chapter 5shodhganga.inflibnet.ac.in/bitstream/10603/73024/12/11... · 2018. 7. 7. · chapter 5...

EVALUATION OF SELECTED PLANT EXTRACTS FOR

ANTI-AGING ACTIVITY

School of Science, SVKM’s NMIMS University Page 95

CHAPTER 5


ANTI-AGING

ACTIVITY


ANTI-AGING ACTIVITY


5. EVALUATION OF SELECTED PLANT

EXTRACTS FOR ANTI-AGING ACTIVITY

5.1 MATERIALS AND METHODS

5.1.1 Antioxidant assays

5.1.2 Enzyme assays

5.2 RESULTS AND DISCUSSION

5.3 CONCLUSION


ANTI-AGING ACTIVITY


5.1 MATERIALS AND METHODS

a) List of Reagents and Chemicals used in the experiments

Sr. No Name Make

1 Nitro blue tetrazolium (NBT) SRL Pvt Ltd

2 Nicotine adenine dinucleotide

reduced (NADH)

SRL Pvt Ltd

3 Phenazine Methosulfate (PMS) SRL Pvt Ltd

4 Tris HCl Qualigens

5 Dimethyl sulfoxide (DMSO) Qualigens

6 Ammonium persulphate (APS) Rankem

7 1. 2,2‟-Azino-bis

(3-ethylbenzothiazoline-6-sulfonic acid)

Diammonium salt (ABTS)

Sigma-Aldrich

8 Gallic acid Sigma-Aldrich

9 Potassium chloride Loba Chemie Pvt Ltd

10 Sodium di-hydrogen phosphate Himedia

11 Di-sodium hydrogen orthophosphate

dihydrate

Himedia

12 Fluorescein sodium salt Sigma-Aldrich

13 2,2-azobis (2-methyl propionamidine)

dihydrochloride [AAPH]

Sigma-Aldrich

14 () 6-hydroxy-2,5,7,8-tetramethyl

chromane-2- carboxylic acid [TROLOX]

Sigma-Aldrich

15 Clostridium histolyticum collagenase

(EC.3.4.23.3)

Sigma-Aldrich

16 N-(3-[2Furyl] acryloyl)-Leu-Gly-Pro-Ala

[FALGPA]

Sigma-Aldrich

17 Tricine Loba Chemie Pvt Ltd

18 Calcium chloride dihydrate Loba Chemie Pvt Ltd

19 Porcine pancreatic elastase

(E.C. 3.4.21.36)

Sigma-Aldrich

20 N-Succinyl-Ala-Ala-Ala-p-nitroanilide

[SANA]

Sigma-Aldrich


ANTI-AGING ACTIVITY


b) List of Instruments uded in the experiments

Sr. No Instrument Make

1 UV Visible

Spectrophotometer

LAMBDA 25 Perkin Elmer

2 Microplate Reader VERSA max, Molecular devices

3 Fluroscence Meter Bio-Tek M Quant, FLx 800

4 Microplate Reader Bio-Tek M Quant, FLx 800

5 Weighing Balance Presica

6 Microplate Reader Bio-Tek M Quant, FLx 800


ANTI-AGING ACTIVITY



A. Superoxide anion scavenging assay

Principle:

The superoxide anion scavenging activity was measured in the phenazine

methosulfate/ Nicotine adenine dinucleotide reduced- Nitro blue tetrazolium

(PMS/NADH-NBT) system. The superoxide anion derived from dissolved oxygen

from PMS/NADH coupling reaction reduces NBT. The decrease of absorbance at 560

nm with antioxidants thus indicates the consumption of superoxide anions in the

reaction mixture (Muruhan et al. 2013).

Sample preparation:

For all samples, a sample stock solution of 1000 µg/ ml was prepared. 10 mg of dried

plant extract was weighed, dissolved in 1000 µl of methanol by sonication and

volume was then made up to 10 ml. Subsequent dilutions for the assay were made as

per the requirement.

Standard solution preparation:

100 µg/ ml stock solution was prepared by dissolving 1 mg of ascorbic acid in 10 ml

of methanol. From this 20 µg/ ml, 40 µg/ ml, 60 µg/ ml, 80 µg/ ml and 100 µg/ ml

ascorbic acid solutions were prepared respectively.

Procedure:

The superoxide scavenging assay was performed according to the method of Lau et al

(Lau et al. 2002). 3 ml Tris HCl buffer (16 mM, pH 8.0) was mixed with 0.75 ml

NBT (50 µM) and 0.75 ml NADH (78 µM) solution. 0.3 ml test extract of different

concentrations were added to the mixture. The reaction was started by adding 0.75 ml

of PMS solution (10 µM). The reaction mixture was incubated at 25 ºC for 5 min, and

the absorbance was measured at 560 nm against the corresponding blank samples.

Ascorbic acid was used as a standard.


ANTI-AGING ACTIVITY


Calculations:

The % inhibition was calculated as follows:

Absorbance (control) – Absorbance (test)

% inhibition = X 100

Absorbance (control)

Statistical analysis:

The EC50 values were expressed as mean ± SD. Statistical analysis was carried out by

ANOVA followed by Bonferroni‟s Test (P < 0.05). All calculations were performed

using Graph Pad Prism (version 5.0).

B. ABTS radical scavenging assay

Principle:

ABTS (2, 2‟-azinobis-3-ethyl- benzothiozoline-6-sulphonic acid) assay is based on

the scavenging of light by ABTS radicals. An antioxidant with an ability to donate a

hydrogen atom will quench the stable free radical; a process which is associated with

a change in absorption can be followed spectrophotometrically. The relatively stable

ABTS radical has a green color and is quantified spectrophotometrically at 734 nm

(Jain & Agrawal 2008)

Sample preparation:

For all samples, a sample stock solution of 2500 µg/ ml was prepared. 12.5 mg of

sample was weighed, dissolved in 500 µl of DMSO and volume was then made up to

5 ml with 10 mM PBS, pH 7.4. Subsequent dilutions for the assay were made as

required in vehicle buffer.


100 µg/ ml stock solutions were prepared by dissolving 1 mg of Gallic acid in 10 ml

of methanol. From this 0.5 µg/ ml, 1 µg/ ml, 1.5 µg/ ml, 2 µg/ ml and 2.5 µg/ ml

ascorbic acid solutions were prepared respectively.

Procedure:

The assay is performed as per the method of Auddy et al (Auddy et al. 2003). ABTS

radical cations were produced by reacting ABTS and Ammonium per suphate (APS)


ANTI-AGING ACTIVITY


and incubating the mixture at room temperature in dark for 16 hours. In brief, the total

reaction volume contained 10 mM PBS (pH 7.4) and positive control or test solutions

of various concentrations. ABTS radical solution was added to a final concentration of

0.219 mM. The reaction mixture was mixed and immediately read at 734 nm using

microplate reader. A control reaction was carried out without the test sample. Gallic

acid was used as a positive control.

Calculations:


Absorbance (control) – Absorbance (test)

% inhibition = X 100

Absorbance (control)





C. Oxygen radical absorbance capacity (ORAC) assay

Principle:

The ORAC assay depends on the free radical damage to a fluorescent probe through

the change in its fluorescence intensity. In the present assay, 2,2-azobis (2-methyl

propionamidine) dihydrochloride[AAPH] is used as free radical generator to reduce

the fluorescence characteristics of Sodium fluorescein, which is used as the

fluorescence probe. The change of fluorescence intensity (reduction in fluorescence)

is an index of the degree of free radical damage. In the presence of an antioxidant,

there is decrease in the change of fluorescence induced by AAPH. In the ORAC

assay, the antioxidant activity of a sample is expressed relative to TROLOX, a water

soluble analog of Vitamin – E (Huang et al. 2005)

Sample preparation:

1 mg/ ml stock of sample was prepared by dissolving 5 mg of sample in 5 ml of 75

mM phosphate buffer pH 7.4. The samples were allowed to settle for 5 min and

supernatant was used for the assay. Further dilutions were made in 75 mM phosphate

buffer pH 7.4 as required.


ANTI-AGING ACTIVITY



A stock solution (100 μM) was prepared by dissolving 5 mg Trolox in 200 ml PBS.

This was further diluted 1:4 v/v in PBS to give a working solution of 20 μM.

Procedure:

The assay was performed as per the method described by Dávalos et al (Dávalos et al.

2004). A pre-incubation mixture of 140 µl contained 20 µl of test solution or

TROLOX of various concentrations and 75 mM Sodium phosphate buffer (pH 7.4).

120 µl of Sodium fluorescein (117 nM) was mixed and incubated at 37 ºC for 10

mins. Following pre-incubation, 60 µl of AAPH (40 mM) was added and mixed for

15 seconds. The reaction was carried out for 90 minutes at 37 ºC. The fluorescence

measurements were taken at 485 nm excitation and 520 nm emissions. Trolox, a

vitamin E analogue, was used as a reference standard. ORAC values were expressed

as moles TROLOX equivalent/ g of substance.

5.1.2 Enzyme Assays

A. Anti-collagenase assay

Principle of the assay:

FALGPA + Collagenase

> FAL +Gly - Pro – Ala

Clostridium histolyticum collagenase (EC 3.4.24.3) is one of the few proteinases

capable of degrading the triple-helical region of native collagen under physiological

conditions. Collagenase cleaves the X-Gly bond of collagen and synthetic peptides at

loci which contain the sequence -Pro-X- Gly-Pro-, where X can be almost any amino

acid, provided that the imino terminus is blocked. FALGPA is used as a substrate and

decrease in absorbance of the substrate after addition of enzyme was measured

spectrophotometrically at 340 nm (Van Wart & Steinbrink 1981).

Sample preparation:

For all samples, a sample stock solution of 10 mg/ ml was prepared. 100 mg of dried

plant extract was weighed, dissolved in 1 ml of Tricine buffer (50 mM) by sonication


ANTI-AGING ACTIVITY


and volume was then made up to 10 ml. Subsequent dilutions for the assay were made

as required in Tricine buffer (50 mM).


1 mg/ ml stock solution was prepared by dissolving 1mg of Catechin in 1ml of

methanol. From this 62.5 µg/ ml, 125 µg/ ml, 250 µg/ ml, 500 µg/ ml and 1000 µg/ ml

solutions were prepared respectively.

Procedure:

Collagenase inhibition assay was performed according to the method described by

Kim et al (Kim et al. 2004). Collagenase from Clostridium histolyticum (0.8 units/ ml)

and synthetic substrate, FALGPA (2 mM) were used for assay. The final reaction

mixture contained 25 μl of 50 mM Tricine buffer, 25 μl of test extract and 25 μl of 0.1

units of Clostridium histolyticum collagenase enzyme. After adding 50 μl of 2 mM

FALGPA substrate, collagenase activity was measured immediately at 340 nm using a

96 well micro plate reader. Catechin was used as a positive control.

Calculations:


Enzyme inhibition activity (%) = [1 - (B / A)] X 100

Where, A = Enzyme activity without test extract,

B = Activity in the presence of test extract.





B. Anti-elastase assay

Principle:

In anti-elastase assay, porcine pancreatic elastase was assayed spectrophotometrically

by using N-Succ-(Ala) 3-p-nitroanilide as a substrate, and the amount of p-nitroaniline

was determined by measuring the absorbance at 410 nm (Lee & Choi 1998).


ANTI-AGING ACTIVITY

School of Science, SVKM’s NMIMS University 4

Sample preparation:

For all samples, a sample stock solution of 10 mg/ ml was prepared. 100 mg of dried

plant extract was weighed, dissolved in 1 ml of Tris-HCl buffer (0.2 M) by sonication

and volume was then made up to 10 ml. Subsequent dilutions for the assay were made

as required in vehicle buffer.


1 mg/ ml stock solution was prepared by dissolving 1 mg of Catechin in 1 ml of

methanol. From this 0.031 mg/ ml, 0.0625 mg/ ml, 0.125 mg/ ml, 0.25 mg/ ml and 0.5

mg/ ml solutions were prepared respectively.

Procedure:

Elastase inhibition assay was performed according to the method described by Lee et

al (Lee et al. 1999). This assay was performed in 0.2 M Tris-HCl buffer (pH 8.0).

Porcine pancreatic elastase (PE – E.C. 3.4.21.36), was dissolved to make a 1 mg/ ml

stock solution in 0.2 M Tris-HCl buffer. The substrate N-Succinyl-Ala-Ala-Ala-p-

nitroanilide (SANA) was dissolved in buffer (0.8 mM). The test extracts were

incubated with the enzyme for 20 minutes before adding substrate to begin the

reaction. The final reaction mixture (Total 250 µl) contained 50 µl plant extract, 160

µl buffer, 20 µl enzyme and 20 µl substrate. Catechin was used as a positive control.

Negative controls were performed using Tris-HCl buffer. Absorbance was measured

immediately at 410 nm,using a 96 well micro plate reader.

Calculations:


Enzyme inhibition activity (%) = [1 - (B / A)] X 100

Where, A = Enzyme activity without sample,

B = Activity in presence of sample.






ANTI-AGING ACTIVITY


5.2 RESULTS AND DISCUSSION


Antioxidant potential of plant extracts was evaluated through their ability to scavenge

the synthetic radicals such as superoxide anion, ABTS and oxygen radicals.

A. Superoxide anion scavenging assay

In vitro antioxidant activity of plant extracts was evaluated by superoxide anion

scavenging assay where, superoxide radicals generated from dissolved oxygen by

PMS-NADH coupling can be measured by their ability to reduce NBT. The decrease

in absorbance at 560 nm with the plant extract and the reference compound Ascorbic

acid indicates their abilities to quench superoxide radicals in the reaction mixture.

Superoxide anion scavenging activity was measured for all extracts and expressed as

percentage scavenging activity.

Superoxide anion scavenging activity of Ascorbic acid is shown in Fig. 5.1. The

inhibition of O2•−

was found to be concentration dependent for Ascorbic acid.

Ascorbic acid exhibited superoxide anion scavenging activity from 26.32 ± 1.16 to

64.62 ± 1.61 %.

20 40 60 80 1000

10

20

30

40

50

60

70

80Ascorbic Acid

Conc (µg/ ml)

% S

cave

ngi

ng

Act

ivit

y

Fig. 5.1: Superoxide anion scavenging activity of Ascorbic acid


ANTI-AGING ACTIVITY


Superoxide anion scavenging activity of OT HAE and OT CAE is shown in Fig. 5.2.

Percentage scavenging activity of OT HAE was ranged from 18.20 ± 0.54 to 61.74 ±

0.54 % whereas For OT CAE, it was from 7.84 ± 2.60 to 54.01 ± 2.24%. Thus, OT

HAE showed strong superoxide scavenging activity than OT CAE.

20 40 60 80 100 1200

10

20

30

40

50

60

70

80 OT HAE OT CAE

Conc (µg/ ml)

% S

cave

ngi

ng

Act

ivit

y

Fig. 5.2: Superoxide anion scavenging activity of OT HAE and OT CAE

Superoxide anion scavenging activity of CR HAE and CR CAE is shown in Fig. 5.3.

CR HAE exhibited higher percentage scavenging activity than CR CAE at all

concentrations.

50 100 150 200 250 300 3500

10

20

30

40

50

60

70

80CR HAE CR CAE

Conc (µg/ ml)

% S

cave

ngi

ng

Act

ivit

y

Fig. 5.3: Superoxide anion scavenging activity of CR HAE and CR CAE


ANTI-AGING ACTIVITY


EC50 values of OT HAE, OT CAE, CR HAE, CR CAE and Ascorbic acid are

expressed in Table 5.1.

Table 5.1: EC50 values of Superoxide anion scavenging assay

Test Sample EC50 Value (µg/ ml)

Ascorbic acid 52.42 ± 1.94

OT HAE 68.55 ± 3.37

OT CAE 129.20 ± 25.04

CR HAE 221.27 ± 11.25

CR CAE 354.57 ± 23.79

EC50 values of OT HAE and CR HAE were found to be lower than their

corresponding cold alcoholic extracts i.e. OT CAE and CR CAE respectively (**P <

0.01, *** P < 0.001) (Fig. 5.4). These results suggest that, all plant extracts possess

superoxide anion scavenging ability and extracts obtained by Soxhlation were found

to be more potent than extracts obtained by maceration.

As Acid OT HAE OT CAE CR HAE CR CAE0

50

100

150

200

250

300

350

400 As Acid

OT HAE

OT CAE

CR HAE

CR CAE

**

***

EC

50

VA

LU

E (

g/

ml)

Fig. 5.4: EC50 values of Superoxide anion scavenging assay

Superoxide anion is a very harmful to cellular components (Hazra et al. 2008). It is a

type of ROS which is a major contributor in skin aging. Antioxidants inhibit the

production of ROS by direct scavenging and decreasing the amount of oxidants in the

cells. This prevents the aging phenomenon (Oresajo et al. 2010). Results

demonstrated all test extracts have the antioxidant potential and ability to scavenge

superoxide anion radical and thus can be incorporate in anti-aging skin care

formulations.


ANTI-AGING ACTIVITY


B. ABTS radical scavenging assay

The ABTS radical scavenging ability of all extracts was calculated from the

decolourization of ABTS

free radical cation, which was measured

spectrophotometrically at 734 nm. Interaction with the extract or standard Gallic acid

decreases the absorbance of the ABTS

free radical cation. ABTS activity was

quantified in terms of percentage inhibition of the ABTS free radical cation by

antioxidants in each sample.

ABTS radical scavenging activity of Gallic acid is shown in Fig. 5.5. Gallic acid

demonstrated the concentration dependent percentage scavenging activity. Gallic acid

showed a very strong percentage scavenging activity ranging from 21.54 ± 0.06 to

79.86 ± 0.66% at very low concentrations i.e. from 0.5 to 2.5 µg/ ml.

0.5 1 1.5 2 2.50

10

20

30

40

50

60

70

80

90

100 Gallic Acid

Conc (µg/ ml)

% S

cave

ngi

ng

Act

ivit

y

Fig. 5.5: ABTS radical scavenging activity of Gallic acid


ANTI-AGING ACTIVITY


ABTS radical scavenging activity of OT HAE and OT CAE is shown in Fig. 5.6. OT

HAE exhibited strong ABTS free radical cation scavenging activity than OT CAE.

2.5 5 10 17.5 25 37.50

10

20

30

40

50

60

70

80

90

100OT HAE OT CAE

Conc (µg/ ml)

% S

cave

ngi

ng

Act

ivit

y

Fig. 5.6: ABTS radical scavenging activity of OT HAE and OT CAE

ABTS radical scavenging activity of CR HAE and CR CAE is shown in Fig. 5.7. It

was found that both CR HAE and CR CAE demonstrated almost similar percentage

scavenging activity at 100 and 200 µg/ ml, CR CAE showed slightly higher activity

(68.29 ± 2.22 and 95.14 ± 1.96%) than CR HAE (67.37 ± 0.09 and 92.04 ± 2.15%).

5 10 25 50 100 2000

10

20

30

40

50

60

70

80

90

100 CR HAE CR CAE

Conc (µg/ml)

% S

cave

ngi

ng

Act

ivit

y

Fig. 5.7: ABTS radical scavenging activity of CR HAE and CR CAE


ANTI-AGING ACTIVITY


EC50 values of Gallic acid, OT HAE, OT CAE, CR HAE and CR CAE are expressed

in Table 5.2. EC50 value of Gallic acid was found to be 1.17 ± 0.04 µg/ ml.

Table 5.2: EC50 values of ABTS radical scavenging assay


Gallic acid 1.17 ± 0.04

OT HAE 16.76 ± 0.31

OT CAE 19.03 ± 1.34

CR HAE 59.16 ± 2.17

CR CAE 59.12 ± 6.21

EC50 values of OT HAE and OT CAE were found to be almost similar hence no

significant difference found in ABTS radical scavenging activity of OT HAE and OT

CAE, Similar results were obtained for CR HAE and CR CAE (Fig. 5.8). These

results suggest that, all plant extracts showed ABTS radical scavenging ability but

Ocimum tenuiflorum (OT) extracts were more potent than Citrus reticulata (CR)

extracts in given conditions.

Gal Acid OT HAE OT CAE CR HAE CR CAE0

10

20

30

40

50

60

70

80 Gal Acid

OT HAE

OT CAE

CR HAE

CR CAE

EC

50

VA

LU

E (

g/m

l)

Fig. 5.8: EC50 values of ABTS radical scavenging assay


ANTI-AGING ACTIVITY


C. Oxygen radical absorbance capacity (ORAC) assay

Since DPPH is not a natural free radical found in biological systems, the plant extracts

were screened for radical scavenging activity against peroxyl radicals. Peroxyl

radicals are a common free radical found in biological systems, where the formation

of peroxyl radicals is the major chain-propagation step in lipid peroxidation. The

ORAC assay is based on the detection of peroxyl radical damage to the fluorescent

probe, fluorescein, where under appropriate conditions, the loss of fluorescence in the

presence of a free radical is an index of the oxidative damage to the molecule. In the

presence of an antioxidant the inhibition of free radical damage is reflected in

protection against the decrease in fluorescence (Esterhuizen et al. 2006) .

In the ORAC assay, the antioxidant activity of a sample is expressed relative to

TROLOX (r2

= 0.995) (Fig. 5.9). The ORAC values are expressed in moles

TROLOX equivalent/gm of substance (Table 5.3). Higher the ORAC value more is

the antioxidant potential. OT HAE has strong antioxidant potential as it showed

highest ORAC value which is then followed by OT CAE, CR CAE and CR HAE.

Fig. 5.9: Standard Curve of Trolox

Tested

extracts

ORAC value

(moles TROLOX

equivalent/ gm of

substance)

OT HAE 5792

OT CAE 3307

CR HAE 1243

CR CAE 1063

Standard curve of Trolox

0 5 10 150

1000000

2000000

3000000

4000000r

2=0.995

Conc (µM)

AU

C

Table 5.3: ORAC Value of plant extracts


ANTI-AGING ACTIVITY


5.2.2 Enzyme assay

A. Anti-collagenase assay

Collagen, the major component of the skin, is degraded by the enzyme collagenase.

Inhibition of collagenase activity delays the process of forming pre-collagen fibres

and subsequently the wrinkling process (Ndlovu et al. 2013). The inhibitory effect of

test extracts on FALGPA hydrolysis catalyzed by collagenase was evaluated in anti-

collagenase assay. FALGPA is a short peptide, N-[3-(2-Furyl) acryloyl)]-Leu-Gly-

Pro-Ala that can be used as synthetic substrate for collagenase activity (Van Wart &

Steinbrink 1981).

Catechin was used as a positive standard. Collagenase inhibition activity of Catechin

is shown in Fig. 5.10. Percentage collagenase activity of Catechin was concentration

dependant and the range was obtained from 59.76 ± 0.42 to 76.79 ± 0.31%.

Catechin

62.5 125 250 500 10000

10

20

30

40

50

60

70

80

Conc (µg/ml)

% C

olla

gena

se I

nhib

ition

Fig. 5.10: Collagenase inhibition activity of Catechin


ANTI-AGING ACTIVITY


Collagenase inhibition activity of OT HAE and OT CAE is shown in Fig. 5.11. OT

HAE exhibited slightly higher anti-collagenase activity (up to 68.39 ± 0.47 %)

compared to OT CAE (up to 66.87 ± 0.22 %).

125 250 500 1000 20000

10

20

30

40

50

60

70

80 OT HAE OT CAE

Conc (µg/ml)

% C

olla

gen

ase

Inh

ibit

ion

Fig. 5.11: Collagenase inhibition activity of OT HAE and OT CAE

Collagenase inhibition activity of CR HAE and CR CAE is shown in Fig. 5.12. CR

HAE showed collagenase inhibition from 56.38 ± 1.77 to 76.55 ± 0.62 % and CR

CAE exhibited collagenase inhibition from 51.30 ± 0.85 to 70.41 ± 0.50 %

250 500 1000 2000 40000

10

20

30

40

50

60

70

80 CR HAE CR CAE

Conc (µg/ml)

% C

olla

gen

ase

Inh

ibit

ion

Fig. 5.12: Collagenase inhibition activity of CR HAE and CR CAE


ANTI-AGING ACTIVITY


EC50 values of Catechin, OT HAE, OT CAE, CR HAE and CR CAE are represented

in Table 5.4.

Table 5.4: EC50 values of collagenase inhibition assay


Catechin 75.60 ± 1.51

OT HAE 194.13 ± 2.56

OT CAE 208.23 ± 2.29

CR HAE 329.33 ± 6.38

CR CAE 466.93 ± 8.04

No significant difference observed for anti-collagenase activity of OT HAE and

OT CAE where as CR HAE was found to be more effective than CR CAE

(*** P < 0.001) (Fig. 5.13).

Catechin OT HAE OT CAE CR HAE CR CAE0

50

100

150

200

250

300

350

400

450

500 Catechin

OT HAE

OT CAE

CR HAE

CR CAE

***

EC

50

VA

LU

E (

g/m

l)

Fig. 5.13: EC50 values of Collagenase inhibition assay

Results suggest that all the tested plant extracts have an ability of collagenase

inhibition but OT HAE and CR HAE were found to be more potent and thus could be

able to protect the ECM and prevent skin aging if incorporated in anti-aging

cosmeceuticals.


ANTI-AGING ACTIVITY


B. Anti-elastase assay

Elastin is the main component of the elastic fibres of the connective tissue. In the

skin, the elastic fibre, together with the collagenous fibres form a network, developing

under the epidermis. Elastase is the only enzyme capable of degrading elastin.

Inhibition of elastase enzyme can retain the elasticity and suppleness of skin (Lee et

al. 1999). In anti-elastase assay, porcine pancreatic elastase was assayed

spectrophotometrically by using N-Succ-(Ala) 3-p-nitroanilide as a substrate, and the

amount of p-nitroaniline was determined by measuring the absorbance at 410 nm.

The inhibitory effects of four plant extracts on elastase activity were investigated and

Catechin was used as a positive control. Elastase inhibition activity of Catechin is

shown in Fig. 5.14. Elastase inhibition activity of Catechin was found to be in

between 75.19 ± 1.50 to 94.74 ± 1.50%.

Catechin

0.031 0.0625 0.125 0.25 0.50

10

20

30

40

50

60

70

80

90

100

Conc (mg/ ml)

% E

last

ase

Inhi

bitio

n

Fig. 5.14: Elastase inhibition activity of Catechin


ANTI-AGING ACTIVITY


Elastase inhibition activity of OT HAE and OT CAE is shown in Fig. 5.15.

Concentration dependent inhibition of porcein pancreatic elastase was shown by both

the test extracts. At initial concentrations, OT HAE exhibited lower percentage of

elastase inhibition than OT CAE but later, the extract showed higher activity than OT

CAE.

0.156 0.312 0.625 1.25 2.5 5 100

10

20

30

40

50

60

70

80

90

100OT HAE OT CAE

Conc (mg/ml)

% E

last

ase

Inh

ibit

ion

Fig. 5.15: Elastase inhibition activity of OT HAE and OT CAE

Elastase inhibition activity of CR HAE and CR CAE is shown in Fig. 5.16. CR HAE

showed up to 80.02 ± 1.71% elastase inhibition and was found to be more effective

than CR CAE, showed up to 72.28 ± 2.76 % elastase inhibition.

0.156 0.312 0.625 1.25 2.5 5 100

10

20

30

40

50

60

70

80

90

100 CR HAE CR CAE

Conc (mg/ml)

% E

last

ase

Inhi

bit

ion

Fig. 5.16: Elastase inhibition activity of CR HAE and CR CAE


ANTI-AGING ACTIVITY


EC50 values of Catechin, OT HAE, OT CAE, CR HAE and CR CAE are expressed in

Table 5.5.

Table 5.5: EC 50 values of anti-elastase assay

Test Sample EC50 Value (mg/ ml)

Catechin 0.012 ± 0.002

OT HAE 2.61 ± 0.13

OT CAE 4.39 ± 0.19

CR HAE 3.22 ± 0.24

CR CAE 5.09 ± 0.30

Catechin OT HAE OT CAE CR HAE CR CAE0

1

2

3

4

5

6Catechin

OT HAE

OT CAE

CR HAE

CR CAE***

***

EC

50

VA

LU

E (

mg

/ml)

Fig. 5.17: EC50 values of elastase inhibition assay

Results suggest that all the tested plant extracts have an ability of elastase inhibition

but OT HAE and CR HAE were found to be more potent in inhibiting the porcein

pancreatic elastase enzyme and thus coul be effectively use in protection of ECM

proteins.


ANTI-AGING ACTIVITY


5.3 CONCLUSION

This study revealed the anti-aging abilities of selected plant extracts through

antioxidant and anti-enzyme activities. Various in vitro antioxidant assays

demonstrated the free radical scavenging potential of plant extracts; can reduce the

oxidative stress and aging of the skin. All four plant extracts showed anti-collagenase

and anti-elastase activity. The results obtained indicate the methanolic extracts of

Ocimum tenuiflorum possesses strong antioxidant and anti-enzyme activities can be

added to cosmetics as plant-based materials having anti-ageing effects.

chapter 5shodhganga.inflibnet.ac.in/bitstream/10603/73024/12/11... · 2018. 7. 7. · chapter 5...

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