-
5. RESULTS AND DISCUSSIONS
5.1. Ash values:
The ash values such as total ash, acid insoluble, water soluble and
sulphated ash of Talinum Cuneifolium and Flemingia Wightiana was determined
and the results were shown in Table: 5.1.
Table: 5.1 Ash values of TC and FW
.
Figure: 5.1. Ash values Talinum Cuneifolium and Flemingia Wightiana
Types of Ash % Yield of TC % Yield of FW
Total ash 20.7 5.7
Acid insoluble ash 14.6 3.1
Water soluble ash 12.5 2.5
Sulphated ash 16.7 10.3
-
5.2. Elemental Analysis:
Now a days the implications of heavy metals are becoming very vital. The
ingestion of these might be beneficial or harmful depending upon the elements
present. The toxic elements might develop cumulative or genetic toxicity and
would also be carcinogenic in nature.
Macronutrients:
Sodium, potassium and magnesium were quantified using a Inductively
Coupled Plasma Optical Emission Spectrometer (ICP-OES), following standard
protocol and the results were tabulated in Table: 5.2.
Table: 5.2 Macronutrients of TC and FW .
Each value represented in ppm
Toxic heavy metal content:
The toxic elements like Arsenic, Lead, Palladium and Mercury were
estimated by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-
OES) and results were tabulated in Table: 5.3. All the observations confirm the
safety for the preclinical and clinical studies.
Metals TC FW
Magnesium 33013.7 24575
Potassium 69235.6 47444
Sodium 42469.8 37897
-
Table: 5.3 Toxic Heavy metal contents of TC and FW
Each value represented in ppm Discussions: Talinum Cuneifolium and Flemingia Wightiana contain ash
content. It is due to the presence of high inorganic content. However, the ash
content is possibly due to the Na+ and Ca2+salts which are not harmful. Heavy
metals are being spoken out vary widely in the global scenario, due to the
recent episodes of a few Indian Ayurvedic formulations which have been found
to have heavy metals more than that of the permissible level as advised by
W.H.O. and F.A.O. of U.S.A.137. The percentage of macronutrients like sodium,
potassium and magnesium were within the stipulated limits. Toxic heavy
metals like arsenic, lead, palladium and mercury were within the limit and
ensure the safety of the study138.
5.3. Extractive values:
The percentage yields of the extractives of Talinum Cuneifolium and
Flemingia Wightiana were as follows (Table: 5.4)
Table: 5.4 Extractive values of Talinum Cuneifolium and Flemingia Wightiana.
Metals TC FW
Arsenic 0.151 0.009
Lead 0.231 0.049
Palladium 0.325 0.015
Mercury 0.212 0.075
-
Figure: 5.2. Extractive values of Talinum Cuneifolium and
Flemingia Wightiana.
Discussions: As a part of this study, initially plant extractives were analysed to
estimate the percentage yield of individual extracts and found that, the yield
was abundant in ethanol rather than chloroform, hexane and water. Due to the
high polarity of ethanol most of the chemical constituents of extracts would be
dissolved in it and thus percentage yield was increased tremendously than
other solvents. The percentage yields of extracts in ethanol are EETC -17.5%
and EEFW -15.3% respectively. Due to higher yield in ethanol, ethanolic
extracts were selected for further studies.
5.4. Preliminary phytochemical screening :
Extractives % Yield of TC % Yield of FW
Hexane 3.7 1.9
Chloroform 2.3 2.6
Ethanol 17.5 15.3
Water 5.76 3.8
-
The preliminary phytochemical studies of extractives of Talinum
Cuneifolium and Flemingia Wightiana were as follows (Table: 5.5)
Table: 5.5 Preliminary phytochemical screening of Talinum
Cuneifolium and Flemingia Wightiana.
Type of Phytochemical Constituents
EETC EEFW
Alkaloids + +
Carbohydrates + +
Flavonoids + +
Glycosides -
Tannins/phenols + +
Proteins + +
Steroids + +
Saponins -
Triterpenoids - +
+ :Indicate presence of PhytochemicalConstituents
: Indicate absence of PhytochemicalConstituents
Discussion: Preliminary phytochemical studies of EETC confirmed the
presence of alkaloids, flavonoids, tannins, steroids, carbohydrates, proteins
and in EEFW demonstrated the presence of alkaloids, triterpenoids, flavonoids,
tannins, steroids, carbohydrates and proteins.
5.5. Antimicrobial activity of EETC and EEFW:
Measurement of zone of inhibition:
Following incubation, the plates were inspected to identify the zone of
inhibition. The diameter of zone of inhibition of each extracts and standard
discs of different concentrations was recorded with the help of zone measuring
-
scale (Hi-Media). Zone of inhibition in different organisms were shown in Table:
5.6.
The EETC showed dose dependent activity in gram positive
microorganisms like Staphylococcus aureus and Micrococcus luteus at 20, 40,
60, 80 and 100 µg/ml but in case of Bacillus subtilis, only at 80 and 100
µg/ml. In gram negative microorganism like Salmonella typhi, Klebsiella
pneumoniae, Escherichia coli and Pseudomonas aeruginosa antibacterial activity
was observed at all the concentrations mentioned.
The EEFW showed good activity in gram positive microorganism like
Staphylococcus aureus, Micrococcus luteus and in gram negative microorganism
like Salmonella typhi, Klebsiella pneumoniae, Escherichia coli and Pseudomonas
aeruginosaat 20, 40, 60, 80 and 100 µg/ml. But in Bacillus subtilis it was
observed only at 80 and 100 µg/ml.
Table: 5.6 Zone of inhibition (in mm) of EETC and EEFW at different concentration in various micro-organisms.
Organism conc.
Sample
Zone of inhibition in mm
20 40 60 80 100
Staphylococcus aureus
EETC
EEFW
9
8
9
9
9
10
13
10
14
11
Control 1 mm
Cefazoline 26 (30 µg)
Micrococcus luteus
EETC
EEFW
9
7
10
9
12
9
12
11
14
13
-
Control 1 mm
Meropenem 24 (10 µg)
Bacillus subtilis
EETC
EEFW
-
-
-
-
-
-
10
8
11
10
Control 1 mm
Cefepime 24 (30 µg)
Salmonella typhi
EETC
EEFW
9
6
11
6
13
10
14
11
17
11
Control 1mm
Kanamycin 25 (30 µg)
Klebsiella pneumonia EETC
EEFW
10
8
12
10
13
12
16
13
19
15
Control 1mm
Amikacin 24 (30 µg)
Escherichia coli
EETC
EEFW
10
10
12
12
13
14
16
15
19
18
Control 1 mm
Ofloxacin 23 (30 µg)
Pseudomonas
aeruginosa
EETC
EEFW
10
10
11
10
13
12
17
14
19
16
Control 1 mm
Levofloxacin 24 (5 µg)
-
Discussions: The present study demonstrates antibacterial effect of ethanolic
extract of Talinum Cuneifolium and Flemingia Wightiana. Antibacterial effect
was mainly observed in gram-negative organisms like E.coli, Pseudomonas
aeruginosa, klebsiella pneumoniae and Salmonella typhimurium. In case of
gram-positive bacteria both the extracts showed good activity against S aureus,
Microccocus luteus, and moderate effect against Bacillus subtilis. Various
concentrations of ethanolic extract was prepared and screened for anti-
microbial activity. As the concentration of plant extract increased the potency
of anti-microbial effect in terms of zone of inhibition was also found to be
increased. These results were in accordance with work of Tanja et.al139 and
Nagesh K.S et.al140 ,
People have extensively applied the use of plants to treat diseases,
including infectious ones. Plant extracts and phytochemicals, with known anti-
microbial properties, can be of great significance in therapeutic treatments. In
the last few years, a number of studies have been conducted by many
researchers to prove such efficiency. Many plants have been used because of
their anti-microbial traits, which are due to compounds synthesized in the
secondary metabolism of the plant. These products were known as active
substances. Various researchers reported different phytoconstituents like
tannins, flavonoids and glycosides were responsible for their potent
antibacterial activity141-143.
-
Antibacterial activity shown by Talinum Cunefolium may be due to the
presence of flavonoids, glycosides and tannins. Whereas, the antibacterial
activity of Flemingia Wightiana may be due to its unique phytoconstituents like
flavonoids, amino sugars and amino acids. This statement was supported by
findings of Iffet S et.al144 reported the invitro antimicrobial activity of various
amino acids like glycine, alanine etc.
The bacterial strains of Bacillus subtilis did not respond to both crude
extracts at the concentration of 20 g, 40 g and 60 g whereas the higher
concentration of both plants i.e. 80 g, 100 g showed good activity. The
variation of antibacterial activity between both extracts might be due to
discrepancy in distribution of anti-microbial substances, which varied from
plant to plant. Similar observations were made by Jansoka et al145 who
screened the ethanolic extracts of 10 medicinal plants against both Gram
negative as well as Gram-positive pathogens.
5.6. Acute toxicity study:
Acute toxicity of ethanolic extract of Talinum Cuneifolium and Flemingia
Wightiana results were shown in Table: 5.7
Table: 5.7 Toxicity studies of Talinum Cuneifolium and Flemingia
Wightiana
-
Treatment Dose
mg/kg
No. of animals
No. of Survival
No. of death
Percentage
of mortality L.D50
Control 1% NaCMC 10 10 0 0 -
EETC &
EEFW 100 10 10 0 0
200 10 10 0 0
400 10 10 0 0
800 10 10 0 0
1600 10 10 0 0
3200 10 10 0 0
6400 10 10 0 0 >>66 6.4g/kg
body Wt.
It has been found that these extracts are safe to use in animals
even at a dose 6.4 gm/kg orally. Body weight before and after administration of
both the extracts as per schedule were noted and any changes in skin, fur,
eyes, mucous membranes, behaviour pattern etc. were observed. No sign of
tremors, convulsions, salivation, diarrhoea and coma were seen.
5.7. Effect of EETC and EEFW on carrageenan inflammation:
The percentage inhibition of edema of standard drug ibuprofen at dose
10 mg/kg at 3, 6, 24 hours were 53.03%, 61.42%, 87.50% respectively. The
percentage inhibition of edema at 200 mg/kg of EETC at 3, 6, 24 hours were
46.96%, 54.28%, 65.62% respectively. At 3, 6, 24 hours, 200 mg/kg EEFW the
percentage inhibition of edema were 45.45%, 52.85%, 62.50% respectively. The
percentage inhibition of edema at 400 mg/kg of EETC at 3, 6, 24 hours were
-
51.51%, 60.00%, 78.13% and with 400 mg/kg of EEFW at 3, 6, 24 hours were
50.00%, 58.57%, 71.87% respectively. From these results it was found that
both the extracts have antiinflammatory activity in dose dependent manner,
but EETC at 400 mg/kg showed comparable antiinflammatory activity with
that of standard drug Ibuprofen.
Table: 5.8 Antiinflammatory effect of EETC and EEFW on
carrageenan induced rat paw edema.
Group Treatment Edema volume (ml)
0 h 3h 6h 24h
1 Control 2 ml of
1% NaCMC
0.8±0.12 1.46±0.44 1.50±0.039 1.12±0.08
2
Ibuprofen
10 mg/kg
0.71±0.02
1.02±0.04*
[53.03]
0.98±0.09**
[61.42]
0.75±0.03*
[87.50]
3 EETC 200 mg/kg 0.86±0.02
1.21±0.12
[46.96]
1.18±0.08*
[54.28]
0.97±0.02*
[65.62]
4
EETC 400 mg/kg 0.82±0.04
1.14±0.04**
[51.51]
1.10±0.02**
[60.00]
0.89±0.03**
[78.13]
5
EEFW 200 mg/kg 0.82±0.01
1.18±0.05**
[45.45]
1.15±0.05**
[52.85]
0.94±0.03*
[62.50]
6 EEFW 400 mg/kg 0.83±0.03 1.16±0.08**
[50.00]
1.12±0.06*
[58.57]
0.92±0.08**
[71.87]
Each value represents the mean ± SEM (n=6).*P
-
Figure: 5.3 Antiinflammatory effect of EETC and EEFW on
Carrageenan induced rat paw edema.
5.8 Effect of EETC and EEFW on Analgesia:
Acetic acid induced writhing method:
Intraperitoneally injected acetic acid produced abdominal contractions
which were characterized by a stretching response. The percentage of inhibition
of writhing of EETC at dose 200 and 400 mg/kg were found to be 48.13% and
67.70% respectively. The percentage of inhibition of writhing of EEFW at dose
200 and 400 mg/kg were found to be 42.33% and 62.07% respectively. The
analgesic effect of EETC and EEFW were comparable with that of standard
drug Aspirin at dose 100 mg/kg and the percentage of inhibition of writhing of
Aspirin was found to be 76.94%. The results were shown in Table: 5.9 and
Figure: 5.4.
-
Table: 5.9 Effect of EETC and EEFW by acetic acid induced writhing
method in mice.
Group Treatment No. of
Writhing
% of
Inhibition
1
Control 2 ml of 1%
NaCMC
66.18±3.66
2
Aspirin 100 mg/kg
15.26± 1.29
76.94
43
EETC 200 mg/kg
34.33±1.30*
48.13
4
EETC 400 mg/kg
21.82±2.20*
67.70
5
EEFW 200 mg/kg
38.16±1.17*
42.33
6 EEFW 400 mg/kg
25.1±1.26*
62.07
Each value represents the mean ± SEM (n=6) *P
-
Figure: 5.4 Effect of EETC and EEFW by acetic acid induced
writhing method in mice.
Discussions:
Pain and inflammation is associated with clinical condition like arthritis,
cancer, and vascular diseases. Various literatures recommend the use of
natural products in various traditional medical systems to treat pain and
inflammation146. In the present study the EETC, EEFW demonstrated analgesic
and anti-inflammatory activity at two different dose levels in carrageenan
induced rat paw edema for inflammation. In present study, both EETC and
EEFW extracts showed significant reduction in writhing response at both the
doses. In acetic acid model, EETC was found more active than EEFW at both
doses and results were comparable to the standard analgesic drug aspirin.
Chemical (acetic acid induced writhing) method was used to detect
central and peripheral analgesics. Acetic acid induced writhing test was used
for detecting both central and peripheral analgesics. In our study, both extracts
-
showed significant analgesic activity. However, the analgesic activity of both
extracts were found to be more significant in acetic acid induced model and
thus it appears that the test drugs acts by inhibiting predominantly the
central pain mechanism.
In carrageenan induced rat paw edema model, the anti-inflammatory
effect of standard drug Ibuprofen 10 mg/kg, EETC and EEFW at two different
doses 200 and 400 mg/kg was observed upto 24 hrs after carrageenan
challenge. Carrageenan is known inflammatory agent and cause increase in
prostaglandins and bradykinins synthesis at various time intervals.
Carrageenan induced paw edema has been reported to have more than one
phase and the initial has been attributed to the release of histamine and
serotonin, the maintenance of edema during the plateau phase is caused by
kinin like substance and the second accelerating phase of swelling is due to
prostaglandin like substances147.
Administration of EETC, EEFW at 200 mg/kg and 400 mg/kg showed
decrease in paw oedema volume from 3 to 24 hrs. Interestingly both dose levels
of both extract exhibited similar pattern in reducing carrageenan induced paw
edema from 3rd hr to till the end of the experiment.
This observation clearly showed that the effect of various
phytoconstituents of both the extracts might interact with the prostaglandins
and leukotrienes spurt. Various researchers reported that flavanoids inhibit
eicosanoids synthesis by inhibiting both cyclooxygenase and lipoygenase
activities148-149, as well as hamper the non enzymatic peroxidation of
-
polyunsaturated fatty acids required for the activation of these oxygenases150.
Quercetin and other flavonoids inhibit leukotrienes synthesis and histamine,
prostaglandins release, as well as acts as superoxide scavengers151,152. These
extracts contain number of chemical constitutents like tannins, phenolic
compounds, alkaloids, among them β-Sitosterol and Quercetin in EETC, 2. (-)-
tetrahydro palmatine in EEFW in both the extracts and the presence of ortho-
dihydro groups at B-ring and –OH substitutent pattern at C5 position of
the A-ring could significantly contribute to the anti-inflammatory activities of
flavonoids and may be responsible for suppressing abdominal writhing,
inhibition of the phases of carrageenan induced paw edema, confirms the
analgesic and antiinflammatory activity of the extracts153,154.
5.9. Spectral Analysis:
IR spectra of isolated compound TC-1:
IR (KBR)
OH : 3426, 1056 cm-1
Hydrogen Skeleton : 2935, 2852, 1706, 1462 cm-1
-CH=C= : 965, 802 cm-1
NMR spectra of isolated compound TC-1:
1H NMR (DMSO-d6).
0.8-2.3 : (46 H)
3.5 : (m, 2H, =CH-CH2-)
-
5.6 : (d, 1H, -CH=C=)
Mass spectrum of isolated compound TC-1:
Mass spectrum (EI. MS): m/z 414 (M+), m/z 396 (M-18), 383 (M-33), 273
(M-side chain), 255 (M-side chain-18), 231 (M-side chain-42), 213 (231-H2O).
All the recorded data such as IR, 1HNMR and mass spectrum
conclusively prove that the isolated steroidal compound may be “β-
Sitosterol”
Figure: 5.5. Structure of Isolated Fraction TC-1 (β-Sitosterol)
H O
-
IR spectra of isolated compound TC-2:
IR (KBR)
-OH : 3450 cm-1
-C=O : 1645 cm-1
-C=C= : 1610, 1505 cm-1
NMR spectra of isolated compound TC-2:
1H NMR (DMSO-d6).
3-3.4 : (BS, 5H, -OH)
6.2 : (d, 1H, -H-6)
6.4 : (d, 1H, -H-8)
6.8 : (d, 1H, -H-5’)
7.5 : (d, 1H, -H-6’)
7.6 : (d, 1H, -H-2’)
Mass spectrum of isolated compound TC-2:
Mass spectrum (EI. MS): m/z 303.11 (M+)
All the recorded data such as IR, 1HNMR and mass spectrum
conclusively prove that the isolated steroidal compound it may be “Quercetin”
O
OH
OH
OH
HO
OH O
Figure: 5.6. Structure of Isolated Fraction TC- 2 (Quercetin)
-
IR s
pectr
a o
f is
ola
ted c
om
poun
d T
C-
2
-
1 HN
MR
spectr
a o
f is
ola
ted c
om
pound T
C-
2
-
EI M
ass s
pectr
a o
f is
ola
ted c
om
pound T
C-
2
-
IR spectra of isolated compound FW-1:
IR (KBr)
C=C : 1610.27, 1513.0 cm-1
C-N : 3610.0 cm-1
C-O : 1280.0, 1257.0 cm-1
NMR spectra of isolated compound FW-1:
1HNMR (DMSO-d6)
From HNMR Data Presence of 25 protons was found. Four different type
of Protons were observed Aromatic CH, Methylene CH2, Methine H, Free Methyl
CH3
Type Number Number Peaks Observed
of groups of Hydrogen
Aromatic CH 04 04 δ 6.77, δ6.73, δ 6.86, δ6.89
Methylene CH2 04 08 δ 2.60, δ 3.16, δ2.69, δ3.28
δ3.24, δ2.87 , δ2.71, δ2.64
Methine H 01 01 δ4.267
Free Methyl CH3 04 12 δ 3.53, δ3.56, δ3.51, δ δ3.77,
δ 3.79 δ3.81, δ3.85, δ 3.86, δ3.89,
δ3.92, δ3.93, δ3.95, δ 3.29
-
Mass spectra of isolated compound FW-1:
From Mass data the molecualr weight of compound was observed to be
355
The Molecular ion peak was observed at m/z 355
The Base peak was observed at m/z 354
The Isotopic peak were observed at m/z 356, m/z 357
All the recorded data such as IR, 1HNMR and mass spectrum
conclusively prove that the isolated compound may be “2. (-)- tetrahydro
palmatine”
Figure: 5.2. Structure of Isolated Fraction FW-1
( 2 (-)- tetrahydro palmatine)
N
H3CO
H3CO
OCH3
OCH3
H
2,3,9,10-Tetramethoxy-5,8,13,13a-tetrahydro-6H-isoquino[3,2-a]isoquinoline
(-)- tetrahydro palmatine2.
-
IR S
pectr
um
of
isola
ted c
om
poun
d F
W1
-
1H
NM
R s
pectr
um
of
com
poun
d F
W1.
-
Discussions:
With the help of Infra red, NMR and mass spectrums the isolated compounds
TC-1 and TC-2 were found to be β-Sitosterol and Quercetin respectively in
Mass s
pectr
um
of
com
poun
d F
W1.
-
Talinum cuneifolium. With the support of Infra red, NMR and mass spectrums
the isolated compounds FW-1 are established to be 2. (-)- tetrahydro palmatine
in Flemingia wightiana.