Basic Civil Engineering first year Notes- Chapter 4 Building.pptx
tooba shafi presentation.pptx
1.
2. Studies on the Chemical constituents and
Pharmacological activities of Fagonia indica
Tooba Shafi
MPCHE-20-15
Supervisor Co-Supervisor
Dr. Tanzila Rehman Dr. Hina Andleeb
Assistant Professor Assistant Professor
Department of Chemistry Department of Biochemistry
The Women University Multan Bahauddin Zakaria University Multan
Department of Chemistry
The Women University, Multan
4. Plan of work
Plant (Fagonia
indica) selection
and identification
Plant extraction
in different
solvents
Biochemic
al analysis
Phytochemical
analysis
Antioxidant
activity
Antibacterial
activity
5. 1. Introduction
Fagonia indica is a member of
wild flowering plants in the
Zygophyllaceae family. This
plant can be found in subtropical,
tropical, and temperate climates
around the world, including
Algeria, Cyprus, Egypt, India,
Morocco, Pakistan, and Saudi
Arabia.
It is commonly known as
Dhamasa.
6. Fagonia indica is a spiky shrub
with whitish powdered or
hairless shrublet or stalkless
glands. Branches are
procumbent or erect cylindrical
and stem is slightly woody at
the base
It has spiny stipules, pulple
petals and it is more or less
bushy.
7. Chemical properties and pharmacological effects
Bitter in taste
Antiseptic, astringent and stimulant
Antioxidant
Hepatoprotective and anti-bacterial
Chemical ingredients
Tannins, alkaloids, saponins, phenolic acids, and
flavonoids
Calcium, potassium, magnesium, chromium and iron
8. Medical importance
Used in the treatment of liver diseases, heart disorders,
allergies, typhoid, stomach issues, urine discharge, toothache,
and skin ailments.
Detoxify the blood
Traditionally used to treat a common fever
Prevent smallpox
Control diabetics' blood glucose levels
aqueous extract is effective in the treatment of some forms of
cancer.
9. Aim of study
On basis of the medicinal importance of Fagonia indica, the
objective of this study was to investigate the composition of
following parameters of Fagonia indica:
Biochemical parameters i.e. reducing sugars and protein contents.
Phytochemical parameters i.e. alkaloids, tannins, flavonoids and
saponins.
Antioxidant properties i.e. total phenolic acids, total antioxidants and
antioxidant activity.
Mineral analysis
Antibacterial properties against the Streptococcus pyogenes bacteria.
10. 2. Experimental procedures
Plant extraction
5g sample
soaked in
methanol
Filtrate evaporated
in rotary evaporator
Methanol
extract dissolve
in water
Using solvents of
different polarity
viz. hexane,
butanol,
chloroform,
ethylacetate
Hexane fraction
Chloroform fraction
Ethylacetate fraction
Butanol fraction
Residual aqueous fraction
11. Biochemical analysis
Reducing sugars
Estimated by 3,5-dinitrosalicylic acid (DNSA) method.
2ml of DNSA was added in 1ml each plant extract.
Heated for 5 minutes and absorbance was taken at 540nm.
Protein contents
estimated by Bradford assay.
mixed 1ml of each plant extract in 5ml Bradford reagent.
Heated for 30 minutes.
Absorbance was determined at 595nm.
(Bradford 1976)(Miller 1959)
12. Phytochemical analysis
Tannins
determined by using the Folin-Ciocalteu method.
Added 7.5ml distilled water, 0.5mL Folin-Ciocalteu reagent and 1ml
of 35% sodium bicarbonate solution in 0.1ml plant extract and heated
it for 30 minutes.
Absorbance was taken at 725nm.
Flavonoids
determined by aluminium chloride colorimetric method.
Dissolved each plant extract in deionized water and added 75µl
sodium nitrite solution.
Then added 150µl Aluminium chloride (AlCl3) and absorbance was
taken at 510nm.
(Ainsworth and Gillespie 2007)
(Park, Jung et al. 2008)
13. Alkaloids
Mixed 5g of each plant extract in 200 ml of 20% CH3COOH in
C2H5OH.
Kept it for 4 hours and then filtered.
Added ammonium molybdate solution drop wise until it
precipitated.
Saponins
Mixed 5g of each plant extract in 100ml ethanol (20%) and
heated for 4 hours in water bath at 55°C.
Added 100ml ethanol for re-extraction and condensed to 40ml
at 90°C.
Added 20ml ethylacetate in concentrate and 2 layers separated.
(Obadoni and Ochuko 2002)
14. Discarded the ethylacetate layer.
Then added 30ml butanol in residual aqueous layer.
Washed the butanol extracts with 10ml NaCl (5%).
Evaporated the remaining solution and then weighed.
Ascorbic acid contents
Mixed 1 g of each plant extract with 10ml of 0.05M oxalic acid
and 0.02M EDTA mixture.
Added meta phosphoric acid (0.5ml), 0.5ml of acetic acid
(CH3COOH),1ml of 5% H2SO4 solution, and 2 ml of
ammonium molybdate solution and absorbance was taken at
760nm.
(Hussain, Ullah et al. 2011)
15. Antioxidant analysis
Total Pheolic acids
Determined by Folin-Ciocalteau method.
Mixed 200µl each plant extract in 1ml distilled water.
Added 10ml Folin reagent and heated.
Added 10ml sodium bicarbonate solution and warmed it.
Absorbance was taken at 765nm.
Total antioxidants
Total antioxidants were extracted in 50ml methanol (75%).
Added 40µl DPPH reagent in 1ml plant sample and heated for
30 minutes.
Absorbance was taken at 517nm.
(Kim, Jeong et al. 2003)
16. Antioxidant activity
determined by the DPPH (1, 1-diphenyl-2-picryl-hydrazyl,
ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) and Superoxide method.
DPPH radical scavenging activity
Ascorbic acid was taken as standard.
Dissolved different concentration of plant extract in methanol.
Mixed 2.4mg DPPH in 100ml methanol. And diluted with
methanol.
Absorbance was taken at 517nm.
ABTS radical scavenging activity
Mixed ABTS solution (7 mM) with 2.45 mM K2S2O8 and kept
it in the dark for 8 hours.
(Mensor, Menezes et al. 2001)
17. Added 1ml ABTS in different concentration (250, 200, 150,
100, 50 and 25 μg/ml in methanol).
Observed the absorbance at at 745nm.
Superoxide radical scavenging assay
Mixed 150µl of plant sample of different concentration (250,
200, 150, 100, 50, and 25g/ml in methanol) with 250µl of
potassium phosphate buffer (0.05M; pH 7.6), phenazine
methosulphate (0.02M, 126µl), nitroblue tetrazolium (0.005M,
50µl), and riboflavin (0.05M, 150µl).
Left the mixture under a fluorescent lamp for 20 minutes.
Absorbance was taken at 560nm.
18. Following equation was used to calculate the antioxidant activity:
𝑫𝑷𝑷𝑯 𝑺𝒄𝒂𝒗𝒆𝒏𝒈𝒊𝒏𝒈 𝒂𝒄𝒕𝒊𝒗𝒊𝒕𝒚 %
=
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒄𝒐𝒏𝒕𝒓𝒐𝒍 − 𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝐜𝐨𝐧𝐭𝐫𝐨𝐥
× 𝟏𝟎𝟎
19. Mineral analysis
Mineral analysis was performed
by flame photometry method.
Three acids HNO3, H2SO4 and
HClO4 with ratio 5:8:0.5 were
used.
Added 0.25g each plant extract
in 50ml of each acid separately
and heated.
Diluted the solution up to the
50ml with water and filtered.
Then analyzed these solutions
with flame photometer
(Mengal, P. W., Samiullah, N., Khan)
20. Anti-bacterial analysis
Antibacterial properties of each plant extract was determined by
the use of two different methods.
1. Disc diffusion method
This method is used to determine the zone of inhibition.
2. Serial dilution method
This method is used to determine the minimum inhibitory
concentration.
(Aslam, Hayat et al. 2021)
(Kouser and Qureshi 2013)
21. 1. Disc diffusion method
2g of Agar was
mixed in 100ml LB
media
Taken 30ml Agar
solution in each
petri-dish
1ml of LB
media
taken in
eppendorf
Cotton bud (take
swab from throat to
collect the bacteria
Streptococcus
pyogenes) dipped in
eppendorf
100µl of solution from
the eppendorf spread
over the surface of Agar
plates.
bacterial
colonies were
observed on
the plates
centrifuged
for 2 min
22. 0.1ml plant
extract mixed in
10ml DMSO
pieces of filter paper
dipped into the solution
of different
concentrations made
from the 1% sample
solution.
pieces of filter paper were
placed on agar plates and
left the plates for 24 hours
23. Plates for standard and control were prepared.
DMSO was used as control.
Standard solution was prepared by mixing the 1 tablet of
Erythromycin in 1ml ethanol.
Pieces of filter paper were dipped into standard and control and
then placed on agar plates.
These portions of plates will serve as standard and control
group.
24. 2. Serial dilution method
Prepared solutions of different concentration from plant
extract.
Added DMSO, fresh LB media and bacterial cultures in it.
Then optical density was determined at 600nm.
25. 3. Results and Discussion
Biochemical composition (mg/g dry weight) of Fagonia indica plant
Biochemical
constituents
Aqueous
fraction
Methanol
fraction
Butanol
fraction
Chloroform
fraction
Ethylacetate
fraction
Hexane
fraction
P
value
Reducing
sugars
5.81±0.012a 4.32±0.09b 5.53±0.08a 5.2±0.07a 5.55±0.015a 5.08±0.04a 0.02
Proteins 60.23±10.33a 108.60±15.6b 80.67±8.23a 105.43±12.34b 123.43±10.67b 110.83±8.79b 0.01
Mean ±SE (n=3). Means not sharing the same letter (a-d) are significantly different at
p<0.05 probability in each column.
26. Total reducing sugars in different extracts of Fagonia indica
0
1
2
3
4
5
6
7
aqueous methanol butanol chloroform ethyleacetate hexane
Concentration
(mg/g)
b
a
a a
a
a
27. Total protein contents in different extracts of Fagonia indica
0
20
40
60
80
100
120
140
160
aqueous methanol butanol chloroform ethyleacetate hexane
Total
Proteins
(mg/g)
a
a
b
b
b b
28. Phytochemical analysis
Phytochemical composition (mg/g dry weight) of Fagonia indica
Phytochemica
l constituents
Residual
aqueous
fraction
Methanol
fraction
Butanol
fraction
Chloroform
fraction
Ethylacetate
fraction
Hexane
extract
P.
value
Tannins 35.875±0.085b 38.345±1.125a 36.9±0.35b 0.00 33.772±0.542c 0.00 0.03
Alkaloids 0.234±0.0059c 0.935±0.0062a 0.345±0.0065c 0.645±0.0077a 0.765±0.0068a 0.934±0.0023a 0.02
Flavonoids 51±0.765a 14±0.991d 32±0.763c 24±0.777c 41±1.055b 0.00 0.04
Saponins 0.923 ±0.005a 0.883 ±0.004a 1.207±0.012b 1.516±0.014b 2.043±0.031c 2.223±0.043a 0.02
Ascorbic acid 0.782±0.002a 0.753±0.0035b 0.744±0.0085b 0.733±0.0022c 0.725±0.0054d 0.733±0.0066c 0.01
Extraction
yield (%)
13.59a 15.56a 10.025a 2.98b 9.25b 4.21b 0.01
Mean ±SE (n=3). Means not sharing the same letter (a-d) are significantly different at
p<0.05 probability in each column.
36. Antibacterial activity
ZOI of bacterial colony of various plates with different concentration of
plant extract: (A) a(0.1mg/L), b(0.2mg/L), c(0.3mg/L), d(0.4mg/L); (B)
e(0.5mg/L), f(0.6mg/L), g(0.7mg/L), h(0.8mg/L); (C) i(standard), j(control)
37. Zone of inhibition of plant extract against Streptococcus
pyogenes bacteria
Sr. No. DMSO (%) Inhibition zone (mm)
1 0.1 3
2 0.2 5
3 0.3 6
4 0.4 4
5 0.5 5
6 0.6 7
7 0.7 6
8 0.8 6
40. Conclusion
Our study reveals that plant Fagonia indica is a medicinal plant
as it contains a variety of biochemical, phytochemical,
antioxidant and antibacterial contents and there is a significant
difference between them.
It has significant amount of reducing sugars and proteins.
Tannins, flavonoids, alkaloids, saponins are some of the
phytochemical components found in abundance in the plant
This plant also contains a lot of antioxidants which shows the
antioxidant activity against the DPPH radicals, ABTS and
superoxide radicals.
41. Fagonia indica also shows antibacterial activity against
Streptococcus pyogenes bacteria, so it is used in medical
textiles to provide adequate protection against microorganisms.
These components reportedly have anticancer, heptoprotective,
antioxidant and antibacterial properties.
Therefore the plant Fagonia indica has traditionally been used
to treat a variety of ailments.
42. Acknowledgement
First and foremost, all praises and thanks to the Almighty Allah. It
is due to the continuous support of the Supreme Power the
Almighty Allah that I have completed my research work.
I am feeling oblige to pay my sincere thanks to Dr. Sara
Musaddiq, The Head of Chemistry Department. It is genuine
pleasure to express my deep sense of gratitude to my supervisor,
guide, mentor and a loving personality Dr.Tanzila Rehman,
Assistant professor and Co-Supervisor Dr.Hina Andleeb,
Assistant professor whose guidance and positive thinking made
this research possible.
May Allah bless you all!
Tooba Shafi
