extraction and phytochemical analysis of medicinal plants

2

Seminar on

Extraction and Phytochemical Analysis

of Medicinal Plantsby

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 AcetoneAnthocyaninsStarchesTanninsSaponinsTerpenoidsPolypeptides

Lectins

TanninsPolyphenolsPolyacetylenesFlavonolsTerpenoidsSterols

Alkaloids

AnthocyaninsTerpenoidsSaponinsTanninsXanthoxyllinesTotarolQuassinoidsLactonesFlavonesPhenones

Polyphenols

Terpenoids

Flavonoids

AlkaloidsTerpenoidsCoumarins

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

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 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 methods

Phytochemicals Tests Reagents Positive resultsAlkaloids Dragendorff test Dragendorff’s reagent Prominent yellow ppt

Wagner test Wagner’s reagent Reddish brown pptMayer test 1% HCl, Mayer’s

reagentTurbid extract is obtained

Flavonoids Ammonia test 1% NH3 Yellow colourSodium 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 pptSaponins Foam test 20 mL distilled water

(mixed vigorously for 15 minutes)

Presence of froth

Qualitative Analysis

Phytochemicals Tests Reagents Positive resultsTerpenoids Salkowski test 0.5 mL chloroform,

1 mL conc. H2SO4

Reddish brown colouration at the interface

Carbohydrates Molish test Conc. HCl Violet ringFehling test Conc. HCl & Mg

turningsYellow & brick red ppt

Proteins Biuret test 4% NaOH, 1% CuSO4 Violet or pink colourGlycosides Legal’s test Pyridine, sodium

nitroprussidePink 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, 2011Assam, India

To carry out qualitative and quantitative phytochemical analysis of selected medicinal plants

Phytochemical Analysis of Some Medicinal Plants

Objective

Methodology

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 Analysis

Qualitative Quantitative

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

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

Methodology

Seeds of Cyamopsis tetragonoloba

Washed, shade dried & powdered mechanically

Preparation of extractsPowdered material was weighed

Soxhlet extraction using methanol, acetone, chloroform & hexane

Solvent was recovered using Rotary Vacuum Evaporator

Phytochemical screening

Subjected to

48 hrs

Phytochemical Analysis

Qualitative Quantitative

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

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

TestsSolvents

Methanol Acetone Chloroform Hexane

1 Carbohydrates Molisch’s test +ve +ve +ve +ve2 Reducing sugars Fehling test +ve +ve -ve -ve3 Monosaccharide Barfoed’s test -ve -ve -ve -ve4 Tannins Ferric chloride test

Lead sub acetate test-ve -ve -ve +ve

5 Saponins Frothing test +ve -ve -ve -ve6 Flavonoids Shinoda’s test +ve +ve -ve -ve7 Terpenes/ steroids Liebermann Burchard’s

test+ve -ve +ve -ve

8 Alkaloids Mayer’s testWagner’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 +ve12 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 contentmg of Catechin equivalents/

200mg dried extract

Total flavonoids contentmg of Catechol equivalents/

200mg dried extractMethanol 14.51 17.34Acetone 9.17 8.75Chloroform 11.32 14.05Hexane 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., 2013Bangladesh

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

Methodology

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 Analysis

Qualitative Quantitative

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)

Table 4. Phytochemical screening of different extractives of Averrhoa bilimbi fruits

TestsExtracts

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., 2014Andhra 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

Methodology

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 Analysis

Qualitative Quantitative

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)

Alkaloids Steroidal compounds Phenolic compounds Flavonoids Saponins Tannins Coumarins Cardiac glycosides

Table 6. Physico-chemical evaluation

SolventInitial 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. TestsExtracts

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. TestsExtracts

Hexane Acetone Methanol Water

Contd...

Table 8. Total phenol content

% of phenol content µg GAE/µgConcentration

(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/µgConcentration

(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., 2014Tamil 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

Methodology

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 Analysis

Qualitative Quantitative

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

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., 2014Karnataka, 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

Methodology

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 Analysis

Qualitative Quantitative

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)

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 speciesTFC (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, 2014Tamil 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

Methodology

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 Analysis

Qualitative

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 analysis

Total phenolics Expressed as tannic acid equivalents

Total flavonoid content Colourimetric method expressed as

rutin equivalent

Carbohydrates Total chlorophyll content Proteins Total lipid content

Total tannins Tannin (%) = Total phenolics (%) – Non tannin phenolics (%)

Primary metabolites Secondary metabolites

Plant constituentExtracts

TestsCholoroform Methanol Aqueous Acetone

Alkaloids - - - - Wagners testFlavonoids - + - + Shimoda

Lead acetate testPhenolics & tannins - + + - Lead acetate test

Ferric chloride testSteroids & sterols + + - + Salkowski testCarbohydrates - + - - Fehlings test

Benedicts testSaponins - - + + Honeycomb test

Foam testGlycosides - + - - Glycoside testProtein & amino acids

+ + + - Biuret testNinhydrin 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., 2014Uttarakhand, India

To screen the phytochemicals present in Cinnamon zeylanicum aqueous bark extract

Phytochemical Screening of Selected Medicinal Plant Cinnamon zeylanicum Bark Extract

Objective

Methodology

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 Analysis

Qualitative

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

ExtractsCold 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, 2014Manipur, 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

Methodology

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 Analysis

Qualitative Quantitative

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

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 extractH. 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 + + +Phlobatannin - - +

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., 2015Karnataka, 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

Methodology

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 Analysis

Qualitative Quantitative

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

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 ± 29.69GAE = 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