Deals with the procedures for herbal extraction and phytochemical analysis of plants

2. 2
Seminar on
Extraction and Phytochemical
Analysis of Medicinal Plants
by
Shameembanu A. ByadgiShameembanu A. Byadgi

3. 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)]

4. 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

5.  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

6. 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

7. 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

8. 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

9. 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

10. General techniques of medicinal plant extraction
 Plant tissue homogenization
 Maceration
 Infusion
 Percolation
 Digestion
 Decoction
 Soxhlet extraction (Hot continuous extraction)
 Sonication (Ultrasound extraction)

11. Plant tissue homogenization

12. 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

13. Infusion

14. 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

15. Decoction
 Suitable for extracting  water-soluble, heat-stable constituents
 Typically used in preparation of Ayurvedic extracts

16. 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

17. ,
• 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

18. Soxhlet Extraction
(Hot Continuous Extraction)

20. 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

21. Effect of extracted plant phytochemicals depends on
• The nature & origin of the plant material
• Degree of processing
• Moisture content
• Particle size

22. 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

23. 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

24. 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....

25. 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

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
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

29. 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)

30. Table 1. Phytochemical constituents of medicinal plants
Results and discussion
L = leaves; S = stem

31. Fig 1. Total phenolic
content
Fig 2. Total flavonoid
content

32. 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

33. 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

34. 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

35. 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)

36. 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

37. 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

38. 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

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 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

41. 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)

42. 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

43. 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

44. Conclusion
 It may be 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 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

47. 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

48. 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

49. 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 + + + +

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. 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

52. 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

53. 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

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 & 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

56. 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

57. 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

58. 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

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
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

62. Table 12. Qualitative phytochemical screening of forest species
Figure 6. Total phenolic content (mg per gram of fresh leaf)
Results and discussion

63. 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 -

64. 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

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 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

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 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

70. 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

71. Conclusion
The results indicates that 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 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

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. 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

76. 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

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, 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

79. 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)

80. 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

81. 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

82. 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

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
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

86. Figure 9. Yield of extracts
Results and discussion

87. 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

88. 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

89. 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