UNIVERSITY OF MAURITIUS

FACULTY OF AGRICULTURE
BSc (Hons) Biotechnology
Plant Biotechnology AGRI 3066Y

Student I.D: 1712892

Date: 10th May 2020

Lecturer Name: Dr Soulange Govinden

1.0 Introduction
According to the World Health Organization, the value of plants as a source of medicinal
compounds is widely recognized today, with about 80 percent of the world's population
relying on herbal medicine as primary health care (Vines 2004). In medicinal and
aromatic plants the active compounds are primarily secondary substances which occur
as complex mixtures. They are widely used in medicinal and cosmetic products and in
food supplements. The analytical techniques used to classify medicinal and aromatic
plants should be appropriate for separation, elucidation of structure and quantification.
The key components are not always the active substances, because most often minor
constituents are more responsible for physiological effects and the nature of these
plants is determined. The chemical structure of secondary compounds, ranging from
monoterpenes to plant resins, the following classes of natural substances are of
importance: sesquiterpenes, diterpenes and polyterpenes, phenylpropanes,
amaroides, glycosides, saponins, alkaloids, glucosinolates, anthracenes, flavonoids,
coumarins, tannins, lipoids and lectins. The genus Psiadia. Form part of the family
Asteraceae which is also known as the largest plant family worldwide and contains over
60 species mostly found In tropical and subtropical regions, this genus is especially well
known in Madagascar and the Mascarene Islands (La Réunion, Mauritius and
Rodrigues).Several Phytochemistry and pharmacology studies on the Psiadia genus
have resulted in the isolation and identification of 73 compounds, including flavonoids,
phenylpropanoids, coumarins, and terpenoids. So far, of all the compounds known, only
the isolated flavonoids are considered as bioactive compounds. Some studies has
shown that some Psiadia plant possess benefits various medicinal properties and are
used for expectorant for treatment of bronchitis and asthma.
Secondary metabolism only occurs under specific conditions and are derived from
primary metabolism. The secondary metabolism is inherited by products of primary
metabolism as illustrated below
Photosynthesis Carbohydrates

Building block Glucose Primary

metabolism

Amino acids Glycolysis

Nucleic acids Acetyl CoA

Secondary metabolites
Figure 1 shows the secondary metabolism pathway which is inherited from primary
metabolites.
Objectives
 To highlight the analytical methods used for extraction of secondary metabolites
from medicinal plant; Psiadia plant.
 Investigating the qualitative and quantitative analysis antimicrobial crude extract
from psiadia plant displaying highest phytochemical composition and antioxidant
property

2.0. Materials and Methodology

2.1 Extraction of solids by Decantation

 50-100g of clean leaves were cut and put in conical flask.

 The leaves were covered with organic solvent; methanol, Methanol:
Dichloromethane 1:1, Dichloromethane, Ethyl Acetate and water. (50/50v/v) of a
total volume of 250ml.
 The leaves were left to macerate for a maximum of 48 hours and occasionally
swirling was done which allowed the liquid to reach homogenously the surface of
the leaves.
 The crude extract was filtered and concentrated to dryness in rotary evaporator
at 40-50°c

2.1.2 Extraction method by Soxhlet extraction & Fractionation of Crude

Extracts
 Fresh Psiadia leaves were oven dried at 60°c for 24 hours and was grinded using
a blender.
  5g of the solid leaves material which was to be extracted was packed into a filter
pater and then a thimble.
 The thimble was placed into the soxhlet apparatus and the whole Soxhlet
extractor on the top of a well which was supported by a round flask containing
the organic solvent 250 ml –Hexane first.
 The reflux condenser was place on the top of the Soxhlet extractor which heated
the flask using electrical heating until the solvent was boiled.
 The material was extracted out of the solid into the hot solvent for about 30
minutes and the solvent was removed from the round bottom flask and was
retained into a conical flask.

2.2. Phytochemical screening of compounds

1. Test for Coumarins
From 2ml of crude plant extract, add 2 drops of concentrated Ammonia and mix well.
View under UV light (366nm).Green Fluorescence of solution under uV indicates
presence of Coumarins
2. Test for Tannins
 Wash the plant crude extract with petroleum Ether and filtrate. Add ferric Chloride:
Potassium hexacyanoferrate iii dropwise. Green precipitate indicates presence of
Hydrolysable Tannins and Blue precipitate indicates the presence of condensed
Tannins.

3. Test for Saponins

Add 5ml of HCl to 0.5g of dried crushed plant. Heat to 100°C and cool. Shake
vigorously. Formation of a froth (1-2ml) above the solution persisting for at least 30
minutes indicates presence of saponins.

4. Test for Anthraquinones

Dissolve crude plant in warm distilled water and add ammonia to extract solution.
Shake and add Ammonium salt to the lower aqueous layer.A red coloration
indicates the presence pf Anthraquinones.

5. Leucoanthrocyanins
Extract the crude plant and extract all the pigments. Add magnesium turnings and
concentrated HCl to the filtrate.10minutes if coloration appears red this means that
leucoanthrocyanins is present. Or add concentrated HCL to the filtrate .Warm for 30
minutes. Red coloration shows presence of Flavonols.

2.3. Quantitative Analysis of plant Extracts

2.3.1 Determination of total Phenol content by the Folin –ciocalteau method

 The three aqueous extract was centrifuged at 3000 rpm for 3 minutes
  0.25 ml of diluted extract was added to 3.5 ml of distilled water in a screw cap
test tubes.
 0.25 lm of Folin-ciocalteau solution was added.
 The contents of the tube was mixed properly.
 After 3 minutes, 1 ml of 20% sodium carbonate was added, the tubes were
allowed to stand for 40 minutes in a water bath at 40°C.
 The tubes were allowed to cool in the dark and the absorption of the blue colour
was read at 685 nm using the spectrophotometer.
 A gallic acid standard curve was made and the total phenolic content was
determined in mg garlic acid/g fresh weight.

2.3.2. Determination of Total Flavonoid content

 2.5ml of crude extract was added to a test tube followed by 0.15 ml of 5% sodium
nitrite.
 After 5 minutes 0.15 ml of 10% of Aluminium chloride was added and after 1
minute, 1 ml of 1M of sodium hydroxide was also added.
 The tubes were properly vortexed and allowed to stand for 5 minutes.
 Absorbance of the yellow colouration was read at 510 nm and the flavonoid
content was calculated after analysing the quercetin curve.

2.4 Biological assays

2.4.1 Antioxidant assay
2.4.2 Antibacterial and antifungal assays
1. Preparation of suspension for antimicrobial test
 Two loops of 3mm diameter of spores of each species were transferred
aseptically to small vials containing 10ml of distilled water, 1 drop of Tween 20
and 7 drops of 1g/L of technical agar and was mixed.
 0.1 ml of 1g/L agar media containing the overnight culture of each
microorganism was inoculated on sterile petri dishes containing 15ml full
strength malt extract agar for antifungal test and 15ml of 50% nutrient agar for
antibacterial tests.

2. Agar well diffusion method for antimicrobial assays

 The antimicrobial agent held in the reservoir diffuses through the agar
medium to form a diffusion gradient to which the microorganisms, growing
on the gar are exposed.
 After inoculation of the test of the microorganisms, three wells of 7 mm
diameter are made on the plate of the agar solidified medium. The wells
are aseptically filled 75 microliter of different microorganisms of plant
extract.
 0.5 mg/L Chloramphenicol and 3mg/L of Nystatin was used as positive
controls while methanol was used a control.

3.0 RESULTS

3.1 Extraction method by Soxhlet extraction

For the Soxhlet extraction, the weight of the extract was estimated by weighing the
round bottom flask empty first and then weighing it with the extract. The results are
shown in table 1 to table 11 for the different solvents used below.
 Table 1: Weight of extracted material for our group which used Hexane.

Apparatus Weight (g)

Empty round bottom flask 271.9

Round bottom flask + extract 272.1

Extract 0.2

3.2 Phytochemical screening

Table 2 shows results for the phytochemical composition on the Psiadia extract.

Extract Coumarins Tannins Saponins Anthraquinones Leucoanthocya

nins/
Flavonoids
Methanol + + + - -

Meth/DCM - +green + - -
+blue
DCM + +blue ++ - +
++green
Ethyl - ++blue ++ - -
Acetate
Water - ++blue ++ + -

3.3 TLC Thin-layer chromatography

The TLC conducted on the Psiadia leaves were sprayed with: LB reagent on plate 1 to
detect for Terpenes/steroids, Follin’s reagent on plate 2 to detect for Phenols and
Dragendorff reagent on plate 3 to detect for Alkaloids. The results are interpreted in
table 3 below and figure 1.

Table 3: the results of the TLC Thin-layer chromatography conducted on the Psiadia
leaves.
 Plate Results

1 Negative

2 Positive: (Blue and Dark spots)

3 Negative

Figure 1: The results of the TLC Thin-layer chromatography conducted on the Psiadia
leaves

3.4 Quantitative Analysis of plant Extracts

For the quantitative analysis, the total phenolic content and the total flavonoid content,
the Quercetin (10mg/ml) stock, Gallic acid stock and crude extract were used and the
absorbance of the several dilutions are shown in table 4 to 7 and figure 5 to 7 shows
interprets the results.
3.4.1 Determination of Total Flavonoid content
Table 4: The results for the absorbance of the total Flavonoid.

Concentration (µg/ml) Absorbance(nm)

 510

0 0.000

10 0.020

20 0.039

30 0.051

40 0.064

50 0.070

Quercetin Standard Curve

0.08
f(x) = 0 x
0.07 R² = 0.99
0.06
Absorbance at 510 nm

0.05
0.04
0.03
0.02
0.01
0
0 10 20 30 40 50 60
Concentration (µg/ml)

Figure 2 Quercetin Standard curve for total flavonoid content.

 V
The total phenolic content of the samples can be calculated using the formula: C = c
m
where, C = total phenolic content mg/g dry extract, c = concentration of quercetin
obtained from calibration curve in µg/mL, V = volume of extract in ml, m = mass of
extract in gram.

10000
Dilution factor = =20 Volume = 10ml Mass of extract = 1.2g
500
0.06
x= =42.8571, y = 0.0014x, Absorbance at 50µg/ml = 0.06
0.0014

c =42.8571 X 20=857.1429

C = (857.1429 X 10)/1.2

C = 7142.86 mg QE/g

Table 5: The results for the absorbance of the total flavonoid content for the other group
which used extract and quercetin.

Concentration (µg/ml) Absorbance(nm)

510

0 0.00

10 0.017

20 0.039

30 0.048

40 0.055

50 0.060
 Total Flavonoid content using extract
0.07
f(x) = 0 x
0.06 R² = 0.98
Absorbance at 510 nm

0.05

0.04

0.03

0.02

0.01

0
0 10 20 30 40 50 60
Concentration (µg/ml)

Figure 3: Total flavonoid content using extract

3.4.2. Determination of total Phenol content by the Folin –ciocalteau method

Table 6: The results for the absorbance of the total phenolic content for the group which
used gallic acid.

Concentration (µg/ml) Absorbance(nm)

510

0 0.00

10 0.381

20 0.885

30 1.146

40 1.721

50 2.266
 Gallic acid standard curve
60

50 f(x) = 22.21 x + 1.31

R² = 0.99
Absorbance at 685 nm

40

30

20

10

0
0 0.5 1 1.5 2 2.5
Concentration of gallic acid at µg/ml

Figure 4: Gallic acid standard curve

Table 7: The results for the absorbance at 685nm for the group which used diluted
extract and gallic acid.

Concentration (µg/ml) Absorbance(nm)

685

X100 0.194

X1000 0.103
3.5 Antibacterial assay
The results were taken in terms of diameter of the zone of inhibition. Table 8 to table 12
and figures 8 and 9 shows the results and the means of the zone of inhibition.

Table 8: shows result for our group and we used hexane extract.

Extract Replicates (mm) Mean (mm)

1 2 3

Extract 1 22 12 10 15

Extract 2 23 21 0 15

Positive control 0 0 0 0

Negative 0 0 0 0
 control

Table 9: shows result for group 2 which used Chloroform:Methanol(1:1) extract.

Extract Replicates (mm) Mean (mm)

1 2 3

Extract 1 20 15 20 18

Extract 2 12 18 16 15

Positive control 20 35 25 27

Negative 0 0 0 0
control

Table 10: shows result for group 3 which used methanol extract.

Extract Replicates (mm) Mean (mm)

1 2 3

Extract 1 6.0 4.0 6.5 5.5

Extract 2 8.0 7.0 7.0 7.3

Positive control 7.5 7.0 6.0 6.8

Negative 0 0 0 0
control
Table 11: shows result for group which used chloroform extract ( informations as per
provided by the group)

Extract Mean (mm)

Extract 1 20

Positive control 22

Negative control 0

Table 12: shows result for group which used Chloroform:Methanol(4:1) extract.

Extract Replicates (mm) Mean (mm)

1 2 3

Extract 1 0 0 0 0

Extract 2 0 0 0 0

Positive control 0 0 0 0

Negative 0 0 0 0
control
Figure 5: The results for the Antibacterial assay for our group.

Figure 6: The results for the Antibacterial assay for the group which used Methanol
chloroform (4:1)

3.6 MICRODILUTION ASSAY

For the microdilution assay, the solvent-extract of different groups were used to
determine the antimicrobial activity of the extracts. Figures 10 to 14 shows the 96-wells
microplates used for the assay.
Figure 7: The 96-wells Microplate for our group which used hexane.

Figure 8: The 96-wells Microplate for the group which used Chloramphenical.
Figure 9: The 96-wells Microplate for the group which used Chloroform.

Figure 10: The 96-wells Microplate for the group which used Methanol.
Figure 11: The 96-wells Microplate for the group which used Chloroform:methanol(1:1).

ANTIOXIDANT ASSAY
The antioxidant assay was conducted to determine the free radical scavenging activity
of the extracts. The table 21 to table 25 show the absorbance of the diluted solutions
and the antioxidant activity (AA%). The antioxidant activity (AA%) was calculated using
the formula:

AA %=
[( Absblank −Abssample )∗100 ]
Absblank

Table 13: The absorbance of the diluted solutions and the antioxidant activity (AA%) for
the group which used Quercetin.

Concentration (µg/ml) Absorbance AA%

 (blank) 0.812

40 0.930 -11.2

50 0.821 123.0
60 0.801 1.35

70 0.711 12.4
80 0.691 14.9

90 0.381 53.1
100 0.231 71.5

Table 14: The absorbance of the diluted solutions and the antioxidant activity (AA%) for
the group which used hexane.

Extract Absorbance AA%

blank 0.00
Rep1 0.465 -46.5
Rep2 0.468 -46.8
Rep3 0.467 -46.7

Table 15: The absorbance of the diluted solutions and the antioxidant activity (AA%) for the
group which used Methanol.
 Extract Absorbance AA%
blank 0.00
Rep1 0.064 -6.4

Rep2 0.068 -6.8

Rep3 0.063 -6.3

Table 16: The absorbance of the diluted solutions and the antioxidant activity (AA%) for the
group which used Chloroform:methanol(1:1).
Extract Absorbance AA%
blank 0.00
Rep1 0.164 -16.4
Rep2 0.146 -14.6
Rep3 0.105 -10.5

4.0. Discussion
Five solvents were tested on Psiadia crude extract which include methanol, Methanol:
Dichloromethane, Dichloromethane, Ethyl acetate and water. It is important to note that
the type of polarity of a specific solvent determine which type of compound it will
dissolve. Both methanol, Methanol: Dichloromethane was able to extract Coumarins,
Tannins and saponins while Dichloromethane solvent was able to exact all except for
Anthraquinones. (Table 2). Flavonoids was extracted only by Methanol:
Dichloromethane solvent. Tannins and saponins were extracted from all the solvents.
However ethyl acetic solvent was not able to extract coumarin, saponin and flavonoids
as illustrated in table 2. Polar molecules tend to move towards polar organic solvents
and stays in the aqueous phrase while non-polar molecules favours the organic phrase.
The total phenolic content and total flavonoid were also determined by the Folin –
ciocalteau method.It was seen that the phenolic content and flavonoid is low total so the
psiadia plant is not a good antioxidant.It was also noted that antimicrobial activity of
psiadia extract was more effective with the use of Chloroform:Methanol(4:1) than the
other extracts.

5.1 Conclusion
The phytochemical study of Psiadia plant has revealed the presence of phytochemical
composition such as tannins, saponin and couramins, phenol and flavonoids. Further
studies can be done on the psiadia plant to isolate the most bio constituent attributing to
the medicinal benefits of psiadia plant such as pain relief.