Ph.

D Synopsis Viva voce - examination

Ref No. 16454/Ph.D.K1/Botany/Full time/October 2017

In vitro organogenesis and pharmacognostical screening

of Oxystelma esculentum R. Br.,

Research Guide Presented by,

Dr. T. Francis Xavier
Assistant Professor
Department of Botany,
St. Joseph’s College (Autonomous)
Tiruchirappalli – 620 002
Ms. R. Sabitha
Research Scholar
Department of Botany,
St. Joseph’s College (Autonomous)
Tiruchirappalli – 620 002
 The thesis comprises

 Chapter I – Introduction

 Chapter II - Review of Literature

 Chapter III - Materials and Methods

 Chapter IV - Results and Discussion

 Chapter V - Summary and Conclusion

2
 Previous reports of the Experimental Plant
Author (Year) Study Area Plant parts used

Maliga and Yoganath Leaf and stem

(2016) Antimicrobial activity
Savitha and
Balamurugan (2016) Leaf

Ashok kumar et al., Aerial parts

(2009) Antioxidant activity
Kamel and Eman (2018) Leaves and Fruits

Paul et al. (2017) Phytochemical studies leaf

Previous reports for Antidiabetic work - None

3
Previous reports for Molecular docking studies – None
4
 INTRODUCTION
 Plants containing more number of medicinal properties and it should be
used to treat many diseases in humans.
 The extraction and characterization of active compounds from medicinal
plants to discover many clinically useful drugs.
 Plant tissue culture technology a great promise for micropropagation,
conservation, and enhancement of the natural valuable secondary
metabolites.
 Contrary to the synthetic drugs, antimicrobial activities of plant origin are
associated with lesser side effects and have an enormous therapeutic
potential to heal many infectious diseases.
 Medicinal herbs are an important source of phytochemicals that offer
traditional medicinal treatment to various
5 diseases.
 Cont…
 Oxystelma esculentum R. Br. is a least concern medicinal plant belongs to
Apocynaceae family and is also known as ‘Jaldudhi’ (Shahzad et. al., 2016;
Panda 2019).
 In India, a single species has existed in this genus (Senthilkumar et. al.,
2009).
 It is one of the plant to contain cardenolides and pregnane glycosides, which
are major classes of therapeutically important phytoconstituents.
 O. esculentum (OE) has been reported to possess good therapeutic actions
like antiseptic, depurative and galactagogue properties and the decoction is
used as a gargle for throat and mouth infections (Poornima et. al., 2009).
 This plant also has been to treat against many ailments of the current world
but, which has not sufficiently explored.
 OBJECTIVES
 To develop suitable protocol for in vitro organogenesis of Oxystelma esculentum R.
Br.,
 To determine the pharmacognostical analysis (Antimicrobial, Antidiabetic and
Antioxidant) of Oxystelma esculentum
 To find out the active compounds of the experimental plant by various qualitative
phytochemical analyses, isolate and characterize the active compound responsible for
the antimicrobial activity through UV-Vis, FT-IR spectroscopy, HPTLC, 1H NMR,
and 13C NMR.
 To synthesize the silver nanoparticles and characterize through spectroscopic methods
viz., UV-vis, FT-IR, PSA, SEM, EDAX and to determine the antibacterial activity of
synthesized nanoparticles
 To study the in silico molecular docking of O. esculentum plant extracts against
pancreatic α-amylase through its phytoconstituents.
Materials and Methods

8
 Experimental plant
Systematic Position
Kingdom : Plantae
Class : Asterids
Order : Gentianales
Family : Apocynaceae
Genus : Oxystelma
Species : esculentum
Author name : R. Br.,
Plant collection and identification
Fresh healthy mature plant material was collected from natural
habitat at Trichy District. It was identified and authenticated (Ref. No.
R.S.001) in Rapinat Herbarium and center for Molecular Systematics, St,
9

Joseph’s College, Tiruchirappalli – 620 002.

Plant Authentication

10
Work Plan -I

11
Steps for In vitro plant tissue culture

Sterilization
Media preparation

Inoculation

Incubation

Hardening
12
 Sterilization of Explants
Healthy Explant

Treated with Teepol Solution

Washed With Distilled water

Followed by70% Alcohol

↓
Sterilized With 0.1% HgCl 2
↓
Washed with Sterile Distilled water
13
Constituents of MS medium (Murashige and Skoog, 1962)
Macronutrients
D.H2O 800ml 400ml Per liter media
NH4NO3 33.0 g 16.5 g
KNO3 38.0 g 19.0 g
50 ml
KH2PO4 3.4 g 1.7 g
MgSO4 7.4 g 3.7 g
CaCl2H2o 8.8 g 4.4 g
Micronutrients
D.H2O 200ml 100ml Per liter media
H3BO3 1240 mg 620 mg
NaMoO4 50 mg 25 mg 1 ml
COCl2 5 mg 2.5 mg
CuSO4 5 mg 2.5 mg
D.H2O 200ml 100ml Per liter media
ZnSO4 1720 mg 860 mg
MnSO4 4460 mg 2230 mg
KI 166 mg 83 mg
Amino acid
D.H2O 20 ml 10 ml Per liter media
Glycine 40 mg 20 mg 1 ml
Vitamins
D.H2O 100 ml 50 ml Per liter media
Thiamine 10 mg 5 mg
Nicotinic acid 50 mg 25 mg
1ml
Pyridoxine HCL 50 mg 25 mg
Myo- inositol 100 mg 50 mg
Iron source
D.H2O 100 ml 50 ml Per liter media
Na2EDTA 745 mg 372.50 mg
5 ml
FeSO4 557 mg 278.50 mg
Carbohydrate
Sucrose 30 g
14
Agar 8.0 g
pH(before autoclave) 5.7
 Dissolving agents of plant growth hormones
S.No
Hormones Dissolving Agent

1. α-Naphthaleneacetic acid (NAA) * 0.1 N NaOH

2. Indole -3- acetic acid (IAA) * 0.1 N NaOH

3. 3-Indolebutyric acid (IBA) * 0.1 N NaOH

4. 6- Benylaminopurine (BAP) ** 0.1 N NaOH

5. Kinetin (Kin) ** 0.1 N NaOH

6. 2,4-Dichlorophenoxyacetic acid (2,4-D) * 50% ethyl alcohol

7. Gibberellic acid (GA3) 50% ethyl alcohol

15
Medium preparation (Murashigie and Skoog, 1962)

30% of Sucrose was added to 1 L standard flask

↓
Add Stock solutions
↓
The solution make up to 1 L
↓
hormones were added with different concentrations
↓
pH was Adjusted to 5.6-5.8
↓
0.8 % of Agar was added
↓
The media were sterilized
↓
After solidification, the media
16
were used for inoculation
Work Plan - 2 Antimicrobial Activity
Oxystelma esculentum R. Br.,

17
 Disc diffusion assay (Maruzella and Henry, 1958)
The rapid determination of the drug or a particular substance on a specific micro organisms.
Sterile liquid Nutrient Agar and Potato Dextrose Agar medium (pH 7.4 ± 2)

100 l of bacterial/Fungal cultures

The sterile discs were impregnated with 10 l of the 3 mg/ml extracts

(30 g/disc)

Negative controls were prepared using the same solvents

Chloramphenicol (30 g/disc) / Amphotericin (100 g/disc)/ were used as positive control

The inoculated plates were incubated at 37°C for 24 hrs. / 37°C for 36-72 hrs.

Zone of inhibition was measured in mm.

18
Each assay was conducted in triplicate.
 Plate hole diffusion method (Leven et. al., 1979)
100 l bacterial culture + 20 ml of Nutrient Agar medium
100 l Fungal culture + 20 ml of Potato Dextrose Agar medium

6 mm diameter wells created

100 l (300 g/well) of plant extract were added to each well

Negative controls were prepared using the respective solvents

100 l of positive control (300 g/well)

Both experimental and control plates were incubated at 37°C for 24 hrs. / 37°C

for 36-72
hrs.
The inhibition zone formed around each well was measured
19
 Anti-Oxidant Activity
Work done at Analytical Service Centre, Department of
Microbiology & Botany, A.V.V.M Poondi Pushpam College ,
Thanjavur

20
 DPPH Scavenging Activity (Blois 1958)
 Take diffefent concentrations of the extracts and make the volume to 100 µL with
methanol.
 Add 5 mL of 0.1 mM methanolic solution of DPPH and incubate for 20 min at 27º
C.
 Measure the absorbance of the solution at 517 nm.

 Methanol alone will serve as blank

 A test tube with 100 µL of methanol and 5 mL of DPPH solution serves as negative
control.
 The mixture of methanol, DPPH and standard (BHT, BHA, quercetin and α-
tocopherol will serve as positive control
 The reduction in purple colour of the DPPH solution to pale yellow will give the
percentage of inhibition.
 Calculate the percentage inhibition from the absorbance of sample and negative
control using the equation.
 % of inhibition = [(Control OD – Sample21 OD) / Control OD] * 100

 IC50 (Inhibitory concentration 50 %) from the concentration and percentage

Phosphomolybdenum Assay (Prieto et. al., 1999)
 Take test tubes and add a standardized concentration of the
sample solution in triplicate
 To the same test tubes add 1 mL of the reagent solution.
 Blank solution served will be a reaction mixture without
sample or standard.
 Incubate the reaction mixture in a water bath at 95ºC for 90
min.
 Cool the mixture now at room temperature and measure the
absorbance of the mixture at 765 nm against a blank.
 The results are expressed in ascorbic acid equivalents per
gram extract (AEAC). 22
Ferric Reducing Antioxidant Power (FRAP) Assay
(Pulido et. al., 2000)
 To the sample in triplicate determination add distilled
water to make equal volume of solution in each test
tube.
 Now add 900µL of FRAP reagent, vortex well and
incubate at 37ºC for 30 min.
 Immediately after the incubation at 593 nm read the
absorbance of the chromophore developed.
 The FRAP value is expressed as mmol Fe (II)
equivalent/mg extract.
23
 Metal Chelating Activity (Dinis et. al., 1994)

 To the extracts in test tubes of triplicate determination add 50 µL of

2 mM FeCl2.
 By adding 0.2 mL of 5 mM ferrozine solution initiate the reaction.

 A test tube with deionized water alone will act as blank and reaction
mixture without standard or sample will be negative control
 Make the solution to a volume of 1-3 mL.

 For 10 min at room temperature shake the reaction mixture well

vigorously and leave to stand
 Measure the absorbance of the solution at 562 nm.

 EDTA equivalence comparing the sample with the standard curve

graph the metal chelating activity is determined.
24
 Superoxide Radical Scavenging Activity
(Beauchamp and Fridovich 1971)
 In a test tube take 100 µL of plant extract and standards (BHT and rutin)
in triplicates.
 Add 3 mL of reaction mixture containing 50-mM sodium phosphate
buffer pH-7.6), 20 µg riboflavin, 12 mM EDTA and 0.1 mg of NBT to the
extracts in the test tubes.
 By illuminating the reaction mixture with samples for 90 sec to start the
reaction.
 The negative control is the illuminated reaction mixture without sample.

 The blank will be un illuminated reaction mixture without plant sample

 The absorbance at 590 nm against the blank is measured immediately

after the illumination.
 Calculate the scavenging activity (%) of superoxide anion generation
25
using the following equation:
Anti-Diabetic Activity

26
 α-Amylase Inhibitory Assay (Miller, 1959)

 The α-amylase with the extract at various concentrations (50-200 µg/mL) is

premixed
 Add starch as a substrate at 0.5% to start the reaction

 At 370 C the reaction is incubated for 5 min and terminated by the addition of 2
ml of DNS (3,5-dinitrosalicylic acid) reagent and incubated.
 The reaction mixture is heated for 15 min at 100 degree Celsius and dilute with
10 ml of distilled water in an ice bath (Miller, 1959).
 By measuring the spectrum at 540 nm the A-amylase inhibitory activity is
measured.
 Calculation

The following formulae we can calculate the A-amylase activity: % Inhibition =

[(Abs control - Abs samples)]
27
/ Abs Control] * 100
 α-Glucosidase Inhibitory Assay (Miller, 1959)
 The extract at various concentrations (50-200 µg/mL) is premixed with α-
glucosidase.
 To the reaction add 3mM p-nitrophenyl glucopyranoside (pNPG) as a substrate to
start the reaction.
 At 370 C the reaction is incubated for 5 min and terminated by the addition of 2 ml
of DNS (3,5-dinitrosalicylic acid) reagent and incubated.
 The reaction mixture is heated for 15 min at 100 degree Celsius and dilute with 10
ml of distilled water in an ice bath (Miller, 1959).
 By measuring the spectrum at 540 nm the A-amylase inhibitory activity is measured.

 Calculation

The following formulae we can calculate the A-amylase activity: % Inhibition = [(Abs
control - Abs samples)] / Abs Control] * 100
28
Work Plan - 3 Phytochemical Analysis & Green synthesis of silver nanoparticle

Oxystelma esculentum R. Br.,

Leaf

Ethnaol, Ethyl Acetate

and Acetone

29
 Synthesis of Silver Nanoparticles
1g of plant powder +100ml of Distilled water at 70̊ C

Filtered with Whatman No 1 Filter Paper

10 ml leaf extract was added into 90 ml of Ag NO3 solution

Once the mixture had a reddish brown color reduction Ag+ ions was
monitored

The Ag NPs were observed using UV and

FT-IR spectroscopy

Then centrifuged at 5000 rpm for 20 min

The purified pellets were collected then dried

Powder was taken and characterized

30 through FE-SEM
 Synthesis of Silver Nanoparticles

Silver – the biochemical reaction of AgNO3 react with plant broth leads
to the formation of AgNPs by following reaction
31
Ag+ NO3 - + Plant extract AgºNPs + by products
 Qualitative Phytochemical Analysis
S.No. Name of the Test Experimental Procedure Observation
1. Test for 2-3ml of extract, add two drops of violet coloration occurs.
Carbohydrates: alpha naphthol solution in alcohol
Molisch’s test shake and add conc. H2SO4 from
sides of test tube.
2. Test for Steroids: 2ml of extract add 2ml chloroform Chloroform layer
Salkowski test: and 2ml conc. H2SO4. Shake well. appears red and acid
layer shows greenish
yellow florescence.

3. Test for Add 10ml of 50% HCl to the 2ml Brick red precipitate is
Glycosides: test solution. Heat on boiling water obtained.
a)General test: bath for 30min. Add 5ml of
Fehling’s solution and boiled for
5min.

b) General test: To test solution adds 1ml of water Yellow coloration

and NaOH. occurs.
32
4. Test for 3ml extract add dil. H2SO4. Boil and Ammoniacal layer turns
Anthraquinone filter. To cold filtrate add equal volume pink or red.
Glycosides: benzene or chloroform. Shake well.
a) Borntrager’s test: Separate organic layer. Add equal volume
dil. Ammonia.

b) Modified 5ml 5% FeCl3 and 5ml dil. HCl. Heat for Ammonia cal layer shows
Borntrager’s 5 min in boiling water bath. Cool and add pinkish red color.
test: benzene or any organic solvent. Shake
well. Separate organic layer. Add dilute
ammonia.

5 Test for Alkaloids: 2-3ml test solution add few drops Orange brown precipitate.
a) Dragendroff’s test: dragendroff’s reagent.

b) Wagner’s test: 2-3ml test solution with few drops Reddish brown precipitate.
wagner’s reagent.
c) Mayer’s test: 2-3ml test solution with few drops of White or pale precipitate.
mayer’s reagent.
33
6 Test for Tannins : 3-5 ml test solution with few drops Red precipitate is
a) Lead acetate test: of 1% lead acetate. obtained.

b) Ferric chloride test: 2-3ml test solution add 2ml of Blue precipitate is
FeCl3. obtained.
7 Test for Phenolic 1-2 ml test solution adds 2ml of Green color solution
Compounds: water and 10% aqueous ferric is obtained.
Ferric chloride test: chloride solution.
8 Test for Flavonoids: Test solution, add 5ml of alcohol.
Pink coloration
Shinoda test: Few drops of conc. HCl and
occurs.
magnesium turnings.
9 Test for Terpenoids: a) Test solutions add 2ml of Reddish brown
chloroform and 1ml of conc. coloration occurs.
H2SO4.
b) Test solution add 1ml of 2,4- Yellow orange
dinitrophenyl hydrazine in 2M HCl. coloration occurs.
10 Test for Saponins: Test solution add drop of sodium Honey comb like
Foam test: bicarbonate. Shake well. froth is obtained.
34
In-silico molecular docking
analysis

35
  In-silico anti-diabetic activity by Molecular docking
studies
 Oxystelma esculentum R. Br
 Preparation of ligand (2D ChemDraw Professional 16.0 software)
mol format.

Acarbose (PubChem CID: 444254)sdf format

ligands - pdbqt format (OpenBabel GUI version 2.4.1 )

Preparation of target protein

Human pancreatic alpha-amylase (PDB ID: 1B2Y) (Nahoum et. al., 2000).
RCSB Protein Data Bank database (Thirumalaisamy et. al., 2018).
Virtual Screening Analysis
AutoDock Vina Wizard (PyRx python prescription version 0.8 software)
(Ivan et. al., 2013)
The binding affinity (kcal/mol) csv file.
Molecular Docking Study
Interpretation done by using EMBL-EBI
36 PDBsum Generate tool.
Results

37
Effect of auxin (BAP) and cytokinin (KIN) (mg/L) on micropropagation on
nodal explant of Oxystelma esculentum R. Br.

S.No PGR No of Explant No of Responded % of Respoded No of shoot per

BAP Inoculated Explant
(mg/L)
1 0.5 20 15 75 1.92±0.61

1.81±0.4
2 1.0 20 14 70
2.06±0.59
3 1.5 20 16 80

4 2.0 20 18 90 2.27±0.57
1.4±0.5
5 2.5 20 11 55
PGR
KIN

1.73±0.66
1 0.5 20 11 55

2 1.0 20 17 85 2.01±0.81
1.73±0.75
3 1.5 20 15 75
1.72±0.78
4 2.0 20 11 55
1.64±0.60
5 2.5 20 38 13 65
Effect of auxin (BAP) and cytokinin (KIN) (mg/L) on micropropagation on nodal
explant of Oxystelma esculentum R. Br.

BAP KIN
100
90
90 85
80
80 75 75
70
70
% of Responded

65
60 55 55 55
50

40

30

20

10

0
1 2 3 4 5

Hormone Concentration

39
Effect of different concentrations of IAA with IBA on root induction of
Oxystelma esculentum R. Br

No. of explants No. of roots /

% of root
Plant Growth Regulators (PGRs) Inoculated / shoot
induction
Response Mean ± SD
S. No.

IAA IBA
Nodal Nodal Nodal
(mg/L) (mg/L)

1. 1.0 0.5 11/20 55 8.0 ± 2.64

2. 1.0 1.0 17/20 85 12.16 ± 3.76

3. 1.0 1.5 15/20 75 9.4 ± 1.14

4. 1.0 2.0 11/20 55 5 ± 1.25

5. 1.0 2.5 13/20 65 5.33 ± 1.15

40
Effect of different concentrations of IAA with IBA on root induction of
Oxystelma esculentum R. Br

90
85

80
75

70
65

60
% of root induction

55 55

50

40

30

20

10

0
1+0.5 1+1 1+1.5 1+2.0 1+2.5

Hormone Concentration IAA + IBA

41
Effect of cytokinins in shoot prolifereation on Oxystelma esculentum R.Br.

A & B - Shoot initiation on MS medium

C,D & E - Shoot multiplication on 1.0+1.0

mg/L (BAP+KIN) supplemented MS
medium

F & G - Elongation of shoots on 1.0 mg/L

KIN supplemented MS medium

H - Average length of shoot (10 cm).

42
Effect of auxins in root prolifereation on Oxystelma esculentum R.Br.

A & B - Root initiation on half-strength

MS medium

C & D - Root multiplication on 0.5 mg/L

(IBA) supplemented half-strength MS
medium

E - Hardened invitro plantlets on

soil+vermicast mixture (1:1 v/v)

F – Measurement of the highest shoot

length (22cm)

43
Effect of hormone concentration on different type of Callus formation from leaf
explants of Oxystelma esculentum R. Br.

PGR
BAP (mg/l) No of
% of Callus
S. No Explant callus formation Type and color
induction
NAA 2,4-D Inoculated
(mg/l) (mg/l)

1 1.0 1.0 12/20 60 Compact yellowish green

callus

2 1.0 2.0 11/20 55 Friable white callus

3 1.0 3.0 17/20 85 Compact white callus

4 1.0 4.0 19/20 95 Compact green callus

5 1.0 5.0 13/20 65 Friable white callus

44
Effect of hormone concentration on different type of Callus formation from stem
explants of Oxystelma esculentum R. Br.

PGR
BAP (mg/l) No of
% of Callus
S. No Explant callus formation Type and color
induction
NAA 2,4-D Inoculated
(mg/l) (mg/l)

1 1.0 1.0 11/20 55 Compact yellow callus

2 1.0 2.0 13/20 65 Compact green callus

3 1.0 3.0 19/20 95 Compact green locular callus

4 1.0 4.0 15/20 75 Compact white callus

5 1.0 5.0 12/20 60 Compact green callus

45
Effect of hormone concentration on different type of Callus formation from leaf
and stem explants of Oxystelma esculentum R. Br.

Leaf Stem

100 95 95
90 85
80
% of Callus induction

75
70 65 65
60 60
60 55 55
50

40

30

20

10

0
1+1 1+2 1+3 1+4 1+5

Hormone Concentration (NAA+2 ,4-D)

46
47
 Indirect Organogenesis from Organogenic callus of Leaf Explants of
Oxystelma esculentum R.Br.

Plant growth regulators

No. of explants
% of Shoots No. of shoots /
S.No. Responded /
induction callus
2,4 –D BAP KIN Inoculated
(mg/L) (mg/L) (mg/L)

1 0.5 1.0 - 5/15 33 3.90 ± 1.22

2 0.5 2.0 - 8/15 53 3.60 ± 1.26

3 0.5 3.0 - 13/15 87 6.33 ± 2.51

4 0.5 4.0 - 11/15 73 3.00 ± 0.81

5 0.5 5.0 - 7/15 47 4.4 ± 1.14

6 0.5 - 1.0 6/15 40 3.77 ± 1.20

7 0.5 - 2.0 10/15 67 6.66 ± 1.52

8 0.5 - 3.0 9/15 60 5.16 ± 2.13

9 0.5 - 4.0 13/15 87 6.83 ± 1.72

10 0.5 - 5.0 8/15 53 3.92 ± 1.38

48
Indirect Organogenesis from Leaf callus Explants of
Oxystelma esculentum R. Br.

49
Effect of auxins on root induction from in vitro shoots obtained through calli of
Oxystelma esculentum R.Br.

No. of explants
Plant growth No. of Roots /
Responded / % of Roots induction
regulators Shoot
Inoculated
S.No.

IAA IBA
(mg/L) (mg/L)

1 0.5 1.0 5/10 50 23.81±3.65

2 0.5 2.0 7/10 70 26.54±5.55

3 0.5 3.0 9/10 90 29.85±6.93

4 0.5 4.0 6/10 60 24.14d±4.94

5 0.5 5.0 4/10 40 23.25±1.70

50
Effect Effect of auxins on root induction from in vitro shoots obtained through calli
of of Oxystelma esculentum R.Br.

90

70
60
% of Response

50
40

1 2 3 4 5

Concentration

51
52
ANTIBACTERIAL ACTIVITY

53
 Antibacterial screening of leaf extracts of Oxystelma esculentum on
pathogenic bacteria (Disc diffusion method)

Inhibition zone diameter in mm (mean  SD)

Test bacteria Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether Positive Control

Experimen Negative Experimen Negative Experimen Negative Experimenta Negative Experimen Negative Chloramphenicol
t control tal control tal control l control (30 g/disc) control (30 mcg/disc)
(30 g/disc) (30 g/disc) (30 (30 g/disc)
g/disc)

Gram-negative

Staphylococcus - - - - - - - - 7.6±1.1 - 15.3  0.57

heamolyticus
Staphylococcus lentus 11.6±1.5 - 7.6±6.8 - 9.6±2.5 - - - - - 20.6  1.55
Staphylococcus aureus 11±2.6 - 8±1 - - - 8±1 - 7.3±0.5 - 10.6  0.57
Bacillus cereus 12.3±2.5 - 7.3±7 - - - 10.3±1.5 - - - 9.0  1.00
Gram-positive

E.coli 13±3 - 12.3±4.7 - 12±6.8 - 11±2 - - - 19.0  1.00

S. marcescens 11±1 - 14±4.5 - 6±5.2 - 10.3±1.5 - - - 18.6  1.51
Enterobacteraerogens 13±2 - 20±2.6 - 8±1.5 - 8.6±2 - - - 19.6 0.57
Klebsiellapneumoniae - - 3±5.1 - - - - - 5±4.3 - 18.6  1.51
Klebsiellaoxytoca 11.3±1.5 - 11±4 - - - - - - - 20.6  1.15
Brevebacterium 11.6±1.5 - 17±3.6 - 10.6±1.1 - 13.6±1.5 - - - 13.3  1.52
paucivorans

‘–’ represents as ‘no inhibition’ 54

 Antibacterial screening of leaf extracts of Oxystelma esculentum on
pathogenic bacteria (Disc diffusion method)
Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

25

20

15
Zones of inhibition in mm

10

0
s us us us i s ns ns
icu t ol en iae ca
lyt len u re
ce
re E.c esc oge on yto ora
o us sa s ar
c
ae
r m ox uc
iv
am cc cc
u illu m er n eu ella a
e o c S. ct i p
sh c co Ba a lap leb
s m
cu hylo ylo
ro
b siel K er
iu
coc ap ph te eb
ac
t
lo St St
a
En Kl eb
phy ev
St
a Br
Tested bacteria

55
56
 Antibacterial screening of stem extracts of Oxystelma esculentum on
pathogenic bacteria (Disc diffusion method)
Inhibition zone diameter in mm (mean  SD)

Test bacteria Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether Positive Control

Experiment Negative Experiment Negative Experimental Negative Experimental Negative Experimen Negative Chloramphenico
(30 g/disc) control al control (30 g/disc) control (30 g/disc) control (30 g/disc) control l
(30 g/disc) (30 mcg/disc)

Gram-negative

Staphylococcus - - - - - - - - - - 15.3  0.57

heamolyticus

Staphylococcus lentus 10.3±1.5 - 10.3±1.5 - 11±1 - - - - - 20.6  1.55

Staphylococcus aureus 4.6±4 - 11±1 - 5±4.3 - - - - - 10.6  0.57
Bacillus cereus 12±1 - 13.6±4.1 - 13±3 - 11.6±1.5 - - - 9.0  1.00
Gram-positive

E.coli 12.3±1.5 - 12.3±2.5 - 14.6±1.5 - 10±1 - - -

19.0  1.00
S. marcescens 11±1 - 14±3 - 12±2 - 11.3±1.5 - - - 18.6  1.51
Enterobacteramnigenus 11.3±1.5 - 14±1 - 13±2.6 - - - - - 19.6 0.57
Klebsiellapneumoniae - - - - - - - - - - 18.6  1.51
Klebsiellaoxytoca - - - - - - - - - - 20.6  1.15
Brevebacterium 14.3±3.5 - 16.3±3.5 - 12.6±1.5 - - - - - 13.3  1.52
paucivorans

‘–’ represents as ‘no inhibition’ 57

Antibacterial screening of stem extracts of Oxystelma esculentumon
pathogenic bacteria (Disc diffusion method)

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

18

16

14
Zones of inhibition in mm

12

10

0
s us us us li ns s ca ns
icu nt re re co ce en iae to ra
yt e u e E. es ro
g on y o
ol sl a c c um ox iv
m cu us lus ar ra
e
lla uc
ea c c c c i l
.m te ne sie pa
h co co Ba S ac lla
p m
us lo ly o b leb iu
cc hy
h ro sie K te
r
co ap ap te leb ac
ylo St St En K eb
h e v
ap Br
St Tested bacteria

58
59
Antibacterial screening of leaf extracts of Oxystelma esculentum on pathogenic bacteria
(Plate hole diffusion method)
Inhibition zone diameter in mm (mean  SD)
Positive
Test bacteria Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether
Control
Experime Negative Experime Negative Experime Negative Experiment Negative Experimen Negative Chloramp
nt control ntal control ntal control al control (30 control henicol
(30 (30 (30 (30 g/disc) g/disc) (30
g/disc) g/disc) g/disc) mcg/disc)
Gram-negative

Staphylococcus 9.3±1.5 - 9±1.7 - 8.3±1.5 - - - - - 15.3  0.57

haemolyticus

Staphylococcus lentus 16.6±3 - 17.6±0.5 - 12.6±2.5 - - - - - 20.6  1.55

Staphylococcus aureus 10.3±1.5 - 15.6±3.7 - 7.3±0.5 - - - - - 10.6  0.57

Bacillus cereus 18±1.7 - 11.3±2 - 15±1 - 9.3±0.5 - - - 9.0  1.00

Gram-positive

E.coli 19.3±1.1 - 15.3±2.8 - 13±2 - 14.3±1.1 - - -

19.0  1.00

S. marcescens 20.3±1.5 - 13.6±1.5 - 13.6±6 - - - - - 18.6  1.51

Enterobacteramnigenus 17±2.6 - 15.6±2 - 13.3±2 - - - - - 19.6 0.57

Klebsiellapneumoniae 16.6±0.5 - 7.3±0.5 - 5.6±4.9 - - - - - 18.6  1.51

Klebsiellaoxytoca 9.6±0.5 - 14±6 - 14.3±6 - - - - - 20.6  1.15

Brevebacterium 11.3±1.5 - 15.6±3.7 - 12.6±2.5 - 14.3±2 - - - 13.3  1.52

paucivorans

‘–’ represents as ‘no inhibition’ 60

 Antibacterial screening of leaf extracts of Oxystelma esculentumon pathogenic
bacteria (Plate hole diffusion method)

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

25

20

15
Zones of inhibition in mm

10

0
s us us s li ns s ca ns
icu nt re eu co ce en iae to ra
yt e u er E. es ro
g on y o
ol sl a sc c um ox iv
m cu us illu ar ra
e
lla uc
ea c c c c .m te ne sie pa
h co co Ba S ac lla
p m
s lo ly o b leb iu
c cu hy
h ro sie K te
r
co ap ap te leb ac
ylo St St En K eb
ph e v
S ta Br
Tested bacteria

61
62
Antibacterial screening of stem extracts of Oxystelma esculentum on pathogenic bacteria
(pate hole diffusion method)

Positive
Test bacteria Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether
Control
Experime Negative Experime Negative Experim Negative Experimen Negative Experime Negative Chlorampheni
nt control ntal control ental control tal control n control col
(30 (30 (30 (30 (30 (30 mcg/disc)
g/disc) g/disc) g/disc) g/disc) g/disc)
Gram-positive

Staphylococcus 14±1 - 14.3±2.8 - 8.6±0.5 - - - - - 15.3  0.57

hemilyticus

Staphylococcus lentus 8.6±0.5 - 13.3±4.7 - - - - - - - 20.6  1.55

Staphylococcus aureus 12.6±2 - 14.6±3 - 11.3±5.7 - - - - - 10.6  0.57

Bacillus cereus 11.3±2 - 16±2.6 - 12.3±2 - - - 7.3±0.5 - 9.0  1.00

Gram-negative

E.coli - - 13.3±2. - 17±1 - - - 7±0 -

19.0  1.00
5

S. marcescens - - 7.3±0.5 - 9±1 - - - 7.3±0.5 - 18.6  1.51

Enterobacter 7.6±0.5 - - - - - - - 8±1 -

19.6 0.57
amnigenus

Klebsiella pneumoniae 9±1 - 7.3±0.5 - 5.3±4.7 - - - - - 18.6  1.51

Klebsiella oxytoca 14±1 - 12.6±3 - - - - - - - 20.6  1.15

Brevebacterium 8±1 - 15.6±0.5 - 8±1 - - - - - 13.3  1.52

paucivorans
63
‘–’ represents as ‘no inhibition’
 Antibacterial screening of stem extracts of Oxystelma exculentum on
pathogenic bacteria (pate hole diffusion method)

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

18

16

14

12

10
Zones of inhibition in mm

0
s us us s li ns s ca ns
icu nt re eu co ce en iae to ra
yt e u ce
r E. es ro
g on y o
ol sl a s c um ox iv
m cu us illu ar ra
e
lla uc
ea c c c c .m te ne sie pa
h co co Ba S ac lla
p m
us lo ly o b leb iu
cc hy
h ro sie K te
r
co ap ap te leb ac
ylo St St En K eb
h e v
ap Br
St Tested bacteria

64
65
 Antifungal activity of leaf extracts of Oxystelma esculentum on pathogenic Fungi
(Disc diffusion method)
Inhibition zone diameter in mm (mean  SD)
Ethyl acetate Ethanol Acetone Chloroform Petroleum ether
Positive
Test Fungi
Control
Experiment N Experiment N Experiment N Experiment N Experiment N C
(30 g/disc) (30 g/disc) (30 g/disc) (30 g/disc) (30 g/disc) (30mcg/disc)

Aspergillus niger - - - - - - - - - - 13±0

Cryptococcus neoformans - - 7.6±0.5 - - - - - 9.6±0.5
7.33±0.57 7.33±0.57
Candida dubliniensis 8.66±1.5 - 11.3±1.15 - 11.3±1.5 - - - - - 14.6±0.5
Candida albicans 6.33±5.5 - 7±0 - 9.6±2.5 - - - - - 13.3±0.5
Candida tropicalis 12±1 - 8±1 - - - 7.33±0.57 - - - 14±1

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

14
12
12 11.311.3
Zones of inhibition in mm

10 9.6
8.66
7.6 8
8 7.33 7.33 7 7.33
6.33
6
4
2
0 0 0 0 0 0 0 0
0
Aspergillus niger Cryptococcus neoformans Candida dubliniensis Candida albicans Candida tropicalis

Tested fungi
66
 Antifungal activity of stem extracts of Oxystelma esculentum on pathogenic Fungi
(Disc diffusion method)
Inhibition zone diameter in mm (mean  SD)
Ethyl acetate Ethanol Acetone Chloroform Petroleum ether Positive
Test Fungi Control
Experiment N Experiment N Experiment N Experiment N Experiment N C
(30 g/disc) (30 g/disc) (30 g/disc) (30 g/disc) (30 g/disc) (30mg/disc)

Aspergillus niger 8±1 - - - - - - - - - 13±0

Cryptococcus neoformans - 12±1 - - 7.3±0.5 - - - 9.6±0.5
- 8±1

Candida dubsliniensis 7.33±0.57 - 11.33±2 - - - - - - - 14.6±0.5

Candida albicans 11.3±1.5 - 12±1 - 8±1 - 9±1 - - - 13.3±0.5
Candida tropicalis 9.3±1.5 - 11.3±2.5 - 9.6±1.1 - - - - - 14±1

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

12
Zones of inhibition in mm

10
8
6
4
2
0

Tested fungi

67
68
 Antifungal activity of leaf extracts of Oxystelma esculentum on
pathogenic Fungi (Plate hole diffusion method)
Inhibition zone diameter in mm (mean  SD)
Ethyl acetate Ethanol Acetone Chloroform Petroleum ether Positive
Test Fungi Control
Experiment N Experiment N Experiment N Experiment N Experimen N C
(30 g/disc) (30 g/disc) (30 g/disc) (30 g/disc) t (30mcg/
(30 g/disc) disc)

Aspergillus niger - - - - - - - - - - 13±1

Cryptococcus - - 9.6±0.5 - - - - - 15±1.5
9.3±0.57 7.0±1.0
neoformans
Candida dubliniensis 6.3±0.57 - 7.3±1.1 - 9.3±0.57 - - - - - 14.6±0.5
Candida albicans - - 11±1.5 - 6.5±0.7 - 8.3±0.5 - - - 14±1
Candida tropicalis 13.0±9.8 - 12.0±8.4 - - - - - - - 12±0.5

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

14
Zones of inhibition in mm

12
10
8
6
4
2
0
Aspergillus niger Cryptococcus neoformans Candida dubliniensis Candida albicans Candida tropicalis

Tested fungi

69
 Antifungal activity of Stem extracts of Oxystelma exculentum on pathogenic Fungi
(Plate hole diffusion method)
Inhibition zone diameter in mm (mean  SD)
Ethyl acetate Ethanol Acetone Chloroform Petroleum ether Positive
Test Fungi Control
Experiment N Experiment N Experiment N Experimen N Experiment N C
(30 g/disc) (30 g/disc) (30 g/disc) t (30 g/disc) (30mg/disc)
(30
g/disc)

Aspergillus niger - - - - - - - - - 13±1

Cryptococcus neoformans - - 12.3±1.5 - 10.6±2 - - 15±1.5
12.0±9.5 10.0±4.2

Candida dubsliniensis 12.6±0.5 - 7.3±0.5 - 10±1 - - - - 14.6±0.5

Candida albicans 11.0±7.0 - 10±1 - 10.6±2 - 8.6±0.5 - - 14±1
Candida tropicalis - - 8± 1 - 9±1 - - - - 12±0.5

Ethyl Acetate Ethanol Acetone Chloroform Petroleum ether

14
Zones of inhibition in mm

12
10
8
6
4
2
0
Aspergillus niger Cryptococcus neoformans Candida dubliniensis Candidaalbicans Candida tropicalis

Tested fungi

70
71
Anti-oxidant Activity

72
 Scavenging Activity
IC50 Value (µg / ml)
Solvents used and Control DPPH SO
Ethanol 40.32288 27.062
Ethyl acetate 16.62041876 30.552
Acetone 103.816 19.838
13.23219
Ascorbic acid 11.347

DPPH SO

120
Ic 50 value of activity

100
80
60
40
20
0
Ethanol Ethyl acetate Acetone Ascorbic acid

73
(Phosphomolybdenum Assay, Ferric Reducing Antioxidant Power
(FRAP) Assay and Metal Chelating Activity)
Gram Equivalent (mg /gE)

Solvents used and MC

Control PHM FRAP
EDTA equivalence / mg
Ascorbic acid equivalence /g extract mmol Fe (II) equivalent/ mg extract
extract

Eethanol 145.70 ± 13.45 1680.90± 322.45 691.00±22.56

Ethyl acetate 129.22 ± 6.26 1525.67± 360.26 751.83± 18.87
Acetone 140±11.34 492.33± 38.90 612.94± 28.44

Ascorbic acid 147.19 ± 0.64 1127.10 ± 11.64 359.33± 0.00

PHM FRAP MC
1800
1600
1400
1200
1000
800
600
400
200
0
Eethanol Ethyl acetate Acetone Ascorbic acid

74
Anti-diabetic Activity

75
α-Amylase activity of Oxystelma esculentum (in percentage)
Concentration ug/ml Acetone Ethyl acetate Ethanol Acarbose

47.34 46.28 44.79 41.71

10 ug/ml

48.83 51.8 53.71 57.82

20 ug/ml

54.14 53.92 57.11 68.44

30 ug/ml

55.2 54.77 61.35 79.14

40 ug/ml

56.05 56.9 62.63 90.76

50 ug/ml

Acetone Ethyl acetate Ethanol Acarbose

100
90
80
percentage of activity

70
60
50
40
30
20
10
0
10 20 30 40 50
α-glucosidase activity of Oxystelma esculentum (in percentage)
Acetone Ethyl acetate Ethanol Acarbose
Concentration ug/ml

39.06 3.6 22.29 42.5

10 ug/ml

44.79 12.31 27.38 58.34

20 ug/ml

51.16 25.26 45.85 70.6

30 ug/ml

57.53 28.66 57.96 82.05

40 ug/ml

78.98 40.97 65.39 92.88

50 ug/ml

Acetone Ethyl acetate Ethanol Acarbose

100
90
80
percentage of activity

70
60
50
40
30
20
10
0
10 ug/ml 20 ug/ml 30 ug/ml 40 ug/ml 50 ug/ml

77
Anti-diabetic activity of Oxystelma esculentum
IC50 Value (µg / ml)

Solvents used and Control

α-amylase α-glucosidase

Acetone 20.28163 25.35105

Ethyl acetate 18.70715 60.56318

Ethanol 17.96143 35.33139

Acarbose 15.28387 14.51514

78
GREEN SYNTHESIS OF SILVER
NANOPARTICLES

79
Fig-20: UV-Spectrum of Synthesized Silver Nanoparticles
80
 100

90
445.15cm-1

2079.06cm-1
80

70
661.50cm-1

60

50
%T

40
1638.45cm-1

30

20
3436.82cm-1

10

0
4000 3500 3000 2500 2000 1500 1000 500 400
cm-1
Name Description
Plant-II-OX-E-

Fig -21: FT-IR Spectrum of Synthesized Silver Nanoparticles

81
Characteristic IR absorption frequencies of organic functional group

Sl. No Wave length Cm-1 Molecular motion Functional Groups

1 446.15 Cm-1 S-S stretch Aryl disulfides

2 661.50 Cm-1 C-H bend Alkyne

3 1638.45 Cm-1 C=C stretching alkene

4 2079.06 Cm-1 N=C=S stretching isothiocyanate

5 3436.82 Cm-1 N-H stretching primary amine

82
 SEM and DLS Analysis of Synthesized Silver Nanoparticles

Fig -22: FE-SEM image Fig-23: DLS (Dynamic Light Scattering)

83
EDAX image of Synthesized Silver Nanoparticles
cps/eV
3.5

3.0

2.5

2.0
Ag
Cl
1.5 C O Cl Ag

1.0

0.5

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
keV

84
 Antibacterial activity by synthesized silvernanoparticle

Disc Diffusion Plat hole Diffusion

Test bacteria
Method Method

Staphylococcus haemolyticus - 12.33 ± 0.5

Staphylococcus lentus 8.66 ± 1.5 8.66 ± 1.1
Staphylococcus aureus 9±1 10.33 ± 0.5
Bacillus cereus - 11 ± 2
Escheritia coli 8±1 11.33 ± 1.52
Serratia marcescens - 7.66 ± 1.15
Enterobacter amnigenous - 9 ±1
Klebsiella pneumonia 9±1 10 ± 1
Klebsiella oxytoca 4.66 ± 4 7.66 ± 0.57
Brevibacterium paucivorans 11 ± 1 11± 1

85
Antibacterial activity by synthesized nanoparticle from leaf extracts of
Oxystelma esculentum

Disc Diffusion Method Plat hole Diffusion Method

14

12

10

86
87
Qualitative Phytochemical Studies

88
 Preliminary phytochemical screening of different leaf extracts of
Oxystelma esculentum R. Br.

S. No PhytoConstituents Ethyl Acetate Ethanol Acetone

1 Carbohydrates + + +

2 Reducing Sugar + + +

3 Proteins + + +

4 Amino acids + + +

5 Steroids + - +

6 Alkaloids + + +

7 Flavonoids + + +

89
(+) Present (-) Absent
 Preliminary phytochemical screening of different leaf extracts of
Oxystelma esculentum R. Br.

8 Phenolic compounds + + +

9 Terpenoids + + +

10 Saponins + - +

11 Glycosides + + +

12 Non-reducing sugars + - -

13 Cardiac glycosides + + +

14 Tannins + + +

15 Saponin glycosides - - -

90
(+) Present (-) Absent
GC-MS chromatogram of O. esculentum
ethanol leaf extract

91
 Phytoconstituents of O. esculentum R. Br.
screened by CG-MS and their biological activity
Sl. Rtn. % Mol. Mol.
Compound Name Biological Activity Reference
No Time Area Formula Wt.
1 P-Chloroamphetamine 6.107 0.38 C9H12ClN 169 - -

Farina et al.,
Antimalarial, antifungal, 2014;
2 1-Heptadecanol (Aliphatic alcohol) 15.921 0.08 C17H36O 256
Antioxidant, Antidiabetic Ramalakshmi,201
9

Yogeswari et al.,
3 Pentadecane (Aliphatic hydrocarbon) 16.096 0.13 C15H32 212 Antibacterial 2012; Rahbar et
al., 2012

Markus et al.,
Antimicrobial, anticancer, 2017; Osaro-
Cycloheptasiloxane, Tetradecamethyl-
4 17.526 0.9 C14H42O7Si7 518 Antioxidant Matthew et al.,
(Organo Silicone compound)
Antiseptic 2020

Osaro-Matthew et
5 Cyclooctasiloxane, Hexadecamethyl- 20.771 1.03 C16H48O8Si8 592 Antimicrobial
al., 2020

Markus et al.,
6 Cyclononasiloxane, Octadecamethyl- 32.324 2.02 C18H54O9Si9 666 Antioxidant
2017

Markus et. al.,

2,2,4,4,6,6,8,8,10,10,12,12,14,14,16,16,18,18,2 C20H60O10Si1 Antioxidant
7 26.07 0.54 740 2017; Bratty et.
0,20-Icosamethylcyclodecasiloxane 0 Antimicrobial
al., 2020
92
 Antibacterial Gavamukulya
antioxidant, et.al., 2015;
hemolytic, anti- Teh et. al.
Hexadecanoic Acid, 2-Hydroxy-1-(Hydroxymethyl)Ethyl Ester 34.6
8 0.3 C19H38O4 330 androgenic, 2017; Tyagi
(Palmitoyl glycerol-amino compound) 6
nematicide and and Agarwal,
pesticidal; 2017
antimicrobial

37.2 Murugesu et.

9 Octadecanoic Acid, 2,3-Dihydroxypropyl Ester 0.29 C21H42O4 358 Antidiabetic
55 al., 2018

Hepatoprotective,
38.9 C24H72O1 Bratty et. al.,
10 Tetracosamethyl-Cyclododecasiloxane 4.34 888 antispasmodic,
18 2Si12 2020
antirheumatic

Yan et.al.,
38.5 89.4 2015
11 Solanesol C45H74O 630 Antibacterial, antiviral
9 2 Li and chase,
2010

Antibacterial, Ogunlesi et.

1,2-Benzenedicarboxylic Acid, Dibutyl Ester (Plasticizer 26.5 Antifouling, al., 2009;
12 0.22 C16H22O4 278
compound) 24 Antimicrobial, Mary and
Pesticide, Repellent. Giri, 2018

Antimicrobial,
1,2-Benzenedicarboxylic Acid, Disooctyl Ester (Plastilizer
34.8 Solvent, Plastilixer, Mary and
13 compound ) 0.35 C24H38O4 Pesticide, Repellent.
72 Giri, 2018

93
 2D confirmations of selected phytoligands

Cl
NH2

a) p Chloramphetamine

HO

b) 1-Heptadecanol

c) Pentadecane

OH
O

OH
O

d) Hexadecanoic acid-2-hydroxy-1-(hydroxymethyl)ethyl ester

O OH

OH

e) Octadecanoic acid-2,3-dihyroxypropyl ester

HO

f) Solanesol
94
Virtual Screening Results using PyRx tool

Rank for
Ligand Binding Affinity best drug

p Chloramphetamine -6.3 3

1-Heptadecanol -5.9 4

Pentadecane -5.7 5

Hexadecanoic acid-2-hydroxy-1-(hydroxymethyl)ethyl ester -5.9 4

Octadecanoic acid-2,3-dihyroxypropyl ester -6.7 2

Solanesol -12.3 1

1b2y_acarbose -7.7 standard

95
Docking pose of the Solanesol-1B2Y
complex

96
LigPlot interactions representing the binding residues
involved the docked

97
Works to be done
 Compound Interpretation

98
Published Articles
R Sabitha, T Francis Xavier, S Balavivekananthan, A Freeda Rose 2021. An

influence of coco peat and vermi compost on successful Ex vitro rooting of

micropropagated plantlets of Oxystelma esculentum R. Br. International Journal of

Botany Studies 6(5): 1520-1526. (web of science - RJIF 5.12)

R Sabitha, T Francis Xavier, S Balavivekananthan, A Freeda Rose 2021. Evaluation

of antibacterial activity of leaf and stem extracts of Oxystelma esculentum R. Br.,

against selected pathogenic bacteria. International Journal of Botany Studies 6(5):

1527-1530. (web of science) - RJIF 5.12

99
Under Review Article

South African Journal of Botany – Impact factor 2.3

R Sabitha, T Francis Xavier, S Balavivekananthan. In vitro pharmacological

investigations of Oxystelma esculentum R.Br. and in silico molecular docking

analysis of its leaf constituents on diabetic related target

100
101
102
103