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.