) Tinospora cordifolia (Leaves/Stem) Xanthium strumarium (Leaves)
  • 28.
    Hot water extraction 5gmof dried finely powdered plant material mixed with 200ml of distilled water Heated on a hot plate with continuous stirring at 30º-40ºC for 20 minutes filtered through filter paper Solvent extraction 20gm powdered plant material packed into a thimble and extracted with 250ml of solvents Extraction continues for 24 hours or till the solvent in siphon tube of an extractor become colourless kept on hot plate and heated at 30-40ºC Preparation of extracts
  • 29.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content FolinCiocalteu reagent expressed as gallic acid equivalent, mg/g of extracted compound  Total phenolic content FolinCiocalteu reagent expressed as gallic acid equivalent, mg/g of extracted compound  Total flavonoid content Aluminium chloride colourimetric method expressed as quercetin equivalent, mg/g of extracted compound  Total flavonoid content Aluminium chloride colourimetric method expressed as quercetin equivalent, mg/g of extracted compound  Proteins (Millon’s & Ninhydrin test)  Carbohydrates (Fehling’s, Benedict’s, Molisch’s & Iodine test)  Phenols & tannins (Ferric chloride test)  Flavonoids (Shinoda & alkaline reagent test)  Saponins (Foam test)  Glycosides (Liebermann’s, Salkowski’s & Keller-kilani test)  Steroids  Terpenoids  Alkaloids (Mayer’s & Wagner’s test)  Proteins (Millon’s & Ninhydrin test)  Carbohydrates (Fehling’s, Benedict’s, Molisch’s & Iodine test)  Phenols & tannins (Ferric chloride test)  Flavonoids (Shinoda & alkaline reagent test)  Saponins (Foam test)  Glycosides (Liebermann’s, Salkowski’s & Keller-kilani test)  Steroids  Terpenoids  Alkaloids (Mayer’s & Wagner’s test)
  • 30.
    Table 1. Phytochemicalconstituents of medicinal plants Results and discussion L = leaves; S = stem
  • 31.
    Fig 1. Totalphenolic content Fig 2. Total flavonoid content
  • 32.
    Conclusion  Results revealedthat extracts from these plants can be used as a good source for drugs  Further work should be carried out to isolate, purify and characterize the active constituents responsible for the activity of these plants
  • 33.
    Badugu, 2012 To screenthe presence of phytochemicals and estimate the amount of total phenols and flavonoids in Cyamopsis tetragonoloba Phytochemical Screening, Quantitative Estimation Total Phenolics and Total Flavonoids, Antimicrobial Evaluation of Cyamopsis tetragonoloba Objective
  • 34.
    MethodologyMethodology Seeds of Cyamopsistetragonoloba Washed, shade dried & powdered mechanically Preparation of extracts Powdered material was weighed Soxhlet extraction using methanol, acetone, chloroform & hexane Solvent was recovered using Rotary Vacuum Evaporator Phytochemical screening Subjected to 48 hrs
  • 35.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content Folin-Ciocalteu reagent expressed in terms of milligrams of catechol per gram of dry weight  Total phenolic content Folin-Ciocalteu reagent expressed in terms of milligrams of catechol per gram of dry weight  Total flavonoid content Aluminium chloride colourimetric method expressed as catechin equivalent, mg catechin/g dried extract  Total flavonoid content Aluminium chloride colourimetric method expressed as catechin equivalent, mg catechin/g dried extract  Carbohydrates, reducing sugars, monosaccharides, alkaloids, saponins & tannins (Evans, 1996)  Flavonoids (Shinoda’s test)  Terpenes/ steroids (Liebermann- Burchard’s test)  Anthraquinones (Borntrager’s test)  Cardiac glucosides (Sodium nitro proside method)  Proteins (Copper sulphate & Folin- Ciocalteau solution)  Amino acids (Ninhydrin test)  Carbohydrates, reducing sugars, monosaccharides, alkaloids, saponins & tannins (Evans, 1996)  Flavonoids (Shinoda’s test)  Terpenes/ steroids (Liebermann- Burchard’s test)  Anthraquinones (Borntrager’s test)  Cardiac glucosides (Sodium nitro proside method)  Proteins (Copper sulphate & Folin- Ciocalteau solution)  Amino acids (Ninhydrin test)
  • 36.
    Table 2. Phytochemicalscreening of Cyamopsis tetragonoloba Sl. No. Secondary metabolites Tests Solvents Methanol Acetone Chloroform Hexane 1 Carbohydrates Molisch’s test +ve +ve +ve +ve 2 Reducing sugars Fehling test +ve +ve -ve -ve 3 Monosaccharide Barfoed’s test -ve -ve -ve -ve 4 Tannins Ferric chloride test Lead sub acetate test -ve -ve -ve +ve 5 Saponins Frothing test +ve -ve -ve -ve 6 Flavonoids Shinoda’s test +ve +ve -ve -ve 7 Terpenes/ steroids Liebermann Burchard’s test +ve -ve +ve -ve 8 Alkaloids Mayer’s test Wagner’s test +ve +ve -ve -ve 9 Cardiac glucosides Sodium nitroproside +ve +ve -ve +ve 10 Proteins Copper sulphate Folin Ciocalteau solution -ve -ve -ve -ve 11 Amino acids Ninhydrin -ve +ve -ve +ve 12 Anthraquinones Borntrager’s test -ve -ve -ve -ve Results and discussion
  • 37.
    Table 3. Totalphenolic and flavonoids content of different extracts of Cyamopsis tetragonoloba Extract Total phenolic content mg of Catechin equivalents/ 200mg dried extract Total flavonoids content mg of Catechol equivalents/ 200mg dried extract Methanol 14.51 17.34 Acetone 9.17 8.75 Chloroform 11.32 14.05 Hexane 5.53 6.41
  • 38.
    Conclusion  The studyindicated that methanol is used as best solvent for extraction of phytochemicals  Other solvents like chloroform, acetone and hexane are less commonly used for the extraction of phytochemicals  The herbal extract can be used for curing diseases without any side effects
  • 39.
    Hasanuzzaman et. al.,2013 Bangladesh To investigate total phenolic content of methanolic extract of Averrhoa bilimbi fruits Evaluation of Total Phenolic Content, Free Radical Scavenging Activity and Phytochemical Screening of Different Extracts of Averrhoa bilimbi Objective
  • 40.
    MethodologyMethodology Fruits of Averrhoabilimbi Washed, sliced & dried under sun Preparation of extracts Powdered fruits (500g) 2500 ml methanol for 15 days at room temperature Extract filtered using filter cloth & Whatman’s filter paper Filtrate evaporated under ceiling fan & in a water bath below 40°C until dried Soaked in Cold maceration technique
  • 41.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content Folin-Ciocalteu reagent (oxidizing agent) & gallic acid (standard) expressed as mg of GAE (gallic acid equivalent) / gm of the extractive  Total phenolic content Folin-Ciocalteu reagent (oxidizing agent) & gallic acid (standard) expressed as mg of GAE (gallic acid equivalent) / gm of the extractive  Alkaloids (Mayer’s & Dragendoff’s test)  Tannins (FeCl3 test)  Saponins (Foam test)  Flavonoids (chip of magnesium & HCl)  Glycosides (NaCl & Fehling’s solutions A & B)  Steroids & triterpenes (Ethylic, H2SO4 & anhydride acetic)  Phenols (FeCl3 & K3Fe(CN6))  Cardiac glycosides (Acetic, FeCl3 & conc. H2SO4)  Carbohydrates (Alcoholic α-naphthol, Benedict’s reagent)  Alkaloids (Mayer’s & Dragendoff’s test)  Tannins (FeCl3 test)  Saponins (Foam test)  Flavonoids (chip of magnesium & HCl)  Glycosides (NaCl & Fehling’s solutions A & B)  Steroids & triterpenes (Ethylic, H2SO4 & anhydride acetic)  Phenols (FeCl3 & K3Fe(CN6))  Cardiac glycosides (Acetic, FeCl3 & conc. H2SO4)  Carbohydrates (Alcoholic α-naphthol, Benedict’s reagent)
  • 42.
    Table 4. Phytochemicalscreening of different extractives of Averrhoa bilimbi fruits Tests Extracts MEF PTSF CTSF CSF AQSF Alkaloids + + - + + Tannins + + + - - Saponins + + - - + Flavonoids + + + + + Cardiac glycosides + + - + + Glycosides + + + + + Phytosterols - - - - - Triterpenes + + - + - Phenols + + + + + Carbohydrates + - + + + MEF = methanolic extract; PTSF = pet-ether soluble fraction; CTSF = carbon tetrachloride soluble fraction; CSF = chloroform soluble fraction; AQSF = aqueous soluble fraction Results and discussion
  • 43.
    Table 5. Totalphenolic content of different extractives of Averrhoa bilimbi fruits Extractives Total phenol content (mg of GAE/g of extractive) Methanolic extract 65.16 ± 0.52 Pet-ether soluble fraction 55.31 ± 1.01 Carbon tetrachloride soluble fraction 52.00 ± 0.90 Chloroform soluble fraction 68.67 ± 0.94 Aqueous soluble fraction 50.23 ± 0.56 GAE = Gallic acid equivalent
  • 44.
    Conclusion  It maybe concluded that Averrhoa bilimbi fruits is a good source of phytochemicals  Different extractives showed significant total phenolic content
  • 45.
    Pranoothi et. al.,2014 Andhra Pradesh, India To carry out qualitative and quantitative phytochemical analysis of aerial parts of Leucas indica (L) Studies on Qualitative, Phytochemical Analysis and Screening of In Vitro Biological Activities of Leucas indica (L) Objective
  • 46.
    MethodologyMethodology Aerial parts ofLeucas indica Cleaned, shade dried, mechanically grinded & coarsely powdered Preparation of extracts Powdered material Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening Subjected to
  • 47.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content FolinCiocalteu reagent expressed as µg of gallic acid equivalents per gram dry mass of extract (µg GAE/gDM)  Total phenolic content FolinCiocalteu reagent expressed as µg of gallic acid equivalents per gram dry mass of extract (µg GAE/gDM)  Total flavonoid content Aluminium chloride colourimetric assay expressed with the rutin equivalents per g of dried fraction)  Total flavonoid content Aluminium chloride colourimetric assay expressed with the rutin equivalents per g of dried fraction)  Alkaloids  Steroidal compounds  Phenolic compounds  Flavonoids  Saponins  Tannins  Coumarins  Cardiac glycosides  Alkaloids  Steroidal compounds  Phenolic compounds  Flavonoids  Saponins  Tannins  Coumarins  Cardiac glycosides
  • 48.
    Table 6. Physico-chemicalevaluation Solvent Initial weight of the powder (g) Final weight of the powder (g) Weight of the crude extract (g) Crude extract (%) Colour of the extract Hexane 50 44.563 5.437 10.874 Dark brown Acetone 50 40.415 9.585 19.17 Dark green Methanol 50 35.552 14.448 28.896 Dark green Water 50 38.621 11.379 22.758 Dark red Figure 3. Yield of extracts Results and discussion
  • 49.
    Table 7. Phytochemicalanalysis of whole aerial part extracts of Leucas indica (L) Sl. No. Tests Extracts Hexane Acetone Methanol Water 1 Alkaloids Mayers - + + + Dragon - + - + Wagners - + + + Hagers - + - + 2 Phenolics FeCl2 test - + + + 3 Flavonoids Lead acetate test - + + + NaOH test - + + - Ethyl acetate test - - - - 4 Anthraquinones Borntrager’s test - - - - 5 Steroids Salkowski’s test + + + +
  • 50.
    6 Tannins FeCl2 test- + + - Lead acetate test - + + - Potassium dichromate test - + + - 7 Saponins Vigorous shaking test + + + + 8 Anthocyanins Ammonia-HCL test - - - - 9 Leuco-Anthocyanin Iso amyl alcohol test - - - - 10 Coumarins NaOH test - - - - 11 Reducing sugars Keller-Kiliani test + + + + Sl. No. Tests Extracts Hexane Acetone Methanol Water Contd...
  • 51.
    Table 8. Totalphenol content % of phenol content µg GAE/µg Concentration (µg/ml) Hexane extract Acetone extract Methanol extract Water extract 100 15.69 18.5 25.6 22.8 200 22.54 26.9 45.86 41.8 300 30.41 38.9 72.8 68.4 400 42.89 54.78 88.96 76.5 500 54.58 65.8 105.68 95.8 Figure 4. Total phenol content
  • 52.
    Table 9. Totalflavonoid content % of phenol content µg GAE/µg Concentration (µg/ml) Hexane extract Acetone extract Methanol extract Water extract 100 00 10.28 17.25 00 200 00 15.56 26.35 00 300 00 32.47 29.12 13.20 400 00 41.58 43.52 18.25 500 00 50.24 62.34 24.69 Figure 5. Total flavonoid content
  • 53.
    Conclusion  The presenceof most general phytochemicals in Leucas indica might be responsible for their therapeutic effects  It further reflects a hope for the development of many more novel chemotherapeutic agents from plants which in future may serve for the production of synthetically improved therapeutic agents
  • 54.
    Rajesh et. al.,2014 Tamil Nadu, India To evaluate the phytochemicals from aqueous, ethanolic and petroleum ether extracts of four fern species Qualitative and Quantitative Phytochemical Analysis in Four Pteridophytes Objective
  • 55.
    MethodologyMethodology Washed & cutinto small pieces Shade dried & then hot air oven drying at 55-60ºC Dried leaves, stems & roots were pulverized using mixer Strained through 25 mm mesh diameter sieve to obtain fine dust Phytochemical screening Actinopteris radiata Pityrogramma calomelanos Drynaria quercifolia Dryopteris cochleata
  • 56.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total tannins Folin-Ciocalteu reagent expressed as mg tannic acid equivalent per 100g of the sample)  Total tannins Folin-Ciocalteu reagent expressed as mg tannic acid equivalent per 100g of the sample)  Total phenols Folin-Ciocalteu reagent method expressed in mg of gallic acid equivalent (GAE) per g of dry weight of plant powdres  Total phenols Folin-Ciocalteu reagent method expressed in mg of gallic acid equivalent (GAE) per g of dry weight of plant powdres  Alkaloids (Mayer’s test)  Anthocyanin & betacyanin (NaOH test)  Cardio glycosides  Coumarins  Flavonoids (NaOH test)  Glycosides  Phenols  Quinones  Saponins (Foam test)  Steroids  Tannins  Terpenoids  Alkaloids (Mayer’s test)  Anthocyanin & betacyanin (NaOH test)  Cardio glycosides  Coumarins  Flavonoids (NaOH test)  Glycosides  Phenols  Quinones  Saponins (Foam test)  Steroids  Tannins  Terpenoids
  • 57.
    Table 10. Qualitativeanalysis of phytochemicals in four fern species Table 11. Quantitative analysis of phytochemicals (mg/g) in four fern species Phytochemicals A.radiata D.quercifolia D.cochieata P.calamelanos Total tannin 12.189 ± 0.258 6.332 ± 0.187 9.405 ± 0.299 17.181 ± 0.441 Total phenol 10.962 ± 0.327 7.131 ± 0.184 8.912 ± 0.310 13.581 ± 0.481 Results and discussion
  • 58.
    Conclusion  The resultsrevealed that ethanolic solvent performed well exhibiting the presence of major phytochemicals compared to aqueous and petroleum ether extracts  Quantitative analysis showed highest content of tannins and phenols in Pityrogramma calomelanos fern extract followed with least content of tannins and phenols in Actinopteris radiata, Dryopteris cochleata and Drynaria quercifolia
  • 59.
    Vastrad et. al.,2014 Karnataka, India To screen the presence of phytochemicals in leaf extracts of C. fistula, P. pinnata, T. grandis & J. curcas and assess total phenolic & total flavonoid content Characterization of Phytoconstituents in Leaf Extracts of Forest Species for Textile Applications Objective
  • 60.
    MethodologyMethodology Cleaned, shade dried,chopped & ground in laboratory mortar & pestle Ground leaf was mixed with the solvent & incubated (200 strokes) in incubator cum shaker for 24 hours at 25ºC The extract was centrifuged (5000 rpm) at room temperature & supernatant separated Residue was re-extracted with the respective solvent The extract was filtered using Whatman filter paper & yield was measured Cassia fistula Pongamia pinnata Tectona grandis Jatropha curcas
  • 61.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent in milligrams per gram of fresh leaf)  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent in milligrams per gram of fresh leaf)  Total flavonoid content Colourimetric method expressed as milligrams of rutin equivalent (mg RE) per gram of sample)  Total flavonoid content Colourimetric method expressed as milligrams of rutin equivalent (mg RE) per gram of sample)  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins
  • 62.
    Table 12. Qualitativephytochemical screening of forest species Figure 6. Total phenolic content (mg per gram of fresh leaf) Results and discussion
  • 63.
    Table 13. Totalflavonoid content (TFC) of extracts in forest species Forest species TFC (mg/g of sample) Ethyl alcohol Methanol Aqueous Cassia fistula 106.4 126.21 9.49 Pongamia pinnata 121.53 148.33 8.96 Tectona grandis 153.52 179.1 16.71 Jatropha curcas 149.70 138.7 -
  • 64.
    Conclusion  The resultsrevealed that phytochemicals viz., alkaloids, flavonoids, tannins and terpenoids were found in appreciable amount in the selected forest species  Methanolic extract of C. fistula, P. pinnata and T. grandis exhibited maximum TPC and TFC content while J. curcas depicted higher amount of TPC and TFC content in ethanolic extract  Therefore it can be concluded that extracts from forest species such as C. fistula, P. pinnata and T. grandis and J. curcas can be used for applying eco friendly and healthy finishes to textile substrates
  • 65.
    Geetha and Geetha,2014 Tamil Nadu, India To carry out qualitative and quantitative phytochemical analysis of lemongrass leaves Phytochemical Screening, Quantitative Analysis of Primary and Secondary Metabolites of Cymbopogan citratus (DC) Leaves Objective
  • 66.
    MethodologyMethodology Leaves of Cymbopogancitratus Washed & air dried under shade Preparation of extracts Powdered leaf Fresh leaf Extracted with chloroform, methanol & acetone using soxhlet apparatus Ground using distilled water & filtered; used as an aqueous extract Concentrated using rotary vacuum evaporator & dried
  • 67.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative Alkaloids (Wagner’s test)  Flavonoids (Shinoda & lead acetate test)  Phenols & tannins (Lead acetate, FeCl3 & NaOH test)  Steroids & sterols (Salkowski’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Saponins (Honey comb & foam test)  Glycosides (Glycoside test)  Protein & amino acids (Biuret & Ninhydrin test)  Anthraquinones (Borntragers test)  Alkaloids (Wagner’s test)  Flavonoids (Shinoda & lead acetate test)  Phenols & tannins (Lead acetate, FeCl3 & NaOH test)  Steroids & sterols (Salkowski’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Saponins (Honey comb & foam test)  Glycosides (Glycoside test)  Protein & amino acids (Biuret & Ninhydrin test)  Anthraquinones (Borntragers test)
  • 68.
    Quantitative analysisQuantitative analysis Total phenolics Expressed as tannic acid equivalents  Total phenolics Expressed as tannic acid equivalents  Total flavonoid content Colourimetric method expressed as rutin equivalent  Total flavonoid content Colourimetric method expressed as rutin equivalent  Carbohydrates  Total chlorophyll content  Proteins  Total lipid content  Carbohydrates  Total chlorophyll content  Proteins  Total lipid content  Total tannins Tannin (%) = Total phenolics (%) – Non tannin phenolics (%)  Total tannins Tannin (%) = Total phenolics (%) – Non tannin phenolics (%) Primary metabolites Secondary metabolites
  • 69.
    Plant constituent Extracts TestsCholoroform MethanolAqueous Acetone Alkaloids - - - - Wagners test Flavonoids - + - + Shimoda Lead acetate test Phenolics & tannins - + + - Lead acetate test Ferric chloride test Steroids & sterols + + - + Salkowski test Carbohydrates - + - - Fehlings test Benedicts test Saponins - - + + Honeycomb test Foam test Glycosides - + - - Glycoside test Protein & amino acids + + + - Biuret test Ninhydrin test Anthraquinone test + + + - Borntragers test Table 14. Preliminary phytochemical screening of lemongrass leaves Results and discussion
  • 70.
    Table 15. Quantificationof primary metabolites of lemongrass leaves Sl. No. Primary metabolites Weight (mg/g dw) 1 Carbohydrates 150.63 ± 26.83 2 Chlorophyll 2.03 ± 0.02 3 Protein 105.4 ± 2.78 4 Lipids 0.03 ± 0.001 Figure 7. Quantification of secondary metabolites
  • 71.
    Conclusion The results indicatesthat lemon grass leaves can be used as a source of useful drugs because of presence of various phytochemical components
  • 72.
    Pandey et. al.,2014 Uttarakhand, India To screen the phytochemicals present in Cinnamon zeylanicum aqueous bark extract Phytochemical Screening of Selected Medicinal Plant Cinnamon zeylanicum Bark Extract Objective
  • 73.
    MethodologyMethodology Barks of Cinnamonzeylanicum Washed, shade dried and powdered Preparation of extracts Powdered material mixed with 150ml distilled water for 1 hour in rotary shaker Extract was filtered using muslin cloth & Whatman filter paper Concentrated by evaporation on water bath The extract was dried & used as powder Maceration technique
  • 74.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitative Alkaloids (Dragendorff’s test)  Steroids (Salkowski test)  Tannins & polyphenols (Ferric chloride test)  Flavonoids (Shinoda test)  Saponins (Froth test)  Glycosides (Legal’s test)  Cardenoloids (Kellar Killani test)  Alkaloids (Dragendorff’s test)  Steroids (Salkowski test)  Tannins & polyphenols (Ferric chloride test)  Flavonoids (Shinoda test)  Saponins (Froth test)  Glycosides (Legal’s test)  Cardenoloids (Kellar Killani test)
  • 75.
    Table 16. Phytochemicalscreening of secondary metabolites of plant extracts Phytoconstituents Extracts Cold water (15ºC) Hot water (70ºC) Warm water (45ºC) Ethanol Methanol Acetone Carbohydrates + + + - - - Steroids + + + + + + Proteins - - - - - - Glycosides - - - - - - Alkaloids + + + + + + Flavonoids + - - + - + Saponins + + + + + + Tannins & phenol - - - + + + Results and discussion
  • 76.
    Conclusion Characterization and isolationof the active chemical components possessed by traditional plants may lead to the development of a potential drug that may treat various kinds of infections and may lead to full utilization by the local community
  • 77.
    Singh and Bag,2014 Manipur, India To identify and compare the bioactive constituents present in Hedychium species and determine total phenolic content Phytochemical Analysis and Determination of Total Phenolics Content in Water Extracts of Three Species of Hedychium Objective
  • 78.
    MethodologyMethodology Hedychium rubrum Cleaned, shadedried, mechanically grinded & coarsely powdered Preparation of extracts Powdered material Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening Subjected to Hedychium spicatum Hedychium coronarium
  • 79.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content Folin-Ciocalteu reagent expressed as gallic acid equivalent (mg of gallic acid equivalent / g of sample)  Total phenolic content Folin-Ciocalteu reagent expressed as gallic acid equivalent (mg of gallic acid equivalent / g of sample)  Alkaloids (Hager’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Proteins (Xanthoproteic test)  Flavonoids (Alkaline reagent test)  Saponins (Foam test)  Phenolic compounds (Lead acetate test)  Tannins (Lead acetate & FeCl3 test)  Steroids & terpenoids (Salkowski’s test)  Saponins (Froth test)  Cardiac glycosides (Keller Killiani test)  Oil  Phlobatannin  Alkaloids (Hager’s test)  Carbohydrates (Fehling’s & Benedict’s test)  Proteins (Xanthoproteic test)  Flavonoids (Alkaline reagent test)  Saponins (Foam test)  Phenolic compounds (Lead acetate test)  Tannins (Lead acetate & FeCl3 test)  Steroids & terpenoids (Salkowski’s test)  Saponins (Froth test)  Cardiac glycosides (Keller Killiani test)  Oil  Phlobatannin T = (C x V) M T = TPC (mg/g plant extract) C = concentration of gallic acid (µg/ml) V = volume of extract (ml) M = weight of plant extract (g)
  • 80.
    Table 17. Comparativeanalysis of phytochemical constituents of three different species of Genus Hedychium Phytochemical constituents Chemical tests Water extract H. spicatum H. coronarium H. rubrum Alkaloids Hager’s test - - - Carbohydrates (reducing sugar) Benedict’s test Fehling’s test - + - + + + Proteins Xanthoproteic test + + + Flavonoids Alkaline reagent test + + + Phenolic compounds Lead acetate test + + + Tannins Lead acetate test Ferric chloride test + - + + + + Steroids & terpenoids Salkowski’s test + + + Saponins Froth test + + + Cardiac glycosides Keller-killiani test + + + Oil + + + Results and discussion
  • 81.
    Table 18. Totalphenolic content in the water extracts of H. Spicatum, H. Coronarium and H. rubrum Water extracts Concentration (mg/ml) mg of gallic acid/g of extract (Mean ± Standard Deviation) H. Spicatum 1 29.39 ± 0.01 H. Coronarium 1 34.93 ± 0.01 H. rubrum 1 66.48 ± 0.01 Figure 8. Callibaration curve of gallic acid
  • 82.
    Conclusion Results revealed thatthe water extracts of three different species of Hedychium contain a good quantity of phenolic compounds These plants can be studied further to know their biological effects which could be a beneficial in the treatment and controlling of various diseases
  • 83.
    Vastrad et. al.,2015 Karnataka, India To screen various bio-active compounds present in the leaf extracts of A. vera, O. tenuiflorum and T. cordifolia and evaluate total phenolic content & total flavonoid content Identification of Bio-active Components in Leaf Extracts of Aloe vera, Ocimum tenuiflorum (Tulasi) and Tinospora cordifolia (Amrutballi) Objective
  • 84.
    MethodologyMethodology Tinospora cordifolia Cleaned, shade dried,mechanically grinded & coarsely powdered Preparation of extracts Powdered material Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening Subjected to Aloe vera Ocimum tenuiflorum
  • 85.
    Phytochemical AnalysisPhytochemical Analysis QualitativeQualitativeQuantitativeQuantitative  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent (GAE) in milligrams per gram of fresh leaf  Total phenolic content Folin-Ciocalteu assay method expressed as gallic acid equivalent (GAE) in milligrams per gram of fresh leaf  Total flavonoid content Aluminium chloride colourimetric method expressed as mg rutin equivalent (mg RE) per gram of fresh leaf  Total flavonoid content Aluminium chloride colourimetric method expressed as mg rutin equivalent (mg RE) per gram of fresh leaf  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins  Terpenoids  Tannins & phenolic compounds  Flavonoids  Alkaloids  Saponins  Terpenoids
  • 86.
    Figure 9. Yieldof extracts Results and discussion
  • 87.
    Table 20. Totalphenolic content (TPC) of the plant leaf extracts Extraction solvent Total phenolic content (GAE* mg/g) A. vera O. tenuiflorum T. cordifolia Aqueous 94.42 ± 4.92 80.82 ± 8.63 465.82 ± 23.04 Ethanol 138.13 ± 6.63 113.07 ± 9.81 264.06 ± 18.41 Methanol 95.20 ± 3.23 114.34 ± 11.86 301.42 ± 29.69 GAE = Gallic acid equivalent Fig 10. Total phenolic content (TPC): Calibration curve
  • 88.
    Table 21. Totalflavonoid content (TFC) of the plant leaf extracts RE = Rutin equivalent Figure 11. Total flavonoid content (TFC): Calibration curve Extraction solvent Total flavonoid content (RE* mg/g) A. vera O. tenuiflorum T. cordifolia Aqueous 72.28 ± 8.70 61.84 ± 7.25 178.43 ± 6.61 Ethanol 76.50 ± 8.57 95.46 ± 4.12 208.36 ± 2.86 Methanol 88.59 ± 8.38 96.34 ± 5.85 132.59 ± 7.59
  • 89.
    Conclusion  The resultsrevealed that alkaloids were found to be present in all the extracts of A. vera, O. tenuiflorum and T. cordifolia.  Flavonoids were present in ethanol, methanol and aqueous extracts of A. vera and T. cordifolia  TPC was high in ethanol extract of Aloe vera, methanol extract of O. tenuiflorum and aqueous extract of T. cordifolia  TFC was high in methanol extract of A. vera, methanol and ethanol extracts of O. tenuiflorum and ethanol extract of T. cordifolia